Comprehensive Inorganic Chemistry III, Third Edition (Comprehensive Inorganic Chemistry, 3) [2, 1 ed.] 0128231440, 9780128231449

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COMPREHENSIVE INORGANIC CHEMISTRY III

COMPREHENSIVE INORGANIC CHEMISTRY III EDITORS IN CHIEF

Jan Reedijk Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands

Kenneth R. Poeppelmeier Department of Chemistry, Northwestern University, Evanston, IL, United States

VOLUME 2

Bioinorganic Chemistry and Homogeneous Biomimetic Inorganic Catalysis VOLUME EDITORS

Vincent L. Pecoraro Department of Chemistry, University of Michigan, Ann Arbor, MI, United States

Zijian Guo School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

Amsterdam • Boston • Heidelberg • London • New York • Oxford Paris • San Diego • San Francisco • Singapore • Sydney • Tokyo

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge MA 02139, United States Copyright Ó 2023 Elsevier Ltd. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-823144-9

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CONTENTS OF VOLUME 2 Editor Biographies

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Volume Editors

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Contributors to Volume 2

xv

Preface

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2.01

Introduction: Bioinorganic chemistry and homogeneous biomimetic inorganic catalysis Vincent L Pecoraro and Zijian Guo

1

2.02

Siderophores and iron transport Rachel Codd

3

2.03

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction Matthew R Jordan, Matias Villarruel Dujovne, Daiana A Capdevila, and David P Giedroc

30

2.04

Metallomics and metalloproteomics Xueting Yan, Ying Zhou, Hongyan Li, Guibin Jiang, and Hongzhe Sun

53

2.05

Biomineralization Yueqi Zhao, Biao Jin, and Ruikang Tang

77

2.06

Iron-sulfur clusters – functions of an ancient metal site Sofia R Pauleta, Raquel Grazina, Marta SP Carepo, José JG Moura, and Isabel Moura

105

2.07

[FeFe]-hydrogenases: Structure, mechanism, and metallocluster biosynthesis Mohamed Atta and Marc Fontecave

174

2.08

Heme-containing proteins: Structures, functions, and engineering Osami Shoji, Yuichiro Aiba, Shinya Ariyasu, and Hiroki Onoda

194

2.09

Engineering of hemoproteins Takashi Hayashi and Shunsuke Kato

215

2.10

The biochemistry and enzymology of zinc enzymes Guillermo Bahr, Pablo E Tomatis, and Alejandro J Vila

231

2.11

Cobalt enzymes Bernhard Kräutler

268

2.12

Biological and synthetic nitrogen fixation Oliver Einsle, Tobias A Engesser, and Felix Tuczek

302

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Contents of Volume 2

2.13

Photosynthesis Junko Yano, Jan Kern, and Vittal K Yachandra

347

2.14

Bio-inspired catalysis Xinyang Zhao, Lu Zhu, Xue Wu, Wei Wei, and Jing Zhao

373

2.15

Imaging Brooke A Corbin, Jacob C Lutter, Susan A White, Enas Al-ani, Elizabeth S Biros, John P Karns, and Matthew J Allen

407

2.16

Phosphorescent metal complexes for biomedical applications Jiangping Liu, Ruilin Guan, Xinlin Lin, Yu Chen, and Hui Chao

460

2.17

Photoactive metallodrugs Huayun Shi and Peter J Sadler

507

2.18

Metallophores: How do human pathogens withdraw metal ions from the colonized host Henryk Kozlowski, Karolina Piasta, Aleksandra Hecel, Magdalena Rowinska-Zyrek, and Elzbieta Gumienna-Kontecka

553

2.19

The role of d-block metal ions in neurodegenerative diseases Yanahi Posadas, Víctor E López-Guerrero, Trinidad Arcos-López, Richard I Sayler, Carolina Sánchez-López, José Segovia, Claudia Perez-Cruz, and Liliana Quintanar

575

2.20

Metal ion interactions with nucleic acids Besim Fazliji, Carla Ferreira Rodrigues, Haibo Wang, and Roland KO Sigel

629

2.21

Metal-mediated base pairs in nucleic acid duplexes Marian Hebenbrock and Jens Müller

664

2.22

Supramolecular metal-based molecules and materials for biomedical applications Angela Casini, Roland A Fischer, and Guillermo Moreno-Alcántar

714

2.23

Metal complexes as chemotherapeutic agents KM Deo and JR Aldrich-Wright

744

2.24

Protein targets for anticancer metal based drugs Tiziano Marzo and Luigi Messori

794

2.25

Platinum anticancer drugs: Targeting and delivery Zhiqin Deng, Houzong Yao, Zhigang Wang, and Guangyu Zhu

808

2.26

Anti-cancer gold compounds Ka-Chung Tong, Pui-Ki Wan, Di Hu, Chun-Nam Lok, and Chi-Ming Che

847

EDITOR BIOGRAPHIES Editors in Chief Jan Reedijk Jan Reedijk (1943) studied chemistry at Leiden University where he completed his Ph.D. (1968). After a few years in a junior lecturer position at Leiden University, he accepted a readership at Delft University of Technology in 1972. In 1979 he accepted a call for Professor of Chemistry at Leiden University. After 30 years of service, he retired from teaching in 2009 and remained as an emeritus research professor at Leiden University. In Leiden he has acted as Chair of the Department of Chemistry, and in 1993 he became the Founding Director of the Leiden Institute of Chemistry. His major research activities have been in Coordination Chemistry and Bioinorganic Chemistry, focusing on biomimetic catalysis, molecular materials, and medicinal inorganic chemistry. Jan Reedijk was elected member of the Royal Netherlands Academy of Sciences in 1996 and he was knighted by the Queen of the Netherlands to the order of the Dutch Lion (2008). He is also lifetime member of the Finnish Academy of Sciences and Letters and of Academia Europaea. He has held visiting professorships in Cambridge (UK), Strasbourg (France), Münster (Germany), Riyadh (Saudi Arabia), Louvain-la-Neuve (Belgium), Dunedin (New Zealand), and Torun (Poland). In 1990 he served as President of the Royal Netherlands Chemical Society. He has acted as the Executive Secretary of the International Conferences of Coordination Chemistry (1988–2012) and served IUPAC in the Division of Inorganic Chemistry, first as a member and later as (vice/past) president between 2005 and 2018. After his university retirement he remained active as research consultant and in IUPAC activities, as well as in several editorial jobs. For Elsevier, he acted as Editor-in-Chief of the Reference Collection in Chemistry (2013–2019), and together with Kenneth R. Poeppelmeier for Comprehensive Inorganic Chemistry II (2008–2013) and Comprehensive Inorganic Chemistry III (2019-present). From 2018 to 2020, he co-chaired the worldwide celebrations of the International Year of the Periodic Table 2019. Jan Reedijk has published over 1200 papers (1965–2022; cited over 58000 times; h ¼ 96). He has supervised 90 Ph.D. students, over 100 postdocs, and over 250 MSc research students. Kenneth R. Poeppelmeier Kenneth R. Poeppelmeier (1949) completed his undergraduate studies in chemistry at the University of Missouri (1971) and then volunteered as an instructor at Samoa CollegedUnited States Peace Corps in Western Samoa (1971–1974). He completed his Ph.D. (1978) in Inorganic Chemistry with John Corbett at Iowa State University (1978). He joined the catalysis research group headed by John Longo at Exxon Research and Engineering Company, Corporate Research–Science Laboratories (1978–1984), where he collaborated with the reforming science group and Exxon Chemicals to develop the first zeolite-based light naphtha reforming catalyst. In 1984 he joined the Chemistry Department at Northwestern University and the recently formed Center for Catalysis and Surface Science (CCSS). He is the Charles E. and Emma H. Morrison Professor of Chemistry at Northwestern University and a NAISE Fellow joint with Northwestern University and the Chemical Sciences and Engineering Division, Argonne National Laboratory. Leadership positions held include Director, CCSS, Northwestern University; Associate Division Director for Science, Chemical Sciences and Engineering Division, Argonne National Laboratory; President of the Chicago Area Catalysis Club; Associate Director, NSF Science and Technology Center for Superconductivity; and Chairman of the ACS Solid State Subdivision of the Division of Inorganic Chemistry. His major research activities have been in Solid State and Inorganic Materials Chemistry focusing on heterogeneous catalysis, solid state chemistry, and materials chemistry. His awards include National Science Council of Taiwan Lecturer (1991); Dow Professor of Chemistry (1992–1994); AAAS Fellow, the American Association for the Advancement of Science (1993); JSPS Fellow, Japan Society for the Promotion of Science (1997); Natural Science Foundation of China Lecturer (1999); National Science Foundation Creativity Extension Award (2000

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and 2022); Institut Universitaire de France Professor (2003); Chemistry Week in China Lecturer (2004); Lecturer in Solid State Chemistry, China (2005); Visitantes Distinguidos, Universid Complutenses Madrid (2008); Visiting Professor, Chinese Academy of Sciences (2011); 20 years of Service and Dedication Award to Inorganic Chemistry (2013); Elected foreign member of Spanish National Academy: Real Academia de Ciencia, Exactas, Fisicas y Naturales (2017); Elected Honorary Member of the Royal Society of Chemistry of Spain (RSEQ) (2018); and the TianShan Award, Xinjiang Uygur Autonomous Region of China (2021). He has organized and was Chairman of the Chicago Great Lakes Regional ACS Symposium on Synthesis and Processing of Advanced Solid State Materials (1987), the New Orleans National ACS Symposium on Solid State Chemistry of Heterogeneous Oxide Catalysis, Including New Microporous Solids (1987), the Gordon Conference on Solid State Chemistry (1994) and the First European Gordon Conference on Solid State Chemistry (1995), the Spring Materials Research Society Symposium on Environmental Chemistry (1995), the Advisory Committee of Intense Pulsed Neutron Source (IPNS) Program (1996–1998), the Spring Materials Research Society Symposium on Perovskite Materials (2003), the 4th International Conference on Inorganic Materials, University of Antwerp (2004), and the Philadelphia National ACS Symposium on Homogeneous and Heterogeneous Oxidation Catalysis (2004). He has served on numerous Editorial Boards, including Chemistry of Materials, Journal of Alloys and Compounds, Solid State Sciences, Solid State Chemistry, and Science China Materials, and has been a co-Editor for Structure and Bonding, an Associate Editor for Inorganic Chemistry, and co-Editor-in-Chief with Jan Reedijk for Comprehensive Inorganic Chemistry II (published 2013) and Comprehensive Inorganic Chemistry III (to be published in 2023). In addition, he has served on various Scientific Advisory Boards including for the World Premier International Research Center Initiative and Institute for Integrated Cell-Material Sciences Kyoto University, the European Center SOPRANO on Functional Electronic Metal Oxides, the Kyoto University Mixed-Anion Project, and the Dresden Max Planck Institute for Chemistry and Physics. Kenneth Poeppelmeier has published over 500 papers (1971–2022) and cited over 28000 times (h-index ¼ 84). He has supervised over 200 undergraduates, Ph.D. students, postdocs, and visiting scholars in research.

VOLUME EDITORS Risto S. Laitinen Risto S. Laitinen is Professor Emeritus of Chemistry at the University of Oulu, Finland. He received the M.Sc and Ph.D. degrees from Helsinki University of Technology (currently Aalto University). His research interests are directed to synthetic, structural, and computational chemistry of chalcogen compounds focusing on selenium and tellurium. He has published 250 peer-reviewed articles and 15 book chapters and has edited 2 books: Selenium and Tellurium Reagents: In Chemistry and Materials Science with Raija Oilunkaniemi (Walther de Gruyter, 2019) and Selenium and Tellurium Chemistry: From Small Molecules to Biomolecules and Materials with Derek Woollins (Springer, 2011). He has also written 30 professional and popular articles in chemistry. He is the Secretary of the Division of Chemical Nomenclature and Structure Representation, International Union of Pure and Applied Chemistry, for the term 2016–2023. He served as Chair of the Board of Union of Finnish University Professors in 2007–2010. In 2017, Finnish Cultural Foundation (North Ostrobothnia regional fund) gave him an award for excellence in his activities for science and music. He has been a member of Finnish Academy of Science and Letters since 2003.

Vincent L. Pecoraro Professor Vincent L. Pecoraro is a major contributor in the fields of inorganic, bioinorganic, and supramolecular chemistries. He has risen to the upper echelons of these disciplines with over 300 publications (an h-index of 92), 4 book editorships, and 5 patents. He has served the community in many ways including as an Associate Editor of Inorganic Chemistry for 20 years and now is President of the Society of Biological Inorganic Chemistry. Internationally, he has received a Le Studium Professorship, Blaise Pascal International Chair for Research, the Alexander von Humboldt Stiftung, and an Honorary PhD from Aix-Maseille University. His many US distinctions include the 2016 ACS Award for Distinguished Service in the Advancement of Inorganic Chemistry, the 2021 ACS/SCF FrancoAmerican Lectureship Prize, and being elected a Fellow of the ACS and AAAS. He also recently cofounded a Biomedical Imaging company, VIEWaves. In 2022, he was ranked as one of the world’s top 1000 most influential chemists.

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Zijian Guo Professor Zijian Guo received his Ph.D. from the University of Padova and worked as a postdoctoral research fellow at Birkbeck College, the University of London. He also worked as a research associate at the University of Edinburgh. His research focuses on the chemical biology of metals and metallodrugs and has authored or co-authored more than 400 peer-reviewed articles on basic and applied research topics. He was awarded the First Prize in Natural Sciences from Ministry of Education of China in 2015, the Luigi Sacconi Medal from the Italian Chemical Society in 2016, and the Outstanding Achievement Award from the Society of the Asian Biological Inorganic Chemistry in 2020. He founded Chemistry and Biomedicine Innovation Center (ChemBIC) in Nanjing University in 2019, and is serving as the Director of ChemBIC since then. He was elected to the Fellow of the Chinese Academy of Sciences in 2017. He served as Associated Editor of Coord. Chem. Rev and an editorial board member of several other journals.

Daniel C. Fredrickson Daniel C. Fredrickson is a Professor in the Department of Chemistry at the University of WisconsinMadison. He completed his BS in Biochemistry at the University of Washington in 2000, where he gained his first experiences with research and crystals in the laboratory of Prof. Bart Kahr. At Cornell University, he then pursued theoretical investigations of bonding in intermetallic compounds, the vast family of solid state compounds based on metallic elements, under the mentorship of Profs. Stephen Lee and Roald Hoffmann, earning his Ph.D. in 2005. Interested in the experimental and crystallographic aspects of complex intermetallics, he then carried out postdoctoral research from 2005 to 2008 with Prof. Sven Lidin at Stockholm University. Since starting at UW-Madison in 2009, his research group has created theory-driven approaches to the synthesis and discovery of new intermetallic phases and understanding the origins of their structural features. Some of his key research contributions are the development of the DFT-Chemical Pressure Method, the discovery of isolobal bonds for the generalization of the 18 electron rule to intermetallic phases, models for the emergence of incommensurate modulations in these compounds, and various design strategies for guiding complexity in solid state structures.

Patrick M. Woodward Professor Patrick M. Woodward received BS degrees in Chemistry and General Engineering from Idaho State University in 1991, an MS in Materials Science, and a Ph.D. in Chemistry from Oregon State University (1996) under the supervision of Art Sleight. After a postdoctoral appointment in the Physics Department at Brookhaven National Laboratory (1996–1998), he joined the faculty at Ohio State University in 1998, where he holds the rank of Professor in the Department of Chemistry and Biochemistry. He is a Fellow of the American Chemical Society (2020) and a recipient of an Alfred P. Sloan Fellowship (2004) and an NSF Career Award (2001). He has co-authored two textbooks: Solid State Materials Chemistry and the popular general chemistry textbook, Chemistry: The Central Science. His research interests revolve around the discovery of new materials and understanding links between the composition, structure, and properties of extended inorganic and hybrid solids.

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P. Shiv Halasyamani Professor P. Shiv Halasyamani earned his BS in Chemistry from the University of Chicago (1992) and his Ph.D. in Chemistry under the supervision of Prof. Kenneth R. Poeppelmeier at Northwestern University (1996). He was a Postdoctoral Fellow and Junior Research Fellow at Christ Church College, Oxford University, from 1997 to 1999. He began his independent academic career in the Department of Chemistry at the University of Houston in 1999 and has been a Full Professor since 2010. He was elected as a Fellow of the American Association for the Advancement of Science (AAAS) in 2019 and is currently an Associate Editor of the ACS journals Inorganic Chemistry and ACS Organic & Inorganic Au. His research interests involve the design, synthesis, crystal growth, and characterization of new functional inorganic materials.

Ram Seshadri Ram Seshadri received his Ph.D. in Solid State Chemistry from the Indian Institute of Science (IISc), Bangalore, working under the guidance of Professor C. N. R. Rao FRS. After some years as a Postdoctoral Fellow in Europe, he returned to IISc as an Assistant Professor in 1999. He moved to the Materials Department (College of Engineering) at UC Santa Barbara in 2002. He was recently promoted to the rank of Distinguished Professor in the Materials Department and the Department of Chemistry and Biochemistry in 2020. He is also the Fred and Linda R. Wudl Professor of Materials Science and Director of the Materials Research Laboratory: A National Science Foundation Materials Research Science and Engineering Center (NSF-MRSEC). His work broadly addresses the topic of structure–composition– property relations in crystalline inorganic and hybrid materials, with a focus on magnetic materials and materials for energy conversion and storage. He is Fellow of the Royal Society of Chemistry, the American Physical Society, and the American Association for the Advancement of Science. He serves as Associate Editor of the journals, Annual Reviews of Materials Research and Chemistry of Materials.

Serena Cussen Serena Cussen née Corr studied chemistry at Trinity College Dublin, completing her doctoral work under Yurii Gun’ko. She then joined the University of California, Santa Barbara, working with Ram Seshadri as a postdoctoral researcher. She joined the University of Kent as a lecturer in 2009. She moved to the University of Glasgow in 2013 and was made Professor in 2018. She moved to the University of Sheffield as a Chair in Functional Materials and Professor in Chemical and Biological Engineering in 2018, where she now serves as Department Head. She works on next-generation battery materials and advanced characterization techniques for the structure and properties of nanomaterials. Serena is the recipient of several awards including the Journal of Materials Chemistry Lectureship of the Royal Society of Chemistry. She previously served as Associate Editor of Royal Society of Chemistry journal Nanoscale and currently serves as Associate Editor for the Institute of Physics journal Progress in Energy.

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Rutger A. van Santen Rutger A. van Santen received his Ph.D. in 1971 in Theoretical Chemistry from the University of Leiden, The Netherlands. In the period 1972–1988, he became involved with catalysis research when employed by Shell Research in Amsterdam and Shell Development Company in Houston. In 1988, he became Full Professor of Catalysis at the Technical University Eindhoven. From 2010 till now he is Emeritus Professor and Honorary Institute Professor at Technical University Eindhoven. He is a member of Royal Dutch Academy of Sciences and Arts and Foreign Associate of the United States National Academy of Engineering (NAE). He has received several prestigious awards: the 1981 golden medal of the Royal Dutch Chemical Society; in 1992, the F.G. Chiappetta award of the North American Catalysis Society; in 1997, the Spinoza Award from the Dutch Foundation for Pure and Applied Research; and in 2001, the Alwin Mittasch Medal Dechema, Germany, among others. His main research interests are computational heterogeneous catalysis and complex chemical systems theory. He has published over 700 papers, 16 books, and 22 patents.

Emiel J. M. Hensen Emiel J. M. Hensen received his Ph.D. in Catalysis in 2000 from Eindhoven University of Technology, The Netherlands. Between 2000 and 2008, he worked at the University of Amsterdam, Shell Research in Amsterdam, and Eindhoven University of Technology on several topics in the field of heterogeneous catalysis. Since July 2009, he is Full Professor of Inorganic Materials and Catalysis at TU/e. He was a visiting professor at the Katholieke Universiteit Leuven (Belgium, 2001–2016) and at Hokkaido University (Japan, 2016). He is principal investigator and management team member of the gravitation program Multiscale Catalytic Energy Conversion, elected member of the Advanced Research Center Chemical Building Blocks Consortium, and chairman of the Netherlands Institute for Catalysis Research (NIOK). Hensen was Head of the Department of Chemical Engineering and Chemistry of Eindhoven University of Technology from 2016 to 2020. Hensen received Veni, Vidi, Vici, and Casmir grant awards from the Netherlands Organisation for Scientific Research. His main interests are in mechanism of heterogeneous catalysis combining experimental and computation studies. He has published over 600 papers, 20 book chapters, and 7 patents.

Artem M. Abakumov Artem M. Abakumov graduated from the Department of Chemistry at Moscow State University in 1993, received his Ph.D. in Chemistry from the same University in 1997, and then continued working as a Researcher and Vice-Chair of Inorganic Chemistry Department. He spent about 3 years as a postdoctoral fellow and invited professor in the Electron Microscopy for Materials Research (EMAT) laboratory at the University of Antwerp and joined EMAT as a research leader in 2008. Since 2015 he holds a Full Professor position at Skolkovo Institute of Science and Technology (Skoltech) in Moscow, leading Skoltech Center for Energy Science and Technology as a Director. His research interests span over a wide range of subjects, from inorganic chemistry, solid state chemistry, and crystallography to battery materials and transmission electron microscopy. He has extended experience in characterization of metal-ion battery electrodes and electrocatalysts with advanced TEM techniques that has led to a better understanding of charge–discharge mechanisms, redox reactions, and associated structural transformations in various classes of cathode materials on different spatial scales. He has published over 350 papers, 5 book chapters, and 12 patents.

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Keith J. Stevenson Keith J. Stevenson received his Ph.D. in 1997 from the University of Utah under the supervision of Prof. Henry White. Subsequently, he held a postdoctoral appointment at Northwestern University (1997– 2000) and a tenured faculty appointment (2000–2015) at the University of Texas at Austin. At present, he is leading the development of a new graduate level university (Skolkovo Institute for Science and Technology) in Moscow, Russia, where he is Provost and the former Director of the Center for Energy Science and Technology (CEST). To date he has published over 325 peer-reviewed publications, 14 patents, and 6 book chapters in this field. He is a recipient of Society of Electroanalytical Chemistry Charles N. Reilley Award (2021).

Evgeny V. Antipov Evgeny V. Antipov graduated from the Department of Chemistry at Moscow State University in 1981, received his Ph.D. in Chemistry in 1986, DSc degree in Chemistry in 1998, and then continued working at the same University as a Researcher, Head of the Laboratory of Inorganic Crystal Chemistry, Professor, Head of Laboratory of fundamental research on aluminum production, and Head of the Department of Electrochemistry. Since 2018 he also holds a professor position at Skolkovo Institute of Science and Technology (Skoltech) in Moscow. Currently his research interests are mainly focused on inorganic materials for application in batteries and fuel cells. He has published more than 400 scientific articles and 14 patents.

Vivian W.W. Yam Professor Vivian W.W. Yam is the Chair Professor of Chemistry and Philip Wong Wilson Wong Professor in Chemistry and Energy at The University of Hong Kong. She received both her B.Sc (Hons) and Ph.D. from The University of Hong Kong. She was elected to Member of Chinese Academy of Sciences, International Member (Foreign Associate) of US National Academy of Sciences, Foreign Member of Academia Europaea, Fellow of TWAS, and Founding Member of Hong Kong Academy of Sciences. She was Laureate of 2011 L’Oréal-UNESCO For Women in Science Award. Her research interests include inorganic and organometallic chemistry, supramolecular chemistry, photophysics and photochemistry, and metal-based molecular functional materials for sensing, organic optoelectronics, and energy research. Also see: https://chemistry.hku.hk/wwyam.

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David L. Bryce David L. Bryce (B.Sc (Hons), 1998, Queen’s University; Ph.D., 2002, Dalhousie University; postdoctoral fellow, 2003–04, NIDDK/NIH) is Full Professor and University Research Chair in Nuclear Magnetic Resonance at the University of Ottawa in Canada. He is the past Chair of the Department of Chemistry and Biomolecular Sciences, a Fellow of the Royal Society of Chemistry, and an elected Fellow of the Royal Society of Canada. His research interests include solid-state NMR of low-frequency quadrupolar nuclei, NMR studies of materials, NMR crystallography, halogen bonding, mechanochemistry, and quantum chemical interpretation of NMR interaction tensors. He is the author of approximately 200 scientific publications and co-author of 1 book. He is the Editor-in-Chief of Solid State Nuclear Magnetic Resonance and Section Editor (Magnetic Resonance and Molecular Spectroscopy) for the Canadian Journal of Chemistry. He has served as the Chair of Canada’s National Ultrahigh-Field NMR Facility for Solids and is a past co-chair of the International Society for Magnetic Resonance conference and of the Rocky Mountain Conference on Magnetic Resonance Solid-State NMR Symposium. His work has been recognized with the Canadian Society for Chemistry Keith Laidler Award and with the Gerhard Herzberg Award of the Canadian Association of Analytical Sciences and Spectroscopy.

Paul R. Raithby Paul R. Raithby obtained his B.Sc (1973) and Ph.D. (1976) from Queen Mary College, University of London, working for his Ph.D. in structural inorganic chemistry. He moved to the University of Cambridge in 1976, initially as a postdoctoral researcher and then as a faculty member. In 2000, he moved to the University of Bath to take up the Chair of Inorganic Chemistry when he remains to the present day, having been awarded an Emeritus Professorship in 2022. His research interests have spanned the chemistry of transition metal cluster compounds, platinum acetylide complexes and oligomers, and lanthanide complexes, and he uses laboratory and synchrotron-based X-ray crystallographic techniques to determine the structures of the complexes and to study their dynamics using time-resolved photocrystallographic methods.

Angus P. Wilkinson

Angus P. Wilkinson completed his bachelors (1988) and doctoral (1992) degrees in chemistry at Oxford University in the United Kingdom. He spent a postdoctoral period in the Materials Research Laboratory, University of California, Santa Barbara, prior to joining the faculty at the Georgia Institute of Technology as an assistant professor in 1993. He is currently a Professor in both the Schools of Chemistry and Biochemistry, and Materials Science and Engineering, at the Georgia Institute of Technology. His research in the general area of inorganic materials chemistry makes use of synchrotron X-ray and neutron scattering to better understand materials synthesis and properties.

CONTRIBUTORS TO VOLUME 2 Yuichiro Aiba Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya, Japan Enas Al-ani Department of Chemistry, Wayne State University, Detroit, MI, United States JR Aldrich-Wright School of Science, Nanoscale Organisation and Dynamics Group, Western Sydney University, Penrith, NSW, Australia; and Western Sydney University, Penrith South DC, NSW, Australia Matthew J Allen Department of Chemistry, Wayne State University, Detroit, MI, United States Trinidad Arcos-López National Institute for Genomic Medicine (INMEGEN), Mexico City, Mexico Shinya Ariyasu Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya, Japan Mohamed Atta University of Grenoble Alpes, CEA, CNRS, CBM-UMR 5249, Grenoble, France Guillermo Bahr Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina; and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Elizabeth S Biros Department of Chemistry, Wayne State University, Detroit, MI, United States Daiana A Capdevila Fundación Instituto Leloir, Buenos Aires, Argentina

Marta SP Carepo LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal Angela Casini Department of Chemistry, Technical University of Munich (TUM), München, Germany Hui Chao MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China Chi-Ming Che Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China; and Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, China Yu Chen MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China Rachel Codd The University of Sydney, School of Medical Sciences, Camperdown, NSW, Australia Brooke A Corbin Department of Chemistry, Wayne State University, Detroit, MI, United States Zhiqin Deng Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, PR China KM Deo School of Science, Nanoscale Organisation and Dynamics Group, Western Sydney University, Penrith, NSW, Australia; and Western Sydney University, Penrith South DC, NSW, Australia Matias Villarruel Dujovne Fundación Instituto Leloir, Buenos Aires, Argentina

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Oliver Einsle Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Freiburg i. Br., Germany

Di Hu Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China

Tobias A Engesser Christian-Albrechts-Universität zu Kiel, Institut für Anorganische Chemie, Kiel, Germany

Guibin Jiang State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

Besim Fazliji Department of Chemistry, University of Zurich, Zurich, Switzerland Carla Ferreira Rodrigues Department of Chemistry, University of Zurich, Zurich, Switzerland Roland A Fischer Department of Chemistry, Catalysis Research Center, Technical University of Munich, München, Germany Marc Fontecave Laboratoire de Chimie des Processus Biologiques, UMR CNRS 8229, Collège de France-CNRS-Sorbonne Université, PSL Research University, Paris, France David P Giedroc Department of Chemistry, Indiana University, Bloomington, IN, United States Raquel Grazina LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal Ruilin Guan MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China Elzbieta Gumienna-Kontecka Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland Zijian Guo Nanjing University, Nanjing, China Takashi Hayashi Department of Applied Chemistry, Osaka University, Suita, Osaka, Japan Marian Hebenbrock Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Münster, Germany Aleksandra Hecel Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland

Biao Jin Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States Matthew R Jordan Department of Chemistry, Indiana University, Bloomington, IN, United States; and Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, United States John P Karns Department of Chemistry, Wayne State University, Detroit, MI, United States Shunsuke Kato Department of Applied Chemistry, Osaka University, Suita, Osaka, Japan Jan Kern Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Henryk Kozlowski Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland; and Institute of Health Sciences, University of Opole, Opole, Poland Bernhard Kräutler Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Hongyan Li Department of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong SAR, China Xinlin Lin MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China Jiangping Liu MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China Chun-Nam Lok Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China

Contributors to Volume 2

Víctor E López-Guerrero Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; and Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico Jacob C Lutter Department of Chemistry and Biochemistry, University of Southern Indiana, Evansville, IN, United States Tiziano Marzo Department of Pharmacy, University of Pisa, Pisa, Italy Luigi Messori Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, Sesto Fiorentino, Italy Guillermo Moreno-Alcántar Department of Chemistry, Technical University of Munich (TUM), München, Germany; and Facultad de Química, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, México Isabel Moura LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal José JG Moura LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal Jens Müller Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Münster, Germany Hiroki Onoda Synchrotron Radiation Research Center, Nagoya University, Nagoya, Japan Sofia R Pauleta Microbial Stress Lab, UCIBIO e Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal; and Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal Vincent L Pecoraro University of Michigan, Ann Arbor, MI, United States Claudia Perez-Cruz Department of Pharmacology, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico

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Karolina Piasta Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland Yanahi Posadas Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; and Department of Pharmacology, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico Liliana Quintanar Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico Magdalena Rowinska-Zyrek Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland Peter J Sadler Department of Chemistry, University of Warwick, Coventry, United Kingdom Carolina Sánchez-López Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico Richard I Sayler Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico José Segovia Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico Huayun Shi Department of Chemistry, University of Warwick, Coventry, United Kingdom Osami Shoji Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya, Japan Roland KO Sigel Department of Chemistry, University of Zurich, Zurich, Switzerland Hongzhe Sun Department of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong SAR, China Ruikang Tang Center for Biomaterials and Biopathways, Department of Chemistry, Zhejiang University, Hangzhou, China; and Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China

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Contributors to Volume 2

Pablo E Tomatis Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina; and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Ka-Chung Tong Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China Felix Tuczek Christian-Albrechts-Universität zu Kiel, Institut für Anorganische Chemie, Kiel, Germany Alejandro J Vila Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina; and Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Pui-Ki Wan Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China Haibo Wang Department of Chemistry, University of Zurich, Zurich, Switzerland Zhigang Wang School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, PR China Wei Wei School of Life Sciences, Nanjing University, Nanjing, China Susan A White Department of Chemistry, Wayne State University, Detroit, MI, United States Xue Wu State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

Vittal K Yachandra Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Xueting Yan Department of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong SAR, China; and State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for EcoEnvironmental Sciences, Chinese Academy of Sciences, Beijing, China Junko Yano Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States Houzong Yao Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, PR China Jing Zhao State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China Xinyang Zhao State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China Yueqi Zhao Center for Biomaterials and Biopathways, Department of Chemistry, Zhejiang University, Hangzhou, China Ying Zhou Department of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong SAR, China Guangyu Zhu Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, PR China Lu Zhu School of Life Sciences, Nanjing University, Nanjing, China

PREFACE Comprehensive Inorganic Chemistry III is a new multi-reference work covering the broad area of Inorganic Chemistry. The work is available both in print and in electronic format. The 10 Volumes review significant advances and examines topics of relevance to today’s inorganic chemists with a focus on topics and results after 2012. The work is focusing on new developments, including interdisciplinary and high-impact areas. Comprehensive Inorganic Chemistry III, specifically focuses on main group chemistry, biological inorganic chemistry, solid state and materials chemistry, catalysis and new developments in electrochemistry and photochemistry, as well as on NMR methods and diffractions methods to study inorganic compounds. The work continues our 2013 work Comprehensive Inorganic Chemistry II, but at the same time adds new volumes on emerging research areas and techniques used to study inorganic compounds. The new work is also highly complementary to other recent Elsevier works in Coordination Chemistry and Organometallic Chemistry thereby forming a trio of works covering the whole of modern inorganic chemistry, most recently COMC-4 and CCC-3. The rapid pace of developments in recent years in all areas of chemistry, particularly inorganic chemistry, has again created many challenges to provide a contemporary up-to-date series. As is typically the challenge for Multireference Works (MRWs), the chapters are designed to provide a valuable long-standing scientific resource for both advanced students new to an area as well as researchers who need further background or answers to a particular problem on the elements, their compounds, or applications. Chapters are written by teams of leading experts, under the guidance of the Volume Editors and the Editors-inChief. The articles are written at a level that allows undergraduate students to understand the material, while providing active researchers with a ready reference resource for information in the field. The chapters are not intended to provide basic data on the elements, which are available from many sources including the original CIC-I, over 50-years-old by now, but instead concentrate on applications of the elements and their compounds and on high-level techniques to study inorganic compounds. Vol. 1: Synthesis, Structure, and Bonding in Inorganic Molecular Systems; Risto S. Laitinen In this Volume the editor presents an historic overview of Inorganic Chemistry starting with the birth of inorganic chemistry after Berzelius, and a focus on the 20th century including an overview of “inorganic” Nobel Prizes and major discoveries, like inert gas compounds. The most important trends in the field are discussed in an historic context. The bulk of the Volume consists of 3 parts, i.e., (1) Structure, bonding, and reactivity in inorganic molecular systems; (2) Intermolecular interactions, and (3) Inorganic Chains, rings, and cages. The volume contains 23 chapters. Part 1 contains chapters dealing with compounds in which the heavy p-block atom acts as a central atom. Some chapters deal with the rich synthetic and structural chemistry of noble gas compounds, low-coordinate p-block elements, biradicals, iron-only hydrogenase mimics, and macrocyclic selenoethers. Finally, the chemistry and application of weakly coordinating anions, the synthesis, structures, and reactivity of carbenes containing non-innocent ligands, frustrated Lewis pairs in metal-free catalysis are discussed. Part 2 discusses secondary bonding interactions that play an important role in the properties of bulk materials. It includes a chapter on the general theoretical considerations of secondary bonding interactions, including halogen and chalcogen bonding. This section is concluded by the update of the host-guest chemistry of the molecules of p-block elements and by a comprehensive review of closed-shell metallophilic interactions. The third part of the Volume is dedicated to chain, ring and cage (or cluster) compounds in molecular inorganic chemistry. Separate

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chapters describe the recent chemistry of boron clusters, as well as the chain, ring, and cage compounds of Group13 and 15, and 16 elements. Also, aromatic compounds bearing heavy Group 14 atoms, polyhalogenide anions and Zintl-clusters are presented. Vol. 2: Bioinorganic Chemistry and Homogeneous Biomimetic Inorganic Catalysis; Vincent L. Pecoraro and Zijian Guo In this Volume, the editors have brought together 26 chapters providing a broad coverage of many of the important areas involving metal compounds in biology and medicine. Readers interested in fundamental biochemistry that is assisted by metal ion catalysis, or in uncovering the latest developments in diagnostics or therapeutics using metal-based probes or agents, will find high-level contributions from top scientists. In the first part of the Volume topics dealing with metals interacting with proteins and nucleic acids are presented (e.g., siderophores, metallophores, homeostasis, biomineralization, metal-DNA and metal-RNA interactions, but also with zinc and cobalt enzymes). Topics dealing with iron-sulfur clusters and heme-containing proteins, enzymes dealing with dinitrogen fixation, dihydrogen and dioxygen production by photosynthesis will also be discussed, including bioinspired model systems. In the second part of the Volume the focus is on applications of inorganic chemistry in the field of medicine: e.g., clinical diagnosis, curing diseases and drug targeting. Platinum, gold and other metal compounds and their mechanism of action will be discussed in several chapters. Supramolecular coordination compounds, metal organic frameworks and targeted modifications of higher molecular weight will also be shown to be important for current and future therapy and diagnosis. Vol. 3: Theory and Bonding of Inorganic Non-molecular Systems; Daniel C. Fredrickson This volume consists of 15 chapters that build on symmetry-based expressions for the wavefunctions of extended structures toward models for bonding in solid state materials and their surfaces, algorithms for the prediction of crystal structures, tools for the analysis of bonding, and theories for the unique properties and phenomena that arise in these systems. The volume is divided into four parts along these lines, based on major themes in each of the chapters. These are: Part 1: Models for extended inorganic structures, Part 2: Tools for electronic structure analysis, Part 3: Predictive exploration of new structures, and Part 4: Properties and phenomena. Vol. 4: Solid State Inorganic Chemistry; P. Shiv Halasyamani and Patrick M. Woodward In a broad sense the field of inorganic chemistry can be broken down into substances that are based on molecules and those that are based on extended arrays linked by metallic, covalent, polar covalent, or ionic bonds (i.e., extended solids). The field of solid-state inorganic chemistry is largely concerned with elements and compounds that fall into the latter group. This volume contains nineteen chapters covering a wide variety of solid-state inorganic materials. These chapters largely focus on materials with properties that underpin modern technology. Smart phones, solid state lighting, batteries, computers, and many other devices that we take for granted would not be possible without these materials. Improvements in the performance of these and many other technologies are closely tied to the discovery of new materials or advances in our ability to synthesize high quality samples. The organization of most chapters is purposefully designed to emphasize how the exceptional physical properties of modern materials arise from the interplay of composition, structure, and bonding. Not surprisingly this volume has considerable overlap with both Volume 3 (Theory and Bonding of Inorganic NonMolecular Systems) and Volume 5 (Inorganic Materials Chemistry). We anticipate that readers who are interested in this volume will find much of interest in those volumes and vice versa Vol. 5: Inorganic Materials Chemistry; Ram Seshadri and Serena Cussen This volume has adopted the broad title of Inorganic Materials Chemistry, but as readers would note, the title could readily befit articles in other volumes as well. In order to distinguish contributions in this volume from

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those in other volumes, the editors have chosen to use as the organizing principle, the role of synthesis in developing materials, reflected by several of the contributions carrying the terms “synthesis” or “preparation” in the title. It should also be noted that the subset of inorganic materials that are the focus of this volume are what are generally referred to as functional materials, i.e., materials that carry out a function usually through the way they respond to an external stimulus such as light, or thermal gradients, or a magnetic field.

Vol. 6: Heterogeneous Inorganic Catalysis; Rutger A. van Santen and Emiel J. M. Hensen This Volume starts with an introductory chapter providing an excellent discussion of single sites in metal catalysis. This chapter is followed by 18 chapters covering a large part of the field. These chapters have been written with a focus on the synthesis and characterization of catalytic complexity and its relationship with the molecular chemistry of the catalytic reaction. In the 1950s with the growth of molecular inorganic chemistry, coordination chemistry and organometallic chemistry started to influence the development of heterogeneous catalysis. A host of new reactions and processes originate from that time. In this Volume chapters on major topics, like promoted Fischer-Tropsch catalysts, structure sensitivity of well-defined alloy surfaces in the context of oxidation catalysis and electrocatalytic reactions, illustrate the broadness of the field. Molecular heterogeneous catalysts rapidly grew after high-surface synthetic of zeolites were introduced; so, synthesis, structure and nanopore chemistry in zeolites is presented in a number of chapters. Also, topics like nanocluster activation of zeolites and supported zeolites are discussed. Mechanistically important chapters deal with imaging of single atom catalysts. An important development is the use of reducible supports, such as CeO2 or Fe2O3 where the interaction between the metal and support is playing a crucial role.

Vol. 7: Inorganic Electrochemistry; Keith J. Stevenson, Evgeny V. Antipov and Artem M. Abakumov This volume bridges several fields across chemistry, physics and material science. Perhaps this topic is best associated with the book “Inorganic Electrochemistry: Theory, Practice and Applications” by Piero Zanello that was intended to introduce inorganic chemists to electrochemical methods for study of primarily molecular systems, including metallocenes, organometallic and coordination complexes, metal complexes of redox active ligands, metal-carbonyl clusters, and proteins. The emphasis in this Volume of CIC III is on the impact of inorganic chemistry on the field of material science, which has opened the gateway for inorganic chemists to use more applied methods to the broad areas of electrochemical energy storage and conversion, electrocatalysis, electroanalysis, and electrosynthesis. In recognition of this decisive impact, the Nobel Prize in Chemistry of 2019 was awarded to John B. Goodenough, M. Stanley Whittingham, and Akira Yoshino for the development of the lithium-ion battery.

Vol. 8: Inorganic Photochemistry; Vivian W. W. Yam In this Volume the editor has compiled 19 chapters discussing recent developments in a variety of developments in the field. The introductory chapter overviews the several topics, including photoactivation and imaging reagents. The first chapters include a discussion of using luminescent coordination and organometallic compounds for organic light-emitting diodes (OLEDs) and applications to highlight the importance of developing future highly efficient luminescent transition metal compounds. The use of metal compounds in photo-induced bond activation and catalysis is highlighted by non-sacrificial photocatalysis and redox photocatalysis, which is another fundamental area of immense research interest and development. This work facilitates applications like biological probes, drug delivery and imaging reagents. Photochemical CO2 reduction and water oxidation catalysis has been addressed in several chapters. Use of such inorganic compounds in solar fuels and photocatalysis remains crucial for a sustainable environment. Finally, the photophysics and photochemistry of lanthanoid compounds is discussed, with their potential use of doped lanthanoids in luminescence imaging reagents.

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Vol. 9: NMR of Inorganic Nuclei; David L. Bryce Nuclear magnetic resonance (NMR) spectroscopy has long been established as one of the most important analytical tools at the disposal of the experimental chemist. The isotope-specific nature of the technique can provide unparalleled insights into local structure and dynamics. As seen in the various contributions to this Volume, applications of NMR spectroscopy to inorganic systems span the gas phase, liquid phase, and solid state. The nature of the systems discussed covers a very wide range, including glasses, single-molecule magnets, energy storage materials, bioinorganic systems, nanoparticles, catalysts, and more. The focus is largely on isotopes other than 1H and 13C, although there are clearly many applications of NMR of these nuclides to the study of inorganic compounds and materials. The value of solid-state NMR in studying the large percentage of nuclides which are quadrupolar (spin I > ½) is apparent in the various contributions. This is perhaps to be expected given that rapid quadrupolar relaxation can often obfuscate the observation of these resonances in solution. Vol. 10: X-ray, Neutron and Electron Scattering Methods in Inorganic Chemistry; Angus P. Wilkinson and Paul R. Raithby In this Volume the editors start with an introduction on the recent history and improvements of the instrumentation, source technology and user accessibility of synchrotron and neutron facilities worldwide, and they explain how these techniques work. The modern facilities now allow inorganic chemists to carry out a wide variety of complex experiments, almost on a day-to-day basis, that were not possible in the recent past. Past editions of Comprehensive Inorganic Chemistry have included many examples of successful synchrotron or neutron studies, but the increased importance of such experiments to inorganic chemists motivated us to produce a separate volume in CIC III dedicated to the methodology developed and the results obtained. The introduction chapter is followed by 15 chapters describing the developments in the field. Several chapters are presented covering recent examples of state-of-the-art experiments and refer to some of the pioneering work leading to the current state of the science in this exciting area. The editors have recognized the importance of complementary techniques by including chapters on electron crystallography and synchrotron radiation sources. Chapters are present on applications of the techniques in e.g., spin-crossover materials and catalytic materials, and in the use of time-resolved studies on molecular materials. A chapter on the worldwide frequently used structure visualization of crystal structures, using PLATON/PLUTON, is also included. Finally, some more specialized studies, like Panoramic (in beam) studies of materials synthesis and high-pressure synthesis are present. Direct observation of transient species and chemical reactions in a pore observed by synchrotron radiation and X-ray transient absorption spectroscopies in the study of excited state structures, and ab initio structure solution using synchrotron powder diffraction, as well as local structure determination using total scattering data, are impossible and unthinkable without these modern diffraction techniques. Jan Reedijk, Leiden, The Netherlands Kenneth R. Poeppelmeier, Illinois, United States March 2023

2.01 Introduction: Bioinorganic chemistry and homogeneous biomimetic inorganic catalysis Vincent L. Pecoraroa and Zijian Guob, a University of Michigan, Ann Arbor, MI, United States; and b Nanjing University, Nanjing, China © 2023 Elsevier Ltd. All rights reserved.

Abstract Arguably, the origins of Biological Inorganic Chemistry can be traced back to the mid 17th century when phosphorous could be isolated from the distillate of urine or to the mid 19th century with the discovery of hemoglobin. However, aggressive interest in the field began in the 1960s and has blossomed over the last 50 years. As a field, its scientific range is enormous covering modern materials to molecular biology and so no single volume is capable of capturing the breadth nor the vitality of such an expansive area. Given that CIC published a volume on this topic in 2013, our job was to both update key developments in well-established areas and highlight interesting new fields of study in bioinorganic chemistry. In this volume, the most recent developments in this area have been covered in 25 excellent chapters.

Arguably, the origins of Biological Inorganic Chemistry can be traced back to the mid 17th century when phosphorous could be isolated from the distillate of urine or to the mid 19th century with the discovery of hemoglobin. However, aggressive interest in the field began in the 1960s and has blossomed over the last 50 years. As a field, its scientific range is enormous covering modern materials to molecular biology and so no single volume is capable of capturing the breadth nor the vitality of such an expansive area. Given that CIC published a volume on this topic in 2013, our job was to both update key developments in wellestablished areas and highlight interesting new fields of study in bioinorganic chemistry. In this volume, the most recent developments in this area have been covered in 25 excellent chapters. As essential as inorganic species are in living systems, they cannot be left alone within cells or tissues without some control for fear of toxic side reactions. For this reason, chapters are included that describe how metals are acquired (see Chapter 2.02) and (see Chapter 2.18) and how they are trafficked in cells (see Chapter 2.03) and (see Chapter 2.04). With this initial understanding of homeostatic control within biological systems, the reader is then prepared for understanding applications of metals in native systems and in medical applications that are subsequently described in this volume. On the other hand, inorganic species can be regulated to form minerals in living organisms, and bio-compatible materials can be rationally designed to fulfill biological functions such as in cell protection, bioenergy production, vaccine modification, etc. A chapter is included to present some of the advances in this discipline (see Chapter 2.05). Another fundamental area of bioinorganic chemistry is the interaction of metal ions with nucleic acids. These metal DNA and RNA complexes not only are important in naturally regulating the structure and function of these biopolymers, but also are primary targets of many metal based pharmaceutical applications. The chapters by (see Chapter 2.20) on metal RNA structure and (see Chapter 2.21) metal DNA species clearly illustrate important considerations for comprehending how ions critically define the properties of these essential systems. Without doubt, the emphasis of most bioinorganic researchers has focused on Metalloproteins and Metalloenzymes. Therefore, we have included 10 chapters that describe such molecules either based on their metal content or on their catalytic function. Among the topics categorized by metal type are contributions on Zn (see Chapter 2.10) and Co (see Chapter 2.11). Such organization allows the reader to see the broad scope of chemistry characterized by a single element. In contrast to single ion systems, some metals are found as co-factors of some complexity such as Fe-S centers (see Chapter 2.06) and heme systems (see Chapter 2.08). These and other complex catalysts are sometimes best understood by seeing their integration within multi-electron reactions. Three critically important reactions for the biosphere and now important targets for commercialization are the production of Dihydrogen (H2), Dioxygen (O2) and ammonia from Dinitrogen (N2). The first two products, H2 and O2, are targets for the highly desired hydrogen economy in which water is split into H2 and O2 and then reformed through reactions that generate significant energy, but which do not generate CO2 as do fossil fuels. Hydrogenase enzymes are models for generating dihydrogen from protons and electrons. These enzymes are described in a chapter focusing on these metalloproteins (see Chapter 2.07). Nature splits water, but instead of generating dihydrogen forms protons and electrons instead. A chapter (see Chapter 2.13) describing the latest thinking on how the calcium-manganese center of the oxygen evolving complex in Photosystem II is included. While hydrogenases and the oxygen evolving complex have significant relevance to alternative energy production, the enzyme nitrogenase has been an inspiration for a room temperature method for converting dinitrogen to ammonia, a critical component of fertilizers. A chapter (see Chapter 2.12) focusing on the most recent advances in this fascinating system has also been included. Every system described in this paragraph is naturally occurring in organisms ranging from bacteria to plants to mammals; however, chemists are often interested in catalyzing reactions that have no direct biological use. None-the-less, using metalloenzymes represents both a strategy to generate stable catalysts that can be used in green chemical processing and providing methods to understand better natural metalloprotein folding and reactivity. A chapter (see Chapter 2.09) illustrates how modern methods allow for chemists to explore new function

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from metalloenzymes. While not necessarily including proteins, metal coordination systems can be used to mimic important chemical reactions. There are many examples of this approach in the literature but a nice summary is provided in a chapter (see Chapter 2.14) devoted to this topic. The final major area considered in this volume examines the role of metals in medicine. Inorganic compounds are widely used in the clinic for the treatment and diagnosis of diseases. Chemotherapy using platinum-based drugs has significantly increased the survival rates of cancer patients, but their wide applications have been limited by severe side effects as well as drug resistance. Photoactive metallodrugs could be applied in photodynamic therapy, photoactivated chemotherapy and photothermal therapy which offer the possibility for on demand activation of drugs at targeted sites (see Chapter 2.17). Another perspective related to the use of photochemistry of metal complexes is to employ their phosphorescent signals to reveal the mechanism of action of potent anticancer drugs (see Chapter 2.16). Molecular design and nano-conjugation of platinum-based anticancer drugs improve the tumortargeting ability and delivery of platinum drugs in vivo, which could potentially lead to higher efficacy and lower toxicity of the drugs (see Chapter 2.25). Unconventional platinum(II) complexes that exert their activity through other biological targets than DNA are also described as an alternative strategy to reduce the side effects of clinical drugs (see Chapter 2.23). One of the most promising non-platinum anticancer therapeutics are gold complexes. An interesting chapter discussing the anti-cancer properties, possible mechanisms of action, and the identification of the engaged molecular targets of gold compounds are included in this volume (see Chapter 2.26). Supramolecular materials including supramolecular coordination complexes and metal organic frameworks enable the rational design and targeted structural modifications of higher molecular weight and extended metal systems for therapy and diagnosis. This offers useful hints to bridge synthetic chemistry with biomedicinal materials (see Chapter 2.22). It is well known that the brain is one of the organs that concentrates metal ions, particularly transition metal ions. Therefore, a chapter is devoted to summarizing the pathological roles of d-block metal ions in neurodegenerative diseases and therapeutic approaches to targeting metal-protein interactions for curing disease (see Chapter 2.19). Protein metalation triggers cellular signaling pathways that eventually lead to cancer cell death. Modern “omics” technologies are applied to identify protein targets of metallodrugs. Innovative chemical proteomics and metalloproteomics approaches are introduced in this volume (see Chapter 2.24). Inorganic chemistry plays a central role in a variety of imaging modalities in clinical diagnosis. In this volume, a nice chapter devoted to the introduction of selective clinical imaging techniques is included (see Chapter 2.15). We have assembled in this volume a wide range of articles that provide a broad coverage of many of the important areas involving metals in biology. Whether the reader is interested in fundamental biochemistry that is assisted by metal ion catalysis or uncovering the latest advances in diagnostics or therapeutics using metal ion probes or agents, there will be contributions that satisfy the interested reader’s needs. We also feel that the chapters are well suited for mid to advanced level instruction in courses focusing on metals in biology.

2.02

Siderophores and iron transport

Rachel Codd, The University of Sydney, School of Medical Sciences, Camperdown, NSW, Australia © 2023 Elsevier Ltd. All rights reserved.

2.02.1 2.02.2 2.02.2.1 2.02.3 2.02.3.1 2.02.3.1.1 2.02.3.1.2 2.02.3.1.3 2.02.3.1.4 2.02.4 2.02.4.1 2.02.4.1.1 2.02.5 2.02.5.1 2.02.6 2.02.6.1 2.02.6.1.1 2.02.6.2 2.02.6.2.1 2.02.6.3 2.02.6.3.1 2.02.6.3.2 2.02.6.3.3 2.02.7 2.02.7.1 2.02.7.2 2.02.7.3 2.02.7.4 2.02.8 2.02.9 2.02.10 Acknowledgments References

Introduction Structures of bacterial and fungal siderophores Sideromycins Characterization of Fe(III)–siderophore complexes X-ray crystallography Hydroxamic acid Catechol Mixed-ligand Pyoverdine A new siderophore functional group N-nitroso-N-hydroxylamine X-ray crystallography Siderophore adaptations Marine organisms Biosynthesis of siderophores Nonribosomal peptide synthetase (NRPS) pathways Pyoverdine chromophore Nonribosomal peptide synthetase-independent siderophore (NIS) pathways Desferrioxamine B Precursor-directed biosynthesis and mutasynthesis Hydroxamic acid Catechol Mixed-ligand Siderophore uptake and transport Gram-negative bacteria Gram-positive bacteria Release of Fe(III) Chirality Siderophores in infection and stealth siderophores Applications of siderophores Conclusion

3 4 4 6 7 7 8 8 9 10 10 10 11 13 13 13 14 15 16 17 17 18 18 18 18 19 20 20 20 21 21 21 21

Abstract Siderophores are low-molecular-weight organic compounds produced by bacteria and fungi for Fe(III) supply. Bacteria and fungi biosynthesize siderophores in response to low iron, which are released into the local environment to sequester Fe(III) to form a high-affinity Fe(III)-siderophore complex. This complex can be avidly recognized by selective proteins at the cell surface of the producing species. Following active transport, the Fe(III)–siderophore complex reaches its destination in the cytoplasm, where the element is released for downstream processing and incorporation into enzymes and proteins essential for growth. This elegant supply mechanism traverses a broad intellectual base across inorganic and coordination chemistry, bioorganic chemistry, molecular microbiology, natural product biosynthesis, medicinal chemistry, and chemical and structural biology. Added interest in the field of siderophores is founded upon the potential of these ligands in metal sequestration in other settings, including environmental metals as contaminants or commodities, and as agents for maintaining metal homeostasis or delivering radiometals for imaging. This chapter seeks to provide the reader with an understanding of foundational knowledge of siderophores and iron transport and to ignite a sense of wonder that has captured many researchers worldwide.

2.02.1

Introduction

The aerobic environment on Earth results in the predominant form of iron existing as Fe(III) oxyhydroxide species, which have limited solubility in water at physiological pH (Ksp ¼ 1039 for Fe(OH)3, giving [Fe(OH2)6]3þ ¼ 1018 M at pH 7).1 The

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Siderophores and iron transport

bioavailability of this essential element is significantly below levels ( 1 mM) required for bacterial growth.2 The low concentrations of available Fe(III) prevents sufficient uptake for growth via passive diffusion, which has led bacteria and fungi to evolve a set of active iron acquisition mechanisms. One of the most successful and widespread of these strategies is the production of lowmolecular-weight organic compounds that coordinate Fe(III) with high affinity, known as “siderophores” (Greek: “iron carrier”).3–13 Iron is an obligate cofactor in a raft of proteins and enzymes, including ribonucleotide reductase, cytochrome oxidase, and iron–sulfur clusters, required to support fundamental physiological processes, including DNA synthesis, cellular respiration and electron transport.14–16 In most gram-negative and low-GC content gram-positive bacteria (e.g., Bacillus sp.), a sufficient level of iron in the cytosol is sensed by the global ferric uptake regulator (Fur) which binds as a Fe(II)–Fur complex to DNA operators to repress the transcription of genes involved in iron acquisition systems. Under iron deficient conditions, apo-Fur dissociates from the DNA operator to activate the transcription of iron acquisition systems, including genes that code for siderophore biosynthesis.17,18 The first siderophore discovered was ferrichrome, which was characterized from the rust fungus Ustilago sphaerogena.19 The discovery of ferrichrome founded a research community set to understand the fundamental coordination chemistry of siderophore complexes and to decipher their role in mechanisms underlying bacterial and fungal iron supply. The impact of these early studiesdparticularly given the available research infrastructuredis immense. The field continues to thrive, with research pushing boundaries in racemic crystallography, discovery through genome mining, siderophore uptake pathways, siderophore biosynthesis, discovering new functional groups, the role of siderophores as virulence factors in bacterial pathogenesis, and applications of siderophores in environmental and biomedical settings. The breadth and depth of the field makes a comprehensive coverage a daunting task. Instead, the chapter aims to represent each of the major classes of siderophores with a focus on one or two sub-topics considered pertinent. The chapter has an emphasis on iron uptake as mediated by siderophores and identifies appropriate literature in relevant sections that detail other iron uptake pathways independent of siderophores.

2.02.2

Structures of bacterial and fungal siderophores

The number of unique siderophore structures is conservatively estimated at between 250 and 50013 and, with the ever-increasing rate of discovery from genome mining, the development of new purification protocols,20,21 and improved analytical methods,22 is likely well beyond this upper limit. The unifying feature of siderophores, classified as low-molecular-weight (Mr 500– 1500 g mol1) bacterial or fungal secondary metabolites, is the presence of an organic scaffold containing functional groups that coordinate Fe(III) with high affinity. Due to the hard acid nature of Fe(III), hard base O0 ,O0 -bidentate oxygen-containing functional groups are common, including a-hydroxycarboxylic acids, hydroxamic acids and catechols, and monodentate phenols derived from salicylic acid. Other functional groups include thiazoli(di)ne and unsubstituted or C-methylated oxazoline heterocycles, as cyclisation products of L-cysteine, L-serine or L-threonine, respectively. The bidentate O0 ,O0 -N-nitroso-N-hydroxylamine functional group has recently been identified in a new class of siderophore isolated from bacteria resident in the rhizosphere.23 These functional groups are incorporated in a myriad of different combinations into different types of linear or macrocyclic organic scaffolds (citric acid, diamine, polyamine, peptide) to deliver, in the majority of cases, a hexadentate ligand that enables the maximum gain in entropy-derived stability upon forming a coordinatively saturated 1:1 complex with Fe(III). A smaller number of tetradentate siderophores form coordinatively saturated 2:3 Fe(III):siderophore complexes at physiologically relevant pH values. A review of the chemistry and structures of siderophores published in 201013 is a “go-to” resource that provides a comprehensive display of the molecular structures of siderophores. The structures of a selection of bacterial and fungal siderophores show the structural diversity of this group of functionallyequivalent secondary metabolites, which include catechol-containing enterobactin, hydroxamic acid-containing desferrioxamine B (DFOB), phenolate-containing mycobactins, mixed-functional group-containing yersiniabactin, and citric acid-based schizokinen (Fig. 1). As a sidenote, the researcher who worked in early career on schizokinen with the founder of siderophore research24 later won (shared) the 1993 Nobel Prize in Chemistry for the discovery of the polymerase chain reaction. Schizokinen has since been isolated from Bacillus megaterium as an imide form,25 as commonly observed from citric acid siderophores isolated from culture.26 The structure of the polycarboxylate siderophore staphyloferrin B, most often depicted as a linear molecule, has been revised to a cyclic hemiaminal structure based on NMR spectroscopic assignments and the match of these data between staphyloferrin B accessed from total synthesis and the native biological isolate.27 The siderophore from Legionella pneumophilia named legiobactin has since been shown to have a structure identical to rhizoferrin,28 which is produced by a range of bacteria and fungi.29 Other siderophores from fungal organisms include members of the fusarinine C, ferrichrome, and coprogen families (Fig. 2).30– 35 These siderophores are assembled from Nd-hydroxy-ornithine units, with variable Nd-acylation patterns. The archetypal fungal siderophore ferrichrome comprises a cyclic hexapeptide assembled from three contiguous Nd-acetyl-Nd-hydroxy-ornithine residues and three glycine residues, resulting in an exocyclic hydroxamic acid molecular architecture. Other members of the ferrichrome class contain different combinations of glycine, alanine or serine residues to meet the balance of the hexapeptide. Ferrichrome A is distinct from other members by its Nd-acylation with a trans-(b-methyl)-glutaconic acid unit.36 The fusarinines are endo-hydroxamic acid trimeric macrocycles with three Nd-acyl-Nd-hydroxy-ornithine residues fused by ester linkages.

2.02.2.1

Sideromycins

Sideromycins are covalently linked siderophore–antibiotic conjugates produced as native compounds by a bacterium that can be actively transported via siderophore uptake pathways for antibacterial action against a phylogenetically-related competitor.37–39 This active transport mechanism increases the concentration of antibiotic delivered to the target bacterial cell by 100 times than

Siderophores and iron transport

Fig. 1

5

Structures of a selection of bacterial and fungal siderophores and the producing organism(s) or a sub-set thereof.

achievable by passive diffusion.38 This is particularly useful for targeting antibiotics toward gram-negative pathogens, which contain an outer membrane that can restrict the passage of low-molecular-weight antibiotics.40 Examples of sideromycins include albomycin as a covalent adduct of ferrichrome and a thioribosyl pyrimidine motif, which elicits antibiotic activity via the inhibition of serylt-RNA synthetase. Salmycin is a sideromycin comprised of a ferrioxamine unit bound to an aminodisaccharide, which inhibits bacterial protein synthesis. Siderophore-microcins comprise conjugates between enterobactin and 10 kDa antimicrobial peptides, which play a role in the infectivity of different strains of pathogenic E. coli in the intestine.41–43

6

Siderophores and iron transport

Fig. 2

Structures of exo- and endo-macrocyclic and linear fungal siderophores.

2.02.3

Characterization of Fe(III)–siderophore complexes

Multiple types of spectroscopic methods, magnetic measurements and X-ray crystallography have been used to characterize Fe(III)– siderophore coordination complexes.5,12 Measurements of magnetic susceptibility (meff  5.8 B.M.),44 and electron paramagnetic resonance (EPR)45 and Mössbauer spectroscopies of Fe(III)–siderophore complexes46,47 report the Fe(III) spin state as high-spin d5. Given there are no spin-allowed d–d optical transitions, the characteristic broad absorption band for a Fe(III)–siderophore complex at 420 nm (3  3  103 M1 cm1) is charge transfer in nature. The catechol-based Fe(III)–enterobactin complex shows a broad absorption band at 500 nm (3  3.2  103 M1 cm1) assigned as a phenolate to Fe(III) charge transfer.48 The absence of crystal field stabilization energy results in Fe(III)–siderophore complexes that are kinetically labile toward ligand exchange and isomerization reactions,49,50 which has made access to crystals suited for X-ray crystallography less routine, with many fewer structures available than the number of siderophores catalogued as free ligands. The property of high Fe(III)-affinity binding of siderophores is reflected in the magnitude of the equilibrium constants (Table 1), expressed as bFeLH values (b110) for a hexadentate triprotic siderophore, according to Eqs. (1) and (2). These equations describe the equilibrium constant for the Fe(III)–siderophore complex formation independent of pH and do not account for the competition between Hþ and Fe(III) toward ligand binding.12 Given the magnitude of many of the formation constants, these measurements

Table 1

Stability constants of complexes between Fe(III)– or Fe(II)– and a selection of siderophores from each class, and the pM valuesa and reduction potentials (mV/NHE).

Siderophore

log b110 (Fe(III))

log b110 (Fe(II))

pM a

Reduction Potential (mV/NHE)

References

Ferrichrome A Desferrioxamine B Coprogen Rhizoferrin Aerobactin Enterobactin Pyoverdine Gramibactin

32.0 30.5 30.2 25.3 22.5 52 30.8 27.6

11.3 9.5 9.6 NDb 3.8 22.2 9.0 ND

25.2 26.6 27.5 19.7 23.3 35.5 27 25

448 468 447 82 336 750 510 ND

51 52 53 54,55 56 57 58 23,59

pM ¼  log[Fe(III)], when [Fe(III)] ¼ 106 M, [L] ¼ 105 M, and pH ¼ 7.4. The pM value reports the equilibrium concentration of Fe(III) at physiological pH (pH 7.4) under given ligand and metal concentrations and is useful for comparing the Fe(III) complexation properties of different siderophores.56 b ND, not determined.

a

Siderophores and iron transport

7

from a technical standpoint are often undertaken by competition experiments of the siderophore free ligand against Fe(III)–EDTA complexes.56,60–62 L3 þ Fe3þ ðaqÞ #FeL b110 ¼ h

2.02.3.1 2.02.3.1.1

½FeL i  L3

Fe3þ ðaqÞ

(1)

(2)

X-ray crystallography Hydroxamic acid

Archetypal hydroxamic acid siderophores are those in the desferrioxamine class, which are produced widely by Streptomyces species and other actinomycetes, and include linear desferrioxamine B (DFOB)63 and macrocyclic desferrioxamine E (DFOE). The X-ray crystal structure of Fe(III)–DFOB (Fig. 3A) displays the 1:1 Fe(III):siderophore stoichiometry and the cis-geometry with respect to the N–O oxygen atoms in the octahedral coordination sphere,64 which is common to the larger set of Fe(III)–tris-hydroxamic acid siderophores, including macrocyclic Fe(III)–DFOE (Fig. 3B)65 and Fe(III)–desferrioxamine D1.66 The restricted conformational flexibility of DFOE and the favorable pre-organization of the set of donor atoms toward Fe(III) coordination, enabled the growth of X-ray quality crystals relatively early in the timeline of siderophore coordination chemistry research. Equivalent data for linear DFOB was more difficult to achieve since its flexibility translates to 16 theoretical isomers (optical: L, D; geometric: cis, trans) with minor energy differences that would predict a population of species present in solution. Ultimately, Fe(III)–DFOB was successfully co-crystallized with perchlorate and a non-standard ethanolpentaaquomagnesium(II) perchlorate species.64 Tetradentate alcaligin, as first isolated from Alcaligenes denitrificans subsp. xylosoxydans,71 and subsequently found native to Bordetella pertussis and B. bronchiseptica,72,73 is one member of a small subset of dihydroxamic acid macrocyclic siderophores,74 including bisucaberin,75–77 putrebactin78 and avaroferrin.79 The X-ray crystal structure of the 2:3 Fe(III)–alcaligin complex (Fig. 3C) shows one ligand each coordinating a discrete Fe(III) center, with a third ligand bridged between the two metal ions to saturate each octahedral coordination sphere.67 The conformation of the terminal alcaligin ligand as bound to Fe(III) is the same as the free ligand, which demonstrates the macrocyclic effect and its contribution to the stability constant (log b110 ¼ 32.2 (per Fe(III)).80

Fig. 3 X-ray crystal structures (H atoms and counter ions omitted for clarity) of complexes between Fe(III) and (A) desferrioxamine B (DFOB),64 (B) desferrioxamine E (DFOE),65 (C) alcaligin,67 (D) N,N0 ,N00 -triacetylfusarinine,68 (E) ferrichrome69 or (F) ferrichrome A.70

8

Siderophores and iron transport

Although evolved in nature as Fe(III) chelators, hydroxamic acid siderophores including DFOB and putrebactin, have a broader coordination chemistry, with complexes characterized in solution with Cr(III), V(IV), Mn(III), Cr(V), Mo(VI) or Zr(IV).81–83 Complexes between Zr(IV) and hydroxamic acid siderophores are relevant to the development of Zr(IV)-89 for positron emission tomography (PET) imaging of cancer or infection.84–88 Tetradentate putrebactin forms 1:1 or 2:2 complexes in solution with a range of (di)oxido metal centers, including V(V), Cr(V) or Mo(VI).89,90 The distinct structures of complexes between Fe(III) and endo- or exo-macrocyclic siderophores is evident from the X-ray crystal structures of Fe(III)eN,N0 ,N00 -triacetylfusarinine (Fig. 3D),68 Fe(III)–ferrichrome (Fig. 3E)69 and Fe(III)–ferrichrome A, which was partially solved91 prior to a complete solution (Fig. 3F).70 While each of Fe(III)–DFOB and Fe(III)–DFOE crystallized as a racemic mixture of the L-N-cis and D-N-cis isomers (with the latter isomer depicted in Fig. 3), some Fe(III)–siderophore complexes have crystallized as preferred isomers. Crystals of Fe(III)eN,N0 ,N00 -triacetylfusarinine formed in the L-cis configuration, with CD measurements showing a preference in solution to form the D-cis diastereoisomer.68 The minor fusarinine-type siderophore neurosporin was isolated from Neurospora crassa (ATCC 10816) and its complex with Fe(III) solved by X-ray crystallography in the L-cis configuration.30 Examples of the first and second coordination shells of these geometries are illustrated in Fig. 4.

2.02.3.1.2

Catechol

Enterobactin is the archetypal tris-catechol-containing siderophore produced by enteric gram-negative bacteria, including Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae. As the first catechol-based siderophore discovered, enterobactin has provided a wealth of knowledge of the fundamental coordination chemistry of siderophores.92,93 The 1:1 Fe(III)–enterobactin complex has the highest known Fe(III) formation constant of any Fe(III)–siderophore complex of Kf 1052.94 The X-ray crystal of a structure of a des-oxidoV(IV)–enterobactin complex showed a pseudooctahedral coordination geometry with close to C3 symmetry (Fig. 5F).95 The long-awaited X-ray structure of the Fe(III)–enterobactin complex, as most relevant to biology, was solved only recently, using an innovative approach involving the co-crystallization of the native complex assembled from L-serine and the complex as its mirror image prepared from D-serine. This racemic crystallography approach was predicted to maximize the number of intermolecular contacts, and together with the selection of the aromatic and high-volume tetraphenylarsonium counter ion, resulted in successful crystallization.96 The Fe(III)–enterobactin complex is isostructural with the V(IV) complex, with the three bidentate catechol groups bound to the metal center with the three ortho-hydroxy groups positioned in a facial orientation (Fig. 5C). The work has allowed the definitive assignment of the D configuration of the natural Fe(III)–enterobactin complex. Similar coordination environments are observed from X-ray crystal structures of complexes between Si(IV), Ge(IV) or Ti(IV) and enterobactin.97,98 Other coordination complexes have been characterized in solution between the bis-catechol siderophore azotochelin from A. vinelandii and Mo(VI).99

2.02.3.1.3

Mixed-ligand

There is a small set of X-ray crystal structures from complexes between Fe(III) or Ga(III) and siderophores with mixed-ligand functional groups. The structure of Fe(III)–yersiniabactin shows a 1:1 stoichiometry with an O3, N3 coordination sphere derived from the aliphatic and phenolate hydroxide ions, the terminal carboxylic acid group, and the three nitrogen atoms in the thiazoline and thiazolidine rings (Fig. 5A). The donor atoms from the carboxylate, hydroxyl and terminal thiazoline groups are meridonally disposed to the plane defined by the remaining donor atoms. The siderophore anguibactin was isolated from the fish pathogen Vibrio anguillarum, with its structure posited from NMR spectroscopy of the parent compound and X-ray crystallographic data from a close analogue anhydroanguibactin.104 An X-ray crystal structure of a 2:2 Ga(III):anguibactin complex shows a binuclear coordination site with each Ga(III) center bound to the ortho-hydroxy group from the catechol, the hydroxamate N–O group, and nitrogen atoms from the thiazoline and imidazole rings (Fig. 5B). The pseudooctahedral coordination sphere is completed by two bridging methoxide ligands.101 Although anguibactin has the capacity to act as a hexadentate ligand to form a theoretical

Fig. 4 Fe(III)–siderophore complexes configured as lambda (L) or delta (D) and N-cis (arrow) isomers from non-H atom coordinates extracted from the X-ray crystal structures of Fe(III)–neurosporin (L)30 or Fe(III)–DFOE (D).65

Siderophores and iron transport

9

Fig. 5 X-ray crystal structures (H atoms and counter ions omitted for clarity) of complexes of (A) Fe(III)–yersiniabactin,100 (B) Ga(III)– anguibactin,101 (C) Fe(III)–enterobactin,96 (D) Fe(III)–amychelin,102 (E) Fe(III)–pyoverdine (pseudobactin),103 or (F) V(IV)–enterobactin.95

complex with 1:1 metal:ligand stoichiometry, the donor atoms are configured in a manner than prevents this, leading instead to the 2:2:2 metal:ligand:solvento complex. The siderophore amychelin was isolated from the actinomycete Amycolatopsis sp. AA4, with the purification protocol conducted in the presence of excess Fe(III) or Ga(III), which was found to minimize the decomposition of the free ligand, and led to the growth of Fe(III)–amychelin crystals. The X-ray crystal structure of the 1:1 Fe(III)–amychelin complex (Fig. 5D) identifies the coordinating groups as the N-terminal 2-hydroxybenzoyl-oxazoline group and two contiguous C-terminal hydroxamic acid groups, which are configured to generate a high-affinity Fe(III) complex (pFe(III) ¼ 30  1.6). This confers the ability of Amycolatopsis sp. AA4 to arrest the development of a subset of Streptomyces sp. in co-culture, through outcompeting for Fe(III).102,105 The Fe(III)–amychelin complex formed in the D-cis configuration, as defined by the three oxygen donor atoms in the O3, N3 coordination sphere.102

2.02.3.1.4

Pyoverdine

Pyoverdines are a major class of siderophores produced by the group of fluorescent Pseudomonas species, which include the human pathogen Pseudomonas aeruginosa. Pyoverdines have an intrinsic yellow-green fluorescence due to the presence of a common (1S)-5-amino2,3-dihydro-8,9-dihydroxy-1H-pyrimido-[1,2-a]quinoline-1-carboxylic acid chromophore.106–108 A similar chromophore is present in the fluorescent siderophore azotobactin produced by the nitrogen-fixing soil bacterium Azotobacter vinelandii.109,110 Pyoverdine I and its role in iron supply is one of at least three virulence factors established for P. aeruginosa infection, with treatment of this opportunistic pathogen posing particular concern for immunocompromized patients, including those with cystic fibrosis.111–114 There are more than 100 members of the pyoverdine class of siderophores, which vary in the number and type (6–14 residues in L- and D-configurations) and architecture (linear, trimeric or tetrameric macrocycle) of the amino acid residues that comprise the peptide region stemming from the carboxylic acid bound to the tricyclic chromophore.62,115 The catechol-type unit in the chromophore together with two hydroxamic acid groups derived commonly from N-formyl-N-hydroxy-ornithine or N-hydroxy-ornithine with different N-acylation patterns provide the six donor atoms for Fe(III) coordination. The exo-amine group in the chromophore contains a pendent group selected from succinic, malic or a-ketoglutaric acid or an amide derivative of these diacids.116 The X-ray crystal structure of Fe(III)–pyoverdine has been solved, with this siderophore named pseudobactin on some occasions in early literature.103 The Fe(III)–pyoverdine complex formed as the L isomer (Fig. 5E), which matched the absolute configuration in solution as determined from CD spectroscopy. The stability constant of the 1:1 Fe(III)-pyoverdine complex has been determined as Ka ¼ 1030.8 M1, with the corresponding value for the 1:1 Fe(II)-pyoverdine complex as Ka ¼ 109 M1.58 which demonstrates the selectivity of siderophores for Fe(III) above Fe(II). The stability constant of complexes formed between Ni(II), Cd(II) or Cu(II) and pyoverdine are reported as Ka ¼ 1010.9 M–1, Ka ¼ 108.2 M–1 or Ka ¼ 1020.1 M1, respectively.58,117,118

10

Siderophores and iron transport

2.02.4

A new siderophore functional group

2.02.4.1

N-nitroso-N-hydroxylamine

Similar with animals, bacteria and fungi, plants also have an obligate requirement for iron for common pathways of oxygen transport and cellular respiration, and specialist pathways of thylakoid biogenesis and chlorophyll development. While mugineic acidbased phytosiderophores describe one iron supply pathway in the plant kingdom, bacterial siderophores also play a role in this ecosphere, particularly in the complex rhizosphere of plant roots and exudates, bacteria and inorganic matter.119 Recent work focused on siderophores in the rhizosphere conducted genome mining of the sequence of a bacterial isolate from maize Paraburkholderia graminis (strain C4D1M) and other bacteria resident in soil and/or the rhizosphere, including Burkholderia spp. The study identified genes coding for TonB-dependent siderophore receptors, iron-hydroxamate transporter ATP binding proteins and associated nonribosomal peptide synthetase (NRPS) gene clusters with high homology coding for a cryptic siderophore. Cultures of P. graminis led to the discovery of the siderophore gramibactin which contains an unusual N-nitroso-N-hydroxylamine functional group (Fig. 6).23 The 1:1 Fe(III)-gramibactin coordination sphere is assembled from two bidentate N-nitroso-N-hydroxylamine groups and one bidentate b-hydroxyaspartic acid group (logb ¼ 27.6). The Nd-nitroso-Nd-hydroxy-D-ornithine motif is a novel non-canonical amino acid named graminine.23 The N-nitroso-N-hydroxylamine (R-N(OH)-NO) functional group, which is also termed a diazeniumdiolate,120 is an isostere of the N-formyl-N-hydroxylamine group, as equivalent to a retro hydroxamic acid group (R-N(OH)-CH(O)). Retro hydroxamic acids, including retro-DFOB analogues, show useful properties distinct from those of the forward or natural siderophore analogues.121,122 The N-nitroso-N-hydroxylamine functional group is present in the synthetic Cu(II) chelator cupferron, and in a limited number of natural products, including the antitumor antibiotic alanosine from Streptomyces alanosinicus ATCC 15710,123,124 the fungicide fragin from Pseudomonas fragi,125 and the signaling molecule rac-valdiazen from Burkholderia cenocepacia H111.126,127 Since the discovery and characterization of gramibactin and its properties,23,59 suites of diazeniumdiolate-containing siderophores, named plantaribactin and megapolibactins have been identified from other plant-associated Burkholderia and Paraburkholderia bacteria,128 together with trinickiabactin from the bacterial plant pathogen Trinickia caryophylli DSM 50341 (Fig. 6).129 Trinickiabactin is the linear form of the depsipeptide gramibactin, and has also been named gramibactin B. Although there is a strong resemblance between the biosynthetic gene clusters of Trinickia (trbHIJ) and Burkholderia/Paraburkholderia (grbHIJ) (63.9% average identity), it appears that linear trinickiabactin (gramibactin B) and cyclic gramibactin are produced as discrete compounds from the respective producing strains. This has been ascribed to potential differences in the biosynthetic function of the thioesterase (TE) accessory enzyme used to release the product in the NRPS assembly line. The pH conditions used to culture and purify trinickiabactin were mild (pH  6.5) and did not correlate with its presence as an acid- or base-mediated hydrolysis product of the cyclic lactone.129 Combined in vitro and in vivo studies have established this class of siderophore liberates nitric oxide in plant roots, which identifies a role as a biofertilizer to partner its function in Fe(III) supply. The discovery of the diazeniumdiolate-containing siderophores signals the rhizosphere as a rich hunting ground for other siderophore candidates likely to be multifunctional in the support of symbiotic plant-bacteria relationships.130

2.02.4.1.1

X-ray crystallography

The presence of the N-nitroso-N-hydroxylamine (R-N(OH)-NO) functional group in cupferron, used historically in analytical chemistry for metal determination, suggests that gramibactin and analogues could coordinate metals other than Fe(III), such as Cu(II), and might play a role in the mechanisms of homeostasis and/or supply for a broader range of metal ions. Structures of Cu(II)–, Fe(III)–, or Zr(IV)–cupferron have been solved by X-ray crystallography (Fig. 7B, E, H, respectively),131–133 with each showing a coordination geometry and a respective metal:cupferron stoichiometry of 1:2, 1:3 or 1:4 typical for these metal ions. The N-nitroso- or N-hydroxyl Zr(IV)-O coordinate bonds average 2.211 Å or 2.168 Å, respectively, in the 1:4 Zr(IV):cupferron complex, which is similar to the average bond lengths for 8-coordinate Zr(IV)-O complexes reported from a survey of the Cambridge Structural Database.134

Fig. 6

Structures of N-nitroso-N-hydroxylamine-containing siderophores.

Siderophores and iron transport

11

Fig. 7 X-ray crystal structures (H atoms omitted for clarity) of complexes between Cu(II) and (A) N-tert-butylacetohydroxamic acid135 or (B) cupferron (N-nitroso-N-phenylhydroxylamine),131 with overlayed structures (to second shell) in (C) (RMS 0.06 Å); or Fe(III) and (D) acetohydroxamic acid,136 or (E) cupferron,132 with overlayed structures in (F) (RMS 0.27 Å); or Zr(IV) and (G) N-methylacetohydroxamic acid,137 or (H) cupferron,133 with overlayed structures in (i) (RMS 0.10 Å).

The relationship between the N-nitroso-N-hydroxylamine (R-N(OH)-NO) and the hydroxamic acid functional group is demonstrated with the overlay of X-ray crystal structures of stoichiometrically equivalent complexes of Cu(II), Fe(III), or Zr(IV) formed with N-acylated hydroxamic acid monomers (Fig. 7A, D, G, respectively).135–137 The coincidence of these structures (Fig. 7C, F, I, respectively) supports the notion of N-nitroso-N-hydroxylamine-containing siderophores coordinating a wider range of metal ions and identifies this functional group of potential interest in other metal sequestration/delivery applications. Recent work has identified oximes as degradation products of the diazeniumdiolate group,125 which poses the question whether natural product oximes might be degradation products of diazeniumdiolate parent and that the diazeniumdiolate motif is more widespread as a functional group in bacterial metabolomes.

2.02.5

Siderophore adaptations

A number of structural features of siderophores enables their function as ligands for Fe(III) supply can be maintained in different environmental niches. The different types of functional groups present in siderophores, including hydroxamic acids, carboxylic acids, and catechol groups have pKa values than span across pKa 3.0 (rhizoferrin; average pKa values of a-carboxylic acid groups) to pKa 4.7 (rhizoferrin; average pKa values of b-carboxylic acid groups)54 to pKa  9 (DFOB (hydroxamic acid)) to pKa  12 (enterobactin, meta hydroxy group (catechol)) (Fig. 8A). This translates to the predominance of citric acid-based siderophores in acidic environments.

12

Siderophores and iron transport

While many bacteria produce a unique siderophore, other species generate a set of siderophores that may or may not be structurally related. This provides the bacteria with the best opportunity for survival in microenvironments (ocean, soil, host) with variable iron availability, including temporal variation. Maps of the hydroxamic acid siderophore metabolome of the marine bacterium Salinispora tropica CNB-400 showed the production of multiple siderophores,138,139 with a distribution that varied with culture conditions used to simulate environmental challenge.139 Mycobacteria species produce multiple siderophores, including mycobactins and exochelins to address the challenge for Fe(III) in different infection niches.140 Some bacteria deploy native siderophores for Fe(III) capture and can also harness siderophores produced by other organisms. This competitive edge is exercised by P. aeruginosa, with this pathogen able to express cell-surface outer membrane transporters that recognize different xenosiderophores, including DFOE, enterobactin, or ferrichrome.141 Three siderophores are produced by pathogenic strains of Acinetobacter baumannii, including acinetobactin, fimsbactin, and baumannoferrin.142–147 Of these, acinetobactin is the only siderophore required for virulence.148 Acinetobactin can be produced from a pH-dependent rearrangement of pre-acinetobactin, which is the dominant siderophore isomer at pH values < 6.145,149 At pH values > 7, an enzyme-independent intramolecular rearrangement occurs to generate acinetobactin, which contains an isoxazolidinone motif (Fig. 8B).149 Both pre-acinetobactin and acinetobactin remained competent Fe(III) ligands under the same pH condition preferred by the free siderophore, which could support A. baumannii growth at infection sites with acidic or basic microenvironments.149 Other pH-dependent siderophore isomerizations have been reported for vibriobactin from Vibrio cholerae150 and staphyloferrin B from Staphylococcus aureus.27 One of several phenomenon that contribute to the thermodynamic stability of Fe(III)–enterobactin is the ability for the catechol Fe(III) coordination mode at physiological pH values to switch to a salicylate mode at more acidic pH values, with each isomer stabilized by a different hydrogen bond network (Fig. 8C).93,151 Studies of siderophore-mediated iron uptake in Campylobacter jejuni152 showed that the C. jejuni periplasmic binding protein CeuE binds Fe(III) complexes of hydrolysis products of enterobactin with greater affinity than Fe(III)–enterobactin itself.153 The Fe(III) complex with tetradentate bis(2,3-dihydroxybenzoyl-L-Ser) (bisDHBS) (Fig. 9) bound to CeuE (Kd ¼ 10.1 nM) more avidly than Fe(III)–enterobactin (Kd ¼ 0.4 mM).153 This supports an adaptive mechanism to scavage iron using xenosiderophore hydrolysis products in addition to the parent xenosiderophore. This notion aligns with a recent study of the X-ray crystal structure of the outer membrane transporter Fiu from E. coli, which indicates the capacity of this protein to transport non-siderophore compounds containing catecholate motifs, as models of enterobactin hydrolysis products.154 Each of these examples highlights the optimal structural evolution of siderophores to meet their function as high-affinity chelators and the ability for these structures to adapt to sustain function under different environmental challenges.

Fig. 8 Range of pKa values for exemplar siderophores from the major classes (A); and a pH-dependent switch in siderophore isomerization (B), or in the Fe(III) binding mode (C).

Siderophores and iron transport

Fig. 9

13

Enterobactin and its hydrolysis products.

2.02.5.1

Marine organisms

Siderophores produced by marine bacteria have molecular adaptations specific to maintaining function in open ocean waters, which contain low concentrations of surface Fe(III) (20 pM to 1 nM),155–157 with further limits on bioavailability posed by the alkaline waters. Many marine siderophores contain acyl chains appended to the parent molecular scaffold.158 These fatty acid structures act as lifeline ropes, enabling the relatively polar siderophore head group to extend into the seawater milieu to coordinate Fe(III), with the acylated tail group embedded in the cell membrane leaflet to prevent the hard-won iron cargo drifting away. Petrobactin was first characterized from the marine bacterium Marinobacter hydrocarbonoclasticus,159 Under UV irradiation that mimics natural sunlight, the Fe(III)–a-hydroxycarboxylic acid motif of the Fe(III)–petrobactin complex undergoes a facile ligand-to-metal charge-transfer reaction that results in the decarboxylation of the internal carboxylic acid group to generate a 3ketoglutarate motif that retains Fe(III) coordination capacity.159 Petrobactin has since been identified to have a key role in the virulence of a selection of bacterial pathogens. Examples of amphiphilic marine siderophores include the amphibactins,160 which are siderophores dominant to ocean bacteria161,162 and b-hydroxyaspartic acid-containing aquachelins,163 alterobactins A and B, marinobactins and synechobactins (Fig. 10).164 The b-hydroxyaspartic acid motif is common to many siderophore structures,165,166 and is also featured in the siderophore pacifibactin identified from the hydrocarbon-degrading marine bacterium Alcanivorax pacificus.167 Despite genome mining predicting for a typical hexadentate siderophore, pacifibactin as isolated was found as an octadentate ligand containing two bhydroxyaspartic acid groups, and one linear and one cyclic L-ornithine-derived hydroxamic acid groups. Naturally occurring octadentate siderophores are few, and include the tetrameric hydroxamic acid macrocycle DFOT1 and malleobactin D.168,169 Analysis of diamagnetic Ga(III)–pacifibactin by NMR spectroscopy correlated with metal ion coordination occurring through the two b-hydroxyaspartic acid groups and the linear hydroxamic acid group. Pacifibactin showed the photoreactivity typical of b-hydroxyaspartic acid- and/or citric acid-based siderophores.164,167 Other classes of siderophores isolated from freshwater bacteria include the variochelins, which were identified using genome mining and found to contain a lipopeptide motif and an a-hydroxycarboxylic acid motif that is photoreactive when coordinated to Fe(III).170 As observed for model Fe(III)–polycarboxylate complexes,171 this promotes the release of more labile Fe(II) into the water to supply iron to algae in exchange for the supply of organic matter to bacteria as part of a bacteria-algae synergistic relationship.172,173

2.02.6

Biosynthesis of siderophores

2.02.6.1

Nonribosomal peptide synthetase (NRPS) pathways

The two major pathways for siderophore biosynthesis include nonribosomal peptide synthetase (NRPS) and NRPS-independent siderophore (NIS) pathways. Many peptide-based antibiotic agents, including bleomycin and vancomycin, are biosynthesized by the NRPS system. The NRPS pathway uses contiguous modules of biosynthetic enzymes that grow the molecular scaffold by appending single units of native or pre-modified amino acids. Tailoring enzymes proximal to the NRPS modules oversee other structural alterations.174 The molecular architecture of the multienzyme NRPS system includes an adenylation (A) domain, a condensation (C) domain, a peptidyl carrier protein (PCP) domain, and cyclisation (Cy) domains. The mature peptide chains are released following an interor intramolecular cyclisation reaction commonly catalyzed by a thioesterase at the C-terminal region of the synthetase.175–177 The NRPS-dependent biosynthesis of enterobactin, yersiniabactin, vibriobactin, mycobactin, pyochelin and pyoverdine has been delineated,175,176,178,179 with the biosynthetic logic in some cases a hybrid of NRPS and polyketide synthase (PKS) pathways.178,180 Yersiniabactin contains one phenol group and three thiazole-based heterocycles derived from the cyclisation of cysteine, with two at the dihydro (thiazoline) and one at the tetrahydro oxidation state (thiazolidine).181 Pyochelin comprises one each of

14

Fig. 10

Siderophores and iron transport

Structures of a selection of siderophores from marine bacteria.

a phenol group, a thiazoline group and a thiazolidine group, the latter which is N-methylated.182 Cyclisation of serine or threonine residues in precursor NRPS peptides generate the oxazoline or C-methylated oxazoline ring systems, respectively, characteristic of mycobactins,183,184 acinetobactin,185 and vibriobactin.186 A simple deconstruction of a siderophore assembled via a NRPS-dependent pathway demonstrates the amino acid assembly line that might be more obscure when absorbing the complete molecular structure. Strains of the actinobacteria Pseudonocardia produce a metabolite, named attinimicin, which acts as an iron-dependent antifungal agent against specific Escovopsis spp. fungal pathogens.187 These pathogens can invade the fungal food source cultivated by fungus-growing attine ants.187,188 The attinimicinproducing bacteria hosted by the ant colony protect their food supply, with a synergistic relationship met by the hosts feeding the bacteria.187 Attinimicin is assembled via a NRPS-dependent pathway from one unit of salicylic acid, two units of serine (Land D-), one unit of b-alanine and two units of N-hydroxy-L-ornithine, one as the linear N-formylated derivative and the other in its intramolecular cyclized form (Fig. 11). The biosynthesis of enterobactin co-opts three enzymes EntABC, which transform the shikimate-derived metabolite chorismate189–191 to 2,3-dihydrobenzoate, which is next processed by EntBDE together with L-serine in ATP-dependent reactions to generate the trilactone-based tris-catechol siderophore.192 The enzyme EntB has been shown to be bifunctional, with upstream isochorismase lyase activity for the production of 2,3-dihydrobenzoate and additional activity as an aryl carrier protein.189,192

2.02.6.1.1

Pyoverdine chromophore

The biosynthesis of the pyoverdine chromophore begins from the intramolecular condensation of the NRPS-assembled tripeptide S2,4-diaminobutyric acid-D-tyrosine-L-glutamic acid.193,194 This resulting ferribactin unit as biosynthesized in the cytoplasm is acylated for transport to the periplasm, and deacylated by the periplasmic hydrolase PvdQ prior to ongoing enzyme-mediated maturation steps.195,196 The periplasmic enzyme PvdP catalyzes the hydroxylation of ferribactin to generate the catechol motif, and its oxidation to the quinone. A pH-dependent intramolecular reaction forms the tricyclic pyoverdine precursor, with tautomerization and an PvdOmediated oxidation step generating the final pyoverdine chromophore (Fig. 12).196,197 The pendent L-glutamic acid residue is further

Siderophores and iron transport

Fig. 11

15

The amino acid assembly line for the NRPS-dependent biosynthesis of attinimycin.

modified by tailoring enzymes to generate different pyoverdine siderophores. The mechanisms of the five periplasmic enzymes involved in pyoverdine biosynthesis are of interest as potential antibiotic drug targets, with several studies identifying compounds as potential inhibitors of PvdQ.195,198,199 The genomes of two Marinobactin species that produce acylated marinobactin siderophores have been found to contain genes homologous to the pvdQ acylase gene in P. aeruginosa.200

2.02.6.2

Nonribosomal peptide synthetase-independent siderophore (NIS) pathways

Many siderophores are biosynthesized by pathways independent of NRPS pathways, as termed NRPS-independent siderophore (NIS) biosynthesis.201–204 Siderophores of the desferrioxamine class, including desferrioxamine B and analogues, and the related

Fig. 12

Biosynthesis of the chromophore unit in pyoverdine siderophores.

16

Siderophores and iron transport

dimeric hydroxamic acid macrocyclic siderophores putrebactin, alcaligin and bisucaberin are produced from NIS biosynthesis pathways.205–213 Siderophores built from a citric acid core, including aerobactin, rhizobactin 1021, rhizoferrin and achromobactin, are also assembled via NIS biosynthesis pathways.214,215 Many of these types of siderophores can be most simply considered as condensation products of diamines or polyamines and dicarboxylic acids, with variable patterns of N- and C-functionalization. The mechanisms of siderophore biosynthetic enzymes are of fundamental interest and present potential targets for the design of antibiotic agents that block these pathways and attenuate iron uptake.183,195,216–219 This underpins structural biology studies, which include recent X-ray crystal structures of the ornithine N-hydroxylase SidA involved in the upstream pathway of the biosynthesis of fusarinine C in Aspergillus fumigatus,220,221 and a range of siderophore synthetases,222–224 including the type B synthetase SbnC involved in the latter stages of the biosynthesis of staphyloferrin B in Staphylococcus aureus.225 The biosynthesis of aerobactin was the first example of NIS biosynthesis.201,226 An E. coli mutant deleted for a putative biosynthetic enzyme IucC showed the accumulation of two hydroxamic acid fragments in the supernatant, which supported the role of IucC in catalyzing the terminal condensation step of these two precursors to produce aerobactin.226 One decade later, the biosynthetic gene cluster AlcABC for alcaligin biosynthesis was identified in Bordetella pertussis and B. bronchiseptica, together with the function of a pyridoxal phosphate (PLP)-dependent ornithine decarboxylase in generating the initial substrate 1,4diaminobutane.227,228 Since these foundational studies, the biosynthesis of many hydroxamic acid siderophores have been shown to use either 1,5-diaminopentane or 1,4-diaminobutane as the initial substrate, as produced from the decarboxylation of L-lysine or L-ornithine, respectively.90,205–213,229–231

2.02.6.2.1

Desferrioxamine B

The biosynthesis of desferrioxamine B (DFOB) begins with the DesA-catalyzed decarboxylation of L-lysine to generate 1,5diaminopentane as the diamine substrate.201,205–209,232,233 A single N-hydroxy group is installed on 1,5-diaminopentane by DesB to generate N-hydroxy-1,5-diaminopentane (HDP), with DesC next in line to catalyze the N-acylation reactions with cosubstrates acetyl- or succinyl-coenzyme A to generate N-acetyl-N-hydroxy-1,5-diaminopentane (AHDP) or N-succinyl-Nhydroxy-1,5-diaminopentane (SHDP), respectively. The final enzyme in the cascade DesD generates an adenylated derivative of SHDP, with this activated precursor primed for condensation with a unit of AHDP to generate the AHDP-SHDP heterodimer. The trimeric DFOB species is produced from the second-round DesD-mediated condensation of a second unit of SHDP onto the AHDP-SHDP heterodimer (Fig. 13). The preferred sequence of steps for DesD in grafting a SHDP monomer onto the AHDP-SHDP heterodimer rather than grafting a SHDP-SHDP homodimer onto a AHDP monomer was delineated from the population of constitutional isomers of dimer precursors and trimeric end-products produced using precursor-directed biosynthesis in culture of Streptomyces pilosus supplemented with a non-native diamine substrate.234 Enzyme homologues involved in the biosynthesis of related hydroxamic acid siderophores, including alcaligin, have been found to have flexible substrate selectivity and are competent in producing a range of linear and macrocyclic analogs.211

Fig. 13

The biosynthesis of desferrioxamine B.207,234

Siderophores and iron transport 2.02.6.3

17

Precursor-directed biosynthesis and mutasynthesis

Understanding pathways of siderophore biosynthesis opens the opportunity to perturb native siderophore structures using precursor-directed biosynthesis or mutasynthesis approaches.177,235 Precursor-directed biosynthesis involves culturing a producing species in medium supplemented with non-native substrates designed to compete against the native substrate during siderophore assembly. The method is attractive in its simplicity and can deliver new siderophore analogues and insight into the nuances of the biosynthetic pathway. The shortcoming of precursor-directed biosynthesis is the compounds are produced in low yield and as a mixture. The use of mutant strains unable to produce the native substrate can address these issues, although a mutasynthetic approach may carry its own complications should the native substrate be obligate for growth and/or involved in multiple biosynthetic pathways.

2.02.6.3.1

Hydroxamic acid

2.02.6.3.1.1 Putrebactin Knowledge of the biosynthesis of hydroxamic acid siderophores206–209 and foundational precursor-directed biosynthesis studies168,236,237 inspired more recent studies aimed to generate new analogues of putrebactin and DFOB. In one study, cultures of the native putrebactin-producer Shewanella putrefaciens78 were supplemented with 1,4-diamino-2-butanone as an inhibitor of ornithine decarboxylase. The attenuated levels of native 1,4-diaminobutane resulted in this species upregulating the production of 1,5-diaminopentane and its assembly of DFOB as a replacement siderophore, in addition to producing low levels of a putative hybrid siderophore assembled from 1:1 1,4-diaminobutane:1,5-diaminopentane.229 This chimeric siderophore was subsequently identified in S. algae B516 cultures and named avaroferrin, with its structure confirmed by X-ray crystallography.79 Cosupplementation of S. putrefaciens medium with 1,4-diamino-2-butanone to decrease native 1,4-diaminobutane and 1,4diamino-2(E)-butene as an alternative substrate resulted in the production of E,E-putrebactene as an unsaturated analogue of putrebactin (Fig. 14A).230 2.02.6.3.1.2 Desferrioxamine B Unsaturated analogues of DFOB have been identified in S. pilosus cultures supplemented with 1,4-diamino-2(E)-butene (Fig. 14A).234 As an asymmetric compound containing flanking N-acetyl or amine groups, analogues of DFOB generated using precursor-directed biosynthesis appear as a suite of constitutional isomers containing either one, two or three exogenous group built

Fig. 14 Unsaturated analogues of (A) putrebactin and DFOB; or of DFOB containing (B) ether or (C) disulfide groups in the backbone, produced using precursor-directed biosynthesis.230,234,238,241

18

Siderophores and iron transport

from a given native or non-native diamine substrate. The incorporation of one or two exogenous groups each delivers a set of three isomers with distinct positional patterns, as defined using a binary naming system,234 and depicted for ether analogues of DFOB produced from S. pilosus medium supplemented with oxybis(ethanamine) (Fig. 14B).238 The tri-ether DFOB analogue was about 45 times more water soluble than DFOB and was a useful synthon in the production of a ligand for Zr(IV) about 3.6 times more water soluble238 that its methylene isostere.84 Mixed-substrate precursor-directed biosynthesis with oxybis(ethanamine) and thiobis(ethanamine) generated 27 O- and Scontaining DFOB analogues, which matched the theoretical maximum (xy) of a trimeric siderophore (y ¼ 3) assembled from three substrates (x ¼ 3), including native 1,5-diaminopentane.239 The use of asymmetric substrates, such as 1,4-diamino-2-fluorobutane, generated a more complex speciation profile of DFOB-type analogues, with the similarity in product distribution between rac-1,4diamino-2-fluorobutane and R-1,4-diamino-2-fluorobutane suggesting one or more enzymes in the DesBCD biosynthetic cascade might operate in an enantioselective fashion.240 The use of cystamine (disulfanebis(ethanamine)) as a non-native substrate competitor against 1,5-diaminopentane in S. pilosus culture produced a DFOB analogue containing a cleavable disulfide bond in the terminal amine region (Fig. 14C).241 This was the single isomer detected in this system, which indicated that while cystamine was a viable DesB substrate for the production of Nhydroxy-cystamine, this intermediate was either unviable for DesC-catalyzed N-acetylation or if this was not the case, N-acetylN-hydroxy-cystamine was unviable for DesD condensation reactions.241 This shows the value of precursor-directed biosynthesis in generating siderophore analogues with new potential applications, such as antibiotic delivery through a reductive-triggered prodrug,241,242 and providing insight into the subtleties of the biosynthetic pathways.

2.02.6.3.2

Catechol

Homologues of the tris-catechol siderophore vibriobactin have been generated using precursor-directed biosynthesis with cultures of Acinetobacter bouvetii DSM 14964. This system exploited the capacity of a VibH homologue in A. bouvetii DSM 14964, as identified from genome mining, to install two 2,3-dihydroxybenzoic acid groups onto diamine or polyamine backbones. In iron-deficient medium, the strain produced three native siderophores featuring 1,3-diaminopropane, 1,4-diaminobutane or 1,5diaminopentane flanked by two units of 2,3-dihydroxybenzoic acid. A precursor-directed biosynthesis approach using medium supplemented with these exogenous diamines modulated the relative concentration of these siderophores. Other non-native substrates that were successfully transformed included diethylenetriamine, propargylamine and allyamine, with the latter compounds of potential use for the development of chemical probes.243 In other work, an E. coli construct with the biosynthetic gene cluster for enterobactin deleted and the biosynthetic gene cluster for vibriobactin inserted was shown to produce dimeric and monomeric analogues of enterobactin assembled from L-threonine rather than L-serine.244 This was understood to be a result of the capacity of the heterologous enzyme VibF to process L-threonine and Lserine, unlike the enzyme homologue for enterobactin biosynthesis, which processes only L-serine. The work proceeded to undertake precursor-directed biosynthesis studies using a library of diamines and polyamines for medium supplementation, which resulted in the production of a range of mono-substituted 2,3-dihydroxybenzoic acid diamine or polyamine compounds.244

2.02.6.3.3

Mixed-ligand

There are limited studies that have produced analogues of mixed-ligand siderophores using either precursor-directed biosynthesis or mutasynthesis. One study focused on Amycolatopsis methanolica and the generation of analogues of amychelin as potential antibiotic agents. The study generated halogenated analogues of amychelin from a A. methanolica mutant deficient in salicylate synthesis cultured in the presence of 4- or 5-fluoro- or 4- or 5-chloro-salicylic acid. This resulted in the production of fluoroamychelin I or chloroamychelin II, assembled from 4-fluoro- or 5-chloro-salicylic acid, respectively, which were active in a screen measuring the rescue of Caenorhabditis elegans from P. aeruginosa infection.245

2.02.7

Siderophore uptake and transport

2.02.7.1

Gram-negative bacteria

The active transport of Fe(III)–siderophore complexes from the extracellular region to the cytoplasm is orchestrated by a set of functionally distinct proteins.246,247 Specific proteins embedded in the outer cell membrane of a gram-negative bacterium can bind a given Fe(III)–siderophore complex with high avidity through a network of intermolecular interactions formed with up to 10 amino acid residues at the surface of the transporter.116 The transport of the Fe(III)–siderophore complex across the outer cell membrane requires an energy input, which is derived from the three-component Ton protein system in the cytoplasmic membrane, comprising TonB, ExbD and ExbB (or homologs).247–252 The TonB and ExbD proteins are anchored to the cytoplasmic membrane and extend into the periplasmic space and the ExbB protein is integral to the cytoplasmic membrane. Proton-motive force drives conformational changes in TonB, which mechanically interacts with the outer cell membrane protein to enable active transport of the Fe(III)–siderophore complex to the periplasm. The Fe(III)–siderophore complex is next shuttled by periplasmic binding proteins to a family of ABC transporter proteins integral to the cytoplasmic membrane. The periplasmic binding proteins are less specific that the outer membrane transporters and function to shuttle multiple Fe(III)–siderophore complexes, with periplasmic FhuD from E. coli shown to bind to DFOB, coprogen, and the sideromycin albomycin.253 The ABC transporters comprise two

Siderophores and iron transport

19

membrane-spanning domains of 10 a helices, in addition to an ATP-binding domain, which supplies the energy for the Fe(III)– siderophore complex translocation via ATP hydrolysis.247,251,252,254 The structures of Ton-B dependent siderophore outer membrane transporters in E. coli, including FhuA, FepA and FecA, have been characterized by X-ray crystallography.255–259 Each of FhuA, FepA and FecA, which transport ferrichrome, enterobactin or ferric citrate, respectively, have b-barrel structures similar with porins. The structure of albomycin complexed to the E. coli outer membrane transporter FhuA260 has also been solved, with a selection of other siderophore-protein complexes solved by X-ray crystallography given in Table 2. The b-strands in the characteristic outer membrane transporters are linked with short peptide regions on the periplasmic region, with long extracellular loops. These proteins are distinguished with a 150-amino acid residue region at the N-terminus which forms a globular structure that acts as a plug to occlude transport across the b-barrel cavity. The plug domain and a set of extracellular loops participate in an array of hydrogen bonding interactions providing the conformation for selective and high-affinity binding of the Fe(III)–siderophore complex. The TonB complex provides the energy required to induce a conformation change to open a pore in the outer membrane b-barrel to enable import into the periplasm. The role of the plug domain in Fe(III)–siderophore transport has been an active research area. Studies have examined transport in plug-less mutants and also in chimeric species that interchange the b-barrel and plug regions of proteins specific to the transport of different Fe(III)–siderophore complexes.247 X-ray crystal structures of Fe(III)–coprogen bound to an E coli outer membrane transporter (Fig. 15A)261 or a periplasmic binding protein (Fig. 15B)253 show the clear structural distinctions between these classes of protein underlying siderophore-mediated transport.

2.02.7.2

Gram-positive bacteria

In contrast to gram-negative bacteria, gram-positive bacterial cells do not have an outer membrane or periplasmic space. In these species, the structural and functional homologue of the periplasmic binding protein in gram-negative species, occurs in a lipoprotein form that is covalently anchored to the plasma membrane. This siderophore binding protein (SBP), also named more broadly as a solute or substrate binding protein, recognizes the Fe(III)-siderophore complex akin to the periplasmic binding protein of gramnegative species, with transport under the operation of an ABC transporter system.16,247,273 A recent study has posed that the siderophore binding protein (SBP) in gram-positive bacteria carries a dual function as a receptor and an enzyme.274,275 Earlier studies demonstrated the formation of non-covalent complexes between a given apo-siderophore and the cognate siderophore binding protein.265 A shuttle mechanism was proposed to form the Fe(III)–siderophore complex, with the Fe(III) sourced from a localized holo-siderophore.272 This challenged the long-held assumption that a siderophore operated as a free Fe(III) sequestering ligand and that only the Fe(III)-siderophore complex was recognized by the siderophore binding protein prior to being transferred to the ABC transporter. The recent work presents the apo-siderophore-SBP complex as a functional enzyme that catalyzes the removal of Fe(III) from a tightly held source, such as Fe(III)-transferrin.275 This would ascribe a function to the apo-siderophore as an enzyme co-factor.275 Table 2

A selection of X-ray crystal structures of siderophore-protein complexes relevant to siderophore-mediated iron uptake and transport.

Type

Name

Class

Source

Kd (nM)

References

OMT SBP OMT OMT OMT OMT OMT OMT OMT OMT SBP SBP SBP SBP SBP SBP SBP SBP SBP SBP SBP SBP

Fe(III)–ferrichrome Fe(III)–coprogen Fe(III)–coprogen Albomycin Fe(III)-DFOB Fe(III)–enterobactin Fe(III)–citrate Pyochelin Fe(III)–DFOE Fe(III)–DFOB Fe(III)–enterobactin Petrobactin Petrobactin Fe(III)-petrobactin Enterobactin Fe(III)-enterobactin Fe(III)-petrobactin petrobactin Fe(III)-petrobactin DFOB Fe(III)-DFOB Fe(III)–acinetobactin

FhuA FhuD FhuE FhuA FhuD2 FepA FecC FptA FoxA FoxA FepB YclQ FpuA YclQ FeuA FeuA FpuA FatB FatB YxeB YxeB BauB

Escherichia coli E. coli E. coli E. coli Staphylococcus aureus E. coli E. coli Pseudomonas aeruginosa P. aeruginosa P. aeruginosa E. coli Bacillus subtilis Bacillus cereus B. subtilis B. cereus B. cereus B. cereus B. cereus B. cereus B. cereus B. cereus Acinetobacter baumannii

100–200 NDa 508 ND 34–50 50 ND 17 178 100 ND 35 23 113 36 12 175 77 127 36 29 160

255,262 253 261 260 263,264 257,258 259 265,266 267 267,268 269 270 271 270 271 271 271 271 271 272 272 252

a

ND, not determined.

20

Siderophores and iron transport

Fig. 15 X-ray crystal structures of Fe(III)–coprogen bound to the E. coli (A) outer membrane transporter FhuE (PDB: 6E4V)261; or (B) periplasmic siderophore binding protein FhuD (PDB: 1ESZ).253

Recasting the function of the SBP as both a receptor and an enzyme, is coupled with broadening the function of the siderophore from a substrate for ABC transport to a SBP ferrichelatase cofactor. Differences between the behavior of linear and macrocyclic hydroxamic acid siderophores DFOB and DFOE were ascribed to the increased conformational flexibility of the linear constructs, which was amenable to forming a pre-organized state with the SBP for the subsequent catalytic step.

2.02.7.3

Release of Fe(III)

The release of iron from the Fe(III)–siderophore complex has been proposed to occur by several mechanisms, including Fe(III)/(II) reduction, pH-dependent dissociation, or by siderophore degradation.55,276 The negative reduction Fe(III)/(II) reduction potential of Fe(III)–enterobactin at pH 7 precludes a reductive mechanism for Fe(II) release at this pH value. Observations of a green Fe(II)– enterobactin complex in methanol at pH 4277 and measurements of the strong pH-dependence of the Fe(III)/(II)–enterobactin redox potential, suggested that Fe(II) release might be possible within the acidic periplasmic space of E. coli,278 although more recent understanding indicates the dominant release mechanism as one mediated by the E. coli Fes-esterase and its hydrolysis of the triester backbone in the Fe(III)–enterobactin complex.279 An esterase PfeE in P. aeruginosa has been shown to hydrolyze Fe(III)–enterobactin, with the release of iron requiring a downstream reductase.280

2.02.7.4

Chirality

The configurational (D, L) isomerism of Fe(III)–siderophore complexes (Fig. 4) and/or the chirality inherent to the siderophore as a free ligand (R, S) provides a further mechanism for biology to modulate iron assimilation pathways.281 Molecular discrimination can conceivably occur upstream at the level of Fe(III)-siderophore-outer membrane transporter recognition and/or at downstream points in the pathway, such as the enzyme-mediated disassembly of the Fe(III)–siderophore complex. The siderophore enantiomers pyochelin (Pch I: 40 R,200 R,400 R) and enantiopyochelin (EPch I: 40 S,200 S,400 S) are produced by P. aeruginosa and P. fluorescens, respectively.282 The Fe(III)-pyochelin or Fe(III)-enantiopyochelin complexes are specifically recognized and transported by the outermembrane transporters FptA (Kd ¼ 2.5  1.1 nM) or FetA (Kd ¼ 3.7  1.1 nM), respectively, with no cross complex-transporter behavior.282,283 An example of chirality affecting a downstream process involves the uptake of D-Fe(III)–enterobactin or L-Fe(III)–bacillibactin complexes by Bacillus subtilis. While the uptake of these complexes was similar, there were differences in the B. subtilis growth rate, which were ascribed to the stereoselective properties of the hydrolytic enzymes, Fes and BesA.284

2.02.8

Siderophores in infection and stealth siderophores

To establish an infection, a bacterial pathogen must meet its requirement for iron. Deprivation of this element describes a state of nutritional immunity and will prevent or attenuate the ability of the infection to establish. The host organism limits the concentration of free plasma iron to about 1018 M, with its own essential iron reserves secured in ferritin or bound to transferrin or lactoferrin. Competition for iron has led pathogenic bacteria to evolve siderophores, in addition to alternative iron uptake systems, including heme acquisition and transferrin/lactoferrin receptors.285–289 Affinity constants for Fe(III)–siderophore complexes (Table 1) are many orders of magnitude greater than for Fe(III)–transferrin/lactoferrin complexes (logb  20), which underpins the trans-chelation function of siderophores in infection. The tug-of-war for iron between host and pathogen is further played out with the production of the human plasma protein siderocalin (also known as lipocalin 2 or neutrophil gelatinase-associated lipocalin (NGAL)), which is produced as part of the innate immune system. Siderocalin recognizes and binds Fe(III) complexes formed with select siderophores produced by bacterial pathogens. This thwarts iron acquisition in the pathogen and attenuates growth. Siderocalin recognizes Fe(III)-enterobactin produced by Escherichia coli but cannot bind salmochelins, which are glycosylated analogues of enterobactin first isolated from Salmonella enterica serotype Typhimurium.290 This has led pathogens to evolve

Siderophores and iron transport

21

the production of partner siderophores that evade recognition by siderocalin, and operate by stealth to restore iron supply to the bacterial cell.291 In addition to being the single known example of a mixed citric acid-catechol siderophore, the substitution pattern of the catecholate groups in petrobactin (3,4-) are different from other catechol-based siderophores, such as enterobactin and bacillibactin (2,3-). The 3,4-catechol motif is unable to be recognized by siderocalin, which provides the potential for a bacterial pathogen to evade the host immune system by producing petrobactin for Fe(III) acquisition. This was established for the causative agent for anthrax, Bacillus anthracis, which produces bacillibactin and petrobactin as native siderophores via independent pathways. While bacillibactin is bound by siderocalin, petrobactin is not recognized by siderocalin either as the free ligand or the Fe(III) complex. This founded the notion of petrobactin as a ‘stealth’ siderophore, which acts in a covert operation to supply iron for growth, with bacillibactin produced as a decoy siderophore rendered ineffective by siderocalin. In addition to specific toxins, this identifies petrobactin as a key virulence factor in B. anthracis.292 Further insight into this system has been provided with the use of combinatorial protein design to reprogram human siderocalin to bind Fe(III)–petrobactin rather than Fe(III)–bacillibactin.291

2.02.9

Applications of siderophores

The ability to coordinate metal ions other than Fe(III) confers value upon siderophores in a range of applications in medicine and the environment,293–300 and in the development of molecular tools, probes, and live-cell imaging.301–303 Siderophores are being developed for use in radiochemistry to complex isotopes suited for therapeutic (Ga(III)-67) or diagnostic (Ga(III)-68, Zr(IV)-89) use against cancer or infection.84,87,304–308 Desferrioxamine B was the first siderophore evaluated as a bifunctional ligand for radiochemistry applications, as labelled with Ga(III)-67 and conjugated to human serum albumin.309 The fungal siderophore fusarinine C has been studied as a bifunctional ligand, with a biological recognition motif conjugated to the free amine groups at the macrocycle periphery.310 Desferrioxamine B has a long history as a therapeutic agent to treat iron overload disease in people with transfusion-dependent anemias.63,311,312 Siderophore conjugates have potential utility antibiotic delivery.39,242,313–315 A fascinating application of the catechol-based exo-macrocyclic siderophore trichrysobactin from the plant pathogen Dickeya chryasanthemi has been revealed in the realm of physical and materials chemistry. Trichrysobactin has adhesive properties akin to 3,4dihydroxyphenylalanine (DOPA)-containing mussel foot proteins used by sessile marine organisms to adhere to wet ocean surfaces.316

2.02.10 Conclusion Siderophore-mediated Fe(III) uptake is an important pathway for bacterial and fungal iron acquisition. This phenomenon impacts the survival of non-pathogenic bacteria and fungi and affects the ability of pathogenic species to establish an infection in a mammalian host. This establishes the significance of the field and the on-going need to drive siderophore research at the level of fundamental discovery and coordination chemistry and protein binding and transport. This understanding will reveal new understanding of bacterial and fungal ecology and opportunities in modulating iron acquisition to improve pathogen control. Siderophore discovery research continues to deliver new molecular architectures and new functional groups as a platform toward deeper knowledge of coordination chemistry relevant to biology. The applications of siderophores in the environment and medicine are expanding. The field promises a rich intellectual picking ground for the next generation of researchers, with never-ending scope for creative and cross-discipline thinking.

Acknowledgments Support from the Australian Research Council (DP180100785) is gratefully acknowledged.

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303. Pfister, J.; Lichius, A.; Summer, D.; Haas, H.; Kanagasundaram, T.; Kopka, K.; Decristoforo, C. Live-Cell Imaging With Aspergillus fumigatus-Specific Fluorescent Siderophore Conjugates. Sci. Rep. 2020, 10. Article 11519. 304. Petrik, M.; Umlaufova, E.; Raclavsky, V.; Palyzova, A.; Havlicek, V.; Pfister, J.; Mair, C.; Novy, Z.; Popper, M.; Hajduch, M.; Decristoforo, C. 68Ga-Labelled Desferrioxamine-B for Bacterial Infection Imaging. Eur. J. Nucl. Med. Mol. Imaging 2021, 48, 372–382. 305. Petrik, M.; Umlaufova, E.; Raclavsky, V.; Palyzova, A.; Havlicek, V.; Haas, H.; Novy, Z.; Dolezal, D.; Hajduch, M.; Decristoforo, C. Imaging of Pseudomonas aeruginosa Infection with Ga-68 Labelled Pyoverdine for Positron Emission Tomography. Sci. Rep. 2018, 8. Article 15698. 306. Rudd, S. E.; Roselt, P.; Cullinane, C.; Hicks, R. J.; Donnelly, P. S. A Desferrioxamine B Squaramide Ester for the Incorporation of Zirconium-89 Into Antibodies. Chem. Commun. 2016, 52, 11889–11892. 307. Boros, E.; Holland, J. P.; Kenton, N.; Rotile, N. J.; Caravan, P. Macrocycle-Based Hydroxamate Ligands for Complexation and Immunoconjugation of 89zirconium for Positron Emission Tomography (PET) Imaging. ChemPlusChem 2016, 81, 274–281. 308. Pandey, A.; Savino, C.; Ahn, S. H.; Yang, Z.-H.; Van Lanen, S. G.; Boros, E. Theranostic Gallium Siderophore Ciprofloxacin Conjugate with Broad Spectrum Antibiotic Potency. J. Med. Chem. 2019, 62, 9947–9960. 309. Yokoyama, A.; Ohmomo, Y.; Horiuchi, K.; Saji, H.; Tanaka, H.; Yamamoto, K.; Ishii, Y.; Torizuka, K. Deferoxamine, a Promising Bifunctional Chelating Agent for Labeling Proteins With Gallium: Ga-67 DF-HSA: Concise Communication. J. Nucl. Med. 1982, 23, 909–914. 310. Zhai, C.; Summer, D.; Rangger, C.; Haas, H.; Haubner, R.; Decristoforo, C. Fusarinine C, a Novel Siderophore-Based Bifunctional Chelator for Radiolabeling With Gallium-68. J. Label. Compd. Radiopharm. 2015, 58, 209–214. 311. Zhou, T.; Winkelmann, G.; Dai, Z.-Y.; Hider, R. C. Design of Clinically Useful Macromolecular Iron Chelators. J. Pharm. Pharmacol. 2011, 63, 893–903. 312. Liu, J.; Obando, D.; Schipanski, L. G.; Groebler, L. K.; Witting, P. K.; Kalinowski, D. S.; Richardson, D. R.; Codd, R. Conjugates of Desferrioxamine B (DFOB) With Derivatives of Adamantane or With Orally Available Chelators as Potential Agents for Treating Iron Overload. J. Med. Chem. 2010, 53, 1370–1382. 313. Liu, R.; Miller, P. A.; Vakulenko, S. B.; Stewart, N. K.; Boggess, W. C.; Miller, M. J. A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria to Commit Suicide With a Gram-Positive Antibiotic. J. Med. Chem. 2018, 61, 3845–3854. 314. Fan, D.; Fang, Q. Siderophores for Medical Applications: Imaging, Sensors, and Therapeutics. Int. J. Pharmaceut. 2021, 597, 120306. 315. Neumann, W.; Sassone-Corsi, M.; Raffatellu, M.; Nolan, E. M. Esterase-Catalyzed Siderophore Hydrolysis Activates an Enterobactin-Ciprofloxacin Conjugate and Confers Targeted Antibacterial Activity. J. Am. Chem. Soc. 2018, 140, 5193–5201. 316. Maier, G. P.; Rapp, M. V.; Waite, J. H.; Israelachvili, J. N.; Butler, A. Adaptive Synergy Between Catechol and Lysine Promotes Wet Adhesion by Surface Salt Displacement. Science 2015, 349, 628–632.

2.03 Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction Matthew R. Jordana,b, Matias Villarruel Dujovnec, Daiana A. Capdevilac, and David P. Giedroca, a Department of Chemistry, Indiana University, Bloomington, IN, United States; b Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN, United States; and c Fundación Instituto Leloir, Buenos Aires, Argentina © 2023 Elsevier Ltd. All rights reserved.

2.03.1 2.03.2 2.03.3 2.03.3.1 2.03.3.1.1 2.03.3.1.2 2.03.3.2 2.03.3.2.1 2.03.3.2.2 2.03.3.2.3 2.03.3.3 2.03.3.3.1 2.03.3.3.2 2.03.3.4 2.03.3.4.1 2.03.3.4.2 2.03.3.4.3 2.03.4 Acknowledgments References

Metal ion homeostasis Nutritional immunity and pathogen adaptation Metalloenzyme “paralogs” Category 1: Metal-independent paralogs Ribosomal C– paralogs DksA/DksA2 Category 2: Obligatory Zn-dependent paralogs QueD/QueD2 PyrC/PyrC2 HisI Category 3: Zn-independent or metal-promiscuous paralogs FolE/FolE2 HemB/HemB2 Others Carbonic anhydrase ThrRS2/CysRS2 Bacterial cell wall remodeling enzymes Conclusions and perspectives

30 32 33 34 34 35 36 36 38 40 41 41 43 44 44 45 47 49 49 49

Abstract Zn is an essential catalytic or structural cofactor for z6–10% of all proteins in a typical proteome. Bacteria often encounter extreme Zn limitation in their native environments and must adapt to the challenge of survival when there is insufficient Zn to metalate all cellular Zn binding sites. As a result, bacteria have evolved multiple mechanisms of cellular adaptation to low Zn to acquire, redistribute, or prioritize Zn for essential processes. One such mechanism is the increased cellular abundance of enzyme “paralogs” that complement the function of obligatory Zn-dependent enzymes that cannot be fully metalated in the Zn restricted environment. Here, we carefully review identified Zur (zinc uptake repressor)-regulated paralogs, compare each to their canonical enzyme counterpart, and infer how the paralog contributes to cellular function under conditions of Zn restriction. These paralogs can be catalogued in three distinct ways: (1) those that dispense of the need for a metal cofactor altogether; (2) those that still utilize Zn but have evolved new structural properties to minimize metal dissociation; and (3) those that replace Zn with a different metal. We evaluate the mechanistic distinctions between canonical protein and paralog and close by placing all known paralogs within the context of bacterial metabolism. We find that paralogs cluster within ancient and essential pathways and processes, notably nucleotide metabolism and the central dogma, as a way to prioritize Zn within the cell and sustain growth in Zn-deplete environments.

2.03.1

Metal ion homeostasis

Transition metals are essential cofactors in enzymes that function in a multitude of physiological pathways.1 Although zinc (Zn) is not technically a transition metal as it features a filled d-shell, we group it here with other immediately adjacent late d-block first-row transition metals that also function in biology, from manganese (Mn) to copper (Cu). ZnII (Zn) is the second most abundant biological transition metal in most organisms; indeed, up to 10% of proteins in a vertebrate proteome are predicted to require Zn for activity.2 The functional roles of Zn within the context of biological macromolecules has been extensively reviewed elsewhere,3,4 but in brief, Zn essentially serves either as a structural or a catalytic cofactor. Structural Zn coordination sites are typically characterized by tetrahedral coordination as classically observed in zinc finger (zf) domains (Fig. 1A). Zinc finger domains often exhibit thiolate coordination from Cys residues but can also contain His imidazole or mono /bidentate carboxylate coordination. Importantly, structural sites are coordinatively saturated and kinetically and/or thermodynamically stable, exhibiting very slow off rates.7 A

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Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

(A)

(B)

(C)

HO–

Substrate

Zn H 2O

31

Hydroxide ionization

Substrate HO

H

B-

Zn

Zn Substrate Zn-bound enzyme Structural treble clef Zn finger domain from IleRS

Catalytic alcohol dehydrogenase Zn binding site bound to EtOH substrate

Zn

Polarization of H2O

Direct coordination of substrate

Fig. 1 Representative Zn coordination sites in the Zn metalloproteins. (A) The tetrathiolate Zn-finger domain of bacterial Ile-tRNA aminoacyl synthetase (IleRS; see Section 2.03.3.4.2) as representative of a coordinatively saturated structural Zn site. Formation of this chelate stabilizes the fold of the enzyme (PDB: 1ffy).5 (B) The active site of an alcohol dehydrogenase as representative of a catalytic Zn site (PDB: 1adc).6 The Zn is coordinated by two Cys and one His, while the open coordination site is occupied by the alcohol group of an ethanol molecule as substrate; ethanol is oxidized to acetaldehyde by oxidized pyridine nucleotide, NADþ. (C) Major functions of coordinatively unsaturated catalytic Zn sites. An open coordination site is bound by a solvent water molecule in the ligand-free state. Top, Zn coordination of a water molecule lowers its pKa to create significant concentration of hydroxide anion in the active site, which functions as a nucleophile to perform a hydrolysis reaction. Middle, Zn coordination of a water molecule engages an active site base (B) to increase the nucleophilicity of the bound water molecule. Bottom, Zn coordinates the substrate (a hydroxyl group, for example) directly (see panel B) to increase the electrophilicity of a directly bonded carbon center to facilitate attack by a nearby active site nucleophile, which can be a Zn-coordinated water molecule.

structural Zn atom often nucleates folding around a small region of the protein, which then forms an interface that often promotes protein-protein or protein-nucleic acid interactions. Structural Zn sites can also play regulatory roles, as seen in the coordinatively saturated sites of Zn metalloregulatory proteins (see below) and the inhibition of tyrosine phosphatase 1B by Zn.8 Here, the Zn coordination site is typically at or very near the protein surface and is ligand exchange-labile where it functions at a distance as an allosteric modulator of protein function. Catalytic Zn sites tend to exhibit far more variation in coordination number, but notably differ from structural sites in the presence of an open coordination site that can engage with a substrate(s) or a water molecule(s) (Fig. 1B). Zn serves its catalytic function as a Lewis acid by accepting a pair of electrons from this non-protein derived ligand.4 In the absence of substrate, water molecules typically fill this open coordination site. Catalytic Zn sites can be broadly distinguished from one another based on what molecule(s) fill the open coordination site during catalysis (Fig. 1C). Zn can function as a Lewis acid that ionizes the bound water molecule or hydroxide anion which can then engage in nucleophilic attack of a substrate in enzyme catalysis; this mechanism lies at the heart of nearly all Zn-dependent hydrolytic enzymes. Alternatively, polarization of the Zn-bound water molecule creates a stronger nucleophile capable of engaging a neighboring protein-derived base. Thirdly, an ordered water molecule is directly replaced by the substrate, activating the substrate for subsequent chemical steps in the reaction. Due to its filled d-shell, Zn gains no ligand-field stabilization energy and is stable in the 2þ oxidation state and thus does not undergo redox cycling. As a result, Zn is not prone to oxidative inhibition and is capable of sampling multiple coordination numbers and geometries throughout a catalytic cycle, making Zn an advantageous, efficacious, and highly versatile cofactor in metalloenzymes.9 As Zn and other transition metals are crucial in biological processes, it is important for metalloproteins in the cell to distinguish between cognate and non-cognate metals for proper metalation, thereby preserving enzymatic function.10 Metalloproteins acquire their metal not directly from solvent as fully hydrated cations, but from exchange-labile transition metal complexes made with components of the cellular milieu.11,12 Many metabolites, including the cellular reductant glutathione, the amino acid histidine and myriad phosphate esters contain solvent-exposed S, O, and N atoms that can weakly coordinate transition metals with moderate affinity. Moreover, these metabolites are at much high concentrations relative to metalloproteins, and thus are central to the “bioavailable pool” of buffered (exchange-labile) transition metals. Total cellular Zn is readily measured to be in the z0.1 mM range, however the intracellular Zn level that is sensed by Zn metalloregulatory proteins in bacterial systems is between 10 fM–1 pM.13–15 This metal buffering process maintains a relatively narrow concentration range of metal activity to ensure that a particular metalloprotein is able to acquire the correct (cognate) metal and only the correct metal under homeostatic conditions to the exclusion of all others. As such, only minor increases (toxicity) and decreases (starvation) in the bioavailable concentration of a transition metal can lead to mismetallation (the replacement of the cognate metal with non-cognate metal) and under-metallation (the loss of metal from a metal binding site), respectively, of metal binding sites which can ultimately become inhibitory toward cellular growth. In order to adapt to changes in the environmental levels of transition metals, bacteria utilize transcriptional regulators to direct responses that maintain metal homeostasis.16–18 The Zn uptake regulator (Zur) is a well characterized transcriptional repressor that governs the bacterial response to changes in bioavailable Zn; Zur binds to DNA at elevated intracellular Zn concentrations to repress genes traditionally associated with Zn uptake (Fig. 2A).13,14,21,22 Zn uptake regulation is conserved in all bacteria. Zur proteins are members of the Fur (ferric uptake repressor) family of transcriptional repressors, while some Gram-positive bacteria encode for a protein that is functionally equivalent to Zur, but structurally distinct, from the MarR (multiple antibiotic resistance regulator) family, denoted AdcR.23 Zur is functionally a dimer with an N-terminal, mostly a-helical, DNA-binding domain and a C-terminal

32

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

(A)

(B)

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Category 2

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50

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Resistance Zn sparing to dissociation/ (alternate metal) Mass action

0 10-18

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active

active

active

Pseudomonas aeruginosa PA01

P CO A553 G0 5 52 3 PA 5 Dk 536 sA 2 PA Un 553 kn 7 ow n PA 55 Am 38 iA

P CO A553 G0 2 52 3 PA Un 5533 ko w PA n DU 553 F1 4 82 6

PA 5 To 531 nB

Zur box

PA 5 Fo 539 lE2 PA Ca 554 an rbon 0 hy dra ic se PA 5 Py 541 rC 2

Zur box

(C)

Fig. 2 Zur-regulated genes, often arranged as operons or clusters of operons and single genes to create regulons, encode proteins that play roles in the Zn limitation response. (A) Graded response of Zur to decreasing Zn bioavailability. The negative cooperativity of Zn binding allows for the sequential derepression of particular Zur operons by differentially metalated Zur forms, thereby expanding an organism’s Zn sensing range. (B) Model defining the different categories of Zur-regulated paralogs. At higher intracellular Zn (gray spheres) concentrations, the canonical enzyme (yellow) can acquire Zn while the paralog (cyan) is repressed by Zn-bound Zur (green). When Zn levels drop within the cell and the canonical enzyme is inactivated by the absence of Zn, the Zur-regulated paralog is expressed where it functions in the absence of any metal (category 1), with Zn (category 2), or with an alternative metal to Zn (light gray sphere, category 3). (C) Part of the P. aeruginosa Zur regulon comprised of two divergently transcribed operons is shown, with locus tags (gene IDs; PAxxxx) and inferred functional assignments. Two known Zur boxes are indicated.19 Genes that encode paralogs as defined here are shaded cyan, COG0523-family candidate Zn metallochaperones shaded red,20 genes encoding proteins involved in Zn uptake are dark blue, and genes of unknown function are shaded gray.

dimerization domain.21 All Zur proteins possess one tetrathiolate, structural Zn-binding site required for folding and stability of the dimer, and one or two additional “sensing” sites that bridge the DNA-binding and dimerization domains, resulting in “allosteric” coupling of the Zn- and DNA-binding sites; Zn binding at the sensing site(s) leads to a “closed” conformation that exhibits high affinity to the DNA operator (Fig. 2A).21 Zur senses fM levels of free Zn, which should not be interpreted as far less than one atom of hydrated Zn per cell, but rather indicative of an over-capacity of the cell to chelate transition metals in exchange-labile, coordinatively unsaturated complexes of modest stability.13,14,22 Interestingly, Zn sensing by most Zur proteins is not well-described by a single-site “on state-off state” two-state model, but instead incorporates a range of Zn affinities on the dimer that allows for a graded transcriptional regulatory response to decreasing concentrations of cellular Zn (Fig. 2A). This is achieved through partial metalation of the sensing sites across the Zur dimer: Streptomyces coelicolor Zur has two pairs of sensing sites and metalation of one pair of sites allosterically activates binding to a subset of DNA operators but not others, which require metalation of both pairs of sensing sites,22 while Bacillus subtilis Zur possesses only one pair of sensing sites and negative homotropic cooperativity of Zn binding to each protomer site activates DNA binding to some operators but not others (Fig. 2A).14 Thus, Zur is potentially capable of orchestrating a transcriptional response to Zn bioavailability over a much wider range of changes in mobile Zn concentration than would be possible with a simple two-state on-off switch (Fig. 2A). Obviously, an acute phase response to severe zinc starvation would rapidly induce the entire Zur regulon, in an effort to maintain the Zn quota required to fill all cellular Zn-binding sites.

2.03.2

Nutritional immunity and pathogen adaptation

While the Zn requirement for organismal growth has long been known, many bacteria often reside in severely Zn-depleted environments. Soil bacteria often live in Zn-limited niches, and even when Zn is present in the soil it is largely insoluble and therefore not

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

33

generally bioavailable.24,25 Marine bacteria encounter the same solubility problem where dissolved Zn is extremely low in the upper ocean.26 Likewise pathogenic bacteria that infect vertebrate hosts are subject to Zn deprivation through the innate immune response.27 Here, neutrophils are recruited to sites of bacterial infection and the multi-metal chelating protein calprotectin (CP) is released into the extracellular space to withhold essential transition metals, notably Zn, and inhibit bacterial growth.28–31 CP is a member of the anti-inflammatory S100 proteins, made up of the S100A8/A9 heterodimer, and possesses two distinct transition metal binding sites within the dimer: one hexaHis site that exhibits broad metal binding specificity for divalent transition metals and a second, largely Zn-specific His3Asp site. When excreted into the high Ca environment of the extracellular space, the CP dimer binds Ca at EF hand motifs and forms a tetramer, which strongly enhances the affinity of both sets of transition metal binding sites. CP-mediated Zn restriction is hypothesized to result in undermetallation of certain Zn-dependent enzymes that function in essential metabolic pathways, although identifying specific undermetallated sites in a proteome has proven challenging. Zurmediated regulation enables cellular persistence in low Zn through several mechanisms that differ in the degree to which metal starvation drives Zur-dependent transcriptional derepression (Fig. 2A). First, Zur controls the expression of dedicated, high affinity Zn uptake systems that compete with CP for extracellular Zn. These include classical ATP-binding cassette (ABC) uptake systems like ZnuABC19,32 and ZnuD, which brings Zn across the outer membrane in Gram-negative bacteria, as well as recently described proteins involved in the biosynthesis, export, and import of small molecule zincophores that, like classic siderophore-FeIII complexes, are deployed to scavenge extracellular Zn.33–37 The second key bacterial response to Zn limitation is Zn prioritization via the upregulation of one or more COG0523 Zn metallochaperones.20,38,39 COG0523 proteins are GTPases that bind Zn with high affinity and are thought to scavenge intracellular Zn, directly interact with an unmetallated and inactive client protein, and deliver the metal to the client active site using the energy of, or conformational changes associated with, GTP hydrolysis.28,39–42 A third important Zur-regulated adaptation mechanism is through the expression of protein “paralogs” of obligatory Zndependent enzymes that function in key physiological pathways. These include ribosomal proteins, enzymes that carry out tRNA modification and aminoacylation, nucleotide (purine, pyrimidine and pyridine) biosynthesis, one-carbon metabolism, transcriptional regulation, heme biosynthesis, carbon fixation, amino acid biosynthesis and bacterial cell wall recycling. These paralogs are widely cited in the literature as a form of “Zn sparing” where the paralog is expected to utilize an alternative metal such that this Zn could be “spared” for other crucial processes. However, as we review here, there is rather little experimental evidence to support these claims (Fig. 2B). In this chapter, we first evaluate the functions of Zn enzymes that are induced in a bacterial cell under conditions of Znlimitation. We then identify key differences in metal coordination and active site architecture of companion “Zur-regulated paralogs” and discuss how they function in mediating an adaptive response to extreme Zn limitation. Understanding these processes at a fundamental level transcends basic science to the potential identification of new avenues for antimicrobial therapeutics development. Indeed, metallo-b-lactamases expressed by clinically important isolates of major pathogens appear to have evolved toward a systematically increased Zn affinity in direct response to host-mediated nutritional immunity.43–46 We note however that these recently evolved metallo-b-lactamases are not paralogs, but simply homologs, as the dual selective pressures of antibiotics stress in the context of nutritional immunity selects variants of a single copy gene. Finally, many of the enzymes we discuss here are bona fide drug targets in a number of bacteria47–50; acquiring structural and mechanistic insights into how Zur-regulated paralogs differ from their constitutively expressed metalloenzyme counterparts may provide a path to the development of designer antimicrobials that target the enzyme paralog that accumulates to high levels during an infection and thus is likely to be more efficacious.

2.03.3

Metalloenzyme “paralogs”

Enzymes identified in Zur regulons, although designated metalloenzyme paralogs, sometimes have little or no sequence identity to their bona fide Zn-dependent counterparts. When sequence identity is found in catalytic or metal-binding domains, there are noticeable variations in the primary structure of the paralog that would seem to negatively impact metal binding. Thus, this nomenclature is somewhat problematic.51 The term “homology” is used for two proteins that are related through evolutionary descent and encompasses both “orthologs” and “paralogs,” each of which is defined by a distinct evolutionary event: speciation and duplication, respectively.51 Orthologs are related genes derived from a last common ancestor typically found in different organisms, while paralogs are related genes derived from a gene duplication event, and may or may not be in the same organism.51 Function is, in fact, completely irrelevant when comparing the evolutionary history of two genes in efforts to define two gene products as homologous, orthologous, or paralogous. However, the term “paralog” is widely used to distinguish two enzymes or proteins with the same fold, derived from a common evolutionary ancestor, but possess distinct functions. This formally incorrect but useful designation derives from the generalization that if there are two copies of a gene derived from a common evolutionary ancestor, the second copy may allow for adaptation if there is a functional distinction relative to the original copy. In the case of Zur-regulated Zn-metalloenzyme paralogs, the expectation would be that a gene duplication event allowed for the paralog to evolve the ability to catalyze the same reaction without a strict requirement for Zn. With this in mind, some of the Zur-regulated proteins that we discuss below may be true paralogs, while others do not appear to have sufficient sequence identity to confidently conclude that they share a common evolutionary ancestor. In this case, the additional copy simply incorporates mutations that provides some evolutionary advantage under conditions of chronic Zn starvation. We use the term “paralog” here to classify all Zur-regulated enzymes because of its historical use in the literature.38,52

34

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

We further use the P. aeruginosa Zur regulon as a model representative of other bacterial Zur regulons and which has been characterized in some detail (Fig. 2C).19 This regulon contains two distinct Zur-boxes that regulate the expression of different sets of genes (an operon). The first divergently transcribed operon encodes for two proteins of unknown function, two COG0523family putative Zn metallochaperones, the DksA2 paralog (Section 2.03.3.1.2), and a TonB transporter component that may be involved in Zn uptake across the outer membrane in this Gram-negative bacterium, through the outer membrane porin ZnuD.53 The second divergently transcribed operon encodes for the paralogs FolE2, PyrC2, AmiA, and a g-carbonic anhydrase, each of which is discussed below. Overall, we conclude that the functional characteristics of paralogs that we currently know of can be classified into three distinct categories (Fig. 2B): (1) paralogs that have lost metal coordinating residues and may or may not dispense of the need for metal altogether (Category 1), (2) paralogs that retain specificity for Zn but may have evolved some new functional characteristic (Category 2), and (3) paralogs that require a metal for catalysis but have evolved to utilize a metal that is not Zn (Category 3). Each paralog category exhibits distinct mechanistic features in adaptation to low Zn, while crucially sustaining core components of the central dogma, and maintaining metabolic flux through major biosynthetic pathways.

2.03.3.1 2.03.3.1.1

Category 1: Metal-independent paralogs Ribosomal C– paralogs

The classical case of Zur-regulated paralogs comes from the modification of structural, not catalytic, Zn binding sites.52,54 This is observed in the Zn sparing phenomenon of Zur-regulated, non-enzymatic ribosomal protein paralogs that lack the CxxC Zn binding motifs (called “C–” ribosomal proteins) that are thought to replace their Zn binding counterparts (“Cþ,” Fig. 3).54 This exchange happens spontaneously upon upregulation of the transcription of C– subunits57 so that Cþ subunits are degraded, releasing Zn for other crucial physiological processes, and enabling the bacteria to resist Zn limitation. Notably, this can account for a massive influx in bioavailable Zn as ribosomes are one of the most abundant molecules in the cell. Seven C– paralogs have been identified in Zur regulons, with a large diversity across bacteria; some organisms encode for no C– paralogs while others possess up to six (Fig. 3A).58 These paralogs are present across the ribosome structure, spanning both large and small subunits of the bacterial ribosome. Although the key distinction between C þ and C– proteins is the loss of Zn binding Cys residues in C– paralogs, some C– subunits also contain insertions, extensions, and divergent sequences compared to their C þ counterparts (Fig. 3). Long hypothesized for

L31 L31B

L33 L33B

L28 L28B

14-VQ.C CGC CGH 14-FQDAATGA

13-LAC CEVC CKHR 12-LRSTAGTGY

36-EVC CSEC CHPFY 52-DVTSDSHPFW

S14 S14B

21-YTRC CNKC CGRPHSVYRKFGLC CRIC CLRE 61-RNRDVVDGRPRGHLRKFGLSRVRVRE

39-KKFC CPNC CGTH 38-RKYDPVLRRH

3-AVC CDIC CGKGP 3-AHCQVTGRGP

50-NAC CTSC CIKA 50-RVSAKGIKV

S18 S18B

18-RKC CVFC CSK 19-RRNQLEAL

54-TGNC CVQH HQRDIA 55-TGLTPQQQRQVA

Fig. 3 C– ribosomal protein paralogs. The M. smegmatis 70S ribosome is colored gray with the indicated ribosomal subunits highlighted in yellow (PDB: 5o61 C þ ribosome and 6dzi C– ribosome55,56). The five Cþ subunits (yellow) that are associated with Zur-regulated C– paralogs (cyan) in M. smegmatis are shown in ribbon representation and overlaid with C– insertions or extensions colored in red. A select sequence alignment of Cþ vs. C– subunits with Zn-binding Cys residues labeled in yellow and highlighted with a yellow sphere.

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

35

their role in Zn redistribution, these ribosomal paralogs have only recently been evaluated for functional differences. Both C þ and C– ribosomes from M. smegmatis have been structurally characterized55,56 and specific C– insertions resist antibiotic inhibition of the ribosome active site.59 C– ribosomes have similar translation and translation error rates as C þ ribosomes and may be more active under Zn-limiting conditions.60 However, C– ribosomes exhibit distinct translation profiles60,61 and have slower initiation rates than Cþ ribosomes.60 C– subunits clearly confer additional functions and characteristics to the ribosome that remain to be fully evaluated, but an exciting hypothesis is that C– ribosomes may selectively upregulate the translation of certain cellular transcripts that directly impact physiological adaptation to Zn limitation.

2.03.3.1.2

DksA/DksA2

A second Zur-regulated paralog that exhibits the loss of key metal binding residues is the paralog of DksA, DksA2. DksA, first identified as a DdnaK phenotype suppressor mutant, is a global transcriptional regulator crucial in the stringent response.62 The stringent response is induced by general nutrient starvation resulting in hyperphosphorylation of nucleotides which signals an alleviation mechanism(s) that involves DksA.63 The major signaling molecules in this response are the alarmones guanosine 50 -triphosphate 30 -diphosphate (pppGpp) and guanosine 50 -diphosphate 30 -diphosphate (ppGpp), collectively referred to as (p)ppGpp, which are synthesized from GTP and GDP. These alarmones severely deplete cellular GTP pools, and function either by inhibiting enzymatic activity directly by binding to a GTP or GDP binding site in competition with this substrate or “signal” within the transcriptional machinery itself.64 DksA can also respond to ROS/RNS in a (p)ppGpp independent manner.65 Although DksA is constitutively expressed, it is known to function only when (p)ppGpp and/or ROS/RNS become cellularly abundant (Fig. 4A).65,68 Under these conditions, DksA binds directly to RNA polymerase and either inhibits or enhances transcription from selected promoters of genes encoding proteins involved in translation or amino acid biosynthesis and uptake (Fig. 4A).66,69,70 The DksA mode of action in transcriptional inhibition is through the destabilization of initiation complexes, while the mechanism of transcriptional activation by DksA is poorly understood.71 The combination of DksA and (p)ppGpp in the stringent response gives rise to inhibition of ribosome production, thereby decreasing amino acid consumption by translation and increasing amino acid biosynthesis, thereby restoring

(B) (A)

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[Zn]

A (p)ppGpp DksA

B

AA biosynthesis proteins

Zn binding site E. coli DksA 110-DFGYC CESC CGVEIGIRRLEARPTADLC CIDC CKTLAE P. aeruginosa DksA2 92-DYGWCQETGEPIGLRRLLLRPTATLCIEAKERQE Disulfide bridge (D) (C)

G

ROS/RNS

(E)

CTH DksA

A (p)ppGpp B ROS/RNS

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Fig. 4 DksA transcription factor. (A) Mechanistic model of DksA and (p)ppGpp/ROS/RNS-mediated transcriptional inhibition of RNA polymerase. Under Zn replete conditions (top), Zn-bound DksA can respond to nutrient starvation in conjunction with (p)ppGpp to inhibit ribosomal RNA and protein transcription and activate amino acid biosynthesis gene transcription. Zn-bound DksA can additionally respond to high levels of ROS/RNS to inhibit transcription of both ribosomal RNA/protein and amino acid biosynthesis gene transcription. Under low Zn (bottom), DksA becomes undermetallated and nonfunctional, therefore upregulation of Zn-less DksA2 restores cellular DksA function. DksA2 can respond to nutrient starvation with (p)ppGpp similarly to Zn-bound DksA, however DksA2 responds to lower levels of ROS/RNS than that of Zn-bound DksA2, and similarly inhibits transcription of both ribosomal RNA/protein and amino acid biosynthesis gene transcription. (B) Ribbon representation of an overlay of the global structures of E. coli DksA (yellow, PDB: 1tjl)66 and P. aeruginosa DksA2 (cyan, PDB: 4ijj)67 with the globular (G), coiled-coil (CC), and C-terminal helix (CTH) domains indicated, and the DksA Zn ion shown as a black sphere. (C) Sequence alignment of the global transcriptional regulator of E. coli DksA and P. aeruginosa DksA2, centered on the DksA metal binding region. DksA Zn-binding residues are labeled in yellow and highlighted with a yellow sphere and the DksA2 disulfide bridge is labeled with a bracket. (D) Tetrathiolate Zn binding site of E. coli DksA with Zn coordinating Cys residues labeled and shown in stick, with the Zn ion shown as a black sphere. (E) Analogous metal binding site region of P. aeruginosa DksA2, with the two conserved Cys that from a disulfide bridge labeled and shown in stick representation.

36

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

nutrient balance in the organism. The DksA-dependent regulatory profile that is independent of (p)ppGpp and connected to ROS/ RNS stress down-regulates both the ribosome biogenesis machinery and amino acid biosynthesis, while also promoting redox buffering and repair of biomolecules, thus resulting in broad cellular resistance to these conditions.65,72 DksA is a small protein with an N-terminal globular (G) domain, an extended coiled-coil (CC) domain, and a C-terminal a-helix (CTH, Fig. 4C).66 All domains make contacts with RNA polymerase within what is known as the secondary channel, where nucleotide triphosphates are predicted to enter the active site for RNA synthesis.68 Structurally, DksA belongs to a family of transcription factors that bind to RNA polymerase within secondary channel, but have distinct regulatory functions.73,74 The G domain is linked to the CTH by a structural, tetrathiolate Zn binding site that exhibits tetrahedral coordination geometry (Fig. 3D). Zn was initially proposed to play a structural role, whereby Zn coordination simply stabilizes the placement of all three structural domains for productive binding to RNA polymerase.66 Indeed, mutation of Zn-coordinating Cys residues disrupts DksA function in cells.67,69,75 However, the picture that has emerged more recently is far more complex than this. For example, the four Zn-coordinating Cys residues have been shown to be susceptible to oxidation by various reactive oxygen and reactive nitrogen species (ROS and RNS) which enhances global DksA-mediated transcriptional inhibition, a finding not fully consistent with a purely structural role.65,72 Reaction with ROS and RNS species results in the release of Zn, and treatment with reducing agent after oxidation results in nearly full restoration of DksA secondary structure. These findings suggest that the primary role of Zn is not that of a structural cofactor, but rather as a redox-sensor; to confirm this, the functionality of reduced, apo-DksA would have to be tested.72 However, oxidation results in a disulfide bond primarily linking C114 and C135, and oxidized DksA retains  60% of its ability to bind RNA polymerase. This oxidation event also leads to potent transcriptional inhibition in the absence of (p)ppGpp, consistent with its assignment as a regulatory metal site that tunes ROS and RNS sensing by DksA.72 The DksA2 paralog is found in Zur regulons of b- and g-proteobacteria38 and has been most extensively characterized in P. aeruginosa (Fig. 2A). PaDksA2 shares 34% sequence identity with PaDksA, and both proteins adopt nearly identical folds (RMSD ¼ 0.82 Å, Fig. 3C). PaDksA2 possesses only two cysteines of the four Zn-coordinating Cys present in DksA (C96 and C117), which are precisely analogous to the redox-sensing cysteines C114 and C135 in EcDksA (Fig. 3B). Although these two remaining DksA2 Cys residues form a disulfide bond that has been structurally captured (Fig. 3E), this disulfide linkage may not be required in vivo and cannot be identified in cell extracts.67 However, mutation of these two Cys residues completely abolishes both DksA and DksA2 activity in cells. Loss of two or three Zn coordinating cysteines (some DksA2 paralogs possess only one of the four Cys observed in EcDksA) would of course abolish Zn binding in the cell, and thus would effectively “spare” this Zn for some other purpose.67 In P. aeruginosa the DdksA strain exhibits a growth phenotype on Zn-replete minimal media since Zur will repress the expression of DksA2 under these conditions.75 As expected, this phenotype is suppressed under low Zn, where DksA2 is upregulated.75 Indeed, DksA2 has been shown to functionally complement DksA in vivo and in vitro, however DksA2 may have attenuated activity relative to DksA.75 Additionally, a P. aeruginosa strain that expresses only DksA (DdksA2 strain) exhibits inhibited growth on Zn-deplete minimal media, clearly indicative of a disruption of DksA function only under conditions of low Zn.75 A consensus model holds that DksA becomes undermetallated under Zn limiting conditions resulting in increased sensitivity to ROS/RNS and an impaired alarmone-dependent adaptive response. Under these conditions DksA2 is upregulated, thus restoring the stringent response, although with perhaps less potency. Why then is DksA a Zn metalloprotein at all? One possibility is that Zn coordination by DksA simply attenuates its redox-sensing activity, reserving the redox-sensing Cys pair to respond only under conditions of chronic ROS stress, where cellular reductant pools become depleted.65 Consistent with this, the Zn-free DksA2 is  50-fold more reactive toward H2O2 in vitro and oxidation-induced transcriptional repression is far more sensitive to low H2O2 in cells expressing only DksA2 vs. cells expressing only DksA.65 Kinetic modeling reveals that the Zn-bound DksA requires significantly higher ROS to enhance transcriptional repression since the major cellular reducing thiol glutathione (GSH) preferentially scavenges ROS under these conditions; in striking contrast, DksA2 is a better ROS scavenger than GSH itself.65 This results in reduced survival in macrophage models of infection of bacterial cells that express only DksA2 compared to those expressing only DksA, as (p)ppGpp-independent transcription inhibition occurs at lower ROS levels. Taken together, dispensing of the Zn binding site in DksA2 is a remarkably complex evolutionary adaptation that appears to integrate the bacterial adaptive response to Zn restriction and chronic ROS exposure that may well be coincident in the infected host.

2.03.3.2 2.03.3.2.1

Category 2: Obligatory Zn-dependent paralogs QueD/QueD2

QueD is a 6-carboxy-5,6,7,8-tetrahydropterin synthase that performs the committed step in the biosynthesis of the transfer RNA (tRNA) modification queuosine (Q).76 The Q modification is specifically installed in the wobble position of the anticodon loop of four tRNAs that decode synonymous NAC and NAU (where N is any nucleotide) codons.77 In the absence of the Q modification, the unmodified tRNA exhibits weaker codon-anticodon pairing to NAU codons (called Q-decoded codons) leading to ribosome stalling, whereas the presence of Q modification alleviates the disruption of translational speed.77–82 In addition, the Q modification prevents second-position misreading of certain codons, enhancing translational accuracy.81–83 While only mild growth phenotypes are typically associated with lack of Q production thus far,84 the importance of maintaining the production of Q-modified tRNA may well be dependent upon the number of Q-decoded codons encoded in an organism’s genome. For example, those genomes with an increased representation of Q-decoded codons in protein-coding open reading frames may well have a significant dependence on the Q modification to maintain translational speed and fidelity. Beyond QueD, it is interesting to note that the Q

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

37

biosynthesis pathway harbors five additional transition metal-dependent enzymes that may be impacted by cellular metal limitation.85–91 QueD converts 7,8-dihydroneopterin triphosphate into 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) and exhibits activity when coordinating a Zn ion, although the transition metal-dependence of this activity has not been extensively evaluated (Fig. 5A).86 QueD is a member of the tunnel fold (T-fold) superfamily characterized by an oligomeric assembly state that forms a central b-barrel that resembles a tunnel.93 This family consists of two b2nan rings (n  2) that join in a head-to-head fashion. Binding to planar substrates and metal ions occurs at the interface of protomers in most T-fold proteins, however this superfamily is characterized by a significant catalytic and sequence diversity.93 E. coli QueD adopts a hexameric oligomerization state composed of two stacked trimeric rings (Fig. 5C). QueD proteins possess a conserved Cx3HGH motif containing the essential catalytic Cys27 residue and two His (His31 and His33) residues that coordinate the Zn (Fig. 5B and C). An additional conserved residue, His16, contributes the third protein-derived coordination ligand, with two water molecules or two oxygen atoms from the substrate, intermediates or product completing the five-coordinate trigonal bipyramidal Zn site (Fig. 5C). The mechanistic cycle, as described for E. coli QueD,92 involves two distinct catalytic triads, both of which employ the catalytic Cys27 of the Cx3HGH motif (Fig. 5A). The first catalytic triad deprotonates Cys27, while the Zn ion coordinates the 10 - and 20 hydroxyls such that the thiolate can more easily abstract the C20 proton for triphosphate elimination. The second catalytic triad again deprotonates Cys27 to activate a water molecule for nucleophilic attack of the C30 carbonyl carbon, where resonance rearrangement then leads to the elimination of acetaldehyde and the formation of the 6-carboyxy-5,6,7,8-tetrahydropterin product. Throughout the mechanistic cycle, Zn coordination orients the substrate for catalysis and withdraws electron density from the substate/intermediate oxygen atoms, causing adjacent carbon atoms to become more electrophilic while also stabilizing the acetaldehyde leaving group. The QueD2 paralog exhibits low (< 20%) sequence homology to QueD and is specifically defined by an insertion in the catalytic Cx3HGH motif of QueD to Cx4HGH motif of QueD2 (Fig. 5B). Despite low sequence conservation, a recent structure of QueD2 from Acinetobacter baumannii reveals a T-fold superfamily architecture that adopts a distinct octameric assembly state (two stacked

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H13 H28

CPH4 14-AAH HRLPHVPEGHKC CGRLH HGH HSF CKRSIH HGH HSY 11-NAHVVRNCTSDRC

68-RLDH.. H33 H31 65-SFDH HAI

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Fig. 5 The 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) synthase QueD. (A) A proposed catalytic mechanism of QueD showing the conversion of dihydroneopterin triphosphate (H2NTP) to 6-carboxy-5,6,7,8-tetrahydropterin (CPH4).92 Important catalytic residues from panels B-D are highlighted in yellow, cyan, and red. (B) Sequence alignment of E. coli QueD and A. baumannii QueD2 in the region of the catalytic Zn coordinating residues. QueD Zn binding residues are highlighted in yellow, while QueD2 Zn binding residues are highlighted in cyan, and the catalytic Cys residue of the Cx3/4HGH motif is highlighted in red. (C) Top-down (top) and side (middle) views of the hexameric E. coli QueD (PDB: 4ntm).92 A single protomer is shaded yellow, with the other protomers shaded gray with the Zn ions (black) shown. Close up view of the E. coli QueD Zn binding site (bottom) with conserved catalytic and metal binding residues labeled and shown in stick, product 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) shown in green stick, and the Zn ion represented by a black sphere. (D) Top-down (top) and side (middle) views of the octameric A. baumannii QueD2 (PDB: 7v0f). A single protomer is colored cyan, the remaining protomers are shaded gray, with the Zn ions (black) shown. Close up view of the A. baumannii QueD2 Zn binding sites 1 and 2 (bottom) with conserved catalytic and metal binding residues labeled and shown in stick, with water molecules shown as red spheres, and the Zn ion represented by a black sphere.

38

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

tetrameric rings) relative to the E. coli QueD hexamer (Fig. 5D).94 A. baumannii QueD2 is not Zur-regulated, however it becomes cell abundant under conditions of Zn-restriction28 and is homologous to other Zur-regulated QueD2.94 With the insertion of the additional amino acid in the QueD2 Cx4HGH motif, the metal coordinating HGH (His28 and His30) residues are shifted further along the b2 strand relative to QueD which positions the HGH motif at the start of b2 (Fig. 5C and D). Surprisingly, the crystallographically determined Zn coordination sphere is also changed, where the third protein-derived ligand is His680 even though QueD2 possesses His13 (analogous to His16 in QueD), and the Zn atom is coordinated by two water molecules (Fig. 5C and D). His680 (reaching up from the lower tetrameric ring) is predicted to function in the first catalytic triad and QueD2 shows Zndependent activity, so the origin of this striking change in the first Zn coordination sphere in QueD2 relative to QueD is unknown. Perhaps more importantly, QueD2 uniquely possesses a second Zn site coordinated by Cys18 which is invariant in QueD2 sequences, Cys23, and His18800 (from a neighboring protomer within the same tetrameric ring, Fig. 5D). Notably, this second site contains the catalytic Cys23 of the Cx4HGH motif that is required for activity; as expected, filling of this site in vitro blocks enzyme turnover.94 Some organisms encode a QueD, while others only encode only the QueD2 paralog that is typically not Zur-regulated, and yet others encode both a constitutively expressed QueD and Zur-regulated QueD2, suggesting a conserved function between the canonical QueD and the QueD2 paralog. Indeed, both Acinetobacter baylii QueD2 and a QueD that has been altered to introduce a QueD2like Cx4HGH catalytic motif, functionally complement an E. coli DqueD strain, although the extent to which this was the case was not quantified.95 Conversely, S. aureus QueD can functionally complement the A. baumannii DqueD2 strain to the same degree as A. baumannii QueD2.94 QueD is maximally active when bound to Zn, but also has significant activity with Mn and FeII. QueD2 on the other hand is strongly activated by FeII but also exhibits activity in the presence of Zn and Mn. These findings suggest that these enzymes may well be somewhat metal promiscuous, which is not so surprising given a general role in substrate positioning and activation (Fig. 5A). The fact that QueD2, relative to QueD, features a unique, highly dynamic, metal-liganding loop that is crystallographically captured as a second Zn site may well allow QueD2 to limit dissociation of the catalytic metal ion as metal activity falls in the cell.94 Indeed, A. baumannii cells that over-express S. aureus QueD exhibit disrupted flux through the Q biosynthesis pathway when stressed with low Zn, while cells over-expressing A. baumannii QueD2 maintain normal levels of Q production while Zn starved. This resistance to undermetallation is specifically due to the presence of the invariant Cys18 in the unique QueD2 Zn site 2 loop, which kinetically slows the release of the catalytic Zn, ion in what we term a “metal retention” model.94 Zur-regulated QueD2 provides an interesting and remarkably complex, multi-modal mechanism of adaptation to cellular Zn restriction. On the one hand, second Zn binding loop containing mutiple potential metal coordinating residues (Fig. 5D), coupled with a displacment of the primary catalytic Zn (Fig. 5C and D), suggests significant coordinative “plasticity” or heterogeneity in the substrate-free QueD2 that collectively inhibit catalytic Zn dissociation, thereby maintaining flux through the Q biosynthesis pathway. This is clearly advantageous for Zur-regulated QueD2 proteins, as QueD turnover is likely impeded by enzyme undermetallation. On the other hand, in niches deplete of Zn and replete of Fe, QueD2 may well utilize the alternative metal cofactor Fe, although this remains to be fully evaluated. Since the A. baumannii genome is significantly enriched in Q-decoded codons, translation may be particularly dependent upon the Q modification given the relative abundance of NAU vs. NAC codons in proteincoding genes; thus, a QueD2 may be better suited to function under conditions of extreme Zn-limitation in A. baumannii. Notably, QueD2s have not evolved a metal selectivity profile that is distinct from that of QueD, which may originate with the requirement for a strong and oxidatively stable Lewis acid to perform this chemistry.

2.03.3.2.2

PyrC/PyrC2

PyrC is a dihydroorotase (DHOase) that catalyzes the reversible interconversion of carbamoyl aspartate (CAA) and dihydroorotate (DHO). DHOase functions as the third step in the pyrimidine biosynthesis pathway, just downstream from the rate-determining or “committed” step catalyzed by another Zn-metalloenzyme, aspartate transcarbamoylase (Fig. 6A and B).99 As such, PyrC is an essential enzyme in many bacteria47,100,101 and is a long-standing antimicrobial target.102 PyrC belongs to the amidohydrolase superfamily that acts upon structurally similar cyclic amide substrates.103 Historically, bacterial DHOases can be grouped into two classes and a representative member of each has been structurally characterized. These are the  45 kDa class I PyrC enzymes largely found in Gram-positive bacteria, e.g., Bacillus anthracis (Fig. 6D)97,104 while the  38 kDa class II enzymes are generally found in Gram-negative bacteria, including the prototypical enzyme from Escherichia coli (Fig. 6E).96,105 Class I and class II PyrCs possess relatively modest (< 30%) sequence identity, and the class I enzyme has an N-terminal domain not found in class II enzymes. Both PyrC classes are characterized by the same amidohydrolase fold (RMSD ¼ 2.82 Å between protomers) and exist as dimers in solution, yet incorporate distinct dimerization interfaces.106,107 They absolutely require divalent metal ion for enzymatic activity, thought to be Zn in the cell; Zn and Co typically exhibit maximal activity while other divalent metals result in inhibition.96,108–110 Both enzyme classes harbor a nearly isostructural binuclear Zn site where each Zn exhibits a trigonal bipyramidal geometry (Fig. 6D and E). Five coordinating residues, including four His and one monodentate Asp, are common to both enzyme classes; while the sixth coordinating and bridging ligand is an Asp in class I PyrC and a post-translationally modified carboxylated Lys in class II DHOases.96,97,104,105 Ligand-free and DHO-bound structures additionally contain an ordered water or hydroxide anion that also bridges the two Zn ions. The reaction direction of PyrC is partly dictated by the pH with the reverse reaction favored at high pH due to the need for a Zncoordinated hydroxide ion (Fig. 6B). At neutral and slightly acidic pH, the forward cyclization reaction toward dihydroorotate (Fig. 6A) is favored. A proposed reaction mechanism is derived largely from studies of the class II E. coli PyrC but anticipated to

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

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Fig. 6 The binuclear zinc metalloenzyme dihydroorotase PyrC. (A) PyrC catalytic mechanism in the forward direction (toward pyrimidine biosynthesis) for the conversion of carbamoyl aspartate (CAA) to dihydroorotate (DHO), with the Zn and the active site base (Asp) shown.96 (B) PyrC catalytic mechanism in the reverse direction for the conversion of CAA to DHO.96 (C) Sequence alignment of class I B. anthracis PyrC, class II E. coli PyrC, class II P. aeruginosa PyrC, class III P. gingivalis PyrC, and Zur-regulated P. aeruginosa PyrC2 comparing the different Zn coordinating residues in each class. Class I Zn coordinating residues are highlighted in red, class II Zn binding residues are highlighted in yellow, and class III residues are highlighted in cyan, with the Zn1 (1) and Zn2 (2) ligands indicated. (D) Global structure (top) of B. anthracis PyrC (PDB: 4yiw)97 with the dimerization interface indicated by the dashed line, and the CAA-bound Zn binding site (bottom) with Zn binding residues labeled and shown in stick, the CAA substrate shown in green stick, and Zn ions shown as black spheres. (E) Global structure (top) of E. coli PyrC (PDB: 1xge)98 with the dimerization interface indicated by the dashed line, and the DHO-bound Zn binding site (bottom) with Zn binding residues labeled and shown in stick, the DHO substrate shown in green stick, water molecules shown as red spheres, and Zn ions shown as black spheres. (F) Global structure (top) of P. gingivalis PyrC (PDB: 2gwn) with the putative dimerization interface based on homology to B. subtilis PyrC indicated by the dashed line, and the Zn binding site (bottom) with Zn binding residues labeled and shown as sticks, water molecules shown as red spheres, and Zn ions shown as black spheres.

40

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

be identical for class I DHOases. The two oxygen atoms of the terminal carboxylate group of CAA form a bridging coordination complex with the binuclear Zn center. Asp250 abstracts a proton from the CAA amide such that the nitrogen is primed for intramolecular nucleophilic attack of the terminal carboxylate group, forming the diol intermediate which then spontaneously dehydrates to form DHO. The reverse reaction (Fig. 6B) begins with a bridging hydroxide ion; DHO then binds the active site, with the 4-keto oxygen atom coordinated by one of the Zn ions. The activated hydroxide acts as a nucleophile to attack the Zncoordinated carbonyl carbon, while the conserved Asp250 engages in proton transfer and resonance stabilization resulting in a both Zn atoms coordinating two oxygen atoms of the intermediate diol. Protonation of the ring amide by Asp250 subsequently results in breaking of the ring, a collapse of the diol into a carboxylate, and the production of CAA. The Zur-regulated paralog PyrC2 is structurally uncharacterized, but is found in Zur regulons of Gram-negative bacteria, e.g., P. aeruginosa (Fig. 2C), B. cenocepacia, where the housekeeping PyrC is a class II enzyme that is not regulated by Zur. P. aeruginosa PyrC2 is distinct from class II P. aeruginosa PyrC, with  10% sequence identity, but notably exhibits z30% sequence identity to the class I B. anthracis PyrC. Furthermore, P. aeruginosa PyrC2 has lost the most N-terminal of the Zn1-coordinating His residues, which is replaced by a Gln that is conserved in other Zur-regulated PyrCs (Fig. 6C).38 Interestingly, the structure of Gram-negative Porphyromonas gingivalis PyrC, a third DHOase denoted class III,111 (Fig. 6F), reveals features of both class I (sequence similarity and an N-terminal extension) and class II (a carboxylated Lys as a bridging ligand, instead of an Asp) enzymes (Fig. 6F). We view this structure as an excellent model for Zur-regulated PyrC2 given the high (50%) sequence identity and the presence of the same site 1 Gln substitution that is found in the P. aeruginosa PyrC2 paralog (Fig. 6C). This suggests that the uncharacterized class III PyrCs may well contain other paralogs that may function better under Zn-limited conditions. There has been limited functional characterization of PyrC2. In P. aeruginosa, PyrC2 can sustain pyrimidine biosynthesis on minimal media in a DpyrC strain, whereas the DpyrC/DpyrC2 strain is auxotrophic and cannot grow unless supplemented with uracil, bypassing the pyrimidine biosynthesis pathway.112 The cognate metal of PyrC has classically been assumed to be Zn and consistent with this, recombinant PyrC co-purifies with two mol protomer Zn equivalents. PyrC may well be undermetallated at low Zn, thereby necessitating the upregulation of PyrC2 to maintain flux through the pyrimidine biosynthesis pathway; however, the ability of a DpyrC2 strain to grow in Zn-limited conditions has not yet been evaluated. As no careful biochemical characterization of a PyrC2 paralog exists to date, we can only speculate on how PyrC2 differs from PyrC. Although the Gln substitution, which introduces carboxamide in the first coordination sphere of Zn1, may relax the strict Zn-dependence of PyrC to allow Mn binding for example,113 the coordination spheres will remain virtually identical to class I and II PyrCs. Another possibility is that this Histo-Gln substitution in the context of the bridging carboxylated Lys raises the pKa of bound water so as to make PyrC2 more irreversible and thus biasing flux toward pyrimidine biosynthesis under conditions of zinc restriction. A third possibility is metal affinities are simply higher, and/or less kinetically labile, like that of the QueD/QueD2 pair. Finally, PyrC2 may simply allow adaptation to low Zn as a form of Zn prioritization into an essential pathway, where the upregulation of PyrC2 simply maintains metabolite flux toward pyrimidines by mass action (Fig. 2B), as has been proposed for a Zur-regulated, Zn-dependent peptidase114 (Section 2.03.3.4.3).

2.03.3.2.3

HisI

HisI is a phosphoribosyl adenosine 50 -monophosphate (PR-AMP) cyclohydrolase that performs the third step in the histidine (His) biosynthesis pathway, opening the PR-AMP purine ring (Fig. 7A).116 HisI lies downstream of the committed step of His biosynthesis, catalyzed by HisG, and upstream of the branchpoint that directs metabolite flux to both de novo purine and His biosynthesis,117 both known virulence factors in some organisms.118,119 Prokaryotic HisI is encoded either as a dimeric single-domain protein,120 or as a fusion protein with HisE or with both HisD and HisE, to form bi- and tri-functional enzymes, respectively, as in some bacteria and higher organisms.115,121 HisI has been structurally characterized as the PR-AMP cyclohydrolase120 and catalyzes the same activity in the bifunctional HisIE.115 In both cases, HisI (and the HisI domain) form nearly structurally identical dimers (RMSD ¼ 0.68 Å) and harbor two, interdigitated metal binding sites located at the dimer interface with a common DCDxD metal binding motif derived from one protomer. One site, assigned as the catalytic Zn1 site, is coordinated by three Cys residues (Cys142, Cys149, and Cys1260 , the C in the DCDxD motif in the M. truncatula sequence; Fig. 7B–D). The second metal site is thought to be a Mg binding site although bound to Zn (Zn2) or Cd in existing crystal structures and is coordinated by the three Asp residues in the DCDxD motif (Asp1250 , Asp1270 , and Asp1290 in the M. truncatula sequence) (Fig. 7B–D). The catalytic cycle has not yet been clearly established for HisI, but has been proposed to include the ionization of a Zn-bound water molecule by a neighboring His residue, that ultimately cleaves the purine ring between positions N1 and C6 (Fig. 6A).122 Mg binding at site 2 is predicted to coordinate the phosphoribosyl group within the active site. Bacterial HisI proteins copurify with Zn bound and require both Zn and Mg for activity.122,123 High concentrations of Zn inhibit activity, likely due to the replacement of Zn within the Mg site 2.123 The site 1 metal selectivity has been difficult to test as reconstitution of the native Zn to the apoprotein recovers less than 50% of the starting activity.123 However, excess Cd is able to outcompete Zn at site 1 and exhibits enzymatic turnover that is 10–80-fold lower than that of the Zn enzyme, depending on reaction buffer pH.120 For site 2 metal specificity, replacement with Mn, Ca, or Zn abolishes enzymatic activity, leading to the its assignment as a true Mg site.123 Pseudomonas protegens encodes for a HisI as well as a Zur-regulated HisI2 paralog of unknown structure.124 These enzymes are 56% identical and HisI2 likely adopts an overall fold that is similar to that of the structurally characterized HisI enzymes (Fig. 7C and D, z45% identity to each). HisI2 maintains the core DCDxC metal binding motif as well as the two additional Cys residues that complete the coordination of Zn1 (Fig. 7B). Therefore, we tentatively assign the HisI2 as a Category 2 paralog

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

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C149

Fig. 7 PR-AMP cyclohydrolase, HisI. (A) Proposed catalytic mechanism of HisI-catalyzed PR-AMP cyclohydrolase activity, including Zn and His base activation of a water molecule for nucleophilic attack and Mg stabilization of ribose. (B) Select sequence alignment of Zn- and Mg-dependent M. truncatula HisI subdomain and uncharacterized P. protegens HisI and HisI2. Zn binding sites are indicated by yellow or cyan (paralog enzyme) spheres and labeled as site 1 (1) or 2 (2), where site 2 is believed to be a Mg binding site although it is structurally captured bound to Zn. (C) Global structure of the dimeric M. truncatula HisI subdomain (PDB: 7bgn).115 (D) Active site architecture of M. truncatula HisI subdomain with important residues labeled. For panels C and D, individual monomers are colored different shades of yellow, Zn coordinating residues are shown as sticks, Zn ions are shown as black spheres, bound AMP molecule is shown as green sticks, and water molecules are shown as red spheres.

that retains Zn-dependent enzymatic activity, where the upregulation of HisI2 under Zn restriction may well represent a cellular strategy to increase flux through the His or purine biosynthesis pathways, rather than a form of Zn sparing. Additional studies will be required to assess the extent to which HisI2 is more resistant to Zn dissociation under conditions of cellular Zn restriction.

2.03.3.3 2.03.3.3.1

Category 3: Zn-independent or metal-promiscuous paralogs FolE/FolE2

GTP cyclohydrolase-IA (GCYH-IA; FolE) is an obligatory Zn-dependent enzyme that catalyzes the remarkable conversion of GTP to 7,8-dihydroneopterin triphosphate (H2NTP, Fig. 8A), the first intermediate and committed step in the de novo biosynthesis of tetrahydrofolate (THF, folate). This enzyme is found in bacteria, fungi, plants, and mammals, although in some eukaryotes it generally performs other functions beyond folate biosynthesis, including the rate limiting step in the tetrahydrobiopterin (BH4) pathway.127 In bacteria, the FolE product, H2NTP represents a true metabolic branch point, since it serves as the substrate for QueD/QueD2 in the biosynthesis of the tRNA modification queuosine (Q) and other tRNA nucleosides (Section 2.03.3.2.1).128 On the other hand, FolE2 (also known as GCYH-IB) has been described as a Zn-independent paralog that can catalyze the same reactions when metalated with another divalent metal (Fig. 2B).129,130 It is interesting to note that FolE2, like QueD2 discussed above, are not exclusive to Zur regulons and in some organisms, e.g., Staphylococcus aureus, only encode FolE2 that is not known to be Zur-regulated. Both FolE and FolE2 are members of the tunnel (T)-fold superfamily,130 but are characterized by very low pair-wise sequence identity and exhibit dramatic differences in oligomeric assembly state, i.e., dimer of pentamers vs. a dimer of dimers double ring-shaped architectures in FolE and FolE2, respectively.

42

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

ZY Zn site T. thermophilus FolE 106–SMC CEH HHLLPFFGKVI N. gonorrhoeae FolE2 145–SLC CPCSKEISQYGAH H Metal site X

X

(C)

(A)

Z H

O

O

H

N

HN

O

Cys108/147

N2

X

Y

H O H

N

N H

Z H H N

HN

-

O

H

(E)

OH

O

Y OH

NH2 N

O O-

O PPPO

OH

Cys108/147 M

X

Y

O H

H

OH

O O-

OH N

HN H2N

X Y OH

H O

PPPO

H

NH

N

N

His’

O

Cys108/147

O M

H2N

OH

HN

H N

X HN

H NH

N

PPPO

(B)

Z

Cys108/147 M

O

O

H2N

(D)

OH

O

H2N

X

+

PPPO PPPO

Cys108/147 M

OH Y

O

His’ H

OH

O

H N

HN

M

N

N

H2N

-

171-VVVEGVHLC CMM 237-VESENFESIH H.

N

N H

OPPP OH

OH

GTP*

C108

GTP*

C147

GTP* C108

M

M

C179 H111 (Y)

C179 (X)

E201’ (Y)

H159 (X)

H111

GTP* C149-SNO E201’

C147 H159

Fig. 8 GTP cyclohydrolase I, FolE. (A) Zn-dependent steps of the FolE catalytic mechanism in the opening of the GTP purine ring before subsequent conversion to the dihydroneopterin triphosphate product.125,126 Z, active site acid (donates a proton); X, Y are metal ligands along with a conserved Cys. (B) Schematic representation of the Zn coordination models in T. thermophilus FolE (left) and Neisseria gonorrhoeae FolE2 (right). (C) Sequence alignment of T. thermophilus FolE and N. gonorrhoeae FolE2 in the active site region. Note that the FolE2 Y (not shown in this region) and X coordinating ligands (panel B) and the Z functional groups in the primary structure of T. thermophilus FolE and N. gonorrhoeae FolE2 are significantly displaced from one another. (D) Top-view (top) and side view (middle) of the global structure of decameric T. thermophilus FolE (PDB: 1wur)85,125 with a single protomer shaded in yellow ribbon, with the remaining protomers shaded gray. Active site region (bottom) with Zn binding residues labeled and highlighted in yellow stick, the catalytic intermediate mimic 8-oxo-GTP (GTP*) shown in green stick, and the Zn ions shown as black spheres. (E) Top-view (top) and the side view (middle) of the global structure of tetrameric N. gonorrhoeae FolE2 (PDB: 3d2o) with a single protomer shaded in cyan ribbon, with the remaining protomers shaded gray. Active site region (bottom) with Zn binding residues labeled and highlighted in cyan stick, the catalytic intermediate mimic 8-oxo-GTP is labeled as GTP* and shown in cyan stick, and the Zn ions shown as black spheres. Snitrosated C149 is also shown in NgFolE2. See text for details.

Both FolE and FolE2 apoproteins are inactive and activity can only be observed by metalation of the active site as revealed by mutagenesis and enzymatic activity assays.125,130 Both enzymes coordinate Zn in a highly distorted tetrahedral geometry as revealed by the structures of E. coli FolE and the Neisseria gonorrhoeae FolE2 (Fig. 8D and E), with three protein-derived ligands and a mimic of the first catalytic intermediate, 8-oxo-GTP (GTP*; Fig. 8B) coordinated to the metal.85,125 In the resting enzyme, this open coordination site is filled by OH, which is poised to attack the electrophilic C8, which itself is activated by a nearby His (Z residue; Fig. 8A). FolE features a Cys2His-OH coordination site while in FolE2, one of the Cys (C179 in FolE; the Y ligand) is replaced with a glutamate (E2010 ) from a protomer in the opposite dimer in this dimer-of-dimers architecture, thus creating a “harder” coordination site. FolE is thought to be an obligatory Zn enzyme, although its activity with other metals has not yet been reported. Characterized FolE2 paralogs from B. subtilis and N. gonorrhoeae generally have lower, but readily detected activity when metalated by Zn (z15% the activity of the Mn enzyme), with maximum activity with Mn, somewhat less activity with FeII and low activity with Ni, all trends consistent with the observed change in the first coordination shell in FolE2 vs. FolE.130 Interestingly, both FolE2s are reported to be active with Mg, to an extent on par with CoII, which is puzzling given the presence of clearly “softer” ligands in both FolE2 coordination shells. It is unknown how Mg is coordinated to FolE2, but this is a subject well worth investigating.

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

43

Both FolE and FolE2 are thought to proceed through the same catalytic cycle which features classic Lewis acid chemistry and proton transfer-catalyzed ring-opening and ring-closing rearrangements that involves several pentacoordinate-Zn intermediates, with the release of formate and H2NTP as products (Fig. 8A). It is interesting to note that the active site regions of both FolE and FolE2 harbor additional candidate metal binding or acid-base functionalities that suggests that posttranslational modification of one or more of these residues might have a regulatory role. Indeed, NgFolE2, as purified, features additional electron density on the non-liganding C149 (Fig. 8C and E, bottom) assigned as an S-nitrosated Cys, SNO, essentially positioned “on top” of the active site.126 Mutation of this Cys to Ala or Ser effectively abolishes activity, which suggests that S-nitrosation here may be a protective mechanism that preserves FolE2 activity under conditions of nitrosative stress. In this context, we point out that peroxynitrite (a reactive nitrogen species) S-nitrosates one of the two coordinating Cys in human GCYH-I, which results in loss of Zn and activity.131 The evolution of FolE2 in bacteria may be reflective of a GCYH that evolved under the influence of dual stressors of oxidative/nitrosative stress and Zn limitation, along the lines of DskA2 vs. DksA (Section 2.03.3.2.2). In any case, the significant activity of FolE2 with divalent metal ions other than Zn (category 3) constitutes a considerable evolutionary adaptation for bacterial survival under conditions of Zn starvation.41,132

2.03.3.3.2

HemB/HemB2

HemB encodes porphobilinogen (PBG) synthase, also known as 5-aminolevulinic acid (ALA) dehydratase, fuses two ALA molecules into a single molecule of PBG (Fig. 9A).135 HemB catalyzes the rate-determining step in heme biosynthesis, and provides the foundational pyrrole precursor that is used in the synthesis of all tetrapyrroles, including heme, vitamin B12, and chlorophyll.136 Inhibition of HemB significantly impairs heme biosynthesis in humans, causing a multitude of heme-deficient diseases.137 HemB function is also required for normal bacterial growth in the absence of exogenous heme supplementation.138 As HemB fulfills important cellular functions in multiple physiological pathways, it is highly conserved from bacteria to humans.136 HemBs are clearly demarcated into two distinct classes: animals utilize a Zn-dependent PBG synthase (termed “Cys-rich”), plants possess a Mg-dependent PBG synthase (termed “Asp-rich”), while bacteria encode one or both class of enzyme.139 Zn-dependent

Fig. 9 Porphobilinogen synthase, HemB. (A) A putative reaction of the conversion of two molecules of 5-aminolevulinic acid (ALA) into porphobilinogen (PBG) by HemB. The contribution to the final product of A-side and P-side substrates are colored red and black, respectively, in the structure of PBG. (B) Selected sequence alignment of Zn-dependent E. coli HemB, predicted Zn-dependent P. putida HemB, Mg-dependent P. aeruginosa HemB, and Zur-regulated P. putida HemB2 with Zn and Mg binding residues highlighted in yellow and cyan, respectively. (C) Global structure of homo-octameric HemB with one protomer shaded yellow, and the others in gray. (D) Overlay of the E. coli HemB (yellow PDB: 5mhb)133 and P. aeruginosa HemB2 (cyan PDB: 2woq).134 (E) Structure of PBG-bound E. coli Zn coordination site with Zn coordinating residues labeled and shown in yellow stick, the PBG product shown in green stick, and Zn ion shown as a black sphere. (F) Structure of alaremycin 2-bound P. aeruginosa HemB Mg coordination site with Mg coordinating residues labeled and shown in cyan stick, alaremycin 2 inhibitor shown as green stick, water molecules shown as red spheres, and Mg ion shown as a gray sphere.

44

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

PBG synthases, E. coli HemB for example, contain three coordinating Cys residues (Cys120, Cys122, Cys130 in E. coli HemB133) in the active site, while Mg-dependent HemB, as characterized in P. aeruginosa, do not conserve the first Cys and replace the second and third Cys residues with Asp residues (Asp131 and Asp139 in P. aeruginosa HemB134), while also recruiting an additional Asp176 from a neighboring loop into the active site (Fig. 9B). Members of both enzyme classes exhibit high degrees of sequence identity outside of the metal coordinating residues in the active site, and thus appear derived from a recent common evolutionary ancestor. Both HemBs exhibit an ab-barrel architecture (Fig. 9C and D), and exist in an equilibrium between hexamers and octamers, although the octameric assembly state tends to dominate and is the most active.140 The binding of substrate, and in some cases, the binding of an allosteric activator, Mg, stabilizes the octameric assembly state.136 The active site of HemB consists of three metal-coordinating ligands with one or two open coordination sites available on the Zn and Mg ions, respectively (Fig. 9C and D). Beyond these dramatic differences in first coordination shell, the structures of the active sites are nearly identical, with Zn adopting a tetrahedral coordination geometry, and Mg adopting a distorted trigonal pyramidal geometry. In addition to the metal coordination site, there are two conserved Lys residues that are crucial for catalysis (see below). The enzymatic mechanism has been nearly exclusively studied in the Zn-dependent, Cys-rich HemB, and despite wholesale replacement of the active site in Mgdependent HemB, the chemistry is thought to be largely unaffected. Although many studies have evaluated distinct aspects of the HemB catalytic cycle,49,141–143 no consensus has yet been reached (Fig. 9A). The binding of ALA substrate to the active site is biphasic in substrate concentration.143 The first mole equivalent of ALA binds in the P-side (named for its contribution to the pyrrole-N of the PBG product), while the second ALA binds to the A-side (contributes the amino/acetyl functionality to the PBG product, Fig. 9A). Zn likely plays an initial role in the proper positioning of the two ALA substrates for Schiff-base formation and couples activation of a hydroxide/water molecule for proton abstraction and donation during cyclization to create PBG (Fig. 9A). The metal selectivity of HemB proteins is entirely dictated by the presence of Cys or Asp residues within the active site. Indeed, a Zn-dependent HemB can be created from a Mg-dependent HemB simply by inserting the Zn-coordinating Cys residues into the existing active site.144,145 E. coli HemB co-purifies with Zn-bound and can bind up to 2 molar equivalents of Zn; the second molar equivalent has since been assigned as bound to the allosteric Mg-binding site.146 Removal of Zn from Cys-rich HemB abolishes enzymatic activity and Zn can be reversibly added to restore activity,146 while substitution with Co or Mg cannot.147 Treatment of bovine HemB with excess Pb inactivates the enzyme by replacement of the active site Zn with Pb, which has been identified as a primary target for Pb poisoning in the blood.148 From the currently available experimental data, the Zn-dependent HemB is an obligatory Zn-dependent enzyme, and under- or mis-metallation disrupts enzymatic activity. Mg-dependent P. aeruginosa HemB, on the other hand, also absolutely requires metal for enzymatic activity. Substitution with Mn and Co fully activates HemB to that observed with Mg bound, while Ni and Zn show measurable, though limited, enzymatic turnover, a finding reminiscent of the metal promiscuity of FolE2 vs. FolE (Section 2.03.3.3.1).149 It is important to note that Mg-dependent Asp-rich HemBs are resistant to mismetallation by thiophilic xenobiotics Pb and Cd.145 HemB2 paralogs are not common in Zur regulons, but have been identified in Pseudomonas putida and some cyanobacteria where the classic HemB encoded in the genome is the Zn-dependent Cys-rich enzyme.38,58 P. putida HemB2 shares 43% sequence identify with P. putida HemB but possesses the conserved Asp residues of Mg-dependent HemB proteins and thus has 59% sequence identity with Mg-dependent P. aeruginosa HemB (Fig. 9B). HemB2 draws many parallels to FolE2 although HemB and HemB2 clearly share a recent common evolutionary ancestor, while this is not so clear with FolE/FolE2 (Section 2.03.3.3.1). The first coordination shell in both cases has evolved in the direction of a “harder” ligand set that can accommodate harder ions, in a way that preserves essential features of the reaction chemistry. More recently, an apparently metal-independent HemB that possesses an Asp-rich active site identified in Rhodobacter capsulatus, which exhibits enzymatic turnover in the absence of metal cofactors and is completely insensitive to Zn or Mg supplementation.150 The origin of this activity is not known but brings to mind a class of ribonucleotide reductases that do not require metal for activity.151 Although much additional work is required, Zur-regulated expression of an Asp-rich HemB2 provides a means of Zn sparing, where Zn from the presumably inactive Cys-rich HemB can be utilized for other Zndependent processes.

2.03.3.4 2.03.3.4.1

Others Carbonic anhydrase

Carbonic anhydrases (CA) are archetypal metalloenzymes found in all living organisms that catalyze the reversible hydration of the gaseous CO2, with the products bicarbonate anion, HCO3, and a proton (Hþ), thus equilibrating CO2 and bicarbonate in the cell (Fig. 10A). The mammalian CAs are representative of the a-class of enzymes where the active site Zn is coordinated by three His and a water molecule at the base of a deep channel (Fig. 10B). Comprehensive studies of the cell-abundant human isoform (type II) have provided detailed insights into how the second Zn coordination shell impacts Zn affinity and turnover,156,157 and these studies have fueled the development of CA-based biosensors capable of quantifying chelatable [Zn] in a complex cellular milieu.158–160 This isoform is one of fifteen known isoforms in humans, which differ in oligomerization state, expression levels in different organs, tissues, and cells, and kinetic properties, but all employ the same active site architecture.48 The critical importance of carbonic anhydrases in global carbon capture, pH regulation, pyrimidine biosynthesis and in providing a universally employed substrate for all biotin-dependent carboxylation reactions that function in fatty acid, amino acid and carbohydrate metabolism161 is exemplified by the remarkable evolutionary diversity of CAs. There are now eight known evolutionarily unrelated gene families that encode structurally divergent classes of enzymes: the a-, b-, g-, d-, z-, h-, q-, and

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

O

(A)

CO2 His H2O H2O H2O

O

H 2O

C +

His-H

OH2 OH2

Zn

-

HO

His-H+

O

(C)

H108

HO Zn

Zn

(B)

OH2 OH2

45

His H2O H2O H2O

O

Zn

HCO3

(D)

C51

H91

H104

H107 H102’

H89

AZM SCN-

C107 H75

Fig. 10 Carbonic anhydrase. (A) Reaction mechanism of mammalian a-carbonic anhydrase II. Zn coordination of a water molecule within a hydrogen bonding network promotes hydroxide formation as the active site nucleophile to convert CO2 to HCO3.152 (B) Global structure (top) of dimeric a-carbonic anhydrase (PDB: 4x5s)153 with the highlighted monomer labeled in red and additional protomer colored gray. a-carbonic anhydrase active site (bottom) with Zn binding residues shown as red sticks, Zn ion as a black sphere, and acetazolamide (AZM) inhibitor shown as green sticks. (C) Global structure (top) of tetrameric b-carbonic anhydrase (PDB: 2a5v)154 with the highlighted monomer labeled in yellow and additional protomers colored gray. b-carbonic anhydrase active site (bottom) with Zn binding residues shown as yellow sticks, Zn ion as a black sphere, and SCN molecule shown as green sticks. (D) Global structure (top) of trimeric g-carbonic anhydrase (PDB: 3kwc)155 with the highlighted monomer labeled in cyan and additional protomers colored gray. g-carbonic anhydrase active site (bottom) with Zn binding residues shown as cyan sticks, Zn ion as a black sphere, and water molecules as red spheres.

ι-CAs.162,163 In bacteria, the major structural classes are the a, b and g enzymes while the ι-CA is present in marine diatoms (Fig. 10B–D).164 Most bacteria encode more than one CA, often but not always derived from all three major classes (b- and g-class enzymes feature prominently in clinically relevant pathogens), with some regulated by nutrient stressors. a-CAs are generally monomers or dimers and closely resemble the mammalian enzymes and thus employ Zn as the Lewis acid cofactor, while b-CAs are typically dimers, tetramers or octamers that are also obligate Zn metalloenzymes. g-CAs are obligate trimers that adopt a left-handed b-helical structure that are reported to be Fe-dependent enzymes, which also function with Zn and Co. One ι-CA has been reported to prefer Mn over Zn, although the extent to which this is broadly the case is not known.165 In b-CAs, the Zn is coordinated by two Cys and His with an open coordination site, while a-CAs and g-CAs employ the same His3-H2O motif, but the metal ligands in the later derive from adjacent protomers within the trimer (Fig. 10). a-CAs are nearly exclusively periplasmic in Gram-negative bacteria, while b- and g-class enzymes are often cytoplasmic but can also localize to the periplasm. b-CAs often exhibit strongly pHdependent activity in which an Asp is recruited into the open coordination site at high pH, thus abolishing activity.154 Interestingly, a-CAs have been detected in outer membrane vesicles, the function of which are poorly understood.166 In the few instances where it has been documented that cellular expression of a CA changes with Zn restriction28 and/or represents a bona fide Zur target19 (see Fig. 2C, in P. aeruginosa) these enzymes tend to belong to the b- and g-classes, with bacterial pathogens often encoding multiple b-CAs with apparently non-redundant functions.164 Overexpression of a b-class enzyme, in for example, Acinetobacter baumannii, which is only known to function with Zn as the cofactor, would seem to point to mass action as a way to capture more metal to ensure sufficient bicarbonate and pH balance to meet cellular needs under these conditions. However, Zur-regulated expression of a g-CA may represent a case of Zn-sparing, since this enzyme is reported to be metalpromiscuous with FeII the preferred metal cofactor when prepared under anaerobic conditions.167,168 The extent to which Zurregulated g-CAs are metalated with another metal in cells is not known, and clearly worthy of additional study.

2.03.3.4.2

ThrRS2/CysRS2

Aminoacyl tRNA synthetases (aaRS) are essential enzymes that charge tRNA with the correct amino acid for protein synthesis (Fig. 11A).172 At least one aaRS exists for every amino acid. Intrinsic to its function in mRNA decoding is the exquisite specificity that each has for both the correct amino acid and tRNA adaptor molecule, thus preserving the integrity of the genetic code during translation. A number of aaRS that charge distinct amino acids have been found to harbor a Zn coordination chelate. IleRS (encoded by ileS, see Fig. 1A) and MetRS both possess a structural Zn binding site that is important in stabilizing the interaction between the

46

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

Val blocked

(A)

Thr stabilized

O

Zn

Zn

O

+

H3N

-

O

O

N

N

O-

OO

N

O

+H N 3

O-

O-

O

OH

HO

tRNA

N N

O O-

Zn binding site Anabaena ThrRS Anabaena ThrRS2

O

N

Nucleobase

tRNA

N HO

O

OH

O P O O

(B)

OH

NH3+

HO

PPi

OH

ThrRS2 insertion (C)

NH2

N

O O O P O P O O-

Nucleobase

tRNA NH2

OH

AMP

O

+H

OH

306-PMNC CPFH 304-PMNC CPFH

OH

O 3N

361-DDAH HIFC 359-DDSH HLFV

484-MIH HRAI 485-MIH HRAP

(D)

tRNA

H517

ATP ThrRS

Thr H387 C336

Fig. 11 Threonyl tRNA synthetase, ThrRS. (A) Catalytic mechanism of ThrRS aminoacylation. Zn enhances the selectivity of Thr over Val by coordination to the Thr hydroxyl group.169,170 (B) Selected sequence alignment of Anabaena ThrRS and ThrRS2 with ThrRS Zn coordinating residues highlighted in yellow and putative ThrRS2 Zn binding residues highlighted in cyan. (C) Global structure of E. coli ThrRS bound to its cognate tRNA (PDB: 1qf6).169 The ThrRS monomer is shown in yellow, tRNA molecule is shown in green, the Zn ion is shown as a black sphere, and AMP is shown as green sticks. The location of the ThrRS2 insertion is highlighted (see text for details). (D) Active site of S. aureus ThrRS bound to Zn, Thr, and ATP (PDB: 1nyr).171 The Zn coordinating residues are represented as yellow sticks, the Zn ion is shown as a black sphere, and Thr and ATP molecules are represented as greens sticks.

synthetase and the anticodon loop of the cognate tRNA.5,173,174 In striking contrast to these structural Zn sites, ThrRS (thrS), CysRS (cysS), and SerRS (serS) employ a Zn chelate located in the active site169,175,176 that discriminate among closely related pairs of small amino acids (Fig. 11A and D). For example, ThrRS employs Zn to directly coordinate the hydroxyl group of Thr allowing for discrimination between Thr and Val.170 Likewise, CysRS discriminates Cys from Ser on the basis of formation of a strong thiolate Zn coordination bond,175 while Zn coordination in SerRS provides selectivity for Ser over Thr by coordinating the hydroxyl group of Ser in such a way that the Thr methyl group would sterically clash with adjacent residues in SerRS.176 Thus, in these cases, Zn plays a critical role in the maintaining the genetic code by enhancing substrate specificity, rather than functioning as an active site catalyst or playing a structural role. Both ThrRS and CysRS have paralogs (ThrRS2, encoded by thrS2 and CysRS2, cysS2) that are present in Zur regulons,38,58 although only ThrRS2 has been functionally characterized in any detail in Anabaena177 (Fig. 11B). The Zur-regulated ThrRS2 has 52% sequence identity with ThrRS, notably preserving the Zn coordinating residues (Fig. 11B), and can complement its aminoacylation function in a heterologous system. Thr aminoacylation levels decrease slightly under conditions of Zn limitation, however this is further exacerbated in the DthrS2 strain which results in disruption of cell growth.177 ThrRS and ThrRS2 share various functional similarities: both enzymes conserve the known Zn binding residues, copurify with Zn, bind Zn with similar affinities, and are active as dimers in solution. Removal of Zn in ThrRS2 abolishes aminoacylation and substitution with Ni, CuII, and Cd does not recover the activity of what appears to be an obligatory Zn enzyme. A key distinction is a 20 amino acid insertion that is present in ThrRS2 (Fig. 11C), but not ThrRS, which paradoxically appears to negatively impact the strict specificity of aminoacylation, as ThrRS2 exhibits a higher rate of misacylation of Ser relative to ThrRS. A recently proposed model posits that the ThrRS2 protomer of a ThrRS2-ThrRS heterodimer functions to periodically misacylate, which is then immediately corrected (edited) by the ThrRS protomer within the dimer.177 In any case, ThrRS2 seemingly represents a Category 2 paralog that appears to have retained the obligatory Zn-dependence of the canonical ThrRS enzyme (Fig. 11B and D), but may have acquired a specific characteristic, e.g., the 20-residue insertion, that allows ThrRS, relative to ThrRS, to resist metal dissociation under conditions of cellular Zn restriction. Further experiments that probe the phenotype of strains that express only ThrRS2 or ThrRS may begin to shed light on the functional role of this Zur-regulated paralog and to what extent this rescue mechanism of the canonical enzyme permits adaptation to Zn starvation.

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction 2.03.3.4.3

47

Bacterial cell wall remodeling enzymes

Maintaining the structural integrity of the bacterial cell is an essential component of survival. Regulation of the process is multifactorial and key to balancing cell wall synthesis with degradation that must occur during cell division, for example, or for Gram-negative bacteria, remodeling the periplasmic space, which is filled with peptidoglycan (PG). The structure ofPG features a repeating disaccharide unit consisting of N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) and a 5-residue “stem” peptide harboring a terminal D-Ala-D-Ala linkage. Stem peptide crosslinking is catalyzed by penicillin-binding proteins (PG X-link; Fig. 12A) that endow the cell wall with its structural integrity. Recycling and remodeling of PG is carried out by cell wall hydrolases (CWH) that cleave the peptide stems (peptidases) and the disaccharide unit (lysozyme, lytic transglycosylases) in specific places (Fig. 12A).178 Cell wall hydrolases work on the degradation arm of the PG homeostasis and largely function in the periplasm (Fig. 12B). Two general families of the Zn-cofactored CWHs that change expression levels under conditions of severe Zn restriction and are known Zur targets include ZrlA from A. baumannii (Fig. 12C)114 and AmiA from P. aeruginosa (Fig. 2C; Fig. 12D).19 ZrlA is an M15 class D,D-carboxypeptidase (D,D-CP) that is anchored to the inner membrane and removes the terminal D-Ala from the pentapeptide stem

(A)

glucosaminidase

lysozyme (muramidase); Lytic

MurNAc

(B)

TGs

GlcNAc

OM

PG

{

X-link

SYNTHESIS

periplasm

Gram-negative bacteria

lipid I

amidase

MraY

DEGRADATION IM

lipid II

AmpG

MurG

UDP-MurNAc

endopeptidases

signaling

L-Ala GlcNAc-anhMurNac

D-Glu

lysostaphin

carboxypeptidases

ZrlA (D,D-carboxypeptidase)

cytoplasm

meso-Dap

L-Ala

D-Ala

D-Glu

D-Ala

meso-Dap

UDP-MurNAc

MurF L-Ala

PG X-link

D-Ala-D-Ala

Cell wall hydrolases (CWH)

meso-Dap

MurA-E

D-Ala

D-Ala

Mpl

D-Glu

UDPMurNAc

AmpD

L-Ala D-Glu

meso-Dap

GlcNAc-anhMurNac NagZ GlcNAc + anhMurNAc

PEP, NADPH UDP-GlcNAc

(C)

(D) Putative Zn binding site A. baumannii A1S_1248 A. baumannii ZrlA

145-SKH HLFNSAID DFR HVFNAALD DFR 148-SRH

201-HID DTQ 204-HID DSQ

Zn binding site B. henselae AmiB P. aeruginosa AmiB P. aeruginosa AmiA

186-PGH HGGIDGGARGVTGILE EKD HGGEDPGALGPGGLHE EKN 183-AGH 161-AGH HGGKDPGAVGSKGERE EKD

255-SIH HAD DTI 252-SIH HAD DAA 230-SVH HAD DAA

288-ESE ENK 286-DSE ENR 264-ERE ENG

D259 E203 E290

H188

H257

Fig. 12 Cell wall hydrolases and Zur-regulated paralogs in peptidoglycan remodeling. (A) Chemical structure of the peptidoglycan in a Grampositive and Gram-negative bacterium. The bonds that are hydrolyzed by ZrlA and the amidases are shown in bold. (B) Schematic rendering of the cell wall recycling, with synthesis and degradation arms indicated. AmpD hydrolyzes the amide linkage between L-Ala and anhydro-muramic acid. (C) Selected sequence alignment (top) of D,D-CP homologs A1S_1248 and Zur-regulated ZrlA from A. baumannii. Putative conserved Zn coordinating residues are highlighted with yellow spheres. A ribbon representation of a ZrlA model on that of another M15-family D,D-CPase from Streptomyces albus (PDB: 1lbu, bottom) with Zn represented as a gray sphere and metal coordinating residues shown as sticks. (D) Select sequence alignment (top) of structurally characterized Bartonella henselae AmiB with Pseudomonas AmiB and Zur-regulated AmiA. Global structure (bottom left) and Zn binding site (bottom right) of B. henselae AmiB (PDB: 3ne8) with the Zn atom represented as a black sphere and metal coordinating ligands shown as sticks.

48

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

(A)

(p)ppGpp

DksA /(p)ppGpp

DNA

NTPs

mRNA

C–

protein

queuosine-tRNA

aa-tRNA ThrRS CysRS aa tRNA

dNTPs

QueD

dTMP

Gln glycine HCO3– Asp

dUMP CTP

UTP

UMP

dATP

GTP

ATP

GMP

IMP AMP

nucleobases PRPP

AICAR

OMP

PRPP

IPG

HisI

orotate

PR-ATP

dihydroorotate

PR-AMP PPi

ATP

(B)

L-His

PR-formaminoAICAR-P

YjiA

(p)ppGpp

PyrC Ribose-5-P

Ribose-5-P

dGTP

FolE

H2NTP

folates

central dogma

dCTP

(p)ppGpp

CPH4

N-carbomyl-Asp

folates

Asp

carbomyl-phosphate

GTP

ZagA HCO3– H2O

CA

CO2

ZigA Q

Gln GS

Glu

flavins

NH4+

Glu-tRNA

HemB

ALA

PBG

heme

Fig. 13 A Zur-regulated paralog-centric view of nucleotide biosynthesis and utilization. (A) PRPP, 5-phosphoribosyl-1-pyrophosphate, is derived from ribose-50 -phosphate synthesized in the pentose phosphate pathway via glucose-6-phosphate and ATP and provides the cellular link between carbon and nitrogen metabolism, and thus purine, pyrimidine and pyridine heterocycles. Glutamate (Glu) is the most abundant metabolite in all cells,186 and provides 88% of the nitrogen quota of the cell, with glutamine (Gln) the remainder. 80% of the flux through PRPP is committed to pyrimidine and purine biosynthesis. Paralogs discussed in this chapter are highlighted by the light purple boxes. Insets, the chemical structures of inosine 50 -monophosphate (IMP) and uridine-50 -monophosphate (UMP) are indicated, with each atom color-coded as to its cellular source, which themselves are color-coded in the same way. Individual folate derivatives are not specified. Two important cellular alarmones, AICAR (5aminoimidazole-4-carboxamide ribonucleotide; acadesine; ZMP) and (p)ppGpp (guanosine-50 ,30 -(penta)tetraphosphate) which function as signaling molecules and become cell-abundant under conditions of nutrient deprivation and antibiotic stress are highlighted.187 Single-headed arrows are not meant to imply irreversible steps. Riboflavin (FAD, FMN) biosynthesis also begins with GTP via the action of GTP cyclohydrolase II (RibA) and is subject to metabolic rewiring under conditions of transition metal starvation (see panel B),28 while bacterial biosynthesis of pyridine nucleotides nicotinamide adenine dinucleotide (phosphate), NADþ and NADPþ, also involves PRPP which is condensed with quinolinic acid to create nicotinic acid mononucleotide (NAMN).188 Deoxyribonucleotides are derived from ribonucleotides by ribonucleotide reductases (RNRs) which are cofactored by distinct metals or no metal at all, depending on the organism.189 C– hexagon, C– ribosomes that become cell abundant under conditions of transition metal starvation. See text for details. Other abbreviations: CA, carbonic anhydrase; OMP, orotidine-50 -phosphate; PR-ATP, phosphoribosylATP; PR-AMP, phosphoribosyl-AMP; IPG, imidazole-glycerol-phosphate; ALA, d-aminolevulinic acid; PBG, porphobilinogen. (B) Schematic representation of the impact of known COG0523 metallochaperones (B. subtilis ZagA,41 A. baumannii ZigA,28 and E. coli YjiA64,190) on GTP catabolism (see text for details).

(Fig. 12A).114,179,180 Loss of ZrlA in A. baumannii increases cell wall permeability and susceptibility to antibiotics; ZrlA also appears to play some role in the assembly of the CsuABCDE chaperone-usher pili system.114,179,180 AmiA, and evolutionarily related enzymes AmiB (48% identical to AmiA) and AmiC in some organisms, on the other hand, are amidases that cleave the amide linkage between the lactate moiety of MurNAc and L-Ala (Fig. 12A and D).181,182 The enzymes generally play roles in PG remodeling during cell division and loss of all Ami enzymes gives rise to hyper-elongated bacterial cells.183 A common feature is that each appears to have evolved a distinct PG binding domain which serves to localize each to appropriate place in the periplasm, e.g., the division septum or the poles of the cells.181 In the case of AbZrlA, this enzyme is homologous to another D,D-CP (Fig. 12C) that appears to be constitutively expressed, A1S_1248; comparative studies of ZrlA and A1S_1248 reveals essentially identical first coordination shells, as determined by Co substitution and electronic spectroscopy, and identical Zn binding affinities, of 2–3  1011 M 1.111 These studies effectively rule out a classical zinc-sparing response and instead suggest a mass action model (Section 2.03.3.2; Fig. 2B) or a model dependent on specific protein-protein or protein-PG interactions that can only be satisfied with ZrlA. This model is reminiscent of a model of various AmpD Zn-metalloenzymes encoded in the same

Metal ion homeostasis: Metalloenzyme paralogs in the bacterial adaptative response to zinc restriction

49

organism; in P. aeruginosa, AmpD, AmpDh2 and AmpDh3 differ from one another by the presence of a separate N-terminal PGbinding domain that imparts functional specificity, with the chemistry of the active site absolutely conserved.184,185 Here, AmpD is the true PG recycling enzyme that functions in the cytoplasm (Fig. 12B), while the others function in the periplasm in cell wall turnover.

2.03.4

Conclusions and perspectives

Zn is a crucial cofactor in enzymes that operate in all forms of life. As a result, the cellular adaptive response to Zn restriction is critical for bacterial survival, whether living in the soil, a marine environment, or an infected vertebrate host. The intracellular Zn activity is held within a narrow range so as to ensure proper proteome metalation; therefore, changes in the extracellular environment that limit Zn availability must be rapidly sensed and transduced into a cellular response. The adaptive response to low Zn is mediated by transcriptional repressor Zur (the functional equivalent of Zur in some Gram-positive bacteria is AdcR23) that senses slight fluctuations in Zn activity and upon Zn dissociation results in unregulated expression of the Zur regulon in a manner that is “tuned” to increasing Zn depletion. Beyond the classical upregulation of extracellular Zn uptake mechanisms, one major function of Zur regulons is the prioritization of a scarce resource (Zn) to a subset of cellular targets through the use of metallochaperones and protein paralogs, the latter of which is the focus of this review. We map the paralogs discussed here on bacterial metabolism, which localizes these enzymes in and around the central dogma of biology (Fig. 13A). First, Zur-regulated paralogs directly contribute to the nucleotide pool required for DNA and RNA synthesis. PyrC2 functions in pyrimidine biosynthesis, while one-carbon units provided by CO2 fixation by carbonic anhydrases and FolE2-derived folate derivatives are used to synthesize purines and pyrimidines. Second, DksA2 and (p)ppGpp collaborate to regulate the transcription of DNA into mRNA by RNA polymerase while also dialing down transcription of ribosomal RNAs, while increasing transcription of genes encoding enzymes required for amino acid biosynthesis. Lastly, C– ribosomal proteins, ThrRS2 and CysRS2, QueD2, and HisI2 contribute to ribosome structure, tRNA modification, tRNA charging, and amino acid biosynthesis, respectively, all core elements of the protein translation machinery. HemB2 ensures sufficient heme to cofactor the aerobic respiratory electron transport chain, while Zur-regulated cell wall biogenesis and remodeling enzymes maintain the structural integrity of cells under conditions of cellular Zn limitation. These Zur-regulated paralogs must have provided clear evolutionary advantages for bacteria to thrive in a severely Zn-restricted environment. Moreover, Zur-regulated COG0523 metallochaperones, as reviewed elsewhere,20 appear to impact the same metabolic neighborhood, in particular those processes that utilize GTP as a substrate (Fig. 13B).20,40 These include riboflavin biosynthesis, which provides electron carriers that function in aerobic respiration and redox chemistry,28 folate biosynthesis,41 and (p)ppGpp binding and signaling.64 The breadth of the evolutionary adaptative responses to what is essentially limitation of a single nutrient exemplified by Zurregulated paralogs across species is remarkable, and likely tuned to the evolutionary pressures experienced by specific organisms in their specific habitats or niches. Category 1 and category 3 paralogs beautifully illustrate this adaptive response as they largely retain the same cellular function of the Zn-utilizing protein or enzyme, while dispensing of the absolute requirement for Zn. In striking contrast to this “zinc-sparing” response, other Zur-regulated paralogs (grouped in Category 2) maintain an obligatory dependence on Zn cofactor, but appear to have evolved unique structural features that allow the paralog to resist Zn dissociation and therefore function under conditions of restricted Zn availability. General features of these Zur-regulated Zn-dependent enzymes are not yet apparent, beyond the possibility that the specific chemistry catalyzed by those enzymes can only be fulfilled by Zn, but may not, in fact, be absolutely necessary. Indeed, up-regulation of the expression of a second copy of an obligatory Zn-dependent enzyme may simply exploit “mass action,” allowing capture of a scarce resource for use in an important metabolic pathway, thus ensuring sufficient flux through that pathway. However, this is likely the case only for those paralogs that catalyze the rate-limiting step in a pathway, which characterizes just a subset of the enzymes discussed here. The study of Zur-regulated paralogs represents an exciting area for future research into general features of bacterial adaptation to low Zn.

Acknowledgments This research is supported by an NIH grant to D.P.G. (R35GM118157) and MinCyT Argentina (PICT 2019-0011; 2019-0385) to D.A.C. M.R.J. was supported by a graduate training fellowship in Quantitative and Chemical Biology at Indiana University (T32 GM109825, T32 GM131994). D.A.C. is a staff member from CONICET, Argentina; M.V.D. is supported by fellowships provided by CONICET, Argentina.

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2.04

Metallomics and metalloproteomics

Xueting Yana,b, Ying Zhoua, Hongyan Lia, Guibin Jiangb, and Hongzhe Suna, a Department of Chemistry and CAS-HKU Joint Laboratory of Metallomics on Health and Environment, The University of Hong Kong, Hong Kong SAR, China; and b State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China © 2023 Elsevier Ltd. All rights reserved.

2.04.1 2.04.1.1 2.04.1.2 2.04.2 2.04.2.1 2.04.2.2 2.04.2.3 2.04.2.4 2.04.2.5 2.04.3 2.04.3.1 2.04.3.2 2.04.3.3 2.04.3.4 2.04.3.5 2.04.3.6 2.04.4 2.04.4.1 2.04.4.2 2.04.4.3 2.04.5 References

Introduction Metallomics Metalloproteomics Technical platform for metallomics and metalloproteomics Separation techniques Detection techniques Identification techniques Structure analysis techniques Computer-aided approaches Application of metallomics and metalloproteomics for metallodrug research Platinum Ruthenium Bismuth Silver Gold Arsenic Application of metallomics and metalloproteomics for environmental health and toxicology Mercury Lead Cadmium Summary and outlook

54 54 55 55 55 58 59 59 60 60 61 62 63 65 65 66 69 69 71 71 71 72

Abbreviations 2D-GE Two-dimensional gel electrophoresis CE Capillary electrophoresis ESI-MS Electrospray ionization mass spectrometry GE-ICP-MS Online coupling of column-type gel electrophoresis with inductively coupled plasma-mass spectrometry ICP-MS Inductively coupled plasma-mass spectrometry IEC Ion-exchange chromatography IEF Isoelectric focusing IMAC Immobilized metal affinity chromatography LA-ICP-MS Laser ablation-inductively coupled plasma-mass spectrometry MALDI-TOF-MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MS/MS Tandem mass spectrometry

Abstract Metal ions play pivital roles in biological processes being involved in many cellular and subcellular functions. Metallomics and metalloproteomics are new frontier interdisciplinary fields of research addressing the entirety of metals and metalloids and their roles, uptake, storage and transport essential for protein functions in organisms, especially in cells and tissues. With the rapid development of metallomic and metalloproteomic techniques, they have been successfully applied in inorganic medicinal chemistry, chemical biology and environmental science with significant progresses being achieved, in particular in understanding the molecular mechanisms of actions of metals and metallodrugs. This chapter introduces the concepts and research techniques in metallomics and metalloproteomics and expatiates their applications in metal-based drugs as well as environmental metallomic studies in biological systems.

Comprehensive Inorganic Chemistry III, Volume 2

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Metallomics and metalloproteomics

2.04.1

Introduction

Metal ions are associated with biological macromolecules such as proteins and nucleic acids and play important roles in fundamental biological processes such as signaling, gene expression, and catalysis. It is estimated that around one third of the proteins are metalloproteins, with the intrinsic metal ions providing catalytic, regulatory and structural role critical to protein function.1 However, it remains elusive on the molecular bases of many metal-dependent biochemical processes such as the molecular mechanisms by which the metal is sensed, stored or incorporated as a cofactor in organisms. Over the past decades, various bioanalytical techniques have been developed to extensively elucidate the biological essentiality and toxicity of the metal elements,2–4 which is an important subject in various scientific fields such as bioscience, medicine, pharmacy, nutrition, agriculture, and environmental science.

2.04.1.1

Metallomics

High-throughput technologies have revolutionized molecular biology research. Addition of “omics” to a molecular term, such as genomics, proteomics and metabolomics, allows a comprehensive or global assessment of a set of biomolecules. “Metallome” was initially proposed by Robert JP Williams (Oxford University), who referred to it as an element distribution, metal ion concentration, and the metal-containing biomolecules or free metal ions present in a cell or biological tissue.5 In 2004, Hiroki Haraguchi (Nagoya University), first described metallomics as “integrated biometal science” and pointed out the future directions of metallomics research.6 Metallomics is an emerging research area, which systematically studies the distribution, content, chemical form and function of all free or complexed metal elements in life.7 The most important research goal of metallomics is to elucidate the physiological role and function of biomolecules bound to metal ions in biological systems. The journal “Metallomics” was launched in January 2009 by the Royal Society of Chemistry, providing a platform to report the recent advances and state of the art techniques in metallomics research.8 The evolution of metallomics is illustrated in Fig. 1. In the early studies, metallomics mainly focused on the distribution and effects of metal elements on the environment and organisms9; while the modern metallomics focus more on the distribution and chemical speciation of metals and metalloids in biological systems, characterization of metal binding biomolecules, especially the structure and function of metal-binding proteins/enzymes.10 Metallomics will not only reveal the interaction between metals and biological molecules in the body, but also open up new avenues for studying the biological functions of metal-binding biomolecules and mechanism of actions of metallodrugs in life science. Only when we master the whole information about total metals or metalloids in organisms can we have an in-depth understanding of the role of metals in the life processes.

Fig. 1

The development of metallomics.

Metallomics and metalloproteomics 2.04.1.2

55

Metalloproteomics

With the cross-development of proteomics and metallomics research, a new interdisciplinary field, metalloproteomics, has gradually formed in recent years. Metalloproteome is defined as all the metalloproteins in a proteome or all the proteins with metalbinding sites.11 As a branch of metallomics, metalloproteomics mainly focus on the structure and function between biometals and proteins in a proteome. A key aspect of metalloproteomics is the annotation of a protein as a metalloprotein and identification of the intrinsic metal, which would be an efficient way to reveal the structure and function of proteins with metal-binding abilities.12 Due to the extraordinarily important role of metalloproteins/metalloenzymes, metalloproteomics have received great attention in modern bioanalytical chemistry. Conventional comparative proteomics is to analyze proteome changes in response to exogenous compounds, while metalloproteomics focus on investigating the distributions, compositions, and alteration of all metalloproteins in a proteome. With the rapid development of high-throughput analytical techniques, unequivocal identification and characterization on a set of metalloproteins including metal-binding proteins, and their metal-binding motifs in a given biological sample could be easily achieved nowadays. Metalloproteomics may offer holistic information on the structure and function, synthesis process of metalloproteins in organisms, as well as the post-translational processes of these proteins, such as modification, assembly, transport, and metal absorption, which unveils the complicated mechanisms and precise roles of metalloproteins/metalloenzymes in cells.13

2.04.2

Technical platform for metallomics and metalloproteomics

Metallomics and metalloproteomics are emerging fields focusing on the entirety of metals/metalloids within biological systems and their uptake, transport and storage, as well as functions when associated with biomolecules.14 Significant progresses have been made in this area since its inception, largely attributed to the development of a great variety of analytical tools.15–19 A great number of well-established proteomic strategies are readily applicable for the field of metalloproteomics, in particular the techniques for protein separation and identification. The protein separation techniques include conventional two-dimensional gel electrophoresis (2D-GE) separation, which serves as a powerful tool for resolving hundreds of proteins in a biological sample, and liquid chromatography (LC), which is usually performed by several fractionation steps to achieve better resolution.20 For quantitative determination of metals or metalloids in biological systems, inductively coupled plasma mass spectrometry (ICP-MS) and atomic spectrometry techniques are the best choices. Recently, the hyphenation of column-type GE to ICP-MS, namely, continuousflow GE-ICP-MS or GE-ICP-MS allows not only metal/metallodrug associated proteins to be tracked on a proteome-wide scale, but also metalloproteins associated metals to be investigated on a metallome-wide scale.21 This technique was further extended to twodimensional separation through combination with LC, namely, LC-GE-ICP-MS,22 enabling large quantity of authentic metalbinding proteins being mined in a whole-cell scale. In addition, immobilized metal ion affinity chromatography (IMAC), a well-known technique commonly used to purify proteins or peptides according to their affinity to specific metal ions,23 has also been frequently used to track metal-binding proteins, in particular for those proteins with low abundance.24 Similar to this approach, a fluorescence-based approach via unique metal-tunable fluorescent probes was recently developed, enabling various endogenous metalloproteins that bind metal ions transiently or weakly to be tracked.16,19,25 For protein identification, a variety of mass spectrometric techniques are frequently coupled with protein separation techniques, such as electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), and tandem mass spectrometry (MS/MS).26 Metallographic distribution techniques mainly include laser ablation (LA)-ICP-MS, synchrotron radiation X-ray fluorescence imaging (SR-XRF), and secondary ion mass spectrometry (SIMS). Typical analytical approaches that are widely used to resolve and analyze metalloproteome derived from cells and tissues, are summarized in Fig. 2.

2.04.2.1

Separation techniques

The commonly used protein separation methods mainly include GE, LC, and capillary electrophoresis (CE). GE is more suitable for the separation of macromolecules and complex samples due to its unique resolution advantage, and has been widely used in proteomics research. The hyphenation of column-type GE to ICP-MS enables simultaneously monitoring metals and their associated proteins.21 The column-type gel adopted the traditional slab gel preparation. Both native and denaturing gels could be prepared and the gel composition could also be varied according to the protein targets of interest. A T-connection was used to split the elutes from the column gel system into two parts, one for online metal measurement by ICP-MS and the other for protein identification through biological mass spectrometry analysis.21 To increase the resolution of protein separation, this method was further improved by combination with LC (LC-GE-ICP-MS) to separate proteins according to their isoelectric points (pIs) prior to GE (Fig. 3A).18,22 The 2D-GE is the primary technique for protein separation, including isoelectric focusing electrophoresis (IEF) and polyacrylamide gel electrophoresis (PAGE), which separates proteins according to charge and size, respectively. IEF is generally performed in immobilized pH gradient (IPG) gels that molecules are separated based on their pIs. The experimental conditions, especially pH values, are vitally important for metal-binding proteins to remain folded. For the second dimension, PAGE can be divided into denaturing sodium-dodecyl sulfate (SDS)-PAGE and native-PAGE. The same caveats as SDS-PAGE is still the loss of metals due to the application of reducing agents (DL-dithiothreitol) and detergent (SDS). Native-PAGE can maintain the original properties of proteins

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Fig. 2

Experimental workflow of metalloproteomics.

and has little effect on metal-ligand interactions, but the resolution is slightly worse than SDS-PAGE. Therefore, the specific techniques are required to not only achieve higher resolution, but also preserve the metal that bound to its associated proteins during gel-based separation. Liquid-based chromatographic techniques employ much mild separation buffers close to physiological conditions, thus potentially preserving the native protein-metal complexes during separation. The commonly used liquid chromatography techniques mainly include size exclusion chromatography (SEC), ion-exchange chromatography (IEC), affinity chromatography (AC), reversed-phase liquid chromatography (RPLC), 2D high-performance liquid chromatography (HPLC), etc. LC is often coupled online/offline with various mass spectrometry techniques for the separation and detection of metal-binding proteins (Fig. 3B).27 One-dimension separation methods are usually less efficient and more suitable for the separation of small molecules and simple matrix samples. Given the inherent complexity of biological systems, the need for more powerful and highly resolving separation methods has grown. Consequently, the development and application of multidimensional chromatography in metallomics and metalloproteomics have thrived over the past few years.28 Multidimensional LC combines two or more forms of LC to improve the selectivity and resolution, thus producing a greater separation power for the fractionate peptides before entering the mass spectrometer. Apart from chromatography methods mentioned above, IMAC is a well-known technique commonly used to purify proteins or peptides according to their affinity to specific metal ions.23 IMAC, which can be performed under denaturing or nondenaturing conditions, relies on the interaction between electron donors on the surface of a protein and metal ion-based electron acceptors chelated to a solid-phase support. The proteins with significant high affinity to the immobilized metal ions (Co2þ, Ni2þ, Cu2þ, and Zn2þ) will be captured on the column, then eluted, separated, and further identified by mass spectrometry. This approach has been used to identify metal-binding proteins/peptides in biological samples, such as Cu-binding proteins in hepatocellular,29 Ni-interacting proteins in human B cells,30 Cu-binding proteins in Arabidopsis roots,31 Bi-binding proteins in Helicobacter pylori,32and Ni- and Co-binding proteins from Streptococcus pneumoniae.33 In a metalloproteomic workflow, IMAC can be designed prior to 2D gel technique or 2D-LC and extensively adopted for large-scale proteomic analyzes and bottom-up global metalloproteome profiling. The benefits of IMAC are low cost, high protein loading, ligand stability, mild elution conditions, simple regeneration and can be implemented in high-throughput liquid-handling workflows. Moreover, pre-enrichment capability of IMAC also allows low abundance proteins that bind to the metallodrugs to be tracked. It is also noteworthy that the false positives (e.g., histidine-rich proteins) and false negatives (e.g., metal-containing proteins) can occur during the binding process. CE is a high-resolution separation technique with short separation time and lower sample and reagent consumption. The hyphenation of CE to ICP-MS was applied to trace metal speciation in proteins, providing information on metal binding and metal exchanges occurred in proteins. The separation and characterization of metallothionein (MT) isoforms were performed on a CEICP-MS method based on surface-modified capillaries.34 With the anionic polymer-coated capillary, nine MT complexes in a commercial preparation from rabbit liver were successfully separated in a natural cytosolic pH environment. Although the determination of metalloproteins can be carried out by CE-ICP-MS, it is still challenging to quantify metalloproteins with high precision, accuracy, and isoform specificity. Using online isotope dilution by continuous introduction of an isotopically enriched, speciesunspecific (mixed 34S, 65Cu, 68Zn, 116Cd) spike solution after the CE separation step, the MT molecular formulae can be obtained simultaneously by determining the sulfur-to-metal ratios.35

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Fig. 3 Emerging strategies in metalloproteomics. (A) Flow chart of LC-GE-ICP-MS. (B) Schematic of 2D-HPLC-UV–ICP-MS instrumentation. (C) “Metal-tunable” fluorescent probe for tracking and editing metalloproteomes inside living cells. Adapted with permission from Wang, H.; Zhou, Y.; Xu, X.; Li, H.; Sun, H. Metalloproteomics in Conjunction With Other Omics for Uncovering the Mechanism of Action of Metallodrugs: MechanismDriven New Therapy Development. Curr. Opin. Chem. Biol. 2020, 55, 171–179, Lai, Y. T.; Yang, Y.; Hu, L.; Cheng, T.; Chang, Y. Y.; KoohiMoghadam, M.; Wang, Y.; Xia, J.; Wang, J.; Li, H.; Sun, H. Integration of Fluorescence Imaging With Proteomics Enables Visualization and Identification of Metallo-Proteomes in Living Cells. Metallomics 2017, 9, 38–47, and Yun, Z.; Li, L.; Liu, L.; He, B.; Zhao, X.; Jiang, G. Characterization of Mercury Containing Protein in Human Plasma. Metallomics 2013, 5, 5821–5827. Copyright 2020 Elsevier, 2013 and 2017 Royal Society of Chemistry.

At present, most separation methods are performed after the proteome is extracted from the whole-cell or tissues, which will mix the proteins that are originally separated. During the process, weakly bound metal ions may dissociate from proteins and transfer to other proteins with higher affinity or abundance. To some extent, this problem can be mitigated by pre-fractionation into subcellular fractions or microanalytical techniques for in situ imaging of metals. Considering the differences in affinity of metalloproteins, special attention should be paid to the separation procedure to avoid loss of the bound metals. To circumvent this issue, a novel fluorescence-based approach based on a metal-tunable fluorescent probe has been developed by conjugation of nitrilotriacetate (NTA) moiety with a fluorophore and arylazide, followed by coordination with metal ions, namely Mn þ-TRACER.25 Incorporation of arylazide is essential for strengthening the binding between the probe and POIs, as well as for fluorescence turn-on effects. The probes, after entering live cells, bind to proteins via Mn þ-NTA moiety and are anchored to the labeled proteins through formation of covalent bond between them upon UV exposure, and the labeled proteins can be subsequently identified by conventional

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proteomics (Fig. 3C).25 Using this approach, a variety of metal-binding proteins including Bi3þ, Ga3þ, Fe3þ have been tracked, providing rich sources for further analysis of their mode of actions.36–38

2.04.2.2

Detection techniques

After the metalloproteins are separated, the containing metal elements need to be detected, identified, and quantified. The highquality bio-mass spectrometry applied in the traditional proteomics are also applicable to metalloproteomics. For quantitative determination of trace metals in biological systems, mass spectrometry and atomic spectrometry techniques are the methods of choice. The commonly used metal detection techniques mainly include ICP-MS, atomic absorption spectrometry (AAS) and atomic fluorescence spectrometry (AFS). ICP-MS is a sensitive multi-element detector and commonly used for the quantitative determination of metals or metalloids in various biological systems. As an elemental specific technique, ICP-MS has been intensively applied as a detector coupled with various chromatographies, including RPLC, SEC or IEC. So far, LC coupled to ICP-MS has been applied to bioinorganic chemistry and clinical fields, providing important information on metalloproteins, such as Cu-, Zn-binding proteins in human blood serum,39,40 metalloproteins contained in rabbit plasma,41 Se-containing proteins in yeast,42 metallothionein and metal/semimetallic binding polysaccharides in organism.43 Other hyphenated ICP-MS techniques for identifying and quantifying metalbinding proteins include CE, GE, and laser ablation (LA). LA-ICP-MS, first developed in the late 1980s, is a powerful technique for in situ imaging of metal distribution in biological samples, as it provides high spatial resolution and excellent sensitivity for quantitative imaging.44 It serves as a microanalytical methodology that uses a focused laser beam to ablate material from the surface of a solid sample.45 LA-ICP-MS has been successfully applied to detect metal (Mn, Fe, Cu, and Zn) distribution in Parkinson’s and Wilson’s disease mouse models.46,47 The systemic quantification of metals related to neurodegenerative diseases provides new insights into the intricate spatial relationship of morphology and chemistry within tissue. A metalloproteomic study involved human blood serum samples from bipolar disorder (BD) patients was performed with LA-ICP-MS and MALDI-TOF MS/MS.48 The differential profile in terms of metals (Na, Mg, Zn, Ca, and Fe) bound to serum proteins was observed for control and BD patients treated with or without Li, therefore opening the door for further discovery of potential biomarkers for BD (Fig. 4).48 In addition, LA-ICP-MS has been used to investigate the distribution of metallodrugs within single-cell,49 tissue,50 or tumor tissue,51 revealing the potential targets of anticancer and antibacterial metallodrugs, which provides bases for further exploring their molecular mechanisms of action. By combining the partially denatured SDS-PAGE with LA-ICP-MS, seven potential Bi-binding proteins in H. pylori treated with colloidal bismuth subcitrate (CBS) were identified.52 Bianga et al. developed a method based on LA-ICP-MS together with MALDI-MSI to explore the penetration and distribution of two platinum-based metallodrugs (cisplatin and oxaliplatin) in human tumors removed from patients diagnosed with colorectal or ovarian peritoneal carcinomatosis.53 LA-ICP-MS has proven to be a fast and fairly robust technology, enabling multi-elemental analysis at trace and ultratrace level, providing elemental distribution maps in diversified biological samples.

Fig. 4 LA-ICP-MS imaging of a 2D electrophoresis gel piece showing the qualitative distribution of metal-containing proteins of blood serum from BD patients treated with Li. Adapted with permission from Sussulini, A.; Kratzin, H.; Jahn, O.; Banzato, C. E. M.; Arruda, M. A. Z.; Becker, J. S. Metallomics Studies of Human Blood Serum from Treated Bipolar Disorder Patients. Anal. Chem. 2010, 82, 5859–5864. Copyright 2010 American Chemical Society.

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Given that ICP-MS serves as a powerful multiple elements analytical tool, multidimensional separation techniques are considered to be coupled with it through online or offline manner. For metalloproteomic studies, the hyphenation of HPLC, GE, LA, ICPMS and MALDI-TOF-MS profoundly promotes the identification and characterization of metal/metalloid-protein complexes, which provides more information on the modes of action of metals and metallodrugs in biological systems. Modern nuclear analytical techniques, including neutron activation analysis (NAA), SR-XRF, particle-induced X-ray emission (PIXE), have been extensively employed for multi-elemental quantification and distribution analysis as well as for structural characterization of metallomes and metalloproteomes in a biological system.54,55 NAA is a multi-elemental quantification technique based on the measurement of characteristic radiation from radionuclides formed directly or indirectly by neutron irradiation of materials.56 However, real-time and on-line analysis are not achievable by this method, and the lower analysis speed and higher cost restrict its wide usage. The X-ray microbeams can penetrate sample in depth (1000 mm) and have high spatial resolution in speciation analysis (0.1–1 mm). With the advances in the third generation synchrotron microprobe beamlines, a growing interest in the use of SR-XRF has emerged for elemental mapping in individual cells or cellular compartments.57 PIXE is an X-ray spectrographic technique, which can be used for the non-destructive, simultaneous multi-element analysis of biological and environmental samples.58 PIXE is often used in combination with chromatographic separation technology to detect trace elements in a proteome.

2.04.2.3

Identification techniques

The identification of biological macromolecules is an important step in the characterization of metalloproteins for further functional studies. Mass spectrometry is an analytical technique for measuring the mass-to-charge ratio (m/s) of ions from small and macromolecular compounds, such as proteins, polypeptides and oligonucleotides.59 Mass spectrometry is constantly improved and innovated, including a variety of soft ionization technologies, such as the fast atom bombardment (FAB), field desorption ionization (FD), SIMS, ESI, thermal spray (TSI), and MALDI, plasma desorption (PD), laser desorption (LD), etc. Among them, MALDI and ESI are commonly used in biological molecules, especially in proteomic analysis. In recent years, MALDI-TOF-MS has emerged as a potential tool for the identification of gel-isolated proteins. A major advantage of MALDI-TOF-MS is the ability to analyze biomolecules in complex matrices whose polypeptide fragments are not disturbed by the presence of salts and buffers in the sample. In addition, MALDI-TOF-MS is highly sensitive and can identify proteins with low concentrations, and the molecular weight of measurable biological macromolecules can be as high as 600 kDa. ESI is a technique to generate ions for mass spectrometry using an electrospray with a high voltage to create an aerosol from a liquid. ESI overcomes the propensity of macromolecules to fragment during ionization. ESI is different from other atmospheric pressure ionization processes (e.g., MALDI), in which it may produce multiple-charged ions, effectively extending the analyzer’s mass range to accommodate the magnitude of kDa-mDa observed in proteins and their associated peptides. It has been demonstrated that ESI-MS is capable of monitoring the flux of metal ions transferred into and out of the metalloprotein and drug-modified metallothionein.60 The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins were determined using ESI-MS under very gentle conditions in the interface region.61 ESIMS is particularly useful for analyzing metalloprotein behavior, and it reveals the metal retention and conformational properties of human serum transferrin under a variety of conditions.62 Since these early studies, the use of ESI-MS has grown rapidly, advancing our understanding of dynamic aspects of the functional properties of metalloproteins endowed by metal binding. Over the past years, multidimensional chromatography coupled to mass spectrometry has demonstrated to be an emerging technique for the analysis of complex protein mixtures. One approach of them, multidimensional protein identification technology (MudPIT), is a non-gel technique for separating and identifying individual components of complex protein and peptide mixtures by electrospray ionization, tandem mass spectrometry, and database searching.63 It consists of two-dimensional separation systemsstrong cation exchange (SCX) and reversed phase (RP)-coupled online in an automated fashion to mass spectrometric detection. The use of biphasic chromatographic columns showed to be beneficial for metalloproteins and metallodrug–protein adducts. The technique has already been successfully applied for the characterization of [(h6-p-cymene)RuCl2(DMSO)] binding sites in E. coli, and cisplatin binding sites in E. coli and human serum proteins respectively.64–66 Using MudPIT for proteomic analysis, Wolters et al. examined the effect of RAPTA-T on human cancer cells, shedding new insight into cellular response mechanisms to metallodrug treatment.67 This method offers the possibility to analyze the highly complex samples necessary for large-scale proteome analysis in a single experiment.

2.04.2.4

Structure analysis techniques

Characterization of the protein structure is the basis for understanding the function of a protein and its structure-function relationship. The traditional methods of structural determination are X-ray crystallography68 and nuclear magnetic resonance (NMR).69 In addition, X-ray absorption spectrometry (XAS), Mössbauer spectroscopy, electron paramagnetic resonance (EPR), resonance Raman spectroscopy (RR) and magnetic circular dichroism (MCD) are employed for the detailed structural characterizations of the metal sites in metalloproteins.70–72 NMR is a powerful technique that allows the determination of the structural and dynamic properties of biological macromolecules such as proteins, DNA, and RNA. NMR was applied to investigate Cu(I, II), Fe(II), Ni(II), and Zn(II) interactions with disordered biomacromolecule, such as neurodegenerative proteins and extracellular fragments of metal transporters, revealing interesting

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information on the corresponding metal coordination sphere, metal affinity, and structure.73 NMR spectroscopy has also been successfully applied in the characterization of metalloproteins, including structural studies, identification of metal coordination sphere by hetro-/homo-nuclear metal NMR spectroscopy.74,75 Paramagnetic NMR, which yields long-range structural information e.g., paramagnetic relaxation enhancement (PRE); pseudocontact shifts (PCS); residual dipolar couplings (RDC) induced by anisotropic paramagnetic centers, offers great potentials to structurally characterize paramagnetic metalloproteins.74,76 In addition, in-cell NMR has also been shown as an emerging method of studying metal-related events in living cells and this technique is anticipated to provide an excellent tool in studying metal associated events in complex native environments of living cells.77,78 In combination with spectroscopic characterizations, X-ray crystallography is a very powerful method to visualize coordination of metal ions and metallocofactors within a protein scaffold. In a systematic assessment of protein structures deposited in the Protein Data Bank (PDB) to date (August 2021), 87.78% structures (out of 181,295) were determined using X-ray crystallography, and about a quarter to one third of them contains metals or metallocofactors. The Metalloprotein Database and Browser (MDB), a web-accessible resource for metalloprotein research, contains quantitative information on geometrical parameters of metal sites available from structures in the PDB. The major disadvantage of X-ray crystallography is that it is difficult to obtain suitable crystals, which is a prerequisite for X-ray crystallography. The storage and transportation of crystals under nonphysiological environments is another concern, which can occasionally lead to functionally irrelevant conformational changes. XAS is a valuable tool for probing changes in the chemical environment of metal centers, such as the oxidation states of metals in cells, its coordination motifs as well as the identity and number of adjacent atoms.79 Since no crystallization is required in sample preparation, XAS has often been used in the structural studies of metalloproteins as another alternative to X-ray crystallography and NMR. The X-ray absorption spectrum is presented as a function of the X-ray energy (XAFS) and can be divided into two regions, the near edge (XANES) and beyond the edge (EXAFS). As a noninvasive technique, XAS has been successfully used to monitor the biotransformation of metallodrugs in biological fluids.80,81

2.04.2.5

Computer-aided approaches

In recent years, the availability of sequence information for metalloproteome in different organisms has opened the door for systematic analysis of the utilization and function of metals in biology. In order to comprehensively interpret the large collections of data, subsequent bioinformatics analysis by incorporating computer tools or statistical methods is crucial. Bioinformatics methods can give valuable support to experimental methods to functionally characterize metalloproteins. In the past several decades, numerous metal-associated proteins have been identified by metalloproteomics, providing sequence database for metalloproteomes in different organisms. A number of computational tools and methods have been developed for the prediction of either metalloprotein genes or metal-binding sites in proteins. For example, the web server MetalDetector82 and CheckMyMetal83 predict metal-binding sites in proteins from sequence or structures, based on structured-output learning predicts metal-binding sites from protein sequence,84 TEMSP (3D TEmplate-based Metal Site Prediction) predicts zinc-binding sites,85 fragment transformation method predicts metal ion-binding sites,86 and Fold-X force field predicts water and metal binding sites and their affinities in proteins.87 Andreini et al. discussed the development of bioinformatics methods focused on the prediction of zinc, nonheme iron, and copper-proteins based solely on protein sequences, and provided hints to understand several properties of these metalloproteins in living organisms.88 With the aid of bioinformatics methods, the molecular functions and biological structure of proteins perturbed by metals will be largely unveiled. Machine learning, an essential component in artificial intelligence (AI), provides a set of tools that can improve data generation and analytics. Very recently, a novel high-throughput zinc-binding residue prediction method, ZnMachine, was gul presented three different developed by combining several intensively trained machine learning models.89 Haberal and O
deep learning architectures (2D Convolutional Neural Network, Long-Short Term Memory, and Recurrent Neural Network) using three different feature sets for predicting metal-binding of histidines and cysteines in proteins.90 A deep learning approach was developed by incorporating both spatial and sequential features of metal-binding sites into a multichannel convolutional neural network (MCCNN) to predict harmful missense mutations occurred at the metal-binding sites of metalloproteins from the human genome (Fig. 5).91 Bioinformatics offers a powerful tool for studying metal-binding sites, metalloproteins, and metal-based drugs, providing significant insights into the general principles of metal utilization and function in biology, and also unveiling potential targets perturbed by a (metallo)drug.

2.04.3

Application of metallomics and metalloproteomics for metallodrug research

As basic components of life, metal ions play an irreplaceable role in the processes of life. They serve as essential cofactors of enzymes, fulfilling various cellular functions that cannot be achieved by organic molecules. Metal ions or metal compounds have also been introduced into the biological system for diagnostic and therapeutic purposes, which has stimulated interest in medicinal inorganic chemistry for many years.92 Throughout history, silver and other metals such as copper and zinc have consistently been used as potential antibacterial agents. The clinical success of cisplatin and other platinum-based drugs has demonstrated the potential of metal-based compounds in cancer therapy.93 Due to drug resistance and the severe side effects of classical platinum-

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Workflow of data collection and feature extraction to train the deep learning model.

based chemotherapy, other metal compounds such as ruthenium, gold and titanium were developed in the search for advanced pharmacological profiles.94–96 However, the mode of action of metallodrugs even for the established treatments has only been partially elucidated, which prohibits further development of metal-based therapies. Knowledge on the physiological processes (i.e., uptake, cellular distribution and biotransformation), molecular targets and functioning pathways will not only enable fully exploiting therapeutic potential of a metallodrug, but also guide in the design of new metallodrugs with higher efficacy and fewer side-effects.22,36,97 Traditionally, the molecular mechanisms of metallodrugs are often obtained from accumulated experimental data, i.e., by investigation of individual putative drug targets (proteins or other biomolecules), and extensive researches are needed to differentiate the real targets from the false ones. Given the inherent complexity of the biological system, an integrated approach is in need to provide a systemic view on the response of a metallodrug. In these circumstances, metallomics and metalloproteomics are ideal tools for obtaining a holistic picture on the mode of action of a metallodrug. With the rapid development of metallomic and metalloproteomic strategies, which has been successfully applied in the field of medicinal inorganic chemistry, notable progresses have been made in the mechanistic study of metallodrugs.15–18,98,99

2.04.3.1

Platinum

Platinum-based drugs are one of the most widely used classes of drugs in cancer therapy. Approximately half of all patients on anticancer chemotherapy regimens are treated with a platinum drug. Since the first generation of platinum antitumor agents, cisplatin, is often associated with serious side effects and drug resistance, new generation platinum anticancer drugs such as carboplatin, oxaliplatin, nedaplatin, and satraplatin,100 which exhibit clinical activity against cisplatin-resistant cancers with fewer side effects, are extensively applied in cancer therapy clinically. Synchrotron radiation-induced X-ray emission (SRIXE) was applied to monitor the in situ cellular distribution of platinum within single ovarian carcinoma cells treated with cisplatin.101 A higher platinum concentration was observed in the cell nucleus than in the surrounding cytoplasm, indicating DNA as the major target of cisplatin.102 Whereas, stable platinum(II)-based metallointercalators accumulated more specifically in the nucleus within single A549 human lung cancer cells compared with that of cisplatin, suggesting relative stable platinum-based compounds have the potential to overcome the cellular toxicity and side effects induced by platinum binding to cytoplasmic proteins.103 The combination of LA-ICP-MS with MALDI-MS imaging analysis provides a fast and sensitive tool to study the penetration and distribution of two Pt-based metallodrugs (cisplatin and oxaliplatin) in human tumor samples.53 The optimized LA-ICP-MS together with the innovative preclinical multicellular tumor spheroid model provided a new tool for studying the distribution of platinum-based compounds in a 3D manner.104 A combined imaging approach consisting of LA-ICP-MS, NanoSIMS, and TEM was applied to kidney and tumor samples upon administration of selected platinum(IV) anticancer prodrugs, showing uneven platinum distribution in the organs.105 Although the formation of stable platinum-DNA adducts has long been considered to be involved in their overall pharmacological profiles, several proteins/enzymes have recently been proposed to be the targets of platinum compounds.106,107 The first cisplatin-binding protein was successfully identified in biological samples by Dyson et al. in 2001.108 By using 1D-SDS-PAGE coupled off-line with LA-ICP-MS, the profile of cisplatin-binding proteins in E. coli was obtained. The protein band corresponding to the most intense platinum peaks was found to contain outer membrane protein A, which acted as an ion channel with potential relevance for cisplatin uptake. Shotgun proteomics with no prior enrichment for Pt-proteins has been performed on cisplatintreated E. coli with successful identification of Pt-bound peptides (Fig. 6).65 Subsequently, cisplatin-binding proteins in rat serum and renal proximal tubule epithelial cells (RPTECs) were identified using GE coupled with LA-ICP-MS.109

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Fig. 6

Proteomics strategies for identification of Pt-bound proteins in treated cells.

The intracellular cisplatin-protein adducts were identified by combination of 2DE, ESI-MS/MS and a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analog (CFDA-cisplatin).110 Furthermore, the optimized 2DE in an alkaline pH range facilitates the identification of intracellular CFDA-cisplatin-protein adducts in ovarian cancer cells.111 Recently, a new method consisting of azide-modified cisplatin derivative, azidoplatin (AzPt) and the Cu-catalyzed azide-alkyne cycloaddition (CuACC) or “click” reaction was developed for Pt-protein enrichment and MS/MS identification (Fig. 6).112 The extracted proteins can be visualized in-gelation after treatment with AzPt, resulting in 152 proteins to be identified in Pt(II)-treated Saccharomyces cerevisiae. Very recently, a fluorescent cisplatin analog BODIPY-cisplatin, 2DE and mass spectrometry were employed to identify the protein binding partners in A2780 and cisplatin-resistant A2780cis ovarian carcinoma, as well as in HCT-8 and oxaliplatin-resistant HCT-8ox colorectal cell lines (Fig. 6).113 Among the binding partners, some have previously been described in connection with drug resistance, such as vimentin, Grb2, and GSTP1. Overall, a variety of metalloproteomics approaches such as GE/LA-ICP-MS, chemically modified Pt probes, and biotinylated pull-down assays have been applied for successfully profiling of global cellular targets of platinum-based drugs, providing new information on platinum-associated proteomes and platinum-targeting cellular processes in cancer cells.

2.04.3.2

Ruthenium

Ruthenium (Ru) anticancer agents are considered to be the most promising alternatives to platinum anticancer drugs, as they are often identified as less toxic and generally more selective to tumors. The Ru(III) complexes, NAMI-A,114 KP1019,115 NKP1339,116 and TLD1443117 are in clinical trials. Ruthenium anticancer compounds show multiple targets (DNA, RNA, and proteins) and diverse mechanisms for their antitumor properties. Therefore, it is particularly important to understand the uptake, distribution, and interaction of ruthenium complexes in cells and organs, which will facilitate the most potent ruthenium complex to be selected for effective therapy. The cellular distribution of Ru in single human neuroblastoma cells treated with KP1019 was studied by XRF imaging at two incident energies, revealing colocalization of Ru with Fe in both the cytosol and nuclear regions.118 In contrast, Ru could not be visualized in cells after treatment with NAMI-A, which is consistent with the proposition that its activity is exerted through a membrane-binding mechanism. LA-ICP-MS was also applied to study the spatially resolved distributions of structurally identical osmium and ruthenium-based organometallic anticancer drug candidates in organs, muscles and tumors of treated mice.119 The interaction between KP1019 and human serum albumin was investigated by X-ray crystallography and ICP-MS, revealing two ruthenium binding sites (histidine residues 146 and 242) located in hydrophobic binding pockets of albumin.120 Over the past

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decades, the most prosperous structural moiety of ruthenium anticancer drugs was half-sandwich Ru-arene complexes containing 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (PTA) ligand, namely RAPTAs, which exhibit remarkable in vitro and in vivo anticancer activities.121 A combined metallomics and proteomics study of human ovarian cancer cells treated with the RAPTA-T revealed striking differences in the intracellular behavior of the complexes between cisplatin-sensitive and resistant cell lines.67 The mode of action of two ruthenium compounds NAMI-A and PAPTA-T in exposure to A2780 human ovarian carcinoma cells was further investigated by 2D-DIGE proteomic analysis.122 Notably, the patterns of protein alterations induced by both ruthenium compounds were quite similar to each other while being significantly different from those caused by platinum compounds, suggesting a different metal-based mode of action. A chemical proteomic approach (“drug pull-down”), involving affinity chromatography, shotgun proteomics and bioinformatics, was developed to identify molecular targets of an antimetastatic RAPTA anticancer agent.123 Through an integrated metallodrug pull-down and target-response profiling approach, Meier and Gerner et al. found an unexpected target selectivity of a Ru(arene) pyridinecarbothioamide compound for plecstatin (Fig. 7A), a scaffold protein and cytolinker.124 Thus, targeting plectin by plecstatins may be considered as a novel and promising anticancer strategy. A time-dependent shotgun proteomics was performed with plecstatins in HCT116 colon carcinoma cells, revealing a correlation of the metallo-prodrug activation status with the changes in drug target selectivity over time.125 According to the target profiling results, up to 450 interactors were identified at 4 h pull-down experiment, while target profiling after 19 h did not reveal significant enrichments, possibly owing to the drug deactivation via arene cleavage. The ruthenium-based anticancer agent BOLD-100/KP1339 has been used as a double-prodrug in several in vitro and in vivo tumor models as well as in early clinical trials.116 By using a multi-omics approach complemented by NanoSIMS and TEM as imaging techniques (Fig. 7B), the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I were found as potential interactors of this ruthenium(III) anticancer agent, which supporting the ribosomal disturbance and concomitant induction of ER stress induced by BOLD-100.126

2.04.3.3

Bismuth

Bismuth drugs such as colloidal bismuth subcitrate (CBS) and ranitidine bismuth citrate (RBC) are being used worldwide in combination with antibiotics for eradicating H. pylori, the leading risk factor for the development of gastric cancer. Moreover, CBS and

Fig. 7 Integrated metallodrug multi-omics approach. (A) Chemical structures of the studied Ru compounds and the derivative with a hydrophilic biotin linker. (B) Workflow of proteomics, transcriptomics and TEM/NanoSIMS to reveal unprecedented details regarding the mode of action of BOLD-100. Adapted with permission from Meier, S. M.; Kreutz, D.; Winter, L.; Klose, M. H. M.; Cseh, K.; Weiss, T.; Bileck, A.; Alte, B.; Mader, J. C.; Jana, S.; Chatterjee, A.; Bhattacharyya, A.; Hejl, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.; Hartinger, C. G.; Keppler, B. K.; Wiche, G.; Gerner, C. An Organoruthenium Anticancer Agent Shows Unexpected Target Selectivity for Plectin. Angew. Chem. Int. Ed. 2017, 56, 8267–8271, and Neuditschko, B.; Legin, A. A.; Baier, D.; Schintlmeister, A.; Reipert, S.; Wagner, M.; Keppler, B. K.; Berger, W.; Meier-Menches, S. M.; Gerner, C. Interaction With Ribosomal Proteins Accompanies Stress Induction of the Anticancer Metallodrug BOLD-100/KP1339 in the Endoplasmic Reticulum. Angew. Chem. Int. Ed. 2021, 60, 5063–5068. Copyright 2017 and 2021 Wiley-VCH.

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related Bi(III) compounds were found to be a class of novel and potent inhibitors of metallo-b-lactamase e.g., NDM-1, via a unique metal displacement mechanism between Bi(III) and Zn(II) ions.127 Recently, Bi(III) drug and compounds have been proved to be potent anti-SAR-CoV-1 and anti-SAR-CoV-2 agents by targeting helicase via an irreversible displacement of zinc(II) ions from the enzyme by bismuth(III) ions.128–130 The uptake of CBS in single H. pylori cells was monitored by time-resolved ICP-MS for the first time.131 Accumulative studies indicated that the binding of bismuth to multiple proteins and enzymes probably contributed to their biological effects.16,132–134 With the assistance of a variety of metallomic and metalloproteomic approaches, the mechanism of action of bismuth drugs against H. pylori have been unveiled at a system level. Using Bi-IMAC in combination with 2DE and MALDI-TOF, seven Bi-binding proteins in H. pylori were identified for the first time.32 The combined use of partial denatured 1D SDS-PAGE with LA-ICP-MS provided more analytical evidence for the presence of Bi-protein complexes in H. pylori cell extracts.52 A robust strategy based on online GE-ICP-MS was established to rapidly acquire the profile of Bi-binding proteins in H. pylori.21 Seven Bi-binding proteins, including five previously reported proteins (UreA, UreB, TsaA, CeuE, and Ef-Tu) and two newly identified proteins (HP1286 and cell binding factor 2) were unequivocally identified (Fig. 8A). The in vitro characterization of binding of these proteins were also made to validate the reliability of the method.32,135 In addition, over 300 nonredundant Bi-binding peptides from 166 proteins in H. pylori were further obtained by Bi-IMAC oncolumn digestion coupled with high-throughput LC-MS, allowing the binding motifs to be identified.136 Small molecule fluorescent probes are powerful tools to visualize biological molecules in living cells and organisms with minimal cell perturbations. A total of 46 bismuth-binding proteins in H. pylori were identified using a newly developed fluorescent

Fig. 8 (A) Profile of Bi-binding proteins in H. pylori measured by continuous-flow GE-ICP-MS. (B) The binding site of urease inhibitors was located at the guanine nucleotide binding pocket. The G1 (P-loop) motif is in magenta, and residues K146 and R179 of UreG are in red, which provide potential hydrophobic interaction with compounds. (C) Bi-influenced H. pylori protein interaction (BiPI) network. Proteins are colored and shaped according to their different properties in the network. Nodes in larger sizes represent hub nodes in the network. (D) The underlying inhibitory mechanisms of bismuth in H. pylori. Adapted with permission from Hu, L.; Cheng, T.; He, B.; Li, L.; Wang, Y.; Lai, Y. T.; Jiang, G.; Sun, H. Identification of Metal-Associated Proteins in Cells by Using Continuous-Flow Gel Electrophoresis and Inductively Coupled Plasma Mass Spectrometry. Angew. Chem. Int. Ed. 2013, 52, 4916–4920, Wang, Y.; Hu, L.; Xu, F.; Quan, Q.; Lai, Y. T.; Xia, W.; Yang, Y.; Yang, X.; Chai, Z.; Wang, J.; Chu, I. K.; Li, H.; Sun, H. Integrative Approach for the Analysis of the Proteome-Wide Response to Bismuth Drugs in Helicobacter pylori. Chem. Sci. 2017, 8, 4626–4633, Yang, X.; Koohi-Moghadam, M.; Wang, R.; Chang, Y. Y.; Woo, P. C. Y.; Wang, J.; Li, H.; Sun, H. Metallochaperone UreG Serves as a New Target for Design of Urease Inhibitor: A Novel Strategy for Development of Antimicrobials. PLoS Biol. 2018, 16, e2003887, and Han, B.; Zhang, Z.; Xie, Y.; Hu, X.; Wang, H.; Xia, W.; Wang, Y.; Li, H.; Wang, Y.; Sun, H. Multi-Omics and Temporal Dynamics Profiling Reveal Disruption of Central Metabolism in Helicobacter pylori on Bismuth Treatment. Chem. Sci. 2018, 9, 7488–7497. Copyright 2013 Wiley-VCH, 2017 and 2018 Royal Society of Chemistry and 2018 PLOS.

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probe-based approach (Bi3þ-TRACER).36 The combination of home-made Bi3þ-TRACER and biochemical techniques enabled the discovery of metallochaperone UreG as a new target for the design of urease inhibitors (Fig. 8B).137 By integration of different metalloproteomics with quantitative proteomics, a comprehensive Bi-binding and Bi-regulated proteome in H. pylori was obtained, with over 60% being annotated with catalytic functions. A giant Bi-influenced protein interaction network in H. pylori was obtained by mapping the bismuth-associated proteins on the protein-protein networks, which gave rise to five hub proteins, i.e., UreA, UreB, RpoA and DnaK (a major heat shock protein); and a down-regulated NusG. UreA and UreB are two subunits of urease, a virulence factor of H. pylori (Fig. 8C).36 This study demonstrated that bismuth disrupts multiple essential pathways in H. pylori, such a unique mode of action may explain the sustainable antimicrobial activity of bismuth drugs against H. pylori and low likelihood to develop resistance to bismuth drugs by H. pylori. Subsequently, other omics approaches, such as transcriptomics and metabolomics were integrated with metalloproteomics to thoroughly examine the cellular responses of H. pylori under the stress of a bismuth drug CBS. It was found that bismuth drug affected multiple metabolic pathways and energy production in H. pylori through disruption of central carbon metabolism.138 Through temporal dynamics profiling, it was found that bismuth initially perturbs TCA cycle prior to urease activity, followed by induction of oxidative stress and inhibition of energy production, inducing extensive down-regulation of H. pylori metabolome (Fig. 8D). Such integrative omics analyzes reveal a system response profile for a deeper understanding of the physiological and pathological roles of metallodrugs.

2.04.3.4

Silver

Silver has been used for the treatment of various maladies or prevention of the transmission of infection for over thousands of years. So far, silver and silver nanoparticles (AgNPs) are being used in a wide range of healthcare, food industry, clinical/pharmaceutical applications, domiciliary applications, and are commonly found in hard surface materials and textiles.139 Based on accumulated evidences, the possible modes of action of silver and AgNPs were proposed, such as cell wall damage, DNA binding, protein interaction, enzyme inhibition, depletion of energy production as well as ROS generation.140 Identification of authentic biomolecular targets of silver could allow a deeper understanding of its mode of action, in turn to extend its therapeutic application including improving the bactericidal efficacy, reducing the toxicity, overcoming the resistance as well as more rationally designing new drugs. Conventional 2DE combined with MALDI-TOF-MS has been extensively applied in the identification of up or down-regulated proteins in different organisms in response to metal compounds. However, only limited information has been obtained due to the compromised separating resolution of 2DE. Using MS-based shotgun proteomics and quantifying peptides with labeling of iTRAQ could be a better option to gain insights into the alteration of protein levels induced by metal compounds.141,142 A GE based method combining native SDS-PAGE, fluorescent staining, and ICP-MS strategy was developed for separation and detection of silver-associated proteins.143 Two well-known silver-associated proteins (GroEL and LDH) and four new silverassociated proteins (AhpC, NADP(þ), ODH, and dThdPase) were successfully identified in P. aeruginosa and S. aureus treated with AgNPs. The molecular mechanisms of antimicrobial activity of AgNPs in P. aeruginosa were investigated using a metalloproteomics approach.144 The results clearly suggested that AgNPs interfere with the cell membrane function and generate intracellular reactive oxygen species (ROS), which are the main pathways perturbed by AgNPs. Both the nanoparticles themselves and the silver ions released from AgNPs play a crucial role. By using GE-ICP-MS, five silver-binding proteins were identified in P. aeruginosa treated with either Agþ or AgNPs, which implies that AgNPs interact with protein targets in vivo through the release of silver ions. In addition, AgNPs were demonstrated to be a new potential trigger of metal allergy, which induced acquired immune responses depended on CD4þ T cells and elicited IL-17A-mediated inflammation.145 To further enhance the resolution of protein separation, LC-GE-ICP-MS was developed by combining one-dimensional GE-ICPMS with LC, allowing the proteins to be separated by LC and GE according to their differences on pIs and molecular weights, respectively.22 In total, 34 Ag-binding proteins (Ag-proteome) in E. coli in the whole-cell scale were separated, identified, and validated for the first time by using LC-GE-ICP-MS approach (Fig. 9A). The combination of metalloproteomics, metabolomics, bioinformatics, and bioassays demonstrated that silver disrupted cellular respiration, including TCA cycle, glycolysis and electron transport chain as well as antioxidant system to induce production of ROS. Among the identified Ag-binding proteins in E. coli, glyceraldehyde-3phosphate dehydrogenase (GAPDH), an essential enzyme in glycolysis, was validated to be a vital target of Ag (Fig. 9B).146 The structure of the Ag-binding protein GAPDH was studied by X-ray crystallography, and it was found that silver ions abolish the enzymatic function through targeting Cys149 in its catalytic site (Fig. 9C). Moreover, detailed characterization of silver binding to its authentic target, malate dehydrogenase (MDH) showed that silver binds MDH at multiple sites including three cysteinecontaining sites, and binding preference to these sites were further delineated.147 Such an integrated-omic approach can be potentially applied to explore the molecular targets and mechanisms of action of other metal-based antimicrobial and anticancer drugs, and thereby facilitating the development of new therapeutics.

2.04.3.5

Gold

Gold-based drugs have been clinically used for the treatment of arthritis decades ago, but there has been a significant resurgence of interest in the anticancer activities of gold compounds in recent years, due to their strong antiproliferative potency. So far, a variety of structurally diverse Au(I) and Au(III) compounds have been investigated for their antitumor properties in vitro and in vivo,

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Fig. 9 (A) Map of Ag-associated proteins in E. coli. (B) GE-ICP-MS electropherograms of purified GAPDH with pre-incubation of Agþ. (C) Crystal structure of Ag-GAPDH with Agþ (gray sphere) binding at Cys149/His176 site. Green, red, blue and yellow represent carbon, oxygen, nitrogen and sulfur atoms, respectively. Adapted with permission from Wang, H.; Yan, A.; Liu, Z.; Yang, X.; Xu, Z.; Wang, Y.; Wang, R.; Koohi-Moghadam, M.; Hu, L.; Xia, W.; Tang, H.; Wang, Y.; Li, H.; Sun, H. Deciphering Molecular Mechanism of Silver by Integrated Omic Approaches Enables Enhancing Its Antimicrobial Efficacy in E. coli. PLoS Biol. 2019, 17, e3000292, and Wang, H.; Wang, M.; Yang, X.; Xu, X.; Hao, Q.; Yan, A.; Hu, M.; Lobinski, R.; Li, H.; Sun, H. Antimicrobial Silver Targets Glyceraldehyde-3-Phosphate Dehydrogenase in Glycolysis of E. coli. Chem. Sci. 2019, 10, 7193–7199. Copyright 2019 PLOS and 2019 Royal Society of Chemistry.

mostly showing promising preclinical results.148,149 Auranofin (Fig. 10A), an FDA-approved antirheumatic drug, has shown potent bactericidal activity against Gram-positive pathogenic bacteria.150 Importantly, this drug has been shown potentials to resensitizing carbapenem and colistin-resistant bacteria to antibiotics owing to its capability of inhibiting both MCR-1 and NDM-1 resistance enzymes via the displacement of Zn(II) cofactors by Au(I) (Fig. 10B).151 It has been proposed that the biochemical mechanisms of these compounds are DNA-dependent, but the interactions with proteins and enzymes (e.g., thioredoxin reductase) in cells are also implicated by experimental evidence.150,152 Au(III) porphyrins were regarded as a promising lead for anticancer development, exhibiting in vitro cytotoxicity against a panel of cancer cell lines, including multidrug- and cisplatin-resistant cancer cells.153 Functional proteomic studies revealed that Au(III) porphyrin 1a induced apoptosis through both caspase-dependent and caspase-independent mitochondrial pathways, and regulated a number of cytoplasmic proteins mainly involved in cellular redox balance and energy production.154 The proteomic alterations induced by two representative gold compounds, Auranofin and Auoxo6, in cisplatin-sensitive and resistant human ovarian cancer cell lines (A2780/S and A2780/R) were analyzed by 2DE and MALDI-TOF-MS.155,156 Proteins involved in cellular redox homeostasis were frequently identified to be regulated by gold in these studies, implying that the cell damage is probably the consequence of severe oxidative stress induced by gold. Another proteomic study identified the glycolytic pathway as the primary target of an Au(III) complex in A2780 ovarian cancer cells.157 Recently, a distinct thiol-targeting property of Au(III) mesoporphyrin IX dimethyl ester (AuMesoIX) was shown to modify reactive cysteine residues and inhibit the activities of anticancer protein targets including thioredoxin, peroxiredoxin, and deubiquitinases (Fig. 10C).158 Photoaffinity labeling with click chemistry is a valuable technique for studying the interaction of ligands with their target proteins. Che et al. synthesized a clickable photoaffinity probe of [Au(TPP)]Cl (probe-1, Fig. 10A) by appending a linker, a clickable tag, and a photoaffinity tag onto the meso-phenyl rings of the porphyrin ligand to isolate the protein binding partner(s) of Au(III).159 The chaperone protein heat-shock protein 60 (Hsp 60) was identified as a direct target of probe-1 a through the combination of a photo-affinity and click chemistry approach. The N-heterocyclic carbene (NHC) ligands have strong donor strengths in stabilizing metal compounds to resist physiological reductants.160 Metal-NHC complexes (Au, Pt and Pd) have been developed as effective alternatives to classical metallodrugs, due to their potent cancer cell cytotoxicity, anti-tumor activities in animal models as well as selective binding to molecular targets.123,161 A chemical probe, Au(III) NHC compound (probe-2 and probe-3, Fig. 10A), was synthesized by introducing a small photoaffinity diazirine group and a clickable alkyne moiety onto NHC, which led to the identification of the cellular targets of the compound (Fig. 10D).162 By using the probe-2, six photoaffinity-labeled (biotinylated) proteins were detected and identified simultaneously in the lysates of HeLa, NCI-H460 and HCT116 cells. The identified proteins have all been described as potential anticancer targets, and the anticancer effects of the Au(III) NHC compound resulting from the multitarget binding were verified by proteomic analysis. Remarkably, the Pt(II) and Pd(II) NHC analogs were found to bind to the Au(III) NHC target proteins, highlighting the particularity of pincer cyclometalated metal–NHC scaffold in their anti-cancer activities. Therefore, metal-NHC complexes are promising candidates for anti-cancer medicines with multiple molecular targets and low drug resistance.

2.04.3.6

Arsenic

Arsenic is one of the double-edged sword elements, in one way, it has been confirmed as a human carcinogen and can cause various health effects with chronic exposure; on the other hand, arsenic trioxide (ATO), is a chemotherapeutic agent for the clinical remission of acute promyelocytic leukemia (APL).163 XRF and XAS analyzes were performed on single HepG2 cells following exposure to arsenite or arsenate, in an effort to determine their intracellular distribution and biomolecular targets.164 The XRF elemental

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Fig. 10 (A) Chemical structures of auranofin and the clickable photoaffinity probes (probe-1, 2 and 3). (B) Structure of the active site of Au-MCR-1S (PDB ID: 6LI6, Au shown as a purple sphere) and Au-NDM-1 (PDB ID: 6LHE, Au shown as yellow spheres). (C) Proposed reaction of AuMesoIX with thiol-containing biomolecules under physiological conditions. (D) Schematic of procedure to identify cellular protein targets with probe-2. Adapted with permission from Sun, H.; Zhang, Q.; Wang, R.; Wang, H.; Wong, Y. T.; Wang, M.; Hao, Q.; Yan, A.; Kao, R. Y. T.; Ho, P. L.; Li, H. Resensitizing Carbapenem- and Colistin-Resistant Bacteria to Antibiotics Using Auranofin. Nat. Commun. 2020, 11, 5263, Tong, K. C.; Lok, C. N.; Wan, P. K.; Hu, D.; Fung, Y. M. E.; Chang, X.; Huang, S.; Jiang, H.; Che, C. M.An Anticancer Gold(III)-Activated Porphyrin Scaffold That Covalently Modifies Protein Cysteine Thiols. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (3), 1321–1329, Hu, D.; Liu, Y.; Lai, Y. T.; Tong, K.C.; Fung, Y. M.; Lok, C. N.; Che, C. M. Anticancer Gold(III) Porphyrins Target Mitochondrial Chaperone Hsp60. Angew. Chem. Int. Ed. 2016, 55, 1387–1391, and Fung, S. K.; Zou, T.; Cao, B.; Lee, P.; Fung, Y. M. E.; Hu, D.; Lok, C. N.; Che, C. M. Cyclometalated Gold(III) Complexes Containing N-Heterocyclic Carbene Ligands Engage Multiple Anti-Cancer Molecular Targets. Angew. Chem. Int. Ed. 2017, 56, 3892–3896. Copyright 2016 Wiley-VCH, 2020 Springer Nature, 2020 National Academy of Sciences and 2017 Wiley-VCH.

mapping clearly showed the accumulation of arsenic in the nucleus, indicating arsenic binding to DNA or proteins involving in DNA transcription. The XANES and EXAFS analyzes demonstrated the predominance of arsenic tri-sulfur species in arsenitetreated cells, providing increased evidence of arsenic coordinating to sulfur-rich proteins. By using ICP-MS, the intrinsic relationship between As accumulation and the corresponding cytotoxicity in single leukemia cells are elucidated,165 and the cell-cycle dependent cellular uptake of arsenic-based drugs was demonstrated.166 The binding of arsenic to proteins may play a key role in its metabolism and modulate the activities of key regulatory proteins.167 Affinity chromatography,168,169 fluorescent arsenical probes,170–173 biotinylated arsenical pull-down approaches174,175 and GEICP-MS176 have been successfully applied to search for arsenic-binding proteins in the cellular environment. These approaches have resulted in large quantities of proteins to be identified as arsenic binding proteins, however, the authentic protein targets remained to be elusive. A targeted proteomic approach involving arsenical affinity chromatography and tandem mass spectrometry was developed to identify specific arsenic-binding proteins in cancer cell proteome.168 50 proteins in the nuclear fraction and 24 proteins in the membrane/organelle fraction of A549 human lung carcinoma cells were successfully identified, adding more candidates to the current list of arsenic-binding proteins. A number of the identified arsenic-binding proteins are of important biological functions, involved in DNA repair, modulating cell redox status and apoptosis. A panel of arsenite-binding proteins were systematically identified from the Chinese hamster ovary cell line CHOA (arsenic-sensitive) and SA7 (arsenic-resistant) using a PAO (p-aminophenylarsine oxide)-agarose matrix.169 19 proteins were differentially expressed in CHOA or SA7 cells, which could be functionally categorized into metabolic, stress and developmental processes. The functional role of sulfhydryl groups on arsenic binding was further examined by choosing four identified proteins with various cysteine residues. An azide-labeled arsenic probe, p-azidophenylarsenoxide (PAzPAO), was designed for the capture of arsenic-binding proteins and identification by shotgun proteomics (Fig. 11A).172 Among the 48 arsenic-binding proteins identified in A549 cells, the two most abundant proteins are thioredoxin and peroxiredoxin-1. GAPDH has also been found to bind to arsenic, which could be

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Fig. 11 (A) Capture and identification of arsenic-binding proteins inside cells by using an azide-labeled arsenic probe. Ribbon diagram of GAPDH monomer (PDB code IU8F). Sulfur atoms in the side chains of three cysteines (C152, C156 and C247) are indicated as yellow balls. C152 and C156 are the two possible vicinal thiol groups that bind to PAzPAO. (B) Schematic of the procedure for detecting arsenic-binding proteins by arsenic–biotin conjugate (Biotin–As) and Cy3-conjugated streptavidin (Cy3–SA). (C) ZIO-101 binds to histone H3.3 in vitro and in cellulo. Binding ratio between ZIO-101 and histone H3.3 analyzed by MALDI-TOF-MS (left). Cellular thermal shift assay of histone H3.3 in NB4 cells after treatment with ZIO-101 (right). Adapted with permission from Yan, X.; Li, J.; Liu, Q.; Peng, H.; Popowich, A.; Wang, Z.; Li, X.; Le, X. C. p-Azidophenylarsenoxide: An Arsenical “Bait” for the In Situ Capture and Identification of Cellular Arsenic-Binding Proteins. Angew. Chem. Int. Ed. 2016, 55, 14051–14056, Zhang, H.; Yang, L.; Ling, J.; Czajkowsky, D. M.; Wang, J.; Zhang, X.; Zhou, Y.; Ge, F.; Yang, M.; Xiong, Q.; Guo, S.; Le, H.; Wu, S.; Yan, W.; Liu, B.; Zhu, H.; Chen, Z.; Tao, S. Systematic Identification of Arsenic-Binding Proteins Reveals That Hexokinase-2 is Inhibited by Arsenic. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 15084–15089, and Xu, X.; Wang, H.; Li, H.; Hu, X.; Zhang, Y.; Guan, X.; Toy, P. H.; Sun, H. S-Dimethylarsino-Glutathione (Darinaparsin®) Targets Histone H3.3, Leading to TRAIL-Induced Apoptosis in Leukemia Cells. Chem. Commun. 2019, 55 (87), 13120–13123. Copyright 2016 Wiley-VCH, 2015 National Academy of Sciences and 2019 Royal Society of Chemistry.

a potential molecular target for arsenic-based drugs, by blocking the hyper-active glycolytic pathway of cancer cells. A human proteome microarray containing 16,368 affinity-purified N-terminally GST-tagged proteins with a biotinylated arsenic molecule was applied for systematic identification of arsenic-binding proteins.173 The arsenic-biotin conjugate, biotinylated

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p-aminophenylarsenoxide (Biotin-As), was incubated with the human proteome microarray, and arsenic-binding proteins were fluorescently revealed by adding Cy3-conjugated streptavidin (Fig. 11B). In this way, 360 arsenic-binding proteins were identified, among which proteins involved in glycolysis are significantly enriched. Arsenic-binding proteins in APL cells were pulled down with streptavidin and identified using LC-MS/MS, which led to more than 40 arsenic-binding proteins were separated and further studied.175 Notably, pyruvate kinase M2 (PKM2), as a high affinity arsenic binding protein, was involved in the arsenic-mediated APL suppressive effects. S-Dimethylarsino-glutathione (darinaparsin, ZIO-101) is a novel organic arsenic agent with broader antitumor activity and less toxicity than ATO. By using GE-ICP-MS strategy, histone H3.3 was identified as the first arsenic-binding protein in the nuclei of leukemia cells treated with ZIO-101 (Fig. 11C).176 Further study revealed that ZIO-101 binds to histone H3.3 via cysteine residue, which disrupts the formation of histone H3.3 dimers and facilitates the denaturation of nucleosomes. The results demonstrated histone H3.3 as a vital target for ZIO-101, providing significant information for the distinct molecular mechanisms and therapeutic effects of organic and inorganic arsenic drugs. Subsequently, it has been shown that ZIO-101 exerted antiproliferative effects against leukemia cells via activating ferroptosis pathway, a newly discovered iron-dependent programmed cell death, at the early stage as evidenced by abnormally elevated intracellular iron contents and lipid peroxidation. The findings on ferroptosis-mediated anti-leukemia activity of darinaparsinÒ based on the dynamic and temporal transcriptomic analysis provide promising approaches to combat drug-resistant leukemia by combination of ZIO-101 with kinase inhibitors, and the methodology might be further exploited for uncovering the modes of action of other drugs.177 Recently, an integrated-omic study showed that arsenene mainly affected the nuclear proteins in the NB4 cells and targeted the nuclear protein TXNL1. Further mass spectrometric and cellular fluorescence studies revealed that the bound proteins of arsenene in the NB4 cells were mainly 106 nuclear nucleotide acid binding proteins (such as HMGN2 and SF3B1) and the arsenene destroyed the nuclei, leading to the formation of nuclear fragments.178

2.04.4

Application of metallomics and metalloproteomics for environmental health and toxicology

Trace elements, including metallic elements and metalloid elements, coordinate with diverse classes of proteins and enzymes as the active or structure centre. The essential transition metals (e.g., Fe, Cu, Zn, Mg, Mo, Ni and Se) provide catalytic, regulatory, and structural roles, while the exogenous ones (e.g., Hg, Pb, Cd, Sn and Cr) usually possess no particular biological function and are toxic to human and other organisms even at low concentrations. The molecular basis of metal mediated biochemical processes has been extensively studied to elucidate the biological essentiality and toxicity of elements. Serious concerns have been raised on the risk assessment of toxic metals, including their high toxicity at a low level of exposure and the safety threshold of metals in environment and commodities.179 Analytical techniques applied to metallomics and metalloproteomics provide the advantage of highthroughput analysis, can be utilized to study the environmental metallomics, including quantification, distribution, speciation and structure.180 In this case, the use and development of comprehensive and global methodologies in environmental metallomics, would provide a systematic and complete view on the metal pollution in environmental health and toxicology.

2.04.4.1

Mercury

Mercury is a widespread environmental contaminant, which can elicit toxic effects to central nervous system, kidney, and liver. Having high affinity for thiol and selenol groups, mercury is likely to bind to various proteins that contain sulfhydryl groups (-SR). Characterization of mercury binding proteins in human body is very important to understand the metabolism and the mechanism of toxication of the ingested mercuric compounds. The Hg-containing protein fractions in the brain cytosol of the maternal and infant rats after exposure to a low-dose of methylmercury were analyzed by a SEC-ICP-MS method with the postcolumn isotope dilution analysis.181 A method based on immobilized mercury ion affinity chromatography was developed for the identification of putative Hg-binding proteins in human neuroblastoma SK-N-SH cells.182 In total, 38 proteins were identified as Hg-binding proteins, in which most of them were mainly involved in protein processing in endoplasmic reticulum, protein folding, and cytoskeleton organization. Human serum albumin (HSA) was first separated and identified as a Hg-binding protein in human plasma by an on-line heartcutting 2D-HPLC-ICP-MS system.27 This 2D separation system used SEC followed by weak anion exchange liquid chromatography (WAX) and the two LC parts were coupled by a six port valve equipped with a storage loop and controlled by the computer. The binding mechanism of Hg2þ to HSA was investigated under simulated physiological conditions utilizing steady-state/time-resolved fluorescence spectroscopy, UV-vis absorption spectroscopy, CD spectroscopy, and X-ray photoelectron spectroscopy (XPS) (Fig. 12A).183 Hg2þ was preferably bound to cysteine and cystine in HSA, where the R–S–S–R structure is responsible for maintaining the protein’s structure by stabilizing the a-helical bundles. Hg-binding protein profiles in rat plasma treated with HgCl2 in vitro and in vivo were systematically investigated using GE-ICPMS (Fig. 12C).184 Hemoglobin, glutathione peroxidase 3, albumin, and selenoprotein P were found to be the main Hg-binding proteins both in vitro and in vivo at different mercury exposure concentrations (100–1000 ng mL 1). At higher HgCl2 concentration (10,000 ng mL 1) in vitro, more proteins other than those four proteins were detected, including apolipoprotein A-I, apolipoprotein E, apolipoprotein A-IV, albumin, and transferrin. In a recent study, 2D-PAGE coupled with graphite furnace atomic absorption spectrometry (GFAAS) and ESI-MS/MS were used for the identification of mercury–bound proteins in samples of muscular and hepatic tissue from fish (Fig. 12B).185 Triosephosphate isomerase A and Protein FAM45A were identified as

70 Metallomics and metalloproteomics Fig. 12 (A) Hg 4f XPS spectra of the HSA-Hg2þ system. (B) 2D-PAGE and GFAAS analysis showing the mercury-bound proteins in liver of dourada. The spots marked in red are the ones that present mercury, along with the spots in 3D (spot selected is green). (C) Detection of mercury-binding proteins in rat blood plasma at different levels of mercury exposure in vitro and in vivo by GE-ICP-MS. Adapted with permission from Cavecci-Mendonça B; de Souza, Vieira JC; de Lima PM; Leite AL; Rabelo Buzalaf MA; Zara LF; de Magalhães Padilha P (2020) Study of proteins with mercury in fish from the Amazon region. Food Chem. 309: 125460, Song, S.; Li, Y.; Liu, Q.; Wang, H.; Li, P.; Shi, J.; Hu, L.; Zhang, H.; Liu, Y.; Li, K.; Zhao, X.; Cai, Z. Interaction of Mercury Ion (Hg2þ) With Blood and Cytotoxicity Attenuation by Serum Albumin Binding. J. Hazard. Mater. 2021, 412, 125158, and Li, Y.; He, B.; Nong, Q.; Qu, G.; Liu, L.; Shi, J.; Hu, L.; Jiang, G. Characterization of Mercury-Binding Proteins in Rat Blood Plasma. Chem. Commun. 2018, 54, 7439–7442. Copyright 2020 and 2021 Elsevier and 2018 Royal Society of Chemistry.

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mercury–bound proteins in fish from the Amazon region, revealing the possibles existing physiological and cellular interactions induced by mercury. In another study, salmon and tuna fish muscles, collected from seven different regions and countries, were analyzed using metallomics and proteomics approaches.186 Beta-actin was separated and identified as a novel Hg-binding protein from the fish muscles by SEC-ICP-MS, and this protein could also bind to other metals, such as Bi, Ag, and Cu. A capillary single-cell ICP-MS online system was developed to track the accumulation of Hg in individual unicellular Tetrahymena.187 This novel method has the advantages of high sensitivity and real-time detection at single-cell level, which can further be used to track the accumulation of other metal compounds.

2.04.4.2

Lead

The level of Pb in the environment, as a notorious mutagenic and teratogenic toxin, has been increased more than 1000 times due to anthropogenic activities over the past three centuries. Metal contaminants are difficult to remove from the environment, and unlike most of organic contaminants, they cannot be chemically or biologically degraded and are ultimately indestructible. To search biological factors against naturally occurring species, including proteins, lipids and genetic basis, which are involved in metal accumulation or biosorption, is especially important to the remediation and detoxification of toxic metals. A new method combined CE with electrothermal atomic absorption spectrometry (ETAAS) was developed to measure the binding sites and equilibrium constants between Pb(II) and DNA, providing a reliable and convenient way to study the interactions between metal ions and DNA.188 Although a large variety of Pb tolerant strains were isolated from various environmental niches, microbial Pb-binding proteomes have not been well characterized. GE-ICP-MS was further utilized for identifying the Pbbinding protein in Pb resistant bacterial strain.189 Flagellin was found to be a novel Pb-binding target, which was helpful in understanding the molecular mechanism of Pb tolerance and biosorption. Understanding the presence and dynamics of chemical pollutants in individual cells is fundamentally important for their trafficking, fate, and toxicity in humans. Based on mass cytometry, a method for the determination of metal content in single cells was established.190 Pb content in individual mature erythrocytes of Pb poisoning patients was analyzed, revealing the distribution and dynamics of metals at the single-cell level of higher organisms.

2.04.4.3

Cadmium

Cadmium is a highly toxic non-essential trace element that is widely distributed in nature due to its use in multiple industries such as battery, pigments, coatings, plating and plastics. Cadmium can accumulate in animals and human beings through the consumption of cadmium containing plants, especially rice. MT is a family of cysteine-rich, low molecular weight proteins, which shows high binding ability to Cadmium.191 Cadmium affinity chromatography coupled with LA-ICP-MS has been used to separate and identify Cd-binding proteins from different parts (root, stem, leaf and grain) of cadmium polluted rice (Oryza sativa L.).192 As a result, seven Cd-binding proteins with low isoelectric points containing relatively few cysteine residues were identified from rice plant. These new Cd-binding proteins could bind Cd2þ via electrostatic interactions under neutral conditions (pH ¼ 7), which may play an important role in accumulation of cadmium from the environment for bioremediation applications. Recently, Cd-binding protein was identified in human plasma by using GE-ICP-MS system, which offers new information for the transportation and accumulation of cadmium in human blood.193 Apolipoprotein A-I was firstly recognized as Cd-binding protein, and presented in both adult human plasma and umbilical cord plasma from normal populations exposed to cadmium at low environmental levels.

2.04.5

Summary and outlook

Metallomics and metalloproteomics are becoming increasingly important areas of research within biology systems and provide information on the identification, quantification and function of metalloproteins. The rapid development of high-throughput analytical techniques has enabled a number of novel metallomics and metalloproteomics approaches available, in combination with biotechnology and bioinformatics tools, environmental science, inorganic chemistry, proteomics and toxicology methodologies, a large number of data could be generated for subsequent validation and further investigation. In recent years, metallomics research has also spawned some new branches of omics, such as environmental metallomics, nano-metallomics and pathological metallomics. The development of these novel interdisciplinary disciplines facilitates the study of not only the biological activity of metal elements, but also metal-related ecological environment system and human health risk assessment. At present, there are still many aspects to be improved in metallomics and metalloproteomics researches, both in methodology and mechanism study. Attention should be paid to the process of separation and analysis, in order to preserve the whole information of metal-protein complexes in situ or under physiological conditions. In future, innovative technologies, such as deep learning-based artificial intelligence methods and integration of multi-omics, should be generalized in a larger scope, so that a holistic picture of the full system response profile of cells or organisms toward metals will be elucidated.

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2.05

Biomineralization

Yueqi Zhaoa, Biao Jinb, and Ruikang Tanga,c, a Center for Biomaterials and Biopathways, Department of Chemistry, Zhejiang University, Hangzhou, China; b Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA, United States; and c Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China © 2023 Elsevier Ltd. All rights reserved.

2.05.1 2.05.2 2.05.2.1 2.05.2.2 2.05.2.2.1 2.05.2.2.2 2.05.2.2.3 2.05.3 2.05.3.1 2.05.3.2 2.05.3.3 2.05.4 2.05.4.1 2.05.4.2 2.05.4.3 2.05.5 2.05.5.1 2.05.5.2 2.05.5.3 2.05.5.4 2.05.5.4.1 2.05.5.4.2 2.05.6 Acknowledgment References

Introduction Crystallization in biomineralization Classical crystallization Nonclassical crystallization Amorphous precursor Phase-transformation-based crystallization Nano attachment Organic matrix and its regulation effect Organic-inorganic interface Template effect Confinement effect Application of biomineralization for tissue regeneration Collagen mineralization Tooth repair Bone repair Organism improvement Artificial shell Bioenergy Environmental protection Biomedical therapy Vaccine improvement Cancer treatment Conclusion

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Abstract The hierarchically structured biominerals with excellent functions are produced by the regulation of organisms via biomineralization. The organic matrix and molecules regulate the inorganic mineralization to fabricate the delicate structural materials with the optimized properties. Inspired by natural biomineralization process, may biomimetic tactics have been developed for applications for materials and biomedicines, such as collagen re-mineralization, tooth and bone repairs. Biomineralization always highlights the control of inorganic materials by using organisms; reversely, the mineralized materials can also regulate or improve the living organisms. By conferring materials on to organism, the rationally designed organism-material hybrids can be created artificially, which are featured by their improved or new functions such as cell protection, bioenergy production, vaccine modification and cell treatment. Such a combination follows a materials-based biological modification, which would contribute a more comprehensive view of biomineralization as well as a new window for biological inorganic chemistry.

2.05.1

Introduction

Since the beginning of life on Earth, living organisms have played an important role in the formation of minerals in nature, this process is known as Biomineralization.1 The products of biomineralization, biominerals, are widespread in the world, such as egg shells, vertebrate bones and teeth, kidney stones, coral skeletons, mollusk shells, pearl, pelagic shells, otolith, pelagic and diatom shells, ivory and antlers, etc.2–5 They can be either physiological or pathological mineralization in the living organisms. The physiological mineralization is the ideal result of biomineralization in bone, tooth and growth plate cartilage.6 For example, the hydroxyapatite is the major inorganic composition in bones, which is mineralized by osteoblast and distributed within collagen fibers.7 The basic building blocks of hydroxyapatite are needle-liked nanoparticles, distributing within collagen fibers.8 The hydroxyapatite-collagen hybrid structure further assembles to a more complex hierarchical structure, endowing the excellent

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mechanical properties of bones.9 However, if excess hydroxyapatite is ectopic calcified in the cartilage, which is a soft and less mineralized tissue in arthrosis, it damages the original structure of cartilage and induces the arthritis.10 This abnormal result of uncontrolled regulation in biomineralization named pathological mineralization.11 The understanding of biomineralization mechanisms helps us to regulate and control the biominerals. In current understanding, the growth of many inorganic biominerals rely heavily on biologically generated organic matrices, and the process includes nucleation,12 growth,13 phase transformation,14 polymorph and orientation evolution.15 The amorphous and crystalline biominerals can be formed with the precise regulation in biological environments, which can be as simple as a few random sediments or as complex as elaborate multi-level structures.16 The precise architecture contributes to their superior strength and toughness, allowing them to construct various inorganic-organic materials with specific functions, such as skeletal support, gravity sensors, optical properties, soft tissue protection and so on.17 Currently, due to the advantages of biomineralization production, more and more biomimetic strategies based on biomineralization are emerged for biological applications, including tooth repair,18 bone repair,2 pathological prevention19 and biomedical therapy.20 Moreover, the nature-inspired materials can be created as well, such as nacre-like materials,21 abiotic tooth enamel22 and so on. These biomimetic artificial materials can be fabricated by imitating the natural biological processes, which also have the similar multiscale architecture and mechanical properties with natural minerals. It is notable that the regulation of organic matrices is essential for the biomimetic mineralization in these approaches.15 Meanwhile, for the living organisms, although some of them can spontaneously mineralize in natural environment, many other organisms with special functions cannot achieve by themselves.23 In order to better utilize their functions more and more tactics of biomimetic mineralization on ling organisms develop to actualize biological improvement.24,25 In recent years, these researches by integration between biological organisms and biomimetic materials gradually advance from initial shellization of single cell to modification of multicellular aggregation, expanding to bioenergy development, environment protection and biomedicine therapy.26–28 Therefore, this chapter systematically classifies and sorts out the mechanism of biomineralization process, applications of biomimetic mineralization and biological improvement by biomimetic materials, finally summarizes and analyzes the current problems and future challenges.

2.05.2

Crystallization in biomineralization

Learning from nature, biomineralization can produce many delicate structures with remarkable functions. Understanding the fundamental principles of biomineralization to design novel biomimetic materials or applications is believed to be a successful and well-employed strategy. Biomineralization can be referred to be a complicated formation process of inorganic minerals involving the crystallization regulation of organic molecules or inorganic ions on inorganic crystals.29 Therefore, the mechanistic understanding of crystal nucleation and growth is critically important to comprehend the biomineralization. Generally, mineral formation process can be divided into the classical and nonclassical pathways based on their basic units: either monomers or particles (Fig. 1). Compared to thermodynamic favorable crystallization pathway, the system prefers to choose a kinetic favorable pathway, as depicted in the energy landscape shown in Fig. 1A, explaining the emergence of metastable precursors and intermediates under kinetically controlled crystallization.

Fig. 1 Crystal nucleation and growth mechanism. (A) Schematic free energy landscape for a variety of possible crystallization pathways from ions in solution. (B) Schematic illustration of proposed mechanisms of the minerals. (I, II) Crystalline nuclei can form and grow into crystals through ionby-ion attachment. (III) Direct formation of amorphous particles via ion attachment. (IV) Counter ions association into prenucleation complexes (PNCs, ionic oligomers), followed by their aggregation into DLP or amorphous particles. (V) Liquid-liquid phase separation to form a DLP, followed by their dehydration or nucleation into solid hydrated amorphous particles. (VI) The phase transformation of amorphous particles into crystals via solid-state transformation. (VII) The phase transformation of amorphous particles into crystals through dissolution–recrystallization. (VIII) The nonclassical crystal growth via particle attachment.

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Classical crystallization

Nucleation is one of the initial crystallization processes whereby nuclei act as embryos.30 Classical nucleation theory is applied to describe the nucleation of many crystal growth processes.31,32 In classic nucleation theory, the total free energy (DG) of a nanoparticle is affected by both the free energy of the bulk (DGv) and the free energy of the interface (DGs). Although DGv is reduced for crystal nucleation in supersaturated solution, DGs is increased because of the crystal-solution interface formation. Although both DGv and DGs are relevant to nuclei size (radius, r), the different powers of DGv (f r3) and DGs (f r2) result in the changes of DG, which is shown in the Fig. 2.30 When r reaches the critical radius of crystal nucleus (rc), the maximum free energy (critical free energy, Gcrit) is reached in the crystal nucleation process. Both the degree of supersaturation and solid-liquid interface energy can affect the formation of nuclear energy barrier, which is the thermodynamic driving factor of crystallization. However, the formation of nuclear energy barrier can be dynamically regulated by crystal-solution interface energy, which plays a key role in the regulation of nucleation.33 According to the condition of heterogeneous solution, it can be divided into homogeneous nucleation and heterogeneous nucleation.34 Homogeneous nucleation occurs in which the crystals uniformly form new nuclei in the parent phase system.35 It is generally believed that the homogeneous nucleation can be considered by thermodynamic model.36 On the contrary, heterogeneous nucleation forms at structural inhomogeneities, which is generally occurring on the surface of different phases, impurities, grain boundaries and dislocation. When the crystalline phase is formed on the substrate, the crystal-solution interface energy is increased, but the matrix-solution interface energy is decreased at the same time. Therefore, the interface free energy should be adopted, which is related to the contact angle (q) of the solution-matrix-crystal interface.30 In biomineralization, the heterojunctions are often organic substrates and the heterogeneous nucleation is more likely to occur in liquid phase due to the presence of a variety of surface prone to nucleation.37 It can be considered that the organic matrix is the heterogeneous nucleation site in the mineralization process, which can induce mineral nucleation and control mineral phase, nucleation spatial distribution and crystal orientation. After crystal nucleation, the crystal will continue to grow in supersaturated solution. The ionic monomers of the mineral crystal firstly diffuse to the crystal surface and then enter into the lattice structure to realize the growth on the crystal surface as the traditional understanding.30 With the improvement of atomic force microscopy (AFM), scientists have confirmed that the growth of crystal surface is not simply a process of ion-bonded layer growth, but actually grows on a step of crystal surface and eventually makes the whole crystal grow through the step growth and movement.38 When ions are close to the crystal surface, there are more bonds formed at kick than at the terrace or step, so that steps are more likely to occur than continuous planes in crystal growth. Because of the fewer bonds among ions at the terrace, the crystal dissolution is relatively easy to occur and the crystal surface will appear etch pits.39 Due to the anisotropy of crystal, there will be exposed different crystal surface. Living organisms regulate the crystal growth and select the corresponding crystal surface, which is actually achieved by the control of step growth.40 Peptides and proteins play an important role in realizing this regulation.41,42 Some chiral organic molecules have been used to regulate the step growth on the surface of calcite crystals (Fig. 3).43 Different from symmetrical step growth in pure solution, steps on the surface of calcite can grow in a specific orientation in the presence of L- or D-aspartic acid (Asp). This microscopic step growth also affects the macroscopic growth of crystals and eventually form calcite single crystals with different orientations and morphologies. The formation of organic-inorganic compound is not observed during this growing process of calcite, and it is confirmed that the enantiomerspecific binding of Asp control morphology and orientation by changing the step-edge free energies.

Fig. 2 The free energy diagram for nucleation to explain the existence of a “critical nucleus.” Reproduced from Thanh, N.T.K.; Maclean, N.; Mahiddine, S., Chem. Rev. 2014, 114, 7610–7630.

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Fig. 3 (A–F) The AFM images to show the effect of amino acids on growth-hillock and dissolution-pit geometry. (A) A pure calcite growth hillock. Growth hillocks following addition of supersaturated solutions with (B) 0.01 M glycine; (C) 0.01 M L-aspartic acid (Asp); and (d) 0.01 M D-Asp. (E and F) Dissolution pits undersaturated with respect to calcite and saturated with respect to L- and D-Asp respectively. The sizes of images are: (A) 3.5  3.5 mm; (B) 3  3 mm; (C) and (D) 15  15 mm; (E) 10  10 mm; and (F) 5  5 mm. (G and H) The SEM micrographs of calcite crystals nucleated on COOH-terminated regions of patterned self-assembled monolayers of alkane thiols and respectively growing in the presence of 0.01 M L-Asp and D-Asp. Reproduced from Orme, C.A.; Noy, A.; Wierzbicki, A.; McBride, M.T.; Deyoreo, J.J., Nature 2001, 411, 775–779.

2.05.2.2

Nonclassical crystallization

Beside the classical crystallization theory through monomer diffusion and adsorption, nonclassical nucleation and growth by particle attachment occurs by the addition of higher-order species ranging from prenucleation clusters (PNCs) or complexes, dense liquid phase (DLP), amorphous particles, primary particles to nanocrystals (Fig. 1B).44–48 Among of them, amorphous phase is one of the most common and important precursor for various minerals, such as amorphous calcium carbonate (ACC),49 calcium sulfate (ACS),50 calcium phosphate (ACP)51 and calcium oxalate (ACO).52 The simplest formation pathway is that amorphous nuclei directly form through the attachment of ions in bulk solution,53 as described by classical nucleation theory. However, different types of intermediate species (PNCs or complexes, DLP and even ionic oligomers) have been proposed before the amorphous phase formations (Fig. 1), which enrich nonclassical nucleation pathway. Meanwhile, nonclassical crystal growth is always described by the attachment of amorphous particles, primary particles or nanocrystals and their subsequent phase transformation, including solid-state transformations and dissolution-recrystallization reactions.44 The physicochemical natures and formation process of amorphous precursors or intermediates provide in-depth mechanistic understandings of crystallization theory, which inspire novel synthetic strategies for directing hierarchical architectures with remarkable functions. Therefore, in this part, we focus on the formation and phase transformation of amorphous phases, as well as particle-based crystal growth.

2.05.2.2.1

Amorphous precursor

Many works have reported that mineral ions can form dynamic, self-associates termed as prenucleation complexes or PNCs before establishing solid/liquid or liquid/liquid interfaces.47,54,55 Such PNCs can exist in undersaturated and supersaturated CaCO3 solutions (Fig. 4A),56 which have also been reported in other mineral systems such as calcium phosphate,57 CaSO4,58 CaC2O4,59 and FeO(OH)60 etc. The formation of PNCs in CaCO3 solution implies that they might be relevant species for the nucleation of CaCO3.56,61 Through the aggregation of PNCs, the nucleation of ACC is initiated.62 A recent work reported another type of PNC, which is named as surface-stabilized (CaCO3)n nanoclusters as the key growth species at the minimum of the nucleation reaction energy.63 Molecular dynamics (MD) simulations show that CaCO3 PNCs are dynamically ordered liquid-like oxyanion polymers (DOLLOPs) composed of alternating Ca2þ and CO32 ions, with a dynamic topology consisting of chains, branches and rings.64 These PNCs are stabilized by balancing ionic coordination and ion hydration,65 and thus the stabilization of solvation water in the PNCs hinder the clusters dehydration, thereby prolonging induction times before nucleation.66 A thorough thermodynamic analysis of the enthalpic and entropic contributions to the overall free energy of PNC formation suggests that solute clustering is driven by entropy.67 This can be quantitatively rationalized by the release of water molecules from ion hydration layers. The key role of the solvation state of ions and water release suggests that the formation of PNCs should be a common phenomenon in their aqueous solutions, which can explain why ion association is not limited to simple ion pairing. Free energy calculations and

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Fig. 4 Various intermediates prior to amorphous phases formation. (A) High-resolution cryo-TEM image of a fresh 9 mM Ca(HCO3)2 solution after image processing in which prenucleation clusters are observed. An arbitrary number of clusters are highlighted by black circles. Inset is nonfiltered images representing the zone delimited by the red square. In the high-magnification image, all particles present are highlighted by black circles. Particle sizes below the detection limit of 0.45 nm (three times the pixel size) are considered noise. (B) High-resolution cryo-TEM image of assemblies of clusters in SBF; the arrow indicates gold particles used as a fiducial marker for tomography. Determination of the true diameter of prenucleation clusters by extrapolation of the diameter measured in High-resolution cryo-TEM images recorded at different defocus values (inset). (C) Time sequential TEM images of ACP nanoparticles formed within the ion-rich liquid phase. I: Zoomed-in TEM image of the blue boxed area in (C) at 3277 s. II: Zoomed-in TEM image of the blue boxed area in (C) at 5382 s. (D) Cryo-TEM image of  60 nm-sized CaCO3 nanoparticle, indicating the 2 nm-sized subunits. (E) Cross-section through the 3D reconstruction of a cryo-TEM image of CaP-PILP, showing that the nanoclusters are homogeneously distributed and separated. Zoomed-in and close-to-focus cryo-TEM images showing that the size of the clusters is z1 nm (inset 1), while the SAED pattern shows that the clusters are amorphous (inset 2). (F) Pair-distance distribution function (P(r)) of the (CaCO3)n oligomers. The inset shows the shape simulation of the oligomer. Error bars represent one standard deviation, n ¼ 20. (G) High-resolution TEM images of (CaCO3)n oligomers grown at different Ca:TEA ratios from 1:100 to 1:2. (H) TEM image of calcium phosphate ion clusters. Inset: DLS size distributions of the calcium phosphate ion clusters in ethanol solution. (A) Reproduced from Pouget, E.M.; Bomans, P.H.; Goos, J.A.; Frederik, P.M.; Sommerdijk, N.A. Science 2009, 323, 1455–1458; (B) reproduced from Dey, A.; Bomans, P.H.; Muller, F.A.; Will, J.; Frederik, P.M.; de With, G.; Sommerdijk, N.A., Nat. Mater. 2010, 9, 1010–1014; (C) reproduced permission from He, K.; Sawczyk, M.; Liu, C.; Yuan, Y.; Song, B.; Deivanayagam, R.; Nie, A.; Hu, X.; Dravid, V. P.; Lu, J., Sci. Adv. 2020, 6, eaaz7524; (D) reproduced from Xu, Y.; Tijssen, K.C.; Bomans, P.H.; Akiva, A.; Friedrich, H.; Kentgens, A.P.; Sommerdijk, N.A., Nat. Commun. 2018, 9, 1–12; (E) reproduced from Yao, S.; Lin, X.; Xu, Y.; Chen, Y.; Qiu, P.; Shao, C.; Jin, B.; Mu, Z.; Sommerdijk, N.A.J.M.; Tang, R. Adv. Sci. 2019, 6, 1900683. (G) reproduced from Liu, Z.; Shao, C.; Jin, B.; Zhang, Z.; Zhao, Y.; Xu, X.; Tang, R., Nature 2019, 574, 394–398; reproduced from Shao, C.; Jin, B.; Mu, Z.; Lu, H.; Zhao, Y.; Wu, Z.; Yan, L.; Zhang, Z.; Zhou, Y.; Pan, H. J. S. A., Sci. Adv. 2019, 5, eaaw9569.

simulations also show that calcium phosphate in supersaturated solution may nucleate through the aggregation of small negatively charged clusters that grow further through a combination of ions attachment and particle attachment.68–70 Both cryo-TEM and nuclear magnetic resonance (NMR) results unravel that stable PNCs with size of approximately 1 nm have already appeared in solution prior to nucleation, and then aggregate into ACP as building units (Fig. 4B).57,71 These PNCs are identified as ion-association complexes consisted of calcium triphosphate complexes with a Ca/P ratio of 1:3.72 Such prenucleation complexes is the most energetically favored and more thermodynamically stable than the free ions, that is seven-coordinated by two water molecules, two bidentate phosphates, and one monodentate phosphate.72 Meanwhile, these calcium triphosphate complexes could be kinetically trapped,73 and thus can form polymeric assemblies by a reaction limited aggregation.74 The binding of extra Ca2þ can subsequently trigger the aggregation of highly charged PNCs into ACP particles.68,74 With increasing supersaturation, free ions and solute clusters start to aggregate, condense and dehydrate, leading to liquid-liquid phase separation to produce mineral-enriched fluidic phases, which might be named as DLP, represented by either liquid droplets or gels.75–82 DLP can be transiently formed during mineral nucleation, even lacking external additives.78,79 Without extrinsic stabilization, the DLP rapidly dehydrate and condense, transforming to amorphous particles. But in the presence of acidic proteins, DLP can stay longer time before solification.83,84 The higher ion diffusivities show that the droplets of a dense ion-rich liquid phase are consisted of CaCO3 clusters,77 which is identified as a DLP with a composition of CaCO3$4–7H2O,76 and then their coalescence

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and solidification lead to the formation of ACC.85,86 Besides, liquid-liquid separation in PNCs forming in homogeneous solutions as fundamental precursors, also contributes to the formation of DLP.87 It is documented that under reaction conditions that enable steady increases in supersaturation, liquid-liquid binodal decomposition of PNC aggregates produces liquid droplets, and then their aggregation into DLP, are followed by the formation of hydrated ACC solids via dehydration.88 When a high level of supersaturation is established, the system can pass the binodal regime and becomes unstable against density fluctuations, so that spinodal decomposition occurs, leading to the appearance of DLP with gel-like structure.75,81,89 However, it should be addressed that while spinodal decomposition may occur under certain conditions, it cannot serve as a general mechanism for calcium carbonate nucleation. In contrast, it is easier to get binodal region to form liquid droplet. Liquid-liquid phase separation process has also been extended to calcium phosphate system.90,91 It is revealed by liquid-cell transmission electron microscopy (LC-TEM) that the nucleation of calcium phosphate in the saliva solution undergoes a liquid-liquid phase separation, which leads to the appearance of ionrich solution in which ACP nanoparticles nucleate (Fig. 4C).92 The DLP is always kinetic stabilized by various additives, such as poly(aspartic acid) (PAsp) and ovalbumin, through increasing the transformation barrier from liquid phase to hydrous amorphous phase.93–98 The stabilization of a dense calcium phosphate/carbonate phase in the presence of acidic polymers led to the emergence of “polymer-induced liquid precursor” (PILP) phase.83,90,91,93 However, detailed physicochemical investigations revealed that PILP is in fact a polymer stabilized liquid phase by depletion stabilization and de-emulsification, rather than ‘induced’ by acidic proteins and polymers during PILP process.84,97 The liquid-like behavior of PILP phases at the macroscopic level is caused by the small size and surface properties of the assemblies. As the literature reports, the microscopic structure of liquid CaCO3 is actually a polymerdriven assembly of ACC clusters (Fig. 4D).97 Correspondingly, calcium phosphate-PILP material also consists of homogeneously distributed ultrasmall (z1 nm) ACP clusters (Fig. 4E).91 Recently, the concept of inorganic polymerization and crosslinking is creatively proposed in Ca-minerals system.99 Beside silica oligomers,100,101 an linear polymer-like ionic oligomers ((CaCO3)n) precursor with controllable molecular weights and fluid-like behavior are successfully prepared, in which triethylamine (TEA) act as a capping agent to stabilize the oligomers. The oligomers are rod-like with a length of 1.2 nm, (Fig. 4F) which corresponds to 3–4 CaCO3 units. The removal of triethylamine in oligomers, accompanied by a volatilization of ethanol solvent, initiates crosslinking of the (CaCO3)n oligomers via a step chain growth (Fig. 4G), and thus resulting in the formation of pure monolithic ACC with continuous and dense structure. The fluid-like behavior of the oligomer precursor enables it to be readily processed or molded into specific shapes, even for materials with structural complexity and variable morphologies. Using a similar strategy, spherical ultra-small calcium phosphate oligomers are synthesized (Fig. 4H), which can also transform into metastable ACP particles.102 With the identification of ionic oligomers for CaCO3 and calcium phosphate, the controlled preparation and crosslinking of inorganic ion oligomers not only provide a promising method to prepare inorganic bulk materials but also offer novel insights to physical understanding of crystallization mechanism.103,104

2.05.2.2.2

Phase-transformation-based crystallization

During biomineralization process, the transformation of amorphous phases as ions reservoirs into crystalline phases is demonstrated as an important growth pathway for biominerals, in which crystal growth can proceed by the attachment of amorphous particles to crystalline surfaces.105–107 For instance, the mineralization of the spicules in sea urchins’ embryos is a biogenic crystallization of accumulating ACC precursor nanoparticles into calcite in the presence of polymetric additives.108 Similarly, new platelets-like apatite mineral within the collagen matrix is delivered and deposited as packages of ACP nanospheres.109 Analogous to the roles of mineral ions, amorphous particles as basic units can attach and organize on crystal surfaces. For example, in situ AFM reveals a layer-by-layer attachment of ACC particles on calcite surface (Fig. 5A).110 These ACC assemblies can undergo restructuration and particle fusion, followed by an interface-coupled dissolution  reprecipitation mechanism, producing smooth calcite surfaces without nanogranular texture. However, in the presence of additives, the ACC nanoparticles transform to calcite preserving the nanogranular texture with initial particle shapes.106,110 Also, coarse-grained (CG) models is developed to understand aggregation induced CaCO3 crystallization process.111 Due to the weak Ca-water interactions, ACC clusters attach and ultimately crystallize into a perfect calcite structure. And calcite can induce the crystallization of ACC clusters when they contact with each other. With the increasing Ca-water interactions, the aggregated ACC clusters could not transform into calcite. Scientists have also found that the HAP crystallization occurs by the attachment of ACP nanoparticles to form organized assemblies/aggregates with triangular and hexagonal morphologies (Fig. 5B).112 Similar ACP clusters with size of 1–2 nm are observed to aggregate into larger particle assemblies within the organized enamel protein matrix, which transform into initial needles-like HAP through lattice- and matrix guided bridging of individual nucleation sites.113 These facts show that amorphous particles can serve as modular building units for generating complex mineral morphologies and superstructures via the coupling of particle attachment with phase transformation. Wherein, the controlled converting of transient amorphous precursors to final crystalline minerals is very important for their applications, which determines their final polymorphous, sizes and shapes. The phase transformation through a stepwise phase transition process, including solid-state transformation and dissolution-recrystallization (Fig. 1B), have been directly observed by in situ TEM observations.114,115 Usually, solid-state transformation through direct rearrangement occurs at the conditions without bulk solution.116 Differing from normal amorphous inorganic materials, hydrated amorphous biominerals containing structural water must be dehydrated prior to via solid-state transformation.46 A higher enthalpy of hydrated ACC particles than dehydrated particles and any crystals suggests that the dehydration and crystallization is thermodynamically favorable. Consequently, the phase transformation of ACC follows an energetically downhill sequence: more metastable hydrated ACC / less metastable hydrated ACC 0 anhydrous ACC  biogenic anhydrous ACC 0 vaterite / aragonite / calcite.117 It should be noted that not all these phases should appear

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Fig. 5 Amorphous phase-based crystallization process through particle attachment and phase transformation. (A) Sequential AFM height images of the attachment on (104) calcite and growth of ACC nanoparticles formed upon contact of the calcite surface. Note the precise alignment of ACC nanoparticles along step edges of the dissolution pits already present on the calcite substrate before solution injection. (B) In situ AFM investigations revealing the growth of HAP crystals by particle attachment and morphology evolution on the HAP (100) surface. (C) In situ TEM images showing the direct transformation of ACC to aragonite (top) and vaterite (bottom). (D) TEM images of mineral at the induction time, showing the nucleation of HAP on ACP surface. (E) TEM images showing the nucleation of HA with the dissolution of ACP in artificial saliva. Green arrows indicate that the HAP crystals are attached to the surface of the ACP substrate. (F) In situ TEM analysis of direct transformation of single CaCO3 nanoparticle in the presence of 5 mM Mg2þ. (G) Chemical and structural model of the synthetic amorphous nanoparticles. Representative cryo-TEM micrograph of a nanoparticle of the synthetic, Amorphous Calcium Magnesium Carbonate (ACMC) sample dispersed in deionized water (left). Also shown is a twodimensional chemical and structural model of the surface region of an amorphous particle of ACMC soaked in water (right). (H) Image sequence after 45 min of (NH4)2CO3 diffusion, showing initial nucleation and growth of a CaCO3 particle inside or on a primary Ca–PSS globule within 4 s (i–viii). (A) Reproduced from Rodriguez-Navarro, C.; Burgos Cara, A.; Elert, K.; Putnis, C. V.; Ruiz-Agudo, E., Cryst. Growth Des. 2016, 16, 1850–1860; (B) reproduced from Li, M.; Wang, L.; Zhang, W.; Putnis, C. V.; Putnis, A., Cryst. Growth Des. 2016, 16, 4509–4518; (C) reproduced from Nielsen, M. H.; Aloni, S.; De Yoreo, J., Science 2014, 345, 1158–1162; (D) reproduced from Jiang, S.; Pan, H.; Chen, Y.; Xu, X.; Tang, R., Faraday Discuss. 2015, 179, 451–461; (E) reproduced from He, K.; Sawczyk, M.; Liu, C.; Yuan, Y.; Song, B.; Deivanayagam, R.; Nie, A.; Hu, X.; Dravid, V. P.; Lu, J., Sci. Adv. 2020, 6, eaaz7524; (F) reproduced from Liu, Z.; Zhang, Z.; Wang, Z.; Jin, B.; Li, D.; Tao, J.; Tang, R.; De Yoreo, J. J., Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 3397–3404; (G) reproduced from Gong, Y. U.; Killian, C. E.; Olson, I. C.; Appathurai, N. P.; Amasino, A. L.; Martin, M. C.; Holt, L. J.; Wilt, F. H.; Gilbert, P., Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 6088–6093; (H) reproduced from Smeets, P. J.; Cho, K. R.; Kempen, R. G.; Sommerdijk, N. A.; De Yoreo, J. J., Nat. Mater. 2015, 14, 394–399.

under a given reaction condition. Dehydration of ACC particles is a key step for the solid-state transformation, because it is associated with a rearrangement of ions within ACC.116 With the loss of water within ACC, a stronger interaction between ion-water molecules increases the activation energy barrier for further dehydration.118,119 The solid-state transformation of CaCO3 is accompanied by a series of dehydration, ordering and crystallization, without changing chemical compositions in these different

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phases.116,120 In contrast, the solid-state transformation of ACP into crystalline calcium phosphate is more complicated relative to CaCO3 system, since dissociation of water and the capture of hydroxyl ions as well as an increase in Ca/P molar ratio accompany the transition.121 It is speculated that atoms in the center of amorphous particles rearrange into a crystalline core where apatite is nucleated.122 And only HAP-resembling ACP can transform into HAP via martensitic, bulk lattice reordering mechanism.123 Additionally, the kinetics analyzes of thermally induced crystallization of ACP show that dehydrated ACP required a high activation energy for the crystallization of ACP to final crystals.124 Therefore, additional triggers, like elevated temperature, humidity and even electron beam, are necessary to promote solid-state transformation.119,124–126 For example, under humid conditions, transformation of ACP spherical particles to HAP platelet-like crystals proceeds via a cluster migration from parent ACP particles and attachment of smaller ACP clusters from environment, where nanocrystalline areas are formed and crystallization advances via migration of nanoclusters by forming steps at the growth front.127 Enhanced kinetic stability of ACC by dehydration118 makes phase transformation tend to occur via a local dissolution/reprecipitation mediated by the physiosorbed water on the particle surface.128,129 It is reported that, even without the bulk solution, the humidity-induced crystallization and the pressure-induced crystallization from ACC to calcite follow a surface-coupled partial dissolution-reprecipitation and a dehydration-assisted local dissolution-precipitation pathway, respectively.130 Such pathway that amorphous phases dissolve for the precipitation of new crystals is termed as dissolution-recrystallization, which has been demonstrated by in situ LC-TEM investigations.114 The observations showed that the nucleation of CaCO3 crystal can be based on ACC precursor which appears first, and then dissolves and recrystallizes into a more energetically favorable vaterite or aragonite through a direct, physical connection between amorphous and crystalline phases (Fig. 5C).114 However, in the case that ACP particles transit to HAP crystals, it is a little difference because a surface nucleation of HAP occurs by functioning ACP as the substrate, accompanied by a dissolution of ACP particles (Fig. 5D).131 It is obvious that the kinetic stability of amorphous phases is controlled by the dissolution rate of amorphous particles and nucleation and growth rate of new crystals. The kinetic stability of amorphous phases can be affected by temperature, pH, additives, confinement environments, particle size and solvent.132–137 For example, CaCO3 crystallization in the droplets proceeds through an ACC intermediate at early stage, which significantly slows the crystal nucleation than in the bulk solution.138 Through altering dissolution kinetics of amorphous phase and crystal nucleation kinetics, the crystalline polymorphs, sizes and shapes can be influenced.133,139,140 For instance, small and larger ACC particles transform into vaterite and calcite, respectively,141 and in the presence of alginate-based additives, a mixture of octa-calcium phosphate (OCP) and HAP from ACP is produced.142 Most amorphous minerals (i.e., ACC, ACP) in nature contain Mg2þ, and acidic proteins with carboxyl or phosphate functional groups. It is suggested that the introduction of additives provides a promising approach to control the amorphous phase mediated phase transition process. Mg2þ, as an effective stabilizer and inhibitor for amorphous phases, has been extensively investigated in the formation of CaCO3 and calcium phosphate.12,143–148 Mg2þ ions increase the amorphous phases stability to retard the formation of crystalline phases because higher dehydration energy of Mg2þ than Ca2þ makes amorphous phases dehydration more difficult before crystallization.145 Recent work also report that the stability effect of Mg2þ is likely to be caused by binding to hydroxide in ACC.149 MD simulation shows that Mg2þ can inhibit phase transformation of ACC by tuning ACC local structure and stability, and endothermic dehydration and exothermic crystallization can be used to determine the whole phase transition process.146 It is documented that ACP-adsorbed Mg2þ is more effective than the incorporated one in stabilizing ACP to retard HAP formation by reducing the ACP solubility,12 and the presence of Sr2þ can significantly enhance the stabilization of Mg2þ on ACP via a synergic effect.144 Briefly, Mg2þ in crystallization control plays an important role, but its detailed mechanism remains unclear. Organic molecules, like citrate, acidic proteins and polymers, also contribute to and effectively regulate the amorphous phase mediated phase transformation process.132,150,151 For example, PAsp, PAA and citrate adsorbed on ACP surface inhibit HAP nucleation,131,152 which in return confirms the heterogeneous nucleation pathway of HAP on the precursor phase surfaces (Fig. 5E).92 Furthermore, certain proteins are putatively used to stabilize ACC, rather than to inhibit mineralization entirely, allowing for a subsequent prompt transformation into aragonitic crystallites.153 Finally, it deserves to address that these additives sometimes promote the phase transformation. For example, during the conversions of ACP to HAP proceeding through dissolution and recrystallization at the ACP–fluid interface, the nonphosphorylated counterpart in adsorbed amelotin promotes the ACP dissolution and HAP formation through a strong complexation of the amelotin protein with calcium ions.154 Similar case is that the recombinant Alv stimulates the transition from ACC to calcite.151 To understand the detailed roles of additives in controlling phase transformation, various advanced techniques are employed, such as LC-TEM and in situ NMR. Recent in situ LC-TEM investigation reveals that Mg2þ switches the pathway from dissolutionreprecipitation to a shape-preserving solid-state transformation (Fig. 5F).115,155 The presence of free Mg2þ ions in bulk solution inhibit dissolution-reprecipitation pathway by decreasing the supersaturation with respect to calcite or vaterite. Further analyzes from MD and in situ ATR-FTIR show that the structural water content in ACC increases with the involvement of Mg2þ, which can destabilize the ionic network thus promoting direct ion rearrangements in ACC. Similarly, a combination of high-resolution imaging and in situ solid-state NMR spectroscopy unravels that the solid-state phase transformation mechanism of ACC in the presence of Mg2þ is accompanied by a fast hydrogens exchange between amorphous nanoparticles inside, surface and the surrounding solution (Fig. 5G).116 Such exchange process promotes the motion and rearrangement of ions within the nanoparticles and thus enables their direct solid-state rearrangement into crystalline structures. In addition, in situ LC-TEM observations indicate that ion binding between polystyrene sulphonate (PSS) and calcium ion is a key step in the formation of metastable ACC (Fig. 5H).156 In the absence of PSS, vaterite crystals directly precipitated from solution. By contrast, the introduction of PSS decreases the supersaturation in bulk solution by sequestering the Ca2þ to form Ca–PSS globules so that no nucleation event occurs

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outside of the globules. And the high supersaturation in the globules enables ACC formation, and PSS can stabilize these ACC particles.

2.05.2.2.3

Nano attachment

The attachment of primary particles or nanocrystals has also been proposed as an important pathway for biomineralization, as well as the rational design of novel biomimetic functional nanomaterials. The preferential precipitation and aggregation of hemihydrate bassanite nanocrystals, followed by phase transformation, lead to the formation of gypsum (CaSO4$2H2O).157 Consistently, in situ and fast time resolved small-angle X-ray scattering unravels the aggregation of sub-3 nm primary species in the solution-based nucleation and growth of gypsum. Using LC-TEM, oriented attachment of gypsum nanocrystals drives the formation of gypsum microneedles (Fig. 6A).158 Besides, the nucleation and growth of magnetite proceed through rapid agglomeration of nanometric primary particles (Fig. 6B),159 in which both the thermodynamics and the kinetics of the process can be described in terms of colloidal assembly (Fig. 6C).160 Another detailed investigation shows that Fe3O4 aggregates are formed through a multistep process involving first the conversion of ferrous hydroxide precursors in  5 nm primary particles that aggregate into  10 nm primary Fe3O4 crystals that finally arrange into the secondary mesocrystal structure.161 Noticing that particle attachment includes oriented attachment and random attachment. Usually, oriented attachment of nanocrystals leads to the formation of mesocrystal or single crystal.162 An example is the formation of hexagonal plates of vaterite mesocrystal caused by the oriented aggregation of primary hexagonal vaterite building blocks in the presence of polymer.163 However, random attachment of vaterite nanocrystals can also result in the formation single-crystalline vaterite by grain-boundary migration (Fig. 6D).164 Specifically, an aggregate with disordered surface layer evolves into single crystals due to the existence of inherent surface stress applied by the disordered surface layer (Fig. 6E). With increasing evidences for the precursors (PNCs, DLP, oligomers) prior to amorphous phases, an in-depth understanding of nonclassical nucleation models is very necessary. Based on these investigations, it is suggested that the nucleation process cannot be deconstructed to a simple ion aggregation into solid particle, but also depends on the configurational and nature of precursors in relation to their environments. So far, little is known about the effects of additives on precursors formation and subsequent phase transformation, especially for the synergic effects of multiple additives. Since an enormous but underexplored potential keeps for connecting crystallization mechanism and the preparation of functional nanomaterials, there is yet plenty of room to elucidate a more completed theoretical description of non-classical nucleation and growth. These advancements require a solid experimental database from advanced techniques. For instance, LC-TEM is promising to reveal microscopic nucleation and growth process of solids owing to its high temporal and spatial resolutions.165

Fig. 6 The attachment of primary particles or crystals. (A) In situ LC-TEM image sequences showing the aggregation of preexisting gypsum nanoparticles into nanoneedles. (B) Cryo-TEM image of primary particles (arrows) attaching to the surface of a magnetite nanoparticle. (C) Cryo-TEM images of primary particles (left) formed after the addition of NO3 on a graphene oxide support and reaction product (right) after the addition of 40% of the total stoichiometric amount of NO3. Inset: Schematic of the formation (left), colloidal assembly (middle) and conversion of the primary particles (right) into magnetite. (D and E) TEM images showing that nanocrystals served as building blocks for the final spindle-shaped vaterite crystal, which always contained a 2–20 nm nonoriented surface layer during the crystal-growth process, and underneath the surface was the singlecrystal bulk (E). (A) Reproduced from He, K.; Nie, A.; Yuan, Y.; Ghodsi, S. M.; Song, B.; Firlar, E.; Lu, J.; Lu, Y.-P.; Shokuhfar, T.; Megaridis, C. M.; Shahbazian-Yassar, R., ACS Appl. Nano Mater. 2018, 1, 5430–5440; (B) reproduced from Baumgartner, J.; Dey, A.; Bomans, P. H. H.; Le Coadou, C.; Fratzl, P.; Sommerdijk, N. A. J. M.; Faivre, D., Nat. Mater. 2013, 12, 310–314; (C) reproduced from Mirabello, G.; Ianiro, A.; Bomans, P. H. H.; Yoda, T.; Arakaki, A.; Friedrich, H.; de With, G.; Sommerdijk, N., Nat. Mater. 2019, 19, 391–396; (D and E) reproduced from Liu, Z.; Pan, H.; Zhu, G.; Li, Y.; Tao, J.; Jin, B.; Tang, R., Angew. Chem. Int. Ed. 2016, 55, 12836–12840.

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2.05.3

Organic matrix and its regulation effect

Compared with synthetic minerals, natural biominerals show incomparable superiorities both in composition and morphology, which is achieved by biological regulation of living organisms in biomineralization.166 These processes of biomineralization can be divided into biologically induced mineralization and biologically controlled mineralization.167 The former refers to the mineral deposition at the cell interface as a result of interaction between the concentration changes of pH, CO2 and excreted metabolites in the metabolism process of the organism and the ions and compounds in the external environment. Due to low degree of biological regulation, the minerals tend to be low crystalline and lack distinct crystal morphology in this process. In biological control mineralization, the biominerals with complex high-level structures and special biological functions are produced in a relatively closed and limited environment. The living organisms regulate the transport of mineral ions and organic molecules through cell activities, which control the nucleation and growth of biominerals in the reaction process.168 Recently, the organic compounds (such as saccharide, protein and polymer) have been confirmed the modulating effect on inorganic crystal growth in biomineralization. More precisely, these organic compounds can provide a structural template for crystal growth, segment mineralization space, stabilize and control nucleation sites and phase transitions of minerals.169 For example, phosphoglycoprotein, bone sialoprotein and polypeptides can regulate calcium phosphate mineralization,170–172 silaffins, polyamines and chitin polysaccharides can control silica mineralization,173–175 and so on. Understanding the regulation of organic matrix on biomineralization can provide important guidance for the study of biomineralization.

2.05.3.1

Organic-inorganic interface

The regulation of organic macromolecules in the mineralization process is the core issue in the study of biomineralization. The inorganic mineral deposition can be controlled at the molecular level through the interaction between organic macromolecules and inorganic ions or clusters at the interface, so that these minerals have special morphologies, multi-stage structures and assembly modes.176–178 Mineralization on organic matrix is a typical heterogeneous nucleation system, which can reduce interfacial energy and nucleation activation energy to control inorganic crystal nucleation. At the same time, the functional groups of biological proteins (such as –COO/NH4þ) can also interact with mineral ions in the solution, so that they are gathered on the surface of organic matter to improve the local supersaturation of ions and lead to deposit on the surface of organic assembly. Therefore, the role of organic-mediated nucleation in biomineralization is to reduce the activation energy through the interaction between functional groups of organic macromolecules and ions in supersaturated solution at the interface.179 The charge matching, polarity, structure and stereochemistry can cause changes in nucleation activation energy in organicinorganic interface recognition model, which can be transformed into regulation of nucleation rate, crystallographic site, mineral phase structure and crystal degree phase. For example, Stephen Mann’s group simulated the calcium carbonate mineralization system on the Langmuir monolayer film to investigate the effects of environmental composition, pH, functional groups, structure and morphology of ordered bodies, electrical properties of membrane surface, and chemical potential on crystal formation and growth.180 The selection of nucleating inorganic ions by the organic macromolecular involves molecular reactions and protein adsorption on the interface, which can recognize the mineralization site, mineral type and orientation on organic-inorganic interface.181 The specific binding of amelogenin with side faces of enamel crystals also has been investigated, which is essential for their proper elongation and thickness.182

2.05.3.2

Template effect

During the process of biomineralization, organic matrix can provide the template to regulate the nucleation and crystallization orientation of minerals. In general, the template effect refers to the geometric or stereochemical match between the surface of crystal and organic matrix.183 For instance, the orientation of the growing mineral is controlled by the self-assembled monolayers acting as a template.184 The structural template effect in precursor phase crystallization (like ACC) can also be helpful to understand the crystallization pathway of biomineralization.185 In addition, the demineralized organic matrix of biominerals is a splendid natural template. Nacre is a frequently investigated and copied biomineral model system,186 which can be artificially prepared by using the insoluble organic nacre matrix as the template (Fig. 7).187 This retrosynthetic nacre morphologically indistinguishable from the natural ones could be achieved through the polymer-stabilized ACC as the precursor phase to be oriented crystallized. At the meanwhile, the demineralized bone or dentin substance and the reconstructed collagen fibrils can also lead to the oriented crystallization of CaP. The highly charged polymer can make ACP precursors liquid-like,188 highly-charged189 and alter the osmotic pressure190 to guide ACP precursors into collagen fibrils, which is important for oriented crystallization inside collagen fibrils. The construction of highly structured multilevel materials in organism begins with the recognition and regulation of inorganic mineral phases by proteins to guide them ordered assembly.191 Thus, proteins and peptides also can be used to prepare ideal templates with multistage structures for controlling the construction of functional composites.192 The polypeptides and polymers with material-specific recognition ability and strong operability can be utilized to fabricate materials with complex structure, which has a broad prospect in the preparation of organic-inorganic composite materials.193 With the rapid development of molecular biology and nanomaterial technology in recent years, the polypeptides with recognition ability to inorganic nanomaterials through genetic modification has various applications, such as tissue repair, biological detectors and sensors, etc.194

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Fig. 7 Using an insoluble organic nacre matrix as the template to retro-synthesize nacre layers. TEM micrographs of (A and B) of highly mineralized parts of synthetic nacre after 24 h reaction time. (C) Electron diffraction pattern of the platelets in panel B. (D) Original nacre from Haliotis laevigata. Reproduced from Gehrke, N.; Nassif, N.; Pinna, N.; Antonietti, M.; Gupta, H. S.; Cölfen, H., Chem. Mater. 2005, 17, 6514–6516.

2.05.3.3

Confinement effect

The process of biomineralization often occurs in confined spaces, such as organic microvesicles of cells. The deposition of inorganic minerals and self-assembly of organic matrix can be achieved through ion exchange with the external environment.17 It can effectively control the reaction concentration in confined spaces and induce nucleation via the organic substrates in the vesicles. The mechanism of nano-confinement has been proposed to comprehend the crystal growth in a confined space, which can be sufficient to produce oriented crystallization.195,196 The gradual ordering crystallization of mollusks shells can affirm this mechanism through detailed micro-structural analyzes. The oriented mineralization inside collagen fibrils can also be regulated by nanoconfinement.188 Meanwhile, the amorphous precursors also play an important role for the oriented crystallization of calcite inside collagen fibrils, which can promote infiltration of mineral precursors into nano-gap space before the crystallization.197 Moreover, some artificial confined spaces had been found to measure the effect of nano-confinement. For example, calcium phosphate in nano-cylindrical pores, which is simulated in collagen fibrils, can be oriented crystallized (Fig. 8).198 A biomimetic prismatic-type calcium carbonate layer has been formed on substrates with a granular transition layer by competing growths on thin films.199 In addition, the amorphous precursors were generated in poly-aspartic acid or poly-acrylic acid,93 which were a granular transition layer on substrates by diffusing (NH4)2CO3 gas into calcium solutions. The precipitate precursor phase can be formed from highly supersaturated solutions and then deposited as films via connecting between soluble inhibitor and insoluble matrix.200 Furthermore, the materials with nacre-like structures can be built through layer-by-layer assembly201,202 and freeze-induced chitosan matrix,21 which can be utilized multilayered organic sheets as the scaffold.

2.05.4

Application of biomineralization for tissue regeneration

Various technologies had been developed to simulate the process of biomineralization, which can fabricate the elaborate structure of biominerals.203 These biomimetic functional materials had been designed for diversified applications, and especially for tissue regeneration, including collagen mineralization and tooth/bone repair.204,205 Meanwhile, the natural materials also had been imitated with superior structural characteristics and mechanical performance.21 In addition, the integration between organic matrix and inorganic biominerals is opening a spectrum of possibilities through the formulation and synthesis of composite materials.206,207

2.05.4.1

Collagen mineralization

Collagen exists in most tissues of vertebrates, which is a preponderant and abundant protein in human body.208 Hence, it provides various important functions, including the capacity of mineralization in bone, cartilage, tendon and dentin.209 Due to the different charge density distribution of the collagen fibers, type I collagen has a triple-helix structure, which is a natural component of connective tissue.210 In mineralized system, collagen with excellent biocompatibility can orientate the growth of biominerals,211 so that it can be applied in tissue engineering, such as repairing bone and cartilage.212,213 It is essential for the mineralization of collagen that there are interactions between the collagen and mineral.214 The collagen can not only induce hydroxyapatite formation with non-collagenous proteins, but also guide the organization and growth of the crystal.215,216 Several investigations have been developed to simplify the intangible process of mineralizing collagen from the atomic level to the micrometer level.197 There are several hypotheses to explain these mineralization processes, including polymer-induced liquid precursor,217 inhibitor exclusion,218 Coulomb gravity-induced mineralization,209 co-assembly mineralization208 and osmotic pressure/charge double equilibrium induced mineralization.190 The potential mechanisms involved in intrafibrillar mineralization had been gained insight by these extensive works on collagen mineralization.74,219,220 The concept of

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Fig. 8 Schematic illustration of (A) the precipitation of CaP within the membrane pores by using a double-diffusion configuration and (B) the formation of HAP crystals within the membrane pores. The ACP particles (yellow) begin to convert into HAP (red), which under competitive growth become preferentially oriented. (C) SEM and TEM (inset) images of rods formed in 200 nm pores after 6 days. (D) Rods formed in 50 nm pores after 1 day, which are composed of small particles (inset). (E) HAP rod isolated after 1 day from 50 nm pores. Reproduced from Cantaert, B.; Beniash, E.; Meldrum, F. C., Chemistry 2013, 19, 14918–14924.

Columbic attraction, as a dominant mechanism for intrafibrillar mineralization, had been improved through the phenomenological observation of polyanion-stabilized mineralization precursors.189,221,222 In the living organism, the charged amino acid residues in collagen can be easily linked with calcium ions, as nucleation sites of biominerals, to achieve biomineralization in vertebrate.223 There many biomolecules can interact with collagen residues, especially citrate, which has a high level of citrate in biological hard tissues.224 It should be noted that the liquid-like amorphous precursors of calcium phosphate are the linchpin for successful collagen mineralization.225 Shao et al. found that the citrate molecules can reduce interfacial energy between collagen and ACP and promote heterogeneous formation of amorphous precursors and intrafibrillar mineralization on collagen (Fig. 9).150 Collagen fibrils and minerals serve as the major components in biological hard tissues, which can be helpful to investigate the fundamental issues in biomineralization.26 The organic matrix and inorganic materials play key roles in the process of collagen mineralization to provide potentiality in biomimetic hard tissue reparation and new implantable materials fabrication.226

2.05.4.2

Tooth repair

The tooth is the hardest one of numerous biological composites with excellent mechanical performance in biomineralization.227 The basic components of enamel are nonstoichiometric fluoridated carbonate apatite crystals, which are closely arranged with specific orientations to ensure a high impacting strength.228,229 In general, the nucleation, growth and features of HAP, as a simplified mineral model, would be used for investigating formation and remineralization of enamel.18,230 For example, the molecular mechanism of enamel matrix proteins in the formation of dentin has been explicated by impairment of ameloblastin self-assembly, which fabricate the enamel with disordered hydroxyapatite crystallites.231 Due to the acellular and scarcely self-repaired features of

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Fig. 9 Mineralization of the collagen fibrils in the (A–D) absence and (E–K) presence of citrate pretreatment. The (A, E) TEM images, (B, F) enlarged images, (C, G) SAED patterns and (D, H) element mapping analysis of mineralized collagen fibrils. The (I) 2D and (J) 3D STORM images of mineralized collagen fibrils. (K) The z-slice of the STORM image of the mineralized collagen fibrils. Scale bars: 200 nm (I–K). (L) Schematic of the functional citrate treatment on the mineralization of collagen fibrils. Reproduced from Shao, C.; Zhao, R.; Jiang, S.; Yao, S.; Wu, Z.; Jin, B.; Yang, Y.; Pan, H.; Tang, R., Adv. Mater. 2018, 30, 1704876.

mature enamel, the dental caries and decay are both a universal chronic disease and a fundamental health problem in the worldwide.232,233 There are various biomimetic mineralization strategies to investigate enamel reconstruction.2 For instance, the ordered enamellike structures of fluoride-substituted apatite could be directly prepared by using ethylenediaminetetraacetic acid disodium salt dehydrate.234,235 At the same time, the design of synthetic amelogenin-inspired peptides and protein with functional amino acid residues had been enabled to mimetics and repair tooth enamel.236,237 Moreover to peptides, amino acids (e.g., glutamic acid, Glu) could also induce the regeneration of enamel-like hierarchical structure by precursor assembly in physiological conditions.204 In addition, the hydrogel-driven mineralization had also been developed for in situ enamel regrowth, such as amelogenin-chitosan hydrogel and glycerine-enriched gelatin gel.238,239 Due to the similar dimensional scale and peripheral functionalities with amelogenin, poly(amido amine) dendrimers could be used for repairing the etched enamel surface, which could alter the aggregation in HAP crystal growth to form oriented HAP crystals.240,241 Bioactive glass materials, such as inorganic amorphous of calcium and sodium phospho-silicate material, have been utilized as tooth and bone graft materials expecting for clinical treatment.242 In order to promote the penetration of precursors in carious lesion effectively, these bioactive materials could be improved to form stable nano-precursors by adding polyacrylic acid (PAA).243 Besides, fluoride also plays a substantial role on the repair and reconstitution of enamel, which can impact the nucleation kinetics of HAP in a simulated body fluid.244 The needle-like fluoridated hydroxyapatite crystals had been remineralized on the surface of etched enamel by using a modified biomimetic deposition.245 These great attempts of enamel remineralization provide a basis for clinical development, and the applicable repair techniques are still expanding. A biomimetic crystalline-amorphous mineralization frontier has been designed for simulating the natural process of epitaxial growth in biomineralization (Fig. 10).102 It can be stabilized by removable small organic molecules, which can promote the coalescence and fusion between particles and maintain original structural complexity of enamel. Although this bioinspired approach supply a new pathway for tooth repair, such layer has been only fabricated in the laboratory scale, the replicated clinical treatment remains improve in future.

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Fig. 10 (A) The SEM image of both acid-etched enamel and repaired enamel. (B) A 3D AFM image of repaired enamel. (C) High-magnification SEM image of the red circle in (A). (D) Cross-sectional view of final repaired enamel, R and IR respectively represent for enamel rod and inter-rod. (E and F) Enamel rods with different orientations. (G) Calculated hardness and elastic modulus of the native, etched and repaired enamel samples. (H) Coefficient of friction of these enamel samples measured under a constant normal force of 500 mN. Reproduced from Shao, C.; Jin, B.; Mu, Z.; Lu, H.; Zhao, Y.; Wu, Z.; Yan, L.; Zhang, Z.; Zhou, Y.; Pan, H. J. S. A., Sci. Adv. 2019, 5, eaaw9569.

2.05.4.3

Bone repair

Bone, a hierarchical hard tissue, is one of the most important products in biomineralization, which consists of collagen fibrils and minerals.246 In the formation of bone, the calcium and phosphate ions are transferred minerals into the collagen.247,248 It is well known that the elongated mineral apatite nanoparticles are aligned and embedded in collagen fibrils possessing periodic gaps to enhance load-bearing characteristics.249 The deposition of hierarchical bone material is established following these features: (i) formation of a highly ordered organic substrate by molecular self-assembly and intermolecular crosslinking; (ii) compartmentalized nucleation and growth of inorganic minerals; and (iii) origination of bone apatite through an intermediate phase of ACP.250–252 Due to the trauma and other diseases, bone defect is not only a common disease in clinic, but also one of the difficult problems in orthopedic treatment.253 Although the best approach of bone repair is autologous bone grafting, there are some risks of secondary surgery and limitations of sources. The problems of allogeneic bone graft also exist, including rejection reaction and virus/bacterial infection.254 Therefore, the researches of bone-fixation materials have raised extensive attention. Currently, the most commonly ideal bone repair materials in clinical practice are those with good tissue tolerance, biocompatibility, mechanical strength and biodegradability.255 To explore the biological effects of apatite, HAP nanoparticles with different size and morphology had been generated to investigate their influence on the proliferation and osteogenic differentiation of cells, which has diverse effects in bone repair.256,257 The bone cement based on calcium phosphate mineral is an important substitute material to induce bone bonding and healing for bone repair.258 Although it has good degradability and bone induction ability,

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the insufficient mechanical properties of CaP limit their clinical applications.259 Moussa et al. found that addition of homochiral L(þ)-tartaric acid could enhance the mechanical properties of bioceramics though decreasing their crystal size, which has potential for bone repair.260 In terms of bone composition, the organic-inorganic composite materials are also the ideal alternative for repairing bone defects.261 Besides the synthetic polymers, some natural bio-polymers with good biocompatibility and excellent mechanical properties, such as bacterial cellulose, have also been used for bone repair.262 Meanwhile, the hydrogels with superior osteoconductivity and biodegradability can also control the growth factor release and apply to cell encapsulation.253 A tough double-network hydrogel hybridized with HAP nanocrystals had been bonded to bone, which can induce the gradient structure at the gel-bone interface in vivo.263 The formation of early bone substantially is a collagen-matrix hydrogel material with a variety of stem cells and functional cells, so that is a “living” material. Inspired by this, the nano-apatite has been directly deposited in supersaturated calcium and phosphate medium with a non-collagenous protein analog, which can embed with osteoprogenitor, vascular, and neural cells in collagen.264 This biomimetic approach could fabricate bone-like tissue models with good biomimicry that might have broad potentialities for disease modeling, drug development, and regenerative tissue engineering. In the process of bone formation, the mineralization precursors would be utilized for mineralization of collagen matrix. Yao et al. fabricated a free-flowing calcium phosphate PILP material with excellent bone inductivity, which can penetrate into collagen fibrils and make oriented growth of hydroxyapatite crystals. This injectable hydrogel is beneficial for minimally invasive bone healing265 (Fig. 11) and osteoporosis treatment.91 Inspired by the natural biomineralization, the biomimetic mineralization technologies had been developed to obtain biological materials, which can achieve bone regeneration and repair from biomineralization system.

