Comprehensive Organometallic Chemistry IV. Volume 15: Applications IV. Bio-Organometallics, Metallo-Therapy, Metallo-Diagnostics, Medicine and Environmental Chemistry [15] 9780128202067

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COMPREHENSIVE ORGANOMETALLIC CHEMISTRY IV

COMPREHENSIVE ORGANOMETALLIC CHEMISTRY IV EDITORS-IN-CHIEF

GERARD PARKIN Department of Chemistry, Columbia University, New York, NY, United States

KARSTEN MEYER Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität, Erlangen, Germany

DERMOT O’HARE Department of Chemistry, University of Oxford, Oxford, United Kingdom

VOLUME 15

APPLICATIONS IV. BIO-ORGANOMETALLICS, METALLO-THERAPY, METALLO-DIAGNOSTICS, MEDICINE AND ENVIRONMENTAL CHEMISTRY VOLUME EDITORS

LENA J. DAUMANN Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany

ESZTER BOROS Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, United States

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 © 2022 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.

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Publisher: Oliver Walter Acquisition Editor: Blerina Osmanaj Content Project Manager: Claire Byrne Associate Content Project Manager: Fahmida Sultana Designer: Christian Bilbow

CONTENTS OF VOLUME 15 Editor Biographies

vii

Contributors to Volume 15

xiii

Preface 15.01

xv

Introduction to Applications IV. Bio-Organometallics, Metallo-Therapy, Metallo-Diagnostics, Medicine and Environmental Chemistry

1

Lena J Daumann and Eszter Boros

15.02

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

3

Gustav Berggren, Starla D Glover, and Mun Hon Cheah

15.03

Nitrogenases and Model Complexes in Bioorganometallic Chemistry

41

Daniel WN Wilson and Patrick L Holland

15.04

Bioorganometallic Chemistry of Vitamin B12-Derivatives

73

Bernhard Kräutler

15.05

Bioorganometallics: Artificial Metalloenzymes With Organometallic Moieties

96

Michela M Pellizzoni and Andriy Lubskyy

15.06

Opportunities for interfacing organometallic catalysts with cellular metabolism

121

Rudy Rubini and Clemens Mayer

15.07

Oligonucleotide Complexes in Bioorganometallic Chemistry

146

Tuomas A Lönnberg, Madhuri A Hande, and Dattatraya U Ukale

15.08

Organometallic Receptors and Conjugates With Biomolecules in Bioorganometallic Chemistry

183

Benjamin Neuditschko, Bernhard K Keppler, Christopher Gerner, and Samuel M Meier-Menches

15.09

Organometallic Chemistry of Anticancer Ruthenium and Osmium Complexes

206

Artem Osypenko, Adnan Ashraf, Valentyn Pozhydaiev, Maria V Babak, and Muhammad Hanif

15.10

Organometallic Chemistry of Drugs Based on Technetium and Rhenium

226

Roger Alberto

15.11

Organometallic Chemistry of Drugs Based on Iron

261

Mziyanda Mbaba, Setshaba D Khanye, Gregory S Smith, and Christophe Biot

15.12

Organometallic Chemistry of Gold-Based Drugs

297

Claudia Schmidt and Angela Casini

15.13

Manganese-Based Carbon Monoxide-Releasing Molecules: A Multitude of Organometallic Pharmaceutical Candidates Primed for Further Biological Analysis

314

Jonathan S Ward

15.14

Organometallic Synthesis in Flow

331

Johannes H Harenberg, Benjamin Heinz, Dimitrije Djukanovic, Niels Weidmann, Rajasekar R Annapureddy, Benjamin Martin, and Paul Knochel

Index

357

v

EDITOR BIOGRAPHIES Editors in Chief Karsten Meyer studied chemistry at the Ruhr University Bochum and performed his Ph.D. thesis work on the molecular and electronic structure of first-row transition metal complexes under the direction of Professor Karl Wieghardt at the Max Planck Institute in Mülheim/Ruhr (Germany). He then proceeded to gain research experience in the laboratory of Professor Christopher Cummins at the Massachusetts Institute of Technology (USA), where he appreciated the art of synthesis and developed his passion for the coordination chemistry and reactivity of uranium complexes. In 2001, he was appointed to the University of California, San Diego, as an assistant professor and was named an Alfred P. Sloan Fellow in 2004. In 2006, he accepted an offer (C4/W3) to be the chair of the Institute of Inorganic & General Chemistry at the Friedrich-Alexander-University ErlangenNürnberg (FAU), Germany. Among his awards and honors, he was elected a lifetime honorary member of the Israel Chemical Society and a fellow of the Royal Society of Chemistry (UK). Karsten received the Elhuyar-Goldschmidt Award from the Royal Society of Chemistry of Spain, the Ludwig Mond Award from the RSC (UK), and the Chugaev Commemorative Medal from the Russian Academy of Sciences. He has also enjoyed visiting professorship positions at the universities of Manchester (UK) and Toulouse (F) as well as the Nagoya Institute of Technology (JP) and ETH Zürich (CH). The Meyer lab research focuses on the synthesis of custom-tailored ligand environments and their transition and actinide metal coordination complexes. These complexes often exhibit unprecedented coordination modes, unusual electronic structures, and, consequently, enhanced reactivities toward small molecules of biological and industrial importance. Interestingly, Karsten’s favorite molecule is one that exhibits little reactivity: the Th symmetric U(dbabh)6. Dermot O’Hare was born in Newry, Co Down. He studied at Balliol College, Oxford University, where he obtained his B.A., M.A., and D.Phil. degrees under the direction of Professor M.L.H. Green. In 1985, he was awarded a Royal Commission of 1851 Research Fellowship, during this Fellowship he was a visiting research fellow at the DuPont Central Research Department, Wilmington, Delaware in 1986–87 in the group led by Prof. J.S. Miller working on molecular-based magnetic materials. In 1987 he returned to Oxford to a short-term university lectureship and in 1990 he was appointed to a permanent university position and a Septcentenary Tutorial Fellowship at Balliol College. He has previously been honored by the Institüt de France, Académie des Sciences as a leading scientist in Europe under 40 years. He is currently professor of organometallic and materials chemistry in the Department of Chemistry at the University of Oxford. In addition, he is currently the director of the SCG-Oxford Centre of Excellence for chemistry and associate head for business & innovation in the Mathematics, Physical and Life Sciences Division. He leads a multidisciplinary research team that works across broad areas of catalysis and nanomaterials. His research is specifically targeted at finding solutions to global issues relating to energy, zero carbon, and the circular economy. He has been awarded numerous awards and prizes for his creative and

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Editor Biographies

ground-breaking work in inorganic chemistry, including the Royal Society Chemistry’s Sir Edward Frankland Fellowship, Ludwig Mond Prize, Tilden Medal, and Academia–Industry Prize and the Exxon European Chemical and Engineering Prize. Gerard Parkin received his B.A., M.A., and D.Phil. degrees from the Queen’s College, Oxford University, where he carried out research under the guidance of Professor Malcolm L.H. Green. In 1985, he moved to the California Institute of Technology as a NATO postdoctoral fellow to work with Professor John E. Bercaw. He joined the Faculty of Columbia University as assistant professor in 1988 and was promoted to associate professor in 1991 and to professor in 1994. He served as chairman of the Department from 1999 to 2002. He has also served as chair of the New York Section of the American Chemical Society, chair of the Inorganic Chemistry and Catalytic Science Section of the New York Academy of Sciences, chair of the Organometallic Subdivision of the American Chemical Society Division of Inorganic Chemistry, and chair of the Gordon Research Conference in Organometallic Chemistry. He is an elected fellow of the American Chemical Society, the Royal Society of Chemistry, and the American Association for the Advancement of Science, and is the recipient of a variety of international awards, including the ACS Award in pure chemistry, the ACS Award in organometallic chemistry, the RSC Corday Morgan Medal, the RSC Award in organometallic chemistry, the RSC Ludwig Mond Award, and the RSC Chem Soc Rev Lecture Award. He is also the recipient of the United States Presidential Award for Excellence in Science, Mathematics and Engineering Mentoring, the United States Presidential Faculty Fellowship Award, the James Flack Norris Award for Outstanding Achievement in the Teaching of Chemistry, the Columbia University Presidential Award for Outstanding Teaching, and the Lenfest Distinguished Columbia Faculty Award. His principal research interests are in the areas of synthetic, structural, and mechanistic inorganic chemistry.

Volume Editors Simon Aldridge is professor of chemistry at the University of Oxford and director of the UKRI Centre for Doctoral Training in inorganic chemistry for Future Manufacturing. Originally from Shrewsbury, England, he received both his B.A. and D.Phil. degrees from the University of Oxford, the latter in 1996 for work on hydride chemistry under the supervision of Tony Downs. After post-doctoral work as a Fulbright Scholar at Notre Dame with Tom Fehlner, and at Imperial College London (with Mike Mingos), he took up his first academic position at Cardiff University in 1998. He returned to Oxford in 2007, being promoted to full professor in 2010. Prof. Aldridge has published more than 230 papers to date and is a past winner of the Dalton Transactions European Lectureship (2009), the Royal Society of Chemistry’s Main Group Chemistry (2010) and Frankland Awards (2018), and the Forschungspreis of the Alexander von Humboldt Foundation (2021). Prof. Aldridge’s research interests are primarily focused on main group organometallic chemistry, and in particular the development of compounds with unusual electronic structure, and their applications in small molecule activation and catalysis (website: http:// aldridge.web.ox.ac.uk). (Picture credit: John Cairns)

Editor Biographies

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Eszter Boros is associate professor of chemistry at Stony Brook University with courtesy appointments in radiology and pharmacology at Stony Brook Medicine. Eszter obtained her M.Sc. (2007) at the University of Zurich, Switzerland and her Ph.D. (2011) in chemistry from the University of British Columbia, Canada. She was a postdoc (2011–15) and later instructor (2015–17) in radiology at Massachusetts General Hospital and Harvard Medical School. In 2017, Eszter was appointed as assistant professor of chemistry at Stony Brook University, where her research group develops new approaches to metal-based diagnostics and therapeutics at the interfaces of radiochemistry, inorganic chemistry and medicine. Her lab’s work has been extensively recognized; Eszter holds various major federal grants (NSF CAREER Award, NIH NIBIB R21 Trailblazer, NIH NIGMS R35 MIRA) and has been named a 2020 Moore Inventor Fellow, the 2020 Jonathan L. Sessler Fellow (American Chemical Society, Inorganic Division), recipient of a 2021 ACS Infectious Diseases/ACS Division of Biological Chemistry Young Investigator Award (American Chemical Society), and was also named a 2022 Alfred P. Sloan Research Fellow in chemistry. Scott R. Daly is associate professor of chemistry at the University of Iowa in the United States. After spending 3 years in the U.S. Army, he obtained his B.S. degree in chemistry in 2006 from North Central College, a small liberal arts college in Naperville, Illinois. He then went on to receive his Ph.D. at the University of Illinois at Urbana-Champaign in 2010 under the guidance of Professor Gregory S. Girolami. His thesis research focused on the synthesis and characterization of chelating borohydride ligands and their use in the preparation of volatile metal complexes for chemical vapor deposition applications. In 2010, he began working as a Seaborg postdoctoral fellow with Drs. Stosh A. Kozimor and David L. Clark at Los Alamos National Laboratory in Los Alamos, New Mexico. His research there concentrated on the development of ligand K-edge X-ray absorption spectroscopy (XAS) to investigate covalent metal–ligand bonding and electronic structure variations in actinide, lanthanide, and transition metal complexes with metal extractants. He started his independent career in 2012 at George Washington University in Washington, DC, and moved to the University of Iowa shortly thereafter in 2014. His current research interests focus on synthetic coordination chemistry and ligand design with emphasis on the development of chemical and redox noninnocent ligands, mechanochemical synthesis and separation methods, and ligand K-edge XAS. His research and outreach efforts have been recognized with an Outstanding Faculty/Staff Advocate Award from the University of Iowa Veterans Association (2016), a National Science Foundation CAREER Award (2017), and a Hawkeye Distinguished Veterans Award (2018). He was promoted to associate professor with distinction as a College of Liberal Arts and Sciences Deans Scholar in 2020. Lena J. Daumann is currently professor of bioinorganic and coordination chemistry at the Ludwig Maximilian Universität in Munich. She studied chemistry at the University of Heidelberg working with Prof. Peter Comba and subsequently conducted her Ph.D. at the University of Queensland (Australia) from 2010 to 2013 holding IPRS and UQ Centennial fellowships. In 2013 she was part of the Australian Delegation for the 63rd Lindau Nobel Laureate meeting in chemistry. Following postdoctoral stays at UC Berkeley with Prof. Ken Raymond (2013–15) and in Heidelberg, funded by the Alexander von Humboldt Foundation, she started her independent career at the LMU Munich in 2016. Her bioinorganic research group works on elucidating the role of lanthanides for bacteria as well as on iron enzymes and small biomimetic complexes that play a role in epigenetics and DNA repair. Daumann’s teaching and research have been recognized with numerous awards and grants. Among them are the national Ars Legendi Prize for chemistry and the Therese von Bayern Prize in 2019 and the Dozentenpreis of the “Fonds der Chemischen Industrie“ in 2021. In 2018 she was selected as fellow for the Klaus Tschira Boost Fund by the German Scholars Organisation and in 2020 she received a Starting grant of the European Research Council to study the uptake of lanthanides by bacteria.

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Editor Biographies

Derek P. Gates hails from Halifax, Nova Scotia (Canada) where he completed his B.Sc. (Honours Chemistry) degree at Dalhousie University in 1993. He completed his Ph.D. degree under the supervision of Professor Ian Manners at the University of Toronto in 1997. He then joined the group of Professor Maurice Brookhart as an NSERC postdoctoral fellow at the University of North Carolina at Chapel Hill (USA). He began his independent research career in 1999 as an assistant professor at the University of British Columbia in Vancouver (Canada). He has been promoted through the ranks and has held the position of professor of chemistry since 2011. At UBC, he has received the Science Undergraduate Society—Teaching Excellence Award, the Canadian National Committee for IUPAC Award, and the Chemical Society of Canada—Strem Chemicals Award for pure or applied inorganic chemistry. His research interests bridge the traditional fields of inorganic and polymer chemistry with particular focus on phosphorus chemistry. Key topics include the discovery of novel structures, unusual bonding, new reactivity, along with applications in catalysis and materials science. Patrick Holland performed his Ph.D. research in organometallic chemistry at UC Berkeley with Richard Andersen and Robert Bergman. He then learned about bioinorganic chemistry through postdoctoral research on copper-O2 and copper-thiolate chemistry with William Tolman at the University of Minnesota. His independent research at the University of Rochester initially focused on systematic development of the properties and reactions of three-coordinate complexes of iron and cobalt, which can engage in a range of bond activation reactions and organometallic transformations. Since then, his research group has broadened its studies to iron-N2 chemistry, reactive metal–ligand multiple bonds, iron–sulfur clusters, engineered metalloproteins, redox-active ligands, and solar fuel production. In 2013, Prof. Holland moved to Yale University, where he is now Conkey P. Whitehead Professor of Chemistry. His research has been recognized with an NSF CAREER Award, a Sloan Research Award, Fulbright and Humboldt Fellowships, a Blavatnik Award for Young Scientists, and was elected as fellow of the American Association for the Advancement of Science. In the area of N2 reduction, his group has established molecular principles to weaken and break the strong N–N bond, in order to use this abundant resource for energy and synthesis. His group has made a particular effort to gain an insight into iron chemistry relevant to nitrogenase, the enzyme that reduces N2 in nature. His group also maintains an active program in the use of inexpensive metals for transformations of alkenes. Mechanistic details are a central motivation to Prof. Holland and the wonderful group of over 80 students with whom he has worked. Steve Liddle was born in Sunderland in the North East of England and gained his B.Sc. (Hons) and Ph.D. from Newcastle University. After postdoctoral fellowships at Edinburgh, Newcastle, and Nottingham Universities he began his independent career at Nottingham University in 2007 with a Royal Society University Research Fellowship. This was held in conjunction with a proleptic Lectureship and he was promoted through the ranks to associate professor and reader in 2010 and professor of inorganic chemistry in 2013. He remained at Nottingham until 2015 when he was appointed professor and head of inorganic chemistry and co-director of the Centre for Radiochemistry Research at The University of Manchester. He has been a recipient of an EPSRC Established Career Fellowship and ERC Starter and Consolidator grants. He is an elected fellow of The Royal Society of Edinburgh and fellow of the Royal Society of Chemistry and he is vice president to the Executive Committee of the European Rare Earth and Actinide Society. His principal research interests are focused on f-element chemistry, involving exploratory synthetic chemistry coupled to detailed electronic structure and reactivity studies to elucidate structure-bonding-property relationships. He is the recipient of a variety of prizes, including the IChemE Petronas Team Award for Excellence in Education and Training, the RSC Sir Edward Frankland Fellowship, the RSC Radiochemistry

Editor Biographies

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Group Bill Newton Award, a 41st ICCC Rising Star Award, the RSC Corday-Morgan Prize, an Alexander von Humboldt Foundation Friedrich Wilhelm Bessel Research Award, the RSC Tilden Prize, and an RSC Dalton Division Horizon Team Prize. He has published over 220 research articles, reviews, and book chapters to date. David Liptrot received his MChem (Hons) in chemistry with Industrial Training from the University of Bath in 2011 and remained there to undertake a Ph.D. on group 2 catalysis in the laboratory of Professor Mike Hill. After completing this in 2015 he took up a Lindemann Postdoctoral Fellowship with Professor Philip Power FRS (University of California, Davis, USA). In 2017 he began his independent career returning to the University of Bath and in 2019 was awarded a Royal Society University Research Fellowship. His interests concern new synthetic methodologies to introduce main group elements into functional molecules and materials.

David P. Mills hails from Llanbradach and Caerphilly in the South Wales Valleys. He completed his MChem (2004) and Ph.D. (2008) degrees at Cardiff University, with his doctorate in low oxidation state gallium chemistry supervised by Professor Cameron Jones. He moved to the University of Nottingham in 2008 to work with Professor Stephen Liddle for postdoctoral studies in lanthanide and actinide methanediide chemistry. In 2012 he moved to the University of Manchester to start his independent career as a lecturer, where he has since been promoted to full professor of inorganic chemistry in 2021. Although he is interested in all aspects of nonaqueous synthetic chemistry his research interests are currently focused on the synthesis and characterization of f-block complexes with unusual geometries and bonding regimes, with the aim of enhancing physicochemical properties. He has been recognized for his contributions to both research and teaching with prizes and awards, including a Harrison-Meldola Memorial Prize (2018), the Radiochemistry Group Bill Newton Award (2019), and a Team Member of the Molecular Magnetism Group for the Dalton Division Horizon Prize (2021) from the Royal Society of Chemistry. He was a Blavatnik Awards for Young Scientists in the United Kingdom Finalist in Chemistry in 2021 and he currently holds a European Research Council Consolidator Grant. Ian Tonks is the Lloyd H. Reyerson professor at the University of MinnesotaTwin Cities, and associate editor for the ACS journal Organometallics. He received his B.A. in chemistry from Columbia University in 2006 and performed undergraduate research with Prof. Ged Parkin. He earned his Ph.D. in 2012 from the California Institute of Technology, where he worked with Prof. John Bercaw on olefin polymerization catalysis and early transition metal-ligand multiply bonded complexes. After postdoctoral research with Prof. Clark Landis at the University of Wisconsin, Madison, he began his independent career at the University of Minnesota in 2013 and earned tenure in 2019. His current research interests are focused on the development of earth abundant, sustainable catalytic methods using early transition metals, and also on catalytic strategies for incorporation of CO2 into polymers. Prof. Tonks’ work has recently been recognized with an Outstanding New Investigator Award from the National Institutes of Health, an Alfred P. Sloan Fellowship, a Department of Energy CAREER award, and the ACS Organometallics Distinguished Author Award, among others. Additionally, Prof. Tonks’ service toward improving academic safety culture was recently recognized with the 2021 ACS Division of Chemical Health and Safety Graduate Faculty Safety Award.

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Timothy H. Warren is the Rosenberg professor and chairperson in the Department of Chemistry at Michigan State University. He obtained his B.S. from the University of Illinois at Urbana-Champaign in 1992 and Ph.D. from the Massachusetts Institute of Technology in 1997. After 2 years of postdoctoral research at the Organic Chemistry Institute of the University of Münster, Germany with Prof. Dr. Gerhart Erker, Dr. Warren started his independent career at Georgetown University in 1999 where he was named the Richard D. Vorisek professor of chemistry in 2014. He moved to Michigan State University in 2021. Prof. Warren’s research interests span synthetic and mechanistic inorganic, organometallic, and bioinorganic chemistry with a focus on catalysis. His research group develops environmentally friendly methods for organic synthesis via C–H functionalization, explores the interconversion of nitrogen and ammonia as carbon-free fuels, and decodes ways that biology communicates using nitric oxide as a molecular messenger. Mechanistic studies on these chemical reactions catalyzed by metal ions such as iron, nickel, copper, and zinc enable new insights for the development of useful catalysts for synthesis and energy applications as well as lay the mechanistic groundwork to understand biochemical nitric oxide misregulation. Dr. Warren received the NSF CAREER Award, chaired the 2019 Inorganic Reaction Mechanisms Gordon Research Conference, and has served on the ACS Division of Inorganic Chemistry executive board and on the editorial boards of Inorganic Synthesis, Inorganic Chemistry, and Chemical Society Reviews.

CONTRIBUTORS TO VOLUME 15 Roger Alberto Department of Chemistry, University of Zurich, Zurich, Switzerland

Dimitrije Djukanovic Ludwig-Maximilians-Universität München, Munich, Germany

Rajasekar R Annapureddy Ludwig-Maximilians-Universität München, Munich, Germany

Christopher Gerner Department of Analytical Chemistry, University of Vienna, Vienna, Austria; Joint Metabolome Facility, University of Vienna and Medical University of Vienna, Vienna, Austria

Adnan Ashraf School of Chemical Sciences, University of Auckland, Auckland, New Zealand Maria V Babak Department of Chemistry, Drug Discovery Lab, City University of Hong Kong, Hong Kong, People’s Republic of China

Starla D Glover Physical Chemistry, Department of Chemistry-Ångström, Uppsala University, Uppsala, Sweden Madhuri A Hande Department of Chemistry, University of Turku, Turku, Finland

Gustav Berggren Molecular Biomimetics, Department of ChemistryÅngström, Uppsala University, Uppsala, Sweden

Muhammad Hanif School of Chemical Sciences, University of Auckland, Auckland, New Zealand

Christophe Biot Université de Lille, CNRS, UMR 8576—UGSF—Unité de Glycobiologie Structurale et Fonctionnelle, Lille, France

Johannes H Harenberg Ludwig-Maximilians-Universität München, Munich, Germany

Eszter Boros Department of Chemie, Ludwig-Maximilians - Universität München, München, Germany; Department of Chemistry, Stony Brook University, Stony Brook, NY, United States

Benjamin Heinz Ludwig-Maximilians-Universität München, Munich, Germany

Angela Casini Chair of Medicinal and Bioinorganic Chemistry, Department of Chemistry, Technical University of Munich, Garching, Germany Mun Hon Cheah Molecular Biomimetics, Department of ChemistryÅngström, Uppsala University, Uppsala, Sweden Lena J Daumann Department of Chemie, Ludwig-Maximilians - Universität München, München, Germany; Department of Chemistry, Stony Brook University, Stony Brook, NY, United States

Patrick L Holland Department of Chemistry, Yale University, New Haven, CT, United States Bernhard K Keppler Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria Setshaba D Khanye Faculty of Pharmacy, Division of Pharmaceutical Chemistry, Rhodes University, Makhanda, South Africa Paul Knochel Ludwig-Maximilians-Universität München, Munich, Germany

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Contributors to Volume 15

Bernhard Kräutler Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innsbruck, Austria

Michela M Pellizzoni Adolphe Merkle Institute, University Fribourg, Fribourg, Switzerland

Tuomas A Lönnberg Department of Chemistry, University of Turku, Turku, Finland

Valentyn Pozhydaiev Institut de Science et d’Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, Strasbourg, France

Andriy Lubskyy Adolphe Merkle Institute, University Fribourg, Fribourg, Switzerland Benjamin Martin Ludwig-Maximilians-Universität München, Munich, Germany Clemens Mayer Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands Mziyanda Mbaba Department of Chemistry, Faculty of Science, University of Cape Town, Cape Town, South Africa Samuel M Meier-Menches Department of Analytical Chemistry, University of Vienna, Vienna, Austria; Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria; Joint Metabolome Facility, University of Vienna and Medical University of Vienna, Vienna, Austria Benjamin Neuditschko Department of Analytical Chemistry, University of Vienna, Vienna, Austria; Institute of Inorganic Chemistry, University of Vienna, Vienna, Austria Artem Osypenko Institut de Science et d’Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, Strasbourg, France

Rudy Rubini Stratingh Institute for Chemistry, University of Groningen, Groningen, The Netherlands Claudia Schmidt Chair of Medicinal and Bioinorganic Chemistry, Department of Chemistry, Technical University of Munich, Garching, Germany Gregory S Smith Department of Chemistry, Faculty of Science, University of Cape Town, Cape Town, South Africa Dattatraya U Ukale Department of Chemistry, University of Turku, Turku, Finland Jonathan S Ward Department of Chemistry, The University of Liverpool, Liverpool, United Kingdom Niels Weidmann Ludwig-Maximilians-Universität München, Munich, Germany Daniel WN Wilson Department of Chemistry, Yale University, New Haven, CT, United States

PREFACE Published 40 years ago in 1982, the first edition of Comprehensive Organometallic Chemistry (COMC) provided an invaluable resource that enabled chemists to become efficiently informed of the properties and reactions of organometallic compounds of both the main group and transition metals. This area of chemistry continued to develop at a rapid pace such that it necessitated the publication of subsequent editions, namely Comprehensive Organometallic Chemistry II (COMC2) in 1995 and Comprehensive Organometallic Chemistry III (COMC3) in 2007. Organometallic chemistry has continued to be vibrant in the 15 years following the publication of COMC3, not only by affording compounds with novel structures and reactivity but also by having important applications in organic syntheses and industrial processes, as illustrated by the awarding of the 2010 Nobel prize to Heck, Negishi, and Suzuki for the development of palladium-catalyzed cross couplings in organic syntheses. Comprehensive Organometallic Chemistry IV (COMC4) thus serves the same important role as its predecessors by providing an indispensable means for researchers and educators to obtain efficiently an up-to-date analysis of a particular aspect of organometallic chemistry. COMC4 comprises 15 volumes, of which the first provides a review of topics concerned with techniques and concepts that feature prominently in current organometallic chemistry, while 5 volumes are devoted to applications that include organic synthesis, materials science, bio-organometallics, metallo-therapy, metallodiagnostics, medicine, and environmental chemistry. In this regard, we are very grateful to the volume editors for their diligent efforts, and the authors for producing high-quality chapters, all of which were written during the COVID-19 pandemic. Finally, we wish to thank the many staff at Elsevier for their efforts to ensure that the project, initiated in the winter of 2018, remained on schedule. Karsten Meyer, Erlangen, March 2022 Dermot O’Hare, Oxford, March 2022 Gerard Parkin, New York, March 2022

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15.01 Introduction to Applications IV. Bio-Organometallics, Metallo-Therapy, Metallo-Diagnostics, Medicine and Environmental Chemistry Lena J Daumann and Eszter Boros, Department of Chemie, Ludwig-Maximilians - Universität München, München, Germany; Department of Chemistry, Stony Brook University, Stony Brook, NY, United States © 2022 Elsevier Ltd. All rights reserved.

The volume “Applications IV. Bio-organometallics, metallo-therapy, metallo-diagnostics, medicine and environmental chemistry” is now established in Comprehensive Organometallic Chemistry IV. However, it is unlikely to have existed for the first volumes of COMC. The application of organometallic drugs and the discovery of natural systems utilizing organometallic moieties is, nevertheless, nowadays a flourishing area of research. This volume assists bioinorganic chemists, coordination and medicinal chemists, and those interested in new-to-nature reactions. It also includes opportunities for green and sustainable organometallic catalysis. Nature’s methods of employing organometallic chemistry for catalysis and human attempts to mimic nature’s techniques by both building and optimizing synthetic analogs are examined in this volume, as well as the utilization of organometallic complexes to diagnose and treat diseases. The first chapter, contributed by Berggren, Glover and Cheah offers an overview of [FeFe]-, [NiFe]-, and [Fe] Hydrogenases and recent efforts to develop model complexes in this field. Enthusiasts of metal hydride chemistry will not be disappointed. Another hot topic in bioinorganic chemistry and enzymes bearing metal-carbide bonds is the chapter by Holland and Wilson on Nitrogenase enzymes. The focus of the chapter is on the active sites, in particular the iron-sulfur clusters FeMoco, FeVco, and FeFeco for the reduction of N2, as well as recent efforts to design functional model complexes. Both Hydrogenase and Nitrogenase are highly relevant for our world, as researchers try to identify sustainable methods for N2 reduction and make use of the unique chemistry of Hydrogenases for biofuel production. Both chapters strongly highlight the importance of these models. With such small complexes, the chemistry of the parent enzymes can be better understood. The third major cornerstone of biological organometallic chemistry is covered in the chapter by Kräutler on Vitamin B12. Here, the structure, reactivity and biochemistry of B12-derivatives are discussed along with the newest developments on antivitamins B12 and B12-mimics. However, not only natural enzymes and organometallic cofactors play a role. The design of artificial metalloenzymes bearing organometallic moieties has opened new ways for organometallic catalysis. The chapter by Pellizzoni and Lubskyy highlights different anchoring strategies along with selected examples on chemical and genetic optimization of the hybrid systems, aiming at improving catalyst (enantio)selectivity, substrate scope and efficiency. This ultimately yields more environmentally friendly applications. Mayer and Rubini describe in their chapter on “Opportunities for interfacing organometallic catalysts with cellular metabolism” new-to-nature reactions and designer microbes for new applications in biotechnology and biomedicine. The potential to access value-added products from renewable resources or the clean-up of pollutants, as well as targeted synthesis of bioactive compounds, make these systems interesting for sustainable and medical applications. The chapter by Lönnberg, Hande and Ukale focuses on various types of interactions between nucleic acids and organometallic compounds, covering organomercury compounds with affinity for nucleic acids, organometallic metallointercalators up to ferrocene-containing nucleoside phosphoramidite building blocks. The synthetic approaches for such compounds with applications spanning synthetic bioorganic chemistry to therapeutic agents are highlighted in particular. To appropriately study and subsequently understand the behavior of organometallic fragments and conjugates in the context of the biological environment of interest, it is essential to develop a specialized toolbox of analytical methods. Meier-Menches describes tools that have been creatively adapted from the metabolomics and proteomics space with specific, relevant and current examples from the field of bioorganometallic chemistry. The contribution by Hanif and co-workers details the chemistry of ruthenium and osmium organometallic complexes. This class of compounds has recently emerged as a highly attractive tool for conventional chemo- and photodynamic cancer therapy, using excitation pathways that are unique to transition metal complexes. This chapter describes ideal compound design criteria, synthesis, and activity profiling; thus providing a perspective for the direction of the field. Unquestionably, as Roger Alberto’s review chapter describes, organometallic Rhenium and Technetium complexes have both provided compounds of imminent biomedical interest to the scientific and, more recently, clinical research community. Technetium-99m remains among the most frequently used radioisotopes in nuclear medicine and, lately, the first organometallic Tc-99m complex has successfully advanced to the clinic. Rhenium, the 3rd row group VII congener of Tc-99m, not only provides means to characterize isotopically stable analogs of Tc-99m compounds, but also shows promise as a potent beta-therapeutic (Re-186, Re-188) and anticancer agent with distinct mechanisms of action, qualifying Re-organometallics as potent cisplatinum alternatives. The exceptional inertness of organometallic iron complexes, in comparison to their coordination chemistry counterparts, renders them excellent building blocks in drug molecules, as Biot and co-workers describe. The modification of approved antimalarials with iron-half and iron sandwich complexes results in potentiation of activity to combat this pathogen and others

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Introduction to Applications IV

that to date have only few clinically available and affordable treatment options. The low cost and wide accessibility of iron precursors is, in addition, an important aspect to the production of potent, small molecule drugs, especially for the developing world. The clinically approved Au-linked rheumatoid arthritis drug Auranofin sets the stage for a host of potential applications of Gold-based organometallic small molecule and protein conjugate systems, as Casini and Schmidt eloquently summarize. This book chapter provides a concise overview over the diverse reactivity of the two prevalent oxidation states of Au-organometallic complexes, Au(I) and Au(III), which unlocks a wide range of possibilities from N-heterocyclic carbenes to cyclometallation as feasible paths of functionalization. Applications range from infectious disease to cancer, with the mechanism of action of these compounds subject to ongoing research. Albeit part of the group VII triad with Tc and Re, manganese remains, in many ways, within a class of its own (reactivity). The more labile coordinative nature of Mn(I) complexes, when compared with its heavier group VII congeners, has given rise to the unique and emerging field of photo-triggered CO release from Mn(I) organometallic complexes, as Ward’s chapter reports. In turn, the selectively triggered release of CO, a potent gasotransmitter and toxin, has vast potential for pharmaceutical applications. The excellent manner in which flow chemistry and organometallic catalysis go together is demonstrated in the chapter by Knochel and co-authors. A more environmentally friendly generation of main group organometallics in continuous-flow-involving microreactors is the focus alongside a demonstration of how conditions, substrate scope, and up-scaling of organometallic reactions can be improved using flow chemistry.

15.02

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Gustav Berggrena, Starla D Gloverb, and Mun Hon Cheaha, aMolecular Biomimetics, Department of Chemistry-A˚ ngström, Uppsala University, Uppsala, Sweden; bPhysical Chemistry, Department of Chemistry-A˚ ngström, Uppsala University, Uppsala, Sweden © 2022 Elsevier Ltd. All rights reserved.

15.02.1 Hydrogenases and models in bioorganometallic chemistry 15.02.1.1 Introduction 15.02.1.1.1 The aim of this book chapter 15.02.1.1.2 General overview 15.02.1.1.3 Metal hydrides 15.02.1.1.4 The role of proton-coupled electron transfer in H2/2H+ interconversion 15.02.1.2 [FeFe] hydrogenases and their model compounds 15.02.1.2.1 Structure and mechanism 15.02.1.2.2 H-cluster assembly 15.02.1.2.3 [FeFe] hydrogenase model chemistry 15.02.1.3 [NiFe] hydrogenase and their model compounds 15.02.1.3.1 Structure and functions of [NiFe] hydrogenases 15.02.1.3.2 Mechanisms for proton and hydrogen conversion in [NiFe] hydrogenases 15.02.1.3.3 Unique oxygen tolerance in [NiFe] and [NiFeSe] hydrogenases 15.02.1.3.4 Biomimetic Ni containing analogs 15.02.1.3.5 Future challenges in developing [NiFe] model complexes 15.02.1.4 [Fe] hydrogenase and their model compounds 15.02.1.4.1 Enzymatic activity, inhibitors and isolatable cofactors 15.02.1.4.2 Early ‘iron free’ hypothesis and refutation 15.02.1.4.3 Spectroscopic studies 15.02.1.4.4 Structure and mechanism of [Fe] hydrogenase 15.02.1.4.5 Model complexes of the FeGP cofactor Acknowledgments References

3 3 3 3 5 5 6 6 8 9 21 21 22 24 25 30 31 31 32 32 32 34 36 36

15.02.1 Hydrogenases and models in bioorganometallic chemistry 15.02.1.1 Introduction 15.02.1.1.1

The aim of this book chapter

Hydrogenases are enzymes involved in H2 metabolism, and provide a blue-print for how efficient H2/H+ interconversion can be achieved utilizing base metals. The societal interest in H2 as a future energy carrier, and a desire for fundamental understanding of how their biologically unique cofactors operate, has promoted intense studies of these enzymes and their related biomimetic analogs. Indeed, hydrogenase research is a striking example of how organometallic (biomimetic) chemistry and biochemistry/ biophysics has come together and enhanced each other in a truly synergistic fashion. In this book chapter, we will present an overview of efforts aimed at preparing structural and functional models of these fascinating enzymes. We will start with a brief overview of hydrogenases, highlighting shared features, and metal hydride chemistry. A more detailed presentation of each individual class and their associated model compounds will then be provided in separate subchapters, following the common division of hydrogenases, i.e. [FeFe]-, [NiFe]-, and [Fe] hydrogenases. The main contributing author to the section on [Fe] hydrogenase is M. H. Cheah, while the subchapters on [NiFe] and [FeFe] hydrogenases are primarily written by S. Glover and G. Berggren, respectively. In order to provide context for the model chemistry, each subchapter starts with a summary of our current understanding of the biochemistry of that specific hydrogenase class, with a focus on catalytic mechanism and key structural features, before moving on to the relevant model chemistry. In the case of [FeFe] hydrogenase we will also briefly describe the biosynthesis the cofactor, and show how our understanding of this process has paved the way for the merging of biochemistry and synthetic organometallic chemistry in the preparation of semi-synthetic hydrogenases.

15.02.1.1.2

General overview

Hydrogen gas (H2) is often put forward as a post-oil fuel, and large H2 infrastructure is currently being developed for the transportation sector in several countries around the globe. If produced through water-splitting it provides a convenient complement to batteries as a means of renewable energy storage e.g. wind and solar energy, thus solving the problems arising from the intermittent nature of the latter energy sources. As is so often the case, our innovations are preceded by evolution, which has already exploited the energy rich nature of the HdH bond and developed a highly efficient hydrogen economy. The interconversion between H2 and H+ is catalyzed with remarkable efficiencies by ancient gas-processing enzymes referred to as hydrogenases.1

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The first reports on the characterization of hydrogenases and their activity was published in the early 20th century,2 and since then these enzymes have been found in all domains of life.3–8 Despite the apparent simplicity of the reaction, evolution has given rise to a range of different cofactors for performing this chemistry. Consequently, hydrogenases are divided into three, phylogenetically distinct, main classes, named after the metal composition of their catalytic cofactors, i.e. [FeFe]-, [NiFe]-, and [Fe] hydrogenase. All three have received extensive attention from the synthetic community, resulting in the development of highly elaborate model compounds. An important distinction between the hydrogenases is the fact that [FeFe] and [NiFe] hydrogenases are redox enzymes catalyzing H+/H2 interconversion (Eq. 1). H2 Ð 2 H + + 2 e −

(1)

The [Fe] hydrogenase instead catalyze hydride transfer from H2 to a specific organic substrate, namely methenyltetrahydromethanopterin (methenyl-H4MPT), releasing one equivalent of H+ in the process (Eq. 2). Consequently, the latter enzyme is also known as H2-forming methylenetetrahydromethanopterin dehydrogenase, or Hmd. H2 + methenyl − H4 MPT Ð H + + methylene − H4 MPT

(2)

In particular the former two classes, in turn display a bewildering amount of structural and functional diversity resulting in their sub-classification into various groups and subclasses.3,4,6 As a case in point, Benoit et al. noted that the genome of the human parasite Trichomonas vaginalis encode no less than 13 different [FeFe] hydrogenases, of which at least 5 are present in the proteome at the same time!4,12 We are still far from exploring the full chemical space of these enzymes, and structural data is available only for a very limited subset of hydrogenases. Still, considering the well-conserved nature of the enzymatic machineries involved in their post-translational maturation we can be relatively confident that they within each main class all employ highly similar catalytic cofactors.7,8 Albeit, as will be further explained below, exceptions to this have been reported in the case of [NiFe] hydrogenase. Representative crystal structures of the three main classes of hydrogenases, highlighting their catalytic cofactors, are shown in Fig. 1. The details of each cofactor will be discussed in more depth below, but already at this stage it is important to note some striking similarities between the three classes. In a remarkable case of convergent evolution all three of these, biologically unique, cofactors contain an FeII ion, coordinated by thiolato and CO ligands. The strong field ligands e.g. CO, and in [FeFe] and [NiFe] hydrogenase also CN−, ensures a low-spin configuration of the metal site. The presence of Fe is not surprising considering the prevalence of this metal in the active-site of redox enzymes. Similarly, thiols are relatively common soft ligands in biology, in particular in the ligation of low valent metals. Conversely, CO ligands are exceptionally rare in a biological context, and only observed in a handful of gas processing enzymes.1 Considering the challenges associated with the controlled production and delivery of these toxic ligands in a living cell, the p-backbonding properties of CO are arguably critical for tuning of the electronic properties of the metals and the stabilization of these electron rich cofactors. In combination, the metals, their primary ligand spheres as well as long-range tuning by their respective active-site pockets ensure that the cofactors are capable of both acid/base and redox chemistry at mild pH and

Fig. 1 Representative examples of [FeFe]-, [NiFe]- and [Fe] hydrogenases. A schematic representation of their catalytic cofactors is shown below the respective enzyme structure with substrate H+/H− and oxidation states highlighted in red. (Panel A) The CpI [FeFe] hydrogenase from Clostridium pasteurianum (Hox state) at 1.63 A˚ resolution (PDB ascension code 4XDC, ref.9); (Panel B) The [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (Ni-R state) at 0.89 A˚ resolution (PDB ascension code 4U9H, ref.10); (Panel C) The [Fe]-hydrogenase (Hmd) from Methanococcus aeolicus in complex with FeGP and methenyl-tetrahydromethanopterin (closed form A, only one monomer of the homodimer shown) at 1.06 A˚ resolution (PDB ascension code 6HAV, ref.11). The protein backbone is colored based on secondary structure, while cofactors and substrates are shown in ball and stick format with carbons shown as pale grey, heteroatom color coding: Fe: orange; S: yellow; O: red; N: blue; P: dark orange.

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

5

potentials. In addition to the structural similarities related to the nature of the metal and its primary coordination sphere, they all feature a proton relay motif in the immediate vicinity of the metal site, resulting in similar heterolytic mechanisms with regards to H2 activation and formation. Considering the chemistry of the hydrogenases, it comes as no surprise that metal hydrides and proton coupled electron transfer processes are of central importance to their reactivity. The fact that evolution has developed highly similar structural motifs not once, but on at least on three separate occasions, underscores the remarkable properties of Fe-thiolatocarbonyls for hydride chemistry and H2 activation.

15.02.1.1.3

Metal hydrides

The activation of H2 by transition metals can occur via two different mechanisms. In classical organometallic hydrogenation catalysts the most commonly proposed mechanism is homolytic cleavage, where oxidative addition of H2 results in the formation of two metal hydrides and the formal oxidation-state of the metal center (M) is increased by +2 (Scheme 1).

Scheme 1 Oxidative addition of H2 to a metal center (M) via homolytic H2 cleavage. Change in metal oxidation state indicated in red.

Alternatively, the binding of H2 to the metal can provide a sufficient increase in the acidity of H2 to enable a heterolytic cleavage, resulting in the addition of one hydride ligand to the metal with concomitant release of a proton (Scheme 2). In this scenario, the oxidation state of the metal remains unchanged in process. The feasibility of this reaction is obviously dependent not only on the electrophilic properties of the metal center, but also the presence of a suitably positioned Brønstedt base capable of extracting the proton. In hydrogenases this reaction is well orchestrated through the positioning of suitable proton relays in close proximity to the site of H2 coordination.

Scheme 2 Addition of a hydride ligand to a metal center (M) via heterolytic H2 cleavage. Change in metal oxidation state indicated in red.

The [NiFe] and [FeFe] hydrogenases are also capable of forming metal hydrides through protonation, i.e. the oxidative addition of a proton to the metal with a concomitant increase in the metal oxidation state by +2 (Scheme 3A). In the case of bimetallic metal sites, it should be noted that hydrides can form in either terminal position, or in a bridging position as outlined in Scheme 3B and C (in which it is assumed that both metal ions become oxidized upon hydride formation).

Scheme 3 Formation of metal hydrides through oxidative addition of a proton, to a mononuclear metal site (A); to a dinuclear metal site to form a terminal hydride ligand (B) or a bridging hydride ligand (C). Changes in metal oxidation state indicated in red.

Albeit formally negatively charged, the reactivity of such metal hydrides can vary greatly, resulting in reactivity more like protons or hydrides. In short, depending on the oxidation state and electron density of the metal they can be considered either electrophilic (facilitating their release as a proton), or nucleophilic (resulting in e.g. H2 formation in the presence of a proton source).

15.02.1.1.4

The role of proton-coupled electron transfer in H2/2H+ interconversion

Proton-coupled electron transfer (PCET) reactions are increasingly recognized for their prevalence in biological and chemical redox reactions.13 Coupling electron and proton transfer in a redox process can bring the advantage of lowering the activation energy and driving force for the reaction, which may result in more favorable reaction rates. This is of particular importance in the catalysis of H2/2H+ interconversion where two PCET events, are possible. PCET can proceed by either a stepwise or a concerted mechanism (Fig. 2). In the stepwise mechanisms, PTET or ETPT, proton transfer precedes electron transfer, or an electron transfer precedes proton transfer, respectively. In the concerted mechanism, CEPT, electron and proton transfer occurs when both particles tunnel through a common transition state in a single kinetic step. Hydrogen atom transfer (HAT) is a special type of concerted reaction where H
is transferred from the same bond of the donor and arrives at the same bond on the acceptor. Distinct intermediates states are formed in the stepwise mechanisms; reaching these states may involve a significantly energetically uphill step to form a very reactive intermediate. CEPT, bypasses the intermediate states, which imparts an energetic and kinetic advantage to the PCET reaction.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 2 A general square scheme showing the different mechanisms of proton-coupled electron transfer (PCET) between a donor, D, and acceptor, A. The stepwise pathways, ETPT and PTET, are shown along the edges. The concerted mechanism, CEPT, bisects the square representing a reaction that proceeds through a single transition state and does not form an intermediate.

The PCET mechanism taken is determined by the free energies of electron and proton transfer, DG ET and DG PT, respectively, as well as the electronic and proton coupling. These parameters can be tuned in the protein environment by changing the types of chemical species acting as electron/proton donors and acceptors, and the distances between them. Specifically, DG ET is defined by the difference in reduction potentials between the electron donor and acceptor, while DG PT is taken from the difference in pKa between proton donor and acceptor. Changing the distance between donor and acceptors will modulate the electronic and proton coupling, which is a determining factor in the tunneling probability.13 The rapid H2/2H+ interconversion in hydrogenases indicates that proton and electron transfer is highly optimized. Hydrogenases are thus fascinating and instructive candidates for mechanistic studies of PCET. To elucidate how hydrogenases use PCET to achieve rapid catalysis makes it possible to apply and test such principles in artificial systems, even for catalysis beyond hydrogen evolution. To date a small number of studies of PCET mechanisms have been undertaken for [NiFe] and [FeFe] hydrogenases.14–16 Given the important role of catalysis in future global energy solutions, further mechanistic investigations into H2/2H+ interconversion of hydrogenases could bring forth a wealth of new knowledge to advance the field of catalysis.

15.02.1.2 [FeFe] hydrogenases and their model compounds 15.02.1.2.1

Structure and mechanism

Identification of the unusual CO and CN− ligands by FTIR spectroscopy predated the crystal structure,17–19 while the definite assignment of the bridgehead amine was later achieved through spectroscopy in combination with biomimetic model chemistry.20,21 Nevertheless, there is no doubt that the first reports of the structure of [FeFe] hydrogenase in the late 1990s had a huge impact on the bioinorganic chemistry and biomimetic organometallic communities, as they revealed the presence of an unprecedented hexanuclear iron cofactor in the active site, the hydrogen activating cluster or “H-cluster”.22,23 In terms of catalytic rates, the [FeFe] hydrogenases are considered the fastest among the hydrogenases, with turn-over-frequencies above 104 s−1 reported. However, it is important to realize that [FeFe] hydrogenase represent a very diverse family of enzymes, serving a range of different functions related to signaling and H2 metabolism, and different members of this enzyme family display very different catalytic activities. Different classification schemes exist, but commonly [FeFe] hydrogenases are subdivided into four main groups A-D. These groups are further divided into different subclasses based on the presence of additional domains beyond the H-cluster containing “H-domain.”5 The H-cluster can be regarded as consisting of a canonical [4Fed4S] cluster ([4Fed4S]H), which in turn is connected via a bridging cysteine thiolate ligand to a diiron complex, denoted the [2Fe]H subsite. The organometallic [2Fe]H subsite is unique in biology and the site of catalysis. More specifically, each Fe ion of the [2Fe]H subsite is coordinated by one cyanide (CN−) and one carbonyl ligand, and the dimeric nature of the complex is enforced by one bridging CO ligand and a bidentate aza-dithiolate ligand (−SCH2NHCH2S−, adt).20,21 The Fe ions of [2Fe]H are further specified as the proximal and distal Fe, respectively, referring to their distance from the [4Fe4S]H cluster. In addition to the primary ligand sphere, a key structural aspect of the H-cluster is the so-called “rotated structure” of the distal Fe, which provides an open coordination site promoting the formation of a terminal hydride (t-H−) during catalysis. This specific structure is stabilized in the active-site, via H-bonding interactions between the CN− ligands and specific amino acids. The latter interaction also serves to tune the electron donating capacity of the anionic CN− ligands, and by extension the reduction potential(s) of the H-cluster. The catalytic mechanism of [FeFe] hydrogenase has been intensively studied by multiple groups over decades. Numerous redox and protonation states of the H-cluster have been identified and characterized, most commonly under pseudo-steady state

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

7

Fig. 3 Proposed catalytic cycle of [FeFe] hydrogenase. All states have been characterized under in vitro conditions, and those also observed under whole-cell conditions are labelled in blue. Substrate H+ and H− species labelled in red. Adapted from Mészáros, L. S.; Ceccaldi, P.; Lorenzi, M.; Redman, H. J.; Pfitzner, E.; Heberle, J.; Senger, M.; Stripp, S. T.; Berggren, G., Chem. Sci. 2020, 11(18), 4608–4617 and Birrell, J. A.; Pelmenschikov, V.; Mishra, N.; Wang, H.; Yoda, Y.; Tamasaku, K.; Rauchfuss, T. B.; Cramer, S. P.; Lubitz, W.; DeBeer, S., J. Am. Chem. Soc. 2020, 142(1), 222–232.

conditions. The most common techniques for probing the metals directly include Electron paramagnetic resonance (EPR) and Mössbauer spectroscopy; while the diatomic ligands (CO and CN−) provide excellent spectroscopic handles for Fourier transformed infrared (FTIR) spectroscopy. Albeit less common, important contributions have also recently been made using e.g. Nuclear Vibrational Resonance Spectroscopy and (NRVS) and NMR. Multiple mechanisms have been proposed around these states, and they are still continuously refined as more structural and spectroscopic data become available.5,24 In parallel to the spectroscopy, detailed kinetic information has been obtained from protein film electrochemistry.25–33 We will summarize the current knowledge around H-cluster chemistry based on what we consider the most commonly adopted mechanism (Fig. 3),34,35 but note that alternative proposals are currently discussed in the literature.36–38 The most oxidized state of the catalytic cycle is the “Hox-state,” which also is the resting state in the vast majority of [FeFe] hydrogenases characterized to-date.24 The Hox-state features a mixed valent [2Fe]H subsite (II,I), and an oxidized [4Fe-4S]H cluster (+2) (paramagnetic with spin, S ¼ ½). One electron reduction of Hox generates the Hred state, with a reduced [4Fe-4S]H cluster (+1) (S ¼ 0).39–41 Protonation of the adt-amine with concomitant re-distribution of the H-cluster electron density results in the HredH+ state, best described as a reduced [2Fe]H subsite (I,I) and an oxidized [4Fe-4S]H cluster (S ¼ 0). Further one-electron reduction generates the “super reduced” state, denoted Hsred. Again, the electron enters the system via the [4Fed4S]H cluster, and the system is best described as a reduced homovalent [2Fe]H subsite (I,I) coupled to a reduced [4Fe-4S]H cluster (S ¼ ½).42,43 The Hhyd state is formed via intramolecular proton transfer from the adt-amine to the distal Fe of [2Fe]H to give a terminal hydride ligand, with concomitant oxidation of the [2Fe]H subsite to II,II (S ¼ 1/2).44–47 Protonation at the adt-amine of the HHyd intermediate results in HHydH+. The latter state is to-date the least characterized of the proposed catalytic intermediates, but the available spectroscopic data supports a model where the [4Fed4S]H cluster remains reduced following the protonation (the S ¼ ½ state is retained following amine protonation).45,48 Nucleophilic attack by the Fe bound hydride on the adt-ammonium proton generates H2, and returns the H-cluster to Hox. During H2 oxidation the same cycle is expected to operate in reverse, with the second sphere amine group playing a critical role in activating the H2 molecule. The adt-amine serves as a Brønstedt base during H2 activation while Fed accepts the hydride ion, i.e. the Fed ion and adt-amine can be regarded as a frustrated Lewis pair promoting heterolytic H2 cleavage. Many of the catalytic intermediates proposed in Fig. 3 have also been observed under whole-cell conditions, supporting their physiological relevance.48 In order to gain deeper insight into the mechanism various photo-triggers have been explored, taking advantage of the intrinsic photochemistry of iron-carbonyls or by coupling the enzyme to artificial photosensitizers.49–51 This has enabled transient spectroscopy studies, which have verified the kinetic competence of many, albeit not all, of the intermediates outlined in Fig. 3.49 In addition to the proposed catalytic intermediates, a number of inhibited states have been reported. Carbon monoxide (CO) can reversibly bind to the [2Fe]H subsite, resulting in the Hox-CO and Hred-CO states,41 and similar observations have been reported for formaldehyde.52 In contrast to CO,

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

O2 is an irreversible inhibitor of [FeFe] hydrogenase, causing degradation of the H-cluster. However, coordination of a sulfide ligand under mildly oxidizing conditions generates the so-called Htrans state, which in turn is reversibly oxidized to the Hinact state. Critically, this form of the H-cluster is insensitive to O2 attack, and can re-enter the catalytic cycle upon reduction under anaerobic conditions.53–56 As outlined above, the mechanism of [FeFe] hydrogenase has been elucidated in great detail, and quite possibly an intermediate corresponding to each elementary reaction step has been identified, with the notable exception of an H2 bound species. Still, numerous challenges remain in this field. In addition to developing conditions for trapping the missing H2 bound intermediate, more detailed time-resolved studies are clearly needed to solidify the proposed mechanism, and to facilitate the identification of any transient states that have so far eluded detection. In parallel, it should be noted that the vast majority of mechanistic studies have been performed on a very limited subgroup of the enzyme family, i.e. the “prototypical” group A [FeFe] hydrogenases.5 Thus, it remains to be verified whether or not the same mechanism holds true for all groups of [FeFe] hydrogenases.

15.02.1.2.2

H-cluster assembly

Unsurprisingly the assembly of the H-cluster is not a spontaneous process, but is instead dependent on a range of accessory proteins.57 The first step in the process is the assembly of the [4Fed4S]H cluster to generate “apo-[FeFe] hydrogenase”. This is handled by the house-keeping FeS cluster assembly machinery of the cell, and the [4Fed4S]H containing form is also what is generally obtained following heterologous over-production of the enzyme in standard expression hosts like E. coli.58,59 A significant breakthrough in the [FeFe] hydrogenase field was the identification of the genes encoding the proteins HydG and HydEF (the latter observed as two different proteins, HydE and HydF, in many organisms) reported in 2004. The HydE, -F, -G proteins are found in all [FeFe] hydrogenase expressing organisms and essential for complete H-cluster assembly. Indeed, when co-expressed together with an [FeFe] hydrogenase-encoding gene an active [FeFe] hydrogenase can be obtained also from E. coli, an organism which does not carry a native [FeFe] hydrogenase and thus lacks the H-cluster assembly machinery.60,61 The first reports on in vitro biochemical characterization of these three enzymes quickly followed,62,63 and it was shown that HydE and HydG belong to the radical S-adenosyl methionine (SAM) enzymes superfamily while HydF is a GTP:ase enzyme featuring a [4Fed4S] cluster. It is now well-established that HydF acts as a chaperone. Through the combined activities of HydE and HydG, a pre-catalyst ([(m-adt) Fe2(CN)2(CO)4]2−, 2.1) is assembled on HydF, and from there transferred to apo-[FeFe] hydrogenase to generate the active enzyme.20,64–68 HydG is the most well-characterized of the two radical SAM enzymes.69–76 It catalyzes the formation of CO and CN− from tyrosine, to form a mononuclear Fe complex, or “synthon,” [Fe(CO2)(CN)(cys)], (2.2). The aforementioned synthon is also the source of the S-atoms of the adt ligand, while the CH2 groups and the amine-bridgehead are derived from serine in a HydE catalyzed reaction.77–80 It is tempting to assume that the fusion of two synthons to form the complete pre-catalyst occurs on HydF in close interaction with HydE, but this remains to be experimentally verified. In a wonderful example of the interdisciplinary nature of hydrogenase research, it has been shown that both the proposed dinuclear pre-catalyst, and the HydG produced mononuclear synthon can be replaced by synthetic analogs, enabling partial as well complete replacement of the HydEFG machinery by synthetic chemists.20,81,82 The possibility to activate apo-[FeFe] hydrogenase using 2.1 has been used as a tool for studying the final steps of H-cluster assembly, i.e. the fusion between 2.1 and [4Fe-4S]H, both spectroscopically and using electrochemistry.83,84 The coupling reaction is spontaneous, and can be considered to occur via an associative mechanism. It requires that the [4Fed4S]H cluster resides in its oxidized +2 state, as a critical step appears to be an electron transfer from the dinuclear Fe complex to the [4Fed4S]H cluster to generate an intermediate similar, or identical, to Hred-CO. Rapid oxidation of the H-cluster at this stage generates the Hox-CO state, followed by relatively slow CO release to form the active-ready Hox state (Fig. 4).

Fig. 4 Schematic representation of the final steps of H-cluster assembly. The pre-catalyst, 2.1, is delivered by HydF and inserted into the active-site of apo-[FeFe] hydrogenase, where it spontaneously fuses with the [4Fed4S]H cluster. The reaction proceeds via an associative mechanism, with binding of the cysteine thiol producing an Hred-CO like intermediate. Subsequent oxidation and dissociation of a CO ligand generates the “active-ready” Hox-state. Oxidation states are indicated in red.

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15.02.1.2.3

9

[FeFe] hydrogenase model chemistry

The structural elucidation of the H-cluster and the low valent iron-dithiolate complex, [2Fe]H, resulted in intense activities in the organometallic community, which continue to this date. However, it is important to note that synthetic efforts related to preparing thiolate coordinated diiron carbonyl complexes ([2Fe]) have been developed since the 1920s, and thus significantly predates the identification of the H-cluster. Key early examples include e.g. the 1928 report from Reihlen et al. on the synthesis of [(m-SEt)2Fe2(CO)6] (2.3),85 and the propanedithiolate (−SCH2CH2CH2S−, pdt) bridged hexacarbonyl diiron complex ([(m-pdt)Fe2(CO)6], 2.4) reported by Seyferth et al in 198786 (Fig. 5). The rich iron carbonyl chemistry, developed largely unrelated to the hydrogenases, will not be covered herein, but is nicely summarized in e.g. COMC(2007) in the chapter by G. Hogarth entitled “Dinuclear Iron Compounds with Iron-Iron Bonds”.87 Here we will instead focus more strictly on the [2Fe]H biomimetic models, i.e. diiron complexes that: (i) reproduce the primary ligand sphere; (ii) reproduce the rotated structure of the “distal-Fe”; (iii) introduce a proton-relay in the second coordination sphere; (iv) introduce a redox active ligand mimicking the [4Fed4S]H cluster; (v) promote the formation of terminal over bridging hydrides; and vi) mimicking the reactivity of hydrogenase. 15.02.1.2.3.1 Di-cyanide containing models and variations of the bridging di-thiolate ligand Much of the early work related to preparation and characterization of biomimetic iron-carbonyl complexes came from the laboratories of Rauchfuss, Pickett and Darensbourg. Indeed, by 1999 all three of the aforementioned groups reported the successful preparation of the dicyanide analog of 2.4, [(m-pdt)Fe2(CO)4(CN)2]2− (2.5), readily obtained by treating 2.4 with two equivalents of CN− (Fig. 5). Shortly thereafter, the related [2Fe] models with an amine (adt) or oxygen (− SCH2OCH2S−, odt) bridgehead were also reported. Similarly to 2.5, the dicyanide complexes [(m-adt)Fe2(CO)4(CN)2]2− (2.1) and [(m-odt)Fe2(CO)4(CN)2]2− (2.6), respectively, were synthesized from their corresponding hexacarbonyls [(m-adt)Fe2(CO)6] (2.7) and [(m-odt)Fe2(CO)6] (2.8) (Fig. 6).88,89 The synthetic community has continued to expand the library of adt-like bridging ligand variants, and the repertoire of [2Fe]H mimics now also include e.g. S, Se and Sn bridgeheads. In parallel, the bridging thiolates have been exchanged, and the diphosphido, diselenoates and ditelluroates analogs have been reported.91–97 The chemistry of these latter variants will be briefly discussed in Section 15.02.1.2.3.2. Apart from the cysteine thiol ligand, complexes 2.1, 2.5 and 2.6 all share a striking resemblance to the [2Fe]H subsite with regards to primary ligand sphere. Moreover, as homo-valent Fe(I)Fe(I) dimers they are identical to the proposed catalytic intermediate HredH+ state in terms of oxidation state. However, it quickly became apparent that these dianionic complexes are not suitable as standalone catalyst for H+/H2 interconversion. Although stable in aqueous buffer, they display very negative reduction potentials. Indeed, comparing 2.4 and 2.5, substituting two CO ligands for CN− causes a shift of >1 V for both oxidation (1.3 V less positive) and reduction (1.2 V more negative).98 Moreover, while the all carbonyl variants 2.4 and 2.7 are too electron poor to readily form metal hydrides in their I,I oxidation state, the cyanide substituted complexes instead rapidly degrade upon protonation in organic solvent.99 In a historical context it is also noteworthy that the nature of the bridgehead group was a matter of debate for at least 10 years following the publication of the crystal structures, and 2.1, 2.5 and 2.6 were all consequently considered as potential representations of the [2Fe]H subsite. The first experimental evidence for the presence of an amine bridgehead was provided in 2009 through HYSCORE spectroscopy, supported by comparisons to model complexes.21,100 In 2013 it was finally unequivocally proven through the development of the artificial maturation technique.20 As will be further discussed below, incorporation of 2.1 into apo-[FeFe] hydrogenase resulted in a semi-synthetic [FeFe] hydrogenase indistinguishable from the native enzyme, while 2.5 and 2.6 afforded enzyme variants locked in specific oxidation states. 15.02.1.2.3.2 Replicating the rotated structure Albeit the ligand sphere of 2.1, 2.5 and 2.6 closely mimic that of the [2Fe]H they proved challenging to stabilize in the rotated structure of the distal Fe, with a bridging CO ligand, (Fig. 7A), and to stabilize in the mixed valent Fe(II)Fe(I) form analogous to the Hox state.101 From a catalyst design perspective the former point is critical, as it positions the open coordination site closer to the apical nitrogen of the adt-bridge in the [2Fe]H cluster. Thereby facilitating proton-shuttling and providing a suitable binding site for H2 to enable heterolytic HdH bond cleavage. The Fe ions of homo-valent Fe(I)Fe(I) complexes like 2.1-2.8 are 18 electron species due to the presence of an FedFe bond that stabilizes the non-rotated “square pyramid/square pyramid” structure generally observed for such complexes (Fig. 7B). One electron

Fig. 5 Representative examples of [2Fe]H mimics predating the crystal structure of [FeFe] hydrogenase (2.3 and 2.4), and the first dicyanide complex reported shortly after the structural elucidation of the H-cluster (2.5).

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Fig. 6 Schematic representation of the synthesis of dicyanide [2Fe] complex featuring amine (2.1) or oxygen (2.6) heteroatom bridgeheads, as reported in.88,89 Alternative synthetic routes have been reported. The synthesis of the common di-m-sulfido precursor was reported by Seyferth et al. in 1981.90

reduction or oxidation weakens the FedFe bond, and thus facilitates formation of the rotated structure. This observation, in combination with the introduction of steric bulk into the ligand sphere, has now enabled the preparation of such rotated structures, as represented by the Hox-state mimics [(m-S2C2H4)Fe2(CO)3(PMe3)(dppv)]+ (2.9, dppv ¼ 1,2-bis(diphenylphosphino)ethene) and [(m-dmpdt)Fe2(CO)4(PMe3)2]+ (dmpdt ¼2.2-dimethyl-1,3-propanedithiolate) 2.10 (Fig. 7C).102–104 The Hox model 2.9 was also found capable of mimicking the reactivity towards CO, by reversibly forming the Hox-CO mimic 2.9-CO in the presence of excess CO gas at low temperatures.103 Parallel studies on Se and Te variants of complex 2.4 has shown that chalcogenide substitution provides the expected increased electron density on the Fe ions, and should thus stabilize oxidized mixed-valent states. However, formation of the corresponding bridging carbonyl species is hindered by the increased FedFe distance imposed by the later chalcogenides.92 As predicted from density functional theory,105 bulky ligands, shielding the vacant coordination site, in combination with unsymmetrical substitution of the two Fe ions subsequently also allowed the generation of rotated structures in the homovalent Fe(I)Fe(I) oxidation state. Representative examples of such models, [(m-depdt)Fe2(CO)4(dppv)] (depdt ¼2.2-diethyl-1,3propanedithiolate, 2.11) and [(m-(SCH2)2NBn)Fe2(CO)4(dmpe)] (Bn ¼ benzyl; dmpe ¼ 1,2-bis(dimethylphosphino)ethane, 2.12), are shown in Fig. 8.106–108 These models show that a rotated structure featuring a (semi-)bridging CO species can indeed be stabilized also in small molecule system, providing a design method for placing the open coordination-site in close vicinity of the bridgehead Brønstedt base. In an enzyme context, this work shows that such bridging CO ligands can be stabilized in both the biologically relevant Fe(I)Fe(I) and Fe(II)Fe(I) oxidation state configurations, as proposed in the mechanism of the H-cluster outlined in Fig. 3. Moreover, the necessity to employ highly unsymmetrical ligand spheres to stabilize these states in the models underscore the crucial role of the active-site pocket to stabilize this “rotated” form of the [2Fe]H subsite.

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Fig. 7 Schematic representation of the [2Fe]H subsite featuring the rotated distal Fe center (A) and the early dicyanide mimic 2.1 featuring a “non-rotated” structure (B), highlighting the structural difference between Fed and the mimic. Panel C shows two representative examples of synthetic mimics (2.9 and 2.10) reproducing the rotated structure of Fed. Adapted from reference Singleton, M. L.; Bhuvanesh, N.; Reibenspies, J. H.; Darensbourg, M. Y., Angew. Chem. Int. Ed. 2008, 47(49), 9492–9495.

Fig. 8 Schematic representation of Hred mimics (2.11 and 2.12) featuring a rotated Fe center.

15.02.1.2.3.3 Mimicking the [4Fed4S]H cluster - redox active ligands The [2Fe]H subsite mimics outlined above replicate a number of critical structural features related to the native cofactor. In order to generate even closer models, attempts have also been reported at introducing a redox active ligand mimicking the function of the [4Fed4S]H cluster. As the latter acts as a non-innocent, redox active, ligand during catalysis, this could potentially provide a path toward even better catalysts operating at more modest over-potentials. Synthetic models of [4Fe-4S] clusters have been extensively studied,109,110 but the first system fusing a [4Fed4S] cluster and a [2Fe]H unit to generate a complete H-cluster model was reported by Pickett and coworkers in 2005.111 The latter model featured a cubane [4Fed4S] cluster capped by a bulky tridentate thiolate ligand (¼ 1,3,5-tris (4,6-dimethyl-3-mercaptophenylthio)-2,4,6-tris (p-tolyl-thio)benzene), providing an open coordination site on one Fe ion to which a [2Fe3S]-model was bound via a thiol ligand to yield complex 2.13 depicted in Fig. 9. Cubane [4Fed4S] clusters are also known to self-assemble in cysteine containing oligopeptides.112–114 This was exploited by Esmieu et al to generate a “miniaturized” [FeFe] hydrogenase by assembling a complete H-cluster model in a 16 amino acid synthetic peptide (Fig. 9, 2.14), via spontaneous fusion of a pre-assembled [4Fe-4S] cluster and complex 2.1, i.e. analogous to the reaction occurring during H-cluster assembly (Fig. 4).115 Dinuclear Fe complexes have also been linked to e.g. fullerenes,116 ferrocene117–119 and phospholes,120 in efforts to exclusively mimic the function, rather than structure, of the [4Fed4S]H cluster. Both 2.13 and 2.14, are capable of catalytic H2 production, in organic and aqueous solution, respectively. In the case of 2.14 the beneficial effects of the [4Fed4S] cluster on the reactivity is readily apparent, as complex 2.1 is catalytically inactive in isolation. However, arguably the most illustrative example of a system directly involving a redox active ligand in H2/H+

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Fig. 9 Representative examples of model complexes that incorporate redox active ligands and display hydrogenase like activity. In 2.14 the cysteine ligands originate from a synthetic ferredoxin maquette (FdM). Both complexes 2.15 and 2.33 display outstanding catalytic properties, attributable in part to the presence of their redox active ligands (FcP and phosphole, respectively).

interconversion is provided by the complex [(m-(SCH2)2NBn)Fe2(CO)3(FcP )(dppv)] (FcP ¼ Cp Fe(C5Me4CH2PEt2)), in which a ferrocene derivative is linked to a dppv coordinated diiron complex via a monodentate phosphine ligand (Fig. 9, 2.15). The catalytic properties of 2.15, as well as the phosphole containing complex (2.33) will be further discussed in Section 15.02.1.2.3.5. 15.02.1.2.3.4 Ligand and metal protonation sites of [2Fe]H models Considering the crucial role of hydrides in the catalytic cycle, and the presence of the bridgehead amine in [2Fe]H, it is not surprising that the protonation chemistry of biomimetic [2Fe] complexes has received extensive attention. For detailed reviews on [2Fe]H mimics and their protonation/hydride chemistry, as well as their spectroscopic properties see e.g. refs.121 and 122. Herein we will provide a brief summary of this chemistry, and highlight representative examples. In contrast to the aforementioned dicyanide complexes, the structurally homologous monocyanide substituted adt complex [(m-adt)Fe2(CO)5(CN)]− (2.16), is catalytically active in both aqueous and organic solutions.123 Similarly, the monocyanide, monophosphine, substituted pdt complex [(m-pdt)Fe2(CO)4(CN)(PMe3)]− (2.17, Fig. 10), is capable of electrocatalytic H+ reduction to form H2.124 In the latter case, addition of a strong acid results in the formation of a bridging hydride species [(m-pdt)(m-H−) Fe2(CO)4(CN)(PMe3)]+ (2.18). Hydride formation results in an increase of the formal oxidation state of the metals to II,II

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Fig. 10 Site-selective protonation of [2Fe]H model complexes controlled by the basicity of functional groups in the vicinity of the di-Fe core. Protonation sites highlighted in red for clarity.