2.05.5

Organism improvement

2.05.5.1

Artificial shell

In nature, many organisms will be affected by the external stresses, especially the lower organisms. The biomimetic materials can be coated on the surface of organisms by forming an artificial shell to protect them in the harsh environment. Inspired by eggshell, Wang et al. firstly realized calcium mineralization on the surface of yeast cells though layer by layer self-assembly (LBL) technique, which provide yeast cells a CaP mineral shell with 700 nm thick (Fig. 12A).266 This yeast cell with artificial shell could be maintained the cell activity and controlled the cell division, leading the cell into a dormant state, which would be preserved in pure water and lytic enzyme for a longer time (Fig. 12B). Then the silica encapsulations were successively established on the surface of yeast cells and HeLa cells by biomimetic silicon mineralization (Fig. 12C–F).267–269 These works indicate that cell modification by biomimetic mineralization is a way to prolong the cell preservation and improve the cell viability (Fig. 12G). The artificial shell also could be built by the nanoparticles to provide an exoskeleton for cells, which could enhance the resistance against endo- and exogenous stimuli, such as the stresses of osmotic pressure, ROS, pH, ultraviolet and toxic nanoparticles.270 The conformation of proteins would be changed or even inactivated in high temperature, so that it is very necessary to improve the thermal stability of biological and protein products.271 Many plants in nature, such as rice, horseradish and cactus, have silica mineral layers on their surfaces, which can not only provide mechanical support and prevent the invasion of pathogenic bacteria, but also retain water and enhance their heat resistance.272 Inspired by the natural phenomenon, a silica nanoshell by electrostatic adsorption of LBL technique had been achieved on the surface of yeast cells.273 During the heat stress, the nanoshell of silicon dioxide could maintain the structure and limiting the deformation of yeast cells to remain cell viability. In addition, the cytocompatible nanoencapsulation of SiO2–TiO2 composites could also be built on Chlorella cells in the solution of their precursors, which could provide cellular thermal protection.274 For the thermal protection of living organisms, the essence is mainly protecting the proteins and enzymes, so that it is valuable for preserving proteins and enzymes of pharmaceutical products. For instance, the ACP nanoparticles could be wrapped on catalase by in-situ mineralization and enhanced the thermal stability on cellular activity and structure.275 More importantly, this tactic could stabilize the structural water molecules in mineralized shell to closely bind with proteins or other biomolecules in the future, which could reduce the structural damage caused by the intense movement of water molecules in high temperature. With the increasing demand for renewable energy, biomass (e.g., photosynthetic microorganisms) has attracted much attention to apply for the primary productivity supply system.276 However, the photosynthesis of microbes in nature is usually at a low level of efficiency by excessive light, which can be significantly inhibited owing to the photooxidation of photosynthetic organ damage.277 The elaborate nano-silica shells on diatoms in nature can provide mechanical protection, photon response and higher photosynthetic efficiency.278 Simulating the diatoms, Xiong et al. artificially endowed cyanobacteria with a nanoscale silica shell on the surface by biomimetic mineralization (Fig. 12H).279 It was found that the shell of silicon dioxide could help the cyanobacteria cells block a large part of the high-light, which can be served as “bio-amours” for organisms against strong light (Fig. 12I, J). Moreover, with the continuous expansion of human activities, the ultraviolet radiation is a serious environmental problem for harming the biological organisms.280 The modification of biological organisms by biomimetic materials also can be used for resist ultraviolet stress. For example, the artificial construction of shell with ceria nanoparticles on the Chlorella cells can improve the scattering of ultraviolet light to realize the mitigation with ultraviolet damage.281 Wang et al. fabricated a suitable shell on the eggs of zebrafish, which was consisted of rare earth materials to absorb ultraviolet light for providing the embryonic protection.282 Therefore, these biological modifications on living organisms with biomimetic materials can be used for various applications, such as cells preservation, enhancement of thermal stability and photosynthetic bioenergy.

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Fig. 11 (A) Schematic illustration of the preparation of the cluster-loaded hydrogel and its application in rat calvarial bone defective regeneration. (B) The bone regeneration of control, PAA/PASP, HAP, P-control, and cluster-loaded hydrogel group after 10 weeks in vivo, including the micro-CT images (Scale bars: 5 mm) and HE staining images (Scale bars: (i) 1 mm; (ii–iii) 50 mm). NB: new bone; O: Original bone; V: vessel; C: cluster-loaded hydrogel. Reproduced from Yao, S.; Xu, Y.; Zhou, Y.; Shao, C.; Liu, Z.; Jin, B.; Zhao, R.; Cao, H.; Pan, H.; Tang, R., ACS Appl. Bio. Mater. 2019, 2, 4408–4417.

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Fig. 12 (A) Schematic representation of calcium mineralization on the surface of yeast cells by layer self-assembly technique. (B) Curves showing the percentage of yeast cells in pure water. (C) Schematic illustration of the artificial shell on yeast cells, which composed with organic poly(norepinephrine) (PN) and inorganic silica. The (D) SEM micrographs and (E) EDX line-scan analysis of coated yeast cells confirming the presence of silica shells. (F) The schematic illustration of the silica nanocoating on HeLa cells through bioinspired silicification, which can protect them to resist harmful substances. (G) The survival ratio native of HeLa cells and HeLa@SiO2 cells under the enzymatic stress of trypsin. (H) Procedure for silica coating on individual cyanobacteria. The (I) photosynthetic O2 evolution and (J) PSII activity of native cyanobacteria and cyanobacteria@SiO2 cells. (A and B) Reproduced from Wang, B.; Liu, P.; Jiang, W. G.; Pan, H. H.; Xu, X. R.; Tang, R. K., Angew. Chem. Int. Ed. 2008, 47, 3560–3564; (C–E) reproduced from Hong, D.; Lee, H.; Ko, E. H.; Lee, J.; Cho, H.; Park, M.; Yang, S. H.; Choi, I. S., Chem. Sci. 2015, 6, 203–208; (F and G) reproduced from Lee, J.; Choi, J.; Park, J. H.; Kim, M. H.; Hong, D.; Cho, H.; Yang, S. H.; Choi, I. S., Angew. Chem. Int. Ed. 2014, 53, 8056–8059; (H–J) reproduced from Xiong, W.; Yang, Z.; Zhai, H.; Wang, G.; Xu, X.; Ma, W.; Tang, R., Chem. Commun. 2013, 49, 7525–7527.

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2.05.5.2

Bioenergy

With the increasing serious environmental problems caused by the extensive use of fossil energy, bioenergy serves as the renewable energy through biological activities playing an important role in energy development.283 Among these, the microbial energy represented is regarded as the most promising bioenergy.284 However, the development and utilization of bioenergy are mainly limited by the functions of biological organisms themselves. Therefore, it is necessary to find the ideal biological organisms or make artificial modification to reform biological organisms. At present, the application of modified organisms by biomimetic materials in bioenergy has been made in preliminary. For example, the hydrogen production can be achieved by photosynthetic microorganisms, which can directly photolysis water to generate hydrogen under the interaction with photosynthetic system and hydrogenase.285 Due to the hydrogenases would be rapidly inactivated from the oxygen production of photosynthesis, it is usually a very short and inefficient process. In order to develop a longtime and efficient hydrogen production, the aggregation of Chlorella cells had been fabricated inducing by biomimetic silicification (Fig. 13A and B).286 It was showed that the photosynthetic hydrogen production of aggregated Chlorella with significantly high rate lasted for at least 48 h under the condition of natural oxygen. Hydrogen and oxygen are distributed in these aggregated Chlorella cells with functional differentiation in space. Surface cells were exposed to the environment, which acted as a protective shell preventing oxygen from permeating into the interior of the aggregated Chlorella cells. These interior cells create a hypoxic microenvironment by using respiration to consume oxygen that could maintain the activity of hydrogenase and photosynthetic systemII for the sustainable photosynthetic production of hydrogen. At the meantime, the hypoxic conditions to enhance photolysis of water for hydrogen production also could be built through incorporation of polydopamine, laccase, and tannic acid (TA) on the surface of Chlorella cells to consume oxygen (Fig. 13C).287 The hydrogen production can be adjusted by controlling the concentration of the substrate TA, which can impact on the oxygen consumption. Moreover, the biomimetic silicon encapsulation also had been used on Escherichia coli (E. coli) by combination with CdS semiconductors for hydrogen photosynthesis to induce aggregation of them and produce aerobic microenvironment.288 These biocompatible CdS nanoparticles in the aggregated E. coli cells, which had light-harvesting capability, could utilize solar energy for hydrogen production. In addition, there were several tremendous progresses has been achieved in integration with biomimetic materials and biological organisms to develop bioenergy. The nonphotosynthetic organisms, such as bacterium Moorella thermoacetica (M. thermoacetica), could be converted into selfphotosensitization through combining with semiconductor nanoparticles289. It could enable the non-photosynthetic bacterium to use photogenic electrons generated by CdS nanoparticles, which could reduce carbon dioxide and obtain acetic acid through Wood-Ljungdahl pathway under the visible light (Fig. 13D–F). Meanwhile, the indium phosphide (InP) nanoparticles with highly efficient light-harvesting property can also be used for the bioenergy production. It could be modified on the genetic engineered yeast cells by polyphenol-based assembly technique to harvest photogenerated electrons (Fig. 13G).290 It was shown that the coated yeast cells by the illuminated InP nanoparticles could promote the cytosolic regeneration of redox cofactors for efficient bioenergy production (Fig. 13H). Besides, the biomimetic materials would not only apply on the surface of biological organisms, but also be used in the interior of them for fuel production. The gold nanoclusters (AuNCs) could be gotten into the interior of M. thermoacetica, which could be considered as a biocompatible intracellular light absorber for this non-photosynthetic bacterium.291 The AuNCs could covert the photo-excited electrons into cytoplasm-distributed redox mediators for production of CO2 and eliminate ROS to remain the cell viability. These researches not only have great significance for the construction of artificial transformation on living organisms, but also expand the application of bioenergy production in new pathways.

2.05.5.3

Environmental protection

During the heavy consumption and utilization of energy resources by human beings, there are various serious problems harming the ecological environment and human health, including water pollution, heavy metal pollution, etc.292 Among them, cyanobacteria bloom is one of the most important water environmental problems in the world.293 It would not only release the algal toxins to seriously harm the health of animals and humans, but also damage the ecosystem of aquatic environment to cause a series of other environmental problems.294 In contrast to the cyanobacteria, diatoms have silica shell with special structures to keep the cells submerged in the bottom, which grow more slowly and are less prone to bloom in the insufficient light and low temperature.295 Inspired by this phenomenon, Xiong et al. proposed a strategy, imitating biological silicon mineralization, to limit the bloom of Microcystis flos-aquae. The Microcystis flos-aquae could be directly composited with SiO2 nanoparticles induced by poly(dimethyl diallyl ammonium chloride) (PDADMAC) coating, which would rapidly sink to the bottom due to the larger size of aggregated cells (Fig. 14A–D).296 It was notable that this approach could delay the photosynthetic growth to inhibit the cyanobacteria blooms and the release of algal toxins for maintaining the oxygen concentration in water. Meanwhile, this method is effective not only in laboratory, but also in lake and field experiments (Fig. 14E). This new strategy for inhibiting blooms was important for the global water environment and water resources protection, which was expected to apply for reducing the costs of biological energy and solving more extended environmental problems in future.

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Fig. 13 (A) Native Chlorella cells and their aggregates. The bar indicates the standard color changes of WO3 in the presence of H2 production. (B) The scheme of aggregated Chlorella cells about the Spatial-functional differentiation. (C) Schematic illustration of the laccase-modulated anaerobic layer around individual Chlorella cells to H2 production under aerobic conditions. (D) Moorella thermoacetica-CdS reaction schematics. The CdS nanoparticles (shown in yellow) on cell surface could promote the conversion of CO2 (center right) to acetic acid (right). (E) Pathway diagram for the M. thermoacetica-CdS hybrid system. (F) Photosynthetic production of acetic acid by M. thermoacetica-CdS hybrids system. (G) InP nanoparticles were functionalized with polyphenol moieties and further assembled on the surface of genetically engineered yeast cells (Saccharomyces cerevisiae). (H) The shikimic acid production profiles under light and dark conditions. (B) Reproduced from Xiong, W.; Zhao, X. H.; Zhu, G. X.; Shao, C. Y.; Li, Y. L.; Ma, W. M.; Xu, X. R.; Tang, R. K., Angew. Chem. Int. Ed. 2015, 54, 11961–11965; (C) reproduced from Su, D.; Qi, J.; Liu, X.; Wang, L.; Zhang, H.; Xie, H.; Huang, X., Angew. Chem. Int. Ed. 2019, 58, 3992–3995; (F) reproduced from Sakimoto, K. K.; Wong, A. B.; Yang, P. D., Science 2016, 351, 74–77; (H) reproduced from Guo, J. L.; Suastegui, M.; Sakimoto, K. K.; Moody, V. M.; Xiao, G.; Nocera, D. G.; Joshi, N. S., Science 2018, 362, 813– 816.

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Fig. 14 (A) Scheme illustration of the direct incorporation with SiO2 nanoparticles on the Microcystis flos-aquae cells using PDADMAC to aggregate them. The optical microscope image and SEM image of (B) native cyanobacteria and (C) aggregated cyanobacteria, which demonstrate the nanosilica particles on their surface. (D) The performance of aggregated cyanobacteria in the beaker (100 mL) at different times. (E) Schematic representation of the prevention on cyanobacterial bloom inducing sedimentation by silica and PDADMAC. Reproduced from Xiong, W.; Tang, Y.; Shao, C.; Zhao, Y.; Jin, B.; Huang, T.; Miao, Y.; Shu, L.; Ma, W.; Xu, X.; Tang, R., Environ. Sci. Technol. 2017, 51, 12717–12726.

2.05.5.4

Biomedical therapy

The modifications of biological organisms by biomimetic materials are also potentially applied in biomedical therapies to keep human healthy, including the vaccine and cell treatment, etc.

2.05.5.4.1

Vaccine improvement

Vaccination is the most typical effective biological product against with infectious diseases, which is of great significance for human beings.297 However, due to the temperature-sensitive of vaccines, the transportations and storages are limited at a low temperature to maintain their structures and functions.298 Therefore, improving the thermal stability of vaccines is essential for reducing the expensive costs of cold chain and high rates of vaccine waste. Inspired by the mineralized state of virus, several biomineralization-based strategies were used to improve the thermostability and immunogenicity of vaccines.299 For example, a CaP mineralized Japanese encephalitis vaccine (JEV) had been successfully prepared through in-situ mineralization (Fig. 15A and B), which could be stored without losing its immunogenicity and activity for 1 week at room temperature (Fig. 15C).300 Obviously, it is viable that the thermostability of vaccines can be enhanced by rational design of biomimetic materials on their surface. But it is difficult for many vaccines to get the shielding shell through direct mineralization, owing to the lack of functional groups on their surface. An engineered vaccine with self-mineralization function had been obtained by genetic engineering, which was inserted the gene fragment of nucleating polypeptide. It could promote spontaneously the formation of CaP mineral layer on the surface of enterovirus 71 (EV71) in a solution that rich in calcium ions (Fig. 15D).301 Importantly, this engineered vaccine had good immunogenicity and significantly enhanced thermal stability (Fig. 15E), which was heritable through genetic engineering. In natural evolution, amorphous silicon minerals have been selected from hot spring bacteria to resist stress of plants. Meanwhile, some studies had shown that silicon could inhibit the fluidity of water molecules by forming hydrogen bonds between hydroxyl groups on surface and water molecules around.302 In order to explore the potential of biomimetic silicification in improving vaccines, Wang et al. investigated the performance of silica minerals on the surface of EV71 (Fig. 15F).303 It had been shown that the intact silica shell would let vaccine inactivated, but the discontinuous silica layer could greatly enhance its thermal stability without affecting the activity. By adjusting the pH of silicic acid, a large number of amorphous silicon nanoclusters formed on the surface of EV71 by in situ silicification, which could be stored for 35 days at room temperature (Fig. 15G). However, for the vaccines that lack mineralization sites on the surface, chemical modifications are usually required. The JEV, as an example, could be successfully biomimetic silicified forming a hydration layer on the surface through PEI modification, which could be preserved at room temperature for at least 15 days.304 Therefore, silicon mineralization is more effective than CaP mineralization in improving the thermal stability of vaccines. In addition, the alumina gel-like coating also could be spontaneously biomineralized on the surface of EV71 by electrostatic interaction, which could improve thermal stability and immunogenicity at the same time.305 Moreover, not only for the inorganic shells, the metal-organic framework (MOF) materials could encapsulate biomacromolecules as well, such as proteins, DNA and enzymes by biomimetic mineralization to provide superior protection.306 The MOFs can be applied on the surface of vaccines.307 For instance, the tobacco mosaic virus (TMV) could be endowed a shell of hydrolytically stable zeolitic

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Fig. 15 The (A) TEM images and (B) EDX analysis of mineralized JEV particles. (C) The thermostability assessments in vitro, which store at room temperature. Squares: native JEV; Circles: mineralized JEV. (D) The design of engineered EV71 carrying nucleating peptides. These peptides may induce in situ biomineralization on the surface of vaccine to form a CaP mineral exterior (gray). (E) Thermal-inactivation kinetics to test the thermostability at room temperature. (F) Schematic illustration of introducing exterior silica shells on EV71 by biomimetic silicification. (G) Thermalinactivation kinetics to test the thermostability of native and silicified EV71 at room temperature. (H) Schematic representation of CaP camouflage to inhibit the antibody recognition under extracellular conditions. Binding affinity between (I) 4G2, (J) 2A10G6 and the virus (DENV or shelled DENV) ranging from 3 to 5 log 10 PFU. (K) Schematic illustration of biomineralized vaccine for intranasal immunization. (C) Reproduced from Wang, G. C.;

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imidazolate framework-8 (ZIF-8) with highly symmetric quaternary structures to protect it from organic solvents and high temperature, which could provide a potential in functionalizing vaccines.308 Some vaccines can be utilized in gene therapy because they can introduce genes into various human cells.309 However, this treatment in biomedicine has been limited through the unnecessary immune responses, which rapidly eliminated the vectors by antibodies in human body.310 Wang et al. realized in-situ CaP mineralization on recombinant adenovirus serotype 5 (rAd5) and proposed the concept of “shell engineering of vaccines based on biomineralization.”311 The mineralized vaccine could circumvent the neutralization effect of antibodies and enhance the specific T cell immune response, which provided a new way of vaccine optimization and modification including HIV vaccine.312 Meanwhile, the CaP mineralization modification could be applied for increasing adhesive property313 (Fig. 15H–J) and modulating the immunity of vaccine to change the method of vaccinations (Fig. 15K).314 Besides, Zhou et al. found that avian influenza virus with CaP mineralized could enhance thermal stability and infectivity at the same time, and might lead to the transmission for humans beings.315 Furthermore, the virus could also be endowed a calcium and manganese carbonates (MnCaCs) biomineral shell, which could not be eliminated by host immune system and specifically replicate in cancer cells for antitumor therapy.316 Therefore, the vaccines can be improved by biomimetic mineralization that can enhance the thermostability, immunogenicity, and potentially functionalization.

2.05.5.4.2

Cancer treatment

There are various lymphocytes in the immune system of human body to keep our healthy, such as T cells, B cells, and natural killer cells.317 Due to the fragility of these lymphocytes in vitro, it is important to protect the cells from during the outside stresses for their operation and preservation.318 For example, a bioinspired TiO2 shell could be endowed for T cell by peptide-modification (Fig. 16A). This rational design would not only maintain the cell viability, juxtacrine interactions and cytokine secretion (Fig. 16B), but also prolong the storage time of T cells for cell therapy.319 Meanwhile, it is essential to track and monitor the objective cells for estimating the effect in cell therapies when the allogeneic host immune rejection occur. Multifunctional nanoparticles are the ideal biomimetic materials to combine with cell surface, which can maintain the activity and functionality of cells. For instance, the mesoporous silica nanoparticles with near-infrared fluorescent could be mildly modified on cell surface by reduction of disulfides.320 This coating of functional nanoparticles on cell surface could not only preserve the performance of ligands and receptors, but also track the position of cells in vivo during the early cell therapy. Furthermore, among the cell treatments, the cancer therapy is one of the most significant issues, which causes a great deal of death worldwide.321 Currently, the cancer treatments are limited to chemotherapy,322 radiotherapy and surgery.323 Unfortunately, it often has side effects on normal cells as well in chemotherapy and radiotherapy, and the limitation in surgical removal. In recent years, the research of biomimetic mineralization has provided many new ideas and methods for cancer treatment. For instance, due to the high expression of folate receptors (FR) in many human cancer cells, the injection of Ca2þ can specifically integrate FR enriched with folic acid to induce nucleation of calcium mineral on tumor (Fig. 16C).324 This specific mineralization of tumor could be achieved in vivo, leading to the dysfunction and death of cancer cells without damage for normal tissues or organs (Fig. 16D). Meanwhile, the in situ calcification targeting at cell membrane also can be used for inhibiting leukemia with the inhibition rate of 85%.325 In addition, based on the unique cytotoxicity mechanism of Ca2þ, the calcium peroxide nanoparticles (CaO2 NPs) with modification of pH-sensitive sodium-hyaluronate are used for making the death of cancer cells (Fig. 16E–H).326 The CaO2 NPs could only slowly decompose into free Ca2þ and H2O2 in the acidic tumoral microenvironment to induce the tumor calcification, but not enrich Ca2þin the normal cells due to the sufficient catalases. These calcium-induced strategies could inhibit survival of cancer cells and further show its great potential in clinical. Moreover, the multifunctional and biocompatible theranostic agents could also be fabricated with the combination of oxidization polymerization into the albumin-templated biomineralization. It demonstrated the good imaging contrast capabilities in MRI and ultrasonic imaging, which can be effectively applied for photothermal therapy in orthotopic tumors.327 The peptide nanotubes (PNTs) could be coated with oxaliplatin prodrug through covalent integration between Cu ions and imidazole groups on the surface of nanotubes. It could establish a versatile nanoplatform for cancer treatment, which can induce the production of reactive oxygen species by electron transfer and Fenton-like reaction to provide obvious hyperthermia effect. These tactics highlights the ability of biomimetic mineralization by modifying biological organisms and the promising potential of the multifunctional and biocompatible nanoplatforms for cancer therapies.

2.05.6

Conclusion

Biomineralization in nature is a process to generate inorganic biominerals through the regulation of biological macromolecules on living organisms. The formation processes of biominerals, including crystal nucleation, growth, morphology, homogenous and

=

Li, X. F.; Mo, L. J.; Song, Z. Y.; Chen, W.; Deng, Y. Q.; Zhao, H.; Qin, E.; Qin, C. F.; Tang, R. K., Angew. Chem. Int. Ed. 2012, 51, 10576–10579; (E) reproduced from Wang, G. C.; Cao, R. Y.; Chen, R.; Mo, L. J.; Han, J. F.; Wang, X. Y.; Xu, X. R.; Jiang, T.; Deng, Y. Q.; Lyu, K.; Zhu, S. Y.; Qin, E. D.; Tang, R. K.; Qin, C. F., Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 7619–7624. (G) reproduced from Wang, G.; Wang, H. J.; Zhou, H.; Nian, Q. G.; Song, Z.; Deng, Y. Q.; Wang, X.; Zhu, S. Y.; Li, X. F.; Qin, C. F.; Tang, R., ACS Nano 2015, 9, 799–808; (I and J) reproduced from Wang, X.; Deng, Y. Q.; Yang, D.; Xiao, Y.; Zhao, H.; Nian, Q. G.; Xu, X.; Li, X. F.; Tang, R.; Qin, C. F., Chem. Sci. 2017, 8, 8240–8246; (K) reproduced from Wang, X.; Yang, D.; Li, S.; Xu, X.; Qin, C. F.; Tang, R., Biomaterials 2016, 106, 286–294.

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Fig. 16 (A) Schematic illustration of the cytoprotective encapsulation of individual T cells with TiO2 shells. (B) Cytokine (IL-2) secretion of native T cells and coated T cells after stimulation. (C) Schematic representation of cancer cell targeted calcification. The folate receptors on cancer cell surface could specifically bind to folic acid molecules, which actively induce calcification owing to its carboxylate residues. (D) It could specifically bind Ca2þ from biological fluids to facilitate calcium mineral nucleation on tumor. (E) TEM images of CaO2 NPs (inset: a TEM electron diffraction image). (F) Time-dependent Ca2þ release from the CaO2 NPs dispersed in different pH buffer solutions. (G) Schematic illustration of the functional CaO2 NPs in tumor region. (H) CT images of mice for a larger tumor after the injection of multiple doses in 12 days. (B) Reproduced from Youn, W.; Ko, E. H.; Kim, M. H.; Park, M.; Hong, D.; Seisenbaeva, G. A.; Kessler, V. G.; Choi, I. S., Angew. Chem. Int. Ed. 2017, 56, 10702–10706; (D) reproduced from Zhao, R. B.; Wang, B.; Yang, X. Y.; Xiao, Y.; Wang, X. Y.; Shao, C. Y.; Tang, R. K., Angew. Chem. Int. Ed. 2016, 55, 5225–5229; (H) reproduced from Zhang, M.; Song, R.; Liu, Y.; Yi, Z.; Meng, X.; Zhang, J.; Tang, Z.; Yao, Z.; Liu, Y.; Liu, X.; Bu, W., Chem 2019, 5, 2171–2182.

heterogeneous modality, are usually strictly regulated by organism matrix of living organisms. Therefore, the morphology, crystallization behavior and material properties of biominerals are significantly different from those of the geologically corresponding minerals. These biominerals with hierarchical structures and marvelous functions could be applied for collagen mineralization, repair of dental tissues and skeletal tissues. For living organisms, they gradually learn to utilize materials by the natural biomineralization in biological evolution, which can change their own functions to adapt the environment. Inspired by the natural evolution process, the biological enhancements by biomimetic mineralization develop to combine the biomaterials and living organisms. The biological improvements can be used for almost all the basic units of organisms, including viruses, prokaryotes, animal cells, etc. Due to the functions and characteristics of specific cells, the corresponding functions and applications through modifications of biomimetic materials can be endowed. However, the current modification of biomimetic materials to improve biological organisms would harm organisms themselves, so that the biocompatibility and biosafety of biomimetic materials are required to be better. In addition, the available types of biomimetic materials for biomimetic mineralization are limited to calcium phosphate, calcium carbonate, silicon dioxide, etc., which give finite functions. Novel material systems and bonding methods are the basis for exploring new functions and applications in this field. Moreover, although there are several applications of biological improvement, they are mainly to realize cell protection, which are similar to the functions of most mineralized shells in nature. The modifications of vaccine are mainly focused on improving their thermal stability and stealth performance, which also belongs to the function of biological protection endowed by biomimetic materials. With the changed modification techniques and material-biological systems, the novel functions and applications, such as bioenergy development, environmental protection and biomedical therapy, have been discovered in recent years. In the future, it not only has further improvement on the original basis, but also has broad prospects in new methods, mechanisms and applications.

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Acknowledgment This study was supported by the National Natural Science Foundation of China (21625105). This work was also supported by the Pacific Northwest National Laboratory (PNNL). PNNL is a multiprogram national laboratory operated for the U.S. Department of Energy by Battelle under Contract DE-AC0576RL01830.

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2.06

Iron-sulfur clusters – functions of an ancient metal site

Sofia R. Pauletaa,b, Raquel Grazinac, Marta S.P. Carepoc, Jose´ J.G. Mourac, and Isabel Mourac, a Microbial Stress Lab, UCIBIO – Applied Molecular Biosciences Unit, Department of Chemistry, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal; b Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, Caparica, Portugal; and c LAQV, REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, Caparica, Portugal © 2023 Elsevier Ltd. All rights reserved.

2.06.1 2.06.2 2.06.2.1 2.06.2.1.1 2.06.2.1.2 2.06.2.1.3 2.06.2.1.4 2.06.2.1.5 2.06.2.2 2.06.2.2.1 2.06.2.2.2 2.06.3 2.06.3.1 2.06.3.1.1 2.06.3.2 2.06.3.2.1 2.06.3.2.2 2.06.3.2.3 2.06.3.3 2.06.3.4 2.06.4 2.06.4.1 2.06.4.2 2.06.4.2.1 2.06.4.2.2 2.06.4.2.3 2.06.5 2.06.5.1 2.06.6 Acknowledgments References

Introduction Type of centers and variability of coordination Basic structures and cluster coordination modes [1Fe] cluster [2Fe-2S] cluster [3Fe-4S] cluster [4Fe-4S] cluster Linear clusters and cluster interconversions Complex iron-sulfur clusters Unique clusters Organometallic and mixed-metal clusters Direct catalysis at iron-sulfur clusters Radical-SAM enzymes Examples of radical-SAM enzymes Iron-sulfur (de)hydratases Aconitase IspG and IspH involved in isoprenoid biosynthesis Pentonate dehydratases ADP-ribosyltransferases (unusual iron-sulfur cluster) Other enzymatic activities Iron-sulfur clusters involved in metabolic regulation Post-transcriptional regulation of iron homeostasis Transcription regulators Rrf2 family CRP-family Other transcription regulators The role of iron-sulfur clusters in DNA processing enzymes DNA repair glycosylases Conclusions

106 106 106 107 108 111 112 113 114 115 118 124 125 126 132 132 133 134 136 137 138 138 140 140 144 146 149 149 151 152 152

Abstract Iron-sulfur clusters are ubiquitous and ancient prosthetic groups that are present in all kingdoms of life. In the 1960s, they were recognized to play a role in electron-transfer reactions, but since then several other functions were identified, which can be attributed to their flexible coordination and redox properties. In here, the canonical iron-sulfur clusters, as well as the ones with other coordinating ligands will be described. The chapter has also been updated to account for the advances in the knowledge of complex iron-sulfur clusters of nitrogenase and hydrogenases. In addition, the role of iron-sulfur clusters in metabolic regulation, as sensors of gases (nitric oxide, oxygen), iron and cellular content of iron-sulfur clusters, cellular redox status, and redox cycling compounds, as well as their role in DNA processing enzymes, and their involvement in catalysis of a wide range of reactions will be described. Iron-sulfur clusters also participate in their biosynthetic and repair pathways. The knowledge in this field as evolved tremendously in recent years, which would require a complete chapter devoted to it by itself, reason why the authors have decided not to include this subject in this chapter. The chapter is an update of the one published in the previous edition, focusing on the recent advances mostly on the ironsulfur clusters involved in new catalytic functions, sensor mechanisms and DNA processing.

Comprehensive Inorganic Chemistry III, Volume 2

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2.06.1

Iron-sulfur clusters – functions of an ancient metal site

Introduction

Iron-sulfur clusters are ubiquitous and evolutionary ancient prosthetic groups that are required by all living organisms.1–4 Their primary role is electron transfer as they can delocalize electron density over both Fe and S atoms.5,6 Iron-sulfur clusters are the redox-active centers found in ferredoxins, one of the largest classes of electron shuttles in biology, and they are the major components of the photosynthetic and respiratory electron transfer chains as electron wires, defining the electron transfer pathways in many soluble redox enzymes and membrane-bound complexes.7 Besides this major function, iron-sulfur proteins have a wider role in nature, which includes catalysis of gases, non-redox catalysis (making use of its strong Lewis acid properties to polarize substrate bonds), radical chemistry (in radical-SAM dependent enzymes), regulation of transcription, and nucleic acid metabolism.3,8,9 Examples of iron-sulfur proteins involved in direct catalysis, regulation, and DNA processing will be described in Section 2.06.3, 2.06.4 and 2.06.5 of this Chapter, while Section 2.06.2 will focus mainly on the structure and properties of the basic iron-sulfur clusters, unique iron-sulfur clusters and of the organometallic and mixed-metal clusters. These examples show that the field of the iron-sulfur clusters has increased with clusters coordinated by different atoms, from the canonic ligands provided by the sulfur atoms of cysteines, such as nitrogen or oxygen atoms from residue sidechains. In this section is also described the structure of clusters with a higher number of iron atoms (up to 8), that are involved in the catalysis of gases (nitric oxide, nitrogen, hydrogen, and carbon monoxide), as well as sulfite. Some of these complex clusters contain iron and sulfur and other metals, and/or organic molecules. The authors would like to make a remark that this chapter is a revised and updated version of one published in 2013, but the biosynthesis of iron-sulfur clusters and complex clusters will not be addressed, since in 2020–2021 a series of comprehensive reviews have been published (on their maturation in plants,10 prokaryotes and eukaryotes, including mammals11–17). The main changes in this chapter are related with the recent discoveries on nitrogenase active site, hydrogenases, other catalytic activities, and enzymes involved in nucleic acid metabolism, including transcription regulators.

2.06.2

Type of centers and variability of coordination

2.06.2.1

Basic structures and cluster coordination modes

Iron-sulfur clusters were first detected as electron paramagnetic resonance signatures in mitochondrial membrane proteins,18 and afterwards in small proteins, such as ferredoxins.19,20 In few years, many other small iron-sulfur proteins were found and characterized.21 During the last decades, several studies implementing X-ray crystallography, mass-spectrometry, chemical synthesis of structural analogs, and several spectroscopic techniques revealed the structural components and the chemical and magnetic properties of the different iron-sulfur clusters.22–25 The four basic or canonical structures of iron-sulfur clusters can be distinguished by the number of iron and inorganic sulfur atoms as: [1Fe], [2Fe-2S], [3Fe-4S] and [4Fe-4S], which are represented in Fig. 1. In addition, there are larger and complex clusters containing up to eight iron atoms26,27 or containing other metals (e.g., nickel and molybdenum) besides iron, that will be discussed in Section 2.06.2.2.26,28 Relative to the atoms (and residues) that coordinate the iron-sulfur clusters, there is a clear preference for thiolate ligation, with cysteinyl sulfhydryl side chains being the most frequently observed ligands.23 However, different coordination has been identified and it is in many cases related with the role played by the iron-sulfur cluster in the protein function. The imidazole of histidine and, to a lesser extent, oxygen from aspartate, glutamate, glutamine, or nitrogen from arginine have been observed as ligands (Table 1). The oxygen atom from serine side-chains can also coordinate iron-sulfur cluster in variant proteins, and coordinating cysteines have been substituted by other residues as a mean to study the spectroscopic properties of the iron-sulfur cluster, though in many cases resulted in apo-proteins.52 In most cases there is two different type of ligands (Table 1), but there is already one report of three different types of ligands coordinating a [2Fe-2S] cluster in RsrR, a redox-sensitive response regulator (see Fig. 4C and Section 2.06.4.2.1.4 and Fi). Thus, it is plausible to hypothesize that different coordination will be identified in the future. This fact increases the difficulty for searching novel iron-sulfur clusters in the genome: (i) diverse and non-canonical coordination motifs are being described, and (ii) other conserved residues, besides cysteines (distantly located in the polypeptide or in different subunits) can coordinate these clusters. As it will be discussed in the following sections, iron-sulfur clusters can also be coordinated by non-protein ligands, such as water molecules, substrates (e.g., aconitase) or S-adenosylmethionine (in radical SAM enzymes). As mentioned, iron-sulfur proteins are widespread in the three kingdoms of life and are essential for life. Their prevalence is higher in anaerobic prokaryotic organisms, and in the majority of these organisms they account for around 5% of the genome.53 Their occurrence decreases to around 2.5% in facultative-anaerobic prokaryotes, and to around 2% in aerobic prokaryotes. In prokaryotes, the most common iron-sulfur clusters bound to proteins are [2Fe-2S] and [4Fe-4S] clusters, with a higher prevalence of [4Fe-4S] clusters. However, the relative abundance of each is dependents on the organism.53,54 The genome of eukaryotes encodes a much lower number of iron-sulfur proteins (around 0.4%), with most of these having a universal common ancestor. Another difference is the relative fraction of each type of iron-sulfur cluster, as in these organisms the number of [4Fe-4S] and [2Fe-2S] clusters is similar and they are mainly bound to proteins located in the mitochondria, cytosol, and nucleus.53

Iron-sulfur clusters – functions of an ancient metal site

(A)

(B)

(C)

(D)

107

Fig. 1 Structure of the basic iron-sulfur clusters. (A) [1Fe] rubredoxin (PDB ID 7RXN), (B) [2Fe-2S] cluster (PDB ID 1M2A), (C) [3Fe-4S] cluster (PDB ID 6FD1) and (D) [4Fe-4S] cluster (PDB ID 1E2U). The carbon, iron and sulfur atoms are represented as gray, orange, and yellow spheres, respectively. The image was created in Discovery Studio Visualizer (BIOVIA).

2.06.2.1.1

[1Fe] cluster

The [1Fe] cluster can be found in small proteins, named rubredoxins (with approximately 55 residues). This center is unique among iron-sulfur clusters, as it does not bind inorganic sulfur. Rubredoxins have a single iron atom tetrahedrally coordinated by four thiolate ligands (Fig. 1A) provided by two Cys-X2-Cys segments located in two symmetrically related loops. The reduction potential of the [1Fe]3 þ/2 þ couple in rubredoxins is within the  100 to þ 200 mV range (Fig. 2),55–58 and its spectroscopic properties showed that the metal center in the native state is a high-spin Fe3þ (S ¼ 5/2) and in the reduced state is a high-spin Fe2þ with an integer spin state (S¼ 2).59,60 The absorption spectrum of oxidized rubredoxin is characterized by two absorption bands with a maximum absorbance at 350 nm, 380 nm, and 490 nm, with a shoulder at 570 nm, corresponding to a ligand to metal charge transfer (LMCT) of the sigma orbital. The electron paramagnetic resonance (EPR) spectrum, typical of a high-spin Fe3þ center with a large zero-field splitting and high rhombic distortion (E/D z 1/3, D > 0), has a resonance at g  9.5 (from the lowest Kramers doublet) and a narrow intense signal at 4.3 (from the middle Kramers doublet).61 These proteins are electron shuttles in several metabolic pathways, such as hydrocarbon oxidation, and protection against reactive oxygen species (superoxide and hydrogen peroxide, as electron donors of superoxide reductase and rubrerythrin).55-57,62,63 These proteins have also been identified in plastids and play a role in electron transfer under certain environmental conditions. Another small protein that also contains a rubredoxin-type center is the Desulfovibrio gigas desulforedoxin.30,63–66 This protein is a homodimer with each monomer presenting a shortened rubredoxin-like fold with one Cys-X2-Cys and one Cys-Cys ligand loop. However, this protein is spectroscopically different from rubredoxin, which has been attributed to the distorted tetrahedral geometry of its iron-center due to the presence of two adjacent cysteines.67 An interesting property of these small proteins containing the simple [1Fe] center is the possibility of replacing the iron atom by other metals. In the case of rubredoxin its iron atom has been substituted by cobalt, copper, nickel, molybdenum and zinc,61,68–74 while desulforedoxin iron atom has been replaced by In3þ, Ga3þ, Cd2þ and Hg2þ.75 The metal-substituted rubredoxins and desulforedoxins have been investigated by visible, EPR and NMR spectroscopy.64,65,76 A longer rubredoxin has been identified in the aerobe Pseudomonas oleovorans and in Pseudomonas putida. These 170 residue-long proteins contain two rubredoxin-like domains connected by a linker of 70 residues.77,78 The rubredoxin-like center (either the “canonical” or the distorted-type center) has been identified in other proteins, that also bind other metal or non-metal co-factors. Superoxide reductase (named desulfoferrodoxin when was first isolated79) contains

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Iron-sulfur clusters – functions of an ancient metal site Table 1

Examples of proteins containing [1Fe], [2Fe-2S], [3Fe-4S] or [4Fe4S] clusters and their coordinating residues, with the unusual ligands highlighted in bold.

Protein [1Fe] Rubredoxin Desulforedoxin Desulfoferrodoxin NarE [2Fe-2S] Plant-and vertebrate-type ferredoxin Aldehyde oxidoreductase Ferrochelataseb Biotin synthase Rieske-type protein mitoNEETb RsrR [3Fe-4S] 3Fe Ferredoxin 7Fe Ferredoxin Aconitase [NiFe]-Hydrogenasec [4Fe-4S] 4Fe Ferredoxin 4Fe Ferredoxin (Pyrococcus furiosus) 8Fe Ferredoxin (2  [4Fe-4S]) HiPIP Hybrid cluster proteind [NiFe]-Hydrogenasee [FeFe]-Hydrogenasee Dihydropyrimidine dehydrogenaseb IspG

Iron-sulfur coordinating residues a

References

Cys6 Cys9 Cys39 Cys42 Cys9 Cys12 Cys28 Cys29 Cys9 Cys12 Cys28 Cys29 His46 His57 Cys67 Cys128

29 30 31 32

Cys41 Cys46 Cys49 Cys79

33

Cys40 Cys45 Cys48 Cys60 Cys100 Cys103 Cys137 Cys139 Cys196 Cys403Cys406 Cys411 Cys97 Cys128 Cys188 Arg290(Nh) Cys140 His142(Nd) Cys170 His173(Nd) Cys72 Cys74 Cys83 His87(Nd) Glu8(O3) His12(N3) Cys90 Cys110

34 35 36 37 38 39

Cys8 Cys14 Cys50 Cys8 Cys16 Cys49 Cys358 Cys421 Cys424 Cys228 Cys246 Cys249

40 41 42 43

Cys8 Cys11 Cys14 Cys50 Cys12 Asp15(Od) Cys18 Cys57

44 45

Cys8 Cys11 Cys14 Cys47 Cys18 Cys37 Cys40 Cys43 Cys41 Cys46 Cys61 Cys75 Cys3 Cys6 Cys15 Cys21 His185(Nd) Cys188 Cys213 Cys219 His94(N3) Cys98 Cys101 Cys107 Cys91 Cys130 Cys136 Gln156(O3)

46

Cys265 Cys 268 Cys 300 Glu307(O3)

51

47 48 43 49 50

a The residue numbering in the ligation mode is related with the protein in the reference. Proteins from other sources can present different coordination motifs. b These proteins have only been identified in eukaryotes. c This protein contains two additional [4Fe-4S] clusters, one of them is represent in this table and presents an unusual coordination motif. d This protein also contains an unusual iron-sulfur cluster. e These proteins contain more than one [4Fe-4S] cluster, but in here it is only represented the one with the unusual coordination mode. HiPIP - high potential iron protein.

a distorted rubredoxin-type center (as the one found in desulforedoxin).31 In addition to this center, it contains a single non-heme iron center, the active center, that is coordinated by four histidine residues and one cysteine ligand in a square pyramidal geometry.31,80 Rubrerythrin, similarly to superoxide reductase, is a non-heme iron enzyme, involved in reduction of hydrogen peroxide. This enzyme is a dimer with each monomer being a four-helix bundle, with a diiron-oxo center in the middle, and a rubredoxin-like center in the C-terminus.81 A rubredoxin-type center has also been identified as an additional domain in some classes of flavodiiron proteins.82 These enzymes, constituted by a two-domain core harboring a diiron center and a FMN (flavodoxin domain), are proposed to have oxygen, nitric oxide, and hydrogen peroxide reductase activities.83,84

2.06.2.1.2

[2Fe-2S] cluster

The proteins belonging to this class constitute a large family with several subgroups.85 The plant-type ferredoxins, that coordinate a [2Fe-2S] cluster (Fig. 1B), are electron carriers between photosystem I and several enzymes. The vertebrate (e.g., adrenodoxin) and

Iron-sulfur clusters – functions of an ancient metal site

[1Fe]

109

4Cys 2Cys, 2His 3Cys, 1His

[2Fe-2S] 3Cys, 1Arg

2Cys, 2Glutathione

4Cys

[3Fe-4S]

3Cys

3Cys, 1His

[4Fe-4S]

3Cys, 1Asp

4Cys Glycosylase

4Cys ferredoxin

-800

-400

4Cys HiPIP

0

400

800

Em (mV) vs S.H.E. Fig. 2 Reduction potentials observed for the different type of iron-sulfur clusters. The shaded region indicates the potential range observed for those clusters in pH dependence experiments. Adapted from Bak, D. W.; Elliott, S. J., Alternative FeS Cluster Ligands: Tuning Redox Potentials and Chemistry. Curr. Opin. Chem. Biol. 2014, 19, 50–58.

bacterial (e.g., putidaredoxin) ferredoxins transfer electrons to hydroxylating enzymes, which are usually P450 cytochromes.86 Another group include the [2Fe-2S] ferredoxins involved in the biosynthesis of iron-sulfur clusters (ISC machinery),3,87 and the XylT-type [2Fe-2S] ferredoxin implicated in the activation of some oxygenases.88 Other [2Fe-2S] ferredoxins have been isolated from hyperthermophile Aquifex aeolicus89 and halobacteria.90 The biotin synthase (SAM enzyme) also contains a [2Fe-2S] cluster that is involved in sulfur donation for the conversion of dethiobiotin to biotin during a single catalytic turnover (this enzyme and the role of the iron-sulfur cluster will be further discussed in Section 2.06.3.1.1.2). In this protein the binding motif of the [2Fe-2S] cluster is atypical, as it includes a nitrogen atom from an arginine side chain (Table 1 and Fig. 4D).36,91,92 Another curious example of [2Fe-2S] proteins is the mammalian ferrochelatase,35 the terminal enzyme of heme biosynthesis. The observation that the cluster is vital for activity and it is readily degraded by NO but it is not present in most of the equivalent bacterial enzymes, has led to the suggestion that it is part of a defense mechanism that prevents the infecting organism from using the heme synthesized by the host.93 The plant and vertebrate [2Fe-2S] ferredoxins are globular proteins (of approximately 100 residues), with the cluster located near the surface, but protected by a long loop that has three of the four coordinating cysteine residues.33,86,88,90,94 The opposite side of the protein consists of a four-stranded b-sheet covered by an a-helix, that together form a ubiquitin-like fold known as the b-grasp.95 The [2Fe-2S] cluster can exist in two oxidation states that differ by a single electron, [2Fe-2S]2þ and [2Fe-2S]1þ. The reduction potential of this redox pair ranges from - 420 to - 250 mV (Fig. 2), revealing the high reducing nature of these clusters.1 In the two oxidation states of the [2Fe-2S] cluster, the formal oxidation state of the two iron atoms are localized, being Fe3þ-Fe3þ and Fe3þFe2þ, as represented in Fig. 3A. In the all-ferric state, [2Fe-2S]2þ, the two Fe3þ are antiferromagnetically coupled, since the spins of the five d electrons on the two iron atoms are oppositely aligned, so that their pairing produces an effective S ¼ 0, diamagnetic ground state (EPR silent). In the reduced form, [2Fe-2S]1þ, the iron atoms have localized valences (S ¼ 5/2 (Fe3þ) and S ¼ 2 (Fe2þ)) and are antiferromagnetically coupled, leaving one net unpaired spin (with a S ¼ 1/2 ground state). The EPR signal has g values around 1.88, 1.94 and 2.04. These proteins have a characteristic dark-brown color and an absorption spectrum with maxima at around 330 nm, 420 nm, 460 nm, with a shoulder at 560 nm, corresponding to metal to ligand charge transfer bands.96 The circular dichroism (CD) spectrum of the oxidized state of [2Fe-2S] proteins presents positive bands at 420 nm and 460 nm, while in the reduced state there are negative bands at 440 nm and 510 nm.96,97 The resonance Raman spectra of these proteins have a few vibration modes in the low frequency region (in both oxidized and reduced state).97,98 2.06.2.1.2.1 Rieske proteins The Rieske proteins are considered a subclass of the [2Fe-2S] proteins. The Rieske clusters were first discovered and characterized as subunits of respiratory and photosynthetic complexes, as well as in small electron transfer proteins, such as ferredoxins.99,100 These proteins contain a [2Fe-2S] center that is coordinated by nitrogen atoms from the imidazole moiety of two histidine sidechains (Fig. 4A), instead of the usual four cysteinyl sulfhydryl. The basic structural framework of Rieske proteins (with approximately 120 residues) consists of three stacked b-sheets, of which the upper one includes the two ligand loops holding the [2Fe–2S] cluster.99,101 These two loops contain one cysteine and one histidine ligand each and are interconnected by a disulfide bond, which contributes significantly to the stability of the protein. The iron

110

Iron-sulfur clusters – functions of an ancient metal site

(A)

(B)

(C)

Fig. 3 The most usual oxidation states and respective spin states of the canonical iron-sulfur clusters: (A) [2Fe-2S] cluster, (B) [3Fe-4S] cluster, and (C) [4Fe-4S] cluster. In (C) the usual redox pair is highlighted by a dotted brown line and the redox pair of HiPIP is highlighted by a dotted red line. Legend: a – iron atoms with localized valences and b – iron atoms with delocalized valences with a formal charge of 2.5 þ.

Fig. 4 Structure of the [2Fe-2S] clusters with non-canonical coordination. (A) Rieske center (PDB ID 3h1J), (B) mitoNEET center (PDB ID 3EW0), (C) [2Fe-2S] cluster in RsrR (PDB ID 6HSE) and (D) [2Fe-2S] cluster in biotin synthase (PDB ID 1R30). The carbon, iron, nitrogen, oxygen, and sulfur atoms are represented as gray, orange, blue, red, and yellow, respectively. The image was created in Discovery Studio Visualizer (BIOVIA).

atom closer to the surface has two solvent-exposed histidine ligands and the other iron is bound to two buried cysteine residues.99,101 The oxidation states of [2Fe–2S]-Rieske cluster are the same as the ones of [2Fe–2S] plant-type ferredoxins, [2Fe–2S]2 þ/1 þ (Fig. 3A), but the histidine coordination causes an increase in the reduction potential. Therefore, many Rieske proteins present positive reduction potentials ( 100 to þ 400 mV)102 (Fig. 2). Moreover, as this cluster is coordinated by histidine sidechains, the reduction potential is pH dependent above pH 8. The pH profile has a slope of  120 mV/pH and is explained by two pKa values in the oxidized state (at 7.8 and 9.6) and one in the reduced state (at 12.5).103,104 This pH dependence was also observed in the visible and CD spectra.103

Iron-sulfur clusters – functions of an ancient metal site

111

The visible spectrum of Rieske proteins has absorption maxima at 325 nm and 460 nm with a shoulder at 560–580 nm. The CD spectra is quite unique, with two positive bands (310 nm and 350 nm) and a negative band at 375–380 nm, in the oxidized state, whist in the reduced state it has a positive band at 314 nm and a negative band at 385 nm and 500 nm.105 The EPR spectrum has a rhombic signal, but it varies significantly between proteins with gx of 1.72–1.834, gy of 1.888–1.92 and gz of 2.008–2.042.105 Electron nuclear double resonance (ENDOR), electron spin echo envelope modulation and hyperfine sublevel correlation (HYSCORE) experiments have also been used to characterize this cluster and to confirm the histidine coordination.105,106 Both oxidation states of Rieske proteins have been characterized by resonance Raman. The spectra show only small differences with shifts in the vibration modes, and when compared with the resonance Raman spectra of other [2Fe-2S] proteins, the spectra presents additional vibration modes corresponding to the histidine ligands (260–261 cm 1 Fe-His bending mode, weak peak at 266–270 cm 1 Fe3þ-His stretching mode).107,108 These spectra have also more bands in the 250–450 cm 1 regions as these clusters are less symmetric then the [2Fe-2S] clusters. The resonance Raman spectra also show a pH dependence but only at values above the pKa of the second histidine, which warrants a rapid proton-coupled electron transfer at physiological pH.108 2.06.2.1.2.2 NEET proteins The mitoNEET (cisd1), NAF-1/Miner1 (cisd2) and MiNT/Miner2 (cisd3) belong to the NEET family of iron-sulfur cluster binding proteins that are involved in iron homeostasis and response to reactive oxygen species (ROS) in eukaryotes.109–111 In humans, these proteins have also been associated with the proliferation of cancer cells.111 These proteins have in common a CDGSH domain, and a C-terminal sequence Asn-Glu-Glu-Thr (that gave their name NEET) and bind a [2Fe-2S] cluster coordinated by 3 cysteines and a histidine residue (Table 1 and Fig. 4B), that is redox active and labile. MitoNEET and NAF-1 are homodimers found in the outer membrane of the mitochondria, and NAF-1 has also been found in the endoplasmic reticulum, while MiNT is co-localized with the mitochondria. MitoNEET and NAF-1 are bound to the membrane by one N-terminal transmembrane helix and have a common fold.112 The CDGSH domain was also found in both bacteria and archaea, but in this case the proteins are monomeric and present two of these domains. The plant homolog (At-NEET) has also a N-terminal transmembrane domain and a single CDGSH domain. The NEET-fold is composed by a cluster binding domain and b-cap domain. The CDGSH domain comprises the cluster binding domain that harbors the [2Fe-2S] cluster. The coordinating histidine is solvent accessible, which has been proposed to confer lability to this iron-sulfur cluster. These proteins present an absorption spectrum with absorption bands with maxima at 340 nm (8.5 mM 1 cm 1), 460 nm (5 mM 1 cm 1) and a less intense band at 530 nm (4 mM 1 cm 1),109,113 in the oxidized state, while in the dithionitereduced spectrum these bands lose intensity. This cluster was shown to be redox active as upon exposure to molecular oxygen, the absorption bands recover the initial intensity. MitoNEET [2Fe-2S] cluster is EPR silent in the oxidized state, while in the reduced state its EPR spectrum, acquired in Ka-band at a microwave frequency of 31 GHz, has a rhombic signal with g values of 2.007, 2.937 and 1.897.114 The X-band EPR spectrum showed splitting in gy and gz, which was explained by a magnetic-dipolar interaction between the two iron-sulfur clusters in the homodimer.114 This interaction was not observed in the first preparations and EPR studies of this protein because the samples were not fully reduced.109 The resonance Raman spectrum shows a pH dependence between 6.2 and 8.0, consistent with protonation of the coordinating histidine, and it is characterized by intense vibrational modes at 330 cm 1, 347 cm 1, 394 cm 1, and less intense modes at 267 cm 1, 284 cm 1, and 293 cm 1 (associated with the coordinating histidine), that are sensitive to pH and phosphate binding.115 The reduction potential of mitoNEET was estimated to be 0 mV, at pH 7.0116,117 (Fig. 2) and has a pH dependence with a slope of  40 mV/pH. Different models were used to adjust the data, but in all these models the pKa of the coordinating histidine was determined to be around 7.117,118 Proteins involved in iron metabolism and biogenesis of iron-sulfur clusters (cluster transfer and repair) have iron-sulfur clusters coordinated by histidine residues.13 One of these is the heterodimeric complex Grx3/4-Bol2, that binds a [2Fe-2S] cluster. The Grx homodimer coordinate a bridging [2Fe-2S] cluster through a cysteine residue in each monomer (found in the conserved CGFS sequence motif) and two glutathione molecules. Upon formation of the heterocomplex Grx3/4-Bol2, the [2Fe-2S] cluster locates in the interface between the two proteins and is coordinated by one histidine and one cysteine from Bol2 (previously known as Fra2) and two cysteines from Grx.119

2.06.2.1.3

[3Fe-4S] cluster

The existence of a [3Fe-4S] cluster (Fig. 1C) was first recognized in ferredoxin I from the anaerobic nitrogen fixing bacterium Azotobacter vinelandii, which is a seven iron (7Fe) ferredoxin that contains, besides the [3Fe-4S] cluster, a [4Fe-4S] cluster.120 In parallel with this discovery, two other proteins played an important role in the identification and understanding of the nature of the [3Fe4S] cluster: D. gigas ferredoxin II and aconitase (see Section 2.06.3.2.1). Several spectroscopic studies on these proteins clearly showed that they should contain a cubic [3Fe-4S] cluster121–126 (Fig. 1C). However, the first structure determined for A. vinelandii ferredoxin I erroneously modeled this center as being an almost planar cyclic [3Fe-3S] core.127 The controversy around the existence of a [3Fe-4S] cluster was later resolved with the reevaluation of A. vinelandii ferredoxin I crystal structure128 and the determination of the crystal structure of D. gigas ferredoxin II at 1.7 Å resolution.129 The presence of a [3Fe-4S] cluster was later identified in other ferredoxins and in several enzymes, such as succinate dehydrogenase,130 fumarate reductase,131 nitrate reductase,132 glutamate synthase,133 and [NiFe] hydrogenases.134

112

Iron-sulfur clusters – functions of an ancient metal site

The magnetic and electronic properties of the [3Fe-4S] cluster, in the þ 1 and 0 oxidation states, have been extensively explored by several spectroscopic (e.g., EPR and Mössbauer,121,124 magnetic circular dichroism (MCD),125 extended X-ray absorption fine structure (EXAFS),135 resonance Raman98) and electrochemical136 techniques. In the oxidized state, [3Fe-4S]1þ, the cluster presents three high-spin ferric atoms (Fig. 3B), which are spin coupled to form an S ¼ 1/2 state and exhibits an almost isotropic EPR signal centered around g ¼ 2.02. Reduction of the cluster by one electron yields a [3Fe-4S]0 with an integer spin (S¼ 2), but the Mössbauer spectrum revealed the presence of two quadrupole doublets with an intensity ratio of 2:1. This suggested that the reduced state involved a coupled, delocalized Fe2þ-Fe3þ unit responsible for the outer doublet, with a single Fe3þ unit responsible for the inner doublet with half the intensity. In the couple Fe2þ-Fe3þ, the two iron atoms are formally in a Fe2.5 þ oxidation state (Fig. 2B). The reduction potential of the redox pair [3Fe-4S]1þ/[3Fe-4S]0 depends on the protein, varying between  460 and  70 mV137 (Fig. 2). Using electrochemical techniques, it was observed that the [3Fe-4S]0 cluster of D. gigas ferredoxin II can accept two additional electrons attaining a [3Fe-4S]2 oxidation state (Fig. 3B). This state is only possible to attain at very low reduction potentials (ca. -690 mV), at an electrode surface, and it was proposed that the three iron atoms are unusually in the ferrous state.136 This allferrous state was also observed for the Pyrococcus furiosus ferredoxin138 and for the [3Fe-4S] cluster of the Desulfovibrio africanus 7Fe ferredoxin III.139 The [3Fe-4S] cluster is coordinated by two cysteines in a Cys-X2-Y-X2-Cys motif with a more remote -CysPro- providing the third cysteine ligand137 (Table 1). In P. furiosus ferredoxin, Y is an aspartate, which coordinates the fourth iron atom.45,140,141 In fact, this ferredoxin is isolated with a [4Fe-4S] cluster that can be converted to a [3Fe-4S] cluster. On the contrary, Desulfovibrio alaskensis G20 7Fe ferredoxin is isolated with a [3Fe-4S] that can be converted to a [4Fe-4S] center.142 Another [3Fe-4S] cluster protein that has been extensively characterized is D. gigas ferredoxin II,129,143 which has a cysteine as the Y residue, coordinating the labile Fe atom in the [4Fe-4S] cluster of these ferredoxins.141,144 D. gigas ferredoxin II can also be isolated with a [4Fe-4S] cluster, and it was designated as ferredoxin I.136,145 The two ferredoxins differ in their reduction potentials and appear to have different metabolic functions in this bacterium.145 Moreover, the two clusters proved to be interconvertible, as when ferredoxin I is oxidized it leads to ferredoxin II, and the treatment of ferredoxin II with iron salts in a reducing environment leads to ferredoxin I (see Section 2.06.2.1.5). Although [3Fe-4S] clusters have been identified in some proteins, these clusters are rare when compared with the ubiquitous [2Fe-2S] and [4Fe-4S] clusters that are present in the three kingdoms of life.137 However, in contrast with the [2Fe-2S] and [4Fe4S] clusters, all the available evidence indicates that biologically relevant [3Fe-4S] clusters are coordinated exclusively by cysteine residues (Table 1).

2.06.2.1.4

[4Fe-4S] cluster

Historically, within this group a strong distinction has been made between ferredoxins with a negative reduction potential ( 700 to  150 mV),24 and the ones with a positive reduction potential (þ 100 to þ 450 mV) (Fig. 2), which are named High Potential Iron Proteins (HiPIPs).146 The HiPIPs have been isolated mostly, but not exclusively from photosynthetic bacteria,114 although their physiological role has only been well-established in photosynthetic pathways.147,148 The [4Fe-4S] clusters are found in numerous microbial, plant, and mammalian redox enzymes, including nitrate reductase,132 sulfite reductase,149 trimethylamine dehydrogenase150 and hydrogenases.151 The first suggestion for the presence of a [4Fe-4S] cluster (Fig. 1D) in a protein occurred in 1968, when a 4 Å resolution crystallographic study indicated the presence of a potentially tetrahedral [4Fe-4S] cluster in the HiPIP from Allochromatium vinosum (formely Chromatium vinosum).152 This fact was only clearly established in 1972, with the high-resolution structure of both A. vinosum HiPIPs.153 Moreover, the structures of both oxidized and reduced HiPIP have been determined, revealing that the [4Fe-4S] cluster remained intact during the redox interconversion.154 Later, other [4Fe-4S] clusters were identified in several crystallographic studies, such as in A. vinelandii ferredoxin I (that also harbors a [3Fe-4S] cluster155), and in the active form of aconitase.42 The [4Fe-4S] clusters are usually bound to the polypeptide chain by four cysteine residues, as shown in Table 1. The protein ligands of these clusters are arranged in a particular motif, since three of the iron atoms are bound to almost adjacent cysteines (Cys-X2-Cys-X2-Cys), while the fourth iron atom is bound to a cysteine from a distant portion of the polypeptide chain.4 The binding of a given cluster by cysteine residues from different segments of the polypeptide chain apparently helps stabilizing the tertiary structure of the protein.156 Besides cysteines, the [4Fe-4S] cluster can be coordinated by other residues, as shown in Table 1 (e.g., [NiFe]43 and [FeFe] hydrogenases,49 dihydropyrimidine dehydrogenase,50 only found in eukaryotes, and IspG51). The thiocubane unit can exist in at least three stable oxidation states (Fig. 2C). This so-called three-state model1,153,157 contrasts significantly with the previously described clusters, in which only two stable oxidation states are observed. It is important to point out that, in strong contrast with the [2Fe-2S] clusters, and similarly to [3Fe-4S]0, the valence electrons are delocalized in the [4Fe-4S] clusters (Fig. 3C). The three oxidation states that can be attained are: [4Fe-4S]3þ, [4Fe-4S]2þ and [4Fe-4S]1þ, corresponding to [2Fe3þ-2Fe2.5 þ], [4Fe2.5 þ] and [2Fe2.5 þ-2Fe2þ] valence-state combinations, respectively. The [4Fe-4S]3þ/[4Fe-4S]2þ pair is the high-potential redox couple characteristic of HiPIPs, while the [4Fe-4S]2þ/[4Fe-4S]1þ pair is responsible for the low-potential process characteristic of the classic ferredoxins. Under physiological conditions, only one of these redox couples appears to be accessible and functional.1,153 The absorption spectrum of [4Fe-4S]-ferredoxin type proteins has a characteristic broad absorption band with a maximum at around 400 nm in the oxidized state, while the HiPIP absorption spectrum has a broad band centered at 388 nm. Both the [4Fe-4S]3þ and the [4Fe-4S]1þ oxidation states of the cluster are paramagnetic and display characteristic EPR spectra. The [4Fe4S]1þ cluster in reduced ferredoxins displays a rhombic EPR signal with g values of 1.88, 1.92, and 2.06. The oxidized form

Iron-sulfur clusters – functions of an ancient metal site

113

([4Fe-4S]2þ state) of low-potential ferredoxins is EPR-silent and attempts to achieve a higher oxidation state, [4Fe-4S]þ 3, led to irreversible cluster decomposition, probably through a [3Fe-4S] intermediate. The [4Fe-4S]þ 3 signal is usually referred to as the HiPIP signal and shows distinct g values at 2.00–2.04 (gt) and 2.08–2.14 (g||). This signal is present in oxidized HiPIP but absent in reduced HiPIP.158–161 Reduction of HiPIP to the [4Fe-4S]1þ oxidation state, occurs in aqueous/DMSO solution but under partially denaturing conditions.157 EPR can also be used to distinguish a [2Fe-2S] cluster from a [4Fe-4S] cluster by performing a temperature dependence of the signal, as they differ in the relaxation time, which usually follows the order [2Fe-2S] < [3Fe4S] < [4Fe-4S]3þ < [4Fe-4S]1þ.162 Nevertheless, enhanced relaxation times can occur due to spin-spin interactions between clusters. The resonance Raman of [4Fe-4S]-ferredoxin type proteins have a predominant feature at 336 cm 1 assigned to vibration modes of the Fe-Sbridging, due to the total symmetry of the cubane structure.98 The CD spectra of HiPIP in the reduced and oxidized state are similar with a positive peak at 450 nm and two negative peaks at 350 nm and 390 nm. Nevertheless, some HiPIPs present quite distinct features.163

2.06.2.1.5

Linear clusters and cluster interconversions

Proteins containing [3Fe-4S] clusters proved to be particularly useful to study cluster conversions and to understand and identify the iron-sulfur cluster present in aconitase (see Section 2.06.3.2.1). The interconversion of [3Fe-4S] into [4Fe-4S] was shown to occur when the coordinating ligand of the fourth iron atom is not a cysteinyl ligand,122,139,140 as is the case of D. desulfuricans 7Fe ferredoxin and P. furiosus ferredoxin (being an oxygen from an aspartate,45,140–142 as mentioned before). Cluster interconversion have also been observed in transcription regulators upon reaction with molecular oxygen (see Section 2.06.4). The pathways of cluster interconversions were extensively probed and enabled the specific isotopic labeling of iron-sulfur clusters124 (Fig. 5). The in vitro interconversions studies were performed under non-physiological and near physiological conditions using D. gigas cell extracts.164 Further insights into the electronic, magnetic, and redox properties of the iron-sulfur clusters, as well as information on the sitespecific properties of the cubane clusters, was achieved based on the preparation of the so-called heterometallic cubane clusters [MFe3S4] by incorporation of a heterometal, M, in the vacant coordination site of a [3Fe-4S] cluster. This pioneer work of Munck, Moura, and co-workers led to the synthesis of heterometallic [CoFe3S4], [ZnFe3S4], [CdFe3S4], [NiFe3S4], [CuFe3S4], and [CrFe3S4] clusters, using D. gigas and P. furiosus ferredoxins as templates.44,165–168 These interconversion studies gathered important information on spin states, localized and delocalized valences, as well as reduction potentials. This same experimental procedure continues

Fig. 5 Iron-sulfur cluster interconversion pathways and isotopic labelling. In panel (A) is shown the possible interconversions starting with ferredoxin II, and in panel (B) the possible interconversions starting with apo ferredoxin II reconstituted with 57Fe. Interconversion pathway after incubation with (i) 57Fe2þ or (ii) 56Fe2þ, in the presence of sodium dithionite, dithiothreitol and sulfide (S2-), or (iii) upon oxidation with ferricyanide. 56 Fe and 57Fe are represented by orange and black circles, respectively.

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to be used to study more complex systems, either introducing other metals or 57Fe for Mössbauer experiments (see Section 2.06.3 and 2.06.4).169,170 The [3Fe-3S] intermediate cluster with a planar hexagonal structure has been proposed to be the intermediate in cluster interconversion and degradation mechanism of the transcription regulator RirA (an iron sensor) and in FNR (an oxygen sensor) upon exposure to molecular oxygen (see Section 2.06.4). A “linear” [3Fe-4S] cluster has been identified in the first spectroscopic studies of aconitase155,171 (Fig. 6). Although its physiological relevance is unclear, this cluster will be mentioned here due to its importance in cluster interconversion, protein-cluster interactions, and synthesis of heterometallic clusters. The [3Fe-4S] linear cluster was observed after subjecting aconitase to alkaline conditions, and its structure was confirmed by comparing the visible, EPR, Mössbauer and MCD data with the one obtained for a mimetic [3Fe-4S] linear cluster.172–174 Besides its linear structure, this cluster differs from the native one as it is coordinated by four cysteines, instead of three: Cys421 and Cys424 that also coordinate the native cluster, and two adjacent cysteine residues, Cys250 and Cys257, in substitution of Cys358155 (Fig. 6). The presence of similar linear clusters have been proposed to occur as intermediate species during the unfolding of ferredoxins,175,176 although there are some evidences that in this case there is formation of iron sulfide species rather than a stable linear cluster.177 A biologic linear [Fe-2S]n has been observed in IssA, an iron-sulfur storage protein from P. furiosus. This protein forms nanoparticles ( 300 nm, comprised of 20 nm spheres) inside the cell when this bacterium is grown in the presence of S0. The thioferratetype linear structure of this cluster has been assigned through Fe and S and K-edge X-ray absorption spectroscopy (XAS) and EXAFS, and EPR. P. furiosus IssA has 179 amino acids, with a N-terminal globular domain of 109 residues (IPR003731) and a cationic glycine-rich tail region in the C-terminus.178 The globular domain binds iron and sulfur spontaneously forming the linear cluster, and oligomerizes, while the tail is proposed to stabilize the structure through electrostatic interactions. In vitro studies showed that holo-IssA can reconstitute the [4Fe-4S] cluster of P. furiosus apo-ferredoxin.178 Although IssA physiological function remains unknown it is phylogenetically distant from NIF-related families (involved in the biosynthesis of nitrogenase cluster).178 Moreover, linear clusters with a similar structure but containing no iron (D. gigas Orange protein with Mo-Cu-Mo coordinated by sulfide179) or containing other metals in addition to iron (Mo or Cu, in the Blue protein from D. alaskensis or Desulfovibrio aminophilus, respectively180,181) have been identified as being non-covalently bound to the polypeptide chain. In fact, the visible spectra of these Blue proteins have a similar signature to the one of aconitase at high pH,172 indicating the presence of a [Fe-S] moiety.

2.06.2.2

Complex iron-sulfur clusters

Besides the classic iron-sulfur clusters already discussed, unique, larger, and more complex iron-sulfur clusters are present in a few proteins and enzymes. Among these are the hybrid cluster of hybrid cluster protein, the active site of sulfite reductases, the [8Fe-7S] (P-clusters) and [Mo-7Fe-9S] (FeMo-cofactor) clusters of nitrogenase, the unique organometallic site (H-cluster) of [FeFe] hydrogenases and the mixed-metal clusters of [NiFe] hydrogenase and carbon monoxide dehydrogenase/acetylCo-A synthase. Two other complex iron-sulfur clusters have been identified: a [8Fe–9S] cluster in the active site of a ATP-dependent reductase from Carboxydothermus hydrogenoformans182and a noncubane [4Fe-4S] cluster in the heterodisulfide reductase from methanogenic archaea.183

Fig. 6

Proposed mechanism for the interconversion of the cubane cluster into a “linear” cluster in aconitase.

Iron-sulfur clusters – functions of an ancient metal site 2.06.2.2.1

115

Unique clusters

The relevant features of unique iron-sulfur clusters will be discussed in this section. The structures of these clusters are shown in Fig. 7. 2.06.2.2.1.1 Hybrid cluster protein In 1989, an unusual iron-sulfur protein was isolated from Desulfovibrio vulgaris Hildenborough.184 The preliminary characterization of this protein showed that it was constituted by six iron atoms and six inorganic sulfur atoms per protein. Upon reduction with sodium dithionite, the protein showed an EPR signal (S ¼ 1/2) distinct from the canonical [2Fe-2S] and [4Fe-4S] clusters, but similar to synthetic compounds containing a [6Fe-6S]3þ cluster and known as the prismane center.184 Based on these observations, this unusual iron-sulfur protein was considered to contain a [6Fe-6S] cluster and was named as prismane protein. Not very long after this first discovery, in 1992, another iron-sulfur protein with similar features was isolated from Desulfovibrio desulfuricans ATCC 27774.185 A detailed spectroscopic study of this protein, which included EPR and Mössbauer spectroscopies, led to the conclusion that the protein contained not one [6Fe-6S] prismane cluster, but two distinct multinuclear iron-sulfur clusters. In 1998, the three-dimensional structure of the D. vulgaris Hildenborough protein was determined at 1.72 Å resolution186 and it was undoubtedly proven that the protein contains two distinct clusters, as previously predicted from the Mössbauer studies on the D. desulfuricans 27774 protein. However, the nuclearity of the clusters was incorrect and the protein was not constituted by [6Fe-6S]

(A)

(B)

Cys433

Glu273

His249

S5

Cys317 Cys

Fe5 O10 Fe7

Fe6

O8 O9

S6

Fe8

Cys458

ThioCys405

S7

Glu492

(C) (D)

Cys88 S1

Cys88

S1 Fe5

Fe5

Fe6

Fe6

Ser188

Ser188

Fig. 7 Structures of the unique iron-sulfur clusters described in this chapter. (A) Hybrid cluster (PDB ID 7DE4), (B) sulfite reductase active site (PDB ID 1AOP), (C) P-cluster (PN) in the reduced state (PDB ID 3MIN) and (D) P-cluster (POX) in the oxidized state (PDB ID 2MIN) of nitrogenase. The carbon, iron, molybdenum, nitrogen, oxygen, and sulfur are represented in gray, orange, purple, blue, red, and yellow, respectively. The images were created in Discovery Studio Visualizer (BIOVIA).

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Iron-sulfur clusters – functions of an ancient metal site

cluster but by two tetranuclear clusters, a cubane-type [4Fe-4S] cluster (cluster 1) and a cluster with a novel and unique structure (hybrid cluster), [4Fe-2S-3O]. The name prismane protein was abandoned since it became clear that the protein does not contain the initially postulated [6Fe-6S] cluster. The name fuscoredoxin was given to the protein by Moura et al.187 due to its brown color and because the protein could stabilize the iron-sulfur cluster in various oxidation states. Nevertheless, it was afterwards decided to name this protein as hybrid cluster protein (Hcp).187,188 Phylogenetic analysis of these proteins revealed three different families that differ in the sequence motif that binds the cubane cluster located in the N-terminus. Family 1 and 3, found in D. vulgaris Hildenborough and D. desulfuricans 27774, in methanogens and in (hyper)thermophilic bacteria and archaea, has the sequence motif Cys-X2-Cys-X7–8-Cys-X5-Cys. Family 2, found in facultative anaerobic Gram-negative bacteria (e.g., Escherichia coli and Salmonella enterica), has the sequence motif Cys-X2-Cys-X11-Cys-X6Cys. Family 3 differs from Family 1 due to 100-residues deletion between the N- and C-terminal domains. In Family 1 and 3, the cubane cluster is a conventional [4Fe-4S] cluster bound to the protein by the unusual sequence motif of the four cysteine residues.188 In Family 2, this sequence motif was initially proposed to bind a [2Fe-2S] cluster,189 but was then shown to also bind a [4Fe-4S] cluster.190 The hybrid cluster, [4Fe-2S-3O], is unique among the iron-sulfur clusters for which structures are available, in that it has both sulfur and oxygen bridges (Fig. 7A).188,190,191 The local environments of the metal atoms can be described as tetrahedral for Fe5 and Fe6, and trigonal bipyramidal for Fe7 and Fe8. Fe5 is coordinated by one protein ligand Cys433, two bridging S atoms (S5 and S6) and an oxygen, O10. Fe6 is also coordinated by one protein ligand, Cys317, which adopts a cis-peptide conformation, and two bridging S atoms (S5 and S6). Thus, this part of the cluster resembles either a [2Fe-2S] moiety or one face of a cubane cluster. However, Fe6 is also coordinated by an oxygen atom (O8), which bridges F6 and Fe8 to give a tetrahedral environment to Fe6. Fe7 is bound to three protein ligands, His249, Glu273 and Cys458, and to two oxygen atoms, O9 and O10, which bridges Fe8 and Fe5, respectively. Fe8 is coordinated by one protein ligand, Glu492, two bridging oxygen atoms (O8 and O9), a bridging sulfur, S6 (bridges Fe5 and Fe6) and a sulfur atom S7. S7 forms a persulfide with Sg of Cys405, producing a thiocysteine moiety. Thus, Fe8 has a distorted trigonal bipyramidal geometry188 (the residue numbering used are according with E. coli Hcp). The thiocysteine is a distinctive iron ligand that has also been observed in catalytic intermediates in sulfur transfer reactions.192 In 2002, Macedo et al. determined the X-ray structure of D. desulfuricans 27774 and D. vulgaris Hildenborough Hcp at 1.25 Å,191 showing that the structures of both proteins are essentially the same, comprising three domains and two iron-sulfur clusters. In that work, it was demonstrated that although the two Hcps were purified under different conditions, D. desulfuricans 27774 Hcp in the absence of oxygen and D. vulgaris Hildenborough Hcp in the presence of oxygen, the nature and the oxidation state of the hybrid cluster remained the same, thus being independent of the presence of oxygen. In the following year, these two Hcps were crystallized in the reduced state.48 The overall structure of the backbone did not differ from the as-isolated form (either under oxic or anoxic conditions), but structural changes in the hybrid cluster were observed, especially in the iron atom bonded to the persulfide. These changes were proposed to reflect the function of this protein, possibly as a reductase, though the nature of the substrate was unknown.48 In fact, to support this hypothesis there is a putative electron transfer pathway, composed by aromatic residues that bridge the hybrid cluster, the [4Fe-4S] cluster 1 and the protein surface. The distance between the two clusters is around 11 Å, which is amenable for electron transfer.48 A similar hypothesis has been proposed based on the E. coli Hcp structure,190 though in this case the redox partner is Hcr (vide infra). [4Fe-4S] cluster 1 is a one-electron accepting [4Fe-4S]2þ cubane that in the reduced state [4Fe-4S]1þ presents an unusual magnetism of a spin-admixed system (S ¼ 3/2). The hybrid cluster can be stabilized in four distinct oxidation states, ranging from the most oxidized state (þ 6), containing four Fe3þ, to the fully reduced state (þ 3), with three Fe2þ and one Fe3þ. Upon successive one-electron reduction and starting from the fully oxidized protein, four oxidation states were observed with spins states of S ¼ 0 (þ 6), S ¼ 9/2 and 1/2 (þ 5), S ¼ 0 and 4 (þ 4) and finally a spin state of S ¼ 1/2 for the fully reduced state (þ 3).185,187,193 For three decades the physiological function of Hcp remained unknown, and initially it was wrongly proposed to be a hydroxylamine oxidoreductase, converting hydroxylamine to ammonia.194,195 However, as mentioned, Hcps are present in several anaerobic and facultative bacteria, as well as in anaerobic archaea. In E. coli and Morganella morganii, hcp was shown to be expressed mainly under anaerobic conditions in the presence of either nitrate or nitrite.189 In fact, hcp is under the transcription regulation of NsrR.196 Moreover, in D. gigas a mutant strain that cannot produce Hcp was shown to be sensitive to nitrosative stress.197 In E. coli, Hcp is encoded by the operon, hcp-hcr, with the second gene coding for a NADH-dependent reductase (Hcr). These two proteins form a complex and together can reduce NO. This led to the hypothesis that Hcp is a high affinity NO reductase, being able to detoxify low concentrations of NO that accumulates in the bacterial cytoplasm during anaerobic growth.198 D. vulgaris Hildenborough Hcp was also shown to be a NO reductase and its catalytic mechanism was studied by EPR, showing the presence of a dinitrosyl Fe intermediate and that N2O binds Hcp in the reduced state.199 2.06.2.2.1.2 Sulfite reductase The reduction of sulfite is a vital reaction both in assimilatory pathways, which leads to the incorporation of sulfur into amino acids and other metabolites, or in dissimilatory pathways that are used by anaerobic microorganisms that respire sulfur compounds.200 Both assimilatory and dissimilatory sulfite reductases (SiRs) contain in the active site a unique combination of co-factors that includes a reduced porphyrin, designated by siroheme, which is coupled through a thiolate to a [4Fe-4S] cluster (see Fig. 7B).201,202

Iron-sulfur clusters – functions of an ancient metal site

117

The assimilatory sulfite reductases (aSiRs) have been extensively investigated and served as a model for this family of proteins since they are structurally well characterized.201 These proteins are monomeric and share low sequence similarity to dissimilatory sulfite reductases (dSiRs). One of the most extensively studied dSiRs is the protein also known as desulfoviridin from Desulfovibrio genus.203–205 dSiRs are large oligomeric proteins with a molecular weight in the order of 200 kDa and composed by two different types of subunits in a a2b2 arrangement. This protein, DsrAB, forms a stable complex with DsrC, which is also essential for its activity. It is proposed that DsrAB catalyzes, not the 6-electron but the 4-electron reduction of sulfite, forming a S0 intermediate. DrsC is the acceptor of this intermediate, forming a persulfide in one of its cysteine residues. After dissociation from DsrAB, DrsC is reduced by a membrane-bound protein complex (DsrMKJOP).206,207 The SiRs enzymes present unusual magnetic and electronic properties, which result from the close association of the paramagnetic siroheme and nominally diamagnetic [4Fe-4S] cluster. Most probably, this direct association of an iron-sulfur cluster to a siroheme has been conserved to provide efficient delivery of multiple electrons to a substrate bound to the other side of the siroheme in a controlled and specific manner.202,208–210 The isolated SiRs have a high-spin ferric (S ¼ 5/2) siroheme and a diamagnetic [4Fe-4S] cluster with a þ 2 charge, with exception of the so-called low molecular weight SiRs (alSiR), which are found in D. vulgaris Hildenborough, Methanosarcina barkeri and Desulfuromonas acetoxidans, that have a low-spin ferric (S ¼ 1/2) siroheme.204,211 For the E. coli SiR each co-factor can be reduced by one electron: the siroheme to a ferrous state (S ¼ 2), and the iron-sulfur cluster to a þ 1 state (S ¼ 1/2). These two co-factors can undergo electron-exchange interactions conferring paramagnetic properties to all the irons in the assembly, even when the [4Fe-4S] cluster is oxidized and nominally diamagnetic.210,212,213 The electron coupling between the two prosthetic groups was extensively characterized for this enzyme using several spectroscopic techniques (e.g. Mössbauer,209 EPR,202 57Fe ENDOR214), leading to the hypothesis that a ligand should be shared between the two cofactors. This assumption was confirmed when the structure of the enzyme was solved by X-ray crystallography, which proved that a cysteine residue bridges the two redox centers.201 The electron-coupling was also observed in the dSiRs and alSiRs, although the electronic and structural properties of the coupled siroheme-[4Fe-4S] unit are not identical and differ from the ones of E. coli SiR. It seems that the different spectral and redox properties of this class of enzymes are induced by variations in the protein environment surrounding each coupled center.204,211,215,216 Novel structural insights have been given for dSiRs with the studies on D. vulgaris Hildenborough dSiR, Desulfomicrobium norvegicum SiR,206,217,218 D. gigas SiR in two active forms219 and A. vinosum SiR.220 2.06.2.2.1.3 Nitrogenase (FeMo-cofactor and P-cluster) Nitrogenase is a two metalloprotein component system that catalyzes the reduction of dinitrogen to ammonia coupled to the hydrolysis of ATP.221–224 The two protein components are the Fe-protein, a homodimer containing a [4Fe-4S] cluster and the MFe protein, an heterotetramer that contains the P-cluster and the active site, the FeM cofactor (FeMco). The active site can be heterometallic (M ¼ Mo, V) or be homometallic (M ¼ Fe) as in the iron-only nitrogenase.225,226 Even though several mechanistic aspects of the catalysis need to be clarified it is a consensus that the process involves association and dissociation between the Fe-protein and the MFe protein and four electrons resulting from four bridging hydrides are essential for reductive hydrogen elimination with concomitant activation and reduction of the N^N triple bond.225,227,228 The MoFe protein is the most extensively studied nitrogenase containing the catalytic FeMoco, a [1Mo-7Fe-9S-1C]-homocitrate cluster.229 However, several diazotrophs under Mo scarcity conditions can activate a different set of genes encoding a vanadiumcontaining protein with a similar FeV cofactor (FeVco) that has important structural and functional differences.230 So far, there are no X-ray structural data for the iron-only nitrogenases, but the FeFe cofactor is thought to be a [8Fe-9S-1C] cluster.231,232 The Fe-protein has two identical subunits that symmetrically coordinates a single [4Fe-4S] cluster. The dimer has a a/b fold that is common in nucleotide binding proteins. The residues Cys97 and Cys132 (in Azotobacter vinelandii residue numbering) from both subunits coordinate the [4Fe-4S] cluster, and in the interface between the subunits are located several nucleotide binding motifs.233,234 Near the N-terminal of the Fe-protein is located a Walker A motif where the ATP or ADP binds, as this protein is a P-loop containing ATPase. The [4Fe-4S] cluster can have three oxidation states of þ 2 (oxidized state), þ 1 (reduced with dithionite) or 0 (reduced with Ti(III)).235,236 In nitrogenase the Fe-protein functions as a reductase that receives electrons, binds, and hydrolyses ATP, transferring an electron to the catalytic protein namely to the P-cluster. The P-cluster is an [8Fe-7S] cluster in the MFe protein that subsequently transfers this electron to the active site of the enzyme, the FeMco where the substrate is reduced. The catalytic versatility associated with nitrogenase that allows the reduction of different substrates (such as, C2H2, CN or CO), and not only N2 or H2 is associated with the ability to shuttle electrons throughout the enzyme cofactors.221,237,238 The MoFe protein is a a2b2 heterotetramer, where the a and b subunits exhibit a similar polypeptide fold consisting of three domains of the a/ b-type, with some extra helices.239,240 This protein contains two copies of two different types of unique metalloclusters: the FeMco and the P-cluster. The P-clusters are [8Fe-7S] clusters (Figs. 7C and D) that are proposed to be involved in electron transfer between the Fe-protein and the substrate reduction site of the FeMoco.241,242 The P-cluster has a unique overall architecture, constructed from two linked [4Fe-4S] subclusters that share a m6-sulfide at one corner, and it is the only known naturally occurring iron-sulfur cluster that contains serinate-O (b-Ser188) and amide-N (a-Cys88) ligands coordinating an iron atom, in addition to the typical cysteinyl-S ligands.243,244 The P-cluster is obtained after the purification process of the MFe protein in all ferrous state, as determined by Mössbauer spectroscopy245,246 with the 8 irons in the þ 2 oxidation state (dithionite is used in the purification) and this form of P-cluster, a [8Fe-7S]2þ, is called the PN-state. X-ray crystallographic data of two different oxidation states of the MoFe protein, the PN-state and

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Iron-sulfur clusters – functions of an ancient metal site

the two electrons oxidized POX-state, revealed that the P-cluster can undergo structural rearrangements.240,247 The P-cluster in the PN-state is highly symmetric and can be described as two [4Fe-4S] linked by a sulfur that is hexacoordinated, whereas in the POXstate the symmetry is lost. In the POX-state (see Fig. 7D), the [4Fe-4S] moieties are distorted, become more open and the Fe5 and Fe6 move away from the central sulfur and coordinate a backbone amide-N (a-Cys88) and a serinate-O (b-Ser188), respectively. In some nitrogenases, like the one isolated from Gluconacetobacter diazotrophicus, instead of Fe6 is Fe8 that is moved and becomes coordinated by a O from a tyrosine (Tyr 98).248 The coordinating Cys88 and Ser188 become deprotonated, and this may stabilize the POX-cluster lowering the reduction potential of the P-cluster and enabling conformational changes that could facilitate the electron transfer to the FeMco.249 The conformational changes observed in the P-cluster in different oxidation states are observed for either Mo or V nitrogenases.230,250 Going from the PN- to the POX-states, two electrons are transferred, though the catalytic mechanism involving N2 reduction is usually identified by a one-electron transfer steps. It is known that the treatment of the MoFe protein with small electron transfer molecules can induce different oxidation states in the P-cluster. In the PN-state, all the Fe atoms are in the ferrous oxidation state.245,246 Using mediators,246,251,252 the PN-state can be sequentially oxidized by three electrons, attaining the P1þ, P2þ (POX), and P3þ oxidation states. The P3 þ/2 þ redox couple is not reversible in vitro, and thus, it is concluded that this couple does not function in vivo. This suggests a model where the P1 þ/N and P2 þ/1 þ couples could be involved in P-cluster electron transfer, presenting the possibility of transferring one or two electrons from the P-cluster to the FeMoco during catalysis. The reduction potential of the couple P2 þ/1 þ is associated with aproton transfer and changes from - 224 mV vs. SHE at pH 6 to - 340 mV vs. SHE at pH¼8.5, whereas the reduction potential of the couple P1 þ/N is - 290 mV vs. SHE and is pH independent.  253 These reduction potentials are similar to the reduction potential of the [4Fe-4S] cluster in the Fe protein ( 420 mV vs. SHE, when MgATP is bound to the Feprotein).254 The PN-state of the P-cluster is EPR silent, while the P1þ and P2þ states are paramagnetic; the P1þ state is a mixed spin system with a spin sate of S ¼ 1/2 and S ¼ 5/2, and the P2þ state is an S > 3 system with a EPR signal in the perpendicular mode.255 Although being EPR active, the P1þ and P2þ states have been difficult to detect during nitrogenase turnover, and there is little information about the oxidation states that the P-cluster attains during turnover.256 In the P1þ state, the Fe5 remains in the same position as in PN-state but the Fe6 is coordinated by Ser188, this implies P1þ can be an intermediate and the P-cluster can accept or donate one or two electrons, as the Fe protein [4Fe-4S] cluster. The transfer of two electrons could represent a 50% ATP saving for the enzyme. The active site of nitrogenases, the FeMco cluster, is a bridged double [4Fe-4S] cubane cluster with a metal M at an apical position. The metal can be Mo, V or Fe and so far, X-ray crystallographic data is available only for the heterometallic nitrogenases, as mentioned. Mo3þ and the V3þ are coordinated by 3 sulfide ligands from one of the cubane clusters.229,230 The FeMco cluster is bound to the polypeptide chain by a histidine residue that also coordinates the metal and a Cys residue that coordinates the Fe1 in the opposite side of the FeMco cluster (Fig. 8A). An homocitrate molecule coordinates bidently the Mo/V atom that are octahedrally coordinated. In both nitrogenases a central ligand (a C4) from a S-adenosyl methionine binds the two cubane clusters and it is hexacoordinated by 6 iron atoms (Fe2, Fe3 and F4 from one cubane cluster and Fe6, Fe7 and Fe5 from the other cubane).240 The main difference observed between the active site of both Mo and V nitrogenases besides the replacement of Mo by V is the nature of an additional ligand bridging the two cubane clusters, between the Fe5 and the Fe4 (Fig. 8B). The FeMoco cluster has a sulfide binding these two irons, whereas in the FeVco cluster this ligand is a carbonate (CO32þ).230 The difference in size between these two ligands is responsible for higher distances between the two irons in the FeV protein and a general distortion observed in all different metal-metal bonds between the two cofactors.230,240 The different metals in the active site have a major impact in the reduction of N2 to ammonia. The catalytic efficiency of the three types of nitrogenases is associated with differences observed for the rate constant concerning the ratio N2 reduction/H2 formation being the Mo nitrogenase the most efficient and the Fe nitrogenase the least.257 The binding of the substrate in still a matter of discussion. The FeMco cluster has no available coordination sites but the sulfide S2B that coordinates the Fe2 and Fe6, have a His and a Gln residues in the vicinity, and can be replaced by CO in both the VFe protein and the MoFe protein (Fig. 8C).250,258,259 The His residue can form a hydrogen bond with 2B sulfide, connecting the active site with the protein surface by accessing to a hydrogen-bonding pathway that is probably involved in proton transfer during catalysis. In the resting state the side chain of the conserved Gln in the vicinity of the FeMco is directed away from the cluster. However, for the VFe protein in a turnover state Gln176 can rotate toward the cluster with the amide oxygen from this residue forming two hydrogen bonds, one with His180 at a 2.84 Å and a second hydrogen bond with a bridging ligand (N/O). Recently a crystal structure of a Mo-nitrogenase obtained in the presence of N2 (turnover conditions) suggested a displacement of the belt-sulfur sites corresponding to S2B, S3A and S5B by N2,260 however this is still a matter of controversy.261,262

2.06.2.2.2

Organometallic and mixed-metal clusters

In this section, the properties of the fascinating and unusual organometallic and mixed-metal clusters will be described. The structure of these clusters is presented in Fig. 9. 2.06.2.2.2.1 Hydrogenases Hydrogenases are a family of enzymes that catalyze the reversible two electron oxidation of hydrogen: H2 4 2Hþ þ 2 e, and many microorganisms use hydrogenase to metabolize H2. They are a heterogeneous group of enzymes that differ in size, subunit composition, metal content and cellular location. However, based on their metal content, two main groups can be distinguished: the [FeFe]

Iron-sulfur clusters – functions of an ancient metal site

(A)

(B)

(C)

Cys257

Cys275 His195

Cys257

His180

His180

1

2

4

2B

3A 6

1

1 3

2

34

6

75

75

2

CO326

Gln176

75

Gln176

His442 homocitrate

34

CO32-

2B

Gln191

119

His423 homocitrate

His423 homocitrate

Fig. 8 The catalytic FeMco of nitrogenase [M:7Fe:9/8S:C] is a complex iron-sulfur cluster bound to an apical metal (M ¼ Mo, V, Fe) coordinated to a homocitrate molecule. In panel (A) is shown the FeMoco (PDB ID 3U7Q), in panel (B) the FeVco (PDB ID 5N6Y) and in panel (C) the FeVco with bound CO in the position of S2B (PDB ID 7ADR). The iron, sulfur, molybdenum, and vanadium atoms are represented in orange, yellow, purple, and green, respectively. The carbon, oxygen and nitrogen atoms are represented in gray, red and blue, respectively. Images were created with the program Discovery Studio Visualizer (BIOVIA).

hydrogenases, which contain only Fe, and the [NiFe] hydrogenases, which contain both Ni and Fe in the active site. The [FeFe] hydrogenases catalyze the reduction of proton as terminal electron acceptor to yield H2 and thus mainly function in H2 production, while the [NiFe] hydrogenases most often catalyze the forward reaction in which H2 is consumed.151,263–266 Hydrogenases have been the subject of great attention and research, which is mainly related with the potential application of these enzymes in green hydrogen production, a future important alternative energy source.267 However, one of the major drawbacks is their sensitivity toward oxygen. All hydrogenases are oxygen sensitive but the [FeFe] hydrogenases are more sensitive toward oxygen than the [NiFe] hydrogenases that can be aerobically purified because the oxygen inhibition is reversible.268,269 Some membrane-bound [NiFe] hydrogenases found in aerobic bacteria can even oxidize H2 and promote hydrogen cycling in the presence of oxygen. The majority of the [FeFe] hydrogenases are irreversibly inactivated by oxygen and even though some exceptions are found in nature, so far only a particular type of [NiFe] hydrogenase can oxidize hydrogen.270–273 2.06.2.2.2.1.1 [FeFe] hydrogenases Several [FeFe] hydrogenases isolated from anaerobic bacteria, such as D. vulgaris and D. desulfuricans, Megasphaera elsdenii and Clostridium pasteurianum (which contains two different hydrogenases, CpI and CpII) have been well characterized using several spectroscopic techniques.151,274–282 Despite some similarities among the various [FeFe] hydrogenases, variations in the metal content exist that translate into differences in their iron-sulfur cluster content. However, all of them have in common the presence of iron-sulfur clusters (termed F clusters), the H-cluster (the catalytic site), and carbon monoxide (CO) as a potent inhibitor. The F clusters usually are [4Fe-4S] cubane-type clusters with exception of C. pasteurianum CpI that also contains a [2Fe-2S] cluster. The structures of two [FeFe] hydrogenases have been solved by X-ray crystallography: the periplasmic [FeFe] hydrogenase from D. desulfuricans283 and the cytoplasmic [FeFe] hydrogenase from C. pasteurianum (CpI).49,283,284 The structures of these enzymes clearly revealed the presence of an unusual active site, the so-called H-cluster, which is constituted by a [2Fe]H subcluster linked to the [4Fe-4S] subcluster via a cysteine thiol group (see Fig. 9A). The binuclear cluster is formed by two iron atoms, the proximal Fe (Fep), which is the one near the [4Fe-4S] cluster, and the distal Fe (Fed) (Fig. 9A), each coordinated by two diatomic, non-protein ligands, CO and CN. This site also carries a bridging CO and an azapropane-1,3-dithiolate (ADT) bridge between the two Fe atoms. In result, the Fep is hexacoordinated whereas the Fed is pentacoordinated and therefore the Fed was identified as the substrate binding site where the catalysis occurs. This was later corroborated by a structure obtained with a CO coordinated to the Fed.285–287 The distal Fe can either bind CO inhibiting hydrogenase activity or a hydride or a hydrogen, as part of the catalytic cycle. The conjugation between the ADT nitrogen, acting as a base, and the low valent Fed provides the conditions for heterolytic splitting of hydrogen.288,289 Furthermore, the CN ligands can provide H-bonding with a serine and a lysine present in the cluster surroundings that helps stabilizing the [2Fe]H cluster that is bound to the [4Fe4S]H cluster by a thiol group from a cysteine residue. Several oxidation states of the H-cluster have already been identified and characterized spectroscopically. The active oxidized state (Hox) is characterized by a mixed valence (Fe1þ-Fe2þ) configuration of the bi-nuclear cluster. These iron atoms are in a low spin state and present an S ¼ 1/2 EPR signal. Reduction of the H-cluster in the Hox state leads to the active reduced state (Hred), which is EPR silent and has both iron atoms of the [2Fe] subcluster most probably in the Fe1þ-Fe1þ formal oxidation state.151 The CO-inhibited state is EPR active due to a Fe1þ-Fe2þ mixed valence state of the bi-nuclear cluster, similar to Hox.290–294 According to Mössbauer studies, large 57Fe hyperfine coupling are observed between [4Fe4S]H and [2Fe]H clusters, suggesting a strong

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(A)

(B)

Fep

(D)

C

X

Fed

(E)

Cys509

FCII

Nid Nip

Fig. 9 Structures of the organometallic and mixed-metal clusters described in this chapter. (A) H-cluster of [FeFe] hydrogenase (PDB ID 1HFE), (B) NiFe active site of [NiFe] hydrogenase in the oxidized form (PDB ID 1FRV), (C) NiFe active site of [NiFeSe] hydrogenase (PDB ID 5JT1), (D) C-cluster (PDB ID 6B6X) and (E) A-cluster (PDB ID 1OAO) of CO dehydrogenase/acetyl Co-A synthase. The iron, sulfur, selenium, and nickel atoms are represented in orange, yellow, pink and green, respectively. The carbon, oxygen and nitrogen atoms are represented in gray, red and blue, respectively. The X identifies the position of a third bridging ligand that can be an oxygenated species in inactive states of the enzyme (Ni-A or Ni-B) or a hydride in active forms (Ni-C and Ni-R). Images were created with the program Discovery Studio Visualizer (BIOVIA).

exchange coupling between them.281 1H NMR spectroscopy was used to identify paramagnetically shifted 1H resonances from Chlamydomonas reinhardtii hydrogenase.295 A signal similar to bacterial ferredoxins was observed for the unmaturated enzyme containing only the [4Fe-4S]H. In the maturated protein in the Hox and Hox-CO states, shifted 1H resonances were identified originated from the methylene protons of the ADT bridging ligand of the [2Fe]H subsite.295 Furthermore, in the Hox state the spin density is shifted toward de distal Fe whereas for Hox-CO states the spin density is more evenly distributed between the two Fe atoms indicating that upon the substrate binding electron density can be modulated between the two subsites.294,296 In the H-cluster the single reduced state can occur with or without protonation of the nitrogen of the ADT ligand depending on the pH. At low pH values, the protonation occurs, and the CO bridging ligand is absent whereas at high pH values the protonation is not observed and the CO bridge is kept. The protonation converts NH into NH2 causing electronic rearrangement moving the reducing equivalent from the [4Fe4S]H to the [2Fe]H subsite and this step is extremely important in catalysis. The reduction potentials associated with the first and second reduction steps are similar to the Hþ/H2 potential and the transition between the Hred state and the HredHþ state has a pKa close to 7.297 The two electrons and the one proton can probably combine and generate a hydride in the Fed and further protonation will generate H2. However, states like HhydHþ and Hox-H2 were not yet observed maybe due to its highly transient nature. 2.06.2.2.2.1.2 [NiFe] hydrogenases [NiFe] hydrogenases can be divided into two major groups according to its sensitivity toward oxygen, the standard O2-sensitive and the O2-tolerant [NiFe] hydrogenases. X-ray crystallographic structures of [NiFe] hydrogenases belonging to the O2-sensitive group are available from D. gigas,43,298 Desulfovibrio vulgaris Miyazaki F,299–301 D. desulfuricans,302 Desulfovibrio fructosovorans303 and Desulfomicrobium baculatum,304 closely related sulfate reducing bacteria. All these hydrogenases have a small (z 30 kDa) and a large subunit (z 60 kDa). The small subunit contains the three iron-sulfur clusters (two [4Fe-4S] clusters and one [3Fe-4S] cluster) that are involved in the electron transport to/from the active site ([NiFe] cluster). In the catalytically active hydrogenases, a proximal

Iron-sulfur clusters – functions of an ancient metal site

121

[4Fe-4S]p2 þ/þ cluster is located near the [NiFe] cluster, flanked by a medial [3Fe-4S]mþ 1/0 cluster. Near the protein surface, a distal [4Fe-4S]dþ 2/þ completes the biological electron transfer pathway in these type of enzymes.305 The reduction potential estimated for these clusters in D. gigas hydrogenase was - 315 mV for the [4Fe-4S]p,  80 mV for the [3Fe-4S]m and  445 mV for the [4Fe-4S]d.306 Upon reduction, the [4Fe-4S]pþ magnetically interacts with the [NiFe] active site151 and the [4Fe-4S]p2 þ/þ controls the electron flow from the buried [NiFe] active site present in the large subunit to the hydrogenase surface in the small subunit, controlling the electrons flow with the redox partner in the enzyme surface.307 The large subunit contains the [NiFe] active site (see Fig. 9B), and the geometry of this site is highly conserved throughout all [NiFe] hydrogenases.308 The nickel and iron atoms are separated by a distance of about 2.5–2.9 Å264 and are bridged by the thiol groups of two cysteines. The nickel atom is coordinated by two additional thiol groups from cysteines bound in a terminal position. The iron atom carries three inorganic diatomic ligands that have been identified by infrared (IR) spectroscopy as two CN and one CO.309–311 The oxidized inactive state in general is a mixture of the so-called “unready” or Ni-A and “ready”-Ni-B states that correspond to a slow or fast catalytic activation state respectively. In these states additional electron density is detectable between nickel and iron, which seems to arise from a third oxygenated bridging ligand.299,312 Both oxidized states, are paramagnetic and characterized by different g values. The bridging ligand in the Ni-B state is an OH and probably the same bridging ligand is present in the Ni-A state. The differences observed in the hyperfine coupling constants between the two states may be due to rotation of Cys549 about Ca–Cb–Sg–Ni dihedral angle. Cys546 rotation can also account for differences observed in the intermediate g value.301,313–315 In the active state of the enzyme (Ni-C/Ni-R) the bridging ligand is a hydride (H).316 In all states of standard hydrogenases, the nickel atom has an open coordination site, which defines an axial direction, and it is therefore believed that the Ni represents the primary hydrogen binding site. This is supported by the fact that the inhibitor CO binds at this position and that the H2 transfer channel ends near the Ni.300,303 It has been shown by X-ray crystallography of single crystals treated with CO,300 that the CO binds at the sixth free-coordination site of the nickel atom (see Fig. 9B). The Ni-CO state is paramagnetic and photosensitive. Upon illumination at low temperatures, the CO molecule photodissociates, resulting in the Ni-L state, the same state formed from Ni-C.317 The H2 molecule can access the buried [NiFe] active site through four hydrophobic tunnels that combine into one, leading to the Ni atom. In the process of enzyme “activation” and during the catalytic cycle, the [NiFe] hydrogenase passes through several intermediate states (Fig. 10) observed and characterized by EPR spectroscopy, which showed that the enzyme cycles between EPR silent and EPRdetectable (paramagnetic) nickel-centered states. The oxidized inactive states Ni-A, Ni-B, mentioned above, and the active Ni-C state are all paramagnetic and EPR active, Ni-L is light-induced and EPR active, Ni-SI is EPR silent, and Ni-R is reduced, and EPR-silent.318 A full characterization has become possible by Fourier-transform infrared (FTIR) spectroscopy by which the IR vibrations of the CN and CO ligands at the iron are monitored. In the IR experiments, both the paramagnetic and EPR-silent states are detected.310 Upon one-electron reduction of Ni-A and Ni-B, the EPR-silent states Ni-SU (silent unready) and Ni-SIr (silent ready) are formed. The Ni3þ is reduced to Ni2þ in both EPR silent states and the oxygenated species is still present as a bridging ligand. Under reducing conditions at temperatures  30  C, the Ni-SIr is converted into another EPR-silent state, Ni-SIa (silent active). This step leads to removal of the OH binding ligand and the Ni2þ is then tetracoordinated leaving a vacant coordination position. The Ni-Sia can be further hydrogen reduced and generate the fully reduced state Ni-R. In this state a hydride is present between Ni2þ (NiH  1.58 Å) and Fe2þ (Fe-H  1.78 Å), and the Cys546 that coordinates the Ni atom becomes protonated. Alternatively, the conserved Arg479 in D. vulgaris Miyazaki F [NiFe] hydrogenase was proposed as proton acceptor. One-electron oxidation of

Fig. 10 Scheme of the oxidation states of [Ni-Fe] hydrogenase. The EPR-detectable states and the EPR-silent states are shown in red and blue, respectively. The states involved in the catalytic cycle of the enzyme are represented in the yellow box. The nomenclature of the states is the following Ni-A (unready state), Ni-B (ready state), Ni-SU (EPR-silent unready state), Ni-SIr (EPR-silent ready state), Ni-SIa (EPR-silent active state), Ni-SCO (EPR-silent CO inhibited state), Ni-C (EPR-detectable reduced state), Ni-L (Light induced state), Ni-CO (EPR-detectable CO inhibited state), and Ni-R (EPR-silent reduced state). The reduction potential of the redox couples is given for pH 7.4, except for the Ni-A/Ni-SU pair, that is given for pH 8.2. Adapted from Pandelia, M. E.; Ogata, H.; Currell, L. J.; Flores, M.; Lubitz, W. Inhibition of the [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F by Carbon Monoxide: An FTIR and EPR Spectroscopic Study. Biochim. Biophys. Acta 2010, 1797(2), 304–313.

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Ni-R leading to the oxidation of Ni2þ to Ni3þ generates the Ni-C EPR active state. In this state the hydride remains a bridging ligand between the Ni and Fe atoms and the terminal Cys546 coordinating the Ni is no longer protonated (Cys546-S). The Ni-C state is light-sensitive. Upon illumination with white light, the characteristic EPR signal disappears, and a new signal named Ni-L emerge. In Ni-L, the light irradiation reduces Niþ 3 to Ni1þ and the hydride is transformed in Hþ. The proton formed is transferred to Cys546. Ni-L can be oxidized and generates Ni-Sia oxidizing the Ni1þ to Ni2þ and releasing a Hþ. The [4Fe-4S]p cluster needs to be oxidized and ready to receive an electron to enable the conversion of Ni-L state in Ni-Sia. At least two subforms have been identified with different g values, Ni-L1 and Ni-L2, depending on the temperature and the duration of light exposure and pH conditions. Throughout the catalytic cycle the Fe2þ does not change its oxidation state and maintains the coordination to two CN and one CO ligands keeping the organometallic nature of the [NiFe] active site of hydrogenases. The different EPR active and EPR-silent oxidation states of D. vulgaris Miyazaki F [NiFe] hydrogenase are depicted in Fig. 10, as well as the reduction potentials determined through spectroelectrochemical titrations for the various steps.311,319 Similar titrations have been performed for D. gigas,320 D. fructosovorans321 and also for 310 [NiFe] hydrogenases. Each electron transfer is accompanied by a proton-transfer step. The two EPR-silent states Ni-SIr and Ni-SIa are in an acid-base equilibrium.311,320 Biohydrogen has gain importance in the discussion of future alternative energy sources to fossil fuel due to the harmless final product, water.322 An effort has been made to produce H2/O2 biofuel cells and artificial hybrid solar fuels combining hydrogenase and dot-in-rod components.266,323–326 One major drawback to further developments in this field is the O2-sensitivity of the standard hydrogenases. Special attention is being given now to hydrogenases that are O2-tolerant and can retain catalytic activity (hydrogen oxidation) for considerable periods of time in oxygenic environments. O2-tolerant hydrogenases have been purified from different microorganisms. Some examples are the membrane-bound hydrogenases form Hydrogenovibrio marinus, E. coli (EcHyd-1), A. aeolicus and Ralstonia eutropha.271-273,327 These O2-tollerant hydrogenases upon reaction with oxygen form only the Ni-B inactive “ready” state.272 X-Ray crystallographic structures revealed that the [4Fe-3S]p cluster is quite unique since it is coordinated by 6 cysteine residues instead of the 4 cysteine residues that coordinate the [4Fe-4S]p cluster of the O2-sensitive enzymes (Fig. 11).327 The structure of the proximal [4Fe-3S]pþ 5/þ4/þ3 cluster enables two consecutive electron transfers with close reduction potentials, being the second electron transfer associated with a proton coupled reaction (þ 230 mV and þ 30 mV for EcHyd-1328). The [3Fe-4S]m1 þ/0 and the proximal [4Fe-3S]p exhibit higher reduction potentials compared with the ones from the O2-sensitive hydrogenases. The [3Fe-4S]m can transfer one electron (þ 190 mV for EcHyd-1328) and the [NiFe] active site can oxidize Ni2þ to Ni3þ supplying another electron. In the O2-tolerant hydrogenases redox structural changes occur at the [4Fe-3S]p. For R. eutropha hydrogenase upon oxidation, the Fe4 position is shifted toward the amide of Cys20 in a similar manner to the Fe6 of the P-cluster of nitrogenase (Fig. 11).270 The Fe4 forms a covalent bond with the deprotonated amide nitrogen from Cys20 stabilizing the super oxidized state (Fig. 11). These results were also observed for super oxidized H. marinus and EcHyd-1 hydrogenases.271,327 Resonance Raman and pulsed EPR spectroscopy detected a OH ligand at Fe1.270 [NiFeSe] hydrogenases are enzymes that belong to the group of [NiFe] hydrogenases but whereas a selenocysteine replaces one cysteine that coordinates the Ni in the NiFe active site. In this type of enzymes, the Ni is coordinated by three sulfur atoms from three cysteine residues and one Se atom from a selenocysteine329,330 (Fig. 9C). The [NiFe] and [NiFeSe] enzymes are structurally similar with identical subunits, with the active site buried in the large subunit. The electron transfer in [NiFeSe] enzymes is also performed by a set of three iron-sulfur clusters present in the small subunit connecting the active site to the surface, however, the medial cluster is a [4Fe4S] cluster instead of the [3Fe4S] cluster present in [NiFe] hydrogenases.304,329–332 The [NiFeSe] hydrogenases have high catalytic activity directed toward H2 production333–336 and is less sensitive to oxygen showing lower reactivation times after oxygen exposure.336 As-purified [NiFeSe] hydrogenases are EPR silent.337–339 The aerobically purified enzymes do not form the inactive states Ni-A and Ni-B observed in [NiFe] hydrogenases characterized by the presence of a bridging oxygenated species between Ni and Fe (Fig. 9B). The oxygen inhibition occurs through different pathways. Computational studies suggest that the oxygen permeation pathways in [NiFe] and [NiFeSe] hydrogenases are different even though

Cys149

Cys149

His229

His229

3

Cys20

4

Cys120

oxidation

1 2

4

Cys120

1 2

reduction

Cys17

Cys17 Cys115

Cys20

3

Cys115

Fig. 11 The redox dependent conformational changes occurring at the [4Fe-3S]p cluster of R. eutropha O2-tolerant [NiFe] hydrogenase (PDB ID 4IUD). The iron and sulfur atoms are represented in orange, yellow and green, respectively. The carbon, oxygen and nitrogen atoms are represented in gray, red, and blue, respectively. Images were created with the program Discovery Studio Visualizer (BIOVIA).

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123

some of the resides are conserved, with the latter having a lower oxygen permeation efficiency.340 The lower oxygen permeation along with the selenocysteine properties in the active site, different structural features like the “cage effect” surrounding the active site corroborated by the high Hþ/Dþ exchange activity341 or differences in proton transfer,342 contributes to the unique catalytic properties of [NiFeSe] hydrogenases and their associated oxygen tolerance. 2.06.2.2.2.2 Carbon monoxide dehydrogenases/acetyl-CoA synthases Carbon monoxide dehydrogenases/acetyl-CoA synthases (CODH/ACSs) are believed to be ancient enzymes, perhaps responsible for the ability of early organisms to live in the CO2-rich atmospheres that existed at the origin of life.343 These enzymes can be divided in two major groups: (i) the monofunctional enzymes, carbon monoxide dehydrogenases (CODHs), which catalyze the reversible oxidation of CO to CO2 and (ii) the bifunctional enzymes, which in the direction of CO2 reduction couple the synthesis of acetyl-CoA. This second group of enzymes is known as the carbon monoxide dehydrogenases/acetyl-CoA synthases, CODH/ACS complex, found in anaerobic bacteria, or as the acetyl-CoA decarbonylases/synthases, ACDS, a multienzyme complex found in archaea.344–347 Nevertheless, based on subunit composition Lindahl et al. have grouped these enzymes into five classes.348 These enzymes participate in one of the known six carbon fixation pathways, the Wood-Ljungdahl pathway, which is used for energy conservation and carbon fixation in bacteria and archaea. The first produce acetate as the end-product, while the second produce methane.347 The monofunctional CODH enzymes have been isolated from anaerobic CO-utilizing bacteria, such as C. hydrogenoformans and Rhodospirillum rubrum. These enzymes are homodimers, and its X-ray structure shows the presence of three domains (a helical domain at the N-terminus, and two a/b Rossmann-like domains). The homodimer binds five metal clusters: each monomer binds one [4Fe-4S] cluster (named B-cluster) and one highly unusual [Ni-4Fe-4S] cluster (named C-cluster), which is the catalytic site (Fig. 9D), and there is another [4Fe-4S] cluster bound between the subunits (named D-cluster).349,350 In the D. vulgaris CODH, the D-cluster is a [2Fe-2S] cluster, which is proposed to be responsible for the enzyme tolerance to oxidative damage by oxygen.351,352 The C-cluster is a [Ni-3Fe-4S] cubane bound to a mononuclear iron atom through one of the sulfur atoms of the cubane, (named the FCII) (Fig. 9D). Each metal of the [Ni-3Fe-4S] is coordinated by a cysteine residue (Fig. 9D), while the iron atom, FCII, is coordinated by a histidine and a cysteine residue, plus an inorganic sulfur in an approximately trigonal planar geometry.348,352–354 The B-cluster has reduction potentials between  390 and  440 mV, and g values of 2.04, 1.94 and 1.90,355 and together with the D-cluster form an electron transfer pathway, connecting the C-cluster to the protein surface. The D-cluster, located near the protein surface, has a very negative reduction potential (below  530 mV). This cluster might play a role in holding the two subunits together,356 and in exchanging electrons with a redox partner, such as ferredoxin.357 As mentioned, the D-cluster is usually a [4Fe4S] cluster bound to the polypeptide chain through the sequence motif Cys-X7-Cys in each subunit, with exception of the D. vulgaris CODH, in which the [2Fe-2S] D-cluster is bound to a Cys-X2-Cys motif.352 The bifunctional CODH/ACS enzymes have been identified in acetogenic bacteria and methanogenic archaea, such as Moorella thermoacetica (previously known as Clostridium thermoaceticum) and M. barkeri. These enzymes are tetramers with the subunits arranged as a2b2. The X-ray structures of M. thermoacetica CODH/ACS complex358 and of C. hydrogenoformans ACS subunit359 revealed that the ACS a-subunits are located toward the outside, while the CODH b-subunits form a central core. The ACS subunit has three structural domains: (i) the N-terminal domain that interacts with the CODH subunit, (ii) the middle domain, and (iii) the C-terminal domain that binds the catalytic site (named A-cluster) (Fig. 9E). The CODH subunit binds similar metal clusters to the ones described above for the monofunctional CODH. The three-domain ACS subunit can adopt different conformations, depending on the reaction catalyzed: (i) for the carbonylation, the CO reduced from CO2 in the C-cluster of CODH travels through the hydrophobic channels, in a diffusion controlled manner, to the A-cluster in ACS, to be assembled with coenzyme (CoA) and a methyl group to form acetyl-CoA, while (ii) for the methylation, the ACS is in an open state, so that a corrinoid iron-sulfur protein can bind, delivering the methyl group.360 The A-cluster, [2Ni-4Fe-4S], is constituted by a [4Fe-4S] cluster bridged by a cysteine side chain to a proximal nickel atom (Nip), and this proximal metal site is in turn bridged by two cysteine side chains to the distal nickel atom (Nid), which is in a square-planar thiolato- and carboxamido-type N2S2 coordination environment.348,353 The overall architecture of the A-cluster is similar to the Hcluster of the [FeFe]-hydrogenases, since both contain a [4Fe-4S] cluster bridged to a binuclear site.361 Thus, the A-cluster is also a unique and one of the most complex cofactors found in biological systems. The proximal nickel atom (Nip) in the A-cluster can be substituted by Cu or Zn, and thus this binuclear part of the A-cluster can be found as Cu-Ni or as Zn-Ni, besides the physiologically relevant Ni-Ni.358,359,362,363 X-ray structures are available for these different clusters, but as the physiologically relevant form of the A-cluster is the one containing Ni-Ni, only this cluster is depicted in Fig. 9E. The structure of A-cluster with bound CO has been solved, which provided further insights into the catalytic mechanism of this enzyme.360 Similarly to iron-sulfur clusters, hydrogenase and nitrogenase, the unique C- and A-clusters have specific biosynthetic pathways that involve dedicated chaperones and proteins. A description of this molecular systems is outside of the scope of this chapter, but more information can be found in.364,365 2.06.2.2.2.2.1 Redox and spectroscopic properties of the C-cluster The C-cluster can be in four oxidation states, named Cox, Cred1, Cred2 and Cint. The inactive Cox state is diamagnetic, but at potentials below  200 mV, it is reduced by one electron to the Cred1 state, with a S ¼ 1/2, exhibiting an EPR signal with a gav of 1.82 (g values

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of 2.01, 1.81 and 1.65).366,367 Mössbauer studies revealed that Cred1 contains four iron atoms, two of which are designated FCII and FCIII (the other two iron atoms could not be individually characterized). FCIII is a high-spin Fe2þ that, along with the uncharacterized iron atoms, probably constitutes the [3Fe-4S] subsite. FCII is a high-spin five- or six-coordinated Fe2þ and it is almost certainly the iron of the [Ni-S-Fe] subsite. ENDOR studies by Hoffman and co-workers indicated that a histidine residue coordinates the FCII site,368 and that cyanide, a potent tight-binding inhibitor of CO/CO2 catalysis, binds to FCII in the Cred1 state and displaces a strongly coupled OH group.357,368,369 The C-cluster exhibits two additional oxidation states named Cred2 and Cint.366,370–372 The two-electron reduced Cint is EPR silent, while Cred2 is a three-electron reduced form, that binds CO (Cred1/Cred2 has a reduction potential of approximately  530 mV). Cred2 has a S ¼ 1/2 spin state and exhibits an EPR signal similarly to that of Cred1 but with a gav around 1.86 (g values of 1.97, 1.87 and 1.75). The Cred1-to-Cred2 conversion is substrate-dependent and occurs under argon atmosphere, presenting a reduction potential close to the CO/CO2 couple ( 558 mV). The spectroscopic properties of Cred2 have been difficult to study since B-clusters (and probably D-clusters) are paramagnetic under conditions where Cred2 is produced.369 However, it is clear, that FCII is present in Cred2 spectra, and that the strongly coupled OH, evident in the ENDOR studies of Cred1 is absent.368,369 Although the electronic structure of Cred1 and Cred2 is not known, the unpaired electron spin density is proposed to be localized on the iron atom, to account for the large 57Fe and small 61Ni hyperfine coupling.366,373 The catalytic mechanism at the C-cluster has been the subject of several studies,28,368,374,375 with one of the most difficult aspects of the cycle, being the location of the two electrons in the most reduced state of the cluster (Cred2). Three hypotheses are currently accepted: (i) dative Ni-Fe bond, (ii) Ni0 atom, and (iii) hydride-bond Ni2þ.348,376,377 Further insights into the structure and catalytic mechanism of the C-cluster have been obtained through inhibition studies, using cyanide and molecular oxygen.378–380 These studies revealed that these enzymes have different susceptibilities to oxygen, and that the C-cluster can adopt different conformations.351,352,378,381 The structure of the oxidized form of D. vulgaris CODH C-cluster showed that the FCII and the Ni atom, as well as some of the residue sidechains in the cluster binding site are shifted from the canonical position. This rearrangement, in which those two metal exchange positions,352 can confer plasticity to the cluster to avoid oxygen damage, and be critical for cluster assembly (Ni incorporation) and show that the metals in these complex clusters are mobile. 2.06.2.2.2.2.2 Redox and spectroscopic properties of the A-cluster The A-cluster can be stabilized in two oxidation states, the Aox state with a S ¼ 0 spin state and the Ared-CO state with a S ¼ 1/2. Conversion of the Aox to Ared-CO involves one-electron reduction and CO binding. The Ared-CO state exhibits the so-called NiFeC EPR signal, (with g values of 2.074 and 2.028) since the incorporation of 61Ni (I ¼ 3/2), 57Fe (I ¼ 1/2), and 13CO (I ¼ 1/2) in the cluster, the hyperfine-lines broadens the signal. These studies conducted by Ragsdale, Wood, and Ljungdahl provided the first evidence for a [NiFeS] cluster in Biology.345,382 The basic structure of the A-cluster was determined using EPR, ENDOR and Mössbauer spectroscopies, model compounds and XAS,345,373,382–384 with the strongest evidence being revealed when methods were developed to isolate the a-subunits from M. thermoacetica CODH/ACS.385,386 This procedure enabled the evaluation of the spectroscopic properties of the A-cluster without interference from the other clusters in the b-subunit. The Mössbauer and UV–visible spectroscopic studies demonstrated that the nickel atom in the Aox state presents a þ 2 oxidation state, while in the Ared-CO state it is in the reduced þ 1 oxidation state. Thus, Aox corresponds to [4Fe-4S]2þ-X-Ni2þ, and Ared-CO corresponds to [4Fe-4S]2þ-X-Ni1þ-CO.385,386 The intensity of the NiFeC signal was also determined and only z0.3 spin/a-subunit was obtained, which is significantly lower than the expected value of 1. This was explained based on the lability of the nickel atom at the Nip site, which can be removed using 1,10-phenanthroline.387 This procedure eliminates acetyl-CoA synthase activity and the ability to generate the Ared-CO state and the NiFeC signal. The Ni reincorporation reactivates the enzyme and restores its ability to generate this state and signal. The amount of nickel removed and replaced (z0.3 Ni/ab) suggested that only z 30% of the A-cluster has labile nickel atoms and are catalytically active, exhibiting the NiFeC EPR signal.385,387 The remaining A-cluster has nonlabile Ni atoms and do not exhibit the NiFeC signal, thus being inactive.

2.06.3

Direct catalysis at iron-sulfur clusters

The focus of this chapter will be the catalysis mediated by iron-sulfur clusters, which include the expanding group of iron-sulfur cluster-mediated radical catalysis, new and old dehydratases, and isomerase activities, and ADP-ribosyltransferases. Most of these enzymes have a [4Fe-4S] cluster in its active site, which is usually bound by three cysteinyl ligands, in opposition to the cubane-type [4Fe-4S] cluster involved in electron transfer that is coordinated by four cysteine residues. The unique fourth iron atom, with an empty or weak coordination, found in catalytic iron-sulfur clusters, is usually the binding site for different substrates and/or involved in the binding of intermediate species.388 A similar feature is also observed in copper or heme binding enzymes.389 Moreover, the catalytic properties of the iron-sulfur clusters are related to their capacity to shift electrons within the cluster and its ligands, leading to the polarization of attached or nearby groups or molecules.

Iron-sulfur clusters – functions of an ancient metal site 2.06.3.1

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Radical-SAM enzymes

The first enzyme shown to be S-adenosylmethionine-dependent was the pyruvate formate-lyase activating enzyme, in 1965 by Knappe and co-workers.390 Five years later, Barker and co-workers reported the isolation and characterization of 2,3aminomutase from Clostridium subterminale SB4.391 These researchers found that the activity of these enzymes was dependent on S-adenosylmethionine (SAM) and Fe2þ. In recent years, the number of enzymes belonging to this family has increased from 650, in the years 2000’s identified by primary sequence alignments in all kingdoms of life,392,393 to over 100,000 using metagenomic data.394 More than 50 different reactions have been recognized to be catalyzed by these enzymes,394,395 which usually involve difficult chemistries. Some examples are sulfur insertion, glycyl radical formation, the cleavage of non-activated CeH and CeC bonds, dehydration of 2-hydroxyacyl-CoA, the reduction of aromatic compounds, unusual methylation, methylthiolation, isomerization and ring formation. Thus, these enzymes catalyze different steps in several pathways,393 such as nucleic acid modification,396,397 antibiotic and herbicide biosynthesis,398,399 synthesis of organic cofactors (e.g., biotin400 and menaquinone (vitamin K)397), and the biosynthesis of the complex iron-sulfur clusters of nitrogenase (FeMoco),401–403 and [FeFe] hydrogenase (H-cluster).404 Some of these enzymes are listed in Table 2 and some of the most studied SAM-dependent enzymes will be briefly described here. A comprehensive list of these enzymes and the reaction they catalyze can be found in.394,395,426 The radical-SAM superfamily of proteins has a conserved cysteine motif, usually at the N-terminus with a consensus Cys-X3-CysX2-Cys motif393 that coordinates the [4Fe-4S] cluster, leaving one iron atom with an incomplete coordination, and the possibility to be coordinated by the amino and carboxylate groups of S-adenosylmethionine. This bidentate ligation of this unique iron was first proposed based on spectroscopic studies427–432 and confirmed afterwards when the X-ray structure of biotin synthase was solved.36 This bidentate ligation is proposed to be pivotal for proper positioning for radical generation.431,433 The mentioned consensus sequence motif is present in 90% of the primary sequences, but variations in the spacing between cysteine 1 and 2 or 2 and 3 have been observed, with the number of residues varying between 3 and 22. Adjacent to the third cysteine, there is also a conserved aromatic residue (His, Phe, Tyr or Trp), which protects the iron-sulfur cluster from the solvent and thus lowers its reduction potential.393,434 Sequence alignments also revealed the presence of a “glycine-rich region” or a “GGE motif” (Gly-X-Ile-X-Gly-X2-Glu) that binds the amino group of adenosylmethionine through H-bonds,393,435 and positions it in the correct orientation to coordinate the unique iron of the iron-sulfur cluster. The active site [4Fe-4S] cluster is oxygen sensitive and can rapidly degrade to a [3Fe-4S] cluster (or be destroyed) during isolation and characterization when oxic conditions are used, which made the initial studies on these enzymes difficult. Nevertheless, the structure of several radical-SAM enzymes has been determined. These structures share a common core fold composed by eight or six b,a-pair motifs arranged in a triose phosphate isomerase (TIM) barrel-fold, that is not completely closed in most of the known structures.36,433,436,437 This TIM-fold has also been found in domains of multidomain proteins, such as viperin.396 The exposed face of the b-sheet harbors the active site residues that bind the essential oxygen-sensitive [4Fe-4S] cluster and the adenosylmethionine. In most of the radical-SAM enzymes, the active site [4Fe-4S] cluster is buried at 7–10 Å from the surface,433 but being somewhat solvent accessible and in an amenable distance to receive electrons from redox partners. Some of these enzymes have other redox active centers, mainly [4Fe-4S] and [2Fe-2S] clusters,438 but cobalamin has also been observed in other structural domains.439 The catalytic mechanism of most of these enzymes shares a common step in which SAM is reductively cleaved by a reduced [4Fe4S]1þ cluster (Fig. 12), giving rise to methionine and to a highly oxidizing and unstable radical, the 50 -deoxyadenosyl radical, which is then used to initiate each specific reaction. The detailed mechanism for the formation of this radical has been a matter of debate for many years, with several spectroscopic and computational studies performed on both enzymes and model compounds. In one of the proposed mechanisms, it is the unique iron of the [4Fe-4S]1þ cluster that mediates the electron transfer to the sulfonium of adenosylmethionine421,428,440 and the formation of the iron-sulfur bond reduces the activation energy for the homolytic cleavage of C-S bond (Fig. 12A). This proposal is based on the binding geometry of the S-adenosylmethionine, which positions its sulfur in a closer distance to the Fe (3.4 Å) than the sulfur (3.8 Å) of the iron-sulfur cluster. Based on ENDOR and Mössbauer experiments another mechanism has been proposed, in which the [4Fe-4S]1þ will function as an electron donor, with an electron being transferred from a sulfide (from the iron-sulfur cluster) to the sulfonium (Fig. 12B).429,431,432,441 Recent quantum mechanical/molecular mechanical (QM/MM) computations on the mechanisms of biotin synthase supports this later proposal, which is now considered to be the accepted mechanism for radical SAM cleavage.421,442 The first step of this mechanism is the one-electron reduction of the [4Fe-4S]2þ cluster to [4Fe-4S]1þ, by a reducing agent. For some of the enzymes their physiological reducing agent has been identified to be single electron donors, such as NADPH,443 flavodoxin443,444 or flavodoxin reductase.445 However, for many enzymes the reductant has still not been established, though in in vitro assays, sodium dithionite or photoreduced 5-deazariboflavin are the electron donors usually used.446 Protein film voltammetry has also been applied to the characterization of some of these enzymes, which avoids the use of electron donors, and enables the determination of the reduction potential of the active site and of the additional redox centers, as well as its pH dependence under turnover and non-turnover conditions.447 Most of the studied radical-SAM dependent enzymes have in common the formation of a radical, 5’-deoxyadenosyl radical, that is used for stereospecific hydrogen abstraction and formation of a carbon radical (Fig. 13A), as mentioned before. However, these enzymes differ on how the radical is used in the reaction cycle. Another group of SAM-dependent enzymes catalyze the adenosylation of substrates by adding the generated 50 -deoxyadenosyl radical to a sp2 carbon (Fig. 13B). These enzymes are involved in natural product biosynthetic pathways and are attracting attention due to its potential in biocatalysis and bioengineering applications.448 Examples of these enzymes are adenosylhopane synthase (HpnH) and aminofutalosine synthase (MqnE). Adenosylhopane synthase is an enzyme involved in the biosynthesis of a bacteriohopanepolyol, aminobacteriohopanetriol, by Streptomyces

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Table 2

Some examples of radical SAM-dependent enzymes. References

Radical SAM Mutases LAM Lysine 2,3-aminomutase PylB Lysine Mutase Radical SAM Chemistry to Cleave C-X (X¼ C,N,P) Bonds SplB Spore photoproduct lyase Sulfur Insertion LipA Lipoyl synthase BioB Biotin synthase Modified Tetrapyrroles Synthesis HemN Coproporphyrinogen III oxidase Glycyl Radical Enzyme Activation PFL-AE Pyruvate formate-lyase activating enzyme ARN-AE Anaerobic ribonucleotide reductase activating enzyme GD-AE Glycerol dehydratase activating enzyme BSS-AE Benzylsuccinate synthase activating enzyme HPD-AE 4-Hydroxyphenylacetate decarboxylase activating enzyme FGS Formyl glycine synthase Methylation and Methylthiolation MiaB Methylthiolation of tRNA RimO RlmN

Methylthiolation of ribosomal protein S12 rRNR methyltransferase

Cfr

rRNR methyltransferase

Complex Rearrangements and Cyclizations ThiC Hydroxymethylpyrimidine phosphate synthase MoaA Molybdopterin biosynthesis protein A Complex Metal Cluster Biosynthesis HydE [FeFe] Hydrogenase maturase protein NifB FeMoco maturase protein Dehydration BtrN Biosynthesis of 2-deoxystreptamine Viperin

Viperin - antiviral activity – 3’-deoxy-3’,4’-didehydro-CTP (ddhCTP) synthase Other chemistries outside SAM superfamily Dph2 Diphthamide biosynthesis protein 2

b-Lysine antibiotics, lysine metabolism Pyrrolysine Biosynthesis

405 406

initiates DNA repair

407

Sulfur Insertion/Lipoic acid biosynthesis Sulfur Insertion/Biotin biosynthesis

408 36

Heme biosynthesis

409

Glycyl radicalization in pyruvate metabolism Glycyl radicalization in dNTP synthesis Glycyl radicalization of glycerol dehydratase Glycyl radicalization in toluene metabolism Formation of p-cresol

410 411 412 413 414 415

Sulfur Insertion/Thiomethylation of isopentenyl adenosine in tRNA Methylthiolation of residue D88 of S12 Methylation of position 2 of adenosine2503 in 23S ribosomal RNA Methylation of position 8 of adenosine2503 in 23S ribosomal RNA

416

Thiamine pyrimidine biosynthesis Molybdenum cofactor (Moco) biosynthesis

419 420

[FeFe] hydrogenase cluster biosynthesis Biosynthesis of the FeMoco of nitrogenase

421 403,422

Conversion of 2-deoxy-scyllo-inosamine into 3-amino2,3-dideoxy- scyllo-inosose Conversion of cytidine triphosphate (CTP) to 30 -deoxy30 ,40 -didehydro-CTP (ddhCTP)

423

diphthamide biosynthesis

417 418 418

424 425

coelicolor, while aminofutalosine synthase is involved in an alternative pathway of menaquinone biosynthesis, which has been identified in some human pathogens, such as Campylobacter jejuni and Helicobacter pylori.

2.06.3.1.1

Examples of radical-SAM enzymes

2.06.3.1.1.1 Radical SAM mutases - lysine 2,3-aminomutase In some enzymes that catalyze isomerizations, such as lysine 2,3-aminomutase (LAM),405 spore photoproduct lyase,407 SAM is used as a reversible source of the 50 -deoxyadenosyl radical, and thus it functions as a coenzyme. Then the 50 -deoxyadenosyl radical mediates hydrogen transfer from the substrate and is regenerated to SAM after each catalytic cycle. LAM isolated from C. subterminale SB4, catalyzes the interconversion of L-lysine and L-b-lysine (Fig. 14) and was the first enzyme to be characterized as a radical-SAM dependent enzyme.391 It was shown that LAM binds an iron-sulfur cluster and requires pyridoxal-50 -phosphate and SAM for full activity.449,450 Several spectroscopic and kinetic studies were used to elucidate LAM kinetic mechanism.451 This mechanism is initiated by the formation of the 50 -deoxyadenosyl radical, which is then used to cleave the C-H bond at C-3 of the lysine bound to the pyridoxal-50 phosphate through an imine linkage, by abstracting a hydrogen atom. The intermediate that is formed, with a carbon radical, undertakes two rearrangement steps with migration of the nitrogen to C-3, and in the last step the 50 -deoxyadenosyl radical is regenerated.452–454 ENDOR has been used to elucidate the mechanism of this enzyme, in which the 50 -deoxyadenosyl radical is never in the free state.455

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Fig. 12 The binding mode of S-adenosyl-L-methionine to the iron-sulfur cluster and the two proposed mechanisms for the formation of the 50 deoxyadenosyl radical through the homolytic cleavage to the C5’-S bond of adenosylmethionine. In (A) the formation of the radical is driven by the formation of the bond between the sulfonium ion and the unique iron, while in (B) there is an electron transfer mediated by the interaction of the sulfide of the iron-sulfur cluster with the sulfonium of adenosylmethionine. This is the accepted mechanism.

Fig. 13 Generic reactions catalyzed by radical SAM-dependent enzymes. In panel (A) is shown the hydrogen abstraction mediated by 50 deoxyadenosyl radical (the canonical reaction). In panel (B) is shown the 50 -deoxyadenosyl radical addition that results in the adenosylation of a substrate.

2.06.3.1.1.2 Catalysis of sulfur insertion - biotin synthase Biotin synthase catalyzes the sulfur insertion in dethiobiotin to form biotin, through the formation of two C-S bonds in nonactivated carbons (C6 and C9). In each catalytic cycle, the enzyme requires two molecules of SAM36,400,456 (Fig. 15). This enzyme, like lysine aminomutase, has also been extensively studied, but some questions remain. For many years, it had been debated what was the source for the sulfur, though now it is generally accepted that it is the [2Fe-2S] center present in the

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Fig. 14

Reaction catalyzed by lysine 2,3-aminomutase.

enzyme. The clarification of the mechanism of biotin synthase was based on experiments that used33S isotopic labeling,457 the observation that the [2Fe-2S] cluster disassembles at each turnover of the enzyme,92,456,458 and that this cluster and dethiobiotin are in close proximity.36 However, the mechanism and intermediates that are formed are still a matter of debate and of extensive study, as it is proven by the several publications on the subject.442,459,460 Since the [2Fe-2S] cluster is destroyed after each turnover cycle, it has been questioned whether this enzyme could be a suicidal enzyme. However, it was later shown that in vivo the enzyme can perform several turnovers, and thus the [2Fe-2S] cluster must be regenerated after each catalytic cycle.461 Lipoyl synthase, like biotin synthase, also requires two equivalents of the 50 -deoxyadenosyl radical (thus two SAM molecules) to activate chemically unreactive C-H bonds of octanoyl group and inserts one mole of sulfur.36,408,456,462 2.06.3.1.1.3 Glycyl radical enzyme activation - pyruvate formate-lyase activating enzyme In this family of SAM-dependent enzymes the substrate is another enzyme, that requires the generation of an active site radical.410,411,413 In this case, stoichiometric SAM is used as an oxidizing substrate to stereospecificaly abstract a proton from a conserved active site-glycine residue, forming a stable glycyl radical, and releasing methionine and 50 -deoxyadenosyl radical, as by-products. This glycyl radical is transferred to a cysteine and the cysteinyl radical is then used to activate the enzyme substrate (Fig. 16). The rational for using a cysteinyl radical in the catalysis is the formation of a weak S-H bond that will then donate H to the product. Pyruvate formate-lyase activating enzyme has been highly studied, and several spectroscopic techniques have been applied to clarify its catalytic mechanism.431,432,463–465 This enzyme catalyzes the formation of a glycyl radical in the partner protein, the pyruvate formate-lyase, through the abstraction of the pro-S hydrogen from Gly734 in a SAM dependent reaction,466 which also requires the presence of reduced flavodoxin (or other artificial one-electron donors)467 (Fig. 17). The pyruvate formate-lyase activating enzyme presents an oxygen sensitive [4Fe-4S] cluster per monomer, that is active in the reduced [4Fe-4S]1þ form in the presence of SAM.468–470 However, even when the enzyme was purified under anaerobic conditions, which increased the content in [4Fe-4S] cluster, Mössbauer data revealed the presence of iron mainly as [3Fe-4S]1þ, with minor amounts of [2Fe-2S]2þ and [4Fe-4S]2þ clusters, in the as-isolated form, which converted to [4Fe-4S]2 þ/1 þ, when reduced with sodium dithionite.469,471 Further in vivo Mössbauer studies of this enzyme showed the presence of a cluster interconversion process in response to higher oxygen levels, which has been proposed to be a process to control the pyruvate formate-lyase activity.472 Although, a similar activity control by cluster interconversion has been observed in aconitase/IRP1 (vide infra), in the case of pyruvate formate-lyase activating enzyme, the cluster is proposed to interconvert between [2Fe-2S]2þ and [4Fe-4S]2þ.472 These in vivo studies also established that this [4Fe-4S]2þ was valence-localized, an unusual feature, while the one in the as-isolated enzyme is valence delocalized, possibly indicating the loss of a ligand during protein purification.472 The identity of this small molecule is still unknown, but Broderick and co-workers showed that addition of adenosyl-based molecules causes valence localization of [4Fe-4S]2þ.472

Fig. 15 Reaction catalyzed by biotin synthase. Adapted from Taylor, A. M.; Stoll, S.; Britt, R. D.; Jarrett, J. T., Reduction of the [2Fe-2S] Cluster Accompanies Formation of the Intermediate 9-Mercaptodethiobiotin in Escherichia coli Biotin Synthase. Biochemistry 2011, 50(37), 7953–7963.

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Fig. 16 Generic mechanism of the catalytic cycle of glycyl radical enzymes. Adapted from Selmer, T.; Pierik, A. J.; Heider, J., New Glycyl Radical Enzymes Catalysing Key Metabolic Steps in Anaerobic Bacteria. Biol. Chem. 2005, 386(10), 981–988.

Fig. 17 Reaction catalyzed by pyruvate formate-lyase activating enzyme (PFL-AE), that through the formation of 50 -deoxyadenosyl radical (DOA) produces the glycyl radical at Gly734 activating PFL. a – active, i – inactive.

Moreover, an important question remains to be answered relative to the mechanism and conformational changes that are required in the complex of the two proteins for the direct and stereospecific abstraction of the hydrogen from the specific glycyl residue. Nevertheless, some insights have been obtained through structural studies on the pyruvate formate-lyase activating enzyme complexed with a 7-mer peptide with the sequence surrounding Gly734,473 which pointed out for the occurrence of a conformational change in the structure of the protein. In fact, other structural studies indicated that Gly734, buried at 8 Å from the surface in the active pyruvate formate-lyase, is more solvent exposed in an open conformation of the inactive enzyme, probably to enable hydrogen abstraction by the activating enzyme.474 A monovalent cation was identified in the X-ray structure of the protein, being coordinated by a backbone carbonyl, the sidechain of two conserved aspartate residues, and an oxygen of SAM’s carboxylate. The role and identity of this cation is still under investigation, though an increase in enzymatic activity of the pyruvate formate-lyase activating enzyme was attained in the presence of K1þ.475 2.06.3.1.1.4 Catalysis of methylations by RlmN and Cfr RlmN and Cfr are methyltransferases (recently proposed to be methyl synthases) involved in the methylation of a specific adenosine nucleotide (adenosine2503) in the peptidyl transferase center of the ribosome.476,477 This methylation is a post-transcriptional modification of 23S rRNA nucleotides and functions as a mechanism to modulate the ribosome’s function and confer bacterial resistance to ribosome-targeting antibiotics.477,478 These enzymes are radical SAM enzymes containing an iron-sulfur cluster bound to the usual motif Cys-X3-Cys-X2-Cys,479 and use S-adenosylmethionine as the source of 50 -deoxyadenosyl radical and methyl donor to the aromatic carbon atoms to form the 2,8-dimethylated product.480 The proposed mechanism includes the formation of a 50 -deoxyadenosyl radical that abstracts hydrogen from a second S-adenosylmethionine molecule to form a SAM-derived radical cation, which is then added to the substrate.481 The reactions catalyzed by these enzymes proceed through a ping-pong mechanism that involves the transient methylation of a conserved cysteine residue that is then transferred to a electrophilic sp2-hybridized carbon of adenosine (Fig. 18), and provide evidences for these enzymes to be renamed as methyl synthases.418,482 Thus, these enzymes use SAM to methylate an electrophilic rather than the usual nucleophilic carbon center.482 2.06.3.1.1.5 Dehydration – synthesis of ribonucleotide - viperin Viperin is a protein discovered, in 1997, to respond to viral infections via the immune response pathway.483 This 42 kDa protein has three domains: a N-terminal amphipathic alfa-helix domain, a radical SAM central domain (homologous to MoaA/NifB) and a C-terminal domain of still undefined function, with the N- and C-terminus being essential for antiviral activity.396,484 The radical SAM containing domain was identified by the presence of conserved motifs common to other radical SAM enzymes: the Cys-X3-CysX2-Cys sequence motif that binds the [4Fe-4S] cluster and the “GGE motif”, that it is responsible for the enzymatic activity.

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Fig. 18 Proposed mechanism of the S-adenosylmethionine activation by Cfr and RlmN. SAM – S-adenosylmethionine, SAH – Sadenosylhomocysteine, DOA – 50 -deoxyadenosyl radical. Adapted from Grove, T. L.; Radle, M. I.; Krebs, C.; Booker, S. J., Cfr and RlmN Contain a Single [4Fe-4S] cluster, which Directs Two Distinct Reactivities for S-Adenosylmethionine: Methyl Transfer by SN2 Displacement and Radical Generation. J. Am. Chem. Soc. 2011, 133(49), 19586–19589.

Viperin is encoded in the genome of both vertebrates and invertebrates, and the crystal structure of the mouse viperin has been solved by X-ray crystallography, showing to adopt a TIM-barrel fold and to bind an iron-sulfur cluster, a SAM, and a CTP molecule.396,485–487 The enzymatic activity of viperin was demonstrated to be the conversion of cytidine triphosphate to 3’deoxy30 ,40 -didehydro-CTP (Fig. 19).424,486,488 This ribonucleotide when incorporated by RNA-dependent RNA polymerase of some virus leads to premature termination of the RNA synthesis.489 2.06.3.1.1.6

•

Atypical SAM-dependent enzymes

Noncanonical radical SAM enzyme - Diphthamide biosynthesis protein 2

A different mechanism of SAM cleavage was observed in diphthamide biosynthesis protein 2 (Dph2), which instead of the 50 -deoxyadenosyl radical generates a 3-amino-3-carboxypropyl radical.425 In addition, this radical also undergoes a different chemistry, an addition and not the classical hydrogen abstraction. Dph2 is responsible for the catalysis of the first step of diphthamide biosynthesis, a rare amino acid.490,491 Diphthamide modification is a post-translational modification of a histidine residue in the translation elongation factor 2 (EF-2) of eukaryotes and archaea, which is essential for ribosomal protein synthesis.492,493 This modified histidine is the target of a toxin secreted by Corynebacterium diphtheriae, the diphtheria toxin, which catalysis the ADP-ribosylation of that histidine.494 Biophysical and structural studies on this enzyme have shown that it is a homodimer in archaea (e.g., Pyrococcus horikoshii Dph2), with the [4Fe-4S] cluster being coordinated by three cysteines Cys59-Xn-Cys163-Xn-Cys287, which are located in the three different domains and not arranged in the conserved motif found in most radical SAM-dependent enzymes.425 In eukaryotes, the enzyme that catalyzes the same reaction is heterodimeric formed by Dph1 and Dph2.495 As mentioned, the catalytic mechanism of Dph2 does not involve the formation of the canonical 50 -deoxyadenosyl radical. Instead, it was observed the formation of a 3-amino-3carboxipropyl radical due to the reductive cleavage of the Cg,Met-S bond of SAM.425,496 This radical also undergoes a different chemistry, instead of hydrogen abstraction in the case of the 50 -deoxyadenosyl radical, the radical generated in Dph2 carries out an addition: the generated 3-amino-3-carboxipropyl (ACP) radical is proposed to be added to the imidazole ring of histidine for the formation of the diphthamide modification.496 This C-C coupling forms an ACPhistidine radical intermediate (an organic radical), that can be protonated, releasing the modified EF2497 (Fig. 20).

Fig. 19 Reaction catalyzed by viperin, as an antiviral ribonucleoside synthase. Adapted from Gizzi, A. S.; Grove, T. L.; Arnold, J. J.; Jose, J.; Jangra, R. K.; Garforth, S. J.; Du, Q.; Cahill, S. M.; Dulyaninova, N. G.; Love, J. D.; Chandran, K.; Bresnick, A. R.; Cameron, C. E.; Almo, S. C., A Naturally Occurring Antiviral Ribonucleotide Encoded by the Human Genome. Nature 2018, 558(7711), 610–614.

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This complex catalytic mechanism has been elucidated by combining rapid-freezing EPR and ENDOR with crystallographic studies using isotopically labelled SAM. This approach enabled the captured of an organometallic intermediate formed between ACP radical and the [4Fe-4S] cluster, with an iron-carbon bond, as depicted in Fig. 20. The electronic structure of the [4Fe-4S] cluster was shown by classical molecular dynamics and quantum mechanics/molecular mechanics to control the reductive cleavage of SAM’s Cg,Met-S bond, through a spin-regulated electron transfer mechanism.498

•

Adenosylation

There is a group of enzymes that catalyze the radical SAM-dependent adenosylation (Fig. 13B) in biosynthetic pathways of natural products.448 These enzymes have also been exploited to catalyze unnatural transformation using synthetic substrates, opening their applications to biotechnology and bioengineering. These applications include the production of nucleoside-containing compounds by modifying different olefins. Examples of these enzymes are adenosylhopane synthase and aminofutalosine synthase.499–501 o Aminofutalosine synthase MqnE The synthesis of menaquinone (vitamin K2) in some pathogenic microorganisms follows an alternative pathway with aminofutalosine as an intermediate. The enzyme responsible for its synthesis, aminofutalosine synthase (MqnE), was reported to bind an iron-

Fig. 20 Proposed catalytic mechanism of Dph2 with the formation of the 3-amino-3-carboxypropyl radical intermediate, followed by the formation of a 3-amino-3-carboxypropyl-histidine radical intermediate in the second step. Adapted from Zhu, X.; Dzikovski, B.; Su, X.; Torelli, A. T.; Zhang, Y.; Ealick, S. E.; Freed, J. H.; Lin, H. Mechanistic Understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] Enzyme Required for Diphthamide Biosynthesis. Mol. Biosyst. 2011, 7(1), 74–81; Feng, J.; Shaik, S.; Wang, B., Spin-Regulated Electron Transfer and Exchange-Enhanced Reactivity in Fe4S4-Mediated Redox Reaction of the Dph2 Enzyme During the Biosynthesis of Diphthamide. Angew. Chem. Int. Ed. Engl. 2021, 60(37), 20430–20436.

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sulfur cluster in 2013.502 In the following years, the intermediates have been identified,503–505 and as this enzyme is essential for some pathogenic bacteria it has been considered to be a target for the development of antibiotics.506 In 2019, a compound, in which the bridging oxygen of 3-[(1-carboxyvinyl)oxy]-benzoic acid was substituted by a methylene group, was identified to be a potent inhibitor of H. pylori MqnE and also to present antimicrobial activity similar to amoxicillin and clarithromycin. This proved that this pathway and in particular this enzyme are promising targets for the development of new antibiotics against certain pathogenic bacteria, such as H. pylori, Chlamydia strains, C. jejuni, and Spirochaetes.

2.06.3.2

Iron-sulfur (de)hydratases

Iron-sulfur (de)hydratases are another group of non-redox enzymes that contain an iron-sulfur cluster in the active site and catalyze the removal of a water molecule from a carbon-carbon bond by converting an alcohol into a vinyl group. These enzymes can be divided into two families that differ in the type of iron-sulfur cluster present in the active site. In one family there is a [4Fe-4S] cluster with a unique iron that is not coordinated by a cysteine side chain and define the substrate binding site. Examples of enzymes that belong to this family are L-serine dehydratase,507 fumarase,508,509 aconitase, a family of enzymes involved in the prokaryotic biosynthetic pathway of non-mevalonate isoprenoid (IspG and IspH), and others, such as quinolate synthase.510–514 For most of these enzymes there has been extensive studies to elucidate their structure,515,516 spectroscopic features (EPR, HYSCORE, Mössbauer), catalytic mechanism, and to identify intermediate species515,517–519 and inhibitors (some with potential antibacterial properties).520,521 In the other family of enzymes, their active site has a [2Fe-2S] cluster, though they share the feature of having one of the iron atoms with an empty coordination site that is involved in the catalytic cycle, and transiently binds a hydroxyl group during catalysis.522 These enzymes have been identified as belonging to a different class of aconitase enzymes and are involved in the metabolism and biosynthetic pathway of carbohydrates,523,524 such as pentonate dehydratases.

2.06.3.2.1

Aconitase

Aconitase is an isomerase that catalysis the reversible and stereospecific dehydration/rehydration of citrate to isocitrate via cisaconitate in the Krebs cycle.172,525 This is a non-redox process, and for some time it was thought that this enzyme contained a simple iron center (with one Fe2þ), proposed to be involved in the Lewis-acid function of facilitating the hydrolytic reaction.78 Aconitase was the first enzyme526,527 for which a catalytic function of an iron-sulfur cluster was identified. In addition, it has been a model system to study the role of the iron-sulfur cluster in non-redox reactions,527 and the subject of extensive studies using different spectroscopic and biophysical methods, to elucidate its structure and catalytic cycle. The aconitase family is composed by the true aconitases, which catalyze the isomerization of citrate to isocitrate, while there are three other groups that are involved in the isomerization of isopropylmalate in the leucine biosynthetic pathway, homocitrate in the lysine and coenzyme B biosynthetic pathways,528–531 and methylcitrate dehydratase in the catabolism of propionate.532,533 In multicellular eukaryotes there are two aconitases, one in the cytoplasm (c_aconitase) and another in the mitochondria (m_aconitase), that share 30% sequence identity, but the iron-sulfur cluster sequence binding motif and other important residues in the active site are conserved.172,534,535 The m_aconitase is one of the enzymes of the Krebs cycle, while c_aconitase is a bifunctional enzyme as in its apo-form it is the iron responsive protein1 (IRP1), a post-transcriptional regulator of the iron metabolism536,537 (see Section 2.06.4.1). The [4Fe-4S] cluster of aconitase active site shares, with the iron-sulfur enzymes described so far, the feature of having one of the iron atoms (Fea or the so-called unique iron) not coordinated by a cysteine residue but interacting with the carboxyl and hydroxyl oxygen atoms of the substrate and solvent species (Fig. 19). In the first studies on aconitase it was reported the isolation of the enzyme in an inactive form, binding a [3Fe-4S] cluster, that could be activated by restoring the [4Fe-4S] cluster by addition of a reducing agent and Fe2þ (see Section 2.06.2.1.5 and Fig. 6).42,172,526,538–540 This has been named the “iron-sulfur cluster switch” (Fig. 21), a cluster assembly/disassembly mechanism that is also responsible for the regulation of the bifunctional activity of c_aconitase/IRP1. The active site of aconitase encompasses 21 residues with different roles: coordination of the iron-sulfur cluster (cysteines), Hbonding the substrate carbonyl moieties, the cysteine, inorganic sulfur atoms, and bound water molecules (that are also involved in the reaction).42,540 These residues are also responsible for the stabilization of intermediates and are involved in the steps of dehydration (the nucleophilic attack performed by Ser642 is represented as: B in Fig. 22) and rehydration.535 In addition, these residues dictate the global positive charge of aconitase active site and contribute to its ability to bind anionic substrates. This positive charge is attributed to the presence of four arginine residues and due to the three-active site histidine residues being paired with aspartate or glutamate residues.172 The fourth iron (Fea), occupying the empty corner of the inactive [3Fe-4S] cluster (Fig. 21A), has a tetrahedral geometry, and is proposed to bind a hydroxyl group. This oxygen ligand has been observed in the X-ray structure of reconstituted aconitase42,540 (Fig. 21B) and identified by ENDOR experiments.541 The [4Fe-4S] cluster of aconitase has been characterized in different oxidation states and bound to the substrate and inhibitors by X-ray crystallography (Fig. 21C) and spectroscopic techniques, such as EPR, ENDOR, MCD and Mössbauer,42,540–545 which in combination with kinetic studies and isotopic labeling experiments546,547 enabled the proposal of the catalytic mechanism presented in Fig. 22.

Iron-sulfur clusters – functions of an ancient metal site

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Fig. 21 Active site of aconitase in the inactive (A) and active (B) and bound to its substrate citrate (C). The atoms are colored according to element: carbon in gray, iron in orange, oxygen in red and sulfur in yellow. Figures A, B and C were prepared in Discovery Studio Visualizer (BIOVIA) using the coordinates from PDB ID 5ACN, 6ACN and 1C96, respectively.

Fig. 22 Proposed catalytic mechanism of aconitase. In the “flip” step the cis-aconitate is released (displaced), and re-binds to the iron-sulfur cluster in an alternate mode. Adapted from Lauble, H.; Kennedy, M. C.; Beinert, H.; Stout, C. D., Crystal Structures of Aconitase with Trans-Aconitate and Nitrocitrate Bound. J. Mol. Biol. 1994, 237(4), 437–451.

This mechanism explains the stereospecificity of the reactions and the requirements for the trans eliminations/addition of a hydroxyl group and a proton at the first and third steps after the binding of citrate. During the catalytic cycle, the unique iron (Fea) is penta-coordinated after the release of and prior to the re-binding of a water molecule, that originates from and is incorporated into the substrate hydroxyl group, respectively (step two and four).172

2.06.3.2.2

IspG and IspH involved in isoprenoid biosynthesis

IspG (4-hydroxy-3-methylbut-2-enyl diphosphate synthase also designated as GcpE) and IspH (4-hydroxy-3-methylbut-2-enyl diphosphate reductase also designated as LytB) are iron-sulfur enzymes that catalyze the reaction of reductive dehydration in non-mevalonate isoprenoid prokaryotic biosynthetic pathway, also named methylerythritol phosphate pathway.548 IspG catalyzes the reductive dehydroxylation opening of the eight-member ring of methylerythritol cyclic diphosphate, yielding 4-hydroxy-3methylbut-2-enyl diphosphate, then the hydroxy group of this compound is reductively removed by IspH, to form isopentenyl

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diphosphate and dimethylallyl diphosphate548 (Fig. 23). This biosynthetic pathway plays an important role in pathogenic bacteria and parasites, and since it is absent in humans, it is an excellent target for drug design.549–551 IspG is a functional homodimer with the two subunits arranged in a “head-to-tail configuration. Each subunit has two domains: the N-terminal domain is a (ba)8 TIM barrel, while the C-terminal domain is a five-stranded b-sheet flanked by helices on both sides, which harbors the iron-sulfur cluster51,551 (Fig. 24A). This [4Fe-4S] cluster is located in between the two subunits, in an interface with the C-terminal domain of the other subunit. It is coordinated by three cysteine residues found in the consensus sequence motif Cys-X-Cys-Cys-X5-Gly-Glu, and in the absence of substrate it is also coordinated by a glutamate (Fig. 24B). This glutamate (Glu307 in A. aeolicus) does not directly participate in the activation of the substrate (neither as proton donor nor nucleophile), but it has been suggested to have three important roles: (i) stabilization of the [4Fe-4S] cluster against loss of the fourth unique iron atom, (ii) binding of the substrate and, (iii) tuning the reduction potential of the [4Fe-4S] cluster.552,553 In addition, the active site cavity has several positively charged residues (four arginine residues and one lysine) that interacts with the diphosphate moiety.554 The unique iron atom of the [4Fe-4S] cluster participate in the catalysis by forming an iron-oxo intermediate complex, which implies an induced-fit mechanism with structural rearrangements between the N- and C-terminal domains.51,553,555 The X-ray structure of IspG solved in the presence of substrate revealed that for substrate binding and product release, a conformational change must occur from the “closed form” to an “open form”, corresponding to a 64 rotation of the C-terminal domain. The “closed form” of IspG corresponds to the catalytically competent conformation of the active site.51,554,556 The catalytic mechanism of IspG has been studied for several years,551,553,555,557–559 combining isotope labeling experiments, modeling studies, structural and spectroscopic data from EPR, ENDOR, and HYSCORE. Although there is still some debate about the identity and lifetime of the intermediate species identified so far.555,560 The first step is the replacement of Glu307 carboxylate group coordinating the fourth iron atom by the substrate. This leads to the conformational change mentioned above that brings another glutamate sidechain close to the substrate C3 hydroxyl group to abstract a proton.51,556,560,561 Thus, the ionized substrate interacts with the [4Fe-4S] cluster forming an alkoxide complex. Then, there is the formation of a series of intermediates,555 in which the C2 alternates between a carbocation and a radical state assisted by electron transfer from the [4Fe-4S]2 þ/3 þ cluster. In the next step, there is another electron transfer event to form a stable intermediate, a monocarbanionic species bound to [4Fe-4S]2þ. The last two steps include two proton transfer, and release of a water molecule. The residue Glu232 is proposed to donate the proton, facilitating the release of the Fe-bound oxygen, and in connecting the active site to a proton channel that is involved in the delivery of the second proton, necessary for the release of the water molecule.555 As mentioned, IspH catalyzes the reductive dehydration of 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) to form a 5:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate (Fig. 23), isoprenoid building blocks.562 IspH is a monomeric enzyme composed of three domains with similar fold, that are related between each other by a pseudo-C3 symmetry in a trefoil-like protein structure563–565 (Fig. 25A). The iron-sulfur cluster is in a hydrophobic pocket in the center of the three domains and is coordinated by three cysteine residues564,565 (Fig. 25B). The iron-sulfur cluster present in the first crystallographic structures was a [3Fe-4S] cluster, which was later proven to be an artifact caused by iron loss during crystallization. The active site is in fact a [4Fe-4S] cluster, as observed in the X-ray structure analysis of the enzyme with bound substrate563 (Fig. 24B), corroborating the kinetic, spectroscopic (Mössbauer and EPR) and other biochemical studies.563,566–568 Similarly to IspG, the catalytic mechanism of IspH has been a matter of discussion,551 but spectroscopic data on the wild-type and key mutants, along with crystallographic data and QM/MM studies elucidated the catalytic mechanism of this enzyme, as a bioorganometallic mechanism,562,563,569,570 as there was no evidence for the involvement of a radical species.571 The active oxidation state is the [4Fe-4S]1þ, which facilitates the rotation of the 4-OH group of HMBPP away from the iron-sulfur cluster, giving rise to a p-complex intermediate with the C2¼ C3 double bond. The 4-OH group interacts with Glu126, which is then protonated, mediated by a conserved water molecule, to remove the 4-OH as a water molecule, and forming a h1-allyl complex (with C2-C3 atoms). Next, the iron-sulfur cluster is reduced by a second electron and there is the formation of another intermediate species containing a metal-carbon bond, with similar structure to nitrogenase bound to allyl alcohol complex, a h3-allyl complex.571 The following step comprises another protonation, which occurs at C2 or C3, giving rise to isopentenyl diphosphate and dimethylallyl diphosphate, respectively. The residues His124 and Glu126 are proposed to be involved in this last protonation step.562,563

2.06.3.2.3

Pentonate dehydratases

Pentonate dehydratases are a family of enzymes with biotechnology value for the bioconversion of biomass into different organic compounds (e.g., D-pantothenic acid), and production of biofuels (e.g., ethanol and isobutanol).572–575 These enzymes are involved in the non-phosphorylative oxidation pathways of pentose sugars, such as the catalysis of dehydration of D-xylonate, by D-xylonate dehydratase, and L-arabinonate, by L-arabinonate dehydratase.576,577 These enzymes contrary to the ones described before have a [2Fe-2S] cluster in its active site,[523,578] that is stable under oxic conditions. Similarly to the other iron-sulfur enzymes, visible and EPR spectroscopies have been employed in their characterization. Moreover, X-ray structure of several enzymes show the presence of a [2Fe-2S] cluster,524,579,580 with a unique iron atom that is coordinated by one cysteine sidechain, instead of two, and having a close by Mg2þ. The unique iron atom has a vacant coordination position to which the intermediate hydroxyl can bind (Fig. 26) and will also function as strong Lewis’ acid facilitating the loss of the substrate hydroxyl group. The Mg2þ is proposed to be important in the stabilization of the carbanion that is transiently formed, and in reducing the electronegativity of the C2 oxygen, which weakens the CeO bond at C3.580

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Fig. 23 The canonical non-mevalonate isoprenoid prokaryotic biosynthetic pathway. Adapted from Grawert, T.; Groll, M.; Rohdich, F.; Bacher, A.; Eisenreich, W. Biochemistry of the Non-Mevalonate Isoprenoid Pathway. Cell. Mol. Life Sci. 2011, 68(23), 3797–3814; Perez-Gil, J.; RodriguezConcepcion, M. Metabolic Plasticity for Isoprenoid Biosynthesis in Bacteria. Biochem. J. 2013, 452(1), 19–25; Rohdich, F.; Zepeck, F.; Adam, P.; Hecht, S.; Kaiser, J.; Laupitz, R.; Grawert, T.; Amslinger, S.; Eisenreich, W.; Bacher, A.; Arigoni, D. The Deoxyxylulose Phosphate Pathway of Isoprenoid Biosynthesis: Studies on the Mechanisms of the Reactions Catalyzed by IspG and IspH Protein. Proc. Natl. Acad. Sci. USA 2003, 100(4), 1586–1591.

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Fig. 24 (A) Structure of IspG dimer, with one monomer colored by secondary structure and the other gray. (B) Structure of the iron-sulfur cluster of IspG. In Panel B the atoms are colored according to element: carbon, iron, oxygen, and sulfur in gray, orange, red and yellow, respectively. Panel A and B were prepared in Discovery Studio Visualizer (BIOVIA) using the coordinates from PDB ID 3NOY.

(A)

(B)

Cys96

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Fig. 25 The active site of IspH. (A) Structure of IspH monomer colored by secondary structure, and (B) structure of the iron-sulfur cluster of IspH bound to substrate HMBPP. The atoms are colored according to element: carbon, iron, oxygen, phosphorus, and sulfur in gray, orange, red, green, and yellow, respectively. Panel A and B were generated with the program Discovery Studio Visualizer (BIOVIA), using the coordinates from PDB ID 3KE8.

These enzymes are tetramers (dimer of dimers) in solution, with each subunit having two domains with a unique complex fold: the N-terminal domain has a a/b structure, and the C-terminal domain a central mixed eight-stranded b-barrel. The cysteine residues that coordinate the [2Fe-2S] cluster are in the N-terminal domain, with the cluster located in a pocket between the two domains, and partially in the interface between the monomers.524,580 The Mg2þ binding site is also located in the N-terminal domain.

2.06.3.3

ADP-ribosyltransferases (unusual iron-sulfur cluster)

The ADP-ribosyltransferases catalyze the ADP-ribosylation, in which an ADP-ribose moiety is transferred from b-nicotinamide adenine nucleotide (NADþ) to an arginine in the target proteins, releasing nicotinamide.581,582 These enzymes constitute a group of exotoxins produced by several bacterial pathogens and viruses that are responsible for diseases, such as gonorrhea, meningitis, whooping cough, cholera, and diphtheria.583 It is known that this type of exotoxins interferes with signal transduction, protein synthesis or modify cytoskeleton functions, by ADP-ribosylation of host proteins, such as GDP-binding proteins, elongation factor-2, or actin, respectively,582 as well as antimicrobial peptides.584 Other targets besides proteins have been identified, such as DNA and antibiotics.583,585,586 NarE from Neisseria meningitidis is a dual enzyme as it has ADP-ribosyl transferase and NADþ-glycohydrolase activities,587 modulated by the presence of its substrate, which include actin and other cytoskeleton-related proteins.588,589 NarE toxin binds an iron center,587,590 with the iron atom coordinated by two cysteine and two histidine side-chains (Cys67, Cys128, His46 and His57), in an atypical rubredoxin-type center.32 The EPR spectrum of this protein has the features of a high-spin ferric iron (with g values of 4.4, 4.3 and 4.2) and a maximum absorption band at 420 nm in the visible spectrum.32,590

Iron-sulfur clusters – functions of an ancient metal site

137

Fig. 26 Reaction mechanism of D-xylonate dehydrogenase. In the last step, the enol intermediate is tautomerized to the ceto form. Adapted from Rahman, M. M.; Andberg, M.; Koivula, A.; Rouvinen, J.; Hakulinen, N. The Crystal Structure of D-Xylonate Dehydratase Reveals Functional Features of Enzymes from the Ilv/ED Dehydratase Family. Sci. Rep. 2018, 8(1), 865; Rahman, M. M.; Andberg, M.; Thangaraj, S. K.; Parkkinen, T.; Penttila, M.; Janis, J.; Koivula, A.; Rouvinen, J.; Hakulinen, N. The crystal structure of a bacterial l-arabinonate dehydratase contains a [2Fe-2S] cluster. ACS Chem. Biol. 2017, 12(7), 1919–1927.

The NarE structure in the apo-form, determined by solution state biomolecular NMR, is similar to other enzymes of this family. The NADþ binding site was modeled using chemical shift perturbation data and a docking software HADDOCK.32,591 The atypical iron-sulfur cluster was shown initially to be essential for the catalytic activity of the enzyme as ADP-ribosyltransferase but not for its glycohydrolase activity.590 The oxidation state of the iron atom modulates the activity of the enzyme: ADP-ribosyl transferase is stimulated by the ferric state, while NAD-glycohydrolase activity is activated by the ferrous state.592 The catalytic mechanism of NarE has not yet been proposed, though NMR data supports the hypothesis that iron coordination is required for the recognition and binding of the target protein.32 However, the coordination of this center, with two cysteine and two histidine sidechains, is more characteristic of a zinc-binding site and therefore, the in vitro role of zinc, iron and of Mg2þ ions (the latest required to activate other bacterial enzymes of this family) in the activity of this enzyme has been assessed, but no effect was observed.592

2.06.3.4

Other enzymatic activities

Besides the catalytic activities reported before, iron-sulfur cluster can also catalyze other reactions, such as the reductive cleavage of a disulfide substrate, by disulfide reductases,593–595 or the a,b-elimination reaction adjacent to a carboxylate group, by the L-cysteine desulfidase.596 The membrane bound elemental sulfur reducing reductase, MBS, is an early ancestor of complex I. The cryo-EM structure of MBS shows the presence of 26 protein subunits, with the monomer consisting of a peripheral cytoplasmic arm anchored (MbsJ, K, L and N) to the membrane by MbsM. There are three [4Fe-4S] clusters (two in MbsN and one in MbsJ), that form an electron transfer pathway from ferredoxin to polysulfide. The proximal iron-sulfur cluster located in MbsJ is coordinated by only 3 cysteines, instead of the usual four, creating a catalytic site,595 where polysulfide reduction can occur. In fact, the role of the unique iron in catalysis was provided using cluster interconversion to generate a [3Fe-4S] cluster140 (Section 2.06.2.1.5), that lost catalytic activity. EPR spectroscopy was also employed to show the involvement of this center in catalysis,595 and a novel mechanism for sulfur reduction was proposed without the formation of H2S since tri- and disulfides are kept stable in the hydrophobic pocket. The L-cysteine desulfidase catalyzes the decomposition of L-cysteine to hydrogen sulfide, ammonia, and pyruvate, and up-to-now has only been isolated from Methanocaldococcus jannaschii,596 and its gene expression and organization has been characterized in Yersinia ruckeri,597 S. enterica and E. coli.598 In common with other enzymes containing an iron-sulfur cluster in the active site, these enzymes present a [4Fe-4S] cluster that can exist in an active and inactive form, due the loss of the unique iron atom that is not coordinated by any residue and is the substrate binding site. Contrary to the disulfidase, disulfide reductases have been more extensively characterized using spectroscopic and other biophysical techniques. This family of enzymes comprise the ferredoxin:thioredoxin reductase (FTR) and the heterodisulfide reductase (HTR) isolated from methanogenic archaea. The former catalyzes the reaction of an active site disulfide, while the second subfamily catalyzes the reduction of the heterodisulfide of the methanogenic thiol-coenzyme and coenzyme B. These enzymes differ from all the other presented here since each of the iron atoms of its active site [4Fe-4S] cluster is coordinated by a cysteine residue. However, the iron-sulfur cluster interacts directly with either the substrate (HTR) or with an active site disulfide (FTR), delivering one of the two electrons required to complete the reaction.593,594 For this reason, these enzymes are not further discussed, though further information can be found from the cited literature.

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Iron-sulfur clusters – functions of an ancient metal site

Iron-sulfur clusters involved in metabolic regulation

Iron-sulfur proteins with a role as sensors and regulators are widespread in both prokaryotic and eukaryotic organisms. Their function includes iron sensing and homeostasis (e.g., IRP1, IscR, RirA), sensing gases, such as oxygen (FNR) and nitric oxide (e.g., WhiB, NsrR), or responding to oxidative stress conditions (e.g., SoxR). The environmental stimulus is sensed by these proteins through disassembly and assembly of their iron-sulfur cluster, modification of the cluster or change in its oxidation state, with most of the proteins studied up-to-now falling into the first categories. Moreover, these proteins differ in the way they transmit the information, some are simultaneously transcription regulators that bind to regulatory DNA sequences, activating the transcription, while in others the information is transmitted to an effector protein through phosphorylation, and it is this protein that either binds to DNA or activates a cascade. Here, these regulatory proteins are divided according to their action as post-transcriptional or transcriptional regulators.

2.06.4.1

Post-transcriptional regulation of iron homeostasis

All living organisms present molecular systems that are involved in different metal homeostasis,599 and are responsible for maintaining a tight control of the intracellular levels of metals, their storage, and their distribution between tissue (in the case of eukaryotes). In the case of iron, the presence of these homeostasis systems is essential since in one hand there are many different enzymes and proteins that require iron or iron-containing cofactors for their function (e.g., hemoglobin, cytochrome c, cytochrome c oxidase) and, on the other hand, free iron can originate, through Fenton chemistry, reactive oxygen species that can lead to cell damage and death.600 In mammalian, iron homeostasis is controlled at the post-transcriptional level by IRP1 and IRP2, iron regulatory proteins. IRP1 and IRP2 bind to iron responsive elements (IRE) in the UTRs (untranslated regions) of mRNA encoding proteins involved in iron use, storage, export, and uptake.601,602 These two proteins sense not only the intracellular iron levels, but also respond to nitric oxide and reactive oxygen species,603,604 providing a link between the iron metabolism and oxidative stress.605 IRP1 is also known as cytoplasmic apo-aconitase (or apo c-aconitase)172,536 and is an example of a bifunctional protein (also known as moonlighting protein606) as mentioned before (Section 2.06.3.2.1). In fact, IRP1 can catalyze the conversion of citrate to isocitrate when an [4Fe-4S] cluster is bound to its polypeptide chain (see Section 2.06.3.2.1). However, when there is a change in the intracellular level of iron, and there is the need for iron uptake, the iron-sulfur cluster present in IRP1 disassembles (Fig. 27A), and this protein is then able to bind to IREs in the 5’UTR of mRNAs of heavy- and light-ferritin polypeptide, and ferroportin, inhibiting ribosome binding and the translation of these mRNAs (Fig. 27B). At the same time, IRP1 inhibits nucleolytic degradation of mRNA coding for the transferrin receptor 1 by stabilizing this mRNA through the binding to its IRE in the 3’UTR607 (Fig. 27B). The regulation of c_aconitase/IRP1 dual function is a complex mechanism that is still not completely understood but seems to be related with the level of iron and synthesis of iron-sulfur clusters in the mitochondria.608,609 For activity, IRP1 requires not just the complete removal of iron-sulfur cluster, but also the concomitant reduction of coordinating cysteine residues.537 However, the disassembly of the [4Fe-4S] cluster is not kinetically favorable, simply as a response to low intracellular levels of iron172 and it has been shown that it can be triggered by reactive oxygen species610,611 and reactive nitrogen species.605,612 In the presence of reactive oxygen species, it was observed the release of the unique iron, Fea, and formation of an intermediate [3Fe-4S]1þ cluster,613,614 while in the presence of NO/peroxynitrite the complete disassembly of the cluster is observed with no stable intermediate species being detected.612,615 The Fea atom is the most solvent exposed iron atom and the one that is not coordinated by any cysteinyl residue (named also unique iron atom) (see Section 2.06.3.2.1). The interconversion of [4Fe-4S] cluster in aconitase to [3Fe-4S] was observed in vivo by whole cell EPR, and it was also demonstrated that phosphorylation can control the response of IRP1 to iron levels by affecting the iron-sulfur cluster stability and turnover of the conversion.614 Moreover, phosphorylation of Ser138 allows the [4Fe-4S]2þ cluster to cycle to [3Fe-4S]0 and thus it seems that regulation of the cluster disassembling can be initiated solely due to iron availability.616,617 The structure of IRP1 bound to the IRE of ferritin has been determined.618 The comparison between this structure and the one of aconitase elucidated the extensive conformational changes that need to occur for IRE recognition and binding (Fig. 28). These conformational changes occur mainly in domain 3 and 4, with its rotation and translation relative to the core domains (domain 1 and 2). These motions separate these two domains, creating a space where the IRE-stem loop will be accommodated. Moreover, the shifts that occur in domain 3 are also accompanied by a conformational change of two other regions in the interface of domains 2 and 3, residues 436 to 442 and 534 to 544, that play an important dual structural-functional role as forming the ligand-binding environment for the [4Fe-4S] cluster (Fig. 28A) or being involved in the interaction with A15 and G16 bases of the IRE-stem loop618 (Fig. 28B). Therefore, as shown in Fig. 28, IRP1 adopts an extended L-shaped conformation that embraces the IRE stem-loop. Contrary to IRP1, IRP2 does not assemble an iron-sulfur cluster and thus it is not a bifunctional protein, but its activity is also dependent on the presence of an iron-sulfur cluster (vide infra). In addition, IRP2 shares 65% of sequence identity with IRP1,619,620 and like IRP1 also functions as a post-transcriptional regulator of genes involved in iron metabolism (Fig. 27B). The activity of IRP2 is dependent on another protein, FBXL5,621,622 that binds an [2Fe-2S] cluster in its C-terminal domain,623 and has a N-terminal hemerythrin-like domain.624–626 The UV–visible spectrum of this protein has absorption bands with maxima at 330 nm and 425 nm, and it is EPR silent in the as-isolated and oxidized state, while in the reduced state presents a signal with g values of 2.042, 1.918 and 1.889 (with an average g-value of 1.950), which are consistent with the presence of a [2Fe-2S]1þ cluster coordinated by 4-cysteine residues.623

Iron-sulfur clusters – functions of an ancient metal site

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Fig. 27 Schematic representation of the mode of action of IRP1 and IRP2 as post-transcriptional regulators. In panel A is represented the interconversion of aconitase-IRP1 that occurs depending on the cellular iron level, due to oxidative disruption by superoxide anion, peroxynitrite and carbonate radical. In panel B is represented the function of IRP1 and IRP2 in translation activation and repression during normal and low iron levels, respectively.

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Fig. 28 Conformational changes observed in cytosolic IRP1 (or holo c_aconitase) upon iron-sulfur cluster disassembly. In panel A is represented the structure of the inactive human holo-cytosolic IRP1, and in panel B the structure of IRP1 (apo c_aconitase) bound to ferritin IRE mRNA. Panel A and B were prepared using the coordinates 2B3Y and 3SNP, respectively. Protein structure is colored according to secondary structure, iron-sulfur cluster is represented as sticks and RNA is colored green. Images in Panel A and B were created with the program Discovery Studio Visualizer (BIOVIA).

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Iron-sulfur clusters – functions of an ancient metal site

The cryoelectron microscopy structure of IRP2 in complex with FBXL5 was determined and shows that IRP2 adopts a L-shape open conformation, like apo-IRP1, and FBXL5 binds in the middle through its C-terminal domain,623 with the iron-sulfur cluster being close but not in the immediate interface. The binding of FBXL5 with its iron-sulfur cluster in the oxidized state, [2Fe-2S]2þ, is required to displace IRP2 from the IRE-stem loop, and it is also critical for ubiquitin ligase binding.623 Therefore, the presence of this cluster explains how IRP2 activity is dependent on iron (through its N-terminal hemerythrin-like domain that binds a diiron center, changing its structure621,624–626) and oxygen, as in the presence of high levels of iron and oxygen there is formation of an oxidized [2Fe-2S]2þ cluster in FBXL5, that then binds to IRP2, displacing it from its IRE. In the following steps, FBXL5 recruits IRP2 for polyubiquitination and degradation (Fig. 27B). The biological relevance of having two IRP proteins is still not completely understood, but it might be related with cell specificity627 and the signals that are sensed, since they are active under different conditions. In fact, these two proteins were shown to be reductant as mice without both alleles of Irp1 and Irp2 are not viable,628 while mice lacking either Irp1 or Irp2 are viable and fertile.629 Moreover, IRP2 has been associated with erythropoietic homeostasis and nervous system, while IPR1 is essential for controlling the equilibrium between iron homeostasis and erythropoiesis, with an important role in the cardiovascular and pulmonary systems.630,631 Therefore, further studies are required to completely understand the role of the IRP1/IRP2 system in human health and disease.

2.06.4.2

Transcription regulators

Several transcription regulators have been shown to bind iron-sulfur clusters, either [2Fe-2S] or [4Fe-4S] type clusters. These transcription regulators are usually composed of two domains, one that is the regulatory domain and usually contains the iron-sulfur cluster and the other that binds to specific DNA sequences (Fig. 29). Primary sequence alignment enabled the grouping of these transcription regulators into two main families, the Rrf2 family and the CRP-family. A third family includes different transcription regulators that have a domain that also binds an iron-sulfur cluster, but either their domain organization or mode of action differs from the ones in the other two families. In this latter family are also included PAS domains of sensor kinases of two-component systems (NreB) and of a s54-dependent activator (OrpR), that bind an iron-sulfur cluster. GAF domains have also been shown to bind [2Fe-2S] cluster, as in the case of Staphylococcus aureus AirS-AirR, a two-component system involved in sensing O2 and oxidative stress (similarly to the NreBC system).632 Moreover, like RirA, Aft1 and Aft2 are involved in the regulation of iron uptake through its [2Fe-2S] cluster in Saccharomyces cerevisiae.633,634 For a more complete list of iron-sulfur protein involved in the regulation of gene expression see Mettert & Kiley.635 These transcription regulators make use of the versatile properties of their iron-sulfur clusters, such as the ability to delocalize electrons over the Fe and S atoms of the cluster, to change oxidation state (by reacting with oxygen, reactive oxygen species and redox-cycling compounds), and to be vulnerable to modification by molecular oxygen and NO. In addition, the presence or absence of the iron-sulfur cluster can be used to sense the iron or even the iron-sulfur cluster cellular level. The sensing mode involves, in many cases, cluster interconversion that leads to conformational changes in the protein and modulates its DNA affinity. This enables these transcription regulators to respond to different stimuli, such as reactive oxygen species, cellular redox potential, nitric oxide, and iron availability, and control transcription of different metabolic pathways.

2.06.4.2.1

Rrf2 family

The Rrf2 family of transcription factors is widespread but still poorly characterized. Four members of this family that bind an ironsulfur cluster will be described here: IscR, RirA, NsrR and RsrR. IscR senses the iron-sulfur cluster level in the cell and regulates the biogenesis of iron-sulfur clusters, while RirA responds to the level of iron and controls iron uptake and storage systems (Fig. 30). NsrR senses a gas, nitric oxide, and regulates the nitric oxide stress response or reactive nitrogen species stress in many prokaryotes, while RsrR senses the cellular redox status. The analysis of the primary sequence of Rrf2 family of transcription regulators shows that there is a winged helix-turn-helix (wHTH) at the N-terminus domain, and a cysteine rich region at the C-terminus located in an insertion segment absent in the proteins that do not bind an iron-sulfur cluster, as in CymR636 (Fig. 31). The analysis of the structures of apo-IscR, holo-NsrR and holo-RsrR, and that of CymR (a non-iron-sulfur cluster binding protein) show that they share a common fold composed mainly by a-helixes and two anti-parallel b-strands, organized in two domains. The wHTH DNA-binding domain, and the dimerization helix are connected through a loop that contains the two/three conserved cysteines. The location of these cysteine residues places the iron-sulfur cluster close to the wHTH motif of an opposing monomer, and thus its presence/absence can modulate the DNA binding properties of the transcription factor. The fourth ligand of the iron-sulfur cluster in these proteins has been confirmed with the characterization of variants of conserved residues or by the comparative analysis of their holo-structures (see Section 2.06.4.2.1.1 to 2.06.4.2.1.4). The nature of the fourth ligand varies between the Rrf2 family members, which can determine the properties of the iron-sulfur cluster and play a role in regulating its function.

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Envionmental Cues

Rrf2 Family IscR

HTH

[2Fe-2S]

Fe-S cluster

RirA

HTH

[4Fe-4S]

Fe

NsrR

HTH

[4Fe-4S]

NO

RsrR

HTH

[2Fe-2S]

Redox status

CRP-Family FNR

[4Fe-4S]

HTH

O2

ANR

[4Fe-4S]

HTH

O2

FNRBs

CRP-like

HTH

[4Fe-4S]

O2

Others

SoxR

HTH

[2Fe-2S]

Redox status

SufR

HTH

[4Fe-4S]

NO, O2

WhiB-like NreB

NO, O2

[4Fe-4S] [4Fe-4S]

kinase

O2

Fig. 29 Schematic representation of the different families of transcription regulators that contain an iron-sulfur cluster. HTH – helix turn helix domain. AAAþ – ATPase associated activity. The domains are not represented to scale. FNRBS is the FNR homolog found in Bacillus subtilis.

RirA

+[Fe]

RirA

IscR

[FeS]

[FeS]

Iron storage ferri ns

Fe-S biosynthesis

[Fe-S] cell status

Iron uptake systems Siderophore uptake

Fig. 30

Fe3+ uptake Fe2+ uptake

Schematic representation of the role of iron-sulfur cluster containing transcription regulators in iron homeostasis.

2.06.4.2.1.1 IscR, iron-sulfur cluster biogenesis regulator The gene encoding IscR is located immediately upstream of the iron-sulfur cluster biosynthesis system (Isc), isc operon, iscRSUAhscBA-fdx.637 The expression of this operon and of sufABCDSE, also involved in iron-sulfur cluster biosynthesis, is induced by oxidants. In fact, under oxidative stress conditions the cell has an increased demand for the maintenance and assembly of ironsulfur clusters,638 which are in many cases oxygen-sensitive. Under anaerobic conditions, IscR is a feedback repressor of the iscRSUA operon as a [2Fe-2S]1þ cluster containing protein,637 while under oxidative stress conditions and iron limitation, apo-IscR is a transcriptional activator of the suf operon and derepresses the isc operon.638 This mechanism enables IscR to regulate the function of Isc and Suf systems and coordinate the consumption of iron and cysteine between these two systems.639 IscR binds an [2Fe-2S] cluster and its presence changes IscR DNA binding specificity. The holo-IscR binds a type I sequence motif with high affinity,640 while both the apo and holo-IscR bind to a type II sequence motif. As mentioned, the regulatory mechanism of IscR depends on the presence of a [2Fe-2S] cluster, as it is proposed that the conformational change that occurs upon loss of the [2Fe-2S] cluster, induces unfavorable interactions of the protein with the type-I motif.641 The position of these motifs in relation to the promoter dictates whether IscR will function as an activator or a repressor. The anaerobically isolated E. coli IscR binds a [2Fe-2S] cluster. The EPR signal of the [2Fe-2S]1þ state has g values at 1.99, 1.94 and 1.88,637 which suggested a full cysteinyl coordination of the cluster. This iron-sulfur cluster has a low relaxation rate and can be reversibly oxidized to [2Fe-2S]2þ, without significant loss of the cluster.637 In the case of IscR from Acidithiobacillus ferrooxidans, its visible spectrum presents maximum absorption bands at 315 nm and 410 nm, and an EPR signal with a gav value at 2.013.642

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Iron-sulfur clusters – functions of an ancient metal site

1 10 20 30 40 50 60 | | | | | | | MRLTSKGRYA VTAMLDVALN SEAGPVPLAD ISERQGISLS YLEQLFSRLR KNGLVSSVRG MRLTTKGRYA VTAMLDLALH AQQGPVSLAD ISERQGISLS YLEQLFAKLR RGNLVTSVRG MKLTTKGRYA VTAMLDLALN QRGGVVTVAD ISQRQQISVA YLEQLFGRLR RFGLVESVRG MKLSGGVEWA LHCCVVLTA- -ASRPVPAAR LAELHDVSPS YLAKQMQALS RAGLVRSVQG MKMSGGVEWA LHCCVVLTV- -SSRPVPAAR LAELHDVSSS YLAKQLQSLA RAGLIHSVQG MRLTKQTNYA VRMLMYCAA- NGEKLSRIPE IARAYGVSEL FLFKILQPLT RAGLVETVRG MRLTKQTNYA VRMLMYCAA- NDGHLSRIPE IARAYGVSEL FLFKILQPLN KAGLVETVRG MQLTSFTDYG LRALIYMASL PEGRMTSISE VTDVYGVSRN HMVKIINQLS RAGYVTAVRG MYLTQHTDYG LRVLIYTAI- NDDALVNIST IAVTYGISKS HLMKVVTALV KGGFLHSVRG * :: :. : : : :: :* .: : . * : . : :*:*

IscR IscR IscR RsrR RsrR RirA RirA NsrR NsrR

Ecoli Psa Ac Sv Mc Sinor Rhiz Ecoli Nm

IscR IscR IscR RsrR RsrR RirA RirA NsrR NsrR

Ecoli Psa Ac Sv Mc Sinor Rhiz Ecoli Nm

PGGGYLLGKD PGGGYQLSRH PGGGYRLAMP KTGGYVLTRP KSGGYALTRA RNGGVRLPRP RNGGVRLGKP KNGGIRLGKP KGGGLRLAAP ** *

ASSIAVGEVI MSGIHVAQVI DSEIPLTRIV AVEITLLDVV PESITLLDVV ASEITLFDVV AADITLFDVV ASAIRIGDVV PDRINIGSVV * : ::

SAVDESVDAT DAVNESVDAT EAVNESISTT QAVDGPDPAF RAVDGPGPAF KVTEDSFAMA RVTEDSFAMA RELEP-LSLV RHLEP-MQLV :

RCQGKGG--RCQGQGD--QCGGDPH--VCTEIRQRGP VCTEIRQRGP ECFEA-G--E ECFEDDG--V NCSSE----ECMGE----N *

-----C-QGG -----C-HSG ---LCCKGDG LATPPEKCTK LATPANACTR ---IDCPLVD ---VECPLVD ----FCHITP ---NECLITP

DKCLTHALWR DTCLTHHLWC QQCLTHDLWE -ACPIARAMG -ACPVARAMW -SCGLNAALR -SCGLNSALR -ACRLKQALS -SCRLTGILG * : :

IscR IscR IscR RsrR RsrR RirA RirA NsrR NsrR

Ecoli Psa Ac Sv Mc Sinor Rhiz Ecoli Nm

DLSDRLTGFL DLSLQIHEFL ELGNRIAEFL AAEAAWRASL TAEEAWREAL KALNAFFEVL KALNAFFAVL KAVQSFLTEL GAMKSFFTYL *

NNITLGELVN SGISLADLVS GGITLGQLVQ ASTTIADLVA AAVTIADLAR QGYTIDDLVK SEYSIDDLVK DNYTLADLVE DGFTLQDLLN :: :*

NQEVLDVSGR RQEVQEVALR KQLHKELMQA TVDDES---DVGTDS---AR-PQINFLL AR-PQINFLL ENQPLYKLLL --KPTYDLLY

QHTHDA---QDERRCSGKT AP-IAMTDKT ---GPDALPG ---GPEALPA GLEEPVRPQT GITGEQPYRK VE-------EPRIPIAVQ-

PRTRTQDAID PRLDKIEASA PLRETHDTAVGAWLIEGLG VRTWLTGASD SAA------PAIVAPAA--------------------

VKLRA ID----------H--------------------

Fig. 31 Primary sequence alignment of the transcription regulators belonging to the Rrf2 family that bind an iron-sulfur cluster, IscR, NsrR and RirA. Legend: Ecoli – E. coli, Psa – Pseudomonas aeruginosa, Ac - Acidothiobacillus ferrooxidans, Sv – Streptomyces venezuelae, Mc – Micromonospora coriariae, Sinor – Sinorhizobium, Rhiz – Rhizobium, Nm – Neisseria meningitidis. Asterisks, colons or stops below the sequence indicate identity, high conservation, or conservation of the amino acids, respectively. Conserved cysteines are highlighted in yellow, the third and/or fourth ligand is in pink, and putative fourth ligand of [4Fe-4S] RirA is in gray.

The primary sequence analysis of IscR revealed the presence of only 3 conserved cysteine residues (Fig. 31), indicating that the [2Fe-2S] cluster could not have only cysteinyl coordination. The fourth ligand of the iron-sulfur cluster was then identified by sitedirected mutagenesis as being His107,643 also a conserved residue (Fig. 31). The mechanism of assembly and disassembly of the [2Fe-2S] cluster into IscR has not been deeply investigated, though is it proposed that the cluster is sensitive to oxygen, enabling IscR to oscillate between the holo and apo-forms.639 Moreover, while the Mössbauer analysis showed that in whole cells over-expressing IscR its iron-sulfur cluster is in the [2Fe-2S]1þ oxidation state,643 this does not influence its DNA binding affinity.643 The structure of apo-IscR shows that it binds to DNA as a dimer with a long helix forming the dimer interface and the two wHTH are in two opposite sides of the dimer.641 The position of two wHTH enables this transcription regulator to bind 23–27 DNA consensus motifs. The position of the proposed fourth ligand is located at the surface of the monomer, an indication that IscR iron-sulfur cluster can be solvent-exposed, and thus prone to the action of reactive oxygen or nitrogen species. As mentioned, the conformational changes that occur in the iron-sulfur cluster bound form, even if small and local, modulate the DNA consensus motifs that IscR recognizes. 2.06.4.2.1.2 Rhizobial iron regulator A (RirA) Another transcription regulator that binds an iron-sulfur cluster is RirA, a rhizobial iron regulator A, that was initially just identified to be an iron-responsive regulator in Rhizobium leguminosarum.644 RirA and its homologs have been found in all members of the a-proteobacteria of the genus Rhizobiaceae. Similarly to the wellstudied E. coli Fur (ferric uptake regulator), RirA is proposed to be a global iron-responsive transcription regulator involved in the regulation of iron uptake and metabolism in Rhizobium644 and Sinorhizobium.645 Proteomic and transcriptomic studies have shown that, in Fe-replete growth conditions, RirA represses the expression of several genes involved in the synthesis and uptake of

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siderophores, uptake of heme, synthesis of iron-sulfur clusters and of genes that code for proteins that probably participate in Fe3þ transport646 (Fig. 30). RirA is proposed to bind an iron-sulfur cluster in analogy to another Rrf2-type proteins (see Sections 2.06.4.2.1.1 to 2.06.4.2.1.4),647 and due to the presence of a triad of conserved cysteines in its C-terminal region,644 which are essential for its function646 (Fig. 29). In fact, bioinformatic analysis of several genomes of a-proteobacteria, identified a palindromic motif with the consensus 50 -TGA(N9)-TCA-30 , as being the RirA-box (IRO-box).648 The heterologously produced RirA after in vitro cluster reconstitution was shown to bind an [4Fe-4S] cluster, having a visible spectrum with a broad absorption band with a maximum below 400 nm.649 The presence of an [4Fe-4S] cluster was supported by its CD spectrum and by its mass spectrum.649 RirA is a dimer in solution and binds DNA with a high affinity in the holoform. In the presence of low concentrations of iron (mimicked by the presence of EDTA), it is observed a red shift in the absorption bands of [4Fe-4S] RirA, consistent with the formation of a [2Fe-2S] cluster, and then there is a decay in intensity of these bands corresponding to the loss of cluster and formation of apo-RirA. These forms of RirA have decreased DNA binding affinity649 when compared with [4Fe-4S] RirA. The mechanism of decomposition of the [4Fe-4S] cluster to a [2Fe-2S] cluster has been studied by CD, EPR and time-resolved mass spectrometry, and two intermediate species were identified: a [3Fe-4S]0 and a [3Fe-3S] cluster.649,650 The first step of this process is the loss of iron with a dissociation constant of around 3 mM, while the following steps in cluster conversion are O2-dependent, with formation of a [3Fe-4S]1þ cluster.650 The dissociation constant for the release of the first Fe atom is within the physiological range, which is consistent with this protein being an iron-dependent transcription regulator.651 The coordinating residues of the intermediate [2Fe-2S] cluster are proposed to be the ones that coordinate the [4Fe-4S] cluster, with one of the coordinating residues not yet identified (in either case). The analysis of the sequence alignment shown in Fig. 31 indicates that Gln6, Asn8, Arg12 (opposing subunit) and Asn109 are conserved and in proximity of the iron-sulfur cluster in the model structure generated using SwissModel (Fig. 32). However, it is also possible that the fourth ligand is a oxygen fom a nonprotein molecule (N. E. LeBrun personnal communication). 2.06.4.2.1.3 Nitrite-sensitive transcription repressor (NsrR) NsrR, a nitrite-sensitive transcription repressor, is also part of the Rrf2 family. NsrR regulates several genes,196,652 and it has been suggested to be a global transcriptional regulator of the bacterial NO stress response.653 One of these genes encodes the hybrid cluster protein, Hcp, indicating that this protein might be part of a defense mechanism against reactive nitrogen species,196 as it is a NO reductase198 (see Section 2.06.2.2.1.1). Another gene that is under the control of NsrR is hmp, which encodes a flavohemoglobin that converts NO to nitrous oxide (N2O) or to nitrate (NO3).654–656 NsrR has been isolated from either Gram-positive (such as, Bacillus subtilis,657 and S. coelicolor 658) or Gram-negative (such as, E. coli,659 S. enterica,660 Neisseria meningitides,661 Neisseria gonorrhoeae,662 and Nitrosomonas spp.663) bacteria. Like other members of the Rrf2 family of transcription regulators, NsrR presents three conserved cysteines (Fig. 31), that are proposed to coordinate an iron-sulfur cluster,658 that is NO-sensitive and essential for its DNA binding properties. The iron-sulfur cluster present in S. coelicolor NsrR was initially identified to be a [2Fe-2S] cluster by different spectroscopic techniques, such as CD, UV–visible and EPR.658 The CD spectrum of NsrR is similar to the one of other proteins containing a [2Fe-2S]2þRieske type cluster, such as BphF664 (with three positive features, with lmax at 324 nm, 445 nm and 490 nm, and two negative features, with lmax at 375 nm and 550 nm).658 The visible spectrum has absorption bands with maxima at 325 nm and 420 nm and shoulders at 460 nm and 550 nm, while being EPR silent is consistent with the presence of an oxidized [2Fe-2S]2þ cluster.658

Asn109

Gln6 Arg12

Fig. 32 Model structure of Sinorhizobium meliloti SM11 RirA obtained with SwissModel and the coordinates of NsrR (PDB ID 5N07), which has 37% primary sequence identity with RirA. The iron and sulfur atoms are represented by orange and yellow spheres, respectively. The putative fourth coordinating residues are represented as sticks colored by element. One of the monomers of RirA is colored in gray and the other by secondary structure. The model was generated in SwissModel, and image prepared in Discovery Studio Visualizer (BIOVIA).

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A similar cluster was proposed to be present in N. gonorrhoeae NsrR, though the evidence was only the 145 Da difference between the expected mass of the apo-NsrR and the isolated NsrR.662 However, the iron-sulfur center present in B. subtilis NsrR was proposed to be a [4Fe-4S] cluster, based on its UV–visible (maximum absorption band at 412 nm) and resonance Raman spectra.657 As in other NsrR proteins, B. subtilis NsrR [4Fe-4S] cluster is NO-sensitive and required to bind the specific DNA sequence.665 The initial discrepancy in the type of iron-sulfur cluster bound to NsrR was clarified by the isolation of S. coelicolor NsrR under anaerobic conditions in the absence of low molecular weight thiols and lead to the identification of a bound [4Fe-4S] cluster.666 In fact, it was shown that these compounds (such as DTT, usually added to the purification and storage buffers of oxygen sensitive proteins) modified the NsrR iron-sulfur cluster,657,666 that decomposes to a [2Fe-2S] cluster. On the contrary, physiological thiols, such as cysteine and mycothiol did not induce this decomposition.666 The [4Fe-4S] cluster is coordinated by three conserved cysteine residues (Fig. 31) and by an oxygen ligand, as suggested by its resonance Raman,666 and later confirmed by its X-ray structure.667 The structure of holo-NsrR identified Asp8, of the opposite monomer, as the fourth coordinating residue. This was the first report of such asymmetrically coordinated iron-sulfur cluster.667 The active form of NsrR is a dimer containing a [4Fe-4S]2þ cluster and its nitrosylated form loses the ability to bind the DNA consensus regions. The mechanism by which NsrR is proposed to sense nitric oxide encompasses the nitrosylation of the ironsulfur cluster, with formation of cysteine thiolate-bound dinitrosyl iron complex species, that loses the ability to bind to DNA.656,668 Earlier studies detected the formation of tetrahedral [FeI(NO)2(SR)2], with SR being cysteinate ligands provided by the protein,658 using EPR spectroscopy, as this cluster has a distinctive signal at g ¼ 2.03.658 Afterwards, with the improvement in protein production and sample handling, as well as novel techniques developed for iron proteins, such as nuclear resonance vibrational spectroscopy, complemented by liquid chromatography electrospray ionization mass spectrometry, enabled the identification of the nitrosylation products that are formed during this complex process.656 In the case of NsrR there is formation of Roussin’s Red Ester, [Fe2(NO)4(SR)2], and a persulfide form [Fe2(NO)4(S-Cys)(Cys)].669–671 2.06.4.2.1.4 Redox-sensitive response regulator (RsrR) Streptomyces venezuelae genome encodes a Rrf2 family transcription regulator that responds to the redox status of the cell and was named redox-sensitive response regulator (RsrR). RsrR regulates the cellular concentrations of NADH and NAD(P)H by controlling the expression of the gene sven6562 (a putative NAD(P)þ binding repressor belonging to the NmrA family (nitrogen metabolite repression)), and genes evolved in glutamine and glutamate metabolism. RsrR binds to a binding motif organized in a 11–3-11 base pairs inverted repeated sequence.672 This dimeric protein binds a [2Fe-2S] cluster that can oscillate between the [2Fe-2S]2þ and [2Fe-2S]1þ oxidation state. This redox process controls RsrR DNA binding activity, with the RsrR-[2Fe-2S]2þ oxidation state having higher affinity than the RsrR-[2Fe-2S]1þ oxidation state.672 The visible and CD spectra of RsrR have the features of a [2Fe-2S] cluster with absorbance bands with a maximum at 460 nm, in the oxidized state, and at 540 nm in the reduced state, with this form being EPR active with g values of 1.997, 1.919 and 1.867.672 The structure of [2Fe-2S] RsrR was determined in different oxidation states.39 The protein backbone adopts the already described conserved fold of Rrf2 family but revealed that the iron-sulfur cluster has a unique coordination being bound by two cysteines, Cys90 and Cys110, located in the C-terminal domain and two other residues in the N-terminal domain of the other subunit, Glu8 and His12 (Fig. 4C). This was the first report of an iron-sulfur cluster coordinated by a glutamate sidechain and having three different types of residues as ligands.39 Additionally, these structures revealed the role of Trp9 in the sensing process, in which a change in the oxidation state of the ironsulfur cluster translates into a conformational change of the DNA binding domain, and alteration in the DNA binding affinity of this transcription regulator.39 The presence of the sequence motif EWXXH is observed in other proteins of the Rrf2 family, which could indicate that this type of cluster is more widely present in nature than initially though, and that the role of Trp9 is not unique to RsrR.

2.06.4.2.2

CRP-family

2.06.4.2.2.1 Fumarate and nitrate reductase (FNR) FNR, a member of the CRP (cAMP receptor) family, is a global transcriptional regulator that senses O2 levels via an iron-sulfur cluster bound to the N-terminal domain of the protein, and that binds to the DNA through its helix-turn-helix motif at the C-terminal domain673,674 (Fig. 33). The FNR isolated from E. coli has been extensively characterized both at the molecular and structural level. FNR is involved in the transcriptional regulation of more than 100 genes, activating genes encoding proteins involved in anaerobic metabolism, such as nar operon (nitrate reductase), dms operon (dimethyl sulfoxide reductase) and frd operon (fumarate reductase) and repressing the ones involved in aerobic metabolism.675,676 The primary sequence of FNR shows that there are four conserved cysteine residues in the motif Cys-X2-Cys-X5-Cys, with the fourth cysteine ligand located around 90 residues toward the C-terminus (Cys20, Cys23, Cys29, and Cys122).677 These cysteine residues bind a [4Fe-4S]2þ cluster per monomer, required for FNR to bind specific DNA consensus sequence, the FNR-box, TTGAT-N4-ATCAA.678 The mechanism of regulation has been extensively characterized using spectroscopic techniques and time resolved electrospray ionization mass spectrometry enabling the identification of several intermediate species in what was thought to be a simple cluster

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conversion from a [4Fe-4S] to [2Fe-2S] cluster.679 Some of these studies employed a variant of E. coli FNR, FNR S24F, for which the initial reactions of the [4Fe-4S] to [2Fe-2S] cluster conversion was determined to be 4 times slower.680 These experiments allowed not only the identification of intermediate species in the cluster conversion but also changes in the protein oligomerization state that occur during this process. The FNR’s [4Fe-4S]2þ cluster (S¼ 0), when exposed to oxygen, is converted to [2Fe-2S]2þ, with a [3Fe-4S]1þ (S ¼ 1/2),673,681–683 and a [3Fe-3S] clusters as intermediate species,684 and release of two Fe atoms. In fact, the primary target of O2 has been proposed to be Cys23 or the Fe bound to this cysteine,680 with the loss of one Fe2þ, but without formation of any oxygen adduct species,668,684 as initially proposed. The exact mechanism for this initial step is still not completely understood and could include an [3Fe-4S]0 cluster in equilibrium with free iron (Fe2þ), that is oxidized to [3Fe-4S]0 by O2.668 Then, there is formation of the [3Fe-3S] intermediate species through the loss of sulfide.684 The decay of this intermediate to the [2Fe-2S] cluster is slow and is the rate-limiting step of the [3Fe-4S] to the [2Fe-2S] conversion.680,684 Single and double persulfide intermediate species have been identified by mass spectrometry and resonance Raman, with a [2Fe-3S] species proposed to be generated by the decomposition of the [3Fe-3S], and [2Fe-4S] from [3Fe-4S]. Thus, this process is not due to the incorporation of S0 but an oxidative process with loss of an iron atom, that can then decay into a persulfide apo-form.684,685 The formation of these persulfide species can be a way to easily revert the O2-inactived FNR to an active form without the intervention of the iron-sulfur biogenesis system.684 Further exposure to oxygen leads to the complete disassembly of the [2Fe-2S] cluster or persulfide versions. This conversion of holo-FNR to apo-FNR has been confirmed to occur also in vivo, with all the cysteine residues in the apo-FNR kept in the thiol state,686 which, as mentioned, facilitates the re-assembly of the [4Fe-4S]2þ cluster upon decrease of O2 availability. Besides the modifications that occur at the level of the iron-sulfur cluster, it was also observed that FNR is only a dimer when binding the [4Fe-4S]2þ cluster, and that the other FNR forms are monomeric and do not efficiently bind to the FNR-box677,687,688 (Fig. 33). This change in the oligomerization state of FNR might be due to conformational changes that occur at the dimer interface, driven by alterations in the iron-sulfur cluster binding site.689,690 The dimerization interface is proposed to be mediated by the formation of a coiled-coil of an a-helix in between the DNAbinding and iron-sulfur domains, which contains mainly hydrophobic residues.689 The analysis of the two FNR structures identified a negatively charged residue in this helix, Asp154, that is in a region that cannot stabilize this charged residue. These factors have been proposed to mediate the charge repulsion monomerization of the FNR under aerobic conditions.690 In addition, Ile151 is involved in coiled-coil interactions that stabilize the dimer in the absence of O2, and there is a salt bridge between Arg140 and Asp130 in opposing monomers that also contributes to this stabilization. The disruption of these interactions upon exposure to O2 will lead to the opening of the coiled-coil and dislocation of the equilibrium toward the monomer.691,692 FNR plays a secondary role by sensing and responding to NO.678,693 The iron-sulfur cluster present in FNR becomes nitrosylated upon exposure to NO, forming a dinitrosyl-iron-cysteine complex, which abolishes its capacity to recognize and bind to the specific DNA promoter sequences.678 The physiological significance of the reaction of NO with FNR has been questioned. However, it has been shown that when E. coli cells are exposed to NO there are several FNR-repressed genes that become activated.694,695 Orthologs of FNR have been isolated from other organisms, such as Aliivibrio fischeri,691 Paracoccus denitrificans (FnrP),696 N. meningitidis,697 A. vinelandii (CydR),698,699 B. subtilis (FNRBc),700 which also bind an oxygen-sensitive iron-sulfur cluster, but the mechanism of disassembly is still unknown. Furthermore, the type of iron-sulfur cluster that is bound to these proteins might be different, since the iron-sulfur cluster binding motif of E. coli FNR is not conserved in these organisms. In fact, in the case of

Fig. 33 Schematic representation of the mode of action of FNR transcription regulator. Adapted from Green, J.; Paget, M. S., Bacterial Redox Sensors. Nat. Rev. Microbiol. 2004, 2(12), 954–966.

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B. subtilis FNRBc, it was identified that there are three conserved cysteines in the C-terminal and the fourth ligand of the [4Fe-4S]2þ cluster is an aspartate residue, Asp141.700 This cluster was characterized using UV–visible (lmax at  420 nm and a shoulder at 320 nm) and Mössbauer (DEQ ¼ 0.92 mm/s and d ¼ 0.42 mm/s) spectroscopies,700,701 and its monomer dimer equilibrium was shown not to be dependent on the presence of the iron-sulfur cluster, in contrast to FNR.701 2.06.4.2.2.2 Anaerobic regulation of arginine deiminase and nitrate reduction (ANR) ANR (anaerobic regulation of arginine deiminase and nitrate reduction) is a FNR/CRP-like transcription activation homologous to FNR that has been identified in Pseudomonas aeruginosa.702,703 In Pseudomonas spp., ANR, involved in the switch between the aerobic and anaerobic way of life, is an oxygen-sensing transcription regulator.704,705 The initial studies showed that ANR regulates nitrate transport and reduction, narK1K2GHJI, and two other transcription regulators NarXL and Dnr.706,707 However, a more global analysis indicates that ANR is a global anaerobic regulator, controlling the expression of around 250 genes, involved in central metabolism and aerobic electron transport chain.708 The primary sequence of ANR presents conservation of the four cysteine residues proposed to bind a [4Fe-4S]2þ cluster and it has been shown that ANR can functionally complement an E. coli fnr knock-out variant.703 ANR, akin to FNR, is composed of two domains, with the helix-turn-helix found at the C-terminus, and the iron-sulfur cluster in the consensus motif Cys-X2-Cys-X5Cys in the N-terminus domain. The type of iron-sulfur cluster was shown by Mössbauer spectroscopy (DEQ ¼ 1.2 mm/s and d ¼ 0.43 mm/s) and visible (lmax at  420 nm and a shoulder at 320 nm) spectroscopies709 to be a [4Fe-4S]2þ cluster, similarly to the one present in FNR.709 This [4Fe4S]2þ cluster is converted to a [2Fe-2S]2þ cluster upon exposure to oxygen or NO, and as a consequence ANR loses its ability to bind to its DNA binding consensus sequence,709 in a mechanism akin to the one described in Section 2.06.4.2.2.1 for FNR. In P. putida there are two other proteins with high sequence homology to ANR, that also bind [4Fe-4S] clusters, judging by its UV–visible and CD spectra, but differ in their O2-sensitivity.710

2.06.4.2.3

Other transcription regulators

2.06.4.2.3.1 Superoxide response regulator (SoxR) In E. coli and other enteric bacteria, SoxRS are two separate transcription activators that participate in a two-step activation process in response to redox-cycling compounds. SoxR (superoxide response regulator) enhances to 100-times the transcription of the gene soxS,711,712 and it is SoxS, a member of the AraC family, that will control the expression of around 100 genes, such as the ones encoding for aconitase, manganese-containing superoxide dismutase, endonuclease IV, and Fur, that are involved in repair or avoiding the oxidative stress damage.713,714 However, in other bacteria, such as Pseudomonas spp., SoxR is itself responsible for the transcriptional activation of genes, such as the ones encoding a putative flavin-dependent monooxygenase, and two multidrug efflux pumps, in response to the presence of phenazines, endogenous redox-active pigments, in a superoxide-independent manner.715,716 These genes have been suggested to be involved in phenazine transport and detoxification.716–718 Moreover, in P. aeruginosa and S. coelicolor, SoxR was shown to regulate genes that are involved in the production and transport of endogenously produced redox-active antibiotics.719,720 Although SoxR from different bacteria respond to different signals and is possibly involved in different regulatory networks, its mode of action is proposed to be similar. In fact, expression of P. putida soxR in E. coli complements a soxR deletion mutant.721,722 SoxR, a transcription regulator of the Mer-family, is a homodimer of 17 kDa that binds a [2Fe-2S]1þ cluster per monomer in the consensus sequence Cys-X2-Cys-X-Cys-X5-Cys near the C-terminus.723 The presence of this iron-sulfur cluster does not change the affinity of SoxR to the target promoter region, though its transcriptional activity is completely dependent on the oxidation state of the [2Fe-2S] cluster.711,724,725 In fact, the iron-sulfur cluster present in SoxR needs to be oxidized to [2Fe-2S]2þ, for SoxR to function as a transcription activator,711 and its affinity is 4-fold higher than when its iron-sulfur cluster is in the reduced state, [2Fe-2S]1 þ.726 Although, an increase in almost 500 mV in the iron-sulfur cluster reduction potential upon DNA binding (from  285  10 mV (pH 7.6)711 to þ 200 mV727) was initially reported, latter a different result was obtained. In fact, using electron transfer mediators and a different experimental approach, it was shown that there is a much smaller impact on the reduction potential upon DNA binding, and that this change corresponds to a negative shift, from  293 mV to  320 mV (pH 7.6).726 This smaller difference can be attributed to the small conformational change observed in the structure of the protein in the two oxidation states.728 Although, many advances of the SoxR activation mechanism have been made it is still poorly understood (Fig. 34). Previous studies pointed out that in E. coli, SoxR was activated in response to superoxide anion, that would be generated when the cells were exposed to redox-cycling agents, such as methyl viologen, quinone or phenazine.729,730 However, the direct reaction of this anion with SoxR had not been shown. In 2011, Gu and Imlay731 demonstrated that SoxR does not respond to superoxide anion, but that redox-cycling drugs trigger its activation, by directly oxidizing its [2Fe-2S]1þ cluster. Therefore, SoxRS is proposed to be responsible for the response against these compounds, which are produced by several bacteria and plants to inhibit the growth of competitors.732,733 However, in vivo SoxR is maintained in the reduced state by SoxR reductases (Rsx/Rse system)734,735 that use NADPH as electron donor, thus SoxRS system could be triggered by an imbalance in the NADPH/NADPþ cellular content, reflecting a change in the redox state of the cell.736 The promoter region under the control of SoxR has a long 19 or 20 bp spacer between the  35 and  10 operator elements that need to be untwisted for transcription activation. This process is accomplished by the conformational change that occurs in SoxR upon the one-electron oxidation of the [2Fe-2S]1þ cluster to [2Fe-2S]2þ, conformational change that is also transmitted to the target promoter region.728,737 The extent of the proposed conformational change was determined by comparing the free and DNA-bound

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SoxR structures.728 SoxR is composed by a DNA-binding domain, a dimerization helix, and an iron-sulfur cluster-binding domain at the C-terminus.728 The dimerization helix forms an anti-parallel coiled-coil that stabilizes the dimer, and the [2Fe-2S] cluster is solvent exposed (with S2 and two Fe atoms being completely exposed) and located in an asymmetric environment that is stabilized by interactions with the other subunit.728 It was proposed that the existence of a single glycine residue, Gly123, in between the two internal cysteine residues is required to induce the SoxR conformational changes coupled with the DNA distortion.728,738 Other residues, such as lysines (namely Lys89 and Lys92) have been shown to be important in the reaction with superoxide anion in vitro and affect the transcription activity in vivo.739 However, how the oxidation state of the [2Fe-2S] cluster influences the conformation of SoxR remains unknown, though it has been proposed that a change in the oxidation state can change the hydrogen bonding network in the protein.737 The presence of the solvent exposed iron-sulfur cluster might facilitate its oxidation and can also explain its nitrosylation by NO.740,741 The nitrosylation of the SoxR [2Fe-2S] cluster has been shown to form a dinitro iron complex by EPR both in vitro and in vivo.740,742 Complementary studies using CD and pulse radiolysis enabled the identification of rate limiting steps and other intermediate species.742,743 The dinitro iron complex-SoxR was shown to have a similar DNA affinity as SoxR [2Fe-2S]2þ, and thus to activate gene transcription.740 However, since there are other transcription regulators that specifically respond to NO (with higher affinity for NO and activate several NO scavenging system) (such as NsrR, see Section 2.06.4.2.1.3), the question remains as whether NO reaction is physiologically relevant for the SoxR system.744 2.06.4.2.3.2 WhiB-family of transcription factors regulators WhiB-like proteins, identified in several Actinomycetales, such as Streptomyces spp., Corynebacterium glutamicum, and pathogenic bacteria, such as Mycobacterium tuberculosis and Corynebcterium diphtheria, are proposed to play critical roles in the biology of these bacteria, including morphological differentiation, virulence, and antibiotic resistance.745,746 WhiB-like proteins have also been identified in the genome of several mycobacteriophages, and these also bind an iron-sulfur cluster that regulates their DNAbinding activity.747 The WhiB-like family of proteins is involved in different functions in Streptomyces, such as slow-down of biomass accumulation and changes that precede sporulation (WhiA), multi-drug resistance (WhiC), septation during sporulation (WhiB), and in later stages of sporulation (WhiD).748,749 Moreover, in M. tuberculosis, proteins of this family (WhiB1 to WhiB7) have been shown to be involved in diverse functions, such as antibiotic tolerance and enabling this bacterium to survive for longer periods within its host, maintaining redox homeostasis750 and virulence.750,751 For instance, WhiB1 was shown to be encoded by an essential gene, whiB1, and bind to specific DNA sequences752 and WhiB3 is essential for optimal growth on tricarboxylic acid cycle intermediates during infection753 (further information on WhiB-like protein classes can be found in679,746). The analysis of the primary sequence of several members of the WhiB-family revealed that these proteins can be grouped into five classes, and the presence of (i) four conserved cysteine residues arranged in the motif Cys-Xn-Cys-X2-Cys-X5-Cys, (ii) a motif named b-turn, G[I/V/L]W[G/A]G, after the last conserved cysteine, and (iii) a patch of positively charged residues (with Lys/Arg) at the Cterminus.746,754 The four cysteine residues are proposed to coordinate an iron-sulfur cluster,754 essential for the function of these proteins, that can sense O2 and NO. The b-turn has been proposed to be important for protein interactions, as these proteins do not present the usual DNA-binding domain. The exception is the class that includes Whib7/WblC, as these have a AT-hook that is known to bind AT-rich sequences in the minor groove,755 though as mentioned all these proteins have a positively charged patch at the C-terminus. The mode of action of WhiB-like proteins is not completely known, and vary between the different classes, but it seems to involve the coordinated action of accessory proteins to provide DNA binding specificity.746 The type of iron-sulfur center bound to WhiB-like proteins was identified by its characteristic spectroscopic features: a broad absorption band with a maximum around 400 nm and positive features at 429 nm and 512 nm in its CD spectrum.752,756–758 WhiD from S. coelicolor, isolated under anoxic conditions, binds an oxygen-sensitive [4Fe-4S]2þ cluster that has a very low reduction potential (<  460 mV at pH 8.0),759 and is essential for its function as transcription regulator.754 In some of these proteins, the [4Fe-4S]2þ cluster reacts with O2 and completely disassembles from the protein in a slow process, with the conversion of [4Fe-4S]2þ to a [3Fe-4S]1þ intermediate, which then decays to the EPR silent [2Fe-2S]2þ intermediate species before complete cluster disassembly.753,754,758 On the contrary, the reaction with NO is more universal in these proteins and their

Fig. 34 Schematic representation of the mode of action of SoxR transcription regulator. Adapted from Green, J.; Paget, M. S., Bacterial Redox Sensors. Nat. Rev. Microbiol. 2004, 2(12), 954–966.

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nitrosylation mechanism seems to be similar. The [4Fe-4S]2þ cluster reacts very rapidly with NO in a multiphasic reaction, with stepwise formation of [4Fe-4S](NO), and [4Fe-4S](NO)2. In the presence of higher concentrations of NO there is formation of [4Fe-4S](NO)4 and of novel nitrosylated cluster [Fe3S3(NO)7].756,757 The nitrosylation of the iron-sulfur cluster activates the protein, enabling it to bind to specific DNA regulatory sequences.752,755 This activation might involve a conformational change, driven by the rearrangement of the iron positions relative to the cysteine thiols due to the formation of the nitrosylated species.756,757 The structure of WhiB1 and WhiB7 has been determined by solution NMR and X-ray crystallography,755,756,760 showing that it is constituted by a 4-helix bundle, with the [4Fe-4S] cluster held by three a-helices. Their mechanism of interaction with sigma factor sA has been proposed based on these structures,755,756,760 and it was also shown that nitrosylation of the iron-sulfur cluster abolish this interaction, allowing the protein to interact with the DNA. In the case of WhiB7, its AT-hook was also proposed to be important for the binding to the  35 element. In conclusion, the WhiB-like proteins are proposed to be intracellular redox sensors that sense the physiological relevant host signaling molecules O2 and NO, and integrate these signals with core intermediary metabolism, essential for survival.761 Nevertheless, their mechanism of action still needs to be completely understood, as well as the role played by the iron-sulfur cluster in their diverse functions. 2.06.4.2.3.3 Sensing oxygen by NreBC two component system NreB (nitrogen regulation), isolated from Staphylococcus carnosus and found in other Staphylococcus species,762 is encoded by the operon nreABC, a novel two-component system, which controls the dissimilatory nitrate/nitrite reduction and simultaneously responds to oxygen and nitrate.762,763 In the regulatory model, the oxygen-sensor NreB phosphorylates NreC that is the transcription regulator and binds to the promoter regions of the narGHJI and nirRBD operons to activate their expression. NreA is a nitrate-sensing protein764,765 that controls the autophosphorylation activity of NreB, as in the absence of nitrate converts NreB from a kinase to a phosphatase.766–768 NreB is a sensory histidine kinase of 347 residues with a N-terminal PAS domain with four conserved cysteine residues arranged in the Cys-X2-Cys-X11-Cys-X2-Cys motif,765 that binds an iron-sulfur cluster identified as a [4Fe-4S]2þ cluster, using spectroscopic techniques. This iron-sulfur cluster is oxygen-sensitive and completely disassembles when exposed to oxygen. A diamagnetic [2Fe2S]2þ cluster is proposed to be an intermediate species in this process.769,770 The presence of the oxygen-sensitive iron-sulfur cluster is required for the kinase activity of NreB but does not seem to be essential for its dimerization.769 2.06.4.2.3.4 SufR transcription repressor The Suf system is involved in the biosynthesis of iron-sulfur clusters in many organisms (prokaryotes and plastids), in response to iron starvation and oxidative stress. Some organisms have both Isc and Suf system while others only have the Suf system. The gene composition and organization of the Suf system differ between species.771 In Actinobacteria and Cyanobacteria these genes are under the regulation of SufR. SufR is a transcription regulator that belongs to the ArsR-family, with a HTH DNA binding domain at the N-terminus and binds a [4Fe-4S] cluster through three conserved cysteines located in the C-terminus (the fourth ligand has not yet been identified)772 (Fig. 29). This protein is a repressor of the suf operon by binding to the palindromic sequence CAAC-N6-GTTG present in the promoter region of sufRBDCSU, which is involved in the biosynthesis of iron-sulfur clusters in Gram-positive bacteria.772,773 In the presence of O2, the absorption band with a maximum at 413 nm decreases rapidly in intensity, without passing through an intermediate species. This might be an indication that this protein is extremely sensitive to O2, and its iron-sulfur cluster decomposes rapidly without formation of a [2Fe-2S] stable intermediate. The loss of [4Fe-4S] cluster abolishes DNA binding.772 The M. tuberculosis SufR is a dimer in solution. Its [4Fe-4S] cluster responds to NO, and upon nitrosylation, the suf operon transcription is activated. SufR [4Fe-4S] cluster was spectroscopically characterized by UV-visible (lmax at 413 nm) and CD (positive peaks at 330 nm and 420 nm) spectroscopy,773 while its reaction with O2, H2O2 and NO was followed by UV-visible and EPR spectroscopies. The presence of NO was proposed to lead to the formation of a dinitrosyl-iron dithiol complex.773 2.06.4.2.3.5 OrpR s54-dependent activator OrpR is a s54-dependent activator found in D. vulgaris Hildenborough and D. alaskensis G20.774,775 The analysis of its primary sequence showed that it is composed by three domains: a N-terminus PAS domain, a central AAA þ domain (that can hydrolyze ATP) and a C-terminus DNA binding domain. The PAS domain binds an oxygen-sensitive [4Fe-4S]2þ cluster, that was identified by EPR and visible spectroscopy.776 This iron-sulfur cluster is coordinated by three conserved cysteines present in a C-X8-C-X3-C motif, and the fourth ligand is proposed to be an aspartate. The mechanism of action has not yet been completely unraveled, but it is proposed to be a redox sensor. In fact, in the presence of O2 the cluster completely disassembles, but in the presence of mild oxidative conditions it is converted to a [3Fe-4S]1þ cluster, that has the same affinity to the specific palindromic DNA sequence as the [4Fe-4S]2þ OrpR.776 OrpR activates the transcription of two divergent operons in D. vulgaris Hildenborough that encodes the Orange Protein (Orp) complex.775 This protein complex was identified by pool-down assays and is composed by a core group of three proteins, Orp and two-ATPases proposed to bind iron-sulfur clusters.775 Besides these, two other proteins encoded by these operons are proposed to be iron-sulfur proteins.774,777 The Orp is a 12 kDa protein that has been isolated from D. gigas and D. alaskensis G20 and binds non-

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covalently a Mo-Cu-Mo linear cluster bridged by sulfur atoms778,779 (Section 2.06.2.1.5). The heterologously produced apo-Orp can be reconstituted in vitro.778–781 However, the physiological function of this non-redox heterometallic cluster remains unknown, as well as what is the function of the Orp complex.

2.06.5

The role of iron-sulfur clusters in DNA processing enzymes

DNA is subjected to modifications due to oxidative stress, radiation, and chemical modification, such as alkylation, and to misincorporation of nucleotides. Therefore, the action of molecular repair systems is essential to correct these DNA damages and mismatches that would change the genomic code and lead to mutagenic effects that can be the cause of diseases, such as cancer. Although, the presence of iron-sulfur clusters in DNA and RNA processing enzymes can be counterintuitive, as iron can generate hydroxyl radicals via Fenton reactions that damage DNA,782 these clusters has been identified in several enzymes, such as Rad3 family helicases,783–788 AddAB helicase-nuclease,789 archaea RNA polymerases,790–792 DNA polymerase,793 DNA primase794–796 and elongator domains.797 In fact, iron-sulfur clusters are present in all replicative DNA polymerases and helicase-nuclease Dna2.793,798 In common, these enzymes bind a [4Fe-4S] cluster through four cysteine residues organized in a sequence motif, which is unique for each enzyme family and different from the canonical motifs described so far (see Table 1 in Ref. 799). The iron-sulfur cluster is a [4Fe-4S]2þ identified by visible, EPR and Mössbauer spectroscopies, and in a few cases by CD, resonance Raman,800,801 FTIR802 and surface enhanced IR.803 Another peculiarity is that the presence of the iron-sulfur cluster is not restricted to prokaryotic DNA processing enzymes, but it is present in bacteria, archaea, and eukaryotic enzymes, and in some cases, it is present in novel iron-sulfur cluster domains.796 In fact, there are several human diseases that are related with iron-sulfur proteins involved in DNA and RNA metabolism.9 As mentioned in Section 2.06.3 and 2.06.4, iron-sulfur clusters can be employed in radical SAM chemistry (methylthiolate tRNA, e.g., MiaB), or as sensors in transcription factors. Nevertheless, in other DNA/RNA processing enzymes the role of the iron-sulfur cluster is still unclear. One exception are glycosylases, in which iron-sulfur clusters have been proposed to be involved in DNA damage recognition (vide infra), via DNA-mediated charge transport. A similar mechanism might be important in other DNA-processing enzymes, thus the question “why this mechanism in not ubiquitous or conserved even in an enzyme family” remains to be answered.804 A detailed characterization of the iron-sulfur cluster in this group of proteins is required to determine its contribution to their function. In most cases, there is no detailed spectroscopic characterization, the reduction potential (in the presence and absence of nucleic acid) and the contribution of the iron-sulfur cluster to the protein function in vivo is unknown. However, the presence of this prosthetic group has been used as an endogenous quencher of fluorescence to study the protein-DNA interaction in Rad3 family helicases.787 Moreover, the location of the iron-sulfur cluster in the three-dimensional structure of these proteins has shed some light into their function, that can go beyond the simple structural role. In some of these proteins, as is the case of Dna2 helicase-nuclease and XPD helicase, the iron-sulfur cluster is in the catalytic domain, while in others, as glycosylases, the iron-sulfur cluster is in a distant domain.805,806 Such locations might correspond to a difference in the role played by this cofactor: (i) sense double helix disruption, or (ii) modulate DNA affinity and processivity. Here, only the DNA repair glycosylases will be described since the contribution of the iron-sulfur cluster for their function is better understood.

2.06.5.1

DNA repair glycosylases

Base excision repair (BER) glycosylases are responsible for identifying in the genome chemically modified bases and mismatches, and catalyze their removal, constituting one of the most common mechanisms of DNA repair.807 In the first step, BER glycosylases need to locate the modified base and then flip it into their catalytic site and catalyze the break of the N-glycosidic bond between the damaged base and the sugar-phosphate backbone.807 These enzymes are divided into six superfamilies,808,809 and up-to-now in two of these some were found to bind iron-sulfur clusters: (i) the helix-hairpin-helix superfamily, containing E. coli endonuclease III (EndoIII) and MutY (including the mammalian homologs hNTH1, MUTYH) and (ii) the uracil DNA glycosylase superfamily (Table 3). In EndoIII and MutY the unique cysteine binding motif Cys-X6-Cys-X2-Cys-X5-Cys coordinates the iron-sulfur cluster, that is located near the surface at the C-terminus domain, in a loop that has been named as the [4Fe-4S]2þ cluster loop (FCL) domain.815,816 The comparison between the structure of the free and DNA-bound EndoIII, shows that the overall structure is very similar, and that there is a loop projecting from the cluster with several positively charged residues, which directly interacts with the DNA phosphate backbone,816–818 which led to the hypothesis that iron-sulfur cluster had a structural role important for DNA binding. In the case of EndoIII the iron-sulfur cluster was identified to be a [4Fe-4S]2þ by EPR and Mössbauer spectroscopies,819 and it was shown that it could not be easily oxidized nor reduced. Further characterization by resonance Raman showed that the presence of oligonucleotides containing a reduced apyrimidinic site or thymine glycol (an inhibitor) resulted in small shifts in the stretching modes assigned to Fe-S(Cys), which also contributed to the assignment of a structural role to this cluster.800 However, later it

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was shown that in MutY, the presence of the iron-sulfur cluster was not required for protein folding and stability, though essential for DNA-binding and activity.810,820 The identification of a redox active iron-sulfur cluster and its importance in the function of these proteins was only demonstrated using electrochemistry. Different electrochemical methods were applied to EndoIII and MutY, showing that the reduction potential of the iron-sulfur cluster in the presence of DNA has a positive value (around 130 mV, depending on the electrode surface) (Fig. 2), consistent with the observation of a [4Fe-4S]2 þ/3þ redox pair,811,821,822,824 as observed in HiPIP iron-sulfur proteins (see Section 2.06.2.1.4). Binding of EndoIII to DNA has an impact of – 50 mV in its reduction potential.824 This negative shift indicates that the enzyme binds more tightly to DNA when its [4Fe-4S] cluster is in the [4Fe-4S]3þ state (KD of 11 nM) than in the reduced [4Fe-4S]2þ state (KD of 6 mM),825,826 as no conformational change was observed between the DNA free and bound forms. A value of 80 mV has been determined for the reduction potential of other repair and replication proteins using DNA modified electrodes (either DNA films on gold surfaces or DNA duplexes on highly oriented pyrolytic graphite),811,827–832 a value that falls within the physiological range. These studies also pointed toward a DNA-mediated charge transport between the electrode and the iron-sulfur cluster. Micro-FTIR microscopy has been used to study the interaction between EndoIII and DNA. The change in the vibration modes, assigned to the iron-sulfur cluster, indicated an increase in the bond lengths, that makes the cluster more stable, and prepares it to a higher oxidation state, [4Fe-4S]3þ, without much energy change, which is required for the damage detection in the DNA802 (vide infra). In the family-4 uracil-DNA glycosylases (4 UDGs) from thermophilic organisms, the cysteine binding motif that coordinates the iron-sulfur cluster is Cys-X2-Cys-Xn-Cys-X(14–17)-Cys, in which n can range from 70 to 100 residues.806 The X-ray structure of Thermus thermophilus 4 UDG shows that the [4Fe-4S]2þ has a distorted cuboidal structure, located 10 Å from its active site.812 In the absence of further experimental data, a structural role was also initially attributed to this iron-sulfur cluster. However, it is clear from the available crystal structures that the distance between the iron-sulfur cluster and the DNA is comparable in all these glycosylases and thus its contribution to the enzyme function must be similar.812 The impact of DNA binding in the reduction potential of these proteins is in the same range as the one reported for EndoIII.811 Therefore, the function of the iron-sulfur cluster in the glycosylases might not be structural, and it has been proposed that the [4Fe-4S]2þ cluster is responsible for the detection of DNA lesions through DNA-mediated charge transport.804 The base-pair p-stack of duplex DNA can mediate charge transport over 200 Å distances, which is disrupted when the DNA is damaged.833,834 Thus, this could be the mechanism used by this type of glycosylases to sense these disruptions and repair the DNA damage. The proposed mechanism for the action of the iron-sulfur glycosylases is the one in which in the DNA unbound form, the [4Fe4S] cluster is in the 2 þ oxidation state, and in the presence of DNA there is a shift in  50 mV in the reduction potential that increases the glycosylase DNA binding affinity (Fig. 35A, (Step1)). The protein is activated toward oxidation, and this electron is going to reduce a distally bound protein in a DNA-mediated charge transport reaction (Fig. 35A, (Step2)). This protein loses affinity for the DNA and is released (Fig. 35A, (Step3)). However, in the presence of a lesion, this charge transport mechanism cannot occur, and the glycosylase stays bound to the DNA and repairs it811,822,823 (Fig. 35B). Moreover, since guanine radicals are the first products of oxidative DNA damage inside the cell, and it has been shown that they can also oxidize the iron-sulfur of MutY glycosylase, these base radicals may provide the in vivo driving force for iron-sulfur glycosylates to initiate DNA-mediated signaling.835 In the case of the 4UDGs family, it has been observed that the presence of the iron-sulfur cluster is not conserved in all the proteins but seems to be ubiquitous for the ones isolated from thermophilic bacteria.806,812 Therefore, this cluster might provide a faster detection of the DNA damage since these organisms need to deal with a higher rate of temperature-induced DNA damage.806 This mechanism might be common to other DNA processing enzymes,828,829 in which a change in the oxidation state of its [4Fe4S] cluster increases the enzyme DNA binding affinity and modulates its activity.

Table 3

Some DNA glycosylases that bind [4Fe-4S] clusters.

Enzyme Name

Substrate

References

MutY/MUTYH Nth (EndoIII)/hNTH1 Thermophilic family-4 UDGs Methanibacterium thermoautotrophicum TDG rpS3 (UV-endonuclease)

OG:A Oxidized pyrimidines; T-T U:A/G T:G T-T; C:OG

810 811 812 813 814

The excised base is highlighted in bold. OG:A - 7,8-dihydro-8-oxo-20 -deoxyguanosine-20 -deoxyadenosine; TDG – thymine DNA glycosylase; rpS3 – human ribosomal protein S3.

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2.06.6

151

Conclusions

Iron-sulfur clusters are ancient metal centers participating in a wide range of biological functions. The tetrahedral Fe(S)4 unit, formed by iron and sulfur, is used as a basic and versatile building block of proteins’ metal centers and enzymes’ active sites. Iron-sulfur clusters are optimized devices for electron transfer due to the multiple oxidation and spin states attainable, the possibility of magnetic coupling, as well as the ability to stabilize localized and delocalized charges. In addition, the modulation of the cluster properties by varying the coordination (not exclusively made of sulfur atoms), inserting other metal atoms (such as, nickel and molybdenum) and having flexible coordination spheres, opens their biological relevance to non-redox catalysis and radical chemistry, gene regulation and nucleic acid metabolism. The ability found in certain iron-sulfur proteins for sulfur transfer and ligand swapping has been exploited in the biosynthesis of iron-sulfur containing proteins. In fact, the complexity involved in iron-sulfur cluster assembly, not described in here, contrasts with the self-assembly chemistry most often observed when apo-forms are reconstituted by addition of iron and inorganic sulfur under reducing conditions. It is worth mentioning that all the chemical and biochemical processes observed in this field were inspiring topics for the generation of biocatalysts mimicking enzyme active sites23 and possible roles in biotechnological oriented processes. Medical aspects related with iron-sulfur cluster containing proteins are out of the scope of this review but represent an emergent and important field,836–841 for which it will be necessary to develop appropriate strategies. It is remarkable the speed of expansion of the field and the new directions to which our understanding of the role of iron-sulfur clusters is moving. In this review we intended to provide an update on our previous work on the same subject, highlighting the main discoveries in the field of iron-sulfur cluster containing proteins in the last 8 years, such as the identity of the X atom in the nitrogenase cofactor, the identification of Fe-hydrogenases that are oxygen-tolerant, the increasing number of radical SAM enzymes, the sensor mechanism in many transcription regulators, and the role played by iron-sulfur cluster in DNA processing enzymes.

Fig. 35 Schematic representation of the mechanism of action of iron-sulfur glycosylases in the absence (A) and in the presence (B) of a DNA lesion. Adapted from Boal, A. K.; Yavin, E.; Barton, J. K., DNA Repair Glycosylases with a [4Fe-4S] Cluster: A Redox Cofactor for DNA-Mediated Charge Transport? J. Inorg. Biochem. 2007, 101(11–12), 1913–1921; Barton, J. K.; Silva, R. M. B.; O’Brien, E., Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication. Annu. Rev. Biochem. 2019, 88, 163–190.

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Acknowledgments This work is financed by national funds from FCT - Fundação para a Ciência e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences - UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy - i4HB, and in the scope of the project UIDB/50006/2020, UIDP/50006/2020 and LA/P/0008/2020 of the Associate Laboratory for Green Chemistry-LAQV. FCT supported SRP through the projects FCT-ANR/BBB-MET/0023/2012 and PTDC/BIA-BQM/ 29442/2017, and JJGM through the project PTDC/BTA-BTA/0935/2020. Author contributions: SRP and RG has planned the manuscript with contributions from JJGM and IM. SRP, RG and MSPC wrote the manuscript, which was critically revised by JJGM and IM.

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J.; David, S. S. A Substrate Recognition Role for the [4Fe-4S]2þ Cluster of the DNA Repair Glycosylase MutY. Biochemistry 1998, 37 (18), 6465–6475. 821. Boon, E. M.; Livingston, A. L.; Chmiel, N. H.; David, S. S.; Barton, J. K. DNA-Mediated Charge Transport for DNA Repair. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (22), 12543–12547. 822. Yavin, E.; Stemp, E. D.; O’Shea, V. L.; David, S. S.; Barton, J. K. Electron Trap for DNA-Bound Repair Enzymes: A Strategy for DNA-Mediated Signaling. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (10), 3610–3614. 823. Barton, J. K.; Silva, R. M. B.; O’Brien, E. Redox Chemistry in the Genome: Emergence of the [4Fe4S] Cofactor in Repair and Replication. Annu. Rev. Biochem. 2019, 88, 163–190. 824. Bartels, P. L.; Zhou, A.; Arnold, A. R.; Nunez, N. N.; Crespilho, F. N.; David, S. S.; Barton, J. K. Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA. Langmuir 2017, 33 (10), 2523–2530. 825. Gorodetsky, A. A.; Boal, A. K.; Barton, J. K. Direct Electrochemistry of Endonuclease III in the Presence and Absence of DNA. J. Am. Chem. Soc. 2006, 128 (37), 12082– 12083. 826. Tse, E. C. M.; Zwang, T. J.; Barton, J. K. The Oxidation State of [4Fe4S] Clusters Modulates the DNA-Binding Affinity of DNA Repair Proteins. J. Am. Chem. Soc. 2017, 139 (36), 12784–12792. 827. Silva, R. M. B.; Zhou, A.; Grodick, M. A.; Barton, J. K. DNA-Mediated Redox Signaling in Bacterial Nucleotide Excision Eepair by UvrC. Biophys. J. 2016, 110 (3), 62a–63a. 828. Bartels, P. L.; Stodola, J. L.; Burgers, P. M. J.; Barton, J. K. A Redox Role for the [4Fe4S] Cluster of Yeast DNA Polymerase Delta. J. Am. Chem. Soc. 2017, 139 (50), 18339– 18348. 829. O’Brien, E.; Holt, M. E.; Thompson, M. K.; Salay, L. E.; Ehlinger, A. C.; Chazin, W. J.; Barton, J. K. The [4Fe4S] Cluster of Human DNA Primase Functions as a Redox Switch Using DNA Charge Transport. Science 2017, 355 (6327). 830. Mui, T. P.; Fuss, J. O.; Ishida, J. P.; Tainer, J. A.; Barton, J. K. ATP-Stimulated, DNA-Mediated Redox Signaling by XPD, a DNA Repair and Transcription Helicase. J. Am. Chem. Soc. 2011, 133 (41), 16378–16381. 831. Sontz, P. A.; Mui, T. P.; Fuss, J. O.; Tainer, J. A.; Barton, J. K. DNA Charge Transport as a First Step in Coordinating the Detection of Lesions by Repair Proteins. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (6), 1856–1861. 832. Ekanger, L. A.; Oyala, P. H.; Moradian, A.; Sweredoski, M. J.; Barton, J. K. Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation. J. Am. Chem. Soc. 2018, 140 (37), 11800–11810. 833. Boal, A. K.; Barton, J. K. Electrochemical Detection of Lesions in DNA. Bioconjug. Chem. 2005, 16 (2), 312–321. 834. Hall, D. B.; Holmlin, R. E.; Barton, J. K. Oxidative DNA Damage through Long-Range Electron Transfer. Nature 1996, 382 (6593), 731–735. 835. Genereux, J. C.; Boal, A. K.; Barton, J. K. DNA-Mediated Charge Transport in Redox Sensing and Signaling. J. Am. Chem. Soc. 2010, 132 (3), 891–905. 836. Ye, H.; Rouault, T. A. Human Iron-Sulfur Cluster Assembly, Cellular Iron Homeostasis, and Disease. Biochemistry 2010, 49 (24), 4945–4956. 837. Sheftel, A.; Stehling, O.; Lill, R. Iron-Sulfur Proteins in Health and Disease. Trends Endocrinol. Metab. 2010, 21 (5), 302–314.

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838. Read, A. D.; Bentley, R. E.; Archer, S. L.; Dunham-Snary, K. J. Mitochondrial Iron-Sulfur Clusters: Structure, Function, and an Emerging Role in Vascular Biology. Redox Biol. 2021, 47, 102164. 839. Kung, W. M.; Lin, M. S. The NFkappaB Antagonist CDGSH Iron-Sulfur Domain 2 Is a Promising Target for the Treatment of Neurodegenerative Diseases. Int. J. Mol. Sci. 2021, 22 (2). 840. Liao, H. Y.; Liao, B.; Zhang, H. H. CISD2 Plays a Role in Age-Related Diseases and Cancer. Biomed. Pharmacother. 2021, 138, 111472. 841. Berndt, C.; Christ, L.; Rouhier, N.; Muhlenhoff, U. Glutaredoxins with Iron-Sulphur Clusters in Eukaryotes - Structure, Function and Impact on Disease. Biochim. Biophys. Acta Bioenerg. 2021, 1862 (1), 148317.

2.07 [FeFe]-hydrogenases: Structure, mechanism, and metallocluster biosynthesis Mohamed Attaa and Marc Fontecaveb, a University of Grenoble Alpes, CEA, CNRS, CBM-UMR 5249, Grenoble, France; and b Laboratoire de Chimie des Processus Biologiques, UMR CNRS 8229, Collège de France-CNRS-Sorbonne Université, PSL Research University, Paris, France © 2023 Elsevier Ltd. All rights reserved.

2.07.1 2.07.2 2.07.2.1 2.07.2.2 2.07.3 2.07.3.1 2.07.3.1.1 2.07.3.1.2 2.07.3.1.3 2.07.3.1.4 2.07.3.1.5 2.07.3.2 2.07.3.2.1 2.07.3.2.2 2.07.3.2.3 2.07.3.3 2.07.3.3.1 2.07.3.3.2 2.07.3.3.3 2.07.3.4 2.07.4 Acknowledgements References

Introduction [FeFe]-Hydrogenases structure and mechanism Structural features of [FeFe]-hydrogenases Reaction mechanism of [FeFe]-hydrogenases H-cluster biosynthetic proteins The radical-SAM enzyme HydG Structure of HydG Leads from sequence alignments: Tyrosine is the substrate of HydG The N-terminal [4Fee4S]RS mediates radical chemistry The C-terminal [4Fee4S]AUX of HydG is a platform for a rich organometallic chemistry, especially for the assembly of a [Fe(CO)2(CN)] species precursor to the [2Fe]H subcluster HydG enzyme mechanism The radical-SAM enzyme HydE HydE structure The radical-SAM enzyme HydE acts on the HydG product CH2NCH2 moiety of the azapropanedithiolate bridge derives from a serine amino acid residue The scaffold HydF protein HydF: A [4Fee4S] protein with ability to bind a precursor of the [2Fe]H subcluster Structure of HydF HydF, a GTP-binding protein H-cluster of [FeFe]-hydrogenase: Mechanism of bioassembly Conclusion

175 177 177 178 180 181 181 182 182 183 184 184 185 185 187 188 188 188 189 189 190 190 190

Abbreviations adt2 L Azapropanedithiolate Cp Clostridium pasteurianum Cr Chlamydomonas reinhardtii Dd Desulfovibrio desulfuricans Df Desulfovibrio fructosovorans DFT Density functional theory Dg Desulfovibrio gigas Ec Escherichia coli ENDOR Electron nuclear double resonance EPR Electron paramagnetic resonance ESI-MS Electrospray ionization mass spectrometry EXAFS Extended X-ray absorption fine structure FTIR Fourier transform infrared GTP Guanosine-5-triphosphate Hmd H2-forming methylenetetrahydromethanopterin dehydrogenases HYSCORE Hyperfine sublevel correlation (spectroscopy) Me Megasphaera elsdenii RS Radical-SAM SAM S-adenosyl methionine Tm Thermotoga maritima

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https://doi.org/10.1016/B978-0-12-823144-9.00117-5

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Tme Thermosipho melanesiensis Tn Thermotoga neapolitana

Abstract [FeFe]-hydrogenases catalyzes the reversible conversion of hydrogen into protons and electrons with astonishing efficiency. The reaction occurs at the H-cluster, which is unique in biology. It consists of an organometallic [2Fe]-subcluster bound to a classical [4Fee4S] cluster by a single cysteine bridging ligand. This book chapter will describe the structure and mechanism of [FeFe]-hydrogenases and focus on recent developments on [FeFe]-hydrogenase maturation, leading to the assembly of the [2Fe]-subcluster, which have revealed a remarkably complex pattern of mostly novel biochemical reactions. This chemical process depends on the concerted action of three metalloproteins. Two radical-SAM enzymes HydE and HydG and a scaffold iron-sulfur containing protein, HydF.

2.07.1

Introduction

It is known that energy from fossil fuels has powered innovations in every domain of modern life. However, concerns about climate change and declining petroleum reserves are fueling a resurgence in the search for alternative, renewable fuels.1 A rapid transition to sustainable energy systems is one of the central challenges facing humanity during this 21st century. Among the possible candidates hydrogen molecule (H2) is seen by many as a potential renewable and carbon-neutral energy carrier whose combustion releases large amounts of free energy (H2 carries three times the energy content per unit mass of petrol) but no greenhouse gases.2–6 The chemical energy present in H2 can also be converted within fuel cells into electrical energy that can power cars for example. It is also interesting to highlight that H2 is a key molecule in the bioenergetic metabolism of several microorganisms. Indeed, its centrality in life stems from the fact that H2 can not only be formed by reduction of protons in order to dispose of deleterious excess reducing power, but also be used as an energy vector in the opposite reaction, which implies its cleavage and release of protons and electrons, thus providing energy to cell metabolism. Because the demand for hydrogen is expected to dramatically increase in the near future,7,8 there is ongoing research for its production, storage and utilization. Unfortunately, current methods for its production rely on fossil fuels (methane) and alternatives are expensive. These include steam reforming of natural gas, which produces high amounts of greenhouse gases in parallel, and water electrolysis into H2 and O2. However, the latter requires noble metals (platinum, iridium) as catalysts, which are expensive and whose resources on earth are limited, and furthermore it will be environmentally acceptable only if fueled with renewable electricity.9,10 Thus, urgent incentive for new and renewable methods are obviously needed for hydrogen production. Bio-hydrogen production systems have a great deal of potential in that respect. This field of research concerns: (i) genetic engineering of microorganisms in order to introduce and optimize the hydrogen generation pathway and to eliminate metabolisms that compete with hydrogen production within these microorganisms; (ii) biochemistry and enzymology of the enzymes, named hydrogenases, involved specifically in hydrogen metabolism; (iii) design and characterization of synthetic biomimetic and bioinspired catalysts for both H2 evolution and consumption. In first place, the development of technological devices (electrolyzers, photo-electrochemical cells and fuel cells) based on bio-electrodes (using genetically modified organisms, enzymes and biomimetics) will indeed depend on our understanding of the structure and mechanism of hydrogenases at the molecular level. Bio-hydrogen production is thus based principally on hydrogenases (H2ases). They are present in all three domains of life, archaea, bacteria and eukaryotes. Hydrogenases are capable of catalyzing hydrogen conversion reversibly and efficiently, under ambient temperature and pressure, according to the Eq. (1): H2 5Hþ þ H 52Hþ þ 2e

(1)

Although most, if not all, known H2ases catalyze the reaction in either direction in vitro, they are usually committed to catalyze either hydrogen uptake or evolution in vivo, depending on the metabolism of the host organism. Research in this field has covered three distinct periods. The early years of research (1930–1990) constitute the first period in which traditional physiological and biochemical studies have provided information on the cellular function of H2ases.11 From 1960 to 1990, H2ases were purified from a variety of organisms and characterized by spectroscopic, analytical and structural methods. This detailed characterization has allowed a classification of these enzymes into two classes, which are defined by the metal composition of their dimetallic active sites.12 The first class comprises the [FeFe]-H2ases, encoded by the hydA gene and named HydA in the following, which contain a diiron site, directly attached to a [4Fee4S] cluster, thus giving a 6Fe-center called the H-cluster in the following.13,14 The [NiFe]-H2ases form the second group with a heterodimetallic active site based on nickel and iron atoms.15 A third enzyme class has been incorrectly reported as hydrogenases. The H2-forming methylenetetrahydromethanopterin dehydrogenases (Hmd),16–18 found only in archaea, notably in methane-producing methanogens, do not catalyze the reaction (1) but instead the transfer of a hydride from H2 to methenyltetrahydromethanopterin following the first step in reaction (1), a process fundamentally different from that catalyzed by

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[FeFe]- and [NiFe]-H2ases.19 To achieve catalysis these enzymes depend on a mononuclear Fe active site whose structure has been unambiguously determined by X-ray crystallography.19–21 The second period, from 1995 to the beginning of the 21st century, was a key step in the development of the H2ases field. During this time, the first crystal structures of both classes of H2ases were obtained, yielding long-expected three-dimensional models. The structure of [NiFe]-H2ase from D. gigas15 and then shortly thereafter of [FeFe]-H2ase from C. pasteurianum13 and D. desulfuricans14 confirmed that both active sites share a dimetallic framework and the unique presence of CO and CN as iron ligands, as anticipated by Fourier-Transformed Infra-Red (FTIR) spectroscopy12,22 (Scheme 1). IR spectroscopy proved, all over the years and until now, a fantastic tool allowing fine characterization of these enzymes since characteristic CO and CN vibrational signals are easy to observe and provide unique probes of the electronic properties of the active sites under various redox states. In both enzymes the two metals are connected through two thiolate bridges which have different origins in the two enzymes: they are provided by two cysteinate residues from the polypeptide chain in [NiFe]-H2ases and by a small dithiolate organic ligand, namely azapropanedithiolate (adt2 ), in [FeFe]-H2ases (Scheme 1). These similarities appear to be requirements of their common catalytic activity. Finally, both types of hydrogenases are iron-sulfur ([FeeS]) enzymes since they harbor, in addition to the H-cluster, one or several [4Fee4S] and [2Fee2S] clusters. The last period in H2ases development, during the last 15 years, has been essentially devoted to the study of hydrogenase biosynthesis, specifically to the understanding of the maturation process leading to the assembly of the active sites and the activation of the enzymes. Considering the complexity and the sophistication of these active sites, it is not surprising that a large number of genes are required for their maturation. With respect to this, our understanding of [NiFe]-H2ases maturation has progressed much earlier than that of [FeFe]-H2ases thanks to Prof. August Böck contribution on the biosynthesis of [Ni-Fe]-H2ases from E. coli.16 During that time, almost all genes committed to the assembly of the NieFe site of [NiFe]-H2ases have been identified, their products characterized, and most of their functions unraveled. This biosynthetic pathway will not be discussed here but has been the subject of recent review articles.18,23 For [FeFe]-H2ases instead, our knowledge of the maturation process is more recent, with the long-expected breakthrough achieved in 2004 through genetic analysis of H2ase-deficient mutants of the green algae C. reinhardtii (Cr).24 Two genes, hydEF and hydG, were identified as critical for activation of HydA and furthermore predicted to encode iron-sulfur [Fe-S] proteins of the Radical S-Adenosyl-L-Methionine (SAM) family.24 However, in most other organisms, hydE and hydF were separate genes and hydE, but not hydF, was shown to encode a Radical-SAM enzyme. The Radical-SAM family is a broad class of [FeeS] proteins which catalyze radical reactions thanks to the controlled decomposition of SAM into the active 50 -deoxyadenosyl radical (50 Ado•) and will be described in more detail below. The key role of the corresponding HydE, HydF and hydG proteins as maturases for HydA activation was established by showing that the structural gene CrhydA could be expressed as an active enzyme form in E. coli, an organism otherwise unable to synthesize an active [FeFe]-H2ase, only when co-expressed together with the three C. reinhardtii hydE, hydG, hydF maturase genes.24 This was corroborated by cell-free in vitro synthetic approaches showing that an E. coli cell lysate containing separately overexpressed HydE, HydG, HydF could activate apo-HydA, defined as a form of HydA lacking the dimetallic center but containing the [FeeS] clusters.25 Finally, a revolution in HydA maturation was achieved with the discovery that a synthetic biomimetic diiron organo-metallic complex [Fe2(adt)(CN)2(CO)4]2, mimicking the enzyme diiron center, can be integrated directly into apo-HydA to form a fully active H-cluster in vitro26,27 (Scheme 2). This novel procedure thus alleviates the need for HydE, HydG and HydF and practically facilitates the production of active HydAs.28 It is remarkable that the same biomimetic complex can be used to transform E. coli and Synechocystis living cells, expressing only HydA in the absence of maturases, into H2-producing bacteria29–31. All the in vitro maturation methods described above in particular allowed selective incorporation of isotopes into HydA active site and proved extremely useful in interpreting the spectroscopic properties of the dimetallic center as well as in identifying the precursors of the atoms present in this center, as discussed in detail below.

Scheme 1 Schematic representation of the [FeFe]-H2ase (left) and [NiFe]-H2ase (right) active site, in the oxidized state. The 6-iron unit (left) is named the H-cluster and the diiron subunit (iron atoms represented as a blue spheres) is called [2Fe]H subcluster.

[FeFe]-hydrogenases: Structure, mechanism, and metallocluster biosynthesis

Scheme 2

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Chemical maturation of HydA using a synthetic analog of [2Fe]H subcluster.

The spectacular development of hydrogenase field provided, in less than 30 years of research, an unprecedented body of data with many details about these highly complex enzymes, which ironically catalyze the simplest of chemical reactions. Many excellent reviews, however quite ancient, have described the biodiversity, the structures, the molecular mechanisms and the biosynthesis of H2ases in general.11,12,16-19,32 However, as indicated above, a number of important clarifications have appeared during the last 4 years regarding [FeFe]-H2ase catalytic mechanism and reaction intermediates as well as the mechanism of [FeFe]-H2ase active site assembly. As a consequence, in the present chapter, we focus on [FeFe]-H2ases describing: (i) the current understanding of the structure and mechanism of the active site; (ii) recent results and current hypothesis regarding the structure and mechanism of the protein machinery dedicated to the assembly of the active site. During the course of the preparation of this chapter two interesting review articles on [FeFe]-H2ase maturation from D. Britt and collaborators33,34 and one on bioinspired diiron catalysts22 have appeared.

2.07.2

[FeFe]-Hydrogenases structure and mechanism

2.07.2.1

Structural features of [FeFe]-hydrogenases

[FeFe]-H2ases are highly modular enzymes and, while many are found in a single monomer form, some enzymes are composed of multi-subunits up to hetero-hexamers.17,32 This modularity emerged from the first sequence determination and, immediately thereafter, has been confirmed when more sequences became available.35,36 In particular, the [FeFe]-H2ases modularity is a consequence of the association of the catalytic domain with other domains, containing auxiliary [FeeS]-clusters, whose number varies from one enzyme to another. These include both [2Fee2S] and [4Fee4S] clusters (generally referred to as the F-clusters). As an example, the D. desulfuricans as well as the Megasphaera elsdenii [FeFe]-H2ases, DdHydA and MeHydA, contain two accessory [4Fee4S] clusters, whereas the Clostridium one contains four accessory clusters, one [2Fee2S] and three [4Fee4S] clusters14,37,38 (Fig. 1). These

Fig. 1 The structure of the [FeFe]-hydrogenases: Left, the X-ray crystal structure of apo-HydA from Chlamydomonas reinhardtii (PDB ID: 3LX4) expressed in the absence of the three maturases reveals an active site containing only the [4Fee4S]H cluster. Middle and right the X-Ray crystal structures of HydA from Desulfovibrio desulfuricans (PDB ID: 1HFE) and Clostridium pasteurianum (PDB ID: 3C8Y) featuring the H-cluster as well as the auxiliary FeeS cluster containing domains ([4Fee4S] and [2Fee2S]) serving as electron transfer wires.

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F-clusters are supposed to participate in the long-range electron-transfer pathway coupling the active site to physiological redox partners at the surface of the enzyme. The green algae C. reinhardtii HydA, CrHydA, constitutes the simplest form of the [FeFe]-H2ases with only a catalytic domain containing the H-cluster and no auxiliary [Fe-S] clusters.39 However, as shown in Fig. 1, only the apoHydA form, lacking the [2Fe]H subcluster, has been structurally characterized.39 It has been proposed that the auxiliary clusters protect the enzyme from oxygen adverse effects since CrHydA is one of the most air sensitive hydrogenase. However, a recent study showed that deletion of the domain containing the two auxiliary clusters in MeHydA did not change the sensitivity of the enzyme to oxygen.40 In all HydAs, the catalytic domain contains a unique active metal center, named the H-cluster (Scheme 1 and Fig. 2). It is formed by a classical [4Fee4S] cubane cluster, referred to as [4Fee4S]H, attached to a very unusual diiron cluster, referred to as the [2Fe]H subcluster, using a bridging sulfur atom from a cysteine residue from the polypeptide chain.13,14 The two Fe atoms of the [2Fe]H subcluster are connected by a small dithiolate ligand, an azapropanedithiolate (adt2  in the following), and the iron atom proximal to the [4Fee4S]H cluster (in the following referred to as Fep) is coordinated by one CN and one CO ligand, while the second Fe atom, named distal (in the following referred to as Fed), is bound also to one CN and one CO ligand (Scheme 1 and Fig. 2). An additional bridging CO ligand completes the first coordination sphere of both Fe atoms. As a consequence, Fed has an open coordination site where hydrogen, H2, can bind. Furthermore, the amino head-group of the dithiolate bridge is assumed to serve as a Brönsted base that shuttles protons between the coordination site and the proton channel of the enzyme which connects the solvent to the active site.41 The amino acid residue closest to the amine, Cys169 in CrHydA or Cys299 in CpHydA, participates in a hydrogen bonding interaction with the amine nitrogen and has been shown to play a central role in proton transfer41 (Fig. 2). A remarkable feature of the [2Fe]H subcluster is its attachment to the protein moiety via only one atom, the sulfur atom of the lateral chain of a cysteine which in addition makes a bridge to the [4Fee4S]H cluster.13,14 As such, the [2Fe]H subcluster of HydA is one of the rare examples of biological organometallic centers, such as adenosylcobalamin, the FeMo cofactor of nitrogenase, the monuclear active site of Hmd and the Ni center of lactate racemase.42

2.07.2.2

Reaction mechanism of [FeFe]-hydrogenases

Since their discovery 80 years ago, the catalytic mechanism of [FeFe]-H2ases has been investigated in considerable detail. However, it is only recently that the catalytic cycle is fully characterized and understood, thanks to, (i) the spectacular soaring of electrochemical, FTIR, EPR and HYSCORE methods applied to the study of [FeFe]-H2ases; (ii) the accessibility of an exceptionally large number

Fig. 2 Active site of the [FeFe]-hydrogenase of Clostridium pasteurianum (CpI). H-cluster ball-and-stick representation with carbon, nitrogen, oxygen, sulfur, and iron atoms colored gray, blue, red, yellow, and orange, respectively (PDB ID: 4XDC). Residues surrounding the [2Fe]H subcluster of the H-cluster are shown as thin sticks.

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of synthetic compounds mimicking the structure of the [2Fe]H subcluster; (iii) the access to crystallographic structures of the enzyme under various states.12,43–46 While functioning reversibly, [FeFe]-H2ases are usually associated with proton reduction because they display a remarkable catalytic efficiency for this reaction with reported turnover frequencies up to 10,000 H2 per second.47 In Scheme 3 we provide a simplified catalytic cycle for proton reduction based on the most recent interpretations of the mechanistic studies on prokaryotic [FeFe]-H2ases.22 In the initial resting Hox state, the H-cluster has an oxidized diamagnetic (S ¼ 0) [4Fee4S]2þH cluster together with a mixed-valent [FeIeFeII]H subcluster, as confirmed by its characteristic rhombic S ¼ 1/2 EPR paramagnetic signal. Thanks to FTIR spectroscopy, the absorption bands of the three CO and the two CN ligands of Hox are also characteristic and provide a clear signature of this state.12 Starting from Hox, a first proton-coupled electron transfer leads to the Hred state with an oxidized [4Fee4S]2þH and a oneelectron reduced [2Fe]H subcluster, thus in the [FeIeFeI]H form. This one-electron reduction is concomitant with a protonation of the bridgehead nitrogen atom of the adt2  ligand. This state is EPR silent and the bridging CO ligand has been shown to be converted into a terminal CO in some [FeFe]-H2ases. The second electron first reduces the [4Fee4S]H cluster leading to the super-reduced state, Hsred, comprising a reduced [4Fee4S]1þH cluster with the [2Fe]H subcluster remaining in the [FeIeFeI]H form. Since the latter is diamagnetic, the EPR signal of Hsred is assigned to the S ¼ 1/2 [4Fee4S]1þH cluster in agreement with the obtained g values that are similar to those of canonical [4Fee4S]1þ clusters. Following electron transfer from the [4Fee4S]H cluster to the [2Fe]H subcluster, migration of the proton from the bridgehead group of adt2  to Fed, the distal Fe of the [2Fe]H subcluster, and its 2-electron reduction into a hydride species, a second proton binds at the N atom. This critical intermediate, named Hhyd, has been the subject of intense studies and debates. In particular whether the hydride anion was bridging the two Fe atoms of the [2Fe]H subcluster or only terminally bound to Fed was carefully investigated. Recently, Hhyd could be stabilized enough, thanks to site-directed mutation of Cys299 to alanine resulting in altered proton transfer kinetics, to be characterized by Mössbauer, NMR, FTIR, EPR and nuclear resonance vibrational spectroscopy.44,46,48–50 There is now convincing evidence for the existence of a terminal hydride at Fed, as shown in Scheme 3. In agreement with a hydride species at Fed, the [2Fe]H subcluster is in the mixed valence [FeIeFeII]H state together with [4Fee4S]2þH. The active site is thus remarkably designed to facilitate H2 formation as it places a proton at a good distance of a hydride for an efficient coupling. In the last step of the mechanism, indeed, the proton from the bridgehead ammonium is transferred to the hydride producing a bound dihydrogen species at Fed within the last intermediate of the reaction cycle. Finally, liberation of H2 regenerates the initial stable Hox state thus closing the catalytic cycle.

Scheme 3 Simplified catalytic cycle of H2 evolution by [FeFe]-H2ase. The cubane represents the [4Fee4S]H subcluster. For the [2Fe]H subcluster, only the iron ions (blue sphere) and bridging CO are represented. The numbers are referring to the oxidation state.

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The catalytic cycle in Scheme 3 can be read in the other direction for oxidation of dihydrogen: H2 substrate first binds to Fed of Hox and then enjoys a heterolytic cleavage leading to Hhyd thanks to an efficient proton abstraction by the adjacent N atom of the adt2  ligand. Then Hhyd converts back to Hox via the liberation of 2 electrons and 2 protons.

2.07.3

H-cluster biosynthetic proteins

While the genes necessary for the biosynthesis, maturation, and processing of [NiFe]-H2ases are relatively well known and their products have been biochemically and functionally well characterized,16,23 the protein machinery required for the maturation of [FeFe]-H2ases has started to be revealed more recently.18,33,34,51 Unlike [NiFe]-H2ases, the structural genes were apparently not located in operons, and thus not in association with putative maturase-encoding genes, which would have facilitated their identification. Furthermore, the genomic organization of the maturase-encoding genes varies from one organism to another with three possible patterns; (i) independent genes scattered in the genome (Clostridium acetobutylicum, Clostridium perfringens); (ii) fused genes (C. reinhardtii); (iii) genes clustered in operons (D. desulfuricans, Bacteroides thetaiotaomicron).11,17 This genetic organization has been the major obstacle that made the discovery of these genes more challenging. The maturation machineries of [NiFe]- and [FeFe]H2ases have very recently been reviewed in detail and compared.12,18,33,34 Briefly, the [NiFe]-H2ases maturation requires at least eight auxiliary proteins referred to as HypA, HypB, HypC, HypD, HypE, HypF and HypX as well as an endopeptidase.16,52,53 Seven genes, HypA-X, are necessary for the biosynthesis and the insertion of the [NiFe(CN)2CO] catalytic cluster into the large subunit of [NiFe]-H2ases. The maturation process ends with the cleavage of a short C-terminal peptide by the endopeptidase producing an active enzyme. As some organisms produce several [NiFe]-H2ases, different endopeptidases have been discovered illuminating that proteolytic cleavage is a specific event.11,54 For [FeFe]-H2ases maturation, only three genes have been identified so far and shown to be essential for H-cluster biosynthesis.24 They are found in all organisms expressing [FeFe]-H2ase showing that the occurrence of an active [FeFe]-H2ase in any genome is intimately linked to the presence of the three maturases. They are named Hyd in the following, HydE, HydG and HydF. Now, after 16 years of research on [FeFe]-H2ases maturation, it is well established that the Fclusters and the [4Fee4S]H subcluster are assembled by the same cellular machineries, named ISC and SUF, in charge of the maturation of any other cellular iron-sulfur protein.39 As a consequence, HydE, HydG, and HydF are specifically and only involved in the assembly of the [2Fe]H subcluster.24 Thus, when HydA is expressed heterologously in E. coli, a microorganism lacking this class of hydrogenases and the corresponding maturase-encoding genes, the resulting hydrogenase contains the [4Fee4S]H cluster in the active site but not the [2Fe]H subcluster, and is totally inactive.39,55,56 In the following, such an inactive form of HydA is named apo-HydA. However, an E. coli strain complemented with hydA and hydEFG genes produces a [FeFe]-H2ase containing a wellassembled H-cluster and with full catalytic activity.39 Whether other proteins, that would be present even in E. coli, are required for an optimal maturation process seems to be excluded so far. The sequences of the three proteins are highly conserved, explaining why the heterologous expression of an active [FeFe]-H2ase from any organism can be successfully achieved using the Hyd machinery from any other organism. As an example, expression of an active HydA enzyme from C. reinhardtii or S. obliquus has been shown to be possible using C. acetobutylicum, another [FeFe]-H2ase synthesizing organism, as the expression host.57,58 Further evidence for the lack of selectivity of the Hyd machinery came from the observation that co-expression of HydE, HydG and HydF from the bacterium C. acetobutylicum with various algal and bacterial [FeFe]-H2ase genes in E. coli resulted in purified enzymes with specific activities that were not very different from those of their counterparts from native sources.59 Also, the bacterium Shewanella oneidensis proved to be an efficient system for expression and maturation of HydA from C. reinhardtii.60 As an additional evidence of non-specificity within the maturases, incubation of E. coli extracts expressing inactive HydA from either Clostridium saccharobutylicum, C. reinhardtii or C. pasteurianum, with E. coli extracts expressing the three maturation proteins from C. acetobutylicum was reported to result in the efficient activation of HydA.61 A first in vitro HydA activation process was reported by Swartz and collaborators, which consists of a cell-free transcription/translation system allowing production of active algal and bacterial [FeFe]-H2ases in an anaerobic cell-free reaction mixture.25 The reaction mixture contains a plasmid expressing HydA, a DNA template and a RNA polymerase, with required substrates for transcription/translation, together with cell extracts from an E. coli strain expressing the HydE, HydG and HydF proteins from S. oneidensis, treated with iron and sulfide for [Fe-S] cluster assembly.25 Later on, in 2012, the cell-free approach was refined by the same group: the purified inactive apo-HydA (for example from C. reinhardtii or C. pasteurianum and harboring only the [4Fee4S]H subcluster and ferredoxin-like clusters), is incubated in a test tube under anaerobic conditions with E. Coli cell lysates, containing the three purified maturase proteins and a cocktail of selected low- molecular-weight cofactors and precursors. As this approach has proven efficient in HydA activation, it has been employed to identify small molecules, cofactors and salts having a stimulatory effect on the [FeFe]-H2ase activity.62 Unambiguously, cysteine, tyrosine, iron, sulfide and S-adenosylmethionine (SAM) were shown to play an important role in the maturation process.63,64,62 While iron and sulfide are likely to participate in [FeeS] cluster assembly, the function of the other additives is now well understood and will be discussed below. Finally, while not identified at that time, serine proved also critical for the assembly of the [2Fe]H subcluster only in 2020 and will be discussed below as well. HydE and hydG gene products were identified as iron sulfur enzymes from the Radical-SAM family, because they contain the wellestablished characteristic CysX3CysX2Cys motif, involved in a [4Fee4S] cluster chelation.65 In general, enzymes belonging to the Radical-SAM superfamily are involved in the formation of radical species, via the reductive homolytic cleavage of S-Adenosyl-

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Methionine (SAM) mediated by a [4Fee4S] cluster. The product of this primary reaction, the 50 -Ado• radical, is used to activate the substrate of the enzyme by H atom abstraction66–68 (Scheme 4). Once radicalized, the substrate has become reactive enough to enjoy conversion to the reaction product through complex radical pathways and electron transfers. Radical-SAM enzymes participate in a large number of metabolic and biosynthetic reactions; for example for the biosynthesis of a variety of cofactors, for the synthesis of deoxyribonucleotides and for DNA repair.69–71 HydF was also identified as an iron sulfur protein, but not from the Radical-SAM family, and, in addition, shown from sequence analysis to contain a nucleotide binding motif, suggesting a function of scaffold/carrier protein during [2Fe]H subcluster assembly.

2.07.3.1 2.07.3.1.1

The radical-SAM enzyme HydG Structure of HydG

HydG contains two distinct [4Fee4S] clusters, both absolutely required for activity.59,72 The first one, [4Fee4S]RS, is chelated by the characteristic “Radical-SAM” CysX3CysX2Cys motif present at the N-terminal domain while the second, [4Fee4S]AUX, uses the cysteines from another conserved C-terminal CysX2CysX22Cys motif. The six amino acid cysteine residues were shown by site-directed mutagenesis to be essential for activity.59,72 Thus, both [4Fee4S] clusters display three Fe atoms each with a cysteine ligand while the fourth Fe atom has a free coordination site, allowing binding of an exogenous ligand. In the case of the [4Fee4S]RS, this fourth Fe binds SAM via its a-amino-carboxylate moiety in a bidentate fashion. One-electron reduction of the [4Fee4S]RS-SAM adduct leads to the reductive cleavage of the adenosyl sulfonium bond of SAM to generate methionine and a 50 -deoxyadenosyl radical (50 -Ado•) (Scheme 4). The [4Fee4S]AUX cluster is much more complex and its structure has been revealed by a combination of X-ray crystallography and spectroscopic studies. A major breakthrough in understanding the structure and the function of HydG indeed came from the high-resolution structure (1.59 Å) of HydG from Thermoanaerobacter italicus (TiHydG).73 HydG thus consists of a complete (ba)8 TIM barrel fold with N- and C-terminal helical extensions.73 While the TIM barrel carries the [4Fee4S]RS cluster, it is the C-terminal extension of  80 amino acids residues that harbors the [4Fee4S]AUX cluster.73 The distance between the two clusters is about 24 Å (Fig. 3). The TihydG structure thus revealed that a fifth Fe atom, so-called “dangling Fe,” was associated with the [4Fee4S]AUX cluster via a sulfide bridge so that it was defined at that time as an unprecedented [5Fee5S]AUX cluster (Fig. 3).73 The fifth iron adopts an approximately octahedral geometry with a highly conserved histidine ligand (His265) coordinating the iron via the N3 of the imidazole ring.73 The coordination sphere of this fifth iron is completed by two H2O molecules and a non-proteinaceous amino acid, bound in a bidentate manner via the a-amino and a-carboxy functions.73 As the latter could not be identified due to structural disorder it was modelled as an alanine in the structure (Fig. 3)73. In agreement with this structure, EPR spectroscopic

Scheme 4 Mechanism of H-abstraction by Radical-SAM enzymes. R-H: substrate; 50 -Ado : 50 -deoxyadenosyl radical; R : radical substrate; P: product; Met: methionine; [4Fee4S]-SAM: [4Fee4S] cluster in complex with SAM; SAM shown in red is a ligand to the fourth iron in red, cysteine residues of the CysX3CysX2Cys motif are shown in green. l

l

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Fig. 3 X-ray crystal structure of HydG from Thermoanaerobacter italicus (PDB 4WCX). Overall structure (left) and close-up views of the [4Fee4S]AUX (top-right) and [4Fee4S]RS (bottom-right) catalytic sites.

characterization of HydG indicated the presence of a S ¼ 5/2 species specifically assigned to the [5Fee5S]AUX cluster.73 Accordingly, the S ¼ 5/2 signal is absent from EPR spectra of HydG mutants that cannot bind the C-terminal cluster, as a consequence of mutation of Cys residues, that coordinate this cluster, into serine.73 Finally, further characterization of the “dangling Fe” by Britt D. and coworkers, with the aim of identifying the amino acid ligand, demonstrated that it has L-Cysteine as a ligand rather than a sulfide and alanine.74 This led to the current model for the auxiliary cluster in which the “dangling Fe,” likely a [(k3-Cys)Fe(His)(H2O)2] unit, with a ferrous ion, is attached to the [4Fee4S]AUX cluster via a sulfur bridge, in agreement with the three-dimensional structure, however with the bridge provided by the cysteine ligand and not by a sulfide. It thus can be defined as a [5Fee4S]AUX cluster. This model can be reconciled with the structure reported by P. Roach and coworkers with a reinterpretation of the electron density around the “dangling Fe”. The absence of connection of the electron density associated with the amino acid and the sulfur atom is just a consequence of the low resolution of the structure but the overall density can easily be accounted for the presence of a cysteine instead.

2.07.3.1.2

Leads from sequence alignments: Tyrosine is the substrate of HydG

The second breakthrough in understanding the function of HydG came from the discovery of its substrate. An amino acid sequence comparison of HydG with other members of the “Radical-SAM” protein family indicated that tyrosine lyases (ThiH) were among the most closely related to HydG with 27% identity. 35% of the identities between HydG and ThiH correspond to strictly conserved residues in all the known amino acid sequences of both families. ThiH catalyzes the conversion of tyrosine into para-cresol and dehydroglycine (DHG), a precursor in thiazole biosynthesis.75 This genomic analysis and the remarkable homology between ThiH and HydG led us to discover that tyrosine is the substrate of HydG.76 This result extends the sequence homology of HydG and ThiH to a remarkable functional similarity, which provided a solid basis for subsequent mechanistic investigations. Immediately after this finding, it was established that CO and CN ligands are produced, together with para-cresol, from HydG-dependent radical cleavage and decomposition of tyrosine.77,78 Furthermore, experiments carried out with labelled tyrosine confirmed that tyrosine is indeed the unique source of both CO and CN.78,79 More specifically, 13CO and C15N were formed when tyrosine was selectively labelled at the carboxylic (with 13C) and the amino (with 15N) group, respectively.80

2.07.3.1.3

The N-terminal [4Fee4S]RS mediates radical chemistry

The pathway from tyrosine to para-cresol in one hand and CO and CN in another hand has been a matter of detailed studies by Britt D. R. and coworkers. Using a combination of physical methods (EPR, HYSCORE and electron nuclear double resonance spectroscopies), 2H-, 13C- and 15N-labelled tyrosine substrate and HydG from S. oneidensis (SoHydGWT) allowed showing the formation of a transient 4-hydroxy-benzyl radical together with dehydroglycine.81 Consistent with HydG being a Radical-SAM enzyme, a mechanism (Scheme 5) was thus proposed in which the 50 -Ado• radical, derived from SAM cleavage at the N-terminal [4Fee4S]RS cluster, first abstracts a H atom from the tyrosine substrate. The site of this initial H-atom abstraction was debated in the literature.81–84 Two possibilities indeed exist since either the phenolic HeO atom or

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Scheme 5 Reactions catalyzed by the Radical-SAM maturase HydG. The N-terminal domain containing a [4Fee4S]RS (light blue cubane) catalyzes the reductive cleavage of SAM into 50 -Ado• radical that is used to fragment tyrosine substrate into p-cresol and dehydroglycine (DHG) which further decomposes into CO and CN (light blue box). The orange box shows the catalytic cycle occurring at the [4Fee4S]AUX center present at the C-terminal domain (orange cubane) generating the organometallic [(k3-Cys)Fe(CO)2(CN)] product.

the amino HeN atom of the tyrosine substrate are candidates. Because both are solvent-exchangeable H-atoms it was impossible to use 2HeO/2HeN labelled tyrosine to differentiate them. However several lines of evidence pointed to the H atom of the amino group.73,85 First, although efforts to crystallize HydG with tyrosine were unsuccessful, computational analysis of the active-site cavity within the TIM barrel identified a putative tyrosine binding site with the a-amino H-atom oriented toward the [4Fee4S]RS cluster.73 Second, one should consider that the strong amino NeH bond strength is better matched with that of 50 -AdoH CeH bond and therefore more consistent with the experimentally observed reversibility of H-atom abstraction.82,85 Taken together all these results led to a mechanism in which the 50 -Ado• radical abstracts the HeN atom of the amino carboxylate moiety of tyrosine (Scheme 5). The resulting radical undergoes homolytical cleavage of CaeCb bond producing the spectroscopically characterized 4-hydroxy-benzyl radical and dehydroglycine. After reduction and protonation of this intermediate, p-cresol is generated as a byproduct of the reaction, while dehydroglycine is activated further for conversion into CO and CN (Scheme 5). As described below the tyrosine-derived CO and CN molecules are likely moving toward the [5Fee4S]AUX cluster and ending up as ligands of the “dangling Fe”. It is still unclear how dehydroglycine decomposes into CO and CN, whether this reaction is assisted by one of the clusters of HydG and which one. However, it is important to note that a HydG mutant lacking the C-terminal cluster produces only cyanide and not CO.86 Formate instead is produced, showing that the auxiliary cluster is essential for controlled concomitant production of CO and CN from dehydroglycine. Furthermore, a HydG mutant, in which binding of the “dangling Fe” is prevented by mutation of the histidine ligand, does not produce any CO (but formate), supporting the idea that this Fe site is critical for CO synthesis.87

2.07.3.1.4 The C-terminal [4Fee4S]AUX of HydG is a platform for a rich organometallic chemistry, especially for the assembly of a [Fe(CO)2(CN)] species precursor to the [2Fe]H subcluster The current model indeed implies that, once formed, both CO and CN are immediately trapped, before leading to toxic reactions, by the “dangling Fe”, which is clearly well organized to receive two CO and one CN ligands to generate a labile [(k3-Cys) Fe(CO)2(CN)] unit with the iron atom remaining in the ferrous state. The latter was proposed to be the final product of the HydG-dependent reaction and the precursor of the [2Fe]H subcluster during HydA maturation. In agreement, 57Fe ENDOR

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spectroscopic studies have shown that Fe in the [2Fe]H subcluster of HydA is derived from HydG.88 Furthermore, Britt and coworkers, using time-resolved FTIR and ENDOR spectroscopy, indeed provided evidence for the formation, during HydG catalysis, when HydG is mixed with tyrosine, SAM and a source of necessary electrons (dithionite), of two distinct Fe(CO)x(CN) species bound to the [4Fee4S]AUX cluster.88 The first one, named complex A with the stoichiometry Fe(CO)(CN) and the Fe coordination sphere completed with the histidine residue and the cysteine ligand, is followed by complex B, with a [(k3-Cys)Fe(CO)2(CN)] unit.88,89 The presence of a second CO ligand in complex B thus implies the requirement for HydG to fragment two tyrosine and the release of one CN. Thus, the CO and CN molecules derived from the first tyrosine fragmentation both bind to the “dangling Fe” generating complex A, while only CO, derived from the fragmentation of the second tyrosine, binds to it, generating complex B. The second CN molecule has been proposed to allow displacement of the [(k3-Cys)Fe(CO)2(CN)] product from complex B. Accordingly, using labelled tyrosine and HYSCORE spectroscopy, formation of an EPR-detectable [4Fee4S]AUX-CN species, as a reaction by-product, was also observed, following formation of complex A and complex B, presumably via substitution and liberation of [(k3-Cys)Fe(CO)2(CN)]74,90 (Scheme 5). The last important contribution to the understanding of HydG function came from experiments aiming at defining whether the cysteine ligand of the [(k3-Cys)Fe(CO)2(CN)] product is a source of atoms for the adt2  ligand of the [2Fe]H subcluster. This was indeed recently addressed by in vitro [2Fe]H subcluster assembly experiments using a synthetic chemical analog of complex B, named syn-B, which proved to be functionally competent for the production of an active [FeFe]-H2ase in the absence of HydG.91 In the absence of a crystal structure of syn-B, FT-IR spectroscopy, electrospray ionization mass spectrometry (ESI-MS) and elemental analyses established that it contained the target [Fe(Cysteine)(CO)2(CN)] species, likely within a cluster containing 3–4 [Fe(Cysteine)(CO)2(CN)(H2O)] units bound to a high-spin Fe(II) center.91 Syn-B was obtained in two steps: (i) synthesis of [FeI2(CN)(CO)3] from either cyanation of FeI2(CO)4 or, preferably, iodination of [Fe(CN)(CO)4]; (ii) reaction of [FeI2(CN)(CO)3] with dipotassium cysteinate. Remarkably, incubation of apo-HydA with syn-B in the presence of HydF and HydE, but in the absence of HydG, resulted into a fully active HydA, with spectroscopic signatures identical to the ones of naturally maturated HydA.91 This semisynthetic HydG-free maturation procedure is reminiscent to the purely synthetic (in the absence of HydE, HydF and HydG) assembly of the H-cluster and maturation of HydA exclusively using with a synthetic diiron organometallic complex, [Fe2(adt)(CO)4(CN)2]2, mimicking the [2Fe]H subcluster26 (Scheme 2). The ability of syn-B to replace complex B was exploited for demonstrating that the cysteine ligand is the source of the bridging sulfur atoms of the 2-azapropane-1,3-dithiolate (adt2 ) ligand.91 Indeed, maturation of apo-HydA with syn-B in which a selenocysteine is used as a substitute for cysteine results in the incorporation of Se into the [2Fe]H subcluster, characterized by EXAFS spectroscopy.91 Furthermore, spectroscopic characterization of semi-synthetically maturated [FeFe]-H2ase conducted with syn-B containing labelled cysteine (syn-B-13C3,15N) and using pulse electron paramagnetic resonance spectroscopy, EXAFS spectroscopy and mass spectrometry, revealed that no 13C or 15N was incorporated into the 2-azapropane-1,3-dithiolate ligand.91 Taken together, these results, with the observation that the cysteine backbone of syn-B is converted into pyruvate, validate that the cysteine ligand of the [(k3-Cys)Fe(CO)2(CN)] complex is just the source of the two sulfur atoms for the biosynthesis of adt2 .91 This thus implies that a chemistry allows a specific cleavage of the SeC bond of that cysteine and that the FeeS bond remains intact throughout the assembly of the H-cluster of HydA.

2.07.3.1.5

HydG enzyme mechanism

Based on the thorough characterization of HydG described above, one can draw the enzyme mechanism shown in Scheme 5. The resting state of the enzyme contains two clusters. The first one, [4Fee4S]RS, binds and activate SAM while the second one, [4Fee4S]AUX, serves to transiently assemble a mononuclear organometallic Fe complex, [(k3-Cys)Fe(CO)2(CN)]. Thanks to these clusters, HydG catalyzes a complex reaction, namely the conversion of tyrosine, cysteine and Fe into [(k3-Cys)Fe(CO)2(CN)], the final product, which, after liberation, is used as a source of Fe, S, CO and CN for the synthesis of the [2Fe]H subcluster of HydA. For that purpose, a source of electrons is required and SAM as a cofactor is essential. In the first step, SAM binds to the [4Fee4S]RS cluster where it receives one electron and tyrosine binds to the adjacent substrate site. At the same time, the [4Fee4S]AUX cluster reacts with Fe2þ and cysteine and binds a [(k3-Cys)Fe(H2O)2(His265)] species, the so called “dangling Fe”, via a sulfur bridge provided by the cysteine ligand, and with two molecules of water and a bond with His265 of the protein to complete the coordination. Upon reductive cleavage of SAM cofactor by the reduced [4Fe-4S]RS cluster, the generated 50 -Ado• radical abstracts the HeN atom of tyrosine producing a transient, and yet to be detected, N-based radical intermediate.92 The latter fragments into the observed EPR-active 4-hydroxy-benzyl intermediate radical, which ends up as p-cresol and dehydroglycine (DHG). DHG then decomposes into one molecule of CO and one molecule of CN, however via a sill ill-defined mechanism. In particular, there is still no clarification regarding the role of the clusters in this decomposition. CO and CN displace the water ligand and bind to the “dangling Fe” of the C-terminal cluster thus forming the first intermediate, complex A. After a second cycle of tyrosine fragmentation, one molecule of CO displaces the His ligand and binds to the dangling Fe, generating complex B, while CN serves to release [(k3-Cys)Fe(CO)2(CN)], the final product. A new enzyme cycle then can restart upon binding SAM at the N-terminal cluster and Fe2þ and cysteine, as well as two molecules of H2O, at the C-terminal cluster.

2.07.3.2

The radical-SAM enzyme HydE

As HydG, HydE was shown to contain the CysX3CysX2Cys motif characteristic of “Radical-SAM” enzymes24 and, based on biochemical and spectroscopic studies of HydE from T. maritima (TmHydE), to harbor two distinct [4Fee4S] clusters.72 The established Radical-SAM [4Fee4S] cluster is chelated by the characteristic motif present at the N-terminal domain and displays a unique Fe atom with a free coordination site allowing binding of SAM cofactor as expected. The second [4Fee4S] cluster was shown to be

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present at the C-terminal domain of the protein and chelated by the three cysteines of the CysX7CysX2Cys motif. Intriguingly, this motif is present in several, but not all, HydE orthologs, and there is thus a second class of HydE proteins, which lack the CysX7CysX2Cys motif. In addition, site-directed mutagenesis experiments of any of the cysteines of this motif into alanine in C. acetobutylicum HydE, belonging to the first class, did not prevent it from activating the [FeFe]-hydrogenase from the same organism, heterologously expressed in E. coli.93 Moreover, in similar experiments, replacement of C. acetobutylicum HydE by B. thetaiotaomicron HydE, which belongs to the subset lacking the cysteine ligands of the second cluster, did not affect hydrogenase maturation.93 All these data clearly indicate that the cysteine-containing sequence that coordinates the second cluster, found in HydE from T. maritima and some of its orthologs, is not essential for maturation, raising the question of the function of the second cluster, if any.

2.07.3.2.1

HydE structure

As for other “Radical SAM” proteins, HydE has the common structural core assembled around a (ba)6 three quarter TIM barrel69,93 (Fig. 4). As was expected from previous amino acid sequence alignments, the HydE structure is very similar to that of BioB93,94 with its C-terminal extension defining a complete (ba)8 TIM barrel.93 HydE is thus classified within the “complete barrel Radical SAM” protein sub-class. This, in turn, suggests that its substrate is a small molecule. Indeed, many of the conserved amino acids in HydE are found on the b-strands that, in BioB and other members of this family, define the substrate-binding cavity inside the barrel, below the [4Fee4S]-SAM complex site. Consequently, mutations of these residues around the inferred substrate-binding site produced HydE mutants with little or no maturation activity assayed via in vivo tests.93 In the HydE crystal structure, three linearly disposed sites bind halide ions as shown by the anomalous difference peaks observed when displacing putative Cl by Br in soaking experiments.93 Two of these sites are found in the putative substrate-binding region mentioned above whereas the third one is located at the bottom of the internal cavity, relatively far from SAM cofactor. Interestingly, additional crystal soaking experiments showed that SCN binds with very high affinity to this latter site, displacing the putative Cl ion of the original structure. Site-directed mutagenesis around the SCN binding site also resulted in a significant drop in maturase activity.93 The in silico screening approach has been used to dock  60,000 small molecules showing that the two anion-binding sites found in the putative substrate-binding region of HydE could also bind two carboxylate moieties.95 For example, Nicolet Y. and collaborators reported a crystal structure of TmHydEDFeS (in which the cysteines of the CysX7CysX2Cys motif have been mutated into serines and the second cluster is absent) containing a 2-methyl-1,3-thiazolidine-2,4-dicarboxylic acid sitting in the substrate binding site96 (Fig. 4). The two carboxylate moieties are located in the previously identified anion-binding sites. While it is not the physiological substrate of HydE, this study showed that HydE catalyzes the reaction of this molecule with SAM, in the presence of a source of electrons, to generate methionine, S-adenosyl-cysteine and pyruvate. The reaction is likely to start with the addition of the 50 -Ado• radical, derived from SAM, on the sulfur atom of the 1,3-thiazolidine moiety thus creating a C50 -S bond followed by fragmentation of the intermediate adduct.96 The product of the reaction shown in Fig. 4C, S-Adenosyl-Cysteine, indeed displays a covalent bond between the 50 -methylene carbon of adenosyl and the sulfur atom derived from the substrate. One should note that there are two precedents for radical-SAM enzymes adding 50 -Ado• to the substrate rather than using it for substrate H atom abstraction: these are the MqnE and HpnH enzymes involved in the biosynthesis of menaquinone (vitamin K) and bacteriohopanepolyols respectively. For both enzymes, a stereo-selective CeC bond formation is obtained through the addition of the 50 -Ado• radical to the substrate double bond.97,98 Taken together these results at that time suggested that (i) the HydE protein structure is designed to bind a small molecule, most likely a sulfur-containing compound carrying negatively charged groups such as carboxylates; (ii) HydE has the ability to catalyze the formation of a CeS bond via addition of 50 -Ado• onto the S atom of the substrate.

2.07.3.2.2

The radical-SAM enzyme HydE acts on the HydG product

Initially, HydE was thought to be involved in synthesizing the bridging azadithiolate ligand of the [2Fe]H-subcluster. However, given the demonstration that HydG provides not only the diatomic ligands and the two iron atoms to form the [2Fe]H subcluster but also the two sulfur atoms of the adt2  ligand, we end up with the hypothesis that HydE is responsible for the generation of the only missing fragment, namely the NH(CH2)2 moiety of the adt2  ligand, needed to assemble a full and active [2Fe]H subcluster. A first breakthrough came from a recent paper by Britt and his collaborators showing that HydE that the [(k3-Cys)Fe(CO)2(CN)] complex, the product of HydG, is likely to be the substrate of HydE.99 On the basis that apo-HydA can be activated, in the absence of HydG, only with HydE, HydF and the syn-B complex (see above), the reaction of syn-B with purified TmHydE enzyme was carried out in the presence of sodium dithionite as a source of electrons and SAM.99 This reaction mixture was incubated for varying times before freeze-quenching in liquid nitrogen for EPR spectroscopy characterization for any paramagnetic intermediates. The first detected EPR intermediate, after about 10 s, is a paramagnetic species with S ¼ 1/2 rhombic signal,99 likely derived from the addition of the 50 -Ado• radical on the sulfur atom of the cysteine ligand of syn-B. This reaction thus implies formation of a CeS bond concomitant with reduction of the one-electron reduction of the FeII ion of synB yielding a low-spin FeI-containing species.99 Further characterization of this first intermediate by ENDOR spectroscopy combined with syn-B complex labelled with 13CN or 13C3/15N-Cys indicates that it consists in an adenylated FeI species containing CO, CN and a modified cysteine ligand in which the S atom is covalently bound to adenosine through a S-C50 bond. This reaction is reminiscent of the reported HydE reaction with 1,3-thiazolidine described above.96 Interestingly, after 10 min. incubation, this first

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Fig. 4 Structure of HydE from Thermotoga maritima. Top: Overall organization of HydE showing the Radical-SAM [4Fee4S]RS cluster and the nonessential accessory [4Fee4S] cluster (cluster 2) (PDB 4JY8). Chlorine atoms are shown as spheres colored in cyan. Middle and bottom: Close-up views of the radical SAM cluster from the crystal structure of HydE in complex with SAM and 2-methyl-1,3-thiazolidine-2,4-dicarboxylate before (middle) and after (bottom) reaction (PDB 5FEP and PDB 5FEW respectively).

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intermediate undergoes further transformation during which the cysteine SeC3 bond is heterolytically cleaved leading to pyruvate and a new S ¼ 1/2 complex, characterized as [FeI(CO)2(CN)(L)] with L being a 50 -thio-deoxyadenosine ligand.99 Thus, the HydG reaction product, [(k3-Cys)Fe(CO)2(CN)], is likely to be the substrate of HydE which serves to cleave away the entire C/N backbone of cysteine and retain only the S atom in the Fe complex (Scheme 6). In the route to the [2Fe]H assembly it is likely, but not yet shown, that this [FeI(CO)2(CN)(L)] complex enjoys further transformation, via ribose release and dimerization, leading to a putative [(FeI)2(CO)4(CN)2S2]4 dimer, for which there are a number of synthetic examples.26,100,101 Obviously, the final step in building the [2Fe]H subcluster is the decoration of this dimer with the CH2NCH2 moiety as discussed in the following section.

2.07.3.2.3

CH2NCH2 moiety of the azapropanedithiolate bridge derives from a serine amino acid residue

The last missing part in the [2Fe]H subcluster assembly puzzle is the identification of the molecular precursor of the CH2NCH2 fragment and the mechanism of its appendage to the putative [(FeI)2(CO)4(CN)2S2] 4 dimer intermediate. Once again, the in vitro cellfree synthesis method described above and developed by the Swartz group proved decisive.102 With the suspicion that the CH2NCH2 fragment could derive from one or more amino acid residues, CrHydA1 maturation was achieved in the presence of the standard cocktail of maturases and low-molecular weight cofactors together with a complete mixture of 13C-, 15N-labelled amino acids.102 Characterization of the active CrHydA1 in the Hox state, using hyperfine sublevel correlation spectroscopy method (HYSCORE), recorded at g ¼ 2.103 (g1 of the Hox state), revealed the presence of a set of two doublet peaks. The first doublet is separated by 1.9 MHz and centered at the Larmor frequency of 15N (n15N ¼ 1.02 MHz at 333 mT) with A 15N ¼ [1.9, 1.6, 1.6] MHz as a hyperfine tensor.102 The second doublet is centered at the 13C Larmor frequency (n15C ¼ 3.56 MHz at 333 mT). Remarkably, these features are in excellent agreement with those reported for CrHydA1 preparations chemically activated with labelled variants of the synthetic [Fe2(adt)(CO)4(CN)2]2 complex, containing 15N-adt2  or 13C-adt2  ligands.103,104 Taken together all these results unambiguously demonstrate that the nitrogen and carbon atoms of the adt2  ligand indeed derive from one or more amino acids. Finally, this study revealed that only labelled serine was able to introduce the labelled atoms into the [2Fe]H subcluster of CrHydA1.102 In addition, it showed that both CH2 groups of the CH2NCH2 fragment were sourced from the C3 methylene of serine, thus implying that two serine molecules are needed for [2Fe]H subcluster assembly.102 Even though all precursors of the [2Fe]H

Scheme 6 Reaction carried out by the Radical-SAM maturase HydE. Top box shows the transformation of HydG product [(k3-Cys)Fe(CO)2(CN)] into a putative diiron [Fe2(CO)4(CN)2S2]4 complex; in the lower box some of the spectroscopically observed intermediates are highlighted.

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[FeFe]-hydrogenases: Structure, mechanism, and metallocluster biosynthesis

subcluster are now identified, it is still unknown how serine is fragmented, what exact roles HydE and HydF play in that process and how the methylene groups and the N atom from serine are installed into the adt2  ligand of the [2Fe]H subcluster.

2.07.3.3 2.07.3.3.1

The scaffold HydF protein HydF: A [4Fee4S] protein with ability to bind a precursor of the [2Fe]H subcluster

HydF is the last protein of the three maturases known to be involved the [FeFe]-H2ases maturation and it is the only one that is not from the Radical-SAM family.24 HydF is proposed to assemble a diiron precursor of the [2Fe]H subcluster, derived from HydG/HydE action, namely [Fe2(adt)(CO)4(CN)2]2, and deliver it to apo-HydA (Berggren 2013, Caserta 2017). In full agreement with a scaffold/carrier protein function, HydF from C. acetobutylicum, overexpressed either in C. acetobutylicum, a microorganism naturally expressing HydE and HydG, or in E. coli expressing the C. acetobutylicum maturases, was shown to contain indeed a [4Fee4S] cluster but also a second, dinuclear, Fe center with CO and CN ligands, as demonstrated by FTIR spectroscopy.105,106 X-ray absorption spectroscopy at Fe K-edge confirmed this extra cluster to be a diiron complex.107 These results indicate that when expressed in the presence of HydE and HydG, HydF is obtained in a form which contains all the requested chemical elements for activating apo-HydA. Furthermore, we have shown that the synthetic analog of the [2Fe]H subcluster, [Fe2(adt)(CO)4(CN)2]2, can bind to TmHydF26 or Tme-HydF.108 Elegant FTIR spectroscopy, pulsed EPR techniques and DFT calculations unambiguously demonstrated that the [4Fee4S] cluster of HydF and the diiron complex shared a cyanide ligand.27 The FTIR spectrum of the hybrid was remarkably similar to that of Ca-HydF purified from the native host. Furthermore, we showed that the diiron component of the hybrid is efficiently transferred to apo-HydA, leading to a fully active HydA.26,27

2.07.3.3.2

Structure of HydF

Tm-HydF from the hyperthermophilic organism T. maritima has been studied in detail and shown, by a variety of spectroscopic methods and crystallography, to bind a unique [4Fee4S] cluster, which is described further below.109 The first crystal structure of Tn-HydF, from the hyperthermophilic organism T. neapolitana, however lacking its [4Fee4S] cluster, refined at 3.0 Å resolution, has been reported in 2011 by Cendron L. and coworkers.110 The asymmetric unit contains a monomer, but the biological unit is a dimer, generated by a crystallographic twofold axis, or a tetramer, produced by the dimerization of dimers.110 The structure shows also that the three cysteines of the CysXHisXnHisCysXXCys motif are involved in intramolecular and intermolecular disulfide bridges giving rise to a supramolecular tetrameric organization (dimer of dimers). Unfortunately, this apo-TnHydF X-ray structure could not provide any information on the iron-sulfur cluster site.110 Finally, the three-dimensional X-ray structure of fully metallated Thermosipho melanesiensis TmeHydF was obtained in our group.108 The structure was refined at 2.8 Å resolution showing that the asymmetric unit contains four monomers and the crystal is formed by a packing of dimers for which the overall structure is depicted in Fig. 5. Careful analysis of this structure indicates that TmeHydF is a homodimeric protein with three domains per monomer as shown in apo-HydF.110 The N-terminal part, residues 7–166, holds the GTPase domain, the central area, residues 172–237, constitutes the dimerization domain, and the C-terminal half, residues 238–395, holds the [4Fee4S] cluster-binding domain108 (Fig. 5). The GTP binding domain is linked to the dimerization domain via a stretch of about 5 amino acid residues which likely provides flexibility.108 The location of the clusters in TmeHydF was determined thanks to the anomalous Fe signal using a dataset collected at the iron edge.108 The [4Fee4S] cluster is solvent-exposed and is coordinated by three cysteines (Cys298, Cys349 and Cys352) that are strictly

Fig. 5 Crystal structure of HydF from Thermosipho melanesiensis (PDB 5KH0). Overall structure of the HydF dimer (left). One monomer is colored as a function of secondary structures elements with strains in orange and helices in green. The second monomer is colored as a function of the domains with the N-terminal GTP binding domain in orange, the dimerization domain in pink and the C-terminal [4Fee4S] cluster binding domain in blue. Close-up view near the [4Fee4S] cluster showing the cavity expected to bind the [Fe2(adt)(CN)2(CO)4]2 complex (right).

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189

conserved among the members of the HydF family.108,109,111 Interestingly, the fourth iron ion is coordinated by the carboxylate group of a glutamate, Glu305 (Fig. 5). Although this position is not strictly conserved when analyzing the alignment of 227 bacterial HydFs, nevertheless it is an acidic residue (Asp or Glu) in the overwhelming majority (98% of the sequences).108 There are few precedents for [4Fee4S] clusters chelated by 3 Cysteines and a glutamate, as in the IspG protein from Aquifex aeolicus involved in the biosynthesis of the universal isoprenoid precursors (isopentenylpyrophosphate and dimethylallylpyrophosphate).112 The four [4Fee4S] clusters observed in the asymmetric unit show some variability with respect to the orientation of the side-chain of Glu305. This flexibility, together with the fact that the [4Fee4S] cluster is solvent-exposed, is consistent with the function of the cluster, that is to transiently bind a diiron complex, through a CN bridge. This indeed requires decoordination of the labile glutamate ligand. The exchangeability of glutamate has been experimentally substantiated. In the case of both TnHydF111 and TmHydF,113 exogenous imidazole has been shown to bind to the cluster of TmeHydF, as monitored by HYSCORE spectroscopy.108 The cluster sits in an area of highly conserved and positively charged amino acid residues. This might serve for specific binding of protein partners (HydA and/or maturases) and, interestingly, it also fits with the need for the protein to bind the negatively charged [Fe2(adt)(CO)4(CN)2]2diiron complex. More specifically, the structure shows that the cluster is adjacent to a crevasse with enough room to accommodate that diiron complex (Fig. 5).

2.07.3.3.3

HydF, a GTP-binding protein

Primary sequence analysis of the N-terminal domain of HydF from T. maritima and all homologous proteins from various [FeFe]H2ases containing microorganisms revealed the presence of several conserved consensus sequences, similar to those involved in guanine nucleotide binding in Small-G proteins. The first motif is the (G/A)X4GK(T/S) sequence responsible for the binding of a- and b-phosphate groups of the nucleotide (the P-loop).24,109 Site-directed mutagenesis experiments conducted on the P-loop motif of the HydF protein from C. acetobutylicum in which either one of the glycine or the serine has been changed into alanine indicates that the modified HydF protein is unable, in combination with HydE and HydG, to activate apo-HydA from the same organism.59 Other remarkable features present in HydF proteins are: (i) a conserved threonine residue that might correspond to the residue of the G2 loop involved in Mg2þ binding; (ii) the DX2G sequence that represents the G3 loop involved in the interaction with the g-phosphate and Mg2þ, and (iii) the G4 loop, (N/T)(K/Q)XD, which is supposed to interact with the nucleotide part of GTP. GTP binding to HydF was indeed demonstrated using fluorescence spectroscopy, with a Kd value of 3 mM for the dissociation of the HydF protein-GTP complex, and furthermore HydF was shown to catalyze GTP hydrolysis to GDP in the presence of Mg2 þ.109 A GTPase activity has also been reported in the case of HydF from C. acetobutylicum.105,106 This activity depends on the nature of the monovalent cation in the reaction buffer with the highest activity obtained with Kþ and Rbþ, suggesting that a binding pocket for Kþ exists near the active site.105 Furthermore, while it is not affected by the presence of HydA, it is stimulated by the presence of HydE and HydG. These results, together with the observed lack of effect of GTP on the activation of HydA by HydF, suggest that the GTPase activity of HydF plays no role in the transfer of the transient diiron complex from HydF to HydA but more likely contributes to assembling this complex on HydF, in concert with HydE and HydG activity.

2.07.3.4

H-cluster of [FeFe]-hydrogenase: Mechanism of bioassembly

Efforts conducted in hosts not capable for synthesizing the complete H-cluster, shows that the insertion of the metal centers occurs in two steps.38,39,56,114,115 First, the accessory clusters and the [4Fee4S]H cluster are assembled thanks to the participation of the ISC and SUF machineries specific for the biosynthesis of canonical FeeS clusters.116,117 Introduction of the [2Fe]H subcluster occurs in a second step with the absolute requirement of three specific maturases, i.e., HydE, HydF and HydG, as discussed above. While an impressive progress during the last years has allowed a better understanding of HydA maturation, there are still a number of unsolved questions, which do not allow drawing a definitive mechanism. The most important ones are: (i) what is the chemistry of two serines decomposition into the CH2NHCH2 fragment of the adt2  ligand and which enzyme catalyzes this reaction?; (ii) by which mechanism and with which enzymes the CH2NHCH2 fragment make bonds with the cysteine-derived S atoms to form the adt2  ligand?; (iii) how dimerization of the putative [Fe(CO)2(CN)S] intermediate occurs and which maturase, HydE or HydF, promotes this reaction? Nevertheless, in Scheme 7 we provide a general view of the maturation pathway. It is now well established that HydF is the true maturase of HydA as it serves to bind the precursor diiron complex [Fe2(adt)(CO)4(CN)2]2 and deliver it to apo-HydA for full activation. This key precursor is assembled in HydF from the [(k3Cys)Fe(CO)2(CN)] complex, which is produced by HydG via decomposition of tyrosine into CO and CN and binding of one ferrous ion and one molecule of cysteine. The conversion of the [(k3-Cys)Fe(CO)2(CN)] complex, likely dependent on HydE, implies: (i) cleavage of the CeS bond of the Cys ligand; (ii) dimerization of the mononuclear complex; (iii) addition of the CH2NHCH2 fragment derived from serine. Activation of HydA likely occurs within a transient HydF–apo-HydA complex, which allows channeling the diiron complex from HydF to the [4Fee4S]H cluster of apo-HydA. During that reaction: (i) a CO ligand is displaced allowing a cysteine ligand of the [4Fee4S]H cluster to bind to one Fe atom of the diiron complex and (ii) a rotation occurs leading to the formation of a CO bridge and liberating an open coordination site at the distal Fe for catalysis. Through these transformations associated with the transfer from HydF to HydA, [Fe2(adt)(CO)4(CN)2]2 is converted into the [2Fe]H subcluster and the hydrogenase is activated.

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Scheme 7

2.07.4

The [FeFe]-H2ase H-cluster biosynthesis and bioassembly pathway.

Conclusion

The field of [FeeFe]-hydrogenases will remain a very active one in the future. Not only because the mechanism of maturation is incompletely understood and novel fascinating chemical reactions are expected regarding biosynthesis of a unique organometallic cofactor, a central theme in bioinorganic chemistry. But also because this increased knowledge has opened new directions of research. First, synthetic maturation using a chemical, without the need for the maturation machinery, allows faster and easier analysis of enzyme mutants as well as of the biodiversity, aiming at finding new very active and more stable hydrogenases for biotechnological applications (bioelectrodes for fuel cells for example). Second, the impressive activity of [FeFe]-hydrogenases is further incentive to continue the efforts aimed at developing bioinspired molecular diiron catalysts22 as well as artificial hydrogenases which are just emerging.118–120 Finally, to become a significant component of the new “hydrogen economy,” production of [FeFe]-hydrogenase enzymes at large scale and understanding the molecular basis of their O2-sensitivity are issues to be addressed. It is very likely that the next review article on this topic will be necessary very soon.

Acknowledgements We are grateful to Dr. Ludovic Pecqueur for the preparation of the Figures describing the three-dimensional structures of the HydA hydrogenases and the HydE, HydG and HydF maturases. This work was supported by the French State Program “Investissements d’Avenir” (Grants “LABEX DYNAMO”,ANR-11-LABX-0011).

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Structural Characterization of HypX Responsible for CO Biosynthesis in the Maturation of NiFeHydrogenase. Commun Biol 2019, 2. 54. Kalia, V. C.; Lal, S.; Ghai, R.; Mandal, M.; Chauhan, A. Mining Genomic Databases to Identify Novel Hydrogen Producers. Trends Biotechnol. 2003, 21 (4), 152–156. 55. Pierik, A. J.; Hagen, W. R.; Redeker, J. S.; Wolbert, R. B.; Boersma, M.; Verhagen, M. F.; Grande, H. J.; Veeger, C.; Mutsaers, P. H.; Sands, R. H.; et al. Redox Properties of the iron-Sulfur Clusters in Activated Fe-Hydrogenase from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 1992, 209 (1), 63–72. 56. Voordouw, G.; Hagen, W. R.; Krusewolters, K. M.; Vanberkelarts, A.; Veeger, C. Purification and Characterization of Desulfovibrio-Vulgaris (Hildenborough) Hydrogenase Expressed in Escherichia-Coli. Eur. J. Biochem. 1987, 162 (1), 31–36. 57. Girbal, L.; von Abendroth, G.; Winkler, M.; Benton, P. M. C.; Meynial-Salles, I.; Croux, C.; Peters, J. W.; Happe, T.; Soucaille, P. Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases With High Specific Activities. Appl. Environ. Microbiol. 2005, 71 (5), 2777–2781. 58. von Abendroth, G.; Stripp, S.; Silakov, A.; Croux, C.; Soucaille, P.; Girbal, L.; Happe, T. Optimized over-Expression of [FeFe] Hydrogenases With High Specific Activity in Clostridium acetobutylicum. Int. J. Hydrogen Energy 2008, 33 (21), 6076–6081. 59. King, P. W.; Posewitz, M. C.; Ghirardi, M. L.; Seibert, M. Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System. J. Bacteriol. 2006, 188 (6), 2163–2172. 60. Sybirna, K.; Antoine, T.; Lindberg, P.; Fourmond, V.; Rousset, M.; Mejean, V.; Bottin, H. Shewanella Oneidensis: A New and Efficient System for Expression and Maturation of Heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii. BMC Biotechnol. 2008, 8. 61. McGlynn, S. E.; Ruebush, S. S.; Naumov, A.; Nagy, L. E.; Dubini, A.; King, P. W.; Broderick, J. B.; Posewitz, M. C.; Peters, J. W. In Vitro Activation of [FeFe] Hydrogenase: New Insights into Hydrogenase Maturation. J. Biol. Inorg. Chem. 2007, 12 (4), 443–447. 62. Kuchenreuther, J. M.; Britt, R. D.; Swartz, J. R. New Insights into [FeFe] Hydrogenase Activation and Maturase Function. PLoS One 2012, 7 (9), e45850. 63. Kuchenreuther, J. M.; Stapleton, J. A.; Swartz, J. R. Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation. PLoS One 2009, 4 (10). 64. Kuchenreuther, J. M.; Shiigi, S. A.; Swartz, J. R. Cell-Free Synthesis of the H-Cluster: A Model for the In Vitro Assembly of Metalloprotein Metal Centers. Methods Mol. Biol. 2014, 1122, 49–72. 65. Sofia, H. J.; Chen, G.; Hetzler, B. G.; Reyes-Spindola, J. F.; Miller, N. E. Radical SAM, a Novel Protein Superfamily Linking Unresolved Steps in Familiar Biosynthetic Pathways with Radical Mechanisms: Functional Characterization Using New Analysis and Information Visualization Methods. Nucleic Acids Res. 2001, 29 (5), 1097–1106. 66. Fontecave, M.; Mulliez, E.; Ollagnier-de-Choudens, S. Adenosylmethionine as a Source of 5 ’-Deoxyadenosyl Radicals. Curr. Opin. Chem. Biol. 2001, 5 (5), 506–511. 67. Wang, S. C.; Frey, P. A. S-Adenosylmethionine as an Oxidant: The Radical SAM Superfamily. Trends Biochem. Sci. 2007, 32 (3), 101–110. 68. Wang, J. R.; Woldring, R. P.; Roman-Melendez, G. D.; McClain, A. M.; Alzua, B. R.; Marsh, E. N. G. Recent Advances in Radical SAM Enzymology: New Structures and Mechanisms. ACS Chem. Biol. 2014, 9 (9), 1929–1938. 69. Broderick, J. B.; Duffus, B. R.; Duschene, K. S.; Shepard, E. M. Radical S-Adenosylmethionine Enzymes. Chem. Rev. 2014, 114 (8), 4229–4317. 70. Atta, M.; Mulliez, E.; Arragain, S.; Forouhar, F.; Hunt, J. F.; Fontecave, M. S-Adenosylmethionine-Dependent Radical-Based Modification of Biological Macromolecules. Curr. Opin. Struct. Biol. 2010, 20 (6), 684–692. 71. Latham, J. A.; Barr, I.; Klinman, J. P. At the Confluence of Ribosomally Synthesized Peptide Modification and Radical S-Adenosylmethionine (SAM) Enzymology. J. Biol. Chem. 2017, 292 (40), 16397–16405. 72. Rubach, J. K.; Brazzolotto, X.; Gaillard, J.; Fontecave, M. Biochemical Characterization of the HydE and HydG iron-Only Hydrogenase Maturation Enzymes from Thermotoga maritima. FEBS Lett. 2005, 579 (22), 5055–5060. 73. Dinis, P.; Suess, D. L. M.; Fox, S. J.; Harmer, J. E.; Driesener, R. C.; De La Paz, L.; Swartz, J. R.; Essex, J. W.; Britt, R. D.; Roach, P. L. X-Ray Crystallographic and EPR Spectroscopic Analysis of HydG, a Maturase in [FeFe]-Hydrogenase H-Cluster Assembly. P Natl Acad Sci USA 2015, 112 (5), 1362–1367. 74. Suess, D. L. M.; Burstel, I.; De La Paz, L.; Kuchenreuther, J. M.; Pham, C. C.; Cramer, S. P.; Swartz, J. R.; Britt, R. D. Cysteine as a Ligand Platform in the Biosynthesis of the FeFe Hydrogenase H Cluster. P Natl Acad Sci USA 2015, 112 (37), 11455–11460. 75. Kriek, M.; Martins, F.; Challand, M. R.; Croft, A.; Roach, P. L. Thiamine Biosynthesis in Escherichia coli: Identification of the Intermediate and by-Product Derived from Tyrosine. Angew. Chem. Int. Ed. Engl. 2007, 46 (48), 9223–9226. 76. Pilet, E.; Nicolet, Y.; Mathevon, C.; Douki, T.; Fontecilla-Camps, J. C.; Fontecave, M. The Role of the Maturase HydG in [FeFe]-Hydrogenase Active Site Synthesis and Assembly. FEBS Lett. 2009, 583 (3), 506–511. 77. Driesener, R. C.; Challand, M. R.; McGlynn, S. E.; Shepard, E. M.; Boyd, E. S.; Broderick, J. B.; Peters, J. W.; Roach, P. L. [FeFe]-Hydrogenase Cyanide Ligands Derived from S-Adenosylmethionine-Dependent Cleavage of Tyrosine. Angew. Chem. Int. Ed. 2010, 49 (9), 1687–1690. 78. Shepard, E. M.; Duffus, B. R.; George, S. J.; McGlynn, S. E.; Challand, M. R.; Swanson, K. D.; Roach, P. L.; Cramer, S. P.; Peters, J. W.; Broderick, J. B. [FeFe]-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide. J. Am. Chem. Soc. 2010, 132 (27), 9247–9249. 79. Kuchenreuther, J. M.; George, S. J.; Grady-Smith, C. S.; Cramer, S. P.; Swartz, J. R. Cell-Free H-Cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN() Ligands Derive from Tyrosine. PLoS One 2011, 6 (5), e20346. 80. Kuchenreuther, J. M.; Myers, W. K.; Stich, T. A.; George, S. J.; NejatyJahromy, Y.; Swartz, J. R.; Britt, R. D. A Radical Intermediate in Tyrosine Scission to the CO and CNLigands of FeFe Hydrogenase. Science 2013, 342 (6157), 472–475. 81. Suess, D. L.; Britt, R. D. EPR Spectroscopic Studies of [FeFe]-Hydrogenase Maturation. Catal. Lett. 2015, 58 (12), 699–707. 82. Duffus, B. R.; Ghose, S.; Peters, J. W.; Broderick, J. B. Reversible H Atom Abstraction Catalyzed by the Radical S-Adenosylmethionine Enzyme HydG. J. Am. Chem. Soc. 2014, 136 (38), 13086–13089. 83. Stich, T. A.; Myers, W. K.; Britt, R. D. Paramagnetic Intermediates Generated by Radical S-Adenosylmethionine (SAM) Enzymes. Acc. Chem. Res. 2014, 47 (8), 2235–2243. 84. Suess, D. L.; Kuchenreuther, J. M.; De La Paz, L.; Swartz, J. R.; Britt, R. D. Biosynthesis of the [FeFe] Hydrogenase H Cluster: A Central Role for the Radical SAM Enzyme HydG. Inorg. Chem. 2016, 55 (2), 478–487. 85. Nicolet, Y.; Pagnier, A.; Zeppieri, L.; Martin, L.; Amara, P.; Fontecilla-Camps, J. C. Crystal Structure of HydG from Carboxydothermus hydrogenoformans: A Trifunctional [FeFe]-Hydrogenase Maturase. ChemBioChem 2015, 16 (3), 397–402. 86. Nicolet, Y.; Martin, L.; Tron, C.; Fontecilla-Camps, J. C. A Glycyl Free Radical as the Precursor in the Synthesis of Carbon Monoxide and Cyanide by the [FeFe]-Hydrogenase Maturase HydG. FEBS Lett. 2010, 584 (19), 4197–4202. 87. Pagnier, A.; Martin, L.; Zeppieri, L.; Nicolet, Y.; Fontecilla-Camps, J. C. CO and CN- Syntheses by [FeFe]-Hydrogenase Maturase HydG Are Catalytically Differentiated Events. Proc. Natl. Acad. Sci. U.S.A. 2016, 113 (1), 104–109. 88. Kuchenreuther, J. M.; Myers, W. K.; Suess, D. L.; Stich, T. A.; Pelmenschikov, V.; Shiigi, S. A.; Cramer, S. P.; Swartz, J. R.; Britt, R. D.; George, S. J. The HydG Enzyme Generates an Fe(CO)2(CN) Synthon in Assembly of the FeFe Hydrogenase H-Cluster. Science 2014, 343 (6169), 424–427.

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89. Rao, G. D.; Tao, L. Z.; Suess, D. L. M.; Britt, R. D. A [4Fe-4S]-Fe(CO)(CN)-L-Cysteine Intermediate Is the First Organometallic Precursor in [FeFe] Hydrogenase H-Cluster Bioassembly. Nat. Chem. 2018, 10 (5), 555–560. 90. Suess, D. L. M.; Pham, C. C.; Burstel, I.; Swartz, J. R.; Cramer, S. P.; Britt, R. D. The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster. J. Am. Chem. Soc. 2016, 138 (4), 1146–1149. 91. Rao, G.; Pattenaude, S. A.; Alwan, K.; Blackburn, N. J.; Britt, R. D.; Rauchfuss, T. B. The Binuclear Cluster of [FeFe] Hydrogenase Is Formed with Sulfur Donated by Cysteine of an [Fe(Cys)(CO)(2)(CN)] Organometallic Precursor. Proc. Natl. Acad. Sci. U.S.A. 2019, 116 (42), 20850–20855. 92. Sayler, R. I.; Stich, T. A.; Joshi, S.; Cooper, N.; Shaw, J. T.; Begley, T. P.; Tantillo, D. J.; Britt, R. D. Trapping and Electron Paramagnetic Resonance Characterization of the 5’ dAdo(Center Dot) Radical in a Radical S-Adenosyl Methionine Enzyme Reaction With a Non-Native Substrate. ACS Central Sci 2019, 5 (11), 1777–1785. 93. Nicolet, Y.; Rubach, J. K.; Posewitz, M. C.; Amara, P.; Mathevon, C.; Atta, M.; Fontecave, M.; Fontecilla-Camps, J. C. X-Ray Structure of the [FeFe]-Hydrogenase Maturase HydE From Thermotoga maritima. J. Biol. Chem. 2008, 283 (27), 18861–18872. 94. Berkovitch, F.; Nicolet, Y.; Wan, J. T.; Jarrett, J. T.; Drennan, C. L. Crystal Structure of Biotin Synthase, an S-Adenosylmethionine-Dependent Radical Enzyme. Science 2004, 303 (5654), 76–79. 95. Nicolet, Y.; Rohac, R.; Martin, L.; Fontecilla-Camps, J. C. X-Ray Snapshots of Possible Intermediates in the Time Course of Synthesis and Degradation of Protein-Bound Fe4S4 Clusters. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (18), 7188–7192. 96. Rohac, R.; Amara, P.; Benjdia, A.; Martin, L.; Ruffie, P.; Favier, A.; Berteau, O.; Mouesca, J. M.; Fontecilla-Camps, J. C.; Nicolet, Y. Carbon-Sulfur Bond-Forming Reaction Catalysed by the Radical SAM Enzyme HydE. Nat. Chem. 2016, 8 (5), 491–500. 97. Mahanta, N.; Fedoseyenko, D.; Dairi, T.; Begley, T. P. Menaquinone Biosynthesis: Formation of Aminofutalosine Requires a Unique Radical SAM Enzyme. J. Am. Chem. Soc. 2013, 135 (41), 15318–15321. 98. Sato, S.; Kudo, F.; Rohmer, M.; Eguchi, T. Characterization of Radical SAM Adenosylhopane Synthase, HpnH, which Catalyzes the 5 ’-Deoxyadenosyl Radical Addition to Diploptene in the Biosynthesis of C-35 Bacteriohopanepolyols. Angew. Chem. Int. Ed. 2020, 59 (1), 237–241. 99. Tao, L. Z.; Pattenaude, S. A.; Joshi, S.; Begley, T. P.; Rauchfuss, T. B.; Britt, R. D. Radical SAM Enzyme HydE Generates Adenosylated Fe(I) Intermediates En Route to the [FeFe]-Hydrogenase Catalytic H-Cluster. J. Am. Chem. Soc. 2020, 142 (24), 10841–10848. 100. Gilbert-Wilson, R.; Siebel, J. F.; Adamska-Venkatesh, A.; Pham, C. C.; Reijerse, E.; Wang, H. X.; Cramer, S. P.; Lubitz, W.; Rauchfuss, T. B. Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [Fe-57(2)(Adt)(CN)(2)(CO)(4)](2-). J. Am. Chem. Soc. 2015, 137 (28), 8998–9005. 101. Li, Y. L.; Rauchfuss, T. B. Synthesis of Diiron(I) Dithiolato Carbonyl Complexes. Chem. Rev. 2016, 116 (12), 7043–7077. 102. Rao, G. D.; Tao, L. Z.; Britt, R. D. Serine Is the Molecular Source of the NH(CH2)(2) Bridgehead Moiety of the In Vitro Assembled [FeFe] Hydrogenase H-Cluster. Chem. Sci. 2020, 11 (5), 1241–1247. 103. Adamska-Venkatesh, A.; Roy, S.; Siebel, J. F.; Simmons, T. R.; Fontecave, M.; Artero, V.; Reijerse, E.; Lubitz, W. Spectroscopic Characterization of the Bridging Amine in the Active Site of [FeFe] Hydrogenase Using Lsotopologues of the H-Cluster. J. Am. Chem. Soc. 2015, 137 (40), 12744–12747. 104. Reijerse, E. J.; Pelmenschikov, V.; Birrell, J. A.; Richers, C. P.; Kaupp, M.; Rauchfuss, T. B.; Cramer, S. P.; Lubitz, W. Asymmetry in the Ligand Coordination Sphere of the [FeFe] Hydrogenase Active Site Is Reflected in the Magnetic Spin Interactions of the Aza-Propanedithiolate Ligand. J Phys Chem Lett 2019, 10 (21), 6794–6799. 105. Shepard, E. M.; McGlynn, S. E.; Bueling, A. L.; Grady-Smith, C. S.; George, S. J.; Winslow, M. A.; Cramer, S. P.; Peters, J. W.; Broderick, J. B. Synthesis of the 2Fe Subcluster of the [FeFe]-Hydrogenase H Cluster on the HydF Scaffold. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (23), 10448–10453. 106. Czech, I.; Silakov, A.; Lubitz, W.; Happe, T. The [FeFe]-Hydrogenase Maturase HydF from Clostridium acetobutylicum Contains a CO and CN- Ligated iron Cofactor. FEBS Lett. 2010, 584 (3), 638–642. 107. Czech, I.; Stripp, S.; Sanganas, O.; Leidel, N.; Happe, T.; Haumann, M. The [FeFe]-Hydrogenase Maturation Protein HydF Contains a H-Cluster like [4Fe4S]-2Fe Site. FEBS Lett. 2011, 585 (1), 225–230. 108. Caserta, G.; Pecqueur, L.; Adamska-Venkatesh, A.; Papini, C.; Roy, S.; Artero, V.; Atta, M.; Reijerse, E.; Lubitz, W.; Fontecave, M. Structural and Functional Characterization of the Hydrogenase-Maturation HydF Protein. Nat. Chem. Biol. 2017, 13 (7), 779. 109. Brazzolotto, X.; Rubach, J. K.; Gaillard, J.; Gambarelli, S.; Atta, M.; Fontecave, M. The [Fe-Fe]-Hydrogenase Maturation Protein HydF From Thermotoga maritima Is a GTPase With an Iron-Sulfur Cluster. J. Biol. Chem. 2006, 281 (2), 769–774. 110. Cendron, L.; Berto, P.; D’Adamo, S.; Vallese, F.; Govoni, C.; Posewitz, M. C.; Giacometti, G. M.; Costantini, P.; Zanotti, G. Crystal Structure of HydF Scaffold Protein Provides Insights into [FeFe]-Hydrogenase Maturation. J. Biol. Chem. 2011, 286 (51), 43944–43950. 111. Berto, P.; Di Valentin, M.; Cendron, L.; Vallese, F.; Albertini, M.; Salvadori, E.; Giacometti, G. M.; Carbonera, D.; Costantini, P. The [4Fe-4S]-Cluster Coordination of [FeFe]Hydrogenase Maturation Protein HydF as Revealed by EPR and HYSCORE Spectroscopies. BBA-Bioenergetics 2012, 1817 (12), 2149–2157. 112. Lee, M.; Grawert, T.; Quitterer, F.; Rohdich, F.; Eppinger, J.; Eisenreich, W.; Bacher, A.; Groll, M. Biosynthesis of Isoprenoids: Crystal Structure of the [4Fe-4S] Cluster Protein IspG. J. Mol. Biol. 2010, 404, 600–610. 113. Berggren, G.; Garcia-Serres, R.; Brazzolotto, X.; Clemancey, M.; Gambarelli, S.; Atta, M.; Latour, J. M.; Hernandez, H. L.; Subramanian, S.; Johnson, M. K.; Fontecave, M. An EPR/HYSCORE, Mossbauer, and Resonance Raman Study of the Hydrogenase Maturation Enzyme HydF: A Model for N-Coordination to [4Fe-4S] Clusters. J. Biol. Inorg. Chem. 2014, 19 (1), 75–84. 114. Asada, Y.; Koike, Y.; Schnackenberg, J.; Miyake, M.; Uemura, I.; Miyake, J. Heterologous Expression of Clostridial Hydrogenase in the Cyanobacterium Synechococcus PCC7942. Bba-Gene Struct. Expr. 2000, 1490 (3), 269–278. 115. Gorwa, M. F.; Croux, C.; Soucaille, P. Molecular Characterization and Transcriptional Analysis of the Putative Hydrogenase Gene of Clostridium acetobutylicum ATCC 824. J. Bacteriol. 1996, 178 (9), 2668–2675. 116. Baussier, C.; Fakroun, S.; Aubert, C.; Dubrac, S.; Mandin, P.; Py, B.; Barras, F. Making iron-Sulfur Cluster: Structure, Regulation and Evolution of the Bacterial ISC System. Adv. Microb. Physiol. 2020, 76, 1–39. 117. Fontecave, M.; de Choudens, S. O.; Py, B.; Barras, F. Mechanisms of iron-Sulfur Cluster Assembly: The SUF Machinery. J. Biol. Inorg. Chem. 2005, 10 (7), 713–721. 118. Papini, C.; Sommer, C.; Pecqueur, L.; Pramanik, D.; Roy, S.; Reijerse, E. J.; Wittkamp, F.; Artero, V.; Lubitz, W.; Fontecave, M. Bioinspired Artificial [FeFe]-Hydrogenase with a Synthetic H-Cluster. ACS Catal. 2019, 9 (5), 4495–4501. 119. Caserta, G.; Roy, S.; Atta, M.; Artero, V.; Fontecave, M. 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2.08

Heme-containing proteins: Structures, functions, and engineering

Osami Shojia, Yuichiro Aibaa, Shinya Ariyasua, and Hiroki Onodab, a Department of Chemistry, Graduate School of Science, Nagoya University, Nagoya, Japan; and b Synchrotron Radiation Research Center, Nagoya University, Nagoya, Japan © 2023 Elsevier Ltd. All rights reserved.

2.08.1 2.08.1.1 2.08.1.2 2.08.1.3 2.08.2 2.08.2.1 2.08.2.2 2.08.3 References

Myoglobin Heme analogs with a different central metal or modified side chain Porphyrinoids with modified heme (porphyrin) skeleton Metal complexes other than porphyrins and porphyrinoids Cytochrome P450 Cytochrome P450s catalyzing monooxygenation Cytochrome P450s catalyzing peroxygenase Heme acquisition protein

195 195 197 198 198 198 204 208 211

Abstract Hemoproteins, metalloproteins containing iron-protoporphyrin IX, are essential for most living organisms. Heme serves as a prosthetic group of hemoproteins, and all hemoproteins lost their function without heme. Accordingly, the function of hemoproteins is highly dependent on the nature of iron-protoporphyrin IX and amino acids located around the heme. This chapter will mainly introduce the structure and engineering of hemeproteins focusing on myoglobin, cytochrome P450, and heme acquisition protein, wherein three approaches for the development of artificial hemeproteins, site-directed and random mutagenesis, heme substitution, and utilization of native substrate mimics (decoy molecules) are briefly covered herein.

Iron is an essential element for almost all living organisms as it serves as a catalytic center of many enzymes. Heme (ironprotoporphyrin IX) is widely used throughout the biosphere and is thus one of the essential metal complexes. It serves as a prosthetic group of hemoproteins that perform diverse functions such as oxygen storage and transport, gas sensing, electron transfer, iron acquisition, and catalysis. Construction of artificial hemeproteins and application of hemeproteins are reviewed in this chapter focusing on the structure and engineering of widely studied hemoproteins, myoglobin, cytochrome P450, and heme acquisition protein.

Fig. 1

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Structure of horse heart myoglobin (PDB: 1WLA).

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Myoglobin

Myoglobin, one of the globular proteins called globin, was the first protein whose structure was solved by X-ray crystallography (Fig. 1).1 This was a significant milestone in protein research, making it possible to discuss the structure at the atomic level. Since then, it has been widely studied as a representative hemoprotein. Unlike the tetrameric hemoglobin, which is also an oxygenbinding protein, myoglobin exists as a monomer and is composed of a single polypeptide chain of 153 amino acids forming eight a-helices, surrounding a heme (iron-porphyrin metal complex), with the imidazole of His coordinated to the heme. The fact that myoglobin is a small protein, which allows for easy recombinant overexpression and crystallization, makes myoglobin a model hemoprotein for protein functionalization studies. The original function of myoglobin is to bind and store oxygen,2 but proteins in the same hemoprotein category have a wide range of functions and play a variety of roles such as electron transfer, catalysis, oxygen transport, heme storage, and sensors. Although these proteins share the typical structure of having heme as a cofactor, they are specialized for specific functions, indicating that the fine structure of the protein (including the second coordination sphere) has a significant influence on its function. In understanding this interesting relationship between protein structure and function, small and stable myoglobin has been a suitable scaffold for various protein modifications and designs. Therefore, myoglobin has been studied from multiple angles, and there are three major strategies to modify myoglobin3–5 (Fig. 2): (1) introduction of mutations by specific amino acid conversion using genetic engineering methods6; (2) chemical modification of specific amino acid residues (especially on the protein surface)7; (3) replacement of heme to non-natural synthetic metal complexes.3,4 Among these, the third “heme substitution” can most drastically modify the function of myoglobin, and thus the details of heme substitution are described hereafter. A reconstituted protein can be obtained by substitution of heme with a heme analog or synthetic metal complex (Fig. 3). To get heme-substituted myoglobin, it is first necessary to prepare apo myoglobin that does not have heme. Myoglobin can be treated under acidic conditions to obtain the apo form, taking advantage of its small and stable structure. Namely, heme can be removed from myoglobin by treating myoglobin with an acidic solution of 0.1N HCl and then extracting heme with an organic solvent such as 2-butanone.8 The UV–Vis spectrum can be used to confirm that it does not contain heme. The aqueous layer is then neutralized, and apo myoglobin is used in subsequent experiments. This is because the heme in myoglobin is heme b, which is bound to the heme pocket by non-covalent interactions and can be easily removed. In some cases, the technique to stop heme biosynthesis in the iron-limited minimal medium during E. coli expression is also used.5,9,10 Various heme-substituted myoglobins have been reported using such reconstitution methods and can be categorized into the following three types depending on the synthetic metal complex used (Fig. 4): (1) heme analogs with a different central metal or modified side chain, (2) porphyrinoids with a modified heme (porphyrin) skeleton, and (3) metal complexes with structures completely different from porphyrins and porphyrinoids. These three types of heme-substituted myoglobins are introduced as follows.

2.08.1.1

Heme analogs with a different central metal or modified side chain

By dropping metal complexes onto the aforementioned apo myoglobin, myoglobins reconstituted with metal substituents and heme analogs with relatively similar structures such as mesoporphyrins have been reported. Protoporphyrin IX (PPIX) with various central metals such as Co, Cu, Mn, and Ag, which are metal complexes with almost the same outline as heme, have been examined in heme-substituted myoglobin.10,11 For example, myoglobins reconstituted with manganese PPIX (Mn-PPIX) were investigated for their peroxidase activity and the effect of distal histidine through mutagenesis.11 Recently, carbene insertion catalysts using

Fig. 2

Three major myoglobin functionalization methods.

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Fig. 3

Heme-containing proteins: Structures, functions, and engineering

Outline of heme substitution method.

metalloproteins have also been investigated intensively.5,10,12,13 The cyclopropanes produced by this reaction are important structural motifs found in drug molecules. Since the cyclopropane contains up to three optically active sites, there are very complex stereoisomers, and enzymes’ selective synthesis attracts attention. To address this demand, efficient mutant searches have been conducted using PPIX and mesoporphyrin IX (MPIX)-myoglobins with Co, Cu, Mn, Ag, Rh, Ru, and Ir as the central metal (Fig. 5).10 In addition to these metal complexes, substitutions with chemically modified hemes via propionic acid have also been reported.14–16 As an example, studies were conducted using modified heme, in which multiple carboxy and amino groups were introduced using chemical modification via propionic acid. Even these porphyrins, which have a significantly different charge from the original heme, are incorporated into the heme pocket. Through these modifications, cationic or anionic clusters are introduced on the myoglobin, changing the interaction properties of the methyl viologen dication and anthraquinone-2,7-disulfonate, and attempts were made to gain insight into biological electron transfer.14 Another example is the heme in which a maleimide group is introduced through a short PEG linker. Using this modified heme in combination with a myoglobin mutant (A125C), the construction of a linear protein polymer has been reported as a result of the conjugation reaction between the thiol and maleimide groups (Fig. 6).

Fig. 4

Metal complexes for heme-substituted myoglobins.

Heme-containing proteins: Structures, functions, and engineering

197

Fig. 5 (a) The active site of myoglobin. Some of the residues targeted for mutagenesis are highlighted in red (His64, Val68, and His93).10,13 (b) Cyclopropanation of styrene with ethyl diazoacetate by the myoglobin-based catalyst.

More specifically, by reacting the modified heme with the holo myoglobin mutant, followed by removal of the original heme, a polymer is formed as the modified heme bond to the myoglobin is incorporated in the heme pocket of another myoglobin.16

2.08.1.2

Porphyrinoids with modified heme (porphyrin) skeleton

In addition to heme-like synthetic metal complexes, porphyrinoids with different skeletons have been investigated.3 Porphycene,17,18 a structural isomer of porphyrin with high symmetry, and corrole,19 a porphyrinoid with one less meso carbon, with a central metal such as Mn, Co, or Ni in addition to Fe, have been reported to exhibit functions different from those of the original

Fig. 6

Protein supramolecular polymer by using the modified heme in combination with a myoglobin mutant (A125C).

198

Heme-containing proteins: Structures, functions, and engineering

myoglobin. Myoglobin reconstituted with Fe-porphycenes showed high oxygen-binding ability, peroxidase and cyclopropanation activities.17 Furthermore, hydroxylation reaction has been reported for Mn-porphycene (Fig. 7).18 Myoglobin reconstituted Cotetradehydrocorrin has also been examined as a model for cobalamin-dependent methionine synthase.20

2.08.1.3

Metal complexes other than porphyrins and porphyrinoids

The synthetic metal complexes mentioned above have porphyrin or porphyrinoid frameworks and are somewhat similar to heme, the original cofactor of myoglobin. In addition, reconstitution of myoglobins with synthetic metal complexes of completely different structures from porphyrins and porphyrinoids has also been reported. Reconstitution of myoglobins with Fe, Cr, Mn, and Co salen/salophen complexes has been achieved.21–23 These complexes were incorporated into the heme-binding pocket of myoglobin. Moreover, the structure of the salophen-myoglobin complex has been confirmed by X-ray crystal structure analysis (Fig. 8).22,23 Myoglobins reconstituted with these non-porphyrin (oid) complexes are useful for catalysis, and sulfoxidation reactions have been achieved.

2.08.2

Cytochrome P450

2.08.2.1

Cytochrome P450s catalyzing monooxygenation

Cytochrome P450 enzymes (P450s) are ubiquitous enzymes in a broad range of organisms, which have the ability of oxidative chemical transformations for biosynthesis, metabolism, and drug detoxification. P450s can insert one oxygen atom into inactivated C–H bonds in substrates (hydroxylation) through reductive activation of molecular oxygen under physiological conditions. In addition, some P450s catalyze other reactions such as epoxidation, sulfoxidation, hydroxylation-mediated demethylation, decarboxylation, and so on (Fig. 9).24–28 Based on the excellent catalytic performances of P450s, P450s can be regarded as a biocatalyst for oxyfunctionalization of substrates.29,30 The catalytic cycle of hydroxylation by P450s is shown in Fig. 10. The resting state of P450s must be activated by binding of appropriate substrates and subsequent elimination of axially coordinated water ligands on the heme. Subsequently, reactive oxygen intermediate (compound I) is generated through reductive activation of molecular oxygen. This intermediate can activate inert C–H bonds in substrates.31 Substrate binding is the essential switch to start this catalytic cycle. Because each P450s has a unique shape of substrate binding pocket, P450s can accept individual substrates having suitable structures toward P450s pockets, resulting in substrate specificities of P450s. This property is essential to appropriately utilize extremely high reactive compound I in complex living organisms. However, the substrate specificities of P450s also hamper industrial application of P450s by limitation of available substrates. It is one of the most important motivations of researchers for engineering of P450s to overcome their substrate specificities. In P450’s catalytic cycle, two electrons are needed to generate compound I. These electrons are typically supplied from NAD(P)H as electron donors through several reductase enzymes, meaning that catalytic reactions of P450s are basically required both P450s and corresponding reductases. On the other hand, some P450s such as the CYP102 subfamily contain reductase domains in their sequence, leading to efficient intramolecular electron transfer from NAD(P)H to P450 domains (Fig. 11). Among the CYP102 series, P450BM3 (CYP102A1) has been attracted as biocatalysts for industrial application because P450BM3 exhibits extremely high hydroxylation efficiency toward long fatty acids.32 In this review, we mainly focused on P450BM3 and describe engineering strategy of P450s such as mutagenesis including directed evolution, heme substitution with artificial cofactors, and use of substrate mimics, decoy molecules (Fig. 12).