(analogous to the reaction outlined in Scheme 3C), with a concomitant a shift of reduction potential by approx. 1 V less negative and a hypsochromic shift of the n(CO) bands by about 60 cm−1. Addition of a second equivalent of acid results in protonation of the cyanide nitrogen, readily observable from a second, smaller, hypsochromic shift of the n(CO) bands by about 10 cm−1, and an additional shift of 100 mV toward less reducing potentials. Protonation of the analogous di-phosphine ([Fe2(pdt)(CO)4(PMe3)2] (2.19), affords the m-hydrido species [(m-pdt)(m-H)Fe2(CO)4(PMe3)2]+) but the complex is not catalytically active. These observations highlight a number of relevant points related to [2Fe] H-cluster mimics and their catalytic properties. In short, this family of diiron complexes generally feature multiple potential protonation sites, including not only the metals (to generate hydrides) but also ligand protonation. With regards to hydride formation it is important to note that replacing p-accepting CO ligands with electron donating ligands (e.g. cyanides, phosphines and carbenes) increases the electron density of the Fe ions to promote protonation and formation of metal hydrides via oxidative addition at the I,I oxidation state. The thermodynamically favored product in these cases are generally bridging hydrides. Moreover, albeit relatively simple [2Fe] complexes like 2.16 and 2.17 are capable of (electro)catalytic proton reduction, such complexes generally require significant over-potentials. Finally, the observation that the cyanide substituted complex 2.17 is catalytically active, while the di-phosphine 2.19 is not, underscores the

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importance of a Brønstedt acid/base functional group in close proximity to the metal for efficient catalysis. As their potential application for H2/H+ catalysis is a major motivation for studying hydrogenase derived biomimetic complexes, minimizing their over-potential requirement, while retaining a high turn-over frequency (TOF), is a key objective. Clearly, this is closely tied to controlling the protonation chemistry. A second representative example of the reactivity of [2Fe]H model complexes toward protons, and their different potential protonation sites, was reported by Ott and coworkers in 2010 (Fig. 10).125 The incorporation of di-phosphine ligands featuring different substituted amines (2.20, 2.21) or lacking an amine (2.22), allowed selective protonation of the amine (2.23), the metals (2.24) or a bridging thiolate ligand (2.25). A related system reported by Sun and coworkers illustrate the influence of second coordination sphere Brønstedt bases also on the reactivity of diiron m-hydride species (Fig. 11).126 The addition of a diphosphine containing an amine bridge, N-nPr, (nPr ¼ CH2CH2CH3) to complex 2.4 gives the unsymmetrically substituted complex [(m-pdt)Fe2(CO)4((PPh2CH2)2N-nPr)] (2.26). Upon treatment with two equivalents of a strong acid in non-coordinating solvent, the m-hydride, ammonium salt species [(m-pdt) (m-H)Fe2(CO)4((PPh2CH2)2NH-nPr)]2+ (2.27) is quantitatively formed. The m-hydride is readily observable by 1H NMR with a signal at d −13.0 ppm (such m-hydrides are commonly observed between −8 and −20 ppm). The latter signal was rapidly lost when 2.27 was treated with an excess of D+, with a new high field signal attributed to the m-deuteride appearing in the 2H NMR. In contrast, no H+/D+ exchange was observed for the analogous [2Fe] complex that lacks a protonatable bridgehead in the phosphine ligand ([(m-pdt)(m-H)Fe2(CO)4((PPh2CH2)2CH2)], 2.28). Moreover, the hydride of 2.27 was readily removed by the addition of a weak base, while 2.28 was inert. A similar dependence on second coordination sphere proton relays on the acid/ base chemistry of the diiron unit has been reported for phosphine substituted variants of 2.4, 2.7 and 2.8, featuring different bridgehead atoms in the dithiolate bridging ligand.127 Thus, not only do these types of diiron complexes feature multiple potential protonation sites, the presence and chemical nature of a second coordination sphere Brønstedt base can significantly alter the reactivity of the metal-bound hydride ligand. 15.02.1.2.3.5 Bridging versus terminal hydrides and their involvement in catalysis As presented in the previous examples, [2Fe]H model complexes feature a range of potential protonation sites. Moreover, the geometry of the metal hydride species is critical in the context of catalysis. As bridging and terminal hydrides display very different reactivity in this family of complexes. The first spectroscopic characterization of dithiolato-diiron hydride complexes was published already in 1976, with Fauvel et al.’s report on three structurally similar m-hydride complexes [(m-SMe)2(m-H)Fe2(CO)4 (PPhnMe3-n)2]+ (n ¼ 0–2, 2.29-2.31).128 The electron donating nature of the phosphine ligands (PPhnMe3-n) enabled their synthesis through protonation of the respective Fe(I)Fe(I) diphospine. In the wake of the crystal structure of [FeFe] hydrogenase, such bridging hydride species have been implicated in numerous [2Fe] biomimetic H2 producing catalytic systems. In many cases high turn-over frequencies for H+ reduction have been estimated from electrochemical experiments.129,130 Two representative mechanistic studies of such systems were reported during 2018–2019.131–133 A key aspect of these latter reports is the direct spectroscopic detection of the catalytic intermediates by transient spectroscopy, via a combination of FTIR spectroscopy and rapid-mixing stopped flow or flash-induced reduction. This ensures a solid experimental foundation for the mechanistic discussion. Moreover, the studies focused on diiron complexes either lacking (Fig. 12), or including (Fig. 13) a Brønstedt base in the second coordination sphere. Thus the two examples elucidate the influence of the protonation chemistry of the bridging dithiolate ligand on overall catalyst performance. A schematic summary of the catalytic cycle of [(m-Cl2-bdt)Fe2(CO)6] (Cl2-bdt ¼ 3,6-dichloro-1,2-dithiolato-benzene; 2.32) is shown in Fig. 12.131,133 As expected from the hexacarbonyl coordination motif the Fe ions of the parent complex are too electron poor to readily protonate. One electron reduction of 2.32 gives 2.32−. The increased electron density on the Fe ions is reflected by a down-shift of the CO ligand vibrations by 40–60 cm−1 due to back-donation into the CO p orbitals, and the increased basicity of the anionic 2.32− relative to 2.32 enables protonation to generate the m-hydride species [2.32(m-H−)] through an oxidative addition (Eq. C in Scheme 3). It is noteworthy that the combined effects on the FTIR spectrum of reduction and protonation practically cancel each other, making the identification of [2.32(m-H−)] to some extent indirect due to the striking spectroscopic similarity to the parent compound 2.32. A second reduction generates [2.32(m-H−)]−, which formally constitutes a homovalent Fe(I) dimer. The hydride is a rather weak nucleophile in this state, but can react with strong acids to form H2, thus closing the catalytic cycle. Under electrochemical conditions or in the presence of a potent chemical reductant the formation of [2.32(m-H−)]− can also proceed via the dianionic intermediate 2.322−.

Fig. 11 Schematic representation of the H+/D+ (and H−/D−) exchange observed for complex 2.27.

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Fig. 12 Schematic depiction of the catalytic cycle for H2 production from 2.32. All proposed intermediate species except [2.32(H2)] (shown in grey) have been observed by spectroscopy, where reported the rate constants for the individual steps are indicated (determined using tosylic acid as proton source and [Ru(bpy)3]+ as reductant). Figure adapted from reference Wang, S.; Pullen, S.; Weippert, V.; Liu, T.; Ott, S.; Lomoth, R.; Hammarström, L., Chem. Eur. J. 2019, 25(47), 11135–11140.

In the case of [(m-adt)Fe2(CO)6], 2.7, the situation is more complex due to acid/base chemistry of the adt-amine and best described by a three dimensional reaction scheme as outlined in Fig. 13.132 In the presence of weak acids, complex 2.7 catalyzes H2 formation via an initial electron transfer step, as observed also for complex 2.32. When stronger acids are employed, the reaction sequence is instead initiated by protonation of the adt-ligand to form [2.7(H+)]+ (pKa ¼ 8, determined in MeCN). This protonation step is readily monitored by FTIR from a hypsochromic shift of 15–20 cm−1 observed for the CO ligand vibrations, typical for protonation of the adt-bridgehead amine in related diiron complexes. However, despite subsequent reduction to Fe(I)Fe(0), the ammonium proton does not shuttle to the metal to form the corresponding [2.7(H−)] hydride, as proposed for the enzyme in the Hsred to the Hhyd transition (Fig. 3). More specifically, the [2.7(H+)] to [2.7(H−)] tautomerization does not occur on a time-scale relevant to catalysis (ktautomerization < 1 s−1, and reported TOF for H2 evolution 103–104 s−1).129,132 This is arguably due to the bridging nature of the resulting hydride, causing steric hindrance to the intramolecular proton transfer. Still, the presence of a protonatable ligand is highly beneficial for catalysis, as protonation shifts the reduction potential to form the species [2.7(H+)] by >300 mV less negative relative to reduction of [2.7] to form [2.7]−. A second protonation step occurs through the expected oxidative addition to the Fe dimer to give the bridging hydride [2.7(H+)(m-H−)]+. Importantly, metal protonation occurs with similar rate constants for the already amine protonated [2.7(H+)] species as for the reduced, but non-protonated, propyldithiolate bridged analog, 2.4−. Finally, and as in the case of 2.32 a second reduction step increases the nucleophilicity of the m-hydride species

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Fig. 13 Schematic depiction of the protonation chemistry of 2.7 and the anionic 2.7−. Intermediate species in black have been observed by spectroscopy. Figure adapted from reference Aster, A.; Wang, S.; Mirmohades, M.; Esmieu, C.; Berggren, G.; Hammarström, L.; Lomoth, R., Chem. Sci. 2019, 10(21), 5582–5588.

enabling formation of the HdH bond and release of H2 to close the catalytic cycle. In short, in the case of hexacarbonyl complexes the formation of catalytic hydrides through oxidative proton addition are generally preceded by reduction of the Fe(I)Fe(I) starting species to either Fe(I)Fe(0) or in some cases all the way to the homovalent Fe(0) dimer. When a Brønstedt base is present in the bridging dithiolate ligand, protonation of this site can facilitate subsequent metal centered reduction. Critically, this can be achieved without a noticeable loss in basicity of the resulting reduced intermediate (e.g. [2.7(H+)] relative to 2.4−) and thus enable catalysis at milder potential without the expected loss of TOF. However, in the context of mechanistic understanding it is important to note that this beneficial effect from the second coordination sphere Brønstedt acid/base can be achieved also without invoking a direct proton-shuttling mechanism. Though the vast majority of catalysis studies have focused on H+ reduction, bridging hydrides have also been proposed as intermediates during H2 activation, as represented by complex 2.27 (see also Section 15.02.1.2.3.4). A mechanism for H2 oxidation has been proposed based on DFT calculations and is presented in Fig. 14.134 Two consecutive one electron oxidations yields a homovalent Fe(II)Fe(II), at which point the Fe center is suitably electrophilic to trigger heterolytic H2 splitting, resulting in a m-hydride intermediate. In contrast to complex 2.7, subsequent deprotonation is modelled to proceed via an intramolecular proton transfer of the m-hydride to the pendant amino-group. This apparent discrepancy in reactivity of m-hydrides in 2.7 and 2.27 is arguably due to the relatively short distance between the amine of the diphosphine ligand and the m-hydride in the latter complex. The catalytic properties of 2.15 has been probed experimentally and computationally, and its reactivity toward H2 and H+ is summarized is Fig. 15.117,135 It shares many structural features with the aforementioned complex, 2.27. However, the addition of a redox active ligand (FcP ) enables catalysis via more biologically relevant oxidations states (compare with Fig. 3). Protonation of 2.15 yields the bridging hydride species [2.15(m-H−)]+ with the diiron unit best described as a homovalent Fe(II)Fe(II) species with the FcP remaining in its reduced Fe(II) state (Fe(II)cp∗Fe(II)Fe(II), analogous to the Hhyd-state). Conversely, one-electron oxidation using Fc+ generates the mixed valent Fe(II)Fe(I) species 2.15+ (Fe(II)cp∗Fe(II)Fe(I), analogous to the Hred-state), with concomitant ligand rearrangement to form a semi-bridging CO ligand and a vacant coordination site. The oxidation of the diiron unit is readily observed by FTIR from the  60 cm−1 hypsochromic of the n(CO) bands. A second equivalent of Fc+ triggers oxidation of the FcP moiety rather than the diiron unit to give 2.152+ ((Fe(III)cp∗Fe(II)Fe(I), analogous to the Hox-state), observable through magnetic susceptibility measurements revealing the presence of two unpaired electrons and in agreement with DFT calculations. Moreover, the formation of 2.152+ results in a modest hypsochromic of  4 cm−1 of the n(CO) bands, as expected from the oxidation event occurring distant from the CO coordinating diiron unit. Critically, the doubly oxidized species 2.152+ reacts quantitatively with H2, and heterolytic cleavage of the HdH bond occurs between the adt-amine and the Fe ions with concomitant intramolecular electron

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Fig. 14 Proposed mechanism for H2 oxidation catalyzed by complex 2.27, proceeding via a bridging hydride intermediate. Intramolecular proton transfer to the second coordination sphere amino-group is proposed as a key aspect of the reaction. Fc+ ¼ Ferrocenium.

Fig. 15 Proposed mechanism for H2 oxidation catalyzed by complex 2.15, in which the Cp unit serves as a redox active ligand analogous to the [4Fed4S]H component of the H-cluster. Fc+ ¼ Ferrocenium.

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Fig. 16 Proposed mechanism for electrocatalytic H+ reduction by complex 2.33. Intermediates in black have been observed by spectro-electrochemistry FTIR in CH2Cl2, and a similar mechanism is proposed to operate also under aqueous conditions.

transfer to generate [2.15(t-H−)(H+)]2+ (Fe(II)cp∗Fe(II)Fe(II)). In the presence of excess oxidant and proton acceptor the reaction with H2 is catalytic, albeit slow. In contrast, a stoichiometric mixture of FeCp and [(m-(SCH2)2NBn)Fe2(CO)3(PMe3)(dppv)]+ was not found capable of catalytic H2 oxidation, underscoring the synergistic effect of covalently linking the two redox active components. A second striking example of a system coupling two redox active units is the aforementioned phosphole substituted bdt derivative 2.33 (Fig. 16), for which impressive rates for electrocatalytic proton reduction has been reported.120 The phosphole unit not only serves as a redox active ligand but also stabilizes additional electron density via ligand protonation. An impressive TOFmax approaching 105 s−1 for H2 production was estimated under aqueous conditions with an over-potential of 660 mV. A proposed mechanism based on a combination of DFT calculations and spectro-electrochemistry FTIR is shown in Fig. 16. Complex 2.33 serves as a pre-catalyst, and enters the catalytic cycle as 2.330 following two proton-coupled reduction steps. During catalysis the di-iron unit cycles between Fe(I)Fe(I) and Fe(I)Fe(II), with an additional electron distributed on the phosphole ligand. The reduced phosphole ligand is in turn stabilized via protonation of the pyridyl substituents. Considering that the enzyme employs terminal hydrides during catalysis, the factors promoting the formation of such species have been thoroughly investigated. The first crystal structure of a di-ferrous di-thioloato complex featuring a terminal hydride was reported in 2005 (i.e. 30 years after the spectroscopic characterization of the m-hydrides 2.29-2.31).136 This was achieved by treatment of the tetraphosphine ligated diferrous complex [(m-edt)(m-CO)Fe2(CO)(PMe3)4(NCMe)]2+ (edt ¼ ethanedithiolate, 2.34) with a suitable hydride donor at low temperature to give [(m-edt)(m-CO)Fe2(CO)(PMe3)4(t-H−)]2+ (2.35), with concomitant release of the labile acetonitrile ligand (Fig. 17). The study also provides an illustrative example of the decreased stability of terminal hydrides relative to their bridging variants. The reaction from 2.34 to 2.35 proceeds smoothly at −40 to −25  C, but the terminal hydride species tautomerizes spontaneously to the thermodynamically favored m-hydride form (2.36) upon heating to room temperature. Moreover, while 2.35 reacts spontaneously with a strong Brønstedt acid to yield H2, complex 2.36 is inert under the same conditions, highlighting the increased reactivity of terminal hydrides during H2 evolution catalysis.

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Fig. 17 The terminal hydride complex 2.35 is formed via hydride addition to 2.34 at low temperature, but converts to the thermodynamically more stable bridging hydride, 2.36, upon warming.

Between 2007 and 2008 parallel reports from multiple groups verified the possibility of promoting the formation of terminal hydrides also through the oxidative addition of protons to [2Fe]H mimics. Generally such terminal hydrides display proton resonances in the range of −3 to −5 ppm, enabling their detection by NMR spectroscopy. A common theme of these systems is the use of bidentate phosphine ligands to increase the electron density of the diiron unit in combination with steric bulk on the ligands. Moreover, and in line with the reactivity reported for 2.35, their synthesis commonly requires low temperatures, as the terminal hydrides spontaneously convert to the bridging hydride tautomer at elevated temperatures (often already at temperatures above −30  C).137–139 A crystal structure of such a diiron complex with a terminal hydride ligand was reported in 2012, following the low-temperature isolation of the doubly protonated di-dppv complex [(m-adtH)Fe2H(CO)2(dppv)2]2+ ([2.37(H+)(t-H−)]2+). Of particular note in the structure is the presence of the biologically relevant bridging CO ligand, and the short distance, 1.88(7) A˚ , between the hydride and an ammonium proton on the adt-ligand (labelled H1 and H3 in Fig. 18).140 In addition to these structural insights, the reactivity of the di-dppv substituted variants of 2.4, 2.7 and 2.8, i.e. [(m-adt) Fe2(CO)2(dppv)2]+ (2.37), [(m-odt)Fe2(CO)2(dppv)2]+ (2.38) and [(m-pdt)Fe2(CO)2(dppv)2]+ (2.39) (Fig. 19), has been studied extensively. Consequently, the latter complexes have provided a wealth of data on the formation and reactivity of terminal vs bridging hydrides.127,139,140 In all three cases, protonation with a strong acid at low temperatures generates the terminal hydride through oxidative addition ([2.37(t-H−)]+, [2.38(t-H−)]+ and [2.39(t-H−)]+); and they convert to their respective bridging hydride tautomers at room temperature ([2.37(m-H−)]+, [2.38(m-H−)]+ and [2.39(m-H−)]+). In addition to their characteristic NMR properties, the ligation mode of the hydrides is discernable also by FTIR spectroscopy. Complex 2.37 displays two distinct bands at 1888 and 1868 cm−1. Upon formation of [2.37(t-H−)]+ these two bands shift to 1965 and 1915 cm−1, with the latter assigned to the semi-bridging CO ligand. In contrast, as the [2.37(m-H−)]+ complex lacks the bridging CO ligand, the bands appear closer together, at ca. 1969 and 1948 cm−1. Subsequent protonation of the amine bridgehead to generate [2.37(H+)(H−)]2+ results in the expected hypsochromic shift of 15 cm−1. The presence of a bridgehead heteroatom enables protonation of the iron using weaker acids, and greatly accelerates the removal of the terminal hydride, in the order -NH- (2.37) > -O- (2.38) > -CH2- (2.39).127 Conversely, the

Fig. 18 Structure of [(m-adtH)Fe2(t-H)(CO)2(dppv)2](BF4)2 ([2.37(H+)(t-H−)]2+) with thermal ellipsoids drawn at 50% probability but those for phenyl carbon atoms omitted for clarity. Counter ions and solvent molecules not shown. Figure reproduced with permission from Carroll, M. E. et al. J. Am. Chem. Soc. 2012, 134(45), 18843–18852.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 19 Schematic representation of 2.37-2.39 (left) and their corresponding bridging (middle) and terminal (right) hydride derivates.

corresponding bridging hydrides were stable in the presence of a Brønstedt base, again verifying their relatively sluggish reactivity. These observations are consistent with the notion that the amino-bridgehead of the adt ligand plays an active role in delivering the proton to the iron and also increase the reactivity of the resulting metal hydride. The bridging thiolato ligands also have a role in promoting the formation of terminal hydrides. As implied from the observation that related di-phosphido bridged complexes do not appear to form terminal hydrides, and rather convert immediately to the bridging hydride form upon protonation.141 The possibility to generate both bridging and terminal hydrides in a controlled fashion in the pdt and adt bridged complexes, 2.37 and 2.39, enabled direct comparisons of the electrocatalytic properties of the two different types of hydrides. A 200 mV decrease in over-potential requirement for H2 production was observed under conditions favoring the terminal hydride intermediate, relative to its bridging hydride tautomer, in the case of 2.39.139 A similar decrease in over-potential was reported also for the adt ligated analog, 2.37. Additionally, a comparative study of the capacity of 2.37 and 2.39 for electrocatalytic H+ reduction highlights the remarkable influence of a proton-relay in the second coordination sphere.140 The incorporation of an amino Brønstedt base in the bridging dithiolate ligand not only enabled catalysis with weaker acids, but also resulted in a dramatic increase in catalytic rate. A TOF of 5000 s−1 was calculated for the singly protonated terminal hydride species [2.37(t-H−)]+, while a thousand fold lower TOF (5 s−1) was observed for the pdt bridged derivative [2.39(t-H−)]+. In fact, the latter complex was outcompeted even by the, intrinsically less active, bridging hydride tautomer of 2.37, [2.37(m-H−)]+, which displayed a TOF of  20 s−1.140 Finally, in the presence of sufficiently strong acids, expected to promote formation of the doubly protonated intermediate [2.37(H+)(t-H−)]2+, a TOF of  58,000 s−1 was achieved, underscoring the catalytic potential of these finely tuned molecular catalysts. 15.02.1.2.3.6 Artificial maturation and semi-synthetic [FeFe] hydrogenases Our understanding of the biological machinery responsible for the biosynthesis and insertion of the [2Fe]H subsite into apo-[FeFe] hydrogenase paved the way for the development of “artificial maturation,” where the biological components are replaced by synthetic analogs. As mentioned in Section 15.02.1.2.2, this has been used as a tool for gaining detailed understanding of H-cluster assembly, but also the preparation of semi-synthetic [FeFe] hydrogenases. Reported first in 2013, the artificial maturation method and the preparation of such semi-synthetic [FeFe] hydrogenases could unequivocally prove the nature of the bridging ligand as adt, rather than pdt or odt which had also been proposed.20 Introduction of complex 2.1 into apo-[FeFe] hydrogenase resulted in a fully active enzyme, spectroscopically indistinguishable from the native form. Conversely, incorporating 2.5 and 2.6, respectively, locked the [2Fe]H subsite in specific oxidations states. More specifically, replacing the protonatable amine-bridgehead with a methyl group, prevents reduction of the [2Fe]H subsite, enforcing an Hox-like Fe(II)Fe(I) mixed valence state. Somewhat surprisingly, the ether analog, 2.6, instead locks the enzyme in an Hhyd-like state with a homovalent Fe(II)Fe(II) [2Fe]H subcluster. Critically, these, and related, “organometallic mutants” have been crystallized, and despite variations of the bridging ligand they are all structurally very similar. This ensures that the observed differences in reactivity are due to fine-tuning of the proton-transfer network rather than more dramatic structural differences.9 Thus, the biochemistry community can now approach the H-cluster similar to a classical molecular catalyst, i.e. making chemical modifications in order to probe the influence of its different components on overall enzyme reactivity. Work using cofactors featuring modifications of the bridgehead amine has contributed to the elucidation of the protonation chemistry of the H-cluster, including e.g. disentangling the Hred and HredH+ states,39,41 the potential protonation chemistry of the [4Fed4S]H-cluster,38 and the first direct verification of a terminal hydride ligand in Hhyd.47 In the context of catalyst design, the reactivity observed for these organometallic mutants underscore the critical importance of a Brønstedt base in the second coordination sphere of the H-cluster, not only for substrate delivery and activation, but also the stabilization of reduced forms of the [2Fe]H subsite via proton-coupled electron transfer. Indeed, this is in nice agreement with the diverging reactivity observed for example for complexes 2.37-2.39 (see Section 15.02.1.2.3.5). Moreover, it highlights the influence of the active-site pocket on the reactivity of these organometallic complexes, with complex 2.1 serving as an excellent example. The complex is catalytically inactive and unstable in isolation, but is transformed into a highly active and robust catalyst upon incorporation into the protein host. This is arguably due to fusion with the [4Fed4S]H cluster providing a redox active ligand, in combination with the stabilization of the rotated structure (Fig. 7) through H-bonding as well as site-isolation with well-tuned substrate delivery channels. Critically, we now know that chemists have a large degree of freedom in the design of these semi-synthetic H-clusters, as Siebel et al. demonstrated in 2015 that the active-site can accommodate a wide range of structurally modified [2Fe] cofactors.142 An overview of the variations reported at the writing of this book chapter is shown in Fig. 20. This strongly suggests that not only improved hydrogenases can be generated through changes to the [2Fe]H subsite, but also hints at the possibility of utilizing the

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21

Fig. 20 Overview of reported chemical modifications of the [2Fe]H subcluster.

[FeFe] hydrogenase as a scaffold for preparing enzymes catalyzing novel reactions. Replacement of the [2Fe]H subsite with non-native metals has also been achieved, with successful maturation of apo-[FeFe]-hydrogenase using Ru analogs of 2.1 and 2.5.143 The Ru complexes are basic enough to form a bridging hydride ligand upon solvation in water to yield [(m-adt)(m-H) Ru2(CO)4(CN)2]− (2.40) and [(m-pdt)(m-H)Ru2(CO)4(CN)2]− (2.41). These m-hydride species spontaneously incorporate into the enzyme, and the resulting semi-synthetic enzymes are stable but not catalytically active. Strikingly, incorporation into the enzyme results in spontaneous rearrangement of the hydride ligand from bridging to terminal binding, underscoring the protein frameworks role in promoting the formation of this key catalytic intermediate. Despite providing a wealth of mechanistic data, to date, no organometallic mutant has outcompeted the catalytic efficiency of the native cofactor. Considering the evolutionary optimized nature of the active-site specifically for the [2Fe]H subsite, any attempt at improving the catalytic activity as compared to the native enzyme is likely to require an element of directed evolution. However exchange of the sulfur atoms in complex 2.1 to selenium resulted in native-like enzymatic activity and a slight shift in catalytic bias favoring H+ reduction over H2 oxidation.144 In 2017 the method for artificial maturation of [FeFe]-hydrogenases was extended also to whole-cell conditions, with the successful activation of an apo-[FeFe] hydrogenase in bacteria through addition of complex 2.1 to the growth media.145 The method is not restricted to generating the native H-cluster, and the formation of organometallic mutants has been verified both with activity measurements and through spectroscopy.145,146 Moreover, the resulting semi-synthetic enzymes couple to the cell metabolism in the host organism,147,148 providing the possibility for new-to-Nature metabolic pathways using hydrogenase based artificial enzymes. Thus, artificial maturation provides the organometallic chemistry community the rare opportunity to study designed synthetic catalysts in an exceptional range of settings going from isolated molecules in organic solvent to incorporation in a protein in aqueous buffer, all the way to their function in the form of a semi-synthetic enzyme inside a living cell. Moving forward, there is clearly rich chemistry waiting to be discovered related to [FeFe] hydrogenases and their synthetic mimics.

15.02.1.3 [NiFe] hydrogenase and their model compounds 15.02.1.3.1

Structure and functions of [NiFe] hydrogenases

The [NiFe] hydrogenase enzymes can be categorized into groups based on evolved structure (molecular phylogeny) leading to some notable differences in overall function24: (i) Group 1 – membrane bound H2 uptake and actinobacterial hydrogenases; (ii) Group 2 – uptake and sensory hydrogenases; (iii) Group 3 – reducing hydrogenases that target F420, NAD(P)+, and methyl viologen; and (iv) Group 4 – energy converting hydrogenases. Group 1 hydrogenases, which will be the focus of the discussion here, are the most studied among the [NiFe] hydrogenases wherein two subgroups, O2-sensitive and O2-tolerant, have been identified. O2-sensitive [NiFe] hydrogenases are found in Nature where anaerobic conditions for life are needed, e.g. in sulfate-reducing bacteria.24 More recently, O2-tolerant [NiFe] hydrogenases have been identified in aerobic bacteria. A special subset of O2-tolerant hydrogenases, [NiFeSe] hydrogenases, contain a selenocysteine residue at the terminal cysteines position at the Ni active site.149 Group 1 [NiFe] hydrogenases, including O2-sensitive, O2-tolerant and [NiFeSe]-hydrogenases, have very similar overall protein structures.151,152 These hydrogenase proteins are composed of a large and small subunit (63 and 29 kD, respectively). The NidFe active site lies in a deeply buried position at the interface of the large and small subunits. The large and small subunit have a total volume of ca. 5 nm3. The active site is surrounded by a highly functional protein environment that efficiently relays electrons, protons and H2 to and from NiFe center. The following paragraphs elaborate on the structure about the NiFe and NiFeSe centers as well as the aspects of the protein environment that support the conversion of H2/H+. The [NiFe] and [NiFeSe] active sites are housed in the large subunit. In [NiFe] hydrogenases the primary coordination sphere consists of a redox-active Ni(II) bound by the thiolates of four cysteine (Cys) residues to give [Ni(S-Cys)4] coordination; two Cys bind in a terminal fashion while two Cys form a m-S bridge between Ni and Fe metal centers. The redox-inactive Fe(II) is additionally coordinated by one CO and two CN− ligands.24,121 The structure of [NiFeSe] is nearly identical with the exception of the substitution of one terminal Cys to a terminal selenocysteine (SeCys).150 A side-by-side comparison of the active site structures of [NiFe] and [NiFeSe] hydrogenases determined by x-ray crystallography are given in Fig. 21A and B, respectively. The [NiFe] structure is shown for the NidR state (as described in Section 15.02.1.3.2) where a hydride ligand bridges the Ni and Fe metal centers. Inspection of the structures in Fig. 21 shows that the Ni center takes on a highly distorted geometry. In Desulfovibrio vulgaris Myazaki F (Fig. 21A)10 the angle between terminal C546 and bridging C84 approaches linearity where ∠C546-S–Ni–S-C84 ¼ 176.3 , while the angle between C546 and terminal C81 is significantly smaller, ∠ C549-S–Ni–S-C81 ¼ 107.9 ).121 This coordination geometry is perhaps more easily seen in the structure of the [NiFeSe] hydrogenase active site of Desulfovibrio vulgaris

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 21 The active sites of (A) [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (Ni-R state) at 0.89 A˚ resolution (PDB ascension code 4U9H, Ref.150); (B) [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough in reduced state (pdb ascension code: 5JSK, ref.,10 dominant occupancy structure shown) The protein backbone is shown in pale blue, while the cofactors and protein derived ligands are shown in ball and stick format with carbons shown as pale grey, heteroatom color coding: Fe: orange; Ni: green; S: yellow; Se: purple; O: red; N: Blue; H−: pink.

Hildenborough,150 Fig. 21B, where the coordination site between Ni and Fe centers is unoccupied. In the unoccupied state the Ni adopts a see-saw geometry and the Fe takes on a distorted square pyramid with the CO at the apical position. The unusual geometry about the Ni center contrasts to what is typically found for Ni(II)-d8 complexes; a square planar geometry is preferred, which is consistent with the observed geometries in the Ni-based artificial hydrogenases described in Section 15.02.1.3.4. When the site between the Ni and Fe metal centers is occupied, as in the NidR state (vide infra) of [NiFe] hydrogenases (Fig. 21A), the Ni and Fe take on distorted square pyramid and octahedron, respectively.10,121 The formation of the bridging hydride is a key intermediate in the mechanism for H2/H+ conversion, and is a sought-after intermediate in the biomimetic hydrogenases described in Section 15.02.1.3.4. In catalytically inactive forms of [NiFe] hydrogenases a hydroxide takes the bridging position. The [NiFe] and [NiFeSe] protein structures have been exquisitely tuned by billions of years of evolution to transport hydrogen, protons and electrons along dedicated pathways to and from the NiFe active site. In the identification of hydrogen and proton transfer pathways, molecular dynamics simulations based on crystallographic protein structures has been particularly helpful.153,154 From such studies it was determined that hydrophobic channels connect to the surface of the protein and converge at the NiFe and NiFeSe active sites. These channels are suited for the permeation of small non-polar molecules.153,154 Proton exchange between the surface of the protein and the active site is facilitated by hydrophilic proton transfer channels. While the exact mechanism of proton transfer along such pathways is not known, there are two prevailing theories. Either protons are exchanged at the active site via a water molecule network supported by glutamate residues, or the liberated proton is initially transferred to an arginine residue that resides 4.5 A˚ from the bridging hydride,155 [Ni-m-H-Fe], and subsequently moved to the surface by a histidine rich region wherein the proton is transferred by a Grotthus type mechanism.156 The small subunit (29 kD) contains redox cofactors based on iron-sulfur clusters that “wire” electron flow between the active site and the surface of the protein.24,157 Electrons that are delivered to, or removed from, the [NiFe] and [NiFeSe] active sites do so by hopping along the chain of three iron-sulfur clusters having an intercluster distance of 12 A˚ .24 For oxidative H2 conversion to 2H+ in [NiFe] hydrogenase, electrons are extracted from the [NiFe] active site by the proximal [4Fed4S], transferred to the medial [3Fed4S] cluster, transferred to the distal [4Fed4S] cluster, and finally to the surface of the small subunit, where the electron can be carried away by a physiological “partner” (e.g. cytochrome).24,158 In the case of [NiFeSe] hydrogenase, the medial cluster is a [4Fed4S] cluster. The number Fe and S atoms and nature of amino acids that anchor the iron-sulfur cluster to the protein matrix is how the enzyme tunes reduction potentials of each cluster; this in turn determines the kinetics of long-range electron transfer between the active site and the surface of the protein. The protein structure of the [NiFe] enzymes contains all of the essential components to carry out the functions necessary for H2/H+ conversion including: well defined substrate channels, electron transfer pathways, and a catalytic active site. [NiFe] hydrogenases manage hydrogen conversion at impressively fast rates (e.g. 1500 - 9000 s−1)159 at low overpotentials with high fidelity, made possible by the optimized protein structure. The following section summarizes what is currently known about the mechanisms at play in [NiFe] hydrogenases.

15.02.1.3.2

Mechanisms for proton and hydrogen conversion in [NiFe] hydrogenases

The interconversion between H2/H+ (Eq. 2) in [NiFe] hydrogenases involves the movement of two protons and two electrons.121,160,161 Movement of proton and electrons by way of a proton-coupled electron transfer (PCET) reaction, may bring the advantage of avoiding high energy intermediates that can cause slow kinetics or damage reaction centers. The mechanism of H2/H+ interconversion in [NiFe] hydrogenases has been investigated over many years of research, in many different groups using a variety of techniques; the cited reviews contain many excellent references of the spectroscopic techniques used.24,121,162–164 Vibrational (FTIR, spectroelectrochemical FTIR, resonance Raman), magnetic spectroscopies (EPR, ENDOR, HYSCORE) and higher energy spectroscopies (XAS, Mössbauer) have been used to establish the oxidation and protonation states, ligand coordination and geometries of the active site catalytic intermediates. Electrochemical and spectroelectrochemical FTIR methods have been particularly useful for mechanistic studies of hydrogenases and artificial systems that mimic hydrogenases. Electrons can be readily

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23

exchanged between the electrode and the active site by the use of an electrode in lieu of a biological redox partner.163 Following the changes in FTIR spectra as different potentials are applied to an enzyme adsorbed to an electrode can give information as to the various oxidation and protonation states in the enzyme active site. In the case of [NiFe] and [NiFeSe] hydrogenases, the two CN− and one CO group on the Fe center are excellent reporters of the oxidation state of both metal centers.162 The Fe(II) center is not redox active during catalysis, however, due to the significant delocalization between the metal centers, the CN− and CO stretching frequencies can report on the different oxidation state of Ni, e.g. Ni(I), Ni(II), or Ni(III), and the protonation state of the bridge. Specifically, n(CN−) and n(CO) stretching frequencies will shift to lower frequencies when greater electron density is present on the Fe metal center due to back-bonding into the CN− and CO p orbitals. The reduction or oxidation of the Ni center will decrease or increase the stretching frequencies, respectively. When a bridging hydride is present unpaired spin density will delocalize onto the hydride, which will result in an increase in the n(CN−) and n(CO) stretching frequencies. Thus, the different redox and protonation states of the [NiFe] center will be diagnostic for the different catalytic intermediates. Some excellent examples of such FTIR spectra for [NiFe] and [FeFe] hydrogenases can be found in several of the references cited in this book chapter.20,162 During catalysis the [NiFe] and [NiFeSe] systems cycle through very similar intermediates, but the oxidized forms the active sites are not necessarily the same. Fig. 22 summarizes the catalytic cycle for [NiFe] and [NiFeSe] hydrogenases.24,160,162,165 The oxidized

Fig. 22 Catalytic mechanism for proton reduction and hydrogen oxidation in [NiFe] and [NiFeSe] hydrogenases involves a series of coupled proton and electron transfer steps. In the catalytic cycle E indicates the presence of a sulfur or selenium from cysteine or selenocysteine, respectively. Adapted from references Yang, X. M.; Darensbourg, M. Y., Chem. Sci. 2020, 11(35), 9366–9377 and Ash, P. A.; Kendall-Price, S. E. T.; Vincent, K. A., Acc. Chem. Res. 2019, 52(11), 3120–3131.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

[NiFe] active site features a bridging hydride or end-on peroxo (NidB or Ni-A, respectively), while the oxidized [NiFeSe] active site forms involves oxygens that bridge the Ni and Se (Fig. 22). Reducing the oxidized forms of both [NiFe] and [NiFeSe] active sites brings the system to NidSI. For example, starting at the NidB inactive form of [NiFe], which contains a bridging m2-hydroxo, reduction by one electron and loss of the m2-hydroxo brings the system into the catalytic cycle (state Nia-SI, 32 e−, S ¼ 0). The description from here follows the catalytic cycle in the oxidative direction (counter clockwise). H2 undergoes a heterolytic cleavage to form a bridging hydride, [Ni(II)-H-Fe(II)], and a proton is transferred to the terminal cysteine or selenocysteine, (state NiadR, 34 e−, S ¼ 0). From NiadR, the stepwise oxidation and loss of the liberated proton from the coordination sphere results in the formation of a [Ni(III)-H-Fe(II)] species (state NiadC, 33 e−, S ¼ 1/2). With the loss of one electron and one proton the Nia-C can return to the Nia-SI state. A second pathway involves a Ni(I) intermediate that is formed by reductive elimination wherein the proton which forms the bridging hydride is transferred to Cys546 and a Ni(I)dFe(II) bond is formed (state Nia-L, 33 e−). Until very recently it was unclear if and to what extent Nia-L is involved in catalysis, however it has now been established that the Nia-L state is present under electrocatalytic and photocatalytic conditions.162 There have been two illuminating mechanistic studies on likely proton transfer pathways between the primary coordination sphere of the [NiFe] active site and outer sphere amino acids. In one study, the proximal arginine residue which sits in the canopy above the bridging site, Arg509 (Escherichia coli Hyd-1 numbering), has been shown to be extremely important for the reactivity of H2/H+ conversion.155 Replacing Arg509 for a lysine residue by site directed mutagenesis, led to a 100-fold lower activity than the native enzyme. The overall protein structure was unperturbed by the lysine mutation, which pointed to the importance of arginine in the mechanism of H2 activation. A second set of studies looked into the proton and electron transfer mechanism associated with the Ni-SI ⇆ Ni-C equilibrium in the soluble hydrogenase 1 from Pyrococcus furiosus.14,15 Steady state and time resolved FTIR measurements were used to track the shifts in n(CN−) and n(CO) stretching frequencies that occurred upon laser excitation of the Nia-C state, which liberates the bridging hydride. The evolution of these vibrational frequencies allowed identification of the species involved in Ni-C ! Ni-SI conversion under different pH and isotope conditions. Using kinetic and thermodynamic arguments the mechanism for Ni-C !Ni-SI conversion is as follows: (1) Photolysis of the Ni-C state liberates the hydride and forms the Ni-L state with the proton residing on a cysteine in the primary coordination sphere. (2) In a concerted PCET step the proton is transferred to a neighboring amino acid, E16, and the electron from Ni+ to an iron–sulfur cluster, thus forming the Ni-SI state.14,15 A significant reduction in H+ reduction activity for the E16Q mutant of the enzyme supported the assignment of E16 acting as a necessary proton acceptor in the PCET of Ni-L !Ni-SI transition. It has been noted that [NiFeSe] hydrogenases have an increased kinetic bias toward H2 production.164 This can be understood by the greater polarizability of Se versus S. The increased polarizability of Se makes selenocysteine a better acid (pKa(SeCys) ¼ 5.2 versus pKa(Cys) ¼ 8.0).121 The increased nucleophilicity of Se likely enables faster H+ shuttling during catalysis, which is consistent with faster H2 production in [NiFeSe] hydrogenases.

15.02.1.3.3

Unique oxygen tolerance in [NiFe] and [NiFeSe] hydrogenases

An intriguing quality of some [NiFe] hydrogenases is their ability maintain catalytic activity in the presence of O2. The evolutionary impetus behind the design of O2 tolerant hydrogenases likely stems from the need for early lifeforms to manage hydrogen catalysis under high atmospheric concentrations of O2. To date, three strategies that protect the active sites of [NiFe] hydrogenases from O2 have been elucidated.166 The hydrophobic gas channels through which H2 travels (permeates) are narrow thus hindering diffusion of the larger O2 molecule. For example, in a study of an O2 tolerant [NiFe] hydrogenase from in Ralstonia eutropha H16, it was found that mutation of residues to expand the gas channel rendered the enzyme oxygen sensitive. Very recently molecular dynamics (MD) simulations were undertaken in order to account for the differences in O2 diffusion in a pair of NiFe and NiFeSe enzymes that are nearly structurally identical.154 The MD simulations predicted different preferred pathways for O2 diffusion whose termini were not the same with regards to the coordination environment of the catalyst center. Thus, it seems very likely that the mechanism for catalyst inactivation by O2 is not the same in [NiFe] and [NiFeSe] hydrogenases. Importantly, it was observed from the MD simulations that O2 permeation was less efficient in the [NiFeSe] enzyme compared to the [NiFe] enzyme, which would boost the oxygen tolerance in the former.154 The second strategy involves the presence of a special case of the proximal FeS cluster from which electrons can be more rapidly transferred to O2, and along with the transfer of protons, O2 can be reduced to water. This avoids the formation of more aggressive partially reduced reactive oxygen species (e.g. H2O2 or O2%−). The typical situation in hydrogenases is that iron-sulfur clusters are coordinated to the protein scaffold through four cysteine residues (or three cysteines and one histidine), e.g. [4Fed4S]4 Cys. However, this special subunit, found in Ralstonia eutropha, features two additional cysteine residues near to the proximal cluster ([4Fed3S]6 Cys). In this exceptional cluster, one of the corner sulfides is absent and two additional cysteine thiolates coordinate Fe atoms.167,168 The ([4Fed3S]6 Cys) cluster is capable of delivering two electrons to O2 in rapid succession, instead of one which is more commonly observed for standard [4Fe-4S] clusters. Such reactivity means that the enzyme can repair itself, by diverting two electrons to the reduction of O2 to form H2O at the active site, thus avoiding oxidative damage.165 The third, and perhaps most significant means of O2 tolerance stems from the incorporation of selenocysteine in lieu of cysteine at the terminal position on the Ni center (Figs. 21 and 22). In both [NiFe] and [NiFeSe] enzymes O2 exposure to the active sites results in oxygenation of sulfur or selenium, respectively. This mechanism of ligand oxidation protects the NidFe core from oxidative damage and degradation.165,166 Subsequent delivery of reducing equivalents and protons to the bound oxygen will lead to

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formation of a water molecule; this repairs the active site and restores catalytic activity. Elemental differences between selenium and sulfur play a role in the superior O2 tolerance of [NiFeSe] hydrogenases.164,165 In addition to selenium’s increased polarizability, nucleophilicity and affinity for oxygen binding, the SedO bond is weaker than SdO because of selenium’s larger size. Thus, the release of bound oxygen will be more facile in [NiFeSe] versus [NiFe] hydrogenases.166 Systematic studies of oxygen damage and repair in NiFe-biomimetic systems using Se and S support this view and are discussed in Section 15.02.1.3.4.2.

15.02.1.3.4

Biomimetic Ni containing analogs

Nature’s choice of a heterobimetallic hydrogen processing catalyst has fascinated researchers for decades.24 Their high activities for H2/H+ interconversion, and prevalence of oxygen tolerant systems makes the [NiFe] hydrogenases an excellent template from which to develop cheap and effective rationally designed catalysts. Since the early crystal structures of [NiFe] hydrogenases were reported in the 1990’s many bioinspired and biomimetic catalysts based on [NiFe] hydrogenases have been realized.152,160 Enzymatic systems outperform the man-made analogs when considering efficiency, rates of catalysis, self-repair, use of Earth abundant materials, and operation in water. Still the efficiencies of such human-designed systems mimicking [NiFe] hydrogenases have improved significantly during the last decade and they been very useful in drawing insight into the behavior of natural systems.152 The following sections describes synthetic hydrogenase mimics based on heterobimetallic complexes of the form [NiM], where M ¼ Fe, Mn, Ru, W, and systems containing a single Ni. 15.02.1.3.4.1 [NiFe] analogs based on heterobimetallic Ni complexes Synthetic biomimetic hydrogenases based on Ni and a second transition metal, M, in very many instances are based on a motif of: (i) one four-coordinate, square planar Ni(II), (ii) two (or sometimes one) bridging-thiolates to M, and (iii) at least one carbonyl or cyanide ligand bound M. Frequently at least two of the coordination sites on Ni(II) are occupied by soft atoms (e.g. S, P, Se). These basic characteristics form a starting point from which to build artificial systems that emulate the structure and/or behavior (function) and reactivity of Nature’s hydrogenases. The first heterobimetallic thiolate bridged NiFe complex was reported in the 1990s with many more examples appearing in the following years, yet catalytic activity remained elusive in these early mimics.169,170 Several examples are shown in Fig. 23, 3.1–3.4. The first of such complexes prepared, 3.1a was significant in that it demonstrated that the preparation of NiFeCO complexes with a bridging thiolate was within the realm of chemically possibility. This first system utilized an N2S2 ligand motif, from which many

Fig. 23 Selected structural mimics of the NiFe hydrogenase active site. Each of these mimics is characterized by a square planar Ni(II) with one or two m2-thiolate bridges to an Fe(II). None of these complexes showed catalytic activity toward H2/H+ conversion. 3.1a can be reversibly oxidized and reduced while keeping the NiFe core intact, but with significant structural changes.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

future biomimetic designs were based upon.169 Other early examples, 3.2–3.4 utilized an tetra-thiolate (S4) binding motif about the Ni(II) and a thiolate-bridged FedCO unit containing at least one other ligand, e.g. CO, CN, cyclopentadienyl (Cp), or PMe3.171,172 The first functional, i.e. hydrogen evolving, biomimetic NiM complexes were based on Ni and organometallic Ru(II). Ruthenium is an appropriate choice given that hydrogen transfer reactions were already described for Ru complexes. Ru(II) can bind hard or soft ligands including H2 and H2−; such reactivity is a necessity for complexes that mimic the behavior of [NiFe] enzymes, and in particular with respect to the ability of Ni-M to form a bridging hydride during catalysis. Compounds 3.5–3.8 show a selection of hydrogen forming artificial [NiRu] hydrogenases.152 Although not strictly biomimetic, informative relationships between structure and catalyst function were validated by the study of these NiRu systems. Firstly, it was observed that more electron rich metal centers made better hydrogen evolving catalysts. As apparent from Fig. 24, complexes 3.7 and 3.8 are nearly identical except for the difference in functionalization on the Cp ring, where Ru(Cp ) is decorated with five electron donating methyl groups.173 This results in a greater electron density on the NiRu core in Ru(Cp ) versus Ru(Cp). As in the case of the [2Fe]H mimics discussed in Section 15.02.1.2, the increased electron density on the Ru metal center is associated with lower n(CO) frequencies due to increased back-bonding. The Ru(Cp ) displayed a lower overpotential (620 mV) and higher turnover frequency. Further, the bulkier Cp ligand offered steric protection to the catalyst and increased the stability of 3.8 during electrocatalytic cycling.173 The first series of the artificial hydrogenases containing a NiFe core was prepared in 2009.121,174 The structures of these complexes, 3.9–3.12 of Fig. 24, are based on a [(dppe)Ni(m-pdt)Fe(CO)3] where the bridging hydride was prepared by protonation with HBF4 to give [(dppe)Ni(m-H)(m-pdt)Fe(CO)3]+. Complexes 3.10–3.12 were prepared by introduction of the appropriate phosphite or phosphine. The complexes shown here, as well as others of similar composition, were active for electrocatalytic hydrogen evolution in methylene chloride with trifluoroacetic acid (TFA) was used as a proton source.121 The overpotential for hydrogen production was approximated at 1 V for 3.9 ([(dppe)Ni(m-H)(m-pdt)Fe(CO)3]+), while for the phosphine derivatized complexes the overpotential was estimated at 260-430 mV under slightly different conditions (acetonitrile solution). The lower overpotential can be understood by the increased electron density introduced into the system by the phosphine ligands on the dppe ligand. 3.9 was the first to faithfully reproduce the oxidation sates for the hydride bridged (i.e [Ni(II)-H-Fe(II)] species as is observed in NidR in [NiFe] hydrogenases.121 Related complexes based on a different tetradentate ligand with S4 coordination about the nickel were prepared by reacting [Ni(xbsms)], where xbsms ¼ 1,2-bis(4-mercapto-3,3-dimethty-2-thiabutyl)benzene), to an Fe(CO) complex, e.g.

Fig. 24 Structures of representative [NiFe] hydrogenase mimics based on Ni and Ru metal centers.

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

27

Fig. 25 [NiFe] hydrogenase mimics based on NiFe, NiMn, and NiW cores that show activity for hydrogen evolution.

[CpFe(CO)2(thf )]+ or [Fe(CO)3(bda)], where bda ¼ benzylidene acetone) to form 3.13 and 3.14, respectively (Fig. 25).175,176 The thiabutyl groups formed a bridge between nickel and iron which mimics the bridging structure of the cysteines in NiFe hydrogenases. 3.13 was electrocatalytically active for H2 evolution in DMF with TFA as the proton source with an overpotential of 730 mV.175 3.13 is quite different structurally from 3.14; in this highly distorted complex one of the thiols (from mercaptobenzene) binds (axially) to the Fe center and a Ni-(m-CO)dFe bridge is formed (Fig. 25). Interestingly, treatment of 3.14 with HBF4 resulted in protonation of the terminal thiol ligand on the Ni center.176 This protonation behavior mirrored the proposed protonation of the cysteine thiol in the catalytic cycle of [NiFe] hydrogenases, however the bridging CO, thiolate, and s NidFe bond precluded the formation of the bridging hydride in this complex. Nonetheless, both unprotonated and protonated complexes evolved H2 from TFA in acetonitrile solution with an overpotential of ca. 550 mV.176 The synthetic [NiFe] analogs discussed thus far show only activity toward H2 evolution.152 The first [NiFe] hydrogenase mimic to display reversible reduction/oxidation of H2 was found in 3.15, which features a Ni(N2S2) environment from N,N0 -diethyl3,7-diazanonane-1,9-dithiolato and an Fe-triethylphosphate [Fe(P(OEt)3)3] that is bridged by the two thiolate groups.177 The phosphate groups of 3.15 promote the binding of H2 to the Fe center, and subsequent addition of a strong base promotes heterolytic cleavage with proton transfer to the base and the formation of an iron-hydride, which is weakly associated with the Ni center. The reduced hydridic complex was primed to react with weak oxidants, such as methyl viologen (MV2+) or ferrocene (Fc+), where the release of the electron to the acceptor was coupled to proton transfer from the Fe center. A single catalytic turnover was observed for this system; the hydride intermediate was prepared chemically and then isolated prior to reacting with oxidants and bases.177 Electrocatalytic behavior was not described for this system. W and Mn were incorporated into models that were structurally similar to 3.9 and 3.13, respectively, to explore catalytic behavior beyond Fe. A representative [NiMn] complex was prepared by reacting [Ni(xbsms)] with [Mn(CO)5Br], to give [Ni(xbsms)Mn(CO)3(H2O)]Br, 3.16.152,178 Ni(II) adopts a square planar geometry, while the Mn(I) has an octahedral geometry

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 26 The possible mechanistic pathways for H2 evolution in complex 3.16. The heterolytic pathways are shown in blue and the homolytic pathways are shown in green. The analysis predicted a heterolytic pathway from the [NidHdMn] species with an electron transfer followed by a proton transfer to the complex (central pathway of the catalytic cycle), with subsequent release of H2.

with a labile water molecule. 3.16 evolved H2 under electrocatalytic conditions with TFA as the proton source in dimethylformamide when an overpotential of 860 mV was applied to the sample.178 The sample produced hydrogen with a Faradic efficiency of 94%, and 16 turnovers in 4 h. The possible pathways involved in H2 evolution are shown in Fig. 26. Detailed mechanistic studies were carried out for 3.16 using a combination of electrochemical data and simulated electrochemical data, and DFT. The analysis concluded that a heterolytic pathway was involved in H2 formation where: (i) a single electron reduction and the loss of the H2O ligand, (ii) a second reduction to produce the [NiMn]− species, (iii) the transfer of a proton from the solution to produce the hydride bridged [Ni-H-Mn]0 species, and (iv) the addition of an electron and a proton to release H2 and generate [NiMn], or (v) the transfer of a proton to release H2 and generate [NiMn]+, and finally vi) reduction by one or two electrons to bring the complex back to the catalytically active state [NiMn]−. The mechanistic analyses presented for 3.16 provide a useful framework for broad use in other electrocatalysts.178 [NiW] dithiolates were readily prepared by binding of a W(CO)4 fragment to [(R2PCH2CH2PR2)Ni(pdt)], where R]Ph, or Cy, to form [Ni(II)W(0)] heterobimetallic complexes 3.17 and 3.18, respectively.179 Protonation did not form the bridging hydride, instead the complexes were rather protonated at the W alone to form terminal tungsten hydrides. Low catalytic activity in these model complexes was attributed to the electron poor nature of the W(II) center as well as the lack of interaction with the Ni metal center. It should be noted that complexes 3.9–3.18, while all active for H2, evolution are not able to mimic the mechanism of proton reduction to hydrogen in hydrogenases. Specifically, in [NiFe] and [NiFeSe] hydrogenases the iron does not change oxidation state during hydrogen processing, while in all of the mimics described here the accompanying metal is also redox active.152 15.02.1.3.4.2 Understanding O2 tolerance in [NiFe] and [NiFeSe] hydrogenases In order to more precisely understand oxygen damage and repair in [NiFe] and [NiFeSe] hydrogenases and also to uncover guiding principles in how to make more oxygen tolerant molecular catalysts, a series of heterobimetallic synthetic analogs incorporating S and Se into the Ni primary coordination environment were prepared.166 The structural analogs represent minimal models of the hydrogenase active sites where the differences in oxidative damage in the presence of S versus Se can be studied in a highly controlled fashion. These models 3.19 and 3.20, feature a N2S tridentate ligand and an aryl substituted chalcogenide (S or Se) bound to the Ni center in a square planar arrangement; the CpFe(CO) unit is bridged by the two chalcogenide units to give a piano stool geometry about the Fe, Fig. 27.165,166 The rates of oxygen insertion were dependent which chalcogenide, Se or S, was present, and the choice para-substitution on the bridging arylthiolate (or arylselenate). The use of Se increased the rates and yields of oxygenation insertion by 4–7 fold and 10–20%, respectively. Increasing the electron donor strength of the aryl-substituted chalcogenide also produced higher rates and yields. The stable oxygen damaged Se incorporated complex could be repaired to recover the original oxygen free complex. First oxygen damaged 3.20b was isolated, then it was treated with two equivalents of reducing agent (CoCp2) followed by the addition of 2 equivalents of HBF4. This process led to 60% recovery of the parent species with water as a side product. The repair process could not be reproduced in the analogous oxygen damaged arylthiolate variant. Studies of oxygen damage and repair in 3.19 and 3.20 suggest: i) that when Se and S are in nearly identical environments Se is more reactive and more effective at scavenging O2 and releasing oxygen and ii) remote electronic affects play a role in rates of O2 uptake.166 The use of structural NiFe and NiFeSe models demonstrated the chemical advantages of having Se over S in the active site; and this understanding translates very clearly into understanding the increased O2 tolerance in [NiFeSe] hydrogenases. 15.02.1.3.4.3 Bioinspired Ni only complexes Mechanistic studies of [NiFe] and [NiFeSe] hydrogenases have shown that Ni is the redox active metal during catalysis and the Fe(II) center acts as a Lewis acid and does not change oxidation state. When the goal is to produce hydrogen as efficiently and at the lowest cost possible, it would save a considerable amount of effort to prepare and study systems based on a single metal center. Catalysts based on Ni are the obvious choice when considering it is the redox workhorse during hydrogen formation or oxidation.

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

29

Fig. 27 Structural models for [NiFe] and [NiFeSe] hydrogenase active sites composed of a Ni bridged to the Fe center through sulfur or selenium. The effects of O2 damage and repair were investigated in all complexes. Repair after oxygen damage was observed in 3.20b.

One family of such bioinspired H2/H+ interconversion catalysts is based on Ni coordinated to two bidentate P2N2 heterocyclic ligands, [Ni(P2N2)2].180–182 A small selection of these systems, 3.21a – 3.21f is shown in Fig. 28. The R and R’ groups on the heterocycle are used to tune the steric and electronic properties of the catalyst. The R-group is expected to have the largest influence on reduction potentials on the Ni-center; more electron donating substituents at R result in more negative reduction potentials for the metal complex, while concomitantly increasing its basicity. The size of R will also exert a steric influence on the complex which affects the P bite angles and dihedral angles about the Ni-center and results in changing the hydride donor ability of the NidH species.183 The pendant amine groups act as proton relays that can deliver proton equivalents to/from the Ni-center. Each pendant amine groups can fluctuate between chair-boat conformations, yielding four possible conformers, some of which will be more advantageous geometries for proton shuttling to from the Ni-center.180 Further, the pKa of the nitrogen proton relay can be modulated by the electron donating or withdrawing nature of the R’-group. For proton-coupled redox processes the increasing or decreasing pKa of the pendant amine can be an important contribution to the rate of hydrogen evolution or oxidation.184 When R¼ Cy and R’ ¼ Bz the [Ni(P2N2)2] behaves as a reversible electrocatalyst for hydrogen oxidation/proton reduction. In acetonitrile with protonated dimethylformamide as the proton source, H2 production proceeds with turn over frequencies of 350 s−1 and at low overpotentials of ca. 300 mV.180 H2 oxidation with this same complex proceeds with a turn over frequency of 10 s−1. The synthetic ease with which the P2N2 ligand can be functionalized with different R and R’ groups has made it possible to explore many different [Ni(P2N2)2] catalyst systems. With the [Ni(P2N2)2]‘s systematic tuning of electronics, sterics, increased flexibility of proton relays, and solubility it has been possible to optimize catalysis and to learn what structural requirements are needed to build better catalysts. One interesting strategy was to increase the biomimetic aspects of the [Ni(P2N2)2] system by introducing amino acids into the proton relay (R’) positions of the complex.181 The electrocatalytic properties of [Ni(P2N2)2] with R ¼ cyclohexyl and R’ ¼ Gly, Arg, Phe, Tyr, or Tym (a decarboxylated derivative of tyrosine) was studied in methanol/water mixtures

Fig. 28 Selected [Ni(P2N2)2] complexes that display activity for H2 oxidation and H+ reduction.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

with protonated bis-triflimide (as the proton source) and 25% H2 in Ar.181,182 Catalytic reversibility was observed when Phe and Tyr are used for R’.181 The system did not display catalytic reversibility under the same buffer/H2 conditions when the aromatic group or the carboxyl was absent; such was the case when Gly, Arg, and Tym were used. The aromatic groups were hypothesized to provide conformational stability that decreased the rate of chair-boat interconversion which improved the ability for the R’-nitrogen to act as a relay.181 The presence of the carboxyl groups enhanced the conformational stability and may also take an active role in relaying protons to the Ni active site. It should be noted that under aqueous buffer conditions the systems with Gly and Arg exhibited reversible electrocatalytic hydrogen oxidation/proton reduction.182 A biomimetic strategy to incorporate amino acids into the second coordination sphere has clear advantages for H2 production/ oxidation. Extending the outer coordination sphere even further into a well-defined protein is a strategy that could alleviate some of the synthetic effort needed to generate a molecular catalyst while providing a mechanistic model for the [NiFe] active site. Rubredoxin (Rd) is a small iron-based electron transfer protein186 which was made to host a tetrahedral Ni(II) bound to four cysteine amino acids (C06, C09, C32, and C35), Fig. 29.185 The resulting NiRd system was an active electrocatalyst for H2 evolution. Observed TOFs for H2 production varied between 30–150 s−1, with only very minor decrease in TOF observed as a function of lower buffer pH. The pH independence of the catalytic rate constant indicates that proton transfer is likely not a part of the rate limiting step.185 Interestingly, the observed turnover frequencies increased significantly when V08 in the secondary coordination sphere was mutated to a histidine. Histidine, unlike valine, can act as a proton relay to assist catalysis. Using both DFT and molecular dynamics simulations it was predicted that two cysteine residues in the primary coordination sphere were involved in proton transfer to the Ni-center.185 Further, it was shown that histidine could adopt conformations that would bring the histidine-N within hydrogen bonding distance of the cysteine-S. The NiRd system provides an excellent example of catalytic enhancement with the incorporation of proton relays. Finally, catalytic activity in NiRd was unchanged when buffers were saturated with O2, even though the reaction of reduced NiRd was thermodynamically possible. This remarkable O2 tolerance was instead attributed to the faster kinetics of H2 evolution which outcompeted an unproductive reaction with O2.185

15.02.1.3.5

Future challenges in developing [NiFe] model complexes

When the goal of catalysis is to be as fast as possible at the lowest overpotential, enzymes win. [NiFe] and [NiFeSe] hydrogenases are still more effective at H+/H2 conversion with respect to the synthetic analogs that have been tested. This is arguably due to the enzymes superior ability to move electrons and substrate to/from the active site as well as optimizing the primary coordination sphere for low over-potentials. In the fields of organometallic and inorganic chemistry impressive advances have been made to replicate the advantageous qualities in structural and functional models of hydrogenase enzymes. Yet, challenges remain. For example, in the enzyme systems the Ni center adopt a highly distorted see-saw geometry, which is most likely a key component in the enzyme reactivity. Such a geometry has thus far proven elusive in among artificial systems, where in nearly all of the known systems the Ni adopts a square planar geometry, with the exception being the NiRd system where the Ni center is tetrahedral. Additionally, the Fe(CO)(CN)2 unit has not been introduced into any of the structural or functional model systems, and it is desirable to gain further insight into the electronic behavior of Fe during catalysis. Finally, O2 in very many cases will compete for electrons intended for H+/H2 conversion, meaning that either stringently anaerobic conditions or O2 tolerance will be required. O2 tolerance is more sustainable in the long term, and has already been demonstrated in biomimetic systems.169,185 Developing O2 tolerant systems would greatly increase the utility of artificial systems for H2 production.

Fig. 29 Model of Ni Rubredoxin (NiRd) based on the crystal structure of Rubredoxin (PDB ascension code: 6RXN), based on ref.185 Heteroatom color coding: Ni: green; S: yellow.

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31

15.02.1.4 [Fe] hydrogenase and their model compounds As mentioned in Section 15.02.1.1.2, unlike [NiFe] and [FeFe] hydrogenase, [Fe] hydrogenase does not catalyze the reversible redox reaction between protons and dihydrogen (Eq. 1). Instead, [Fe] hydrogenase is involved in the reversible hydrogenation/ dehydrogenation of N5,N10-methenyltetrahydromethanopterin (methenyl-H4MPT, or CH^H4MPT) and N5,N10methylenetetrahydromethanopterin (methylene-H4MPT, or CH2]H4MPT) intermediate in the methanogenesis cycle of CH4 from CO2 in methanogenic archaea (Eq. 2 and Fig. 30).187 Hydrogenation of CH ^ H4MPT in the methanogenesis cycle under normal condition is catalyzed by both F420-dependent methylene-H4MPT dehydrogenase (Mtd) and F420-reducing [NiFe] hydrogenase (Frh). Under limiting nickel condition ([Ni2+] < 0.2 mM), biogenesis of [Fe] hydrogenase enzyme and its associated metabolic counterparts are upregulated and the hydrogenation is catalyzed by [Fe] hydrogenase instead.

15.02.1.4.1

Enzymatic activity, inhibitors and isolatable cofactors

[Fe] hydrogenase (Hmd, N5,N10-methylenetetrahydromethanopterin dehydrogenase, EC 1.12.99) isolated from Methanobactor marburgensis was first reported by Thauer188,189 to exhibit hydrogenase activity by catalyzing reversible dehydrogenation of CH2]H4MPT to CH^H4MPT and dihydrogen gas. [Fe] hydrogenase does not require presence of electron acceptors such as methyl viologen, methylene blue or coenzyme F420 during dehydrogenation. The purified enzyme exhibit reversible hydrogenation (Km ¼ 40 mM, kcat ¼ 1462 s−1 at pH 6.5) and dehydrogenation (Km ¼ 50 mM, kcat ¼ 975 s−1 at pH 7.5) activity at 60  C. Unlike [NiFe] and [FeFe] hydrogenases (Sections 15.02.1.2 and 15.02.1.3), [Fe] hydrogenase does not catalyze isotopic exchange of 3H/1H+ in absence of electron acceptor. However, the enzyme does catalyze this isotope exchange reaction in presence of its substrates CH2] H4MPT and CH ^ H4MPT.189 This seemingly contradictory observation is rationalized by obligatory 3H+/1H+ release during hydrogenation of CH ^ H4MPT. In contrast to [NiFe] and [FeFe] hydrogenases, acetylene, nitrite and azide, which are good ligands to transition metals, do not inhibit [Fe] hydrogenase activity. Only cyanide189 and CO190 inhibit enzyme activity, but with low affinity (Ki ¼ 0.1 mM); CO inhibition is reversible upon exposure to N2 or Ar. The apparent insensitivity of the enzyme toward common inhibitors of [NiFe] and [FeFe] hydrogenases led to earlier erroneous conclusions of a ‘metal-free’ hydrogenase (see Section 15.02.1.4.2). Similar to [FeFe] hydrogenases and many [NiFe] hydrogenases, the enzyme is not tolerant toward exposure to O2. The catalytic cofactor of [Fe] hydrogenase, the Fe-guanylylpyridinol (FeGP) cofactor, was first reported by Buurman et al to be isolatable from Hmd by extraction with high concentration of urea.191 The structure of the FeGP active site (verified by crystallography, Section 15.02.1.4.4) consists of a mononuclear Fe center with approximate octahedral coordination geometry: two CO ligands cis to each other, a pyridine N-donor and a methylene-acyl moiety from a pyridinol guanosine monophosphate (GMP) group, a thiolate from a cysteine residue and a water molecule occupying the putative H2 binding site. It is now known that intact FeGP can be extracted from [Fe] hydrogenase by denaturation of the protein with a combination of acetic acid or 2-hydroxyethanethiolate and either methanol or urea.192 The extracted FeGP coordinates either acetate or 2-hydroxyethanethiolate, depending on extraction method, with concomitant loss of two native ligands to satisfy octahedral coordination environment of the Fe center (Fig. 31). The isolated FeGP can be re-constituted into inactive, heterologously produced Hmd to recover enzymatic activity.

Fig. 30 Hydrogenation of N5,N10-methenyltetrahydromethanopterin (CH ^ H4MPT) to N5,N10-methylenetetrahydromethanopterin (CH2]H4MPT) catalyzed by Hmd. Figure adapted from Ref. Huang, G.; Wagner, T.; Wodrich, M. D.; Ataka, K.; Bill, E.; Ermler, U.; Hu, X.; Shima, S., Nat. Catal. 2019, 2(6), 537–543.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 31 Structure of the FeGP (left) and the extracted FeGP isolated in the presence of acetate or 2-hydroxyethanethiolate (right). Figure adapted from Ref. Huang, G.; Wagner, T.; Ermler, U.; Shima, S., Nat. Rev. Chem. 2020, 4(4), 213–221.