Fig. 7 Superimposed structures of Fe-porphycene-myoglobin (cyan; PDB 2D6C) with Mn-porphycene-myoglobin (magenta; PDB 3WI8).18 Structures of Fe-porphycene-myoglobin and Mn-porphycene-myoglobin are similar to each other.

Heme-containing proteins: Structures, functions, and engineering

Fig. 8

199

Crystal structure of Fe-salophen incorporated in myoglobin mutant (A71G: PDB 1UFJ).22

Mutagenesis including directed evolution is the established conventional engineering strategies of not only P450s, but also all proteins. At present, there are abundant reports of P450 variants for alteration of substrate specificity, enhancement of catalytic properties, changing stereo- or regioselectivities and improvement of thermal stabilities.33 When crystal structures of P450s of interest have been solved, rational design of mutants through site-directed mutagenesis or saturation mutagenesis are possible.34 For example, mutation of Arg47 in P450BM3, which is a key residue of recognition of carboxyl groups in long fatty acids, enable to change their substrate specificities toward non-carboxyl molecules.35 Mutation of amino acid residues close to Cys providing axial ligand of heme leads to critical effect on catalytic properties of P450s. For example, P450BM3 I401P mutant exhibited improved catalytic properties due to negative shift of the reduction potential of the heme.36 Site-directed mutation is also an effective method to introduce anchors for chemical modification of P450s. Cheruzel et al. reported introduction of a Cys residue by sitedirected mutation and anchoring ruthenium complexes as photosensitizers to create photo-driven P450BM3 (Fig. 13b).38,41 Even if crystal structures of P450s are not available, directed evolution based on random mutagenesis are promising way. As one of the most successful stories of directed evolution of P450BM3 is development of P450PMO by Prof. Arnold group (Fig. 13c) .39,40 They repeatedly performed various mutagenetic methods including random mutation, DNA shuffling, site-saturation mutation, site-directed mutation and so on toward P450BM3 to change their substrate specificity from long fatty acids to small alkanes, and they finally obtained P450PMO (propane monooxygenase) that can hydroxylate propane efficiently. Mutagenesis is promising strategies for not only P450BM3 but also other P450s. Prof. Wong group has reported ethane hydroxylation by highly engineered P450cam variants (Fig. 13a).37 At present, various enzymatic transformations including tandem enzymatic reactions, total synthesis of complex natural products and so on have been achieved by mutagenesis of P450s. Alteration of catalytic performance of P450s by mutagenesis is mainly attributed to rearrangement of shape of substrate pocket in each P450s. Different from mutagenesis, our group has developed a unique strategy for alteration of substrate specificities of P450s by just addition of substrate analogues that possess shorter chain length than native substrates, which are called to “decoy molecules” (Fig. 14a).45 Decoy molecules can be misrecognized as native substrate by P450s. However, small space should be

Fig. 9

Representative reactions catalyzed by P450s.

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Heme-containing proteins: Structures, functions, and engineering

Fig. 10

Catalytic cycle of P450.

remained around heme because of short chain of decoy molecules. Such space can be used as artificial substrate pocket, contributing to hydroxylation of small substrates. At beginning, decoy molecules for H2O2-dependent P450s (peroxygenases) had been developed.46–48 Inspired by such initial decoy molecules, our group and Prof Reetz group independently discovered perfluorinated carboxylic acids (PFCs) can be responded as decoy molecules by wild-type P450BM3 without any mutations, resulting in achievement of hydroxylation of small hydrocarbons such as cyclohexane and benzene (Fig. 14b).42,49 However, catalytic efficiencies in hydroxylation of non-native substrates were not so sufficient compared with those in hydroxylation of long fatty acids because of insufficient binding affinities of PFCs against P450BM3. Such weak binding properties of PFCs (1st generation decoys) have been solved by simple conjugation of amino acid moieties. Expectedly, amino acid linked PFCs (2nd generation decoys) exhibits sufficiently strong binding affinities, responding to improvement of catalytic performance in hydroxylation of cyclohexane and propane. In the crystal structure of P450BM3 with PFC9-Trp, the Trp moiety is tightly fixed at entrance of substrate pocket of

Fig. 11

Structure of the heme domain and FMN domain of P450BM3 (PDB code: 1BVY) and FAD domain (PDB code: 4DQK).

Heme-containing proteins: Structures, functions, and engineering

Fig. 12

201

Representative approaches of engineering of P450s.

P450BM3 through several hydrogen-bonding interactions.50 This crystal structures suggest that decoy molecules containing amino acid moieties are hardly attacked by compound I. Based on this hypothesis, non-fluorinated decoy molecules (3rd generation decoys) were developed. As expected, simple amino acid-linked fatty acids such as C9-Phe can also work as decoy molecules toward P450BM3 and more effectively induce hydroxylation of benzene than corresponding 2nd generation decoys such as PFC9-Phe.43

Fig. 13 Representative examples of P450s engineered via mutagenesis. (a) Ethane hydroxylation by P450cam variants.37 (b) Photo-driven hydroxylation of long fatty acids by P450BM3 heme domains bearing Ru photosensitizers.38 (c) Directed evolution of wild-type P450BM3 to P450PMO.39,40

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Heme-containing proteins: Structures, functions, and engineering

Fig. 14 Schematic images of substrate-misrecognition system using decoy molecules (a) Schematic image of the proposed mechanism of the substrate-misrecognition system. (b) History of the evolution of decoy molecules for the hydroxylation of benzene.42–44

More interestingly, not only simple fatty acids-amino acids conjugates, but also N-substituted dipeptides including C7-Pro-Phe, whose structures are totally different from those of native substrates, can be used as decoy molecules. In addition, their catalytic efficiencies of benzene hydroxylation were higher than those by simple 3rd generation decoys such as C9-Phe. Compared with 2nd generation decoys, 3rd generation decoys can be easily synthesized, and the variation of their subunits is abundant, suggesting that extremely broad range of synthetic and/or natural carboxylates has potential of decoy molecules or their subunits. Because the shape of artificial reaction space reconstructed by decoy molecules in P450BM3 must be influenced by chemical structures of decoy molecules, substrate specificity, catalytic efficiency and regio
/stereo-selectivity of oxyfunctionalization of non-native substrates by P450BM3 can be controlled by just changing decoy molecules. To find optimal decoy molecules from vast area of possible candidates of decoy molecules, it was performed stepwise and fragment optimization (chemical evolution) of N-substituted dipeptides that can be considered as combination of three substructures that are N-substituents and two amino acids. Combining with combinatorial peptide synthetic techniques, several new 3rd generation decoy molecules from the range of ca. 100,000 structural diversities was found through just ca. 600 trials. The discovered decoy molecules exhibited promising catalytic efficiencies in benzene hydroxylation (turnover frequency (TOF): 405 min
1).44 Furthermore, by using HPLC-based high pressure reactor, P450BM3 with decoy molecules can efficiently hydroxylate propane and ethane.51 The TOF values reached 2200 min
1 for propane, and 83 min
1 for ethane, respectively, which are fastest values in all reported P450 chemistry. Because of structural diversity of 3rd generation decoys, suitable decoy molecules can be easily selected. Control of stereoselectivity in hydroxylation of indene by P450BM3 was also achieved by 3rd generation decoy molecules.52 In addition, some decoy molecules can uptake by bacterial cells and activate P450BM3 in E. coli to achieve whole-cell reaction including benzene hydroxylation.53 Different from reaction by purified P450BM3, catalytic efficiencies in whole-cell reaction should be affected by uptake-efficiency of decoy molecules into E. coli cells. To overcome this limitation, engineered outer membrane protein F (OmpF) that involve uptake of decoy molecules at outer membrane of E. coli was developed and succeeded in broadening acceptability of various decoy molecules in whole-cell

Heme-containing proteins: Structures, functions, and engineering

203

Fig. 15 Representative examples using decoy molecules. (a) Whole-cell biotransformation of benzene to phenol via the substrate-misrecognition system in E. coli.54 (b) Enantioselective cyclopropanation of styrene by P450BM3 variants with decoy molecules.55

transformations (Fig. 15a).54 Apart from viewpoints of engineering of P450s, it was recently reported that one of decoy molecules, N-abietoyl–tryptophan (AbiA-Trp) can dramatically facilitate crystallization of P450BM3 with extremely high quality for X-ray crystallography, while AbiA-Trp does not work as decoy molecules. More importantly, microcrystal of P450BM3 with AbiA-Trp can be applied to cross-microseeding approach, which strongly contribute to structural analyses of interaction of P450BM3 and decoy molecules.56 One of the most important advantages of utilization of decoy molecules in viewpoints of P450’s engineering is applicability of wild-type P450s to non-native enzymatic conversions. However, this technique can be also adopted to P450 mutants and improvement of catalytic efficiencies of hydroxylation of non-native substrates by combination of P450BM3 mutants and decoy molecules was reported.57 In addition, this hybrid engineering strategy of P450s are effective for not only hydroxylation reaction but also non-native enzymatic reactions such as carbene insertions (Fig. 15b).55 Different from the mutagenesis approach, decoy molecules can more easily provide non-canonical functional groups around the catalytic center of P450s, expecting more flexible redesigning reaction environment in P450s to realize desired functions. Although above two engineering strategies, mainly reshaping of substrate pockets of P450s, are one of effective ways for changing substrate specificity, catalytic efficiency and regio/stereo selectivity of P450s, heme substitution is expected to drastically change catalytic properties of P450s. Heme substitution strategies have been extensively studied in various hemeproteins such as myoglobin and cytochrome b to construct artificial metal enzymes.3 On the other hand, examples of heme substitution in P450s are limited,58 possibly due to their heme binding sites located deep inside of the protein. Although treatment with acid/ organic solvent is established way for removal of heme from hemeproteins, such harsh conditions cause to irreversible denaturation of P450s. To avoid harsh conditions, an alternative method of heme proteins using apo-myoglobin as a scavenger of heme from P450s was developed.59 In addition, it was also reported that the incorporation of non-native heme analogue such as Mn PPIX (PPIX: protoporphyrin IX) into heme domain of P450BM3 by culturing E. coli cells under iron-limiting condition (Fig. 16a).9 By using this heme substitution strategies, methyliridium mesoporphyrin (Ir(Me)PPIX) containing CYP119 (Ir(Me)PPIXCYP119), which is thermophilic P450 from Sulfolobus solfataricus, was successfully constructed. The protein matrix of CYP119 can provide an enantioselective environment around the iridium complex to achieve enantioselective amination of C–H bonds (Fig. 16b).60,61 Because P450BM3 consists of two domains, P450 and reductase domains, heme-substitution of full-length P450BM3 had not been achieved. However, because preparation of heme-substituted heme domain of P450BM3 was possible, Mn-PPIX containing full-length P450BM3 was successfully constructed by post-ligation between MnPPIX containing heme domain and reductase domains which were separately expressed and purified in advance. Full-length Mn-PPIX-P450BM3 can also show abilities of hydroxylation of substrate using molecular oxygen as oxidant.9 Because the impact on catalytic performance of P450s by heme-substitution must be more drastic, heme substitution of P450s has a considerable potential to improve the catalytic performance of P450s.

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Heme-containing proteins: Structures, functions, and engineering

Fig. 16 Reported heme-substitution methods for P450s. (a) Incorporation of MnPPIX into wild-type P450BM3h (heme domain) cultured in ironlimiting medium.9 (b) Non-native chemical transformation by Ir(Me)PPIX-CYP119.60

2.08.2.2

Cytochrome P450s catalyzing peroxygenase

Monooxygenation reaction by P450s consumes two electrons supplied with NAD(P)H for the generation of actives species, Compound I (Fig. 9). NAD(P)H is capable of being exchanged by available energy currency in vivo. However, these reagents are not readily available at a low price. Cytochrome P450s possess shunt pathway utilizing hydrogen peroxide, which is two electron reduced molecular oxygen or two electrons oxidized water. In modern times, hydrogen peroxide is commercially produced from molecular oxygen and molecular hydrogen in the presence of anthraquinone as catalyst. Recently hydrogen peroxide can be generated from water and molecular oxygen by photoelectrocatalyst62 and electrode.63 Peroxygenase reactions have high expectation for

Fig. 17

Fatty acid hydroxylation mechanism of CYP152 peroxygenases.

Heme-containing proteins: Structures, functions, and engineering

205

the bioreaction because of low environmental loading. P450 monooxygenases were not stable under the hydrogen peroxide, although the shunt pathway requires excess amount of hydrogen peroxide.64 CYP152A1 (P450BSb)65 and CYP152B1 (P450SPa)66 utilize hydrogen peroxide for the oxidation reaction.67 CYP152A1 has a high affinity to hydrogen peroxide compared with other peroxides such as cumene hydroperoxide.68 CYP152 family enzymes exclusively catalyze the oxidation of long-chain fatty acids66 and classified as a fatty acid peroxygenase (EC. 1.11.2.4). The crystal structure of the palmitic acid bound-form of CYP152A169 suggests a unique catalytic mechanism (Fig. 17): (1) The catalytic reaction begins with the interaction of the terminal carboxylate of the long-chain fatty acid with Arg242 above the heme; (2) A general acid– base function of the fatty acid-Arg242 salt bridge allows the generation of the Compound I (heme oxyferryl porphyrin p-cation radical); (3) The long-chain fatty acid is oxidized by Compound I. In this reaction mechanisms, CYP152 family enzymes recognize the carboxylate of long-chain fatty acid, therefore, CYP152 family enzymes have high substrate specificity.66 This carboxylatearginine salt bridge is crucial for the utilization of hydrogen peroxide. In fact, a similar carboxylate-arginine salt bridge is observed in a peroxygenase from Agrocybe aegerita (AaeUPO)70 and a dye-decolorizing peroxidase from Auricularia auricula-judae (AauDyP)71 (Fig. 18). CYP152A1 catalyzes mixed selectivity (a:b ¼ 43:57) on fatty acid hydroxylation, although CYP152B1 selectively produces a-hydroxy fatty acid (a > 99%).72 The whole structures of CYP152A1 (PDBID: 1IZO)69 and CYP152B1 (PDBID: 3WSP)72 exhibit high structure similarly (Ca-RMSD ¼ 0.74 Å). However, the sequence similarity of these peroxygenases is only 44%. Several amino acids in the reaction field were not conserved between CYP152A1 and CYP152B1. Accordingly, the chimeras of CYP152A1 and CYP152B1, such as CYP152A1 G290F and CYP152B1 L87F were prepared.72 CYP152A1 G290F catalyzes a selective (a:b ¼ 95:5) hydroxylation in contrast to the wild type.72 There are over 30 CYP152 family enzymes73 and over 3500 preCYP152 family enzymes, which possess amino acid sequence identity of more than 40% toward CYP152A1 judged by BLAST.74 CYP152A2 (P450CLA) also catalyzes selectively to produce a-hydroxy fatty acid.75 a-keto acid76 and non-natural amino acid77 were produced from fatty acids under the cascade reaction using CYP152A2. Although CYP152 family enzymes had been considered to solely play a role in fatty acid hydroxylation reactions78 (Fig. 19a), the paradigm came in 2011 with the discovery of OleTJE (CYP152L1). CYP152L1 catalyzes terminal olefins biosynthesis from fatty acids.73,78 The terminal olefins are produced via b-decarboxylation reactions of fatty acids27,79 (Fig. 19b), which can be initiated by C–H abstraction80 in the same way as fatty acid hydroxylation by CYP152A1. The crystal structure of CYP152L1 (PDBID: 4L40) was high similarly to that of CYP152A1 and CYP152B1.73 At the same time as CYP152L1 discovery, it was reported that CYP152A1 also produced a trace amount of terminal olefin,78 while CYP152A2 cannot produce any terminal olefin.81 Alkenes are more ideal biofuel than bio-ethanol and bio-diesel (fatty acid methyl ester, FAME).82 Owing to the low peroxygenation activity of CYP152L1 (TOF < 10 min
1),78 NAD(P)H-dependent reductases and electron transfer proteins such as CamAB81 and RhFRED64 were used for the reductive activation of CYP152L1. Especially, an artificial fusion protein of CYP152L1 and BMR (Cytochrome P450BM3 reductase) produces 1-heptadedene 73% yield from octadecanoic acid.83 Under the optimized condition using catalase and re-generation system of NADPH, 2.5 g of heptadecane was generated from a 1 L reaction mixture.83 Alkenes production in the bacteria has also studied.64 However, most produced alkenes were long-chain alkenes owing to the bacterial abundance of fatty acid. CYP152N1 (P450Exa) produces shorter-alkyl-chain fatty acids, including tridecanoic acid from myristic acid, upon increasing of the hydrogen peroxide concentration. The shorter-alkyl-chain fatty acids was produced by the H2O2 clacking of a-keto acid, which is produced by overoxidation of a-hydroxy fatty acid. The overall reaction is a-oxidative decarboxylation. The alkyl chain length of yielding shorter-alkyl-chain fatty acids was controlled by the reaction time. The combination of a-oxidative decarboxylation and b-oxidative decarboxylation might be effective. CYP152A1 possesses mixed selectivity both of hydroxylation and decarboxylation at a- and b-position. Furthermore, CYP152K6 produces a,b-dihydroxydodecanoic acid. Intriguingly, 3- and d-

Fig. 18 Crystal structure of CYP152A1 (a, PDBID: 1IZO), AaeUPO (b, PDBID: 2YOR) and AauDyP (c: 4AU9). Proteins were shown as the cartoon model. Heme complex and several residues were shown as stick model.

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Heme-containing proteins: Structures, functions, and engineering

Fig. 19

Mechanism of fatty acid hydroxylation (a) and fatty acid decarboxylation (b) catalyzed by CYP152 family peroxygenases.

selective hydroxylation of trans-a,b-unsaturated fatty acids was catalyzed by CYP152L1, although g-selective hydroxylation proceeded for the oxidation of cis-a,b-unsaturated fatty acids. It was reported that CYP152P1 (CYP-MP),84 CYP152A8 (CYPAa162)85 and CYP152L2 (CYP-Sm46)85, CYP152AC1 (P450Ja)86 catalyzed fatty acid oxidation reactions. These results show that CYP152 family enzymes possess the potential to perform reactions other than fatty acid oxidation, however, the formation of Compound I had been strictly controlled by the recognition of carboxy group. CYP152 family enzymes such as CYP152A1, CYP152B1, CYP152N1 can catalyze over a thousand hydroxylation of fatty acid within 1 min using hydrogen peroxide.28,72 CYP152 family enzymes were regarded as possible candidates for biocatalysts for organic synthesis. However, CYP152A1 and CYP152B1 cannot oxidize non-native substrates.66 Recently, it was reported that

Fig. 20

Mechanism of epoxidation of styrene catalyzed by CYP152 family peroxygenases in the presence of short chain fatty acid (decoy molecule).

Heme-containing proteins: Structures, functions, and engineering Table 1

207

One electron oxidation rate of CYP152A1 in the presence of 20 mM short chain fatty acid as decoy molecule.87

TOF (min
1)

Peroxidation reaction 87

Sulfoxidation 46

Epoxidation 87

Benzylic hydroxylation 87

Aromatic hydroxylation 47

Fatty acid None Acetic acid (C2) Propionic acid (C3) Butanoic acid (C4) Pentatonic acid (C5) Hexanoic acid (C6) Heptanoic acid (C7) Octanoic acid (C8) Nonanoic acid (C9) Decanoic acid (C10) Myristic acid (C14)

Guaiacol N.D. N.D. 26 230 1900 2420 3750 2490 2380 2360 14

Thioanisole 24  2
270  20 260  80 270  50 390  50 510  70 250  20




Styrene N.D.

140  30 317  14 330  60 290  40 150  30




Ethylbenzene N.D.

11  1 20  2 24  2 28  4 10  3




1-MeO-Nap 1.7  0.2 8.0  0.3 11.6  0.5 10.8  0.3 34  2 61  3 103  2 76  3




CYP152A1 could catalyze the oxidation of non-native substrates such as guaiacol,87 thioanisole,46 styrene,88 ethylbenzene,87 and 1methoxy- naphthalene89 in the presence of n-alkyl carboxylic acids with a medium-chain length such as heptanoic acid and hexanoic acid (Fig. 20, Table 1). Furthermore, oxidation of various non-native substrates using wild-type CYP152A1 and CYP152B1 by simply increasing the concentration of acetate anion in the reaction buffer. The system using substrate mimics is called “substrate misrecognition system,” and these substrate mimics called “decoy molecules.” Cytochrome P450 peroxygenase such as CYP152A1 and CYP152B1 efficiently utilize H2O2 for the long-chain fatty acid oxidation. The peroxide-dependent oxidation reactions of fatty acid specific peroxygenases were activated only by the supplementation of carboxylate such as acetate anion.47 In general, cytochrome P450 consists of heme and 20 natural amino acids.90 Glutamic acid and aspartic acid, which are acidic amino acids, possess carboxylate at the side chain.91 It was reported that these carboxylate groups of the side chain also activate CYP152B1 for non-natural substrate oxidation.48 Especially, the glutamic acid residue close to the distal arginine (A245E) was the best for mutation to rebuild the carboxylate arginine salt bridge (Fig. 21a and b).48 It was reported that CYP152L1 P246D mutant also possesses the peroxygenation activity (Fig. 21c and d).92 These carboxylate rescue methods should be studied in several CYP152 family enzymes. In the case of most P450 monooxygenase the location of Glu245 of CYP152B1 A245E corresponds to a highly conserved threonine (Table 2), which is critical for the reductive activation of molecular oxygen. The typical exceptions of the conserved threonine were serine or alanine in bacterial P450 family enzymes. The side chain of serine possesses a role in oxygen activation in the same way as threonine. The alanine converted families such as CYP107 and CYP158 rescue the oxygen activation on the hydroxy grope of substrate.93 The peroxygenation activity can be transferred by the mutation of alanine to glutamic acid. To examine whether the mutation of the conserved threonine to glutamic acid effectively introduces peroxygenation activity into P450s, the corresponding mutants of CYP101A1 T252E, CYP119 T213E, and CYP102A1 (P450BM3) T268E48 were prepared. These mutations evoke the peroxygenation activities of native substrates and styrene. The crystal structure of CYP199A4 T252E shows the carboxylate of mutated glutamic acid was inserted into I-helix and accommodate above the heme.94 Two crystal structure of glutamic acid possessing P450 (CYP1232A24 and P450GcoA) were reported (Fig. 22b and c), although only 0.2% of over 39,000 P450 sequences have glutamate at this position judged by the analysis of a 3DM P450 superfamily database.95 The glutamic acid of CYP1232A24 is located at the flipped I-helix like the T252E mutant of CYP199A4

Fig. 21 Crystal structure of CYP152B1 wild type (a, PDBID: 3AWM), CYP152B1 A245E mutant (b, PDBID: 3VOO) and CYP152L1 wild type (c, PDBID: 4L40), and predicted structure of CYP152L1 P246D mutant prepared by LocalColabFold v1.0 (D). Heme, 6-corinated water, fatty acid and several residues were shown as stick models. Proteins were shown as cartoon model (a, b).

208

Heme-containing proteins: Structures, functions, and engineering Table 2

Highly Conserved amino acid on I-helix of CYP101-CYP200.

Conserved amino acid

Count CYPs

T S

75 9

A P E Others

5 4 2 5

CYP112, CYP121, CYP122, CYP128, CYP144, CYP149, CYP161, CYP181 CYP107, CYP152, CYP158, CYP177, CYP186 CYP134, CYP156, CYP157, CYP198 CYP104, CYP193 CYP118 (pseudogene), CYP165 (Q), CYP171 (G), CYP176 (N), CYP182 (I)

(Fig. 22a). The multiple sequence alignment reveals the glutamic acid possessing family was existed in typical bacterial P45096 (Table 2). Unexplored P450 peroxygenase might be exist in cytochrome P450 super family enzymes.

2.08.3

Heme acquisition protein

HasA proteins are extracellular proteins that can strongly bind heme and member of heme acquisition system (Has) in some bacterial strains. HasA proteins are expressed from some Gram-negative bacteria such as Serratia marcescens,97 Pseudomonas aeruginosa (P. aeruginosa),98 Pseudomonas fluorescens and Yersinia pestis99 under iron limiting conditions to acquire essential iron ions for keeping various physiological phenomena in the cells (Fig. 23a). Because of strong binding affinity of HasA toward heme, apo-HasA, which does not contain heme, can acquire heme from other heme proteins in host organisms to afford holo-HasA.100,102 Subsequently, holo-HasA selectively interacts with its corresponding receptor, HasR, leading to exchange of heme from HasA to HasR (Fig. 23b).101,103 The heme bound in HasR can be internalized into bacterial periplasm region to be utilized iron sources

Fig. 22 Crystal structure of CYP199A4 T252E mutant (a, PDBID: 7REH), CYP1232A24 (b, PDBID: 6G71) and P450GcoA (c, PDBID: 5NCB). Heme and glutamic acid residues were shown as stick models. Proteins were shown as cartoon model.

Fig. 23 (a) Crystal structure of holo-HasA from P. aeruginosa. PDB: 3ELL.100 (b) Crystal structure of the complex of HasA and HasR from Serratia marcescens. PDB: 3CSL.101 (c) Schematic images of iron acquisition pathway in P. aeruginosa.

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209

(Fig. 23c). Because HasA is important to heme acquisition in several notorious pathogens such as P. aeruginosa, it can be regarded as promising targets in antibacterial treatment.104 Crystal structures of several HasA proteins have been clarified (Fig. 23a). The heme binding site is located close to the surface of HasA and thus the heme of HasAs is partially exposed to solvent. Tyrosine and/or histidine are coordinated to the iron of heme as axial ligands. The overall protein matrix is folded as a compact globular shape, which might contribute to exceptional stability of HasAs against such as organic solvent. The position of heme-binding site of HasA would be important for original function of HasAs that are acquisition of heme from heme proteins and heme transportation to HasR (Fig. 23b and c). This unique heme-binding feature of HasA leads to other benefit that is heme replacement. Although heme substitution is one of the most promising strategies of construction of artificial metal proteins,3 the heme binding sites of most hemoproteins are optimized for the heme binding. Therefore, heme analogues whose structures are close to heme can be replaced with heme in the case of typical hemeproteins. In contrast, HasAs were found to be able to capture not only heme analogues, but also various synthetic metal complexes (Fig. 24). At beginning, complexation of synthetic metal complexes having plane structure such as iron phthalocyanine (FePc) and iron salophen with apo-HasA from Pseudomonas aeruginosa (HasAp) was reported.105 These synthetic metal complexes are captured in the heme-binding position. The structures of artificial HasAp having phthalocyanine (FePc) and salophen are essentially identical to the holo-HasA (heme-bound HasA) except for metal complexes captured. Interestingly, FePc-HasAp inhibits the growth of P. aeruginosa (Fig. 25a). It was expected that FePc-HasAp interact with the outer membrane receptor HasR of P. aeruginosa in the same manner as holo-HasA. Because the molecular size of FePc is larger than that of heme, FePc molecules might stuck in the channel of HasR, leading to inhibition of heme-acquisition. Xue and Wilks et al. reported Ga-salophen-HasAp as dual inhibitor against P. aeruginosa.107 Ga-salophen-HasAp compete with holo-HasAp in interaction with HasR, resulting in growth inhibition. In addition, because Ga-salophen-HasAp is not so stable, Ga-salophen can be gradually eliminated from its HasA conjugates. The released Ga-salophen can be internalized through iron uptake system and disrupt iron homeostasis which contribute to decreasing virulence of P. aeruginosa. Although FePc-HasAp in the initial study shows growth-inhibition effect, P. aeruginosa should be still alive after treatment of FePc-HasAp. To develop bactericidal treatment using artificial HasAs, Ga phthalocyanine (GaPc) was selected as photosensitizers that can generate cytotoxic singlet oxygen, which is one of the reactive oxygen species, upon photoirradiation of red light (Fig. 25b).106 GaPc was incorporated into HasAp like heme. After incubation of GaPc-HasAp with P. aeruginosa, almost all P. aeruginosa (99.99%) was effectively sterilized by light irradiation at 680 nm for just 10 min. In addition, even in the case of P. aeruginosa acquiring various drug resistance, this photo-sterilization method can be adapted. Because human cells do not express HasR, this photo-sterilization strategy may avoid side effects. Furthermore, because each bacteria have their specific HasAHasR pair, the antibacterial method using artificial HasA would have promising potential of pathogen-specific treatment. At present, there are countless reports of synthetic porphyrins because of their attractive properties including rigid planar structures, photophysical and electrochemical properties and catalytic performance. Because most synthetic porphyrins possess bulky substituents orthogonal to porphyrin plane, for example, meso-substituted porphyrins, hemeproteins usually cannot capture

Fig. 24

Scheme of heme substitution of HasA and chemical structures of synthetic metal complexes that can be incorporated into HasA.

210

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Fig. 25

(a) Growth inhibition of P. aeruginosa by FePc-HasA.105 (b) Photosterilization of Pseudomonas aeruginosa by GaPc-HasA.106

synthetic porphyrins. It was examined whether meso-substituted porphyrins can be captured into HasAs. For this purpose, various porphyrin complexes such as diphenyl porphyrin (DPP), triphenyl porphyrin (TriPP), ethynyl diphenyl porphyrin, bis(ethynyl) diphenyl porphyrin were synthesized and tested complexation with apo-HasAp.108 Reconstitution yields were largely affected by structures and hydrophobicity of each metal complexes. The crystal structures of obtained artificial HasAp are almost identical to the holo-HasAp (heme-binding HasA), indicating high acceptability of HasAp against even meso-substituted porphyrins. More interestingly, it was demonstrated that complexation of Fe-tetraphenylporphyrin (TPP) with HasA through optimization of buffer condition in complexation step.109 In the case of other meso-substituted porphyrins such DPP and TriPP, substituents at meso positions does not locate at inside of heme binding pocket to avoid steric repulsion with protein matrix of HasA. However, in the case of TPP, several perturbations such as flipping of loop region of HasA was observed, possibly due to avoidance of steric repulsion of one phenyl ring located at inside of heme binding pocket. During investigation of mutants of HasA to more suitably capture FeTPP, it was found that HasA mutant (V37G/K59Q) with FeTPP can form higher-order structure in crystal. In this crystal, one loop region, which normally cover the space above FeTPP and provide His residue as the axial ligand, are filliped out from FeTPP, resulting in formation of a vacant coordination site on FeTPP. This unexpected space in crystals can be used as catalytic center for enantioselective chemical conversions, cyclopropanation of styrene (Fig. 26). Compared with other proteins, HasA proteins including its mutants and artificial HasAs tend to be easily obtained good crystals for X-ray crystallography. Therefore, HasA proteins are potent scaffold of artificial metalloproteins and metalloenzymes rationally designed through selecting suitable metal complexes and fine tuning of HasA by mutagenesis. HasA also exhibits ability of complexation with other types of metal complexes. For example, iron and cobalt complexes of tetraphenylporphycene, which is less symmetric structures than FeTPP, have also been captured with HasA.110 Recently, it has also reported successful reconstitution of a cobalt 5-oxaporphyrinium cation and HasA. These artificial HasAs show growth inhibition

Fig. 26

Enantioselective cyclopropanation of styrene catalyzed by crystals of FeTPP-HasA variants.109

Heme-containing proteins: Structures, functions, and engineering

Fig. 27

211

Representative approaches of engineering of HasAs.

effect against P. aeruginosa.111 Since porphycene complexes and 5-oxaporphyrinium cation have totally different properties such as photophysical and catalytic properties from porphyrin complexes, such new types of artificial HasAs can be expected to open new application. One of the most promising features of HasA proteins is the broad availability of synthetic metal complexes in heme substitution because metal complexes in metalloproteins frequently play an important role in their functions (Fig. 27). Apart from metal complexes, protein scaffold of HasA itself can be also targeted as engineering for alteration and/or control of their functions. Mutagenesis methods, which are conventional strategy of protein engineering, are also available to HasAs.112 Especially, mutation at amino acid residues providing axial ligands of heme is attractive way to change coordination environment of heme or other synthetic metal complexes in HasAs, resulting in change of affinity of heme and so on.113 For example, mutation of the His working as the axial ligand of HasAs to Ala provides 5-coordination environment around metal centers that has the potential for artificial enzymatic reactions using created vacant sites on metal centers. Combining mutagenesis and chemical modification on amino acid residues in HasA is also potent way for introduction of artificial functions to HasAs. For example, HasA probes which can monitor replacement of metal complexes based on the fluorescence changes106 was prepared by combination of mutation to cystein and labeling of fluorescent dyes to the cysteine residue. Combination of these conventional strategies of protein engineering and unique heme substitution strategy in HasAs would be expected to open new research areas of artificial metalloproteins in future.

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105. Shirataki, C.; Shoji, O.; Terada, M.; Ozaki, S.; Sugimoto, H.; Shiro, Y.; Watanabe, Y. Angew. Chem. Int. Ed. Engl. 2014, 53 (11), 2862–2866. https://doi.org/10.1002/ anie.201307889. 106. Shisaka, Y.; Iwai, Y.; Yamada, S.; Uehara, H.; Tosha, T.; Sugimoto, H.; Shiro, Y.; Stanfield, J. K.; Ogawa, K.; Watanabe, Y.; et al. ACS Chem. Biol. 2019, 14 (7), 1637–1642. https://doi.org/10.1021/acschembio.9b00373. 107. Centola, G.; Deredge, D. J.; Hom, K.; Ai, Y.; Dent, A. T.; Xue, F.; Wilks, A. ACS Infect. Dis. 2020, 6 (8), 2073–2085. https://doi.org/10.1021/acsinfecdis.0c00138. 108. Uehara, H.; Shisaka, Y.; Nishimura, T.; Sugimoto, H.; Shiro, Y.; Miyake, Y.; Shinokubo, H.; Watanabe, Y.; Shoji, O. Angew. Chem. Int. Ed. Engl. 2017, 56 (48), 15279–15283. https://doi.org/10.1002/anie.201707212. 109. Shisaka, Y.; Sakakibara, E.; Suzuki, K.; Stanfield, J. K.; Onoda, H.; Ueda, G.; Hatano, M.; Sugimoto, H.; Shoji, O. e202200095. Chembiochem 2022. https://doi.org/10.1002/ cbic.202200095. 110. Sakakibara, E.; Shisaka, Y.; Onoda, H.; Koga, D.; Xu, N.; Ono, T.; Hisaeda, Y.; Sugimoto, H.; Shiro, Y.; Watanabe, Y.; et al. RSC Adv. 2019, 9 (32), 18697–18702. https:// doi.org/10.1039/c9ra02872b. 111. Takiguchi, A.; Sakakibara, E.; Sugimoto, H.; Shoji, O.; Shinokubo, H. Angew. Chem. Int. Ed. Engl. 2022, 61 (7), e202112456. https://doi.org/10.1002/anie.202112456. 112. Kumar, R.; Matsumura, H.; Lovell, S.; Yao, H.; Rodriguez, J. C.; Battaile, K. P.; Moenne-Loccoz, P.; Rivera, M. Biochemistry 2014, 53 (13), 2112–2125. https://doi.org/ 10.1021/bi500030p; Kumar, R.; Qi, Y.; Matsumura, H.; Lovell, S.; Yao, H.; Battaile, K. P.; Im, W.; Moenne-Loccoz, P.; Rivera, M. Biochemistry 2016, 55 (18), 2622–2631. https://doi.org/10.1021/acs.biochem.6b00239. 113. Jepkorir, G.; Rodrıguez, J. C.; Rui, H.; Im, W.; Lovell, S.; Battaile, K. P.; Alontaga, A. Y.; Yukl, E. T.; Moenne-Loccoz, P.; Rivera, M. J. Am. Chem. Soc. 2010, 132, 9857– 9872; Dent, A. T.; Brimberry, M.; Albert, T.; Lanzilotta, W. N.; Moenne-Loccoz, P.; Wilks, A. Biochemistry 2021, 60 (33), 2549–2559. https://doi.org/10.1021/ acs.biochem.1c00389.

2.09

Engineering of hemoproteins

Takashi Hayashi and Shunsuke Kato, Department of Applied Chemistry, Osaka University, Suita, Osaka, Japan © 2023 Elsevier Ltd. All rights reserved.

2.09.1 2.09.2 2.09.3 2.09.4 2.09.4.1 2.09.4.2 2.09.4.3 2.09.4.4 2.09.5 2.09.5.1 2.09.5.2 2.09.6 2.09.6.1 2.09.6.2 2.09.6.3 2.09.7 2.09.7.1 2.09.7.2 2.09.8 References

Introduction Hemoproteins Modification of hemoproteins Oxidation Modification of the heme pocket of myoglobin Modification of heme-propionate side chains Modification of the heme framework: Reconstitution with an iron porphyrinoid Insertion of a non-porphyrinoid metal complex into apomyoglobin Hydroxylation Conversion of myoglobin to hydroxylase Modification of substrate specificity of cytochrome P450BM3 Carbene and nitrene transfer reactions Genetic engineering of hemoproteins toward abiological reactions Metal substitutions of heme cofactor Modification of the heme framework Reactions by Co and Ni porphyrinoids in hemoproteins Hemoprotein reconstituted with cobalt porphyrinoid Hemoprotein reconstituted with nickel porphyrinoid Conclusion

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Abstract Hemoproteins are versatile metalloproteins involved in a variety of important biological processes, such as oxygen storage/ transfer, gas molecule sensing, electron transfer, and catalysis. Chemical modification and genetic engineering of these hemoproteins bring great challenges and opportunities for understanding the molecular mechanisms of their original functions. In particular, replacement of the native heme cofactor with an artificial metal porphyrinoid is an extremely effective strategy to alter the physiological and chemical properties of the hemoproteins. As a result, artificial hemoproteins with a non-native cofactor will serve as new models of native metalloenzymes with complicated structures as well as new artificial metalloenzymes capable of catalyzing non-biological reactions. This chapter summarizes recent progresses in engineering of hemoproteins to modify their catalytic properties such as oxidation, hydroxylation, carbene and nitrene transfer reactions, and model reactions of cobalamin- and cofactor F430-dependent enzymes.

2.09.1

Introduction

Metal ions are known to play essential roles in biological systems such as metabolism, regulation, growth, energy conversion, and signaling. In particular, first-row transition metals such as iron, cobalt, copper, manganese and zinc are common reaction centers in protein matrices, where the metal ions are ligated to amino acid residues.1 Many proteins containing metal complexes act as biological catalysts known as metalloenzymes. These enzymes catalyze important reactions under mild conditions which are challenging to reproduce using laboratory-level synthetic organic synthesis. In most cases, the reactivities of the metal ions are controlled by directly coordinated ligands as well as non-coordinated protein matrices operating as a second coordination sphere. Therefore, it is known that the positions and conformations of amino acid residues near metal ions have an important influence on the activity and selectivity of metalloenzymes in biological reactions. Metal ions bound to the protein matrix are known as metal cofactors. In general, metal cofactors belong to two main categories: (i) inorganic metal ions in a protein matrix bound to amino acid residues and (ii) metal ions stabilized by organic molecules forming prosthetic groups bound to a protein matrix. Focusing on iron-containing enzymes, for example, various mononuclear and dinuclear nonheme iron enzymes, are found in monooxygenases or dioxygenase families of the first category. Furthermore, many different types of iron-sulfur clusters are involved in oxidoreductases. In contrast, a family of heme enzymes is a representative of the second category. Heme cofactors consisting of iron porphyrin derivatives are ubiquitous in biological systems and promote electron transfer, dioxygen storage and transport, gas molecule sensing and enzymatic reactions such as oxidation or oxygenation.2 Crystal structures for myoglobin3 and hemoglobin4 were first obtained, followed by thousands of studies of structure-function

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relationships to understand metalloproteins and metalloenzymes in bioinorganic chemistry.5 This chapter describes efforts to modify hemoproteins by substitution of the heme prosthetic group to generate artificial enzymes, and outlines recent genetic engineering approaches for catalyzing abiological reactions.

2.09.2

Hemoproteins

Hemoproteins are versatile metalloproteins in biological systems. Several hemoproteins operate as biocatalysts. Many different types of heme structures exist in nature and differ in peripheral substituents at various positions of the porphyrin framework (Fig. 1). However, most heme molecules are relatively flat and fit into hollow concave heme pockets in protein matrices. In particular, heme b (also known as iron protoporphyrin IX complex), is present in various hemoproteins and bound via dominant coordination of amino acid residues (e.g. His, Cys, Met, Tyr) and multiple noncovalent interactions such as hydrophobic contact and hydrogen bonding using two propionate side chains at the position 6 and 7. Heme iron in proteins typically adopts a penta- or hexacoordinate structure with axial ligation which provides a major effect with respect to protein function. Penta-coordination exists in globins and many heme enzymes, although one water molecule is often weakly bound to the heme iron as a sixth ligand. In the latter case, the water molecule is typically replaced by small molecule ligands such as O2, CO and NO or, in the case of heme enzymes, by a substrate which penetrates the heme pocket. As described above, heme b is not covalently linked to a protein matrix, but is usually stabilized in the heme pocket with a binding constant ranging from 1010 M–1 to 1015 M–1.6 In contrast, heme can be released from the heme pocket of some hemoproteins under acidic conditions or upon addition of denaturing reagent.7 Release of heme makes the corresponding apoprotein available for experimentation. Furthermore, it is well known that the addition of hemin 1, ferric iron protoporphyrin IX complex with chloride ligand, into a solution of the apoprotein provides a reconstituted protein which is essentially identical to the native protein, as confirmed by UV-vis and 1H NMR spectra.8 In contrast, c-type heme, heme c, is another representative prosthetic group in the hemoprotein family. This heme is covalently linked to the protein via two thioether bonds involving thiol groups of cysteine residues. Cytochrome c is the best-known example of a heme c-dependent protein, which has the sequence motif CeXeYeC where the two cysteine residues are linked to 2- and 4-positions of the peripheral side chains of heme in a reaction supported by cytochrome c heme lyase.9 The bioinorganic roles of hemoproteins have been studied for over 50 years and many hemoproteins have been characterized by various spectroscopic analyses.2 Protein matrices possessing the heme cofactor play important roles not only on fixation of heme but also control of the outer sphere of heme reactivity. Most reactions catalyzed by hemoproteins cannot be similarly catalyzed by the heme molecule itself in the absence of the applicable protein matrix, although heme itself has redox and Lewis acidic properties. Therefore, it is important to characterize the molecular interactions between heme and protein matrices to understand the reaction mechanisms and dynamics of hemoproteins in various functions ranging from electron transfer to gas molecule storage, transport, sensing and catalysis. Metal porphyrinoids other than heme are used as cofactors in natural biological systems. Well known examples are cobalamin,10 a cobalt corrinoid (derivative of vitamin B12) and the F430 cofactor,11 a nickel corphinoid (Fig. 2). The framework of cobalamin is formed from a corrin derivative which differs from the porphyrin framework in lacking one of the four meso carbons of the porphyrin ring. Cobalamin dependent enzymes are mainly divided into three categories; adenosylcobalamin-dependent isomerase, methylcobalamin-dependent methyltransferase and B12-dependent reductive dehalogenase. The intermediate of the enzymatic reactions involves organometallic CoeC bonding structures. On the other hand, the F430 cofactor, which has a highly hydrogenated porphyrin framework, has been identified in methyl-coenzyme M reductase. The F430 cofactor in the enzyme is responsible for methane generation in the last step of energy-producing synthesis of methane from CO2 by autotrophic archaebacteria.

Fig. 1

Chemical structures of heme a, heme b and heme c.

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Fig. 2 Chemical structure of cobalamin (R ¼ 5’-deoxyadenosyl: adenosylcobalamin R ¼ CH3: methylcobalamin, and R ¼ CN: cyanocobalamin) and cofactor F430.

2.09.3

Modification of hemoproteins

Three main techniques are used in engineering of hemoproteins: (i) chemical modification of a functional group at the terminal of a side chain of an amino acid located on the protein surface, (ii) a selective amino acid mutation at the target of a residue in protein matrix by mutagenesis, and (iii) replacement of the native heme cofactor with an artificial metal complex (Fig. 3). Method (i) is a functional group conversion familiar to organic chemists. A side chain of Cys or Lys or an N-terminal site is a target for functionalization of proteins. For example, when a photosensitizer is introduced into a side chain, electron transfer to heme occurs with light irradiation.12 Method (ii) is a well-known technique for modifying protein functions. In the case of hemoproteins, genetic engineering at the heme pocket has an influence on the reactivity of heme as well as providing stereoselectivity and/or regioselectivity for substrates in heme-dependent enzymatic reactions.13 In contrast, it is challenging to enhance catalytic activity of heme enzymes only by a mutagenesis approach because naturally occurring enzymes have evolved over a very long time period. However, the range of substrates, regio/stereo selectivity and protein stability can be changed relatively easily by genetic engineering around the heme pocket, based on crystal structure and MD calculation results.14 Method (iii) is quite effective and unique way for engineering of hemoproteins, because heme works as a reaction center.15 The molecular structure of porphyrin side chains and/or the porphyrin framework can be easily modified by synthetic organic chemistry, and this approach has the potential to significantly influence the reactivity of the metal ligated by macrocyclic porphyrinoids. It is of particular interest to modify a series of hemoproteins understand underlying biochemistry as well as coordination chemistry. Myoglobin is a well-known, relatively small stable globin consisting of 153 amino acid residues and heme b. Myoglobin functions as an oxygen storage protein in muscle tissue. The amino acid residues of the heme pocket, particularly distal His64, stabilize dioxygen bound to heme and the entire hydrophobic heme pocket protects the oxygenated complex (Fe(II)eO2 or Fe(III)eO2–
)

Fig. 3

Modification of hemoproteins.

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from autoxidation. In contrast, myoglobin exhibits only low level enzymatic activity even though it has heme b as a cofactor which is widely used by many catalytically powerful heme-dependent enzymes such as cytochrome P450 (P450) and horseradish peroxidase (HRP). Therefore, it is challenging to convert myoglobin to an artificial metalloenzyme by chemical modification. We discuss recent examples of engineering of myoglobin and other simple hemoproteins to construct artificial metalloenzymes mainly by method (iii) in this chapter.

2.09.4

Oxidation

HRP, one of the best-known heme peroxidases, has been investigated for many years.16 This enzyme has proximal and distal histidines in the heme pocket and is responsible for small molecule one-electron oxidation reactions. The resting state of the high-spin Fe(III) porphyrin is converted by H2O2 to an intermediate, two oxidative states higher consisting of an oxoferryl species (Fe(IV)]O) with a porphyrin p-cation species. This intermediate, known as Compound I, oxidizes a substrate such as phenol or phenol derivatives via one-electron transfer, and then the next intermediate, Compound II, an oxoferryl species, is formed. Furthermore, Compound II is converted to the resting state by an additional one-electron oxidation of substrate. Myoglobin, an oxygen storage protein, also has the same heme b cofactor and two key histidine residues in the proximal and distal sites of the heme pocket as seen in HRP. However, myoglobin has poor peroxidase activity (Fig. 4). One of the reasons for the poor peroxidase activity is that myoglobin does not have an H2O2 activation system provided by several amino acid residues in the heme pocket. Another reason for the poor peroxidase activity of myoglobin is that it does not have a substrate-binding site on its surface. Therefore, it has been of particular interest to enhance peroxidase activity of myoglobin by mutagenesis and/or by cofactor modifications as described below.

2.09.4.1

Modification of the heme pocket of myoglobin

Compared to the X-ray crystal structure of HRP with myoglobin, the distal His42 residue of HRP is located in an optimized position to work as a general base and acid for the bound H2O2, in the catalytic reaction which releases one water molecule with generation of compound I is via OeO bond heterolytic cleavage (Fig. 4). The cationic Arg38 near His42 is also known to assist the heterolytic cleavage. Furthermore, the proximal His170 strongly coordinates to the heme iron, providing a push effect to assist the generation of compound I from the bound H2O2. In contrast, the distal His64 in myoglobin plays a role in stabilization of heme-bound O2 and the strength of the proximal ligation of His93 is moderate, as indicated by resonance Raman spectroscopy (HiseFe bands for HRP and myoglobin are located at 243 and 220 cm–1, respectively).17 Myoglobin variants have been prepared where His64, Leu29 and/or Phe43 are replaced with different amino acid residues to modify the distal site of the heme pocket toward activation of H2O2 to produce a compound I species similar to compound I of HRP. As a result, the compound I species was successfully detected in H64X variants of myoglobin such as H64A and H64D, although it is known that the compound I species is generally not detectable in native myoglobin upon addition of H2O2 or mCPBA.18 Furthermore, the L29H/H64L and F43H/H64L double variants of myoglobin were found to clearly accelerate thioanisole sulfoxidation and styrene epoxidation with significant enantioselectivity as shown in Table 1.19 These results indicate that protein engineering can convert myoglobin to an artificial metalloenzyme.

2.09.4.2

Modification of heme-propionate side chains

To construct a substrate-binding site in native myoglobin, benzene moieties were introduced at the termini of the heme-propionate side chains located near the surface of the protein. After removal of native heme from myoglobin, synthetic heme derivatives 2–4 were incorporated into the obtained apoprotein to produce the corresponding reconstituted myoglobins, rMb(2)–rMb(4)

Fig. 4 (a) Crystal structure of HRP (PDB: 1ATJ), (b) Crystal structure of myoglobin (PDB: 1MBN), (c) Catalytic cycle of the HRP-mediated peroxidation reaction.

Engineering of hemoproteins Table 1

Asymmetric sulfoxidation and epoxidation catalyzed by myoglobin variants.

Wild type R Sulfoxidation Epoxidation

Fig. 5

219

Me Et H Me

L29H/H64L

F43H/H64L

Rate (Ton/min)

ee (%)

Rate (Ton/min)

ee (%)

Rate (Ton/min)

ee (%)

0.25 0.46 0.015 0.076

25 (R) 7.6 (R) 9 (1R) 39 (1R, 2R)

5.5 6.5 0.14 0.29

97 95 80 83

47 26 4.5 16

85 (R) 54 (R) 68 (R) 96 (1R, 2R)

(R) (R) (R) (1R, 2R)

Synthetic heme cofactors 2–4 with modified propionate side chains.

(Fig. 5).20 The rMb(2) protein represents the first example of accelerating the H2O2-dependent one-electron oxidation of small molecules such as catechol, hydroquinone and guaiacol. Km value for the rMb(2)-catalyzed oxidation has clearly decreased to 3.4 mM compared to native myoglobin (Km ¼ 54 mM), which suggests the binding of the aromatic substrates within the artificially modified side chain of 2. In addition, thioanisole sulfoxidation is catalyzed by rMb(2) with a kcat/Km value of 0.34 s–1 mM–1 which is approximately 6.9-fold higher than the corresponding value of native myoglobin at 20  C, pH 7.0. Next, a dual modification of myoglobin by mutagenesis and synthetic approaches was prepared to produce rMbH64D(2) where native heme is replaced with 2 and His64 was replaced with Asp64. Focusing on guaiacol oxidation, the kcat/Km value of rMbH64D(2) was determined to be 23,000 s–1 M–1 and then rMbH64D(3) which includes the second generation artificial cofactor 3 was found have catalytic efficiency similar to that of native HRP.

2.09.4.3

Modification of the heme framework: Reconstitution with an iron porphyrinoid

Porphyrin is a highly symmetric tetrapyrrole macrocycle. Examples of non-natural porphyrinoids with decreasing symmetric structures have been prepared by organic chemists primarily to characterize various physicochemical properties. Based on this knowledge of porphyrinoid chemistry, metal porphyrinoids have been used as artificial cofactors for making dramatic changes to the reactivity of hemoproteins. For example, an artificial cofactor consisting of iron porphycene 5, a constitutional isomer of iron porphyrin, was inserted into apomyoglobin to obtain a reconstituted myoglobin, rMb(5) (Fig. 6).21 Interestingly, deoxy rMb(5) was found to have extremely high O2 affinity, with an increase by 2300-fold compared to that of native myoglobin; Ka (rMb(5)) ¼ 1.6 x 109 M–1 at 25  C.21a One of the reasons for the high affinity is the high Lewis acidity of the iron center in 5 due to the decrease symmetry of the porphycene macrocycle relative to the porphyrin macrocycle. In fact, the pK1/2, which represents the pH value where 50% of heme molecules of a sample are released from the heme pocket, has been determined to be 3.1, while native myoglobin has a of pK1/2 value of 4.5. One-electron oxidation of 2-methoxyphenol (guaiacol) is promoted by native myoglobin and by rMb(5) upon addition of H2O2.21b The initial turnover number of rMb(5) for the guaiacol oxidation monitored by UV-vis absorption at 480 nm is calculated to be 0.23 s 1, which is 11-fold higher than that of native myoglobin at 7.0 at 20  C. This result clearly indicates that the artificial

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Fig. 6

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(a) Chemical structure of iron porphycene 5. (b) Crystal structure of rMb(5) (PDB: 2D6C).

Table 2

Sulfoxidation of thioanisole catalyzed by rHRP(5).

Protein

Thioanisole oxidation (Ton/min)

Guaiacol oxidation (Ton/min)

HRP rHRP(5)

1.4  0.1 17  2

2400  100 2300  100

cofactor 5 accelerates the peroxidase reaction. Furthermore, 5 can also be incorporated into apo-HRP after removal of heme from native HRP to yield reconstituted HRP, rHRP(5).22 Sulfoxidation of thioanisole was found to be significantly accelerated by rHRP(5) compared to the native enzyme as shown in Table 2. In addition, the two-electron oxidative intermediate of peroxidase reaction was detected using a stopped-flow apparatus. The spectral changes which occur in the near IR region upon addition of mCPBA into a solution of rHRP(5) indicate the characteristic profile of a porphycene p-cation species within 40 ms. This finding indicates that the intermediate is a compound I-like species, in this case, an oxoferryl porphycene p-cation species. Corrole has a framework resembling the porphyrin framework, with the exception of the lack of one meso-carbon. Corrole is also a useful artificial cofactor, because the trianionic ligand is expected to stabilize the key high-valent iron intermediate.23 Myoglobin reconstituted with iron corrole 6, rMb(6), was found to accelerate one-electron oxidation of guaiacol upon addition of H2O2, while HRP reconstituted with 6 has decreased activity toward the guaiacol oxidation (Fig. 7). The as-isolated form of 6, the Fe(III) species combined with a macrocycle p-cation radical, was confirmed by spectroscopic analyses. Furthermore, 6 was found to be reduced to Fe(III) corrole after its incorporation into the myoglobin matrix. In contrast, an NMR study suggests that 6 in HRP still attains the þ 4 metal oxidation state.

2.09.4.4

Insertion of a non-porphyrinoid metal complex into apomyoglobin

The heme pocket is an attractive reaction scaffold because there is sufficient space for binding of a substrate and a small metal complex and the heme-coordinated axial histidine may be a candidate of a ligand for the new metal complex. Thus, it is of interest to insert a relatively flat metal complex. For example, 3,3’-dimethylsalen Cr(III) complex (7) and Mn(III) complex (8) were inserted

Fig. 7 (a) Chemical structure of iron corrole 6 form as isolated. (b) Catalytic activities of rMb(6) and native Mb for guaiacol oxidation. (c) Catalytic activities of rHRP(6) and native HRP for guaiacol oxidation.

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221

Fig. 8 (a) Chemical structure of Cr(III)- and Mn(III)-salen complex 7 and 8. (b) Crystal structure of rMb(7) (PDB: 1J3F). (c) Crystal structure of rMb(8) (PDB: 1V9Q).

Fig. 9

(a) Chemical structure of Mn(III)-salen complex 9. (b) Asymmetric sulfoxidation of thioanisole catalyzed by rMb(9) variants.

into mutant apomyoglobin(A71G) to obtain the reconstituted metalloproteins, rMbA71G(7) and rMbA71G(8), respectively (Fig. 8).24 The rate of thioanisole sulfoxidation catalyzed by rMbA71G(8) in the presence of H2O2 is 1.3 Ton min 1, which is 20fold higher than that of the corresponding metal salen (0.062 Ton min 1) under the same conditions. In addition, modification of the 3- and 3’-positions of the salen substituents was found to control the enantioselectivity for the thioanisole sulfoxidation. Furthermore, engineered myoglobin (L72C/Y103C) with a manganese complex of salen derivatives (9) formed via covalent anchoring by methane thiosulfonate groups at the termini of two side chains of 9 has also been reported (Fig. 9).25 The engineered myoglobin rMbL72C/Y103C(9) enhances the catalytic activity toward H2O2-dependent sulfoxidation of thioanisole by 160-fold compared to 9 without protein. The enantioselectivity was found to be increased by covalent anchoring of 9 to the protein matrix.

2.09.5

Hydroxylation

The cytochrome P450 monooxygenase family includes heme b and promotes several different reactions such as hydroxylation of alkanes, oxidative dealkylation, amine and sulfide oxidation, and deformylation. For example, cytochrome P450cam from Pseudomonas putida has been the subject of P450 research for many years and is well known for its selective conversion of d-camphor to 5hydroxycamphor via NADPH-dependent O2 activation.26 Furthermore, the reaction mechanism of P450 hydroxylation has also been thoroughly investigated and its radical rebound mechanism which occurs via a compound I-type species has been characterized by spectroscopic measurements.27 Research has also focused on re-engineering of P450s by mutagenetic approaches to alter substrate specificity and stereoselectivity as well as enhancing tolerance to organic solvents.28 This section summarizes recent efforts to modify hemoproteins to enhance hydroxylase activities by addition of synthetic organic molecules such as artificial cofactors and decoy molecules.

2.09.5.1

Conversion of myoglobin to hydroxylase

P450s and myoglobin use the same cofactor, but myoglobin does not catalyze alkane hydroxylation. It has been surmised that the heme-thiolate of P450s is essential to catalyze the hydroxylation reaction. The heme axial ligand of myoglobin is histidine. However, myoglobin reconstituted with manganese porphycene 10, rMb(10), was found to promote the H2O2-dependent hydroxylation of alkanes such as ethylbenzene and cyclohexane to yield a-hydroxyethylbenzene (Ton ¼ 13) and cyclohexanol (Ton ¼ 0.9), respectively (Fig. 10).29 In contrast, myoglobin reconstituted with manganese protoporphyrin IX and iron porphycene 10 does not exhibit hydroxylation activity for ethylbenzene. Additionally, the addition of H2O2 to a solution of rMb(10) can promote an artificial hydroxylation reaction. The natural hydroxylation reaction catalyzed by P450s requires two electrons transferred from NADH to activate O2. Moreover, spectroscopic studies using UV-vis-NIR and EPR measurements suggest that the key reaction intermediate is a Mn(V)-oxo species.30

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Engineering of hemoproteins

Fig. 10 (a) Chemical structure of Mn porphycene 10. (b) Crystal structure of rMb(10) (PDB: 5YL3). (c) rMb(10)-catalyzed hydroxylation of ethylbenzene and cyclohexane.

2.09.5.2

Modification of substrate specificity of cytochrome P450BM3

The name “cytochrome P450” refers to a superfamily of heme-containing oxidoreductases which catalyze monooxygenation reactions of aliphatic substrates, such as fatty acids and steroids. Since P450s exhibit remarkable catalytic activities toward the catalytically challenging hydroxylation of the inert alkanes, P450s have great potential to be exploited as a biocatalysts for industrial synthetic chemistry. However, the strict substrate specificity of P450s often causes problems in developing P450s as customized biocatalysts. Furthermore, substrate-induced conformational changes play a crucial role in initiating the catalytic cycle of monooxygenation reactions. As a result, P450s rarely provide adequate catalytic activities toward non-native substrates with different structures. To address this problem, P450s have been extensively engineered in attempts to alter their substrate specificity. One of the most common strategies is genetic engineering of the substrate binding site. By repeating the iterative cycles of mutagenesis, substrate specificity of P450s has been altered for a broad range of non-native substrates.28 Another strategy is to provide a reaction system which uses a “decoy molecule” for P450BM3 from Bacillus megaterium.31 The addition of appropriate perfluorinated carboxylic acids as a decoy molecule has been found to efficiently promote catalytic hydroxylation of various non-native alkane substrates (Fig. 11a). In this reaction system, the decoy molecules serve as structural analogues for the original substrates of P450BM3 and are misrecognized by the substrate binding site. This misrecognition induces structural changes of the active site, thereby generating a reactive compound I species responsible for catalyzing hydroxylation of small gaseous alkanes. In addition, the same group has applied this strategy for direct C(sp2)eH bond hydroxylation of benzene to produce phenol.32 After screening over 600 dipeptides in a library, a new decoy molecule 3CPPA-Pip-Phe was found to increase the turnover number of P450BM3 for benzene hydroxylation up to 54,500 (Fig. 11b).33

2.09.6

Carbene and nitrene transfer reactions

Transition metal-catalyzed carbene and nitrene transfer reactions have emerged as powerful synthetic processes for constructing structurally complex molecules with diverse functional groups. In organic chemistry, it is well known that metal-carbenoid and metal-nitrenoid species are key intermediates for these transfer reactions which have the potential to react with a broad range of inert chemical bonds, such as alkyl CeH bonds and alkenyl C]C bonds.34 Inspired by this knowledge, a first example of a P450-catalyzed nitrene transfer reaction was demonstrated in 1985.35 Since the metal-nitrenoid species has a structure similar to that of iron-oxo species in P450 monooxygenase, P450-LM3 from rabbit liver was found to accommodate an iron-nitrenoid species through a reaction between heme and iminoiodinanes. However, its catalytic activity towards a nitrene CeH insertion reaction was found to be much lower than the original monooxygenation activity of P450s (Ton ¼ 0.31). Starting with this report,

Fig. 11

Hydroxylation of (a) propane and (b) benzene catalyzed by P450BM3 with decoy molecules.

Engineering of hemoproteins

223

a number of P450s and other hemoproteins have been engineered to improve the catalytic performance of such enzymatic carbene and nitrene transfer reactions.

2.09.6.1

Genetic engineering of hemoproteins toward abiological reactions

The genetic engineering strategy based on amino acid mutagenesis is one of the most common methods to expand the catalytic repertoire of heme-containing enzymes. For example, a P450BM3-catalyzed cyclopropanation reaction via carbene transfer between styrenes and alkyl diazoacetates has been reported (Fig. 12a).36 Although the wild-type P450BM3 demonstrated modest catalytic activity (Ton ¼ 5) with low diastereo- and enantioselectivities for the cyclopropanation (cis:trans ¼ 37:63, 2% ee for trans products), a T268A mutation at the heme pocket, was found to clearly increase the activity and selectivity up to Ton ¼ 323, cis:trans ¼ 1:99, and 96% ee for trans products. After this finding, further engineering of P450BM3 variants based on the genetic engineering strategy, including the directed evolution techniques, resulted in development of P450 catalysts for a variety of the carbene and nitrene transfer reactions,37 such as cyclopropenation,38 bicyclobutanation,39 carbene CeH insertion,40 aziridination,41 and nitrene CeH insertion42 (Fig. 12). Other hemoproteins have been exploited as carbene and nitrene transfer catalysts. For example, a series of genetically-engineered myoglobin variants from Physeter macrocephalus were found to efficiently catalyze cyclopropanation reactions from styrenes and ethyl diazoacetate.43 In particular, amino acid substitutions at the distal heme pocket were found to significantly improve the catalytic activity and diastereo- and enantio-selectivity up to > 99% conversions, 99.9% de, and 99.9% ee. Moreover, these engineered myoglobin variants were also found to be capable of catalyzing nitrene CeH insertion,44 carbene XeH insertion (X]N and S),45 Doyle-Kirmse reactions,46 and aldehyde olefination.47 Furthermore, carbene SieH and BeH insertion reactions catalyzed by cytochrome c from Rhodothermus marinus (Rma cyt c) have also been reported.48 Through the iterative cycles of directed evolution, Rma cyt c was refined to produce chiral organosilanes and organoboranes which are not known in the natural biological world. In addition to these representative reports, several research groups have also developed carbene and nitrene transfer reactions using a variety of engineered hemoproteins, such as protoglobin,49 nitric oxide dioxygenase,50 and dye-decolorizing peroxidase.51 De novo hemoproteins are also suitable for development as catalysts for carbene transfer reactions. An artificially created supramolecular heme protein, in which hemin 1 was incorporated into the hydrophobic pore of lactococcal multidrug resistance

Fig. 12 Engineered P450BM3-catalyzed (a) cyclopropanation, (b) bicyclobutanation, (c) carbene C–H insertion, (d) aziridination, and (e) nitrene C–H insertion.

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Engineering of hemoproteins

Fig. 13 (a) Crystal structure of heme-bound LmrR (PDB: 6FUU). The bound heme is highlighted in yellow. (b) Cyclopropanation of styrene catalyzed by LmrR(1).

regulator (LmrR), was developed as a biocatalyst.52 Although the original functions of LmrR are not related to the binding to heme,53 the resulting artificial enzyme LmrR(1) was found to efficiently accommodate a heme molecule. The resulting protein exhibits high catalytic activity (up to 449 Ton) and moderate enantio-selectivity (up to 51% ee) for cyclopropanation reactions (Fig. 13).

2.09.6.2

Metal substitutions of heme cofactor

In inorganic chemistry, various types of metal-porphyrin complexes other than iron porphyrins have been synthesized and exploited as catalysts for carbene and nitrene transfer reactions.34 In particular, porphyrin complexes of late transition metals such as Ru, Rh and Ir are well known to have the potential to promote challenging chemical transformations that cannot be catalyzed by simple Fe-porphyrin complexes. Based on this knowledge, an artificial metalloenzyme containing an Ir-mesoporphyrin IX complex 11 within the heme-binding cavity of myoglobin from Physeter macrocephalus was constructed (Fig. 14).54 Notably, the resulting artificial metalloenzyme rMb(11) and its mutants were found to efficiently catalyze intramolecular carbene C–H insertions to produce dihydrobenzofuran derivatives with high diastereo- and enantio-selectivity (Ton ¼ 134, e.r. ¼ 85:15). In addition, a series of myoglobin-based artificial metalloenzymes was produced with native heme replaced by various metal-porphyrins (M ¼ Mn, Co, Ru, Rh, and Ir).44,55 These artificial metalloenzymes exhibit unique catalytic activities and different chemoselectivities toward carbene transfer reactions compared to native myoglobin. This metal substitution strategy has been also extended to the cytochrome P450 variants. In one example, complex 11 was incorporated into the heme-binding pocket of Sulfolobus solfataricus cytochrome P450, which is also known as CYP119.56 The resulting artificial metalloenzyme rCYP119(11) was subjected to iterative mutagenesis and investigated as a catalyst for cyclopropanation, intra- and inter-molecular carbene CeH insertion, and nitrene CeH insertion.57 Furthermore, rCYP119(11) has been recently investigated in a whole-cell catalysis in E. coli.58 By combining the rCYP119(11)-mediated cyclopropanation with a heterologous biosynthetic pathway for synthesis of limonene, an unnatural chiral cyclopropyl limonene was produced in a one-pot tandem procedure (Fig. 15).

2.09.6.3

Modification of the heme framework

Chemical modification of the porphyrin framework provides another strategy to alter the catalytic performance of hemoproteins for carbene and nitrene transfer reactions. Myoglobin reconstituted with iron porphycene cofactor 5 was found to exhibit higher catalytic activity toward cyclopropanation reactions.59 Compared to native myoglobin, the kcat/KM value of rMb(5) was increased 26fold (Table 3). Kinetic studies and DFT calculations revealed that rMb(5) particularly accelerates the formation of metal carbenoid species due to the strong ligand field of porphycene framework. The second order rate constant of the reaction between rMb(5) and ethyl diazoacetate was found to be 0.25 mM 1 s 1 which represents 615-fold enhancement relative to the native myoglobin.

Fig. 14

(a) Chemical structure of Ir-mesoporphyrin IX 11, (b) carbene C–H insertion and cyclopropanation catalyzed by rMb(11) variants.

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225

Fig. 15 (a) In vivo assembly of rCYP119(11) in E. coli harboring the HUG transport system. (b) Whole-cell catalysis of rCYP119(11) combined with limonene biosynthesis. Table 3

Michaelis-Menten parameters for rMb(5)-catalyzed cyclopropanation.

Protein

kcat (s 1)

Km (mM)

kcat/Km (mM 1 s 1)

rMb(5) Mb

1.7  0.3 0.06  0.01

1.3  0.4 1.2  0.5

1.3  0.5 0.05  0.02

Fig. 16 Chemical structure of (a) Fe-chlorin e6 complex 12 and (b) Fe-2,4-diacetyl deuteroporphyrin IX complex 13. (c) Crystal structure of Mb(H64V/V68A/H93NMH) variant with heme (PDB: 6F17).60

A myoglobin-based artificial metalloenzyme containing Fe-chlorin e6 complex 12 as a synthetic metal cofactor was also investigated as a catalyst for the carbene transfer reaction (Fig. 16a).61 In general, myoglobin is known to exhibit less carbene transfer activity under aerobic conditions. However, the H64V/V68A mutant of the artificial metalloenzyme rMb(12), was found to promote cyclopropanation reactions even in the presence of dioxygen. A number of chiral cyclopropanes were synthesized with excellent yields (> 99%) under aerobic conditions. In addition, the artificial metalloenzyme rMb(13) reconstituted with Fe-2,4-diacetyl deuteroporphyrin IX complex 13 was designed. The porphyrin framework of this metalloenzyme was directly modified with electronwithdrawing acetyl groups to enhance the electrophilic reactivity of the metal-carbenoid species (Fig. 16b).62 Moreover, genetic incorporation of N-methyl histidine (NMH) at the His93 heme axial position was found to further improve the cyclopropanation activity of rMb(13) up to > 1,000 Ton (Fig. 16c). Notably, the resulting rMb(13)(H64V/V68A/H93NMH) variant exhibits sufficient catalytic activity even for the less-reactive electron-deficient alkene.

2.09.7

Reactions by Co and Ni porphyrinoids in hemoproteins

In nature, metal coordinated porphyrinoid complexes other than iron complexes are common cofactors in proteins. Representative metal cofactors are cobalamin and the F430 pigment, representing cobalt and nickel complexes, respectively (Fig. 2). The structures

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Engineering of hemoproteins

of enzymes containing these metal cofactors are relatively complicated and some of them are unstable under aerobic conditions. In addition, the structures of the naturally occurring cofactors are also complicated. Therefore, simplified structural and functional models of these enzymes are required to understand their catalytic mechanisms.

2.09.7.1

Hemoprotein reconstituted with cobalt porphyrinoid

The best known cobalt-containing porphyrinoid in nature is vitamin B12, a cyanocobalamin within the cobalamin family. Cobalamin consists of a cobalt complex of a partially hydrogenated corrin derivative. Corrin is a well-known porphyrinoid which has a structure similar to that of porphyrin but lacks one of the four meso carbons of porphyrin. Corrin is a mono anionic ligand. Thus, the corrin ligand is can stabilize low-valent metal species. In fact, the reactions catalyzed by cobalamin-dependent isomerases and methyltransferases include the low-valent Co(I) species as a reaction intermediate.63 Therefore, a tetradehydrocorrin cobalt complex has been investigated as a model complex for structurally complicated cobalamin. For example, methionine synthase is a representative cobalamin-dependent enzyme responsible for synthesis of methionine from homocysteine via a methyl transfer reaction. In the catalysis, there are two key intermediates, a Co(I)-cobalamin complex with tetracoordinated structure and organometallic species involving a Co(III)eCH3 bond.64 The former species has strong nucleophilicity to generate the methylated cobalt complex from methylfolic acid. The latter species acts as a methyl group donor to provide methionine via an SN2-like reaction. However, the enzymatic mechanism has not completely been clarified due to challenges imposed by the large size of the protein composites. Myoglobin has been investigated as a simplified protein model for methionine synthase because the cobalamin-binding domain of the enzyme is known to have a critical histidine residue which acts as an axial ligand and because myoglobin also has an axial histidine ligand.65 As shown in Fig. 17a, cobalt tetradehydrocorrin 14, simplified from cobalamin, was found to be suitable for use as an artificial cofactor of myoglobin because the X-ray crystal structure analysis reveals that the Co(II) complex is located in the normal heme pocket without significant perturbation of the overall structure of myoglobin.65a The reconstituted protein rMb(14) was found to promote the following reactions in a manner similar to the methionine synthase reaction; (i) generation of the Co(I) species of 14 in the protein matrix, (ii) Co(III)eCH3 bond formation upon the addition of a methyl group doner such as methyl iodide, and (iii) methyl group transfer to the N-atom of imidazole in the distal histidine (Fig. 17b and c).65b In particular, the structure of the tetracoordinated cobalt complex in the protein was directly detected by X-ray crystal structure analysis. In this structure, the axial histidine is deviated from its normal position as a result of cleavage of the Co(II)eHis93 bond induced by soaking of dithionite into Mb(14) crystals. Furthermore, it was found that the cobalt-bound methyl group gradually shifts toward the imidazole ring of His64. This system provides a useful model of the methyl group transfer reaction catalyzed by methionine synthase and is expected to provide important insights into understanding its mechanism.

Fig. 17 (a) Chemical structure of cobalt tetradehydrocorrin 14. (b) Crystal structure of rMb(14) with four-coordinated cobalt (I) species. (c) rMb(14)-mediated methyl group transfer to distal His93 residue.

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227

Fig. 18 Chemical structure of (a) nickel tetradehydrocorrin 15 and (b) nickel didehydrocorrin 16. (c) rMb(15)-promoted methane generation from methyl iodide. (d) 16-meditated methane generation from Met7 residue in Cyt b562.

2.09.7.2

Hemoprotein reconstituted with nickel porphyrinoid

Nickel porphyrinoid F430 is found in methyl-coenzyme M reductase (MCR) which is responsible for methane generation and its reverse reaction, the anaerobic oxidation of methane.66 MCR contributes to the final step of energy metabolism in methanogenic bacteria, where methyl-coenzyme M reacts with coenzyme B to yield methane and the corresponding heterodisulfide species. The F430 framework consists of hydrocorphin, a highly hydrogenated and monoanionic porphyrinoid to stabilize the nickel species. The key reaction intermediate is expected to be the Ni(I) complex, which has been characterized by spectroscopic and theoretical studies.67 In contrast, several model complexes such as a nickel cyclam derivative have been reported as models of the F430 complex, however, these nickel complexes require a strong reductant to yield the Ni(I) species. Even in this case, myoglobin is a suitable protein for modeling MCR with the nickel cofactor.68 For example, nickel tetradehydrocorrin 15 and nickel didehydrocorrin 16 (Fig. 18) have been investigated as models of the F430 model cofactor. Myoglobin reconstituted with 15, rMb(15), was found to be easily reduced by dithionite, enabling methane to be generated upon addition of methyl iodide instead of methyl-coenzyme M with 1.6 Ton.69 Furthermore, cytochrome b562 reconstituted with 16, rCyt b562(16), also generates methane gas upon irradiation in the presence of photocatalyst Ru(bpy)3 and ascorbate to reduce the nickel species, because CeS bond cleavage of the side chain of Met7 in the heme pocket will be initiated by the Ni(I) species of 16.70 Although the reaction mechanism of the native enzyme continues to be investigated, our study indicates that the Ni(I) species is essential for generating methane gas in the model system.

2.09.8

Conclusion

This chapter has described the modification of myoglobin, a simple oxygen-storage protein with the heme b cofactor, to convert to artificial metalloenzymes. In a series of the studies, it was confirmed that substitution of the native cofactor with an artificiallycreated metal complex in myoglobin or other heme b-dependent proteins is a highly effective strategy for regulating these physicochemical properties and reactivities. Furthermore, myoglobin reconstituted with metalloporphyrinoids can provide new models to represent the complex structure of metalloenzymes as well as new artificial biohybrid catalysts capable of promoting non-biological reactions. In addition, the use of mutagenesis to optimize the amino acid residues in the heme pocket and control the structure and reactivity of the artificial metal cofactor will contribute to the generation of more efficient artificial enzymes. The amino acid residues of the heme pocket act as a second coordination sphere which facilitates catalytic reactions and plays an important role in substrate specificity, and regioselectivity and stereo-selectivity. The present body of research indicates that hemoproteins reconstituted with artificial cofactors will generate new artificial metalloenzymes capable of catalyzing significant transformations of organic molecules and providing insights into understanding the catalytic mechanisms of native enzymes.

References 1. Bertini, I.; Gray, H. B.; Stiefel, E. I.; Valentine, J. S. Biological Inorganic Chemistry: Structure and Reactivity, University Science Books, 2007. 2. Chapter 9: Iron in Heme and Related Proteins. In Handbook on Metalloproteins; Turano, P., Lu, Y., Bertini, I., Sigel, A., Sigel, H., Eds., Marcel Dekker, 2001; pp 269–356. 3. Kendrew, J. C.; Bodo, G.; Dintzis, H. M.; Parrish, R. G.; Wyckoff, H.; Phillips, D. C. A Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis. Nature 1958, 181, 662–666. 4. Perutz, M. F.; Rossmann, M. G.; Cullis, A. F.; Muirhead, H.; Will, G.; North, A. C. T. Structure of Hæmoglobin: A Three-Dimensional Fourier Synthesis at 5.5-Å. Resolution, Obtained by X-Ray Analysis. Nature 1960, 185, 416–422. 5. Phillips, G. N.; Paoli, M.; Nagai, K. Heme Proteins: Oxygen Storage and Oxygen Transport Proteins. In Handbook of Metalloproteins; Messerschmidt, A., Huber, R., Poulos, T., Wieghardt, K., Eds.; 1; John Wiley, 2001; pp 3–30. 6. Hargrove, M. S.; Barrick, D.; Olson, J. S. The Association Rate Constant for Heme Binding to Globin Is Independent of Protein Structure. Biochemistry 1996, 35, 11293– 11299. 7. (a) Teale, F. W. J. Cleavage of the Haem-Protein Link by Acid Methylethylketone. Biochim. Biophys. Acta 1959, 35, 543; (b) Yonetani, T. Studies on Cytochrome c Peroxidase: X. Crystalline APO- and Reconstituted Holoenzymes. J. Biol. Chem. 1967, 242, 5008–5013; (c) Wagner, G. C.; Perez, M.; Toscano, W. A.; Gunsalus, I. C. Apoprotein Formation and Heme Reconstitution of Cytochrome P-450cam. J. Biol. Chem. 1981, 256, 6262–6265.

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2.10

The biochemistry and enzymology of zinc enzymes

Guillermo Bahra,b, Pablo E. Tomatisa,b, and Alejandro J. Vilaa,b, a Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET-UNR), Rosario, Argentina; and b Area Biofísica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina © 2023 Elsevier Ltd. All rights reserved.

2.10.1 2.10.2 2.10.3 2.10.3.1 2.10.3.2 2.10.4 2.10.4.1 2.10.4.2 2.10.4.3 2.10.4.4 2.10.5 2.10.5.1 2.10.5.1.1 2.10.5.2 2.10.5.2.1 2.10.5.2.2 2.10.5.3 2.10.5.4 2.10.5.5 2.10.5.6 References

Introduction Zinc is an essential transition metal ion for life Cell biology of zinc Distribution and ubiquity of zinc proteins in the proteomes Zinc homeostasis Chemistry of zinc enzymes Chemical properties of zinc Zinc ligands and their role in modulating the activity of catalytic zinc centers The impact of second-shell ligands in zinc reactivity The pKa of zinc-bound water molecules Zinc-dependent enzymes Zinc lyases Carbonic anhydrases Zinc hydrolases Mononuclear zinc hydrolases Binuclear zinc hydrolases Zinc alcohol dehydrogenases and other zinc-dependent oxidoreductases Zinc transferases Zinc isomerases Zinc ligases

232 233 234 234 235 236 236 237 238 239 241 241 241 244 244 247 253 254 256 256 258

Abbreviations aa-AMP Aminoacyl-adenylate aaRS Aminoacyl-tRNA synthetase AAS Atomic Absorption spectroscopy ADH Alcohol dehydrogenase APD Aminopeptidase BTZ Bisthiazolidine CA Carbonic anhydrase CPD Carboxypeptidase DFT Density functional theory EC Enzyme class EPR Electron paramagnetic resonance EXAFS Extended X-ray absorption fine structure spectroscopy F6P D-fructose 6-phosphate FTase Farnesyltransferase GGTase-I Geranylgeranyltransferase ICP-MS Inductively coupled plasma mass spectrometry LAP Leucine aminopeptidase MBL Metallo-b-lactamase MDR Medium-chain dehydrogenase/reductase MMP Matrix metalloproteinase MMTZ Mercaptomethylthiazolidine NMR Nuclear magnetic resonance PDB Protein data bank Pfam Protein family

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00148-5

231

232

The biochemistry and enzymology of zinc enzymes

PMI Phosphomannose isomerase PNPA para-Nitrophenyl acetate SCR Short-chain dehydrogenases/reductase QM/MM Hybrid quantum mechanics/molecular mechanics calculations XANES X-ray absorption near edge structure spectroscopy

Abstract Zn2þ is an essential transition metal ion for the life cycle of all living organisms. This metal ion plays key roles in stabilizing proteins, as a cellular messenger, but it is mostly present as an essential cofactor in enzymes. The intracellular levels of Zn2þ are tightly regulated to prevent adventitious zinc binding to other metalloproteins, to limit its toxic effects at high concentrations, and (most importantly) to ensure the proper delivery to metalloproteins that require zinc for their function. Zinc plays catalytic roles in enzymes from all six classes (oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases), being mostly present in hydrolases. The key factor in the biochemical action of zinc resides is its role as Lewis acid by three main mechanisms: (1) activating a water ligand for deprotonation thus leading to formation of a hydroxide (a potent nucleophile) at neutral pH, (2) polarizing the enzyme substrate or (3) stabilizing negatively charged reaction intermediates. This simple role is tuned according to the specific physiological requirements by the zinc ligands and by a hydrogen bond network (outer sphere ligands) in the protein structure.

2.10.1

Introduction

Zinc is essential for life, being the second most abundant transition metal ion after iron in living organisms, from bacteria to mammals.1–6 In particular, zinc is present in ca. 10% of human proteins, while there are more than 300 enzymes that exploit the properties of the Zn2þ ion for its biochemical function. These metalloproteins have Zn2þ either for catalytic or structural roles.7 Zinc was not biologically available before the Great Oxidation event (ca. 2.6 billion years ago). However, the Zn2þ concentration was low in an early sulfidic ocean.8 The conversion of metal sulfides into metal oxides upon the oxygenation of deep waters led to a dramatic increase in the bioavailability of zinc.8–10 Since eukaryotes contain much more zinc binding proteins than prokaryotic organisms, development of the cell nucleus has been linked in terms of evolutionary age with a dramatic rise in the availability of zinc ions.9 It has been suggested that many current zinc proteins may were originally cobalt proteins, despite some authors have contested this hypothesis.11,12 The concentration of zinc is now estimated at ca. 30 ppb in seawater and 75 ppm in the earth’s crust.13 Wolfgang Maret has masterfully defined different phases in the history of the study of zinc in life sciences,6 based on selected findings that changed the approach to study zinc biochemistry. The first study of zinc in biology dates back to Pasteur’s days. Jules Raulin, trained by Louis Pasteur in the study of microorganisms, was interested in studying the growth of the fungus Aspergillus niger. He experimented different chemical media with defined composition that allowed him to identify the optimal growth conditions for this fungus. In doing so, he discovered that zinc was essential for this microorganism, as reported in 1869. This seminal article, entitled “Études chimique sur la végétation, et recherches sur le développement d’une Mucédinée, dans un milieu artificial”, was the first evidence of the role of zinc in life.14 In 1934, Todd, Hart and coworkers reported that zinc was an essential growth factor for rats,15 with zinc deficiency leading to irreversible effects on the animals. This was the first evidence of the essentiality of zinc in nutrition. Another milestone was achieved by David Keilin and T. Mann in Cambridge in 193916 who purified carbonic anhydrase from 10 L of blood and identified zinc as a main cofactor of this enzyme, this being the first physiological role attributed to zinc. They concluded: “Like iron and copper, zinc forms probably with different proteins several compounds having different properties and function”. The identification of copper and iron-containing proteins preceded the characterization of a zinc-dependent enzyme, due to its colorless nature. In this regard, the early identification of zinc as a cofactor in carbonic anhydrase (being colorless) was a biochemical tour-de-force. Carbonic anhydrase has been the most widely studied zinc enzyme, with more than 20,000 articles devoted to its study, and > 1200 crystal structures of different isozymes, mutants and inhibited variants available in the Protein Data Bank. Beyond its undeniable physiological relevance, carbonic anhydrase has been a paradigmatic enzyme to understand the mechanistic bases of zincdependent hydrolases in general.17–21 Works in carbonic anhydrase also helped to develop methods to study the metal site of a d10 metal ion (not amenable to most spectroscopic techniques),22,23 to understand the role of second shell ligands in metalloenzyme catalysis,24,25 and to provide the bases for the inhibition of many zinc enzymes.26 Carboxypeptidase was characterized as a zinc-dependent enzyme in 1954 by Vallee and Neurath.27 The first crystal structure of a zinc enzyme was that of bovine carboxypeptidase A, by William Lipscomb and coworkers.28–30 Indeed, bovine carboxypeptidase A was the third protein to have its structure solved, after myoglobin and lysozyme. The structure of the native, unbound form of the enzyme, was followed by a series of reports on the structure of carboxypeptidase complexed to inhibitors, substrate mimics and transition state analogues.31,32 This seminal approach set the bases for the structural dissection of the catalytic mechanism of an enzyme.

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233

The field of zinc enzymology flourished in the 1960s to the 1980s with the works of the groups of Bert Vallee, William Lipscomb, Joseph Coleman, Sven Lindskog, Ivano Bertini, that helped dissecting the role of the zinc in different types of enzymes.22,23,30,33–39 Even after these efforts, in the early 1990s Auld and Vallee claimed: “there is a great need to inspect the chemical properties of zinc complex ions containing combinations of nitrogen, oxygen, and sulfur ligands, as well as those that only contain sulfur ligands”.4 In this work, the authors analyzed for the first time the presence of characteristic zinc ligand motifs and spacings in the protein sequence, before bioinformatics was an established field. Synthetic efforts played a key role in the understanding of the chemistry of zinc enzymes, particularly regarding water activation. The work of Eichi Kimura, Gerard Parkin, Heinrich Vahrenkamp, R.S. Brown, Steve Lippard, among others, was essential in this regard.40–45 Finally, spectroscopy played a key role in the study of the mechanism of zinc enzymes. Since Zn2þ is a d10 metal ion, it can only be studied by X-ray absorption techniques such as XANES and EXAFS, providing details on the donor atom and metal-ligand distances.46,47 However, metal substitution of the native metal ion by Co2þ and Cd2þ has (and still is) a powerful approach to study the metal sites of zinc enzymes in the resting state and during turnover.23,36,48–52 Despite some studies have indicated that Co2þ derivatives tend to expand their coordination sphere by binding of one additional water molecule in some occasions,53,54 this strategy enables the use of electronic absorption spectroscopy, CD, MCD, EPR and NMR for zinc enzymes.55–60 The coupling of these techniques with stopped-flow or rapid-quench-flow devices has enabled trapping otherwise elusive reaction intermediates.60–63 In any case, the understanding of the catalytic mechanism of the different zinc enzymes herein described has been achieved by the combination of all these approaches: structural, enzymological, spectroscopic, and (finally) site-directed mutagenesis to assess the role of active site ligands in substrate recognition and catalysis. The knowledge built along several decades have also enabled the de novo design of zinc sites in non-native protein scaffolds, as witness by the early efforts of Bill de Grado,64,65 Maurizio Sollazzo66 and more recently by Vince Pecoraro,67–69 David Baker,70 and Akif Tezcan.71 Upon the availability of better and faster sequencing techniques that provided a huge amount of genomic information, a bioinformatics approach developed by Rosato and Bertini provided a larger perspective of the zinc metallome in different organisms.72–74 They analyzed the information available in the genomes and proteome databases looking for characteristic zinc binding patterns. This revealed that ca. 10% of the human genome encodes zinc-binding proteins. Half of them are putative zinc finger proteins, most of them with unknown function. Eukaryotes have a larger number of zinc proteins (9%) than bacteria and archaea (5–6%), due to the enormous number of zinc-binding proteins involved in gene regulation (see below).

2.10.2

Zinc is an essential transition metal ion for life

Zinc is an essential micronutrient for cells since it is the driving force of the chemistry occurring in many key reactions in the life cycle of a cell.3,13,75,76 The impact of zinc deficiency in humans was first demonstrated in 1963 by Ananda Prasad.77 Nowadays, zinc deficiency is an emerging global health problem that affects almost 2 billion people in the world, mostly in developing countries, with a severe impact on development.78 This has led to the development of different zinc dietary supplementation strategies.79 Zinc is also an essential trace element for plant growth, and as a result, zinc deficiency has a detrimental impact in agriculture.80,81 Zinc is the only metal ion that is present in enzymes belonging to all six classes of enzymes according to the classification of the International Union of Biochemists. Despite most zinc-dependent enzymes are hydrolases,73 there are zinc-dependent lyases, transferases, ligases, isomerases and even oxidoreductases, although zinc is a non-redox cofactor. Table 1 summarizes some relevant enzymes belonging to the different groups, as well as their metal binding sites. In this chapter, we aim to cover the role of zinc as a catalytic cofactor in enzymes. The multifaceted roles of zinc in biochemical processes reveal that, in addition to its chemical Table 1

Examples of Zn2þ enzymes, indicating their Zn2þ content, coordinating residues and reaction catalyzed.

Enzyme Class I: Oxidoreductases Alcohol dehydrogenase Class II: Transferases Farnesyl Transferase Class III: Hydrolases Alkaline Phosphatase Aminopeptidase Carboxypeptidase Matrix metalloproteinase Metallo-b-lactamasea Phospholipase C Purple acid phosphatase

Metal ions

Coordination sphere

Function

1  Zn

1  His, 2  Cys, 1  H2O

Oxidation of primary and secondary alcohols

1  Zn

1  His, 1  Cys, 1  Asp, 1  H2O

Transfer of prenyl groups to sidechain of Cys residues

2  Zn 2  Zn 1  Zn 1  Zn 2  Zn 3  Zn

3  His, 3  Asp, 1  Ser, 1  H2O 3  Asp, 1  Lys, 1  Glu, 1  H2O 2  His, 1  Glu, 1  H2O 3  His, 1  H2O 4  His, 1  Cys, 1  Asp, 2H2O 5  His, 2  Asp, 1  Glu, 1  Trp (main chain), 3  H2 O 2  His, 1  Asn, 1  Asp, 1  H2O

Hydrolysis of phosphate monoesters Cleavage of N-terminal amino acids from peptides Cleavage of C-terminal residues of proteins Degradation of extracellular matrix proteins Hydrolysis of b-lactam antibiotics Hydrolysis of phospholipids, yielding diacylglycerol and phosphorylated head groups Hydrolysis of phosphate monoesters

1  Zn

(Continued)

234

The biochemistry and enzymology of zinc enzymes Examples of Zn2þ enzymes, indicating their Zn2þ content, coordinating residues and reaction catalyzed.dcont'd

Table 1 Enzyme

Metal ions

Coordination sphere

Function

Thermolysin

1  Zn

2  His, 1  Glu, 1  H2O

Class IV: Lyases Carbonic anhydrase

Hydrolysis of peptide bonds containing hydrophobic residues

1  Zn

3  His, 1 H2O

Reversible interconversion of carbon dioxide and carbonic acid

1  Zn

2  His, 1  Glu, 1  Gln, 1  H2O

Reversible isomerization of D-mannose-6 phosphate to D-fructose 6-phosphate

1  Zn

2  His, 1  Cys, 1  H2O

Ligation of tRNA and cognate aminoacid

Class V: Isomerases Phosphomannose isomeraseb Class VI: Ligases tRNA synthetase

Only catalytic Zn2þ ions have been included. a Subclass B1 of Metallo-b-lactamases. b C. albicans enzyme (CaPMI).

versatility, there is a fine tuning of its physicochemical properties through their direct environment (zinc ligands, see Section 2.10.4.2) and different shells of residues surrounding the zinc active sites (see Section 2.10.4.3). In addition to these already diverse functions, zinc is also ubiquitous as a structural element, such as in superoxide dismutase, a copper-dependent enzymes, and in zinc fingers. Moreover, these transcription factors represent the largest family of zinc-containing proteins in eukaryotic proteomes.73 Zinc also plays a key role in signal transduction.6 Zinc is the second most abundant transition metal ion (after iron) in the human body, represented by more than 300 different enzymes using this metal ion as an essential cofactor.6 In addition, zinc is highly ubiquitous in eukaryotic organisms due to their presence in transcription factors (zinc fingers), playing a structural role. There is an estimate of 2 g of zinc per 70 kg of human body weight, but the total zinc concentration in human cells is ca. 0.2– 0.3 mM,82 which almost compares to ATP levels in certain circumstances. While the measurement of total zinc can be achieved by classical analytical techniques such as ICP-MS or AAS, addressing the distribution of zinc among protein-bound, non-protein bound Zn2þ and “free” zinc has represented a methodological challenge in the last two decades. Different names, such as labile zinc, loosely bound zinc, free zinc and other terms have been employed to represent the transport, flux and storage of zinc that is not tightly bound to proteins. Binding to small molecules in the cell, such as GSH, ATP, or free amino acids should also be considered since, despite the binding affinities of these molecules towards Zn2þ are much smaller than those observed in zinc enzymes, this may be overridden by the higher concentration of these molecules in the cell. The analysis of the involved equilibria requires a precise description of the thermodynamics and kinetics of zinc binding to the pool of potential metal-binding molecules in the different cellular compartments. This should also consider the competition with other metal ions (see Section 2.10.3). Recent advances in Mass Spectrometry have paved the way for the speciation of zinc (and other metal ions) in cells.83–86 The determination of the levels of non-protein bound Zn2þ is more challenging since it requires the use of specific fluorescent probes or sensors coupled with fluorescence microscopy.82,87 These values also change with the cell metabolism. Thus, measuring the zinc fluxes in the cell has represented a great challenge.82,87–89 Many experiments performed in cell cultures may be also misleading since the in vivo availability of Zn is highly dependent on the metal buffering capacity of the environment. Indeed, in the case of zinc-dependent b-lactamases (MBLs, Section 2.10.5.2.2), these enzymes are metal loaded in the periplasmic space, where the zinc levels are not regulated and mostly depend on the metal availability in the external milieu. The zinc cargo of these enzymes is critical, since bacterial survival in the presence of b-lactam antibiotics depends on the fact that MBLs are active by binding of the essential metal ion, thus being able to hydrolyze the antibiotic. A native immune response of mammalian hosts elicits a massive metal ion starvation, that affects zinc levels in the periplasm, ultimately determining the fate of the bacterial cell in the presence of antibiotics. As a result, many estimations of bacterial resistance in the clinical laboratory are misleading since they may not represent the in vivo situation in which the zinc availability is highly variable.90–95

2.10.3

Cell biology of zinc

2.10.3.1

Distribution and ubiquity of zinc proteins in the proteomes

The annotation of the metalloproteomes is not straightforward, since it requires a bioinformatics approach that considers information from protein structure databases and protein domains, as well as a detailed curated analysis of the data. This can result in the prediction or annotation of metalloproteins with known folds and metal binding domains up to putative proteins with novel folds and new metal binding motifs. The first approach from Rosato and Bertini dates back to 2005.96 In this work, they analyzed the human genome97 using three complementary approaches: structure-, annotation and domain-based. The structural approach relied in the identification of metal

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235

binding patterns based on the zinc structures available at that moment in the protein data bank, while the domain-based approach relied on the Pfam domain database.98 In total, 314 different domains from the Pfam library were identified as zinc binding domains. However, the use of the Pfam approach only leads to the identification of many false positives. The combined use of the domain analysis with structural data results an efficient filtering strategy for discarding wrong predictions. This approach also allowed the identification of Pfam zinc binding domains that were not originally annotated as metalloproteins. A similar search performed over 57 different genomes, encompassing bacterial, plant and animal species7,73,74 revealed that the amount of zinc binding proteins represented from 4% to 10% of the proteome. In general, eukaryotes present a significant larger number of zinc proteins (8.8%) compared to bacteria and archaea (from 5% to 6%). This different is mostly due to the larger repertoire of zinc-dependent regulatory proteins in eukaryotes. Among the studied microorganisms, Rhodopirellula baltica, a marine bacterium, was identified as that with the lowest amount of zinc-binding proteins (3.9%). Archaea such as the marine thermophile Pyrobaculum aerophilum contains 5% of zinc proteins. In contrast, 7.9% of the 682 proteins of the obligate parasite Mesoplasma florum (with a small genome) were identified as zinc-binding proteins. The zinc-proteome in these organisms is expected to reflect the bioavailability of this metal ion in their natural environment. In Archaea and Bacteria, the size of the zinc proteome correlates with the average growth temperature of these organisms: the proteome of hyperthermophiles contains an average of 7% of zinc proteins, versus 6% for thermophiles, 5.3% in mesophiles and 4.5% in psychrophilic organisms. This can be attributed to a larger presence of zinc ions playing structural roles, stabilizing proteins at higher temperatures. In contrast, there are no significant differences in the zinc protein content between anaerobic and aerobic microorganisms. Among Eukarya, the human zinc proteome was 9.2%, being intermediate between a minimal value of 8% found for the worm Caenorhabditis elegans and the model plant Arabidopsis thaliana, and 10.2% for Drosophila melanogaster.7 When the zinc proteins related to gene regulation are removed, the number of zinc-binding proteins is similar to that found in prokaryotes. As a result, most zinc binding proteins in Archaea and Bacteria are zinc-dependent enzymes. In this regard, the distribution of putative zincbinding enzymes among the six enzymes classes described in Table 1 is similar in Archaea and Bacteria, with hydrolases representing > 50% of the zinc proteome. Instead, in Eukarya, there is a larger number of ligases, a fact that is related to the tight control of gene expression present in these organisms. The rise of the first multicellular organisms is linked to the second large increase in zinc bioavailability,74 requiring zinc fingers as transcription factors and the role of zinc as messenger.

2.10.3.2

Zinc homeostasis

In the cells, there is a tight competition for the binding of metal ions of the different proteins that require metal cofactors. In general, the levels of transition metal ions are limited since they may be toxic at high concentrations. The Irving-Williams series defines the affinities of divalent metal ions towards a given ligand99: Mg2þ < Mn2þ < Fe2þ < Co2þ < Ni2þ < Cu2þ > Zn2þ Thus, Zn2þ ions are second to Cu2þ in terms of affinity. Since this trend reflects a thermodynamic equilibrium between bound and unbound forms, it holds strictly when all metal ions have the same concentration, which is not the case within the cell and even in the natural extracellular milieu. In addition, the metalation site must be considered for each case. If all metalloproteins bound their native metals coupled to their synthesis in the ribosome, the nascent polypeptide would be unable to discriminate among the different metal ions, and metal binding would be dominated by the preferences dictated by the Irving-Williams series. Also, the redox properties of some of these metal ions must be considered, since Cu2þ is reduced to Cu1þ by specific proteins in some cellular environments, and iron is more commonly found as Fe3þ. In this regard, the non-redox nature of the Zn2þ makes it easier to describe its cellular homeostasis. This is, however challenged by its silent spectroscopic nature. Protein metalation is a tightly controlled process within the cell. On one hand, transition metal ions are essential for many biological functions, but if their levels are too high, they are toxic. This requires a fine control not only of the intracellular levels of the different metal ions in all cellular compartments, but also a balance in the mechanisms of import, export, storage, transport and (in some cases) specific delivery of the metal ions to their target proteins. As a result, cells can react upon excess or deficiency of certain metal ions in the environment. On the other hand, mismetalation can be deleterious for the cell.100,101 In general, this is avoided by several mechanism that override the trend imposed by the Irving-Williams series: (1) the presence of specific metallochaperones, that are responsible of delivering the correct metal ion to the target protein or (2) the different metal ion concentrations in distinct cellular compartments, that can drive proper metalation processes.102 Computational studies have revealed that, in the absence of metallochaperones, the metal specificity of a given ligand set in a protein framework is mostly determined by the metal ion concentration in the native cellular compartment.103 All these processes have been thoroughly discussed and review elsewhere,101,104–110 and here we aim to provide a succinct discussion of zinc homeostasis in different organisms. Cellular zinc homeostasis is maintained by Zn sensors that act by repressing uptake or efflux mechanisms. Bacteria react to metal ion changes by activating the expression of specific genes that are generally controlled by the so-called metalloregulatory proteins, which are transcription factors that experience a conformational change upon binding of defined metal ions.101,104 In the specific case of zinc, there is a battery of specific zinc sensors that regulate gene transcription as a response to metal binding. The affinity of these metalloregulators against zinc results in buffering the available metal ion pool. In general, cytosolic zinc enzymes display metal binding affinities in the picomolar range. Upon an excess of metal ions (sensed by these regulators), cells respond by repression of uptake or derepression of efflux and storage proteins. The total zinc concentration in the bacterial cytoplasm can range between 0.1 mM and 1 mM, but the high binding affinities of these metalloregulatory proteins is so high that there is no free Zn2þ in the cells.111 The specific Zn2þ uptake transcriptional

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The biochemistry and enzymology of zinc enzymes

regulator (Zur) is sensitive to changes of intracellular Zn2þ at femtomolar levels. When Zn2þ levels are high, Zur is metalated and binds the Zur boxes in in the DNA promoter region of its regulon, repressing gene transcription (Fig. 1). Instead, when Zn2þ concentrations are limited, Zur is in the apo form and its conformation leads to detachment of the DNA, derepressing the Zur regulon. In Gram-negative bacteria, this results in the induction of genes encoding the high-affinity ZnuABC Zn2þ transporters. The periplasmic protein ZinT is responsible of recruiting Zn2þ in the periplasm to deliver it to ZnuA. There is also a fine interplay of zinc with many small ligands in different cellular compartments at high concentrations, mostly thiol-containing molecules such as glutathione and bacillithiol. The zinc speciation among the different proteins and small molecules is still unknown, and requires the development and improvement of mass spectrometry-based approaches.83–86 In contrast to this tightly controlled regulation in the cytosol, periplasmic Zn2þ levels are not regulated in most Gram-negative bacteria, depending on the availability of this metal ion in the near environment. Zinc uptake takes place through non-selective porins, such as OmpF and OmpC in E. coli. Bacteria can also secrete small molecules able to bind extracellular zinc and transfer it to membrane transporters. Bacteria are usually challenged by zinc starvation. During an infection, an innate immune system response elicits a massive chelation of Mn2þ and Zn2þ, by releasing proteins with a high metal binding affinity, such as calprotectin. As a result, bacteria are adapted to react to zinc limitation.112 In the proteobacterium Cupriavidus metallidurans, the number of putative zinc binding sites was shown to exceed the total zinc in the cell, revealing that there are many empty (or mismetalated) zinc binding sites.113 A total of 582 zinc-binding proteins were predicted in the yeast proteome (most of them already known).114 Under zinc-replete conditions, mass spectrometry identified 229 zinc proteins over a total of 2500 proteins, that represent 9 million zinc binding sites within the cell. Instead, upon zinc restriction, this value decreased to ca. 5 million zinc binding sites per cell. On the other hand, zinc levels dropped by one order of magnitude (from 20 million to 1.7 million zinc atoms per cell). In other words, zinc replete cells store the metal ion surplus, while under zinc deprivation, many zinc-binding sites are expected to be non-metalated or mismetalated. In eukaryotic cells, organelles act as reservoirs of the excess zinc.107 This role is fulfilled by vacuoles or the endoplasmic reticulum in yeast cells, synaptic vesicles in neurons,115 and secretory vesicles in pancreatic116 and mast cells,117 as representative examples. There are also reports of membrane-bound vesicles that store zinc that have been called zincosomes.118,119 Mammalian zinc transporters have been thoroughly studied from the biochemical and structural aspect to their cellular regulation.120

2.10.4

Chemistry of zinc enzymes

2.10.4.1

Chemical properties of zinc

Zinc is found in nature in its only stable redox state, Zn2þ. This metal ion presents a closed-shell d10 electronic configuration, with an ionic radius of 0.74 Å. As a result, Zn2þ is diamagnetic and colorless, and cannot be examined by most of the spectroscopic

Fig. 1 Mechanisms of Zn2þ homeostasis in Gram-negative bacteria, including Zn2þ import by the ZnuABC transporter, replacement of Zn2þdependent ribosomal proteins, Zn2þ passive entry into the periplasm through porins and through active transport by the action of ZnuD, and the acquisition of this metal ion by the use of zincophores and zinc piracy. Proteins belonging to the Zur regulon are represented in light blue.

The biochemistry and enzymology of zinc enzymes

237

techniques employed in biological inorganic chemistry. In addition, its most abundant isotopes 64Zn (48.63%) and 66Zn (27.9%) are NMR-inactive (I ¼ 0). X-ray absorption-based spectroscopies such as EXAFS or XANES are an exception to this, and these techniques have been widely employed to interrogate zinc binding sites in enzymes. These studies have been complemented by replacement of the Zn2þ ions by other surrogates that reproduce (at least partially) the catalytic properties of the native metal ion. In this regard, Co2þ and Cd2þ have been the most widely used ions for metal substitution. Co2þ has a d7 electronic configuration, and Cd2þis also a d10, but its most ubiquitous isotopes are NMR-active. Zinc as d10 is invisible, but this nature makes it useful for many chemical roles without the risk of undergoing redox processes, in contrast to the two other most abundant transition metal ions in nature: iron and copper. Zn2þ is a strong Lewis acid, i.e., is able to accept an electron pair, and this is its most common role in enzymes. Unlike other firstrow transition metals, its d10 electronic configuration makes it an excellent Lewis acid without undergoing a redox reaction. Zn2þ is frequently involved in catalyzing hydrolytic reactions by polarizing substrates and water molecules. When binding water molecules, this polarization lowers the water pKa. This can result in the availability of a hydroxide group (a potent nucleophile) at physiological pH within the enzyme active site, or in favoring deprotonation of a water molecule for a protonation event. In the absence of zinc enzymes, these reactions only occur at extreme pH values that are not met at physiological conditions. In most of the cases, the activity of zinc enzyme relies in the presence of a zinc-bound water, but other protein residues or exogenous ligands can bind the zinc center. The most common function of zinc in enzymes is to act as a potent Lewis acid favoring: 1) activation of a water ligand for deprotonation and/or nucleophilic attack to the substrate, 2) polarization of the cleavable bond in the substrate, and 3) stabilization of the negative charge developed in a transition state or a reaction intermediate. These diverse roles (Fig. 2) can be present in different combinations, particularly in hydrolases or lyases (such as carbonic anhydrase). Zn2þ, as a d10 metal ion, does not show any preferred geometry stabilized by the ligand field. This fact does not only result in zinc binding sites with coordination numbers ranging mostly from 4 to 6, but it also enables the interconversion between different geometries with no energy cost, favoring the use of zinc in catalyzing reactions that require conformational changes. This is particularly favored by the fast exchange rate of ligands with zinc, that enable binding and detaching of exogenous ligands (and even protein ligands) during catalytic turnover.

2.10.4.2

Zinc ligands and their role in modulating the activity of catalytic zinc centers

In terms of the hard-and-soft acid/base theory formulated by Pearson,121 Zn2þ is a “borderline” metal. In other words, it is intermediate between being “soft” (highly polarizable) or “hard” (not polarizable) and thus, it does not have a strong preference for binding with oxygen (hard), nitrogen (hard) or sulfur (soft) atoms. Zinc binding sites in proteins display a highly variable combination of ligands, donor atoms, and coordination geometries, within a large variety of sequence motifs and protein superfamilies. This results in a broad range of functions that can be tuned by the immediate protein environment. Exhaustive analyses of the crystal structures of zinc centers and quantum mechanical calculations have revealed different trends that provide information on how the coordination chemistry defined by the protein scaffold determines the chemistry of the zinc binding sites.73,122–133 The main difference between structural and catalytic zinc sites is that structural centers lack an exogenous, non-protein ligand, which is generally a water molecule that enables the activation of a substrate or the water itself. More than 90% of the structural sites are four-coordinated, with at least two Cys ligands. There are exceptions, such as structural zinc sites defined at the interface of multimers, that present a geometry resembling a catalytic zinc site with a water ligand, such as in interferon134 and insulin.135 On the other hand, the metal ion in a Zn(Cys)4 site in the Ada protein activates one of the cysteine ligands for

Fig. 2 Basic mechanisms of the chemical role of zinc as a Lewis acid in enzymes. The zinc ion can (1) activate a water molecule, inducing its ionization to generate a nucleophilic hydroxide, (2) orient a water molecule so that it is polarized and activated by a general base to generate a hydroxide, or (3) experience the displacement of the water by the substrate as an exogenous ligand that is polarized by zinc binding.

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The biochemistry and enzymology of zinc enzymes

methylation, repairing methylphosphotriesters in DNA.136–139 Despite these exceptions, there are defined trends that help defining the features of catalytic zinc sites and understanding their chemistry. Moreover, these trends have been of help to guide the de novo design or redesign of catalytic zinc centers in proteins and peptides.68–71,140–144 Negatively charged ligands decrease the electronegativity of a Zn2þ ion by transferring charge to the metal ion, therefore tuning its strength as Lewis acid. In particular, Cys ligands transfer more charge than Asp, Glu or hydroxide ligands.129 This accounts for the larger abundance of Cys ligands in structural zinc sites, in which the electronegativity of the Zn2þ ion is decreased, therefore decreasing its Lewis acid acidity.145 Cys ligands would therefore prevent unwanted side-reactions at the zinc site. This would explain the absence of structural zinc sites with a His4 ligand set. Based on these results, Lee and Lim have proposed, as a rule of thumb that a zinc center with more than two Cys ligands is a poor electron acceptor and can only play a structural role.129 In contrast, Cys ligands are less ubiquitous in catalytic zinc sites, endowed with a higher positive charge and an enhanced acidity of the metal ion that enable the activation of exogenous ligands. There are a few catalytic zinc sites with two Cys ligands, all of them with a coordination number of four. One notable exception is blasticidin S deaminase, with three cysteines and one water molecule as zinc ligands.146 Structural zinc sites are mostly four-coordinated. Instead, when the zinc plays a catalytic role, the metal binding sites have at least one exogeneous ligand, and their coordination number and geometry are very diverse, according to the structural requirements of the protein fold and the catalytic function. The most common protein ligands are His (47%) > Asp/Glu (36%) [Cys (13%) which, in contrast to structural sites are less common in catalytic zinc centers. His ligands bind this metal ion in the neutral form of their imidazole ring by the N32 or the Nd1 atoms, with a 3:1 preference for the former, that provides a sterically less restricted coordination mode (Fig. 3). The N binding atom depends on the ligand binding motif, that imposes specific geometric restrictions.125 Carboxylate binding to Glu and Asp residues can take place either with syn or anti conformations (Fig. 3). The syn conformation is more frequent, with the metal ion positioned in the same plane of the carboxylate moiety.147–149 The metal-ligand distances in structural zinc sites resemble more closely those from synthetic zinc coordination compounds, as results from a comparison of data from the Protein Data Bank (PDB) with the Cambridge Structural Database (CSD).129 Catalytic zinc sites show longer ZneN distances (e.g., 2.16 Å vs. 2.05 Å for tetrahedral geometries) and ZneO distances (Table 2).129 The ZneO distances are particularly longer for Zn-Wat moieties in catalytic centers (in tetrahedral centers, 2.28 Å vs. 2.00 Å for model complexes). This can be attributed to the network of hydrogen bonding interactions present in the Zn-bound water molecules in the enzyme active sites that contribute to activate and orient the water ligand. Four-coordinated catalytic zinc sites are the most common, with three protein ligands and a water molecule (in 49% of the cases), generally adopting a tetrahedral geometry. Then, 43% of the sites contain four protein ligands, while 8% have five protein ligands.123 DFT calculations predict that tetrahedral zinc sites in proteins have the least strained coordination geometry, while 5and 6-coordinated sites would be the result of the protein strain, despite the null ligand field stabilization energy.150 Andreini and coworkers have shown that ca. 80% of the zinc sites identified from genomic data can be described based in 10 structural motifs conserved in a large group of protein superfamilies.123 Zinc proteins span ca. 300 protein superfamilies, with two thirds of them corresponding to structural sites. Instead, catalytic zinc binding sites are distributed in 68 superfamilies. Polynuclear zinc sites have protein ligands bridging the zinc ions, usually carboxylate moieties from Glu or Asp residues. The zinc-zinc distance decreases upon larger number of bridging oxygen atoms, since the electrostatic zinc-oxygen interactions compensate the repulsion between the positively charged Zn2þ ions. Binuclear metallo-b-lactamases are an exception, since they only have a bridging water/hydroxide ligand, with no bridging protein ligands. Consequently, these enzymes show the largest ZneZn distances among binuclear enzymes (ca. 3.5 Å, vs. 3.0 Å) and are endowed with a large flexibility (see specific section below).

2.10.4.3

The impact of second-shell ligands in zinc reactivity

The definition of second-shell ligands dates back to 1913, when Werner defined them as “groups directly coordinated to a ligand in the first sphere”.151 The first (obvious) criterion involves residues making hydrogen bonds with the metal ligands. However, other aspects such as steric restrictions, hydrophobic contacts and solvation effects should also be included into this category.152 The environment of zinc ligands is defined by a cluster of charged residues that are generally involved in hydrogen bonds with the zinc ligands, tuning their electronic structure and ultimately, the chemical properties of the zinc site. There is a 2:1 ratio of polar vs. non-polar residues in the second shell,125 being mostly buried residues, in contrast to the first coordination sphere of the zinc ion. The specific role of second-shell ligands in modulating the pKa of zinc-bound water ligands is described in detail in the following section.

Fig. 3

Different metal coordination modes for His (binding through Nd1 and N32) and carboxylates (syn- and anti-conformations).

The biochemistry and enzymology of zinc enzymes Table 2 Zn type L4 L4 L5 L5 L6 L6

CSD Cat CSD Cat CSD Cat

239

Distances between Zn2þ and its coordinating atoms, in structures from the Cambridge Structure Database (CSD) and catalytic sites in PDB structures (Cat). Zn-S

Zn-N

Zn-O

Zn-O(water)

2.32  0.05 2.38  0.18 2.42  0.13

2.04  0.04 2.16  0.12 2.10  0.08 2.08  0.07 2.16  0.05 2.10  0.10

1.97  0.03 2.09  0.17 2.09  0.04 2.16  0.19 2.10  0.06 2.19  0.17

2.00  0.04 2.28  0.23 2.01  0.04 2.24  0.25 2.11  0.06 2.27  0.11

Tetracoordinated (L4), Pentacoordinated (L5) and Hexacoordinated (L6) Zn2þ sites are shown separately. Distances are shown as mean  standard deviation. Adapted from Lee, Y.; Lim, C. Physical Basis of Structural and Catalytic Zn-Binding Sites in Proteins. J. Mol. Biol. 2008, 379 (3): 545–553. doi: 10.1016/j.jmb.2008.04.004.

As previously discussed, His residues are the most frequent ligands in catalytic zinc sites. The Zn2þ-His-carboxylate(Asp/Glu) triad and the Zn2þ-His-backbone triad (Fig. 4) are two common motifs present in zinc-dependent lyases, hydrolases and oxidoreductases, i.e., enzymes with quite diverse functions and coordination sites.126,153 In the case of the carboxylic acids, the effect depends on whether the Asp/Glu residue is protonated or not, that ultimately defines the protonation state of the imidazole ligand. An interaction with a carboxylate group increases the imidazolate character of the His, impacting on the acidity and nucleophilicity of a zinc-bound water and the zinc binding affinity of the ligand set. DFT calculations have revealed that the solvent accessibility of the metal site plays a key role in tuning the impact of this conserved triad on the metal site reactivity.154 Specifically, a carboxylate group increases the HOMO energy of the zinc core and the reactivity of the attacking nucleophile.155 The interaction with backbone residues, instead, stabilizes anionic, buried, zinc cavities.155

2.10.4.4

The pKa of zinc-bound water molecules

The coordination environment of most zinc enzymes involves a metal-bound water molecule. The Lewis acidity of the Zn2þ ion enhances the Brønsted acidic properties of the bound water, i.e., it lowers its pKa from the normal value of 14 (Fig. 5). A zincbound hydroxide is a potent nucleophile, despite being bound to a potent Lewis acid. In this regard, binding of a water molecule to two Zn2þ ions in a bridging mode would lower even further the water pKa, but the nucleophilicity of the resulting bridging hydroxide would be compromised.3 Thus, even if several structures of binuclear zinc enzymes show a bridging water/hydroxide, it is very likely that this moiety needs to be detached from one of the zinc ions, becoming a terminal zinc-bound hydroxide with a good nucleophilicity.

Fig. 4 triad.

Typical second-shell ligands for zinc-bound histidines, giving rise to the Zn2þ-His-carboxylate(Asp/Glu) triad and the Zn2þ-His-backbone

Fig. 5

Activation of water by Znþ 2 ions, lowering its pKa below 14.

240

The biochemistry and enzymology of zinc enzymes

The pKa of [Zn(H2O)6]2þ is 9.0, thus being unable to provide a relevant concentration of hydroxide at neutral pH. The pKa values of hexaaquo metal complexes in solution generally correlate with the Irving-Williams series of decreasing ionic radii and increased effective nuclear charge. Instead, the zinc-bound water molecule in carbonic anhydrase is ca. 7.0 (dependent on the isozyme), revealing that the immediate environment can exert a large effect on tuning the acidity of the water and therefore in the reactivity of the zinc center at physiological pH.43,156 The synthesis of a model compound in 1975 by Paul Woolley demonstrated that the coordination environment could lower the pKa of a zinc-bound water to 8.7, supporting the hypothesis postulated for carbonic anhydrase.157 This early work stimulated the synthesis of a large series of biomimetic zinc complexes aiming to reproduce a pKa as low as that of carbonic anhydrase (Fig. 6).40,43,158,159 Different water-soluble scaffolds, such as the Tris(pyrazolyl)hydroborato, the macrocyclic triaza- and tetraaza- cyclododecanes have been successfully exploited to synthesize zinc complexes able to polarize a water molecule eliciting pKa values close to 7 (Table 3), and to hydrolyze CO2 with rates as high as 103 M 1 s 1, which are remarkable for these synthetic model systems. The works from Kimura and Parkin are remarkable in this regard.40,41,43 The role of the metal ligands and the second-shell ligands has been dissected in the case of human carbonic anhydrase II.19,156 A larger negative charge at the zinc ligands disfavors a metal-bound hydroxide, therefore increasing the pKa of the zinc-bound water molecule. This charge is determined by the coordination number of the zinc site, the identity of the metal ligands, and the secondshell (mainly hydrogen bonding) interactions of these ligands with other residues in the enzyme structure. This was postulated by early ab initio calculations in 1990 by Bertini, Luchinat and coworkers.167 More recent theoretical studies refined and supported this seminal work,168 being able to reproduce pKa data from different zinc enzymes based on their crystal structures. In general, it can be stated that the pKa and the reactivity of a zinc-bound hydroxide is determined by the hydrogen bonding interactions subtended by the hydroxide, and the coordination environment of the zinc ion. The direct hydrogen bonding interactions of the OH not only help modulating its nucleophilicity, but also contribute to orient the active nucleophile in a fixed orientation, decreasing the entropic penalty for the nucleophilic attack.169 This is exemplified by the hydrogen bond network in the active site of human carbonic anhydrase II (Figs. 7 and 8) that locks the hydroxide group with its lone pair pointing towards the region where the substrate is bound (see below specific section discussing the mechanism of carbonic anhydrase). This is of particular relevance in mononuclear zinc enzymes with an activated water molecule, and when the substrate is small (such as CO2), lacking defined anchoring patches. In 1989, Christianson proposed that the hydrogen bonding interaction of His ligands at zinc sites with carbonyl and carboxylate groups could tune the electronic structure of the zinc site, and therefore the pKa and nucleophilicity of the zinc-bound water.153 This hypothesis has been exhaustively tested in carbonic anhydrase, in which the metal ligands, second-shell residues, and the hydrogen bond network of the zinc-bound water/hydroxide have been replaced by other residues. Basically, the identity of direct and secondshell ligands impacts on the pKa by tuning the ability of the zinc ion to stabilize a negatively charged bound hydroxide. In carbonic anhydrase, replacement the His ligands by Cys or Glu/Asp ligands results in an increase of the pKa of the zinc-bound water, due to the negative charge of these ligands compared to the imidazole ring that counteract the positive charge of the zinc ion (Table 4). Some of these effects, with large increases of the pKa values, imply a destabilization of the zinc/hydroxide interaction. The impact of second shell ligands also depends on the electrostatics, but the interpretation may be less straightforward in some cases. In the case of ligand His119, removing the hydrogen bond formed with Glu117 has little effect on the pKa, except when a Gln residue is introduced into this position (Table 4). On the other hand, introduction of a negative charge replacing the neutral Gln92 by a Glu residue, results in an increase of the pKa value by 1 pH unit, which implies a destabilization of the zinc/hydroxide group by 1.2 kcal/mol. Instead, impairing the hydrogen bonding by introducing a neutral Leu decreases the pKa. In this regard, the impact of these mutations in the water pKa provides an indirect measurement of the electrostatic contribution of the hydrogen bonds to the ability of a zinc ion to polarize a bound water molecule. However, in some cases, a larger effect would be expected when a non-polar residue is introduced, such as in most of the Gln92 mutants. The crystal structures of these mutants have revealed that the introduction of these smaller residues creates a cavity where water molecules can be located, also providing a hydrogen bonding interaction with the His ligand.173 The pKa values of the zinc-bound water molecules in different zinc enzymes therefore depend on many features (Fig. 9). In the case of carboxypeptidase (see section 2.10.5.2.1.1), the presence of a Glu residue would be expected to destabilize the ability of the zinc ion to polarize a water molecule compared to carbonic anhydrase. Instead, the presence of a glutamate residue in the active site interacting with the zinc-bound water molecule lowers the pKa. Binuclear zinc enzymes (section 2.10.5.2.2) have a water/hydroxide moiety bridging the two zinc ions (in addition to terminal water molecules bound to only one of the zinc ions). The simultaneous

Fig. 6

Model compounds presenting different pKa values for Zn2þ-bound water molecules.

The biochemistry and enzymology of zinc enzymes Table 3

241

pKa values of zinc-bound water molecules in model complexes.

Complex

Donor Set

pKa

H2O Zn(HP[12]aneN3) H2O2þ Zn([14]aneN4) H2O2þ Zn(DMAM-PMHD)H2O2þ Zn(H2O)62þ Zn(CR)(H2O)2þ Zn([11]aneN3) H2O2þ Zn([12]aneN4) H2O2þ Zn([12]aneN3) H2O2þ Zn(C-PMHD)H2O2þ Zn[Tpt-Bu,Me] H2O2þ Zn[THB] H2O2þ

– N3O2 N4 O N3O2 O6 N4 O N3 O N4 O N3 O N3O2 N3 O N3 O

15.74 10.7 9.8 9.2 9.0 8.7 8.2 8.0 7.3 7.1 6.5 6.2

References –

160 158 161 162 157 158 158 158,163 164 165 166

DMAM-PMHD ¼ 1-[(((6-dimethylamino)methyl)-2-pyridyl)methyl]hexahydro-1,4-diazepin-5-one; C-PMHD ¼ 1-[((6carboxy)-2-pyridyl)methyl]hexahydro-1,4-diazepin-5-one; [12]aneN3 ¼ 1,5,9-triaza-cyclododecane; HP[12]aneN3 ¼ 2-(2hydroxyphenylate)-1,5,9-triaza-cyclododecane; CR ¼ Schiff base between 2,6-diacetylpyridine and bis(3-aminopropyl)amine; Tp ¼ tris(pyrazolyl)borate; THB ¼ Tripodal histidine benzyl ligand.

binding of the water molecule to the two zinc ions further lowers the pKa, but it also provides a weaker nucleophile. Therefore, in all these cases, the hydroxide is expected to detach from one of the zinc ions before the nucleophilic attack. Many binuclear zinc hydrolases also possess many carboxylate ligands, some of them bridging the two zinc ions. These negatively charged residues offset the effect of the presence of two zinc ions and can result in higher pKa values, such as in aminopeptidase. Finally, in the case of metallob-lactamases, the lack of bridging carboxylate ligands decreases the transfer of negative charge, resulting in very low pKa values (see Fig. 9).

2.10.5

Zinc-dependent enzymes

2.10.5.1

Zinc lyases

2.10.5.1.1

Carbonic anhydrases

Carbonic Anhydrases (EC 4.2.1.1) are the prototype of a zinc-dependent lyase, that catalyze the water-mediated interconversion between CO2 and bicarbonate, playing critical roles in pH regulation, cellular respiration and ion transport. Carbonic anhydrases are metalloenzymes widespread in all kingdoms of life and can be classified into four evolutionary unrelated families: the a-CAs (found mostly in vertebrates), b-CAs (in higher plants and bacteria), g-CA (mostly in archaea), d-CA, z-CA (only in some marine diatoms), h-CA (in protozoa),174 and ι-CA (in marine phytoplankton).175 Most CAs are zinc-dependent, but enzymes from marine diatoms use other metal ions, due to the differential availability of transition metal ions in their native environment. This is the case of z-CA from Thalassiosira weissflogii, dependent on Cd2 þ,176,177 and ι-CA from Thalassiosira pseudonana, active with Mn2 þ.175 Here we will discuss the structural and mechanistic details of a-CAs. So far, 16 a-CAs isozymes have been described in mammals, distributed in different organs and tissues, playing different essential physiological roles related with CO2 hydration. Among them, carbonic anhydrase II (CA II) is one of the most efficient known enzymes, with catalytic efficiencies close to the diffusion limit, with kcat/Km ¼ 1.5  108 M 1 s 1.17–21 Of note, carbonic anhydrase was the first zinc enzyme for which inhibitors were developed. The best-known CA inhibitors are related to the treatment of glaucoma, since inhibition of this enzyme controls the accumulation of water in the eye, relieving eye pressure.

Fig. 7 Hydrogen bond network of the zinc-bound hydroxide in the active site of carbonic anhydrase. The hydrophobic region where the CO2 substrate is bound is also indicated.

242

The biochemistry and enzymology of zinc enzymes

Fig. 8 First- and second-shell ligands in the active site of carbonic anhydrase. The structured used to generate the image (PDB 3KKX) was obtained via neutron diffraction, and presented the positions of H atoms, but only those of His residues participating in H-bonding and the Zn2þ-bound hydroxide are shown. The Zn2þ ion and the hydroxide are shown as spheres, while coordination and H-bond interactions are shown as yellow and cyan dashes, respectively. First and second-shell residues are shown as sticks, (bb) indicates that main chain atoms are shown.

Carbonic anhydrase has been the most thoroughly studied zinc enzyme for many decades by means of enzymology, metal substitution approaches, crystallography, interaction with small anions mimicking the linear CO2 substrate and extensive mutagenesis studies of the metal ligands, second-shell ligands and active site residues. The early work from Lindskog, Bertini, Liljas, Coleman was followed later by Fierke and Christianson.18,19,22,23,33–38,178–180 The basic mechanistic outline came from studies on the bovine and human isoenzymes I and II, later extrapolated to other a-CAs. This overwhelming body of evidence not only provided the foundations for the mechanistic understanding of other, more complex zinc enzymes, but also enabled the use of carbonic anhydrase as a template for protein engineering and its redesign as a fluorescent-based zinc sensor. Since it is challenging to measure accurately the CO2 hydratase activity, most activity assays on carbonic anhydrase are based in its moonlighting esterase activity using the chromophoric substrate p-nitrophenyl acetate (PNPA).181 The active site in a-CAs contains a single zinc ion in the bottom of a deep cavity (ca. 15 Å, with a volume of volume of 900 Å3) coordinated to His94, His116, His119 and a water molecule, adopting a distorted tetrahedral geometry (Fig. 10). This water molecule displays a pKa ca. 7, depending on the isoenzyme, and can therefore be described as a hydroxide ion, depending on the working pH. The first structure of a carbonic anhydrase was solved in 1972 at 2.0 Å, and further refined 15 years later.182–184 His94 and His96 bind the metal ion through their N32, while His119 is coordinated by using the imidazolic Nd1. This zinc binding pattern, with a His-X-His motif, and a more distant His ligand located upstream, has been found in many zinc enzymes, with the same binding mode, with the metallo-b-lactamases being an exception.125

Table 4

Modulation of the zinc-bound water pKa in Carbonic Anhydrase by site-directed mutagenesis.

Variant

pKa

Wild type 6.8 Zinc ligands His94Cys >9.5 His94Asp >9.6 His96Ala 8.4 His96Cys 8.5 His119Asp 8.6 Hydrogen bond network with zinc-bound water Thr199Ala 8.3 Thr199Ser 7.3 Thr199Val 8.7 Thr199Pro 9.2 Second shell ligands (metal ligand indicated in parentheses) Glu117Gln (His119) >9.0 Glu117Ala (His119) 6.4 Glu117Asp (His119) 6.7 Gln92Ala (His94) 6.8 Gln92Leu (His94) 6.4 Gln92Asn (His94) 6.9 Gln92Glu (His94) 7.7

References 170 171 171 171 171 171 25 172 172 172 24 25 25 25 25 25 25

The biochemistry and enzymology of zinc enzymes

243

Fig. 9 pKa values of prototypical zinc enzymes with an activated metal-bound water molecule. In the case of metallo-b-lactamases, the active site geometry shown corresponds to enzymes of the B1 subclass (see below).

The active site cavity can be described as composed by two halves: one containing hydrophobic residues, while the other is mostly composed by polar residues (Fig. 11). The hydrophobic patch is most likely the region driving CO2 binding, and where different inhibitors are located. This has been confirmed by studying the interaction of the enzyme with the bicarbonate and formate ions by crystallography and NMR.184–186 Mutagenesis of residues Val 121 and Val143 in this hydrophobic pocket revealed that the hydrolysis of PNPA depends on the hydrophobicity of the amino acid at these positions.170,187,188 Residues in this pocket are relevant for CO2 and PNPA binding and steering the proper orientation of the substrate towards the catalytic zinc-hydroxide.170,189 The zinc-bound water is oriented by a well-defined hydrogen bond network in which the carboxylate group from Glu106 is a proton acceptor from the Og1 of Thr199 which, on its turn, is a hydrogen bond acceptor from the ZneOH reactive moiety. This network orients the lone electron pair of the zinc-bound water/hydroxide for the nucleophilic attack (Fig. 7).169,190 These residues are highly conserved in the a-CA family. Indeed, disruption of this hydrogen bond network results in an increase of the pKa value of the water molecule up to 9.2, with a concomitant reduction of the catalytic efficiency by two orders of magnitude.172,191 The hydroxyl from Thr199 is also involved in the stabilization of the transition state by hydrogen bonding, destabilizing bicarbonate binding and favoring product release. The rate-determining step of the reaction is a proton transfer that takes place by means of a hydrogen bond network involving several water molecules in the active site cavity that acts as “proton wire” connecting the zinc-bound hydroxide with His64, the proton shuttle to the bulk solvent.190,192,193 When protonated, His64 experiences a conformational change so that is pointing outside the active site to facilitate proton transfer to the solvent.

Fig. 10 Active site structure of carbonic anhydrase (PDB 3KKX), showing residues participating in metal coordination and other positions important for catalytic activity. Zn2þ is shown as a grey sphere, and the Zn2þ-bound hydroxide and water molecules as thin sticks. Coordination and H-bond interactions are shown as yellow and cyan dashed lines, respectively, while the proposed path for proton transfer to His64 (which requires reorganization of water molecules to form the adequate H-bonds) is shown as orange dashed lines.

244

The biochemistry and enzymology of zinc enzymes

Fig. 11 Global structure of carbonic anhydrase (PDB 6LUX). Residues belonging to the hydrophobic and hydrophilic pockets are shown as yellow and cyan spheres, respectively, while Zn2þ-coordinating residues are shown as green sticks. His-64 is shown as orange sticks, and displays two conformations. The protein crystals were pressurized under 20 atm CO2, allowing CO2 to be observed bound in the active site (red and white spheres). The Zn2þ ion and its bound hydroxide are shown as grey and red spheres, respectively.

Carbonic anhydrases act by a two-step ping-pong mechanism in which the zinc-bound hydroxide attacks the CO2 carbon atom, forming a zinc-bound bicarbonate. This anion is then displaced by a water molecule (Figs. 12 and 13). In a second step, the zincbound water is ionized to regenerate the active hydroxide by means of His64, acting as a proton shuttle in the rate-determining step of the reaction, with a rate to 106 s 1.17,194,195 This mechanism involves the catalytic zinc ion undergoing conformational changes, starting from a tetrahedral coordination, then becoming penta-coordinated in a complex in which the bicarbonate acts as a bidentate ligand, and finally restoring the tetrahedral geometry. Metal substitution experiments using Co(II) as a spectroscopic probe were foundational in the 1960s and 1970s to propose these geometry changes during turnover at the zinc site. Since many small anions are CA inhibitors, since they can mimic the linear CO2 substrate or the bicarbonate products, these anions have been exploited to demonstrate these geometry changes at the metal site. The second-shell residues in the active site of carbonic anhydrase form a well-defined hydrogen bond network that orients the His ligands to adopt the required coordination mode of the zinc ion: the proton bound to the Nd1 of His94 is a hydrogen bond donor to Gln92, the proton bound to the Nd1 of His94 interacts with the backbone carbonyl group of Asn244, while the N32-H of His119 forms a hydrogen bond with Glu 117 (Fig. 8). These interactions have been extensively characterized by site-directed mutagenesis and have been shown to modulate the pKa and the reactivity of the zinc-bound hydroxide. An example of the role of zinc ligands and second shell residues in modulating the pKa of the attacking nucleophile has been provided in a previous section. Acetazolamide was the first inhibitor for carbonic anhydrase, approved in 1953 by the FDA. To the moment, there are ca. 20 CA inhibitors approved. All these inhibitors share a sulfonamide/sulfamate/sulfamide moiety, that binds the zinc ion displacing the zinc-bound water, at the same time accommodating in the active site cavity. This strategy, designing an efficient zinc binding group that can accommodate into the enzyme active site and displaces or blocks the active nucleophile, has been employed to design inhibitors for many zinc enzymes. However, the differences across the reactivities and active site topology pose different challenges for inhibitor design.

2.10.5.2 2.10.5.2.1

Zinc hydrolases Mononuclear zinc hydrolases

2.10.5.2.1.1 Carboxypeptidases Carboxypeptidases (CPDs) are zinc-dependent exopeptidases that hydrolyze peptide bonds and cleave amino acids from the carboxyl terminus of polypeptides. Enzymes from the CPD family are widely distributed in different types of organisms, participating in the digestion of food and assimilation of dietary proteins, digestion of misfolded proteins, in the processing and regulation of hormone precursors and in the regulation of protein binding, among other events.196–198 These enzymes can be classified based on their substrate preference, their active site mechanism or based on their sequence similarities. Three groups can be defined according to their substrate specificities: carboxypeptidase A-like enzymes (that preferentially

Fig. 12 Scheme displaying the two stages in the reaction catalyzed by carbonic anhydrase: formation of bicarbonate from CO2 followed by displacement by a water molecule (top), and regeneration of the active site hydroxide by the action of His64 as a proton shuttle (bottom).

The biochemistry and enzymology of zinc enzymes

Fig. 13

245

Reaction mechanism for carbonic anhydrase.

cleave hydrophobic residues at the C-terminus) and carboxypeptidase B-like enzymes (that selectively cleave the C-terminal at basic residues arginine or lysine). The enzymes from the CPD family are also named according to the essential catalytic residues in the active site that give rise to different reaction mechanisms: metallo-carboxypeptidases (EC number 3.4.17), serine carboxypeptidases (EC number 3.4.16) and thiol carboxypeptidases (EC number 3.4.189). Carboxypeptidase A (A1) is the prototypical zinc carboxypeptidase and the canonical form of the M14 family of peptidases. It was the first zinc metalloenzyme solved at high resolution structure by Lipscomb and coworkers.28,29 In the active site, the zinc ion is bound to His69, Glu72, His196 in a typical HXXE..H motif, and a water molecule (Fig. 14).3,197,199 This water molecule forms a hydrogen bond with the conserved Glu 270, which serves to orient this ligand. The presence of a Glu residue in the ligand set of the metal ion makes it a weaker Lewis acid compared with the zinc center in carbonic anhydrases. Binding of the substrate to the zinc ion occurs in a bidentate fashion through the carbonyl oxygen and the C-terminus, displacing the water molecule. Many crystal structures with substrate and transition state analogues and slowly hydrolyzed substrates have provided snapshots of the catalytic mechanism31,200–204 that ultimately led to the accepted mechanism nowadays. The active nucleophile is the zinc-bound hydroxide, which is deprotonated by Glu 270. After substrate binding and the nucleophilic attack, an intermediate is formed that is stabilized by the positive charges of Arg127 and the zinc ion that now is pentacoordinated (Fig. 15). In this regard, the role of Arg127 is equivalent to the oxyanion hole which stabilizes the development of negative charge in serine proteases.205,206 Glu270 then transfers the proton accepted from the zinc-bound water molecule to the leaving amino group of the scissile peptide bond. In addition, there are amino acids important for substrate binding in five subsites: S1, S1’, S2, S3 and S4. The specificity of different carboxypeptidases is dictated by the residue in position 255, that is an isoleucine in A-type enzymes or an aspartic acid in B-type enzymes.3,197,198,207 Pancreatic carboxypeptidase is produced as zymogens for proper activity control, with an activation domain, a connecting segment and the enzyme domain (Fig. 16). Structures from other CPDs show several similarities with Carboxypeptidase A. Carboxypeptidases B, T, G and A2 are homologous to Carboxypeptidase A1 despite they have differences in substrate preferences.197,207

Fig. 14 Active site structure of Bos taurus carboxypeptidase A1 (PDB 5CPA). Metal coordination interactions are displayed as yellow dashed lines, and the catalytic Zn2þ ion and its bound water are shown as grey and red spheres, respectively.

246

The biochemistry and enzymology of zinc enzymes

Fig. 15

Catalytic mechanism proposed for in Carboxypeptidase A.

2.10.5.2.1.2 Matrix metalloproteinases Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that degrade proteins in the extracellular matrix.208–210 MMPs have a role in several cell processes: proliferation, differentiation, migration, apoptosis, angiogenesis, tissue repair, and immune response. As a result, MMPs are involved in tumor invasion and metastasis. MMPs can also modulate various cellular and signaling pathways. They are present in most kingdoms of life, under different forms. For example, there are 23 MMP paralogs in humans. These enzymes are produced as zymogens, which are subsequently processed by proteolytic enzymes to generate the active forms.211,212 From a structural point of view, MMPs (or matrixins) are members of the metzincin superfamily of proteases. These metalloproteinases have in general 4 domains: a propeptide region, the catalytic metalloproteinase domain where the active zinc is found and that also contains a calcium-binding site, a linker peptide or hinge region and the hemopexin-like domain (PEX) that defines the substrate specificity. Some MMPs can have a signal peptide of ca. 20 amino acids, while others can have additional domains. Based on structural domains and substrates, MMPs are classified into gelatinases, collagenases, matrilysins, stromelysins, membrane-type (MT)-MMPs, and other MMPs.213,214 The propeptide domain is critical for the regulation of the activity of MMPs. It consists of three helices forming a hydrophobic core that occludes the active site (Fig. 17). This region contains a PRCGXPD motif, in which the Cys residue binds the zinc ion, thus preventing the access of a water molecule as a metal ligand. The “cysteine switch” mechanism consists in the proteolysis of this propeptide region, that removes the Cys ligand, thus making a solvent molecule (and the substrate) accessible to the active site, converting the enzyme from an inactive latent form to the catalytically active species. The zinc ion in the catalytic site is bound to three histidine residues present in a conserved HEXXHXXGXXH motif and an axially coordinated water molecule, thus resembling the metal binding site of carbonic anhydrase (Fig. 17). The water ligand is hydrogen bonded to a conserved glutamate and a methionine from the XBMX Met-turn motif that acts as a hydrophobic base. The catalytic domain also contains a structural zinc ion coordinated to three histidine and one aspartic acid, with no water nor exogenous ligands. Up to six pockets of different depth are flanking the catalytic zinc center.212,213,215,216 In the currently accepted reaction mechanism, binding of the substrate to the metal ion through the carbonyl oxygen gives rise to a penta-coordinated metal center. Then, the water/hydroxide molecule activated by the Glu residue is responsible of the nucleophilic attack to the substrate. Then, the substrate is cleaved and the water molecule is released.213,214

Fig. 16 Protein fold of human procarboxypeptidase A2 (PDB 1AYE). The activation peptide is shown in orange, while the mature protein is shown in green. The catalytic Zn2þ ion is displayed as a grey sphere.

The biochemistry and enzymology of zinc enzymes

247

Fig. 17 (A) General fold of human pro-collagenase 1 (MMP1, PDB 1SU3). The propetide, catalytic domain and hemopexin domain are shown in orange, green and cyan, respectively, while the catalytic Zn2þ ligands, including the inhibitory Cys from the propeptide, are shown as sticks. The catalytic and structural Zn2þ ions are displayed as grey and blue spheres, respectively. Ca(II), Na(I) and chloride ions are shown as orange, purple and green spheres, respectively. (B) and (C) Active site structures of human pro-collagenase 1 (PDB 1SU3) and mature collagenase 1 (PDB 2CLT). TheZn2þ ion and its bound water are shown as grey and red spheres, respectively. Metal coordination and H-bond interactions are displayed as yellow and cyan dashed lines, respectively. Note that Glu200 is mutated to Ala in PDB 2CLT, the mutation was reverted in silico and the orientation of this sidechain set to that observed in other structures of MMP1.

2.10.5.2.2

Binuclear zinc hydrolases

2.10.5.2.2.1 Aminopeptidases Peptidases can be classified into endo- or exopeptidases, depending on the site where the enzyme cuts the peptide. The latter, that cleave at the end of a protein or a peptide substrate, can be aminopeptidases and carboxypeptidases. Aminopeptidases (APD) cut off N-terminal amino acid residues. APDs are important enzymes in the processing, catabolism and degradation of proteins, having different roles in a tissue- specific manner. They are found in animals and plants, as well as in prokaryotes.3,217 There are various classes of aminopeptidases. In this section, will focus on bi-zinc metalloenzymes that belong to the M17 LAPs group (EC3.4.11.1), to compare their active site features and activity with the previously discussed mononuclear hydrolases. Leucine aminopeptidase (LAP) was one of the first peptidases to be characterized. LAP is an homohexameric protein peptidase M17, with two zinc ions in the active site (Zn1 and Zn2) separated by ca. 3.0 Å.218 Both zinc atoms are pentacoordinated, despite adopting an octahedral geometry, that leaves a vacant binding site. The binuclear site is structured by three ligands bridging the two zinc ions: a water molecule, Asp255 (monodentate) and Glu 334 (bidentate) (Fig. 18). The coordination sphere of Zn1 is completed by Asp332, that binds the metal ion through one of the carboxylate oxygens and the backbone carbonyl (at a short distance of 2.1 Å). In the case of Zn2, the remaining ligands are Lys250 and Asp273. There are several inhibitors reported for LAPs: bestatin, amastatin, L-leucinal, and leupeptin.3,219 In particular, the hydrated form of the aminoaldehyde L-leucinal inhibits the enzyme by binding of the gem-diol moiety to Zn1 and as a bridging ligand, displacing the bridging water molecule, and by a direct interaction of the amine group to Zn2 (Fig. 18). Based on a series of structural studies of

Fig. 18 (A) and (B) Active site structures of substrate-free bovine leucine aminopeptidase LAP3 (A, PDB 1LAM) and of this protein bound to the inhibitor L-leucinal (B, PDB 1LAN). Zn2þ ions and the active site water molecule are shown as grey and red spheres, respectively, while metal coordination interactions are shown as yellow dashed lines. “bb” indicates that the backbone atoms are shown for the residue. (C) Chemical structure of the hydrated (gem-diol) form of L-Leucinal.

248

The biochemistry and enzymology of zinc enzymes

Fig. 19

Reaction mechanism scheme for leucine aminopeptidase.

complexes of LAP with different inhibitors, a catalytic mechanism has been proposed (Fig. 19). The (poly)peptide substrate is expected to bind at the empty ligand position of Zn2 through its terminal amino group, and to the Zn1 ion by means of its carbonyl group. The bridging water molecule is the purported nucleophile attacking the carbonyl, leading to a dioxygen species bound resembling the interaction of the diol moiety from inhibitors such as L-leucinal. Binding of this water molecule to two zinc ions simultaneously would provide the driving force for lowering its pKa and eliciting a hydroxide, counteracting the high concentration of negatively charged ligands. As already mentioned, a since a bridging hydroxide is not expected to be a potent nucleophile, it might detach from one of the two zinc ions for the nucleophilic step.220 DFT calculations, instead, have supported the notion that the attacking nucleophile could be bound to both zinc ions.221 Residue Lys262 and water molecules interacting with Arg336 have been proposed to stabilize the transition state.3,218,222,223 Bovine LAP, Escherichia coli aminopeptidase A (PepA, PDB 1GYT), and LAPs from Leishmania donovani,224 Pseudomonas putida (PDB 3H8G), Staphylococcus aureus (PDB 3H8E), Coxiella burnetii (PDB 3IJ3), C. elegans (PDB 2HC9) and Francisella tularensis (PDB 3PEI) are homohexamers containing two trimers stacked on top of one another (Fig. 20), have an analogous active site and an equivalent catalytic mechanism.223 Remarkably, the N-terminal domain of E. coli PepA has an alternative function as a DNA-binding protein in Xer recombination and in transcriptional control.225 Some bacterial aminopeptidases (such as the ones from Aeromonas proteolytica, Vibrio proteolyticus and Streptomyces griseus) are monomeric enzymes with a single a/b globular domain. These enzymes represent a large family of binuclear hydrolases that follow the same mechanistic scheme, with different residues playing equivalent roles as general bases and stabilizing the development of negative charges in the intermediates and transition states.

Fig. 20 Structure of bovine leucine aminopeptidase LAP3 displaying its hexameric state. The three subunits in the foreground are colored in green, cyan and yellow, while those in the background are colored light grey. Zn2þ ions are shown as orange spheres.

The biochemistry and enzymology of zinc enzymes

249

2.10.5.2.2.2 Metallo-b-lactamases Metallo-b-Lactamases (MBLs) are zinc-dependent hydrolases which act as important resistance determinants against b-lactam antibiotics.226–229 In order to protect the peptidoglycan synthetic machinery from these drugs, MBLs are periplasmic enzymes in Gramnegative bacteria, while they are secreted to the extracellular milieu in Gram-positive bacteria. MBLs use Zn2þ ions to bind their substrates and to activate a hydroxide to attack the carbonyl carbon of the substrate b-lactam ring, leading to ring opening and inactivation of the antibiotic. These enzymes are phylogenetically unrelated to the mechanistically distinct serine-b-lactamases (SBLs), which employ a catalytic serine residue to hydrolyze b-lactam antibiotics. All three major bicyclic b-lactam classes are inactivated by MBLs, namely penicillins, cephalosporins and carbapenems (Fig. 21). Only monocyclic b-lactams, such as the monobactam drug aztreonam, are spared by MBLs, due to the formation of an unproductive enzyme-substrate complex.230 While MBLs are highly divergent, with sequence identities below 15% for the most distantly related members,226 they all consist of a single domain presenting a characteristic ab/ba fold with the active site located in a cleft between the two ab halves of the protein (Fig. 22). This is known as the MBL fold and is conserved not only among MBLs but across a wide range of proteins with diverse functions, which constitute the MBL superfamily.228,231–233 In addition to the general fold, MBLs share a series of conserved motifs which are for the most part associated to Zn2þ binding. Due to the large degree of sequence divergence, these and other important residues are generally identified via a consensus numbering scheme termed BBL,234,235 in which structurally conserved residues are assigned common numbers across all MBLs. Accordingly, BBL numbering will be used for all MBL residues referenced in the current text. MBLs are divided into three subclasses based on their sequence identity: B1, B2 and B3226,227 (Fig. 22). Enzymes from these groups differ in their substrate spectrum and occupancy of the two potential zinc-binding sites, located next to each other in the active site cavity and termed Zn1 and Zn2. Both sites are occupied in subclass B1 and B3 MBLs, which are wide spectrum enzymes that can degrade most b-lactam antibiotics. The metal at the Zn1 site is coordinated in enzymes of both subclasses by three His residues (His116, His118 and His196) and a hydroxide ion, with tetrahedral geometry, resembling the active site of carbonic anhydrase. The hydroxide is a bridging ligand which is also bound to the Zn2 ion, although it is positioned asymmetrically and closer to Zn1 than to Zn2 (1.9 Å vs 2.2–2.5 Å, respectively). In B1 MBLs, the coordination sphere of the Zn2 site is completed by Asp120, Cys221, His263 and an additional water molecule, resulting in a bipyramidal trigonal geometry. Meanwhile B3 enzymes share most Zn2 ligands with B1 MBLs but lack the Cys residue, which is replaced by His121. On the other hand, B2 enzymes are active in the mono-zinc form and have a narrow substrate spectrum, as they are only effective in hydrolyzing carbapenem antibiotics. Only Zn2 is occupied in the active form of these enzymes, with the same coordination as for the corresponding site in B1 MBLs. Proteins from this subclass have weaker metal binding affinity in the Zn1 position due to the replacement of His116 by Asn, and are inhibited when this site is occupied. As can be observed, most metal ligands in MBLs cluster within the conserved motif [H/ N]116-X-[H]118-X-[D]120-[H/X]121, which is also present in proteins of the MBL superfamily.228,231,232 Some MBLs present exceptions to the metal coordination residues previously discussed, such as the B3 enzyme GOB. In this protein, His116 is replaced by Gln (similarly to B2 MBLs), and the enzyme was found to be active both in the binuclear form and in the mono-zinc form, with only site Zn2 occupied.236,237 In contrast to what may be expected from their substrate spectrum and zinc stoichiometry, B1 and B2 MBLs are phylogenetically much closer to each other than to B3 MBLs, which have higher homology to other members of the superfamily than to enzymes from the former subclasses.238,239 As a result, the B1 þ B2 and B3 enzymes form distinct clades within the superfamily, and there is evidence indicating that b-lactamase activity would have evolved independently for both groups.239,240 The active sites of MBLs have some notable features compared to other metallohydrolases. In contrast to most other binuclear metallohydrolases, such as the previously discussed aminopeptidases, MBLs do not possess a protein residue acting as a bridging ligand between the two metal ions, a characteristic that is present in proteins of the MBL superfamily.228,232 This may enable

Fig. 21 (A) Chemical structures of the four main classes of b-lactam antibiotics: penicillins, cephalosporins, carbapenems and monobactams, with the b-lactam ring in each of them shown in blue. The numbering for atoms in the core of each antibiotic class is shown in orange. (B) Reaction catalyzed by b-lactamases, exemplified by the hydrolysis of a penicillin antibiotic.

250

The biochemistry and enzymology of zinc enzymes

Fig. 22 Active site structure (top) and general fold (bottom), for enzymes belonging to each MBL subclass: B1: VIM-1 (PDB 5N5G), B2: SfhI (PDB 3SD9), B3: BJP-1 (3LVZ). Zn2þ ions and active site waters/hydroxides are shown as grey and red spheres, respectively, while metal coordination interactions are shown as yellow dashed lines.

a greater active site flexibility, which could be important to accommodate and hydrolyze a wide range of substrates with different steric requirements. Additionally, B1 and B2 MBLs feature a Cys residue as a metal ligand, an infrequent ligand in catalytic zinc sites103,241 and which is specially rare for zinc proteins located outside the cytoplasm, due to its potential to become oxidized when not in a reducing environment. In the case of B1 enzyme BcII, it has been demonstrated that while Cys221 can be replaced by Asp and maintain enzyme activity in vitro, this requires supplementation with excess Zn2þ as it reduces the metal affinity of the Zn2 site.242 Without added Zn2þ, the capability to confer resistance of BcII Cys221Asp to its bacterial host is lower than the wild type enzyme,242 suggesting that the presence of Cys is required for optimal Zn2þ acquisition by MBLs within their native environment. The active site of B1 MBLs is delimited on opposite sides by two extended loops, namely loops L3 and L10, which contribute to substrate binding and have been shown to influence the enzyme activity.226,243 In particular, loop L3 has been shown to possess significant mobility in various B1 enzymes and would close over the active site upon substrate binding, with a generally conserved aliphatic or aromatic residue at the tip of the loop contributing to interactions with different substituents in the bicyclic structures of the substrates. Meanwhile, loop L10 includes conserved residues Lys224 and Asn233, which respectively aid in substrate binding by interacting with the b-lactam C7/C8 carbonyl oxygen and with the carboxylate present at C3/C4 position in all bicyclic b-lactam antibiotics (Fig. 21). On the other hand, B2 enzymes possess a much shorter L3, and have instead an extended helix (a3) forming one of the active site walls. This leads to a narrower active site cavity, which would contribute to the restricted substrate spectrum of these b-lactamases. B3 enzymes possess a protruding loop next to their active site (a3-b7 loop) that would act similarly to L3 in B1 MBLs, and which has been proposed to participate in substrate binding and catalysis. While Zn2þ ions are essential for MBL function, the biologically relevant metal stoichiometry for B1 and B3 MBLs has long been a matter of debate. The first high-resolution crystal structure of an MBL, that of B1 enzyme BcII by Carfi and coworkers,244 showed a single Zn2þ ion bound at the Zn1 position. It was only after subsequent structures of BcII and CcrA that both metal sites were observed to be filled.245–248 Additionally, Cys221 of B1 MBLs is prone to oxidation, preventing Zn2þ binding to the Zn2 site, and leading to structures of BcII, VIM-2 and SPM-1 with only site Zn1 occupied.249–251 This oxidation was interpreted as a consequence of Zn2 being easily dissociated, which casted doubt on its importance on the catalytic mechanism. Furthermore, MBLs generally have lower metal-binding affinity than other Zn2þ enzymes, with KD values in the nM range92 while these are pM for thermolysin and carbonic anhydrase.252,253 Different groups have reported varying behaviors regarding metal binding cooperativity in binuclear MBLs, leading to discussions of the viability of obtaining the bimetallic species of the enzymes in their native environments. However, the key role of Zn2 was ascertained by analysis of BcII mutant Cys221Asp, in which metal binding to this site is weaker and thus it remains vacant in absence of excess Zn2 þ.242 While the mutant maintained similar activity to the wild type enzyme when supplemented with Zn2þ, it was unable to confer resistance to its bacterial host in unsupplemented growth media. Furthermore, the Zn2 site is a common denominator across all MBLs, and the role of this Zn2þ in substrate binding would be a key conserved feature in the mechanism of hydrolysis of all these enzymes. As such, the general consensus is that the optimally functional form of B1 and B3 MBLs is as binuclear enzymes.

The biochemistry and enzymology of zinc enzymes

251

The reaction mechanism for MBLs was the subject of intense study over more than two decades,63,243,254–256 using diverse approaches that included steady-state and pre-steady-state kinetics, X-ray structures with bound products or intermediates and QM/MM simulations. Elucidation of the reaction mechanism of MBLs was complicated by the heterogeneity of substrates hydrolyzed by these enzymes, in combination with their varying ability to function bound to one or two metal ions. In spite this, hydrolysis of all substrates across all MBLs shares considerable similarities. All in all, the reaction mechanism consists of three basic steps: (1) nucleophilic attack by a hydroxide on the b-lactam carbonyl, (2) breaking of the CeN bond of the b-lactam ring, (3) protonation of the resulting species, followed by product release. Here we present a consensus view on the catalytic mechanism of binuclear and mononuclear MBLs. Substrates bind to the active site of binuclear MBLs such that Zn1 interacts with the C7/C8 carbonyl oxygen, polarizing the CeO bond, while the C3/C4 carboxylate coordinates with Zn2 (Fig. 23). Substrate binding also triggers an increase in the ZneZn distance, in which the bridging hydroxide becomes a terminal ligand attached to Zn1 only. This boosts the nucleophilicity of the hydroxide, which would otherwise be impaired by simultaneous coordination to the two positively charged Zn2þ ions. The now terminally-bound hydroxide performs a nucleophilic attack on the C7/C8 of the substrate, which would result in a tetrahedral intermediate that has not been experimentally observed to accumulate, in contrast to serine-b-lactamases. This suggests that such intermediate would constitute a high energy species that is insufficiently stabilized in MBLs and would rapidly decay. Subsequently, the b-lactam bond breaks and yields a ring-opened intermediate with a negative charge on the nitrogen atom, that is stabilized by interaction with Zn2. Accumulation of such negatively charged species was first observed for the chromogenic cephalosporin nitrocefin,257–259 for which a characteristic band at 665 nm is observed to arise in pre-steady-state kinetics experiments, distinct from the absorption bands of the intact (390 nm) and hydrolyzed (490 nm) species. The relevance of this anionic intermediate for the general catalytic mechanism was a matter of debate, as the p-conjugated system provided by the C3 substituent of nitrocefin would aid in distributing the negative charge and stabilizing the intermediate, but that may not be the case for clinically useful cephalosporins. While this intermediate has not been experimentally observed for non-chromogenic cephalosporins, DFT calculations of the mechanism for cefotaxime and cephalexin hydrolysis by binuclear enzymes support its stabilization by Zn2 as a general step in the reaction.260,261 Additionally, experiments performed with carbapenems as substrates also identified the accumulation of a negatively charged intermediate,63,262–264 and thus point to the protonation of this species as the rate-limiting step in the reaction. The proton donor for this step would be a water molecule, possibly that which initially is bound apically to Zn2 and is postulated to become a bridging ligand during the reaction, occupying the vacant position left by the attacking hydroxide. This would also regenerate the nucleophile for subsequent reaction cycles. There is also the possibility that a water molecule from the bulk solvent acts as a proton donor, after entering the active site and occupying the bridging position between the two metals. In the reaction mechanism of B2 MBLs, Zn2 retains the same role while the nucleophile OH is oriented and activated by the ligand residues of the vacant Zn1 site, including His118 and His19663 (Fig. 23). A similar scheme would be followed by the mono-Zn2þ form of B3 enzyme GOB, with only the Zn2 site occupied. In contrast to that observed for carbapenem hydrolysis with binuclear MBLs, significant accumulation of enzyme-substrate adducts was observed for these mono-zinc enzymes in presteady-state kinetics experiments.63 This suggests the presence of a weaker nucleophile leading to a slower first step in the reaction, which is consistent with the attacking nucleophile being activated by protein sidechains instead of a Zn2þ ion. In contrast to serine-b-lactamases, for which various inhibitors exist that show effectiveness against different subsets of these enzymes, there are currently no clinically useful inhibitors for MBLs. While development of inhibitors targeting MBLs is a pressing issue, it is complicated by the lack of a covalently-bound intermediate, heterogeneity in active site topology and metal content, in addition to a general lack of conserved substrate binding features apart from the Zn2þ ions. Thousands of MBL inhibitors with various chemical characteristics have been assayed, and this topic has been extensively reviewed elsewhere.265–270 As such, we will only summarize the main strategies pursued for inhibiting these b-lactamases. Many inhibitors target Zn2þ ions as their primary mode of binding to the enzyme, often through the use of thiol groups, which can be supplemented by hydrophobic side groups that mimic the varying substituents bound to the core scaffold of each b-lactam antibiotic class. Examples of these include classic MBL inhibitors such as mercaptocarboxylates,271 thiomandelic acid272 and captopril.273 Some thiol-containing inhibitors imitate the geometry and features of substrates, such as bisthiazolidine (BTZ) inhibitors,274,275 or those of hydrolysis products, as is the case for mercaptomethylthiazolidine (MMTZ) compounds.276 In all of these drug classes the thiol group localizes between the two Zn2þ ions in binuclear MBLs. BTZ and MMTZ inhibitors have demonstrated inhibition of MBLs from subclasses B1, B2 and B3, and feature a carboxylate that imitates the corresponding group present in bicyclic b-lactams, which helps binding by interacting with active site residues such as Lys224 and Asn233. Boronates are a distinct class of MBL inhibitors in which numerous developments have occurred in the last decade. Monocyclic boronates such as vaborbactam have already been approved to counteract SBLs but lack inhibitory activity against MBLs.277 The development of bicyclic boronates which not only target SBLs but also MBLs, such as taniborbactam278 (formerly VNRX-5133) and QPX7728,279 shows the potential for “ultrabroad-spectrum” inhibitors targeting most clinical b-lactamases. Both compounds are currently undergoing clinical trials, and thus may constitute the first MBL inhibitors to be used in healthcare. Irreversible inhibitors that bind covalently upon reacting with active site residues such as Cys221 or Lys224 have also been reported.280–282 Given that Zn2þ ions are essential for MBL activity, strategies such as Zn2þ removal or metal replacement have also been pursued for MBL inhibition. Examples of the former include the use of Ca-EDTA or the natural product Aspergillomarasmine A, both which showed effectiveness in animal models to restore b-lactam sensitivity to bacterial strains producing MBLs.283,284 On the other hand, replacement of Zn2þ ions with other metals that yield an inactive enzyme has also shown promise for potential clinical MBL inhibition, given the effectiveness demonstrated by metal-based drugs such as Bismuth subcitrate and the Au(I) compound auranofin to inhibit MBL

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The biochemistry and enzymology of zinc enzymes

Fig. 23 (A) Reaction mechanism for cephalosporin hydrolysis by binuclear MBLs. The metal coordination shown corresponds to B1 MBLs. (B) and (C). Reaction mechanism for carbapenem hydrolysis by binuclear (B) and mononuclear (C) MBLs. The mechanism for carbapenem hydrolysis is branched, leading to two different tautomeric forms products according to the site where the intermediate is protonated during the reaction (for more details, see reference63). The intermediate may be protonated at N4 by the water molecule attached to Zn2 (reaction path indicated in green), leading to the D2 tautomer as a product. After release from the enzyme, this product in turn spontaneously decays to an equal mixture of the more stable D1a and D1b tautomers (not shown). Alternatively, the reaction intermediate may be protonated by a water molecule from the bulk solvent (path indicated in red), generating the D1b tautomer as a product.

function.285,286 An advantage of these drugs is that they are already approved for other clinical treatments, which would facilitate their transition to use as clinical MBL inhibitors.

The biochemistry and enzymology of zinc enzymes

Fig. 24

2.10.5.3

253

Reaction catalyzed by alcohol dehydrogenases.

Zinc alcohol dehydrogenases and other zinc-dependent oxidoreductases

Alcohol dehydrogenases (ADH) catalyze the oxidation of primary or secondary alcohols (either branched or cyclic) to aldehyde or ketone groups, respectively (Fig. 24). ADHs are oxidoreductases belonging to the SCR/MDR (short/medium-chain dehydrogenases/ reductases) protein superfamily. Some ADHs can catalyze the reverse reaction.287,288 ADHs are widespread in all living organisms, being present in animals (including humans), yeast and many bacteria, fungi, and plants. They serve to break down alcohols that otherwise would be toxic, and they also participate in the generation of useful aldehyde, ketone, or alcohol groups during the biosynthesis of various metabolites or during fermentation. In plants, ADH is crucial during fruit ripening, and also for seedlings and pollen development. ADHs may have several clinical significances, as can be implicated in alcoholism, drug dependence, poisoning and some drug metabolism.289 From an historical point of view, ADH was also one of the first oligomeric enzymes that had its amino acid sequence and threedimensional structure determined.290 ADHs are dimeric or tetrameric enzymes, each unit being composed of two domains. Each subunit (size about 40 kDa) can bind one or two zinc ions. The subunits are structured as a coenzyme-binding domain and a catalytic domain, with the active site located between these two domains.288,291 As an example, horse liver ADH (LADH) is a dimeric enzyme in which each monomer unit binds two zinc ions (Fig. 25). One zinc ion fulfills a structural role and influences the interactions between the subunits, while the other is essential for catalysis. In animals (including humans) there are multiple isoforms of these dimers, and each polypeptide binds two zinc ions and is encoded by seven different genes.289 The structural zinc is tetracoordinated by four cysteines (residues 97, 100, 103, 111 in horse liver alcohol dehydrogenase), i.e., with a typical coordination sphere of a structural site without any open coordination position.291,292 The catalytic zinc adopts interacts a distorted tetrahedral geometry with Cys 46, His 67, Cys 174 and a water ligand (Fig. 25). After binding of NAD, this water molecule is displaced by the deprotonated alcoxide group of the substrate. Therefore, in ADHs the catalytic role of the zinc ion does not consist in the activation of a water molecule, as described previously for the lyases and hydrolases, but the activation of the substrate that displaces the zinc-bound water molecule. ADHs have an ordered catalytic mechanism (Fig. 26). Binding of the coenzyme (the nicotinamide ring of NADþ) triggers a large conformational change in the enzyme, which brings the catalytic zinc about 1 Å closer to the NADþ binding domain. This NADþ is then in a position to accept a hydride from the zinc-bound substrate, as the aldehyde or ketone product forms. Deprotonation of the coordinated alcohol yields a zinc alkoxide intermediate, which then undergoes hydride transfer to NADþ to give the zinc-bound aldehyde and NADH. A water molecule then displaces the aldehyde to regenerate the original catalytic zinc center, and finally NADH is released to complete the catalytic cycle.293 In the reverse reaction, Zn2þ polarizes the carbonyl bond and promotes the transfer of a hydride ion from NADH to the carbonyl group.

Fig. 25 (A) Catalytic zinc site in horse liver alcohol dehydrogenase (PDB 8ADH). The Zn2þ ion and its associated water molecule are shown as grey and red spheres, respectively, while metal coordination interactions are shown as yellow dashed lines. (B) Dimeric structure of horse liver alcohol dehydrogenase (PDB 4DXH), bound to trifluoroethanol (green sticks) and NADþ (cyan sticks). The two monomers are colored yellow and orange, while their catalytic and structural Zn2þ ions are shown as grey and blue spheres, respectively.

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The biochemistry and enzymology of zinc enzymes

Fig. 26

Catalytic mechanism of liver alcohol dehydrogenase.

Human sorbitol dehydrogenase is an enzyme participating in the metabolic pathway that interconverts glucose and fructose,294,295 that is evolutionary related to the canonical liver alcohol dehydrogenases,296 with one catalytic zinc center47,297 in which the metal ion is coordinated to a His, a Cys and Glu residues and a water molecule.298,299 The zinc ion has been proposed to play a role similar to that in canonical ADH enzymes, i.e., binding and polarizing the substrate priming the redox reaction. Other related enzymes are L-Histidinol dehydrogenase, that catalyzes the NAD-mediated four electron oxidation of L-Histidinol to L-Histidine, present in plants, archaea and bacteria, with one zinc center with a Gln, two His and an Asp ligand.300 This zinc center was previously suggested to play a catalytic role,301,302 but later crystal structures revealed that it contributes to bind and orient the Lhistidinol substrate.300,303

2.10.5.4

Zinc transferases

Transferases catalyze the transfer of functional groups from a donor molecule to an acceptor. In particular, prenyl-transferases add lipidic isoprenoids by forming stable thioether bonds to proteins that act at cell membranes. Post-translational lipidation events as prenylation, allow cytosolic proteins to interact with cell membranes.304 Protein farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase-I) are two examples of zinc dependent prenyltransferases. Both FTase and GGTase are present in all eukaryotic cells and are responsible of attaching the corresponding isoprenoid moiety to a specific cysteine residue included in a typical CAAX motif at the C-terminal region of the protein substrate (Fig. 27). In the CAAX box, C is a Cys, A corresponds to an aliphatic amino acid, and X can be a range of amino acids (mainly Ala, Ser, Met or Glu).305 The crystal structure of a mammalian farnesyltransferase306 showed that prenyl-transferases are heterodimeric zinc enzymes. FTase and GGTase share a common a subunit, but b subunits have a similarity of only 30%. Both subunits are mainly composed of a-helices, but with different layouts (Fig. 28). The a subunit is a crescent-shaped super helix made of 13 or 14 a helices, whereas the b subunit is made of 12 a helices giving rise to an a-a barrel conformation.307,308 Structural analysis of FTases shows an active site formed in the junction of two clefts, a cavity detected at the interface of a and b subunits.309 The catalytic Zn2þ ion is

Fig. 27 Reaction catalyzed by a prenyl-transferase, specifically protein farnesyltransferase. The protein undergoing post-translational modification is represented as an orange sphere. A1 and A2 are aliphatic aminoacids, while the position indicated by X can be occupied by various residues depending on the specific prenyl-transferase, mainly Ala, Ser, Met or Glu.

The biochemistry and enzymology of zinc enzymes

255

Fig. 28 (A) Active site structure of rat protein farnesyltransferase (PDB 1FT1). (B) and (C) Active site (B) and global (C) structure of rat protein farnesyltransferase (PDB 1JCS), bound to a substrate peptide (green sticks) and a farnesyl diphosphate analogue (cyan sticks). In each panel, the catalytic Zn2þ ion and its bound water is shown as grey and red spheres, respectively, and metal coordination interactions are represented as yellow dashed lines. In (C), the a subunit is shown in light blue while the b subunit is shown in pink.

coordinated to 3 residues from the b subunit (Asp297, Cys299 and His362) plus a water molecule306 (Fig. 28). The crystal structure of a ternary complex of rat FTase with a CaaX peptide and a substrate analogue shows that the lipid isoprenoid binds to a hydrophobic cavity near the catalytic Zn2þ ion and the coordination of the sulfur-containing peptide with the active-site zinc310 (Fig. 28). Zinc can activate thiols for the nucleophilic attack in several enzymes involved in sulfur alkylation, as was shown by using metal substitution in FTase.311 Replacement of the native metal ion by Co(II) revealed formation of a charge transfer band in a FTaseisoprenoid-peptide ternary complex and the time-evolution of the sulfide-metal interaction during turnover. Although the zinc is essential for catalysis and binding of the CaaX peptide in FTase, its role in farnesyl binding has not been definitively addressed.312 The presence of a Mg2þ ion is fundamental for FTase activity, but not for GGTase.313 The chemical step of the catalysis by FTase was debated for a long time.314,315 Currently, the accepted catalytic mechanism for FTase include two states interchanged by a carboxylate-shift: the zinc ion interacts with residues Asp297, Cys299, His362, and with a water molecule, in a distorted tetrahedral coordination; and a second state where there is a second interaction with Asp297 in replacement of the zinc-water bond still having the same geometry of coordination. These two states with similar energies and coordination numbers allow an efficient mechanism of catalysis. The carboxylate group from Asp 197 can control ligand entrance. It has been proposed that both transferases, FTase and GGTase follow equivalent catalytic mechanisms and that these enzymes recognize substrates based on the structure of their C terminus.304,315 Prenyltransferases proteins substrates are miscellaneous in nature, but probably the most relevant are small GTPases of the Ras superfamily, where the prenylation is indispensable for GTPases activity.316 Inhibition of FTase activity is of great interest for cancer therapeutics, parasitic diseases, antiviral agents and neurological diseases.305,309 As already discussed, the E. coli Ada repair protein is an outlier, since it contains a Zn(Cys)4 site, that looks like a canonical zinc finger site, but the metal ion plays a catalytic role. The Ada protein is a DNA repair protein that catalyzes the transfer of a methyl group from the DNA methylphosphotriester to the sulfur of a Cys ligand.137,317 Methylation of the Ada protein induces a conformational change137 that converts it into a transcription factor that induces its own gene expression as well as the expression of other genes that repair DNA methylation. The zinc ion lowers the pKa of the bound Cys thus enhancing its reactivity.139

256 2.10.5.5

The biochemistry and enzymology of zinc enzymes Zinc isomerases

The first report of a zinc-dependent phosphomannose isomerase (PMI) comes from foundational works from Gracy and Noltman in 1968.318–320 Metal-dependent phosphomannose isomerases (E.C. 5.3.1.8), also known as Mannose-6 phosphate isomerase, catalyze the reversible isomerization of D-mannose 6-phosphate (M6P) and D-fructose 6-phosphate (F6P) (Fig. 29). PMIs can be found both in prokaryotic and eukaryotic cells, from bacteria to fungi to mammals. These aldose-ketose isomerases are divided in three unrelated families: Type I, Type II, and Type III. Type I PMIs are monofunctional zinc-dependent isomerases found in some bacterial and all eukaryotic enzymes. Type II PMIs are bifunctional enzymes with guanosine-diphosphate-D-mannose pyrophosphorylase plus the PMI activities. There is only one type III PMI, identified in Rhizobium meliloti.321,322 M6P is an essential precursor of mannosylated molecules as glycoproteins, the fungal cell wall and bacterial exopolysaccharides. PMIs are needed for the survival, virulence and/or pathogenicity of bacteria and protozoan parasites, as well as for the integrity of some fungi. As a result, inhibition of PMI is an attractive strategy to control fungal infections that can be potentially lethal, in particular for immunodeficient individuals.323,324 The first structure of a PMI from Candida albicans was reported in 1996. This work identified the zinc binding site as well as the active site.324 PMI is a monomeric protein with three domains, two of which are beta-domains while the third is almost exclusively alfa-helical. PMIs have an overall mixed a/b structure with the active site containing one zinc ion. This catalytic Zn2þ is pentacoordinated in the resting state, with Gln111, His113, Glu138, His285 and a water molecule defining the ligand set (Fig. 30). The presence of glutamine as a zinc ligand is an unusual characteristic of this PMI. The resulting coordination geometry is a distorted trigonal bipyramid. In addition, Arg304 is conserved in PMI type I enzymes, and it is proposed to be involved in substrate binding. The crystal structure of the C. albicans PMI (CaPMI) complexed with the competitive inhibitor 5-phospho-D-arabinonhydrazide (5PAHz) (PDB: 5NW7), revealed more details about the role of the zinc ion and regarding substrate binding features.322 5PAHz is an analogue of the 1,2-cis-enediolate high-energy reaction intermediate. The structure of this complex has provided information about the mechanism of the reversible isomerization catalyzed by PMIs. The CaPMI- 5PAHz structure identifies Glu294 as the general base in the mechanism. Inhibitor binding replaces the water molecule from native PMI. The structure of the PMI of Salmonella Typhimurium shows a different picture compared to that obtained for the fungal enzyme. The main difference is that the Zn2þ ion in this bacterial enzyme displays a distorted tetrahedral coordination, with His99, Glu134, His255 and a water molecule as metal ligands325 (Fig. 30). Despite the Gln ligand observed in the PMI from C. albicans is conserved, the distance between the amide O atom of Gln97 and the zinc ion 3.8 Ǻ, i.e., indicating the absence of a coordination bond from the Gln residue to the zinc ion. The isomerization process catalyzed by PMI involves a general acid/base catalysis with a stereospecific proton transfer between the C1 and C2 atoms of the substrate (Fig. 31). There is evidence supporting the notion that isomerization probably follows the cisenediol mechanism.325 The specific role of zinc is not well understood, as it can participate in the catalytic reaction or might stabilize the active site at the needed conformation for catalysis. In a posterior work by Murthy326 the structural analysis of certain mutants shows that the zinc ion have a fundamental role for M6P (substrate) binding in active site. They also proved that, in contrast with previous ideas; Glu 264 is not the catalytic base in the isomerization reaction but they support the role of a Lys residue as the catalytic base. The L. Salmon group322 proposed a dual role in catalysis for zinc: acting as a Lewis acid by polarizing the CeO bond for the substrate deprotonation, and stabilizing 1,2-cis-enediolate binding. This supports the hypothesis that F6P/M6P substrate forms a bidentate ligand by O1 and O2 oxygen atoms with zinc. Despite several efforts, there is no clear final picture of which residues are the essential ones for proper catalysis in PMI metalloenzymes.322,326,327

2.10.5.6

Zinc ligases

Aminoacyl-tRNA synthetases (aaRS), or tRNA-ligases, are enzymes that covalently attach specific amino acids (AA) onto its corresponding tRNA for the following RNA translation and protein biosynthesis. Additionally, aaRS are involved in some metabolic and signaling pathways.328,329

Fig. 29

Scheme of the reaction catalyzed by mannose-6 phosphate isomerase.

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Fig. 30 (A) and (B) Active site structure of mannose-6 phosphate isomerase from C. albicans (A, PDB 1PMI) and from S. Typhimurium (B, PDB 3H1M). (C) Global structure of mannose-6 phosphate isomerase from C. albicans (PDB 1PMI). In each panel, the Zn2þ ion and its associated water molecule are shown as grey and red spheres, respectively, while metal coordination interactions are shown as yellow dashed lines.

Aminoacyl-tRNA synthetases catalyze a two-step aminoacylation reaction, in order to add the specific amino acid to the 30 end of the corresponding tRNA by forming an ester bond, using energy from the hydrolysis of ATP, yielding aminoacyl-tRNA, AMP, and PPi. In the amino acid activation step, both substrates (AA and ATP) bind the aaRS catalytic site. The in-line nucleophilic attack of the a-carboxylate oxygen of the AA to the a-phosphate group of the ATP, yields the aminoacyl-adenylate (aa-AMP) and inorganic pyrophosphate. In the second step, the carbonyl carbon of the adenylate is attacked by a hydroxyl from a 30 -terminal adenosine of tRNA, to produce aminoacyl-tRNA and AMP as the leaving group. In addition to this main reaction, the proofreading and editing steps are present in various aaRS to eliminate incorrect amino acids recognized by a noncognate aaRS.330,331

Fig. 31

Reaction mechanism for mannose-6 phosphate isomerase proposed by Salmon group.322

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Fig. 32 (A) Active site Zn2þ coordination for E. coli threonyl-tRNA synthetase (PDB 1QF6). The Zn2þ ion and its associated water molecule are shown as grey and red spheres, respectively, while metal coordination interactions are shown as yellow dashed lines. (B) Global structure of E. coli threonyl-tRNA synthetase (PDB 1QF6) bound to its cognate tRNA (orange sticks) and AMP (cyan sticks).

These universally distributed enzymes are divided into two distinct classes (Class I and Class II) without similarities on sequence or structure. For class I enzymes the aminoacyl-tRNA release is the rate-limiting step, but for class II it is the amino acid activation rate instead.329 Translation fidelity is key for organismal survival, so tRNA synthetases must unequivocally recognize and pair a tRNA with their associated amino acid. This property relies on the appropriate identification of both substrates (tRNA and AA). In particular, AA recognition and discrimination is quite difficult based on the limited contacts that the enzyme can make with these small molecules. Aminoacyl-tRNA synthetases deployed various strategies for selectively, such as charge and size recognition. Also, some aaRSs use the ability of metal ions for binding specific chemical features and strict geometry according to ligands, as detailed below.329,330 Structural studies of threonyl-tRNA synthetase from E. coli disclosed the presence of a zinc ion in the active site that mediated amino acid discrimination versus the noncognate valine. The zinc in active site of this ThrRS is coordinated to Cys334, His385, His511 and a water molecule in a tetrahedral coordination332 (Fig. 32). After substrate (threonine) binding, the molecule of water is displaced and a pentacoordinated state adopts a square-based pyramidal geometry. The steric constraints imposed by the geometry of the three protein ligands and zinc, abolish valine binding. As ThrRs cannot activate valine, there is no need for an additional editing mechanism in ThrRS, opposite to other aaRSs where this mechanism is mandatory.333 Here, the zinc ion has a distinct role for amino acid recognition in aaRSs, not being strictly a catalytic nor structural zinc site. Other examples of cognate/noncognate discrimination thanks to zinc in the active site are some CysRS and SerRS enzymes. In the monomeric class I CysRS from E. coli, the zinc is coordinated to residues Cys31, Cys215 and His240. The coordination sphere is completed with a strong bond with the thiolate group of the cognate substrate cysteine in a stringent mechanism. Likewise, as in ThrRS, this property does not require an extra editing step.334 Atypical SerRS from methanogenic archaea have one zinc coordinated to Cys306, Glu355, Cys461 and a water molecule that is displaced when the serine substrate is bound. The zinc coordination geometry remains tetrahedral after serine binding. Non-cognate AA threonine have steric impediments to enter the active site properly.335 Alanyl-tRNA synthetase class II aaRS have a zinc ion in the editing site, coordinated to His316, His617, Cys717 and a water molecule. Apparently, AlaRSs use a different mechanism compared to ThrRS described before.336 In summary, these zinc-dependent amino acid recognition mechanisms differ fundamentally from other aaRSs, highlighting the versatility of the zinc ion in fulfilling different roles in essential enzymes.

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Chem. 1968, 243 (20), 5410–5419. https://doi.org/10.1016/S0021-9258(18)91963-0. 321. Proudfoot, A. E. I.; Turcatti, G.; Wells, T. N. C.; Payton, M. A.; Smith, D. J. Purification CDNA Cloning and Heterologous Expression of Human Phosphomannose Isomerase. Eur. J. Biochem. 1994, 219 (1–2), 415–423. https://doi.org/10.1111/j.1432-1033.1994.tb19954.x. 322. Ahmad, L.; Plancqueel, S.; Dubosclard, V.; Lazar, N.; Ghattas, W.; Li de la Sierra-Gallay, I.; Tilbeurgh, H.; Salmon, L. Crystal Structure of Phosphomannose Isomerase from Candida albicans Complexed with 5-phospho- D -arabinonhydrazide. FEBS Lett. 2018, 592 (10), 1667–1680. https://doi.org/10.1002/1873-3468.13059. 323. Proudfoot, A. E. I.; Payton, M. A.; Wells, T. N. C. Purification and Characterization of Fungal and Mammalian Phosphomannose Isomerases. J. Protein Chem. 1994, 13 (7), 619–627. https://doi.org/10.1007/BF01890460. 324. Cleasby, A.; Wonacott, A.; Skarzynski, T.; Hubbard, R. E.; Davies, G. J.; Proudfoot, A. E. I.; Bernard, A. R.; Payton, M. A.; Wells, T. N. C. The X-Ray Crystal Structure of Phosphomannose Isomerase from Candida Albicans at 1.7 Å Resolution. Nat. Struct. Mol. Biol. 1996, 3 (5), 470–479. https://doi.org/10.1038/nsb0596-470. 325. Sagurthi, S. R.; Gowda, G.; Savithri, H. S.; Murthy, M. R. N. Structures of Mannose-6-Phosphate Isomerase from Salmonella Typhimurium Bound to Metal Atoms and Substrate: Implications for Catalytic Mechanism. Acta Crystallogr. D Biol. Crystallogr. 2009, 65 (7), 724–732. https://doi.org/10.1107/S0907444909013328. 326. Bangera, M.; Gowda, K. G.; Sagurthi, S. R.; Murthy, M. R. N. Structural and Functional Insights into Phosphomannose Isomerase: The Role of Zinc and Catalytic Residues. Acta Crystallogr. D Struct. Biol. 2019, 75 (5), 475–487. https://doi.org/10.1107/S2059798319004169. 327. Roux, C.; Bhatt, F.; Foret, J.; de Courcy, B.; Gresh, N.; Piquemal, J.-P.; Jeffery, C. J.; Salmon, L. The Reaction Mechanism of Type I Phosphomannose Isomerases: New Information from Inhibition and Polarizable Molecular Mechanics Studies: Type I Phosphomannose Isomerases Mechanism. Proteins 2011, 79 (1), 203–220. https://doi.org/ 10.1002/prot.22873. 328. Cusack, S. Aminoacyl-TRNA Synthetases. Curr. Opin. Struct. Biol. 1997, 7 (6), 881–889. https://doi.org/10.1016/S0959-440X(97)80161-3. 329. Kaiser, F.; Krautwurst, S.; Salentin, S.; Haupt, V. J.; Leberecht, C.; Bittrich, S.; Labudde, D.; Schroeder, M. The Structural Basis of the Genetic Code: Amino Acid Recognition by Aminoacyl-TRNA Synthetases. Sci. Rep. 2020, 10 (1), 12647. https://doi.org/10.1038/s41598-020-69100-0. 330. Rubio Gomez, M. A.; Ibba, M. Aminoacyl-TRNA Synthetases. RNA 2020, 26 (8), 910–936. https://doi.org/10.1261/rna.071720.119. 331. Ibba, M.; Soll, D. Aminoacyl-TRNA Synthesis. Annu. Rev. Biochem. 2000, 69, 617–650. https://doi.org/10.1146/annurev.biochem.69.1.617. 332. Sankaranarayanan, R.; Dock-Bregeon, A.-C.; Romby, P.; Caillet, J.; Springer, M.; Rees, B.; Ehresmann, C.; Ehresmann, B.; Moras, D. The Structure of Threonyl-TRNA Synthetase-TRNAThr Complex Enlightens Its Repressor Activity and Reveals an Essential Zinc Ion in the Active Site. Cell 1999, 97 (3), 371–381. https://doi.org/ 10.1016/S0092-8674(00)80746-1. 333. Sankaranarayanan, R.; Dock-Bregeon, A.-C.; Rees, B.; Bovee, M.; Caillet, J.; Romby, P.; Francklyn, C. S.; Moras, D. Zinc Ion Mediated Amino Acid Discrimination by ThreonylTRNA Synthetase. Nat. Struct. Mol. Biol. 2000, 7 (6), 461–465. https://doi.org/10.1038/75856. 334. Zhang, C.-M.; Christian, T.; Newberry, K. J.; Perona, J. J.; Hou, Y.-M. Zinc-Mediated Amino Acid Discrimination in Cysteinyl-TRNA Synthetase. J. Mol. Biol. 2003, 327 (5), 911–917. https://doi.org/10.1016/S0022-2836(03)00241-9. 335. Bilokapic, S.; Maier, T.; Ahel, D.; Gruic-Sovulj, I.; Söll, D.; Weygand-Durasevic, I.; Ban, N. Structure of the Unusual Seryl-TRNA Synthetase Reveals a Distinct Zinc-Dependent Mode of Substrate Recognition. EMBO J. 2006, 25 (11), 2498–2509. https://doi.org/10.1038/sj.emboj.7601129. 336. Sokabe, M.; Ose, T.; Nakamura, A.; Tokunaga, K.; Nureki, O.; Yao, M.; Tanaka, I. The Structure of Alanyl-TRNA Synthetase with Editing Domain. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (27), 11028–11033. https://doi.org/10.1073/pnas.0904645106.

2.11

Cobalt enzymes

Bernhard Kra¨utler, Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innsbruck, Austria © 2023 Elsevier Ltd. All rights reserved.

2.11.1 2.11.2 2.11.2.1 2.11.2.2 2.11.3 2.11.3.1 2.11.3.2 2.11.3.3 2.11.4 2.11.4.1 2.11.4.1.1 2.11.4.1.2 2.11.4.1.3 2.11.4.1.4 2.11.4.2 2.11.4.2.1 2.11.4.2.2 2.11.4.2.3 2.11.4.3 2.11.4.3.1 2.11.4.3.2 2.11.4.4 2.11.5 2.11.5.1 2.11.5.2 2.11.5.3 2.11.6 2.11.7 References

Introduction Structures of the B12-derivatives “Incomplete” and “complete” corrinoids The “base-on/base-off” switch of “complete” corrinoids Organometallic and redox-chemistry of B12-derivatives On the homolytic cleavage and formation of the CoeC bond On the nucleophile-induced heterolysis and formation of the CoeC bond On the radical-induced abstraction of cobalt-bound methyl groups Cobalt-corrins as cofactors and intermediates in enzymes B12-dependent methyl transferases Cobamide-dependent methionine synthase Corrinoid methyl group transferases in anaerobic methane metabolism Corrinoid methyl group transferases in bacterial acetate metabolism B12-dependent radical-SAM methyl group transferases Enzymes dependent on coenzyme B12 and related adenosylcobamides Carbon-skeleton mutases Coenzyme B12-dependent isomerases Coenzyme B12-dependent ribonucleotide reductases B12-processing enzymes Adenosyltransferases Cobalamin-deligase CblC B12-dependent dehalogenases B12-derivatives as ligands of proteins and nucleic acids B12-binding proteins for uptake and transport in mammals and bacteria Cobalamins as gene-regulatory RNA-ligandsdB12-riboswitches Coenzyme B12 as light-sensitive ligand in photo-regulatory proteins Why cobalt?dB12-analogs with other metals and antivitamins B12 Summary and outlook

268 269 269 271 271 274 274 275 275 275 275 276 277 278 279 282 284 287 288 288 289 290 291 291 291 291 292 293 293

Abstract Cobalt enzymes come in two major categories: widespread B12-dependent enzymes and lesser known non-corrin cobalt enzymes. We focus here on B12-enzymes, which are grouped further in three classes according to the nature of their stable B12cofactor forms. Hence, these enzymes either use organometallic B12-cofactors, adenosylcobamides (AdoCbas) or methylcobamides (MeCbas), or primarily redox-active cobamides, such as Co(II)cobamides (Cbas(II)). The AdoCbas catalyze enzymatic radical reactions with “difficult chemistry,” initiated by the reactive 50 -deoxy-50 -adenosyl radical from homolysis of their CoeC bond; MeCbas help catalyze enzymatic methyl group transfer reactions, involving either nucleophilic or radical type reaction partners; “non-ligated” Cbas(II) are the typical redox-cofactor forms observed in enzymes dealing with dehalogenation and related reduction reactions. Additional “borderline” cobalt-enzymes are involved in AdoCba-dependent photo-regulatory processes, in B12-processing and B12-biosynthesis. The natural Cbas have evolved a rich spectrum of biological catalysis and regulation as the result of the special interplay of their helical, ring-contracted corrin ligand and the tightly bound cobalt-center. Clearly, enzyme catalysis with cobalt-corrins and other cobalt-centers has become indispensable in the evolved world, where cobalt-corrins play essential roles in most spheres of Life.

2.11.1

Introduction

The element cobalt, named after the mischievous goblins (kobolds) that bothered German miners, is an important and puzzling “bio-metal” essential for life.1,2 Cobalt enzymes play central roles in most living organisms and occur in two categories.3–5 Besides the B12-dependent enzymes,6–9 other cobalt-enzymes are known that use directly protein-bound cobalt centers to catalyze hydration/dehydration processes,3,4 and to insert cobalt into cyclic tetrapyrrole intermediates of the B12-biosynthesis.10–12 This chapter

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focusses on the widespread B12-dependent enzymes.13,14 In fact, with the exception of higher plants, organisms in all kingdoms of life depend upon B12-enzymes.15–18 In the living world the cobalt-corrin enzymes catalyze unique reactions making direct use of the organometallic reactivity and redox-activity of the bound B12-cofactors.19 The B12-coenzymes fascinate as the most complex of the known cofactors, yet “early” forms of the cobalt-corrins may have played decisive roles in the evolution of life.20 The human metabolism needs two of the B12-dependent enzymes for catalyzing essential reactions: B12 is, thus, classified as a vitamin.16,18

2.11.2

Structures of the B12-derivatives

Over 70 years ago the (extrinsic) anti-pernicious anemia factor21,22 was isolated as a red cyanide containing cobalt complex called vitamin B12, opening up the B12-field to biomedicine and the natural sciences. Crystals of vitamin B12 (cyanocob(III)alamin, CNCbl) were subjected to a pioneering X-ray structure analysis by Hodgkin and her collaborators.23,24 It revealed CNCbl as the cobalt complex of the unique corrin ligand conjugated to a remarkable dimethylbenzimidazole (DMB) pseudo-nucleotide (see Fig. 1). In fact, the precise structure of the corrin ligand of vitamin B12 was not fully clarified before the first crystallographic analyses of some “incomplete” corrinoids were completed,24 among them Coacyano,Cobaquo-cobyric acid (CN,H2O-Cby), the entire corrin moiety from partial degradation of CNCbl.25 The discovery of the corrin ligand with the exceptional structure of a ring-contracted, helical tetrapyrrole, suggested its special architecture to be essential for strong binding of cobalt-ions20,26 and for encasing them in an entatic state, promoting their specific organometallic reactivity and redox-catalysis.27

2.11.2.1

“Incomplete” and “complete” corrinoids

The “incomplete” corrinoids constitute the intact core part of vitamin B12 and lack its pseudo-nucleotide moiety.18 The Co(III)forms of cobyric acid (Cby) and of the cobinamides (Cbis) are their best-known representatives (Fig. 2). CN,H2O-Cby has served as starting material for the first partial synthesis of CNCbl28 and it also represented the final corrinoid synthesis target for the total synthesis of vitamin B12 in the laboratories of Eschenmoser and of Woodward.29,30 Cbis are generated from Cby by attachment, at the f-side chain, of the (R)-isopropanol amine linker section of the Cbl pseudo-nucleotide segment.28 In a related approach, the “incomplete” organometallic B12-biosynthesis intermediate 50 -deoxy-50 -adenosylcobyrate (AdoCby)31 was used for a chemical synthesis of coenzyme B12 (AdoCbl).32 Hepta-esters of the hepta-acid cobyrinic acid (Cbin), semisynthetic lipophilic B12-derivatives, have played a role as B12-model compounds,20 most prominent among them the crystalline dicyano-heptamethylcobyrinate (“cobester”), which was a key relay compound in the course of the total synthesis of vitamin B12.29 While crystal structures of “cobester”33 and of its Co(II)-form, heptamethyl-cob(II)yrinate (“cob(II)ester”),34 became measured, related analyses of cobinamides, or of a Co(I)-corrin, have remained unavailable. Most investigations with vitamin B12-derivatives have used biotechnologically produced cobalamins (Cbls), among them vitamin B12 (CNCbl), in particular. The crystal structure of the “complete” corrinoid CNCbl not only provided insights into the CN

H2NOC

CONH2 CH3

H2NOC

+

CNCbl N

CONH2 H 3C

O

Co+ N N

H H2NOC

N

N CN N

H3C

CH3

CH3

+

MeCbl N

H 3C CONH2 O

HN H3 C

HO

H

N O

N

O

H 2C

CH3 O

P OH

O

N

N

N N

NH2

III

Co N

O

OH

HO

CH3

N

+

O

CoIII

CH3

H3C

O

CoIII

CH3

AdoCbl

N O

Fig. 1 Structural formula of vitamin B12 (CNCbl) (left) and symbolic presentations (right) of CNCbl, methylcobalamin (MeCbl) and coenzyme B12 (50 -adenosylcobalamin, AdoCbl).

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Fig. 2 Structural formulae of natural cob(III)alamins and of “incomplete” corrinoids. Left: cobalamins: vitamin B12 (CNCbl), coenzyme B12 (AdoCbl), methylcobalamin (MeCbl) and hydroxycobalamin (HOCbl). Right: “incomplete” natural corrinoids: Coacyano,Cobaquo-cobyric acid (CNa,H2Ob-Cby), dicyano-cobinamide (CN2-Cbi) and the lipophilic dicyano-heptamethylcobyrinate “cobester.”

structure of the natural corrin ligand, but also into the unique 3D-architecture of the mutually interacting pseudo-nucleotide and corrin moieties.24,35 The Co(III)-center of CNCbl and of other Co(III)-corrins is a d6-ion that has a high tendency for coordinating two axial ligands to constitute a (nearly) octahedral arrangement.36 In the Cbls, a 5,6-dimethylbenzimidazole (DMB) heterocycle coordinates the cobalt ion from the so-called “lower” (or a) axial side, making the CN ligand of CNCbl occupy the “upper” (or b) axial side.13,36–38 Remarkably, CNCbl, the most important commercially available form of the naturally occurring Cbls, has no direct physiological function, itself, in humans.39 The very light-sensitive organometallic cofactors, coenzyme B12 (50 -deoxy-50 -adenosylcobalamin, AdoCbl)40 and methylcobalamin (MeCbl)41,42 (see Fig. 2) were shown instead to represent the biologically primarily relevant Cbls. The crystal structure analysis of AdoCbl led to the discovery of its puzzling organometallic nature,43 focusing further interest on specifically describing the structural features and the reactivity of organometallic B12-derivatives.9,38,44 In fact, the crystal structure of AdoCbl revealed two relatively long axial bonds (Co–C: 2.030 Å and Co–N: 2.237 Å), consistent with the weak CoeC bond of this biologically important organocobalamin.45 Interest in the chemistry of MeCbl eventually led to an analysis of its crystal structure, showing both its axial bonds to be shorter than the ones in AdoCbl, as expected.46 The oxygen-sensitive cob(II)alamin (Cbl(II)), the product of CoeC bond homolysis of coenzyme B12 (AdoCbl), displayed a remarkably similar structure as the Co(III)-corrin moiety of AdoCbl.47 The crystal structures of a range of “inorganic complete” corrinoids were also studied,48 among them aquocob(III)alamin (H2OCblþ),49 the conjugate acid to hydroxocob(III)alamin (HOCbl). The solution structures of CNCbl and of coenzyme B12 (AdoCbl) in water were also investigated with increasing detail.50,51 Extensive heteronuclear NMR-studies were key in deriving information on such structures, which was mostly consistent with the X-ray crystallographic structure data. The NMR-analysis of AdoCbl, furthermore, revealed the dynamic nature of the organometallic 50 -deoxyadenosyl group, which explores various conformations around the CoeC bond.51 Surprisingly, the original crystal structure of MeCbl46 and the NMR-derived structure in aqueous solution differed,52 indicating significant restructuring of the nucleotide loop of MeCbl by the solvent. Hence, organo-Cbls are cobalt-corrins carrying rather flexibly attached organic “upper” ligands53 and non-rigid “lower” pseudo-nucleotide moieties.37 The broad interest in the structures of organometallic Cbls induced a number of crystal structure analyses,37,44 such as of vinylcobalamin and of cis-chlorovinylcobalamin, examples with sp2-hybridized carbon ligands studied in the context of the B12-dependent dehalogenases.54 The structures of arylcobalamins,55 and arylalkynylcobalamins,56 helped to characterize the structures of B12-derivatives with an organometallic CoeC bond to aromatic sp2- or to spcarbons. Interestingly, while the Co-Csp bonds were shown to be rather short (1.86 Å), as expected, the Co-Csp2 bond of the phenyl-Cbls was only marginally shorter (1.98 Å) than the one of the Co-Csp3 bond of MeCbl (1.99 Å), an observation rationalized by steric strain between the corrin and aryl moieties in the organometallic phenyl-Cbls.55,57 In addition to Cbls, cobamides (Cbas) with nucleotide bases other than DMB have been analyzed by crystallography. The analyses of Cob-cyano-70 -adeninyl-Cba (also named pseudovitamin B12) and Cob-cyano-(70 -[20 -methyl]-adeninyl)-Cba (factor A) furnished a similar 3D-structure and coordination geometry around the cobalt-center in these two cyano-purinyl-Cbas,58 as was known for CNCbl itself. These structures were also similar to the one of nor-pseudovitamin B12 (Cob-cyano-70 -adeninyl-176norcobamide), a natural 176-nor-cobamide (nCba) discovered as cofactor of dehalogenases.58 The nCbas present a natural variation in the linker part of a Cba by lacking the methyl group C176 of the cob(in)amide moiety (Fig. 3).58 The methyl group at C176 of

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vitamin B12 and of other Cbas, while stabilizing the “base-on” form with respect to its “base-off” counterpart, displayed an insignificant structural effect in the cobalt-coordinated “base-on” form of CNCbl in comparison to the 176-nor-cobalamin (nCbl) norvitamin B12.59

2.11.2.2

The “base-on/base-off” switch of “complete” corrinoids

The cobalamins (Cbls) exhibit a highly intriguing structure as a result of the covalent linkage of the cobyric acid core and a pseudonucleotide with a DMB-heterocycle. Cbls are chiral and asymmetric in 3D-space and their “base-on” form displays a non-planar graph, making it chiral even topologically.13,60 The pseudo-nucleotide moieties of Cbls and other cobamides (Cbas) coordinate the cobalt ion from the “lower” (or a) axial side and also feature an exceptional a-configuration at the anomeric carbon of the ribofuranose. This rare specific configuration is crucial for the stable intramolecular coordination of the pseudo-nucleotide heterocycles in the “base-on” forms of the Cbas, generating a nearly strain-free, 19-membered macrocycle, including the Co(III)-center.24,59 Among the Cbas, the Cbls stand out by their relatively strong intramolecular cobalt-coordination of a DMB-heterocycle and their stable “base-on” forms.13 The purine heterocycles in adeninyl-Cbas are more weakly coordinated at the central cobalt-ion of the corrin moiety, and the “base-off” and “base-on” forms of the organometallic 50 -adenosyl-adeninyl-Cbas are comparably stable, pairwise, in neutral aqueous solution.61 De-coordination of the DMB-base in Cbls furnishes their alternative “base-off” forms (see Fig. 4).8,19 Cbls and Cbas are, thus, natural “molecular switches.”62 Protonation of the DMB-heterocycle induces a complete switch to “base-off.” The associated acidity of the protonated “base-off” form (as expressed by its pKa) reflects, quantitatively, the strength of the intramolecular DMBcoordination.8,48 Hence, the proton-assisted de-coordination is inhibited significantly when cobalt-coordination is strong, as in CNCbl8 and in alkynyl-Cbls,63 which exhibit pKa’s near 0 for their protonated “base-off” forms. On the other hand, the (protonated) “base-off” forms of the B12-cofactors AdoCbl and MeCbl are readily accessible, exhibiting pKa’s of 3.67 (AdoCbl-Hþ) and 2.9 (MeCbl-Hþ).8 The global and local structure of Cbls (and of other Cbas) is, obviously, a critical determinant for the selective and tight binding by B12-binding macromolecules.9,64 Hence, the “base-on” forms of the “complete” corrinoids may (or may not) be structured correctly for strong binding by specific B12-apoenzymes (see below). In order to generate a tight interaction with the Cba-ligand, a protein or an oligonucleotide environment may switch the bound B12-cofactors from “base-on” to “base-off,”65 or, in the reverse sense, from “base-off” to “base-on.”64,66,67 Since the two forms (“base-off” or “base-on”) of Cbas differ significantly as to their reactivity, this type of restructuring may affect the crucial reactivity of the bound B12-cofactors (see below).68 The coordinating DMBnucleotide function also steers the face-selectivity at the corrin-bound cobalt center. By coordinating to the “lower” face in Cbls, the DMB directs alkylation (and other ligation) reactions to the “upper” (or b) axial face, thermodynamically.68 This stereo-directing effect may be particularly relevant, kinetically, in the very rapid (re)combination reaction between Cbl(II) and organic radicals.55,69

2.11.3

Organometallic and redox-chemistry of B12-derivatives

The discovery of coenzyme B12 (AdoCbl) has induced a rapid surge of syntheses of organometallic Cbls, of AdoCbl in particular, accompanied by investigations of their organometallic chemistry. The formation and cleavage of the CoeC bond, two key reactions

H2NOC

H2NOC

CONH2 CH3 CH3

H2NOC

CONH2 CH3

CH3

H2NOC

CONH2

CONH2 H3 C H3 C H H2NOC

N CNN Co+ N N

CH3 CH3

H3 C

H3 C H3 C H H2NOC

CH3

N CNN Co+ N N

H3 C

CH3

CONH2 HN

O

176

HO

H3C H

N

N

O

O

O

HN

O

176

HO

N

N

NH2

O

O

CONH2 N

NH2 O

P

P O

N

N N

O

CH3 CH3

OH

pseudovitamin B12

O

O

OH

nor-pseudovitamin B12

Fig. 3 Structural formulas of two natural purinyl-Cbas differing by the covalent linker to their nucleotide moiety: Co-cyano-70 -adeninyl-Cba (pseudovitamin B12, left) and Co-cyano-70 -adeninyl-176-norcobamide (nor-pseudovitamin B12, right).

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R +

R Kon

CoIII

+

CoIII

in H2O H2 O

N

N N

N

O

O RCbl "base-on" + H+

+H+, H2O

RCbl "base-off" R +

CoIII

H2 O

protonated "base-off" form of RCbl

N NH O Fig. 4 Cobalamins as “molecular switches.” The DMB-base is cobalt-coordinated in the “base-on” Cbls and de-coordinated in their less stable “base-off”-form. In aqueous solution, the “base-on” form of AdoCbl (R ¼ 50 -deoxyadenosyl) is more stable with Kon ¼ 72 at 25  C; protonation of the DMB-base of AdoCbl leads to the stable protonated “base-off” form that is a weak acid, featuring a pKa of 3.67.

of organometallic B12-derivatives, are essential for the catalysis by B12-dependent enzymes (see below).8,13,14,70 Organometallic Cbls were mostly prepared by applying alkylation of their Co(I)-form Cbl(I) by typical reagents undergoing nucleophilic substitution reactions readily.28,42 In contrast, for the efficient syntheses of aryl-Cbls55 (see Fig. 5) and of alkynyl-Cbls,56 radical generating conditions and the Co(II)-corrin Cbl(II) were employed. The 50 -deoxy-50 -adenosyl-Co(III)-corrin AdoCbl decomposes in acidic aqueous solutions by proton-induced heterolysis and fragmentation of its deoxyadenosine moiety, furnishing H2OCblþ.71 Alkynyl-Cbls are also cleaved slowly in acidic aqueous medium by proton-induced heterolysis of the CoeC bond and formation of H2OCblþ.63 In contrast, the cleavage of the CoeC bond of MeCbl by CH3-protonation is not documented. However, the polarizable electrophilic HgII-ions readily abstract the methyl group of MeCbl in an environmentally important path to the poisonous HgII-CH3 ion.72,73 In all of these heterolytic reactions the cobalt centers of the involved Cbls retain their formal oxidation level Co(III). The organometallic reactivity of the natural Cbas is firmly intertwined with their basic redox-chemistry. Under physiological conditions, vitamin B12-derivatives occur as Co(III)-, Co(II)-, or Co(I)-corrins.13,14 Each of these (formal) oxidation states possesses strongly differing spectra, reactivity, and coordination properties.13,74 Electrochemistry has allowed not only the determination of the crucial redox-potentials of Cbls in solution,74 but also the efficient electro-synthesis of intricate organometallic B12-derivatives,75–77 as well as the analytical generation of electrode-bound B12-derivatives78 and the preparation of reduced forms of protein-bound B12-derivatives.79 The formal oxidation state of the cobalt ion controls axial coordination at the corrin-bound cobalt center, and the number of axial ligands generally decreases along a decreasing cobalt oxidation state.74 In the thermodynamically predominating forms, the diamagnetic Co(III)-center of cobalt corrins carries two axial ligands (coordination number 6), the paramagnetic (low spin) Co(II)corrins feature coordination number 5, and axial ligands are absent in the diamagnetic Co(I)-corrins, which are inferred to exhibit coordination number 4 (see Fig. 5). In consequence, electron transfer reactions involving B12-derivatives are accompanied in a thermodynamically and kinetically relevant way by a change in the number of axial ligands.74,80 Along these lines, electroanalytical studies of organometallic B12-derivatives indicate a rapid and (an effectively) irreversible reaction upon reduction.74,81,82 As analyzed with MeCbl, in particular, the CoeC bond is weakened considerably by the one-electron reduction.74,83 However, the standard potentials of the typical Co(III)-/Co(II)-redox pairs of organometallic B12-derivatives are strikingly more negative than the one of the Cbl(II)/Cbl(I) couple. Hence, the further reduction of organometallic Co(III)corrins does, typically, not occur at the potentials needed for electro-generation of the highly nucleophilic Co(I)-corrins (and is out of reach of the biological reductants).74,80 Thus, the selective electro-synthesis of Co(I)-corrins in the presence of suitable alkylating agents represents an excellent preparative method to alkyl-corrinoids and more complex organo-corrinoids.62,76,77,80 Strongly electron-withdrawing substituents in the organometallic ligand make them more easily reducible. In such cases, the nucleophilic Co(I)-corrins may interfere with the effective preparation of these organo-corrinoids.83–85 In cobalt-corrin enzymes, three main organometallic reaction modes are known, which involve a change of the (formal) oxidation level of cobalt (see Figs. 6 and 7): (i) Thermally induced CoeC bond homolysis, essential cofactor reactivity of AdoCbas: 50 -deoxyadenosyl-CoðIIIÞ-corrin %CoðIIÞ-corrin þ 50 -deoxyadenosyl radical

Cobalt enzymes

273

Fig. 5 Examples of basic organometallic and redox processes with cobalamins. AdoCbl is prepared (left) by the reaction of the highly nucleophilic Cbl(I) with (e.g.,) 50 -iodo-50 -deoxy-adenosine (Ado-X, X ¼ I) and is homolytically cleaved to Cbl(II) and the 50 -adenosyl radical (Ado) upon thermolysis; MeCbl is demethylated by mercuric ions furnishing methylmercury and H2OCbl(III)þ (right), or by the radical R$ to furnish R-CH3 and Cbl(II) (center); the aryl-Cbl EtPhCbl is generated from Cbl(II) in a radical reaction (center). The basic redox-reactions in water involve (bottom, from left to right) the reversible one-electron oxidation of Cbl(I) (4-coordinate Co(I)-center) to Cbl(II) (5-coordinate Co(II)-center), which may be further oxidized at more positive potential to H2OCblþ (6-coordinate Co(III)-center).

(ii) Nucleophile-induced heterolysis and formation of the CoeC bond, e.g., of MeCbas: methyl-CoðIIIÞ-corrin þ nucleophile %CoðIÞ-corrin þ methylating agent

(iii) Radical-induced abstraction of the cobalt-bound methyl group of MeCbas: methyl-CoðIIIÞ-corrin þ radical/CoðIIÞ-corrin þ methylated radical

Ado +

O

+

CoIII

Ado

N

CH3

N

CH3

coenzyme B12 (AdoCbl)

+

O

CoII N

CH3

N

CH3

cob(II)alamin (Cbl(II))

Fig. 6 The readily induced thermal homolysis of the CoeC bond of coenzyme B12 (AdoCbl) furnishes the 50 -deoxy-50 adenosyl radical (Ado) and the “radical trap” cob(II)alamin in a reversible manner.

274

Cobalt enzymes

Nu X

X = Nu -

H

C H HH

CoI +

+

CoIII N

Co(I)Cbl X=R

+

R

N O

N N

O

H H C H

H

C H H

-

CoII

+ N N

MeCbl O

Co(II)Cbl Fig. 7 Abstraction of the cobalt-bound methyl group of MeCbl by nucleophiles (Nu) (top) or by an organic radical (R) (bottom), as biologically important modes of substrate-induced cleavage of the CoeC bond of MeCbl and other MeCbas.

2.11.3.1

On the homolytic cleavage and formation of the CoeC bond

Coenzyme B12 (AdoCbl) has been classified as a “reversible carrier of an alkyl radical” or as a “reversibly functioning radical source,”86 as it readily undergoes selective thermal homolysis of its CoeC bond (see Fig. 6).87 Indeed, the homolytic cleavage of the CoeC bond of AdoCbl is particularly important for its cofactor role (see below). The homolytic bond dissociation energy (BDE) of the CoeC bond of AdoCbl in aqueous solution is about 30 kcal/mol (from kinetic analyses of its thermal decomposition88,89). In a similar way, the slightly higher homolytic Co-C BDE of MeCbl has been derived as about 37 kcal/mol.90 The homolytic Co-C BDEs of the “incomplete” organocorrinoids adenosylcobinamide (AdoCbi) and methylcobinamide (MeCbi) were determined by mass spectrometric experiments as being somewhat higher (in the gas-phase), at 41.5 and 44.6 kcal/mol, respectively.91 The intramolecular coordination of the nucleotide base of some thermally labile organocobalamins was suggested to cause a weakening of their CoeC bond, as their nucleotide coordinated “base-on” forms decomposed considerably faster than their (protonated) “base-off” forms, or than the corresponding “incomplete” organocobinamides.92,93 However, the specific ease of homolysis of AdoCbl is only marginally affected by the DMB coordination, which was derived to weaken the CoeC bond by only 0.7 kcal/mol.68,94 Furthermore, the intramolecular coordination of the nucleotide in MeCbl even increases the homolytic Co-C BDE slightly by about 0.3 kcal/mol.68,94 Consistent with the reversible nature of the homolysis of the CoeC bond of many organocobalamins, the latter are (also) accessible by the reaction between the persistent radicaloid Cbl(II) and organic radicals.19,55 Cbl(II) carries a penta-coordinated Co(II)center and features the structural criteria of a highly efficient “radical trap.”47,69 Indeed, the reactions of Cbl(II) with alkyl radicals are nearly diffusion controlled and may occur with negligible restructuring of the cobalt corrin moiety. The retained cobalt-corrin structure not only supports a remarkably high reaction rate of Cbl(II) with radicals, but also induces a diastereo-face specific reaction at the (“upper”) b-face. AdoCbl and other organo-Cbls are, hence, directly formed from the trapping of (even some very short-lived) organic radicals by Cbl(II).69,88,95 The CoeC bond of most organocorrinoids also undergoes homolysis very effectively upon absorption of visible light.36,96,97 Hence, exposure of typical organocobalamins to irradiation by sunlight furnishes organic radicals conveniently and selectively under mild conditions.98 However, visible light does not induce the CoeC bond cleavage of (phenyl)-alkynyl-Cbls,56,99,100 and aryl-Cbas, such as ethylphenyl-Cbl (EtPhCbl),55 undergo the photo-induced decomposition with only a low quantum yield.99

2.11.3.2

On the nucleophile-induced heterolysis and formation of the CoeC bond

Nucleophilic substitution reactions represent biologically important alternative paths of formation and cleavage of the CoeC bond (see Fig. 7). The highly nucleophilic (“supernucleophilic”) Cbl(I) reacts readily with alkylating agents producing organocobalamins.19,101 This heterolytic reaction type does not involve free methyl (or alkyl) cations and adheres to an SN2-reaction, a particularly relevant reaction mechanism in typical enzyme-catalyzed methyl-transfer reactions,70,102 as well as in the biosynthetic adenosylation of Cbl(I) furnishing AdoCbl.103,104 Whereas typical alkylation reactions with Cbl(I) proceed via the “classical” bimolecular nucleophilic substitution (SN2) mechanism, alkylation with Co(I)-corrins may, in certain cases, occur via a two-step electron transfer path involving Co(II)-corrin and radical intermediates, where the strongly reducing Co(I)-corrins first induce a one-electron reduction of the alkylation agents.68,105 Related electron-transfer induced radical processes have also been inferred in the synthesis of aryl-Cbls,55,106 where geometric boundary conditions exclude an adequate backside attack of the nucleophile as required by genuine SN2-processes.107

Cobalt enzymes

275

The nucleophile-induced demethylation of methyl-Co(III)-corrins is the mechanistic basis for the typical B12-dependent biological methylation steps. Formally a reductive trans elimination at cobalt,108 this step (re)generates a strongly reducing Co(I)-corrin.109 Due to the stabilization of MeCbl by about 4 kcal/mol by the coordinated DMB-nucleotide,68,94 the demethylation of MeCbl by nucleophilic thiolates is approximately 1000 times slower than that of the “incomplete” MeCbi.110 Hence, the axial coordination of a(n imidazole) ligand (or of DMB) is also critical for enzymatic methyl-group transfer reactions involving proteinbound MeCbl. In some of these methyl group transferases, a histidine ligand replaces the DMB moiety and plays a significant kinetic role.102,111 However, the nucleophilic reactivity of the 4-coordinate Co(I)-centers is virtually unaffected by the presence of the cobalt-coordinating DMB-nucleotide, and Cbl(I) and “incomplete” Co(I)-corrins react, both, with typical alkylation agents with similar rates. Interestingly, the 4-coordinate corrin-bound Co(I)-ion preferentially reacts with alkylation agents at its “upper” b-face, which is, therefore, the more nucleophilic of the two diastereo-topic faces.68,105

2.11.3.3

On the radical-induced abstraction of cobalt-bound methyl groups

The radical-induced abstraction of a cobalt-bound alkyl group (see Fig. 7) is an effectively irreversible mode of cleavage of the CoeC bond of organometallic B12 derivatives.112,113 Since the abstraction of the cobalt-bound methyl group of MeCbl by an alkyl radical was very favorable not only thermodynamically, but it was found to be remarkably effective also kinetically, it seemed to provide the basis for a “second” type of biological role of methylcorrinoids.113 Indeed, biosynthetic methylations at inactivated carbon centers are suspected nowadays to be achieved by the S-adenosyl-methionine (SAM)-dependent radical enzymes114–116 that harbor a Cba as second cofactor. The mechanistically related direct intramolecular attack of a carbon-radical at a B12eCoeC bond, generating cycloalkanes,112 may serve as model for some other unusual biological CeC bond-forming reactions.117

2.11.4

Cobalt-corrins as cofactors and intermediates in enzymes

MeCbl and AdoCbl, or more broadly, methyl- and adenosyl-cobamides (MeCba and AdoCba) represent the two basic types of organometallic B12-cofactors essential in most functioning B12-dependent enzymes.8,9,13,14,118 In the case of enzymes depending upon (“complete”) methyl-Co(III)-corrinoids, only “base-off” (or “base-off/his-on”) forms, but no “base-on” forms have been observed by crystallography.70,111,119 In the case of adenosyl-corrinoids, such as AdoCbl, their enzyme-bound cofactor forms have been detected “base-on,”120 as well as in “base-off/his-on” form.9,121,122 The catalytically important protein-bound cofactor intermediate Cbl(II) has been studied by crystallography (see e.g., Ref. 123) as well as by EPR-spectroscopy (see e.g., Refs. 124,125). However, the typically transient and highly reduced Cbl(I)-form has merely been observed by absorption spectroscopy, and its structure was inferred indirectly126,127 (a collection of pictured protein structures, published by 2012, is found in Ref. 13).

2.11.4.1

B12-dependent methyl transferases

Cobamide-dependent methyltransferases are widespread and important organometallic enzymes that catalyze methyl group transfer via one of two main biochemical mechanisms.70,114 The abundant B12-dependent enzyme methionine synthase (MetH) carries a MeCba as cofactor and undergoes nucleophile-induced heterolytic methyl group transfer steps.70 Related microbial methyltransferases are central catalysts of methyl group transfer in methanogenesis,128 in acetogenesis (as part of an anaerobic CO2 fixation pathway129),130 and in acetic acid degradation to CH4 and CO2.131 The “super-nucleophilic” reactivity of Co(I)-corrins and the unique activity of MeCbas as methyl group donors toward nucleophiles are the key to the enzymatic catalysis of methyl-group transfer.19,102 In addition, alternative bifunctional B12- and S-adenosyl-methionine (SAM)-methyl transferases (classified as “class B” radical SAM) have turned out to be notably important in some biosynthetic methylations involving non-nucleophilic species,115,132,133 which mostly operate via radical methyl group transfer.113,114

2.11.4.1.1

Cobamide-dependent methionine synthase

Cobamide-dependent methionine synthase (MetH) is a particularly widespread organometallic enzyme, and the most extensively studied B12-dependent methyl-transferase.14,70,109 The enzyme MetH from E. coli has become a particularly thoroughly studied B12dependent methionine synthase.70,134 The methyl group transfer catalyzed by MetH involves the nucleophile-induced, heterolytic cleavage/formation of the CoeCH3 bond, and free methyl cation intermediates do not occur. It represents a methyl “cation” transfer only in a strictly formal sense.102 Methyl group transfer, catalyzed by MetH, basically follows a two-step ping-pong mechanism (see Fig. 8).135 As initial MetH step, Zn-bound, activated homocysteine de-methylates the protein-bound MeCbl, furnishing protein-bound Cbl(I) and methionine. In the second step, the reactive protein-bound Cbl(I) is methylated by activated N-methyl-tetrahydrofolate,136 generating tetrahydrofolate and regenerating MeCbl.102 The two steps proceed with an overall retention of configuration at the methyl group carbon, consistent with two SN2-type nucleophilic displacement steps, each inferred as occurring with inversion of configuration.137 During turnover, striking structural changes accompany the transitions of the B12-cofactor between its state with a (tetra-coordinate) Co(I)-ion bound and the one with a methylated (hexacoordinate) “base-off/his-on” Co(III)-center (see Fig. 8). The protein environment plays a crucial role in controlling substrate positions, as well as in providing access to the catalytic center.70

276

Cobalt enzymes

+

+

NH3

NH3

(CH2)2CHCO2

ZnII +

(CH2)2CHCO2

ZnII

S

S

CH3

CH3 +

SN2

CoIII

CoI

N DMB

N O H

H N N N H

Ar N H

O HN

H N

NH2 NH

Ar N H

O

CH3 +

DMB

Enz

DMB

Enz

SN2

CoIII

N

NH2 NH

CoI Enz

DMB Enz

N

CH3 O

N N O H

N------H+

O

HN

N------H+

Fig. 8 Cbl-dependent methionine synthase (MetH) catalyzes the formation of methionine from homocysteine (top), coupled with the liberation of tetrahydrofolate from demethylation of N5-methyltetrahydrofolate (bottom). MetH engages protein-bound MeCbl in a “base-off/His-on”-state alternating with Cbl(I) (the imidazole ring symbolizes the residue His759). The heterolytic methyl group transfer occurs via two nucleophilic substitution (SN2) steps, made possible in MetH by large mutual domain reorganizations, symbolized here by vertical correlations.

The pioneering crystallographic analysis of the B12-binding domain of MetH by Drennan and coworkers has provided a first insight into the structure of a B12-dependent enzyme.111 It showed the position of MeCbl in the core of a “Rossmann fold” of the B12-binding domain of MetH and revealed the displacement of the pseudo-nucleotide of the MeCbl cofactor by the histidine of a conserved His-Asp-Ser-triad,109 i.e., B12-binding in the characteristic “base-off/His-on” mode.65 Interestingly, the 5-coordinate “base-off” form of MeCbl (where an external axial ligand, or coordination of the DMB-base or of His were absent) has also been observed by crystallography in a corrinoid methyltransferase involved in acetyl-CoA synthesis,138 and in the B12-processing enzyme CblC139 (see below). Clearly, as these two cases exemplify, the abstraction of a methyl group by a nucleophilic acceptor is activated particularly strongly when the methyl is bound at a 5-coordinated Co(III)-center.19 The His-Asp-Ser-triad helps to position the protein-bound MeCbl in MetH for methyl group transfer, and the His-coordination also plays a significant thermodynamic role in the methyl group transfer reactions of MetH.65 Indeed, the DMB-coordination in MeCbl,94 as well as the coordination of an imidazole in the analogous methyl-imidazolyl-cobamide (Me-ImCba),140 exert a significant thermodynamic trans-effect on heterolytic methyl group transfer reactions in aqueous solution. The His-Asp-Ser-triad may also function as “relay” for Hþ-uptake/release accompanying the enzymatic methylation/demethylation cycles.65,141 Hence, the “triad” activates the methyl group of MeCbl for abstraction by a nucleophile and also promotes the re-reduction of adventitiously oxidized Cbl(II) to the catalytically active, protein-bound Cbl(I).127

2.11.4.1.2

Corrinoid methyl group transferases in anaerobic methane metabolism

Chemoautotrophic methanogens gain energy from the reduction of carbon dioxide to methane.142 Methyl-Co(III)-corrinoids are central methyl group transfer cofactors in anaerobic metabolism involving methane.128,143 Originally, methyl-Co(III)-corrinoids were considered to have a direct role in methane formation via protonation of their cobalt-bound methyl group.144 This view was eventually refuted as methane is generated by methyl coenzyme M reductase, an enzyme that carries a unique nickel porphyrinoid as cofactor145,146 that was named coenzyme F430 (Fig. 9).147 Methanogens use corrinoid methyl transferases for the methylation of the thiol coenzyme M with methyl groups provided by N5-methyltetrahydropterins, methanol or methylamines.148,149 Both substrates, the methyl group donor methanol and the methyl group acceptor coenzyme M, are activated by coordination to zinc ions in the dedicated methyltransferase from Methanosarcina barkeri.150,151 The methylamine methyltransferase from this methanogen houses a unique natural amino acid called “pyrrolysine,” as

Cobalt enzymes

277

CH4 (+ 2H2O)

CO2 + 4H2

H3C-CoM reductase CH3 CH3

CoIII

THP

L

H3C

S

CoM

base

CoI

H

CoM

HS

THP base

Fig. 9 In methanogens, the multistep reduction of CO2 with hydrogen gives CH4 and provides energy. A corrinoid methyltransferase accepts a methyl group (e.g., from an N-methyl-tetrahydropterin, H3C-THP) and transfers it to coenzyme M (HS-CoM ¼ 2-mercaptoethanesulfonate), giving methyl-coenzyme M [H3C-S-CoM ¼ 2-(methyl-thio)-ethanesulfonate], the substrate of the CH4 producing H3C-S-CoM reductase (HS-CoB ¼ 7mercaptoheptanoyl-threonine-phosphate ¼ coenzyme B).

part of the protein backbone in its active center.152 The formation of methyl-coenzyme M by methyl group transfer from methylated substrates typically takes two SN2-reaction steps,153 both involving the protein-bound corrinoid cofactor.102 The core section of the methyltransferase Mta from Methanosarcina barkeri, which transfers the methyl group of methanol to the corrinoid cofactor (a 50 -hydroxybenzimidazolyl-Cba typical of methanogens,154) has been analyzed by crystallography.151 Its B12binding unit shows sequence homology to the B12-binding domain of MetH and also harbors an Asp-X-His-X-X-Gly sequence that provides a histidine for B12-binding in a “base-off/His-on” form. The coordination of MeOH to the Zn-center activates MeOH for methyl transfer and positions its methyl group at a distance of about 4.5 Å above the corrin-bound cobalt-ion, assisting a nucleophilic attack of the Co(I)-corrin on the methyl group donor.151

2.11.4.1.3

Corrinoid methyl group transferases in bacterial acetate metabolism

B12-dependent methyl transferases play central roles in the acetate metabolism of methanogens and of some anaerobic bacteria.130,155,156 Various chemoautotrophic anaerobes can fix carbon dioxide reductively into the acetyl group of acetylcoenzyme A (acetyl-CoA), via the so called “Wood-Ljungdahl” pathway (see Fig. 10). The biological assembly of the acetyl group and the industrial organometallic production of acetic acid show remarkable chemical similarities.157 Indeed, this acetyl-CoA pathway depends heavily upon oxygen-sensitive organometallic enzymes.13,158 Initially, free CO and a methyl group bound and activated by cobalt-corrins (such as MeCbl) were inferred as the crucial components for the assembly of the acetyl group.159 Indeed,

O CH3

X

+

CO

+

HS

H3C

CoA

C

SCoA

+

HX

CH3

O

CoIII

H3C

C

SCoA

Ni

H-THP base

HSCoA CoI

H3C THP

CH3

O Ni

Ni base

C

CH3

CO

Fig. 10 Organometallic steps of the “Wood-Ljungdahl” pathway of autotrophic carbon dioxide fixation. The reduced B12-cofactor of the corrinoid iron sulfur protein (CoFeSP) is methylated by an activated N5-methyltetrahydropterin (CH3-THP). The methylated CoFeSP (CH3-X), in turn, transfers its methyl group to acetyl-CoA synthase forming a methylated Ni-center (H3C-Ni) of the latter. Carbon monoxide-insertion into the Ni-methyl bond gives a Ni-acetyl intermediate (H3C-CO-Ni). Nucleophilic abstraction of the acetyl group by coenzyme A (HSCoA) furnishes acetyl-CoA and a reduced Ni-center.

278

Cobalt enzymes

MeCbl could cleanly be photo-carbonylated to Cob-acetyl-Cbl.95 However, specific “corrinoid-iron-sulfur” proteins (CoFeSP) actually catalyze the transfer of methyl groups from a methyl group donor, such as N5-methyltetrahydrofolate, to the nickel center of the acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) complex, which also assembles the acetyl group.130,156,160 The transfer of the methyl group via CoFeSP, probably, involves a heterolytic path with methylation of a protein-bound Cba(I) and demethylation of the so formed Me-Cba(III) by the nucleophilic bimetallic Ni-Ni-center of the ACS/CODH-complex.156 The overall stereo-chemical course of the methyl group transfer has long been known to be consistent with nucleophilic displacement steps related to those in other methyl transferases.161,162 In the CoFeSP from the bacterium Carboxydothermus hydrogenoformans, the corrinoid cofactor was revealed as a “base-off” Cbl(II) carrying a water ligand at its penta-coordinate Co(II)-center but lacking any cobalt-coordinated protein-derived ligand.138,163 This coordination pattern is significant as it shifts the reduction potential of corrinoid Co(II)/Co(I)-redox couples to more positive values, i.e., into a biologically more accessible range.74 By a mechanism that apparently relates to the path of autotrophic fixation of CO2 via acetyl CoA, but which operates in the “reverse” sense, acetate is degraded to CH4 and CO2 in acetate-metabolizing methanogens.131,156 There, an acetyl-CoA-decarbonylase/CO-dehydrogenase complex (ACD/CODH) splits acetyl-CoA into CO and a methyl group, which is transferred to the corrinoid center of the corrinoid iron sulfur protein (CoFeSP). In these methanogens, enzyme-bound methyl corrinoids are, thus, indicated to serve twice as intermediates of methyl group transport, once from ACD/CODH to a N5-methyltetrahydropterine, and also in the characteristic methylation of coenzyme M on the way to methane.158,160,164

2.11.4.1.4

B12-dependent radical-SAM methyl group transferases

In the 1980s, several biosynthetic investigations provided evidence for apparently unprecedented biological methylation reactions at saturated and, presumably, un-activated (non-nucleophilic) carbon centers.165,166 According to these studies, intact methyl groups, originating from methionine, were attached with overall retention of their configuration. Related puzzling methylation reactions were also indicated in biosynthetic studies on the assembly of (branched) alkyl side chains in bacteriochlorins167 and of the antibiotic fosfomycin (1R,2S-epoxypropylphosphonic acid).168 In the course of the biosynthesis of fosfomycin, its precursor, 2-hydroxyethylphosphonate, is methylated as the 50 -cytidyl-conjugate by the methyltransferase Fom3 to give the cytidyl-conjugate of (S)-2-hydroxypropyl-phosphonate (Fig. 11).169 The methylation reaction at C-2 of the phosphonate would be indicative of proceeding with stereo-chemical inversion of the methyl group. Presumably, such biological methylation processes could be accomplished by the thermodynamically very favorable and also kinetically remarkably effective abstraction of the cobalt-bound methyl group of methyl-Co(III)-corrins by an organic radical (see Fig. 5).113,114 As speculated on the basis of such a detailed mechanism,13 the methyltransferase Fom3 features a remarkable stereo-specificity of the methyl transfer with respect to both, the methyl group and the substrate indicate.169 Considering the broad importance of “radical” SAM-enzymes170 in the biosynthetic generation of radicals,171–175 the beefed-up synthetic capacity of the B12-dependent (so called type B) “radical” SAM enzymes by their two cofactors appears perfectly suitable for the proposed type of methyl group transfer catalysis via radical methylation by methyl-Co(III) corrins.13 Strikingly, however, a first crystal structure analysis of a representative of this class of B12- and “radical” SAM-dependent methyl group transferases, of a tryptophan-2C-methyltransferase, gave structural insights for this enzyme that were strongly supportive of a “classical” (i.e., heterolytic) mechanism for the methyl group transfer.176 Clearly, while methylation of radicals via the methylCo(III)-corrin cofactor113 of B12-dependent “radical” SAM-enzymes is now an accepted mechanism for biosynthetic methylation reactions at saturated carbon positions,169,177,178 the mechanistic features of the numerous B12-dependent “radical” SAM-

SAM

Met

+e

Ado

Ado

H

H

Ado

HR HO

O

O

CMP HO

P

HS

O

CMP O O

P

HO

O O

H

P H

CH3

HEP-CMP

CoIII

O O

HPP-CMP CH3

CH3 +

CMP

(S)

+

CoIII

+

CoII

Fig. 11 2-Hydroxyethylphosphonate (HEP) is methylated as the (50 -cytidylyl)-2-hydroxy-ethylphosphonate (HEP-CMP) to the (50 -cytidylyl)-2(S)hydroxypropylphosphonate (HPP-CMP) by the B12-dependent (class B) radical SAM methyltransferase Fom3. Stereospecific abstraction of HR at C-2 and the generation of (2S)-HPP-CMP are rationalized by a stereo-chemically correlated abstraction of the H-atom at C-2 of HEP and methylation of the intermediate radical from the opposite face.

Cobalt enzymes

279

enzymes warrant further detailed studies.179 Another B12- and SAM-dependent radical enzyme actually catalyzes a puzzling biosynthetic ribose ring contraction to the 4-membered oxacyclobutane part of oxetanocine, a potent antitumor, antiviral and antibacterial compound.180

2.11.4.2

Enzymes dependent on coenzyme B12 and related adenosylcobamides

The organometallic B12-derivative AdoCbl, now called coenzyme B12,181 was discovered in the Barker labs soon after Cob-50 adenosyl-adeninyl-Cba (Ado-adeCba) was identified there as a coenzyme related to pseudovitamin B12 (Cob-cyano-70 -adeninylCba) and essential for clostridial glutamate metabolism.182 As has become apparent, following the discovery of AdoCbl, AdoCba-dependent enzymes occur in many organisms, where they catalyze biologically important reactions that rely on “difficult radical chemistry.”173,183–185 The AdoCba-dependent enzymes come in three families, classified as carbon skeleton mutases (see Tables 1 and 2), B12-dependent isomerases (see Tables 3 and 4) and B12-dependent ribonucleotide reductases.118,186,187 AdoCbas also play other physiological roles in microorganisms,188 as described in the later sections of this chapter. The common mechanistic feature of the AdoCba-dependent enzyme reactions is their initial chemistry, the substrate-activated homolytic cleavage of the CoeC bond of the protein-bound AdoCba.87 Hence, AdoCbl is, first of all, a reversible source for the 50 deoxy-50 -adenosyl radical,86 or a structurally highly sophisticated “pre-catalyst” (catalyst precursor).13 Indeed, the homolysis of the CoeC bond of coenzyme B12 (AdoCbl) occurs readily in aqueous solution, and yields cob(II)alamin (Cbl(II)) and a 50 -deoxy-50 adenosyl radical (Ado-radical) selectively (see Section 2.11.4).88,189 Amazingly, the homolysis of the CoeC bond of the proteinbound AdoCbl is accelerated by a factor of about 1012, compared to the rate of thermal CoeC bond homolysis of AdoCbl, as deduced from the observed reaction rates of the AdoCbl-dependent enzymes.86,89

Table 1

Adocobamide-dependent acyl-CoA mutases.

R)-Methylmalonyl-CoA / succinyl-CoA O HO2C

HO2C

S

S CoA

CoA

CH3

H

O

(R)-Ethylmalonyl-CoA / (2S)-2-methyl-succinyl-CoA O HO2C

H

S CH2

H

CoA

CH3

HO2C

S CoA O

CH3 Isobutyryl-CoA / n-butyryl-CoA O H 3C S CoA

O H3C

S CoA

CH3 Pivalyl-CoA /

iso-valeryl-CoA CH3

O

O

H3C S CoA H3C

H3C

S CoA

CH3

3-Hydroxy-iso-butyryl-CoA / 3-hydroxybutyryl-CoA O

OH

O

H3C S CoA HO

CH3

H3 C

S CoA

280 Table 2

Cobalt enzymes Adocobamide-dependent glutamate and methyleneglutarate mutases.

(A) Glutamate mutase H

NH2

HO2C

NH2

H

(S)-glutamic acid

CO2H

HO2C

CO2H

H

CH3

(2S,3S)-3-methylaspartic acid

(B) Methylene glutarate mutase CH2

CH2

CO2H HO2C

CO2H

HO2C CH3

2-methyleneglutarate

H

(R)-3-methylitaconate

The dramatic labilization of the organometallic bond of the protein-bound AdoCbl has been much discussed.120,122,185,190,191 Clearly, there is no covalent restructuring of the bound AdoCbl in the carbon skeleton mutases, except for the formation of its “baseoff/His-on”-form. Likewise, a significant conformational “butterfly” deformation of the corrin moiety of the bound AdoCbl was not observed, a proposed mode of activation toward CoeC bond homolysis.86,192 On the contrary, X-ray analyses of crystalline AdoCbl, of AdoCbl structures in enzymes and of pristine Cbl(II),47 the “complete” corrinoid fragment from CoeC bond homolysis of AdoCbl, have revealed extensive structural similarity between (the cobamide part of) these corrins.193 Hence, the puzzling protein (and substrate) induced homolysis of the CoeC bond of AdoCbl would not come about, primarily, by a corrin deformation, but, alternatively, from strong binding of the separated homolysis fragments.47 Indeed, in the carbon skeleton mutases, the protein provides a binding interface in which an “adenine-binding pocket” is positioned well separated from the corrin moiety of the bound AdoCbl cofactor. Only a “stretch” of the CoeC bond of the B12-cofactor would, hence, accommodate both of the (developing) homolysis fragments with stabilizing binding interactions to the protein.122,194 To test this hypothesis, the “stretched” homolog of coenzyme B12, Cob-(50 -deoxy-50 -adenosylmethyl)-Cbl (“homocoenzyme B12”) was prepared and studied as a covalent transition state model mimicking “activated” AdoCbl structurally.195 As roughly expected, the distance between the cobalt-center and C50 of the 50 -adenosylmethyl moiety of “homocoenzyme B12” was increased to 2.99 Å. This would be close to the observed distance Table 3

Reactions catalyzed by Ado-Cba-dependent isomerases/eliminases.

1.2-Propanediol / propanal H

OH

H

OH

+ H 2O

O Glycerol / 3-hydroxypropanal H HO

OH OH

H

HO

+ H 2O O

Ethanolamine / acetaldehyde O HO

NH2

H

+ NH3

Cobalt enzymes Table 4

281

Reactions catalyzed by AdoCba-dependent aminomutases.

between the cobalt-center and C50 in the “activated” coenzyme B12 in the crystal structure of some AdoCbl-dependent enzymes.122,196 Hence, binding of “homocoenzyme B12” should inhibit the AdoCbl-dependent enzymes. In experiments with AdoCbl-dependent diol dehydratase this was observed.197 A largely common basic mechanism of the transformations catalyzed by AdoCba-dependent enzymes has meanwhile emerged from a wealth of intricate studies.118,120,173,185 Accordingly, the tightly controlled Ado-radical generated from AdoCbl directly abstracts a hydrogen atom from the substrate to furnish a “substrate radical” that isomerizes to a “product radical,” which reabstracts a hydrogen atom from 50 -deoxyadenosine to give the rearranged product.13,120,198–201 Recombination of Cbl(II) and the Ado-radical then regenerates the B12-cofactor (see e.g., Fig. 12). In the B12-dependent ribonucleotide reductases (type-II RNRs), the radical sequence initiated by the Ado-radical (that originates from the homolysis of AdoCba) proceeds along a different path, by generating an enzyme-bound thiyl radical first, as relay for the further enzymatic radical and reduction steps (see below).187,202 In all AdoCba-dependent enzymes the proper control of the individual radical steps for the prevention of unwanted side reaction is intriguing, and particular attention has been given to the control of the highly reactive primary Ado-radical.200,203 However, the possible participation of Cbl(II), the “other” fragment from homolysis of AdoCbl, has also been discussed: would Cbl(II) remain a mere “spectator” of the radical steps or participate as a “conductor” in the AdoCbl-dependent radical enzymes?204,205

X R

C

X

1,2-rearrangement C

substrate radical

R

C

C product radical

Ado-H

H-abstraction

H-abstraction Ado +

R

X

H

C

C

Cbl(II) R

substrate

H

X

C

C

product AdoCbl

Fig. 12 steps.

Generalized catalysis scheme for AdoCba-dependent enzymes, showing the “forward” direction only of the typically reversible enzyme

282

Cobalt enzymes

2.11.4.2.1

Carbon-skeleton mutases

The biological catalysis of the carbon skeleton rearrangements appears to be an exclusive terrain of AdoCba-dependent enzymes.185,204 Five B12-dependent mutases are now known (two additional AdoCbl-dependent acyl-CoA mutases have provisionally been added to this list).196 They catalyze a reversible rearrangement of the carbon skeleton (see Table 1) by exchange of the positions, in a formal sense, of two vicinal groups, an organic moiety and a H-atom, in a (pseudo)intramolecular process. In these mutases, AdoCbl is situated at the interface between the B12-binding and substrate-activating modules, and is bound in the striking “base-off/His-on”-form, as discovered in MetH.206 Methylmalonyl-CoA-mutase (MCM), ethylmalonyl-CoA-mutase (ECM)207 and 2-methylene-glutarate mutase (MGM)208 consist of a single peptide chain. Isobutyryl-CoA mutase (ICM)196,209 glutamate mutase (GM)210,211 are built up from two subunits. The B12-binding domains in MCM, ECM and MGM, as well as the B12-binding subunits of GM and ICM, all exhibit considerable sequence homology and display the motif ..Asp–X–His–X–X–Gly..,65,196 diagnostic and essential for the “base-off/His-on” mode of Cba binding. Other AdoCba-dependent enzymes exist, which bind AdoCbl in the “base-on” form, and displaying characteristically different sequences and topology.186,212 2.11.4.2.1.1 Methylmalonyl-CoA-mutase and other acyl-CoA mutases Methylmalonyl-CoA-mutase (MCM) interconverts (R)-methylmalonyl-CoA (MC) and succinyl-CoA (SC) (see Fig. 13 and Table 1).118,213,214 MCM is a widely occurring coenzyme B12-dependent enzyme, important in the metabolism of branchedchain amino acids, cholesterol and odd-chain fatty acids, yet is the only one relevant for humans and higher animals.14,16,215 Surprisingly, MCM plays a role even in a pathway of CO2-fixation in Archaea.216 Binding of the acyl-CoA substrates triggers the homolysis of the CoeC bond of the enzyme-bound AdoCbl.196,217 In this process the tyrosine group Tyr-89 of MCM may help to displace the Ado-ligand of the bound AdoCbl-cofactor.218,121 The increased rate of Co-C homolysis of the bound AdoCbl appears to primarily come about by a decrease (roughly 16 kcal/mol) of the enthalpy of activation.219 In the next (formal) step, the Ado-radical then abstracts an H-atom from the methyl group of the methylmalonate to give the 2methyl-malon-20 -yl-CoA radical, which undergoes a(n intramolecular) rearrangement to the succin-30 -yl-CoA radical (see Fig. 13).185 The latter radical then abstracts an H-atom from 50 -deoxyadenosine to produce succinyl-CoA and an Ado-radical, which combines with the bound Cbl(II) to regenerate AdoCbl.185 The H-atom abstractions and the radical rearrangement are highly (stereo)-selective and tightly controlled by the protein environment in the active site.13,199,200

O HC

S-CoA CH2

CO2H

O

S-CoA C

H

CH2

O 1,2-rearrangemt.

H

CO2H substrate radical

C

S-CoA CH2

CO2H

product radical

Ado-H

H-abstraction

H-abstraction Ado + O H

S-CoA C

CH3

H

CO2H (R)-methylmalonyl-CoA

S-CoA

O H

Cbl(II)

C

CH2

CO2H AdoCbl

succinyl-CoA

Fig. 13 Methylmalonyl-CoA mutase (MCM) interconverts (R)-methylmalonyl-CoA and succinyl-CoA and is induced by Co-C homolysis of AdoCbl and formation of Cbl(II) and of the 50 -deoxy-50 -adenosyl radical (Ado). The proposed mechanism involves H-atom abstraction by Ado, generating the 2-methyl-malon-20 -yl-CoA radical. This substrate radical undergoes C-skeleton rearrangement to the succin-30 -yl-CoA radical via a hypothetical cyclopropyloxyl radical; H-atom abstraction by the succin-30 -yl-CoA radical from 50 -deoxyadenosine (Ado-H) provides succinyl-CoA and regenerates Ado (the sequence of steps of the reversible MCM reaction is shown only in the “forward” direction).

Cobalt enzymes

283

Interestingly, the 50 -deoxy-50 -adenosyl radical, the postulated “active” radical species in these enzymes, is only fleetingly existent and has, so far, evaded all pertinent, direct observations. In recent work from the Banerjee group with human and Mycobacterium tuberculosis MCM, the Ado radical could, however, be trapped as a relatively stable adduct with itaconyl-CoA,220 a known suicide inhibitor of MCM. The X-ray crystal structure of MCM from Propionibacterium shermanii (pMCM) was the first of a coenzyme B12-dependent enzyme.207 Crystal structures of MCM from humans (hMCM) are also known, which forms a homodimer with two active chains.221 In contrast, pMCM is a heterodimer consisting of two protein chains, one of them an active, AdoCbl-binding subunit, the other one inactive and lacking a B12-cofactor.214 The crystal structure of pMCM gave first insights into the binding-interaction of AdoCbl with the B12-binding domain in the “base-off/His-on” mode119 as in MetH.111 The AdoCbl-cofactor is also positioned at the domain interface, exposing its Ado-ligand to the catalytic domain, a TIM-barrel housing a funnel for the access of the substrate methylmalonyl-CoA. The crystal structures revealed a rather “flat” corrin ligand without indication of an “upwards conformational distortion,” speculated to activate AdoCbl for the homolysis of its CoeC bond.86 In the crystal structures of MCM and of other acylCoA mutases reconstituted with AdoCbl, two conformations of the partially homolyzed Ado-ligand were deduced, similar as discovered in glutamate mutase (GM). A radical shuttling mechanism, likewise first proposed for GM,122 appears to be common for the acyl-CoA mutases.196 Isobutyryl-CoA mutase (ICM)222 and Ethylmalonyl-CoA mutase (ECM)223 are bacterial members of the acyl-CoA mutases, showing sequence homology to the acyl-CoA mutases (see Table 1). ICM catalyzes the rearrangement of isobutyryl-CoA and nbutyryl-CoA (reversibly), relevant in the course of the biosynthesis of polyketide antibiotics in Streptomyces.224 ECM interconverts (R)-ethyl-malonyl-CoA and (S)-2-methyl-succinyl-CoA and plays a central role in the assimilation of acetyl-CoA in Rhodobacter sphaeroides.223 Both enzymes (ICM and ECM) bind AdoCbl in a “base-off/His-on” form to their smaller subunit.225 and display high sequence homology of their B12-binding subunits of with those of GM, MCM and MetH, and the substrate activating subunits are homologous among the acyl-CoA mutases. Indeed, the large coenzyme A moiety of the substrates of the AdoCba-dependent acyl-CoA mutases represents a spacious carrier stuck in the funnel of the commonly found TIM barrel motif, positioning the rearranging acyl-function close to the AdoCba-cofactor in the enzyme’s active site.196,226 There is good evidence for two further acyl-CoA mutases, the pivalyl-CoA mutase and the 2-hydroxyisobutyryl-CoA mutases, which interconvert the isomeric pivaloyl and isovaleroyl groups,227 or the 2-hydroxyisobutyryl-CoA stereoisomers and 3hydroxybutyryl-CoA, respectively.228,229 Additional Acyl-CoA mutases have been implied in the degradation of indole acetic acid and of short chain hydrocarbons.230 2.11.4.2.1.2 Glutamate mutase Glutamate mutase (GM), the first discovered enzyme requiring a B12-cofactor,182 catalyzes the reversible interconversion of (S)glutamate and (2S,3S)-3-methylaspartate (see Table 2). GM was studied in greater detail in Clostridium tetanomorphum and Clostridium cochlearium198 and plays a key role in glutamate metabolism of various bacteria.199 In halo-archaea, GM appears to participate in acetate assimilation.231 The natural AdoCba cofactors in the two clostridia are the purinylcobamides pseudocoenzyme B12 (Ado-adeCba, Cob-adenosyl-adeninyl-cobamide) and adenosyl-factor A (Cob-adenosyl-20 -methyladeninyl-cobamide),182 which predominately exist “base-off” in aqueous solution.61 However, AdoCbl also functions as B12-cofactor of GM.210 Binding of the substrate to GM triggers homolysis of the CoeC bond of the AdoCba cofactor. The so generated 50 -Ado radical (when going from (S)-glutamate to (2S,3S)-3-methylaspartate) abstracts an H-atom from glutamate to furnish a 40 -glutamyl radical and 50 -deoxyadenosine. The substrate radical isomerizes to a 3-methyl-3-aspartyl radical in a 1,2-rearrangement step, involving radical fragmentation and re-addition (see Fig. 14).185,232 The so formed 3-methyl-aspartyl radical abstracts an H-atom from 50 deoxyadenosine, furnishing (2S,3S)-3-methylaspartate and the 50 -Ado radical for recombination with Cba(II) and regenerating the intact AdoCba cofactor. Functional GM is a hetero-tetramer (of about 140 kDa) consisting of two copies each, a smaller s-subunit and a larger 3-subunit. Two AdoCba molecules bind in a “base-off/His-on” form, sandwiched between the 3- and s-subunits of the dimeric enzyme.232,233 The s-subunit harbors the histidine residue (His-16) that coordinates the cobalt ion at the “lower” a-side of the B12-cofactor (as shown by crystallography233) and is a crucial part of the consensus sequence (DxHxxG) characteristic for proteins binding B12 in the “base-off/His-on” mode.210 The s-subunit features a deep pocket between a central b-sheet and two of the flanking a-helices of the Rossmann-type fold, which binds the de-coordinated nucleotide tail.233,234 The X-ray crystal structure of GM from C. cochlearium has been thoroughly analyzed.122,232,233 In GM, reconstituted with coenzyme B12 and with a tartrate molecule as pseudo-substrate bound in the active site, the CoeC bond is partially broken and the 50 deoxy-adenosyl group is observed in two conformations that differ in the puckering of their ribose rings (C20 -endo and C30 endo).122,232 In the first conformation, the 50 -carbon atom is located above the metal center, but at a distance of about 3.2 Å, i.e., more than 1 Å farther than in an unstrained CoeC bond. In the second deduced structure, the 50 -carbon atom has moved by about 1.7 Å toward the substrate and to a distance of 4.2 Å from the cobalt center, ideally positioned for hydrogen atom abstraction. The observation of two 50 -deoxyadenosyl conformers suggests a mechanism for the structurally restricted and energetically facile translocation of the 50 -carbon radical center from cobalt to the substrate along the ribose pseudo-rotation path.9,122 While the barrier between the two minimum-energy conformations C20 -endo and C30 -endo is very low, the trajectory of the 50 -carbon is restricted. The overall position and orientation of the ribose ring is additionally restrained by hydrogen bonds to the side chains of residues Lys-326 and Glu-330. Together with the tight binding of the substrate, the restricted dynamics of the ribose helps to

284

Cobalt enzymes

H

Hre +

HO2C

H HO2C

NH2

NH2 Hre 1,2-rearrangemt. CO2H

substrate radical

Ado-H

CO2H

NH2 H CO2H HO2C Hre H 2C product radical

H-abstraction

H-abstraction Ado +

H HO2C

NH2 Hsi H re

glutamic acid

Cbl(II)

H

NH2

HO2C

CO2H

H 3C AdoCbl

CO2H H

methylaspartic acid

Fig. 14 Glutamate mutase (GM) interconverts (S)-glutamate and (2S,3S)-3-methyl-aspartate and is induced by Co-C homolysis of AdoCbl, furnishing Cbl(II) and the 50 -adenosyl radical (Ado). The proposed mechanism involves H-atom abstraction by Ado, and formation of a 4-glutamyl radical; this substrate radical undergoes C-skeleton rearrangement (presumed to take place via fragmentation/recombination) to the 3-methyl-3aspartyl radical; H-atom abstraction by this product radical from 50 -deoxyadenosine (Ado-H) provides (2S,3S)-3-methylaspartate and regenerates Ado (the sequence of steps of this reversible enzyme reaction is shown only in the “forward” direction).

prevent unwanted side reactions of the high energy intermediates (a feature of enzymes enhancing selectivity that is known as “negative catalysis”200,235). The adenine ring of the apparently activated AdoCbl in the GM structure is positioned above C5 of the corrin ring and forms specific polar interactions with the protein and the c-side chain of the corrin ring. It is located in an “adenine binding pocket,” the structure of which is similar in GM reconstituted with CNCbl and MeCbl.233 Since the adenine moiety cannot reach this predefined pocket as long as the CoeC bond of AdoCbl is still intact, the bound AdoCbl undergoes an elongation of its Co-C toward Cbl(II) and Ado, basically providing a “mechanochemical” path for Co-C homolysis.236 In complementary studies, the solution structures of the cofactor free s-subunits of GM from C. tetanomorphum and C. cochlearium was investigated by heteronuclear NMR-spectroscopy.237,238 A dynamic equilibrium between a more and a less structured fold of this B12-binding protein was found, which shifted toward the more structured state in the complex with the separate DMB-nucleotide of AdoCbl as specifically bound ligand.234 A structural model for B12 binding to GM was deduced, in which the apo-form of the s-subunit may first bind the nucleotide tail of a “base-off” B12-molecule and thereby inducing helix formation.234 This model appears to have a more general relevance for binding of B12-cofactors in their “base-off/His-on” form.13 2.11.4.2.1.3 Methyleneglutarate mutase Methylene Glutarate Mutase (MGM) catalyzes the (reversible) rearrangement of 2-methylene-glutarate to (R)-3-methylitaconate (see Table 2) as part of a degradative path of nicotinic acid in Clostridium barkeri.208 Coenzyme B12 (AdoCbl) is the authentic cofactor of MGM, which binds AdoCbl in a “base-off/His-on” form.185 A radical rearrangement, induced by H-atom abstraction is proposed, as similarly described for MCM and GM. Taking into account that the isomerization catalyzed by MGM involves an sp2-hybridized carbon center, its mechanism is presumed to involve a fast intramolecular rearrangement via a methylcyclopropyl radical intermediate, related (topologically) to the cyclopropyloxyl radical critical in the MCM isomerization (see Fig. 13).185,239,240

2.11.4.2.2

Coenzyme B12-dependent isomerases

2.11.4.2.2.1 Diol dehydratase and ammonia lyase The AdoCba-dependent diol dehydratases (DDH) and ethanolamine ammonia lyase (EAL) catalyze isomerizations in which the migrating group (“X” in Fig. 12) is either a hydroxyl or an amino group to generate a typically cryptic 1,1-diol or 1-amino-1-ol intermediate, which loses H2O or NH3 to liberate an aldehyde function (see Table 3).173,186,241 Besides the here discussed AdoCbldependent combined isomerases/eliminases, exist functional analogs with SAM as source of the 50 -deoxy-50 -adenosyl radical.198,241

Cobalt enzymes

OH

HO R Hb substrate radical

285

OH

1,2-rearrangemt.

R Hb

Ha

Ha OH product radical

Ado-H

H-abstraction

H-abstraction Ado + HO

OH

R Hb 1,2-diol

Cbl(II)

Ha Ha

Ha R Hb

AdoCbl

OH Ha OH

1,1-diol

Ha R Hb

O + H2 O Ha

aldehyde

Fig. 15 Diol dehydratase (DDH) catalyzes the conversion of 1,2-propanediol, first to 1,1-propanediol, which loses one molecule of water to yield propanal. The isomerization step is induced by Co-C homolysis of AdoCbl and formation of a 50 -deoxy-50 -adenosyl radical and Cbl(II). The proposed mechanism involves H-atom abstraction by the 50 -deoxy-50 -adenosyl radical, generating a substrate 1,2-dihydroxyalkyl radical (R ¼ CH3), which undergoes a 1,2-migration of the 20 -OH-group to give a 1,1-dihydroxy-20 -alkyl radical; H-atom abstraction by this product radical from 50 deoxyadenosine (Ado-H) provides a 1,1-diol and regenerates the 50 -deoxy-50 -adenosyl radical. The 1,1-diol, generated as product of the isomerization, loses water (reversibly) to give an aldehyde.

DDH catalyzes the conversion of, e.g., 1,2-propanediol to 1,1-propanediol, which loses one molecule of water (in a second step) to yield propanal (see Fig. 15).242 It also affords the dehydration of ethane-1,2-diol to acetaldehyde, while its isofunctional analog glycerol dehydratase preferentially transforms glycerol to 3-hydroxypropanal.186 In fact, DDH also accepts glycerol as substrate, but has about a twofold preference for conversion of propane-1,2-diol, whereas glycerol dehydratase has a reversed preference for glycerol.186 The crucial radical process is the isomerization of a 1,2-diol to a geminal (or 1,1) diol, followed by a highly selective (among the two hydroxyl groups of the 1,1-diol intermediate) loss of water. Overall, the dehydratase reaction is significantly exothermic and is considered to be effectively irreversible.204 In contrast to the B12-dependent carbon skeleton mutases, the B12-cofactor in the dehydratases is bound in a “base-on” form. This binding mode was first indicated by the analysis of the protein sequence of diol dehydratase, which lacked the diagnostic “B12-binding” (Gly-X-X-His-X-Asp)-sequence motif. Evidence for B12-binding in a “base-on” form, first provided by ESRinvestigations of DDH,243,244 was confirmed by X-ray crystal structure analyses of a (fully or partially) reconstituted DDH.245 The structure of DDH from Klebsiella oxytoca.246 displays a hetero-hexamer, with two copies each of three subunits (a, b and g). The a-subunit shows a TIM-barrel fold and houses the active site of the enzyme; the b-subunit binds the B12-cofactor in a “base-on” form and at the interface with the a-subunit. The upper face of the bound AdoCbl interacts with the a-subunit that binds the diol substrate above the corrin cofactor. The g-subunit appears to contribute neither to cofactor nor to substrate binding.120 The diol bound by DDH is also coordinated by a calcium ion, in addition to making hydrogen bonds with surrounding amino acid residues.247 The relative position of cofactor and substrate requires a radical transport pathway differing from the path taken in the C-skeleton mutases, such as MCM and GM. Instead of the ribose pseudo-rotation first observed in GM,122 in DDH a complete displacement of the ribose moiety occurs by rotation around the gylcosidic bond of the adenosyl ligand.186 By reconstitution of DDH with the structural AdoCbl mimic adeninyl-pentylcobalamin a preformed adenine binding site could be identified and located (above ring C of the corrin ring).246 Replacement of AdoCbl by the more homolysis-labile 50 -deoxy-30 ,40 -dehydroAdoCbl resulted in rapid formation of Cbl(II), which was oxidized to Cbl(III) by the 50 -deoxy-30 ,40 -dehydro-Ado radical, inducing suicide inactivation of DDH under anaerobic conditions.248 The derived location of the adenine binding site in DDH suggested a “mechanochemical” mechanism for activation of AdoCbl toward CoeC bond homolysis and homocoenzyme B12, the “stretched” homolog of AdoCbl, inactivated DDH.197 Ethanolamine ammonia lyase (EAL) binds the AdoCba cofactor “base-on” and catalyzes the conversion of ethanolamine (or of homologous vicinal amino alcohols) to acetaldehyde (or its homologs), with loss of ammonia (see Table 3).249 EAL was identified and studied in various microorganisms, specifically in some clostridia, in E. coli and in Salmonella typhimurium.186 Several AdoCbas, among them AdoCbl, are accepted as B12-cofactors. Similarly to the proposed mechanism of DDH, a radical mechanism accounts for the isomerization of the vicinal amino alcohol ethanolamine, starting with the abstraction of an H-atom from its (C-1)-position. Rearrangement to a geminal 1-amino-1-hydroxy-2-ethyl radical250,251 and H-atom re-abstraction furnish 1-amino-1-hydroxyethane, which loses ammonia to give acetaldehyde. The crystal structure of EAL from E. coli showed the substrate bound at a distance above the corrin ring. However, as the g-subunit is missing, the overall structure of EAL differs from that of DDH. The structure of EAL with adeninyl-pentyl-Cbl bound indicated a preformed adenine binding site.120,252

286

Cobalt enzymes

2.11.4.2.2.2 Adenosylcobamide-dependent aminomutases Two AdoCba-dependent aminomutases have been identified, which use an AdoCba and pyridoxal-phosphate (PLP) as cofactors, in order to catalyze the isomerization of diamino-acids by migration of an u-amino group to the (u-1)-position.120,173,253 One of these aminomutases, D-lysine/L-b-lysine-5,6-aminomutase (5,6-LAM), has the capacity to catalyze the isomerization of two substrates. D-lysine or L-b-lysine are both accepted as substrates and are isomerized to 2,5-diaminohexanoic acid or to (3S,5S)3,5-diaminohexanoic acid, respectively (see Table 4). The other known B12-dependent aminomutase, ornithine-4,5aminomutase, interconverts D-ornithine and (2R,4S)-diaminovaleric acid.254 The mechanism of the migration of the amino group in B12-dependent aminomutases has been studied in detail (see Fig. 16) and radical intermediates have been identified.173,255 The structures of lysine-5,6-aminomutase (5,6-LAM)256 and of ornithine-4,5aminomutase254 from Clostridium sticklandii were analyzed by X-ray crystallography. These two aminomutases bind the B12-cofactor “base-off/His-on” and the pyridoxal-phosphate (PLP) assists the migration by forming a Schiff base involving the migrating amino function and by stabilizing an aziridine-type radical intermediate from intramolecular migration of a PLP-conjugated imine group (Fig. 16). Strikingly, lysine-2,3-aminomutase (2,3-LAM), which catalyzes the apparently related isomerization of L-a-lysine to L-b-lysine is dependent upon SAM as source of an adenosyl-radical (but not on AdoCba-cofactor) and pyridoxal-phosphate (PLP).257–260

H (CH2)2

CH H2 C

CO2

C NH3

N CH OH

HO3PO N H

CH3

substrate radical

product radical H

H 2C

(CH2)2

C H

N

C

H CO2

NH3

CH

H2 C 1,2-rearrangemt.

CH

N H

C

CO2

NH3

N

OH

HO3PO

(CH2)2

CH OH2

HO3PO

CH3

N H

Ado-H

CH3

H-abstraction

H-abstraction Ado + H

H H 2C

C H

(CH2)2

N

C

H

Cbl(II) CO2

H 3C

NH3

CH

CH3

PLP-conjugated D-lysine

C

CO2

NH3 CH

AdoCbl N H

(CH2)2

N

OH

HO3PO

CH

OH

HO3PO N H

CH3

PLP-conj.of 2,5-diamino-hexanoic acid

Fig. 16 Proposed mechanism for the isomerization of D-lysine by D-lysine/L-b-lysine 5,6-aminomutase (5,6-LAM). The enzyme catalyzed process starts by formation of a Schiff base between the terminal amino group of D-lysine and PLP, also inducing homolysis of the (CoeC)-bond of AdoCbl. The Ado radical (Ado) abstracts an H-atom from C-5 of the Schiff-base-conjugate. The PLP-conjugated 5-D-lysyl radical undergoes a 1,2-shift via a stabilized aziridinyl-methyl radical to the isomeric 6-D-lysyl radical. H-atom abstraction by the latter from 50 -deoxyadenosine (Ado-H) gives the PLPconjugated 2,5-diaminohexanoic acid and the Ado radical, which recombines with Cbl(II) to regenerate AdoCbl.

Cobalt enzymes

P* O

base

H O

P* O

287

base

H O

H

H HO

HO OH

H

Fig. 17 Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotide-diphosphates (NDPs, P*¼ P2O63 L) and ribonucleotidetriphosphates (NTPs, P* ¼ P3O95 L) to the corresponding 20 -deoxy-ribonucleotides.

2.11.4.2.3

Coenzyme B12-dependent ribonucleotide reductases

Ribonucleotide reductases (RNRs) catalyze the conversion of the nucleoside di- or tri-phosphates of standard RNA to the corresponding 20 -deoxyribonucleotides, and are found in all living organisms requiring DNA.261–263 (Fig. 17). To achieve this central process in primary metabolism, RNRs are radical enzymes using three different ways to initiate a basically common radical reaction.262,264 The so-called “class II” RNRs depend on AdoCbas, which are bound in the “base-on” form.9,212,265 In AdoCbadependent (class-II) RNRs, as well as in class-I RNRs, two cysteines are oxidized to a disulfide to furnish the reducing equivalents (whereas in class-III RNR formate is the directly oxidized external reducing agent).262,263 AdoCba-dependent RNRs are known from a range of microorganisms, but the reductase from Lactobacillus leichmannii (Ll-RNR) is studied best.266,267 The proper substrates of the reductase Ll-RNR are nucleoside triphosphates (NTPs), while 20 -deoxynucleoside triphosphates (dNTPs) are its allosteric effectors.267,268 In Ll-RNR, the B12-cofactor is bound “base-on” (as first indicated by ESRspectra265) and the “classical” B12-binding motif Gly-X-X-His-X-Asp is not part of the protein sequence.212 As in the other RNRs, a protein-centered thiyl-radical is first generated by H-atom abstraction (in Ll-RNR by the Ado-radical originating from AdoCba).202 Homolytic cleavage of the CoeC bond of AdoCbl is accelerated by about 1011-fold in RNR compared to the homolysis of AdoCbl in aqueous solution (and the free enthalpy of activation of AdoCbl, when bound to GTP-activated but substrate-free Ll-RNR, is lowered in the enzyme by about 13–15 kcal/mol).269 The residue Cys-408 of Ll-RNR, suspected to be the thiol critical for the radical process, apparently accelerates the cleavage of the organometallic bond of the AdoCba-cofactor, as cleavage of the CoeC bond, formation of 50 -deoxyadenosine, and the generation of the thiyl radical are kinetically coupled.270 However, in mutants C408A and C408S of Ll-RNR isotopic labeling at C-50 of AdoCbl led to stereo-chemical scrambling of the label due to the reversible cleavage of the CoeC bond of AdoCbl (when Ll-RNR was complete with the allosteric activator dGTP).271 In the subsequent enzyme step, the Cys-408 thiyl radical abstracts the H-atom at C-30 of the bound substrate to induce the reductive dehydration of the bound NTP to a dNTP (see Fig. 18), which involves the oxidation of the thiol groups of two cysteines (suggested to be Cys-119 and Cys-419 in Ll-RNR) to a disulfide unit.267

P* O

base

H O

P* O

H HO

H

HO RS

OH

base

O

H 2O

OH

RSH

Cys-408

RNA

P* O Cbl(II) + Ado

AdoCbl

base

O O

Ado-H

H HS

P* O

base RS

H O

S

RSH Cys-408

P* O

base

O

H HO H DNA

S

SH

Cys-119 / Cys-419

H HO H

Fig. 18 Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides (RNA) to deoxyribonucleotides (DNA) (P* stands for di- or triphosphate. Cys-residues numbered as in Ll-RNR). In type-II RNR, the proposed mechanism involves generation of the protein thiyl radical via H-atom abstraction by a 50 -deoxy-50 -adenosyl radical (Ado) from homolysis of the Co-C-bond of AdoCbl. The subsequent abstraction of an H-atom from the 30 -position of the ribonucleotide activates loss of water from the 20 -position. Transfer of an H-atom from Cys-419 and a successive one-electron reduction furnishes the 20 -deoxyribonucleotide radical and a Cys-119/419 disulfide unit. Back transfer of a H-atom from Ado-H furnishes the reduced 20 -deoxyribonucleotide and the Ado radical (Ado), ready for recombination with Cbl(II) and reconstitution of protein-bound AdoCbl.

288

Cobalt enzymes

The crystal structures of dGTP-free AdoCbl-dependent Ll-RNR in the apo-form and complexed with Cob-adeninylpentylcobalamin showed the bound corrinoid “base-on.”212 The crucial cysteine Cys-408 is located at a distance of about 10 Å from the Co(II)-center of the bound corrinoid and in a region of space that is also well conserved in the three classes of RNRs.261 However, the position of the organometallic ligand of the structural mimic of AdoCbl was strongly disordered, and the crystal structures of both B12-dependent RNRs from L. leichmanii212 and from Thermotoga maritima (Tm-RNR)190 display a less well defined electron density for the bound AdoCbl cofactor. As with the mutases and isomerases, in the B12-dependent RNRs investigated, the adenine ring of the adenosyl moiety of the B12-cofactor is positioned in a preformed binding site.190 The crystallographically observed distances between cobalt and the 50 -carbon atom of the adenosyl ligand of 2.6 and 2.9 Å, respectively, indicate considerable lengthening of the CoeC bond from its normal value (2.03 Å) and a similar “mechanochemical” activation mechanism as deduced in diol dehydratase.120 In many regards, the AdoCbl-dependent RNRs appear to be less complex than members of the two other classes of RNRs.262,263 Indeed, the corrinoid cofactor of the AdoCbl-dependent RNRs replaces, functionally, the entire protein subunits that are needed for radical generation in the class I and III RNRs.263,272

2.11.4.3 2.11.4.3.1

B12-processing enzymes Adenosyltransferases

The enzymatic adenosylation of cobalt-corrins occurs as a critical step, not only when AdoCba cofactors are (re)generated from “complete” Cba(II)-precursors, but also in the course of both pathways (anaerobic and aerobic) of the de novo B12-biosynthesis. The adenosylation of the common biosynthesis intermediate cobyrinic acid a,c-diamide by adenosyl-transferases CobA (in anaerobes) or CobO (in aerobes) uses the highly nucleophilic Co(I)-form of the corrinoid substrate and ATP to generate 50 -deoxy-50 adenosyl-cobyrinic acid a,c,-diamide as the next common intermediate.10 Two other bacterial adenosyl-transferases (ATRs), classified as PduO and EuT ATRs, required in “1,2-propanediol utilization” (pdu) or ethanolamine utilization (eut) pathways, respectively, are both involved in the (re)generation of AdoCbas from their Co(II)Cba forms. These latter arise occasionally as catalysis-inactive species during the turnover of the AdoCba-dependent enzymes DDH and EAL.10,120 The transfer of a 50 -deoxyadenosyl moiety from ATP to Cbl(I) by the homologous mammalian PduO-type ATR in the mitochondrion of humans and mammals that is encoded by the cblB gene273–275 and comes up for the formation of AdoCbl from Cbl(II). The underlying mechanisms of this exceptional biosynthetic step catalyzed by an ATP:Cbl(I) ATR was first elucidated by UV–Vis and ESR (electron spin resonance) spectroscopy,274 and later by crystallographic snapshots of the trimeric PduO-type ATR from Lactobacillus reuteri (LrPduO) loaded with ATP alone in the active site,276 with Cbl(II) and ATP and then with “baseoff” AdoCbl as direct reaction product.277 The PduO-type LrPduO, in complex with ATP and Cbl(II), also showed the Cbl(II) in the active site of the enzyme in a four-coordinate “base-off” form.277 The displaced DMB nucleotide becomes solvent-exposed, suggested to be critical for recognition and direct transfer of AdoCbl to the target enzyme MCM in humans.278 Similar crystallographic “snapshots” have also become available for an EuT-type103 and some PduO-type ATRs123 (Fig. 19). The enzyme-catalyzed adenosyl transfer implies the intermediate formation of the highly nucleophilic Cbl(I), which substitutes a triphosphate at the 50 -carbon of ATP. However, the reduction to Cbl(I) would ordinarily require a reduction potential beyond the capacities of in vivo reducing agents.74 The ATP:Cbl(I) ATRs solve this problem by binding Cbl(II) in a “base-off” form, harboring an easily reducible four-coordinate Co(II)-center. Furthermore, the substrate ATP is placed directly above the bound Cbl(II), perfectly pre-oriented for the nucleophilic attack. The favorable substrate orientation in the customized protein environment and the deduced stabilization of the transition state lower the reaction energy barrier and enable the transfer of the 50 -deoxyadenosyl moiety to the readily oxidized, fleetingly existing Cbl(I). Indeed, the postulated Cbl(I) intermediates have not yet been directly observed in the ATRs. Ado-transfer was inhibited by nibalamin, the redox-inactive, isoelectronic Ni(II)analog and structural

Cbl(II) from CblC

O O O HO O-P-O-P-O-P-O O O O

OH N O

N

N N

NH2

SN2

H2C

+e+

CoII

HO

+

CoI

OH N O

N

N N

NH2

CoIII

- PPPi5AdoCbl

N N O

N N

N N

O

O for MCM 

Fig. 19 Mechanism of the adenosylation step catalyzed by the ATP:Cbl(I) adenosyl-transferase (ATR). Protein-bound Cbl(II) in its four-coordinate “base-off” form is activated toward the reduction to the highly nucleophilic Cbl(I), which substitutes triphosphate at the 50 -carbon of ATP in an SN2process that leads to “base-off” AdoCbl (which is subsequently used by MCM).

Cobalt enzymes

289

mimic of Cbl(I) (see below).279 Strikingly, the remarkable enzymatic adenosylation could not be mimicked in aqueous solution by the reaction of electrochemically generated Cbl(I) with ATP.280

2.11.4.3.2

Cobalamin-deligase CblC

Vitamin B12 (CNCbl) has, in fact, no direct physiological functions in humans. It is a provitamin281,282 that healthy human cells transform into the organometallic cofactors MeCbl and AdoCbl.104 Among eight human Cbl-deficiency genes283 two were deduced to be directly responsible for intracellular organometallic processing of Cbls. The genetic locus, named cblB, encodes the adenosyltransferase ATR producing AdoCbl from Cbl(II) (see above),284 the other one (cblC) encodes for the Cbl-processing enzyme CblC, also referred to as the MMACHC gene (for methylmalonic aciduria type C and homocystinuria), as its lack of activity is responsible for the accumulation, in parallel, of methylmalonate (MMA) and homocysteine (Hcys).285 The intact enzyme CblC “tailors” cob(III)alamins entering the cell to Cbl(II), the physiological precursor of the two B12-cofactors MeCbl and AdoCbl.104 Hence, processing by the human CblC protein is essential for the production of MeCbl and AdoCbl from the ingested Cbls. The lack of active CblC due to critical mutations results in the inactivity of both B12-dependent enzymes in humans and in the diagnostic accumulation of MMA and Hcys as biomarkers of “functional” Cbl-deficiency.286 Depending on its B12-substrate, CblC employs its cosubstrate glutathione (GSH) in two different ways for the production of Cbl(II) (Fig. 20): When presented with CNCbl, the enzyme catalyzes the reductive de-cyanation to Cbl(II),287 whereas binding of alkylcobalamins (such as MeCbl) initiates the nucleophilic substitution of the upper ligand by glutathione, furnishing Cbl(I), which undergoes subsequent oxidation to Cbl(II).288,289 The crystal structures of human CblC139 and of its homolog from Caenorhabditis elegans,289 both in complex with MeCbl, revealed the 3D-structure of this “B12-tailoring” enzyme.104 The corrinoid substrate MeCbl is bound “base-off” at a cavity formed at the domain interface, with the nucleotide tail buried in a crevice of the N-terminal domain. With a five-coordinate Co(III)-center, the bound corrinoid is activated for reduction (in the case of CNCbl) or for nucleophilic substitution (of the methyl group of MeCbl). With knowledge of the role of CblC, non-natural Cbls were designed that were resistant to the tailoring by CblC, aborting their possible subsequent cellular transformation to the B12-cofactors AdoCbl and MeCbl, and causing “functional” B12-deficiency in a highly controlled way (e.g., in mice).55,290,291 When mimicking the (outside) structure of vitamin B12, but differing by their cobalt-centered chemical reactivity from natural Cbls (i.e., behaving like a wolf in a sheep’s clothing), such analogs of vitamin B12 would constitute antivitamins B12.290 Aryl-Cbls with a substitution and reduction inert Co-Csp2 bond were produced for this purpose by radical arylation of Cbl(II) (Fig. 5), the 4-ethylphenyl-Cbl (EtPhCbl)55 and phenyl-Cbl (PhCbl).106 With a substitution and reduction inert Co-Csp bond, suitably structured light- and heat-resistant alkynyl-Cbls are also potential antivitamins B12.56 The

CN +

GS

GS

H2 O

.

+

CoIII

CoII

- CN

DMB

DMB

O

O II

Cbl bound 'base-off'

CNCbl bound 'base-off'

ox. (O2)

alkyl +

DMB

GSH

GS-alkyl

CoIII

CoI

DMB O alkyl-Cbl bound 'base-off'

O CblI - is 'base-off'

Fig. 20 Outline of the reduction mechanisms of the Cbl-deligase CblC. The common enzyme product Cbl(II) is formed either by reductive decyanidation of CNCbl or by nucleophile substitution of alkyl-Cbls furnishing Cbl(I), followed by oxidation to Cbl(II). The Cbl(II) is subsequently handed over in the cytosol to MetH (which converts Cbl(II) into MeCbl), or to the mitochondrial ATP:cob(I)alamin adenosyltransferase (ATR) to generate the cofactor AdoCbl for its function in MCM (GSH: glutathione).

290

Cobalt enzymes

F

Et F

C C +

+

CoIII N

CoIII N

N

N O

O

EtPhCbl

F2PhEtyCbl

Fig. 21 Symbolic formulae of the two Cbl-based antivitamins B12 EtPhCbl and F2PhEtyCbl (which have been shown to resist tailoring by the enzyme CblC).

specifically hydrolysis-resistant difluorophenyl-ethynylcobalamin (F2PhEtyCbl, see Fig. 21) is an inert substrate and inhibitor of human CblC that turned out to be helpful for a crystal structure of the enzyme fully assembled with the co-substrate GSH.63

2.11.4.4

B12-dependent dehalogenases

Some methanogens and anaerobic acetogens can drive their energy metabolism by reductive dehalogenation of chloromethanes and other halogenated organic compounds.292 This capacity of these microorganisms has long been proposed to involve reduced metal cofactors, such as reduced corrinoids.293 Besides dechlorination of chloromethane,294,295 a variety of other environmentally relevant microbiological dehalogenation reactions have been studied,296 such as those of chloroethenes,297,298 of halogenated phenols,299,300 and of various chloroalkanes, e.g., trichloroethane301 and hexachlorocyclohexane.302 The anaerobic bacterium Sulfurospirillum multivorans (formerly called Dehalospirillum multivorans) dechlorinates tetrachloroethene by the Cba-dependent tetrachloroethene reductive dehalogenase (TCED) which functions as the terminal electron acceptor of anaerobic (dehalo)-respiration.294,298 The dehalogenase TCED reduces tetrachloroethene to trichloroethene (first) and (then) to cisdichloroethene (see Fig. 22). The membrane-bound TCED uses a reduced form of a nor-cobamide (nCba), isolated as a nor-pseudovitamin B12 (see Fig. 3).58 The corrinoid cofactor from D. multivorans was about 50 times more active than CNCbl in an in-vitro reduction of trichloroacetate.297 Nor-cobamides and nor-cobalamins have a lesser preference for the B12 “base-on” forms,59 enabling redox reactions to occur at less negative potentials.303 Indeed, TCED from Sulfurospirillum multivorans showed such a less negative redox potential too, and binds its corrinoid cofactor in a “base-off” form, as was clearly shown by an X-ray crystal structure.298 In the dehalogenating enzymes, instead of organometallic B12-cofactors like AdoCbl or MeCbl, nCba(I) s are relevant for catalysis, reducing and nucleophilic corrinoid cofactors that interact with the halogenated substrates. The available data from crystal structure analyses of TCED298,300 and of a dehalogenase that uses ortho-halogenated phenols (in particular, 3,5-dibromo-4hydroxybenzoic acidda breakdown product of the herbicide bromoxynil)299 are consistent with a first electron transfer from

Cl

Cl

Cl C

Cl

Cl C

C

C Cl

H

+ Cl CoI

Cl C

Cl

Cl

4e 2H

Cl

e /H

C

?

CoII

2e / H e - Cl

H

- 2 Cl Cl Cl

Cl C

H

C

Cl C

C H

CoIII

Cl

Cl

Cl C

H

C H

Fig. 22 Possible mechanisms of the reduction of tetrachloroethylene (TCE) to trichloroethylene and further to cis-1,2-dichloroethene, catalyzed by Cba-dependent TCED.

Cobalt enzymes

291

the nCba(I) to the halogenated substrates (either via a long range300 or with transfer of a halogen in an “inner-sphere” mechanism299). These crystallographic studies provide no evidence for organo-cob(III)amide intermediates, from which (partially) dehalogenated hydrocarbons may then be set free under reducing conditions, as also considered earlier.13,14 The enzymatic reductive dechlorination of tetrachloroethene was studied in model chemistry using Cbl(I) as reductant, also indicating a free radical process as the first step of the abiotic reduction.54,304 In the later stages of the further reduction of trichloroethylene organo-corrinoid intermediates may occur. Thus, depending on the halogenated substrate, organometallic Cbas represent enzyme intermediates.54 Another type of Cba-dependent reductase, the epoxyqueuosine reductase (QueG), generates the exceptional RNA base queuosine by reduction of its epoxide.305 At first sight, the biological role of QueG appears to be rather different from that of the Cbadependent dehalogenases. However, the reductase QueG is actually remarkably related to the dehalogenases (as well as to the Cba-deligase CblC) by both, the protein fold, and its reducing role in the surprising biosynthetic transformation of a modified nucleoside at a complete tRNA loop. Like the dehalogenase TCED, QueG binds its Cba-cofactor in a readily reduced “base-off”form. Based on a crystal structure, the bound reduced Cba(I) has been suggested to open up the epoxide ring of epoxiqueuosine by a nucleophilic attack, generating a short-lived b–hydroxyalkyl-Cba intermediate that releases queuosine by regio-specific b-elimination.306 The reducing dehalogenases, CblC, and the RNA-modifying enzyme QueG may represent members of a new class of Cba-dependent enzymes,306 which use reduced cobalt-corrins rather than organometallic B12-cofactors.

2.11.5

B12-derivatives as ligands of proteins and nucleic acids

2.11.5.1

B12-binding proteins for uptake and transport in mammals and bacteria

Since B12-derivatives are (bio)-synthesized exclusively by specific micro-organisms, other forms of life (from B12-dependent bacteria to humans) that depend upon Cbas, acquire them by selectively absorbing, transporting and processing the minute amounts of natural corrinoids available to them. In the human body, the delivery of dietary Cbls to the cells is accomplished by three transport proteins, haptocorrin (HC), intrinsic factor (IF), and transcobalamin (TC), which all strongly select for intact “base-on” Cbls.16,307,308 The three transport proteins, HC, IF and TC, have a common topology, molecular weights of about 45 kDa, and their crystal structures with Cbls bound are available.48,309,310 HC is secreted in saliva, binds free Cbls even under the acidic conditions of the stomach, delivering the Cbl-cargo to the duodenum, where HC is degraded. Intact (“base-on”) Cbls are trapped by IF with high affinity and selectivity, and transported to the terminal ileum, to be released into the enterocytes, which dispatch Cbls into the portal circulation. There, Cbls are bound by TC giving the TC-Cbl complex that is absorbed from plasma by the specific cellular TC-receptor and internalized (reviewed in Refs. 13,307). In the cells, the Cbl-deligase enzyme CblC adapts the imported Cbls for their further intracellular processing (see above).39 Bacteria also possess highly intricate B12-uptake and transport systems. Most detailed studies have been performed with E. coli, which biosynthesizes its entire B12-cofactors from cobinamides or other Cbls.311 The import of corrinoids into E. coli is managed by the “B-twelve-uptake” (Btu) system, which involves the outer membrane transporter BtuB,312 the periplasmic binding protein BtuF,313 and the ATP-dependent inner membrane permease BtuC2D2.314

2.11.5.2

Cobalamins as gene-regulatory RNA-ligandsdB12-riboswitches

Riboswitches are sequence elements of mRNAs that regulate gene expression upon binding of a specific ligand.315 Riboswitches occur in mRNA as part of the 50 -untranslated region (50 -UTR) and are up to about 300 nucleotides long. Their discovery was actually linked to puzzling observations relating to the expression of BtuB, the outer membrane B12-transporter of the bacterium E. coli, in response to AdoCbl.316 Strikingly, coenzyme B12 was observed to directly bind to the E. coli btuB mRNA, then named a B12-riboswitch, as it was refolded (“switched”) by the bound AdoCbl in a way inhibiting the further translation of the BtuB protein.317 AdoCbl binding to the BtuB riboswitch occurred with a roughly 100 nM affinity, outperforming significantly the binding of alternative B12-ligands with related structures, such as CNCbl, Ado-adeCba, and AdoCbi.67 AdoCbl binds B12-riboswitches (such as btuB) in their intact “base-on” form, and the riboswitch adapts its structure to the ligand by undergoing a conformational “switch.”318 Other B12-riboswitches may prefer binding MeCbl and feature a binding interface that discriminates between the different Cbls structurally.319,320 Several B12-riboswitches have been analyzed by X-ray crystallography.320,321 Since the discovery of the BtuB B12-riboswitch of E. coli, a vast number of bacterial riboswitches have been identified that sense Cbls or other metabolites, expanding the gene-regulatory capacity of the non-coding RNA.322

2.11.5.3

Coenzyme B12 as light-sensitive ligand in photo-regulatory proteins

Photo-regulation of gene expression is a broadly relevant phenomenon that is of particular importance in photosynthetic organisms.323 Photoreceptors respond to light via light-sensitive ligands, e.g., flavins, bilins, and retinal.323–325 Coenzyme B12 (AdoCbl) has joined these light-absorbers lately and acts as a photoreceptor bound to DNA-binding enzymes that make use of the light sensitive AdoCbl for regulating gene expression.326 The so revealed striking expansion of the biological portfolio of the B12-cofactors exploits the efficient light-induced cleavage of the CoeC bond of AdoCbl.96,97 As first observed with the AdoCbl-based photoreceptor CarH of Myxococcus xanthus,327 which regulates the biosynthesis of the photo-protecting carotenoids in this bacterium,

292

Cobalt enzymes

the class of B12-based photo-regulators appear to be abundant in bacteria.328 Thus, this group has been joined by the AdoCbldependent photo-regulator AerR of Rhodobacter capsulatus,329 which helps regulate the tetrapyrrole biosynthesis in this photosynthetic bacterium.330 Crystallographic and biochemical studies with CarH have largely clarified the question how it could repurpose AdoCbl for a broadly relevant light-sensing gene-regulatory role.331,332 AdoCbl is not directly involved in DNA binding and its gene regulatory activity relies on its capacity to modulate the structure of the protein CarH through coordinative interaction with the bound AdoCbl. In the absence of light, CarH binds intact AdoCbl in a dimer-of-dimers-type tetramer that inhibits the transcription of genes coding for carotenoid biosynthesis. A heterolytic light-induce cleavage of the Co-C bond of “base-off/His-on” AdoCbl attached to CarH leads to the unreactive 40 ,50 anhydroadenosine and to a bis-His-coordinated “base-off” Cbl333 (Fig. 23), but not to an Ado-radical, produced by the photolysis of AdoCbl in solution.97,334 The photo-decomposition of AdoCbl and the induced restructuring of CarH loosens its DNA-binding and sets the stage for carotenoid biosynthesis.328 The question how CarH “reprograms” the path of the light-triggered cleavage of the Co-C bond of AdoCbl from homolysis to heterolysis has been studied by a range of biochemical,335 photo-physical,334,336 computational,337 and structural338 investigations.188

2.11.6

Why cobalt?dB12-analogs with other metals and antivitamins B12

As delineated here, natural cobalt-corrins are the typical cofactors of the known cobalt-enzymes, reflecting the biological importance of the partnership of cobalt-ions and the corrin ligand of vitamin B12.24 These insights have sparked the B12-field and the question: What makes the natural corrins and cobalt such unique and biologically important partners? (see e.g., Refs. 20,339). It also raised an interest in the fundamental consequences of replacing one of these components, cobalt or corrin, by another metal or a different porphyrinoid ligand.20,27,339,340 The replacement of cobalt would, first of all, furnish “debilitated” B12-mimics, among them potential antivitamins B12 that specifically counteract the metabolic activity of Cbas, inducing “functional B12 deficiency.”290 This intriguing topic gave metal analogs of Cbls (metbalamins, Metbls) a particularly desirable role. It also increased a specific interest in rhodium,282 as it has related coordination properties as its group-IX homolog cobalt. Indeed, AdoCbl and its Rh-analog, 50 adenosyl-rhodibalamin (AdoRhbl) (see Fig. 24), have very similar structures.340 However, the RheC bond of AdoRhbl is presumed to be stronger than the CoeC bond of AdoCbl, so that AdoRhbl would have the biological features of an antivitamin B12.282 Indeed, AdoRhbl inhibited a bacterial diol-dehydratase and the growth of Salmonella enterica very effectively.340 In the 1970s, Koppenhagen and coworkers reported the preparation of incompletely characterized rhodibalamins (Rhbls), among them AdoRhbl,341 which behaved as a B12 antimetabolite in microorganisms and in human cell cultures.342 Interestingly, the RheC bond of AdoRhbl is stable under irradiation with sunlight, again contrasting with AdoCbl.340,341 A chemical-biological methodology for the synthesis of Rhbls was opened up by a newly bioengineered preparative route to the metal-free B12-ligand hydrogenobyric acid (Hby).27 The natural metal-free corrin Hby (see Fig. 24) represents an amazing helical ligand structure that fails to meet the coordinative preferences of cobalt, enhancing the loss of axial ligands, like a “Procrustean Bed.”27 Hby is a rational starting material for the partial synthesis of hydrogenobalamin (Hbl), the complete metal-free ligand of the Cbls.279 From Hbl specific Metbls can be made accessible, a long-standing dream in the B12-field.340,343–345 So far, the syntheses and the detailed structural characterization of zincobalamin (Znbl) and nibalamin (Nibl) were reported, the Zn(II)and Ni(II)-analogs of vitamin B12279,346 (see Fig. 24): The redox-inactive penta-coordinate (“base-on”) Znbl constitutes a luminescent structural mimic346 of the penta-coordinate “base-on” Cbl(II). The largely redox-inactive tetra-coordinate diamagnetic (“baseoff”) Ni(II)-corrin Nibl is a structural mimic of the highly reactive “base-off” Cbls, tetra-coordinate Cbl(II) and Cbl(I).279

Fig. 23 AdoCbl, the chromophore of the photoreceptor protein CarH, is cleaved by visible light with liberation of 40 ,50 -anhydroadenosine, significant restructuring of the protein environment and subsequent formation of a histidine-bis-coordinated Cbl(III).

Cobalt enzymes

H2NOC H2NOC

H2NOC CH3

H2NOC

CONH2 CH3

H3 C H3C H

i) and then ii) H3C H 3C H

CONH2 CH3 CH3

N HN NH N

H2NOC H 3C f

CONH2 or CH3 CH3

ii) and then i)

H 3C H

Hby

O

O

N

CH3

O

O

N

N

Met N

CH3

CONH2 O HO

O

CONH2

CH3

HN H

CONH2 CH3

CH3

f

H3 C

N

N

H2NOC H3 C

CH3

O

N

CH3

N

CH3

O

P

P O

H 3C H3 C H

CONH2

HO O

CH3

CH3

HN CONH2

CONH2

CH3

f

CH3

H2NOC

N

H2NOC H3 C

CH3

CO2

N Met(L) N N

H2NOC

293

OH

Metbl ("base-on")

O

O

OH

Metbl ("base-off")

Fig. 24 Metbalamins (Metbls) can be prepared from hydrogenobyric acid (Hby) by (i) metal incorporation and (ii) attachment of the B12-nucleotide or in the reversed sequence. This approach has furnished the “base-on” Metbls zincobalamin (Met(L) ¼ Zn(II), Znbl) and chlororhodibalamin (Met(L) ¼ Rh(III)Cl, ClRhbl) and the “base-off” Metbl nibalamin (Nibl).

2.11.7

Summary and outlook

Nature makes use of the capacities of the cobalt-corrins in remarkable ways. Increasing insights into the B12-dependency of organisms from most kingdoms of life reveal a fascinating versatility of the complex B12-derivatives in an unbelievable range of biological functions,260,347 often in very specific and important ecological roles.17,348,349 The discovery of the organometallic nature of coenzyme B12 has opened the exciting field of bio-organometallic chemistry. Indeed, the B12-cofactors are exceptional catalysts on their own. When bound and controlled by proteins, they are specifically promoting and targeting cellular metabolism. Their many biological roles in the B12-dependent enzymes are based on the coordination chemistry and redox-chemistry of cobalt, modulated by the natural corrin ligand of B12. The exceptional reactivity and structure of B12-derivatives impact life processes by extending the range of biological catalysis and by allowing for exceptional regulatory mechanisms. The ever growing knowledge concerning the roles of B12-cofactors in living cells is due to continue keeping B12 “in the spotlight.”188,350–352 Bio-structural, biochemical and chemical biological studies with B12-derivatives will clearly continue to fascinate the interdisciplinary B12-fraternity, as well as the neighboring bioinorganic and biological research communities.

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Biol. 2003, 10 (5), 394–401. 313. Borths, E. L.; Locher, K. P.; Lee, A. T.; Rees, D. C. The Structure of Escherichia coli BtuF and Binding to its Cognate ATP Binding Cassette Transporter. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (26), 16642–16647. 314. Rees, D. C.; Lee, A. T.; Borths, E. L.; Locher, K. P. Structure of BtuCD, the ABC Transporter for Vitamin B12. FASEB J. 2003, 17 (5), A1185. 315. Deigan, K. E.; Ferre-D’Amare, A. R. Riboswitches: Discovery of Drugs That Target Bacterial Gene-Regulatory RNAs. Acc. Chem. Res. 2011, 44 (12), 1329–1338. 316. Nou, X. W.; Kadner, R. J. Adenosylcobalamin Inhibits Ribosome Binding to btuB RNA. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (13), 7190–7195. 317. Nahvi, A.; Sudarsan, N.; Ebert, M. S.; Zou, X.; Brown, K. L.; Breaker, R. R. Genetic Control by a Metabolite Binding mRNA. Chem. Biol. 2002, 9 (9), 1043–1049. 318. Nahvi, A.; Barrick, J. E.; Breaker, R. R. Coenzyme B12 Riboswitches Are Widespread Genetic Control Elements in Prokaryotes. Nucleic Acids Res. 2004, 32 (1), 143–150. 319. Polaski, J. T.; Webster, S. M.; Johnson, J. E.; Batey, R. T. Cobalamin Riboswitches Exhibit a Broad Range of Ability to Discriminate Between Methylcobalamin and Adenosylcobalamin. J. Biol. Chem. 2017, 292 (28), 11650–11658. 320. Johnson, J. E.; Reyes, F. E.; Polaski, J. T.; Batey, R. T. B12 Cofactors Directly Stabilize an mRNA Regulatory Switch. Nature 2012, 492, 133–137. 321. Peselis, A.; Serganov, A. Structural Insights Into Ligand Binding and Gene Expression Control by an Adenosylcobalamin Riboswitch. Nat. Struct. Mol. Biol. 2012, 19, 1182–1184. 322. Serganov, A.; Nudler, E. A Decade of Riboswitches. Cell 2013, 152 (1–2), 17–24. 323. Moeglich, A.; Yang, X. J.; Ayers, R. A.; Moffat, K. Structure and Function of Plant Photoreceptors. Annu. Rev. Plant Biol. 2010, 61, 21–47. 324. Purcell, E. B.; Crosson, S. Photoregulation in Prokaryotes. Curr. Opin. Microbiol. 2008, 11 (2), 168–178. 325. Rockwell, N. C.; Su, Y. S.; Lagarias, J. C. Phytochrome Structure and Signaling Mechanisms. Annu. Rev. Plant Biol. 2006, 57, 837–858. 326. Padmanabhan, S.; Jost, M.; Drennan, C. L.; Elias-Arnanz, M. A New Facet of Vitamin B12: Gene Regulation by Cobalamin-Based Photoreceptors. Annu. Rev. Biochem. 2017, 86, 485–514. 327. Ortiz-Guerrero, J. M.; Polanco, M. C.; Murillo, F. J.; Elias-Arnanz, M.; Padmanabhan, S. Light-Dependent Gene Regulation by a Coenzyme B12-Based Photoreceptor. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (18), 7565–7570.

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328. Elias-Arnanz, M.; Padmanabhan, S.; Murillo, F. J. Light-Dependent Gene Regulation in Nonphototrophic Bacteria. Curr. Opin. Microbiol. 2011, 14 (2), 128–135. 329. Cheng, Z.; Li, K. R.; Hammad, L. A.; Karty, J. A.; Bauer, C. E. Vitamin B12 Regulates Photosystem Gene Expression via the CrtJ Antirepressor AerR in Rhodobacter Capsulatus. Mol. Microbiol. 2014, 91, 649–664. 330. Cheng, Z.; Yamamoto, H.; Bauer, C. E. Cobalamin’s (Vitamin B12) Surprising Function as a Photoreceptor. Trends Biochem. Sci. 2016, 41 (8), 647–650. 331. Jost, M.; Fernandez-Zapata, J.; Polanco, M. C.; Ortiz-Guerrero, J. M.; Yang-Ting Chen, P.; Kang, G.; Padmanabhan, S.; Elias-Arnanz, M.; Drennan, C. L. Structural Basis for Gene Regulation by a B12-Dependent Photoreceptor. Nature 2015, 526, 536–541. 332. Padmanabhan, S.; Perez-Castano, R.; Elias-Arnanz, M. B12-Based Photoreceptors: From Structure and Function to Applications in Optogenetics and Synthetic Biology. Curr. Opin. Struct. Biol. 2019, 57, 47–55. 333. Jost, M.; Simpson, J. H.; Drennan, C. L. The Transcription Factor CarH Safeguards Use of Adenosylcobalamin as a Light Sensor by Altering the Photolysis Products. Biochemist 2015, 54 (21), 3231–3234. 334. Kutta, R. J.; Hardman, S. J. O.; Johannissen, L. O.; Bellina, B.; Messiha, H. L.; Ortiz-Guerrero, J. M.; Elias-Arnanz, M.; Padmanabhan, S.; Barran, P.; Scrutton, N. S.; Jones, A. R. The Photochemical Mechanism of a B12-Dependent Photoreceptor Protein. Nat. Commun. 2015, 6. 335. Fernandez-Zapata, J.; Perez-Castano, R.; Aranda, J.; Colizzi, F.; Polanco, M. C.; Orozco, M.; Padmanabhan, S.; Elias-Arnanz, M. Plasticity in Oligomerization, Operator Architecture, and DNA Binding in the Mode of Action of a Bacterial B12-Based Photoreceptor. J. Biol. Chem. 2018, 293 (46), 17888–17905. 336. Miller, N. A.; Kaneshiro, A. K.; Konar, A.; Alonso-Mori, R.; Britz, A.; Deb, A.; Glownia, J. M.; Koralek, J. D.; Mallik, L.; Meadows, J. H.; Michocki, L. B.; van Driel, T. B.; Koutmos, M.; Padmanabhan, S.; Elías-Arnanz, M.; Kubarych, K. J.; Marsh, E. N. G.; Penner-Hahn, J. E.; Sension, R. J. The Photoactive Excited State of the B12-Based Photoreceptor CarH. J. Phys. Chem. B 2020, 47, 10732–10738. 337. Toda, M. J.; Mamun, A. A.; Lodowski, P.; Kozlowski, P. M. Why is CarH Photolytically Active in Comparison to Other B12-Dependent Enzymes? J. Photochem. Photobiol. B Biol. 2020, 209, 111919. 338. Gruber, K.; Kräutler, B. Coenzyme B12 Repurposed for Photo-Regulation of Gene Expression. Angew. Chem. Int. Ed. 2016, 55, 5638--5640. 339. Pratt, J. M. The Roles of Co, Corrin and Protein. I. Co-Ligand Bonding and the Trans Effect. In Chemistry and Biochemistry of B12; Banerjee, R., Ed., John Wiley & Sons, 1999; pp 73–112. 340. Widner, F. J.; Lawrence, A. D.; Deery, E.; Heldt, D.; Frank, S.; Gruber, K.; Wurst, K.; Warren, M. J.; Kräutler, B. Total Synthesis, Structure, and Biological Activity of Adenosylrhodibalamin, the Non-Natural Rhodium Homologue of Coenzyme B12. Angew. Chem. Int. Ed. 2016, 55 (37), 11281–11286. 341. Koppenhagen, V. B.; Elsenhans, B.; Wagner, F.; Pfiffner, J. J. Methylrhodibalamin and 5’-Deoxyadenosylrhodibalamin, Rhodium Analogs of Methylcobalamin and Cobalamin Coenzyme. J. Biol. Chem. 1974, 249 (20), 6532–6540. 342. Carmel, R.; Koppenhagen, V. B. Effect of Rhodium and Copper Analogs of Cobalamin on Human Cells in vitro. Arch. Biochem. Biophys. 1977, 184 (1), 135–140. 343. Koppenhagen, V. B. Metal-Free Corrinoids and Metal Insertion. In B12; Dolphin, D., Ed.; vol. 2; John Wiley & Sons, 1982; pp 105–150. 344. Holze, G.; Inhoffen, H. H. The 1st Chemical Partial Synthesis of the Nickel-Complex of a Cobyrinic Acid-Derivative. Angew. Chem. Int. Ed. 1985, 24 (10), 867–869. 345. Brenig, C.; Prieto, L.; Oetterli, R.; Zelder, F. A Nickel(II)-Containing Vitamin B12 Derivative With a Cofactor-F430-type p-System. Angew. Chem. Int. Ed. 2018, 57 (50), 16308–16312. 346. Kieninger, C.; Baker, J. A.; Podewitz, M.; Wurst, K.; Jockusch, S.; Lawrence, A. D.; Deery, E.; Gruber, K.; Liedl, K. R.; Warren, M. J.; Kräutler, B. Zinc Substitution of Cobalt in Vitamin B12: Zincobyric acid and Zincobalamin as Luminescent Structural B12-Mimics. Angew. Chem. Int. Ed. 2019, 58 (41), 14568–14572. 347. Chan, W.; Almasieh, M.; Catrinescu, M. M.; Levin, L. A. Cobalamin-Associated Superoxide Scavenging in Neuronal Cells Is a Potential Mechanism for Vitamin B12-Deprivation Optic Neuropathy. Am. J. Pathol. 2018, 188 (1), 160–172. 348. Croft, M. T.; Lawrence, A. D.; Raux-Deery, E.; Warren, M. J.; Smith, A. G. Algae Acquire Vitamin B12 Through a Symbiotic Relationship With Bacteria. Nature 2005, 438 (7064), 90–93. 349. Degnan, P. H.; Taga, M. E.; Goodman, A. L. Vitamin B12 as a Modulator of Gut Microbial Ecology. Cell Metab. 2014, 20 (5), 769–778. 350. Braselmann, E.; Wierzba, A. J.; Polaski, J. T.; Chrominski, M.; Holmes, Z. E.; Hung, S.-T.; Batan, D.; Wheeler, J. R.; Parker, R.; Jimenez, R.; Gryko, D.; Batey, R. T.; Palmer, A. E. A Multicolor Riboswitch-Based Platform for Imaging of RNA in live Mammalian Cells. Nat. Chem. Biol. 2018, 14 (10), 964–971. 351. Krchlikova, V.; Mikesova, J.; Geryk, J.; Barinka, C.; Nexo, E.; Fedosov, S. N.; Kosla, J.; Kucerova, D.; Reinisova, M.; Hejnar, J.; Elleder, D. The Avian Retroviral Receptor Tva Mediates the Uptake of Transcobalamin Bound Vitamin B12 (Cobalamin). J. Virol. 2021, 95, e02136–20. 352. Kainrath, S.; Stadler, M.; Reichhart, E.; Distel, M.; Janovjak, H. Green-Light-Induced Inactivation of Receptor Signaling Using Cobalamin-Binding Domains. Angew. Chem. Int. Ed. 2017, 56 (16), 4608–4611.

2.12

Biological and synthetic nitrogen fixation

Oliver Einslea, Tobias A. Engesserb, and Felix Tuczekb, a Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Freiburg i. Br., Germany; and b Christian-Albrechts-Universität zu Kiel, Institut für Anorganische Chemie, Kiel, Germany © 2023 Elsevier Ltd. All rights reserved.

2.12.1 2.12.2 2.12.2.1 2.12.2.1.1 2.12.2.1.2 2.12.2.1.3 2.12.2.1.4 2.12.2.1.5 2.12.2.2 2.12.2.2.1 2.12.2.2.2 2.12.2.2.3 2.12.2.2.4 2.12.2.2.5 2.12.2.3 2.12.2.3.1 2.12.2.3.2 2.12.2.3.3 2.12.2.3.4 2.12.2.3.5 2.12.2.3.6 2.12.2.4 2.12.2.4.1 2.12.2.4.2 2.12.3 2.12.3.1 2.12.3.1.1 2.12.3.1.2 2.12.3.2 2.12.3.3 2.12.3.3.1 2.12.3.3.2 2.12.3.4 2.12.3.5 2.12.3.5.1 2.12.3.5.2 2.12.3.5.3 2.12.3.5.4 2.12.3.5.5 2.12.3.5.6 2.12.3.6 2.12.4 References

Introduction Biological nitrogen fixation (by O. Einsle) Nitrogenase enzymes The role of Fe protein Mo-dependent nitrogenase V-dependent nitrogenase Fe-only nitrogenase Biogenesis of nitrogenase cofactors Properties and function of nitrogenase cofactors The Lowe-Thorneley model Electronic structure of resting state FeMo cofactor Hydride formation and unproductive H2 release Reductive elimination of H2 generates a super-reduced state A Dinuclear binding site for substrates CO reduction by V-dependent nitrogenase Requirements for binding different substrates Provision of electrons and protons in nitrogenase cofactors CO-bound structures are dead-end adducts CO is activated by insertion of a hydride Continuous electron and proton supply in three phases Product release Summary and conclusion N2 binds to the E4 state Reductive elimination of H2 is linked to N2 binding Synthetic nitrogen fixation (by T. A. Engesser and F. Tuczek) Mononuclear molybdenum systems The Schrock catalyst The Chatt cycle Dinuclear molybdenum systems Mononuclear iron systems Peters’ systems Nishibayashi’s systems Dinuclear iron systems Systems with other transition metals Cobalt Ruthenium and osmium Titanium Vanadium Rhenium Chromium Lessons from small-molecule models Summary: Toward a comprehensive understanding of biological and synthetic nitrogen fixation

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Abstract This article deals with biological and synthetic nitrogen fixation. The first part gives an overview over the structure and function of nitrogenase, with an emphasis on discussing the available mechanistic information on the N2-to-NH3 conversion in the enzyme. In the second part, the available, catalytically active model small-molecule model systems of nitrogenase are reviewed. Again, the focus is put on describing the available mechanistic information regarding to the conversion of dinitrogen to ammonia. Having considered the enzyme and the available inorganic models, common trends in the bonding,

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activation, and conversion of N2 to ammonia are identified and lessons derived from the complementary study of these systems are formulated.

2.12.1

Introduction

The element nitrogen is interconverted between its various modifications by a network of natural and biotic reactions summarized as the biogeochemical nitrogen cycle. In it, the extraordinary inertness of the N2 molecule makes molecular dinitrogen a major atmospheric sink that comprises more than 99% of all nitrogen cycling through Earth’s biosphere. As an essential constituent of all classes of biological macromolecules, the availability of nitrogen is the most frequent limiting factor for organismic growth. The conversion of dinitrogen (N2) into a bioavailable form thus is a key reaction of nature. Bound to operating at ambient pressure and temperature, the nitrogenase enzymes must achieve the activation and reduction of the N2 molecule with a physiological source of energy, the hydrolysis of adenosine-50 -triphosphate (ATP) and electrons deriving from central metabolism. Such restrictions could not be further from the reaction conditions in the industrial Haber-Bosch process, a heterogeneous gas-phase reaction, and yet the underlying thermodynamic problem remains the same. To reduce N2, a bond dissociation energy of 946 kJ mol 1 must be overcome, and the required reducing power is not only far outside the electrochemical water window but is also not achievable by any known biological reductant.1 The challenges for enzymatic N2 reduction are substantial, and despite the fundamental role of nitrogen fixation for organic life we find that evolution has only found a single solution for the problem, the nitrogenase system. Nitrogenases are large and dynamic metalloprotein complexes that exist in three isoforms (classes), which all follow the same mechanistic principles and will be discussed in detail below.2,3 They consist of a  240 kDa dinitrogenase, a catalytic component with two large metal clusters, P-cluster and the active site cofactor (vide infra) that interacts with a specific reductase, the homodimeric Fe-protein that is also the site of ATP binding and hydrolysis (Fig. 1).2,3 For the transfer of a single electron to the active site of nitrogenase, the Fe-protein obtains an electron from a low-potential ferredoxin or flavodoxin and binds two ATP before forming a transient complex with the dinitrogenase. The interaction of the two component proteins first triggers electron transfer within the dinitrogenase that is then followed by ATP hydrolysis in the Fe-protein, then the reduction of the dinitrogenase by Fe-protein and eventually the release of ADP and Pi that leads to the dissociation of the complex. Fe-protein is the only functional reductase for the enzyme, and as it transfers a single electron in each cycle concomitant with the hydrolysis of 2 ATP, this process must be repeated multiple times for each N2 molecule. The minimal overall stoichiometry of the reduction of dinitrogen as catalyzed by the enzyme is. N2 þ 10Hþ þ 8 e þ 16 ATP / 2 NH4þ þ H2 þ 16 [ADP þ Pi] In this reaction, NH4þ is the predominant product at physiological pH. Note that the reaction involves the stoichiometric formation of 1 H2 per N2, although the enzyme in most cases diverts a far greater proportion of the electron flux toward H2 formation. The distinction between stoichiometric and adventitious H2 formation is of high mechanistic relevance. Although the reaction conditions are mild, the energy requirements of the biological reaction are close to those of the Haber-Bosch process, but every individual step is highly regulated, and the sequential transfer of electrons and protons imposed by the interaction with the Fe-protein indicates that the reaction proceeds through a series of defined intermediates. The recurring complex formation and dissociation of the two components of the nitrogenase system makes the otherwise fast electron delivery the rate-limiting step of the reaction that occurs at an overall rate of less than one N2 molecule per second, and the reaction is generally envisioned as a sequence of eight alternating electron and proton transfer steps. Understanding the mechanism of nitrogenase thus implies the identification and analysis of such intermediates, a decade-long endeavor that has led to a multi-pronged approach involving biochemical, spectroscopic, structural, and theoretical analyses that have provided a wealth of data. From this, a comprehensive mechanism for the action of nitrogenase enzymes begins to emerge.

Fig. 1 The nitrogenase system. The organism provides an electron on a low-potential ferredoxin (Fdx) or flavodoxin (Fld) to reduce the dimeric Feprotein. After binding of 2 ATP, Fe-protein forms a complex with the dinitrogenase, triggering electron transfer from the [8Fe:7S] P-cluster to the active site cofactor. Only then, P-cluster is again reduced from the [4Fe:4S] cluster in Fe-protein, ATP is hydrolyzed, and the complex dissociates for another round of reduction of Fe-protein. Figure based on Mo-nitrogenase (PDB 1N2C).

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Biological and synthetic nitrogen fixation

Relevant information regarding the conversion of N2 to NH3 via a series of protonation and reduction steps can also be obtained from the investigation of small-molecule model systems in solution. From its very beginning, the research area of ‘synthetic nitrogen fixation’ also aimed at performing this reaction in a catalytic fashion. During the early phase of these studies, several catalytic systems were established, some of which also reached significant turnover numbers. However, in most of these the reaction mechanism was not understood or elucidated on a molecular level. On the other hand, various transition-metal dinitrogen complexes were synthesized that subsequently were found to mediate the stepwise conversion of N2 to NH3. The earliest of these systems, established by the groups of Chatt and Hidai, were based on mononuclear bis(dinitrogen) phosphine-Mo/W dinitrogen complexes supported by diphosphine ligands such as dppe (Ph2PCH2CH2PPh2) or depe (Et2PCH2CH2PEt2). Until recently the associated reaction scheme, the Chatt cycle, could not be turned into a catalytic mode, which has been attributed to number of mechanistic drawbacks, such as the presence of anionic coligands that cause disproportionation at the Mo(I) stage. This problem could be overcome by novel multidentate phosphine ligands (see below).4,5 The first evidence of a catalytic conversion of N2 to NH3 based on a well-defined transition-metal dinitrogen complex was provided by Schrock and co-workers in 2003.6 Based on a molybdenum complex with a sterically shielding HIPTN3N ligand (HIPTN3N ¼ [{3,5-(2,4,6-iPr3C6H2)2-C6H3-NCH2CH2}3N]3), decamethylchromocene as reductant and (lutidinium) (BArF4) (BArF4 ¼ tetrakis[3,5-bis(trifluoro-methyl)phenyl]borate) as proton source, ammonia was generated from dinitrogen in four cycles with an overall yield of 66%. This discovery marked the starting point of an impressive scientific race leading to new catalytic models of nitrogenase with ever increasing activities. A few years after Schrock’s discovery, Nishibayashi et al. established a dinuclear molybdenum dinitrogen complex supported by a PNP pincer ligand. Using a combination of cobaltocene and lutidinium triflate, this system produced 11.6 equiv. NH3 per molybdenum center.7 In 2013, Peters et al. demonstrated for the first time that catalytic synthesis of ammonia from N2 is also possible based on iron complexes. Using KC8 and HBArF4$Et2O at low temperature ( 78  C), up to 64 equiv. NH3 were generated per Fe center.8 The last years have witnessed further significant advancements in terms of alternative metal centers, novel catalyst designs, increased catalytic activities and deeper mechanistic insight into these systems which have pushed the area of synthetic nitrogen fixation to one of the topics at the forefront of organometallic and bioinspired chemistry. The present review is organized as follows: the first part gives an overview over the structure and function of nitrogenase, with an emphasis on discussing the available mechanistic information on the N2-to-NH3 conversion in the enzyme. In the second part, the available, catalytically active model small-molecule model systems of nitrogenase are reviewed. Again, the focus is put on describing the available mechanistic information regarding to the conversion of dinitrogen to ammonia. Tables 2–4 give an overview over all currently known homogeneous catalytic systems for N2-to-NH3 conversion. Having considered the enzyme and the available inorganic models, common trends in the bonding, activation, and conversion of N2 to ammonia are identified and lessons derived from the complementary study of these systems are formulated.

2.12.2

Biological nitrogen fixation (by O. Einsle)

2.12.2.1

Nitrogenase enzymes

Nitrogenases are large and dynamic metalloprotein complexes that exist in three isoforms (classes), which all follow the same mechanistic principles. They consist of a  240 kDa dinitrogenase, a catalytic component that interacts with a specific reductase, the homodimeric Fe-protein that is also the site of ATP binding and hydrolysis. Fe-protein is the only functional reductase for the enzyme, and it transfers a single electron in each cycle to associate with and dissociate from the catalytic component, concomitant with the hydrolysis of 2 ATP. All components of the three isoforms have been studied and will be discussed in the following.

2.12.2.1.1

The role of Fe protein

Nitrogenases are only one of several families of redox enzymes that use electron-transferring reductases. The Fe-proteins are members of the large class of P-loop NTPases that is present in all kingdoms of life and comprises crucial members such as actin/FtsZ, myosin or the G-proteins. P-loop NTPases bind nucleoside triphosphates and typically have a very low to low hydrolytic activity for the third phosphodiester bond that often requires the interaction with an activating protein as a trigger. A conserved region, the P-loop (or Walker A motif) binds the nucleotide together with a Mg2þ cation, and the change from an NTP-bound to an NDP-bound state triggers a reversible conformational change in two regions termed ‘switch I’ and ‘switch II.’ The consequence of this structural rearrangement is typically mechanical in nature: G-proteins dissociate their a-subunit to initiate cellular signaling processes, myosin executes the power stroke that forms the basis for muscle movement and actin filaments can polymerize or depolymerize. In the Fe-proteins of nitrogenase, this conformational switching is complemented with a second function as an electron donor to the catalytic component. Fe-proteins form homodimers that coordinate a cubane-type [4Fe:4S] cluster at the dimer interface, with two cysteine residues from each monomer as ligands (Fig. 2). One of these cysteines is part of the switch II region of each chain, and the conformation of the Fe-protein dimer changes from a rather closed ADP-bound state to a more flexible and open form with bound ATP. However, ATP hydrolysis only occurs after complex formation with the dinitrogenase component, and structural data of these complexes revealed that the binding to its redox partners brings the two monomers of Fe proteins closer together, allowing for a residue in monomer to trigger ATP hydrolysis in the P-loop region of the other. The closure of the dimer also pushes the metal cluster closer to the surface, shortening the distance to the receiving P-cluster, a [8Fe:7S] site in the dinitrogenase from where an electron is then transferred to the active site cofactors.

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Fig. 2 Architecture of NifH, the Fe-protein of molybdenum nitrogenase. (A) Cartoon representation of A. vinelandii NifH in the ATP-bound state in front view (above) and top view (below). (B) Detail of the dimer interface of NifH with the bridging [4Fe:4S] cluster and a Mg-ADP ligand bound to each monomer. (C) ADP binding to the NifH monomer. The phosphate groups of the ligand bind to the P-loop that also holds a Mg2þ cation (gray), which is essential for ATP hydrolysis. Conformational changes upon ATP binding are transmitted via the switch I and II regions to the bridging [4Fe:4S] cluster.

During catalysis, each individual electron transfer involves (i) the reduction of Fe protein by a ferredoxin or a flavodoxin in the cytoplasm, (ii) the exchange of bound ADP for ATP in both monomers of Fe protein, (iii) the complex formation of Fe protein with the dinitrogenase, (iv) ATP hydrolysis in the Fe protein, (v) the transfer of one electron from P-cluster to the active site cofactor, (vi) the reduction of P-cluster by the [4Fe:4S] cluster of Fe protein, and (vii) the dissociation of the two protein components (Fig. 1).9 Note that in this sequence of events an electron is transferred within the dinitrogenase, from P-cluster to the cofactor, before being replenished from Fe protein.10 What has been described as a ‘deficit-spending mechanism’ is a functional necessity, as P-cluster is already highly reduced (all-ferrous) when both protein components form a complex.11 As the cycle is repeated at least eight times for a single turnover of N2, this implies that every electron reaches the active site sequentially and at isopotential. However, even in assays with a high excess of Fe-protein this only ever led to a 1-electron-reduced cofactor in the absence of substrate, a state that is not yet capable of binding or activating substrates. It remains under debate how ATP hydrolysis is utilized to drive catalysis. ATP hydrolysis was reported to be slower than electron transfer from P-cluster to the cofactor, while at the same time the redox potential for reduction of the cofactor is lower than that of reduced Fe protein in its free form. Along the way, the free enthalpy of ATP hydrolysis must be translated into a lower redox potential of the electron transferred to the cofactor, but the mechanistic basis for this process remains to be elucidated. Each of the three nitrogenase isoforms described in the following paragraphs has a dedicated Fe-protein and all of these follow the same mechanistic principles. In essence, the reaction at the active site cofactor of any nitrogenase is driven by a sequential supply of electrons at isopotential from the [8Fe:7S] P-cluster. The intricacies of the Fe-protein cycle outlined above can be largely disregarded when considering events at the active site, except that electron transfer occurs at such slow rates (ca. 5 s 1) that reaction intermediates must be long-lived to persist. This can be a challenge for catalysis, and the adventitious H2 production described above is one of its symptoms.12

2.12.2.1.2

Mo-dependent nitrogenase

Among the three classes of nitrogenase enzymes, the Mo-containing variant is the most widespread and by far the best-studied. All known diazotrophs possess a Mo-dependent nitrogenase and only some additionally contain one or both other types.13 The genes encoding this enzyme form the nif cluster (‘nitrogen fixation’), and the catalytic dinitrogenase is the MoFe-protein, a NifD2K2 heterotetramer that works in conjunction with its Fe-protein, NifH (Fig. 1). This variant of nitrogenase was first identified as an ATPdependent two-component system in 1966,14 to be purified to homogeneity and used for extensive spectroscopic and functional studies. In 1992, three-dimensional structures for the MoFe protein and for NifH were reported by Rees (Fig. 3A), followed in 1997 by the architecture of the complex of both components.15 MoFe protein contains the two signature iron-sulfur clusters unique to nitrogenases. The [8Fe:7S] P-cluster bridges the interface of the phylogenetically related NifD and NifK subunits and is symmetrically coordinated by three cysteine residues from each polypeptide. In its reduced state (‘PN’), the cluster exhibits C2 symmetry and has a total spin S ¼ 0 and an overall charge of þ 2 (Fig. 3C). When oxidized by one (‘Pþ 1’) or two (‘POx’) electrons, one or two of the central Fe ions of the cluster are changing position, exchanging their coordinative interaction with the central sulfide of the cluster for one with a harder ligand, the hydroxyl group of Ser188K and the backbone amide of Cys88D, respectively. As a reductant for the dinitrogenase the Fe-protein is assumed to be a 1-electron donor, so that likely only the first of these transitions (PN/Pþ 1) is of physiological relevance. P-cluster is located at 21 Å distance (center-to-center) from the second metal site, the FeMo-cofactor or M-cluster (Fig. 3B). Of

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Fig. 3 Architecture of molybdenum nitrogenase. (A) The NifD2K2 heterotetramer in cartoon representation. The positions of the two clusters are only indicated in one half of the enzyme. (B) FeMo cofactor is the catalytic site of MoFe protein. Mo, the seven iron ions and the m2-sulfides are numbered. (C) P-cluster, the [8Fe:7S] electron transfer site at the NifD-NifK interface. All panels from PDB 3U7Q.

similar size and shape as P-cluster, this cofactor is extensively modified, making it the largest and most complex biological metal center known to date. Like P-cluster it is formed by two cubane-type subclusters, but instead of a bridging sulfide as in the electron transfer site, the cofactor contains a central light atom that was only discovered 10 years after its initial structural characterization and was identified as a novel interstitial m6-carbide only another decade later.16,17 The two cubane subclusters are additionally bridged by three m2-sulfides, and in one of the cubanes the apical Fe ion is replaced by molybdenum. FeMo cofactor of Monitrogenase thus has an overall composition of [Mo:7Fe:9S:C], complemented by an organic homocitrate ligand to the Mo ion. Only the apical positions of the cofactor are coordinated to the protein chain via Cys275D to Fe1 and His442D to Mo. The protein matrix surrounding the cofactor not only provides a medium for electron transfer from P-cluster, but also features defined access pathways for protons, substrates, and products, so that nitrogenase catalysis is highly controlled, both temporally and spatially. Nevertheless, the functionality of the enzyme crucially depends on the unique properties of its metal cofactors that will be discussed below. Fe protein and the dinitrogenase dynamically form a complex that again dissociates for re-reduction of the Fe protein and the exchange of nucleotides. Rees and coworkers reported a structure of a NifH-NifDK complex that they stabilized by using ADP $ AlF 4 as an inhibitory transition state analog of ATP hydrolysis (Fig. 1).15 In this structure, the NifH dimer bound with its two-fold symmetry axis precisely along the twofold pseudosymmetry axis relating subunits NifD and NifK, thus placing the [4Fe:4S] cluster of the reductase directly above P-cluster, in a position to ideally facilitate electron transfer. Several other complex structures with and without nucleotides and other analogs by Tezcan and others have since confirmed this mode of interaction but showed that other conformations exist that are thought to represent early encounter complexes.18

2.12.2.1.3

V-dependent nitrogenase

Early studies of nitrogen fixation by the diazotroph Azotobacter vinelandii hinted toward the presence of dinitrogenase activity that was independent of Mo,19 and Bishop and Eady subsequently identified a vanadium-dependent enzyme that carried this activity.20 Vanadium-dependent nitrogenase is distinct from its Mo-containing counterpart and is encoded in a separate gene cluster, vnf (‘vanadium-dependent nitrogen fixation’), but its biogenesis requires several accessory factors from the nif cluster.21 The first isolations of V-nitrogenase from A. vinelandii and A. chroococcum were reported in 1986 and 1987 by Hales and Eady, respectively.22 Isolated V-nitrogenase only showed about 30% of the N2-reducing activity of Mo-nitrogenase and typically diverted more than three times the number of electrons toward unproductive H2 formation than the Mo-dependent variant.23 Due to its substantially lower catalytic activity and effectiveness, V-nitrogenase is generally considered a backup system for environments with limited Moavailability, but the enzyme is sufficient to allow for diazotrophic growth with N2 as a sole nitrogen source at very similar rates as the Mo-dependent system.24 In 2010, Ribbe and Hu reported the catalytic reduction of carbon monoxide by V-nitrogenase.25 CO is a known, non-competitive inhibitor for any nitrogenase substrate other than protons, but the vanadium-dependent enzyme also reduces the gas. A re-examination of Mo-nitrogenase found a similar reaction, although at an 800-fold lower activity,26 unless an active site variant was employed.27 Interestingly, CO, in contrast to the isoelectronic N2, is not fully reduced by the enzyme. Rather, the main product is ethylene, implying that the reaction involves a C-C coupling step.26 The V-nitrogenase system includes a dedicated Fe-protein, VnfH, which shows > 90% sequence identity to NifH and is fully cross-compatible in activity assays. Our three-dimensional structure of VnfH revealed a nearly identical architecture and a fully conserved interaction interface with the dinitrogenase, as well as an ADP-bound conformation of the homodimer that corresponds to the one found for NifH.28 The V-dependent dinitrogenase is the VFe-protein that assembles a 230 kDa VnfD2K2G2 heterohexamer (Fig. 4).29 In addition to the structurally conserved D- and K-subunits, VnfG is a small, a-helical protein in exclusive contact with VnfD that may play a role in the maturation of the enzyme.30 It does not contain any metal cofactors and it is not implied in catalysis. For substrate reduction, VnfH and VFe-protein dynamically form a complex for electron transfer and ATP hydrolysis, in full analogy with the molybdenum-dependent ortholog described above. At the interface of the VnfD and VnfK subunits, VFeprotein possesses a P-cluster with the exact same [8Fe:7S] architecture as the one known from MoFe-protein. However, while the

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Fig. 4 Vanadium-dependent nitrogenase from A. vinelandii. (A) Cartoon representation of the VnfD2K2G2 heterohexamer, with the cluster positions indicated in one VnfDK protomer. (B) The catalytic FeV cofactor contains a bridging carbonate in place of sulfide S3A and has vanadium replacing the apical molybdenum of FeMo cofactor. (C) P-cluster is highly similar to the site in MoFe protein but has a more stable 1-electron oxidized Pþ 1 state. All panels from PDB 5N6Y.

latter was initially only characterized in the PN and POx states (see Section 2.12.2.2),31 the functional Pþ 1 form was more easily formed in VFe-protein than in MoFe-protein, where its crystallization required the potentiometric titration of entire crystals.32 Despite this difference, the P-clusters in both variants are assumed to have identical functionality. The catalytic site of VFeprotein is a large cofactor cradled in the VnfD protein. This FeV-cofactor shares the same basic architecture with FeMo cofactor of MoFe protein, but its structural analysis at high resolution revealed some alterations. As expected, the apical Mo ion of FeMocofactor is exchanged for vanadium in FeV-cofactor, with only minor changes to bond distances and cofactor geometry. The cofactor contains a central carbide and m2-bridging sulfides at a trigonal prism of six central Fe ions, but one of the m2-sulfides in the ‘belt’ of FeMo cofactor, atom S3A, was exchanged for another bridging ligand in FeV-cofactor. We identified this unexpected ligand as a divalent carbonate anion in the electron density maps, an assignment that was later confirmed by theoretical studies. The role of this carbonate remains unclear to date, but there are no indications that it exchanges during catalysis or that it participates otherwise except by possibly altering the electronic structure of the cofactor. The homocitrate ligand found in MoFe protein is also present in VFe protein, completing the structure of FeV cofactor as a [V:7Fe:8S:C:CO3]:homocitrate moiety. While the initial structural analysis of VFe protein showed the enzyme in a resting state, further studies revealed that in FeV cofactor one of the remaining belt sulfides, atom S2B, was easily and reversibly replaced by a light atom, concomitant with a conformational change of a nearby conserved glutamine residue, Glu176. We suggested this change to a ‘turnover state’ to be of high mechanistic relevance, and it has since been included into functional models for nitrogenase catalysis (vide infra).

2.12.2.1.4

Fe-only nitrogenase

Some diazotrophic organisms retain the ability to fix N2 even in the absence of both Mo and V, and this observation eventually led to the discovery of a third class of nitrogenases that utilize iron as the only transition metal in their dinitrogenase component. The model diazotroph A. vinelandii contains this ‘alternative nitrogen fixation’ system (Anf) in addition to the Mo- and V-dependent enzyme, but genomic analyses have shown that this ‘Fe-only’ nitrogenase is the only backup system beside Mo-nitrogenase in most organisms. It catalyzes N2 reduction again with only about half the specific activity of the V-dependent enzyme, but its sole dependence on the highly available iron has made it an intensely studied object with a perspective for biotechnological applications and for the development of novel catalysts that dispense of rare metals without sacrificing catalytic efficiency. Accordingly, the ortholog from the Gammaproteobacterium Klebsiella oxytoca was the first to be successfully produced in a recombinant form in E. coli as a heterologous host. In A. vinelandii, the Fe-only nitrogenase is encoded by a distinct set of genes in an anf gene cluster that also includes a dedicated Fe-protein, AnfH. We reported a three-dimensional structure of AnfH in complex with ADP, which showed that although the sequence identity with the analogs NifH and VnfH is only around 60%, the overall architecture of this dimeric iron-sulfur protein was highly conserved.33 Nevertheless, a matrix of cross-reactivities of the three Fe protein with the three dinitrogenases revealed that MoFe protein would reach its full activity with either NifH or VnfH as a redox partner but would not interact well with AnfH. In reverse, FeFe-protein was strictly required AnfH for activity and did not function with either NifH or VnfH (Fig. 5). Interestingly, VFe-protein is itself more similar to FeFe protein but employs a reductase, VnfH, that is almost identical to NifH. The activity profile showed that VFe-protein is the only nitrogenase isoenzyme that does not discriminate significantly between the three Fe-proteins.33 FeFe-protein is a 240 kDa heterohexamer with composition AnfD2K2G2 and is indeed more similar in both sequence and architecture to VFe-protein than to MoFe-protein (Fig. 6A). Like the former, it carries a cofactor-free G subunit that is in exclusive contact with the D subunit. In our most recent crystal structure of the enzyme, the P-cluster at the AnfD-AnfK interface was positioned exactly as in the other isoforms and was in the all-ferrous, C2-symmetric PN state. The active site, FeFe cofactor, is a [8Fe:9S:C] moiety that has the m2-sulfide-bridged dicubane architecture with a central carbide known from the FeMo- and FeV-cofactors,

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Fig. 5 The three Fe proteins of A. vinelandii. (A) Selectivity of the 9 possible Fe protein:dinitrogenase pairings. NifH and VnfH behave very similarly and operate with MoFe and VFe protein, but not with Fe-only nitrogenase. In contrast, FeFe protein only works with AnfH, while VFe protein accepts all three reductases. (B) Structure of NifH (PDB 1FP6). All Fe proteins are shown in the ADP-bound state. (C) Structure of VnfH (PDB 6Q93). (D) Structure of AnfH (PDB 7QQA).

Fig. 6 The FeFe protein of Fe-dependent nitrogenase from A. vinelandii. (A) Cartoon representation of AnfD2K2G2, with the cluster positions indicated in one protomer. (B) FeFe cofactor is a [8Fe:9S:C] cluster with three m2-bridging sulfides. (C) The P-cluster of FeFe protein was in the allferrous state in the crystal structure and corresponds to its counterparts in the other nitrogenase isoenzymes.

with the apical heterometal replaces by iron, as was expected (Fig. 6B). A P-cluster was also present, in the same position and with the same structural features as in the other isoenzymes (Fig. 6C) FeFe-cofactor also bound a homocitrate ligand to the apical Fe8, which was anticipated and which together with a histidine ligand of the protein, H423D, conveys an octahedral ligand field to this metal ion, while all other irons remain tetrahedral. In the immediate proximity of FeFe cofactor, key amino acid residues are largely conserved, including the points of attachment to the protein at the apical positions to C257D and H423D, the histidine and glutamine, H180D and Q176D, above the Fe2-Fe6 edge of the cofactor and the valine and phenylalanine that bound this edge on both sides. In contrast to FeV-cofactor, the active site of iron-nitrogenase did not contain a carbonate ligand. The structure furthermore revealed that a specific m-sulfide of the cofactor, S2B, was again partially replaced by a light atom, in line with the observed ‘turnover state’ in VFe-protein. The three nitrogenases share a common biosynthetic precursor (see Section 2.12.2.1.5), and FeFe-cofactor is closest to this precursor, adding only the homocitrate ligand. Their common feature is the fusion of two cubane subclusters via a central carbide inserted during biosynthesis, leading to the stable core formed by the trigonal prism of Fe2-Fe7 and the m6-carbide in its center.34 FeMo-cofactor is the most widely distributed version of this cluster and shows the highest catalytic activity toward dinitrogen,

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suggesting that the coupling of Mo to the iron core promotes its potential to activate N2. As an evolutionarily ancient enzyme, nitrogenase is believed to have evolved in the oceans before life made its landfall, and molybdate indeed has a far higher bioavailability in seawater than it has in terrestrial environments, where in turn vanadate is more accessible. It seems tempting to connect the emergence of an alternative, V-based nitrogenase to the migration of diazotrophs to a terrestrial lifestyle but note that Mo-dependent nitrogenases remained the most abundant isoform in all diazotrophs to date. Vanadium is a suitable replacement for molybdenum in terms of its coordination geometry and ligand preferences, but it may not have been as effective in the cofactor to promote nitrogen fixation. Possibly the insertion of a carbonate at the Fe4-Fe5 edge of this cofactor was a measure to salvage a part of the lost activity, but so far, a version of FeV cofactor without a carbonate is not available for comparing catalytic efficiency. FeFecofactor features the simplest architecture of the three and exhibits the lowest catalytic activity toward N2. Nevertheless, its reliance solely on the highly available iron and the lower complexity of the required biosynthetic apparatus make it an important target for genetically engineering the fixation of atmospheric N2 into food crops.35

2.12.2.1.5

Biogenesis of nitrogenase cofactors

The two characteristic metal clusters of nitrogenases, P-cluster and the respective MoFe-, VFe- or FeFe-cofactors, represent the largest and most complex iron-sulfur clusters known to date. Their initial assembly, subsequent maturation, and insertion into the catalytic dinitrogenase component requires a large number of accessory proteins and reflects their highly specific properties (Fig. 7). The basic building block for both clusters is a canonical cubane-type [4Fe:4S] cluster that also serves as the metal center in the Feproteins. It is assembled by the proteins NifS and NifU, close relatives of the iron-sulfur biogenesis machinery ISC in eukaryotic mitochondria. NifS is a cysteine desulfurase that mobilizes the sulfur component required for cluster formation from the amino acid cysteine and forms an internal persulfide on a flexible loop. By a conformational rearrangement the persulfide moves from the active site of the pyridoxal phosphate dependent NifS to its partner NifU. As a homologue of IscU, NifU serves as a scaffold for the assembly of a [2Fe:2S] unit that swiftly dimerizes into a cubane-type [4Fe:4S] unit. As such, it is transferred to the Feprotein in a process that also requires the scaffold protein NifM, or to apo-nitrogenase where two adjacent clusters are inserted as precursors of the electron-transferring P-cluster. All further steps of P-cluster maturation then occur in situ on the enzyme. Key accessory factors are the NifW and NifZ proteins that act successively on each side, as well as the (mature) Fe-protein, whose role may at least in part be in the provision of electrons. When during maturation the two precursor [4Fe:4S] clusters are fused into the unique [8Fe:7S] moiety, a single sulfur atom is removed, and the details of this event remain to be understood. With both Pclusters completed, the enzyme remains deplete of the active site cofactors and attains an open state, where parts of the D-subunit are rearranged to allow for the insertion of the respective FeMo-, FeV- or FeFe-cofactors. Contrary to the one of P-cluster, the entire assembly of the catalytic cofactors takes place ex situ, and the actual insertion of the moiety that completes the functional assembly of the dinitrogenase is the very last step of the process. Again, the basic building blocks of all cofactors are [4Fe:4S] clusters provided by the NifSU system. For cofactor assembly, two such units are transferred to the radical/SAM enzyme NifB, where in a complex rearrangement reaction a carbon atom (from S-adenosyl methinonine) and an additional sulfide are inserted to form a precursor

Fig. 7 Simplified overview of the biosynthesis of Mo-nitrogenase. The cysteine desulfurase NifS mobilizes sulfur from cysteine and together with an iron ion a [2Fe:2S] unit is assembled on the scaffold NifU, where it is also dimerized into the [4Fe:4S] clusters that serve all other assembly pathways. The Fe protein NifH is stabilized as a complex with the chaperone NifM and dimerizes upon cluster insertion to then play multiple roles during assembly and function of nitrogenase. Cofactor biogenesis occurs ex situ, starting with the radical/SAM enzyme NifB that fuses two [4Fe:4S] clusters under insertion of a carbide and an additional sulfur into a [8Fe:9S:C] precursor termed L-cluster or NifBco. This then is transferred by the chaperone NifX to the second maturase, the NifEN complex, where the apical Fe8 is exchanged for Mo and homocitrate is attached. Meanwhile, other [4Fe:4S] from NifU are inserted into the subunits NifD and NifK of the enzyme that then form the NifD2K2 heterotetramer. They are matured into Pcluster in situ, with the successive involvement of NafH, NifW, NifZ and Fe protein. Only in the final step, the chaperone NafY delivers the finished cofactor to complete the assembly of active nitrogenase.

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cluster, termed L-cluster or NifB-cofactor. This D32-symmetric [8Fe:9S:C] cluster additionally requires reducing equivalents provided by a designated ferredoxin, and sulfite was reported as a source for the ninth sulfur atom. In A. vinelandii, NifB possesses a C-terminal domain that is homologous to the NifX protein, a potential precursor chaperone, and both likely serve to shuttle Lcluster to the next station of this complex assembly line. However, at this point, due to the different architecture of the cofactors of the three nitrogenase isoforms, the maturation pathways diverge. The FeFe cofactor of Fe protein corresponds to the L-cluster produced on NifB except for the additional requirement of the organic homocitrate ligand, and accordingly the remaining assembly steps occur directly on apo-FeFe-protein. For the Mo- and V-dependent nitrogenases, further modifications of L-cluster involve the exchange of an apical iron ion, Fe8, for the respective heterometal. For this purpose, the gene respective clusters contain the NifEN and VnfEN systems. The most striking feature of these maturation enzymes is their structural similarity to dinitrogenases. The EN complexes are a2b2-tetramers that show the same three-domain architecture in each chain and even coordinate a subunit-bridging [4Fe:4S] cluster in the exact place where the actual enzymes have their P-clusters. A crystal structure of NifEN by Rees showed that the cofactor precursor binds near the respective site in the dinitrogenases, albeit with an obvious access pathway to the protein surface. Only upon completion of cofactor maturation on the EN complexes, FeMo-cofactor and FeV-cofactor are then transferred to the apo-dinitrogenases to complete the assembly of the enzymes.

2.12.2.2

Properties and function of nitrogenase cofactors

The basic catalytic ability of all isoforms of nitrogenase is to mediate a stepwise multi-electron reduction of unreactive substrates. The mechanistic challenge lies in the generation of a sufficiently strong reducing power to functionalize highly inert small molecules such as N2 or CO while using biochemical energy sources and operating in aqueous solution. To sustain the reduction of N2 at the high rates required to allow organismic growth, electrons must be drawn from central metabolism, e.g., the complete oxidation of glucose to CO2 in glycolysis and the Krebs cycle. In this process, electrons are stored in NADH before being fed into the aerobic respiratory chain, but already at the stage of NADH their redox potential is too positive to activate N2 or CO. In fact, the theoretical overpotential required to break the N2 triple bond is around  1.5 V vs. SHE, and this is not straightforward to achieve in an aqueous milieu where  0.815 V are sufficient to reduce the ubiquitous water. The essence of nitrogenase functionality thus is to use metabolic energy in the form of ATP to lower the effective midpoint potential of electrons required for substrate reduction to a level far below that of NADH, and to create such reducing species precisely at the time and place where they are needed. The reduction of a single N2 molecule requires eight single-electron transfers from Fe-protein to the dinitrogenase, each accompanied by the hydrolysis of two ATP. Consequently, every single electron reaches the active site cofactor at the same potential. Nevertheless, the enzyme must acquire multiple electrons to generate a super-reduced state not only to activate, but already even to bind its substrate.

2.12.2.2.1

The Lowe-Thorneley model

2.12.2.2.2

Electronic structure of resting state FeMo cofactor

Before structural data for MoFe protein first became available in 1992, extensive kinetic and spectroscopic studies had been carried out with the enzyme from K. pneumoniae that culminated in a comprehensive kinetic model presented by Lowe and Thorneley in four seminal contributions.12,36 Describing the six-electron reduction of N2 to ammonia, this Lowe-Thorneley (LT) model included the mandatory and stoichiometric formation of one molecule of dihydrogen for every N2 reduced. Importantly, Lowe and Thorneley recognized that the release of H2 was conconmitant with the binding and activation of N2 and elaborated on the necessity of extensive reductive activation of the enzyme before its inert substrate would bind. Describing an eight-electron process, the LT model comprised eight intermediates, E0–E7, and outlined the requirement for transferring four electrons (and protons) from the resting state E0 to an E4 state where the H2/N2 exchange takes place (Scheme 1). Every single electron transfer step involves the formation of a complex of the dinitrogenase with its cognate Fe-protein, the actual electron transfer event, hydrolysis of two equivalents of ATP and the subsequent dissociation of the two nitrogenase component proteins. The sequential nature of this slow process means that every single intermediate state in the LT model must be sufficiently stable to persist until the next electron is delivered and catalysis can proceed. A further important implication is that while electrons accumulate at the active site cofactor when progressing from the E0 to the E4 state, reducing equivalents are provided at isopotential although each reduction should become successively harder to achieve. While the LT model thus was uniquely powerful in integrating most if not all the available biochemical and kinetic data, its translation into a molecular mechanism that must include a description of every intermediate state remained far from straightforward.