15.02.1.4.2

Early ‘iron free’ hypothesis and refutation

Early studies on the [Fe] hydrogenase enzyme concluded that it is free from iron or an iron-sulfur cluster. This led to the confusing nomenclature in the literature for a period of time where, what is now known definitively as [Fe] hydrogenase, Hmd is labelled as “Fe-free” or “metal-free” hydrogenase while [FeFe] hydrogenase was also known as “[Fe]-hydrogenase” or “[Fe]-only hydrogenase”. Earlier assignment of Hmd as “Fe-free” or “FeS cluster free” was based on analysis of the peptide sequence of Hmd, which do not show closely spaced cysteine residues, a motif that is necessary for binding iron sulfur clusters and present in both [NiFe] and [FeFe] hydrogenases. The “Fe-free” assignment was further supported by earlier observations that CO, a common inhibitors of hydrogenase enzymatic activity, has apparently little to no inhibitory effect on Hmd activity as the Ki for [Fe] hydrogenase is orders of magnitude larger when compared to many [NiFe] and [FeFe] hydrogenase. In addition, samples of isolated Hmd were colorless and did not contain iron-sulfur clusters, nickel or Flavin, lending further support to the metal free hypothesis. However, the “iron free” hypothesis was later reexamined in light of FTIR193 and Mössbauer,194 observations that the Hmd enzyme and an isolated cofactor from the enzyme is inactivated by UV-A and blue light.190 The light sensitivity of [Fe] hydrogenase was not fully recognized or appreciated prior to 2004 and contributed toward ambiguity or overlooking of the single Fe site in [Fe] hydrogenase in earlier studies. It is now known that light inactivation of the [Fe] hydrogenase results in release of the single iron atom and the associated two CO ligand. The resulting inactivated cofactor was shown to be (6-carboxymethyl-3,5-dimethyl-2pyridone-4-yl)-(5’guanosyl) phosphate by NMR and mass spectrometry.195

15.02.1.4.3

Spectroscopic studies

The FTIR spectrum of [Fe] hydrogenase shows two distinct n(CO) stretching bands of equal intensity at 2011 and 1944 cm−1,193 providing compelling evidence for the presence of Fe carbonyl species. Analysis of the n(CO) stretching bands indicate that the two CO ligands are coordinated in cis fashion and later confirmed with nuclear vibrational resonance spectroscopy (NVRS).196 Analogously, the isolated FeGP cofactor with acetic acid (2029, 1957 and 1697 cm−1) or 2-hydroxyethanethiolate (2004, 1934 and 1680 cm−1) shows a distinct set of bands192 in agreement with a metal carbonyl binding motif. The high frequency region around 2000 cm−1 is consistent with linear metal carbonyl binding while the low frequency around 1600 cm−1 corresponds to the acyl iron C]O stretching frequency. The synchronized shift in all three bands corresponds to the difference between acetate and 2-hydroxyethanethiolate ligand binding in extracted FeGP and is consistent with acyl coordination to the iron center also in intact FeGP. Mössbauer and XANES studies of the enzyme, in comparison with model complexes, indicate that the FeGP cofactor contains a low spin Fe(II) center.194,197,198

15.02.1.4.4

Structure and mechanism of [Fe] hydrogenase

Unlike the [NiFe] and [FeFe] hydrogenases where a wealth of spectroscopic data of intermediate states are available to provide insights to their respective mechanisms, the only available spectroscopy information for [Fe] hydrogenase is from the resting open state. Thus, the current prevailing mechanism for hydrogenation of CH^H4MPT by [Fe] hydrogenase is based on DFT calculations199–203 and high resolution crystallographic data.11 High-resolution crystallographic structures of the [Fe] hydrogenase in the resting form (‘open conformation’) and active form (‘closed conformation’) are available.11,204 The enzyme forms homodimers where two C terminal segments intertwine to provide a central domain. The active site with its FeGP cofactor and CH^H4MPT/CH2]H4MPT substrate is located in the two clefts between the central domain and N terminals (Fig. 32). As suggested from the extraction experiments and spectroscopy, the FeGP cofactor is covalently linked to the enzyme via a single cysteine thiol (cys176), in addition to non-covalent interactions between the N-terminal polypeptide and the GMP group. The methylene-acyl moiety was previously postulated to be a carboxylate group in earlier studies,204 but was later revised based on

Hydrogenases and Model Complexes in Bioorganometallic Chemistry

33

Fig. 32 Crystal structures of Hmd in the open (pdb code: 6HAC) and closed (pdb code: 6HAV) form. The structures of the FeGP and CH ^ H4MPT substrate shown on the right (truncated for clarity). The protein backbone is colored based on subunits to highlight the dimeric nature, while cofactors and substrates are shown in ball and stick format with carbons shown as pale grey, heteroatom color coding: Fe: orange; S: yellow; O: red; N: Blue; P: Dark orange. Adapted from reference Huang, G.; Wagner, T.; Ermler, U.; Shima, S., Nat. Rev. Chem. 2020, 4(4), 213–221.

studies with the C176A mutant.205 In the C176A mutant, the thiolate ligation of FeGP is replaced by a dithiothreitol (DTT) moiety that spatially precludes modelling of the putative carboxylate group into the observed electron density but allows inclusion of an acyl ligation. This lead to revision of the wild type crystal structure from carboxylate to acyl ligation. Binding of a CH^H4MPT substrate to the resting open conformation induces a structural change where the N-terminal domain rotates by approximately 25 closing the cleft to form the active closed conformation (Fig. 32). The weakly bound water ligand to the FeGP is lost resulting in a vacant coordination site. The closure of the cleft also brings the CH^H4MPT substrate closer to the FeGP cofactor. The site of hydrogenation on CH^H4MPT (C14a) to Fe distance is 3.8 A˚ in the active closed form compared to over 9 A˚ in the inactive open form. As outlined in Fig. 33 the pyridinol moiety of the FeGP is then deprotonated at the 2-OH group to give a pyridonate group. This deprotonation is evidenced by the C2dO distance of 1.33 A˚ in the crystal structure of the active closed form, which lies between the distance of a CdO bond of phenol (1.36 A˚ ) and a C]O bond within an aromatic ring (1.2 A˚ ).11 This pyridonate group is proposed to function as an internal base to facilitate proton removal and heterolytic H2 cleavage. H2 binds at the now vacant site on the Fe center to give a Fe(Z2dH2) species that then undergoes heterolytic cleavage to give an FedH hydride species, with the nearby pyridonate accepting a proton. Subsequent hydride transfer from Fe to C14a of CH^H4MPT to give CH2]H4MPT, followed by release of product, water coordination to FeGP and structural rearrangement completes the catalytic cycle. It should be noted that the metal oxidation state remains at Fe(II) throughout catalytic cycle, in contrast to the redox active cofactors of [FeFe] and [NiFe] hydrogenases. Albeit, it is noteworthy that the Fe center remains Fe(II) throughout the [NiFe] catalytic cycle. Still, we consider it important to reiterate that despite the apparent differences in chemistry between [Fe] hydrogenases and its [FeFe] and [NiFe] counterparts, there are a number of structural and functional parallels between all three classes of hydrogenase. Firstly, the active site of all three classes of hydrogenases contains Fe centers that are ligated by CO ligands. Coordination of CO (and in the case of [FeFe] and [NiFe] hydrogenases also CN−), while unusual in biological systems, stabilizes a low spin Fe center which

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 33 Proposed mechanism of [Fe] hydrogenase. The resting state of the enzyme is shown in the top right.

facilitates coordination of H2 to form a Fe(Z2dH2) species. Second, the presence of a vacant coordination site for H2 binding and a nearby basic residue for proton abstraction is a shared motif in all three hydrogenases. In the [Fe] hydrogenase, the enolate group on the pyridinol ligand is positioned close (3.2 A˚ ) to the vacant site on the FeGP cofactor. In [FeFe] hydrogenase, the amino group on the adt bridging ligand is proposed to function as base for proton abstraction while a nearby Cys546 or Arg509 provides the same function in [NiFe] hydrogenase.24,121 These common motifs, low spin Fe, vacant site and a nearby base facilitate binding and heterolytic cleavage of H2 in all three classes of hydrogenase.

15.02.1.4.5

Model complexes of the FeGP cofactor

The following section summarizes major developments in synthetic model complexes of the FeGP cofactor and its contributions toward a mechanistic understanding on the reactivity of [Fe] hydrogenase. The specific complexes discussed herein are summarized in Fig. 34. These systems have also been the topic of a number of excellent reviews during the last few years.24,121,171,187,206 Synthetic modelling of the FeGP active site began in earnest with the first publication of the enzyme crystal structure in 2008.204 Early efforts were focused on reproducing the structural motifs of the FeGP active site, namely a mononuclear dicarbonyl Fe(II) center. One of the early model complexes is the cis carbonyl Fe(II) complex 4.1 used as model for comparison of XANES and Mössbauer spectroscopy of the native enzyme.198 After publication of the first crystal structure204 containing the FeGP active site, synthetic modelling began focusing on installing a bidentate pyridone ligand (N and O/S ligation) on a cis carbonyl Fe(II) center (4.2 and 4.3).207,208 Subsequent revision and confirmation of an acyl ligand in the crystal structure205 inspired synthesis of model complexes containing an acyl ligand (4.4 and 4.5).209,210 Recent advances in synthetic structural model complexes incorporate multiple structural features of the FeGP such as pyridone, acyl and thiolate ligation to an Fe(II) center with two cis carbonyl ligands, for example complexes 4.5 to 4.7 and 4.10 (Fig. 34).211–213 While progress in synthetic structural models is approaching faithful reproduction of the FeGP cofactor ligation environment, the above structural models (Fig. 34) do not bind H2 or exhibit any H2 activation activity. One approach toward more functionally relevant model complex is to replicate the closed active form of the FeGP cofactor with a vacant site. An early report of a five coordinate square pyramid complex (4.8) has ligation environment that is less faithful to the FeGP cofactor and does not exhibit H2 activation activity.214 Later efforts to produce five coordinate square pyramid complexes (4.9 and 4.10) resulted in complexes that more closely resemble the FeGP cofactor (Fig. 34) in terms of ligation of the Fe center, yet no H2 activation activity is reported.215,216 Despite the close structural similarity to the closed form FeGP cofactor, the lack of H2 activation may be due to missing crucial secondary interactions, such as the presence of a suitable base near the vacant site. Xu et al. reported a complex incorporating a proximal base to the vacant site via a bidentate Et2PCH2N(Me)CH2PEt2 (PNP) ligand ([4.13(I)]) that is capable of activating H2 as evident by H/D exchange activity.217 Additionally, complex [4.13(I)] is capable of performing hydrogenation of an aldehyde although the reaction is not catalytic as the complex is degraded in the process (Fig. 35). Activation of H2 is hypothesized to proceed by loss of an iodide ligand to reach a five coordinate intermediate (4.13+) which reacts with H2 to give the hydride species [4.13(H−) (H+)]+ although no direct evidence for such species has been reported. The loss of iodide from [4.13(I)] to form 4.13+ is substantiated by the observation that rate of both H/D exchange and hydrogenation of aldehyde are slower in the presence of

Fig. 34 Examples of structural models of the FeGP cofactor. Top row: model complexes with cis carbonyl only and cis carbonyl with acyl motif. Middle row: model complexes with acyl and thiolato motif. Bottom row: model complexes with vacant coordination site.

Fig. 35 Examples of functional models of FeGP and their respective hydrogenation/dehydrogenation reactions.

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Hydrogenases and Model Complexes in Bioorganometallic Chemistry

excess iodide. Complex 4.13+ demonstrate the functional importance of a proximal base provided by a non-biomimetic PNP ligand in H2 activation. The role of the proximal base is further highlighted by reconstitution experiments of apo-[Fe] hydrogenase with model complexes 4.11 and 4.12, analogous to the semi-synthetic approach in [FeFe] hydrogenase (Section 15.02.1.2.3.6).218 Reconstitution of complexes 4.11 and 4.12 is achieved by loss of an iodide and a CO ligand as evidenced by FTIR, which closely resembles that of the wild type enzyme. Reconstituted [Fe] hydrogenase using complex 4.11 exhibit reversible hydrogenation/dehydrogenation of CH^H4MPT/CH2]H4MPT while the methoxy derivative (4.12) do not exhibit any activity. This study provides strong evidence that deprotonation of the pyridinol moiety to pyridonate that function as proximal base is crucial for H2 activation. Additionally, this study also provides experimental evidence to disfavor earlier suggestions that a cysteine derived thiolate would act as proximal base during catalysis.201,203 The reconstituted [Fe] hydrogenase using complex 4.11 exhibit approximately 1% of wild type activity and this is attributed to subtle electronic differences between the synthetic pyridinol and biological GP ligands. Indeed, reconstitution experiments in presence of exogenous GMP and complex 4.11 exhibit two-fold activity compared to experiments in absence of GMP. As the native FeGP cofactor has extensive non-covalent interactions between the GMP moiety and the protein, it is likely that there are subtle geometric differences around the Fe center between the reconstituted 4.11 and 4.11+ GMP assays that influences the reaction rates. This reconstitution study provides an elegant demonstration of the utility of model complexes, allowing selective tuning of substituents on metal active site by synthetic organometallic chemistry, toward understand the mechanism of the native enzyme. Remarkably a reconstitution study using Mn based complexes also demonstrated enzymatic activity,219 and a subsequent Mn based model complex also showed hydrogenation reactions in absence of the protein environment.220 Demonstration of dehydrogenation activity was achieved by model complexes (4.14) utilizing an anthracene scaffold that was reported221,222 to exhibit H2/D2 activation, hydride transfer to H+/D+ and CdH hydride abstraction (Fig. 35). The reactivity of these complexes is attributed to a stable fac arrangement of the C, S, N ligands, analogous to the FeGP cofactor, imposed by the anthracene scaffold, with the putative reactive site trans to the acyl moiety. The corresponding complex with mer arrangement of these C, S, N ligands do not exhibit any activity. Progress on modelling the FeGP cofactor by synthetic organometallic complexes has steadily progressed from reproducing a one or two ligation environment to near faithful replication of the structural feature. Beyond producing structural model complexes, the field of organometallic chemistry producing functional mimics that are inspired by the FeGP cofactors is advancing beyond the infancy stage. The current generation of functional mimics have demonstrated both hydrogenation and dehydrogenation reactions, albeit the reactions have low activity or are non-catalytic. Nonetheless, the field is moving forward and current goals are focused on two major areas of development in structural and functional mimics: (i) characterization and isolation of detectable reactive intermediates and (ii) development of more active and robust catalysts for hydrogenation/dehydrogenation reactions.

Acknowledgments Research in the authors laboratories is supported by the Swedish Research Council (S.G. SDG project number 2017-04992); the Swedish Energy Agency (GB project number 48574-1, MHC project number 50529-1), the Carl Trygger Foundation (S.G. and G.B.); the Horizon 2020 program, ERC StG (G.B. contract number 714102) and Horizon2020 program, ITN (G.B., eSCALED, contract number 765376).

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15.03

Nitrogenases and Model Complexes in Bioorganometallic Chemistry

Daniel WN Wilson and Patrick L Holland, Department of Chemistry, Yale University, New Haven, CT, United States © 2022 Elsevier Ltd. All rights reserved.

15.03.1 Introduction 15.03.2 Nitrogenase 15.03.2.1 The Fe protein and the F-cluster 15.03.2.2 The MoFe protein 15.03.2.2.1 The P-cluster 15.03.2.2.2 Atomic and electronic structure of the FeMo cofactor resting state 15.03.3 Interaction of FeMoco with substrates 15.03.3.1 Nitrogen 15.03.3.1.1 The kinetic model of N2 reduction 15.03.3.1.2 The E1 state 15.03.3.1.3 The E2 and E4 states 15.03.3.1.4 The E7 and E8 states 15.03.3.1.5 The alternating and distal mechanisms 15.03.3.2 Alternative substrates 15.03.3.2.1 Protons, H+ 15.03.3.2.2 Acetylene 15.03.3.2.3 Carbon monoxide and selenocyanate 15.03.3.2.4 Cyanide 15.03.3.2.5 The surroundings of the FeMoco, and extracted FeMoco 15.03.3.3 Alternative nitrogenases 15.03.3.3.1 Reactivity of V-nitrogenase towards carbon monoxide 15.03.4 Biosynthesis of FeMoco 15.03.4.1 Radical-SAM enzymes and alkylated Fe4S4 clusters 15.03.5 Model complexes 15.03.5.1 Functional models of catalytic N2 reduction 15.03.5.2 Iron-sulfur clusters 15.03.5.3 Iron complexes with sulfur and N2 ligands 15.03.5.4 Iron complexes with sulfur and NxHy ligands 15.03.5.5 Iron carbides 15.03.5.6 Iron complexes with other carbon ligands 15.03.5.7 Iron hydrides 15.03.5.8 Modeling second-sphere effects 15.03.6 Outlook 15.03.7 Note Added in Proof Acknowledgments References

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15.03.1 Introduction Nitrogen is found in many industrially and biologically important molecules and is one of the cornerstones of life. The most plentiful source of N is dinitrogen (N2), which constitutes 78% of Earth’s atmosphere. However, the nitrogen atoms in N2 are nearly inaccessible in such an inert molecule and must be “fixed,” i.e., converted to a solid or liquid form, such as ammonia, in order to engage the subsequent reactions needed in biological or industrial uses. Approximately equal amounts of N2 are fixed by microorganisms and by the Haber-Bosch process, each producing approximately 2  1013 g year−1.1–3 Despite over a century of study, the mechanism of nitrogen fixation, by either human catalysts or biological enzymes, has yet to be fully elucidated. Improved understanding of the biological mechanism of N2 reduction could inform the design of new catalysts for production of ammonia or other N products.4 Additionally, the development of new technologies could enable fertilizers to be produced locally.5 The reduction of N2 requires breaking the strong N^N bond (944 kJ mol−1). In addition to this thermodynamic challenge, there are kinetic challenges because N2 is a non-polar molecule that interacts weakly, if at all, with most compounds. Considering these difficulties, it is amazing that enzymes have evolved to fix N2 under ambient temperature and pressure. This article highlights the developments in understanding how nitrogenases reduce N2 from the perspective of organometallic chemistry. Particular attention

Comprehensive Organometallic Chemistry IV

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is given to the metal cluster which is the active site of reduction and the current understanding of the mechanism by which it reduces substrates. After a description of relevant structural, spectroscopic, kinetic, and other mechanistic data on the enzyme, we describe synthetic model complexes and the insights they contribute. A more detailed view of current N2 research, including several articles on nitrogenases, can be found in a recent special edition of Chemical Reviews.6–13

15.03.2 Nitrogenase Nitrogenases are enzymes that catalyze the reduction of N2 to ammonia using protons, a reducing agent, and ATP. The reaction is shown in Eq. (1), where ATP and ADP are adenosine tri- and di-phosphate, respectively, and Pi is inorganic phosphate (PO43−, HPO42−, and/or H2PO4− depending on pH). N2 + 8e + 8H + + 16ATP ! 2NH3 + H2 + 16ADP + 16Pi

(1)

Nitrogenases are produced by anaerobic and aerobic bacteria and archaea. Prior to the advent of the Haber-Bosch process, these bacteria were responsible for the majority of the bioavailable nitrogen.14 These diazotrophs may live symbiotically with leguminous plants or may be free-living in soils and oceans. The three known isozymes of nitrogenase are distinguished by the composition of their cofactors—the Mo-dependent, V-dependent, and Fe-only nitrogenases.15 Each isozyme is encoded by its own set of genes, but they have similar overall structure and layout of cofactors. All nitrogenases contain a “Fe protein,” (FeP) a  60 kDa homodimer that is encoded by nifH, vnfH, or anfH, and a catalytic protein (MoFe, VFe, or FeFe, which we collectively term MFe) that accepts electrons from FeP.16,17 This latter protein is in turn made up of subunits, encoded by the nifD and nifK genes in an a2b2 tetramer in MoFe and an a2b2d2 heterohexamer in the VFe and FeFe proteins.18,19 Each ab subunit of the MFe proteins contains its own copy of the P-cluster and FeM cofactor (FeMco: FeMoco, FeVco, or FeFeco), which are the metal sites responsible for the activity of the protein. Each pair of clusters is sufficiently separated (>70 Å) that the pairs can each function independently, though there may be cooperativity (discussed below). The MFe and FeP proteins are numbered 1 and 2, respectively, with an abbreviation of the organism name. For example, the Fe protein from A. vinelandii is termed Av2 and the MFe protein from C. pasteurianum is named Cp1. The P-cluster is an [Fe8S7] cluster responsible for transferring electrons to the FeMco cluster (M ¼ Mo, V, Fe) which serves as the active site of the enzyme.20,21 The FeMco, also known as M-clusters, are some of the most complex metalloclusters found in nature, comprising of a [MoFe7S9C] cluster for the FeMo cofactor (FeMoco) and a [VFe7S8C(CO3)] cluster for FeVco. The exact composition of FeFeco is yet to be determined, but extended X-ray absorption fine structure (EXAFS) and Mössbauer results indicate that it has a similar constitution as the other cofactors.22 The role of the FeP is to bind to the MFe protein, hydrolyze ATP, and transfer electrons to the MFe protein.23 Most research conducted on nitrogenases has focused on the MoFe nitrogenase, which is the most active towards N2 fixation. It is preferentially produced when the enzyme is grown with sufficient Mo present.24 The V and Fe nitrogenases are only expressed under Mo-poor conditions, or when the genes for expressing the preferred nitrogenase are knocked out, with the order of preference being Mo > V > Fe.24,25 As such, these latter two nitrogenases are termed “alternative nitrogenases.” The bulk of this article discusses MoFe nitrogenase, with comparisons to VFe nitrogenase where its reactivity differs significantly and passing mentions to the lesser understood Fe-only variant. It is the FeMco that binds and reduces N2 and as such it is this site which is of particular interest to organometallic chemists. Therefore, we introduce the structure and function of the metal clusters within nitrogenases with a particular focus on the spectroscopy and reactivity of the FeMoco. This is followed by describing the progress made by organometallic chemists towards modeling the structure and reactivity, and outlining the key challenges that remain.

15.03.2.1 The Fe protein and the F-cluster The iron protein, referred to as FeP or NifH in the case of the Mo-dependent system after the gene that encodes it, houses a [Fe4S4] cluster shaped like a cube with Fe and S at alternating vertices (Fig. 1). This F-cluster lies at the interface between the 2 units of the homodimer and is held in place by the sulfur atoms of two cysteine sidechains from each protein subunit. During catalysis the F-cluster accesses [Fe4S4]2+ (FePox) and the [Fe4S4]1+ (FePred) oxidation states, which have mixtures of iron(II) and iron(III). The FeP functions as a specialized reducing agent towards the MFe protein (M ¼ Mo, V, Fe) during catalysis, and is also needed during key steps in the biosynthesis of the FeMoco and P-clusters.26 After FePred donates an electron, the resultant FePox must be re-reduced, and these electrons come from flavodoxin hydroquinone (FldHQ) or ferredoxin in vivo, though in most in vitro studies the terminal reductant is dithionite (see below). Electron transfer (ET) in nitrogenase is intimately linked to structural changes in the protein. Fig. 2A outlines the ET between cluster sites in nitrogenase, while Fig. 2B shows the binding and structural changes of the two proteins during ET. The reduced FeP binds two equivalents of ATP,27 which changes the redox potential of the [Fe4S4]2+/1+ couple (−0.30 V in the absence of nucleotides, −0.43 V in the presence of ATP, and −0.49 V in the presence of ADP).28–32 Additionally, the reduced FeP binds to MoFe, giving an encounter complex (ATP)2FeP:MoFe which places the F-cluster in a hydrophobic environment, making it a stronger reducing agent and providing additional driving force for ET from the F-cluster (in the FeP) to the P-cluster (in the MoFe protein). In addition, there is a hydrogen-bonding network connecting the MoFe peptide backbone to the sulfide sites on the cluster. Combined, these effects

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Fig. 1 The clusters of Mo nitrogenase. FeP is shown in green while MoFe is shown in blue. The location of the three iron-sulfur clusters, denoted M, P and F, is indicated. Below, the structure of the three clusters are shown. Color legend: yellow, S; orange, Fe; gray, C; teal, Mo. Used with permission from Van Stappen, C.; Decamps, L.; Cutsail, G. E.; Bjornsson, R.; Henthorn, J. T.; Birrell, J. A.; DeBeer, S. Chem. Rev. 2020, 120, 5005–5081.

(A)

(B)

Fig. 2 (A) A simplified scheme showing electron transfer between metal clusters in a nitrogenase enzyme. Flavodoxin (Fld) is a physiological reductant, while dithionite is frequently used during in vitro study. n is the number of electrons accumulated on FeMoco prior to the cycle beginning. (B) Scheme representing the binding and structural changes of the two proteins during the electron transfer. Numbers in circles correspond to steps which include electron transfer to/from metal clusters; note that step 3 precedes step 2 (“deficit spending mechanism”). Figure used from Rutledge, H. L.; Tezcan, F. A. Chem. Rev. 2020, 120, 5158–5193, with permission.

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result in an efficient electron transfer pathway between the two metal clusters and explains why the FeP protein is able to induce N2 reduction in the MoFe protein while artificial reducing agents with lower redox potentials cannot.23 The electron is then transferred from the P-cluster to the FeMco, and finally the ATP bound to the FeP is hydrolyzed, breaking the encounter complex apart.27,33 Dissociation of phosphate entails recovering the energy that previously went into burying the cluster and providing the driving force for ET (there is no free lunch!) and not surprisingly is a slow step in catalysis.33,34 Work remains to establish a molecular level understanding of how ATP hydrolysis and Pi release destabilize the complex and how this enables dissociation of the spent FeP. When studying the enzyme in vitro, the terminal reductant can be varied but FeP is still needed because of its specific ability to induce N2 reduction. The number of electron equivalents available for ET to FeMco is typically modulated by changing the ratio of %− FeP to MFe.8 The most commonly used reductant is dithionite, S2O2− 4 , which is convenient because it dissociates to SO2 which in 2− turn releases one electron and gaseous SO2. However, it was recently proposed that SO3 can act as a source of one of the belt sulfides in FeMoco.35 A recent crystal structure of FeVco grown under deficient S2O2− 4 showed partial substitution at one of the belt sulfides.36 Thus, the various roles of dithionite in catalysis must be re-evaluated in the future. FePox exhibits a diamagnetic S ¼ 0 ground state, which comes from antiferromagnetic coupling between high-spin iron subsites. 57 Fe Mossbauer studies have characterized two Fe environments, with a 3:1 ratio.37 This was analyzed using spatially resolved anomalous dispersion (SpReAD) analysis, which uses X-ray crystallography at several wavelengths near the Fe X-ray absorption edge to assign oxidation state at individual metal sites. SpReAD indicated the Fe sites of FePox to all be Fe2.5+, where some valence electrons are equally shared between iron sites.38 FePred, on the other hand, formally has three Fe2+ sites and one Fe3+ site, which are coupled as an S ¼ 1/2, 3/2 mixture. The ratio of the two spin states depends on the solvent and the presence of bound ADP or ATP.37–42 For example, FePred prepared in 50% ethylene glycol produces  90% S ¼ 1/2, while the presence of 0.4 M urea instead results in  85% S ¼ 3/2. A combination of electron paramagnetic resonance (EPR) and EXAFS suggest that the cluster maintains its geometry and it is the surrounding protein which is influenced by the presence of ATP.37,43 A third oxidation level, [Fe4S4]0 with all Fe2+ subsites, can be accessed through reduction of [Fe4S4]1+ with Ti3+, Cr2+, or radiolytic reduction with gamma rays.44–46 Flavodoxin hydroquinone, a physiological reductant, is also capable of reducing the P-cluster to its all ferrous state.11,47 Whether the [Fe4S4]0 state is involved in physiological nitrogenase activity is still an open question.

15.03.2.2 The MoFe protein 15.03.2.2.1

The P-cluster

The MFe protein contains an [Fe8S7] cluster (“P-cluster”), which has a structure that has been observed nowhere outside the nitrogenases. In the two structurally characterized proteins (MoFe and VFe) the topology of the P-cluster is the same.48 The role of the P-cluster is to accept incoming electrons from FePred and to donate electrons to FeMco.20,21 Interestingly, the electron transfer steps do not occur in the order one might expect. Upon docking of the FeP to the MoFe protein, the P-cluster transfers an electron to the FeMoco, which is followed by an electron transfer from the FeP to the now oxidized P-cluster to regenerate the reduced state. This “deficit-spending” mechanism is outlined in Fig. 2.11,20,31,34,49 Recent studies have shown that during the FeP to MoFe electron transfer the two ab subunits in Mo-dependent nitrogenase display negative cooperativity,29 and thus electron transfer in one half of the heterotetramer suppresses the electron transfer reaction in the other half. The significance of this observation is not yet clear. The reduced state of the P-cluster, termed PN, has all Fe2+ sites. It can be oxidized by two electrons to give the P2+ state (often called POx), and under special conditions can access the intermediate P1+ oxidation level.50–52 The P2+/P1+ redox potential is about −0.3 V, but varies with pH.11,53 The redox changes are accompanied by significant structural changes.54,55 The PN state has all-thiolate coordination, with a pseudo-2-fold symmetry axis through the central sulfide (Fig. 3). In P1+, the Fe6 atom moves away from the central S1 to bind to a nearby Ser188, while Fe5 remains in the same location as in the PN state.54 Upon oxidation to P2+ there is movement of two Fe ions, Fe5 and Fe6, away from S1 with binding of different protein sidechains. This structural flexibility is likely a necessity of the deficit spending mechanism, altering the midpoint potential for reduction, and communicates information about the oxidation state of the P-cluster to the FeP to trigger ATP hydrolysis. However, the reason for the ability to

Fig. 3 Structural changes to the P-cluster upon oxidation (PDBs 3MIN, 6CDK, 2MIN). Color legend: yellow, S; orange, Fe; gray, C; red, O; blue, N. Figure used from Van Stappen, C.; Decamps, L.; Cutsail, G. E.; Bjornsson, R.; Henthorn, J. T.; Birrell, J. A.; DeBeer, S. Chem. Rev. 2020, 120, 5005–5081.

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access three oxidation levels from PN to P2+ is not yet explained, since N2 reduction is expressed as sequential one-electron reductions. Some have argued that this is connected with the possibility of two-electron changes using the [Fe4S4]0 level of FeP.56 The PN state is diamagnetic, with a featureless EPR spectrum and no response of its Mössbauer signal to a magnetic field.57 The P1+ state displays an S ¼ 1/2, 5/2 mixed spin state, potentially arising from mixed isomers in which the proximal Ser188 residue is bound or not.52 The P2+ state displays an S ¼ 3 or 4 signal depending on the method of oxidation.51 Finally, a more highly oxidized form can be observed with an S ¼ 7/2 signal and has been assigned as a P3+ state. Computational studies on the PN, P1+, P2+, and P3+ states of the cluster have been used in an effort to elucidate their protonation states and conformation.58 The lowest energy state, termed BSb11, has good agreement with X-ray data and features antiferromagnetic coupling of local spins between Fe sites. However, a separate study using a density matrix renormalization group approach suggested a different description of the low-lying electronic states of P1+ and P2+.59 Additional work is required to understand the differing signals displayed for the electronic states, and likely will require improved theoretical methodologies rather than advancement in spectroscopic techniques.

15.03.2.2.2

Atomic and electronic structure of the FeMo cofactor resting state

The M-clusters of nitrogenases are some of the largest metalloclusters that have been identified in nature, and are the only natural cofactors known to contain a carbide (C4−). The long quest to determine the atomic-level details of these natural organometallic clusters required Herculean efforts of both spectroscopy and crystallography. The first crystal structure of Mo-nitrogenase, from A. vinelandii, came from X-ray data to a resolution of 2.7 Å and suggested a model of the FeMoco as linked [Fe4S3] and [MoFe3S3] subclusters m2-bridged by three ligands, two of which were modeled as sulfides and the third designated as an unknown “Y”.60 Better resolution of the A. vinelandii MoFe protein to 2.2 Å enabled the assignment of “Y” as a third m2-sulfide.61,62 Encased by the two subclusters was an internal cavity surrounded by six Fe ions, which was initially identified as a possible substrate binding site for N2. However, in 2002 a crystal of suitable quality for 1.16 Å structural resolution finally showed that the central cavity was occupied by a m6-coordinated light atom, “X”, consistent with carbide (C4−), nitride (N3−), or oxide (O2−).63 Initially, the light atom “X” was suggested to be N3− from the cleavage of N2, an extrapolation of the earlier theory that the cavity is a substrate binding site. However, later spectroscopic studies detected neither evidence for the exchange of N nor the presence of any nitrogen in the FeMoco.64,65 The unequivocal assignment of “X” came in 2011 using Fe Kb X-ray emission spectroscopy, which showed far better agreement with the calculated signature of an interstitial C than that of an N or O.66 This assignment was corroborated by a 1.0 Å resolution structure of the MoFe protein, with electron spin echo envelope modulation (ESEEM) studies of the MoFe protein labeled with 15N and 13C, and with 14C labeling studies which all are consistent with a central C4−.67–69 The known properties of synthetic clusters with C4− ligands are outlined in Section 15.03.5.5. The consensus atomic-level model of the FeMoco is shown in Fig. 4. Each Fe and S site is labeled based on its position in the protein structure; this notation will be used throughout this article. Each of the metal sites in the resting state is fully coordinated: 4-coordinate iron is commonplace in iron-sulfur clusters, and the Mo site is octahedral. Though Fig. 4 implies a pseudo-threefold symmetry around the long axis of the cluster, the protein surroundings differentiate the sides. The two positively charged guanidine groups of Arg96 and Arg359 lie along the Fe3-S5a-Fe5 side, and there is a hydrogen bonding interaction between His195 imidazole and the m2-sulfide S2B in FeMoco (the same is true in the FeVco).70 Crystal structures of Av and Cp FeMoco at lower pH indicate that S3A or S5A, the other two m2-sulfides in the “belt” region, can be protonated as well.71 The FeMoco in its resting state has an S ¼ 3/2 ground state.72 The Mo ion was initially assigned as Mo4+ based on electron nuclear double resonance (ENDOR) spectroscopy,73 but recent high energy resolution fluorescence-detected (HERFD) X-ray absorption spectroscopy (XAS) at the K-edge of Mo gave pre-edge and rising edge features more consistent with a Mo3+ (4d3) assignment.74 The Mo3+ state is proposed to be in a non-Hund electronic configuration in which the local spin configuration is stabilized through Mo-Fe interactions (Fig. 5). The seven Fe have high-spin electronic configurations as indicated by Mössbauer spectroscopy.75 SpReAD analysis of the nitrogenase resting state in the NifD2K2 heterotetramer suggests that three Fe sites are more reduced (Fe1, Fe3, and Fe7), and four more oxidized (Fe2, Fe4, Fe5, Fe6).76 The presence of the P-cluster with all Fe2+ was used as an internal standard and aligned with the profiles of Fe1, Fe3, and Fe7, allowing for an assignment of 4Fe3+:3Fe2+ which together with Mo3+ can rationalize the observed S ¼ 3/2 ground spin state of FeMoco from EPR spectroscopy. Single crystal EPR spectroscopy indicates that the Fe2 and Fe6 sites are the most oxidized in the resting state, supporting the idea that FeMoco has asymmetric surroundings that influence its electronic structure.77

Fig. 4 Molecular structure of the FeMo cofactor with atom labels. The nearby His195 residue is also shown.

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Nitrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 5 Occupied localized t2g orbitals of FeMoco [MoFe7S9C]1− displaying overlap between Mo and Fe d-orbitals. Mo is positioned in the bottom left of the cluster. Color legend: yellow, S; red, Fe; black, C. Figure used from Bjornsson, R.; Lima, F. A.; Spatzal, T.; Weyhermüller, T.; Glatzel, P.; Bill, E.; Einsle, O.; Neese, F.; DeBeer, S. Chem. Sci. 2014, 5, 3096–3103.

Fig. 6 Model of the BS7-235 spin state of FeMoco, the lowest energy electronic structure of the resting state of FeMoco. Color legend: yellow, S; orange, Fe; gray, C; teal, Mo; red, O; blue, N. Figure used from Benediktsson, B.; Bjornsson, R. Inorg. Chem. 2017, 56, 13417–13429.

The magnetic coupling between the eight spin-bearing metal centers in the FeMoco is complex. Spin-polarized broken-symmetry density functional theory (BS-DFT) model initially defined ten potential coupling schemes,78–80 and BS7 has repeatedly been the best supported, containing four Fe sites in the spin-up configuration coupled to three spin-down sites, which maximizes the antiferromagnetic coupling (Fig. 6). However, it is important to note the limitations of the BS-DFT method, which has failed to reproduce the spin coupling in much simpler mixed valent iron-sulfur clusters due to the number and energy density of low-lying states.81 The use of advanced multiconfigurational computations to model the electronic structure of the FeMoco with appropriate treatment of electron-electron correlation is an area of current development.82

15.03.3 Interaction of FeMoco with substrates 15.03.3.1 Nitrogen 15.03.3.1.1

The kinetic model of N2 reduction

As outlined in Eq. (1), reduction of N2 to NH3 by nitrogenase requires a total of eight e− and eight H+ equivalents, because two protons and electrons are inevitably lost as a molecule of H2. The generation of H2 is proposed to be a requisite of the N2 reduction mechanism, as H2 is observed alongside NH3 even at 50 atm of N2.83 Each reducing equivalent delivered to FeMoco is assumed to be accompanied by a proton to balance charge, which explains why it is possible to accomplish multiple-electron reduction using the same reductant repeatedly in series. The combined kinetic data for N2 reduction under varied conditions were modeled by Lowe and Thorneley, resulting in the eponymous Thorneley-Lowe scheme (Fig. 7).84–87 In this scheme, the E states are numbered to represent the number of electrons residing on the cofactor relative to its resting state, E0. The values inside the parentheses are the number of N and H atoms present. The key features of the Thorneley-Lowe scheme are as follows. (i) Several reductions of the FeMo protein occur before N2 binds. Consequently, the crystallographically characterized resting state of FeMoco (E0) is not directly relevant to N2 binding. (ii) Electrons are added one at a time and accompanied by a proton, noted as [e−/H+]. Whether these protons are associated with the metal centers as hydrides or the sulfide/carbide/homocitrate ligands as protons is not specified by the model and remains a point of debate. (iii) N2 displaces H2 at the E4 state in a reductive elimination/oxidative addition equilibrium (re/oa).83,88,89 The re/oa equilibrium explains why H2 is an inhibitor of activity in nitrogenases (see Section 15.03.3.1.3). (iv) Hydrazine can be released at the E4(2N2H) state, as shown by the detection of hydrazine upon quenching the enzyme under specific conditions.90

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Fig. 7 A depiction of the Thorneley-Lowe scheme. The number of electrons added is represented in the En subscript, and the number of added protons in the system is in parentheses. The protonation states of the intermediates up to E4(2N2H) have been evaluated spectroscopically, and ones after this are speculative.

There has not yet been any definitive crystallographic characterization of a species with N2 or its partial reduction products on the enzyme. Over the years, some papers have claimed the identification of N-containing species on the FeMoco, but in each case, crystallographic problems such as disorder and/or misassignment of atoms have emerged.36,63,91 This leaves the frustrating situation where we have a relatively detailed kinetic model that relates the intermediates, but no clear view of the structures corresponding to these intermediates. (It is also a reminder to the community about the limitations of X-ray crystallography, which is often given undue mechanistic significance despite the disconnect between the transient nature of reaction intermediates vs. the slow timescale of crystal growth.) While structural characterization of no E state other than E0 has been achieved, advances in experimental techniques have enabled spectroscopic detection of the E1, E2, and E4 states. As probing the various E states has predominantly used EPR and ENDOR spectroscopy, which are most effective for odd-electron species, characterization of the even-electron states E3 and E5 lags behind. Aspects of the E7 and E8 states have been proposed from reactivity studies with alternative substrates hydrazine and diazene, as described below.

15.03.3.1.2

The E1 state

The E1 state is the result of the first electron and proton transfer to the FeMoco. As with all intermediates during N2 turnover, the E1 state has not been generated as a pure species. However, information about this state has come from spectroscopic and computational analysis. Significant populations of E1 can be generated during turnover in the absence of N2 and with the correct ratio of MoFe:FeP. By decreasing the ratio of FeP to MoFe, the steady-state concentrations of different intermediates can be adjusted.92 With the appropriate ratio, the only states significantly populated are E0 and E1. Though the E1 state is inaccessible to EPR, the ratio of E0 to E1 present in a sample can be determined through quantification of the remaining E0 concentration. Studies performed in this way using Mo and Fe K-edge XAS and 57Fe Mössbauer revealed the E1 reduction to be Fe centered.93 The Mo and Fe K-edge EXAFS reveal the coordination sphere of Mo to be similar, and a mild expansion of average Fe-S is accompanied by contraction of the Fe-Fe distances.93,94 Computational studies in conjunction with the Fe K-edge XAS data attempted to identify the most likely site of protonation in E1(1H), concluding that the either S2B or S5A is the most energetically favorable position with the best fit to the experimental data.95

15.03.3.1.3

The E2 and E4 states

The E4(4H) state is the result of four [H+/e−] deliveries and is proposed to be the species at which N2 binding occurs. As such, E4(4H) has received considerable spectroscopic and computational attention.8 This state sits at the transition point of the Thorneley-Lowe cycle, where it can relax back towards E0 through liberation of H2 or it can move forward by binding N2.96 The E4(4H) state was first detected by EPR spectroscopy in the Val70Ile mutant of the Av Mo nitrogenase, and later in the wild type protein.96,97 The position of E4(4H) on the Thorneley-Lowe cycle was assigned through kinetic and EPR studies through a step-annealing technique.98 During these experiments, samples held at 77 K were rapidly warmed to 253 K for various fixed times before being cooled back to 77 K or lower for EPR spectroscopy (for quantification) and ENDOR spectroscopy (for elucidating electron-nuclear coupling for insight into structure). Even though the sample stays frozen during this whole process, the increased dynamics of the protein at 253 K allow loss of H2 over time. These studies showed the S ¼ 1/2 signal, which corresponds to the E4(4H) state, converting to a new S ¼ 1/2 EPR signal assigned as the E2(2H) state. Further step-annealing allowed the relaxation of the E2(2H) state to E0. Both relaxation processes were shown to involve the release of H2, by observing substantial kinetic isotope effects when using D2O. A 1H ENDOR study of E4(4H) revealed two strongly coupled hydrogen signals that are rhombic, which is consistent with bridging hydrides but not with terminal hydrides (which would give an axial tensor).99,100 Later 57Fe ENDOR and 95 Mo ENDOR data indicated that these hydrides are bound to iron, and not to molybdenum.75 The similarity of the coupling to the metals in the different states was extrapolated to speculate that only two oxidation levels of the FeMoco are accessed during the Thorneley-Lowe scheme, with added electrons lying formally in hydrides (in E2-E4) or on the partially reduced substrate (beyond E4).

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15.03.3.1.3.1 The oxidative addition/reductive elimination model of the E4 state The binding of N2 to the E4(4H) state liberates an equivalent of H2, and the resulting state is termed E4(2N2H). The formation of E4(2N2H) is reversible, exposure of the E4(2N2H) state to H2 liberates N2 to produce E4(4H), and likewise exposure of the E4(4H) state to partial pressures of N2 liberates H2 and yields the E4(2N2H) state (Fig. 6).88,89 This equilibrium is associated with the oxidative addition of H2 and the reductive elimination of two Fe hydrides, respectively, and is termed the oxidative addition/ reductive elimination (oa/re) model (Fig. 8). Important early evidence for this mechanism came from isotopic labeling experiments. It was found that mixtures of H2 and D2 do not scramble to form HD in the absence of N2, however under turnover conditions with N2, D2 scrambles to form HD.101–104 This implies that formation and cleavage of HdH bonds occurs only with the E4(2N2H) state, after N2 binding. Further, reduction of acetylene to ethylene (discussed below) can incorporate added D2 only in the presence of N2. These results imply that the mechanism does not follow the organometallic chemists’ expectation that H2 reductive elimination would form a reduced, unsaturated site which subsequently binds N2. These data indicate instead that the presence of the hydrides somehow facilitates N2 binding, and that N2 binding drives reversible H2 reductive elimination. The ability to interconvert between the states has allowed the spectroscopic study of E4(2N2H), which has confirmed the above model.104 Exposure of E4(4H) to N2 produces a new S ¼ 1/2 signal with a g-tensor comparable to the hydrazine- and diazene-derived reaction intermediates of nitrogenase (see Section 15.03.3.1.5). This has resulted in the proposal that once N2 binds to FeMoco it is promptly hydrogenated to give an intermediate at the diazene oxidation level (i.e., the N2 complex is unstable with respect to both N2 loss and N2 protonation).95 Using EPR and ENDOR, it was possible to monitor the oa/re equilibrium, which showed that the conversion of E4(4H) + N2 to E4(2N2H) + H2 to be nearly thermoneutral (DG ¼ −2 kcal mol−1), suggesting that reductive elimination of H2 provides the driving force for trapping the normally unreactive N2.97 A series of cryogenic photolysis experiments accompanied by DFT calculations have recently added more intermediates to the E4 region of the Thorneley-Lowe cycle. Irradiation of the E4(4H) state at 20 K liberated H2 and allowed for the detection of a new state by ENDOR and EPR spectroscopy, labeled E4(2H) (Fig. 7), which is not on the catalytic cycle but can relax to re-enter the cycle.105–108 Examination of the progress curves for the loss of E4(4H) and appearance of E4(2H) indicated an EPR silent intermediate species, assigned as the H2 complex E4(2H;H2).107,109 The proposed states E4(4H) and E4(2H;H2) have not been observed directly. E4(2H) displays an S ¼ 1/2 signal that has been detected by EPR and ENDOR spectroscopy.108 The E4(2H) state is two electrons more reduced than E4(4H) with the protons remaining on the sulfides. Upon warming, this state binds H2 and relaxes back to the E4(4H) state. The proposed implication for the reduction of N2 is that the E4(4H) state is able to interconvert with a more reactive dihydrogen complex, termed E4(2H;H2), and N2 is then able to displace the H2 in a concerted manner (Fig. 9).

15.03.3.1.3.2 Computational models of the E4 state The kinetic and spectroscopic evidence above does not assign specific structures to the species. However, the ENDOR data obtained for the E4(4H) state have been used to validate broken-symmetry DFT calculations that assess the feasibility of potential structural models.108 These computations are very challenging, because they must include nearby amino acid residues as well as the metal cluster.109 The results should be viewed with caution, because a systematic assessment of different functionals shows that there are large discrepancies in outcome depending on the details of the methodology.110,111 With this caveat in mind, models of the E4 state have assessed protonation of S2B and S5A and bridging hydrides between Fe2 and Fe6 and Fe3 and Fe7, which gave four low-energy structures (E4(4H)a; Fig. 10), with the relative orientation of the SdH bonds and their interaction with the surrounding protein residues influencing the stability of the state.110 This E4(4H)a configuration is also the one that fits best to the observed ENDOR parameters.112 It is important to keep in mind that other models (for example E4(4H)b), including ones in which a belt sulfide bridge has opened to form of a terminal sulfhydryl group (E4(4H)c and E4(4H)d) have similar energies, as found by several groups.109-111,113–115 It is feasible that several isomeric forms are populated under ambient conditions, and that the hydride/SH protons are dynamic. Since interconversion is rapid, any of these isomers could be the gateway to N2 reactivity, though of course an H2 complex looks particularly reasonable.

Fig. 8 The reductive elimination/oxidative addition step of the Thorneley-Lowe cycle.

Fig. 9 Irradiation of the E4 state of the Thorneley-Lowe cycle.

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Fig. 10 E4(4H) models predicted to be energetically feasible by DFT.

Fig. 11 Bjornsson’s proposed mechanism with conversion of proposed E4(4H) to E4(2N2H) states.

In an independent study, Björnsson and co-workers also proposed E4(4H) states with bridging hydrides and dissociated sulfhydryl groups (E4(4H)c and E4(4H)d) to be energetically feasible as part of a N2 reduction mechanism (Fig. 11).115 These E4(4H) states led to two potential E4(2N2H) states involving an initial binding of N2, followed by reductive elimination of H2 and finally proton transfer to form a bound diazene. Interestingly, N2 binding to Fe6 rather than Fe2 resulted in significantly lower transition-state barriers and a more stable E4(2N2H) state, supporting the idea that the asymmetry of the cluster plays a role in stabilizing bound N2. In another independent computational study on the transition of the E4(4H) to E4(2N2H) state, Dance predicts that dissociating a protonated S2B from Fe6 can allow N2 to coordinate in its place (in an unprecedented m-1,1 binding mode).116 This unhooking mechanism is further aided by the local His195 residue which stabilizes the unhooked hydrosulfide through hydrogen bonding interactions (Fig. 12). Following this, the terminal N2 could move to bridge between Fe2 and Fe6 and would be well positioned to receive a H atom from the pendent SH group. All in all, though the different computational models have some differences, they are converging on models with opening of FedS bonds to S2B, which is also indicated by some crystallographic results described below.

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Fig. 12 Dance’s computed mechanism of N2 binding indicating a possible role of the histidine residue.

15.03.3.1.4

The E7 and E8 states

In early studies hydrazine, N2H4, was detected upon acid quenching of MoFe under turnover conditions.90 This observation led to the proposal that the reduction of N2 involved a semireduced substrate bound to the cofactor, equivalent to diazene or hydrazine. Methyldiazene, dimethyldiazene and the parent diazene (N2H4) are each substrates for FeMo nitrogenase.117–119 The reduction of N2H4 is inhibited by H2 in the same manner as N2 due to the re/oa mechanism. This implies that N2H4 can somehow enter the catalytic cycle to give the E4(2N2H) state outlined in Section 15.03.3.1.3.120 Utilizing the Val70Ala mutant, which reduces the steric bulk near the Fe2/3/5/6 cluster face, enables the two-electron reduction of N2H4 to NH3.121 It is assumed that opening the cofactor’s Fe2/3/5/6 face allows access of the larger substrates. It was possible to detect an S ¼ 1/2 species during this nitrogenase reaction, and isotope labeling experiments using 15N2H4 and 14N2H4 revealed a single 1.5 MHz ENDOR signal. The same species was detected during reduction of methyldiazene, hydrazine, and diazene, implying that the species contains NH2, the only common part of these three substrates, or the reduced dNH3.117–121 This was supported by selective 15N labeling on methyldiazene which showed that the NdN bond is cleaved. Later analysis of the same system by Q-band CW-EPR and ESEEM spectroscopy revealed an additional integer-spin non-Kramers doublet, with a proposed S ¼ 2.120 This signal was assigned as an dNH2 species, corresponding to the E7 state of the Thornley-Lowe cycle, while the S ¼ 1/2 signal was assigned as an dNH3, corresponding to E8.

15.03.3.1.5

The alternating and distal mechanisms

The region between E4 and E7 has not been probed directly, because no methods have yet been found to trap these intermediates. However, some mechanistic distinctions arise from considering the different possible sites of protonating N2 and its partially reduced congeners. The first possibility, known as the distal or Chatt mechanism, features successive protonation of the N atom distal to the metal center with decreasing NdN bond order and increasing MdN bond order until ammonia is liberated to yield the terminal nitride (Fig. 13).99,122 Further [H+/e−] steps then place protons on the nitride to liberate a second equivalent of ammonia. The first Mo based catalyst for N2 reduction to ammonia used this mechanism of reduction.123,124 Another pathway has been observed in a catalytic system based on Fe, which was termed the alternating pathway.125 This mechanism involves protonations that alternate between distal and proximal N atoms, and circumvents a metal nitride intermediate. Only this mechanism entails a “diazene-level” intermediate such as that proposed above for the E4(2N2H) state, and therefore it is currently favored.106,117 However, it has been noted that these are not the only possibilities. For example, a hybrid between the distal and alternating mechanisms is possible, based on a model system in which an intermediate on the distal pathway can be converted to a FedN2H4 complex upon addition of two [H+/e−] equivalents.126 At this time, there are still gaps in our knowledge about the structures corresponding to different steps in the Thorneley-Lowe cycle for nitrogenase-based reduction of N2. However, this section has displayed the substantial progress that has come from a combination of spectroscopy, computations, and consideration of mechanistic logic.9,120,127 Additional spectroscopic tools and trapping protocols are needed to query the structures of the states that cannot be probed using EPR and ENDOR spectroscopies.

Fig. 13 The distal and alternating pathways of N2 reduction as well as the recently proposed hybrid mechanism (gray).

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15.03.3.2 Alternative substrates Nitrogenases are prodigious reducing agents capable of reducing a range of substrates besides N2. These alternative substrates include acetylene, carbon monoxide, hydrogen cyanide, diazenes, azides, nitrite, nitric oxide, and others.9,10 Such versatile reactivity makes nitrogenases attractive to organometallic chemists who aim to learn how natural systems harness such reactivity, so that these principles may be applied in synthetic catalysts. In addition, the results gained with some of the alternative substrates also provide benefit by putting constraints on the possible mechanism of N2 reduction, as described below. Finally, the reader is referred to recent work on electrochemistry with nitrogenases.128

15.03.3.2.1

Protons, H+

All three types of nitrogenase (MoFe, VFe, FeFe) reduce protons to hydrogen regardless of the presence of N2. As outlined in Section 15.03.3.1.3, in the absence of N2 the E4(4H) state loses H2 to go to the E2(2H) state, and then loses another H2 to return to the E0 state. Since N2 binds only after the FeMoco reaches the E4(4H) state, it is clear that any failure to provide saturating N2 gives the FeMoco the opportunity to waste [H+/e−] equivalents that could have been spent on N2 reduction. While generating one equivalent of H2 per molecule of N2 reduced is a requirement for the observed stoichiometry (Eq. 1), further generation of H2 can be considered a parasitic reaction (it is not advisable to compare this reaction to the action of hydrogenase enzymes, because unlike hydrogenase, nitrogenase hydrolyzes 2 equivalents of ATP for every molecule of H2 produced). The efficiency of nitrogenase, i.e., the number of molecules of N2 reduced per molecule of H2 produced, decreases in the order MoFe > VFe > FeFe.15,129 It was previously assumed that this was because of differences in the inherent selectivity of the different active sites, but recent kinetic studies indicate that the primary reason is that KM is higher for the VFe and FeFe nitrogenases (in other words, it requires higher N2 pressures to saturate the rate of N2 reduction with alternative nitrogenases).130

15.03.3.2.2

Acetylene

All three nitrogenases reduce acetylene to ethylene, and ethylene production is used as an assay of nitrogenase activity because it is less prone to being an adventitious product than ammonia.131,132 The reduction of acetylene to ethylene is facile relative to N2 reduction, requiring only two [e−/H+] equivalents. Acetylene reduction by the MoFe suppresses H+ reduction, with >90% of the electron flux being utilized for the production of ethylene.133 Thus, it is likely that acetylene binds at an earlier stage of the Thorneley-Lowe scheme (perhaps E2). Interestingly, the formation of ethylene is stereoselective.133,134 When C2H2 was reduced in D2O cis-deuterated ethylene was produced, and reduction of C2D2 in H2O yielded the same product. Rapid freezing of the Ala70 nitrogenase during the reduction of propargyl alcohol gave an S ¼ 1/2 signal in EPR and ENDOR spectra.135 This signal was assigned as the 2-alkene complex of the FeMoco.135,136 Kinetic studies of the reduction of acetylene by Mo-nitrogenase, in conjunction with DFT studies, support a mechanism in which acetylene has two binding sites separated spatially and temporally, with varying substrate affinity.137–139 In the proposed mechanism, acetylene has different binding sites in the E1 and E2 intermediates. The Michaelis constant (KM) reported for the low-affinity binding site was found to be similar to that of the Gly69Ser mutant of MoFe, a variant which is acetylene resistant due to the substitution of a serine for a glycine making the Fe2/3/5/6 face of the FeMoco inaccessible (this is the space between the S2B and S5A sulfides). This face of FeMoco has been proposed as the high-affinity binding site by computations as well.139 The observation that N2 reduction is not affected by this mutation has led to the proposal that it shares the location of the low-affinity C2H2 binding site. Conversely, increasing the size of the cavity next to the Fe2/3/5/6 face by substitution of the valine to the smaller alanine allows for the reduction of larger alkynes.140,141 Thus these studies also support the side of the FeMoco near S2B as a key substrate-binding location.

15.03.3.2.3

Carbon monoxide and selenocyanate

Carbon monoxide (CO) blocks the reduction of all other nitrogenase substrates, with the exception of protons.142 The inhibition of N2 reduction by CO is non-competitive, implying that each has a separate binding site to which the other cannot bind.143 In this case the separation is temporal, with CO binding to nitrogenase in the E2 state, before the cluster has been reduced enough to interact with N2.144 Two unique EPR signals can be detected upon exposure of nitrogenase to CO during turnover. The first species could be detected under a stoichiometric excess of CO and is termed hi-CO, while exposure of a sub-stoichiometric amount of CO allows for detection of the lo-CO signal.145–147 The signals are thought to be the result of two and one molecules of CO bound to the cofactor, respectively. How these signals relate to the inhibition of nitrogenase is unclear, as the time taken to develop EPR signals is significantly longer than the inhibition of N2 reduction by CO, implying that hi- or lo-CO may not be the species responsible for the inhibition.148 IR studies under lo-CO conditions identified a single feature at 1904 cm−1 associated with the CO stretching vibration.149,150 This feature is correlated to a second, slower forming, feature at 1715 cm−1. These data support a mechanism in which the CO initially binds terminally and slowly interconverts to a bridging mode. Under hi-CO conditions features can be observed at 1958, 1936, 1906, and 1880 cm−1. The band at 1906 cm−1 is transient, while the bands at 1936 and 1958 cm−1 increase in intensity over time. ENDOR studies assign hi-CO as having two terminal FedCO groups, while lo-CO contains a bridging CO moiety.145-147,151,152 A crystal structure of the lo-CO state was obtained by inhibiting the enzyme under turnover and subsequently isolating the protein (Fig. 14, left).153 The 1.5 Å resolution structure showed the loss of m2-bridging sulfide S2B with a CO ligand in its place bridging Fe2 and Fe6. When the CO was removed under continued turnover, the sulfide returned to its original position to give the

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Fig. 14 Crystallographically characterized structures of FeMoco with substitution at the S2B site.

resting state of the cofactor. The location of the displaced sulfide is unclear. This was the first structural evidence that bonds to the belt sulfide groups may break. However, it should be remembered that the observed CO species are not on the pathway to N2 reduction. SeCN− is another substrate for FeMoco that has yielded crystals after turnover.154 X-ray crystallography showed that the selenide anion, Se2−, occupies the former site of S2B in the resting state of the nitrogenase. Upon turnover of acetylene by the modified enzyme, further crystal structures showed the intriguing result that some of the selenide migrates into the positions of S3A and S5A. This exchange suggests that the FeMoco can somehow undergo rotation of the two halves that scrambles the belt sulfide/selenide positions. Whether this motion has any relevance for N2 reduction is unclear, but it is interesting to speculate that the cluster may rotate through different faces that are adapted to different steps along the eight-[H+/e−] pathway for N2 reduction.

15.03.3.2.4

Cyanide

Hydrogen cyanide, HCN, is impressively reduced to methane and ammonia by nitrogenases.155,156 Cyanide does not bind to the resting state of FeMoco in the wild type MoFe, but the Arg96Leu mutant, which opens up the Fe2/3/5/6 face of the cofactor, can bind cyanide to produce new EPR-active species.155 Spectroscopy with 13C ENDOR confirmed the interaction between CN−, as well as acetylene, and the E0 state of FeMoco of this mutant. This is further evidence of substrates binding at this face of the FeMoco.

15.03.3.2.5

The surroundings of the FeMoco, and extracted FeMoco

The His195, Val70, and Arg96 residues lie adjacent to the Fe2/3/5/6 face of FeMoco, which is believed to be the active site for many of the substrates reduced by the cluster (Fig. 15). As such, substitution of these amino acid residues influences the reaction outcome. A number of Val70 mutations have already been discussed, as substitution of this residue influences the steric crowding around the proposed active site. Increasing the size of the residue (which increases steric crowding) reduces the N2 and C2H2 reduction activity of MoFe, and reducing the size of the residue allows the reduction of larger alkynes.141,157 The His195 residue has demonstrated hydrogen bonding interactions with the S2B sulfur in the resting state of the cluster, and to CO in the structure of lo-CO (Section 15.03.3.2.3). This implies that it may play a role in hydrogen bonding to bound N2 or N-derived intermediates.153 When the His195 residue is substituted for an glutamine, the N2 reduction rate is reduced by more than 95%.70,158,159 However, in this variant N2 is able to inhibit the reduction of H+ and acetylene, implying that N2 is able to bind to the cluster but cannot be reduced.160 More recently, it has been proposed that the His195 may play a role in the dissociation of the S2B sulfide to open an active site on a Fe atom.119 The Arg96 residue is also thought to participate in hydrogen bonding with substrates and its substitution primarily influences the reactivity of MoFe towards C2H2.161 Another way to assess the steric, electrostatic, and non-covalent influences of the surroundings is to separate the cluster from the protein. Amazingly enough, the FeMoco cluster can be extracted from the protein by acid denaturation or by binding the protein to DEAE-cellulose.162–164 This “extracted FeMoco” is stable in N-methylformamide (NMF), dimethylformamide (DMF), and acetonitrile. IR studies suggest that NMF replaces the His and Cys ligands.165 The extracted FeMoco in NMF solutions displays an S ¼ 1/2 EPR signal comparable to that of the E0 state of the holo enzyme. This signal sharpens upon addition of thiophenol, which presumably ligates Fe1 in place of the cysteine.166,167 Most importantly, the extracted FeMoco can be inserted back into FeMoco-free

Fig. 15 Figure showing the amino acid residues which surround FeMoco in the resting state. C ¼ cysteine, H ¼ histidine, V ¼ valine, R ¼ arginine. PDB: 3U7Q. Figure used from Hoffman, B. M.; Lukoyanov, D.; Yang, Z. Y.; Dean, D. R.; Seefeldt, L. C. Chem. Rev. 2014, 114, 4041–4062.

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MoFe protein, which causes it to regain its full activity.168 Therefore, any structural changes caused by extracting the protein are minor and/or reversible. However, no other structural information is known about the extracted FeMoco. Extracted FeMoco is not active for the reduction of N2. However, it is able to reduce protons to H2 electrochemically.169 It can bind two equivalents of cyanide and reduce acetylene to ethene and ethane in the presence of a Zn or Eu amalgam reducing agent.170,171 Cyclic voltammetry of FeMoco displays a reversible redox event at −0.27 V (E1/2) and an irreversible reduction at −0.94 V, though the nature of these redox events is not yet known.172 Electrochemical investigation of this extracted cofactor in the presence of cyanide and CO does indicate binding of these substrates.173 Synthetic clusters can also be placed into the cavity occupied by the cofactor in the holo enzyme by using a cofactor deficient form of the protein. This was demonstrated using the NifKD protein, which does not contain FeMoco. It was instead possible to insert a synthetic [Fe6S9(SEt)2]4− cluster, which, when compared to FeMoco, lacks a Mo and two Fe atoms and has a bridging m4-S2− rather than a m6-C4−.174 This artificial enzyme can reduce acetylene and CN−, the latter of which is reduced to C1dC3 hydrocarbons.

15.03.3.3 Alternative nitrogenases While the lion’s share of the research into nitrogenases has been conducted on the FeMo cofactor, two “alternative nitrogenases” are known which feature V or Fe in place of Mo. The gene clusters that regulate the alternative nitrogenases are distinct from those of the FeMo cofactor and their production is only favored under conditions in which Mo is limited, or if the gene expressing the Mo cofactor is turned off.7,10 The resting state of the FeV cofactor (FeVco) has been crystallographically characterized.48 It contains a V in the position of Mo in the FeMoco, but also the S3A m2-sulfide ligand is replaced by a carbonate (CO2− 3 ) moiety (Fig. 16). Neither the function nor the source of the carbonate is known. An insightful crystal structure of the vanadium-nitrogenase has been reported, which came from limiting the concentration of the dithionite reductant during protein crystallization.36 This structure features the loss of the S2B sulfide ligand from the cofactor, which is replaced by a bridge with a lighter atom (most likely hydroxide).175,176 While the location of this structure in the Thorneley-Lowe scheme is unclear, it joins the CO- and Se-substituted FeMo cofactors in featuring a substituted S2B sulfide site. The lability of this S suggests a mechanism of N2 reduction in which the sulfide partially or completely dissociates from the Fe center to open a binding site for N2 (see Section 15.03.3.1.3.1). The Fe-only nitrogenase is the nitrogenase of last resort, as an active Fe-only nitrogenase only accumulates when neither Mo nor V has sufficient concentration to support maturation of the Mo- or V-nitrogenases.177,178 It is the least well characterized of the nitrogenases, with no crystal structure reported. In the presence of dithionite, the EPR spectrum of the Fe-only protein is featureless, which implies that it has a different oxidation level of the cluster than the other nitrogenases.179 Mössbauer spectroscopy is consistent with the active site FeFeco having eight Fe centers, four Fe2+ and four Fe3+, in addition to an all-ferrous P-cluster. Whether this cluster has structural differences (cf. carbonate in FeVco) is not yet clear.

15.03.3.3.1

Reactivity of V-nitrogenase towards carbon monoxide

The most dramatic differences in reactivity between VFe and MoFe nitrogenases regards the reduction of CO and CO2. As mentioned earlier, CO acts as a non-competitive inhibitor for the reduction of N2 by the MoFe protein.141 Interestingly, the extracted Av VFe protein is able to reduce CO to methane, ethane, and propane.180,181 The efficiency of Av VFe for CO reduction is much lower than for reduction of N2 or C2H2, with the majority of reducing equivalents lost to H2 formation.177 The mechanism of hydrocarbon formation is unclear, but the observation of multiple CO bound in hi-CO with FeMoco (see above) suggests that a multimetallic mechanism may be reasonable. This reaction, and the use of multiple metal sites, are reminiscent of the heterogeneous Fischer-Tropsch reaction.182 However, this differs from a hypothetical electrochemical Fischer-Tropsch reaction because it has substantial added thermodynamic driving force from ATP hydrolysis. Other recent reports have shown that all three nitrogenases (MoFe, VFe, and Fe-only) can reduce CO2 to methane, ethylene, or even propene.183–186 Overall, the ability of nitrogenase metalloenzymes to transform C1 substrates will lead to continued cross-fertilization between organometallic and bioinorganic chemistry.

Fig. 16 Crystal structures of FeMoco and FeVco. PDBs: 3U7Q and 5N6Y. Color legend: yellow, S; orange, Fe; gray, C; teal, Mo; purple, V; red, O; blue, N. Figure used from Tanifuji, K.; Ohki, Y. Chem. Rev. 2020, 120, 5194–5251.

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15.03.4 Biosynthesis of FeMoco Part of the organometallic chemistry of nitrogenase is the central carbide, which has six FedC bonds in the resting state. There are no known examples of synthetic clusters in which such a carbide lies between high-spin iron centers, and thus the natural system is using unprecedented organometallic chemistry. In this section, we briefly present the state of knowledge about carbide insertion, which starts with fundamental chemistry of FedC bonds to iron-sulfur clusters. The details of FeMoco biosynthesis have been reviewed recently.6

15.03.4.1 Radical-SAM enzymes and alkylated Fe4S4 clusters The radical-SAM enzymes function to generate alkyl radicals for a variety of biological transformations.187 They require S-adenosylmethionine (SAM) and a reduced Fe-S cluster as cofactors.188 Upon electron transfer from the cluster to the sulfonium, an SdC bond is cleaved, which typically generates an adenosyl radical (50 -dA
) that undergoes reactions reminiscent of natural cobalamins. More recently, ENDOR studies have demonstrated that this alkyl radical can form a bond to the cluster to generate an organometallic intermediate (Fig. 17).189,190 However, the SdC bond to the adenosyl group is not the only one that can be broken.191 Selectivity for cleaving different SdC bonds at the sulfonium has been observed, for example the enzyme PhDph2 generates the 3-amino-3-carboxypropyl radical over the 50 -deoxyadenosyl radical.189 Recent studies have demonstrated that the third sulfonium SdC bond can also be cleaved to generate methyl radicals under photolytic conditions, including the detection of a short-lived clusterdCH3 intermediate.192 Thus the combination of a reduced Fe4S4 cluster and SAM is a recipe for alkylated iron-sulfur clusters. Attempts to prepare synthetic alkylated [Fe4S4] clusters were not successful until recently. In an important step, [Fe4S4]3+ clusters featuring methyl functionalities were stabilized through use of an anionic scorpionate ligand scaffold.193 Larger alkyl functionalized clusters (benzyl and octyl) could be stabilized by three 1,3-dimesitylimidazol-2-ylidene (IMes) ligands coordinated to three of the Fe sites. The alkylated clusters were found to liberate alkyl radicals upon addition of Lewis basic pyridine derivatives.194 This brings us to FeMoco assembly, which occurs on a scaffold protein encoded by nifE and nifN genes, known as NifEN, which has a pair of [Fe4S4] cubane clusters.195–197 This is acted upon by NifB (a radical-SAM enzyme) to transfer a methyl group to one of these clusters, which is converted to carbide on NifEN (Fig. 18). This was demonstrated through X-ray emission spectroscopy, and by labeling SAM with a 14CH3 group and following the radiolabel into the FeMoco.67,69 NifB continues to act using a second molecule of SAM that generates this time a 50 -deoxyadenosyl radical, which can abstract an H atom from the methyl group, presumably forming some sort of CH2 group on the cluster, though the nature of this apparent alkylidene intermediate is unknown. Eventually, two further hydrogen atoms are removed, either through abstraction or deprotonation, resulting in the C4−. The order of these processes is yet to be confirmed, however an initial report claims a [Fe8S8C] cluster (absent a belt sulfide) during maturation of FeMoco.35 There is little precedent for these steps on iron-sulfur clusters, and this is an area for future study.

Fig. 17 Alkylation of [Fe4S4] cubanes by SAM enzymes and the subsequent dissociation into an alkyl radicals and the oxidized cluster.

Fig. 18 Simplified illustration of the carbide insertion into FeMoco.

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35 The next step is addition of the ninth sulfide, which may come from SO2− Finally, an Fe site is substituted for a 3 , at least in vitro. 196 Mo to yield the FeMoco. The complete cofactor is then transferred from NifEN into the catalytic nitrogenase enzyme, in the culmination of the complex biosynthetic pathway.6

15.03.5 Model complexes The sections above have shown that the structural and functional understanding of the enzyme is in need of more development. One way of gaining insight is through synthetic model complexes, which can illustrate the possible ways for metalloenzymes active sites to interact with substrates, enable systematic variation of the environment of the metal sites, and provide spectroscopic benchmarks to compare with data obtained from the enzymes. In the following section we outline a number of metal complexes and clusters which give insight into the structure and/or reactivity towards N2-related substrates of FeMoco. The advances in synthetic analogues of the F- and P-clusters have been discussed in a number of recent reviews, and rarely involve organometallic compounds.12,198–200 As such, information regarding their chemistry will not be discussed further here.

15.03.5.1 Functional models of catalytic N2 reduction At the nitrogenase FeMco clusters, neither the location nor the mode of N2 binding is definitively known. Though the current data point towards N2 binding at an Fe site, much of the early work attempting to replicate the cofactor function focused on Mo-based model systems.201 This led to a substantial number of Mo complexes that bind and cleave N2, and the first systems capable of catalytic N2 reduction were based on Mo.124,201 However, given the advances in understanding of nitrogenases, in particular the confirmation that the alternative nitrogenases have negligible amounts of Mo, we do not discuss molybdenum systems here.131 A review of catalytic N2 reduction by homogeneous systems recently appeared.13 Iron-based systems are most relevant here, and a number of them can catalytically produce ammonia from N2 through the addition of strong reducing agents (often KC8) and strong acids (often HBArF4, where ArF ¼ 3,5-bis(trifluoromethyl)phenyl). These reagents are much more energetic (negative redox potential; low pKa) than what is possible in the enzymatic systems, and this requires the use of aprotic solvents (often Et2O). Examples of catalytically active iron complexes are shown in Fig. 19.125,202–210 The most active and most thoroughly studied catalytic Fe system uses three phosphine arms on a borane anchor that behaves as a Z-type ligand (1).125 The electronic flexibility of the FedB interaction is an important feature of these complexes, and enables it to span formal oxidation states from −2 to 4, including a terminal nitride complex having two FedN p-bonds (18). This system has enabled the isolation and/or observation of numerous NxHy species relevant to N2 reduction, and these are summarized in Fig. 20.207,211–216 A number of key mechanistic insights have emerged, which may be relevant to potential nitrogenase mechanisms. First, protonation occurs at the distal nitrogen, and it has been possible to detect a metastable FeNNH species. This compound can take two pathways: in the presence of excess acid it can be protonated a second time at the distal nitrogen to give an isolable FeNNH2 compound, but without additional acid, it can undergo bimolecular loss of H2 to return to an oxidized FedN2 complex. This result is reminiscent of the prompt “pinning” of N2 on the FeMco, and is representative of the extremely weak NdH bond in

Fig. 19 Homogeneous Fe complexes reported to mediate catalytic N2 reduction.

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Fig. 20 Interconversion of different nitrogenase-relevant intermediates. R ¼ t Bu, HBArF ¼ [H(OEt2)2][B(C6F5)4], HOTf ¼ trifluoromethanesulfonic acid.

the FeNNH species.215,216 Second, the resting state during catalysis is an off-cycle species (13): it has a borohydride and an iron hydride, thus incorporating an extra equivalent of H2 that can be lost to return to the catalytic cycle. This has a relationship to the H2 loss from the E4 state(s) in nitrogenase, including the use of a bridging hydride. In comparing these results to the nitrogenase, notice that in the natural systems double reduction/protonation of the bound N2 is vital for driving the enzymatic reaction forward.