Among the obstacles for understanding the mechanism of nitrogenases was the complexity of the large iron-sulfur clusters of the enzymes. The electron-transferring P-cluster today is assumed to cycle between the all-ferrous [8Fe:7S]2þ state PN and the oneelectron oxidized [8Fe:7S]1þ state Pþ 1. With its architecture reminiscent of two fused cubane-type [4Fe:4S] clusters, P-cluster also maximizes the antiferromagnetic coupling between its iron-centers, leading to the PN state being diamagnetic and the Pþ 1 state showing a total apparent spin of S ¼ 1/2. In MoFe protein, a two-electron oxidized Pþ 2 state was observed spectroscopically and structurally but is not considered to be of relevance during catalysis. The active site FeMo-, FeV- and FeFe- cofactors present a far more complicated picture. Here, once more, the Mo-dependent nitrogenase is the most thoroughly characterized system. The enzyme is typically isolated in the resting state E0 of the LT scheme (Scheme

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Scheme 1 The kinetic model of Lowe and Thorneley. For each N2 molecule reduced, the enzyme progresses through eight distinct intermediates as electrons are delivered sequentially by the Fe-protein. From the resting state E0, an initial charging phase eventually leads to the storage of four electrons at the active site, in the form of two surface hydrides. From the critical E4 state, these can either be lost unproductively through protonolysis, or undergo reductive elimination of H2, leaving the enzyme in a super-reduced state able to bind and reduce N2.

1) in the presence of a chemical reductant such as S2O42 or Ti3þ. The E0 state has a characteristic, broad and rhombic EPR spectrum with a total spin of S ¼ 3/2 and apparent g-values of gx ¼ 2.03, gy ¼ 3.65 and gz ¼ 4.31. With tetrahedral geometry, the seven iron ions are in a high-spin configuration and broken-symmetry (BS) DFT calculations by Noodleman and Case had pointed toward a particular coupling scheme, BS7, that maximizes antiferromagnetic coupling within the cluster.37 BS7 has four Fe-sites in a spin-up configuration and three in spin-down configuration (Fig. 8). The molybdenum ion was highly reduced according to 95 Mo ENDOR spectroscopy and was therefore originally assigned as Mo4þ, the most common redox state found for the metal in biological systems.38 To accommodate an overall spin of S ¼ 3/2 for the cluster, Burgess and Münck proposed a configuration of 4Fe2þ:3Fe3þ based on Mössbauer spectroscopy,39 but combinatorially, configurations of 2Fe2þ:5Fe3þ or 6Fe2þ:1Fe3þ would also be feasible.40 The ambiguitiy could not be lifted at the time and the electronic structure of the cluster consequently remained under debate. First progress toward clarifying the properties of FeMo cofactor came from an investigation of the Mo ion using high energy resolution fluorescence-detected X-ray absorption spectroscopy (HERFD-XAS). Björnsson and DeBeer compared the enzyme to a series of known model complexes and found that the pre-edge and rising-edge features of the enzyme were only consistent with Mo3þ, implying a 4d3 configuration. Due to the coupling of the ion with the adjacent iron sites, they additionally identified an unusual 2 downd1 up ground state that did not follow Hund’s rule.41 With this unprecedented finding the combinatorics for the electronic structure of the E0 state of FeMo cofactor had to be reconsidered. Solution XAS was not a suitable method for studying individual iron sites, but instead it was possible to exploit the fact that the anomalous diffraction behavior of an element close to an X-ray absorption edge is proportional to its absorption cross-section for X-ray, leading to a wavelength-dependent modulation of diffraction data that is strictly element-specific. In addition, it also contains the spatial information from diffraction on a crystal lattice, so that a series of diffraction data sets collected around the K-edge of iron allowed for the refinement of the individual absorption properties of every single iron ion. We established this spatially refined anomalous dispersion (SpReAD) analysis first for a thioredoxin-type [2Fe:2S] ferredoxin from Aquifex aeolicus to

Fig. 8 The electronic structure of FeMo cofactor in the E0 state. The blue and red arrows denote the coupling of the high-spin d5 Fe atoms 1–7 according to the broken-symmetry DFT scheme BS7 that maximizes antiferromagnetic coupling. Listing the spin-down irons, this solution is designated (346). The apical molybdenum is a unique Mo3þ in a non-Hund ground state that together with a SpReAD analysis that revealed that Fe1, Fe3 and Fe7 are reduced to formal Fe2þ, yielding an overall spin of S ¼ 3/2 and a total charge of 1.

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identify a localized Fe2þ site in an antiferromagnetically coupling [2Fe:2S] cluster by a shift of the X-ray absorption edge of the more reduced ion by 2 eV toward lower energy.42 A similar analysis for MoFe protein had to account for the edge positions of 15 Fe ions rather than the two of the ferredoxin but nevertheless revealed that three sites, Fe1, Fe3, and Fe7, appeared to be more reduced than the other four. At the same time, the P-cluster in its all-ferrous PN state provided an excellent internal reference for Fe2þ that matched the reduced sites in the cofactor very well.43 Indeed, a distribution of 3Fe2þ:4Fe3þ:Mo3þ results in an overall S ¼ 3/2 system with a total charge of  1 that is also in line with DFT calculations and Mössbauer data.44 According to the BS7 coupling scheme, Fe3 and Fe4 and Fe5 and Fe7 form ferromagnetically coupling pairs within the cluster, implying that in the E0 state of FeMo cofactor iron sites Fe2 and Fe6 are the most highly oxidized positions in the cluster (Fig. 8). To further characterize FeMo-cofactor, single crystals of MoFe-protein were examined by X-band EPR spectroscopy to determine the relative orientation of the principal axes of the apparent g-tensor of a S ¼ 3/2 system. In this analysis, the longest principal axis gz aligned very well with the pseudo-threefold axis of the metal cluster. Expectations for gx and gy were less clear, as the rhombic g tensor did not obviously relate to the symmetry of the cluster.45 The EPR analysis revealed that the gx component was in a plane formed by Fe1, Fe2, Fe6, and Mo, which indicates an electronic distinction between the core irons Fe2 and Fe6 in this plane from the other four, Fe3, Fe4, Fe5, and Fe7. The subsequent findings about the distinct role of Fe2 and Fe6 during catalysis may well be reflect in these properties, but to date detailed information on the changes in the electronic structure of FeMo-cofactor as it progresses through the different states of the LT model remains scarce.

2.12.2.2.3

Hydride formation and unproductive H2 release

2.12.2.2.4

Reductive elimination of H2 generates a super-reduced state

Arguably the most crucial feature of the Lowe and Thorneley model is a finding that dates to Chatt’s finding that N2 is bound in exchange for H2,46 which in turn is formed by the reduction of protons. This turns the six-electron reduction of N2 into a net eight-electron process. The LT model includes a requirement for four subsequent reduction steps to promote nitrogenase from its resting state to the E4 state that is uniquely able to productively mediate this N2/H2 exchange, breaking the stable triple bond of the substrate. The LT model furthermore implied that as soon as two electrons are available at the cofactor, i.e., from state E2 onward, the enzyme can release H2. This gas release sets the catalytic cycle back by two positions and is obviously unproductive with respect to dinitrogen catalysis, distinguishing it fundamentally from the stoichiometric N2/H2 exchange in E4. Factors such as electron flux in the system (reflected in the Fe:MFe-protein ratio used in activity assays), temperature, and most significantly also the isoform of the nitrogenase strongly influence unproductive H2 release. For unknown reasons, VFe-protein and FeFeprotein divert a far larger proportion of electron flux toward H2 formation than MoFe-protein, but in a recent study Seefeldt and coworkers found that for high N2 partial pressure the H2/N2 ratio for all three classes of nitrogenases converges toward the same minimal value of 1 that was found for MoFe-protein. Essentially, H2 is a competitive inhibitor for N2 reduction by nitrogenase but does not affect other substrates or protons. Intriguingly, Burgess, Stiefel and co-workers reported47 that N2 is required for the enzyme to catalyze the formation of HD in an exchange of D2 and protons according to D2 þ 2Hþ þ 2 e / 2 HD A rationale for this finding was offered by Chatt, who first suggested that the enzyme forms hydride ligands that are displaced as H2 upon binging of N2.46 The above reaction may then represent the replacement of bound N2 by D2 to yield bound deuterides that subsequently are lost as HD upon protonation.48

Three decades after Chatt’s considerations, Seefeldt, Hoffman, Dean and co-workers expanded on the concept of hydride formation, suggesting that whenever two electrons were transferred to the cofactor, they would combine with a single proton to form a surface hydride.49 As the enzymatic reaction proceeds slowly, the authors favored the more stable bridging hydrides over terminal ones. ENDOR spectroscopy identified intermediates that were interpreted to represent the (paramagnetic) E4 state.50 As a high-energy state, E4 could either undergo unproductive protonation of one or both bound hydrides to fall back in the catalytic cycle of the LT model, or it could exchange H2 for N2 and initiate substrate reduction. Starting from two bound hydrides, the exchange reaction would constitute the reductive elimination of H2 that initially leaves the cofactor in a two-electron reduced state. As in this scheme electron transfer from Fe-protein will only reduce the cofactor by one electron and form a surface hydride in the next state, the reductive elimination of H2 from two hydrides in E4 is the only path to create a two-electron-reduced state that then is sufficiently reactive to bind the substrate N2 and immediately reduce it by two electrons (Scheme 1).51 This hypothesis helps to rationalize several key mechanistic questions surrounding the nitrogenases. It explains the interplay of H2 and N2 at this active site, because the rigid and electron-rich cofactor will only accept a single electron in the transitions to the odd E-states,52 while a further reducing equivalent can only be accommodated as a surface hydride that then is prone to protonation and unproductive H2 formation. Reductive elimination of H2 furthermore requires two hydrides at the cofactor, which is only achieved in the E4 state. The formation of a superreduced state, E*4, thus is how nitrogenase achieves the activation of the inert N2 molecule at ambient pressure and temperature, and contrary to the surface catalysis of the Haber-Bosch process this must be a highly controlled process.53 The E*4 state must be highly reactive and will likely reduce water, so that the active site cavity must be designed to exclude the ubiquitous solvent. As this state is also expected to have a short lifetime, the enzyme must ensure that the substrate N2 is available and ideally close to the active site before H2 is eliminated from E4. Beyond the mere presence of hydrides, an understanding of nitrogenase catalysis thus necessarily requires a detailed picture of the precise sites where hydrides are bound, the interactions of the metal cluster with the substrate and the role of the highly conserved protein environment of nitrogenase cofactors during the catalytic cycle. Spectroscopic studies have

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identified a series of activated states that were assigned to E-states along the catalytic cycle, but in these studies the positions of the formed hydrides remained hypothetical.54

2.12.2.2.5

A Dinuclear binding site for substrates

Aiming for a molecular mechanism of biological N2 fixation, one of the most enigmatic features of all nitrogenase cofactors is that all metal sites are coordinatively saturated. Surrounded by sulfides and the central carbide, Fe1–Fe7 are tetrahedrally coordinated, while the apical heterometals or Fe8 in Fe-nitrogenase are in an octahedral coordination that is completed by the bidentate ligand homocitrate.34 No particular position on the pseudo-D3 symmetric clusters seems to be outstanding, which has not only complicated the analysis and prediction of its mechanistic features but has also left unclear how inspiration for novel synthetic N2 fixation catalysts can be drawn. It was therefore highly significant when Rees and colleagues in 2014 reported the structure of a CO-inhibited FeMo cofactor in Mo-nitrogenase.55 This was the first ligand-bound structure of the enzyme, and the formation of the adduct required the addition of CO under turnover conditions. CO-bound MoFe-protein was not catalytically active but could be reactivated under continuous turnover conditions and removal of CO in the headspace of the assay. Rather than binding end-on to one of the iron sites on the cofactor, CO attained a m2-bridging position at Fe2 and Fe6, the site that in the resting state cofactor is occupied by sulfide S2B (Fig. 9A and B). We designate this ligand mCO. Using anomalous diffraction data and working at high resolution, the analysis showed unambiguously that the sulfide was fully replaced by CO but was reinstated upon reactivation of the enzyme. The environment of the cofactor did not undergo substantial changes, and the geometry and bond distances of the mCO were in excellent agreement with other metal-bridging carbonyls. The S2B site at Fe2 and Fe6 was also the position that was replaced by a light atom in the turnover state structure of FeV cofactor in V-nitrogenase,53 and the structure of FeFe cofactor revealed a dual conformation of a resting and a turnover state at the same site. In 2020, we also reported a mCO-bound structure for FeV cofactor at 1.0 Å resolution (Fig. 9C).56 Here, CO binding at the Fe2-Fe6 edge corresponded exactly to the binding mode observed in MoFe protein, but the removal of CO under continued turnover now led to an enzyme in the turnover state, i.e., with a light atom rather than S2B reinserted into the cluster. With the turnover state, this distinction was straightforward, as the smaller bridging ligand led to a rearrangement of the sidechain of nearby Glu176 that then formed a short H-bond to His180 directly above the Fe2-Fe6 edge of the cofactor. In this, Glu176 vacated the position it occupies in the resting state and the place of its two carboxylate oxygens there was taken by a water molecule and the displaced sulfide S2B, as evidenced by the anomalous signal of sulfur. The turnover state of FeVand FeFe-cofactor thus reveals a holding site for the sulfide, presumably, as HS, suggesting that the reversible removal of this bridging ligand is indeed part of the catalytic mechanism. In line with the observed mode of CO binding, the cofactor after release of S2B appears as a dinuclear site, allowing for modes of association that do not change the coordination number of the iron sites

Fig. 9 CO adducts and ligand binding sites at the cofactors. (A) Structure of the low-CO state of MoFe protein generated under turnover conditions (PDB 4TKV, 1.5 Å resolution). (B) The high-CO state of MoFe-Protein with a mCO bridging Fe2 and Fe6 and a tCO at Fe6 (PDB 7JRF, 1.33 Å resolution). (C) The low-CO state of VFe protein (PDB 7ADR, 1.0 Å resolution). (D) The high-CO state of VFe-protein with analogous mCO and tCO ligands (PDB 7AIZ, 1.05 Å resolution).

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and do not require any distortions of the site that would induce geometric strain. The reversible removal of sulfide S2B is unusual, but flexibility of iron-sulfur clusters is not unprecedented, with the redox-dependent rearrangements of the nearby P-cluster as the most prominent example.31 Considering nitrogenase cofactors as dinuclear binding sites for substrates represents a paradigm shift that has the potential to integrate a multitude of experimental data. Importantly, all ligand binding events in the cluster should occur at the Fe2-Fe6 edge, highlighting the role of the fully conserved histidine and glutamine residues situated above this edge, possibly including the formation of hydrides for storing electrons in the early stages of the catalytic cycle. Spectroscopic investigations of the interaction of nitrogenase with CO not only revealed the association of the ligand, but also showed that in addition to a ‘low-CO’ state with a single molecule bound, a form of the enzyme can be generated that holds two CO molecules in a ‘high-CO’ state.57 Other than the low-CO state, the structural characterization of a high-CO state was not possible with enzyme crystallized from turnover conditions. Instead, it required crystals of nitrogenase in the low-CO state to additionally be pressurized with CO gas before being flash-frozen in liquid N2 for diffraction data collection. Structures of MoFe-protein (Fig. 9B) and VFe-protein (Fig. 9D) in the high-CO state then showed identical binding modes.58,59 In addition to the bridging CO at Fe2 and Fe6 of the respective cofactor, the second CO molecule, designated tCO, bound terminally to Fe6 with a bond distance of 1.9 Å. While the tCO ligand was not bound at full occupancy in either structure, the analyses showed that the simultaneous binding of a bridging and a terminal ligand to the cluster can be accommodated and that only at Fe6 the active site cavity is sufficiently spacious to hold a terminal diatomic ligand. Experimental data to date has identified two modes of ligand binding to the nitrogenase cofactors, and in discussing the mechanistic implications of this finding the two positions in the following will be designated the ‘m-site’ for bridging binding at Fe2 and Fe6 and the ‘t-site’ for a terminal ligand to Fe6 (Fig. 10).

2.12.2.3 2.12.2.3.1

CO reduction by V-dependent nitrogenase Requirements for binding different substrates

The definition of two distinct binding sites at the open Fe2-Fe6 edge of the nitrogenase cofactors is supported by the observed binding of two CO in the high-CO state of MoFe and VFe proteins. Nevertheless, CO is not generally considered a physiological substrate of the enzyme, with reports on a secondary metabolism in A. vinelandii based on VFe-protein-mediated CO reduction remaining controversial.60 The reductions of CO and N2 show at least two further important differences that are mechanistically relevant. First, N2 reduction proceeds to the fully reduced products in the form of two NHþ 4 ions, while the equivalent CH4 is only a minor product of CO reduction by V-nitrogenase, amounting for less than 1% of product compared to 93% of the bulk product ethylene.61 CO reduction thus not only is incomplete, but it also involves a C-C coupling step. Second, although the bond energy of CO at 891 kJ mol 1 is only slightly lower than the one of N2 (946 kJ mol 1), only N2 reduction requires the enzyme to be charged to the E4 state for the reductive elimination of H2 as outlined above. CO binds to the enzyme in the E2 state, and although its reduction does not seem to be a favored process even in V-nitrogenase, the enzyme is able to activate the inert gas without requiring a super-reduced E*4 state.

2.12.2.3.2

Provision of electrons and protons in nitrogenase cofactors

The transfer of electrons from the Fe protein to the dinitrogenase is a strictly sequential process, in which ATP hydrolysis likely acts to lower the effective midpoint potential of an electron that is transferred from P-cluster to the FeMo cofactor. This occurs four times to reach the E4 state, where reductive elimination of H2 then provides an additional potential boost to bind and reduce N2 from the reactive E*4 state. Binding of CO, however, is already possible at E2, which is reflected in a non-competitive inhibition of N2 reduction by CO. In general, the transfer of a proton is suggested to accompany each individual reduction step, so that theoretical approaches frequently assumed an actual proton transfer to the cofactor with each electron transfer. However, considering the protein environment of the metal cluster and the rearrangements outlined by structural data, the early reduction steps can be put into a model that accounts for protons and electron within the framework of available experimental data. The steps outlined are discussed based on FeMo-cofactor but are readily applicable to all three isoenzymes. In E1, protonation of a m2-sulfide was suggested, placing the focus on potential proton sources. Several proton pathways have been suggested for nitrogenases, and in the highly conserved environment of the cofactors they terminate in two separate places: The

Fig. 10 Ligand binding sites at the nitrogenase cofactors. Sulfide S2B is labile in all three isoforms of the cofactor and can dissociate to reveal the m-site for a ligand bridging Fe2 and Fe6 (green). We propose that proton supply to the m-site is provided from the N32 atom of the active site histidine. In addition, the high-CO state structures of MoFe and VFe protein revealed a t-site for the terminal binding of a second ligand (blue). Here, we propose a second proton supply via the 30 -OH group of the homocitrate ligand.

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Scheme 2 The initial steps of cofactor activation. In the resting state E0, the protonated H180 forms a hydrogen bond to S2B, Q176 points away from the active site. Upon reduction to E1, Fe6 is reduced and the proton of H180 is transferred to S2B, leading to reprotonation of Nd1 of H180. With the subsequent electron transfer to E2, a terminal hydride is formed at Fe6 and S2B becomes a dangling thiol. The tH at Fe6 then migrates into a bridging position at Fe2 and Fe6, S2B is released as HS-, and Q176 rotates towards H180, simultaneously blocking proton access to the bound hydride and opening a holding site for S2B.

N32 atom of the histidine residue (H195D in MoFe, H180D in VFe and FeFe) and the 30 -OH group of homocitrate. In the resting state, the histidine is in H-bonding distance to sulfide S2B (Scheme 2, (1)), but the formation of a terminal hydride in the t-site at Fe6 in E2 more likely draws a proton from homocitrate. Theoretical studies indicate that the protonation of S2B might make the bridging ligand more labile (Scheme 2, (2)), possibly inducing its release from one of the cofactor iron ions. At the same time, the complete dissociation of S2B that we find in the turnover state is mostly not observed. To reconcile these findings, we have suggested that in E2 a terminal hydride is initially formed at the t-site (Scheme 2, (3)), but then migrates into a more stable bridging position at the m-site, leading to the complete dissociation of S2B as HS. As the sulfhydryl ion moves to the holding site 7 Å away, the glutamine side chain rotates in and forms a short hydrogen bond to the histidine that requires the N32 atom of the latter to be protonated. This conformation prevents proton transfer from histidine to a bound bridging hydride and therefore contributes crucially to extending the lifetime of the intermediate (Scheme 2, (4)). In the next step, reduction would take place at E3, but this step can be challenged by CO binding. As for the hydrides, the initial point of contact for CO will be Fe6, and from here the ligand can migrate into the m-site, resulting in the low-CO state that has been structurally characterized. Most notably, both CO and hydride are strong-field ligands with a very similar bond distance to Fe, and here both follow the same, recurring pattern of cluster reductiondbinding to the t-sitedmigration to the m-site. This sequence also explains why CO binding inhibits the reduction of all substrates (that bind to the m-site) except for protons, where unproductive H2 release can uniquely occur by protonation of a terminal hydride in the t-site.

2.12.2.3.3

CO-bound structures are dead-end adducts

Forming the low-CO state of nitrogenase requires turnover conditions, which is explained by the requirement to reach E2 to release S2B and accommodate a CO ligand. From here, however, the high-CO state was reached by pressurization of CO-bound crystals, i.e., without further provision of electrons. While the m- and t-binding modes of CO are apt models for the position of two hydrides, they are not suggested to represent actual intermediate states of CO reduction by V-nitrogenase: Starting from state E2 with hydride at the m-site, a t-CO binding would prevent the reorientation of the glutamine and leave the m-H less protected, so that it is more likely to be lost through protonation. We suggest that this has happened in the low-CO structures, implying that here the enzyme has returned to the electronic level of E0, but with bound CO as an inhibitor in place of S2B. Accordingly, the removal of CO not only requires an exchange of the headspace, but also turnover conditions. Pressurization of the low-CO state with CO then forms the high-CO state but electronically the system remains at E0. As in most instances, semi-stable intermediates are less likely to be caught in crystal structures than stable off-pathway adducts. However, the information gained from the CO-bound nitrogenase cofactors is that two binding sites are present that are suitable for CO and hydrides, but also for other substrates and intermediates, and from this, a mechanistic proposal arises.

2.12.2.3.4

CO is activated by insertion of a hydride

The difference between the formation of dead-end CO adducts and the reductive catalysis observed for VFe protein is that to initiate catalysis, the reducing equivalents must not be lost. If CO binds to the E2 state, its reduction can only be triggered if it inserts into the hydride bound to the m-site (Fig. 11). The resulting 2-electron reduction leads to a bound formyl intermediate where the CO triple bond is broken and the formal E2 state is preserved. Rather than ending in an inhibited state, the formyl adduct can react further with the enzyme, cycling through the same recurring steps. The insertion of CO is followed by another round of reductions, i.e., the sequential transfer of two electrons from Fe protein via the P-cluster. With it, two protons are transferred from the two sites at the histidine and the homocitrate. While the latter is required for the formation of another t-H, the former binds to histidine and forms a hydrogen bond to the bound formyl group. The t-H then once again inserts into the bound intermediate (formally a hydrogenation) to reach a methanol adduct. Following the same steps for another cycle then leads to water release, leaving a bound methyl group at the cluster. As the product profile of the reaction shows, this intermediate is not released from the enzyme, but the active site is now primed for the second CO molecule that will initially bind to Fe6, attack the bound methyl group, and form a CeC bond to yield an acetyl intermediate that then follows the exact same steps of reduction and hydrogenation as the C1 species in the first

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Fig. 11 Steps of CO reduction by V-nitrogenase. (A) The enzyme is activated to the E2 state as described (Scheme 2), when CO can bind terminally to Fe6. The reduction of CO is initiated if the ligand inserts into the bound mH. (B) In a repeating sequence of events, electron transfer from Feprotein leads to reduction of Fe6 and the subsequent formation of a tH that then inserts into the bound intermediate. Oxygen interacts with H180 and is released as water after two [H] transfers to yield bound methane. (C) Methane does not readily dissociate, but a second CO molecule can bind to the t-site. Its insertion into bound methane represents the C-C coupling step. The C2-intermediate then is reduced stepwise, in full analogy to (B). (D) The final reduction steps lead to bound ethane, and a chain elongation is straightforward if another CO molecule is bound here. Alternatively, the C2product ethane now can undergo a b-hydride elimination to yield ethylene, the main product.

half of the reaction sequence. After the transfer of eight electrons and the release of two water molecules, the enzyme remains in a formal E2 state with a bound ethyl group. From here it is easy to envisage that the next step again could consist of CO binding to Fe6, followed by insertion into the ethyl group and a sequence of reduction/hydrogenation events that would eventually lead to a bound propyl group. VFe protein was indeed found to produce 2.5% of propane and 0.2% of propylene, so the formation of longer-chain hydrocarbons can take place in the enzyme but is strongly disfavored over the C2 product, likely due to steric restrictions within the active site cavity.

2.12.2.3.5

Continuous electron and proton supply in three phases

The multi-electron reduction of CO remains a complex process, but the outlined series of events in Section 2.12.2.3.4 implies that the enzyme fundamentally functions by cycling through three distinct phases. A reduction step (i) technically occurs as two sequential, proton-coupled electron transfers, but in summary leads to (ii) the formation of a t-H at Fe6 and the reprotonation of the active site histidine. It is followed by the migratory insertion of the hydride into the bound intermediate (iii), at which point the proton bound at the histidine is also transferred to the substrate. For a bound CO, the reduction steps are symmetric, with electrons

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alternatingly transferred to C and O, so that water is released after the transfer of four and eight electrons, respectively, following CO binding to the E2 state. To reach an odd E-state an electron is required to reduce Fe6, and a corresponding Hþ re-protonates the N32 atom of the histidine. The subsequent even E-state then leads to hydride formation at the t-site, with a proton provided by homocitrate. With the hydride inserting into the intermediate bound at the m-site the sequence is completed, and the system is reset for the next 2-electron cycle.

2.12.2.3.6

Product release

Although the reduction of N2 by nitrogenases leads to ammonium as a sole product, CO reduction by VFe protein only yields a minor fraction of methane or ethane but rather prefers the unsaturated ethylene.61 The suggested mechanism for CO reduction involves bound methane and ethane after the transfer of four and eight electrons, respectively, so that the s-alkyl compounds are formed, but the alkyl group is not released.59 Methyl complexes of transition metals are typically quite stable, and their most common reactivity is the insertion of a variety of metal-ligated groups into the MeC bond. The stability of such complexes decreases rapidly for any larger alkyl chain, as the olefin can be released by b-hydride elimination. This same chemistry is likely at play in COreducing nitrogenases, where the bound methyl group after transfer of four electrons is more prone to undergo a migratory insertion of a further t-CO at Fe6 than its release. Once an ethyl group is formed, however, b-hydride elimination is possible and favored, releasing ethylene and leaving the enzyme with a m-H in the bridging position, corresponding to the E2 state of the LT scheme. As the substrate spectrum of CO reduction shows, there is a small propensity for the insertion of a third CO instead of a b-hydride elimination and ethylene release, and this is more pronounced when studying the reactivity of isolated cofactors. Nevertheless, the finding that ethylene appears as a main product can be traced back to fundamental principles of metalloorganic reactivity. CO reduction differs from the one of N2, but the high stability of the transition metal complexes of the fully reduced compounds is noteworthy and should be relevant in either case.

2.12.2.4

Summary and conclusion

Nitrogenases are arguably nature’s most potent catalysts for multi-electron reductions of inert, often gaseous substrates. However, their natural substrate is the N2 molecule that stands out as an even more challenging catalytic task that others, including CO. However, although rates, affinities, and product spectrum for CO and N2 reduction differ substantially, the basic mechanistic steps outlined above are fully compatible with the data available for the two reactions. CO reduction holds many clues for the mechanism of nitrogen fixation, but a key difference is that the N2 molecule is a much weaker metal ligand than CO and will not show any reactivity with the enzyme in the E2 state, even if a reactive hydride is already bound. Parallels of the two reduction reactions must be worked out with caution and many details of N2 reduction remain hypothetical to date, but recent years have seen significant progress in understanding in particular the initial steps of enzyme activation and N2 binding that are most critical for this reaction.

2.12.2.4.1

N2 binds to the E4 state

CO and N2 are isoelectronic stable gases, but their reductive conversion by nitrogenases highlights a series of important differences, including the inability of MoFe protein to catalytically reduce CO. Mechanistically, a crucial difference is that N2 activation requires the enzyme to reach the E4 state of the LT scheme, while CO can be bound and reduced already at E2. This reflects the fact that CO readily forms various terminal or bridging transition metal complexes, while binding of N2 to metal centers is known but typically requires the transition metal to be in a far more reduced state. As outlined above, the reduction of nitrogenase according to the LT scheme most likely leads to the release of sulfide S2B and the formation of a bridging m-H at Fe2 and Fe6 (Scheme 3, (1)). Proceeding along the kinetic scheme in the absence of CO then leads to the diamagnetic E3 state where Fe6 is reduced from Pcluster (Scheme 3, (2)), followed by a fourth reduction step that generates the highly reactive E4 state in which the stage is set for the reductive elimination of N2 (Scheme 3, (3)). A m-H and a t-H in this state may re-arrange to form a Fe2(m-H)2 diamond core structure for increased stability that then also opens the t-site for ligand binding (Scheme 3, (4)). Note that model compounds with this very arrangement have been reported by Rittle and Peters that bind N2 with high affinity upon reduction.90 Lowe and Thorneley have reported N2 binding already to the E3 state, but from here the reduction of the substrate was not initiated before another electron was transferred to reach E4, and the significance of E3 as a state able to bind N2 remains under debate.

2.12.2.4.2

Reductive elimination of H2 is linked to N2 binding

Reaching the E4 state is an obvious effort for the enzyme that takes its toll in the observed, unwanted release of H2 if a hydride formed in E2 or E4 is accidentally protonated (Scheme 3, (5)). In E4, the presence of two hydrides then allows for the reductive elimination of H2, and the considerations made above strongly suggest structures for the intermediates involved. This leaves the enzyme in the highly reactive E*4 state, but this intermediate can only be of productive use if the substrate N2 is within reach and can be attacked during its presumably very short lifetime (Scheme 3, (6)). To coordinate the sequence of events at this critical point of nitrogenase catalysis, it therefore is likely that the reductive elimination is set up to be triggered by the presence ordideallydbinding of N2 to or near the active site, leading immediately to the cleavage of the triple bond and reduction to a diazene-level intermediate (Scheme 3, (7)). The proposed mechanism for CO reduction by vanadium-dependent nitrogenase implies that the key elementary step occurring at the cofactor is the insertion of the terminal hydride at Fe6 into an intermediate bound terminally to Fe2 or bridging to Fe2 and Fe6. The reductive elimination in this sense is then the insertion of the t-H into the m-

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Scheme 3 The critical E4 state and N2 activation. (A) Activation steps from E2 to E4. In the third reduction step of the LT scheme, the E2 state with bound m-H (2) is reduced at Fe6, leading to E3 (2). The following reduction to E4 again leads to the formation of a t-H at Fe6 (3) that can rearrange to a Fe2(m-H)2 core, possibly triggered by (or causing) N2 binding to Fe6. (B) Reductive elimination of H2 from the diamond core structure (5) leaves the enzyme in a unique, 2-electron reduced state, E*4 (6) that binds nitrogen and immediately reduces it to a diazene-level intermediate (7). The binding mode of N2 in (7) is hypothetical.

H, in line with calculations by Raugei and Hoffman. We suggest that all substrates first reach Fe6, and accordingly the approximation or binding of N2 to Fe6 may be the trigger to eliminate H2, leaving the substrate precisely in place to be activated by the resulting E*4.59 In its quest for new nitrogen-fixing catalysts, model chemistry has largely focused on mononuclear systems, leading to several breakthroughs employing either Mo or Fe. However, a survey of non-catalytic models inspired by the functionality of nitrogenases reveals a series of compounds that gain astounding actuality when combined with the concept of hydride formation. The first isolable, diiron-hydride complexes with a Fe2(m-H)2 core were reported by Holland in 2003 and were based on sterically demanding b-diketiminate ligands that assembled a Fe2(m-H)2 diamond core very similar to the proposed E4 state of nitrogenase (Fig. 12 top). Compounds of this type were frequently found to reductively eliminate H2 upon binding of a ligand and also insert hydrides across multiple bonds of a substrate, i.e., the exact type of reactivity suggested for the nitrogenases. Building on the concepts established with Holland’s models, Peters and Rittle reported a diiron compounds with a Fe2(m-H)2 core that upon reduction to a mixed-valent Fe(II)-Fe(I) state would show a 106-fold enhanced affinity to bind N2 terminally to the metal ions (Fig. 12 bottom).62 These complexes would not eliminate H2 or turn over N2, but the structural analogy to the Fe2-Fe6 edge of the enzyme cofactors is quite remarkable.

Fig. 12 Diiron complexes of Holland (top) and Peters (bottom) showing the formation of a bis(m-hydrido) core capable of adding a dinitrogen ligand to one of the iron centers.

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Fig. 13

319

Structure of [MoIII(N2)(HIPTN3N)] (1).

2.12.3

Synthetic nitrogen fixation (by T. A. Engesser and F. Tuczek)

In the following sections the known catalysts for the conversion of dinitrogen to ammonia in homogeneous solution are reviewed and an overview is given in the Tables 2–4. Although most of these systems are based on mono- and dinuclear transition metal complexes whose structures largely differ from the active site of nitrogenase, the FeMoco, they mediate the same reaction and face similar mechanistic challenges as the enzyme deriving from the extreme inertness of the dinitrogen molecule and thus constitute functional models of nitrogenase. As the first breakthroughs in the area of synthetic nitrogen fixation, both in terms of stoichiometric and catalytic N2-to-NH3 conversion, have been achieved with molybdenum complexes, these systems are reviewed first. The second key element in this research area is iron, due to the fact that it is the main constituent of the FeMoco. Correspondingly, ammonia synthesis from N2 catalyzed by iron-based catalysts is presented subsequently. Both in the molybdenum and in the iron section, mono- and dinuclear systems are treated separately. Afterwards, other transition metal complexes that are catalytically active toward the N2-to-NH3 conversion in homogeneous solution are considered, and the general implications of these studies on the mechanism of N2 reduction in homogeneous solution and in the enzyme are discussed.

2.12.3.1

Mononuclear molybdenum systems

2.12.3.1.1

The Schrock catalyst

Scheme 4

Structural formula of the Schrock catalyst 1 and the derived catalytic cycle. Bold: experimentally found and characterized species.64

In 2003 Yandulov and Schrock presented the first catalytic model system of nitrogenase proceeding through a series of well-defined intermediates.6 Using 48 eq.s [LutH](BArF4) (Lut ¼ 2,6-dimethylpyridine, BArF4 ¼ [(3,5-(CF3)2C6H3)4B]) as a proton source and as reductant their [MoIII(N2)(HIPTN3N)] complex (1; 36 eq.s of decamethylchromocene (CrCp*2) HIPT ¼ hexaisopropylterphenyl, Fig. 13) was shown to mediate the conversion of N2 to NH3 with a TON of 4 (7.56 equivalents of NH3 generated).6,63 Detailed information regarding the reaction course was obtained from isolation and, respectively, characterization of a series of intermediates by means of single crystal structure analysis, cyclic voltammetry and 15N NMR spectroscopy. Some of these intermediates were found to act as catalysts as well.37,63 Based on these results, a mechanism of the catalytic cycle was derived (Scheme 4).64

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Fig. 14

Free energy profile of the Schrock cycle with [LutH]BArF4 and CrCp2* as protonating and reducing agent respectively.69

With a well-characterized, working example for a small-molecule model of nitrogenase being available, it appeared of interest to obtain an energy profile of this system. In 2005, our group presented the first comprehensive theoretical approach to this problem, based on isodesmic calculations of free reaction enthalpies for all protonation and reduction steps. To this end, the [Mo(HIPTN3N)] catalyst (with the HIPT substituents being replaced by H-atoms) as well as the proton source (lutidinium) and the reductant (decamethylchromocene) were treated by DFT (B3LYP and LANL2DT/TZVP including a solvent correction).65 From these calculations three important conclusions could be drawn: (1) Protonations and reductions proceed in a strictly alternating fashion; i.e., two sequential protonation or reduction steps are always energetically unfavorable; (2) the energetically most demanding (i.e., most endergonic) step is the first protonation of the N2-complex: and (iii) the most exergonic step is the cleavage of the NeN bond, proceeding at the level of the NNH3 (hydrazidium-) complex. Notably, the described calculations postulated a direct protonation of coordinated N2, leading to the diazenido() intermediate. On the other hand, there was experimental evidence that this reaction proceeds differently. Based on calculations of the full catalyst, Reiher and coworkers identified an alternative pathway, involving initial protonation of one of the amide nitrogen atoms.66 This avoids the energetically unfavorable first protonation of the dinitrogen ligand and enables a more favorable reaction path. Furthermore, a six-coordinate intermediate was shown to be involved in the final step of the cycle, the NH3-N2 ligand exchange, and characterized computationally.67 These results are in agreement with the experimental results and contributed to a deeper understanding of the reactivity of the catalyst.68 Exploiting the advances in computational power and efficiency several groups set out to improve the model chemistry in terms of system size, basis set size and solvation treatment.66,69–71 In particular, it became evident that, in order to account for all steric and electronic effects present in the system, the full catalyst needs to be modeled.72 This way, several important advancements could be made over the years. Our group repeated the theoretical treatment of the Schrock cycle in 2015, employing the same methodology as 2005 (see above), but employing a full model of the catalyst.69 Notably, the derived energy profile was almost identical to that obtained with the truncated model (Fig. 14), which can be ascribed to the orthogonality of the phenyl rings bearing the tris(isopropyl)phenyl substituents to the amide lone pairs of the HIPN3N ligand which (along with the corresponding s-bonds) determine metal-ligand bonding in 1. This actually renders replacement of the HIPT substituents by H-atoms a good approximation. The theoretically derived energy profile on the basis of the free reaction enthalpy is given in Fig. 1. Notably, the Mo oxidation number varies between þ III to þ VI over the cycle and the energy consumption required per mole of N2 is calculated as approx. 190 kcal/mol (see below).65 Regarding the reduction steps, excellent agreement with the available experimental data and the theoretical results is obtained. For all protonation steps, on the other hand, the predicted free reaction enthalpies are consistently too negative by about 10 kcal/mol (Table 1). We ascribe this deficiency to the formation of ion pairs between the lutidinium cation and the BArF4 anion, the dissociation of which requires energy (which is not accounted for by the calculation). A further factor may be the lack of explicit solvent modeling, which was addressed by Magistrato and coworkers who determined that this concerns mostly the protonating agent and its conjugate base [LutH]þ/Lut.70 Importantly, these authors showed that providing the acid/base pair with a first sphere of explicit solvent molecules (n-heptane) reduces the deviation from experiment to less than 5 kcal/mol.

Biological and synthetic nitrogen fixation Table 1

321

Gibbs Free Enthalpies of the [Mo(HIPTN3N)] catalytic cycle of which experimental values are available. All values are in kcal/mol.

Reaction

Thimm et al. 97 expt.

12.6 [Mo-NNH] þ [LutH]þ / [Mo-NNH2]þ þ Lut [Mo-NNH2]þ þ CrCp*2 / [Mo-NNH2] þ [CrCp*2]þ 5.5 [Mo-N] þ [LutH]þ / [Mo-NH]þ þ Lut 13.1 [Mo-NH]þ þ CrCp*2 / [Mo-NH] þ [CrCp*2]þ [Mo-NH3]þ þ CrCp*2 / [Mo-NH3] þ [CrCp*2]þ [Mo-NH3] þ N2 / [Mo-N2] þ NH3

7.2 0.5 0.9

0.28 4 to 7 þ1.35 5 to 6 0 to þ1 1.4

Taken from W. Thimm, C. Gradert, H. Broda, F. Wennmohs, F. Neese, F. Tuczek, Inorg. Chem. 2015, 54, 9248.

2.12.3.1.2

The Chatt cycle

A second mechanistic scenario of dinitrogen reduction based on mononuclear molybdenum complexes is the Chatt cycle (Scheme 5). In the 1970’s it was found by the groups of Chatt and Hidai that octahedral molybdenum(0) and tungsten(0) dinitrogen complexes of the type [M(N2)2(diphos)2] (M ¼ Mo, W; diphos ¼ depe, dppe) mediate the stepwise protonation and reduction of coordinated dinitrogen, ultimately leading to the formation of NH3.73 Starting from the bis(dinitrogen) complexes, diazenido() (N2H), hydrazido(2-) (N2H2) and hydrazidium (N2H3) intermediates were generated through addition of mineral acids (HBF4, HCl, H2SO4), whereby one of the N2 ligands is exchanged against the conjugate base of the employed acid. These systems were studied by infrared, Raman and UV/Vis absorption spectroscopies and DFT calculations.74 According to their NeN stretching frequencies, the parent Mo(0) and W(0) dinitrogen complexes are moderately activated.75 Based on normal coordinate analysis, a reduction of NeN bond order from  3 to  1 upon stepwise protonation from N2 to NNH3 was determined,76 whereby the metal-N bond order increases up to a triple bond. This acts to prevent loss of the partly reduced intermediate from the metal center and induces cleavage of the NeN bond. The kinetics of the latter process was investigated based on protonation of the fivecoordinate complexes [M(NNC5H10)(depe)2] (M ¼ Mo,W) in nitrile solvents, generating the corresponding nitrido complexes and piperidine.77 Although strongly exothermic, this process is subject to appreaciable activation barriers which have been determined experimentally and theoretically for the dialkylhydrazido complex [Mo(NNC5H10)(depe)2] and analyzed using DFT.78 The second half of the Chatt cycle commences with protonation and reduction of the Mo(IV) nitrido complex, leading to the Mo(II) amido complex. This process has been investigated based on electro- and spectroelectrochemical studies of the Mo(IV)ethylimido complex [Mo(NEt)(CH3CN)(depe)2](OTf)2.78 One-electron reduction of this complex generates the Mo(III) intermediate [Mo(NEt)(CH3CN)(depe)2]OTf which is immediately protonated and further reduced to [MoII(HNEt)(CH3CN)(depe)2]OTf. These results support the central paradigm of alternating single reduction and protonation steps for the biomimetic conversion of dinitrogen to ammonia. However, while this scenario is strictly followed in the Schrock cycle (see above), the first part of the classic

Scheme 5

Chatt cycle. ([M] ¼ [M(P4)(X)]; M ¼ Mo or W, P4 ¼ tetraphosphine environment, X ¼ 6th additional ligand/donor).

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Fig. 15

pentaPod ligand and derived Mo(N2) pentaPod complex [Mo(N2)(P2MePP2Ph)] (2).

Chatt cycle exhibits deviations like a double protonation (i.e., from the dinitrogen complex to the hydrazido(2-) complex) and a double reduction (i.e., from the hydrazido(2-) intermediate to the nitrido complex), going along with NeN cleavage. After studying the elementary reactions of the Chatt cycle experimentally, it appeared of interest to also obtain an impression of the energetics of this cycle by computational methods, in analogy to the Schrock cycle. Again, the employed acid, the reductant and the intermediates of the ‘classic’ Chatt cycle were treated by DFT. In analogy to the Schrock cycle, decamethylchromocene was considered as a reductant; for the protonation reactions two acids were examined, HBF4/diethylether and lutidinium ([HLut]þ). The free reaction enthalpy changes of all protonation and reduction steps were calculated.79 The derived energy profile is very similar to that of the Schrock cycle, although certain differences exist. In contrast to the Schrock cycle, no catalytic action of the Chatt cycle toward the formation of NH3 from N2 could be achieved until recently. In an early report, a cyclic production of ammonia has been reported on the basis of this system, although with very small yields.80 From detailed investigations of the Chatt cycle, two major mechanistic problems could be identified, possibly accounting for this observation; i.e., (i) ligand exchange in trans-position to the dinitrogen ligand that gets protonated, and (ii) disproportionation at the level of Mo(I)-intermediates with one X-ligand (e.g., X ¼ halide) to Mo(II)X2 and Mo(0) species. In order to suppress these side reactions, it appeared meaningful to include the donor in trans-position of the bound dinitrogen into a polydentate ligand. Initially, tridentate and tripodal phosphine ligands were employed in combination with simple diphos ligands (dmpm, dppm) to create Mo(0) dinitrogen complexes with a P5 environment.81 However, the P-donor in trans-position to N2 proved to be labile in these systems as well, rendering a more robust attachment of this moiety to the metal center necessary. Ultimately, this problem could be solved by creating the pentaPod ligand (Fig. 15) which represents the fusion of a tripodal and a tridentate ligand. In order to enable a well-defined, stepwise coordination of this ligand to molybdenum centers, the tripod part was furnished with dimethylphosphine and the trident part with diphenylphosphine endgroups. This way, the corresponding ligand Ph 3 PMe 2 PP2 was first coordinated to Mo(III)X3 precursors (X ¼ Cl, Br, I), leading to facially k -coordinated Mo(III) complexes [MoX3(P2 Me Ph Me Ph PP2 )] which were subsequently reduced to the corresponding Mo(0) dinitrogen complex [Mo(N2)(P2 PP2 )] (2) supported by the k5-coordinated pentaPod ligand.82 Ph The [Mo(N2)(PMe 2 PP2 )] complex (2) was characterized by single crystal X-ray structure determination as well as by NMR and vibrational spectroscopy. Importantly, 2 was found to exhibit the lowest NeN stretching frequency of all Mo(0) dinitrogen complexes with phosphine ligands, reflecting the strong activation of the bound N2 ligand. In agreement with its unique properties, 2 was recently shown to be the first Chatt-type complex with a single coordination site catalytically converting N2 to ammonia.5 Employing 180 eq.s SmI2/H2O as reductant and proton source 26 equivalents of ammonia were produced from N2. The analogous Ph F F hydrazido(2-) complex [Mo(NNH2)(PMe 2 PP2 )](BAr )2 was generated by protonation with [H(OEt2)2](BAr 4) in ether and characterized by NMR and vibrational spectroscopy. Importantly, it was shown to be catalytically active as well. Along with the fact that the structure of 2 precludes dimerization this demonstrates that the corresponding catalytic cycle follows a mononuclear pathway. The implications of a PCET mechanism on this reactive scheme were considered. Besides 2, a number of classic Chatt complexes have been examined with regard to catalytic NH3 formation in the presence of SmI2/water.87 Among these, only [Mo(N2)2(PMePh2)4] exhibited a catalytic activity comparable to 2, but in view of the fact that this complex is prone to phosphine ligand exchange retention of the molybdenum tetraphosphine ligation during the entire cycle is doubtful. Employing more stable [Mo(N2)2(dppe)2], on the other hand, led to a drastic decrease of catalytic activity, presumably due to ligand exchange processes at the trans position of the protonated N2 ligand which are precluded in the single-site catalyst 2.

2.12.3.2

Dinuclear molybdenum systems

In 2011 Nishibayashi and coworkers published a dinitrogen-bridged dinuclear molybdenum complex containing a PNP pincer ligand which represented the first catalytic molybdenum system for nitrogen fixation since the discovery of the Schrock system.7 [{Mo(N2)2(PNP)}2(m-N2)] (3) (PNP ¼ 2,6-bis(di-tert-butyl-phosphinomethyl)pyridine) generated 23.2 equivalents of ammonia (11.6 equivalents/Mo) using 288 equivalents of [LutH]OTf as proton source and 216 equivalents of CoCp2 as reductant at room temperature (Scheme 6). The amount of ammonia was found to depend on the used equivalents of acid and reductant: The more [LutH]OTf (max. 216 equiv.) and CoCp2 (max. 288 equiv.) were applied the more ammonia could be detected. The formation of ammonia was always accompanied by the evolution of hydrogen whereby the H2:NH3 ratio increased with an increasing amount of acid and reductant. To optimize the catalytic reaction different electron and proton sources were examined. CrCp*2 as well as CoCp2 were found to be

Biological and synthetic nitrogen fixation

Scheme 6

323

Conversion of dinitrogen into ammonia using [LutH]OTf, CoCp2 and [{Mo(N2)2(PNP)}2(m-N2)] (3) as catalyst. P ¼ P(tBu)2.7

suitable candidates for the reduction of dinitrogen whereas CrCp2, possessing a lower reducing ability, was found to be ineffective. By variation of the proton source the necessity of employing an acid with a (weakly) coordinating conjugate base was evidenced. In particular, [LutH](BArF4), as employed in the Schrock system (see above), was not found to be efficient in the case of [{Mo(N2)2(PNP)}2(m-N2)] (3). Instead, use of [LutH]OTf led to excellent catalytic activities. To improve the performance of the catalyst different PNP ligands substituted in 4-position of the pyridine ring were synthesized and investigated regarding their influence on the turnover numbers of NH3 formation.88 Most of these groups are electron donating; due to the introduction of such a group in the 4-position of the pyridine ring of the ligand the electron density at the molybdenum center is enhanced and p-backdonation into empty p* orbitals of the dinitrogen ligand should increase. Consequently, the protonation steps in the catalytic cycle are accelerated, and the yield of ammonia is higher or comparable to the parent PNP ligand (R ¼ H). In the range of the new ligands that with the highly electron donating methoxy group leads to the highest yield of ammonia (34 equiv.). The introduction of ferrocenyl residues to the pyridine ring exhibits a comparable effect on the catalytic formation of ammonia,89 i.e., using 216 equiv. of CoCp2 and 288 equiv. of [LutH]OTf 39 equiv. of ammonia could be generated. In contrast to the PNP ligands with alkyl, aryl, or silyl substituents the reason for the increased catalytic activity of the ferrocenyl-substituted PNP ligands was not ascribed to the promotion of the protonation steps. Instead, these ligands were considered as facilitating the reduction steps due to a possible electron transfer from the iron to the molybdenum centers. As a consequence the turnover number as well as the turnover frequency were found to be higher than in case of PNP ligands with non-redox active substituents.83,88,89 Finally, the nature of the substituents was also found to influence the selectivity of the catalysts; i.e., the evolution of dihydrogen could be partially reduced using ligand backbones with electron donating groups.88,89 As nitrido complexes are known to be important intermediates in a distal pathway of N2 reduction (see above), two mononuclear five-coordinated nitrido complexes [Mo(N)Cl(PNP)] and [Mo(N)Cl(PNP)]OTf were synthesized and investigated for nitrogen fixation as well. In fact the nitrido complexes were found to also function as effective catalysts ([Mo(N)Cl(PNP)]: 6.6 equiv. NH3, 36 equiv. CoCp2 and 48 equiv. [LutH]OTf; [Mo(N)Cl(PNP)]OTf: 7.1 equiv. NH3, 36 equiv. CoCp2 and 48 equiv. [LutH]OTf).90 Supported by DFT calculations (see below) Nishibayashi and coworkers undertook first attempts to elucidate the reaction pathway of the N2-to-NH3 conversion in their dinuclear catalytic systems. Importantly, they determined that the first protonation of a dinitrogen ligand takes place at one of the terminal N2 ligands of a molybdenum subunit. The other molybdenum subunit can be considered as a (potentially dissociable) ligand that provides additional electron density to the active Mo(0) site.83,84,90 By adding 4 equiv. [H(OEt2)2]BF4 instead of [LutH]OTf in tetrahydrofuran to the dinuclear Mo(0) complex followed by addition of pyridine, the mononuclear hydrazido(2-) complex [MoF(PNP)(NNH2)(pyridine)]BF4 could be generated and characterized. This hydrazido(2-) complex, however, is not catalytically active.7 Consequently, the dinuclear structure was found to be important for the catalytic activity, supporting the hypothesis that the first step in the reaction pathway is the protonation of a dinitrogen ligand in the dinuclear complex (see above). Further reactions along a dinuclear pathway were inferred from mass spectrometric identification of the dinuclear ammine and nitrido complexes [Mo(NH3)(PNP)-N^N-Mo(N2)2(PNP)] and [Mo(^N)(OTf)(PNP)-N^N-Mo(N2)2(PNP)], respectively.83,84,90 Following publication of the first dinuclear molybdenum catalyst for nitrogen fixation in 2011 by Nishibayashi and coworkers (Scheme 6),7 theoretical investigations of the mechanism of its catalytic cycle were performed by Batista et al. on one hand and Yoshizawa et al. on the other and published almost simultaneously.85,90 Whereas the mechanism proposed by Yoshizawa et al. involved dissociation of the dinuclear catalyst after initial protonation, Batista and coworkers postulated a mechanism in which the catalyst remains intact throughout the entire cycle. In a later publication, however, Nishibayashi and Yoshizawa theoretically derived another mechanism in which the dinuclear complex never dissociates, in agreement with Batista’s original proposal.91 This mechanism is shown in Scheme 7. Notably, the authors calculated activation barriers (in brackets) along with the free energy changes associated with the elementary steps of the cycle. The first protonation of the dimeric complex proceeds at one of the terminal N2 ligands in agreement with the classic Chatt cycle; i.e., the N2 ligand in trans position is exchanged with the conjugate base of the employed acid, triflate. The resulting diazenido() intermediate 3-NNH-OTf opens the actual catalytic cycle and, after two alternating protonation/reduction steps and cleavage of the NeN bond, generates the nitrido complex 3-N-OTf along with one molecule of NH3. Subsequent alternating protonation and reduction steps lead to ammine complex 3-NH3-OTf which releases NH3 and rebinds N2, forming Mo(I)/Mo(0) dinitrogen complex 3-N-OTf which by protonation and reduction reforms 3-NNH-OTf, closing the cycle. Notably, the triflate anion remains bound to the ‘active’ Mo center during the entire cycle.

324

Biological and synthetic nitrogen fixation

Scheme 7 Free energy changes at 298 K and free energies of activation (in parentheses) for individual steps were calculated at the B3LYP-D3 level of theory (units in kcal/mol). NB represents that the corresponding reaction has np activation barrier.91

The next experimental step taken by Nishibayashi and coworkers toward optimization of their catalytic system involved replacement of the pyridine ring of the PNP pincer by a third phosphorus donor. The advantage of a phosphine as compared to a pyridine moiety is the lower Brønsted basicity; i.e., protonation of the central phosphine donor during the catalytic cycle is less probable than protonation of the nitrogen atom of the pyridine ring. Furthermore, the stability of electron-rich intermediates in the catalytic cycle should be increased by using p-accepting phosphine donors.92 However, formation of a dinitrogen-bridged dimolybdenum complex supported by PPP ligands, analogous to 3, could not be evidenced. Instead, the neutral molybdenum(IV) and cationic Mo(V) complexes [Mo(N)X(PPP)] (4) and [Mo(N)X(PPP)](BArF4) (5) (PPP ¼ PhP(C2H2P)2, P ¼ PtBu2; X ¼ Cl), respectively, were synthesized and investigated regarding their performance in nitrogen fixation (Fig. 16). The ability of nitrido complexes to catalyze the conversion of dinitrogen into ammonia had been demonstrated before, using the parent PNP ligand (see above). As expected, complex 4 and 5 were also found to be very effective catalysts. Using 540 equiv. CoCp2* instead of CoCp2 and 720 equiv. [ColH]OTf (Col]2,4,6-trimethylpyridine) instead of [LutH]OTf up to 63 equiv. ammonia (and no hydrazine) could be generated. Again, the formation of ammonia was accompanied by the evolution of dihydrogen, but in contrast to the PNP-based molybdenum catalysts (see above) lower amounts of dihydrogen were generated. Although these new nitrido complexes 4 and 5 exhibited an increased catalytic activity compared to earlier molybdenum catalysts the reaction pathway is unclear92 but in case of a very similar PPP system (4 with P ¼ PCy2 and X ¼ I, Fig. 16) and the catalytic formation of NTMS3 instead of NH3, a mechanism via an imido derivative was proposed by Mézailles et al.86 In 2017 Nishibayashi et al. published new molybdenum complexes supported by PCP pincer ligands which turned out to be even more active catalysts in synthetic nitrogen fixation than their predecessors.93 The combination of two phosphorus donor atoms and one N-heterocyclic carbene unit leads to dinuclear molybdenum complexes that are strongly activated due to the high electron

Biological and synthetic nitrogen fixation

325

Fig. 16 The nitrido complexes [Mo(N)X(PPP)] (X ¼ Cl (4)) and [Mo(N)Cl(PPP)](BArF4) (5) work as effective catalysts in nitrogen fixation. P ¼ PtBu2.92

donating properties of the carbene. Molybdenum dinitrogen complexes could be obtained based on benzimidazole- (6, 7, Fig. 17) and imidazole-PCP ligands (8, Fig. 17). Employing 6 as catalyst and cobaltocene, decamethylcobaltocene or decamethylchromocene as reductant 5.7 (CoCp2), 11.8 (CoCp*2) and 17.8 equiv. (CrCp*2) ammonia were generated. With 8 used as catalyst the formation of ammonia was not nearly as efficient as in case of 6 (with CoCp2, CoCp*2 and CrCp*2 1.4, 2.9 and 3.2 equiv. of ammonia, resp., were generated). Interestingly, complex 8 was able to generate 1.5 equiv. ammonia without a reducing agent.15 Moreover, the influence of the various proton sources was studied. To this end, larger amounts of both CrCp*2 (360 equiv.) and the respective acid (480 equiv.) were employed. Starting with [LutH]OTf as a proton source 79 equiv. of ammonia could be generated. This number drastically decreased to 19 equiv. if [PicH]OTf (Pic]2-methylpyridine) was used; employing [H(OEt2)2](BArF4) as a proton source only led to 15 equiv. of ammonia. The investigation on the influence of the proton source also included the utilization of [ColH]OTf which led to 61 equiv. of ammonia based on the catalyst.93 By further tweaking the reaction conditions the authors were able to generate even higher amounts of ammonia. If excessive amounts of [LutH]OTf (1920 equiv.) and CrCp*2 (1440 equiv.) were employed in the presence of 6, 200 equiv. of ammonia were formed. This result was even surpassed in the presence of 7 as catalyst; i.e., under the same reaction conditions 230 equiv. of ammonia were formed. The reason for the higher catalytic activity could be the presence of two additional methyl groups in the PCP pincer ligand, leading to an improved backdonating ability of molybdenum to the coordinated dinitrogen ligands. Time profiles of the catalytic formation using catalysts 6, 7 and [{Mo(N2)2(PNP)}2(m-N2)] (3) were recorded in order to compare the catalytic performance of the respective compounds. Initial values after 1 h revealed a turnover frequency of 53 h 1 for catalyst 7 and 42 h 1 for catalyst 6 in comparison to [{Mo(N2)2(PNP)}2(m-N2)] (3) which gives rise to a TOF of 17 h 1. These results prove the higher catalytic activity as well as a higher rate of ammonia formation of 7 as compared 6.93 The year 2017 witnessed an important breakthrough in the accessability of pincer-based molybdenum complexes in nitrogen fixation. As shown by Nishibayashi et al.,94 simple trihalogenido complexes of the PNP ligand can be employed as precatalysts to generate, along with metallocene reductants (CoCp*2, CrCp*2) and suitable protonating agents ([LutH]OTf, [ColH]OTf), significant amounts of ammonia from N2. Moreover, the formation of dihydrogen in this system was greatly reduced in comparison with earlier systems. Using 180 eq.s of CoCp*2 and 240 eq.s of [ColH]OTf, the highest yields (50 eq.s) were obtained with [MoI3(PNP)] (9). The yield could be further increased by increasing the amount of acid and reductant. Notably, employing the corresponding nitrido complex or the dinuclear, N2-bridged complex led to lower turnover numbers. Nevertheless, a central role of the nitrido complex could be evidenced by two stoichiometric experiments: (i) reaction of the [MoI3(PNP)] precursors (e.g., 9) with two eq.s of CoCp*2 in toluene under N2-atmosphere afforded the Mo(IV) nitrido complex [Mo(N)I(PNP)] (10), which implicates the presence of a N2-bridged Mo(I) complex that spontaneously cleaves the NeN bond; and (ii) the nitrido complex, along with 3 eq.s of CoCp*2 and 4 eq.s of [ColH]OTf generates 60% of NH3 (Scheme 8). These experiments led to a revised mechanistic scenario of Nishibayashi’s system not involving a dinuclear Mo(0) complex any more, but instead a dinuclear Mo(I)-PNP complex with one bridging N2 ligand and two iodo coligands which mediates spontaneous NeN cleavage. The corresponding mechanistic scenario was supported by DFT calculations (Scheme 9).91 Reduction of the precatalyst [MoI3(PNP)] (9) leads to trans-[MoI(N2)2(PNP)] (11). This intermediate undergoes NeN cleavage, leading to the nitrido complex [Mo(N)I(PNP)] (10) which is converted by three protonation/reduction steps to the Mo(I) ammine complex [MoI(NH3)(PNP)] (12). At this stage N2 is bound which initiates dimerization. Along with these processes, NH3 is released.

Fig. 17

Dinitrogen molybdenum complexes containing benzimidazole (left) and imidazole (right) PCP pincer ligands by Nishibayashi et al.93

326

Biological and synthetic nitrogen fixation

Scheme 8 ligand.95

Formation of a nitrido complex via direct cleavage of the bridged-dinitrogen ligand on a molybdenum complex bearing a PNP pincer

Scheme 9

Plausible reaction pathway via direct cleavage of the bridged dinitrogen ligand.

Biological and synthetic nitrogen fixation

327

A final increase in catalytic activity, leading to the most active small-molecule model system of nitrogenase in homogeneous solution to date, was established in 2019 when Nishibayashi and coworkers discovered that SmI2 in conjunction with water or alcohols can be employed as protonating/reducing reagent in their pincer-based systems to convert N2 to NH3.95 This way, up to 4350 equivalents of ammonia could be produced (based on the molybdenum catalyst), with a turnover frequency of around 117 per minute. The high reactivity was ascribed to a PCET mechanism enabled by the SmI2/water or SmI2/alcohol system. More recently, these PCET reagents were also applied to [MoCl3(BimPCP)], leading to 90–92 eq.s of NH3, depending on the actual conditions.97

2.12.3.3

Mononuclear iron systems

Due to the fact that dinitrogen binds to iron in the FeMoco of nitrogenase, a range of iron complexes have been synthesized and analyzed during the first two decades of this millennium that convert dinitrogen into ammonia.101 Later Peters and coworkers synthesized and characterized mononuclear iron complexes that are able to perform this reaction in a catalytic fashion, thus extending the range of catalytic nitrogenase models to this element.8,102–109 More recently, other additional mononuclear iron catalysts containing an anionic PNP ligand for synthetic nitrogen fixation have been synthesized by Nishibayashi et al.110 These systems are the subject of the following sections.

2.12.3.3.1

Peters’ systems

An overview of the iron complexes by Peters et al. is given in Table 3. Most of these systems are coordinated by tetradentate tripodal triphosphine ligands with a central donor atom (Si, B, and C) in a trigonal-pyramidal fashion (Fig. 18).102,107,111 The position trans to Si, B, or C is occupied by a dinitrogen ligand. In 2013 the complex [Fe(N2)(TPB)][Na(12-crown-4)2] (13; TPB ¼ tris(phosphine)borane, Fig. 6) was shown to be an effective catalyst for nitrogen fixation.8 Before using 13 as catalyst, its structure and properties had been investigated and characterized in detail.107,112,113 In particular, the iron-TPB unit was shown to bind different nitrogen species; i.e., N2, NH2, NH3, N2H4 or imido derivatives. The high activation of N2 mediated by 13 was further demonstrated by functionalization of the coordinated dinitrogen ligand at the Nb atom using silyl electrophiles.112 As a first step toward the conversion of dinitrogen to ammonia, the reaction of 13 with an excess of [H(OEt2)2](BArF4) was investigated at low temperature ( 78  C). Importantly, this reaction generated the ammine complex [Fe(NH3)(TPB)](BArF4) (30–35%), the substrate-free complex [Fe(TPB)](BArF4) (40–45%) and two minor paramagnetic iron species, demonstrating the protonation and reduction of the coordinated dinitrogen ligand. One of the paramagnetic species was assumed to be the hydrazido(2-) complex [Fe(^N-NH2)(TPB)](BArF4). In fact, this species could be generated selectively in a later study by adding an excess of [H(OEt2)2](BArF4) to the N2-complex and was characterized at low temperatures ( 136  C and  78  C, respectively) by EPR, ENDOR and EXAFS spectroscopy and theoretical calculations.104,108 This result demonstrated the stepwise protonation of the N2 ligand in the iron-TBP system.8

Fig. 18 Iron complexes for nitrogen fixation developed by Peters et al. and the corresponding maximal number of produced equivalents of NH38,102,103,104.

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Scheme 10

Catalytic reduction of dinitrogen into ammonia at 78  C using an iron catalyst.

To further explore the potential of the iron catalyst 13 in nitrogen fixation, different proton (HCl, HOTf, [H(OEt2)2](BArF4), [LutH][BArF4]) and electron sources (Na/Hg, KC8, CoCp*2, CrCp*2 and potassium) were applied in various solvents (THF, dimethoxy ethane, Et2O, toluene). Using 58 equiv. of KC8 and 48 equiv. of [H(OEt2)2](BArF4) in diethyl ether at  78  C a catalytic conversion of dinitrogen to ammonia yielding up to 8.5 equiv. NH3 per Fe could be achieved (Scheme 10).8 In contrast to the iron catalyst 13, complex 14 containing the SiPiPr3 ligand was only able to produce substoichiometric amounts of ammonia under the given reaction conditions (58 equiv. reductant, 48 equiv. acid).8 Consequently, in 2013, the catalytic generation of ammonia was considered to be an exclusive property of the iron complex 13 supported by the tetradentate ligand TPB. It is of significant interest to relate this model chemistry to the structure and reactivity of the iron molybdenum cofactor in nitrogenase. While the central atom of the iron molybdenum cofactor could be identified as a carbide (C4) anion,16,17 the specific role of this interstitial carbon atom has not finally been resolved. In order to obtain further information on this issue Peters et al. designed and synthesized an iron complex supported by a tetradentate triphosphine ligand with a central carbon atom, [Fe0(N2)(CSiPPh3)][K(benzo-15-crown-5)2] (15; cf. Fig. 18).102,104,114 The interactions between the iron center and the carbon atom were studied in detail. In particular, the FeeCligand bond was investigated as a function of the nitrogen intermediate coordinating to the metal center and the oxidation state of iron.8,102,114 Focusing on the latter aspect Peters et al. synthesized three iron-carbonyl complexes [FeII(CO)(CSiPPh3)]þ, [FeI(CO)(CSiPPh3)] and [Fe0(CO)(CSiPPh3)] which are more stable than corresponding dinitrogen complexes.114 Notably, upon reduction from iron(II) to iron(0) the Fe-C bond length increases from 2.138(2) Å in [FeII(CO)(CSiPPh3)]þ over 2.236 Å in [(CSiPPh3)FeI(CO)] to 2.303 Å in [(CSiPPh3)Fe0(CO)]. These experimental results were supported by theoretical calculations. Thereby, it was found that the s-bond interactions between the metal center (iron (0)) and the carbon atom exhibit a distinctly ionic character with the negative charge being located at the carbon atom whereas the character of the FeeSi s-bond in [Fe(N2)(SiPiPr3)] (14) is clearly covalent.114 The relevance of these findings to the iron molybdenum cofactor of nitrogenase becomes evident from the fact that several theoretical studies of the FeMoco115,116 identified iron as the binding site of dinitrogen trans to the central (carbon) atom and predicted an increase of the iron-carbon bond upon N2 bonding. Based on their data Peters and coworkers suggested that during the catalytic cycle of nitrogenase, especially during reduction of the system, the FeeC bond is elongated. As a consequence one iron atom slips closer into the plane generated by the three sulfide atoms (Scheme 11).114,117 The other belt iron atoms can stabilize the negative carbon during the cycle due to their positive charge by shortening the iron-carbon distance.116 Although the iron complexes supported by the tetradentate ligand CSiPPh3 provided significant insight into the possible interaction of the iron center and the central carbon atom of the FeMoco, the derived complex 15 was not found to catalyze the conversion of dinitrogen into ammonia.102 In order to establish a catalytically active iron complex bearing a carbon atom trans to the dinitrogen ligand complex [Fe(N2)(CPiPr3)][K(Et2O)0.5] (16) was synthesized (Fig. 18). Delocalization of the negative charge located at the central carbon atom of the ligand CPiPr3 was achieved by incorporation of phenylene groups.102 In analogy to the iron complexes supported by TPB or SiPiPr3 ligands the series [FeII(N2)(CPiPr3)]þ, [FeI(N2)(CPiPr3)] and [Fe0(N2)(CPiPr3)] (16) was prepared. Again, the FeeC bond length increases in the three different oxidation states of the iron center from iron(II) (2.081(3) Å) over iron(I) (2.152(3) Å) to iron (0) (2.1646(17) Å). In contrast, the FeeSi bond length in the triad [FeII(N2)(SiPiPr3)]þ (2.298(7) Å), [FeI(N2)(SiPiPr3)] (2.2713(6) Å) and [Fe0(N2)(SiPiPr3)] (14) (2.2526(9) Å) decreases upon reduction of the metal center. This difference is due to the electropositive character of silicon and electronegative character of carbon. The flexibility of the FeeX bond (X ¼ B, Si, C) bond increases in the sequence Si < C < B.102 Just like the FeeC bonding interaction in the complex [Fe0(CO)(CSiPPh3)] exhibiting a distinctly ionic character the s-bond in the complex [Fe0(N2)(CPiPr3)] (16) is strongly polarized. This ensures the flexibility of the FeeC bond which is required for nitrogen fixation.102 Accordingly, 16 was shown to catalyze the conversion of N2 to ammonia; i.e., addition of 38 equiv. of [H(OEt2)2](BArF4) and 40 equiv. of KC8 to this complex at  78  C in Et2O gave 4.6  0.8 equiv. of NH3. Investigation of the solution after addition of acid and reductant revealed that 70% of 16 are converted into the hydrido species [FeH(N2)(CPiPr3)] which is catalytically inactive.102 Notably, no

Scheme 11 During electron and substrate binding the iron-carbide bond length increases and the respective iron slips closer to the plane of three sulfide atoms.

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free ligand was observed in the reaction solution after the catalytic run indicating that the CPiPr3 binds strongly to the metal center. This behavior of the tetradentate ligands CPiPr3 and TPB was particularly important regarding the optimization of the catalytic reduction to ammonia.8,102 Besides the mentioned iron complexes 15 and 16 supported by carbon-centered tripod ligands Peters et al. also investigated twocoordinate iron complexes containing cyclic alkyl amino carbene ligands (Fig. 18). The motivation behind this study was the idea that due to the polarizability of the CAAC ligand, compared to other N-heterocyclic carbene ligands, binding and functionalization of dinitrogen should be possible.103 In fact, the iron(0) complex [(CAAC)2Fe] (17) was able to bind dinitrogen at temperatures below  80  C which could be monitored by UV/vis spectroscopy. The resulting three-coordinate dinitrogen-iron(0) complex was reduced to an iron(-I) species at low temperatures using KC8. The dinitrogen ligand coordinated in an end-on manner to the iron center and to the counterion [K(18-crown-6)]þ. A similar structural coordination motif was established by the Holland group with the complex [{Fe(N2)(b-diketiminato)}2Mg(thf)4] where two iron-dinitrogen units are associated through coordination of the N2 ligands to a common magnesium cation.118 The activation of the dinitrogen ligand in the complex [Fe(N2)(CAAC)2][K(18-crown-6)] was first explored by using the silylating reagent triethylsilylchloride, leading to the product [Fe(N2SiEt3)(CAAC)2]. These results appeared promising regarding the reaction of the N2 ligand with a proton and electron source. In fact, reaction of the iron(0) complex 17 with 50 equiv. KC8 and 50 equiv. [H(OEt2)2](BArF4) under an N2 atmosphere at  95  C yielded a maximum of 4.7 equiv. ammonia (average 3.3  1.1 equiv. ammonia). As dinitrogen only binds to the iron center at low temperatures this catalytic reaction of N2 to NH3 is strongly temperature dependent. Peters et al. assumed that the catalytic activity of complex 17 compared to the Holland system originates from the flexibility of the catalyst; i.e., the possibility of generating a twoand threefold coordination environment facilitates the formation of Fe-Nx multiple bonds.103 In 2016 Peters and coworkers published a modified protocol significantly increasing the catalytic activities of the iron complexes supported by TPB, CPiPr3, and SiPiPr3 (Fig. 18).104 Investigation of the reaction solution after the catalytic run had shown that active catalytic species remained in solution. Therefore, after addition of [H(OEt2)2](BArF4) and KC8  to the catalyst in diethyl ether, stirring for an hour at - 78  C and freezing the solution to  196  C additional acid and reductant were given to the reaction mixture; this procedure was repeated several times.104 Since the catalytic species is stable in the reaction mixture the limiting factor of ammonia formation is the competitive reduction of protons provided by [H(OEt2)2](BArF4) to dihydrogen.104 Due to the high stability of (some of) the catalysts further optimization of the reaction conditions was envisaged. Addition of a large excess of reductant and acid led to the formation of ammonia with much higher yields than reported earlier.8,102,104 In Table 3 the optimized reaction conditions at  78  C are listed. By using 1500 equiv. of [H(OEt2)2](BArF4) and 1800 eq, of KC8 at the beginning of the catalytic reaction complex [Fe(N2)(CPiPr3)][K(Et2O)0.5] (16) generated a maximum of 47 equiv. of ammonia. Complex [Fe(N2)(TPB)][Na(12-crown-4)2] (13), on the other hand, produced a maximum of 64 equiv. of ammonia, respectively 94 equiv. NH3105 under irradiation (see below). Even [Fe(N2)(SiPiPr3)][Na(12-crown-4)2] (14) which previously had been found to be catalytically inactive was shown to exhibit a small catalytic activity under these reaction conditions (4.4 equiv. NH3).104 Again, the fact that 14 is catalytically less active catalyst than [Fe(N2)(TPB)][Na(12-crown-4)2] (13) or [Fe(N2)(CPiPr3)] [K(Et2O)0.5] (16)21,23 was ascribed to the distinct covalent character and the lower flexibility of the SieFe bond as compared to the FeeB and FeeC bonds of 13 and 16, respectively (see above), which seems to play a key role in the N2-to-NH3 conversion. Moreover, to find out whether the catalytic conversion of dinitrogen into ammonia can only be effective with KC8 Peters et al. performed catalytic runs using sodium amalgam. Specifically, employing complex 13 as catalyst along with 150 equiv. of acid and 1900 equiv. wt10% sodium amalgam 5.0  0.2 equiv. of ammonia were detected. The application of KC8 thus is not necessary for the generation of ammonia but leads to higher yields of NH3 as compared to sodium amalgam.104 As mentioned above, Peters and coworkers were able to characterize several intermediates that are relevant to a catalytic cycle for the conversion of dinitrogen into ammonia. Protonation of, e.g., catalyst 13 leads to the hydrazido(2-) complex which is one of the first intermediates in a distal or Chatt-type pathway (see above). Further reaction generates a few products with spin state S]3/2 which is compatible with the formation of [Fe(NH3)(TPB)]þ, [Fe(NH2)(TPB)] and [Fe(TPB)]þ.108 These intermediates can be found in the distal as well as in the alternating pathway. It cannot be excluded that the NNH2-complex transforms to the diazene complex [Fe(NH]NH)(TPB)]þ. Another possibility is the (formal) reaction of Na of the NNH2 complex with two hydrogen atoms to the hydrazine complex [Fe(N2H4)(TPB)]þ (Scheme 12, center). This complex, which is an intermediate in the alternating pathway, was characterized in a further study of Peters et al., and the decay to the ammine complex [Fe(NH3)(TPB)]þ was monitored.108,109,112 Apart from the intermediates which are relevant to the reaction pathway, Peters and coworkers identified the hydridoborohydrido complex [FeH(N2)(TPB)(m-H)] (Scheme 13) as an important species formed under the catalytic conditions using the catalyst [Fe(N2)(TPB)] (13). Notably, as determined by Mössbauer spectroscopy, this complex is the predominant species in frozen solution after 5 min of catalytic reaction. During the further reaction the starting complex 13 was regenerated (measured after 25 min).104 In contrast to earlier investigations8 the hydrido-borohydrido complex itself exhibited catalytic activity (5.6  0.9 equiv. NH3) if the reaction was performed in a mixture of Et2O and toluene (3:1). In accordance with the results of the catalytic run (see above), stoichiometric conversion of the hydrido-borohydrido complex [FeH(N2)(TPB)(m-H)] into the parent catalyst [Fe(N2)(TPB)] (13) could be achieved (Scheme 13).104 As a consequence of these results Peters et al. interpreted the complex [FeH(N2)(TPB)(m-H)] as a resting state of the catalytic cycle. Whereas this complex is not directly involved in the reaction pathway, it can be converted in a species which participates in the catalytic cycle.104 Based on the existence of a hydrido species in the N2-to-NH3 conversion using [Fe(N2)(TPB)] (13) as a catalyst, Peters et al. investigated the catalytic behavior under irradiation with a mercury lamp. Photoinduced elimination of dihydrogen should

330

Biological and synthetic nitrogen fixation

Scheme 12 Distal and alternating pathway for the catalytic formation of ammonia. The complexes shown in black were characterized by Peters et al. Complexes in gray haven’t been characterized yet.120,121

Scheme 13 The hydrido-borohydrido complex was generated under catalytic conditions as a resting state. Using the proton and electron source catalyst 2 could be regenerated.104

promote transformation of the hydrido-borohydrido complex into the parent catalyst 13. Accordingly, the yield of ammonia could be increased to (on average) 88.1  8.0 equiv. at low temperatures ( 78  C) under very high acid and electron loading (1500 equiv. [H(OEt2)2](BArF4), 1800 equiv. KC8) as well as photoirradiation (Hg-lamp; Table 3).105 In contrast to the known molybdenum-based catalysts,6,7,92,93 the iron systems established by Peters8,102–104 and Nishibayashi (see below)110 require the strong reductant KC8 and the strong acid [H(OEt2)2](BArF4) to mediate the N2-to-NH3 conversion. Recently, Peters et al. published new results concerning this topic.119 Former studies had revealed that the iron system [Fe(N2)(TPB)] (13) indeed produces 2.1 equiv. of ammonia employing KC8 and the weaker acid (2,6dimethylanilinium)(OTf) and 0.6 equiv. NH3 using CoCp*2 along with [H(OEt2)2](BArF4), respectively.119 In 2017 the authors further reduced the thermodynamic driving force of NH3 formation by employing [Fe(N2)(TPB)](BArF4) (13) along with

Biological and synthetic nitrogen fixation

331

CoCp*2 and the anilinium cations Ph2NH2þ or PhNH3þ.120 Specifically, using this catalyst, 54 equiv. of CoCp*2 and 108 equiv. of [Ph2NH2]OTf 12.8  0.5 equiv. of NH3 (72% in relation to added reductant) could be detected at  78  C. Increasing the amount of reductant and acid gave up to 34  1 equiv. NH3 and traces of hydrazine; however, the overall yield of ammonia decreased to 63%. On the other hand, upon re-loading of reductant and acid up to 84  8 equiv. NH3 could be obtained.119 Performing freeze-quench Mößbauer spectroscopy, some iron species in the mixture of catalyst, CoCp*2 and [Ph2NH2]OTf could be identified. In contrast to catalytic runs in the presence of KC8 and [H(OEt2)2](BArF4),104 no hydrido-borohydrido species (‘resting-state,’ see above) was detected. As a consequence, a PCET mechanism for the conversion of dinitrogen into ammonia was invoked, formally corresponding to transfer of hydrogen atoms to the dinitrogen ligand of the catalyst. Protonation of the applied decamethylcobaltocene, leading to endo- and exo-[CoCp*(h4-C5Me5H)]þ, possibly generates a source of such hydrogen atoms.120 Considering the calculated NeH bond strength of the resulting intermediate, Peters et al. argue that the reaction of the PCET reagent [CoCp*(h4-C5Me5H)]þ with the catalyst, leading to intermediates like [Fe(NNH)(TPB)] and [Fe(NNH2)(TPB)]þ, respectively, is thermodynamically feasible. They conclude that a PCET scenario should also be investigated in other catalytic systems, e.g., the Schrock catalyst, which generate ammonia in the presence of metallocene reductants.118 A new development published by Peters et al. in 2018 involved the electrocatalytic generation of NH3 mediated by Fe-TBA complex (13).120 Importantly, the authors observed a correlation between pKa of the proton source and the efficiency of N2-toNH3 conversion. Stoichiometric studies indicated that the used anilinium triflate acids only mediate the formation of early stage intermediates of N2 reduction (e.g., Fe(NNH) or Fe(NNH2)) in the presence of Cp*2Co. DFT studies identified protonated Cp*2Co as a strong PCET donor capable of forming the NeH bonds of these intermediates. Furthermore, these studies also suggested that the observed pKa effect on N2RR efficiency can be attributed to the rate and thermodynamics of Cp*2Co protonation by the different anilinium acids. Addition of [Cp*2Co]þ as a cocatalyst in controlled potential electrolysis experiments led to improved yields of NH3 (up to 6.7 equiv. of NH3 per Fe at  2.1 V vs. Fc þ/0). In 2021 new Fe-based complexes based on P3Al and P3Ga ligands were presented and employed as catalysts for the conversion of N2 to ammonia (cf Fig. 18).121 As inferred from spectroscopic, structural, electrochemical and DFT studies, the series of [Fe(N2)(XP3)]0/ 1 compounds (X ¼ B, Al, Ga) possess similar electronic structures, degrees of N2 activation, and geometric flexibility. However, treatment of [Fe(N2)(XP3)][Na(12-crown-4)2] (X ¼ Al (18), Ga (19)) with an excess [H(OEt2)2](BArF4) and KC8 generates only 2.5  0.1 and 2.7  0.2 equiv. of NH3 per Fe, respectively. Similarly, the use of [H2NPh2]OTf/Cp*2Co leads to the production of 4.1  0.9 (X ¼ Al) and 3.6  0.3 (X ¼ Ga) equiv. of NH3. These results are attributed to a higher propensity of the P3Al and P3Ga complexes for proton than for dinitrogen reduction compared to the parent P3BFe system.

2.12.3.3.2

Nishibayashi’s systems

In 2016, the Nishibayashi group synthesized iron complexes containing a pincer ligand with a central pyrrolide unit and terminal di-tert-butylphosphine groups which along with a terminal coligand L exhibited a square-planar coordination geometry (Fig. 19).110 Reduction of [FeCl(PNP)] using KC8 yielded an iron(I) complex with an end-on terminally bound dinitrogen ligand (20-N2) whereas complexes 20-CH3 and 20-H could be obtained by reaction of the iron(II) chlorido complex with MeMgCl and KBHEt3, respectively. All three complexes were investigated regarding their catalytic properties in synthetic nitrogen fixation. By using the previously reported combination of CoCp2 as reductant and [LutH]OTf (2,6-lutidinium trifluoromethanesulfonate) as proton source7 no catalytic activity was observed. However, when using complex 20-N2 as catalyst along with 200 equiv. KC8 and 184 equiv. [H(OEt2)2](BArF4) in diethyl ether at  78  C up to 14.3 equiv. of ammonia (along with 1.8 equiv. od N2H4) were obtained. On the other hand, when this reaction was performed at room temperature, no formation of ammonia was observed but molecular hydrogen was generated. Complexes 20-CH3 and 20-H were also found to be active in N2-to-NH3 conversion. The yield of ammonia was, however, markedly lower (3.7 equiv. using 20-CH3, 3.0 equiv. using 20-H) as compared to 20-N2 (Fig. 19).110 In order to elucidate a possible mechanistic pathway 20-N2 was reacted with one equiv. of [H(OEt2)2](BArF4), leading to an iron complex with a protonated pyrrole moiety, [Fe(N2)(PNP-H)](BArF4). An N-H stretching vibration of the protonated dinitrogen ligand could not be observed, suggesting that protonation occurred at the PNP ligand. Subsequent reduction of the product using KC8 led to the starting complex 20-N2 and free protonated PNP ligand. Notably, the protonated complex [Fe(N2)(PNP-H)](BArF4) exhibits a lower catalytic activity than catalyst 7-N2 under the same reaction conditions (2.6 equiv. NH3 vs. 4.4 equiv. NH3 using 40 equiv. KC8 and 38 equiv. [H(OEt2)2](BArF4)). It was therefore concluded that the complex [Fe(N2)(PNP-H)](BArF4), is a deactivated species in the catalytic reaction.110

Fig. 19

Iron catalysts for synthetic nitrogen fixation by Nishibayashi et al.110 L ¼ N2, Me, H.

332

Biological and synthetic nitrogen fixation

Scheme 14 Two reaction pathways for the conversion of dinitrogen into ammonia. The generation of hydrazine in the Nishibayashi iron systems cannot be explained based on a purely distal pathway.110

The formation of hydrazine in the reduction and protonation of N2 mediated by 20-N2 cannot be explained in terms of a distal reaction pathway (Scheme 14, left). The authors therefore also considered an alternating scenario (Scheme 14, right) involving a hydrazine intermediate. This may in part dissociate, explaining the observation of free hydrazine in the reaction solution. A new dinitrogen ligand can bind to the free coordination site, regenerating the starting complex. Alternatively, bound hydrazine further reacts to ammonia.110 Whereas the formation of hydrazine implicates the presence of an alternating pathway, the generation of ammonia can proceed along a distal, alternating or a hybrid pathway. This question could not be finally resolved.

2.12.3.4

Dinuclear iron systems

The diiron(I) complex [{FeH(P2P’Ph)}(m-N2)] (21, Fig. 20) with two terminal hydrido ligands and one bridging N2-ligand was synthesized by reaction of the iron bromido precursor [FeBr2(P2P’Ph)] with two equivalents of NaHBEt3 at low temperature. The solid-state structure was determined by single crystal X-ray structure analysis, however, the hydrido ligands could not be located. Coordination of these ligands was then proven by IR spectroscopy. Spectroscopic data revealed a partial decomposition of complex 21 in solution. The resulting mononuclear iron complex [FeH(N2)(P2P’Ph)] could be identified as a minority species by Mössbauer, (ESE-)EPR and ENDOR spectroscopy.105 Reaction of 21 with 300 equiv. [H(OEt2)2](BArF4) and 360 equiv. KC8 under a dinitrogen atmosphere at  78  C in Et2O led to the formation of 7.5  0.8 equiv. ammonia. In contrast to Nishibayashi’s iron system,110 no hydrazine was generated. The authors

Fig. 20

Dinuclear dinitrogen bridged iron catalyst 21 by Peters et al.105

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333

attempted to increase the turnover number by performing the catalytic run under irradiation with a mercury lamp. They assumed that conversion of the dinuclear complex into a more active mononuclear species is facilitated by photolysis. In fact, using the same amount of acid and electron source (see above) the yield of ammonia could be increased up to 18.1  0.8 equiv. under irradiation with a Hg-lamp. Increasing the equivalents of acid (up to 3000 equiv.) and reductant (up to 3600 equiv.) under irradiation even led to 66.7  4.4 equiv. NH3. On the other hand, a catalytic run without irradiation but with the same loading of acid and reductant only gave 24.5  1.2 equiv. NH3.105 The formation of intermediates during catalysis was investigated by performing experiments with 10 equiv. of acid and 12 equiv. of reductant, leading to the dihydrido complex [Fe(H)2(N2)(P2P’Ph)]. In comparison with the dinuclear system 21 the catalytic activity of [Fe(H)2(N2)(P2P’Ph)] (2.6  0.1 equiv. NH3, 150 equiv. [H(OEt2)2](BArF4), 180 equiv. KC8). is lower. Nevertheless, the turnover number could be enhanced by irradiation with a Hg lamp (8.9  0.9 equiv. NH3). This was attributed to the fact that H2-elimination occurs from the complex [Fe(H)2(N2)(P2P’Ph)] and a more active species, [Fe0(N2)(P2P’Ph)], is generated. The aforementioned possible explanation for a higher catalytic activity under photolysis by irradiation was examined. Contrary to the original assumption, irradiation of a solution of complex 21 only a increased the concentration of the monomeric complex [FeH(N2)(P2P’Ph)]. Consequently, the increased amount of ammonia by performing the catalytic experiments under photolysis conditions cannot be ascribed to a photodissociation.105

2.12.3.5

Systems with other transition metals

Since the first discovery of a catalytic conversion of dinitrogen to ammonia with molybdenum by Schrock et al. in 20036 and 10 years later also with iron by Peters et al.,8 various transition metals were used for this purpose. Until now, reports on catalysis with Ti, V, Cr, Mo, Re, Fe, Ru, Os, and Co complexes can be found in the literature (Tables 2–4). The focus on molybdenum systems, which numerically dominate the field of catalytic systems, was based on several facts. The discovery of the first N2 complex [Ru(NH3)5(N2)]X2 (X ¼ Cl, BF4) in 1965 by Allen and Senoff showed that it is possible to activate N2 with a transition metal, but Chatt et al. could show that it is incapable of forming NH3 under protonation.122 Starting his work in the mid 60s, a time where it was already known that Fe and Mo are present in nitrogenase, he later still successfully protonated N2 bound to a metal by using bis-N2 molybdenum complexes.123 This made it seem logical to use this metal for further investigations. Furthermore, Shilov et al. could show that it is possible to reduce N2 in solution with Mo precursors.124,125 As the electronic situation on the metal center mainly determines the ability to activate N2, it is necessary to understand the different contributions affecting the activation for a rational optimization of catalysts. Furthermore, it could be shown that it is in deed possible to mediate catalytic N2-to-NH3 conversion by not biologically relevant metals. Starting with cobalt in 2016,126 investigation of such systems helped to understand the entire nitrogen fixation process in more detail. Over the years many different N2 complexes were characterized127 and the activation of N2 was assessed from NeN bond lengths and stretching frequencies. Interestingly, the ‘early’ transition metals in group 4–6 often form N2 complexes with weaker, longer NeN bonds, whereas the ‘late’ transition metals of group 8–10 have short and strong NeN bonds. This behavior can be understood by p-backbonding; i.e., due to the lower energy of the d electrons of the electronegative ‘late’ metals the contribution of backbonding is low. Based on the body of information regarding synthesis, activation, impact of the electronic situation and compatibilities with reductants and acids many new catalysts for N2-to-NH3 conversion were prepared over the last 10 years. With the presentation of a rhenium catalyst in 2021,128 all groups between IV and IX have now at least one catalytically active element (Fig. 21). Remarkably, in group VIII all three metals are catalytically active, and very recently with chromium the second group VI element and lighter homologue of the intensively studied molybdenum was presented. These first examples of these systems, all well-defined coordination compounds, supported by chelate ligands including boranes,8 carbenes,129 anionic silicon ligands,130 ligands with pincer design,125,130 polydentate amines132 or imides,6 are shown in Fig. 22 and will by considered in the next sections.

2.12.3.5.1

Cobalt

The influence of the electronic configuration and the ligand environment on the capability of a transition-metal complex to mediate N2-to-NH3 conversion is also clearly observable in the case of cobalt. [CoH(N2)(PPh3)3], as one of the earliest of all dinitrogen complexes was discovered in 1967 by Yamamoto et al.,133 only 2 years after the discovery of [Ru(N2)(NH3)5]X2 (X ¼ Cl, BF4) by Allen and Senoff.134 With a NeN stretching frequency of 2088 cm 1 the contained N2 ligand is only weakly activated and not protonable, which also due to the d8 configuration of Co(I). Correspondingly, no ammonia could be obtained by reaction 

with acids.122 However, enhancing the backdonation by reduction led to several tetrahedral, stronger activated (v ¼ 1840– 1920 cm 1) Co(-I) compounds. All of these include the anionic complex [Co(N2)(PPh3)3] the N2 ligand of which can be successfully converted into hydrazine and ammonia.135 The activation of the N2 ligand can also be strengthened by increasing the energy of the d orbitals. As shown by Holland et al. in 2009, this can be achieved by providing a low-coordinate, e.g., trigonal, geometry.136 Besides the octahedral and low-coordinated trigonal complexes, square-planar cobalt complexes supported by pincer ligands were also investigated intensively toward their properties in nitrogen fixation. This development started in 2001 with [Co(N2)(BIP)] reported by Gibson et al.,137 followed by works of Gambarotta et al.,138 Chirik et al.139 and Fryzuk et al.140 The first truly catalytic cobalt system containing a PNP-type pincer

334 Table 2

Biological and synthetic nitrogen fixation Molybdenum systems for catalytic N2-to-NH3 conversion.

Precatalyst/Catalyst

Reductant (equiv.)

Proton source (equiv.)

NH3 (equiv. per Mo (per catalyst))

Conditions

n(NN) a [cm 1]

Author et al. (year Lit. of publ.)

[Mo(N2)(HIPTN3N)] (1)

CrCp*2 (36)

7.56  0.11

Heptane, 23–25  C

1990

6

Schrock (2003)

[Mo(N2H)(HIPTN3N)] [MoN(HIPTN3N)] [Mo(NH3)(HIPTN3N)](BAr4F) [MoH(HIPTN3N)] [MoN(pBrHIPTN3N)]

7.73  0.15 7.97  0.23 8.06  0.21 7.65  0.03 6.4–7.0

ˮ ˮ ˮ Heptane, 22–26  C ˮ

1587 – – – –

ˮ ˮ ˮ 98

ˮ ˮ ˮ Schrock (2004)

2.6–2.9

ˮ

–

[MoN(HIPTN3N)]

ˮ ˮ ˮ ˮ CrCp*2 (31– 36) CoCp2 (36– 38) CoCp2 (36)

3.6

ˮ

–

[MoN(Ar2N3)(O-t-Bu)]

CoCp*2 (36)

4.4/3.6

Et2O, 78  C

–

99 Schrock (2017)

ˮ

CoCp*2 (54)

5.2

ˮ

–

ˮ

ˮ

ˮ

ˮ

7.9/7.6

ˮ

–

ˮ

ˮ

ˮ

CoCp*2 (108)

8.2

ˮ

–

ˮ

ˮ

ˮ

ˮ

10.3

ˮ

–

ˮ

ˮ

ˮ

ˮ

4.8

ˮ

–

ˮ

ˮ

ˮ

CoCp*2 (162)

5.4

ˮ

–

ˮ

ˮ

[Mo(N2)2(PNP)]2(m-N2) (3)

CoCp2 (72)

[LutH](BAr4F) (48) ˮ ˮ ˮ ˮ [LutH](BAr4F) (48–50) [LutH](BAr4F) (44–49) [LutH](BAr4F) (48) [Ph2NH2]OTf (48) [Ph2NH2]OTf (60) [Ph2NH2]OTf (108) [Ph2NH2]OTf (120) [Ph2NH2]OTf (140) [Ph2NH2]OTf (162) [Ph2NH2]OTf (322) [LutH]OTf (96)

5.9 (11.8)

Toluene, RT

ˮ

10.0 (20.1)

ˮ

Nishibayashi (2011) ˮ

11.6 (23.2)

ˮ

ˮ

ˮ

ˮ

4.5 (9.1)

ˮ

ˮ

ˮ

ˮ

ˮ [MoN(PNP)(Cl)]

CoCp2 (144) [LutH]OTf (192) CoCp2 (216) [LutH]OTf (288) CoCp2 (72) [2-PicH]OTf (96) ˮ [PyH]OTf (96) CoCp2 (72) [LutH]OTf (96)

1936(t)/ 7 1890(b) ˮ ˮ

1.9 (3.9) 6.6

ˮ Toluene, RT

ˮ –

[MoN(PNP)(Cl)]OTf [Mo(N2)2(tBuPNPAd)]2(m-N2)

ˮ CoCp2 (72)

ˮ Toluene, RT, 20 h

– –b

[Mo(N2)2(4-Ph-PNP)]2(m-N2)

10.5 (21)

ˮ

[Mo(N2)2(4-Me3Si-PNP)]2(m-N2)

CoCp2 (216) [LutH]OTf (288) ˮ ˮ

11.5 (23)

ˮ

[Mo(N2)2(4-tBu-PNP)]2(m-N2)

ˮ

ˮ

14 (28)

ˮ

[Mo(N2)2(4-Me-PNP)]2(m-N2)

ˮ

ˮ

15.5 (31)

ˮ

[Mo(N2)2(4-MeO-PNP)]2(m-N2) ˮ

ˮ ˮ

ˮ ˮ

17 (34) 18 (36)

ˮ

CoCp2 (360) [LutH]OTf (480) CrCp*2 (216) [LutH]OTf (288) CrCp*2 (216) ˮ ˮ ˮ

26 (52)

ˮ Toluene, RT, 20 h, 5 h CoCp2 addition ˮ

1950(t)/ 1880(b) 1947(t)/ 1886(b) 1939(t)/ 1899(b) 1939(t)/ 1896(b) 1932(t) ˮ

ˮ ˮ 90 Nishibayashi (2014) ˮ ˮ 88 Nishibayashi (2014) ˮ ˮ

15 (30)

ˮ

12 (24) 16.5 (33)

ˮ Toluene, RT, 20 h, 5 h CrCp*2 addition

ˮ

ˮ ˮ

[Mo(N2)2(4-Me-PNP)]2(m-N2) [Mo(N2)2(4-MeO-PNP)]2(m-N2) ˮ

ˮ 7.1 [LutH]OTf (96) 7 (14)

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ ˮ

ˮ ˮ

ˮ

ˮ

1939(t)/ ˮ 1896(b) 1932(t) ˮ ˮ ˮ

ˮ

ˮ

ˮ ˮ

Biological and synthetic nitrogen fixation Table 2

Molybdenum systems for catalytic N2-to-NH3 conversion.dcont'd

Precatalyst/Catalyst [Mo(N2)2(PNP)]2(m-N2)

Reductant (equiv.)

Proton source (equiv.)

NH3 (equiv. per Mo (per catalyst))

Conditions

Author et al. (year Lit. of publ.)

[Mo(N2)2(PNP)]2(m-N2) (3) [Mo(N2)2(EtFc-PNP)]2(m-N2)

ˮ

ˮ

15 (30)

FeCp2 (2 equiv. to cat.) was added ˮ

[Mo(N2)2(PhFc-PNP)]2(m-N2)

ˮ

ˮ

5 (10)

ˮ

[Mo(N2)2(Rc-PNP)]2(m-N2)

ˮ

ˮ

12.5 (25)

ˮ

[MoN(PPP)(Cl)] (4)

CoCp*2 (36) [ColH]OTf (48) 11.0

Toluene, RT

1936(t)/ ˮ 1890(b) 1944(t)/ 89 1888(b) 1936(t)/ 1890(b) 1939(t)/ 1894(b) 1951(t)/ 1876(b) 1944(t)/ 1888(b) – 92

[MoN(PPP)(Cl)](BArF4) (5) ˮ ˮ

ˮ [LutH]OTf (48) 6.1 ˮ [ColH]OTf (48) 9.6 63 CoCp*2 (540) [ColH]OTf (720) CoCp2 (72) [LutH]OTf (96) 5.7  0.6

ˮ ˮ ˮ

– – –

ˮ ˮ ˮ

ˮ

1978(t)

17.6  0.7 11.8  1.0 39.5  2.0

ˮ ˮ ˮ

ˮ ˮ ˮ

93 Nishibayashi (2017) ˮ ˮ ˮ ˮ ˮ ˮ

9.5  0.5

ˮ

ˮ

ˮ

ˮ

30.5  0.5

ˮ

ˮ

ˮ

ˮ

7.5  2.5

ˮ

ˮ

ˮ

ˮ

100  10

ˮ

ˮ

[Mo(N2)2(Fc-PNP)]2(m-N2)

[{Mo(N2)2(Bim-PCP)}2(m-N2)] (6)

Toluene, RT, 20 h

n(NN) a [cm 1]

CoCp2 (216) [Me-LutH]OTf 5 (10) (288) ˮ [LutH]OTf 18.5 (37) (288) ˮ ˮ 10.5 (21)

ˮ

ˮ Nishibayashi (2015) ˮ ˮ ˮ ˮ Nishibayashi (2015) ˮ ˮ ˮ

CrCp*2 (72) ˮ CoCp*2 (72) ˮ CrCp*2 (360) [LutH]OTf (480) ˮ ˮ [PicH]OTf (480) ˮ ˮ [ColH]OTf (480) ˮ ˮ [LutH](BArF4) (480) [LutH]OTf ˮ CrCp*2 (1440) (1920) [{Mo(N2)2(Me-Bim-PCP)}2(m-N2)] (7) CrCp*2 [LutH]OTf (1440) (1920) [{Mo(N2)2(Im-PCP)}2(m-N2)] (8) CrCp*2 (72) [LutH]OTf (96) ˮ CoCp*2 (72) ˮ [Mo(N2)2(PNP)]2(m-N2) (3) CrCp*2 (72) ˮ [MoI3(PNP)] (9) CoCp*2 (36) [ColH]OTf (48)

115

ˮ

1969(t)

1.60  0.15 1.45  0.50 12.2 10.9  0.2

ˮ ˮ ˮ ˮ

1911(t) ˮ 1936(t) –

ˮ ˮ ˮ 94

[Mo(N)I(PNP)] (10) [Mo(N2)2(PNP)]2(m-N2) (3)

ˮ ˮ

ˮ ˮ

12.2  0.1 7.1  0.4

ˮ ˮ

ˮ ˮ

[MoI3(PNP)] (9) ˮ ˮ ˮ [MoBr3(PNP)] [MoCl3(PNP)] [MoI3(tBuPPP)] [MoCl3(tBuPPP)] [Mo(N)Br(PNP)] [Mo(N)Cl(PNP)] [MoI3(PNP)]

CrCp*2 (36) CoCp*2 (36) ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ CoCp*2 (2160) SmI2(thf)2 (180) ˮ ˮ ˮ

ˮ ˮ ˮ [LutH]OTf (48) [ColH]OTf (48) ˮ ˮ ˮ ˮ ˮ [ColH]OTf (2880) HOCH2CH2OH (180) MeOH (180) EtOH (180) iPrOH (180)

10.3  0.2 4.2 3.1  0.2 50.7  0.4 40.5 24.4 6.3 6.4 11.1 8.4 415

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

– 1936(t)/ 1890(b) – – – – – – – – – – –

ˮ ˮ ˮ Nishibayashi (2017) ˮ ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

42.8  1.5

THF, RT, 18 h

–

17.2  1.1 14.5 11.8

ˮ ˮ ˮ

– – –

ˮ ˮ ˮ

[MoI3(PNP)] ˮ ˮ ˮ

335

96 Nishibayashi (2019) ˮ ˮ ˮ ˮ ˮ ˮ (Continued)

336 Table 2

Biological and synthetic nitrogen fixation Molybdenum systems for catalytic N2-to-NH3 conversion.dcont'd

Precatalyst/Catalyst

Reductant (equiv.)

Proton source (equiv.)

NH3 (equiv. per Mo (per catalyst))

Conditions

n(NN) a [cm 1]

Author et al. (year Lit. of publ.)

ˮ ˮ

ˮ ˮ

7.7 13.8

ˮ ˮ

– –

ˮ ˮ

ˮ ˮ

ˮ ˮ

ˮ ˮ

16.5 14.4

ˮ ˮ

– –

ˮ ˮ

ˮ ˮ

[Mo(N)I(PNP)]

ˮ

50.0  0.1

ˮ

–

ˮ

ˮ

[MoO(OH2)(PNP)] [{Mo(N2)2(Bim-PCP)}2(m-N2)] (6) [MoCl3(Bim-PCP)] ˮ

ˮ ˮ ˮ SmI2(THF)2 (14,400) ˮ SmI2(THF)2 (36) SmI2(THF)2 (180) SmI2(THF)2 (36) ˮ SmI2(THF)2 (180) SmI2(THF)2 (36) SmI2(THF)2 (180) ˮ

tBuPH (180) CF3CH2OH (180) PhOH (180) [ColH]OTf (180) HOCH2CH2OH (180) ˮ ˮ ˮ HOCH2CH2OH (14,400) H2O (14,400) HOCH2CH2OH (36) HOCH2CH2OH (180) HOCH2CH2OH (36) ˮ HOCH2CH2OH (180) HOCH2CH2OH (36) HOCH2CH2OH (180) ˮ

44.1 53.3  2.1 55.0  0.9 3650  250

ˮ ˮ ˮ ˮ

– 1978(t) – –

ˮ ˮ ˮ ˮ

ˮ ˮ ˮ ˮ

4350  150 9.2

ˮ ˮ

– 1926100

25.2

ˮ

ˮ

ˮ ˮ 87 Nishibayashi (2019) ˮ ˮ

5.1

ˮ

ˮ

11.2 54.3

ˮ ˮ

2030/ ˮ 1975100 – ˮ – ˮ

11.1

ˮ

–

ˮ

ˮ

–

ˮ

ˮ

2.7–10.8

ˮ

–

ˮ

ˮ

ˮ trans-[Mo(N2)2(PMePh2)4] ˮ trans-[Mo(N2)2(dppe)2] [MoCl3(Bim-PCP)] ˮ [MoCl3(PNP)] ˮ [MoI3(thf)] þ L L ¼ PMePh2, PEtPh2, PnPrPh2, PPh3, dppm, dppe, dppp, dppb, dpppe, dpph, dppf [MoCl3(Bim-PCP)]

50.8

ˮ ˮ

H2O (360)

91  3

Mag. stirrer, 15 mL THF

–

ˮ

SmI2(THF)2 (360) ˮ

ˮ

92

–

ˮ

ˮ

ˮ

90

–

ˮ

ˮ

[Mo(N2)(PentaPod)] (2) [Mo(NNH2)(PentaPod)](BAr4F)2

SmI2 (180) ˮ

H2O (180) ˮ

25.73  0.37 26.14  0.32

Mech. Stirrer, 15 mL THF 0.005 mmol catalyst Mech. Stirrer, 300 mL THF 0.1 mmol catalyst THF ˮ

97 Nishibayashi (2019) ˮ ˮ

1929 1490 (calc)

5 ˮ

Tuczek (2020) ˮ

In case of bridged dinculear complexes, t ¼ terminal and b ¼ bridging N2 ligand. Not provided.

a

b

ligand with a central pyrrolide moiety was presented by Nishibayashi in 2016.126 An earlier anionic cobalt complex supported by the tripodal tetradentate tris(phosphine)borane ligand developed by Peters et al., which was already employed for the first catalyt

ically active iron system (see above),8 led to a relatively high activation (v ¼ 1978 cm 1, cf. [Co(N2)(PP3)]þ with 2125 cm 1 by Biachnini et al.141). With 2.4 equivalents only a slightly superstoichiometric conversion of N2 to ammonia was achieved.142 Whereas the borane system ([Co(N2)(TPB)]) is slightly catalytic, the isoelectronic neutral systems [Co(N2)(CP3)] and [Co(N2)(SiP3)] with carbon or silicon in the apical position are totally inactive under the same conditions (KC8 and [H(OEt2)2](BArF4)), which underscores the importance and role of the Co-E bond trans to the N2 ligand. The highly reduced form of ([Co(N2)(TPB)]) is usually also favorably correlated to catalytic ammonia production, which also applies to the analogous, catalytically active ruthenium and osmium systems.130 Cobalt dinitrogen complexes [Co(N2)(RPNP)] (22, 23, Fig. 22) supported by pincer-type ligands (R ¼ tBu; 2,5-bis(di-tert-butylphosphinomethyl)pyrrolide, R ¼ Cy; 2,5-bis(dicyclohexylphosphinomethyl)-pyrrolide) were presented by Nishibayashi et al. in 2016. They combine the approach of lowering the symmetry and coordination with moderately activating PNP ligands, which led to sufficient but not too strong activation (2016 and 2020 cm 1), which is discussed to be essential for the catalytic activity.126

Biological and synthetic nitrogen fixation Table 3

337

Iron systems for catalytic N2-to-NH3 conversion.

Precatalyst/Catalyst a [Fe(N2)(TPB)][Na(12-c4)2] (13) [Fe(TPB)](BAr4F) [Fe(N2)(CPiPr3)][K(OEt2)3] (15) [Fe(N2)(CPiPr3)][K(12-c4)2] [Fe(CAAC)2] (17)

Et2O, 78  C

1905

8

6.2  0.7 4.6  0.8

ˮ ˮ

– 1870

ˮ ˮ 102 Peters (2014)

3.5  0.3

ˮ

1905

ˮ

3.3  1.1

Et2O, 95  C

–

103 Peters (2015)

3.0  0.7 3.4  1.0 7.3  0.5

Et2O, 113  C Et2O, 95  C Et2O, 78  C

– – 1905

ˮ ˮ ˮ ˮ 104 Peters (2016)

12  1

ˮ

ˮ

ˮ

ˮ

17.4  0.2

ˮ

ˮ

ˮ

ˮ

43  4

ˮ

ˮ

ˮ

ˮ

59  6

ˮ

ˮ

ˮ

ˮ

5.0  0.2

10 wt% NaXHg

ˮ

ˮ

ˮ

14  3

ˮ

1870

ˮ

ˮ

19  4

ˮ

ˮ

ˮ

ˮ

36  7

ˮ

ˮ

ˮ

ˮ

3.8  0.8

ˮ

1920

ˮ

ˮ

4.4  0.2

Et2O, 78  C

1964

110 Nishibayashi (2016)

6.7 10.9  0.4 14.3  0.4 2.9  0.2 3.0  0.9 3.7  0.5 2.6  0.2 12.8  0.5 34  1 26.7  0.9 56  9

ˮ ˮ ˮ ˮ – – 2034 – – – –

ˮ ˮ ˮ ˮ ˮ ˮ ˮ 118 ˮ ˮ ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ Peters (2017) ˮ ˮ ˮ

–

ˮ

ˮ

81

ˮ ˮ ˮ THF, 78  C Et2O, 78  C ˮ ˮ Et2O, 78  C ˮ ˮ Et2O, 78  C Add. substrate Et2O, 78  C Add. substrate Et2O, 78  C

–

ˮ

ˮ

71 16  3 7.5  0.8

ˮ ˮ Et2O, 78  C

– – –b

ˮ ˮ ˮ ˮ 105 Peters (2017)

8.7  0.7 18.1  0.8 24.5  1.2

Et2O, 78  C, overnight Et2O, 78  C, Hg lamp Et2O, 78  C

ˮ ˮ ˮ

ˮ ˮ ˮ

ˮ ˮ ˮ

66.7  4.4

Et2O, 78  C, Hg lamp

ˮ

ˮ

ˮ

NH3 (equiv. per Fe) Conditions

KC8 (50)

[H(OEt2)2](BAr4F) (46) ˮ [H(OEt2)2](BAr4F) (38) ˮ

7.0  1.0

[H(OEt2)2](BAr4F) (50) ˮ ˮ [H(OEt2)2](BAr4F) (48) [H(OEt2)2](BAr4F) (96) [H(OEt2)2](BAr4F) (150) [H(OEt2)2](BAr4F) (720) [H(OEt2)2](BAr4F) (1500) [H(OEt2)2](BAr4F) (150) [H(OEt2)2](BAr4F) (220) [H(OEt2)2](BAr4F) (750) [H(OEt2)2](BAr4F) (1500) [H(OEt2)2](BAr4F) (1500) [H(OEt2)2](BAr4F) (38) ˮ ˮ ˮ ˮ ˮ ˮ ˮ [H2NPh2]OTf (108) [H2NPh2]OTf (322) [H2NPh2]OTf (638) [H2NPh2]OTf (2  322) [H2NPh2]OTf (3  322) [H2NPh2](BAr4F) (108) [H3NPh]OTf (108) [H3NPh]OTf (322) [H(OEt2)2](BAr4F) (300) ˮ ˮ [H(OEt2)2](BAr4F) (3000) ˮ

ˮ KC8 (40) ˮ KC8 (50) ˮ ˮ KC8 (58)

ˮ

KC8 (185)

ˮ

KC8 (860)

ˮ

KC8 (1800)

ˮ

NaXHg (1900)

KC8 (58)

[Fe(N2)(CPiPr3)][K(OEt2)3] KC8 (230) (16) ˮ KC8 (810) ˮ

KC8 (1600)

[Fe(N2)(SiP3)][Na(12-c4)2] (14) [Fe(N2)(PNP)] (20-N2)

KC8 (1800) KC8 (40)

ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ CoCp*2 (54) CoCp*2 (162) CoCp*2 (322) CoCp*2 (2  162) CoCp*2 (3  162) CoCp*2 (54)

ˮ ˮ [FeH(P2P
Ph)]2(m-N2) (21)

ˮ CoCp*2 (162) KC8 (360)

ˮ ˮ ˮ

ˮ ˮ KC8 (3600)

ˮ

ˮ

ˮ

Author et al. (year of Lit. publication)

Proton source (equiv.)

ˮ [Fe(CAAC)2][BAr4F] [Fe(N2)(TPB)][Na(12-c4)2] (13) ˮ

ˮ ˮ ˮ ˮ [FeH(PNP)] (20-H) [FeMe(PNP)] (20-Me) [Fe(N2)(HPNP)](BAr4F) [Fe(TPB)](BAr4F) ˮ ˮ ˮ

n(NN) [cm 1]

Reductant (equiv.)

84  8

Peters (2013)

ˮ

(Continued)

338

Biological and synthetic nitrogen fixation

Table 3

Iron systems for catalytic N2-to-NH3 conversion.dcont'd n(NN) [cm 1]

Author et al. (year of Lit. publication)

Et2O, 78  C

ˮ

ˮ

ˮ

8.9  0.9 60.0  3.7

Et2O, 78  C, Hg lamp Et2O, 78  C

ˮ 1905

ˮ ˮ

ˮ ˮ

88.1  8.0 7.3  0.1 8.6  0.7

Et2O, 78  C, Hg lamp Et2O, 78  C ˮ

ˮ – –

ˮ ˮ 120 Peters (2018) ˮ ˮ

10.7  0.1

ˮ

–

ˮ

ˮ

13.9  0.7

ˮ

–

ˮ

ˮ

13.8  0.9

ˮ

–

ˮ

ˮ

12.8  0.4

ˮ

–

ˮ

ˮ

3.6  0.1

ˮ

–

ˮ

ˮ

2.6  0.3

Et2O, 35  C, 2.1 V vs. Fc/Fcþ Et2O, 35  C, 2.1 V vs. Fc/Fcþ 1 equiv. [CoCp*2][BAr4F] Et2O, 35  C, 2.1 V vs. Fc/Fcþ 5 equiv. [CoCp*2][BAr4F] ˮ

–

ˮ

ˮ

–

ˮ

ˮ

–

ˮ

ˮ

–

ˮ

ˮ

ˮ

ˮ

Precatalyst/Catalyst a

Reductant (equiv.)

Proton source (equiv.)

NH3 (equiv. per Fe) Conditions

[FeH2(P2P
Ph)]

KC8 (180)

2.6  0.1

ˮ [Fe(N2)(TPB)][Na(12-c4)2] (13) ˮ [Fe(TPB)](BAr4F) ˮ

ˮ KC8 (1800)

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

ˮ

–

[H(OEt2)2](BAr4F) (150) ˮ [H(OEt2)2](BAr4F) (1500) ˮ [PhNH3]OTf (108) [2,6-MePhNH3]OTf (108) [2-ClPhNH3]OTf (108) [2,5-ClPhNH3]OTf (108) [2,6-ClPhNH3]OTf (108) [2,4,6-ClPhNH3]OTf (108) [per-ClPhNH3]OTf (108) [H2NPh2]OTf (50)

ˮ

–

ˮ

4.0  0.6

ˮ

–

ˮ

4.0  0.6

ˮ

–

5.5  0.9

ˮ

–

[H2NPh2]OTf (2  50) [H2NPh2]OTf (50)

[Fe(N2)(AlP3)][Na(12-c4)2] (18) ˮ [Fe(N2)(GaP3)][Na(12-c4)2] (19) ˮ

KC8 (50)

ˮ CoCp*2 (54) ˮ

CoCp*2 (50) KC8 (50) CoCp*2 (50)

[H(OEt2)2](BAr4F) (46) [Ph2NH2]OTf (46) [H(OEt2)2](BAr4F) (46) [Ph2NH2]OTf (46)

2.5  0.1

Et2O, 35  C, 2.1 V vs. – Fc/Fcþ 10 equiv. [CoCp*2][BAr4F] Et2O, 78  C 1914

121 Peters (2021)

4.1  0.9 2.7  0.2

ˮ ˮ

ˮ 1912

ˮ ˮ

ˮ ˮ

3.6  0.3

ˮ

ˮ

ˮ

ˮ

41

12-c-4 ¼ 12-crown-4; TPB ¼ BP3 ¼ tris(o-diisopropylphosphinophenyl)borane; CP3iPr¼ tris(o-diisopropylphosphinophenyl)methyl; SiP3 ¼ SiP3iPr¼ tris(o-diisopropylphosphinophenyl) silyl; AlP3 ¼ tris(o-diisopropylphosphinophenyl)alane; GaP3 ¼ tris(o-diisopropylphosphinophenyl)galane. b Not provided. a

The complexes were synthesized by reduction of the paramagnetic precursors [CoCl(tBuPNP)] and [CoCl(CyPNP)], respectively, with KC8 under dinitrogen and examined in detail with regard to the conversion of dinitrogen into ammonia under ambient reaction conditions. The catalytically most active system with R ¼ tBu produced 15.9 equiv. of NH3 and thus clearly exceeds the just slightly ovestoichiometric NH3 production of the earlier tripodal system (see above).

2.12.3.5.2

Ruthenium and osmium

Ruthenium is known as component in Haber-Bosch systems such as the Kellog Advanced Ammonia Process (KAAP).143 Nevertheless, it is not found in biology but plays an important role in biomimetic chemistry as ruthenium complexes are often more stable than the iron analoga. After the discovery of [Ru(N2)(NH3)5]X2 (X ¼ Cl, BF4),134 it seemed likely that the bound N2 ligand could be used for synthetic nitrogen fixation, but already 4 years later Chatt showed that it is impossible to obtain NH3 upon protonation of 

this complex due to the low activation (v ¼ 2133 cm 1).122,144 One disadvantage of both ruthenium and osmium is that low redox states, which are necessary for a functional catalytic process, are difficult to access. This is also reflected by the fact that only a few ruthenium(0) dinitrogen complexes are known,145 such as trigonal bipyramidal [Ru(N2)(P2P3R)] (R ¼ Cy, iPr) of Field et al., which forms an octahedral hydrido complexes under acidic conditions and is therefore also not suitable for NH3 production.146 Nevertheless, if the electron density is more ligand-localized, which is often the case in d7 systems, this leads to lower activation and

Biological and synthetic nitrogen fixation Table 4

339

Transition metal systems of groups IV-XI (without Mo and Fe) for catalytic N2-to-NH3 conversion.

Metal M Precatalyst/Catalyst Group IV Ti [{Ti(Tren™S)}2(m-h1:h1:h2:h2N2K2)] (26) ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ Group V V [VCl(OXyl)(tBu-PNP)] [VCl(OPh)(tBu-PNP)] [VCl(OAr)(tBu-PNP)] [VCl(Xyl)(tBu-PNP)] [VCl(OXyl)(tBu-PNP)] [V(OXyl)(tBu-PNP)] (27) ˮ [V(OXyl)(tBu-PNP)]2(m-N2) [V(NH2)(OXyl)(tBu-PNP)] [{V(TrenMeBn)}2(m-N2)] Group VI Cr [CrCl(N)(PCP)](BArF4) ˮ ˮ [CrCl2(PCP)] [CrI2(PCP)] [CrCl(PCP)](BArF4) [{Cr(N2)(PCP)}2(m-N2)] [CrI(PCP)](BArF4) (29) ˮ Group VII Re [ReCl(N2)(PNP)]2(m-N2) (28) ˮ

Author et al. (year of Lit. publication)

Et2O,  78  C

1201

132 Liddle (2018)

11.91 10.81 17.77 5.82 3.89 8.95 2.45 4.77 11.73 7.37

ˮ ˮ ˮ Pentane, 78  C Toluene, 78  C THF, 78  C Et2O,  78  C ˮ ˮ ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

4.0  0.5

Et2O,  78  C

–

131 Nishibayashi (2018)

3.6  0.1 3.2  0.1 3.0  0.4 3.5  0.4

ˮ ˮ ˮ ˮ

– – – –

ˮ ˮ ˮ ˮ

ˮ ˮ ˮ ˮ

6.8  0.2

ˮ

–

ˮ

ˮ

12  0

ˮ

–

ˮ

ˮ ˮ

NH3 (equiv. per Conditions Proton source (equiv.) M)

KC8 (120)

[Cy3PH]I (120)

6.41

KC8 (300) ˮ KC8 (600) KC8 (300) ˮ ˮ ˮ ˮ ˮ ˮ

[Cy3PH]I (300) [Cy3PH]I (400) [Cy3PH]I (600) [Cy3PH]I (300) ˮ ˮ [Cy3PH]Cl (300) [Cy3PH](BArF4) (300) [nBu3PH]I (300) [tBu3PH]I (300)

ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

[H(OEt2)2](BArF4) (38) ˮ ˮ ˮ ˮ ˮ ˮ KC8 (200) [H(OEt2)2](BArF4) (184) [H(OEt2)2](BArF4) KC8 (40) (38) [H(OEt2)2](BArF4) KC8 (200) (184) KC8 (40) [H(OEt2)2](BArF4) (38) ˮ ˮ K[C10H8] (40) [LutH]OTf (40)

4.6  0.1

ˮ

(unstable) ˮ

6.6  0.1 3.4

ˮ THF, 78  C

– 1394

ˮ ˮ 169 Masuda (2018)

KC8 (40) ˮ ˮ ˮ ˮ ˮ ˮ ˮ KC8 (200)

[PCy3H]OTf (36) [PCy3H](BArF4) (36) [PCy3H](BF4) (36) ˮ ˮ ˮ ˮ ˮ [PCy3H](BF4) (180)

3.98  0.97 2.98 5.71  0.63 2.16  0.34 4.21  0.33 5.77  0.67 2.29  0.08 4.87  0.39 11.60  1.27

Et2O,  78  C ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

– – – – – – 1897(t) – –

129 ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

KC8 (400) ˮ

[Cy3PH](BArF4) (400) [H(OEt2)2](BArF4) (400) [Cy3PH](BArF4) (800) [H(OEt2)2](BArF4) (800) [tBu3PH](BArF4) (800) [nBu3PH](BArF4) (800) [Cy3PH](BArF4) (800) [Cy3PH](BArF4) (800)

3.9  0.2 3.7  0.4

Et2O,  78  C ˮ

1890(b) ˮ

128 Nishibayashi (2021) ˮ ˮ

8.4  0.9 4.9  0.1

ˮ ˮ

ˮ ˮ

ˮ ˮ

ˮ ˮ

7.5  6.0

ˮ

ˮ

ˮ

ˮ

6.0  1.2

ˮ

ˮ

ˮ

ˮ

3.9  0.6 3.6  0.7

ˮ ˮ

– –

ˮ ˮ

ˮ ˮ

[H(OEt2)2](BArF4) (46) [H2NPh2]OTf (46) [H2NPh2]OTf (150) [H2NPh2]OTf (500) [H2NPh2]OTf (800)

4.3  0.3

Et2O,  78  C

1960

130 Peters (2017)

7.1  0.6 18  1 50  3 86  5

Et2O,  78  C ˮ ˮ ˮ

1931 ˮ ˮ ˮ

ˮ ˮ ˮ ˮ

KC8 (40)

ˮ ˮ

KC8 (800) ˮ

ˮ

ˮ

ˮ

ˮ

[ReCl3(PNP)] [Re(N)Cl2(PNP)] Group VIII Ru [Ru(N2)(P3Si)][K(THF)2] (24)

ˮ ˮ

Os

CoCp*2 (50) CoCp*2 (180) CoCp*2 (600) CoCp*2 (960)

[Os(N2)(P3Si)][K(THF)2] (25) ˮ ˮ ˮ

n(NN) [cm 1]

Reductant (equiv.)

KC8 (50)

Nishibayashi (2022) ˮ ˮ ˮ ˮ ˮ ˮ ˮ ˮ

(Continued)

340

Biological and synthetic nitrogen fixation

Table 4

Transition metal systems of groups IV-XI (without Mo and Fe) for catalytic N2-to-NH3 conversion.dcont'd

Metal M Precatalyst/Catalyst ˮ ˮ ˮ ˮ Group IX Co [Co(N2)(TPB)][Na(12-c-4)]

Reductant (equiv.)

NH3 (equiv. per Proton source (equiv.) M) Conditions

[H2NPh2]OTf (1500) CoCp*2 (1800) CoCp*2 (50) [H3NPh]OTf (46) CoCp*2 (50) [H3N-2,5-Cl2C6H3] OTf (46) CoCp*2 (50) [N-Me-H2NPh]OTf (46) KC8 (60)

[CoH(tBuPNP)]

KC8 (40)

[Co(N2)(tBuPNP)] (22) ˮ [Co(N2)(CyPNP)] (23) [Co(N2)(tBuPNP)]

ˮ ˮ ˮ KC8 (200)

[H(OEt2)2](BArF4) (47) [H(OEt2)2](BArF4) (38) ˮ ˮ ˮ [H(OEt2)2](BArF4) (184)

n(NN) [cm 1]

Author et al. (year of Lit. publication)

120  11

ˮ

ˮ

ˮ

7.9  0.3 7.9

ˮ ˮ

ˮ ˮ

ˮ ˮ

6.3  0.5

ˮ

ˮ

ˮ

2.4  0.3

Et2O,  78  C

1978

142 Peters (2015)

2.2  0.1

ˮ

–

126 Nishibayashi (2016)

4.2  0.1 4.0  0.3 3.1  0.1 15.9  0.2

ˮ MTBE, 78  C Et2O,  78  C ˮ

2020 ˮ 2016 2020

ˮ ˮ ˮ ˮ

ˮ ˮ ˮ ˮ

Ar ¼ 2,6-di-isopropylphenyl; TrenMeBn ¼ (tris(2-((4- methylbenzyl)-amino)ethyl)-amine); 12-c-4 ¼ 12-crown-4; TPB ¼ P3B ¼ tris(o-diisopropylphosphinophenyl)borane

Fig. 21

Timeline of the first examples of catalytic systems with different transition metals of group IV–IX.

Fig. 22

The first examples of catalytically active systems with different transition metals.6,8,126,128–132

Biological and synthetic nitrogen fixation

341

successful functionalization of the coligand N2 is possible. Pincer dinitrogen complexes of Ru generally display weak activation and can easily lose their N2 ligands in ligand substitution reactions.147 An interesting dinuclear ruthenium(II) system, which actually came close to N2-to-NH3 conversion, contains two bridged porphyrin-based ligands and can bind N2, N2H2, N2H4 and ammonia. The oxidation of 2 NH3 to N2 was also possible but not the reversed process.148 Dinitrogen chemistry with osmium is rare. [Os(N2)(NH3)5]2þ as heavier homologue of Allen and Senoff’s ruthenium complex was prepared by Allen in 1967.149 Remarkably, this compound can be converted photolytically into the only monomeric side-on N2 complex being known to date.150 The photochemical properties are also relevant to nitrogen fixation as electrolysis of [Os(N2)(NH3)5]2þ leads to the bridged system [(NH3)5OsII(m-N2)OsIII(NH3)5]5þ, which can be converted via NeN cleavage into the imido complex [OsVI(N)(NH3)5]3þ.151 Notably, the reverse reaction of this process involving NeN bond formation to N2 is known as well.152 It was also proposed that hydrolysis of photochemically formed [OsVI(N)(NH3)5]3þ leads to ammonia formation, which would represent a rare example of stoichiometric N2-to-NH3 conversion mediated by osmium, also being described for a few other examples.153 The first and only examples of catalytic N2-to-NH3 conversion with ruthenium and osmium were presented in 2017 by Peters et al. (24, 25, Fig. 22).130 The employed tris(phosphino)silyl complexes are not only isostructural to each other, but also to the earlier presented first catalytically active iron system, which contains boron as apical atom instead of silicon.8 The key features of these exceptional systems are the local threefold symmetry, the negative charge and the flexible ligand trans to N2, a formal silicon anion in case of Ru and Os. For group VIII to X metals, low oxidation states (d7–9 systems) are more stable in a trigonal coordination sphere as compared to other geometries, in particular octahedral. Accordingly, no octahedral M(0)-N2 (M ¼ Ru, Os) complexes are known. Compared to the neutral complexes, the charge density on the terminal Nb is increased in the catalytically active negatively charged complexes which favors protonation of the N2 ligand over protonation of the metal center; therefore, no unwanted hydrido complexes are formed.

2.12.3.5.3

Titanium

Despite the fact that a multitude of N2 containing complexes with titanium can be found in the literature there is only one early and one recent example of nitrogen fixation related catalysis with N(TMS)3 production, which can be seen as NH3 analogon.154 The first catalytic NH3 production was presented in 2018. In analogy to the first catalytically active system by Schrock,6 Liddle et al. used a triamidoamine ligand (TrenTMS ¼ N(CH2CH2NSiMe3)3), but in combination with titanium (26, Fig. 22). In a reaction with N2 a dinuclear bridged complex with moderate activation of the N2 ligand (1.700 cm 1) was obtained which can be reduced to its strongly activated dianionic derivative,132 the N2 ligand of which can be described as [N2]2. Reaction with HCl already showed the capability of nitrogen fixation as N2H4 (0.88%) and NH3 (0.13%) are formed, albeit in substoichiometric yields. Using KC8 and phosphonium acids led to catalytic formation of NH3 (up to 17.77 equivalents). Interestingly, [R3PH]þ compounds as proton sources seem to be a good alternative to the commonly used [H(OEt2)2](BArF4), especially in combination with KC8, which often leads to unwanted side reactions. This was already successfully shown by Nishibayashi et al. with the first rhenium catalysts in 2021 where the phosphonium acids led to higher TONs than [H(OEt2)2](BArF4), both in combination with KC8.128 After the discovery of Liddle et al., one additional example of NH3 production was reported by Lee et al. but in this case in a stoichiometric fashion.155

2.12.3.5.4

Vanadium

2.12.3.5.5

Rhenium

Following the report of Shilov et al. that V(OH)2 can mediate dinitrogen fixation when supported with Mg(OH)2,124,156 a multitude of studies on dinitrogen vanadium chemistry were published over the years,157–159 often referring to the ‘alternative nitrogenase’ which contains vanadium.160 Some of these systems are able to produce NH3, but only in a stoichiometric fashion.158,159,161 In 2017, Nishibayashi presented the first catalytic system for N(TMS)3 production162 and 1 year later the first for NH3.131 The catalyst was supported by a pincer type PNP ligand which had already been used in iron and cobalt systems (see above) and contained an aryloxide coligand (27, Fig. 22). Using an excess of KC8 and [H(OEt2)2](BArF4) at low temperature led to a good yield (51%) with a low TON (6.8) or a low yield (18%) with higher TON (12), depending on the conditions.

In the last years, rhenium was intensively studied especially for its ability to cleave the NN bond in N2 to form nitrido complexes.163,164 Aiming at NH3 formation, protonation of these complexes was attempted, but turned out to be difficult due to protonation of the coligand.163 Still, in one case stoichiometric NH3 formation was achieved.165 The first catalytic system was discovered only recently. Inspired by their dinuclear Mo(0) systems with pincer ligands Nishibayashi et al. successfully used an isoelectronic (d6) dinuclear Re(I) complex, [Re(N2)Cl(PNP)]2(m-N2) (28, Fig. 22), for catalytic N2-to-NH3 conversion.128 Interestingly, this system is incompatible with SmI2/H2O and CoCp2/[LutH]OTf or CoCp2*/[ColH]OTf as proton source/reductant at room temperature and requires KC8 as reductant in combination with [H(OEt2)2](BArF4) or phosphonium acids as proton sources at  78  C. Use of [HPR3]þ was introduced by Liddle et al. in their report of the first catalytic activity of a titanium complex (see above).132 With different types of phosphonium acids ([HPR3](BArF4), R ¼ Cy, tBu, nBu) between 6.0 (nBu) and 8.4 (Cy) equivalents of NH3 were obtained. The tested mononuclear Re(I), Re(III) and Re(V) systems were either not active or only barely catalytic with 3.9 equiv. (ReCl3(PNP)) and 3.6 equiv. (Re(N)Cl2(PNP)) of NH3.

342

Biological and synthetic nitrogen fixation

2.12.3.5.6

Chromium

‘The N2 chemistry of chromium (Cr) is exceptional for its diversity, rarity, and richness of depth.’166 Nevertheless, there are not many systems for nitrogen fixation in terms of N2-to-NH3 conversion in the literature. Besides several chromium systems that were able to produce stoichiometric amounts of NH3,167 there are only a few examples of catalytic systems for N(TMS)3 production,168 which is often described as analogue of NH3. N(SiMe3)3 can also be hydrolyzed to NH3, such that one equivalent of N(SiMe3)3 leads to one equivalent of NH3. Very recently, Nishibayashi et al. presented the first examples of catalytic N2-to-NH3 conversion with chromium complexes.129 A chromium(V) nitrido and different chromium(II) halide complexes, all supported by a PCP pincer ligand (29, Fig. 22), were able to produce between 2.16 and 5.77 equivalents of NH3 by using the conditions introduced by Liddle et al.132 They include the combination of KC8 as reductant with phosphonium acids, which has also led to successful catalytic conversion in case of dinuclear rhenium complexes. With higher amounts of reductant and acid [CrI(PCP)](BArF4) (29) even led to 11.60 equivalents of NH3. Interestingly, the analoguous dinuclear chromium complex was not able of mediating the catalytic conversion and with 2.29 equivalents, only a slightly overstoichiometric amount of NH3 was produced. Furthermore, the combination of SmI2 and H2O as reductant and proton source, which lately has very successfully been used in case of the molybdenum complexes96 did not lead to catalytic behavior. In the above-mentioned catalytic systems, a square-planar [Cr(N2)(PCP)] complex has been discussed as possible intermediate, being responsible for the catalytic activity.129

2.12.3.6

Lessons from small-molecule models

While the industrial counterpart of biological nitrogen fixation, the Haber-Bosch process, has been conceived and realized more than a century ago, the goal of converting dinitrogen to ammonia at room temperature and ambient pressure, in analogy to nitrogenase, has fascinated and occupied inorganic and organometallic chemists for the last five decades. As described herein, this problem has been solved in the meantime, leading to a considerable number of catalytic model systems of nitrogenase thatdat least in partdallow converting N2 to NH3 in a catalytic fashion, achieving high turnover numbers and yields. Moreover, significant insight into the reactive pathways of many of these systems has been achieved and a number of mechanistic scenarios for the conversion of dinitrogen to ammonia in homogeneous solution has been identified. Unfortunately, the impact of these studies on the question of where and how exactly N2 is bound and reduced in nitrogenase has been limited so far. This is mostly due to the fact that the active site of this enzyme is a polynuclear iron-molybdenum-sulfur cluster whereas the majority of the smallmolecule systems are either mononuclear or, if dinuclear, exhibit bonding modes of N2 (e.g., linear bridging) which are incompatible with the structure of the FeMoco. Thus, a close synthetic model for the binding of N2 to this cluster which would reproduce both structural as well as electronic features of the FeMoco and mediate (catalytic) conversion to NH3 is not available to date. As mentioned above, dinuclear model systems of nitrogenase, preferably with two iron centers, could significantly improve this situation, potentially providing mechanistic information of high relevance to the enzymatic system; however, none of these systems has been shown to mediate catalytic N2-to-NH3 conversion so far.

2.12.4

Summary: Toward a comprehensive understanding of biological and synthetic nitrogen fixation

Historically, biological and synthetic nitrogen fixation are two research areas that have developed independently. Nevertheless, the structure and function of nitrogenase have always been a major source of inspiration for inorganic, coordination and organometallic chemists dealing with synthetic nitrogen fixation in homogeneous solution, whereas results of small-model chemistry regarding the (catalytic) N2-to-NH3 conversion have provided clues with respect to the molecular mechanism applying to the enzyme. Apart from the common reactiondthe conversion of dinitrogen to ammonia under ambient conditionsda second aspect ‘uniting’ these two research areas has been the distinct difference to the industrial process which performs the same reaction at high temperature and pressure. In the latter, extreme reaction conditions cause N2 to adsorb to an iron catalyst and dissociate into surface-bound nitrides. In a similar way, metal hydrides are formed from H2 in the syngas mixture, and while such hydrides are highly mobile on the catalyst surface, they are stabilized by the high partial pressure of H2 until they combine with the nitrides to eventually yield ammonia as a product. The enzyme, on the other hand, must build up reducing power at ambient conditions at its active site that far exceeds the potentials that are sustainable in an aqueous environment, generating a species that is a sufficiently strong reductant to bind an inert N2 molecule and break its stable triple bond. Exactly at this point small-model chemistry can provide relevant mechanistic insight without being restricted to an aqueous environment. The reaction mechanism of the nitrogenase enzyme is still a story in the making, but the recent advances outline how all these requirements can indeed be fulfilled at a biological metal site. The reaction starts with some of the most strongly reducing electron carriers available in ‘regular’ cellular metabolism, [2Fe:2S] ferredoxins and flavodoxins at a midpoint potential of about  475 mV vs. SHE that transfer electrons to the Fe proteins. Upon docking to the dinitrogenase, the hydrolysis of ATP then triggers an electron to be transferred from P-cluster to the cofactor at an even lower potential. This transfer step occurs at a potential that is sufficiently low to reduce the catalytic cofactor, which in turn is a highly reduced compound to start with. It will only accept a single electron into its core, so that every second electron transfer step leads to the formation of a surface hydride. In nitrogenase, a single hydride is not sufficient to promote either the binding or activation of N2, but at least in the V-dependent isoenzyme it can trigger a reduction cascade for CO that leads to the Fischer-Tropsch-like chemistry outlined above. For N2 reduction, a second hydride must be formed

Biological and synthetic nitrogen fixation

343

to reach the E4 state, and here the reductive elimination of H2 as a stoichiometric byproduct now generates a super-reduced state that is otherwise not accessible to the enzyme and must have an effective midpoint potential substantially below  1 V to achieve its task of reducing N2. The identification of binding sites for substrates, intermediates and/or hydrides at the m-site at the Fe2-Fe6 edge and the t-site at Fe6 helps to rationalize the atomic structure of the intermediates involved in this process and the additional steps that the enzyme takes to stabilize the increasingly reactive intermediates on the path from the resting state E0 to the critical E4 state during the slow-sequential one-electron transfer from the Fe protein via P-cluster to the cofactor. In spite of great differences between the constitution of small-molecule catalysts and the reactive center of nitrogenase, the study of the small-molecule model systems has established a number of mechanistic scenarios for the conversion of dinitrogen to ammonia in homogeneous solution; e.g., the distinction between ‘distal’ and ‘alternating’ reaction modes, which are also relevant to the enzyme. Moreover, small-molecule model systems have provided insight into fundamental parameters of the reduction and protonation of N2 under ambient conditions. Just like in nitrogenase, a key parameter for the catalytic activity of a particular model system is the activation imparted to the bound N2 molecule by the transition-metal center(s).75 Although dinitrogen binds to one of the iron-centers of the FeMoco (see above), the range of transition metals and coordinating ligands available to small-molecule model chemistry allows tuning this activation in an almost continuous fashion, from very weak to very strong. Notably, an increasing degree of activation requires an increasing free energy input (see below), and on this background it needs to be remembered that nitrogenase uses iron centers in the FeMoco which in iron-sulfur clusters attain redox states of þ 3 and þ 2, only weakly activating N2.75 Correspondingly, no catalytic activity toward N2-reduction has been achieved in small-molecule model systems based on iron in these redox states. In this context, the multinuclear constitution of the FeMoco comes into play, as well as its ability to store redox equivalents in the form of hydrides (see above). Reductive elimination of H2 from a dihydrido intermediate appears to be the (only) way for the enzyme to reach the activation of N2 necessary for its protonation, and model chemistry may be able to provide further insight into the intriguing interplay between hydride formation and dinitrogen activation/reduction. The second key parameter of biological and synthetic nitrogen fixation refers to the necessary energy input. Contrary to a widespread notion of industrial ammonia synthesis based on the Haber-Bosch process being particularly energy-consuming due to the high temperatures and pressures involved,170 performing this reaction at room temperature and normal pressure requires a much higher energy input which in nature is provided by coupling N2-reduction to an infinite source of energy, photosynthesis. As a matter of fact, the energy expenditure of biological nitrogen fixation and a synthetic model system have been shown to be comparable (ca. 180 kcal/mol per N2 molecule being converted to 2 NH3).65 Third, biological and synthetic nitrogen fixation both mediate a multi-electron/multi-proton conversion of a highly inert substrate. In order to perform this task efficiently, a controlled/gated delivery of both protons and electrons is necessary. In synthetic systems, failure to meet this condition invariably leads to hydrogen evolution becoming dominant. As shown by many experimental and theoretical investigations, the multi-electron/multi-proton conversion of a small-molecule substrate like dinitrogen proceeds in a strictly alternating fashion and thus is favorably mediated by using PCET reagents. One of the remaining challenges of synthetic nitrogen fixation, efficiently performing this reaction in an electrocatalytic fashion,171 may be limited by the requirement to realize such PCET conditions in an electrochemical environment. This problem has recently been addressed by employing a PCET reagent as redox mediator in the electrochemical conversion of dinitrogen to ammonia.119 Whereas synthetic nitrogen fixation aims at totally suppressing hydrogen evolution as a side reaction, it is an integral part of the biological process. On the other hand, nitrogenase has to tolerate water as a solvent, and the generation of a critical overpotential thus must be precisely timed and coordinated with the presence of a suitable substrate other than a water molecule. The reductive elimination of H2 from two bridging hydrides can achieve just this and is widely known from model chemistry. Nitrogenases offer the possibility for bridging and terminal binding of adjacent hydrides and/or substrates at a dinuclear site, combined with the rigid iron-carbide prism at the core of the cofactors. Furthermore, they tightly control access for protons to this site as a key prerequisite to stabilize bound hydrides and prevent or limit the unwanted formation of H2 before a four-electron reduced state has been reached. The mechanistic chemical basis of nitrogenase action thus also holds the potential to inspire a new generation of homogeneous or even heterogeneous catalysts for challenging reduction reactions at ambient conditions and in an aqueous environment.

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Imayoshi, R.; Nakajima, K.; Nishibayashi, Y. Chem. Lett. 2017, 46, 466. Klopsch, I.; Finger, M.; Würtele, C.; Milde, B.; Werz, D. B.; Schneider, S. J. Am. Chem. Soc. 2014, 136, 6881. a van Alten, R. S.; Wätjen, F.; Demeshko, S.; Miller, A. J. M.; Würtele, C.; Siewert, I.; Schneider, S. Eur. J. Inorg. Chem. 2020, 2020, 1402; b Schendzielorz, F.; Finger, M.; Abbenseth, J.; Würtele, C.; Krewald, V.; Schneider, S. Angew. Chem. Int. Ed. 2019, 58, 830; c Lindley, B. M.; van Alten, R. S.; Finger, M.; Schendzielorz, F.; Würtele, C.; Miller, A. J. M.; Siewert, I.; Schneider, S. J. Am. Chem. Soc. 2018, 140, 7922; d Klopsch, I.; Kinauer, M.; Finger, M.; Würtele, C.; Schneider, S. Angew. Chem. Int. Ed. 2016, 55, 4786. Bruch, Q. J.; Connor, G. P.; Chen, C.-H.; Holland, P. L.; Mayer, J. M.; Hasanayn, F.; Miller, A. J. M. J. Am. Chem. Soc. 2019, 141, 20198. Kendall, A. J.; Mock, M. T. Eur. J. Inorg. Chem. 2020, 2020, 1358. a Egbert, J. D.; O’Hagan, M.; Wiedner, E. S.; Bullock, R. M.; Piro, N. A.; Kassel, W. S.; Mock, M. T. Chem. Commun. 2016, 52, 9343; b Mock, M. T.; Pierpont, A. W.; Egbert, J. D.; O’Hagan, M.; Chen, S.; Bullock, R. M.; Dougherty, W. G.; Kassel, W. S.; Rousseau, R. Inorg. Chem. 2015, 54, 4827; c Mock, M. T.; Chen, S.; O’Hagan, M.; Rousseau, R.; Dougherty, W. G.; Kassel, W. S.; Bullock, R. M. J. Am. Chem. Soc. 2013, 135, 11493; d Sobota, P.; Jez_ owska-Trzebiatowska, B. J. Organomet. Chem. 1977, 131, 341. a Yin, J.; Li, J.; Wang, G.-X.; Yin, Z.-B.; Zhang, W.-X.; Xi, Z. J. Am. Chem. Soc. 2019, 141, 4241; b Kendall, A. J.; Johnson, S. I.; Bullock, R. M.; Mock, M. T. J. Am. Chem. Soc. 2018, 140, 2528; c Shiina, K. J. Am. Chem. Soc. 1972, 94, 9266. Kokubo, Y.; Wasada-Tsutsui, Y.; Yomura, S.; Yanagisawa, S.; Kubo, M.; Kugimiya, S.; Kajita, Y.; Ozawa, T.; Masuda, H. Eur. J. Inorg. Chem. 2020, 2020, 1456. Capdevila-Cortada, M. Nat. Catal. 2019, 2, 1055. Singh, A. R.; Rohr, B. A.; Schwalbe, J. A.; Cargnello, M.; Chan, K.; Jaramillo, T. F.; Chorkendorff, I.; Nørskov, J. K. ACS Catal. 2017, 7, 706.

2.13

Photosynthesis

Junko Yano, Jan Kern, and Vittal K. Yachandra, Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States © 2023 Elsevier Ltd. All rights reserved.

2.13.1 2.13.2 2.13.3 2.13.3.1 2.13.3.2 2.13.3.3 2.13.3.3.1 2.13.3.3.2 2.13.4 2.13.4.1 2.13.4.2 2.13.4.3 2.13.4.3.1 2.13.4.3.2 2.13.4.3.3 2.13.4.3.4 2.13.4.3.5 2.13.4.4 2.13.5 2.13.5.1 2.13.5.2 2.13.5.3 2.13.5.4 2.13.6 2.13.7 2.13.8 2.13.8.1 2.13.8.2 2.13.8.3 2.13.8.4 2.13.8.5 2.13.8.6 2.13.9 References Further reading

Introduction Photosynthetic reaction centers Function of photosystem II Architecture of photosystem II Electron transfer chain Energetics of the water oxidation reaction Redox potential Quantum efficiency S-state transition of the oxygen evolving complex Kok cycle Capturing intermediate S-states The OEC structure The S1 state The S2 state The S3 state The S0 state Structural/Spin isomers in each S-state and its functional role Structural changes during the S-state transitions Channels Identifying channels Oxygen channel Water channel Proton channel Mechanism of photosynthetic O2 evolution Light-driven assembly of the manganese cluster Several techniques that are fundamental to the PSII research EPR Mass spectroscopy X-ray spectroscopy Infrared spectroscopy X-ray crystallography at X-ray free electron lasers Cryo-electron microscopy Perspective

348 349 349 349 350 351 351 352 352 352 354 354 355 356 356 356 356 357 357 357 359 359 359 359 360 363 363 365 365 368 368 369 369 370 372

Abstract Among many processes occurring in oxygenic photosynthesis, the water oxidation reaction catalyzed by the Mn4Ca cluster provides various types of insights into the field of the metal coordination chemistry. The water oxidation reaction in nature is carried out by Photosystem II (PS II), a multi subunit membrane protein complex. This light-driven reaction is made possible by a spatially separated, yet temporally connected series of cofactors along the electron transfer chain of PS II over 40 Å, through the donordthe Mn4CaO5 catalytic center, the reaction center chlorophylls, to the mobile quinone electron acceptors. Such chemical architecture provides an ideal platform to investigate how to control multi-electron/proton chemistry, using the flexibility of metal redox states, in coordination with the protein and the water network. Understanding the insights of nature’s design gives inspiration of how to build artificial photosynthetic devices, where the controlled accumulation of charge and high-selectivity of products is currently challenging. The electronic and geometric structure of this catalyst have been extensively investigated, but its step-wise water oxidation mechanism is not yet completely understood. In this chapter, we summarize our current understanding of the water oxidation reaction in nature.

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00162-X

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Photosynthesis

Introduction

O2, that supports our life, was largely generated by photosynthetic water oxidation in plants, algae, and cyanobacteria. This process evolved probably 2–3 billion years ago in the precursors to present-day cyanobacteria.1–4 Oxygen could have been a pollutant that made anaerobic life less likely, but it enabled oxygenic life that led to the current diverse and complex life on earth by dramatically increasing the metabolic energy that became available from aerobic respiration. Oxygen produced by this process was also key for the development of the protective ozone layer, that allowed life to transition from marine forms to terrestrial life. A critical component in the evolution of oxygenic photosynthesis was a Mn4CaO5 cluster, that stores oxidizing equivalents to catalyze the four-electron oxidation of H2O to O2.5–7 Then, the water derived electrons travel along the photosynthetic electron transfer chain, where they gain further reducing power by the photoreaction. These electrons, together with the proton gradient produced through these reactions are used to store energy in the form of ATPs (adenosine triphosphate), the energy currency of cells, that are eventually used for the reduction of CO2 in the Calvin–Benson cycle to make compounds (carbohydrates) which serve as precursors for synthesis of the biological molecules needed by the organism. Part of such biomass was converted by geological processes into coal, oil and natural gas, which we have been using as major energy sources. In the photosynthetic energy conversion reaction process, light energy is converted to chemical energy in two types of membrane protein complexes, Photosystem I (PS I) and Photosystem II (PS II) (Fig. 1).8–11 These are located in the thylakoid membrane inside the chloroplasts. They form an assembly with light-harvesting antenna complexes that collect the light and transfer the excitation energy to the reaction centers in PS I and PS II. In the reaction center, a charge separation occurs and a positive (hole) and a negative charge (electron) are created. This initial charge separated state needs to be stabilized by a sequence of electron transfer reactions that reduce the free energy difference and increase the distance between the two opposite charges. This stabilization allows the reaction centers to couple the fast one-electron charge separations with chemical reactions that are several orders of magnitude slower and involve multiple electrons and protons. In PS II, the energized electron is transferred from the excited chlorophyll molecule through an electron transfer chain(s) to an electron acceptor. The holes are used to oxidize the Mn4CaO5 cluster in a stepwise-manner. This metal complex embedded in PS II, called the oxygen-evolving complex (OEC), seems to be preserved in oxygenic photosynthetic organisms through cyanobacteria, algae, up to higher plants. It suggests that the earlier incorporation of the Mn4CaO5 cluster never changed through the evolution tree. Nature has thus evolved an elegant way to store energy from sunlight in the form of chemical energy. Presently, researchers are on their way to unraveling the details of this unique biological energy conversion process. The need to develop renewable energy resources, that are carbon neutral, has highlighted the importance of learning how nature accomplishes the photosynthetic process.12 This knowledge could be critical for creating artificial photosynthetic systems to facilitate building artificial devices that, by employing the same principles, are able to store solar energy within fuels.13 One of the key questions is how nature manages the uphill photo-induced water oxidation reaction. In this chapter, we describe the bioinorganic chemistry of the water oxidation reaction that occurs in PS II. PS II serves as fertileground for bioinorganic chemists to understand multielectron/multiproton catalysis, the role of the protein environment, spectroscopy, and crystallography. At the same time, we hope to highlight the concepts that may be important for developing artificial systems for converting solar energy into fuels.

Fig. 1 A schematic view of photosynthetic protein complexes embedded in the thylakoid membrane. Except for ATP synthase, all the complexes are required for the light-dependent linear electron transfer from water to NADP þ. Under some conditions, electrons on the electron acceptor side of PS I cycle back toward Cytochrome b6f complex and then again toward PS I, thus performing a cyclic electron transfer. PQ: plastoquinone, PQH2: plastoquinole, PC: plastocyanin, FNR: ferredoxin-NADPþ reductase. Adapted from Shevela, D.; Kern, J. F.; Govindjee, G.; Whitmarsh, J.; Messinger, J. In eLS; John Wiley & Sons, Ltd, Ed.; Wiley, 2021; pp 1–16 with a permission.

Photosynthesis

2.13.2

349

Photosynthetic reaction centers

The regulation and fine tuning of cofactor properties by the interaction with the protein environment are key to direct the flow of electrons in photosynthetic protein assemblies.14,15 The proteins that drive photosynthesis are structurally analogous across prokaryotes and eukaryotes. It has been hypothesized that eukaryotes gained the ability to perform photosynthesis through a symbiosis with photosynthetic bacteria. There are two classes of photosynthetic reaction centers, Type-1 uses iron sulfur cluster as electron acceptors, and Type-2 uses quinones. The former includes green sulfur bacterial reaction center,16 heliobacterial reaction center,17 and Photosystem I.18 The latter includes purple bacteria and Photosystem II. The oxygenic photosynthetic organisms need both PS II and PS I. Light is initially absorbed by the chlorophylls at the core of PS II (P680) or the antenna complexes that surround it.19 This oxidized form of P680 (P680þ) is the strongest oxidizing agent in biological systems, and is used to oxidize water at the oxygen evolving complex, while reducing the terminal electron acceptor, plastoquinone to plastiquinol. Besides charge separation and electron transfer, the process generates protons, and a proton gradient is thereby formed across the thylakoid membrane. This proton gradient drives ATP synthesis, that produces ATP, which is a chemical form of the energy currency in biological systems. Thus, the light energy is converted to chemical energy. Plastiquinol further shuttles electrons into PS I and the reduced ferredoxin at the stromal side of PS I drives reduction of NADPþ to NADPH. The ATP and NADPH are then used in the Calvin–Benson cycle for the conversion of CO2 to biomass.20 In these reaction centers, the redox potential is tuned to ensure an efficient forward electron transfer and minimizing deleterious or wasteful back reactions by subtle changes in the cofactor environment. Upon the formation of the charge separated state, the protein environment responds in the ultrafast time-scale. This rearrangement, starting from shifts in H-bonding, but also extending to overall movements of the ligands and backbone participating in the specific binding pocket, is thought to be essential for stabilizing the charge separated state and guiding the forward reaction. The limiting time for generating a primary radical pair in both type I and type II reaction centers is around 1 ps. This limit may be connected to the protein relaxation dynamics that have to provide the free energy difference that drives the energy trapping in the forward direction, as without dynamic energy relaxation of the early radical pairs no efficient photosynthetic charge separation would be possible.

2.13.3

Function of photosystem II

2.13.3.1

Architecture of photosystem II

PS II is a membrane pigment-protein complex, where as described above, charge separation by absorption of light and successive electron transfer occurs via vectorially arranged pigment molecules along the thylakoid membrane. Fig. 2 shows the structure of PS II and its electron transfer chain. The overall structure of PS II is well understood through the X-ray crystallography studies over the years since the first structure reported in 2001 and significant improvements in the data quality since then. PS II is present as a dimer with a molecular weight of  750 kDa. Each monomer contains 19 protein subunits, and cofactors that include 36 chlorophyll a (Chl), 7 carotenoids (b-carotene), and the oxygen evolving complex (OEC). The subunits are the reaction center core proteins D1 and D2, core antenna proteins CP43 and CP47, cytochrome b559, and other small proteins that are less than < 10 kDa. Despite a wide span of evolution from cyanobacteria to higher plants, the core of PS II is highly conserved from

Fig. 2 Arrangement of cofactors of the electron transfer chain in photosynthetic reaction centers and reaction times for electron transfer processes along the co-factor chains in PS II (left) and PS I (right). OEC: Oxygen evolving complex, Chl: chlorophyll, QA and QB, primary and secondary plastoquinone electron acceptors; PQ, mobile plastoquinone molecules, Pheo: pheophytin. In PS I, A0 is the primary electron acceptor and A1 is the pair of phylloquinone (vitamin K) molecules, and FX, FA, and FB are the bound iron-sulfur clusters. Chl eCA1, eCB2, eCA3, and PhQA show one side of the electron transfer chain, and Chl eCB1, eCA2, eCB3, and PhQB show the other side.

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cyanobacteria to higher plants, with some minor differences. For example, the extrinsic subunits are PsbO, PsbP, PsbQ, and PsbV in cyanobacteria, which are proximal to the OEC. Higher plants in contrast contain PsbO, PsbP, PsbQ, and PsbR.21,22 PS II is surrounded by peripheral antenna pigment-binding proteins, containing chlorophylls and carotenoids. The optical absorption cross-section of the reaction centers like PS I and PS II is increased by an outer antenna system composed of lightharvesting complex (LHC) subunits. The absorption of a photon by one of the chlorophylls in the pigment-binding proteins results in the formation of an excited state of a chlorophyll, that is, an electron is excited from the highest occupied molecular orbital to the lowest unoccupied orbital of higher energy. This excitation energy hops from one pigment to another and then is funneled to the reaction center. Unlike the reaction centers, these antenna structures and its assemblies are highly different in various species. In cyanobacteria, it is made of phycobilisomes, located on the cytoplasmic side of PS II.23 On the other hand, the higher plant PS II has light-harvesting chlorophyll a/b-binding complex II (LHCII, composed of LHCB 1, LHCB 2, and LHCB 3) that are membrane proteins, together with monomeric antennae LHCB 4, LHCB 5, and LHCB 6.24,25 The core of PS II is composed of two homologous polypeptides (D1 and D2), each of which consists of five transmembrane helices. It binds various redox cofactors that take part in charge separation, transfer and stabilization, which includes a chlorophyll dimer (PD1PD2), two chlorophyll monomers (ChlD1 and ChlD2), two pheophytins (PheoD1 and PheoD2), and two quinones QA and QB. The Mn4CaO5 cluster at the luminal side of the membrane is the catalytic center for the water oxidation reaction. The redox active tyrosine (TyrZ), located between P680 and the OEC, couples the photochemistry at P680 and four-electron redox chemistry at the OEC.

2.13.3.2

Electron transfer chain

The light-induced charge separation occurs at the reaction center in PS II, with the excitation energy funneled into it from the antenna proteins. PS II monomer consists of two symmetrical branches of cofactors with a pseudo C2 symmetry axis perpendicular to the membrane plane, similar to other reaction centers. The electron transfer, however, only proceeds in one of these two branches. Such symmetry in the electron transfer chain, but asymmetry in function is commonly observed in all the reaction centers (PS II, PS I, bacterial reaction centers).15 The chlorophyll a moiety P680, consisting of the four excitonically coupled chlorophylls, PD1, PD2, ChlD1, and ChlD2, absorbs visible light with maximum absorption at  680 nm, leading to primary charge separation on a picosecond timescale (Fig. 2).26 To suppress side effects and back reactions, nature has placed secondary donor/acceptor pigments in close proximity to P680 (Fig. 3). The negative charge (electron) is rapidly transferred to the acceptor side toward the cytoplasm, and the positive charge (hole) is transferred to the donor side for charge stabilization. Located  10 Å from ChlD1 is an electron acceptor PheoD1, yielding the   26 charge-separated radical pair state (P680þ PheoD1 ), with the radical cation P680þ and the radical anion PheoD1 . This state is

Fig. 3 Co-factor distances of PS II from the room temperature crystallography. Distances for central cofactors are shown. OEC: Oxygen evolving complex, Chl: chlorophyll, QA and QB: primary and secondary plastoquinone electron acceptors, PQ: mobile plastoquinone molecules, TyrD and TyrZ: the redox-active tyrosine residues. Adapted from Young, I. D.; Ibrahim, M.; Chatterjee, R.; Gul, S.; Fuller, F. D.; Koroidov, S.; Brewster, A. S.; Tran, R.; Alonso-Mori, R.; Kroll, T.; Michels-Clark, T.; Laksmono, H.; Sierra, R. G.; Stan, C. A.; Hussein, R.; Zhang, M.; Douthit, L.; Kubin, M.; de Lichtenberg, C.; Vo Pham, L.; Nilsson, H.; Cheah, M. H.; Shevela, D.; Saracini, C.; Bean, M. A.; Seuffert, I.; Sokaras, D.; Weng, T.-C.; Pastor, E.; Weninger, C.; Fransson, T.; Lassalle, L.; Bräuer, P.; Aller, P.; Docker, P. T.; Andi, B.; Orville, A. M.; Glownia, J. M.; Nelson, S.; Sikorski, M.; Zhu, D.; Hunter, M. S.; Lane, T. J.; Aquila, A.; Koglin, J. E.; Robinson, J.; Liang, M.; Boutet, S.; Lyubimov, A. Y.; Uervirojnangkoorn, M.; Moriarty, N. W.; Liebschner, D.; Afonine, P. V.; Waterman, D. G.; Evans, G.; Wernet, P.; Dobbek, H.; Weis, W. I.; Brunger, A. T.; Zwart, P. H.; Adams, P. D.; Zouni, A.; Messinger, J.; Bergmann, U.; Sauter, N. K.; Kern, J.; Yachandra, V. K.; Yano, J. Nature. 2016, 540 (7633), 453–457.

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further stabilized by successive electron transfer steps to the two plastoquinones, QA and QB. QA is fixed and mediates a single electron transfer process. On the other hand, QB is a mobile electron carrier; after double reduction and protonation, QB leaves PS II (as a QBH2) and is replaced by another plastoquinone molecule. Thus, two reducing equivalents are stored as QBH2 generated after two light absorption/charge separation and protonation events. The radical cation P680þ is reduced back to P680 by the redox active tyrosine residue, TyrZ (or D1-Y161 or Yz), that donates an electron to the reaction center on a nanosecond (20–250 ns) time scale. TyrZ is, in turn, reduced by the electron extracted from the water oxidation reaction in the OEC. TyrZþ is stabilized by shifting a proton to the neighboring D1-His190 in a reversible fashion, forming a neutral tyrosine radical (TyrZ). Yz, then oxidizes the OEC in the ms to sub ns time scale. The electrons originating from water molecules travel from PS II, Cyt b6f, and PS I along the photosynthetic electron transfer chain (Fig. 1).8 In the process, gaining additional reducing power by the reaction center of PS I. This reducing power is finally used at the Calvin–Benson cycle for reducing carbon dioxide. Thus, water oxidation reaction and CO2 reduction reaction are coupled in the natural photosynthetic systems with energy originating from visible light absorption.

2.13.3.3 2.13.3.3.1

Energetics of the water oxidation reaction Redox potential

Water is a stable compound under physiological conditions, and oxidizing it requires a standard potential of 1.23 V (vs NHE), that requires strongly oxidizing species (at pH 7.0 the midpoint potential of the H2O/O2 couple is 810 mV per electron). PS II is this strongly oxidizing entity in nature, where the oxidizing power is generated by light absorption by the reaction center, P680, converting this energy into electrochemical potential and leading to the accumulation of the oxidizing potential at the OEC to be used for the water oxidation reaction.12 In this mechanism, a tyrosine residue YZ, located between the reaction center (P680) and the OEC, plays a critical dual role. After charge separation, P680þ rapidly extracts an electron from the neighboring YZ, going back to P680. The tyrosine radical cation Yzþ is stabilized by losing a proton to the neighboring D1-His190 in a reversible fashion, forming a neutral tyrosine radical, Yz. Then Yz$oxidizes the Mn cluster at each step. Effectively, the OEC couples the one-electron photochemistry occurring at the P680 reaction center to the four-redox chemistry required for the oxidation of water to dioxygen. In this manner, the enzyme prevents the formation and the release of reactive one-electron intermediates of the water oxidation reaction, such as hydroxide radicals or peroxide or superoxide in free form, which could potentially damage the protein/lipid environment. It is interesting to compare the requirement of Gibbs energy for oxidizing water in aqueous solution and in the OEC in PS II.27 The water oxidation reaction step is unique in the OEC as it stores oxidizing equivalents by oxidizing four Mn in a step-wise manner, instead of directly oxidizing water. Then in the last step the four-electron chemistry occurs by using the redox power stored in the OEC. The energetic cost is significantly lowered in this case and as close to the thermodynamic minimum as possible, as the fourelectron redox process is nearly equally leveled, and the high-energy first step, seen in aqueous solution is avoided (Fig. 4). Not oxidizing water directly also helps avoid releasing harmful chemical intermediates, such as H2O2 and OH into the biological environment as described above.

Fig. 4 Gibbs energy for oxidizing water in aqueous solution and in the OEC of PS II. In aqueous solution, the removal of the first electron takes the highest in energy. In the biological system, the requirement of such high-energy step is avoided; by oxidizing the Mn4CaO5 cluster and storing oxidizing equivalents through the four consecutive steps that occur with the absorption of four photons at the reaction center chlorophylls, the redox process is leveled. Copyright from Messinger, J.; Renger, G. Chapter 17 Photosynthetic Water Splitting, In Primary Processes of Photosynthesis, Part 2: Principles and Apparatus; The Royal Society of Chemistry, 2008; Vol. 9, pp 291–349.

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2.13.3.3.2

Quantum efficiency

The solar energy conversion efficiency, hsolar, is defined as the fraction of the incident solar energy absorbed by the antenna pigments that is eventually stored in the form of chemical products. For PS II, Dau and Zaharieva estimated that theoretically  34% of the incident solar energy can be used for creation of the photochemistry-driving excited state, P680* at high concentrations28 with an excited-state energy of 1.83 eV (Fig. 5). Subsequent electron transfer to create TyrZþ/QA radical pair state happens within 1 ms. Separating positive and negative charge in distance along the membrane axis reduces its recombination loss and stabilizes the radical pair. At this level, the maximal hsolar is  23%. At the water oxidation step, this number becomes  16%.

2.13.4

S-state transition of the oxygen evolving complex

2.13.4.1

Kok cycle

The PS II accomplishes a complex task of converting the one-electron photoexcited state of chlorophyll at the reaction center into the concerted four-electron oxidation of two water molecules at the Mn4CaO5 catalytic center. Since the insightful experiment and hypothesis by Pierre Joliot and Bessel Kok based on the kinetics of oxygen evolution as a function of flashes number in the water oxidation reaction in the 1970s2930, it has been known that water oxidation reaction requires four photons to complete its reaction cycle. Illumination of dark-adapted PS II samples with saturating flashes of light leads to the production of O2 in a characteristic pattern peaking on the 3rd flash, and then repeating every four flashes (Fig. 6). It was therefore concluded that O2 production takes place at a catalytic center and that each flash advances the oxidation state of the catalytic site by the removal of one electron (Fig. 7). O2 is formed and released only after four oxidation equivalents are accumulated. The stoichiometry of the inorganic cofactors that make up the OEC consists of four Mn and one Ca. This was determined by various spectroscopic and biochemical studies, and confirmed by X-ray crystallography.31–33 Fig. 7 shows the redox cycle of the Mn4CaO5 cluster (Kok cycle),29 together with the charge separation at P680, redox change of Tyr, and the oxidation state changes of the acceptor side quinone (QA/QB). PS II carries out the reaction by coupling the one-electron photochemistry occurring at the reaction center with the four-electron oxidation of water at the OEC. 2H2O / O2 þ 4Hþ þ 4e The metal center that catalyzes this reaction consists of an oxo-bridged structure with four Mn and one Ca atom (Mn4CaO5 cluster), where O2 is a biproduct of this reaction. The OEC cycles through five intermediate S-states (S0 to S4) that correspond to the abstraction of four successive electrons from the OEC via the redox-active tyrosine residue (Yz). Once four oxidizing equivalents accumulate at the OEC (metastable or transient S4-state), the release of O2 and the formation of the most reduced S0-state take place

Fig. 5 Estimated energy levels and energetic efficiency of Photosystem II. Gray box: fractional energy yield, Orange box: hSOLAR (the overall efficiency of solar energy conversion). Black arrows show the forward reactions, and the red dotted arrows show loss paths. The 6G-axis indicates the drop in free energy (in meV). The excited state of P680* is 1.83 eV above the ground state, and is chosen as the zero point of the 6 G-axis. Estimation of the lifetime of the state are also shown in the figure. Adapted from Dau, H.; Zaharieva, I. Principles, Efficiency, and Blueprint Character of Solar-Energy Conversion in Photosynthetic Water Oxidation. Acc. Chem. Res. 2009, 42 (12), 1861–1870.

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Oxygen yield per flash

Photosynthesis

Flash number Fig. 6 Flash-induced oxygen evolution pattern of dark-adapted spinach thylakoids induced by a train of saturating xenon flashes. Adapted from Messinger, J., Berlin, T.U. Untersuchungen u¨ber die reaktiven Eigenschaften der verschiedenen Redoxzusta¨nde Si der Wasseroxidase Ho¨herer Pflanzen, 1993.

spontaneously. In this way, the energetically demanding one-electron oxidation step of water to the hydroxyl radical and the formation of other reactive species is circumvented. After four-fold oxidation, the cluster splits two water molecules into one molecule of dioxygen, and distributed over the reaction cycle a total of four protons are released. The protons are released into the luminal side of the membrane. The intrinsic protons released from substrate water molecules, and the stoichiometry has been estimated to be 1:0:1:2,34 for the S0 to S1, S1 to S2, S2 to S3, and S3 to S0 transitions, respectively. Additional information about the kinetics of the S-state advances, and the stoichiometry of proton release is given in Table 1.

Fig. 7 Kok cycle. Center: The Kok S-state cycle for water oxidation by PS II. Grayddonor side: each of the four photons absorbed by the PS II P680 reaction center oxidizes the tyrosine YZ intermediate, which in turn oxidizes the Mn4CaO5 complex. Bluedacceptor side: QA and QB are reduced on the acceptor side. Left top: A best case steady state S-state population as a function of laser-flash illuminations. 10% miss hit is assumed. Insets: The evolution of the populations of the S-states as a function of time are shown after 1flash (1F), 2 flashes (2F) and 3 flashes (3F). Adapted from Alonso-Mori, R.; Asa, K.; Bergmann, U.; Brewster, A. S.; Chatterjee, R.; Cooper, J. K.; Frei, H. M.; Fuller, F. D.; Goggins, E.; Gul, S.; Fukuzawa, H.; Iablonskyi, D.; Ibrahim, M.; Katayama, T.; Kroll, T.; Kumagai, Y.; McClure, B. A.; Messinger, J.; Motomura, K.; Nagaya, K.; Nishiyama, T.; Saracini, C.; Sato, Y.; Sauter, N. K.; Sokaras, D.; Takanashi, T.; Togashi, T.; Ueda, K.; Weare, W. W.; Weng, T.-C.; Yabashi, M.; Yachandra, V. K.; Young, I. D.; Zouni, A.; Kern, J. F.; Yano, Towards Characterization of Photo-Excited Electron Transfer and Catalysis in Natural and Artificial Systems Using XFELs. J. Faraday Discuss. 2016, 194, 621–638 with a permission.

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S-state transition kinetics and proton release pattern during the water oxidation reaction. Stoichiometry of proton release b

Transition S0 S1 S2 S3

/ S1 / S2 / S3 / S0

Half-time (T1/2) a/Time constant (s) (msec)

pH 7.4

pH 6.3

30 (40) 85 (130) 240 (350) 1300 (1870)

0.5 1.0 1.5 1.0

1.5 1.1 0.5 0.9

From G. Renger, Ref. 35 Note that the 1st digit was rounded, and s was calculated from T1/2. From W. Junge, et al. (Ref. 34) Modified from Junge, W.; Haumann, M.; Ahlbrink, R.; Mulkidjanian, A.; Clausen, J. Electrostatics and Proton Transfer in Photosynthetic Water Oxidation. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2002, 357 (1426), 1407–1418, Renger, G. Photosynthetic Water Oxidation to Molecular Oxygen: Apparatus and Mechanism. Biochim. Biophys. Acta - Bioenerg. 2001, 1503 (1–2), 210–228. a

b

2.13.4.2

Capturing intermediate S-states

When PS II is dark-adapted, it relaxes to the dark stable S1 state. The dark S1 state can be advanced to higher S-states with a laser flash(es) at room temperature. By cryo-trapping the sample after the laser flash, each S-state (S0, S2, S3, in addition to the dark S1 state) has been characterized with various methods. As the S-states do not decay rapidly, freeze-quenching of the illuminated samples at liquid N2 temperature within a couple of seconds is sufficient for trapping these stable intermediates. The yield is never 100% due to the intrinsic limitations in PS II S-state advancement because of misses and double-hits by visible light photons. In the best case, a S-state distribution after each flash is similar to what is shown in the inset table in Fig. 7. If the T1/2 of each S-state reported by Renger35 is used (T1/2 of  30 msec for S0 to S1,  85 msec for S1 to S2,  240 msec to S2 to S3, and  1.3 msec from S3 to S0), the population changes of the S-states after each laser flash looks like the graphs shown in Fig. 7. Note that the T1/2 number somewhat changes depending on the species (plants vs cyanobacteria, for example) and sample preparations.36,37 In addition, more detailed studies by several spectroscopic techniques show that each S-state transition, in particular, S2 to S3 and S3 to S0, cannot be explained by a single exponential curve, and multiple time-constant will be required to explain the accurate kinetics of the Sstate transitions.38 Interestingly, it should be noted that although the time-constants of each of these transitions are in the 30 ms to 1.1 ms timescale, if one wants to generate these states by multiple flash illumination, one needs to wait for considerably longer intervals of 0.5–1 s for the subsequent steps to occur on the acceptor sides, such as for the quinone acceptor QA to transfer the electron to the acceptor QB and for QB to be replaced. The detailed structure of the complete PS II protein is currently available from X-ray crystallography studies as well as from some recent cryo EM studies. The dark S1 structure was obtained first at high resolution under cryogenic temperature.9 This study determined the overall structure of PS II, and also clearly showed the structure of the OEC with some radiation-induced damage (estimated to be  25%) to the Mn4Ca cluster, as the X-ray dose was monitored and minimized. More recently, the introduction of X-ray free electron lasers (XFELs) enabled researchers to not only determine the completely radiation-damage free structure of the OEC, but also to study the higher S-state intermediate structures, as well as the time-resolved structural changes from one state to another. Using this approach, room temperature structures of PS II in the dark ((S1), 1F (S2-rich), 2F(S3-rich), and 3F(S0-rich)) states are reported with resolutions of 2.04–2.08 Å.39 The data from flash-induced states were collected with visible light excitation of PS II crystals in situ. Another technique that was extensively used for structural studies of PS II is the Extended X-ray Absorption Fine Structure (EXAFS) method,40–42 which is used to determine the metal-metal (Mn-Mn and Ca-Mn) or metal-ligand (Mn-ligand and Caligand) distances of the Mn4CaO5 catalytic center. Below is a summary of each S-state structure of the OEC, based on the X-ray crystallography and EXAFS studies.

2.13.4.3

The OEC structure

As exampled in Fig. 7, S-states are mixed after laser illumination, in particular after 2 or 3 flashes and in the higher S-states. Using the characteristic S2-state multiline EPR spectrum, however, one can estimate S-state population in each flash state (0F, 1F, 2F, and 3F), and deconvolute the pure S-state components (S1, S2, S3, and S0). The section below describes the S-state structural information obtained with this approach. We will focus mostly on more recent structural studies, and especially, on the room temperature X-ray crystal structures using the X-ray free electron laser.

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Fig. 8

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The OEC structure with surrounding ligands in the resting S1 state.

2.13.4.3.1

The S1 state

The Mn4CaO5 cluster forms an open cubane-like structure (Fig. 8), in which Mn-Mn and Mn-Ca are oxo-bridged. Three di-m-oxo bridged Mn-Mn distances (Mn1-Mn2, Mn2-Mn3, and Mn3-Mn4) are in the 2.7–2.9 Å range (Fig. 9). The Mn1-Mn3 distance is about 3.2 Å, and Mn1-Mn4 distance is about 4.9 Å. Ca is oxo-bridged with three Mn; Mn1, Mn2, and Mn3 ( 3.4 Å). A di-m-oxo bridged Mn3 and Mn4 is anchored by a bidentate D1-Gly333. Oxo-bridged Mn1-Mn2-Mn3 and Ca forms the open cubane-like structure, with no bond between Mn1 and O5, while Mn4-O5 is covalently bonded: the Mn4-O5 distance is  2.2 Å, while Mn1-O5 is  2.7 Å, leaving Mn1 penta-coordinated. Such open, non-cubane-like structures with a bridged Ca atom had been proposed based on EXAFS studies of PS II solutions, and, especially, by the single crystal polarized EXAFS studies which proposed a structure which is very similar to that from XFEL-based crystallography.43 The general consensus is that the formal oxidation state

Fig. 9 (a) The OEC structure of S-state intermediates. Top panel show the 2mFo-DFc and the isomorphous Fo–Fo difference maps of the OEC in the dark (S1) and 1F (S2-rich) states. (b) Atomic distances in the OEC in each S state in ångström. Standard deviations for metal–metal, metal-bridging oxygen and metal–ligand distances are 0.1, 0.15, and 0.17 Å, respectively. Adapted from Kern, J.; Chatterjee, R.; Young, I. D.; Fuller, F. D.; Lassalle, L.; Ibrahim, M.; Gul, S.; Fransson, T.; Brewster, A. S.; Alonso-Mori, R.; Hussein, R.; Zhang, M.; Douthit, L.; de Lichtenberg, C.; Cheah, M. H.; Shevela, D.; Wersig, J.; Seuffert, I.; Sokaras, D.; Pastor, E.; Weninger, C.; Kroll, T.; Sierra, R. G.; Aller, P.; Butryn, A.; Orville, A. M.; Liang, M.; Batyuk, A.; Koglin, J. E.; Carbajo, S.; Boutet, S.; Moriarty, N. W.; Holton, J. M.; Dobbek, H.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Zouni, A.; Messinger, J.; Yano, J.; Yachandra, V. K. Structures of the Intermediates of Kok’s Photosynthetic Water Oxidation Clock. Nature 2018, 563 (7731), 421–425.

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of Mn is Mn4(III2,IV2) in the dark S1 state, with an integer spin state, with Mn2 and Mn3 in þ 4 and Mn1 and Mn4 in þ 3 oxidation states. These assignments are based on X-ray spectroscopy studies (XANES and XES), analysis of the distances observed in EXAFS and high-resolution cryogenic crystal structures, and the EPR exchange coupling scheme of the S2 state. Note that there is a discussion in the literature about the directionality of the Jahn-Teller axis of the Mn3 þ and their potential variations. Four Mn and one Ca of the Mn4CaO5 cluster are ligated with 7 ligand residues (6 carboxyl and one histidine ligands) and 4 waters (W1-W4) as shown in Fig. 8. W1 and W2 are ligated to Mn4, and W3 and W4 are to Ca. D1 subunit provides most of the ligands to the Mn4CaO5 cluster, except for one from the antenna subunit, CP43. D1-Glu 189 is ligated between Mn1 and Ca in the S1 state. As described in the later section, this ligand moves away from Ca upon the insertion of a new oxygen ligand to Mn1 in the later S-state, thus suggested to play important functional role. Mn1 is also ligated with D1-His332, and this is the only nitrogen ligand to Mn in the OEC. D1-Asp170 bridges between Mn4 and Ca, the C-terminal of D1-Ala344 bridges Mn2 and Ca. D1-Glu333 is a bidentate ligand to Mn4 and Mn3, and D1-Asp342 is to Mn1 and Mn2. Mn2 is ligated with D1Asp342. Mn1 is clearly five coordinate and the structure is ‘open’ and retains this motif in the S2 and S0 states.

2.13.4.3.2

The S2 state

2.13.4.3.3

The S3 state

Upon the S1 to S2 transition, one Mn is oxidized from þ 3 to þ 4. As is expected based on the similarity of the S1 and S2 state EXAFS spectra44, the geometry of the cluster remains fundamentally the same as that in the dark S1 state in the crystallography data,39 with the coordination numbers of all the metals preserved. This ‘right-open’ geometry is consistent with the S2 state model, total spin (Stotal) 1/245,46 with Mn4 oxidized from þ 3 to þ 4. The Mn4-O5 distance is longer than what is observed in typical sixcoordinated MnIV environment in inorganic complexes and suggests an open coordination site. From EPR data obtained at cryogenic temperature, it is known that there are two S2 forms, the low spin Stotal ¼ 1/2 and the high-spin Stotal ¼ 5/2 form.47 The low spin Stotal ¼ 1/2 corresponds to the right-open geometry. For the high-spin Stotal ¼ 5/2 form, on the other hand, there has been a debate on its structure. There are three proposals from the DFT calculations, that include (i) a closed cubane structure in which O5 is ligated to Mn1 (þ 3), and Mn4 (þ 4) is five-coordinated (left-open structure), (ii) right-opened geometry with a different protonation state of oxo ligand, and (iii) the structure with the 6th water ligand inserted in Mn1 already in the S2 state. 46,48–52 No clear evidence for the left-open structure is so far observed in the crystallography data in the literature up to now.

Upon a second illumination (2F), the majority of the reaction centers advance from S2 to S3. In the S3 state, all four Mn are proposed to be formally in the þ 4 oxidation state with all hexacoordinated. This is the last stable intermediate prior to the O–O bond formation process. The O5 omit electron density map, in which the O5 atom was omitted from the structural model, overlaid with the 2Fo-Fc map (Fig. 9) shows evidence of additional density observed near Mn1, that can be assigned to an inserted ligand (hydroxo or oxo). In the 0F and 1F data, there is only one envelop of density observed, that corresponds to the O5 position. In the 2F data, however, an additional omit map feature is observed near Mn1, that can be assigned to an inserted water (hydroxo/oxo). This Ox is also bound to Ca (2.46 Å). The Ca coordination number, however, remains eight, as D1-Glu189 that is ligated to Ca in the S1 and S2 states (2.78 and 2.69 Å, respectively) moves away from Ca in the S3 state (3.01 Å) in order to make space for Ox. These atomic movements are also accompanied by changes in the positions of several ligands near or coordinating the catalytic cluster. The O5-Ox distance is about 2.0 Å.39,53 Note that the shorter distance can be interpreted as a peroxide-like bond formation to occur in the S3 state, which would have to be accompanied by the reduction of two Mn from þ 4 to þ 3. This conflicts with the various spectroscopic observations up to now3,23,34, as well as in situ XES data collected simultaneously with the diffraction data, which does not show any such reduction of Mn. Therefore, no peroxo-bond formation between O5 and Ox is likely in the S3 state. The S2 to S3 state transition is accompanied by noticeable Mn-Mn distance changes,41 and several factors such as Ca-depletion, site-specific mutations, and chemical treatments (for example, with fluoride) are known to block this transition.47,54–56 The requirement for a structural change, and its susceptibility to many chemical and biochemical treatments, makes the S2 to S3 transition one of the critical steps for the water oxidation reaction during the S-state cycle.57

2.13.4.3.4

The S0 state

2.13.4.3.5

Structural/Spin isomers in each S-state and its functional role

With a third visible laser flash (3F), the major fraction of PS II goes from the highest-oxidized S3 to the most reduced S0 state by releasing O2 and acquiring one water molecule and resetting the catalytic cycle. In the 3F data collected 200 ms after the 3rd flash, we estimated the S0 population to be 60%, in line with observation of residual density for Ox at  40% of the level observed in the 2F state. The deconvoluted S0 structure shows the loss of Ox and the return to the ‘right-open’ structure similar to the dark resting state (Fig. 9). In the S0 state, the oxidation state of the four Mn is Mn(III)3Mn(IV). While there is no definitive assignment, it is suggested that Mn1, Mn3, and Mn4 are þ 3 and Mn2 is þ 4, based on the EPR data.45,58,59

It has been observed that there are spin isomers in the S-states, as reported using EPR spectroscopy (reviewed in Refs. 47,55). The most discussed one is the high-spin and low-spin states of the S2 state, and its functional role has been discussed actively in the literature. Yet, the structural reasons, if any, for this change, and whether such isomers play an important role in the catalytic mechanism has

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357

not been understood. For example, the importance of the isomorphism observed in the S2 state has been suggested in recent studies,45,46,60 in relation to the transition to the S3 state. Similarly, there have been suggestions of isomorphism in the S1 and S3 states.45,48,61,62 The difficulty to prove the role of isomers, however, stems from the fact that each set of experimental data contains its own uncertainty and they are often collected under different experimental conditions. Thus, extracting the structural and electronic heterogeneity that is functionally important, while eliminating others that might be due to variations in experimental conditions and uncertainty of the experimental data becomes important. From EPR studies of the S2 state, it is clear that there is a high-spin (Stotal ¼ 5/2) (HS) and a low spin (Stotal ¼ 1/2) (LS) form.63 As discussed above, it has been proposed that the proposed geometric and electronic structural flexibility in S2 may play a role in the formation of the S3 state through water binding.46,60,64–66 However, there are no structural studies, either from X-ray crystallography or EXAFS studies, that indicate that there are any major structural changes, thus different protonation states of the oxo-bridges may be the likely cause of these spin-state changes.

2.13.4.4

Structural changes during the S-state transitions

All the stable intermediates of PS II described above serves as the steppingstone for understanding the catalytic process. In the next step, sequence of events that occurs during each S-state transition can be looked at for understanding why one state proceeds to another by following a certain pathway. Snapshots of crystallography data taken at timepoints during the transition helps capture key events during the process.67,68 Here we use the S2 to S3 transition as an example, since this process involves an insertion of the first substrate water into the OEC. Four transient points, 50, 150, 250, and 400 msecs after the second flash are reported. Fig. 10 shows the schematics of structural changes around the OEC. The earlier event that is observed is the motion of tyrosine residue (TyrZ) at 50 msec, which is located between the reaction center chlorophylls (P680) and the OEC. Oxidation of TyrZ after the charge separation at P680 occurs with a T1/2 of in the nsec to msec range. The strong hydrogen bonds between the O of TyrZ and Ns of His190 makes PCET possible upon charge separation.69 The motion at TyrZ is accompanied by the ligation environment changes of the Glu189 residue that is about 4 Å away from TyrZ. This residue, which is ligated to Mn1 as well as weakly interacting with Ca in the S1 and S2 state, moves away from Ca, but remains a monodentate ligand to Mn1 prior to the insertion of Ox between Mn1 and Ca. The changes at the OEC starts becoming clear at 150 ms and the elongation of the Mn1-Mn4 distance from  4.8 to 5.2 Å is observed.67 Followed by the Mn1-Mn4 elongation, the Ox density starts to appear as a positive electron density between Ca and Mn1. At 400 msec, the OEC structure is similar to that of the stable S3 state, indicating that in most of the centers the changes at the OEC are complete around this time point. The Ox insertion kinetics roughly matches with the observation of the Mn oxidation kinetics obtained from the Kb1,3 XES. A similar effort can be taken to understand the S3 to S0 step, where the O–O bond formation, O2 release, and the resetting of the catalytic center occurs. However, the S-states are more mixed in the later state, and therefore higher resolution structures will be required to capture changes in this last step. This effort is in progress.

2.13.5

Channels

2.13.5.1

Identifying channels

The spatially controlled transport of substrate (water) and products (protons, dioxygen) to and from the catalytic center to the lumenal side of the protein is essential for efficient catalysis, especially for a multi-electron reaction like water oxidation. PS II likely uses multiple channels that are spatially separated, to direct the flow of water/oxygen/protons to and from the Mn4CaO5 catalytic site. As the Mn4CaO5 cluster is embedded inside the protein close to the lumenal side of the membrane, it was postulated earlier that water channels and proton exit pathways most likely exist within the complex to ensure proper substrate supply and removal of reaction products (protons and oxygen). The flexibility of water network(s) and its interaction with amino acid residues likely play an important role for this function. Initial work was performed based on the search for cavities and channels using the lower resolution crystal structures.70–73 Later, the improved resolution of crystal structures contributed largely to identify potential channels (different studies and nomenclature summarized in Table 2). In the crystal structure, eight potential water and dioxygen channels have been identified, that lead away from the Mn4CaO5 cluster toward the lumen. Channels A1, A2, C, and D are formed largely by conserved residues from subunits D1 and CP43, with few conserved residues contributed by PsbO and D2. Three channels, A1, A2, and B, have a minimum van der Waals diameter of  2.7 Å, that allow water or dioxygen to pass through. Another five narrower channels that initiate from the OEC are mostly enclosed by hydrophilic groups with their minimum van der Waals diameter being 1.3 Å, and these might be important for proton transfer. Computational approaches are also powerful for predicting channels, based on the structural information. Applying molecular dynamics (MD) simulations gave new insights into, e.g., identifying new channels, characterizing water permeation energetics, and investigating water diffusion from the bulk. These channels match with some paths identified in the earlier crystal structures. Four channels are identified that lead to the lumenal surface from the OEC.75,77,79–81 These channels match with some paths identified in the crystal structure. The studies further suggest a strict spatial separation of water and oxygen fluxes and proton fluxes to and from the Mn4CaO5 cluster.

Fig. 10 S2 to S3 sequence. Schematic summarizing the structural changes in the O1, Cl1, and O4 channels leading to the first water insertion and proton release during the S2 / S3 transition. Mn1 and Mn4 oxidations from (III) to (IV) are shown as a color change from pink to purple. The gray arrow represents the possible proton pathway, while the blue dashed arrow represents the potential stepwise water insertion pathway. From Hussein, R.; Ibrahim, M.; Bhowmick, A.; Simon, P. S.; Chatterjee, R.; Lassalle, L.; Doyle, M.; Bogacz, I.; Kim, I.-S.; Cheah, M. H.; Gul, S.; de Lichtenberg, C.; Chernev, P.; Pham, C. C.; Young, I. D.; Carbajo, S.; Fuller, F. D.; Alonso-Mori, R.; Batyuk, A.; Sutherlin, K. D.; Brewster, A. S.; Bolotovsky, R.; Mendez, D.; Holton, J. M.; Moriarty, N. W.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Dobbek, H.; Messinger, J.; Zouni, A.; Kern, J.; Yachandra, V. K.; Yano, Structural Dynamics in the Water and Proton Channels of Photosystem II During the S2 to S3 Transition. J. Nat. Commun. 2021, 12 (1), 6531.

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Frankel et al. utilized an X-ray footprinting method to experimentally detect the water and oxygen channels.82 In this method, radiolysis of water by synchrotron X-ray radiation produces OH-, and oxidized amino acid residues that are in contact with water (OH-) in the protein can be identified by mass spectroscopy. They have observed a number of buried residues, that lead from the surface of the complex to the Mn4O5Ca cluster, are modified in the proteins. In a similar approach, Weisz et al.78 looked at a release pathway of reactive oxygen species (hydrogen peroxide and hydroxyl radical) produced at the OEC, by detecting the modified amino acid residues with high-resolution tandem mass spectroscopy. The idea is that water and OH* have similar size and hydrophilicity, and therefore it likely diffuses using a water pathway. Among the three water pathways identified by this method, two of them (O1A and Cl1A) overlap with the pathways proposed from crystallography. The main channels further discussed below are shown in Fig. 11.

2.13.5.2

Oxygen channel

To identify possible hydrophobic pathways for dioxygen, Guskov et al.72 used Xe, a dioxygen diffusion/X-ray scattering-analog, by studying the PS II crystal structure under pressure. No Xe was located in the channels described above, and all Xe sites are located in a hydrophobic environment formed by fatty acids from lipids, phytol chains from Chls, carotenoids or hydrophobic amino acids. Guskov et al. speculated that the hydrophobic patches in the interior, together with lipids, could allow diffusion of the dioxygen through PS II to the cytoplasmic side, thereby channeling dioxygen away from the reaction center region and preventing oxidative damage to P680 as well as accumulation of dioxygen in the lumen of the thylakoid membrane.

2.13.5.3

Water channel

During one cycle of the catalytic reaction, the OEC consumes two water molecules; one is introduced into the cycle during the S2 / S3 transition and the second during the S3 / S0 transition. The movement of water and changes in hydrogen bonding networks are observed in room temperature crystallography.67,68 The water mobilities in the channels are represented by the higher root-mean-square deviation values (RMSD, the average deviation between the corresponding atoms of two proteins), higher Bfactor or lower occupancy of oxygen atoms in the crystallography data. Fig. 12 shows that the RMSD values of the O1 channel waters are noticeably higher than those in the other proposed channels. From this data, it has been suggested that the O1 channel acts as a substrate intake channel during the water oxidation reaction. In the S2 to S3 transition in which the additional ligand Ox39 (or O653,83), bridged between Ca and Mn likely as hydroxo (Fig. 9), appears at the open coordination site of Mn1, and the exact pathway of water to this position is not yet solved. However, a theoretical study suggested that the formation of the S3 state is coupled to the movement of a Ca-bound hydroxide (W3) from the Ca to a Mn (Mn1 or Mn4) in a process that is triggered by the formation of a tyrosyl radical (TyrZ) and its protonated base, His-190.84 Note that there is another proposal that suggests the Cl1 channel as being a water channel based on the water exchange studies using site-specific mutants.85

2.13.5.4

Proton channel

In addition to four electrons, four protons are released from the catalytic reaction in the pattern of 1:0:1:2 for the S-state transitions, S0 / S1 / S2 / S3 / S0, respectively (see Table 1).34,86–89 There are several suggested pathways based on crystallography, Fourier transform infrared spectroscopy, and theory, that are formed by hydrogen-bonded waters and amino-acid residues.68,90–92 In a combination of QM/MM and MD simulations, Sakashita et al. proposed that the O4 channel likely serves as a proton release pathway during the S0 to S1 transition.91,93 They described the requirement of Hþ channel vs water channel, by showing that residues facing the channel that acts as H-bonded partners of water molecules predominantly determined the proton-transfer ability. Upon the formation of the S2 state, however, the electron density of W20 located near the OEC in the O4 channel disappears,39,53,83 and it is not observed until the recovery of the S0 state. This suggests that the H-bond network between the OEC and the O4 channel is disconnected during the S2 to S3, and S3 to S0 transitions. For the S2 to S3 transition, it has been proposed that the Cl1 channel may serve as a proton release channel, based on the motion of waters and amino-acid residues from the time-resolved crystal structure data for this transition. The reversible motion of D1-D65 that is located at the bottleneck of the Cl1 channel seems to suggest that this residue together with D1-R334 serves as a gate for the proton transfer reaction, and opening and closing of this gate may be controlled by the electronegativity changes at the OEC. Although further structural investigation is necessary to determine the proton pathways, different proton release pathways may be used during different S-state transitions.

2.13.6

Mechanism of photosynthetic O2 evolution

There has been active discussion of the O–O bond formation mechanism over the decades.5,64,94–99 Since the availability of the high-resolution crystallography data of the resting state,9 there have been efforts to merge the structural knowledge, spectroscopic information, and theory, to solve the reaction mechanism (some of the methods that have been important for the mechanistic studies of the water oxidation reaction are summarized in the next Section). Recent progress of the crystallography information on intermediate S-states, in particular, the S3 structure, made it possible to narrow down the possible O–O bond formation sites.

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Table 2

Channel nomenclature in literature. Ho and Styring 74

Method

Murray and Barber 71

Surface contact calculation 2AXT

PDB ID

Gabdulkhakov et al. 72 Xe 3BZ1

Resolution 3.00 Å O1 Channel A large

channel ii

2.90 Å B1

O1 Channel B large

channel ii

B2

O4 Channel

NA

E, F

narrow

Umena et al. 9 H-bond network analysis 3ARC (2WU2) 1.90 Å

4.c

–

G

Cl1 Channel B

channel iii

C, D

channel i

A1, A2

Cl2 network

back

Ogata et al. 76

3ARC (2WU2) 1.90 Å 4.A

2

4.c 3.b

Weisz et al. 78 ROS

3ARC 3ARC (2WU2) (2WU2) 1.90 Å 1.90 Å O1-water chain Path 3

Channel X 4.b

Sakashita et al. 77

MD simulation

4B

D Cl1 Channel A broad

Vassieliev et al. 75 6/26/22 10:16:00 PM

1 3

Path 2

5

Path 1

O4-water chain E65/E312 channel

Arm 2 Arm 2 Arm 3 Arm 1 Arm 3

There are multiple names used for identifying the water and proton channels in PS II. The table summarizes their correspondence. Adapted from Hussein, R.; Ibrahim, M.; Bhowmick, A.; Simon, P. S.; Chatterjee, R.; Lassalle, L.; Doyle, M.; Bogacz, I.; Kim, I.-S.; Cheah, M. H.; Gul, S.; de Lichtenberg, C.; Chernev, P.; Pham, C. C.; Young, I. D.; Carbajo, S.; Fuller, F. D.; Alonso-Mori, R.; Batyuk, A.; Sutherlin, K. D.; Brewster, A. S.; Bolotovsky, R.; Mendez, D.; Holton, J. M.; Moriarty, N. W.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Dobbek, H.; Messinger, J.; Zouni, A.; Kern, J.; Yachandra, V. K.; Yano, J. Nat. Commun. 2021, 12 (1), 6531.

As described in Section 2.13.4.3.3, a new ligand (Ox, which is either O or OH) is inserted in the open coordination site of Mn1 in the S3 state. This ligand is bridged between Mn1 and Ca, and located around 2.1 Å from O5, but without a peroxide bond formation at this stage. This configuration makes the O–O bond formation with Ox and O5 during the next S3 to S0 transition a highly likely candidate. An alternative idea is that Ox is placed between Ca and Mn1 to replace O5 during O2 formation or release. In such a case, O–O bond formation may occur between O5 and W2 or O5 and W3. Fig. 13 shows some of the options that could happen during the final S3 / S0 transition, including possible order of (1) electron and proton release; (2) O–O bond formation and O2 release; and (3) refilling of the empty substrate site (Fig. 13A), and chemistry that leads to the O–O bond formation (Fig. 13B). These are by no means a list of all the proposed mechanisms but a subset of the mechanisms that are being discussed. The central question for the chemistry of the mechanism is whether the O–O bond formation occurs via a nucleophilic attacktype mechanism in which the electrophilic water attacks nucleophilic high-valent Mn (in this case, Mn(V)), or via a radical coupling type mechanism in which the reaction occurs between an oxo-group and a terminal water ligand.100 In the former case, for example, a nucleophilic water bound to Ca attacks an oxyl radical (or oxo group), and such a mechanism with the water on Mn4101 has been proposed. In the latter case, an oxo-group (O5) and Mn1-coordinated OH has been proposed.98 A possible starting of the O–O bond formation already in the S3 state has also been proposed.48 However, this proposal does not match with the various spectroscopy-based evidence for the S3 state that shows that all the Mn in this state are in the þ 4 oxidation state.41,102 The critical information that is required for determining the exact O–O bond formation mechanism is to capture the structural intermediate specie(s) that will appear during the S3 to S0 transition, that will then be used to identify the O–O bond formation site, understand the geometric and electronic structural changes of the cluster during the catalysis, the water intake process to the cluster, and the reformation of the metal catalyst after the cycle to start over the reaction.

2.13.7

Light-driven assembly of the manganese cluster

All metalloenzymes with complex metal active sites must correctly assemble its center so that proper function of the enzymes is ensured. In many cases, assembly factors and chaperon proteins are employed that allows assembly and insertion of a (pre) form of the metal co-factor and maturation of the active site (see e.g., for assembly of the cofactors in nitrogenase).103–106 In

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Fig. 11 Proposed channels that extend from the OEC. (Left): The position of proposed water and proton channels that extend from the luminal side of bulk water to the oxygen evolving complex. (Right) A detailed view of the water channels showing the waters within each channel (O1 Channel red dotted, O4 Channel blue dotted, and Cl1 Channel green dotted). The region highlighted with solid yellow represents the Yz network. Residues involved in forming bottlenecks in the channels are shown in black. Adapted from Hussein, R.; Ibrahim, M.; Bhowmick, A.; Simon, P. S.; Chatterjee, R.; Lassalle, L.; Doyle, M.; Bogacz, I.; Kim, I.-S.; Cheah, M. H.; Gul, S.; de Lichtenberg, C.; Chernev, P.; Pham, C. C.; Young, I. D.; Carbajo, S.; Fuller, F. D.; Alonso-Mori, R.; Batyuk, A.; Sutherlin, K. D.; Brewster, A. S.; Bolotovsky, R.; Mendez, D.; Holton, J. M.; Moriarty, N. W.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Dobbek, H.; Messinger, J.; Zouni, A.; Kern, J.; Yachandra, V. K.; Yano, Structural Dynamics in the Water and Proton Channels of Photosystem II During the S2 to S3 Transition. J. Nat. Commun. 2021, 12 (1), 6531.

contrast, PS II utilizes a system of self-assembly of the Mn4CaO5 cluster that requires no additional protein cofactors or energy input except light. The photoassembly of the Mn4CaO5 cluster was initially described by Cheniae and Martin.107 One important driving force for developing a self-assembly process for the OEC was that PSII is very susceptible to photo-damage and the primary target is the D1 protein, the location of the Mn cluster,108 see also109 for an overview of photodamage to thylakoid proteins. The D1 replacement occurs roughly every 20–30 min, depending on the light conditions. The subsequent repair process requires a partial disassembly and reassembly of PS II, including the synthesis of the metal cluster. The PS II assembly and repair processes are not well-understood up to now, as the fully functional PS II requires the sequential assembly of  20 polypeptides plus approximately 50 cofactors and around 20 auxiliary proteins are involved in this process110–112 While the initial steps of PSII biogenesis are still not

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Fig. 12 Root-mean-square values of water molecules in the proposed channels. Adapted from Ibrahim, M.; Fransson, T.; Chatterjee, R.; Cheah, M. H.; Hussein, R.; Lassalle, L.; Sutherlin, K. D.; Young, I. D.; Fuller, F. D.; Gul, S.; Kim, I.-S.; Simon, P. S.; de Lichtenberg, C.; Chernev, P.; Bogacz, I.; Pham, C. C.; Orville, A. M.; Saichek, N.; Northen, T.; Batyuk, A.; Carbajo, S.; Alonso-Mori, R.; Tono, K.; Owada, S.; Bhowmick, A.; Bolotovsky, R.; Mendez, D.; Moriarty, N. W.; Holton, J. M.; Dobbek, H.; Brewster, A. S.; Adams, P. D.; Sauter, N. K.; Bergmann, U.; Zouni, A.; Messinger, J.; Kern, J.; Yachandra, V. K.; Yano, Untangling the Sequence of Events During the S 2 / S 3 Transition in Photosystem II and Implications for the Water Oxidation Mechanism. J. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (23), 12624–12635.

well understood it is thought that in cyanobacteria the first step is insertion of the D1 precursor into the plasma membrane via the SecYEG translocase complex followed by binding of Chl to the precursor protein and binding of the PsbI subunit. Formation of the first reaction center intermediates that include D1 and D2, occurs in a region of the membrane called PratA defined Membrane (PDM), which is connecting the plasma membrane with the thylakoid membrane. The subsequent steps that involve the addition of the CP43 and CP47 subunits, are thought to happen in the thylakoid membrane.113–115 Recent structures of assembly intermediates obtained by cryo-EM methods provide some important insights. For example, one recent report of a PS II complex with the Psb27 protein indicated that it could be part of a repair cycle and is important in regulation of binding of the extrinsic subunit during repair/assembly of the complex.116 Two other studies describing intermediates with either Psb28 and Psb34117 or Psb27, Psb28 and Psb34118 led to the conclusion that in these assembly intermediates the electron acceptor side was modified to prevent PS II from photodamage during the assembly process and that binding of these factors helped to optimize the position of the C-terminal end of the D1-protein for optimum incorporation of Mn in the subsequent photoactivation.

Fig. 13 (A) Three likely location (1, 2 and 3) of the O–O bond formation site during the final S3 / S0 transition, including possible order of (1) electron and proton release; (2) O–O bond formation and O2 release; and (3) refilling of the empty substrate site. (B) Nucleophilic attack (a) vs radical coupling (b) mechanism. Panel (A): From Kern, J.; Chatterjee, R.; Young, I. D.; Fuller, F. D.; Lassalle, L.; Ibrahim, M.; Gul, S.; Fransson, T.; Brewster, A. S.; Alonso-Mori, R.; Hussein, R.; Zhang, M.; Douthit, L.; de Lichtenberg, C.; Cheah, M. H.; Shevela, D.; Wersig, J.; Seuffert, I.; Sokaras, D.; Pastor, E.; Weninger, C.; Kroll, T.; Sierra, R. G.; Aller, P.; Butryn, A.; Orville, A. M.; Liang, M.; Batyuk, A.; Koglin, J. E.; Carbajo, S.; Boutet, S.; Moriarty, N. W.; Holton, J. M.; Dobbek, H.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Zouni, A.; Messinger, J.; Yano, J.; Yachandra, V. K. Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature. Nature 2018, 563 (7731), 421–425, Panel (B): Adapted from Cox, N.; Pantazis, D. A.; Lubitz, W. Current Understanding of the Mechanism of Water Oxidation in Photosystem II and Its Relation to XFEL Data. Annu. Rev. Biochem. 2020, 89 (1), 795–820.

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In both de novo assembly and the repair process, the chaperon-free, light-driven assembly of the Mn4CaO5 cluster is a crucial process and unique to PS II.114,119–121 In the recent research, different models about this mechanism have been proposed.111 Nickelsen and Rengstl have compared two de novo assembly models for both plants and cyanobacteria exhibiting the common main disassembly phases during the repair. The assembly of the Mn4CaO5 cluster requires Mn2þ, Ca2þ, Cl, and light.122 The process of photoactivation involves the stepwise incorporation of the Mn and Ca ions into the ‘apo-PSIIcc,’ that is the PS II core complex without any metal ions bound at the OEC ligand pocket.118,123,124 Its mechanism has remained elusive, despite the recent progress in the structure elucidation and mechanistic PSII research in general. Below we summarize the current view of the Mn4CaO5 cluster assembly. Assembly of the cluster starts from a Mn free apo-PSII and recent structural studies have provided some insights into the details of this apo-structure. Starting from a fully active crystallized PSII complex from the thermophilic cyanobacterium Thermosynechococcus vestitus and developing a procedure for careful removal of the Mn cluster in-crystallo allowed Zhang and co-workers for the first time to structurally characterize the apo-PSII.123 Interestingly, no indication for a larger structural change in the binding pocket was evident upon removal of the Mn in the 2.55 Å apo-PSII structure. Nearly all of the coordinating amino acids were found to be in the same place compared to the native structure, indicating that the environment of the cluster is already present before binding of the metal center. The only exception is CP43-Glu354, a ligand to Mn2 and 3, which carboxylate moiety could not be located as well as other ligands and most likely can adopt various conformations in the apo-structure. The interaction between TyrZ and His190 was also maintained although with a slightly elongated Tyr-O-N-His distance. The place of the Mn cluster was filled by two additional water molecules that are roughly located at the positions of O2 and O3 bridging Mn2 and Mn3 of the assembled cluster. Based on the distance found between the ligand groups it was concluded that 2–3 additional carboxylate residues are protonated in the apo-structure. Based on this assumption it was suggested that stepwise deprotonation of these H-bonding interactions in the apo-protein can allow for coordination of two Mn2þ ions to the carboxylate groups without a change in net charge of the binding pocket or major movements of the His and Glu/Asp side chains.123 Recently Gisriel and colleagues observed the structure of an apo-PSII complex from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 using cryo-EM.124 In this structure the extrinsic proteins that usually shield the site of the OEC from the lumen were not present and a more open structure of the active site was observed. Specifically, the residues at the C-terminal end of the D1 subunit starting from residue 332 to the terminus (344) were shifted from their position in the mature PSII and highly disordered between residue 337 and 344. Therefore, it is unclear what structure the C-terminal domain of the D1 subunit assumes in vivo during Mn cluster assembly. DFT and MD calculations using the thermophilic cyanobacterial apo-structure as a starting point examined the protonation state of the apo binding pocket in more detail.125 This modeling approach suggested that the apo-PSII has a flexible structure with several different possible protonation patterns, always giving 5–6 additional protons in the site compared to the fully assembled structure. When trying to force a third water molecule into the apo-site it was found that this water is not stable within the MD trajectories, indicating that two waters are sufficient to fill the cavity in the absence of the OEC.125 In addition to observing the apo-PSII structure in T. elongatus Zhang et al. also reported on the structure of a partially assembled and a partially disassembled cluster.123 A region of high electron density was identified in both cases close to the positions of Mn1 and Mn2 in the active cluster, but due to the limited resolution of the available data it was not possible to conclude if one or two Mn ions are contributing to this density and to properly model the metal ligation in this structure. Nevertheless, an interpretation with two Mn bound at the Mn1 and Mn2 sites seems reasonable based on analogy to a di-u oxo bridged Mn-Mn unit proposed as an intermediate in heat induced disassembly of the cluster126 as well as an intermediate in photoactivation.127 The initial steps of the photo-activation process can be illustrated in the two quantum model.107,120,121,128,129 The absorption of light by PS II produces the oxidant TyrZox, which oxidizes the Mn2þ ions. The first oxidation of a Mn2þ has a high quantum yield, unlike the following steps which are needed for the oxidation-induced assembly of the stable Mn4CaO5 cluster, leading to a low quantum yield of  1% for the overall process. First, a Mn(II) ion binds in the dark at the high-affinity side of the apo-PSIIcc and is oxidized after absorption of a first light quantum, forming an unstable Mn(III) intermediate (Fig. 14). In the next step, a light-independent rearrangement occurs. Then a second light quantum drives the oxidation of a second Mn(II) ion to Mn(III), resulting in a next assembly intermediate, a binuclear Mn complex, possibly involving two di-m-oxo bridged Mn(III) ions.127 The incorporation of the other two missing Mn ions to complete the metal-cluster has not been kinetically resolved.

2.13.8

Several techniques that are fundamental to the PSII research

X-ray diffraction (XRD), electron paramagnetic resonance spectroscopy (EPR), X-ray absorption/emission spectroscopy (XAS/XES), infrared spectroscopy, isotope-labeled water exchange coupled to mass spectroscopy, and theoretical studies have all provided valuable insights into the structure and mechanism of the Mn4CaO5 cluster in PS II. These studies have all benefitted from the availability of well characterized samples made possible by improved biochemical methods. Below is a brief summary of some of these methods.

2.13.8.1

EPR

Electron Paramagnetic Resonance spectroscopy has been a workforce for the study of the Mn4CaO5 cluster, due to the rich EPR signal of Mn that has a nuclear hyperfine I ¼ 5/2 of 55Mn nuclei.130 The total spin state of the cluster is determined by the oxidation

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Fig. 14 Possible Model describing the initial reassembly of the Mn4CaO5 cluster. The scheme represents the binding and the light driven relocation/ oxidation of the first two Mn ions into the apo-PSII. The first Mn2þ (light orange) moves to the Mn4 position of the apo-PSII (green) and binds at this high affinity site (Step 1). After light driven oxidation with low quantum efficiency a rearrangement takes place where the oxidized Mn3þ (orange) migrates from the Mn4 position to the Mn1 position (Step 2). The same rearrangement also applies to the second Mn2þ which binds at the Mn4 position and then relocates as the oxidized Mn3þ to the Mn2 position (Steps 3, 4). Both Mn3þ ions form a stable Mn1-(m-O)2-Mn2 assembly intermediate (Step 4). Binding of the two remaining Mn ions and the Ca ion (blue) into the apo-OEC has not been resolved yet (Step 5). Note that Ca2þ could also bind in an earlier step, e.g., in step 3, 4 or 5. Adapted from Zhang, M.; Bommer, M.; Chatterjee, R.; Hussein, R.; Yano, J.; Dau, H.; Kern, J.; Dobbek, H.; Zouni, A. Structural Insights Into the Light-Driven Auto-Assembly Process of the Water-Oxidizing Mn4CaO5-Cluster in Photosystem II. Elife. 2017, 6, e26933, 10.7554/eLife.26933.

state of Mn, their coupling geometry and type of ligands. Among the catalytic intermediates, the S0 and S2 states are paramagnetic, and have unique EPR signals with characteristic hyperfine splittings. The S1 and S3 states, have integer number of spins and have broad less-defined signals, and have been studied with parallel-mode EPR spectroscopy. In the S0 state, there is a multiline signal with 24–26 hyperfine lines with spacings of 80–90 G, due to the S ¼ 1/2 ground state with exchange coupling within a Mn(III)3Mn(IV) cluster. This S0 signal has been produced in two ways, one is by visible laser flash (forward direction of the Kok cycle), and the other is by chemical reduction with hydroxylamine (backward direction of the cycle).58,131,132 The S2 state is the most studied state with EPR, as nearly 100% of the centers can be converted to this state, upon illumination of the dark stable S1 state. In the S2 state, two types of EPR signals have been assigned to the Mn cluster (Fig. 15). The multiline signal (MLS) centered at g ¼ 2, exhibiting at least 18 partially resolved hyperfine lines at X-band ( 9 GHz), is a low spin (Stotal ¼ ½, i.e., MnIII/MnIV and MnIV/MnIV are anti-ferromagnetically-coupled, respectively) ground state, S2 LS state.47,132–135 Another broad featureless EPR signal at g  4.1, attributed to a higher spin multiplicity (Stotal ¼ 5/2, i.e., ferromagnetically-coupled three MnIV with anti-ferromagnetically-coupled one MnIII), S2 HS state, is also observed under different experimental conditions.47,136,137 Together with density functional theory calculations and the EPR and double resonance spectroscopy results, the oxidation state of each Mn and coupling pathways have been proposed. The S1 and S3 states with integer spin states have been studied using parallel mode EPR techniques. The S1 spin state has been determined to be a Stotal ¼ 1 state.138 and the S3 spin state has been shown to be Stotal ¼ 3.45,62,139 More advanced EPR techniques, such as ENDOR (Electron Nuclear Double Resonance) and ESEEM (Electron Spin Echo Envelop Modulation) methods, have been used to study the interaction of spins and metal nuclei, and a S3 state structure has been proposed on the basis of these data.98 Also, EPR has been used to gain insights of the substrate water binding using ammonia as a substrate analog. One of the important proposals from EPR studies is the role of spin isomorphism in the reaction, that has provoked consideration of structural isomerization within the S-states, and/or the possible structural intermediates upon the S2 to S3 transition. Another powerful approach one can use with EPR is to use its sensitivities to certain isotopes of N and O, and use isotope labeling of substrate water (or NH3)to monitor the interaction of labeled water injected into the system. Cox et al. used 17O EDNMR (ELDOR-detected NMR, where ELDOR is electron-electron double resonance) method with 17O isotope labeled water.45 17O has

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Fig. 15 Representative EPR signals of each S-state. From Geijer, P.; Peterson, S.; Åhrling, K. A.; Deák, Z.; Styring, S. Comparative Studies of the S0 and S2 Multiline Electron Paramagnetic Resonance Signals From the Manganese Cluster in Photosystem II. Biochim. Biophys. Acta - Bioenerg. 2001, 1503 (1–2), 83–95.

a spin of I ¼ 5/2, and therefore has a magnetic moment unlike EPR silent 16O and 18O waters. 17O labeled water is injected into the system at Dt after illumination, and hyperfine couplings of coordinating 17O nuclei to Mn due to the exchange of waters is measured. The result showed that all terminal waters (W1-W4) plus bridging water like O5 can be exchangeable with bulk water.98,140

2.13.8.2

Mass spectroscopy

One of the major questions of the water oxidation mechanism in OEC is which two oxygens form an O–O bond during the S3 to S0 transition. To identify substrate waters, water exchange kinetics in the OEC has been studied by several different techniques. Two primary methods are membrane inlet mass spectroscopy (MIMS)141 and EPR spectroscopy (as described above).98 In both cases, isotope labeled waters are used to differentiate between substrate waters and background waters. In MIMS, the mass difference between different oxygen isotopes (16O vs 18O) are used to monitor substrate water uptake/exchange, by adding 18O-labeled water. In EPR, 17O-labeled water that has a magnetic nuclear spin of I ¼ 5/2 is used. MIMS has identified the slow and fast exchange waters and its exchange rates at each S-state. These waters are expected to be the substrate waters for the dioxygen formation, and the result has been used to propose candidates for the O–O bond formation site and the possible water insertion mechanism. A significant advantage of MIMS with isotope-labeled water plus flash sequence is that it detects the exchange rate relevant only to substrate waters. In the time-resolved MIMS (Fig. 16),65 isotope labeled water is injected at a Si state, and the O2 molecules (Z ¼ 34 (16O18O) or 36 (18O18O)) released from PS II are detected. 36O2 requires both substrate waters to be exchanged, while 34 O2 requires one of them to be exchanged. The level of isotopic enrichment of the product as a function of various incubation times, thus, becomes a measure of the substrate exchange rate of the Si state. More precisely, after the injection of H218O in the Si state, the equilibration of bulk H218O into the substrate binding sites of the OEC occurs during the incubation time. Then, by giving remaining flashes, the isotopic composition of the O2 released during the S3 to S0 transition is measured. Time-resolution is determined by the mixing time of isotope labeled water into the PS II solution. The biphasic behavior of 34O2 pattern indicates there are two types of substrate water, the fast (Wf) and slowly (Ws) exchanging waters. The monophasic behavior of 36O2, on the other hand, is the indication of homogeneous samples, and that rate should match with the slower phase of 34O2 pattern. The exchange kinetics of each S-state reported by Lichtenberg et al. is summarized in Table 3.142 Based on the advancement of structural studies, there has been active efforts to assign Wf and Ws to specific waters at the OEC or in the vicinity of it.

2.13.8.3

X-ray spectroscopy

X-ray absorption spectroscopy (XAS) allows us to study the local structure of the element of interest without interference from absorption by the protein matrix. X-ray absorption spectroscopy (XAS), both EXAFS and XANES have been very important structural tools for the study of the Mn4Ca cluster in PS II. It was EXAFS that first determined the presence of Mn-Mn distances that indicated

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Fig. 16 Substrate-water exchange kinetics measured by time-resolved membrane-inlet mass spectrometry at 16O16O (m/z ¼ 34, A) and 18O18O (m/ z ¼ 36, B) in the S3 state of spinach thylakoids at 10  C and pH 6.8. Black symbols are data, and blue lines are biexponential (34O2, A) and monoexponential (36O2, B) fits. (C) Flash-injection protocols for measuring substrate water-exchange in PS II by time-resolved isotope-ratio membrane-inlet mass spectrometry. Black vertical lines indicate excitations of the PS II sample with single turnover flashes, the blue arrow shows the time of rapid H218O injection into the sample. Variation of the delay between H218O injection and the O2 evolving flash sequence (incubation time) allows to point wise probe the kinetics of the substrate water exchange reaction. From Cox, N.; Messinger, Reflections on Substrate Water and Dioxygen Formation. J. Biochim. Biophys. Acta - Bioenerg. 2013, 1827 (8–9), 1020–1030.

oxo-bridges between them, the occurrence of an unusual hetero-nuclear MnCa cluster where Ca is bridged to Mn atoms by oxobridges, and the changes in the electronic and geometric structure as the cluster advanced through the four-step light induced catalytic cycle. The inter metal, Mn-Mn and Mn-Ca, and also the metal-ligand distances were determined with high-accuracy that are better than any that can be accessed by X-ray diffraction. Using X-ray Mn K-edge and EXAFS on oriented membranes and later Table 3

Substrate water exchange rate from MIMS experiment. H2O exchange rate (/sec) a

Transition

Fast exchange water (Wf)

Slow exchange water (Ws)

S1 S2 S3

– 132 14 353

0.33 0.01 2.60.2 1.15  0.06

a

From WT-PSII core complexes from Synechocystis sp. 6803.142 Modified from de Lichtenberg, C.; Avramov, A. P.; Zhang, M.; Mamedov, F.; Burnap, R. L.; Messinger, J. The D1-V185N Mutation Alters Substrate Water Exchange by Stabilizing Alternative Structures of the Mn4Ca-Cluster in Photosystem II. Biochim. Biophys. Acta - Bioenerg. 2021, 1862 (1), 148319.

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single-crystals of PS II, this method was used to determine the orientation of the metal-metal vectors and proposed a model structure for the Mn4Ca complex that is similar to the eventual cluster structure determined using high-resolution X-ray diffraction. Fig. 17 shows the Mn K-edge spectrum of each S-state of spinach PS II after deconvolution of the spectra obtained from consecutive flash illumination into pure S-state spectra, and their second derivative spectra.143,144 The edge positions for each of the Sstates have been quantitated by measuring the inflection point energy (IPE), given by the zero-crossing of the second derivative. When Mn is oxidized by one electron in a set of Mn model compounds with similar ligands, the IPE shifts 1–2 eV to higher energy.145 In the EXAFS, the first peak around 1.8 and 2.0 Å attributable to N and O atoms that are directly ligated to Mn, and the 2nd peak at  2.7–2.8 Å is di-m-oxo bridged Mn to Mn interactions. The 3rd peak arises from mono-m-oxo bridged Mn-Mn at  3.2 Å as well as Mn-Ca interactions around 3.4 Å. The Mn EXAFS spectra changes upon the S-state transitions, particularly from the S2 to S3 state transition, that reflects the structural changes caused by the insertion of additional ligand to the Mn1 open coordination site.41 The S-state catalytic cycle has also been studied with Ca XAS. Prior to the availability of crystallography data, XAS was used to show that Ca is a part of the OEC cluster.146 In general, the requirements of X-ray spectroscopy place some restrictions with respect to sample preparation and experimental conditions. For elements like Ca and Cl, which can occur in a wide variety of environments in biological materials, it is particularly challenging to remove sources of background signals that greatly complicate interpreting the results. Another strategy to study the role of such light element co-factor(s) is to replace it with heavier element(s). Ca can be replaced chemically or biosynthetically with Sr without losing its enzymatic activity. Similarly, Cl can be substituted with Br. XAS measurements at the Sr K-edge ( 16,200 eV)31,147 or Br K-edge ( 13,600 eV)148 compared to those at lower-energy Ca K-edge ( 4050 eV) or Cl Kedge ( 2850 eV) are less prone to X-ray damage. A series of studies using Ca EXAFS, Sr EXAFS and on both solution and oriented membranes and unequivocally established that Ca is bridged to three Mn atoms via oxo bridges and that there is a clear rearrangement in the S2 to S3 transition. Recent XFEL work is demonstrating that Ca coordination environment changes during this transition, and that Ca may be a critical element in transporting the substrate to the Mn center. A complementary technique to XAS is X-ray emission spectroscopy (XES). In this case, the photons that are emitted after the creation of a core hole in the 1s shell form the K emission spectrum. Among such emission processes, Ka lines originate from 2p to 1s transitions and Kb lines from the 3p to 1s transitions (Kb1,3/Kb0 ) and valence level to 1s transitions (Kb2,5/Kb00 ). These spectra change depending on the chemical environment of the metals of interest. In particular, Kb spectra have been used in PS II for probing the Mn oxidation states (Fig. 17c) and identifying the ligand environment. Kb1,3/Kb0 spectra arise predominantly from the exchange interaction between the metal 3p and the net electron spin in the metal valence shell, i.e., the effective number of unpaired metal 3d electrons. Therefore, the spectrum is sensitive to the spin state of the metals, which indirectly reflects the oxidation state and covalency of the metal site, and has often been used for determining the oxidation states of metal catalytic sites. Kb1,3 arises from the constructive spin interactions, while Kb0 from destructive spin interactions.

Fig. 17 (a) XANES and (b) EXAFS spectra of PS II. (c) X-ray emission spectroscopy of Mn in PS II. Panel (A) and (B): Adapted from Yano, J.; Yachandra, V. K. Photosynth. Res. 2009, 102 (2–3), 241–254. Panel (C): Adapted from Messinger, J.; Robblee, J. H.; Bergmann, U.; Fernandez, C.; Glatzel, P.; Visser, H.; Cinco, R. M.; McFarlane, K. L.; Bellacchio, E.; Pizarro, S. A.; Cramer, S. P.; Sauer, K.; Klein, M. P.; Yachandra, V. K. J. Am. Chem. Soc. 2001, 123 (32), 7804–7820

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The higher energy emission peaks above Kb1,3 originate from valence-to-core transitions just below the Fermi level and can be separated into Kb00 and the Kb2,5 emission. Kb2,5 emission is predominantly from ligand 2p (metal 4p) to metal 1s, and the Kb00 emission is assigned to a ligand 2s to metal 1s; both are referred to as cross-over transitions.149 Therefore, only direct ligands to the metal of interest are probed with Kb,2,5/Kb00 emission; i.e., other C, N, and O atoms in the protein media do not contribute to the spectra. This method was used to directly detect the presence of oxo-bridges in the Mn4Ca cluster. In particular, the energy of the Kb00 transition depends on the difference between the metal 1s and ligand 2s binding energies, which reflects the environment of the ligand owing to orbital hybridization. Therefore, the spectrum can be used to detect protonation state of bridging ligands,150,151 which we expect to happen in the OEC during the catalytic cycle.

2.13.8.4

Infrared spectroscopy

Fourier-transform Infrared (FTIR) spectroscopy, often in combination with site-directed mutagenesis of putative carboxylate ligands and/or isotopic substitutions, provided insights of important ligands around the OEC and along the potential proton and substrate channels.54,152,153 FTIR difference spectra have been often used to detect light-induced changes of cofactors and amino acid residues. The data can be collected at physiological temperatures, and time-resolved infrared (TR-IR) spectroscopy has also been used to monitor the movement of electrons and protons during the S-state transitions of OEC.38 Fig. 18 shows the flash-induced FTIR difference spectra during the S-state transitions. The changes observed in the difference spectra of the mid-frequency region (1800–1000 cm 1) represent structural changes in each S-state transition. The amide I bands (the CO stretch of backbone amide) region in 1700–1600 cm 1 indicate that the secondary structures of the proteins around the catalytic center are affected. The changes are also visible in the amide II (the NH bend coupled to the CN stretch of backbone amide) and asymmetric COO  stretching bands in 1600–1500 cm 1. The bands in the 1450–1300 cm 1 region arise from the symmetric COO stretching vibrations of carboxylate groups coupled to the Mn4CaO5 cluster.154 The carboxylate bands are also sensitive to perturbations of the Mn4CaO5 cluster and its environment, such as Ca depletion, Sr substitution to Ca,155–157 and addition of small molecules (ammonia, methanol, etc.).158–160

2.13.8.5

X-ray crystallography at X-ray free electron lasers

X-ray-crystallography has been a method of choice to look at molecular structures of protein complexes and its assemblies. It has been widely applied using laboratory X-ray sources, at synchrotron radiation facilities, and recently at more powerful X-ray sources, such as the X-ray free electron laser (XFEL) facilities. One of the most challenging limitations of X-ray techniques, both crystallography and spectroscopy, for studying biological systems is X-ray-induced changes. The X-ray interaction with the biological matter is of course required as it gives us important fingerprints to understand the nature of the chemical environment. At the same time, the interaction of the X-rays with biological matter could change both the native chemical states and the structures of proteins. Therefore, one needs to understand the effect of X-ray-induced changes, and thereby control, and minimize the degree of changes. In biological systems such as proteins, radiation damage occurs mainly due to the migration and diffusion of radicals and solvated electrons to sensitive areas, when X-rays interact with water in protein. The diffusion of these radicals and solvated electrons often leads to harmful reactions with proteins and lipids. At synchrotron X-ray facilities, both X-ray crystallography and X-ray spectroscopy are typically carried out at cryogenic

Fig. 18 Flash-induced FTIR difference spectra of the PS II core complexes from T. elongatus in the high-frequency (3800–2200 cm 1) and midfrequency (2200–1200 cm 1) regions during the S-state cycle of OEC. From Noguchi, T.; Sugiura, M. TIR Detection of Water Reactions during the Flash-Induced S-State Cycle of the Photosynthetic Water-Oxidizing Complex. Biochemistry. 2002, 41 (52), 15706–15712.

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temperature in the frozen state to slow down the diffusion of radicals or solvated electrons and minimize radiation damage effects. Since the first report in 2001,33 various crystallography studies of PS II from the thermophilic cyanobacteria Thermosynechococcus (T.) elongatus and T. vulcanus have been published at resolutions up to 1.9 Å,9 and provided critical structural information of the enzyme, in spite of radiation-induced damage. Since the introduction of XFELs, it has become possible to collect X-ray data at room temperature without incurring any damageinduced from intense fluxes of X-rays. Shot-by-shot data collection with ultrashort X-ray pulses allows us to collect diffraction or spectroscopy data prior to the onset of radiation-induced sample changes. This approach also allows in situ triggering of reaction (laser illumination in case of the light-driven reaction in PS II), and the method has been used for the data collection of the intermediate S-state structures and timepoint data between the S-state transitions (Section 2.13.4.4) at room temperature. X-ray crystallography at XFELs has also been combined with non-resonant X-ray emission spectroscopy (XES) (Fig. 19).161 The X-ray wavelength used for typical crystallography measurements is  1 Å, corresponding to an energy that is above the 1s binding energy of 3d transition metals. Therefore, X-ray crystallography and X-ray emission spectroscopy data can be collected simultaneously. The important advantage of this approach is that one can get structural information of proteins together with the chemical information (oxidation state of Mn) at the catalytic centers. Thus, the method is highly suitable for following dynamically changing structural and chemical changes that occur under physiological temperature.

2.13.8.6

Cryo-electron microscopy

More recently, cryo-electron microscopy (cryo-EM) has been actively used for the structural studies of PS II.118,124,162,163 The resolution one can achieve with cryo-EM has been increased dramatically in the recent years, thus becoming a highly attractive tool in the field of structural biology. It is expected that such single protein environment, without the artificial arrangement of proteins in unit cells may represent the physiological states of proteins more closely. However, damage to the samples caused by the electron beam irradiation during the data collection has been an issue, in particular, to the redox active Mn4CaO5 center.162 Nevertheless, the method is highly beneficial to compare PSII from different species, site-specific mutants, as well as assembly intermediates, all of which are difficult to study in crystal as crystallizing these samples with high resolution is challenging. Thus, crystallography and cryo EM provide complementary and unique information, and cryo-EM is becoming a highly attractive method for enzyme structural studies.

2.13.9

Perspective

Through the longstanding efforts of many groups with various approaches, the chemistry involved in water oxidation is emerging slowly. However, there are still many fundamental questions including the mechanism of the O–O bond formation that are not yet resolved. Moreover, the knowledge gained in the recent years has provoked discussions and leading to the asking of many new questions, such as, how the events that occur in spatially separated locations are temporarily coordinated, and what is the structural and chemical sequence of events, how the environment including hydrogen bonding network plays a role in controlling kinetics, and how it accommodates the electronic states of the OEC during the multiple electron/proton reaction. Many of these questions are

Fig. 19 Schematics of the shot-by-shot simultaneous X-ray diffraction (crystallography) and X-ray emission spectroscopy data collection at X-ray free electron lasers.161

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fundamental to the enzymes related to solar energy conversion, as well as artificial photosynthetic systems. Light-driven reaction of PS II provides a unique platform to tackle these questions.

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116. Huang, G.; Xiao, Y.; Pi, X.; Zhao, L.; Zhu, Q.; Wang, W.; Kuang, T.; Han, G.; Sui, S.-F.; Shen, J.-R. Proc. Natl. Acad. Sci. U. S. A. 2021, 118 (5), e2018053118. 117. Xiao, Y.; Huang, G.; You, X.; Zhu, Q.; Wang, W.; Kuang, T.; Han, G.; Sui, S.-F.; Shen, J.-R. Nat. Plants 2021, 7 (8), 1132–1142. 118. Zabret, J.; Bohn, S.; Schuller, S. K.; Arnolds, O.; Möller, M.; Meier-Credo, J.; Liauw, P.; Chan, A.; Tajkhorshid, E.; Langer, J. D.; Stoll, R.; Krieger-Liszkay, A.; Engel, B. D.; Rudack, T.; Schuller, J. M.; Nowaczyk, M. M. Nat. Plants 2021, 7 (4), 524–538. 119. Nixon, P. J.; Michoux, F.; Yu, J.; Boehm, M.; Komenda, J. Ann. Bot. 2010, 106 (1), 1–16. 120. Becker, K.; Cormann, K. U.; Nowaczyk, M. M. J. Photochem. Photobiol. B 2011, 104 (1–2), 204–211. 121. Oliver, N.; Avramov, A. P.; Nürnberg, D. J.; Dau, H.; Burnap, R. L. Photosynth. Res. 2022. https://doi.org/10.1007/s11120-022-00912-z. 122. Nickelsen, J.; Rengstl, B. Annu. Rev. Plant Biol. 2013, 64 (1), 609–635. 123. Zhang, M.; Bommer, M.; Chatterjee, R.; Hussein, R.; Yano, J.; Dau, H.; Kern, J.; Dobbek, H.; Zouni, A. Elife 2017, 6, e26933. 124. Gisriel, C. J.; Zhou, K.; Huang, H.-L.; Debus, R. J.; Xiong, Y.; Brudvig, G. W. Joule 2020, 4 (10), 2131–2148. 125. Han, R.; Rempfer, K.; Zhang, M.; Dobbek, H.; Zouni, A.; Dau, H.; Luber, S. ChemCatChem 2019, 11 (16), 4072–4080. 126. Pospısil, P.; Haumann, M.; Dittmer, J.; Sole, V. A.; Dau, H. Biophys. J. 2003, 84, 1370–1386. 127. Barra, M.; Haumann, M.; Loja, P.; Krivanek, R.; Grundmeier, A.; Dau, H. Biochemistry 2006, 45 (48), 14523–14532. 128. Dasgupta, J.; Ananyev, G.; Dismukes, G. Coord. Chem. Rev. 2008, 252 (3–4), 347–360. 129. Bao, H.; Burnap, R. L. Front. Plant Sci. 2016, 7, 578. 130. Geijer, P.; Peterson, S.; Åhrling, K. A.; Deák, Z.; Styring, S. Biochim. Biophys. Acta - Bioenerg. 2001, 1503 (1–2), 83–95. 131. Åhrling, K. A.; Peterson, S.; Styring, S. Biochemistry 1998, 37 (22), 8115–8120. 132. Kulik, L. V.; Epel, B.; Lubitz, W.; Messinger, J. J. 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R.; Wydrzynski, T.; Hillier, W. Photosynth. Res. 2009, 102 (2–3), 511–522. 142. de Lichtenberg, C.; Avramov, A. P.; Zhang, M.; Mamedov, F.; Burnap, R. L.; Messinger, J. Biochim. Biophys. Acta - Bioenerg. 2021, 1862 (1), 148319. 143. Messinger, J.; Robblee, J. H.; Bergmann, U.; Fernandez, C.; Glatzel, P.; Visser, H.; Cinco, R. M.; McFarlane, K. L.; Bellacchio, E.; Pizarro, S. A.; Cramer, S. P.; Sauer, K.; Klein, M. P.; Yachandra, V. K. J. Am. Chem. Soc. 2001, 123 (32), 7804–7820. 144. Yano, J.; Yachandra, V. K. Photosynth. Res. 2009, 102 (2–3), 241–254. 145. Visser, H.; Anxolabéhère-Mallart, E.; Bergmann, U.; Glatzel, P.; Robblee, J. H.; Cramer, S. P.; Girerd, J.-J.; Sauer, K.; Klein, M. P.; Yachandra, V. K. J. Am. Chem. Soc. 2001, 123 (29), 7031–7039. 146. Cinco, R. M.; McFarlane Holman, K. L.; Robblee, J. H.; Yano, J.; Pizarro, S. A.; Bellacchio, E.; Sauer, K.; Yachandra, V. K. Biochemistry 2002, 41 (43), 12928–12933. 147. Pushkar, Y.; Yano, J.; Sauer, K.; Boussac, A.; Yachandra, V. K. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (6), 1879–1884. 148. Haumann, M.; Barra, M.; Loja, P.; Löscher, S.; Krivanek, R.; Grundmeier, A.; Andreasson, L.-E.; Dau, H. Biochemistry 2006, 45 (43), 13101–13107. 149. Glatzel, P.; Bergmann, U. Coord. Chem. Rev. 2005, 249 (1–2), 65–95. 150. Lassalle-Kaiser, B.; Boron, T. T.; Krewald, V.; Kern, J.; Beckwith, M. A.; Delgado-Jaime, M. U.; Schroeder, H.; Alonso-Mori, R.; Nordlund, D.; Weng, T.-C.; Sokaras, D.; Neese, F.; Bergmann, U.; Yachandra, V. K.; DeBeer, S.; Pecoraro, V. L.; Yano, J. Inorg. Chem. 2013, 52 (22), 12915–12922. 151. Pushkar, Y.; Long, X.; Glatzel, P.; Brudvig, G. W.; Dismukes, G. C.; Collins, T. J.; Yachandra, V. K.; Yano, J.; Bergmann, U. Angew. Chem. Int. Ed. 2010, 49 (4), 800–803. 152. Noguchi, T. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008, 363 (1494), 1189–1195. 153. Chu, H.-A. Front. Plant Sci. 2013, 4, 146. 154. Noguchi, T.; Sugiura, M. Biochemistry 2003, 42 (20), 6035–6042. 155. Strickler, M. A.; Walker, L. M.; Hillier, W.; Debus, R. J. Biochemistry 2005, 44 (24), 8571–8577. 156. Suzuki, H.; Taguchi, Y.; Sugiura, M.; Boussac, A.; Noguchi, T. Biochemistry 2006, 45 (45), 13454–13464. 157. Kimura, Y.; Hasegawa, K.; Ono, T. Biochemistry 2002, 41 (18), 5844–5853. 158. Fang, C.-H.; Chiang, K.-A.; Hung, C.-H.; Chang, K.; Ke, S.-C.; Chu, H.-A. Biochemistry 2005, 44 (28), 9758–9765. 159. Hou, L.-H.; Wu, C.-M.; Huang, H.-H.; Chu, H.-A. Biochemistry 2011, 50 (43), 9248–9254. 160. Tsuno, M.; Suzuki, H.; Kondo, T.; Mino, H.; Noguchi, T. Biochemistry 2011, 50 (13), 2506–2514. 161. Kern, J.; Alonso-Mori, R.; Tran, R.; Hattne, J.; Gildea, R. J.; Echols, N.; Glöckner, C.; Hellmich, J.; Laksmono, H.; Sierra, R. G.; Lassalle-Kaiser, B.; Koroidov, S.; Lampe, A.; Han, G.; Gul, S.; DiFiore, D.; Milathianaki, D.; Fry, A. R.; Miahnahri, A.; Schafer, D. W.; Messerschmidt, M.; Seibert, M. M.; Koglin, J. E.; Sokaras, D.; Weng, T.-C.; Sellberg, J.; Latimer, M. J.; Grosse-Kunstleve, R. W.; Zwart, P. H.; White, W. E.; Glatzel, P.; Adams, P. D.; Bogan, M. J.; Williams, G. J.; Boutet, S.; Messinger, J.; Zouni, A.; Sauter, N. K.; Yachandra, V. K.; Bergmann, U.; Yano, J. Science 2013, 340 (6131), 491–495. 162. Kato, K.; Miyazaki, N.; Hamaguchi, T.; Nakajima, Y.; Akita, F.; Yonekura, K.; Shen, J.-R. Commun. Biol. 2021, 4 (1), 382. 163. Li, M.; Ma, J.; Li, X.; Sui, S.-F. Elife 2021, 10, e69635.

Further reading Blankenship, R. E.; Tiede, D. M.; Barber, J.; Brudvig, G. W.; Fleming, G.; Ghirardi, M.; Gunner, M. R.; Junge, W.; Kramer, D. M.; Melis, A.; Moore, T. A.; Moser, C. C.; Nocera, D. G.; Nozik, A. J.; Ort, D. R.; Parson, W. W.; Prince, R. C.; Sayre, R. T. Science 2011, 332 (6031), 805–809. Cox, N.; Pantazis, D. A.; Lubitz, W. Annu. Rev. Biochem. 2020, 89 (1), 795–820. Glatzel, P.; Bergmann, U. Coord. Chem. Rev. 2005, 249 (1–2), 65–95. Hussein, R.; Ibrahim, M.; Bhowmick, A.; Simon, P. S.; Chatterjee, R.; Lassalle, L.; Doyle, M.; Bogacz, I.; Kim, I.-S.; Cheah, M. H.; Gul, S.; de Lichtenberg, C.; Chernev, P.; Pham, C. C.; Young, I. D.; Carbajo, S.; Fuller, F. D.; Alonso-Mori, R.; Batyuk, A.; Sutherlin, K. D.; Brewster, A. S.; Bolotovsky, R.; Mendez, D.; Holton, J. M.; Moriarty, N. W.; Adams, P. D.; Bergmann, U.; Sauter, N. K.; Dobbek, H.; Messinger, J.; Zouni, A.; Kern, J.; Yachandra, V. K.; Yano, J. Nat. Commun. 2021, 12 (1), 6531. Oliver, N.; Avramov, A. P.; Nürnberg, D. J.; Dau, H.; Burnap, R. L. Photosynth. Res. 2022. https://doi.org/10.1007/s11120-022-00912-z. Siegbahn, P. E. M. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (19), 4966–4968.

2.14

Bio-inspired catalysis

Xinyang Zhaoa, Lu Zhub, Xue Wua, Wei Weib, and Jing Zhaoa, a State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China; and b School of Life Sciences, Nanjing University, Nanjing, China © 2023 Elsevier Ltd. All rights reserved.

2.14.1 2.14.1.1 2.14.1.2 2.14.1.2.1 2.14.1.2.2 2.14.1.3 2.14.1.4 2.14.1.5 2.14.1.6 2.14.2 2.14.2.1 2.14.2.1.1 2.14.2.1.2 2.14.2.1.3 2.14.2.1.4 2.14.2.1.5 2.14.2.2 2.14.2.2.1 2.14.2.2.2 2.14.2.2.3 2.14.2.2.4 2.14.2.3 2.14.2.3.1 2.14.2.3.2 2.14.2.3.3 2.14.2.3.4 2.14.2.3.5 2.14.2.3.6 2.14.3 2.14.3.1 2.14.3.2 2.14.3.2.1 2.14.3.2.2 2.14.3.3 2.14.3.4 2.14.3.5 2.14.3.5.1 2.14.3.5.2 2.14.3.5.3 2.14.3.5.4 2.14.3.5.5 2.14.3.6 References

Bioinspired oxidation Introduction CeH bond oxidations Alkanes and cycloalkanes Alkyl benzenes C]C oxidation Alcohol oxidation Ketone oxidation Conclusion Bioinspired energy-relevant catalysis Bioinspired oxygen reduction reactions Introduction Fe-related metal complexes Co-related metal complexes Cu-related metal complexes Conclusion Bioinspired carbon dioxide reduction Introduction Mimics of FDH Mimics of CODH Conclusion Bioinspired hydrogen evolution reaction Introduction Mimics of [NiFe] hydrogenase Mimics of [FeFe] hydrogenase Metal chlorin Biohybrid systems Conclusion Bioinspired bond-forming reactions Introduction CeC bond formation CeC bond-forming enzymes Bioinspired CeC bond-forming reactions CeN bond formation CeO bond formation NeN bond formation N2O-forming enzymes Bioinspired N2O-forming reactions N2-forming enzymes Bioinspired N2-forming reactions Other enzyme-mimicking catalysts Conclusion

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Abstract “Bioinspired catalysis” is a newly-added section since the second edition “Comprehensive Inorganic Chemistry IIdFrom Elements to Applications” in 2013. In the 2013 edition, there were four related sections in “Volume 6, Part 4: Biologically Inspired Catalytic Processes”, which included “Biocatalysis by Metalloenzymes” by Torres and Ayala, “Biomacromolecules as

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00140-0

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Bio-inspired catalysis Ligands for Artificial Metalloenzymes” by Hamels and Ward, “Hydrocarbon Oxidations Catalyzed by Bio-Inspired Nonheme Iron and Copper Catalysts” by Oloo and Que., and “Artificial Metalloproteases and Metallonucleases” by Suh. As of this writing in June 2022, striking advances have taken place in such a wide topic as bioinspired catalysis. Based on the catalytic applications, we selected three sub-topics from the scientific discoveries and updates: oxidation, energy-relevant catalysis, and bond-forming reactions. Since each of the three sub-topics is a big research field itself, naturally, there are many excellent reports that we failed to include or mention.

2.14.1

Bioinspired oxidation

2.14.1.1

Introduction

Efficient and direct oxygenation of organic compounds under mild conditions has been a challenge for decades both in academic and applied research. The gradual understanding of the mechanism of action of natural systems has enabled oxygenation to be performed at the laboratory and industrial levels. For example, inspired by the oxidative metabolism of cytochrome P-450 enzymes discovered in 1958 by Garfinkel1 and Klingenberg2, chemists have successfully mimicked several types of oxidation reactions using metalloporphyrin analogs as catalysts. In addition to metalloporphyrin enzymes, bioinspired nonheme iron oxygenases are often used as catalysts. Que3 and Ray4 have reviewed the progress of bioinspired nonheme iron oxidation catalysis from several perspectives. The use of many non-porphyrin complexes based on other metals, such as Mn5 and Cu6, in bioinspired catalytic oxidation have also been reported. Vast amounts of work in this field based on macrocyclic metal complexes, such as metalloporphyrin complexes, have been reported. The effect of catalytic oxidation are influenced by many factors, such as the structure of the biomimetic metalloporphyrin enzymes (different central metals and ligand structures), reaction conditions (temperature, pressure, time, catalyst concentration, solvent, etc.), the reaction systems (homogeneous or heterogeneous), and the type of oxidants and catalysts (including the addition of co-oxidants and/or co-catalysts). The typical metal core ions of metalloenzymes are Fe(III), Mn(III), and Co(III). The ligands of metalloporphyrin enzymes often bear different electron-withdrawing or electron-donating substituents at the periphery; these peripheral groups include nitro attracting groups and methoxy donor groups. Metalloenzyme catalysts can be homogeneous or heterogeneous, and the yields of homogeneous systems are comparable to those of heterogeneous systems in most available cases. However, heterogeneous catalytic systems seem to be more friendly to the environment, and efforts have been made to reduce the use of highly polluting co-oxidants and/or co-catalysts. Inorganic supports are also used to improve the reutilization of the catalyst. Several oxygen donors have been used, including iodosylbenzene (PhIO)7, hydrogen peroxide (H2O2), and molecular oxygen (O2). Among the several types of oxygen donors mentioned above, molecular oxygen (O2) has become the most attractive because of its abundance and easy acquisition. The catalytic oxidation of organic compounds mainly includes the oxidation of CeH, C]C, CeO and C]O bonds. To better understand how the various properties influence the reactivity of metalloenzymes, we herein review the research progress of catalytic oxidation of some common compounds (such as alkanes/cycloalkanes, olefins, alcohols and ketones) catalyzed by metalloporphyrin enzymes in homogeneous and/or heterogeneous systems over the past 5 years.

2.14.1.2

CeH bond oxidations

The high dissociation energy and non-polarity of CeH bonds make the catalytic oxidation process of such bonds challenging, especially for unreactive primary CeH bonds. To improve catalytic oxidation, the following measures can be taken. First, a highly reactive oxidant can be used to overcome the large activation barrier.8 Second, the selectivity must be finely controlled with this highly reactive oxidant species, and overoxidation of the first formed oxidation products should be limited because alcohols are more reactive than CeH bonds and are more likely to be further oxidized to ketones, aldehydes or acids9. In comparison, the oxidation of secondary and tertiary CeH bonds is more likely to occur under mild experimental conditions, and selective oxidation can also be observed. The catalytic oxidation of CeH bonds can be roughly categorized into alkyl benzenes, alkanes and cycloalkanes.

2.14.1.2.1

Alkanes and cycloalkanes

The reaction of light alkanes is one of the most difficult classes of substrates in aliphatic CeH oxidation. Costas et al.10 reviewed the oxidation of primary CeH bonds of light alkanes catalyzed by Fe- and Mn- complexes bearing non-porphyrin ligands. The oxidation of cycloalkanes is one of the most fundamental reactions in organic synthesis. The ability of metalloporphyrin complexes to catalyze the oxidation of primary CeH bonds of cycloalkanes has been extensively investigated. Many works using homogeneous metalloporphyrins as catalysts for the biomimetic catalytic oxidation of cycloalkanes have been reviewed.11 However, it seems that heterogenization of the catalyst system is likely a better oxygenation strategy. Itoh et al.12 reported an oxido-iron(IV) porphyrin p-radical cation species FeIV(TMPþ•)(O)(Cl) with an increased (720 times) lifetime by employing a,a,a-trifluorotoluene (TFT) as a solvent. The reaction efficiency of FeIV(TMPþ•)(O)(Cl) toward inert alkane substrates was improved by increasing the lifetime. Higuchi et al.13 presented a Ru porphyrin-pyridine N-oxide system for the selective direct modification of alkanes. When eight bulky aryl groups were introduced at the 2,6-positions of the meso-phenyl groups, the Ru porphyrin complex showed unique u-1

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selectivity and provided a much higher turnover number in the oxidation of linear alkanes compared to the reaction catalyzed by conventional Ru porphyrins. Che et al.14 designed a series of highly active and selective Ru(III) porphyrin complexes for rapid hydrocarbon oxidation under mild conditions. At room temperature, using [RuIII(TDCPP)(Ph)(OEt2)] as catalyst and m-CPBA as oxidation, they obtained the yield of CeH bond oxidation to alcohol/ketone up to 99%. Using [nBu4N]IO4 as a mild alternative oxidant can help avoid the formation of cyclic ketones into lactones. In addition, the catalytic epoxidation of terminal and internal alkenes gave yield up to 99% and showed high selectivity with no aldehydes as side product. Yang et al.15 reported two porphyrin-conjugated organic polymers, Mn(III)P-CMP and Fe(III)P-CMP. The two polymers were used as heterogeneous catalysts for the oxidation of CeH bonds in cyclohexane (Scheme 1a). In the oxidation reaction of cyclohexane with Fe(III)P-CMP as a catalyst, the yield of cyclohexanol and cyclohexanone was 19.2%, and the conversion of cyclohexane was 27.5% after 4 h of reaction. Mn(III)P-CMP exhibited a better catalytic effect with a yield of 21.6% and a conversion of 29.9% after 3.5 h of reaction. Both Mn(III)P-CMP and Fe(III)P-CMP exhibited excellent reusability, and the conversion rates of cyclohexane exceeded 25% and 23% after being recycled 5 times, respectively. Tabor et al.16 studied the effect of substituents on the catalytic activity of iron m-oxo porphyrins in the oxidation of cycloalkanes with molecular oxygen. Several Fe-porphyrin complexes bearing different meso-aryl substituents were synthesized to investigate their electrochemical properties and catalytic activity. The introduction of either electron-donating or electron-withdrawing substituents into the porphyrin rings improved the catalytic performance over that of the unsubstituted porphyrins. Nam et al.17 reported a Co-porphyrin complex, CoII(TPP), for the photocatalytic oxygenation reactions of hexamethylbenzene to afford pentamethylbenzyl alcohol and hydrogen peroxide as products with a turnover number of > 6000.Recently, the same group reported three Mn(III)-iodosylarene porphyrins complexes, [MnIII(ArIO)(TDCPP)]þ, [MnIII(PhIO)(TDFPP)]þ and [MnIII(PhIO)(TPFPP)]þ, with capability of activating the CeH bonds of hydrocarbons and sulfoxidation reactions, such as unactivated alkanes such as cyclohexane. The reactivities and mechanisms of Mn(III)-iodosylarene porphyrins complexes are well studied and compared with MnIV(Porp)(O) complex. This is the first example of highly reactive Mn(III)-iodosylarene porphyrins for CeH bond activation and OAT reactions.18 In addition, in 2021, they reported a series of Mn(IV)-oxo porphyrins bearing different ERP/EDPs, [MnIV(O)(TMP)], [MnIV(O)(TDMPP)], [MnIV(O)(TMePP)] with ERP and [MnIV(O)(TDCPP)], [MnIV (O)(TDFPP)], [MnIV(O)(TPFPP)] with EDP. All of the six Mn(IV)-oxo porphyrins are highly reactive CeH activation reactions and the reactivity and kinetic studies of Mn(IV)-oxo porphyrins were carried out.9 Huang et al.19 reported a series of heterogeneous M TCPP/ZnS (M TCPP ¼ metal tetrakis(4-carboxyphenyl)porphyrins, M ¼ Co, Fe, or Mn) catalysts. ZnS was used as a thiolate ligand resource, and M TCPP was immobilized on ZnS. These catalysts exhibited good stability and activity, and they could be reused three times for cyclohexane oxidation (Scheme 1b). Compared to the corresponding unsupported metalloporphyrins, each ZnS-immobilized M TCPP provided better catalytic efficiency. Among them, Co TCPP/ZnS exhibited the best catalytic performance with a cyclohexanol and cyclohexanone yield of up to 27.0% and a cyclohexane conversion of up to 79% (Fig. 1).

2.14.1.2.2

Alkyl benzenes

The oxygenation of CeH bonds of alkyl benzenes, some of the least expensive and most readily available raw materials, is one of the most fundamental and extensive transformation pathways of alkyl benzene alkylation in the manufacture of pharmaceuticals and fine chemicals. Efforts have been made to infer the effect of the different peripheral substituents and central metals on the oxidation progress of many alkyl benzenes, including p- and o-cresol20–22, toluene22, and p-xylene23. Regardless of the different central metals, metalloporphyrins bearing electron-donating substituents show higher activity; however, the central metals greatly influence the selectivity.20 Among metalloporphyrins with the same ligand structure, Co porphyrins often exhibit relatively high catalytic activity, and the mechanisms of Co porphyrin reactions may involve an autooxidation process;24 this process differs from the mechanisms of Fe- and Mn-porphyrins catalyst interactions, which may be prone to form oxo-metal(IV) porphyrin cation radicals.21 Yang et al.25 synthesized a porous alkynyl porphyrin conjugated polymer, MnE-TPP, by Sonogashira coupling of [p-ethynyl]4PMn and [p-Br]4PMn as building blocks. With a porous structure and large BET surface area, MnE-TPP was developed as a heterogeneous catalyst for the oxidation of toluene under mild conditions (Scheme 2a) (O2 as an oxidant, 160  C under

(A)

(B)

Scheme 1

Oxidation of cyclohexanes.

376

Fig. 1

Bio-inspired catalysis

Structures of metalloporphyrin catalysts for the oxidation of alkanes and cycloalkanes.

0.8 MPa for 5 h) with a conversion up to 10.2% and selectivity for benzaldehyde (42.2%) and benzyl alcohol (27.8%). The MnETPP could be reused for 5 cycles with similar conversions.

Bio-inspired catalysis

(A)

(B)

(C)

(D)

377

(E)

(F)

(G)

Scheme 2

Oxidation of alkyl benzenes.

Huang et al.26 reported a heterogeneous Mn(III)-porphyrin catalyst, Mn TCPP/pd-CTS. To reduce the environmental impact of the production of acetophenone and phenethyl alcohol, tetrakis(4-carboxyphenyl)porphyrin manganese chloride (Mn TCPP) was grafted onto powdered chitosan (pd-CTS) after acylation. The grafted material was used for the oxidation of ethylbenzene with molecular oxygen as the oxidant. The effects of temperature, pressure, and amount of catalyst on the oxidation of ethylbenzene catalyzed by Mn TCPP/pd-CTS were investigated. The catalytic activity and efficiency of Mn TCPP increased greatly because the electron cloud densities of the Mn ion changed after the ligation of the nitrogen atom in the amino groups of pd-CTS to Mn TCPP. Mn TCPP/pd-CTS provided a conversion of 20.74%, selectivity for acetophenone of 53.91% and catalyst turnover numbers (TON) up to 2.47  105 (Scheme 2b). However, the conversion by Mn TCPP/pd-CTS decreased to 12.64% after recycling three times, which can be attributed to the instability of chitosan at high temperatures. Ji et al.27 developed a novel heterogeneous system comprised of metalloporphyrins M TPPCl (M ¼ Fe, Co, Mn) and cyclohexene for the oxidation of toluene and its derivatives (Scheme 2c), where cyclohexene served as the key activator for dioxygen. The catalytic system exhibited good functional group tolerance, selectivity and broad substrate scope. In the biomimetic catalytic system, it was found that MnTPPCl and cyclohexene were the best choices and aldehydes were the main products. Two cationic metalloporphyrins, CoTEtPyP and MnTEtPyP(OAc), were synthesized and immobilized on the surface of SiO2.28 Porphyrin-SiO2 (PSC1 and PSC2) were obtained as heterogeneous catalysts for the oxidation of ethylbenzene. The activities for ethylbenzene catalysis indicated that both metalloporphyrins exhibited high selectivity for acetophenone (99%) with conversions of 87.6% (PSC1) and 93.0% (PSC2), respectively. The catalytic performance of PSC2 was better than that of PSC1 due to the excellent activity of the Mn-porphyrin. Lu et al.29 developed seven metalloporphyrin-based porous polymers CP1-CP7 (1-Fe, 2-Co, 3-Ni, 4-Cu, 5-Zn, 6-Mn, 7-Pb) based on H2TZP (TZP ¼ 5,10,15,20-tetrakis[4-(2,3,4,5-tetrazolylphenyl)] porphyrin). These coordination polymers exhibit effective catalytic activities, and selectivities (81–99%) (except CP3) for the oxidation of ethylbenzene to ketones and metal atoms had a great influence on the catalytic activities (Scheme 2d). Among the seven polymers, CP1 showed the best catalytic activity with a high yield (89%) and selectivity (99%) for acetylbenzene. CPs can be reused after filtration with a slight decrease in catalytic activity. A series of metalloporphyrin catalysts T(p-R)PPM (R]NO2, CN, F, Cl, Br, Me, Et, OMe, OH, M]Mn, Fe(Cl), Co, Ni, Cu, Zn) was synthesized for the oxidation of 4-ethylnitrobenzene to 4-nitroacetophenone with O2 as a clean oxidant (Scheme 2e).30 The catalytic activities and reaction selectivities could be significantly affected by the central metal ions and the substituted groups. Fe(III)and Mn(II)-porphyrins exhibited high activities with selectivities over 90.0% for the desired ketone, and 1-(4-nitrophenyl)ethanol and the overoxidation product 4-nitrobenzoic acid were also observed in this study. In addition, the catalytic oxidation of 4methylnitrobenzene by metalloporphyrin has also been reported recently. Yang et al.31 prepared a cobalt porphyrin polymer (Co/PCP) by hybrid polymerization of a mixture of CoTBPP and TBPP in a ratio of 3:7 with 1,4-phenylenebisboronic acid as the coupling reagent. The obtained polymer was further carbonized to the final

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catalyst (Co3O4@PNC-400). The cobalt catalyst exhibited higher catalytic activity and selectivity for the aerobic CeH bond oxidation of toluene and cyclohexane (Scheme 2f). The Co3O4@PNC-400 catalyst exhibited synergy between the Co3O4 nanoclusters and the nitrogen-carbon framework generated after the heat treatment. Ji et al.32 established an efficient system for the oxidation of primary benzylic CeH bonds with cobalt porphyrin and NHPI (Nhydroxyphthalimide) and scCO2. In this protocol, the conversion of toluene was 21.6% and the benzoic acid selectivity reached 81.2%, and this protocol exhibited good substrate tolerance (Fig. 2).

2.14.1.3

C]C oxidation

C]C bonds are also relatively unreactive compared to most functional groups. The oxidation of olefins usually yields epoxides, and further reactions can produce dihydroxylated products. However, selective oxidation sometimes gives unexpected hydroxylation or ketone products. Mn(III) and Fe(III) porphyrins are common catalysts for the epoxidation of alkenes, though they sometimes require the addition of co-reducing agents. Mn protoporphyrin IX (MnPPIX) reconstituted metalloproteins exhibited catalytic reactivity similar to that of peroxidase; however, it was unable to catalyze oxygen atom transfer reactions.33 Zhang et al.34 reported the first example of the epoxidation of the C]C bond (styrene) catalyzed by MnPPIX recombinant myoglobin (MnIIIMb (L29H/F43H)) using oxone (2KHSO5$KHSO4$K2SO4) as an oxidant (Scheme 3a). Interestingly, His3 MnIIIMb with H2O2 as an oxidant showed no reactivity in the epoxidation of styrene because the heterocleavage of the OeO bond generated more active [MnIV ¼ O]þ• intermediates using oxone as an oxidant. Wu et al.35 reported a Ln-metalloporphyrin-POM hybrid material combined with metalloporphyrins and inorganic polyoxometalates (POMs) in the framework structure as a heterogeneous catalyst for the epoxidation of olefins (Scheme 3b). It exhibited high efficiency for the epoxidation of olefins under mild conditions with very high turnover numbers (TON up to 220,000) and turnover frequency (TOF up to 22,000 h 1). The synergistic action of metalloporphyrins and POMs can achieve superior efficiency in the epoxidation of olefins. Rayati et al.36 reported the synthesis of the heterogeneous catalyst Fe(THPP)Cl@MWCNTs for the oxidation of olefins and sulfides by immobilizing Fe(THPP)Cl (Fe-porphyrin complexes) onto carbon nanotubes with O2/isobutyraldehyde (IBA) as the oxygen donor (Scheme 3c). The catalyst showed good catalytic performance and superior reusability with substrate conversion and selectivity of epoxidation up to 100% under mild conditions. Wu et al.37 reported a porous covalent porphyrin framework Co-CPF-2 for heterogeneous catalysis (Scheme 3d) of the aerobic epoxidation of olefins with conversion over 99%, epoxide selectivity up to 93%, TON ¼ 29,215, and TOF ¼ 3434 h 1. Compared to its homogeneous counterpart CPF-2, the new heterogeneous catalyst Co-CPF-2 exhibited higher stability and could be reused in up to 15 cycles without loss of activity/selectivity. Fe3O4-[Mn(TPyP)tart], a chiral Mn-porphyrin, was synthesized for enantioselective epoxidation of olefins using molecular oxygen as an oxidant and isobutyraldehyde (IBA) as a co-reductant.38 The chiral catalyst was obtained by immobilizing Mn(TPyP)OAc onto magnetite nanoparticles and stabilized with chiral L-(þ)-tartaric acid (tart). The cationic system showed excellent activity, selectivity and reusability for the oxidation of olefins with enantiomers up to 97% (Scheme 3e). Two hybrid Cu-porphyrin-based magnetic nanomaterials, MNP@SiO2-bNH-Cu-TDFPP and MNP@SiO2-bNH-Cu-TDCPP, were efficiently synthesized as reusable biomimetic catalysts for the CeH oxidation of cyclohexene in the presence of O2 (0.4 MPa) at 100  C for 4 h (Scheme 3f).39 Cyclohex-2-en-1-one (69%) and 2-cyclohexen-1-ol (21%) were the main oxidation products, and the highest TON reached 201,015. Pereira et al.40 reported the Mn-porphyrin-based hybrid material MNP@SiO2[4-NH-Mn-TDCPP], which was efficient in the epoxidation of cyclooctene in the presence of O2(bubbling) as the oxidant and isobutyraldehyde (IBA) as the co-reductant (Scheme 3g). The heterogeneous catalyst showed high conversion (99%) and selectivity for epoxidation (98%) and was able to be reused in five runs without loss of activity or epoxide selectivity. Tian et al.41 investigated the epoxidation of propylene via O2 with acrolein by Mn(p-CH3)TPPCl and Fe(p-CH3)TPPCl. Through density functional theory (DFT) calculations, peroxyacyl radicals and peroxy acids were proposed as active species, and the center ion of metalloporphyrins played a principal role in the initiation of the reaction. Mo2TCPP, a new metalloporphyrin with a 3D network structure, was synthesized and used for the epoxidation of cyclohexene with cumene hydrogen peroxide and hydrogen peroxide as oxidants (Scheme 3h).42 The conversion and selectivity of epoxidation were both over 99%, and the catalyst exhibited excellent stability and reusability for ten runs without obvious degradation. Matsubara et al.43 described the development of a mild and selective catalytic aerobic oxidation process of olefins. By using a ferric porphyrin complex and pinacolborane, a ferric boroperoxo porphyrin complex intermediate was in situ generated as an oxidizing species for the catalytic aerobic oxidation of alkenes under ambient conditions to form oxidation products. Gross’s group has done a lot of research on the epoxidation of olefins by ruthenium porphyrin complexes in the early years. In 1996, they reported the first example of a homochiral ruthenium porphyrin as enantioselective epoxidation catalyst and proposed that the enantioselectivity of chiral ruthenium complexes is sensitive to solvents and oxidants.44 Then, they investigated the effects of metals, solvents, and oxidants on metalloporphyrin-catalyzed enantioselective epoxidation of olefins.45 In 1999, they designed a series of chiral ruthenium porphyrin complexes for the asymmetric catalytic epoxidation of olefins.46

Bio-inspired catalysis

Fig. 2

379

Structures of metalloporphyrin catalysts for the oxidation of alkyl benzenes.

Che et al. reported the ruthenium porphyrin-catalyzed oxidation of aryl alkenes to aldehydes with high selectivity under mild conditions in 2004 and 200847, and then they reviewed the Ru porphyrin complexes for the oxidation of alkenes in 2009.48 In 2018,

380

Bio-inspired catalysis

(A)

(B)

(C)

(D)

(E)

(F)

(G)

Scheme 3

Oxidation of olefins.

they described the first computational study of Ru porphyrin-catalyzed oxidation of styrenes to aldehydes. The results of DFT indicated that in the oxidation progress, monooxoruthenium porphyrin participates in the epoxide isomerization (E-I) to selectively yield an aldehyde and generate a dioxoruthenium porphyrin, triggering new oxidation reaction cycles. The catalytic abilities of ruthenium porphyrins with different axial ligands were also investigated, and the ruthenium porphyrin featuring a chlorine axial ligand was found to be more reactive (Fig. 3).49

2.14.1.4

Alcohol oxidation

The oxidation of alcohols is much easier than that of CeH and C]C bonds. Depending on the nature of the alcohol substrates, their oxidation typically produces aldehydes and ketones. Recent progress in the catalytic oxidation of alcohol compounds catalyzed by metalloporphyrins has been mostly based on manganese porphyrins. Yang et al.50 reported a series of conjugated Mn-porphyrin polymers (MnP-AMPs) with different surface areas. MnP-AMPs exhibited high catalytic activity for the aerobic oxidation of alcohols using isobutyraldehyde as a co-reductant (Scheme 4a), with the conversion and selectivity of benzyl alcohol to benzaldehyde in the range 95–98%. The catalytic performance of MnPAMPs for alcohol oxidation was improved with increasing specific surface area, and these catalysts also remained stable and active after several cycles. A series of Mn-porphyrin-based polymers (Mn/TFP-DPM, Mn/TFP-DPM-2, Mn/TFP-DPM-3 and Mn/TFP-DPM-4) with different morphologies was designed and served as heterogeneous catalysts for the selective oxidation of alcohols (Scheme 4b)51. These porphyrin-based polymers were synthesized by a one-pot method, and the morphology was greatly affected by the synthesis conditions. In contrast, the reaction rate and activity of Mn/TFP-DPM-3 were significantly better than those of Mn/TFP-DPM due to the

Bio-inspired catalysis

Fig. 3

381

Structures of metalloporphyrin catalysts for the oxidation of olefins.

larger surface area of Mn/TFP-DPM-3. Both Mn/TFP-DPM and Mn/TFP-DPM-3 can be reused six times without any obvious loss of catalytic activity.

382

Bio-inspired catalysis

(A)

(B)

(C)

Scheme 4

Oxidation of alcohols.

Zhou et al.52 developed a homogenous system comprised of Mn-porphyrin and cyclohexene for the aerobic oxidation of alcohols to carbonyl compounds, where cyclohexene was used to generate Mn-oxo species by propagating free radicals generated from itself (Scheme 4c). The comparison between MnTPPCl and other metalloporphyrins that have the same ligand but different metal ions revealed that Mn-porphyrin MnTPPCl exhibited higher catalytic activity than Fe, Cu, Co and Zn porphyrins when benzyl alcohol was the substrate. The synergistic action of MnTPPCl and cyclohexene enabled the complete oxidation of benzyl alcohol, and the catalytic system exhibited excellent catalytic performance and selectivity for most primary and secondary alcohols under mild conditions (Fig. 4).

2.14.1.5

Ketone oxidation

The transformation of ketones to the corresponding lactones mostly using heterogeneous catalysts has been reported in the past decade; however, there seem to be few reports on the oxidation of ketones during the last 5 years. Yang et al.53 synthesized a series of metalloporphyrin polymers with a hyperconjugated mesoporous reticulate structure (MPPAMNPs) using T(p-NH2)PPMnCl and p-dibromobenzene as building blocks. These polymers were developed to promote the aerobic oxidation of cyclic ketones toward lactones with benzaldehyde as a co-reductant (Scheme 5) under mild conditions. The yield of cyclic ketone substrates was in the range of 75–98% after 5 h of reaction, and the stability and activity of the catalyst were maintained for at least 10 cycles (Fig. 5).

2.14.1.6

Conclusion

Biomimetic metalloporphyrin catalysts have wide application prospects in the catalytic oxidation of organic substrates, but their use remains one of the largest challenges; thus, research will continue over the next decade. The design of metalloporphyrin catalyst systems is affected by several factors: (i) the nature and position of the substituents on the periphery of the porphyrin ligands;

Fig. 4

Structures of metalloporphyrin catalysts for the oxidation of alcohols.

Bio-inspired catalysis

Scheme 5

383

Oxidation of cyclic ketones.

(ii) the choice of core metal ions for certain types of compounds; (iii) the addition of co-reducing agents for better efficiency and selectivity; and (iv) heterogenization of the catalyst system. It is likely that more efficient, stable, selective and reusable catalytic oxidation systems will be developed in the future.

2.14.2

Bioinspired energy-relevant catalysis

2.14.2.1

Bioinspired oxygen reduction reactions

2.14.2.1.1

Introduction

The oxygen reduction reaction (ORR) plays an important role in life activities and energy-converting systems. It mainly occurs via two pathways in aqueous solution (Table 1): the direct four-electron pathway of O2 reduction to H2O and the two-electron pathway

Table 1

Oxygen reduction reaction pathways. ORR reactions

4e Pathway O2 þ 4Hþ þ 4e / H2O O2 þ H2O þ 4e / 4OH  2e Pathway O2 þ 2Hþ þ 2e / H2O2 H2O2 þ 2Hþ þ 2e / 2H2O O2 þ H2O þ 2e / HO2 þ OH HO2 þ H2O þ 2e / 3OH  1e Pathway O2 þ e / O2 O2- þ e / O22

Fig. 5

Structures of metalloporphyrin catalysts for the oxidation of cyclic ketones.

Electrolyte Acidic aqueous solution Basic aqueous solution Acidic aqueous solution Basic aqueous solution Nonaqueous aprotic solvents

384

Bio-inspired catalysis

of O2 reduction to H2O2, which is subsequently reduced to H2O. Additionally, a one-electron reduction pathway from O2 to O2 can occur in basic solutions or nonaqueous aprotic solvents.54,55 Fuel cells, a promising clean and sustainable energy source, have been greatly developed in recent years to mitigate global warming and the energy crisis.56 In fuel cells, the ORR occurs at the cathode. For example, in proton exchange membrane (PEM) fuel cells, H2 is oxidized into protons and electrons at the anode. The newly generated protons migrate through the polymer electrolyte membrane with external circuit-produced electrons to reduce O2 to H2O at the cathode.57 Due to the slow kinetics of this reaction, an electrocatalyst is needed to lower the energy barrier, accelerating the reaction. Among the existing technologies, Pt-based catalysts are the most efficient for the ORR.58 However, due to its high price and scarcity, Pt is not suitable for the manufacture of commercial fuel cells. To solve this problem, nonprecious metal catalysts have undergone considerable development.59 The iron porphyrin complex exists as heme (Fig. 6) in numerous metalloenzymes and proteins, such as cytochrome c oxidase, cytochrome P450, and hemoglobin, which participate extensively in oxygen reduction, activation, and binding. For example, cytochrome c oxidase contains two heme structures (one cytochrome a and one cytochrome a3) and two copper centers (CuA and CuB). Cytochrome a3 and CuB form a dual core center that acts as a reduction site converting O2 to H2O through a four-electron pathway.60,61 Overall reaction: Inspired by these biological complexes, many coordination compounds have been designed and synthesized as efficient ORR electrocatalysts. They are mostly based on macrocyclic ligands such as porphyrins, corroles and phthalocyanines (Fig. 7). In this section, we briefly introduce the application of Fe, Co, and Cu catalysts in the electrocatalytic ORR.

2.14.2.1.2

Fe-related metal complexes

Heme plays several important roles in biology, including in oxygen storage, transportation, and catalysis.62 The different substituents on its porphyrin ring lead to a series of derivatives with different functions. Many Nature-inspired heme/porphyrin-based electrocatalysts have been developed. Starting from the catalytic mechanism, Kushal et al. used surface-enhanced resonance Raman spectroscopy coupled to rotating disk electrochemistry (SERRS-RDE) to achieve the in situ capture of O2-derived intermediates on the electrode surface. Based on the results of that study, an oxygen-reducing mechanism by an iron porphyrin was proposed (Fig. 8). SERRS-RDE showed that heterolytic OeO bond cleavage of the intermediate FeIII-OOH was the rate-determining step of the ORR catalyzed by simple iron porphyrin complexes. Many studies have focused on the effect of distal residues on the rate and selectivity of the ORR.63 For example, Sarmistha et al. attached distal basic groups to porphyrins to obtain three substrates (Fig. 9). After the substrates were loaded onto the electrode, the catalytic efficiency of selectively reducing O2 to H2O under rate-limiting electron transfer conditions at pH 7 was greater than 90%. The O2 reduction rate is 100 times faster than that in the heme/Cu system. Remarkably, the kcat of the FeL2 obtained on the EPG electrode surface was (1.80  0.07)  107 M 1 s 1. The experimental results showed that the basic group can form a hydrogen bond with the intermediate FeIII-OOH to prevent its hydrolysis and can activate the OeO bond of such peroxide by pulling to promote the transfer of protons to the remote oxygen.64 Asmita et al. studied the effect of pendant phenol and quinol groups on the iron porphyrin-catalyzed ORR (Fig. 10). The data showed that all the complexes could reduce O2 to H2O by the four-electron pathway with similar rates but through different mechanisms. FeQMe2 inhibited the hydrolysis of the FeIII-OOH group, causing the 4e ORR reaction to proceed through an electron

(B)

(A)

Fig. 6 (A) Chemical structure of iron protoporphyrin IX (active site of heme proteins). (B) The structure and cofactors of mammalian cytochrome c oxidase Copyright.60

Bio-inspired catalysis

Fig. 7

385

Chemical structures of porphyrin (left), phthalocyanine (middle), and corrole (right).

transfer-proton transfer (ETPT) pathway. In addition, FeQH2 reduced O2 through two consecutive hydrogen atom transfer (HAT) pathways to intermedia FeIII- O2 because of the low reduction potential of quinol. In addition, the low pH of pendant phenol (FePh) allowed O2 to be reduced to FeIII- O2 species via a proton coupled electron transfer (PCET) pathway. In summary, pendant phenol and quinol can stabilize the FeIII-OOH intermediate through hydrogen bonding, resulting in over 95% selectivity for the 4e ORR under slow electron transfer flux.65 In addition to iron porphyrin, iron phthalocyanine (FePc) has attracted attention due to its unique FeN4 active sites and low ORR energy barrier. Different catalytic effects can be obtained by loading FePc on different nanocarbon materials in different ways. All FePc/nanocarbon catalysts have higher activity for the ORR than commercial Pt/C.66 For instance, carbon black has become the most commonly used electrocatalyst carbon carrier due to its low cost, large specific surface area, and high conductivity. Zhang et al. loaded FePc on Mn2þ-modified graphitized carbon black (GCB), and additional Pc molecules were used to regulate the coordination environment of iron by the delocalized p coordination effect. The ORR catalytic activity of Pc-FePc/Mn-GCB reached 2.26 mA cm 2, which is 3.6 times greater than that of commercial Pt/C. Its durability is also excellent; after 3000 potential cycles, only a 3-mv half-wave potential shift occurred. The additional Pc group protects the FePc from intervention with SO42, Br, and SCN, thus enhancing the stability.67 Carbon nanotubes (CNTs) have also become popular support materials due to their highly crystalline graphite surface and high electrical conductivity. Yang et al. prepared a multiwalled carbon nanotube/iron phthalocyanine (MWNT/FePc) composite catalyst for the ORR in basic media by simply shaking and heating it. Furthermore, MWNT/FePc was used as a cathode in a zinc-air battery, obtaining excellent performance (185 mW cm 2) power density at 0.8 Subsequent density functional theory (DFT) calculations revealed that the geometry and electronic structure of MWNT/FePc deposited on the surface of MWNTs was notably different due to the p-p interactions that the electrocatalytic activity and durability.68 It is well known that graphene has a large specific surface area, good thermal conductivity, and excellent chemical and thermal durability. An iron(II) tetra-aminophthalocyanine (FeTAPc)-modified carboxyl-functionalized graphene oxide (CFGO) catalyst was synthesized by a simple amidation reaction (Fig. 11). The data showed that the peak potential was  0.12 V vs. SCE in 0.11 M NaOH solution. Chronoamperometric tests showed that after 10,000 s, the FeTAPc-CFGO catalyst still retained 83.5% of the original current, while Pt/C retained at only 40.5%. This novel catalyst showed good ORR activity, stability, and methanol tolerance (3 M methanol with a slight peak current change). In another report, a colloid chemical reaction was first used to synthesize Fe(II)Pc/Fe(III)Pc/graphene nanostructures. Experimental and DFT results revealed that Fe(II) and Fe(III) have synergistic effects on ORR catalysis.69

Fig. 8

Proposed mechanism of oxygen reduction by an iron porphyrin.

386

Fig. 9

Bio-inspired catalysis

Chemical structures of FeL1, FeL2, and FeL3.

2.14.2.1.3

Co-related metal complexes

In 1964, cobalt phthalocyanine was first used as a nonnoble metal catalyst in H2/O2 fuel cells.70 Since then, various cobalt-related electrocatalysts have been synthesized and used for the ORR. Like iron porphyrin, cobalt porphyrin (CoP) has been extensively studied, and it has better stability and reactivity. Covalently attaching CoP to carbon nanotubes is a practical preparation method. Zhang et al. functionalized multiwalled carbon nanotube (MWCNT) sidewalls via an SN2 substitution reaction (Fig. 12a). The resulting catalyst could reduce O2 to H2O through a 4e pathway within a large pH range (0.0–5.0) at room temperature. The ORR rate was one order of magnitude higher than was achieved when Co porphyrin was adsorbed onto MWCNTs by dip coating.71 Hijazi et al. used polymerization to surround CoPs in a covalent network around MWCNTs (Fig. 12b), which showed higher ORR activity than monomeric porphyrins physisorbed on MWCNTS. The average number of transferred electrons was close to four, indicating that O2 was completely reduced to H2O. Furthermore, MWNT-CoP was highly stable due to the multiple p-stacking interactions between MWCNTs and porphyrins and the porphyrin covalent network.72 In addition to CoP loaded on a carbon base, individual multicore CoP particles are also practical. Functionally modified binuclear CoP was proven to have high selectivity and activity (Fig. 13a). The results indicated that CoII is the O2 binding and reduction site. When CoIII is generated from the oxidation of CoII, it may assist O2 binding and activation as a Lewis acid. The through-space charge interactions could occur between positively charged CoIII and negatively charged O2 adduct units, causing a reduction in the ORR activation energy barrier.73 Coordination-driven self-assembly was used by Oldacre et al. to form a cofacial Co prism organized by two cobalt(II) tetra(meso-4-pyridyl)-porphyrinate (CoTPyP) and four arene-Ru clips (Fig. 13b). This method required no chromatographic purification and had a high yield (53%). The Co prism was 90% selective for the ORR, and the rate constant of the overall homogeneous catalysis exceeded that of CoTPyP by an order of magnitude.74 Corroles, as cyclic tetrapyrrole aromatic compounds, consist of 19 carbon atoms, which is one less meso-carbon atom than porphyrin. The unique structure makes corrole a trianion ligand, resulting in better stabilization of high-valence metal ions. Kadish and coworkers synthesized a series of cobalt corroles to investigate their ORR activity by coating them on an edge-plane pyrolytic graphite electrode in acid media (Fig. 14a). The results revealed that the cobalt b,b’-tetrabutano-substituted triarylcorroles could catalyze O2 to H2O2 by a 2e pathway with high selectivity. Furthermore, they studied the effects of different substituents on the catalytic ORR efficiency. Catalysts with electron-withdrawing substituents showed better catalytic activity than those with electron-donating groups on the three meso-phenyl rings.75 A 3D polymeric cobalt complex (Fig. 14a, R]NH2) was synthesized by Friedman et al. using tris(4-aminophenyl)corrole (CoTAC). When polymerized polyCpTAC was inside high-surface-area carbon, the resulting catalyst was highly selective for the 4e/4Hþ pathway. Compared to the commercial catalyst, in an acidic environment, the novel catalyst exhibited significantly better performance with a higher onset potential (approximately 100 mV higher) and limiting currents. However, in a basic environment, the onset potential was lower and limiting currents were higher, indicating that the novel catalyst had faster reaction kinetics.76

Fig. 10

Chemical structures of FeQH2, FePh, and FeQMe2.

Bio-inspired catalysis

Fig. 11

387

The monomer structure of FeTAPc.

Recently, Han et al. combined the N,N-di(2-picolyl)ethylenediamine unit with the meso-phenyl ring (Fig. 14b). The pyridyl groups and one tertiary amine unit were partially protonated under acidic conditions, and then the multiple positively charged sites were used to stabilize negative intermediates and promote proton transfer. This strategy improved the activity and 4e selectivity of the ORR. This work showed that functional groups can effectively affect the ORR selectivity and activity of Co corrole.77

2.14.2.1.4

Cu-related metal complexes

In addition to heme, multicopper oxidase enzymes, such as laccase, can efficiently catalyze the ORR through a 4e pathway. As shown in Fig. 15a, the type 1 Cu center takes an e from the substrate and then transfers it through a Cys-His pathway to the trinuclear Cu clusters (known as active sites), where O2 is reduced to H2O. Inspired by Nature, various kinds of copper complexes have been applied in the ORR.78 Thiyagarajan and coworkers used a tricopper cluster complex to functionalize reduced carbon black (Fig. 15b). The onset oxygen reduction potentials were  0.92 V at pH 13 and  0,77 V at pH 7. The kinetic studies indicated that the ORR was accomplished through the 4e pathway.79 A copper-phenolate complex that mimics the active center of galactose oxidase was synthesized by Gentil et al. (Fig. 16a). The complex was immobilized at the surface of MWCNTs by supramolecular p-stacking. The combined catalyst exhibited 4e/4Hþ activity. The redox potential was 0.6 V vs. RHE at pH 5. The rich redox performance of phenol-based ligands provides new inspiration for ORR catalyst design.80 In a recent study, Goff and coworkers immobilized unprotected phenolatocopper complexes containing pyrene groups on MWCNTs, which promoted their dimerization (Fig. 16b). The dinuclear complex showed better ORR activity (12.7 mA cm 2 ORR activity and 0.78 V onset potential versus reversible hydrogen electrode) than the mono electrode.81 Wang et al. placed copper 5-nitrophenanthroline complexes sandwiched between polyvinylimidazole layers and then wrapped them on CNTs (Fig. 17a). When used as an ORR electrocatalyst, the results exhibited high selectivity (approximately 97%) for the 4e reduction of O2 to OH in 0.1 M KOH solution. The onset potential was 1.046 V vs. RHE, and the half-wave potential is 0.859 V. The current density was stable, retaining 94% of its initial value after 90,000 s at 0.60 V, and the methanol tolerance reached 2.0 M; thus, this catalyst outperformed Pt/C in terms of stability. However, the catalytic activity of this catalyst was comparable to that of Pt/C.82 Covalently bonding copper to polymer-coated carbon is a new, simple and low-cost way to construct electrocatalysts. A 1,10phenanthroline copper complex was bonded to poly(pyrrole-3-carboxylic acid)-coated carbon to form an efficient ORR catalyst (Fig. 17b). The composite catalyzed the ORR through the 4e pathway at 0.62 V vs. RHE onset potential in a neutral phosphate buffer solution. Remarkably, under the same conditions, the copper-based composite exhibited better stability and ORR activity than the iron-based composite (onset potential ¼ 0.56 V vs. RHE). In another report, a 1,10-phenanthroline-modified nitrogenrich polymer (PHTPP) was synthesized on the electrode. Then, a copper sulfate solution was used to obtain the polymer-Cu complex (Fig. 17c), which could electrocatalyze a 4e ORR at pH 7. The polymer without copper coordination could reduce O2 to H2O2 following a 2e pathway. Additionally, this complex showed excellent performance in microbial fuel cells as a cathode catalyst.83

(A)

(B)

Fig. 12 (a) Chemical structure of CoP-functionalized MWCNTs synthesized by Zhang et al. (b) Chemical structure of poly-CoPs surrounded by MWCNTs synthesized by Hijazi et al.

388

Bio-inspired catalysis

(A)

Fig. 13

(B)

(a) Chemical structure of multicore CoP synthesized by et al. (b) Chemical structure of CoTpyP and the Co prism.

2.14.2.1.5

Conclusion

Inspired by Nature, nonprecious metal macrocyclic complexes with metals such as Fe, Co, and Cu have been widely used in oxygen reduction reactions. The macrocyclic ligands are mainly porphyrins, phthalocyanines, and corroles. By modifying the macrocyclic ligands or changing the axial ligands, the ORR selectivity and activity can be improved. The complex can either be loaded directly onto the electrode or onto carbon nanomaterials such as graphene and carbon nanotubes by covalent binding to optimize the catalytic performance. Many of the catalysts described in this review showed higher ORR activity, durability and methanol resistance than commercially available Pt/C in basic or even strongly acidic media. Although there has been much research, many studies have only focused on half reactions, and only some of these reactions are used in fuel cells. In practical applications, the molecular integrity of these catalysts is gradually be destroyed, so durability is a major challenge. It is believed that in the future, through the modification of ligands and the change of the loaded carbon material itself, it is possible to design oxygen reduction catalysts that undergo mechanisms similar to those of natural enzymes and that are more stable during actual use.

2.14.2.2 2.14.2.2.1

Bioinspired carbon dioxide reduction Introduction

In addition to oxygen reduction reactions, the reduction of carbon dioxide into valuable hydrocarbons is another way that organisms convert energy. CO2 reduction is challenging due to the high activation energy and chemical inertness of CO2. In Nature, carbon monoxide dehydrogenase (CODH) can convert CO2 into CO and formate dehydrogenase (FDH) for CO2/HCOOH conversion. Inspired by the active sites of these enzymes, a number of efficient, selective catalysts have been developed, providing a foundation for the further production of carbon-based fuels with high energy density.84 In Nature, bioinspired small molecules and biomolecular catalysts often imitate Ni-containing CODH, which contains a nickeliron-sulfur cluster, during CO2/CO conversion (Fig. 18). A dinuclear MoeCu active center where Mo is coordinated with the molybdopterin ligand is used as a template. During CO2/HCOOH conversion in FDH, two molybdopterin ligands chelated with a mononuclear Mo or W are the active sites. In this section, we briefly introduce the progress of mimicking various enzymes.85

2.14.2.2.2

Mimics of FDH

In the chemical simulation of FDH, researchers often use Mo as the metal center and optimize the catalytic function through the ligand structure. Quinoxaline-pyran-fused dithiolene (qpdt) was synthesized to mimic molybdopterin. In 2015, (Bu4N)2 [MoIV O(qpdt)2] was synthesized by Fontecave and coworkers for CO2 reduction (Fig. 19a). Unfortunately, under both electrochemical and photochemical conditions, the Mo complex only reduced Hþ to H2. In a subsequent study, they slightly changed the structure (A)

(B)

Fig. 14 (a) Chemical structure of cobalt corroles synthesized by Kadish et al. and Friedman et al. (b) Chemical structure of pyridyl group-modified Co corrole synthesized by Han et al.

Bio-inspired catalysis

(A)

389

(B)

Fig. 15 (a) The structure of resting multicopper oxidase. Copyright78. (b) Chemical structure of the tricopper cluster synthesized by Thiyagarajan and coworkers.

of the qpdt ligand to obtain K2[MoIVO(H-qpdt)2] and (Bu4N)[MoVO(2H-qpdt)2] (Fig. 19b). The results showed that both complexes have CO2 reduction ability. Among them, (Bu4N)[MoVO(2H-qpdt)2] was more effective, with a total turnover number of 210, and the yield of the desired products was approximately 60% (with 19% CO and 39% formate) under photochemical conditions, while the K2[MoIVO(H-qpdt)2]-catalyzed products were mostly hydrogen (53%).86

2.14.2.2.3

Mimics of CODH

There are two types of CODH. One of them is MoeCu CODH, derived from Oligotropha Carboxidovorans. Its unique heterobimetallic active site inspired the synthesis of numerous Mo/W-Cu complexes. Tatsumi and coworkers synthesized a [MoVI(O)(bdt) S2CuICN]2 (bdt ¼ benzene-1,2-dithiolate) catalyst (Fig. 20a), which was further applied in CO2 electroreduction by the Mougel group.87 In a later study, the catalyst could reduce CO2 to formate but not to CO. This is also the first study in which a MoeCu mimic showed catalytic potential for CO2 reduction.88 The other type of CODH is Ni-containing CODH. At the active site, the nickel-iron-sulfur cluster, Ni is the only metal with redox activity. As a result, a few mononuclear Ni catalysts have been synthesized for CO2 reduction. In 2017, Hong et al. synthesized a Ni(II) complex (Fig. 20b), [Ni(bpet)]2 þ (bpet ¼ bis(2-pyridylmethyl)-1,2-ethanedithiol), for photoreduction by using [Ru(bpy)3]2 þ (bpy ¼ 2,20 -bipyridine) as the photosensitizer. The Ni catalyst had a turnover number of over 700 and > 99% selectivity for CO2 reduction to produce CO.89 Fontecave and coworkers reported a Ni(III) complex with qpdt as the ligand (Fig. 20c). This complex could catalyze the electroreduction of CO2 to HCOOH as the major product (> 60%) with small amounts of CO and hydrogen as minor products.90 They synthesized and tested a series of Ni(III) compounds using a modified qpdt ligand in subsequent studies. Although the results indicated a lack of selectivity, they provide a good reference for further design of the catalyst structure.91 At present, the selective capture of CO2 in the atmosphere and subsequently selective reduction are urgently needed for our sustainable future. Various efforts have been made to establish novel biomimetic CO2 reduction systems.92 In Nature, the precise location of lysine and histidine in NieFe CODH is critical to establish the secondary sphere, where CO2-metal intermediate could be stabilized by hydrogen bond and proton relay groups.93 Inspired by this, several pioneering reports explored the effect of the

(B) (A)

Fig. 16 (a) Chemical structure of the copper-phenolate complex synthesized by Gentil et al. (b) Chemical structure of the phenolatocopper complexes synthesized by Goff and coworkers.

390

Bio-inspired catalysis

(A) (B)

(C)

Fig. 17 (a) Chemical structure of copper 5-nitrophenanthroline complexes containing polymer. (b) Chemical structure of the 1,10-phenanthroline copper complex-containing polymer. (c) Chemical structure of PHTPP.

secondary coordination constructed by hydrogen bond donor or acceptor groups and demonstrated its ability to lower overpotential and improve selectivity. Aukauloo and coworkers designed an iron porphyrin with four urea functions (FeTPP-Ur, Fig. 21a) as the multipoint hydrogen bond donor, which demonstrates a 302 mV drop of catalytic potential with excellent catalytic performance for selective reduction of CO2 into CO.94 Liu et al. reported a serious of iron porphyrins bearing an amino group in the phenyl ring. Ortho-substitution (Fe-1; Fig. 21b) could improve turn over frequency (104 s 1) and CO selectivity while reducing overpotential when compared to para-substitution. DFT results revealed that the hydrogen bonds in Fe-1 could promote the formation [Fe– CO2]2 adduct.95 Recently, Dey and coworkers constructed an iron chlorin complex with a pendant amine group (FeTEsCCl, Fig. 21c), whose second sphere could be protonated for CO2 binding and reduction. The complex could reduce CO2 to HCOOH at 97% yield with no H2 and CO at very low overpotentials.96

2.14.2.2.4

Conclusion

Mimicking the active sites of FDHs and CODHs for CO2 reduction remains challenging. Existing research has made good progress, and the structural simulation of molybdopterin provides active catalysts for CO2 reduction. However, the selectivity of the reduction products remains poor, and hydrogen, a competitive product, accounts for a large proportion of the products. A better understanding of the respective roles of metal ions in the active sites of metalloenzymes is indeed needed. A better understanding and application of second coordination sphere is also of great importance. These studies will guide us in designing more effective and selective bioinspired catalysts for CO2 reduction.

2.14.2.3

Bioinspired hydrogen evolution reaction

2.14.2.3.1

Introduction

Hydrogenases are a class of metalloenzymes that can reversibly catalyze the hydrogen evolution reaction and the reverse reaction, the conversion of hydrogen into protons. They can rapidly catalyze the interconversion of H2/Hþ at low overpotentials and under mild conditions, which makes them good models for designing fuel cells and H2 production catalysts. Hydrogenases are widely distributed in bacteria, archaea, and some eukarya. They can be categorized into [NiFe], [FeFe], and [Fe] hydrogenases according to the active site metal ions.97 The chemical structures of the active sites are shown in Fig. 21. The synthesis of hydrogenase-mimicking substances began immediately after the publication of the crystal structure.55,98 Since hydrogen is a clean energy source and has potential for energy transport, research on the hydrogen evolution reaction is growing rapidly.99 In this section, we briefly introduce some catalysts mimicking hydrogenases for the hydrogen evolution reaction (HER).

2.14.2.3.2

Mimics of [NiFe] hydrogenase

The [NiFe] hydrogenase is the most common of all hydrogenases and tends to absorb and oxidize H2. In 1995, the first characterization of the [NiFe] hydrogenase structure was published.100 Since then, many mimics have been fabricated with this active site. At

(A)

Fig. 18

(B)

(C)

(a) Active site of FDHs: M ¼ Mo/W, R1 ¼ S/O, and R2 ¼ S-Cys/Se-Cys. (b) Active site of CODHs. (c) Chemical structure of molybdopterin.

Bio-inspired catalysis

(A)

Fig. 19

(B)

391

(C)

(a) Chemical structure of (Bu4N)2 [MoIVO(qpdt)2]. (b) Chemical structures of K2[MoIVO(H-qpdt)2] and (Bu4N)[MoVO(2H-qpdt)2].

the active site, four cysteines are coordinated to Ni ions through four sulfurs, where two of them connect Fe ions as bridges and the other two chelate Ni ions in a terminal form. Interestingly, unlike in Nature, chemical mimics are apt to evolve hydrogen. In 2009, Rauchfuss et al. reported the first [NiFe] hydrogenase mimic carrying a hydride bridge (Fig. 22a). A subsequent study showed that this model can evolve H2 in strong acid conditions near  1.46 V vs. Fc0/þ.101 After this study was published, many models showed good H2 evolution activity. In 2013, Ogo et al. reported the first model of an analog [NiFe] hydrogenase that could undergo a reversible reaction (Fig. 22b).102 However, this complex required a strong base to split H2 and a strong acid to release H2; however, the yield and turnover number were low and it had a large overpotential. The main disadvantage of most mimics is that their reactivity is mainly concentrated on the Fe ions.103 Based on this limitation, Brazzolotto et al. reported a LN2S2NiIIFeII complex (Fig. 22c) where reduction occurs in nickel ions in acetonitrile medium with [Et3NH]þ[BF4] as the proton source.104 This model showed high H2 evolution rates with turnover frequency values of 250 s 1 under mildly acidic (10 mM Hþ) conditions. In a recent study, Hayami and coworkers synthesized a novel NiIIFeII complex that is capable of electron transfer, H2 evolution, and proton transfer in different forms.105

2.14.2.3.3

Mimics of [FeFe] hydrogenase

[FeFe] hydrogenase is the most active enzyme in Nature for hydrogen evolution. Its active site contains a dinuclear Fe cofactor, which is bridged by a 2-aza-propane-1,3-dithiolate ligand ([2Fe-2S] cluster), and one of them is covalently linked to a [4Fe-4S] cluster. Interestingly, in the early 20th century, before the crystal structure was determined, a [2Fe-2S] mimic was already described, which gave a foundation for further mimicking. Most [FeFe] hydrogenase mimics are based on the [2Fe-2S] cluster by changing the bridging ligand.106 A typical model uses azadithiolate (ADT) as the bridge. In 2017, Dey et al. reported a complex that used N-(bromo-phenyl)-ADT as the bridge (Fig. 18a, left) and was then mounted on graphene to electrocatalyze H2 evolution under strong acid conditions. This catalyst showed a 6.4  103 s 1 turnover frequency at a 500 mV overpotential.107 In another study, a series of ADT analogs was used to bridge the Fe cofactor (Fig. 23a, right). These models reduced the overpotential of the HER to 160–180 mV by increasing the pKa of the bridging ligands. The catalytic activity could be performed at near neutral pH and in the presence of O2.108 These complexes had already been applied in the anode of a H2/O2 fuel cell device.109 In addition, PNP as a bridge or chelate ligand has also been investigated in recent years (Fig. 23b).110 The PNP-chelated complex showed a higher turnover number than the PNP-bridged complex for H2 evolution in acetonitrile solution using acetic acid as the proton source. In addition, a novel [FeFe]-metallopolymer using benzenedithiolate (BDT)-containing polymer sidechains as bridges was published (Fig. 23c).111 This complex showed rate of H2 evolution that was an order of magnitude higher and O2 tolerance that was better than those of [FeFe] hydrogenase. In a subsequent study, they further demonstrated the pivotal role of the polymers. In a recent study, Weigand and coworkers designed a photosensitizer-catalyst dyad (PS-CAT) containing a silanedithiolate bridge (Fig. 23d), which could photocatalytically evolve H2 under visible light irradiation with remarkable long-term activity. Both

(A)

Fig. 20

(B)

(C)

Chemical structures of [MoVI(O)(bdt)S2CuICN]2, [Ni(bpet)]2 þ, and (Bu4N)[NiIII(qpdt)2].

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Bio-inspired catalysis

Fig. 21

Chemical structures of FeTPP-Ur, Fe-1, and FeTEsCCl.

operando EPR spectroscopy and theoretical calculations revealed the generation of an active [FeIFe0] species that drives H2 generation (Fig. 24).112

2.14.2.3.4

Metal chlorin

2.14.2.3.5

Biohybrid systems

Chlorin is reduced at b-periphery of the macrocycle compared to porphyrin. The similarity with porphyrin in structure and chemical properties gives it potential to be applied in energy conversion. The electrochemistry of Ni and Fe chlorins were first studied in the 1980s.113 Some recent studies have also applied chlorins in HER as newly interesting catalysts.114 Some studies also revealed that proton coupling electron transfer through secondary coordination sphere is crucial for improving HER activity.115 Consists with this finding, the conjugation interruption caused by reduced pyrrole distorts the porphyrin ring, causing a larger binding space and a more electron rich metal center (Fig. 25a), which could make chlorin superior to porphyrin in some respects. Zhang and coworkers synthesized a Ni(II) chlorin (Fig. 25b), when compared to Ni(II) porphyrin, it possessed less negative potential and 24 times greater turnover frequency. The DFT results revealed that the barrier of intermediates for Ni(II) chlorin is 3.3 kcal/mol lower and the protonation of Ni hydride to generate H2 is exothermic by 31.9 kcal/mol.116 Another work demonstrated a b-hydroxyl tunichlorin mimic (Fig. 25c) could accelerate HER rate 56-fold compared to porphyrin form. The b-hydroxyl group could form hydrogen bond network with water, which is key to its higher catalytic activity.117 Chlorin, as an emerging metal cofactor, provides a new direction in designing better catalysts.

Biohybrid systems combine inorganic photosensitizers with living cells, and they have great potential for light-driven hydrogen production.118 The new trend for this system is to use semiconductor nanomaterials as light receivers and bacteria containing hydrogenase as H2 producers.119 This biological process, compared to electrochemical processes, is more environmentally friendly and consumes less energy, and it is a promising way to sustainably generate clean energy. In 2018, Zhao and coworkers developed a surface-display biohybrid system. The PbrR protein was displayed on the surface of E. coli cells as the photosensitizer to biosynthesize CdS nanoparticles. In addition, the engineered E. coli cells expressed [NiFe] hydrogenase for H2 production. Additionally, biomimetic silica was used to encapsulate the system to protect it from O2. This system could produce a maximum H2 of 0.52  0.01 mmol/108 cells under light in aerobic conditions (Fig. 26).120 In a recent study, Luo et al. designed a periplasmic photosensitized biohybrid system by introducing CuInS2/ZnS quantum dots into Shewanella oneidensis MR-1. This system showed great H2 production of 491.8  26.6 mmol under visible light irradiation; this production was 8.6 times as much as that of bare quantum dots.121 Xiao et al. modified iodine-doped hydrothermally carbonized carbon (I-HTCC) on the surface of E. coli. Photoelectrons were captured by I-HTCC and further delivered into the cell for H2 production. The H2 production efficiency reached 2.12 mM/2000 W m 2.122 In another study, Pereira and coworkers compared the H2 production capacity of three organisms (Desulfovibrio desulfuricans, Citrobacter freundii, and S. oneidensis) with E. coli using CdS as the photosensitizer. The results revealed that D. desulfuricans was the most efficient biological catalyst. The H2 production time could  1  1 123 last more than 10 days at a rate of 36 mmolgdcw h .

Fig. 22

Actives of [FeFe] and [NiFe] hydrogenases.

Bio-inspired catalysis

(A)

Fig. 23

(B)

393

(C)

Chemical structures of related [NiFe] hydrogenase mimics.

2.14.2.3.6

Conclusion

Great advances have been made in mimicking [NiFe] and [FeFe] hydrogenases. In some models, via enzyme-like mechanisms, their catalytic activity is comparable to that of natural enzymes. Ingenious catalyst design also allows H2 evolution reactions to occur under mildly acidic conditions and can overcome the high O2 sensitivity of natural enzymes. However, some challenges remain. For example, reversible catalysts for H2-Hþ conversion are still rare. Better mimics will be designed when enzyme-relevant mechanisms are better understood. Combining theoretical calculations and practice will play a key role in the future. Notably, new metal cofactors like chlorin provide opportunities for designing better catalysts. Furthermore, biohybrid systems are thriving and expected to be promising platforms for photosynthetic hydrogen production.

2.14.3

Bioinspired bond-forming reactions

2.14.3.1

Introduction

Bioinspired catalysis is a catalytic technology with both chemical and biocatalytic features that can selectively convert reactants into products under environmentally friendly mild conditions. Bioinspired catalysis involves the following three factors: a structural unit similar to the functional structure of the enzyme (coenzyme), which serves as the catalyst parent structure; a similar catalytic process as that of enzyme catalysis; and a similar reaction environment and reaction effect as that in enzyme catalysis. Metalloporphyrinbased carbon-hydrogen bond air oxidation is considered the most representative form of bioinspired catalysis. The formation of CeC bond124, CeN bond125, CeO bond126 also plays a crucial role in the synthesis of many natural products in Nature. It is well known that the formation and breaking of NeN bonds plays an important role in Nature, as these bonds are related to important activities such as microbial metabolism and N fuel supply. Lancaster et al.127 published related reports on the formation of NeN bonds in 2020.

(A)

(C)

Fig. 24

(B)

(D)

Chemical structures of related [FeFe] hydrogenase mimics.

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Bio-inspired catalysis

Fig. 25

Chemical structures of chlorin and Ni-chlorin complexes.

2.14.3.2

CeC bond formation

2.14.3.2.1

CeC bond-forming enzymes

Many drugs possess 1,2-dihydro or tetrahydronaphthalene skeletons in their structures and oxidase can catalyze the oxidative coupling of phenols to prepare such phenol dimers. The selectivity of these oxidative coupling could be tuned to improve the reaction efficiency. In 1994, Harri et al.128 reported that Me-Sin was oxidatively coupled by phenol in acetone-water solvent with the horseradish peroxidase at pH 4, and the yield was 41% (Fig. 27). While in traditional methods, when aryl dihydronaphthalene reacts in a methanol-water system, the main product obtained usually is a mixture with a yield of only 14%. In 2018, Yokoyama et al.129 reported the use of free-radical SAM enzymes to catalyze the formation of CeC bonds. In this paper, the function and mechanism of free-radical SAM enzymes in CeC bond formation were reviewed, and their important role in the synthesis and biological application of natural organic molecules was emphasized. The mechanism of CeC bond formation by the free-form SAM enzyme involves three important processes: free radical initiation, receptor substrate activation and free radical

Fig. 26

Proposed biohybrid system from Zhao and coworkers. Copyright.120

Fig. 27

Structure of Me-Sin Oxidative Coupling Products.

Bio-inspired catalysis

Fig. 28

395

Structures of SalA-K and cyclobutene compounds.

quenching. Understanding the functions and mechanisms of action of these characteristic enzymes can advance our understanding of free radical-type SAM enzymes and the biosynthesis of natural products and cofactors.

2.14.3.2.2

Bioinspired CeC bond-forming reactions

Sage has been used since ancient times to treat various diseases. Today, sage is still a herbal medicine to help reduce saliva production, control night sweats, relieve cavity and throat inflammation. In 1999, Lu et al.130 isolated two compounds from sage: the known SalA-K and a new cyclobutene compound (Fig. 28). SalA-K is formed by the partial oxidative cross-coupling of rosmarinic acid and caffeic acid. The cyclobutene compound forms a Cb-Cb bond through the phenol oxidative coupling of two rosmarinic acid units and then undergoes intramolecular Michael addition to form a closed ring CeC bond. In 2021, Hamley et al. reported131 peptide (and peptide-coupled) a-helix and b-sheet structures and rotational or disordered peptides in biological catalytic applications. Compared with the structures of folded proteins, peptide structures have simpler design rules and thus can mimic the binding pockets of natural enzymes from the initial design or simply present catalytic motifs at high density on nanostructured scaffolds. Different classes of polypeptides have been found to be highly active in catalytic reactions. Advances in peptide design and synthesis methods mean that they hold great potential for the future development of efficient bioinspired and biocompatible catalysts. Enzymes exhibit inherent selectivity and reactivity through host-guest interactions, but many reactions remain elusive compared to transition metal catalysts. Scientists introduced the behavior of enzymes into transition metal catalytic systems to obtain biocatalytic transition metal catalysts with high enantioselectivity and high reactivity, thereby producing enantiomeric compounds with high efficiency. In 2022, Tsuda et al.132 reported a rhodium-cyclodextrin complex using a self-contained strategy of biomimetically catalyzing CeC bond formation to promote asymmetric aldehyde arylation. By combining bifunctional components

Fig. 29

Bioinspired inclusion strategies.

396

Bio-inspired catalysis

with metal coordinating groups and substrate acceptors, this system achieved highly enantioselective and reactive asymmetric arylation (Fig. 29).

2.14.3.3

CeN bond formation

Aminoallyl cations are a three-carbon species for the preparation of complex amine-containing carbocycles; however, methods for generating and utilizing these active species are limited compared to oxallyl cations. In 2020, Cobin et al.133 reported a bioinspired strain-driven ring-opening of bicyclic methyleneaziridines to 2-aminopentadienyl cationic intermediates that are amenable to Nazarov cyclization. The advantages of this strategy featured facile generation and enhanced reactivity, control over the final position of the alkene, the potential for high dr between adjacent stereocenters, and the ability to further refine the preparation of fully substituted aminocyclopentanes. Artificial metalloenzymes (ArMs), an emerging class of catalysts for unnatural reactions, are prepared by introducing synthetic cofactors into protein scaffolds. In 2021, Gu et al.134 reported the combination of the P450 enzyme CYP119 mutant and the cofactor Ir(Me)MPIX in vivo. This process involved directed evolution in whole cells containing Ir(Me) by co-expression with the heme transporter encoded by a repressible operon to form the artificial metalloenzyme MPIX. Using this platform, an artificial metalloenzyme was developed to catalyze the insertion of the acyclic carbene bis-aziridine (Me-EDA) into the NeH bond of Nalkylaniline, forming a CeN bond (Fig. 30). The design of functional metalloenzymes plays an important role in biosynthesis. In 2021, Lin et al.135 used myoglobin as a model protein and introduced a CXXC artificial motif through double mutation of F46C/L49C to construct a new disulfide bond in the heme distal vesicle of myoglobin. They discovered that the triple mutant F43Y/F46C/L49C myoglobin showed PHslike activity and its catalytic efficiency exceeded that of native metalloenzymes. During the study, 1,6-disulfonic acid-2,7diaminophenazine derivatives were found as a potential pH indicator.

2.14.3.4

CeO bond formation

Laccase is a polyphenol oxidase that can treat wastewater and remove natural phenol from oil. In 2001, Carunchio et al.136 used laccase as a catalyst to couple furan with phenol at pH 6 in an ethanol-water system and obtained two main products: the benzofuran dimer (a) and the b-O-4 coupling product (b) (Fig. 31). In 2017, Wu et al.137 reported the synthesis of catechol using an iridium catalyst. Catechol plays an important role in human metabolism and physiological activities, but its synthesis is tedious, the reaction conditions are harsh, and the yield is low. This method realized the one-step oxidation-reduction synthesis of catechol under mild conditions. In this mechanism, oxyacetamide acts as the oxygen source, which enables the reaction to proceed without the addition of other oxidants. Here, the bionic intramolecular oxygen transfer strategy was also adopted. Rifamycin antibiotics are a class of antibiotics produced by Streptomyces mediterranean that have broad-spectrum antibacterial effects and have strong effects on gram-positive bacteria such as Mycobacterium tuberculosis, Leprosy, Streptococcus, and Pneumococcus, especially drug-resistant Staphylococcus aureus. It is also effective against some gram-negative bacteria. However, the industrially important final steps of rifamycin synthesis are still unknown. In 2018, Qi et al.138 reported the reaction steps of rifamycin biosynthesis. It was found that the double subunit transketolase can mediate the formation of CeO bonds in rifamycin, while cytochrome P450 can induce atypical ester-ether conversion.

2.14.3.5

NeN bond formation

2.14.3.5.1

N2O-forming enzymes

NO reductase is an enzyme in the heme copper oxidase superfamily. As a part of prokaryotic denitrification, it can catalyze the reduction and conversion of NO to N2O.139Heme copper oxidase is a diverse integrated oxidoreductase involved in the respiration and detoxification processes in living organisms.140 The structure of heme copper oxidase has a conserved core catalytic subunit, and some of them even have secondary subunits involved in electronic operation or regulation. The heme copper oxidase family is mainly classified according to its catalytic reaction. Heme copper oxidase can couple the four-electron reduction of oxygen with proton conversion in the form of an electron-chemical cross-membrane gradient, thereby promoting the aerobic respiration of living organisms. To date, NO reductase and its homologs have not been observed to send protons from the negative side to the positive side, and because the formation of water requires protons to be absorbed from the negative side, some NO reductases are electrotropic.

Fig. 30

Ir(Me)MPIX catalyzes the formation of CeN bonds.

Bio-inspired catalysis

(A)

Fig. 31

397

(B)

Structure of the phenolic oxidative coupling reaction product of furan.

Until now, through experiments and calculations, the catalytic mechanism of heme copper oxidase-type NO reductase to convert NO to N2O has been hypothesized to be one of three mechanisms: a trans mechanism, a homeopathic heme b3 mechanism, or a cis FeB mechanism. (Fig. 32). Through Raman spectroscopy, it was found that in the resting state, a m2-oxo ligand binds the nonheme FeB and heme b3.141 In this resting state, the m2-oxo ligand pulls the heme b3 iron from the porphyrin plane, causing the proximal histidine to dissociate from the heme b3 iron.142 In this trans mechanism, the m2-oxo ligand dissociates when the protein is completely reduced, and the proximal histidine associates with heme b3, which also leads to the extension of the distance between ferrous centers. Then, the nonheme FeB and heme b3 combine with NO to form two Fe nitrosyl compounds.143 Protons in the solvent promote NeO bond cleavage to generate N2O and H2O, and the heme b3 iron is linked by proximal histidine, leaving the two Fe centers in the iron state.144 Then, the abovementioned turnover method is repeated, and finally, NO provides oxo groups to return to the oxygen-bridged iron species and end the cycle. In the second cis-FeB mechanism, nonheme Fc is the only substrate binding site catalyzing the formation of dinitro iron complexes. Heme b3 simultaneously mediates electron transfer and forces FeB-bound NO molecules to reorient themselves, and the NeO bond breaks.145 In the final mechanism, that of cis-heme b3, NO binds to heme b3, NO combines with b3, and FeB coordinates with oxygen atoms, resulting in the attack of the electrostatically polarized red blood cell-bound nitrosyl by a second NO molecule to generate nitrite, which asymmetrically connects the diimine center bridge to the nonheme FeB through dioxygen coordination.146 In addition to the aforementioned heme copper oxidase, a P450 NO reductase reduces NO to N2O. Experimental data show that the main contribution of global environmental N2O synthesis is fungal denitrification, and fungal NO reductase belongs to the cyt P450 enzyme family.147 Shoun et al.148 reported the first purification of P450 NO reductase and later reported denitrification of the enzyme.149 P450 NO reductase is a soluble B-type heme enzyme coordinated axially to a cysteine ligand. Fungal P450 NO reductase has a high catalytic effect on a single metal cofactor.150 In the resting state, the iron in P450 NO reductase exists in a low-spin state, and the resting enzyme can combine with NO at a higher speed to form a six-coordinate iron nitrosyl compound. This complex has the ground state electronic configuration of Fe2þ-NOþ and oxidizes NO during coordination, where the nitroxyl ion (NOþ) bends and prepares for nucleophilic attack.151 Intermediate I accumulated and was produced under the conditions of stopping flow and stable turnover, but the protonation state of its iron oxide core hydroxyl amide is still controversial. Vincent et al.152 and Lehnert et al.153 suggested that Intermediate I was present in the form of Fe4þ-NHOH, while Riplinger et al.154 suggested that Intermediate I was present in the form of Fe3þ-NHOH. In addition, Cyt P460 can also generate N2O. Cyt P460 is a soluble homodimeric enzyme that exists in the genome of metabolically diverse microorganisms. It can catalyze the oxidation of hydroxylamine (NH2OH) to N2O.155 The purification of this enzyme was first reported by Erickson and Hooper in 1972156, and it was extracted from prototype ammonia-oxidizing bacteria. Cyt P460 is related to the biological nitrification process, so it is usually present in the prototype ammonia-oxidizing bacteria and catalyzes the complete oxidation of ammonia to (NO3).157

Fig. 32

Three catalytic mechanisms.

398

Bio-inspired catalysis

Initially, it was believed that based on the activity analysis of prototype ammonia-oxidizing bacteria, NH2OH was oxidized to NO2 through a single enzymatic process.158 In an aerobic environment, NH2OH can be oxidized to NO2 with a yield of 60%. In an anaerobic environment, the oxidation of NH2OH is indeed a mixture of NO and N2O. This experimental result shows that the presence of O2 is important for the formation of NO2. In addition to the above three enzymes that can catalyze the formation of N2O, there are also several enzymes that have the same effect. For example, the initial discovery of Cyt c554 was proposed by Yamanaka and Shinra159, but at that time, they believed that the structure of the enzyme had two hemes; however, in 1986, Andersson and his colleagues160 proposed a new point of view that c554 has four c heme, and two pairs of magnetic interactions occur between them. Flavonoid imide protein can also catalyze the production of N2O. Flavonoid imide protein is a soluble cytoplasmic protein. They obtained an isolate from Desulfovibrio, and the substance can couple O2 reduction and NADH oxidation in vitro and act as a NO reductase.

2.14.3.5.2

Bioinspired N2O-forming reactions

Nitric oxide reductase (NOR) is a membrane-bound enzyme that reduces nitric oxide (NO) to nitrous oxide (N2O), a necessary step for the continuous reduction of nitrate to nitrous oxide. In 2008, the first functional diimide diiron model of NO reduction was reported by Solomon.161 The structure of this model has a dual-functional ligand “L” that can coordinate with heme and nonheme metals. At room temperature, the ligand forms by reacting LFe2þ with 1 equivalent of Fe(OTf)2MeCN in tetrahydrofuran to generate LFe2þH/Fe2þB under the protection of nitrogen. In 2009, Xu et al.162 reported a N2O22 bridging Fe porphyrin complex [(OEP) Fe]2(m-N2O2). Hydrochloric acid is added to the toluene solution of the complex to react to generate N2O. The mechanism of the reaction is that Hþ attacks an oxygen atom in the N2O22 ligand, causing the OeN bond to break to form N2O. Heme-assisted nitric oxide (NO) coupling to form Fe{N2O2} intermediate plays an important role in the reduction of NO to N2O. Nitric oxide (NO) is a signaling molecule in mammals involved in blood pressure control and neural signal transduction, and a key immune defense agent produced by macrophages. Flavonoid nitric oxide reductases (FNORs) exist in many pathogenic bacteria and can reduce NO to N2O for detoxification. In 2013, the first N2O model suitable for the reduction of flavin diimine NO was reported.163 Each Fe center in the [Fe2(BPMP)-(OPr)(NO)2](BPh4)2 complex is coordinated with two pyridine units and a bridged dinitro group to form a pseudo-octahedral geometric configuration, and then the complex is subjected to twoelectron reduction by electrochemical methods to generate N2O.164 In 2017, Jana et al.165 reported a mononitrosodiimide complex [Fe2(Et-HPTB)(NO)-(DMF)3](BF4)3. The complex can also catalyze the reduction of NO to N2O. One Fe center of the complex is coordinated with NO and DMF, and the other Fe center is coordinated with two DMFs. This substance reacts to generate N2O when it is treated electrochemically or with cobaltene at room temperature. An innovative synthetic strategy was employed that circumvents the long-standing problem that hinders the preparation of mononitrosyl diiron(II) complexes within the symmetric ligand platform. In 2018, Dong et al.166 reported another complex model, [Fe2(Py2PhO2)MP(OPr)2](OTf) (Fig. 33). The complex interacts with cobaltocene to directly generate N2O without forming any intermediates. Non-heme dinitroso complexes can not only induce fast and efficient NeN coupling and N2O generation through reduction, but also activate the NO reduction pathway through iron redox potential. In addition to iron complexes that can build NO reduction models, copper complexes can also be used. In 1990, Paul et al.167 reported a Cu2eNO substance [Cu2(XYL)(NO)]2þ (XYL ¼ 2,6-bis[N,N-bis(2-pyridin-2-ylethyl)aminomethyl]phenolate), which decomposes to produce N2O and Cu2þ-O-Cu2þ. The key intermediates in the reduction of nitric oxide (NO) to nitrous oxide (N2O) is N2O22, which is the product of the reductive coupling of two NO molecules. In 2016, Lionetti et al.168 reported the use of an yttrium copper core to reduce NO to produce N2O. After reacting for 6 h at  78  C, gaseous NO can combine with the metal in the yttrium copper core to form the N2O22intermediate, and then N2O is generated, but this reaction further generates N2.

Fig. 33

The structure of [Fe2(Py2PhO2)MP(OPr)2](OTf).

Bio-inspired catalysis

399

In 2017, Wijeratne et al.169 reported the first example of [Cu1þ(tmpa)(MeCN)](B(C6F5)4) that can stoichiometrically reduce NO to N2O. The stability of this substance is dependent on the solution. The addition of methanol and excess NO to this substance generates the [{Cu2þ(tmpa)}2(m-N2O22)](B(C6F5)4)2 intermediate, and the addition of excess tetrahydrofuran causes N2O22 to disproportionate, generating N2O. The hydrogen bonding of methanol is crucial for the formation and stabilization of nitrite complexes. The complex exhibits a reverse redox process in an aprotic solvent to generate CuI þ NO(g), resulting in CuI-mediated NO(g) disproportionation.

2.14.3.5.3

N2-forming enzymes

2.14.3.5.4

Bioinspired N2-forming reactions

The nitrogenase protein complex was first reported in 1986 and can catalyze the reduction of N2O to N2.170 In 2000, Christiansen et al.171 reported another nitrogenase protein complex, and in 2010, Fernandes et al.172 reported that thermophilic archaea can also catalyze the reduction of N2O. These discovered nitrogenase protein complexes and thermophilic archaea are soluble, so they are collectively referred to as soluble N2O reductase. Soluble N2O reductase is a dimeric copper protein complex encoded by the highly conserved nosZ gene. All soluble N2O reductases have common features: a head-to-tail homologous dimer structure, a dimerization interface composed of conserved residues, and a Cu center and two domains in each monomer. The N-terminal b-propellers of these N2Os all carry a Cuz tetranuclear center, while the copper ions at the C-terminal contain a CuA binuclear redox center.173 Subsequently, the catalytic mechanism of soluble N2O reductase was studied. First, the [1CuZ2þ:3CuZ1þ] state was assumed to be a nonphysiological state because research has found that this state is redox inert under mild conditions; CuZ* was found to be present.174 Therefore, in 2003, Ghosh et al.175 assumed that the coordination of the substrate molecule occurred at the edge of the CuZ1/CuZ4 interface, and then through density functional theory calculations, they found that N2O can bind to the inactive [1CuZ2þ:3CuZ1þ] as a linear N-terminal pair, and this binding is similar to the morphological structure of N2O. The CuZ* center preferentially combines with N2O, resulting in geometric bending. The bond energy between CuZ1-N and CuZ4-O is greater than that of NeO bonds and NeN bonds176 and can compensate for the energy loss caused by N2O deformation, activate inert N2O molecules, and cause NeO bond cleavage, resulting in the dissociation of N2.177 In addition, under physiological conditions, the heme protein complex hydrazine dehydrogenase can also produce N2, and hydrazine dehydrogenase can complete the last step of anaerobic ammonia metabolism.178 In this reaction, hydrazine dehydrogenase can catalyze N2H4 to release 50% of N2 back to the atmosphere. In 2016, Maalcke et al.179 reported the working mechanism of hydrazine dehydrogenase-catalyzed oxidation of N2H4 (Fig. 34).

In 2009, Bar-Nahum et al.180 reported a tri-copper disulfide cluster, which can reduce N2O to N2 by forming a double-copper complex. In 2014, Johnson et al.181 reported a complete complement model with a Cu center: [(m2-dppa)4Cu4(m4-S)](PF6)2. This model has an asymmetric center, and the fourth copper atom is replaced by other elements. In 2014, Jayarathne et al.182 reported a bimetallic N-heterocyclic carbene compound that can yield N2 in stoichiometric amounts. In 2016, Johnson et al.183

Fig. 34

The catalytic mechanism of hydrazine dehydrogenase.

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reported a Cu4(m4-S) compound that can reduce N2O through [3Cu1þ: Cu2þ] to generate N2 and [2Cu1þ:2Cu2þ]. In addition, in 2019, Gwak et al.184 reported a NieNO3 compound prepared by treating [(PNP)Ni](OTf) with NaNO2:(PNP)Ni-(ONO2). The coordination layer around Ni was composed of a PNP ligand and an oxygen atom from the NO3 group.

2.14.3.5.5

Other enzyme-mimicking catalysts

In 2021, Zhao et al.185 reported a family of two-domain enzymes that are widespread in bacteria. Methionyl tRNA synthetases can catalyze the ATP-dependent condensation of two amino acid substrates to form labile ester intermediates, which are subsequently captured by zinc-bound cupin proteins and undergo redox-neutral intramolecular rearrangements to generate products containing NeN bonds.

2.14.3.6

Conclusion

In this section, several enzymes and reactions that can form CeC bonds, CeN bonds, CeO bonds, and NeN bonds are outlined. In the construction of these bonds, we found that most of the reactions were designed to transfer protons and electrons, and the clever use of new catalysts also has many advantages that improve the reaction rate and yield. The mechanisms of reactions catalyzed by some enzymes are still obscure, though they could inspire the discovery of new technologies, and the construction of some of these bonds could eventually help alleviate atmospheric pollution caused by greenhouse gases.

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A.; Ghosh, S.; Ohta, T.; Solomon, E. I. A Functional Nitric Oxide Reductase Model. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (41), 15660–15665. https://doi.org/10.1073/pnas.0808606105. 162. Xu, N.; Campbell, A. L. O.; Powell, D. R.; Khandogin, J.; Richter-Addo, G. B. A Stable Hyponitrite-Bridged Iron Porphyrin Complex. J. Am. Chem. Soc. 2009, 131 (7), 2460– 2461. https://doi.org/10.1021/ja809781r. 163. Zheng, S.; Berto, T. C.; Dahl, E. W.; Hoffman, M. B.; Speelman, A. L.; Lehnert, N. The Functional Model Complex [Fe-2(BPMP)(OPr)(NO)(2)](BPh4)(2) Provides Insight into the Mechanism of Flavodiiron NO Reductases. J. Am. Chem. Soc. 2013, 135 (13), 4902–4905. https://doi.org/10.1021/ja309782m. 164. Feig, A. L.; Becker, M.; Schindler, S.; vanEldik, R.; Lippard, S. J. Mechanistic Studies of the Formation and Decay of Diiron(III) Peroxo Complexes in the Reaction of Diiron(II) Precursors with Dioxygen. Inorg. Chem. 1996, 35 (9), 2590–2601. https://doi.org/10.1021/ic951242g. 165. Jana, M.; Pal, N.; White, C. J.; Kupper, C.; Meyer, F.; Lehnert, N.; Majumdar, A. Functional Mononitrosyl Diiron(II) Complex Mediates the Reduction of NO to N2O with Relevance for Flavodiiron NO Reductases. J. Am. Chem. Soc. 2017, 139 (41), 14380–14383. https://doi.org/10.1021/jacs.7b08855. 166. Dong, H. T.; White, C. J.; Mang, B.; Krebs, C.; Lehnert, N. Non-Heme Diiron Model Complexes Can Mediate Direct NO Reduction: Mechanistic Insight into Flavodiiron NO Reductases. J. Am. Chem. Soc. 2018, 140 (41), 13429–13440. https://doi.org/10.1021/jacs.8b08567. 167. Paul, P. P.; Tyeklar, Z.; Farooq, A.; Karlin, K. D.; Liu, S. C.; Zubieta, J. Isolation and X-Ray Structure of a Dinuclear Copper Nitrosyl Complex. J. Am. Chem. Soc. 1990, 112 (6), 2430–2432. https://doi.org/10.1021/ja00162a060. 168. Lionetti, D.; de Ruiter, G.; Agapie, T. A Trans-Hyponitrite Intermediate in the Reductive Coupling and Deoxygenation of Nitric Oxide by a Tricopper-Lewis Acid Complex. J. Am. Chem. Soc. 2016, 138 (15), 5008–5011. https://doi.org/10.1021/jacs.6b01083. 169. Wijeratne, G. B.; Hematian, S.; Siegler, M. A.; Karlin, K. D. Copper(I)/NO(g) Reductive Coupling Producing a Trans-Hyponitrite Bridged Dicopper(II) Complex: Redox Reversal Giving Copper(I)/NO(g) Disproportionation. J. Am. Chem. Soc. 2017, 139 (38), 13276–13279. https://doi.org/10.1021/jacs.7b07808. 170. Jensen, B. B.; Burris, R. H. N2O as a Substrate and as a Competitive Inhibitor of Nitrogenase. Biochemistry 1986, 25 (5), 1083–1088. https://doi.org/10.1021/bi00353a021. 171. Christiansen, J.; Seefeldt, L. C.; Dean, D. R. Competitive Substrate and Inhibitor Interactions at the Physiologically Relevant Active Site of Nitrogenase. J. Biol. Chem. 2000, 275 (46), 36104–36107. https://doi.org/10.1074/jbc.M004889200. 172. Fernandes, A. T.; Damas, J. M.; Todorovic, S.; Huber, R.; Baratto, M. C.; Pogni, R.; Soares, C. M.; Martins, L. O. The Multicopper Oxidase from the Archaeon Pyrobaculum Aerophilum Shows Nitrous Oxide Reductase Activity. FEBS J. 2010, 277 (15), 3176–3189. https://doi.org/10.1111/j.1742-4658.2010.07725.x. 173. Zumft, W. G.; Kroneck, P. M. H. Respiratory Transformation of Nitrous Oxide (N2O) to Dinitrogen by Bacteria and Archaea. In Advances in Microbial Physiology; Poole, R. K., Ed.; 2007, vol. 52; pp 107–227. https://doi.org/10.1016/S0065-2911(06)52003-X; Pauleta, S. R.; Dell’Acqua, S.; Moura, I. Nitrous Oxide Reductase. Coord. Chem. Rev. 2013, 257 (2), 332–349. https://doi.org/10.1016/j.ccr.2012.05.026. 174. Dell’Acqua, S.; Pauleta, S. R.; Monzani, E.; Pereira, A. S.; Casella, L.; Moura, J. J. G.; Moura, I. Electron Transfer Complex between Nitrous Oxide Reductase and Cytochrome c(552) from Pseudomonas Nautica: Kinetic, Nuclear Magnetic Resonance, and Docking Studies. Biochemistry 2008, 47 (41), 10852–10862. https://doi.org/10.1021/ bi801375q. 175. Ghosh, S.; Gorelsky, S. I.; Chen, P.; Cabrito, I.; Moura, J. J. G.; Moura, I.; Solomon, E. I. Activation of N2O Reduction by the Fully Reduced Mu(4)-Sulfide Bridged Tetranuclear Cu-Z cluster in Nitrous Oxide Reductase. J. Am. Chem. Soc. 2003, 125 (51), 15708–15709. https://doi.org/10.1021/ja038344n. 176. Gorelsky, S. I.; Ghosh, S.; Solomon, E. I. Mechanism of N2O Reduction by the Mu(4)-S Tetranuclear Cu-Z Cluster of Nitrous Oxide Reductase. J. Am. Chem. Soc. 2006, 128 (1), 278–290. https://doi.org/10.1021/ja055856o. 177. Chen, P.; Cabrito, I.; Moura, J. J. G.; Moura, I.; Solomon, E. I. Spectroscopic and Electronic Structure Studies of the Mu(4)-Sulfide Bridged Tetranuclear Cu-Z Cluster in N2O Reductase: Molecular Insight into the Catalytic Mechanism. J. Am. Chem. Soc. 2002, 124 (35), 10497–10507. https://doi.org/10.1021/ja0205028; Dell’Acqua, S.; Pauleta, S. R.; Paes de Sousa, P. M.; Monzani, E.; Casella, L.; Moura, J. J. G.; Moura, I. A New CuZ Active Form in the Catalytic Reduction of N2O by Nitrous Oxide Reductase from Pseudomonas Nautica. J. Biol. Inorg. Chem. 2010, 15 (6), 967–976. https://doi.org/10.1007/s00775-010-0658-6. 178. Kartal, B.; Maalcke, W. J.; de Almeida, N. M.; Cirpus, I.; Gloerich, J.; Geerts, W.; den Camp, H. J. M. O.; Harhangi, H. R.; Janssen-Megens, E. M.; Francoijs, K.-J.; et al. Molecular Mechanism of Anaerobic Ammonium Oxidation. Nature 2011, 479 (7371), 127–159. https://doi.org/10.1038/nature10453. 179. Maalcke, W. J.; Reimann, J.; de Vries, S.; Butt, J. N.; Dietl, A.; Kip, N.; Mersdorf, U.; Barends, T. R. M.; Jetten, M. S. M.; Keltjens, J. T.; et al. Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle. J. Biol. Chem. 2016, 291 (33), 17077–17092. https://doi.org/10.1074/ jbc.M116.735530. 180. Bar-Nahum, I.; Gupta, A. K.; Huber, S. M.; Ertem, M. Z.; Cramer, C. J.; Tolman, W. B. Reduction of Nitrous Oxide to Dinitrogen by a Mixed Valent Tricopper-Disulfido Cluster. J. Am. Chem. Soc. 2009, 131 (8), 2812–2814. https://doi.org/10.1021/ja808917k. 181. Johnson, B. J.; Lindeman, S. V.; Mankad, N. P. Assembly, Structure, and Reactivity of Cu4S and Cu3S Models for the Nitrous Oxide Reductase Active Site, Cu-z*. Inorg. Chem. 2014, 53 (19), 10611–10619 https://doi.org/10.1021/ic501720h. 182. Jayarathne, U.; Parmelee, S. R.; Mankad, N. P. Small Molecule Activation Chemistry of Cu-Fe Heterobimetallic Complexes Toward CS2 and N2O. Inorg. Chem. 2014, 53 (14), 7730–7737. https://doi.org/10.1021/ic501054z. 183. Johnson, B. J.; Antholine, W. E.; Lindeman, S. V.; Mankad, N. P. A Cu4S Model for the Nitrous Oxide Reductase Active Sites Supported only by Nitrogen Ligands. Chem. Commun. 2015, 51 (59), 11860–11863. https://doi.org/10.1039/c5cc04675k. 184. Gwak, J.; Ahn, S.; Baik, M.-H.; Lee, Y. One Metal is Enough: a Nickel Complex Reduces Nitrate Anions to Nitrogen Gas. Chem. Sci. 2019, 10 (18), 4767–4774. https:// doi.org/10.1039/c9sc00717b. 185. Zhao, G. Y.; Peng, W.; Song, K. H.; Shi, J. K.; Lu, X. Y.; Wang, B. J.; Du, Y. L. Molecular Basis of Enzymatic Nitrogen-Nitrogen Formation by a Family of Zinc-Binding Cupin Enzymes. Nat. Commun. 2021, 12 (1). https://doi.org/10.1038/s41467-021-27523-x.

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Imaging

Brooke A. Corbin, Jacob C. Lutter*, Susan A. White, Enas Al-ani, Elizabeth S. Biros, John P. Karns, and Matthew J. Allen, Department of Chemistry, Wayne State University, Detroit, MI, United States © 2023 Elsevier Ltd. All rights reserved.

2.15.1 2.15.2 2.15.2.1 2.15.2.2 2.15.2.3 2.15.2.4 2.15.3 2.15.3.1 2.15.3.2 2.15.3.2.1 2.15.3.2.2 2.15.3.2.3 2.15.3.3 2.15.3.4 2.15.3.5 2.15.4 2.15.4.1 2.15.4.2 2.15.4.3 2.15.4.4 2.15.5 2.15.5.1 2.15.5.2 2.15.5.3 2.15.5.4 2.15.6 2.15.6.1 2.15.6.2 2.15.6.3 2.15.6.4 2.15.6.5 2.15.6.6 2.15.6.7 2.15.6.8 2.15.7 2.15.7.1 2.15.7.2 2.15.7.3 2.15.8 References Further reading Relevant websites

Introduction X-ray computed tomography Targeted imaging Multimodal imaging Combined imaging and therapy- image-guided therapy Conclusions Optical and near-IR imaging Optical imaging with inorganic compounds and materials Trivalent lanthanide-based luminescence and imaging Targeted imaging using LnIII luminescent probes Packaging systems for LnIII imaging probes Other applications of LnIII luminescence for bioimaging Imaging with 4d and 5d transition metal complexes Cherenkov radiation with inorganic lumiphores Conclusions Magnetic particle imaging (MPI) Magnetic particle imaging Iron-cobalt nanoparticles for MPI Variation on nanoparticle coatings and construction in MPI Conclusions Ultrasound and photoacoustic imaging Imaging with sound waves Ultrasound imaging Photoacoustic imaging Conclusions Magnetic resonance imaging (MRI) Contrast agents GdIII-containing contrast agents and alternatives Iron oxide agents Chemical exchange saturation transfer (CEST) PARASHIFT probes 19 F probes Responsive contrast agents Conclusions Positron emission tomography (PET) and single photon emission computed tomography (SPECT) Nuclides of interest and relevant properties Chelators for complexation and targeting Conclusions Summary and outlook

408 408 409 410 411 411 412 412 412 412 414 416 419 422 425 426 426 427 427 430 431 431 431 434 436 436 436 436 440 441 442 442 443 444 444 445 446 448 448 449 459 459

Abstract Inorganic chemistry has had a large impact on both clinical and preclinical imaging, and this chapter describes the contribution of inorganic chemistry to various imaging modalities. The chapter is divided into sections by modality. Each section introduces an imaging technique then describes recent highlights from 2017 through early 2021 and important historical

*

Current affiliation: Department of Chemistry and Biochemistry, University of Southern Indiana, Evansville, IN, United States

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00157-6

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Imaging examples of the impact of inorganic chemistry on the imaging technique in the section. Imaging modalities covered in this chapter include X-ray computed tomography, optical and near-IR imaging, magnetic particle imaging, ultrasound and photoacoustic imaging, magnetic resonance imaging, and positron emission tomography and single-photon emission computed tomography. This chapter is intended to be an introduction to the use of inorganic chemistry in imaging, and also provides references to thorough reviews on specific subtopics within each section.

2.15.1

Introduction

Imaging is a wide-reaching field that involves the interplay between chemistry, biology, physics, and engineering to visualize a widerange of structures, including the surfaces of nanoparticles and the inside of living organisms. This chapter focuses on imaging modalities used in clinical and preclinical studies, with an emphasis on the impact of inorganic chemistry on these modalities. Imaging modalities covered in this chapter include X-ray computed tomography, optical and near-IR imaging, magnetic particle imaging, ultrasound and photoacoustic imaging, magnetic resonance imaging, and positron emission tomography and singlephoton emission computed tomography. This chapter is not intended to be an exhaustive review but instead is intended to serve as an introduction to the area. The chapter is divided into sections by imaging modality with a brief introduction to each modality followed by select examples that are of historical significance to the field or demonstrate current examples of where recent contributions of inorganic chemistry have occurred. Readers interested in further information are encouraged to read the Further Reading List and Relevant Websites listed at the end of this chapter.

2.15.2

X-ray computed tomography

X-rays have been a tool for medical imaging since the late 19th century1; however, the first X-ray computed tomography (CT) imaging devices did not appear until the 1970s.2 CT instruments produce high-resolution three-dimensional isotropic images within minutes, and CT imaging is one of the most commonly used medical procedures, accounting for 80% of diagnostic scans.3 The mechanism for standard X-ray CT involves X-ray beams (0.1–10 nm electromagnetic radiation) and detectors moving in tandem around a sample to collect data at multiple angles. Images are produced by attenuation of the X-rays based on the density of the material the beam is passing through, enabling different structures to be distinguishable from one another. Computer software is used to construct cross-sectional images from the data. Most CT scanners are calibrated to the attenuation density value of water (0 Hounsfield units). Because soft tissue can have similar attenuation density values to water (30–100 Hounsfield units), imaging adjacent tissues can be difficult.3 To enhance contrast in X-ray computed tomography, exogenous compounds called contrast agents that contain atoms with large electron densities are used to modify how X-rays pass through structures. For an atom to enhance contrast well in CT, the K-edge, or the sudden increase in energy at the K-shell binding energy due to the photoelectron effect, needs to be within the range of the X-ray spectrum. When designing contrast agents for CT, many factors are important, including K-edge values, mole percent of contrast-enhancing element, tissue retention, biodistribution, solubility, toxicity, and clearance.1,3,4–12 These contrast agents vary in design, ranging from iodinated organic molecules to nanoparticles (Fig. 1). With the emergence of new CT technology including multi-energy CT, micro-CT, and improved algorithms, the need for new and tunable contrast agents for CT continues to expand.13 Clinically used X-ray contrast agents include barium sulfate suspensions and iodinated organic compounds. Barium sulfate suspensions predominantly are used to study the gastrointestinal system, including tracheostomies. Introduced to the clinic in the 1950s, iodinated organic compounds have are the predominate agents of contrast-enhanced imaging.7,8,14 Although a wellestablished and successful field, efforts have been made to optimize iodinated agents to improve safety by avoiding risks such as nephropathy and renal toxicity and to increase specificity using targeting moieties,7,8,15 One targeting strategy involves the addition of carboxylic acids on the periphery of agents to promote chelation of Ca2þ ions for the purpose of characterizing bone microdamage using micro-CT.16–18 Currently, the field of new contrast agents for CT is dominated by nanoparticles. Nanoparticles vary in size from 1 to 1000 nm, and the tuneability in size alters imaging-relevant properties, such as biodistribution.1,3,19 For example, larger nanoparticles ranging from 15 to 200 nm mainly accumulate in the liver and spleen (Fig. 2), and smaller nanoparticles (< 15 nm) remain in the vasculature longer.20 With regards to the effect of size on contrast enhancement, reports vary on the X-ray attenuation of gold nanoparticles.21–24 Another key feature of nanoparticle formulations is the propensity to carry a large payload of contrast-enhancing atoms. Large payloads are easier on renal excretion because they result in fewer discrete excretion events than an equal number of contrastenhancing atoms distributed over more particles with smaller payloads. The design of slow-eroding nanoparticles also has been studied, reducing acute stress on the renal system.25,26 Furthermore, some nanoparticles are biocompatible, and there are currently liposomal nanomedicines clinically approved for cancer treatment, encouraging the design of new nanoparticulate agents.27 The first uses of nanoparticle-based formulations for CT involved emulsions and liposomes containing iodinated small molecules or iodinated lipids.1,28,29 Polymer-coated bismuth sulfide nanoparticles also found early success, but a lack of morphological control and challenges in surface modification led to other formulations.5,11 In the last decade, the field has flourished to include a wide range of other inorganic nanomaterials including gold, tantalum, iron–platinum alloys, alkaline earth elements, lead–uranyl acetate, silver, iron oxides, lanthanides, tungsten, semiconductor quantum dots, and transition-metal dendrimers.1,3,9,30

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Examples of clinically used iodinated contrast agents for X-ray computed tomography.

Of the many formulations examined, one of the most prominent and well-studied inorganic nanomaterials for CT contrast are gold nanoparticles.31–33 Gold nanoparticles are commercially available for research purposes as an X-ray contrast agent. In addition to being chemically inert and nontoxic within imaging-relevant concentrations and timeframes, gold has a larger electron density than iodine and, consequently, better X-ray attenuation per atom in CT.34,35 Further, gold nanostructures are able to pack a larger concentration of gold per unit compared to iodinated nanostructures that are commonly synthesized by covalent surface modifications of existing particles or polymerization of iodinated monomers.11 From a synthetic perspective, the preparation of gold nanoparticles can be modified to result in a variety of different morphologies, including rods, cubes, hollow-shapes, spheres, and clusters.36–39 Additionally, the surfaces can easily be modified to suit the needs of CT. Because the main focus of inorganic chemistry with respect to CT imaging involves nanoparticles, the remainder of this section highlights important examples of inorganic chemical contributions to CT imaging that involve advanced areas of imaging including target imaging, multimodal imaging, and combined imaging with therapy. For exhaustive reviews, readers are referred to the Further Reading List.