15.03.5.2 Iron-sulfur clusters Only very recently has an iron-sulfur cluster been identified that has interactions with N2 (see Note Added in Proof ). However, some structural analogues of the FeMoco, FeVco, and FeFeco have been prepared. Some of these start by building eight-metal scaffolds such as m2-sulfide bridged [Fe8S9] (19), [Fe6Mo2S9] (20), and [Fe7MoS9] (21) clusters, which are prepared through addition of Li2S or Na2S to the chloride functionalized cubanes (Fig. 21).217,218 Closer topological similarity to FeMoco can be achieved through oxidation of the coordinatively unsaturated dinuclear Fe2+ complex (22), resulting in a [Fe8S10] cluster that resembles the shape of the FeMo cofactor with a central m6-sulfide in place of a

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Fig. 21 Coupling [Fe4S4] cubanes with sulfide.

carbide (23).219 Note that it has thiolate ligands rather than sulfides around the belt region, and thus has a closer resemblance to the P-cluster (Section 15.03.2.2.1). The size of the interstitial sulfur prevents it from attaining threefold axial symmetry as observed in the FeMco clusters. A related cluster is formed upon oxidation of the related diiron(II) species 24 with water and sulfur in toluene.220 In the resulting [Fe8S8O2] cluster (25), an oxygen atom occupies the central cavity to form an asymmetric bridge between the two [Fe4S3] fragments. The asymmetry of the oxygen atom creates two Fe sites supported by Fe-aryl interactions. The structure of this cluster suggests that a central atom can have hemilabile interactions with the belt iron atoms, with a rearrangement of the environment of the interstitial atom to produce a coordinatively unsaturated Fe site. However, the substantial differences between the electronegativity, bond lengths, and bonding preferences of O vs. C make these analogies somewhat distant (Fig. 22). The failure of iron-sulfur clusters to bind or reduce N2 may be because their iron sites have four donors already. In order to test this hypothesis, an unusual, planar [Fe4S3] cluster supported by b-diketiminate ligands has been prepared which contains a central three-coordinate Fe atom with exclusively m3-sulfide ligands.221 This central site replicates the purported Fe sites that would be produced by cleavage of hemilabile FedC bonds in FeMoco. The cluster is more reduced (3Fe2+1Fe1+) than any characterized FeMoco species, which might be expected to enhance its ability to bind N2 through p-backbonding. However, this compound does not react with N2, indicating that it lacks some structural/electronic features that help to trap N2. One-electron oxidation of this cluster yields an all-Fe2+ cluster (26), which reacts with hydrazine to yield an Fe-amide complex (27), demonstrating that a sulfide-supported Fe2+ can at least cleave NdN single bonds (Fig. 23). Finally, it is worthwhile to refer back to the previously mentioned synthetic [Fe4S4] clusters (Section 15.03.4.1), which differentiate one iron that can react at its fourth position.193,194 These offer an exchangeable iron site with a very biomimetic environment and flexible electronics, and hold substantial promise for learning more about reactivity at unsaturated iron-sulfur clusters.

15.03.5.3 Iron complexes with sulfur and N2 ligands The iron complexes that bind and reduce N2 (Section 15.03.5.1) often feature strong-field ligands such as phosphines, which results in low-spin configurations at the metal centers. The preparation of Fe complexes supported by sulfur-based ligands enables the

Fig. 22 Clusters with topological similarity to FeMoco. Mes ¼ mesitylene.

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Fig. 23 Activation of hydrazine by an [Fe4S3] cluster (L ¼ 3-methyl-2,4-bis(2,6-xylylimido)pentyl, which is drawn for the right-hand sites).

study of Fe in a more biologically relevant coordination environment. In order to evaluate the influence of sulfur donors, a series of Fe phosphine complexes has replaced one or two of the phosphine donors with thioether donors.222 An Fe2+ complex with one thioether group, two phosphines and an axial Si was found to bind N2. The product (28) is paramagnetic, with S ¼ 1. The bound N2 is lost under vacuum, and attempts to reduce this complex caused cleavage of the SdC(alkyl) bond, dissociation of the thioether, or formation of a bridging sulfide. When a second phosphine was substituted with a thioether, the Fe2+ complex did not bind N2. Thus, the incorporation of an S donor seems to be detrimental towards ligand binding. The same trend was observed in a series of b-diketiminate complexes with S donors, in which observation of N2 products was not observed with sulfur donors as with analogous carbon donors.223,224 It is not certain whether this is due to a difference in inherent N2-binding ability, or whether instead the S-containing compounds are more prone to decomposition upon reduction. Notably, reduction of the phosphine-thioether complexes can give the mixed valence end-on bridging N2 complexes 29 and 30, and here the inherently weak N2 binding is overcome by the formation of a bridge. In order to encourage bimetallic binding with anionic sulfur donors, the scaffold used for 28–32 was also modified to have a bridging thiolate ligand.225 Reduction to the Fe1+Fe1+ oxidation state facilitates the binding of one equivalent of N2 to each Fe site to yield the diamagnetic complex 33. Stepwise oxidation allows access to the Fe1+Fe2+ and Fe2+Fe2+ oxidation states. Exposure of 33a to KC8 in the presence of [H(OEt)2] BArF4 produces 1.8  0.3 equivalents of ammonia, and the Fe2+Fe2+ form is capable of catalytic disproportionation of the NdN bond in N2H4 to yield NH3. The ability to isolate a large number of structurally similar complexes elucidates some structure-function relationships in this series. A summary of bond metrics involving N2 can be found in Table 1. In all the cases where multiple oxidation states were accessible (33a-c, 34a-b, 35a-b) the more reduced the metal centers, the shorter the FedN bond and the lower the NdN stretching frequency. This is in keeping with the established trend that a lower oxidation state of the metal gives more electron density available to back donate into the NdN p -orbital. Another feature of these series of complexes is the introduction of a hydride ligand. In the bis(thioether) complex, the incorporation of hydrides facilitates N2 binding (31 and 32), perhaps because of the influence of the strong field hydride donors (see Section 15.03.5.7). In the dinucleating system, it was also possible to access the homo- and mixed-valent Fe1+Fe2+ and Fe2+Fe2+ hydrides (34a-b). The monometallic, terminal FedN2 complex supported by a similar scaffold featuring a thiolate ligand (35a-b) was stable only in the presence of a hydride ligand.226 In order to understand the influence of more biomimetic ligand environments containing only elements found in the FeMoco without P or N donors, the Fe2+ tris(thiolate) complex 36 was prepared.227 Reduction with one equivalent of KC8 yielded the Fe1+ complex (37) with the ligand scaffold still intact and no N2 bound, while further reduction to the Fe0 complex (39) induced

Table 1

Selected bond parameters for terminal FedN2 complexes bearing S-containing supporting ligands.

Complex

Oxidation state(s)

FedN (Å)

NdN (Å)

nNN (cm−1)

References

+ [Fe(SiPiPr 2 SAd)(N2)] 28 [Fe(SiPiPr(SAd)2)(H)(N2)] 31 [Fe(S(iPiPr)2SAd)(H)(N2)] 32 [Fe(N2)(m-SAr)Fe(N2)]− 33a [Fe(N2)(m-SAr)Fe(N2)] 33b [Fe(N2)(m-SAr)Fe(N2)]+ 33c [Fe(N2)(H)(m-SAr)Fe(N2)]− 34a [Fe(N2)(H)(m-SAr)Fe(N2)] 34b − [Fe(SiPiPr 2 S)(H)(N2)] 35a iPr [Fe(SiP2 S)(H)(N2)] 35b

Fe2+ Fe2+ Fe2+ Fe1+, Fe1+ Fe2+, Fe1+ Fe2+, Fe2+ Fe2+, Fe1+ Fe2+, Fe2+ Fe2+ Fe3+

1.954(3) 1.828(2) NA 1.808(1)1.822(1) 1.889(3)1.917(3) 1.854(7) 1.804(3)1.819(3) 1.8392(9) 1.810(4) 1.882(3)

1.037(5) 1.116(3) NA 1.128(1)1.122(2) 1.048(5)1.034(5) 1.05(1) 1.124(4)1.120(6) 1.110(1) 1.117(6) 1.077(4)

2156 2060 2055 20171979 20701983 2129 20441981 20362093 1971 2123

222 222 222 225 225 225 225 225 226 226

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Fig. 25 Coordination of N2 to a carbon/sulfur supported Fe center. Inset: a proposed binding mode of N2 to FeMoco.

dissociation of the arylthiolate ligand and coordination of N2, with additional support provided by an Z2-interaction with the aryl ring. The IR spectrum was consistent with a significantly weakened N2 ligand (nNN ¼ 1880 cm−1). 39 was only stable at low temperatures, and N2 dissociation occurred upon warming. Addition of 3.6 equivalents of KC8 to 36 in the presence of N2 gives rise to a new species as identified by Mössbauer spectroscopy, thought to be the Fe1− complex. Treatment of this highly reduced complex with the weak acids H2O and 2,6-di-tert-butyl-4-methylphenol (BHT) produces small amounts of hydrazine and ammonia, respectively.228 The binding of N2 to the sulfur-bearing Fe site is again weak, and can only be induced to occur at a lower oxidation state than proposed in the FeMoco, leaving questions about how the FeMoco can accomplish N2 binding in the assumed Fe2+/3+ states that are biologically accessible (Fig. 25).

15.03.5.4 Iron complexes with sulfur and NxHy ligands While few sulfur-supported iron complexes have been reported to bind N2, numerous Fe complexes supported by sulfur-based ligands can bind nitrogenase-relevant substrates such as hydrazine, diazene, and ammonia. These complexes are themselves valuable structural and reactivity models for the later Thorneley-Lowe states, potentially representing aspects of E4(2N2H) through to E8. In one example, sulfide bridged Fe complexes 40 and 41 were obtained by exposure of the monosulfide-bridged bimetallic Fe complex to ammonia or hydrazine, respectively.229,230 These compounds show that it is geometrically feasible to have either terminal or bridging binding modes for N2-derived species on the FeMoco (Fig. 26). Sellman and coworkers have reported a series of complexes supported by thiolate donors with additional thioether, amine, and/or pyridine groups.231–233 Two complexes from this series, 42 and 43, feature bridging trans-diazene bridges, which were generated through air oxidation of the hydrazine species or through in situ generation and trapping of hydrazine from potassium azodicarboxylate or benzenesulfonic acid hydrazide.234–237 The diazene protons form hydrogen bonds to the proximal thiolates. Supplementary DFT calculations suggest that the hydrogen bonding interactions significantly stabilize the complex.238,239 This suggests that FeMoco may similarly stabilize partially reduced N2 during turnover using hydrogen bonds. An additional stabilizing factor is a significant degree of p-backbonding, resulting in a lower NdN bond stretching frequency (nNN ¼ 1382 cm−1) with respect to free diazene (nNN ¼ 1529 cm−1).231 Oxidation of 25 with two equivalents of ferrocenium at −78  C formed a purple species

Fig. 26 Sulfide bridged Fe complexes featuring nitrogenase-relevant substrates.

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Fig. 27 Fe-diazene complexes in sulfur-rich environments 24 and 25. Bottom: Two-electron oxidation of 25, with the proposed product shown in two tautomeric forms 26i and 26ii.

which upon warming above −40  C liberated N2 to yield a green Fe2+ complex.240 It was proposed that this species has a bridging N2 ligand between two Fe2+ centers with the protons formerly associated with the diazene moving onto the sulfides (tautomeric species 44i and 44ii). This process would model the reverse of the nitrogenase N2 binding and reduction process, the prompt “pinning” of N2 during the E4 state.120 Unfortunately, the instability of the purple species prevented sufficient characterization of the complex to support this idea (Fig. 27). Several complexes with thiolate and pentamethylcyclopentadienyl (Cp ) donors are capable of catalytic cleavage of the NdN bond in alkyl- and aryl-substituted hydrazines.241 A bis-alkylthiolate bridged bimetallic Fe complex reacts with hydrazine to form the cis diazene complex 45.242 In the presence of reductant and acid, this complex liberates NH3. DFT calculations indicate a mechanism in which protonation of the diazene involves an intermediate with a bound (m-NH)–NH2 ligand.243,244 Two similar complexes were reported featuring a methyldiazene (HN]NdCH3) or methyldiazenido (N]NCH−3) moiety bound side-on between the two iron centers (46 and 47).245 These complexes also feature NdN bond cleavage upon reduction and protonation. Placing a 1,2-bis(thiolato)benzene linker between the two bridging thiolates enabled the isolation of two additional intermediates during the reduction of the parent hydrazine (Fig. 24). Protonation of the m-Z1:Z1 cis diazene complex 48 resulted in electron transfer to the diazene ligand to form a diferrous complex in which the hydrazido (N2H−3) is bound asymmetrically m-Z1:Z2 between the two Fe2+ sites (49). The bridging thiolate ligand moves to ligate only one metal center. Addition of a further two reducing and proton equivalents liberates NH3 resulting in the bridging amido complex 50, with the bis(thiolate) ligand moving back to its symmetrical coordination mode. This process models the latter stages of the Thorneley-Lowe cycle, and demonstrates that if N2 has been reduced to the diazene-level intermediate, two Fe centers are capable of the final reduction steps while maintaining biologically accessible oxidation states. The flexibility of the bridging thiolate, which moves between Fe centers, is reminiscent of the m2-sulfides in FeMoco, which could dissociate and re-associate to their respective Fe sites to facilitate the binding of reduced forms of N2 (Section 15.03.3.1.3) (Fig. 28). As this section has demonstrated, the latter stages of the reduction of N2 can be replicated by Fe complexes featuring sulfur-rich ligand environments comparable to that of FeMoco, and oxidation states known to be accessible to FeMoco under biological conditions. However, the early stages of N2 reduction (the binding and reduction of N2) by model complexes often requires the presence of strong field ligands and/or Fe sites in oxidation states lower than Fe2+. Models more closely structurally related to FeMoco may be required to fully understand the initial E4(4H) to E4(2N2H) transition. Compeling directions include the inclusion of carbide (C4−) ligands or analogies thereof; the preparation of higher nuclearity model complexes; further understanding of Fe-hydrides; and, attempting to model second-sphere hydrogen bonding and/or charge.

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Fig. 24 Iron complex featuring sulfur-based ligands based on a multidentate phosphine ligand. Where multiple oxidation levels are accessible, alphabetical notation is used, starting with the most reduced state as “a”. Ad ¼ 1-adamantyl; R ¼ isopropyl.

Fig. 28 Fe-diazene complexes stabilized by Cp and thiolate ligands.

15.03.5.5 Iron carbides The discovery of the interstitial carbide in the nitrogenase cofactors represents one of the most intriguing discoveries in bioinorganic chemistry. It is the only carbide found in nature and has specialized cellular machinery in place for its construction, as described above in Section 15.03.4.1.6 In materials chemistry, metal carbides display increased strength and hardness over their carbide-free alternatives, for example carbides are present in steel.246 This behavior suggests the potential for a structural role of the carbide in FeMoco and would explain why this cluster is stable to extraction from the MoFe protein, and can survive catalytic conditions (in contrast with synthetic FeS clusters, where FedS bonds are often broken upon reduction).247 Alternatively, mechanisms have been proposed that include protonation of the FeMoco carbide to induce a conformational change that allows N2 binding to occur.248 The first synthetic Fe m6-carbide was found within [Fe6C(CO)2− 16] (51), which is prepared by refluxing NaMn(CO)5 with Fe(CO)5 in diglyme (presumably forming Mn oxide byproducts).249,250 X-ray emission studies indicate significant covalency in the FedC(carbide) bonds.251 Since 51 can be reduced by two electrons to yield a cluster with an overall 4− charge, the ability to isolate such a highly reduced cluster may indicate an electronic communication role for the carbide.252,253 The bonding between the carbide in 51 and the surrounding Fe atoms was found to be highly covalent in nature. Notably, 51 exclusively features coordinatively saturated iron centers with strong-field carbonyl ligands, which induce low-spin configurations, significantly differing from the weak-field environment created by the sulfide ligands in FeMoco. A recent study on high-spin Fe-alkyl and

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-alkylidene complexes (see Section 15.03.5.6) also determined these FedC bonds to have similar covalency as FedS bonds.224 Beyond covalency, little is known about the ligand properties of the C4− in FeMoco. Nuclear resonance vibrational spectroscopic (NRVS) analysis of FeMoco gave a FedC force constant of about 1.3 N cm−1. This value indicates a fairly strong interaction, and accordingly the carbide complexes are much more stable than the synthetic complexes in the previous section.254–258 The treatment of 51 with HCl resulted in quantitative formation of [HFe5(m6-C)(CO)−14], and addition of a further three equivalents of HCl fragmented the cluster into the neutral HFe4(m2-CH)(CO)12 (52), in which the carbide is singly protonated.259–261 The same product was also obtained upon oxidation of 51 in the presence of H2.262,263 Increasing the relevance of these clusters to FeMoco is synthetically challenging due to the difficulty in removing the CO ligands, with attempts to do so frequently resulting in Fe carbonyl clusters of other sizes. The functionalization of 51 to include a sulfide ligand was recently achieved through stepwise deoxygenation of SO2, resulting in the m3-sulfide containing cluster 53 which was the first iron cluster to have both sulfide and carbide.264 It has also been demonstrated that reaction of 51 and lower nuclearity Fe carbide clusters with S2Cl2 yields sulfide-linked Fe carbide clusters, some of which have been characterized (for example 54).265 These compounds are important stepping stones to carbide sulfide clusters with cluster and iron site geometries more similar to those in the FeMco clusters. Up to now, no reactivity of any of these carbonyl-supported Fe carbide clusters towards nitrogenase-relevant substrates has been reported, possibly due to the coordinatively saturated environments of the Fe sites, which all feature multiple p-acidic CO ligands (Fig. 29). An additional synthetic route to an Fe carbide was reported during the synthesis of the linear m2-carbide complex 55.266–271 55 was prepared by addition of tetraiodomethane, CI4, to the Fe2+ tetraphenylporphyrin in the presence of iron powder, which acts as a reductant. This reactivity diverges from the analogous reactions with CCl4 and CBr4, which instead give Fe(porphyrin)CX2, where X ¼ Cl, Br. The use of carbon tetrahalides to produce iron carbides in other systems has not been reported in the literature, and in the authors’ hands has not been successful. In general, methodologies to produce molecular carbides are underdeveloped, with relatively few examples of carbides across the periodic table and, with the exception of the metal carbonyl clusters and 55, exclusively involve 5d and 6d metals.272–275 Consequently, no examples of Fe carbide clusters in weak field ligand environments are known. NRVS analysis of FeMoco gave a force constant that is much smaller than for a [Fe4(m4-C)(CO)12]2− cluster, suggesting that the CdFe bonds could be substantially weaker.254–258 Synthetic methodologies that give access to C4− ligands, particularly in biomimetic weak-field environments, remain a key challenge for organometallic chemists.

Fig. 29 Fe complexes featuring carbides.

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15.03.5.6 Iron complexes with other carbon ligands The first report of an iron complex capable of catalytic homogeneous N2 reduction was a tetradentate complex 1, which features three equatorial phosphines and an axial borane (Fig. 30).125 Upon exposure of the complex to N2, KC8 (50 equiv.) and HBArF (46 equiv.) at −78  C the complex produced 7.0  1 equivalents of NH3 per metal center. While 1 does not contain donor atoms that are common to the FeMoco, it is possible to substitute the boron for a carbon atom, increasing the biomimetic relevance.209,276 The analogous tris(2-(diisopropylphosphino)phenyl)methyl complex 2 contains equatorial phosphine groups and an axial R3C− moiety, and thus gives insight into the potential roles of FedC interactions during N2 reduction. With the alkyl-supported ligand, N2 binding occurs upon reduction with Na under an N2 atmosphere, yielding the low-spin Fe1+dN2 complex 2b. Single electron oxidation of 2b gives access to the more nitrogenase-relevant Fe2+dN2 complex 2c. In the Fe2+ oxidation state the N2 ligand is labile in solution and is lost under reduced pressure. Comparison of bond lengths across the Fe0, Fe1+, and Fe2+ series shows a stepwise contraction of the FedC bond length, indicating that the FedC bond is responsive to the oxidation state, and supporting the idea that the carbide could be capable of stabilizing various oxidation states differentially. The most successful approach to designing Fe complexes for nitrogen binding and reduction has been to utilize strongly s-donating ligands, and as such many of the complexes discussed throughout feature phosphine ligands.13 Carbenes are another class of strong s-donors that notably coordinate through a carbon atom, making them useful tools when designing FeMoco model complexes. The most frequently employed are the N-heterocyclic- and cyclic(alkyl)(amino)-carbenes (NHCs and cAACs, respectively), and complexes featuring one or more carbenes have been established to bind N2 (Fig. 31).210,277–280 Using the highly stabilizing ligand environment provided by two cAAC ligands enabled the isolation of the two-coordinate Fe0 species (cAAC)2Fe which can reversibly bind N2 in a terminal, end-on fashion (61a).210 Further reduction with KC8 results in the Fe1− complex 61b which displays an NdN stretching frequency of 1850 cm−1, consistent with a weakened NdN bond. 61b reacts with silyl chlorides to yield silyldiazenido complexes. In the presence of excess KC8 and [H(OEt2)2]BArF4 at −95  C, 61a can also catalytically reduce N2 to give 3.3 equiv. of ammonia. While this system is remarkable, being the sole example of a two-coordinate Fe complex reducing N2, the Fe0 state is lower than conventionally imagined for the FeMoco (Fig. 32).

Fig. 30 The first Fe complex capable of catalytic reduction of N2 to NH3 and its carbon analogue. Inset: a proposed binding mode of N2 to FeMoco.

Fig. 31 Select examples of FedN2 complexes supported by carbon-based ligands.

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Nitrogenases and Model Complexes in Bioorganometallic Chemistry

Fig. 32 Iron complexes relevant to the reduction of N2 to NH3.

Dinuclear iron complexes were recently reported that feature bridging alkylidenes (m2-CH(SiMe3)) (62), or a combination of alkylidene and sulfide ligands (63).224,281 Comparing the reactivity of 62 and 63, only the coordinatively-unsaturated Fe centers in 62 are capable of reducing N2 to NH3. However, no N2-bound species could be detected and the mechanism of NH3 formation is unknown. The bridging alkylidene ligand in 62 and 63 facilitated electronic communication between the Fe centers. Further, computational analysis of 63 revealed the FedC bonds to similar covalency as the FedS bonds, and play a significant role in electronic communication between the two Fe sites.

15.03.5.7 Iron hydrides As discussed in Section 15.03.3.1.3, the presence of hydrides or bound H2 is a requirement of the oa/re model of the E4(4H)Ð E4(2N2H) equilibrium.9,96,282 Hydrides are strong field ligands and, if present in the E4(4H) state, would represent the only strong field ligands in FeMoco (with the possible exception of the carbide, whose position in the spectrochemical series is not yet understood).100,275 The introduction of hydrides may facilitate a change in the relative orbital energies on the iron, to allow access to electron configurations with lower spin states. Complexes in a lower spin state generally form stronger metal-ligand bonds, and therefore may be a way for the cofactor to bind N2 more strongly. Another trend worth noting is the effect of hydride on NdN bond strength. The introduction of a hydride changes the overall coordination number, charge, and geometry at the Fe site, all of which may influence the ability of an iron atom to bind N2. Complexes 28 and 32 both feature Fe2+ metal centers and differ primarily in the presence of a hydride ligand.222 The hydride has a significant impact on the N2 stretching frequency (nNN ¼ 2156 cm−1 without hydride (28), 2055 cm−1 with hydride (32)). The bimetallic Fe2+/Fe2+ complexes 33c and 34b show a similar degree of N2 activation upon hydride introduction (nNN ¼ 2129 cm−1 without hydride (33c), 2036 and 2093 cm−1 with hydride (34b)).225,226 The mixed valence Fe2+/Fe1+ complexes 33b and 34a display a smaller change in N2 stretching frequency, but still indicate a modest weakening of the NdN bond, indicating that the effect of strong field hydride ligands is more pronounced at the biologically relevant Fe2+ oxidation state. Thus, the hydrides in FeMoco could be pivotal for encouraging the binding of N2. To superimpose this onto the oa/re mechanism, it would imply an intermediate species which features both bound N2 and hydride ligands which then liberates H2, according to the computational and spectroscopic ideas presented in Section 15.03.3.1.3. A challenge for future synthetic complexes with biomimetic environments is to test this idea, establishing its feasibility. So far, there has been an example in which reductive elimination of H2 is tied to NdN double bond cleavage in a diazene. Upon exposure of the high-spin high-spin Fe2+ hydride dimer (64) to diphenyldiazene the N]N bond is cleaved to yield the bridging imido complex 65.283 In this system, computational and experimental evidence indicated a mechanism in which diazene binding precedes H2 elimination, and thus the hydrides behave as reducing equivalents that can be stored for breaking the NdN bond after substrate binding (Fig. 33). The reductive elimination of H2 can also facilitate subsequent N2 binding. For example, the monometallic Fe complexes supported by tridentate ligands, which are catalysts for the reduction of N2 to ammonia (Fig. 19), are prepared from the respective dihydride complex.207,284 This work was expanded through the synthesis of 66, which features a bridging alkylidyne, and hydride between two Fe sites further supported by a bulky hexaphosphine ligand.285,286 In the absence of a H atom source, 66 shows no reactivity towards N2. However, the presence of a H source in the form of an acid (HCl or TEMPO-H) or H2 66 is activated and can bind N2.

Fig. 33 Reductive elimination of H2 facilitates N]N bond cleavage by the Fe hydride 43.

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Fig. 34 Hydrogen-dependent binding of N2 to a bimetallic Fe complex (L ¼ H2, N2). Insets: hypothetical binding modes of N2 to FeMoco.

Exposure of 66 to mixtures of N2 and H2 results in protonation of the bridging alkylidyne to a methyl group with binding of multiple hydrides and N2 ligands to the Fe sites (69).285 It was possible to observe and independently prepare two intermediates during the reaction, the first of which features an asymmetrically bound alkylidene ligand bound to an Fe center, with both Fe sites now able to bind N2 (67). Further exposure of this complex to a N2/H2 mixture results in conversion of the bridging carbon to a methyl group, and it incorporates a bridging N2 ligand and a hydride on each Fe entre (68). This series of reactions suggests the feasibility of a nitrogenase mechanism in which the carbide in FeMoco forms CdH bonds, opening the interstitial pocket to enable N2 binding (Fig. 34; right inset). This mechanism of N2 binding to FeMoco is related to one proposed computationally based on the observation that protonation of the carbide and belt sulfides is energetically competitive.248 However, more complete computational models of FeMoco have found protonation of the C4− to be less favorable than protonation of the belt sulfides,108,110,111,114,115 and the computational paper has other implications that are unrealistic (e.g., it claims that the active intermediates are lower in energy than the resting state). Most of the isolable Fe hydride complexes in the literature are supported by strong field ligands such as CO or phosphine, which are dissimilar from the ligand environment of FeMoco.287 Few examples of Fe hydrides supported by weak field sulfur-based ligands have been reported. The diiron complexes 70a-b feature a bridging hydride ligand in addition to a bridging bisthiolate and flanking Cp ligands.288 The 1e− oxidized form 70a features an Z2-aryl interaction, which rapidly moves between Fe sites in solution. A bimetallic Fe hydride complex bridged by a sulfide and supported by b-diketiminate ligands has also been prepared (71).289 Upon photolysis, 71 can release both the H and S atom to yield an N2 complex, although the yield is low and the mechanism is unknown. Clearly, more remains to be learned about the interplay of sulfur-based ligands and hydrides in multimetallic iron compounds. Ideally, these studies will address the dynamics of S, C, and H ligands, and the interplay of different spin states as a function of the iron coordination environment (Fig. 35).

15.03.5.8 Modeling second-sphere effects Section 15.03.3.2.5 noted that substituting the amino acid residues close to the proposed belt sites of FeMoco hinders the ability of MoFe to reduce N2. In addition, the FeMoco is inactive outside the nitrogenase proteins. Since most of the amino acids near the belt are not covalently linked to the FeMoco, this indicates that the protein provides non-covalent interactions that are vital for the cluster to reduce N2. These even influence the electronic structure of the resting state, for example the positively charged guanidine groups of the Arg96 and Arg359 residues (which flank the Fe3-S5a-Fe5 cluster edge) generate electronic asymmetry.78 The localized

66

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Fig. 35 Fe hydride complexes featuring sulfur ligands.

positive electrostatic environment may also play a role in polarizing bound N2 for rapid trapping at the E4 level (see Section 15.03.3.1.3.1). Interactions with N2 in FedN2 complexes are known with alkali metal cations and Lewis acids like B(C6F5)3, which can weaken the NdN bond.290,291 Thus it is reasonable that both the charge and the hydrogen bond donors in the local environment of FeMoco can influence and contribute to its reactivity towards N2. It is likely that the later intermediates, of the types N2Hx and NHy, can interact more strongly both through formation of hydrogen bonds and through interaction with local Lewis acidic residues, but little is known about these stages in the enzyme. In addition to the work of Sellmann highlighted above (Section 15.03.5.4) a growing number of ligands incorporate hydrogen-bonding elements. For example, the pyrazole containing pincer complex 72 can catalytically reduce hydrazine to ammonia (Fig. 36).292 During catalysis, the pyrazole ligands protonate a bound hydrazine to release two equivalents of ammonia, with the second equivalent being exchanged at the metal site by a second hydrazine. A series of proton transfers from hydrazine to the pyrazole ligand then occurs to regenerate the original, protonated pyrazole and diazene and the cycle can continue. Analogous proton shuttling has been proposed by the His195 residue in nitrogenases, which has been proposed to transfer a single proton equivalent from the e-NH to a bound N2 or N2 derivative.161 There have also been efforts to incorporate NH groups into the tris(phosphine) systems that have been so successful for catalytic N2 reduction. One important aspect to the design is that it must be constrained enough to place the NH group near N2, but not so

Fig. 36 Catalytic reduction of hydrazine to ammonia and diazene with a non-innocent ligand. Phosphines and charge excluded in the cycle for clarity.

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67

Fig. 37 Fe complexes with second sphere interactions.

rigid that the desired chemistry is inhibited. An example is the FedN2 complex 73, in which the pendent groups adopt a chair conformation with no interaction between the amine sites and the N2 ligand.293 However, upon replacement of the N2 with ammonia the pendent arms rearrange into a boat conformation with hydrogen bonds between the protons and amine sites (74). These compounds point the way towards incorporating different geometries and numbers of acid/base groups near N2 (Fig. 37).

15.03.6 Outlook The understanding of N2 reduction by nitrogenases has improved dramatically over the past decade. The identification of an interstitial carbide within FeMoco has highlighted a gap in our understanding as organometallic chemists: how does C4− behave as a part of a weak-field cluster, and how does it influence the reactivity of the metals to which it is connected? Synthetic methodologies for the insertion of hypervalent carbon into metal clusters present a key synthetic challenge. Creating a variety of complexes of this type should open the door to new catalysts that harness the structural features that underlie the activity of the amazing natural cofactors. The interplay of hydrides, H2, and nitrogen activation is another area where natural systems have lessons to teach us, as they are using novel organometallic choreography to achieve catalysis under mild conditions. Using H2 elimination to drive N2 activation has received some initial attention in coordination chemistry, and promises a route to N2 activation in the absence of strong reductants.294 Nitrogenase uses weak N2 binding for catalysis, which is reasonable because rapid catalytic cycles require the intermediates to avoid large energy changes.295 Another strategy of nitrogenase is to use nearby charged groups, which probably also engage in hydrogen-bonding interactions at various steps in the catalytic cycle. Yet another strategy is the use of highly polarizable, mixed-valence iron-sulfur clusters, which can be likened to complexes of redox-active ligands that can buffer changes in redox state. All in all, deeper understanding in nitrogenases connects with many emerging trends in organometallic chemistry, and the insights from each area will enhance the other.

15.03.7 Note Added in Proof A very recent report describes an iron-molybdenum cluster that can bind N2.296

Acknowledgments We acknowledge financial support from the National Institutes of Health (GM-065313). We thank Prof. Dennis Dean, Dr. Majed Fataftah, Alexandra Nagelski, and Linda Zuckerman for careful reading of this manuscript, and Prof. Lance Seefeldt for insight on the F-cluster.

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A.; Pink, M.; Chen, C. H.; Smith, J. M. Chem. Commun. 2017, 53, 412–415. Lissel, F.; Schwarz, F.; Blacque, O.; Riel, H.; Lörtscher, E.; Venkatesan, K.; Berke, H. J. Am. Chem. Soc. 2014, 136, 14560–14569. Ouyang, Z.; Cheng, J.; Li, L.; Bao, X.; Deng, L. Chem. Eur. J. 2016, 22, 14162–14165. Reesbeck, M. E.; Grubel, K.; Kim, D.; Brennessel, W. W.; Mercado, B. Q.; Holland, P. L. Inorg. Chem. 2017, 56, 1019–1022. Hoffman, B. M.; Lukoyanov, D.; Yang, Z. Y.; Dean, D. R.; Seefeldt, L. C. Chem. Rev. 2014, 114, 4041–4062. Bellows, S. M.; Arnet, N. A.; Gurubasavaraj, P. M.; Brennessel, W. W.; Bill, E.; Cundari, T. R.; Holland, P. L. J. Am. Chem. Soc. 2016, 138, 12112–12123. Schild, D. J.; Peters, J. C. ACS Catal. 2019, 9, 4286–4295. Arnett, C. H.; Agapie, T. J. Am. Chem. Soc. 2020, 142, 10059–10068. Arnett, C. H.; Bogacz, I.; Chatterjee, R.; Yano, J.; Oyala, P. H.; Agapie, T. J. Am. Chem. Soc. 2020, 142, 18795–18813. Gargano, M.; Giannoccaro, P.; Rossi, M.; Vasapollo, G.; Sacco, A. J. Chem. Soc. Dalton Trans. 1975, 9–12. Yang, D.; Li, Y.; Wang, B.; Zhao, X.; Su, L.; Chen, S.; Tong, P.; Luo, Y.; Qu, J. Inorg. Chem. 2015, 54, 10243–10249. Arnet, N. A.; Dugan, T. R.; Menges, F. S.; Mercado, B. Q.; Brennessel, W. W.; Bill, E.; Johnson, M. A.; Holland, P. L. J. Am. Chem. Soc. 2015, 137, 13220–13223. Geri, J. B.; Shanahan, J. P.; Szymczak, N. K. J. Am. Chem. Soc. 2017, 139, 5952–5956.

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Connor, G. P.; Holland, P. L. Catal. Today 2017, 286, 21–40. Umehara, K.; Kuwata, S.; Ikariya, T. J. Am. Chem. Soc. 2013, 135, 6754–6757. Creutz, S. E.; Peters, J. C. Chem. Sci. 2017, 8, 2321–2328. Ballmann, J.; Munhá, R. F.; Fryzuk, M. D. Chem. Commun. 2010, 46, 1013–1025. Kozuch, S.; Shaik, S. Acc. Chem. Res. 2011, 44, 101–110. McSkimming, A.; Suess, D. L. M. Nature Chem. 2021, 13 in press https://doi.org/10.1038/s41557-021-00701-6.

15.04

Bioorganometallic Chemistry of Vitamin B12-Derivatives

Bernhard Kräutler, Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innsbruck, Austria © 2022 Elsevier Ltd. All rights reserved.