2.15.2.1

Targeted imaging

Targeted imaging refers to the modification of a contrast agent to make it enhance contrast in a specific location, usually through accumulation or activation. Additionally, for CT, targeted contrast agents offer opportunities to improve the sensitivity of CT. This point is an important area of research because although convenient, cost-effective, and widely available, contrast agents for CT have

Fig. 2 Effect of gold nanoparticle size (listed above each image) on contrast enhancement in different areas of mice. Images taken in CT at a voxel size of 100 mm, window level of 1090 HU and window width of 930 HU. Images were taken 2 h post-injection of gold nanoparticles. Contrast from injected nanoparticles is displayed from red to orange-yellow.20 Reprinted from Open Access Reference Dong, Y. C.; Hajfathalian, M.; Maidment, P. S. N.; Hsu, J. C.; Naha, P. C.; Si-Mohamed, S.; Breuilly, M.; Kim, J.;Chhour, P.; Douek, P.; Litt, H. I.; Cormode, D. P. Effect of Gold Nanoparticle Size on Their Properties as Contrast Agents for Computed Tomography. Sci. Rep. 2019, 9, 14912.

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less sensitivity ( 10 3 M) compared to contrast agents for other noninvasive imaging modalities, such as MRI ( 10 5 M).40 In addition to increasing the diagnostic capabilities of CT by targeting specific biological regions, targeted agents also ideally decrease the concentration of contrast agent needed for accurate imaging. Currently, 2 mg of contrast-enhancing element per cm3 of tissue is necessary for general CT, leading to approximately 30–50 g of contrast-enhancing element for diagnostic scans of humans.13 This limitation is especially relevant for CT contrast agents containing Z ¼ 75 (rhenium) through Z ¼ 79 (gold) that have excellent X-ray attenuation but are expensive. Therefore, recent studies of inorganic materials involve the addition of targeting capabilities to increase local concentrations while minimizing accumulation of contrast agent in off-target locations, thus decreasing the overall amount of agent needed for imaging.41,42 Targeted contrast agents can accumulate in tissues either passively or actively. Passive targeting is a result of general morphology leading to different retention times and clearance pathways. For example, nanoparticulate blood pool contrast agents can detect defects from thrombi or atherosclerosis in the peripheral vasculature system.1 Additionally, these agents can passively detect tumors by accumulating in the tumors due to the enhanced permeability and retention effect.43 Antibiofouling polymer coatings, such as polyethylene glycol or silane derivatives, and other coatings, such as polydopamine, have been reported to alter the biodistribution of nanoparticles and increase circulation times by increasing colloidal stability.44–46 For example, Hyeon and coworkers studied tantalum-oxide nanoparticles containing polyethyleneglycol–silane that targeted the reticuloendothelial system with no noticeable toxicity observed after histological staining of major organs.45 Active or direct targeting contrasts agents include the addition of specific surface modifications that incorporate targeting moieties that result in the accumulation of contrast agent in the desired location. Towards the study of actively targeted contrast agents, many modifications have been pursued, including antibodytagging, that leads to tumor deposition in cancers that overexpress the corresponding antigen. For example, Kopelman and coworkers examined gold nanorods conjugated to UM-A9 antibodies that induced 3–4 times the local contrast in two separate squamous cell carcinoma head and neck cancer cell lines that overexpress the A9 antigen corresponding to metastasis.47 Another example from Offen and coworkers involves the use of gold-nanoparticle-labeled mesenchymal stem cell-derived exosomes.48 Exosomes are lipid-nanovesicles ranging in size from 40 to 150 nm and are an emerging therapy-delivery device. Mesenchymal stem cell-derived exosomes can cross the blood-brain barrier, thereby enabling gold-nanoparticle conjugates of these exosomes to noninvasively image specific brain regions to study neuroinflammation associated to pathologies such as ischemic stroke, neurodegeneration, and even neuropsychic disorders. Further, a similar system from Zhu and coworkers used  32.5 nm glucose-modified gold nanoparticles conjugated to  97.7 nm mesenchymal stem cell-derived exosomes to study the effect of the exosomes on the heart repair of damaged myocardium in mice models.49 Although more studies are needed to examine the exact effect of the mesenchymal stem cell-exosomes on heart repair, the use of contrast-enhanced CT is an important tool in this emerging field.

2.15.2.2

Multimodal imaging

Multimodal probes are contrast agents or cocktails of agents that enhance contrast in several different imaging modalities, including CT, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), and optical imaging.50 The main goal of multimodal imaging is to merge the advantages of different imaging modalities to maximize diagnostic benefits by using orthogonal modalities to overcome the limitations of each other. CT is widely studied in multimodal imaging probes because the modality provides anatomical information and because the mode of contrastenhancement is chemically straightforward: the inclusion of a high-payload of atoms with large electron densities. Examples of multimodal imaging probes include FePt alloy nanoparticles for dual MRI/CT and 64Cu-doped gold nanoparticles for PET/ CT.51,52 Lanthanide-containing nanoparticles, like gold nanoparticles, are popularly studied as contrast agents for CT, most notably for multimodal applications to use the unique properties of lanthanide ions commonly studied for other noninvasive imaging modalities.30 For example, a variety of nanoparticles doped with YbIII and TmIII have been studied as upconversion nanophosphors because of their near-IR photophysical properties enabling bright contrast-enhancement upon proper excitation.53–55 However, photoluminescent imaging has limitations regarding tissue depth penetration of light, and therefore, combining it with a second modality like CT provides visual information not available by photoluminescence imaging alone. Further, Gd-containing dendrimers with entrapped gold nanoparticles have been studied for MRI and CT.56,57 In one study, GdIII influences the contrast in T1-weighted MRI, the presence of both gadolinium and gold increase the contrast in CT imaging, and the addition of folic acid on the periphery of the dendrimer increased tumor uptake in a human epithelial carcinoma cell line and tumor models, as shown by Shi and coworkers.56 However, the presence of the gold nanoparticles decreased the T1 contrast enhancement., emphasizing the importance of careful consideration of balancing features within the design of multimodal probes. Consequently, an important chemical facet of the design of multimodal imaging probes is the need to limit unwanted interactions between corresponding imaging moieties within the same probe. For example, the presence of GdIII within a nanoprobe is not expected to have the same effect on contrast enhancement in T1-weighted imaging, because of the distance dependence of the dipolar interaction between GdIII and water that is different between GdIII ions in the interior and exterior of nanoparticles.58 Additionally, activators in nanophosphors, such as ErIII or TmIII, best avoid nonradiative decay when located in the inner shells of a nanoparticle formulation.59,60 To this end, NaLuF4:Yb,Tm@NaGdF4-(153Sm) was designed by Li and coworkers as a tetramodal probe: a NaLuF4 host co-doped with 20% YbIII and 1% TmIII for upconverting luminescence and CT imaging and a 4 nm NaGdF4 shell doped with 153 SmIII for MRI and gamma photon emissive radioactivity. Using all four imaging modalities, researchers were able to display quantitative and qualitative information of tumor angiogenesis (Fig. 3).61As previously discussed, the main point of multimodal probes is a delicate balance of different modalities to outweigh deficiencies of one modality by including another. Nevertheless, care

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must be exercised when designing multimodal probes to avoid unproductive interactions between modalities, consider the concentration needed for different modalities, and weigh the relative benefits of the different modalities, if there is a desire to have biomedical relevance.

2.15.2.3

Combined imaging and therapy- image-guided therapy

In addition to using CT contrast agents in partnership with other imaging modalities for diagnostics, CT contrast agents have also been studied in theranostic applications.62 There are several clinical and preclinical advantages of theranostics, most notably the real-time imaging of both the therapeutic capabilities and excretion process of different treatments. Nanomaterials are specifically apt for theranostics due to their tunable morphology and surfaces, carrier abilities, and biodistribution.63 There are three main pathways for the design of CT agents for combined imaging and therapy: (1) using a contrast-agent nanocarrier as a vehicle to deliver a therapeutic drug; (2) the design of a nanomaterial capable of both contrast enhancement and therapy; or (3) some combination of both. For drug delivering contrast agents, the success of these agents heavily relies on the addition of targeted nanoparticles to ensure the drug is delivered to the desired tissue. For example, the addition of aptamers as targeting ligands for prostate specific membrane antigens to  12 nm gold nanoparticles was studied by Jon and coworkers as an anticancer delivery system of doxorubicin to LNCaP prostate epithelial cells.64 The conjugated system was able to differentiate between the cancerous cells and the control cells with a fourfold difference using CT and targeted the cancerous cells to deliver the doxorubicin at a statistically significant level compared to control cells that did not express the targeted antigen. However, aptamer degradation and general diffusion did lead to undesired cytotoxicity and fast drug release. Radiation therapy is used in 50% of cancer treatment, and targeted nanoparticle radiation-dose-amplification agents have been studied to increase local efficacy and limit damage to non-cancerous tissue.65–68 Heavy elements useful in radiation-dose amplification overlap with the elements required for CT contrast agents, making CT image-guided radiation therapy chemically straightforward. Examples of materials studied include gold-mesoporous silica nanoparticles, silica-based gadolinium and bismuth nanoparticles, and CuS nanoparticles doped with iodide.65,68,69 Another therapy commonly studied in conjunction with CT is photothermal therapy. Photothermal therapy, an extension of photodynamic therapy involving the release of heat upon electromagnetic irradiation. Inorganic nanoparticles studied for photothermal therapy include metal sulfides, gold nanostructures, and NaYF4 nanomaterials with formulations that include CT contrast including lanthanide or iodine doping.69–71 The success of theranostic agents using CT requires proper attention to the distinct properties required for both imaging and therapy. For example, the imaging modality must be possible within the timeframe of the therapeutic activity for suitable evaluation of drug performance. Additionally, the relative concentrations need to be taken into consideration in the design of theranostic agents; For contrast agents in CT, a large payload of contrast-enhancing atoms are required for high-quality images, whereas for many therapies, much lower concentrations are considered safe.

2.15.2.4

Conclusions

There are many contributing factors to the expansive research into inorganic contrast agents for CT, many of which stem from the morphological tunability of nanomaterials that influences biocompatibility, payload of contrast-enhancing elements, blood circulation half-lives, and functionalization with targeting moieties. Although still a dominant form of imaging, no new contrast agents for CT have been clinically approved in the last several decades.13 There are many factors contributing to this absence of new agents in the clinic, including cost-effectiveness of metals in preclinical nanomaterials, like gold, compared to iodinated contrast agents.

Fig. 3 The use of NaLuF4:Yb,Tm@NaGdF4-(153Sm) with all four imaging modalities: (a) upconverting luminescence; (b) X-ray CT; (c) SPECT; (d) MRI, and (e) confocal imaging inset from (a). (f) Schematic representation of experiment depicting tail-vein injection. Reprinted with permission from Sun, Y.; Zhu, X.; Peng, J.; Li, F. Core-Shell Lanthanide Upconversion Nanophosphors as Four-Modal Probes for Tumor Angiogenesis Imaging. ACS Nano 2013, 7, 11,290–11,300. Copyright 2013 American Chemical Society.

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Additionally, there is a need for thorough investigations into the long-term safety of nanomaterials.3 However, these nanoparticles offer valuable information in preclinical studies, and possibilities for important niche functionalities for specific clinical applications remain. Additionally, strides in new CT methodologies could assist the progress of the further development and use of nanomaterials in CT.13

2.15.3

Optical and near-IR imaging

2.15.3.1

Optical imaging with inorganic compounds and materials

Optical imaging uses electromagnetic waves within the visible and near-IR range of the spectrum arising from electronic transitions of probes. These electronic transitions are generally referred to as luminescence and occur in two well-understood forms: fluorescence and phosphorescence. Fluorescence is the release of a photon from the relaxation of an excited electron without a change in spin multiplicity. This process is fast with lifetimes on the order of nanoseconds and only results in energy loss between excitation and emission due to internal conversion. Phosphorescence undergoes intersystem crossing to change spin multiplicity between the excited- and ground-state electronic configurations. This process is forbidden and, therefore, slower than fluorescence with lifetimes on the order of milliseconds to seconds, and phosphorescence results in loss of energy between excitation and emission. For bioimaging, both processes see use for assays, deep tissue imaging, and as visual markers for surgeons.72–76 There have been many examples of lumiphores reported over the years, ranging from small organic molecules to massive compounds such as the green fluorescent protein.77–84 At the interface of inorganic chemistry, optical imaging involves compounds that feature metal complexes and semiconductor nanoparticles known as quantum dots. In this chapter, detailed discussion of quantum dots is omitted because recent breakthroughs with quantum dots feature surface modifications with non-inorganic molecules for advanced functions. Readers interested in quantum dots are referred elsewhere.85–88 There are a wide range of compounds that sense biorelevant metals such as iron, copper, calcium, magnesium, and zinc using luminescence. Major highlights from this field involve work from Tsien and coworkers, Chang and coworkers, and Lippard and coworkers. Early on, Tsien and coworkers explored sensors for calcium due to its role in cellular signaling, but since then, other metals have gained interest.89 Zinc is found in many catalytic enzymes, and examples of zinc sensors including Zinpyr from Tsien, Lippard, and coworkers or porphyrins from Lippard and coworkers demonstrate how basic inorganic chemistry principles may be used to tailor such sensors for specific metals.90–92 Their fluorescein-based probe has proven useful for mobile zinc as well as zinc in neurochemistry.93,94 In addition, the role of other metals including copper, nickel, cobalt, and iron have been explored by Chang and coworkers using specifically designed ligands containing macrocyclic binding sites or compounds that are sensitive to certain redox events related to metals, such as iron and cobalt.95,96 As a result, the role of metals such as copper in neurochemistry as well as questions of homeostasis have been elucidated. Readers that are interested in Further Reading on this topic are referred elsewhere.97–102 This section focuses on metal-ion complexes using both d- and f-block elements. First, progress on lanthanide-ion compounds with regard to bioimaging is described. Because trivalent lanthanide-ion compounds dominate the optical bioimaging field compared to other oxidation states, readers interested in chemistry related to divalent lanthanide-ion compounds are referred elsewhere.103,104 Next, recent reports of compounds featuring 4d or 5d metal ions are described with a focus on ruthenium(II) and iridium(III). Finally, a method for deep-tissue excitation using a phenomenon known as Cherenkov radiation is described with respect to excitation of inorganic lumiphores.

2.15.3.2

Trivalent lanthanide-based luminescence and imaging

Trivalent lanthanide ions (LnIIIs) have valence electrons in the 4f orbitals that are shielded from their surroundings by fully occupied 5s and 5p orbitals.105 This sheltered configuration results in interesting effects on LnIII electronic structure and photophysics. For instance, photoemission from a LnIII is characteristic to the identity of the ion because each ion has a unique electronic structure that is perturbed minimally by the ligand field. In addition, the bandwidths for LnIII emission are narrow, atom-like peaks because environmental factors are shielded from the valence electrons. In conjunction with the sheltered valence electrons, 4f–4f electronic transitions are Laporte forbidden. This property contributes to luminescence lifetimes on the order of microseconds to milliseconds, but also has the side effect of being associated with small native extinction coefficients for LnIIIs ( 1 M 1 cm 1 on average). As a result, “antennae” are often adorned onto LnIIIs to sensitize the ions and circumvent the scant native absorbance. Additionally, antenna often sequester LnIIIs from energetic oscillators such as OeH, NeH, and CeH bonds that contribute to non-radiative decay, thus increasing emission intensity. For more in-depth explanation of trivalent lanthanide photophysics, readers are encouraged to read more detailed literature.105,106 Recent growth within the field has shown the potential for LnIII-containing complexes as luminescent probes for biomedical imaging.

2.15.3.2.1

Targeted imaging using LnIII luminescent probes

The Parker group developed a LnIII complex with the capability of bioconjugation with a peptide.107 The compound is a derivative of 1,4,7-triazacyclononane conjugated to the peptide AcCFFKDEL. Two different conjugation strategies using thiols were used: direct substitution of an aryl p-nitro group on the EuIII complex and maleimide-thiol Michael addition on a pendant maleimide. Fluorescent probes from both conjugation strategies were examined in murine dermal fibroblasts (NIH-3T3). The direct

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substitution strategy showed emission from endoplasmic reticulum, which the conjugated polypeptide should target, and the maleimide-addition strategy showed emission from lysosomes (Fig. 4). The authors attributed this behavior to lability of the CeS linkage in the thiosuccinamide of the maleimide that is not present in the directly substituted probe. This finding suggests that these probes can target various organelles based on the selectivity of the targeting group when inert conjugation strategies are used. Starck, Parker, and coworkers developed pH-responsive EuIII complexes as turn-on sensors for acidic conditions, such as inside lysosomes.108 Their compound was based on 1,4,7-triazacyclononane with alkynyl additions onto a picolinate or methylpyridylphosphate arms to encapsulate the LnIII. These arms were functionalized with aromatic amines to act as proton acceptors. These compounds show a marked increase in EuIII emission as pH is decreased from 8 to 4 (Fig. 5), supporting potential applications as turn-on sensors in acidic environments. Imaging experiments using the complexes on murine dermal fibroblasts (NIH-3T3) and co-staining with Lysotracker green shows colocalization of emission from both the EuIII and the Lysotracker green (Fig. 5). This observation suggests that the EuIII emission is localized in lysosomes, which tend to encase more acidic conditions. In addition to the localization of emission from lysosomes, cell division was able to be tracked, and over time a net increase in brightness is observed as cell division occurs. This tendency was attributed to the net decrease in pH in aging endosomes or lysosomes. Based on these findings, this technology could be extended into internalization assays for acidic organelles. Eliseeva, Pecoraro, Petoud, and coworkers demonstrated an example of a coordination complex within the metallacrown archetype that sensitize LnIII in cellulo. Their metallacrown is an “encapsulated sandwich” structure where two 12-metallacrown-4 species bind a LnIII ion to form a “sandwich” that is encapsulated by 24-metallacrown-8.109 The structure is built using pyrazine hydroxamic acid and zinc(II) to form the metallacrown structures that bind the lanthanide. Both YbIII and NdIII could be sensitized in the metallacrown with quantum yields of 1.112  10 2 (Yb) and 7.7  10 3% (Nd) and lifetimes of 5.57 (Yb) and 0.214 (Nd) ms in water. In addition, these compounds are able to achieve cell fixation and selectively internalize into necrotic cells.109,110 Cell fixation was confirmed using confocal Raman spectroscopy comparing the metallacrown to more common fixation methods including paraformaldehyde and methanol. The Raman band at 752 cm 1 was used as an indicator of fixation because that band, corresponding to cytochrome c, decreases with fixation. In all three treatments, the band at 752 cm 1 disappears (Fig. 6) suggesting fixation. Selective imaging of necrotic cells was achieved using a glucose-starved cell medium for incubation, which results in a mixture of living and necrotic HeLa cells. These cells were incubated with the metallacrown and propidium iodide (a dye that stains the nucleus of necrotic cells) and imaged with epifluorescence. The metallacrown and propidium iodide signals colocalize in the necrotic HeLa cells, and the living cells remained unstained (Fig. 7). These results demonstrating selective epifluorescent imaging are potentially useful in flow cytometry assays.

Fig. 4 Colocalization between EuIII containing compounds and Lysotracker Green in the case of CeS bond lability (top) and ER-Tracker Green in the case of conjugated peptide (bottom) in confocal microscopy. Reprinted with permission from Starck, M.; Fradgley, J. D.; Di Vita, S.; Mosely, J. A.; Pal, R.; Parker, D. Targeted Luminescent Europium Peptide Conjugates: Comparative Analysis Using Maleimide and para-Nitropyridyl Linkages for Organelle Staining. Bioconjugate Chem. 2020, 31, 229–240. Copyright 2020 American Chemical Society.

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Fig. 5 pH response of EuIII emission shows significant change (left) and colocalization is observed between the EuIII emission and LysoTracker Green in confocal microscopy (right).108 Reprinted from Open Access Reference Starck, M.; Fradgley, J. D.; Pal, R.; Zwier, J. M.; Lamarque, L.; Parker, D., Synthesis and Evaluation of Europium Complexes that Switch on Luminescence in Lysosomes of Living Cells. Chemistry 2021, 27 (2), 766–777.

2.15.3.2.2

Packaging systems for LnIII imaging probes

Petoud and coworkers also demonstrated a method for loading LnIII-containing compounds into polystyrene beads for in cellulo imaging.111 Their system involved loading Yb(CF3SO3)3 and dihydroxyanthraquinone antennae into amine-functionalized polystyrene beads that were then adorned with polyethyleneglycol via peptide linkages using surface amines (Fig. 7). The system displayed YbIII sensitization via the antenna excitation and has excellent photostability over the course of 1 h with regard to both the antenna and the YbIII ion.111 Cell experiments were performed with HeLa cells to see if the functionalized beads enter cells. Confocal microscopy using anthraquinone emission showed general distribution within the cellular cytoplasm. Epifluorescent microscopy using both the anthraquinone and YbIII emissions also were performed, demonstrating the usefulness of the beads in both visible and near-infrared detection (Fig. 8). To study the mechanism of uptake into cells, flow cytometry was performed using different uptake inhibitors to distinguish between active and passive transport. The authors concluded that multiple mechanisms are active; however, passive uptake is likely the dominant pathway because cold (4  C) temperatures had the greatest suppression compared to chemical inhibitors. In essence, the bead system is a straightforward method for LnIII luminescent dyes with excellent photostability. Metal-organic frameworks (MOFs) are also capable of use for optical imaging in cellulo as demonstrated by Petoud, Rosi, and coworkers in 2020.112 The challenges presented for bioimaging and MOFs are water stability, low density of emitting species, and tuning of excitation and emission wavelengths to the biological-transparency window. Some MOFs use large conjugated systems as a part of the structure that tend to increase lattice voids and channels that exacerbate the density issue. The authors designed a MOF with the intention of incorporating conjugated systems that reside in the voids and channels of the parent MOF. In this case, Yb4(OH)4 and 2-aminoterephthalic acid linkers were combined to form MOF-1114 and MOF-1140 (Fig. 9).

Fig. 6 (Left) Raman spectra examine the presence or absence of the 752 cm 1 band in cells with (a) no fixation, (b) methanol, (c) paraformaldehyde, and (d) metallacrown. (Right) HeLa cells incubated with propidium iodide and metallacrown: (a) brightfield image, (b) metallacrown emission, (c) propidium iodide emission, (d) merged (b) and (c), and (e) merged (a) and (d). Reprinted from Open Access Reference Cotruvo, J. A., Jr.; Aron, A. T.; Ramos-Torres, K. M.; Chang, C. J. Synthetic Fluorescent Probes for Studying Copper in Biological Systems. Chem. Soc. Rev. 2015, 44, 4400–4414; Martinic, I.; Eliseeva, S. V.; Nguyen, T. N.; Foucher, F.; Gosset, D.; Westall, F.; Pecoraro, V. L.; Petoud, S., Near-infrared luminescent metallacrowns for combined in vitro cell fixation and counter staining. Chem. Sci. 2017, 8 (9), 6042–6050 and with permission from Martinic, I.; Eliseeva, S. V.; Nguyen, T. N.; Pecoraro, V. L.; Petoud, S. Near-Infrared Optical Imaging of Necrotic Cells by Photostable Lanthanide-Based Metallacrowns. J. Am. Chem. Soc. 2017, 139, 8388–8391. Copyright 2017 American Chemical Society.

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Fig. 7 Packing strategy employed to contain Yb(CF3SO3)3 and anthraquinone derivatives into polystyrene beads. Reprinted with permission from Martinic, I.; Eliseeva, S. V.; Collet, G.; Luo, T.-Y.; Rosi, N.; Petoud, S. One Approach for Two: Towards the Creation of Near-Infrared Imaging Agents and Rapid Screening of Lanthanide(III) Ion Sensitizers Using Polystyrene Nanobeads. ACS Appl. Bio. Mater. 2019, 2, 1667–1675. Copyright 2019 American Chemical Society.

The MOFs were postsynthetically modified by reacting the aryl amines with methyl propiolate resulting in a dense tangle of short conjugated chains. This modification changes the appearance of the crystals of each MOF from colorless to red indicating a red shift in absorbance. Spectrophotometry confirms the red shift of the absorbance edge from about 400 to > 450 nm. Excitation spectra in dimethylformamide show signal to 700 nm after the postsynthetic modification, which is compatible with the biological transparency window for maximum tissue penetration for optical imaging. Near-IR epifluorescence of each MOF with RAW 264.7 cells showed that the MOFs withstand incubation in cell media and maintain YbIII-based emission (Fig. 10). The MOFs do not penetrate cell membranes, but stick to cell surfaces. These findings speak to the capability of MOF compounds for deep-tissue luminescent imaging.

Fig. 8 Epifluorescence images of HeLa cells: (a) brightfield image, (b) anthraquinone emission, and (c) YbIII emission. Reprinted with permission from Martinic, I.; Eliseeva, S. V.; Collet, G.; Luo, T.-Y.; Rosi, N.; Petoud, S. One Approach for Two: Towards the Creation of Near-Infrared Imaging Agents and Rapid Screening of Lanthanide(III) Ion Sensitizers Using Polystyrene Nanobeads. ACS Appl. Bio. Mater. 2019, 2, 1667–1675. Copyright 2019 American Chemical Society.

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Fig. 9 2-aminoterephthalic acid forms MOF-1114 (A and B) and MOF 1140 (C and D). Reprinted with permission from Muldoon, P. F.; Collet, G.; Eliseeva, S. V.; Luo, T. Y.; Petoud, S.; Rosi, N. L. Ship-in-a-Bottle Preparation of Long Wavelength Molecular Antennae in Lanthanide Metal–Organic Frameworks for Biological Imaging. J. Am. Chem. Soc. 2020, 142, 8776–8781. Copyright 2020 American Chemical Society.

2.15.3.2.3 III

Other applications of LnIII luminescence for bioimaging

Ln complexes have been studied in combination with quantum dots for imaging applications. Huang, Hildebrant, and coworkers reported a tunable Förster resonance energy transfer system between LnIII and quantum dots for cell barcoding.113 Of note in this system is that LnIII such as TbIII and EuIII were used to sensitize the quantum dots with coatings SiO2 (6 or 12 nm) that separated the quantum dots from the LnIII ions. The combination of TbIII or EuIII with coatings of 6 or 12 nm led to four systems with similar photoluminescence decay curves (Fig. 11). The decay curves were divided into red (0.05–0.5 ms), green (0.5–1.0 ms), and blue (1–3 ms) time windows, enabling identification of each probe using the ratio of luminescence. To test the barcode identification potential of the luminescence ratios, HeLa cell groups were incubated with different combinations of the four systems, and timegated luminescence imaging was used to assign a unique color to each label. A mixture of labeled cells was examined, and each of

Fig. 10 YbIII emission observed on the edge of RAW264.7 cells: (C) brightfield image, (D) YbIII emission, and (E) merged images. Reprinted with permission from Muldoon, P. F.; Collet, G.; Eliseeva, S. V.; Luo, T. Y.; Petoud, S.; Rosi, N. L. Ship-in-a-Bottle Preparation of Long Wavelength Molecular Antennae in Lanthanide Metal–Organic Frameworks for Biological Imaging. J. Am. Chem. Soc. 2020, 142, 8776–8781. Copyright 2020 American Chemical Society.

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the four labels was identified for each cell (Fig. 12). The authors postulated that this technology is expandable to more combinations of LnIII and quantum dots for higher-order barcoding despite the similarities in overall lifetime. The de Bettencourt-Dias group reported a tris(dipicolinate) LnIII complex that can be used to image HeLa cells using epifluorescence and generate singlet oxygen via light activation for controlled cytotoxicity.114 Two different ligands were investigated (Fig. 13) with EuIII and YbIII as the ions for visible and near infrared emission. Quantum yields in an aqueous environment for complexes of 1Tdpa2  were measured and the EuIII quantum yield was much larger than that of YbIII (33% and 0.003%, respectively), which is not an uncommon observation. Measurement of 2Tdpa2  complexes with YbIII gave a quantum yield of 0.0007% in water. EuIII with 2Tdpa2  quantum yields were not reported. Live HeLa cells were imaged using [Eu(1Tdpa)3]3 , Hoechst 33342, and MitoTracker Green FM (Fig. 14). Colocalization of emission from EuIII and Hoechst 33342 confirm that the compound penetrates the cell membrane, and colocalization of signal from EuIII and MitoTracker Green FM suggest that the compound aggregates around the mitochondria of the cells. For investigation of singlet oxygen generation, each ligand and complexes with GdIII, EuIII, and YbIII were measured using phosphorescent detection of 1O2 emission at 1270 nm in 95% ethanol. All of the experiments showed some singlet oxygen generation; however, measurable quantum yields were determined for [Gd(1Tdpa)3]3  (13%), H22Tdpa (23%), and [Gd(2Tdpa)3]3  (45%). Cell viability assays on HeLa cells were performed using a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay in both light and dark conditions for H22Tdpa, [Gd(2Tdpa)3]3 , and [Yb(2Tdpa)3]3 . Phototherapy indexes determined from the ratio of light to dark IC50 were 4.5 for H22Tdpa, 9.5 for [Gd(2Tdpa)3]3 , and 7.0 for [Yb(2Tdpa)3]3 . So, all three compounds induce cell death in the light more than in the dark, suggesting that each compound is a photoactivated toxin in the HeLa cells. The authors found evidence for both necrotic and late-stage apoptotic cells using flow cytometry when determining how the cells die with exposure to the compounds in the light. Overall, this report describes a set of compounds that can be used to image HeLa cells and have the potential to act as photoinduced therapy agents.

Fig. 11 (a) Decay curves of quantum dots are (b) divided into three regions with unique ratios. Reprinted with permission from Chen, C.; Ao, L.; Wu, Y. T.; Cifliku, V.; Cardoso Dos Santos, M.; Bourrier, E.; Delbianco, M.; Parker, D.; Zwier, J. M.; Huang, L.; Hildebrandt, N. Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots. Angew. Chem., Int. Ed. 2018, 57, 13686–13690. Copyright 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Fig. 12 Time-gated imaging of HeLa cells barcoded with different nanoparticles. (a) Red time gate, (b) green time gate, (c) blue time gate, (d) merged image, and (e) brightfield image. Arrows indicate cells with different barcodes. Reprinted with permission from Chen, C.; Ao, L.; Wu, Y. T.; Cifliku, V.; Cardoso Dos Santos, M.; Bourrier, E.; Delbianco, M.; Parker, D.; Zwier, J. M.; Huang, L.; Hildebrandt, N. Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots. Angew. Chem., Int. Ed. 2018, 57, 13686–13690. Copyright 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A methodology for single-protein imaging using LnIII has been introduced recently by Hildebrant and coworkers.115 Their system uses rolling circle amplification Förster resonance energy transfer (FRET) to examine b-actin proteins. Rolling circle amplification uses a circular DNA fragment to form long chains of labeled oligomers typically adorned with fluorescent probes. Some limitations of the technique include the need for multiple washing steps, limited color choice, and problems with autofluorescence. The authors addressed these limitations by designing FRET pairs for the product from rolling circle amplification, including LnIII species. Because the FRET pairs only produce signal if they are in close proximity (for example, placed in close proximity via rolling circle amplification), there is no need to wash away excess probe, eliminating the need for washing steps. The use of LnIII with long luminescence lifetimes enabled time-gated assays to circumvent issues with autofluorescence. The specific lumophores examined in the study include Lumi4-Tb, Cy3.5, and AF647. Cell experiments were performed with HaCaT cells and an enzyme-linked immunosorbent assay for detection of b-actin proteins. The first setup used Cy3.5 to sensitize AF647 to show the proof-of-concept for washing-free rolling circle amplification. FRET emission was observed using 510 nm excitation and 720 nm emission bands both with and without washing steps. To demonstrate the ability for time-gated experiments, TbIII was used to excite Cy3.5 with 349 nm excitation and 660 nm emission wavelengths, and FRET emission specific to actin bound probes was observed without washing steps (Fig. 15). In summary, the authors developed a rolling circle amplification FRET technique that could include TbIII as a FRET donor to enable washing-free assays and time-gated imaging.

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Fig. 13 Representations of the compounds of interest for singlet oxygen generation. Complex with 1Tdpa2  (left) and 2Tdpa2  (right). Reprinted with permission from Rodrigues, C. V.; Johnson, K. R.; Lombardi, V. C.; Rodrigues, M. O.; Sobrinho, J. A.; de Bettencourt-Dias, A. Photocytotoxicity of Thiophene- and Bithiophene-Dipicolinato Luminescent Lanthanide Complexes. J. Med. Chem. 2021, 64, 7724–7734. Copyright 2021 American Chemical Society.

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Fig. 14 Images of HeLa cells with emission from MitoTracker Green FM (top left), Hoechst 33342 (top center), and EuIII complex (top right). Merged image is on the bottom. Reprinted with permission from Rodrigues, C. V.; Johnson, K. R.; Lombardi, V. C.; Rodrigues, M. O.; Sobrinho, J. A.; de Bettencourt-Dias, A. Photocytotoxicity of Thiophene- and Bithiophene-Dipicolinato Luminescent Lanthanide Complexes. J. Med. Chem. 2021, 64, 7724–7734. Copyright 2021 American Chemical Society.

2.15.3.3

Imaging with 4d and 5d transition metal complexes

Phosphorescence imaging with 4d and 5d transition metal complexes has been of interest for several decades. Reports of compounds with PtII, RuII, ReI, IrIII and AuIII have shown the viability of these metal ions for bioimaging.81,116 These compounds tend to feature bright phosphorescence and remarkable tunability based on ligand field to adjust excitation and emission into the biocompatible optical window. This tunability combined with massive absorbance inherent to metal-to-ligand change transfer transitions have shown potential. Ligand modifications also lead to adjustment of solubility and provide opportunities for installation of specific markers or functional groups. Within the past few years, these ions have shown potential not only for bioimaging but also for biosensing. Chao and coworkers reported an IrIII probe in 2021 that included a hydroxyquinone/quinone redox-active moiety. Their intention was to use this moiety to monitor peroxynitrite, a naturally occurring oxidizer, and glutathione, a naturally occurring reductant, that are both associated with mitochondrial activity.117 The hydroxyquinone reacts with peroxynitrate to form the quinone that quenches the phosphorescence of the compound at 704 nm. Glutathione converts the quinone back to the hydroxyquinone, restoring phosphorescence. Selectivity for peroxynitrate and glutathione was confirmed by measuring phosphorescence of the compound upon exposure to other oxidants (1O2, H2O2, superoxide, perchlorate, hydroxy radical, nitric oxide, and nitrite) and reductants (cystine, homocysteine, histidine, tyrosine, vitamin C, hydrogen sulfide, and bisulfite). Only peroxynitrate and glutathione modulated phosphorescence of the IrIII compound. The compound was incubated into HepG2 cells with different concentrations of peroxynitrite and glutathione (Fig. 16), and confocal microscopy revealed that increasing concentration of peroxynitrite reduces phosphorescence and increasing concentration of glutathione leads to more intense phosphorescence. Further, intraperitoneal injections into mice demonstrated how phosphorescence changes in vivo. Acetaminophen was used to induce peroxynitrite formation and acetylcystine was used to induce glutathione production. Their probe turned off and on based on both concentration of acetaminophen and acetylcystine as well as incubation time of the system (Fig. 17). These results demonstrate the capability of luminescent probes to monitor in vivo oxidative stress and recovery especially for cases of overdose of acetaminophen. The ability to generate singlet oxygen is described in the section regarding trivalent lanthanide compounds; however, this property is not exclusive to such compounds. Xie, Xhu, Bryce, and coworkers designed a porphyrin derivative that is attached to one or

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Fig. 15 Images of rolling circle amplification on b-actin: (top left) brightfield, (top right) time-gated TbIII emission, (bottom left) emission from Cy3.5, and (bottom right) FRET emission. Reprinted with permission from Francés-Soriano, L.; Leino, M.; Dos Santos, M. C.; Kovacs, D.; Borbas, K. E.; Söderberg, O.; Hildebrandt, N. In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging. Anal. Chem. 2021, 93, 1842–1850. Copyright 2021 American Chemical Society.

four IrIII complexes.118 These molecules were loaded into polymeric nanoparticles comprised of 1,2-distearoyl-sn-glycero-3phosphorylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) coblock polymers that were adorned with HIV-1 Tat for cell penetration. UV–visible absorbance of the loaded nanoparticles in water shows absorbance to 675 nm, and

Fig. 16 Phosphorescence imaging from the IrIII complex at increasing concentration of peroxynitrite (ONOO) and glutathione (GSH). Reprinted with permission from Wu, W.; Liao, X.; Chen, Y.; Ji, L.; Chao, H. Mitochondria-Targeting and Reversible Near-Infrared Emissive Iridium(III) Probe for In Vivo ONOO/GSH Redox Cycles Monitoring. Anal. Chem. 2021, 93, 8062–8070. Copyright 2021 American Chemical Society.

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Fig. 17 Optical imaging of mice demonstrating that signal from the IrIII complex is lost as more acetaminophen is introduced and signal is gained with more acetylcystine. Reprinted with permission from Wu, W.; Liao, X.; Chen, Y.; Ji, L.; Chao, H. Mitochondria-Targeting and Reversible NearInfrared Emissive Iridium(III) Probe for In Vivo ONOO/GSH Redox Cycles Monitoring. Anal. Chem. 2021, 93, 8062–8070. Copyright 2021 American Chemical Society.

phosphorescence bands are observed between 640 and 800 nm that are amenable for use with biological samples. The authors describe two phenomena that were observed with their system: singlet oxygen generation and photothermal activity. To measure singlet oxygen generation, 1,3-diphenylisobenzofuran was used as an indicator, and singlet oxygen quantum yields were 72% for nanoparticles with the mononuclear IrIII compound and 89% for nanoparticles with the tetranuclear IrIII compound. To quantify the photothermal abilities of the system, aqueous solutions at room temperature were irradiated with a 635 nm laser for 5 min. Solutions of nanoparticles with the mononuclear compound reached 46.9  C and solutions of the tetranuclear compound reached 59.1  C. The authors examined their system in cellulo using HeLa cells. Confocal microscopy confirmed that the nanoparticles penetrate cell membranes with even distribution about the cell. Cell viability was measured both in the dark and under irradiation with a 635 nm laser for 5 min using an MTT assay. In the dark, cell viability was maintained; however, in the light, IC50 values of 0.145 mM for the mononuclear compound and 0.057 mM for the tetranuclear compound were measured, suggesting that this system might be useful for phototherapy. Finally, the tetranuclear variant was examined using a murine model with U14 tumors. Intratumoral injection of the nanoparticles that were irradiated for 10 min with 635 nm light showed an increase in local temperature from 38.3 to 61.9  C and a reduction in tumor size over 2 weeks. Intravenous injection of the nanoparticles labeled with IR780 resulted in accumulation within tumors within 12 h. Irradiation for 10 min with 635 nm light showed an increase in local temperature from 37.0 to 53.0  C, and tumor volume decreased over the course of 2 weeks. These murine experiments confirm the viability of the proposed system for phototherapy applications. Multiphoton excitation is another technology that has the potential to enhance tissue penetration. Shen, Pei, Chao and coworkers reported a set of derivatives of an IrIII compound that was able to achieve two-photon excitation and three-photon excitation.119 HeLa cells were used to demonstrate the potential of the compounds in cellulo. The cells were incubated with the complex and MitoTracker Green and were imaged with 405 nm light for single-photon excitation, 750 nm light for two-photon excitation, and 980 nm light for three-photon excitation of the IrIII agent. Colocalization of signal in confocal microscopy of both IrIII and MitoTracker Green suggest that the IrIII agents target the mitochondria. To demonstrate the power of three-photon excitation imaging, zebrafish embryos were placed under various thicknesses of synthetic tissue. With no tissue, zebrafish were able to be imaged using all three techniques; however, 200 mm of tissue attenuates signal from 405 nm excitation, and 1000 mm of tissue attenuates sign from 750 nm excitation, but 980 nm excitation remains observable in all cases. Finally, three-dimensional models of murine brain vasculature were obtained on DSPE-PEG2000 nanoparticles loaded with the IrIII agents. Both two-photon and threephoton excitation techniques were explored, and the model created by three-photon excitation with 980 nm light was much deeper and better resolved than the model created with two-photon excitation (Fig. 18). This report describes a promising technology for deep-tissue imaging with optical probes. In 2019, the Peng group reported an innovative metallopolymer containing RuII that was packaged into a polystyrene nanoparticle.120 Their system used a modified polystyrene to incorporate a bipyridine moiety that was used to bind Ru(bpy)2Cl2 to form the metallopolymer (Fig. 19). This polymer was precipitated with polystyrene as well as polystyrene functionalized with polyethylene glycol terminated by a carboxylic acid to form the polystyrene nanoparticle. Finally, (3-aminopropyl)triphenylphosphine bromide was attached to the surface via amide linkages with terminal carboxylates on the nanoparticle for the purpose of targeting the mitochondria of a cell for intracellular monitoring of oxygen content. The nanoparticles retain similar spectroscopic properties to [Ru(bpy)3]2 þ, with a metal-to-ligand charge transfer absorbance band centered at 450 nm and a phosphorescence band centered at 608 nm in an aqueous environment. In addition, Stern–Vollmer plots show a linear relationship where more emission quenching is observed with greater dissolved oxygen content. These results encouraged exploration of the possibility of in vivo oxygen sensing. The first step was confocal microscopy of the nanoparticle and MitoTracker Green in HepG2 cells. The resulting images show that the nanoparticle is internalized by HepG2 cells and colocalizes with MitoTracker Green in the mitochondria. Next, dynamic response of the nanoparticle to oxygen content was explored using rotenone to inhibit mitochondrial function (increasing oxygen content) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to promote mitochondrial function (decreasing oxygen content) in HepG2 cells. As expected, cells treated with rotenone showed quenching while cells treated with FCCP became

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Fig. 18 (a) Three-dimensional reconstruction from two-photon excitation and (b) three-photon excitation of murine cerebral blood vessels.119 Reprinted from Open Access Reference Jin, C.; Liang, F.; Wang, J.; Wang, L.; Liu, J.; Liao, X.; Rees, T. W.; Yuan, B.; Wang, H.; Shen, Y.; Pei, Z.; Ji, L.; Chao, H., Rational Design of Cyclometalated Iridium(III) Complexes for Three-Photon Phosphorescence Bioimaging. Angew. Chem. Int. Ed. 2020, 59 (37), 15987–15991.

brighter over the course of 10 min (Fig. 20). These results are encouraging, and demonstrate strong potential for oxygen sensing in more complex tissues.

2.15.3.4

Cherenkov radiation with inorganic lumiphores

Cherenkov radiation was first described in the 1930s by Pavel Cherenkov and Sergey Vavilov as a peculiar source of photoemission.121,122 The phenomenon occurs when a charged particle such as an electron or positron travels through a medium faster than a photon in the same medium. In such a case, electromagnetic waves are perturbed by the particle in a similar manner as supersonic aircraft do to sound waves. A cone-shaped wavefront formed from interference of circular “ripples” ultimately results in the emission of light. This light is typically in the ultraviolet to blue range, so escape of these photons from deep tissue is difficult because the biological transparency window is in the red to near-IR range. Positron emitting isotopesdincluding 18F, 64Cu, 68 Ga, 89Zr, 90Y, 99mTc, and 131Idthat are useful in positron emission tomography are candidates for in vivo Cherenkov radiation generation because the positrons emitted from these isotopes take the role of the faster-than-light charged species in water.123,124 Recently, Cherenkov radiation has shown promise for bioimaging, not only as its own mechanism, but also as an excitation method for other compounds with emission in the biological optical transparency window. One example of Cherenkov sensitized emission was performed on an IrIII complex studied by Yang, Yang, Wang, and coworkers in 2019.125 This group placed luminescent iridium(III)(2-phenylquinoline)2(bipyridine)chloride in liposomes, for biocompatibility, and investigated the ability of Cherenkov radiation from 18F-fluorodeoxyglucose (FDG) and Na99mTcO4 to excite the Ir

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Fig. 19 Schematic of construction of the RuII containing polymeric nanoparticles. Reprinted with permission from Zhou, C.; Zhao, W.-x.; You, F.-t.; Geng, Z.-x.; Peng, H.-s. Highly Stable and Luminescent Oxygen Nanosensor Based on Ruthenium-Containing Metallopolymer for Real-Time Imaging of Intracellular Oxygenation. ACS Sens. 2019, 4, 984–991. Copyright 2019 American Chemical Society.

complex to an emissive state. Emission from the IrIII complex was about 29-fold greater with FDG compared to Na99mTcO4, so FDG was used for more in-depth study. The authors examined the phosphorescence of the Ir complex with no stimulation, the Cherenkov-stimulated emission from both compounds, and the Cherenkov emission from FDG on its own under tissue slices with thicknesses of 1, 2, and 3 mm to test light penetration. While the native phosphorescence of the Ir-containing complex and FDG signal were lost at 3 mm of tissue, the Cherenkov-stimulated emission was still visible at that tissue depth. A similar experiment was performed in a murine model with a 4T1 xenograft tumor. Intratumoral injection was examined using the using the same three concoctions, and overall luminescence intensity was greatest for the native phosphorescence, followed by the Cherenkovstimulated emission, and least intense for the Cherenkov emission in the absence of Ir. However, comparison of the tumor with muscle tissue that was not stained showed that the stimulated emission had the best signal-to-noise ratio (Fig. 21). This enhanced signal-to-noise ratio suggests that the technique is potentially attractive for in vivo applications. The Boros group has also shown the potential for LnIII excitation with a Cherenkov radiation source. One example from that group is a DO3A derivative with a picolinic acid arm to act as a LnIII antenna.126 This compound was metalated with TbIII, EuIII, or LaIII (as a dark control) to test their system. Radiance observed between 515 and 540 nm for each compound in the presence of FDG confirms that some of these compounds can be stimulated, and that screening assays are possible to find more candidates (Fig. 22). For both TbIII and EuIII, there is clearly observable radiance when the antenna is present with a 89Zr Cherenkov radiation source. This experiment suggests that the system functions as intended, and Cherenkov radiation excites the picolinic acid that sensitizes the LnIII. Overall quantum yields were determined using the relative method to be 47% for TbIII and 1.5% for EuIII. In addition, the authors attached a chelator for 89Zr as well as the picolinic acid antenna onto DO2A to make the system intramolecular.127 The intramolecular system displayed radiance in the same region of interest, and the quantum yield was 63%, suggesting that the intramolecular system was more efficient than the intermolecular system for LnIII luminescence. Another example from the Boros group describes a system for a turn-on sensor for tumor imaging.128 TbIII DO3A with the picolinic acid antenna was used, as well as an EuIII DO2A or DO3A with a phenanthroline antenna as the LnIII emissive species. These species were functionalized with 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid that is a bioconjugate for prostate-specific

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Fig. 20 Imaging of HepG2 cells with the RuII system. (top) Mitochondrial inhibition, (center) control in phosphate-buffered saline (PBS), and (bottom) mitochondrial stimulation. Reprinted with permission from Zhou, C.; Zhao, W.-x.; You, F.-t.; Geng, Z.-x.; Peng, H.-s. Highly Stable and Luminescent Oxygen Nanosensor Based on Ruthenium-Containing Metallopolymer for Real-Time Imaging of Intracellular Oxygenation. ACS Sens. 2019, 4, 984–991. Copyright 2019 American Chemical Society.

membrane antigen (PSMA), which is associated with cancerous prostate cells. FDG was introduced as a Cherenkov radiation source and is known to associate with the GLUT-1 protein in many cancers. To achieve emission, both FDG and the LnIII must be in close proximity, which is only possible with specific pancreatic cancer cells containing both PSMA and GLUT-1 (Fig. 23). In vivo imaging of a murine model with a xenografted PSMA-expressing tumor demonstrated the ability of the EuIII complex to be used in conjunction with the FDG radiation source (Fig. 23). TbIII was not used in vivo because the emission was attenuated by tissue, but the emission from EuIII was able to penetrate tissue. This system is promising as an example for targeted optical imaging for specific cancers. In addition to using Cherenkov radiation to excite trivalent lanthanides, Decréau and coworkers demonstrated how to use Cherenkov radiation energy transfer with ZnII phthalocyanine (ZnPc).129 The challenge with ZnPc is the inefficiency of the 370 nm Soret band for excitation of near-IR fluorescence, so direct excitation with Cherenkov radiation is difficult. To amend this shortcoming, as well as the poor water solubility of ZnPc, pyranine was appended to ZnPc. To examine the proposed system, first phantom images were taken comparing wells with Cherenkov radiation generated by FDG and wells containing FDG and the pyranine-appended ZnPc with 620, 670, and 710 nm filters. Overall, a maximum of five-fold increase in emission for wells with ZnPc was observed with the 710 nm filter suggesting the ability to sensitize ZnPc with light from FDG. To be sure pyranine was an intermediate antenna, another ZnPc analog was synthesized for water solubility but no absorbance in the Cherenkov radiation emission range. This analog did not show a pronounced increase in emission like the pyranine-functionalized ZnPc. Additionally, thin slices of meat

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Fig. 21 Images of a mouse comparing signal from a tumor (yellow/red circle) and muscle (white circle). (left) Phosphorescence from IrIII, (center) Cherenkov-stimulated IrIII, and (right) FDG signal. Republished with permission from The Royal Society of Chemistry from Iridium Complex Nanoparticle Mediated Radiopharmaceutical-Excited Phosphorescence Imaging by Yutong Hou, Chenchen Wang, Ming Chen, Mingwei Wang, Guang Deng, Hong Yang, Zhiguo Zhou, and Shiping Yang. Chem. Commun. 2019, 55, 14442–14445. Copyright 2019. Permission conveyed through Copyright Clearance Center.

were placed over wells containing FDG or FDG and the modified ZnPc to a maximum thickness of 2.4 mm. Increasing tissue thickness resulted in decreased emission from FDG, but emission from the FDG and ZnPc wells remained. This experiment showed how using near-IR emission from ZnPc enhances tissue penetration compared to the UV or blue Cherenkov radiation on its own. Finally, emission from Cherenkov radiation of 90YCl3 or from a mixture of 90YCl3 and ZnPc was studied in murine models with xenografted HCC1954 cells (Fig. 24). Both solutions were injected into the tumors then imaged, and the presence of ZnPc showed enhanced signal compared to the Cherenkov radiation on its own due to better tissue penetration.

2.15.3.5

Conclusions

Over the course of this section, recent progress in optical bioimaging at the interface of inorganic chemistry was described. Trivalent lanthanide-ion compounds demonstrate versatility as pH sensors, targeted probes, barcoding, phototherapy, and rolling circle amplification. In addition, recent reports with RuII and IrIII described the potential uses of compounds tuned for the biological transparency window with respect to oxygen sensing, multiphoton excitation imaging, and phototherapy. Finally, Cherenkov radiation was introduced, and the ability to use this light source to excite various inorganic systems was outlined. In all, these contributions highlight the strength of inorganic chemistry at the cutting edge of optical imaging.

Fig. 22 Luminescence from TbIII DOTA complexes stimulated by FDG. Reprinted with permission from Cosby, A. G.; Quevedo, G.; Boros, E. A High-Throughput Method To Measure Relative Quantum Yield of Lanthanide Complexes for Bioimaging. Inorg. Chem. 2019, 58, 10611–10615. Copyright 2019 American Chemical Society.

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Fig. 23 (left) Schematic for the system with Cherenkov stimulated LnIII emission. (right) Image of EuIII radiance in a mouse. Reprinted with permission from Martin, K. E.; Cosby, A. G.; Boros, E. Multiplex and In Vivo Optical Imaging of Discrete Luminescent Lanthanide Complexes Enabled by In Situ Cherenkov Radiation Mediated Energy Transfer. J. Am. Chem. Soc. 2021, 143, 9206–9214. Copyright 2021 American Chemical Society.

2.15.4

Magnetic particle imaging (MPI)

2.15.4.1

Magnetic particle imaging

One of the newer entries into the field of imaging is magnetic particle imaging (MPI) that has been making significant progress since its inception in 2001. First introduced by Gliech and coworkers, MPI relies on the magnetization relaxation of superparamagnetic nanomaterials, typically iron oxides.130 Superparamagnetism is the minimization of individual magnetic domains to individual nanoparticles or molecules. Superparamagnetic materials also have a small barrier to magnetic relaxation that is measured using a small alternating-drive field to induce spin flips.131 The deviation between the drive field and the magnetization is described as the component of the superparamagnetic spin vector that is orthogonal to the drive field (also called the imaginary or out-ofphase susceptibility, c00 ). This barrier to relaxation is used to quantify superparamagnetic materials by mass.132 In MPI, strong magnetic-field gradients are swept across samples generating what is known as “field-free zones” as the spins of superparamagnetic nanomaterials are inverted (Fig. 25). The field-free zone enables spontaneous oscillation of magnetic spin and generates an electronic signal proportionate to the mass of the superparamagnetic species.130 These electronic signals are used to produce images. As with any imaging technique, MPI has unique advantages and limitations. For example, MPI offers facile quantification, excellent sensitivity, and little to no background signal. However, spatial resolution depends on uniformity of tracer size, and MPI does not offer the ability to display anatomical information.133 Still, due to sensitive and fast acquisition, MPI has found niches in imaging of blood flow, internal hemorrhaging, and cell tracking.134–143 The workhorse for MPI tracers has been superparamagnetic iron-oxide nanoparticles (SPIONs), including a clinically approved SPION, Resovist.133 A large body of research describes the optimization of SPIONs for MPI, typically via variation of core size. In essence, core size must be paired with a particular frequency of the drive field in the field-free zone for optimal signal.144–148

Fig. 24 Images of mice with radiance from the ZnPc analog stimulated by 90YCl3 both with and without 710 nm filters. Reprinted with permission from Lioret, V.; Bellaye, P.-S.; Arnould, C.; Collin, B.; Decréau, R. A. Dual Cherenkov Radiation-Induced Near-Infrared Luminescence Imaging and Photodynamic Therapy Toward Tumor Resection. J. Med. Chem. 2020, 63, 9446–9456. Copyright 2020 American Chemical Society.

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Fig. 25 Depiction of MPI scanning across spins of a superparamagnetic species. Reprinted with permission from Springer Nature: Springer, Cham from Magnetic Particle Imaging by Bo Zheng, Kuan Lu, Justin J. Konkle, Daniel W. Hensley, Paul Keselman, Ryan D. Orendorff, Zhi Wei Tay, Elaine Yu, Xinyi Y. Zhou, Mindy Bishop, Beliz Gunel, Laura Taylor, R. Matthew Ferguson, Amit P. Khandhar, Scott J. Kemp, Kannan M. Krishnan, Patrick W. Goodwill, and Steven M. Conolly in Design and Applications of Nanoparticles in Biomedical Imaging; Bulte, J. W. M., Modo, M. M. J., Eds. Copyright Springer International Publishing Switzerland 2017.

Additionally, focus has been placed on the effect of nanoparticle composition and coating to improve MPI tracers from a chemical perspective and is described in this section.

2.15.4.2

Iron-cobalt nanoparticles for MPI

Although SPIONs function as MPI agents, some research groups have expanded the field of MPI by exploring materials that are a composite of metal ion species, specifically iron and cobalt. The possibility of using CoFe2O4 rather than Fe3O4 was first suggested by Viereck and coworkers in 2019 as a preferable material for determining the viscosity dependence of MPI signal.149 Their investigation of solutions of water with increasing concentrations of glycerol revealed a notable change in the frequency-dependence of alternating-current susceptibility (c“) for CoFe2O4 nanoparticles capped with citrate (Fig. 26), where greater viscosity induced c“ peak maxima at smaller frequencies of drive-field oscillation. This trend in c“ maxima correlated with expanded magnetic hysteresis loops and suppression of magnetization signal intensity (Fig. 27). However, these results were not the only attractive properties of a mixed-metal nanoparticle. Another rationale for new MPI agents was presented in 2020 by Rao and coworkers, who reported graphite-coated FeCo nanoparticles with which larger magnetic saturation values were observed for FeCo nanoparticles (215 emu g 1) compared to Fe3O4 nanoparticles (21–80 emu g 1).150 The larger values were expected to result in enhanced MPI signal. Their investigation explored metal composition and nanoparticle core sizes with respect to changes in MPI signal intensity. First, the Fe/Co ratio of the alloy was varied to 2:1, 1:1, and 1:2. One mg of nanoparticle was normalized to the signal of 1 mg of VivoTrax (a commercially available MPI tracer), and the ratio of 1:1 resulted in the strongest relative signal that was 2.4-fold more intense than the signal from VivoTrax. They also systematically adjusted core size to average diameters of 5, 7, 10, 12, and 16 nm. Of these five core sizes, the 10 nm nanoparticles performed the best with 3.5-fold signal enhancement compared to VivoTrax, and 8.6-fold enhancement compared to Feraheme (a clinically approved MRI contrast agent, Fig. 28). Not only was the MPI signal enhanced, but the FeCo@C nanoparticles showed improvement in r2 over Feraheme. The gravimetric transverse relaxivity of both nanoparticles was measured at 1.5 T and 37  C where FeCo@C nanoparticles had r2 ¼ 11.74 mg 1 s 1 L and Feraheme had r2 ¼ 1.15 mg 1 s 1 L. These results suggest that FeCo-alloy nanoparticles have potential not only as MPI tracers but also as contrast agents for MRI.

2.15.4.3

Variation on nanoparticle coatings and construction in MPI

Another branch for rational chemical alteration of SPIONs in MPI focuses on alteration of the coatings around nanoparticles. In some cases, these alterations attempt to target specific cells, such as brain glioma cells as reported by Krishnan and coworkers.151 Their investigation involved lactoferrin coating on SPIONs and C6 gliomal cells to observe targeted uptake of MPI tracers. Their study used optical microscopy to examine uptake into the C6 cells (Fig. 29). First, Prussian blue was used to stain the SPIONs

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Fig. 26 Measured AC susceptibility where the in-phase susceptibilities (c0 ) are represented as dashed lines and out-of-phase susceptibilities (c00 ) are represented as solid shows c00 peak maxima at decreasing frequency with increasing viscosity. Reprinted with permission from Draack, S.; Lucht, N.; Remmer, H.; Martens, M.; Fischer, B.; Schilling, M.; Ludwig, F.; Viereck, T. Multiparametric Magnetic Particle Spectroscopy of CoFe2O4 Nanoparticles in Viscous Media. J. Phys. Chem. C 2019, 123, 6787–6801. Copyright 2019 American Chemical Society.

in the cells, and nanoparticles with lactoferrin conjugation showed more staining compared to unconjugated nanoparticles after 24 h of incubation. To be sure the nanoparticles migrated into the cells, the near-IR dye Cy5.5 was affixed to the lactoferrinlabeled nanoparticles that were incubated with C6 cells for 24 h. Confocal microscopy showed signal inside the cells, confirming uptake of the SPIONs. MPI of C6 cells with the lactoferrin-coated nanoparticles after 2, 6, 12, and 24 h was compared to MPI of C6 cells with unlabeled nanoparticles, and enhancement was observed in the case of the lactoferrin-coated nanoparticles (Fig. 30). In addition, alteration of nanoparticle coating has also been used to ameliorate rapid clearance of SPIONs. Krishnan, Khandhar, and coworkers demonstrated use of a polyethylene glycol (PEG)-coated SPIONs for this reason (Fig. 31).152 Their investigation revealed two key findings, one was the relation of colloidal stability to PEG loading; the other was change in blood half-life (t1/ 2) with respect to PEG molecular mass and loading. For colloidal stability, PEG-coated SPIONs with PEG molecules with molecular masses of 5, 10, and 20 kDa were monitored over 7 days by measurement of hydrodynamic radius using dynamic light scattering in HCl and KH2PO4 solutions with pH values of approximately 1.2 and 4.3, respectively, at room temperature. These pH values were selected to accelerate decomposition into reasonable timeframes for experimentation. As the molecular mass of the PEG increased, the hydrodynamic radius changed more slowly over time, indicating less aggregation and greater colloidal stability in aqueous KH2PO4. (Fig. 32) However, the t1/2 values followed the opposite trend, and as the molecular mass of PEG increased, the SPIONs were cleared more rapidly: 155 min for 5 kDa PEG and 58 min for 20 kDa PEG. To develop PEG-coated SPIONs that are both stable and cleared slowly, the loading of 20 kDa PEG was reduced from the initial value of 25% to values of 18.8 and 12.5%. The particles with 12.5% PEG loading cleared rapidly (t1/2 ¼ 26 min) compared to the particles with 25% loading, but the particles with 18.8% loading cleared less rapidly with a t1/2 ¼ 105 min. In addition, the particles with 18.8% 20 kDa loading were stable over the course of 7 days. As an end result, the investigation revealed a strategy for optimizing SPION MPI agents to be more stable and cleared more slowly over time than current clinical agents such as Resovist.

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Fig. 27 Measured magnitude spectra (left) and dynamic M(H) curves (right) of the viscosity series show expanded hysteresis and more less suppression of signal for a 500 Hz field (top) vs a 10 kHz field (bottom). Reprinted with permission from Draack, S.; Lucht, N.; Remmer, H.; Martens, M.; Fischer, B.; Schilling, M.; Ludwig, F.; Viereck, T. Multiparametric Magnetic Particle Spectroscopy of CoFe2O4 Nanoparticles in Viscous Media. J. Phys. Chem. C 2019, 123, 6787–6801. Copyright 2019 American Chemical Society.

MPI performance is impacted by SPION core size, but nanoparticle coating is also an important factor. Groll and coworkers adorned SPIONs with Pluronics F127 (PF127), as well as an acrylate-functionalized PF127 (PF127DA).153 These functionalized PF127DA SPIONs were adorned with polyglycidol that was linked via a Michael addition between the acrylate and the thiol PF127DAPG. Micelles of this coating are known to swell or shrink with regard to temperature, so preservation of this property was examined for the SPION. Incubation of PF127DAPG-coated SPIONs from 4 to 25  C showed an overall decrease in hydrodynamic radius of about twofold. For MPI, imaging with PF127DAPG-coated SPIONs showed an increase in signal-to-noise ratio compared to PF127-coated SPIONs as well as enhanced spatial resolution (Fig. 33). Another investigation by Nair and coworkers led to SPION cores coated with gold shells.154 Direct comparison of SPIONs to gold-coated SPIONs showed an increase in MPI signal (Fig. 34), likely due to a change in anisotropy from the gold coating. Furthermore, compositional adjustments have been applied such that MPI tracers can act as fluorescent dyes. Rao and coworkers developed a system in which SPIONs were embedded into a semiconducting polymer (PFODBT) such that only one hemisphere of the SPION was exposed.155 These amalgamations were termed Janus nanoparticles, and they displayed properties of both the PFODBT and SPION components. The absorbance profile was a combination of both components and there was an emission

Fig. 28 FeCo nanoparticles show enhanced MPI signal compared to Feraheme and Vivotrax. Reprinted with permission from Springer Nature: Elsevier from Song, G.; Kenney, M.; Chen, Y. S.; Zheng, X.; Deng, Y.; Chen, Z.; Wang, S. X.; Gambhir, S. S.; Dai, H.; Rao, J. Carbon-Coated FeCo Nanoparticles as Sensitive Magnetic-Particle-Imaging Tracers with Photothermal and Magnetothermal Properties. Nat. Biomed. Eng. 2020, 4, 325–334, Copyright The Author(s), under exclusive licence to Springer Nature Limited 2020.

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Fig. 29 (A) Prussian blue staining shows greater uptake for Lf-IONPs compared to PMAO-IONPs. (B) Confocal images without nanoparticles and with Cy5.5 labeled Lf-IONPs (white arrow). Red spots represent the Lf-IONPs labeled by Cy5.5. Republished with permission from The Royal Society of Chemistry from Lactoferrin Conjugated Iron Oxide Nanoparticles for Targeting Brain Glioma Cells in Magnetic Particle Imaging by Asahi Tomitaka, Hamed Arami, Sonu Gandhi and Kannan M. Krishnan. Nanoscale 2015, 7, 16890–16898. Copyright 2015. Permission conveyed through Copyright Clearance Center.

centered at 680 nm from an excitation of 540 nm, which are within useful ranges for in vivo fluorescence (Fig. 35). In addition, the Janus nanoparticles displayed equivalent MPI signal compared to the non-Janus SPIONs (Fig. 35). The authors also explored the limits of sensitivity of MPI by imaging as few as 250 HeLa cells with distinguishable signal-to-noise ratios. In contrast, T2-weighted MRI and fluorescence imaging could only show distinguishable signal with 2500 cells with the same loading of the Janus nanoparticle tracer.

2.15.4.4

Conclusions

Magnetic particle imaging has made great strides over the past 20 years, and is useful for quantitative imaging to count cells or measure bloodflow. SPIONs have dominated this field and a clear relationship between core size and oscillation frequency has been elucidated. The chemistry interface of this technique has been showing potential to provide more stable tracers with enhanced signal-to-noise ratios by altering the nanoparticle composition, coating with surfactants, or both. These improvements demonstrate the growing potential of MPI for biomedically relevant imaging.

Fig. 30 MPS signals and their normalized data (inset) (A) incubation with PMAO-IONPs for 2, 6, 12, and 24 h, and (B) incubation with Lf-IONPs for 2, 6, 12, and 24 h. Republished with permission from The Royal Society of Chemistry from Lactoferrin Conjugated Iron Oxide Nanoparticles for Targeting Brain Glioma Cells in Magnetic Particle Imaging by Asahi Tomitaka, Hamed Arami, Sonu Gandhi and Kannan M. Krishnan. Nanoscale 2015, 7, 16890–16898. Copyright 2015. Permission conveyed through Copyright Clearance Center.

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Fig. 31 Schematic depicting SPIONs coated with PMAO-PEG polymer. Republished with permission from The Royal Society of Chemistry from Evaluation of PEG-Coated Iron Oxide Nanoparticles as Blood Pool Tracers for Preclinical Magnetic Particle Imaging by A. P. Khandhar, P. Keselman, S. J. Kemp, R. M. Ferguson, P. W. Goodwill, S. M. Conolly, and K. M. Krishnan. Nanoscale 2017, 9, 1299–1306. Copyright 2017. Permission conveyed through Copyright Clearance Center.

2.15.5

Ultrasound and photoacoustic imaging

2.15.5.1

Imaging with sound waves

There are two main imaging modalities that involve the use of soundwaves to noninvasively render images: ultrasound imaging and photoacoustic imaging (also known as multispectral optoacoustic tomography). Although the two modalities differ in stages of development and specific modes of activity, both techniques involve similar instrumentation due to their both involving image construction from sound waves. In this section, the inorganic contributions and directions of both distinct fields are described using general and select examples. For more exhaustive reviews of each modality, please refer to the Further Reading list.

2.15.5.2

Ultrasound imaging

Ultrasound imaging is a commonly used, fast, noninvasive modality that characterizes anatomical structures, including the measuring of distances, volumes, and areas.156–159 In medicine, ultrasound imaging is commonly used in gynecology; soft tissue imaging of the heart, tendons, and muscle; and imaging of the gastrointestinal system.160–164 Reasons for the success of this imaging modality stem from the portability of the instrumentation, cost-effectiveness, and relative safety compared to imaging modalities that require the use of ionizing radiation. Ultrasound imaging uses ultrasound transducers that send pulses of high frequency sound (commonly 3–20 MHz) into a medium.165–167 When ultrasound pulses strike objects that reflect ultrasound waves back to the transducer, the amplitudes of the echoes are translated to pixel intensities on the ultrasound image. For example, bones and other tissue with large concentrations of calcium appear bright, or hyperechoic.168 If an ultrasound wave passes through an object without producing a reflection, it is called anechoic and appears dark in an ultrasound image. Fluids, like those found in the bladder,

Fig. 32 Hydrodynamic radius (dH) and saturation magnetic moment (m) describe the colloidal stability in KH2PO4 solution (pH z 4.3), (a) and (b), and HCl with NaCl (pH z 1.2), (c) and (d), respectively. Republished with permission from The Royal Society of Chemistry from Evaluation of PEGCoated Iron Oxide Nanoparticles as Blood Pool Tracers for Preclinical Magnetic Particle Imaging by A. P. Khandhar, P. Keselman, S. J. Kemp, R. M. Ferguson, P. W. Goodwill, S. M. Conolly, and K. M. Krishnan. Nanoscale 2017, 9, 1299–1306. Copyright 2017. Permission conveyed through Copyright Clearance Center.

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Fig. 33 SNR (left) and spatial resolution (right) improve for PF127DAPG-coated SPIONs compared to PF127-coated SPIONs.153 Reprinted from Open Access Reference Horvat, S.; Vogel, P.; Kampf, T.; Brandl, A.; Alshamsan, A.; Alhadlaq, H. A.; Ahamed, M.; Albrecht, K.; Behr, V. C.; Beilhack, A.; Groll, J., Crosslinked Coating Improves the Signal-to-Noise Ratio of Iron Oxide Nanoparticles in Magnetic Particle Imaging (MPI). ChemNanoMat 2020, 6 (5), 755–758.

commonly appear anechoic.168 The usefulness of ultrasound imaging is heavily influenced by the ability of sound waves to penetrate tissue, leading to a reduction of resolution in deeper tissue due to the inverse relationship between wavelength and frequency. Faster frequency sound waves lead to higher resolution because they enable narrower beams, but faster frequency sound waves also do not penetrate tissue as deeply: a 1-MHz ultrasound wave penetrates 2.3 to 5 cm into tissue, and a 3-MHz wave penetrates  1.6 cm into tissue.169 Resolution in ultrasound imaging can be improved with contrast agents, enabling visualization of features at depths that would be otherwise be too low of resolution to be useful without contrast agents. Contrast agents enhance ultrasound backscatter, or the reflection of signals back to the transducer, thus enabling better visualization of objects that would be difficult to detect clearly using ultrasound imaging without contrast agents.165 Contrast agents for ultrasound imaging display both resonance and harmonic oscillation; whereas, biological tissues only oscillate at resonance frequencies.165 This phenomenon enables differentiation between biological tissue containing contrast agents, such as within the vasculature, and the surrounding areas. In the rational design of contrast agents, the properties of materials alter the extent of contrast enhancement, and these properties include density, compressibility, size, and specific chemical materials.165,170–172 Contrast agents for ultrasound imaging are largely biocolloids. The contrast-enhancing ability of gaseous microbubbles was serendipitously discovered in 1968, when researchers realized an injection via a small-bore needle or catheter leads to saline bubbles that inherently display a contrast effect during echocardiography.173,174 This discovery cumulated in a plethora of research towards stabilizing microbubbles with surfactants and thin shells to overcome limitations such as short lifetimes and inhomogeneous sizes to maximize the utility of ultrasound imaging.175–179 For example, perfluorocarbons largely replaced traditional air

Fig. 34 (a) MPI images and (b) MPI signals of the SPION (MNPs) and gold-plated SPION (MNP@Au) show enhanced signal with gold plating. Republished with permission from The Royal Society of Chemistry from Dynamic Magnetic Characterization and Magnetic Particle Imaging Enhancement of Magnetic-Gold Core–Shell Nanoparticles by Asahi Tomitaka, Satoshi Ota, Kizuku Nishimoto, Hamed Arami, Yasushi Takemura, and Madhavan Nair. Nanoscale 2019, 11, 6489–6496. Copyright 2019. Permission conveyed through Copyright Clearance Center.

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Fig. 35 UV–vis absorption spectra of PFODBT in THF, and Fe3O4–COOH and Fe3O4@PFODBT-COOH in PBS (left), fluorescence spectrum of Fe3O4@PFODBT-COOH (lex ¼ 540 nm, center) two-dimensional projection MPI scanning of Fe3O4–COOH and Fe3O4@PFODBT-COOH with the same amount of iron (right). Reprinted with permission from Song, G.; Chen, M.; Zhang, Y.; Cui, L.; Qu, H.; Zheng, X.; Wintermark, M.; Liu, Z.; Rao, J. Janus Iron Oxides @ Semiconducting Polymer Nanoparticle Tracer for Cell Tracking by Magnetic Particle Imaging. Nano Lett. 2018, 18, 182–189. Copyright 2018 American Chemical Society.

microbubbles due to the lack of interactions with both hydrophobic and hydrophilic molecules.180,181 Over the last several decades, many microbubble formulations received clinical approval, including four microbubbles currently used in clinical practice that use different gases (octafluropropane, perfluorobutane, and sulfur hexafluoride) and shell structures.170 The most common shell structures are composed of phospholipids, cross-linked proteins, and monomolecular membranes. However, there have been numerous studies using inorganic silica carriers.182–184 Additionally, iron-oxide-coated microbubbles have been studied for dual-purpose contrast agents.185 Because the iron-oxide coating fragments with violent oscillations, these contrast agents enhance contrast at low pressure and deliver therapeutic agents upon bursting at high pressures.185 Gold nanoshell microcapsules have also been studied for theranostic applications using ultrasound because gold is biocompatible and offers unique properties useful in other modalities such as photothermal ablation.186–188 Inorganic chemistry also has been studied for ultrasound drug delivery relying on CuII-ligand interactions in metallo-supramolecular block copolymer micelles that break when agitated with high-oscillation sound waves, but these methodologies are not used for imaging purposes.189 Although dominated by research into organic microbubbles and nanobubbles, other formulations being researched include liposomes, nanodroplets, and solid nanomaterials.190–193 There is a significant difference between the acoustic impedance variations of solid nanoparticles and biological material that leads to contrast enhancement from nanoparticles relative to physiological media in ultrasound experiments.194 The use of solid nanomaterials for ultrasound enables the enhanced permeability and retention (EPR) effect to enhance contrast in tumors.195 The tumor uptake of nanoparticles and longer retention times of solid nanoparticles juxtaposes with microbubbles that terminate quickly and are limited to the vasculature. Additionally, researchers can tailor the structure of the material to enhance contrast in ultrasound by maximizing reflective capabilities of the material for soundwaves. For example, Gigli and coworkers showed that the size of spherical silica nanoparticles was found to be linearly positively correlated to the level of contrast enhancement in ultrasound when examining nanoparticles of different diameters (160, 330, and 660 nm).194 However, it is important to note that specific biological applications impose size limitations, depending on the designed use of the nanoparticle. Another example demonstrating the importance of structure on ultrasound contrast enhancement is the design and use of various mesoporous and nanoporous nanostructures.182,196,197 For example, Shi and coworkers examined a rattle-type mesoporous silica nanomaterial that was studied specifically due to the presence of structural interfaces that lead to double-scattering and reflection and, in turn, higher intensity in contrast enhancement compared to other silica nanostructures that do not possess multiple interfaces.182 Hybrid systems also have been studied that involve solid silica cores and porous silica shells: for example, a microparticle of this type involving cross-linked pH-sensitive thiol-functionalized multilayered polymer film between the two silica layers was studied for pH responsive ultrasound imaging due to the swelling and shrinking of the polymer layer at different pH values resulting in a changing of the number of silica interfaces interacting with sound waves.198 The pH effect of the particle was studied at pH 5, 6, and 7 with a two-fold increase in contrast enhancement described with each changing pH value, with largest enhancement being observed at the lowest pH value. Porous nanoparticles also can be functionalized to impart new characteristics such as the addition of temperature-dependent phospholipids to the exterior that melt at temperatures indicative of necrosis, triggering a change in contrast enhancement.199 Another example from Caruso and coworkers of peripheral functionalization involves a targeted system using nanoporous metal-phenolic particles that include FeIII and phenolic-ligand chelates that target H2O2 by catalytically converting H2O2 to oxygen at biologically relevant pH values (Fig. 36).200 Notably, the contrast enhancement of this agent stems from the formation of gaseous oxygen microbubbles in vivo. It is important to note that a large portion of research into solid silica nanoparticle for ultrasound contrast enhancement requires high frequencies, limiting usefulness to specific physiological targets.

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Fig. 36 Cartoon representation of (a) the synthetic procedure for the FeIII-phenolic nanoporous (np) replica particles (RPs) through replication of npCaCO3 template particles and sequential loading of FeIII-tannic acid (TA) complexes and (b) the catalytic use of FeIII-phenolic nanoporous particles via splitting of H2O2 into H2O and O2 microbubbles for ultrasound imaging. Reprinted with permission from Guo, J.; Wang, X.; Henstridge, D. C.; Richardson, J. J.; Cui, J.; Sharma, A.; Febbraio, M. A.; Peter, K.; de Haan, J. B.; Hagemeyer, C. E.; Caruso, F. Nanoporous Metal–Phenolic Particles as Ultrasound Imaging Probes for Hydrogen Peroxide. Adv. Healthcare Mater. 2015, 4, 2170–2175. Copyright 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2.15.5.3

Photoacoustic imaging

Photoacoustic imaging, also known as optoacoustic tomography, uses laser-induced sound waves to produce images. Photoacoustic imaging involves the use of nanosecond pulsed lasers to induce local heating of molecules that result in rapid expansions and compressions that generate oscillating pressures similar to sound waves.201–205 Ultrasound transducers detect these pressure changes to generate images upon digital reconstruction. Because photoacoustic imaging involves similar instrumentation as ultrasound imaging, photoacoustic imaging shares some of the advantages of ultrasound imaging, including a lack of ionizing radiation and portability. Another benefit of photoacoustic imaging is that the wavelength of incident light is tuned to the specific molecule or macromolecule being imaged. Because of this tunability, agents can be selected that do not have overlapping absorption spectra with other molecules within the physiological area being examined. Ideal agents for photoacoustic imaging involve molecules that excite in the low-energy visible or near-IR range because of the tissue penetration at these wavelengths: for example, light within the near-IR window penetrates several centimeters of tissue.206–208 Optical agents that excite in the far-IR or microwave range of light have also been studied in thermacoustic imaging with good tissue penetration and diverse targets and resulting translations into the study of different pathologies.209–211 There are several naturally occurring absorbers that can be studied for photoacoustic imaging including hemoglobin, melanin, lipids, and water.208,212–217 The distinct geometric and electronic change in hemoglobin upon oxidation leads to a shift in absorption and, therefore, can be used to track oxygenation in tissue to image disease states such as ischemia or hypoxia.218,219 For example, photoacoustic imaging at 750 nm and 850 nm has been used to study diethylnitrosamineinduced hepatic fibrosis in rats to quantify oxygenated and deoxygenated hemoglobin and, sequentially, oxygen saturation and overall hemoglobin concentration.220 Similar photoacoustic imaging studies have been performed on a variety of cancers that display altered hemoglobin concentrations, including breast, prostate, and ovarian cancer.208,221–224 A study of 49 ovarian masses from 33 patients used hemoglobin monitoring via photoacoustic imaging to calculate percent oxygen maps in combination with the diagnostic scores from radiologists to improve accuracy of diagnosis to an area under the curve value of 0.93.208 However, the imaging depths with the current instrumentation do not enable full imaging (< 95%) of all of the ovaries in this study. Additionally, acoustic spectrum compensations recently were reported for the photoacoustic imaging of hemoglobin in efforts to lower discrepancies in data collection.225 To use photoacoustic imaging to probe specific physiological conditions, exogenous contrast agents have been pursued.203,204,206 There are many critical properties to consider in the design of contrast agents for photoacoustic imaging, including high molar extinction coefficients; low energy (commonly near-IR) absorption profiles distinct from surrounding biological media; stability against photobleaching; low quantum yields to maximize nonradiative decay; low toxicity; water solubility; and desirable circulation times and excretion pathways.206 Additionally, interactions within the bloodstream, and not just the eventual target, must be considered when designing any agent that must surpass clinical approval. Recent contrast agents incorporate targeting properties to localize in specific regions of medical interest, including the addition of small molecules and monoclonal antibodies.226–228 For example, gold nanostars (hydration radii of  87 nm) were tagged with monoclonal antibody CD147 and studied for photothermal therapy and real-time monitoring via photoacoustic therapy.229 CD147 is overexpressed on cancerous

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liver cell membranes and is known to regulate growth and proliferation. Addition of CD147 antibodies to the gold nanostars (hydration radii of  102.5 nm) led to accumulation of the tagged nanostars in the tumors of mice after 6 h, as shown by photoacoustic imaging with 808 nm irradiation.229 It is important to note that the addition of larger proteins to the periphery of contrast agents leads to larger overall sizes that alter biodistribution and circulation times. Consequently, antibody fragments have been studied in combination with contrast agents for photoacoustic imaging to control localization and biocompatibility.206,230,231 Small-molecule dyes for photoacoustic imaging are predominantly organic and include large aromatic systems and or conjugated olefins.232,233 Because some commercially available organic dyes are FDA-approved for other medical applications, the safety of these agents at certain concentrations is already known. For example, indocyanine green, which is used clinically for diagnostic applications such as fluorescent angiography in ophthalmology, absorbs at 780 nm and has a low quantum yield (0.027 nm), making it an ideal candidate for photoacoustic imaging.234 Importantly, the modification of small-molecule dyes can be challenging to overcome constraints such as hydrophobicity, low molar extinction coefficients, and photobleaching.206 One such modification involves the incorporation of small-molecule dyes into nanoparticles.235–237 Nanoparticulate photoacoustic agents maximize the payload of contrast-enhancing moieties per agent and are chemically versatile. For nanoparticle formulations, size and shape dictate the optical-to-acoustic conversion efficiency that relates to the number of incident photons absorbed and converted and the speed of that resulting wave generation.209,238 Several types of nanoparticles have been studied including iron oxides, carbon nanotubes, polymer nanoparticles, and gold nanoparticles.239–242 In addition to being biologically inert and nontoxic, gold also possesses unique electronic properties that make it suitable for photoacoustic contrast enhancement and other optical techniques.243 Specifically, gold displays localized surface plasmon resonance that leads to high molar extinction coefficients due to the presence of a plasmon on the surface of gold particles.209,244 A plasmon is a pooled oscillation of free electrons that can be described as a displaced, negatively charged electron cloud within the Fermi liquid model.245 Excitation via irradiation at the proper resonance frequency creates, in essence, a large dipole parallel to the electric field of the incident wave: the large number of displaced electrons after excitation results in a positive lattice, and the positive lattice pulls the electrons back to the lattice.245 Localized surface plasmon resonances are tuned via morphological adjustments including size and shape. Examples of gold nanoparticle formulations studied for photoacoustic imaging include spheres, rods, shells, cages, clusters, and stars.242,246–251 The specific structures of gold nanomaterials change the strategies necessary for optimizing properties for photoacoustic imaging. For example, gold nanospheres absorb at  520 nm, which is unideal for biological use because it coincides with the absorbance of blood. Altering the size of gold nanospheres only shifts absorbance  50 nm, but synthesis of gold nanovesicles via polymer-graft-regulated ordered assembly shifts absorbance to biologically relevant values (650–800 nm).242,252,253 For gold nanorods, the level of absorbed irradiation versus scattered irradiation is a function of size; however, the wavelength of absorbance is dependent on aspect ratio.238,254 Therefore, both size and aspect ratio are critical in the design and utility of agents of this type. Additionally, for use in biological media, photothermal stability must be carefully considered because some gold

Fig. 37 (left) Pictures of mice with 1 cm prostate-cancer tumors, indicated in red boxes, and (right) photoacoustic color maps overlaid on ultrasound images; photoacoustic imaging used 1064 nm irradiation and was completed 24 h after tail vein injection with (top) large gastrin-releasing peptide receptor tagged (GRPR) gold nanorods (AuNRs) and (bottom) small GRPR AuNRs. The scanning volume for both images is 23 mm (x)  19 mm (y)  16 mm (z). Reprinted with permission from Springer Nature: Elsevier from Chen, Y.-S.; Zhao, Y.; Yoon, S. J.; Gambhir, S. S.; Emelianov, S. Miniature Gold Nanorods for Photoacoustic Molecular Imaging in the Second Near-Infrared Optical Window. Nat. Nanotechnol. 2019, 14, 465–472, Copyright The Author(s), under exclusive licence to Springer Nature Limited 2019.

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nanostructures melt upon the introduction of pulsed near-IR radiation.255 To this end, miniature ( 8 by 49 nm) gold nanorods that absorb in the near-IR window were studied by Emelianov and coworkers and found to generate 3.5 times the contrast enhancement in photoacoustic imaging compared to similar larger ( 18 by 120 nm) nanorod configurations, while being three times more thermally stable.256 The gastrin-releasing peptide receptor tagged form of miniature gold nanorods was studied in tumor-bearing mice in comparison to the larger configurations. The tagged nanorods displayed brighter photoacoustic signal (Fig. 37) and were not associated with a decrease in signal upon experimental completion, like larger nanorods.256

2.15.5.4

Conclusions

Advances in the imaging techniques available using soundwaves is an exciting frontier in modern medicine, and inorganic contributions hold important roles in the design and application of contrast enhancement for both ultrasound imaging and photoacoustic imaging. The design of future contrast agents requires balancing the properties that lead to bright signals with the properties necessary for biocompatibility and minimization of toxicity. Additionally, imaging agents must aim to fill specific physiological functions to prompt investment into new imaging modalities over currently successful techniques. Because photoacoustic imaging is a relatively new imaging modality, technological advances in instrumentation are a limiting factor in clinical implementation. However, advances are being made to improve instrumentation, such as with optical-resolution photoacoustic endoscopes, to offset high spatial resolution with clinically necessary imaging speeds.222,257 These advances in instrumentation coupled with new inorganic contrast agents are likely to greatly advance the utility of photoacoustic imaging.

2.15.6

Magnetic resonance imaging (MRI)

Magnetic resonance imaging (MRI) is a noninvasive, widely used technique in medical diagnostic imaging and preclinical biomedical research. MRI generates images using pulsed radiofrequency radiation (nonionizing) in magnetic fields. MRI is essentially a spectroscopy experiment: radiofrequency pulses are absorbed by nuclear spins that are first oriented in a magnetic field, and upon stopping of the applied pulse, the nuclear spins reorient with the magnetic field (relaxation), releasing radiofrequency pulses that are detected. Three-dimensional images are produced using gradients of the magnetic field. Much information is learned from MRI using only radiofrequency pulses and magnetic fields, but additional contrast enhancement in MRI can be achieved by altering the relaxation rate of the nuclear spins using unpaired electronsdoften associated with inorganic complexes or nanoparticlesdthat tend to be effective at changing relaxation rates. Consequently, MR images are often enhanced using paramagnetic inorganic complexes as contrast agents. This section is not meant to be an exhaustive survey of the area, but instead it is meant to be an introduction to the different areas including select examples. For more exhaustive reports readers are referred to the Further Reading Section.

2.15.6.1

Contrast agents

MRI often involves the nuclei of water protons, and greyscale images that are acquired by weighting longitudinal (T1) or transverse (T2) relaxation rates or proton density using different sequences of radiofrequency pulses.258 Clinically approved contrast agents influence these relaxation rates. Contrast agents for MRI have been used clinically since 1988 with approximately 30 million procedures per year performed across the world,259 and in the United States, an estimated 30% of all MRI procedures use contrast agents.260 Over the past few decades, other uses of MRI have emerged that involve imaging protons that are not on water (magnetic resonance spectroscopy), exchanging with water protons (chemical exchange saturation transfer), or paramagnetically shifted (PARASHIFT); nuclei other than protons; and hyperpolarized nuclei.261,262 For each of these other MRI techniques, contrast agents or probes have been developed. This section focuses on contrast agents and probes for MRI that involve inorganic chemistry with an emphasis on cutting edge developments, including complexes with large relaxivity values, alternatives to gadolinium, and responsive imaging of protons and other nuclei.

2.15.6.2

GdIII-containing contrast agents and alternatives

The most common inorganic ion found in contrast agents for MRI is GdIII. The GdIII ion has seven unpaired electrons in a symmetric ground state that effectively relaxes the nuclear spins of protons. Clinically used contrast agents for MRI contain GdIII complexed by octadentate ligands (Fig. 38).263 Multidentate ligands are used because unchelated GdIII is toxic, and it is important that complexes of GdIII be kinetically inert over the course of imaging to avoid dissociation of GdIII, which is linked to nephrogenic systemic fibrosis.260,264 Nephrogenic systemic fibrosis is a potentially fatal disease that conventionally occurs in patients with late-stage renal failure. Because GdIII-containing contrast agents typically are excreted through the kidneys, it was discovered in 2006 that less kinetically inert clinically approved GdIII-containing agents, specifically containing linear chelates, were linked to the onset of nephrogenic systemic fibrosis in patients with renal failure, likely due to transmetallation of GdIII.265 This observation was alarming due to the popularity, at the time, of Omniscan and Magnevist (Fig. 38) that contain linear chelates. However, due to the critical importance of MRI contrast enhancement in the diagnosis of disease and because nephrogenic systemic fibrosis is only pertinent to patients suffering from renal disease, black-box warnings and pre-screenings for renal disease have enabled the use of these agents

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Fig. 38 GdIII-based clinically approved MRI contrast agents. Below each structure is the abbreviation, trade name, generic name, and ligand structure (macrocyclic or linear).

to continue in healthy patients with no proven adverse side effects. Moving forward, most preclinical studies of GdIII-containing agents focus on tuning the coordination chemistry of GdIII to favor kinetically inert complexes. To prevent toxicity, ligands are designed to promote kinetic inertness for Gd-based contrast agents. Gadolinium interacts electrostatically with ligands and prefers hard donor atoms, such as oxygen, over softer donors. Additionally, GdIII tends to form nine-coordinate complexes, so octadentate ligands leave one coordination site available for coordination to water. This innersphere water molecule is relaxed by the GdIII ion and also rapidly exchanges with other water molecules to make the relaxation of nuclear spins catalytic in GdIII. Innersphere relaxation contributes approximately 50–60% to the total relaxivity of most small-molecule GdIII-based contrast agents for MRI, with the rest of the enhancement arising from second-sphere and outer-sphere interactions.266 To maximize the contrast-enhancing properties of GdIII while minimizing toxicity, the relaxivity of contrast agents have been a focus of research. Relaxivity is a measure of the ability of a molecule to enhance contrast as a function of concentration. Contrast agents with larger relaxivity values enhance contrast more than the same concentration of contrast agents with smaller relaxivity values. Because of the inherently low sensitivity of MRI relative to other imaging modalities, grams of contrast agent are often needed to perform clinical imaging. Large relaxivity values enable both the use of small concentrations of contrast agents and also imaging of biologically important targets that are present in small concentrations. To produce contrast agents with large relaxivity values, ligands have been designed to tune the hydration number (q), rotational correlation time, and water residency time of

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Fig. 39

Structure of gadopiclenol.

GdIII-containing complexes because these parameters directly influence relaxivity.266 The inertness of new complexes in biologically relevant conditions is important to study with any new contrast agent. An example of a large-relaxivity contrast agent was recently reported by Fries and co-workers, gadopiclenol, also known as P03277 (Fig. 39).267 The T1 relaxivity of gadopiclenol was measured to be nearly 2.5 times larger than gadobutrol at clinically relevant field strengths in human plasma at 37  C (12.8 vs 5.2 mM 1 s 1, respectively, at 1.5 T and 11.6 vs 5.0 mM 1 s 1, respectively, at 3.0 T) and 1.8 times larger at larger field strengths (8.6 vs 4.7 mM 1 s 1, respectively, at 9.4 T).267 These differences in relaxivity were visualized in a rat tumors at 9.4 T. Gadopiclenol demonstrated similar contrast and pharmacokinetics to gadobutrol in these studies,267,268 with an inertness that exceeded several clinical contrast agents (dissociation half-life ¼ 20  3 days).268 The large relaxivity observed with gadopiclenol is due to its water hydration number, q ¼ 1.7, that is larger than the water hydration number of gadobutrol and other clinically approved contrast agents that all have a water hydration number of one.267,269 To achieve the increase in water hydration, and consequently relaxivity, gadopiclenol uses a heptadentate coordinating ligand. In addition, gadopiclenol is designed to have a three-dimensional structure with a larger hydrodynamic radius compared to other small-molecule GdIII-based contrast agents, and large radii are associated with slow molecular tumbling rates that contribute to large relaxivity values.267–269 Several other studies have focused on water coordination numbers of 2 or larger as a means to increase relaxivity, including GdIIIcontaining complexes with q ¼ 2 using heptadentate ligands including PCTA, AAZTA, CyPic3A, and aDO3A and hexadentate ligands such as TACN-3,2-HOPO to create complexes with q ¼ 3 (Fig. 40).270–274 Although incorporation of large water coordination numbers often results in large relaxivity values, this design path carries a risk of lability as well as a risk of anion binding to inhibit the coordination of water; therefore, the risks and benefits of the inner-hydration sphere must be balanced in contrast agent design.274,275 Similar to the effect of increasing hydrodynamic radius, other approaches to increase relaxivity focused on slowing the rotational correlation time to increase relaxivity. Rotational motion can be reduced by increasing the molecular weight of a contrast agent or through interactions of contrast agents with macromolecules, like proteins, peptides, or nanoparticles. Clinically approved contrast agents generally have molecular weights of approximately 550–900 Da leading to rotational correlation times of approximately 60– 120 ps at room temperature.258 For low molecular weight contrast agents, relaxivity is largely dominated by the effect of the fast rotation, particularly at high field strengths, rather than water exchange and electronic relaxation.276 Molecular tumbling can be slowed by increasing the molecular weight thereby increasing the relaxivity, but the rigidity of the link between the increased molecular weight and the contrast enhancing ion influences local rotations that diminish the effect of increased molecular weight.258,266 As a general trend, relaxivity increases with increasing molecular weights that are associated with longer rotational correlation times. For example, the GdIII complex MS-325 has a larger relaxivity than Gd-DTPA (6.6 and 4.0 mM 1 s 1, respectively at 0.47 T and 37  C) due to the larger molecular weight of MS-325.277–279 This difference is enhanced in human blood when MS-325 binds to the human serum albumin (HSA), increasing relaxivity to 48.9 mM 1 s 1.280 MS-325 noncovalently interacts with HSA to

Fig. 40 Ligand structures of PCTA, AAZTA, CyPic3A, aDO3A, and tacn(1-Me-3,2-hopo)3. Reprinted with permission from Wahsner, J.; Gale, E. M.; Rodríguez-Rodríguez, A.; Caravan, P. Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem. Rev. 2019, 119, 957–1057. Copyright 2019 American Chemical Society.

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slow molecular rotation,280 but macromolecular contrast agents for MRI also can be synthesized by covalent conjugation to macromolecules281 or embedding GdIII-based contrast agents in proteins like a GdP3W, gadolinium metallopeptide.282 Attaching multiple contrast agents together can increase relaxivity both by increasing the number of GdIII ions and molecular weight.281 The connectivity between GdIII ions also influences relaxivity. A linear connectivity of GdIII complexes increases molecular weight but allows relatively fast rotation from internal motion of linker, whereas connecting multiple GdIII complexes around a spherical carrier minimizes internal rotations to increase relaxivity.276 To further restrict rotation, rigid linkers can be used or GdIII can be placed at the barycenter of a molecule.276 A more recent example is the GdIII-containing protein-based contrast agent, ProCA32, that has a large relaxivity and can be functionalized with target-specific peptides.283–286 Recent studies by Yang and coworkers reported ProCA32 (1) with a peptide targeting prostate-specific membrane antigen (PSMA) to identify PSMA-positive tumors in mice284; (2) for use to visualize liver fibrosis targeted by a type I collagen specific peptide (Fig. 41)285; and (3) by targeting chemokine receptor 4 to reveal liver tumors (Fig. 41).286 Additionally, this approach has been employed by Straub and co-workers to measure the progression of Duchenne muscular dystrophy in mice.287 The MR probe in this study was EP-3533 that contains four GdIII-based complexes and a type I collagen targeting peptide.287 These studies adapt GdIII-based contrast agents to specific goals, enabling molecular imaging and providing a noninvasive, selective method of using the paramagnetic image enhancing abilities of GdIII to detect disease and characterize disease progression. Strategies to optimize the water residency times for contrast agents to improve relaxivity are not effective if the rotational correlation time is short.288 Therefore, adjusting this parameter is limited to slow tumbling complexes. Coordination chemistry can be used to tune water-exchange rates by modifying the geometry of the complex to favor square antiprismatic structures over twisted square antiprismatic structures, increasing steric bulk at the water-binding site, changing the number of pendant side arms and their chemical nature (for example, hydrophobicity or hydrogen-binding ability), or organizing the second hydration sphere.258,289 GdDOTA has a water residency time of 244 ns at 25  C and two derivatives of Gd-DOTA, Gd-DO3A-pyNox and Gd-DO3APABn (Fig. 42), have water residency times of 39.0 and 16.2 ns, respectively, and these relatively short times are the result of attaching groups to sterically inhibit the water exchange site.290,291 Although, GdIII-based contrast agents are prevalent in clinical practice, there are some limitations to their use.292 The relaxivity of III Gd -based contrast agents is small enough to require large concentrations (mid-mM to low mM) to enhance contrast in images, making it difficult to perform molecular imaging of targets that often are present at much lower concentrations. Furthermore, the relaxivity of GdIII-based contrast agents decreases with increasing magnetic field strength, exacerbating the issue.292 One strategy to address this limitation is through the use of nanoparticle-based GdIII-containing contrast agents that either embed GdIII into a nanostructure, such as nanoparticulate metal oxides like Gd2O3, or attach lanthanide complexes to existing nanoparticle scaffolds, like Gd-DOTA-functionalized silica nanoparticles.293 The use of nanoparticles enables tuning of composition, size, shape, the hydration number, rotational correlation time, and water residency time of the nanoparticle-based contrast agents.293 These nanoparticulate contrast agents often achieve large relaxivity values by combining multiple GdIII ions with slow tumbling.261 GdIII-based nanoparticles for MRI have been reported using gold, silicon, dendrimers, carbon nanotubes, fullerenes, polymers, liposomes, micelles, viral particles, and nanodiamonds. In addition, nanoparticulate contrast agents are often designed to be multimodal by incorporation of features that enhance contrast in other imaging modalities in addition to MRI.293–298 In addition to GdIII, paramagnetic MnII- and FeIII-containing contrast agents have been studied for MRI, and two MnII-based contrast agents have been clinically approved: manganese dipyridoxyl diphosphate (mangafodipir trisodium) and liposomal

Fig. 41 Model structure and development of ProCA32.collagen1 by engineering collagen type I targeting moiety (GGGKKWHCYTYFPHHYCVYG, red) at C-terminal of ProCA32 using a flexible hinge (green) and PEGylation.285 Reprinted from Open Access Reference Salarian, M.; Turaga, R. C.; Xue, S.; Nezafati, M.; Hekmatyar, K.; Qiao, J.; Zhang, Y.; Tan, S.; Ibhagui, O. Y.; Hai, Y.; Li, J.; Mukkavilli, R.; Sharma, M.; Mittal, P.; Min, X.; Keilholz, S.; Yu, L.; Qin, G.; Farris, A. B.; Liu, Z.-R.; Yang, J. J. Early Detection and Staging of Chronic Liver Diseases with a Protein MRI Contrast Agent. Nat. Commun. 2019, 10, 4777.

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Fig. 42

Chemical structures of (left) Gd-DO3A-pyNox and (right) Gd-DO3APABn.

encapsulated magnesium chloride (LumenHance). Both MnII and FeIII have half-filled d orbitals that promote nuclear relaxation similar to GdIII. MnII-containing complexes for MRI generally have coordination of six or seven including an exchangeable water ligand. Recently a MnII complex, Mn-PyC3A (Fig. 43), has been proposed as a potential alternative to GdIII-based contrast agents.299 PyC3A is a hexadentate ligand, and the Mn-PyC3A complex is 7-coordinate with one coordinated water, q ¼ 1.299 The inertness of Mn-PyC3A was tested using 25 mol equiv. of Zn at pH 6.0 and 37  C and determined to be more inert under those conditions than Gd-DTPA.299 Gale, Caravan, and co-workers studied Mn-PyC3A in vivo and determined that Mn-PyC3A (r1 ¼ 3.8 mM 1 s 1 at 1.4 T and 37  C in bovine plasma) enhanced contrast in a mouse breast cancer tumor similarly to Gd-DOTA (r1 ¼ 3.6 mM 1 s 1, respectively at 1.5 T and 37  C in bovine plasma) with better elimination.299,300 Subsequent tests have demonstrated that MnPyC3A is more efficiently eliminated than Gd-DOTA or manganese dipyridoxyl diphosphate.301

2.15.6.3

Iron oxide agents

Superparamagnetic iron oxide nanoparticles (SPIONs, often referred to as iron oxide nanoparticles, IONPs) enhance contrast in MRI, in addition to being the foundation of MPI. Superparamagnetism is described in Section 2.15.4 Magnetic Particle Imaging. Several acronyms are used for iron oxide nanoparticles and often these acronyms represent different variations of nanoparticles, so care should be taken when comparing reported studies. For example, micro-sized iron oxide particles (MPIOs), superparamagnetic iron oxides (SPIOs), ultrasmall superparamagnetic iron oxides (USPIOs), and monocrystalline iron oxide nanoparticles (MIONs) have diameters of > 1000, > 50, < 50, 10–30, and 7–9 nm, respectively. In the context of MRI, iron oxide nanoparticles shorten T1 and T2 relaxation times of water protons similar to GdIII-based contrast agents, but they tend to have a greater effect on T2, especially for larger nanoparticles. Superparamagnetic iron oxide nanoparticles are composed of an iron oxide core of either magnetite (Fe3O4) or maghemite (g-Fe2O3).302 The core is coated with dextran or a similar substance to provide protection and stabilization and to enable colloidal suspensions of the nanoparticles.302 Some of the beneficial features of superparamagnetic iron oxide nanoparticles include the ability to adapt the size and shape to change functionality, a large concentration of paramagnetic ions in each particle, low toxicity, biodegradability, biocompatibility, and the option for additional functionality by attaching molecules to the surface of the coatings. A challenge with iron oxide nanoparticles can be synthesis with uniform size and shape, but despite this challenge, many examples of iron oxide nanoparticles (as well as other types of paramagnetic nanoparticles) have been reported as contrast agents for MRI.261,292–298,303,304 Early first generation iron oxide nanoparticles used in MRI were relatively large in size with large polydispersities and large T2 relaxivities like the dextran-coated SPION Feridex (diameter 80–150 nm; r1 ¼ 40 mM 1 s 1; r2 ¼ 160 mM 1 s 1 at 0.47 T and 37  C).305,306 Several iron oxide nanoparticle-based contrast agents have been approved for clinical use.306 For iron oxide nanoparticles, the size of the core influences relaxation properties because magnetization decreases with decreasing core size. Large superparamagnetic iron oxide particles contrast agents are T2 MRI contrast agents and USPIO nanoparticles act as T1 MRI contrast agents. Decreasing particle size affects not only r1, but also r2/r1, leading to T1 enhancement with USPIO nanoparticles around 3.6 nm in diameter.307,308 As the ability to synthesis smaller particles developed, researchers have used USPIO nanoparticles as T1 MRI contrast agents as well as T2/T1 switchable contrast agents.308 Choi and Cheon reported a supramolecular amorphous-like iron oxide (SAIO) T1 MRI contrast agent with the ability to image blood vessels in a rat with better image enhancement compared to Gd-DOTA (Fig. 44).309 Iron oxide nanoparticles continue to be used a wide variety of applications that include imaging tumors,

Fig. 43

Structures of Mn-PyC3A and Mn-DPDP.

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Fig. 44 SAIO-enhanced MRI images of peripheral regions and whole-body images obtained with SAIO and Gd-DOTA. (a) MRI of rat feet scanned after injection of SAIO with inset showing small peripheral vessels with diameters of 100 mm. (b) SAIO-enhanced (left) and Gd-DOTA-enhanced (right) whole-body images demonstrating enhancement of the vascular system of the animals. Images were obtained at 9.4 T. Reprinted with permission from Springer Nature: Elsevier from Shin, T.-H.; Kim, P. K.; Kang, S.; Cheong, J.; Kim, S.; Lim, Y.; Shin, W.; Jung, J.-Y.; Lah, J. D.; Choi, B. W.; Cheon, J. High-Resolution T1 MRI via Renally Clearable Dextran Nanoparticles with an Iron Oxide Shell. Nat. Biomed. Eng. 2021, 5, 252–263, Copyright The Author(s), under exclusive licence to Springer Nature Limited 2021.

inflammation, vascular pathology, and other disease processes.308 A great deal of research is focused on adding functionality to particles via the coatings,306,308,310,311 but because that is not relevant to the inorganic chemistry of the cores, it is not described in detail here.

2.15.6.4

Chemical exchange saturation transfer (CEST)

Clinically approved GdIII-containing contrast agents for MRI shorten the relaxation times of water, resulting in altered image contrast. Contrast also is altered by chemical exchange saturation transfer (CEST) agents that transfer saturated magnetization from their exchangeable protons to the bulk water.312,313 CEST MRI is performed using either endogenous or exogenous CEST probes to detect biomolecules, such as proteins, and cellular conditions like pH.313 Many CEST probes are based on organic or biological molecules,313–318 but inorganic chemistry is used in combination with CEST when non-isotropic paramagnetic ions, like most of the non-f7 lanthanide ions, shift the resonance of the exchanging proton away from the bulk water signal. Probes that paramagnetically shift the signal arising from exchanging protons are referred to as PARACEST agents. Early PARACEST examples involved trivalent lanthanide ions.319–322 Recently, Morrow and co-workers reported several PARACEST agents that involve the transition metal ions.323–326 These PARACEST probes include complexes of CoII, FeII, NiII and low-spin FeIII with imidazole-appended macrocyclic ligands,323,324 CoII macrocyclic complexes with fluorophores,325 and CoII liposomal probes (Fig. 45).326 Similar to how the chemical shifts arising from different lanthanide ions are vastly different from each other,327 using

Fig. 45 (Left) Schematic of the di-imidazole complex. (Right) CEST spectrum of both the Co2þ (blue) and Ni2þ (green) analogs. Reprinted with permission from Burns, P. J.; Cox, J. M.; Morrow, J. R. Imidazole-Appended Macrocyclic Complexes of Fe(II), Co(II), and Ni(II) as ParaCEST Agents. Inorg. Chem. 2017, 56, 4545–4554. Copyright 2017 American Chemical Society.

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different transition metals also shifts the peaks arising from exchangeable protons. This point is exemplified by the CoII and NiII complexes of an imidazole-containing ligand that display CEST peaks that differ by tens of ppm from each other (Fig. 45).323 In addition to protons, other nuclei have been used for CEST imaging. Of interest to inorganic chemists is CEST with inclusion complexes of 129Xe. Hyperpolarized 129Xe can be used to image lungs,328 and like 1H, the chemical shift of 129Xe, depends on chemical environment. If the gas is trapped in an environment that enables exchange to a different environment, then CEST can be observed. An example of hyperpolarized CEST with 129Xe, was reported by Deng, Zhou, and coworkers, in which 129Xe was entrapped inside of metal-organic frameworks.328

2.15.6.5

PARASHIFT probes

Similar to probes that paramagnetically shift CEST signals, shift reagents can be used to shift other signals that do not exchange with bulk water. These other signals can be imaged directly in what is known as PARASHIFT imaging. PARASHIFT probes are complexes with lanthanide ions or transition metals, including Fe and Co.329–331 An example of PARASHIFT probe is a DyIII-containing complex with a nitrogenous cyclic ligand with two chemically equivalent tert-butyl groups  6.5 Å from the DyIII ion.329 The chemically equivalent protons of the tert-butyl groups were used for in vivo imaging of mice at 7 T. The probe was detectable in vivo at a low tissue concentrations (23 mmol dm 3) and acted as a tissue temperature probe due to the 0.28 ppm K 1 temperature dependence of the paramagnetic shift of the complex (Fig. 46).329

2.15.6.6

19

F probes

In addition to 1H, other nuclei can be imaged with MRI. One useful nucleus for MRI is 19F. 19F has a large natural abundance, is spin ½ like 1H, and has a similar gyromagnetic ratio to 1H, causing its imaging to be fairly straightforward relative to other nuclei. Additionally, unlike 1H, 19F has no inherent signal in biological systems, causing the background to be nonexistent. However, the sensitivity of 19F is low, so large amounts of probes must be used for imaging, because unlike contrast agents for 1H that catalytically shorten the relaxation times of water protons that are then imaged, 19F probes are directly imaged. To generate detectable signals, probes include a large number of equivalent 19F atoms; however, because fluorine is hydrophobic the incorporation of the fluorine atoms must be balanced with other functional groups to enable aqueous solubility or inclusion in emulsions. A variety of 19F probes are reported including fluorinated nanoparticles, fluorinated dendrimers, fluorinated polymers, liquid perfluorocarbon-based 19F probes, and discrete paramagnetic metal complexes.332 Several types of perfluorocarbon-based 19F probes have been studied including unmodified perfluorocarbons, perfluorocarbon nanoparticles, and perfluorocarbon nanoemulsions. Perfluorocarbon systems have been combined with inorganic chemistry to have an impact on contrast enhancement. For example, Kikuchi and co-workers reported fluorine accumulated silica nanoparticle for MRI contrast enhancement that have a liquid perfluorocarbon core surrounded by a silica shell.333 This technology was modified to include different perfluorocarbons, resulting in three variations of the probes with different chemical shifts, enabling in vivo tricolor images of a mouse (Fig. 47).334 Additionally, Ahrens and co-workers formulated an FeIII-containing lipid-based nanoemulsion using perfluoro-15-crown-5 ether that detects inflammation in vivo.335 Fluorinated metal complexes have been investigated for use as 19F probes using GdIII, EuIII, EuII, DyIII, TmIII, ErIII, HoIII, TbIII, YbIII, CoII, NiII, FeIII, and FeII.332,336–341 Pierre and co-workers investigated a series of 10 metallated complexes (M ¼ LaIII, EuIII, GdIII, TbIII, DyIII, HoIII, ErIII, TmIII, YbIII, and FeII) coordinated individually to the same ligand with 12 equivalent fluorine atoms.339 The complexes were imaged in both water and blood with the FeII-containing complex demonstrating the largest T2/T1 ratio (0.57), a single fluorine signal, good water solubility, and the lowest limit of detection of the series (300 mM).339 Que. and co-workers

Fig. 46 (Left) Schematic of the tert-butyl containing PARASHIFT probe. (Right) PARASHIFT images demonstrate sensitivity at 7 T in mice.329 Reprinted from Open Access Reference Senanayake, P. K.; Rogers, N. J.; Finney, K.-L. N. A.; Harvey, P.; Funk, A. M.; Wilson, J. I.; O’Hogain, D.; Maxwell, R.; Parker, D.; Blamire, A. M. A New Paramagnetically Shifted Imaging Probe for MRI. Magn. Reson. Med. 2017, 77, 1307–1317.

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Fig. 47 Tricolor imaging is demonstrated by simultaneous injection into a murine model. Reprinted with permission from Akazawa, K.; Sugihara, F.; Nakamura, T.; Matsushita, H.; Mukai, H.; Akimoto, R.; Minoshima, M.; Mizukami, S.; Kikuchi, K. Perfluorocarbon-Based 19F MRI Nanoprobes for In Vivo Multicolor Imaging. Angew. Chem., Int. Ed. 2018, 57, 16742–16747. Copyright 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

reported fluorinated Co, Fe, and Ni complexes as dual CEST and 19F probes.340 In those probes, exchangeable amide protons were the CEST functionality, and 18 equivalent fluorine atoms per complex enabled 19F MR imaging.340 In addition to the applications discussed in this section, 19F probes have been designed as responsive probes that are described in Section 2.15.6.7.

2.15.6.7

Responsive contrast agents

MRI provides anatomical information, but since the late 1990s, probes and contrast agents have been studied that enable MRI to be used for molecular imaging in addition to anatomical imaging.342–346 The ability to use MRI to noninvasively monitor biochemical conditions has the potential to add functional information to anatomical images for applications including monitoring of responses to therapies. In the design of responsive probes, chemical attributes of probes that lead to signal or contrast enhancement are linked to a molecular switch that toggles those attributes. The specific attributes vary based on the type of contrast agent but examples include water coordination number, water-exchange rate, tumbling rate, metal oxidation state, and chemical shift. The molecular switches that trigger the change in attributes, and consequent change in signal or contrast, include temperature, pH, redox-active molecules, enzymes, and the presence of specific cations and anions. Meade and co-workers modulated water-coordination number using b-galactosidase activity to report the activity in a mouse in vivo with MRI (Fig. 48).347 The contrast agent consisted of a GdIII complex with a pendant galactose moiety.347 When the sugar is present, water is blocked from coordinating with the GdIII ion resulting in an off state of minimal 1H-MRI signal. Cleavage of the sugar by b-galactosidase removes the group that impedes water coordination, resulting in a water-coordination number of one and

Fig. 48 b-Galactosidease cleaves the linker leaving [Gd(N-propylaminoDO3A)]3 þ with bright T1 weighted contrast. Reprinted with permission from Lilley, L. M.; Kamper, S.; Caldwell, M.; Chia, Z. K.; Ballweg, D.; Vistain, L.; Krimmel, J.; Mills, T. A.; MacRenaris, K.; Lee, P.; Waters, E. A.; Meade, T. J. Self-Immolative Activation of b-Galactosidase-Responsive Probes for In Vivo MR Imaging in Mouse Models. Angew. Chem., Int. Ed. 2020, 59, 388– 394. Copyright 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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contrast enhancement in 1H-MRI from the change in relaxivity between the GdIII-containing complex before and after being exposed to enzyme activity.347 Chemical shift is another attribute that modulates contrast in response to changes in a molecular switch. For example, Morrow and co-workers reported FeII- and CoII-containing chemical shift probes that respond to temperature and pH.331,348 One of those FeII PARASHIFT agents demonstrated shifted methyl proton resonances (105 and  46 ppm) with temperature coefficients of  0.29 ppm  C 1 and 0.22 ppm  C 1, respectively, with small line broadening and a large temperature-dependent response.331 Other examples include the CoII-containing complex of the 1,8-isomer of 1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11tetraazacyclotetradecane that displays a change in chemical shift in response to changes in temperature.348 This complex also produces ratiometric readouts of pH from two CEST peaks. Additionally, Pierre and co-workers reported thulium-based PARACEST probes to sense copper and zinc.349 The metal oxidation state of a contrast agent can be altered due to an interaction with a redox-active molecule, and this change in oxidation state can lead to dramatic differences in contrast enhancement. For example, divalent europium is isoelectronic with GdIII and enhances contrast in 1H-MRI, but trivalent europium does not to a measurable extent at imaging-relevant concentrations.350 This oxidation switch was used in a multimodal fluorinated EuII-containing redox-responsive contrast agent.341 The f7 EuII ion enhances contrast in 1H-MRI and suppresses CEST and 19F signals.341 However, in the presence of oxygen, EuII is oxidized to EuIII, resulting in no measurable enhancement in 1H-MRI and the presence of signal in both CEST and 19F-MRI (Fig. 49).341 Other metals have been used as redox switches, including Cu, Co, Mn, and Fe.351–355 In the Eu example, line broadening was used as the contrast attribute that was changed by the switch (oxygen) for CEST and 19F-MRI. Line broadening is also used with other ions, including GdIII. Kikuchi and coworkers used GdIII to suppress 19F-MRI using 19F-containing silica nanoparticles functionalized with GdIII-containing complexes with enzyme-cleavable linkers.356 Caspase-1 activity was the trigger that released GdIII from the surface of the nanoparticles, where it ceased to suppress 19F signal. This study demonstrated feasibility to detect caspase-1 activity in a living mouse. There are many other examples of responsive MRI probes, and interested readers are referred to the Further Reading Section.

2.15.6.8

Conclusions

Advances in contrast enhancement for MRI heavily rely on inorganic chemistry. This impact has been present since the early studies of how coordination chemistry of GdIII relates to contrast enhancement, and it is present in cutting edge research involving other metal ions and types of MRI involving nuclei other than 1H. Future advances in this area are likely to continue involving inorganic chemistry.

2.15.7

Positron emission tomography (PET) and single photon emission computed tomography (SPECT)

Nuclear medicine is an essential facet of modern medicine that is used in both therapeutic and diagnostic applications.357–360 From the diagnostic perspective, nuclear medicine involves the use of radioactive compounds to generate images. The radioisotopes of interest for diagnostic nuclear imaging are contrapositive from those required for treatment applications, although there is some overlap between the two fields; imaging applications require isotopes that either emit gamma rays or annihilation photons from positrons interacting with electrons. Nuclear imaging can be partitioned into two main imaging techniques, inherently centered on the type of emitter being employed: positron emission tomography (PET) or single-photon emission computerized tomography (SPECT). Fundamentally, the process for PET involves injection of a radioactive nuclide that decays by releasing a positron. Annihilation of the positron via collision with an electron releases two 511 keV gamma rays 180 apart from each other. Subsequent detection of these gamma rays by a scanner results in images after employment of computer algorithms.361 SPECT is an imaging method that combines the use of injected radiotracers with CT image construction techniques. SPECT differs from X-ray CT because the source of radiation comes from within the patient or sample rather than from an external source. Essentially, SPECT imaging involves the injection of a low-

Fig. 49 Fluorinated redox switchable probe changes from 1H-MRI active with EuII to 19F and CEST MRI active with EuIII.341 Reprinted from Open Access Reference Basal, L. A.; Bailey, M. D.; Romero, J.; Ali, M. M.; Kurenbekova, L.; Yustein, J.; Pautler, R. G.; Allen, M. J. Fluorinated EuII-based Multimodal Contrast Agent for Temperature- and Redox-Responsive Magnetic Resonance Imaging. Chem. Sci. 2017, 8, 8345–8350.

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energy gamma emitter (commonly 100–250 keV) and relies on direct detection and collimation of the gamma rays for image production.362–364 PET and SPECT imaging techniques have been used to study a wide variety of physiological conditions including cardiac function after acute myocardial infarction, cerebral blood flow, and hypoxia in cancer.365–367 Although PET imaging is 10-fold more sensitive than SPECT, SPECT is less expensive. Consequently, both modalities have routine clinical uses. Of the imaging modalities readily used in clinical practice, PET and SPECT imaging are of specific interest due to their intrinsic sensitivities, culminating in the ability to image a plethora of physiological parameters and functionalities including perfusion, vascularity, tissue oxygenation, enzymatic function, glucose metabolism, neurotransmitter function, and the real-time understanding of the uptake and efficiency of some drugs.368–374 Unlike other imaging techniques, nuclear imaging cannot function without the injection of the radioactive isotopes and, consequently, only produces images of areas where the isotopes are present, active, and at the required imaging concentrations. Therefore, nuclear imaging commonly necessitates bioconjugates to direct the imaging probe towards the relevant areas.375 Ideally, the conjugated vector needs to target the area of interest selectively with minimal interaction with other tissues. Additionally, nuclear imaging techniques lack anatomical context. To rectify this limitation, PET and SPECT imaging is commonly completed in conjunction with another spatially detailed imaging technique, such as MRI or X-ray CT.376–378 The information gathered from the combination of nuclear and anatomical imaging techniques leads to more accurate results than either imaging technique alone. For example, perfusion SPECT-CT imaging using 99mTc-labeled macro aggregated albumin has been used in diagnosing pulmonary embolisms in mild-to-moderate COVID-19 and improving therapeutic recommendations.379 In another example of dual-modality nuclear imaging, Caravan and coworkers studied a pH-sensitive GdIII-containing MRI agent that can be tagged with 18F or 19F to track changes in T1 measurements (MRI) and concentration calculations (PET) to directly quantify relaxivity and pH within a set of samples (pH 6–8.5).380 The combined results and the popularity of the dual-modalities has spurred the optimization of dual-imaging parameters. For instance, strives have been made towards the development of innovative detectors systems for PET that are functional within MRI instrumentation.381 The popularity of nuclear imaging largely is due to the success of 18F-fluorodeoxyglucose (FDG) in oncology as a probe for PET imaging. Because FDG is taken into the cells via a similar pathway to glucose, and because tumors have a higher rate of glucose metabolism, FDG accumulates in tumors. The sensitivity of PET imaging (10 11–10 12 M) enables detection of metastases not visible using other imaging modalities, spurring the use of FDG for PET imaging in routine clinical imaging for high-resolution images of a variety of cancers and other diseases.382–386 The pharmacokinetics of FDG (30–60 min for trapping and eventual clearance) being compatible with the half-life of 18F (110 min) is of critical importance to the intended efficacy.382 However, the desire to study biological processes with timeframes of activity different than the half-life of 18F permits has pushed the field towards studies using other radioisotopes, including many organic radiotracers (11C, 13N, 15O, and 123I) and metal radiotracers.387–391 Nuclear imaging is a large field that involves many contributions from inorganic chemistry. This section is not meant to be a comprehensive list of the multitude of inorganic complexes previously and currently used clinically and preclinically, but rather an introduction into the important properties, factors, and roles that inorganic chemistry plays in designing and using probes of this type. For more detailed information regarding the different radiotracers studied and subsequent coordination chemistry, see the Further Reading List.

2.15.7.1

Nuclides of interest and relevant properties

Radionuclides relevant to the medical field are created using one of three production systems: cyclotrons, nuclear reactors, and generator systems. The recent prevalence of small, biomedical cyclotrons (commonly < 20 MeV) within hospitals or academic institutions is one of the driving factors behind the clinical popularity of PET and SPECT imaging.392–394 The ability to produce radioactive isotopes as necessary within a hospital setting is a valuable development that limits early and unwanted decay prior to use. Longer half-life radionuclides can be produced using a reactor and transported to medical facilities. Some biologically relevant isotopes are created using nuclear generator systems that produce desired radionuclides from parent radionuclide decay and are separated via resin-loading and eluting.393 A benefit of the generator methodology stems directly from not requiring a cyclotron or reactor system, which widens clinical availability. For example, 99mTc, a workhorse of nuclear imaging, is generated using a 99Mo/99mTc generator system.395 However, Mo shortages have prompted research into new methods of 99mTc generation and spurred interest into other radioisotopes.394 One of the most critical properties in the selection of a radioactive isotope is the half-life. The radioactive half-life of an element is the time it takes for half of the radioactive elements within a sample to decay. The half-life of an element is independent of initial quantities and environmental factors. Although the half-lives of radioactive elements can span upwards of millions of years, medically applicable radioactive elements must decay within a relatively short timeframe. Within the field of nuclear medicine, many inorganic radioactive elements are considered useful for medical imaging including main group elements (for example, 66Ga, t1/2 ¼ 9.49 h; 67Ga, t1/2 ¼ 78.2 h; 68Ga, t1/2 ¼ 1.13 h; 110mIn, t1/2 ¼ 1.15 h; and 111In; t1/2 ¼ 67.2 h), transition metals (for example, 99m Tc, t1/2 ¼ 6 h; 45Ti, t1/2 ¼ 3.1 h; 51Cr, t1/2 ¼ 665 h; 51Mn, t1/2 ¼ 46.2 min; 52Mn, t1/2 ¼ 134 h; 55Co, t1/2 ¼ 17.5 h; 64Cu, t1/2 ¼ 12.7 h; 63Zn, t1/2 ¼ 38.5 min; 89Zr, t1/2 ¼ 78.4 h; 90Nb; t1/2 ¼ 14.6 h; 105Rh, t1/2 ¼ 35.4 h; and 195mPt, t1/2 ¼ 96 h), and rare earth elements (for example 44Sc, t1/2 ¼ 4.04 h; 47Sc, t1/2 ¼ 80.4 h; 86Y, t1/2 ¼ 14.7 h; 177Lu, t1/2 ¼ 159 h; 149Tb, t1/2 ¼ 4.12 h; 152Tb, t1/2 ¼ 17.5 h; and 155Tb, t1/2 ¼ 128 h).393,394 The selection of a radioisotope is vital in determining the usefulness for imaging biological targets because nuclear imaging experiments involve timing considerations for radiopharmaceutical production, storage, and transport; biodistribution, target uptake, and clearance; and imaging protocols. For example, the half-life of 89Zr (78.4 h) makes

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radiopharmaceuticals containing 89Zr suitable for the study of monoclonal antibodies using PET imaging.396 Additionally, 68Ga has a half-life of 1.13 h and is often used to produce PET radiotracers based on small molecules and peptides.397

2.15.7.2

Chelators for complexation and targeting

With the existence of many options for nuclides in PET and SPECT imaging, ligand chemistry is critical to sequestering these ions and preventing side effects in clinical applications. Because PET radionuclides are often short-lived, ligands must complex with the ions rapidly and form inert complexes with respect to the half-life of the isotope. Extensive research has been performed on these topics.391,393,394,398–404 Within the past few years, focus has been placed on introducing the possibility of functionalization of complexes for targeted imaging.405 A few examples are summarized in this section. In 2019, Holland and Gut reported a system based on the ligand diethylenediamine penta-acetic acid that undergoes lightactivated bioconjugation to antibodies.406 The bioconjugation follows one of two pathways: one has the nuclide (either 68Ga3þ or 111In3þ) coordinated first followed by in situ transformation of an aryl azide into an azepin linkage to the antibody (Trastuzumab) and the other is the converse process with bioconjugation first and nuclide binding second (Fig. 50). Little difference was reported in overall output of radio-labeled bioconjugates between the approaches, suggesting their system is versatile in applicability. Additionally, preliminary kinetic studies demonstrated that the bioconjugation step is first-order in antibody concentration, which is ideal given the expense of nuclides for imaging. One specific target for PET imaging that has been investigated are amyloid-b plaques associated with Alzheimer’s disease. Donnelly and coworkers reported a thiosemicarbazone pyridylhydrazone chelator for 64Cu2þ in 2019 that featured a vinyl pyridine moiety to bind the compound to amyloid-b plaques.407 The copper complex is straightforward to synthesize and amenable to kit-based formulations popular in PET. To examine the affinity of the compound for amyloid-b, a competitive fluorescence assay was performed with thioflavin T and synthetic amyloid-b. The data showed moderate affinity with a Ki of 4.3  1.6 mM. The compounds were introduced to amyloid-containing brain tissue of 7 mm thick slices to study the specificity of the compound. Fluorescence of the ligand was compared to a 1E8 amyloid-b specific antibody stain, revealing excellent colocalization (Fig. 51). These results suggest the compound has the potential to image the plaques selectively. PET chelating agents have also been investigated for prostate specific proteins and tumors. Donnelly and coworkers describe a set of compounds that have a part to chelate either 68Ga3þ or 89Zr4þ as well as a part known to target Zn2þ sites in prostate-specific membrane antigen (PSMA, Fig. 52).408 The compound sequesters both nuclides, and the specificity was examined in mice with

Fig. 50 Bioconjugation follows either a one-pot or two-step synthetic route. Reprinted with permission from Gut, M.; Holland, J. P. Synthesis and Photochemical Studies on Gallium and Indium Complexes of DTPA-PEG3-ArN3 for Radiolabeling Antibodies. Inorg. Chem. 2019, 58, 12,302–12,310. Copyright 2019 American Chemical Society.

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Fig. 51 Epifluorescence from the ligand (a) colocalizes with a 1E8 stain (b) of amyloid-b plaques. Republished with permission from CSIRO Publishing from A Copper Complex of a Thiosemicarbazone-Pyridylhydrazone Ligand Containing a Vinylpyridine Functional Group as a Potential Imaging Agent for Amyloid-b Plaques by Lachlan E. McInnes, Asif Noor, Peter D. Roselt, Catriona A. McLean, Jonathan M. White, and Paul S. Donnelly. Aust. J. Chem. 2019, 72, 827–834. Copyright 2019. Permission conveyed through Copyright Clearance Center.

LNCaP prostate tumor xenografts. Intravenous injections of each nuclide analog show uptake in the tumor as well as accumulation in the kidneys (PSMA is also expressed in murine kidneys, Fig. 52), demonstrating that the complex targets PSMA. Bartholomä and coworkers described a different prostate specific PET chelator.409 Their chelator was based on 1,4,7triazacyclononane with two azaheterocyclic arms and one arm that formed an amide linkage with a bioconjugate, in their case PSMA-7 (Fig. 53). The authors metalated the pentadentate ligand with 68Ga3þ and investigated performance in mice. The compound was injected into nude mice and monitored over time in the blood, liver, kidneys, and bladder (Fig. 53). The authors state that the accumulation and clearance times are similar to other PSMA-containing agents, suggesting that the compound could have targeted accumulation in forthcoming experiments. In addition, the ability to track the compound through its clearance from mice suggests excellent in vivo stability. This PSMA targeting technique was also applied to a chelator for 47Sc3þ and 177Lu3þ by Boros and coworkers.410 The agent is based on 1,4,7-triazacyclononane with a picolinic arm and acetate arms, one of which is attached to a PMSA targeting moiety (Fig. 54). The authors put forward two interesting observations. One is how DFT optimization of the binding site for both metals leads to homologous seven-coordinate structures for both nuclides. The second was the ability of the agent to target PSMA-

Fig. 52 Schematic of the ligand of interest (left) that has a metal binding motif on the left of the squaramide, and PSMA-targeting groups to the right. Combined PET and X-ray CT images of the 89Zr and 68Ga analogs in a murine model (right). Reprinted with permission from Noor, A.; Van Zuylekom, J. K.; Rudd, S. E.; Waldeck, K.; Roselt, P. D.; Haskali, M. B.; Wheatcroft, M. P.; Yan, E.; Hicks, R. J.; Cullinane, C.; Donnelly, P. S. Bivalent Inhibitors of Prostate-Specific Membrane Antigen Conjugated to Desferrioxamine B Squaramide Labeled with Zirconium-89 or Gallium-68 for Diagnostic Imaging of Prostate Cancer. J. Med. Chem. 2020, 63, 9258–9270. Copyright 2020 American Chemical Society.

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Fig. 53 Schematic of the ligand of interest (left) and time-lapse PET images of the 68Ga compound in mice (left). Reprinted with permission from Schmidtke, A.; Läppchen, T.; Weinmann, C.; Bier-Schorr, L.; Keller, M.; Kiefer, Y.; Holland, J. P.; Bartholomä, M. D. Gallium Complexation, Stability, and Bioconjugation of 1,4,7-Triazacyclononane Derived Chelators with Azaheterocyclic Arms. Inorg. Chem. 2017, 56, 9097–9110. Copyright 2017 American Chemical Society.

20

177 47

N O O

N

O

15

O

N M3+ N

H N

H N

O

O

OH

5

O

%ID/g

O

O

Lu-(picaga)-DUPA 2 h ns

10

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N H

N H

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5 0 od lo B

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Fig. 54 Schematic of the ligand of interest (left) and a chart of biodistribution of the 47Sc and 177Lu analogs in mice. Reprinted with permission from Vaughn, B. A.; Koller, A. J.; Chen, Z.; Ahn, S. H.; Loveless, C. S.; Cingoranelli, S. J.; Yang, Y.; Cirri, A.; Johnson, C. J.; Lapi, S. E.; Chapman, K. W.; Boros, E. Homologous Structural, Chemical, and Biological Behavior of Sc and Lu Complexes of the Picaga Bifunctional Chelator: Towards Development of Matched Theranostic Pairs for Radiopharmaceutical Applications. Bioconjugate Chem. 2021, 32, 1232–1241. Copyright 2021 American Chemical Society.

expressing tumors. The compound was injected into mice bearing PC-3 PiP (PSMA þ) and PC-3 Flu (PSMA-) xenograft tumors on opposite flanks. Biodistribution showed accumulation in the PSMA þ tumor, with some accumulation in the liver and kidneys for both nuclide analogs (Fig. 54). These results demonstrate the ability to use ligands to extend prostate targeting imaging to more nuclides in PET.

2.15.7.3

Conclusions

The field of nuclear imaging is an important facet of clinical medicine of specific importance to diagnostic applications. Recent advances in PET and SPECT imaging include the development of new instrumentation, scanners and technologies involved in both the production of relevant radionuclides and acquisition of imaging data, and use of radioactive analogs of MRI contrast agents to image biodistribution.411 Like other imaging modalities, the frontiers of inorganic chemistry pertaining to nuclear medicine involve the addition of targeting moieties to accumulate probes in areas of interest to investigate diseases and biological processes from new perspectives and to find targets that garner the interest of physicians.

2.15.8

Summary and outlook

The use of inorganic chemistry has had a tremendous impact on almost every modality of biomedical imaging. Fundamental principles of coordination chemistry and the ability to control the properties of nanoparticles enable tuning of the properties of inorganic probes to match the specifications of each imaging modality with respect to enhancing signal and contrast as exemplified throughout this chapter. The ability to use imaging to study events on the molecular scale and to target specific areas of interest selectively in vivo remain grand challenges across all modalities that will likely require inorganic chemistry to overcome. Additionally, there is likely space for inorganic chemistry to contribute to improving limits of detection for many of the modalities described in this chapter.

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H.; Weisman, G. R.; Anderson, C. J. Coordinating Radiometals of Copper, Gallium, Indium, Yttrium, and Zirconium for PET and SPECT Imaging of Disease. Chem. Rev. 2010, 110, 2858–2902. 405. Chomet, M.; van Dongen, G. A. M. S.; Vugts, D. J. State of the Art in Radiolabeling of Antibodies with Common and Uncommon Radiometals for Preclinical and Clinical Immuno-PET. Bioconjug. Chem. 2021, 32, 1315–1330. 406. Gut, M.; Holland, J. P. Synthesis and Photochemical Studies on Gallium and Indium Complexes of DTPA-PEG3-ArN3 for Radiolabeling Antibodies. Inorg. Chem. 2019, 58, 12302–12310. 407. McInnes, L. E.; Noor, A.; Roselt, P. D.; McLean, C. A.; White, J. M.; Donnelly, P. S. A Copper Complex of a Thiosemicarbazone-Pyridylhydrazone Ligand Containing a Vinylpyridine Functional Group as a Potential Imaging Agent for Amyloid-b Plaques. Aust. J. Chem. 2019, 72, 827–834. 408. Noor, A.; Van Zuylekom, J. K.; Rudd, S. E.; Waldeck, K.; Roselt, P. D.; Haskali, M. B.; Wheatcroft, M. P.; Yan, E.; Hicks, R. J.; Cullinane, C.; Donnelly, P. S. Bivalent Inhibitors of Prostate-Specific Membrane Antigen Conjugated to Desferrioxamine B Squaramide Labeled with Zirconium-89 or Gallium-68 for Diagnostic Imaging of Prostate Cancer. J. Med. Chem. 2020, 63, 9258–9270. 409. Schmidtke, A.; Läppchen, T.; Weinmann, C.; Bier-Schorr, L.; Keller, M.; Kiefer, Y.; Holland, J. P.; Bartholomä, M. D. Gallium Complexation, Stability, and Bioconjugation of 1,4,7-Triazacyclononane Derived Chelators with Azaheterocyclic Arms. Inorg. Chem. 2017, 56, 9097–9110. 410. Vaughn, B. A.; Koller, A. J.; Chen, Z.; Ahn, S. H.; Loveless, C. S.; Cingoranelli, S. J.; Yang, Y.; Cirri, A.; Johnson, C. J.; Lapi, S. E.; Chapman, K. W.; Boros, E. Homologous Structural, Chemical, and Biological Behavior of Sc and Lu Complexes of the Picaga Bifunctional Chelator: Toward Development of Matched Theranostic Pairs for Radiopharmaceutical Applications. Bioconjug. Chem. 2021, 32, 1232–1241. 411. Le Fur, M.; Rotle, N. J.; Correcher, C.; Jordan, V. C.; Ross, A. W.; Catana, C.; Caravan, P. Yttrium-86 Is a Positron Emitting Surrogate of Gadolinium for Noninvasive Quantification of Whole-Body Distribution of Gadolinium-Based Contrast Agents. Angew. Chem. Int. Ed. 2020, 59, 1474–1478.

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Further reading X-ray computed tomography further reading Yeh, B. M.; FitzGerald, P. F.; Edic, P. M.; Lambert, J. W.; Colborn, R. E.; Marino, M. E.; Evans, P. M.; Robert, J. C.; Wang, Z. J.; Wong, M. J.; Bonitatibus, P. J., Jr. Opportunities for New CT Contrast Agents to Maximize the Diagnostic Potential of Emerging Spectral CT Technologies. Adv. Drug Deliv. Rev. 2017, 113, 201–222. Lusic, H.; Grinstaff, M. W. X-Ray-Computed Tomography Contrast Agents. Chem. Rev. 2013, 113, 1641–1656. Attia, M. F.; Wallyn, J.; Anton, N.; Vandamme, T. F. Inorganic Nanoparticles for X-Ray Computed Tomography Imaging. Crit. Rev. Ther. Drug Carrier Syst. 2018, 35, 391–431. Hsu, J. C.; Nieves, L. M.; Betzer, O.; Sadan, T.; Noël, P. B.; Popovtzer, R.; Cormode, D. P. Nanoparticle Contrast Agents for X-Ray Imaging Applications. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2020, 12, e1642. Optical and near-IR further reading Bünzli, J.-C. G.; Eliseeva, S. V. Basics of Lanthanide Photophysics. In Lanthanide Luminescence; Hänninen, P., Härmä, H., Eds.; Springer Series on Fluorescence; Springer: Berlin, 2010; pp 1–45. Cherenkova, E. P. The Discovery of the Cherenkov Radiation. Nucl. Instrum. Methods Phys. Res. Sect. A 2008, 595, 8–11. Coogan, M. P.; Fernández-Moreira, V. Progress with, and Prospects for, Metal Complexes in Cell Imaging. Chem. Commun. 2014, 50, 384–399. de Bettencourt-Dias, A., Ed.; Luminescence of Lanthanide Ions in Coordination Compounds and Nanomaterials, Wiley: Chichester, U.K., 2014. Shen, J.; Rees, T. W.; Ji, L.; Chao, H. Recent Advances in Ruthenium(II) and Iridium(III) Complexes Containing Nanosystems for Cancer Treatment and Bioimaging. Coord. Chem. Rev. 2021, 443, 214016. Magnetic particle imaging further reading Bulte, J. W. M. Superparamagnetic Iron Oxides as MPI Tracers: A Primer and Review of Early Applications. Adv. Drug Deliv. Rev. 2019, 138, 293–301. Dadfar, S. M.; Roemhild, K.; Drude, N. I.; von Stillfried, S.; Knüchel, R.; Kiessling, F.; Lammers, T. Iron Oxide Nanoparticles: Diagnostic, Therapeutic and Theranostic Applications. Adv. Drug Deliv. Rev. 2019, 138, 302–325. Knopp, T.; Gdaniec, N.; Möddel, M. Magnetic Particle Imaging: From Proof of Principle to Preclinical Applications. Phys. Med. Biol. 2017, 62, R124–R178. Saritas, E. U.; Goodwill, P. W.; Croft, L. R.; Konkle, J. J.; Lu, K.; Zheng, B.; Conolly, S. M. Magnetic particle imaging (MPI) for NMR and MRI researchers. J. Magn. Reson. 2013, 229, 116–126. Wu, L. C.; Zhang, Y.; Steinberg, G.; Qu, H.; Huang, S.; Cheng, M.; Bliss, T.; Du, F.; Rao, J.; Song, G.; Pisani, L.; Doyle, T.; Conolly, S.; Krishnan, K.; Grant, G.; Wintermark, M. A Review of Magnetic Particle Imaging and Perspectives on Neuroimaging. AJNR Am. J. Neuroradiol. 2019, 40, 206–212. Zheng, B.; Lu, K.; Konkle, J. J.; Hensley, D. W.; Keselman, P.; Orendorff, R. D.; Tay, Z. W.; Yu, E.; Zhou, X. Y.; Bishop, M.; Gunel, B.; Taylor, L.; Ferguson, R. M.; Khandhar, A. P.; Kemp, S. J.; Krishnan, K. M.; Goodwill, P. W.; Conolly, S. M. Magnetic Particle Imaging. In Design and Applications of Nanoparticles in Biomedical Imaging; Bulte, J. W. M., Modo, M. M. J., Eds., Springer: Switzerland, 2017; pp 69–93. Ultrasound and photoacoustic imaging further reading de Leon, A.; Perera, R.; Nittayacharn, P.; Cooley, M.; Jung, O.; Exner, A. A. Ultrasound Contrast Agents and Delivery Systems in Cancer Detection and Therapy. Adv. Cancer Res. 2018, 139, 57–84. Frinkling, P.; Segers, T.; Luan, Y.; Tranquart, F. Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements. Ultrasound Med. Biol. 2020, 46, 892–908. Mallidi, S.; Luke, G. P.; Emelianov, S. Photoacoustic Imaging in Cancer Detection, Diagnosis, and Treatment Guidance. Trends Biotechnol. 2011, 29, 213–221. Li, W.; Chen, X. Gold Nanoparticles for Photoacoustic Imaging. Nanomedicine 2015, 10, 299–320. Weber, J.; Beard, P. C.; Bohndick, S. E. Contrast Agents for Molecular Photoacoustic Imaging. Nat. Methods 2016, 13, 639–650. Magnetic resonance imaging further reading Chen, C.; Ge, J.; Gao, Y.; Chen, L.; Cui, J.; Zeng, J.; Gao, M. Ultrasmall Superparamagnetic Iron Oxide Nanoparticles: A Next Generation Contrast Agent for Magnetic Resonance Imaging. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2021;, e1740. Shuvaev, S.; Akam, E.; Caravan, P. Molecular MR Contrast Agents. Invest. Radiol. 2021, 56, 20–34. Gupta, A.; Caravan, P.; Price, W. S.; Platas-Iglesias, C.; Gale, E. M. Applications for Transition-Metal Chemistry in Contrast-Enhanced Magnetic Resonance Imaging. Inorg. Chem. 2020, 59, 6648–6678. Hu, H. Recent Advances of Bioresponsive Nano-sized Contrast Agents for Ultra-High-Field Magnetic Resonance Imaging. Front. Chem. 2020, 8, 203. Hequet, E.; Henoumont, C.; Muller, R. N.; Laurent, S. Fluorinated MRI Contrast Agents and their Versatile Applications in the Biomedical Field. Future Med. Chem. 2019, 11, 1157–1175. Li, H.; Meade, T. J. Molecular Magnetic Resonance Imaging with Gd(III)-based Contrast Agents: Challenges and Key Advances. J. Am. Chem. Soc. 2019, 141, 17025–17041. Wahsner, J.; Gale, E. M.; Rodríguez-Rodríguez, A.; Caravan, P. Chemistry of MRI Contrast Agents: Current Challenges and New Frontiers. Chem. Rev. 2019, 119, 957–1057. Pierre, V. C., Allen, M. J., Eds.; Contrast Agents for MRI: Experimental Methods; New Developments in NMR No. 13, The Royal Society of Chemistry: Croydon, U.K, 2018. Positron emission tomography and single photon emission computed tomography further reading Wadas, T. J.; Wong, E. H.; Weisman, G. R.; Anderson, C. J. Coordinating Radiometals of Copper, Gallium, Indium, Yttrium, and Zirconium for PET and SPECT Imaging of Disease. Chem. Rev. 2010, 110, 2858–2902. Kostelnik, T. I.; Orvig, C. Radioactive Main Group and Rare Earth Metals for Imaging and Therapy. Chem. Rev. 2019, 119, 902–956. Boros, E.; Packard, A. B. Radioactive Transition Metals for Imaging and Therapy. Chem. Rev. 2019, 119, 870–901.

Relevant websites https://www.nibib.nih.gov/science-education/science-topics/ultrasounddNational Institutes of Health. https://www.nibib.nih.gov/science-education/science-topics/magnetic-resonance-imaging-mridNational Institutes of Health. https://www.nibib.nih.gov/science-education/science-topics/optical-imagingdNational Institutes of Health. https://www.nibib.nih.gov/science-education/science-topics/computed-tomography-ctdNational Institutes of Health. https://www.nibib.nih.gov/science-education/science-topics/nuclear-medicinedNational Institutes of Health.

2.16

Phosphorescent metal complexes for biomedical applications

Jiangping Liu*, Ruilin Guan*, Xinlin Lin, Yu Chen*, and Hui Chao*, MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou, PR China © 2023 Elsevier Ltd. All rights reserved.

2.16.1 2.16.2 2.16.2.1 2.16.2.2 2.16.2.2.1 2.16.2.2.2 2.16.2.2.3 2.16.2.2.4 2.16.2.2.5 2.16.2.2.6 2.16.2.3 2.16.2.3.1 2.16.2.3.2 2.16.2.3.3 2.16.2.3.4 2.16.2.4 2.16.3 2.16.3.1 2.16.3.2 2.16.3.3 2.16.3.4 2.16.4 2.16.4.1 2.16.4.1.1 2.16.4.1.2 2.16.4.1.3 2.16.4.1.4 2.16.4.1.5 2.16.4.1.6 2.16.4.2 2.16.4.2.1 2.16.4.2.2 2.16.4.2.3 2.16.4.2.4 2.16.4.3 References

Introduction Phosphorescent metal complexes for bioimaging Advantages of phosphorescent metal complexes as bioimaging agents Organelle imaging and tracking Nucleus and nucleolus Mitochondria Lysosomes Endoplasmic reticulum (ER) and Golgi apparatus Cytoplasm Other cell organelles Cellular molecule labeling and cellular physical state detection Metal ions Intracellular oxygen and hypoxic environment Intracellular redox small molecule Intracellular biomacromolecule Conclusion Phosphorescent metal complexes for chemotherapy Phosphorescence in chemotherapy Phosphorescent ruthenium complexes as chemotherapeutic agents Phosphorescent iridium complexes as chemotherapeutic agents Other phosphorescent metal complexes as chemotherapeutic agents Phosphorescent metal complexes for photodynamic therapy Phosphorescent Ru(II) complexes for PDT Elongating excited-state lifetime Enhancing light-harvesting ability Extending absorption profile to phototherapeutic window Promoting performance in hypoxia Imparting tumor targeting and uptake ability Multimodal therapies for enhanced cancer therapy Phosphorescent Ir(III) complexes for PDT Promoting photophysical performance for PDT Targeted PDT by Ir(III) complexes Reinforcing phototherapeutic potency in hypoxia Multimodal therapy Other phosphorescent metal complexes/polymetallic complexes for PDT

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Abstract Benefiting from the application of state-of-the-art imaging techniques, phosphorescent species are gaining a surge of research interest. Phosphorescent metal complexes is an interesting emerging branch that is currently being intensively investigated, since they are distinctively different from fluorescent chromophores regarding the nature and lifetime scale of photoluminescence. Alongside the success of fundamentally non-emissive cisplatin-based compounds in anticancer therapeutics, the phosphorescent signals of the metal complexes raise an intriguing possibility of revealing the mechanism of action of the potent candidates from another perspective. Under the inherent difference in the photophysical processes, these metal complexes have been successfully exploited to a wide range of biomedical applications involving biosensing/cell staining, visualized chemotherapy (also known as theranostic), and phototherapy (especially photodynamic therapy), revealing promising potentials in serving as candidates for their organic counterparts. This review article overviews the most recent

*

These authors have contributed equally to this work.

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Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00061-3

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works on phosphorescent metal complexes for biomedical applications and concentrates on phosphorescent metals such as ruthenium and iridium.

2.16.1

Introduction

Phosphorescent metal complex is an exciting realm of bioinorganic chemistry with applications in new materials (including sensors and tissue staining dyes), healthcare (diagnosis and therapy), biological function, and beyond. Complexes of the d6 and d8 metal ions, such as Re(I), Ru(II), Os(II), and Ir(III), as well as some other platinum group metals, are readily detectable by their phosphorescence in cells by confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and flow cytometry (including two-/three-photon absorption). It is widely accepted that metal complexes exhibit distinct properties from the organic counterparts on physicochemical, photophysical, and pharmacokinetic facets, and remain largely underexplored aside from cisplatin-based derivatives. The phosphorescence will undoubtedly provide more insights to unravel this mysterious field. Phosphorescent metal probes with large Stokes shifts are capable of conveying detailed information on the biological dynamics of the microenvironment.1 Besides, the mechanism of action of phosphorescent therapeutic metal complexes can be further explored by leveraging their intrinsic phosphorescence properties to determine and track their intracellular localization. Moreover, the inherent heavy atom effect of phosphorescent metal complexes bolsters the possibility of efficient intersystem crossing (ISC) and paves way for application in photodynamic therapy (PDT). In this chapter, the advances of phosphorescent metal complexes, especially Ru(II) and Ir(III) complexes, for biomedical applications involving bioimaging/biosensing, visualized chemotherapy, and PDT are introduced at length (Scheme 1). Their advantages for pertinent applications are highlighted. Also, the challenges and future development of the phosphorescent metal complexes are envisioned.

2.16.2

Phosphorescent metal complexes for bioimaging

2.16.2.1

Advantages of phosphorescent metal complexes as bioimaging agents

Phosphorescent metal complexes show various advantageous photophysical properties for the development of bioimaging agents. Firstly, due to the synthetic versatility of metal complexes, their photophysical properties are easily tunable.2 Complicated excited states can be formed with the metal centers and/or ligands. Different excited states have different energy levels, which are mainly dependent on the electronic structure of the central metal atom and the chemical structures of ligands. Therefore, a metal complex library can be easily constructed by combining different metal centers and ligands. To date, the tunable excitation and emission wavelength over the whole visible (even near-infrared) range have been achieved.3 This is conducive to the development of suitable metal complexes for different targets and research purposes. Secondly, compared to purely organic emissive agents, phosphorescent metal complexes-based probes can achieve high signalto-noise ratio results. Due to the heavy atom effect of the metal center, the intersystem crossing (ISC) process is efficient for metal complexes and therefore triplet-state metal-to-ligand charge-transfer (3MLCT) is the most commonly observed one. Owing to spinforbidden transition, the existence of triplet excited states endows phosphorescent metal complexes with high emission quantum

Scheme 1

Schematic illustration of phosphorescent metal complexes for biomedical applications.

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Phosphorescent metal complexes for biomedical applications

yields, large Stokes shifts, and long phosphorescence lifetimes. The phosphorescence lifetimes of metal complexes usually range from hundreds of nanoseconds to tens of microseconds while that of background auto-fluorescence is less than 10 ns.4 Using time-resolved fluorescence imaging technology can conveniently avoid the interferences from the background. Phosphorescent metal complexes also exhibit significant Stokes shifts. The emission wavelength is usually red-shifted by more than 50 nm than that of the exciting light, thus preventing the fluorescence quenching induced by self-absorption. Thirdly, it is easy to construct dual signal ratiometric probes because of the complicated excited states of metal complexes. In addition to the MLCT, the other possible excited states include intraligand charge-transfer (ILCT), ligand-to-ligand chargetransfer (LLCT), metal-centered (MC) excited states, metal-metal-to-ligand charge-transfer (MMLCT), ligand-to-metal-metal charge-transfer (LMMCT) and metal-to-ligand-ligand charge-transfer (MLLCT) states.3 Compared with qualitative probes, quantitative probes are better suited to improve the understanding of biological processes and disease development. The use of a single emission probe for accurate detection of analytes is defective in practice. To conquer the drawbacks of single-emission probes depending on accurate quantitative measurements of signal intensities, dual-signal ratiometric probes use the dual-signal intensity ratio of two sources as the output and establish a relationship of the ratio vs. analyte concentration.5 In addition to the above common advantages, phosphorescent metal complexes also stand them in good stead for the application in two-photon excitation, stochastic optical reconstruction, and stimulated emission depletion microscopy.

2.16.2.2

Organelle imaging and tracking

The imaging of organelles by metal complexes can be divided into two ways: one is to accumulate in the organelle and use its emission; the other is to utilize the organelle characteristic (e.g. the acidity of lysosomes, etc.) to turn on its emission. The former has strict requirements for the localization of organelles, while the latter does not. The latter can also be defined as organelle molecule labeling. Introducing organelle-targeting chemical groups or organelle-penetrating peptides are the common approaches to achieve the organelle localization of metal complexes. Although these two have been successful within a certain scope, they can still be limited by disadvantages, including off-target, synthetic difficulties, lacking a common relationship between the amino acid sequence and translocation pathway and so on.6,7 It is a better strategy to achieve the organelle accumulation of metal complexes by harnessing their intrinsic characteristics. On the other hand, organelles are not static. Their dynamics play crucial roles in physiological and pathological processes. Therefore, dynamic tracking is more important than static imaging. It brings higher demands to the dyes, especially in low cytotoxicity and high photostability. However, because there is no distinction between these two in the available studies, we put them together in this book.

2.16.2.2.1

Nucleus and nucleolus

The cell nucleus is the brain in the eukaryotic cell. It houses the chromosomes and controls metabolism, heredity, and reproduction.3 Separated from the cytosol by a double lipid bilayer (the nuclear envelope), substances that enter the nucleus are stringently controlled. Therefore, only a few phosphorescent metal complexes have been reported for nucleus staining. So for this reason, nucleoli, the nuclear sites for ribosomal RNA transcription processing and ribosome assembly, whose imaging is also described in this section. It should be pointed out that, due to the nucleus is full of DNA, RNA, and histones, almost existing nuclear dyes have enabled nuclear imaging by binding to these macromolecules. Nuclear DNA is generally accepted to be the main target of cisplatin (Fig. 1). Since its clinical success, a large number of nucleustargeting Pt-based anticancer drugs have been developed. Nucleus imaging can easily be obtained when some of them are emissive. For example, in 2010, Koo and co-workers developed a Pt(II) complex (1) as an efficient RNA transcription inhibitor.8 Interestingly, with a mitochondrial-targeted group triphenylphosphonium (TPP), this complex was found to localize in the nucleus. It, to some extent, indicates the unreliability of the organelle targeting group. In addition to the profound cytotoxicity, complex 1 is also a twophoton bioimaging agent. With a two-photon absorption cross-section of 28.0 GM, the nucleus staining was achieved in cells. A year later, Prof. Guo and co-workers reported a monofunctional chelated Pt(II) complex (2, Fig. 1).9 Bearing a 7-nitro-2,1,3benzoxadiazole fluorophore, this complex can be visualized in vitro and in vivo (Fig. 2). After 3 h of incubation, small bright spots appear especially around the nucleus, suggesting that the complex can stride into the nucleus. Due to their cytotoxicity, this class of complexes will be described later in the section on Antitumor Agent. In contrast, nontoxic Pt(II) complexes for nuclear imaging are rare. By taking advantage of two-photon time-resolved emission imaging microscopy (TP-TREM), Baggaley and co-workers showed a Pt(II) complex (3a-d, Fig. 1) with large emission lifetimes could identify cell nuclei from the surrounding matrix proteins.10 However, the small TPA cross-sections (3.5 GM) somewhat limit its further applications. Ir(III) complexes have been applied to stain the nucleus. In 2011, Li et al. synthesized a cationic solvated Ir(III) complex (4, Fig. 1), in which the two DMSO were coordinated to the Ir(III) metal center.11 As a first reaction-based fluorescence-turn-on agent for the nuclei of living cells. Complex 4 can rapidly and selectively light-up the nuclei of living cells over fixed cells, giving rise to a significant luminescence enhancement (200-fold), and shows very low cytotoxicity at the imaging concentration. Subsequently, to explore the structure-activity relationship of the analogous non-emissive probes, a series of non-emissive iridium(III) complexes were designed and synthesized in 2013.12 The results showed that variation of coordinated solvent ligands, and counter anions, does not affect the localization of the complexes. And then Li and co-workers further reported a structurally similar Ir(III) complex but containing two hydro-solvated ligands instead of DMSO (5). Different from complex 4, complex 5 could not be up-taken by the living cells, whereas it could stain the nucleus of dead HeLa cells. The weak membrane penetration ability of complex 5 in living

Phosphorescent metal complexes for biomedical applications

Fig. 1

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The chemical structures of cisplatin and some metal complexes for nucleus and nucleolus staining.

4h

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In Vitro

Bright Field

Fig. 2 The in vitro and in vivo imaging application of complex 2. In vitro: MCF-7 cells; In Vivo: 50 h-zebrafish larva. Reprinted with permission from Ref. Wu, S. D.; Zhu, C. C.; Zhang, C. L. et al. In Vitro and In Vivo Fluorescent Imaging of a Monofunctional Chelated Platinum Complex Excitable Using Visible Light. Inorg. Chem. 2011, 50, 11847–11849. Copyright 2011, American Chemical Society.

cells was attributed to its high hydrophobicity.13 Another example was developed by Zhao, Huang, and co-workers (6 and 7, Fig. 1). By introducing an organic DNA intercalator as the main ligand, complex 6 could target the nucleus in bioimaging studies.14 Some other metal complexes, including ruthenium, ytterbium, europium, organotin complexes have also been designed as nuclear bioimaging agents. Since we cannot cover all the reported work here due to the space limitation, we here provide a list of representative papers recently published by different research teams in the reference section for interested readers to retrieve.15–19

2.16.2.2.2

Mitochondria

Due to the proton-pumping effect induced by mitochondrial oxidative phosphorylation, the mitochondrial outer membrane possesses a strong negative potential as high as 180–200 mV. Because cationic species are natively attracted by this large mitochondrial membrane potential, it is relatively easy to develop metal complexes-based dyes for mitochondrial imaging.20,21 The characteristics and design methods of the mitochondria-targeting metal complexes are summarized and reviewed.6 Iridium complexes predominate in this area. In 2012, Lo and co-workers prepared a simple cyclometalated Ir(III) complex [Ir(bt)2(en)]þ (en ¼ ethylenediamine, bt ¼ 2-phenylbenzothiazole) (8, Fig. 3). The majority of overlap between 8 and commercial dyes indicated the complex was mainly localized inside the mitochondria.22 To the best of our knowledge, this is the first Ir(III) complex that specifically targets mitochondria due to its intrinsic properties, without incorporating mitochondria-targeting groups. Our group has performed detailed research in this area. In 2013, four Ir(III) complexes with the general formula:

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8

9

10a

10

9a

9b

10b

10c

9d

9c

10d

10e

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In Vitro t=0s

t = 30 s

t = 60 s

t =90 s

t=0s

t = 30 s

t = 60 s

t =90 s

t=0s

t = 30 s

t = 60 s

t =90 s

Fig. 3 The chemical structures of Ir(III) complexes for mitochondria staining. Complex 10 has been applied to track mitochondrial fission/fusion in vitro/vivo. In vitro: HeLa cells; In Vivo: C. elegans. Reprinted with permission from Ref. Huang, H. Y.; Yang, L.; Zhang, P. Y. et al. (2016c). RealTime Tracking Mitochondrial Dynamic Remodeling with Two-Photon Phosphorescent Iridium (iii) Complexes. Biomaterials 2016c, 83, 321–331. Copyright 2016, Elsevier.

[Ir(C^N)2(PhenSe)]þ were synthesized as multi-colorful mitochondrial dyes (9a-d, Fig. 3). Inductively coupled plasma mass spectrometer (ICP-MS) results indicated that their organelle specificity is independent of the size of the planar C^N ligand.23 On this foundation, several series of Ir(III) complexes were synthesized to investigate the influence of charge, hydrophilicity, electronic effects of the substituents on their mitochondria-targeting ability, including dinuclear Ir(III) complexes bearing 1,3-bis(1substituted-imidazo[4,5-f][1,10]phenanthroline-2-yl)benzene bridging ligands, mononuclear Ir(III) complexes containing different sizes of conjugated aromatic rings and so on.24–28 Results showed that the mitochondrial specificities could be related to increased charge and lipophilic. Among these Ir(III) complexes-based dyes, complexes 10 has been applied to track mitochondrial fission and fusion, morphological changes during apoptosis and autophagy (Fig. 3). Aggregation-induced emission (AIE) groups were also introduced to construct superior photostable mitochondrial imaging agents. With these AIE-active Ir(III) complexes, mitochondrial images can be performed without a washing step.29,30 With similar cationic and lipophilic property to that of Ir(III) complexes, some Ru(II),31 Re(II),32 Au(III),33 Cu(II),34 Zn(II),35 Ln(III),36 Ru(III)37 and Pt(II)38–40 complexes also were demonstrated to accumulate in mitochondria and acted as mitochondria

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selective probe for bioimaging. However, among the above examples, all but Re(II) complex are toxic and, in fact, are theranostic agents.

2.16.2.2.3

Lysosomes

2.16.2.2.4

Endoplasmic reticulum (ER) and Golgi apparatus

Lysosomes are recycling factories for cells. Low pH and more than 60 types of acid hydrolases are the characteristics of lysosomes.41 It helps to degrade biomacromolecules.42 Different from the nucleus and mitochondria that contain lipid bilayer membranes, the lysosomes are monolayered. Lysosomes are also involved in endocytosis, which is a general cell uptake mechanism for various extracellular materials.43 Thus, probes targeting lysosomes are relatively easy to achieve. The characteristics and design methods of the lysosome-targeting metal complexes are also summarized.44 In 2020, Williams and co-workers developed an Ir(III) complex bearing an imidazole derivative ligand (11, Fig. 4).45 Existed as either an anionic or a neutral ligand with pKa ¼ 6.6, a significant phosphorescence change was observed, which is useful for the targeted visualization of an acidic lysosome. With a similar mechanism, Aoki and Mao prepared Ir(III) complex having basic diethylamino (pKa z 7, complex 12, Fig. 4) and b-carboline groups (pKa ¼ 5.4, complex 13, Fig. 4), respectively.46,47 The pH-induced tautomerization led to the turn-on phosphorescence of complex 11–13, enabled their selective staining of lysosomes (pH ¼ 5.5) over mitochondria (pH ¼ 7.5). However, it should be noted that there was no direct evidence in these three reports that the complexes were accumulated in lysosomes. Their selective lysosomes imaging depends on the low pH of lysosomes. Unlike the above examples, our group has reported lysosome-accumulated Ir(III) complexes (14, Fig. 4).48 The introduced morpholine ring, an often-used lysosome targeting moiety, accounts for its lysosomal specificity. 14 exhibited pH-dependent emission due to the protonation or deprotonation of the morpholine ring. The emission intensity decreased as the pH value increased (pH 3– 9). 14 was capable of tracking the lysosomes specifically and retaining its green emission after four passages of cell culture (Fig. 4), and this robustness in lysosome-luminescence tracking was successfully applied to some long-term cellular processes, such as cell migration and apoptosis.49 In the same year, to make clear the relationship between lipophilicities and the lysosome targeting, our groups prepared another series of Ir(III) complexes bearing morpholine rings (15, Fig. 4). The organelle targeting of the complexes was turned by the N^N ligands with different lipophilicities. Colocalization experiment results showed that the hydrophobic complexes preferably stained the mitochondria, while the hydrophilic ones were mainly localized in the lysosomes.50 This conclusion was evidenced by a case of Ru(II) complexes.51 Due to the high positive charge (þ 8), these Ru(II) complexes were extremely hydrophilic. These complexes entered cells by endocytosis and then localized in lysosomes. Because of their photocytotoxicity, they acted as photosensitizers, which will be discussed later in the photodynamic therapy section. Except for the Ir(III) and Ru(II) complexes, lysosome imaging agents based on other metal complexes have been sporadically reported. Yam, Wong, and Lam developed tridentate platinum(II) complex to selectively stain lysosomes, respectively.52–54 Containing hydroxyl, pentaethylene glycol, or other protonatable groups, these Pt(II) complexes were hydrophilic and cationic in the acidic interior of lysosomes. It prevents the complexes from crossing the membrane and leaving the lysosomes. Baker et al. reported that a dinuclear gold(I) complex with N-heterocyclic carbene moiety and could preferentially stain the lysosomes.55 Massi et al. developed a Re(I) complexes with a 3-pyridyltetrazolate ligand as lysosome-targeting two-photon emissive probes.56 Zhang et al. employed ZnSalen applied in the lysosome imaging of a model living organism.57,58 The electronic states of the diamine moieties can effectively modulate the photophysical properties and subcellular localization of the complexes.59 Gasser et al. presented the use of bis(dipyrrinato)-Zn(II) complexes for lysosome monitor.60

The ER is a crucial organelle of the eukaryotic cell’s perinuclear region and contains half of the total membranes of the cell. The Golgi apparatus is a phospholipid membrane-based organelle which like the ER is made up of cisterna. Due to the similarity in the composition of ER and Golgi apparatus, meanwhile, there are few reports on metal-based probes that target these two organelles, we discuss them together. Because of the lipophilic membrane of the ER and Golgi apparatus, metal complex with the feature of lipophilicity and positive charge is thought to favor its enrichment at these two organelles.37 However, the relationship between the structure and the achievement of the ER/ Golgi apparatus targeting has not yet been summarized. By the addition of ER-targeting signal peptides, metal complex can avoid lysosomes uptake via the clathrin-independent pathway.61 To the best of our knowledge, there is no phosphorescent metal complex for the ER-targeted imaging agent only. Reports on ERtargeted phosphorescent metal complexes are therapeutic agents in reality. In 2011, Lo et al., report four cyclometalated iridium(III) diimine complexes (16, Fig. 5).62 Among them, the complex with 4,7-diphenyl-1,10-phenanthroline (DIP) (16d) was found to stain the ER. Its IC50 value was smaller than that of cisplatin. Subsequently, they prepared another three iridium(III) diimine complexes of endoplasmic reticulum imaging with higher antitumor activity (17, Fig. 5).63 In 2013, Fei’s group presented cationic iridium(III) complexes with an ancillary N^N ligand of DIP (18, Fig. 5).64 The complex accumulated in the ER and exhibited high cytotoxicity. It caused ER stress and initiated the intrinsic apoptotic pathway by inducing the cytosolic release of calcium and cytochrome c, finally leading to cell death. The first ER-targeting Ru(II) complex was reported in the same year by Thomas and coworkers.65 This dinuclear Ru(II) complex showed a turn-on emission upon the addition of liposomes and comparable cytotoxicity to cisplatin, acting as an ER imaging agent as well as an antitumor drug. The Che’s group developed a class of emissive Pt(II) and Ir(III) complexes bearing N-heterocyclic carbene ligands that can selectively localize to the ER domain and were cytotoxic to cancer cells.66,67 In addition to these tradition antitumor agents, ER-targeting Ir(III) complexes for PDT were also reported by Soumik, Nam and our group, respectively.66,68,69 Furthermore, our group presented a cyclometalated iridium(III) complex that can specifically

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Fig. 4 The chemical structures of metal complexes for lysosome staining. Complex 14 has successfully applied to long-term cellular processes (cell migration) tracking. Reprinted with permission from Ref. Qiu, K. Q.; Huang, H. Y.; Liu, B. Y. et al. Long-Term Lysosomes Tracking with a WaterSoluble Two-Photon Phosphorescent Iridium(iii) Complex. ACS Appl. Mater. Interfaces 2016c, 8, 12702–12710. Copyright 2016, American Chemical Society.

accumulate in the ER and induce immunogenic cell death (ICD) in non-small cell lung cancer (NSCLC).70 This Ir(III) complex is the first iridium(III) complex to induce ICD in non-small cell lung cancer. Since these ER targeted metal complexes are all theranostic agents, they will be discussed in detail in the subsequent antitumor section. Fewer Golgi targeted metal complexes have been reported. In 2012, Wong and co-workers developed iridium(III) complex that could localize in the Golgi apparatus (19, Fig. 6).71 Possessing low cytotoxicity, high emission intensity, and a large two-photon absorption cross-section, this complex was a potential Golgi apparatus imaging agents in vitro. In the same year, Policar et al. synthesized a rhenium(II) tris-carbonyl derivative as a single core multimodal probe for the Golgi apparatus imaging (20, Fig. 6).72 Lo and co-workers also presented two dendritic cyclometalated Ir(III) complexes that were found in the Golgi apparatus. Different from the

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The chemical structures of Ir(III) complexes for endoplasmic reticulum staining.

Golgimarker

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20 Fig. 6 The chemical structures of Ir(III) and Re(I) complex for Golgi apparatus staining. Reprinted with permission from Ref. Ho, C. L.; Wong, K. L.; Kong, H. K. et al. A Strong Two-Photon Induced Phosphorescent Golgi-Specific In Vitro Marker Based on a Heteroleptic Iridium Complex. Chem. Commun. 2012, 48, 2525–2527 and ref. Clède, S.; Lambert, F.; Sandt, C. et al. A Rhenium Tris-Carbonyl Derivative as a Single Core Multimodal Probe for Imaging (Scompi) Combining Infrared and Luminescent Properties. Chem. Commun. 2012, 48, 7729. Copyright 2012, Royal Society of Chemistry.

above two reports, these Ir(III) complexes exhibited higher cytotoxicity than cisplatin, indicating the complexes were potential as anticancer agents.73

2.16.2.2.5

Cytoplasm

The cytoplasm is the jelly-like fluid that fills the gap between the nucleus and the plasma membrane and consists of a transparent substance called hyaloplasm or cytosol.3 The above organelles such as mitochondria and lysosomes all exist in the cytoplasm. To date, many kinds of phosphorescent complexes, such as Pt(II),74,75 Ru(II),76–78 Ir(III),79–84 Re(I),85 Rh(III),86 Ln(III),87 Zn(II),88 Cd(II)88 and Au(I)55 complexes, have been exploited for cytoplasm staining.

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As mentioned above, since the clinical success of cisplatin, a large number of Pt(II) complexes have been developed. Although expected to target is nuclear DNA, some of these Pt(II) complexes could not enter the nucleus, but localized in the cytoplasm. In 2009, Che and Lam prepared cyclometalated Pt(II) complexes (21 and 22, Fig. 7), respectively.74,75 Possessing a leaving group, however, the hydrolysis of these two complexes was not explored. Complex 21 showed a turn-on emission upon the presence of bovine serum albumin (BSA). Confocal microscopy experiment results demonstrated that 21 and 22 could accumulate in the cytoplasm rather than the nucleus. Ru(II) complexes are another type of metal complexes that have been extensively studied. At the end of the last century, Barton began to investigate the interaction of Ru(II) complexes with DNA while studying the cellular uptake and intracellular distribution of Ru(II) complexes.76,78 Ru(II) complexes containing the DNA intercalating ligand dipyrido[3,2-a:20 ,30 -c]phenazine (dppz) but with different ancillary ligands were synthesized. These complexes show turn-on emission when bound to DNA or otherwise protected from water. All these complexes are found to stain the cytoplasm. Increasing the lipophilicity of the ancillary ligands can significantly enhance cellular uptake. Our group also reported a class of dinuclear Ru(II) complex for cytoplasmic imaging. It is interesting that bearing the same 1,3-bis(1-substituted-imidazo[4,5-f][1,10]phenanthroline-2-yl)benzene bridging ligands, dinuclear Ir(III) complexes localized in the mitochondria24 but dinuclear Ru(II) complexes accumulated in the cytoplasm (23–24, Fig. 8).77 The cationic and lipophilic Ir(III) complexes have a high ability to penetrate the cell membrane. In 2008, Huang and co-workers presented two iridium(III) complexes with bright green and red phosphorescence emissions by variation of the diimine ligand structure. The exclusive staining in cytoplasm, low cytotoxicity, and reduced photobleaching make them promising candidates for the design of specific phosphorescence bioimaging agents.83 Based on these, a series of cationic iridium(III) complexes, [Ir(dfpy)2L]þ (dfpy ¼ 2-(2,4-difluorophenyl)pyridine; L denotes a series of N^N ligands with different conjugated lengths) were synthesized by the same team (25–26, Fig. 9).84 By variation of the N^N ligands, the emission colors of these complexes were turned from blue to red, implying the potential of being multi-color cytoplasmic dyes. In 2014, Chen and Zhao prepared two water-soluble cyclometalated Ir(III) complexes. Different from the dyes using single molecules, the emission of these two complexes comes from their self-assembly nanoparticles.81 Ir(III)/Eu(III) dinuclear complex was investigated. Due to the d / f energy transfer, characteristic emission peaks coming from the Eu(III) component were observed in the total emission profile. Since the difference in the lifetime for the Ir(III) and Eu(III) motif, this Ir(III)/Eu(III) complex was able to carry out the cytoplasm imaging in time-gated detection mode.79,80 In addition to these non-toxic probes, many cytoplasm-targeting Ir(III) complexes exhibited high cytotoxicity. For example, Lo’s group reported a phosphorescent cationic Ir(III) complex functionalized with an N-methylamino-oxy group. Confocal microscopy revealed that this complex localized in the cytoplasm.82 This kind of cytoplasm targeting theranostic agents will be introduced in detail in the antitumor section.

Complex 21

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21 Complex 22

22 Fig. 7 The chemical structures of Pt(II) complexes for cytoplasm staining. Reprinted with permission from Ref. Wu, P.; Wong, E. L. M.; Ma, D. L. et al. Cyclometalated Platinum(ii) Complexes as Highly Sensitive Luminescent Switch-On Probes for Practical Application in Protein Staining and Cell Imaging. Chem. A Eur. J. 2009, 15, 3652–3656. Copyright 2009, John Wiley and Sons. Reprinted with permission from Ref. Koo, C.-K.; Wong, K.-L.; Man, C. W.-Y. et al. A Bioaccumulative Cyclometalated Platinum(ii) Complex with Two-Photon-Induced Emission for Live Cell Imaging. Inorg. Chem. 2009, 48, 872–878. Copyright 2009, American Chemical Society.

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Fig. 8 The chemical structures of Ru(II), Ir(III) complexes for cytoplasm and mitochondria staining, respectively. Reprinted from Ref. Xu, W.; Zuo, J.; Wang, L.; Ji, L.; Chao, H. Dinuclear Ruthenium(ii) Polypyridyl Complexes as Single and Two-Photon Luminescence Cellular Imaging Probes. Chem. Commun. 2014, 50, 2123 and Ref. Chen, Y.; Xu, W. C.; Zuo, J. R.; Ji, L. N.; Chao, H. Dinuclear Iridium(iii) Complexes as Phosphorescent Trackers to Monitor Mitochondrial Dynamics. J. Mater. Chem. B 2015b, 3, 3306–3314. Copyright 2014 and 2015, Royal Society of Chemistry.

Fig. 9 The chemical structures of Ir(III) complexes for cytoplasm staining. Reprinted with permission from Ref. Zhao, Q.; Yu, M. X.; Shi, L. X. et al. Cationic Iridium(iii) Complexes with Tunable Emission Color as Phosphorescent Dyes for Live Cell Imaging. Organometallics 2010b, 29, 1085–1091. Copyright 2010, American Chemical Society.

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2.16.2.2.6

Other cell organelles

It is well known that the cell membrane is the first barrier of a cell and controls the movement of substances in and out of the cell. Quite a few phosphorescent metal complexes were developed for membrane imaging. Recently, our group presented five new fluorinated Ru(II) complexes containing fluorinated ligands that exhibit excellent two-photon absorption properties (27, Fig. 10).89 The complexes were able to stain the cell membrane within 30 min. However, possessing good 1O2 quantum yields, these complexes acted as photosensitizers and killed cells effectively. Bioorthogonal labeling is a potentially feasible approach to achieve membrane localization of metal complexes. Lo and co-workers reported three Ir(III) complexes featuring a nitrone group, which could bioorthogonal react with the strained cyclooctyne and turned on the emission of the cycloaddition product.90 In the same year, by replacement of nitrone group with the 2,4,5-tetrazine motif, the same group synthesized four new Ir(III) complexes for bioorthogonal labeling. However, with strained cyclooctyne-modified BSA, the visible intracellular emission was observed in the cytoplasm rather than the cell membrane.91 Subsequently, the complex containing the 2,4,5-tetrazine motif was utilized to interact with the N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz)-modified glycans in the CHOK1 cells. Although the complex was initially labelled on the cell membrane by the orthogonal reaction, with the prolongation of incubation time, the complex gradually entered into the cytoplasm. Vacuoles are membrane-bound sacs that play roles in intracellular digestion and the release of cellular waste products.3 In 2007, Williams’ research team reported a Re(I) anionic complex with bathophenanthroline sulfate and hydroxymethylpyridine moieties. This complex was reported to digestive vacuoles (28, Fig. 10).85 Re(I) complex developed by Massi and Shandala et al. has also been used as a lipid droplets imaging agent (29, Fig. 10).56

2.16.2.3

Cellular molecule labeling and cellular physical state detection

Intracellular substances are traditionally divided into inorganic small molecules and biological macromolecules. The distribution of these substances varies in organelles. Organelle targeting molecule probes can be conveniently obtained by introducing corresponding recognition groups to the organelle-targeted metal complexes. For example, based on the mitochondrial-targeted Ir(III) complexes, our group reported detection probes for mitochondrial NO,92 HClO,93 SO294,95 and so on.

2.16.2.3.1

Metal ions

Iron, zinc, and copper are the three most abundant essential trace element in the human body and plays important roles in many fundamental physiological processes in organisms. The disruption of metal ions homeostasis can lead to various diseases, such as Alzheimer’s disease, Menkes and Wilson diseases, familial amyotrophic lateral sclerosis, and even cancer. Reports on cellular metal ions probes mainly focus on organic molecules. Metal complexes applied as metal ions sensors are rarely developed while those for intracellular detection are scarcer. Lo and co-workers reported three tricarbonyl Re(I) complexes (30, Fig. 11) as the first example of phosphorescent metal complexes that respond to intracellular Zn2þ.96 However, due to their high cytotoxicity, the application of

Fig. 10 The chemical structures of metal complexes for membrane, digestive vacuoles, and lipid droplets staining. Reprinted with permission from Ref. Qiu, K. Q.; Wang, J. Q.; Song, C. L. et al. Crossfire for Two-Photon Photodynamic Therapy with Fluorinated Ruthenium (ii) Photosensitizers. ACS Appl. Mater. Interfaces 2017, 9, 18482–18492. Copyright 2017, American Chemical Society. Reprinted with permission from Ref. Amoroso, A. J.; Coogan, M. P.; Dunne, J. E. et al. Rhenium Fac Tricarbonyl Bisimine Complexes: Biologically Useful Fluorochromes for Cell Imaging Applications. Chem. Commun. 2007, 3066–3068 and Ref. Bader, C. A.; Brooks, R. D.; Ng, Y. S. et al. Modulation of the Organelle Specificity in Re(i) Tetrazolato Complexes Leads to Labeling of Lipid Droplets. RSC Adv. 2014, 4, 16345–16351. Copyright 2007 and 2014, Royal Society of Chemistry.

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Fig. 11 The chemical structures of Re(I) and Ru(II) complex for cellular metal ions bioprobes. Reprinted with permission from Ref. Zhang, P. Y.; Pei, L. M.; Chen, Y. et al. A Dinuclear Ruthenium(ii) Complex as a One- and Two-Photon Luminescent Probe for Biological Cu2 þ Detection. Chem. A Eur. J. 2013, 19, 15494–15503. Copyright 2020, John Wiley and Sons.

these three Re(I) complexes are limited. In 2013, our group synthesized a dinuclear Ru(II) complex that could serve as a one- and two-photon phosphorescent probe for biological Cu2þ detection (31, Fig. 11).97 This complex showed a remarkable turn-on emission in the presence of Cu2þ ions. Importantly, it could select copper ions out of many other abundant cellular cations, such as Naþ, Kþ, Mg2þ and Ca2þ. High selectivity and low cytotoxicity make this complex as a sensing probe for the detection of Cu2þ in living cells and zebrafish. In addition to these essential trace element, environmentally important metal ions detection by metal complexes were reported. Huang and co-workers previously presented two phosphorescent Ir(III) complexes for Hg2þ detection in solution. Subsequently, they further developed an Ir(III) complex as a ratiometric probe for monitoring intracellular Hg2þ ions. An emission intensity increase was observed when HeLa cells were pretreated with the Ir(III) complex and further incubated with 10 equiv. of Hg2þ ions in the media for 30 min.3

2.16.2.3.2

Intracellular oxygen and hypoxic environment

Oxygen is a critical component for many physiological and pathological processes in live cells.3 Hypoxia refers to a low oxygen environment, which is one of the characteristics of solid tumors, also discussed here. As mentioned above, 3MLCT is the most commonly observed excited state for metal complexes. Generally speaking, the triplet-state is sensitive to oxygen. Therefore, in a certain sense, the phosphorescent complexes can all act as oxygen probes. In 2015, Tobita et al. developed five cytoplasmtargeting Ir(III) complexes. The emission intensity of the phosphorescent images was enhanced as the oxygen pressure of the incubation condition changed from normoxia mode (volume ratio: 21%) to hypoxia mode (2.5%). Importantly, the intracellular oxygen level could also be detected via the phosphorescence lifetime measurement.98 In 2016, Zhao and co-workers team reported a dinuclear Ir(III) complex that specifically localizes in the cytoplasm for the sensing of oxygen in solutions and the time-resolved luminescence imaging of intracellular oxygen level.99 Subsequently, they reported another three examples.100–102 In addition to these probes based on single-molecule, nanosensors for oxygen were also reported. Dutta et al. presented the synthesis of tris(2,20 -bipyridyl) Ru(II) chloride within the super-cages of a highly hydrophobic zeolite Y for monitoring intracellular oxygen in human monocyte-derived macrophages.103 There are other reports of metal complexes encapsulated or combined with nanomaterials for intracellular oxygen probe.104–106 But since oxygen generally quenches triplet state emission, this class of probes makes little sense for applications. Unlike the above-mentioned probes that depend on oxygen-quenching triplet state emission, our group developed a series of Ir(III) complexes to detect intracellular hypoxia based on their oxygen-sensing capability and azo-reduction switch (32, Fig. 12).107 Featuring an azo functional group in the bpy-based N^N ligand, these complexes are non-emissive. The oxygen-switch-on mechanism was triggered by the manual azo-reduction (or nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase in natural

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Fig. 12 The chemical structures of Ir(II) complexes for cellular hypoxic environment sensors. Reprinted from Ref. Sun, L. L.; Li, G. Y.; Chen, X. et al. Azo-Based Iridium(iii) Complexes as Multicolor Phosphorescent Probes to Detect Hypoxia in 3d Multicellular Tumor Spheroids. Sci. Rep. 2015b, e14837. Copyright 2015, Springer Nature. Reprinted with permission from Ref. Kuang, S.; Sun, L. L.; Zhang, X. R. et al. A Mitochondrion-Localized Two-Photon Photosensitizer Generating Carbon Radicals Against Hypoxic Tumors. Angew. Chem. Int. Ed. 2020b, 59, 20697–20703. Copyright 2020, John Wiley and Sons.

cells), in which this reaction converted the non-emissive azo-based complexes into the oxygen-sensitive amino-based analogs.49 Similarly utilizing the reductase in natural cells under hypoxic conditions, Ir(III) complexes with anthraquinone moiety were synthesized for hypoxia detection (33 and 34, Fig. 12).108,109 The anthraquinone group can be reduced through a two-electron reduction process and the Ir(III) complexes re-emissive.

2.16.2.3.3

Intracellular redox small molecule

The redox state balance is essential for every aspect of cell life. Cells produce reactive oxygen/nitrogen species through respiration or metabolism. Some of them also serve as intra- and intercellular signaling molecules, play important roles in the cardiovascular, immune, and nervous systems. Lysosomal hydrogen peroxide (H2O2) is closely associated with autophagy and apoptosis in normal and pathological processes. Imaging H2O2 in lysosomes is a powerful tool to elucidate its diverse roles. In 2013, Zhang et al. designed a Zn-Salen to detect H2O2 in cells.110 This probe targeted lysosomes and exhibited great reactivity and selectivity toward the transformation of “H2O2-Cl” to HClO by myeloperoxidase, which could light up the probe even at a pH of 4.5–6 (lysosomal pH range), while HClO itself could only light it up at pH > 8, far from lysosomal conditions. The production of hypochlorous acid (HClO) and ClO also results from the activity of myeloperoxidase. A mitochondrialtargeted Ir(III)-based HClO sensor was developed by our group (35, Fig. 13). After reacted with HClO, the highly phosphorescent compound, [Ir(ppy)2(mbpy-monoacid)]Cl (Ir-COOH) could be obtained. The thick zebrafish embryo image stained with the complex and LPS/PMA demonstrated the superiority of the two-photon excitation technique over one-photon excitation. Living zebrafish were also employed, and bright-emitted signals were observed in the zebrafish liver.111 As an important signaling molecule, nitric oxide (NO) is involved in a wide range of biological activities in living cells and tissues. Lippard and coworkers developed the first NO-sensitive reaction-based phosphorescent probes based on Cu(II) complex.112 The introduction of Cu(II) quenched the fluorescence of the probe, which could be restored by reaction with cellular NO. This probe was subsequently applied to visualize NO in vasculature with two-photon microscopy.113 The first phosphorescent probe for mitochondrial NO imaging in vivo was synthesized by our group (36, Fig. 13). Bearing 1,10-phenanthroline-5,6-diamine moiety that can rapidly respond to NO, the emission of the Ir(III) complex was restored after reacted with NO. This probe was successfully applied to detect endogenous NO inside live zebrafish.92 Recently, our group also reported a phosphorescent Ir(III) complex for bimodal visualization of endogenous NO in lysosomes (37, Fig. 13).114 Phosphorescent metal complex-based probes for detection of other redox small molecules and physical states have also been reported. Since we cannot cover all the reported work here due to the space limitation, we here provide a list of representative papers recently published by different research teams, including H2S,115 CO,116,117 SO2,118 ONOO,119 Hcy,120,121 Cys,120,121 GSH,122 viscosity,123 temperature124,125 and pH.126–129

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37 Fig. 13 The chemical structures of Ir(II) complexes for cellular HClO and NO bioimaging. Reprinted with permission from Ref. Li, G. Y.; Lin, Q.; Sun, L. L. et al. A Mitochondrial Targeted Two-Photon Iridium(iii) Phosphorescent Probe for Selective Detection of Hypochlorite in Live Cells and In Vivo. Biomaterials 2015b, 53, 285–295 and Ref. Chen, X.; Sun, L. L.; Chen, Y. et al. A Fast and Selective Two-Photon Phosphorescent Probe for the Imaging of Nitric Oxide in Mitochondria. Biomaterials 2015a, 58, 72–81 Copyright 2015, Elsevier. Reprinted with permission from Ref. Wu, W. J.; Guan, R. L.; Liao, X. X. et al. Bimodal Visualization of Endogenous Nitric Oxide in Lysosomes with a Two-Photon Iridium(iii) Phosphorescent Probe. Anal. Chem. 2019, 91, 10266–10272. Copyright 2019, American Chemical Society.

2.16.2.3.4

Intracellular biomacromolecule

The exploitation of optical probes capable of direct imaging of DNA in living cells has attracted a great of attention. Thomas et al. presented a dinuclear Ru(II) complex, which is non-emissive in water but displays intense luminescence when bound to DNA. Confocal microscopy result demonstrated that this complex can target nuclear DNA and visualize characteristic structural changes in nuclear DNA as cells progress through the cell cycle.79,80,130 RNA bioimaging agent based on metal complexes was also developed. Turro et al. introduced a phenanthridine moiety to the Ru(II) complex and found that it could act as an RNA probe for cell imaging.131 In addition to the normal structured DNA/RNA, probes for the secondary structure of DNA, the mismatched and abasic DNA have been developed.132

2.16.2.4

Conclusion

In summary, phosphorescent metal complexes have been widely applied in biological imaging, and greatly help people better understand life. However, due to space limitation, the above are only some examples. To provide more information in a limited space, we summarize the fundamental properties of these complexes in Table 1.

2.16.3

Phosphorescent metal complexes for chemotherapy

Cancer remains one of the most vital diseases despite all the great effort trying to find its cure for decades. Over the past 50 years, cisplatin has been the first-line clinical drugs for chemotherapy, which leads to continuous interest in the development of inorganic cytostatic anticancer drugs.155 Cisplatin, carboplatin, and oxaliplatin is the first generation of platinum-base anticancer drugs, but their clinical use is limited by their severe side effects, e.g. gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal pain), renal tubular injury, neuromuscular complications, and ototoxicity, as well as the acquired drug-resistance by cancer cells.156 Other transition metal-base anticancer agents are therefore developed, trying to solve the aforementioned problems. Among the most popular drug candidates, ruthenium and iridium compounds attracted growing attention as anticancer agents.49,157 Phosphorescent ruthenium and iridium complexes as chemotherapeutic agents are emphasized herein, as well as other phosphorescent metal anticancer agents.

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2.16.3.1

Summary of phosphorescent metal complexes as bioimaging probes.

Complexes

Localization or detection target

Imaging Level

References

1 2 3 4 5 6–7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25–26 27 28 29 30 31 32 33 34 35 36 37

Nucleolus Nucleus Nucleus Nucleus Nucleus Nucleus Mitochondria Mitochondria Mitochondria Lysosome Lysosome Lysosome Lysosome Lysosome/Mitochondria Endoplasmic reticulum Endoplasmic reticulum Endoplasmic reticulum Golgi Golgi Cytoplasm Cytoplasm Cytoplasm Mitochondria Cytoplasm Cell membrane / Mitochondria Vacuoles Lipid Droplet Zn2þ Cu2þ Hypoxia Hypoxia Hypoxia HClO NO NO

Monolayer Cell Zebrafish Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell C. elegans Monolayer Cell Monolayer Cell Monolayer Cell 3D Spheroid Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell Monolayer Cell 3D Spheroid Monolayer Cell Monolayer Cell Monolayer Cell Zebrafish 3D Spheroid 3D Spheroid 3D Spheroid Zebrafish Zebrafish Zebrafish

133 134 135 136 137 138 139 140 64 70 141 142 143 144 145 146 147 148 149 150 25 151 152 153 154 85 56 96 97 107 109 141 111 92 114

Phosphorescence in chemotherapy

The luminescence of metal complexes allows the visualization of the distribution of the complexes in cells and real-time monitoring of the interactions between the complexes and biomolecules, which might give a hint to their molecular mechanism of action.25,150,151 The phosphorescence of the metal complexes could easily show their cellular uptake and help determine the preference and selectivity of the anticancer agents toward different cell lines.136 In many cases, the phosphorescence indicates the initial location of the complex, which can be an important evidence or thread of the molecular target of the complex and its anticancer pathway.138,140,145 The mode of action of the anticancer agent can also be revealed by monitoring the phosphorescence that reflects the localization of the complex.141 We summarize the localization of phosphorescent chemotherapeutic agents in Table 2.

2.16.3.2

Phosphorescent ruthenium complexes as chemotherapeutic agents

Ruthenium compounds are popular candidates as anticancer agents, for plenty of ruthenium-base agents have been synthesized and tested for potential antitumor activity.157 Ruthenium is a transition metal of the platinum group. Hence, the anticancer mechanism of ruthenium complexes was first expected to be DNA interaction as one of the platinum drugs. Further investigation, however, indicates several differences from traditional platinum complexes: (1) ruthenium appears preference for neoplastic accumulation rather than normal tissues, probably due to transferrin transportation. Ruthenium compounds can be actively transported into neoplastic tissues with a high density of transferrin receptors;158 (2) some ruthenium agents demonstrate greater efficacy against cancer metastases than against primary tumors.159 These differences drive unique patterns of antitumor activity and clinical toxicity distinct from those of platinum drugs.160

Phosphorescent metal complexes for biomedical applications Table 2

475

Summary of phosphorescent metal complexes as chemotherapeutic agents.

Complexes

Localization determined by phosphorescence

References

38–41 42 43a-45c 46 47 48a-48d 49a-49b 50a-50b 51 52 53 54 55 56a-56d 57a-57d 58a-58d 59 60 61a-61b 62 63 64 65a-65b 66a-66c 67

Nucleus Cytoplasm / nucleus Cellular membrane Mitochondria Mitochondria and endoplasmic reticulum Mitochondria Cytoplasm Cellular membrane Endoplasmic reticulum Endoplasmic reticulum Lysosome / mitochondria Lysosome Lysosome Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Protein-protein interaction Protein-protein interaction Protein-protein interaction Lysosome and mitochondria Lysosome and mitochondria Lysosome / mitochondria

133 134 135 136 137 138 139 140 64 70 141 142 143 144 145 146 147 148 149 150 25 151 152 153 154

Several ruthenium-base chemotherapeutic agents have entered clinical trials (Fig. 14). NAMI-A, with a formula [ImH][transRuCl4(dmso-S)(Im)] (dmso-S ¼ sulfur-bonded dimethyl sulfoxide, Im ¼ imidazole), is the first ruthenium compound that enters clinical trial, and also the only one that reaches the phase II stage.161,162 NAMI-A was developed for the inhibition of tumor metastasis but was ultimately eliminated due to its low therapeutic efficacy and the progression of disease in clinical investigation.162 KP1019, which is transported into the cell via transferrin cycle and activated by reduction, is the second Ru-based anticancer agent to enter clinical trials.163 After both of them ended the trials, KP1339, also named KP-1339, or NKP1339, or IT139, is the only Rubased chemotherapeutic agent currently under clinical trials.163–165 To date, KP1339 has completed phase I clinical trial that evaluates the safety, tolerability, maximum tolerated dose, pharmacokinetics, and pharmacodynamics. KP1339 was found to induce immunogenic cell death (ICD) signature, which can initiate a prolonged immune response against the tumor.166,167 Owing to the in vitro success of NAMI-A, ruthenium compounds belonging to NAMI-A class were developed, and loaded in liposomes, to cross cellular membranes.168 The mechanism of action of many Ru-based agents relies on the interaction with the traditional target, DNA.133,169–172 Wang reported a series of nuclear-targeted ruthenium(II)-arene complexes of the type [(h6arene)-Ru(N^N)(X)]n þ, in which X is a monodentate ligand (chloride or pyridine, N^N are chelating ligands (Fig. 15, 38-41).133 Though emissive, these Ru(II) compounds were supposed to emit fluorescence by the dppn group rather than phosphorescence. Other Ru-based agents that combat cancer cells by different mechanisms were reported, including the ones that target the hypoxia conditions in solid tumors,173,174 or exhibit strong anti-angiogenesis effect by down-regulation of vascular endothelial growth factor receptor-2.175,176 Some ruthenium complexes exert different modes of cell death, i.e. paraptosis, and necroptosis.177,178 Some highlight their inhibitory effects of proteins, such as topoisomerase,178,179 vascular endothelial growth factor receptor-2,175 and p53 and p21, two important proteins of many cell death pathways.134 Many of the aforementioned compounds are non-phosphorescent, which we will not discuss much here, but among the proteinregulating agents, the one developed by Chao and coworker exhibited strong phosphorescent property (Fig. 15).134 The ruthenium(II) b-carboline complex [Ru(tpy)(Nh)3]2 þ (tpy ¼ 2,20 :60 ,200 -terpyridine, Nh ¼ Norharman, 42) induced apoptosis via the mitochondrial pathway, and cause accumulation of p53 proteins from phosphorylation at Ser-15 and Ser-392 correlated with an increase in p21 and caspase activation and suppressed MCF-7 and HepG2 tumor growth in mouse. Subcellular targeting properties were shown by Ru-based agents. A series of ruthenium polypyridyl complexes containing N,Nchelating ligands were synthesized by Chen et al. (43a-45c, Fig. 16).135 45c accumulated in the cell membrane and induce mitochondria-mediated and caspase-dependent apoptosis in human cancer cells, which can be suppressed by z-VAD-fmk, a general caspase inhibitor. In 2018, Chen reported a multifunctional mitochondria-targeted ruthenium-based conjugate, 46, with rational design (Fig. 16).136 By the conjugation of biotin, 46 can be selectively internalized by tumor cells, thus minimizing side effects toward normal organs. The pH-response of the Ru conjugate in the tumor acidic microenvironment causes ligand substitution

476

Phosphorescent metal complexes for biomedical applications

Fig. 14

Ruthenium chemotherapeutic agents that entered clinical trials.

Fig. 15

Chemical structures of complexes 38–42.

Fig. 16

Chemical structures of complexes 43a-46.

Phosphorescent metal complexes for biomedical applications

Fig. 17

477

Chemical structures of complexes 47-48d.

and release of the therapeutic complex. The phosphorescence of 46 allows the real-time tracking and imaging of the drug and revealed its selective accumulation in tumor tissue in vivo to realize enhanced theranostic effects. Song and co-worked presented a novel ruthenium(II) triazine complex, 47, which effectively distributed into mitochondria and endoplasmic reticulum (ER) (Fig. 17).137 47 significantly reduced the protein levels of glucose-regulated protein 78 (GRP78), a protein associated with drug resistance, and executed ROS-mediated calcium-dependent apoptosis in drug-resistant cancer stem cells (CSCs). Li et al. reported a series of polypyridyl ruthenium(II) complexes, 48a-48d, which mainly distributed in mitochondria and induced the dissipation of mitochondrial membrane potential in HepG2 cells (Fig. 17).138 Interestingly, Li and coworker showed the anti- proliferation property of 48a by the suppression of tumor growth in a zebrafish xenograft model.

2.16.3.3

Phosphorescent iridium complexes as chemotherapeutic agents

Iridium complexes as chemotherapeutic agents are of great interest.180–184 Some of the very first designs of iridium chemodrugs started with the mimicking of the structure or mechanism of action, of cisplatin, where the hydrolysis of the chloride ligands leads to the direct binding of the complexes and DNA. Giraldi et al. focused on the d8 square-planar 1,5-cyclooctadiene Ir(I) complexes due to their structural similarity to cisplatin and these complexes did show some anticancer activity.185,186 However, more early attempts for the design of Ir(III) complexes failed. For example, cytotoxic activity of the bis(imidazole) tetrachloride complexes [Ir(Im)2Cl4] and [Ir(Im)(DMSO)Cl4] (DMSO ¼ dimethylsulfoxide) was significantly less than that of their Ru(III) analogs [Ru(Im)2Cl4] and [Ru(Im)(DMSO)Cl4].187,188 As a result, iridium complexes were usually considered “inert” in terms of biomedical activity due to their prevailing chemical inertness.155 “Half-sandwich” cyclometalated Ir(III) complexes were later developed, and exhibiting considerable cytotoxicity against a range of cancer cell lines.181 The half-sandwich iridium complexes following this formula [(Cpx)Ir(L^L’)Z]0/n þ (with Cp* or extended Cp* and L^L’ ¼ chelated C^N or N^N ligands) can process potent cytotoxicity and catalytic activity which usually affects the redox balance of the cells. Sadler and coworkers have nicely reviewed these organoiridium complexes as both catalysts and anticancer agents,181 but unfortunately, many of them are nonluminous, which we will not discuss much here. On the other hand, octahedral cyclometalated Ir(III) complexes are getting increasing attention and applied in bioimaging and cancer treatment due to their luminescence properties.189–191 Unlike ruthenium and platinum complexes, the active sites of many octahedral cyclometalated Ir(III) complexes are not the nuclei, as revealed by their phosphorescence using a confocal laser scanning microscope (CLSM). Mao reported two cytoplasm-localized iridium(III)-b-carboline complexes (49a-49b) as potent autophagy-inducing agents via the inhibition of mTOR signaling (Fig. 18).139 In contrast to the caspase-dependent apoptosis induced by cisplatin with autophagy as a cytoprotective response, 49b induces caspase-independent autophagic cell death in the absence of apoptosis.

Fig. 18

Chemical structures of complexes 49a-49b.

478

Phosphorescent metal complexes for biomedical applications

A new family of cyclometalated Ir(III) oligocationic peptides (50a-50b) with remarkable cytotoxicity was presented by Salvado et al. (Fig. 19).140 The luminescent properties of 50a-50b allowed the observation of their aggregation as particular spots on the cellular membrane and a membrane-disrupting mechanism of action was shown by trypan blue exclusion assay and model experiments in fluorogenic artificial vesicles. Endoplasmic reticulum (ER) can be a subcellular target for Ir(III) complexes. The endoplasmic reticulum (ER) is an essential organelle with multiple functions. Based on the phospholipid membrane, ER can be divided into the rough ER and the smooth ER. The rough ER is coated with ribosomes for the folding and functionalization of proteins, while the smooth ER is responsible for lipid synthesis and signal transfer including calcium ion, enzymes, cholesterol, and ROS.192,193 [Ir(ppy)2(N^N)]þ (ppy ¼ 1phenyl-pyridine; N^N ¼ 4,7-diphenyl-1,10- phenanthroline (DIP, 51) was developed by Fei and coworkers (Fig. 20).64 51 was localized in the membrane structures in cells and accumulated in the ER, inducing ER stress and mitochondria-mediated apoptosis. ER-targeting iridium(III) complex (52) which induces immunogenic cell death in nonsmall cell lung cancer was reported by Chao (Fig. 20).70 52 contains an N,N-bis(2-chloroethyl)-azane derivate, which was inspired by another ICD inducer cyclophosphamide. The characteristic discharge of damage-associated molecular patterns (DAMPs), i.e. cell surface exposure of calreticulin (CRT), extracellular exclusion of high mobility group box 1 (HMGB1), and ATP, were generated by 52 in non-small cell lung cancer cells (A549). A vaccination assay using 52-treated dying cancer cells significantly suppressed the tumor growth in mice, indicating the occurrence of ICD effect in vivo. 52 is the first iridium-based complex that is capable of developing an immunomodulatory response by immunogenic cell death. Lysosomes can be popular targets of iridium complexes. Lysosomes are the cellular waste disposal units involved in protein degradation and waste recycling. Activators and soluble hydrolytic enzymes are packed inside their one-layer membrane, whose damage might cause the leakage of the content and leads to severe degradation of biomolecules and activation of the cell death pathway.194 Chao reported lysosome-targeting iridium(III) prodrug (53), which can be activated by iron(III) in cells (Fig. 21).141 The meta-imino catechol group in the complexes can be oxidized by free Fe(III) inside the gastric cancer cell, and release Fe(II) as well as the aminobipyridyl Ir(III) complex that migrates to mitochondria and 2-hydroxybenzoquinone, enhancing its phosphorescence and toxicity. Many half-sandwich Ir(III) complexes were also developed as lysosome-targeting anticancer agents by Liu, but only two of them are emissive.142,143,195–202 The luminescence of 54 easily indicated their subcellular localization, which is in lysosomes. 54 can convert the coenzyme 1,4-dihydronicotinamide adenine dinucleotide (NADH) to NADþ.142 The annexin V/propidium iodide dual-staining in A549 cells after treated with 54 indicated the apoptosis/necrosis dual-induction, accompanied by overproduction of reactive oxygen species, and disruption of the mitochondrial membrane potential. Liu also reported an N-heterocyclic carbene-modified half-sandwich iridium(III) complex (55), which had potent cytotoxicity studies toward various cancer cell lines (Fig. 21).143 Caspase-associated apoptosis initiated by the lysosomal-mitochondrial pathway may be involved in the death mechanism of 55-treated cells.

Fig. 19

Chemical structures of complexes 50a-50b.

Fig. 20

Chemical structures of complexes 51–52.

Phosphorescent metal complexes for biomedical applications

Fig. 21

479

Chemical structures of complexes 53–55.

Mitochondria are another popular subcellular targets of iridium complexes. Mitochondria take part in the regulation of cell metabolism, biomolecule synthesis, and cell division, the dysfunction of which can result in ischemia-reperfusion injury, diabetes, neurodegenerative disease, and even cancer.203 Over 80% of ATP are produced in these “power plant” of cells and mitochondria are also responsible for calcium metabolism, electron transport, reactive oxygen regulation, and the initiation of apoptotic pathways. Chao and Chen synthesized a series of mitochondria-targeted iridium(III) complexes (56a-56d), which can initiate mitochondriamediated apoptotic cell death in cisplatin-resistant lung cancer cells (A549R) (Fig. 22).144 Chao presented a series of mitochondriatargeting cyclometalated Ir(III) complexes, which activated the oncosis-specific protein porimin and calpain in cisplatin-resistant cell line A549R, and determined their cytotoxicity against a wide range of drug-resistant cancer types (57a-57d, Fig. 22).145 It is indicated that 57a-57d induced oncosis, a non-apoptotic mode of cell death, in cancer cells. Mitochondria swelling, plasma membrane bleb, and cytosol vacuolization, the other three typical phenomena of oncosis, were characterized by confocal microscopy and transmission electron microscopy (TEM). Over-generation of ROS, loss of mitochondrial membrane potential, leakage of lactate dehydrogenase (LDH), and ATP depletion were also observed. Later in 2020, Fei reported structure-tuned membrane-active Ir-complexed oligoarginine which induces oncosis and triggers immune responses in mice.204 Suntharalingam reported a series of mitochondria-targeted iridium(III) complexes with high charge as mitochondriotropics to cancer stem cells (58a-58d, Fig. 22).146 59 was presented by Liu, as an apoptosis and autophagy inducer in B16 cells through inhibition of the AKT/mTOR pathway.147 Similar to 56a-56d, complexes 60-61b generates mitochondrial dysfunction apoptosis way of cell death in cancer cells.148,149 Protein-protein interactions are important targets of the therapeutic intervention for the treatment of human diseases. Collaborating with Wang and Cai, Ma and Leung reported 62 as the first metal-based, irreversible inhibitor of bromodomain-containing protein 4 (BRD4)-acetylated histone protein-protein interaction (Fig. 23).150 They presented an iridium(III) complex (63) in 2016, to regulate the p53/hDM2 protein-protein interaction (Fig. 23).25 63 is the first reported organometallic p53/hDM2 protein-protein interaction inhibitor. In 2017, Ma and Leung collaborated with Cai again and developed an inhibitor (64) of the Ras/Raf interaction (Fig. 23).151 D-enantiomer of 64 showed superior potency in the biological assays compared to L-64 or racemic 64. Both 62 and 64 showed repression in cancer xenografts in mice. Some other works regarding phosphorescent iridium complexes were published in recent years, focusing on the cytotoxicity, anti-migration property, and antitumor activity in vitro or in a zebrafish model.205–208 To improve the biochemical characteristics and increase the biocompatibility of iridium(III) complexes, liposomes are widely used as the vehicle (Fig. 24). The Liu group developed two iridium(III) complexes (65a-65b) and loaded them into liposomes.152 The loaded complexes showed increased cytotoxicity as expected and induced apoptosis in B16 through ROS-mediated lysosomalmitochondria dysfunction, inhibition of polymerization of microtubules, and induce cell cycle arrest at S phase as well as in vivo antitumor efficacy. Liu then designed a series of liposome-encapsulated iridium(III) complexes (66a-66c), which also increase the cytotoxicity and induce apoptosis in A549 cells through a ROS-mediated lysosome-mitochondria dysfunction pathway.153 Another liposome loaded iridium(III) complex was reported by Liu (67).154 The liposome 67 generated intracellular ROS that regulates lysosomal-mitochondrial dysfunction, followed by microtubule disruption that subsequently led to a G0/G1 phase of cell cycle arrest.

480

Phosphorescent metal complexes for biomedical applications

Fig. 22

Chemical structures of complexes 56a-61b.

Fig. 23

Chemical structures of complexes 62–64.

2.16.3.4

Other phosphorescent metal complexes as chemotherapeutic agents

Many designs of platinum chemotherapeutic agents are based on the primitive structure of the first generation of the platinum-base anticancer drugs such as cisplatin, carboplatin, and oxaliplatin. Most of these new platinum drugs inherit the non-emissive property of cisplatin. There are some designs where certain luminophores are linked to the platinum complex to make the whole complex emissive, e.g. the nucleolus-targeted Pt(IV) complex reported recently by Zhu.209 As delicate as these designs are, the emission of those luminophores is often fluorescence, instead of phosphorescence. Zhu et al. reported dual-emissive platinum(II) metallacage with a sensitive oxygen response.210 A Pt(II) complex with red phosphorescence in a deaerated atmosphere is chosen. The metallacage emits blue fluorescence in both normoxia and hypoxia, but the red phosphorescence is highly dependent on the oxygen level and therefore becomes a sensitive ratiometric probe for the imaging of hypoxia and imaging-guided chemotherapy.

2.16.4

Phosphorescent metal complexes for photodynamic therapy

Photodynamic therapy (PDT) is a promising treatment modality with minimal invasiveness for localized cancers that are accessible by using fiber optics, such as cancers of the skin, esophagus, lung, and bladder, etc., and non-cancer diseases, such as bacterial

Phosphorescent metal complexes for biomedical applications

Fig. 24

481

Chemical structures of complexes 65a-67.

infection, viral infection, and fungal atherosclerosis, etc.211 As shown in Fig. 25, in a PDT process, the essentially non-toxic photosensitizer (PS) is excited to the triplet excited state by the irradiation of light, and subsequently transfers its energy to O2 (type II) or interacts with intermediate molecules in an electron transfer manner (type I), generating reactive oxygen species (ROS). The mainstream focus of the PS development for the last half-century has been on organic molecules which are primarily represented by porphyrin derivatives, while transition metal complexes have long been underappreciated, and limited to the field of chemotherapy. The past decade witnessed the emerging interest in extending the use of transition metal complexes to the realm of phototherapy (including PDT) by their distinct appealing characteristics over their organic counterparts in many facets. Particular interest has been devoted to the transition heavy metals of low spin d6, and d8 electronic configuration, such as Ru(II), Ir(III), Pt(II), Os(II), and Re(I), etc.183,211–217 The reason for a growing interest in transition heavy metal complexes as PSs is that they meet several basic needs for PDT. While ISC could be a serious impediment to the development of organic molecule-based PDT therapeutics, the most salient inherent feature for heavy metal complexes is represented by their heavy-atom effect whereby a singlet excited state of the complex is easily induced to a relatively long-lived triplet excited state, which would, therefore, provide ampler time for the excited states to interact with oxygen (or other intermediates) generating more ROS. Compared to that of singlet excited states, emission from triplet excited states allows a higher quality of PDT-involved phosphorescent theranostics by limiting the extent of concentration quenching as well as avoiding “cross-color interference.”1 Unlike many organic molecules that tend to suffer from photo-bleaching,218 seldom (if at all) in current reports are there any evident indications of the photo-bleaching of robust transition metal complexes, which

Fig. 25

Jablonski energy level diagram for type I and type II PDT.

482

Phosphorescent metal complexes for biomedical applications

favors their use in PDT. Added to these attractive photophysical properties is the relative ease with which transition metal complexes can be synthesized, where judiciously selected ligands and metal ions can be combined in a combinatorial manner, dramatically enriching their fine-tuning potential for desired properties. There are four metal-based PSs (i.e., WST11, PdII, approved for vascular-targeted prostate cancer; Lutex, LuIII, for cervical intraepithelial neoplasia; Purlytin, SnIV, for age-related macular degeneration; TLD1433, RuII, for non-muscle invasive bladder cancer) under ongoing clinical PDT investigation.219 Transition phosphorescent metal complexes encompass various metal centers with distinct reactivities ranging from kinetically labile to substitutionally inert. Among them, kinetically labile species possess chemotherapeutic potency generally via the release of the metal centers to interact with protein or nucleic acids to trigger biological responses, which deviates from the benign benchmark of PSs without the light input. As such, many kinetically reactive Pt(II) complexes are eliminated from consideration in regard of PDT agents. Moreover, Pt(II) adopts planar coordination geometry which facilitates the interactions between the Pt(II) complexes and the photogenerated 1O2, readily leading to the oxidation of the ligand or even the formation of Pt(IV). Re(I) tricarbonyl lowspin d6 centers are substitutionally inert and robust motif in the context of biological environments. However, they show less promise and are relatively understudied in terms of PSs because of their high energetic absorption profiles which in most cases locate in the UV region and generally shorter excited state lifetime compared with their electronic congener Ir(III) complexes. Of note, the lability of axial halogen ligand in Re(I) complexes also poses problems of unwanted toxicity by ligand substitution. Indeed, Ru(II) polypyridyl complexes are the most widely investigated metal complexes as PDT agents. Tris-diimine homoleptic Ru(II) complexes are the earliest reported species with long-lived 3MLCT excited states in the scope of microsecond. The MLCT charge transfer in Ru(II) polypyridyl complexes results in a moderate absorption in the visible region of around 450 nm which is advantageous over other metal species in the realm of PDT. Nevertheless, Ru(II) complexes are prone to cross membrane via passive diffusion and most tris-diimine homoleptic Ru(II) complexes are unfortunately hindered by poor cellular uptake. Exchanging Ru(II) to Os(II) generally results in a lowering of MLCT energy level. By judicious ligand selection, Os(II) complexes can absorb throughout visible region and even the NIR, albeit weakly. Unlike many Ru(II), Os(II) and Re(I) complexes that are dominated by 3MLCT states, Ir(III) complexes can possess ligand-to-ligand charge-transfer (3LLCT), metal-to-ligand chargetransfer (3MLCT) and ligand-centered triplet states (3LC). The excited state properties of iridium complexes can also be susceptible to changes in biological microenvironment which endows them additional utility in designing smart drugs.220 In addition, cyclometalated Ir(III) complexes often demonstrate intrinsic preferential localization patterns in cellular compartments, such as mitochondria, ER, Golgi apparatus, etc., making them applicable to targeted PDT. PDT therapeutic efficacy in most cases is achieved by the collaboration of O2, PSs, and light, the absence of any of which would render the whole therapeutic platform invalid. Made much effort as scientists have to push the envelope for PDT development, there have been recurring challenges in the clinic concerning the simultaneous assembly of these three components in diseased tissues. In the present chapter, the counterstrategies to overcome these challenges will be emphasized. However, the present section doesn’t intend to make an overall coverage of metal-complexes-based PDT agents. Instead, it will underscore the progress of some classical phosphorescent metal complexes such as Ru(II), and Ir(III) complexes, and meanwhile some high-impact advances on other phosphorescent heavy metal complexes will be reviewed.

2.16.4.1

Phosphorescent Ru(II) complexes for PDT

Ru(II) complexes have revealed an amazing range of potential biological applications ever since F. P. Dwyer et al. pioneered the biological activity study of Ru(II) complexes in the 1950s.221 Of note, one phosphorescent inert polypyridyl Ru(II) complex, TLD1433,222 has entered phase IIB clinical trials for bladder carcinoma PDT therapeutics (Fig. 26). In most cases, Ru(II) complexes exhibit short retention time in live organisms, which is a favorable property for PDT application.223–225

Fig. 26

Chemical structure of TLD1433.

Phosphorescent metal complexes for biomedical applications 2.16.4.1.1

483

Elongating excited-state lifetime

Most PSs undergo a type II process wherein the interaction between excited-state PSs (PSs*) and O2 takes place to give rise to the generation of ROS. Because of this, a longer PSs* lifetime should ensure more efficient conversion of O2 into ROS. Indeed, the relationship can be described as the Stern-Volmer equation226: s0 =s ¼ 1 þ ks0 ½O2  where s0 and s are the lifetime of PS* in the absence and presence of O2, respectively, and k is the rate constant for the diffusionlimited reaction. Given the above, molecules with a longer lifetime would be more preferable. An elegant strategy to elongate the excited-state lifetime of Ru(II) complexes is by combining the metallic coordination moiety with appropriate organic chromophores, such as BODIPY (4-bora-3a,4a-diaza-s-indacene), anthracene, pyrene, coumarin, and naphthalimide, etc., forming metal-organic dyads/conjugates with excited-state equilibration. Of note, some reviews have already nicely introduced and summarized this field.227–229 The McFarland group reported a series of similar pyrenylethynylene-appended polypyridyl Ru(II) dyad, [Ru(bpy)2(L)]2 þ (68a-68e, Fig. 27, bpy ¼ 2,20 -bipyridine), and presented a comprehensive study on their physiochemical properties and PDT efficacy in a metastatic melanoma model.230 In their study, an unprecedented long-lived 3ILbased excited states for Ru(II)-based dyads containing one organic chromophore with lifetimes spanning from 22 to 270 ms in fluid solution and from 44 to 3440 ms in glass at 77 K were recorded. These excited states were proven to be extremely sensitive even to a trace amount of oxygen due to the long lifetimes and very low radiative rates. In the anticancer PDT therapeutics by these Ru(II) complexes, the photocytotoxicity index (PI) reached a record high level (1747) toward pigmented metastatic melanoma cells where the presence of melanin, as well as low oxygen tension is PDT compromising factor. This work underlines the PDT potency of metalorganic dyads as a promising strategy to combat malignant melanoma by elongation of the PSs* lifetime. In their later work, they combined the PIP (2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline) scaffold with a pyrene moiety constructing a bichromophoric system where the low-lying 3IL state could act alone or in concert with 3MLCT state in Ru(II) polypyridyl complexes, [Ru(L)2(ippy)]2 þ (L ¼ bpy, phen, dmb, dtbb; ippy ¼ 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]-phenanthroline, phen ¼ [1,10]-phenanthroline, dmb ¼ 4,40 -dimethyl-2,20 -bipyridine, dtbb ¼ 4,40 -di-t-butyl-2,20 -bipyridine).231 The resulting dyads exhibited very high lethality toward HL-60 and Streptococcus mutans bacteria under irradiation even in a low oxygen tension, which can be attributed to the long-lived 3IL state.

2.16.4.1.2

Enhancing light-harvesting ability

The absorption intensity of light alongside singlet oxygen quantum yield decides the upper limit of the gross ROS production in a PDT therapeutic regimen. A strong light-harvesting ability in the visible range is, therefore, especially sought after when one wants to design efficient PDT agents. The most adopted strategy for metal complexes to enhance light-harvesting performance is by coupling an organic chromophore to the main framework. When the metal centers are directly conjugated to the p system of light-harvesting chromophores, the metal atom generally functions as a heavy atom substituent of the chromophore to prompt ISC228 Such a manner is more facile in synthesis and charge manipulation in comparison to the direct use of halogen substituents as the heavy atom. However, when the metal center and chromophores are linked via a long-range non-p-conjugated spacer, the insufficient electronic communication between the subunits shapes an antenna-like dyad wherein the electronic properties of the individual units retain. Intramolecular charge transfers between the two subunits could take place if the organic chromophores possess a higher energy level 1IL state than the 1MLCT state of the metal center. Under this circumstance, the dyads are capable of funneling the energy to metal centers, giving rise to enhanced light harvesting. In particular, organic chromophores like pyrene, coumarin and naphthalimide have proved to be effective in funneling excitedstate energy to the [Ru(phen)3]2 þ moiety and thus enhance the absorption intensity of the dyads.232–234 However, some chromophores displayed the opposite trend because of the reverse order of energy levels. For instance, PBI (perylene bisimide) derivative exhibits a very low-lying p-p* transition excited state, which is unlikely to help pool the MLCT state of [Ru(bpy)2(phen-PBI)]2þ, but, on the contrary, is very likely to eclipse the MLCT transition.235 BODIPY was also found to possess a higher energy level 1IL state than the MLCT state of Ru(II) center in [Ru(tpy)(tpy-BODIPY)]2þ framework.236 Of note, Drapper and coworkers reported a bimetallic Ru(II) polypyridyl BODIPY complex via acetylene linkers (69, Fig. 28), and found that the enhanced absorption is imparted

Fig. 27

Ruthenium(II) dyads (68c-68e) with long-lived excite state lifetime, and reference complexes (68a, 68b) reported by the McFarland group.

484

Phosphorescent metal complexes for biomedical applications

Fig. 28

Structure of binuclear complex 69.

by the inclusion of the second Ru(II) center but not the BODIPY moiety.237 The resultant complex showed dual phosphorescence at room temperature (assigned to 3IL* and 3MLCT*), indicating that the Ru(II) centers not only play the role of the spin converter but also are excited-state energy donators in the photophysical process. The dual triplet-excited states combined prompted an efficient PDT therapeutic outcome in HeLa cells.

2.16.4.1.3

Extending absorption profile to phototherapeutic window

NIR biological spectral window is beyond reach for traditional phosphorescent Ru(II) complexes of which the penetration depth of light through tissues is therefore highly limited. There are several empirical strategies to extend the absorption profile to phototherapeutic window in addition to the method of direct metal chelation to red/NIR-light-absorbing organic chromophores.238–241 One potent access to realize red-shifted absorption profile lies in extending the p system of the ligand. Gasser and coworkers launched a detailed study on the extension of [Ru(bpy)3]2 þ core aiming to shift the MLCT absorption toward the red region.242 The [Ru(bpy)3]2 þ core extended with vinyl dimethylamino groups outperformed the one with methyl groups or the [Ru(bpy)3]2 þ core alone by a 65 nm red shift in the maximum of MLCT transition as compared to the other two complexes. Of note, the absorption tail of the optimal compound is in the desired phototherapeutic window (600–900 nm). This character is tentatively concluded as the result of an extended p system along with electron-donating dimethylamino moieties. In their recent work, they rationally designed a series of long-wavelength absorbing Ru(II) polypyridyl complexes (70a-70g, Fig. 29) under the guidance of DFT calculation.243 Whist 70e and 70g showed desired red-shift, their inferior photophysical property (1O2 production, luminescence) and poor stability hurdled further biological application. The biological activity of compound 70f of which the absorption tail extended toward biological spectral window was fully investigated under irradiation at clinically relevant 595 nm in both 2D and 3D models. Heteroleptic Ru(II) polypyridyl complexes featured extended p system ligand were also observed with longwavelength absorption.244 Two-photon excitation (TPE) may serve as another way to enable red/NIR excitation of phosphorescent Ru(II) complexes in deep-embedded tumors. In a TPE process, a PS simultaneously adopts two lower-energy photons in high instantaneous photon densities to realize excitation. The concept and theoretical framework of TPE were established by Maria Goeppert-Mayer in 1931.245 Meanwhile, the potential medicinal applications of TPE have been suggested for a long time.246,247 However, the formidable challenges that limit the application and temper enthusiasm are the scarcity of TPE-active PDT drugs and the lack of necessary

Fig. 29

Long-wavelength absorbing compounds and reference compounds reported by the Gasser group.

Phosphorescent metal complexes for biomedical applications

485

optical technologies rendering TPE impractical. It is not until the advent of commercially available femtosecond tunable Ti:sapphire lasers that TPE became a practical tool for bio-imaging and phototherapy. While the theoretical know-how for the design of TPE agents remains elusive, some explorations indicated that several factors such as p system, terminal group, structural rigidity, and structure symmetry, etc., appear to exert influence on the two-photon absorption (TPA) cross-sections.248 A comprehensive account of transition metal-based TPE PDT agents can be found in some recent reviews.226,249 The Lemercier group has launched a lot of explorations on the potent application of Ru(II) complexes.250–253 They firstly demonstrated the in vitro TPE PDT efficacy of an ethylene-glycol decorated Ru(II) polypyridyl complex with a TPA cross-section (d2) of ca. 40 GM under the irradiation of 740 nm pulsed lasers in a confined space of F98 glioma cells.250 Wong and coworkers reported several series of TPE-active porphyrin-motif Ru(II) complexes for PDT and scrutinized their intracellular localization in HK-1 cells.254,255 Their Ru(II) exhibited significant d2 values which enable the TPE PDT at 850 nm, but the complexes were probably confronted with poor solubility, limited cellular intake, and overlong retention time. The Chao group has been devoted to the development of efficient and versatile TPE agents for a long time.31,51,89,248,256 They reported a series Ru(II) polypyridyl complexes decorated with triphenylphosphine group to impart mitochondria localization ability, and compound 71a (Fig. 30) was found to excel over other compounds in 1 O2 quantum yield (0.81), d2 value (198 GM), and mitochondria accumulating extent (88%).31 Of note, 3D multicellular tumor spheroids (MCTS) was adopted to evaluate the TPE PDT efficacy (lirr ¼ 810–830 nm) for the first time, and the therapeutic outcome was compared to that in the traditional 2D monolayer screening model. This work underlined the merits of MCTS as a superior screening model over a 2D cell model for PDT to bridge the gap between in vitro and in vivo tests. In their later work in collaboration with the Gasser group, a series of highly positively charged Ru(II) polypyridyl complexes were developed as TPE PDT agents.51 These tertiary ammonium groups decorated compounds feature favorable water solubility, excellent photo-stability, low toxicity in the dark (EC50 > 300 mM), and lysosome sequestration, which promise great potential for lysosome-targeting PDT therapeutics. Compound 71b (Fig. 30) showed the most potent PDT activity under the irradiation at 450 nm with an EC50 value as low as 1.5 mM (PI ¼ 313), and its superior TPE PDT efficacy was also observed in MCTS. Under a closer scrutiny of the PDT procedure, 71b was found to swiftly escape lysosome by membrane oxidation upon irradiation (within 15 s), and penetrate into the nucleus to interact with DNA, and initiate cell necrocytosis. Recently, Gasser and Chao rationally designed a series of Ru(II) polypyridyl complexes with (E,E0 )-4,40 -bisstyryl-2,20 -bipyridine ligands for 1- and 2-photon PDT to overcome the limitations of poor aqueous

Fig. 30

TPE-active Ru polypyridyl complexes reported by Chao (71a-71b) and Gasser (71c-71i).

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Phosphorescent metal complexes for biomedical applications

solubility, aggregation, photo-bleaching, slow clearance, lack of significant absorption in NIR therapeutic window when treating large tumors that seated beneath deep tissues.257 The compounds were designed under the guidance of DFT calculations, and their 1-photon absorption profiles were remarkably red-shifted. In addition, unprecedented significant d2 values were recorded for these complexes (up to a magnitude higher than the ones published so far) among which 71d showed a TPA cross section of astonishing  6800 GM. Such an efficient TPA properties prompted their further in vivo evaluation in xenografted model in addition to the 3D MCTS experiments.

2.16.4.1.4

Promoting performance in hypoxia

Due to the unsound vasculature and rapid proliferation, the interior of tumor has the reputation of hypoxia which challenges PDT efficacy. While the mainstream hold the opinion that in both type I and II PDT oxygen is indispensable to renew the catalysts (i.e., PSs),226,258 some believe otherwise.259–261 From a material perspective, oxygen-independent type I analogous PDT process is readily accessible.262,263 In contrast, molecular PSs that undergo an oxygen-independent type I process remain scarce but are gaining momentum. Collectively, covalent modification and metal coordination are routine strategies employed for transformation from type II to type I. The most classic example to instill type I PDT activity is the covalent incorporation of a-oligothiophenes as the organic chromophores, which leads to Ru(II) polypyridyl systems (i.e., TLD1433 and its derivatives) possessing type I and II dual mechanisms of actions and gaining possibilities to treat hypoxic tumors.261 The Brewer group has made some important contributions in oxygen-independent DNA photo-binding/photo-cleavage.264–266 Whereas type I PDT is an electron transfer process resulting in the generation of ROS, type III PDT process involves the electron transfer from PS* to cellular targets as well but results in a new mechanism of action which is derived from photo-cleavage/photobinding of biomolecules. The oxygen-free mechanism of type III PDT implicates that it could be another way out for the hypoxia phototherapy.249,267 However, due to the non-catalytic nature of type III procedures, they are usually classified as photoactivated chemotherapy (PACT) rather than PDT.268 There are excellent reviews concerning this field.267–270 However, the majority of PACT agents are not emissive at room temperature, because the excited states primarily contribute to the dissociation of the labile coordination structure.

2.16.4.1.5

Imparting tumor targeting and uptake ability

Rational molecular engineering to impart specificity of PSs toward cancer cells is of significant importance to guarantee the sufficient presence of PSs in the diseased tissues, improve PDT treatment, and alleviate systemic toxicity as well as peripheral photo-induced lesions. Tumor delivery vectors, such as peptides, antibodies, molecular substrates to the cancer-cell overexpressed receptors, etc., or tumor-microenvironment (acidity, hypoxia, reducibility, etc.) responsive groups are often adopted to help achieve this goal. Since the rapidly proliferating cancer cells are notoriously fueled by low efficient glycolysis, they grow much more avid for glucose. The overexpressed glucose transporters (GLUTs) is responsible for the enhanced uptake of glucose and can serve as a valid target for specific cancer therapy. Ru(II) complexes in combination with glucose were reported by the Chao and Bonnet groups, respectively.256,271 The work on glucose appended Ru(II) polypyridyl complexes [Ru(dip)2(N^N-glucose)]2 þ (72a-72d, Fig. 31, dip ¼ 4,7-diphenyl-1,10-phenanthroline) revealed the enhanced uptake of Ru(II) complexes can be attributed to a major proportion of active transport by sodium-dependent glucose cotransporters and a minor proportion of passive transport. These Ru(II) complexes integrated cancer cells specifically enhanced uptake and mitochondria-targeted localization. Of note, 72b was able to eradicate HeLa tumor xenograft in a multicourse in vivo TPE PDT treatment. Whereas the photophysical and biological properties of [Ru(tpy)(dppn)(L-glucose)]2 þ (72e, Fig. 31, dppn ¼ benzo[i]dipyrido-[3,2-a:20 ,30 -c]phenazine) wherein a glucose monodentate ligand was coordinated to the Ru(II) center via a photo-labile thiolether bonding are different. The labile glucose ligand failed to navigate the compound to cancer cells despite that they are still mitochondria-targeted. The Ru(II) complex was very toxic (EC50 < 1 mM) even in a low dose of irradiation (3.1 J/cm2) because it may undergo dual modes of actions to induce cell death in the light regime: (1) a PACT process via the photosubstitution of the labile monodentate ligand forming an aquo-complex to interact with DNA; (2) the generation of 1O2 under irradiation leads to an efficient photodynamic DNA cleavage. Brain cancer phototherapy requires not only high spatiotemporal precision of excitation but also cancer-cell specific affinity of PDT agents to alleviate peripheral nerve lesions. Taurine is the most abundant amino acid in the brain but the cellular and biochemical mechanisms mediating taurine are incomprehensive. Work on the combination of taurine with the [Ru(bpy)3]2 þ core (72f-72g, Fig. 31) showed that the modified Ru(II) complexes were endowed with enhanced cancerous brain cell targeting ability, lysosome-specific sequestration, and improved PDT efficacy.272 Compound 72f which was modified with the most taurine substituents showed the best targeting ability and PDT performance. Tamoxifen is a competitive substrate to the estrogen receptors which are highly expressed in breast cancers. Work on the tamoxifen modified TPE-active Ru(II) complex (72h, Fig. 31) achieved breast cancer MCF-7 cell-targeted accumulation, lysosome sequestration, and destruction, and boosted breast cancer-specific TPE PDT therapeutics.273 Similarly, a conjugation of Ru(II) complex with biotin (72i, Fig. 31) enabled the selectively enhanced uptake of the PSs in biotin receptor overexpressed cisplatin-resistant lung cancer A549R cells and triggered cancer-specific ablation despite its chemoresistance.274 The efficient conjugation of a PDT-active Ru(II) complex with the cancer-cell targeted peptide/antibody would yield biohybrids that are highly selective as well as easily taken by target cells with better biocompatibility. Since somatostatin receptors (SSTRs) are often overexpressed in tumor cells and neovasculature of tumors, they can be the target for tumor vessels. The endogenous peptide hormone somatostatin (SST) can specifically bind to SSTRs in the nanomolar regime. Because of this, Wang et al. presented a biohybrid consisted of the [Ru(bpy)3]2 þ core and SST (73a, Fig. 32) to achieve cancer-targeting accumulation.275 The bioactivity of

Phosphorescent metal complexes for biomedical applications

Fig. 31

487

Ru(II) complexes (72a-72i) coupled with molecular substrates to the cancer-cell overexpressed receptors.

these two moieties was not compromised when they were conjugated together. 73a retained the PDT efficacy of the [Ru(bpy)3]2 þ core after modification making cancer-targeted PDT possible. Epidermal growth factor receptor (EGFR) is overexpressed in a variety of solid tumors, and is, therefore, a crucial target for targeted therapy. Zhao et al. devised an RGD-peptide conjugated polypyridyl Ru(II) complex (73b, Fig. 32) which proved to be TPE-active and targeted mitochondria of integrin avb3-rich tumor cells.276 Karges et al. validated a series of Ru(II) polypyridyl-nanobody conjugate (73c, Fig. 32) as EGFR-targeting agents in A431 cell line.277 However, the biohybrid demonstrated an elusive ROS generating mechanism. It showed an appreciable 1O2 quantum yield in solution but was nontoxic in the light regime in cellulo rendering the targeted PDT impractical.

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Fig. 32

Biohybrids containing Ru(II) complexes and peptide (73a, 73b)/nanobody (73c).

2.16.4.1.6

Multimodal therapies for enhanced cancer therapy

With all of the above suggestions, rational design and fine-tuning of the Ru(II)-based PS structures can push the envelope of effective and practical PDT therapeutics. Another surge of interest in realizing enhanced tumor suppression is represented by the incorporation of PDT with other therapeutic modalities wherein a synergistic effect is often observed. The most adopted strategy to realize dual-mode of therapeutics originates from the possibility of the dual actions of ligand exchange and 1O2 generation upon excitation of some engineered Ru(II) complexes. Such a photophysical process would allow the complex to uncage bioactive drugs by ligand exchange and unleash their chemotherapeutic damages in a light-triggered manner in addition to the PDT mechanism. This strategy could “hide” the bioactivities of the uncaged species before the activation by light, and thus dramatically reduce off-target toxicity. There are many excellent reviews in the field of PDT/PACT combined with photouncaging of therapeutic toxins.278,279 Due to the non-emissive characteristic of most PACT agents, the present section doesn’t give any further introductions to this field. Another molecular design strategy to achieve synergistic cancer inhibition and expand the PDT therapeutic window is represented by the covalent conjugation of the chemotherapeutic drugs with PDT-active moiety.280 A work on heteroleptic Ru(II) polypyridyl complex conjugated with chlorambucil revealed mitochondria-targeted chemo-photodynamic dual action against HeLa cells.281 While Pt-based anticancer agents are the mainstay of the chemotherapeutics, polymetallic complexes that consist of Ptbased drugs with Ru(II) complexes will be discussed in polymetallic section. Combination of PDT with chemotherapy is a much easier strategy to implement in the clinic because it requires no delicate molecular engineering, but, unlike the photo-uncaging strategy, is probably confronted with aggravated off-target toxicity coming from divergent modals of therapeutic drugs. Gasser and colleagues found that HeLa cells could become more sensitive to a Ru(II)based PDT with a combinatorial CDK1 inhibition treatment to synchronize the cells to the mitotic phase.282 The EC50 value for phase-synchronized cells was 3.6-fold stronger than that of the unsynchronized counterpart. Despite the increased number of preclinical reports, the drawback for combination therapy is apparent, and there are considerable efforts to undertake to properly make the risk-benefit tradeoff.

Phosphorescent metal complexes for biomedical applications 2.16.4.2

489

Phosphorescent Ir(III) complexes for PDT

The preliminary studies on the anticancer potency of Ir complexes date back to the 1970s during which the focus was on d8 squareplanar 1,5-cyclooctadiene Ir(I) complexes (74a, Fig. 33) because of their similar coordination geometry with cisplatin.183 More recently, highly potent Ir(III) “half-sandwich” complexes of the formula [(h5-Cp)Ir(X^Y)Z]n þ (74b, Fig. 33, n ¼ 0, 1, Cp is the pentamethylcyclopentadienyl Cp* or its phenyl or biphenyl derivative, XY is a chelating N^N or N^C bidentate ligand, and Z is halogen anion or neutral monodentate ligand) were extensively investigated by the groups of Sadler and Sheldrick.283 Meanwhile, octahedral cyclometalated Ir(III) complexes of the formula [Ir(C^N)2(N^N)]þ (74c, Fig. 33) have revealed an astonishing range of biological applications. Compared to the other types, octahedral cyclometalated Ir(III) complexes feature kinetic inertness and tunable phosphorescence and are garnering attention on PDT application in recent years.284 Unlike Ru(II) complexes, octahedral cyclometalated Ir(III) complexes possess more abundant excited-state electronic configurations and can encompass a wide range of emission colors in the visible and NIR region by fine-tuning the structure, whereas their specific biological properties are still difficult to rationalize, and generally correlated with molecular attributes, such as the intrinsic charge of the complex, lipophilicity, and water solubility.213 These peculiar properties suggest the potential of octahedral cyclometalated Ir(III) complexes as alternative PDT agents.

2.16.4.2.1

Promoting photophysical performance for PDT

Like Ru(II), elongation of the excited-state lifetime of Ir(III) complexes can be realized by tethering appropriate chromophores. Recently, work on pyrene appended Ir(III) cyclometalated complex (75a, Fig. 34) for the first time unveiled a TPE “ping-pong” type energy transfer process wherein the energy transferred from the singlet excited state of the tpy-pyrene unit to the Ir(III) center and returned to the triplet excited state of the tpy-pyrene ligand.285 Such a back and forth energy transfer fashion resulted in a 230fold longer triplet excited-state lifetime of 75a (15 ms) compared to 75b (65 ns) as well as an enhanced 1O2 quantum yield (FD from 0.26 to 0.98 in MeOH). In addition, work on [Ir(C^N)2(N^N)]þ indicated that extension of p-system of the N^N ligand can give rise to long-lived 3IL excited states.286 Since the inclusion of organic chromophores or the extension of the p-system would undermine the hydrophilicity, Ir(III) polypyridyl complexes with higher charge could be a solution but were largely unexplored. Recently, the Sun and McFarland groups presented a series of novel pyrene-modified Ir(III) N6 polypyridyl complexes which possess a þ 3 charge and long-lived triplet excited states (ca. 3.3–33 ms), thus facilitating their PDT application.287 The absorption bands of Ir(III) complexes are generally located in the ultraviolet region with a very weak absorption tail extending to the visible range, which either brings in undesired photo-damages to biological architectures under UV excitation or limits the PDT performance in the visible regime. The combination of Ir(III) center with chromophore scaffold opens an avenue to the enhancement of visible-light-harvesting ability. The Zhao group presented a series of coumarin-modified biscyclometalated Ir(III) complexes (76a-76d, Fig. 35) among which 76c and 76d both showed over 10-fold larger visible-light extinction coefficients than their unmodified counterparts.288 Recently, Zhou et al. reported a rhodamine-decorated Ir(III) complex (76e, Fig. 35) which

Fig. 33

Geometries of the organometallic Ir complexes as anticancer agents.

Fig. 34

Chemical structures of 75a and 75b.

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Phosphorescent metal complexes for biomedical applications

Fig. 35

Light-harvesting Ir(III) complexes modified with coumarin (76a-76d), rhodamine (76e), and BODIPY (76f-76h).

exhibited an intense absorption in the visible region, and demonstrated the effect PDT efficacy by populating energy in 3IL excited state.289 Intriguingly, 76e accumulate in ER, and readily elicited ER stress after PDT treatment. Likewise, BODIPY has proven to be able to boost strong absorption at a longer wavelength by either conjugation to the ligand or direct chelation to the biscyclometalated Ir(III) complexes.290 Interestingly, an efficient manner to funnel energy from visible light is by the fluorescence resonance energy transfer (FRET) whereby more units of chromophores are allowed to instill excited-state energy to the Ir(III) centers.

Phosphorescent metal complexes for biomedical applications

491

Zhao and coworkers reported a new kind of dual-emissive semiconducting polymer nanoparticles (SPNs) containing BODIPY derivatives and NIR emitting Ir(III) moiety (76f-76h, Fig. 35).291 In 76f, the BODIPY units serve as energy donors in the FRET process enhancing the light absorption of the SPNs and NIR Ir(III) complexes as the energy acceptors and efficient PSs. Compared with 76 h that lacks spin converter and 76f that lacks light-harvesting group, 76f demonstrated the superior PDT efficacy (FD 0.96), and was utilized in PDT treatment of HeLa cells xenograft zebrafish and tumor-bearing mice. They found that the self-assembly of 76f into nanoparticles facilitates tumor targeting ability, shields the luminophores from photobleaching, and enables versatile function as an in vivo O2 imaging as well as an efficient PDT theranostic agent. Similar visible-light-harvesting Ir(III) complex designs based on FRET were reported by the same group.292 In terms of extending the absorption profile of Ir(III) complexes to the biological window, conjugation with chromophore is the most routine method. Zhao and colleagues reported a series of biscyclometalated Ir(III) complexes with styryl-BODIPY ligand (77a77d, Fig. 36) featuring NIR absorption (644–729 nm)/emission (700–800 nm).293 These complexes are strongly fluorescent (1IL), despite the p-conjugation between BODIPY and Ir(III) center. Ir(III) center functions as a moderate spin converter due to the longrange conjugated spacer between metal and the BODIPY moiety as well as an energy contributor to the 3IL excited state which accounts for the PDT activity. Secondly, TPE is another elegant manner to achieve NIR excitation because many cyclometalated Ir(III) was found to be TPEactive.183 Nam et al. rationally developed a series of biscyclometalated Ir(III) complexes with different energy levels (78a-78d, Fig. 37) for PDT study.69 Among these complexes, 78c (FD 0.95) and 78d (FD 0.78) effectively triggered cell death via an ERspecific PDT even under low concentration (EC50  2 mM) and weak excitation energy ( 1 J/cm2). Also, 78c was found to be active in TPE PDT treatment. Interestingly, in proteins near endoplasmic reticulum (ER) and mitochondria, protein oxidation and protein cross-linking were verified in the PDT process. The catalytic activity of the Ir(III) complexes in protein photo-cross-linking indicates an oxygen-independent type I-analogous mechanism of the Ir(III) complexes, thus fostering targeted photo-catalytic therapeutic potential beyond PDT. At the same time, Bryant and Weinstein reported a series of small molecular Ir(III) complexes as viable TPE PDT agents (at 760 nm) with mitochondrion and lysosome localization (78e-78f, Fig. 37).294 Cyclometalated Ir(III) complexes for TPE PDT were also reported by another group.295

2.16.4.2.2

Targeted PDT by Ir(III) complexes

It is a growing recognition that many organometallic Ir(III) complexes show intrinsic affinity toward specific organelle/cellular compartment/biomacromolecules, such as mitochondria,39,296–299 ER,69 lysosome,300 nucleus,11,301 proteins,302 etc., thus making organelle-specific PDT possible. However, it is still challenging to design Ir(III) complex with specific targeting ability because even a simple fine-tuning of the scaffold of Ir(III) complexes often alters target preference of which there is barely a mechanism-based rationale. There are many excellent reviews on the advances of organelle-specific PDT based on Ir(III) complexes wherein the importance of organelle targeting in enhancing PDT effect has been highlighted.183,284 hence the present section doesn’t intend to undertake a repeated introduction in this field. Instead, the strategies to obviate drawbacks in targeted therapy and tumor-targeting strategies will be emphasized.

Fig. 36

Ir(III) complexes with NIR absorption reported by the Zhao group.

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Phosphorescent metal complexes for biomedical applications

Fig. 37

TPE-active Ir(III) complexes for ER-(78a-78d)/ mitochondrion and lysosome-targeted (78e-78f) TPE PDT.

For many Ir(III) complexes, the intrinsic characteristic of organelle-targeted accumulation may lead to aggregation caused quenching (ACQ) effect, and thus compromising PDT efficacy and greatly restricting practical application. To address this issue, the Chao group proposed the introduction of aggregation-induced emission (AIE) active moiety to organometallic Ir(III) complexes, constructing a series of mitochondria-targeting aggregation-induced PDT agents (79a-79c, Fig. 38).298 These compounds showed identical recovery trajectories of emission intensity and 1O2 quantum yield and were viable for TPE PDT therapeutics. The same strategy was recently played by Zhang et al. in a series of multinuclear AIE-active Ir(III) complexes, and the further encapsulation of the complexes by biocompatible functionalized liposome enabled an enhanced in vivo PDT.303 Yi et al. proposed another method to prevent ACQ of Ir(III) complexes.304 They introduced quaternary ammonium groups functionalized with different lengths of saturated carbon chains to the complexes (79d-79i, Fig. 38), thus imparting them amphiphilic characteristics which gives rise to their self-assemble into vesicles in a Gemini fashion in water. Of note, 79f with an amphiphilic substituent in the 4-position of C^N ligands can overcome the ACQ effect and reside in mitochondria, and showed excellent PDT performance in inhibiting the HepG2 tumor xenograft under PDT treatment. Whist the molecular engineering on Ir(III) complexes to achieve tumor-specific targeting has been barely investigated, strategies regarding nanomaterial to fulfill this goal have been reported. Recently, Yang et al. designed an Ir(III)-cyanine conjugate (80a, Fig. 39), and fabricated F127-encapsulating IrCy nanoparticles (NPs) via a solvent evaporation-induced self-assembly strategy.305 These hydrophilic NIR-absorbing NPs manifested a long circulation half-lifetime ( 18 h) which guarantees the passive targeted uptake by tumors by EPR effect and were applied to photoacoustic (PA) imaging for tumors and imaging-guided PDT under irradiation at 808 nm. Xiang et al. reported two GSH activatable versatile organometallic Ir(III) complexes (80b-80c, Fig. 39) which could release chemotherapeutic drug camptothecin (CPT) in the presence of GSH, and fabricated F127-encapsulating Ir(III) micelles via noncovalent self-assembly which was further decorated with folic acid to impart tumor-targeting ability.306 The micelles showed appreciably stronger cellular uptake in folate receptor (FR) overexpressed HeLa cells than FR deficient MCF-7 cells, leading to a highly effective tumor-specific lethality due to the synergistic effect of PDT and GSH-stimulated CPT chemotherapy.

2.16.4.2.3

Reinforcing phototherapeutic potency in hypoxia

Due to their abundant photocatalytic potentials and elusive biological activities, Ir(III) complexes are full of subtleties in reinforcing the PDT potential in hypoxia. Recently, Novohradsky et al. reported a far-red-emitting coumarin derivative modified

Fig. 38

Ir(III) complexes designed to conquer ACQ effect.

Phosphorescent metal complexes for biomedical applications

Fig. 39

493

Chemical structure of 80a and 80b-80c.

biscyclometalated Ir(III) complex (81a, Fig. 40) functioning as an effective oxygen-independent type I PDT agent. 81a generates superoxide anion radicals upon visible-light irradiation, and is toxic toward HeLa cells with proximate EC50 and PI values between hypoxia and normoxia.307 This complex can be exploited to overcome some of the drawbacks in traditional PDT agents, such as poor tissue penetration depth and O2-tension dependency. More recently, the Chao group reported a series of mitochondriatargeted Ir(III) complexes with ligand functioned with different chromophores (81b-81e, Fig. 40), among which complex 81e with Ir(III) center conjugated to anthraquinone exhibited hypoxia-responsive activation of emission and PDT lethality.108 In particular, the anthraquinone moiety in 81e proves to be specifically reduced by hypoxia microenvironment to anthracene diol. The reduced product is capable of generating carbon radicals and keeps PDT competency even in hypoxic tumors. The potential photoredox catalysis activity of Ir(III) complexes may provide an alternative way to PDT to address the hypoxia issue that hampers clinical translation. A series of simple Ir(III) complexes that undergo type I analogous process has been introduced previously.69 Recently, the groups of Sadler, Chao, and Gasser reported a mitochondria-targeted Ir(III) complex (82, Fig. 41) that undergoes distinct photoredox catalysis mechanisms beyond PDT under normoxia and hypoxia to achieve photolethality.308

Fig. 40

Structures of Ir(III) complexes for type I PDT agents.

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Phosphorescent metal complexes for biomedical applications

Fig. 41

Chemical structure of compound 82.

Under normoxia, compound 82 photocatalytically oxidizes 1,4-dihydronicotinamide adenine dinucleotide (NADH) which is an important coenzyme in living cells, thus generating NAD• radicals in biological media. Moreover, compound 82 and NADH synergistically photoreduce cytochrome c under hypoxia. Since 82 is localized in mitochondria where cytochrome c resides, the compound is capable of disrupting electron transport via NADH photocatalysis resulting in NADH depletion, redox imbalance, and apoptosis. It’s noteworthy that these reactions can proceed without the participation of oxygen. Directing the intracellular localization can also make a difference in the hypoxia PDT. Lv et al. developed two biscyclometalated Ir(III) complexes (83a-83b, Fig. 42) with the distinct organelle-targeting ability and evaluated their PDT efficacy under hypoxia.39 HeLa cells treated with the mitochondria-targeted compound (83a) maintained a slower respiration rate, leading to a higher intracellular O2 level under hypoxia. As a result, 83a showed an improved PDT effect in comparison with lysosome-targeted compound (83b) in both hypoxia and normoxia. Their findings suggest that PSs targeting mitochondria would have higher practicable PDT potential.

2.16.4.2.4

Multimodal therapy

Ir(III) complexes have been extensively investigated as anticancer agents for a long time.49 The advent of phototherapeutic capability opens an avenue to multimodal therapy. Pracharova et al. found that the anticancer toxic effect of a series of phosphorescent biscyclometalated Ir(III) complexes can be significantly potentiated by additional visible-light irradiation, suggesting the underlying synergistic effect of chemotherapy and PDT in these Ir(III) complexes.309 The Mao group has made some contributions to this field.

Fig. 42

Chemical structures of 83a and 83b, and the process of mitochondria- or lysosome-targeted PDT.

Phosphorescent metal complexes for biomedical applications

495

Histone deacetylases (HDACs) regulate the expression and activity of many proteins concerning tumorigenesis. The inhibition of HDACs leads to the potent anticancer outcome by promoting the acetylation level of histones and non-histone proteins and giving rise to detrimental transcriptional events that undermine cell viability. In this regard, Mao and colleagues developed a series of cyclometalated Ir(III) complexes with HDACs inhibitor pendant (84a-84d, Fig. 43), and found that an additional PDT would greatly aggravate their chemotherapeutic lethality toward HeLa cells.310 In their later work, they reported a series of Ir(III) complexes (84e-84h, Fig. 43) that can. inhibit several critical cancerous events, including migration, invasion, colony formation, and in vivo angiogenesis, and, meanwhile, exert lysosome-damaged PDT efficacy. In addition, MELK, PIK3CA, and AMPK were validated as the target of compound 84h in an in vitro screening assay against 70 kinds of kinases, which might partly account for its supreme PDT therapeutic outcome. In their recent work, a mitochondria-targeted Ir(III) complex appended with a photoacid generator (84i, Fig. 43) was designed.311 Upon irradiation, 84i generates photoacid and is gradually decomposed to 84j and 84k both of which are PDT-active, thus initiating dual-mode phototherapy which warrants their anticancer efficacy in hypoxia. The Chao group reported a series of TPE-active Ir(III) PDT agents (84l-84o, Fig. 43) bearing dichloroacetate (DCA) via esterase-labile ester bonding.311 The avenue to mitochondria was opened to DCA by conjugation with the Ir(III) vectors, thus efficiently prompting the DCA inhibiting activity on pyruvate dehydrogenase kinase 1 (PDK1) which resides in mitochondria. PDK1 inhibition gives rise to the reversion of glycolysis metabolism phenotype of cancer cells, and subsequently reopens the defective mitochondrial machinery, leading to selective sensitization of cancer cells to PDT therapeutics. As a result, the combination of TPE PDT originated from the Ir(III) moiety with DCA-associated chemotherapy converged a synergistic effect in triggering cancer cell death.

2.16.4.3

Other phosphorescent metal complexes/polymetallic complexes for PDT

Aside from Ru(III) and Ir(III) complexes, other phosphorescent metal complexes have also made promising progress in PDT application. In the following section, some of the most exciting cases will be briefly exemplified. Alongside monometallic PSs, polymetallic complexes seem to promise the even greater potential to overcome the drawbacks confronting their mononuclear counterparts in PDT application by, for instance, promoting optical absorption under ether a one-photon or two-photon excitation process, changing internal quantum, tuning lipophilicity, or even redirecting the target of the supramolecular complex in cellulo, etc.312 Of particular, mix-metal polynuclear complexes are powerful systems to achieve multimodal therapy and synergistic effect taking advantage of the distinct properties of each metals.268 Recently, Johannes Karges et al. reported a series of novel homoleptic bis(dipyrrinato) Zn(II) complexes (85a-85d, Fig. 44) that exhibited exceptionally long lifetimes (207–559 ns) for the application of PDT.313 85c was further encapsulated in a polymer matrix for PDT evaluation in 3D MCTS and showed impressive photocytotoxicity upon irradiation at 500 nm. Evidence suggests that compact combination of hetero metals in a complex system might give rise to a red-light-absorbing characteristic.314 The Brewer group designed a. Ru(II)-Pt(II) supramolecular complex, [(dip)2Ru(dpp)PtCl2]2þ (dpp ¼ 2,3-bis(2-pyridyl)pyrazine), and found that it can photobind DNA via a 3MLCT excited state by using red light in the therapeutic window.315 Their further investigation on this complex revealed synergistic effects between the metal centers, enabling multiple pathways to provoke lethality toward F98 rat malignant glioma cells (i.e., blue light triggered platination and oxidative stress).316 A similar structure was later studied by Zheng et al.317 In addition, symmetric polymetallic complexes with extended conjugation systems can boost significant two-photon absorption in the NIR window. Zhou et al. developed a Ru(II)-Pt(II) metallacycle (85e, Fig. 44) that manifested a significant TPA property (d2 ¼ 1371 GM at 800 nm), NIR emission, significant 1O2 quantum yield, and dual-targeted accumulation in cellulo, and found that 85e showed significant inhibition to A549 tumor xenograft model after a TPE PDT treatment.318 Later, they constructed a series Ru(II)-Pt(II) polymetallic complexes with different dimensionality by coordination-driven self-assembly (85f-85g, Fig. 44) and found that both of them showed dramatic enhanced d2 values compared to the building blocks.319 Of particular, 85g with 5468 GM (at 800 nm) TPA cross-section was encapsulated with liposome for further biological study. The resultant micelles showed lysosome sequestration which incurs efficient lysosome destruction in the two-photon laser excitation regime, and the TPE PDT efficacy was proved in both 3D MCTS and tumor xenograft models. The interactions between metal centers facilitate the design of activatable polymetallic complexes, thus achieving targeted enhanced therapy. Meade and colleagues reported a type III analogous light-activatable Ru(II)-Co(III) bimetallic complex (86a, Fig. 45) for protein inhibition.320 86a undergoes a photo-induced electron transfer from Ru(II)* to Co(III) upon excitation at 455 nm and trigger a dissociative axial ligand exchange of the Co(III) center, resulting in the coordination to histidine residues of a-thrombin and enhanced inhibition rate of a-thrombin. Recently, the Chao group developed an Ir(III)-Ru(II) bimetallic complex (86b, Fig. 45) as a prodrug for photo-activated PDT and chemotherapy against cisplatin-resistant A549R and SGC7901/DDP cells.321 86b is found to be localized in mitochondria and exert synergistic dual therapeutic efficacy in mtDNA damaging and mitochondria dysfunction, and showed no appreciable toxicity discrepancy between cisplatin-resistant cell lines and nonresistant peers. By contrast to light activation strategy, tumor-associated stimuli-responsive complexes enable tumor theranostics. The Chao group designed a homonuclear bimetallic Ru(II) polypyridyl complex wherein the metal centers were bridged via an

496

Phosphorescent metal complexes for biomedical applications

Fig. 43 Chemical structures of Ir(III) complexes that integrate PDT with HDAC inhibition (84a-84d)/antimetastasis activity (84e-84h)/photoacid generation (84i-84k)/metabolic alteration (84l-84o) multimodal therapy.

Phosphorescent metal complexes for biomedical applications

85a 85b

85c

85d

497

85e

= =

=

[2 + 2]

85f Fig. 44

[4 + 6]

85g

Structures of complex 85a-85g for TEP PDT.

azo group (86c, Fig. 45).322 The excited state of the complex is quenched by the very fast conformational change around azo moiety, thus restricting their PDT capacity. When exposed to the microenvironment of cancer cells, the azo can be specifically reduced by a high level of GSH leading to the recovery of the excited state of the complex and boost PDT efficacy therein under two-photon laser irradiation. Recently, Karges et al. developed a Ru(II)-Pt(IV) conjugate prodrug (86d, Fig. 45) that is responsive to the reductive microenvironment of cancerous cells.323 The specific reduction of Pt(IV) to Pt(II) unleashes phenylbutyrate axial ligand which acts as histone deacetylase inhibitor and can de-condense the chromatin, potentiating the Pt-based chemotherapy. The released Ru(II) moiety accumulates in Golgi apparatus and exerts a long-wavelength PDT effect. These multimodal therapies were found to function in a synergistic way to trigger cancer cell death. The oxygen dependency of PDT by polymetallic complexes could be decreased by the emerging oxygen-independent mechanism of action introduced by the new metal centers. Conti et al. designed a highly charged Ru(II)-Cu(II) complex (87, Fig. 46) that is localized around the outer membrane of nuclei, and found that upon irradiation, Ru(II) center generates 1O2 by a type II mechanism, whereas the Cu(II) center is involved in Fenton–like reactions with H2O2 (namely, CuII þ H2O2 / CuI þ OH þ $OH), and plays a synergistic role providing additional/alternative mechanisms for oxidative damage of biological targets.324

498

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Fig. 45

Polymetallic complexes for light-activated (86a, 86b) and redox-responsive (86c, 86d) PDT.

Fig. 46

Polymetallic complexes for enhanced PDT in hypoxia.

Phosphorescent metal complexes for biomedical applications

499

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Photoactive metallodrugs

Huayun Shi and Peter J. Sadler, Department of Chemistry, University of Warwick, Coventry, United Kingdom © 2023 Elsevier Ltd. All rights reserved.

2.17.1 2.17.2 2.17.2.1 2.17.2.2 2.17.2.3 2.17.3 2.17.3.1 2.17.3.2 2.17.3.2.1 2.17.3.2.2 2.17.3.3 2.17.3.3.1 2.17.3.3.2 2.17.3.3.3 2.17.3.3.4 2.17.3.3.5 2.17.3.4 2.17.3.4.1 2.17.3.4.2 2.17.3.5 2.17.4 2.17.4.1 2.17.4.1.1 2.17.4.1.2 2.17.4.2 2.17.4.3 2.17.5 2.17.6 2.17.6.1 2.17.6.1.1 2.17.6.1.2 2.17.6.2 2.17.7 Acknowledgments References

Introduction Phototherapy Photodynamic therapy (PDT) Photoactivated chemotherapy (PACT) Photothermal therapy (PTT) Photophysics and photochemistry of metallodrugs Absorbance and luminescence Activation wavelengths One-photon activation Multi-photon activation Photoactivation mechanisms and pathways Photocatalysis Photoreduction Photosubstitution Photoactivation of ligands Combinations of mechanisms Photoreactions with biomolecules Nucleotides and DNA Amino acids, peptides and proteins DFT and TD-DFT calculations Photoactive anticancer metallodrugs Photodynamic therapy (PDT) PDT metallodrugs entered clinical trials Candidate PDT metallodrugs Photoactivated chemotherapy (PACT) Photothermal therapy (PTT) Photoactive antimicrobial metallodrugs Drug delivery systems for photoactive metallodrugs Organic nanocarriers Natural polymeric nanocarriers Synthetic polymeric nanocarriers Inorganic nanocarriers Summary and perspectives

508 509 509 510 510 511 511 511 512 512 513 513 513 513 514 514 514 514 514 514 515 515 515 516 525 535 535 537 537 537 538 540 542 546 546

Abstract Photoactive metal complexes are emerging as promising clinical drug candidates for anticancer and antimicrobial treatments. Phototherapy, mainly as photodynamic therapy (PDT), photoactivated chemotherapy (PACT) and photothermal therapy (PTT), is attractive due to its high efficiency, precise localization, and novel mechanism of action. The introduction of photoactive metallodrugs has promoted the further development of phototherapy due to their rich biological and chemical diversity. Two photoactive metallodrugs have been approved for clinical use, while more are in clinical trials or development. In this Chapter, we aim to give a comprehensive description of photoactive metallodrugs, including their history, photophysics, photochemistry and mechanisms of action, as well as the design of novel photoactive metallodrugs, and drug delivery systems.

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00037-6

507

508

Photoactive metallodrugs

2.17.1

Introduction

Pharmacologically inactive prodrugs that undergo transformation in vivo to release active drugs are attracting attention in the clinic.1–3 Ideally, prodrugs can overcome drug delivery barriers, allow target-specific activation and improve physiochemical, pharmaceutical, and pharmacokinetic properties.1,2 Prodrug activation triggers are classified into two types, endogenous stimuli-based on the intrinsic properties of the target biological system, and exogenous stimuli applied externally to patients.1 Endogenous stimuli, including pH, oxygen, and enzymes, rely completely on intracellular parameters, and the fate of prodrugs cannot be controlled by physicians after administration.1 In contrast, exogenous stimuli, including light, microwaves, magnetic field, ultrasound, electric pulse, and temperature, can provide accurate remote control of prodrug activation spatially and temporally to reduce side effects and systemic toxicity.1 Light as a safe and non-invasive trigger, exhibits high efficiency and precise localization, and thus has been widely applied in the treatment of various diseases.4,5 Sunlight was first used in combination with plant extracts to treat vitiligo more than 3500 years ago in ancient Egypt, while modern phototherapy was started with Finsen, the Physiology or Medicine Nobel Prize winner in 1903, who used concentrated light radiation to treat lupus vulgaris.5 PUVA therapy, combination of psoralen and UVA, began in 1974 and made phototherapy a standard treatment for skin diseases.6 Hematoporphyrin derivatives (HpDs) underwent first systematic human trials in 1978, by Dougherty et al. at the Roswell Park Cancer Institute, and the most important HpD, Porfimer sodium, was approved in 1993 to treat bladder cancer in Canada as the first photodynamic therapy (PDT) agent, under the brand name PhotofrinÒ, the beginning of the modern PDT era.7 Nowadays, PDT is the most widely applied clinical phototherapy, and PhotofrinÒ is still being investigated in one third clinic trials.7–10 Two other types of phototherapies are photoactivated chemotherapy (PACT) and photothermal therapy (PTT), which are still under development.11,12 Although organic molecules have dominated the photo-therapeutic market, significant progress has been achieved for photoactive metallodrugs with their rich biological and chemical diversities (Scheme 1).1,13–16 The serendipitous discovery of cisplatin as antiproliferative agent by Rosenberg et al. in 1967 has revolutionized cancer treatment and stimulated the development of metallodrugs for various diseases.17–19 Since FDA approval in 1978, cisplatin and its analogs carboplatin and oxaliplatin are now used in more than 40% of cancer chemotherapy treatments.20,21 Since then, a few other metallodrugs have received clinical approval or have been undergoing clinical trials based on their novel mechanisms of action.15 Pd-based TookadÒ soluble (Fig. 1A) was the first metal-based PDT agent approved by EMA to treat localized prostate cancer (2017), and is under consideration by the FDA.22–24 Compared with organic molecules, metal complexes display the following potential advantages as photoactive prodrugs.1,13,25,26 (1) Metal complexes possess a wide range of structures with diverse oxidation states, coordination numbers, and geometries, thus offering extensive possibilities for novel mechanisms of action to overcome current drug resistance. (2) Metals can improve the stability and photocytotoxicity of the photoactive agents, while the ligands also have a considerable influence on their pharmacological activities. (3) The introduction of a metal allows quantification and localization of the agents in cells and tissues using metal-specific methods, e.g. ICP-MS and XAS. Some metals are also suitable for applications in MRI and radionuclide tracing and imaging. (4) Luminescent metal complexes show unique photophysical properties, including tunable and intense emission, large Stokes shifts, long emission lifetimes, and high signal-to-noise ratios, allowing real time monitoring of the agents. (5) Interactions between metal complexes and biomolecules can be investigated with the help of advanced mass spectrometry, especially utilizing characteristic isotope patterns. In this Chapter, we aim to provide a comprehensive description of photoactive metallodrugs (mostly candidate drugs) with a focus on photoactive anticancer and antimicrobial complexes. The discussion begins with an introduction to phototherapy, followed by

Niels Finsen won Nobel Prize for light irradiation to treat lupus vulgaris

1904

BC 3000

1903 Sunlight used with plant extracts to treat vitiligo in Egypt Scheme 1

Photosensitiser Photofrin® approved in Canada

2009

2004

1993

“Photodynamic action” proposed by Hermann von Tappeiner

2008

Clinical trials on Tookad® (WST-09) photodynamic therapy

Development timeline of photoactive metallodrugs.

Metal-based photosensitiser Tookad® soluble (WST-11) approved by EMA

Clinical trials of AuroLase® photothermal therapy

2018

2017 “Photoactivated chemotherapy” introduced

TLD-1433 successfully completed Phase Ib clinical trial

Photoactive metallodrugs

(A)

(B) -

SO3-

O3S

4-

SO3-

OH

O

N Pd

N N

O

N

N

N

N

S H N

N

N

S

N N

O O-

-

SO3

O3S

N Ru

O O

N -

S N

N

N

Al

N

2+

2-

HN

N N

509

®

Tookad® soluble

Photosens

TLD-1433 OH

(C)

O O

N N

N Zn

N

N Cl N Sn Cl

N N

N

OCOCH3 N

N

N

Fig. 1

O

O

O

OCH3

N

O

O

O

OCH3

N

N

OCOCH3

N

Purlytin®

CGP 55847

N Lu

OH

Antrin®

Photoactive metallodrugs (A) clinically approved, (B) in clinical trials, and (C) entered clinical trials which were terminated.

brief descriptions of the photochemistry, photobiology and photopharmacology of a wide range of metal complexes, including the choice of activation wavelengths and drug delivery systems. In conclusion, feasible strategies for the future clinical use of photoactive metallodrugs are discussed together with a perspective of phototherapy.

2.17.2

Phototherapy

Phototherapy, including PDT, PACT and PTT, is based on the dual application of light and phototherapeutic agents, which exert efficacy with high spatio-temporal precision (Table 1).11,12,27,28 Ideally, these phototherapeutic agents are nontoxic and stable in the dark. Upon light irradiation, PDT agents generate reactive oxygen species (ROS) in the presence of oxygen, PACT agents undergo chemical changes to form active species, and PTT agents induce temperature increases.28,29

2.17.2.1

Photodynamic therapy (PDT)

In the 1900s, Raab observed that incubation of paramercia with acridine orange killed this aquatic microorganism upon exposure to sunlight and hypothesized that acridine orange converted light into a form of active chemical energy, which formed the basis of PDT.30 The term “photodynamic action” (“photodynamische wirkung”) was proposed by his supervisor Tappeiner in 1904, who also showed the essential role of oxygen in PDT.31 PDT agents are usually called photosensitisers (PS), and PDT mechanisms traditionally fall into two categories.32 The excited Type-I photosensitisers interact with biomolecules by electron or hydrogen atom exchange to generate cytotoxic radicals, while the excited Type-II photosensitisers convert ground state 3O2 to active 1O2 by energy transfer.33 Both mechanisms are dependent on oxygen, but some Type-I photosensitisers are able to generate superoxide anion radicals (O2
) under hypoxic conditions.13,34–36

Table 1

Comparison between PDT, PACT and PTT.

Photo-therapy

Mechanism

Active species

Advantages

Disadvantages

PDT

Photocatalysis

ROS

Oxygen-dependent; Skin photosensitivity

PACT

Photochemical change

Photo-products

PTT

Photocatalysis

Heat

Clinically approved; High photoefficacy; Activation with red/NIR light Oxygen independent; Tunable and multiple mechanisms of action Oxygen independent; NIR activation with deep penetration

Short activation wavelength; Limited photoefficacy Nanomaterials required; Dark cytotoxicity

510 Table 2

Photoactive metallodrugs PDT anticancer metallodrugs approved for the clinic or entered clinical trials.a

Agent

Metal

Stage

Area

lex (nm)

Cancer types

Photosens® Tookad® soluble TLD-1433 CGP55847 Purlytin® Antrin®

Al Pd Ru Zn Sn Lu

Russia approved EMA approved Phase II Terminated Terminated Terminated

Russia EU, Israel, Mexico Canada Switzerland USA USA

675 753 525 670 664 732

Skin, breast, lung, head and neck, gastrointestinal tract Prostate Bladder Upper aerodigestive tract Breast Prostate, breast, cervix

a

Data from https://www.clinicaltrials.gov/.

PDT has wide applications in the clinic to treat cancer, microbial infections, and non-malignant dermatological, ophthalmic or vascular diseases.7,8,37,38 An ideal photosensitiser should possess the following properties: intense absorption band in the PDT window (ca. 620–850 nm, low tissue absorption, high enough energy to excite the PS), effective ROS generation, an appropriate retention time in the body, low photosensitivity, no dark cytotoxicity, high photostability, aqueous solubility for formulation, and feasible synthetic routes with high yield.13,39 The first-generation PDT photosensitiser, PhotofrinÒ, is a mixture of oligomeric HpDs that can be activated by 630 nm light and was approved to treat bladder cancer, early-stage lung cancer and advanced obstructive esophageal cancer.40,41 The prodrug 5aminolevulinic acid (LevulanÒ) and its methyl derivative (MetvixÒ) were approved by the FDA as second-generation photosensitisers. These can form protoporphyrin IX in vivo and be activated by light at 635 nm.41 Most PDT photosensitisers possess a heterocyclic tetrapyrrole structure resembling that of chlorophyll or heme in hemoglobin, such as clinically approved chlorin derivatives FoscanÒ, RadachlorinÒ and LaserphyrinÒ.40,41 Metal-based photosensitisers, TookadÒ soluble approved by EMA, PhotosensÒ approved in Russia, and AntrinÒ, PurlytinÒ and CGP55847 entered clinical trials, are all based on the tetrapyrrole structure (Table 2, Fig. 1).40 However, the Ru-based photosensitiser TLD-1433 (Fig. 1B) has an octahedral structure with three N,N-chelating bipyridyl/phenathroline ligands, and has completed a Phase 1b clinical trial for non-muscle invasive bladder cancer (NMIBC).13 The structural diversity of metal complexes provides the foundation for the development of next-generation photosensitisers,42 including octahedral Ru(II),43–46 Ir(III)47,48, Os(II)43 and Re(I)49 complexes, and square-planar Pt(II)50 complexes.

2.17.2.2

Photoactivated chemotherapy (PACT)

Despite the clinical approval of PDT, its application in hypoxic environments is limited by the high oxygen dependence of its mechanism of action, especially in hypoxic tumors, typically oxygen concentrations < 1%.51 PACT relies on photoinduced chemical changes of prodrugs to exert efficacy, and therefore is independent of oxygen.11,29 The desirable features of PACT agents include large photoselectivity indices, long excitation wavelengths, effective photochemical processes, and efficacy in hypoxia.29 Photochemotherapy began in the early 1970s when psoralens activated by UVA irradiation were used to treat psoriasis, binding covalently to DNA base pairs to form crosslinks, called PUVA therapy.6 Photoactive rhodium(III) compound [Rh(phen)2Cl2]Cl that underwent photosubstitution of chlorides by water with UV light then formed DNA adducts was reported in the 1990s as early PACT metallodrugs.52,53 The term “photoactivated chemotherapy” was used by Bednarski and Sadler et al. in 2009, and became the modern term for “photocaged” compounds, and inorganic photocaging strategies.11,29 Chemical changes to photoactivated metal-based PACT agents mainly involve photoreduction, photosubstitution, and photocleavage or photoswitch of ligands.11,25 Pt(IV)54,55 and Co(III)56 complexes tend to be photoreduced and release cytotoxic Pt(II) and Co(II) species, respectively. Ru(II),46,57 Rh(III),58 and Re(I)49 complexes can undergo photosubstitution by solvent. Ligand-based photocleavage and photoswitch reactions are often related to the behavior of bonds as found in the organic parts photocaged compounds.11

2.17.2.3

Photothermal therapy (PTT)

Similar to PACT, PTT is based on an oxygen-independent mechanism of action.59 However, metal complexes alone seldom act as PTT agents, and usually need to be conjugated to near-infrared (NIR) absorbing photothermal nanomaterials to be PTT agents.12,59 The history of hyperthermia in cancer treatment dates back to the 19th century, and global interest was initiated in 1975 by the first hyperthermic oncology international congress in Washington.60 Photothermal therapy allows controlled and localized heating, and thus has proven to be an effective strategy.61 Ideally, PTT agents show NIR light-triggered mild hyperthermia (rise to ca. 43  C), which is sufficient for treatment, but causes no undesirable thrombocytopenia and damage.62,63 The first photothermal nanoparticles which entered clinical trials, PEGylated silica-cored Au nanoshells (AuroShellsÒ), were developed by Halas and West from Rice University in 1998,64 and near-infrared thermal therapy of tumors was investigated in 2003.65 AuroLaseÒ Therapy ablates prostate tumors effectively in humans as demonstrated in clinical trials.66 Other than the direct effect of nanomaterials, loading with photoactive prodrugs can enhance the photothermal efficacy of PTT agents.67 Conjugates of PTT nanomaterials with Ru(II)68,69 or Ir(III)70,71 PDT complexes can exhibit enhanced photothermal stability and efficacy.

Photoactive metallodrugs

2.17.3

511

Photophysics and photochemistry of metallodrugs

The photophysical properties and photochemical reactivities of metal complexes can be tailored by altering the metal and its oxidation state, as well as ligands, since they determine the structural and electronic properties of metal complexes.13 The complexes in light-induced excited states show different electronic distributions compared with those in ground states, leading to luminescence and photoreactivity.29,72 Unlike the p-p* excited states in organic PDT agents, photoactive metallodrugs offer a wider range of excited-state electronic configurations, which enriches their photophysics and photochemistry and gives rise to photobiological mechanisms which are impossible for organic agents.13,72

2.17.3.1

Absorbance and luminescence

The UV–vis absorbance of metal complexes arises from a variety of electronic transitions.73 Photoexcitation occurs only when the energy difference between any excited states and the ground state equals the energy of the incident photon (EES -EGS ¼ hy).74 The electronic transitions of metal complexes are classified into three groups: metal-centered (MC) transitions, ligand-centered (LC) transitions, and charge-transfer (CT) transitions.29 MC transitions, commonly d-d or ligand-field (LF) transitions, are symmetry (Laporte)-forbidden, giving modest absorption intensity (3  5–100 M 1 cm 1).75 They can also be spin-forbidden if the spin state changes, and then have very low intensity (3 < 0.1 M 1 cm 1), but can be enhanced for complexes with heavy atoms.75 Ligandcentered (LC) transitions, or intraligand (IL) transitions, within only single ligand-centered orbitals are generally seen in large delocalized systems with high intensity (3 [ 103 M 1 cm 1).29,75 CT transitions can be metal-to-ligand (MLCT), ligand-to-metal (LMCT) and to-solvent (TS) transitions, which typically give rise to very intense bands (3  103–105 M 1 cm 1) since they are parity-allowed.29,73 These transitions sometimes occur in the UV region, have high energy, and are able to lead to redox reactions and homolytic bond cleavage.29,76 Light excitation leads to excited states through various electronic transitions and pathways (Scheme 2).29 Excited states of metal complexes with the same multiplicity but different electronic distributions as compared to the ground state have higher energy levels, and are able to undergo not only radiative processes (e.g. fluorescence and phosphorescence), but also radiationless processes (e.g. intersystem crossing (ISC), internal conversion (IC), intramolecular vibrational redistribution and solvation dynamics), ultimately leading back to the ground-states.29,72 Metal complexes usually generate phosphorescence from excited triplet states of 3 MLCT origin, which are formed through ISC from excited singlet states and can be enhanced by the large spin-orbit coupling caused by heavy metals.77 Thus, luminescent metal complexes often have large Stokes shifts and long emission lifetimes, which allows them to be distinguished from short-lived background autofluorescence, sometimes a major problem for organic fluorescent molecules, and to be investigated by highly sensitive phosphorescence lifetime imaging microscopy (PLIM).78

2.17.3.2

Activation wavelengths

The choice of activation wavelength in phototherapy is restricted by the absorption spectra of photoactive prodrugs and based on a compromise between tissue penetration by the light and photon energy. According to the Planck–Einstein relation: E ¼ hnʋ ¼ hc / l, light with shorter wavelength possesses higher energy.79 Light penetration is dependent on the optical properties of tissue and the wavelength.10,80 The tissue-scattering coefficient of cells decreases monotonically as the wavelength increases, while absorption coefficient is affected by chromophores in tissues, mainly hemoglobin and melanin, and varies significantly over tissue and light wavelength.80 The efficacy of shorter wavelength light is limited by chromophore absorption, while longer wavelength light is absorbed by water.10 Therefore, the ideal activation wavelength range for phototherapy is

Absorption Fluorescence (~ ns)

E

Phosphorescence (~ µs) Internal conversion

S2

Intersystem crossing Nonradiative relaxation

S1

e-/H+

PDT type I

T1 PTT

1

energy

hv

PDT type II

~ 1270 nm ~ 1 eV

Photoproducts

S0 Scheme 2

O2

PACT

Reactive pathways for photoactive metallodrugs (Jabłonski diagram).

3

O2

512

Photoactive metallodrugs

28 0

– 32 320 0 – 40 40 0 0 – 47 47 0 0 – 55 55 0 0 – 60 60 0 0 – 65 65 0 0 – 10 10 00 00 +

Wavelength (nm)

0 1 2 Tissue Penetration (mm) 3 4 5 Fig. 2 Tissue penetration depth of irradiance, Reprinted with permission from Ruggiero, E.; Alonso-de Castro, S.; Habtemariam, A.; Salassa, L. Dalton Trans. 2016, 45, 13012–13020. Copyright 2016 Royal Society of Chemistry.

between 620 and 850 nm, the so called “phototherapeutic window,” in which photons have enough energy for photoactivation, and light penetration is sufficiently deep (Fig. 2).81

2.17.3.2.1

One-photon activation

2.17.3.2.2

Multi-photon activation

Traditional photoactive prodrugs are activated by one-photon excitation, including clinically approved photosensitisers.42 Onephoton activation can be achieved by both lasers with narrow spectral width and by broadband lamps (e.g. LEDs).82 The relatively low price, small size, larger irradiation area and convenience to operate mean that LEDs are the mainstream light sources for skin phototherapy.83 However, to reach internal organs, lasers with high power output and easy connection to optical fibers and endoscopes are required for precise light delivery.84 The common one-photon light wavelengths in phototherapy vary from UV radiation (100–400 nm), visible light (400– 750 nm) to NIR radiation (750–2500 nm).81 The majority of reported photoactive metallodrugs are investigated with UVA and visible light.1,85 UV radiation is divided into UVC (100–280 nm), UVB (280–320 nm) and UVA (320–400 nm), each having different physiological effects.86 UV radiation has been applied in dermatology alone or in combination of photoactive prodrugs for decades, mainly acting by its local and systemic immunosuppression.87 However, due to the UV damage and poor tissue penetration, its medical application is limited to skin and other superficial diseases.88 Visible light is less harmful with deeper tissue penetration than UV radiation and therefore widely used to activate clinically approved photosensitisers, especially red light.40 Light of 600–700 nm possesses a 50– 200% deeper penetration compared with that of 400–500 nm in most of tissue models.82 Despite the wide application of red light, its deeper penetration might cause unwanted side effects. PhotofrinÒ was first approved with red light (630 nm) to treat bladder cancer, but abandoned due to skin photosensitivity and compromised bladder function.40 In contrast, green light (525 nm) is used to activate TLD-1433 for NMIBC to reduce PDT-induced damage to the bladder wall.13 Activation of simple metallodrugs by NIR radiation with satisfying tissue penetration is relatively rare. TookadÒ soluble is the only approved photosensitiser that is activated by NIR (753 nm). In contrast, NIR radiation has been widely used in nanomaterial-based phototherapy, especially PTT.67 Notably, photoactive metallodrugs that are NIR-inactive can be activated with NIR radiation when loaded into upconversion nanoparticles (UCNP), owing to their ability to absorb and convert low-energy NIR photons into higher energy UV–visible light.89

Multi-photon activation is a promising strategy to red-shift activation wavelengths to the NIR region, and two-photon activation is the most frequently investigated among them.42,90,91 Two-photon absorption was proposed by Göeppert-Mayer, a Physics Nobel Prize winner in 1963,92 and subsequently two-photon fluorescence microscopy was invented in 1990.93 Different from one-photon excitation (OPE), two photons are absorbed simultaneously, leading to two-photon excitation (TPE), with each photon contributing half of the required energy.42 Thus, TPE can populate the same excited state normally resulting from OPE with twice the energy, e.g. blue light (440 nm) activatable prodrugs can be excited by two-photon NIR (880 nm) radiation.91 To achieve TPE of a prodrug, a large TPA cross-section is necessary, which is usually determined by z-scan and two-photon excited fluorescence (TPEF).91 Traditional TPE investigations focus on organic dyes with strong conjugation and rigidity, the first TPE metal complex dipyridinium Tl(III) pentachloride was reported in 1974, followed by Ru(II) and Ir(III) TPE photosensitisers.42 Compared with one-photon activation, multi-photon activation can achieve deeper penetration and cause less photodamage.90 However, to allow absorption of multi photons simultaneously, high power femtosecond solid-state lasers are required, which increases the expense of multi-photon phototherapy.82 In addition, the small irradiation volumes significantly slow down the treatment time, which makes multi-photon activation less practicable.85

Photoactive metallodrugs 2.17.3.3

513

Photoactivation mechanisms and pathways

Phototherapy is typically classified into PDT, PACT and PTT according to the active factors, while the mechanism of action in phototherapy can be divided into photocatalysis, photoreduction, photosubstitution, photoactivation of ligands, and the combination of two or more mechanisms (Fig. 3).

2.17.3.3.1

Photocatalysis

Metallodrugs can behave as catalysts in photocatalytic mechanisms upon irradiation, which can generate high phototherapeutic efficacy at low drug doses.94 The photocatalytic nature of PDT photosensitisers makes photocatalysis the most important clinically relevant mechanism of photoactivation (Scheme 2).32 A photosensitiser in the ground singlet state S0 absorbs energy from light photons at a specific wavelength and is converted to an excited singlet state S1. This is usually unstable and partially releases energy by fluorescence radiation to return to S0, while the remaining energy converts the photosensitiser to excited triplet state T1 through ISC. A T1 photosensitiser is in its therapeutic form. It can either radiate energy as phosphorescence or undergo Type-I or Type-II pathways to interact with tissue oxygen. In the Type-I pathway, T1 photosensitiser transfers energy to surrounding biomolecules by hydrogen or electron transfer to form radicals, which can further react with oxygen to produce ROS, e.g. superoxide, hydrogen peroxide, and hydroxyl radicals, leading to oxidative stress. In the Type-II pathway, T1 photosensitiser interacts directly with triplet oxygen (3O2, ground state) by energy transfer to form singlet oxygen (1O2), the major cytotoxic species in PDT, leading to the facile oxidation of most biological organic molecules that are usually in lower energy singlet states. Generally, the Type-II pathway is assumed to be the dominant PDT mechanism, while Type-I starts to prevail when oxygen runs out; the ratio of these mechanisms can be affected by many factors, e.g. oxygen concentration, pH and photosensitiser structure.95 Oxygen is indispensable in both pathways, although the Type-I PDT process is less oxygen dependent than Type-II.33 Ideally, photosensitisers should return to their original ground state and be ready for the next photocatalysis cycle, namely possess high photostability.96 For PDT metallodrugs, such as polypyridyl Ru(II) and cyclometalated Ir(III) complexes, light irradiation usually populates the 1MLCT excited states, which can undergo ISC to reach 3MLCT excited states.13,47 3MLCT excited states are sensitive to oxygen, and can be emissive or interconvert to other excited states, e.g. 3IL states for potent PDT, which is significantly influenced by the coordinated ligands.13 The mechanism of action of PTT prodrugs is also based on photocatalysis (Scheme 2). PTT agents absorb a photon to reach an excited singlet state S1 similar to PDT, but they undergo nonradiative relaxation processes to release heat and raise the surrounding temperature directly, and thus are not dependent on oxygen.97

2.17.3.3.2

Photoreduction

Metal oxidation states can affect the stability and reactivity of the complexes. Photoreduction is a promising PACT mechanism to covert the inert prodrugs into active drugs with light control. Generally, photoreductive metallodrugs possess dissociative excited states with LMCT nature, and thus irradiation leads to the dissociation of ligands with electron transfer to the metal.98 Ligand dissociation and a redox process in an excited metal complex can occur at any stage during the decay back to the ground state.29 Pt(IV)/ Pt(II) is a commonly studied photoreduction system, owing to the kinetic inertness of Pt(IV) and higher reactivity of Pt(II).54

2.17.3.3.3

Photosubstitution

Photosubstitution is another common PACT mechanism. Irradiation into the dissociative LMCT bands in Pt(IV) complexes can also lead to non-redox photosubstitution of photolabile ligand by solvent molecules without electron transfer.98,99 However, photosubstitution of polypyridyl Ru(II) and tricarbonyl Re(I) complexes with sterically bulky ligands typically starts from the formation of 1 MLCT excited states upon irradiation, and leads to the population of 3MLCT excited states, followed by dissociative triplet metal-

(A)

BM + O2 hv

MLn

ROS + MLn

3O2

MLn*

1

O2 + MLn

heat + MLn

Photocatalysis e-

(B) M

e

X

-X

2S

(C)

hv

M

M

X

2X

Photoreduction

(D) M

L X

hv

X

M

hv

M

S S

2X

Photosubstitution L

M

X X M

hv M

X X

M

X

Ligand photoactivation Fig. 3

Common pathways involved in the photoactivation of metallodrugs (M: metal; L: ligand; BM: biomolecule; S: solvent; X: photoactive moiety).

514

Photoactive metallodrugs

centered (3MC) or ligand-field (3LF) states.13,49,100 3MC and 3LF states are favorable for ligand dissociation with substitution by solvent molecules.100

2.17.3.3.4

Photoactivation of ligands

Ligands play an important role in the photoactivation. They can not only affect the photoactivation mechanisms of metal complexes by modulating their absorption and emission, and steric strain, but also undergo photoactivation to generate active species.11,13,54,100 Organic molecules can be photosensitive,32 photocleavable,101,102 or photoswitchable.103 These photoactive molecules have been applied as pro-fragments in metallodrugs.11,54 The inclusion of photosensitive ligands in metal complexes can give rise to enhanced photocytotoxicity of PDT agents.104 Photocleavage of bonds in the ligands via photon absorption by a nearby metal center is a common strategy to re-activate “photocaged” complexes.105 Photoswitchable ligands can alter the cytotoxicity of dinuclear metal complexes upon irradiation by changing the configuration, usually as bridging ligands.106

2.17.3.3.5

Combinations of mechanisms

Common combinations of mechanisms include photocatalysis (PDT) with photoreduction or photosubstitution (PACT), and photocatalysis (PDT) with PTT, since PDT is oxygen dependent, while PACT and PTT are not.107–110 The combined photoactive components can be both metal complexes, or metal complexes together with organic molecules or nanomaterials. The combination of mechanisms can circumvent current drug resistance, relieve the hypoxia effect, red-shift activation wavelength, and enhance the photocytotoxicity. Combinations with chemotherapeutic agents, usually platinum complexes with hydrolysis or redox activation mechanisms, are also reported to be effective against cisplatin-resistant cancer cells and allow cellular imaging.111,112

2.17.3.4

Photoreactions with biomolecules

Photoproducts of metallodrugs often include ROS and radicals, metal complex fragments with active binding sites, e.g. Pt(II), Ru(II), and Rh(III) species, and released ligands, which are able to interact with nucleotides, DNA, amino acids, peptides and proteins, to mediate the photocytotoxicity of metallodrugs.85

2.17.3.4.1

Nucleotides and DNA

Cisplatin reacts at the N7 position of guanine and adenine residues of DNA preferentially to form intrastrand or interstrand crosslinks, which disrupt DNA structure in cell nuclei (as well as mitochondrial DNA), ultimately leading to cell death.113 As a result, DNA is an important target for metallodrugs. The major interactions between photoactive metallodrugs and DNA include intercalation, cleavage, oxidation and crosslinking.1,13,46,54,114 These interactions target different parts of DNA. Intercalation is noncovalent insertion between parallel planar base pairings, cleavage breaks the backbone of DNA strands, while oxidation and crosslinking involve the nucleobases. Generally, light-activated PDT agents generating ROS can induce DNA cleavage and oxidation.13,115 The PDT metallodrugs with a planar structure (e.g. Pt(II) photosensitisers), or a large p-conjugated ligand (e.g. dipyridophenazine (dppz)), can efficiently intercalate into a DNA double helix in the dark, allowing ROS production close to DNA.115–117 The combination of DNA interaction and cleavage can effectively increase the photocytotoxicity. Covalent crosslinking is more frequently observed in PACT agents due to the presence of active metal binding sites after irradiation, including interstrand and intrastrand crosslinking, analogous to cisplatin.45,54 DNA intercalation, photocleavage and oxidation can also be detected for some PACT metallodrugs.45,54

2.17.3.4.2

Amino acids, peptides and proteins

Amino acids, peptide and proteins are highly susceptible to photooxidation induced by ROS generated by PDT. This can lead to cleavage of the polypeptide chain and formation of cross-linked protein aggregates.118,119 On the other hand, metal binding to amino acid residues in peptides and proteins has been reported for PACT metallodrugs.54,115 Also, DNA-protein crosslinks can be induced by PACT photoproducts.120

2.17.3.5

DFT and TD-DFT calculations

A better understanding of the photophysics and photochemistry of metallodrugs is crucial for future development. However, numerous excited states lying in a small energy range and their short lifetimes, as well as forbidden electronic transitions and spectroscopic dark states make the spectra of metallodrugs complicated and limit the experimental characterization of their excited states.121 Computational methods, especially density functional theory (DFT) and time-dependent DFT (TD-DFT), can provide precise information on excited-state electronic and geometrical features, and the energy gap between excited states and ground states.121 In combination with experimental data, DFT gives insight into the assignment of the absorption and emission spectra of photoactive metallodrugs, therefore aiding the analysis of their photophysics and photochemistry.72,121

Photoactive metallodrugs

2.17.4

515

Photoactive anticancer metallodrugs

Cancer is traditionally treated by surgery, radiation therapy, and chemotherapy. Phototherapy is emerging as a new anticancer treatment, owing to its high spatio-temporal controllability and minimal invasiveness.28 The majority of clinically approved photosensitisers are applied in the treatment of cancer and precancer.40,41 The first FDA-approved anticancer photosensitiser, PhotofrinÒ, suffers from poor cancer selectivity, low red-light absorption and long-term photosensitivity.28 The introduction of a metal into the tetrapyrrole structure can significantly enhance ISC, therefore population of excited triplet states, favorable for efficient ROS generation, and red-shifting the absorption band.42 The EMA has approved Pd(II) TookadÒ soluble, and Russia has approved Al(III) PhotosensÒ, which has encouraged the development of photoactive anticancer metallodrugs.25,28,40,42 In this section, anticancer photoactive metallodrugs will be classified according to the phototherapy types and discussed in atomic number order. The PDT sub-section contains agents with merely a PDT (O2-dependent) mechanism; combinations with other photoactivity mechanisms will be classified in the corresponding subsections.

2.17.4.1

Photodynamic therapy (PDT)

PDT has long been a routine treatment for superficial skin cancers and precancerous lesions in dermatology, and a developing therapy for internal tumors.7 PDT destroys tumors not only by killing cancer cells directly with ROS, but also by causing vascular damage to obstruct oxygen supply to tumors and induce an immune response to eliminate metastatic tumors.122 Increasingly, metallodrugs are entering clinical trials as anticancer photosensitisers, with further photosensitive metal complexes under development.13,42

2.17.4.1.1

PDT metallodrugs entered clinical trials

Most anticancer PDT metallodrugs that have entered clinical trials contain a heterocyclic structure with a large p system to give rise to absorption in the red-NIR region and consequent deep tissue penetration.40 Al(III). PhotosensÒ (AlPcSn, Fig. 1A) developed at the General Physics Institute of the Russian Academy of Sciences is a mixture of Al(III) sulfonated phthalocyanines bearing different degrees of sulfonation, and received a series of approvals in Russia between 2001 and 2008.40,123 The average sulfonation degree of PhotosensÒ is 3; sulfonation significantly improves the aqueous solubility.124 PhotosensÒ can be administered by intravenous or direct lesion injection and by aerosol spray, then activated by red light at 675 nm.41 It has entered clinical trials for skin, breast, lung, head and neck, and gastrointestinal tract malignancies, as well as agerelated macular degeneration.124 However, PhotosensÒ failed to gain worldwide acceptance due to a purity issue, since single agents rather than mixtures are preferred as drugs. Pd(II). TookadÒ soluble (Padeliporfin dipotassium, WST-11, Fig. 1A) was approved by the EMA in 2017, while use of TookadÒ (Padoporfin, WST-09) was terminated due to its poor water solubility.40 They are both Pd(II)-substituted bacteriophorbide derivatives developed by Scherz and Salomon in Israel.41 WST-09 can be activated by NIR at 763 nm, while the activation wavelength of WST-11 is 753 nm, and light is delivered using interstitial transperineal optical fibers inserted into prostate tumors.24 Both of them are vascular-targeting PDT agents that are administrated intravenously and activated in blood vessels via a Type-I PDT mechanism to cause rapid vascular shutdown.125 They have entered clinical trials for localized prostate cancer, and the efficacy and safety of WST11 were demonstrated in randomized phase III trials.22,23 The rapid clearance of WST-11 allows illumination shortly after infusion and limits the cutaneous photosensitivity.126 Ru(II). TLD-1433 (Fig. 1B) is a racemic Ru(II) dyad with two 4,40 -dimethyl-2,20 -bipyridine ligands and a imidazo[4,5-f][1,10]phenanthroline ligand appended to an a-terthienyl organic chromophore synthesized by McFarland et al. in Canada.13 Chloride was chosen as a counterion for enhanced aqueous solubility and biocompatibility. It is the only photoactive metallodrug to advance to clinical trials based on an octahedral structure rather than macrocyclic structure with a large p system.40 Thus, the absorption spectrum of TLD-1433 peaks at ca. 425 nm in aqueous solution and green light at 525 nm is employed for photoactivation.127 The excited TLD-1433 possesses 3MLCT states for luminescence diagnostic imaging, 3IL states for cytotoxic 1O2 generation, and a 3ILCT state on the a-terthienyl group (in theory) for intermolecular charge-transfer reactions, so called Type-I/II PDT.13,127 A phase Ib clinical trial was performed in BCG refractory high-risk NMIBC patients with satisfactory results.13 TLD-1433 can be administrated into the bladder by intravesical instillation and an optical fiber with a spherical diffuser designed by Lilge is placed in the bladder through the urethra.13,128 The 200 photocytotoxicity selectivity of bladder tumors over normal, healthy urothelial tissue, allows illumination of the entire bladder treated by TLD-1433.13 Zn(II). CGP55847 (zinc phthalocyanine, Zn-PC, Fig. 1C) developed by Ochsner et al. in Switzerland, is a liposomal Zn(II)phthalocyanine.129 Its absorption spectrum peaks at 670 nm with a large extinction coefficient (3  2.7  105 M 1 cm 1). CGP55847 entered Phase I/II clinical trials for squamous cell carcinomas of the upper aerodigestive tract before being terminated, without the details being reported.40 Sn(IV). PurlytinÒ (Rostaporfin, SnET2, Fig. 1C) synthesized by Morgan et al. in the USA is a Sn(IV) complex based on ethyl etiopurpurin.130 PurlytinÒ in a Cremophor-EL emulsion is injected intravenously and activated by red laser light at 664 nm to exert direct cancer cell photocytotoxicity.131 PurlytinÒ entered phase II/III clinical trials for recurrent cutaneous metastatic breast cancer and was certified to be effective without significant systemic toxicity.132,133 However, the development of PurlytinÒ was discontinued mainly due to skin photosensitivity.130

516

Photoactive metallodrugs

Lu(III). AntrinÒ (Motexafin lutetium, Lutrin, Fig. 1C) is a water-soluble Lu(III) texaphyrin derivative developed by Sessler et al. in the USA.134,135 AntrinÒ bears a Hückel aromatic periphery of 22 p-electrons, which allows its photoactivation at 732 nm for deep tissue penetration.136 Notably, AntrinÒ exhibited a selective accumulation in tumor tissue and microvasculature of female DBA/2 N mice bearing a fast-growing spontaneous subcutaneous mouse mammary tumor, limiting damage to the surrounding normal tissue and vascular system.137 AntrinÒ entered phase I clinical trials for locally-recurrent prostate cancer using intravenous administration and interstitial light delivery.138 Large but transient increases in serum prostate-specific antigen (PSA) levels in patients were induced by AntrinÒ PDT.139 Inter- and intra- patient heterogeneity in light, oxygen, photosensitiser and optical properties of prostate tissue were detected by an integrated PDT dosimetry system, which resulted in significant variability in tissue necrosis throughout the prostate.138 The uncertainty and inconsistency caused by these variabilities may hinder the continuation of AntrinÒ in oncological PDT clinical trials.

2.17.4.1.2

Candidate PDT metallodrugs

V(IV) and V(V). Vanadium complexes are well known as insulin mimetics,140 and as candidate anticancer drugs, with oxovanadium(IV) and dioxovanadium(V) complexes giving rise to DNA photocleavage upon UVA irradiation.141–144 Oxovanadium(IV) complexes are stable under physiological conditions and have a low energy d-d absorption band near 700 nm, allowing photocleavage of DNA and proteins with red light.141,142,145,146 Chakravarty et al. have developed a series of photoactive oxovanadium(IV) complexes by introducing N-donor chelating ligands with large delocalized p systems.141,142 Complex V-1 (Fig. 4) chelated by N-salicylidene-L-methionate in a meridional binding mode is a red-light activatable oxovanadium(IV) complexes that shows a d-d band in 694 nm with a shoulder at 840 nm in DMF.142 The planar heterocyclic dppz ligand in V-1 allows ct-DNA intercalation in the dark with an intrinsic equilibrium DNA binding constant (Kb) value of 7.2  105, highest in this series. Efficient DNA cleavage induced by V-1 is observed upon irradiation with both UVA (365 nm) and red light (l > 750 nm) via a 1O2-involved mechanism due to photosensitisation of the phenazine moieties. Since dppz and vanadyl sulfate alone are photocleavageinactive, the DNA cleavage is assumed to be metal-promoted with MLCT and d-d sources. Other than DNA, oxovanadium(IV) complexes also exhibit photocleavage of proteins.146 Complete degradation of BSA and lysozyme in a non-site-specific manner is induced by V-2 (Fig. 4) upon UVA irradiation via an hydroxyl radical pathway.146 With visible light, V-2 gives an IC50 value

2+

O S

N

O

N

N

N

N

N

HN

V O

O

N N

N

N

N

O

V-1

V-2 H3CO

F N F B N

OH

O

N

NH2 O

HN

V N

V-4

+

H2N

O

N

O

O

N

H N

N

N

V

N

O O

V-3

H3CO

V-5

OH

OH H N

3+ N

N

N

N

O V

O

O

N

N

N

Cr N

N

O

V-6 Fig. 4

Photoactive V(IV) and Cr(III) complexes studied as anticancer PDT agents.

N

Cr-1

N

N

N N

N

N

N H

N

V N

N

V N

+

O

Cl

Photoactive metallodrugs

517

of 17 mM towards A549 lung cancer cells, while the dark IC50 is 175 mM. An apoptotic mode of cell death is indicated by the fragmented or highly condensed nuclei, cell shrinkage and membrane blebbing. V-3 (Fig. 4) with two dppz ligands, responsible for the DNA intercalation and ROS generation, exhibits promising photocytotoxicity in HeLa cells with both UVA and visible light exposure.141 Di-iodinated borondipyrromethene (BODIPY) can generate 1O2, while curcumin can generate hydroxyl radicals upon irradiation.145 The combination of these two moieties into oxovanadium(IV) complex V-4 (Fig. 4) not only increases the physiological stability of curcumin, and enhances the photocytotoxicity with visible light, but also allows cellular imaging.145 V-4 exhibits high dark stability, preferentially localises in mitochondria, generates ROS and potent photocytotoxicity with an apoptotic mechanism. The pyrene pendant in V-5 (Fig. 4) enhances the photoinduced ROS generation, and thus improves its photocytotoxicity with a photocytotoxicity index (PI) of 16.147 In contrast to V(IV), the PDT applications of V(V) complexes are limited, owing to their UVA activation.143,144 Dioxovanadium(V) complex V-6 (Fig. 4), with a Vitamin-B6 Schiff base, exhibits damage to nuclear DNA and ROS-mediated apoptotic photocytotoxicity with visible light.148 Cr(III). Polypyridyl Cr(III) complexes are potential photosensitisers in biological applications due to their long-lived excited states and promising photoredox catalytic activity.149–151 Highly water-soluble and photostable Cr-1 (Fig. 4) exhibits strong luminescence and a high 1O2 quantum yield.152 The average PIs of Cr-1 in different cancer cell lines are ca. 5 upon irradiation at 450 nm, while satisfactory dark stability is observed.153 Fe(II) and Fe(III). Iron is the most abundant bio-essential transition metal and plays an important role in cancer therapy as well as being recognized for the clinical success of Fe–bleomycin.154 High biocompatibility and rich photochemistry make Fe(II) and Fe(III) complexes promising photoactive anticancer agents.155 The earliest well-documented photochemical reaction of iron complexes is the photo-assisted Fenton reaction (H2O2/Fe(II)), in which the ROS generation is significantly accelerated by UV radiation due to the rapid regeneration of Fe(II) through photoreduction of Fe(OH)2þ.156 Fe-1 (Fig. 5) and its derivatives with 9aminoacridine or 1,8-naphthalimide pendants are reported as DNA photocleavage agents with a ROS-involved mechanism.157 Although Fe(II) complexes coordinated to three dipyridoquinoxaline (dpq) ligands are DNA photocleavage-inactive with visible light, polypyridyl Fe(II) complexes with visible light absorption and emission have been designed through extension of the p-system.158 These complexes exhibit satisfactory low cytotoxicity in the dark. Fe-2 (Fig. 5), with DNA intercalating pyrenyl fragments, localizes in the nucleus, generates ROS and photocleaves DNA efficiently with visible light, thus inducing significant photocytotoxicity in HeLa cancer cells with an IC50 of 7.4 mM.159 Fe-3 (Fig. 5) with highly fluorescent BODIPY displays red emission with a long lifetime (624 ns) and a high 1O2 quantum yield (68%) in acetonitrile.160 It is a mitochondrial-targeting photosensitiser that is significantly photocytotoxic to HeLa cancer cells (1.05 mM), while relatively non-toxic to healthy MRC5 lung cells (36.2 mM) with green light (500 nm). In contrast to the terpyridine Fe(II) complexes, Fe-4 and Fe-5 (Fig. 5) with three N,N-bidentate ligands show weak red luminescence and poor 1O2 quantum yields, and thus extremely low photocytotoxicity in cancer cells with a PI of ca. 1.161,162 Other photoactive Fe(II) complexes are ferrocene derivatives, which are anticancer ROS generators via redox catalysis.163 Ferrocene conjugate Fe-6 (Fig. 5) with an imidazophenanthroline substituent on the Cp ring exhibits intercalative and groove binding to DNA in the dark, DNA photocleavage and photocytotoxicity towards HeLa cells with visible light irradiation.164 In contrast, the ferrocene-free control, 2-phenylimidazophenanthroline is less photoactive than Fe-6. Photoactive ferrocene derivatives alone are less reported, while multinuclear conjugates with ferrocene moieties have been widely investigated. Ferrocene with its low-energy visible absorption bands is a convenient fragment to change lipophilicity and excited state behaviors of metal complexes and facilitate the generation of cytotoxic species upon irradiation.163 Fe(III) complex Fe-7 (Fig. 5) with a pendant ferrocene and an esculetin ligand hydrolyses in aqueous solution and localises mostly in the mitochondria.165 The LMCT transitions (esculetin / Fe(III)) at 720 nm allows photoactivation of Fe-7 using visible light, which leads to cellular ROS generation and apoptosis. Notably, Fe-7 displays selectivity to cancer cells with low photocytotoxicity towards normal cells. Ferrocene conjugates with other metal complexes will be discussed in the Multi-metal section. Photosensitive Fe(III) complexes comprise two main classes.166 The first, Fe(III) complexes Fe-8 to Fe-11 (Fig. 5), are stabilized by a 2,2-bis(3,5-di-tert-butyl-2-hydroxybenzyl)aminoacetate ligands, the N,N-bidentate ligand serves as DNA intercalator and photosensitiser.167–170 The formation of hydroxyl radicals in a redox pathway is proposed as a DNA photocleavage mechanism.167 The increase in planar aromatic groups from Fe-9 to Fe-10 results in significant enhancement in the DNA photocleavage, cellular accumulation and photocytotoxicity.169 The appended biotin moiety in Fe-11 led to an increased photocytotoxicity in HepG2 cells with overexpressed sodium-dependent multi-vitamin transporters.170 The second type of Fe(III) complexes Fe-12 to Fe-15 (Fig. 5), contain a chelated 2,20 -dipicolylamine ligand instead.171–174 The low-energy LMCT band from a phenolate p orbital to the dp* orbital of Fe(III), facilitates red light-induced DNA cleavage and photocytotoxicity.172 The pendant glucose in Fe-13 improves the internalization into HeLa cells and visible light-induced cytotoxicity.172 Fe-14 with a triphenylphosphonium (TPP) group localizes in mitochondria specifically, while its TPP-free analog distributes uniformly throughout the cells, while no significant difference in photocytotoxicity is observed.173 Conjugate Fe-15 with BODIPY exhibits both in vitro and in vivo inhibition in growth of human breast adenocarcinoma (BT474luc) cells with red light exposure, and a photo-Fenton induced ROS-involved apoptotic cell-death mechanism has been proposed.174 Co(III). Cobalt is an essential metal found in vitamin B12.175 Octahedral Co-1 (Fig. 6) with two dppz ligands and a curcumin ligand displays a curcumin-based p / p* absorption band at 400–500 nm and a green emission at 525 nm.176 Co-1 is stable towards oxygen in the presence and absence of ascorbic acid, while releasing curcumin under hypoxia. Co-1 selectively localizes in mitochondria, then induces an ROS increase and apoptotic photocytotoxicity with visible light. The high affinity of human serum albumin (HSA) for Co-1 allows its delivery in the blood. Replacement of curcumin by esculetin to give Co-2 (Fig. 6) gives rise to a photoredox Type-I pathway involving hydroxyl radicals which can photocleave supercoiled DNA, and shows promising visible light photocytotoxicity.177

518

Fig. 5

Photoactive metallodrugs

Photoactive Fe(II) and Fe(III) complexes studied as anticancer PDT agents.

Zn(II). Zn(II) phthalocyanines (Zn-Pcs) are important anticancer photosensitisers with excitation wavelengths > 630 nm and Zn-Pc CGP55847 has entered clinical trials.178 However, the poor solubility of Zn-Pcs limits their applications, which can be overcome by introduction of peripheral (b position) and non-peripheral (a position) substituents in the Pc framework (Fig. 6). Substituted Zn-Pcs include sulfonated, phosphonated or carboxylated anionic derivatives; hydroxylated or fluorinated derivatives;

Photoactive metallodrugs

2+ H3CO

2+

OH

N

N

N

N N

N

N

O

N

Co O

N N

N H3CO

OH

Co-1 R

Co-2 R

R

N

N N

N N

Zn N

N

R

R

α-tetrasubstituted Zn-Pcs

N

N

N I

I

N R

N Zn

N N

N

I

I N

Zn

N

N

Fig. 6

O

N

N

N

O

O

N

N

N

O Co

N

R

519

R

β-tetrasubstituted Zn-Pcs

Zn-1

Photoactive Co(III) and Zn(II) complexes studied as anticancer PDT agents.

alkylated derivatives; and derivatives with tumor-targeting vectors (e.g. lactose, folic acid and peptides).123,178 Other than Zn-Pcs with a macrocyclic p system, novel homoleptic bis(dipyrrinato)Zn(II) complexes have been developed with iodine substituents to enhance intersystem crossing to excited triplet states by the heavy atom effect, and thus improve 1O2 generation, and peripheral bulky aryl groups to improve their photophysical properties.179 The leading complex Zn-1 (Fig. 6) shows an intense absorption band at 517 nm, emission at 535 nm with a luminescence quantum yield of 4.5% and a 1O2 quantum yield of 57% in methanol with 505 nm excitation. Polymer matrix-encapsulated Zn-1 is highly photocytotoxic upon irradiation at 500 nm in both monolayer cells and 3D spheroids, while non-toxic in the dark. The polymeric encapsulation improves the aqueous solubility of Zn-1 and protects it from quenching effects in water. Ru(II). The archetypal photoactive polypyridyl Ru(II) complex [Ru(bpy)3]2 þ (Ru-1, Fig. 7) was synthesized by Burstall in 1936,180 and the toxicity and antimicrobial activity of such compounds was first reported by Dwyer et al. in 1952.181 Electron transfer from Ru-1 in the excited triplet state was discovered by Adamson and Demas in 1971, which inspired the rapid development of polypyridyl Ru(II) complexes as photosensitisers.182 Nowadays, polypyridyl Ru(II) complexes are a research focus as anticancer PDT metallodrugs, and TLD-1433 has become the first Ru(II) complexes to enter clinical trials as a PDT agent.13 The photophysics and photochemistry of polypyridyl Ru(II) derivatives of Ru-1 are tunable by modification of bipyridine ligands, including solubility, excitation wavelength, luminescence, 1O2 generation, dark- and photo-stability, cellular accumulation and localization, photocytotoxicity, and mechanism of action.39,44–46 Extension of the ligand p system and functionalization with electron-donating or -withdrawing substituents are straightforward strategies to tune the excitation wavelength and photoactivity of polypyridyl Ru(II) complexes.183 DFT calculations reveal the significant reduction of the HOMO  LUMO energy gap by introduction of a vinyl dimethylamine electron-donating group in Ru-3 and Ru-5 (Fig. 7).183 Although changing the ligand scaffold from phen (Ru-2, Fig. 7) to bphen (Ru-4, Fig. 7) does not result in a large difference in the gap, Ru-4 exhibits red-shifted absorption tails for photoactivation with 595 nm light. Notably, despite the intense absorbance of Ru-3 and Ru-5, they have poor photophysical properties (luminescence and 1O2 quantum yield) and poor stability. Ru-4 localizes in the cytoplasm and displays high photocytotoxicity in both monolayer cells and 3D spheroids with 595 nm light exposure by disturbance of mitochondrial respiration and glycolysis. Generally, octahedral polypyridyl Ru(II) complexes are þ 2 positively charged, and the introduction of extra charge in ligands can alter their cellular localization substantially.184,185 The highly positively-charged complex Ru-6 (Fig. 7) carrying six tertiary ammonium groups, raising the overall charge to þ 8, has better aqueous solubility and affinity for negatively-charged cell membranes, which thus facilitates its internalization.184 Compared with unsubstituted Ru-1, Ru-6 displays higher luminescence

520

Photoactive metallodrugs

2+

2+

2+

N N

Ru

Ru N

N

N

Ru-1

N

N N

N

N

N

N

N

N Ru

R

N

R

N

N

N

R

R

R = CH3

Ru-2

R = CH3

Ru-4

R = CH2=CH2NH(CH3)2

Ru-3

R = CH2=CH2NH(CH3)2

Ru-5

SO3-

4-

2+ N

8+ -

O 3S

N N

SO3-

N

N

N

N

N

N Ru

Ru

Ru N N

N

N

N

N

N

N

N

N N

N

N

-

O 3S

N

SO3-

N -

O 3S

Ru-6

Ru-7

Ru-8 COO-

2+

2+

N N

N

N

N

N

N

N

N

Ru N

N

N

N N

N

Ru N

+

N

N

N

N

C

N N

N N

Ru

Ru

N

N

N

N

N

N N

N

N

COO-

Ru-9 Fig. 7

Ru-10

Ru-11

Ru-12

Photoactive Ru(II) complexes studied as anticancer PDT agents.

and 1O2 quantum yield, as well as significantly enhanced cellular accumulation and photocytotoxicity. Once it has entered cancer cells, Ru-6 localizes in lysosomes first, then escapes and generates ROS upon irradiation, and finally penetrates the nucleus causing cell death and morphological changes. Compared to its þ 2 charged archetypal complex, Ru-7 (Fig. 7) has higher lipophilicity (log P ¼ 1.8  0.02), and Ru-8 (Fig. 7) with six sulfonate substituents is  4 negatively-charged and more hydrophilic (log P ¼  2.2  0.12).185 Ru-7 localizes in lysosomes and mitochondria in the dark, while Ru-8 is mainly found in the cytosol. Upon irradiation, Ru-7 damages nuclear membranes, while Ru-8 mainly localizes in lysosomes with the nuclear membranes remaining intact. As a result, Ru-7 induces necrotic cell death with high photocytotoxicity, while Ru-8 shows low dark cytotoxicity and kills cells with light via an apoptosis mechanism. The DNA intercalative dppz ligand is frequently used in anticancer Ru(II) photosensitisers.186 Ru-9 (Fig. 7) was reported in 1990 as a “light switch” probe for DNA. The complex displays > 104 increase in luminescence in the presence of double-helical DNA.187 Binding to DNA allows proximate ROS generation, an effective strategy to enhance DNA photocleavage. The electron/energy transfer efficiency of short-lived excited Ru-9 is not sufficient as a photosensitiser, but its analog Ru-10 (Fig. 7) with p-expansive benzodipyridophenazine (dppn) ligand has low-lying and long-lived 3IL excited states and is suitable for PDT at various wavelengths from blue-green light to NIR with a high 1O2 yield of 81% (420 nm).188

Photoactive metallodrugs

521

Ru-11 (Fig. 7) is a cycloruthenated analog of Ru-9, which displays remarkably enhanced cellular accumulation and photocytotoxicity compared with Ru-9.189 Notably, nearly 90% of Ru-11 accumulates in nuclei within a 2 h incubation and leads to effective disruption of cellular transcription by inhibiting binding of transcription factor NF-kB to DNA sequences. Ru-12 (Fig. 7) bearing two tridentate N-heterocyclic carbene (NHC) ligands displays enhanced visible light absorbance, superior excited-state lifetimes and higher 1O2 generation compared with its terpyridine analogs. Consequently, low micromolar photocytotoxicity is observed for Ru12 with no dark cytotoxicity, while its terpyridine analogs show no photocytotoxicity.190 Conjugation with cancer-targeting vectors can enhance the selective accumulation and photocytotoxicity of Ru(II) complexes.39 Ru-13 (Fig. 8) containing mitochondria-targeting TPP, shows an IC50 value of 9.6 mM in one-photon PDT (450 nm) and 1.9 mM in two-photon PDT (830 nm) in 3D spheroids.191 Glucose conjugate Ru-14 (Fig. 8) preferentially targets both tumor cells and mitochondria as an effective two-photon photosensitiser.192 Ru-15 to Ru-17 (Fig. 8) combine a hydrophobic porphyrin and hydrophilic Ru(II)-polypyridyl complex and exhibit enhanced aqueous solubility and two-photon absorption characteristics.193 Interestingly, the bridging ligand can alter the subcellular localization of these compounds: Ru-15 targets lysosomes, Ru-16 targets mitochondria,

Fig. 8

Photoactive Ru(II) conjugates studied as anticancer PDT agents.

522

Photoactive metallodrugs

while Ru-17 locates in the cytoplasm. Dinuclear Ru(II) complex Ru-18 (Fig. 8) bridged by a rigid tetrapyridophenazine ligand, primarily localizes in the nucleus and binds to duplex and quadruplex DNA with high affinity.194 An increase in cytotoxicity is observed for C8161 melanoma cells upon irradiation at 405 nm. Notably, two-photon activation at 900 nm results in death of cells and spheroids treated with Ru-18.195 In addition to polypyridyl Ru(II) complexes, porphyrins are also frequently used as ligands in other Ru(II) complexes since porphyrins are present in several clinically approved photosensitisers.45 Gianferrara et al. have reported a series of Ru-porphyrin conjugates and the leading compounds (e.g. Ru-19, Fig. 8) show high photocytotoxicity with low-dose visible light exposure.196 Ru(arene)-porphyrin complexes have also been investigated.45 Notably, porphyrins can be delivered by hexa- and octanuclear Ru(arene)-based cages with different release mechanisms. The hexanuclear cages must be disturbed before porphyrin release, while octanuclear cages allow porphyrin diffusion from their sides.197 Rh(II). Reports on Rh(II) PDT agents are quite rare. Dinuclear Rh(II) complexes bridged by acetate (m-O2CCH3; e.g. Rh-1, Fig. 9) can photocleave DNA in a ROS-mediated mechanism and exhibit 6-24  enhanced cytotoxicity upon irradiation.198 Re(I). Tricarbonyl Re(I) complexes have been widely investigated as anticancer agents and some of them are photocytotoxic.199 Tricarbonyl indolato Re(I) complex Re-1 (Fig. 9) has an intense absorption at 512 nm, giving rise to strong green light (505 nm)induced anticancer activity with a photocytotoxicity index of 1000 via high ROS generation.200 Porphyrin-Re conjugate Re-2 (Fig. 9) shows enhanced cytotoxicity with visible light. Although the Re(I) fragment did not contribute to ROS production upon irradiation, it improves the cellular accumulation of the conjugate.201 Os(II). Compared with Ru(II) complexes, polypyridyl Os(II) complexes tend to show lower luminescence quantum yields, but absorption bands at longer wavelengths and stronger resistance to photobleaching.202 Panchromatic polypyridyl Os(II) complex Os-1 (Fig. 9) is photoactivatable from 200 to 900 nm with high photostability.203 Satisfactory photocytotoxicity in glioblastoma cells is observed under normoxic and hypoxic conditions with both red and NIR light irradiation. Notably, Os-1 exhibits good in vivo efficacy in a murine colon cancer model using 635 nm light. Lysosome-targeting Os-2 (Fig. 9) exhibits NIR emission and photocytotoxicity upon red light exposure in A549 lung cancer cells.204 Os-3 (Fig. 9) based on a Os(phen)2-scaffold and a imidazo[4,5-f][1,10]phenanthroline tethered to four thiophene rings, has a PI exceeding 106 in normoxia and PI > 90 in hypoxia with visible light (400–700 nm) irradiation.205 Photocytotoxicity with green (523 nm), red (633 nm) and NIR (733 nm) light is also observed. A maximum tolerated dose (MTD) in mice > 200 mg kg 1 indicates the potential for Os-3 for in vivo applications. Ir(III). Cyclometalated Ir(III) anticancer photosensitisers can exhibit high photostability and energy-transfer activity due to their highly oxygen-sensitive triplet excited states, and therefore can be efficient Type-II photosensitizsers.48 The targeting of cellular organelles and proteins by Ir(III) complexes is an important feature which affects their photocytotoxicity.48 Ir-1 (Fig. 10) with high 1O2 generation ability (0.95), localizes in the endoplasmic reticulum (ER) primarily, as detected by confocal imaging (lem ¼ 560 nm).206 Photocytotoxicity in SK-OV-3 cells is observed for Ir-1 with low-dose “sunlight” exposure. Notably, twophoton activation (860 nm) of Ir-1 leads to apparent cell shrinkage. Photoinduced ROS cause photo-oxidation and photocrosslinking of proteins near the ER and mitochondria, which trigger cell death eventually. Ir-2 (Fig. 10) localizes in nuclei first by microtubule-dependent endocytosis to cross the plasma membrane, then cleaves DNA and migrates to mitochondria during two-photon irradiation, which results in double damage.207 Thus, Ir-2 is able to inhibit the growth of a solid tumor in mouse models with a similar inhibition (41.6%) as the PDT agent Chlorin e6 (40.8%). O ^O chelated complex Ir-3 (Fig. 10) displays potent photocytotoxicity towards monolayer cancer cells with low-dose visible light and towards tumor spheroids with two-photon activation at 750 nm.208 Liquid chromatography-tandem mass spectrometry studies indicated the specific oxidation of histidine residues to 2-oxo-His in the key proteins, aldose reductase and heat-shock protein-70 (Hsp-70), by irradiated Ir-3. The malfunction of cancer mitochondria and oxidative stress induced by irradiated Ir-3 increase glycolysis and contribute to cell death. Highly photooxidative Ir-4 (Fig. 10) localizes in mitochondria and exerts phototoxicity towards both normoxic and hypoxic cancer cells by disturbing electron transport through catalyzing photooxidation of NADH synergistically with photoreduction of cytochrome c.209 Aggregation may result in quenched emission and reduced photocytotoxicity of organelle-targeting photosensitisers. However, Ir-5 (Fig. 10) aggregates in mitochondria and displays aggregation-induced emission and 1O2 generation upon irradiation (405 nm), thus potent photocytotoxicity.210 The TPP-conjugated Ir-6 (Fig. 10) and its TPP-free analog Ir-7 (Fig. 10) specifically accumulate in mitochondria and lysosomes, respectively.211 Notably, Ir-6 reduces the respiration rate of HeLa cells under hypoxia, leading to a higher intracellular oxygen level, thus exhibiting a high PDT effect under hypoxia, whereas Ir-7 does not show similar properties. Ir(III) anthraquinone complex Ir-8 (Fig. 10) is a mitochondria-localizing carbon radical initiator, which displays turnedon emission under hypoxic conditions after reduction by a reductase.212 Reduced Ir-8 induces loss of the mitochondrial membrane potential and cell death upon irradiation at 405 nm, and significantly reduced tumor volume in mice bearing A549 xenografted tumors with two-photon activation at 730 nm. Ir-9 (Fig. 10) bearing a far-red-emitting coumarin-based fluorophore, displays high cellular accumulation in the cytoplasm of HeLa cells, and high photocytotoxicity with green and blue light, even under hypoxia.213 Superoxide O2
, is believed to be the only ROS generated by Ir-9 upon irradiation and is responsible for cell death. Thus Ir-9 shows no photocytotoxicity towards cells pre-treated by the $O2 scavenger tiron. Ir-10 (Fig. 10) with intense absorbance, localizes in lysosomes and mitochondria, induces an intracellular redox imbalance through the unique photocatalytic oxidation of NADPH and generation of ROS.214 Blue- and green-light-activated photocytotoxicity against drug resistant cancer cells, zebrafish and mouse cancer models was observed. Pt(II). Due to the clinical success of cisplatin, platinum complexes have attracted intense research interest as anticancer agents.98,115,215 Cyclometalated Pt(II) complexes can be luminescent and generate 1O2 upon irradiation.98 Pt-1 (Fig. 11) shows an extremely high 1O2 quantum yield (0.95, 355 nm), and binds to DNA and BSA.216 Notably photocytotoxicity is observed for

Photoactive metallodrugs

523

3+ N 2+ N O

N

O

Rh

O

Rh

O

N

H N

O

N

O

NH

N

N N

N N

N N OC

N

N Re

CO

N

N

N

CO

N H

Re-1

O

N

H N

O

O

Re-2 2+ S

2+

2+ N

N N N

H N

Os

S

N

N Os N

N N

N

N

N

S

N N

N

H N

N

N

Os

N

N

N

N

O OH

Os-1 Fig. 9

Fig. 10

CO CO Re

O

Rh-1

HN

Os-2

Photoactive Rh(II), Re(I) and Os(II) complexes studied as anticancer PDT agents.

Photoactive Ir(III) complexes studied as anticancer PDT agents.

Os-3

S

CO SCN

524

Photoactive metallodrugs

O H2N

H N

NH HN

HN

NH O

+

OH

O

H N

O

HN

O PEG NH

OH

O S

O PEG

O

N N

Pt N

N

S

O

N HN

NH

O

O O

O N

Pt-1 Cl H3N Pt NH3 N

Pt Cl

N

Pt H3N

Pt-3

Pt-2 4+

H2 N

O

N NH N

N HN

+

Pt N H2

O

NH3 Cl Pt N NH3

NH3

NH3 N Pt Cl NH3

N

N

N N Pt Cl N

O F B F

O

O H2 N

O

N

N B F F

Pt O

N H2

N H2N Pt NH3 Cl

Pt-4 Fig. 11

Pt-5

Pt-6

Photoactive Pt(II) complexes studied as anticancer PDT agents.

Pt-1 in HeLa cells after visible light exposure, with predominant cellular localization in nuclei and mitochondria. RGD vectorguided Pt-2 (Fig. 11) accumulates selectively in avb3 integrin-rich U87 cells, with an intense luminescence signal.217 A moderate IC50 concentration of Pt-2 (ca. 50 mM) with blue light exposure was observed for AY27 cells. Non-luminescent Pt(II) complexes are often conjugated to highly fluorescent photosensitisers, such as porphyrins and BODIPY. Pt(II) complexes with PEG-derivatized hematoporphyrins (e.g. Pt-3, Fig. 11) display varied dark and photocytotoxicity with red light (600–730 nm), affected by the solubility, type of platinum fragments and the corresponding porphyrin ligands.218 Tetraplatinated porphyrin Pt-4 (Fig. 11) has IC50 values down to 19 nM with indigo light (420 nm) irradiation, with phototoxicity indices up to 5000 in a cisplatin-resistant cell line CP70, showing a high synergism compared with the simple mixture of porphyrins and cisplatin.104 Notably, a 30  higher nuclear Pt concentration of Pt-4 was observed for HeLa cells compared to cisplatin when incubated at the same concentration. Diplatinated BODIPY Pt-5 (Fig. 11) is lysosome-targeting and photocytotoxic with red light (600– 720 nm) via ROS generation and an apoptotic mechanism.219 Mononuclear BODIPY-derived Pt(II) complex Pt-6 (Fig. 11) is sequestered in lysosomes via endocytosis in the dark, but escapes from lysosomes upon irradiation and accesses the nucleus due to its photoinduced ROS generation, giving distinct photocytotoxicity with IC50 values of ca. 3–6 mM in various cancer cells.220 Ln(III). The unique photophysics of Ln(III) complexes is related to the shielding of the 4f orbitals and Laporte-forbidden electronic transitions. Lack of any directional bonding character and large ionic sizes result in the high coordination number (> 6) of Ln(III) complexes.221 The absorption spectra of Ln(III) complexes usually show characteristic ligand-centered absorption bands and strong remaining photophysical properties of the ligands.221 Lutetium-texaphyrin complexes (e.g. Lu-1, Fig. 12) photocleave DNA upon NIR irradiation (> 700 nm), and thus are potential anticancer PDT agents.222 The lutetium–texaphyrin complex AntrinÒ has entered clinical trials. Non-macrocyclic Ln(III) complexes are less explored than macrocyclic texaphyrin complexes and usually contain p-conjugated N-donors and bidentate O-donors.221 The eight-coordinated core {LnN2O6} in Eu-1 (Fig. 12) adopts a distorted square-antiprism geometry with bidentate ligands, a N-donor dpq and three O-donors 4,4,4-trifluoro-1-(2-napthyl)-1,3-butanedione (tfnb).223 The dpq 3T state is well located energetically to allow efficient energy transfer (ET) to the emissive 5D0 states of Eu(III), leading to a high luminescence quantum yield and the detection of cytosolic and nuclear localization of Eu-1. ROS-induced DNA photocleavage upon UVA irradiation results in the enhanced photocytotoxicity of Eu-1. The replacement of Eu(III) by Tb(III) in Eu-1 causes no significant difference in the photocytotoxicity. Generally, non-macrocyclic Ln(III) complexes exhibit photocytotoxicity upon UVA irradiation.221 Polycyclic aromatic hydrocarbons attached to Tb(III)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetate complex can extend the excitation wavelength into the visible region.224 Tb-1 (Fig. 12) with pyrene appended, has an absorption band at

Photoactive metallodrugs

OH

525

2+ F3 C O

N N N

R

N

CF3 O

N

R

N

O

O

Eu

Lu N

O

N

O

N

N

O N

O

O

Tb N N

CF3

O

O O

O OH

Lu-1 Fig. 12

Eu-1

Tb-1

Photoactive Ln(III) complexes studied as anticancer PDT agents.

406 nm (3 ¼ 7600 M 1 cm 1) and a 1O2 quantum yield of 15% in PBS when excited at 420 nm. Photocytotoxicity is observed with 350 nm (IC50 12.8 mM) and 420 nm (IC50 14.3 mM) irradiation in HeLa cells, with a high dark stability. Multi-metal. Heterometallic multinuclear complexes have been designed as anticancer PDT agents to enhance photocytotoxicity and overcome drug resistance with novel mechanisms due to their varied properties.225 Ferrocene derivatives are one of the most common organometallic fragments used in heterometallic photosensitisers.115,163,225 Transition metal complexes with two ferrocenyl terpyridine ligands show significant visible light-induced DNA photocleavage.226 Among them, Fe-Zn-1 (Fig. 13) displays photocytotoxicity in HeLa cells, while its ferrocene-free analog is non-toxic with visible light. The ferrocenyl terpyridine ligands can also be attached to Nd(III) complexes to form Fe-Nd-1 (Fig. 13), which localizes in the mitochondria of HeLa cells specifically and causes DNA photocleavage and apoptosis via visible light-induced ROS generation.227 Enhanced cellular accumulation induced by ferrocene also contributes to the high efficacy of Fe-Nd-1. Complex Fe-Pt-1 (Fig. 13) is stable, but able to kill HaCaT cells by ROS generation upon irradiation.228 Ferrocene-appended Cu(II) complex FeCu-1 (Fig. 13), predominantly localizes in the ER, minimizing undesirable effects on nuclear DNA and has satisfactory dark stability.229 The d–d band near 600 nm of Fe-Cu-1 gives rise to its photocytotoxicity with visible light. Combination of photosensitive polypyridyl Ru(II) complexes with DNA-binding Pt(II) complexes is a practical strategy to direct the photosensitisers to targeting sites and generate 1O2 in the proximity of DNA, and thus enhance the photocytotoxicity and overcome cisplatin resistance.230 Complex Ru-Pt-1 (Fig. 13) selectively accumulates in mitochondria and induces platination and damage to mt-DNA upon irradiation.112 This complex causes mitochondrial dysfunction and apoptosis, and almost eliminates A549 solid tumors in nude mice. Similarly, the covalent binding to DNA of Pt in Ru-Pt-2 (Fig. 13) leads to higher DNA photocleavage and photocytotoxicity with the synergistic effects of ROS.231 Inert Pt(IV) complexes are used as prodrugs for cytotoxic Pt(II) complexes to reduce side effects.215 Complex Ru-Pt-3 (Fig. 13) consists of polypyridyl Ru(II) and Pt(IV) with cancer-targeting phenylbutyrate, and undergoes Pt(IV) reduction to release Pt(II), Ru(II) and phenylbutyrate.111 Upon irradiation with up to 595 nm irradiation, Ru-Pt-3 with multi-target and multi-action effects exhibits low nanomolar range activity in various cancer cells and tumor spheroids, including cisplatin-resistant A2780cis ovarian cancer cells. The supramolecular heterometallic metallacycle Ru-Pt-4 (Fig. 13) displays NIR luminescence, a large two-photon absorption cross-section, and high 1O2 generation efficiency, and is thus a strong PDT candidate.232 The metallacycle accumulates in mitochondria and nuclei selectively, induces simultaneous damage to mitochondrial function and nuclear DNA upon irradiation. The complex also inhibits tumor growth in mice.

2.17.4.2

Photoactivated chemotherapy (PACT)

PACT is being developed to overcome the high dependence of PDT on oxygen concentration, but is still in its infancy.11 Although no PACT metallodrug has entered clinical trials yet, much effort is being invested in this field.29 Mn(I). Manganese is an essential metal in chloroplasts for photooxidation of water, and fac-{Mn(CO)3}-based complexes are able to release CO upon irradiation.233 Ideal photo CO releasing molecules (PhotoCORMs) are biocompatible with high dark stability and release CO only in targeted sites with an appropriate excitation wavelength.233 Tridentate tris(pyrazolyl)methane was the first ligand used in the fac-{Mn(CO)3} system to construct Mn-1 (Fig. 14) that releases two equiv. of CO and exhibits photocytotoxicity upon UVA exposure.234 2-Phenylazopyridine with superior p-acidity promotes a MLCT transition of Mn-2 (Fig. 14) in the visible range, and thus allows rapid CO release and photocytotoxicity with visible light.235 The bromide ligand in Mn-2 contributes to an increased CO release and red-shifted MLCT transition. Mn(I) tricarbonyl complexes with bromide and benzimidazole coligands have been reported recently, showing CO release with UVA irradiation.236 Among them, Mn-3 (Fig. 14) displays satisfactory photocytotoxicity, and the intense luminescence of Mn-4 (Fig. 14) allows cell imaging to monitor its cellular accumulation. The biocompatible Mn-5 (Fig. 14) with a dansyl fluorophore, releases CO upon irradiation (405 nm), which is accompanied by turn-on luminescence at 514 nm. Similar activation can also be achieved by H2O2, constructing a co-registered logic “OR” gate. Although cytotoxicity is not enhanced by blue light (455 nm), the CO release can be monitored by luminescence.237 Mn-6 and

526

Photoactive metallodrugs

Fig. 13

Photoactive multi-metal complexes studied as anticancer PDT agents.

Mn-7 (Fig. 14) are both Mn(I) tricarbonyl complexes containing luminescent dansylimidazole for bioimaging and exhibit photoinduced dose-dependent apoptotic cell death due to CO release.238,239 Lipophilic Mn-7 preferentially accumulates in the cellular membrane.239 Fe(II) and Fe(III). Di-Fe(III) complexes are important PACT candidates with high dark stability and photocytotoxicity.155 The DNA photocleavage oxo-bridged di-Fe(III) complex Fe-16 (Fig. 15) releases CO2 upon irradiation to form an intermediate that generates hydroxyl radicals from oxygen.240 Similarly, the carboxylates in Fe-17 (Fig. 15) undergo decarboxylation to produce Fe(II) in situ, which reduces oxygen to release hydroxyl radicals.241 Both complexes exhibit enhanced ROS generation and cytotoxicity upon visible light irradiation.241 The introduction of curcumin into the di-Fe(III) complex made Fe-18 (Fig. 15) luminescent, and thus its cytosolic localization was monitored.242 The release of curcumin upon visible light irradiation resulted in photocytotoxicity and ROS generation. Fe(II) carbonyl complex Fe-19 (Fig. 15) releases CO by solvent substitution and displays enhanced cytotoxicity upon UVA irradiation.243 Fe-20 (Fig. 15) is a o-nitrobiphenyl photocaged complex that releases cytotoxic aminoferrocene upon UV irradiation at 355 nm to increase cellular ROS levels.244 Co(III). Anticancer Co(III) complexes are important hypoxia-activated prodrugs, which can undergo in vivo reduction to Co(II) with release of cytotoxic ligands. The release of curcumin from Co-3 (Fig. 15) is accompanied by Co(III) reduction and luminescence increase upon irradiation at 470 nm. This is thought to be a key factor for its enhanced photocytotoxicity.56 TPP guides Co-4 (Fig. 15) to release curcumin and generate ROS in mitochondria with visible light exposure, and thus results in photocytotoxicity.245 Ru(II). Polypyridyl Ru(II) complexes are promising photosensitisers with satisfactory photostability, while the introduction of sterically hindering ligands increases the distortion of the coordination octahedron and lowers the energy of dissociative 3MC

Photoactive metallodrugs

HN

+ N

HC

N N

N

Br

N

N

CO

N

Mn N

Br

N

HN

CO

Br N

CO Mn

CO

HN

CO

Mn-1

N

Mn N

CO CO

Mn-2

N

CO

N

HN

CO

Mn-4 + N

N

S N O

S

S N CO Mn

N

CO CO

N

N CO

N Mn N

Mn-5

O

N

O

CO CO

Fig. 14

O

O N

CO CO

Mn-3

N +

CO Mn

+

O

527

Mn-6

N

CO Mn

N

CO CO

Mn-7

Photoactive Mn(I) complexes studied as anticancer PACT agents.

excited states, making them accessible from 3MLCT and leading to their photosubstitution by solvent molecules.45 6,60 -Dimethyl2,20 -bpy in Ru-20 (Fig. 16) is a typical bulky methylated ligand that can undergo rapid photoejection, while the rigidity of 2,9dimethyl-dpq in Ru-21 (Fig. 16) enhances rechelation, and slows down the photoejection by 30 .246 Strained complexes Ru20 and Ru-21 undergo photo-induced binding to DNA, and Ru-21 also causes DNA photocleavage, while unstrained control

Fig. 15

Photoactive Fe(II), Fe(III) and Co(III) complexes studied as anticancer PACT agents.

528

Photoactive metallodrugs

N

N N

N

N

N

N

N

Ru-20

N

N

N

N

Ru N

N

N

Ru N

N

N

N

N N

N Ru

Ru

2+

2+

2+

2+

Ru-21

N

N

N

N

Ru-22

Ru-23

2+

2+

2+

+ N

N

N

N

N

N

N

N

N

Ru

N Ru

N

N N

O N

Ru-24

COOH

S O

N

N

N

Ru

O

S

Ru N

N

N

N

N

N COOH

N

N

N

N

Ru-26

Ru-25

Ru-27

N

O

H N

2+ O

2+ 2+

N

NH

N N

N N N

Ru

N

N

Ru N

N Ru

N N

O

N

N N

NH

S N

N

N H

O HN

O

NH

N O

Ru-28 O

Ru-29

Ru-30

+ 2+

HN O

COOH

S

+ N

S N

N

N

N

N

Ru N HN

N Ru N

N

N NH

HN

N

N Ru

NH N

N

HO HO

OH O

-

OOC

S

Cl

O

O

O

S

OH

NH O

Fig. 16

Ru-31

Ru-32

Ru-33

Photoactive polypyridyl Ru(II) complexes studied as anticancer PACT agents.

Ru-22 (Fig. 16) displays only photocleavage as a photosensitiser. As a result, high photocytotoxicity of Ru-20 and Ru-21 towards A549 cells and spheroids is observed when irradiated with visible light (> 450 nm) with high PIs, while that of Ru-22 is much lower. Notably, the photo IC50 value of Ru-20 in A549 spheroids is 3 lower than Ru-21, indicating the importance of photoejection in the mechanism. Compared with methylated ligands, biquinoline (biq) can not only deform the structure of Ru(II) complexes, but also red-shift their absorption bands.247 The low energy MLCT absorption of archetypal Ru-1 is 450 nm. Replacement of one bpy by biq shifts the MLCT band to 525 nm, and incorporation of two biq shifts MLCT of Ru-23 (Fig. 16) to 550 nm with a tail extending to 900 nm. Ru23 induces DNA photo cross-linking, with the highest photoreactivity observed upon blue light irradiation. Photocytotoxicity

Photoactive metallodrugs

529

towards HL-60 cells was detected with blue, red and NIR exposure, with PIs > 9.2. Both monodentate acetonitrile ligands dissociate from Ru-24 (Fig. 16) in 3LF excited states upon irradiation, while a 3pp* excited state of Ru-24 allows photosensitization of 1O2.248 Ru-24 gives a 2.5 higher PI than the photostable Ru-10. Besides the released ligands, auxiliary ligands play an important role in the photocytotoxicity by affecting the cellular accumulation and localization. Among four complexes with py-2-sulfonate as dissociative ligand, only Ru-25 (Fig. 16) with 4,7-diphenyl1,10-phenanthroline displays notable photocytotoxicity despite its low photodissociation quantum yield, owing to its high DNA binding ability and cellular accumulation, especially in the nucleus.249 As well as bidentate ligands, tridentate and quadridentate auxiliary ligands have been studied. Ru-26 and Ru-27 (Fig. 16) with coordinated terpyridine release pyrazine upon irradiation.250 Notably, Ru-26 with 2,20 -biquinoline-4,40 -dicarboxylic acid displays no cytotoxicity even after irradiation, and thus can be an ideal photocage for drug delivery, while Ru-27 with 2-benzothiazol-2-yl-quinoline shows enhanced cytotoxicity with light irradiation (PI > 3.6), and thus provides a scaffold for dual action agents. Ru-28 (Fig. 16) which has a quaterpyridine ligand, possesses two pyrazines in trans positions and can eject one pyrazine upon irradiation.251 In contrast, its pyridine analog is photostable. Aquated Ru(II) species are not the only cytotoxic photoproducts of PACT polypyridyl Ru(II) complexes, the released ligands may also exert efficacy after activation. Photoreleased 6,60 -dimethyl-2,20 -bpy from Ru-20 is responsible for the photocytotoxicity, while Ru(II) bis-aqua photoproduct of Ru-29 (Fig. 16) is the only cytotoxic species caged by the non-toxic ligand.252 Ru-30 (Fig. 16) containing three cytotoxic 5-cyanouracil (5CNU) ligands, is able to release one or two axial 5CNU ligands to form DNA-binding aquated Ru(II) species as a dual action agent upon visible light exposure, and exhibit photocytotoxicity similar to the cytotoxicity of free 5CNU.253 A quadridentate ligand is coordinated in the basal plane of Ru-31 (Fig. 16) that has two dissociable axial monodentate sulfur ligands in trans positions.254 Upon green light irradiation, Ru-31 releases one axial ligand, while its analog Ru-32 (Fig. 16) with more labile axial ligands undergoes hydrolysis of chloride in the dark then releases DMSO when irradiated. Therefore, Ru-32 displays higher photoinduced DNA-binding and photocytotoxicity via an apoptotic mechanism. The thioether-glucose ligand improves the hydrophilicity of Ru-33 (Fig. 16) and is released upon irradiation to give hydrophobic active Ru(II) species.255 Ru-33 is taken up passively by cells and selectively accumulates in mitochondria. Upon irradiation, Ru-33 generates a significant amount of 1O2 and releases aquated Ru(II) species that bind to DNA and induce DNA photocleavage. Replacement of the thioether-glucose ligand by chloride results in > 30  reduced 1O2 generation and > 13  lower photocytotoxicity in A549 lung cancer cells.256 Ru(II) arene complexes can selectively photodissociate a monodentate ligand upon irradiation to form DNA-binding aquated species. Photodissociative Ru(II) arene complex Ru-34 (Fig. 17) has an absorbance tail in the 400 nm region that is composed mainly of mixed partially dissociative 1MC–1MLCT transitions, allowing photodissociation of pyridine with visible light (400– 600 nm), followed by binding to 9-ethylguanine (9-EtG) through N7.257 Introduction of a highly delocalized 2,3-bis(2-pyridyl)benzoquinoxaline (dpb) ligand into Ru-35 (Fig. 17) red-shifts the absorption bands to 600 nm for visible light activation and results in a long-lived 3LC excited state for 1O2 generation and DNA photocleavage.258 The bulky dpb ligand also distorts the coordination structure, making a 3MC excited state accessible and leading to dissociation of py and dpb with a 3.4: 1 ratio, which turns on the emission and DNA binding. The dual-action mechanism results in a 6.9  enhanced cytotoxicity in A549 cells upon

2+

2+

2+ Ru

Ru

N

N

N

Ru

N

N

N

N

N

N

N

N

N

N N

Ru-34

Ru-35

Ru-36 2+

2+ N

Ru

N

N N N

O

Ru N H

N Peptide = Dicarba-octreotide = Arg-Gly-Asp-CONH2

Fig. 17

O

Peptide

O

N

N

N

N

O

Ru-37 Ru-38

Photoactive Ru(II) arene complexes studied as anticancer PACT agents.

Ru-39

F B F

530

Photoactive metallodrugs

irradiation (l > 400 nm). Notably, Ru-35 and/or released dpb selectively accumulate in the nucleus. A decrease in the basicity of the monodentate ligand enhances the ligand photodissociation and photoinduced DNA binding capabilities of Ru(II) arene complexes. Ru-36 (Fig. 17) with pyrazine (pKa ¼ 0.6) displays a 76% ligand dissociation yield, DNA photobinding and an IC50 of 16.5 mM towards A549 lung cancer cells upon irradiation, while its analog with pyridine (pKa ¼ 5.2) exhibits negligible ligand dissociation, DNA binding and an IC50 of 60.3 mM under the same conditions.259 Tumor-targeting peptides dicarba-octreotide and Arg-Gly-Asp (RGD) have been attached to photodissociative pyridine in Ru-37 and Ru-38 (Fig. 17), respectively, and lead to selective accumulation of these complexes in cancer cells with overexpressed receptors.260 Interestingly, Ru-37 and Ru-38 form monofunctional adducts with 9-EtG, and a new bifunctional Ru(II) adduct involving two bound guanines in the presence of oligonucleotides due to the loss of p-cymene from monofunctional adducts. Luminescent BODIPY red-shifts the absorption band of Ru-39 (Fig. 17) with an absorption maximum at 504 nm, which allows photodissociation of the py-BODIPY ligand and binding with 9-EtG upon irradiation at l > 500 nm.261 Re(I). The tricarbonyl Re(I) PhotoCORM Re-3 (Fig. 18) containing the water-soluble phosphine ligand tris(hydroxymethyl) phosphine (thp) exhibits a red shift in its emission from 515 to 585 nm after exhaustive photolysis due to the release of CO and formation of aquated Re(I) species. The blue luminescence within PPC-1 cells treated with Re-3 indicates its accumulation in the cellular cytosol, while the green luminescence after 405 nm exposure suggests photolysis.262 The effect of ancillary ligands on Re(I) PhotoCORMs has been investigated.263,264 The emission, photolysis and photocytotoxicity of tricarbonyl phen Re(I) complexes are dependent on the sixth ligand.263 In acetonitrile, an emission maximum at 532 nm with the highest intensity in the series was detected, but no CO photorelease was observed. In contrast, Re-4 (Fig. 18) containing a PPh3 ligand, displays sensitivity towards low-energy UVA light and a much faster CO release rate. Notably, Re-4 exhibits a moderate nuclear localization in addition to the cytosolic distribution. Tricarbonyl Re(I) complexes bearing thp or 1,4-diacetyl-1,3,7-triaza-5-phosphabicylco[3.3.1] nonane (dapta) ligand exhibit triplet state luminescence with quantum yields ranging from 3.4 to 11.5%. They undergo CO photosubstitution and 1O2 photosensitization upon irradiation at 365 nm, while their analogs with 1,3,5-triaza-7-phosphaadamantane (PTA) exhibit no luminescence and photosubstitution.264 Re-5 (Fig. 18) gives rise to the highest photocytotoxicity in the series with an IC50 value of 6 mM in HeLa cells. Re-6 (Fig. 18) derived from 2-(2-pyridyl)-benzothiazole, releases CO upon irradiation (302 nm) with a luminescence change from orange to deep blue, allowing trackable CO delivery and photocytotoxicity in cancer cells.265 Tricarbonyl Re(I) complexes can be delivered to cancer cells selectively by being caged with a photolabile protecting group conjugated to a peptide, such as a nuclear localization sequence (NLS) or a bombesin peptide in Re-7 and Re-8 (Fig. 18), respectively.266 The released Re(I) complex shows comparable photocytotoxicity to that of cisplatin towards HeLa cells. Twenty-five percent of Re-7 accumulates in the nucleolus and induces severe cell stress in the dark and upon irradiation.

+ CO N CO Re CO N P

+

+ CO N CO Re CO N PPh3

CO N CO Re CO N P(CH2OH)3

N

N

N

Re-3

Re-4

Re-5

O

O

R S

+

+ CO CO Re CO N OH2

O

N S

N O2N

O O

Re-6

+

O

N

N H

N

CO CO Re CO N

R = NLS R = Bombesin

Fig. 18

Photoactive Re(I) and Ir(III) complexes studied as anticancer PACT agents.

Re-7 Re-8

N

HN N

Ir N

Ir-11

N HN

Photoactive metallodrugs

531

Ir(III). Compared with Ru(II) complexes that can populate dissociative 3MC and 3LF states and release ligands, Ir(III) PACT agents remain relatively unexplored due to their high photostability.47,267 The monodentate five-membered heterocyclic ligands dissociate from Ir-11 (Fig. 18) upon irradiation at 425 nm, accompanied by 1O2 generation, and thus induce cellular ROS production, caspase activation, and eventually apoptosis.268 Notably, a higher PI is observed for Ir-11 towards cisplatin-resistant A549R compared to parent A549 lung cancer cells. Pt(II) and Pt(IV). Transplatin (Pt-7, Fig. 19), the non-toxic isomer of cisplatin, displays cytotoxicity comparable with cisplatin in the presence of UVA irradiation, which can promote the loss of the second chloride from transplatin, leading to the formation of increased interstrand DNA crosslinks.120 The photodecomposition of cyclometalated Pt-8 (Fig. 19) can be monitored by the decreased emission intensity and absorbance.269 1O2 generation is observed upon irradiation, leading to a 28-fold improved photocytotoxicity of Pt-8 towards HeLa cells. Curcumin-derived cisplatin analog Pt-9 (Fig. 19) releases DNA-binding Pt(II) species and photosensitive curcumin that produces hydroxyl radicals in the cytosol upon visible light irradiation, showing apoptotic photocytotoxicity with IC50 values of 12–18 mM.270 Replacement of curcumin with an acetylacetone (Hacac) derivative of heptamethine cyanine results in Pt-10 (Fig. 19), which can release Pt(II) species and the photodetachable ligand on NIR exposure.271 Significant enhancement of the 1O2 quantum yield and 30–60  cytotoxicity enhancement are observed for Pt-10 in the presence of NIR radiation. Interestingly, photooxidation of the released ligand by 1O2 is detected. Pt-11 (Fig. 19) undergoes two-photon-absorption decomposition when irradiated with a focused femto-second (fs) laser at 600, 650 and 700 nm, and releases the MOPEP ligands.272 Pt-12 (Fig. 19) intercalates into DNA in the dark, releases the azide ligand, and generates 1O2 with blue light (420 nm) exposure.273 The photoactivation of squaramide-based Pt-13 (Fig. 19) is regulated by oxygen; a cytotoxic Pt(II) species with an amino-sulfide fragment is generated in the absence of oxygen, which can be oxidized by oxygen, leading to non-toxic species.274 Therefore, Pt13 displays photocytotoxicity only in hypoxic conditions. Photoactivation of Pt(II) PACT agents can also be ligand-centered. Pt14 and Pt-15 (Fig. 19) are both coordinated by photosensitive o-nitrobenzyl alcohol derivatives.275,276 Pt-14 undergoes a photoinduced internal redox process with its nitro group reduced to a nitroso group and the alcohol oxidized to a carbonyl group.275 Pt-15 exhibits photoinduced backbone breakage to release the active Pt(II) species and nitroso by-products.276 Photoswitching of 1,2dithienylethene derivatives in Pt-16 (Fig. 19) between open and closed forms alters its cytotoxicity. The closed form resulting from UVA irradiation, is less voluminous and more planar with a greater p conjugation, and thus exhibits stronger DNA binding and eventual cytotoxicity.106

+ H3 N

N S

Cl

H3N

N

Pt Cl

Pt NH3

+

NH3

H3N

O

2+

NH3 Pt

Pt O

O

O

N

N N

HO

OH O

Pt-7

N

O

Pt-8

Pt-9

N

Pt-10

O O

HN

Cl

N

O

NH

Pt N

N

Cl

Pt N3

N S

Pt-11

O

Pt-12

F F O

NH3

N

Pt

O2N O

O

Pt-14 Fig. 19

O

NH3

N

N Pt

Cl

Pt-13

NO2

O

S

Pt Cl

N

Pt-15

Photoactive Pt(II) complexes studied as anticancer PACT agents.

Cl S Pt N O Cl

F F S

Pt-16

F F S

Cl N Pt S Cl O

532

Photoactive metallodrugs

Kinetically-inert Pt(IV) complexes dominate attempts to design Pt PACT metallodrugs due to their high dark stability compared with their Pt(II) analogs.54 Diiodo-Pt(IV)-ethylenediamines (e.g. Pt-17, Fig. 20) were the first-generation photoactive Pt(IV) complexes, possessing strong broad LMCT bands centered at ca. 400 nm with a tail extending out into the visible range.277 They can be activated with light lirr > 375 nm, and their photochemical properties are modified by the axial ligands. However, their poor dark stability, ascribed to facile bio-reduction, limits their application as photoactive agents.278 Replacement of iodides by azides has led to second-generation photoactive Pt(IV) complexes with improved dark stability and photobiological properties.54 Diazido Pt(IV) complexes Pt-18 and Pt-19 (Fig. 20) are stable under physiological conditions in the dark, while able to form PtGMP and Pt-GpG adducts upon visible light irradiation.279 Significantly enhanced cytotoxicity in both wild-type 5637 and

OH

H2 N

I

OH H 2 N Pt N H2 OH

N3

Pt I

N H2

OH

N3

Pt-17

H3N

NH3

N3

OH N

N3

N

Pt

Pt N3

N3

N

N3

N

OH

Pt-21

Pt-20

OH

N

N3

N3 OH

Pt-19

Pt

OH

H3N

OH

N

Pt

NH3 Pt

N3

OH N3

N

OH N3

NH3 Pt

Pt-18

OH N3

OH N3

OH

OH

Pt-22

Pt-23

Pt-24 O O

OH

O N

N3

N H

NH

OH

N3

N

Pt

Pt N3

N O O

O

N O

N H

N3

c(RGDfK)

O

O

O

NH

H N

O

OH

O

Pt-25

Pt-26

O OH Cl2HC N3

O N

N3

N3 O

N

Pt N O

Pt

N N

Pt O O

O

H N

N H

O

N O

O

N3 O

O

N3

O

N

N3

OH

O O

N3

N Pt

N

N

O

N3 OH

Pt-27

Pt-28

Pt-29 N

O

O O

O NH NH2CH3

N

N

NH2CH3 Cl

H2 N

O

O

O

O

Pt-30 Fig. 20

O

O

O

O

H N

O

Pt N H2

O

HN

Pt N H2

O

O

O

H2 N

HN

O

Pt N

N

2+

Cl

O

O N 8H

OH O

OH

Pt-31

Photoactive Pt(IV) complexes studied as anticancer PACT agents.

H2N

Pt-32

NH2

O

Photoactive metallodrugs

533

cisplatin-resistant 5637 bladder cancer cells treated with Pt-18 and Pt-19 is observed upon UVA exposure.280 Dramatic morphological changes in 5637 cells treated with Pt-19 and light are in accord with its photocytotoxicity. Contrary to the early structure-activity relationship that cis diam(m)ine geometry is preferred for anticancer platinum complexes, Pt-20 (Fig. 20) with all-trans configuration, displays higher aqueous solubility and a more intense and red-shifted LMCT band, and higher photocytoxicity, compared with its cis isomer.281 Therefore, rapid photoreaction to form a bis(5’-GMP) adduct and higher photocytotoxicity towards cancer cells (as toxic as cisplatin) under UVA are detected for Pt-20. Introduction of one pyridine ligand generates the highly phototoxic Pt-21 (Fig. 20), showing a 13–80  higher photocytotoxicity compared with cisplatin and a different mechanism of action.282 Two of seven nude mice bearing OE19 esophageal cancer xenograft treated with Pt-21 and blue light (420 nm) survived at 35 days, while none of control mice without drug or irradiation survived.283 The presence of two trans-pyridine ligands in Pt-22 (Fig. 20) also gives rise to its photoactivation and photocytotoxicity upon visible light irradiation.284 One methyl substituent at the 2-position of pyridine causes steric hindrance in Pt-23 (Fig. 20), leading to its rapid photodecomposition and dark cytotoxicity.285 However, Pt-24 (Fig. 20) with a methyl substituent at the 4-position of both pyridines displays the highest photocytotoxicity in the series, while Pt-23 is comparatively less photocytotoxic. The photoreleasable axial ligands in octahedral Pt(IV) complexes can not only alter the reduction potential, but also improve their cancer targeting ability and photocytotoxicity.54 Conjugate Pt-25 (Fig. 20) containing cyclic RGD-containing peptide c(RGDfK) displays enhanced cellular accumulation in cancer cells overexpressing avb3 and avb5 integrins.286 The disubstituted Pt-26 (Fig. 20) with two axial suberoyl-bis-hydroxamate (Sub) ligands, a histone deacetylase (HDAC) inhibitor, releases SubH and Pt(II) species upon UVA irradiation to exert photocytotoxicity with a low resistance factor compared with cisplatin and its Pt(IV) analogs containing inactive axial ligands.287 This indicates a different mechanism of action, in which the photoreleased SubH inhibits HDAC activity and allows Pt(II) to access chromatin DNA and form DNA adducts. The presence of two different axial ligands in Pt-27 (Fig. 20) increases its reduction potential to  0.886 V from  1.699 V for the unsubstituted complex Pt-22.288 Cellular accumulation is enhanced 568  and photocytotoxicity 26  in A2780 cells for Pt-27 compared with Pt-22. The presence of 1,8-naphthalimide red-shifts the absorbance of Pt-28 (Fig. 20) to 450 nm, allowing photoactivation with green light.289 The derivatives with less bulky substituents on 1,8-naphthalimides can pre-intercalate into DNA, resulting in enhanced photoinduced DNA crosslinking and photocytotoxicity. Dinuclear Pt-29 (Fig. 20) shows similar photocytotoxicity in A2780 ovarian cancer cells and in cisplatin-resistant A2780cis cells, but remains non-toxic towards normal MRC-5 cells.290 Dichlorido Pt(IV) complexes are also potential PACT agents. Pt-30 (Fig. 20) can undergo photoisomerization then photoreduction to release Pt(II) species and CH3NH2, accompanied by the formation of HOCl.291 The oxaliplatin-based Pt(IV) prodrug Pt-31 (Fig. 20) with pyropheophorbide is ascorbic acid-stable in the dark and red light activatable to release cytotoxic oxaliplatin and pyropheophorbide.292 Pt-31 displays up to 1786  greater photocytotoxicity than oxaliplatin in cancer cells, which is higher in cisplatin-resistant A2780cis than A2780 cancer cells, suggesting a novel mechanism of action. Reductions of 67% in tumor volume and 62% in tumor weight were observed in BALB/c mice bearing the 4 T1 tumor model treated with Pt-31 and red light compared with the control group. Cell-penetrating nucleolus-targeting peptide R8K leads Pt-32 (Fig. 20) to accumulate selectively in the nuclei (68.2%), while the coumarin-based ligand allows photo-switch-on fluorescence.293 Pt-32 displays up to 2 orders of magnitude higher photocytotoxicity than oxaliplatin, as well as a novel mechanism to overcome drug resistance by inducing cell senescence, p53-independent cell death, and immunogenic cell death along with T cell activation. Au(III). The dark-stable cyclometalated Au(III) complex Au-1 (Fig. 21) dissociates the hydride ligand efficiently to release H2 and generates an Au(III) species with visible light.294 The photoactivated Au(III) species forms Au-thiol adducts and potently inhibits thioredoxin reductase, exhibits photocytotoxicity > 100 the dark cytotoxicity in HCT116 cells without deactivation by serum albumin, and strong inhibition of angiogenesis in zebrafish embryos. Notably, two-photon photoactivation of Au-1 is as effective as blue light. Multi-metal. A ferrocenyl terpyridine ligand has been coordinated to Pt(II) to form Fe-Pt-2 (Fig. 21), which has an intense absorption band near 640 nm, providing photodegradation in red light, releasing ferrocenium ions, DNA-binding Pt(II) species and biotinylated ligands, and generating ROS.295 A photo-IC50 of 7.7 mM was observed for Fe-Pt-2 in BT474 human breast cancer cells by ROS-mediated apoptosis. Trimetallic supramolecular complex Ru-Rh-1 (Fig. 21) contains two Ru(II) light absorbers and a central Rh(III) bridged by communicative ligands that allow electronic communication between subunits.296 DNA photocleavage was observed for Ru-Rh1 involving the lowest lying MMCT excited state. In contrast, its Ir(III) analog possesses MLCT instead and fails to cleave DNA. Growth inhibition of cells treated with Ru-Rh-1 at low concentration (< 10 mM) and visible light (> 460 nm) is observed, and cell death is seen with increased Ru-Rh-1 concentration.297 Notably, the importance of Ru(II) is emphasized by the lack of cell death induced by its Os(II) analog, although it is also capable of photocleaving DNA due to the MMCT states.298 Ru-Rh-2 (Fig. 21) also induces DNA photocleavage upon irradiation (l  475 nm).298 Compared to these trimetallic complexes, their bimetallic analogs are smaller and less positively charged, and thus might exhibit better cell-membrane permeability. Rh-Os-1 (Fig. 21) binds to and photocleaves DNA with red light exposure in an oxygen-independent manner.299 The bimetallic complex can bind to DNA via the Rh(III) site since it is more sterically accessible than trimetallic complexes. Upon irradiation at 450 nm, mitochondria-targeting RuIr-1 (Fig. 21) releases photosensitive Ir(III) species that can generate 1O2 under 405 nm only and DNA-binding Ru(II) species.107 As a result, the combination of 405 and 450 nm light exposure enhances photocytotoxicity significantly. Notably, Ru-Ir-1 displays higher photocytotoxicity in cisplatin-resistant cancer cell lines with an apoptotic mechanism via mt-DNA damage and mitochondrial dysfunction.

534

Photoactive metallodrugs

+ N N Au H

O

N Pt Fe

N H

N

S

H N

NH HN

O

Au-1

O

Fe-Pt-2

5+ N

N Ru

N N

Cl N

Rh

N

N

Cl

N

N N

N

Cl

Ru

N

N

N

N

N

Cl

N

N Ru

N

3+

N

Rh

N

N

N

Ru-Rh-1

Cl

Cl N

Ru

N

N

N N N

N

Ru-Rh-2 3+

N

N Os

N

Cl N

N

N

N

N

Cl Rh

N

2+

N

N

N

N

N

Ir

N N

Cl

Rh-Os-1

O N

Eu N

O

+

O OH2

O

O

N

N

O N

N

NH O

O

N

N

OH2 N N

NH O

O Cl N Pt H3N NH3

N

Ru N

Eu N

O Cl

N

Ru-Eu-1 Fig. 21

N

+

N

O

N

Ru-Ir-1

O O

Ru

N

N

N

Eu-Pt-1

Photoactive Au(III) and multi-metal complexes studied as anticancer PACT agents.

The non-emissive Ru-Eu-1 (Fig. 21) exhibits no cytotoxicity in the dark, but releases emissive Eu(III) species and DNA-binding Ru(II) species under irradiation at 488 nm.300 The long-lived red Eu(III) luminescence can be monitored when excited by onephoton 350 nm or two-photon 700 nm irradiation, and reveals the real time drug delivery quantitatively within cells. An enhancement of 8.5 photocytotoxicity in HeLa cells and 77.3% tumor volume decrease of SW480 xenografts in BALB/c nude mice were observed with 488 nm exposure compared to dark conditions. Notably, red emission is only detected when cancer cells and tumors are treated with irradiated Ru-Eu-1, indicating controllable drug release by light. The same Eu(III) fragment is also combined with cisplatin in Eu-Pt-1 (Fig. 21), which can release luminescent Eu(III) and cytotoxic Pt(II) species, but the drug activation light has the same wavelength as the luminescence excitation light.301 In addition, Eu-Pt-1 shows high dark cytotoxicity and a low PI (1) with 488 nm light, since no photoactivation can be induced by light at 488 nm.300

Photoactive metallodrugs 2.17.4.3

535

Photothermal therapy (PTT)

In contrast to PDT and PACT agents, PTT metallodrugs need to be bound to nanomaterials or form nanomaterials on their own. Nanomaterials are the key component for photothermal effects.12 The introduction of metal complexes can increase the absorption intensity in NIR region, improve the photothermal effect, stabilize the nanomaterials, affect the localization, and probe hypoxia.69,70,109,110 This section covers only PTT nanomaterials formed by metallodrugs, while examples of metallodrugs loaded in PTT nanomaterials will be discussed in Section 2.17.6. Fe(III). Fe(III)-gallic acid nanoparticles are not stable in neutral conditions, but remain intact in mildly acidic environments (pH  5).302 Therefore, the nanoparticles can be easily decomposed and metabolized in normal tissue, while exerting a PTT effect in acidic tumors. Fe(III)–gallic acid nanoparticles show a wide absorption from 400 to 900 nm, peaking at 575 nm. When irradiated by 808 nm laser light for 10 min, a 53  C temperature increase is observed for the nanoparticles, displaying photothermal ablation of 4T1 cancer cells and a 4T1 tumor model in BALB/c nude mice. Notably, Fe(III)–gallic acid nanoparticles have longer retention times in tumors than in the liver and spleen due to their pH sensitivity, also preferential accumulation in large tumors than in small ones owing to the enhanced permeability and retention (EPR) effect. Ni(II). The aqueous-soluble pegylated Ni(II)-bis(dithiolene) complex Ni-1 (Fig. 22) displays a strong absorbance at 936 nm, allowing a 65  C temperature increase after 10 min irradiation with a 940 nm laser (5 W/cm2).303 Ni-1 induced death of > 80% of 786–0 renal cancer cells in combination with 940 nm laser light at 60 mM.

2.17.5

Photoactive antimicrobial metallodrugs

Antimicrobial resistance is currently of global concern for health and the economy.304 Penicillin discovered by Alexander Fleming in 1928 was the world’s first broadly effective antibiotic, but bacteria have evolved to resist this and new drugs.304 Phototherapy using narrow wavelength light is a promising alternative antimicrobial treatment.305 The efficacy of phototherapy can be improved when photoactive agents are used in combination with light.306 Metal complexes have a 10  higher hit-rat towards ESKAPE pathogens than purely organic molecules.307 Metal complexes also have a high efficacy against microbes growing as biofilms, which have important roles in antimicrobial resistance.308 Despite the rapid development of photoactive metallodrugs, the field of photoactive antimicrobial metallodrugs is relatively less explored and none of them has been clinically approved as antimicrobials. In this section, antimicrobial photoactive metallodrugs will be discussed in atomic number order without classification by mechanism due to the limited number of reports. Cr(III). Polypyridyl Cr(III) complex Cr-2 (Fig. 23) and its derivatives with different substituents on 1,10-phenanthroline ligands display DNA photocleavage via an electron transfer mechanism (Type-I PDT).309 The substituents can affect their DNA photodamage and antibacterial efficacy. Cr-2 displays a growth inhibition of 11% towards Escherichia coli (E. coli) XL1-Blue strain upon exposure to 457 nm light, while with a chloride substituent in each ligand at the 5-position, the inhibition increases to 37%. Mn(II). Aqueous soluble Mn-8 (Fig. 23) is stable in the dark, but activated by UVA (365 nm) to release toxic CO, which can significantly inhibit NADH-supported respiration rates, restrict ATP generation, and bind to cytoplasmic heme proteins.310 The Mn(II) species resulting from CO release reacts with hydrogen peroxide avidly to produce hydroxyl radicals. Thus, site-specific and time-controlled reductions in viability and growth of E. coli EC958 strain are observed for Mn-8. Fe(II), Co(II) and Cu(II). Fe-21 bearing two heterocyclic dppz ligands binds to DNA via groove-binding with a binding constant of 8.1  104 M 1.311 UVA (365 nm) irradiation induces 1O2 generation by Fe-21 (Fig. 23) via a Type-II PDT mechanism, which photocleaves DNA and causes a 72.5% reduction of E. coli ATCC-25922 colony-forming units (CFU) at 25 mM. Replacement of the Fe(II) by Co(II) gives Co-5 (Fig. 23) that does not show a notable difference from Fe-21 as a photoactive antimicrobial.312 Photoactive Cu-1 (Fig. 23) with dppz generates 1O2 and kills E. coli ATCC-25922 strain on irradiation with a 500 W halogen–tungsten lamp.313 Ru(II). Photoactive anticancer polypridyl Ru(II) complexes have also been investigated as antimicrobial agents. TLD-1433 which entered Phase II clinical trials as an anticancer PDT agent, exerts photodynamic inactivation of bacterial strains in both normoxic

O O

3

2-

O 3O

O

O S

3

O

O

O

O

3

S Ni

S O

3O

O

S

O

O

3

Ni-1 Fig. 22

3

O

Photoactive Ni(II) complex studied as an anticancer PTT agent.

3

536

Photoactive metallodrugs

+ N

+

N

3+ +

OH2

N

N N

N

N

Cr N

N

N

N

CO

N

N

N N

N CO

N

N

N

N

Cu

M

Mn

N

S

S

N

CO N N

Cr-2

Fe-21 Co-5

M = Fe Co

Mn-8

Cu-1

2+ 2+ 2+

O

O

N

O

N N

N N

N

N

N

N

O

N

N

S O2

N

O

N

Ru

N

N

O

N

Ru

Ru N

N

O2 S

Ru N

N

N

Cl

O

+

N

O O

Ru-40

Ru-41

O

Ru-42

Ru-43

2+ 2+

O

N

N

N

N N

N

N

N

H N

N

N

N

Cl

Ru

Ru

Ru N

N H

N

N N

NH2

N NH2

Cl N

O

Ru-44

Ru-45

Ru-46

4+ N

Cl N

O

M N O

N

N M

Cl

NH

N

N

N HN

N

Cl M

N

O N

N M

N

Fig. 23

Cl

M = Pd Pt

N

N Ir

N

N N B F F

O

N Cl N Sn N Cl N

Pd-1 Pt-33

N

N

Sn-1

Ir-12

Photoactive metal complexes studied as antimicrobial agents.

and hypoxic conditions with 530 nm light via a combination of Type-I and Type-II photoprocesses.127 Ru-10 is another Ru(II) photosensitiser displaying both anticancer and antimicrobial activity.188,314 The positive charges and the lipophilic intercalating dppn ligand allow Ru-10 to bind to the negatively charged outer membrane of E. coli, leading to significant phototoxicity towards Gramnegative bacteria. Ru-40 (Fig. 23) is an antimicrobial Ru(II) photosensitiser that generates 1O2 via a long-lived triplet excited state activated by visible light (400–500 nm).315 Ru-40 is photocyotoxic towards a series of bacteria, while its unsubstituted analog Ru-1 displays no apparent toxicity upon irradiation. Photosensitiser Ru-41 (Fig. 23) shows a higher 1O2 quantum yield compared with

Photoactive metallodrugs

537

Ru-42 (Fig. 23) upon 420 nm irradiation, thus a higher PI (80) towards HeLa cells.316 However, Ru-42 is photoactive against both Gram-positive S. aureus and Gram-negative E. coli, while Ru-41 is completely inactive towards E. coli. The Cl-7-IVQ (5-chloro7-(2(1,3,3-trimethyl-3H-indol-1-ium-2-yl)vinyl)quinolin-8-olate) ligand in Ru-43 (Fig. 23) contributes to the MLLCT transition (Ru(dp)-Cl-7-IVQ(p) orbital to the p* (Cl-7-IVQ) orbital) and pp* transition of the merocyanine framework, giving an intense absorption band centered at 649 nm and allowing efficient hydroxyl radical-mediated DNA photocleavage.317 Notably, Ru-43 can preferentially accumulate in and selectively inactivate E. coli bacterial cells over HeLa cells upon red light irradiation, and thus is a promising antimicrobial PDT candidate. Ru-44 (Fig. 23) shows mild antibacterial effects in the dark, but strongly enhances efficacy upon irradiation, despite its low photoinduced 1O2 generation, while its analog with two or three substituents does not show antibacterial activity.318 As well as being PDT agents, PACT Ru(II) complexes also show antimicrobial activity. Two mol. equivalents of anti-tuberculosis isoniazid can be rapidly released from Ru-45 (Fig. 23) upon blue light (465 nm) irradiation, accompanied by the formation of aquated Ru(II) species.319 A 5.5 higher phototoxicity towards M. smegmatis is observed for Ru-45 compared with isoniazid alone. Notably, Ru-45 displays no activity in the dark and no photocytotoxicity towards normal human MRC-5 cells. Although Ru-46 (Fig. 23) releases 4-benzoylpyridine upon irradiation (453 nm) then binds to DNA, no enhancement in antimicrobial ability is observed after irradiation.320 It is assumed that DNA damage is not related to its antimicrobial activity. Pd(II) and Pt(II). Tetra-Pd(II) porphyrin Pd-1 (Fig. 23) displays high photostability, a 0.51 1O2 quantum yield in DMSO with red light exposure, and satisfactory white light MIC values towards bacteria that are controllable by ROS scavengers.321 Similar to Pd-1, its Pt(II) analog Pt-33 (Fig. 23) shows photodynamic inactivation of rapidly growing mycobacterial strains via ROS generation.322 However, the MIC values only increase in the presence of 1O2 scavenger ascorbic acid, indicating the importance of 1O2 in the mechanism. Pt-33 induces significant morphological changes in mycobacteria with light. Moreover, both nanomechanical and electrostatic adhesion properties of mycobacteria treated with Pt-33 decrease, especially after irradiation. Sn(IV). In Sn(IV) porphyrin Sn-1 (Fig. 23), the axial chloride ligands contribute to the high molar absorption coefficient, while C8 alkoxy chain substituents allow a high luminescence quantum yield and increase the difficulty of reduction.323 Sn-1 shows higher activity against B. subtilis than E. coli bacteria. Groove binding to DNA and photodamage of DNA caused by 1O2 appear to be possible mechanisms of action. Ir(III). Cyclometalated Ir(III) complex Ir-12 (Fig. 23) has an intense BODIPY-based 1IL/1MLCT absorbance at 530–543 nm and 1,3 IL/1,3CT emission at 582–610 nm.324 Aqueous soluble Ir-12 displays 135  and > 15  enhanced cytotoxicity towards SKMEL28 cancer cells and S. aureus bacteria, respectively, with visible light, owing to effective in vitro ROS generation. Multi-metal. The antibacterial ability of cisplatin towards E. coli promoted its anticancer applications, and cisplatin analogs can act as antibacterial agents due to their DNA binding ability.17 Ru-Pt-2 exerts a multi-mode of interaction with DNA, including thermal-induced coordination, photobinding and photocleavage, owing to the photo-facilitated chloride loss from the cis-PtCl2 fragment and ROS generation by Ru(II).325 Thus, 10 mM Ru-Pt-2 inhibits growth and kills an entire population of E. coli after photolysis.

2.17.6

Drug delivery systems for photoactive metallodrugs

Numerous nanocarriers have been developed as drug delivery systems for photoactive metallodrugs.1,54,326 Common drug delivery nanocarriers include organic (proteins, hydrogels, micelles, vesicles and other polymers) and inorganic (carbon-based nanomaterials, silica nanoparticles, gold nanoparticles, magnetic nanoparticles, metal-organic frameworks and UCNPs) materials. An ideal nanocarrier for photoactive metallodrugs should deliver the drug efficiently without premature leakage, then release the payload in the targeted site upon irradiation.326 Elaborate nanocarriers allow not only accurate drug delivery, but also specific administration, red-shifted activation wavelength and novel mechanisms of action.

2.17.6.1

Organic nanocarriers

Organic nanocarriers are generally based on polymers, and the metallodrugs can be loaded on the polymers either non-covalently or covalently. Covalent conjugation is mainly classified into three types: (1) metal centers linked to the backbone via an organic linker, (2) coupled to the backbone, or (3) directly on the backbone. Polymers are classified into natural and synthetic nanocarriers based on their origin. Common natural polymeric nanocarriers include proteins and polysaccharides, while the synthetic polymers are diverse and usually employed in the delivery of metallodrugs as amphiphilic block copolymers. Polymeric nanocarriers are capable of forming hydrogels, micelles, vesicles, and other morphologies.

2.17.6.1.1

Natural polymeric nanocarriers

Proteins. Delivery of metallodrugs by proteins with upregulated transmembrane transporters in tumors is a well-known strategy to improve their cancer selective accumulation.25 Fe–S cluster Fe-22 (Fig. 24) shows high affinity towards horse spleen apoferritin cavities.327 The cooperation with apoferritin improves its stability in PBS buffer and cellular accumulation by 14% after 1 h incubation in HeLa cells. This nanocomposite releases NO, increases cellular ROS levels and exerts photocytotoxicity with an IC50 of 5.4 mg Fe/ mL upon irradiation with white light. Rutherrin, the adduct of TLD-1433 and apo-transferrin, displays a 4.3 x enhanced absorbance at 525 nm and a 15% lower photobleaching over TLD-1433.328 Enhanced cellular accumulation, photoinduced ROS generation

538

Photoactive metallodrugs

CD

2+

O

N HN 2+ N

N

NO S Fe S ON NO S Fe Fe NO ON Fe ON NO

N

H N

N

N

Ru N

NH2

N

N

N

H N

N

CD

Ru

O N

N N

N N NH

Fe-22

Ru-47

CD = b-cyclodextrin

Ru-48

O CD + O O

O

N

OH

OH N

O N

Pt

Pt

Ir

N N

N3

O

N3

N3

S NH

OH O

O

N

O

NH3 Pt

O

O

O

N

OH N3

N

N3

N

N3

O

HN

O

NH3 O O

OH O

N O

Ir-13 Fig. 24

Pt-34

Pt-35

Pt-36

Photoactive metal complexes functionalized for delivery by natural polymeric nanocarriers.

and photocytotoxicity are also observed for Rutherrin compared with TLD-1433. Conjugation to a chemically-modified HSA by covalent attachment of mitochondria-targeting triphenylphosphonium groups and photosensitive Ru-47 (Fig. 24) units, as well as polyethylene(oxide) side chains, produces a highly active photodynamic system, displaying photo IC50 values in the nanomolar range.329 Supramolecular assembly of photosensitive b-cyclodextrin-functionalized Ru-48 (Fig. 24) and cancer-targeting adamantane-functionalized transferrin displays higher photocytotoxicity and > 18 preferential accumulation in transferrin receptor-positive A549 lung cancer cells over normal 293T cells.330 The monodentate pyridines functionalized with a maleimide substituent in Ir-13 (Fig. 24) allow its reaction with Cys34 thiol to form adducts with HSA, which increases its luminescence and photoinduced 1O2 generation.331 Upon irradiation, one monodentate ligand is released from Ir-13 and replaced by His39 in HSA. The photoinduced adduct preferentially accumulates in the nucleus and shows significant photocytotoxicity towards cancer cells and spheroids with blue light. HSA nanocarriers carrying photoactive Pt-34 (Fig. 24) and an apoptosis-sensitive probe containing a caspase-3 activatable peptide-bridged fluorescence resonance energy transfer (FRET) pair, a far-red fluorescence donor Cy5 and a NIR quencher Qsy21, trigger apoptosis with UVA irradiation.332 Caspase-3, a key enzyme in programmed cell death, cleaves the peptide, turns on the Cy5 emission and allows imaging of cellular apoptosis. Biotinylated Pt-35 (Fig. 24) induces dramatic changes in cellular morphology of A2780 cells upon irradiation, including damaged membranes and fragmented nuclei.333 Notably, when co-incubated with avidin, Pt-35 exhibits enhanced photocytotoxicity and cellular accumulation. Polysaccharides. Curcumin has been encapsulated into nanoparticles self-assembled by amphiphilic conjugates consisting of hydrophobic Pt-36 (Fig. 24) and hydrophilic dextran.334 Photoactivation of the nanoparticles with UVA leads to the release of ROS from curcumin and Pt(II) species from Pt-36, which results in a 17 enhanced cytotoxicity in HeLa cells compared to those in the dark and high in vivo antitumor efficacy with low systemic toxicity.

2.17.6.1.2

Synthetic polymeric nanocarriers

Synthetic polymeric nanocarriers include organic polymers containing simple organic monomers, and polymetallodrugs with metallodrugs as monomers. Metallodrugs are usually attached to the side chains of organic polymers, while located in the backbone of polymetallodrugs. Organic polymers. Amphiphilic block copolymers are common organic polymers that can self-assemble into micelles, which generally have a core–shell structure with insoluble polymeric blocks forming the core and soluble blocks forming the shell.335 Metallodrugs can be encapsulated in the cores to reduce unfavorable interactions during delivery. Porphyrin-based Co(II) complex Co-6 (Fig. 25) has been encapsulated into the cavities of heptakis(2,3,6-tri-O-methyl)-b-cyclodextrins (TM-b-CDs) to prevent the formation of its m-oxo-dimer, and thus the imidazole groups of a heptapeptide and an oxygen molecule can bind as the axial ligands.336 The complex is loaded into the hydrophobic core of micelles self-assembled by PEG-b-PLys, which exhibit good biocompatibility

Photoactive metallodrugs

539

4-

SO3-

2+

2O

O

3

N -

S

SO3-

Co

O3S

N

S

S

N

N

N

N

Ru

Ni

N

N

N

3

N

N

S

COOH

N 3

O

O

3

SO3-

Ni-2

Co-6

Ru-49 +

N

N

O +

O N

O

S

O

S

N

O

O

N

O

O

N

Ir

Ir N

O

N

O

S

S

O

O O

N O

O

N

N

O

Ir-14

OH H 2 N Pt N H2 O

N3 O

N3

HO O

Pt-37 Fig. 25

N H

N

Ir-15

OH

OH N3

N3

NH3 N3 O

Pt-38

N3 O

OH O

OH

Pt

H3N

O

N

N3

Pt

Pt H3 N

OH N O

N OH

O

N3 O O

Pt-39

OH

O N H

Pt-40

Photoactive transition metal complexes functionalized for delivery by synthetic organic polymeric nanocarriers.

and cellular uptake, and thus work as a promising artificial O2 carrier in vivo. Photothermally NIR-active Ni-2 (Fig. 25) has been loaded in the core of micelles constituted by amphiphilic PEG-b-PMLABe.337 The micelles induce a drastic temperature increase (ca. 75  C) and up to 80% MDA-MB-231 cell death at 50 mg/mL with NIR (940 nm) exposure. Complex Ir-14 (Fig. 25) consisting of a luminescent Ir(III) complex and chemotherapeutic drug camptothecin, through GSHresponsive disulfide bond linkages, assembles with amphiphilic pluronic F127 to afford micelles, which have been further decorated with cancer-targeting folic acid on the surface.338 The micelles display a 258 enhanced ROS generation in HeLa cells with visible light, which results in the increased cytotoxicity. The triblock copolymer mPEG114-b-PCL20-PLL10 self-assembles in aqueous solution with polycaprolactone and poly-L-lysine forming the hydrophobic core with Pt(IV) complexes Pt-37, Pt-38 or Pt-39 (Fig. 25) encapsulated inside the core via an amide linkage.339–341 Micelles with Pt-37 display up to a 13  enhancement in UVA photocytotoxicity towards SKOV-3 ovarian cancer cells and enhanced H22 murine hepatocarcinoma tumor growth inhibition with reduced systemic toxicity over oxaliplatin.339 The encapsulation of Pt-38 can also protect it from potential deactivation in blood circulation.340 Notably, micelles with Pt-38 show similar photocytotoxicity as cisplatin and are capable of overcoming cisplatin resistance in ovarian cancer cells with a resistance-fold of 0.65 upon UVA irradiation. Micelles loaded with sterically hindered Pt-39 are > 100 times more effective than cisplatin with UVA exposure.341 Compared with block copolymers, homopolymers as drug delivery systems are less reported. Poly(PEGA) homopolymer displays a higher loading of Ru-49 (2.99 wt%, Fig. 25) compared with block copolymer (1.49 wt%). Ru-49-loaded micelles assembled by homopolymers quickly disperse in water and show no cytotoxicity to normal cells, but 3  and 12  increased cytotoxicity towards SK-HEP-1 liver cancer cells in the dark and upon irradiation, respectively.342 Ir-15 (Fig. 25) has been encapsulated into NIR

540

Photoactive metallodrugs

photothermally-active polydopamine nanoparticles functionalized with b-CD substitutions and further assembled with adamantane-modified RGD.343 The resulting nanoparticles can be applied in targeted cancer photothermal-chemotherapy and thermal/photoacoustic/two-photon phosphorescence lifetime imaging. Hydrogels, consisting of cross-linked polymer networks and water, are biocompatible due to their similarity with tissues, and are capable of encapsulating hydrophilic drugs easily.344 G4Kþ hydrogels have been applied in drug delivery systems for topical medicines to treat superficial tumors.345 Dopamine appended Pt-40 (Fig. 25) forms photoactive Pt-G4Kþ B hydrogels, which display selective photocytotoxicity towards cisplatin-resistant A2780cis ovarian cancer cells over normal MRC-5 cells.346 Polymetallodrugs. Loading metallodrugs in micelles can result in a low drug content compared to polymers assembled from photoactive metal complexes.347 For example, amphiphilic Ru-containing triblock copolymer Ru-50 (Fig. 26) with hydrophilic PEG blocks self-assembles into nanoparticles, which accumulate in tumors via the EPR effect, then release anticancer aquated Ru(II) species and generate cytotoxic 1O2 upon red light irradiation.347 No pathological tissue damage was observed in mice treated with Ru-50 and light, while their tumor growth was inhibited significantly, indicating the good performance of Ru-50 as a controllable photoactive agent. An oxygen-responsive phosphorescent Pt(II) porphyrin has been introduced into a polyfluorene-based hyperbranched conjugated polyelectrolyte to improve its aqueous solubility and stability.348 Förster resonance energy transfer (FRET) from polyfluorene units to Pt(II) porphyrin Pt-41 (Fig. 26) allows accurate ratiometric O2 detection. The promising photocytotoxicity of Pt-41 with green light (532 nm) exposure is attributed to its high 1O2 quantum yield. Photoactive diazido Pt(IV) prodrug-backboned block copolymer Pt-42 (Fig. 26) self-assembles into micelles that decompose with wide range light exposure to release cytotoxic Pt(II) species, effectively reducing A549 xenografts in BALB/c nude mice with low weight loss.349 Poly-Pt(IV) prodrug Pt-43 (Fig. 26) allows light-controllable codelivery of Pt(IV) prodrug and siRNA for synergistic cancer therapy.350 The released azidyl radicals facilitate endosomal/lysosomal escape, and the concurrent Pt(II) release and siRNA unpacking result in synergistic therapeutic efficacy in cisplatin-resistant ovarian cancer cells upon blue light irradiation. Except for covalent polymerization, metallodrugs can self-assemble into nanoparticles as monomers via non-covalent interactions. Pt-44 (Fig. 26) with one axial hydrophilic lactose ligand self-assembles into micelles, while its analog Pt-45 (Fig. 26) with two lactose ligands forms vesicles.351 Vesicles formed by Pt-45 can behave not only as a self-delivery system, but also as a nanocarrier to deliver curcumin, showing synergistic photocytotoxicity in vitro.

2.17.6.2

Inorganic nanocarriers

Carbon-based nanomaterials. Carbon-based nanomaterials, such as carbon nanotubes, graphene oxide and carbon dots (CD), have been widely used in drug delivery.352 Ru-51 (Fig. 27) has been loaded into single-walled carbon nanotubes via noncovalent p  p interactions and exerts a combined photothermal and photodynamic effect upon NIR 808 nm irradiation both in vivo and in vitro.110 Reduced nanographene oxide sheets have been decorated with Ru-52 (Fig. 27) via p–p stacking and hydrophobic

Fig. 26

Photoactive polymetallodrugs.

Photoactive metallodrugs

541

2+

2+

O

N

N

N

N

N

N

Ru

N

NH2

O

N H

O n N3

Ru

N

N

N

N

N

N

H N

N

Ru-51

N Pt

N3

O n

O

O

Fig. 27

OH

NH3 OH

Ru-52

Pt-46

Photoactive metal complexes functionalized for delivery by carbon-based nanomaterials.

interactions.109 The release of Ru-52 is pH dependent, and can be facilitated by light. Irradiation at 405 nm of the nanohybrid leads to ROS generation, while NIR 808 nm irradiation causes a photothermal effect. The combined mechanism enhances the anticancer activity via apoptosis and lysosomal damage. Carbon dots can be decorated with the photoactive diazido Pt(IV) prodrug Pt-46 (Fig. 27) and folic acid (FA) molecules via amide coupling, showing selective accumulation in the folate receptor of FR-positive HeLa cancer cells.353 Mesoporous silica nanoparticles (MSN). The acid-sensitive PEGylated Zn-2 (Fig. 28) can be used as a gatekeeper to block the nanopores of histidine-grafted MSN by metallo-supramolecular-coordinated interaction between Zn-2 and histidine, which is stable in healthy tissue, to prevent drug-leaking.354 However, the cis-aconitic anhydride linker cleaves at cancer extracellular pH (ca. 6.8), and thus the Zn-porphorin will have a positively charged amino group to facilitate cell internalization. The MSN disassembles in intracellular acidic microenvironments (pH ca. 5.3) to release loaded drug and photosensitive Zn-porphyrin to allow combined chemotherapy and PDT. Ru-53 (Fig. 28) has been loaded to pores of MSN covalently through different ligands or by a simple physisorption.355 However, no significant photocytotoxicity was observed after 10 min irradiation with 350 nm light, which might be due to the low drug loading (up to 7.5% of Ru wt%). Oxygen-sensitive Ru-54 (Fig. 28) displays phosphorescence in hypoxic environments, which can be quenched by oxygen.356 Immobilization in the MSN pores can protect Ru-54 from direct interaction with intracellular biomolecules and reduce its photocytotoxicity, since 1O2 photoinduced by Ru-54 is deactivated before leaking

COOH

O

O

HN

O n

O 2+ N N

N

N O

N

N

N

N

N

N

Ru

NH

Zn

N

OH

N COOH

O

n

O O O

NH

O n

O

O

HOOC

Ru-53

Zn-2

2+ N 2+

N

O N

N Ru

N

N

N

N

N

N

Ru

N

N

N

N O

N O Si O O

Ru-54 Fig. 28

O N H

N H

Ru-55

Photoactive Zn(II) and Ru(II) complexes functionalized for delivery by mesoporous silica nanoparticles.

542

Photoactive metallodrugs

from the pore. Ru-55 (Fig. 28) modified MSN undergo rapid cellular uptake, and release DNA-binding aquated Ru(II) species after irradiation.357 Paclitaxel stored in the MSN pores blocked by Ru-55 is also released after the photodecomposition of Ru-55 to allow combined therapeutic effects. Gold nanoparticles. Gold nanoparticles have been widely used in phototherapeutic drug delivery due to their good biocompatibility, feasible modification, and NIR photothermal efficacy.67 The co-self-assembly of the photosensitive Zn(II)-phthalocyanine Zn-3 (Fig. 29) and a PEG derivative onto gold nanoparticles significantly improves the drug retention in subcutaneous transplanted B78H1 amelanotic melanoma and leads to 40% of the treated mice showing no tumor regrowth and complete survival.358 Photothermally-active gold nanorods coated with dipicolyl amine form stable complexes with Zn(II), which exhibit strong complexation with anti-polo-like kinase I siRNA (siPLK) to form Zn-4 (Fig. 29).359 The nanosystem Zn-4 displays significant antitumor activity in a PC3 tumor mouse model via a combined gene/photothermal therapy. Ru-56 (Fig. 29) can significantly enhance the two-photon luminescence intensity and PTT efficiency of gold nanospheres, which allows real-time luminescence imaging-guided PTT in live cancer cells and in vivo tumor ablation upon irradiation with a diode laser (808 nm).69 The electron-withdrawing azo group quenches the emission of Ir-16 (Fig. 29) conjugated to gold nanorods and acts as a bio-reducible switch to release a luminescent Ir(III) complex in hypoxia, allowing hypoxia imaging.70 Ir-16-loaded gold nanorods selectively accumulate in mitochondria and exhibit NIR PTT efficacy with low side effects. Polydopamine coating allows high cis-{Pt(NH3)2}2þ loading efficiency, RGD conjugation, and chelator-free iodine-125 labelling in gold nanorods to form a robust platform Pt-47 (Fig. 29), which selectively accumulates in tumors and tumor angiogenic vessels, thus ablating tumors and inhibiting tumor relapse via an image-guided chemo-photothermal (NIR) therapy.360 Magnetic nanoparticles. Magnetic nanoparticles are promising contrast agents for magnetic resonance imaging (MRI). Ru-57 (Fig. 30A) has been conjugated to magnetic Gd2O3 cores encapsulated by a polysiloxane shell; the resulting nanoparticles can kill HEK293 cells with 470 nm light excitation owing to an effective 1O2 quantum yield of 0.33.361 Re-9 (Fig. 30A) is covalently anchored to a superparamagnetic iron oxide core coated by a compact silica shell, and the resulting nanoparticles are further coated with a PEG layer to improve their stability.362 1O2 generation is maintained for Re-9 when encapsulated in the nanoparticles, which are easily internalized and accumulate in the perinuclear region of the cells as investigated by two photon excitation confocal microscopy, and display promising photocytotoxicity. Upon NIR irradiation, magnetic Fe3O4 nanoparticles decorated by Ir-17 (Fig. 30A) increase the localized temperature to 42  C, resulting in the accelerated hydroxyl radical production from H2O2, and thus mild photothermal therapy.71 MRI images suggest the accumulation of the nanoparticles in a HeLa tumor in mice, which results in an inhibition of the tumor without significant mouse body weight loss in combination with NIR. Metal-organic frameworks (MOFs). The sizes of Zr(IV)-based porphyrinic metal  organic framework (MOF) nanoparticles Zr-1 (Fig. 30B) are tunable over a broad range.363 Among them, 90-nm nanoparticles display preferential cellular uptake and higher PDT efficacy over other sizes. UCNPs. UCNPs are lanthanide-doped crystals that convert low-energy NIR photons into higher energy UV–vis light, and have been widely employed as nanocarriers for photoactive metallodrugs to allow deeper tissue penetration.89 Zn-5 (Fig. 31) has been covalently conjugated to amino-functionalized NaYF4:Yb3þ,Er3þ UCNPs to shorten their distance, thus enhancing energy transfer and NIR activation.364 Low-dose 980 nm NIR radiation causes inhibition of liver tumor volume by ca. 80% in combination with Zn-5 loaded UCNPs, causing no pathological changes or inflammatory response. PACT agent Ru-20 has been loaded into HSAcoated lanthanide-doped UCNPs.365 The HSA coating improves the biocompatibility and aqueous solubility and emits green fluorescence, while the UCNPs emit blue up-conversion fluorescence, which allows dual-fluorescence cell imaging. White light-activated Ru-20 is released from the UCNPs and binds with DNA to exert photocytotoxicity. Amphiphilic PDT photosensitiser Ru-58 (Fig. 31) is efficient and photostable, and can be encapsulated in NaYF4:Yb3þ,Tm3þ UCNPs coated with phospholipids and cholesterol.366 The resulting system generates ROS under irradiation with a 969 nm laser. Upon NIR irradiation at 980 nm, Re-10 (Fig. 31) loaded in NaYF4:Yb3þ,Tm3þ UCNPs coated by poly(acrylic acid) (PAA) and cholesterol is activated by upconverted UV radiation emitted from UNCPs, releasing CO and inhibiting growth of both A2780 and A2780cis ovarian cancer cells.367 LiYF4:Tm3þ,Yb3þ UCNPs are capable of producing strong UV emissions when excited at 980 nm, which can activate the conjugated Ir-18 (Fig. 31) to generate ROS.368 Hydrophobic photosensitive Ir-19 (Fig. 31) is polymerized with the hydrophilic PEG, forming block copolymer with lapatinib at the end.369 The block copolymer self-assembles into micelles, which include UNCPs to permit NIR excitation. The nanosystem displays considerable cytotoxicity against HER-2-positive SKOV3 cancer cells with 980 nm irradiation, as well as tumortargeting ability and significant antitumor efficacy in vivo. In addition to HSA, Pt-34 has also been attached to UCNPs with a FRET pair (Cy5 and Qsy21), which allows photocytotoxicity with 980 nm NIR irradiation.370 PEGylated phospholipid functionalized Pt48 (Fig. 31) can decorate the surface of NaYF4:Yb3þ,Tm3þ@NaYF4 core-shell UCNPs that convert 980 nm NIR to UV light, leading to the decomposition of Pt(IV) in Pt-48 and the release of cytotoxic cisplatin.371 Pt-49 (Fig. 31)-containing Yb/Tm-codoped UCNPs exhibit higher tumor inhibition with NIR irradiation than direct UV irradiation, and can be used as a multimodality bioimaging contrast agent.372 Amphiphilic oligomer Pt-50 (Fig. 31) self-assembles into micelles with UCNPs co-assembled to convert NIR to UV, allowing the photoactivation of Pt-50 as an O2-self generating PACT-PDT agent which exhibits promising photocytotoxicity in hypoxic environments.108

2.17.7

Summary and perspectives

Phototherapy represents a new generation treatment that has the potential to provide accurate spatial and temporal control over activity to reduce side effects and introduce novel mechanisms of action to circumvent drug resistance. The introduction of metal

Photoactive metallodrugs

C6H13

543

Dipicolyl amine

N C6H13

N

C6H13

N

Zn(II)

N

Zn

N

SH

N

N

C6H13

SiRNA

N

Gold nanorods

C6H13 C6H13

Zn-3

Zn-4 O 2+ N N

N

H N

H N

N

N

N

N

N

N

Ir

+ (CH2)10SH

N N N

Ru N

N

N N

Ru-56

Ir-16 HO O

H3N

Pt

O

NH3

NH O

O

O

*

O *

OH

*

NH

N

n

NH2

H

O

H N

O

HN O

O

*

o

P

NH

HO

O

S

COO

NH HN

HN

O

HN H2 N

NH

Iodine-125

Pt-47 Fig. 29 Photoactive metallodrugs delivered by gold nanoparticles. Figures for Zn-4 and Pt-47 are modified from Min, K. H.; Kim, Y.-H.; Wang, Z.; Kim, J.; Kim, J. S.; Kim, S. H.; Kim, K.; Kwon, I. C.; Kiesewetter, D. O.; Chen, X. Theranostics 2017, 7, 4240–4254 and Zhang, L.; Su, H.; Cai, J.; Cheng, D.; Ma, Y.; Zhang, J.; Zhou, C.; Liu, S.; Shi, H.; Zhang, Y.; Zhang, C.A. ACS Nano 2016, 10, 10404–10417 with permission from Ivyspring International Publisher, Copyright 2014; and American Chemical Society, Copyright 2016, respectively.

544

Photoactive metallodrugs

(A)

Ru-57

Re-9

Ir-17

(B)

Fig. 30 (A) Photoactive metallodrugs delivered by magnetic nanoparticles; (B) A cubic unit of Zr-1 and schematic illustration of spherical Zr-1 based MOF nanoparticles with tunable size. Reproduced from Park, J.; Jiang, Q.; Feng, D.; Mao, L.; Zhou, H. C. J. Am. Chem. Soc. 2016, 138, 3518– 3525 with permission from American Chemical Society, Copyright 2016.

complexes into photoactive agents offers multiple advantages, including diverse structures, improved stability and photocytotoxicity, novel mechanisms of action, unique photophysical and photochemical properties, and specific metal-based detection methods. In this Chapter, we have discussed the history and classification of phototherapy, the photophysics and photochemistry of photoactive metallodrugs, including light absorption and luminescence, activation wavelengths, activation mechanisms and photoreactions with biomolecules, and described selected examples of photoactive anticancer and antimicrobial agents with various mechanisms of action. Rapid development has been achieved with some photoactive metallodrugs, especially polypyridyl Ru(II) and cyclometalated Ir(III) PDT agents, Pt(IV) PACT agents, and gold nanoparticle-based PTT agents (summarized in Table 3). However, PhotosensÒ and TookadÒ soluble are the only two photoactive metallodrugs which have been approved for clinical use. Major challenges remain to be addressed before clinical applications of photoactive metallodrugs are more widespread. (1) To ensure sufficient tissue penetration and minimize side effects, the ideal activation wavelength range for phototherapy, the “phototherapeutic window,” is between 620 and 850 nm. However, a wide range of photoactive metallodrugs display no activity with light within this range, especially PACT agents. Incorporation of an extensive p system can red-shift the absorption bands, but may result in hydrophobic complexes with poor aqueous solubility. UNCPs can convert NIR to UV, and thus activate attached metallodrugs, but these may encounter biocompatibility problems. Multiphoton activation can provide NIR photocytotoxicity, however its clinical applications are limited by the lack of inexpensive dispersed laser light sources. X-ray activation is a new option. However, intense X-rays may induce side effects. Thus, novel strategies to allow practical NIR photoactivation are urgently required. It should be noted, however, that less deep penetration (and use of shorter wavelength light) may be an advantage for minimizing damage where tissues are thin (e.g. the bladder). (2) Current clinically approved photoactive metallodrugs are all based on oxygen-dependent PDT, which limits their efficacy in hypoxic tumors. PACT and PTT agents do not require oxygen to exert efficacy, but are still under development and often have complicated mechanisms of action. (3) Despite phototherapy being a localized treatment, enhancement of tumor-specific accumulation of administered photosensitisers is an important issue to address, which can improve the efficacy and reduce side effects significantly.

Photoactive metallodrugs

545

HOOC +

2+

COOH N N N

N

N N

Zn

N

O

N

N

O

CO CO

N

Re

Ru

N

N

N

CO

N

5

N

N

N HOOC COOH

Cl

Zn-5

Ru-58

Re-10

O +

S

COOH

N

N

N Ir

N

N

N

N

Ir N

N

COOH S

N OH

O

Ir-18

Ir-19 O

O HO

HO

O O

O O

NH3

Cl

N3

O

O

N3 OH

O

O

Pt-48

N3 O

OH

H N

O

H3N

NH3 O

Cl

N H

O

N Pt

Pt

Fig. 31

+

OH

Pt-49

O

NH3 O

N O

Pt N3

n

H N

HO NH3

N H N

O O

OH

OH

Pt-50

Photoactive metallodrugs delivered by UCNPs.

(4) Higher photocytotoxicity indices are also desired for new photoactive metallodrugs to decrease their systemic toxicity. More efficient photochemical reactions, better defined reaction pathways, and improved quantum yields are likely to improve photocytotoxicity indices. (5) As a localized treatment, photoactive metallodrugs are unable to treat metastatic diseases (e.g. late stage tumors) effectively. Immunomodulation induced by phototherapy may be an effective strategy to overcome this limitation. In addition, the possibility of inducing immunogenic cancer cell death has the potential to prevent recurrence of the same tumor. (6) Biocompatible drug delivery systems are important for clinical applications to allow effective administration, improve efficacy and reduce systemic toxicity. (7) Efficient synthetic routes with commercially-available and cheap raw materials are important for drug candidates to decrease the cost and guarantee the supply. Despite the long history of light application in medicine, modern phototherapy is still in its infancy. The development of novel photoactive metallodrugs will contribute to the evolution of phototherapy. Only now is the photochemistry of a wide range metal coordination complexes beginning to be explored, aided by advances in light technology and instrumentation that allows photochemical pathways to be probed on a subnanosecond timescale and the photoproducts to be identified. We have seen above how the photophysics, photochemistry and photobiology of metal complexes can be controlled by the metal, its oxidation state, the types and number of ligands, the coordination geometry, and subtle changes in the substituents on ligands and their electronic structures. Although we have focussed on photoactivated anticancer and antimicrobial agents because these are the most highly active areas of current research, the future potential in other areas of therapy is evident. A wide range of biologically-active signalling molecules can be delivered by photoactivation with wider potential applications, not only CO, but also for example H2S (ischemiareperfusion),373 NO (vasodilation),374,375 and CS2 (cell growth, neurotransmission).376

546

Photoactive metallodrugs

Table 3

Summary of the main types of phototherapeutic metal complexes described here, their modes of photoactivation and mechanisms of biological action.

Complex

Metal ion

EC a

Radiation

Phototherapy

Mechanism of Activation

Active species

Oxovanadium(IV) Peroxovanadium(V) Polypyridyl Cr(III) Tricarbonyl Mn(I) Polypyridyl Fe(II) Ferrocene derivatives Mononuclear Fe(III) Oxo-bridged diiron(III)

V(IV) V(V) Cr(III) Mn(I) Fe(II) Fe(II) Fe(III) Fe(III)

3d1 3d0 3d3 3d6 3d6 3d6 3d5 3d5

UVA–NIR UVA– Vis Vis UVA– Vis Vis UVA– Vis Vis Vis

PDT PDT PDT PACT PDT PDT, PACT PDT PACT

ROS ROS ROS CO ROS ROS ROS Fe(II); ROS

Co(III) curcumin

Co(III)

3d6

Vis

PDT, PACT

Ni(II) bis(dithiolene) Zn(II) phthalocyanines Polypyridyl Ru(II) Arene Ru(II) Dinuclear Rh(II) Mononuclear Rh(III) Pd(II) bacteriophorbides Tricarbonyl Re(I)

Ni(II) Zn(II) Ru(II) Ru(II) Rh(II) Rh(III) Pd(II) Re(I)

3d8 3d10 4d6 4d6 4d7 4d6 4d8 5d6

NIR Red Vis–NIR Vis Vis UV NIR UVA– Vis

PTT PDT PDT, PACT, PTT PACT PDT PACT PDT PDT, PACT

Polypyridyl Os(II) Cyclometalated Ir(III)

Os(II) Ir(III)

5d6 5d6

UVA–NIR Vis–NIR

PDT PDT, PACT

Cyclometalated Pt(II) Diiodo Pt(IV) Diazido Pt(IV) Cyclometalated Au(III) Ln(III) texaphyrin Non-macrocyclic Ln(III)

Pt(II) Pt(IV) Pt(IV) Au(III) Lu(III) Eu(III); Tb(III)

5d8 5d6 5d6 5d8 4f14 4f6; 4f8

UVA– Vis UVA– Vis UVA–NIR Vis NIR UVA– Vis

PDT, PACT PACT PACT PACT PDT PDT

Photocatalysis Photocatalysis Photocatalysis Photosubstitution Photocatalysis Photocatalysis; Photosubstitution Photocatalysis Photoreduction; Photosubstitution; Ligand photoactivation Photocatalysis; Photoreduction Photocatalysis Photocatalysis Photocatalysis; Photosubstitution Photosubstitution Photocatalysis Photosubstitution Photocatalysis Photocatalysis; Photosubstitution Photocatalysis Photocatalysis; Photosubstitution Photocatalysis; Photosubstitution Photoreduction Photoreduction Photosubstitution Photocatalysis Photocatalysis

Co(II); ROS Heat ROS Ru(II); ROS; Heat Ru(II) ROS Rh(III) ROS ROS; Re(I); CO ROS ROS Pt(II); ROS Pt(II) Pt(II); ROS; azidyl radicals Au(III) ROS ROS

EC ¼ Electronic configuration.

a

A future need in this field is to identify in more detail the molecular mechanisms of action of photoactivated metallodrugs. As well as concluding that, for example, ROS production is the cause of cell death, the types of ROS need to be identified and their specific target sites, for example, proteins or DNA. Modern methods of metallomics need to be applied, recognizing that a systems pharmacology approach is often needed to understand cell metabolism rather than simple emphasis on single pathways. Also, it is important to recognize that experiments with cells are not often predictive of the behavior of drugs in animals where a range of other uptake, transport, metabolism, and excretion processes can occur. These are all major challenges, but are exciting for future research with the potential to generate truly novel phototherapies (and diagnostic agents) based on the design of metal complexes.

Acknowledgments We acknowledge financial support for our recent research on photoactive metal complexes from the EPSRC (EP/G006792, EP/F034210/1), The Royal Society (Newton Fellowships), Wellcome Trust, Anglo American Platinum and University of Warwick. We are also grateful for stimulating discussions on this topic with our research group, collaborators, and colleagues.

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2.18 Metallophores: How do human pathogens withdraw metal ions from the colonized host Henryk Kozlowskia,b, Karolina Piastaa, Aleksandra Hecela, Magdalena Rowinska-Zyreka, and Elzbieta Gumienna-Konteckaa, a Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland; and b Institute of Health Sciences, University of Opole, Opole, Poland © 2023 Elsevier Ltd. All rights reserved.

2.18.1 2.18.1.1 2.18.1.1.1 2.18.1.1.2 2.18.1.1.3 2.18.1.1.4 2.18.1.1.5 2.18.1.1.6 2.18.1.2 2.18.1.2.1 2.18.1.2.2 2.18.2 Acknowledgments References

Introduction Siderophores in the microbial battle for iron and their role in homeostasis of other metals Environmental aspects of siderophore production Siderophore transport systems Implications of siderophores secretion for social relations between microorganisms and with the host Interactions of siderophores with other metal ions Metallophore biomimetics Metal transport in vivo and lighting up metallophore–metal–metal transporter interactions and infection Peptide/protein-based zincophores in the tug-of-war over zinc Fungal zincophores Bacterial zincophoresdSubstrate-binding proteins (SBPs) Conclusions

554 555 555 556 558 560 562 563 563 564 566 569 570 570

Abbreviations ABC-transporter ATP-binding cassette transporter ABN Aerobactin ACN/pACN Acinetobactin/pre-acinetobactin BFN A/B Baumannoferrin A/B CBD C-terminal domain CF Cystic fibrosis ENT Enterobactin FBN Fimsbactin FC Ferrichrome Fit protein Facilitator of iron transport protein FOX B/E Ferrioxamine B/E FSC Fusarinine C GPCR G-protein coupled receptor IM Inner membrane IPA Invasive pulmonary aspergillosis MRSA Methicillin-resistant Staphylococcus aureus NBD Nucleotide-binding domain NDM-1 New Delhi metallo-beta-lactamase 1 NTD N-terminal domain OM Outer membrane PBP Periplasmic binding protein PCH Pyochelin PET Positron emission tomography PVD Pyoverdine QD Quantum dot RIA Reductive iron assimilation ROS Reactive oxygen species SBD Substrate binding domain SBP Siderophore/substrate binding protein SFN A/B Staphyloferrin A/B

Comprehensive Inorganic Chemistry III, Volume 2

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SIT Siderophore iron transporter SOD Superoxide dismutase STP Staphylopine TAFC Triacetylfusarinine C TBDT TonB-dependent transporter TMD Transmembrane domain TRAP-transporter Tripartite ATP-independent periplasmic transporter TTT Tripartite tricarboxylate transporter YBT Yersiniabactin ZIP Zrt/Irt-like protein ZnT Zinc transporter

Abstract The increasing interest in metallophoresdmetal chelating molecules which are excreted outside the pathogen in order to efficiently bind a given metal ion and in their interactions with appropriate metals is one of the results of the dramatic increase of antimicrobial resistancedthe resistance of a microorganism to an antimicrobial drug that was originally effective for treatment of infections caused by it. Resistant microorganisms, e.g., bacteria and fungi, are able to withstand the attack of antibiotics and antifungals, making standard treatments ineffective. Novel, effective treatments and ways to specifically deliver them to antibiotic resistant bacteria and to drug resistant invasive mycoses are being actively sought. One of the biggest obstacles in finding such effective, pathogen-specific therapeutics that will not cause severe side-effects in patients arises from the fact that bacteria and fungi share essential metabolic pathways with humans (especially fungi, since they are both eukaryotes). In order to design a highly specific antifungal drug, it is crucial to understand and aim at differences in the metabolism of humans and pathogens. Although pathogen-selective targets are scarce, there is at least one significant difference between the microbial and mammalian cells: the transport system of transition metal ions. In this chapter, we focus on siderophore and zincophoredbased metal transport system, explaining their biological inorganic chemistry, showing metal binding sites, complex geometries and thermodynamic stabilities. Although chemically versatiledsiderophores are most commonly small metal chelating agents, while zincophores are more often protein or peptidedbased, both classes of metallophores (i) chelate the metal ion in a specific binding mode, (ii) require a binding partner on the pathogen surface, (iii) are a potentially powerful tool for building a Trojan horse strategy based drugs and (iv) are a fascinating phenomenon for bioinorganic chemists.

2.18.1

Introduction

The frequency of drug resistant microbes has increased significantly over the past three decades, becoming an increasingly serious threat to global public health that requires action across all government sectors and society.1,2 Half a million cases of multidrug-resistant tuberculosis each year, abnormally high percentages of hospital-acquired infections caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Gram-negative bacteria pose serious threats to human health. The incidence of drug resistant invasive fungi has increased dramatically, in both immunosuppressed and non- immunosuppressed patients.3 New resistance mechanisms emerge and spread globally, making common infectious diseases untreatable, resulting in death or disability of patients. Pathogens resistant to standard forms of treatment are becoming a serious threat and novel, effective treatments and ways to specifically deliver them to antibiotic resistant bacteria and to drug resistant invasive mycoses are being actively sought. Finding effective, pathogen-specific and side effect-free therapeutics is a great challenge, since microbes share essential metabolic pathways with humans. One of the specific differences between human and microbial cells is the transport system of transition metal ions. Effective acquisition of Fe(III) and Zn(II) is crucial for the survival and virulence of bacteria and fungi, being substantial for metalloproteins, such as metalloenzymes, storage proteins and transcription factors. From the functional point of view, such metal ions have a structural role, take part in noncatalytic reactions, redox or non-redox catalysis. In the scope of this chapter, we will focus on the metallophore-mediated transport of two biologically indispensable metal ionsdFe(III) and Zn(II). Their effective acquisition is often considered as a virulence factor. Iron, due to its ability to assume multiple oxidation states, is an essential metal for a wide range of cellular events, such as oxygen metabolism, and electron-transfer processes, synthesis of DNA and RNA; therefore, it is an indispensable micronutrient for almost all living organisms. On the other hand, an excess of iron is toxic, as it may induce oxidative damage by the classical Fenton reaction.4 Zinc, the second most abundant transition metal in living organisms (after iron),5 is also a crucial survival and virulence factor for pathogens. It is present in superoxide dismutases (SODs), central enzymes in bacteria and fungi associated with the detoxification of ROS generated by host cells during host-pathogen interactions.6 Zinc-binding metalloproteases are involved in pathogen

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invasion; they are secreted by distinct species of pathogenic fungi7 such as the ADAM metalloproteinases or deuterolysin (with two zinc-binding histidines and a catalytic glutamate in its catalytic center).8 It is worth mentioning that one group of enzymes that require Zn(II) as a cofactor are metallo-beta-lactamases (e.g., the New Delhi metallo-beta-lactamase 1 (NDM-1) protein),9 able to inactivate beta-lactam antibiotics, used to treat multidrug-resistant infections. Both pathogens and hosts are aware of the importance of the acquisition of substantial metal trace elements.10 Bacteria and fungi have developed highly efficient transport systems, which rely on metallophores. This can serve either as metal acquisition systems (metal-bound metallophores are transported back to the pathogen) or as a way of protecting themselves against the toxic excess of metal ions in their surroundings. On the other hand, vertebrate hosts have developed mechanisms which restrict the bioavailability of metal ions for pathogens via a process called nutritional immunity.11 This process describes the competition between the pathogen and the host for an important resource (which, in this case, is metal ions), during which both the pathogen and the host make huge efforts to control its availability. Originally, this term used to refer to the host mediated restriction of iron availability, but recently, has also been applied to mechanisms for withholding other essential transition metals or to mechanisms which direct the toxicity of these metals against microbial invaders.12 In the scope of this chapter, we focus mainly on Fe(III) and Zn(II), trying to explain their roles at the pathogen–host interface and providing insight into the thermodynamics and coordination chemistry of their interactions with specific metallophores and metal transporters.

2.18.1.1

Siderophores in the microbial battle for iron and their role in homeostasis of other metals

Microorganisms, bacteria and fungi, require iron concentrations of ca. 10
6 M for optimal growth,13 and the natural abundance being in the range of 10
18 to 10
24 M14 due to host nutritional immunity, is insufficient to support microbial needs. To acquire iron, microorganisms employ a number of transport mechanisms,15 including iron acquisition from heme due to their hemolytic activity, removing iron from ferritin, transferrin and lactoferrin, ferric ions uptake by siderophores or Fe(II) utilization by ferrous ions transport systems.16 Among others, an intricate iron acquisition and trafficking system involving low molecular weight molecules termed siderophores or iron carriers (from greek ‘sidero’ for iron) is the most common way for microorganisms to acquire this nutrient.17–19 Low iron concentrations trigger a “signal” to start biosynthesis of the appropriate siderophores and the proteins involved in siderophore uptake machinery. More than 500 siderophores have been isolated from prokaryotic and eukaryotic microbes, and more than 270 were structurally characterized.20,21 These compounds have remarkably diverse structures considering that they all perform the same biological functiondthe chelation and transport of ferric iron. Despite their structural diversity, many siderophores possess at least one of three different ferric chelating functional groups: hydroxamates (e.g., ferrichrome, FC); a-hydroxycarboxylates (e.g., achromobactin) or catecholates (e.g., enterobactin, ENT). Others contain combinations of phenolate, carboxylate and nitrogen heterocycle ligands (e.g., pyochelin, PCH and yersiniabactin, YBT), often called “mixed-type” iron carriers (Fig. 1).22 The majority of siderophores are hexadentate chelators, and their ferric complexes are among the most stable coordination compounds of Fe(III) known in nature. Tetradentate siderophores are less efficient chelators, as two or three ligands are needed to satisfy the octahedral geometry of ferric ions (with 2:1 and 3:2 ligand-to-metal stoichiometries). Siderophore chelating moieties may be arranged in diverse topologies: linear, cyclic or a template pendant with chelating “arms.” Cyclization was shown to improve the siderophore complex thermodynamic and chemical stability, ultimately improving resistance to enzymatic degradation.23 Excreted into the environment, siderophores strongly and specifically bind ferric ions; the proton independent binding constants (logb) for Fe(III)–siderophore systems range from  1020 to 1049, reflecting the diversity of chemical composition and structural arrangement of siderophores.24,25 Despite their usefulness, logb values are not always possible to compare, e.g., due to differences in ligand dissociation constants or metal-to-ligand binding stoichiometries. Therefore a pH-dependent pFe(III) parameter was developed,26 in which the pFe(III) value is defined as
log[Fe(III)aq], and calculated with fixed concentration of metal, ligand and proton, i.e., [Fe(III)]total ¼ 1 mM and [L]total ¼ 10 mM at pH ¼ 7.4.26 Log of stability constants, as well as pFe values for selected siderophores are shown in Table 1.

2.18.1.1.1

Environmental aspects of siderophore production

Siderophores can be classified not only according to the chemical properties of functional groups that chelate ferric iron, and their structural arrangement (molecular architecture), but also according to their producing organisms (bacterial, fungal or plant). Catecholate siderophores are for example mainly produced by Gram-negative bacteria (primarily the Enterobacteriaceae and the genus Vibrio), while hydroxamate based siderophores are produced commonly by fungi (the Ascomycetes and Basidiomycetes) and some Gram-positive bacteria (e.g., Streptomycetes). Carboxylate siderophores are produced by few bacteria (e.g., Rhizobium) and fungi (Mucorales, belonging to the Zygomycota).23 These selections often stem from ecological conditions. Complex stability, high environmental pH, lipophilicity and weak nitrogen metabolism foster catechol production, while acid-stability and versatile nitrogen metabolism foster hydroxamate production. Organic acid secretion by fungi is probably the reason why most fungal siderophores are hydroxamates (e.g., the fusarinine C (FSC) and FC), which are stable down to pH 2.17,23 Carboxylate siderophores such as staphyloferrin A (SFN A) or rhizoferrin also exhibit a superior affinity for ferric iron at acidic pH and hence, likely contribute to enhanced fitness of microbes in more acidic milieus. In contrast, in physiologic environments, catecholate siderophores such as ENT and salmochelin (Fig. 1) reveal higher affinity for ferric iron, relative to carboxylate or hydroxamate siderophores. The ability to utilize a number of siderophore systems operating at different pH and iron conditions enhances efficacy of iron uptake by an organism,

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Fig. 1 Structures of selected siderophores discussed in this chapter. The hydroxamic groups were marked in blue, catechol in green, a-hydroxycarboxylic in red, and phenolothiazoline in purple.

facilitating its adaptation to various environments. Indeed, most pathogens are able to produce several structurally different siderophores.33

2.18.1.1.2

Siderophore transport systems

The siderophore-mediated iron uptake occurs via active escort through siderophore receptor systems, which differ between Gramnegative or Gram-positive bacteria and fungi (Fig. 2). In case of Gram-negative bacteria, the presence of two membranes and a peptidoglycan cell wall requires the use of a multi-stage mechanism for ferrisiderophore uptake. Firstly, Fe(III)-siderophore system is recognized by ligand-gated TonB-dependent transporters (TBDTs), localized in outer membrane (OM).34–36 TBDTs are generally composed of two main subunits: C-terminal antiparallel amphiphilic transmembrane b-barrel, which allows TBDTs to be classified into the porin superfamily, and structurally distinct N-terminal globular domain, called the plug.37 The 22 b-strands in C-domain are anchored in the periplasmic side of Table 1

Log of stability constants and pM values for selected siderophores.

Siderophore

Stability constant a

pFe

Pyoverdine, PVD Pyochelin, PCH Enterobactin, ENT Ferrichrome, FC Ferrioxamine B, FOX B Ferrioxamine E, FOX E Acinetobactin, ACN Pre-Acinetobactin, pACN Staphyloferrin B, SFN B, (hemiaminal form) Yersiniabactin, YBT

logb110 ¼ 30.8 27 logb120 ¼ 28.8 28 logb110 ¼ 49 17 logb110 ¼ 29.1 17 logb110 ¼ 30.6 17 logb110 ¼ 32.2 29 logKApp120 ¼ 27.4 30 logKApp120 ¼ 26.2 30

27 27 16 28 34.3 17 25.2 17 26.6 17 27.7 26

a

logKPI ¼ 36.6 32

23.6 31

The conditions under which the constants were determined differ from each other and are described in the given references.

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Fig. 2 Schematic representation of Fe(III)-siderophore transport systems in Gram-negative (A) and Gram-positive (B) bacteria, and fungi (C). (A) Two membranes and a peptidoglycan cell wall in Gram-negative bacteria require a multistep mechanism for Fe(III)-siderophore uptake. Firstly, Fe(III)siderophore system is recognized by ligand-gated TonB-dependent transporters (TBDTs), localized in outer membrane (OM). Next, the Fe(III)siderophore is transferred by a periplasmic binding protein (PBP) to ATP-binding cassette (ABC) transporter or proton-motive permeases localized in the inner membrane (IM). The release of iron ions in the cytoplasm requires dissociation of the Fe(III)-siderophore complex, reduction of Fe(III) ion or degradation of the siderophore molecule. Aposiderophore may be then recycled, relocated outside the cell and reutilized. (B) Gram-positive bacteria require relatively simpler uptake system, involving siderophore-binding protein (SBP), one to two transmembrane protein permeases and an ATPase providing energy to the process. All those components form the ABC-type transporter. (C) Yeasts and filamentous fungi use the unique siderophore iron transporters (SITs) localized in the membrane. The fungi cell wall may also contain the facilitator of iron transport (Fit) proteins, facilitating ferric siderophores uptake by SITs. Following the uptake, Fe(III)-siderophore complexes are hydrolysed, the aposiderophores are excreted and Fe(III) may be stored in a siderophore-based deposits in vacuoles or delivered to the specific metabolic pathways.

OM and the expansive loops, involved in ligand recognition, are located on the outer surface. The N-domain region contains a sequence of 7-11 residues, called TonB-box, whose movement is induced by a series of changes in the position of extracellular loops upon ligand binding, mediating signal transition to TonB.38–40 It has been shown, that binding FC to the FhuA results in some changes in loops propagated through the plug, which alter the position of the TonB-box.41,42 TonB-box biding to the TonB, localized in the inner membrane (IM) allows iron to pass through the channel. Furthermore, N-terminal region restricts the direct passage of apo- or ferrisiderophore through the porin.35 The binding site consists of groups derived from both the barrel and the globular domain. Depending of the nature of the binding site amino acids, each TBDT can recognize and translocate specific Fe(III)-siderophore system, or less frequently few structurally related siderophores complexes, 35,40 for example FecA of E. coli K-12 recognizes ferric-citrate41 and P. aeruginosa FptA and FpvA transport ferric complexes of OPCH43 and pyoverdine (PVD),44 respectively. After passing the TBDT, ferrisiderophore is adsorbed on periplasmic binding protein (PBP)45,46 and transferred to ATP-binding cassette (ABC) transporter or proton-motive permeases, localized in IM. ABC transporters usually consist of five domains, which include: a binding protein, a transmembrane channel of two polypeptides, and two nucleotide-binding subunits responsible for the hydrolysis of ATP. This organization is for example observed in the ferric Ent ABC transporter in E. coli, FepBC2D2 (FepB being the PBP, and the dimers FepC2 and FepD2 forming the permease and the ATPases, respectively).47 ABC transporters for complexes of other siderophores may have slightly different organization and stoichiometry of subunits.36 In contrast, permeases act as single subunit transporters.36 Iron transport through the IM is less specific than through the OM due to the higher “elasticity” in ABCtransporters ligand specificitydi.e., though FC, aerobactin (ABN) and ferrioxamine B (FOX B) require specific TBDT (FhuA, Iut and FhuF, respectively) to pass the OM, they use the same ferric hydroxamate uptake system FhuBCD in E. coli during transport by IM.48 The release of iron ions in the cytoplasm requires dissociation of the Fe(III)-siderophore complex, reduction of Fe(III) ion or degradation of the siderophore molecule.49,50 Aposiderophore may be then recycled, relocated outside the cell and reutilized.33 In some cases, iron can be released when the complex passes through the periplasmic space, as is the case, for example, in P. aeruginosa. Free iron ion is then transported to the cytoplasm and aposiderophore is released outside the cell.16 When sufficient amount of iron is transported and accumulated inside the cell, the iron acquisition systems are turned off.

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The lack of OM and a periplasmic space in Gram-positive bacteria requires a different ferrisiderophore uptake mechanism, relatively simpler than described above. Internalization of iron involves siderophore binding protein (SBP), localized on the external membrane surface, one to two transmembrane protein permeases and an ATPase providing energy to the process. All those components form the ABC-type transporter.16,51 In case of S. aureus, ferrisiderophore uptake occurs by ABC transporters HtsABC and sirABC for SFN A and staphyloferrin B (SFN B), respectively. Both of those receptors contain heterodimeric permease components and ferri-SFNs transport is powered by the activity of FhuC ATPase.33,52–54 S. aureus can also utilize a range of different heterologous siderophores, i.e., catecholate ENT and “pseudosiderophore” hormones, including epinephrine and dopamine mediated by SstABCD transporter, or hydroxamate FC, FOX B, coprogen and ABN by FhuCBG, FHUD1 and FhuD2 receptors.51,55,56 Some SBPs may bind not only ferrisiderophores but also free siderophores with similar affinity. This phenomenon can increase the iron uptake ratio due to metal exchange mechanism between Fe(III)-siderophore complexes and aposiderophores facilitated by SBP.57,58 Yeasts and filamentous fungi use the unique siderophore iron transporters (SITs) localized in the membrane, absent in other eukaryotes and prokaryotes, to transfer ferrisiderophore systems into the cell. The active transport is powered by energy obtained from pumping ions through the membrane, which allows SITs to be classified as proton symporters.59 SIT-based systems are present and common in fungal species that produce siderophores (Aspergillus spp.), as well as in non-producer, for example Candida spp.59,60 Most fungi produce a variety of SITs, different by the means of siderophore specificity. It is predicted, that A. fumigatus may use up to ten different SITs61 in A. nidulans MirA and MirB transporters are responsible for ENT and triacetylfusarinine (TAFC) transport, respectively.61,62 In addition, the fungi cell wall may contain the facilitator of iron transport (Fit) proteins, appearing to trap ferrisiderophores and facilitate their uptake by SITs, as in case of Fit1, Fit2 and Fit3 of S. cerevisiae.63 Another mechanism used by fungi to siderophore mediated iron acquisition is the transport of Fe(III) ions from extracellular siderophore complexes to intracellular aposiderophores. Such a process can be observed in Rhodotula spp. for ferric-rhodotulate uptake.17,64 This phenomenon is unusual, as intracellular siderophores are mostly used to accumulate iron and not to assimilate it.65 Except some of Mucorales, producing ferritin-type protein mycoferritin for iron storage, the majority of fungi store iron in vacuoles and siderophore-based deposits.59,66

2.18.1.1.3

Implications of siderophores secretion for social relations between microorganisms and with the host

Apart ecological aspects, also social relationships in microbial community impact the adaptation of microorganisms choices to produce certain siderophores and their dynamics. These further translate to microbiota interactions with the host, both beneficial and harmful. In this section, we address selected principles illustrated by most prominent examples for bacterial and fungal pathogens, especially the ones relevant to the subject of the colonization of the host. The first puzzling question relates to why microorganisms relay on compounds that excreted into the environment might be diffused away and possibly lost? This physical process may have several advantages, i.e., an increase of the solubility of iron, its more homogenous distribution which further makes the nutrient more available in the local pool (especially important for the bacteria attached to surfaces and/or operating with membrane-embedded chelators).67 A range of factors can limit the loss of siderophores, like their own structure and physico-chemical properties (for example amphiphilic siderophores that can be embedded in the membranes) or the structure of the habitat (e.g., viscosity of the mucus of cystic fibrosis patients68 or a biofilm.69 Of importance, as in biofilms, but also other environments, microorganisms rarely function alone. Developing in groups, they can use siderophore– iron complexes produced by other bacterial/fungal species (xenosiderophores). This process of siderophore sharing, typically occurring between microorganisms operating via the same siderophore systems, may partially compensate the possible loss of the excreted and diffused compounds, and may be therefore regarded as cooperation between siderophore producers and nonproducers.67,70 Still, based on the same opportunity of siderophores being a public good, other social interactions may be developed in microbial communities. When cooperation is excessively used by non-producers, cheating scenario is implemented.71 There are many factors that regulate the balance between producers and non-producers, limit access of cheaters to produced siderophores, and contribute to the preservation of the cooperative siderophore production not to deplete their pool. Among others, these are changes in spatial structure, genetic architecture, changes in cell density.67 The studies are however limited to only a few bacterial species, like P. aeruginosa72 or E. coli73, and laboratory conditions. In case of P. aeruginosa, those evolutionary aspects have been studied for two of its siderophores: large, mixed chelating units PVD with dihydroxyquinoline and 6-14 L- or D-amino acids, sometimes cyclized, and smaller PCH containing two thiazolidine and hydroxyphenyl rings.27,28,74 These bacteria, as opportunistic antibiotic-resistant pathogens causing lung infections, especially in patients with cystic fibrosis (CF), provide an excellent research model to study interactions between microorganisms. Studies conducted on bacterial strains collected from the lungs of infected patients show that with the prolonged duration of infection, the production of PVD decreases, while the ability of bacteria to form biofilm does not change. This clearly indicates that P. aeruginosa loses some of its social characteristics over time.68 On the other hand, co-evolutionary studies show, that in the case of PVD-producers, they are capable of coexisting with non-producers for up to 150 generations. Coexistence is fostered by both the mutual adaptations of microorganisms to each other (i.e., by reducing the production of PVD, resulting in a reduction in the level of cooperation) and to the environment (by reducing mobility, which can potentially limit the mixing of producers and non-producers, and thus reduce social interactions and sharing of PVD).75 At the same time, studies by Weigert and Kümmerli have shown that significant amount of PVD can be captured by non-producers, even if there is no direct contact between the microcolonies. In such mixed cultures, non-producers have been shown to grow much faster than producers and their colonies grow into higher number of bacterial cells. Interestingly, non-producers can benefit from the

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presence of PVD-producing bacteria, whether they are in direct contact or not, and as the distance between cooperators and cheaters increases, producers cease to experience negative effects resulting in decreased growth.76 One of the mechanisms allowing for the reduction of PVD use by non-producers is the huge diversity of pyoverdine synthesis. This allows P. aeruginosa to switch to the production of PVD, the ferric complexes of which can only be recognized and taken up by cells of the same strain.68 One can easily imagine that such interactions dominate host-related habitats.68,77 As it was already mentioned, in intraspecific competition, the adversary siderophore producers can compete for iron tuning (i) the amount78 and (ii) dynamics79 of compounds being produced and excreted or (iii) their chelating power.80 In iron deficient conditions siderophore production is upregulated, creating an evolutionary advantage for non-producers. Under real conditions of infection, pathogens must coexist with other microorganisms as well, and inter-species competition also affects the cost/benefit ratio of siderophore production. In case of P. aeruginosa coexisting with S. aureus in the lungs of CF patients, the competition between both pathogens in iron limiting conditions results in upregulation of siderophore production in P. aeruginosa and, simultaneously, there is an increase in the number of non-producers in this bacterium. In iron supplemented environment this effect is not observed.78 Interesting interspecific interactions can also be observed in the case of the coexistence of P. aeruginosa and B. cenocepacia. In the early phase of the competition, P. aeruginosa increases production of PVD in iron deficient conditions, and this process is fine-tuned as a result of the level of competition against B. cenocepacia. It allows for accessing and binding ferric ions before the competitor, locking it away and allowing for “uptake reservation” for cells with matching receptor.79 Bacteria can also incorporate a mechanism called “the loner effect” to allow for coexisting of producers and cheaters together. In iron limited environments, when typically defection is favored, the presence of an independent “loner” strain, not involved in social interactions, allows for producers and non-producers cooperation. This phenomenon is based on the fact that the “loner” produces the mechanism of iron acquiring, e.g., through a siderophore, which allows it to outrun the non-producer, while the initial producer, due to more efficient iron uptake mechanisms, outcompete the “loner.” This specific “rock-paper-scissors” mechanism can only function if the sharing of the producer’s siderophore takes place on a local scale and the bacterial cells from the same strains are in close proximity to each other. The presence of a “loner” in the space between producer and non-producer cells leads to separation of the base siderophore from cheaters, reducing non-producer’s uptake of iron complexes, which in turn leads to direct profit for producers.80 Interestingly, for the competition with other species and with the host, the same microorganism can produce a range of structurally different siderophores. E. coli recovered from patients with urinary tract infections often synthesize different siderophore types spanning three chemical families: the prototypical, genetically conserved ENT, ABN and/or YBT (Fig. 1).81 This multiplicity raises the question of why bacteria would commit scarce cellular resources to synthesize chemically distinctive siderophores with a common recognized function? One reason emerged from the discovery that mammalian hosts produce siderocalin (also known as lipocalin 2, NGAL, or 24p3), a 25 kDa protein that tightly binds ferric-tris-catecholate complexes such as ferric Ent, which as a consequence, are intercepted and never reach the uptake system on the cell surface of the pathogen.82,83 Ferric ABN (as well as YBT) are not bound by siderocalin, and therefore are categorized as “stealth siderophores.” An additional rationale for synthesizing ABN is its better iron scavenging ability in low pH environments where protonation diminishes the ferric ion affinity of catecholate siderophores.84,85 Moreover, numerous publications over the years indicate that ABN is an important virulence determinant in the pathogens that produce it, and therefore it is an attractive target for antibiotic treatment. Moreover, the creation of distinct siderophores may render a producer more competitive as it will be less probable that a rival can exploit the same siderophore collection. The YBT system is encoded on a pathogenicity island and is functional in a number of different Gram-negative pathogens.86 In Yersinia pestis, the YBT system is essential in the early dermal/lymphatic stages of bubonic plague, irrelevant in the septicemic stage, and critical in pneumonic plague.87 Early research on the YBT system focused on Y. enterocolitica and Y. pestis. The mechanism for triggering and secretion of Yersinia YBT has not been elucidated. Once in the environment, YBT can remove iron from transferrin and lactoferrin under in vitro conditions and has a calculated proton-independent stability constant for Fe(III) of 4  1036 M
1 with three hydroxyl oxygen atoms, two thiazoline and one thiazolidine nitrogen atoms coordinated.32,88 However, it should be emphasized that the given constant was determined using protonation constants, the values of which were not correctly determined, but only estimated on the basis of literature data for individual functional groups present in the ligand structure.32 The crystal structure of Fe(III)-YBT has been characterized.89 Although uptake of Fe from YBT has been demonstrated, translocation of the siderophore into the bacterial cytoplasm has not.90 Uptake requires the TonB-dependent outer membrane receptor FyuA and two fused-function inner membrane permease-ATPases, YbtP and YbtQ.73 Where or how Fe is removed from the YBT siderophore also remains unresolved.77 Above discussed P. aeruginosa also uses the diversification of own siderophores to ensure growth and development under altered conditions. While PVD binds Fe(III) with higher affinity (logb ¼ 30.8),27,91 PCH forms complexes with Fe(III):PCH 1:2 molar ratio and with considerably lower affinity (logb120 ¼ 28.8).28 However, this siderophore is capable of displacing ferric ions from transferrin under acidic pH. Also, expression of fewer genes is required for its synthesis, so when extracellular iron levels decrease under micromolar concentration, P. aeruginosa produces PCH in first order.92 For biosynthesis of PCH, P. aeruginosa involves proteins encoded by PchDCBA and PchEFGHI operons utilizing salicylic acid and two cysteines93 and overall process is regulated by a transcriptional regulator PchR and PCH itself, acting as an effector molecule.94 Ferric PCH complexes are transported to the periplasm through specific FptA TBDT, which binding site consists mainly aromatic and hydrophobic residues. Interestingly, for the transporter to recognize the complex, it is enough to have only one PCH molecule bound, the remaining coordination sites may be occupied by another chelator, i.e., different siderophore.95

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PVD not only capture iron ions, but also regulates synthesis of itself, exotoxin A and the protease Prp, with the latter two being extracellular virulence factors.37 Ferric PVD is bound to TonB-dependent transporters FpvAI and FpvB, and then transited to periplasmic space.91 Next, in contrary to the most of Gram-negative bacteria, instead being internalized through IM to the cytoplasm, the complex is bound to FpvC or FpvF and reduced by FpvG, releasing both free Fe(II) and aposiderophore to traverse the IM. The free ferrous ion is transported to the cytoplasm by FPvdE transporter.96 Interestingly, depending on whether the infection is acute or chronic, P. aeruginosa alters preferred iron source in order to minimize the metabolic cost, and adjusts the mechanisms of ferric uptake.97 Another example of the necessity to develop many mechanisms of iron acquisition in order to increase the chances of survival and pathogenesis is Acinetobacter baumannii. To overcome iron-deficiency, A. baumannii upregulates genes involved in iron acquisition and secretes atypical siderophores: acinetobactin (ACN), fimsbactin (FBN) and baumannoferrins (BFNs).37,98 ACN is the native, mixed chelate siderophore of A. baumannii, containing hydroxamate, imidazole and catecholate groups. ACN exists in two isomeric forms: pACN containing isooxazolidinone ring, which undergoes isomerization to an oxazoline ring in ACN influenced by pH. Both isomers form ferric complexes with Fe(III):pACN/ACN 1:2 molar ratio and both promote A. baumannii growth.99 The selectivity of the pertinent OM receptor BauA for ferric complexes of pACN and ACN is not known, though the PBP BauB is able to bind FeACN and FepACN with comparable affinity.100 ACN synthesis is essential for A. baumannii pathogenesis, with biosynthetic protein BasD and OM receptor BauA being the virulence factors. Another unique ferric uptake system is based on mixed catechol-hydroxamate siderophores FBN A-F, with predominant FBNA and FBNB-F being most probably biosynthetic intermediates or byproducts.101 Contrary to ACN, FBN forms equimolar 1:1 ferric complexes due to single hydroxamate and two catecholate groups in the structure. The FBN biosynthetic cluster contains genes for an efflux pump FbsOQ, a TBDT FbsN, and FbsP, which is presumably a reductase secreted to the periplasm. After biosynthesis, FBN is exported in part by FbsOQ, and extracellular FeFBN is imported by FbsN to the periplasmic space, where FbsP reduces iron. Next, Fbm is released to efflux and ferrous iron for import via the ferrous transport system FeoABC.100 This model suggests, that FBN might be utilized by A. baumannii as the periplasmic siderophore and above-mentioned pACN/ACN as the cytoplasmic siderophores. It is in agreement with the fact that analogs of FBN conjugated with antibiotics with periplasmic targets, like i.e., vancomycin, show potent activity against A. baumannii while there is no antibacterial activity when the same siderophores are conjugated to antibiotics with cytoplasmic targets.101 As FBN gene cluster does not encode any PBP, accumulation in the periplasm may lead to the competition between FBN and pACN/ACN for BauB and competitively antagonize ferric ACN uptake.102 In 2015, another siderophore, structurally distinct from ACN and FBN produced by A. baumannii, BFN, was discovered. The lipophilic nature of the decenoic acid side chain in BFN structure suggest that this siderophore might be membrane associated.103 Both BFN A and B are chemically related to acinetoferrin from another opportunistic human pathogen Acinetobacter haemolyticus.104 Specific for BFN porine is BfnH.105 The role of BFNs in pathogenesis is currently undefined. Of importance, in order to gain a competitive growth advantage, some microorganisms develop receptors able to recognize and transport xenosiderophores.66,106–108 This scenario may be implemented by species operating on completely different siderophore systems, which under iron-depleted conditions without matching receptors have the metal ions locked away.109,110 This situation is especially difficult for siderophore non-producers, which have to rely on alternative iron assimilation systems, often less efficient than siderophore-based ones. In case of P. aeruginosa, there are more than 30 porins encoded in its genome, with many of them intended for the transport of xenosiderophores, for example ferric Ent in transmitted by PfeA and PirA, FOX B by FoxA, and FiuA, FemA and FecA transporters are meant for transport of FC, mycobactin and citrate ferric complexes, respectively.37 Also A. baumannii changes its OM protein composition in order to increase the uptake of its own and foreign siderophores.37,98 Besides BauA, BfnH and FbsN recognizing FeACN, FeBFN and FeFBN, respectively, encodes FepA transporter, allowing for assimilation of FeENT, though it lacks the ability to produce ENT. Bacteria of this genus are also able to acquire iron from monocatecholate compounds by PirA and PiuA. A. baumannii might also assimilate FeFC complexes, except FeFC A.37 In contrast to bacterial and some fungal pathogens (e.g., Candida albicans), Aspergillus fumigatus, the most common cause of invasive pulmonary aspergillosis (IPA),60 lacks specific uptake systems for host iron sources such as heme. To acquire iron, it uses primarily siderophore-mediated means, through production of hydroxamate siderophores FSC and TAFC, and reductive iron assimilation (RIA).111 While other siderophores such as FC can be taken up by A. fumigatus, elimination of siderophore production results in an avirulent strain, while a deficiency in siderophores attenuates virulence. In contrast, the inactivation of RIA imposes no consequences on A. fumigatus virulence. Another fungus, Rhizopus oryzae (family Mucorales) which causes mucormycosis, utilizes both RIA and siderophore-mediated uptake to acquire iron in the host; this ability is a key virulence determinant.112 Although it secretes rhizoferrin, a polycarboxylate siderophore, Rhizopus uses FOX B as a xenosiderophore.113 Clinical reports have shown that patients treated with FOX B are more susceptible to mucormycosis.114 C. albicans is a fungal commensal and pathogen that persistently associates with its mammalian hosts. Although it remains unclear if it synthesizes its own siderophores,115 it is able to exploit the use of exogenous FC-type siderophores, importing them via the outer membrane Sit1.19 Deletion of Sit1 abrogates C. albicans virulence in a reconstituted human epithelial infection model, but not in a bloodstream infection model.

2.18.1.1.4

Interactions of siderophores with other metal ions

Despite their preference for iron, siderophores have been proved to chelate numerous other metal ions with variable stoichiometry and stability, including those both with biologically relevant and with no known biological function (toxic to living cells). There is growing evidence that metals other than iron can trigger or suppress the production of siderophores by bacteria, thus connecting

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siderophores with the homeostasis of metals other than iron, however, the molecular mechanisms involved in these processes are not enough understood.116–118 Binding of certain non-iron, biologically relevant metals may well play a role in their delivery to bacterial cells to counter metal limitations imposed by the growth environment. On the other hand, the concept that siderophores play a role in heavy metal tolerance and protection of bacteria against metal toxicity has been recognized. This aspect might be of importance during nutritional immunity, when the host uses withdrawal of, or poisoning with, metals as defence approaches against microbial invaders.11,119 The YBT system is the most frequently carried, genetically non-conserved siderophore system in uropathogenic E. coli.120 Although its primary role in ferric iron binding, Cu(II)-YBT complexes were detected in urine from patients infected with YBTproducing E. coli, demonstrating the physiologic relevance of copper binding by YBT.121 Interestingly, the Cu(II)-YBT/Fe(III)-YBT molar ratio is above 1, suggesting that YBT binds host derived copper at least as extensively as Fe(III) during infections. Fe(III)–YBT competitively inhibited non-ferric YBT uptake, consistent with a shared transport mechanism. Cu(II)–YBT, however, did not competitively inhibit Fe(III)–YBT import and exhibited maximal import at low (0.1 mM) extracellular concentrations. The distinctive ability of FyuA to maintain Fe(III)–YBT import in the presence of excess Cu(II)–YBT was explained as adaptation of YBT system to copper-rich intracellular compartments, and the way to distinguish between nutritionally valuable iron and toxic copper.122 Indeed, it has been demonstrated that host immune cells, like macrophages, are able to increase copper concentration and expression of copper-transporting machinery in response to bacterial, mycobacterial,123,124 and fungal125 infections, most probably with the purpose to force pathogens to deal with copper excess. Copper resistance systems, including efflux proteins, oxidases and copper sequestering molecules, have been described in a number of invading pathogens.126 Siderophores seem to serve as one of these defensive mechanisms. Of importance, catecholate Ent produced by E. coli sensitize bacteria to copper toxicity through reduction of extracellular Cu(II) to more toxic Cu(I), and the formation of stable Cu(II)-YBT complex may prevent this process.78 Moreover, the Cu(II)-YBT complex has been shown to perform SOD-like activity (via Cu(II)-YBT þ O2
/ Cu(I)-YBT þ O2 and Cu(I)-YBT þ O2
þ 2Hþ / Cu(II)YBT þ H2O2 reactions),127 protecting pathogens from the respiratory burst within copper-containing phagosomes, during which superoxide anion is produced by the NADPH oxidase system.128 Analogous SOD-like behavior was evidenced for methanobactin,129 a small copper chelator found in aerobic methane oxidizing bacteria, or methanotrophs.130 The physiological function suggested for methanobactin is that of copper binding and transport, thus being a chalkophore because its peptidic nature resembles many iron siderophores (from greek ‘chalko’ for copper). Methanobactin binds Cu(I) with high affinity, through the oxazole nitrogen and thioamide sulfur atoms (logb ¼ 20.8),131 and is hypothesized to mediate copper acquisition from the environment via TonB-dependent mechanism.132 Although methanobactin is able to coordinate Cu(II), it gets quickly reduced to Cu(I) via an unknown mechanism.133 Very recently, the Henderson group demonstrated non-Fe(III) metal interactions with YBT and its TonB-dependent outer membrane Fe(III)-YBT importer FyuA. YBT was shown to form stable complexes with physiologically relevant metal ions like Cu(II), Ni(II), Co(III), and Cr(III), and all the complexes were imported in E. coli by FyuA. Outside of endosomal compartments where copper availability and YBT concentrations are low, the YBT system functions as a chalkophore for nutrient acquisition.134 Thus, virulence-associated YBT, regarded previously as a secondary Fe(III) scavenging siderophore, emerges as a triple-function defence metabolite engaged in copper nutritional passivation, a strategy of minimizing a metal ion’s toxicity (protecting pathogens from phagocytic killing via toxic metal binding and its exploitation for catalytic SOD-like activity) while preserving its nutritional availability (under low Cu(II) conditions). The nature of the interactions between the FyuA receptor and Cu(II)-YBT versus Fe(III)-YBT constitutes an intriguing problem,79 and a better mechanistic understanding of their transport is necessary to discern precisely how Cu(II)–YBT-specificity is achieved. Moreover, YBT’s ability to form stable, FyuA-importable complexes with physiologically relevant nickel, cobalt, and chromium ions was demonstrated and may suggest YBT supply trace nutrients for pathogens beyond iron. The lack of stable zinc and manganese YBT complexes are notable;89,134 however, a Dirp2 znu double mutant, unable to synthesize YBT and lacking functional ZnuABC zinc transporter, was unable to grow in 1 mM iron and 0.6 mM zinc supplemented medium. Growth was restored by complementation with the cloned irp2 gene, zinc supplementation to 2.5 mM or addition of purified apo-YBT. These findings suggest that YBT is involved in high affinity zinc acquisition and may serve as zincophore (vide infra). The Zn(II)-YBT complex is taken up into the cytoplasm by an inner-membrane protein, YbtX, a member of the Major Facilitator Superfamily.106 Therefore, YBT seems to be the first siderophore system with demonstrated involvement in iron and zinc acquisition by completely independent uptake systems. Explicit studies of the zinc coordination chemistry of YBT are lacking in the literature.106 In addition, the antibiotic micacocidin, produced by Pseudomonas spp. and R. solanacearum, with a chemical structure very similar to YBT, is able to bind iron, zinc and copper ions, and has been crystallized as a zinc complex.135,136 The crystal structure revealed a coordination geometry resembling that of ferric–YBT.28 One key difference was that the deprotonated secondary alcohol in the structure of the Fe(III) complex remains protonated in the Zn(II) structure, resulting in charge neutrality for both compounds. The Zn(II)-bound structure of micacocidin confirms a zinc-related non-classical function for YBT, and highlights the need for detailed investigations of the zinc coordination chemistry of the latter siderophore. The role of siderophores in the uptake of metals other than iron was studied also in P. aeruginosa. It is suggested, that distinctly low PCH affinity for ferric ions and the presence of additional potential binding sites beyond oxygen atoms may indicate its probable role in other metallic ions uptake. Indeed, PCH has been found to form stable complexes with biologically relevant Co(II), Ni(II), Cu(II), Zn(II) and Mo(VI)137 as well as Ga(III) or Tb(III).138 Furthermore, PCH complexes with nonferric metal ions can be transported by the PCH pathway in P. aeruginosa, but 23–35 times less effective than that for Fe(III) and this process occurred

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only for Co(II), Ni(II), Ga(III) and Mo(VI).137–139 Interestingly, nonferric metal complexes of PCH are recognized by TBDT FptA and their affinity for this receptor is similar to that of free PCH and Fe(III)-PCH complexes and binding of metal-PCH complexes to FptA inhibits Fe(III)-PCH uptake of around 25% or more. Surprisingly, despite the high receptor specificity for PCH, FptA appears to have low specificity for the metal ion coordinated to PCH. Moreover, it is only one PCH molecule present in the complex required to receptor recognition, the second chelator can be any different molecule and does not interact with FptA. In fact, only one molecule of PCH is a part of complex that is bound to FptA binding site, and none of its residues can interact with second chelator or metal ion present in complex.138 Additionally, it has been shown, that non-ferric metal ions are able to regulate siderophore production, regardless of iron concentration. In case of P. aeruginosa, in iron-limited conditions, addition of Cu(II), Ga(III), Ni(II) or Mn(II) stimulate PVD production and inhibit production of PCH.138,140 Although the cause of this phenomenon remains unclear, one of the explanations is the reduction in the concentration of free siderophores in the medium due to the formation of metal complexes, which may be sufficient to activate the synthesis process. Another potential mechanism for the stimulation of PVD synthesis may also be the metal-PCH interaction with the FpvA receptor on the cell surface, which may trigger a FpvR/PvdS signaling cascade.139 Precise metal specificities of other very recently discovered, nicotianamine-like metallophores staphylopine (STP)141–143 or pseudopaline144 are also of particular interest due to their presence in various pathogens. Apart iron, STP seems to transport nickel, cobalt, zinc and copper in vivo, while pseudopaline favors zinc and nickel over other metal ions. A putative metallophore activity was also ascribed to several other siderophores, like coelichelin, coelibactin,145 FOX B, coprogen, or desferrithiocin; unfortunately, transport of these complexes has not been demonstrated in vivo. However, the roles of these (often secondary) transporters await discovery.

2.18.1.1.5

Metallophore biomimetics

The difficulties in synthesis of structurally complicated natural siderophores have prompted researchers, to consider biomimetic compounds as structural probes for the understanding of microbial iron uptake processes.146–149 The biomimetic approach relies on the identification of minimal essential features of a complex biological system and their incorporation into the simplest possible synthetic molecular structure. The goal is to reproduce biological activity, rather than the detailed structure of a substrate. Rationally engineered synthetic siderophores, recognizable and utilizable by the same uptake system as the natural siderophores, provide a unique platform to introduce key structural factors affecting the recognition and species specificity. FC150 is a prototype of the natural hydroxamate siderophores. It is composed of a chiral hexapeptide ring template, nonsymmetrically extended by three identical arms that are terminated by hydroxamate moieties. FC, similarly to most siderophores, is not species-specific; it exhibits broad-spectrum activity and thus can be recognized by various types of microorganisms.151 Among the various proteins that control FC transport inside the cell, the outer membrane transporter is responsible for the selective recognition of FC.152 FhuA is a FC transporter from the outer membrane of E. coli and is among the few membrane transporters with a resolved crystal structure.153,154 Therefore, it is an attractive model for studies, allowing researchers to focus attention on the parameters important for transporter recognition. Our recent data obtained for tripodal analogs of FC indicates that recognition domains of specific transporters may differ considerably between different species of bacteria and fungi.65,155 A wide range of siderophore activity was observed in a series of FC biomimetic analogs varying in length and polarity of the amino acid chain separating between the tripodal scaffold and the pendent Fe(III) chelating hydroxamic acid groups (Fig. 3): from a rare case of a species specific growth promotor in P. putida, but not to

OH

HO

HO

O

O O

( )n

( )n

( )n

R'

R'

Reversed hydroxamic acids for Fe(III) chelation

N

N

N

NH

HN

R'

HN O

O O

Polipeptide chain replaces ring structure in FC R' allows introduction of peripheral groups and chirality

O O

O

R

Fig. 3

Schematic structure of FC biomimetic analogs.

Symmetric, tripodal structure R to be used for a functional group binding

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E. coli, to analogs with FC-like activity causing maximal growth promotion in both E. coli and P. putida. Other analogs fall in between, exhibiting some activity in both strains. It seems that the recognition domain in P. putida is more tolerant (possibly less crowded) than that of E. coli, what allows us to establish the minimal essential structural parameters for broad-spectrum recognition; an elusive target that siderophore researchers have been pursuing for years. The governing rules for designing analogs with multiple-uptake capabilities are: (i) to mimic the transporter with the most rigorous requirements, such as the FhuA transporter in E. coli; (ii) to develop a library of analogs in which each building group can be systematically and gradually modified until optimization is obtained, as was demonstrated through step-by-step structural adjustments of the modular system described in our work. It should be noted that minor modification may abolish broad-spectrum activity, so inducing the formation of narrowspectrum analogs utilizing practically the same platform. Although we have not identified an artificial analog targeting exclusively E. coli, we believe that understanding the unique requirements of FhuA gained during our recent work will facilitate its preparation, and we challenge it in our further studies. Moreover, we suggest that the interactions between FC and its transporter may serve as a prototype for other siderophores, especially the ones for which the detailed X-ray structures are lacking, like ABN. As discussed above, this siderophore with the mixed set of binding groups, namely hydroxamates and a-hydroxycarboxylates, is of particular importance mainly because it is secreted and utilized by pathogenic bacteria; it appears to be an important contributor to extracellular pathogenesis of E. coli strains causing septicaemia and urinary tract infections. Further work on biomimetics of other siderophores, like YBT, STP, should be performed in order to fully understand M(II)/(III)dligand thermodynamics and interactions of YBT complexes with the corresponding receptors.

2.18.1.1.6

Metal transport in vivo and lighting up metallophore–metal–metal transporter interactions and infection

The approach of using synthetic analogs of siderophores allows to incorporate, at will, external side groups (at the template apical site) for possible integration of “signaling” elements like fluorescent chromophores,156,157 surface-adhesive groups for solid surface based sensors,158,159 and recently, in a related Zn binding analog, an antibody targeting a highly specific-MMP receptor.160 Linking fluorescent probes to bioactive analogs, at sites not interfering with iron binding or receptor recognition, allows monitoring the time course, pathways, as well as the localization of complexes within the microbial cells. Earlier results demonstrated the usefulness of these fluorescent tools in studying the FC uptake system in bacterial Pseudomonas sp.161,162 and fungal Ustilago maydis,19 or FOX B uptake pathways in Y. enterocolitica.163 The fluorescent conjugate of the broad-range analog, studied by Nudelman et al., visualized siderophore destination in bacteria (periplasmic space) vs. fungi (cytosol) mapping new therapeutic targets.157 Divergent fluorescent behavior is displayed depending on the nature of the probe, enabling to choose the most suitable probe for specific process to be monitored. For example, fluorescence quenching upon iron(III) binding, regained upon ligand exchange with a competing chelator or via iron removal by the microbial cell, observed for 7-nitrobenz-2-oxa-1,3-diazole and fluorescein,19 allowed to follow the fate of the iron carriers after iron delivery. On the other hand, the lack of effect of iron binding, characteristic for lissamine rhodamine B,19 facilitated monitoring of the iron uptake pathways in fungal cells. Moreover, we have recently shown that Quantum Dots (QD) decorated with the most potent FC analog provided a tool for immobilization of FC-recognizing bacteria.157 Bacterial clusters formed around QDs may provide a platform for their selection and concentration. Siderophores can bind not only Fe(III) but also some other metals with high affinity, particularly Ga(III). As the coordination chemistry of Ga(III) is similar to that of Fe(III), Ga(III) can serve as an iron analog, additionally possessing anti-microbial properties by replacing iron from its binding site on siderophores.164 Moreover, 68Ga(III) can serve as tracer for imaging infections by means of Positron Emission Tomography (PET).165,166 Radiolabeling with 68Ga as a surrogate marker for Fe(III) allows to perform quantitative uptake studies and to characterize essential pharmacokinetic properties and biodistribution for selection of compounds.167,168 In numerous studies it was shown that a variety of siderophores such as PVD, FSC, TAFC, FC- and FOX-type displayed excellent 68 Ga-radiolabelling.169–172 We have documented this feature also in our recent data performed for FOX E biomimetic analogs.173,174 Moreover, recent findings175–177 documented high contrast imaging of fungal and bacterial pulmonary infection with 68Ga-TAFC and -FOX B in a rat model (achieved using Micro-PET/CT technology). The studies exhibited pronounced accumulation of 68Ga-TAFC in infected areas, thereby being highly specific with no accumulation in bacterial and C. albicans infections, sterile inflammations, or tumor cells.

2.18.1.2

Peptide/protein-based zincophores in the tug-of-war over zinc

Zinc is an essential nutrient for almost all living organisms. On average, 10% of bacterial and fungal proteomes consist of zincbinding proteins, with one fourth of them being involved in transcriptional regulation.178 A variety of zinc-binding proteins are crucial for the virulence of pathogens in humans, such as superoxide dismutases (SODs), responsible for the detoxification of ROS generated by host cells during host-pathogen interactions, or the zinc-binding metalloproteases involved in pathogen invasion, such as ADAM metalloproteinases or deuterolysin.7 Proper zinc homeostasis and acquisition are critical for the survival and virulence of pathogens, which have to overcome the nutritional immunity of the host. Zn(II) uptake is not trivial, with the total host tissue zinc concentration being in the micro to nanomolar range, and the concentration of free Zn(II) being as low as picomolar.12 Bacteria and fungi have adapted to the low zinc availability by expressing a variety of chemically versatile zincophores; in this chapter, we discuss the proteineous ones.

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2.18.1.2.1

Fungal zincophores

The tug of war over zinc(II) between the fungus and its host can be regarded as a potential target for new antifungal therapies which aim at the differences in human and fungal Zn(II) transport.179,180 This is a challenging task, since, because both fungi and humans are eukaryotes, they share many basic metabolic pathways (many more than with prokaryotic bacteria). Fungi rely on two classes of zinc transporters: (i) ZIPs (Zrt/Irt-like proteins), which transport zinc from outside the cell into the cytoplasm, and (ii) ZnTs (zinc transporters) which transport Zn(II) from the cytoplasm out of the cell and also from the cytoplasm into intracellular vesicles. The main difference in the human and fungal zinc transport is related to zincophores - small zinc binding proteins secreted by fungi. The most well-described zincophores are those found in Candida albicans and Aspergillus fumigatus. Candida albicans encodes two membrane zinc importers, which transport Zn(II) into the cell: the high affinity Zrt1 and the low affinity Zrt2. The expression of both of them is regulated by zinc availability,181 via the Zap1 regulator,182 and by pH via Rim101.183 Aspergillus fumigatus orthologues of Zrt1 and Zrt2 are zrfA and zrfB. Their expression is up-regulated by ZafA at low Zn(II) availability, and down-regulated by PacC at slightly basic pH, inducing the expression of an additional Zn(II) transporter, ZrfC.184,185 Low availability of Zn(II) ions and increasing pH, which further lowers the availability of zinc(II), triggers the expression of a different Zn(II) import systemdone base on zincophoresdin this case, small proteins/ large polypeptides, able to sequester zinc from the host tissue. The best described peptide-based zincophore is the 299 amino acid Pra1 (pH-regulated antigen 1), expressed by C. albicans; it is released from the hyphal surface into the host environment, from where it acquires Zn(II)dboth the free one and the one bound to other biological ligands. It then delivers the metal nutrient to the fungus via a physical interaction with the Zrt1 transporter (Fig. 4).186 The pra1 and zrt1 genes share the same promoter, and their deletion prevents C. albicans from using host zinc at low Zn(II) availability and slightly basic pH.186,187 In A. fumigatus, the Pra1 and Zrt1 orthologues are Aspf2 and ZrfA, respectively).186 Pra1 and Aspf2 share 43% of sequential identity, while Pra1 from C. albicans and Pra1 from B. dermatitidis188 share 37% of sequential identity (Fig. 5). The fungal transporters that interact with zincophores are also very well conserved, with Zrt1 and ZrfC sharing 48% identity. It is highly probable for both zincophores and Zn(II) transporters have similar zinc binding motifs.184 Pra1 zinc(II) binding sites were determined on peptide models, which were chosen based on three criteria: (i) were suspected to have a high affinity towards Zn(II) ions, containing numerous potential zinc binding sitesdHis, Cys and acidic residues; (ii) were located in unstructured regions of Pra1, based on the predictions of Phyre2 (a remote homology recognition technique, able to regularly generate reliable protein models)189 and (iii) were sequentially conserved in Pra1 (C. albicans) and Aspf2 (A. fumigatus).190 The thermodynamically most stable Zn(II) binding site turned out to be the C-terminal part of Pra1, SHQHTDSNPSATTDANSHCHTHADGEVHC (residues 281-299), which binds Zn(II) via His288, His290, His292, and His298).190 Most likely, this is also the region responsible for the interactions with the Zrt1 zinc transporter. A similar approach was used to point out the Zn(II) binding site on the N-terminal, extracellular region of the Zrt1 transporter, a similar experimental approach was useddthe potential sites were chosen from the extracellular, unstructured regions, conserved with respect to A. fumigatus. At physiological pH, the region able to bind Zn(II) with the highest affinity turned out to be KKCHFHAGVEHCVDDNNHDA (residues 151-170), with His156, His161 and His168 imidazoles and the Cys162 thiol being the Zn(II) coordination sites.191 Comparing the binding constants of the Zn(II) complexes of the above mentioned Pra1 zincophore and the Zrt1 transporter zinc binding regions, it can be stated that Zn(II) is very likely to be passed from the Pra1 C-terminal part to the Zrt1extracellular region

Fig 4 Scheme of C. albicans zincophore-based Zn(II) acquisition: (i) Pra1 is expressed at slightly alkaline pH and low Zn(II) concentration; (ii) Pra1 is secreted from the fungal cell surface; (iii) binds host cellular zinc (either free cytosolic or the one bound to host protein) and (iv) returns to the fungal cell, interacting with the Zrt1 transporter in order to deliver the bound Zn(II) ions. From Citiulo, F.; Jacobsen, I. D.; Miramon, P.; Schild, L.; Brunke, S.; Zipfel, P.; Brock, M.; Hube, B.; Wilson, D. PLoS Pathog 2012, 8.

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Fig. 5 The alignment of zincophore sequences from C. albicans Pra1, A. fumigatus Aspf2 and B. dermatitidis Pra1 (Uniprot accession numbers P87020, D3W9Z7 and T5BRU7, respectively).

(Fig. 6); at pH 7.4, about 85% of the available Zn(II) would be bound to the extracellular region of Zrt1, and only 15%dto Pra1, enabling efficient Zn(II) transport. From the functional point of view, the Pra1/Zrt1 zinc uptake system and the bacterial ABC transporter system are analogous to each other; both involve a secreted zinc scavenger (Pra1/ZnuA) and a transmembrane transporter (Zrt1/ZnuB), which is why the zinc(II) sequestration strategy seems to be well conserved in pathogens.179,192 In fungal species, the Pra1 zincophore orthologues were identified in about 85% of all sequenced fungi.193 Several species (e.g., C. glabrata, H. capsulatum, or C. neoformans) have lost the pra1 gene, most likely (i) because of their adaptation to acidic environmentsdthe Pra1 zincophore based system is functional not at acidic, but at physiological and at mildly basic pH,194 or (ii) due to the fact that the Pra1 zincophore could influence the pathogens’ interactions with host immunity, being a neutrophil ligand and thus triggering their migration. This is also observed in C. albicans Pra1 and A. fumigatus Aspf2, which interact with components

Fig. 6 Schematic representation of Zn(II) transfer between Pra1 and Zrt1. Reproduced with permission from Loboda, D.; Rowinska-Zyrek, M. Dalton Trans. 2018, 47, 2646–2654.

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Metallophores: How do human pathogens withdraw metal ions from the colonized host

of the complement cascade.195 Without the zincophore system, the fungus could avoid the unwanted attention of the immune system of the host. Understanding the fungal uptake of Zn(II) opens new therapeutic possibilities, based on antifungal agents bound to the C-terminal fragment of the Pra1 zincophore, which would deliver the drug to the fungus in a selective way. The most common antifungal agents are those that (i) interfere with the functions of the cell membrane (polienols or pirridone derivatives), (ii) are inhibitors of ergosterol biosynthesis (azoles, triazoles, alliloamines and phenylomorfolines), (iii) are nucleic acid synthesis inhibitors (cytosine analogs), and (iv) are cell wall synthesis inhibitors (echinocandins).196 Another reasonable option would be to search for new classes therapeutics, targeting metabolic pathways, which standard therapies do not aim at.

2.18.1.2.2

Bacterial zincophoresdSubstrate-binding proteins (SBPs)

Substrate-binding proteins (SBPs) and substrate-binding domains (SBDs) form a well-organized complex, mostly associated with metal ions transport and signal transduction. SBPs are found in a wide variety of protein-membrane complexes such as (i) prokaryotic ATP-binding cassette (ABC)-transporters,197–200 (ii) TRipartite ATP-independent periplasmic (TRAP)-transporters and the tripartite tricarboxylate transporters (TTTs),201–204 (iii) prokaryotic two-component regulatory systems,205 (iv) eukaryotic guanylate cyclase-atrial natriuretic peptide receptors,206,207 (v) G-protein coupled receptors (GPCRs) and ligand-gated ion channels.208 According to the structural fold and substrate specificities, recently updated classification reveals that eight different classes (“clusters A-H”) of SBPs can be distinguished.209–211 Generally, despite the low sequence similarity and diverse function, SBPs retain a conservative structure fold. The overall SBPs structure consists of two rigid a/b N- and C-terminal domains (NTD and CTD) linked by a flexible hinge region. Both NTD and CTD domains have a b-sheet core surrounded by a-helices.198 In the absence of substrate, the SBPs are flexible with the two separated domains rotating around the hinge212 and presents mainly the open conformation,198 while ligands binding between the two tightly packed domains at the interface, stabilizes a closed conformation of the proteins. This process has been called the “Venus Fly-trap” mechanism.213 2.18.1.2.2.1 ATP-binding cassette (ABC) transporters According to the SBPs classification, only one class (cluster A) is representative of Zn(II)-related chelators, which constitute extramembrane components of the ATP-binding cassette (ABC) transporters, high-affinity uptake systems present in bacteria. In Gramnegative bacteria, the SBP is located in the periplasm, while in Gram-positive bacteria, it is anchored to the membrane. The SBPs, included in cluster A, are identified by a single a-helix functioning as the hinge region that links the two globular, pseudo symmetrical a/b domains. The cluster A SBPs are all part of ABC import systems214 and bind transition metal ions (Cluster A-I proteins) and transition metal complexes (Cluster A-II proteins). ABC transporters have an extremely important function in metal homeostasis, withdrawing and delivering the substrate to the transmembrane domains (TDMs) of the transport system. Based on the transport direction, ABC transporters can be classified as exporters or importers. They are comprised of two nucleotide-binding domains (NBD) and two transmembrane domains (TMD)200,215 (Fig. 7A). ABC transporters have a common mechanism for substrate delivery across the membrane via an ATP-switch model (by binding and hydrolysing ATP). The transport process starts by the substrate binding to the TMDs, which cause structural changes to the NBDs, leading to ATP hydrolysis and closed dimer formation of the NBDs. Further, the closed NBD dimer initiates a major conformational change in the TMDs, inducing release of the substrate through a rotation of the TMDs. TMDs form an open conformation towards the extracellular space. In the end, ATP is hydrolysed to ADP and Pi with a simultaneous destabilization of the closed dimer conformation of NBDs. The return of TDMs and NBDs to their initial states is essential for another cycle to begin (Fig. 7B).216–219 ABC importers, found in prokaryotic organisms only, contain a fifth domain, which is part of the functional unit. This is an a-helical hinge region, which makes the overall structure relatively rigid and is probably responsible for the delivery of different substrates in a cluster A-I SBPs, which significantly differs from the common “Venus fly-trap” identified in SBPs with non-metal cognate substrate.210,211 In case of cluster A-I SBPs, only slight structural changes between apo (metal-free) and holo- (metalbound) forms are observed.220 The long a-helix hinge connecting the two a/b domains disfavours large-scale domain rotations, which provide only small changes in the spatial conformation near the metal-binding site. This may suggest that metal uptake and release is likely mediated by the structural rearrangements of secondary elements in metallophores. Understanding how exactly the metal ions are recruited and how these zincophores deliver them to their binging partners further remains a challenge. The overall mechanism of the substrate transport is based on the high affinity ligand binding by SBPs and delivering them to the translocator (the TMDs), where the ligand is released in the extracellular space upon hydrolysis of ATP in the NBDs.221,222 One of the key features in the structure of peptide-based metallophores is the presence of a flexible unstructured loop near the metal-binding site. The flexible loop is rich in histidine and acidic residues in the case of G-negative bacteria (group II) and short with or without histidine residues in G-positive bacteria (group I). Their role in zinc acquisition and transport has not been established yet, however, it seems to (i) act as a fishing net to capture zinc ions,223,224 (ii) be recognized by the membrane permease component,223,225 (iii) be involved in specific protein–protein interaction and potential Zn2þ acquisition from additional metallochaperones.226 2.18.1.2.2.2 ZnuA and ZinT periplasmic SBPs ZnuA periplasmic protein is a part of the Zn(II)-specific uptake system ZnuABC, which is responsible for the distribution and excretion of zinc(II) ions in Gram-negative bacteria.227,228 ZnuA is included in a group II of cluster A-I and contains a long N-terminal

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Fig. 7 (A) The schematic structure of an ATP-binding cassette (ABC) transporter. ABC transporter with two transmembrane domains (TMDs), TMD1 (green) and TMD2 (blue), and two nucleotide-binding domains (NBDs), NBD1 (orange) and NBD2 (yellow). (B) The ATP-switch model for the substrate transport across the membrane. (1) binding of the substrate (purple circle) to TDMs; (2) the structural changes of NBDs (orange and yellow), ATP binding (brown hexagon), closed dimer formation of the NBDs, conformational change in the TMDs, substrate translocation; (3) ATP hydrolysis and ADP (gray hexagon) and Pi (gray triangle) releasing; (4) destabilization of the closed dimer conformation, open dimer configuration for another cycle. Redrawn from Wu, C.; Chakrabarty, S.; Jin, M. H.; Liu, K.Y.; Xiao, Y.T. Int. J. Mol. Sci., 2019, 20, 18.

His-rich loop located near its high-affinity primary Zn(II) binding site. Its main role is Zn(II) uptake and delivery to the ZnuB component. The crystal structure of Zn(II)-ZnuA from Escherichia coli shows two metal anchoring sites.224,229,230 The first Zn(II) ion is coordinated to Glu59, His60, His143, and His207. The primary metal binding site, His143, is situated in the proximity of the His-rich loop (residues 116
138), located in the N-terminal domain and plays a significant role in Zn(II) acquisition.224,229–231. The histidine-rich loop has a role in the capture of zinc(II), which is then further delivered into other regions of the protein. The second metal binding site in ZnuA is situated approximately 12 Å from the main metal binding site, and its affinity for Zn(II) is slightly lower than that of the first site. The second zinc(II) ion is bound to His224, but other ligands have not been identified. The available crystal structures reveal that the binding residues and the surrounding environment are highly conserved among different bacterial species. After metal coordination, slight differences in the structure, in comparison to the apo form, are observed. The flexible Hisrich loop is shifted and shields the metal substrate from the solvent. The subtle rearrangements of the helices around the binding pocket, resulting in the flipping of one bound histidine, are also noticeable.224 It is worth to mention, that the relatively long Hisrich loop of ZnuA, besides the obvious His residues, provides additional donor atoms that may be involved in metal coordination (Glu and Asp residues), significantly increasing the protein’s efficiency of zinc acquisition.224,231,232 The molecular mechanism of zinc(II) acquisition and delivery is not completely understood yet, but the data available so far suggest that metal uptake and release is likely mediated by the structural rearrangements of secondary elements in SBPs proteins. Although the exact role of the flexible region rich in acidic and histidine residues near the metal binding site has not been established yet, most probably, the presence of the highly plastic loop can facilitate the release of the zinc ion.233 Several studies suggest that this unstructured loop may influence the stability of the protein scaffold,233 facilitating the capture and/or transfer of the zinc ions to the binding pocket between the two ZnuA domains and also play role as metal scavenger or chaperone during zinc-limited conditions.224,232,234

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In some bacterial species, the ZinT protein helps ZnuA to recruit Zn(II) from the periplasmic space,235 suggesting its function as an additional Zn(II)-chaperone for the ZnuABC system. ZinT is a lipocalin/calycin-like protein, consisting mainly of the b-barrel domain, which is structurally related to this protein family. The interaction of ZinT with the ZnuABC transporter has been demonstrated in Salmonella enterica serovar Typhimurium.235 ZinT is an essential component of the ZnuABC complex and contributes to metal transport by interacting with ZnuA in zinc uptake within the periplasmic space. Although the apo-form of ZinT could be secreted outside the cells to acquire zinc ions as well,236 the interaction between ZinT and ZnuA may help to increase zinc capture under zinc limiting conditions.235 It is worth to mention that ZnuA and ZinT do not interact when both are present in the apo form, but form a stable complex only when zinc is coordinated to their binding sites.235 The ZinT protein most probably has two Zn(II)-binding sites. The first is located in the calycin like-domain, containing three histidine residues in positions 167, 176 and 178,237,238 forming a well-known tetrahedral (3His, H2O) zinc complex.238 The second Zn(II) binding site, highly conserved in ZinT homologs from different bacterial species, contains a histidine-rich loop (HGHHXH) at the N-terminus,235,239 typical of zinc-specific SBPs, which has a high affinity for zinc(II) ions and involves 3 or 4His and a water molecule in its coordination sphere.240 The mechanism of zinc transport between ZinT and ZnuA is still under debate. Studies on Salmonella indicate that Zn(II) binding sites of both proteins are close to each other at the binary complex interface. It shows therefore that SeZinT and SeZnuA are involved in a structural interactions that allows Zn(II) to be transported from Zn(II)SeZinT to SeZnuA, likely via the flexible His-rich loop.226 The His-rich loop of ZnuA seems to be a leader in this interaction and has a higher metal affinity than the ZinT binding sites; this makes the flexible ZnuA loop a hypothetical metal transfer link between the two proteins. 2.18.1.2.2.3 AdcA and AdcAII zincophores Zinc is also essential for virulence and survival of S. pneumoniae, in which it is acquired and delivered by an ABC transporter, AdcCB, and two Zn(II)-specific cluster A-I SBPs, AdcA and AdcAII.210,241,242 Both proteins have a conserved structural fold, consisting of the N- and C-terminal (b/a)4 domains, connected by a a-helical linker.243,244 The metal binding site is located in the pocket, which is exposed on the protein surface. The first visible difference in the sequences of the AdcA and AdcAII proteins is the presence a C-terminal domain called AdcAC in the AdcA protein, which is linked to the N-terminal domain via an 11 amino acid linker. This domain is structurally related to ZinT protein from E. coli and Salmonella.226,237 The second difference is the presence of a region enriched with histidine and glutamate residues (His-rich loop), located at the N-terminal domain of AdcA (AdcAN). In vitro studies indicated a higher zinc affinity for the N-terminal domain compared to the AdcAC domain.241 The N-terminal domain exhibits high homology with ZnuA protein, suggesting that AdcAN may play the same or similar biological role in the process of zinc capture. At the same time, the similarity of the AdcAC domain with the ZinT protein may indicate its role in metal storage and/or metal delivery to the AdcBC transporter.245 The similarity to both proteins should also be seen in the metal binding sites of AdcA, however it has not been developed yet. The probable metal binding residues in the ZnuA-like and ZinT-like domains may correspond to the three conserved histidines typical of cluster A-I (a tetrahedral geometry with acidic residues as a fourth coordination ligand). Recent computational and structural studies reveal that both the N- and C-terminal domains of AdcA interact with the flexible His-rich loop between them, which allows to speculate that the Zn(II) bound to AdcAC is transported to AdcAN Zn(II) binding site via the dynamic mobility of the His-rich loop.246 The AdcAII protein contains only one metal binding site which corresponds to the canonical amino-terminal Zn2þ-binding domain.242 Interestingly, because of the lack of His-rich loop in AdcAII sequence, this protein requires the presence of polyhistidine triad proteins for the uptake of zinc in vivo,247 while AdcA can successfully acquire zinc in the absence of Pht proteins. The N-terminal domain of the polyhistidine triad protein PhtD of Streptococcus pneumoniae has been shown to transfer zinc to AdcAII in vitro,248 but the mechanism of transfer is still unknown. It is worth of mention here that two homologs of AdcAII, Lsp and Lmb, can also be an important part of the zinc uptake system. Both Lmb expressed by Streptococcus agalactiae and Lsp/Lbp expressed by Streptococcus pyogenes display laminin-binding properties and are responsible for adhesion and invasion during infections.249 The structural organization of both proteins reveals similarity to other ABC-type solute-binding proteins, where the related globular domains interact with each other to form a metal-binding pocket at the interface. Zinc(II) is tetrahedrally coordinated by three histidines and a glutamate from both domains.250 In the structure of both proteins, long, flexible and non-His, containing loop can also be distinguished. The absence of histidines in the loop suggests that it might have a more structural than a zinc acquisition role, in contrast to ZnuA. The mechanism of zinc transport by the two homologs has not been deduced yet, but taking into account that zinc plays an important role in the structure and function of laminin, there is a possibility that the interaction between Lmb and laminin may be mediated through Zn(II)251 to maintain the proper protein fold of Lmb.252 2.18.1.2.2.4 Mn(II)/Zn(II) ABC transporters AztABCD is a zinc transport system recently identified in a large group of diverse bacterial species. Instead of classical SBP AztC, the zinc-uptake system transport involves also a periplasmic metallochaperone (AztD), which is able to transfer zinc directly to AztC. Like ZnuA, the AztC protein belongs to the cluster 9 family of bacterial ABC transporters, and is classified as group III of cluster A-I SBPs. This protein differs from ZnuA and AdcA/AdcAII zincophores, by having an abbreviated His-rich loop (three His in the sequence) and containing only a few charged residues (Table 2).253–255 These three His residues in the loop appear to temporarily coordinate zinc. After metal binding to apo-form, the reorientations of loops and helices near the metal-binding site take place. AztC can acquire zinc either from solution or from the metallochaperone AztD. The mechanism of binding zinc from AztD and from

Metallophores: How do human pathogens withdraw metal ions from the colonized host Table 2

569

Sequences of cluster A-I SBPs highlighting the flexible loop region.

********* Flexible loop regions are shown in red (crystal structure is known); predicted flexible loop regions are shown in green (no crystal structure so far).

solution are most likely similar, which suggests that the His-loop from AztC can access the zinc site of AztD, coordinate the metal via the His residues, and deliver it to the high-affinity site of AztC.255 Another example of SBP, which shares several significant structural similarities with ZnuA and AztC, is TroA. TroA exhibits coordination geometries identical to that of AztC, but shows one essential differencedthe lack of a flexible loop close to its metal binding pocket. TroA is a cation-specific binding protein that belongs to the troABCDR system in Treponella and Streptococcus suis species. In vitro, TroA binds divalent metal ions such as zinc, manganese and iron and in vivo its biosynthesis depends on Zn2þ and Mn2þ concentrations in T. pallidum or Mn2þ and Fe2þ concentrations in T. denticola.256,257 As mentioned before, TroA does not contain an unstructured flexible loop, thus only one metal binding site, present in the inter-domain pocket, was identified. The crystal structure of Treponema pallidum TroA revealed four residuesdHis68, His133, His199 and Asp279 involved in Zn(II) coordination,258 while in vivo and in vitro data indicated that manganese was likely to be the natural ligand.228 Moreover, TroA does not show any detectable differences between its apo- and Zn(II)-forms.259 TroA, together with the previously described ZnuA, are simultaneously expressed in T. pallidum, but in contrast to ZnuA, TroA could also bind other metal ions (e.g., Mn(II) and Fe(II)), not only Zn(II).259 At this point, it is also worth to mention the pneumococcal surface adhesin A (PsaA), which is a surface-exposed lipoprotein of ABC permease encoded by the psaBCA locus found in all known serotypes of Streptococcus pneumoniae. This lipoprotein belongs also to the ABC-type transport protein complex and it is responsible for Mn(II) transport.260–263 Although Mn(II) is the high-affinity substrate for PsaA, this protein can also bind Zn(II) ions, however with a difference in affinity of nearly two orders of magnitude. Despite different metal ion affinities, structures of PsaA in complex with Mn(II) or Zn(II) do not reveal any significant differences. Zn(II) and Mn(II)-binding is favored by 2His and 2 acidic residues (His67, His139, Glu205 and Asp280). Interestingly, because Zn(II)-PsaA is significantly more thermodynamically stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible and it is not transported by the Mn(II) permease.264 The second Mn(II)/Zn(II) ABC transporter for S. aureus and S. pneumoniae is MntABC with MntC, acting as membrane-anchored solute-binding protein, which interacts with metal ions.261 The crystal structure of Mn(II)-MntC shows that MntC protein reveals the same metal binding mode as PsaA. MntC binds Zn(II) and Mn(II) with almost equal affinity and at the same binding site.265 Not surprisingly, the mechanism in which MntC binds Mn(II)/Zn(II) and delivers it to its respective permease has not been fully explained. 2.18.1.2.2.5 Other SBP-metallophores Bacterial peptide metallophores are probably not only specific for zinc, however other metals’ interactions are still insufficiently investigated and little functional information is available. Several other non-zinc-ABC importers and their orthologues have been identified in some bacterial species. The Ni(II) ABC-type importers belong to the same family as the peptide ATP-binding cassette ABC-importers, and according to the SBPs classification, are included in cluster C, which contains proteins that can bind a several types of different ligands (di- and oligopeptides, arginine, nickel and cellobiose). In this cluster, SBPs are larger than other ABC-type transporter receptors, due to the presence of an extra domain, possibly binding large ligands such as oligopeptides. The NikABCDE system of Escherichia coli belongs to the nickel/peptide/opine ABC transporter family and consists of the periplasmic binding protein NikA, two integral membrane components (NikB and NikC), and two ATPases (NikD and NikE).266 The related Ni(II)-ABC system was also found in other pathogens such as Yersinia pseudotuberculosis,267 Brucella suis,268 Staphylococcus aureus.269 The mechanism of selective high-affinity binding of Ni(II) and transport by NikABCDE system still remains unclear. The only Ni-SBP that has been structurally characterized so far is NikA from E. coli (EcNikA).270 Like other SBPs, EcNikA consists of two different domains connected by a hinge region, located close to the ligand binding site. The crystal structure of NikA protein indicates that it does not bind Ni(II) directly, because the only direct contact between the protein and the metal ion is formed via one histidine residue (His416),271 suggesting that protein may form a complex with a metallophore, required to complete the coordination sphere of the metal ion.270,272,273 The putative Ni-binding site of NikA suggests that Ni(II) could be bound in a hydrophobic pocket, rich in aromatic and arginine residues,270 and two models of “auxiliary nickelophores” have been proposed so far (a tricarboxylated molecule273 or two free histidines274,275). Heddle et al.270 showed that NikA behaves as a “classical” periplasmic binding protein. In contrast to other binding proteins, the ligand remains accessible to the solvent and is not completely enclosed, what may suggest their role in further uptake and delivery of metal ions.

2.18.2

Conclusions

To summarize, the numerous metallophore examples presented in this chapter point to their central role in iron, zinc, and other metal ions, homeostasis, and the evolution of important human pathogens.

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As it was highlighted, the properties and biological activity of siderophores are dictated by their structure, chirality, and the extent by which the shape of Fe(III)-siderophore complex fits the binding sites of specific receptor proteins inside the membrane.276 Because of the large variety of siderophores and their corresponding receptors, as well as discoveries of new roles of siderophores previously regarded as secondary chelators, the process of microbial siderophore recognition is not yet fully understood. For YBT, for example, the nature of the interactions between the FyuA receptor and Cu(II)-YBT versus Fe(III)-YBT constitute an intriguing problem.89 The findings support the idea of additional roles of siderophores in non-iron metal acquisition, and defence against metal toxicity. Although TonB-dependent transporters have been the subject of multiple structural analyzes, a better mechanistic understanding of their transport is necessary to discern precisely how metal ion–siderophore specificity is achieved. Moreover, explicit studies on the coordination chemistry of “old chelators with a new roles” (represented here by YBT, but also ABN and many other siderophore-type compounds), and their binding affinity towards Zn(II), Cu(II) and Cu(I) (both forms possibly present within the phagosome), as well as other cations, appear to be lacking in the literature. In this context, further studies are needed to clarify the role of these chelators. Zinc is another key player in host-pathogen interactions. With a variety of sophisticated acquisition systems, it also remains a desirable target for the tug-of-war between the host and its fungal or bacterial pathogens. Peptide based zincophores, Zn(II) importers and transport systems are a clever way of securing appropriate levels of this metal for bacteria and fungi in order to overcome extreme zinc(II) limitation within the host environment. The multiplicity of siderophore and zincophore transfer systems further underline the importance of these nutrients for the survival of bacteria and fungi. Keeping in mind, that such transport systems are crucial for microbial virulence, and are absent or differ significantly from the ferric and zinc transport in the host cells, they are tempting targets for microbial imaging and future antimicrobial therapies. Studies based on fluorescent/radiolabeled siderophores and their analogs are an important contribution towards proper and effective diagnostics of bacterial/fungal diseases. They could also allow identifying critical microbial compartments in which metallophores accumulate and thus illuminate key targets for specific drugs.

Acknowledgments Financial support by the Polish National Science Centre (UMO-2017/26/A/ST5/00363) is gratefully acknowledged.

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Dalton Trans. 2020, 49, 9393–9403. Plumptre, C. D.; Eijkelkamp, B. A.; Morey, J. R.; Behr, F.; Counago, R. M.; Ogunniyi, A. D.; Kobe, B.; O’Mara, M. L.; Paton, J. C.; McDevitt, C. A. Mol. Microbiol. 2014, 91, 834–851. Loisel, E.; Jacquamet, L.; Serre, L.; Bauvois, C.; Ferrer, J. L.; Vernet, T.; Di Guilmi, A. M.; Durmort, C. J. Mol. Biol. 2008, 381, 594–606. Woo, J. S.; Zeltina, A.; Goetz, B. A.; Locher, K. P. Nat. Struct. Mol. Biol. 2012, 19, 1310–1315. Korkhov, V. M.; Mireku, S. A.; Hvorup, R. N.; Locher, K. P. FEBS Lett. 2012, 586, 972–976. Bayle, L.; Chimalapati, S.; Schoehn, G.; Brown, J.; Vernet, T.; Durmort, C. Mol. Microbiol. 2011, 82, 904–916. Luo, Z. Y.; Morey, J. R.; Deplazes, E.; Motygullina, A.; Tan, A.; Ganio, K.; Neville, S. L.; Eleftheriadis, N.; Isselstein, M.; Pederick, V. G.; Paton, J. C.; Cordes, T.; Harmer, J. R.; Kobe, B.; McDevitt, C. A. MBio 2021, 12. Plumptre, C. D.; Hughes, C. E.; Harvey, R. M.; Eijkelkamp, B. A.; McDevitt, C. A.; Paton, J. C. Infect. Immun. 2014, 82, 4315–4324. Bersch, B.; Bougault, C.; Roux, L.; Favier, A.; Vernet, T.; Durmont, C. PLoS One 2013, 8, e81168. Moulin, P.; Patron, K.; Cano, C.; Zorgani, M. A.; Camiade, E.; Borezee-Durant, E.; Rosenau, A.; Mereghetti, L.; Hiron, A. J. Bacteriol. 2016, 198, 3265–3277. Ragunathan, P.; Spellerberg, B.; Ponnuraj, K. Acta Crystallogr. Sect. D Struct. Biol. 2009, 65, 1262–1269. Sridharan, U.; Ragunathan, P.; Spellerberg, B.; Ponnuraj, K. J. Biomol. Struct. Dyn. 2019, 37, 714–725. Ragunathan, P.; Sridaran, D.; Weigel, A.; Shabayek, S.; Spellerberg, B.; Ponnuraj, K. PLoS One 2013, 8. Handali, M.; Roychowdhury, H.; Neupane, D. P.; Yukl, E. T. J. Biol. Chem. 2015, 290, 29984–29992. Handali, M.; Neupane, D. P.; Roychowdhury, H.; Yukl, E. T. J. Biol. Chem. 2015, 290, 11878–11889. Neupane, D. P.; Avalos, D.; Fullam, S.; Roychowdhury, H.; Yukl, E. T. J. Biol. Chem. 2017, 292, 17496–17505. Schreur, P. J. W.; Rebel, J. M. J.; Smits, M. A.; van Putten, J. P. M.; Smith, H. E. J. Bacteriol. 2011, 193, 5073–5080. Saraithong, P.; Goetting-Minesky, M. P.; Durbin, P. M.; Olson, S. W.; Gherardini, F. C.; Fenno, J. C. J. Bacteriol. 2020, 202. Lee, Y. H.; Deka, R. K.; Norgard, M. V.; Radolf, J. D.; Hasemann, C. A. Nat. Struct. Biol. 1999, 6, 628–633. Desrosiers, D. C.; Sun, Y. C.; Zaidi, A. A.; Eggers, C. H.; Cox, D. L.; Radolf, J. D. Mol. Microbiol. 2007, 65, 137–152. Berry, A. M.; Paton, J. C. Infect. Immun. 1996, 64, 5255–5262. Dintilhac, A.; Alloing, G.; Granadel, C.; Claverys, J. P. Mol. Microbiol. 1997, 25, 727–739. Lawrence, M. C.; Pilling, P. A.; Epa, V. C.; Berry, A. M.; Ogunniyi, A. D.; Paton, J. C. Structure 1998, 6, 1553–1561. McAllister, L. J.; Tseng, H. J.; Ogunniyi, A. D.; Jennings, M. P.; McEwan, A. G.; Paton, J. C. Mol. Microbiol. 2004, 53, 889–901. McDevitt, C. A.; Ogunniyi, A. D.; Valkov, E.; Lawrence, M. C.; Kobe, B.; McEwan, A. G.; Paton, J. C. PLoS Pathog. 2011, 7. Lim, K. H. L.; Jones, C. E.; vanden Hoven, R. N.; Edwards, J. L.; Falsetta, M. L.; Apicella, M. A.; Jennings, M. P.; McEwan, A. G. Infect. Immun. 2008, 76, 3569–3576. Navarro, C.; Wu, L. F.; Mandrandberthelot, M. A. Mol. Microbiol. 1993, 9, 1181–1191. Sebbane, F.; Mandrand-Berthelot, M. A.; Simonet, M. J. Bacteriol. 2002, 184, 5706–5713. Jubier-Maurin, V.; Rodrigue, A.; Ouahrani-Bettache, S.; Layssac, M.; Mandrand-Berthelot, M. A.; Kohler, S.; Liautard, J. P. J. Bacteriol. 2001, 183, 426–434. Hiron, A.; Posteraro, B.; Carriere, M.; Remy, L.; Delporte, C.; La Sorda, M.; Sanguinetti, M.; Juillard, V.; Borezee-Durant, E. Mol. Microbiol. 2010, 77, 1246–1260. Heddle, J.; Scott, D. J.; Unzai, S.; Park, S. Y.; Tame, J. R. H. J. Biol. Chem. 2003, 278, 50322–50329. Cavazza, C.; Martin, L.; Laffly, E.; Lebrette, H.; Cherrier, M. V.; Zeppieri, L.; Richaud, P.; Carriere, M.; Fontecilla-Camps, J. C. FEBS Lett. 2011, 585, 711–715. Cherrier, M. V.; Martin, L.; Cavazza, C.; Jacquamet, L.; Lemaire, D.; Gaillard, J.; Fontecilla-Camps, J. C. J. Am. Chem. 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2.19

The role of d-block metal ions in neurodegenerative diseases

Yanahi Posadasa,b,*, Vı´ctor E. Lo´pez-Guerreroa,c,*, Trinidad Arcos-Lo´pezd, Richard I. Saylera, Carolina Sa´nchez-Lo´peza, Jose´ Segoviac, Claudia Perez-Cruzb, and Liliana Quintanara, a Department of Chemistry, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; b Department of Pharmacology, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; c Department of Physiology, Biophysics and Neurosciences, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico; and d National Institute for Genomic Medicine (INMEGEN), Mexico City, Mexico © 2023 Elsevier Ltd. All rights reserved.

2.19.1 2.19.2 2.19.2.1 2.19.2.1.1 2.19.2.1.2 2.19.2.1.3 2.19.2.1.4 2.19.2.1.5 2.19.2.1.6 2.19.3 2.19.3.1 2.19.3.1.1 2.19.3.1.2 2.19.3.1.3 2.19.3.2 2.19.3.2.1 2.19.3.2.2 2.19.3.2.3 2.19.3.2.4 2.19.3.2.5 2.19.3.2.6 2.19.3.2.7 2.19.3.3 2.19.3.3.1 2.19.3.3.2 2.19.3.3.3 2.19.3.3.4 2.19.3.4 2.19.3.5 2.19.4 2.19.4.1 2.19.4.1.1 2.19.4.2 2.19.4.2.1 2.19.4.2.2 2.19.4.2.3 2.19.4.2.4 2.19.4.3 2.19.5 2.19.5.1 2.19.5.2 2.19.5.3 2.19.5.4 2.19.5.5 2.19.6 Acknowledgements References

*

Introduction Prion diseases The prion protein Copper binding to prion protein and its biological implications Zinc binding to prion protein and its biological implications Manganese binding to prion protein and its biological implications Proteolytic processing of cellular prion protein and its impact in metal-binding properties Metal ions and aggregation of the prion protein Metal ions as a therapeutic target in prion diseases Alzheimer’s disease The amyloid precursor protein Copper binding to the amyloid precursor protein and its biological implications Zinc binding properties to the amyloid precursor protein and its biological implications Metal ions and the proteolytic processing of amyloid precursor protein The amyloid-b peptide Copper binding properties to the amyloid-b peptide and its biological implications Zinc binding properties to the amyloid-b peptide and its biological implications Iron binding to the amyloid-b peptide and its biological implications N-truncation of amyloid-b and its impact in metal-binding properties Ab (4-x) and Ab (11-x) fragments Ab (p3-x) and Ab (p11-x) fragments Metal ions and Ab aggregation and its pathological implications The tau protein Copper-binding properties of tau protein and its biological implications Zinc-binding properties of tau protein, aggregation, and toxicity Iron and tau hyperphosphorylation Metal ions and tau kinases The prion protein in Alzheimer’s disease Metal ions as therapeutic target for Alzheimer’s disease Parkinson’s disease DJ-1 protein Metal-binding properties of DJ-1 protein a-Synuclein Calcium-binding properties of a-synuclein and its biological implications Iron-binding properties of a-synuclein and its biological implications Copper-binding properties of a-synuclein and its biological implications Posttranslational modification a-synuclein and its impact on its metal-binding properties Metal ions as therapeutic targets in Parkinson’s disease Huntington’s disease Copper in Huntington’s disease Iron in Huntington’s disease Manganese in Huntington’s disease Zinc in Huntington’s disease Metal ions as therapeutic targets in Huntington’s disease Concluding remarks

578 579 581 584 586 587 587 588 588 590 590 591 593 593 595 596 598 598 598 600 600 601 601 601 602 603 603 604 605 605 606 606 607 608 609 609 610 610 611 611 612 612 613 613 613 614 614

These authors contributed equally to this work.

Comprehensive Inorganic Chemistry III, Volume 2

https://doi.org/10.1016/B978-0-12-823144-9.00115-1

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The role of d-block metal ions in neurodegenerative diseases

Abbreviations 8HQ 8-hydroxyquinoline Ac Acetylated AcD Acidic domain AD Alzheimer’s disease ADAM A-disintegrin-and-metalloprotease AICD APP intracellular domain AMPAR a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors APLP Amyloid precursor-like protein APP Amyloid precursor protein AS Alpha-synuclein ATCUN Amino-terminal copper and nickel binding motif ATP7B Copper-transporting P-type ATPase Ab Amyloid-beta peptide BACE1 b-secretase BBB Brain-blood barrier Bca Bicinchoninic acid Bcs Bathocuproine disulfonate BDNF Brain derived neurotrophic factor CCS Copper-chaperone for superoxide dismutase CD Circular dichroism CDK5 Cyclin-dependent kinase-5 CJD Creutzfeldt-Jakob disease CN Caudate nucleus CNS Central nervous system Cryo-EM Cryogenic electronic microscopy Ctr1 Copper transporter 1 CuBD Copper-binding domain CV Cyclic voltammetry DEER Pulsed double electron-electron resonance DFT Density functional theory DMT-1 Divalent metal transporter 1 ENDOR Electron-nuclear double resonance EPR Electronic paramagnetic resonance ESEEM Electron spin echo envelope modulation ESI-MS Electron-spray ionization mass spectrometry EXAFS Extended X-ray absorption fine structure FRET Fluorescence resonance energy transfer Fs Ferrene S FTIR Fourier-transformed infrared spectroscopy Fz Ferrozine GFLD Growth factor-like domain GP Globus pallidus GPI Glycosylphosphatidylinositol GSK-3 Glycogen synthase kinase-3 HAP1 Huntingtin-associated protein 1 hAb Human amyloid beta peptide HD Huntington’s disease HPLC High-performance liquid chromatography Hst5 Histatin-5 Htt Huntingtin

The role of d-block metal ions in neurodegenerative diseases

HYSCORE Hyperfine sublevel correlation IDE Insulin-degrading enzyme IMAC Immobilized-metal-affinity chromatography IRE Iron response element IRP Iron regulatory protein ITC Isothermal titration calorimetry KA Kainate LPR-1 Low-density lipoprotein receptor-related protein 1 MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight MAP Microtubule associated proteins MS/MS Tandem mass spectrometry MSA Multiple system atrophy MSN Medium spiny neurons MT-3 Metallothionein-3 MTBR Microtubule binding region Mt-Htt Mutant huntingtin (with extended polyglutamine region) NAC Non-amyloid component NEP Neprilysin NHE Normal hydrogen electrode NMDAR N-methyl-D-aspartate receptor NMR Nuclear magnetic resonance NO Nitric oxide PD Parkinson’s disease pE Pyroglutamate PolyQ Extended polyglutamine PRD Proline-rich domain PrPC Cellular prion protein PrPSc Scrapie prion protein PSEN Presenilin PTMs Post-translational modifications ROS Reactive oxygen species rPrP Recombinant prion protein SOD Superoxide dismutase SPR Surface plasmon resonance Wt-Htt Wild-type huntingtin (with normal polyglutamine region) XAS X-ray absorption spectroscopy XAS-EC X-ray absorption spectroscopy under in situ electrochemical control ZFPs Zinc finger proteins ZIP Zrt-/Irt-like proteins ZnT3 Zinc-transporter 3 l

Abstract d-block metal ions (Cu1þ, Cu2þ, Fe2þ, Fe3þ, Mn2þ, and Zn2þ) are essential for the brain; however, disruption of metal ion homeostasis is closely linked to neurodegenerative diseases. Interestingly, many of the proteins that play a key role in neurodegeneration can bind metal ions and, in some cases, impact metal homeostasis. This chapter reviews the role of d-block metal ions in different neurodegenerative diseases, including Prion, Alzheimer’s, Parkinson’s, and Huntington’s diseases. For each pathology, the metal-binding properties of the proteins involved are discussed, attempting to link the bioinorganic chemistry of these proteins with the role of metal ions in function and disease. Therapeutic approaches that target metal-protein interactions for each disease are also discussed.

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2.19.1

The role of d-block metal ions in neurodegenerative diseases

Introduction

In the brain, alkali/alkaline-earth metal ions, such as Naþ, Kþ, Ca2þ, and Mg2þ, are necessary for the establishment of membrane potentials, as well as polarization and depolarization of neurons.1 On the other hand, d-block metal ions (Cu1þ, Cu2þ, Fe2þ, Fe3þ, Mn2þ, and Zn2þ) mainly function as cofactors of enzymes that are essential for the function of the central nervous system (CNS). Zn2þ can also play structural roles, and both Cu2þ and Zn2þ can modulate neurotransmitter receptors.2–4 The specific chemical properties of alkali/alkaline-earth and d-block metal ions are exploited for the different roles they play in the brain.3,5,6 Naþ, Kþ, Ca2þ, and Mg2þ are highly soluble in water, facilitating their role as electrical signals, as they are able to move rapidly in an aqueous environment.3,7,8 In contrast, d-block metal ions display lower solubility in water; hence, their trafficking is controlled by a network of metal-binding proteins.3,7,8 Interestingly, during neuronal transmission, Zn2þ and Cu2þ are released into the synapse,3,9–22 where they can interact with a variety of metal-binding proteins (Fig. 1).23 The transfer of d-block metal ions between proteins is dictated by metal ion availability, binding affinities, and the specific coordination preferences of each metal ion.

Fig. 1 Metal-binding proteins at the synapse. The scheme shows a tripartite synapse where all metal-binding proteins involved in neurodegenerative diseases reviewed in this chapter are included. In the presynaptic neuron, a-synuclein (a-syn) is contained in vesicles and is represented as a purple vibrio-shaped structure. Huntingtin (Htt) is shown as two merged blue and orange circles that surround microtubules in presynaptic and postsynaptic neurons. As a result of synaptic activity, the presynaptic neuron releases neurotransmitters (green and purple tips) which in some cases can be coreleased with zinc ions (gray spheres). In the synaptic cleft, other metals such as iron and manganese are represented as red and black spheres, respectively. On the other hand, copper ions (represented as blue spheres) are released from the presynaptic neuron as a consequence of the activation of NMDAR activation. ATP7A fills vesicles with copper, which is released to the synaptic cleft through the secretory pathway. Metal ions can interact in the synaptic cleft with different metal-binding proteins: in the extracellular space, amyloid precursor protein (APP), the amyloid-beta peptide (Ab), the cellular prion protein (PrPC); and in the cytosol, Htt, a-syn, and tau protein (Tau), the latter represented as an orange structure anchored to the microtubules in the postsynaptic neuron.

The role of d-block metal ions in neurodegenerative diseases

579

Although redox-active metal ions such as iron, copper, and manganese are required for their catalytic roles, they may also engage in uncontrolled redox reactions that lead to oxidative stress.23 Alteration of Zn2þ homeostasis can also damage brain tissue, even though this metal is not redox-active.24 Hence, an intricate network of proteins is involved in metal ion uptake, trafficking, distribution, storage, and export to assure the correct homeostasis of d-block metals (for review, see 23,25–27). In particular, metal ion trafficking to the brain requires crossing the blood-brain barrier (BBB).23 Thus, altered metal homeostasis in the blood may impact the ability of a metal ion to cross the BBB. An example of the latter is observed in patients with Alzheimer’s disease (AD), where the copper speciation is altered: the concentration of copper bound to the multicopper oxidase human ceruloplasmin (Cp) decreases, while the non-Cp bound copper levels are increased.28–33 Interestingly, brain tissue from AD patients displays a decrease in copper levels, supporting a possible relationship between metal speciation in blood and brain metal content (Table 1). Other neurodegenerative diseases such as Prion and Parkinson show alterations in blood and brain metal levels; however, changes in metal speciation remain unclear (Table 1). Metal ion distribution and abundance in the brain is region-specific. Iron is the most abundant d-block metal in the brain, followed by zinc in second place, and copper in third place.73 The trafficking of these essential metal ions to the brain has been reviewed in 23,25–27. High levels of iron are found in the substantia nigra (SN), hippocampus, striatum, the interpeduncular nuclei, and myelin.73,74 Iron acts as cofactor of several important enzymes involved in respiration and neurotransmitter production, and it is contained in metalloenzymes as either heme, iron-sulfur or non-heme iron active sites. On the other hand, Zn2þ is most abundant in the cerebral cortex and hippocampus.73 While most of it is contained in metalloenzymes, synaptic vesicles accumulate up to 8– 18% of total Zn2þ.21,75,76 using the transporter ZnT3 to load this metal ion (Fig. 1).77,78 Vesicular Zn2þ is released from presynaptic terminals during neuronal stimulation, along with glutamate.10–22 Copper is most abundant at the choroid plexus, the subventricular zone, 61,62,73 the striatum, frontal cortex, olfactory bulb, hippocampus, cerebellum, and hypothalamic nuclei.61,73 Copper is an essential cofactor of enzymes involved in cellular respiration, neurotransmitter biosynthesis, neuropeptide maturation, and radical detoxification. In the hippocampus, activation of synaptic NMDA receptors by glutamate leads to release of copper into the synapse,9 where it can be bound by several proteins and peptides, including the cellular prion protein (PrPC), the amyloid precursor protein (APP), and the amyloid-b peptide (Fig. 1).23 At the synapse, both, zinc and copper can modulate the activity of several ionotropic receptors, such as NMDA and AMPA/Kainate receptors.63–69 Hence, d-block metal ions are not only important as essential active sites in metalloenzymes, but they also play key roles in neurotransmission and cell signaling in the brain.70 While studying the role of d-block metal ions in neurodegenerative diseases, it is important to keep in mind their modulatory signaling roles in the brain. Manganese is abundant in iron-rich regions, including basal ganglia (SN, caudate, putamen, globus pallidus, and subthalamic nuclei) and the visual cortex.71 Although manganese is less abundant than other d-block metal ions in the brain,72 the catalytic function of this metal ion cannot be substituted by iron or copper. Manganese is cofactor of enzymes involved in energy metabolism, radical detoxification, immune function, development, and neurotransmitter homeostasis. An example of the latter is glutamine synthetase, an enzyme that catalyzes the conversion of glutamate into glutamine in astrocytes.79,80 Alterations in manganese homeostasis have been associated with prion diseases,34,81 while exposure to this metal ion causes manganism, a neurodegenerative disorder with PD-like symptoms.82 Overall, several studies show that distribution of metal ions such as copper, iron, zinc, and manganese is altered in AD, PD, prion diseases and Huntington’s disease (HD) (Table 1), although metal speciation remains unclear. Clearly, disruption of metal ion homeostasis in the brain is closely linked to neurodegenerative diseases (Table 1). Interestingly, many of the proteins associated with the onset of neurodegeneration can bind metal ions and impact metal homeostasis in some cases. In this chapter, the metal-binding properties of the main proteins involved in different neurodegenerative diseasesdincluding prion diseases, AD, PD, and HDdwill be discussed, attempting to link the bioinorganic chemistry of these proteins to the role of metal ions in these diseases. Therapeutic approaches that target metal-protein interactions for each disease are also discussed.

2.19.2

Prion diseases

Prion diseases are a set of rare and fatal neurodegenerative disorders that affect humans and other mammalian species (such as sheep, goats, cattle, deer, and moose).83,84 Human prion diseases include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia, Gerstmann-Straussler-Scheinker disease, and Kuru; while bovine spongiform encephalopathy, transmissible mink encephalopathy, and scrapie are some examples in non-human mammals.84 Clinical features of prion diseases are highly heterogeneous: some subtypes impair mainly cognitive functions, while others cause motor disorders.83 A brain with prion disease is characterized by spongiform appearance caused by vacuole formation inside gray matter, neuronal loss, neuroinflammation (microgliosis/astrogliosis), and deposition of amyloid plaques mainly formed by a misfolded isoform of cellular prion protein (PrPC) that is named prion scrapie (PrPSc).84 PrPC is converted into PrPSc through a posttranslational process in which its secondary structure change from a mainly a-helical to a predominant b-sheet content.85,86 The formation of PrPSc can be induced by genetic mutations in the PRNP gene, sporadic factors, or infection.83 Among infectious diseases, prions are unique because the infectious agent is a protein instead of a virus or bacteria.87 PrPSc is able to propagate cell-to-cell and, in some cases, to a new host by interacting with PrPC and converting it into a nascent PrPSc molecule.88 Even though the crucial role of PrPC and PrPSc in prion diseases is well established, it remains unclear how the loss of PrPC function or the gain of PrPSc neurotoxicity contribute to these pathologies. Indeed, the function of PrPC is not well understood, being

580 Table 1

The role of d-block metal ions in neurodegenerative diseases Neurodegenerative diseases and metal ions.

ND Prion diseases (CJD)

Levels of metal ions in patient/ models Patients Copper

[ Blood Y Frontal cortex Y Cerebellum

Zinc

/ Blood / Frontal cortex

Iron

/ Blood

Metal-binding proteins PrPC

Accumulation of metal ions in Proteins involved in metal homeostasis aggregates

PrPC KO mice Cu2þ Copper YSynaptosomes Cuþ Y Membrane-enriched Zn2þ fractions Mn2þ

Amyloid plaques from infected hamster Y Copper [ Manganese

Zinc

[ Blood Manganese [ Frontal cortex [ Cerebellum Mouse model Copper / Blood Y Brain Manganese [ Blood [ Brain Alzheimer’s disease Patient Copper Y Hippocampus Y Amygdala Y Cerebellum Y Cingulate gyrus Y Entorhinal cortex Y Motor cortex Y Middle temporal gyrus Y Sensory cortex [ Plasma (non-Cp) Zinc

Iron

Parkinson’s disease Copper

PrPC overexpression in cells Zinc [ Uptake Iron APP

Cu2þ Zn2þ APP

Ab

Cu2þ Cuþ Zn2þ Fe2þ Cuþ Cu2þ Zn2þ Fe2þ

BACE1 Tau

[ Intracellular

Copper Zinc Iron Copper BACE1 Zinc Iron

[ Putamen [Y Cortex [ / CFS

[ SN [ SN pars compacta Y Plasma

Iron

[ SN [ Putamen [ Pallidum / Plasma

Amyloid plaques from mice models [ Copper [ Iron [ Zinc

Neurofibrillary tangles (No reported)

[ Hippocampus [ Amygdala [ Putamen [ CSF Y / Cortex [ / Blood a-syn Y SN Y Locus coeruleus / Plasma Y Plasma (non-Cp)

Zinc

Amyloid plaques from human brains [ Copper [ Iron [ Zinc

DJ-1

Ca2þ Parkin mutations in C. elegans Cu2þ [ Zn2þ absorption Cuþ Zn2þ Fe2þ Fe3þ Cu2þ Cuþ Zn2þ

Lewi bodies [ Iron

The role of d-block metal ions in neurodegenerative diseases Table 1

581

Neurodegenerative diseases and metal ions.dcont'd Levels of metal ions in patient/ models

ND Huntington’s disease

Patient Copper

[ Putamen / Blood

Zinc

[ Pallidum [ Putamen [ Blood

Iron

[ Pallidum [ Putamen [ Blood

Manganese

/ Basal ganglia / Blood

Metal-binding proteins Huntingtin

Accumulation of metal ions in Proteins involved in metal homeostasis aggregates Huntingtin (Fe)

Huntingtin aggregates (Not reported)

References: Prion diseases,34–37,81,100,101,104 Alzheimer’s disease,28-33,38–49 Parkinson’s disease,50–57 Huntington’s diseases,56,58–60

this a challenge in the understanding of prion diseases. Nevertheless, several studies demonstrated that PrPC is an essential player in neuronal processes, such as: synaptic transmission, plasticity, calcium homeostasis, neuronal excitability, neuritogenesis, myelin maintenance, neuroprotection, glutamatergic receptor modulation, and metal ion homeostasis (as reviewed in 89). The latter is also supported by spectroscopic studies that demonstrate the ability of recombinant PrP (rPrP) to bind metal ions90,91 and is consistent with the alteration of metal ion levels observed in both CJD patients and Prion models (Table 1). In the following sections, we review the metallobiochemistry of PrPC and how it is impacted by PrPSc infection. Moreover, the metal-binding properties of PrPC and its possible role in PrPC functional and pathological roles are also discussed. Finally, the therapeutical strategies that target metal ions are critically analyzed.

2.19.2.1 C

The prion protein

PrP is a cell-surface glycoprotein of 209 amino acids that is anchored to the cell membrane by a glycosylphosphatidylinositol (GPI).92 PrPC has a largely unstructured N-terminal domain comprising residues from 23 to 127, and a structured C-terminal domain formed by three a-helices (residues 144–154, 173–194, and 200–228, human numbering) and two short anti-parallel b-sheets (Fig. 2A). The tertiary folding of the C-terminal is stabilized by a disulfide bridge formed between Cys179 from the second a-helix and Cys214 from the third a-helix (Fig. 2A).93 PrPC is mainly expressed in the CNS, and to a lesser extent in peripheral tissues, such as the gut, skin, heart, kidney, liver, pancreas, and secondary lymphoid organs.94,95 In the brain, PrPC is found in glial cells, and neurons, where it is detected in endosomes, axons, and dendrites, including pre- and post-synaptic compartments.94,96 Early studies demonstrated that Prion knockout mice (Prnp/) are resistant to PrPSc infection; however, no alterations in behavior were detected, failing to identify the function of PrPC.97 Ablation of PrPC produces electrophysiological abnormalities in the hippocampus but no histological changes or neurodegeneration.98 Synchrotron-based X-ray fluorescence imaging studies that compare wild type, PrPC-overexpressing, and knockout (Prnp/) mice show changes in the amount and distribution of metals in specific regions of the CNS (Table 1), being the most significant effect for iron and copper.37 Particularly, depleting PrPC in mice causes a significant decrease in copper content in synaptosomal and endosome-enriched subcellular fractions.99 Indeed, PrPCablated mice show a 50% decrease in synaptosomal copper concentrations compared to wild-type mice.100 Although PrPC has a clear impact on metal homeostasis, the mechanisms by which it might regulate metal distribution are not clearly understood. Overexpression of PrPC in neuronal cells increases intracellular iron, upregulates ferritin (the iron storage protein), and decreases the expression of transferrin and transferrin receptor (both iron uptake proteins).101 Similarly, high cytosolic iron levels are detected in non-neuronal cells that overexpress PrPC.102 Interestingly, DMT-1 is upregulated in these cellular models, suggesting that PrPC might promote the iron uptake through DMT-1.102 Pioneer studies show that exposure of neuronal cells to high concentrations of Cu2þ or Zn2þ, but not Mn2þ, induces rapid endocytosis of PrPC (Fig. 3A).103 However, the role of this mechanism in Cu2þ and Zn2þ homeostasis remains unclear.103 Cells expressing PrPC display enhanced Zn2þ uptake, as compared to cells lacking the protein.104 The uptake of Zn2þ does not require endocytosis of PrPC, and instead, it is mediated by direct interaction with the a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, as demonstrated by immunoprecipitation assays (Fig. 3C).104 Interestingly, the sequence and the structure of PrPC are homologous to the Zn2þ transporters family termed Zrt-/Irt-like proteins (ZIP).105,106 Indeed, PrPC seems to have evolved from the ZIP family, and PrP-like N-terminal sequences are found in the extracellular domain of some ZIP proteins, including ZIP5, ZIP6, and ZIP10.105 In addition, it has been suggested a connection between PrPC and ZIP14 in iron homeostasis.107 On the other hand, the role of PrPC in manganese homeostasis has been less explored. PrPC

582

The role of d-block metal ions in neurodegenerative diseases

Fig. 2 Structure, metal-binding regions, and proteolytic processing of PrPC. (A) Top: Linear representation of PrPC, highlighting the secondary structure at C-terminal and the metal-binding regions at the N-terminal domain: the site at the free-NH2 group (orange), the octarepeat region (green), His96 (pink), and His111 (purple) sites in the non-octarepeat region. Metals that can bind each region are represented by circles: green for Cuþ, blue for Cu2þ, gray for Zn2þ, and black for Mn2þ. Bottom: tridimensional structure of PrPC showing the His-anchoring residues at the intrinsically disordered N-terminal domain and the secondary structure of the C-terminal domain (PDB:1QM3). (B) Linear representations of PrPC, indicating the cutting sites of a- and b-cleavages in the human sequence.

The role of d-block metal ions in neurodegenerative diseases

583

Fig. 3 PrPC functions and metal ions. (A) High concentrations of Cu2þ and Zn2þ induce the lateral movement of PrPC outside of lipid rafts allowing the assembly of the endocytic machinery and promoting PrPC endocytosis. These mechanisms require two regions involved in metal binding: the motif 23KKRP26 and the octarepeat region. It has been proposed that PrPC endocytosis participate in metal homeostasis. (B) PrPC can modulate the opening of NMDAR in a Cu2þ-dependent manner involving the octarepeat and non-octarepeat sites. Although molecular details of the PrPC and Cu2þdependent modulation of NMDAR remains unclear, two mechanisms have been proposed. First, the formation of a PrPC-NMDAR complex induced by Cu2þ, as illustrated in (B.1). Second, the S-nitrosation of Cys residues at NMDAR, as shown in (B.2); herein, Cu2þ might. S-nitrosation of NMDAR is reduced in mice infected with PrPSc. (C) PrPC forms a complex with AMPAR and promotes the Zn2þ influx. This mechanism requires the presence of the octarepeat region.

expression protects cells from manganese neurotoxicity, reducing the intracellular accumulation of this metal,108 while manganese exposure increases membrane-bound and cytosolic PrPC levels by extent the half-life of the protein.109 Beyond the clear role of PrPC in metal homeostasis, other cellular processes require PrPC and metal ions such as neuronal plasticity.110 In this context, PrPC modulates the activity of one of the major classes of Glu receptorsdthe N-methyl-D-aspartate receptor (NMDAR).111–113 PrPC immunoprecipitated with NMDAR, and this interaction is disrupted by Cu2þ chelators.113 Formation of the PrPC-NMDAR complex reduces Ca2þ entry in neurons when NMDARs are overactivated.113 This mechanism is known as desensitization and protects neurons from neurotoxicity (Fig. 3B.1). 113 Although the molecular details of the Cu2þ- and PrPC-dependent modulation of NMDAR remain unclear, both Cu2þ and PrPC are necessary for the posttranslational modification of NMDA receptors termed S-nitrosation (Fig. 3B.2).114 The latter occurs by the covalent binding of a nitric oxide (NO•) molecule with the thiol group of a Cys residue. 114 Transition metal-catalyzed S-nitrosation could facilitate the selectivity and reactivity of this Cys modification that is unlikely to occur under diffusion-controlled conditions.115 PrPC interacts with other Glu receptors such as AMPA and KA receptors, but the impact of metal ions in these interactions is not clearly understood.104,116 Consistent with the role of PrPC in metal ion homeostasis and NMDAR modulation, conversion to PrPSc alters both metal distribution and NMDAR activity. Before the clinical onset of symptoms, copper is decreased in the brain of PrPSc-infected mice, while copper is increased in the blood of CJD patients, as was observed in other neurodegenerative diseases.34,81 Although the functional implications of dysregulation of copper in brain pathology are not clearly understood, mice infected with PrPSc show a significant decrease in S-nitrosation of NMDA at both pre-symptomatic and terminal stages, suggesting that the PrPC and Cu2þ-dependent modulation of NMDA receptors is a key event in prion pathology.117 The impact of PrPSc in iron homeostasis is less understood. Iron accumulation and deficiency have been reported in brain tissue from prion models, as well as changes in the expression levels of key proteins involved in iron homeostasis such as transferrin and ferritin.118,119 Interestingly, ferritin has been proposed as a key player for prion infection through ingestion of contaminated material.120 PrPSc can form a complex with ferritin, and then it crosses

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The role of d-block metal ions in neurodegenerative diseases

through the intestinal barrier.120 For the case of manganese, it has been reported that this metal ion is increased in blood and brain of PrPSc-infected mice and CJD patients.34,81 Moreover, in cell culture, PrPSc infection reduces Mn2þ uptake and increase expression of DMT-1.121 Although no changes in Zn2þ brain levels have been observed in prion brain CJD familial associated mutations abolish PrPC-mediated Zn2þ uptake through AMPAR and increase AMPAR-mediated neurotoxicity.104,122 Interestingly, at its intrinsically disordered N-terminal domain, PrPC binds Cuþ, Cu2þ, Zn2þ, and Mn2þ but not iron (Fig. 2A).123–128 For the case of Cu2þ, seven binding sites have been identified using rPrP and peptide fragments.123,124,129–131 One involving the free NH2 group at the N-terminal and the first three amino acids of the sequence, four at the region named octarepeat that embraces residues 60–91, and two additional sites in the adjacent region (His96 and His111) termed the non-octarepeat region (Fig. 2A).123,124,132–134 Cuþ and Mn2þ bind both, the octarepeat and non-octarepeat sites, while Zn2þ only coordinates to the octarepeat region (Fig. 2A).125,126 In the following sections, the metal-binding properties of PrPC and its biological implications are discussed.

2.19.2.1.1

Copper binding to prion protein and its biological implications

The metal-binding site at the beginning of the full-length PrPC has been elucidated by Viles and coworkers (Fig. 2A.1).123 Using CD and EPR, they have reported how Cu2þ loads onto the N-terminal NH2 group displaying a coordination mode 3N1O or 2N2O at pH 7.5 with a Kd  60 nM.123 However, no further characterization of this binding site has been done. On the other hand, the octarepeat region, formed by a sequence of eight amino acids (PHGGGWGQ) that is repeated four times, can anchor one Cu2þ ion per each His residue depending on the relative metal/protein concentrations (Fig. 2A.1). At a low Cu2þ/PrP ratio, a multi-His coordination mode termed component 3 is formed, involving His61, His69, His77, and His85 (Fig. 4A.1). ESEEM, EPR, and structure calculations studies disclosed that three or four of these His residues are coordinated to the central Cu2þ.124 Due to its sensitivity to multiple imidazole coordination, ESEEM spectroscopy was key to demonstrating multi-His binding.124 At high Cu2þ/protein ratios, Cu2þ bound to PrP yields a 3N1O coordination mode termed component 1 (Fig. 4A.2).124 This Cu2þ-PrP complex involves a His residue, two deprotonated amides from Gly residues, and one amide carbonyl oxygen from the second Gly, as determined by X-ray crystallography, EPR, and ESEEM studies. 124,135 The equatorial coordination shell of component 1 involves the residues that follow His in the sequence due to the Pro forces the coordination mode towards the C-terminal.124,135 Additionally, one water molecule acts as an axial ligand, forming a hydrogen bonding lattice with the NH of indole ring from Tryptophan,124,135 At intermediate Cu2þ/PrP ratios, a Cu2þ species named component 2 is observed (Fig. 4A.2), where one His, one deprotonated amide nitrogen and two water molecules are likely the ligands for the metal ion. In this coordination mode, unlike component 1, the deprotonated amide nitrogen that binds the Cu2þ ion is provided by the His residue.124 Thus, at the octarepeat region, the affinity of PrP for Cu2þ depends on Cu2þ-PrP speciation. Components 1 and 2 display a Kd in the mM range (7–12 mM), while component 3 binds Cu2þ with a Kd  10 nM (Fig. 4A and Table 2).136 In contrast to the octarepeat region, Cu2þ coordination to the non-octarepeat region does not change at different Cu2 þ/PrP ratios, but it is highly dependent on pH (Fig. 4A.3).129,132–134 For the His96 site, GGTH has been proposed as the minimal sequence to reproduce Cu2þ-binding. The pKa for the Cu2þ-GGTH complex is 7.8, involving the equilibrium of two species with coordination modes 3N1O and 4N.129,134 The 3N1O coordination sphere includes the His, two deprotonated amides, and one backbone carbonyl; while in the 4N species, a third deprotonated backbone amide enters into the sphere coordination, replacing the backbone carbonyl.129 A similar scenario is found for the His111 binding site, where the pKa of the complex is 7.5, and it is associated with an equilibrium between a 3N1O and a 4N coordination modes.133 The coordination features of the Cu2þ-His111 complex also involve the imidazole of His, deprotonated amides of residues that precede the His, and a backbone carbonyl for 3N1O mode; while for the 4N mode, a third deprotonated amide enters the coordination sphere (Fig. 4A.3).133 In contrast to the case of the His96 site, the His 111 site has two methionine residues nearby that could act as metal ligands. Specifically, Met109 in the sequence KTNMKHMAGA has been proposed as a weak axial ligand for Cu2þ bound to His111.130,131 According with ITC studies, the 4 N modes formed at His96 and His111 display similar metal-binding affinities: a Kd of 0.4 mM for the His111 site and 0.7 mM for His96 (Table 2).131 Consistently, the substitution of Met109 by Ile induces small changes in the Cu2þ-binding affinity to the His111 site (from a Kd 0.4 mM to a Kd 0.9 mM).131 Thus, these coordination sites, His96 and His111, are independent sites and display two protonation states at physiological pH. However, at pH 5.5, Cu2þ can bind both His residues, one deprotonated amide nitrogen, and one sulfur atom from methionine residues, as the EXAFS studies evidenced.137 Recently, NMR studies demonstrated that Cu2þ binding to rPrP promotes a long range interaction between N- and C-terminal domains (cis-interdomain interaction).138 Using cross-linking, MS/MS, and NMR, it has been identified the three critical metalbinding regions at the N-terminal domain are involved in the interdomain interactions: the N-terminal polybasic region (23  31), the octapeptide repeats (59–90), and the central region (99–127).139 When the metal promotes the cis-interdomain interaction, Cu2þ bound to His96/His111 sites becomes proximal to helix-1 (residues 144–147), and Cu2þ bound to the octarepeat region (60–91) becomes proximal to helix-2 (174–185).138 Consistently, a study combining NMR and copper-nitroxide pulsed double electron-electron resonance (DEER) demonstrated that Cu2þ bound to the octarepeat sites interacts with the negatively charged globular region at the C-terminal domain, involving helices 2 and 3.138 Interestingly, this cis-interdomain interaction yields a multi-His coordination mode similar to component 3, involving three His residues from the octarepeat region and one His residue from C-terminal (His139 and His176).140 Overall, these studies underscore a possible role of Cu2þ-promoted cis-interdomain interaction as a conformational switch between physiological and pathological states of the protein.139 Copper is a redox-active metal, and it can cycle between Cu2þ and Cuþ oxidation states. Early turnover studies of Cu2þ bound to the octarepeat region of PrP, using reducing agents (ascorbate and glutathione) and oxygen, showed that all Cu2þ-octarepeat

The role of d-block metal ions in neurodegenerative diseases

585

586

The role of d-block metal ions in neurodegenerative diseases

complexes are reduced, but Component 3 does not display catalytic activity towards oxygen.141 Consistently, electrochemical studies demonstrated that Component 3 displays a reduction potential of 323 mV vs. NHE. Thus it can oxidize ascorbic acid   (E AA ¼ 52 mV vs. NHE) to form a Cuþ-octarepeat complex, but it cannot oxidize molecular oxygen to generate H2O2 (E 142 H2O2 ¼ 296 mV vs. NHE). In contrast, Component 1 and the non-octarepeat sites (H96 and His111) have reduction potentials that allow O2 activation by their corresponding Cuþ-PrP complexes. Cyclic voltammetry experiments showed a reduction potential of 172 mV vs NHE for Component 1 in a peptide modeling the octarepeat region;142 while the full-length mouse rPrP protein loaded with five Cu2þ ions displays a reduction potential of 267 mV vs NHE.143 Similarly, the Cu2þ-3N1O complex at the His96 site shows a quasi-reversible reduction potential of 263 mV vs NHE;129 while Cu2þ bound to the mouse PrP(91–110) fragment displays a redox potential of 110 mV vs. NHE.142 Hence, redox cycling of Cu2þ bound to PrP depends strongly on coordination mode. Even though the Cu2þ coordination properties of PrP have been elucidated, the study of Cuþ binding has been limited to the non-octarepeat sites. Early copper K-edge XAS studies of Cuþ bound to the 91–126 and 106–114 fragments of PrP indicated a (N/ O)2S2 coordination sphere at pH 7.4.144 Consistently, the formation of a four-coordinate Cuþ-(His)2(Met)2 complex with a Kd ¼ 10 15–10 12 M was proposed, based on NMR studies of human PrP(91–124/127).125,145 Finally, a combined spectroscopic (NMR and XAS) and theoretical study showed that Cuþ coordination to His111 in the (106–115) fragment is highly dependent on pH: only two Met residues are coordinated to Cuþ at pH < 5; in a pH range of 5–8, a 1N1O2S species is formed, where both Met residues, His111 and a backbone carbonyl oxygen or water molecule bind to Cuþ (Fig. 4B); and at pH > 8, a Met is replaced by a nitrogen based ligand, possibly a deprotonated backbone amide, to form a 2N1O1S species (Fig. 4B).125 While further work is needed to understand Cuþ binding to PrP, it is clear that the His111 site is unique, as it has two Met residues (MKHM) that participate in Cuþ coordination. Particularly, Cuþ binding to the His111 site could play an important role under the reducing and low pH conditions that PrP would encounter upon endocytosis.125 Furthermore, it is evident that redox cycling of Cu2þ bound to PrP must involve large changes in coordination sphere and geometry, with a subsequent cost in reorganization energy and electron transfer rates.125 Hence, though it has been proposed that the redox activity of some Cu2þ-PrP complexes (Component 1 and the nonoctarepeat sites) can generate ROS,142,144,146,147 the amount of H2O2 produced would be considerably lower, as compared to that by free Cu2þ ions.148 This low generation of H2O2 has been proposed to serve as a cellular signal to trigger important cellular processes.149 The ability of PrPC to bind Cu2þ using sites coordination modes with different redox properties and metal-binding affinities is a challenge to link the proposed functional roles of PrPC with a specific coordination mode.110 Cell culture studies using high metal concentrations ( 100 mM of CuSO4) demonstrated that the metal-promoted endocytosis of PrPC requires the presence of two Cu2þ-binding regions: the polybasic motif 23KKRP26 and the octarepeat region. PrPC lacking the region 23KKRP26 cannot exhibit Cu2þ-promoted endocytosis, whereas depletion of the octarepeat region indirectly impairs the PrPC endocytosis by inhibiting the Cu2þ-promoted lateral movement of PrPC out of the lipid rafts (Fig. 3A).150 These studies suggest that the high occupancy modes (components 1 and 2) at the octarepeat region and metal-binding to the region 23KKRP26 could act as a switch that promotes PrPC endocytosis at high metal concentrations. Consistently, mutations of His residues at the octarepeat region inhibit Cu2þpromoted endocytosis.103 On the other hand, at intermediate metal concentrations ( 0.1–0.5 mM), His residues at octarepeat and non-octarepeat regions are necessary to the Cu2þ-dependent modulation of NMDA receptors by PrPC (Fig. 3B).151 Depending on the PrPC expression, at these Cu2þ levels, two Cu2þ ions could be coordinated at the His96 and His111 sites and one at the octarepeat region, possibly yielding the cis-interdomain interaction. Nonetheless, further studies are necessary to demonstrate what Cu2þ-PrPC interactions are necessary for lateral movement outside lipid rafts, endocytosis, and modulation of NMDA receptors. Finally, although Cu2þ-promoted S-nitrosylation of NMDA receptors has not been associated with a specific metal-binding region of PrPC, the redox-active Cu2þ-PrPC species could play an important role in this mechanism.

2.19.2.1.2

Zinc binding to prion protein and its biological implications

After Cu2þ, the metal ion with the next highest affinity for the prion protein is Zn2þ, binding to the octarepeat region with a Kd  200 mM.126 High Zn2þ concentrations ( 300 mM) can alter the distribution of Cu2þ among the octarepeat and non-octarepeat regions, as observed by EPR spectroscopy.126 Zn2þ is thought to shift the Cu2þ coordination by interacting directly with the imidazole side chains of the octarepeat region. Although initially thought to only interact with the N-terminus, Zn2þ can facilitate global changes to the structure of the prion protein.152 Zn2þ induces an interaction between the octarepeat region of the flexible Nterminus and structured helix 2 and 3 of the C-terminus.152 Several point mutations associated with familial prion disease, including P101L and D177N, disrupt this Zn2þ dependent structural change.152 Using Cd2þ as an NMR active surrogate of

=

Fig. 4 Coordination modes proposed for Cu2þ, Cuþ, and Zn2þ bound to PrPC. (A) Cu2þ-PrP complexes formed at the octarepeat and nonoctarepeat regions. (A.1) At a low Cu2þ/PrP ratio, a multi-His coordination mode termed low occupancy mode or component 3 is formed. This metal complex displays a high affinity with a Kd around 10 nM (A.2) At intermediate and high Cu2þ/PrP ratios, two coordination modes named component 1 and 2 or high occupancy modes are formed. These coordination modes have a low affinity for Cu2þ (Kd 7–12 mM). (A.3) At the non-octarepeat region, Cu2þ binds the His96 and His111 sites, displaying two coordination modes (termed 3N1O and 4N modes) per each site. These coordination modes have a higher affinity than components 1 and 2 (Kd 0.4–0.7 mM). (B) Cuþ-binding after Cu2þ reduction in the His111 site at different pH values. (C) Zn2þ is bound to the octarepeat region displaying a multi-His coordination mode with low affinity (Kd 200 mM). (D) Cu2þ binding at a-cleaved PrP yields two coordination modes. At low Cu2þ/PrP ratio is formed a histamine-like coordination mode termed Mode I, while Mode II is observed at high Cu2þ/PrP ratio.

The role of d-block metal ions in neurodegenerative diseases Table 2

587

Dissociation constants for metal-binding sites of prion protein.

Metal

Metal-binding site

Coordination mode

Kd

Method

Cu2þ

NH2-KKR Octarepeat Octarepeat

60 nM 10 nM 7 mM

Competition with glycine followed by CD Competition with Cu2þ chelators followed by EPR Trp Fluorescence quenching

12 mM

Trp Fluorescence quenching

His96 His111 Octarepeat

3N1O or 2N2O Component 3 (3 or 4N) His Component 1 (3N1O) One His and two deprotonated amides Component 2 (2N2O) One His, a deprotonated amide, and two water molecules 4N mode 4N mode 4N or 3N1O (3 or 4N) His

0.7 mM 0.4 mM 200 mM

Octarepeat Non-octarepeat

– –

3 mM 200 mM

ITC ITC Diethyl pyrocarbonate (DEPC) modification and mass spectrometry Competition with metal chelators Competition with metal chelators

Octarepeat

Zn2þ Mn2þ

References: Cu2þ bound to the octarepeat136; Cu2þ bound to NH2-KKR123 and non-octarepeat regions;131 Zn2þ bound to the octarepeat region;126 Mn2þ bound octarepeat and non-octarepeat regions.127

Zn2þ, the cis-interaction between the C- and N-terminus of prion is observed, where 3 or 4 His make up the metal coordination sphere.153 Zn2þ alters the availability of prion at the synapse, by promoting its rapid endocytosis (Fig. 3A). Zn2þ binding to the octarepeat region is a key event for prion endocytosis, since this activity is abolished upon the deletion of the octarepeat region, or the mutation of the His 68, His 76 dyad.103. While Zn2þ induced endocytosis of prion has been proposed to contribute to Zn2þ transport, the prion protein greatly enhances Zn2þ uptake into neuronal cells through a different mechanism involving a direct interaction with a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (Fig. 3C).104 The latter was demonstrated by coimmunoprecipitation assays with both GluA1 and GluA2 AMPA receptor subunits.104 Prion mediated Zn2þ uptake is prevented by selective antagonists of the AMPA receptors, or deletion of the octarepeat region (Fig. 3C).104 Finally, cells infected with PrPSc or cells expressing PrPC with mutations related to familial prion disease also display reduced Zn2þ influx through this mechanism. P101L and D177N stand out as mutations that disrupt Zn2þ induced structural changes, and prevent the direct PrP-AMPA receptor interaction, underscoring the key role of such PrP conformational changes in AMPA receptor mediated Zn2þ uptake.104

2.19.2.1.3

Manganese binding to prion protein and its biological implications

2.19.2.1.4

Proteolytic processing of cellular prion protein and its impact in metal-binding properties

It has been proposed that Mn2þ can bind both the octarepeat and non-octarepeat regions. At low pH ( 5.7), Mn2þ interacts with the fragment PrP(106–126) involving the carbonyl groups of Gly124, Leu125, Gly126, and a H/bonding from metal-bound water to His111.154 The binding affinity of Mn2þ to PrP(91–231), which lacks the octarepeat region, is several orders of magnitude weaker (Kd  200 mM). Mn2þ can also bind the octarepeat region (52–98), but it has an even weaker binding affinity (Kd  3 mM).127,128 Although it has been proposed the participation of His111 in Mn2þ binding, chemical modification of His residues does not prevent the interaction of this metal with rPrP.155 Mn2þ impacts the extent of a-helical folding of PrPC,156,157 and it is proposed that this altered conformation causes the release of Cu2þ ions.157 Thus, although Mn2þ may not be competing with Cu2þ for the same binding sites, it may displace Cu2þ from rPrP through an allosteric effect. In cultured cells, Mn2þ ions can replace Cu2þ ions from PrPC,158 but in contrast to Cu2þ, Mn2þ does not promote PrPC endocytosis.103 Indeed, the Mn2þ binding to PrPC has been associated to pathological but not functional roles.36,159–162

Post-translational modifications (PTMs) of PrPC play a fundamental role in its function. PrPC undergoes removal of the N-terminal signal peptide, N-glycosylation at two asparagine residues (Asn181 and Asn197), formation of a disulfide bond (Cys179-Cys214), attachment of the GPI (glycosyl-phosphatidylinositol) anchor at C-terminal and proteolytic cleavages.163,164 Two of these posttranslational cleavages, a- and b-cleavage, occur in the flexible N-terminal of PrPC; g-cleavage occurs in the vicinity of the sites of glycosylation165 and the shedding close to the GPI anchor (Fig. 2B). a-cleavage cuts PrPC between Lys110 and His111 in the human sequence producing two fragments: N1 (23  110) and C1 (111  231) (Fig. 2B). On the other hand, b-cleavage cuts PrPC in the N2 (23–89) and C2 (90–231) fragments (Fig. 2B).164 a-cleavage is the main proteolytic event. C1 fragment from this cleavage accumulates at the plasma membrane and contributes up to 45% of total PrP levels.164,166 The N1 and C1 fragments have been extensively studied due to their biological activity. A neuroprotective function has been suggested for the N1 fragment because it blocks neurotoxic signaling pathways, protecting the cell against toxic oligomeric species. While the C1 fragment, due to the loss of the neurotoxic domain, is not capable of folding into the pathogenic isoform, so it is not a substrate for the conversion of PrPC to PrPSc.167–169 It has been suggested that ADAM10, 17, 9, and 8 (A-disintegrin-and-metalloproteinase) are the main enzymes responsible for this a-proteolytic cleavage.167 Moreover, there is evidence that metal ions such as Cu2þ affect this type of

588

The role of d-block metal ions in neurodegenerative diseases

post-translational modification. Although McDonald et al. demonstrated that direct binding of Cu2þ to PrPC suppresses the activity of ADAM8, since it is not a good substrate for the metalloprotease, the mechanism involved is still unknown.168 On the other hand, b-cleavage is related with oxidative stress events and has been associated with pathogenic conditions. The C2 fragment has been detected in the post-mortem brains from patients with CJD.164,167 However, despite this categorization, it has been proposed that b-proteolytic cleavage is a mechanism by which PrPC protects the cell against oxidative stress.165 Several studies have shown that b-proteolytic cleavage is induced by reactive oxygen species (ROS) in the presence of Cu2þ.170,171 although it can also be generated by the enzymatic activity.168 It has been reported that PrPC undergoes this proteolytic cleavage from the generation of ROS promoted in the presence of Cu2þ in a Fenton-type reaction, consequently when the copper ion binds to PrPC, a redox reaction can locally generate ROS, which in turn could react at specific sites in the protein, resulting in a cleavage.170 Although the molecular mechanism by which it does remains unknown. Interestingly, in vitro studies using recombinant mouse PrP showed that the N2 fragment undergoes additional proteolytic cleavages near to each His residue at the octarepeat region and this cutting is decreased in the presence of Cu2þ and Zn2þ.168 The study of metal-binding to the different fragments that remain after post-translational modifications of PrPC in its N-terminal domain has been little explored. Recently Sanchez-Lopez et al. reported the study of a synthetic peptide that models Cu2þ binding to the C1 fragment that remains anchored to the cell membrane after the a proteolytic cleavage of PrPC, and it mantains the ability to bind Cu2þ ions through the His111 site.172 Using the HMAGA fragment, it was shown that the His111 and the free NH2-terminal group are the main anchoring sites for Cu2þ, resulting in coordination modes that wildly different from those characterized for the His111 site in the full length protein (Fig. 4D).172 The speciation of Cu2þ at the C1 fragment would also be dependent on proton and copper concentrations.

2.19.2.1.5

Metal ions and aggregation of the prion protein

2.19.2.1.6

Metal ions as a therapeutic target in prion diseases

A pathological hallmark of prion disease is the accumulation of amyloid aggregates mainly composed by fibrillar PrPSc.84,85,173–175 Although PrPC binds Cu2þ with higher affinity than Mn2þ, the aggregates from prion patients have low Cu2þ and high Mn2þ concentrations,36 suggesting different metal-binding properties of PrPSc in amyloid plaques or a significant imbalance of copper and manganese concentrations in the prion brain. Although the structural details of PrPSc remain unknown, early studies using techniques with low structural resolution (Electron microscopy and NMR, CD, and FTIR spectroscopy) demonstrate that PrPSc fibrils are characterized by a high b-sheet content, including b-helix, b-spiral, and extended in-register b-sheets.176 Structural models of fibrillar PrPSc propose that monomers are organized in an intermolecular b-sheet structure to form the amyloid fibril; however, there is no consensus on the conformational changes that yield the b-sheet structure (for review see 176). Some studies indicate that these conformational changes occur mainly at the intrinsically disordered region, where the metal-binding sites are located, suggesting an important perturbation of the metal-binding properties of fibrillar PrP.176 In contrast, a recent Cryo-EM study using recombinant human PrP(23–270) shows that amyloid fibril formation requires a C-terminal refolding into b-strands (Fig. 5B).177 In this structure, the His176 residue involved in the cis-interdomain interaction is embedded in a b-sheet (Fig. 5B).177 Similarly, another CryoEM study using the fibrillogenic peptide model PrP 94–178 (in the human sequence) shows amyloid fibrils formed by two b arch protofilaments with apolar residues tightly packed within the fibril core (Fig. 5C).178 In the latter, His111 and His139 are part of b-strands, where they would not be available to bind metal ions (Fig. 5C). 178 Although high-resolution techniques are available, the structural diversity of PrP fibrils remains a challenge to understand the structure of PrPSc in amyloid plaques. Based on its metal binding properties of PrPC, a plausible role of metal ions in the conversion of PrPC to PrPSc has been proposed.179–181 In vitro studies using peptide models and rPrP suggest that Mn2þ promotes PrP aggregations, while both inhibitory and promoting effects have been reported for Cu2þ.159-162,182–185 However, it has been recently proposed that Cu2þ promotes the cis-interdomain interactions without inducing changes in b-sheet content.138 On the other hand, redox cycling of Cu2þ bound to PrP can trigger chemical modifications, such as oxidation of Met125 and His residues, which in turn may impact PrP structure and aggregation.186 Selective oxidation of His residues at the octarepeat region by Cu2þ and ascorbate was demonstrated on syrian hamster PrP(29–231), leading to subtle structural changes that can trigger PrP aggregation.187 In the case of Mn2þ, some studies have shown that this metal helps the conversion process from PrPC to PrPSc, and it induces the formation of larger aggregates; however, in the presence of Cu2þ, Mn2þ-induced aggregation of PrP is blocked, suggesting an interplay between these two metals and their impact in PrP aggregation.162

Although prion diseases have silent incubation periods of up to 50 years in humans, their progression is very accelerated after diagnosis, as compared with other neurodegenerative diseases.188 All prion diseases are fatal and there is no available treatment to delay their progression, in spite of several therapeutic attempts to inhibit PrPSc conversion, including chemical agents as polyanionic compounds, peptides, cyclic molecules and antibodies to prevent protein aggregation, as well as metal chelation-based therapy.189 Metal chelation targeting metal imbalances in prion diseases are attractive strategies. Low levels of Cu2þ have been observed in the brain of PrPSc-infected mice, while the levels of this metal are increased in blood of CJD patients.34 Thus, chelator-based therapy with D-penicillamine has been studied in prion diseases. The presence of this chelator can extend the incubation phase of the disease and reduce copper levels in brain and blood in treated mice.190 Another study in rodents has shown that clioquinol, a metal chelator antibiotic, has an effect on the long-term maintenance of brain functions.191 Likewise, chelation-based therapy targeting Mn2þ ions has been proposed, as increased Mn2þ levels have been observed in blood and brain of PrPSc-infected mice and CJD patients.34 Mn2þ chelation therapy, using cyclohexanediaminetetra acetic acid, reduces PrPSc levels and extends survival of PrPSc-

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Fig. 5 Comparison between secondary structure of the human prion protein and the Cryo-EM structure of prion fibrils. (A) Full-length prion protein. His-anchoring residues are highlighted in green, pink, and purple at the intrinsically disordered N-terminal domain, while His residues involved in the cis-interaction are highlighted in red at the C-terminal domain. The secondary structure of the C-terminal domain resolved by NMR is shown in purple for a-helices and yellow for b-strands (PDB:1QM3). (B) The amyloid fibrils produced with recombinant PrP(23 231) are characterized by the presence of a hydrophobic core from 170 to229 residues at the C-terminal domain (PDB: 6LNI). The a-helices 2 and 3 are converted into five b-strands (yellow), and the His176 residue (red) involved in the Cu2þ-promoted cis-interdomain interaction is part of a b-sheet. The prion protein used to form these fibrils has intact the N-terminal domain, but these residues are not part of the amyloid core. (C) The amyloid fibrils formed by the fragment PrP(94–178) formed the amyloid core involving the residues 106–145 (PDB: 6UUR). The three a-helices are converted in b-strands, and the His111 residue is embedded in a b-sheet.

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The role of d-block metal ions in neurodegenerative diseases

infected mice,192 suggesting a therapeutic value for the removal of this metal ion and attenuation of Mn2þ imbalance in prion disease. While metal-chelation therapy for prion diseases may have implications in the functional features of metal-PrPC interactions, understanding how metal imbalance promotes alterations in the prion brain will establish the basis for this type of treatment of these neurodegenerative diseases.

2.19.3

Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive neuronal loss, mainly in the cortex, hippocampus, and amygdala.193 Clinically, AD patients present a gradual impairment of memory that progresses to a severe cognitive decline, affecting behavior, speech, visuospatial orientation, and motor system in the late stages of the disease.193 Brain tissue from AD patients is characterized by extracellular deposition of amyloid plaques formed by amyloid-beta (Ab) peptide and intracellular accumulation of neurofibrillary tangles composed of hyperphosphorylated tau protein.194 Ab is a metal-binding peptide produced by the endoproteolytic cleavage of a transmembrane proteindthe amyloid precursor protein (APP)dby b-secretase (BACE1) and the g-secretase complex, while tau is a microtubule-binding protein that plays an important role in axonal transport.195,196 Although amyloid plaques and neurofibrillary tangles have been known as AD hallmarks for more than a century, the role of Ab and tau in the pathogenesis of AD is not well understood.197 Mutations in the genes that encoded APP and two proteins of the g-secretase complex (Presenilin-1 and -2) are enough to cause AD.198 Patients carrying these mutations develop an aggressive form of the disease known as early-onset Alzheimer’s disease (EOAD). However, less than 1% of AD patients have these mutations, and the etiology of the rest of the AD cases is unknown.198 Interestingly, proteins that play a key role in the pathology of AD (APP, BACE1, presenilins, Ab peptide, and tau protein) are either regulated by metal ions, involved in metal homeostasis or are metal-binding proteins. Indeed, disruption of metal homeostasis has been documented in AD patients and AD animal models (Table 1).28-33,38–49 These studies indicate that copper is decreased in the AD brain, while Zn2þ and iron are increased.38–45 Moreover, in blood, AD patients display high levels of copper non-bound to Cp in comparison with healthy patients.28–33 Recently, mutations on the copper transporter ATP7B that produce the substitutions K832R y R952K have been recognized as risk factors to display high levels of copper non-bound to Cp in AD patients.32 In the next sections, the role of APP, Ab peptide, and tau in metal homeostasis is discussed, as well as its metal-binding properties and its physiological/pathological implications. Finally, therapeutical strategies that target metal ions are critically analyzed.

2.19.3.1

The amyloid precursor protein

The amyloid precursor protein (APP) is a transmembrane protein formed by 695–770 amino acids depending on the splicing isoform. APP belongs to a gene family of proteins that also includes amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2, respectively).199 APP and APLP-2 are mainly found in intracellular compartments of neurons, while APLP-1 is mostly in the cell membrane.200 Interestingly, Cu2þ but not Zn2þ promotes APP trafficking to the cell surface by inducing phosphorylation of Thr668.201,202 APP family members are multifunctional proteins involved in transcriptional regulation, signaling, and cellular adhesion. The latter two functions are associated with the fact that APP and APLPs can form dimers, where the two proteins can be in the same cell membrane (cis-dimers) or in membranes from two different cells (trans-dimers).203,204 Genetic studies with single and combined knockouts of APP, APLP1, and APLP2 have demonstrated that the APP family is essential to CNS development, particularly in synapse formation, neuronal differentiation, and synaptic plasticity.199 Double knockout mice studies (APPþ/–APLP2/ and APP/–APLP2þ/) showed an increase of copper and iron in the brain cortex, pointing to an important role of these proteins in metal homeostasis.205 Consistently, APP depletion induces intracellular accumulation of copper and iron in cell culture.206 Indeed, APP expression is regulated by iron levels through an iron-responsive element (IRE).207 IREs are sequences located in the 50 -untranslated region of the mRNA, where iron regulatory proteins (IRPs) bind to control the translation of proteins involved in iron uptake and efflux. The APP mRNA binds IRP-1, and this interaction is modulated by an iron-sulfur switch mechanism. Upon increasing intracellular iron levels, IRP-1 binds a [4Fe-4S] cluster, adopting a conformation that prevents its interaction with IREs and promotes APP expression.208 Although APP has a clear impact on iron homeostasis, the mechanisms by which it regulates the levels of this metal remain unclear. Ferroxidase activity of APP has been proposed; however, isolated APP does not show this catalytic activity.206 On the other hand, APP binds the iron export protein ferroportin, and it has been proposed that APP increases the presence of ferroportin in the cellular membrane, favoring iron export indirectly.206,209 APP, APLP1, and APLP2 have a large extracellular N-terminal domain, a hydrophobic transmembrane domain, and a short cytosolic tail (Fig. 6). Although the structural organization of the full-length proteins remains unclear, individual domains of APP have been resolved with high resolution by X-ray crystallography (Fig. 6).210–213 The N-terminal of APP has two subdomains independently folded: E1(Leu18-Ala190) and E2(Ser295-Asp500) connected by a highly flexible acidic domain (AcD). Notably, the E1 domain is divided into the growth factor-like domain (GFLD) and the copper-binding domain (CuBD), which have a tight interaction between them (Fig. 6). The E2 domain does not seem to interact with E1, and it is linked to the transmembrane helix through a disordered region that contains the N-terminal region of the amyloid-b peptide (Fig. 6).210 A sequence alignment of APP, APLP1, and APLP2 shows that the latter two share the E1 and E2 structural domains with APP, while their disordered region does not contain the amyloid b peptide sequence.214

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Fig. 6 Schematic representation of amyloid precursor protein (APP). The linear scheme shows the extracellular E1 domain with its two independently folded domains, the growth factor like-domain (GFDL) (PDB: 4JFN) and the copper-binding domain (CuBD) (PDB: 2FK1); the acidic domain (AcD); the E2 domain (PDB: 3UMK); and the cytosolic C-tail. The proteolytic cleavage of APP by b and g-secretases to yield the full-length Ab(1–40) is represented by red-dotted lines. Below, linear representation of APP where metal-anchoring His are remarked, and circles represent the metals that each site can accommodate. Blue (Cu2þ), green (Cuþ), and gray (Zn2þ). Secondary structures of the protein are denoted as follows: yellow (b-sheet), violet purple (a-helix), and gray cord for intrinsically disordered regions.

APP is not only involved in metal homeostasis; it also binds metal ions. APP displays three Cu2þ-binding sites at its GFLD, CuBD, and E2 domains, which may also bind Cuþ.212,215,216 and display two Zn2þ-binding sites at CuBD and E2 domains (Fig. 6). On the other hand, while there is a clear role of APP in iron homeostasis, there is no spectroscopic evidence for the direct binding of iron by APP.206 Although the metal-binding properties of APP were discovered in 1993, the nature and physiological role of its metal-binding sites are still unclear.217,218 In the next paragraphs, a summary of the nature of the metal-binding properties of APP is provided, followed by a discussion of their biological relevance.

2.19.3.1.1

Copper binding to the amyloid precursor protein and its biological implications

Cu2þ binding to CuBD has been studied by X-ray crystallography, EPR, EXAFS, and ITC.212,219 CuBD binds one Cu2þ ion with a Kd ¼ 18 nM.219 The crystal structure shows a square pyramidal geometry with Cu2þ bound to N32 of His147, Nd1 of His151, the hydroxyl group of Tyr168, and two water molecules in axial and equatorial orientations (Fig. 7B). Consistently, the EPR signal, with a g|| ¼ 2.296 and A|| ¼ 176  10 4 cm 1, indicates an equatorial 2N2O coordination, while the best fit for the EXAFS spectrum includes two His residues and Tyr one in the coordination sphere.212 On the other hand, Cu2þ binding to GFLD has only been studied by X-ray crystallography and ITC.219 GFLD binds one Cu2þ ion, with a Kd ¼ 28 nM, in a square pyramidal geometry using an oxygen from Asp23 of a symmetry-related molecule in the crystalline structure, two water molecules, and the N32 atoms from His108 and His110, which are separated by a rigid Pro (Fig. 7A). Mutations of the His108 and His110 residues to Ala abolish Cu2þ binding.219 Finally, the E2 domain binds one Cu2þ ion with a Kd ¼ 13 nM, as determined by ITC. The crystal structure shows that metal binding involves four His residues: His382, His313, His432, and His436 (Fig. 7C), consistent with EPR data that suggest a nitrogen-rich coordination environment.211,215 Interestingly, surface plasmon resonance (SPR) studies show that Cu2þ binding induces conformational changes, which have been assigned to the rotation of the a-B and a-C helices, restricting the movement of these coiled-coil helices. The preference of Cu2þ for a square planar geometry may induce the rotation of the helices, which favors the proper orientation of His313 and His382 towards the metal center. Additionally, Cu2þ binding to E2 increases the thermal stability (Tm) of its a-helical structure, as observed by circular dichroism (CD).211 Despite having different coordination modes, the three Cu2þ-binding sites of APP (CuBD, GFLD, and E2) have similar Cu2þ-binding affinities (Table 3).

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Fig. 7 Metal-binding sites of APP. (A) Left: Pymol solid-state structure for Cu2þ binding to GFLD (PDB: 4JFN); right: Proposed coordination sphere for Cu2þ to the GFLD.; (B) Pymol solid-state structure for Cu2þ binding to CuBD (PDB: 2FK1); right: Proposed coordination sphere for Cu2þ in the

The role of d-block metal ions in neurodegenerative diseases

593

In contrast to Cu2þ, the coordination of Cuþ to APP has been less explored. An early study suggests that APP can reduce Cu2þ to Cu involving Cys residues.225 Soaking crystals of Cu2þ-CuBD complexes were reduced with ascorbate, and the formation of Cuþ was demonstrated by X-ray fluorescence. Herein, the coordination Cuþ has some similarities with Cu2þ: it involves nitrogen ligands from His147 and His151 with small changes in the orientation of His147, the hydroxyl oxygen of Tyr168, and only one water molecule, giving place to a distorted square planar geometry for Cuþ instead of the square pyramidal observed for Cu2þ (Fig. 7B).212 Although competition assays with probe ligands of Cuþ such as ferrene S (Fs), ferrozine (Fz), bicinchoninic acid (Bca), and bathocuproine disulfonate (Bcs) suggests tight binding sites at GLFD and E2 domains, the formation of ternary complexes with these probes is also demonstrated.215,216 For the Cu2þ-E2 complex, the redox activity was evidenced by the loss of its EPR signal in the presence of ascorbate.215 The role of copper-binding in the function of APP is not well understood. However, copper impacts the formation of APP cisdimers that are involved in cellular signaling and APP trans-dimers that are implicated in cell-adhesion, synaptogenesis, and synapse stability (Fig. 8).219,226 Cu2þ increases cis-APP dimerization in cellular cultures, while mutations in the Cu2þ-binding sites at GFLD and CuBD drastically reduce this effect.219 Moreover, studies using resin-anchored recombinant APP have shown that Cu2þ can induce trans-dimerization, which has been associated with the ability of this metal to promote synaptogenesis in cell culture.227 Deletion of GFLD, but not CuBD, reduces synaptogenesis, while point mutations H108A and H110A confirm that Cu2þ binding to the GFLD site is associated with trans-dimerization of APP and synaptogenesis.219 Notably, the Cu2þ binding properties of the E1 domain do not seem to be conserved in the APLPs: the metal anchoring His residues in the GFLD site are conserved in APLP1, while only the CuBD site is present in APLP2.227 In contrast, the metal-anchoring His residues of the E2 domain are highly conserved in the APLPs, pointing to an important functional role for Cu2þ coordination to E2 in the APP family that remains to be explored.211 þ

2.19.3.1.2

Zinc binding properties to the amyloid precursor protein and its biological implications

2.19.3.1.3

Metal ions and the proteolytic processing of amyloid precursor protein

An early study using a Zn65 radioisotope revealed that APP binds this metal with a Kd ¼ 765 nM, suggesting the presence of a Cysrich Zn2þ binding site in the E1 domain.217 An spectroscopic study of a model peptide APP169–187 showed that Zn2þ binding may involve two adjacent cysteine residues (Cys186 and Cys187), which are conserved in all APLP members.228 However, the crystal structure of APP shows that these Cys residues are in a trans- orientation; thus, they would not be available for metal-binding mode in the full-length protein.229 A recent ITC study demonstrates that Zn2þ is bound by the E1 domain, but not by the individual GFLD and CuBD domains,227 suggesting a distinct metal-binding site, as compared to Cu2þ. Thus, further studies are needed to elucidate the Zn2þ binding properties of the E1 domain of APP. X-ray crystallography reveals a Zn2þ binding site in the E2 domain, encompassing three His residues (His382, His432, His436), and a water molecule in a nearly tetrahedral geometry (Fig. 7C). ITC data reveals a significantly weaker affinity for Zn2þ (Kd ¼ 4 mM), as compared to Cu2þ (Table 3). Moreover, Zn2þ coordination at E2 does not involve His313 of helix a-B, which is consistent with the smaller conformational changes observed by SPR and X-ray crystallography in comparison with Cu2þ. Still, Zn2þ binding to E2 increases the thermal stability (Tm) of its a-helical structure, as observed by CD.211 In terms of the functional implications of Zn2þ binding to APP, the E1 domain is involved in Zn2þ dependent trans-directed dimerization on all APP members, as demonstrated by a study using resin-anchored recombinant proteins (Fig. 8).227 However, the nature of the Zn2þ binding in the E1 domain, and its role in synaptogenesis remains to be elucidated. On the other hand, FRET experiments in cell cultures demonstrated that Zn2þ binding to the E2 domains of APP and APLP1/2 induces both, homophilic and heterophilic, cis-dimerization between APP members.230 Finally, it is interesting to note that both, E1 and E2 domains of APP display metal-binding sites that could be competitive for Cu2þ and Zn2þ ions. However, the Zn2þ-binding properties of APP need further characterization. This is important, as metal-binding to APP may play a central regulatory role for APP dimerization, synaptogenesis, and the interaction of APP with specific binding partners.211

APP undergoes proteolytic cleavage at extracellular and transmembrane regions by a-, b- and g-secretases, releasing several fragments involved in cellular signaling, metal homeostasis, and neurotoxicity.199 Some proteolytic products preserve intact metalbinding sites observed in the full-length APP, while others display new metal-binding sites, as in the case of the amyloid b-peptide. At the extracellular region, APP family members are cleaved by a-secretase or b-secretase. The a-secretase ADAM10/17 (disintegrin and metalloproteinase domain-containing protein 10/17) cleaves APP at Gln686-Lys687 or Lys687-Leu688 (a-site), producing two fragments: the soluble sAPP-a and the transmembrane APP-C83.199 While no metal-binding sites have been identified in APP-C83, the fragment sAPP-a is released into the extracellular space and preserves intact the metal-binding sites at E1 (GFLD, CuBD) and E2 domains. On the other hand, b-secretases 1/2 (BACE1/2) cleaves APP at Met671-Asp672 (b-site), providing the soluble sAPP-b and the transmembrane APP-C99 fragments.199 All the metal-binding sites at E1 (GFLD, CuBD) and E2 domains are intact in the sAPP-b, whereas a metal-binding site, identical to that of Ab, is formed at the N-terminal of APP-C99. Although

=

CuBD; (C) Left: Pymol solid-state structure for Cuþ binding to CuBD (PDB: 2FK2); right: Proposed coordination sphere for Cuþ in the CuBD; (D) Left: Pymol solid-state structure for Cu2þ binding to E2 domain (PDB: 3UMK); right: Proposed coordination sphere for Cuþ in the E2 domain; (E) Left: Pymol solid-state structure for Zn2þ binding to E2 domain (PDB: 3UMI); right: Proposed coordination sphere for Cuþ in the E2 domain. Cu2þ, Cuþ, and Zn2þ ions are represented by marine blue, green, and gray spheres. Secondary structures are colored in yellow, while secondary structures are colored in yellow (b-sheet) and violet purple (a-sheet).

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The role of d-block metal ions in neurodegenerative diseases Table 3

Dissociation constants for copper binding proteins involved in AD.

Metal Protein

Coordination mode

Kd

Method

Cu2þ APP (CuBD)

2N (His) 1O (Tyr) 2O (water molecules) 2N (His) 1O (Asp) 2O (water molecules) 4N (His)

18 nM

ITC (Competitive chelation)

28 nM

ITC (Competitive chelation)

13 nM

ITC

APP (GFLD)

Cuþ

APP (E2 domain) Ab (1–16) – Ab (3–16) 1N (His) 1N (amide) 1N (N-term) 1O (CO-backbone) Ab (p3–16) 2N (His) 1N (Amide) 1O (Carboxylate or CO-backbone) Ab (4–16) 1N (N-term) 1N (His) 2N (amide) 2N (His) 1O (CO-backbone) 1O (water molecule) Ab (11–16) 1N (N-term) 1N (His) 2N (amide) APP 2N (His) (E2 domain) 1O (Tyr) 1O (water molecule) Ab (1–16) 2N (His)

0.1–10 nM Tyr fluorescence 3.3 nM CD (Competitive chelation)

80 mM

CD (Competitive chelation)

30 fM

Competitive chelation

0.19 mM

Competitive chelation

35 fM

CD (Competitive chelation)

–

–

7.5 mM

Electronic absorption (Competitive chelation)

References: APP,211,219 Ab,220–224

both sAPP-a and sAPP-b preserve the metal-binding sites at E1 and E2 domains, the role of Cu2þ and Zn2þ in the function of these fragments has not been explored. The transmembrane APP fragments (APP-C83 and APP-C99) are splintered by the g-secretase complex in a series of cleavage events, beginning with the endoproteolytic cleavage at Leu672-Val673 (3-site) that produce the APP intracellular domain (AICD), and the consecutive exoproteolytic cuts towards the N-terminal at Val711/Ile712, Ala713/Thr714 or Thr714/Val715, from which p3 fragments are released from APP-C83, while Ab(1–40/42/43) peptides are generated from APP-C99.199 Although the molecular mechanisms that determine the length of Ab fragments produced by g-secretase complex are unclear, the dimerization of APP-C99 before the cleavage has been proposed as a pivotal event to the formation of longer fragments, such as Ab42/43, which are associated with higher toxicity in comparison with Ab40.231 Strikingly, Zn2þ induces the dimerization and oligomerization of APP-C99 in a dose-dependent manner, in spite of the fact that this transmembrane fragment does not display the metalbinding sites associated with APP dimerization at GFLD and CuBD. The Zn2þ-induced dimerization has been associated with a decrease in the proteolytic cleavage of APP-C99 (but not APP-C83) by g-secretase, reducing secreted levels of Ab and AICD and shifting Ab production towards the longest fragment Ab(1–43).232 Despite lacking His residues involved in Zn2þ binding at APP, APP-C99 contains the His residues that bind Zn2þ in Ab (His-6, His13, and His14), which may drive metal-dependent APP-C99 interactions. On the other hand, decreased APP-C99 cleavage by g-secretase is also observed in the presence of copper; however, unlike Zn2þ, Cu2þ cannot induce the dimerization of APP-C99.232 The link between metal ions and the proteolytic processing of APP is beyond its metal-binding properties. The proteases that generate amyloid-b peptidedb-secretase (BACE1) and the g-secretase complexdhave also been implicated in metal homeostasis. BACE1 (beta-site amyloid precursor protein cleaving enzyme) is a type I transmembrane protein with a large extracellular domain (where its catalytic site is found), a single-pass transmembrane domain, and a short C-terminal. The transmembrane domain of BACE1/2 is a unique characteristic that distinguishes BACE1/2 from other aspartyl proteases.233 The formation of BACE1 trimers in cellular membranes was recently demonstrated by single-molecule fluorescence, and it is consistent with previous reports that observed oligomerization of the transmembrane domain of BACE1.234,235 Interestingly, this domain of BACE1 has an MxxxCxxxMxxxCxMxC motif that resembles metal-binding sites found in metal transporters, such as Ctr1. Molecular dynamic simulations suggest that Cuþ binds in the transmembrane region of the BACE1 trimer and translocates from the N- to the C-terminal domain.236 Although the copper-binding motifs of BACE1 resemble the architecture of transporter Ctr1, the role of BACE1 is mainly

The role of d-block metal ions in neurodegenerative diseases

595

Fig. 8 Role of metals on APP function. Synapse components are represented by gradient green color, and APP/APLPs are illustrated intra and extracellularly. The binding of Cu2þ to GFLD and CuBD has an impact on cis-dimerization between APP partners, which triggers cell signaling. While both sites of the E1 domain are important for cis-dimerization, only GFLD has been demonstrated to be important for trans-dimerization, influencing cell adhesion, synaptogenesis, and synapse stabilization. On the other hand, accommodation of Zn2þ to the E2 domain of APP and APLP1/2 enhances homophilic and heterophilic cis-dimerization.

in the organelles, impacting copper compartmentalization inside the cell in a similar fashion to ATP7B.234 Consistent with its plausible role in metal transport, the intracellular region of BACE1 binds the domain I of the copper chaperone for superoxide dismutase (CCS), and it may also bind Cuþ;237 detailed structural studies are needed to elucidate the copper-binding features of full-length BACE1, and their role in its interaction with CCS. Additionally, deficiency of CCS increases Ab production, revealing a role for copper homeostasis in the proteolytic processing of APP.238 Indeed, CCS inhibits Ab production through binding to X11a, a scaffold protein for APP and the g-secretase complex, which is formed by five proteins: Nicastrin, APH-1, PEN2, and the catalytic subunits presenilins (PSEN) isoform 1 and 2. Interestingly, knockout and knockdown of PSEN result in a reduction of Cu2þ and Zn2þ uptake in cell-culture.239 Hence, while there is clearly an interplay between proteolytic processing of APP and metal homeostasis, the mechanisms remain to be elucidated.

2.19.3.2

The amyloid-b peptide

Amyloid beta (Ab) is a peptide of 40–43 amino acids produced by the sequential cleavage of APP by b-secretase at the extracellular region and g-secretase complex at the transmembrane region (as was described in Section 2.19.3.1.3 and Fig. 6). Ab is mainly produced by neurons, but it is also generated from other brain cells that express APP, such as astrocytes and microglia, as well as peripherical cells from the adrenal gland, kidney, heart, liver, spleen, pancreas, muscles, blood, and endothelium.240 In the CNS, Ab levels are dynamic and directly linked with normal neuronal activity.241 Although the final cut for Ab production occurs in endosomes inside the cell, the produced Ab is found intracellularly in organelles such as mitochondria and lysosomes, or extracellularly where it forms the amyloid plaques.242 The N-terminal of Abdthat corresponds to the extracellular region of APPdis highly hydrophilic and binds metal ions, such as Cu2þ, Fe2þ, and Zn2þ (Fig. 9A).195 On the other hand, the C-Terminal of Abdthat

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The role of d-block metal ions in neurodegenerative diseases

comes from the transmembrane region of APPdis highly hydrophobic and promotes the oligomerization and fibrilization of this peptide in aqueous solution, as well as its interaction with membrane-like environments.243 According to NMR data, the N- and C-terminals of Ab monomers lack secondary structure in aqueous solution, while the central region from His13 to Asp23 adopts a-helical structure that interacts with the unstructured N- and C- terminal through hydrophobic residues (Fig. 9A).244,245 In cognitively normal individuals, the production rate of Ab is lower than its elimination rate, yielding low Ab concentrations in the brain.241 In contrast, there is an accumulation of Ab in AD patients, which is associated with overproduction or reduced clearance.246 There are several elimination mechanisms of Ab in the CNS: (i) Degradation by glial cells (microglia and astrocytes); (ii) Proteolytic processing by neprilysin (NEP), the insulin-degrading enzyme (IDE) and matrix metalloproteinases; (iii) Binding to the low-density lipoprotein receptor-related protein 1 (LPR-1) or P-glycoprotein to cross the blood-brain barrier, followed by elimination in the peripherical circulation.240 Interestingly, postmortem analysis of brain tissue from aging mice and transgenic mice APPsw/0 (AD model) shows an increase of copper in brain capillaries, which is associated with the down-regulation of LPR-1 and a reduction of Ab elimination in the brain.247 Consistently, a recent in vivo study, using a fluorescence probe specific for Cu2þ, demonstrated that this metal ion is accumulated in the brain of AD mice, showing an intense fluorescence signal around cortical blood vessels.44 Several X-ray fluorescence studies show co-localization of Ab with copper, iron, and Zn2þ in amyloid plaques from different brain regions of AD mice models and AD patients;42,43,45-49,248 however, copper accumulation is higher in amyloid deposits around blood vessels in comparison with parenchymal amyloid plaques in human AD brain.45 Overall, these studies indicate that copper accumulation at BBB might play a role in alterations of Ab clearance observed in AD. Ab peptide has been linked with several neurotoxic mechanisms, such as ROS production, formation of Ab oligomers that bind membrane receptors, and aggregation in extracellular amyloid plaques.242 Although for several years research of Ab has been focused on pathological mechanisms involved in AD, the evolutionary selection of Ab peptide for at least 430 million years suggests a functional role.249 In the next sections, the metal-binding properties of Ab are described, and its physiological and pathological implications are discussed.

2.19.3.2.1

Copper binding properties to the amyloid-b peptide and its biological implications

Ab binds Cu2þ at its N-terminal region with high affinity (Kd  0.1–10 nM), see Table 3.220 NMR studies indicate that Cu2þ binding to Ab(1–42) affects mainly the first 18 residues.250 Consistently, the equatorial Cu2þ-coordination to Ab fragments 1–16, 1–28, and 1–40 is almost identical.251 CD and EPR studies show that Cu2þ binding to Ab depends on pH, yielding two coordination modes (Mode I and II) at physiological pH with a pKa of 7.8.252–255 Mode I displays a set of EPR signals with g|| ¼ 2.27 and A|| ¼ 180  10 4 cm 1, while Mode II is a complex with g|| ¼ 2.23 and A|| ¼ 154  10 4 cm 1, suggesting an equatorial 3N1O coordination for both complexes.254–256 Acetylation of the terminal NH2 group significantly impacts CD and EPR signals associated with Mode I and Mode II complexes, demonstrating the participation of the free-NH2 group in the coordination sphere of these complexes.252,254 NMR studies elucidated the participation of a deprotonated amide from Ala-2 in Mode II.257 The pKa values of the amide deprotonation and the equilibrium between Mode I and Mode II are  7.8, indicating that deprotonation of Ala-2 drives the formation of Mode II.254,255 His to Ala substitutions and 15N labeling reveals that all three His residues (His-6,  13, and  14) are involved in Cu2þ binding, although ESEEM studies indicate that only one imidazole is present in Mode II, and two His residues are simultaneously bound in Mode I.252,256,258 Consistent with the important role of His residues in Cu2þ binding to Ab, both the mutant H6R associated with early-onset familial AD or murine Ab(1–16) characterized by lacking His13 yield Cu2þ complexes with different spectroscopic features.255,259 Altogether these studies identified the three nitrogen ligands that yield the equatorial 3N1O coordination in Cu2þ coordination to human Ab (hAb). In Mode I, one nitrogen ligand is provided by the free-NH2 group and two by a couple of His residues (His6, 13, or 14), whereas a His residue is replaced by a deprotonated amide in Mode II (Fig. 9B). Conversely, the nature of the oxygen ligand in the Cu2þ-hAb complex remains unclear. Although a pioneer Raman study suggests Tyr10 as the plausible oxygen ligand in Cu2þ-hAb complex, NMR and EPR studies using Tyr to Phe mutants and 17O Tyr labeling discard this possibility.251,252,256,260 NMR signals associated with side chains of Asp1, Asp7, Glu3, and Glu11 were broadening in the presence of Cu2þ, suggesting that these residues participate in metal-binding.257 Mutations of D1N, D7N, E3Q, and E11Q have no impact on the EPR parameters of Mode I and Mode II.255 However, the mutant D1N displays a drastic reduction in the pKa associated with the equilibrium between Mode I and II from 7.8 to 6.0.253,255 In contrast, the mutants E3Q and E11Q show only subtle changes from 7.8 to 7.6 and 7.5, respectively.255 HYSCORE experiments using a 15N and 13C-Asp1 labeled peptide indicate that the carboxylate group from Asp1 participates as an equatorial ligand in Mode I, but not in Mode II, where the oxygen is provided by the carbonyl from Ala-2 (Fig. 9).256,261 DFT calculations support the notion of an equatorial carboxylate ligand in Mode I.262 However, another HYSCORE study suggests that Asp1 may act as an axial ligand, while a backbone carbonyl completes the equatorial coordination shell in Mode I and II.263 Alternatively, ENDOR and DFT studies point to the participation of an axial water molecule that may form a hydrogen bonding with the carboxylate group from Asp1.264 Despite the numerous spectroscopic studies of Cu2þ binding to hAb, no consensus has been reached regarding the nature of the oxygen-based ligands. Moreover, a recent X-ray absorption study proposed the formation of more than two coordination modes at physiological pH, where the predominant species involves the coordination of two His residues.265 Early studies indicate that Ab can produce reactive oxygen species by Cu2þ reduction, showing different reactivity between fragments and species: the most reactive is the hAb(1–42), followed by the hAb(1–40), and the less reactive is rat Ab(1–40).266,267 This behavior has been associated with the aggregation state of Ab and the absence of the His-His motif in the sequence of murine Ab (Fig. 9). 268 It has been proposed that the Cu2þ-Ab complexes accept electrons from Tyr10 or Met35 or reducing agentsdsuch as

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Fig. 9 Ab peptide and its metal-binding properties. (A) Top: Linear representation of Ab peptide, highlighting the metal-binding region at the N-terminal, the hydrophobic region at C-terminal, and the a-helical structure between N- and C-terminal. Ab peptide can bind Cuþ (green circle), Cu2þ (blue circle), and Zn2þ (gray circle) involving the first 16 amino acids. Interestingly, the amino acid sequence of human and murine Ab peptides are different: murine sequence has a Gly5, Phe10 and Arg13 instead of Arg5, Tyr10 and His13. Bottom: NMR structure of Ab in an aqueous environment (PDB: 2lfm), highlighting His-anchoring residues in blue and a-helix in purple. (B) Cuþ-Ab complex formed after reduction of Cu2þ-Ab complex. (C) Coordination mode proposed for Zn2þ-Ab complex.

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Vitamin C (ascorbate), dopamine, 6-hidroxidopamine, and cholesteroldyielding Cu1þ-Ab complexes. Consistently, Met35 oxidation has been observed in AD brains.269 Moreover, XAS studies reveal that Ab coordinates Cuþ after Cu2þ reduction with ascorbate involving two His residues in quasi-linear coordination (Fig. 9).270 Acetylated fragments Ab(6–14), Ab(10–14), and the mutant Y10F also display a His-Cuþ-His coordination and produce H2O2 with similar yields and rates in the absence of ascorbate, suggesting that His13 and His14 are the essential residues for redox activity but no Tyr10 and His6.271 However, considering that the H2O2 production from O2 requires two electrons, the second electron might be provided by other Cu1þ-Ab complexes or by oxidation of the Tyr10 residue.271 According to XANES studies, ascorbate and 6-hidroxidopamine are more potent reducing agents of the Cu2þAb complexes than dopamine, while cholesterol does not show reductive activity.272 In monomeric Ab(1–16), the redox potential of the complexes Cu2þ-Ab is strongly associated with the coordination mode: while Mode I has an E  0.255 V vs normal hydrogen electrode, Mode II displays an E  0.153 V vs normal hydrogen electrode.254 Amyloid fibrils incubated with Cu2þ can also produce H2O2 in the presence of ascorbate by reducing the paramagnetic Cu2þ to the diamagnetic Cu1þ, as demonstrated by NMR studies.273 The Cuþ-Ab(1–40) complex formed with fibrils involves His13 and His14, suggesting similar coordination to those observed in the redox cycling of monomeric Cu2þ-Ab complexes.270,273 Although the molecular details of the reduction mechanism are not entirely understood, it has been proposed that the redox cycling between Cu2þ-Ab/Cu1þ-Ab complexes occurs by a pre-organization electron transfer that involves the formation of a low populated complex termed electrochemical in-between state.274

2.19.3.2.2

Zinc binding properties to the amyloid-b peptide and its biological implications

2.19.3.2.3

Iron binding to the amyloid-b peptide and its biological implications

2.19.3.2.4

N-truncation of amyloid-b and its impact in metal-binding properties

Structural studies of Zn2þ-Ab complexes have been limited because adding Zn2þ to either Ab(1–16) or full-length Ab results in precipitation.275,276 Zn2þ coordination to Ab in solution is likely different from the aggregates since Ab aggregates display a higher metal binding affinity (Kd  100–300 nM) than its monomeric form (Kd  5–10 mM).277,278 Although the coordination mode of Zn2þ bound to Ab aggregates is not clear, it has been proposed that Zn2þ induces cross-linked peptide structures that are metalbridged via His sites.260 Unlike Cu2þ, which prefers the N-terminal group of Ab as its leading anchoring site, Zn2þ preferentially binds to the 8–16 amino acidic region.279 NMR, CD, XAS, and binding affinity studies of Ab(1–16) mutants indicate that Zn2þ coordination to Ab(1–16) monomer at physiological pH involves four to five ligands, including the participation of His6, His13, and His14, as well as one carboxylate side chain from Glu11.280–282 Alternatively, a recent study points to more dynamic behavior, where there may be ligand exchange in a Zn2þ binding site with two imidazole and two carboxylate ligands, namely: His6, Glu11, His13/His14, and Asp1/Glu3/Asp7 (Fig. 9C).283 A protective neurosecretory role for Zn2þ binding to Ab has been proposed in glutamatergic neurons since precipitation of surplus Ab would turn it into nontoxic redox-inert aggregates.284 Moreover, excess Zn2þ would displace Cu2þ from the redox-active site at His13 and His14, limiting Cu2þ coordination to the first six residues, forming ternary Cu2þ-Ab-Zn2þ complexes that potentially prevent damage linked to oxidative stress.285,286

Iron has been observed to co-localize with Ab in senile plaques of AD patients.46 Even though a direct interaction between iron and APP has not been observed (see above), Fe2þ directly binds to the Ab(1–16) fragment, as demonstrated by spectral broadening in 1 H and 13C NMR of Asp1, Glu3, and three His residues (His6, His13, and His14). The proposed Fe2þ binding site is similar to mode I in the Cu2þ-Ab complex.287 In contrast, Fe3þ does not bind to Ab(1–28) under a variety of pH and salt conditions as observed by ESI-MS and 1H NMR, possibly due to the fast precipitation of Fe3þ as a hydroxide that competes for the interaction with Ab.288 Heme metabolism is altered in AD patients: it is observed an increased bioavailability of heme-b and a decreased bioavailability of heme-a. This alteration could be associated with Ab’s ability to coordinate Heme through one of its His residues. Interestingly, hemoglobin is observed to co-precipitate with Ab in senile plaques of AD patients.289 In vitro, Ab(1–16) binds Fe2þ-Heme and promotes redox chemistry with Na2S and molecular oxygen to generate ROS.290 These results indicate that direct iron-Ab interactions are possible in AD; however, this is only one component of a complex alteration of iron homeostasis throughout the brain (see Sections 2.19.2.1 and 2.19.3.1).

Although Ab(1-x) is the most studied, N-truncated Ab peptides, mainly Glu3 (3-x), Phe4 (4-x), or Glu11 (11-x), were identified in amyloid plaques extracted from AD brains (Fig. 10A). Early protein sequencing studies found that the Ab(4-x) isoform accounts for approximately 60% of AD plaques.291,292 Further immunoprecipitation and mass spectrometry analysis confirmed that the Ab(4-x) isoform is present in similar proportions to Ab(1–42), Ab(1–40) and Ab(p3–42), in the cerebellum, cortex, and hippocampus of AD patients.293 Most of the N-terminal truncations of Ab are generated by enzymes that take part in the Ab degradation processes. Many Zn2þ-dependent proteases and peptidases can cleave Ab in vitro or in vivo, such as NEP and the insulin-degrading enzyme (IDE).294,295 Indeed, NEP and full-length Ab levels are inversely correlated, while decreased mRNA levels of NEP are found in brain areas in which amyloid plaques are abundant.296 Incubation of Ab(1–16) with human NEP yields cleavage products, including the Ab(3-x), (4-x), and (11-x) fragments.297 Interestingly, NEP activity is impacted by metal ions, such as copper, displaying a noncompetitive inhibition in vitro (Ki ¼ 1.0 mM).297 In the following sections, how Ab N-truncations impact Cu2þ-coordination is discussed.

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Fig. 10 Copper coordination to N-truncated fragments of Ab. (A) Cleavage sites of Ab(1–42) by NEP or IDE endopeptidases to yield mostly: Ab(3x) purple, Ab(4-x) light blue, and Ab(11-x) green-colored Ab fragments. (B) additionally, the resulting fragments, which harbors N-terminal Glu

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2.19.3.2.5

Ab (4-x) and Ab (11-x) fragments

The metal coordination properties of Ab(4-x) and Ab(11-x) fragments are dictated by their N-terminal sequence (Fig. 10C and F), H2N-Xaa-Yaa-His-, which constitute an amino-terminal copper and nickel (ATCUN) motif,298 as those found in several metalbinding proteins, such as human serum albumin (HSA), histatin-5 (Hst5) and Ctr1. The presence of a His residue in the third posiHis ) coordination sphere, yielding a highly stable tion enables a high-affinity Cu2þ binding via the typical (H2NXaa, N-Yaa, N-His, NIm 299 (5,5,6)-membered chelate ring. The Ab(4–42) isoform displays two copper-binding sites, one with high femtomolar affinity (Kd ¼ 30 fM), and a second site with Kd ¼ 0.19 mM. CD and EPR studies show that the first site forms an ATCUN-type complex at the N-terminal sequence, with EPR parameters (g||: 2.183 and A||: 215  10 4 cm 1) that are consistent with Cu2þ bound to the terminal NH2, two deprotonated backbone amides (Arg5 and His6), and the His imidazole group (Fig. 10F).298 Furthermore, the best fit for the XANES and EXAFS spectra corresponds to a square pyramidal complex with an axial water ligand at 2.36 Å.300 This high-affinity ATCUN motif is able to effectively compete with Ab(1-x) peptides for Cu2þ ions, and it is a redox-inert copper-binding site because it harbors negligible hydroxyl radical production, as compared with the full-length Ab.298 Moreover, the high-abundance of Ab(4–42) in the brain supports a physiological role for Ab(4–x) species as a Cu2þ scavenger that prevents ROS formation in the synaptic cleft.301 Indeed, metallothionein-3 (MT-3), a protein involved in neuroprotection against antioxidant injuries, cannot compete for copper ions with the Ab(4–40/42) isoforms.301 The generation of Ab(4-x) fragments with a femtomolar affinity for Cu2þ would also prevent NEP inhibition by copper ions,297 and it has been demonstrated that the presence of the Ab(4–40) fragment has an impact on the aggregation profile of the full-length Ab(1–40).302 The second copper-binding site appears only after saturation of the ATCUN site. It is proposed to have an equatorial coordination with two His imidazole ligands and backbone amide of His14. These coordination properties resemble those of Ctr1 and Hst5 proteins, which display a bis-His site next to an ATCUN motif. 300 Although Cu2þ coordination to this low-affinity site has not been completely explored, its reduction to Cuþ has been assessed by X-ray absorption spectroscopy under in situ electrochemical control (XAS-EC) experiments. EXAFS experiments at fixed potential of  0.45 V suggest that Cuþ is bound in a four-coordinate quasi-tetrahedral N2O2 complex, with two imidazole groups in a trans arrangement, a backbone carbonyl group from His13, and a water molecule (Fig. 10F). 300 In spite of these findings, the physiological redox relevance of this site remains elusive. Ab(11-x) fragment, which is especially abundant in cerebrospinal fluid Ab(11-x),269 displays a Cu2þ binding-site with Kd ¼ 35 fM, which is comparable with that of Ab(4-x), and within the typical range for ATCUN motifs. EPR and CD studies showed that only the first N-terminal residues of Ab(1–40) are involved in Cu2þ coordination, yielding a square-planar/tetragonal Cu2þ complex with a g|| of 2.17 and a Cu2þ hyperfine splitting of A|| ¼ 193  10 4 cm 1. The N-terminal amino group, two backbone amide nitrogens, and the imidazole of His13 participate in metal binding.303 It is important to note that, in contrast to Ab(4-x), the shorter fragment Ab(11-x) does not display a second metal-binding site, as the bis-His motif is engaged in the ATCUN-binding site. While His13 participates in Cu2þ equatorial coordination, it has been proposed that His14 contributes to metal-binding affinity, and it may participate as an axial ligand, yielding a square-pyramidal Cu2þ complex.303

2.19.3.2.6

Ab (p3-x) and Ab (p11-x) fragments

Having an N-terminal Glu residue, the Ab(3-x) and Ab(11-x) fragments can undergo glutamate cyclization, yielding the Ab(p3–42) and Ab(p11-x) species with an internal lactam named pyroglutamate (pE); this reaction is catalyzed by the glutaminyl cyclase, a highly abundant enzyme in mammalian brain (Fig. 10B). The Ab(p3–42) and Ab(p11-x) isoforms have been isolated from senile plaques, though the latter is less abundant.269,304 The formation of pE from glutamine or glutamate precursors is a common posttranslational event in the processing of bioactive neuropeptides,305 yielding N-term protected species that are resistant to the proteolytic action of aminopeptidases, and thus, remain intact in tissues for a longer period, as compared to their free N-term analogs.304 Most importantly, glutamate cyclization has a profound impact in the metal-coordination properties of Ab fragments, as the terminal NH2 group is no longer available as an anchoring site for the metal ion. The Cu2þ coordination properties of Ab(3-x) and Ab(p3-x) have been studied by CD, EPR, XAS, and NMR.306 Cu2þ binding to Ab(3-x) yields an ATCUN-like complex, where the terminal NH2 group and His6 act as the main anchoring sites (Fig. 10D). In contrast, having its N-terminal NH2 blocked by the pyroglutamate group, Ab(p3-x) coordinates Cu2þ via its His13/His14 motif, yielding two physiologically relevant species. In one of them, the Cu2þ ion is coordinated by His13, His14, a deprotonated backbone amide, and a carboxylate group from Asp7 or Glu11; while a pKa of 7.7 is associated with the deprotonation of a second backbone amide that replaces His13 to yield the second species (Fig. 10E). Consistent with the loss of an ATCUN-like domain, the Cu2þ binding affinity of Ab(p3-x) is two orders of magnitude lower, as compared to the Ab(3-x) isoform. In contrast, Cuþ binding is not impacted by pyroglutamate formation, as the terminal NH2 group does not participate in Cuþ binding. Indeed, both, Ab(3-x) and Ab(p3-x) form linear bis-His Cuþ complexes using His13 and His14.306 Ab(p3-x) is the most abundant N-terminal pyroglutamate species in plaques, and it is localized in diffuse sections, which are formed in the first stages of mature senile plaque formation. Hence, Ab(p3-x) may be involved in early aggregation seeding events associated with the initial

=

residue, undergo Glu cyclization, yielding the Ab(p3–42) purple and Ab(p11-x) green-colored species. These species display a blocked N-term metalanchoring site by an internal lactam named pyroglutamate (pE). (C) Coordination mode of Cu2þ-Ab(11-x). (D and E) There are two coordination modes for Cu2þ-Ab(3-x) and Cu2þ-Ab(p3-x). (F) Coordination mode for Cu2þ-Ab(4-x) in its high-affinity site (left cartoon) and its Cu2þ/Cuþ low-affinity site (right cartoon).

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pathological cascades in the development of AD. The inherent resistance of Ab(p3-x) to degradation by proteases would allow it to remain intact for longer periods of time, as compared to the free N-terminal fragments.307 Finally, Ab(11-x) also undergo glutamate cyclization, yielding Ab(p11-x). Although this isoform is abundant in senile plaques, its metal coordination properties have not been studied.

2.19.3.2.7

Metal ions and Ab aggregation and its pathological implications

Significant effort has been invested in elucidating the structural features of Ab aggregates. The Cryo-EM structure of synthetic Ab fibrils shows that the hydrophilic N-terminal region of the peptide is engaged in the b-sheet structure (Fig. 11).308 CryoEM of Ab fibrils from AD brains shows that morphology is diverse and that fibrils are more intercrossed, as observed in vitro for fibrils in the presence of metal ions.309 Indeed, there is the possibility that metal ions could bridge between fibrils, as proposed for Zn2þ by Raman spectroscopy.260 Interestingly, a study exploring Cu2þ coordination to aggregated Ab shows that it can form both, components 1 and 2 (Fig. 9), depending on the metal/peptide ratio.310 Another study combining EPR and ESEEM found that the His13/His6 or His14/His6 pairs participate in Cu2þ coordination binding by Ab(1–40) fibrils forming mostly component 1.311 Paramagnet induced relaxation in 13C solid-state NMR of Ab(1–40) fibrils with Cu2þ ions confirm N3 coordination by His13 and His14, as well as coordination by the carbonyl groups of Val40 and Glu3, 11, and/or 22.312 EPR, CD and fluorescence studies indicate similar Cu2þ binding affinities of monomeric and fibrillar Ab(1–42).313 Moreover, an EPR study using a modified fluorescent probe of Ab(1–16) estimated a higher Cu2þ binding affinity of Ab aggregates as compared to soluble monomers, such that it would strip Cu2þ ions from human serum albumin.314 Overall, these studies indicate that fibrillar Ab retains the ability of the monomeric peptide to coordinate metal ions. Cuþ binding to aggregated states of Ab has been probed by XAS, showing that the metal ion displays a tetrahedral geometry and is susceptible to oxidation to Cu2þ.315 In contrast, XAS studies of Cuþ binding to Ab oligomers of the English mutation H6R showed a linear bis-His coordination, which showed resistance to metal oxidation.316 Redox cycling of Ab-bound copper, via incubation with ascorbate and oxygen, leads to the formation of di-tyrosine cross-linked Ab dimers.317 A theoretical study shows that Cu2þ bridging between two peptides facilitates the formation of di-tyrosine cross-linked Ab dimers,318 while an ATCUN Cu2þ chelator prevents their formation of dityrosine-bridged.319 However, the impact of these di-tyrosine species in Ab aggregation is not clear. Multiple studies have focused on understanding the impact of Zn2þ and Cu2þ ions in Ab aggregation, as reviewed recently.320 Conflicting results have been reported, as the study of Ab aggregation poses many experimental challenges. Several conditions can impact the study of Ab aggregation, including the length of the Ab fragment, pH, ionic strength, buffer, metal ion concentration, among others. However, there is convergence towards the notion that Cu2þ ions impact Ab aggregation by stabilizing soluble Ab oligomers;320 Also, it is evident that the copper/peptide ratio impacts the lag time of amyloid aggregation.319 On the other hand, Zn2þ ions seem to induce the formation of larger, amorphous, and less toxic aggregates;320 Insights into how these two metal ions stabilize the formation of Ab oligomers have been afforded by recent theoretical studies.321Differential effects of Zn2þ and Cu2þ ions in the hydrophobicity of the peptide may explain the impact of these ions in Ab aggregation.322

2.19.3.3

The tau protein

The tau proteins are a group of six isoforms of MAP (microtubule-associated proteins), which act to promote the assembly and stability of microtubules. Tau proteins are most abundant in neurons of the CNS. Though these proteins are highly soluble and unstructured in vitro, they form structured units when associated with microtubules in neurons during normal function.323,324 Tau proteins are composed of an N-terminal domain, a proline-rich domain, a microtubule-binding region (MTBR), and a C-terminal domain (Fig. 12).325 Of the six isoforms of tau, three contain three MTBR domains, while the other three isoforms contain four MTBR domains such as HTau40. These binding domains are referred to as R1-R4 and are positively charged and located at the C-terminal (Fig. 12). Positively charged semi-repeat domains are associated with the negatively charged microtubules (Fig. 13A). While phosphorylation of tau clearly alters its ability to associate with microtubules, metal ions may also impact this process, by direct binding to the MTBR domain and/or by modulation of kinases involved in the phosphorylation of tau. Both, metal binding to tau and its phosphorylation state are altered in AD. Hence, in this section, the role of metals and metal homeostasis on the function and phosphorylation of tau are discussed.

2.19.3.3.1

Copper-binding properties of tau protein and its biological implications

Copper dyshomeostasis is observed in AD patients and thus can alter copper-tau interactions. Tau is located intracellularly, where copper ions are mostly in their reduced form Cuþ, yet all studies on copper binding to tau have involved Cu2þ ions. Zweckstetter first reported Cu2þ binding to a 198 amino acid fragment of the protein tau, using nuclear magnetic resonance, circular dichroism, light scattering, and microcalorimetry.326 NMR spectroscopy showed that two short stretches of residues, 287VQSKCGS293 and 310 YKPVDLSKVTSKCGS324, are primarily involved in copper binding.326 Indeed, these two regions are contained in the R2 and R3 fragments of tau (Fig. 12). Moreover, most studies have used peptide fragments R1 to R4, where the N- and C- terminal groups are not protected, which leads to Cu2þ coordination modes that would not be physiologically relevant, at least for the R1, R2, and R4 domains. An NMR and ESI-MS study showed Cu2þ binding to each of the individual R1 to R4 fragments.327 Redox cycling has been observed in complexes of Cu2þ and the R1 or R3 fragments.328 The R1 and R3 complex redox is consistent with an earlier electrochemical study using immobilized tau, reporting a redox potential for the Cu2þ/Cuþ pair of 339  5 mV versus NHE. Though there is no consensus

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Fig. 11 Ab monomers and fibrils, showing the secondary structure and the metal-anchoring residues. (A) Crystal structure of Ab in an aqueous environment (PDB: 2lfm), highlighting His-anchoring residues in blue and a-helix in purple. (B) Cryo-EM structure of Ab fibrils formed with recombinant peptide (PDB: 5oqv) without metals, showing in cyan the metal anchoring residues His6, His13, and His14 engaged in a b-sheet structure. (C) Cryo-EM structure of Ab fibrils purified from meningeal AD tissue (predominant morphology, PDB: 6SHS). Unlike fibrils formed with recombinant Ab, His13 and His14 (cyan residues) are not engaged in the b-sheet structure.

on the nature of the Cu2þ binding sites, it is clear that the His residues are key anchoring sites for Cu2þ ions (Fig. 12),327,328 and that the Cys residues in R2 and R3 fragments likely provide alternative coordination environments and redox activity (Fig. 12). The aforementioned Cys291 and Cys322 in R2 and R3 respectively have been shown to form disulfide bridged dimers, as demonstrated by ESI/MS, which can be catalyzed by Cu.327 While Cu2þ-induced dimerization is an interesting phenomenon, this event would only be physiologically relevant once the cells are dead, and the neurofibrillary tau tangles are exposed to extracellular Cu2þ ions. More studies on Cuþ binding to full-length tau are needed for a better understanding of the role of this interaction in the early stages of tau dimerization and aggregation.

2.19.3.3.2

Zinc-binding properties of tau protein, aggregation, and toxicity

In vivo studies provide a link between the impact of Zn2þ in tau aggregation and toxicity. A Drosophila model of tauopathy shows that Zn2þ contributes to tau toxicity by increasing tau phosphorylation and by directly binding to tau. Reduction of Zn2þ levels,

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Fig. 12 Depiction of unstructured HTau40. The N-terminal domain is highlighted in green, the proline-rich domain in yellow is, and the four MTBR domains in orange. These four domains are responsible for the binding of Tau protein to the microtubules, as illustrated at the bottom of the figure. The figure also illustrates the sequence of the MTBR domains. The metal-anchoring histidine residues are represented in blue and cysteine residues in red.

through genetic or dietary changes, rescues Drosophila tauopathy.329 Additionally, a study performed in neuronal cells overexpressing a pathological mutant DK280 of human tau showed that Zn2þ significantly accelerates tau fibrillization, causing neurotoxicity.330 Consistently, in vitro studies show that Zn2þ accelerates Tau non-fibrillar aggregation via binding/bridging to Cys291 and Cys322, as shown in a Tau244–372 fragment.331 ITC studies show that tau displays one Zn2þ binding site with a low micromolar Kd.330,331 Mutation of Cys291 and Cys322 decreases Zn2þ binding affinity.330 The elimination of the Zn2þ binding site by substitution of these Cys residues eliminates tau toxicity in both, the Drosophila329 and neuronal cell culture model.330 Similar Zn2þ-Cys coordination has also been observed by NMR with the R3 and R4 peptides.332 Zn2þ can induce changes in folding in the R1 and R4 peptides as determined by CD, which agrees with increased aggregation of R1 and R4 peptide fragments in the presence of Zn2þ.333

2.19.3.3.3

Iron and tau hyperphosphorylation

2.19.3.3.4

Metal ions and tau kinases

As tau is found intracellularly, it is likely that any direct interaction between tau and iron will be with Fe2þ. Although iron has been shown to induce precipitation of tau, it has not been shown to directly interact with tau. The addition of Fe2þ induces precipitation of both tau and hyperphosphorylated tau, but it is not clear if this effect is caused by Fe2þ ions and not Fe3þ, as the study was performed under aerobic conditions.334 Indeed, Fe3þ ions induce oligomerization335 and the formation of granular aggregates of hyperphosphorylated tau.336 However, given the intracellular localization of tau, the physiologically relevant ion would be Fe2þ and not Fe3þ. The more relevant interaction involving Fe2þ and tau is likely related to metal-induced changes in the phosphorylation state of tau.

Deposits of highly phosphorylated tau protein are a key pathological feature of AD.337,338 Many kinases phosphorylate tau and their dysregulation in AD results in tau hyperphosphorylation. Interestingly, some tau kinases display metal-dependent activity. Zn2þ has been shown to stimulate proline-directed kinases glycogen synthase kinase-3 (GSK-3) which leads to higher Tau phosphorylation levels.339 Also, Zn2þ chelation was shown to decrease the phosphorylation activity of tyrosine-dependent Syk kinase.340 Fe/H2O2 induced oxidative stress induces an imbalance in the function of cyclin-dependent kinase-5 (CDK5) in hippocampal neurons, impacting tau phosphorylation at Ser202, Thr205, and Ser235 residues.341 Another study found that iron induces the activity of CDK5 and GSK3, causing tau phosphorylation at Thr205, Thr231, and Ser396, while this effect was abolished upon treatment with an iron chelator.342 Metal dyshomeostasis, Tau fibril formation, and tau hyperphosphorylation are all key pathologies of AD, though it is not known the extent to which these pathologies correlate. Some key features of metal binding to Tau, metal-

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Fig. 13 Tau protein interacting with microtubules (A), and tau filaments from AD patients (B-C). (A) Cryo-EM structure of the R2 fragment of tau protein interacting with microtubules (PDB: 6cvn). The putative metal-anchoring residues Cys291 and His299 are shown in red and cyan. (B) CryoEM structure of the common form of paired helical filaments of tau that are observed in patients with sporadic and inherited AD (PDB: 6hrf). The amyloid core is characterized by forming a C shape that embraces R3, R4, and C-terminal residues, where the putative metal-anchoring residues His329, His330, His362, His 374, and Cys322 are highlighted in cyan and red. (C) Cryo-EM structure of the amyloid core of paired helical filaments of tau from the cerebral cortex of a female 74-years-old patient (PDB: 5o3l).

induced aggregation, and metal-dependent phosphorylation have been elucidated; however, this picture is still incomplete. Further insight is needed regarding interactions of Fe2þ and Cuþ with Tau, namely full-length Tau.

2.19.3.4 C

The prion protein in Alzheimer’s disease

PrP dwhich plays a critical role in prion disease (Section 2.19.2)dis also present in amyloid plaques from AD patients,343–348 where it co-immunoprecipitates with Ab and tau.348 Interestingly, the expression levels of PrPC change during the evolution of

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the AD pathology: it increases in the early stages of the disease and decreases in the late stages, correlating with tau expression.347 On the other hand, PrPC acts as a receptor of the different aggregation stages Ab involving residues from the metal-binding regions pf PrPC.349 Ab monomers bind the non-octarepeat region (93–113) and the structured C-terminal (123–166) of PrPC;350 soluble Ab oligomers bind to regions N-terminal region (23–27) and part of the non-octarepeat region (95–110);349,351 and fibrillar Ab oligomers bind to the regions the non-octarepeat region 95–113 and the structured C-terminal 122–231.352 Soluble Ab oligomers have been recognized as playing a pivotal role in nearly pathogenesis in AD, as they are neurotoxic353 and impair memory.354 Several in vitro and in vivo studies suggest that Ab oligomers can act as neurotoxic agents by their interaction with PrPC, causing the neuronal damage observed in AD mice.349,355–366 Although most of the studies point that the Ab-PrPC interaction is responsible for the cognitive impairment in AD, it has been also demonstrated that this interaction neutralizes the toxicity of Ab oligomers when it occurs with the shed-PrPC or the N1 (23–110) fragment outside the membrane.367–371 Although the interaction between Ab and PrPC requires residues involved in metal-binding, the role of metals in this interaction has not been deeply studied.110 Recently, it has been demonstrated that Cu2þ can induce ternary complexes between Ab and peptide fragments of PrP.372 An alternative copper-dependent interaction of PrPC protein in the AD context is with the N-methyl-D-aspartate receptors (NMDARs).113 NMDARs are ligand-gated Ca2þ channels that are activated by glutamate and glycine. Signaling mediated by Ca2þ influx through NMDARs plays an essential role in memory and learning acquisition.373 Overactivation of NMDAR can lead to neuronal damage due to Ca2þ overload.374 Under persistent NMDAR agonist exposure and physiological concentrations of glycine, desensitization is observed in cultured rodent hippocampal neurons.375,376 PrPC binds NMDAR and promote NMDAR desensitization, avoiding excitotoxicity damage in a mechanism that depends on copper ions.113,151 A recent study show that NMDAR desensitization by PrPC requires His residues from octarepeat and non-octarepeat sites.151 Both, the Ab monomers and oligomers inhibit this modulation mechanism.113 recent works have shown that Ab species can alter the Cu2þ binding of PrPC protein by the chelation of Cu2þ from specific Cu2þ binding sites from PrP.372 Additionally, it is proposed that copper and PrPC promote desensitization by a mechanism that is also nitric oxide (NO•) dependent, opening the door for the possibility of a PrP-Cu2þ catalyzed S-nitrosation.114 The connection between PrP protein, Ab, copper, NO, and NMDAR suggests an interesting role for all of these players in AD and an important relationship between PrP protein and Ab monomers and soluble Ab oligomers.

2.19.3.5

Metal ions as therapeutic target for Alzheimer’s disease

Unbalance in metal homeostasis has been observed in AD mouse models and patients (Table 1).38–45 AD patients display changes in copper speciation in blood;28–33 in AD brain, copper is decreased while Zn2þ and iron are increased;38–45 and amyloid plaques accumulate copper, zinc, and iron.46–49 Hence, chelating agents have been proposed as a therapeutic approach to restore metal homeostasis. Some synthetic chelating drugs have been tested in pre-clinical and clinical phases, such as D-penicillamine, clioquinol, and PBT2.33,377–388 D-penicillamine is a copper chelating agent, which cannot easily cross the blood-brain barrier. In a clinical pilot phase II, AD patients treated with D-penicillamine orally display a decrease in the superoxide dismutase activity in erythrocytes and serum, as well as an increase in the excretion of copper through urine; however, these patients do not show cognitive benefits.33,377,378 Recently, a hydrogel nasal formulation of D-penicillamine showed promising results, improving cognitive performance in an AD mouse model,379 possibly due to its higher bioavailability in the brain in comparison with oral penicillamine administration. On the other hand, clioquinolda Cu2þ and Zn2þ chelating agentdhas been evaluated in several neurodegenerative diseases, including AD.381 In a clinical phase II study, clioquinol treatment decreases Ab levels in the blood; however, it has no effects on cognitive function.381 Finally, PBT2 emerged as a Cu2þ and Zn2þ chelating agent of second generation with the same metal-binding motif as clioquinol.389 PBT2 display enhanced therapeutic effects in vitro and in vivo in comparison with clioquinol.386 Recent evidence suggests that PBT2 regulates copper homeostasis by using an alternative mechanism different from clioquinol.390 Despite the alleviating effects of PBT2 in AD, this type of therapy is still under controversy due to the role of metals in learning and memory processes, including the understanding of the interaction of d-block metals with Ab under physiological and pathological conditions.391 Several mechanisms have been proposed for PBT2, such as: increasing the availability of metal ions in the cytosol,392 preventing accumulation of metal ions in extracellular amyloid aggregates, inhibiting glutamate-induced neurotoxicity,393 and increasing Ab degradation.394 However, further studies are required to elucidate the pharmacodynamics of this compound.

2.19.4

Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder estimated to affect > 6 million people worldwide.395 PD is a progressive neurodegenerative disorder with clinical manifestations that include bradykinesia, resting tremor, rigidity, loss of postural stability, sleep disorders, and cognitive impairment. PD was first described by Dr. James Parkinson in 1817,396 and it is defined by the progressive destruction of nigrostriatal dopaminergic neurons, triggering dopamine deficiency.397 However, neuronal loss has also been identified in other regions within the CNS.398 The etiology of PD has been proposed as a combination of genetic and environmental factors.399 It is estimated that 5–10% of all PD cases are attributed to genetic autosomal dominant and autosomal recessive forms of the disease, involving 13 genetic loci, PARK1-PARK13.400,401 The rest of the cases are considered idiopathic, involving genetic susceptibility and environmental factors.402 Environmental factors associated with PD include: pesticides, such as paraquat, rotenone, and organochlorines; metals exposition to iron, copper, manganese, lead and mercury and solvents.402

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PD, multiple system atrophy (MSA) and dementia with Lewy bodies, are part of a group of neurodegenerative disorders termed a-synucleinopathies.403 A hallmark of a-synucleinopathies and PD are cytoplasmic inclusions known as Lewy bodies; a dense core encircled by a halo of 10-nm-wide radiating fibrils composed mainly of misfolded a-synuclein.404 a-synuclein is a cytosolic protein of 140 amino acids, belonging, together with beta- and gamma-synuclein to the synuclein family.405 The aggregation of a-synuclein is associated with synucleinopathies404,406 Three point mutations, A53T, A30P and E46K, in a-synuclein have been linked to dominant early-onset familial PD.407–410 Locus duplication and triplication have been described in certain forms of familial PD, indicating that increased expression of alpha-synuclein is also directly involved in the synucleinopathies.411 Besides a-synuclein, other proteins, such as parkin and synphilin-1 are also found as Lewy bodies, along with metal ions, such as iron.412,413 A brief summary of some proteins implicated in PD is provided below. Synphilin-1 is a 919-amino-acid protein that is expressed predominantly in presynaptic nerve terminals; it has been identified as a component of the central core of Lewy bodies in the brains of sporadic PD patients. The function of Synphilin-1 is unknown, but it can bind to synaptic vesicles, probably exerting a concerted synaptic function with a-Syn.414,415 Synphilin-1 can interact, in vitro and in vivo, with parkin and the 1-60N-terminal region of a-Syn; it enhances phosphorylation of Ser129 in a-Syn, impacting its aggregation and degradation.416–418 Indeed, synphilin-1 could act as a modulator of the ubiquitin-proteasome system, and it may play a role in other neurodegenerative diseases.415 Parkin protein is a multifunctional E3 ubiquitin ligase, encoded by the PARK2 gene, involved in ensuring the quality control of protein conformation and mitochondrial function. Overexpression of parkin provides protection against cellular stress and loss of dopaminergic neurons in cell culture.419 Recessive mutations in the PARK2 gene are the most common causes of familial early-onset PD.420 Interestingly, PD patients with PARK2 loss-of-function alleles exhibit heightened vulnerability to Cu2þ and Cd2þ cytotoxicity, but no difference in sensitivity to Mn2þ or methylmercury relative to control subjects.421 Parkin may also be implicated in Zn2þ homeostasis, as genetic models of PD in C. elegans show that mutations of orthologs of PARK9 and PARK2 cause increased Zn2þ absorption.53 Indeed, several studies point to a role of loss of Zn2þ homeostasis in PD. Increased Zn2þ levels are observed in SN and SNpar of the PD brain,56 while decreased Zn2þ levels are found in serum and plasma of PD patients.57 Consistently, mitochondrial inhibitor models of PD induce Zn2þ accumulation,422 while a synergistic effect of Zn2þ and dopamine in cell death has been observed in vivo.423 Moreover, loss of function mutations in the PARK9 gene, coding for a vesicular Zn2þ ATPase transporter (ATP13A2), induces loss of Zn2þ homeostasis and mitochondrial dysfunction.424 Finally, another protein implicated in PD is DJ-1, a member of the DJ-1/ThiJ/PfpI superfamily, encoded by PARK7 gene. Mutations causing a substitution of proline for leucine at residue 166 (L166P) in the DJ-1 are linked to an autosomal recessive form of familial early-onset PD425,426 Interestingly, although DJ-1 has not been linked to metal homeostasis, it is reported as a metalbinding protein, as discussed below. Among the proteins implicated in PD, only a-synuclein and DJ-1 are metal-binding proteins. In the next sections, the metallobiochemistry of these two proteins, and their physiological and pathological implications are discussed. Finally, therapeutic strategies are analyzed.

2.19.4.1

DJ-1 protein

DJ-1 is ubiquitously expressed in body tissues, including gastrointestinal tract, pancreas, and brain areas.425 In the organism, DJ-1 forms a homodimer, where each monomer consists of 189 residues. Structurally DJ-1 folds into 11 b-strands (b1-b11), which form a central core, and eight flanked a-helices (a1-a8). The dimer interface is formed mainly by the interaction of a1, a7, a8 and b4, with the a1 helix of the two subunits running parallel to one another and coming into close contact (Fig. 14A).427 Human DJ-1 has three cysteine residues, Cys46, Cys53, and Cys106 of which Cys106 is highly conserved.428 Cys106 is situated in a distinctive strand-turnhelix motif and in a major surface dent (Fig. 14A).427 The pKa of Cys106 is around 5.4, this makes this residue very sensitive to oxidation.429 It is now well accepted that DJ-1 possesses a direct antioxidant and glyoxalase function and could serve as a sensor for oxidative stress inside cells, where Cys106 appears essential for DJ-1 antioxidant activity.430 It also serves as chaperone protein to prevent protein aggregation and ROS formation.431,432. Interestingly, the oxidation state of Cys106 could be regulated by the chaperone ability of DJ-1.433 The partially oxidized DJ-1 has the ability to sequester a-synuclein monomers, blocking the early stages of a-synuclein aggregation.434

2.19.4.1.1

Metal-binding properties of DJ-1 protein

DJ-1 protein can coordinate copper, zinc, and mercury ions. An ITC study exploring the metal binding properties of DJ-1 revealed a Zn2þ binding site with a Kd in the micromolar range, and a 1:1 stoichiometry.54 The highly conserved Cys106 is essential for Zn2þ binding to DJ-1. The crystal structure of Zn2þ bound to DJ-1 shows a distorted tetrahedral binding site where the metal ion is coordinated by Cys106, Glu18, and two water molecules (Fig. 14C).54 On the other hand, an X-ray fluorescence study identified Cu2þ, Cuþ and Hg2þ as metal ions that can bind to DJ-1, with Kd values of 3.1 mM, 5.6 mM, and 60 nM, respectively.435 The C106A mutation abolishes Cu2þ and Hg2þ binding to DJ-1. Expression of DJ-1 protects cells from copper and mercury-induced cytotoxicity, while the hereditary PD mutations A104T and D149A impact the metal-binding properties of DJ-1 and its protective effect; however, the involved mechanisms might not be limited to metal binding and require further investigation.435 DJ-1 displays two binding sites for copper ions, one located at Cys53 where Cuþ binds, and a second site where Cys106 can anchor both, Cuþ and Cu2þ ions. A study of Cuþ binding to DJ-1 by absorption spectroscopy, mass spectrometry, X-ray fluorescence spectroscopy and X-ray crystallography reveals that the metal ion bridges between two protein molecules in the DJ-1 homodimer via

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Fig. 14 Copper and zinc coordination to the DJ-1 protein. (A) Linear representation of each DJ-1 monomer. Violet segments represent a-helix and yellow represents b-sheets secondary structures. Metal-binding residues are also represented: Cys48, Cys53 and Cys106. Metal ions are represented as colored circles as depicted in the captions. (B) Assembly of DJ-1 monomers to give DJ-1 homodimers. (C) Zn2þ-coordination to DJ-1. (D) Copper-coordination to DJ-1. (d.1) Cuþ-coordination to homodimer interface. (d.2) Cu2þ-coordination to DJ-1 monomer.

Cys53.436 This binding site displays a high affinity for Cuþ (Kd of 6.4  10 16).436 (Fig. 14B–D) On the other hand, Cu2þ binding to DJ-1 has been studied by X-ray crystallography, X-ray Fluorescence, EPR, electronic and atomic absorption spectroscopy. Each DJ1 dimer binds two Cu2þ ions, with a Kd ¼ 3.97  10 4 M.437 The crystal structure shows a Cu2þ ion in a trigonal geometry around Cys106 residue in each monomer. The coordination sphere in the two sites involves the same ligands but differs on the Cu2þ-Glu distance. The Cu2þ is coordinated to a sulfur atom from Cys106 at 2.09 Å, a water molecule at 2.23 Å and a carboxylic oxygen from Glu18 at 2.00 or 2.10 Å; Cu deviates only 0.4 Å from the Cys106/Glu18/H2O plane (Fig. 14D). Indeed, EPR spectroscopy confirms the presence of two slightly different Cu2þ species in DJ-1, showing two signals with g|| ¼ 2.25, A|| ¼ 155.46  10 4 cm 1 and gII ¼ 2.22, A|| ¼ 186.56  10 4 cm 1, indicative of coordination spheres with nitrogen and oxygen-based ligands. Despite the fact that His126 residue is located in close proximity to the Cys106 side chain426, it does not appear to coordinate the metal ion in the reported crystal structure, where the trigonal geometry is likely a result of photoreduction to Cuþ.430,437 Regardless of metal oxidation state, Cys106 is clearly an anchoring residue for Cu ions, and it has been proposed to confer DJ-1 with a chaperone function for SOD1.437 Interestingly, Cys106 is essential for all the enzymatic activities associated with DJ-1 that have been reported,430 while the redox state of Cys106 determines its interaction with AS and its ability to prevent AS aggregation.434 Overall, DJ-1 is a metal-binding protein and Cys106 is one of the metal anchoring residues that confers this protein with its ability to act as a redox buffer and chaperone. Further studies of the metal binding properties of DJ-1 should provide insights into its physiological functions and its role in PD.

2.19.4.2

a-Synuclein

Alpha-synuclein (AS) protein is predominantly expressed in presynaptic terminals of neurons of the CNS. Physiological roles proposed for AS include uptake, storage, recycling of neurotransmitter vesicles, auxiliary chaperone in the synapses as well as maintenance of dopamine levels.438–441 AS structure contains three different regions: a N-terminal region (residues 1–60) that is involved in lipid binding and contains imperfect repeats of the sequence; the non-amyloid component region (NAC, residues 61–95), which is highly hydrophobic and

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fibrillogenic; and the C-terminal region (residues 96–140), which is rich in Pro, Asp and Glu residues (Fig. 15). Although AS is an intrinsically disordered protein, its association with membranes promotes an a-helical structure at its N-terminal region, involving residues 1–37 and 45–92.442,443 On the other hand, long range intramolecular contacts of its C-terminal with the NAC and N-terminal regions have been identified to stabilize monomeric AS.444,445 Pioneering studies revealed that AS is a metal-binding protein, since essential metal ions such as Cu2þ and Fe3þ impact AS amyloid aggregation.446 Further studies evaluated the impact of physiologically relevant micromolar concentrations of divalent metal ions, including Mn2þ, Zn2þ, Fe2þ, Co2þ, Ni2þ and Cu2þ, and found that Cu2þ ions exert the most significant increase in amyloid fibrillation of AS.447,448 Consistently, 2D heteronuclear nuclear magnetic resonance (NMR) and mutagenesis studies revealed site specific interactions of Cu ions with AS, that are distinct from all other metal ions. The Asp and Glu residues contained in the C-terminal 119-124 region, with sequence DPDNEA, constitute the main anchoring site for metal cations, including Ca2þ, Fe2þ, Co2þ, Ni2þ, Mn2þ, Zn2þ, Fe3þ, and Cu2þ. This region is a non-specific and low binding affinity (conditional Kd  10 3 M) metal anchoring site, where electrostatics dominate the nature of the metal-protein interaction.447,448 In contrast, Cu2þ and Cuþ ions coordinate with high affinity at two other specific binding sites at the N-terminal region: a site involving the first six residues of AS, MDVFMK; and the only His residue of AS, His50.448–452 His50 is also a weaker anchoring site for other imidazole-binding metal ions, such as Fe2þ and Zn2þ. An NMR study demonstrated that Zn2þ binds to His50 with an apparent dissociation constant in the millimolar range.453 This low binding affinity challenges the biological relevance of AS-Zn2þ interactions. In contrast, a higher binding affinity of AS for iron ions has been described. Hence, the site-specific interactions of AS with the essential copper and iron ions are discussed in detail below. Also, given that recent work supports a role for the interaction of Ca2þ ions with AS in vesicular trafficking, a discussion of Ca2þ binding to AS is also presented. The discussion of the metal binding properties of AS is accompanied by a summary of how the homeostasis of each metal ion is impacted in PD.

2.19.4.2.1

Calcium-binding properties of a-synuclein and its biological implications

Calcium homeostasis is perturbed in all neurodegenerative diseases, including PD, usually manifested by disruption of the Ca2þ buffering capacity, disruption of mitochondrial Ca2þ homeostasis, and deregulation of Ca2þ channels.454 Indeed, alterations in the Ca2þ buffering capacity are observed in transgenic mice expressing human WT a-synuclein, a model of synucleinopathy, suggesting that a-synuclein may impact Ca2þ dynamics.455 Moreover, significant levels of Ca2þ have been detected in Lewy bodies of PD patients.456 AS binds Ca2þ ions through its C-terminal region, as demonstrated by several biophysical methods. Early microdialysis studies showed that AS binds Ca2þ at its C-terminal region with a Kd ¼ 2–300 mM,457 A study combining ion mobility mass spectrometry, small angle X-ray scattering, transmission electron microscopy and molecular dynamics, demonstrated that AS can bind up to four Ca2þ ions, and that Ca2þ can induce a structural transition of AS monomers to extended conformations where the NAC region is exposed, triggering AS fibrillation.458 Another NMR and mass spectrometry study showed that AS can

Fig. 15 Metal coordination to a-synuclein. Schematic linear representation of a-synuclein. N-terminal, NAC and C-terminal domains are highlighted in different colors. Post-translational modifications are also depicted in the upper part of the strip. a-synuclein contains three anchoring sites for metal ion binding. In the lower part of the strip are represented the sequences of each site and the metals that can bind according to the metal-ion caption (right square). The coordination spheres around Cu2þ and Cuþ are also represented in the bottom part of the figure inside of the dotted squares.

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bind up to 6–8 Ca2þ ions with a Kd of 21 mM,459 a binding affinity that lies within the physiological pre-synaptic Ca2þ concentration fluctuations. Ca2þ binding to AS impacts its ability to associate with membranes and results in its dissociation from the membrane surface.460 Indeed, Ca2þ-AS interactions are proposed to impact AS lipid-binding capability. A solution-state NMR study showed that AS interacts with isolated synaptic vesicles at its N-terminus and via its C-terminal, the latter being regulated by Ca2þ binding to AS.459 Hence, under physiological conditions AS might act as a Ca-dependent modulator of presynaptic vesicle homeostasis, while AS localization at the presynaptic terminal is mediated by this metal ion. Conversely, under pathological conditions, Ca2þ or AS imbalance, as observed in PD, can lead to synaptic vesicle clustering and neuronal toxicity.459 In summary, Ca2þ binding to AS emerges as an important interaction that modulates AS binding to synaptic vesicles, underscoring the key role of Ca2þ binding to AS in health and disease.

2.19.4.2.2

Iron-binding properties of a-synuclein and its biological implications

Meta-analysis of brain metal levels in PD patients show significant iron accumulation in SN, putamen, nucleus caudatus and globus pallidus;50 while no changes in serum iron levels are found.51 Furthermore, mitochondrial iron transport, via the transferrintransferrin receptor (Tf/TfR2) complex is disrupted in PD,461 while genetic variation analysis supports the notion that this complex plays an important role in the etiology of PD, through iron dysregulation in dopaminergic neurons.462 Moreover, AS-knockout mice display lower levels of ferritin expression, as compared to WT mice, underscoring a link between AS and iron homeostasis.463 Indeed, AS can bind Fe2þ and Fe3þ ions at His50 and at its non-specific C-terminal site, as mentioned above. An isothermal titration calorimetry (ITC) study revealed a single binding site for Fe3þ with an apparent Kd  10 5 M;464 while the possibility of AS having two independent binding sites for Fe3þ has also been suggested.465 On the other hand, the formation of a 1:1 complex of AS with Fe2þ was revealed by a study combining mass spectrometry, fluorescence and cyclic voltammetry (CV).466 A reduction potential  0.025 V vs. Ag/AgCl was determined for the AS-Fe2þ complex, which can reduce O2 to H2O2, yielding a putative AS-Fe3þ complex. A recent seminal study has shown that this redox activity has an impact on the conformation of the acetylated form of AS and the structural properties of its aggregates.467 While there is evidence for Fe binding to AS, a structural characterization of these redox active sites is needed to understand the physiological and pathological implications of Fe-AS interactions.

2.19.4.2.3

Copper-binding properties of a-synuclein and its biological implications

Although occupational exposure to copper is linked to an increased risk to develop PD,468 a meta-analysis found no significant variations in serum copper concentrations between PD patients and healthy controls.51 However, copper speciation changes in blood serum of PD patients have been observed, namely, decreased ceruloplasmin-bound copper levels,52 which in turn could impact iron homeostasis. Moreover, decreased Cu and Ctr1 levels are found in surviving neurons of SN and locus coeruleus, suggesting a loss of Cu homeostasis in the PD brain.469 AS, the PD protein, displays three different binding sites for Cu2þ (Fig. 15). The lowest affinity site (site 3) was identified using two-dimensional heteronuclear NMR spectroscopy at 119–124 residues of the C-terminal region. EPR spectroscopy of Cu2þ complex at C-terminal region shows a signal with g|| ¼ 2.316 and A|| ¼ 176  10 4 cm 1, indicative of a 4O coordination, corroborating the involvement of carboxylates of Asp-119, Asp-121, Asn-122, and Glu-123 as major contributors to metal binding. This site displays low specificity for metal cations and a low affinity with a Kd in the millimolar range, as determined by NMR, UV-vis spectroscopy, and equilibrium dialysis experiments.447,448 In contrast, the N-terminal region of AS displays two high sites of Cu2þ binding. The presence of two independent sites was demonstrated by protein mutations and modification by diethyl pyrocarbonate (DEPC), MALDI-TOF mass spectrometry, NMR, EPR, UV-Vis and CD spectroscopies. Site 2 was identified at around His50 the only His site in the whole protein and site 1 at the a-NH2 group of Met1 at 1-6 region of AS. The Cu2þ binding affinity of these two sites are significantly higher than that for the C-terminal site, with conditional Kd values in the nanomolar range.432,448,470 At His50, the EPR parameters show a g|| ¼ 2.228 and an A|| ¼ 179  10 4 cm 1, indicating a ligand set of 3N1O. The metal ion is anchored by the imidazole ring and two deprotonated amide groups to yield a 3N1O coordination mode, with an oxygen atom from a carbonyl group or a water molecule.449,471,472 Using isothermal titration calorimetric (ITC) the apparent dissociation constant of this site was determined, obtaining a value of 3.5  0.4  10 5 M.473 At the N-terminus of AS is located the highest affinity site for Cu2þ with an apparent Kd ¼ 1.0  0.1  10 7 M.473 The EPR signals associated with this site are gII ¼ 2.250 and A|| ¼ 189  10 4 cm 1, indicating a 2N2O equatorial coordination mode. The ligand donor set is composed of the a-NH2 group of Met1, a deprotonated backbone amide, the carboxylate group belonging to Asp2 and a water molecule.443,473 In addition, the participation of Met1 as an axial ligand in site 1 has been proposed. 474 On the other hand, the participation of His50 in the high affinity N-terminal site forming a macrochelate has been proposed by the results of X-band electron spin-echo modulation (ESEEM) experiments.475 An EPR study using high-affinity competitors shows a conditional Kd ¼ 1.0  0.1  10 7 M for this site. However, direct mutations, CD and NMR experiments clearly show evidence of two independent sites at the N-terminal region of AS.449,476 On the other hand, copper is a redox-active metal, indeed the reactivity of AS-Cu2þ complexes can yield the formation of reactive oxygen species (ROS). An NMR study demonstrated that the redox cycling of copper bound to AS can trigger the site-specific metal-catalyzed oxidation on methionine residues.477. Met1 and Met5 residues can be oxidized rapidly (Met1 faster than Met5) to a sulfoxide species, whereas Met116 and Met127 remain unaffected.477,478 A consequence of the Cuþ/O2 chemistry of the AS-copper complex is the chemical modification, as was demonstrated by HPLC-mass spectrometry analysis, in which the sulfoxide species formed at the N-terminal of AS suffer elimination of methanesulfenic acid, rendering a species with no thioether moiety.474 The mechanism of O2 activation by AS-Cuþ remains to be elucidated, however the structural details of Cuþ bound to AS have been extensively studied. The Cuþ binding to AS was preliminary studied using NMR spectroscopy. Three different sites have been identified. The site 3, or the lowest affinity

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site, is located at the C-terminal region, Cuþ is bound through Met116 and Met127 with a conditional Kd ¼ 88  10 9 M. The sites 2 and 1 are located at the N-terminal region. The His50 acts as a Cuþ anchoring site, with a higher affinity for Cuþ with a conditional Kd ¼ 16  10 9 M. Finally, the highest affinity Cuþ binding site (conditional Kd ¼ 7  10 9 M) involves the first five residues MDVFM, where Met1 and Met5 are the main metal coordinating groups.451,477,479 The site 1 was studied using X-ray absorption spectroscopy. The Cuþ complex with the AS(1–15) fragment at pH 7.5 has been proposed as a tetra coordinated complex with a 2S2N/O coordination, where the sulfur-based ligands are provided by Met1 and Met5 and the NH2 terminal group could be a nitrogen-based ligand.480 Finally, a NMR study provides evidence that Asp2 can provide one oxygen-based ligand for Cuþ.481 NMR studies have described that the binding of Cuþ in the site 1 at the N-terminal region stabilizes local conformations with a-helical secondary structure and restricted mobility of AS.482 This event might be relevant for the association of the protein to membranes and its physiological function in vesicle trafficking. The Cuþ coordination to AS associated to membranes was studied using the 1–15 fragment, in this environment the formation of 2:1 AS:Cuþ complex has been proposed, where Cuþ bridges between two helical peptide chains via Met residues.483

2.19.4.2.4

Posttranslational modification a-synuclein and its impact on its metal-binding properties

On the other hand, AS can undergo a variety of posttranslational modifications like phosphorylation and acetylation. AS contains numerous putative and confirmed phosphorylation sites: four serine, four tyrosine and 10 threonine residues, however, the most established are Ser87 and Ser129.484 In brains of patients and in animal models of synucleinopathies AS is mostly phosphorylated on Ser129.485–487 Ser129 is located at the C-terminal region of AS; thus, this phosphorylation may regulate the interactions with biological relevant ligands such as proteins and metal ions.488 On the other hand, AS protein isolated from brain tissue, erythrocytes or mammalian cell lines is ubiquitously acetylated at the N-terminus.489–492 Interestingly, it has been demonstrated that N-terminal acetylation of AS increases its helicity capability, its affinity to lipid vesicles, as well as modulates the Cu binding.445,491,493–496 How post-translational modifications may impact the key AS-metal interactions and the influence on its aggregation and toxicity has been recently reviewed by González et al. in 55. The addition of negatively charged phosphate groups to a protein may alter not only the metal ion affinity and specificity, but also may modify hydrogen-bonding patterns influencing the folding, structure, and functional properties of protein–metal complexes. In AS several putative phosphorylation sites occurs at the C-terminal region spanning residues 120–140 (Y133, Y136, S129 and Y125), which contains the site for the binding of metal ions.497 Using C-terminal AS fragments spanning the region 107–140 and its mono-phosphorylated forms (pY125 and pS129) was demonstrated that the phosphorylation at either Y125 or S129 influences not only the affinity features but also the location of binding sites. The Kd values of Cu2þ, Pb2þ, and Fe2 þ complexes with phosphorylated 107–140 AS were lower than those with the non-phosphorylated peptide, indicating that phosphorylation increases the binding affinities for metal ions, led to the shift of the binding sites of divalent metal ions from the N-terminus to C-terminus.498 This suggests that phosphorylation at the C-terminus might play an important role in regulating metal ion binding, influencing both AS structure and aggregation. On the other hand, it has been demonstrated that the N-terminal acetylation of AS abolishes Cu2þ binding at the high-affinity N-terminal site (site1), however site 2, at His50 and site 3, Asp121 are preserved.496,499 At the C-terminal site, the Cu2þ ions distribute differently when Met1 is acetylated; however, no changes were observed for Cu2þ binding at the His50 site upon N-terminal acetylation of AS.496 Thus, the acetylation of the N-terminus seems to modulate the extent of independence or cooperativity between N- and C-terminal sites. Cooper ions are predominantly found in their Cuþ state inside the reducing environment of living cells. In this sense, acetylation of the N-terminus helps to modulate the Cuþ binding to the high-affinity Met1-X3-Met5 site, while the coordination of this metal ion at His50 and Met116 and Met127 are preserved. The coordination of Cuþ at the high affinity site is through Met1 and Met5 residues that act as the main anchoring moieties; additionally, the N-terminus acetyl group acts as an oxygen ligand source. The conditional affinity for Cuþ binding at this site was reported to be 3.9  1.0  10 9 M.481,500 Furthermore, the formation of AcAS-Cuþ complex at the N-terminal site, induced the stabilization of local conformations with a-helical secondary structure and restricted motility.482 Thus, N-terminal acetylation stabilizes the reduced form of the AcAS-Cu complex and modulates the structural conformation of the protein.

2.19.4.3

Metal ions as therapeutic targets in Parkinson’s disease

Current available treatments for PD are mainly dopamine-replacement therapies (e.g., L-Dopa, dopamine agonists) that treat symptoms of PD, but do not hinder progression of neurodegeneration.501 PD is characterized for the loss of metal ion homeostasis and accumulation of specific proteins, mainly AS; hence, they have been molecular targets for the rational design of novel therapeutic agents.502 Due to the central role of AS in PD pathology, several studies have aimed at reducing AS levels or eliminating AS aggregates.503–507 Therapeutic strategies involving metal chelation, targeting of metalloproteases and other several metal-interfering molecules have also been explored.504,508,509 Several molecules have been developed to target metal-AS interactions. INHHQ, an 8-Hydroxyquinoline (8HQ) derivative, is able to cross the BBB, disrupt Cu2þ-AS interactions, and inhibit AS oligomerization.510,511 On the other hand, clioquinol, an 8HQ metal chelator tested for its therapeutic potential in AD, prevents Fe-induced aggregation of AS, reduction in dendritic spine density of medium spiny neurons of hippocampus and dopaminergic cell loss in transgenic mice overexpressing AS with the A53T mutation, improving motor and cognitive function.512 These results suggest that it is worthwhile to further pursue the targeting of metal-AS interactions for PD therapeutics.

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Clioquinol and other 8HQ derived chelators are also effective at preventing Fe accumulation, reducing oxidative stress, striatal dopamine loss and dopaminergic cell death in MPTP-intoxicated mice.513,514 Some Fe chelators also help reducing AS accumulation, aggregation, oxidative stress and toxicity.515–517 Notably, the Fe chelator PBT434 also rescues motor performance in mice exposed to Parkinsonian toxins (6-OHDA and MPTP) and in a transgenic model (hA53T a-synuclein) of PD; its effects are associated with increased expression of ferroportin-1 and DJ-1, the latter being another PD-related metal-binding protein (vide supra).518 Another chelating agent proposed for the treatment of neurodegenerative disorders associated to iron accumulation is deferiprone (1,2-dimethyl-3-hydroxy-4(1H)-pyridinone), a molecule that is able to cross the BBB and redistribute excess intracellular iron to the extracellular apotransferrin.519,520 In phase I clinical trials a 12-month administration of this drug decreased disease progression in PD patients compared to the placebo group.521 In a phase II clinical trial, deferiprone caused a decrease of dentate and caudate nucleus iron levels.522 Such promising results underscore the importance of restoring iron homeostasis as a therapeutic strategy for PD.

2.19.5

Huntington’s disease

Huntington’s disease (HD) is an inherited neurodegenerative disease that involves impaired movement (chorea), cognitive deterioration, psychological and behavioral disturbances. These symptoms reflect neurodegeneration in the basal ganglia of the brain, especially the striatum. HD is caused by a CAG repeat expansion in the huntingtin coding gene, which is translated into an extended polyglutamine (PolyQ) tract in the huntingtin protein (Htt).523,524 Htt is an ubiquitous 350-kDa cytosolic protein highly conserved from flies to mammals, containing an expanded polyQ stretch at its N-terminal region, which is preceded by 17 amino acids and followed by a proline-rich domain (PRD).525 Both, the polyQ stretch and the PRD are polymorphic in the human population. Htt protein, with a polyQ stretch larger than 35 residues, is considered pathological and it is referred to as a mutant huntingtin (mt-Htt), while Htt protein with 35 Gln residues or less, is called wild type Htt (wt-Htt). (Fig. 16).524 The C-terminal segment is important for protein-protein interactions that are implicated in diverse cellular functions proposed for Htt that include transcription, RNA splicing, endocytosis, cell trafficking, and cellular homeostasis. Interestingly, the interactions of Htt with its partners are modified by the polyQ stretch. 524,526 Expansion of the polyQ stretch results in abnormal interactions between Htt and other proteins, prompting toxicity in the caudate and putamen regions of the basal nuclei by several mechanisms,527 including misfolding and aggregation processes,528 transcriptional repression,529 oxidative damage,530 imbalanced redox signaling,531 and mitochondrial impairment.532 A magnetic resonance imaging (MRI) study with HD patients showed a correlation between increased concentrations of metals in the basal ganglia (pallidum, caudate and putamen) and HD progression.58 However, while there is some evidence for altered metal homeostasis in HD, the exact mechanisms linking metal imbalance and neurodegeneration are still elusive.

2.19.5.1

Copper in Huntington’s disease

Increased copper concentrations have been reported in the striata (putamen) of post-mortem human HD brains,56 while other studies did not report significant differences in copper levels in brain tissue 58, nor in blood samples of HD patients.59 However,

Fig. 16 Human Huntingtin protein. Top: Linear representation of the N-terminal region of huntingtin protein, highlighting the 17 residues (sky blue) that precede the expandable polyQ region (purple), the proline-rich domain (PRD) (orange), and the rest of the N terminus (gray). A short polyQ region (35) is considered wild-type huntingtin (wt-Htt), while a larger region is denoted as mutant huntingtin (mt-Htt). Bottom: Schematic structural representation of wt-Htt using the same color code for each region of the protein. The first 17 amino acids and the polyQ region display an a-helix conformation when bound to a maltose binding protein (PDB: 3lO4). PRD was adapted from the proline-rich domain bound to profilin (PDB: 1CJF). The rest of the protein is represented with no secondary structure, as there are no structural studies of this region. Met8, His82, and His98 are highlighted as the putative anchoring sites for Cu2þ.525

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increased copper levels are clearly observed in a transgenic HD mouse model expressing mt-Htt.533 Remarkably, this HD mouse model also shows decreased levels of APP and APPL2, which may contribute to increased brain copper levels. Immobilizedmetal-affinity chromatography (IMAC) experiments with human HD brain fractions showed that both, wt- and mt-Htt elute as copper binding proteins. Cu2þ ions bind to the N-terminal of Htt and enhance its aggregation in vitro. Although the nature of the Cu2þ binding sites in Htt has not been elucidated, point mutations of His82 and His98 in mt-Htt result in loss of metal binding, while wt-Htt may interact with Cu2þ in the absence of His98.533 Hence, the presence of an expanded polyQ has an effect in the His residues involved in Cu2þ binding, perhaps by changing Htt protein conformation. Cu2þ binding to wt-Htt causes oxidation of N-terminal Cys residues, enhancing the formation of oligomeric species.534 Clearance and degradation of these oxidized species are slower compared to monomeric Htt. Oligomerization of mt-Htt in cell and mouse HD models is modulated by copper and glutathione, and it can be blocked by mutating all Cys residues. Thus, the Cu2þ-Htt complex may display redox activity and contribute to Htt protein aggregation and oxidative stress in HD. Indeed, decreased striatal CuZnSOD activity is observed in late stages of transgenic HD mice.535 On the other hand, in vitro cell studies show that overexpression of metallothionein, or treatment with clioquinol (a Cu2þ and Zn2þ chelating agent), significantly reduce Htt aggregation and toxicity, pointing to a role of Cu2þ and Zn2þ ions in these processes. 536 Moreover, dietary copper reduction in a Drosophila melanogaster model decreases the level of oligomerized and aggregated mt-Htt, while silencing of CTR1 and overexpression of ATP7 leads to lowered copper levels and decreased mt-Htt aggregation and toxicity. Mutation of Met8 and His82 abolishes Cu2þ-induced toxicity of mt-Htt, highlighting the role of these residues as a putative ligand for this metal ion.537 Overall, these results indicate an important role of the interaction of Cu with Htt in protein aggregation, toxicity and HD progression, underscoring the importance of understanding the Cu2þ coordination properties of Htt.

2.19.5.2

Iron in Huntington’s disease

Increased iron levels are observed in blood of HD patients,59 also in striatum, pallidum and putamen of postmortem.56,58,538 Moreover, neuroimaging studies show increased iron signal in the basal ganglia of HD patients (as reviewed in 539) primarily as Fe2þ, as shown by synchrotron X-ray fluorescence analysis.538 Consistently, increased iron levels are found in soluble and membrane fractions of HD mouse brain.538 On the other hand, several studies suggest a functional role for Htt in iron homeostasis. In a zebrafish embryo model, silencing the gene that codes for Htt (or partial knock-down) results in loss of iron bioavailability for hemoglobin and altered Fe endocytosis.540 Cells treated with iron-specific chelators display increased Htt protein expression, suggesting that Htt is an iron-regulated protein.541 Increased iron levels in pathological HD conditions may increase the labile iron pool, enhancing oxidative damage. Indeed, cells expressing polyQ-expanded Htt display aggregate inclusion bodies and oxidative stress, in a CAG-repeat dependent manner; while iron chelation by deferoxamine decreases Htt aggregation.542 Overall, it is clear that iron homeostasis is altered in HD, while there may be an interplay between Fe, Htt aggregation and oxidative stress that deserves further investigation.

2.19.5.3

Manganese in Huntington’s disease

The brain regions affected in HD, namely caudate nucleus (CN), globus pallidus (GP) and putamen, display higher manganese levels when compared to healthy brain structures.60 However, no significant changes in manganese concentrations have been observed in the basal ganglia of postmortem HD brains,56,58 nor in the bloodstream of HD patients.59 Upon manganese exposure, transgenic HD mice (YAC128) expressing mt-Htt (128 Gln residues) show less manganese accumulation in the striatum, when compared to WT mice.543 These mice also show decreased striatal dopamine levels and perturbation of dendritic architecture of medium spiny neurons (MSNs), which are the most affected population of neurons in HD. Striatal-derived cell lines expressing mt-Htt (111 Gln residues) exhibit improved resistance to manganese-induced toxicity and less accumulation of Mn2þ, as compared to those expressing wt-Htt.543 Overall, these findings suggest that mt-Htt prevents striatal manganese accumulation and toxicity; possibly by decreasing Mn2þ uptake, increased Mn2þ export or decreased Mn2þ storage capacity, although Mn2þ trafficking pathways have not been studied in HD models. Different neuropathological mechanisms involving manganese have been related to the context of HD, pointing to this metal as a putative environmental modifier of the disease. One of these mechanisms involves glutamate-induced excitotoxicity; it has been proposed that glutamate released from corticostriatal neurons overstimulates striatal medium spiny neurons (MSN). Primary cultures of MSN from the YAC128 HD mice model are more sensitive to glutamate-induced excitotoxic death, when compared to WT or YAC18 mice;544 while Mn-exposed GABAergic cell lines exhibit increased intra- and extracellular glutamate levels.545 Furthermore, the activity of glutamine synthetase (GS), the most abundant manganese-containing enzyme in the brain that synthesizes glutamine from glutamate,546 is decreased in caudate and putamen of HD patients.547 These findings suggest that transient disruptions in brain manganese levels during the disease could result in increased Glu release from corticostriatal projections, which overstimulate striatal MSN,544 causing excitotoxicity. This manganese-induced damage may explain the selective striatal degeneration observed in HD. Another mechanism of Mn-induced neuronal damage is associated with htt-dependent trafficking of the brain derived neurotrophic factor (BDNF). BDNF belongs to a family of neurotrophins that regulates synapsis, promotes cell survival and increases spine complexity in adulthood.548 In the context of HD, wt-Htt acts as a molecular switch for the transport of BDNF from cortical neurons to striatum through the microtubular cytoskeleton. Htt phosphorylation at S421 promotes the transfer of BDNF from the

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neuronal soma to the nerve endings, favoring synaptic development, while non-phosphorylated wt-Htt enhances transport in the opposite direction. 526,549,550 In vivo manganese exposure to mice increases total wt-Htt protein expression, mostly in its nonphosphorylated form;551 while manganese exposure to primates and mice also leads to a significant decrease in BDNF levels. Moreover, in a HD mice model expressing mt-Htt (109 Gln residues), the polyQ expansion impacts its interaction with HAP1 (Huntingtin-associated protein 1), a protein associated with the microtubule-based motor complex.526 Reduction of spine density and dendritic length of MSN exposed to manganese552 are in line with a mechanism were manganese exposure or transient manganese perturbations, lead to decreased BDNF transport from cortical to striatal neurons. Manganese has been the most studied metal ion in HD. Current findings support the notion of a tight link between manganese, Htt expression, phosphorylation and function, and MSN pathology in the HD brain. However, no direct interaction between Mn2þ and Htt has been reported. Further research could shed light into the molecular details of this connection and how Mn2þ trafficking pathways are affected in HD.

2.19.5.4

Zinc in Huntington’s disease

2þ

Zn is increased in the blood of HD patients,59 and in pallidum and putamen of postmortem HD brains,58 suggesting that mt-Htt might impair Zn2þ homeostasis. Transgenic HD mice expressing mt-Htt show reduced ZnT3 expression, a Zn2þ transporter that regulates vesicular Zn2þ levels in the brain.553 A decline in vesicular Zn2þ may result in synapse dysfunction and cognitive impairment of glutamatergic synapses, which are also affected in HD. Mitochondrial dysfunction and increased oxidative stress are also associated with HD. Indeed, Cu/ZnSOD activity is increased in the striata of HD mice in the early stages of disease.535 Moreover, mitochondrial inhibitor models of HD induce Zn2þ accumulation.422 Overall, there is clear evidence for a loss of Zn2þ homeostasis in HD, though further investigation of the role of this metal ion in HD is needed.

2.19.5.5

Metal ions as therapeutic targets in Huntington’s disease

Although increased oxidative stress is a central feature of HD that is intimately linked to disease progression; antioxidant supplementation, including small molecules (uric acid, GSH, ascorbic acid), precursors of antioxidants (N-acetyl cysteine) or cofactors of antioxidants (e.g., selenium), have shown insufficiently results in human clinical trials. Perhaps, this is because oxidative damage may not be the primary cause contributing to HD pathology.554 Since elevated concentrations of copper and iron are found in the striata of HD patients, metal-binding compounds, such as deferoxamine538 and 8-hydroxiquinolinic derivatives, such as clioquinol 555 and PBT-2,556 have also been proposed as therapeutic agents. Clioquinol selectively reduces expanded polyQ levels and decreases cell death in in vitro HD models; while in vivo, it decreases mt-Htt aggregation in the striatum and reduces behavioral abnormalities in HD mice.555 PBT-2 diminishes abnormal phenotype in the C. elegans model, although its effect is not associated with decreased aggregation of wt-Htt. In HD mice model, PBT-2 improves rotarod performance, prolongs survival and alleviates pathologic phenotype.556 Another therapeutic approach is based on delivering zinc finger proteins (ZFPs) to repress the selective expression of toxic mt-Htt. Delivery of coding sequences for ZFPs using viral vectors reduces expression and aggregation of mtHtt in cell culture and in HD mice model. Targeting the mt-Htt gene at the transcriptional level also favors rotarod performance and improves pathological phenotype.557 Finally, as discussed above, decreased BDNF levels in the striatum is one of the hallmarks of HD;552 hence, the supply of BDNF as potential adjuvant for the treatment of HD using gene therapy has been proposed. The longterm expression of this factor results in a delayed onset of the motor phenotype observed in transgenic HD mice.558 However, further understanding of the disease is required to develop therapeutic strategies that can multi-target the different facets of HD, including loss of metal homeostasis and specific metal-Htt interactions.

2.19.6

Concluding remarks

This chapter provides a critical review of the role of essential d-block metal ions in the etiology of neurodegenerative diseases, including prion diseases, Alzheimer’s, Parkinson’s, and Huntington’s diseases. The bioinorganic chemistry of the metal-binding proteins involved in these diseases has been discussed in detail, revealing that there is still much to be discovered about these systems; particularly as their metal-binding properties likely relate to both, their functional and pathological roles. While the functional implications of these metal-protein interactions are poorly understood, they may play key roles in metal ion homeostasis, which is disturbed in neurodegenerative diseases. On the other hand, the fact that metal ions impact the ability of these proteins to form oligomers and amyloid aggregates has drawn great attention in the field, while the mechanisms involved in the neurotoxicity of such protein aggregates have been less explored and they may involve direct interactions with neuroreceptors at the synapse. Clearly, further research is required to gain a deeper insight in the metal-protein interactions involved in neurodegenerative diseases, in order to guide the design of effective therapeutic strategies that target essential metal ions in the brain.

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Acknowledgements Authors are grateful to Prof. Paolo Carloni (Institute for Advanced Simulation, Forschungszentrum Jülich) for providing molecular simulationderived structures of the N-terminal of PrP that were used for the schematic representation of PrPC in Fig. 2. Authors would like to thank Fatima Trejo-Arroyo for assistance in preparing Section 2.19.4.3. This research was supported by SEP-Cinvestav funds from the Ministry of Education; the National Council for Science and Technology in Mexico (CONACYT) through PhD fellowships to Yanahi Posadas (308512) and Victor E. LópezGuerrero (849060); and COMEXUS through a postdoctoral Fulbright García-Robles fellowship to Richard Sayler.

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2.20

Metal ion interactions with nucleic acids

Besim Fazliji*, Carla Ferreira Rodrigues*, Haibo Wang*, and Roland K.O. Sigel, Department of Chemistry, University of Zurich, Zurich, Switzerland © 2023 Elsevier Ltd. All rights reserved. This chapter is an update of Chapter 3.21 of Comprehensive Inorganic Chemistry II, 2013, by Roland K. O. Sigel and Helmut Sigel. In this updated chapter we now concentrate solely on metal ion binding to RNA, i.e., their role in structure, folding, and mechanism. Numerous text passages are thus copied from our earlier publication, with new findings added to the text. For the original sections on the acid base properties of less common and artificial nucleobases, as well as on the metal ion binding properties of phosphates, sugar-hydroxyl groups, nucleobases, nucleosides, nucleotides, and dinucleotides, we refer the reader to the original Chapter 3.21 from 2013 (Sections 2.2.3. to 7.2). It should be noted that species written in the text without a charge either do not carry one or represent the species in general (i.e., independent of their protonation degree); which of the two possibilities applies is always clear from the context.

2.20.1 2.20.2 2.20.2.1 2.20.2.2 2.20.2.2.1 2.20.2.2.2 2.20.3 2.20.4 2.20.4.1 2.20.4.2 2.20.4.2.1 2.20.4.2.2 2.20.4.2.3 2.20.4.2.4 2.20.4.3 2.20.4.3.1 2.20.4.3.2 2.20.4.3.3 2.20.4.3.4 2.20.4.3.5 2.20.4.3.6 2.20.4.3.7 2.20.4.3.8 2.20.4.3.9 2.20.4.3.10 2.20.4.4 2.20.4.4.1 2.20.4.4.2 2.20.4.4.3 2.20.4.4.4 2.20.4.5 2.20.4.5.1 2.20.4.5.2 2.20.4.6 2.20.4.7 2.20.4.7.1 2.20.4.7.2 2.20.4.7.3 2.20.5 2.20.6

*

Introduction General considerations Relevant metal ions and some of their properties Potential liganding atoms on RNA Acid-base considerations on potential binding sites Micro acidity constants, intrinsic basicities, and tautomeric equilibria Metal ion affinities of individual sites of single-stranded nucleic acids Metal ion binding to RNAs Solvation content of metal ions Thermodynamics of metal ion binding to RNA Indirect methods Hydrolytic cleavage experiments Oxidative cleavage experiments Spectroscopic methods Metal ion binding motifs in RNA by Mg2þ Classification of Mg2þ binding sites Tandem GC base pairs GU wobble pairs GA mismatch base pair Sheared GA base pair Loop E motive or metal ion zipper Mg2þ clamp Y-clamp G-N7 macrochelation and purine N7-seat Further Mg2þ binding motifs Metal ion binding motifs in RNA of monovalent metal ions GU wobble AA platform GG stacking Nucleobase tetrads Binding of kinetically inert metal ions Binding of Pt2þ to RNA Binding of other inert metal ions Metal ion binding in the helix center Binding of metal ion complexes Hexammine complexes with Co3þ and other metal ions Ruthenium complexes Further complexes Metal ions and their role in folding and dynamics of RNA Metal-ion sensing by riboswitches

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These authors contributed equally to this work.

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Metal ion interactions with nucleic acids

2.20.7 2.20.7.1 2.20.7.2 2.20.7.3 2.20.8 Acknowledgments References

Metal ions and their role in RNA catalysis General effects of metal ions on the observed catalytic rate Two-metal ion mechanism Electrostatic influence of metal ions Concluding remarks and future directions

Nomenclature 7,9DiMeGua 7,9-Dimethylguanine (Fig. 3) 9MeAde 9-Methyladenine 9MeGua 9-Methylguanine 9MeHpx 9-Methylhypoxanthine Ade Adenine (Fig. 1) Ado Adenosine (Fig. 1) Cyd Cytidine (Fig. 1) Cyt Cytosine (Fig. 1) ENDOR Electronnuclear double resonance spectroscopy EPR Electron paramagnetic resonance ESEEM Electron-spin echo envelope modulation FT-IR Fourier-transform infrared spectroscopy GMP2L Guanosine 50 -monophosphate GTP4L Guanosine 50 -triphosphate Gua Guanine (Fig. 1) Guo Guanosine (Fig. 1) HDV Hepatitis delta virus HIV-1 TAR Human immunodeficiency virus transactive response (RNA) HMQC Heteronuclear multiple-quantum correlation Hpx Hypoxanthine (Fig. 1) HQS 8-Hydroxyquinoline-5-sulfonic acid Ino Inosine (Fig. 1) ISTAR Intrinsic STAbilities of RNA complexes J Coupling constant ka Micro acidity constant Ka Acidity constant KI Intramolecular dimensionless equilibrium constant L General ligand M2D General divalent metal ion MeRNA Metals in RNA database MINAS Metal Ions in Nucleic AcidS database MRD Magnetic relaxation exchange dispersion NB Nucleobase NBR Nucleobase residue NMR Nuclear magnetic resonance Ns Nucleoside salen N,N0 -Ethylenebis(salicylimine) siRNA Small interfering RNA Thy Thymine ¼ 5-methyluracil (Fig. 1) U2S 2-Thiouridine U2S4S 2,4-Dithiouridine U4S 4-Thiouridine U4SMP2L 4-Thiouridine 50 -monophosphate

654 654 655 656 656 657 657

Metal ion interactions with nucleic acids

631

UDP3L Uridine 50 -diphosphate UMP2L Uridine 50 -monophosphate Ura Uracil (Fig. 1) Urd Uridine (Fig. 1) UTR Untranslated region (of mRNA) XAS X-ray absorption spectroscopy

Abstract This chapter focuses on the interaction of metal ions mainly with RNA. It is an update of the previous Chapter 3.21 in the 2nd Edition of Comprehensive Inorganic Chemistry II (2013) but focusing solely on RNA. Metal ions are key to folding, structure, and function of any nucleic acid. These interactions are generally of a weak and highly dynamic nature as they concern mostly Kþ and Mg2þ in living organisms. Aside from the large excess of loosely bound ions for charge compensation, a network of inner-sphere and outer-sphere interactions holds more specifically bound ions in place. Hence, metal ion binding to larger RNAs is rather complicated and has many facets. After a few general considerations on the basic properties of metal ions and the potential coordination sites on the RNA, the thermodynamics of metal ion binding to RNA and known metal ion binding motifs in RNA are described. This is followed by today’s knowledge on the role of metal ions in folding, dynamics, sensing, and/or catalysis of riboswitches and ribozymes, respectively, is summarized.

2.20.1

Introduction

Nucleic acids, due to the presence of negatively charged phosphate groups, cannot exist without the association of cationic counter ions. Since these counter ions are mostly metal ions, the interactions between metal ions and nucleic acids as well as their constituents have attracted considerable attention during the past six decades. Short historical accounts regarding the constituents are found in Refs.1–3 and reviews on nucleic acid-metal ion interactions,4 covering RNA5–9 and DNA,10–13 exist. The focus of this chapter is on metal ions playing a role in RNA chemistry, i.e., Mg2þ, Ca2þ, Mn2þ, Cu2þ, and Zn2þ as well as in part also on Cd2þ and Pb2þ. In the cited references in many instances also information about further metal ions like Sr2þ and Ba2þ or Co2þ and Ni2þ, etc., may be found. The analytical procedures and their methodology will only be covered if needed to understand a certain “problem” because the “analysis” of metal ion-nucleic acid interactions was previously reviewed.14–16 Metal ions coordinate to the basic sites of ligands, i.e., there commonly is a competition between metal ions and protons for binding. Hence, we first briefly review the acid-base properties of ligands (L), described via the individual stepwise acidity constants of their protonated species (Eqs. 1a and 1b): Hn ðLÞnþ #Hðn1Þ ðLÞðn1Þþ þ Hþ h

H KH n ðL Þ

i Hðn1Þ ðLÞðn1Þþ ½Hþ    ¼ Hn ðLÞnþ

(1a)

(1b)

Commonly, n equals 1, 2 or 3, thus referring to the species H3(L)3þ, H2(L)2þ, and H(L)þ. A neutral nucleobase residue (NBR) may also lose a proton as defined by Eqs. (2a) and (2b) NBR#ðNBR  HÞ þ Hþ

(2a)

½ðNBR  HÞ ½Hþ  ½N

(2b)

H ¼ KNBR

The expression (NBRH) is read as “NBR minus H”, meaning, e.g., that a N(H) site of a NBR is deprotonated. The stabilities of complexes are defined via association or stability constants, that is, by Eqs. (3a) and (3b), and the formation of hydroxo complexes by Eqs. (4a) and (4b) (charges are omitted): M þ L#ML

(3a)

½ML ½M½L

(3b)

M KML ¼

MðH2 OÞn#MðOHÞðH2 OÞðn1Þ þ Hþ

(4a)

632

Metal ion interactions with nucleic acids h

M KMðH 2 OÞ

n

i MðOHÞðH2 OÞðn1Þ ½Hþ    ¼ MðH2 OÞn

(4b)

There are various compilations17–20 of metal ion complexes of nucleotides and their constituents including acidity constants of the ligands. These compilations are very helpful for finding access to the literature, whereas the given (e.g., in Ref. 20) so-called recommended values have to be considered with great care to prevent being misguided (see the final paragraph in Section 2.20.5.4 of Ref. 21 and note 43 of Ref. 22).

2.20.2

General considerations

2.20.2.1

Relevant metal ions and some of their properties

As an important basis to understand the differing coordination properties of metal ions to RNA (and any other ligand) we summarize the more important physico-chemical properties of metal ions in Table 1. As often metal ions substitute for others of the same size and also alkali and alkaline earth ions exhibit variable coordination numbers without a strong directionality in bonding, their ionic radius is given in dependence on the coordination number (columns 2–4).23–25,30 For example, Co2þ and Zn2þ exhibit similar radii in all coordination numbers and indeed, Co2þ may substitute for Zn2þ in many proteins.32 Size may affect selectivity and this also holds for metal ion-nucleic acid interactions, that is, metal ion-fitting into preformed pockets.33 Other points are complex stabilities according to the Irving-Williams series (see also Section 2.20.3),34 Martin’s Stability Ruler,24,29,35–37 and the relative affinities of metal ions for oxygen versus nitrogen ligands.38,39 One may note that Ca2þ shares the preference of Mg2þ for phosphate oxygen ligands and indeed, Ca2þ can inhibit Mg2þ-dependent ribozymes (e.g.,40–43). Regarding the selectivity of metal ions often the Pearson classification44,45 is applied, though this has its limits. For example, Fe3þ (being hard) forms insoluble hydroxide precipitates (hydroxide also being hard), partly even in acidic solution, whereas other hard ions (including the alkali and alkaline earth ions) do not form hydroxo complexes easily (Table 1, column 5). From the

Table 1

Properties of several metal ions of biological interest.a Ionic Radii (pm) for CN b

Metal Ion

4

6

8

pKa/aq c

Seawater [mM] d,e

Extracellular Space [mM] e,f

Mammalian Cell [mM] d

Naþ Kþ Mg2þ Ca2þ Sr2þ Ba2þ Pb2þ Mn2þ Fe2þ Co2þ Ni2þ Cu2þ Zn2þ Cd2þ Fe3þ

99 137 57

102 138 72 100 118 135 119 83 78h 74 69 73 74k 95 65l

118 151 89 112 126 142 129 96 92 90

14.18 14.46 11.44 12.85 13.29 13.47 7.71 10.46 9.5 9.65 9.86 >8j 8.96 10.08 2.2m

470 10 50 10

145 5 1 4

10 140 30g 1

2  103 3  103 3.1  106 1  106 5  106 1  104

2  104 2  102 1  104 1  104 1.8  102 2  102

a

98 66 63 58 55i 57 60 78 49

90 110 78

Given are the varying coordination numbers (CN) with their effective ionic radii (pm), and the pKa/aq values of the bound water molecules, as well as their concentrations (in mM) in seawater, the extracellular space, and in mammalian cells. b Values taken from Refs.23 and 24 (see also Ref. 25 and Wikipedia). The radii of the more common coordination numbers are printed in italics. c From Ref. 26 (see Eqs. 4a and 4b). d From Ref. 27; the value for Cu2þ is from Ref. 28 e For the redox-active metal ions, like Cunþ or Fenþ, the oxidation state is not defined. f The first four entries are from Ref.27. The remaining six entries (for plasma concentration are from Ref. 29) g The free concentration of Mg2þ is about 1 mM.16 h High-spin value; the low-spin value is 61 pm. i Value for a tetrahedral coordination sphere; the value for a square-planar one is 49 pm.23 j At pH 7 a CuO precipitate exists.29 k 2þ Zn has a chameleon-type coordination sphere; coordination number 4 (tetrahedral) is common as well.30 l High-spin value; the low-spin value is 55 pm. m From Ref. 31; the value for the Fe3þ low-spin state is pKa/aq ¼ 0.7.31 At pH 7 a FeO(OH) precipitate exists.29

Metal ion interactions with nucleic acids

633

hydrolysis constants, pKa/aq Eqs. (4a) and (4b), it is further evident that some borderline (or soft) ions form hydroxo complexes close to the neutral pH range (Cu2þ, Pb2þ) while others do not (Ni2þ). Table 1 also lists the metals essential for mammals (and humans).16,24,27,29 The first four are often addressed as bulk metals as they occur to about 20 g or more in a normal human adult.29 From the remaining 6 metals 4 are found only in mg amounts and therefore they are often addressed as trace metals. Iron and zinc occur in amounts in between (ca 2–4 g).29 It is interesting to compare the concentration of the metals in seawater (column 6) and in the extracellular space or plasma (column 7). The four bulk metals are present in a high concentration in seawater, yet for Fe, Co, Ni, Cu, and Zn an enrichment occurs in the human body. Furthermore, the concentrations of the metal ions inside and outside of the cell differ significantly (Table 1, columns 7 and 8). In summary, Mg2þ and Kþ are the most important counter ions inside the cell and indeed they play an important role for ribozymes.5,6,8,9,16,46

2.20.2.2 2.20.2.2.1

Potential liganding atoms on RNA Acid-base considerations on potential binding sites

The nucleobases, the ribosyl ring, and the phosphodiester unit of the backbone are the basic structural units of RNA. Since the metal ions of life are mostly “hard”,24,29 they have a remarkable affinity towards O donors. Of primary interest are the oxygens of the phosphodiester bridge, especially the negatively charged non-bridging ones.39 These are freely accessible for metal ion binding because their pKa is ca. 1.47,48 Similarly, exocyclic nucleobase oxygens (Fig. 1)49–53 do also not suffer from Hþ competition, though C(O) is only a weak metal ion binder.54–57 The sugar hydroxyl groups are poor metal ion-binding sites in the physiological pH range, but they may participate in coordination if supported by a suitable and sterically correctly orientated primary binding site.33 In contrast, the endocyclic nitrogens of the nucleobases are preferred targets, especially for the more softer metal ions.39 This is not true for the exocyclic amino groups.47 The acid-base properties of the nucleobases (column 4) and nucleosides (column 5) are summarized in Table 2, and their comparison shows that the nucleosides are throughout more acidic than the nucleobases. Evidently, the hydroxyl groups of the ribose ring (Fig. 1) change the solvation around the nucleobase thereby facilitating the release of the protons at a lower pH. A decreasing solvent polarity, e.g., due to addition of 1,4-dioxane to water, or within a cellular context, makes charge separation more difficult and thus, the pKa values for neutral (N)H sites,71 but also for P(O)(OH)2 and P(O)2(OH) groups,50 increase. In contrast, for (N)Hþ sites deprotonation is facilitated, that is, the pyridinium unit becomes less stable.38,72 Such effects are of biological relevance, as the intrinsic dielectric constant (permittivity) in an RNA fold, especially within a cellular environment, is expected to change.9

2.20.2.2.2

Micro acidity constants, intrinsic basicities, and tautomeric equilibria

The acidity constants given in Table 2 are so-called macro acidity constants, which however, do not always reflect the true basicity of a certain site. For example, in twofold protonated adenosine (Table 2) (N1)Hþ will exercise a charge repulsion on (N7)Hþ and thus facilitate its deprotonation. Hence, the basicity of N1 is relatively well described, yet the one of N7 is not.63 Nevertheless, it is crucial to know the intrinsic basicity of N7 to understand metal ion binding of the Ado residue.

Fig. 1 Chemical structures of the nucleobases (NB) (R ¼ H) occurring in RNA: adenine (Ade), guanine (Gua), cytosine (Cyt), uracil (Ura). Hypoxanthine (Hpx) is a rare nucleobase and shown for comparison with guanine. Thymine (Thy), which occurs in DNA, corresponds to 5methyluracil. The corresponding nucleosides (Ns) (R ¼ ribosyl residue) are adenosine (Ado), guanosine (Guo), inosine (Ino), cytidine (Cyd), and uridine (Urd). The dominating conformation of the nucleosides is anti, with the Watson-Crick face of the nucleobases pointing away from the sugar.49–53 In the case of thymidine (dThd) R is the 20 -deoxyribosyl residue.

634 Table 2

Metal ion interactions with nucleic acids Negative logarithms of the acidity constants (pKa) of (Protonated) nucleobases and nucleosides (25  C).a

Base (NB)

Nucleoside (Ns)

Site

Base (NB)

Nucleoside (Ns)

Ref.

Cytosine Uracil Thymine Adenine

Cytidine Uridine Thymidine Adenosine

(N3)Hþ (N3)H (N3)H (N7)Hþ (N1)Hþ

4.7 9.33  0.05 9.82  0.05 0.4b 4.2

4.14  0.02 9.18  0.02 9.69  0.03 1.50  0.15 3.61  0.03

49,58 59–61 59,62 63,64

Guanine

Guanosine

(N7)Hþ (N1)H

3.29  0.03 9.36  0.01

2.11  0.04 9.22  0.01

22,65

Hypoxanthine

Inosine

(N7)Hþ (N1)H

2.1 8.9c

1.06  0.06 8.76  0.03

22,49

a The error limits given are three times the standard error of the mean value or the sum of the probable systematic errors, whichever is larger. So-called practical, mixed or Brønsted constants are listed.66 For most entries it holds I ¼ 0.1 M (NaNO3). In the case of pKa < 1, i.e., especially for values with a negative sign, I is of course larger than 0.1 M; in these instances the H0 scale was applied.67,68 In column 6 the first reference refers to the nucleobase and the next one to the nucleoside. b There is a N7,N9 dichotomy for H binding.64,69 c From Ref. 70.

It has been repeatedly shown that a H atom may be replaced by a CH3 group without a significant alteration of the acid-base properties of a nearby site.63,65,73,74 With the help of such methyl derivatives so-called micro acidity constant schemes can be developed,63,73,74 allowing the determination of intrinsic pKa values (micro acidity constants). The results are summarized for adenosine in Fig. 2 (for details see Ref. 63): (i) all three endocyclic N’s of Ado show a remarkable basicity, (ii) the basicity decreases in the series N1 > N7 > N3, and hence (iii) one expects that all three sites are able to bind metal ions. Furthermore, the three monoprotonated

Fig. 2 Summary of the acid-base properties of adenosine (25  C).63 The constants with a negative sign (upper part) are based on the H0 scale as conceived by Hammet and Deyrup;67,68 otherwise I ¼ 0.5 M (NaNO3) applies.

Metal ion interactions with nucleic acids

635

Ado tautomers occur with formation degrees of about 96.1%, 3.2%, and 0.7% for N7(Ado)N1$Hþ, þH$N7(Ado)N1, and N1,N7(Ado)N3$Hþ, respectively, as also confirmed by DFT calculations.63 Similarly, it is interesting to note how the deprotonation of (N1)H, e.g., in guanine, affects the basicity of N7: What is the acidity constant of (N7)Hþ when (N1)H is deprotonated and the guanine residue exists in its zwitterionic form? At the top of Fig. 3 the zwitterionic form of 9-methylguanine, 9MeGua, is shown, and in the lower part at the left its 7-methylated species, 7,9-DiMeGuaþ is given, which mimics monoprotonated 9MeGua. The respective micro acidity constant scheme is found in Ref. 73 and the corresponding results73,74 are summarized in Table 3 together with the directly measured macro acidity constants for the 9-methyl derivatives of hypoxanthine (9MeHpx), guanine (9MeGua), and adenine (9MeAde). These micro acidity constants allow to calculate the position of the following tautomeric Equilibrium (5), where 9MeGua is used as an example: 9MeGua #9MeGua0

(5) þ

The micro acidity constants of Table 3 allow further to obtain the ratio, RT, for the isomeric species having H in one tautomer at N1 and in the other at N7 (Eq. 6): kN7N1 ½N7ð9MeNBÞN1,H H,N7ð9MeNBÞN1  ¼ N7N1 RT ¼ þ H,N7ð9MeNBÞN1 kN7ð9MeNBÞN1,H

(6)

These tautomeric ratios appear in column 6 of Table 3 (for details see Ref. 73) and they show that for the 9-methyl derivatives of hypoxanthine and guanine the neutral tautomers with the proton at (N1) strongly dominate, with formation degrees close to 100% (Eq. 7): %½N7ð9MeNBÞN1$H0 ¼ 100$RT =ð1 þ RT Þ

(7)

73

The situation is somewhat less one-sided for the adenine derivatives; e.g., for monoprotonated 9-methyladenine it follows from RT ¼ 12.9 and Eq. (7) that about 93% of H(9MeAde)þ occur as N7(9MeAde)N1$Hþ and about 7% as þH$N7(9MeAde) N1. The corresponding tautomeric distribution for adenosine is 96 versus 4% (RT ¼ 24.5  12.4).73 However, for adenosine another tautomeric equilibrium, one between an amino and an imino form, is expected to occur (for details see Ref. 73).74,75 The amino form of Ado strongly dominates: Among 1000 000 Ado molecules about one is present in the imino form, but this is enough for initiating mutations.74

2.20.3

Metal ion affinities of individual sites of single-stranded nucleic acids

Understanding the role of metal ions in nucleic acids does not only require knowledge of the exact positioning and coordination sphere of each specifically bound metal ion, but also of its intrinsic site affinity. Yet, the quantification of M2þ metal ion affinities towards certain sites in a single-stranded (though folded) nucleic acid is challenging. Some years ago, a tool has been developed to estimate the binding affinity of a given M2þ, based on its ligating sites within the nucleic acid.39 The available stability constants for binding of Ca2þ, Mg2þ, Mn2þ, Cu2þ, Zn2þ, Cd2þ, and Pb2þ to nucleobase residues were collected and the stability constants for

Fig. 3 The upper part shows the zwitterionic form of 9-methylguanine, i.e., 9MeGua, sometimes also written as þHN7(9MeGua)N1 to indicate that Hþ is at N7 and that N1 carries a negative charge. The structures in the lower part emphasize the close similarity between 7,9-dimethylguanine, H 7,9DiMeGuaþ, and monoprotonated 9-methylguanine, H(9MeGua)þ, which allows the use of pK7,9DiMeGua ¼ 7.22  0.01 (deprotonation of (N1)H), H , N7  N1 þ 73,74 for the micro acidity constant pkH , N7(7, 9MeG)N1 , H of H(MeGua) .

636 Table 3

Metal ion interactions with nucleic acids Micro acidity constants for the (N7)H and (N1)H sites of some 9-methylated purine derivatives.a

protonated 9MeNB (see Fig. 1)

H pKH(9MeNB) ¼ pKa/(N7)H

H pK9MeNB ¼ pKa/(N1)H

- N1 pkHN7$ N7(9MeNB)N1

N7 - N1 pkN7(9MeNB)N1 $H

RT ¼ ½N7ð9MeNBÞN1•H ½H•N7ð9MeNBÞN1

H(9MeHpx)þ H(9MeGua)þ H[H(9MeAde)]2þ

1.87  0.01 3.11  0.06 0.64  0.06

9.21  0.01 9.56  0.02 4.10  0.01

4.62  0.02 5.45  0.06 2.96  0.10

9.21  0.01 9.56  0.02 4.07  0.08

38,900  2000 12,900  1900 12.9  3.8

a

Also given are the ratios, RT (Eq. 7), quantifying the formation degrees of the connected tautomers. The measured macro acidity constants, pKa/(N7)H and pKa/(N1)H (Eqs. 1a, 1b, 2a and 2b), are given for comparison. All values apply to 25  C and I ¼ 0.1 M (NaNO3) with the exception of the value in the second column of the third entry. The constants are collected from Tables 1 and 2 of Ref. 73. For the error limits (3s) see footnote “a” of Table 2; the error limits of the derived data were calculated according to the error propagation after Gauss. RT is calculated with Eq. (6); for its meaning see also Equilibrium (5), where 9MeGua corresponds to þH$N7(9MeNB)N and 9MeGua0 to N7(9MeNB)N1$H.

coordination of these M2þ ions to the phosphodiester bridge were estimated. In these estimates the assumption was included that the four nucleobases of RNA and DNA occur in about equal amounts; if so, then the anionic phosphodiester bridge has a 4-fold

Fig. 4 Upper part: Logarithms of the individual M2þ affinities for the various binding sites within nucleotide residues in single-stranded nucleic acids. Lower part: M2þ-affinity sequences for single-stranded nucleic acids with the phosphodiester groups highlighted in red. “” means the logarithm of the complex-stability difference is below 0.2 log unit, “>” indicates a difference larger than 0.2 log unit, and “> > ” a stability difference of more than 0.5 log unit. For details Ref. 39 should be consulted. This figure is reproduced from Ref. 39 by permission of the American Chemical Society.

Metal ion interactions with nucleic acids

637

excess towards each nucleobase residue. This statistical factor of 4 (¼ 0.6 log unit) was added to the stability constants estimated for the M[(RO)2PO2]þ species.39 In this way stability increments for each ligand site were obtained reflecting a clear selectivity of the ligating atoms, as well as a discrimination by different metal ions (Fig. 4). On the basis of these data a concept is proposed that allows to estimate the intrinsic stabilities of nucleic acid-binding pockets for the given metal ions.39 By adding up the individual increments, like building blocks, one obtains an estimate not only for the overall stability of a given coordination sphere, but also for the most stable complex if an excess of ligating atoms is available in a binding pocket saturating the coordination sphere of the metal ion. For example, Mg2þ binding to a phosphodiester unit and a guanine residue, 1.05 þ 0.75 are added up to yield the intrinsic stability constant of 1.8 log units for a pGpG site in a nucleic acid sequence (Fig. 5). However, a hexacoordinating Mg2þ still has two to three further binding sites. For statistical reasons (and steric constraints or preorientations) it is estimated that this additional stability increment only amounts to about 0.5 log unit (i.e., about 3/5 from 0.75; for details see Ref. 39). This gives an overall micro stability constant of 2.3 log units, which agrees well with experimental data obtained from 1H NMR shift experiments:77,78 The different Mg2þ-binding sites in branch-domain 6 of a group II intron ribozyme vary in their affinities between 2.14  0.03 and 2.38  0.06 log units,78 possibly indicating that the Mg2þ coordination sphere is not always completely filled by RNA binding sites. For further and more complicated examples Ref. 39 should be consulted.

2.20.4

Metal ion binding to RNAs

RNAs fulfill many functions in a living cell: they serve as information carriers between genes and their encoded proteins (mRNA), they are directly involved in protein synthesis (tRNAs and ribosome), participate in post-transcriptional RNA modification (spliceosome and related RNAs), are a crucial part in the replication cycle of viral RNAs, and play a role in gene regulation (riboswitches and miRNAs), among many other functions. In order to achieve this wealth of different functions, RNAs occur in lengths of a few nucleotides up to several thousands and thereby adopt complicated three-dimensional architectures. Whereas some of them function in combination with proteins and/or cofactors, based on structural changes, some also catalyze reactions, i.e., they are “enzymes” composed solely of RNA, so-called ribozymes. In all cases, metal ions are a strict requirement and take part in the RNAs function in some way: They stabilize local and global structures, tertiary contacts, mediate ligand interactions, and/or take directly part in the

Fig. 5 Three- and four-point interaction of a Mg2þ with two consecutive guanines (Gua641 and G642) as is known for Mg2þ in the ribosome.76 Mg2þ is innersphere coordinated to a phosphodiester-oxygen and five water molecules. Hydrogen bonding, i.e., outersphere coordination, is observed to the two N7 atoms (broken lines), thus forming a three-point interaction. A third water molecule forms a hydrogen bond to (C6)O (broken line) resulting in a four-point interaction. The phosphate atoms as well as the coordinating atoms are shown as spheres. This figure is adapted from Ref. 39 and prepared with MOLMOL168 and the PDB file 1S72.76

638

Metal ion interactions with nucleic acids

catalytic mechanism. According to todays knowledge, in vivo only Mg2þ and Kþ are associated with RNAs, but based on recent findings the interaction with other divalent metal ions cannot be excluded anymore. Other metal ions are often applied in vitro15,16 and sometimes even enhance the functional rate,79–81 but an interaction in living systems still has to be proven. In contrast to metalloproteins, metal ions associated with nucleic acids are kinetically labile, rather loosely bound and only occupy a few distinct binding sites. Metal ions are mostly associated with charge compensation of the negatively charged phosphate-sugar backbone and thus the folding of the RNA backbone is strongly driven by the association with metal ions. In addition to this more electrostatic interaction, metal ions at distinct sites stabilize local structures and are placed correctly to enable and support catalysis directly. In the following sections, first the binding of metal ions and their complexes will be discussed including the description of known binding motifs. Second, the folding and dynamics of RNAs with respect to the role of metal ions will be summarized before the distinct roles of metal ions in riboswitches and catalysis will be highlighted.

2.20.4.1

Solvation content of metal ions

Mg2þ is rather small with an ionic radius of 0.72 Å,23 has a strict octahedral coordination sphere and a strong preference for oxygen ligands. In aqueous solution, this ion is thus present in its hexaaqua form with a first pKa of the coordinated water molecules of pKa ¼ 11.44  0.1 (Ref. 26) and a ligand exchange rate of 6.7  105 s1.82,83 While other alkaline earth metal ions used with RNA are larger and have faster ligand exchange rates, d-elements and lanthanoids employed in numerous in vitro experiments differ in many physico-chemical properties such as size, electronic properties, preference of N/O ligands and more. A comprehensive summary of the physico-chemical properties of such metal ions associated with RNA in some way is provided in Refs 15 and 16. Due to the relatively high hydration enthalpy DHHydr ¼ 1858 kJmol1.84 which needs to be overcome when coordinating to another ligand, Mg2þ is mostly not fully dehydrated in the context of nucleic acids. Binding of Mg2þ to nucleic acids is thus mostly a combination of innersphere (i.e., direct) and outersphere (i.e., mediated by H2O) coordination (Fig. 6). In addition, providing the size and shape of the binding pockets allow an equilibrium between different liganding sites may also exist. Such rather dynamic systems hamper the investigation and characterization of Mg2þ binding sites in larger nucleic acid structures and consequently many unanswered questions regarding the metal ion coordination to RNA (and DNA) still exist. An evaluation of the 88 identified Kþ/Naþ and 116 Mg2þ binding sites within the large ribosomal subunit of Haloarcula marismortui reveals some interesting facts: (i) assuming these metal ions are the specifically bound ones, they only account to roughly 11% of the charge compensation needed by the phosphate-sugar backbone; (ii) nine ions are fully hydrated, one is completely dehydrated, but all the others show a mixture between inner- and outersphere coordination; (iii) 106 Mg2þ are coordinated to a phosphate oxygen, 82 of which by one, and 43 by two or more innersphere contacts.85 These numbers reflect well the importance of oxygen ligands on the one hand and on the other also the crucial involvement of water-mediated contacts to place a Mg2þ correctly. Two databases provide more information on the importance and placement of water molecules within nucleic acid structures, both in the presence and absence of metal ions. The Solvation Web Service (SwS)86 provides a statistical survey of water molecules of the first solvation sphere around nucleobase pairs, as identified from nucleic acid structures deposited in the NDB. The just

Fig. 6 A Mg2þ bound via two innersphere interactions to N7 sites of two guanines, bridging two strands, as well as displaying numerous outersphere interactions via their coordinated water molecules Mg2þ N 11 in the large ribosomal subunit of Haloarcula marismortui.76 This figure has been prepared with MOLMOL168 and is adapted from Ref. 85

Metal ion interactions with nucleic acids

639

established Metal Ions in Nucleic AcidS (MINAS) database87 compiles the first and second shell coordination sphere of all metal ions in the context of nucleic acid structures deposited in the PDB. A multitude of search options allows to gain insights into the coordination spheres of metal ions and the involvement of water molecules in their binding to nucleic acids.

2.20.4.2

Thermodynamics of metal ion binding to RNA

Metalloproteins bind their metal ions with high affinity, usually in the order of 1010 M1.27 Mg2þ binding to RNA (and nucleic acids in general) is by about 6-8 orders of magnitudes weaker and in most cases rather unspecific. A large part of charge compensation is achieved by high ionic strengths through monovalent ions as well as a “sea” of Mg2þ ions. Only about 10% are estimated to bind more specifically,85 although only with affinities around 103 M1.9,33 The rather low affinity, fast ligand exchange rates, as well as the presence of a large number of equivalent ions in the surrounding make a localization and characterization of their thermodynamic properties very challenging.15,16 In addition, metal ion binding is mostly accompanied by folding and structural changes of the RNA making it very difficult to distinguish between and separate the two effects. Methods to detect and quantify metal ion binding to RNA range from classical chemical (hydrolytic or radical cleavage), over biochemical (metal ion switch) to spectroscopic methods (EPR, luminescence, NMR, etc.). For a detailed summary of all these methods please see refs.15,16,88 Several of these methods can be used to determine apparent, averaged, or even intrinsic binding constants and shall be briefly summarized in the following.

2.20.4.2.1

Indirect methods

2.20.4.2.2

Hydrolytic cleavage experiments

The thermodynamic stability of RNA structures can be measured in dependence of metal ion concentration, e.g., by UV melting 89,90 or isothermal calorimetry,91,92 yielding first ideas about metal ion affinities. A further indirect method widely used is to measure the observed rate of ribozyme catalysis in dependence on metal ion concentration.93–95 The number of more strongly and specifically bound metal ions to a folded RNA structure can be determined by applying a high background of monovalent ions. For example, the folding of the P4-P6 metal ion core of a group I intron has been examined at 2 M NaCl by looking at the degree of protection from hydroxyl radicals. Fitting to a classical Hill equation leads to the stoichiometry of two Mg2þ needed to fold the core with a KD of about 0.5 mM.96 The extent of metal ion binding to RNA can also be assessed by determining the amount of free metal ions in solution. In such “ion counting” methods, fluorescent chelators that bind to certain metal ions yield the free metal ion concentration (but also disturb the equilibrium).97 For example, the fluorescence of 8-hydroxyquinoline-5-sulfonic acid (HQS) is strongly increased when bound to Mg2þ (K ¼ 1.13  104 M1).97–99 Based on the known RNA concentration and the stability of the Mg(HQS) complex, the amount of bound metal ions can be calculated. Equilibrium dialysis experiments yield equivalent information upon determination of the metal ion concentrations (e.g., by atomic emission spectroscopy) after reaching the equilibrium between an RNA-containing and an RNA-free cell connected by a membrane.97,100

Due to the presence of the 20 -OH group of the ribose, RNA is much more prone to hydrolytic cleavage of the phosphate sugar backbone than DNA. However, the presence of this OH group is not enough. In order for efficient cleavage to occur four prerequisites need to be fulfilled: (i) the attacking nucleophile, i.e., the 20 -OH, needs to be activated; (ii) the nucleophile and the leaving group must be positioned correctly, i.e., ideally in a 180 angle; (iii) the transition state should be stabilized; (iv) the leaving group must be stabilized, i.e., protonated (Fig. 7).101 The corresponding mechanism is employed by the so-called small ribozymes, i.e. catalytic RNAs, like the Hammerhead, hairpin, VS, Hepatitis Delta virus, glms, but also other ribozymes.104 While the activation of the nucleophile and the stabilization of the leaving group is still under debate for most of these ribozymes, from structures it became clear that geometric restraints in the active site always lead to the required 180 between the two decisive groups, i.e., the attacking 20 -OH and leaving 50 -O (Fig. 7).101 Metal ion promoted hydrolytic cleavage has been used repeatedly to identify metal ion binding sites in complex RNA structures. Originally, Pb2þ has been used to probe for metal ion binding sites in PhetRNA.105–109 The leadzyme, an in vitro selected ribozyme, has been optimized such that it is highly selective for Pb2þ to cleave RNA.110,111 Otherwise, mostly other metal ions are used nowadays that resemble better the assumed natural cofactor Mg2þ. This includes especially lanthanide(III) ions,7,112–114 but in general also transition metal ions have been used repeatedly.114,115 The ability to cleave the RNA backbone is mainly dependent on the pKa value of the hydrated metal ion, the reaction being more rapid the more acidic the coordinated water is.15,26 Due to the different coordination properties of the employed metal ions also different binding/cleavage sites are detected, making it often difficult to draw conclusions about the Mg2þ binding sites.79,85 Mg2þ itself can also be used, but requires long incubation times of >24 h and an elevated pH. The latter experiments are usually called “in-line” probing and are used to investigate structural changes of the three-dimensional RNA architecture.15,116–119 Generally, such hydrolytic cleavage experiments are to a large part straight forward and can be applied to RNAs of any size. On the other side, only metal ions close to the 20 -OH will be detected, i.e., those sitting in the minor groove and/or non-helical regions.

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Fig. 7 Hydrolytic cleavage of the RNA backbone. (A) Mechanism of hydrolytic cleavage: The attacking nucleophile 20 -OH needs to be activated, the nucleophile and the leaving group arranged in an 180 angle, and the leaving group stabilized by either a proton or metal ion. Alternatively, also an external nucleophile, e.g., a water molecule, can be used. (B) Catalytic core of the glms ribozyme with the attacking and the leaving group correctly arranged: The attacking 20 -O (in this case methylated to prevent cleavage during crystallization), the phosphorous and the 50 -oxygen form a 180 angle.101,102 (C) Crystal structure of the transition state of the hairpin ribozyme with vanadate as the mimic of the five-coordinate transition state (aquamarine sphere).103 Possible hydrogen bonds of a protonated adenosine and a deprotonated guanosine are indicated by dotted lines.101,103 This panel has been prepared with MOLMOL.168 Adapted from Ref. 101 and based on the PDB files 2NZ4 (glms ribozyme)102 and 1M5O (hairpin ribozyme).103

2.20.4.2.3

Oxidative cleavage experiments

2.20.4.2.4

Spectroscopic methods

In order to circumvent some of the caveats occurring with hydrolytic cleavage, the backbone can also be cleaved oxidatively.15 The major advantage is that no geometric restraints apply and thus that also metal ion binding sites in the major groove are detected. By Fenton chemistry, short lived and highly reactive hydroxyl radicals, $OH, are formed through reduction of H2O2 mediated by the oxidation of Fe2þ to Fe3þ.120 Originally, this method was used to probe for protected regions in protein and RNA structures by applying the Fe(EDTA)2 complex, which is only found at the outside of the three-dimensional structure.121,122 In the form of its free hexaaqua ion, the octahedral Fenþ presumably occupies Mg2þ binding sites in complex RNA structures, the formed $OH radicals cleave then the backbone in close vicinity.123 To minimize the iron(II/III) concentration, the process is made catalytic by the addition of glucose. Similarly, also Fe(II)-bleomycin complexes cleave RNA in the presence of O2.124,125

A variety of spectroscopic methods have been developed to investigate metal ion binding to nucleic acids. These include electron paramagnetic resonance (EPR), lanthanide(III) luminescence, X-ray absorption spectroscopy (XAS), vibrational spectroscopies, as well as a large diversity of nuclear magnetic resonance (NMR) methods. These methods have all been thoroughly summarized and described in a couple of recent reviews.15,16,88,126–128 Thus in the following only a brief overview on every method is provided. EPR has been applied to investigate folding of RNAs by attaching spin labels at two different domains,129, but it can also be used to investigate metal ion binding directly.15,128 As EPR depends on unpaired electrons, Mg2þ has to be replaced by, e.g., Cu2þ.130 the vanadyl ion,131 or in most cases Mn2þ.128 Mn2þ binding to RNA can be quantified because upon binding the perfect symmetry of the Mn(H2O)62þ ion is broken, leading to severe line broadening.132–134 Applying more advanced techniques like electron nuclear

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double resonance spectroscopy (ENDOR), also the hyperfine interaction with other nuclei on the RNA can be detected, allowing for the identification of liganding sites.135 Electron-spin echo envelope modulation (ESEEM) allows to detect nitrogen coordination and to quantify the level of hydration of Mn2þ.136–138 Lanthanide(III) luminescence is strongly dependent on the state of hydration of the metal ion.26 This effect has been used to study the binding of Eu3þ and Tb3þ to the hammerhead ribozyme.139 The whole system turned out to be highly complex and hence, the much needed basis first needed to be established.126,140,141 An important prerequisite for the investigation of metal ion binding by X-ray absorption spectroscopy is that the metal ion is tightly bound. For example, the Mo(IV) center in the polyoxometalate [Mo7O24]6 has been shown to be the active site for phosphodiester hydrolysis,142 and a Cu(III)salen complex has been investigated when cleaving the HIV-1 TAR RNA.143 Bond vibrations are highly sensitive to ligand or metal ion binding, making Fourier-transform infrared spectroscopy (FT-IR) in principle a perfect method for the quantification of metal ion binding to RNA. However, as usual, a crucial problem is the dynamic nature and weak strength of binding and hence again, only a few examples exist with more tightly bound ions like Pt2þ.144 or Agþ 145 as well as As2O3.146 The related Raman signal can be used to distinguish between innersphere and outersphere binding as has been demonstrated in the Mg2þ/HDV ribozyme system.147 By this method metal ion identity and quantification is possible.148 Lastly, NMR has been applied in uncountable studies and variations to detect and quantify metal ion binding to RNAs and is thus certainly one of the best methods. As has been recently summarized in a number of reviews, this method yields structural as well as kinetic and thermodynamic information at the same time.15,16,127 Metal ion binding is classically detected by chemical shift mapping, preferably of non-exchangeable protons,78,149,150 or the 31P resonance.151–155 Line broadening effects of the paramagnetic Mn2þ or the fast exchanging Mg2þ can be used to distinguish local and more distant effects.15,78,156 By applying twodimensional NMR, e.g., [1H,15N]-HMQC (heteronuclear multiple-quantum correlation), to look at the 2J coupling between nitrogen and the proton at an adjacent carbon, direct coordination to the N7 position can be monitored.157,158 Exchange kinetics can be determined by magnetic relaxation exchange dispersion (MRD) experiments using quadrupolar nuclei at different field strengths.159,160 Such, the life times of 23Naþ (50 ns) and 85Rbþ (200 ns) in the minor groove of A-tracts in DNA could be determined.161,162 Much slower exchange rates are observed in a G-quadruplex, being in the lower microsecond range for 23 Naþ (90 ms) and 85Rbþ (17 ms).163 Longer life times of up to 150 ms were found for 205Tlþ.164 Similarly, also NH4þ and Co(NH3)63þ can be used in cross-relaxation experiments to gain upper and lower limits of exchange rates.165 Binding affinities of the metal ion can be calculated from titration experiments using the change in chemical shift of nonexchangeable protons (or also of nitrogen and carbon nuclei) as a measure. It is imperative to use non-exchangeable protons as the chemical shift of imino protons is also strongly affected by the proton exchange rate with the solvent. KD values obtained are usually in the lower millimolar range.15,16,78,149 An important aspect to be taken into account is the fact that RNA usually offers various binding sites with similar affinities competing for the metals and being filled up simultaneously. As a consequence the total metal ion concentration does not correspond anymore to the available one. This can be taken into account by applying an iterative procedure to calculate the intrinsic affinities at each site, e.g., by using a recent algorithm, ISTARdIntrinsic STAbilities of RNA complexes.77,78

2.20.4.3

Metal ion binding motifs in RNA by Mg2D

It is estimated that roughly 10% of the total negative charge of a larger RNA is compensated by more strongly coordinated metal ions.15,76,79,85 These metal ions occupy specific binding pockets, which have been classified into binding motifs according to their coordinated nucleotides. The Metals in RNA database “MeRNA” lists these motifs together with a number of structures where they have been found and described.166 After a brief description of a proposed classification these motifs are shortly summarized in the following.

2.20.4.3.1

Classification of Mg2þ binding sites

A detailed list of all RNA-metal ion interactions and a proposed classification for a strictly octahedral coordination sphere is available at http://www.csgid.org/metalnas/.167 As an example, the binding mode of the Mg2þ depicted in Fig. 8 will be used: Only coordinating ligands other than water are considered in a first place. The Mg2þ is bound to one inner sphere phosphate oxygen which is designated as Oph and to a second inner sphere contact to the uracil O4. An inner sphere contact to a base oxygen is designated as Ob. The full inner sphere classification for the inner sphere is therefore: Oph$Ob. In the second coordination sphere, two outer sphere contacts to a guanine nucleobase are present. In this case, the coordinating atom is irrelevant, the important parameter being the number of interactions, not the number of interactors: Two outer sphere interactions with nucleobases yields a 2Bout classification.

2.20.4.3.2

Tandem GC base pairs

Two or more adjacent GC base pairs offer a binding site for Mg2þ in the major groove of an A-form helix. This finding is not surprising considering that the N7 position of purines is a well-known metal ion binding site in general. The guanine N7 is thereby favored over adenine N7 because of the (C6)O carbonyl. The carbonyl oxygen is a perfect additional coordination site for either innersphere or outersphere coordination of a Mg2þ being bound to N7 of the same nucleobase. The adenine (C6)NH2 in contrast offers no such additional stabilization properties for metal ion binding,170 but rather inhibits coordination.171 Examples of such a metal ion binding motif are domains 5 and 6 of the group II intron ribozyme ai5g from Saccharomyces cerevisiae (Sc.ai5g),78,114,150

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Metal ion interactions with nucleic acids

Fig. 8 An example of a Mg2þ coordination site for the visualization of the classification nomenclature. The Mg2þ is inner sphere bound to a phosphate oxygen and uracil O4. The Mg2þ center is shown as a green sphere, the coordinating nitrogen atoms as dark blue spheres, and the coordinating oxygen atoms as red spheres. This figure has been prepared by MOLMOL168 based on the PBD file 2YIE.169

as well as the stem loop C of a Mg2þ riboswitch.172 By chemical shift experiments, the helical GC regions have been identified to bind Mg2þ in all these cases, showing an intrinsic affinity of, e.g., log KA ¼ 2.33  0.03 in the case of domain 6.78 Crystal structures reveal that metal ion coordination takes place through an intricate network of direct coordination and hydrogen bonding to the purine moieties as well as phosphate oxygens, as is for example seen in the large ribosomal subunit of H. marismortui (Fig. 9).76

Fig. 9 Mg2þ binding to tandem GC base pairs. The Mg2þ is bound in a pure outersphere fashion to two consecutive GC base pairs, as is found in the large ribosomal subunit of Haloarcula marismortui. The Mg2þ center is shown as a green sphere, the coordinating nitrogen atoms as dark blue spheres, and the coordinating oxygen atoms as red spheres. This figure has been prepared by MOLMOL168 based on the PBD file 1S72.76

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Fig. 10 GU wobble pairs are well-known metal ion binding sites. (A) Major groove site of the branch region within domain 6 of the Sc.ai5g group II intron from yeast mitochondria. The two GU wobble pairs flanking the branch adenosine are shown in blue. Potential coordinating atoms in this metal ion binding pocket are shown as spheres, whereby N7 of the branch adenosine (light blue) has been shown not to coordinate to Mg2þ.157 (B) Mg2þ binding to a GU wobble pair. The N7 position is coordinated in an innersphere fashion. Atoms involved in binding are shown as spheres. (C) Cobalt(III)hexammine binding to tandem GU wobble pairs in a purely outersphere fashion. The Co3þ center is shown as cyan sphere, the ammines and coordinating nitrogen atoms as dark blue spheres, and the coordinating oxygen atoms as red spheres. The panels have been prepared with MOLMOL based on the PDB files 2AHT (A); adapted from Refs.150,157 1D4R (B) 176 and 1GID (C) 177.

2.20.4.3.3

GU wobble pairs

GU wobble pairs are the most common non-canonical base pairs in RNA. They often occur in regular A-form helices and can act as a recognition site for proteins or tertiary interactions on the minor groove site as the exocyclic guanine NH2 group sticks out into the minor groove. Consequently these pairs are not only often conserved, but are also important for RNA function: For example, in group II intron ribozymes, a highly conserved GU wobble is part of the catalytic AGC triad in domain 5.173,174 In the same introns two GU wobble pairs flank the branch point adenosine in domain 6 being important for the efficiency of branching.175 In both of these regions, metal ion binding has been shown to occur,114,150,157 and in the latter case both N7 atoms of the two guanosine flanking the branch adenosine have been shown to coordinate to Mg2þ (Fig. 10A).157,176 Although this binding pocket in the major groove of the branch adenosine offers a large variety of possibly coordinating atoms (Fig. 10A), the coordination to the two guanine residues is supported by the intrinsic binding affinity of Mg2þ to this site (log KA ¼ 2.38  0.06).77,78 Using the intrinsic stability increments39 for a coordination to one phosphate oxygen as well as to two guanine moieties (Fig. 10A), an affinity of log KA ¼ 2.30 is obtained (see also Section 2.20.3). Later on, it has additionally been shown that both phosphate oxygens of U19 are involved in Mg2þ coordination, while U21 only coordinates to Mg2þ with the oxygen pointed into the major groove.155 Crystal structures of Mg2þ ions bound to GU wobble pairs show a similar picture: The Mg2þ ion with its surrounding first coordination shell is mostly rather poorly defined (Fig. 10B), but it is likely that mostly coordination to guanine N7 and the neighboring

Fig. 11 Mg2þ binding to the major groove site of a GA sheared base pair. (A) The (C6)O oxygen of the guanine moiety is innersphere coordinated, whereas the N7 and the adjacent phosphate group is linked via outersphere interactions. (B) Three stacked sheared GA base pairs within the large ribosomal subunit bind a metal ion cluster of two Mg2þ corresponding to the loop E motif (Fig. 12). An additional Naþ (magenta) takes the place of a further Mg2þ (magnesium(II) D) in the E. coli 5S rRNA loop E. This figure has been prepared by MOLMOL168 showing Mg204 within the E. coli 5S rRNA (PDB ID 354D)181 as well as Mg43, Mg105, and Na68in the Haloarcula marismortui large ribosomal subunit (PDB ID 1S72).76

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Metal ion interactions with nucleic acids

(C6)O carbonyl takes place. Cobalt(III)hexammine binding has been observed to tandem GU wobble pairs in the P4-6 domains of a group I intron,177 mimicking Mg2þ in a completely outersphere manner (Fig. 10C).

2.20.4.3.4

GA mismatch base pair

2.20.4.3.5

Sheared GA base pair

Next to GU wobble pairs, GA mismatches are very common non-canonical base pairs.178,179 Especially in the large ribosomal subunit the number of GA mismatches is comparable to the number of GU wobble base pairs.76,180 GA mismatches occur in various conformations: The most common type is the Hoogsteen/Sugar-edge interaction, which is also known as the sheared GA base pair. Other types include the cis Watson-Crick/Watson-Crick, the cis Sugar-edge/Sugar-edge, the cis Watson-Crick/Hoogsteen, and the trans Sugar-edge/Sugar-edge. As a Mg2þ ion binding motif, GA mismatches usually occur in pairs (Fig. 11A).182,183

As mentioned above, sheared GA base pairs are the most common type of GA mismatch base pairing. The G minor groove edge is connected to the major groove edge via two hydrogen bonds, i.e., GN3$$$AN6H and GN2H$$$AN7. These GA pairs occur in A-form helices but are best known as the closing base pair in GNRA tetraloops (N ¼ any nucleobase; R ¼ purine). The GNRA tetraloop motif is stabilized into a more compact structure by coordinating Mg2þ at the terminal base pairs.184 GNRA tetraloops are wellknown Mg2þ binding sites, but the exact number of Mg2þ ions and their coordination pattern is still a matter of debate: Alternatively, two more specifically bound ions or one rolling over the surface have been described.77 In the MeRNA database, a Mg2þ binding site is described in which a pentahydrated Mg2þ coordinates directly to (C6)O of the guanine moiety as well as outersphere to phosphate oxygen atoms via a tight network of hydrogen bonds (Fig. 11B). This coordination pattern to one sheared GA base pair does not seem to be distinct as in other instances, e.g., with Mn2þ, different patterns are observed. In the large ribosomal subunit of H. marismortui, one example of three stacked sheared GA base pairs has been found that coordinate two Mg2þ and one Naþ ion (Fig. 11B). The two Mg2þ form a cluster identical to the loop E motif described below.

2.20.4.3.6

Loop E motive or metal ion zipper

The crystal structure of a 62 nt long fragment of the E. coli ribosomal 5S RNA, which contained helices I, IV, and the so-called loop E was solved in 1997 together with a shorter 11 bp long region of the loop E at high resolution.181 The loop E motif contains seven non-Watson Crick base pairs and a number of “cross-strand A stacks”, overall leading to a narrow major groove and a wider minor groove. Most importantly, this motif is always associated with up to five (identified) Mg2þ ions, stabilizing the central three noncanonical base pairs. Two of these ions are hexahydrated (metals A and D) and three pentahydrated having one direct contact each to either a non-bridging phosphate oxygen or a guanine (C6)O, respectively (metals B, C, and E). The Mg2þ ions B and C are most interesting as they form a unique binuclear cluster bridged by three water (or hydroxide) molecules (Fig. 12). Both ions are hexacoordinated, each coordinates one phosphate oxygen directly, both have three water ligands in common and are otherwise connected by a tight network of hydrogen bonds to the RNA. The four metal ions B-E lie within the loop E sequence and thus might well narrow the major groove (Fig. 12), opening the minor grove to interact with protein cofactors. This motif has consequently also been named the “metal ion zipper”.181 It is unclear if this binuclear cluster also exists permanently in solution or if it is only partially formed, as molecular dynamics simulations suggest.185 However, the same molecular dynamics simulations,185 chemical and enzymatic probing,186 as well as NMR studies187,188 have demonstrated that the Mg2þ ions are essential for the structural stability of the loop E motif.

2.20.4.3.7

Mg2þ clamp

The magnesium(II) clamp motif has been first described by Dumas and colleagues in 1999,189 based on the crystal structure of the HIV-1 RNA dimerization initiation site. One Mg2þ ion bridges the backbones of two RNA strands on the major groove side right

Fig. 12 Loop E motif within the E. coli 5S rRNA, showing four Mg2þ ions zipping the backbones of two adjacent strands. Mg2þ ions B and C form a binuclear cluster bridged by three water/hydroxide molecules, whereas ions D and E are separated from the others. All four metal ions show a mixture of innersphere and outersphere coordinations. The Mg2þ ions B, C, D, and E are displayed from left to right. This figure has been prepared with MOLMOL168 based on the PDB file 354D.181

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Fig. 13 The Mg2þ clamp with the metal ion bridging two strands via two trans innersphere coordinations to phosphate oxygens, as it has been observed in the HIV-1 RNA dimerization initiation site (HIV DIS; PDB ID 462D).189 This figure has been prepared with MOLMOL168.

next to two bulged nucleotides (Fig. 13). The metal ion is tetrahydrated with the contacts to the non-bridging RP phosphate oxygens being trans to each other. As a consequence of this metal ion, the major groove becomes very narrow and deep with the two bridged phosphate oxygen atoms being only 4.2 Å apart. Whether the neighboring nucleotides are bulged because of Mg2þ coordination (five further Mg2þ are located around the two bulges) or due to crystal contacts is unclear.189

2.20.4.3.8

Y-clamp

The Y-clamp is a very common motif capable of stabilizing RNA structures in a similar manner as the magnesium clamp. Until now 238 Y-clamps have been observed in both ribosomal subunits, as well as in various riboswitches and ribozymes.167 This motif anchors two distant parts of the same RNA strand, thereby maintaining the RNA structure similar to disulfide bridges in proteins. A Mg2þ bridges the backbone phosphates of three nucleotides from two different strands, creating a three-way configuration (Fig. 14). The Mg2þ is thereby trihydrated with three innersphere contacts to the RP phosphate oxygens in a mer isoform.

Fig. 14 The Y-clamp bridges distant nucleotides of the same strand through a Mg2þ via three innersphere interactions to phosphate oxygens, as it has been observed in the glmS ribozyme of T. tengcongensis. This figure has been prepared with MOLMOL168 based on the PDB file 2Z75.190

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Metal ion interactions with nucleic acids

Fig. 15 (A) Mg2þ binding in a N7 macrochelating manner in a purely outersphere fashion. (B) The Mg2þ is embedded in a N7 purine seat with two innersphere coordinations to guanine N7 and two outersphere interactions to four phosphate oxygens. Additionally, all four nucleobases are stacked, leading to a greater stabilization of this motif. The coordination sites involved in binding are shown as spheres. This figure has been prepared with MOLMOL168 based on the PDB files 4V4Q191 and 3PDR192 respectively.

2.20.4.3.9

G-N7 macrochelation and purine N7-seat

Both motifs have been exclusively found in the ribosome, chelating a purine-N7 by Mg2þ with an outer-sphere phosphate moiety (Fig. 15A). The G-N7 macrochelation is a simplified motif including only sequential nucleobases but has been known from mononucleotides for several decades.193 The N7 macrochelate motifs can occur either with an (I) inner-sphere or (II) outer-sphere coordination to guanine N7 (Fig. 15A) and an additional outer-sphere interaction with the phosphate moiety of the same nucleobase. Such a macrochelate formation was first introduced by Scheller et al.194 and later characterized in adenine-nucleotides by Szabó using NMR spectroscopy.195 In RNA structures the N7 macrochelation motif is usually found in two guanine bases but also in adenine-guanine neighbors.167 The purine N7-seat on the other hand coordinates two inner-sphere N7 nucleobases to two outer-sphere phosphate moieties (Fig. 15B).

2.20.4.3.10 Further Mg2þ binding motifs It is obvious that the above list cannot be comprehensive. The rapid technological progress and the concurrent strong increase in high resolution crystal structures automatically leads to the identification of many more metal ions bound to RNA and thus the possibility to understand the coordination structures in more detail. In addition, in many cases a much more rigorous and indepth analysis of existing structures is needed. For example, a putative motif, the so-called G-phosphate, has been found in a crystal structure of the large ribosomal subunit, two examples of a Mg2þ ion have been found within the 23S RNA, where the Mg2þ bridges a non-bridging pro-SP phosphate oxygen with a guanine (C6)O of an adjacent strand.76 In addition, the N7 position is close enough to be coordinated by outersphere interaction. The following points need to be taken into account when considering such a putative motif: Phosphate groups are the most prominent binding sites of Mg2þ ions, e.g., 106 out of 116 identified Mg2þ in the large ribosomal subunit are coordinated to phosphate oxygens. N7 is the next important site with 50 coordinations, followed by (C6)O with 31 occurrences.76,85 Consequently, the bridging of a guanine nucleobase and a phosphate is highly likely to occur and indeed is also found 48 times in the large ribosomal subunit. Hence, this so-called “G-phosphate” motif might just be a subclass of a much larger family of binding motifs involving the N7/(C6)O site as well as a phosphate oxygen. Similar to the “G-phosphate” motif, a new “U-phosphate” motif has been detected mostly in the ribosome. Databases like MINAS, Metal Ions in Nucleic AcidS, which allows the detailed characterization of the first and second coordination sphere of any metal ion,87 or the Solvation Web Service, SwS,86 will thereby be of great help to identify and classify new binding motifs.

2.20.4.4

Metal ion binding motifs in RNA of monovalent metal ions

Monovalent cations, usually alkaline metal ions, have different coordination properties towards RNA than Mg2þ, despite all being so called hard metal ions. The coordination geometry of monovalent metal ions relevant in nature, mostly Naþ and Kþ, is more flexible than the octahedral geometry of Mg2þ and the coordination distance are distinctly larger for the monovalent cations (Table 1).196 The typical distances of inner sphere coordination of Kþ and Naþ to its ligands exceeds 2.8 Å, as opposed to about 2.0 Å for Mg2þ. Additionally, binding of monovalent metal ions such as Kþ is kinetically even more labile than Mg2þ, meaning

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Fig. 16 GU wobble pairs known to be also monovalent metal ion binding sites. In this example, a Kþ bound to guanine O6 as well as to a phosphate oxygen on the opposite strand in an innersphere fashion. In addition, the GU wobble stacks to a GC base pair. The Kþ is shown in pink, while all other coordinating atoms are depicted by red spheres. This figure has been prepared with MOLMOL168 based on the PDB file 3PDR.192

that they are more transiently bound.196,197 The weaker binding interaction makes the recognition of a specific motif more difficult. In the following sections, we will take a look at some motifs that bind to monovalent metal ions.

2.20.4.4.1

GU wobble

2.20.4.4.2

AA platform

In the last years, it has been shown that GU wobble pairs can also bind monovalent cation such as Kþ.198 Coordination of the Kþ has been observed in the major groove (Fig. 16) coordinating to O4 of uracil and O6 of guanine in an inner sphere manner with simultaneous outer sphere coordination to a phosphate. This binding pattern can be extended into the neighboring base pair of any kind. In this example, the extended coordination sphere of Kþ allows further binding to guanine O6, as well as cytosine N4.

The AA platform is a structural motif that is formed by two consecutive coplanar adenosine moieties, first observed in a crystal structure of a group I intron ribozyme domain.177,199,200 This motif is a crucial part of a standard tetraloop receptor region where the two coplanar As stack onto the incoming adenosines of the tetraloop,177 but is also found in other local non-helical structures.76 AA platforms were observed to be stabilized by monovalent as well as divalent ions.76,199,201Coordination of monovalent ions occurs more often due to the high degree of dehydration within such docked RNA tertiary interactions and the smaller energetic penalty compared to Mg2þ.201 The metal ion sits above the AA plane and is coordinated to the phosphate groups of the two adenosines as well as carbonyl oxygens of adjacent guanosine and uracil residues (Fig. 17).

Fig. 17 AA platform with a Naþ ion lying on top of the two coplanar nucleobases. The potential coordination sites are shown as red (oxygens) and blue (nitrogen) spheres on the RNA. This Figure has been prepared with MOLMOL168 based on the PDB file 1S72.76

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Metal ion interactions with nucleic acids

Fig. 18 The interaction of Kþ in GG stacking can be classified into two types: Type I encompases the interaction with two successive guanines on the same strand, while Type II encompases the interactions between guanines on different strands. (A) Type I stabilization of GG stacking by an interaction between the backbone phosphates. (B) Type I interaction of the Kþ with the guanine bases. (C) Type II stabilization via coordination of a backbone phosphate and a base. In this case, it is an interaction with the 2-hydroxyl of the ribose ring. Additionally coordinating water molecules are not shown. Additionally, Kþ is shown as pink sphere. The panels have been adapted from Ref. 202 and prepared with MOLMOL168 based on the PDB file 6QNR.202

2.20.4.4.3

GG stacking

2.20.4.4.4

Nucleobase tetrads

Two guanines stacking on top of each other is the most abundant interaction stabilized by Kþ in the ribosome (Fig. 18). There are two types of GG stacking: (i) two successive guanines on the same strand are coordinated either via the backbone or the two purines, or (ii) stabilization occurs between two distant guanines from different strands, where the Kþ coordinates either to both bases or one base and one backbone. Type II stabilization seems to be preferred according.202

Nucleobase tetrads, mostly G-quadruplexes are the most well-known nucleic acids structures, being stabilized best by as well as being dependent on monovalent ions. First proposals for four guanosine 50 -monophosphates forming so-called G-tetrads came up already in 1962,203 closely followed by similar proposals for poly-guanylic acids stabilized by Naþ ions.204 Today, DNA Gquadruplexes are structurally rather well characterized,205 whereas corresponding RNA quadruplex structures have been discovered much later and are thus less well characterized.206 Many potential G-quadruplex forming sequences in RNA have been identified today, the most prominent ones being the telomeric repeats. Mostly they are proposed to be regulatory elements, often occurring in the 50 -UTR of mRNAs, e.g., as translation inhibitors or in post-transcriptional regulation (for recent reviews, please see Refs 207–209). Aside from G-quadruplex structures, similar quartets were also found for uracil,210,211 thymine,212 as well as adenine,213 although the latter has so far not been experimentally confirmed (Fig. 19). While in a first NMR solution structure of a U4 quartet,214 the metal ions were not included, in a corresponding X-ray structure, monovalent ions or Pt(II)-NH3 groups were shown to stabilize such a structure.210–212

Fig. 19 Two corresponding U4 quartets as found in the top plane of a G/U quadruplex structure (A) as well as stabilized by two Pt(IV)-NH3 groups of two Pt(NH3)2Cl4 complexes sitting below and on top of four 1-methyluracil nucleobases (B). The H atoms involved in hydrogen bonds of the U quartet are shown as small grey spheres, nitrogens in dark blue, oxygens in red, the Pt4þ centers in light yellow and the chloride ligands in light green. The two panels have been prepared with MOLMOL168 based on the PDB file 1RAU214 and CCDC 182/505.210

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Fig. 20 G quadruplex structure shown from the side (A) and the top (B). The coordinating Kþ ions are lined up in the middle of the tube and are fully dehydrated. This figure has been prepared with MOLMOL168 based on the PDB file 1P79.218

A central feature of all these quartet structures is their essential stabilization by metal ions in their center. The metal ions sit between the nucleobase quartet planes coordinating to the carbonyl oxygens above and below. The overall geometric restrictions imposed by hydrogen bonding within a quartet and stacking interactions between the quartet planes explain the distinct stability sequence for different monovalent and divalent cations in DNA G-quadruplexes: Kþ > > Naþ  Rbþ  Csþ  Liþ and Sr2þ > > Ba2þ > Ca2þ > Mg2þ > Mn2þ > Co2þ > Zn2þ.206,215–217 All metal ions are expected to sit in a octacoordinate, fully dehydrated environment as is depicted in Fig. 20 with Kþ as an example.218 RNA G-quadruplex staructures are much more stable compared to their analogous DNA counterparts due to the presence of the 20 - hydroxyl group.217 While the stability in DNA G-quadruplexes is dependent on the metal ion species, in RNA G-quadruplexes Kþ stabilizes distinctly better than all other monovalent and divalent ions. In fast, all other metal ions result in melting temperatures to the G-quadruplex similar to water or buffer environment only. The melting temperature of RNA G-quadruplexes is usually 10– 30  C higher in the presence of Kþ than of Naþ, the next best stabilizing ion. The stabilizing effect of divalent metal ions is more diverse, i.e., dependent on the quadruplex sequence: while the 18 nt-NRAS sequence displays the same dependence following the dehydration energies (as in DNA G-quaduplexes), a 24 nt-TERRA G-quadruplex is stabilized only by Sr2þ.217 Under high Kþ concentrations and/or a “supramolecular crowding” environment mimicking the cellular medium, melting temperatures of >95  C can be reached, which speaks for stable quadruplex structures also in vivo.206 A further notable difference between DNA and RNA quadruplexes is the fact that RNAs only adopt an all-parallel topology, whereas DNAs occur in various topologies. This restriction in possible topologies results from steric hinderance of the 20 -OH group in RNA, which forces the guanines into an anti-conformation limiting the RNA to only attaining parallel topologies.219

2.20.4.5

Binding of kinetically inert metal ions

To the best of our today’s knowledge, only kinetically labile metal ions coordinate to RNA in a healthy cell. However, numerous medical or analytical applications and conditions lead to the binding of inert metal ions, the most prominent one being platinum(II).

2.20.4.5.1

Binding of Pt2þ to RNA

Pt2þ binding to DNA is well established: After the discovery of the anticancer properties of cisplatin, cis-[(NH3)2PtCl2],220 and the realization of its binding to DNA, half a century of intense research not only on Pt(II)-based complexes, but also on other metalbased drugs was initiated.221,222 Aside from the classical and well-known N7 coordination,170 also a multitude of other coordination sites in all nucleobases has been explored.223–227 Metal ion coordination thereby has a strong influence on the acid-base and hydrogen bonding properties of the nucleobase.47,224,228–230 We concentrate solely on RNA in this chapter. Nuclear DNA is still considered the main target for cisplatin and its analogs.231–233 However, only a small fraction of the drug enters the nucleus, the majority being in the bloodstream.232 The consequent possible interaction of cisplatin with biomolecules other than DNA has been named as one possibility for the numerous side effects during chemotherapy, and the interaction of cisplatin with proteins has been subject of intense studies.221,232,234 RNA, being highly abundant in the cell and involved in uncountable processes,235 has lately also evolved as possible target for cisplatin and related drugs. Comparison of Ptnþ accumulation in RNA and DNA of Saccharomyces cerevisiae shows 4- to 20-fold higher Ptnþ accumulation in the total cellular RNA than in genomic DNA.236 Cisplatin was shown to interfere with RNA-dependent processes like splicing and translation.237,238 Triggered by these studies, RNA has lately moved increasingly into focus. Cisplatin seems to show a generally higher reactivity and salt-dependence towards RNA compared to DNA.239 The detailed investigation of cisplatin binding to a 41 nucleotide long purine rich RNA derived from spliceosomal U2 and U6 strands showed

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that a large array of various adducts is possible due to the more complex local and three-dimensional structures as well as intrinsic dynamics of RNA.240,241 In one case, GU wobble pairs were shown to be a preferred binding site.242,243 Research is now directed towards interactions with more specific RNAs, e.g., ribosomal RNA244 or siRNA, the latter showing an increased silencing activity upon platination.245 Ribosomes are among the largest RNA structures in a cell and hence a natural target for cisplatin. Platinum(II) adducts from cisplatin treatment have been observed in ribosomal RNA from E. coli and S. cerevisiae and have been mapped to single nucleotide resolution using targeted primer extension.236,246–248 Further study using a high-throughput sequencing of Pt-RNA adducts showed that a majority of Pt2þ-RNA sites were located in ribosomal RNA of S. cerevisiae.249 The strongest cisplatin-derived stops are located primarily in the highly conserved core of the ribosome, such as G2824 that is near the peptidyl transferase center and numerous eukaryotic-specific antibiotics bind in this region. Platinum(II)-induced stops are also observed in tRNAs and cytoplasmic RNAs. Resolving structures of cisplatin-RNA adducts (or derivatives thereof) is a challenge and only a limited number of structures have been described.87 In one study by Dumas and colleagues, crystals of the HIV-1 RNA dimerization initiation site were soaked with PtCl4.250 Although the complete octahedral coordination sphere of the bound Pt4þ could not be resolved, the Pt4þ unit seems to coordinate to an adenine-N7 (innersphere) as well as outersphere to adenine-N6, a phosphate oxygen as well as the exocyclic amine of the neighboring cytosine-N4. A more recent 2.6 Å resolution X-ray structure of the cisplatin-modified 70S ribosome251 revealed in the unbiased (Fo–Fc) and anomalous difference electron density maps nine cisplatin-derived Pt2þ species in the entire ribosome. Three of the observed moieties are coordinated to adenine-N7 and five of them to guanine-N7 within the 16S and 23S rRNA. One cisplatin moiety is coordinated to the N-terminus of the ribosomal protein L9. Two of the Pt2þ-binding sites are located in the conserved functional centers of the ribosome, i.e., the mRNA-channel and the GTPase center, which is consistent with the above mentioned high-throughput sequencing result. In the mRNA-channel, cisplatin intercalates between the ribosome and the mRNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA translocation (Fig. 21).

2.20.4.5.2

Binding of other inert metal ions

In the cell, RNAs are associated exclusively with kinetically labile metal ions. Consequently, there are hardly any studies with inert metal ions. For example, in the above-mentioned study of the crystal structure of the HIV-1 RNA dimerization initiation site, the authors also applied AuCl3. The Au3þ cation linearly coordinates within a GC Watson-Crick base pair deprotonating the guanineN1H forming a N1-Au3þ-N3 bridge (Fig. 22).250 The MINAS database enables a detailed and always updated search for any metal ions found in nucleic acid structures.87 A search for classical inert metal ions, like Ru3þ, Ir3þ, Osnþ, or Co3þ gives up to several hundred hits for a given metal ion. However, the metal ions are hexacoordinated by NH3, as these ions are normally used for solving the phase problem in crystal structure determination. In principle, such metal-hexammine complexes, e.g., cobalt(III)hexammine, can (and are) used to probe for coordination sites of the magnesium(II)hexaaqua ion (see also Section 2.20.4.7.1).252 However, one has to consider that Mg2þ is usually partly dehydrated and only a small fraction of Mg2þ ions are coordinated exclusively through outersphere interactions.85

2.20.4.6

Metal ion binding in the helix center

Metal ion binding to Watson-Crick base paired regions takes place at the Hoogsteen phase and/or the phosphate-sugar backbone. In the past years, strongly increasing intense efforts have been made to place a diverse variety of metal ions into the helix center of DNA and RNA. Such doped oligonucleotide duplexes are in the focus of nanosciences, i.e., as materials with specific magnetic and/or electronic properties.253 Metal ions can be placed in the helix center in the form of a metallated base pair using either natural or artificial nucleobases. As early as 1952, Katz observed the incorporation of Hg2þ in between two thymine bases.254,255 The suggested deprotonation of

Fig. 21 Cisplatin coordination to the mRNA-channel within the ribosome. The cisplatin unit is mono-coordinated to N7 of adenine. This figure has been prepared with MOLMOL168 based on the PDB file 5J4B.251

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Fig. 22 A gold(III) ion inserted into a GC base pair linking the N1 and N3 positions of the two nucleobases. In addition, also the (C6)NH2 amine is coordinated (and deprotonated) by the Au(III). This Figure has been prepared with MOLMOL168 based on the PDB file 2OIJ.250

the N3H position was later confirmed,256,257 and today many examples of DNAs carrying T-Hg2þ-T base pairs are known (Fig. 23).262–264,278 Very recently, it has also been shown that a DNA polymerase incorporates T-Hg2þ-T base pairs.279 Similarly, also U-Hg2þ-U base pairs can be constructed and indeed, using T7 RNA polymerase, long stretches of Us can be transcribed, which upon addition of Hg2þ, form duplexes with an array of up to 20 metal ions in its helix center.261 Theoretical calculations have shown that the stabilizing effects of such U-Hg2þ-U base pairs are due to metallo-base and metallophilic interactions.280 Several further metallated pyrimidine base pairs have been identified, e.g., C-Agþ-C 258,278 and also with modified bases like 5-fluoro-U. For a comprehensive review on such metallated pyrimidine base pairs see Ref. 278. Aside from the natural nucleobases mentioned above, also modified and artificial nucleotides have been extensively applied to construct metal-carrying oligonucleotides. For example, with modified purines, like 1-deazaadenine, Hoogsteen-type metallated base pairs with Agþ and thymine were constructed,281–283 and with pyridyl carrying purines, new metallated base pairs were formed.273,284,285 Aside from purines, many other artificial nucleotides and base pairs have been constructed, using, e.g., hydroxypyridones,271,286 salen,276,287,288 imidazole,158 bipyridine,274 and triazole.289 Note that all these nucleotides are actually 20 -deoxynucleotides, i.e., metallated DNAs are formed upon oligomerization. For comprehensive reviews see, e.g., Refs 253,290–294. With some of these nucleotides longer oligonucleotides containing stretches of metal ions can be formed, e.g., three Agþ ions between two imidazoles (Fig. 24),158 different metal ions site-specifically bound between different base pairs,287 and stacked Cu2þ ions that are antiferromagnetically coupled.288

2.20.4.7

Binding of metal ion complexes

Several classes of metal ion complexes exist that bind to RNA: Platinum(II) and platinum(IV) based complexes, metal ionhexammine complexes, fluorescent Ru2þ/3þ and Rh2þ/3þ complexes, as well as mono- and dinuclear Zn2þ complexes. The first class has been covered in Section 2.20.4.5.1. and the others will be briefly discussed below.

2.20.4.7.1

Hexammine complexes with Co3þ and other metal ions

2.20.4.7.2

Ruthenium complexes

Cobalt(III)hexammine is a common mimic for hexahydrated Mg2þ ions, as its ammine ligands are relatively tightly coordinated.252,295 This complex is mainly used in NMR experiments as its protons are directly observable and can be used to obtain distance information to the RNA.296–301 Similarly, also NH4þ has been used as a mimic for monovalent ions.302 The cobalt(III)hexammine complex is rapidly tumbling in the binding pocket giving rise to only a single resonance line for all protons. Based on NOEs, structures of such complexes bound in a complete outersphere manner to the major groove, e.g., tandem GU wobbles, as well as tetraloops have been determined.298,299 Aside from Co3þ also hexammine complexes of other metal ions, e.g., Ru3þ, Ir3þ, Rh3þ, and Osnþ, are regularly employed. For example, hundreds of hits are given when searching for osmium in the MINAS database.87 However, these complexes are employed exclusively in X-ray experiments: The heavy atoms are needed to gain the crucial phase information, but the ammine ligands prevent direct binding to the RNA.

Ruthenium and rhodium complexes with large aromatic ligands, e.g., Ru(bpy)2(dppz)2þ (bpy ¼ 2,20 -bipyridine; dppz ¼ dipyrido [3,2-a:20 ,30 -c]phenazine) have been used for many years to study electron transport in DNA.303 Intrinsically non-luminescent in aqueous solution, upon intercalation of the large aromatic ligand into DNA and loss of water interaction luminescence is restored.304 Their change in luminescence upon undergoing redox chemistry can thus be used to measure electron transfer in oligonucleotides, e.g., it has been put forward that repair enzymes recognize lesions in DNA by a change in conductivity.305 However,

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Metal ion interactions with nucleic acids

Fig. 23 A selection of metal modified base pairs as described in the literature. In some cases, the same base pairs are also known with other metal ions as displayed. The structures are based on the following (selected) references: 1,258,259 2 (X ¼ Br, F, CN;260 X ¼ H;260,261 X ¼ CH3) (Refs. 254–257,262–264), 3,265 4 (X ¼ Y ¼ CH;158,266 X ¼ N, Y ¼ CH and X ¼ Y ¼ N267), 5 (X ¼ O, NH2),268–270 6,271,272 7,273 8,274 9,275 10.276,277 This figure is adapted from Ref. Clever, G. H.; Shionoya, M., Met. Ions Life Sci. 2012, 10, 269–294. For a more detailed description of such metal modified base pairs see Refs. 253285288290

despite the long standing research in electron transfer within DNA, RNA has been neglected and only very little information is available.306 Ru complexes with large aromatic ligands like [Ru(bpy)2(dppz-Br)]2þ and [Ru(dmb)2(dppz-Br)]2þ interact with an RNA poly(U)$poly(A)*poly(U) triplex by intercalation mode effectively stabilizing the triplex, as shown by an increased melting temperature. Both complexes bind to all three strands with the ancillary ligands bpy and dmb as the main ligands to interact with the RNA.307,308 The complex [Ru(phen)2dppz-idzo]2þ (phen ¼ 1,10-phenanthroline, dppz-idzo ¼ dppz-imidazolone) was developed to act as a colorimetric molecular “light switch” for the RNA triplex poly(U)$poly(A)*poly(U) RNA.309 Similarly, a red emissive bisheteroleptic Ru2þ compound and its perchlorate analogs of the 4,7-dichloro phenanthroline ligand rapidly stains the nucleolus of HeLa cells by interacting with nucleolar rRNA. The aggregation-induced emission is believed to originate from the CeCl$$$O

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Fig. 24 Metal modified base pairs. (A) Central section of a DNA duplex containing three consecutive imidazole-Agþ-imidazole base pairs as solved by NMR solution spectroscopy.158 (B) The middle imidazole-Agþ-imidazole base pair as seen from the top. The Agþ ion is shown as light green sphere, nitrogens as dark blue spheres, and hydrogen atoms as dark grey spheres. Agþ coordination could be proven by [1H,15N]-HMQC showing the 1JNAg coupling in the 15N dimension of the H4-N3 correlation.158 The structure has been prepared with MOLMOL168 based on the PBD ID 2KE8.158

halogen bonding interaction.310 A further series of p-extended, acridine-based Ru-complexes have been used as selective light-up probes for DNA and RNA quadruplexes in a both comparative and competitive manner.311

2.20.4.7.3

Further complexes

An uncountable number of mono- and dinuclear metal ion complexes, e.g., of Mn2þ, Cu2þ, Zn2þ, or lanthanide(III) ions, have been designed and synthesized over the past decades with the aim to develop a “perfect” mimic of RNA and DNA cleaving phosphodiesterases.312 Aside from such hydrolytically cleaving complexes, also Fe(II)-bleomycin complexes in the presence of O2 have been thoroughly investigated.125 Again, to the best of our knowledge, no detailed structures of these complexes in combination with RNA exist. Dinuclear complexes thereby are superior to either mononuclear or trinuclear species.313–316 A detailed search of metal ions in RNA also leads to some more exotic complexes, e.g., K6(P2W18O62)$14H2O, which has been used to obtain better diffracting crystals of the small ribosomal subunit of Thermus thermophilus upon soaking.317

2.20.5

Metal ions and their role in folding and dynamics of RNA

RNA folding to the active structure is considered hierarchical; after formation of the secondary structure, the various helical parts and domains come together forming local contacts mediated by hydrogen bonding and metal ions to end up in the final tertiary structure.318–323 The first step of base pair formation is usually fast, yielding a mixture of helices, junctions, and loops. In the second step, slower and spatially wider than the first, these preformed secondary structures come together to form long-range interactions. In some cases, here also some smaller rearrangements in base pairing can take place.104,324 Both processes depend on the presence of metal ions. Secondary structures are mostly stable under a large variety of conditions, requiring a minimum of about 1 mM Mþ, but usually being applied in the 50–100 mM range. Tertiary structures require much higher ionic strengths and also almost exclusively divalent metal ions.325 The reason for the higher charge density required for tertiary structure formation is the accumulation of negative charges in close neighborhood. The small hairpin ribozyme performs catalysis in the presence of either 0.5 mM Co(NH3)63þ, 10 mM Mg2þ, or 1 M Naþ, exemplifying the different interactions and effects of these ions.326 Small ribozymes are an exception in the sense that some of them also function under (unphysiologically) high concentrations of monovalent salts (replacing Mg2þ)dan effect long known, e.g., for the hydrolysis of ATP.2,327 Large ribozymes are strictly dependent on divalent metal ions, with Mg2þ being exclusively employed for folding as the most abundant divalent metal ion in the cell (Table 1). In vivo, the free concentration of Mg2þ is 2–3 mM in bacteria, and 0.5–1 mM in eukaryotes. The high Mg2þ concentrations usually applied

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Metal ion interactions with nucleic acids

in vitro experiments (10 mM) are thus far from physiological conditions. Remarkably, in the cell, also metabolite-bound Mg2þ interacts with RNA to support folding and function, again explaining the need for higher free Mg2þ concentrations in vitro.328 As part of the folding process, metal ions induce kinetic heterogeneity in chemically identical RNAs: Ion coordination in the binding pockets is transient, resulting in RNAs that are loaded to varying degrees with Mg2þ and hence contributing to folding heterogeneity.329–331 The energetic folding landscape of RNAs is rugged and consists of many local minima. Some large RNAs, like the Tetrahymena group I intron ribozyme, collapse rapidly into local minima and then need to rearrange slowly from these off-pathway intermediates to the active state.332–337 The rate of unfolding from these kinetic traps determines the overall folding rate.338 In the case of group II intron ribozymes and RNase P RNA, no kinetic trap exists and the first step of folding from the secondary to the tertiary structure is the slowest.339,340 For the group II intron Sc.ai5g, two fast steps follow the first slow one, the intermediate being the thermodynamically stable, but the final one being the active state, which is only transiently reached.341 This linear folding pathway is independent of the exchange of large parts of Mg2þ with the naturally associated Mss116 protein.342,343 Nevertheless, the folding pathway is strongly dependent on the nature of the divalent ions, as, e.g., Ca2þ leads to the formation of two distinct subpopulations that do not interchange, one being probably the catalytically inactive one.40,43 Molecular crowding agents additionally decrease the need of Mg2þ in RNA folding. For example, with the large group II intron ribozymes, the addition of PEG in various concentration and molecular weight, showed an optimum, under which folding and activity was reached under almost physiological Mg2þ concentrations.344,345 Obviously, molecular crowders, either artificial in vitro or as the natural cellular environment, support the structural integrity of complex RNA folds at low concentrations of 1 mM of free Mg2þ, or even lower. For more details we refer the reader to a recent review on this topic.346

2.20.6

Metal-ion sensing by riboswitches

Riboswitches are RNA sensors that occur mostly in the 50 -untranslated region of mRNAs. These sensor sequences switch their threedimensional structure upon specific ligand binding, leading to the regulation of gene expression of those ligands.347,348 Riboswitches consist of two main regions: the aptamer region recognizing and binding the ligand, and the expression platform responsible for the structural change to regulate gene expression. Employing such a mechanism, riboswitches regulate enzyme cofactors, numerous nucleotide-based signaling molecules, nucleobases, amino acids, as well as metal ions.349,350 Up to today 56 different riboswitch classes have been identified, nine of which respond to elemental (metal) ions and three to metal ion co-factors.351 The latter are the adenosylcobalamin,352,353 the molybdenum cofactor,354,355 and the tungsten cofactor riboswitch.356 Confirmed elemental ion responsive riboswitches include Mg2þ.357,358 Mn2þ.359,360 F.361 Liþ.351 Naþ.362 and the NiCo.363 The last riboswitch has been shown to bind Ni2þ and Co2þ, but recently, it has also been proposed to recognize Fe2þ.364 For a more detailed discussion of the role, folding, recognition, and mechanism of these metal ion (complex) responsive riboswitches we refer the reader to a recent comprehensive review.350

2.20.7

Metal ions and their role in RNA catalysis

2.20.7.1

General effects of metal ions on the observed catalytic rate

Due to their high negative electric charge, RNAs are always closely associated with uncountable metal ions for charge compensation.9 It is therefore very challenging to elucidate the effect and role of single metal ions in catalysis.16 One prominent approach is to substitute part (or all) of the naturally applied Mg2þ with other ions. Based on their different chemical and physicochemical properties, conclusions regarding their coordination and involvement in the catalytic mechanism are drawn.15,16 In many cases, Mg2þ ions are the natural cofactor in the sense that also the maximum catalytic rate is observed in their presence.85 Computational studies on a group I intron from the Azoarcus bacterium highlight how site-specific Mg2þ coordination can promote structural stability as well as catalytic activity.365 Already at low a concentration (0.1 mM), two Mg2þ promote the formation of key tertiary motifs. A coarse-grained model demonstrates that Ca2þ can also stimulate the formation of main helical domains, but their correct arrangement requires Mg2þ due to its higher charge density. For some cases it is known that other metal ions actively inhibit catalysis, meaning that their affinity towards the RNA is considerably higher than that of Mg2þ, as has been observed for the yeast mitochondrial group II intron Sc.ai5g.40 In this case, the folding pathway is disturbed by the cooperative binding of Ca2þ ions, leading to a misfolded structure.43 In other cases, e.g., a group I intron ribozyme, the originally Mg2þ-dependent ribozyme could switch to a Ca2þ preference by in vitro selection.366–368 Similarly, the HDV ribozyme prefers Mg2þ in its genomic and Ca2þ in its antigenomic form, a specificity which can be switched by the mutation of a single nucleotide.369 Mostly for such large ribozymes no clear dependence on the nature of the metal ion can be deduced. This is not surprising, considering that a large RNA with its manifold local structures offers most diverse binding pockets for various kinds of metal ions. It has been shown that for some small catalytic RNAs like the hammerhead ribozyme, a distinct dependence on metal ions can be found. Along the Irving-Williams series, the catalytic rate goes in parallel to the affinity of the individual metal ions to phosphate groups.79 As Cu2þ can usually not be applied due to degradation of the RNA, the maximum rate (and affinity) is observed for Mn2þ. This suggests that the coordination ability to a phosphate group is essential to perform catalysis. Again, this does not seem to be

Metal ion interactions with nucleic acids

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a general rule as another small ribozyme, the glms ribozyme/riboswitch shows no such dependence on phosphate affinity.370 Folding of glms ribozyme/riboswitch is independent of cation identity. Molar amounts of monovalent ions can fold the glms RNA into its native state but fail to promote efficient ligand binding and catalysis. Divalent metal ions are required to fix the ligand phosphate moiety into its binding pocket. For this task Mn2þ, Mg2þ, and Ca2þ can be used in a respective manner.371 Computational studies with the glms ribozyme/riboswitch have investigated the potential effects of Mg2þ in the active site. While Mg2þ positioned in the active site may inhibit catalysis, the presence of Mg2þ near the Hoogsteen face of G40 possibly promotes catalysis by lowering its pKa.372

2.20.7.2

Two-metal ion mechanism

Aside from the ribosome, natural ribozymes catalyze site-specific cleavage of the backbone or ligation of the same. RNA is intrinsically prone to hydrolysis of the phosphate-sugar backbone because of the 20 -OH group, which can act as a nucleophile for phosphate ester hydrolysis. However, alcohol functionalities are poor nucleophiles and need to be activated to obtain significant rates. In the case of the so-called small ribozymes, mostly an acid-base mechanism is put forward, which is related to the well-known mechanism of RNAse A.9,373 The caveat with RNAs is that in contrast to proteins with, e.g., histidine as pH active group at ambient pH, RNA does not possess any equivalent functionality (see also below Section 2.20.2). Although pH dependences are proven by experiment, it is unclear how a shift in pKa of the various nucleobases is achieved within a complex structure. Most likely, metal ions are strongly involved, as they are well known to perturb pKa values upon coordination.47,224,228–230,374–377 At least in the hammerhead and the HDV ribozyme, non-obligatory roles of metal ions have been proposed, although with controverse discussion, e.g., the role of Mg2þ as a general base in the HDV ribozyme.374–383 More subtle effects have also been put forward, i.e., the effect of metal ions over long distances on transition state stabilization.9 Molecular dynamics simulations propose ribozyme activation by two Mg2þ that mediate catalysis in separate steps of the reaction pathway involving a conformational switching. The first ion supports a base pair flip required to achieve the catalytic fold, while the second ion facilitates nucleophile activation.384 Large ribozymes are believed to follow a so-called two-metal ion mechanism. The two metal ions, or actually in some cases three, are used to activate the nucleophile, and to stabilize the transition state with its additional negative charge as well as the leaving group. The involvement of two metal ions has been put forward already very early based on ATP hydrolysis as well as enzyme kinetics.1,2,385,386 In the case of RNA, the best-examined example is the group I intron ribozyme,93,387,388 but group II introns perform catalysis through this general mechanism as well (Fig. 25).94,391–393 The metal ions fulfill several functions: In group I introns, metal ion MA coordinates to the 30 -oxygen of the leaving group stabilizing it. MB activates the nucleophilic 30 -oxygen of the exogenous GMP, and MC the nucleophilic 20 -oxygen of the exogenous GMP. At the same time, MA and MC also coordinate the pro-SP oxygen of the scissile phosphodiester.387 In such a concerted effort, the nucleophile(s) are activated as well as the transition state and the leaving group is stabilized. In general, such a mechanism resembles the one used by enzymes.

Fig. 25 “Two” metal ion mechanism as proposed for group I intron splicing.93,389 Three metal ions have been proposed based on biochemical experiments,93,389 and MA and MC have been confirmed by crystal structures.390 The latter are bound to the pro-SP oxygens of the phosphate groups of C208, A304, A306, and C262.390 MA and MC both stabilize the transition state and MA is additionally responsible to stabilize the leaving group.

656 2.20.7.3

Metal ion interactions with nucleic acids Electrostatic influence of metal ions

As a polyanion at neutral pH, the negative charge of RNAs needs to be neutralized to the greatest part that the oligonucleotide can compact and fold to the active state. In a folded RNA, the negative electrostatic surface potential is around 15 kT/e in the major groove, but can reach also 100 kT/e at specific metal ion binding sites.394 The required positive countercharge is mainly provided by metal ions, which are either rather tightly and specifically bound,85 or are transiently bound and thus poorly localized.104,325,395,396 The latter ones are estimated to make up for about 90% of charge compensation and form something like a dynamic ion atmosphere around the RNA that is mostly responsible for global stabilization of the RNA fold.85,104,325 Both, monovalent (typically Naþ or Kþ) and divalent (Mg2þ) metal ions stabilize secondary and tertiary structures, whereby the latter are much more efficient: For example, short hairpins and duplexes are equally stabilized in either 10 mM Mg2þ or 1 M Naþ.396–398 This difference in concentration is due to the differing intrinsic charge density and affinities to the nucleic acid, and is well known: For example, the accelerating effect of Mg2þ and Zn2þ in the hydrolysis of ATP can be equalized by a 500 times higher concentration of Naþ.2,79,327 Along the same line, small ribozymes are active in the presence of unphysiologically high concentrations of monovalent metal ions,326 but they are much more active if small amounts of Mg2þ are present.81,369 In vivo, molecular crowding of the cellular environment, as well as metabolites can also partially replace the role of Mg2þ and other metal ions.344,345 Proteins can operate as macromolecular counterions to locally charge screen with the same efficiency as molar salt concentrations, contributing to the functionality of ribozymes in low metal ion concentrations.399,400 Classically, diffuse ion binding to RNA has been described by a Hill-type equation based on folding experiments, yielding a small number of specific binding sites and a cooperativity factor.401,402 However, due to the lack of complete charge compensation such a simple model cannot possibly describe the full system, and hence, the non-linear Poisson-Boltzmann theory has been applied successfully to describe such diffuse binding events.402–406 However, the non-linear Poisson-Boltzmann theory neglects ion correlation and fluctuation effects, which often leads to an underestimation of the efficiency of multivalent ions. Even though models to address the shortcomings of the Poisson-Boltzmann theory have been developed,407–410 ion dehydration calculations and distribution sampling remain challenging especially for larger RNA molecules.411 Another commonly used application to predict metal ion effects in RNA folding is the Counterion condensation (CIC) theory.412,413 The CIC theory assumes a uniform distribution of the condensed ions, and also ignores ion correlation similarly to the Poisson-Boltzmann theory. Still, recent efforts in molecular dynamic simulations improved modeling accuracy for the ion effects in RNA folding.414–416 The electrostatic influence of charges on catalysis is well documented for enzymes. For example, in subtilisin, two charged amino acids on the surface roughly 15 Å away from the active site are well known to fine-tune the active histidine residue.417 Similar to certain side chains of proteins, charged nucleobases in ribozymes can take over the role to promote catalysis, either directly, or from the distance. The nucleobases G and U become anionic when deprotonated, whereas nucleobases A and C are catiotic upon protonation. Table 2 lists their pKa values, illustrating that they are all far from the neutral/physiological pH. However, numerous cases have been reported with shifted pKa values of A and C towards neutrality, the most prominent probably being the HDV ribozyme.418–420 For most cases, the origin of such shifted acid-base properties are unknown, but for the catalytic group II intron domain V, stabilization of a protonated adenine moiety through hydrogen bonding has been reported.421 In any case, A and C have the potential to exhibit histidine- and lysine/arginine-like behavior to promote catalysis.422,423

2.20.8

Concluding remarks and future directions

This comprehensive review on metal ion interactions with nucleic acids is an update of Chapter 3.21 of the 2nd Edition of Comprehensive Inorganic Chemistry from 2013.193 The latter chapter from 2013 also included extensive Sections on the basic acid-base and metal ion binding properties of the nucleic acid building blocks, i.e., nucleobase, nucleosides, nucleotides, and dinucleotides, including numerous artificial and natural modifications thereof, but which are not covered here anymore. The interaction of metal ions with the most common nucleobases, nucleosides, and mononucleotides is rather well investigated and well understood in many respects. These basic properties of the building blocks determine also the function and mechanisms of the larger complex nucleic acid structures. For any information on the building blocks, we thus refer the reader to the previous Chapter 3.21.193 In the current update, we concentrate on metal ion binding to larger nucleic acids, i.e., mostly RNA, as RNA adopts a wealth of complex three-dimensional structures and different functions. Consequently, metal ion binding to RNA is much more diverse and complex than binding to mostly double-stranded DNA. and its constituents contain a wealth of information. However, as it is impossible to include all the available data into such a review, many aspects are only mentioned scratching the surface of today’s knowledge, and we thus strongly encourage the interested reader to refer to the cited literature and references therein. Despite the eminent progress over the past decade since publishing the 2nd edition of Comprehensive Inorganic Chemistry, we are still far from understanding how nucleic acids fold and function on the atomic level by employing the crucial assistance of metal ions: (i) Many rare and uncommon nucleotides are not at all or only little investigated. RNA employs probably more than 100 modified nucleotides and modified nucleotides play important roles in medicine. It will be crucial to understand their basic properties as well as their power to trigger and tweak specific functions and roles of natural RNAs. Research in this field will

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broaden our understanding of nucleic acids and will certainly expand the today known functions of RNA to unknown and unexpected borders. (ii) The knowledge on the coordination of metal ions to dinucleotides or even short oligonucleotides is still scarce or nonexistent. The binding equilibria are generally more complicated than with mononucleotides and hence research in this field is very challenging. Nevertheless, such investigations are a crucial basis to understand the function of larger and more complex nucleic acids structures. (iii) Metal ion interactions with larger nucleic acids, like ribozymes, are highly complex. While a wealth of details is known on specific systems, we are only at the beginning of understanding them in a more general manner. The effects of metal ions on the global structure (and also catalysis) of RNA is already rather well investigated for some ribozymes and other functional RNAs. Nevertheless, the effects on the local structure and the interactions on the atomic level are often poorly understood. At the same time, it turns out that, e.g., ribozymes of the same class, but from different organisms or gene loci, differ considerably in folding, metal ion binding properties, and mechanism on the atomic level. Hence, we are far from understanding the general picture or even predicting how an RNA sequence folds and exhibits a certain function. Intrinsic major problems are the large excess of metal ions around the polyanion nucleic acid, the rather low binding affinities, the spectroscopic silence of most metal ions involved, the multitude of coordinating atoms, the small diversity of building blocks, and the large size of the ligand, i.e., the nucleic acid, itself. For example, (a) not for a single ribozyme, it is really understood, why certain metal ions inhibit, and others accelerate catalysis, and (b) acid-base catalysis in small ribozymes is still to large parts a mystery. Proposed shifts in pKa values of the involved nucleobases can so far not be explained convincingly in a single case, despite the origin of a pKa shift has now been described for at least one case within the catalytic center of a ribozyme.421 Structural biology is crucial to understand the function of nucleic acids. In the past years, the number of high-resolution structures of large RNAs has significantly increased. Nevertheless, the exact coordination sphere of different metal ions is still often not completely resolved due to dynamics of the ligand exchange and lack of data from the electronic density map. Considerable progress is still needed, and only in combination with solution studies performed by various techniques, can we move more towards the atomic level. Another factor complicating our understanding of complex RNA structures (and biomacromolecules in general) having surfaced in the past years is that single molecule techniques have revealed molecules to be individuals: They do not behave uniformly, but rather like individuals, sometimes splitting up into distinct subpopulations with distinctly different behavior. At the same time, single molecule spectroscopy in combination with other experimental and computational methods has proven extremely powerful to understand how metal ions govern the structure and equilibria of nucleic acids by their coordination properties.330 The averaged signal observed by most conventional techniques does not necessarily reflect the true pathway or mechanism of a given process; the outcome of any process is governed by kinetic and thermodynamic factors, which are not necessarily the most advantageous for the major species. Single-molecule spectroscopy helps to reveal the importance of minor species and the interplay of species within a large ensemble of molecules. In addition, it has become increasingly clear in the past decade that the behavior of nucleic acids under classical in vitro conditions do not reflect the behavior of the same under in cellulo conditions. This concerns to a large part the interactions of nucleic acids to metal ions, which are much enhanced under molecular crowding and conditions of lower dielectrostatics (compared to water), and hence also folding and mechanism. Consequently, the large field of Coordination Chemistry in RNAs will continue to be a crucial factor in understanding the role and function of nucleic acids in living systems.

Acknowledgments R.K.O.S. is very grateful for financial support over the past years for his research on the interaction of metal ions and metal ion complexes with nucleic acids, namely, from the Swiss National Science Foundation, the European Research Council (ERC Starting Grant MIRNA-259092) as well as within the COST Action CM1105 from the Swiss State Secretariat for Education and Research. We also acknowledge gratefully the continuous support from the University of Zurich.

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2.21

Metal-mediated base pairs in nucleic acid duplexes

Marian Hebenbrock and Jens Mu¨ller, Westfälische Wilhelms-Universität Münster, Institut für Anorganische und Analytische Chemie, Münster, Germany © 2023 Elsevier Ltd. All rights reserved.

2.21.1 2.21.1.1 2.21.1.2 2.21.1.3 2.21.2 2.21.2.1 2.21.2.1.1 2.21.2.1.2 2.21.2.2 2.21.2.2.1 2.21.2.2.2 2.21.2.2.3 2.21.2.3 2.21.2.4 2.21.2.4.1 2.21.2.4.2 2.21.3 References

Introduction Nucleic acids and metal ions in general What are metal-mediated base pairs? Early metal-mediated base pairs Overview of ligands reported in metal-mediated base pairing Pyrimidine and its derivatives (Functionalized) thymine or uracil (Functionalized) cytosine Purine and its derivatives (Functionalized) adenine (Functionalized) guanine Other purine derivatives Artificial nucleobases Structures of oligonucleotides bearing metal-mediated base pairs Metal-mediated base pairs involving canonical nucleobases Nucleic acids involving artificial nucleosides Summary and outlook

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Abstract Metal-mediated base pairs can be considered conjugates of nucleic acids with metal complexes. They are formed by a formal substitution of hydrogen bonds between complementary nucleobases by coordinate bonds. As a result, metal ions are introduced into nucleic acid helices along the helix axis. Metal-mediated base pairs can hence be used for the site-specific decoration of nucleic acids with transition metal ions. They have found applications in DNA nanotechnology, in sensors, and in responsive nucleic acid systems, to name just a few. Typical ligands in metal-mediated base pairs can be canonical nucleobases (particularly thymine and cytosine), but in addition numerous examples exist of the use of artificial nucleobases. Moreover, metal-mediated base pairs are not limited to the naturally occurring nucleic acids DNA and RNA, but have also been established with synthetic nucleic acid analogs. This article summarizes the efforts made in the field of metal-mediated base pairing since the discovery of the first metal-mediated base pair in the 1960s. It lists all ligands reported in the context of metal-mediated base pairing and correlates them with their preferably coordinated metal ions. A particular focus is given to the spectroscopic and spectrometric characterization of the metal-modified nucleic acids. For the first time, a comprehensive overview is given of all metal-mediated base pairs, the experimental conditions under which they were established, and the experimental techniques used to prove their existence. In addition, all experimental nucleic acid duplex structures involving metal-mediated base pairs are presented and discussed. The article covers metal-mediated base pairs reported until early 2021. It is intended to aid scientists interested in this fascinating field at the border of bioinorganic chemistry and supramolecular coordination chemistry in developing it even further.

2.21.1

Introduction

2.21.1.1

Nucleic acids and metal ions in general

Natural nucleic acids consist of three parts: a sugar moiety, different nucleobases and linking phosphodiester bridges. Each part can interact with metal ions in a unique way due to their intrinsic chemical properties. Those interactions occur in nature to maintain the structural integrity of nucleic acids or provide functionalities such as catalysis. The phosphate groups of the nucleic acid backbone are deprotonated at physiological pH and negatively charged. Therefore, nucleic acids can be considered polyanions and the interaction with cations is electrostatically favored. Without any charge compensation, the structural integrity of nucleic acids might not be maintained. Due to the importance of the structure for the function of complex nucleic acids, many reviews discuss this topic in a detailed manner, especially in the context of RNA folding.1–8 Considering nucleic acids as polyelectrolytes in solution, the polyelectrolyte theory provides the fundamental aspect that counterions will always be in close proximity of the phosphates to reduce the charge density of the nucleic acid.9–11 In a physiological context, alkali and alkaline earth metal ions such as Na(I), K(I), Mg(II) or Ca(II) are considered, but also other mono- and divalent metal ions are

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able to stabilize nucleic acid structures.3,8,12,13 Beside their importance for the tertiary structure, metal ions also play a role in catalytic processes. Great importance in this context show hydrolytic processes of metal ions bound to the phosphate groups.14–16 In addition to magnesium ions17,18 also other metal ions catalyze reactions when bound to the backbone of the DNA.19–21 Metal ions bound to the sugar moiety are rare. Already on the nucleotide level these interactions are considered to be weak and only possible if geometrical constraints allow binding in a chelating form.22 Still there are nucleic acids know in which Na(I) or some transition metal ions are chelated by the oxygen atoms of the sugar moiety.2,23 Selected examples of metal ions bound to nucleic acids will now be briefly presented to elucidate special roles of metal ions for nucleic acids. B-DNA as the biologically most relevant DNA conformation is best represented by the archetype of a DNA duplex, the Dickerson-Drew dodecamer.24,25 The grooves of this duplex are not solitarily occupied by water molecules (the so-called spine of hydration),26 but also by sodium ions.27 This spine of hydration is two layers deep, where the sodium ions are localized on the inner layer and exhibit some sequence specificity.28 Based on MRD (magnetic relaxation dispersion) studies, the minor groove of the central adenine-thymine region of the Dickerson-Drew dodecamer is heavily occupied by sodium ions, while only a few are found in the vicinity of guanine and none around cytosine.27,29 While monovalent cations are rarely located in the major groove of DNA, divalent cations, for example calcium(II) and magnesium(II), can be found in that groove.30–32 The occurring inner-shell interaction of divalent metal ions selectively with GC-rich parts of DNA can also cause distortions of the curvature of the helix.30,33 Higher-order structures with essential metal ions include G-quadruplexes. Tetrads of guanine within four-stranded helixes are highly stabilized in the presence of monovalent cations. The guanine nucleobases interact with each other via the Watson-Crick and the Hoogsteen face. The affinity for the cations decreases in the order K(I) > Rb(I) > Na(I) [ Li(I), Cs(I), as determined experimentally34–37 and confirmed by dispersion-corrected DFT calculations.38,39 While sodium ions are located within the tetrad planes,40 potassium ions are located between the layers of the tetrads.41 The monovalent ions are coordinated by the O6 atom of the guanine bases and form a square-planar geometry in case of sodium ions and a distorted square antiprism in case of potassium ions.8 When the non-canonical nucleobase isoguanine is used, nucleobase quintets are formed.42,43 While the hydrogenbonding pattern between the nucleobases changes, the O6 atom remains the donor-atom for the metal ion coordination. The size of the ion channel increases in these pentaplex structures and hence the specificity of metal coordination changes from potassium(I) ions to cesium(I) ions.42

2.21.1.2

What are metal-mediated base pairs?

Metal-mediated base pairs are interstrand interactions of nucleic acids, in which coordinate bonds of the nucleobases to a central metal ion formally substitute the naturally linking hydrogen bonds between the strands. While in the beginning, an increased amount of research was performed utilizing canonical nucleobases to form metal-mediated base pairs, soon artificial nucleobases were synthesized for this purpose. The increasing use of solid-phase synthesis for oligonucleotides44 made defined oligonucleotides with artificial nucleosides easily available. In the beginning, it was proposed to name this kind of DNA “M-DNA” due to its distinct own behavior, which was not merely “a minor variant of duplex DNA.”45 Later, terms like “metal-assisted base pairing”46 or “metal-mediated interstrand binding”47 were used to describe the binding mode of “ligandosides” with metal ions. Nowadays these complexes are typically referred to as “metalmediated base pairs.” For artificial nucleosides, rules were quickly established that would guarantee that the incorporated nucleobase could serve as a ligand for metal complexes within nucleic acids47: (a) the nucleobase has to be compatible with standard DNA synthesis, (b) the affinity of metal ions to the artificial nucleobase has to be higher than to the canonical nucleobases, (c) the size of the formed complex should not exceed the dimensions of natural base pairs.47 In the beginning, the coordination behavior towards metal ions was investigated on the basis of the nucleobases.48 Initially, metal ions with a planar coordination geometry were favored, in particular linear, square-planar or trigonal-planar geometries, and also octahedral and tetrahedral coordination spheres were tested. The first example of an artificial nucleoside coordinating metal ions was reported in 1999, where phenylenediamine served as an artificial nucleobase for the coordination of palladium(II) ions.49 However, this nucleoside was never incorporated into DNA. The first structurally characterized metal-mediated base pair with artificial nucleobases demonstrated the compatibility with the helical structure of these complexes.50 Metal-mediated base pairs are not limited to natural nucleic acid such as DNA or RNA. Also artificial backbones are compatible with metal-mediated base pairs and provide intrinsic benefits over other backbones.51 Nevertheless, this article only focuses on summarizing metal-mediated base pairs in nucleic acids bearing a phosphate-based backbone, i.e. DNA, RNA, GNA (glycol nucleic acid) and LNA (locked nucleic acid). It does not cover the field of metal-mediated base pairs in synthetic nucleic acids with a neutral backbone, such as PNA (peptide nucleic acid). In the case of metal-modified PNA, the interested reader is referred directly to some original articles focusing of the site-specific decoration of PNA with metal complexes.52–58

2.21.1.3

Early metal-mediated base pairs

One year before a structural model of DNA was elaborated, the first, more unintended experiments on metal-mediated base pairs were published by S. Katz in 1952.59 The titration of ct-DNA (calf thymus DNA) with HgCl2 led to a decrease in viscosity and to increased turbidity and was explained by the formation of “an incomplete mercuric salt of nucleic acid”.59,60 The DNA duplex model by Watson and Crick based on the X-ray data of Franklin61 supported the understanding on the binding mode of mercury(II)

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ions with nucleic acids. Thomas stated that “certain hydrogen bonds between the polynucleotide chains will be destroyed” upon binding of HgCl2.62 The interaction of mercury(II) ions was later also investigated with RNA.63 For the mercury-mediated base pairs, a stoichiometry of two bases per mercury(II) ion was suggested.64–67 The actual structure remained undiscovered, while Katz proposed the complexation by two deprotonated thymine bases upon an axial chain shift of the Watson-Crick AT-base pair (Fig. 1A). The fact that mercury(II) selectively binds to AT-rich nucleic acids and the release of two protons upon binding60,68 also led to the proposal of other structures, with mercury being coordinated by a deprotonated thymine and adenine (Fig. 1B),68 or based on the X-ray structure of a uracil mercury(II) complex, via the oxygen atom of thymine.70 The structure proposed originally by Katz67 was later confirmed by the X-ray structure of a complex of 1-methylthymine with mercury(II) ions, where mercury(II) is coordinated by two deprotonated thymine bases (Fig. 1C).69 Despite the transoid arrangement of the thymidine bases, which would be an unsuitable orientation within a DNA duplex, this became a first structural model for the coordination of mercury(II) by nucleic acids. This originally proposed binding mode was later confirmed by NMR spectroscopy also in solution due to the loss of the imine proton signal of thymidine upon binding of mercury(II).71 A more detailed structural elucidation came only decades later, when the interest in metal-mediated base pairs was awakened again. A study on the melting behavior of short DNA double helices with T:T mispairs showed that these duplexes are selectively stabilized by mercury(II) ions, leading to an increase of the melting temperature.72 The structural features of the double helix were verified by circular dichroism and mass spectrometry. The details of the mercury(II) complex within the T:T mispair were resolved by single-crystal X-ray diffraction analysis73 and NMR spectroscopy74 of suitable oligonucleotides. The B-form of the double helix was stabilized by complexation of the metal ions and a similarity of the metal-mediated base pair with normal Watson-Crick base pairs in regard to the placement within the helix and the overall size demand was detected (vide infra). The early interest in the metal-DNA interaction also led to the investigation of the complexation of silver(I) ions by nucleic acids.75 Quickly it turned out that even though comparable nucleic acids were used, the properties of complexes with mercury(II) or silver(I) differ from each other. While the overall structure of the DNA upon the addition of silver(I) ions remained unaltered, no protons were released upon to metal binding, contrasting the behavior in the presence of mercury(II).75 Further, silver(I) ions show a high affinity to GC-rich nucleic acids, while mercury(II) tends to bind to AT-rich nucleic acids.76,77 Binding of silver(I) to RNA, unlike zinc(II), does not induce degradation of the nucleic acid, suggesting that the metal ion does not bind to the phosphate backbone.77,78 The structural details of the complex in nucleic acids remained nebulous for decades. A stoichiometry of one silver(I) ion per base pair was determined,75,77,79 but three different binding modes were proposed. The first of these related to binding without deprotonation of the base pair, the second included deprotonation of the base pair at higher pH values, and the third binding mode involved the binding of a silver(I) ion to a single nucleotide.77 The first model complexes of a C–Ag(I)–C base pair was obtained in analogy to the mercury(II)-mediated base pair by the reaction of 1-methylcytosine or a combination of 1-methylcytosine and 9-methylguanine with AgNO3.80 The structure obtained for the cytosine-silver(I) adduct comprised a complex of two silver(I) ions with two cytosine ligands binding in a transoid fashion (Fig. 2C). Detailed investigation of the interaction of C:C with silver(I) ions started with the discovery of the increased stability of DNA duplexes upon metal binding.82 The higher melting temperature after incorporation of one silver(I) ion per C:C mispair was in line with the initially proposed 1:1 ratio per base pair of GC-rich nucleic acids (Fig. 2B). The higher-order structure of the duplex remained unaltered upon metal ion binding and the integrity of the duplex was shown by mass spectrometry.82,83 The first insight of the structure of the metal complex in nucleic acids was obtained by single-crystal X-ray diffraction analysis of RNA oligonucleotides in the presence of silver(I) ions (Fig. 2A).81 The ions were coordinated by the N3 atom of each cytosine base in a C:C mispair. The great similarity to normal Watson-Crick base pairs means that the overall RNA structure is not particularly influenced by the metal ion coordination. Shortly after the elucidation of the cytosine-silver complex in RNA, also the structure in DNA was unraveled by NMR spectroscopy (vide infra).84 Beside the most prominent examples, silver(I) and mercury(II), of metal ions binding to nucleic acids, the effect of the addition of several other metal ions was investigated.85,86 These became a tool to modify the structural properties of nucleic acids, due to inor decreasing the overall stability or inducing re- and unwinding of the helical structure or even the depolymerization.

Fig. 1 (A) T–Hg(II)–T base pair proposed by Katz (R, R’: DNA backbone)67; (B) suggested structure of a mercury(II)-mediated base pair in AT-rich nucleic acids (R, R’: DNA backbone)68; (C) Lewis structure based on a single-crystal X-ray diffraction analysis of the complex of 1-methythymine and mercury(II).69

Metal-mediated base pairs in nucleic acid duplexes

667

Fig. 2 (A) Structure of the C–Ag(I)–C base pair in RNA (R, R’: RNA backbone)81; (B) proposed type-II binding mode of silver(I) ions in GC-rich nucleic acids (R, R’: DNA backbone)77; (C) Lewis structure based on a single-crystal X-ray diffraction analysis of the complex of 1-methylcytosine and silver(I).80

2.21.2

Overview of ligands reported in metal-mediated base pairing

2.21.2.1

Pyrimidine and its derivatives

2.21.2.1.1

(Functionalized) thymine or uracil

Since the T–Hg(II)–T base pair is the first studied metal-mediated base pair, it is not surprising that it is the best investigated and most widely applied one. In addition, the properties of the base pair provide an excellent basis for specialized applications. The effect of the mercury(II)-mediated base pair on the overall stability of DNA duplexes with a T:T mispair exceeds even that of the canonical A:T base pair, and the T:T mispair specifically binds mercury(II) ions (Table 1).72 The increasing interest in metalmediated base pairs led to the development of improved methods to prove binding of the metal ions. Detection of mercurymediated base pairs by ESI mass spectrometry quickly became a generalized tool for the investigation of metal-mediated base pairs.87 The binding of the metal ion can be directly observed by 199Hg NMR spectroscopy94 and by the presence of a 2JNN coupling across the metal,88 verifying the binding to the N3 atom of the thymine bases. Additional information of the nature of this bonding by Raman spectroscopy revealed an ionic character.95 Further investigations by DFT calculations targeted the photophysical and electronic properties of mercury-mediated base pairs (either individually or as dimers), predicting an interaction between the metal ions of neighboring base pairs.96 Visualization of the ion in the T–Hg(II)–T pair was achieved by AFM (atomic force microscopy) of specifically designed DNA frames.97 The mechanism of the binding process was derived based on the thermodynamic parameters of the binding process as measured by ITC (isothermal titration calorimetry) and was supported by calculations.89,98 A positive reaction entropy is caused by the dehydration of the mercury ion, and the proton of the N3 atom of thymine is released. This deprotonation results in the first Hg–N bond formation, while the second bond is formed through a water-assisted step.98 Cooperative behavior was observed for the incorporation of Hg(II) into two adjacent T:T mispairs. The binding of the first mercury ion resulted in a positive cooperativity where the affinity for the second mercury ion is larger than that for the first one.99 Metallophilic interactions between the two mercury(II) ions were studied by DFT calculations, suggesting a stabilizing interaction.100 The structural properties of two consecutive T–Hg(II)–T base pair became available with an NMR solution structure as well as the crystal structure (vide infra).73,74 The metal-mediated base pairs fit into the B-DNA topology by mimicking the features of a canonical Watson-Crick base pair. The formation of metal-mediated base pairs is not limited to DNA duplexes and was expanded to more complex structures such as G-quadruplexes or even the loops of DNA hairpin structures.101,102 Further, the formation of mercury(II)-mediated base pairs was demonstrated in parallel-stranded duplexes using covalently linked DNA strands.103 The capability of thymine to bind other metal ions was investigated experimentally for silver(I) ions in the T–Ag(I)–C base pair.104 DFT-based studies on 1methylthymine also suggest possible T–Ag(I)–T pairs105 or U–Ag(I)–U pairs based on 1-methyluracil106 as well as copper(II)mediated base pairs, based on studies on uracil-copper(II) complexes.107 The metal-ion specificity of the mercury-mediated base pair led directly to applications as sensors, where the change in luminescence of a fluorophore- and quencher-decorated DNA strand signals the presence of mercury(II) ions in aqueous solution.90 This approach resulted in a sensitive probe with a detection limit of 40 nM and robust detection even in the presence of several other metal ions. Further development led to more easily readable colorimetric probes, based on gold nanoparticles coated with DNA singles strands.108,109 Based on the increased melting temperature of nanoparticle agglomerates108 or the size growth of coated nanoparticles,109 assays with a limit of detection 3 nM were developed. When combined with MOFs, DNA-based dual sensors can be used to detect mercury(II) and iodide by quenching or restoring the luminescence of the fluorophore.110 Aside from that, impedance-based probes were developed, where simultaneously lead(II), silver(I) and mercury(II) can be detected.111 On the other hand, the high specificity for the T:T mispair resulted in the development of sensors for single-nucleotide polymorphisms. The varying stabilization of a DNA duplex upon mercury(II)-binding, depending on the base opposing thymine, was easily accessible by temperature-dependent UV-spectroscopy.112,113 The mercury(II)-mediated base pairs of thymine and cytosine91 and of thymine and guanine114 were further investigated and also structurally explored. The structural features of the T–Hg(II)–C base pair were obtained by single crystal X-ray diffraction analysis,115 while the ability of a conformational switching between A- and B-form DNA was observed for the T–Hg(II)–C base pair in solution (vide infra).92 A helical wire of mercury(II) ions was observed in the crystal structure of a DNA duplex bearing T–Hg(II)–T and T–Hg(II)–G base pairs (vide infra).114 Altering the electronic properties of the T:T mispair upon metal binding resulted in an increasing interest in this motif in chargetransfer applications. Initial studies implied a deniable effect on the radical cation hopping in a DNA duplex upon the formal

668

Metal-mediated base pairs in nucleic acid duplexes

Table 1

Metal-mediated base pairs of thymine.72,87–93

Nucleobase (structure/ number/pKa)

(T) pKa: 9.8

Sequence 5’-GTG ACC ATA GCA GTG-3’ 3’-CAC TGG TTT CGT CAC-5’ 5’-GCC CTG CCT GTC TCC CAG ATC ACT G-3’ 3’-CGG GAC GGA CAG TGG GTC TAG TGA C-5’ 5’-GCT TGC-3’ 3’-CGT TCG-5’ 5’-CGC GTT GTC C-3’ 3’-GCG CTT CAG G-5’ 5’-D-TTC TTT CTT CCC CTT GTT TGT T-F-3’d 5’-CG TYT CAT GAT ACG-3’ 3’-GC ATA GTA CTY TGC-5’ 5’-CGT CTC ATG ATA CG-3’ 3’-GCA TAG TAC TCT GC-5’ 5’-CTT TCT XTC CCT C-3’ 3’-GAA AGA YAG GGA G-5’

(14) pKa: n.d.

5’-CTT TCT XTC CCT C-3’ 3’-GAA AGA YAG GGA G-5’

Metal Backbone Isomer Y ions

Nuclearity a pH

DTm/


Cb

Melting UV CD curve

Other MS measurements

72

72 NMR72

DNA

b

– Hg(II) 1

7.1 9c

DNA

b

– Hg(II) 1

6.8 4.1

DNA

b

– Hg(II) 1

4

DNA

b

– Hg(II) 1

6.0

NMR88

DNA

b

– Hg(II) 1

7.0

Fluorescence90

DNA DNA DNA DNA DNA

b b b b b

T T C C –

Hg(II) Ag(I) Hg(II) Ag(I) Hg(II)

1 1 1 1 1

7.0 7.0 7.0 7.0 7.0

19.4 –1.7 4.2 8.9

91 91 91 91

91 91 91 91

DNA DNA DNA DNA

b b b b

T T 86 86

Hg(II) Hg(II) Hg(II) Hg(II)

1 1 1 1

6.8 6.8 6.8 6.8

0.1 16.9e 7.9 15.5e

93 93 93 93

93 93 93 93

DNA DNA

b b

86 Hg(II) 1 86 Hg(II) 1

6.8 5.3 6.8 4.5e

89 89

ITC89

87 MS/MS87

NMR92

93 93 93 93

(15) pKa: n.d. a

Nuclearity: number of metal ions per designated base pair. DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2.0 equiv. of metal ions. d D: dabcyl F: 6-fluorescein. e Upon irradiation. Conditions: 2 mM duplex, 10 mM MOPS, 100 mM NaNO372; 1 mM duplex, 10 mM sodium cacodylate, 100 mM NaClO489; 2 mM DNA duplex, 100 mM NaClO4, 1.0 mM sodium cacodylate, 4.8 mM Hg(ClO4)288; 10 nM DNA, 10 mM MOPS, 25 mM NaCl, 500 mM NaNO3, 0.1 mM ethylene diamine90; 10 mM duplex, 200 mM NaClO4, 50 mM sodium cacodylate91; 1 mM duplex, 200 mM NaClO4, 50 mM cacodylic acid (in 9:1 H2O:D2O)92; 1 mM duplex, 150 mM NaClO4, 2.5 mM Mg(ClO4)2, 5 mM MOPS.93 b

replacement of the thymine N3 imine protons by mercury(II) ions,116 supported by DFT-based calculations.117 Consecutive T:T mispairs even reduced the hole transport efficiency upon metal binding.118 On the other hand, the computationally predicted influence of the formation of T–Hg(II)–T base pairs on the excess electron transfer due to a shared LUMO of both metal ions117 was confirmed by time-resolved microwave conductivity measurements of a triazole-modified DNA.119 The charge mobility increased threefold with the artificial, neutral backbone compared to canonical bases without metal-mediated base pairs. When using the phosphate backbone instead of triazole-linked nucleic acids, two stacked T–Hg(II)–T base pairs inhibit the electron transfer completely.120 The complexity of these investigations was revealed by scanning tunneling microscopy investigations of break junctions, which resulted in three different conductance values possibly due to different conformations.121 While the conductance along the DNA duplex is inhibited by the metal-mediated base pair,120 DFT calculations suggest that an interstrand transfer is enhanced due to metal binding.122 Despite the high toxicity of heavy metal ions, DNA polymerases are able form T–Hg(II)–T base pairs upon primer elongation in the presence of mercury(II) ions and can even recognize the new base pair and extend the primer further.123 Even the combination with other metal-mediated base pairs is possible,124 and several consecutive metal-mediated base pairs were formed enzymatically.125 Beside this, controlling the activity of aptamer-mediated DNA polymerases was achieved by altering the secondary structure of aptamers upon metal binding.126 In combination with silver-mediated base pairs, logic gate devices were developed, where

Metal-mediated base pairs in nucleic acid duplexes

669

a metal-ion-activated nuclease was regulated by the formation of metal-mediated base pairs, and a colorimetric naked-eye readout was possible.127 To expand the structural variety of metal-mediated base pairs, several nucleobase derivatives were synthesized. Replacing the 5methyl group of the thymine residue by halogen substituents leads to a drastic change of the pKa values (Table 2).128 While thymine has a pKa value of 9.8, the fluoro derivative 1, the bromo derivative 2 and the pseudo-halogen derivative 3 with a cyanido group have significantly lower pKa values. All derivatives are able to bind mercury(II) ions like the parent nucleobase thymine. The mercury(II) complexes of the cyanide derivative 3 are less stable over the entire pH range. At higher pH values, the stability of base pairs with the fluoro derivative 1 are reduced, too. The authors attribute this observation to the effect of the substituents on the electron density of the heteroaryl moiety. The formation of silver(I)-mediated base pairs was also investigated. It mainly occurs at elevated pH values. Furthermore, the concomitant binding of two silver(I) ions was suggested, with the first silver(I) ion coordinated by the N3 atoms and the second one by the O4 atoms of the opposing thymine derivatives. The mechanism of formation of these metalmediated base pairs was investigated by DFT calculations, resulting in three essential steps: (1) binding and deprotonation, (2) silver ion transfer, and (3) dimerization.129 The binding of two silver(I) ions was proposed for the cyanide derivative. An intensive study of alkyl- and aryl-substituted thymine derivatives revealed the influence of the character of the substituents on the melting temperature and hence on the stability of the resulting metal-mediated base pairs.130 The acyl- or alkyl-substituted nucleobases 4 and 5 form stable metal-mediated base pairs, comparable to those of the thymine (Table 3). Alkyne-substituted derivatives 6, 7 and 8 show a hysteresis in the temperature-dependent UV experiments, maybe caused by an interaction of the mercury(II) ions with the triple bonds. When introducing sterically demanding groups at the terminal position of the alkyne as in nucleoside 9, the mercury(II)-mediated base pairs are not only stable, but also exceed the stabilization of the nucleobases with small alkyl substituents. The introduction of aryl substituents in 10 and 11 leads to an increased stability of the non-metalated duplexes, but no stabilizing mercury(II)-mediated base pairs were formed. Expanding the backbone of thymine by annulation of an additional aromatic ring, the fluorescent dioxoloquinazoline derivative 12131 and the dimethylaniline derivative 13132,133 were obtained (Table 4). The fluorescence of 12 is quenched upon the formation of a 12–Hg(II)–12 base pair.131 Selective base pairing with thymine and mercury(II) ions was possible, while cytosine can bind mercury(II) and silver(I) ions. In case of 13, the luminescent properties of the thymine surrogate were used to evaluate the binding process of mercury(II).132 Based on the experimentally determined association and dissociation rates, an equilibrium constant Kd in the order of 8–50 nM was calculated, and the 13–Hg(II)–13 base pair was found to be kinetically stable (t1/2 ¼ 0.3 – 1.3 h).132 The process of metal binding can be controlled by light utilizing the photo-caged thymine-derivative 14 (Table 1).93 Upon irradiation of a 14:14 mispair in the presence of Hg(II), a T–Hg(II)–T pair is formed, whereas without light, no stabilization was observed (Fig. 3). Surprisingly, when 14 was paired with the artificial phenanthroline-derived nucleobase 86 (vide infra), a stabilization upon metal ion addition was observed even prior to irradiation, which increased further upon irradiation. To study the effect on the trapped tautomeric form of thymine in 14, a protected yet light-insensitive derivative 15 was used, and a stabilization of the 15:86 pair was found upon mercury(II) addition. Interestingly, the trapped enol tautomer resembles cytosine, and despite the bulk of the protecting group, it is able to bind mercury(II) ions.93

Table 2

Metal-mediated base pairs of 5-substituted uracil derivatives with (pseudo-)halogen groups.128

Nucleobase (structure/ number/pKa)

(1) pKa: 7.7

(2) pKa: 8.4

(3) pKa: 6.5

Sequence

Metal Backbone Isomer Y ions

Melting

Other MS measurements 128 ICP128 128 ICP128

Nuclearity a pH

DTm/
C b UV CD curve

5’-GTG ACC AXT GCA DNA GTG-3’ 3’-CAC TGG TXA CGT CAC-5’

b

– Ag(I) Hg(II)

1–2 1

9.0 9.0

14c 6c

128 128

5’-GTG ACC AXT GCA DNA GTG-3’ 3’-CAC TGG TXA CGT CAC-5’

b

– Ag(I) Hg(II)

1–2 1

9.0 9.0

14c 10c

128 128

5’-GTG ACC AXT GCA DNA GTG-3’ 3’-CAC TGG TXA CGT CAC-5’

b

– Ag(I) Hg(II) Hg(II)

1–2 1 1

9.0 9.0 7.1

14c 1c 6c

128 128 128

Conditions: 2 mM duplex, 10 mM Na cacodylate (pH 7.1) or boric acid (pH 9.0), 100 mM NaNO3. a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2.0 equiv. of metal ions.

670 Table 3

Metal-mediated base pairs in nucleic acid duplexes Metal-mediated base pairs of 5-substituted uracil derivatives with alkyl groups.130

Nucleobase (structure/ number/pKa)

(4) pKa: 8.8

(5) pKa: 9.9

(6) pKa: 8.5

(7) pKa: 8.6

(8) pKa: 8.8

(9) pKa: 8.7

(10) pKa: 9.0

Sequence

Metal Backbone Isomer Y ions

Nuclearity a pH

DTm/
C b

Melting UV CD curve

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

–1.0 6.5 3.5

130

130 130 130

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

5.5 6.5 6.5

130

130 130 130

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

1.5 6.0 5.5

130

130 130 130

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

6.5 6.0 6.5

130

130 130 130

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

1.0 5.0 7.0

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

1 1 1

6.0 7.0 9.0

9.0 9.5 8.5

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

0 0 0

6.0 7.0 9.0

1.0 –2.5 –3.0

5’-TAG GTC XAT ACT-3’ 3’-ATC CAG XTA TGA-5’

DNA

b

– Hg(II) Hg(II) Hg(II)

0 0 0

6.0 7.0 9.0

–2.5 –2.5 –1.5

(11) pKa: 8.4 Conditions: 5 mM duplex, 10 mM MOPS, 100 mM NaNO3. a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

130 130 130

130

130 130 130

130 130 130

130

130 130 130

Other MS measurements

Metal-mediated base pairs in nucleic acid duplexes

671

Fluorescent thymine analogs for metal-mediated base pairing.131,133

Table 4

Nucleobase (structure/number/ pKa)

(12) pKa: n.d.

Sequence

Metal Backbone Isomer Y ions Nuclearity a pH DTm/
C b

Melting UV CD curve MS Other measurements

5’-CGT CCG TAX TAC DNA GCA CGC-3’ 3’-GCA GGC ATT ATG CGT GCG-5’

b

– Hg(II) 1

7.0 5.2

131

5’-CCC TAA CCC TAA DNA XCC TAA CCC-3’ 3’-GGG ATT GGG ATT TGG ATT GGG-5’

b

– Hg(II) 1

7.4 4.8

133 133

Fluorescence,131 Life time131

133 Fluorescence,133 Life time133 Quantum yield133

(13) pKa: 9.5 Conditions: 2 mM duplex, 10 mM MOPS, 100 mM NaClO4131; 5 mM duplex, 200 mM Na2HPO4, 100 mM citric acid, 100 mM NaCl or NaNO3.133 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Fig. 3

Deprotection of 14 by irradiation yielding thymine; light-insensitive derivative 15.93 (R ¼ 2’-deoxyribose).

Substituting the oxygen atoms by sulfur led to a variety of thiopyrimidine derivatives.134 4-Thiothymine 16 and 2-thiothymine 17 were able to bind up to two silver(I) ions (Table 5). In the case of 16, the structural properties of a duplex with two consecutive 16–Ag(I)2–16 base pairs could even be studied by single-crystal X-ray diffraction analysis (vide infra).135 Here, the four Ag(I) ions exhibit short Ag$$$Ag distances ranging from 280 to 320 pm, suggesting argentophilic interactions between the metal ions. To study the effect of the acidification of the nucleobase by the initially bound silver(I) ion, the uracil derivative 18 was incorporated into DNA and alkylated in situ with methyl iodide to yield nucleobase 19. While 18:18 pairs are still able to bind two silver(I) ions, 19:19 mispairs are not stabilized by excess Ag(I).134 The authors tested a variety of different other metal ions, but besides Ag(I), Hg(II) and Cd(II) they only observed a stabilization of a duplex with 16:16 mispairs in case of Cu(II). Introduction of a protic substituent in the five-position of uracil adds an additional potential metal-binding site to the canonical Watson-Crick face, utilizing the carbonyl oxygen O4 and the introduced substituent. In the case of 5-hydroxyuracil 20, hydrogenbonded base pairing with adenine remains possible via the Watson-Crick face, but added metal ions were found to bind via the oxygen atom of the deprotonated 5-hydroxy substituent and the O4 carbonyl group (Table 6).136,137 The duplexes were only stabilized when three consecutive 20:20 mispairs were present per strand. Beside Zn(II) (Fig. 4A), several lanthanides were able to form metal-mediated base pairs, including Gd(III) and Y(III), which was used in an NMR study.137 In the case of Zn(II), a strong dependence of the stability of the formed base pair on the pH was discovered, while Gd(III) complexes did not respond strongly to a change in pH.136 The capability of a carboxylate at the 5-position was demonstrated by nucleobase 21.138 While it forms a hydrogen-bonded base pair with adenine via the Watson-Crick face, 21–Hg(II)–T and 21–Ag(I)–C are possible, too. In addition, 21 engages in 21–Cu(I)–21 and in 21–Cu(II)–G base pairing (Fig. 4B), involving the O4 carbonyl oxygen atom and the carboxylate group, hence acting as an O^O-bidentate ligand.138 A change in the binding site of metal-mediated base pairs was further achieved by metal–carbon bond formation involving the nucleobase.139 Reaction of a DNA strand containing a single uracil with mercuric acetate yielded the 5-mercuriuracil derivative. Its stabilizing effects on the formation of triple helices was investigated, but only minor effects were found for 5-mercuriuracil.139 Modification of the core of the pyrimidine bases was demonstrated by using 6-azauridine.140 Its interaction with Zn(II) was investigated by precipitation assays as well as by ethidium bromide binding studies.

2.21.2.1.2

(Functionalized) cytosine

The specific interaction of Ag(I) ions with C:C mispairs to form C–Ag(I)–C pairs (Table 7) was discovered many years after the T– Hg(II)–T base pair.82 Studies on the binding behavior showed that a 1:1 complex between a DNA duplex bearing a C:C mispair and

672

Metal-mediated base pairs in nucleic acid duplexes

Table 5

Metal-mediated base pairs of thiopyrimidine derivatives.134

Nucleobase (structure/ number/pKa)

(16) pKa: 8.2

(17) pKa: 8.8

(18) pKa: n.d.

Sequence

Metal Backbone Isomer Y ions

Nuclearity a pH DTm/
C b

Melting UV CD curve

5’-GTG ACC AXT GCA DNA GTG-3’ 3’-CAC TGC TXA CGT CAC-5’

b

– Ag(I) Hg(II) Cd(II)

2 1 1

5’-GTG ACC AXT GCA DNA GTG-3’ 3’-CAC TGC TXA CGT CAC-5’

b

– Ag(I) Hg(II)

2 1

c

5’-TTT TTT TXT TTT DNA TTT-3’ 3’-AAA AAA AXA AAA AAA-5’

b

– Ag(I)

2

7.0 > 30e

134

5’-TTT TTT TXT TTT DNA TTT-3’ 3’-AAA AAA AYA AAA AAA-5’

b

18 Ag(I)

0

7.0 > 0e

134

Other MS measurements

c

7.0 23 20 9.0 4d

134 134 134 134

134 134 134

7.0 23 10

134 134 134

134 134

(19) pKa: n.d. Conditions: 2 mM duplex, 10 mM MOPS (pH 7.0) or 10 mM sodium borate (pH 9.0), 100 mM NaNO3. a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Not mentioned. d Value determined in the presence of 1.1 equiv. of metal ions. e Estimated value.

Table 6

5-Substituted thymine derivatives with protic properties.136–138

Nucleobase (structure/ number/pKa)

(20) pKa: 7.7

(21) pKa: 4.08, 9.98

Sequence

Metal Backbone Isomer Y ions

5’-CAC ATT XXX GTT DNA GTA-3’ 3’-GTG TAA XXX CAA CAT-5’

b

5’-CAC ATT XGT TGT DNA A-3’ 3’-GTG TAA YCA ACA T-5’ 5’-CAC ATT XXX GTT DNA GTA-3’ 3’-GTG TAA YYY CAA CAT-5’

b

b

Nuclearity a pH

DTm/
C b

– Zn(II) Gd(III) Gd(III) Y(III) Gd(III)

1 1 1 1 1

9.0 9.0 8.0 8.0 7.0

21.6 21.9 18.3 20 23.4

21 C 21 T 21 G 21 A

1 1 1 1 1 1 1 1

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

1.4 15.5 10.6 10.7 30.7 8.3 19.4 –6.0

Ag(I) Ag(I) Hg(II) Hg(II) Cu(II) Cu(II) Gd(III) Gd(III)

Conditions: 2 mM duplex, 10 mM HEPES (pH 7.0 and 8.0) or 10 mM CHES (pH 9.0), 100 mM NaCl or NaNO3. a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Melting UV CD curve 136 136 137 137 136 137 137 137 136

Other MS measurements

136

138 138 138 138 138 138 138 138 138 138

137 137 NMR137

138 138 138 138 138 138

Metal-mediated base pairs in nucleic acid duplexes

Fig. 4

673

Proposed binding motifs of (A) the 20–Zn(II)–20 base pair137 and (B) the 21–Cu(II)–21 base pair.138 (R, R’: DNA backbone).

Table 7

Metal-mediated base pairs of cytosine.83,91,103,104,141–144

Nucleobase (structure/number/ pKa) Sequence

DTm/
C b

UV

Melting Other CD curve MS measurements

5’-CGT XTC ATG AYA CG- DNA 3’ 3’-GCA YAG TAC TXT GC-5’ 5’-AGC AAA XAA CGC-3’ DNA 3’-TCG TTT YTT GCG-5’ RNA RNA

b-C a-C b-C a-C b-T b-T

Ag(I) Ag(I) Ag(I) Ag(I) Ag(I) Hg(II)

1 1 1 1 1 1

7.4 7.4 7.4 7.4 7.0 7.0

7.5 14.0 15.0 2.5 8.9c 4.2c

b b b

b-C b-C b-C;

Ag(I) 1 Ag(I) 1 Ag(I) 1

7.1 7.1 7.1

12.0 10.0 9.0

DNA

b

–

Ag(I) 1

7.0

12.0

DNA

b

–

Ag(I) 1

6.8

3.5

DNA

b

–

Ag(I) 1

7.1

13f

103

DNA

b

–

Ag(I) 1

7.1

8f

103

DNA

b

–

Ag(I) 1

7.1

8.4

104

DNA

b b

– –

Ag(I) 1 Ag(I) 1

6.8 6.8

0.9 7.4g

5’-TAG GXC AAT ACT-3’ 3’-ATC CAG TTA TGA-5’ 5’-CTC AGA TCC TGC XCT TCA AAA ACA A-3’ 3’-GAG TCT AGG ACG XGA AGT TTT TGT T-5’ 3’-AAA AAX AAA AATddTTT TTT XTT TTT-5’e 3’-AAA AAX AAA AATddTTT TTT XTT TTT-3’e 5’-GAC GTX CTA CG-3’ 3’-CTG CAT GAT GC-5’ 5’-GAG GGA CAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’

DNA

Metal ions Nuclearity a pH

b b a a b b

5’-TAG GTX AAT ACT-3’ 3’-ATC CAY TTA TGA-5’

(C) pKa: 4.2

Backbone Isomer Y

RNA

144 144 144

144 144 91 91 91

NMR91 NMR91

142 142 142 142 142

MM142

143d 83 83

141 141 141 141

83 ITC,83 NMR83

103

HPLC141 HPLC141

(29) pKa:n.d. Conditions: 5 mM duplex, 100 mM NaOAc, 10 mM Mg(OAc)2144; 10 mM duplex, 200 mM NaClO4, 50 mM cacodylic acid91; 2 mM duplex, 100 mM NaClO4, 10 mM MOPS142; 5 mM duplex, 100 mM NaOAc, 10 mM Mg(OAc)2143; 1 mM duplex, 100 mM NaNO3, 10 mM sodium cacodylate83; 2 mM duplex, 100 mM NaCl, 10 mM MOPS103; 5 mM duplex, 10 mM MOPS, 1 M NaClO4104; 1 mM duplex, 5 mM MOPS, 150 mM NaClO4.141 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2.0 equiv. of metal ions. d At pH 8.0. e TddT represents ethyl-linked thymine nucleobases. f Value determined in the presence of 1.2 equiv. of metal ions. g Upon irradiation.

674

Metal-mediated base pairs in nucleic acid duplexes

a Ag(I) ion is formed with a binding constant in the order of 106 M–1.83 Early DFT-based predictions on the structural properties of C–Ag(I)–C base pairs focused on trans-oriented cytosine moieties.145–147 An intensive approach, by synthesizing and characterizing a set of complexes of (N1-unsubstituted) cytosine with silver(I) ions and comparing the structures based on DFT calculations revealed a bent-transoid structure as the global minimum conformation.148 However, the authors pointed out that this might be caused by the N1–H hydrogen bond donor site. The first crystal structure of a C–Ag(I)–C base pair in an RNA duplex exhibited a cis-conformation (vide infra).81 The NMR solution structure of a corresponding DNA duplex also featured a C–Ag(I)–C base pair with cis-oriented glycosidic bonds (vide infra). The base pair was well characterized by the NMR coupling constant between the 15N3 atom and the 107/109Ag(I).84 The binding process of the Ag(I) ion to the C:C mispair could be visualized by AFM of specially designed nanostructures with a central DNA duplex, where the X-shape formation of the duplex upon metal binding indicates the formation of the metal-mediated base pair.97 To be able to control and trigger externally the metal-binding properties of C:C mispairs, photo-caged cytosine derivative 29 was developed and successfully paired with other artificial nucleobases.141 The addition of excess silver(I) ions led to the formation of a different DNA structure with a distinct CD spectrum, an increased electrochemical overpotential and a modified charge-transfer resistance.149,150 Different backbone modifications were tested to modulate the stability of the C–Ag(I)–C base pair. Introduction of the a-anomer showed that the hetero chiral base pair is more stable than any homo chiral analog.149 The use of methylene-bridged nucleic acids (known as BNA or LNA) increased the stability of the duplexes with a C–Ag(I)–C base pair compared to the respective DNA or RNA duplex, due to their predefined conformation and the improved p stacking interactions with adjacent base pairs.142 The utilization of a 2’-fluorinated deoxyribose backbone improved the stability of the duplexes with C–Ag(I)–C base pairs as judged by the melting temperature.151 Depending on the degree and the configuration of the fluorination, stereoelectronic effects modulate the structure of the base pair and might even alter the number of silver(I) ions taken up by the base pair. Investigations on the overall strand orientation were performed with covalently linked duplexes and revealed that the C–Ag(I)–C base pair is more stable in antiparallel-stranded rather than parallel-stranded duplexes.103 Introducing multiple C–Ag(I)–C base pairs into nucleic acids became an attractive way for the construction of DNA nanostructures due to the enhanced stability and the defined formation of these base pairs. For origami-like DNA structures, the framework was constructed by DNA comprising metal-mediated base pairs, with the central nucleic acids being mediated by dynamic WatsonCrick base pairs.152 For the formation of triplex DNA, the proton involved in the C-N3–H$$$G-N7 hydrogen bond at the Hoogsteen-face can be substituted by a Ag(I) ion, giving rise to a C–Ag(I)–G:C base triple.153 As a result, triplex formation was possible even under neutral conditions. This resulted in the application as fluorescent probe for specific DNA sequences based on the formation of triplex DNA.154 When incorporated into hairpins, a repetitive sequence of cytosine residues can also function as a matrix for the formation of silver nanoclusters upon reduction.155 This strategy was applied in the detection of mutations in CDH1, a gene featuring a sequence of five consecutive cytosine bases. Further, the potential of C–Ag(I)–C pairs in modulating the conductance of DNA duplexes was investigated. An early example applying scanning tunneling microscopy on a sequence of eleven C–Ag(I)–C base pairs showed an increased conductance compared to the canonical Watson-Crick base pairs.156 MD simulations suggested that only parallel-stranded duplexes with C– Ag(I)–C base pairs are stable and structurally altered compared to B-DNA due to additional hydrogen-bonding interactions between the nucleobases.157 This kind of distortion has also been seen in the crystal structures of model complexes of silver(I) with cytidine.158 Even though these structures bear short Ag$$$Ag distances, their electrical conductance is low. To increase the conductivity, arrays of Ag(I) ions in model complexes of 1-methylcytosine with silver(I) were reduced with cold hydrogen plasma.159 The increased conductivity of the resulting material was confirmed by EFM (electric force microscopy) and c-AFM (conductive atomic force microscopy), while powder X-ray diffraction analyses confirmed the preservation of structural characteristics upon reduction. Compared to these findings, the hole migration through a nucleic acid duplex is suppressed by the formation of C–Ag(I)–C base pairs.118 The specificity of the C:C mispair to bind silver(I) ions was used to detect polymorphism in DNA sequences. While the melting temperature of a duplex bearing a C:C mispair is increased in the presence of silver(I) ions, the addition of Ag(I) ions only shows a minor influence on melting temperature of Watson-Crick base pairs or G:G mispairs.113 On the other hand, the Ag(I) ions can be selectively detected and quantified. In designed tweezers with poly-C tails, the bond-rupture forces correlate with the silver(I) concentration, so that concentrations down to 1 nM silver(I) ions can be detected in distilled water.160 Devices based on impedance measurements of suitably modified oligonucleotides were able to simultaneously detect Pb(II), Hg(II) and Ag(I) ions in solution, with a detection limit of 10 nM for silver(I) ions.111 The ability of different metal ions to bind to cytosine depends largely on the opposing nucleobase. Binding of mercury(II) was detected for an opposing thymine base.91 While the thermal stability of the duplex was not impacted by the formation of the T– Hg(II)–C pair, the directly measured affinity of the T:C mispair for mercury(II) ions was high. This kind of metal-mediated base pair was also observed in a crystal structure of a DNA duplex115 as well by NMR spectroscopy in a dynamical transition of A- and B-DNA duplexes (vide infra).92 The T–Hg(II)–C pair was further used in primer extension experiments, where in the presence of silver(I) ions thymine was incorporated opposite to a template cytosine and vice versa.124 Even a combination with T–Hg(II)–T base pairs was possible. Utilizing C:C mispairs in a polymerase-regulating aptamer led to a silver(I)-controlled polymerase activity.126 DFTbased investigations also suggest a possible formation of copper(I) complexes with cytosine in an i-motif-like structure.161 Based on EPR (electron paramagnetic resonance) studies on the interaction of copper(II) with DNA, a crosslink between the N7 atom of guanine and the N3 position of cytosine mediated by copper(II) ions was proposed.162 Surprisingly, in primer extension experiments with Klenow fragment polymerase, adenine was incorporated opposite to cytosine in the presence of silver(I) ions.163 Despite

Metal-mediated base pairs in nucleic acid duplexes

675

the possible formation of C–Ag(I)–A base pairs, a crystal structure of a self-complementary DNA strand with C:A mispairs showed arrays of Ag(I) ions coordinated in C–Ag(I)–C and G–Ag(I)–G pairs (vide infra).164 The scope of cytosine-containing metal-mediated base pairs was increased by modification of the heterocycle. Introduction of a 5-methyl or 5-hydroxymethyl group in 22 and 23, respectively, enabled investigations of epigenetic markers in metal-mediated base pairs (Table 8).165 While the stability of a C–Ag(I)–22 hetero base pair with cytosine and 22 was reduced compared to the corresponding C–Ag(I)–C base pair, the hydroxymethyl group in 23 stabilized the duplex with a C–Ag(I)–23 pair at least to the same extent as a C–Ag(I)–C base pair.165 This trend was altered upon changing the salt concentration. When bulkier groups as 5-iodo or 5-octadiynyl were introduced, the melting point of the oligonucleotides showed a high dependence on the conformers of the base pair.166 In case of the 5-iodocytosine derivative 24, the melting points of oligonucleotide duplexes with the metalmediated base pairs a-24–Ag(I)–b-C and b-24–Ag(I)–a-C were higher than those with a-24–Ag(I)–a-C and b-24–Ag(I)–b-C. The same trend was observed for the 5-octadiynyl nucleobase 25. Ag(I)-mediated base pairing of the nucleobases 24 and 25 with 5-aza-7-deazaguanine 53 inverted the trend regarding the conformers, so that the base pair combinations a:a and b:b became slightly more stable.168 Fluorination of the ribose led to minor effects on the stability compared to unmodified cytidine.151 Also the 5-phenylacetylene derivative of cytosine did not show a notable stabilization upon addition the addition of Ag(I).151 The

Table 8

5-Substituted cytosine derivatives.104,165–167

Nucleobase (structure/ number/pKa) Sequence

(22) pKa: n.d.

(23) pKa: n.d.

(24) pKa: n.d.

(25) pKa: n.d.

(26) pKa: 3.7 0.4

Backbone Isomer Y

Metal ions Nuclearity a pH

DTm /
C b UV CD curve

Melting

5’-AAT AAA ATA XTA TAA A-3’ 3’-TTA TTT TAT CAT ATT T-5’

DNA

b

–

Ag(I) 1

7.1

12.6

165

5’-AAT AAA ATA XTA TAA A-3’ 3’-TTA TTT TAT CAT ATT T-5’

DNA

b

–

Ag(I) 1

7.1

15.2

165

5’-TAG GTX AAT ACT- DNA 3’ 3’-ATC CAY TTA TGA5’

a a b b a b

b-C a-C b-C a-C b-24 a-24

Ag(I) Ag(I) Ag(I) Ag(I) Ag(I) Ag(I)

1 1 1 1 1 1

7.4 7.4 7.4 7.4 7.4 7.4

12.0 –2.0 2.5 13.0 8.5 7.5

166 166 166 166 166 166 166 166 166

5’-TAG GTX AAT ACT- DNA 3’ 3’-ATC CAY TTA TGA5’

a a b b a b

b-C a-C b-C a-C b-25 a-25

Ag(I) Ag(I) Ag(I) Ag(I) Ag(I) Ag(I)

1 1 1 1 1 1

7.4 7.4 7.4 7.4 7.4 7.4

14.5 0.5 3.0 11.0 11.5 11.0

166 166 166 166 166 166 166 166 166

5’-mCGA GmCX mCTG DNA GmC-3’ 3’-GCT CGT GAC CG-5’

b

–

1

7.4

21.3

167

5’-GAC GTX CTA CG-3’ DNA 3’-CTG CAY GAT GC-5’

b

T C 27

Ag(I) 1 Ag(I) 1 Ag(I) 1

7.1 7.1 7.1

12.5 18.9 9.6

104 104 104

Other MS measurements

(27) pKa: n.d. Conditions: 1 mM duplex, 100 mM KNO3, 10 mM MOPS,165 2.5 mM duplex, 100 mM NaOAc, 10 mM Mg(OAc)2,166 3 mM duplex, 100 mM NaClO4, 20 mM sodium cacodylate,167 5 mM duplex, 10 mM MOPS, 1 M NaClO4.104 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

676

Fig. 5

Metal-mediated base pairs in nucleic acid duplexes

Proposed structures of (A) the 26–T base pair167 and (B) the 26–G base pair.167 (R, R’: DNA backbone).

organomercurated cytosine derivative 26 formed a stable metal-mediated base pair with thymine, while still being able to bind guanine in a Watson-Crick like fashion (Fig. 5A and B).167 The ability of 26 to coordinate the N7 atom of adenine via the 5mercuri modification was used in the controlled triplex formation within a 26–A–T trimer.139 The artificial isoform 5methylisocytosine 27 was also able to coordinate Ag(I) ions, even exceeding the stabilization of a duplex with a C–Ag(I)–C pair via the formation of C–Ag(I)–27, T–Ag(I)–27 and even 27–Ag(I)–27 base pairs.104 An additional hydrogen bond in the Ag(I)mediated base pairs involving 27 and cytosine or thymine may be the reason for the additional stabilization of these base pairs. The luminescent cytosine analogue 28 was introduced to study the C-Hg(II)-T base pair, which had been undetected before (Table 9).91 While no mentionable increase in melting temperature was observed upon the addition of Hg(II) ions to the 28:T mispair, its high affinity for Hg(II) was detected by the luminescent properties of the nucleobase 28. A series of pyrrolo and imidazolo derivatives of cytosine expanded the family of luminescent nucleobases (Table 9). The phenyl-substituted pyrrolocytosine 34 was used to bind up to two Ag(I) ions in a 34–Ag(I)2–34 pair in an antiparallel-stranded DNA duplex, and with cytosine mononuclear 34–Ag(I)–C pairs were formed.175 Utilizing 6-pyridinyl-substituted pyrrolocytosines 30 and 31, also dinuclear homo base pairs of the type 30–Ag(I)2–30 and 31–Ag(I)2–31 were formed. However, the duplex stabilization achieved with 31 exceeded the effects of 30 and 34.169,170 Furthermore, 30 and 31 were tested in parallel-stranded DNA, where 30 stabilized the DNA duplex more than twice as much as 31 after Ag(I) ion binding. The Ag(I) binding process was monitored by fluorescence spectroscopy to determine the stoichiometry of the base pairs involving 30 and 31.169 Simplification of the phenyl substituent led to 6-methyl pyrrolocytosine 32, which still forms dinuclear 32–Ag(I)2–32 pairs.170 While one silver(I) ion was included in the 32–Ag(I)–C hetero base pair, the Ag(I) binding quenched the luminescence selectively in the case of base pairing with cytosine, so that this strategy was used in the detection of biologically relevant thiols.174 When the Ag(I) binding properties of 32 were compared to those of 34, the nuclearity for both Ag(I)-mediated homo base pairs was identical and the resulting stabilization of the DNA duplex was similar.171 Consecutive base pairs of 34–Ag(I)2–34 are more stable than the corresponding 32–Ag(I)2–32 pairs. The stability of the complexes was probed upon the addition of halide anions. Indeed, the Ag(I) ions from 34–Ag(I)2–34 base pairs were successfully removed by addition of iodide, while the DNA melting point after the addition of bromide or chloride remained unaltered.171 Modifying the pyrrolo cytosine nucleobase by introducing a triazole moiety, the melting point of a duplex containing a 33:33 mispair increased upon silver(I) addition by 36
C, which is higher than for any other pyrrolo cytosine derivative.170 The impact on the stability by Ag(I)-binding was only topped by the use of imidazolo cytosine derivatives. The homo mispair of unsubstituted imidazolo cytosine 35 is able to bind two silver(I) ions to form 35–Ag(I)2–35, stabilizing the duplex by 39.0
C.172 When a phenyl group is introduced to the imidazole cytosine, the resulting nucleobase 36 exhibits almost identical stabilizing effects upon Ag(I) binding as 35 does. The melting temperature increased in antiparallel- and parallel-stranded DNA duplexes to the same extent, so that the phenyl group has neither a positive nor a negative effect on the overall duplex stability.172 In a direct comparison, the stabilizing effect of the imidazolo derivative with a phenyl substituent 36 exceeds that of the pyrrolo derivative 34 (Fig. 6A and B).175 The lower pKa value of imidazolo cytosine could be an origin of this stabilization. Surprisingly, the substitution of the phenyl group by a furan moiety (37) did not change the nuclearity of the Ag(I)-mediated base pair of 37, i.e. 37–Ag(I)2–37, but the thermal stability upon binding two silver(I) ions increased by almost 50
C in antiparallel- and 40
C in parallel-stranded DNA duplexes.172 The benefits on the Ag(I) binding behavior of nucleobases with an expanded p system were further investigated in nucleobases derived from 1,3-diaza-2-oxophenoxazines.176 The homo mispair of 3H-benzo[b]pyrimido[4,5-e][1,4]oxazin-2(10H)-one 38 was able to bind one Ag(I) ion with a moderate overall stabilization of the duplex (Table 10). When used in a parallel-stranded DNA duplex, the homo mispair of 38 was still able to bind one silver(I) ion, giving rise to the formation of a 38–Ag(I)–38 pair, as confirmed by various spectroscopic techniques, including fluorescence spectroscopy.177 DFT calculations suggest the presence of synergistic hydrogen bonds, stabilizing the base pair, even upon metal binding, which was confirmed by MD simulations. The stability of the hetero base pair 38–Ag(I)–C showed a strong sequence dependence, but its luminescence increased irrespective of the sequence, making it suitable for the development of biosensors.177 The related artificial nucleobase 3H-pyrido[3,2-b]pyrimido[4,5-e][1,4]oxazin-2(10H)-one 39 bound three silver(I) ions in the 39–Ag(I)3–39 homo base pair (Fig. 6C, Table 10).176 This is the only trinuclear metal-mediated base pair reported to date. Further mononuclear metal-mediated base pairing was achieved in the 39–Ag(I)–C and 39–Hg(II)–T base pairs. 4-Aryl derivatives of cytosine bear options to form metal-mediated base pairs with different metal ions (Table 11). The nucleobase derivative 4-(pyridin-2-yl)pyrimidin-2(1H)-one 40 contains a 2-pyridyl substituent and is able to form stable 40–Ni(II)–40 and 40–Co(II)–40 base pairs and to some extent also binds copper(II) ions.178 Further extending the substituent by a 2,2’-bipyridyl unit led to the artificial nucleobase 4-([2,2’-bipyridin]-6-yl)pyrimidin-2(1H)-one 41.179 Based on DFT calculations, Ag(I)-mediated

Metal-mediated base pairs in nucleic acid duplexes Table 9

677

Luminescent cytosine derivatives.91,169–172

Nucleobase (structure/ Sequence number/pKa)

(28) pKa: 4.5173

(30) pKa: 3.0, 11.1

(31) pKa: 4.1, 11.0

(32) pKa: n.d.

(33) pKa: n.d.

(34) pKa: 2.7, 11.5175

(35) pKa: 8.8

Back Metal bone Isomer Y ions Nuclearity a pH DTm/
C b

Melting Other UV CD curve MS measurements

5’-CCC TAA CCC TAA CCX DNA b TAA CCC-3’ 3’-GGG ATT GGG ATT GGT ATT GGG-5’

– Hg(II) 1

7.4 n.d.

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ 5’-TAiG iGTX AAT Ai DNA b CT-3’ 5’-ATC CAY TTA TGA-3’

30 Ag(I) 2

7.4 21.5

170 169

170 Fluorescence169

30 Ag(I) 2

7.4 21.0

169

Fluorescence169

31 Ag(I) 2

7.4 26.0

169

169

Fluorescence169

31 Ag(I) 2

7.4 10.0

169

169

Fluorescence169

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ DNA b

C Ag(I) 1 32 Ag(I) 2

7.4 7.0 7.4 18.5

171 170 170 171

Fluorescence174

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ DNA b

C Ag(I) 1 33 Ag(I) 2

7.4 9.0 7.4 36.0

170 170 170 170

170

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ DNA b

34 Ag(I) 2 C Ag(I) 1

7.4 22.5 7.4 3.5

171

171

171 Fluorescence171

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ 5’-TAG GTX AAT ACT-3’ DNA b 5’-ATiC iCAY TTA TiGA-3’

35 Ag(I) 2

7.5 39.0

172

Fluorescence172

35 Ag(I) 2

7.5 27.0

172

172

Fluorescence172

36 Ag(I) 2 C Ag(I) 1 36 Ag(I) 2

7.4 38.5 7.4 8.5 7.5 27.0

175

175

172

172

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ 5’-TAiG iGTX AAT Ai DNA b CT-3’ 5’-ATC CAY TTA TGA-3’

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ 5’-TAG GTX AAT ACT-3’ DNA b 5’-ATiC iCAY TTA TiGA-3’

Association/ dissociation constant91

Fluorescence172

(36) pKa: 2.4, 7.9175 (Continued)

678 Table 9

Metal-mediated base pairs in nucleic acid duplexes Luminescent cytosine derivatives.91,169–172dcont'd

Nucleobase (structure/ number/pKa) Sequence

Back Metal bone Isomer Y ions Nuclearity a pH DTm/
C b

5’-TAG GTX AAT ACT-3’ DNA b 3’-ATC CAY TTA TGA-5’ 5’-TAG GTX AAT ACT-3’ DNA b 5’-ATiC iCAY TTA TiGA-3’

Melting Other UV CD curve MS measurements

37 Ag(I) 2

7.5 48.0

172

172

Fluorescence172

37 Ag(I) 2

7.5 38.0

172

172

Fluorescence172

(37) pKa: 2.5, 7.4 Conditions: 5 mM duplex, 200 mM NaClO4, 100 mM citric acid, 100 mM NaCl or NaNO391; 5 mM duplex, 100 mM NaOAc, 10 mM Mg(OAc)2.169 –172 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Fig. 6 Proposed structures of (A) the 34–Ag(I)2–34 base pair175; (B) the 36–Ag(I)2–36 base pair175 and (C) the 39–Ag(I)3–39 base pair.176 (R, R’: DNA backbone).

Table 10

Phenoxazine derivatives of cytosine.176,177

Nucleobase (structure/number/ Sequence pKa)

(38) pKa: 2.3, 9.7

Metal Backbone Isomer Y ions Nuclearity a pH

DTm/
C b

Melting Other UV CD curve MS measurements

5’-GCG TTX TTT GCT-3’ DNA 3’-CGC AAY AAA CGA-5’ 5’-AAA AAA AAA ATA DNA XTT TTA AAT ATT T-3’ 5’-TTT TTT TTT TAT YAA AAT TTA TAA A-3’

b

38 Ag(I) 1

7.1

9.0

176

b

38 Ag(I) 1

6.8

5.3

177

C Ag(I) 1

6.8

7.9

177

5’-GCG TTX TTT GCT-3’ DNA 3’-CGC AAY AAA CGA-5’

b

38 Ag(I) 3 C Ag(I) 1 T Hg(II) 1

7.1 7.1 7.1

33 13a 9a

176 176 176 176 176 176 176

(39) pKa: 1.4, 8.2 Conditions: 2 mM duplex, 100 mM NaClO4, 10 mM MOPS176; 3 mM duplex, 150 mM NaClO4, 5 mM MOPS.177 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Fluorescence,177 MD177 Fluorescence,177 MD177 176 Fluorescence176 Fluorescence176 Fluorescence176

Metal-mediated base pairs in nucleic acid duplexes Table 11

679

Substituted pyrimidine derivatives.178,179

Nucleobase (structure/ number/pKa)

Sequence

Metal Backbone Isomer Y ions

Nuclearity a pH

DTm/
C b

5’-CTT TCT XTC CCT-3’ 3’-GAA AGA YAG GGA-5’

DNA

b

38 Ni(II) Co(II) Cu(II)

1 1 1

7.0 7.0 7.0

16.5c 5.2c 2.1c

5’-CTT TCT XTC CCT-3’ 3’-GAA AGA YAG GGA-5’

DNA

b

78 77 G A

1 1 1 1

7.0 7.0 7.0 7.0

12.9d 4.5d 5.4d 5.0d

(40) pKa: n.d.

Ag(I) Ag(I) Ag(I) Ag(I)

Melting UV CD curve

Other MS measurements

178 178 178

179

(41) pKa: n.d. Conditions: 2.5 mM duplex, 50 mM NaCl, 10 mM NaH2PO4,178 2.5 mM duplex, 50 mM NaNO3, 10 mM HEPES.179 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 4 equiv. of metal ions. d Value determined in the presence of 2 equiv. of metal ions.

base pairing of 41 can be achieved opposite to the pyridin-4-yl nucleobase 78 or the pyridin-3-yl nucleobase 77. In a DNA duplex, a stable mononuclear silver(I)-mediated base pair of 41 was obtained with 78, while Ag(I)-mediated base pairs of 41 with 77 were of reduced stability, reminiscent to those with guanine or adenine.179

2.21.2.2 2.21.2.2.1

Purine and its derivatives (Functionalized) adenine

Adenine is the least established canonical nucleobase in metal-mediated base pairing. It has mainly been probed as a complementary ligand opposite an entirely artificial nucleobase.46,180,181 Only a few stable Ag(I)-mediated base pairs have been reported, with C–Ag(I)–A being the most important one. This pair has been established not only in DNA,143,182 but also in a 2’-O,4’-C-methylenebridged nucleic acid analog.142 Computations on the Ag(I)-mediated dimerization of d(CpA) dinucleotides suggest a preferred complexation via the Hoogsteen edge, i.e. involving the N7 atom of adenine rather than N1.145 Studies on the enzymatic formation of Ag(I)-mediated base pairs indicate that the formation of the C–Ag(I)–A is thermodynamically favored over the C–Ag(I)–C pair, as deoxyadenosine is incorporated at the site opposite cytosine.163 It is interesting to note that in a crystal structure of a DNA duplex bearing continuous Ag(I)-mediated base pairs of canonical nucleobases, adenine is the only nucleobase not involved in metalmediated base pairing. Instead, it is bulged out of the duplex, thereby avoiding base pair formation.183 Derivatives of adenine are much more prominent in metal-mediated base pairing than the parent nucleobase is. In particular, a variety of (substituted) deazaadenine moieties have been investigated. Table 12 summarizes some relevant results. In 1deazaadenine (42), a C–H group formally replaces the N1 atom on the Watson-Crick edge of the nucleobase. As a result, metalcoordination must involve the Hoogsteen edge. Its ability to engage in metal-mediated base pairing has been probed with a complementary thymine residue. In this study, a duplex bearing 19 contiguous 42–Ag(I)–T base pairs was reported.184 To prohibit a possible steric hindrance arising from alternating metal-mediated Hoogsteen and canonical Watson-Crick base pairs, duplex sequences were chosen in a way that would allow duplex formation with Hoogsteen base pairing only. 1,3-Dideazaadenine (43) was evaluated with respect to its ability to form 43–Ag(I)–T base pairs, too. The formal replacement of the N3 atom by a C–H group influences the overall basicity of the nucleobase, so that a somewhat different base pairing behavior was observed. When incorporating a single 43:T pair into a regular B-DNA duplex with Watson-Crick base pairing of the canonical nucleobases, a slightly stabilizing 43–Ag(I)–T base pair bearing one Ag(I) ion is formed.185 At elevated pH, the stabilizing effect is more pronounced, indicating the necessity of deprotonation of the thymine residue. Interestingly, in duplexes exclusively composed of 43:T pairs, two Ag(I) ions are incorporated per base pairs.186 DFT calculations indicated that non-planar 43–Ag(I)2–T pairs are formed. Apparently, the absence of structure-determining Watson-Crick base pairs allows the adoption of a duplex topology with non-planar base pairs. According to these calculations, the interatomic Ag$$$Ag distance within the dinuclear base pairs amounts to 288 pm, and the Ag$$$Ag interaction contributes ca. 16 kcal mol–1 to the overall base pair stability.186 The formation

680

Metal-mediated base pairs in nucleic acid duplexes

Table 12 Nucleobase (structure/ number/pKa)

(42) pKa: n.d.

Metal-mediated base pairs involving various deazaadenine derivatives.143,184–189 Metal Backbone Isomer Y ions Nuclearity a pH

DTm/
C b

Melting Other UV CD curve MS measurements

DNA

b

– Ag(I) 1

6.8

>36

184 184 184

DNA

b

– Ag(I) 1

6.8

>18

184

DNA

b

– Ag(I) 1

DNA

b

– Ag(I) 2

6.8 9.0 6.8

4.0 8.2 n.a.

185 185 185 185 186 186 186

186 DLS, DFT

DNA

b

– Ag(I) 2

6.8

n.a.

186 186 186

186 DLS, DFT

DNA

b

– Ag(I) 1

6.8

33

187 187 187

187

DNA

b

52 Ag(I) 1

8.5–9.0

n.d.

188 188

DNA

b

– Ag(I) 1

7.0

3.5

143

DNA

b

– Ag(I) 1

6.0

2.5

143

DNA

b

52 Ag(I) 1

7.4

n.d.

DNA

b

– Ag(I) 1

7.0

3.0

DNA

b

– Ag(I) 1

6.0

3.0

DNA

b

– Ag(I) 1

7.0

2.5

DNA

b

– Ag(I) 1

6.0

3.0

5’-TAG GTX AAT ACT-3’ 3’-ATC CAC TTA TGA-5’

DNA

b

– Ag(I) 1 2

7.4

4 5

189

189

5’-TAG GTX AAT ACT-3’ 3’-ATC CAX TTA TGA-5’ 5’-TAG GTX AAT ACT-3’ 3’-ATC CAC TTA TGA-5’ 5’-XTX TXT XTX TXT-3’ 3’-TXT XTX TXT XTX-5’

DNA

b

7.4

189

b

189

189

DNA

b

4 5 6 8 n.d.

189

DNA

– Ag(I) 1 2 – Ag(I) 1 2 – Ag(I) 1

189

189

Sequence 5’-XXX XXX XXX XXX XXX XXX XA-3’ 3’-TTT TTT TTT TTT TTT TTT TT-5’ 5’-AXA AXA AXA-3’ 3’-TTT TTT TTT-5’

5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT TTC TTT C-5’ 5’-XTX TXT XTX TXT XTX TXT-3’ 3’-TXT XTX TXT XTX TXT (43) XTX-5’ pKa: 0.8  0.2; 5’-XXX XXX XXX TTT TTT 186 4.12  0.03 TTT-3’ 3’-TTT TTT TTT XXX XXX XXX-5’ 5’-TXT XTX TXT XTX TXT XTX-3’ 3’-XTX TXT XTX TXT XTX TXT-5’ 5’-YCX TYC XTY CXT YCX (44) TYC-3’ pKa: n.d. 3’-CYT XCY TXC YTX CYT XCY-5’ 5’-TAG GCC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ 5’-TAG GGC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ 5’-TXY YTC XXT XCT-3’ 3’-XTC CXY TTX TYX-5’ 5’-TAG GCC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ 5’-TAG GGC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ (45) pKa: 5.3143 5’-TAG GCC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ 5’-TAG GGC AAT ACT-3’ 3’-ATC CXG TTA TGA-5’ (46) pKa: 4.2143

(47) pKa: 3.9189

(48) pKa: 5.0189

7.4 7.4

189

189 143

143

Conditions: 1 or 3 mM duplex, 5 mM MOPS, 1 M NaClO4184; 1–5 mM oligonucleotide, 5 mM buffer (MOPS pH 6.8 or borate pH 9.0), 150 mM NaClO4185,186; 2 mM duplex, 5 mM MOPS, 100 mM NaClO4187; 2 mM duplex, 5 mM phosphate, 100 mM NaClO4188; 5 mM duplex, 100 mm NaOAc, 10 mm Mg(OAc)2.143,189 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Table 13

Metal-mediated base pairs involving adenine derivatives.191–199

Nucleobase (structure/number/pKa) Sequence

(49) pKa: n.d.

(51) pKa: 4.17, 9.49

Isomer Y

Metal ions Nuclearity a pH DTm/
C b UV

CD

Melting curve MS Other measurements

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 n.a.

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 7.2

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 n.a.

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 1.4

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 n.a.

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 5.2

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 10.2

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 8.7

20 -O-Methyl-RNA b

–

Cu(II)

1

7.4 15.2

DNA

b

–

Ag(I)

2

6.8 12

194 194 194

DNA

b

–

DNA

b

–

Ag(I) Cu(II) Ag(I)

2 1 1

5.5 16 6.8 3 6.8 15

193 193 198 198 198

DNA

b

–

Ag(I)

2

5.5 20

198 198 198

DNA

b

–

Ag(I)

2

6.8 5

DNA

b

–

Hg(II)

2

DNA

b

–

Hg(II)

1

DNA

b

51 Ag(I)

2

6.8 9.0 6.8 9.0 6.8

DNA

b

–

Ag(I)

2

6.8 23.4

199 199

DNA

b

–

Ag(I)

1

6.8 12.3

199 199

DNA

b

50 Ag(I)

2

6.8 5.4

199 199

8.0 2.0 3 8 5.4

192 192

NMR

197 197 195 195 195 196 196 199 199 DFT, MD, ITC, luminescence

681

Conditions: 3 mM duplex, 20 mM cacodylate, 100 mM NaClO4191,192; 3 mM duplex, 5 mM MOPS, 150 mM NaClO4194,197–199; 3 mM duplex, 5 mM MES (pH 5.5) or MOPS (pH 6.8) buffer, 500 mM NaClO4193,198; 3 mM duplex, 3 mM Mg(ClO4)2, 5 mM MOPS (pH 6.8) or borate (pH 9.0) buffer, 500 mM NaClO4.195,196 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Metal-mediated base pairs in nucleic acid duplexes

(50) pKa: 4.18193

5’-GCG CXC CGG-3’ 3’-CGC GAG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GCG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GGG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GUG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GSG GCC-5’ 5’-AGC GCX-3’ 3’-XCG CGA-5’ 5’-CGC GCX-3’ 3’-XCG CGC-5’ 5’-GGC GCX-3’ 3’-XCG CGG-5’ 5’-UGC GCX-3’ 3’-XCG CGU-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 5’-CTC CCT XTC TTT C-3’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT CTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 5’-CTC CCT CTC TTT C-3’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT TTC TTT C-5’ 5’-iGAiG iGiGA TAiG AAA iG-3’ 5’-CTC CCT XTC TTT C-3’ 5’-GAG GGA TAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA YAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT CTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT YTC TTT C-5’

Backbone

682

Metal-mediated base pairs in nucleic acid duplexes

of a dinuclear 43–Ag(I)2–T base pair was confirmed outside the DNA duplex context by using two-color IR dissociation spectroscopy.190 In 7-deazaadenine (44, Table 12), metal ions cannot bind via the Hoogsteen edge, so that metal-mediated base pairing is anticipated to involve the Watson-Crick edge. B-DNA duplexes containing a single 44 residue are weakly stabilized upon the formation of singly metalated 44–Ag(I)–C or 44–Ag(I)–G base pairs.143 However, when the oligonucleotide sequence is chosen in a way that allows the formation of a duplex composed of 44–Ag(I)–T base pairs only, i.e. Watson-Crick base pairs in which a proton is formally replaced by an Ag(I) ion, then a significant stabilization of that duplex is observed upon the addition of one Ag(I) per base pair.187 Fascinatingly, even duplexes entirely composed of Ag(I)-mediated Watson-Crick-type base pairs involving 7deazaadenine–Ag(I)–T (44–Ag(I)–T) and the analogous 7-deazaguanine–Ag(I)–C (52–Ag(I)–C, vide infra) are possible.188,189 Substituted 7-deazaadenine derivatives such as 7-cyclopropyl-7-deazaadenine (45) and 7-iodo-7-deazaadenine (46) show essentially the same Ag(I)-mediated base pairing behavior as 7-deazaadenine (44) does (Table 12).143 8-Aza-7-deazaadenine represents another fascinating adenine derivative. It has been evaluated with respect to its ability to form Ag(I)-mediated base pairs in two isomeric forms, namely as nucleoside with either an N9-glycosidic bond (47) or an N8-glycosicid bond (48).189 The regularly glycosylated nucleoside forms an Ag(I)-mediated base pair with cytosine. Based on the UV absorbance changes upon the addition of Ag(I) to a duplex containing such a base pair and on the thermal stabilization in the presence of Ag(I), it is difficult to discern the stoichiometry of this metal-mediated base pair. The data are in agreement with a mononuclear 47–Ag(I)–C pair with low Ag(I) affinity, but could also be interpreted assuming the formation of a dinuclear 47–Ag(I)2–C pair, in which the second Ag(I) ion does not contribute much to the overall thermal stabilization. Table 12 lists both possibilities with their respective DTm values. Structures proposed for a tentative dinuclear base pair involve either a coordination by the exocyclic amino groups of the nucleobases or an external binding of the Ag(I) ion to the N8 position of 47 without additional ligands from the complementary strand.189 48 with its unusual glycosidic bond allows unfamiliar coordination patterns. It has been reported to engage in the formation of dinuclear base pairs of the type 48–Ag(I)2–48 and 48–Ag(I)2–C.189 Moreover, a sequence composed of alternating 48 and thymine residues, i.e. a self-complementary sequence under the assumption of the formation of 48:T pairs, binds one Ag(I) ion per base pair. All these deazaadenine derivatives do not contain an additional metal-binding site with respect to the parent nucleobase. In contrast, 2-(3,5-dimethylpyrazolyl)adenine (49, Table 13) contains a dimethylpyrazolyl substituent, thereby providing the possibility of chelate formation. This nucleobase was studied in the context of a possible application of metal-mediated base pairs, namely in an attempt to discriminate the canonical nucleobases via metal-mediated base pairing.191,192,200 Hence, 49 was placed opposite either guanine, adenine, cytosine or uracil. The most stabilizing Cu(II)-mediated base pairs were observed with the pyrimidine nucleobases, i.e. 49–Cu(II)–C and 49–Cu(II)–U.192 Nevertheless, it was not possible to apply this nucleobase for a clear discrimination of all natural nucleobases. 1,N6-Ethenoadenine (50, Table 13) is an exocyclic etheno adduct of adenine with interesting coordination properties. Due to the alignment of the lone pairs of its two nitrogen donor atoms in an almost parallel fashion, it can be used to build dinuclear metal complexes with closely spaced metal ions.193 The homo pair 50:50 binds two Ag(I) ions, irrespective of whether the duplex is antiparallel- or parallel-stranded.193,194 In contrast, only a mononuclear 50–Cu(II)–50 base pair is formed with Cu(II), which may be a direct consequence of the higher charge of this ion, rendering it more difficult to bring two of these cations into close distance.193 Introduction of a (deprotonatable) thymine residue in the position opposite to 50 should therefore facilitate the formation of a dinuclear base pair bearing two divalent metal ions. In fact, a stabilizing 50– Hg(II)2–T base pair is formed in parallel-stranded DNA (Fig. 7B).195 Its formation appears to be strongly sequence-dependent, as a mononuclear 50–Hg(II)–T pair was observed in a similar antiparallel-stranded duplex.196 Interestingly, Ag(I) ions prefer the formation of a dinuclear 50–Ag(I)2–T base pair.197 A fascinating feature was observed for the incorporation of Ag(I) ions into a 50:C pair. In a regular antiparallel-stranded duplex, the mononuclear 50–Ag(I)–C pair is formed, whereas a dinuclear 50– Ag(I)2–C base pair exists in a parallel-stranded duplex.198 7,8-Dihydro-8-oxo-1,N6-ethenoadenine (51, Table 13) represents an oxidation product of 50 and exists in a lactam/lactim tautomeric equilibrium. Its ability to engage in Ag(I)-mediated base pairing resembles that of 50, as it forms a mononuclear 51–Ag(I)–C pair and a dinuclear 51–Ag(I)2–51 homo base pair in antiparallelstranded DNA.199 Hence, the exocyclic oxygen atom does not provide additional donor functionality to form a trinuclear metalmediated base pair. Nevertheless, the 51–Ag(I)2–51 pair is significantly more stabilizing than the 50–Ag(I)2–50 pair, which was

Fig. 7 Proposed structures of (A) the 50–Ag(I)2–50 base pair193,194; (B) the 50–Hg(II)2–T base pair in parallel-stranded DNA,195 and (C) the 51– Ag(I)2–51 base pair in antiparallel-stranded DNA (hydrogen-bonded water molecules not shown for clarity).199 (R, R’: DNA backbone).

Metal-mediated base pairs in nucleic acid duplexes

683

explained by the involvement of two hydrogen-bonded water molecules in metal-mediated base pair formation (Fig. 7A and B). The 51–Ag(I)2–50 hetero base pair is less stabilizing than any of its analogous homo base pairs.199

2.21.2.2.2

(Functionalized) guanine

Unmodified guanine residues have been investigated with respect to their ability to engage in metal-mediated base pairing, too. As confirmed by single-crystal X-ray diffraction analysis of suitable oligonucleotides, G–Ag(I)–G, G–Ag(I)–C and G–Ag(I)–T base pairs can be formed.164,183 In the homo base pair, Ag(I)-binding occurs via the Hoogsteen edge, whereas the Watson-Crick edge is involved in the latter two pairs. In addition, a gold-mediated G:C pair with formal replacement of the central proton of the canonical base pair, a Hoogsteen-type Cu(II)-mediated G:C pair in a left-handed DNA duplex and even a triplex bearing a C–Ag(I)– G–C triple were also reported.12,153,162 Ag(I)-mediated base pairs involving guanine can be applied in the detection of Ag(I) ions,201 even though the stoichiometric formation of such base pairs in solution was never explicitly proven spectroscopically. Several computational studies exist with respect to Ag(I)-mediated guanine homo base pairs in DNA.147,202,203 A common feature of all these metal-mediated base pairs involving guanine is that they are entirely composed of canonical nucleobases. Deaza derivatives of guanine have been probed in metal-mediated base pairing, too. In analogy to the 44–Ag(I)–T base pair involving 7-deazaadenine (44) as reported above, 52–Ag(I)–C base pairs involving 7-deazaguanine (52, Table 14) can be formed.188 By combining these two Ag(I)-mediated analogs of the canonical Watson-Crick base pairs, duplexes entirely composed of Ag(I)-mediated base pairs can be created. Interestingly, the 52:C pair seems to be able to accommodate more than one Ag(I) ion

Table 14

Metal-mediated base pairs of guanine and inosine derivatives.168,188,192,204

Nucleobase (structure/ number/pKa)

(52) pKa: n.d.

(53) pKa: 3.7204

(54) pKa: n.d.

Sequence

Backbone Isomer Y

5’-XXC XXC-3’ DNA 3’-CCX CCX-5’ 5’-XCY TXC YTX DNA CYT XCY TXC-3’ 3’-CXT YCX TYC XTY CXT YCX-5’ 5’-TAG GTC XAT ACT-3’ 3’-ATC CAG YTA TGA-5’ 5’-TAG GTC XAT ACT-3’ 3’-ATC CAG CTA TGA-5’ 5’-TAG GTX AAT ACT-3’ 3’-ATC CAC TTA TGA-5’ 5’-GCG CXC CGG-3’ 3’-CGC GAG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GCG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GGG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GUG GCC-5’

Metal ions Nuclearity a pH

DTm/
C b

Melting Other UV CD curve MS measurements

b

–

Ag(I) 1–2

8.5–9.0

n.d.

188 188 188

b

44

Ag(I) 1–2

8.5–9.0

n.d.

188 188

DNA

a

a

Ag(I) 1

7.4

5

168

DNA

b

–

Ag(I) 1

7.4

7

DNA

b

–

Ag(I) 2

7.4

n.a.

20 -Ob MethylRNA

–

Cu(II) 1 Zn(II) 1

7.4 7.4

8.5 6.5

20 -Ob MethylRNA

–

Cu(II) 1 Zn(II) 1

7.4 7.4

6.7 5.7

20 -Ob MethylRNA

–

Cu(II) 1 Zn(II) 1

7.4 7.4

5.9 6.9

20 -Ob MethylRNA

–

Cu(II) 1 Zn(II) 1

7.4 7.4

5.2 7.0

-dC

168

168

204 204 204

Conditions: 2 mM duplex, 5 mM phosphate, 100 mM NaClO4188; 2.5 mM duplex, 10 mM Mg(OAc)2, 100 mM NaOAc168; 5 mM duplex, 10 mM Mg(OAc)2, 100 mM NaOAc204; 3 mM duplex, 20 mM cacodylate, 100 mM NaClO4.192 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

684

Metal-mediated base pairs in nucleic acid duplexes

Table 15

Metal-mediated base pairs of various 6-substituted or 2,6-disubstitued purines.205–211

Nucleobase (structure/ number/pKa)

(55) pKa: n.d.

(56) pKa: n.d.

(57) pKa: n.d.

DTm/
C b

Melting UV CD curve

b

– Cu(II) 1

7

23

5’-CTT TCT XTC CCT-3’ 3’-GAA AGA YAG GGA-5’ 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TYA CAA CAT-5’ 5’-CAC ATT AYT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-CTT TCT XTC CCT-3’ 3’-GAA AGA XAG GGA-5’ 3’-AAT ATT AYT ATT TTA-2’ 2’-TTA TAA TXA TAA AAT-3’

DNA

b

77 Cu(II) 1

7

22.1

DNA

b

77 Cu(II) 1

7

14.5

DNA

b

77 Cu(II) 1

7

15.0

DNA

b

– Ni(II) 1 Co(II) 1

7.0

18c 10c

207

57 Ni(II) 1 82 Cu(II) 1

7.0

17.9c 37.1c

208

GNA

5’-CTT TCT YTC CCT-3’ 3’-GAA AGA XAG GGA-5’

DNA

b

77 Ag(I)

1

7.0

16.5c

YTC

DNA

b

78 Ag(I)

1

7.0

11.1c

DNA

b

78 Ag(I)

1

7.0

12.5c

5’-GAG GGA XAG AAA DNA G-3’ 5’-CTC CCT XTC TTT C-3’ 5’-GAG GGA XAG AAA DNA G-3’ 3’-CTC CCT XTC TTT C-5’

b

– Ag(I)

1

5.5

14.5

b

– Ag(I)

1

6.8

2.0

5’-CTT TCT CCT-3’ 3’-GAA AGA GGA-5’ 5’-CTT TCT CCT-3’ 3’-GAA AGA GGA-5’

(60) pKa: n.d.

Nuclearity a pH

5’-CAC ATT AXT GTT DNA GTA-3’ 3’-GTG TAA TXA CAA CAT-5’

(58) pKa: n.d.

(59) pKa: n.d.

Metal Backbone Isomer Y ions

Sequence

Other MS measurements

XAG XTC YAG

211 211 211

DFT

210 210

Conditions: 2.5 mM duplex, 10 mM HEPES, 50 mM NaNO3205–207,209; 2 mM duplex, 10 mM Na phosphate, 100 mM NaNO3208; 3 mM duplex, 5 mM MES, 500 mM NaClO4211; 1 mM duplex, 5 mM MOPS, 150 mM NaClO4.210 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2 equiv. of metal ions.

Table 16

Metal-mediated base pairs of 6-pyrazolylpurine derivatives.191,192,212,213

Nucleobase (structure/number/pKa)

(61) pKa: n.d.

(62) pKa: n.d.

Backbone

Isomer

Y

Metal ions

Nuclearity a

pH

DTm/
C b

UV

CD

Melting curve

5’-GAG GGX XXG AAA G-3’ 3’-CTC CCX XXC TTT C-5’ 5’-GAG GGX XXG AAA G-3’ 3’-CTC CCY YYC TTT C-5’ 5’-GAG GGY YYG AAA G-3’ 3’-CTC CCX XXC TTT C-5’

DNA

b

–

Ag(I)

1

6.8

11.0

212

212

212

DNA

b

63

Ag(I)

1

6.8

16.5

213

DNA

b

5’-GAG GGA XAG AAA G-3’ 5’-CTC CCT YTC TTT C-3’ 5’-GAG GGA YAG AAA G-3’ 5’-CTC CCT XTC TTT C-3’ 5’-GAG GGX XXG AAA G-3’ 3’-CTC CCY YYC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 5’-CTC CCT YTC TTT C-3’

DNA

b

62 63 64 63

Ag(I) Ag(I) Ag(I) Ag(I)

1 1 1 1

6.8 6.8 6.8 5.5

17.3 17.4 22.2 10.0

213 213 213 213

DNA

b

64

Ag(I)

1

5.5

16.7

213

DNA

b

61

Ag(I)

1

6.8

17.3

213

DNA

b

61

Ag(I)

1

5.5

>18

213

5’-GAG GGX XXG AAA G-3’ 3’-CTC CCX XXC TTT C-5’ 5’-GAG GGX XXG AAA G-3’ 3’-CTC CCY YYC TTT C-5’ 5’-GAG GGY YYG AAA G-3’ 3’-CTC CCX XXC TTT C-5’ 5’-GAG GGA YAG AAA G-3’ 5’-CTC CCT XTC TTT C-3’

DNA

b

–

Ag(I)

1

6.8

16.5

DNA

b

61

Ag(I)

1

6.8

17.4

213

DNA

b

DNA

b

61 64 61

Ag(I) Ag(I) Ag(I)

1 1 1

6.8 6.8 5.5

10.5 22.4 10.0

213 213 213

DNA

b

DNA

b

61 63 61 63

Ag(I) Ag(I) Ag(I) Ag(I)

1 1 1 1

6.8 6.8 5.5 5.5

22.2 22.4 16.7 9.0

213 213 213 213

5’-GAG GGX XXG AAA G-3’ 3’-CTC CCY YYC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 5’-CTC CCT YTC TTT C-3’

212

212

MS

Other measurements

212

(64) pKa: n.d. (Continued)

Metal-mediated base pairs in nucleic acid duplexes

(63) pKa: n.d.

Sequence

685

Metal-mediated base pairs of 6-pyrazolylpurine derivatives.191,192,212,213dcont'd

Nucleobase (structure/number/pKa)

(66) pKa: n.d.

Sequence

Backbone

Isomer

Y

Metal ions

Nuclearity a

pH

DTm/
C b

5’-GCG CXC CGG-3’ 3’-CGC GAG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GCG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GGG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GUG GCC-5’

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

1.4

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

3.2

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

3.3

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

6.8

20 -O-Methyl-RNA

b

–

20 -O-Methyl-RNA

b

–

20 -O-Methyl-RNA

b

–

20 -O-Methyl-RNA

b

–

20 -O-Methyl-RNA

b

–

Cu(II) Zn(II) Cu(II) Zn(II) Cu(II) Zn(II) Cu(II) Zn(II) Cu(II)

1 1 1 1 1 1 1 1 1

7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4 7.4

9.5 8.5 13.4 7.6 2.8 2.0 6.5 3.2 18.6

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

18.6

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

22.5

20 -O-Methyl-RNA

b

–

Cu(II)

1

7.4

27.6

5’-GCG CXC CGG-3’ 3’-CGC GAG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GCG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GGG GCC-5’ 5’-GCG CXC CGG-3’ 3’-CGC GUG GCC-5’ 5’-AGC GCX-3’ 3’-XCG CGA-5’ 5’-CGC GCX-3’ 3’-XCG CGC-5’ 5’-GGC GCX-3’ 3’-XCG CGG-5’ 5’-UGC GCX-3’ 3’-XCG CGU-5’

UV

CD

Melting curve

191

191

MS

Other measurements

Metal-mediated base pairs in nucleic acid duplexes

(65) pKa: n.d.

686

Table 16

Conditions: 1 mM duplex, 5 mM MOPS (pH 6.8), 150 mM NaClO4212,213; 1 mM duplex, 5 mM MES (pH 5.5), 150 mm NaClO4, 2.5 mM Mg(ClO4)2213; 3 mM duplex, 20 mM cacodylate, 100 mM NaClO4.191,192 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

Metal-mediated base pairs in nucleic acid duplexes

687

in duplexes containing contiguous pairs of this type. It was proposed that one Ag(I) ion is incorporated to form the Watson-Cricktype 52–Ag(I)–C pair, whereas the second Ag(I) ion then coordinates to donors sites of sequential nucleobases.188 5-Aza-7deazaguanine (53) represents another interesting derivative of guanine. Due to the transposition of N7 to the bridgehead position 5, the sequence of its hydrogen bond donors and acceptors changes, so that it resembles isocytosine rather than guanine. This has a profound effect on the Ag(I)-mediated base pairs formed by 53 (Table 14), as they can now be considered analogs of C–Ag(I)–C pairs. For example, an Ag(I)-mediated base pair of a-53 and a-C can be incorporated into a B-DNA duplex.168 Similarly, stabilizing 53–Ag(I)–C base pairs are formed.168,204 The nuclearity of this base pair is not fully clear. It has been reported to include either one or two Ag(I) ions in very similar sequence contexts (Table 14).168,204 Further studies are required to investigate why particularly 7deazaguanine derivatives appear to be prone to coordinate more than one Ag(I) ion. 2-(Dimethylpyrazolyl)inosine (54) has been introduced to evaluate its potential selectivity in metal-mediated base pairing with respect to the canonical nucleobases,192 similar to 2-(dimethylpyrazolyl)adenine (49) discussed above. As summarized in Table 14, this nucleobase is rather unselective, as its Cu(II)- and Zn(II)-mediated base pairs with the four natural nucleobases all display the same extent of thermal stabilization.

2.21.2.2.3

Other purine derivatives

6-Substituted and 2,6-disubstituted purine derivatives represent the largest group of purine-based nucleobases in metal-mediated base pairing. They are summarized in Tables 15 and 16. In 6-carboxypurine (55), the presence of a carboxylate moiety in principle allows the formation of metal-mediated base pairs via the Watson–Crick edge or via the Hoogsteen edge. DFT calculations on isolated base pairs suggest that in the case of 55–Cu(II)–55 pairs, the Watson–Crick edge is involved in metal-ion complexation (Table 15).205 The disubstituted purine-2,6-dicarboxylate (56) acts as a tridentate ligand, so that a square-planar coordination geometry can be obtained with a monodentate pyridin-3-yl (77) in the complementary position. In fact, the resulting 56– Cu(II)–77 base pairs are highly stabilizing in a variety of DNA duplexes (Table 15).206 The 56:77 mispair can also bind Zn(II) and Ag(I) ions.206 It is interesting to note that the dNTP (deoxyribonucleoside triphosphate) of 56 can be introduced enzymatically opposite a 77 residue in the primer strand in the presence of Cu(II), i.e. this Watson-Crick-type base pair is recognized by polymerases.206 Other donor moieties attached to a purine ring in the 2 and or 6 position are pyridine and bipyridine. 6-Pyridylpurine (57) forms stabilizing metal-mediated homo base pairs with Ni(II) and Co(II), with Ni(II) binding being preferred.207 The resulting 57– Ni(II)–57 was suggested to coordinate the metal ion via the Watson-Crick edge of the purine residue. 57 can also be introduced into oligonucleotides enzymatically, namely opposite another 57 and in the presence of Ag(I), suggesting the relevance of metalmediated base pairing in this process.214 6-Pyridylpurine is one of the few artificial nucleobases that were also introduced into GNA (glycol nucleic acid). In the GNA context, it was shown to form 57–Ni(II)–57 pairs (putting forward essentially the same stabilization to the duplex despite the different backbone, Table 15), but in addition also extremely stabilizing 57–Cu(II)–82 pairs with hydroxypyridone (82).208 2,6-Bis(pyridyl)purine (58) is a tridentate ligand with an N^N^N set of donor atoms. It was shown to form Ag(I)-mediated base pairing with a complementary pyridin-3-yl (77) moiety, giving rise to 58–Ag(I)–77 base pairs.209 Essentially the same arrangement of donor atoms is present in 6-bipyridylpurine (59). However, due to the different orientation of the nitrogen atoms in 59 compared to 58, it forms stable Ag(I)-mediated base pairs with pyridin-4-yl (78) rather than pyridin-3-yl (77).209 In otherwise identical duplexes, the 58–Ag(I)–77 is slightly more stabilizing than the 59–Ag(I)–78 base pair (Table 15). 6-Furylpurine (60) contains a furyl substituent and can therefore potentially act as a bidentate N^O donor ligand. However, the 60– Ag(I)–60 homo base pairs in regular antiparallel-stranded DNA are only poorly stabilized (Table 15), indicating that the furyl moiety may not be involved in metal-ion coordination at all.210 6-Thienylpurine is likewise not a good ligand for metalmediated base pairing (data not shown).210 However, highly stabilizing 60–Ag(I)–60 base pairs are formed in parallel-stranded DNA.211 According to DFT calculations on isolated base pairs, this remarkable difference could be due to the different sizes of the base pairs with metal-binding via the Watson–Crick (antiparallel-stranded duplex) and Hoogsteen (parallel-stranded duplex) edges.211 6-Pyrazolylpurine (61) and its deaza derivatives 1-deaza-6-pyrazolylpurine (62), 7-deaza-6-pyrazolylpurine (63) and 1,7dideaza-6-pyrazolylpurine (64) were investigated to evaluate whether N1 or N7 is preferably involved in metal-mediated base

Fig. 8 Proposed structures of (A) the 64–Ag(I)–61 (X ¼ N) and 64–Ag(I)–63 (X ¼ CH) base pair in antiparallel-stranded DNA213 and (B) the 61– Ag(I)–61 (X ¼ N) and 61–Ag(I)–63 (X ¼ CH) base pair in parallel-stranded DNA.213 (R, R’: DNA backbone).

688

Metal-mediated base pairs in nucleic acid duplexes

Table 17

Metal-mediated base pairs of imidazole and its derivatives.155,217–233

Nucleobase (structure/number/ Sequence pKa)

(67) pKa: 6.01217

(68) pKa: 6.61226

(69) pKa: 6.50226

(70) pKa: 1.95; 5.36227

Backbone Isomer Y

5’-TTA ATT TXX XAA ATT DNA AA-3’ 3’-AAT TAA AXX XTT TAA TT-5’ 5’-GAG GGA YAG AAA G-3’ DNA 3’-CTC CCT XTC TTT C-5’

Metal ions Nuclearity a pH

DTm/
C b

Melting Other UV CD curve MS measurements

b

–

Ag(I) 1

7.2

NMR structure218,219

b

Ag(I) 1

6.8 7.9

233

Ag(I) Ag(I) Ag(I) Ag(I)

6.8 6.8 6.8 6.8

DNA

b

87 (GNA) 71 76 74 –

DNA

b

–

Ag(I) 1

6.8 25

224 224

EIS, SECM

DNA

b

–

Ag(I) 1

6.8 14.5

222 222

ITC

DNA

b

6.8 10

225 225

QM/MM

DNA

b

86 Ag(I) 1 (GNA) 75 Ag(I) 1

6.8 12.6

DNA

b

–

Ag(I) 1

6.8 8

220 220

DNA

b

–

Ag(I) 1

6.8 19

155 155

DNA

b

–

Ag(I) 1

6.8 10

223 223

DNA

b

–

Ag(I) 1

6.8 9.0

226 226

5’-TTT GTT TGT TTG XTT DNA GTT TTT TTT TT-3’ 3’-AAA CAA ACA AAC XAA CAA AAA AAA AA-5’

b

–

Ag(I) 1

6.8 8.0

226 226

5’-GAG GGT XTG AAA G-3’ DNA 3’-CTC CCA XAC TTT C-5’ 5’-CAC ATT AXT GTT DNA GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-GAG GGT XTG AAA G-3’ DNA 3’-CTC CCA YAC TTT C-5’

b b

– – –

Ag(I) 1 Cu(II) 1 Cu(II) 1

6.8 17.4 9.0 19.9 7.0 35.2

227 227 227 227 234

GNA

85

Cu(II) 1

10.0 26

232 232

5’-TTT GTT TGT TTG XTT GTT TTT TTT TT-3’ 3’-AAA CAA ACA AAC XAA CAA AAA AAA AA-5’ R-5’-TTT GXT TGT TTG TXT GTT TTT XTT TT-3’ 3’-AAA CXA ACA AAC AXA CAA AAA XAA AA-5’ 5’-TTT GTT TGT XTG TTT GTX TTT TTT TT-3’ 3’-AAA CAA ACA XAC AAA CAX AAA AAA AA-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT YTC TTT C-5’ 5’-AGA AAG XGA GGG A-3’ 3’-TCT TTC YCT CCC T-5’ 5’-TAC AGG TCC AXT GGG ATC TGA-3’ 3’-ATG TCC AGG TXA CCC TAG ACT-5’ 5’-GTT TGT TTG XXX XXX TGT TTT TT-3’ 3’-CAA ACA AAC CCC CCC ACA AAA AA-5’ F-5’-ACC XGG GGG AGT ATT GCG GAG GAX GGT-3’ 5’-TTT GTT TGT TTG XTT GTT TTT TTT TT-3’ 3’-AAA CAA ACA AAC XAA CAA AAA AAA AA-5’

1 1 1 1

19.9 4.7 6.0 6.0

228 231 231 229 221

228 231 229 221

ITC

230 SMFS

Metal-mediated base pairs in nucleic acid duplexes Table 17

689

Metal-mediated base pairs of imidazole and its derivatives.155,217–233dcont'd

Nucleobase (structure/number/ pKa) Sequence

Backbone Isomer Y

5’-GAG GGA XAG AAA G-3’ DNA 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ DNA 3’-CTC CCT YTC TTT C-5’

Metal ions Nuclearity a pH

DTm/
C b

Melting Other UV CD curve MS measurements

b

–

Ag(I) 1

6.8 17.4

228 228

b

67

Ag(I) 1

6.8 19.9

228 228

(71) pKa: n.d. Conditions: 3 mM duplex, 5 mM MOPS (pH 6.8), 150 mM NaClO4233; 1 mM duplex, 5 mM MOPS (pH 6.8) or 5 mM CHES (pH 9.0) or 5 mM CHES (pH 10.0), 150 mM NaClO4155,220–222,224–232 (R: –(CH2)6–S–S–(CH2)6OH); 1 mM duplex, 5 mM MOPS (pH 6.8), 50 mM NaClO4223 (F: fluorescein); 2 mM duplex, 10 mM HEPES (pH 7.0), 100 mM NaCl.234 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

pairing (Table 16). By introducing their homo and hetero base pairs into the same oligonucleotide duplex, conclusions could be drawn with respect to the most likely coordination pattern. In general, a strong sequence dependence of the thermal stabilization was observed, both in antiparallel- and in parallel-stranded duplexes.212,213 Strongly stabilizing Ag(I)-mediated base pairs were observed when an N1 donor atom is available for coordination, i.e. when one of the nucleobases is either 61 or 63, suggesting the involvement of the Watson–Crick edge in base pair formation.212 This is intriguing, considering the fact that complexation studies with metal nucleobases had suggested a preferred N7-binding.215 It appears as if the overall DNA duplex geometry cannot be neglected when predicting possible coordination patterns. This Ag(I)-mediated base pairs are particularly stabilizing when the nucleobase bearing the endocyclic N1 atom is located in the pyrimidine-rich strand of a duplex composed of one purine- and one pyrimidine-rich strand. Hence, it was proposed that the nucleobase located in the purine-rich strand only contributes its pyrazolyl nitrogen atom to the coordination environment of the Ag(I) ion, even if an additional N1 and/or N7 nitrogen atom is available.213 Such a coordination pattern also explains the large increase in melting temperature upon the formation of 64–Ag(I)–61 and 64– Ag(I)–63 base pairs (Fig. 8A and B, Table 16).213 Artificial nucleobases with appended 3,5-dimethylpyrazolyl (rather than unsubstituted pyrazolyl) moieties were investigated to evaluate their ability to differentiate the canonical nucleobases via metal-mediated base pairing. 6-(3,5-Dimethylpyrazolyl)purine (65) forms moderately stabilizing Cu(II)-mediated base pairs with all natural nucleobases, with 65–Cu(II)–A being the least stabilizing and 65–Cu(II)–U the most stabilizing one in a 20 -O-methyl-RNA duplex environment.192 In the presence of Zn(II) rather than Cu(II), essentially no change in melting temperature is observed with respect to the metal-free duplexes.192 In contrast, when applying 2,6-bis(3,5-dimethylpyrazolyl)purine (66) instead of 65, then strongly stabilizing Cu(II)- and Zn(II)-mediated base pairs are formed with the complementary nucleobases, albeit with a different order of stabilization compared to 65 (Table 16).191 66 was also investigated in the context of possible Pd(II)-mediated base pairing, but no stabilizing effect was observed upon the addition of [PdCl4]2– as Pd(II) precursor.191,216

2.21.2.3

Artificial nucleobases

Imidazole (67) is a prominent artificial nucleobase in metal-mediated base pairing. The imidazole 2’-deoxyribose can be considered the nucleoside analog of histidine. Its ability to engage in metal-mediated base pairing was first investigated using model nucleobases,217 indicating that it is ideally suited to accommodate linearly coordinating metal ions. Nucleic acids bearing 67–Ag(I)–67 base pairs were investigated in numerous contexts (Table 17). For example, they were included in the first experimental structure determination of any B-DNA duplex bearing metal-mediated base pairs.218,219 Single-molecule force spectroscopy was applied to evaluate the mechanical stability of a DNA duplex bearing one 67–Ag(I)–67 pair, indicating that the mechanical stabilization exerted by the formation of this metal-mediated base pair exceeds the respective thermal stabilization.220 The association constant for the specific binding of Ag(I) to a DNA duplex with a 67:67 mismatch is in the order of 106 M–1.221,222 Interestingly, isothermal titration calorimetry also indicates that neighboring 67–Ag(I)–67 pairs are formed cooperatively, with the second associating constant being ca. 25-fold higher than the first one.221 This is an important finding, because it suggests that the formation of contiguous stretches of metal-mediated base pairs is thermodynamically favored. In an effort to develop applications for nucleic acids with 67–Ag(I)–67 base pairs, 67 was introduced into an ATP-binding aptamer. As anticipated, the function of the resulting modified aptamers was found to depend on the presence of Ag(I), particularly when the 67:67 mispairs are located in direct vicinity of the ATP-binding pocket.223 In the context of a different potential application, the charge transfer resistance of surface-deposited DNA films containing 67–Ag(I)–67 pairs was probed by electron impedance spectroscopy and scanning electrochemical microscopy.224 The data suggested that the incorporation of such base pairs into DNA duplexes leads to an increase in charge transfer resistance

690

Metal-mediated base pairs in nucleic acid duplexes

Table 18

Metal-mediated base pairs of triazole and its derivatives.217,229–231,238

Nucleobase (structure/ number/pKa)

(72) pKa: 1.32217

Nuclearity a pH

DNA

b

– Ag(I)

1

6.8

5’-AGA AAG XGA GGG A-3’ DNA 3’-TCT TTC YCT CCC T-5’

b

75 Ag(I)

1

6.8 7.4

5’-AAA AAA AXX XTT TTT TT-3’ 3’-TTT TTT TXX XAA AAA AA-5’

(73) pKa: n.d.




Cb

Melting UV CD curve 238 238

Other MS measurements DLS

5’-AGA AAG 3’-TCT TTC 5’-GAG GGA 3’-CTC CCT

YGA XCT XAG YTC

GGG A-3’ DNA CCC T-5’ AAA G-3’ DNA TTT C-5’

b

67 Ag(I)

1

6.8 6.4

b

67 Ag(I)

1

6.8 6.0

5’-AGA AAG 3’-TCT TTC 5’-GAG GGA 3’-CTC CCT

YGA GGG A-3’ DNA XCT CCC T-5’ XAG AAA G-3’ DNA YTC TTT C-5’

b

67 Ag(I)

1

6.8 12.6

230

b

67 Ag(I)

1

6.8 13.4

230

5’-GAG GGA XAG AAA G-3’ DNA 3’-CTC CCT YTC TTT C-5’

b

67 Ag(I)

1

6.8 4.7

(74) pKa: n.d.

(75) pKa: n.d.

DTm/

Metal Backbone Isomer Y ions

Sequence

229 229

231 231 231

(76) pKa: n.d. Conditions: 1–6.4 mM duplex, 5 mM MOPS (pH 6.8), 150 mM NaClO4238; 1 mM duplex, 5 mM MOPS (pH 6.8), 150 mM NaClO4.229–231 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

rather than a decrease. This increase was explained by a larger thickness of the Ag(I)-containing DNA film, so that no conclusions could be drawn with respect to a potentially modified charge transfer ability of the metal-modified DNA.224 The imidazole-based nucleoside was also investigated in hetero base pairs, e.g. with the canonical nucleobase cytosine. The resulting 67–Ag(I)–C base pairs are more stabilizing than related C–Ag(I)–C pairs (vide supra) and can be chemically reduced to form DNA-templated silver nanoclusters.155 An interesting finding was made during the investigation of DNA duplexes bearing 67–Ag(I)–86 base pairs, i.e. metal-mediated base pairs composed of one small (67) and one large (86, 1H-imidazo[4,5-f][1,10]phenanthroline, vide infra) artificial nucleobase. The thermal stabilizing effect exerted by the formation of this base pair shows an extreme sequence dependence and ranges from 1 to 12
C in highly similar duplexes.225 As was shown by QM/MM calculations of the respective duplex structures, the asymmetry of this base pair is the fundamental reason for the large sequence dependence of its stability.225 The introduction of methyl substituents, i.e. the use of 2-methylimidazole (68) or 4-methylimidazole (69), leads to an increase in basicity of the ligand and to a concomitant stabilization of the resulting 68–Ag(I)–68 and 69–Ag(I)–69 base pairs with respect to the parent system 67– Ag(I)–67 (Table 17).226 NTPs of these (methyl)imidazole-based nucleosides, including 2,4-dimethylimidazole, were also investigated with respect to their ability to be incorporated into oligonucleotides enzymatically.235,236 Some substituted imidazole-derived bidentate ligands were probed as artificial nucleosides, too. 1H-Imidazole-4-carboxylate (70) represents a negatively charged bidentate ligand with N^O donor ability.227,234,237 It engages in highly stabilizing 70–Ag(I)–70 base pairing, but can also bind Cu(II) to form 70–Cu(II)–70 pairs (Table 17).227 The latter base pair was successfully applied in the development of a DNAzyme whose

Metal-mediated base pairs in nucleic acid duplexes Table 19

Metal-mediated base pairs of pyridine and its derivatives.46,50,179,209,239–242

Nucleobase (structure/ number/pKa)

(77) pKa: n.d.

(79) pKa: n.d.

(80) pKa: n.d.

DTm/

Metal Backbone Isomer Y ions

Nuclearity a pH

DNA

b

58 Ag(I)

1

DNA

b

79 Cu(II) 1

7.0

46

DNA

b

– Ag(I)

7.0 3.8 6.8

239

DNA

b

DNA

b

82 Hg(II) 1 (X) Cu(II) (Y) 59 Ag(I) 1

7.0 11.1c

DNA

b

59 Ag(I) 41

7.0 12.5c 12.9c

5’-CAC ATT AYT GTT GTA-3’ DNA 3’-GTG TAA TXA CAA CAT-5’ 5’-CGC GXA TYC GCG-3’ DNA 3’-GCG CYT AXG CGC-5’

b

77 Cu(II) 1

7.0

b

77 Cu(II) 1

7.0

5’-CAC ATT AXT GTT GTA-3’ DNA 3’-GTG TAA TYA CAA CAT-5’

b

77 Cu(II) 1

7.0 15d

5’-CAC ATT AXT GTT GTA-3’ DNA 3’-GTG TAA TYA CAA CAT-5’

b

81 Ag(I) 77

7.0 19.1 11.5

Sequence 5’-CTT TCT XTC CCT-3’ 3’-GAA AGA YAG GGA-5’ 5’-CAC ATT AYT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-TTT TTT TTT TXT TTT TTT TTT-3’ 3’-AAA AAA AAA AXA AAA AAA AAA-5’ 5’-GYX YC-3’ 3’-CYX YG-5’

5’-CTT TCT 3’-GAA AGA 5’-CTT TCT 3’-GAA AGA (78) pKa: n.d.

691

YTC CCT-3’ XAG GGA-5’ XTC CCT-3’ YAG GGA-5’

1

1 1

1 1




Cb

Melting UV CD curve

Other MS measurements

7.0 12.0c

7.0

240 240

EPR46

240

179

46 50

EPR46 X-ray50

(81) pKa: n.d. Conditions: 2.5 mM duplex, 10 mM HEPES, 50 mM NaNO3209; 2 mM duplex, 5 mM NaH2PO4, 50 mM NaClO446; 1.2 mM duplex, 10 mM MOPS, 100 mM NaNO3239; 2.1 mM duplex, 10 mM HEPES, 50 mM NaNO3179,240; 1 mM duplex, 5 mM NaH2PO4, 50 mM NaClO4.241,242 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2 equiv. of metal ions. d Value determined in the presence of 15 equiv. of metal ions.

activity can be triggered externally (here: via the addition or removal of Cu(II)).234 An N^N set of donor atoms in an imidazole derivative is realized by 4-pyridylimidazole (71).228 This artificial nucleobase forms stable 71–Ag(I)–71 homo base pairs, but even more stabilizing 71–Ag(I)–67 hetero base pairs, in which the Ag(I) ions are expected to adopt a [2 þ 1] asymmetric coordination environment.228

692

Fig. 9

Metal-mediated base pairs in nucleic acid duplexes

Proposed structures of (A) the 79–Cu(II)–77 base pair46 and (B) the 82–Cu(II)–82 base pair.243 (R, R’: DNA backbone).

Table 20

Metal-mediated base pairs of 3-hydroxy-2-methylpyridin-4(1H)-one208,240,243–245 and quinolin-8-ol.246,247

Nucleobase (structure/ number/pKa)

(82) pKa: n.d.

(83) pKa: n.d.

Sequence 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-GXX XXX C-3’ 3’-CXX XXX G-5’ 5’-GXY XC-3’ 3’-CXY XG-5’ 5’-XXX X-3’ 3’-XXX X-5’ 3’-AAT ATT AXT ATT TTA-2’ 2’-TTA TAA TYA TAA AAT-3’ 5’-AGT CAG TAX TGA CTG A-3’ 3’-TCA GTC ATX ACT GAC T-5’ 5’-TAC AAC XAA TGT G-3’ 3’-ATG TTG XTT ACA C-5’ 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’

DTm/

Metal Backbone Isomer Y ions

Nuclearity a pH

DNA

b

Cu(II)

1

7.0 13.1

243 243 243

DNA

b

Cu(II)

1

7.0

244 244

244 EPR244

DNA

b

1

7.0

240 240

240

DNA

b

77 Cu(II) (X) Hg(II) (Y) Fe(III)

1

7.0

245 245

245

82 Cu(II) 57

1 1

7.0 33.2c 37.1c

GNA




Cb

Melting UV CD curve

Other MS measurements EPR243

208

DNA

b

Cu(II)

1

n.a. 22.0c

246

DNA

b

Cu(II)

1

n.a. 22.0d

EPR246

DNA

b GNA

Cu(II)

1

7.0 28.9 n.d.

247 247 247

(84) pKa: n.d. Conditions: 2 mM duplex, 10 mM Na phosphate, 50 mM NaCl243; 2–8 mM duplex, 10 mM HEPES, 50 mM NaCl244; 2.1 mM duplex, 10 mM HEPES, 50 mM NaNO3240; 50 mM oligonucleotide, 25 mM MOPS, 50 mM NaCl245; 2 mM duplex, 10 mM Na phosphate, 100 mM NaNO3208; 2.5 mM duplex, 10 mM Na phosphate buffer, 250 mM NaCl246; 2 mM duplex, 10 mM Na phosphate, 50 mM NaClO4.247 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 2 equiv. of metal ions. d Value determined in the presence of 1.1 equiv. of metal ions.

In addition to imidazole, a series of triazole-based nucleoside derivatives were probed in metal-mediated base pairing. The 2’deoxyribonucleoside based on 1,2,4-triazole (72) is less basic than its imidazole analog.217 In agreement with this finding, its metalbinding ability is reduced, too. Nevertheless, it was shown to form 72–Ag(I)–72 base pairs (Table 18).238 Bi- and tridentate 1,2,3triazole derivatives were obtained from an azido sugar and a suitable alkyne via 1,3-dipolar cycloaddition. The bidentate nucleosides based on 4-((1H-pyrazol-1-yl)methyl)-1H-1,2,3-triazole (73), 4-((1H-1,2,4-triazol-1-yl)methyl)-1H-1,2,3-triazole (74) and 2-(1H-1,2,3-triazol-4-yl)pyridine (75) form a variety of Ag(I)-mediated homo and hetero base pairs.229,230 Table 18 presents a brief overview of the most stabilizing Ag(I)-mediated base pairs involving these nucleosides. The Ag(I)-mediated hetero base pair formed

Metal-mediated base pairs in nucleic acid duplexes Table 21

693

Metal-mediated base pairs of the GNA nucleoside analogs 3H-imidazo[4,5-f]quinolin-5-ol, 1H-imidazo[4,5-f][1,10]phenanthroline and dipicolylamine.225,232,233,256–261

Nucleobase (structure/ number/pKa) Sequence

(85) pKa: 2.72(9), 5.2(2), 9.7(2)232

(86) pKa: 1.3(2), 3.6(2), 5.4(2)261

DTm/

Metal Backbone Isomer Y ions

Nuclearity a pH

5’-GAG GGT XTG AAA G-3’ 3’-CTC CCA YAC TTT C-5’

DNA

GNA GNA

85 Cu(II) 70

1

10.0 38 21

232 232 232 232

5’-GAG GGA YAG AAA G-3’ 3’-CTC CCT XTC TTT C-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT YTC TTT C-5’

DNA

GNA

67 Ag(I)

1

6.8 10

225 225

QM/MM225

DNA

6.8 1 225 225 4.3 258 258 258 14.5 258 258 6.8 23 256 256

QM/MM225

DNA

67 Ag(I) X Hg(II) T Ag(I)

1

5’-GAG GGT XTG AAA G-3’ 3’-CTC CCA XAC TTT C-5’

DNA

GNA GNA GNA (R)GNA (S)GNA (R)GNA (S)GNA GNA

DNA

5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT YTC TTT C-5’ 5’-AAA AAA AAA XTA ATT TTX AAT ATT T-3’ 5’-TTT TTT TTT YAT TAA AAT TTA TAA A-3’ 5’-AAA AAA AAA XTA ATT TTX AAT ATT T-3’ 5’-TTT TTT TTT XAT TAA AAX TTA TAA A-3’ 5’-GAG GGA AGA XAA G-3’ 3’-CTC CCT TCT YTT C-5’ 5’-GAG GGA XAG AAA G-3’ 3’-CTC CCT YTC TTT C-5’

1




Cb

Melting UV CD curve

Other MS measurements

16 Cu(I)

23

257 257

23

GNA

C Ag(I) 1 T T Hg(II) 1 C Ag(I)/ Hg(II)

6.8 7 –5 5.5 14 12

DNA

GNA

Zn(II)

1

6.8 9

DNA

GNA

67 Ag(I)

1

6.8 5.7

DNA

GNA

67 Ag(I)

1

6.8 7.9

261 261

Beacon261

260 260

259 259 259

233

(87) pKa: n.d. Conditions: 1 mM duplex, 150 mM NaClO4, 5 mM CHES232; 1 mM duplex, 150 mM NaClO4, 5 mM MOPS225,256,261; 1 mM duplex, 150 mM NaClO4, 2.5 mM Mg(ClO4)2, 5 mM MOPS258; 1 mM duplex, 20 mM NaClO4, 50 mM CH3CN, 5 mM MOPS257; 2 mM duplex, 500 mM NaClO4, 2.5 mM Mg(ClO4)2, 5 mM MES260; 3 mM duplex, 500 mM NaClO4, 2.5 mM Mg(ClO4)2, 5 mM MOPS259; 0.5 mM duplex, 150 mM NaClO4, 5 mM MOPS.233 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated.

between the tridentate 6-(1H-1,2,3-triazol-4-yl)-2,2’-bipyridine (76) and 67 exerts a comparatively small stabilizing effect upon its formation (Table 18).231 Not surprisingly, 76 does not form Ag(I)-mediated homo base pairs, as this would require the incorporation of an octahedrally coordinated metal complex into the DNA back stack. Another relatively simple nitrogen donor ligand investigated in the context of metal-mediated base pairing is pyridine (Table 19). Two isomeric 2’-deoxyribonucleosides of pyridine were evaluated, namely pyridin-3-yl (77) and pyridin-4-yl (78). The former is the first artificial nucleobase reported with a linearly coordinated Ag(I) ion.239 With an appropriately chosen oligonucleotide sequence, even an Ag(I)-mediated base triple within a triple helix can be adopted by 77.239 In addition, 77–Hg(II)–77 base pairs were established.240 In contrast, 77 does not seem to form Cu(II)-mediated base pairs.240 The monodentate 77 can also be applied in metal-mediated base pairs with a four-coordinate central ion, as demonstrated in the 58–Ag(I)–77 base pair containing a tridentate 2,6-bis(pyridyl)purine moiety (vide supra) or the 79–Cu(II)–77 pair in the complementary position.46,209 Because of its differently attached glycosidic bond, pyridin-4-yl (78) is not capable of forming metal-mediated homo base pairs. Nevertheless, it represents a useful artificial nucleobase when located opposite a suitably orientated tridentate ligand, e.g. in 59–Ag(I)–78 with 6-

694

Metal-mediated base pairs in nucleic acid duplexes

Fig. 10

Stereoisomers of the 86–Ag(I)–86 base pair: (A) L-isomer and (B) D-isomer.256 (R, R’: nucleic acid backbone).

bipyridylpurine (vide infra) or in 41–Ag(I)–78 with 4-bipyridylpyrimidinone (vide supra).179,209 Symmetrically 2,6-disubstituted purine derivatives represent attractive ligands for metal-mediated base pairing (Table 19). In fact, the first artificial metalmediated base pair reported inside a DNA duplex was based on such a nucleobase.46 Pyridine-2,6-dicarboxylate (79) is a negatively charged ligand with an O^N^O set of donor atoms. It has a high affinity towards Cu(II) and therefore forms a stabilizing 79– Cu(II)–77 base pair with pyridin-3-yl in the complementary position (Fig. 9A).46 This base pair was also included in the first crystal structure of a DNA duplex containing a metal-mediated base pair (see Section 2.21.2.4).50 Because of the successful generation of the 79–Cu(II)–77 pair, several derivatives of 79 were developed and evaluated with respect to their ability to form metal-mediated base pairs. A formal substitution of the carboxylate moieties by carboxamides gives rise to pyridine-2,6-dicarboxamide (80) with an N^N^N donor set, which forms weakly stabilizing 80–Cu(II)–77 base pairs.241 In contrast, N-methylated N2,N6-dimethylpyridine2,6-dicarboxamide does not engage in metal-mediated base pairing at all.241 The introduction of softer donor atoms was achieved by synthesizing 2,6-bis((methylthio)methyl)pyridine (81), in which the carboxylates of 79 are formally replaced by dimethylsulfane.242 This tridentate S^N^S ligand forms highly stabilizing Ag(I)-mediated 81–Ag(I)–81 homo base pairs with unknown coordination geometry, but likewise engages in the formation of a square planar complex such as 81–Ag(I)–77.242 These tridentate pyridine-derived ligands represent the first family of artificial nucleobases whose metal-binding affinity was fine-tuned via a modification of their substituents. Hydroxypyridone (more precisely, 3-hydroxy-2-methylpyridin-4(1H)-one, 82) is one of the best-investigated ligands in metalmediated base pairing (Table 20).243 After deprotonation of the hydroxyl group, it provides an N^O set of donor atoms. The resulting monoanionic ligand is ideally suited to bind a divalent metal ion in a metal-mediated base pair. The 82–Cu(II)–82 pair (Fig. 9B) was the first metal-mediated base pair to be incorporated into a DNA duplex consecutively. In the resulting arrays of Cu(II) ions, these are ferromagnetically coupled with one another, giving rise to the formation of magnetic chains.244 This coupling can be explained by Cu–O bonds between neighboring 82–Cu(II)–82 pairs, as demonstrated in a computational study.248 The 82– Cu(II)–82 pair is one of the very few examples of metal-mediated base pairs that were incorporated with a different metal-mediated base pair within the same DNA duplex, in this case with 77–Hg(II)–77 (vide supra).240 Short oligonucleotides d(77n) (n ¼ 2, 3, 4) can be applied to generate Fe(III)-mediated triple helices.245 In these helices, the helicity seems to depend on the number of artificial nucleosides, as the tetranuclear helix shows a CD spectrum with essentially opposite signs of its minima and maxima compared to the dinuclear and trinuclear helices. The 82–Cu(II)–82 pair was the first metal-mediated base pair bearing an artificial nucleoside to be investigated with respect to its ability to modulate charge transfer along DNA duplexes. In a sophisticated set of experiments, DNA duplexes with up to three consecutive 82–Cu(II)–82 base pairs were placed between surface-deposited electrically conducting carbon nanotubes and covalently attached to them. A series of single-molecule measurements clearly indicated a higher drain current in the presence of Cu(II) compared with the metal-free 82:82 mispair, suggesting that it may be possible to enhance the electrical conductance of DNA duplexes via the incorporation of metal-mediated base pairs.249 After showing that 82 can be incorporated into oligonucleotides enzymatically,250–252 the formation of 82–Cu(II)–82 was applied in the development of Cu(II)responsive DNAzymes.252–254 Last but not least, 82 was shown to form metal-mediated base pairs not only in DNA, but also in GNA duplexes. In this respect, both the homo base pair 82–Cu(II)–82 and the hetero base pair involving 6-pyridylpurine, 82– Cu(II)–57, were established.208 Attempts to modify hydroxypyridone in a way to increase its affinity for soft metal ions via a formal O/S substitution led to the synthesis of deoxyribonucleosides bearing 3-mercaptopyridone or 3-hydroxypyridine-4-thione nucleobases.255 While the nucleosides form the anticipated Pd(II) and Pt(II) complexes outside a DNA duplex context,255 their incorporation into oligonucleotides was not reported yet. 8-Hydroxyquinoline is another ligand with an N^O set of donor atoms (upon deprotonation). It was introduced as an artificial nucleobase in two isomeric forms (Table 20), i.e. as quinolin-8-ol-7-yl (83)246 and quinolin-8-ol-6-yl (84).247 Both form highly stabilizing Cu(II)-mediated homo base pairs.246,247 While 83–Cu(II)–83 base pairs were investigated with respect to their ability to modulate the charge transfer capability of DNA,246 84–Cu(II)–84 represents the first metal-mediated base pair in GNA.247 In fact, the addition of Cu(II) to a GNA duplex bearing a central 84:84 mismatch is even more stabilizing than its addition to an analogous DNA duplex.247 A few artificial nucleoside analogs were investigated with a GNA backbone only, even though being incorporated into a DNA duplex. 3H-Imidazo[4,5-f]quinolin-5-ol (85) can be considered a merger of the well-established imidazole nucleobase with an 8-hydroxyquinoline moiety (Table 21). In analogy to the 8-hydroxyquinoline-containing nucleosides 83 and 84, 85 forms highly stabilizing Cu(II)-mediated base pairs of the type 85–Cu(II)–85 under alkaline conditions, facilitating the required deprotonation

Metal-mediated base pairs in nucleic acid duplexes Table 22

Metal-mediated base pairs of selected bidentate artificial nucleosides.47,265–268

Nucleobase (structure/ number/pKa)

Sequence

Metal Backbone Isomer Y ions

5’-AGT CGX CGA CT-3’ DNA 3’-TCA GCX GCT GA-5’ 5’-XX-3’ DNA 3’-XX-5’ (88) pKa: n.d.

(89) pKa: n.d.

695

5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-CAC ATT XXT GTT GTA-3’ 3’-GTG TAA XXA CAA CAT-5’ 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’

(90) pKa: n.d. 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’

Nuclearity a pH

DTm/


Cb

b

– Cu(II)

1

7.8 7.2

a

– Ag(I)

1

DNA

b

– Cu(II)

1

9.0 9266

DNA

b

– Cu(II)

1

9.0 32

DNA

b

– Cu(II)

1

9.0 15c,268

DNA

b

– Cu(II)

Melting UV CD curve 47

Other MS measurements

47

265

265

267 267

267 267 267

9.0 12.4c,268

(91) pKa: n.d. Conditions: 1 mM duplex, 400 mM Tris acetate47; 1 mM duplex, 150 mM NaCl, 10 mM CHES267; 3 mM duplex, 150 mM NaCl, 10 mM CHES.268 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c Value determined in the presence of 1.3 equiv. of metal ions.

of the hydroxyl group.232 In combination with 1H-imidazole-4-carboxylate (70), it also forms a Cu(II)-mediated hetero base pair.232 A formal combination of imidazole with 1,10-phenanthroline yields the artificial nucleobase 1H-imidazo[4,5-f][1,10]phenanthroline (86). Its GNA-based nucleoside analog was investigated in numerous contexts (Table 21). It forms Ag(I)- and Cu(I)mediated homo base pairs of the type 86–M(I)–86.256,257 It is interesting to note that 86 prefers to coordinate Cu(I) over Cu(II), as was shown by a series of oxidation and reduction experiments.257 Nevertheless, it binds other divalent metal ions such as Hg(II) and Zn(II), as shown in the formation of 86–Hg(II)–86 and 86–Zn(II)–86 base pairs.258,259 Obviously, Cu(II) preferably coordinated the anionic deprotonated 85 rather than the neutral 86. In case of the 86–M(I)–86 pair, a possible influence of the stereogenic center of the GNA backbone on the geometry of the resulting metal-mediated base pair within the DNA duplex was investigated (Fig. 10A and B). By combining this stereocenter with the helical chirality of the distorted tetrahedral coordination environment around the metal ion, two diastereomers may in principle be formed. Similar to the established preferred intercalation of D-configured octahedral complexes into right-handed B-DNA,262 the experiments showed that the same diastereomer of the base pair is present in the B-DNA helix, irrespective of whether the (S)-GNA or the (R)-GNA building block is introduced.256,257 It can be speculated that the helical metal-based chirality of this diastereomer fits better into the right-handed helix. Such a speculation is in good agreement with the differential stabilization exerted by the formation of the 86–Ag(I)–86 base pair (Table 21).256 The nucleobase 86 can also form metal-mediated hetero base pairs. Among these, the 67–Ag(I)–86 pair, i.e. the metal-mediated base pair composed of one small (67) and one large (86) nucleobase, was already discussed in the context of the imidazole base pairs (vide supra).225 In combination with the canonical nucleobases cytosine and thymine, their different metal-binding preferences can be exploited to incorporate Ag(I) and Hg(II) ions site-selectively into the same DNA duplex despite using only one artificial nucleobase. When 86:C and 86:T mispairs are present within one duplex, the Ag(I) selectively forms 86–Ag(I)–C base pairs, whereas Hg(II) selectively inserts into the 86:T pair to generate a 86–Hg(II)–T base pair.260 Similarly, the reluctance of thymine to coordinate Ag(I) ions was exploited in the development of a molecular beacon capable of discriminating thymine from cytosine. A duplex

696

Metal-mediated base pairs in nucleic acid duplexes

Table 23

Metal-mediated base pairs of salena240,267,268,270–272

Nucleobase (structure/ number/pKa)

(92) pKa: n.d.

Sequence 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’ 5’-CGG AXG ACX AGC G-3’ 3’-GCC TXC TGX TCG C-5’ 5’-GCG CGX XXX XXX XXX GGC CG-3’ 3’-CGC GCX XXX XXX XXX CCG GC-5’ 5’-CGG CCT XXX XTT TTX CGC GC-3’ 3’-GCC GGT XXX XTT TTX GCG CG-5’ 5’-CAC XTT AYT GTX GTA-3’ 3’-TAC XAC AYT AAX GTG5’ 5’-CAC ATT AXT GTT GTA-3’ 3’-GTG TAA TXA CAA CAT-5’

Metal Backbone Isomer Y ions

Nuclearity b pH

DTm/


Cc

Melting UV CD curve

Other MS measurements

DNA

b

– Cu(II) 1 Mn(III)

9.0 42.5d 270 270 268 23.3 268 268

270 EPR271

DNA

b

– Cu(II) 1

9.0 48.7

272

DNA

b

– Cu(II) 1 Mn(III)

9.0

DNA

b

– Cu(II) 1

9.0

240

240

DNA

b

89 Cu(II) 1

9.0

267 267

267

DNA

b

– Cu(II) 1

9.0 31.1 35.9d

272 272

272 272 e

(93) pKa: n.d. Conditions: 3 mM duplex, 150 mM NaCl, 10 mM CHES (with Cu(II)) or HEPES (with Mn(III)), excess ethylenediamine267,268,270,272; 15 mM duplex, 20 mM NaNO3, 10 mM CHES, 1 mM ethylenediamine.240 a For this base pair, X:X represents one salen base pair. b Nuclearity: number of metal ions per designated base pair. c DTm ¼ Tm, metalated – Tm, unmetalated. d With respect to two opposing salicylic aldehyde nucleosides. e Duplex also contains five T–Hg(II)–T base pairs.

containing a 86:C pair is stabilized by the addition of Ag(I) due to the formation of a 86–Ag(I)–C base pair, whereas a 86:T pair is destabilized under the same conditions (Table 21).261 The latter destabilizing effect was proposed to occur by assuming that the bidentate 86 binds Ag(I) and forces the complementary thymine to bulge out of the duplex.261 The sequence of the resulting molecular beacon can be modified to enable the sensing of medicinally relevant single-nucleotide polymorphisms.263 N,N-Dipicolylamine (87) is another GNA-based artificial nucleoside to be investigated in a DNA sequence context (Table 21).233 Being a tridentate ligand, it was placed opposite a monodentate ligand such as imidazole (67), 1,2,4-triazole (72) and tetrazole. Stabilizing Ag(I)-mediated base pairs were formed with imidazole only, giving rise to 87–Ag(I)–67 pairs.233 The sequence dependence of the stabilization observed upon formation of this base pair was found to be negligible. 2,2’-Bipyridine is another bidentate ligand well-established in coordination chemistry. As a result, it was also among the first ligands to be introduced as artificial nucleobases for metal-meditated base pairing. A nucleoside with 2,2’-bipyridine attached directly to the deoxyribose is not flexible enough to form stabilizing metal-mediated base pairs.264 In contrast, 5-methyl-2,2’-bipyridine with a glycosidic bond involving the methylene group (88) engages in stabilizing Cu(II)-mediated 88–Cu(II)–88 base pairs (Table 22).47 Similarly, a dinucleotide based on the a anomer of 88 was shown to form a short duplex solely composed of 88– Ag(I)–88 base pairs.265 The biaryl ligand 2-(1H-pyrazol-1-yl)phenol (89) provides an O^N set of donor atoms upon deprotonation. It was shown to form stabilizing Cu(II)-mediated base pairs of the type 89–Cu(II)–89 at elevated pH values, so that the hydroxyl moiety is deprotonated (Table 22).266,267 The necessity of deprotonation of the phenolic OH group becomes obvious when comparing 2-(1H-pyrazol-1-yl)phenol (89) with 1-(2-methoxyphenyl)-1H-pyrazole, which does not form stabilizing metal-mediated base pairs under otherwise identical experimental conditions.266,267 When a DNA duplex contains 89:89 mispairs and a salen ligand (92, vide infra), both of which are capable of binding Cu(II), then initially 89–Cu(II)–89 pairs are formed, followed by the incorporation of Cu(II) into 92.267 DNA comprising five contiguous 89–Cu(II)–89 was probed as a potential Lewis acidic catalyst in a Diels-Alder reaction of 2-azachalcone and cyclopentadiene.267 Previously, Cu(II) complexes non-covalently

Metal-mediated base pairs in nucleic acid duplexes Table 24

Metal-mediated base pairs with artificial organometallic nucleobases.275–278

Nucleobase (structure/ number/pKa)

(94) pKa: n.d.

(95) pKa: n.d.

697

Sequence

Metal Backbone Isomer Y ions

Nuclearity a pH

DTm/


Cb

Melting UV CD curve

5’-TTT TTT TTT TTT TTT-3’ 5’-AAA AAA AXA AAA AAA-3’ 3’-TTT TTT TTT TTT TTT-5’

DNA

b

– Hg(II) 2

7.4 24.2c

275 275

5’-mCGA GmCX mCTG Gm C-3’ 3’-GCT CGT GAC CG-5’

DNA

b

– Hg(II) 2

7.4 7.3

276 276

5’-mCGA GmCX mCTG Gm C-3’ 3’-GCT CGY GAC CG-5’

DNA

b

G Hg(II) 1 T A C

7.0 25.1 28.5 2.0 21.2

279 279

5’-CGA GCX CTG GC-3’ 3’-GCT CGY GAC CG-5’

DNA

b

G Pd(II) 1 T C

7.4 2 1 –2

5’-CGA GCX CTG GC-3’ 3’-GCT CGY GAC CG-5’

DNA

b

G Pd(II) 1 T A C

7.4 40.6d 45d 39.3d 38.2d

(98) pKa: n.d.

(96) pKa: n.d.

Other MS measurements

19

F NMR melting curves279

277 277 277

278 278

(97) pKa: n.d. Conditions: 1 mM triplex, 100 mM NaClO4, 20 mM cacodylate275; 1 mM duplex, 100 mM NaClO4, 20 mM cacodylate.276,277 a Nuclearity: number of metal ions per designated base pair. b DTm ¼ Tm, metalated – Tm, unmetalated. c With respect to the melting of the metal-free duplex. d Based on “high” Tm in the presence of Pd(II), denaturation curves.

Fig. 11 Proposed structures of (A) the T–94–T trimer (relative polarities of the DNA strands are indicated by (–) and (þ))275 and (B) the 97–G base pair (L, L’: unspecified neutral or anionic ligand).278 (R, R’: DNA backbone).

698

Metal-mediated base pairs in nucleic acid duplexes

(A)

(B)

(C)

(D)

O O

O N N R

O

Hg

N R

N O

N R'

O

O

O

N Hg

N

N O

N R'

N R

O

Sr Hg

O N O

N R'

Fig. 12 Experimentally determined structures of DNA duplexes containing T–Hg(II)–T base pairs. (A) NMR structure, pdb access code 2RT874; (B) crystal structure, pdb access code 4L2473; (C) crystal structure, pdb access code 5WSR115; (D) crystal structure, pdb access code 5GSK115; The crystal structures may contain additional aqua ligands (not depicted). Golden spheres represent Hg(II) ions. The grey spheres in Fig. 12D represent Sr(II) ions. The nucleic acid figures were created using UCSF Chimera.283

bound to DNA had been shown to be catalysts with excellent enantioselectivity.269 When using DNA with covalently attached 89– Cu(II)–89 complexes, some chirality transfer was observed.267 The observation that both pH and counter ion of the copper salt influence the enantioselectivity indicates a complex process. Nevertheless, these experiments indicate that DNA with Cu(II)mediated base pairs may serve as DNA-based asymmetric catalysts. Salicylic aldehyde (90) is a bidentate ligand with an O^O set of donor atoms, provided that the hydroxyl group is being deprotonated. Accordingly, it was shown to form stabilizing 90– Cu(II)–90 base pairs at elevated pH values (Table 22).268 Via a condensation reaction with methylamine, 90 yields the Schiff base 2-((methylimino)methyl)phenol (91). Under otherwise identical experimental conditions, the 91–Cu(II)–91 base pair with two nitrogen and two oxygen donor atoms coordinated to the metal ion is slightly less stabilizing than the 90–Cu(II)–90 with four oxygen donor atoms (Table 22).268 Unprecedented fascinating metal-mediated base pairs are formed from the tetradentate salen ligand (92, Table 23). The salen is generated in situ via condensation of two oppositely located salicylic aldehyde nucleosides (90) with excess ethylene diamine.268 The stabilizing effect of the 92–Cu(II) pair exceeds those of the related 90–Cu(II)–90 and 91–Cu(II)–91 base pairs by far (Table 23), a fact that can likely be attributed to the additional covalent linkage between the complementary oligonucleotide strands.268,270 A characterization of DNA duplexes with neighboring 92–Cu(II) base pairs by EPR spectroscopy indicated an antiferromagnetic coupling of the Cu(II) ions,271 i.e. a coupling different from the ferromagnetic one observed between the Cu(II) ions of neighboring 82–Cu(II)–82 pairs.244 The antiferromagnetic coupling can be accounted for based on the relative orientation of the adjacent Cu(II)-mediated base pairs.248 The formation of 92–Cu(II) base pairs was found to be mechanically stabilizing, too, with the binding of the metal ion evoking a doubling of the experimentally determined rupture force of the DNA duplex.273 The 92– Cu(II) pair can be combined with other metal-mediated base pairs within the same duplex, as exemplified by the simultaneous incorporation of either 92–Cu(II) and T–Hg(II)–T base pairs or 92–Cu(II) and 89–Cu(II)–89 pairs.240,267 In addition to Cu(II) ions, 92 can bind a variety of other metal ions, too, most prominently Mn(III).268,270 In a typical experiment, Mn(II) is added to a DNA duplex containing 92. Upon formation of the complex, it is oxidized, so that a 92–Mn(III) base pair is formed. For both 92–Cu(II) and 92–Mn(III), long contiguous stretches of up to 10 metal-meditated base pairs were incorporated into DNA

Metal-mediated base pairs in nucleic acid duplexes

699

duplexes.272 When the salen ligand is connected to the deoxyribose via a different carbon atom, another salen-based nucleoside results (93, Table 23). The Cu(II)-mediated base pairs formed from 93 are less stabilizing that those obtained from 92, again indicating the relevance of an optimal design of the artificial nucleoside so that it geometrically matches the surrounding canonical Watson–Crick base pairs.270 The orthogonality of the 92–Cu(II) pair was additionally verified by the fact that it can be replicated enzymatically and is even replicated by polymerase chain reaction.274 In addition to the organometallic nucleobases derived from cytosine and uracil (vide supra), a series of artificial organometallic nucleobases were developed, too. Particularly intriguing are the dinuclear mercurated nucleobases 2,6-dimercuriphenol (94) and 1,8-dimercuri-6-phenyl-1H-carbazole (95). The first of these was studied in the context of metal-mediated base triples (Table 24).275 2,6-Dimercuriphenol was shown to form stable dinuclear Hg(II)-mediated base triples with adenine, thymine, and cytosine. In the case of thymine, both melting transitions (arising from the triplex / duplex and from the duplex / single strands transitions) were stabilized in comparison with phenol alone.275 As a result, the first organometallic base triple was established by using 94 (Fig. 11A). In contrast, the different orientation of the two metal ions in 95 allows the formation of the first dinuclear organometallic base pair using this artificial nucleobase. As a result, base pairs such as 95–Hg(II)2–T can be formed.276 A possible field of application for organometallic mercurated nucleobases is the development of oligonucleotide probes with optimized hybridization properties, as these do not lose their respective metal ion even at low oligonucleotide concentration.280 3-Fluoro-2-mercuri-6methylaniline (98) represents a nucleobase developed for such an application. It forms strongly stabilizing base pairs with thymine, guanine and cytosine (Table 24), with the order of duplex stabilization being T > G > C >> A.279 Based on temperature-dependent fluorescence measurements of a molecular beacon containing one residue of 98 in its recognition loop, a clear discrimination of all canonical nucleobases was achieved.281 In the context of oligonucleotide probes, other organometallic nucleobases may be of interest, too. For example, artificial nucleobases bearing a C–Pd(II) bond were evaluated with respect to their ability to differentiate between the canonical nucleobases (Table 24). The C–palladated phenylpyridine (96) was reported to form metal-mediated base pairs with cytosine, thymine and guanine, but not with adenine.277 Unexpectedly, the respective duplexes do not experience any change in their melting temperature upon incorporation of Pd(II) (Table 24). Nevertheless, based on the observation that the ellipticity of the duplexes as well as their absorptivity do not decrease even at 90
C, the authors concluded that the Pd(II)-mediated base pairs are of superior stability and do not even dissociate at high temperature.277 Incorporation of the more flexible C-palladated benzoylaminomethyl moiety (97) as an artificial nucleobase into a DNA duplex results in the formation of highly stabilizing

(A)

(B)

(C)

H2N

NH2 N N R

O

Ag

N O

N R'

Fig. 13 Experimentally determined structures of nucleic duplexes containing C–Ag(I)–C base pairs. (A) NMR structure, DNA, pdb access code 2RVP84; (B) crystal structure, RNA, pdb access code 5AY281; (C) crystal structure, DNA, pdb access code 6NIZ.285 Golden spheres represent Ag(I) ions or atoms. The nucleic acid figures were created using UCSF Chimera.283

700

Metal-mediated base pairs in nucleic acid duplexes

97–Pd(II)–X base pairs, were X represents any of the canonical nucleobases (Fig. 11B, Table 24).278 The resulting duplexes show a biphasic melting behavior, where the authors attribute the lower transition to the melting of the canonical base pairs and the higher melting transition to the dissociation of the Pd(II)-mediated base pair.278 These publications show that the experimental data obtained from organometallic metal-mediated base pairs may not necessarily be as easily interpretable as those derived from Werner-type metal complexes.

2.21.2.4 2.21.2.4.1

Structures of oligonucleotides bearing metal-mediated base pairs Metal-mediated base pairs involving canonical nucleobases

The structures of most metal-mediated base pairs are being proposed based either on chemical intuition (in combination with the experimentally obtained knowledge of the metal-binding stoichiometry of the base pair) or on energy optimization via DFT calculations. A few metal-mediated base pairs were structurally characterized by single-crystal X-ray diffraction analysis of crystals obtained from the respective DNA duplexes. In addition, NMR spectroscopy was applied in some cases to determine the solution structure of DNA with metal-mediated base pairs. This section gives a brief overview of these experimental structures. A more extensive review, including the structures of model complexes, can be found elsewhere.282 Entirely computational structures have been reported, too, but are beyond the scope of this review. Several experimental structures were reported of metal-mediated base pairs involving canonical nucleobases. Fig. 12 shows selected DNA duplex structures reported as yet that contain a T–Hg(II)–T base pair. It becomes immediately clear that several versions exist of this base pair that go beyond the simplified view of one Hg(II) ion symmetrically embedded within a T:T mispair. Fig. 12A and B show the NMR solution and solid-state structures of different B-DNA duplexes, both carrying two central contiguous T–Hg(II)–T base pairs.73,74 These structures show that T–Hg(II)–T pairs can be introduced into a B-DNA duplex without significant distortion of the helix. The Hg$$$Hg distance amounts to 403–417 pm in the ensemble of NMR structures and 330 pm in the crystal structure. Considering a van-der-Waals radius of mercury of 175 pm,284 the latter value would be in agreement with a stabilizing

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Fig. 14 Experimentally determined structures of DNA duplexes containing T–Ag(I)–C base pairs. (A) NMR structure, pdb access code 6FY692; (B) NMR structure, pdb access code 6FY792; (C) crystal structure, pdb access code 5WSQ.115 Golden spheres represent Hg(II) ions. The nucleic acid figures were created using UCSF Chimera.283

Metal-mediated base pairs in nucleic acid duplexes

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Fig. 15 Other experimentally determined nucleic acid duplex structures containing metal-mediated base pairs composed of canonical nucleobases. (A) Crystal structure, RNA, pdb access code 2OIJ12; (B) crystal structure, DNA, pdb access code 6IUE114; (C) crystal structure, DNA, pdb access code 5XJZ164; (D) crystal structure, DNA, pdb access code 5XJZ290; (E) crystal structure, DNA, pdb access code 5XJZ.183 Golden spheres represent Au, Ag(I) or Hg(II) ions. The nucleic acid figures were created using UCSF Chimera.283

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contribution by a metallophilic interaction, diminishing the contribution of an electrostatic repulsion of two closely arranged cations.100 As the electrostatic repulsion is often overestimated in NMR structures,219 it is advisable to consider the Hg$$$Hg derived from the NMR structures an upper limit of the actual distance. In case of the NMR structure, the site of complexation of the Hg(II) ion was confirmed by the observation of a 2J(15N,15N) coupling of 2.4 Hz across the T–Hg(II)–T base pair.74 When individual T–Hg(II)–T pairs were introduced into a DNA duplex, as in the self-complementary sequence 5’d(GGTCGTCC), slightly different geometries of this base pair were observed, depending on the crystallization conditions.115 Fig. 12C shows such a structure. Interestingly, the DNA duplex crystallizes in the A-DNA topology. As can be seen from the chemical structure displayed below, the metal-mediated base pair is not symmetric. Instead, the Hg(II) ion is coordinated by the N3 atom of one thymine residue and by a keto oxygen atom of the complementary thymine moiety. A disorder of Hg(II) was observed in the crystal structure, with some Hg(II) ions being coordinated by N3 and O2 and others by O4 and N3 (only one structure shown). In both cases, the respective Hg(II) sites showed an occupancy of only 20–25%. It is interesting to note that such an asymmetric coordination was proposed in a computational study as a stable intermediate during the formation of the symmetric T–Hg(II)–T base pair.98 Alternatively, it may be speculated that the asymmetric metal-mediated base pairs were formed by a substitution of the protons in the T:T wobble mispair observed in the Hg(II)-free duplex by a Hg(II) ion.115 Using different crystallization conditions, a symmetric T–Hg(II)–T pair was observed in the same duplex. The duplex again adopts the A-type topology, but interestingly the metal-mediated base pair also contains an additional alkaline earth metal ion.115 Essentially the same structure was obtained with either Sr(II) or Ba(II).115 As the former showed a higher occupancy (80% vs. 30%), the duplex with the Sr(II)-containing base pair is depicted in Fig. 12D. The fact that divalent metal ions from the buffer can (at least partially) be incorporated site-specifically into a metal-mediated base pair may help explain why some metal-mediated base pairs are found to be stabilizing only under particular buffer conditions. Next to T–Hg(II)–T, C–Ag(I)–C is one of the well-established metal-mediated base pairs involving canonical nucleobases. It is therefore not surprising that experimentally determined duplex structures containing C–Ag(I)–C base pairs were reported, too. Fig. 13A shows the NMR solution structure of a DNA duplex bearing one such Ag(I)-mediated base pair.84 The N3–Ag(I)–N3 coordination was confirmed by the observation of 1J(15N,109Ag) couplings of the N3 atoms (83–84 Hz) and by the appearance of the resonance of the exocyclic amino group 15NH2 as a triplet, ruling out any involvement of the N4 atom in Ag(I) coordination.84 In the C–Ag(I)–C base pair, the cytosine moieties are oriented at an angle of –18  3 degree, probably to avoid any steric clash of the opposing amino groups in the cisoid orientation of the nucleobases.84 A propeller twist of ca. –28
was observed in the crystal structure of an RNA duplex containing C–Ag(I)–C pairs (Fig. 13B). Considering the fact that even the canonical base pairs adopt a propeller twist of ca.  12 degree in A-RNA duplexes, it is interesting to see that this metal-mediated base pair seems to exceed the “normal” angle by about the same amount in DNA and RNA duplexes.81 The crystal structure nicely demonstrates that metal-mediated base pairs can assume the same structure in A-type and B-type duplexes. The lower half of the DNA structure depicted in Fig. 13C shows an interesting arrangement of three adjacent C–Ag(I)–C base pairs. They are part of a fluorescent DNA-templated Ag8 nanocluster,285 likely to be composed of Ag(I) ions and Ag atoms.286 Its geometry was described as being reminiscent of the Big Dipper,285 containing a trapezoidal Ag5 cluster connected to the three linearly arranged Ag(I) ions of the contiguous C–Ag(I)–C base pairs. The Ag$$$Ag distance between the metal-mediated base pairs amounts to ca. 310 pm and is therefore shorter than the sum of their van-der-Waals radii,287 which could be indicative of stabilizing argentophilic interactions.288 In contrast to the C–Ag(I)–C pairs discussed before, the ones in the silver nanocluster adopt a (heavily tilted) transoid orientation of the glycosidic bonds. This arrangement avoids any steric clash of the exocyclic amino groups of the cytosine rings. Regular antiparallel DNA or RNA duplexes cannot incorporate such C–Ag(I)–C pairs with transoid glycosidic bonds, because they are forced into a cisoid orientation by the presence of neighboring canonical base pairs.289 The transoid orientation can be adopted in the nanocluster structure because of the unusual parallel-stranded alignment of the oligonucleotide strands templating that cluster. The fact that DNA-templated silver nanoclusters can comprise Ag(I)-mediated base pairs suggests that is may be possible to synthesize these fascinating metal-bioconjugates from duplexes bearing suitable Ag(I)-containing base pairs.155 Thymine and cytosine can also form a Hg(II)-mediated hetero base pair, i.e. T–Hg(II)–C. Fig. 14 summarizes the experimental structures of DNA duplexes bearing such base pairs. According to NMR studies, the self-complementary oligonucleotide sequence 5’-d(CGTCTCATGATACG) adopts a duplex structure in solution, in which the underlined nucleobases form a T–Hg(II)–C base pair in the presence of Hg(II) ions.92 More precisely, a dynamic equilibrium is observed between a B-type duplex in which both pyrimidine residues coordinate the Hg(II) via their endocyclic N3 atoms (Fig. 14A) and a duplex with increased A-type character in which the cytosine binds the Hg(II) ion via its singly deprotonated amino group whereas the thymine coordination remains unchanged (Fig. 14B). The first of these structures represents the major species, accounting to ca. 75% of all duplexes. Interconversion rates were determined to be k1 ¼ (4.3  0.6) s 1 and k 1 ¼ (8.8  0.9) s 1.92 The respective binding sites were verified based on 15N,199Hg coupling constants. Interestingly, the 1J(15N,199Hg) coupling between Hg and thymine N3 is approximately 10-fold larger than that between Hg and cytosine N3 (1095 Hz vs. 114 Hz), indicating a stronger bonding interaction with thymine.92 In contrast, the respective coupling constants observed for the minor species are highly similar (1063 Hz for the Hg/N4 coupling of cytosine), which can likely be in part attributed to the fact that both nucleobases are anionic in this case.92 The authors suggest that the fact that the predominant species is the one with lower N–Hg bonding energy is likely due to the structural perturbation required to form the minor species with the higher N–Hg bonding energy.92 Noteworthy, the T–Hg(II)–C coordination pattern observed in the minor solution species was also observed in a separate crystal structure (Fig. 14C).115 Just as in the solution case, that duplex adopts an overall A-type geometry. The crystal structure also indicates the presence of a stabilizing hydrogen-bonded water molecule. Relatively short distances between the Hg(II) ion and the exocyclic keto groups of two flanking guanine residues of ca. 290 pm

Metal-mediated base pairs in nucleic acid duplexes

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may additionally contribute to the overall stability of the metal-mediated base pair. Interestingly, the occupancy of the Hg site amounts to only 80% in the crystal structure, which could be indicative of a dynamic interaction similar to the one observed in solution, albeit with a different state of equilibrium.115 Fig. 15 shows all experimental structures of nucleic acid duplexes bearing metal-mediated base pairs of the canonical nucleobases not discussed yet. It confirms the structural variability of nucleic acids and their ability to form non-canonical structures in the presence of suitable transition metal ions. The sole structure of a nucleic acid with a gold-mediated base pair is presented in Fig. 15A. It was obtained in a crystallographic study on the binding of different metal ions to RNA duplexes.12 Soaking of a crystal of an RNA duplex in a AuCl3 solution gave rise to the incorporation of the gold cation into a G:C base pair, formally replacing a proton involved in the hydrogen bonds. Unfortunately, the occupancy of the gold ion was only ca. 40%, so that the observed base pair geometry likely is a superposition of the regular G:C pair and the gold-mediated one.12 It therefore remains unclear if

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Fig. 16 Experimentally determined DNA duplex structures containing Ag(I)-mediated base pairs of artificial nucleobases. (A) NMR structure, pdb access code 2KE8218; (B) NMR structure, pdb access code 2M54219; (C) crystal structure, pdb access code 5XUV.135 Golden spheres represent Ag(I) ions, grey spheres K(I) ions. The nucleic acid figures were created using UCSF Chimera.283

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Metal-mediated base pairs in nucleic acid duplexes

the gold cation remained in its þIII oxidation state, requiring a square-planar coordination environment, or whether it was reduced to the linearly coordinating Au(I) ion. The duplex shown in Fig. 15B here is that of the pentanucleotide d(TTTGC) in the presence of Hg(II) ions.114 The crystal structure is composed of a continuous arrangement of this double helix. Each duplex contains T–Hg(II)–T and G–Hg(II)–T base pairs. Some neighboring duplexes are interconnected by water-mediated C:C mispairs involving the terminal cytosine residues. With respect to the T–Hg(II)–T pairs, it is interesting to note that the Hg(II) ions do not bind two thymine moieties located directly opposite one another. Instead, the T–Hg(II)–T base pairs involve the thymine residue on one strand and the thymine residue next to the one located directly opposite the first one. The authors describe this unusual arrangement as “two pairs of consecutive T residues in two facing strands” which “are staggered and each strand kinks at two consecutive T residues to form two T–Hg(II)–T base pairs on the same plane.”114 The G–Hg(II)–T pair in this duplex is unique and was not (yet) observed in other contexts. The nucleobases are heavily tilted, allowing an additional weak coordination of the Hg(II) ion by the keto group of a neighboring thymine. The Hg$$$Hg distances in this duplex range from 380 to 460 pm, so that none of the Hg(II) ions are within a distance from one another that is shorter than twice the van-der-Waals radius, ruling out the presence of metallophilic interactions in this arrangement. This nucleic acid structure is interesting, because it shows the ability of oligonucleotides to arrange metal ions in wire-like arrays that are structurally distinct from the canonical duplex structures. Such arrays could be of relevance for the study of the electron transfer properties of metal-modified DNA.291 In this particular case, CD-spectroscopic and mass spectrometric studies suggest the presence of a double-stranded tetranuclear species in solution, too.114 Fig. 15C presents the solid state structure of the oligonucleotide 5’-d(GCACGCGC) in the presence of Ag(I) ions. Rather than adopting a duplex structure composed of canonical Watson-Crick base pairs and metal-mediated C–Ag(I)–A pairs (vide supra), a non-helical topology is adopted, containing one C–Ag(I)–C and one G–Ag(I)–G base pair.164 Both metal-mediated base pairs display a transoid alignment of the glycosidic bonds, which appears to be the favored orientation of the metal complexes but is disfavored in B-type duplex structures. The guanine residues are coordinating to the Ag(I) ion via their Hoogsteen edge, i.e. involving their N7 atom in metal ion coordination. Both Ag(I)-mediated base pairs show a large propeller twist (128–132
)

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Fig. 17 Experimentally determined nucleic acid duplex structures containing Cu(II)-mediated base pairs of artificial nucleobases. (A) Crystal structure, DNA, pdb access code 1JES50; (B) crystal structure, DNA, pdb access code 2XY5274; (C) crystal structure, GNA, pdb access code 2JJA.293 Golden spheres represent Cu(II) ions. The nucleic acid figures were created using UCSF Chimera.283

Metal-mediated base pairs in nucleic acid duplexes

705

and a non-linear N–Ag–N angle (164–166
). The large structural deviations from an ideal planar base pair enable additional hydrogen-bonding contacts to neighboring nucleobases.164 A third Ag(I) ion is displayed faintly at the bottom right-hand side of Figure 15c. This metal ion is involved in another C–Ag(I)–C base pair, connecting neighboring DNA structures.164 The Ag$$$Ag distances in this structure amount to 303 and 317 pm and may therefore contribute to its stability by means of argentophilic interactions.288 A CD spectroscopic analysis of this oligonucleotide in the presence of Ag(I) shows a significant negative peak at ca. 275 nm, which might be due to the unusual base pairing patterns adopted by this sequence.164 The non-canonical DNA duplex shown in Fig. 15D is formed by a self-complementary DNA oligonucleotide containing one central C:C mismatch composed of 5-methylcytosine residues with a 2’-O,4’-C-methylene-bridged deoxyribose moiety (“locked nucleic acid”, LNA).290 This oligonucleotide was expected to adopt a duplex structure of canonical Watson-Crick base pairs and one central C–Ag(I)–C pair. Instead, an unprecedented structure was formed containing unique A–Ag(I)–A pairs with Ag(I) coordination via the Watson-Crick edge of adenine and G–Ag(I)–G pairs with Ag(I) coordination via the Hoogsteen edge. Even though the base pairs are strongly tilted, the orientation of the glycosidic bonds can best be described as transoid. A close inspection of the guanine residues involved in metal-mediated base pair formation shows that they both remain hydrogen-bonded to cytosine moieties. In this structure, the interatomic Ag$$$Ag distance of 350 pm is too large to account for argentophilic interactions.288 Interestingly, the CD spectrum of this oligonucleotide in the presence of excess Ag(I) ions displays a strongly negative peak at 280–290 nm (depending on the buffer),290 similar to the one of the structure shown in Fig. 15C. Other reports had attributed such a negative signal to the formation of Ag(I)-mediated base pairs in non-canonical structures, too.147,292 The most spectacular structure of any nucleic acid containing metal-mediated base pairs is shown in Fig. 15E. Just as the three previously discussed structures, the oligonucleotide sequence of this structure had been devised to stabilize one particular Ag(I)mediated base pair. However, rather than forming a B-DNA duplex with two C–Ag(I)–C and surrounding Watson-Crick base pairs, a duplex composed of one G–Ag(I)–C, two C–Ag(I)–C, one T–Ag(I)–T and one G–Ag(I)–G base pair was adopted.183 In addition, adjacent duplexes are interconnected via a G–Ag(I)–G base pair involving dangling guanine residues. All base pairs in this double helix display a cisoid orientation of their glycosidic bonds. The guanine residues in the G–Ag(I)–G pairs coordinate the Ag(I) ion via their Hoogsteen edge. As a result of the connection of neighboring duplexes, a continuous array of Ag(I) is formed in the crystal structure, spanning the entire crystal.183 The only nucleobases not involved in metal-mediated base pair formation are the adenine residues, which are found to be extruded from the duplex. A closer look at the G–Ag(I)–C pair shows that the N–Ag–N angle is nonlinear, allowing one of the three hydrogen bonds originally present in a G:C pair to remain in the metal-mediated pair. In analogy to some of the non-canonical structures discussed above, a strong propeller twist of the base pairs enables the formation of (potentially stabilizing) hydrogen bonds between adjacent nucleobases. The Ag$$$Ag distance between neighboring Ag(I)-mediated base pairs varies between 320–340 pm and would therefore be in agreement with rather weak argentophilic interactions.288 The results of NMR studies of this oligonucleotide sequence in the presence of excess Ag(I), such as the observation of broad signals and decreased diffusion coefficients, are in agreement with the formation of oligomeric metalated species in solution, too.183

2.21.2.4.2

Nucleic acids involving artificial nucleosides

Fig. 16 summarizes experimental structures reported for DNA duplexes bearing Ag(I)-mediated base pairs of artificial nucleobases. A duplex comprising three consecutive 67–Ag(I)–67 base pair involving imidazole (67) is depicted in Fig. 16A and B. Both structures were obtained from the same set of experimental NMR-derived interatomic distance constraints.218,219 The duplex shown in Fig. 16A is a traditional NMR structure,218 whereas additional computationally derived Ag$$$Ag distance information was included during the structure determination of the double helix displayed in Fig. 16B.219 The site of metalation was confirmed by means of a 1J(15N,107/109Ag) coupling constant of 86 Hz involving the nitrogen atom of the imidazole moiety.218 As had already been stated in the discussion of the NMR structure of a duplex bearing T–Hg(II)–T base pairs (Fig. 12A, vide supra), the electrostatic repulsion of the positively charged metal ions is typically overestimated in traditional NMR structures.219 For that very reason, the Ag$$$Ag distances between neighboring base pairs are quite large in the duplex structure given in Fig. 16A, amounting to 379– 451 pm.218 When this traditional NMR structure was used as a starting point for the computational structure determination by means of QM/MM calculations, much shorter Ag$$$Ag distances were obtained, because the quantum mechanical treatment also takes into account the attractive dispersive interactions.219 Addition of these computationally derived distance constraints into the re-refinement of the original NMR structure yielded the duplex shown in Fig. 16B.219 Here the interatomic Ag$$$Ag distances amount to (345  2) pm, placing them at the upper limit of a possible argentophilic interaction.219 These structures of a DNA duplex with three contiguous 67–Ag(I)–67 base pairs were the first experimental proof that metal-mediated base pairs can be incorporated into a B-DNA double helix without a major distortion of its structure. Fig. 16C depicts a duplex with two neighboring 16–Ag(I)2–16 base pairs, involving the pyrimidine derivative 4-thiothymine (16).135 It is the only structure yet of a double helix comprising dinuclear metal-mediated base pairs. In fact, the 16–Ag(I)2–16 pairs can actually be considered trinuclear in this case, because a potassium ion from the crystallization buffer is also included sitespecifically. Titration studies typically performed to elucidate the stoichiometry of metal-mediated base pairs do not give any information about the possible involvement of alkali or alkaline earth metal ions from the buffer in base pair formation, so this structure (and the one depicted in Fig. 12D) presents important information on the relevance of the buffer conditions. In this duplex, four Ag(I) ions are located at close distance from one another. The Ag$$$Ag distance within a base pair amounts to 280 pm, those between neighboring base pairs are in the range of 300–320 pm. All these values are below the sum of the van-der-Waals radii of two silver atoms, so that a significant contribution of argentophilic interactions to the stabilization of this structure can be assumed. In a different dinuclear Ag(I)-mediated base pair containing 1,3-dideazaadenine (43–Ag(I)2–T), where the Ag$$$Ag

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distance had been calculated to be 288 pm, the argentophilic interaction was computed to contribute ca. 16 kcal mol–1 to the base pair stability.186 Hence, a similar contribution can be assumed for each 16–Ag(I)2–16 pair. Nucleic acid duplexes containing Cu(II)-mediated base pairs with artificial nucleobases are depicted in Fig. 17. The 79–Cu(II)– 77 base pair containing pyridin-2,6-dicarboxylate (79) and pyridin-3-yl (77) shown in Fig. 17A was the first metal-mediated base pair to be structurally characterized within an oligonucleotide duplex.50 The structure is special in many ways. It is the only one with an asymmetric metal-mediated base pair composed of two different artificial nucleosides. Moreover, it is the only nucleic acid structure containing a metal-mediated base pair adopting a Z-DNA topology. It is tempting to speculate that the coordination preferences of the Cu(II) contribute to this topology. Cu(II) ions prefer a tetragonally distorted octahedral coordination environment with elongated metal–ligand bonds along one axis. Hence, two distant axial ligands are required in addition to the squareplanar environment provided by 79 and 77. In the case of the structure shown in Fig. 17A, the keto group of a neighboring guanine residue and the O4’ oxygen atom of an adjacent 2’-deoxyribose represent these additional axial donor atoms, with Cu–O distances of 310 pm each.50 As the Z-DNA topology is the only one in which deoxyriboses and nucleobases can be located in the center of the helix, the presence of the metal-mediated base pair appears to force the duplex to adopt this topology. Noteworthy, the CD spectrum of this duplex can be considered at the midpoint between the spectra of B-DNA and Z-DNA.50 It is therefore likely that a conformational equilibrium exists and that the Z-DNA duplex crystallized preferentially. The salen–Cu(II) base pair (92–Cu(II)) comprising a tetradentate ligand and a covalent linkage between the complementary strands was also crystallized within a DNA double-helical context.274 The structure of the resulting duplex is presented in Fig. 17B. The duplex adopts a slightly distorted B-DNA topology. The minor distortion is not necessarily due to the presence of the metal-mediated base pair. It rather seems to be an artefact caused by the fact that this duplex structure was obtained by cocrystallizing the metal-containing DNA with a polymerase.274 In this structure, no explicit axial ligands are detected, to that the coordination environment of the Cu(II) ion can be considered square-planar. Interestingly, the structure of the metal-free duplex bearing opposing salicylic aldehyde nucleosides (90:90 mispair) was obtained as well.274 This mispair induces only very minor distortion with respect to a canonical base pair, as does the 92–Cu(II) pair. Finally, Fig. 17C shows the structure of a GNA duplex bearing a 82–Cu(II)–82 base pair with hydroxypyridone (82) as the artificial nucleobase.293 As a result of the acyclic three-carbon backbone, the duplex topology is quite distinct from that of DNA or RNA duplexes. It nevertheless is able to incorporate metal-mediated base pairs. The hydroxypyridone residues are only slightly twisted in the base pair with a propeller twist of ca. 15
.208 Hence, the Cu(II) ion is embedded in an almost ideal square-planar arrangement. No axial ligands are present, which contrasts the results of a computational study of the 82–Cu(II)–82 pair in a DNA duplex.248 Therefore, the lack of axial ligands is likely due to the GNA duplex topology rather than representing an intrinsic property of the 82–Cu(II)–82 base pair. Even though the C1’$$$C1’ distance in the 82–Cu(II)–82 GNA base pair is significantly larger by ca. 200 pm compared to those of the neighboring canonical base pairs, no major duplex distortion is evoked by the presence of the metal-mediated base pair.208 This finding can be explained by a different conformation around the glycolic C–O bond in 82– Cu(II)–82.

2.21.3

Summary and outlook

This article presents a compilation of all metal-mediated base pairs in DNA, RNA, and GNA reported until early 2021. It offers for the first time a comprehensive overview of the spectroscopic techniques applied to characterize each one of these base pairs and summarizes the respective experimental conditions under which their existence was proven. While many metal-mediated base pairs are composed of canonical nucleobases (or close derivatives thereof), others comprise nucleoside analogs with entirely artificial nucleobase surrogates. As an inspection of the tables included in this article shows, the formation of a metal-mediated base pair is typically deduced by an increase in the melting temperature of the respective nucleic acid duplex upon the addition of the transition metal ion. However, it is important to note that some metal-mediated base pairs are not accompanied by any significant thermal duplex stabilization, e.g. when the metal-free mispair is already stabilized by significant p stacking interactions or when the formation of a technically stabilizing metal-mediated base pair is accompanied by a destabilizing distortion of the duplex. Therefore, additional spectroscopic and spectrometric methods are becoming increasingly important in the characterization of metal-mediated base pairs. As can be seen from the various tables, frequently used techniques used towards this end are titrations of the oligonucleotides followed by UV or CD spectroscopy. In addition, other methodologies such as mass spectrometry, fluorescence spectroscopy, homo- and heteronuclear NMR spectroscopy, EPR spectroscopy, isothermal titration calorimetry, dynamic light scattering, single-molecule force spectroscopy, atomic force spectroscopy, and even sophisticated computational methods involving molecular dynamics, molecular mechanics and/or density functional theory were applied. Obviously, the determination of a crystal structure or an NMR solution structure of a nucleic acid bearing one or more metal-mediated base pairs represents an optimal method for its characterization, even though only a few metal-mediated base pairs could be characterized this way. The data compiled in this article shall aid scientists in the comparison of their own results with those previously reported. Particularly the experimental conditions may play a major role in understanding seemingly different results when investigating essentially identical metal-mediated base pairs (cf. the DTm values reported for the T–Hg(II)–T base pair, ranging from 4.1 to 19.4
C, Table 1). Moreover, as can be seen from the crystal structures of the T–Hg(II)–T base pair shown in Fig. 12D and the 16–Ag(I)2–16 pair depicted in Fig. 16C, alkali or alkaline earth metal ions can be present site-specifically in metal-mediated base pairs. Such a binding event is likely to contribute to the stabilizing effect exerted by the formation of a metal-mediated base pair, but it is not detectable by

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standard spectroscopic techniques. It may therefore very well be possible to modulate the stabilizing effect of the formation of a metal-mediated base pair by an appropriate choice of the buffer conditions. Similarly, the proper choice of the pH can turn out to be important for the formation of a metal-mediated base pair. While this statement may be obvious for ligands that require a deprotonation event prior to the complexation (e.g. the phenolic OH groups of the Cu(II)-binding artificial nucleobases), it is less trivial for ligands in which the desired metalation may compete with ligand protonation. It is therefore advisable to study the relevant pKa values of artificial nucleosides in order to determine the optimal pH range for the formation of the metal-mediated base pair. While initially, the development of new artificial nucleosides for metal-mediated base pairing was a prominent research focus, more recently the creation of sophisticated applications of the metal-modified nucleic acids is becoming more important. This becomes evident from the increasing number of publications reporting the use of metal-mediated base pairs in the contexts of switching or triggering the activity of metal-containing nucleic acids, of oligonucleotides developed for the recognition of other nucleic acids, and of nucleic acids with unusual properties derived from their metal content. Nevertheless, the design of new artificial nucleobases with tailored metal-binding properties remains an important field of research, in particular when it comes to an expansion of the variety of metal ions that can be incorporated into metal-mediated base pairs. May the compilations presented in this review article help scientists in their future endeavors in the field of metal-mediated base pairing!

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Cooperative Formation of Silver(I)-Mediated Base Pairs. Chem. Commun. 2012, 48, 11844–11846. 222. Schweizer, K.; Léon, J. C.; Ravoo, B. J.; Müller, J. Thermodynamics of the Formation of Ag(I)-Mediated Azole Base Pairs in DNA Duplexes. J. Inorg. Biochem. 2016, 160, 256–263. 223. Heddinga, M. H.; Müller, J. Incorporation of a Metal-Mediated Base Pair Into an ATP AptamerdUsing Silver(I) Ions to Modulate Aptamer Function. Beilstein J. Org. Chem. 2020, 16, 2870–2879. 224. Léon, J. C.; She, Z.; Kamal, A.; Shamsi, M. H.; Müller, J.; Kraatz, H.-B. DNA Films Containing the Artificial Nucleobase Imidazole Mediate Charge Transfer in a Silver(I)Responsive Way. Angew. Chem. Int. Ed. 2017, 56, 6098–6102. 225. Scharf, P.; Jash, B.; Kuriappan, J. A.; Waller, M. P.; Müller, J. Sequence-Dependent Duplex Stabilization Upon Formation of a Metal-Mediated Base Pair. Chem. Eur. J. 2016, 22, 295–301. 226. Hensel, S.; Megger, N.; Schweizer, K.; Müller, J. Second Generation Silver(I)-Mediated Imidazole Base Pairs. Beilstein J. Org. Chem. 2014, 10, 2139–2144. 227. Sandmann, N.; Defayay, D.; Hepp, A.; Müller, J. Metal-Mediated Base Pairing in DNA Involving the Artificial Nucleobase Imidazole-4-Carboxylate. J. Inorg. Biochem. 2019, 191, 85–93. 228. Schweizer, K.; Kösters, J.; Müller, J. 4-(20 -Pyridyl)imidazole as an Artificial Nucleobase in Highly Stabilizing Ag(I)-Mediated Base Pairs. J. Biol. Inorg. Chem. 2015, 20, 895–903. 229. Richters, T.; Müller, J. A Metal-Mediated Base Pair With a [2þ1] Coordination Environment. Eur. J. Inorg. Chem. 2014, 437–441. 230. Richters, T.; Krug, O.; Kösters, J.; Hepp, A.; Müller, J. A Family of “click” Nucleosides for Metal-Mediated Base Pairing: Unravelling the Principles of Highly Stabilising MetalMediated Base Pairs. Chem. Eur. J. 2014, 20, 7811–7818. 231. Litau, S.; Müller, J. A tridentate “click” nucleoside for metal-mediated base pairing. J. Inorg. Biochem. 2015, 148, 116–120. 232. Sandmann, N.; Bachmann, J.; Hepp, A.; Doltsinis, N. L.; Müller, J. Copper(II)-Mediated Base Pairing Involving the Artificial Nucleobase 3H-imidazo[4,5-f]quinolin-5-ol. Dalton Trans. 2019, 48, 10505–10515. 233. Seubert, K.; Fonseca Guerra, C.; Bickelhaupt, F. M.; Müller, J. Chimeric GNA/DNA Metal-Mediated Base Pairs. Chem. Commun. 2011, 47, 11041–11043. 234. Takezawa, Y.; Hu, L.; Nakama, T.; Shionoya, M. Sharp Switching of DNAzyme Activity Through the Formation of a CuII-Mediated Carboxyimidazole Base Pair. Angew. Chem. Int. Ed. 2020, 59, 21488–21492. 235. Röthlisberger, P.; Levi-Acobas, F.; Sarac, I.; Marlière, P.; Herdewijn, P.; Hollenstein, M. On the Enzymatic Incorporation of an Imidazole Nucleotide Into DNA. Org. Biomol. Chem. 2017, 15, 4449–4455. 236. Flamme, M.; Levi-Acobas, F.; Hensel, S.; Naskar, S.; Röthlisberger, P.; Sarac, I.; Gasser, G.; Müller, J.; Hollenstein, M. Effect of Metal Shielding on the Enzymatic Construction of Artificial Base Pairs. ChemBioChem 2020, 21, 3398–3409. 237. Röthlisberger, P.; Levi-Acobas, F.; Sarac, I.; Marlière, P.; Herdewijn, P.; Hollenstein, M. Towards the Enzymatic Formation of Artificial Metal Base Pairs With a CarboxyImidazole-Modified Nucleotide. J. Inorg. Biochem. 2019, 191, 154–163. 238. Böhme, D.; Düpre, N.; Megger, D. A.; Müller, J. Conformational Change Induced by Metal-Ion-Binding to DNA Containing the Artificial 1,2,4-Triazole Nucleoside. Inorg. Chem. 2007, 46, 10114–10119. 239. Tanaka, K.; Yamada, Y.; Shionoya, M. Formation of Silver(I)-Mediated DNA Duplex and Triplex through an Alternative Base Pair of Pyridine Nucleobases. J. Am. Chem. Soc. 2002, 124, 8802–8803. 240. Tanaka, K.; Clever, G. H.; Takezawa, Y.; Yamada, Y.; Kaul, C.; Shionoya, M.; Carell, T. Programmable self-assembly of metal ions inside artificial DNA duplexes. Nat. Nanotechnol. 2006, 1, 190–194. 241. Zimmermann, N.; Meggers, E.; Schultz, P. G. A second-generation copper(II)-mediated metallo-DNA-base pair. Bioorg. Chem. 2004, 32, 13–25. 242. Zimmermann, N.; Meggers, E.; Schultz, P. G. A Novel Silver(I)-Mediated DNA Base Pair. J. Am. Chem. Soc. 2002, 124, 13684–13685. 243. Tanaka, K.; Tengeiji, A.; Kato, T.; Toyama, N.; Shiro, M.; Shionoya, M. Efficient Incorporation of a Copper Hydroxypyridone Base Pair in DNA. J. Am. Chem. Soc. 2002, 124, 12494–12498. 244. Tanaka, K.; Tengeiji, A.; Kato, T.; Toyama, N.; Shionoya, M. A Discrete Self-Assembled Metal Array in Artificial DNA. Science 2003, 299, 1212–1213. 245. Takezawa, Y.; Maeda, W.; Tanaka, K.; Shionoya, M. Discrete Self-Assembly of Iron(III) Ions inside Triple-Stranded Artificial DNA. Angew. Chem. Int. Ed. 2009, 48, 1081–1084. 246. Ehrenschwender, T.; Schmucker, W.; Wellner, C.; Augenstein, T.; Carl, P.; Harmer, J.; Breher, F.; Wagenknecht, H.-A. Development of a Metal-Ion-Mediated Base Pair for Electron Transfer in DNA. Chem. Eur. J. 2013, 19, 12547–12552. 247. Zhang, L.; Meggers, E. An Extremely Stable and Orthogonal DNA Base Pair With a Simplified Three-Carbon Backbone. J. Am. Chem. Soc. 2005, 127, 74–75. 248. Mallajosyula, S. S.; Pati, S. K. Conformational Tuning of Magnetic Interactions in Metal–DNA Complexes. Angew. Chem. Int. Ed. 2009, 48, 4977–4981. 249. Liu, S.; Clever, G. H.; Takezawa, Y.; Kaneko, M.; Tanaka, K.; Guo, X.; Shionoya, M. Direct Conductance Measurement of Individual Metallo-DNA Duplexes within SingleMolecule Break Junctions. Angew. Chem. Int. Ed. 2011, 50, 8886–8890. 250. Kobayashi, T.; Takezawa, Y.; Sakamoto, A.; Shionoya, M. Enzymatic synthesis of ligand-bearing DNAs for metal-mediated base pairing utilising a template-independent polymerase. Chem. Commun. 2016, 52, 3762–3765. 251. Takezawa, Y.; Kobayashi, T.; Shionoya, M. The Effects of Magnesium Ions on the Enzymatic Synthesis of Ligand-Bearing Artificial DNA by Template-Independent Polymerase. Int. J. Mol. Sci. 2016, 17, 906. 252. Nakama, T.; Takezawa, Y.; Shionoya, M. Site-specific polymerase incorporation of consecutive ligand-containing nucleotides for multiple metal-mediated base pairing. Chem. Commun. 2021, 57, 1392–1395. 253. Takezawa, Y.; Nakama, T.; Shionoya, M. Enzymatic Synthesis of Cu(II)-Responsive Deoxyribozymes through Polymerase Incorporation of Artificial Ligand-Type Nucleotides. J. Am. Chem. Soc. 2019, 141, 19342–19350. 254. Nakama, T.; Takezawa, Y.; Sasaki, D.; Shionoya, M. Allosteric Regulation of DNAzyme Activities Through Intrastrand Transformation Induced by Cu(II)-Mediated Artificial Base Pairing. J. Am. Chem. Soc. 2020, 142, 10153–10162.

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255. Takezawa, Y.; Tanaka, K.; Yori, M.; Tashiro, S.; Shiro, M.; Shionoya, M. Soft Metal-Mediated Base Pairing With Novel Synthetic Nucleosides Possessing an O,S-Donor Ligand. J. Org. Chem. 2008, 73, 6092–6098. 256. Jash, B.; Neugebauer, J.; Müller, J. Enantiospecific Formation of a Metal-Mediated Base Pair Inside a DNA Duplex. Inorg. Chim. Acta 2016, 452, 181–187. 257. Jash, B.; Müller, J. Stable Copper(I)-Mediated Base Pairing in DNA. Angew. Chem. Int. Ed. 2018, 57, 9524–9527. 258. Jash, B.; Müller, J. Stable Hg(II)-Mediated Base Pairs With a Phenanthroline-Derived Nucleobase Surrogate in Antiparallel-Stranded DNA. J. Biol. Inorg. Chem. 2020, 25, 647–654. 259. Jash, B.; Müller, J. A Stable Zinc(II)-Mediated Base Pair in a Parallel-Stranded DNA Duplex. J. Inorg. Biochem. 2018, 186, 301–306. 260. Jash, B.; Müller, J. Concomitant Site-Specific Incorporation of Silver(I) and Mercury(II) Ions Into a DNA Duplex. Chem. Eur. J. 2018, 24, 10636–10640. 261. Jash, B.; Scharf, P.; Sandmann, N.; Fonseca Guerra, C.; Megger, D. A.; Müller, J. A Metal-Mediated Base Pair That Discriminates Between the Canonical Pyrimidine Nucleobases. Chem. Sci. 2017, 8, 1337–1343. 262. Barton, J. K.; Danishefsky, A. T.; Goldberg, J. M. Tris(phenanthroline)ruthenium(II): Stereoselectivity in Binding to DNA. J. Am. Chem. Soc. 1984, 106, 2172–2176. 263. Jash, B.; Müller, J. Application of a Metal-Mediated Base Pair to the Detection of Medicinally Relevant Single Nucleotide Polymorphisms. Eur. J. Inorg. Chem. 2017, 3857–3861. 264. Brotschi, C.; Leumann, C. J. Transition Metal Ligands as Novel DNA-Base Substitutes. Nucleosides Nucleotides Nucleic Acids 2003, 22, 1195–1197. 265. Weizman, H.; Tor, Y. Oligo-Ligandosides: A DNA Mimetic Approach to Helicate Formation. Chem. Commun. 2001, 453–454. 266. Su, M.; Tomás-Gamasa, M.; Serdjukow, S.; Mayer, P.; Carell, T. Synthesis and Properties of a Cu(II) Complexing Pyrazole Ligandoside in DNA. Chem. Commun. 2014, 50, 409–411. 267. Su, M.; Tomás-Gamasa, M.; Carell, T. DNA Based Multi-Copper Ions Assembly Using Combined Pyrazole and Salen Ligandosides. Chem. Sci. 2015, 6, 632–638. 268. Clever, G. H.; Polborn, K.; Carell, T. A Highly DNA-Duplex-Stabilizing Metal-Salen Base Pair. Angew. Chem. Int. Ed. 2005, 44, 7204–7208. 269. Boersma, A. J.; Megens, R. P.; Feringa, B. L.; Roelfes, G. DNA-Based Asymmetric Catalysis. Chem. Soc. Rev. 2010, 39, 2083–2092. 270. Clever, G. H.; Söltl, Y.; Burks, H.; Spahl, W.; Carell, T. Metal–Salen-Base-Pair Complexes Inside DNA: Complexation Overrides Sequence Information. Chem. Eur. J. 2006, 12, 8708–8718. 271. Clever, G. H.; Reitmeier, S. J.; Carell, T.; Schiemann, O. Antiferromagnetic Coupling of Stacked CuII–Salen Complexes in DNA. Angew. Chem. Int. Ed. 2010, 49, 4927–4929. 272. Clever, G. H.; Carell, T. Controlled Stacking of 10 Transition-Metal Ions inside a DNA Duplex Angew. Chem. Int. Ed. 2007, 46, 250–253. 273. Gaub, B. M.; Kaul, C.; Zimmerman, J. L.; Carell, T.; Gaub, H. E. Switching the Mechanics of dsDNA by Cu Salicylic Aldehyde Complexation. Nanotechnology 2009, 20, 434002. 274. Kaul, C.; Müller, M.; Wagner, M.; Schneider, S.; Carell, T. Reversible Bond Formation Enables the Replication and Amplification of a Crosslinking Salen Complex as an Orthogonal Base Pair. Nat. Chem. 2011, 3, 794–800. 275. Ukale, D. U.; Lönnberg, T. 2,6-Dimercuriphenol as a Bifacial Dinuclear Organometallic Nucleobase. Angew. Chem. Int. Ed. 2018, 57, 16171–16175. 276. Ukale, D. U.; Tähtinen, P.; Lönnberg, T. 1,8-Dimercuri-6-Phenyl-1H-Carbazole as a Monofacial Dinuclear Organometallic Nucleobase. Chem. Eur. J. 2020, 26, 2164–2168. 277. Maity, S. K.; Lönnberg, T. Oligonucleotides Incorporating Palladacyclic Nucleobase Surrogates. Chem. Eur. J. 2018, 24, 1274–1277. 278. Räisälä, H.; Lönnberg, T. Covalently Palladated Oligonucleotides Through Oxidative Addition of Pd0. Chem. Eur. J. 2019, 25, 4751–4756. 279. Aro-Heinilä, A.; Lönnberg, T.; Virta, P. 3-Fluoro-2-mercuri-6-methylaniline Nucleotide as a High-Affinity Nucleobase-Specific Hybridization Probe. Bioconjugate Chem. 2019, 30, 2183–2190. 280. Ukale, D.; Lönnberg, T. Organomercury Nucleic Acids: Past, Present and Future. ChemBioChem 2021, 22, 1733–1739. 281. Aro-Heinilä, A.; Lönnberg, T.; Virta, P. Covalently Mercurated Molecular Beacon for Discriminating the Canonical Nucleobases. ChemBioChem 2021, 22, 354–358. 282. Müller, J. Nucleic Acid Duplexes With Metal-Mediated Base Pairs and Their Structures. Coord. Chem. Rev. 2019, 393, 37–47. 283. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF ChimeradA Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605–1612. 284. Pyykkö, P.; Straka, M. Ab initio studies of the Dimers (HgH2)2 and (HgMe2)2. Metallophilic Attraction and the van der Waals Radii of Mercury. Phys. Chem. Chem. Phys. 2000, 2, 2489–2493. 285. Huard, D. J. E.; Demissie, A.; Kim, D.; Lewis, D.; Dickson, R. M.; Petty, J. T.; Lieberman, R. L. Atomic Structure of a Fluorescent Ag8 Cluster Templated by a Multistranded DNA Scaffold. J. Am. Chem. Soc. 2019, 141, 11465–11470. 6þ 286. Petty, J. T.; Ganguly, M.; Rankine, I. J.; Baucum, E. J.; Gillan, M. J.; Eddy, L. E.; Léon, J. C.; Müller, J. Repeated and Folded DNA Sequences and Their Modular Ag10 Cluster. J. Phys. Chem. C 2018, 122, 4670–4680. 287. Bondi, A. van der Waals Volumes and Radii. J. Phys. Chem. 1964, 68, 441–451. 288. Schmidbaur, H.; Schier, A. Argentophilic interactions. Angew. Chem. Int. Ed. 2015, 54, 746–784. 289. Müller, J. Metal-Mediated Base Pairs in Parallel-Stranded DNA. Beilstein J. Org. Chem. 2017, 13, 2671–2681. 290. Nakagawa, O.; Aoyama, H.; Fujii, A.; Kishimoto, Y.; Obika, S. Crystallographic Structure of Novel Types of AgI-Mediated Base Pairs in Non-canonical DNA Duplex Containing 2’-O,4’-CMethylene Bridged Nucleic Acids. 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2.22 Supramolecular metal-based molecules and materials for biomedical applications Angela Casinia, Roland A. Fischerb, and Guillermo Moreno-Alca´ntara,c, a Department of Chemistry, Technical University of Munich (TUM), München, Germany; b Department of Chemistry, Catalysis Research Center, Technical University of Munich, München, Germany; and c Facultad de Química, National Autonomous University of Mexico, Ciudad Universitaria, Mexico City, México © 2023 Elsevier Ltd. All rights reserved.

2.22.1 2.22.2 2.22.2.1 2.22.2.2 2.22.3 2.22.3.1 2.22.3.2 2.22.3.3 2.22.4 2.22.5 2.22.5.1 2.22.5.2 2.22.5.3 2.22.6 References

Introduction Supramolecular coordination complexes (SCCs) Synthesis Synthesis of helicates Biomedical applications of SCCs Anticancer therapy Drug delivery Imaging Synthesis of metal-organic frameworks (MOFs) Biomedical applications of MOFs Drug delivery Imaging Combined therapy and theranostics Conclusions and perspectives

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Abstract The design and development of metallo-supramolecular systems has resulted in the construction of a myriad of fascinating structures with highly diverse properties and potential applications in biology and medicine. Assessment of the biomedical applications of such metal-based assemblies is an emerging field of research that stems from the recently demonstrated promising results on such systems. In this article, we will present selected examples among two main families of metallosupramolecular systems, namely the discrete supramolecular coordination complexes (SCCs) and the metal organic frameworks (MOFs), respectively. Thus, we will introduce the main synthetic strategies and structural features for each family and discuss in more details the properties that make them suitable for applications as new generation therapeutic and imaging agents, as well as drug delivery systems. Overall, we aim at delineating a useful set of guidelines to help synthetic chemists broadening the biomedical uses of such supramolecular systems, while enabling their rational design and targeted structural modifications.

2.22.1

Introduction

In the last decades, supramolecular chemistry has developed at a tremendous rate. This expansion has been driven by the growing knowledge regarding synthetic and characterization methods for complex structures. Interest in these complex systems lies in the scientific fields of chemistry (recognition and selective transformations), materials science (construction of macroscopic architectures and devices on the molecular level) as well as biomedicine (multifunctional drug delivery systems). In this context, supramolecular self-assembled metal-based structures occupy an important place. The covalent coordination bonds formed by organic ligands to transition metal centers are of intermediate strength relatively to the typical non-covalent weak interactions (hydrogen bonding, van der Waals forces, Coulombic interactions, and dipole-dipole interactions) and the strong covalent carbon-carbon bonds of most organic compounds. In this field, two main types of nanoarchitectures have been described: Metal Organic Frameworks (MOFs) and Supramolecular Coordination Complexes (SCCs). While MOFs are porous polymers formed by coordination bonds between metal ions or clusters and organic linkers,1,2 SCCs are defined and discrete two- (2D) or three-dimensional (3D) structures.3 In each case, by regulating the functions of the individual building blocks and the geometry of their linkages, diverse nanomaterials with unique and enhanced properties can be prepared. In addition, the bioactive nature of the metals or metal complexes also determines the final functions of the resulting supramolecular entities. Here, we will present selected examples of SCCs and MOFs as anticancer and/or imaging agents or as drug delivery systems for chemotherapy, emphasizing the versatility and tunability of these scaffolds.4–12 When available, examples of the applications of these systems in vivo will be discussed, which validate the concept and pave the way to their clinical application. We also include

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information on recently developed hybrid-supramolecular materials for biomedical applications. Overall, this article is not intended to present details on the synthetic strategies to achieve various supramolecular metal-based molecules, but rather to summarize some general design principles focusing on the most recent examples from those systems having applications in the aforementioned areas of medicine. Recent thematic reviews have already described the exponential progress made in the design, synthesis, and possible applications of these discrete nanostructures,13–18 and we refer the reader to this literature for further information. Finally, we aim at providing the future outlook for this exciting research area, which, in defining the various challenges, will hopefully stimulate new ideas within the supramolecular, bioinorganic, and medicinal chemistry communities.

2.22.2

Supramolecular coordination complexes (SCCs)

2.22.2.1

Synthesis

The synthesis of SCCs has been described for several decades and proceeds via the choice of a combination of multi-dentate ligands (Lewis-bases) and metal ions (Lewis-acids), leading to predictable and reproducible supramolecular scaffolds with defined coordination geometry. Since coordination bonds are the driving force to complex formation, this process is often referred to as coordination-driven self-assembly. In most cases, the self-assembly proceeds under mild conditions to form 2D metallacycles and 3D metallacages of general formula [MxLy]n (M ¼ metal, L ¼ ligand, n ¼ charge) in high yields. Various synthetic strategies to obtain SCCs of different shapes have been developed, the earliest originating under the theme of “directional bonding,” including edge- (mainly developed by Stang et al.19,20) and face-directed (firstly reported by Fujita and coworkers in the late 1990s21) approaches, whereby a metal acceptor and a ligand donor are mixed in specific ratios to form highly symmetrical polygons and polyhedra.13 Raymond et al. significantly progressed both approaches, extending the investigations towards the dynamic behavior22 and chirality23 of SCCs, and their applications in catalysis.18,24 Accordingly, numerous 2- and 3D supramolecular architectures based on Pd2þ, Pt2 þ, and Ru2þ ions have been obtained. For example, Fujita reported on a [Pd(N1,N1,N2,N2-tetramethylethane1,2-diamine)]6L4 (L ¼ ligand: 2,4,6-tri(pyridin-4-yl)-1,3,5-triazine) cage accessible by face-directed self-assembly between four equivalents of the planar tridentate ligand (acting as a panel) and six equivalents of the Pd2þ precursor.25 Edge-directed approaches to form metallacages have been described mainly based on bidentate “banana-shaped” pyridyl ligands and Pd2þ and Pt2þ metal precursors.26 In general, the simplest examples of 3D metallacages are built on M2L4 type of scaffolds, but numerous other structures have been reported (M2L3,27 M4L4,28 M4L627,29,30M8L12,29 M12L24,31 M24L48,32 etc.). Recently, Fujita and coworkers demonstrated the scalability of the size of SCCs reporting the synthesis and characterization of Pd48L96, the largest discrete selfassembled edge-directed polyhedron obtained so far.33 Of note, Nitschke et al. introduced the concept of ‘subcomponent selfassembly’34 whereby the actual linker is formed in situ (e.g., by imine formation out of aldehydes and amines) and also enable covalent post-assembly modifications of the SCCs.35 Other methodologies to form SCCs also include symmetry-adapted and weak-link approaches.36 A schematic representation of the main types of “directional bonding” methodologies is given in Fig. 1. In the edge-directed selfassembly approach, the polyhedra are formed using stoichiometric ratios of ligand (often bidentate) to metal precursors (Fig. 1A). In the face-directed approach, also referred to as the “paneling method,” rigid pre-formed organic multi-topic ligands act as “panels” to guide coordination complex precursors to form 3D SCCs (Fig. 1B).43 In the latter strategy, modifying the availability of the coordination sites of the metal induces an increased degree of directionality to form the final assembly. In particular, cis-protected square planar metal fragments [e.g., [Pd2þ(en)], en ¼ ethylenediamine] are shown to be very useful to panel molecules. Metal-assembled cages, bowl, tube, capsule, and polyhedral have been efficiently constructed by this approach. Finally, the weak-link method uses a hemilabile ligand to form firstly a “weak link” (weak metal-heteroatom bond) with a metal precursor, generating a condensed metallacycle, which can then be opened by selective introduction of an ancillary ligand with a higher metal binding affinity (Fig. 1C).39,44 In addition to SCCs assembly via classical coordination chemistry between metal nodes and organic linkers featuring heteroatom donors, organometallic fragments have also been introduced, whereby a carbon donor can be implemented both in the linker molecules as well as in the capping ligands of the metal nodes. Examples of these compounds have been reported by various groups.38,45–48 If the carbon donor is part of the linker-node connection, the (organic) linker is forming the organometallic bond to the metal node; therefore, producing supramolecular organometallic complexes (SOCs). In contrast to the case of organometallic metal nodes, in this case, the carbon-metal bond is now structurally decisive for the “organometallic assembly” of the resulting SOC. Multi-dentate linkers used to build up such assemblies are mainly based on alkynyl, metal-arene,19 or NHCdonor groups. Of note, multi-dentate NHC ligands have undergone an enormous development in the past years, pioneered by the groups of Bielawski,49 Peris,50 and Hahn,51 also towards implementation in SOCs.52,53 The synthesis of discrete heterometallic self-assemblies has also been achieved.54 In general, the synthetic strategies towards such heterometallic architectures, either metallacycles or metallacages, can be summarized into two categories: (i) the metalloligand (metal-containing ligand) approach and (ii) the self-sorting approach. While the former is based on the step-wise assembly of metalloligands used as building blocks and offers precise control over the placement of appended functional groups, the latter approach can generate systems with enhanced complexity, but with scarce control on their resultant architecture. Compared with homometallic architectures, these assemblies possess large internal cavities that have potential as reactors, host-guest frameworks, or as platforms for the design of materials with tailored properties, including controlled redox reactivity.

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Fig. 1 Schematic representations of the main synthetic approaches to form supramolecular complexes by self-assemblydhomometallic: (A) edgedirected self-assembly, with a Pd2L4 example37; (B) face-directed self-assembly, with the example of a capsule containing six Ru2þ ions38; (C) weak link method, with an example of interchangeable Rh-based metallacycle39;dheterometallic: (D) example of a heterometallic M1-M2 rectangle (M1 ¼ Rh, Ir; M2 ¼ Zn, Ni, Cu)40; (E) X-ray structure (CCDC no 1,035,264) and corresponding schematic representation of interlocked cages [3BF4@Pd4L8](BF4)5.41 The X-ray structure was depicted using the Mercury42 software, the hydrogen atoms and extra-cavity BF4 are omitted for clarity.

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In this context, a metalloligand can be defined as a coordination or organometallic complex containing one or several binding groups through which it can be tethered to ions or molecules. It should be noted that a coordination complex with two appended groups is the simplest metalloligand, and this type of scaffold has been widely used to build heterometallic assemblies. For example, bidentate metalloligands with 4-pyridyl groups are known to generally favor the self-assembly of heterometallic macrocycles. Using this approach, several families of gold complexes decorated with crown-ether, amide, pyridine, bipyridine, terpyridine, carboxylate, amino acid, or p-alkyne binding sites have been reported, leading to the formation of simple dinuclear complexes to self-assembled supramolecules, coordination polymers, or solids.55 Lees and coworkers prepared a series of heterometallic square complexes of different transition metals, including Pd-Ru and Re-M (M ¼ Fe, Ru, or Os) multinuclear complexes (Fig. 1D.56 The same strategy was used for the preparation of hexanuclear heterometallic metallarectangles by the reactions of the metalloligands [ML2]2 þ (M ¼ Zn2þ, Ni2þ and Cu2þ, L ¼ 40 -(4-pyridyl)-2,20 :60 ,200 -terpyridine(4-pyterpy)) with half-sandwich organometallic units [(Cp*2M2(l-DHNA)Cl2] (M ¼ Ir, Rh; DHNA ¼ 6,11-dihydroxy-5,12-naphthacenedione) (Fig. 1E).40 Exploiting the self-sorting approach, in an effort to prepare metal-organic metallacycles and cages with higher complexity and diversity, the groups of Nitschke57,58 and Schmittel59 have published a series of supramolecular heterometallic complexes with different elegant architectures. For example, a single heterotopic ligand that can coordinate to Fe2þ (as a tris(pyridylimine) complex), as well as to Pt2þ/Pd2þ ions (through its terminal pyridine group), was used to form soluble heterometallic [8Feþ 6Pt/ Pd] cubic structures in one-pot reactions.57,58 Cubic cages employing labile Fe2þ ions and pyridylimine ligands were also synthesized, which incorporate Ni2þ ions immobilized by TAPP (tetrakis(4-aminophenyl)porphyrin) moieties.60 Another type of recent and more sophisticated system based on SCCs is the interpenetrated (or interlocked) double (or more) metallacages. These are usually formed using less bulky and lengthier ligands than in the classical homoleptic metallacage, but still banana-shaped.61 In this category, the first example was described by Kuroda et al., who reported on a double and interpenetrated cage of the general formula [Pd4L8], in which L is a conformationally flexible bidentate ligand.62 However, it was shown that this dimeric structure was only slightly favored thermodynamically compared to the monomer. Later on, Clever and coworkers reported on the formation of interlocked double cages based on a more rigid bidentate ligand bearing a central dibenzosuberone.63 The investigation showed that the self-assembly first proceeds to form a monomeric cage as a kinetically favored intermediate, which then fully converts, upon heating for a few hours, to form an interpenetrated dimer, whereby three BF4 anions (coming from the Pd2þ precursor) are sandwiched between the four metal centers.63 The dimer was also found to be a strong receptor for halide anions, with a significant preference for chlorides.64 More recently, the same group reported on modifications of the ligand backbone (including the use of acridone-based ligands, Fig. 1E) to change the size and steric hindrance of the internal cavity between the two monomers, and showed that the affinity of the sandwiched anions can be predicted and adjusted with judicious ligand design.41,61,65

2.22.2.2

Synthesis of helicates

Within the 3D SCCs family, helicates represent a chiral example of assemblies between rigid coordinating ligands and metal ions. The term “helicate” (inorganic double helices) was given in the late 1980s to polymetallic helical multi-stranded complexes by JeanMarie Lehn, for their resemblance to a-helices in terms of their diameter, charge, and chirality.66 Usually, these structures feature bipyridine ligands linked by flexible alkyl or alkylether chains, introducing a high degree of flexibility into the helical structure. The various synthetic methodologies and examples of helicates described so far have been extensively reviewed by Hopfgartner et al. in 1997,67 and later on by Tsukube and Albrecht.68,69 Briefly, to obtain optically pure helicates, two main types of approaches have been developed: in the first case, a rigid and rather short linker mechanically allows the coupling of the metal configurations (thus, the enantiomers are chemically different); in the second option, an optically pure ligand and a rigid linker lead to a diastereomeric excess of one helicity based on the increased thermodynamic stability of one isomer compared to the other(s).70 Several complementary strategies have been reported to isolate optically pure helicates, including the resolution of racemic helicates,70 either by crystallization71 or chromatography,72,73 and the use of chiral counter ions, to introduce an enantiomeric enrichment in the architectures.74 Noteworthy, the generation of multinuclear helicates leads to metal-based macrocyclic (two-stranded helicate) or macro-bicyclic (three-stranded helicate) cavities, which are in most cases chiral. The latter property leads to selective molecular recognition processes for small binding partners, which in turn may serve as templates to maintain the structure of the helicates and/or the chirality by blocking the kinetic racemization process.67,75,76 Within the helicates family, Hannon and coworkers developed the synthesis of ‘cylinders’, dinuclear triple-helical compounds which are prepared in a single step from a pyridyl-aldehyde, a diamine, and an octahedral metal (usually Fe2þor Ni2þ) (Fig. 2).77 The cylinders generally differ from the helicates since they are endowed with a certain rigidity along the length of the structure, due to p-stacking interactions between the rings of the diphenylmethane “spacer.”

2.22.3

Biomedical applications of SCCs

2.22.3.1

Anticancer therapy

Several small-molecule transition metal complexes have been studied over the years for their anticancer properties. Among them, the FDA-approved Pt2þ drug cis-diammino-dichlorido-Pt(II) (cisplatin) is used to treat a range of cancers.78,79 Taking inspiration

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Fig. 2 (A) X-ray structure (CCDC no 722,438) and schematic representation of a [Ni2L3]4 þ cylinder.77The X-ray structure was depicted using the Mercury software.42

from the clinical success of cisplatin, supramolecular metal-containing complexes are also under investigation as experimental cytotoxic anticancer agents. In this section, we summarize some of the most investigated systems, including coordination and organometallic supramolecular scaffolds, discussing their main features and design principles. The potency of the tested SCCs is similar to and in some cases even better than the clinically used cisplatin and other classical chemotherapic agents. The anticancer mechanisms of these SCCs can be very diverse and mainly include membrane damage, autophagy, DNA damage, cell apoptosis and increased p53 expression. Concerning 2D SCCs, dinuclear Pt2þ and Pd2þ metallacycles coordinated to amide-based dipyridyl ligands and 1,10 -bis(diphenylphosphino) ferrocene ligands, have been investigated as cytotoxic agents in vitro.80 Interestingly, both metallacycles displayed increased anti-proliferative effects compared to their metal precursors and organic ligands, as well as with respect to cisplatin in different human cancer cells, while being less toxic against non-cancerous cells80 The mechanism of action of both metallacycles was investigated in vitro against the T98G brain tumor cell line, and both compounds resulted to be easily internalized by the cancer cells and to induce oxidative stress eventually leading to cell death by apoptosis.80 Concerning 3D supramolecular architectures, a positively charged [Pt6L4]12þ metallacage was studied for its anti-proliferative activity,81 and it displayed similar cytotoxicity as cisplatin towards a range of human cancer cell lines, while it was markedly less toxic towards normal lung cells. Moreover, the cage was found to be localized inside the cell nucleus using atomic absorption spectroscopy.81 It was also shown that the mechanism of action involves the compound’s non-covalent binding to DNA via intercalation, promoting DNA condensation. Metallacages of general formula PdxLy tethering a cytotoxic compound have also been reported as experimental anti-proliferative agents. As an example, a supramolecular [Pt3L3]6þ hexagon was formed by self-assembly between a dinuclear Pt4þ precursor and a bidentate ligand conjugated to organoplatinum species. The resulting supramolecular hexagons were shown to be effectively internalized by cancer cells, delivering up to three equivalents of cisplatin upon reduction of the Pt4þ prodrug in the intracellular environment, following cell death by apoptosis (Fig. 3A).82 With the aim to exploit SCCs to increase the anticancer potential of an adamantly-based Pt4þ prodrug, Lippard and coworkers used a tridentate ligand and a [Pt2þ(ethane-1,2-diamine)] precursor to achieve a water-soluble cationic [Pt4L6]12þ cage able to encapsulate the Pt4þ complex.84 The hydrophobic adamantyl moiety of the prodrug molecule was postulated to be securely encapsulated within the hydrophobic cavity of the hexanuclear cage, as suggested by 1D and 2D NMR spectroscopy. The cage encapsulating the Pt4þ complex was moderately cytotoxic in A549 human lung cancer cells, compared to the Pt4þ prodrug.84 It was also hypothesized that the Pt4þ complex is reduced intracellularly, thus, releasing cisplatin, as suggested by NMR spectroscopy and mass spectrometry methods. To improve the anticancer activity and overcome drug resistance, other therapeutic modalities were introduced into the SCCs via covalent and non-covalent approaches. For example, metallacycles containing porphyrin-based, Ru complex-based, or BODIPYbased photosensitizer were designed for photodynamic therapy (PDT) applications to enhance the intrinsic anticancer activity of the metallacycles.85,86 PDT is a non-invasive treatment based on the generation of reactive oxygen species (ROS) in the tumoral regions by the activity of photosensitizers (PS) that generate ROS as a response to photoexcitation, ideally by red or IR irradiation. In a recent example, Stang and Chao synthesized an octahedral Ru-Pt metallacage using coordination-driven self-assembly, combining six Ru(II)-based photosensitizers and four Pt(II)-based acceptor building blocks in a single supramolecular ensemble.87

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Fig. 3 (A) Schematic representation of a [Pt3L3]6 þ hexagon exo-functionalized with three moieties of a Pt4þ prodrug82; (B) Multinuclear Pt2þ metallacycle acting as quadruplex binder and telomerase inhibitor and its adduct with a G4 structure studied by molecular modeling.; (C) Schematic representation of a Ru8-cage featuring porphyrin ligands studied as nucleic acid binder.83 Reprinted with permission from reference Kieltyka, R.; Englebienne, P.; Fakhoury, J.; Autexier, C.; Moitessier, N.; Sleiman, H. F. A Platinum Supramolecular Square as an Effective G-Quadruplex Binder and Telomerase Inhibitor. J. Am. Chem. Soc. 2008, 130, 10040–10041, Copyright 2018 American Chemical Society.

The resulting SCC displays deep-red emission, a large 2-photon absorption cross-section, and high ROS generation efficiency upon activation by 2-photon light irradiation. By encapsulating the metallacage with an amphiphilic polymer, nanoparticles were formed which showed selective accumulation in lysosomes. Thus, highly effective 2-photon PDT mediated by the metallacage was achieved in cancer cells and in 3D multicellular spheroids. Furthermore, a significant antitumor efficacy of 2-photon PDT using the metallacage nanoparticles was shown in vivo in A549 tumor xenografted nude mice.87 Yang and coworkers have reported on a particularly interesting SCCs whereby the generation of singlet oxygen was intelligently controlled by light using a dual-stage metallacycle as the theranostic core, realizing precise photodynamic therapy.88 In detail, the metallacycle contained both porphyrin photosensitizers and diarylethene moieties and was further loaded into polymeric nanoparticles to favor tumor accumulation. The excellent photoisomerization property of diarylethene enabled the resultant metallacycle to act as a supramolecular switch, whereby the efficient generation of 1O2 from the porphyrin photosensitizer was observed only when

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the diarylethene unit is in the ring-open form. The latter can be formed starting from the ring-closed form of the metallacycle by light irradiation in vitro and in vivo.88 Concerning helicates structures, the effect of different ligands (functionalized trispyridyl scaffolds with rigid alkene linker vs. benzotriazoles, hexane-triazoles and PEG-triazoles-bisfunctionalized phenyl rings) on the biological activity of quadruplystranded [Pd2L4]4þ helicates have been investigated and showed a direct correlation between their stability in biological media and their anti-proliferative effects.89 Furthermore, mechanistic studies indicate that the helicate does not cause cell death by interacting with DNA, rather it seems to disrupt the cell membrane. Similar [Pd2L4]4þ (L ¼ 1,3-bis-hexanetriazole phenyl) helicates endowed with sufficient stability in aqueous environment were found to be markedly more toxic than cisplatin against the cisplatin-resistant MDA-MB-231 breast cancer cell line,89 and cell death was related to the disruption of the cell membrane.89 With the aim to develop tumor-directed SCCs, the chirality of the helicates scaffolds was also exploited to direct molecular recognition of specific biological targets, namely nucleic acids. A first study investigated the mode of binding of a binuclear Fe2þ triplestranded “cylinders” to a DNA model by NMR spectroscopy and computational modeling techniques, suggesting that the cylinder binds to the DNA major groove.90 It was also revealed that, although a racemic mixture of the chiral helicate was introduced to the double-stranded oligonucleotide, only the M-enantiomer was able to bind DNA altering its conformation.90 The mode of binding of the Fe2þ cylinder was further studied and revealed that the helix preferentially binds to short (8–10 base pairs) purine-pyrimidine sequences within the DNA strand.91 The latter specificity proved a promising feature to target cancer cells via binding of the helicates to oncogenes.91 Later on, Fe2þ cylinders have a high specificity for RNA 3-way junctions,92 as well as for certain non-canonical secondary DNA structures, such as DNA bulges93,94 and G-quadruplex DNA (G4).95 Thus, water-soluble enantiomeric Fe2þ helicates were synthesized, and their affinity for human telomeric G4s was assessed.95 The P-enantiomer Fe2þ helicate was found to bind strongly and selectively to the G4, whereas the M-enantiomer showed no association. Furthermore, the strong binding affinity to G-quadruplex DNA translated into strong inhibition of telomerase activity. A number of studies have shown that binuclear metallahelicates (Fe2þ and Ru2þ-based) can target triple-stranded “Y-shaped” junctions.91,94,96–100 The latter are another example of a non-canonical DNA structure, forming during DNA transcription and replication, and whose regulation may allow to achieve anti-proliferative effects and, most importantly, cell cycle control. In 2010, Hannon and coworkers showed that the stabilization of Y-shaped junctions by supramolecular Ru2þ cylinders importantly inhibits the function of polymerase enzymes, accounting for the observed cytotoxic effects.98 These results provide evidence that the non-covalent binding of the cylinders to DNA can alter the ability of proteins to process the DNA information. Further studies of DNA binding by Fe2þ helicates revealed that to facilitate strong binding to the major groove of duplex DNA, as well as cytotoxic activity, a rigid helicate is preferred over the flexible analogous.101 In addition to Fe-based helicates, the Pt2þ molecular square [Pt(en)(4,40 -dipyridyl)]4 (en ¼ ethylenediamine) has been reported to be an efficient G-quadruplex binder and telomerase inhibitor (Fig. 3B).102 Molecular modeling studies combined with molecular dynamics (MD) calculations suggested that a number of factors contribute to the observed strong G4 binding affinity, including: the square arrangement of the four bipyridyl ligands, the highly electropositive nature of the overall complex, as well as hydrogen bonding interactions between the ethylenediamine ligands and phosphates of the DNA backbone. More recently, a supramolecular [Pt2L2]6þ binuclear metallacycle with large, planar 2,7-diaza-pyrene-based ligands was studied for its DNA binding properties,103 leading to induction of DNA bending, which in turn prevented DNA processing and replication. Moreover, the metallacycle exhibited anti-proliferative effects in cancer cells and different spectrum of activity with respect to cisplatin.103 Supramolecular Pt2þ quadrangular boxes with L-shaped 4,40 -bipyridine ligands were also shown to bind duplex and G-quadruplex DNA motifs in a size-dependent fashion.104 In detail, three dinuclear Pt2þ molecular squares of distinct size (ranging between 110 and 220 Å) inhibited cancer cells’ growth and heavily influenced the expression of genes known to form G-quadruplexes in their promoter regions. Interestingly, the smallest Pt2þ-box displayed less activity, but enhanced selectivity for binding to the G4 promoter c-Kit.104 Supramolecular 2D Ru2þ metallacycles were already reported in the late 1990s and displayed properties, such as water solubility and stability, which make them suitable for biological applications, including as anticancer agents.105 Interestingly, these complexes have been shown to cause cell death via different pathways with respect to cytotoxic metallodrugs, including by triggering excessive autophagy, the controlled process of recycling dysfunctional or destroyed proteins and organelles via lysosome digestion.106 Within a series of Ru2þ-arene metallarectangles with different paneling linkers, one derivative was shown to be moderately cytotoxic in vitro against multidrug-resistant human colon cancer cells compared to cisplatin and doxorubicin (DOX).107 Similarly to metallacycles, 3D Ru2þ-arene metallabowls have also been designed and tested for their anti-proliferative properties in vitro against a range of cancer cell lines.106,108,109 In particular, a metallabowl featuring 8-dihydroxy-1,4-naphthaquinonato ligands was twofold more active than cisplatin and DOX against HCT-15 cells In particular, a metallabowl featuring 8-dihydroxy-1,4-naphthaquinonato ligands was twofold more active than cisplatin and DOX against HCT-15 cells.108 Further investigations showed that, upon metallabowl exposure, overexpression of two known colorectal cancer suppressors, p53 and the Adenomatous polyposis coli (APC) gene, increased.108,109 By introducing tridentate, planar ligands to the binuclear Ru2þ-arene “clips”, a range of hexanuclear 3D metallacages were reported including variation of the paneling linker ([Ru2(p-iPrC6H4Me)2(OOXOO)][CF3SO3]2 (OOXOO ¼ 2,5-dioxydo-1,4benzoquinonato [dobq], 5,8-dihydroxy-1,4-naphthaquinonato (donq), and 6,11-dihydroxy-5,12-naphthacenedionato [dotq] etc.)).110–112 In this series, the dinuclear “clip” containing donq as the bridging ligand was a moderate inhibitor of cell viability against a range of cancer cell lines, whereas all the other tested metallacages were non-cytotoxic.105,113 Other types of 3D Ru-based SCCs, including boxes and cubes, have been reported to be able to interact with nucleic acids. For example, porphyrin-based scaffolds were designed and

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linked via ruthenium-arene moieties used as bridging blocks able to connect two porphyrin units and create octa-ruthenium supramolecular cubes (Fig. 3C).83 The cubes showed strong interactions with different G4 models, as well as scarce selectivity with respect to duplex DNA, as assessed by fluorescence intercalation displacement (FID) and surface plasmon resonance. Although the field of SCCs as anticancer therapeutics is still in its infancy, a few preliminary in vivo experiments have been carried out. For example, the anticancer activity of two cytotoxic Ru2þ-arene metallacycles, one with a 2D rectangular geometry and one featuring a metallabowl geometry (Fig. 4A-B), was studied in vivo using a hollow fiber assay, whereby a semipermeable fiber impregnated with the human colorectal carcinoma HCT-15 cells were implanted into the intraperitoneal and subcutaneous compartments of nude mice.106 The two SCCs were then administered to the impregnated nude mice, and the tumors examined after 7 days. The obtained results revealed that the metallabowl-type metallacycle was a more potent inhibitor of cancer cells growth than the metallarectangle. However, both the ruthenium-arene scaffolds were not as effective inhibitors of cell proliferation as cisplatin.106 Concerning the mechanisms of action, the study revealed that both metallacycles induced autophagy in HCT-15 cells, with the metallabowl being more potent than the metallarectangle, in line with the observed enhanced anticancer activity.106 Concerning 2D SCCs, a luminescent Pt2þ metallacycle of rhomboidal geometry was studied in vitro and in vivo for its anticancer activity (Fig. 4C).114 Preliminary in vitro studies against lung (A549) and cervical carcinoma (HeLa) cells confirmed rapid cellular uptake of the intact platinum metallacycle.114 Afterwards, a mouse tumor xenograft model, generated using nude mice injected in the subcutaneous region with MDA-MB-231 human breast cancer cells, was selected for the in vivo study. Mice were treated with a solution of Pt-metallacycle (0.6 mg/mL, administered via intraperitoneal injection every 3 days for 30 days) and, at the end of the treatment, a 64% median tumor volume reduction was observed in treated mice with respect to controls. Furthermore, the tumor growth inhibition, measured by the change in volume of the tumor throughout the length of the experiment and defined by the T/C ratio (in %, corresponds to the ratio between the Treatment (T) over the Control (C)),115 was calculated as 36%, well below the National Cancer Institute standard.116 Very recently, Chen, Stang and coworkers designed a porphyrin-based metallacage through multicomponent coordination-driven self-assembly, acting as a theranostic platform to fabricate metal nanoparticles (MNPs).117 In details, a discrete Pt2þ metallacage was synthesized, using therapeutic cis-(PEt3)2Pt(OTf)2 (cPt), 5,10,15,20-tetra(4-pyridyl)porphyrin (TPP) and disodium terephthalate (DSTP) as the building blocks, with the idea achieving synergistic anticancer efficacy (Fig. 5A). Of note, both the fluorescence emission and 1O2 generation quantum yield of the porphyrins were dramatically increased upon formation of MNPs, which was favorable for

Fig. 4 Schematic representations of two arene-Ru2þ metallacycles with (A) 2D rectangular geometry and (B) “metalla-bowl” geometry,106 as well as a (C) rhomboidal Pt2þ metallacycle,114 studied in vivo for their anticancer properties.

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both NIRFI and PDT.117 Furthermore, the nano-formulation was functionalized by two amphiphilic di-block polymers (mPEG-b-PEBP and RGD-PEG-b-PEBP), one of them featuring integrin binding RGD ligands. The resultant MNPs exhibited long blood circulation time and high tumor accumulation benefiting from the enhanced permeability and retention (EPR) effect118 and active targeting ability. Indeed, superior tumor suppression with respect to separate cisplatin treatment and light irradiation, was realized without recurrence after single-dose injection of the targeted MNPs in xenograft models of tumors from avb3 integrin overexpressing U87MG cells and cisplatin-resistant human ovarian cancer A2780cisR cells.117 Moreover, in order to verify the in vivo anti-tumor performance and anti-metastasis effect of photochemotherapy, 4 T1 (breast cancer) tumors were orthotopically inoculated in the mammary fat pads to produce spontaneous metastases in the lung, which was an experimental animal model for stage IV human breast cancer. Interestingly, the combination of chemotherapy and PDT using the supramolecular MNPs exhibited superior anti-tumor efficacy with a 93.5% reduction in tumor volume, with respect to the least effective, and undergoing tumor recurrence during treatment, separate chemotherapic and PDT treatments.117 Excellent anti-metastatic effect was also achieved, which was attributed to the synergistic photochemotherapy. In addition, by chelating a positron emitting metal ion (64Cu) or a paramagnetic Mn ion, the 64Cu@MNPs (or Mn@MNPs) were shown to be excellent PET imaging and MRI agents, enabling precise diagnosis of tumor and real-time monitoring of delivery, biodistribution and excretion of the MNPs (Fig. 5B-D).117 Significant tumor accumulation was clearly visible in the U87MG tumorbearing mice administered with MNPs at 6 h post injection, and exceptionally intensive signal was visible in the tumor area for more than 24 h in comparison with other tissues.

2.22.3.2

Drug delivery

Some of the 3D SCCs are suitable as drug delivery systems (DDS) due to their rigid porous structure, where small drug molecules can be encapsulated and protected from metabolism. Drug encapsulation is driven by hydrophobicity of the cargo drug molecule and the host cavity, and non-covalent interactions within the host cavity (e.g., H-bonding, van der Waals). Furthermore, the possibility to modify the ligand structure both pre- and post- self-assembly enables the covalent linkage of a prodrug species to the SCC’s architecture. In the latter case, the prodrug can then be released via external chemical stimuli in a controlled manner. Despite these attractive features, the use of SCCs for drug delivery is still in its infancy.5,6 Within this framework, pioneering work by Therrien and coworkers designed water-soluble hexaruthenium metalla-prisms able to encapsulate lipophilic molecules.38 In details, the cationic hexanuclear metalla-prism [(p-cymene)6Ru6(tpt)2(dhbq)3]6 þ (tpt ¼ 2,4,6trispyridyl-1,3,5-triazine; dhbq ¼ 2,5-dihydroxy-1,4-benzoquinonato) was shown to encapsulate two hydrophobic Pd2þ and Pt2þ complexes [M(acac)2] (M ¼ metal, acac ¼ acetylacetonato) (Fig. 6A).38 While the [M(acac)2] complexes are inactive due to their inherent lack of solubility in water, the encapsulated [Pd(acac)2] – ([Pd(acac)2] 3 [(p-cymene)6Ru6(tpt)2(dhbq)3]6 þ]) – was 20-fold more cytotoxic (IC50 ca. 1 mM) than the empty metalla-prism against human ovarian A2780 cancer cells.38 This study provided the proof-of-concept for the so-called “Trojan horse strategy”, whereby protection of a cytotoxic agent in the cavity of a SCC is achieved until it can be released and perform its antiproliferative activity. Following these initial results, a water-soluble hexaruthenium metallacage [Ru6(p-iPrC6H4Me)6(tpt)2(C6H2O4)3]6þ was investigated for the release mechanism of encapsulated fluorescent pyrene derivatives and for its anticancer properties in vitro.48 The obtained results showed that, while the free pyrene derivative and the cage complex alone were scarcely cytotoxic, the host-guest complex was considerably more active in cancer cells.48 The increased cytotoxicity of the cage-pyrene complex was due to an increased uptake as shown by fluorescence microscopy. Interestingly, the fluorescence of the pyrene moiety is quenched upon encapsulation, allowing for the release of the molecule to be monitored by fluorescence spectroscopy.48 In further studies, the encapsulation properties of the same hexaruthenium metallacage with a series of functionalized fluorescent pyrene derivatives were characterized using NMR (1H, 2D, DOSY) spectroscopy and electrospray ionization mass spectrometry.121 The antiproliferative properties of the pyrene-cage complexes were studied in A2780 ovarian cancer cells, showing enhanced activity in cancer cells in comparison to the free cage121. The effect of the portal size of the hexaruthenium metallacage complex on the retention of the planar guest molecules, [Pd(acac)2] and 1-(4,6-dichloro-1,3,5-triazin-2-yl)pyrene, was also investigated by NMR and fluorescence spectroscopy.122 Further Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and fluorescence microscopy studies showed that the cages deliver the host to intracellular organelles and the mechanisms of uptake involve endocytosis/macropinocytosis rather than passive diffusion across the cell membrane.122 Other SCCs as drug delivery systems, based on different transition metals, include surface functionalized porous coordination nanocages of Cu2þ and 5-(prop-2-ynyloxy)isophthalic acid (pi), featuring a water solubilizing polymer (PEG5k), which were synthesized using a “click chemistry” approach.123 The Cu(pi)-PEG5k scaffold is composed of 12 di-copper paddlewheel clusters and 24 isophthalate moieties, with 8 triangular and 6 square windows that are roughly 8 and 12 Å across, respectively. The internal cavity has a diameter of ca. 15 Å and the cage is highly stable in aqueous solution. In addition, the drug loading and release capacity of the cage was evaluated for the anticancer drug 5-fluorouracil (5-FU).123 Interestingly, around 20% of the loaded drug was released during the first 2 h, while a flatter release curve can be observed up to 24 h. The slow release has been associated to the slow diffusion rate of 5-FU caused by the strong interaction between Lewis acid sites in Cu(pi) and basic coordination sites of 5-FU. Based on previous work by Fujita and coworkers,124 and within the M2L4 cage family, Crowley et al. designed a cationic [Pd2L4]4 þ cage using 2,6-bis(pyridin-3-ylethynyl)pyridine as the bidentate ligand, and characterized the system by various methods, including 1H NMR spectroscopy, ESI-MS and XRD.37 Interestingly, the encapsulation of the anticancer drug cisplatin within the metallacage cavity was demonstrated by XRD studies, revealing that two molecules of the metallodrug could occupy the cavity, lined with the nitrogen atom from the central pyridine of the ligand.37 The release of cisplatin was facilitated by the

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Fig. 5 (A) Schematic diagrams of the MNPs serving as a multifunctional theranostic platform. Structures of TPP, cPt, DSTP, M, mPEG-b-PEBP, and RGD-PEG-b-PEBP.117 (B) Ex vivo image of the main organs separated from U87MG tumor-bearing mice at 24 h post injection of MNPs. (C) PET image of U87MG tumor-bearing nude mice at 2, 4, 6, 12, 24 and 48 h post injection of 64Cu@MNPs (150 mCi). The white circle denotes the tumor site. (D) In vivo T1-weighted axial MRI images (7 T) of the mice pre-injection and after injection of Mn@MNPs. The white circle denotes the tumor site. Adapted with permission from reference Yu, G.; Yu, S.; Saha, M. L.; Zhou, J.; Cook, T. R.; Yung, B. C.; Chen, J.; Mao, Z.; Zhang, F.; Zhou, Z.; Liu, Y.; Shao, L.; Wang, S.; Gao, C.; Huang, F.; Stang, P. J.; Chen, X. A Discrete Organoplatinum(II) Metallacage as a Multimodality Theranostic Platform for Cancer Photochemotherapy. Nat. Commun. 2018, 9, 4335. Springer Nature, Copyright 2018, the authors.

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Fig. 6 (A) Schematic representation and corresponding X-ray structure (CCDC no 673,229) of a [[Ru2L’]3L2]6 þ cage encapsulating [Pt(acac)2] (acac ¼ acetylacetonato).38 (B) Schematic representation and corresponding X-ray structure (CCDC no 1,431,657) of an exo-functionalized [Pd2L4]4 þ metallacage encapsulating two equivalents of cisplatin.119(C) Schematic representation and corresponding X-ray structure (CCDC no 902,397) of a [Pd2L4]4 þ capsule encapsulating two equivalents of corannulene.120 The X-ray structures were visualized using the Mercury software.42

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introduction of competing ligands (4-dimethylaminopyridine or Cl) to disassemble the cage, as shown by 1H NMR and ESI-MS. Unfortunately, while the cisplatin-cage host-guest complex formed in acetonitrile and DMF, no host-guest interactions could be observed in more hydrogen bond competitive solvents (water and DMSO).125 Additionally, the parent Pd2þ cage decomposed rapidly in the presence of nucleophiles. It should be noted that the stability of [Pd2L4]4 þ cages when exposed to biological nucleophiles highly depends on the ligand structure, with triazole-based ligands leading to significantly more stable metallacages in comparison to pyridine-based scaffolds.89 More recently, Casini and coworkers explored similar cationic [Pd2L4]4þ systems featuring bis(pyridyl) ligands – of general scaffold 3,5-bis(3-ethynylpyridine)phenyl) – and developed the exo-functionalization of the ligands to add different functionalities, including fluorescent tags facilitating the study of the cellular accumulation of these systems by fluorescence microscopy.126,127 Structural studies by 1H NMR ad XRD were performed demonstrating encapsulation of two cisplatin molecules (Fig. 6B).119 It is worth mentioning that, at variance with the aforementioned Pd2þ cages by Crowley et al., the cavity of the [Pd2L4]4þ cages is more hydrophobic in this case, and therefore, cisplatin encapsulation was favored over occupancy of the cavity by water molecules or other polar solvents. Notably, most of the reported metallacages and their precursors were non-toxic in healthy rat liver tissue ex vivo, making them suitable for application as drug delivery systems.128 Water solubility and stability under physiological conditions are crucial for the biological application of SCCs. Unfortunately, despite their positive charge, [Pd2L4]4þ cages of this type are generally scarcely soluble in water. Therefore, different strategies have been applied to achieve water-soluble SCCs, with the most straight-forward approach relying on a simple anion exchange. For example, in the case of the organometallic pillarplexes, it was shown that their hexafluorophosphate salts, soluble in organic solvents like acetonitrile or dimethylformamide, can be easily converted into the corresponding acetates, which are well watersoluble (> 1 g/mL water).129 However, it should be noted that further exchange with physiologically available anions can further affect the solubility. When the anion exchange is not applicable or not leading to the desired effect, covalent modifications of the ligand’s exo-position can be performed to introduce polar functional groups, e.g., sulfonates130 or water-soluble moieties in the resulting cage scaffold, e.g., PEG.131 The control of the host-guest properties of the cavity defined by the SCC is another essential feature to enable the most efficient drug encapsulation. For example, anthracene-based Pt2þ- and Pd2þ-linked coordination capsules provide a characteristic spherical cavity – with a diameter of ca. 1 nm and a volume of ca. 600 Å3 – contoured by polyaromatic frameworks (Fig. 6C),120,132 and that can accommodate various neutral molecules, through hydrophobic and p-stacking interactions, in aqueous solution.120,132,133 Fluorescence microscopy studies allowed investigation of the intracellular accumulation of the capsules.133 However, these systems, even without their guest molecules, manifest very pronounced cytotoxic effects, which make them unsuitable for drug delivery. Interestingly, the observed trends in the anticancer activity of the capsules and their host-guest complexes correlate with their different stabilities towards glutathione, estimated by NMR-based kinetic experiments.133 The data suggest the glutathionetriggered disassembly of the capsular structures in cells as a potential activation pathway for their cytotoxic activity. For supramolecular metallacages of the [Pd2L4]4þ type, with a molecular weight of ca. 2–3 kDa and diameter of ca. 10–15 Å, passive tumor targeting via the EPR effect is not likely to influence their delivery.134 In this context, active tumor targeting mechanisms are crucial to achieve selectivity of metallacages for cancerous cells, for example via the conjugation of cancer-cell-specific ligands. However, this concept has been scarcely explored so far and few examples of bioconjugated cages are available in the literature.135 In 2017 Casini and coworkers reported on the first example of bioconjugation of self-assembled [Pd2L4]4þ cages via amide bond formation between the eCOOH (or eNH2) exo-functionalized ligand/cage and a complementary residue on a model linear peptide.136 It should be noted that tethering of the peptide to the metallacages also enabled to increase their solubility in aqueous environment. Afterwards, in a proof-of-concept study, [Pd2L4]4 þ cages conjugated via amide bond to integrins binding ligands were synthesized and studied for their integrin recognition properties, showing to maintain high binding affinity and selectivity.137 Cage formation and encapsulation of cisplatin was proven by 1H NMR, 1H DOSY and 195Pt NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing avb3 integrins, while it was not active against A549 human lung cancer cells lacking this specific integrin.137 Moreover, ex vivo studies showed reduced toxicity of cage-encapsulated cisplatin towards healthy rat liver and kidney tissues. ICP-MS studies suggested that such reduced toxicity is due to the lower metal accumulation of the encapsulated drug in these organs compared to the ‘free’ cisplatin.137 Certainly, other types of exo-functionalization, other than amide bond formation, for tethering metallacages to peptides or antibodies should be investigated, including click chemistry approaches.138,139 Isaacs and Stang conjugated 4,40 -bipyridinium to the metallacages and successfully used these platforms as supramolecular guests for cytotoxic drugs/prodrugs such as DOX and curcumin.140,141 For example, a water-soluble hexagonal Pt(II) metallacycle, cucurbit[8]uril, encapsulating hydrophobic curcumin, exhibited enhanced anticancer activity against melanoma and breast cancer cells compared with the corresponding precursors.142 Finally, concerning PDT applications, a few examples were reported on the use of 3D Ru2þ and Pt2þ metallacages to host porphyrin-based photosensitizers.142,143 The latter are protected from light during the transport, but also facilitated in their delivery to cancer cells. Thus, discrete Pt2þ metallacages containing a platinum-based anticancer drug encapsulated a photosensitizer through noncovalent interactions. The host-guest complex was further embedded in an amphiphilic copolymer, forming nanoparticles. Finally, a targeting ligand was introduced in the nanoparticles through an in situ copper-free click reaction, enabling the

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Fig. 7 (A) Structure of BODIPY-cage ref.144 and (B) Confocal Laser Scanning Microscopy (CLSM) images of fixed human A375 melanoma cells pre-treated with ligand or cages (C1.BF4 and C1.NO3) for 2 h at 37  C. Scale bar represents 20 mm. Data represents maximum projection images. (C) Scheme of the Ru-based BODIPY rectangle reported in reference145 and its monocrystal X-ray structure (CCDC no 1,542,145). The X-ray structure was depicted using the Mercury software.42 Adapted with permission from reference Woods, B.; Döllerer, D.; Aikman, B.; Wenzel, M. N.; Sayers, E. J.; Kühn, F. E.; Jones, A. T.; Casini, A. Highly Luminescent Metallacages Featuring Bispyridyl Ligands Functionalised with BODIPY for Imaging in Cancer Cells. J. Inorg. Biochem. 2019, 199, 110781.Copyright 2019 Elsevier Inc.

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concomitant delivery of cytotoxic Pt2þ and PS to cancer cells overexpressing avb3 integrins.142 The nanoparticles exhibited a superior antitumor performance on a drug-resistant tumor model by combining chemotherapy and PDT.

2.22.3.3

Imaging

Imaging probes can be incorporated into SCCs for in vitro and in vivo imaging to trace their cellular accumulation, sub-cellular distribution, biodistribution and excretion. To this aim, various strategies have been exploited, such as chemical modification of the cage scaffold, direct coordination self-assembly, physical encapsulation, and host-guest recognition. As an example, fluorophores with high quantum yield could be conjugated to the coordination building block, thus, introducing imaging capability into the SCCs. For example, Casini and coworkers exo-functionalized 3,5-bis(3-ethynylpyridine)phenyl ligands with highly emissive boron dipyrromethene (BODIPY) moieties (Fig. 7A and B).144 Using a similar approach, the same group also explored the exo-functionalized with different luminescent groups, including anthracenyl groups and Ru(II) polypyridyl ligands.126,127 However, while the former did not display sufficient luminescence due to the lowered probability of p-p* excitation upon conjugation of the fluorophore to the ligand scaffold,126 the latter achieved strong emission only upon excitation in the UV region,127 which is not ideal for conventional microscopy analysis. Fluorescent probes with rigid structures are inherent coordination donors when modified with carboxylate or pyridine groups, such as BODIPY, tetraphenylethane (TPE), and porphyrins.146,147 As a representative example, thiophene-based BODIPY Ru(II) rectangles were reported by Gupta, Mandal, Lee and coworkers constructed using dinuclear ruthenium-arene precursors and a thiophene-functionalized dipyridine BODIPY ligand (Fig. 7C), and the intracellular distribution of the resulting metallacycle was studied by fluorescence microscopy.145 A highly emissive Pt(II) supramolecular triangles bearing a pyridine-functionalized BODIPY ligand via coordination-driven self-assembly was also reported by Cook and coworkers for theranostic studies, whereby the platinum acceptors were toxic chemotherapeutics and the BODIPY donor was the imaging probe and photo-sensitizer.148 In vitro studies demonstrated that the formation of metallacycles improved the anticancer efficacy of the Pt2þ moieties, and the combination of PDT and chemotherapy showed excellent synergistic effect. Metallacages with hollow cavities are able to complex diagnostic guests, making the host-guest recognition an effective strategy to visualize the metallacages. For example, Therrien et al. constructed water-soluble arene ruthenium metallacages and used them as supramolecular hosts to encapsulate hydrophobic porphyrins.149,150 The cellular endocytosis of the supramolecular complexes and the porphyrin’s release after cellular internalization were investigated by fluorescence microscopy in vitro. The possibility to exploit the host-guest chemistry of SCCs for imaging purposes has been demonstrated by Lusby and Archibald and coworkers, who used a kinetically robust [Co4L6]11þ tetrahedron (L ¼ 5-(5-bipyridin-2,20 -yl)-2,20 -bipyridine or 50 -(40 -amino(2,20 -bipyridin)-5-yl)-(2,20 -bipyridin)-4-amine)) to encapsulate the g-emitting [99mTc]TcO4 anion under conditions compatible with in vivo administration.151 Subsequent single-photon emission computed tomography (SPECT) imaging of the caged-anion in mice revealed a marked change in the biodistribution of the host-guest system compared to the thyroid-accumulating free oxo-anion (Fig. 8A and B). Further optimization of the concept has been recently published by Casini and coworkers, whereby [Pd2L4]4þ cages (L ¼ 3,5-bis(3-ethynylpyridine)phenyl ligand) tethered to a blood brain barrier (BBB)-translocating peptide were synthesized by a combination of solid phase peptide synthesis and self-assembly procedures.152 The cage translocation efficacy was assessed by Inductively Coupled Mass Spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4] 3 cage] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[TcO4] 3 cage] complex (Fig. 8C).152 This study constitutes another proofof-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.

2.22.4

Synthesis of metal-organic frameworks (MOFs)

Closely related to the classical coordination polymers pioneered, the MOF concept was based on the work of Robson,153 who introduced the concept of coordination networks and reticular synthesis, and established by Yaghi, O’Keeffe and collaborators in the 90s154 for a microporous cobalt 1,3,5-benzenetricarboxylate as first categorized as a MOF.155 The idea was shortly after expanded to other 3D frameworks.156 As the discrete SCCs, MOFs are coordination-driven self-assembled entities157,158; however, in MOFs, the combination of metal center nodes and multi-dentate ligands with defined structure allows the formation of polymeric 3D extended networks.159 These materials have attracted attention firstly as platforms with remarkable and tunable porosity.160–162 By the choice of the building units, the size and shape of the pores within the scaffold can be ideally designed and controlled.163 Differently from other porous materials, such as zeolites and silica, the ligands used to form MOFs can be tailored in order to control not only the size and shape,164 but also the chemical character (i.e., acidity, hydrophobicity, charge) of the pores.165,166 These characteristics have been exploited in the early application of MOFs in separation, and storage.167 Furthermore, this freedom in the chemical composition has allowed the inclusion of additional functionalities, making of MOFs excellent potential multifunctional materials with current prospective applications in catalysis,168–170 environmental science,171 energy,172,173 sensing,174–179 electronics180 and biomedicine.9,181–183 This rising interest in MOFs is reflected in the growing number of systems reported to date, the November 2020 release of the Cambridge Structural Database includes more than 105,922 structures characterized as

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Fig. 8 (A) Structure of anion-binding CoIII4L6 cages shown exemplarily at the X-ray structure of a tetrahedral SCC, featuring an encapsulated ReO4 (CCDC no 1,864,366)151 The structure was visualized using the Mercury software.42 (B) Comparison of free [99mTc]TcO4 uptake in naïve mice (left) vs SCC-encapsulated [99mTc]TcO4 (right) monitored by SPECT imaging.151 Encapsulation results in reduced thyroid and stomach uptake, and increased liver uptake. Images are maximum intensity coronal projections. S ¼ Stomach, Th ¼ Thyroid, L ¼ Liver. (C) The optimized structure of a model [Pd2L4]4 þ metallacage encapsulating the TcO4 anion, obtained by DFT methods. The cage is shown in stick representation and TcO4 is shown in VDW representation (C ¼ grey, N ¼ light blue, O ¼ red, Pd ¼ dark blue, Tc ¼ teal. Hydrogens omitted for clarity). (B) Adapted with permission from Burke, B. P.; Grantham, W.; Burke, M. J.; Nichol, G. S.; Roberts, D.; Renard, I.; Hargreaves, R.; Cawthorne, C.; Archibald, S. J.; Lusby, P. J. Visualizing Kinetically Robust CoIII4L6 Assemblies in Vivo: SPECT Imaging of the Encapsulated [99mTc]TcO4–Anion. J. Am. Chem. Soc. 2018, 140, 16877–16881. Copyright 2018 American Chemical Society.; (C) Adapted with permission from Woods, B.; Silva, R. D. M.; Schmidt, C.; Wragg, D.; Cavaco, M.; Neves, V.; Ferreira, V. F. C.; Gano, L.; Morais, T. S.; Mendes, F.; Correia, J. D. G.; Casini, A. Bioconjugate Supramolecular Pd2þ Metallacages Penetrate the Blood Brain Barrier In Vitro and In Vivo. Bioconjug. Chem. 2021, acs.bioconjchem.0c00659. Copyright 2021 American Chemical Society.

MOFs.184 As in all emerging knowledge branches, the development of a common language encounters some difficulties; moreover, MOFs are highly diverse materials. As a consequence, the nomenclature of the MOFs is in most cases arbitrary, frequently composed by an acronym referring to the material type simply as MOF, IRMOF (isoreticular MOF),185 or ZIF (zeolitic imidazolate framework),186,187 in other cases the discovery place is indicated such as in CAU (Christian-Alberchts University),188 DUT (Dresden University of Technology),189 HKUST (Hong Kong University of Science and Technology),190 MIL (Matériaux Institut Lavoisier),191,192 NOTT (Nottingham University),193 NU (Northwestern University)194 UiO (University of Oslo).195 Those acronyms are commonly followed by a number. In some cases, when isostructural networks are formed by different metal centers the same framework name is retained clarifying the included metal unit (i.e., Bi-NU-901196). Attempting a universal system of nomenclature, classification, identification, and retrieval of these topological structures, O’Keeffe et al. developed a system of symbols for the identification of three periodic nets of interest, and this system is now in wide use.197 Although the choice of the MOFs’ building units and the use of the directional metal center coordination are first principles in designing the MOF structure, in practice, the synthetical conditions and the crystallization kinetics play also a key role in the final structure of the networks.166,198,199 The first synthetical approach to MOFs relied upon non-solvothermal classical direct crystallization. By these techniques, several representative MOFs were firstly achieved: MOF-5, MOF-74, MOF-177, HKUST-1, or ZIF-8. Solvothermal methods, whereby the MOF formation is carried out into an autogenously pressurized vessel at temperatures above the solvent boiling point, have shown to be able to favor the formation of porous networks which are not kinetically preferred and with improved thermal stability when compared to low-temperature direct crystallization which often leads to densely packed, interpenetrated or unstable arrangements.163,198 The MOFs’ network formation depends on diverse parameters: reagents molar ratio, solvent, pH, temperature, pressure, and reaction times. Thus, the optimization of experimental parameters leading to materials with desirable properties is an extenuating work. In order to accelerate the discovery of promising materials and optimize synthetical constraints the use of high-throughput (HT) synthesis is an extended practice.163,199–201 The rise of automatization and machine learning in combination with these screening methodologies has recently opened new horizons in speeding up the discovery of MOFs.202–204 Alternative sources of energy have been explored to generate MOFs. How energy is provided to the systems also affects the output of the reactions. So far, it has been demonstrated that some MOFs can be synthesized using methodologies other than conventional induction heating, in particular microwave, mechanical, ultrasonic, and electrochemical synthesis have been reported.163,185,199,205 Once again, the different methods may lead to different material properties, in particular regarding the reaction times, particle size,

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Fig. 9 (A) Multi-stimuli-responsive nanoplatform for drug delivery based on MIL-101. (B) In vivo tumor growth inhibition and (C) body-weight change upon treatment with the DOX@MIL-101 platform (TTMOF) in comparison with controls. Reproduced from Ref. Wang, X. G.; Dong, Z. Y.; Cheng, H.; Wan, S. S.; Chen, W. H.; Zou, M. Z.; Huo, J. W.; Deng, H. X.; Zhang, X. Z. A Multifunctional Metal-Organic Framework Based Tumor Targeting Drug Delivery System for Cancer Therapy. Nanoscale 2015, 7, 16061–16070, with permission from The Royal Society of Chemistry.

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energetic efficiency, scalability, and cleanliness of the process (avoiding the use of solvents).206 The use of microwave-assisted synthesis for producing MOFs has as advantage a more precise control of the temperature and pressure of the reaction media and shorter reaction times. Additionally, fast nucleation processes have been reported, making this method suitable for the generation of nanosized crystals that are attractive for applications such as drug delivery and imaging (vide infra).207,208 Mechanical synthesis is typically carried out in the absence or with trace quantities of solvents in relation to the conventional solutionphase methodologies.209 Thus, the former represents a greener alternative to the synthesis of chemical products. Ultrasounds can also be used as an energy source in MOF synthesis they act by generating local points of extremely high pressure and temperature in which reactive species can be formed leading to chemical reactions such as the MOF formation.199 The application of this method for the synthesis of MOF is attractive due to the easiness and accessibility of ultrasonic sources. To date, some successful protocols for the use of ultrasound in MOF synthesis have emerged, but, differently to the previously mentioned methods, they normally do not lead to unknown phases and are most effective for the synthesis of frameworks readily synthesized under conventional precipitation at room temperature. As mentioned above, one of the main advantages of MOFs is their virtually infinite possible structures and properties. Nevertheless, the majority of the existent systems are formed by a handful of ligand types, i.e. carboxylates, phosphonates, imidazolates, and amines.198,199 When it comes to the synthesis of MOFs for biomedical applications, the need for diversifying building blocks arises great interest.9,181 Two main strategies have been followed in the design of MOFs for biomedical purposes. The first and probably more natural approach was the creation of innocent MOF platforms as host materials for the carrying and delivery of drug and diagnosis agents.8 To this end, the use of low toxicity precursors as building blocks for the MOFs constructions was explored. The application of bio-metals (Fe, Co, Mn, Cu, Zn, etc.,) and biomolecules (nucleobases, peptides, proteins, carbohydrates, etc.,) as polytopic ligands produces platforms that are a priori biocompatible, granting fast clearance or reabsorption times for the generated materials.8 As a second design strategy, the selection of bioactive substances for the construction of MOFs makes them suitable not only as potential drug carriers but also as therapeutic agents. Moreover, this kind of more flexible design allows the inclusion of coadjuvant therapeutic agents or imaging reporters into the same platform.210 In the design of MOFs for drug delivery, the sizes and shapes of the materials is also relevant as they are determinant of the uptake, clearance, and sub-cellular delivery properties of the resulting material.211 The construction of nanosized MOF structures can also be achieved by choosing the proper synthetical strategy.181,212 Moreover, the improvement of the material’s chemical and colloidal stability in the biological medium is another challenge that can be readily approached in terms of the surface functionalization of nanoMOFs.213 Post synthetical modification of MOF platforms has been proposed as a strategy both for the modification of the complete MOF structure or for the partial substitution of ligands allowing the introduction of additional functionalities into the MOFs.214,215 Finally, nanocomposites and hierarchical hybrid materials have been recently proposed for the improvement of MOF-based biomedical applications.216–218

2.22.5

Biomedical applications of MOFs

2.22.5.1

Drug delivery

As mentioned in previous sections, the focalized efficient transport, protection, and controlled release of active pharmaceutical principles is a topic of interest. Porous materials such as MOFs have potential in this area, the tunable physicochemical character of the pores of MOFs has been consistently stated as one of the strengths of these materials in drug delivery applications.219,220 Such tunability was recently systematically studied by Ni, Zang, and coworkers,221 who analyzed the effect of different functionalization on the MOF-5 structure over the loading and release capabilities using oridonin as a cargo drug,222 showing important differences depending on the functionalization of the MOF ligand units. Another desirable characteristic of MOFs as DDS is the possibility to design them with stimuli-responsive behavior, whereby the responsiveness of the material to environmental stimuli can generate controlled delivery of the cargo. In this framework, the groups of Yang and Qiang reported a cationic MOF-based nanocarrier (ZJU-101, ZJU ¼ Zhejiang University),223 obtained by the post synthetical N-methylation of the zirconium-based MOF-867224; in contrast with the starting material, ZJU-101 is able not only to load 0.546 g/g of diclofenac sodium (DS) but also shows pH-responsive release. At acidic pH, characteristic of inflamed tissue (pH ¼ 5.4) the release of DS is nearly 3 times faster than at normal physiological pH (7.4). This feature, along with low observed cytotoxicity, has led the authors to propose the system as a potential anti-inflammatory DDS.223 To ensure the colloidal stability of nanosized MOFs under physiological conditions, surface functionalization with polymeric moieties, such as PEG, is commonly applied. In some cases, this functionalization is innocent, although it is known to modify the biodistribution. Horcajada and Férey have shown a longstanding interest in a promising family of non-toxic, biocompatible iron carboxylate MOFs, members of the MIL family,225 and in 2010 a study was reported regarding the potential of PEG-coated MIL nanoparticles as drug carriers for some challenging anticancer and antiviral drugs (busulfan, azidothymidine triphosphate, cidofovir, and DOX) and other molecules of cosmetic interest. Due to their innocent composition, these systems are highly biocompatible. Moreover, the systems show a sponge-like behavior, loading the tested drugs, with efficiency up to 80%, by simple soaking and achieving loadings considerably exceeding those of other nanocarriers. Further, the progressive degradation of the material by the disassembly of the MOF allows a slow release of the drugs in physiological conditions both in vitro and in vivo.225 That the surface PEGylation of MOFs can provide more than just colloidal stability to MOF-based DDS has been shown by the group of Forgan.226 Indeed, UiO-66195 nanoparticles were subjected to selective Cu-catalyzed click surface PEGylation.226 The

Supramolecular metal-based molecules and materials for biomedical applications

Fig. 10 (A) SEDM images of the rod-shaped Gd3þ nanoMOFs. (B) MRI contrast of Gd3þ nanoMOFs compared with the commercial agent Omniscan™. (C) Visible emitted light due to the doping of the nanoMOFs with 5% emissive rare-earth (Eu or Tb) compared to the pure Gd formulation. Adapted with permission from Rieter, W. J.; Taylor, K. M. L.; An, H.; Lin, W.; Lin, W. Nanoscale Metal-Organic Frameworks as Potential Multimodal Contrast Enhancing Agents. J. Am. Chem. Soc. 2006, 128, 9024–9025. Copyright 2006 American Chemical Society.

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resultant PEG-UiO-66 NPs release calcein, a model drug similar to DOX, slower at pH ¼ 7.4 in comparison with more acidic conditions (pH ¼ 5.5).226 Studies of this phenomenon suggest that at pH ¼ 7.4, a phosphate corona stabilized by the PEG is formed. At pH 5.5 this process is not favored, allowing a fast carboxylate protonation and disassembly of the MOF.226 Another example of the improvement of MOFs as DDS by surface functionalization was shown for the MIL-100 Fe3þ trimesate nanoparticles, which, due to heparin surface coating, have been turned stealth,227 and were able to escape the immune cells, enhancing the MOFs circulation time. These results confirmed that the heparin coating endowed the MOFs with other improved biological properties, such as lack of complement activation and reactive oxygen species production. Furthermore, the photosynthetic functionalization has been done following a one-pot highly efficient and biocompatible protocol.227 Overall, the ability to coat the surface of the MOFs using a simple and straight-forward approach could be taken as a way to enhance their versatility and, thus, the potential of porous MOF nanoparticles in biomedicine. Completely functional drug delivery systems must ensure not only efficiency and control in the loading and release of the cargo drugs, but precise biodistribution and targeting capabilities are also desirable. A smart nanoplatform for targeted anticancer drug delivery based in MIL-101 was reported by the groups of Zhang and Deng,228 whereby, using the azide functionalized MOF (MIL-101-N3(Fe)), the carrier synthesis and loading were performed in a one-pot reaction avoiding non-biocompatible solvents (Fig. 9). After the loading of the MOF with DOX, the particles’ surface was functionalized with a disulfide bearing a b-cyclodextrin (b-CD) using copper-free click chemistry. Finally, adamantane functionalized avb3 integrin targeting peptide polymer including a benzoic imine bond (K(ad)RGDS-PEG1900) was supramolecularly coupled to the particles by the adamantane-b-CD hostguest interaction.228 The inclusion of the stimuli-responsive redox (disulfide) and pH (imine) sensitive linkers were designed to provide the system with a smart behavior. The cleavage of the imide bonds is accelerated at acidic pH, as observed in tumors, once the imine is broken the integrin-targeting peptide gets exposed and the uptake increased selectively in the tumoral region.228Once inside the cell, the higher concentration of glutathione is expected to cleave the disulfide linker, eliminating the b-CD functionalization that, until that moment and combined with the PEG corona prevented the extracellular leaking of the cargo drug. The system inhibited tumor growth in vivo similarly to free DOX but presented significantly reduced systematic toxicity, as indicated by the changes in bodyweight.228 This is in line with other studies on glutathione-responsive cyclodextrin-nanosponges encapsulating DOX.229 Even if MOFs are known to be outstanding porous materials and the pore sizes can be modulated with the used linkers, the generation of cavities ranging above 10 nm carries instability problems.230 This is limiting MOFs applications as DDS, particularly when higher quantities of cargo or large molecules have to be encapsulated and carried. The rise of hollow structures constructed by templated methods231–233 with MOFs opens new opportunities, enabling the obtainment of larger cavities within the MOF structures. Examples of these systems have been explored by the group of Liu and Damarin,234 who, using competitive coordination control of the MOF formation, reported the construction of hollow Fe-MOF-based spherical microcapsules (MBMs). The MBMs, portraying inner cavities of ca. 200 nm and walls of ca. 50 nm, were loaded with 5-fluorouracil, demonstrating a payload capability by impregnation in solution of 77%. The release of the drug was also pretty efficient, reaching 83%.234 In vitro, the system showed important selective inhibition of A2780 human ovarian cancer cells when compared to HL7702 non-tumorigenic ones. Similarly, in vivo tumor growth inhibition was observed when the MBMs were used as DDS carriers for 5-fluorouracil, administrated by injection via the bilateral armpits of the forelimb.234 Besides therapeutic drug delivery, MOF platforms have attracted attention as vehicles for encapsulating, protect, and transport vaccine immunogens. In 2016, Zhang et al. the first example of a MOF-based vaccine platform.235 In detail, they utilized ZIF-8236 to encapsulate ovalbumin (OVA), a well-established model antigen. The OVA@ZIF-8 particles were electrostatically coated with cytosine-phosphate-guanine oligodeoxynucleotides, to increase the biocompatibility and exert adjuvant effects. The system induced strong cellular and humoral immunostimulatory activity, furthermore, immune memory was observed in a second exposure event.235 This study has pioneered the use of MOFs as vaccine platforms endowed with excellent protection to immunogens.237,238

2.22.5.2

Imaging

The versatile character of MOFs has also inspired their use in imaging and diagnosis. The application of these systems as markers for optical imaging (OI), computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and photoacoustic imaging (PAI) has been reported.239 In general, the utilization of MOFs for these applications could be achieved by (i) exploiting their carrier capabilities to load and protect fluorophores and contrast agents, and (ii) designing MOFs bearing building blocks with intrinsic imaging capabilities. Benchmark contrast agents (CA) for CT are typically based on heavy elements, such as iodine and barium, and are usually administrated in high doses in order to get good contrast causing undesirable side effects.240 Iodine-BODIPY functionalized UiO-66 MOF nanoparticles195 reported by Zhang et al. have been evaluated as CT contrast agents due to their content of iodine as attenuating atoms.241 The MOF nanoparticles showed excellent attenuating capabilities, deriving in good contrast, particularly in tumoral regions and more important nonobvious toxicity was detected in vivo241 More recently, exchanging Zr by Bi, the latter is known as one of the safer heavy metals for humans, the group of Farah242 has synthesized a structural analogous of the NU-901 MOF.196 The system gives sevenfold better attenuation than the commercial contrast agent iodixanol, under 70 kV X-ray irradiation tested in agarose suspension.242 The translation of the material to the practice requires the creation of protocols for the synthesis of nanosized particles of the material and detailed biodistribution and biostability studies.

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Fig. 11 (A) Synthesis of the biomimetic UiO-66-based combined PDT/O2 therapeutic system. (B) In vivo tumor growth inhibition and (C) bodyweight change and (D) representative tumor size photography upon treatment with the platform and control groups of partial therapy. Adapted with permission from reference Gao, S.; Zheng, P.; Li, Z.; Feng, X.; Yan, W.; Chen, S.; Guo, W.; Liu, D.; Yang, X.; Wang, S.; Liang, X.-J.; Zhang, J. Biomimetic O2-Evolving Metal-Organic Framework Nanoplatform for Highly Efficient Photodynamic Therapy against Hypoxic Tumor. Biomaterials 2018, 178, 83–94. Copyright 2018 Elsevier Inc.

Contrast agents for MRI applications are normally composed of paramagnetic nuclei, capable of reducing the relaxation times of water protons detected in the experimental setup. Gadolinium chelates are the usual CA for MRI. The substitution of molecular CA by nanomaterials is pursued in order to increase the local concentration of contrast nuclei and extend the retention times in vivo, and recently, MOFs have attracted attention in this sense.243,244 Boyes and coworkers synthetized Gd-MOF rod-like nanoparticles by reverse emulsion method, showing control of the particle’s morphology by the inclusion of hydrotopes.245 Further, the Gd-MOF particles showed 20-fold higher relaxivity (R) values when compared to the commercial agent (MagnevistÔ). The effects of the particle morphology over the MRI-R enhancement were also studied finding a general trend to the increase of R when the rod length decreases. The same group has previously shown that also surface functionalization of this type of particle can enhance their potential as MRI contrast agents.246 The interest in the incorporation of Gd in MOF nanoplatforms for MRI imaging has been reflected in the recent filling of two patents in this regard.247,248 On the other hand, the encapsulation of Gd-based MRI-CA into MOFs has shown recently an important enhancement of the performance.249

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Fig. 12 (A) Synthesis of the DOX & ICG@H-PMOF NPs@membrane multifunctional platform (DIHPm) for cancer therapy in vivo. (B) Action mechanisms proposed by the system: the homologous membrane functionalized particles enter the cell where, by a combination of pH and thermal activation, they release the cargo chemotherapeutic and photothermal drugs. (C) In vivo and ex vivo (at 24 h) NIR fluorescence imaging showing the preferential accumulation of DIHPm in the tumor. Sp, Ki, He, Lu, Li, and Tu stand for spleen, kidney, heart, lung, liver, and tumor, respectively. Adapted with permission from Sun, X.; He, G.; Xiong, C.; Wang, C.; Lian, X.; Hu, L.; Li, Z.; Dalgarno, S. J.; Yang, Y. W.; Tian, J. One-Pot Fabrication of Hollow Porphyrinic MOF Nanoparticles with Ultrahigh Drug Loading toward Controlled Delivery and Synergistic Cancer Therapy. ACS Appl. Mater. Interfaces 2021, 13, 3693. Copyright 2021 American Chemical Society.

Photoacoustic imaging is an emerging technique based on the detection of acoustic waves generated upon pulsed light-induced thermal expansion and contraction of chromophores.250,251 Although, the use of endogenous molecules (particularly melanin and hemoglobin) as chromophores is possible, to gain resolution in the image exogenous CA can be followed.251 Tumor targeting Porphyrin-Pd Hydride MOF nanoparticles (96 nm) were applied in photoacoustic imaging by the group of He.252 The studied particles with photothermal activity showed also an important photoacoustic effect, motivating their application in vivo as PAI CA, due to the passive targeting effect, 1 h after the injection the particles accumulate in the tumoral region giving a sustained signal for 24 h. In situ hydrogen thermal reduction was used as a complementary therapy.252

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Finally, optical imaging, relying on the use of visible light emitters has a limited application due to the poor penetration of most of the visible and UV-light, needed for the sample excitation, into tissues.253 In this context, the introduction of fluorophores into MOF platforms is desirable to facilitate its tracking without additional labeling in vitro and ex vivo. The number of examples and the strategies followed to generate luminescent MOF materials have been extensively revised recently.254–258 Most of these systems are not designed only as OI labels, but instead, the luminescent response is accessorial to more complex multimodal platforms. For example, Rieter et al.259 synthesized 1,4-benzene dicarboxylate Gd3þ rod-shaped nanoMOFs (100 nm  40 nm and 1 mm  100 nm) as MRI contrast agents with large relaxivities, when compared against the commercial contrast agent OmniscanÔ. They also demonstrated the possibility of rendering these systems also photoluminescent by introducing during the synthesis 5% mol of Eu3þ or Tb3þ, whose metal-centered emission enabled multimodal imaging MOF-based platforms (Fig. 10).259

2.22.5.3

Combined therapy and theranostics

Given the resourcefulness scenario given by MOFs as functional platforms for imaging and drug delivery and the virtually limitless possibilities of modification and creation towards new functional materials, MOFs are ideal platforms for the construction of a multipurpose system.8,165,182 In this regard, many studies showing the combination of distinct functionalities in MOF-based materials have appeared in recent years,239,260 which will be discussed in this section. Next, some examples of the synergistic therapeutic and theranostic approaches that emerged lately are presented. One of the most promising theranostic platforms for drug delivery and imaging is the iron carboxylate nanoMOFs, whereby the presence of Fe3þ as paramagnetic nuclei makes them good MRI CA225 Besides the excellent porosity and cargo loading capabilities, these materials have shown remarkable biocompatibility.261 One of these MOFs (MIL-100(Fe)) was recently used as a platform for multifunctional phototheranostic nanoparticles.262 The nanoparticles were surface-functionalized with hyaluronic acid to grant them targeting towards CD44 cancer cells. In order to provide photothermal activity, together with the possibility of integrating PAI and fluorescence into the system indocyanine green was loaded into the platform. Guided by the accessorial OI, PAI, and MRI the applied photothermal therapy showed an efficient suppression of MCF7 tumor growth in vivo.262 MOF-based nanoparticles have also been applied as photosensitizers (PS) or PS hosts for PDT.263 Usually, PDT is applied synergistically with chemotherapy or other treatments. While PDT is effective for spatially localized therapy, it’s inefficient for the treatment of metastasis. The group of Webin Lin studied the combination of MOF-based PDT with immunotherapy achieving tumor regression in vivo both in the primary PDT target and in distant untreated tumors.264 The PS platform, a chlorin-based nanoscale MOF containing homometallic hafnium secondary building units was loaded with a blockade immunotherapeutic Indoleamine 2,3-dioxygenase inhibitor (IDOi). Once released by the MOF, the IDOi disturbs the immunosuppressive environment in the tumors presenting PDT-induced cellular death. The massive cellular death triggers a tumor-specific T-cell response which affects both treated and untreated tumors.264 A common contrast agent for PET is 18F-fluorodeoxyglucose, featuring increased concentration in regions with enhanced metabolic activity (e.g., tumors) enabling their targeted detection. In order to generate MOF-based alternatives, the use of 89Zr-UiO-66 has recently been proposed by the Hong’s group.265 The particles were functionalized with Polyglycolic acid-PEG bearing a peptide ligand for targeting triple-negative breast tumors. Next, the platform was loaded with DOX, generating a chemotherapeutic PET CA platform. In vivo and ex vivo studies confirm an efficient targeting along with low toxicity for the material and its capability to deliver DOX to the tumors.265 An interesting core-shell Prussian blue (PB)-MOF system for multiple imaging-guided cancer therapy was presented by Wang et al.,266 showing the possibility of integrating MOFs with different properties in a single nanoplatform. The system was constructed by growing a 20 nm shell of ZIF-8236 over preformed PB nanoparticles 100 nm) the porous shell structure was charged with DOX, allowing loading of 0.857 mg per gram of material.266 The high iron density on the PB core allowed the use of the system as MRI CA. Additionally, responsive photothermal and pH-dependent chemotherapeutic drug release was proved both in vitro and in vivo. The presented chemo-thermal anti-tumoral therapy demonstrates a synergetic tumor inhibition (V/V0 ¼ 0.62) showing the potential application of core-shell systems in multifunctional theranostics.266 In the last years, “gas therapy” has shown promising effects in cancer treatment, including improving the efficacy and diminish undesirable side effects.267 In particular, the delivery of oxygen can decrease the tumoral hypoxia, incrementing the efficacy of oxygen-mediated therapies.267,268 MOFs excel in gas absorption and transport,167 presenting a first-choice platform for the direct transport of oxygen. UiO-66 was recently used to prepare a biomimetic nanoplatform with oxygen transport and photodynamic therapy applications. To this end, Indocyanine green sulfonic acid was coordinated to the surface of the nanoMOF.268 After loading with O2, the nanoparticles were encapsulated inside red blood cells membranes in order to improve their biocompatibility and render them invisible to the immune system. The material presents an increased circulation time which ensures an enhancement on the EPR passive targeting effect. The system is activated by 808 nm NIR irradiation, which firstly degrades the cell membrane. Afterward, the photothermal effect triggers the release of O2 together with the PDT pathways (Fig. 11). The nanoplatform showed almost full tumor ablation without an obvious change in the body weight, and with no-apparent organ damage, indicating good biocompatibility of the therapy.268 Using a similar immune camouflage strategy, enabling passive EPR-induced accumulation at tumor site, the group of Qu and Ren prepared a glucose oxidase nanoreactor for dual starvation and chemotherapeutic.269 The RBC disguised nanomaterial consists of 120 nm ZIF-8 nanoparticles embedded with glucose oxidase (GOx) and tirapazamine (tpz) a prodrug that generates highly toxic radicals under the hypoxia conditions usually found in the tumors. The action of GOx diminishes the availability of glucose and

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consumes oxygen starving the tumoral cells and increasing the hypoxia that activates the TPZ. The MOF nanoreactor showed to maintain the GOx. While separated therapy models (GOx@ZIF-8 or TPZ@ZIF-8, respectively) presented modest tumor growth inhibition in vivo (63.5% and 77.2% respectively), the combined system showed 97.6% inhibition of growth when studied in colon CT26 cancer.269 As mentioned before, porphyrin-based metal-based platforms are promising due to the photophysical properties of porphyrins. In this sense, a high-performance multitherapeutic porphyrinic-platform has been constructed very recently.270 The system consists of a hallow structure synthesized by a one-pot self-sacrificial digestion of ZIF-8 NPs in the presence of tetrakis(4-carboxyphenyl) porphyrin (TCPP). The hallow porphyrinic MOF (H-PMOF) enables very high (635%) drug loading (DOX and ICG). The inclusion of DOX as a chemotherapeutic drug and ICG as a photothermal agent, together with the capabilities of the TCPP as a photosensitizer for the production of ROs and NIR marker makes this system a very complete platform. Furthermore, the drug-loaded H-PMOFs were coated with murine mammary carcinoma (4 T1) cell membranes providing the system with homologous tumor-targeting ability (Fig. 12).270 The multifunctional platform (DOX & ICG@H-PMOF NPs@membrane, DIHPm) was tested in vivo, showing first by NIR fluorescence imaging a preferential targeted accumulation in the tumor, in contrast with the non-membrane bearing particles (DIHP) whose EPR-promoted accumulation is considerably lower. Moreover, the combined (PDT/PTT/chemotherapy) platform performs superiorly to the control groups of partial functionality in inhibiting the tumoral growth and preventing distant metastases.270

2.22.6

Conclusions and perspectives

The past decades have witnessed the progress of supramolecular-based molecules in biomedical applications, especially in cancer theranostics. By regulating the functions of the individual building blocks and the geometry of their linkages, diverse nanomaterials with unique and enhanced properties could be achieved. The bioactive nature of the metals or metal complexes also determines the final functions of the SCCs, for example, the widely used Pt and Ru precursors can hold excellent intrinsic anticancer activity. Certainly, the unique structural property of most of the metallo-supramolecular assemblies is the presence of a discrete cavity that renders a whole range of additional applications resulting from specific host-guest interactions. Thus, not only drugs can be loaded in the supramolecular guests’ cavities, but also imaging probes can be incorporated into the materials, making their delivery and distribution trackable in cells/tissues. The therapeutic performance of supramolecular scaffolds is substantially improved by introducing other modalities through covalent and non-covalent methods, which provide potential ways to overcome drug resistance. In the case of SCCs, a major challenge is to synthesize heteroleptic scaffolds featuring different ligand combinations to achieve multimodal imaging and theranostic systems. Another synthetic challenge concerns the possibility to encapsulate different drug molecules in the same supramolecular metallacage. Notably, multi-cavity architectures have the potential to allow the binding of multiple different guests within a single assembly and could open up new applications to achieve combination therapy of different cytotoxic agents.271 In the case of SCCs, their water solubility and stability in physiological media are the main obstacles for their clinical uses. While the problem of solubility can be relatively easily overcome by the conjugation of the SCCs to biomolecules, including peptides, concerning stability some benefits can be achieved by the introduction of organometallic units into the supramolecular scaffolds. In general, metal-coordinated supramolecular self-assembly occurs through noncovalent interactions; thus, the nanomedicines may decompose in physiological environment, which can lead to early drug release and side effects to normal tissues. Therefore, in order to move forward in this fascinating research area, chemists should demonstrate the possibility to control the speciation of SCCs in physiological environment. A possible strategy could be the introduction of organometallic bonds in the overall scaffold to obtain supramolecular organometallic complexes (SOCs).5 In fact, metal-carbon bonds are endowed with increased stability with respect to classical coordination bonds. On the other hand, MOFs are rapidly gaining attention in the biomedical field, the initial concerns about their potentially high toxicity have been diminished by the use of non-toxic metals and ligands which has proven to be a strategy to generate biocompatible materials. As an undesirable consequence, most of the studies and platforms investigating in vivo applications of MOFs are nowadays limited to a handful of systems, in particular, MIL-100 and UiO-66 concentrate the efforts of several research groups. Unfortunately, such focalization in a few systems avoids the full exploitation of the rich diversity of the MOFs structures. Besides, the exclusive use of fully bio-compatible materials carries the disadvantage of instability and fast clearance, which are detrimental to the effectiveness of the material platforms. Currently, some of the main drawbacks on the development of MOFs for biomedical applications, relay on the limited availability of strategies for their post-synthetical modification. Due to the coordination-driven selfassembled nature of the frameworks, covalent functionalization of the surface is challenging. Controllable covalent surface functionalization could increase the stability and circulation times of MOF-based platforms. More importantly, by such functionalization, active targeting units able to direct the therapeutic agents to the action sites, can be included. Recently the post synthetical inclusion of folic acid (FA) units on the surface of nanoMOFs was demonstrated by both a coordinative (using the carboxylate of FA)272,273 and click chemistry strategies,274 both providing increased tumor targeting. Remarkably, functionalization has shown to be able to direct nanoMOFs straight to mitochondria, displaying the specificity that can be reached through targeting.275,276 Lately, the inclusion of aptamers has also attracted attention with the aim of exploiting the capabilities of those nucleic acids to recognize specific targets.277,278 These first advances open new horizons on the creation of systems with higher selectivity towards therapeutic

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targets. Nevertheless, a deeper understanding of the long-term effects of the use of MOFs nanostructures and their metabolites is necessary in order to translate from the proof of concept to the technological exploitation of MOF-based theranostic tools. Another challenge in this area of SCCs and MOFs includes not only achieving a size- or shape-selective dynamic molecular recognition, but also detecting and amplifying guest-bonding events to produce a measurable output, i.e., to implement imaging modalities.279 Concerning future possible developments, hybrid supramolecular materials hold great promise. The proof-of-concept for exofunctionalization of metallo-supramolecules with peptides has already been demonstrated and is expected to open additional prospect to construct hybrid systems with a larger size that can benefit from both the targeting properties of the tethered biomolecules and the EPR effect. Meanwhile, water-soluble hybrid [Pt4L6]12 þ metallacages, forming drug-loaded nanoparticles with an anionic polymer, have already been reported and revealed that the fluorophore fluorescein could also be encapsulated within the cavity.280 Thus, the fluorescein moiety was conjugated to a Pt4þ prodrug and encapsulated in the metallacage, and the cellular uptake and release of the guest prodrug could be studied in HeLa cells in vitro by fluorescence microscopy.280 The necessity of optimizing the formulation of SCCs for drug delivery via formation of nanoparticles has also been elegantly addressed by Stang and coworkers using covalent conjugation of a polymer to formulate a Pt2þ supramolecular metallacycle into 50 nm nanoparticles, formed by selfassembly, for the delivery of DOX.281 The impact of the morphology and size of the resulting amphiphilic polymer on its endocytic pathways, uptake rates, and efficiency, as well as cytotoxic effects were also investigated in vitro.281 Furthermore, the polymer antitumor efficacy was studied in vivo in HeLa tumor-bearing mice and showed enhanced tumor reduction with respect to free DOX, while presenting low systemic toxicity.281 Alongside ruthenium-arene complexes exhibiting anti-proliferative effects per se, the synthesis of three new pyrenyl-containing dendrimers and their encapsulation into a water-soluble hexaruthenium(II)-arene metallaprisms was achieved, and the resulting host-guest systems were shown to possess enhanced anti-proliferative effects in cancer cells in vitro with respect to the free components.282 Finally, a consideration should be made on the pivotal role of the second coordination sphere of supramolecular metal-based scaffolds in most biological and biomedical applications, as clearly evidenced in the discussed examples of helicates as nucleic acids binders. Therefore, the introduction of functional groups that can generate weak interactions with biomolecules on the supramolecular scaffold’s building blocks or the guest molecules is essential for the development of biologically active metalla-assemblies.12 Designing metalla-assemblies with second coordination sphere interactions in mind is a challenge, but it could provide the next generation of tailored bioactive supramolecular metal-based materials. In conclusion, we hope that the herewith included examples will inspire many synthetic and biological chemists to explore and fully reveal the multifaceted potential of the metal-based supramolecular molecules and materials.

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Engineering Phototheranostic Nanoscale Metal-Organic Frameworks for Multimodal Imaging-Guided Cancer Therapy. ACS Appl. Mater. Interfaces 2017, 9, 2040–2051. 263. Lismont, M.; Dreesen, L.; Wuttke, S. Metal-Organic Framework Nanoparticles in Photodynamic Therapy: Current Status and Perspectives. Adv. Funct. Mater. 2017, 27, 1606314. 264. Lu, K.; He, C.; Guo, N.; Chan, C.; Ni, K.; Weichselbaum, R. R.; Lin, W. Chlorin-Based Nanoscale Metal-Organic Framework Systemically Rejects Colorectal Cancers via Synergistic Photodynamic Therapy and Checkpoint Blockade Immunotherapy. J. Am. Chem. Soc. 2016, 138, 12502–12510. 265. Chen, D.; Yang, D.; Dougherty, C. A.; Lu, W.; Wu, H.; He, X.; Cai, T.; Van Dort, M. E.; Ross, B. D.; Hong, H. In Vivo Targeting and Positron Emission Tomography Imaging of Tumor with Intrinsically Radioactive Metal-Organic Frameworks Nanomaterials. ACS Nano 2017, 11, 4315–4327. 266. Wang, D.; Zhou, J.; Shi, R.; Wu, H.; Chen, R.; Duan, B.; Xia, G.; Xu, P.; Wang, H.; Zhou, S.; Wang, C.; Wang, H.; Guo, Z.; Chen, Q. Biodegradable Core-Shell Dual-MetalOrganic-Frameworks Nanotheranostic Agent for Multiple Imaging Guided Combination Cancer Therapy. Theranostics 2017, 7, 4605–4617. 267. Jin, D.; Zhang, J.; Huang, Y.; Qin, X.; Zhuang, J.; Yin, W.; Chen, S.; Wang, Y.; Hua, P.; Yao, Y. Recent Advances in the Development of Metal–Organic Framework-Based GasReleasing Nanoplatforms for Synergistic Cancer Therapy. Dalton Trans. 2021, 50, 1189–1196. 268. Gao, S.; Zheng, P.; Li, Z.; Feng, X.; Yan, W.; Chen, S.; Guo, W.; Liu, D.; Yang, X.; Wang, S.; Liang, X.-J.; Zhang, J. Biomimetic O2-Evolving Metal-Organic Framework Nanoplatform for Highly Efficient Photodynamic Therapy against Hypoxic Tumor. Biomaterials 2018, 178, 83–94. 269. Zhang, L.; Wang, Z.; Zhang, Y.; Cao, F.; Dong, K.; Ren, J.; Qu, X. Erythrocyte Membrane Cloaked Metal-Organic Framework Nanoparticle as Biomimetic Nanoreactor for Starvation-Activated Colon Cancer Therapy. ACS Nano 2018, 10, 10201–10211. 270. Sun, X.; He, G.; Xiong, C.; Wang, C.; Lian, X.; Hu, L.; Li, Z.; Dalgarno, S. J.; Yang, Y. W.; Tian, J. One-Pot Fabrication of Hollow Porphyrinic MOF Nanoparticles with Ultrahigh Drug Loading toward Controlled Delivery and Synergistic Cancer Therapy. ACS Appl. Mater. Interfaces 2021, 13, 3693. 271. Vasdev, R. A. S.; Preston, D.; Crowley, J. D. Multicavity Metallosupramolecular Architectures. Chem. – An Asian J. 2017, 12, 2513–2523. 272. Dong, H.; Yang, G.-X.; Zhang, X.; Meng, X.-B.; Sheng, J.-L.; Sun, X.-J.; Feng, Y.-J.; Zhang, F.-M. Folic Acid Functionalized Zirconium-Based Metal–Organic Frameworks as Drug Carriers for Active Tumor-Targeted Drug Delivery. Chem. – A Eur. J. 2018, 24, 17148–17154. 273. Gao, X.; Zhai, M.; Guan, W.; Liu, J.; Liu, Z.; Damirin, A. Controllable Synthesis of a Smart Multifunctional Nanoscale Metal–Organic Framework for Magnetic Resonance/ Optical Imaging and Targeted Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9, 3455–3462. 274. Alves, R. C.; Schulte, Z. M.; Luiz, M. T.; Bento da Silva, P.; Frem, R. C. G.; Rosi, N. L.; Chorilli, M. Breast Cancer Targeting of a Drug Delivery System through Postsynthetic Modification of Curcumin@N 3 -Bio-MOF-100 via Click Chemistry. Inorg. Chem. 2021, 60, 11739–11744. 275. Gong, M.; Yang, J.; Zhuang, Q.; Li, Y.; Gu, J. Mitochondria-Targeted Nanoscale MOFs for Improved Photodynamic Therapy. ChemNanoMat 2020, 6, 89–98. 276. Haddad, S.; Lázaro, I. A.; Fantham, M.; Mishra, A.; Silvestre-Albero, J.; Osterrieth, J. W. M.; Schierle, G. S. K.; Kaminski, C. F.; Forgan, R. S.; Fairen-Jimenez, D. Design of a Functionalized Metal–Organic Framework System for Enhanced Targeted Delivery to Mitochondria. J. Am. Chem. Soc. 2020, 142, 6661–6674. 277. Chen, W.-H.; Sung, S. Y.; Fadeev, M.; Cecconello, A.; Nechushtai, R.; Willner, I. Targeted VEGF-Triggered Release of an Anti-Cancer Drug from Aptamer-Functionalized Metal–Organic Framework Nanoparticles. Nanoscale 2018, 10, 4650–4657. 278. Alijani, H.; Noori, A.; Faridi, N.; Bathaie, S. Z.; Mousavi, M. F. Aptamer-Functionalized Fe3O4@MOF Nanocarrier for Targeted Drug Delivery and Fluorescence Imaging of the Triple-Negative MDA-MB-231 Breast Cancer Cells. J. Solid State Chem. 2020, 292, 121680. 279. He, C.; Wu, X.; Kong, J.; Liu, T.; Zhang, X.; Duan, C. A Hexanuclear Gadolinium–Organic Octahedron as a Sensitive MRI Contrast Agent for Selectively Imaging Glucosamine in Aqueous Media. Chem. Commun. 2012, 48, 9290–9292. 280. Yue, Z.; Wang, H.; Bowers, D. J.; Gao, M.; Stilgenbauer, M.; Nielsen, F.; Shelley, J. T.; Zheng, Y.-R. Nanoparticles of Metal-Organic Cages Designed to Encapsulate PlatinumBased Anticancer Agents. Dalton Trans. 2018, 47, 670–674. 281. Yu, G.; Zhang, M.; Saha, M. L.; Mao, Z.; Chen, J.; Yao, Y.; Zhou, Z.; Liu, Y.; Gao, C.; Huang, F.; Chen, X.; Stang, P. J. Antitumor Activity of a Unique Polymer That Incorporates a Fluorescent Self-Assembled Metallacycle. J. Am. Chem. Soc. 2017, 139, 15940–15949. 282. Pitto-Barry, A.; Barry, N. P. E.; Zava, O.; Deschenaux, R.; Dyson, P. J.; Therrien, B. Double Targeting of Tumours with Pyrenyl-Modified Dendrimers Encapsulated in an Arene– Ruthenium Metallaprism. Chemistry (Easton) 2011, 17, 1966–1971.

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K.M. Deo and J.R. Aldrich-Wright, School of Science, Nanoscale Organisation and Dynamics Group, Western Sydney University, Penrith, NSW, Australia; and Western Sydney University, Penrith South DC, NSW, Australia © 2023 Elsevier Ltd. All rights reserved.

2.23.1 2.23.2 2.23.2.1 2.23.2.1.1 2.23.2.2 2.23.2.2.1 2.23.2.2.2 2.23.2.2.3 2.23.2.2.4 2.23.2.2.5 2.23.2.2.6 2.23.2.2.7 2.23.3 2.23.3.1 2.23.3.2 2.23.3.2.1 2.23.3.2.2 2.23.3.2.3 2.23.3.2.4 2.23.3.2.5 2.23.3.2.6 2.23.3.2.7 2.23.3.2.8 2.23.3.2.9 2.23.3.3 2.23.3.4 2.23.4 2.23.4.1 2.23.4.2 2.23.5 References

Introduction Platinum(II) complexes as anticancer agents Conventional platinum(II) complexes Clinically used anticancer agents Unconventional platinum(II) complexes Multi-nuclear platinum complexes G-quadruplex targeted Cancer stem cell targeted Monofunctional complexes Non-conventional mechanism of action Immunogenic cell death stimulators Non-covalent mechanism of action Platinum(IV) prodrugs as anticancer agents Clinically trialed prodrugs Multi-action prodrugs Histone deacetylase inhibition Cyclooxygenase inhibition Pyruvate dehydrogenase kinase inhibition Glutathione S-transferase inhibition Tumor microenvironment regulators Immunostimulators Cancer stem cell targeted DNA damage response disrupters Prodrugs with unconventional cytotoxic cores Photoactivatable prodrugs Challenges and future perspectives of platinum chemotherapeutics NON-platinum anticancer agents Ruthenium complexes Gold complexes Conclusions

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Abstract Clinically used platinum (II) chemotherapeutics continue to dominate cancer treatment regimens 40 years after the approval of cisplatin. Despite their remarkable success at treating certain types of cancers, their efficacy is still limited against others due to resistance and severe side-effects. Advances in understanding not only their mechanisms of action but also the process of carcinogenesis has made it possible to address these shortcomings through the rational design and development of fundamentally different platinum complexes. This chapter endeavors to showcase the innovative approaches researchers have undertaken in designing complexes that can act upon different cellular targets and/or disrupt biological processes that are integral to tumorigenesis. Unconventional platinum (II) complexes that deviate from the canonical structure and mechanism of cisplatin and its analogs are discussed, including monofunctional and non-covalent binders, multi-nuclear complexes and those that do not specifically bind to DNA. Multi-action platinum (IV) prodrugs that incorporate various inhibitors, exert their activity through other biological pathways, such as immunostimulatory mechanisms are also explored, in addition to photoactivatable platinum (IV) prodrugs. Some notable examples of ruthenium and gold complexes are also highlighted. With the promising outcomes exhibited by many of the metallodrugs investigated, it is envisaged that research within the field of medicinal inorganic chemistry will continue to advance, generate new knowledge and produce chemotherapeutic agents that can further improve on the current limitations of clinically used drugs.

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Metal complexes as chemotherapeutic agents

2.23.1

745

Introduction

The use of metals for remedies and medical applications can be traced back for thousands of years to ancient civilizations. Despite this, the field of medicinal inorganic chemistry as a discipline is considered to be relatively young, with interest surging over the past
60 years, since the discovery of the therapeutic potential of cisplatin.1 Countless metallodrugs have been developed since then as therapeutic and diagnostic agents for various ailments including cancer, rheumatoid arthritis, diabetes, microbial conditions, cardiovascular and neurodegenerative diseases.2,3 The field of inorganic chemistry promotes structural uniqueness, rich coordination chemistry and variable oxidation states which has, and still is, producing complexes of great significance that continues to rapidly emerge. The numerous geometries and oxidation states offered by metallodrugs can be exploited to modulate their pharmacokinetic properties and thus, facilitate alternate mechanisms of action.4 Despite this, only a small number of platinum(II) complexes have gained approval for clinical use over the years (Fig. 1). Advances in genomics, transcriptomics, proteomics, metabolomics and radiomics have enabled the rational design and development of metal complexes to optimize their therapeutic potential.5 Accordingly, fabrication of more sophisticated drugs have allowed for a multifunctional approach. While there is a diverse range of applications of metal-based complexes, this chapter highlights the promising advancements in the development of platinum-based complexes as anticancer agents, among ruthenium and gold.

2.23.2

Platinum(II) complexes as anticancer agents

2.23.2.1

Conventional platinum(II) complexes

2.23.2.1.1

Clinically used anticancer agents

In this chapter, conventional platinum complexes are defined as those that conform to the specific structural requirements that were originally proposed by Cleare and Hoeschele to be essential for exhibiting anticancer activity.6,7 To summarize, complexes that are neutral with a square-planar geometry that follow the general formula [PtA2X2], where A is two ammine ligands or one bidentate chelating diamine and X is two monodentate or one bidentate anionic ligand in a cis configuration.6–8 To date, all clinically approved platinum(II) anticancer drugs adopt this configuration and satisfy these structural requirements. The most well-known example of this is cisplatin (cis-diamminedichloridoplatinum(II), Fig. 2). While cisplatin was known as Peyrone’s chloride since the 1840s, it was not until 1965 when its antiproliferative activity was fortuitously discovered by Rosenberg and co-workers after observing the effects of electric fields on bacterial cell division while using platinum electrodes.9–11 Subsequent FDA approval, in 1978, initially introduced cisplatin to clinics in Canada and then worldwide, where it has been used routinely to treat testicular, ovarian, cervical, bladder, breast and head and neck cancers among others.12,13 It was this discovery that arguably stimulated the evolution of the modern era of medicinal inorganic chemistry. It has been reported that approximately half of all chemotherapeutic treatments constitute of platinum-based agents, emphasizing their clinical importance.5 The antiproliferative activity exhibited by cisplatin and its analogs is attributed to its covalent interactions with DNA, as determined using multinuclear NMR (15N, 19F and 195Pt).14–21 Although a conclusive mechanism of action of cisplatin has not yet been established, decades of ongoing investigations have elucidated several fundamental steps. These include cellular uptake, aquation and DNA binding, followed by processing of the Pt-DNA lesion.22–24 The accepted notion is that cisplatin uptake occurs via passive diffusion and is also mediated by membrane proteins, such as copper transporter 1 (CTR1) or organic cation transporters (OCT)

Fig. 1

Timeline of platinum(II) chemotherapeutics when global or regional approval was granted.

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Fig. 2

Metal complexes as chemotherapeutic agents

Chemical structures of clinically used platinum(II) complexes. * indicates a stereocenter, either S or R.

(Fig. 3).25,26 Upon entering the cell, aquation of cisplatin is facilitated due to the lower chloride concentration in the cytoplasm (4– 20 mM) compared to blood (
100 mM).22 The resulting mono- and di-aqua species, [PtCl(NH3)2(H2O)]þ and [Pt(NH3)2(H2O)2]2þ respectively, are highly electrophilic and can readily to bind to DNA, forming intra/interstrand adducts.

H 3N H 3N

H 3N H 3N

H3 N

Pt

H3 N

H3 N H3 N

Cl Pt Cl

Cl Pt Cl

OH2 Cl

Pt

OH2 OH2

Fig. 3 Schematic representation of the mechanism of action of cisplatin to induce cell death. Cisplatin can also be actively exported from cells by copper exporters ATP7A and ATP7B or glutathione S-conjugate export pump, GS-X (also known as MRP2 or ABCC2). DNA and protein images were acquired from PDB files (2NPW, 1D86 and 1MHU).

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Experiments monitoring the rate of hydrolysis of 15N labelled cisplatin to [Pt(15NH3)2(H2O)2]2þ concluded that the nitrogen donor atoms of DNA were the major targets, primarily the N7 atoms guanine and adenine.27–30 The most predominant adducts were identified as 1,2-d(GpG) (
65%) and 1,2-d(ApG) (
25%) intrastrand cross-links, followed by 1,2-d(GG) (
6%) interstrand crosslinks.31,32 Consequently, these adducts cause significant distortion to the double helix of DNA, resulting in cell cycle arrest and inhibition of transcription, which ultimately leads to apoptosis.33–37 Despite the clinical success of cisplatin, persisting side-effects (including neurotoxicity, nephrotoxicity and ototoxicity) and resistance mechanisms (intrinsic or acquired) limit its applicability.38–42 Several factors have been associated in the development of resistance including, reduced drug influx and increased drug efflux from cells; enhanced repair of cisplatin-DNA adducts; loss of function of upregulated sequence specific binding factors; modulation of the pathways that control regulated cell death and increased concentrations of glutathione and metallothioneins.13,43–46 The clinical limitations exhibited by cisplatin provided the impetus for developing platinum-based chemotherapeutic agents that address the aforementioned drawbacks. Second and third generation platinum(II) complexes deviate slightly from the cisplatin framework by incorporating a wider range of mono /bi-dentate ligands that modify the electronic steric and basicity effects; however, these are still reported to form coordinate bonds with DNA. Of the thousands of analogs synthesized and screened for activity, only two other platinum(II) complexes have gained global approval. Carboplatin (diamine(1,1-cyclobutanedicarboxylato)platinum(II), Fig. 2) was the next to gain global approval for clinical use in 1989 and is primarily used to treat ovarian cancer.47–49 Owing to the more stable bi-dentate leaving group, it exhibits significantly lower levels of nephrotoxicity and neurotoxicity compared to cisplatin, although myelosuppression and thrombocytopenia still hinder its efficacy.49 Oxaliplatin ([1R,2R-diaminocyclohexane][ethanedioato-O,O0 ] platinum(II), Fig. 2) received worldwide approval in 2002 and is used in conjunction with 5-fluorouracil (5-FU) to treat colorectal cancer.50 Unlike cisplatin and carboplatin, it is comprised of two bidentate ligands and exhibits are broader spectrum of activity.50,51 Of particular significance to the activity of oxaliplatin is the chirality of the 1,2-diaminocyclohexane (DACH) ligand, where the R,R isomer exhibits superior activity compared to the S,S or S,R configurations.52 Oxaliplatin exhibits reduced nephrotoxicity and myelosuppression and is not cross-resistant with cisplatin; however, its activity is limited by neurotoxicity.13,51,53–55 It also displays lower levels of hematological toxicity compared to carboplatin.56–58 The pharmacological differences observed for oxaliplatin have recently been attributed to its ability to cause cell death by inducing ribosome biogenesis stress, rather than a DNA damage-response mechanism.59 Studies on oxaliplatin have also indicated that it can promote immunogenic cell death (ICD) by facilitating a T-cell dependent immune response.60,61 It can be reasoned that such differences in the mechanism of action of oxaliplatin allows it to exhibit activity in cell lines resistant to cisplatin. In addition to the globally approved platinum(II) chemotherapeutic agents, nedaplatin (diammine[(hydroxy-kO)acetato(2-)-kO] platinum(II)) and miriplatin (cis-[((1R,2R)-1,2-cyclohexanediamine-N,N0 )bis(myristato)]platinum(II) monohydrate) have been approved in Japan, while heptaplatin ([SP-4-2-[4R-(2a,4a,5b)]]-[2-(1-methylethyl)-1,3-dioxolane-4,5-dimethanamine-N,N0 ][propanedioato(2-)-O,O0 ]-platinum(II)) and lobaplatin ([1R,2R-2-(aminomethyl)cyclobutyl] methanamine-2-hydroxypropanoic acid platinum(II)) have received approval in South Korea and China, respectively (Fig. 2).62 Nedaplatin is significantly more water soluble and displays lower nephrotoxicity than cisplatin.63,64 It has been approved for use in Japan for the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) and head and neck cancers, however, its efficacy is limited by thrombocytopenia and neutropenia.65,66 Lobaplatin is employed in China to treat chronic myelogenous leukemia (CML), SCLC and metastatic breast cancer.62 It is administered as a diastereomeric mixture of S,S and R,R isomers of the carrier ligand and does not induce neuro-, otoor nephrotoxicity, though it is limited by thrombocytopenia.62,67 Heptaplatin has been approved for use in the Republic of Korea. It exhibits high stability in solution, potent anticancer activity against cisplatin-resistant cells and is used for the treatment of gastric cancer; however, it is limited by myelosuppression, nephro- and hepatotoxicity.62,68 Miriplatin, suspended in lipiodol, gained marketing approval in Japan for unresectable hepatocellular carcinoma.69–71 It is reported to cause lower systemic toxicity and is not cross-resistant with cisplatin, although its efficacy is hindered by neutropenia.72

2.23.2.2

Unconventional platinum(II) complexes

The apparent limitations exhibited by all clinically approved platinum(II) complexes may be attributed to their related structural characteristics and consequently, their mechanism(s) of action. It is evident that other innovative strategies are an essential strategy when designing chemotherapeutic agents to circumvent the current limitations and enhance their overall efficacy. The development of platinum(II) complexes that deviate from the conventional framework has resulted in fundamentally new coordination complexes that exhibit cytotoxicity that rivals or betters cisplatin, especially against cisplatin-resistant cell lines. This improved cytotoxicity has been ascribed to different mode(s) of action, which presumably allows the complexes to circumvent the repair mechanisms that inhibit the activity of cisplatin and its analogs. These promising results highlight the significance of exploring alternate avenues for designing and developing novel platinum(II)-based anticancer agents. In this chapter, “unconventional” platinum(II) complexes are those that do not conform to the structural framework described by Cleare and Hoeschele for cisplatin and its analogs but instead, exert their antineoplastic activity via mechanism(s) other than exclusively binding covalently to DNA.

2.23.2.2.1

Multi-nuclear platinum complexes

Early studies into the development of multi-nuclear platinum(II) complexes by Farrell and co-workers produced a class of di- and trinuclear complexes that demonstrated encouraging results. These complexes incorporated two or three units of trans-[Pt(NH3)2Cl]

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Metal complexes as chemotherapeutic agents

that are coupled together with flexible diaminoalkane linkers of selected lengths, which facilitate groove binding (PtII-1 and PtII-2, Fig. 4).73 Additionally, the labile terminal chloride ligands may be aquated and are therefore capable of forming covalent adducts with DNA that span further apart than those of cisplatin, forming 1,2-, 1,3- and 1,4-interstrand crosslinks.73–75 The distortion to DNA caused by such adducts is less prominent (than those caused by cisplatin) and as a result, repair proteins are unable to recognize these distinct lesions, leading to improved activity in cisplatin-resistant cell lines.76,77 Moreover, the cationic nature of these complexes is ascribed to enhance both cellular accumulation and affinity toward DNA.76 From this class of complexes, BBR3464 ([| trans-PtCl(NH3)2}-m-trans-Pt(NH3)2 |NH2(CH2)6NH2}2]4þ, PtII-2, Fig. 4) has been extensively studied in in vitro and in vivo experiments where it exhibited exceptional activity; however, its maximum tolerated dose was limited due to myelosuppression and gastrointestinal toxicities.78–82 It is also the first multi-nuclear platinum(II) complex to progress through to phase I and II clinical trials. Although some of the initial investigations indicated promising results, it did not advance any further due to a poor toxicological profile and insufficient improvements to antitumor activity.83–86 Other trinuclear platinum(II) complexes from this group were designed to incorporate ammine or extended amine ligands by replacing the terminal chloride ligands (PtII-3 and PtII-4, Fig. 4).87 The resulting complexes are unable to bind covalently to DNA; instead, the increased positive charges mediate DNA binding through electrostatic interactions while H-bonding selectively promotes the formation amine-phosphate-ammine motifs with DNA (termed “phosphate-clamps”).87–89 Binding to DNA through this mode induces conformational changes of B-DNA to Z-DNA and B-DNA to A-DNA and has also shown to condense DNA.87 Similar to their covalently binding counterparts, these trinuclear complexes also exhibit activity against cisplatin-resistant cell lines, stemming from increased cellular accumulation and unique mechanism of action.

2.23.2.2.2

G-quadruplex targeted

DNA comprising of guanine-rich sequences are able to form tetrameric structures known as G-quadruplexes (G4). These consist of G-quartets (which are four guanines held together via Watson-Crick and Hoogsteen hydrogen bonding) that fold and stack on one another to form a diverse range of topologies.90 G4 structures are prevalent in human telomeres and oncogene promoter regions and thus, implicated in the progression of cancer. Stabilization of G4s by small molecules has been reported to inhibit telomerase activity, leading to cell senescence and apoptosis.91 As such, exploring avenues that develop chemotherapeutic agents that can selectivity bind to G4 has the potential to improve antitumor efficacy. G4-targeted chemotherapeutics typically incorporate extended planar aromatic ligands that are conducive to p-p interactions with terminal G-quartets.92 However, based on the findings that the non-covalently binding multinuclear complexes could stabilize triple-helical DNA and RNA triplexes; interrupt DNA synthesis by DNA polymerase and inhibit reverse transcription, Farrell, Brabec and coworkers expanded the series of non-covalently binding multinuclear complexes (PtII-5–PtII-8, Fig. 5) and evaluated their binding properties toward different G4 DNA.93–95 The ability of PtII-3–PtII-8 to inhibit DNA synthesis in DNA templates containing the oncogenic promotor G4s, c-myc, c-kit2 and hTelo, was determined by evaluating their effectiveness at inhibiting DNA extension catalyzed by Taq polymerase. All complexes were more effective at inhibiting DNA synthesis in c-myc and c-kit2 templates, which adopt parallel topologies (in Kþ containing solutions), compared to hTelo, which is capable of forming a hybrid of parallel and antiparallel topologies.96 This suggests that PtII-3–PtII-8 have a preference for G4s with parallel topologies. The stabilizing activity was greater for complexes carrying a higher overall charge, where PtII-8 was the most effective. For equally charged complexes, those with more platinum(II) cores and extended amine ligands were more efficient at stabilizing G4 DNA, ascribed to their ability to form additional hydrogen bonds. The binding mode of PtII-3–PtII-8 toward c-myc G4 occurs primarily via interactions with the loops and groove regions rather than binding at the external G-quartets; with the exception of PtII-6, which also exhibited binding at the external G-quartets. Additional studies of PtII-4 revealed its ability to reduce c-myc, and to a lesser extent, c-kit expression at

Fig. 4 Chemical structures of di- and trinuclear platinum(II) complexes: [(trans-PtCl(NH3)2)(N2H(CH2)4NH2)]2þ (PtII-1); BBR3464 (PtII-2); TriplatinNC-A (PtII-3) and TriplatinNC (PtII-4).

Metal complexes as chemotherapeutic agents

Fig. 5

749

Chemical structures of multinuclear G4-targeted platinum(II) complexes (PtII-5–PtII-8).

mRNA protein levels in HEK293 human embryonic kidney cells.96 Although this class of compounds does not exhibit the typical structural features of G4-binding ligands, it is apparent that they demonstrate the capacity to stabilize G4s and terminate DNA polymerization on templates containing G4-forming sequences. With the focus on developing G4-targeted platinum(II) complexes, a family of multinuclear complexes have been synthesized that show promising results (PtII-9–PtII-14, Fig. 6). Fluorescence resonance energy transfer (FRET) assays demonstrated that the porphyrin-bridged tetranuclear platinum(II) complexes, PtII-9 and PtII-10, were substantially more effective at stabilizing HTG21 (human telomere) and Pu27, c-kit and bcl2 (promoter G4s) compared to dsDNA, suggesting higher selectivity toward G4 structures.97 Additional studies revealed that both complexes have a preference for parallel conformations, binding through end-stacking rather than intercalation. PtII-9 and PtII-10 also inhibited telomerase and down-regulated the expression of c-myc in HeLa human cervix carcinoma cells. PtII-10 was more effective than PtII-9 at inhibiting telomerase activity, with IC50 values of 0.25 and 1.46 mM, respectively. This also corresponded with observed cytotoxicity in HeLa cells, where PtII-10 exhibited higher potency than PtII-9, with IC50 values of 4.8 and 24.4 mM, respectively. The biological activity was attributed to the high cationic charge, inhibition of telomerase and oncogene expression. The trinuclear complexes, PtII-11 and PtII-12, displayed high stabilization of human telomeric G4, hTel, compared to promoter G4 (c-kit1, c-myc and bcl2) and dsDNA, indicating greater selectivity toward hTel.98 Both complexes demonstrated high binding affinity toward hTel G4, although it was slightly higher for PtII-11, as established by isothermal titration calorimetry (ITC). Circular dichroism (CD) spectroscopy revealed that while PtII-11 induces a hybrid (unassigned) topology of hTel G4, PtII-12 induces the coexistence of parallel and anti-parallel conformations. PtII-12 was more effective than PtII-11 at inducing the formation of hTel G4 in HeLa cells, which was attributed to its 3-fold higher cellular accumulation. Subsequent analysis of the inhibition of telomerase activity by PtII-11 and PtII-12 illustrated similar results, with IC50 values of 2.13 and 2.78 mM, respectively. Antiproliferative activity assays against telomerase-positive (HeLa, A549 (lung) and HTC75 (fibrosarcoma)) and telomerase-negative (SAOS2 (bone), U2OS (bone) and VA13 (transformed lung fibroblast)) cancer cell lines demonstrated comparable activity between PtII-12 and cisplatin, whereas PtII-11 was up to 4-fold less potent. PtII-12 was found to elicit a strong telomeric DNA damage response and shorten telomere length, leading to cancer cell senescence. High selectivity toward hTel G4 was also exhibited by PtII-13 and PtII-14, characterized by higher melting temperatures from FRET assays.99 Remarkably, PtII-14 displayed 122-fold greater selectivity for hTel G4 compared to duplex DNA, while a 10-fold greater affinity was demonstrated by PtII-13. Both, PtII-13 and PtII-14 were found to induce and stabilize the anti-parallel topology of hTel. Subsequent docking simulation of PtII-13 and PtII-14 with anti-parallel hTel G4 suggested similar binding modes, in which intercalation occurs at the large groove. Evaluation of telomerase activity revealed PtII-14 was more effective at inhibiting telomerase than PtII-13, with IC50 values of 2.67 and 6.41 mM, respectively. Diverging from cationic multinuclear G4-targeted complexes, a neutral, partially planar, mononuclear platinum(II) complex was developed, which demonstrates impressive selectivity toward G4 sequences over dsDNA (PtII-15, Fig. 6).100 Higher affinity was observed for Tel26, wtTel26 and VEGF G4s compared to c-myc, c-kit, bcl2 and dsDNA, although the highest binding specificity was observed toward VEGF G4, as indicated by NMR studies. Upon binding, significant structural changes occur for both VEGF G4 and PtII-15; the chlorido ligand dissociates, rotation of the monodentate ligand occurs and a new PteN bond is formed with the N atom on the pyridine, resulting in a positively charged complex that is completely planar. This configuration enhances the binding interaction with all four G-quartet bases. The dissociation of the chlorido ligand was considerably faster in the presence of G4 than dsDNA, suggesting that DNA binding of PtII-15 is controlled by kinetics. Quantitative real-time polymerase chain reaction (RT-qPCR) indicated PtII-15 was able to reduce VEGF gene expression. Treatment of is5Tg zebrafish with PtII-15 resulted in almost complete suppression of blood vessel growth, whereas cisplatin showed no distinct differences. The anti-angiogenesis activity of PtII-15 was ascribed to stem from its interactions with VEGF G4.

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Fig. 6

Metal complexes as chemotherapeutic agents

Chemical structures of mono-, tri- and tetranuclear G4-targeted platinum(II) complexes incorporating aromatic ligands (PtII-9–PtII-15).

Metal complexes as chemotherapeutic agents 2.23.2.2.3

Cancer stem cell targeted

2.23.2.2.4

Monofunctional complexes

751

The complexity of certain tumors and their microenvironments can result in cancers that are highly resistant to chemotherapy.101 This renders some chemotherapeutic agents ineffective at eradicating an entire population of cancerous cells, which can lead to a relapse.102,103 It has been suggested that recurrence may be related to the presence of cancer stem cells (CSCs); these are a subpopulation of tumorigenic cells that reside in specialized microenvironments (known as niches) and are characterized by their ability to self-renew, differentiate and propagate the cancer.102–105 CSCs also exhibit multi-factorial resistance mechanisms including, efflux pumps, an enhanced DNA damage response and induction of anti-apoptotic mechanisms.106 This confers greater resistance to CSCs against conventional chemo- and radiotherapy techniques. As such, designing CSC-targeted anticancer agents may demonstrate improved therapeutic potential compared to currently available therapies. A trinuclear platinum(II) complex (PtII-18) was synthesized comprising of three alternating units each of [Pt(1,1bis(diphenylphosphino)ethylene)] and benzotriazole in a triangular formation (Fig. 7).107 PtII-18 exhibited comparable activity to cisplatin against HMLER breast cancer cells (2.24 and 2.57 mM, respectively); however, it demonstrated 4.5-fold greater activity than cisplatin against CSC-enriched HMLER-shEcad breast cancer cells (1.26 and 5.65 mM, respectively), suggesting selectivity toward CSC-enriched HMLER-shEcad cells, similar to a known breast CSC-active agent, salinomycin.108 While PtII-16 and PtII17 demonstrated micromolar potency against both cell lines, no significant levels of selectivity were observed for either complex. Accompanying cytotoxicity studies of PtII-18 against “healthy” breast MCF710A resulted in 2-fold lower activity, supporting its selective nature toward CSCs compared to bulk cancer cells and non-malignant cells.107 Enhanced tumor inhibition was also observed in 3D breast CSC spheroids compared to cisplatin, carboplatin and salinomycin. PtII-18 exhibits increased cellular uptake with preferential accumulation in the nucleus, where it interacts non-covalently with DNA to cause damage, leading to caspasedependent apoptosis. These findings highlight the therapeutic potential of multinuclear platinum(II) complexes in selectivity targeting CSCs to reduce the potential for cancer recurrence.

Platinum(II) complexes that include only a single labile ligand has resulted in the development of a structurally similar but mechanistically diverse class of chemotherapeutic agents. DNA is still considered the main target for most cationic complexes from this class of compounds; however, the adducts formed are distinct from cisplatin and its analogs. As such, their unique mode of action has the potential to circumvent typical resistance mechanisms. Early studies of pyriplatin (Fig. 8) identified monofunctional adducts with DNA that did not elicit significant distortion to the double helix, inhibited transcription and evaded repair, although its activity was significantly lower than cisplatin and oxaliplatin.109–112 Evaluation of the role of the pyridine ligand suggested steric hindrance is pertinent to its activity, which led to the development of phenanthriplatin. The increased steric hindrance exhibited by phenanthriplatin results in improved inhibition of RNA polymerase II and DNA synthesis. Consequently, the in vitro cytotoxicity is drastically improved (up to 40-fold more active) across a broad range of cell lines and also demonstrates higher selectivity toward malignant cells.113 Continued research into monofunctional platinum(II) complexes has aided in explicating their mechanisms of action and guided the way for developing complexes that display added functionality. Protein tyrosine phosphatases (PTPs) constitute a superfamily of enzymes involved in the intracellular signal transduction pathway by hydrolytically removing phosphate groups from proteins.114 PTP activities are dysregulated in several human diseases including cancer, diabetes and immune disorders. Elevated expression levels in certain cancers imply their involvement in cancer development and progression.115 PtII-19 exhibited 30-fold greater activity than cisplatin against MCF-7 breast cancer cells and was also more effective at inducing apoptosis, although its ability to bind with DNA was weaker, suggesting it acts upon other cellular target(s).116 The cytotoxicity of PtII-19 was also enhanced against HepG2 liver cancer (high PTP1B expression) compared

Fig. 7 Chemical structures of mono-, di- and trinuclear platinum(II) complexes as potential chemotherapeutic agents for targeting CSCs (PtII-16– PtII-18).

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Fig. 8

Metal complexes as chemotherapeutic agents

Chemical structures of pyriplatin and phenanthriplatin.

to A549 lung cancer (low PTP1B expression), which was attributed to its selective and potent inhibition of PTP1B. Consequently, cellular phosphorylation levels and thus, intracellular signal transduction pathways are substantially altered, indicating a distinct mechanism of action compared to cisplatin and its analogs. Hexokinase (HK) is an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate and is found in the outer mitochondrial membrane. HK is significantly upregulated in malignant cells compared to healthy cells and is also associated with protecting cancer cells from apoptosis.117 Complexes PtII-20–PtII-22 incorporate lonidamine as a HK inhibitor and demonstrate modest activity against several cancers.118 The most active complex from this group, PtII-22, exhibited slightly better selectivity toward MCF-7 and MDA-MB-231 TNBC cells compared to cisplatin. PtII-22 accumulates predominantly in the mitochondria, where it interferes with mitochondrial bioenergetics by altering the mitochondrial membrane potential, inhibiting glycolysis and disrupting mitochondrial respiration. Furthermore, PtII-22 causes significant mtDNA damage, arrests the cell cycle in the G0/G1 phase and induces caspase-dependent mitochondria-mediated apoptosis. Also implicated in the mechanism of activity is the ability to perturb signal pathways associated with cell death, including DNA damage, metabolic process and transcription regulator activity. A series of monofunctional platinum(II) complexes comprising of naphthalene imide derivatives have demonstrated potent activity across several cell lines (PtII-23–PtII-26, Fig. 9). PtII-23 and PtII-25 displayed either comparable or slightly better activity than cisplatin. However, PtII-24 and PtII-26 were respectively 13- and 120-fold more cytotoxic than cisplatin against NCI-H460 NSCLC cells. Additional studies demonstrated PtII-24 and PtII-26 significantly inhibited telomerase activity and downregulated hTERT and c-myc.119 Induction of apoptosis was significantly increased in NCI-H460 cells, which was attributed to a combination of telomerase inhibition and mitochondrial dysfunction. In vivo studies of PtII-26 against NCI-H460 xenografts in mice demonstrated superior tumor inhibition than cisplatin with no significant toxicity.120 Three derivatives of pyriplatin were prepared by anchoring triphenylphosphonium (TPP) in the ortho-, meta- or para- position of the pyridine ring to target mitochondria (PtII-27–PtII-29, Fig. 9). The substitutional position of TPP profoundly impacted the pharmacokinetics of the resulting complex. PtII-27 exhibited comparable antiproliferative activity to cisplatin, especially against A549 lung cancer and also demonstrated some selectivity toward cancerous cell over “healthy” cells.121 In contrast, PtII-28 and PtII-29 displayed modest to low activity, although they were still more active than their precursor, pyriplatin. Mitochondrial cellular accumulation was highest for PtII-27, while PtII-28 and PtII-29 accumulate preferentially in the nucleus. Consequently, PtII-27 causes significant mitochondrial dysfunction by altering the ultrastructure and membrane, interrupting glycolysis and oxidative phosphorylation and promoting the release of cytochrome c (pro-apoptotic protein). Furthermore, PtII-27 can perturb mtDNA and nDNA, which also contribute to its activity. In vivo investigations of PtII-27 to treat A549 xenografts in mice revealed its ability to suppress tumor growth better than cisplatin, with a slightly greater reduction in body weight, although this was attributed to its mitochondrion-disrupting effect. Highest levels of platinum were detected in the liver and kidneys, suggesting uptake of PtII-27 is mediated by organic cation transporters, which are elevated in these organs. PtII-30 also incorporates TPP as a targeting vector for mitochondria to perturb cancer bioenergetics (Fig. 9). Antiproliferative activity of PtII-30 was 3-fold greater than cisplatin against Caov3 ovarian cancer cells, while it was slightly less toxic to “healthy” HK-2 kidney cells, suggesting some selectivity toward malignant cells.122 PtII-30 accumulates equally in mitochondria and nuclei, although the cytotoxic activity primarily stems from damage to mitochondrial morphology since no significant damage was observed for nDNA. The inhibition of mitochondrial thioredoxin reductase (TrxR) leads to suppression of mitochondrial respiration and glycolysis, inducing cells to enter a hypometabolic state and also weakens cellular ROS-defense mechanisms. Additional studies demonstrated PtII-30 could promote autophagy and mitophagy due to the impairment of mitochondrial morphology and trigger cell death via a caspase-3-independent, non-apoptotic pathway.

2.23.2.2.5

Non-conventional mechanism of action

As exemplified in Section 2.23.2.2.1, the developments made in the design of anticancer agents have produced complexes that can interact with their biological target through non-covalent mechanisms. This approach has the potential to overcome the limitations observed with conventional chemotherapeutics, such as cisplatin. In particular, complexes exhibiting alternate mechanism(s) of action are envisioned to circumvent the common resistance mechanisms arising from the use of current anticancer agents.123 Complexes incorporating electron deficient, planar, heterocyclic ligands such as acridine, bipyridine, phenanthroline, quinoline and terpyridine have been reported to interact with DNA via intercalation.124–128 Unlike the covalent adducts formed by cisplatin and its analogs, this mode of action results in a reversible interaction with DNA where the heterocyclic ligand is stabilized by p-p

Metal complexes as chemotherapeutic agents

753

Fig. 9 Chemical structures of monofunctional complexes that can inhibit HK (PtII-20–PtII-22), telomerase (PtII-23–PtII-26) or target mitochondria (PtII-27–PtII-30).

stacking between the intercalating moiety and the base pairs of DNA.129 This causes the DNA to unwind, stiffen and lengthen in order to accommodate the complex; however, the extent of this effect is dependent on the depth of insertion.58,128,129 Numerous platinum(II)-acridine complexes developed by Bierbach and co-workers have been reported to exert their cytotoxicity through a covalent/intercalation dual binding mode and have demonstrated promising results in a range of solid tumor cell lines.

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Metal complexes as chemotherapeutic agents

The platinum center constitutes of a single labile chlorido ligand while the other coordination sites are occupied by non-labile ligands, which includes variations of the ligand N-[2-(acridin-9-ylamino)ethyl]-N-methylacetimidamide (PtII-31–PtII-33, Fig. 10). The DNA binding mechanisms proceeds through the formation of a monofunctional adduct while the acridine moiety intercalates adjacently.130,131 Interestingly, it has been shown that the monofunctional-intercalative adducts formed by this class of complexes can be readily excised; however if unrepaired, poses a greater threat to cell survival than conventional platinum(II)-DNA adducts.132 These distinct lesions disrupt DNA synthesis by causing double-strand breaks, stalled replication forks and inhibition of RNA polymerase II-mediated transcription.133 These complexes demonstrate sub-micromolar to nanomolar levels of cytotoxicity against highly resistant cell lines, such as non-small cell lung carcinoma (NSCLC), although subsequent in vivo studies have demonstrated severe toxicities, attributed to higher platinum concentrations in healthy tissue compared to tumor tissue.134–138 Substitution of the original intercalating moiety with benz[c]acridine (PtII-34 and PtII-35) resulted in a more favorable pharmacological profile, albeit with slightly lower activity being observed.137 Continued advancements into the development of platinum(II)-acridine complexes through structure-activity relationship (SAR) studies have produced numerous variations and expanded this series of complexes (PtII-36–PtII-44, Fig. 10).138–141 PtII36 was identified as one of the most active derivatives to date, where cytotoxicity studies of PtII-36 against the NCI-H460 (largecell lung carcinoma) cell line revealed an IC50 value of 8 nM, while that of cisplatin was 1.2 mM.140,142 Additional SAR studies highlighted the significance of the stereochemistry of the non-leaving groups incorporated into complexes PtII-41–PtII-44.141 Interestingly, PtII-41 (R isomer) was non-active against A549 lung adenocarcinoma (IC50  100 mM), while PtII-42 (S isomer) was considerably more potent (IC50 ¼ 2.6 mM). In contrast, PtII-43 (S isomer) was almost 90-fold more potent than PtII-21 (R isomer) in the same cell line, exhibiting IC50 values of 0.017 and 1.5 mM, respectively. To probe the potent activity of platinum(II)-acridine complexes, gene expression profiles were correlated with results from NCI-60 cytotoxicity screening studies using PtII-36 as the test compound.143 The membrane transporter, hMATE1 (human multidrug and toxin extrusion protein), was found to be essential in the uptake of platinum(II)-acridine complexes. It is possible that increased expression of hMATE1 in NSCLC cell lines contributes to the potent activity of this class of complexes.143 Despite the promising cytotoxicity of these complexes against cisplatin-resistant cell lines in vitro, the systemic toxicities exhibited in vivo limits their applicability in clinical settings. To address this, several liposomal formulations were prepared to encapsulate PtII-36.144 In vivo studies carried out in athymic nude mice with A549 xenografts demonstrated reduced platinum levels in liver, kidney and lung tissue compared to the free complex. The effectiveness of liposomal-encapsulated-PtII-36 in tumor reduction displayed some encouraging results and was shown to be more efficacious against tumors of larger sizes.144 These results illustrate the potential application of liposomal formulations of platinum(II)acridine complexes as drug delivery vehicles.

2.23.2.2.6

Immunogenic cell death stimulators

Reports of oxaliplatin inducing immunogenic cell death (ICD) prompted the investigation of existing and novel platinum-based chemotherapeutics in their potential to also promote ICD. Hallmarks of ICD include: (i) translocation of calreticulin (CRT) from the endoplasmic reticulum (ER) to the cell surface; (ii) secretion of ATP and (iii) extracellular release of high-mobility group box 1 (HMGB1) from the nucleus during the late stages of apoptosis.145 A series of luminescent platinum(II) N-heterocyclic carbene (NHC)-based complexes were synthesized, which exhibited exceptional antiproliferative activity against a panel of malignant cell lines (PtII-45–PtII-50, Fig. 11). Notably, PtII-47 displayed up to 60-fold greater cytotoxicity than cisplatin against H1975 lung carcinoma, with an IC50 value of 0.45 mM.146 Furthermore, complexes PtII-48–PtII-50 demonstrated photoactivity, with up to 33-fold

Fig. 10 General structures of selected platinum(II) complexes comprising of the intercalating moieties, acridine (left, PtII-31–PtII-33 and right, PtII36–PtII-44) and benz[c]acridine (center, PtII-34 and PtII-35). Counter-ions are shown in parentheses; en ¼ 1,2-diaminoethane, pn ¼ 1,3diaminopropane; Me2pn ¼ 2,2-dimethyl-1,3-diaminopropane and DACH ¼ 1,2-diaminocyclohexane (either S,S or R,R configuration).

Metal complexes as chemotherapeutic agents

Fig. 11

755

Chemical structures of platinum(II) complexes that can elicit ICD (PtII-45–PtII-52). OTf ¼ triflate anion.

increase in cytotoxicity after irradiation with light. PtII-47 demonstrated weak interactions with DNA, suggesting DNA binding is not a decisive factor in its activity. Selective localization in the ER domain was found to result in ER stress and mitochondrial dysfunction, ultimately leading to apoptosis. A separate study demonstrated that PtII-47 could trigger reactive oxygen species (ROS)-mediated ER stress; expose CRT; secrete ATP and HMGB1, establishing it as the first type II ICD inducing immuno-chemotherapeutic agent.147 Structural modifications to the above-mentioned cyclometalated complexes expanded the library and produced another complex capable of inducing type II ICD (PtII-51, Fig. 11). PtII-51 (1.5 mM) exhibited greater cytotoxicity than PtII-47 (5.8 mM) and cisplatin (4.0 mM) against CT26 murine colorectal carcinoma.148 Compared to PtII-47, PtII-51 displayed enhanced induction of phagocytosis and release of damage associated molecular patterns (DAMPs), which are associated with antitumor immunological memory. Additionally, PtII-51 can activate ecto-HSP90 proteins that can bind to macrophages to communicate and strengthen the “eat me” signal. The efficiency of this ICD induction was correlated with ER-localized ROS generation and dependent on the accumulation levels of PtII-51 in the ER. A series of aminophosphonate-based platinum(II) complexes were developed, where the lead compound, PtII-52, demonstrated better cytotoxicity and selectivity toward malignant cell lines compared to cisplatin and oxaliplatin (Fig. 11).149 Preferential

756

Metal complexes as chemotherapeutic agents

accumulation in the ER and generation of ROS results in ER stress, consequently triggering the hallmarks of ICD and activating DAMPs. MB-49 murine urothelial carcinoma cells treated with either oxaliplatin or PtII-52 were injected into the flanks of C57BL/6 mice as a tumor vaccine. After 1 week, re-inoculation of untreated MB-49 cells in the opposite flank of the mice showed a significant delay in tumor formation, indicating activation of immunity to prevent tumor growth. Additional in vivo studies in immunocompetent mice displayed superior tumor inhibition of PtII-52 (z 60%) compared to oxaliplatin (z 49%) with significantly lower acute toxicity. Increased CD3þ and CD8þ T lymphocytes in tumor tissue after treatment with PtII-52 support its ability to activate the immune system to synergistic eradicate cancer cells.

2.23.2.2.7

Non-covalent mechanism of action

An extensive series of potent platinum(II) complexes of the type [PtII(HL)(AL)]2 þ, where HL is a bidentate heterocyclic ligand and AL is an ancillary bidentate diamine that can be chiral or achiral have been developed. A comprehensive range of HLs and ALs have been utilized to investigate the SAR relationship of this family of complexes including 1,10-phenanthroline (PHEN), 2-(20 -pyridyl)quinoxaline, 2,20 -bipyridine (bpy), dipyrido[3,2-f:20 ,30 -h]quinoxaline and their functional group variants as the HL and 1,2diaminoethane (en), 1,2S/R-diaminopropane (1,2-pn), 2S/R,3S/R-diaminobutane (2,3-bn), 1S/R,2S/R-diphenyldiaminoethane (dpen), 1S/R,2S/R-diaminocyclopentane (DACP) and 1S/R,2S/R-diaminocyclohexane (DACH) and the AL (Fig. 12).150–157 As expected, the choice of HL and AL can influence the cytotoxicity of the complex, although a more profound effect is observed when chiral ALs are used compared to achiral ligands. Also noteworthy is that considerable changes to the cytotoxicity are observed depending on the enantiomer of the AL that is used.156,158 The most potent complex from this library of complexes is [(5,6dimethyl-1,10-phenanthroline)(1S,2S-diaminocyclohexane)platinum(II)]2þ (PtII-23, 56MESS(II)), while its RRDACH analog (56MERR(II)) is less potent across a range of cell lines tested. For example, against Du145 prostate cancer cell line, 56MESS(II) exhibited 58-fold greater activity than 56MERR(II) with IC50 values of 7 nM and 0.41 mM, respectively.159 The mechanism of action of these complexes have not yet been fully elucidated, although it is expected to be substantially different when compared to cisplatin and its analogs. The activity observed for 56MESS(II) against numerous cell lines in several orders of magnitude higher than cisplatin, particularly in cisplatin-resistant cell lines.150,160,161 While ethidium bromide displacement assays have demonstrated the ability of these complexes to intercalate with DNA, it may not be the sole source of their potent cytotoxicity. For instance, complexes with variations to the HL have demonstrated comparable DNA binding constants, yet their activity was considerably different in vitro.151 In addition to the AL having a greater influence in overall cytotoxicity, these findings suggest that intercalation with DNA is not a major factor in causing cell death and that there are other more significant biological targets.

Fig. 12 General structure of [PtII(HL)(AL)]2 þ type complexes. * indicates a chiral center, either S or R. bpy ¼ 2,20 -bipyridine; 2pq ¼ 2-(20 -pyridyl) quinoxaline; dpq ¼ dipyrido[3,2-f:20 ,30 -h]quinoxaline; DACP ¼ 1,2-diaminocyclopentane; dpen ¼ diphenyldiaminoethane.

Metal complexes as chemotherapeutic agents

757

Changes in gene expression of Saccharomyces cerevisiae after treatment with 56MESS(II) were analyzed using a transcriptomics approach.161 Genes pertaining to iron and copper homeostasis were significantly up-regulated, attributed to transcription factor Aft1p, due to the apparent reduction in intracellular concentration of iron and copper. Depletion of both of these metal ions has detrimental effects to overall cellular function as they are cofactors for enzymes, involved in respiration and scavenging reactive oxygen species and insufficient iron levels can result in termination of the cell cycle process at G1 phase.162–165 Additionally, the biosynthesis of sulfur-containing methionine and cysteine were down-regulated along with the peptide transporter gene PTR2. Since PTR2 is a di- or tri-peptide transporter gene, this may also contribute to the reduced uptake of glutathione.161 As such, it can be reasoned that cellular resistance mechanisms to 56MESS(II) do not involve deactivation by thiol-containing biomolecules as they do for cisplatin.166 Down-regulation was also reported for genes that facilitate an oxidative stress response including CCP1, GRX4 and TPS2; resulting in weakening of cellular defense mechanisms.161 Although 56MESS(II) perturbed several genes that regulate various cellular process, none were implicated in DNA repair mechanisms, further supporting that DNA binding is not significant factor in its cytotoxicity. Additional studies of 56MESS(II) against a range of cell lines, including MDA-MB-231 triple negative breast cancer cells, demonstrated 17-fold greater activity than oxaliplatin (0.24 and 4.2 mM, respectively).167 MDA-MB-231 cells are highly resistant to numerous chemotherapeutic agents; however, the sub-micromolar activity exhibited by 56MESS(II) in this cell line supports a mode of action that is fundamentally distinct from cisplatin and its analogs. While mechanistic studies of these complexes are ongoing, several molecular processes have thus far been implicated in their mode of action. This includes a reduction in the mitochondrial membrane potential; induction of epigenetic processes; changes to cytoskeletal structure and to some extent, modification of nuclear DNA.167 This is the first reported instance of a platinum-based chemotherapeutic that is capable of altering cytoskeletal architecture within cells. Even though this class of complexes exhibit exceptional cytotoxicity in vitro, subsequent in vivo studies have produced mixed outcomes. PC3 prostate cancer xenografts in mice (Female Specific Pathogen Free Swiss nude mice) treated with PHENSS(II) (PtII-22, Fig. 12) showed a comparable reduction in tumor weight compared to those treated with cisplatin. Furthermore, no obvious signs of toxicity were observed for the mice treated with PHENSS(II), while 3 out of 6 mice that were treated with cisplatin did not survive.168 In contrast, the treatment of BD-IX rats bearing peritoneal carcinomatosis (colon cancer) with 56MESS(II) resulted in high levels of nephrotoxicity.169 This is surprising since 56MESS(II) is more potent than PHENSS(II) and significantly more potent than cisplatin in vitro, although it is less efficient at tumor reduction in vivo. Taken collectively, this class of complexes demonstrates a great potential for further development and underpins the idea of synthesizing chemotherapeutics that deviate from the canonical structure and mechanism of cisplatin and its analogs.

2.23.3

Platinum(IV) prodrugs as anticancer agents

2.23.3.1

Clinically trialed prodrugs

The design and synthesis of platinum(IV) complexes has been gaining increasing attention due to their potential pharmacokinetic advantages over their platinum(II) counterparts. In contrast to platinum(II) complexes (four-coordinate square-planar geometry), platinum(IV) complexes adopt a six-coordinate octahedral geometry (Fig. 13).170 This configuration enhances the overall kinetic stability, which decreases the possibility of non-specific interactions prior to reaching the intended biological target.171,172 It has been demonstrated that the increased kinetic inertness of platinum(IV) complexes can enable the drug to be administered orally, rather than intravenously.29,173 Moreover, the additional coordination sites afford the prospect of modulating multiple pharmacological properties including redox stability, increased bioactivity, tumor selectivity and lipophilicity.23,173–175 Although the mechanism by which platinum(IV) complexes exert their antiproliferative activity has not been fully elucidated, they are generally regarded as prodrugs since it is believed that reduction to platinum(II) must occur, extracellularly or preferably intracellularly, before they are able to interact with their biological target (Fig. 13).176 Numerous analogs of cisplatin have been oxidized to platinum(IV) and some have been clinically trialed, where they have demonstrated improvements to certain aspects of therapy such as few side-effects and oral availability.177,178 Iproplatin (ctc-dichloridodihydroxidobis(isopropylamine)platinum(IV), PtIV-1, Fig. 14) was the first platinum(IV) complex to enter clinical trials, eventually progressing to phase III. However, it was considered to be less effective than cisplatin and carboplatin due to lower activity and further studies were abandoned.179,180 Tetraplatin (tetrachlorido(1,2-cyclohexanediamine-N,N0 )platinum(IV), also known as

Fig. 13 General 6-coordinate octahedral geometry of platinum(IV) prodrugs. Y and Z ¼ equatorial ligands; X ¼ axial ligands. Reduction (activation) of platinum(IV) results in dissociation of the axial ligands from the cytotoxic square-planar platinum(II) precursor.

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Metal complexes as chemotherapeutic agents

Fig. 14 Structures of clinically investigated platinum(IV) prodrugs. Iproplatin (PtIV-1); tetraplatin (PtIV-2); satraplatin (PtIV-3) and LA-12 (PtIV-4). * indicates a chiral center, either S or R.

ormaplatin, PtIV-2, Fig. 14) exhibited encouraging in vitro and in vivo results against cisplatin-resistant cancers; however, subsequent phase I clinical trials revealed high levels of neurotoxicity and further investigations were discontinued.179,181 The severe neurotoxicity was attributed to the rapid reduction of tetraplatin (reported to have a half-life of 3 s in rat blood plasma) to its platinum congener.181 Satraplatin (bis(acetato-O)amminedichlorido(cyclohexylamine)platinum(IV), PtIV-3, Fig. 14) is perhaps the most promising platinum(IV) prodrug candidate to be assessed in clinical trials thus far. Preclinical studies of satraplatin revealed favorable pharmacokinetics and activity in cisplatin-resistant cell lines.182 Another advantage of satraplatin is its enhanced stability, which allows for oral administration.183 In vivo evaluation of satraplatin to treat xenograft models of ovarian cancer revealed comparable activity to cisplatin and carboplatin with reduced signs of gastro- and hepatotoxicity.184 After successfully completing phase I and phase II clinical trials, satraplatin advanced to phase III clinical trials where it was used in combination with prednisone to treat hormonerefractory prostate cancer. Despite some success in the phase III trials, the overall improvement to patient survival was deemed insignificant and it did not gain approval.185,186 LA-12 ((OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloridoplatinum(IV), PtIV-4, Fig. 14) is another orally administrable form of a platinum(IV) prodrug that has displayed some encouraging results in preclinical studies.187–189 It is structurally related to satraplatin, with the exception that the cyclohexylamine ligand is substituted by 1-adamantylamine, which confers greater lipophilicity to the complex.188,190 This subtle change in structure results in better activity compared to cisplatin and satraplatin; improved pharmacokinetics (with lower acute toxicity exhibited in vivo) and no cross-resistance with cisplatin.191 In vivo studies also showed no indications of nephro- or hepatotoxicity, although higher doses did cause thrombo- and leukocytopenia.191 There have been reports of LA-12 entering phase I and II clinical trials; however, detailed findings have yet to be published.187,190 Nevertheless, the improvements exhibited by satraplatin and LA-12 in alleviating certain side-effects to chemotherapy highlight the potential of platinum(IV) prodrugs and the necessity for one to be approved for treatment regimens.

2.23.3.2

Multi-action prodrugs

All clinically trialed platinum(IV) prodrugs to date have comprised of simple hydroxido, chlorido or acetato axial ligands. While such ligands are not considered to impart any additional activity, it is apparent that they can significantly alter the pharmacokinetics of the resulting prodrug. To improve the efficacy of platinum(IV) prodrugs, increasing focus has been placed on identifying more sophisticated axial ligands that provide a wider range of functionalities; ideally, ligands that synergistically enhance the effectiveness of the prodrug.192 The most common examples of platinum(IV)-ligand conjugates encompass bioactive ligands that are known to inhibit various enzymes and molecular pathways implicated in the proliferation of cancer cells. The added benefit to this approach, as opposed to conventional combination therapy, is that the prodrugs have a single pharmacokinetic profile; meaning that all components are released simultaneously in the same location. This greatly increases the chances of the bioactive ligand(s) and the cytotoxic platinum(II) payload working synergistically to improve the overall efficacy of chemotherapy.

2.23.3.2.1

Histone deacetylase inhibition

Histone deacetylases (HDAC) are a class of enzymes that are pivotal in mediating transcription and gene expression through modification of histones, where they catalyze the removal of acetyl groups.193,194 Inhibition of HDAC leads to an increase in the acetylation level of histones, which results in expansion of the highly condensed chromatin architecture.194 Consequently, nuclear DNA becomes more exposed and accessible to chemotherapeutics that can exert their cytotoxicity through DNA binding pathways. Additionally, HDACs are upregulated in multiple cancers which make them viable targets for chemotherapy.195–197 Numerous platinum(IV) derivatives of cisplatin (or its analog) and oxaliplatin comprising of valproate (VPA) and 4phenylbutyrate (PhB) HDAC inhibitors have been reported and have shown exceptional activity (PtIV-5–PtIV-12, Fig. 15).198–202 PtIV-9, which consists of two PhB axial ligands, displayed potent activity across the range of cell lines tested. Lower activity was

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Fig. 15 Chemical structure of platinum(IV) derivatives of cisplatin and oxaliplatin comprising of HDAC inhibitors, VPA (PtIV-5–PtIV-8 and PtIV-12) and PhB (PtIV-9–PtIV-11).

observed for the cisplatin derivatives comprising of VPA (PtIV-5–PtIV-7). Almost 100-fold greater activity was exhibited by PtIV-9 compared to cisplatin against MCF-7 breast cancer cells, with IC50 values of 0.18 and 17.4 mM, respectively. More notably, PtIV9 was equally as potent in both A2780 (ovarian cancer) and A2780cisR (cisplatin-resistant ovarian cancer) cell lines, with IC50 values of 0.14 and 0.12 mM, respectively; while that of cisplatin were 2.8 and 13.8 mM, respectively.202 This indicates that PtIV-9 may be able to overcome the resistance mechanisms that hinder the cytotoxicity of cisplatin. Interestingly, the platinum(IV) derivatives of cisplatin, PtIV-5 and PtIV-9, displayed better activity than their platinum(IV) oxaliplatin counterparts, PtIV-8 and PtIV-11, even though oxaliplatin itself was more potent than cisplatin in the tested cell lines. These results may be substantiated by recent findings in which oxaliplatin exerts its activity through ribosome biogenesis stress rather than a DNA-damage response, thus inhibiting HDAC activity and exposing nuclear DNA to oxaliplatin could be considered inconsequential.59 Cellular accumulation studies were also performed in the MCF-7 cell line, although no correlation could be drawn between lipophilicity, uptake and cytotoxicity. Overall, the platinum(IV) complexes with di-substituted PhB or VPA exhibited better HDAC inhibition than their mono-substituted analogs, while no significant inhibition was observed for cisplatin, oxaliplatin or platinum(IV) derivatives lacking HDAC inhibitors.202 Other platinum(IV) derivatives of cisplatin have been synthesized, comprising of PhB and octanoate (OA) axial ligands (PtIV-13 and PtIV-14, Fig. 16). OA is reported to inhibit DNA methyltransferase, which leads to the hypermethylation of DNA. The combination of PhB and OA in the axial positions of PtIV-14 resulted in improved cytotoxicity with an IC50 value of 9 nM against HCT-116 (human colorectal carcinoma) cell line, which is approximately 890-fold more potent than cisplatin.203 Similar to PtIV-9, PtIV-14 does not show any significant cross-resistance with cisplatin in A2780cisR cells, exhibiting an enhanced IC50 value of 32 nM. Against “healthy” MRC-5-pd30 (lung tissue) cells, PtIV-14 demonstrated approximately 2–14-fold lower cytotoxicity (as compared to the other tested cell lines), suggesting some degree of selectivity toward malignant cells over non-malignant cells. Additional cytotoxicity assays carried out in 3D spheroid cell cultures derived from MCF-7 cells also demonstrated the potent activity of PtIV-14, where it was over 300-fold more effective than cisplatin. Annexin V/propidium iodide (PI) dual staining assays in MDA-MB-231 cells treated with PtIV-14 revealed the induction of cell death through apoptotic and necrotic pathways, although more of the cell populations constitute of early necrotic cells.203 PtIV-14 demonstrated markedly higher uptake and DNA platination levels than cisplatin even though the majority was present in the membrane/particulate fraction, similar to cisplatin. Cisplatin did not show HDAC inhibition nor increased DNA methylation; PtIV-10 showed HDAC inhibition but no increase in DNA methylation; PtIV13 did not show HDAC inhibition but showed increased DNA methylation and PtIV-14 showed both inhibition of HDAC and increase in DNA methylation.203 It is apparent that the PhB and OA axial ligands can simultaneously contribute to the overall potency of PtIV-14 through epigenetic changes. This library of complexes was further expanded with the development of platinum(IV) derivatives of oxaliplatin and 56MESS(II) bearing similar combinations of PhB and OA in the axial positions (the development of platinum(IV) derivatives of 56MESS(II) are described in more detail in Section 2.23.3.3). Antiproliferative studies revealed that platinum(IV) derivatives of 56MESS(II) (PtIV18– PtIV-20) were considerably more potent than their analogous platinum(IV) oxaliplatin derivatives (PtIV-15– PtIV-17).167 The cytotoxicity of 56MESS(II) was comparable to its platinum(IV) derivatives, while the platinum(IV) derivatives of oxaliplatin

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Fig. 16 Chemical structures of platinum(IV) derivatives of cisplatin (PtIV-13–PtIV-14), oxaliplatin (PtIV-15–PtIV-17) and 56MESS(II) (PtIV-18–PtIV-20) incorporating the HDAC inhibitors, PhB and/or OA.

demonstrated either comparable or superior activity than oxaliplatin, especially in the case of PtIV-17. This suggests that the bioactive ligands, PhB and OA, do not have a substantial influence on the cytotoxicity of the platinum(IV) derivatives of 56MESS(II), most likely since increasing evidence suggests that DNA is not the prime target of 56MESS(II). Nevertheless, 56MESS(II) and its derivatives, namely PtIV-20, displayed remarkable activity in MDA-MB-231 and HCT-116 (colon carcinoma) cancer cells, with IC50 values of 60 and 64 nM, respectively; approximately 7.5- and 4-fold more active than the most active oxaliplatin derivative, PtIV-17. These results are promising since both of these cell lines express mutations in the RAS gene (most prevalent oncogene in human cancer), which is responsible for increasing their resistance to chemotherapeutic agents.167 No trends were observed between lipophilicity, cellular uptake and cytotoxicity. Oxaliplatin and its platinum(IV) derivatives were shown to accumulate preferentially in the membrane, accounting for 54–71% of internalized platinum, which is comparable to cisplatin and its derivatives. In contrast, 56MESS(II) and its platinum(IV) derivatives favored accumulation in the cytoskeleton, accounting for 73–76% of platinum taken into the cell.167 As such, it is not unreasonable to presume that 56MESS(II) and its derivatives induce cell death through pathways distinct from clinically used complexes. Cytotoxicity studies against CHO-K1 (wild-type Chinese hamster ovary, DNA-repair-proficient) and its mutant MMC-2 (DNArepair-deficient) cell lines demonstrated comparable activity for 56MESS(II) and its derivatives in both cell lines, further supporting that DNA damage is not a significant factor in the cytotoxicity of 56MESS(II)-derived complexes.167 56MESS(II) and its platinum(IV) derivatives caused cell cycle arrest in the G2/M phase, which is consistent with interruption to the cytoskeleton, while oxaliplatin and its derivatives halted cells in the S phase and slightly in the G2/M phase. Accompanying flow cytometry studies identified tubulin (key components of microtubules) as the main target of 56MESS(II) and its platinum(IV) derivatives. HDAC inhibition was more pronounced for the complexes incorporating PhB (PtIV-15, PtIV-17, PtIV-18 and PtIV-20) and DNA methylation levels were higher for complexes comprising OA (PtIV-16, PtIV-17, PtIV-19 and PtIV-20). Generally, derivatives of oxaliplatin were slightly more effective at inhibiting HDAC activity and increasing DNA methylation than their analogous 56MESS(II) counterparts.167 These results indicate that the bioactive ligands, PhB and OA, can work synergistically with the cytotoxic platinum(II) payloads to improve its antiproliferative activity, provided that the platinum(II) core can interact with DNA. In contrast, the

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Fig. 17 Chemical structures of platinum(IV) derivatives of cisplatin (PtIV-15–PtIV-23), [Pt(RRDACH)Cl2] (PtIV-24–PtIV-26) and kiteplatin (PtIV-27–PtIV29) incorporating the HDAC inhibitor, 2-(2-propynyl)octanoate (POA). * indicates a chiral center, either S or R stereochemistry.

activity of platinum(IV) derivatives of 56MESS(II) was largely unaffected by such bioactive ligands, presumably owing to the fact that their cytotoxicity stems from disrupting the cytoskeletal architecture of cells, namely microtubules. Another series of platinum(IV) complexes incorporating the HDAC inhibitor, 2-(2-propynyl)octanoate (POA), have been developed (PtIV-21–PtIV-23, Fig. 17). All derivatives demonstrate exceptional cytotoxicity across several cell lines, with nanomolar to submicromolar IC50 values.204 PtIV-21 was approximately 80-fold more active than cisplatin against HCT-116 colorectal cancer cells, with IC50 values of 2.3 and 0.029 mM, respectively, while PtIV-23 displayed the lowest IC50 value of 3.7 nM against NT2/D1 metastatic testicular cancer. No discernible difference in activity was observed based on the enantiomer of the POA ligand contained within the derivatives. Annexin V/PI assays indicated a dose-dependent mode of cell death for PtIV-21; either late apoptotic or necrotic when incubated at 4 and 10 mM, respectively. PtIV-21–PtIV-23 demonstrate more efficient internalization than cisplatin based on accumulation ratios (AR, ratio of intra- and extracellular platinum concentrations), although it was shown that uptake can be reduced by efflux mechanisms. PtIV-21 forms DNA adducts more effectively than cisplatin over a longer period, presumably due to intracellular reduction occurring overtime which releases the cytotoxic platinum(II) core to bind with DNA. Surprisingly, HDAC inhibition of cisplatin was only slightly lower than that of PtIV-21–PtIV-23, although fluorescence microscopy did reveal decondensation of chromatin, while cisplatin displayed condensation of chromatin. Additionally, PtIV-21 was able to induce apoptosis through induction of caspase-3/7 more effectively than cisplatin and upregulated expression levels of genes targeted by HDAC inhibitors including p21 and COX-2 (cyclooxygenase-2). Remarkably, PtIV-21 (administered orally) displayed superb tumor inhibition (94%) of syngeneic murine Lewis lung carcinoma (LLC), which is known to be a highly aggressive solid tumor model with lower toxicity compared to cisplatin (75%, administered intraperitoneally) and PtIV-5 (69%). The tumor regression ability of PtIV-21 was attributed to the combination of POA (lipophilic) and acetate (hydrophilic) axial ligands improving its biodistribution. POA has also been coordinated to the axial position of the platinum(IV) derivative of [Pt(RRDACH)Cl2] (PtIV-24–PtIV-26, Fig. 17). PtIV-26 recorded the lowest IC50 value of 6.3 nM against CT26 mouse colon carcinoma cells. The lipophilic axial ligand, POA, resulted in substantially higher ARs than cisplatin and inhibition of HDAC activity, characterized by the relaxation of chromatin. The lipophilic axial ligand, POA, resulted in substantially higher ARs and inhibition of HDAC activity compared to cisplatin, characterized by the relaxation of chromatin. Oral and iv administration of PtIV-24 to treat BALB/c mice implanted with syngeneic CT26 mouse colon cancer displayed comparable tumor inhibition; the former resulted in severe toxicity characterized by > 20% reduction in body weight and cachexia, while the latter produced more favorable outcomes of reduced nephro- and hepatotoxicity compared to oxaliplatin. Analysis of the tumor tissue following iv treatment of PtIV-24 revealed the invasion the medullary region of the tumor mass by cytotoxic CD8þ T lymphocytes; to a greater extent than oxaliplatin, indicating PtIV-24 is capable of inducing immunogenic cell death similar to oxaliplatin.205 An analogous group of platinum(IV) complexes were synthesized comprising of POA as the axial ligand and kiteplatin as the cytotoxic platinum(II) core (PtIV-27–PtIV-29, Fig. 17). Cytotoxicity studies demonstrated greater activity than their platinum(II) precursors across all tested cell lines. Superior activity was also observed compared to oxaliplatin (IC50 ¼ 15.5 mM) in oxaliplatin-resistant LoVoOXP colorectal cancer with IC50 values ranging from 50 to 180 nM, suggesting they were able to circumvent resistance mechanisms that hinder the efficacy of oxaliplatin. In most instances, platinum(IV) derivatives of [Pt(RRDACH)Cl2] (PtIV-24–PtIV-26) were slightly more potent than analogous platinum(IV) derivatives of kiteplatin (PtIV-27–PtIV-29). PtIV-24–PtIV-29 exhibited 7–8-fold higher uptake compared to their platinum(II) precursors. Interestingly, PtIV-27–PtIV-29 (kiteplatin derivatives) exhibited slightly higher accumulation than PtIV-24–PtIV-26 ([Pt(RRDACH)Cl2] derivatives), although they were slightly less active in vitro. Oral administration of PtIV-24 in C57BL mice implanted with syngeneic murine LLC displayed slightly better tumor reduction (86%) compared to cisplatin

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(78%, administered intraperitoneally), although it was still not as effective as the previously investigated cisplatin derivative, PtIV-21 (94%). Suberoylanilide hydroxamic acid (SAHA, Vorinostat) is another potent HDAC inhibitor that has been granted FDA approval for clinical use, where combination therapy with cisplatin has demonstrated synergistic activity.206–209 Several examples of molecules that are related to SAHA have been reported to be conjugated to the axial positions of cisplatin and its analogs. Often, significant structural modifications to SAHA are required or specific linker ligands are necessary to allow the molecule to be coordinated to the platinum(IV) complex. Consequently, when the prodrug is reduced intracellularly, it is not SAHA that is being released but rather a derivative that is still conjugated to the linker ligand, which is usually less potent. Thus, the expected synergistic activity of the overall prodrug is diminished. To improve on this, steps have been made to develop novel synthetic procedures to allow SAHA to be conjugated to platinum(IV) prodrugs in such a way that reduction leads to release of the linker ligand from platinum core and SAHA (or its derivative) as well. The use of DSC (bis(2,5-dioxopyrrolidin-1-yl)carbonate) to activate the axial OH of platinum(IV) complexes has been shown to yield the electrophilic mono(2,5-dioxopyrrolidin-1-yl)-carbonate, which can then react with primary, secondary or anilinic amines to afford carbamates.210 Also proposed is DSC-activation of the amine before reaction with the axial OH of platinum(IV) complexes, although this method was generally less efficient with lower yields and longer reaction times. Using these methods, a closely related SAHA derivative (consisting of a p-amino group on the phenyl ring) was conjugated to a platinum(IV) derivative of cisplatin through a carbamate linker (PtIV-30, Fig. 18). Reduction studies of PtIV-30 with excess ascorbic acid demonstrated the release of the SAHA derivative from the cisplatin core and rapid dissociation from the linker. Although PtIV-30 exhibited two-fold greater activity than cisplatin against NSCLC PC9 cells (5.14 and 11.24 mM, respectively), it was generally less potent than cisplatin against A375 melanoma, A2780 and A2780cisR cell lines.210 This suggests that there is no synergistic activity, possibly be due to the fact that the derivative of SAHA used in this study generally had lower activity than SAHA across the tested cell lines. A similar approach was used to couple SAHA to the platinum(IV) derivative of cisplatin either directly through a carbonate linker or an extended 4-aminobenzyl alcohol carbamate linker (PtIV-31–PtIV-33, Fig. 18).211 Both strategies resulted in the release of unmodified SAHA from the cytotoxic platinum(II) core. In vitro studies against A2780 cells displayed slightly lower activity for PtIV-31 (4.2 mM), PtIV-32 (7.2 mM) and PtIV-33 (2.3 mM) compared to cisplatin (1.1 mM). However, cisplatin (15.8 mM) exhibited 14-fold lower activity against A2780cisR cell lines, while PtIV-31–PtIV-33 demonstrated IC50 values ranging from 4.7–7.6 mM.211 This suggests that PtIV-31–PtIV-33 are able to overcome the resistance mechanisms in A2780cisR cells. Evident from both studies, subtle modifications to the chemical structure of known biologically active molecules and/or how they are released after reduction can severely impede their function. These findings provide novel protocols that may allow numerous other bioactive molecules to be tethered to platinum(IV) complexes.

2.23.3.2.2

Cyclooxygenase inhibition

Non-steroidal anti-inflammatory drugs (NSAIDs) have also been investigated as attractive avenues in the development of platinum(IV) prodrugs. NSAIDs exert their activity on cyclooxygenase (COX), which are enzymes that catalyze the biosynthesis of

Fig. 18 Chemical structures of platinum(IV) derivatives of cisplatin incorporating a variant of SAHA and PhB (PtIV-30) or SAHA and acetate (PtIV31–PtIV-33) in the axial positions.

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prostanoids. COX exists in two isoforms, COX-1 and COX-2; the former is constitutively expressed in most cells, while the latter is induced by multiple factors, including inflammatory stimuli.212,213 Of particular significance to chemotherapy is COX-2 since it is involved in the initiation, promotion and progression of tumors.213,214 Furthermore, it is overexpressed in many cancers relative to healthy cells. As such, inhibition of COX-2 could be promising in producing efficient chemotherapeutic regimens. Some earlier examples of COX inhibitors that have been tethered to the axial positions of platinum(IV) prodrugs include aspirin, indomethacin and ibuprofen (PtIV-34–PtIV-38, Fig. 19). PtIV-34 (also known as asplatin and platin-A) encompasses aspirin in the axial position of a platinum(IV) derivative of cisplatin and has exhibited activity that is either comparable or superior to cisplatin in several malignant cell lines.215,216 In particular, 10-fold greater activity was observed in HeLa cervical cancer cells compared to cisplatin with IC50 values of 0.45 and 4.51 mM, respectively. It has also demonstrated slightly better activity than a combination of cisplatin and aspirin (equimolar ratio) in LNCaP androgen-sensitive prostate cancer cells, suggesting a synergistic effect of the prodrug once reduce in the cell.216 COX-inhibition assays revealed the ability of PtIV-34 to inhibit both COX-1 and COX-2, comparable to aspirin.216 The cellular uptake of PtIV-34 in HeLa cells was notably higher (3-fold greater) than cisplatin, while DNA platination levels were at least 2-fold higher.215 In vivo studies of PtIV-34 against HepG2 hepatocellular carcinoma xenograft mice models demonstrated superior antitumor activity than cisplatin with reduced signs of toxicity (lower reduction of body weight). The promising results obtained from such studies have inspired the use of other COX inhibitors as potential axial ligands. Complexes PtIV-35–PtIV-38 (Fig. 19) incorporate either indomethacin or ibuprofen in both axial positions and exhibit potent activity against a panel of malignant cell lines. The platinum(IV) derivatives of cisplatin and oxaliplatin that incorporate ibuprofen (PtIV-37 and PtIV-38, respectively) demonstrated better antiproliferative activity compared to their analogous derivatives containing

Fig. 19 Chemical structures of platinum(IV) derivatives of cisplatin, oxaliplatin and kiteplatin incorporating COX inhibitors aspirin (PtIV-34), indomethacin (PtIV-35 and PtIV-36), ibuprofen (PtIV-37–PtIV-39), ketoprofen (PtIV-40) and naproxen (PtIV-41).

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indomethacin (PtIV-35 and PtIV-36, respectively) despite greater COX inhibition exhibited by PtIV-35 and PtIV-36.217,218 For example, against highly aggressive MDA-MB-231 (COX-2 expression) breast cancer cells, PtIV-37 was the most potent, exhibiting 33-fold greater than PtIV-35 and 400-fold greater activity than cisplatin with IC50 values of 0.05, 1.65 and 20 mM, respectively.217 It is also noteworthy that PtIV-35 (indomethacin derivative of cisplatin) was highly selective for COX-2, while PtIV-36 (indomethacin derivative of oxaliplatin) was highly selective for COX-1, suggesting that the equatorial ligands may contribute to the binding mechanism of the enzyme.218 Combination treatment of cisplatin and ibuprofen or indomethacin (1:2 ratio) showed no apparent improvements to activity, indicating conjugation of the NSAID to the platinum core is necessary for enhanced cytotoxicity; however, this was ascribed to improved lipophilicity rather than COX-2 inhibition.217 Similar findings were reported for the platinum(IV) derivative of kiteplatin, PtIV-39, which is comprised of two ibuprofen axial ligands.219 In vitro antiproliferative studies were carried out against HCT-15 and HCT-116 colon cancer cell lines, where PtIV-39 exhibited 24- and 38-fold greater activity than kiteplatin and cisplatin alone, respectively. These cell lines however, do not have substantial COX-2 expression levels and thus the enhanced cytotoxicity was not attributed to the synergistic activity of COX-2 inhibition and the cytotoxic platinum(II). Instead, increased lipophilicity of the prodrug was postulated to be the major factor in the improved potency.219 Akin to the previously discussed platinum(IV)-COX inhibitor conjugates, platinum(IV) prodrugs containing ketoprofen and naproxen, PtIV-40 and PtIV-41 (Fig. 19) respectively, have also demonstrated similar trends.220 For example, both complexes displayed slightly better activity in SW480 cells (extremely low COX-2 expression levels) than HT29 colon cancer cells (slightly higher COX-2 expression levels), signifying that COX-2 inhibition is not crucial to their mechanism of action. Again, the higher accumulation levels of PtIV-40 and PtIV-41 were attributed to the respective NSAIDs enhancing the overall lipophilicity of the prodrugs.220 Di-substitution of the NSAID, flurbiprofen, to the axial positions of the platinum(IV) derivative of cisplatin produced complex PtIV-42 (Fig. 19), which can inhibit COX-2 activity better than free flurbiprofen. Reduction of PtIV-42 in the presence of ascorbic acid releases axially bound flurbiprofen and cisplatin which can form bifunctional adducts with 20 -deoxyguanosine 50 -monophosphate sodium salt hydrate (50 -GMP).221 Cytotoxicity assays of PtIV-42 revealed higher activity than cisplatin or a combination of cisplatin and flurbiprofen (1:2). Notably, against SW480 colon carcinoma cells, PtIV-42 exhibited an IC50 of 0.6 mM, which is 82- and 49-fold greater activity than cisplatin and combination of cisplatin and flurbiprofen (1:2). Moreover, PtIV-42 is equally potent in A549/ A549DDP (cisplatin-sensitive and cisplatin-resistant lung cancer, respectively) and BEL7404/BEL7404-CP20 (cisplatin-sensitive and cisplatin-resistant liver cancer, respectively) cell lines, indicating that it can overcome resistance mechanisms associated with cisplatin.221 The enhanced potency of PtIV-42 was ascribed to the flurbiprofen axial ligands increasing its lipophilicity, consequently allowing the spontaneous formation of nanoparticles which results in improved cellular accumulation, DNA platination and apoptosis inducing ability compared to cisplatin. To broaden the range of platinum(IV) prodrugs that contain NSAIDs, naproxen was coordinated to the platinum(IV) derivative of cisplatin, carboplatin and oxaliplatin to form the mono- and di-substituted complexes, PtIV-43–PtIV-49 (Fig. 20). PtIV-43–PtIV-47 demonstrated modest IC50 values, with mono-substituted derivatives (PtIV-43–PtIV-45) generally exhibiting better activity than their di-substituted counterparts (PtIV-46 and PtIV-47).222 The prodrugs containing carboplatin (PtIV-45 and PtIV-47) as the platinum(II) were the least active overall. The lowest IC50 was exhibited by PtIV-43 (0.2 mM) against CT26 murine colon cancer. Also noteworthy is that PtIV-44 displayed equivalent activity in A549 (cisplatin-sensitive human lung cancer) and A549R (cisplatinresistant human lung cancer) cell lines, with IC50 values of 5.2 and 4.8 mM, respectively; indicating its potential in overcoming resistance mechanisms associated with cisplatin. The enhanced cellular accumulation of PtIV-44 did not correlate with DNA platination, which may be explained by the differences in mechanism of action of cisplatin and oxaliplatin, since DNA may not be the prime target for oxaliplatin. Treatment of BALB/c mice bearing CT-26 homografts with PtIV-44 demonstrated tumor suppression comparable to cisplatin and oxaliplatin, with lower systemic toxicity.222 Immunohistochemical analysis of the tumor tissue revealed the ability of PtIV-44 to downregulate the expression of matrix metalloproteinase-9 (MMP-9); which is an enzyme that is associated with tumor angiogenesis, invasion and metastasis.223–225 Wound healing assays further supported this finding as PtIV-44 inhibited the migration of cancer cells, comparable to oxaliplatin.222 PtIV-44 exhibited relatively modest inhibition of COX-2 activity, suggesting it is not a crucial factor in the observed antitumor activity. A separate study synthesized similar platinum(IV) derivatives of cisplatin with mono- and di-substituted naproxen ligands (PtIV48 and PtIV-49, Fig. 20) and tested their efficacy in triple-negative breast cancer (TNBC). In this case, the di-substituted derivative, PtIV-49, was more potent than the mono-substituted derivative, PtIV-48.226 PtIV-49 was 187-fold more potent than cisplatin against MDA-MB-231 cells, displaying IC50 values of 0.16 and 29.98 mM, respectively. Intriguingly, combination treatment of cisplatin and naproxen (1:1 or 1:2 ratio) resulted in drastically reduced activity in MDA-MB-231 and MDA-MB-435 cell lines compared to cisplatin, suggesting an antagonistic relationship between the pair.226 The lipophilic axial ligands contributed to enhanced cellular uptake of PtIV-48 and PtIV-49, resulting in preferential accumulation in the cytosol. PtIV-49 exhibited exceptional tumor inhibition in female BALB/c mice with MDA-MB-231 xenografts with very little toxicity compared to cisplatin and PtIV-48. The enhanced efficacy can be attributed to its distinct multimodal mechanism of action that downregulates COX-2 expression, leading to suppression of prostaglandin E2 (PGE2) and programmed death ligand 1 (PD-L1), consequently minimizing cancer cell migration and evasion from the immune response.227 Downregulation of bromodomain-containing protein 4 (BRD4) and extracellular regulated kinases 1/2 (Erk1/2) further inhibits PD-L1, which results in mobilization of T lymphocytes from immunosuppression. Simultaneously, PtIV-49 exerts DNA damage via the formation of non-covalent adducts and/or generation of ROS, as evident by

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Fig. 20 Chemical structures of platinum(IV) derivatives of cisplatin, carboplatin and oxaliplatin incorporating COX inhibitors flurbiprofen (PtIV-42) and naproxen (PtIV-43–PtIV-49).

upregulated g-H2AX (DNA damage marker). Furthermore, inhibition of pro-inflammatory cytokines (interleukin-1b (IL-1b) and IL-6) secreted by macrophages reduces tumorigenesis induced by inflammation.

2.23.3.2.3

Pyruvate dehydrogenase kinase inhibition

Pyruvate dehydrogenase kinases (PDK) are mitochondrial enzymes that promote aerobic glycolysis (also known as the Warburg effect) by inhibiting the catalytic activity of the pyruvate dehydrogenase complex, consequently suppressing mitochondrial oxidation of glucose.228,229 This altered metabolism causes changes in the tumor microenvironment (TME), which is implicated in enhanced survival and proliferation of malignant cells.230,231 Several studies have demonstrated that the small molecule dichloroacetate (DCA) is able to target mitochondria and inhibit the activity of PDK, shifting cellular metabolism back to glucose oxidation, effectively reversing the Warburg effect.232–235 To explore the possibility of improving the potency of chemotherapeutic agents, platinum(IV) prodrugs have been designed to incorporate DCA as axial ligands. One of the earliest reported examples coordinated DCA in both axial positions of a platinum(IV) derivative of cisplatin to produce PtIV-50 (mitaplatin, Fig. 21). The cytotoxicity of PtIV-50 was comparable to that of cisplatin against several cancer cell lines and was less toxic against “healthy” MRC-5 lung fibroblasts, suggesting some selectivity.236 Mechanistic studies revealed that the reduction of PtIV-50 released cisplatin and two equivalents of DCA; where cisplatin can then bind to genomic DNA and DCA could alter the mitochondrial membrane potential of malignant cells, causing a cascade of events to trigger apoptosis. Additional studies of PtIV-50 in cisplatin-resistant KB-CP 20 (human epidermoid adenocarcinoma) and BEL 7404-CP 20 (hepatoma) cell lines demonstrated better activity than cisplatin or cisplatin in combination with DCA (1:2), suggesting the prodrug is able to circumvent resistance mechanisms. The improved activity was attributed to DCA in increasing lipophilicity to enhance accumulation and inducing mitochondria-dependent apoptosis.237 Follow up studies encapsulated PtIV-50, within PLGA-PEG (poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol)) nanoparticles for in vivo studies against MDA-MB-468 breast cancer xenografts in mice. Compared to unencapsulated-PtIV-50, encapsulated-PtIV-50 displayed prolonged retention in the bloodstream and reduced accumulation in the kidneys, although higher accumulation was observed in the liver. Additionally, long term tumor growth inhibition

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Fig. 21

Chemical structures of platinum(IV) prodrugs incorporating different PDK inhibitors (PtIV-50–PtIV-64).

was comparable to unencapsulated-PtIV-50.238 Optimization of the nanoparticle formulation and encapsulation may reduce common dose-limiting side-effects to chemotherapy, such as nephrotoxicity. The absence of nucleotide excision repair (NER) pathway in mitochondria was used as a rationale to develop platinum(IV) prodrugs functionalized with triphenyl phosphine (TPP) to promote accumulation in the mitochondria and DCA or PhB mitochondrial sensitizers to improve efficacy. Two series of prodrugs were synthesized based on the platinum(IV) scaffolds of cisplatin (PtIV51–PtIV-54) and oxaliplatin (PtIV-55–PtIV-58, Fig. 21).239 The oxaliplatin-based derivatives (PtIV-55–PtIV-58) were generally more active than the cisplatin-based derivatives (PtIV-51–PtIV-54). From both series, those incorporating PhB (PtIV-54 and PtIV-58) or DCA (PtIV-53 and PtIV-57) were more potent than the other analogs, suggesting these ligands impart synergistic activity with the platinum core. PtIV-53, PtIV-54, PtIV-57 and PtIV-58 demonstrated preferential accumulation in the mitochondria with higher platinum-mtDNA adducts compared to adducts with nDNA, further supporting that the axially bound ligands work synergistically to enhance mitochondrial accumulation and damage. Additional studies in A2780 cells demonstrated that these prodrugs could completely inhibit mitochondrial respiratory processes, increase pyruvate dehydrogenase (PDH) activity (thus inhibit PDK), induce reactive oxygen species (ROS) and alter the mitochondrial membrane potential, releasing cytochrome C and apoptosis inducing factor (AIF) to facilitate apoptosis. In the case of PtIV-54 and PtIV-I, necrotic cell death was also observed. PtIV-53, PtIV-54, PtIV57 and PtIV-58 also resulted in upregulation of p53 protein and caspase 3, suggesting p53-mediated mitochondrial caspase 3 dependent apoptosis.239 PtIV-53 demonstrated superior tumor inhibition than cisplatin in BALB/c mice inoculated subcutaneously with CT26 colon adenocarcinoma even though platinum content in tumor tissues were identical, suggesting enhanced tumor reduction

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efficacy of PtIV-53 was not related to accumulation levels. A PEGylated liposomal formulation (DPPC, cholesterol and DSPEPEG2000 (1.85:1:0.15)) was used to encapsulate PtIV-53, which remarkably not only lowered renal toxicity, but also achieved complete tumor remission. Another series of cisplatin-based platinum(IV) prodrugs have been developed incorporating estramustine (EM) in one axial position with a HDAC, COX or PDK inhibitor in the opposite axial position (PtIV-59–PtIV-64, Fig. 21).240 Estramustine is a microtubule-targeting agent that disrupts microtubule assembly by binding to microtubule-associated proteins (MAP) and tubulin. Moreover, estramustine can downregulate the expression of androgen receptors, which may be effective in suppressing prostate cancers. All complexes exhibited nanomolar to sub-micromolar activity across a panel of cancerous cell lines with higher selectivity toward malignant cells compared to cisplatin. The prodrug treatments were 7–12-fold more potent than their respective combination treatment (cisplatin þ EM þ bioactive ligand (1:1:1)), exemplifying the efficacy of prodrugs. Mechanistic investigations revealed cell cycle arrest predominantly in the G2/M phase, while cellular uptake and DNA platination corresponded with higher lipophilicity. Intracellular reduction releases cisplatin, which can bind to DNA, while estramustine inhibits tubulin polymerization and disrupts microtubule assembly in addition to downregulating, expression levels of androgen receptors (overexpression leads to resistance to androgen receptor targeted therapy) and prostate-specific antigen (a glycoprotein enzyme often associated with prostate cancer). The bioactive ligand in the opposite axial position also contributes to the mechanism of action where: (i) the addition of PhB (PtIV-61) or VPA (PtIV-62) inhibits HDAC activity; (ii) the addition of aspirin (PtIV-63) results in induction of autophagy; and (iii) the addition of DCA (PtIV-64) decreases the uptake of glucose. As demonstrated, the combination of bioactive ligands to a cytotoxic platinum core can work synergistically to enhance the overall potency of the prodrug by interacting with multiple biological targets.

2.23.3.2.4

Glutathione S-transferase inhibition

Glutathione S-transferases (GSTs) are overexpressed in many cancer cells and are reported to contribute to cisplatin-resistance. GSTs are classed as detoxification enzymes which are found in the cytosol and catalyze the conjugation of glutathione to electrophilic substrates, such as cisplatin.241 The resulting adducts are usually water soluble, which facilitates their excretion via the mercapturic acid pathway and reduces the intracellular concentration of platinum. Ethacrynic acid (EA) is a known inhibitor of GST that can sensitize resistance cancer cells and has been conjugated to the axial position(s) to the platinum(IV) derivative of cisplatin (PtIV65 and PtIV-66, Fig. 22). Both PtIV-65 and PtIV-66 exhibit comparable or superior cytotoxicity than cisplatin against several cell lines, while PtIV-65 comprising of two EA moieties, was most potent at inhibiting GST activity.242 PtIV-66 was significantly more susceptible to reduction due to the free axial hydroxido ligand, possibly diminishing its viability as a prodrug. However, subsequent in vivo

Fig. 22

Chemical structures of platinum(IV) derivatives of cisplatin bearing GST inhibitors; EA (PtIV-65 and PtIV-66) or NBDHEX (PtIV-67).

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studies of PtIV-66 in chicken embryo implanted with A2780 ovarian cancer demonstrated inhibition of tumor growth comparable to cisplatin with lower toxicity, as evident by increased embryo survival.243 A different GST inhibitor, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was tethered to the axial position of a platinum(IV) derivative of cisplatin (PtIV-67, Fig. 22). PtIV-67 exhibited greater antiproliferative activity than cisplatin in several neoplastic cell lines, especially against cisplatin-resistant A549cisR NSCLC, where it was approximately 42-fold more potent, effectively overcoming cisplatin-resistance.244 The improved cytotoxicity was attributed to the axially bound NBDHEX ligand that seemingly increased the lipophilicity to improve cellular accumulation and inhibited GST to enhance DNA platination levels. Additionally, the downregulation of Bcl-2 and upregulation of cleaved PARP contribute to the observed activity. In vivo studies in A549 xenografts of mice revealed greater inhibition of tumor growth compared to cisplatin, NBDHEX (free ligand) and combination treatment of cisplatin and NBDHEX (1:1). Furthermore, PtIV-67 displayed lower systemic toxicity as evident by histological analysis of tissues and no reduction in body weight of mice.

2.23.3.2.5

Tumor microenvironment regulators

Apart from proliferating tumor cells, the tumor microenvironment (TME) is heavily influenced by the tumor stroma, which includes (but not limited to) blood vessels, infiltrating inflammatory cells, blood cells and endothelial cells.245 The tumor stroma is mainly a product of cell-host interactions and results in the characteristic hypoxic and acidic environment of solid tumors. These factors not only contribute to the growth, invasion and metastasis of tumors but also hinder the efficacy of chemotherapeutics.246 Hypoxia inducible factor 1 (HIF-1) is a transcription factor that regulates numerous hypoxia-inducible genes. Its subunit, HIF-1a, dimerizes with HIF-1b to activate the transcription of genes associated with tumorigenesis.246 HIF-1a is overexpressed in 70% of cancers, making it a promising target to regulate the TME and improve the efficacy of chemotherapeutic agents. Platinum(IV) derivatives of cisplatin were designed to incorporate 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) as a potent inhibitor of HIF-1a (PtIV-68 and PtIV-69, Fig. 23).247 Both complexes exhibited better activity than cisplatin across a panel of cell lines, including cisplatin-resistant gastric cancer (SGC7901/CDDP), with IC50 values of 1.45 and 2.78 mM for PtIV-68 and PtIV-69, respectively and 13.09 mM for cisplatin. Lower cytotoxicity was observed against “healthy” human liver (LO2) cells for PtIV-68 (7.64 mM) and PtIV-69 (8.03 mM) compared to cisplatin (6.02 mM), indicating they can overcome cisplatin resistance and are more selective toward malignant cells. Under hypoxic conditions, PtIV-68 and PtIV-69 demonstrated further improvements to cytotoxicity. Notably, PtIV-69 was 38- and 93-fold more potent in A549 and HCT-116 cancer cells, respectively, compared to cisplatin. Mechanistic studies revealed the enhanced ability of PtIV-69 to induce apoptosis under hypoxic conditions more prominently than under normoxic conditions, which is likely attributed to improve cellular accumulation under hypoxic conditions. Furthermore, treatment of HCT-116 with PtIV-69 under hypoxia resulted in marked inhibition in the expression levels of HIF1a, suggesting that YC-1 is essential to the observed cytotoxicity. Subsequent in vivo studies of PtIV-69 in HCT-116 xenografts in mice demonstrated comparable tumor inhibition to cisplatin and oxaliplatin; however, displayed less systemic toxicity. The functionalization of platinum(IV) derivatives of cisplatin has also been carried out using 2-(p-chlorophenoxy)-2methylpropionic acid (clofibric acid, CA). CA activates peroxisome proliferator-activated receptor a (PPARa), which in turn promotes the degradation of HIF-1a (PtIV-70 and PtIV-71, Fig. 23).248 Both complexes were more potent than cisplatin across numerous cell lines, especially PtIV-71, which was 80-fold more active against cisplatin-resistant MM98R sarcomatoid mesothelioma, indicating its ability to bypass cisplatin resistance mechanisms. Moreover, PtIV-71 is equally active under normoxic and hypoxic conditions, while the activity of cisplatin was reduced under hypoxia. The cellular uptake and DNA platination of PtIV71 was significantly enhanced compared to PtIV-70 and cisplatin owing to higher lipophilicity, which also resulted in its ability to trigger apoptosis more effectively through caspase-dependent pathways. Additional studies demonstrated cell cycle arrest in G2/M phase under both normoxic and hypoxic conditions, while increased degradation of HIF-1a was attributed to the released CA moieties, which synergistically improves efficacy under hypoxic conditions. Carbonic anhydrase IX (CAIX) is a cell-surface glycoprotein that is involved in regulating intra- and extracellular pH of the TME and is transcriptionally activated by HIF-1. It is highly overexpressed in malignant tissue, while its expression levels are significantly reduced in normal cells, making it a relevant target for chemotherapeutic agents. Accordingly, a known CAIX inhibitor, benzene sulfonamide, has been coordinated to platinum(IV) derivatives of cisplatin and oxaliplatin (PtIV-72 and PtIV-73, Fig. 23).249 PtIV-72 and PtIV-73 exhibit potent activity and exceptional selectivity toward TNBC cells under hypoxia compared to “healthy” cells; up to 81-fold more selective. In contrast, cisplatin and oxaliplatin are equally active in malignant and non-malignant cells, while their activity is reduced under hypoxic conditions. The potent activity of PtIV-72 and PtIV-73 stems from the axially bound CAIX inhibitor, benzene sulfonamide, which essentially alters the TME and metabolic pathways in favor of the prodrugs. Inhibition of CAIX results in increased cellular O2 levels, reduced HIF-1a expression and reduced extracellular acidification, suggesting it can reverse hypoxia and the acidic TME. Furthermore, downregulation of biomacromolecular synthesis and energy supply to cells effectively suppresses tumor proliferation, metastasis and angiogenesis. In vivo investigations of PtIV-72 and PtIV-73 in BALB/c nude mice bearing MDA-MB-231 xenografts displayed better tumor reduction than cisplatin and oxaliplatin with considerably lower systemic toxicity.

2.23.3.2.6

Immunostimulators

In addition to TME alterations suppressing the immune response, the loss of antigenicity of tumor cells also enables them to evade immune surveillance.250 Restoring the immune system by designing chemotherapeutic agents that can activate the immune response may help inhibit tumor progression. A class of platinum(IV) derivatives of cisplatin were developed as immune-chemotherapeutic

Metal complexes as chemotherapeutic agents

Fig. 23

769

Chemical structures of platinum(IV) prodrugs that can regulate the TME (PtIV-68 and PtIV-73).

agents, which encompass immunostimulating peptides including, annexin-1, WKYMVm (m ¼ D-Met) and fMLFK (f ¼ formyl), that bind to formyl peptide receptors (FPRs) (PtIV-74–PtIV-78, Fig. 24).251 FPR1 and FPR2 are a part of the family of G-protein-coupled receptors that are overexpressed in immune cells, such as monocytes and natural killer (NK) cells and many metastatic tumors. Apart from PtIV-77, all other complexes displayed antiproliferative activity comparable to cisplatin in FPR1/2-overexpressing U-87MG (glioblastoma) and MCF-7 cancer cell lines. The WKMYMVm-conjugated derivatives, PtIV-76 and PtIV-78 supported selective uptake mediated by FPR1/2 receptors in U-87MG cells. Additional studies of PtIV-78 revealed its ability to activate monocytes and NK cells, resulting

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Fig. 24 Chemical structures of platinum(IV) derivatives of cisplatin incorporating immunostimulating peptides (PtIV-74–PtIV-78); IDO inhibitor (PtIV79 and PtIV-80) or TDO inhibitor (PtIV-81 and PtIV-82).

in significantly increased secretion of tumor necrosis factor alpha (TNF-a) and interferon gamma (IFN-g), which are key regulators of the innate immune system. Interestingly, p53 mutant MDA-MB-231 cells were resistant to DNA-damage-induced cell death, although they were sensitive to immune-mediated cytotoxicity, indicating the therapeutic synergy between immunotherapy and chemotherapy. Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme that inhibits T cell and NK cell proliferation and activation, thus allowing tumors to evade the immune response.252 Its overexpression in several cancer types is characteristic of poor prognosis. Prodrugs of cisplatin were developed by conjugating (D)-1-methyltryptophan as an inhibitor of IDO (PtIV-79 and PtIV-80, Fig. 24). Both complexes demonstrated antiproliferative activity, although PtIV-80 was significantly more potent than cisplatin across the tested ovarian cancer cell lines, especially in cisplatin-resistant A2780/CP70 cells, where it was 40-fold more active.253 Mechanistic studies confirmed the ability of PtIV-80 to enhance proliferation of T cells by downregulating aryl hydrocarbon receptor (AHR) and interleukin-6 (IL-6), which are involved in the auto-regulation of constitutive IDO expression. Furthermore, DNA

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damage also contributes to the antiproliferative activity, resulting in G1 cell cycle arrest. PLGA-PEG nanoparticles were used encapsulate PtIV-80 before being injected in BALB/c mice, which demonstrated better stability than cisplatin and satraplatin in blood. Similar to IDO, some tumors overexpress tryptophan 2,3-dioxygenase (TDO) as a way to evade the immune response.254 Two analogous platinum(IV) derivatives of cisplatin were prepared incorporating a TDO inhibitor in one axial position and either a chlorido or hydroxido ligand in the opposite position (PtIV-81 and PtIV-82, Fig. 24). Both complexes demonstrated comparable activity to cisplatin against neoplastic cells but were less toxic to “healthy” LO2 liver cells, suggesting better selectivity.255 In particular, PtIV-82 demonstrated the highest activity against HepG-2 cells, which display elevated expression of TDO. Intracellular reduction results in the concomitant release of the TDO inhibitor and cisplatin, leading to cell death via mitochondrial-dependent apoptosis and arrest of the cell cycle at S phase. Further studies confirmed the inhibition of TDO expression in HepG-2 cells, which blocks kynurenine production and suppresses AHR expression levels. Consequently, the proliferation of T-cells is markedly enhanced, resulting in improved efficacy of the prodrug and also indicates its ability to regulate the tumor immune microenvironment.

2.23.3.2.7

Cancer stem cell targeted

As described in Section 2.23.2.2.3, incomplete eradication of cancer stem cells (CSCs) can result in relapse of cancers. The inefficiency of currently used chemotherapeutics at killing CSCs has prompted the development of platinum(IV) prodrugs that can eradicate both CSCs and bulk cancer. The naturally occurring compound cinnamic acid (CA) commonly exists as the trans isomer and has been reported to exhibit anticancer activity, with the ability to potentiate the differentiation of CSCs, sensitizing them to anticancer agents.256,257 Accordingly, two platinum(IV) derivatives of cisplatin that encompass mono- or disubstituted trans-CA molecules have been developed (PtIV-83 and PtIV-84, Fig. 25).258 Antiproliferative assays against a panel of cell lines demonstrated superior activity compared to cisplatin, especially in the case of PtIV-84, which was approximately 567-fold more potent in RD rhabdomyosarcoma cells (IC50 ¼ 17 and 0.030 mM, respectively). Against “healthy” MRC5 pd30 cells, PtIV-84 was up to 30-fold less active, suggesting higher selectivity toward malignant cells. Furthermore, combination treatment of cisplatin and trans-CA (1:1 or 1:2) showed no difference in activity compared to free cisplatin against all tested cell lines, indicating that conjugation of trans-CA to the platinum core is crucial for enhanced cytotoxicity. The enhanced cellular accumulation of PtIV-84 in RD cells corresponded with increased DNA adducts than PtIV-83 and cisplatin. Additional in vitro studies of PtIV-83 and PtIV-84 in CHO-K1 and MMC-2 cell lines demonstrated greater activity in MMC-2 cells, supporting significant contribution of DNA damage to the biological activity. PtIV-84 exhibited sub-micromolar activity in 3D spheroid models of MCF-7CD44- and RDCD133- (CSC-depleted) and MCF-7CD44 þ and RDCD133 þ (CSC-enriched). Notably, PtIV-84 was 74-fold more potent than cisplatin in CSC-enriched RDCD133 þ spheroids, indicating its ability to effectively kill both differentiated and cancer stem cells. Elevated levels of MyoD and myogenin (differentiation markers) indicated differentiation of RDCD133 þ stem cells, while the transcription factor homeobox protein NanoG (oncogene that is overexpressed in CSCs) was downregulated. These findings support that both trans-CA molecules, once disassociated from the platinum after reduction, contribute to the differentiation and thus, sensitization of CSCs to cisplatin, resulting in synergistic activity to improve the overall potency. A separate study focused on improving the efficacy of chemotherapeutics against aggressive HER2-positive (epidermal growth factor receptor 2) breast cancers. HER2 is overexpressed in 20–25% of breast cancers and is associated with poor prognosis, higher recurrence rate and increased mortality.259 PtIV-85 was designed to incorporate oleic acid (OLA) to suppress HER2 overexpression and trans-CA to sensitize CSCs to cisplatin upon intracellular reduction (Fig. 25).260 PtIV-85 was 38–253-fold more potent than cisplatin against the tested breast cancer cell lines, where activity directly correlated with HER2 expression levels. Lower activity against “healthy” MRC5 pd30 cells suggests selectivity toward cancer cells over non-malignant cells. 3D spheroid model of SKBR-3 (highest HER2 expression) also demonstrated greater activity of PtIV-85 compared to cisplatin, with IC50 values of 1.4 and 33.6 mM, respectively. Enhanced cellular accumulation and DNA platination by PtIV-85 in SK-BR-3 (HER2-positive) cells compared to MCF-7 (HER2-null) cells were correlated with the increased lipophilicity. Intracellular reduction releases the OLA axial ligands which contribute to the significant reduction of HER2 expression, while the cytotoxic core binds to DNA and triggers

Fig. 25

Chemical structures of platinum(IV) derivatives of cisplatin designed to eradicate cancer stem cells (PtIV-83–PtIV-85).

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Metal complexes as chemotherapeutic agents

apoptosis. PtIV-85 was also significantly more potent than cisplatin in 2D and 3D models of SK-BR-3 and CSC-enriched SK-BR3CD44 þ 24-, indicating that it can kill CSCs, similar to PtIV-83 and PtIV-84. Although the exact mechanism by which PtIV-85 suppresses HER2 expression has not been elucidated, it is apparent both OLA and trans-CA ligands exert their activity synergistically with the cisplatin core upon intracellular reduction to exhibit potent cytotoxicity.

2.23.3.2.8

DNA damage response disrupters

2.23.3.2.9

Prodrugs with unconventional cytotoxic cores

DNA damage response (DDR) mechanisms are responsible for detecting, signaling and promoting the repair of DNA lesions.261 In particular, the nucleotide excision repair (NER) pathway is implicated in conferring greater resistance to cells from platinum chemotherapeutics, such as cisplatin, by excising the DNA cross-links formed.262 A NER inhibitor (NERi), (E)-2-(((8a,9a-dihydro-9H-fluoren-9-ylidene)hydrazono)methyl)benzoic acid, was anchored to the axial position of a platinum(IV) derivative of cisplatin to overcome cisplatin resistance (PtIV-86, Fig. 26). Compared to cisplatin, PtIV-86 exhibited 34- and 88-fold increase in cytotoxicity against cisplatin-resistant A2780cisR and A549cisR cell lines, respectively, indicating its ability to overcome resistance mechanisms.263 Mechanistic investigations revealed that PtIV-86 can accumulate more efficiently in cells than cisplatin, attributed increased lipophilicity. Subsequent intracellular reduction results in simultaneous release of the NERi moiety from the cisplatin core, which attenuates the NER pathway from repairing DNA lesions caused by cisplatin, ultimately leading to apoptosis. The synergistic activity between the two components of the prodrug results in improved activity against cisplatin-resistant cell lines. Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme that is pivotal to processes involving DNA replication, transcriptional regulation and DNA damage repair. Following DNA damage, the activity of PARP is significantly increased and displays high affinity toward cisplatin cross-links.264,265 Several PARP-1 inhibitors were conjugated to the axial positions of platinum(IV) derivatives of cisplatin to mitigate the DNA damage response (PtIV-87–PtIV-93, Fig. 26). Upon coordination to platinum, the inhibition of PARP1 was enhanced compared to the respective free ligands.266 On average, PtIV-87–PtIV-90 displayed comparable or slightly lower activity than cisplatin, while PtIV-91–PtIV-93 were highly active across a range of cell lines, especially against A2780cisR cells, suggesting they can overcome resistance mechanisms. PtIV-92 and PtIV-93 can accumulate in cells more efficiently than cisplatin, bind to DNA and disrupt the cell cycle distribution in both A2780 and A2780cisR cells. Moreover, PtIV-91 and PtIV-92 trigger apoptosis by inducing the cleavage of PARP-1.

As evident with the vast majority of platinum(IV) prodrugs discussed here, the cytotoxic platinum(II) core is either cisplatin or oxaliplatin. While these studies have demonstrated differences in their mechanisms of action, DNA remains to be a crucial target upon intracellular reduction. Efforts have been made to develop platinum(IV) prodrugs that encompass platinum(II) cores that are structurally and mechanistically distinct. A few examples have been examined in Section 2.23.3.2.1, bearing axial PhB and/or OA ligands on 56MESS and demonstrated fundamentally different modes of action. Oxidation of [PtII(HL)(AL)]2 þ type complexes (where HL ¼ PHEN or methyl substituted PHEN and AL ¼ SSDACH) discussed in Section 2.23.2.2.7 led to the synthesis of [PtIV(HL)(AL)(OH)2]2þ complexes, which retain their potent cytotoxicity against an extensive range of cell lines (Fig. 27).159 Other derivatives of 56MESS(IV) have been prepared using a combination of different bioactive and non-bioactive axial ligands including, acetate, octanoate, palmitate, phenylbutyrate and/or valproate (PtIV-94–PtIV-100, Fig. 27). The derivatives demonstrate approximately 20-fold greater activity than oxaliplatin against LoVo OxPt oxaliplatin-resistant colon cancer, suggesting they can circumvent resistance mechanisms. However, unlike platinum(IV) prodrugs of cisplatin and oxaliplatin that display enhanced cytotoxicity compared to their respective precursors, the derivatives of 56MESS(IV) did not exhibit improved antiproliferative activity compared to 56MESS(II) across most cell lines.267 The exception was against BxPC3 pancreatic adenocarcinoma, where PtIV-99 and PtIV-100 exhibited improved activity with IC50 values of 0.56 and 0.44 mM. It is possible that the potent cytotoxicity of 56MESS(II) masks any synergistic activity imparted by the axial ligands. Treatment of mice bearing LLC with 56MESS(IV), PtIV-99 or cisplatin displayed tumor inhibition of 61, 73 and 75%, respectively. Cisplatin treatment resulted in slightly greater reduction in body weight compared to 56MESS(IV) and PtIV-99, although some systemic toxicities were observed for all treatments. The potent activity of 56MESS(II), ascribed to its unique mechanism of action, prompted the design of a dinuclear complex incorporating cisplatin and 56MESS(II) as the cytotoxic platinum(II) cores, with DCA and PhB as bioactive ligands (PtIV-103, Fig. 28). PtIV-103 was more cytotoxic across all tested cell lines than its individual precursor complexes, PtIV-101 and PtIV-102 and its analog lacking bioactive ligands (PtIV-104), suggesting that the enhanced activity is a result of the synergistic effect of all components.268 Remarkably, PtIV-103 was most potent in KRAS mutated LoVo colorectal cancer, where it was 456-fold more potent than cisplatin, with IC50 values of 20.0 nM and 9.12 mM, respectively. In 3D spheroid models, PtIV-103 was again most effective, particularly in KRAS mutated PSN1 pancreatic cancer. Furthermore, PtIV-103 was less toxic against “healthy” HEK293 human embryonic kidney cells, indicating selectivity toward neoplastic cells, especially KRAS mutated cells. The cellular accumulation of PtIV-103 was significantly enhanced compared to other complexes, including the analogous PtIV-104, suggesting DCA and PhB contribute to uptake mechanisms. Even though the nuclear DNA platination levels exhibited by PtIV-103 were comparable to cisplatin and PtIV-104, it demonstrated superior inhibition of HDAC and glycolytic activity, stemming from PhB and DCA. Furthermore, the translocation of cytochrome c (proapoptotic mediator) from the mitochondria to the cytosol indicated mitochondrial-mediated apoptosis. The multifaceted biological activity exhibited by PtIV-103 highlights the importance of appropriate ligand choice as they can substantially influence the overall potency of the prodrug. Importantly, PtIV-103 displays remarkable cytotoxicity and selectivity toward KRAS mutated cell lines, which are notoriously difficult to treat and often considered “undruggable.”

Metal complexes as chemotherapeutic agents

Fig. 26

Chemical structures of platinum(IV) derivatives of cisplatin incorporating NERi (PtIV-86) or different PARP-1 inhibitors (PtIV-87–PtIV-93).

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Fig. 27 General structure of [Pt(HL)(AL)(OH)2]2þ type complexes and chemical structures of platinum(IV) derivatives of 56MESS(IV) containing nonbioactive and bioactive axial ligands (PtIV-94–PtIV-100).

Based on the rationale that increased lipophilicity results in enhanced cellular accumulation and cytotoxicity, derivatives of PHENSS(IV) and 56MESS(IV) were developed to encompass short, medium and long chain fatty acids as axial ligands to modulate their lipophilicity (Fig. 29).269,270 Free PHENSS(IV) and 56MESS(IV) are highly hydrophilic and coordination of shorter chained ligands did not significantly alter their solubility. In contrast, addition of longer axial ligands resulted in profoundly different solubilities. For instance, the increased lipophilicity facilitated the spontaneous formation of self-assembled nanoparticles, which could be exploited to assist their delivery to tumor tissues in vivo, owing to the enhanced permeability and retention effect.271 Overall, all derivatives demonstrated better cytotoxicity than cisplatin and their respective di-hydroxido counterparts, either PHENSS(IV) or 56MESS(IV), against a panel of cell lines. In particular, PtIV-105 exhibited 1100-fold greater activity than cisplatin against HT29 colon cancer cells, while PtIV-106 displayed the lowest IC50 value of 3.4 nM against Du145 prostate cancer cells. The lipophilic derivatives accumulated more efficiently in HT29 and A2780 cells compared to their respective platinum(II) and platinum(IV) precursors; however, there was no clear correlation between increasing lipophilicity resulting in enhanced cellular uptake and cytotoxicity. The lack of correlation suggests an active transport mechanism such as endocytosis, rather than passive diffusion.

2.23.3.3

Photoactivatable prodrugs

Another area that has been gaining increasing attention is the development of photoactivated platinum(IV) chemotherapeutic agents. The earliest example of a photoactivatable platinum(IV) complex, trans-[PtIV(N3)2(CN)4]2, was reported in 1978. Irradiation at 302 nm in aqueous solution led to the formation of [PtII(CN)4]2 and release of two azidyl radicals, which rapidly decomposed to form N2.272 Using this approach, platinum(IV) complexes are designed so that their reduction (and hence their activation) is achieved via light irradiation of specific wavelengths, rather than relying on intracellular reductants. The prospect of controlling

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Fig. 28 Chemical structures of platinum(IV) derivatives of cisplatin incorporating DCA and GABA (PtIV-101); 56MESS(II) incorporating PhB and succinate (PtIV-102); combination of both platinum species to form the dinuclear complex, PtIV-103. The dinuclear complex lacking bioactive ligands, PtIV-104, was prepared to compare the influence of bioactive ligands on overall cytotoxicity.

temporal and spatial activation of the platinum(IV) prodrug can result in improved selective activity toward tumor tissue and thus, reduce systemic toxicities.273

Fig. 29

General chemical structures of PHENSS(IV) and 56MESS(IV) with axial ligands of increasing lipophilicities.

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Metal complexes as chemotherapeutic agents

The first generation of photoactive platinum(IV) complexes were reported in 1996, incorporating iodide as the photoactivating ligand, with the general formula ctc-[Pt(en)(X)2(I)2] (where X ¼ Cl, OH, OCOCH3, OSO2CH3 or OCOCF3).274,275 Although these complexes demonstrated photoactivity after irradiation, they were also proved to be less stable in the dark due to reduction by thiolcontaining biomolecules. The development of second generation photoactive platinum(IV) complexes sought to improve upon these drawbacks. Replacement of iodide with azide ligands resulted in [Pt(N3)2(OH)2(L)(L0 )] type complexes (where L and L0 ¼ cis or trans am(m)ine ligands) which exhibited improved stability in the dark and lower reactivity toward biological reducing agents.276–278 In contrast to cis-based analogs of cisplatin that demonstrate enhanced activity than their trans counterparts, complexes from this class of photoactivatable platinum(IV) complexes display higher potency in the trans configuration rather than cis configuration.279 One of the more potent complexes from this series is PtIV-107 (Fig. 30), demonstrated significantly enhanced activity after irradiation, with phototoxicity indices (PI ¼ IC50 after irradiation/IC50 in the dark) > 151 against A2780 ovarian cancer cells (IC50(irr) ¼ 1.4 mM; IC50(dark)  212 mM) and HaCaT keratinocytes (IC50(irr) ¼ 1.4 mM; IC50(dark)  212 mM).280 Significant contributions to the advancement of this class of complexes have been made by Sadler and coworkers over the years and excellent reviews are available on their earlier works.281–284 This section highlights more recent developments in the field of photoactivatable platinum(IV) prodrugs. A series of dinuclear photoactivatable platinum(IV) prodrugs have been prepared which incorporate a derivative of PtIV-107 as the platinum core, where one axial hydroxido ligand is replaced by a succinate. The axial succinate groups are linked via amide bonds with different diamine ligands to form PtIV-108–PtIV-111 as dinuclear complexes (Fig. 30).285 These complexes are nonactive in the dark, although irradiation (465 nm) results in the generation of azidyl and hydroxyl radicals and significantly enhances cytotoxicity. The most potent complex from the series, PtIV-111, exhibited IC50 values of approximately 17 mM against both A2780 and A2780cisR ovarian cancer cell lines and 8.8 mM against OE19 esophageal cancer cells. Additionally, all complexes were nontoxic against “healthy” MRC5 lung fibroblasts (> 100 mM), both in the dark and after irradiation. Under the conditions used for cytotoxicity testing (1 h incubation time), cisplatin was non-active in all cell lines, even after irradiation. The aromatic linker ligand of PtIV-110 resulted in enhanced cellular accumulation attributed to increased lipophilicity. After irradiation, of PtIV-110 and PtIV111, cells arrested mainly in the G2/M phase, as opposed to G0/G1 phase in the dark suggesting they exert a mechanism of action that is distinct from cisplatin and may explain the improved activity of PtIV-111 against A2780cisR cancer cells. Mechanistic studies revealed that photoactivated dinuclear complexes results (PtIV-110 and PtIV-111) release the bridging ligand before binding to DNA and that they were twice as effective at forming interstrand cross-links and unwinding DNA compared to the photoactivated mononuclear complex (PtIV-107). Transcription mapping and DNase I footprinting suggested preferential binding of PtIV-110 and PtIV111 to G/A-rich sequences, similar to PtIV-107. These findings are consistent with photodecomposition of the dinuclear complexes into the two mononuclear platinum(II) precursors before intercellular reactions. Two photoactivatable platinum(IV) prodrugs were synthesized incorporating biotin (vitamin H or B7) and the pyruvate dehydrogenase kinase (PDK) inhibitor, dichloroacetate (DCA) (PtIV-112 and PtIV-113, Fig. 30). Both complexes are stable in the dark and release azidyl radicals and singlet oxygen upon irradiation at 463 nm.286 Photoreactions (420 nm) of PtIV-112 and PtIV-113 demonstrated the formation of interstrand cross-links with ct-DNA. Avidin bound adducts of PtIV-112 (PtIV-112-avi) and PtIV113 (PtIV-113-avi) were also prepared to assess their biological activity. Avidin is a tetrameric protein with a high binding affinity for biotin and has been used in targeted chemotherapy.286,287 All complexes were non-toxic in the dark against A2780, A549 lung cancer, PC3 prostate cancer and “healthy” MRC5 cell lines. Upon irradiation (465 nm), all complexes demonstrated enhanced activity, where PtIV-113 was the most potent (1.3 mM against A2780) and displaying the greatest PI of > 38.5. The avidin-bound PtIV-112-avi was approximately 2.7-fold more phototoxic in A2780 cells compared to the unbound PtIV-112, while PtIV-113-avi was slightly less phototoxic than PtIV-113. Cisplatin was inactive in all instances due to the short 2 h incubation time.286 The biotinylated complexes (PtIV-112 and PtIV-113) did not show any obvious increase in accumulation compared to the unmodified complex (PtIV-107), suggesting that the biotin ligands did not contribute to cellular uptake through receptor mediated pathways. PtIV-113 exhibited 33- and 53-fold higher uptake than PtIV-107 and PtIV-112, respectively, ascribed to increased lipophilicity due to the additional DCA ligand. Interestingly, the uptake of PtIV-112-avi was 10-fold higher than PtIV-112, while no discernible difference was observed between PtIV-113-avi and PtIV-113. Photoactivation of PtIV-107 has been reported to produce azidyl and hydroxyl radicals, nitrenes and singlet oxygen in addition to the cytotoxic platinum(II) core.280,281,288,289 Moreover, it exhibits potent activity in several malignant cell lines, with a high PI and causes DNA damage that results in a cascade of cellular effects, distinct from cisplatin. The ability of oxaliplatin to trigger immunogenic cell death (ICD) prompted re-examination of the mechanism of action of PtIV-107 since ROS and reactive nitrogen species (RNS) can also induce ICD.290 Several characteristic features of ICD were examined, including translocation of calreticulin to the cell surface, extracellular secretion of ATP, release of high-mobility group box 1 protein (HMGB1) and an increase in tumor cell phagocytosis. No apparent differences were observed in the dark; however, irradiation (420 nm) resulted in higher levels of calreticulin on the cell surface along with extracellular secretion of ATP and HMGB1 from the nuclei. Interestingly, irradiation of oxaliplatin or doxorubicin (known ICD inducer) was detrimental to their ability to induce ICD as lower levels of calreticulin were translocated to the cell surface and secretion of ATP and HMGB1 was reduced. In CT26 colon carcinoma cells, PtIV-107 was 2-fold more effective at promoting phagocytosis after photoactivation, while cisplatin and oxaliplatin only marginally promoted phagocytosis. These results support the multi-modal mechanism of action for PtIV-107 that synergistically causes DNA damage and induces ICD. A photoactivatable platinum(IV) derivative of oxaliplatin comprising of an axial pyropheophorbide-a (PPA) ligand that is capable of being activated with red light exhibits promising antitumor activity (PtIV-114, phorbiplatin, Fig. 30).291 The

Metal complexes as chemotherapeutic agents

Fig. 30 Chemical structures of diazido-based (PtIV-107–PtIV-113), oxaliplatin-based (PtIV-114–PtIV-116) and carboplatin-based (PtIV-117) photoactivatable platinum(IV) prodrugs.

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photoreduction (650 nm) of PtIV-114 releases PPA from the oxaliplatin core, which is then able to bind to DNA. PtIV-114 accumulates more efficiently than oxaliplatin and localizes primarily in the cytoplasm, while oxaliplatin by itself typically associates with the cell membrane, which suggests that the axially bound PPA significantly influences the accumulation properties of PtIV114.167,291 Low cytotoxicity (> 10 mM) is exhibited in the dark, although irradiation (650 nm) results in a marked increase in antiproliferative activity. In particular, PtIV-114 displayed a PI of > 227; a 1786-fold increase in activity compared to oxaliplatin (78.6 mM) against MCF-7 cells. Additionally, PtIV-114 was non-toxic to “healthy” MRC5, suggesting selectivity toward malignant cells over healthy cells. PtIV-114 also demonstrated the generation of hydroxyl radicals and singlet oxygen upon irradiation at 650 nm, which contribute to its phototoxicity and induces cell death through apoptotic pathways. Irradiation (660 nm) of tumor sites for 10 min in BALB/c mice bearing 4T1 murine mammary adenocarcinoma xenografts demonstrated better tumor suppression and lower systemic toxicity than oxaliplatin or a combination of oxaliplatin and PPA, exemplifying the improved efficacy of the prodrug may be due to the synergistic action of PPA and oxaliplatin after photoreduction compared to its constituents being administered in combination. These findings highlight the applicability of PtIV-114 in in vivo settings to elicit tumor inhibition with minimized side-effects. Using a platinum(IV) derivative of oxaliplatin as the cytotoxic scaffold, a coumarin-based ligand (CMN) was conjugated to the axial positions to form the photocaged complexes, PtIV-115 and PtIV-116 (Fig. 30). Photoreduction (450 nm) proceeds in a protic environment and generates oxygen by oxidizing water. Addition of an octaarginine peptide (R8K) in the axial position opposite to CMN (PtIV-116) significantly improved cellular penetration and localization in the nucleolus, which directly correlates with the amount of platinum-DNA adducts formed and increased expression of g-H2AX.292,293 Photoactivation of PtIV-116 results in release of CMN accompanied by a 101-fold increase in fluorescence, which may allow the accumulation and reduction of PtIV-116 to be visualized within cells. Upon irradiation (450 nm), the potency was markedly increased with PI values ranging from 7 to 62. Comparable phototoxicity in HCT116 p53þ/þ (p53 wild type colon cancer) and HCT116 p53/ (p53 null colon cancer) cancer cell lines suggests that PtIV-116 may exert its activity via a p53-independent pathways. PtIV-116 demonstrated low resistance factors (RFs) of 1.3 and 0.6 in ovarian (A2780cisR/A2780) and lung (A549cisR/A549) cancer cell lines, indicating its ability to overcome resistance mechanisms. Compared to oxaliplatin, PtIV-116 demonstrated superior tumor penetration in 3D A549cisR tumor spheroids, reaching the necrotic core more efficiently. Irradiation (450 nm) of PtIV-116 A549cisR cells significantly increased the expression levels of senescence-associated b-galactosidase and p16, which are biomarkers of cell senescence and caused translocation of calreticulin to the cell membrane, release of HMGB1 and secretion of ATP. Consequently, PtIV-116 triggers T-cell proliferation and induces ICD more efficiently than oxaliplatin, which contribute to its distinct mechanism of action, allowing it to circumvent resistance mechanisms that hinder clinically used platinum chemotherapeutics. The addition of boron dipyrromethene (BODIPY) to the axial position of a platinum(IV) derivative of carboplatin resulted in the photoactivatable prodrug, PtIV-117 (Fig. 30). Upon irradiation (495 nm), PtIV-117 reduces completely within 10 mins, releasing carboplatin, BODIPY and acetic acid.294 It is noteworthy that an analogous cisplatin prodrug was synthesized with BODIPY; however, it was found to have poor stability in the presence of ascorbate and further studies of it were discontinued. The formation of DNA adducts by PtIV-117 were increased upon irradiation, although still lower than that of cisplatin, possibly due to the slower rate of hydrolysis of the carboxylate group. Photoactivation of PtIV-117 demonstrated the generation of singlet oxygen, albeit less efficiently than free BODIPY. Compared to carboplatin, PtIV-117 was more efficient at entering cells and mainly localized in the cytoplasm with approximately 5-fold higher levels of DNA adducts upon irradiation. In vitro cytotoxicity assays revealed the low toxicity of PtIV-117 in the dark, while irradiation (495 nm) resulted in a notable increase in cytotoxicity across the tested cell lines, with 6.5–43-fold greater activity than carboplatin. PtIV-117 was most potent in MCF-7 cells; 39-fold more active than carboplatin. Mechanistic investigations revealed the percentage of cells arrested in the G2/M phase increased to 28.3% when irradiated, compared to the control group in which 12.5% of cells were at the G2/M phase. Confocal imaging suggested that PtIV-117 induced cell death via oncosis, as evident by decreased expression levels of b-actin and a-tubulin, indicative of disruption to the cytoskeleton. In contrast, PtIV-117 in the dark and carboplatin could not produce these alterations supporting a unique mechanism of action that ultimately results in superior activity than carboplatin.

2.23.3.4

Challenges and future perspectives of platinum chemotherapeutics

The diversity and complexity of cancer as a disease undoubtedly presents numerous challenges in developing metallodrugs as chemotherapeutic agents. While there is much that remains to be explored to understand the exact molecular mechanisms which underlie drug-resistance mechanisms and severe systemic toxicities, considerable efforts have been placed in addressing these drawbacks through rational drug design of metallodrugs. The selection of platinum-based drugs discussed here highlight the innovative techniques numerous researchers have undertaken, with several examples exhibiting greater activity in resistant cell lines and improved selectivity toward cancerous cells. As is often the case however, the results obtained from preclinical studies do not always translate into clinical studies. A majority of preclinical studies rely heavily on 2D cell cultures to obtain IC50 values as a means to quantify the activity of a drug. While there is some merit to this, it should be recognized that such methods are overly simplistic and more robust methods should also be considered to quantify drug activity, which would allow for a better understanding of drug interactions within the body. An increasing number of studies have employed 3D spheroid models to better mimic the tumor microenvironment. While this technique is also less complex than the human body, it does provide a better means of preclinical evaluation of a drug than 2D models. Having a better understanding of how a drug interacts in 3D models brings it closer to in vivo studies; however, this is still far from the ideal model. The inherent differences between animal models and human cancers remains

Metal complexes as chemotherapeutic agents

Fig. 31

779

Chemical structures of ruthenium complexes, NAMI-A and KP1019.

a major challenge in predicting the efficacy of a drug in clinical settings. Improving the models used to screen the activity of potential metallodrugs as chemotherapeutics is critical in accurately evaluating its efficacy if it is to proceed to clinical trials with a greater chance of being successful.295–297 It has been almost two decades since the global approval of oxaliplatin and it is hoped with the advancements in technology and the process of rational drug design that another metallodrug will gain clinical approval within the coming years.

Fig. 32 General chemical structures of “piano stool” type ruthenium complexes based on RAED and RAPTA scaffolds and chemical structure of RAPTA-C.

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Metal complexes as chemotherapeutic agents

Fig. 33

Chemical structures of selected ruthenium-based polypyridyl complexes as PDT agents.

2.23.4

NON-platinum anticancer agents

While platinum complexes have been the focus of this chapter, there are considerably more metals with characteristics that make them ideal for medical applications. Within the volumes and chapters of this edition of Comprehensive Inorganic Chemistry, the

Metal complexes as chemotherapeutic agents

Fig. 34

781

Chemical structures of heteronuclear ruthenium and platinum complexes.

breadth of that knowledge will be available to you. Of course, this accrued knowledge is because of the work of many chemists that infuse their passion for inorganic chemistry into every unique complex that they craft; they are truly artists on a molecular level. Here, we have provided a small sample of the many that can be included when reviewing metal complexes that elicit biological activity.

2.23.4.1

Ruthenium complexes

The legacy of years of coordination chemistry research, where it was predicted that octahedral ruthenium complexes had significant promise as anticancer agents,298–302 has been realized with several complexes entering clinical trials.303 Ruthenium(III) indazolium complexes, such as imidazolium[trans-tetrachlorido(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)], (NAMI-A, Fig. 31), ([trans-tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019, Fig. 31) and derivatives created to improve the solubility of KP1019 are prominent examples which exert potent antitumor activity.303–307 NAMI-A and KP1019 are both octahedral complexes, where the ruthenium core is surrounded equatorially by four chlorido ligands. There is no change in the octahedral geometry however the RueCl bond becomes more labile, facilitating a chlorido- aqua ligand exchange.307,308 NAMI-A is axially coordinated to S- and N- ligand while for KP1019 two N- ligands are coordinated in the axial positions (Fig. 31). Although similar, modest structural differences illicit unique cancer profiles.309 NAMI-A reduces the formation of metastases309 although no definitive mode of action has been reported. NAMI-A has been used in a phase I clinical trial where toxicity profile, maximum tolerated dose and pharmacokinetics were determined.308,310 Subsequently, a phase I trial of KP1019 is reported to be selectivity to cancer cells because reduction to the active ruthenium(II) form occurs in the hypoxic environment of tumor tissues.301 “Piano stool” ruthenium(II) complexes exhibit a pseudo-octahedral geometry (Fig. 32).301,311 The hydrophobic p-bound arene ligand facilities the complex cross cell membranes and stabilize the ruthenium(II) complex, while X, Y and Z, can be various bioactive (including anionic or neutral N/O/S-based chelating) ligands312 or labile leaving (e.g. Cl- or H2O) groups (Fig. 32).301,311 Thoughtful choice of ligands can influence stability, ligand exchange kinetics, solubility, cell uptake, distribution, toxicity, detoxification and biomolecular interactions.301,311 Selectively derivatizing the h6-arene is an additional strategy used to modulate biological activities of the resulting complexes313,314 that are activated by ligand exchange reactions. Sadler and Dyson defined the architecture the complexes bases upon [Ru(h6-arene)(1,2-ethylenediamine)Cl]þ (RAED) and [Ru(h6-arene)(1,3,5-triaza-7-phosphaadamantane)Cl2] (RAPTA), respectively (Fig. 32).315–317 Despite the structural similarity, RAPTA and RAED complexes exert different modes of action and spectra of activity.315,317–320 Individual structural features of RAED complexes play crucial roles in the structure-activity relationship studies; for instance the surface area of the arene has been reported to be proportional to the cytotoxicity in cancer cells (Fig. 32).298,311,321 Controlling each features would advance the development of the design for more effective and potent antitumor agents.298 The reported in vivo antimetastatic activity of

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Metal complexes as chemotherapeutic agents

Fig. 35

Chemical structures of examples of gold-based anticancer complexes.

RAPTA-C (Fig. 32)317 have good water solubility is attributed to the coordinated phosphaadamantane ligand.317 RAPTA complexes are inactive toward primary tumors but are potent toward metastatic tumors.322Aquation is similar to cisplatin317 but the mechanism of action studies suggested that RAPTA complexes exert their cytotoxicity via a non-DNA-binding mechanism in contrast to cisplatin and it derivatives.322 There have been a large number of complexes that have been derived from the architectural blueprints of RAED and RAPTA.313,317,323,324 Ruthenium(II) polypyridyl complexes have been a very familiar structure when exploring the interactions with DNA and their potential as scaffolds for photodynamic therapy (Fig. 33).298,299,301,305–307,310–313,315,318–320,322,323,325–331 Ruthenium photosensitizers/photodynamic therapy (PDT) complexes322,327,328 with distinctly different structures have been reported to exhibit considerable cytotoxicity once activated with light. There have been numerous contributors, where each structure has been modulated to tune the irradiation wavelength required to produce potency. Selectivity is realized through localization of irradiation.329–331

Metal complexes as chemotherapeutic agents

Fig. 36

783

Chemical structures of gold-based complexes.

Heteronuclear ruthenium platinum also contribute in this space332,333 with a recent derivative with potential for photo dynamic therapy coupled to a platinum(IV) derivative of cisplatin with bioactive axial ligands to exploit the attributes of each once activated.333 The advantages of such structures are that each component has different mechanisms of action and as such, are able to exert the effects on multiple cellular targets once activated (Fig. 34).

2.23.4.2

Gold complexes

The propensity of gold for applications in medicine has been appreciated for thousands of years with reports of gold preparations used by many ancient cultures in India, Egypt, and China to treat various ailments.302 Gold complexes have rich and accessible oxidation chemistry although there is a predominance gold(I) complexes in the literature and only few of gold(III) complexes. The range of possible complexes is considerable when coupled together with variable oxidation states and a broad selection of coordination ligands; however, a balance must be accommodated with attention to the possible toxic effects.334 Gold complexes are considered prodrugs because they require ligand exchange before they become potent. In general, gold(III) complexes are more reactive than gold(I) complexes and have evoked widespread interest for anticancer therapy.335–337 There are no signs of gold losing any of its “shine” in the years to come with the number and diversity of gold complexes reported in the literature (Fig. 35).334–336,338–345 The gold(I) complexes, 2,3,4,6-tetra-o-acetyl-L-thio-b-D-glucopyranosato-S-(triethyl-phosphine) gold(I) (auranofin)346 was initially approved for the treatment of rheumatoid arthritis,302 however numerous in vitro and in vivo antitumor studies have

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Metal complexes as chemotherapeutic agents

demonstrated that induced apoptosis and is more effective at decreasing cell viability than cisplatin. It has a well-known toxicity profile and is considered safe for human use.342 Various examples of gold(I) complexes reported include the patented gold(I) bis-diphenylphosphino)ethane complexes,347 gold(I) lansoprazole complexes,334,348 alkynylgold(I)(phosphane) complexes,338 and gold(I)carbenes complexes349 together with luminescent gold(I)-heterocyclic carbene complexes,350 gold(I) chalcone complexes,340 gold(I) erlotinib triphenylphosphane complexes,339 and gold(I) phosphine naphthalimide complexes (Fig. 36).351 Gold(III) complexes exhibit d8 electronic configuration and similar structural and reactivity to platinum(II) complexes, but the mechanism of action is unlike that of platinum(II) complexes.341 Those with reported anticancer cytotoxicity include gold(III) porphyrins,352,353 gold(III) dithiocarbamates,354,355 cyclometalated gold(III) complexes,356 dinucleargold(III) complexes.357,358

2.23.5

Conclusions

The development of metallodrugs in medicinal inorganic chemistry has grown exponentially over the past
60 years. Despite this, cisplatin, carboplatin and oxaliplatin continue to dominate the treatment regimens for various cancers. While DNA is considered the main target for cisplatin, increasing research has been devoted in identifying other potential targets that can mediate cancer cell death in hopes of circumventing the limitations that reduce the efficacy of cisplatin and its analogs. The paradigm shift toward rational design and development of fundamentally distinct platinum(II) and platinum(IV) chemotherapeutics has demonstrated tremendous potential. These complexes encompass a variety of ligands and/or structural motifs that not only target DNA but can also confer additional bioactivity to the drug. This chapter highlights those innovative strategies numerous researchers have explored, either within their own group or through international collaborations, culminating in new knowledge and deepening our understanding in ways to improve drug design. In this chapter, the design of unconventional platinum(II) scaffolds that can elicit biological effects different from cisplatin and its analogs were explored. The rational design of combining two or more functional entities as single platinum(IV) prodrugs demonstrated encouraging outcomes as opposed to single agents, with notable improvements to in vivo efficacy. However, even with these advances, the challenges of overcoming resistance mechanisms and off-target toxicity still remain. Understanding the precise mechanisms of action of these drugs are essential to continually improve drug design. While there are several other transition metals that are being pursued as alternatives to platinum chemotherapeutics, some existing and novel examples of ruthenium- and gold-based complexes have been highlighted here. It is envisaged that research within the field of medicinal inorganic chemistry will continue to grow and thrive to produce innovative chemotherapeutic agents that will one day enter clinical trials and ultimately gain approval.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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An Erlotinib Gold(I) Conjugate for Combating Triple-Negative Breast Cancer. J. Inorg. Biochem. 2020, 203, 110910. 340. González, J. J.; Ortega, E.; Rothemund, M.; Gold, M.; Vicente, C.; de Haro, C.; Bautista, D.; Schobert, R.; Ruiz, J. Luminescent Gold(I) Complexes of 1-Pyridyl-3anthracenylchalcone Inducing Apoptosis in Colon Carcinoma Cells and Antivascular Effects. Inorg. Chem. 2019, 58, 12954–12963. 341. Gamberi, T.; Magherini, F.; Fiaschi, T.; Landini, I.; Massai, L.; Valocchia, E.; Bianchi, L.; Bini, L.; Gabbiani, C.; Nobili, S.; Mini, E.; Messori, L.; Modesti, A. Proteomic Analysis of the Cytotoxic Effects Induced by the Organogold(iii) Complex Aubipyc in Cisplatin-Resistant A2780 Ovarian Cancer Cells: Further Evidence for the Glycolytic Pathway Implication. Mol. Biosyst. 1653-1667, 2015, 11. 342. Roder, C.; Thomson, M. J. Auranofin: Repurposing an Old Drug for a Golden New Age. Drugs R&D 2015, 15, 13–20. 343. Meier-Menches, S. M.; Aikman, B.; Döllerer, D.; Klooster, W. T.; Coles, S. J.; Santi, N.; Luk, L.; Casini, A.; Bonsignore, R. Comparative Biological Evaluation and G-Quadruplex Interaction Studies of Two New Families of Organometallic Gold(I) Complexes Featuring N-Heterocyclic Carbene and Alkynyl Ligands. J. Inorg. Biochem. 2020, 202, 110844. 344. Thomas, S. R.; Casini, A. N-Heterocyclic Carbenes as “Smart” Gold Nanoparticle Stabilizers: State-of-the Art and Perspectives for Biomedical Applications. J. Organomet. Chem. 2021, 938, 121743. 345. Meier-Menches, S. M.; Neuditschko, B.; Zappe, K.; Schaier, M.; Gerner, M. C.; Schmetterer, K. G.; Del Favero, G.; Bonsignore, R.; Cichna-Markl, M.; Koellensperger, G.; Casini, A.; Gerner, C. An Organometallic Gold(I) Bis-N-Heterocyclic Carbene Complex with Multimodal Activity in Ovarian Cancer Cells. Chem. Weinheim Bergstr. Ger. 2020, 26, 15528–15537.

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346. Marzano, C.; Gandin, V.; Folda, A.; Scutari, G.; Bindoli, A.; Rigobello, M. P. Inhibition of Thioredoxin Reductase by Auranofin Induces Apoptosis in Cisplatin-Resistant Human Ovarian Cancer Cells. Free Radic. Biol. Med. 2007, 42, 872–881. 347. Berners-Price, S. J.; Mirabelli, C. K.; Johnson, R. K.; Mattern, M. R.; McCabe, F. L.; Faucette, L. F.; Sung, C. M.; Mong, S. M.; Sadler, P. J.; Crooke, S. T. In Vivo Antitumor Activity and In Vitro Cytotoxic Properties of Bis[1,2-Bis(Diphenylphosphino)Ethane]Gold(I) Chloride. Cancer Res. 1986, 46, 5486–5493. 348. Serratice, M.; Bertrand, B.; Janssen, E. F. J.; Hemelt, E.; Zucca, A.; Cocco, F.; Cinellu, M. A.; Casini, A. Gold(i) Compounds with Lansoprazole-Type Ligands: Synthesis, Characterization and Anticancer Properties In Vitro. Med. Chem. Commun. 2014, 5, 1418–1422. 349. Messori, L.; Marchetti, L.; Massai, L.; Scaletti, F.; Guerri, A.; Landini, I.; Nobili, S.; Perrone, G.; Mini, E.; Leoni, P.; Pasquali, M.; Gabbiani, C. Chemistry and Biology of Two Novel Gold(I) Carbene Complexes as Prospective Anticancer Agents. Inorg. Chem. 2014, 53, 2396–2403. 350. Citta, A.; Schuh, E.; Mohr, F.; Folda, A.; Massimino, M. L.; Bindoli, A.; Casini, A.; Rigobello, M. P. Fluorescent Silver(i) and Gold(i)–N-Heterocyclic Carbene Complexes with Cytotoxic Properties: Mechanistic Insights. Metallomics 2013, 5, 1006–1015. 351. Bagowski, C. P.; You, Y.; Scheffler, H.; Vlecken, D. H.; Schmitz, D. J.; Ott, I. Naphthalimide Gold(i) Phosphine Complexes as Anticancer Metallodrugs. Dalton Trans. 2009, 10799–10805. 352. Sun, R. W.-Y.; Che, C.-M. The Anti-Cancer Properties Of Gold(III) Compounds with Dianionic Porphyrin and Tetradentate Ligands. Coord. Chem. Rev. 2009, 253, 1682–1691. 353. Sun, R. W.-Y.; Li, C. K.-L.; Ma, D.-L.; Yan, J. J.; Lok, C.-N.; Leung, C.-H.; Zhu, N.; Che, C.-M. Stable Anticancer Gold(III)–Porphyrin Complexes: Effects of Porphyrin Structure. Chem. A Eur. J. 2010, 16, 3097–3113. 354. Ronconi, L.; Giovagnini, L.; Marzano, C.; Bettìo, F.; Graziani, R.; Pilloni, G.; Fregona, D. Gold Dithiocarbamate Derivatives as Potential Antineoplastic Agents: Design, Spectroscopic Properties, and In Vitro Antitumor Activity. Inorg. Chem. 2005, 44, 1867–1881. 355. Milacic, V.; Chen, D.; Ronconi, L.; Landis-Piwowar, K. R.; Fregona, D.; Dou, Q. P. A Novel Anticancer Gold(III) Dithiocarbamate Compound Inhibits the Activity of a Purified 20S Proteasome and 26S Proteasome in Human Breast Cancer Cell Cultures and Xenografts. Cancer Res. 2006, 66, 10478–10486. 356. Cinellu, M. A.; Zucca, A.; Stoccoro, S.; Minghetti, G.; Manassero, M.; Sansoni, M. Synthesis and Characterization of Gold(III) Adducts and Cyclometallated Derivatives with 6Benzyl- and 6-alkyl-2,20 -Bipyridines. J. Chem. Soc. Dalton Trans. 1996, 4217–4225. 357. Gabbiani, C.; Casini, A.; Messori, L.; Guerri, A.; Cinellu, M. A.; Minghetti, G.; Corsini, M.; Rosani, C.; Zanello, P.; Arca, M. Structural Characterization, Solution Studies, and DFT Calculations on a Series of Binuclear Gold(III) Oxo Complexes: Relationships to Biological Properties. Inorg. Chem. 2008, 47, 2368–2379. 358. Casini, A.; Cinellu, M. A.; Minghetti, G.; Gabbiani, C.; Coronnello, M.; Mini, E.; Messori, L. Structural and Solution Chemistry, Antiproliferative Effects, and DNA and Protein Binding Properties of a Series of Dinuclear Gold(III) Compounds with Bipyridyl Ligands. J. Med. Chem. 2006, 49, 5524–5531.

2.24

Protein targets for anticancer metal based drugs

Tiziano Marzoa and Luigi Messorib, a Department of Pharmacy, University of Pisa, Pisa, Italy; and b Laboratory of Metals in Medicine (MetMed), Department of Chemistry “U. Schiff”, University of Florence, Sesto Fiorentino, Italy © 2023 Elsevier Ltd. All rights reserved.

2.24.1 2.24.2 2.24.3 2.24.4 2.24.5 2.24.5.1 2.24.5.2 2.24.6 Funding References

Anticancer metal-based drugs: An overview 794 Mechanistic aspects: Proteins as alternative targets for anticancer metal-based drugs 797 The metalation process of individual proteins: a hyphenated ESI-MS/XRD investigative protocol for adducts characterization 798 Proteins as targets for anticancer metal-based drugs: Auranofin and thioredoxin reductase 800 Emerging technologies for target identification in metallodrugs’ research 801 A chemical proteomics approach to disclose the protein targets for the ruthenium complex RAPTA 802 Metallomics studies disclose the main Bismuth Binding Proteins in bacteria 804 Conclusions 805 805 805

Abstract Owing to the extensive investigations carried out on cisplatin soon after its discovery, anticancer metal-based drugs- as a category - were believed to target nuclear DNA selectively and cause cancer cell death primarily through a direct DNA damage, according to the so called “DNA paradigm.” In contrast to this concept, it is now widely accepted that proteins, beyond nucleic acids, play an essential role in the mode of action of anticancer metal drugs. Notably, for certain classes of metal-based drugs, e.g. the cytotoxic gold and ruthenium compounds, proteins rather than nucleic acids are the nearly exclusive targets, an opposite situation with respect to the DNA paradigm. Investigating proteins as targets for anticancer metallodrugs and elucidating the associated protein metalation processes is not trivial owing to the intrinsic high complexity of the biological systems and of the cellular proteomes. However, thanks to a research strategy recently developed in our laboratory, mostly grounded on the combined use of electrospray ionization mass spectrometry (ESI MS) and X-ray crystallography, it is possible to characterize in the atomic detail the metalation of a variety of individual proteins. A few instructive examples of metallodrug-protein adducts analyzed according to this strategy are herein provided. Protein metalation may result into protein’s loss of function triggering a cascade of cellular processes eventually leading to cancer cell death. Yet, describing in detail protein metalation taking place within the real cellular environment where thousands of proteins -instead of a single one- are simultaneously present, remains a very ambitious and challenging goal for researchers. The emerging omics technologies are starting to shed some light on these issues; a few relevant examples featuring the smart implementation of innovative Chemical Proteomics and Metalloproteomics approaches in the search of the true targets for metallodrugs are herein presented. The perspectives for future work in the area are delineated.

2.24.1

Anticancer metal-based drugs: An overview

Metals and metal complexes have been used for centuries in Medicine and still nowadays represent an essential and rich source of molecules for a wide array of biomedical applications.1,2 For instance, Fe, Zn, Ca, Mg, Se, Co, Cu and other essential metals are often administered to patients as supplements in the case of deficiency. Indeed, the lack of an essential element in the appropriate concentration may lead to the insurgence of a pathological condition.3 Certainly, this is among the simplest and well-known applications of inorganic compounds in medicine. However, beyond the physiological metals, even a few exogenous ones can be exploited for the synthesis of coordination compounds featured by specific and favorable pharmacological properties. In this view, Gd chelated compounds are extensively used in magnetic resonance imaging (MRI) as contrast agents and diagnostics. Owing to the strong chelation of macrocyclic DOTA ligands toward the Gd(III) center, the potentially toxic effects of the free metal are avoided while the diagnostically useful paramagnetic relaxation enhancement is maintained given the high number of unpaired electrons.4 However, the paradigmatic event highlighting the huge potential of inorganic molecules for clinical applications has been the serendipitous discovery of the anticancer properties of cisplatin by Barnett Rosenberg and Loretta Van Camp at the Michigan State University during the 1960s.5,6 The subsequent approval of cisplatin by the FDA in 1978 triggered huge efforts from the international research community in the search of metallodrugs with improved biochemical profiles.7 Indeed, the use of cisplatin, despite its very positive impact on the overall therapeutic approach and the management of cancer patients, implies major drawbacks such as systemic and acute toxicity as well as insurgence of resistance leading to treatment failure.8 Accordingly, on the one hand, the second and third generation of cisplatin analogs, i.e. carboplatin and oxaliplatin respectively, were subsequently approved worldwide.7,9,10 On the other hand, metals different from platinum started to attract growing interest in the scientific community. This

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interest was primarily driven by the evidence that transition metals represent a rich source for the discovery of novel anticancer agents and, more in general, of molecules with medicinal applications. Moreover, in the last four decades, a growing awareness has matured that inorganic chemistry has the potential for the development of compounds with properties that cannot be reproduced by simple organic molecules. Accordingly, coordination chemistry offers a unique opportunity for the development of improved agents in the treatment of cancer and several other diseases.11 In fact, through the appropriate design of ligands, and choosing different metal centers it is possible to finely tune the metal’s reactivity toward different targets (including nongenomic ones) thus widening the potential applications and the pharmacological effects. In this view, an emblematic case is represented by the several Pt-based anticancer complexes that have been approved worldwide -i.e. carboplatin and oxaliplatin- or locally in specific countries (Fig. 1).1 All the above Pt-complexes were initially designed as cisplatin analogs. However, due to the different chemical structures and kinetics of activation, they possess different and sometime improved biochemical profiles compared with the parent drug cisplatin.12 For instance, Pt(IV) compounds are less reactive compared with the Pt(II) counterparts and could ensure less offtarget interactions, and in turn a higher tolerability.13 Typically, for anticancer platinum prodrugs, the mechanism responsible for the pharmacological effects relies on the ability of the platinum center to bind DNA thus triggering a complex cascade of events ultimately resulting in the activation of the apoptosis pathways.14 Recently, alternative mechanisms and DNA-independent pathways have been proposed for some approved anticancer platinum drugs. An important case is oxaliplatin. This drug is used almost exclusively to treat colorectal cancer patients, while cisplatin and carboplatin only possess limited effects against this kind of tumor. In this frame, Lippard and coworkers reported an innovative mechanism of action for oxaliplatin.15 Their results pointed out as the DNA binding of oxaliplatin is not the only key event triggering apoptosis. Rather, direct evidence that induction of ribosome biogenesis stress is a key feature for oxaliplatin to induce cell death was gained by these authors. However, it should be stressed that induction of ribosome biogenesis stress by oxaliplatin does not preclude the possibility that oxaliplatin may produce relevant DNA lesions that inhibit the expression of proteins involved in ribosome biosynthesis and consequently cause the ribosome biogenesis stress. The case of oxaliplatin is important also for other reasons. Firstly, it demonstrates that a unique target or pathway is unlikely to exist for the mechanism of action of metal-based drugs. Rather, coordination toward multiple “binding partners” may concur to

Fig. 1 Chemical Structures of relevant platinum-based anticancer drugs that have been approved worldwide (cisplatin, carboplatin, oxaliplatin) or locally (nedaplatin, heptaplatin and lobaplatin). Ormaplatin, satraplatin and iproplatin entered clinical trials.

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determine the observed pharmacological action for metallodrugs, owing to the fact these metal centers are inherently capable to bind several different biomolecules.11 Additionally, it appears clear as advanced experimental methods are of paramount importance to unveil the actual -and often extremely complex- fate of metallodrugs in the biological environment.16 In this frame, integrated multi-omics approaches (including metalloproteomics) and various biophysical methods enable the comprehensive understanding of the cellular responses to metallodrugs, also allowing the rational design of new drugs and the modification/optimization of currently used ones.17 The advent of pioneering investigation technologies further spurred the mechanistically oriented design of metal-based anticancer complexes bearing different metal centers. Among the others, gold, a noble metal used for medicinal applications since very ancient times18 has a peculiar redox chemistry associated with the three main oxidation states (0, þ 1, þ 3) determining the geometry of the corresponding species. Depending on the ligands and the specific oxidation state, gold complexes can adopt square-planar or linear coordination geometries.19 This versatility can be exploited for the preparation of various Au-based anticancer complexes.20–22 Two aspects of the chemistry and solution behavior of gold complexes are particularly important for the medicinal applications: the facile interchange between the oxidation states in the cell, and the evidence that the primary targets for the pharmacological action are non-genomic, and mostly enzymes bearing exposed aminoacidic residues with aurophilic character.19,23 The reference complex for this family of metallodrugs is undoubtedly auranofin (RidauraÒ, Fig. 2). Auranofin is approved for the treatment of rheumatoid arthritis, but in the frame of various drug reprofiling programs, it is currently investigated in depth for its promising properties in different fields of medicine ranging from anticancer and antimicrobial uses to antiparasitic ones.24 As an anticancer agent it entered various clinical trials (see ClinicalTrials.gov website for details).24 Auranofin is a prodrug undergoing activation in the biological milieu. The activation consists in the formation of the cationic species [AuPEt3]þ after the release of the thiosugar moiety. [AuPEt3]þ is the actual pharmacophore showing a high reactivity with biomolecules and in particular enzymes such as those belonging to the thioredoxin system (i.e. thioredoxin reductase) for which auranofin is a potent inhibitor being capable of strong coordination at the level of the key selenium-containing amino acid selenocysteine. In the last decades, other important gold(I) and gold(III) compounds have been developed and tested against cancer and other diseases expanding the studies and the potentialities on this family of metal compounds.24–28 In the same years some ruthenium compounds also attracted a significant attention. This success is mainly attributable to two complexes that entered clinical trials, i.e. NAMI-A and KP1019 (Fig. 2).29 NAMI-A ([ImH][trans-RuCl4(dmso-S)(Im)] (dmso-S ¼ sulfur-bonded dimethyl sulfoxide, Im ¼ imidazole), synthesized for the first time by Prof. Enzo Alessio at the University of Trieste, is well known for its antimetastatic properties. NAMI-A has very limited or no cytotoxic effects in the 60-cell-line NCI panel.30 The mechanism of action for the antimetastatic properties remains largely unknown; however it has been ascertained that it is capable of binding toward several biological substrates including DNA, RNA and to interfere with angiogenesis induced by vascular endothelial growth factor (VEGF).1 KP1019 (indazolium [transRuCl4(1H-indazole)2]), has been developed by the group of Bernhard Keppler and entered clinical trials as an anticancer drug. In contrast with NAMI-A, phase I trials revealed a quite good tolerability.31 For KP1019 the precise mechanisms underlying the anticancer properties have been not clarified as well. Nevertheless, for both NAMI-A and KP1019, the interaction with transferrin (hsTf) has been highlighted as an important step involved in cell internalization. Yet, the real role of transferrin is still debated. In fact,

(A)

(C) (B)

Fig. 2

Chemical structures of (A) Auranofin, (B) NAMI-A and (C) KP1019.

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beyond hsTf, NAMI-A and KP1019 react efficiently with a variety of other proteins e.g. lysozyme as well as human serum albumin (HSA).32–34 Despite this large interest in Au and Ru compounds, in the last 40 years, many other metals -but also metalloids- have been exploited and tested for the synthesis of novel anticancer agents.35 Overall, it is fascinating to observe as relatively simple inorganic molecules such as cisplatin, auranofin, NAMI-A or arsenic trioxide -being this latter a reference drug for the treatment of promyelocytic leukemia- may have an extraordinary therapeutic potential in cancer treatment. Altogether, these considerations well support the upsurge of efforts toward the development of innovative metallodrugs in cancer chemotherapy. In this frame, beyond platinum, gold and ruthenium, novel inorganic molecules bearing different metal centers such as Ir, Ti, Cu, Fe, Ag and others, may represent a suitable alternative for the improvement of anticancer chemotherapy regimens.20,36

2.24.2

Mechanistic aspects: Proteins as alternative targets for anticancer metal-based drugs

Traditionally, genomic DNA is recognized as the main pharmacological target for cisplatin and its Pt analogs. This idea was a logical consequence of the pioneering studies carried out by Barnett Rosenberg et al. showing that platinum compounds produce important DNA alterations, interfere with its replication and ultimately cause the filamentous growth in E. Coli.5,6 This experimental evidence was reputed sufficient to state that DNA is the “true target” of cisplatin. Substantial support in favor of this hypothesis was later gained by Steve Lippard and coworkers, who documented, through several illuminating studies, the large damage produced in the DNA double helix by cisplatin binding.7 A detailed structural description of the distortions of the DNA double helix caused by bidentate platinum binding to adjacent guanine bases was achieved (Fig. 3). Such DNA damage, if not repaired, eventually leads to cancer cell death through apoptosis. These arguments well define the so called “DNA paradigm” for the mechanism of action of cytotoxic Pt drugs.37 Though well-grounded and well justified in the case of cisplatin and of many platinum analogs this concept, i.e. DNA as the main and nearly exclusive target,38,39 has monopolized the research field of anticancer metal-based drugs for decades becoming in a way an obstacle to the study of other modes of actions and to the identification of alternative anticancer metal based drugs possessing different molecular mechanisms. Only in recent times a growing awareness has developed of the importance of the protein interactions of metal based anticancer agents in producing their overall pharmacological effects.40 This gradual change of paradigm is one of the results of the huge technological progresses recorded in the last 20 years in the field of biomedical research,15 in particular of the advent of so-called omics methods. For a few classes of metal-based drugs, a peculiar and far greater reactivity with proteins in comparison to nucleic acids could be unambiguously demonstrated. This behavior is strictly linked to the nature of the metal center and its coordination preferences. Thus, it is possible to assume that proteins rather than DNA will mediate the biological actions and the pharmacological profiles of selected families of inorganic compounds.41,42 To this regard, the cases of Ru and Au -based anticancer compounds are highly instructive. A number of cytotoxic Ru and Au compounds (especially Au(I) compounds) with remarkable anticancer properties were reported to react very weakly -or even not to react- with nucleic acids; on the other hand, the same Ru and Au compounds turned able to form stable adducts with proteins. These observations lend strong support to the idea that the cytotoxic actions produced by these Ru- and Au-based drugs, differently from Pt-based drugs, mostly depend on their direct binding to protein targets.43,44 Remarkably, metal-based drugs having proteins as targets may present a significant therapeutic advantage over drugs targeting DNA. Indeed, the interactions of metallodrugs with proteins may imply a higher selectivity than the interactions with genomic targets. This may result into higher and improved tolerability and lower side effects. It follows that it is convenient to design metallodrugs capable of hitting specific protein targets involved in cellular pathways that are particularly important for cancer cells. Also, the targeting of proteins that are overexpressed in cancer tissues where they are essential for tumor growth and progression can be

Fig. 3 The DNA paradigm of cisplatin. Duplex DNA containing cisplatin 1,2-d(GpG) (left), 1,3-d(GpTpG) intrastrand (middle) and interstrand crosslinks (right). The black boxes indicate the cisplatin binding sites. Reproduced and adapted with permission from reference Jung, Y.; Lippard, S.J. Direct Cellular Responses to Platinum-Induced DNA Damage. Chem. Rev. 2007, 107, 1387–1407.

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conveniently exploited. These targeting strategies are now made easier and more feasible owing to advanced investigative strategies allowing to gather large amount of functional information concerning the biology of cancer cells.23,44–46 Yet, the study of the protein interactions of metal-based drugs in terms of the actual molecular mechanisms remains very complicated. Indeed, while it is now well established that metal-based drugs may bind and metalate hundreds of proteins, it is very likely that most of those binding events may not have major functional consequences. Probably, only a few protein interactions capable of altering selected signaling or metabolic pathways are relevant for determining the observed cytotoxic and anticancer effects while the majority of the other metallodrug protein interactions will turn just detrimental and toxic or simply ineffective. These arguments pose the big problem of target identification and validation when analyzing the interactions of metal-based drugs with proteins; this implies identifying those proteins whose metalation produces major cancer cell perturbations ultimately leading to cancer cell death.47 In this regard several studies have been deserved to the analysis of the interactions of metal-based drugs with proteins and to the characterization of the resulting metallodrug protein adducts at the molecular level. Specifically, a well-defined investigative protocol has been developed in our laboratory to characterize the metalation of individual proteins by a variety of metal based drugs as it will be detailed below.40,48

2.24.3 The metalation process of individual proteins: a hyphenated ESI-MS/XRD investigative protocol for adducts characterization To investigate systematically the interactions taking place between metal based drugs and proteins it is convenient to start from the analysis of the interactions occurring between a single metallodrug and a single protein.1,49 In fact, this approach may offer detailed insight on the coordination preferences of the investigated metal center and its preferred anchoring sites on the protein. Though there are numerous examples in the literature of studies concerning metallodrug-protein interactions grounded on a variety of biophysical methods, we realized that those interactions had been studied so far just in a fragmentary way so that precise structural information on the metallodrug–protein adducts was often lacking. This led us to elaborate a few years ago a systematic approach to elucidate details of protein modifications induced by the binding of anticancer metallodrugs1,50 (“protein metalation”); this approach was mostly reserved to metallodrugs behaving as prodrugs. The experimental strategy we have developed involves the joint application of electrospray ionization mass spectrometry (ESI MS) and X-ray crystallography (XRD) measurements on appropriate model systems. This implies that the model proteins eligible for these studies must produce well resolved ESI MS spectra and must crystallize quite easily. Several studies documenting the feasibility and the efficacy of this strategy have now been published.40,50–52 ESI MS is an optimal tool to disclose the interactions taking place between metallodrugs and small proteins and to determine the nature of the resulting adducts.40 Indeed, the soft ionization induced by the ESI source allows conservation of the metal to protein coordinative bonds in the gas phase. The method is cheap, quick and straightforward; valuable information is typically achieved on adduct formation, on the metal/protein ratio in the adducts and on the nature of protein bound metallic fragments.13,53 As an example, a few ESI MS results concerning the reactions of RNase A with cisplatin, carboplatin, and oxaliplatin, are reported below (Fig. 4).54 As one can see, analysis of the deconvoluted ESI MS spectra offers precise information on the nature of the adducts formed in those reactions. In all cases, various peaks with a mass larger than the free protein are observed. These signals are indicative for the adduct formation. Most peaks show a well-defined mass shift corresponding to protein binding of a single Ptcontaining fragment. Basically, ESI MS spectra provide detailed information on the following issues: (1) the amount of the formed adducts compared to the free protein; (2) the degree of protein metalation; (3) the precise nature of the protein-bound fragments. In addition, specific insight may be achieved on the kinetics of adducts formation and on the time-dependent transformation/evolution of protein-bound metallic fragments. In the proposed investigative protocol, the information arising from the ESI MS measurements may be nicely complemented and integrated with the information emerging from the crystallographic studies conducted on the same systems.1 The XRD experiment is essentially based on the following steps: (1) formation of metallodrug- protein adducts; (2) preparation of single crystals; (3) collection of the X-ray diffraction data; (4) structure determination and refinement through the analysis of the electron density maps. The XRD studies are of course more laborious and time consuming -and uncertainthan the ESI MS studies but allow, if successful, the obtainment of detailed structural information on the metallodrug protein adducts.40 Indeed, analyzing the crystal structure allows to identify the precise aminoacidic residues of the protein capable to coordinate the platinum fragments beyond the nature of the metal fragment itself. From the crystallographic experiment, details on the interactions that govern the metallodrug-protein recognition process and on the interactions established by metal ligands with surrounding protein residues can be also achieved. However, crystallography has some limitations mainly relying on the crystal packing, which limits the dynamics of the adduct. Also, the assignment of the metal ligands from the simple inspection of the electron density map is not always straightforward, because of the likely low occupancy of the metal bound fragment, of the possible heterogeneity of the fragments bound to a specific site and/or the flexibility of the region involved in the metal fragment/protein recognition.40 In our case, the details obtained through ESI MS experiments on the adducts formed upon incubation of Pt-based drugs and the RNase A were combined with those coming from X-ray investigations on the same adducts.54 In spite of the different conditions in the mass and crystallographic experimental environment, the integration of these two techniques allows to obtain consistent and

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suitable information, confirming that the integrated approach offers important advantages with respect to the single technique. Accordingly, the crystallographic structures for the reaction of cisplatin, carboplatin and oxaliplatin with RNase A (see for example Fig. 5), were solved to a good resolution (i.e. a resolution between 1.85 and 2.27 Å). These structures are consistent with the solution data obtained with ESI MS both in terms of number of formed adducts and type of bound metal fragments; indeed Met, Asp

Fig. 4 ESI-MS spectra recorded at increasing time intervals of cisplatin (top), carboplatin (middle), and oxaliplatin (bottom) incubated with RNase A (A ¼ 24 h, B ¼ 72 h, C ¼ 168 h, protein concentration 10 4 M, metal to protein ratio 3:1, ammonium acetate buffer pH 6.8). The label “RNase” indicates the signal corresponding to the native protein (non-metalated) while the labels corresponding to the metal-protein adducts are indicated with the only bound fragments.

800

Fig. 4

Protein targets for anticancer metal based drugs

(continued).

and His side chains represent the main Pt coordination sites. The same experimental setup was also used to study and characterize the interactions occurring between cisplatin and oxaliplatin with the model protein lysozyme, with very interesting results (Fig. 6).55

2.24.4

Proteins as targets for anticancer metal-based drugs: Auranofin and thioredoxin reductase

As stated above, for anticancer metal based drugs, the target identification and validation problem represents a formidable and very ambitious research goal; a lot of work still needs to be done and more efficient and improved research protocols to be established.40 Yet, there are some cases where the probable targets for specific anticancer metallodrugs have been disclosed - with a rather high degree of confidence- not through systematic studies but just through empirical and/or serendipitous research approaches taking advantage of independent information coming from biological studies. The initial findings were then validated through more sophisticated and robust investigative methods. To this regard we will present here the case of thioredoxin reductase which is now considered as the primary target for the gold drug auranofin.25 The case of thioredoxin reductase is emblematic: thioredoxin reductase was first proposed as a target for auranofin thanks to a variety of biochemical and pharmacological experiments including the demonstration of the strong inhibition of its catalytic activity by auranofin. This concept has been validated very recently thanks to the implementation of potent investigative strategies relying on omics methods.17,56 Thioredoxin reductase (TrxR) is a homodimeric flavoenzyme containing a C-terminal selenocysteine that is directly involved in the catalytic activity. TrxR is primarily involved in the reduction of the small cytosolic protein thioredoxin and in the control of the redox state of the cell. TrxR is an ubiquitous protein performing many cellular functions such as antioxidant defense, redox homeostasis and cell proliferation. TrxR is often overexpressed in many human tumor cell lines; this makes TrxR an excellent target for anticancer drugs.57,58 The crystal structure of TrxR has been solved (Fig. 7).59 To note, the presence of the accessible site selenocysteine in the active site of in TrxR implies a high affinity of this enzyme for soft metal ions -specifically for gold(I) compounds- that are known to form tight adducts with thioredoxin reductase and to efficiently its catalytic activity. Accordingly, TrxR is reputed as the most likely primary target for auranofin. Indeed, the potent inhibition of this selenoenzyme caused by gold drug results in severe oxidative stress, dysregulation of the mitochondrial function and eventual apoptotic cancer cell death. Though no crystal structure of the adducts formed between thioredoxin reductase and auranofin has been solved so far, detailed spectrometric studies carried out on the C-terminal dodecapeptide revealed the nature of the interaction of auranofin with the active site selenocysteine.60 It was found that the [AuPEt3]þ fragment originating from auranofin activation upon release of the thiosugar ligand first binds to the selenocysteine residue; a second [AuPEt3]þ fragment then coordinates to the adjacent free thiol. In the light of these results and of more recent cellular and functional studies reported by Arner in 2021 thioredoxin reductase may now be considered – with a certain degree of confidence- as the primary target of this gold drug; the strong inhibition of TrxR activity

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Fig. 5 Details of the binding sites of Pt(II) in the RNase A (molecule A)–oxaliplatin structure. The drug is coordinated to the protein through the interaction with side chain of His119. The [Ptdach]2 þ fragment is involved in several hydrogen bonding interactions with water molecules. Additionally, a Pt ion is bound to Met29 and Asp14 sidechains. A minor Pt binding site is close to the side chain of His105. 2Fo–Fc electron density maps are contoured at 0.8s level (gray) and 4.0s level (red). Adapted and reproduced with permission from reference Messori, L.; Marzo, T.; Merlino, A. Interactions of Carboplatin and Oxaliplatin with Proteins: Insights from X-Ray Structures and Mass Spectrometry Studies of their Ribonuclease A Adducts. J. Inorg. Biochem. 2015, 153, 136–142.

brought about by auranofin nicely accounts for its remarkable biological and anticancer effects.61 The proposed mechanism of action of proapoptotic gold compounds is schematically depicted in Fig. 8.62

2.24.5

Emerging technologies for target identification in metallodrugs’ research

The protein targets of metallodrugs are often elusive despite the intense analytical and biochemical work carried out so far in order to identify them.11 This may be traced back to the broad reactivity of metallodrugs in physiological solution and to the ligand exchange reactions that may take place depending on the actual solution conditions; this situation typically results into extensive binding of metallodrugs or metal containing fragments to a large number of biomolecules and formation of a large number of adducts.7,20 To overcome these problems and perform a more systematic search of the “true” protein targets for a certain metallodrug some authors developed a few smart investigative strategies that turned out to be very effective.1 We like to remind here two distinct approaches developed in the last few years that turned out to be particularly promising and effective, more precisely the “Drug pull-down” strategy reported by Christian Hartinger, Bernhard Keppler et al. that allowed identification of the molecular

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Fig. 6 Representation of the Pt coordination site for oxaliplatin reacted with lysozyme. Pt is bound to the Asp119 side chain (cyan). The cyclobutandicarboxylate ligand is inserted into a cavity formed by Trp62, Trp63, Asp101 and Asn103 residues of a symmetry related molecule. Adapted and reproduced with permission from reference Messori, L.; Marzo, T.; Merlino, A. The X-Ray Structure of the Complex Formed in the Reaction between Oxaliplatin and Lysozyme. Chem. Commun. 2014, 50, 8360–8362.

Fig. 7 Cartoon representation of human TrxR (hTrxR1 – PDB entry 2ZZC). Each monomer is depicted to differentiate the domains/sub-domains, according to the color code reported on the right of the frame. The overall structure is a homodimer where a 2-fold symmetry axis was across the dimerization domain. Auranofin likely binds the enzyme at the level of Sec/Cys - Cys/Cys redox active motif. Reproduced and adapted from reference Saccoccia, F.; Angelucci, F.; Boumis, G.; Carotti, D.; Desiato, G.; Miele, A.; Bellelli, A. Thioredoxin Reductase and Its Inhibitors. Curr. Protein Pept. Sci. 2014, 15, 621–646 under the Creative Commons 3.0 Attribution, Non-Commercial License.

targets of the ruthenium drug RAPTA and a Metalloproteomics strategy proposed by Hongzhe Sun et al. that allowed detection of variety of bismuth binding proteins in bacterial cells.63,64 More details on these two investigative strategies are given below.

2.24.5.1

A chemical proteomics approach to disclose the protein targets for the ruthenium complex RAPTA

RAPTA complexes form an attractive group of organometallic ruthenium(II) compounds that manifest a remarkable antimetastatic activity in vivo, accompanied by a very modest cytotoxicity.63,65 The general formula of RAPTA compounds is [Ru(arene)(PTA)X2] (Fig. 9), where PTA is 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane and X is a halide or a biscarboxylate. DNA was initially reputed as the main target for RAPTAs. However, more recently, the focus of mechanistic studies of RAPTAs has moved from the study of RAPTA–DNA interactions to that of RAPTA-protein interactions. As a matter of fact, it was observed that RAPTA compounds (Fig. 9) preferentially bind to proteins even in the presence of DNA. The molecular description of the interactions of RAPTAs with their cellular targets may improve our understanding of their modes of action. The screening of RAPTA–protein interactions is very laborious because of the huge number of possible biomolecular targets for these organoruthenium(II) compounds in cells.66 Moreover, additional complexity emerges from the

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Fig. 8 The mechanism of action of cell death induction by gold(I/III) compounds. The mitochondrial respiratory chain produces superoxide anion that dismutes to hydrogen peroxide and oxidizes thioredoxin in a reaction mediated by peroxiredoxin. TrxR, inhibited by gold(I/III) complexes, is unable to reduce back oxidized thioredoxin that in turn accumulates together with hydrogen peroxide leading to the opening of the mitochondrial permeability transition pore and/or to an increase of the permeability of the outer membrane. H2O2 is then released to the cytosol causing oxidation of Trx1, that, similarly, to mitochondrial thioredoxin (Trx2), cannot be reduced back by the gold(I/III)-inhibited thioredoxin reductase. Oxidized Trx stimulates the MAP kinases pathways leading to cell death. Reproduced with permission from reference Bindoli, A.; Rigobello, M.P.; Scutari, G.; Gabbiani, C.; Casini, A.; Messori, L. Thioredoxin Reductase: A Target for Gold Compounds Acting as Potential Anticancer Drugs. Coord. Chem. Rev. 2009, 253, 1692–1707.

Fig. 9

General structure of RAPTA compounds.

transformations of these metal complexes in aqueous solutions due to hydrolysis. There is also the chance that non-selective binding to amino acid side chain donor atoms may take place. Therefore, a suitable and robust analytic methodology is needed. To this end these authors developed a Chemical Proteomics method named “drug pull-down,” involving sequential application of affinity chromatography, shotgun proteomics and bioinformatics, to disclose the molecular targets of RAPTAs. To the best of our knowledge, such an approach is unprecedented in the field of metal based drugs.63 The workflow of the pull-down experiment is shown in Fig. 10. At first, a RAPTA-analog appropriate for immobilization on streptavidin beads is prepared by linking a biotin molecule to the Ru-coordinated h6-arene. The immobilized metallodruganalog is then challenged with cancer cell lysates, and the metal-binding proteins identified through high-resolution MS. To assess the selectivity of the observed interactions further experiments are carried out in competition with the free RAPTA complex. Bioinformatic analysis of the data coming out from the drug pull-down experiments led to the identification of a total of 184 proteins and of 29 enriched proteins. A significant amount of these 184 proteins were ribosomal proteins (18) and zinc finger proteins (16). Notably, comparison of the obtained dataset with that arising from the competition experiment with free RAPTA eliminated most of the identified proteins so that only 5 ribosomal proteins and 2 zinc finger proteins resulted to be probable target proteins for RAPTA. Out of the enriched proteins, 15 were found to be cancer related. Authors analyzed the localization and the function of these proteins and found that they are localized in the nucleus, cytoplasm, particulate fraction and extracellular region. These proteins may be the “true” targets of RAPTAs and may account for their in vitro antimetastatic properties as well as for other actions such as the inhibition of angiogenesis. In principle, the described methodology has a broad applicability to several classes of metal-based drugs beyond RAPTA complexes and allows the direct identification of the intracellular interactions of metallodrugs with proteins.

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Fig. 10 Schematic representation of the workflow developed by Hartinger and coworkers for the so-called pull-down approach. In the noncompetitive pathway (data set 1) proteins can bind only to modified beads, whereas in the competitive pathway (data set 2) proteins can bind to modified beads and competitive binder 3. Adapted and reproduced from reference Babak, M. V.; Meier, S.M.; Huber, K.V.M.; Reynisson, J.; Legin, A.A.; Jakupec, M.A.; Roller, A.; Stukalov, A.; Gridling, M.; Bennett, K.L.; Colinge, J.; Berger, W.; Dyson, P.J.; Superti-Furga, G.; Keppler, B.K.; Hartinger, C.G. Target Profiling of an Antimetastatic RAPTA Agent by Chemical Proteomics: Relevance to the Mode of Action. Chem. Sci. 2015, 6, 2449–2456 under Creative Commons Attribution-NonCommercial 3.0 License.

2.24.5.2

Metallomics studies disclose the main Bismuth Binding Proteins in bacteria

The identification of the protein targets of metallodrugs is indeed a very difficult task. Beyond the pull-down experiments described above, this problem may also be faced through alternative approaches such as untargeted Metallomics (or Metalloproteomics) experiments. Indeed, implementation of advanced metallomics strategies may allow direct identification of metalated proteins within cells. We like to remind here the pioneering studies conducted by Hongzhe Sun in Hong Kong concerning the identification and characterization of the bismuth proteome in bacteria.64,67 Though not involving directly anticancer metallodrugs nor cancer cells, this study is very instructive as it defines an investigative strategy of general validity that may be adapted in the future to anticancer metallodrug research. More in detail, Sun et al. have developed a general metalloproteomics platform to characterize the bismuth proteome in bacteria.68 Using conventional approaches, such as immobilized-bismuth affinity chromatography (Bi-IMAC), in combination with two-dimensional gel electrophoresis (2-DE)/ matrix-assisted laser desorption/ionization time-of-flight MS and 1D SDSPAGE coupled with LA-ICP-MS, seven Bi-binding proteins could be identified in Helicobacter pylori extracts. Recently, the same authors have developed a new device, i.e. continuous-flow gel electrophoresis coupled with ICP-MS (GE-ICPMS), that permits detection of both Bi and its associated proteins. Seven Bi-binding proteins, including five previously reported and two newly identified proteins (HP1286 and cell binding factor 2) were revealed through this experimental setup (Fig. 11).17 However, one of the limit of this system is a quite low resolution in the separation of the proteins, this being the consequence of the use of a mono-dimensional column.

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Fig. 11 Profile of Bi-binding proteins in H. pylori analyzed by GE-ICP-MS. Reproduced and adapted with permission from reference Wang, H.; Zhou, Y.; Xu, X.; Li, H.; Sun, H. Metalloproteomics in Conjunction with Other Omics for Uncovering the Mechanism of Action of Metallodrugs: MechanismDriven New Therapy Development. Curr. Opin. Chem. Biol. 2020, 55, 171–179.

2.24.6

Conclusions

It was originally believed that anticancer metal-based drugs should act invariantly on genomic DNA similar to the case of platinum drugs as postulated by the so called DNA paradigm. In contrast to this early view, it is now widely accepted that proteins, beyond nucleic acids, may play a crucial role in the mode of action of anticancer metal drugs. We have shown here that various anticancer metal-based drugs perform their biological actions mainly by interacting with protein targets. The study of the interactions of metallodrugs with proteins is very complex due to the numerosity of the cellular proteins and to their large structural variety. However, an investigative protocol grounded on joint ESI MS and XRD experiments was recently established in our laboratory that allows characterizing in the atomic detail the metallodrug protein adducts that are formed when reacting a metal-based drug with individual model proteins. Thanks to this approach the structures of several metallodrug protein adducts were disclosed and the binding preferences of the various metal centers clarified. Of course, the analysis of the metalation processes occurring in the real cellular environment and the identification of the effective protein targets for the various metallodrugs is far more complicated and requires huge research efforts. As a consequence of that, only in few cases the probable protein targets for selected metallodrug were identified with a good degree of certainty; an emblematic example has been reported above. At the same time, we have shown that advanced proteomics methodologies such as chemical proteomics approaches as well as the new metallomics technologies combined with bioinformatics analysis and targeted biochemical experiments, hold a lot of promise to solve these difficult research issues in the next future.

Funding TM thanks the University of Pisa under the “PRA - Progetti di Ricerca di Ateneo” Institutional Research Grants - Project no. PRA_2020_58 “Agenti innovativi e nanosistemi per target molecolari nell’ambito dell’oncologia di precisione.” Also, the financial support of the Beneficentia Stiftung Foundation, Vaduz (BEN2019/48) is acknowledged. The continuous financial support of AIRC to LM is gratefully acknowledged. Thanks are expressed to the Italian Interuniversity Consortium CIRCMSB and its members for valuable scientific discussions.

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55. Messori, L.; Marzo, T.; Merlino, A. The X-Ray Structure of the Complex Formed in the Reaction between Oxaliplatin and Lysozyme. Chem. Commun. 2014, 50, 8360–8362. 56. Kinoshita, H.; Shimozato, O.; Ishii, T.; Kamoda, H.; Hagiwara, Y.; Tsukanishi, T.; Ohtori, S.; Yonemoto, T. The Thioredoxin Reductase Inhibitor Auranofin Suppresses Pulmonary Metastasis of Osteosarcoma, But Not Local Progression. Anticancer Res. 2021, 41, 4947–4955. 57. Lu, J.; Chew, E.-H.; Holmgren, A. Targeting Thioredoxin Reductase Is a Basis for Cancer Therapy by Arsenic Trioxide. Proc. Natl. Acad. Sci. 2007, 104, 12288–12293. 58. Schmidt, E. E.; Arnér, E. S. J. Thioredoxin Reductase 1 as an Anticancer Drug Target. In Selenium Its Molecular Biology and Role in Human Health, 4th ed; 2016; pp 199–209. 59. Saccoccia, F.; Angelucci, F.; Boumis, G.; Carotti, D.; Desiato, G.; Miele, A.; Bellelli, A. Thioredoxin Reductase and Its Inhibitors. Curr. Protein Pept. Sci. 2014, 15, 621–646. 60. Marzo, T.; Massai, L.; Pratesi, A.; Stefanini, M.; Cirri, D.; Magherini, F.; Becatti, M.; Landini, I.; Nobili, S.; Mini, E.; Crociani, O.; Arcangeli, A.; Pillozzi, S.; Gamberi, T.; Messori, L. Replacement of the Thiosugar of Auranofin with Iodide Enhances the Anticancer Potency in a Mouse Model of Ovarian Cancer. ACS Med. Chem. Lett. 2019, 10, 656–660. 61. Arnér, E. S. J. Effects of Mammalian Thioredoxin Reductase Inhibitors. Handb. Exp. Pharmacol. 2020, 264, 289–309. 62. Bindoli, A.; Rigobello, M. P.; Scutari, G.; Gabbiani, C.; Casini, A.; Messori, L. Thioredoxin Reductase: A Target for Gold Compounds Acting as Potential Anticancer Drugs. Coord. Chem. Rev. 2009, 253, 1692–1707. 63. Babak, M. V.; Meier, S. M.; Huber, K. V. M.; Reynisson, J.; Legin, A. A.; Jakupec, M. A.; Roller, A.; Stukalov, A.; Gridling, M.; Bennett, K. L.; Colinge, J.; Berger, W.; Dyson, P. J.; Superti-Furga, G.; Keppler, B. K.; Hartinger, C. G. Target Profiling of an Antimetastatic RAPTA Agent by Chemical Proteomics: Relevance to the Mode of Action. Chem. Sci. 2015, 6, 2449–2456. 64. Wang, Y.; Hu, L.; Xu, F.; Quan, Q.; Lai, Y.-T.; Xia, W.; Yang, Y.; Chang, Y.-Y.; Yang, X.; Chai, Z.; Wang, J.; Chu, I. K.; Li, H.; Sun, H. Integrative Approach for the Analysis of the Proteome-Wide Response to Bismuth Drugs in Helicobacter Pylori. Chem. Sci. 2017, 8, 4626–4633. 65. Holtkamp, H. U.; Movassaghi, S.; Morrow, S. J.; Kubanik, M.; Hartinger, C. G. Metallomic Study on the Metabolism of RAPTA-C and Cisplatin in Cell Culture Medium and its Impact on Cell Accumulation. Metallomics 2018, 10, 455–462. 66. Lin, K.; Zhao, Z. Z.; Bo, H. B.; Hao, X. J.; Wang, J. Q. Applications of Ruthenium Complex in Tumor Diagnosis and Therapy. Front. Pharmacol. 2018, 9, 1323. 67. Yuchuan Wang, H. W. H. L.; H.S.. Metallomic and Metalloproteomic Strategies in Elucidating the Molecular Mechanisms of Metallodrugs. Dalt. Trans. 2015, 44, 437–447. 68. Ge, R.; Sun, X.; Gu, Q.; Watt, R. M.; Tanner, J. A.; Wong, B. C. Y.; Xia, H. H.; Huang, J.-D.; He, Q.-Y.; Sun, H. A Proteomic Approach for the Identification of Bismuth-Binding Proteins in Helicobacter Pylori. JBIC J. Biol. Inorg. Chem. 2007, 126. 2007, 12, 831–842.

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Platinum anticancer drugs: Targeting and delivery

Zhiqin Denga,*, Houzong Yaoa,*, Zhigang Wangb, and Guangyu Zhua, a Department of Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, PR China; and b School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen, PR China © 2023 Elsevier Ltd. All rights reserved.

2.25.1 2.25.1.1 2.25.1.1.1 2.25.1.1.2 2.25.1.1.3 2.25.1.2 2.25.1.2.1 2.25.1.2.2 2.25.2 2.25.2.1 2.25.2.1.1 2.25.2.1.2 2.25.2.1.3 2.25.2.1.4 2.25.2.1.5 2.25.2.2 2.25.2.2.1 2.25.2.2.2 2.25.2.2.3 2.25.2.2.4 2.25.2.2.5 2.25.2.2.6 2.25.2.3 2.25.2.3.1 2.25.2.3.2 2.25.3 2.25.3.1 2.25.3.2 2.25.3.3 2.25.3.4 2.25.4 2.25.4.1 2.25.4.2 2.25.4.3 2.25.5 References

Introduction of platinum anticancer drugs Platinum(II) anticancer drugs The development of platinum(II) drugs The action mechanism of platinum(II) drugs The limitations of Pt(II) drugs Novel platinum complexes Non-conventional platinum(II) anticancer complexes Platinum(IV) prodrugs Tumor-targeted platinum complexes Tumor-targeted small molecule-platinum conjugates Estrogen-platinum conjugates targeting estrogen receptors Glucose-platinum conjugates targeting glucose transporters Folate-platinum conjugates targeting folate receptors Biotin-platinum conjugates targeting SMVT Phosphonate-platinum conjugate targeting bone cancers Tumor-targeted platinum-peptide conjugates RGD-platinum conjugates targeting integrin NGR-platinum conjugates targeting aminopeptidase N (APN) TPP-platinum conjugates targeting memHSP70 CTX-platinum conjugates targeting chlorotoxin receptors EGFR peptide-platinum conjugates targeting EGFR AHNP-platinum conjugates targeting HER2 Delivery of platinum drugs by proteins Delivery of platinum drugs by albumin Delivery of platinum drugs by antibodies Organelle-targeted platinum complexes Nucleus-targeted Pt complexes Mitochondria-targeted Pt complexes ER-targeted Pt complexes Lysosome-targeted Pt complexes Platinum drug-based nano-delivery systems Platinum-incorporated nano-systems Platinum-self-assembled nano-systems Platinum-conjugated nano-systems Conclusions and perspectives

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Abstract The emergence of platinum drugs has significantly increased the survival rates of cancer patients. The therapeutic outcomes of platinum drugs, however, are still limited by serious side effects that arise from the poor selectivity between cancer and normal cells as well as resistance from cancer cells. Improving the tumor-targeting ability and achieving the targeted delivery of platinum drugs in vivo have been regarded as promising strategies to overcome these limitations. In this chapter, we summarize the strategies for the development of targeted platinum anticancer complexes and delivery systems for carrying platinum complexes to the tumor region, with a focus on their targeting property, antitumor activity, and other advantages over conventional platinum drugs. In addition, we discuss the strengths and challenges of current strategies and proposed

*

These authors contributed equally to this work.

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perspectives for further development. In summary, this chapter may bring researchers new ideas and help them to design next-generation platinum anticancer complexes.

2.25.1

Introduction of platinum anticancer drugs

2.25.1.1

Platinum(II) anticancer drugs

2.25.1.1.1

The development of platinum(II) drugs

Cancer is the common name for a disease caused by the uncontrollable growth of abnormal cells. Currently, the most common cancer treatments include surgery, chemotherapy, and radiotherapy. Platinum drugs, including cisplatin, carboplatin, and oxaliplatin, are used as chemotherapeutic drugs globally in clinics to treat various types of cancer. The antitumor activity of cisplatin [cisdiamminedichloridoplatinum(II), Fig. 1] was discovered serendipitously by biophysicist Barnett Rosenberg in 1965 when he was studying the influence of electric or magnetic fields on bacterial cell division. Inadvertently, platinum electrodes were used in the early experiments. Escherichia coli showed very long filaments that were 200–300 times longer when the field was turned on (Fig. 2). This effect was finally attributed to the electrolysis products of the platinum electrodes. One of the electrolysis products is the cisisomer of [Pt(NH3)2Cl2], which is known as cisplatin.1,2 Cisplatin was subsequently proven to be active against cancer and was approved by the US Food and Drug Administration (FDA) in 1978 for the treatment of metastatic testicular, ovarian, and advanced bladder cancers.3 Particularly, the cure rate from cisplatin treatment for testicular cancer has exceeded 95%.4,5 Carboplatin (Fig. 1), with a cyclobutane-1,1-dicarboxylate (CBDCA) ligand replacing the dichloride in cisplatin, is the second generation platinum(II) drug. Carboplatin is more stable and less toxic than cisplatin, partially due to the slower hydrolysis of the CBDCA leaving group, which hydrolyzes with a rate constant 1000 times lower than the Cl atoms in cisplatin.6,7 After aquation, although the rate of adduct formation is slower, carboplatin yields the same products and forms the same DNA adducts as cisplatin; thus carboplatin may not be effective against cisplatin-resistant cancers.8 In 1989, carboplatin was approved by the FDA for the treatment of ovarian carcinoma. Oxaliplatin (Fig. 1), with an oxalate leaving group and an R,R-diaminocyclohexane (DACH) non-leaving group, is the latest platinum(II) drug that has obtained international approval. The DNA adducts formed with activated oxaliplatin are different from those of cisplatin. In addition, the bulky DACH group can prevent DNA repair proteins from binding to the impaired DNA in an electrophoretic mobility shift assay.9 These properties make oxaliplatin capable of overcoming cisplatin resistance. The slower hydrolyzed oxalate ligand also reduces the side effects when compared with cisplatin.10 The R,R-stereoisomer is more effective than the S,S-isomer because the R,R-isomer preferentially forms a hydrogen bond between the NH of DACH ligand and the O6 atom of deoxyguanosine (dG).11,12 In 2002, oxaliplatin was approved by the FDA for the treatment of metastatic colorectal cancer in combination with fluorouracil and leucovorin. Currently, it is also approved as an adjuvant treatment for stage III colon cancer.13 The milestones in the development of anticancer platinum drugs are shown in Fig. 3. In addition to the three platinum drugs that are approved worldwide, three more platinum drugs are approved in some Asian countries. Nedaplatin [cis-diammineglycolatoplatinum(II), Fig. 1] has the same cis-diammine non-leaving ligand as cisplatin and carboplatin. The glycolate leaving group confers nedaplatin a higher water solubility of 10 mg/mL. Nedaplatin has been demonstrated to be as efficient as cisplatin, and the nephrotoxicity of nedaplatin is lower than that of cisplatin.14–16 Since being approved in 1995, nedaplatin has been exclusively used to treat various cancers, such as head and neck, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and esophageal cancer in Japan.17–19

Fig. 1

Chemical structures of platinum(II) anticancer drugs.

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Fig. 2 Normal (left panel) and filamentous (right panel) forms of E. coli bacteria. The figure is adapted from Ref. Hoeschele, J. D., Biography of Professor Barnett Rosenberg: A Tribute to His Life and His Achievements. Anticancer Res. 2014, 34(1), 417–421. Copyright ©2014 International Institute of Anticancer Research.

Fig. 3

Milestones in the development of platinum drugs for cancer therapy.

Heptaplatin (Fig. 1) features a 2-(1-methylethyl)-1,3-dioxolane-4,5-dimethanamine non-leaving group that forms a sevenmembered ring with platinum. Heptaplatin showed significant efficiency in killing various cancer cells, including cisplatinresistant cells, and an antitumor effect comparable to cisplatin in vivo and in clinical trials.20–24 Furthermore, similar to oxaliplatin, the two stereocenters in the non-leaving group also have R stereochemistry. Heptaplatin was approved by the Korean Food and Drug Administration in 1999 to treat advanced gastric cancer. When compared with heptaplatin, lobaplatin (Fig. 1) has a cyclobutane ring fused to the seven-membered ring instead of the dioxolane in heptaplatin. The marketed lobaplatin is a racemic mixture of the R,R and S,S enantiomers. The advantage of lobaplatin is that it does not induce significant alopecia, nephrotoxicity, ototoxicity, or neurotoxicity after intravenous injection.25–28 In 2003, lobaplatin received approval from the Chinese Food and Drug Administration for the treatment of chronic myelogenous leukemia (CML), metastatic breast cancer, and SCLC in China.29 A summary of the approved platinum(II) drugs is summarized in Table 1.

2.25.1.1.2

The action mechanism of platinum(II) drugs

2.25.1.1.2.1 Circulation of platinum(II) drugs In the clinic, cisplatin is intravenously injected in the form of a 0.9% saline solution. After bolus or infusion administrations, it decays monoexponentially, with a plasma half-life of around 20–30 min. The Cl atoms of cisplatin are easily displaced by nucleophiles like sulfhydryl groups. Therefore, 3 h after bolus injection, 90% of platinum in the plasma is protein bound.30 The major cisplatin-HSA (human serum albumin) adducts are monofunctional adducts with S from methionine (Met) or SH from cysteine

Platinum anticancer drugs: Targeting and delivery Table 1

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Marketed platinum(II) anticancer drugs.

Drugs

Distribution

Used to treat

DLTs

Cisplatin Carboplatin Oxaliplatin Nedaplatin Heptaplatin Lobaplatin

International (1978) International (1989) International (2002) Japan (1995) South Korea (1999) China (2003)

Testicular, ovarian, bladder Ovarian Colorectal, colon Head and neck, NSCLC, SCLC, esophageal Gastric Breast, SCLC, CML

Nephrotoxicity Myelosuppression Neurotoxicity Myelosuppression Alopecia, neurotoxicity, intra-abdominal bleeding Thrombocytopenia

NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; CML, chronic myelogenous leukemia; DLT, dose-limiting toxicity.

(Cys34); a bifunctional macrochelate containing S and N donor ligands is also formed (Fig. 4). The strong trans effect of S from Met and Cys leads to the eventual loss of the NH3 ligand that is trans to them.31 The concentration of cisplatin is higher in the liver, kidney, and prostate than that in other organs following administration at 20–120 mg/m2. Finally, cisplatin is mainly secreted actively by the kidney.32–34 By replacing the two Cl atoms in cisplatin, the CBDCA leaving group that is more slowly hydrolyzed makes carboplatin more stable in circulation than cisplatin. Furthermore, inactivated carboplatin can hardly bind plasma proteins as cisplatin does.35 In patients with creatinine clearances larger than 60 mL/min, carboplatin in the blood decays in a biphasic manner after a 30-min intravenous infusion. The initial plasma half-life (alpha) is about 1.1–2 h, and the post-distribution plasma half-life (beta) is found to be 2.6–5.9 h.36 For oxaliplatin, at the end of a 2-h infusion, approximately 85% of the drug is rapidly distributed to tissues or eliminated by the kidney; only 15% of the administered oxaliplatin is left in the systemic circulation. The decline of platinum concentrations after oxaliplatin administration occurs in a triphasic manner, including two distribution phases (t1/2a: 0.43 h and t1/2b: 16.8 h) and a terminal elimination phase (t1/2g: 392 h). Similar to cisplatin, more than 90% of platinum binds to plasma proteins, including albumin and gamma-globulins, or accumulates in the erythrocytes following oxaliplatin administration.37 Same as cisplatin, the major route of elimination for carboplatin and oxaliplatin is renal excretion.36

Fig. 4

Reactions of cisplatin with human serum albumin (HSA).

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2.25.1.1.2.2 Cellular uptake of platinum(II) drugs After circulation in the blood and distribution in the tumor tissues, the generalized mechanism of action of cisplatin to kill cancer cells involves four steps: (1) cellular uptake; (2) activation in cancer cells (aquation); (3) binding to genomic DNA; 4) initiation of DNA damage-related cell death (Fig. 5). There is still an argument as to how cisplatin enters cancer cells. It was generally believed that cisplatin enters cells largely by passive diffusion, based on the observation that the accumulation of cisplatin is proportional to the drug concentration,38 the accumulation is not inhibited by cisplatin analogs,39 and the accumulation is not saturable.40 However, other studies suggested that the accumulation of cisplatin can be mediated by transporters. For example, Dornish et al. found that the protein synthesis inhibitor benzaldehyde inhibits the accumulation level of cisplatin by 50%.41 The uptake of cisplatin is also affected by other signaling mechanisms, such as protein kinase A (PKA), protein kinase C (PKC), and the calmodulin pathway.42–44 In addition, the accumulation level of cisplatin was also related to the expression level of copper transporters.45 For oxaliplatin, the uptake process is associated with organic cation transporters (OCTs).46 In summary, both passive diffusion and active transport contribute to the uptake of platinum(II) drugs, although the main contributor remains to be determined. 2.25.1.1.2.3 Aquation, DNA binding, and cell death After circulating in the blood and entering cancer cells, platinum(II) drugs are activated. The remarkably lower concentration of cytosolic chloride (4–20 mM) than that in the bloodstream (approximately 100 mM) facilitates the transformation of cisplatin to aquated products, such as cis-[Pt(NH3)2Cl(OH2)]þ and cis-[Pt(NH3)2(OH2)2]2þ.47 The half-life of cisplatin for aquation is approximately 2 h as determined by 195Pt NMR spectroscopy.48 Carboplatin and oxaliplatin are more resistant to aquation, because the CBDCA and oxalate leaving groups are significantly more stable than the Cl of cisplatin. Carboplatin is stable for 60 days in water,49 and the aquation of oxaliplatin is also much slower than that of cisplatin, with a rate constant of 1.2  10 6 s 1 in HEPES buffer at 37  C.50 After aquation, the positively charged aquated products of platinum(II) drugs can be attracted into the nucleus of cancer cells by the negatively charged genomic DNA. Then, the water molecules on the platinum can be substituted by DNA bases, most likely purine residues, i.e., guanine and adenine, at the N7 position. 195Pt NMR experiments have proved that cisplatin forms monofunctional adducts with DNA first, before the substitution of another chloride ligand by a second guanine base, thus forming crosslinks with DNA.51 Such crosslinks are formed on the same strand or different strands, giving rise to intrastrand and interstrand DNA crosslinks, respectively. Along with the most prevalent intrastrand 1,2-d(GpG) crosslinks (65%), intrastrand 1,2-(ApG) (25%), 1,3d(GpTpG) (10%), and a small amount of interstrand 50 -d(GpC)/50 -d(GpC) crosslinks are also formed between cisplatin and DNA.52 Similar DNA crosslinks are formed by carboplatin and oxaliplatin, but the rate of crosslink formation is slower, and higher concentrations of drugs are needed to achieve the same level of crosslinks as cisplatin.53,54 The platinum-DNA crosslinks distort the structure of DNA in different manners including bending and unwinding of the double helix.55 After DNA damage induced by cisplatin, a common response by cancer cells is the arrest of the cell cycle at the G2/M phase, allowing cells to repair the DNA damage before it passes to daughter cells.56 The main DNA repair mechanism is nucleotide excision repair (NER), where proteins are used to remove the oligonucleotide that contains the platinated crosslinks, and a new oligonucleotide is synthesized to replace the damaged one. If the damage cannot be repaired, the cells will initiate a process of programmed cell death, known as apoptosis. After DNA damage, the expression of pro-apoptotic genes increases, leading to the release of cytochrome c from the mitochondria, the cleavage of procaspase 9, and the activation of caspases 3, 6, and 7 to induce apoptosis.57

Fig. 5

The mechanism of action of cisplatin.

Platinum anticancer drugs: Targeting and delivery 2.25.1.1.3

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The limitations of Pt(II) drugs

Despite the fact that platinum(II) drugs are widely used in the clinic for the treatment of testicular, ovarian, bladder, lung, head and neck, colon, and colorectal cancers, the severe side effects due to their poor selectivity over normal tissues still limit their clinical use. The development of drug resistance, either by instinct or acquired mechanisms, also greatly limits the clinical effectiveness of platinum(II) drugs.58 Mechanistic studies suggest that the resistance derives from multifactorial epigenetic alterations, including reduced cellular accumulation of platinum, detoxification of platinum by thiol-containing molecules, and elevated levels of DNA damage repair.51 2.25.1.1.3.1 Side effects of platinum(II) drugs The common side effects of platinum(II) drugs include nephrotoxicity, ototoxicity, neurotoxicity, cardiotoxicity, hepatotoxicity, nausea, and vomiting. The dose-limiting side effect is nephrotoxicity for cisplatin, myelosuppression for carboplatin, and neurotoxicity for oxaliplatin (Table 1).59 Nephrotoxicity (kidney damage) can be caused by all the three platinum drugs approved worldwide because they are all mainly excreted by the kidney. The most predominant ototoxicity of platinum drugs is irreversible hearing loss, which is probably due to the damage to the inner ear via generating reactive oxygen species (ROS), downregulating important antioxidant enzymes in cochlear tissue, and activating potassium channels which leads to follicle apoptosis.60 Neurotoxicity was found in both oxaliplatin- and cisplatin-treated patients.61–63 The oxalate leaving group in oxaliplatin has an effect on the Na and K channels and was found to contribute to acute peripheral sensory neuropathy (PSN).64 Chronic neurotoxicity is thought to be derived from the inhibition of rRNA synthesis by oxaliplatin.65 Cardiotoxicity is mainly found in patients after administration with cisplatin rather than carboplatin and oxaliplatin.66 Some hematological side effects, including those affecting the function of bone marrow and the production of blood cells, may also occur after the administration of all three platinum drugs.67,68 Hepatotoxicity arises because the body tries to metabolize and detoxify the platinum drugs in the liver, giving the platinum drugs the opportunity to be taken up by the liver cells. Cisplatin and oxaliplatin are known to damage the sinusoids of the liver by the generation of ROS in the mitochondria of epithelial cells lining the sinusoids.69–71 The creation of ROS also occurs when platinum drugs attack the CYP450 enzymes, further promoting apoptosis of healthy liver cells.72 Furthermore, gastrointestinal toxicity, including nausea, vomiting, and dyspepsia, is very common in cisplatin-treated patients and is moderate in patients undergoing carboplatin and oxaliplatin treatment.73,74 2.25.1.1.3.2 Drug resistance of platinum(II) drugs 2.25.1.1.3.2.1 Reduced cellular accumulation One of the most noteworthy characteristics of cells that are resistant to platinum(II) drugs is the reduced platinum accumulation compared with the parental cells.75,76 The reduced accumulation may result from the decreased influx and increased drug efflux. For a long time, platinum(II) drugs were thought to enter cells through passive diffusion across the plasma membrane, as the uptake of the drug was linear, non-saturable, and concentration-dependent.77 It is now clear that platinum(II) drugs are also being actively transported in and out of cells by multiple membrane transporters, including copper transporters, Naþ/Kþ-ATPase, and multidrug resistance proteins (MRPs).78,79 A reduced expression level of human copper transporter 1 (hCtr1) was observed in cisplatinresistant lung cancer cells, whereas improved sensitivity to cisplatin was observed in hCtr1-transfected cells.80 Negative association between cisplatin sensitivity and the expression levels of ATPase copper transporting alpha (ATP7A) and ATPase copper transporting beta (ATP7B) further proved that the proteins involved in Cu homeostasis also contribute to cisplatin resistance.81 Inhibition of Naþ/Kþ-ATPase by ouabain reduced platinum accumulation in parental cells but did not alter the level of platinum in resistant cells, suggesting that active transport of cisplatin by Naþ/Kþ-ATPase does not occur in cisplatin-resistant cells.79 A recent study revealed that increased expression of MRP1 and MRP4 was related to notably reduced accumulation of cisplatin.78 These investigations suggest that the reduced cellular accumulation and resistance of platinum(II) drugs are highly associated with the expression levels of membrane transporters. 2.25.1.1.3.2.2 Increased inactivation by thiol-containing molecules Aquated platinum(II) drugs bind to cytoplasmic thiol-containing molecules, including glutathione (GSH), methionine, metallothioneins (MT), and other types of cysteine-rich proteins. The reaction of platinum(II) drugs with GSH expends platinum(II) drugs in the cytosol and causes reduced levels of Pt-DNA adducts in the nucleus, which induces the cellular resistance of platinum drugs. Elevated levels of GSH were observed in association with cisplatin resistance in vitro.82–85 The improved levels of GSH, as a redox regulator, may protect cells from the attack by ROS, and GSH can also serve as a cofactor in facilitating the efflux of cisplatin, which is mediated by multidrug resistance proteins.86 Metallothioneins (MT) are low molecular-weight cysteine-rich proteins, which are involved in the detoxification of metals and regulation of oxidative stress. The binding of aquated platinum(II) drugs with MT results in drug inactivation. Increased expression levels of MT were observed in cisplatin-resistant prostate, lung, and ovarian cancer cell lines.87–89 These thiol-containing molecules regulate the activity of platinum(II) drugs not only in cells but also in the tumor microenvironment. A recent study revealed that fibroblasts in the tumor microenvironment reduced platinum content in cancer cells and induced resistance to platinum by releasing GSH and cysteine.90 2.25.1.1.3.2.3 Elevated DNA damage repair The relationship between DNA damage repair and the resistance of platinum drugs has been extensively investigated for many years. Cisplatin-resistant cells show a higher removal rate of Pt-DNA cross-links and enhanced tolerance to unrepaired DNA lesions.91 As

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Platinum anticancer drugs: Targeting and delivery

aforementioned, cisplatin lesions are primarily removed by the NER machinery, and about 20 NER proteins are involved in this process. Increased NER capacity was observed in cisplatin-resistant ovarian and non-small cell lung cancer cells.92,93 Cisplatin showed enhanced cytotoxicity in cancer cells with the knockdown of NER components.94,95 Among the proteins in the NER process, excision repair cross-complementing group 1 (ERCC1) constitutes the rate-limiting factors for NER.96 The mRNA level of ERCC1 in ovarian cancer was found to correlate with the response to platinum-based chemotherapy.97 Clinical results suggested that ERCC1 level is a promising biomarker for the prediction of cisplatin responsiveness in NSCLC patients.98 Mismatch repair (MMR) also participates in the detection of cisplatin-induced DNA damage. Mutation or under-expression of mismatch repair components MSH2 or MSH6 was reported to be associated with cisplatin resistance.99 Besides NER and MMR, homologous recombination (HR), which is involved in the repair of interstrand cross-links (ICLs), the most formidable cisplatin-induced DNA lesion, also plays a role in cisplatin resistance.100 BRCA1 and BRCA2 genes encode two important components of the HR system. BRCA1/2-deficient ovarian cancers are generally more sensitive to platinum drugs.101 Secondary intragenic mutations in BRCA2 that restore the wildtype BRCA2 reading frame have been shown to favor cisplatin resistance in breast cancer cells.102 In addition, cells are able to synthesize DNA to bypass the cisplatin-induced DNA damage through the so-called translesion synthesis (TLS) system. Taken together, these studies indicate that DNA damage repair regulates the activity of platinum drugs in a multifactor mode, and it is greatly related to the resistance of platinum drugs.

2.25.1.2

Novel platinum complexes

Much effort has been devoted to designing novel platinum-based anticancer drugs, with an aim to achieve high therapeutic efficacies, reduced side effects, increased stability, and reduced resistance. Several strategies have been utilized in the rational design of new platinum anticancer agents, such as the development of non-conventional platinum(II) compounds with novel mechanisms of action, the conversion of platinum(II) drugs into more inert platinum(IV) derivatives, and the application of nano-delivery systems.

2.25.1.2.1

Non-conventional platinum(II) anticancer complexes

The initial development of platinum anticancer agents focused on the screening of platinum agents with structures similar to that of cisplatin. This method led to the discovery of carboplatin and oxaliplatin which show reduced side effects and improved activity in cisplatin-resistant cancer cells, respectively.103 However, the cytotoxicity and anticancer spectrum of many cisplatin analogs are still similar to those of cisplatin. This is attributed to the fact that the cisplatin analogs form an analogous pattern of DNA adducts as cisplatin. As a result, scientists focus on the synthesis of non-classical platinum complexes with different Pt geometries. These complexes are expected to form distinctive DNA adducts to yield a different anticancer profile than cisplatin. 2.25.1.2.1.1 Trans-platinum compounds Transplatin (Fig. 6), the trans isomer of cisplatin, is inactive in cancer cells because of the kinetic instability that accelerates its deactivation and the limited ability to form intrastrand crosslinks with DNA.104 Later, it was noted that the substitution of amines in transplatin to bulky heteroaromatic, iminoether, or asymmetric aliphatic ligands resulted in increased cytotoxicity.105–107 The bulky ligands slow down the hydrolysis rate of chloride ligands so as to increase the cytotoxicity. More importantly, trans-platinum complexes often show a mechanism of action different from that of cisplatin, therefore overcoming cisplatin resistance. For example, Natile and co-workers reported a trans-platinum compound containing one acetonimine (Fig. 6), which was very active against cisplatin-resistant ovarian cancer cells.108 Recently, it was noted that transplatin showed the same level of cytotoxicity as

Fig. 6

Chemical structures of recently developed non-conventional platinum anticancer complexes.

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cisplatin when the treated cells were irradiated with UVA light, due to the enhanced formation of interstrand crosslinks and DNAprotein crosslinks.109 In general, trans-platinum compounds show a different anticancer spectrum than cisplatin. 2.25.1.2.1.2 Monofunctional platinum(II) anticancer agents Cisplatin exerts its cytotoxicity by forming intrastrand or interstrand crosslinks as it has two coordination sites after hydrolysis. Monofunctional platinum(II) complexes refer to a class of complexes binding to DNA through only one coordination site. Recently, the antitumor activity of pyriplatin (Fig. 6), which has an anticancer spectrum distinct from cisplatin and inhibits RNA polymerase II, has been re-discovered.110–112 The synthesis of pyriplatin was conducted in 1989 by E. W. Stern and co-workers, although its anticancer activity was not identified.113 Mechanistic studies revealed that pyriplatin binds to DNA as efficiently as cisplatin and inhibits transcription as strongly as cisplatin. Its DNA repair mechanism is also NER, which is similar to that of cisplatin.114 Much effort has been devoted to the design of novel monofunctional platinum complexes after the re-discovery of pyriplatin.115–118 Notably, phenanthriplatin (Fig. 6) displays higher in vitro cytotoxicity than cisplatin against a panel of cancer cells, and it is currently being developed by Blend Therapeutics for its potential clinical applications.116 2.25.1.2.1.3 Multi-nuclear platinum(II) anticancer complexes Multi-nuclear platinum complexes contain two or more platinum centers connected by designed linkers.119 These complexes are capable of binding to DNA to form DNA adducts that are completely different than cisplatin.120 By generating DNA lesions with distinguished structures, these complexes might display a broader spectrum of anticancer activity than cisplatin.121 Multinuclear complexes represent one of the approaches to systematically alter the cellular response induced by cisplatin, and are promising new anticancer agents to overcome cisplatin resistance.120 Among the multi-nuclear platinum anticancer complexes tested, complex BBR3464 (Fig. 6), a trinuclear bifunctional DNA-binding agent, is the first one that entered phase II clinical trials.120 The anticancer profile of BBR3464 from the NCI-60 test differed from those of established drugs, and the IC50 values of BBR3464 were at least 20-fold lower than that of cisplatin.122 BBR3464 was also very active against cisplatin-resistant cells and cisplatin-refractory xenografts.122 Unfortunately, phase II studies on BBR3464 in patients with gastric and gastroesophageal adenocarcinoma and those with sensitive and resistant small cell lung cancers showed limited activity but severe systemic toxicity. Thus, the clinical investigation of BBR3464 was terminated.123,124

2.25.1.2.2

Platinum(IV) prodrugs

The conversion of platinum(II) drugs into more inert platinum(IV) derivatives is one of the promising strategies to reduce the limitations of platinum(II) drugs including severe side effects and drug resistance. Unlike the square-planar platinum(II) drugs, which face the challenge of deactivation by human serum albumin (HSA) in the blood,125 platinum(IV) compounds (Fig. 7) with a sixcoordinate octahedral geometry are much more inert during circulation, thus minimizing undesired premature activation prior to their entrance into cancer cells. Moreover, the two axial positions of the platinum(IV) center can be used to impart and fine-tune biological properties such as cancer-cell targeting, improved cellular accumulation, and accelerated activation. Certain types of platinum(IV) compounds are regarded as prodrugs because, once inside cancer cells, they will be reduced by internal stimuli such as reducing agents or activated by external stimuli such as light to form active platinum(II) species, which will be further aquated, enter the nucleus, and react with genomic DNA.

Fig. 7 Composition of platinum(IV) prodrugs. The figure is adapted from Ref. Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J., The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116(5), 3436–3486. Copyright © 2016, American Chemical Society.

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2.25.1.2.2.1 Platinum(IV) drugs in clinical trials Several platinum(IV) prodrugs, including tetraplatin, iproplatin, satraplatin, and LA-12 (Fig. 8), have been assessed in clinical trials. Unfortunately, none of them has been approved for clinical use. Tetraplatin, also known as ormaplatin [tetrachlorido(1,2diaminocyclohexane)platinum(IV)], is one of the earliest platinum(IV) prodrugs to be tested in clinical trials. It can be quickly reduced to dichlorido(1,2-diaminocyclohexane)platinum(II) in RPMI 1640 tissue culture medium, with a half-life of 5– 15 min.126 This platinum(II) product forms DNA adducts similar to oxaliplatin, indicating that tetraplatin may also be active in some cisplatin-resistant cancer cells. Six phase I trials were performed using tetraplatin at different dosages and various administration modes; however, no phase II clinical trials were announced due to its severe neurotoxicity.127–129 This severe side effect is thought to be attributed to the fast reduction of tetraplatin. Iproplatin, [dichloridodihydroxidobis(isopropylamine)platinum(IV), Fig. 8] is another platinum(IV) drug that entered clinical trials. Iproplatin contains two hydroxide axial ligands, which makes it harder to be reduced than tetraplatin by biological reducing agents. Many clinical trials ranging from Phase I to III have been conducted using iproplatin. In Phase I trials, the dose-limiting toxicity (DLT) was found to be myelosuppression.130 The phase II trials were performed in patients with many kinds of cancers including ovarian, testicular, colorectal, breast, and urothelial cancers.131–135 Phase III trials were conducted in patients with ovarian and head and neck cancers.136 Iproplatin was ultimately abandoned because the overall effectiveness of iproplatin did not surpass that of cisplatin and carboplatin. Satraplatin [trans,cis,cis-bis(acetato)amminecyclohexylaminedichloridoplatinum(IV), Fig. 8] is the first platinum(IV) prodrug that can be administered orally. In the bloodstream, satraplatin can be reduced to six different platinum(II) species, among which ammine(cyclohexylamine)dichloridoplatinum(II), without the two axial acetate ligands, is the most active and abundant product.137 The cyclohexylamine non-leaving group of satraplatin makes the induced DNA adducts unrecognizable by the DNA repair proteins. Therefore, satraplatin may circumvent the drug resistance of some classical platinum(II) drugs like cisplatin and carboplatin.138,139 However, satraplatin was rejected by the FDA because the overall survival was not significantly improved when compared to cisplatin, and many of the patients in the Phase III trials have previously received docetaxel treatment.140,141 LA-12 [trans,cis,cis-bis(acetato)ammineadamantylaminedichloridoplatinum(IV), Fig. 8] is a derivative of satraplatin, where the cyclohexylamine group is replaced by a bulky hydrophobic adamantylamine ligand. The higher lipophilicity endows LA-12 faster and elevated cellular accumulation than cisplatin. In addition, LA-12 is significantly efficient in cisplatin-resistant ovarian cancer cells.142 This favorable anticancer effect renders LA-12 to enter Phase I clinical trials.143 2.25.1.2.2.2 Reduction of platinum(IV) prodrugs in cells It is believed that platinum(IV) prodrugs need to be reduced to the active platinum(II) species by reducing agents before they can kill cancer cells. Normally, along with platinum(II) species, the axial ligands are also released after reduction (Fig. 9).144 In some cases, the equatorial ligands rather than the axial ligands are released.145,146 The reduction potential of platinum(IV) is a very important parameter to predict its reduction rate. For cisplatin-based platinum(IV) complexes, the reduction potentials correlate with their rates of reduction, and the influence of the axial ligands on the reduction potential is stronger than the equatorial ligands.147 Platinum(IV) complexes with axial OH ligands have lower reduction potentials, and they are reduced more slowly than complexes with axial carboxylate and chloride ligands.148 However, the situation is different for oxaliplatin-based platinum(IV) complexes. Gibson et al. found that oxaliplatin-based platinum(IV) complexes with axial OH ligands were reduced quicker than those with axial diacetate ligands, which is probably because the OH ligands have a higher ability than the carboxylate ligands to form a bridge with the reducing agents to facilitate electron transfer.149 The reduction of platinum(IV) complexes includes two steps: electron transfer from the reducing agents to the platinum center and the breaking of the Pt-ligand bonds. In cyclic voltammetry experiments, the electron transfer is very quick from the cathode to the platinum(IV) complexes, and the reduction potential mainly reflects the ease with which the Pt-ligand bond is broken. However, for platinum(IV) complexes with only carboxylate and amine ligands, the rate-determining step of reduction is the electron transfer from the reducing agents to the platinum, rather than the breaking of the Pt-ligand bonds. In this case, the reduction potential is probably not consistent with the actual reduction rate.

Fig. 8

Structures of platinum(IV) prodrugs in clinical trials.

Platinum anticancer drugs: Targeting and delivery

Fig. 9

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Reduction of platinum(IV) results in the release of platinum(II) and the loss of the two axial ligands.

It is generally assumed that small-molecule reducing agents like glutathione (GSH) and ascorbic acid are responsible for the reduction of platinum(IV) complexes in cells. Ascorbic acid is a two-electron reducing agent, and GSH is a one-electron reducing agent (Fig. 10). The intracellular concentrations of ascorbic acid and GSH are approximately 1 mM and 2 mM, respectively.150–152 Except for these reducing agents with low molecular weights, proteins with reducing ability may also contribute to the reduction of platinum(IV) complexes. For instance, metallothionein with 20 cysteines and one acetylated methionine was found to reduce the platinum(IV) complex iproplatin,153 while McKeage et al. found that metal-containing redox proteins like hemoglobin, cytochrome c, and liver microsomes can reduce the platinum(IV) drug satraplatin in the presence of NADH.154 Salassa et al. also demonstrated that flavoenzyme NADH oxidase (NOX) could catalytically activate platinum(IV) prodrugs in the presence of NADH.155 Furthermore, Gibson et al. found that the platinum(IV) complex [Pt(NH3)2(OAc)2Cl2] was reduced by the high molecular weight fractions of cell extracts at a rate similar to that in whole extracts, but the low molecular weight fractions containing GSH and ascorbic acid were not efficient in reducing the platinum(IV) complex.156 Recently, our study indicated that proteins with molecular weights between 10 and 100 kDa contribute more to the reduction of a carboplatin-based platinum(IV) complex in cell extracts.157 There are many reducing agents in cancer cells, and it is highly possible that several reducing agents work together to reduce platinum(IV) prodrugs in cells. 2.25.1.2.2.3 Photoactivatable platinum(IV) complexes Photoactivatable platinum(IV) complexes are compounds that can be selectively activated by light at the tumor site, with an aim to reduce the side effects associated with the original Pt(II) drugs. The novel action mechanism of these photoactivatable platinum(IV) complexes may also lead to reduced drug resistance. The first generation of photoactivatable platinum(IV) complexes is the diiodoplatinum(IV) complexes (Fig. 11). Due to the electronegativity of iodine atoms, diiodo-platinum(IV) complexes showed a ligandto-metal charge-transfer (LMCT) band at around 400 nm.158 After irradiation under light at 410 nm, complex 1–1 (Fig. 11) was reduced to a species that can bind to DNA. However, the stability of complex 1–1 in the dark under physiological conditions was inadequate. To alleviate this problem, complexes 1–2 and 1–3 (Fig. 11) were prepared, with OH or OAc groups at the axial positions.159 Although the stability of complexes 1–2 and 1–3 in the dark was improved, they were still too easy to be reduced to platinum(II) species by sulfur-rich reducing agents via an inner-sphere mechanism.160 Therefore, diiodo-platinum(IV) complexes are not suitable to be further developed as photoactivatable drugs. Diazido-platinum(IV) complexes are the second-generation photoactivatable platinum(IV) complexes. In the 1980s, these complexes were found to be photoreduced under light.161,162 Sadler et al. later designed and developed the cis-diazidoplatinum(IV) complexes 1–4 and 1–5 (Fig. 11).163 Unlike the diiodo-platinum(IV) complexes, these diazido-platinum(IV) complexes were not easily reduced by GSH in the dark. Although photosubstitution and photoisomerization might take place during the photochemical process, photoreduction products that can bind to the N7 position of guanine were clearly found by

Fig. 10

Cellular reducing agents that may activate platinum(IV) prodrugs: GSH and ascorbic acid.

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Platinum anticancer drugs: Targeting and delivery

Fig. 11

Chemical structures of diiodo-platinum(IV) complexes 1–1 to 1–3 and diazido-platinum(IV) complexes 1–4 to 1–8.

NMR spectroscopy. Moreover, the cytotoxicity of complexes 1–4 and 1–5 was quite low in the dark, but they exhibited a significantly improved cytotoxicity in the presence of light at 366 nm. The IC50 values of complexes 1–4 and 1–5 in cisplatin-resistant 5637 cells were also comparable to that in cisplatin-sensitive 5637 cells. The morphological changes of 5637 cells after the treatment of complexes 1–4 under irradiation showed no typical signs of apoptosis. These results indicate that the diazido-platinum(IV) complexes have the potential to reduce the drug resistance of platinum(II) drugs. Based on the fact that some of the trans-diamine platinum(IV) complexes display comparable cytotoxicity with cisplatin,164 many trans-diazido-platinum(IV) complexes, such as 1– 6, 1–7, and 1–8 (Fig. 11), have been synthesized.165–168 Their LMCT bands were red-shifted to the visible region when compared with the cis-diazido-platinum(IV) complexes. The trans-diazido-platinum(IV) complexes 1–6, 1–7, and 1–8 were also stable toward hydrolysis, could be photoreduced to the platinum(II) species that readily binds to DNA, and showed significant cytotoxicity in both cisplatin-sensitive and cisplatin-resistant cell lines after irradiation. Platinum(IV) complexes with a photoabsorber that can transfer electrons from light to the platinum(IV) center are considered the third-generation photoactivatable platinum(IV) prodrugs. We functionalized an oxaliplatin-based platinum(IV) complex with a widely used photosensitizer pyropheophorbide A (PPA) at the axial position and obtained the first example of a red-light activatable platinum(IV) prodrug, designated as phorbiplatin (Fig. 12).169 Phorbiplatin was stable in the dark, while under irradiation with red light (650 nm, 7 mW/cm2) for 10 min, 81% of phorbiplatin was reduced to release oxaliplatin and the PPA ligand. The IC50 values of phorbiplatin in human ovarian cancer A2780 and cisplatin-resistant A2780cisR cells were 0.13 and 0.19 mM, respectively, which were 523 and 974 times higher than that of oxaliplatin under the same condition, respectively. Owing to the deep tissue penetration of red light, phorbiplatin exhibited great antitumor activity in mice bearing 4 T1 breast cancer. Another example of a photoactivatable platinum(IV) prodrug that contains a photosensitizer as a photoabsorber is coumaplatin (Fig. 12).170 This oxaliplatin-based platinum(IV) complex contains a coumarin dye as the axial photoabsorber to transfer electrons from light to photoreduce the platinum(IV) center; a cell-penetrating peptide R8K was included at the other axial position to achieve nucleolus targeting. Coumaplatin is very stable in the dark and can be reduced to oxaliplatin under irradiation by blue light (450 nm, 8 mW/cm2). In addition, during the reduction process of a coumaplatin precursor, water was oxidated, and oxygen was subsequently released, implying a novel photoreduction mechanism for platinum(IV) complexes. The photocytotoxicity of coumaplatin proved to be two orders of magnitude higher than that of oxaliplatin. Furthermore, the cell death mode induced by coumaplatin in Pt-resistant cells under light is cell senescence via both p53-dependent and -independent pathways, and immunogenic cell death was induced, which is totally different from that of oxaliplatin. Later on, an intramolecular photoswitch was introduced to Pt(IV) prodrugs to significantly promote their photoactivation. The Pt(IV) prodrug, designated as rhodaplatin 2 (Fig. 12), displayed significantly increased photoconversion efficiency in reducing the Pt(IV) prodrug to active Pt(II) species and highly improved photocytotoxicity compared with the conventional “photocatalyst þ Pt(IV) substrate” platform.171 In addition, rhodaplatin 2 could efficiently accumulate in the mitochondria and attack mitochondrial but not nuclear DNA after photoactivation, and the prodrug was able to overcome drug resistance. A boron dipyrromethene (BODIPY) ligand was also conjugated to carboplatin-based platinum(IV) complexes to accelerate their activation. The BODIPY-conjugated platinum complex (BODI-Pt, Fig. 12) could be quickly activated under green light to release carboplatin and the axial ligands.172 The photocytotoxicity of BODI-Pt was up to 39 times higher than that of carboplatin, and the cell death mode was proved to be oncosis, which is different from platinum(II) drugs.172 Furthermore, the photoactivation rate of BODI-Pt

Platinum anticancer drugs: Targeting and delivery

Fig. 12

819

Chemical structures of phorbiplatin, coumaplatin, rhodaplatin, and BODI-Pt.

was further promoted after optimization of the axial ligands.173 These pieces of work confirm that the utilization of photosensitizers as the photoabsorbers at the axial position of platinum(IV) prodrugs is a promising strategy to construct photoactivatable platinum(IV) complexes.

2.25.2

Tumor-targeted platinum complexes

As mentioned above, the severe side effects due to the poor selectivity over tumor tissues are one of the major limitations of platinum drugs. The dose-limiting side effect was found to be nephrotoxicity for cisplatin, myelosuppression for carboplatin, and neurotoxicity for oxaliplatin. A promising strategy to reduce the side effects and improve cancer selectivity of platinum drugs is to obtain targeted platinum complexes by attaching targeting units, which can direct platinum drugs selectively to cancer cells by interacting with the receptors that are overexpressed on the surface of cancer cells.174 Moreover, the targets can be extended to the tumor area, instead of tumor cells, by seeking proteins that are expressed on angiogenic blood vessels or by allowing selective activation within the acidic or hypoxic tumor microenvironment.175

2.25.2.1 2.25.2.1.1

Tumor-targeted small molecule-platinum conjugates Estrogen-platinum conjugates targeting estrogen receptors

Estrogen receptors (ERs) are overexpressed on the surface of many cancer cells, especially breast and ovarian cancer cells.176,177 This overexpression makes ERs a good target for platinum complexes to achieve improved selectivity for cancer cells. There are mainly two types of ERs: estrogen receptor alpha (ERa) and estrogen receptor beta (ERb). ERa mediates the proliferation of estrogen, while ERb seems to have antiproliferative and antiangiogenic properties.178,179 Estrogens with high lipophilicity and strong affinity to the ERs can be conjugated with platinum centers to selectively direct platinum drugs to cancer cells. In addition, estrogen attached to platinum complexes increases the cellular accumulation and anticancer activity of platinum drugs by interacting with the ERs. Some examples of ER-targeted platinum complexes are discussed below.

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Platinum anticancer drugs: Targeting and delivery

Platinum(II) units have been conjugated to estrogen at some distance in order not to impede the interaction between estrogen and the ERs. Most of the estrogen-platinum(II) complexes formed, such as complex 2–1 with estradiol conjugated through a spacer to the N-functionalized 2-aminoalkylpyridyl chelate of platinum(II) (Fig. 13), had a very high affinity for ERa, and some also displayed a high affinity for ERb.180,181 However, their cytotoxicity in ER-positive cells is not improved when compared with that in ER-negative cells.182 In addition, the solubility of these complexes decreased as the length of the alkyl chain between estrogen and platinum increased. A poly(ethylene glycol) (PEG) chain instead of an alkyl chain was used in the estrogen-conjugated complex 2–2 (Fig. 13), in which the various length of the chain did not affect the solubility.183,184 In addition, a platinum(II) compound [Pt(ethylenediamine)Cl2] was tethered to the estrogen residue via a carbamate linker (complex 2–3, Fig. 13). After modification, the ligand maintained a 28% binding affinity to the ER compared to the positive control 17b-estradiol. Complex 2–3 showed higher cytotoxicity in ER-positive MCF-7 breast cancer cells than in ER-negative MDA-MB-231 cells. Furthermore, complex 2–3 was more cytotoxic than the control compound without the estrogen ligand in the ER-positive CAOV3 ovarian cancer cells. These results indicate that both the expression level of the ER and the presence of the estradiol ligand are very important for complex 2–3 to exhibit good cytotoxicity.185 Several novel estrogen-tethered platinum(IV) complexes have also been synthesized. For example, two molecules of estradiol were linked to diamminedichloridodisuccinatoplatinum(IV) with different lengths of polymethylene chain (complex 2–4, Fig. 13). These estrogen-tethered platinum(IV) complexes were reduced in cancer cells to afford cisplatin and two equivalents of estrogen ligands. The released cisplatin can bind to the genomic DNA to form cisplatin-DNA crosslinks, and the estrogen ligands can upregulate the expression of high mobility group box protein B1 (HMGB1), which shields cisplatin-DNA crosslinks from repair. The cytotoxicity of one of the complexes 2–4 (m ¼ 3) in ER-positive MCF-7 cells was 1.8 times higher than that in ER-negative HCC1937 cells, suggesting that the estrogen-tethered platinum(IV) complexes have the potential to specifically target ER-positive malignancies.186

2.25.2.1.2

Glucose-platinum conjugates targeting glucose transporters

To supply the energy required for cell proliferation, the rapidly-growing malignant cells mainly metabolize in a glycolysis manner and require much more glucose than normal cells.187 This improved uptake of glucose relies on the overexpression of glucose transporters (GLUTs) like GLUT1–3 on the membrane of many cancer cells.188–191 Therefore, glycoconjugation becomes a promising strategy for the targeted delivery of platinum drugs to cancer cells that overexpress glucose transporters. Some examples of platinum(II) and platinum(IV) complexes conjugated with glucose for cancer targeting are given below. Lippard et al. investigated the influence of the glucose substitution position on the anticancer activity of glucose-conjugated platinum complexes (Fig. 14).192 A platinum(II) complex was conjugated to D-glucose at all the possible positions in the sugar, and the biological activities of these glucose-tethered platinum complexes were evaluated both in vitro and in vivo. The complex C2-Glc-Pt, with platinum linked at the C2 position of glucose, accumulated in the cancer cells more efficiently than the other complexes, and the cytotoxicity of C2-Glc-Pt was also higher than the other complexes. In addition, complex C2-Glc-Pt was found to have the best cancer-targeting ability and the highest specificity against GLUT1 after comparing the cytotoxicity of these glucose-conjugated platinum complexes in GLUT1-expressing DU145, GLUT1-knockdown DU145, and normal MRC-5 cells and evaluating their cellular uptake in the presence or absence of cytochalasin B as a GLUT1 inhibitor. Finally, complex C2-Glc-Pt could selectively accumulate in the tumors where GLUT1 receptor is overexpressed, and it exhibited high antitumor activity and no observable toxicity in mice.

Fig. 13

Chemical structures of ER-targeting estrogen-platinum conjugates.

Platinum anticancer drugs: Targeting and delivery

Fig. 14

821

Chemical structures of glucose-platinum conjugates at different positions.

The interaction between the glucose-tethered platinum(II) complexes and glucose receptors was confirmed by using the complexes 2–5 (Fig. 15), which showed lower cytotoxicity in the presence of phlorizin, a glucose receptor inhibitor.193 These glucose-conjugated platinum complexes also exhibited up to 155 times higher aqueous solubility, 11 times higher cytotoxicity, and 38 times improved therapeutic index than oxaliplatin. Attaching platinum at the 6th position of glucose is also a good choice, because this OH is the only one that does not form a hydrogen bond with the side chains of transporters.194 The resulting complex 2–6 (Fig. 15) preferentially accumulated in cancer cells rather than normal cells. Further experiments proved that both glucose transporters and OCTs contributed to the uptake and cytotoxicity of this glucose-conjugated platinum complex.195 Many glycosylated platinum(IV) complexes with glycosyl groups in the axial positions have been developed in the research groups of Xin Wang and Peng George Wang. For instance, glycosyl moieties with different lengths of aliphatic chains were incorporated at the axial position of platinum(IV) moieties to obtain glucose transporter-targeting platinum(IV) complexes such as 2–7 (Fig. 15).196 These glycosylated platinum(IV) complexes could be reduced in cancer cells to active platinum(II) species, which further damages genomic DNA and induces cell death. The glycosylated platinum(IV) complex 2–7 exhibited great cytotoxicity, with IC50 values (0.24–3.97 mM) up to 166 times lower than oxaliplatin. The hexadecenoic chain in another axial position not only improved the stability of the platinum(IV) prodrug but also facilitated drug delivery by binding to HSA. Moreover, the preferential accumulation, DNA platination, and good selectivity of these glycosylated platinum(IV) complexes in cancer cells suggested that they had the potential for clinical therapeutic use. The authors also found that both the length of the alkyl chain between the glycosyl moieties and the platinum core and the length of another axial alkyl ligand in complexes 2–8 affect the cytotoxicity of the glucose-conjugated complexes.197

Fig. 15

Chemical structures of glucose-platinum conjugates.

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2.25.2.1.3

Folate-platinum conjugates targeting folate receptors

Many types of cancer cells overexpress folate receptors (FRs), and the uptake of folate is enhanced in these cancer cells to satisfy the need for high-speed cell growth.198 Thus, folate can be used to carry platinum complexes to cancer cells that overexpress FRs.199 Carboplatin-based complex 2–9 (Fig. 16) that contains a conjugated folate unit was developed to target FRs, although the low water solubility of this complex prohibited its use in biological tests. The solubility of folate-conjugated platinum complex 2–10 was improved by inserting a PEG spacer between the platinum and the folate ligand (Fig. 16). Mechanistic studies proved that this complex was taken up in a manner of FR-mediated endocytosis.200 Unfortunately, these conjugates were less cytotoxic than carboplatin, possibly due to the reduced levels of platinum-DNA adducts. These findings suggest that folate-tethered platinum conjugates may not be optimal platinum drug candidates. In contrast, FR targeting has been successfully applied to direct platinum prodrugloaded nanotubes to FR-expressing cancer cells. In complex 2–11 (Fig. 16), a cisplatin-based platinum(IV) prodrug was attached to single-walled carbon nanotubes (SWNTs) via the succinate ligand at one axial position, while a folic acid ligand was included at the other axial position.201 The solubility and biocompatibility of the platinum(IV) complex were modulated by incorporating a PEG spacer between the folic acid and the platinum center.202 The IC50 value of this SWNT-tethered platinum(IV) complex was as low as 10 nM in human nasopharyngeal carcinoma KB cells, which was 8.6 times lower than that of cisplatin.201

2.25.2.1.4

Biotin-platinum conjugates targeting SMVT

Biotin is a promoter of cellular growth and plays an important role in gene regulation and cell signaling.203 To fulfil the high demand for biotin from rapidly-proliferating cancer cells, biotin-specific receptors are overexpressed on the surface of many cancer types.204 The main biotin-specific receptors are sodium-dependent multi-vitamin transporters (SMVTs), which are overexpressed in breast, lung, ovarian, and renal cancer cells.205,206 Thus, biotin conjugates can deliver platinum drugs to the tumor sites and improve the uptake of platinum drugs in cancer cells that overexpress SMVTs. The biotin-conjugated platinum complex 2–12 (Fig. 17) had high selectivity for breast cancer cells but not for normal epithelial cells. In addition, this mono-biotinylated platinum(IV) complex 2–12 was more cytotoxic than the di-biotinylated complex 2–13 (Fig. 17), probably due to their faster reduction in cancer cells.207 Biotin was also linked to a series of platinum(IV) complexes with a Cl ligand at the opposite axial position. For example, complexes 2–14 (Fig. 17) exhibited cytotoxicities that were 2.0–9.6 times higher than that of cisplatin and cellular accumulation levels 1.3–6.3 folds higher, confirming that the introduction of cancer-targeting biotin could improve the cellular accumulation and cytotoxicity of platinum drugs. Biotin was also used to deliver platinum(IV) complexes containing dichloroacetate or indomethacin ligands (complexes 2–15–2-17, Fig. 17) to cancer cells.208–210 The selective accumulation and enhanced antitumor activity of these biotinylated-platinum complexes could be at least partially attributed to the targeting ability of biotin.211

Fig. 16

Chemical structures of FR-targeting folic acid-platinum conjugates.

Platinum anticancer drugs: Targeting and delivery

Fig. 17

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Chemical structures of SMVT-targeting biotin-platinum conjugates.

2.25.2.1.5

Phosphonate-platinum conjugate targeting bone cancers

Bisphosphonates (BPs) have the ability to chelate calcium ions and show a high affinity to the bone.212 BPs are also found to have antitumor activities in preclinical models.213 The bone-targeting properties and the antitumor effects make BPs suitable bonetargeting carriers for platinum drugs. The introduction of BPs may also improve the solubility and uptake of platinum drugs.214 Complex 2–18 is an example of BP-platinum conjugates (Fig. 18), where an analogue of picoplatin is included. This BPplatinum conjugate was quite soluble in water, and its cytotoxicity was lower than that of cisplatin. The action mechanism studies proved that the cell death pathway of complex 2–18 was different from apoptosis induced by cisplatin. Thus, BP-conjugated platinum complexes are novel non-classical platinum antitumor agents with promising bone-targeting properties.215 Platinum complexes comprising monoaminophosphonate ester moieties as the bone-targeting groups have also been developed.216,217 For example, Wang et al. developed a series of platinum(IV) complexes bearing a monoaminophosphonate ester moiety. The complexes displayed up to 6.1–30.2 times better cytotoxicity in cisplatin-sensitive and -resistant SKOV3 cancer cells but 4.8– 23.1 times lower cytotoxicity in normal HL-7702 liver cells when compared with cisplatin and oxaliplatin. The most effective complex 2–19 (Fig. 18) also proved to have a good in vivo antitumor activity in an NCI-H460 xenograft model. Overall, these monoaminophosphonate-conjugated platinum(IV) complexes can effectively improve the antitumor effect and reduce the side effects of platinum drugs.218

2.25.2.2

Tumor-targeted platinum-peptide conjugates

2.25.2.2.1

RGD-platinum conjugates targeting integrin

Peptides are also widely used for the targeted delivery of platinum complexes to tumors. Integrins like aVb3 and aVb5 are overexpressed in endothelial tumor cells; thus, platinum(IV) complexes with peptides that specifically bind to integrins are capable of selectively targeting angiogenic tumor cells. Several platinum(IV) complexes functionalized with aVb3 and aVb5 integrins-

Fig. 18

Chemical structures of bone-targeting phosphonate-platinum conjugates.

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Platinum anticancer drugs: Targeting and delivery

targeting peptides, such as RGD (Arg-Gly-Asp), cyclic (CRGDC)c, and (RGDfK)c, have been prepared. Cyclic peptides are chosen because they can more efficiently target tumor angiogenic endothelial cells than linear peptides. Cytotoxicity study showed that the RGD-Pt(IV) conjugates 2–20 (Fig. 19) were specifically and highly cytotoxic to BCE and HMVEC cancer cells that overexpress the integrins aVb3 and aVb5, and their IC50 values ranged from 2.1 to 5.5 mM.219 Furthermore, the antiproliferative effect of the RGDPt(IV) conjugates 2–20 decreased following co-incubation with integrin-specific peptides and transfection with siRNA for b3 integrin, confirming that integrin aVb3 mediated, at least in part, the anticancer effect of the RGD-Pt(IV) conjugates 2–20. The selectivity with respect to cancer cells was further enhanced by linking four copies of (RGDfK)c peptide at the axial position of the picoplatinbased platinum(IV) complex through triazoles and a cyclodecapeptide scaffold.220 The formed conjugate 2–21 (Fig. 19) was able to accumulate much more efficiently in cells overexpressing aVb3 and aVb5 integrins than in control cells without overexpression of these integrin proteins, leading to 20-time higher cytotoxicity than picoplatin in integrins-overexpressing SK-MEL-28 cancer cells. The integrin targeting (RGDfK)c peptide was also conjugated to a photoactivatable platinum(IV) complex. Upon irradiation by visible light (l ¼ 420 nm, 5 J/cm2), the phototoxicity of complex 2–22 (Fig. 19) increased preferentially in the SK-MEL-28 cancer cells overexpressing aVb3 integrin with a phototoxic index (PI) of 3.6 compared to the control DU-145 cells (PI ¼ 1.3).221

2.25.2.2.2

NGR-platinum conjugates targeting aminopeptidase N (APN)

In addition to integrins, aminopeptidase N (APN) is also overexpressed on the surface of endothelial tumor cells. Thus, NGR (AsnGly-Arg)-containing peptides that specifically bind to APN can also be used to deliver platinum complexes to cancer cells. After conjugation with platinum, the formed NRG-platinum(IV) complexes 2–23 (Fig. 20) displayed IC50 values of 5.1–7.1 mM in BCE and HMVEC cells, which were 3.7–10.8 times more active than the nonspecific peptide-platinum controls.219 The cyclic CNGRC peptide was also linked to platinum through a malonyl linker and a miniPEG spacer. The biological study revealed that these CNGRD-containing platinum(IV) complexes 2–24 (Fig. 20) but not carboplatin selectively accumulated in the APNpositive PC-3 cells.222 Furthermore, platinum(IV) complexes 2–24 containing CNGRD can kill PC-3 cells more efficiently than carboplatin in an apoptosis manner. These results indicate that the NRG-containing peptides can selectively bind to APN and deliver the platinum complexes to the APN-positive cancer cells.

2.25.2.2.3

TPP-platinum conjugates targeting memHSP70

HSP70 is a 70-kilodalton heat shock protein that maintains protein homeostasis in normal cells, and it is upregulated during a stress response.223 The plasma membrane HSP70 (memHSP70) is found to be selectively expressed in many cancers.224 In healthy tissues, however, memHSP70 is not expressed.225 It has been reported that a 14-mer tumor penetrating peptide (TPP, TKDNNLLGRFELSG) can be rapidly internalized by the memHSP70-positive tumor cells, because the TPP matches an epitope in the oligomerization

Fig. 19

Chemical structures of integrin-targeting RGD-platinum conjugates.

Platinum anticancer drugs: Targeting and delivery

Fig. 20

825

Chemical structures of APN-targeting NGR-platinum conjugates.

domain of the memHSP70. Therefore, TPP has the potential for targeted delivery of platinum drugs to tumors that overexpress memHSP70. The oxaliplatin-based platinum(IV) complexes were conjugated with TPP at one or two axial positions. The formed conjugates 2–25 (Fig. 21) had higher cytotoxicity in colorectal cancer cells that expressed high levels of memHSP70 than both oxaliplatin and the control complexes without TPP.226,227

2.25.2.2.4

CTX-platinum conjugates targeting chlorotoxin receptors

2.25.2.2.5

EGFR peptide-platinum conjugates targeting EGFR

Chlorotoxin (CTX) has 36 amino acids and is a component found in the venom of the scorpion.228 It can bind to proteins like matrix metalloproteinase 2 (MMP2), chloride ion channels, and annexin A2, which are overexpressed on the surface of some cancer cells but not normal cells. Thus, CTX has been conjugated in a 1:1 ratio with a platinum(IV) complex for targeting purposes. The CTX-conjugated platinum(IV) complex 2–26 (Fig. 22) exhibited 7.7 times higher cytotoxicity in HeLa cells than the control platinum(IV) complex without the CTX peptide.229 This improved cytotoxicity was attributed to the targeting of the CTX-platinum(IV) complex to the CTX receptors annexin A2 and MMP2, which are expressed on the surface of HeLa cells.230

Epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer, including colon, breast, and NSCLC.231 One of the approaches for EGFR targeting is the use of EGFR-binding peptides, including CMYIEALDKYAC,232,233 YHWYGYTPQNVI (also known as “GE11”),234–237 and LARLLT (also known as “D4”).238–241 These peptides have been used to deliver antitumor drugs such as paclitaxel and doxorubicin to enhance the tumor specificity and cellular accumulation via EGFR-mediated endocytosis. EGFRtargeting peptide-conjugated platinum(IV) complexes 2–27 and 2–28 (Fig. 23) were developed by Keppler et al., in which cisplatinand oxaliplatin-based platinum(IV) complexes bearing maleimide groups were linked to the EGFR-targeting peptide LARLLT.242 Biological studies showed that neither the cytotoxicity nor the cellular accumulation level correlated with the expression level of

Fig. 21

Chemical structures of memHSP70-targeting TPP-platinum conjugates.

826

Platinum anticancer drugs: Targeting and delivery

Fig. 22

Chemical structure of CTX-platinum conjugate.

Fig. 23

Chemical structures of EGFR-targeting LARLLT-platinum conjugates.

EGFR in cancer cells. These data prove that LARLLT may not be suitable for enhancing the selectivity of platinum complexes against EGFR-overexpressing cancer cells.

2.25.2.2.6

AHNP-platinum conjugates targeting HER2

The human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of breast cancer cells (approximately 20– 30%) and gastric cancer cells (20%), and HER2 tyrosine kinase is associated with the initiation, progression, and metastasis of cancer cells.243,244 A famous example that utilizes the overexpression of HER2 to selectively kill cancer cells is the FDA-approved drug HerceptinÒ (trastuzumab), a HER2-targeting monoclonal antibody.245 Besides trastuzumab, an anti-HER2 peptide AHNP with a high affinity to HER2 was also developed based on the structure of the CDR-H3 recognition loop of trastuzumab.246,247 This HER2-targeting AHNP peptide was tethered to cisplatin-based platinum(IV) complexes in order to achieve selectivity for cancer cells that overexpress HER2.248 The AHNP-platinum(IV) complex 2–29 (Fig. 24) selectively killed HER2-overexpressing NCI-N87 cancer cells but not the normal A2780 cells in a co-culture experiment; the percentage of killed NCI-N87 cells was approximately 3 times higher than that of the killed A2780 cells. In addition, the incorporation of the AHNP peptide rendered the resulting platinum complexes a unique biphasic mode of action including necrosis and delayed cell death. This high selectivity for HER2-

Fig. 24

Chemical structure of HER2-targeting AHNP-platinum conjugate.

Platinum anticancer drugs: Targeting and delivery

827

overexpressing cancer cells and the different cell death pathways indicate that these AHNP-platinum(IV) complexes have the potential to reduce the side effects and overcome the drug resistance problem of platinum drugs at the same time.

2.25.2.3

Delivery of platinum drugs by proteins

To deliver platinum drugs to cancer cells, one option is “active targeting” which is based on the cancer-targeting properties of molecules such as sugars, folic acid, and peptides, as described above. Besides, the “passive targeting” strategy, based on the enhanced permeability and retention (EPR) effect, is also widely used. The EPR effect mainly occurs because tumor vasculatures are leakier than those in normal tissues, and the tumor tissues are inefficient in removing fluids via the lymphatic networks.249 The combination of these effects warrants macromolecules like nanoparticles and proteins with sizes ranging from 50 to 200 nm readily extravasate into the tumor sites (permeation) and remain there for a long time (retention). There are many excellent reviews on the delivery of platinum drugs based on nanoparticles.4,174 The following are some examples of delivery strategies based on proteins like albumin and antibodies rather than nanoparticles.

2.25.2.3.1

Delivery of platinum drugs by albumin

2.25.2.3.1.1 Platinum complexes covalently bound to albumin One of the most promising methods to exploit the EPR effect is the use of albumin as a carrier to deliver drugs into cancer cells. Human serum albumin (HSA), the most abundant protein in human blood, is known to accumulate in cancer tissues and can be taken into cancer cells by endocytosis.250–252 Anticancer drugs AbraxaneÒ and aldoxorubicin are two successful examples of drugs delivered by albumin.253–255 Of these, the latter contains a maleimide group, which can couple with the free -SH group of cysteine-34 in HSA. Platinum(IV) complexes with maleimide groups in both axial positions for HSA binding were also prepared.256 The resulting maleimide-functionalized platinum(IV) complexes 2–30 and 2–31 (Fig. 25) enabled fast coupling with the thiolcontaining amino acid cysteine in tumor-targeting HSA. The albumin-bound complex 2–31 was proved to be accumulated in the malignant tissues via clathrin- and caveolin-dependent endocytosis.257 However, only one maleimide group was able to bind to HSA, leaving another maleimide group free to react with the other thiol-containing molecules or proteins, which may cause undesired pharmacological activities. Therefore, cisplatin- and oxaliplatin-based mono-maleimide platinum(IV) complexes like 2– 32 (Fig. 25) were synthesized.258 The platinum concentration of these mono-maleimide platinum(IV) complexes in the plasma largely increased, and the tumor accumulation levels were significantly higher than that from oxaliplatin. In vivo experiments in CT-26-bearing mice also proved that the oxaliplatin-based mono-maleimide platinum(IV) complexes had better antitumor activity than the free oxaliplatin drug. Moreover, this maleimide-mediated albumin binding strategy was applied to achieve the selective delivery of platinum(IV) prodrug 2–33 (Fig. 25) that contains an immunomodulatory ligand 1-methyl-d-tryptophan

Fig. 25

Chemical structures of maleimide-platinum conjugates that covalently bind to albumin.

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Platinum anticancer drugs: Targeting and delivery

(1-D-MT).259 These examples indicate that the maleimide-mediated binding of albumin is a promising strategy to improve the anticancer activity of platinum drugs. However, one of the main drawbacks of drug conjugation through the reaction between maleimide and thiol is the possibility of retro-Michael reactions with GSH, after which the original thiols and GSH-conjugated maleimides are formed. To prevent the retro-Michael reaction and stabilize the thiosuccinimide group of maleimide-thiol conjugates, one of the strategies is to form a locked six-membered ring via a transcyclization reaction (Scheme 1).242,260–263 The require-

Scheme 1

Locking the thioether conjugation bond in a 6-membered ring via a transcyclization reaction

ment of this transcyclization reaction is the presence of an amino group at the b-position of sulfur. The absence or protection of the amino group will prevent the transcyclization reaction. This method enables the preparation of these quite stable conjugates for the targeted delivery of platinum drugs to cancer cells. 2.25.2.3.1.2 Platinum complexes non-covalently bound to albumin HSA has also been exploited as a delivery device that can load platinum(IV) complexes non-covalently. By mimicking the amphiphilic structure of fatty acids that are transported by HSA, long linear alkyl groups were functionalized at the axial position of platinum(IV) to facilitate the association of platinum drugs with HSA.264 The most cytotoxic platinum(IV) complex 2–34 (Fig. 26), with a hexadecyl chain at the axial position, was found to interact with HSA at a 1:1 ratio to form a platinum-protein complex. Computational investigations suggested that the platinum(IV) complex 2–34 was buried under the surface of the HSA protein. Additionally, the association of HSA enhanced the stability of complex 2–34 in human blood and protected it from reduction by reducing agents such as ascorbic acid. This drug delivery strategy, based on the ability of the long linear alkyl chain to noncovalently bind to HSA, was also successfully applied to deliver a platinum(IV) prodrug 2–35 (Fig. 26) containing an indoleamine-2,3-dioxygenase (IDO) inhibitor at the axial position to tumor sites.265

2.25.2.3.2

Delivery of platinum drugs by antibodies

Antibody-drug conjugates (ADCs) are now attracting tremendous attention for drug targeting and delivery. ADCs consist of monoclonal antibodies and potent cytotoxic drugs, with linkers between them. The monoclonal antibodies deliver anticancer prodrugs into targeted cancer cells that express the corresponding antigens on the cell surface. The prodrugs can then be converted to the active drugs in cancer cells, while normal cells are unaffected.266 As mentioned above, HER2 is overexpressed on the surface of breast and gastric cancer cells, and the HER2-targeting monoclonal antibody trastuzumab has been approved by the FDA for the treatment of patients with HER2-positive breast cancers. Therefore, trastuzumab is quite suitable for the construction of antibody-platinum conjugates. Sun et al. developed a cisplatin-based platinum(IV) complex 2–36 (Fig. 27) functionalized with trastuzumab antibodies at the axial positions.267 There are 6.8 mol of platinum complexes loaded per mole of trastuzumab. The trastuzumabcisplatin conjugate maintained a high binding affinity to HER2 and HER2-overexpressing SK-BR-3 breast cancer cells. In addition, the trastuzumab-cisplatin conjugate showed promising cytotoxicity in SK-BR-3 cells rather than HER2-negative MCF-7 and MDAMB-231 cancer cells. Furthermore, a similar trastuzumab-oxaliplatin conjugate was also developed to deliver platinum drugs to

Fig. 26

Chemical structure of platinum complexes with long alkyl chain for non-covalent binding to albumin.

Platinum anticancer drugs: Targeting and delivery

Fig. 27

829

Chemical structures of antibody-platinum conjugates.

HER2-positive cancer cells.268 These examples show that trastuzumab is a promising targeting carrier for the delivery of platinum drugs to cancer cells. In addition to trastuzumab, other monoclonal antibodies, including an anti-c-Met antibody (anti-c-Met IgG) and an anticytokeratin-8 antibody (6D7), were also conjugated with oxaliplatin or carboplatin. c-Met is a receptor tyrosine kinase that is overexpressed on the surface of hepatocellular carcinoma (HCC). The c-Met targeting antibody (anti-c-Met IgG) was conjugated to an oxaliplatin-based platinum(IV) complex to obtain complex 2–37 (Fig. 27) for HCC targeting. Approximately 4.4 mol of platinum have been incorporated into one mole of the antibody. The resulting ADC had selectivity and high binding affinity for c-Met and cMet-positive HepG2 liver cancer cells. Furthermore, this ADC showed better antitumor activity and lower side effects in vivo when compared with oxaliplatin. Cytokeratin-8 is the most abundant type of cytokeratin in malignant cancer cells.269 An anti-cytokeratin8 antibody 6D7-carboplatin conjugate was proven to accumulate more and remain longer in OVA-1 tumors than carboplatin. The Pt content in the tumor increased until 30 min after the administration of 6D7-carboplatin conjugate, whereas the Pt level started to decrease 7 min after the administration of carboplatin. The tumor-suppressing effect of the 6D7-carboplatin conjugate was also better than that of carboplatin and the nonspecific control IgG-carboplatin conjugate.270

2.25.3

Organelle-targeted platinum complexes

2.25.3.1

Nucleus-targeted Pt complexes

It is widely agreed that nuclear DNA is the main target of conventional Pt drugs; however, the amount of Pt drugs that finally bind to DNA in cells is low. Take cisplatin as an example, only less than 10% of cisplatin can finally enter the nucleus and covalently bind to DNA, whereas 75–85% of the drug is captured by various biomolecules (e.g., proteins).271 These “off-target” Pt drugs will not only induce various side effects but also contribute to the drug resistance of cancer cells and compromise the therapeutic outcomes. Therefore, enhancing the accumulation level of Pt complexes in the nuclear region may increase the Pt-DNA binding efficiency and amplify DNA damages to improve anticancer activities, overcome drug resistance, and decrease the induced side effects. With this goal, several strategies have been applied to develop nucleus-targeted Pt complexes. Mieszawska et al. designed a carboplatin-like Pt(II) complex (Pt-NLS) by introducing a nuclear localization sequence (NLS) peptide (PKKKRKV) to the carboxylate ligand through click chemistry (Fig. 28A).272 The NLS peptide can be recognized by protein carriers (importins) on the nuclear membrane and then transport the cargo into the nucleus.273 The authors first examined the nuclear transport ability of the NLS peptide by monitoring the fluorescence of fluorescein-labeled NLS in cancer cells. The result showed that NLS could successfully penetrate the nuclear envelope and localize in the nucleus. Next, the authors investigated the anticancer activities of Pt-NLS in platinum-sensitive and -resistant cancer cell lines. Compared with carboplatin, Pt-NLS presents up to 2-fold increased cytotoxicity in several cancer cell lines, including platinum-resistant cell lines, suggesting that increasing the nuclear accumulation of platinum drugs is a practical way to improve their anticancer potential and overcome drug resistance. Mao’s group conjugated different Pt(II) moieties to the para-position of pyridines in triphenylamine to obtain a class of Pt(II)triphenylamine complexes (Figs. 28B and C).274,275 The obtained Pt(II) complexes exhibited high DNA affinity and could bind to DNA through intercalation. Upon blue light irradiation, these complexes were able to generate a large amount of ROS to trigger DNA damage and initiate cell apoptosis. Interestingly, the author noticed that only the Pt(II) complexes coordinated at the para-position were able to effectively enter the nucleus, whereas those conjugated at the meta-position preferred to localize in the cytoplasm. Consequently, higher DNA damage efficiency, enhanced photocytotoxicity, and a strengthened ability to overcome drug resistance was observed in the Pt(II) complexes coordinated at the para-position, suggesting the advantage of the nucleustargeting strategy for platinum complexes. Recently, our group developed an oxaliplatin-based and photoactivatable Pt(IV) prodrug, coumaplatin (Fig. 12), that can effectively accumulate in the nucleolus of cancer cells.170 Coumaplatin was non-toxic in the dark, whereas upon low-dose visible light

830

Platinum anticancer drugs: Targeting and delivery

(A)

(B) (C)

Fig. 28

The chemical structures of nucleus-targeted Pt(II) complexes.

irradiation, the prodrug was effectively reduced to oxaliplatin, and then it interacted with nuclear DNA and certain nucleolus proteins to eliminate cancer cells. Although oxaliplatin was released upon visible light irradiation, the mechanism of action of coumaplatin was different: oxaliplatin mainly killed cancer cells through apoptosis; whereas coumaplatin mainly induced senescence to eliminate cancer cells. Consequently, the photoactivated coumaplatin presented more than 96-fold increased cytotoxicity than oxaliplatin and could effectively overcome drug resistance, suggesting that delivering platinum drugs into the nucleolus was an effective way to improve the therapeutic outcome.

2.25.3.2

Mitochondria-targeted Pt complexes

The potent ability of cancer cells to repair nuclear DNA damage induced by Pt drugs contributes majorly to developed or intrinsic Pt resistance.91 Developing Pt complexes that can target other organelles besides the nucleus may provide a chance to bypass the genomic DNA damage-repair-mediated platinum resistance. Mitochondria are the central organelle for cell respiration. Most of the cell’s adenosine triphosphate (ATP) is generated in the mitochondria. Emerging evidence suggests that mitochondria are a potential target for anticancer agents due to their important roles in cell survival.276 Several important mitochondrial metabolism processes, such as ROS homeostasis, redox-regulation homeostasis, and tricarboxylic acid cycle, can be interrupted by Pt complexes, resulting in cell death.208,277,278 Importantly, mitochondrial DNA (mtDNA) lacks nucleotide excision repair (NER) and protective histones,279,280 which makes mitochondrial DNA a valuable target for Pt complexes. In addition, inducing mitochondrial DNA damage has proven to be a practical method to initiate cell death.281,282 Therefore, delivering Pt complexes to mitochondria to overcome Pt resistance is a promising approach.

Platinum anticancer drugs: Targeting and delivery

831

Kelley et al. developed a cisplatin-like Pt(II) complex (mtPt, Fig. 29A) that can specifically accumulate in the mitochondria of cancer cells with the help of a conjugated mitochondria-targeting peptide.283 Unlike cisplatin, mtPt did not induce nuclear DNA damage; consequently, no cell cycle arrest was observed. Instead, mtPt effectively damaged mtDNA and triggered mtDNA damage signaling pathways to initiate cell apoptosis. Since the departure rate of mtPt is slower than that of cisplatin, mtPt is not as effective as cisplatin in A2780 cells (e.g., IC50 of 7.5 vs. 0.6 mM). However, mtPt exhibited equal activities toward platinum-sensitive and -resistant cancer cells, suggesting that delivering platinum drugs into mitochondria to overcome drug resistance is a practical approach. Pyriplatin is a monofunctional Pt(II) complex that can bind to DNA in a monodentate manner and possesses an anticancer spectrum different from cisplatin and oxaliplatin.112 Guo and colleagues modified pyriplatin with a mitochondria-targeting group, triphenylphosphonium (PPh3), and obtained three Pt(II) complexes.278 Intriguingly, the substitution position of PPh3 has a significant effect on the cellular uptake and intracellular distribution of these Pt(II) complexes. Only the ortho-substituted complex (OPT, Fig. 29B) could effectively accumulate in the mitochondria and exhibit higher anticancer activities than the other two analogs and cisplatin. Further studies revealed that OPT was able to disrupt mitochondrial oxidative phosphorylation and glycolysis, and the complex interacted with mtDNA to cause mitochondria damage and cell apoptosis. This study suggests that Pt complexes have the potential to disturb mitochondrial energy metabolism, which may provide an opportunity to overcome the drug resistance of cancer cells toward conventional Pt drugs. Chao et al. used an Ir(III) complex as the cell penetration and mitochondria-targeting moiety and obtained a binuclear Ir(III) ePt(II) complex (IrePt, Fig. 29C).284 IrePt entered cancer cells through both passive diffusion and energy-dependent active transport, resulting in 8.2-fold higher cellular accumulation level than cisplatin. Besides, the reduced efflux of IrePt compared to cisplatin was also favorable for executing the cancer cell inhibition effect. Approximately 80% of IrePt was detected in the mitochondria, and the complex effectively induced mtDNA damage, metabolism alteration, ROS accumulation, and necrosis to kill cancer cells. Consequently, compared with cisplatin, IrePt was found to be 5.8- and 32.1-fold more cytotoxic in A549 and A549cisR cells, highlighting the anticancer activities of IrePt, especially toward platinum-resistant cancer cells. Recently, a class of photoactivatable and mitochondria-targeted Pt(IV) prodrugs, namely rhodaplatins, was developed by our group (Figs. 12 and 29D).171 Rhodaplatins could effectively accumulate in the mitochondria of cancer cells. Upon low-dose white light irradiation, they could be rapidly reduced to the corresponding clinical Pt(II) drugs. Subsequently, the released Pt(II) drugs bound to mitochondria DNA, triggered mitochondria DNA damage, and initiated mitochondria damage-mediated apoptotic pathways to kill cancer cells. Rhodaplatins successfully overcame platinum resistance, with the resistance factor (RF) values as low as 0.8; whereas the RF value of Pt(II) drugs was as high as 3.5, suggesting that guiding platinum drugs to the mitochondria is a practical way to overcome drug resistance.

(A) (B)

(D) (C)

Fig. 29

The chemical structures of mitochondria-targeted platinum complexes.

832 2.25.3.3

Platinum anticancer drugs: Targeting and delivery ER-targeted Pt complexes

The endoplasmic reticulum (ER) is the largest organelle where various proteins are synthesized and assembled. Although ER is a central organelle for cell survival, its role as an anticancer drug target has not been well studied, especially compared with other organelles such as the nucleus and mitochondria. Recent studies found that metal complexes that can target ER are generally more cytotoxic toward cancer cells than normal cells, which may be due to the relatively higher ER-resident protein levels in cancer cells.285 Since increasing studies have indicated that platinum complexes show high affinity toward many proteins,286–288 ERtargeted Pt complexes may be able to interact with ER-resident proteins and disrupt the function of the ER to induce cell death. Indeed, emerging studies suggest that ER-targeted metal complexes can trigger ER stress to result in cell death.285,289 In addition, recent studies suggest that metal complex-triggered ER stress is frequently associated with immunogenic cell death (ICD), which can activate the immune response to further improve anticancer efficiency.289,290 Che’s group developed a class of luminescent Pt(II) complexes coordinated with N-heterocyclic carbene ligands (Fig. 30A).291 These complexes selectively accumulated in the ER, generated a large amount of ROS to trigger intense ER stress and activated caspase-mediated pathways to result in cell apoptosis. Consequently, this complex showed 5.2- to 60-fold increased cytotoxicity toward a number of cancer cell lines compared to cisplatin, and the complex did not show cross-resistance with cisplatin, suggesting the potential of the ER-targeting strategy for platinum drugs. Ang and co-workers synthesized a cyclometalated Pt(II) complex, PlatinER (Fig. 30B), which showed an impressive ability to trigger ICD in cancer cells.292 PlatinER localized in the ER and acted as an ICD inducer by generating ER-localized ROS, triggering the unfolded protein response (UPR) to induce ICD. Besides inducing the exposure of calreticulin (CRT), a marker of ICD that serves as an “eat me” signal to recruit immune cells, PlatinER could also activate HSP90 to amplify this signal. Consequently, cancer cells treated with PlatinER were much easier to be recognized and phagocytosed by macrophages than cells treated with other reported ICD inducers.293

2.25.3.4

Lysosome-targeted Pt complexes

The lysosome is a digestive compartment of cells with a low pH (approximately 4.5). To digest endocytosed poisonous species into small molecules, more than 60 types of acid hydrolases are involved.294 The lysosome also participates in many key life activities such as apoptosis, intracellular transportation, and cholesterol homeostasis.295 Damaging lysosome is another choice to conquer the drug resistance problem. A photoactivable monofunctional platinum complex, Pt-BDPA (Fig. 31), that can be sequestered in lysosomes was developed by Guo and co-workers.296 Upon photoactivation, the generated ROS damaged the membrane of the lysosome and endowed the platinum complex with the ability to escape from the lysosome. The released platinum can further access the nucleus and react with genomic DNA. In addition, the ROS decreased the intracellular GSH level, which lowered the possibility of platinum being deactivated by thiol-containing GSH. This novel complex displayed significant cytotoxicity in a couple of cancer cell lines after photoactivation, with IC50 values ranging from 3 to 6 mM, which were much lower than those in the dark.296 This first example of photoactivated lysosomal escape of platinum complex provides a new opportunity to alleviate the drug resistance and side effects of platinum drugs.

2.25.4

Platinum drug-based nano-delivery systems

The emergence of nanomaterials provides a great platform for platinum drug delivery. Compared with small-molecule drugs, nanomaterial delivery systems possess various advantages, e.g., better tumor targeting ability, greater biocompatibility, improved pharmacokinetic properties, and the ability to overcome biological barriers.297 Therefore, numerous strategies have been proposed to develop platinum-drug-based nano-delivery systems for cancer treatment, which has been well summarized in many other reviews.298–301 In this chapter, we will review these nano-systems from three aspects according to the loading and releasing method of platinum drugs: platinum drug-incorporated nano-systems, platinum drug self-assembled nano-systems, and platinum drugconjugated nano-systems, with a focus on the publications in the past couple of years.

(A)

Fig. 30

The chemical structures of ER-targeted Pt(II) complexes.

(B)

Platinum anticancer drugs: Targeting and delivery

Fig. 31

2.25.4.1

833

The chemical structures of lysosome-targeted Pt(II) complex Pt-BDPA.

Platinum-incorporated nano-systems

As aforementioned, the therapeutic effect of platinum drugs is greatly limited by the serious side effects, relatively short circulation half-life, and resistance from cancer cells. The promising potential of nanomaterials for drug delivery has been well demonstrated,302 and using nanomaterials to load platinum drugs could effectively enhance their circulation half-life, tumor targeting ability and cellular uptake level, and improve the treatment effect. Polymers and liposomes are some of the firstly approved drug delivery systems in the clinic, and they have large drug-loading efficiency. Platinum drugs can be directly loaded into these systems, and after administration, these nanoparticles present higher tumor accumulation ability than conventional platinum drugs due to the increased permeability and retention of macromolecules.303 After reaching the tumor region, these nanoparticles can be decomposed steadily, then they can release the loaded platinum drugs to kill cancer cells and therefore improve the anticancer efficacy and decrease the side effects. For example, several polymer-based and platinum drug-loaded nanoparticles have entered clinical trials (Table 2).304–306 Compared with the free platinum drugs, the hydrophilic outer shell of these platinum-loaded nanoparticles contains polyethylene glycol that can prevent the nanoparticles from being arrested by the reticuloendothelial system and significantly increase the circulation half-life. Consequently, these nanoparticles presented 6- to 15-fold increased circulation half-life, better tumor selectivity, and reduced dose-limiting toxicities (DLT).304–306 Liposome is another widely used drug delivery system in the clinic, and many platinum drug-based liposome nanoparticles have been developed. The most successful one, lipoplatin,307 is a liposome consisting of soy phosphatidylcholine (SPC-3), cholesterol, dipalmitoyl phosphatidylglycerol (DPPG), and methoxypolyethylene glycol-distearoyl phosphatidylethanolamine (mPEG2000-DSPE), with cisplatin formulated into it. Although compared with cisplatin, lipoplatin possessed a shorter elimination half-life (11.0 h vs. 24.5 h), the plasma concentration (Cmax) was 1.5-fold higher than that from cisplatin, which warrants the anticancer effect of lipoplatin. In addition, the shorter half-life of lipoplatin brought better body clearance and lower toxicity than cisplatin. As the most effective liposomal platinum drug, lipoplatin is currently in phase III clinical trials (Table 2).308 Besides these polymer-based nano-delivery systems, inorganic nanoparticles are also extensively applied for platinum drug delivery. For example, layered double hydroxide (LDH) is a well-known biocompatible nanomaterial with high cancer selectivity and safety, and therefore it is widely used in drug delivery. Our group used LDH to co-load Pt(IV) prodrugs with other therapeutic agents to improve the anticancer effects and overcome the limitations of conventional platinum drugs. For example, we co-loaded a cisplatin-based Pt(IV) prodrug with an immunotherapy agent, indoleamine-2,3-dioxygenase inhibitor (IDOi), in the LDH nanoparticles (Fig. 32A).309 Compared with cisplatin, this nanosystem presented 17-fold increased cytotoxicity toward HeLa cells.

Table 2

Representative platinum drug nanodelivery systems in clinical trials.

Name

Loaded drug

Carrier

Benefits

Clinical trail phase

NC-6004

Cisplatin

Polymer

Phase III

AP5346

Oxaliplatin

Polymer

NC-4016

Oxaliplatin

Polymer

Lipoplatin

Cisplatin

Liposome

10 h circulation half-life, comparable cytotoxicity to cisplatin, lower neuro-, oto-, or nephrotoxicity 23 h circulation half-life, substantially better therapeutic index than oxaliplatin, higher tumor accumulation and DNA-binding level than oxaliplatin Excellent tolerability, comparable or superior anticancer activity than oxaliplatin 11 h circulation half-life, enhanced or similar efficacy to cisplatin, improved tumor selectivity, decreased toxicity

Phase II Phase I Phase III

834 Platinum anticancer drugs: Targeting and delivery

Fig. 32 Strategic overview of LDH nano delivery systems co-loaded platinum drug with (A) IDO inhibitor, (B) p53 activator, and (C) PDT agent. (A) The figure is obtained from Ref. Wang, N.; Wang, Z.; Xu, Z.; Chen, X.; Zhu, G., A Cisplatin-Loaded Immunochemotherapeutic Nanohybrid Bearing Immune Checkpoint Inhibitors for Enhanced Cervical Cancer Therapy. Angew. Chem. Int. Ed. 2018, 57 (13), 3426– 3430, copyright © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim; (B) The figure is obtained from Ref. Ma, R.; Wang, Y.; Yan, L.; Ma, L.; Wang, Z.; Chan, H. C.; Chiu, S.-K.; Chen, X.; Zhu, G., Efficient Co-Delivery of a Pt(iv) Prodrug and a p53 Activator to Enhance the Anticancer Activity of Cisplatin. Chem. Commun. 2015, 51(37), 7859–7862, with permission from the Royal Society of Chemistry; (C) The figure is obtained from Ref.Wang, Z.; Ma, R.; Yan, L.; Chen, X.; Zhu, G., Combined Chemotherapy and Photodynamic Therapy Using a Nanohybrid Based On Layered Double Hydroxides to Conquer Cisplatin Resistance. Chem. Commun. 2015, 51(58), 11587–11590, with permission from the Royal Society of Chemistry.

Platinum anticancer drugs: Targeting and delivery

835

Fig. 33 Strategic overview of self-assembled platinum drug nano delivery systems. (A) Scheme illustrating of PolyPt/Ru nanoparticles. (B) Schedule of Pt(IV)-ADD nanoparticles. (C) Schematic illustration of self-assembled lipid nanoparticles to co-deliver DSCP and XPF-targeted siRNA. (A) The figure is obtained from Ref. Zeng, X.; Wang, Y.; Han, J.; Sun, W.; Butt, H. J.; Liang, X. J.; Wu, S., Fighting Against Drug-Resistant Tumors Using a Dual-Responsive Pt(IV)/Ru(II) Bimetallic Polymer. Adv. Mater. 2020, 32(43), e2004766, copyright ©2020 Wiley-VCH GmbH. (B) The figure is obtained from Ref. Yang, C.; Tu, K.; Gao, H.; Zhang, L.; Sun, Y.; Yang, T.; Kong, L.; Ouyang, D.; Zhang, Z., The Novel Platinum(IV) Prodrug with Self-Assembly Property and Structure-Transformable Character Against Triple-Negative Breast Cancer. Biomaterials 2020, 232, 119751, copyright ©2019 Elsevier Ltd. (C) The figure is obtained from Ref. The figure is obtained from Ref. Li, C.; Li, T.; Huang, L.; Yang, M.; Zhu, G., Self-Assembled Lipid Nanoparticles for Ratiometric Codelivery of cisplatin and siRNA Targeting XPF to Combat Drug Resistance in Lung Cancer. Chem. Asian J. 2019, 14(9), 1570–1576, copyright ©2019 Wiley-VCH GmbH.

836

Platinum anticancer drugs: Targeting and delivery

Notably, in the presence of human peripheral blood mononuclear cells, the Pt(IV)-IDOi LDH nanoparticles successfully stimulated the proliferation of T cells, which can synergistically kill the cancer cells to further improve the therapeutic results. Besides, we also co-loaded Pt(IV) prodrugs with a p53 activator or a photosensitizer into LDH nanoparticles (Fig. 32B and C). The p53 activator can activate p53 to amplify the apoptosis signal induced by the platinum drug, and the photosensitizer can generate ROS to simultaneously damage cancer cells.310,311 As a result, the Pt-p53 activator and Pt-PDT agent systems presented up to 213- and 190-fold increased cytotoxicities compared with cisplatin, respectively, suggesting the great potential of using LDH nanoparticles for platinum drug delivery.

2.25.4.2

Platinum-self-assembled nano-systems

The main advantage of Pt(IV) complexes is their flexibility for further modification. Pt complexes can be conjugated with certain polymers (e.g., polyethylene glycol, PEG) and self-assemble into nanoparticles to improve their tumor targeting, delivery, and treatment effects. Wu et al. used a hydrophobic PEG to conjugate a Ru(II) anticancer agent with a cisplatin prodrug, and the obtained building block can self-assemble into nanoparticles (PolyPt/Ru, Fig. 33A).312 PolyPt/Ru effectively entered cisplatin-resistant cancer cells; upon red-light irradiation at 671 nm, this nanoparticle served as a PDT agent to generate singlet oxygen (1O2) and trigger the degradation of the polymer building block, releasing the Ru anticancer agent and the Pt(IV) moiety. Next, the Pt(IV) moiety was easily reduced to cisplatin in the intracellular reduction environment. Thus, the released cisplatin, the Ru agent, and the generated 1 O2 synergistically killed cancer cells and overcame cisplatin resistance. Compared with cisplatin, this dual-responsive PolyPt/Ru could induce more intense DNA damage to overcome cisplatin resistance, and it presented a more than 5-fold increased antitumor effect in vivo, suggesting the potential of this self-assembled nano-system. Zhang and co-workers conjugated a cisplatin prodrug with a chemosensitizer adjudin (ADD) at one axial position, and the other axial position was modified with alkyl chains with various lengths to obtain a class of Pt(IV) conjugates (Fig. 33B).313 In the presence of PEG, these conjugates can self-assemble into different nanoparticles with very high drug loading efficiency (84.0%–86.5%). The authors found that when the carbon number was 4 or 6, the obtained conjugates exhibited structure-transformable ability, and they could be self-assembled into a wire or sheet structure and presented significantly higher anticancer activity than their analogs. Notably, the obtained nanoparticles effectively eliminated triple-negative breast cancer (TNBC) cells with up to 266-fold increased cytotoxicity than cisplatin in vitro, whereas TNBC cells showed high resistance to cisplatin treatment. In a tumor-bearing mice model, these nanoparticles presented 2-fold enhanced antitumor efficiency compared with cisplatin, indicating their potential for further development. Our group recently developed a self-assembled nanosystem to deliver cisplatin and siRNA to combat cisplatin resistance (Fig. 33C).314 A cisplatin-based prodrug, disuccinatocisplatin (DSCP), and siRNA were self-assembled into nanoparticles in the presence of cationic lipids. The obtained nanoparticles effectively entered cancer cells to release the encapsulated DSCP and siRNA; the former could be easily reduced to cisplatin in the intracellular reduction environment to damage DNA, and the latter could target the endonuclease xeroderma pigmentosum group F (XPF), an important protein involved in DNA damage repair process in drug-resistant cells, to enhance the DNA damage and apoptosis induced by cisplatin. Consequently, this nanoparticle showed 85-fold increased cytotoxicity compared with DSCP itself and successfully inhibited the nucleotide excision repair (NER) pathway to overcome cisplatin resistance.

2.25.4.3

Platinum-conjugated nano-systems

Pt(IV) prodrugs possess two additional axial ligands, which can be utilized for conjugation with nanoparticles. After entering cancer cells, upon certain stimulation, Pt(IV) prodrugs can be reduced to release Pt(II) drugs to kill cancer cells. Compared with the platinum-incorporated system, which releases the platinum complexes by decomposition of the nanomaterials, the platinumconjugated systems mainly release platinum complexes through the reduction of Pt(IV) prodrugs upon certain stimuli in cancer cells. In this way, platinum-conjugated systems can not only reduce the leakage of cargoes during the circulation but also release platinum drugs more precisely in the cancer cells to further improve the anticancer efficiency and reduce the side effects. Lin and co-workers developed Fe3O4-based nanoparticles that contain siRNA to silence zeste homolog 2 (EZH2), an important gene for cisplatin resistance.315 The authors utilized LHRH peptide as the tumor-targeting group, and a cisplatin-based Pt(IV) prodrug was conjugated to the functionalized nanoparticles. The obtained platinum-loaded nanosystem could effectively accumulate in the LHRH receptor-rich cells (e.g., A2780 ovarian cancer cells) and presented a more than 3-fold increased cellular uptake level than that from cisplatin. After entering cancer cells, the Pt(IV) prodrug was reduced by the intracellular reducing agents to release cisplatin, and the nanoparticles could release siRNA to inhibit the expression of EZH2 to sensitize the cancer cells to cisplatin (Fig. 34A). Consequently, this Pt-nanosystem successfully overcame cisplatin resistance in vitro and exhibited a more than 2fold increased tumor inhibition effect compared with cisplatin in vivo, providing a promising strategy for effective tumor targeting and delivery of platinum drugs.

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837

Fig. 34 Strategic overview of platinum drug-conjugated nano delivery systems. (A) Folate receptor (FR)-mediated targeting and SWNT-Pt delivery system. (B) Strategic illustration of the RBCs-delivered and NIR light activatable Pt(IV) nanoprodrug. (A) The figure is obtained from ref. Yu, C.; Ding, B.; Zhang, X.; Deng, X.; Deng, K.; Cheng, Z.; Xing, B.; Jin, D.; Ma, P.; Lin, J., Targeted Iron Nanoparticles with Platinum(IV) Prodrugs and Anti-EZH2 siRNA Show Great Synergy in Combating Drug Resistance In Vitro and In Vivo. Biomaterials 2018, 155, 112–123, copyright ©2018 Elsevier Ltd. (B) The figure is obtained from ref. Wang, N.; Deng, Z.; Zhu, Q.; Zhao, J.; Xie, K.; Shi, P.; Wang, Z.; Chen, X.; Wang, F.; Shi, J.; Zhu, G., An ErythrocyteDelivered Photoactivatable Oxaliplatin Nanoprodrug for Enhanced Antitumor Efficacy and Immune Response. Chem. Sci. 2021, 12(43), 14353–14362 with permission from the Royal Society of Chemistry.

Recently, our group developed a red blood cell (RBC)-bound and NIR light-activatable Pt(IV) nanoprodrug (Fig. 34B).316 A red light-activable Pt(IV) prodrug was conjugated to the surface of upconverting nanocrystals (NCs), and then the conjugate was functionalized with a peptide that can bind to the surface of the RBCs, which could significantly improve the pharmacokinetic properties. Compared with the free Pt(IV) prodrug, the Pt(IV) nanoprodrug presented a 1149-fold increased circulation half-life and

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a 16.8-fold increased tumor accumulation level. After entering cancer cells, upon 808 nm NIR light irradiation, the nanoprodrug could rapidly release oxaliplatin to kill the cancer cells. Consequently, compared with the oxaliplatin-treated group, the tumor volume in the nanoprodrug-treated group decreased by more than 98.7% after irradiation; compared with the saline-treated group, a 7.2- and 14.5-fold increased T cell number was observed in the tumor tissue from treatment with free Pt(IV) prodrug and nanoprodrug, respectively, indicating that this nanosystem not only significantly improved the anticancer activity but also effectively evoked the immune system to strengthen the treatment outcome.

2.25.5

Conclusions and perspectives

Since the latest global approval of oxaliplatin in 2002, no platinum drugs have been approved within the past 20 years. Several platinum(IV) prodrugs have been tested in clinical trials, but no prodrug has been clinically approved. In general, the limited selectivity between cancer and normal cells, severe side effects, unsatisfactory antitumor effects, and drug resistance limit the further applications of platinum coordination complexes in clinics and clinical trials. Improving the targeting ability of platinum drugs and optimizing their delivery in vivo will increase the accumulation of platinum drugs in the target area to enhance the therapeutic effects and decrease their toxicity toward normal cells to reduce side effects. Therefore, various platinum complexes have been developed with the aim of improving the clinical performance of the original platinum drugs. To improve the tumor-targeting ability and delivery of platinum drugs in vivo, several cancer-targeting small molecules or peptides have been conjugated with platinum drugs. These cancer-targeting moieties increase the accumulation of platinum complexes in cancer cells via interactions with overexpressed acceptors on cancer cells. The enhanced cytotoxicity of platinum complexes was observed in vitro after conjugation; however, limited studies have been conducted to evaluate the tumor selectivity and antitumor effects of these cancer-targeted platinum complexes in vivo. To alleviate drug resistance, novel platinum complexes that can target certain intracellular organelles have been developed. These platinum complexes can specifically accumulate in the target area to improve the interaction between platinum drugs and target biomolecules, with an aim to improve the antitumor effects and overcome drug resistance. In most cases, however, these complexes do not address the poor selectivity of platinum complexes between cancer and normal cells, and studies on their in vivo anticancer performance are rare. With the rise of nanomaterials for biological applications, applying nanoparticles as delivery vehicles to optimize the in vivo pharmacokinetic properties and further improve the tumor accumulation of platinum complexes has become a promising strategy. The tumor accumulation level of platinum complexes could be significantly increased by the EPR effect and/or active targeting. Consequently, these platinum-nanosystems show dramatically improved antitumor effects than conventional platinum complexes in vivo. In conclusion, platinum drugs represent some of the most successful small-molecule drugs in the clinic and have dramatically increased the survival rates of cancer patients. Their therapeutic outcomes, however, are greatly limited by the poor selectivity, serious side effects, and drug resistance of cancer cells. Strategies to improve the targeting and delivery efficiency of approved platinum drugs can overcome these limitations. Although there are still deficiencies in the current strategies, developing novel approaches that can specifically deliver platinum complexes into cancer cells and accumulate the drugs in the targeted areas will maximize the anticancer efficacy of platinum complexes and hopefully expand their further applications in the clinic.

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W.; Cao, Q.; Shen, Y.; Zhao, Y.; Qin, P. Z.; Ji, L. N. A Platinum(II)-Based Photosensitive Tripod as an Effective Photodynamic Anticancer Agent Through DNA Damage. Chem. A Eur. J. 2017, 23 (65), 16442–16446. 276. Armstrong, J. S. Mitochondria: A Target for Cancer Therapy. Br. J. Pharmacol. 2006, 147 (3), 239–248. 277. Wang, K.; Zhu, C.; He, Y.; Zhang, Z.; Zhou, W.; Muhammad, N.; Guo, Y.; Wang, X.; Guo, Z. Restraining Cancer Cells by Dual Metabolic Inhibition with a MitochondrionTargeted Platinum(II) Complex. Angew. Chem. Int. Ed. 2019, 58 (14), 4638–4643. 278. Zhu, Z.; Wang, Z.; Zhang, C.; Wang, Y.; Zhang, H.; Gan, Z.; Guo, Z.; Wang, X. Mitochondrion-Targeted Platinum Complexes Suppressing Lung Cancer Through Multiple Pathways Involving Energy Metabolism. Chem. Sci. 2019, 10 (10), 3089–3095. 279. Fang, E. F.; Scheibye-Knudsen, M.; Chua, K. F.; Mattson, M. P.; Croteau, D. L.; Bohr, V. A. Nuclear DNA Damage Signalling to Mitochondria in Ageing. Nat. Rev. Mol. Cell Biol. 2016, 17 (5), 308–321. 280. Alexeyev, M.; Shokolenko, I.; Wilson, G.; LeDoux, S. The Maintenance of Mitochondrial DNA IntegritydCritical Analysis and Update. Cold Spring Harb. Perspect. Biol. 2013, 5 (5), a012641. 281. Van Houten, B.; Hunter, S. E.; Meyer, J. N. Mitochondrial DNA Damage Induced Autophagy, Cell Death, and Disease. Front. Biosci. (Landmark edn.) 2016, 21, 42. 282. Laberge, R.; Adler, D.; DeMaria, M.; Mechtouf, N.; Teachenor, R.; Cardin, G.; Desprez, P.; Campisi, J.; Rodier, F. Mitochondrial DNA Damage Induces Apoptosis in Senescent Cells. Cell Death Dis. 2013, 4 (7), e727. 283. Wisnovsky, S. P.; Wilson, J. J.; Radford, R. J.; Pereira, M. P.; Chan, M. R.; Laposa, R. R.; Lippard, S. J.; Kelley, S. O. Targeting Mitochondrial DNA with a Platinum-Based Anticancer Agent. Chem. Biol. 2013, 20 (11), 1323–1328. 284. Ouyang, C.; Chen, L.; Rees, T. W.; Chen, Y.; Liu, J.; Ji, L.; Long, J.; Chao, H. A Mitochondria-Targeting Hetero-Binuclear Ir(iii)-Pt(ii) Complex Induces Necrosis in CisplatinResistant Tumor Cells. Chem. Commun. 2018, 54 (49), 6268–6271. 285. Huang, C.; Li, T.; Liang, J.; Huang, H.; Zhang, P.; Banerjee, S. Recent Advances in Endoplasmic Reticulum Targeting Metal Complexes. Coord. Chem. Rev. 2020, 408, 213178. 286. Margiotta, N.; Denora, N.; Ostuni, R.; Laquintana, V.; Anderson, A.; Johnson, S. W.; Trapani, G.; Natile, G. Platinum(II) Complexes with Bioactive Carrier Ligands Having High Affinity for the Translocator Protein. J. Med. Chem. 2010, 53 (14), 5144–5154. 287. Pinato, O.; Musetti, C.; Sissi, C. Pt-Based Drugs: The Spotlight Will Be on Proteins. Metallomics 2014, 6 (3), 380–395. 288. Cunningham, R. M.; DeRose, V. J. Platinum Binds Proteins in the Endoplasmic Reticulum of S. cerevisiae and Induces Endoplasmic Reticulum Stress. ACS Chem. Biol. 2017, 12 (11), 2737–2745. 289. King, A. P.; Wilson, J. J. Endoplasmic Reticulum Stress: An Arising Target for Metal-Based Anticancer Agents. Chem. Soc. Rev. 2020, 49 (22), 8113–8136. 290. Wang, L.; Guan, R.; Xie, L.; Liao, X.; Xiong, K.; Rees, T. W.; Chen, Y.; Ji, L.; Chao, H. An ER-Targeting Iridium(III) Complex That Induces Immunogenic Cell Death in Non-SmallCell Lung Cancer. Angew. Chem. Int. Ed. 2021, 133 (9), 4707–4715. 291. Zou, T.; Lok, C.-N.; Fung, Y. M. E.; Che, C.-M. Luminescent Organoplatinum(ii) Complexes Containing bis(N-Heterocyclic Carbene) Ligands Selectively Target the Endoplasmic Reticulum and Induce Potent Photo-Toxicity. Chem. Commun. 2013, 49 (47), 5423–5425. 292. Tham, M. J. R.; Babak, M. V.; Ang, W. H. PlatinER: A Highly Potent Anticancer Platinum(II) Complex that Induces Endoplasmic Reticulum Stress Driven Immunogenic Cell Death. Angew. Chem. Int. Ed. 2020, 59 (43), 19070–19078. 293. Kepp, O.; Galluzzi, L.; Martins, I.; Schlemmer, F.; Adjemian, S.; Michaud, M.; Sukkurwala, A. Q.; Menger, L.; Zitvogel, L.; Kroemer, G. Molecular Determinants of Immunogenic Cell Death Elicited by Anticancer Chemotherapy. Cancer Metastasis Rev. 2011, 30 (1), 61–69. 294. Jhaveri, A.; Torchilin, V. Intracellular Delivery of Nanocarriers and Targeting to Subcellular Organelles. Expert Opin. Drug Deliv. 2016, 13 (1), 49–70.
295. Cesen, M. H.; Pegan, K.;
Spes, A.; Turk, B. Lysosomal Pathways to Cell Death and Their Therapeutic Applications. Exp. Cell Res. 2012, 318 (11), 1245–1251.

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296. Xue, X.; Qian, C.; Fang, H.; Liu, H. K.; Yuan, H.; Guo, Z.; Bai, Y.; He, W. Photoactivated Lysosomal Escape of a Monofunctional PtII Complex Pt-BDPA for Nucleus Access. Angew. Chem. Int. Ed. 2019, 58 (36), 12661–12666. 297. Mitchell, M. J.; Billingsley, M. M.; Haley, R. M.; Wechsler, M. E.; Peppas, N. A.; Langer, R. Engineering Precision Nanoparticles for Drug Delivery. Nat. Rev. Drug Discov. 2021, 20 (2), 101–124. 298. Deng, Z.; Wang, N.; Ai, F.; Wang, Z.; Zhu, G. Nanomaterial-Mediated Platinum Drug-Based Combinatorial Cancer Therapy. View 2021, 2 (1), 20200030. 299. Jia, C.; Deacon, G. B.; Zhang, Y.; Gao, C. Platinum(IV) Antitumor Complexes and their Nano-Drug Delivery. Coord. Chem. Rev. 2021, 429, 213640. 300. Xiao, H.; Li, C.; Dai, Y.; Cheng, Z.; Hou, Z.; Lin, J. Inorganic Nanocarriers for Platinum Drug Delivery. Mater. Today 2015, 18 (10), 554–564. 301. Xie, P.; Wang, Y.; Wei, D.; Zhang, L.; Zhang, B.; Xiao, H.; Song, H.; Mao, X. Nanoparticle-Based DRUG DELIVERY systems with Platinum Drugs for Overcoming Cancer Drug Resistance. J. Mater. Chem. B 2021, 9 (26), 5173–5194. 302. Manzari, M. T.; Shamay, Y.; Kiguchi, H.; Rosen, N.; Scaltriti, M.; Heller, D. A. Targeted Drug Delivery Strategies for Precision Medicines. Nat. Rev. Mater. 2021, 6 (4), 351–370. 303. Oberoi, H. S.; Nukolova, N. V.; Kabanov, A. V.; Bronich, T. K. Nanocarriers for Delivery of Platinum Anticancer Drugs. Adv. Drug Deliv. Rev. 2013, 65 (13–14), 1667–1685. 304. Nowotnik, D. P.; Cvitkovic, E. ProLindac™(AP5346): A Review of the Development of an HPMA DACH Platinum Polymer Therapeutic. Adv. Drug Deliv. Rev. 2009, 61 (13), 1214–1219. 305. Subbiah, V.; Grilley-Olson, J. E.; Combest, A. J.; Sharma, N.; Tran, R. H.; Bobe, I.; Osada, A.; Takahashi, K.; Balkissoon, J.; Camp, A. Phase Ib/II Trial of NC-6004 (Nanoparticle Cisplatin) Plus Gemcitabine in Patients with Advanced Solid Tumors. Clin. Cancer Res. 2018, 24 (1), 43–51. 306. Mochida, Y.; Cabral, H.; Kataoka, K. Polymeric Micelles for Targeted Tumor Therapy of platinum anticancer Drugs. Expert Opin. Drug Deliv. 2017, 14 (12), 1423–1438. 307. Boulikas, T. Clinical Overview on Lipoplatin™: A Successful Liposomal Formulation of Cisplatin. Expert Opin. Investig. Drugs 2009, 18 (8), 1197–1218. 308. Casagrande, N.; Celegato, M.; Borghese, C.; Mongiat, M.; Colombatti, A.; Aldinucci, D. Preclinical Activity of the Liposomal Cisplatin Lipoplatin in Ovarian Cancer. Clin. Cancer Res. 2014, 20 (21), 5496–5506. 309. Wang, N.; Wang, Z.; Xu, Z.; Chen, X.; Zhu, G. A Cisplatin-Loaded Immunochemotherapeutic Nanohybrid Bearing Immune Checkpoint Inhibitors for Enhanced Cervical Cancer Therapy. Angew. Chem. Int. Ed. 2018, 57 (13), 3426–3430. 310. Ma, R.; Wang, Y.; Yan, L.; Ma, L.; Wang, Z.; Chan, H. C.; Chiu, S.-K.; Chen, X.; Zhu, G. Efficient Co-Delivery of a Pt(iv) Prodrug and a p53 Activator to Enhance the Anticancer Activity of Cisplatin. Chem. Commun. 2015, 51 (37), 7859–7862. 311. Wang, Z.; Ma, R.; Yan, L.; Chen, X.; Zhu, G. Combined Chemotherapy and Photodynamic Therapy Using a Nanohybrid Based on Layered Double Hydroxides to Conquer Cisplatin Resistance. Chem. Commun. 2015, 51 (58), 11587–11590. 312. Zeng, X.; Wang, Y.; Han, J.; Sun, W.; Butt, H. J.; Liang, X. J.; Wu, S. Fighting Against Drug-Resistant Tumors Using a Dual-Responsive Pt(IV)/Ru(II) Bimetallic Polymer. Adv. Mater. 2020, 32 (43), e2004766. 313. Yang, C.; Tu, K.; Gao, H.; Zhang, L.; Sun, Y.; Yang, T.; Kong, L.; Ouyang, D.; Zhang, Z. The Novel Platinum(IV) Prodrug with Self-Assembly Property and StructureTransformable Character Against Triple-Negative Breast Cancer. Biomaterials 2020, 232, 119751. 314. Li, C.; Li, T.; Huang, L.; Yang, M.; Zhu, G. Self-Assembled Lipid Nanoparticles for Ratiometric Codelivery of cisplatin and siRNA Targeting XPF to Combat Drug Resistance in Lung Cancer. Chem. Asian J. 2019, 14 (9), 1570–1576. 315. Yu, C.; Ding, B.; Zhang, X.; Deng, X.; Deng, K.; Cheng, Z.; Xing, B.; Jin, D.; Ma, P.; Lin, J. Targeted Iron Nanoparticles with Platinum(IV) Prodrugs and Anti-EZH2 siRNA Show Great Synergy in Combating Drug Resistance In Vitro and In Vivo. Biomaterials 2018, 155, 112–123. 316. Wang, N.; Deng, Z.; Zhu, Q.; Zhao, J.; Xie, K.; Shi, P.; Wang, Z.; Chen, X.; Wang, F.; Shi, J.; Zhu, G. An Erythrocyte-Delivered Photoactivatable Oxaliplatin Nanoprodrug for Enhanced Antitumor Efficacy and Immune Response. Chem. Sci. 2021, 12 (43), 14353–14362.

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Ka-Chung Tonga, Pui-Ki Wana, Di Hua, Chun-Nam Loka, and Chi-Ming Chea,b, a Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong, China; and b Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Hong Kong, China © 2023 Elsevier Ltd. All rights reserved.

2.26.1 2.26.2 2.26.3 2.26.3.1 2.26.3.1.1 2.26.3.2 2.26.3.3 2.26.3.4 2.26.3.5 2.26.4 2.26.4.1 2.26.4.2 2.26.4.3 2.26.4.4 2.26.4.5 2.26.4.6 2.26.5 2.26.5.1 2.26.5.2 2.26.6 Acknowledgment References

Introduction Anti-arthritic gold(I) drugs with anti-cancer activities Anti-cancer gold(I) complexes Gold(I)-phosphine complexes Coordination of N-heterocyclic carbene ligand(s) Gold(I)-NHC complexes Gold(I)-thiourea complexes Gold(I)-alkynyl complexes Gold(I)-dithiocarbamate complexes Anti-cancer gold(III) complexes Gold(III) porphyrins Pincer-type gold(III) complexes Gold(III) complexes with the coordination of various p-conjugated aromatic ligands Bidentate N^ N-type gold(III) complexes Bidentate C^N-type gold(III) complexes Gold(III)-dithiocarbamate complexes Formulations of gold complexes with improved anti-cancer potency Gold(I) complexes Gold(III) complexes Conclusion

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Abstract Gold is a precious metal that has been used for medicinal purposes as far back as ancient times. For instance, the gold compound, auranofin, was used as anti-rheumatic agent to treat rheumatoid arthritis. Over the past few decades, there has been a surge of interest in the development of gold(I) and gold(III) complexes as potential chemotherapeutics. Some of these novel complexes have been shown to be stable under physiological conditions, overcome cisplatin resistance, exhibit a broad range of cancer cell killing properties, and display effective anti-tumor activity through distinct modes of action. In this book chapter, we summarize the state-of-the-art in research on the development of gold(I) and gold(III) complexes, including the anti-cancer properties of these complexes against different cancers, possible mechanisms of action, and the identification of the engaged molecular targets. We also describe some formulation strategies that have been adopted for the delivery of gold complexes with an intention to modulate anti-tumor efficacy and ameliorate systemic toxicity.

2.26.1

Introduction

The medicinal use of gold against diseases has been recognized since ancient times. Starting in the early 20th century, the discovery of anti-arthritic properties of gold(I) complexes (sodium gold(I) thiopropanol-sulfonate; Allochrysine) led to the development of clinically useful gold(I)–thiolate drugs, such as sodium aurothiomalate (Myochrysine) and the acetylated glucose derivative of the gold(I)–phosphine complex (Auranofin) for the treatment of rheumatoid arthritis (RA). With the success of gold therapy in treating RA, gold complexes have been further examined for anti-cancer applications due to their ability to inhibit cancer cell proliferation. The serendipitous discovery of the therapeutic potential of cisplatin has led to cisplatin-based chemotherapy for the treatment of various types of cancer. Extensive studies on the mechanisms of action have demonstrated that cisplatin covalently interacts with DNA, activates the DNA damage response, inhibits DNA repair mechanisms, ultimately resulting in apoptosis. However, the drawbacks of dose-limiting toxicity and chemoresistance to cisplatin remain challenging in clinical practice, driving the development of novel metal-based therapeutic candidates with alternative mechanisms of action. Numerous gold(I) and gold(III) complexes have been reported to be effective against a wide range of cancers including multidrug resistant cancers. However, the facile reduction of gold(III) ion into gold(I) or gold(0) through redox reactions under

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physiological conditions hampers the therapeutic applicability of gold(III) compounds. With a judicious choice of strong donor ligand(s), such as porphyrins, pincer-type and bidentate cyclometalated ligands with mixed C and N donor atoms, N-heterocyclic carbene (NHC), dithiocarbamate, thiourea, among others, complexes of gold(III) or gold(I) have been found to display decent to good physiological stability and remain active against different types of cancer. Distinct from the cisplatin-based chemotherapeutic agents that target DNA via non-repairable interactions, the unique structural scaffolds and reactivity allow the gold complexes to interact with protein target(s) relevant to cancer cell survival and proliferation. Such biomolecular interactions can result in functional inhibition, imbalance of redox homeostasis, and/or activation of stress response signalling pathways, hence leading to cancer cell death. With the aid of state-of-art chemoproteomic, multi-omics, and bioinformatics approaches in the identification of the molecular targets of anti-cancer gold complexes, work from over the last decades, particularly from structure-activity relationships, can aid in the structural optimization of potential lead compounds to achieve selective targeting to cancer cells. Additionally, chemical modification or formulation of the gold complexes with tumor-targeting properties are envisaged to improve anti-tumor efficacy and selectivity as well as minimize systemic side effects.

2.26.2

Anti-arthritic gold(I) drugs with anti-cancer activities

Starting in the 1970s, the anti-arthritic gold(I) complexes (Fig. 1) were re-examined for anti-cancer properties. Auranofin had been found to inhibit cancer cell growth in vitro and effectively increase the lifespan of mice inoculated with lymphocytic leukemia P388 cells.1 Mechanistic studies revealed that treatment with auranofin inhibits the redox function of thioredoxin reductase through cysteine modification,2 inhibits proteasome-associated deubiquitinase activity, induces the production of intracellular reactive oxygen species (ROS), and activates p38 mitogen-activated protein kinase (MAPK), all of which are associated with auranofininduced apoptosis and tumor growth inhibition.3–5 Nonetheless, auranofin was observed to be inactive in vivo when administered intravenously,6 which was attributed to the rapid covalent binding of the gold ion to Cys-34 of serum albumin via ligand exchange reaction, thus preventing the active gold species from reaching the tumor target site. The other anti-arthritic gold(I) drugs, aurothiomalate and aurothioglucose, can inhibit tumor growth and extend the survival of mice inoculated with syngenic cancer cells.7 These gold complexes block the interaction of protein kinase C with its downstream Par6 scaffold protein to elicit a cytostatic effect in cancer cells.8

2.26.3

Anti-cancer gold(I) complexes

2.26.3.1

Gold(I)-phosphine complexes

Gold(I)-phosphine-based, anti-cancer agents have been extensively studied for over two decades.9 Among these compounds, twocoordinated gold(I) complexes bearing phosphine ligand(s), as exemplified by auranofin, have been the most rigorously studied. Mirabelli and co-workers performed a structure–activity relationship study on these types of compounds revealing the influence of both the phosphine and thiolate ligands on anti-cancer activity.10 Barrios and co-workers demonstrated that the steric variation of phosphine ligands can not only markedly influence the cellular uptake process and biodistribution of auranofin analogs, but also affect the binding affinity of these complexes to target proteins and their inhibition of enzymes, such as the cathepsin family of lysosomal cysteine proteases.11 In addition to the linear coordination structures, a number of tetrahedral bis-chelated gold(I)–phosphine complexes have also been reported to display potent anti-cancer properties. Sadler and Berners-Price et al. first reported the anti-tumor activity of [Au(dppe)2]þ (1) (dppe ¼ bis(diphenylphosphino)ethane).12 [Au(dppe)2]þ is inert in the presence of the biological reductant, glutathione, under physiological conditions. This complex exhibited significant inhibition of tumor growth in different mouse models, including leukemia and solid tumors. However, preclinical toxicological studies identified an associated severe toxicity

O Na

Au

OH

ONa

NaO

SO3

S

S

n

O

Au

Allochrysine

n

Aurothiomalate

OH

OAc AcO AcO

O

S Au P

OAc Auranofin

Fig. 1

Anti-arthritic gold(I) drugs.

HO HO

O

S Au

OH

Aurothioglucose

n

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849

to heart, liver and lung in dogs and rabbits, which was attributed to the complex’s high lipophilicity and stability, thereby resulting in a non-selective accumulation of gold species in mitochondria and subsequent mitochondrial dysfunction.13 Later studies focused on developing anti-cancer gold(I)–phosphine complexes with decreased lipophilicity and higher selectivity against cancer cells over normal cells. For instance, by replacing the phenyl substituents with pyridyl groups, a panel of structural analogs of [Au(dppe)2]þ with lipophilicity parameters spanning a wide range was synthesized. Complex [Au(d2pype)2]þ (2) (d2pype ¼ 1,2-bis(di-2pyridylphosphino)ethane), containing an ethyl-bridged 2-pyridyl phosphine ligand, displayed intermediate lipophilicity and promising anti-tumor activity. The lipophilic/hydrophilic balance was further fine-tuned by using the propyl-bridged 2-pyridyl phosphine ligand (d2pypp).14 Complex [Au(d2pypp)2]þ (3) displayed selective cytotoxicity against breast cancer cells, but negligible toxicity against normal breast cells.15 Additional four-coordinated gold(I) complexes containing phosphine ligands with promising anti-cancer activity were developed by Koide and co-workers.16 Complex [Au[P(CH2OH)3]4]þ (4) is cytotoxic against various cancer cell lines and significantly prolonged the survival of mice inoculated with Meth/A sarcoma cells. Some gold(I)–phosphine complexes, such as [Au(PPh3) Cl] (5), [Au2(dppe)(Cl)2] (6), and [Au3(dpmp)(Cl)3] (7), have been reported to induce autophagy,17 which was reported in association with cell death.18 Interestingly, Contel and co-workers reported the synthesis, characterization and stability studies of heterometallic titanocene-gold(I) phosphine complexes [(h-C5H5)2TiMe(m-mba)Au(PR3)] (wherein mba ¼ S–C6H4–COO) as potential chemotherapeutics for renal cancer.19 Compound 8 is stable in physiological media, highly cytotoxic against human renal cancer cell line, and exhibits effective in vivo anti-tumor activity with an average tumor size reduction of 67% in Caki-1 renal cancer xenograft-bearing mice after a 28-day treatment.

2.26.3.1.1

Coordination of N-heterocyclic carbene ligand(s)

Over the past decades, the coordination of NHC ligands in transition metal complexes has stimulated tremendous interest in the design of potent chemotherapeutic agents based on metal–NHC complexes.20–24 In contrast to the toxic phosphines used in earlier studies, NHCs are stable with strong s-donor strength, which can stabilize gold complexes against demetalation, reduction and ligand exchange under physiological conditions.25 Imidazol-2-ylidene is one of the most extensively studied NHCs. The sp2-hybridized carbene carbon atom (C2 carbon) with adjacent nitrogen atoms display both p-donating and s-inductive effects by donating pelectrons to the empty p-orbital (LUMO) of the C2 carbon atom, while the s-electron withdrawing property of the N atoms further lowers the HOMO energy. The partial aromaticity of the heterocyclic rings is also beneficial to the stability of NHC ligands.26

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The versatility in modification of N-substituents of NHC ligands provides diverse scaffolds for biomolecular targeting and biological activity. The introduction of functional groups, such as fluorophores, cancer-targeting groups and/or affinity groups, can influence the reactivity and binding interactions of the metal complexes with biomolecules.27 Taking the advantages of NHC ligand, a number of metal–NHC complexes of Au(I) and Au(III) with decent stability under physiological conditions have been developed and found to exhibit effective anti-cancer properties, with mechanisms of action distinct from that of cisplatin.

2.26.3.2

Gold(I)-NHC complexes

Gold(I)–NHCs are usually stable under ambient and physiological conditions. The unique structural scaffolds of gold(I)–NHCs may enable specific biomolecular interactions with potential druggable targets.28–30 With facile modification of the imidazolium ring and versatile N-substituents, a number of anti-cancer gold(I)–NHC complexes with tunable lipophilicity, reactivity, and therapeutic and diagnostic properties have been reported and extensively studied.25 Berners-Price and co-workers first reported the anti-cancer properties of a panel of cationic mononuclear [Au(NHC)2]þ (9) and dinuclear [Au2(bisNHC)2]2þ (10 and 11) complexes.31,32 The mononuclear [Au(NHC)2]þ complexes bear different N-substituents and exhibit a wide range of lipophilicity. Some of these complexes display higher cytotoxicity in breast cancer cells than in normal breast cells. [Au(NHC)2]þ complexes are lipophilic cations. Mechanistic studies have revealed that [Au(NHC)2]þ complexes can accumulate in the mitochondria, trigger Ca2þ sensitive mitochondrial swelling, ultimately leading to mitochondria-dependent apoptosis. [Au(NHC)2]þ complexes were proposed to selectively inhibit TrxR activity through a two-step reaction mechanism involving the successive substitution of the two NHC ligands to form either [Au(Cys)2]– or [Au(Sec)2]–, with the formation rate of the latter being much faster.20 The thiol reactivity of gold(I)–NHCs is influenced by the N-substituted groups, where gold(I)– NHCs with bulkier groups have the gold(I) center shielded from thiol attack.

Ott and co-workers described a series of gold(I)–NHC chloride complexes, [Au(NHC)Cl] (12) bearing benzimidazole-derived NHC ligands. The mechanisms of anti-cancer action of these complexes are attributable, at least in part, to anti-mitochondrial effects and TrxR inhibition.33 The binding of gold(I)–NHC to cysteine and selenocysteine residues has recently been characterized.34 Replacing the anionic chloride ligand with other neutral ligands, such as NHC or PPh3, can afford cationic [Au(NHC)L]þ species.35 Compared with the neutral [Au(NHC)Cl], the cationic complexes 13 and 14 displayed decreased inhibition of TrxR activity due to a lower thiol and/or selenol reactivity. However, the cationic gold species exhibited increased cellular uptake and greater accumulation in mitochondria, as well as enhanced anti-mitochondrial activity.

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Functionalization of NHC ligand with a DNA intercalating naphthalimide (Nap) moiety instills the complex [Au(NHCNap)Cl] (15) with an enhanced DNA intercalative binding ability, effective inhibition of thioredoxin reductase and anti-cancer activity.36 Appending structures derived from bioactive natural products to the NHC ligands can improve the biomolecular targeting of gold(I)–NHC complexes. For instance, Casini and co-workers reported the use of the aromatic caffeine ligand to prepare dicarbene gold(I) complex [Au(9-methylcaffeine-8-ylidene)2]þ (16) as a selective G-quadruplex binder that targets and stabilizes the telomeric G4 (Tel23) secondary structure through p–p stacking and possibly electrostatic interactions as illustrated by electrospray ionization mass spectrometry, X-ray diffraction and metadynamics simulations approaches.37,38 The complex displayed selective cytotoxicity against ovarian cancer cell lines and lower toxicity in non-tumorigenic cells.39 As supported by proteomics studies, cancer cells treated with 16 triggered protein regulation related to stress-induced transcription and telomere function. The anti-cancer activity of a xanthine-derived alkynyl phosphine gold(I) complex [Au(PMe3)(alkynyl)] (17) in colorectal cancer was also demonstrated by Ott and co-workers to be associated with the induction of cytotoxicity, aneuploidy with chromosomal instability, and impairment of glycolysis, mitochondrial respiration, and thioredoxin reductase activity.40

Taking the combined coordination of a bridging bis(NHC) and a diphosphine ligands, Che and co-workers reported a dinuclear gold(I) complex [Au2(dcpm)(bisNHC2C4)]2þ (dcpm ¼ bis(dicyclohexylphosphino)methane) (18) displaying appropriate chemical stability and reactivity toward cysteine thiols.41 Complex 18 was found to be moderately stable in the presence of blood thiols and serum albumin, but reactive through coordination with the cysteine thiol and selenocysteine selenol of thioredoxin reductase accompanied by the release of the bis(NHC) ligand. The resultant thiol and/or selenol coordination(s), as well as the tight-binding mode of inhibition, with an IC50 value of 38 nM, are distinct from the action of auranofin under the same experimental conditions.41 Treatment of HeLa cervical cancer cells with this dinuclear gold(I) complex elicited an inhibition of selfrenewal (sphere-forming) ability of cancer stem-like cells and showed significant suppression (81%) of tumor growth in a mouse model bearing cervical cancer xenografts after 9 days of treatment. In addition, no death or body weight loss was observed in the mice during the three-times-weekly administration of 18. Immunohistochemical analysis of the angiogenesis marker, CD31, in the tumor tissues revealed diminished staining in the group of mice treated with 18, implying an inhibition of tumor angiogenesis.

Arambula and co-workers described a panel of redox-active gold(I) bis-NHC complexes containing quinone or ferrocene moieties (19 and 20) that exhibit anti-cancer action by targeting the cancer anti-oxidant network.42,43 The 1,4-naphthoquinone or ferrocene-appended NHC ligands in gold(I) complexes behave as redox modulators to potentiate intracellular oxidants, presumably undergoing ligand exchange reactions with selenium-containing thioredoxin reductase, inhibiting its activity to increase cellular ROS, resulting in activation of the endoplasmic reticulum (ER) stress response pathway, as evidenced by RNA microarray gene expression assays. In the literature, the ROS-mediated ER stress response in cancer cells has been reported to trigger immunogenic cell death (ICD). In colon carcinoma, the redox-active quinone fused gold(I) complex, 21, elicited characteristic signatures of ICD, including relocation of the ER-resident chaperone protein calreticulin, secretion of adenosine triphosphate (ATP), and

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Anti-cancer gold compounds

extracellular release of high mobility group box 1 protein (HMGB1) from apoptotic cells, all of which indicating the gold(I)–NHC complex as effective inducer of ICD.44

Richeter and co-workers reported the preparation of porphyrin conjugate with a peripheral thiolato gold(I) complex (22) as photosensitizer for photodynamic therapy.45 Following the modification of the thiolate ligand with a cancer-targeting mannose unit, complex 22 not only displayed improved solubility in water but, more importantly, enhanced cytotoxicity relative to the unconjugated compounds in human breast cancer cells (MCF-7) that over-expressed mannose receptors, upon UV light irradiation. The elevated intracellular ROS (e.g., 1O2) levels after irradiation were proposed to oxidize the sulfur atom of the coordinated thiolate ligand, resulting in AueS bond cleavage, as supported by mass spectrometric analyses, two-dimensional DOSY NMR experiments, and density functional theory (DFT) calculations.

By integrating the anti-cancer properties of gold(I)–NHC complexes with an aggregation-induced emission (AIE) luminogen, Tang and co-workers described a panel of gold(I)–NHC compounds able to achieve theranostic outcomes against cancer.46 The introduction of the bulky tetraphenylethene to the NHC framework endows the complexes with light-up luminescence for bioimaging of cancer cells, and enhanced bimolecular binding and inhibition of thioredoxin reductase in cervical cancer cells. By investigating the electronic and steric variations of other coordinated ligands (triphenylphosphine, NHC, acetylide and labile chlorido ligands), the gold(I) complexes with a positive charge exhibited targeting specificity to cancer cells and negligible toxicity to normal cells, resulting in effective inhibition of TrxR, which is highly expressed in cancer cells, impairment of cellular redox homeostasis under elevated oxidative stress, and apoptosis. In particular, complex 23, with triphenylphosphine, was shown to be an effective radiosensitizer to boost cytotoxicity to neoplastic cells upon X-ray irradiation, in comparison to treatment with the compound alone. This investigation led to further optimization and the design of luminescent gold(I)–NHC complexes for in vivo imaging and therapy.

Anti-cancer gold compounds

853

Transition metal-mediated bioorthogonal activation in living cells via spatial and temporal control is a feasible approach for the conversion of inactive prodrugs into active forms for therapeutic action. With the aid of a palladium(II)-triggered transmetallation reaction, Zou and co-workers recently described the novel bioorthogonal activation of a NHC–gold(I)–phenylacetylide complex (24) into active gold(I) species inside living cells for anti-cancer activity.47 Prior to being activated by a palladium(II) salt, complex 24 remains inactive and displays good stability against physiological thiols. The introduction of Pd(II) salts allows the efficient transfer of the phenylacetylide ligand to the Pd(II) center and results in the in situ formation of thiol-reactive gold(I)–NHC species that exhibits enhanced suppression of the activity of thioredoxin reductase, inhibits cancer cell proliferation, and decreases angiogenesis in zebrafish models.

2.26.3.3

Gold(I)-thiourea complexes

Thiourea and the similar thiosemicarbazone with N,N 0 -disubstituted ligands have been employed to stabilize the gold(I) ion. The gold(I)–thiourea complex, [Au(TU)2]þ (25), can inhibit the activity of thioredoxin reductase via gold(I) coordination with the active sites’ cysteine thiol/selenocysteine selenol.48 The tight-binding inhibition results in an inhibitory constant of cellular TrxR activity in the nanomolar regime. Complex 25 is cytotoxic to different human cancer cells (nonsmall cell lung, cervical, nasopharyngeal and hepatocellular carcinoma) with IC50 values in the low micromolar range (3.7–17.4 mM). A further in vivo anti-tumor study revealed that nude mice bearing non-small cell lung cancer xenografts receiving twice-weekly intraperitoneal injections of 25 (100 mg/kg) for 28 days exhibited a 38% reduction in tumor size with no weight loss or mortality.

2.26.3.4

Gold(I)-alkynyl complexes

A series of [Au(PPh3)(alkynyl)] complexes was reported to exhibit anti-cancer properties. These complexes, such as 26, can selectively inhibit TrxR activity with little inhibition of the structurally related enzyme, glutathione reductase.49 Treatment of MCF-7 breast cancer cells with complex 26 was observed to affect tumor cell metabolism and mitochondrial respiration. Complexes 26 significantly inhibited the formation of blood vessels in a zebrafish embryo model. A dinuclear gold(I) species, [Au2(PPh3)2(bis-alkynyl)], with a diethynylfluorene linkage (27), is cytotoxic to different cancer cell lines, with IC50 values at low micromolar levels.50 Interestingly, Hep3B hepatocellular carcinoma-bearing mice receiving intraperitoneal administration of 27 exhibited significant reductions in tumor size with limited adverse effects on vital organs, such as the liver and kidney.

854

2.26.3.5

Anti-cancer gold compounds

Gold(I)-dithiocarbamate complexes

Altuwaijri and co-workers presented a series of mononuclear and dinuclear gold(I) complexes containing mixed phosphine and dialkyl-diaryl dithiocarbamate ligands (28 and 29). Compared with cisplatin, all these gold(I) complexes exhibited 5–13 times greater in vitro cytotoxicity in IC50 values toward HeLa, HCT15 and A549 cancer cells.51 Tiekink and co-workers described the preparation of three triorganophosphine gold(I) dithiocarbamato complexes [Au(PR3)S2CN(iPr)CH2CH2OH] (where R ¼ phenyl, cyclohexyl and ethyl) (30) and their anti-cancer cytotoxicity to doxorubicin-resistant breast cancer cells (MCF-7R).52 Among these complexes, the phenyl-substituted gold(I) complex was shown to be the most potent and more effective than doxorubicin or cisplatin. Mechanistic studies demonstrated that the phenyl-substituted gold(I) dithiocarbamato complex can induce apoptosis, activate the tumor suppressor p53 gene, and inhibit the action of topoisomerase I. Recently, a panel of phosphine gold(I) dithiocarbomato complexes, [Au(PR3)S2CNR’2] (where R ¼ methyl, ethyl, isopropyl and R’ ¼ methyl, ethyl) (31), has been developed and their cytotoxicity to human A549 lung and HepG2 liver cancer cell lines was examined.53 Molecular docking results showed that these gold(I) complexes bind to the adenine–thymine residues in the minor groove of DNA, inducing the structural distortion of the DNA double helix.

2.26.4

Anti-cancer gold(III) complexes

2.26.4.1

Gold(III) porphyrins

Che and co-workers established a gold(III) porphyrin system, as exemplified by a gold(III) meso-tetraphenylporphyrin complex denoted [Au(TPP)]þ (32), that exhibits promising anti-cancer properties with potential for clinical applications. The use of a porphyrin ligand can stabilize the electrophilic gold(III) ion against demetalation under physiological conditions and reduction by the biological reductant glutathione.54 Extensive studies revealed a marked anti-cancer potency of gold(III) porphyrin complexes at low micromolar or even nanomolar levels against a broad spectrum of cancer cell lines, including ovarian, breast, colorectal, lung, neuroblastoma, melanoma, and nasopharyngeal cancers.55–58 In addition, in vivo studies have demonstrated a promising tumor growth inhibitory activity of the gold(III) porphyrins in different mouse models of human cancer.59–65 Compared to the clinically used cisplatin, gold(III) porphyrins display high cytotoxicity, with IC50 values significantly lower than those of cisplatin, and show comparable cytotoxicity against both cisplatin-sensitive and -resistant cancer cells.64 Moreover, complex 32 was reported to elicit a blockade of the self-renewal of cancer stem-like cells,66 inhibition of angiogenesis in vitro and in vivo,63 suppression of cancer cell migration, invasion, and metastasis, and prolongation of survival in nasopharyngeal carcinoma metastasis-bearing mice.62

Anti-cancer gold compounds

855

The lack of cross-resistance to cisplatin suggests that the gold(III) porphyrin exerts different anti-cancer mechanisms of action from cisplatin. Based on various biochemical analyses, transcriptomics, and proteomics, several lines of evidence have revealed that 32 targets mitochondria, inducing a rapid depletion of mitochondrial transmembrane potential, and subsequent caspase-dependent and -independent apoptotic pathways.67 Treatment of cancer cells with 32 triggers cellular oxidative stress and shifts the balance between pro-apoptotic and anti-apoptotic proteins. Additionally, 32 treatment results in cell cycle arrest at the G0/G1 phase, p38 MAPK activation and inhibition of thioredoxin reductase activity.55,60 To obtain deeper insight into the mechanisms of action of gold(III) porphyrins, identification of the direct molecular target of 32, with the aid of a chemoproteomic approach using a clickable photoaffinity probe of gold(III) porphyrin containing a benzophenone moiety (33), was undertaken.68 Using this strategy, a mitochondrial chaperone, heat shock protein 60 (Hsp60), was identified as one of the engaged molecular targets of 32. The proposed non-covalent biomolecular interactions between [Au(TPP)]þ and Hsp60, as well as the associated inhibitory activity, has also been validated by various binding studies, including cellular thermal shift assay, protein fluorescence quenching, saturation-transfer difference NMR, and chaperone activity assay. Structure-activity relationship studies based on the analogous gold(III) and platinum(II) complexes revealed that the gold(III) ion center with monocationic charge character and the planar porphyrin scaffold of gold(III) porphyrins, play crucial roles in the inhibition of the chaperone activity of Hsp60.

With meso-unsubstituted porphyrin ligand, a previously unknown thiol reactivity of gold(III) porphyrins harboring reactive and sterically accessible meso-carbons atoms was reported as a new modality for cysteine targeting by anti-cancer gold(III) complexes.69 In the literature, the coordination of cysteine thiol with metal complexes usually occurs through M–S bond formation. The gold(III) mesoporphyrin IX dimethyl ester [Au(MesoIX)]þ (34) is unique in that the reactivity of the peripheral meso carbon atoms of the porphyrin ligand is activated by the gold(III) ion due to its inductive effect. The increased electrophilicity of the porphyrin ring of 34 is susceptible to nucleophilic aromatic substitution with the cysteine thiol of amino acids, peptides, and cancer-associated proteins such as thioredoxin. The cellular thiol-targeting property of 34 was demonstrated by nanoscale secondary ion mass spectrometry showing the tight association of the gold metal from 34 with the sulfur-rich cellular proteins in cellulo. Additionally, the combined approach of thermal proteome profiling, cellular thermal shift and independent biochemical assays identified two cellular thiol proteins, peroxiredoxin III and deubiquitinase (ubiquitin carboxyl-terminal hydrolase isozyme L3), involved in the cellular target engagement of 34, resulting in oxidative stress-mediated cytotoxicity and the accumulation of polyubiquitinated proteins. Importantly, 34, by virtue of its quasiphysiological mesoporphyrin IX ligand, displayed effective anti-tumor activity in two independent tumor xenograft mouse models as well as favorable metabolism, biodistribution and clearance.

856 2.26.4.2

Anti-cancer gold compounds Pincer-type gold(III) complexes

Apart from tetradentate porphyrins, the electrophilic gold(III) ion can also be stabilized by pincer-type ligands containing deprotonated C-donor atom(s). Che and co-workers reported the synthesis and anti-cancer activities of a series of cyclometalated gold(III) complexes bearing a dianionic tridentate C-deprotonated C ^N ^C ligand [Aum(C ^N ^C)mL]n þ (wherein m ¼ 1–3; n ¼ 0–3; H2C ^N ^C ¼ 2,6-diphenylpyridine).70 These gold(III)–pincer complexes exhibit good stability against reduction or demetalation under physiological conditions, and the complexes’ mechanisms of action and anti-cancer potency can be modulated by varying the auxiliary ligand L. For example, the pincer ([Au (C^N^C)(PPh3)]þ complex (35) is cytotoxic against both cisplatin-sensitive and -resistant nasopharyngeal carcinoma. With a bridging bis(diphenylphosphino)propane (m-dppp) ligand, the dinuclear gold(III) complex [Au2(C^N^C)2(m-dppp)]2þ (36) displayed enhanced cytotoxicity to a panel of cancer cell lines, particularly on cervical and hepatocellular carcinoma, with IC50 values down to the submicromolar range.71 Importantly, complex 36 significantly suppressed tumor growth in nude mice bearing primary liver cancer xenografts, eliciting a 77% reduction in tumor size after 4 weeks of treatment, and in rats bearing hepatocellular carcinoma orthografts after 14 days of treatment. In addition, no significant body weight loss, mortality, or any other adverse side effects were observed in the mice treated with 36. The survival of tumor-bearing rats was also prolonged upon treatment with 36. Mechanistic studies with the aid of gene expression profiling, connectivity map analysis, and biochemical experiments, indicated that induction of ER stress and inhibition of thioredoxin reductase activity are associated with the anti-cancer action of this dinuclear gold(III) phosphine complex.

Besides, Che and co-workers described the use of a strong s-donating NHC as an auxiliary ligand to prepare a panel of [Au(C^N^C)(NHC)]þ complexes with good physiological stability and promising anti-cancer properties. These compounds exhibited in vitro cytotoxicity against a broad spectrum of human cancer cell lines with IC50 values at the low micromolar level. In particular, complex 37, with a NHC ligand bearing two N-methyl substituents, exhibited robust cytotoxic selectivity, with a 167-fold lower IC50 value in non-small cell lung cancer cells compared to normal lung fibroblasts.72 Mechanistic studies revealed that complex 37 interacts with DNA through intercalation and induces cell death presumably through inhibition of TopoI-mediated DNA relaxation. In vivo anti-tumor study revealed that nude mice bearing primary liver cancer (PLC) tumor xenografts, receiving intraperitoneal administration of 37 at 10 mg/kg twice weekly for 28 days, displayed significant suppression of tumor growth, with no observable mortality or weight loss. By varying the N-methyl substituents on NHC into N-butyl chains (38), a significant disintegration of cellular spheroids derived from human HeLa cervical cancer cells was realized, with a 70% growth suppression after 72 h of treatment and effective in vivo anti-tumor activity in two-independent xenograft mouse models of cervical and lung carcinoma.73 Through a photo-affinity labelling-based chemoproteomics approach, multiple engaged molecular targets associated with anti-cancer functions were identified and verified, using two clickable probes of a pincer gold(III)–NHC scaffold equipped with a photoaffinity diazirine (39) or benzophenone moiety (40). Following UV-light activated covalent crosslinking between the gold(III)–NHC-based probe and potential targets, the conjugates can be labelled with an azido-containing reporter (fluorescent cyanine) via a copper(I)catalyzed click reaction followed by visualization after two-dimensional gel electrophoretic separation. Based on the MALDI-TOF tandem mass spectrometric analysis, the proteins labelled (biotinylated) with the diazirine-based probe (39) included mitochondrial heat shock protein 60 (Hsp60), nucleoside diphosphate kinase A (NDKA), vimentin (VIM), nucleophosmin, peroxiredoxin I (PRDX1) and nuclease-sensitive element binding protein (Y box binding protein, YB-1), all of which are plausible anti-cancer targets. For the benzophenone-based probe (40), four of these proteins were labelled and identified under the same experimental conditions.

Anti-cancer gold compounds

857

With the judicious choice of strongly fluorescent pincer ligands, Che and co-workers reported a panel of gold(III)–NHC complexes containing N^N^N (2,6-bis(imidazol-2-yl)pyridine (41) or 2,6-bis(benzimidazol-2-yl)pyridine) (42) ligands that can exhibit switchable fluorescent property in response to thiols in biological systems.74 Upon coordination with the gold(III) ion, the low energy 5dx2–y2 orbital of Au(III) renders the complexes non-emissive in solution. However, in the presence of physiological thiols or in cellulo, reduction of the gold(III) to gold(I) species, accompanied by the release of the strongly fluorescent N^N^N ligand was observed. The resultant gold(I) species, stabilized by the coordinated NHC ligand(s), elicited an effective suppression of tumor growth in a mouse model.

The gold(III)–NHC complexes were recently reported to function as switchable cycloaurated putative anion transporters under reductive stimuli conditions.75 By harnessing the known anion transport activity of the pincer 2,6-bis(benzimidazol-2-yl)pyridine ligands, the gold(III) complexes supported by the chloride (43) or NHC (44) ligand can function as switchable moieties, liberating the anion transport pincer ligand upon reduction by glutathione, hence facilitating the binding to chloride (Cl) and nitrate (NO3) in liposomal membrane models for transmembrane transport. This work provides a new strategy in the design of anionophores that impair ion channel function associated with cancer proliferation and induce cellular osmotic stress.

+ N

O

Au N

HN N

NH

O O

= [Au(C^N^C)(NHC-biotin)]+

+

S Avidin

Gold(III)–NHC-Biotin-avidin bioconjugate 45

Modified from Tsai J.L. A luminescent Cyclometalated Gold(III)–Avidin Conjugate with a Long-Lived Emissive Excited State that Binds to Proteins and DNA and Possesses Anti-Proliferation Capacity. Chem. Commun. 2015, 51, 8547.

Taking the advantage of the biotin–avidin interaction as a bioconjugation strategy, Che and co-workers designed a luminescent cyclometalated gold(III)–NHC–avidin bioconjugate (45).76 Functionalization of the NHC ligand with biotin equips the complex with a high affinity for bioconjugation with avidin. Protected by the protein scaffold, the luminescence quenching of the gold(III)– NHC complex is attenuated and the bioconjugate displays emission enhancement with selectivity to single-stranded DNA and aggregated bovine serum albumin. In addition, such bioconjugation allows the luminescent gold(III)–NHC complex to be delivered by the protein carrier into cancer cells for anti-cancer and diagnostic functions.

Controllable activation of bio-active metal complexes in living systems is highly desirable so as to minimize off-target binding and improve specificity. Recently, a series of photo-activatable cyclometalated gold(III)–hydride complexes were reported to act as prodrugs with controllable reactivity toward thiols.77 Among which, complex 46 is stable in the presence of bio-relevant N-acetyl cysteine thiol in the dark, while reactive to form a gold-thiol adduct with concomitant dissociation of the auxiliary hydride moiety

858

Anti-cancer gold compounds

under visible light irradiation. The photo-activated gold(III) complexes potently suppressed thioredoxin reductase activity with IC50 values down to nanomolar level, exhibited up to a greater than 400-fold increase in cytotoxicity, and demonstrated strong antiangiogenic activity in a zebrafish model, compared with the effects of the complexes under dark conditions. Meanwhile, this complex evades deactivation by serum albumin, showing little difference in photocytotoxicity to hepatocellular carcinoma in the presence of this highly abundant plasma protein. Importantly, the activation of this gold(III) complex by two-photon laser irradiation is as effective as blue light irradiation, suggesting the possibility of in vivo application in deep tumor tissues via the spatial and temporal control of light irradiation afforded by two-photon laser technology.

2.26.4.3

Gold(III) complexes with the coordination of various p-conjugated aromatic ligands

Exploring additional aromatic tetrapyrrolic marcocycles, Gross and co-workers reported a water-soluble gold(III) corrole (2,17-bissulfonato-5,10,15-trispentafluorophenylcorrole), containing bis-sulfonate groups (47), that displayed marked cytotoxicity in cisplatin-resistant cancer cell lines.78 Compared with the gallium(III) analog, the lower binding affinity of the gold(III) corrole to human serum albumin (HSA), a highly abundant drug-sequestering serum protein, as assessed by emission quenching and mass spectrometry analysis, may account for its greater in vitro cytotoxicity.

Munro and co-workers described a class of cationic gold(III) marcocycles that act as nucleotide-specific catalytic inhibitors of topoisomerase IB (TopoI).79 The planar scaffold composed of a gold(III) ion and two pyrrole-imine units linked to a quinoxaline moiety on one side and an alkyl chain bridge on the opposite side allows complex 48 to bind with DNA duplexes at the enzyme’s 50 TA-30 dinucleotide target sequence through p–p stacking intercalation and an Au$$$O electrostatic interaction, involving a thymine carbonyl group. Macromolecular simulations of the ternary non-covalent 48–DNA–TopoI complex revealed that DNA–bound 48, with its observed base pair specificity, may account for the blockade of substrate recognition by TopoI through steric repulsion.

Anti-cancer gold compounds 2.26.4.4

859

Bidentate N^ N-type gold(III) complexes

Messori and co-workers reported a panel of dioxo-bridged dinuclear gold(III) complexes [Au2(m-O)2(N^N)2]2þ (wherein N^N ¼ 2,20 -bipyridine, a substituted 2,20 -bipyridine or 2,9-dimethyl-1,10-phenanthroline) (49), displaying anti-proliferative activities against a broad range of cancer cell lines, including both cisplatin-sensitive and -resistant cancer cells.80–82 Mechanistic studies using the COMPARE algorithm and electrospray ionization mass spectrometric analyses suggested reduction of the gold(III) complexes to gold(I) species, followed by the formation of coordinative adducts of Au(I) with the protein targets (likely involving histone deacetylase) and the concomitant release of the phenanthroline ligands.

In addition, Casini and co-workers described a series of anti-cancer gold(III) complexes containing nitrogen donor ligands [Au(N^N)Cl2]þ (wherein N^N ¼ 1,10-phenanthroline, 2,20 -bipyridine, 4,40 -dimethyl-2.20 -bipyridine,and 4,40 -diamino-2,20 -bipyridine) (50).83 These complexes display good aqueous solubility and inhibition of aquaporins (AQPs), which is a plausible chemotherapeutic target involved in the transport of water and glycerol.84

2.26.4.5

Bidentate C^N-type gold(III) complexes

Che and co-workers reported a panel of cyclometalated gold(III) complexes bearing a C-deprotonated bidentate C^N (HC^N ¼ 2phenylpyridine) ligand and biguanide as the auxiliary ligand [Au(R-C^N)(biguanide)]þ (51).85 The presence of the polar amino groups on the biguanide ligand renders the gold(III) complex soluble in water (> 5 mg/mL). As revealed by ESI-MS analysis, complex 51 is able to undergo ligand exchange reaction to form gold(III)–thiolato adducts in the presence of the biological reductant, glutathione. Complex 51 displayed in vitro cytotoxicity to different cancer cell lines, which was associated with the induction of ER stress.

860 2.26.4.6

Anti-cancer gold compounds Gold(III)-dithiocarbamate complexes

Dithiocarbamates (DTCs) have emerged as one of the bidentate ligands (–NCSS) that stabilize gold complexes under physiological conditions via gold–sulfur coordination. Earlier studies demonstrated the use of DTC ligands as metal carriers for the delivery of gold ion to the intended target site(s) for therapeutic actions. This approach was intended to not only improve the solubility and biocompatibility of the gold complexes, but also minimize off-target reactivity so as to lessen side effects in vivo.86 In the last decade, a number of DTC-coordinated gold complexes have been synthesized and the anti-cancer activities of these complexes have been extensively studied. By varying the biguanide moiety of bidentate C^N-type gold(III) complexes [Au(nBuC^N)(biguanide)]þ to a dithiocarbamate-containing ligand, Che and co-workers described [Au(nBuC^N)(dedt)]þ (wherein dedt ¼ diethyldithiocarbamate; 52), which showed potent anti-proliferation activity in MCF-7 breast cancer cells, with negligible toxicity to non-tumorigenic MIHA hepatic cells.87 Mass spectrometric analysis revealed that this complex can form covalent adducts with cysteine thiol-containing peptides and proteins such as deubiquitinases. Indeed, microarray gene expression, connectivity map analysis, together with cell-based activity assays, indicated that deubiquitinases may be cellular targets of this anti-cancer gold(III) complex.

Modification of the bidentate phenylpyridine C^N ligand into benzylpyridine affords the gold(III) complex alternative mechanisms of anti-cancer action. Awuah and co-workers described a gold(III)–dithiocarbamate complex with the incorporation of a bidentate benzylpyridine cyclometalated ligand [Au(C^N)S2CN(Me)2]þ (53) as a mitochondrial respiration modulator.88 As evidenced by NMR spectroscopy, mass spectrometry and cyclic voltammetry, in the presence of glutathione, complex 53 was demonstrated to reduce into the neutral gold(I) disulfide adduct [Au(C^N)(DTC)–GSH], suggesting reactivity to cysteine thiols of protein target(s) in cellulo. Based on transcriptomics analysis and verification by various biochemical experiments, the response of triple negative MDA-MB-231 breast cancer cells treated with 53 includes mitochondrial processes involving oxidative phosphorylation, cell cycle and organelle fission processes. The selective inhibition of mitochondrial respiration by complex 53 in cancer cells over normal cells may also account for its anti-proliferative selectivity in vitro. Together with the biological consequences observed, including ROS production, mitochondrial membrane depolarization, cell cycle arrest and apoptotic cell death, these findings suggest that this class of gold(III)–dithiocarbamate complexes may be useful as targeted therapeutics for neoplasms that rely on mitochondrial oxidative phosphorylation for proliferation.

The anti-cancer potency of gold(III) complexes coordinated with diverse dithiocarbamate ligands, such as N,N-dimethyldithiocarbamate (DMDT) and ethylsarcosinedithiocarbamate (ESDT), has also been demonstrated in a wide range of human tumor cell lines by Fregona and co-workers.89 Complexes [Au(DMDT)X2] (54) and [Au(ESDT)X2] (55) (X ¼ Cl, Br) showed a 1–4 fold increase in anti-cancer potency compared to cisplatin and demonstrated comparable in vitro cytotoxicity to both cisplatin-sensitive and -resistant leukemic cells, overcoming intrinsic and acquired cisplatin resistance.90 As for in vivo, treatment of nude mice bearing PC3 prostate cancer xenografts with the chlorido derivative of 54 [Au(DMDT)Cl2] elicited a significant inhibition of tumor growth without any histologically observable cytotoxicity.91 Changing the chloride ligand to bromide to give [Au(DMDT)Br2] also yielded an effective suppressor of tumor growth in nude mice inoculated with MDA-MB-231 breast cancer cells.92 These gold(III)–dithiocarbamates inhibited RNA and DNA synthesis with a faster kinetics than cisplatin and evaded the cross-resistance phenomenon, indicating a different mechanism of action from cisplatin.89 The mechanisms of action of these gold(III)–dithiocarbamates have been extensively investigated and are associated with the inhibition of proteasomal (20S and 26S) activity in highly metastatic MDA-MB-231 breast cancer cells,92,93 promoting mitochondrial membrane permeabilization and cytochrome-c release, elevation

Anti-cancer gold compounds

861

of oxidative stress, deregulation of the thioredoxin redox system, and activation of the extracellular signal-regulated kinase (ERK) pathway.91,94

Isab and coworkers reported a panel of bipyridine gold(III)–dithiocarbamate complexes [Au(bpy)S2CN(Me)2]þ that exhibits potent in vitro anti-cancer activities against prostate, breast, and ovarian cancer cell lines, and Hodgkin lymphoma cells, with IC50 values lower than that of cisplatin by up to 82-fold.95 In particular, treatment of cells with 56 augmented intracellular ROS accumulation, induced mitochondrial depolarization, triggered the release of cytochrome-c from mitochondria, and activated caspase-dependent apoptosis. Notably, this complex showed comparable cytotoxicity to cancer cells with varying tumor suppressor p53 status, suggesting the potentials for future in vivo anti-tumor evaluation.

A

A T

A C

T

A

G

G G G C

3´

T A C G G T T A G A [T T T T T T]

A

Cl Dye

Au

spacer

Sgc8c aptamer

N CG TCA A TC TA T

C G

G C C G

C

5´

linker

N

HN O

[Au(NHC)CI] aptamer conjugate 57 Modified from Niu W. N-Heterocyclic Carbene–Gold(I) Complexes Conjugated to a Leukemia-Specific DNA Aptamer for Targeted Drug Delivery. Angew. Chem. Int. Ed. Engl. 2016, 55, 8889.

2.26.5

Formulations of gold complexes with improved anti-cancer potency

2.26.5.1

Gold(I) complexes

To improve the anti-cancer potency and to achieve targeted therapy with gold complexes, bioconjugation is one of the strategies to be employed. This approach modulates therapeutic efficacy by directing the conjugates to cancer cell-specific receptors, while minimizing off-target effects. For instance, the introduction of a leukemia-specific DNA aptamer (sgc8c) to the NHC ligand of a gold(I) complex has been demonstrated to afford cancer cell-specific cellular uptake and cytotoxicity.96 With regard to the high binding affinity interactions between the sgc8c aptamer and the receptor, protein tyrosine kinase 7 (PTK-7), to which it binds, the gold(I)–NHC aptamer conjugate (57) showed enhanced cytotoxicity with selectivity against the targeted CCRF-CEM leukemia cells by a factor of 30, compared to K562 leukemia cells that do not overexpress PTK-7.

Taking advantage of t