15.04.1 Introduction 15.04.2 The structure of organometallic B12-derivatives 15.04.2.1 Cobalamins and other “complete” B12-derivatives 15.04.2.2 “Incomplete” B12-derivatives 15.04.2.3 Cobamides as molecular switches 15.04.3 Organometallic chemistry of B12-derivatives 15.04.3.1 Formation and cleavage of the (CodC)-bond in B12-derivatives 15.04.3.2 Thermally induced CodC bond homolysis 15.04.3.3 The nucleophile induced heterolysis and formation of the CodC bond 15.04.3.4 Radical induced abstraction of cobalt-bound alkyl groups 15.04.4 Redox-chemistry of B12-derivatives 15.04.5 Enzymatic organometallic processing of cobalamins 15.04.6 Enzymatic reactions catalyzed by organometallic B12-cofactors 15.04.6.1 B12-dependent methyl transferases 15.04.6.1.1 B12-dependent methionine synthase 15.04.6.1.2 B12- and S-adenosylmethionine-dependent radical methyl transferases 15.04.6.2 Organometallic chemistry of enzymes dependent on coenzyme B12 15.04.6.2.1 Coenzyme B12-dependent isomerases 15.04.6.2.2 Coenzyme B12-dependent ribonucleotide reductase 15.04.6.3 B12-dependent dehalogenases 15.04.7 Gene-regulatory roles of organometallic B12-derivatives 15.04.7.1 B12-riboswitches 15.04.7.2 Photo-regulation of gene expression by coenzyme B12 15.04.8 Organometallic cobalamins as antivitamins B12 15.04.9 Metbalamins: Transition-metal analogues of cobalamin 15.04.10 Summary and outlook Acknowledgments References

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15.04.1 Introduction The discovery of the red cobalt corrin vitamin B12 (cyanocobalamin, CNCbl)1,2 and its crystallographic characterization as an “awfully” complex natural tetrapyrrole,3 has provoked many questions concerning its biological formation,4 chemistry and biochemistry,5–7 as well as biological and physiological roles.8–12 However, when coenzyme B12 was identified as the vitamin B12 derivative 50 -deoxy-50 -adenosyl-cobalamin (AdoCbl, see Fig. 1) in the early 1960s,3 the importance of organometallic chemistry in life processes became apparent for the first time. Nowadays we classify B12-cofactors as Nature’s, perhaps, most broadly relevant organometallic biocatalysts.7 Whereas B12-cofactors are indispensable for a wide range of organisms, including humans,13 only some microorganisms have the capacity to biosynthesize B12 and other natural corrinoids.14 All other B12-dependent organisms rely on B12-derivatives as their vitamins11 and their metabolism depends on the uptake and selective cellular import of useful B12derivatives,15 their metabolic transformation to relevant B12-cofactors,16 as well as the catalysis by B12-dependent enzymes.17–20 In spite of over 70 years of research and the remarkable scientific advances made in the B12-field that have solved many of the earlier major “B12-mysteries,” important physiologic effects of B12 in humans are still amazingly puzzling.21 Remarkably, vitamin B12 (CNCbl), the most common cobalamin (Cbl) vitamin form, has no physiological function itself and, actually, plays the role of a provitamin.22 Indeed, only the organometallic analogues coenzyme B12 (AdoCbl) and methylcobalamin (MeCbl) are the physiologically directly relevant B12-cofactors in humans and other mammals. Thus, the metabolic synthesis and the organometallic chemistry of methyl- and adenosylcorrinoids, most notably of MeCbl and AdoCbl, have become a prime topic in the B12-field.7,16 By making use of the reactivity of their (CodC)-bond the organometallic B12-cofactors help to catalyze exceptional enzymatic reactions.17,23,24

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Fig. 1 Left: Structural formula of coenzyme B12 (50 -deoxy-50 -adenosylcobalamin, R ¼ 50 -deoxy-50 -adenosyl, AdoCbl); Right: Structural model from the X-ray structure of AdoCbl, symbol for AdoCbl (R ¼ 50 -deoxy-50 -adenosyl) and representation of the corrin macroring with standard notation of its 5-membered rings.

15.04.2 The structure of organometallic B12-derivatives The detailed structures of the most important B12-derivatives are known from X-ray crystallography,3 complemented by modern spectroscopic studies, most notably by heteronuclear NMR spectroscopy.25,26 Vitamin B12 (CNCbl), coenzyme B12 (AdoCbl) and methylcobalamin (MeCbl) represent important cobalamins (Cbls), which belong to the larger class of the cobamides (Cbas). The Cbas are classified as “complete” corrinoids in which a pseudo-nucleotide function is attached to the f-side chain of the cobyric acid moiety, the common cobalt-corrin core of all functional natural B12-derivatives. The nucleotide moiety of the Cbls comprises a cobalt-coordinating a 5,6-dimethylbenzimidazole (DMB) heterocycle (Fig. 1).12 In typical other Cbas different cobalt-coordinating heterocycles are present (see Fig. 2).7,11,27–31 Hence, other (substituted) benzimidazolyl-cobamides and purinyl-cobamides pseudovitamin B12 (a 70 -adeninyl-cobamide) and factor A (a 70 -[2-methyl]-adeninyl-cobamide) are widely occurring Cbas that are produced and selectively used by various specific micro-organisms, such as, e.g. some methanogens, acetogens or clostridia.9,32,33

Fig. 2 Structural formulas of CN-Co(III)-forms representing basic types of natural “complete” corrinoids. Left: the benzimidazolyl-cobamides vitamin B12 (R1 ¼ R2 ¼ CH3: the 50 ,60 -dimethylbenzimidazolyl-cobamide cyanocobalamin, CNCbl, a cobalamin), 50 -methylbenzimidazolyl-cobamide (R1 ¼ CH3, R2 ¼ H), 50 hydroxybenzimidazolyl-cobamide (“factor III,” R1 ¼ OH, R2 ¼ H) and 50 -methoxybenzimidazolyl-cobamide (R1 ¼ OCH3, R2 ¼ H); center: the adeninyl-cobamides pseudovitamin B12 (R ¼ H) and 20 -methyl-adeninyl-Cba (“factor A,” R ¼ CH3); right: cobamides with a non-coordinating aryl-pseudonucleotide moiety, dicyanophenolyl-cobamide (R ¼ H) and dicyano-40 -methyl-phenolyl-cobamide (R ¼ CH3).

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Unusual Cbas with a regular non-coordinating aromatic functionality (such as a phenyl- or tolyl-group) also occur naturally. Such phenolyl-Cbas were characterized structurally as the corrinoids from an acetogenic bacterium.33 Semisynthetic Cbas, in which an imidazole substitutes for the DMB-base of the Cbls, as in Cob-cyano-imidazolylcobamide, were prepared by “guided” biosynthesis.34 Such Cbas are of interest in relation to the observation of the astounding mode of binding of B12-cofactors in some B12-dependent enzymes, where a protein-derived histidine-imidazole substitutes and displaces the DMB-base of the bound MeCbland AdoCbl-cofactors.35

15.04.2.1 Cobalamins and other “complete” B12-derivatives Most investigations on organometallic B12-derivatives were carried out with the commercially produced Cbls. The effect of the variations in the nucleotide bases in other Cbas36 and of the linker connecting these with the cobalt-corrin moiety37 on the reactivity have only rarely been addressed. The crystal structure of vitamin B12 (CNCbl) has first revealed the unique 3D-architecture of the mutually interacting nucleotide loop and corrin moieties in a paramount “complete” corrinoid, in which the DMB heterocycle coordinates the cobalt ion from the “lower” axial (or a) side.3,38 The Cbls are chiral and asymmetric in 3D-space, and their cobalt-coordinated (base-on)-forms also represent topologically chiral molecules.12 The crystal structures of two cyano-purinylCbas Cob-cyano-70 -adeninyl-Cba (also named pseudovitamin B12) and Cob-cyano-70 -[20 -methyl]-adeninyl-Cba (also named factor A)39 furnished a similar 3D-structure and coordination geometry around the cobalt-center as is known for CNCbl itself. The solution structure of coenzyme B12 (AdoCbl) in water, derived from extensive heteronuclear NMR-studies,40 provided data consistent with the earlier X-ray crystal structure analysis,41 but revealed the conformationally dynamic position of the organometallic 50 -deoxyadenosyl group. A crystal structure of MeCbl42 and the NMR-derived structure in aqueous solution43 differed to a surprising extent, indicating significant restructuring of the nucleotide loop of MeCbl by the solvent. Hence, Cbls represent cobalt-corrins with rather flexibly attached organometallic ligands and also non-rigid pseudo-nucleotide moieties.6,25 The natural 176-nor-cobamide (nCba) nor-pseudovitamin B12 (Cob-cyano-70 -adeninyl-176-norcobamide) features an unprecedented variation of the linker part of a Cba, as this nCba lacks the methyl group C176 of the cob(in)amide moiety (Fig. 3).39 Studies with the semi-synthetically produced nor-cobalamin (nCbl) nor-vitamin B12 indicated the methyl group C176 relevant in stabilizing the cobalt-coordinated (base-on) form in comparison with its (base-off ) isomer with a de-coordinated pseudo-nucleotide base.37 The crystal structure of the nor-70 -adeninyl-Cba indicated a strikingly marginal conformational effect of the (missing) C176 methyl group when compared to the structure of the corresponding (70 -adeninyl-)coabamide.39 Extensive crystallographic studies have focused on specifically describing the features of the axial coordination in organometallic Co(III)-corrins, with particular emphasis on the acquisition of structural insights concerning the weak CodC bonds of the biologically important organocobalamins AdoCbl and MeCbl.6,24,38,44,45 A crystal structure of coenzyme B12 (AdoCbl) revealed two relatively long axial bonds in [(CodC) (2.030 A˚ ) and (CodN) (2.237 A˚ )].3,44,46 The crystal structure of methylcobalamin (MeCbl) showed both axial bonds shorter than the ones in AdoCbl.38,42 The oxygen sensitive cob(II)alamin (CblII), the product of (CodC)-bond homolysis of coenzyme B12 (AdoCbl) occurring during the catalytic cycle of some coenzyme B12-dependent enzymes, displayed a remarkably similar structure as the Co(III)-corrin moiety of AdoCbl.44,47

Fig. 3 Structural formulas of the natural “complete” corrinoids differing by the covalent linker to their nucleotide moiety: Left: the cobamide vitamin B12; right: the 176-nor-cobamide nor-vitamin B12.

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Among the crystal structures of other organometallic Cbls analyzed, the structures of vinylcobalamin and of cis-chlorovinylcobalamin were the first examples with sp2-hybridized carbon ligands and were of interest in the context of the B12-dependent dehalogenases.48 The structures of the newly available arylcobalamins,49,50 and of arylalkynylcobalamins,51 helped to characterize the structures of B12-derivatives with an organometallic bond between aromatic sp2- or sp-carbons and cobalt. Interestingly, while the CodCsp bonds were shown to be rather short (1.86 A˚ ), as expected, the CodCsp2 bond of the phenyl-Cbls surprised with a length (1.98 A˚ ) only marginally smaller than the one of the CodCsp3 bond of MeCbl (1.99 A˚ ), an observation rationalized by the proposed steric strain between the corrin and aryl moieties in the organometallic phenyl-Cbls.49,52

15.04.2.2 “Incomplete” B12-derivatives In contrast to Cbas, the “incomplete” corrinoids lack a nucleotide moiety, and are represented, first of all, by cobyric acid (Cby) and the cobinamides (Cbis). Cbis are generated from Cby by attachment of an iso-propanol amine group at the f-side chain as the first part of the linker section of the pseudo-nucleotide moiety of the Cbas (see Fig. 4). The “incomplete” corrinoid Coacyano, Cobaquo-cobyric acid (CNa,H2Ob-Cby) crystallizes as a monomer structure playing important roles in the B12-history, as its X-ray investigation provided the first unambiguous structure of the corrin ligand3 and it also represented the direct corrinoid target in the total synthesis of vitamin B12.53–55 Remarkably, the C13-epimer of CNb-Cby crystallized as a dimer, in which the f-carboxylate function of one molecule coordinated to the cobalt-center of the other.56 50 -Deoxy-50 -adenosylcobyrate (AdoCby), an organometallic biosynthetic precursor of coenzyme B12,14 was recently characterized by heteronuclear NMR and was also used in a partial chemical synthesis of AdoCbl.57 The fully esterified lipophilic B12-model compound dicyano-heptamethylcobyrinate (“cobester”)54,58 has been analyzed similarly.59 The crystal structure of heptamethyl-cob(II)yrinate perchlorate (“cob(II)ester”) allowed first detailed insights into the structure of a paramagnetic Co(II)-corrin and confirmed the five-coordinate nature of its Co(II)-ion, as expected.60 Crystal structures of an “incomplete” organometallic B12-derivative or of a Co(I)-corrin are as yet unavailable.

15.04.2.3 Cobamides as molecular switches The ribose linker of the nucleotide moieties of cobamides (Cbas) feature an anomeric carbon with unique a-configuration, which is crucial for the stable intramolecular coordination of the benzimidazole- or purin-type heterocycles to the “lower” a-axial coordination site of the corrin-bound cobalt center.3,39 The DMB-nucleotide function of the Cbls undergoes relatively strong intramolecular cobalt-coordination in the “base-on” form, generating a nearly strain-free large substituted cycle.37,54 The alternative “base-off” form is the direct result of the de-coordination of the DMB-base (see Fig. 5).6,7,24

Fig. 4 Structural formulae of natural and other organometallic cobalamins and of “incomplete” natural corrinoids. Left: cobalamins: coenzyme B12 (AdoCbl, L ¼ 50 -deoxy-50 -adenosyl); methylcobalamin (MeCbl, L ¼ CH3); 4-ethylphenyl-Cbl (EtPhCbl, L ¼ 4-ethylphenyl); 2-phenyl-ethynyl-Cbl (PhEtyCbl, L ¼ 2phenylethynyl). Right: “Incomplete” natural corrinoids Coacyano,Cobaquo-cobyric acid (CNa,H2Ob-Cby), dicyano-cobinamide (CN2-Cbi) and of the unnatural lipophilic dicyano-heptamethylcobyrinate “cobester.”

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Fig. 5 Cobalamins (Cbls) 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 coenzyme B12 (AdoCbl, R ¼ 50 -deoxyadenosyl) is more stable with Kon ¼ 72 at 25oC; protonation of the DMB-base of AdoCbl furnishes the stable protonated “base-off” form that is a weak acid featuring a pKa of 3.67.

Cobalamins and the related cobamides are, thus, natural “molecular switches.”12,61 A complete “base-on” to “base-off” switch results from protonation of the nucleotide base and de-coordination from the corrin-bound cobalt-ion. The associated acidity of the protonated “base-off” form (as expressed by its pKa) reflects quantitatively the strength of the intramolecular DMB-coordination. The (protonated) “base-off” form is readily accessible in AdoCbl and MeCbl, with pKa’s of 3.67 for AdoCbl-H+ and 2.9 for MeCblH+.6, 61 On the other hand, the proton-assisted de-coordination is inhibited significantly and cobalt-coordination is stronger in CNCbl6 and in alkynyl-Cbls,62 whose protonated “base-off” forms exhibited pKa’s near 0. The purine heterocycles in adeninyl-Cbas are less strongly cobalt-coordinated than the DMB-nucleotide moiety of corresponding Cbls. In consequence, the organometallic adeninyl-Cbas 50 -deoxy-50 -adenosyl-Factor A and pseudo-coenzyme B12 (50 -deoxy-50 -adenosyl-7-adeninyl-Cba) may feature an even slightly predominating “base-off” form in neutral aqueous solution.62 The particular structure of Cbls (and other Cbas) is an effective determinant for the selective and tight binding by B12-binding macromolecules.63 This effect is crucial for discriminating Cbls from various other natural Cbas by the human B12-uptake and transport system.64 In mammals the latter recognizes and binds its Cbl-load in the “base-on” form.15,64 In contrast, the “base-on” forms of the “complete” corrinoids may (or may not) be structured correctly for binding by specific B12-apoenzymes (see below).24 Hence, a protein or an oligonucleotide environment may be pre-structured to switch the bound B12-cofactors from “base-on” to “base-off”35 (or—vice versa—in the reverse sense, from “base-off” to “base-on”64–66). The two forms (“base-off” or “base-on”) of Cbas differ strongly not only by their structures, but also by their concomitantly modified reactivity in biologically relevant organometallic reactions. First of all, by stabilizing the “base-on” form of (alkyl)-Cbls significantly, a coordinating DMB-base may impede the loss of a Cob-coordinated methyl group.6,67 The coordinating DMB-nucleotide function also steers the face-selectivity at the corrin-bound cobalt center.68 By coordinating to the “lower” face, it may direct alkylation (and other ligation) reactions (in cobalamins) to the “upper” (or b-face), an effect that may be particularly relevant in the very rapid (recombination) reaction between CblII and organic radicals.68,69

15.04.3 Organometallic chemistry of B12-derivatives 15.04.3.1 Formation and cleavage of the (CodC)-bond in B12-derivatives Formation and cleavage of the (CodC)-bond are the key reactions of organometallic B12-cofactors, and are the essential to catalysis by B12-dependent enzymes.6,7,17,23,70–73 Soon after the discovery of coenzyme B12 (AdoCbl) organometallic Cbls were mostly prepared by applying alkylation of the Co(I)-form CblI − in presumed nucleophilic substitution reactions.5,12 In contrast, radical generating conditions and the Co(II)-corrin CblII were employed for the recent syntheses of aryl- and alkynyl-Cbls (see Figs. 6–8).49,51 Interestingly, the cleavage of the (CodC)-bond of MeCbl by protonation is not documented, whereas polarizable electrophilic metal ions, such as HgII-ions, may readily abstract it, providing an environmentally important path to the poisonous HgIICH3 ion.74 However, the proton-induced heterolysis of the 50 -deoxy-50 -adenosyl-Co(III)-corrin AdoCbl does occur and accompanies the slow fragmentation of its deoxyadenosine moiety in weakly acidic aqueous solution and furnishes H2OCbl+.75

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Fig. 6 Typical partial synthesis of organocobalamins may make use of readily prepared reduced form of vitamin B12, i.e. CblII in a radical reaction (left: for the aryl-Cbl EtPhCbl) or highly nucleophilic CblI in an efficient substitution reaction (right: for the alkyl-Cbl MeCbl).

Fig. 7 The (CodC)-bond of coenzyme B12 (AdoCbl) cleaves by thermally induced homolysis, reversibly furnishing the 50 -desoxy-50 adenosyl radical (Ado%) and the “radical trap” cob(II)alamin (CblII).

Fig. 8 Biologically important modes of substrate-induced cleavage of the (CodC)-bond of methylcorrinoids, such as MeCbl. Abstraction of the cobalt-bound methyl group by nucleophiles (Nu−) or by radicals (R%).

Alkynyl-Cbls are likewise cleaved slowly in acidic aqueous medium by proton-induced heterolysis of the (CodC)-bond and formation of H2OCbl+.76 In all of specific cases, the cobalt-center of the involved Cbls remains on the oxidation level Co(III). Three main organometallic reaction modes are relevant in enzymes, which all involve a change of the (formal) oxidation level of cobalt (see Figs. 7 and 8): (i) Thermally induced CodC bond homolysis—essential for the cofactor role of AdoCbl: 50 -deoxyadenosyl-Co(III)-corrin > Co(II)-corrin + 50 -deoxyadenosyl radical (ii) Nucleophile induced heterolysis and formation of the CodC bond—typical of MeCbl: methyl-Co(III)-corrin + nucleophile > Co(I)-corrin + methylating agent (iii) Radical induced abstraction of the cobalt-bound alkyl group, such as of MeCbl: methyl-Co(III)-corrin + radical ! Co(II)-corrin + methylated radical

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15.04.3.2 Thermally induced CodC bond homolysis Coenzyme B12 (AdoCbl) readily undergoes selective thermal homolysis of its CodC bond and has been classified as a “reversible carrier of an alkyl radical” or as a “reversibly functioning radical source” (see Fig. 7).45 Indeed, this homolytic mode of the cleavage of the (CodC)-bond of AdoCbl is particularly important for its cofactor role (see below). The homolytic bond dissociation energy (BDE) of the (CodC)-bond of AdoCbl has been derived as about 30 kcal/mol by using detailed kinetic analyses of its thermal decomposition in aqueous and glycerol solutions.45,77 The quantitative determination of the homolytic (CodC)-BDE of AdoCbl is hampered by cage effects and the presence of both “base-on” and “base-off” forms. In a similar way, the slightly higher homolytic (CodC)-BDE of MeCbl has been determined at 37 kcal/mol.78 In mass spectrometric experiments the homolytic (CodC)-BDEs of the “incomplete” organocorrinoids adenosylcobinamide (AdoCbi) and methylcobinamide (MeCbi) in the gas-phase were determined and were found to be somewhat higher, at 41.5 and 44.6 kcal/mol, respectively.79 The nucleotide coordinated “base-on” forms of some organocobalamins decomposed considerably faster than their (protonated) “base-off” forms, or than the related “incomplete” organocobinamides.80 Therefore, the intramolecular coordination of the nucleotide was associated with a weakening of the (CodC)-bond of organometallic B12-derivatives.80,81 However, the contribution of the nucleotide coordination to the ease of homolysis of AdoCbl is relatively small: On the basis of available thermodynamic data concerning the coordination of the nucleotide in AdoCbl and of the homolysis product cob(II)alamin (CblII), the coordination of the nucleotide was derived to weaken the (CodC)-bond by only 0.7 kcal/mol.67,68 With MeCbl, in contrast, the intramolecular coordination of the nucleotide increases the homolytic (CodC)-BDE slightly by about 0.3 kcal/mol.67,68 The homolysis of the CodC bond of organo-Cbls is reversible and organo-Cbls are, in turn, conveniently accessible by the reaction between the persistent radicaloid CblII and organic radicals.7,49 The penta-coordinated Co(II)-center of CblII fulfills all the structural criteria of a highly efficient “radical trap.”47,69 Indeed, the reactions of CblII with alkyl radicals are indicated to occur with negligible restructuring of the cobalt corrin moiety. This structural feature supports the remarkably high reaction rate of CblII with organic radicals (such as the 50 -deoxy-50 -adenosyl radical), as well as the diastereo-specificity for the reaction at the (“upper”) b-face. AdoCbl and other organo-Cbls are, hence, directly formed from the recombination of an organic radical with CblII.69,82 CblII even traps the very short lived acetyl-radicals very efficiently, providing, e.g. a remarkable synthetic route to acetyl-cobalamin.83 The (CodC)-bond of most organocorrinoids has long been known to be sensitive to visible light,5 which induces homolytic cleavage of the (CodC)-bond of AdoCbl and MeCbl very effectively.82,84,85 Hence, exposure of organocorrinoids to irradiation by sunlight furnishes organic radicals conveniently and under mild conditions.86 By exception, (phenyl)-alkynyl-Cbls are strikingly stable when irradiated by visible light.87,88 Aryl-Cbas, such as of the antivitamin B12 ethylphenyl-Cbl, also undergo the photo-induced decomposition with only a low quantum yield.87

15.04.3.3 The nucleophile induced heterolysis and formation of the CodC bond An alternative, biologically broadly relevant mode of formation and of cleavage of the (CodC)-bond occurs via nucleophilic substitution reactions (see Fig. 8). The highly nucleophilic (“supernucleophilic”) Co(I)-corrins react readily with alkylating agents to furnish organometallic Co(III)-corrins.7,89 This heterolytic reaction type follows the mechanism of an SN2-reaction without any evidence for the existence of a free methyl (or alkyl) cation, and is particularly relevant in typical enzyme-catalyzed methyl-transfer reactions,23,90 as well as in the biosynthetic adenosylation of highly activated Co(I)-corrinoids.16,91 Whereas typical alkylation reactions with cob(I)alamin (CblI) proceed via the “classical” bimolecular nucleophilic substitution (SN2) mechanism, in certain cases alkylation with Co(I)-corrins may occur via a two-step (“inner-sphere”) electron transfer path, where the strongly reducing Co(I)-corrins induce a one-electron reduction of the alkylation agents, so that the alkylation process involves Co(II)-corrin and radical intermediates.68,92 Related electron transfer induced radical processes have been inferred in the synthesis of aryl-Cbls, where typical SN2-processes would not be feasible.49,50 The nucleophile-induced demethylation of methyl-Co(III)-corrins is the important basis for the biological methylation of a range of nucleophilic substrates. This process, formally a reductive trans elimination at cobalt,93 (re)generates a strongly reducing Co(I)-corrin.90,94 The strongly nucleophilic thiolates de-methylate the “incomplete” MeCbi+-ion approximately 1000 times faster than MeCbl,95 a consequence of the stabilization of MeCbl by about 4 kcal/mol by the coordinated DMB-nucleotide.67 This effect is of relevance also for enzymatic methyl-group transfer reactions involving protein bound methyl-Co(III)-corrins, where a histidine ligand replaces the DMB moiety and plays a significant kinetic role.96 However, the nucleophilic reactivity of Co(I)-corrins, like CblI, is indicated to be virtually unaffected by the presence of the cobalt-coordinating (DMB) nucleotide: “complete” and “incomplete” Co(I)-corrins react both with typical alkylation agents with similar rates and with preference for their b-face, which, therefore, is the more nucleophilic of the two faces of the 4-coordinate corrin-bound Co(I)-ion.68,92

15.04.3.4 Radical induced abstraction of cobalt-bound alkyl groups The radical-induced substitution at the cobalt-bound alkyl group is a recently recognized and practically irreversible mode of cleavage of the (CodC)-bond of organometallic B12 derivatives (see Fig. 8).7,12,97,98 This type of a substitution reaction was first documented in the reaction of a malonyl-methyl-radical with MeCbl.98 The thermodynamically very favorable and also kinetically remarkably effective abstraction of the cobalt-bound methyl group of MeCbl by an alkyl radical has been suggested to be the basis of previously unprecedented, alternative biological roles of methylcorrinoids.98 Indeed, biosynthetic methylations at inactivated

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carbon centers by B12- and S-adenosyl-methionine (SAM) dependent radical enzymes have been reported.99–101 A mechanistically related intramolecular radical attack at the cobalt-bound saturate carbon atom generates small ring cycloalkanes,97 a potential model reaction for a group of unusual biological (CdC)-bond forming reactions.102 The abstraction of the cobalt-bound methyl group of the methyl-Co(III)-corrin MeCbl, by the “incomplete” Co(II)-corrin CbiII furnished MeCbi and CblII, providing a formal precedence for a radicaloid methyl transfer process between two cobalt-centers.67 This reaction does not involve free methyl radicals and is not sensitive to the presence of molecular oxygen, when carried out in aprotic solvents.103

15.04.4 Redox-chemistry of B12-derivatives Under physiological conditions vitamin B12-derivatives exist in the form of stable and spectroscopically observable Co(III)-, Co(II)-, or Co(I)-corrins.5,12 Each of these oxidation states possesses strongly differing reactivity and coordination properties.5,12,94 Electrochemistry has been used not only for the determination of the crucial redox-potentials of Cbls in solution,94 but also for the controlled potential electro-synthesis of intricate organometallic B12-derivatives,104–107 for the generation of reduced forms of protein bound B12-derivatives,108 and of electrode-bound B12-derivatives for analytical applications.109 Axial coordination to the corrin-bound cobalt center depends on the formal oxidation state of the cobalt ion, so that the number of axial ligands generally decreases along a decreasing cobalt oxidation state.12,94 In the thermodynamically predominating forms of cobalt corrins, the diamagnetic Co(III)-center carries two axial ligands (coordination number 6), the paramagnetic (low spin) Co(II) has one axial ligand bound (coordination number 5) and axial ligands are absent in diamagnetic Co(I)-corrins (coordination number 4) (see Fig. 9). Therefore, electron transfer reactions involving B12-derivatives are characteristically accompanied by a change in the number of axial ligands, which influences the thermodynamic and kinetic features of their redox-processes.94 Electroanalytical studies of organometallic B12-derivatives may be complicated due to a rapid and (an effectively) irreversible loss of the organic ligand upon reduction.94,110,111 Organo-corrinoids are labile to strong one-electron reducing agents, and the (CodC)-bond is weakened considerably by the one-electron reduction of MeCbl.94,112 As the standard potential of the typical Co(III)-/Co(II)-redox pair of organometallic B12-derivatives is strikingly more negative than that of CblII/CblI it is out of the reach of biological reductants,94 and the further reduction of organometallic Co(III)-corrins typically does not occur at the potentials needed for electro-generation of the highly nucleophilic Co(I)-corrins.104 Thus, the selective electro-synthesis of Co(I)-corrins in the presence of suitable alkylating agents represents an efficient and selective preparative method to alkyl-corrinoids104 and more complex organo-corrinoids.61,106,107,113 However, strongly electron-withdrawing substituents in the cobalt-bound organic ligand of organo-corrinoids can render them more easily reducible and, thus, difficult to prepare via the strongly reducing Co(I)-corrins as nucleophilic cobalt-corrin species.114,115 The unique structures and exceptional thermally, photo-chemically and electro-chemically exploitable organometallic reactivity of the enantiomerically pure vitamin B12 derived cobalt-corrins furnishes them with a range of properties of interest in organic synthesis,59,111,116 in stereo-selective105,117 and bioinspired catalysis,86 in environmentally benign and useful “green” chemistry in aqueous solution,118,119 as well as in biomedical applications.120,121

15.04.5 Enzymatic organometallic processing of cobalamins Vitamin B12 (CNCbl), the best known commercial B12-vitamin form, has no direct physiological functions in humans, but is used as a provitamin by healthy human cells to biosynthesize the organometallic cofactors methylcobalamin (MeCbl) and coenzyme B12 (AdoCbl).16 Despite of their proper supply with CNCbl, some people show signs of Cbl-deficiency due to a deranged Cbl metabolism. In such patients the B12-deficiency was correlated with eight relevant genes.122,123 Among these human genes were two that were deduced to be directly responsible for intracellular organometallic processing of Cbls. The genetic locus named cblB

Fig. 9 Reversible one-electron reduction of HOCbl converts its 6-coordinate Co(III)-center to the 5-coordinate Co(II)-center of CblII, which may be reduced reversibly at more negative potential to the Co(I)-corrin CblI− with a 4-coordinate Co(I)-center. The reduction steps are accompanied by the de-coordination of one axial ligand (oxidations steps involve the corresponding addition of one axial ligand).

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encodes the adenosyltransferase ACA, which produces AdoCbl from a bound reduced Cbl.124 The other one (cblC) was responsible for a malfunction that led to accumulation of methylmalonate and homocysteine, also referred to as the MMACHC gene (for methylmalonic aciduria type C and homocystinuria), encodes for the cobalamin processing enzyme CblC.125 This protein is a Cbl-deligase that “tailors” different cob(III)alamins to CblII, which is then processed further in the cell to the physiologically active cofactors MeCbl and AdoCbl.16 The removal of the “upper” axial ligand to produce CblII employs two different mechanisms that depend on the Cbl substrate (Fig. 10).12,16,126 When CNCbl is bound, CblC catalyzes a reductive decyanidation by NADPH via flavin cofactors.127 Upon binding of alkylcobalamins, such as MeCbl, the removal of the upper ligand by glutathione (GSH) occurs via the alternative mechanism of a nucleophilic substitution.128 The crystal structure of human CblC in complex with MeCbl (but inactive due to lack of GSH) provided detailed structural insights into the “base-off” binding of MeCbl, as well as the three-dimensional structure of this unique 26 kDa protein.129 The substrate “base-off” MeCbl features an atypical five-coordinated Co(III)-center with the nucleotide tail buried in a crevice of the N-terminal domain. Clearly, the 5-coordinate Co(III)-center of the bound “base-off” MeCbl is highly activated for the nucleophile-induced demethylation. An abortive complex of CblC with the nucleophilic co-substrate GSH bound, but inhibited by the “antivitamin B12” difluorophenyl-ethynylcobalamin (F2PhEtyCbl), allowed for an X-ray analysis that revealed the important further structuring of CblC by GSH as well as the positioning of the nucleophilic thiol-function of GSH in a completely assembled CblC enzyme.76 The specific cblB type disorders, on the other hand, are due to deactivating mutations of the Cbl-ligase ATP: CblI adenosyltransferase (ACA), responsible for the biosynthesis of AdoCbl from the precursor corrinoid CblII.130,131 The enzyme-catalyzed adenosyl transfer is based on the intermediate formation of the nucleophilic CblI, which attacks the 50 -carbon of correctly bound ATP. Indeed, a structural mimic of CblI, the isoelectronic Ni(II)-analogue nibalamin, inhibited a bacterial version of the adenosyltransferase.132 As the reduction of “base-on” CblII to CblI requires a reduction potential beyond the capacities of in vivo reducing agents, ACA activates CblII for reduction by binding it in a highly activated four-coordinate (completely) “base-off” form. Remarkably, since the adenosylation of chemically generated CblI with ATP in solution has, so far, not been observed, ACA appeared to pose even more puzzles. As revealed by crystallographic snapshots of its structure with CblII and ATP and the partially formed reaction product AdoCbl bound in the active site, the customized protein environment facilitates not only the reduction to CblI, but it also lowers the energy barrier for adenosylation by binding ATP in an activated conformation.130

15.04.6 Enzymatic reactions catalyzed by organometallic B12-cofactors Methyl- and adenosyl-cobamides, such as MeCbl and AdoCbl, are the two basic types of organometallic B12-cofactors relevant in functioning B12-dependent enzymes,6,17,24 The protein-bound “complete” adenosyl-corrinoid AdoCbl has been observed in its “base-on,”133 or in the more recently discovered, rather intriguing “base-off/his-on” form.134,135 In the case of enzymes carrying the

Fig. 10 The Cbl-deligase CblC is a dual performance enzyme. The common product CblII is formed in “base-off”-form either by reductive decyanation of (“baseoff”) CNCbl or by nucleophile dealkylation of (“base-off”) alkylcobalamins (such as MeCbl) by glutathione (GSH), followed by oxidation of the directly resulting CblI −.

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methyl-corrinoid MeCbl only a range of “base-off” forms have crystallographically been observed.35,129 Protein-bound species of the catalytically important cofactor form CblII have also been studied by crystallography (see e.g. Ref. 136). However, for reasons of its thermodynamic instability and inaccessibility under typical physiological conditions, the structure of the typically transient CblI-form has only been inferred indirectly.17,137

15.04.6.1 B12-dependent methyl transferases B12-dependent methyltransferases are widespread and important organometallic enzymes. Two basic biochemical mechanisms of methyl group transfer are known.23,99 Cobamide-dependent methionine synthases, which occur in many organisms, including humans, employ nucleophile induced heterolytic methyl group transfer steps.23 Related methyltransferases are key methyl group carriers in one-carbon metabolism in microorganisms,138 most thoroughly studied for methyltransferases in methanogenesis,139 in acetogenesis140 as pathway of anaerobic CO2 fixation,141 and in acetic acid catabolism to methane and CO2 in some anaerobic microbes.142 The “supernucleophilic” reactivity of Co(I)-corrins and the excellent methyl group donor activity of the organometallic methyl-Co(III)-forms of the B12-cofactors are the key to the catalysis of these enzymatic methyl-group transfer reactions.6,7,12,23 However, in a range of biosynthetic methylations, a second group of bifunctional B12- and SAM-dependent radical methyl transferases (now classified as “class B” radical SAM methyltransferases) has turned out to be remarkably important,100,143,144 which operate via the alternative mechanism of radical methyl group transfer reactions.98,99

15.04.6.1.1

B12-dependent methionine synthase

B12-dependent methionine synthase (MetH) is a particularly widespread organometallic enzyme.90 MetH is, probably, the most extensively studied B12-dependent methyltransferase,17,90 which has also served as a basic biochemical model for other methyl transferases that operate via a nucleophile induced methyl group transfer.12,23 The enzyme MetH from E. coli is has been a particularly useful representative of B12-dependent methionine synthase.23 Methyl group transfer, catalyzed by MetH, basically follows a two-step ping-pong mechanism (see Fig. 10). In a first step, the protein-bound MeCbl is demethylated by activated homocysteine, furnishing protein-bound CblI and methionine. In the second step, protein-bound CblI is methylated by activated N-methyl-tetrahydrofolate, generating tetrahydrofolate and regenerating the protein-bound MeCbl.96 The two steps proceed with an overall retention of configuration at the methyl group carbon, consistent with two SN2-type nucleophilic displacement steps, each occurring with inversion of configuration.145 The methyl group transfer catalyzed by MetH involves heterolytic (and nucleophile-induced) cleavage/formation of the (CodCH3)-bond. Only in a formal sense, it represents a methyl “cation” transfer, since free methyl cations (or radicals) are excluded as intermediates. 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 (hexa-coordinate) “base-off/his-on” Co(III)-center in MeCbl (see Fig. 11). The protein environment plays a crucial role in controlling substrate positions, as well as in providing access to the catalytic center.96 The crystal structure analysis of the B12-binding domain of MetH provided the first insight into the three-dimensional structure of a B12-dependent enzyme.35,146 This work revealed the displacement of the cobalt-coordinating DMB-nucleotide tail of the protein-bound organometallic cofactor MeCbl by the histidine of a conserved His-Asp-Ser-triad and bound by the core of a “Rossmann fold” of the protein.146 Consequently, MeCbl is bound in MetH and in most known B12-dependent methyltransferases in the typical “base-off/His-on” mode.146 Interestingly, a contrastingly different complete “base-off” form (i.e. with neither a crystallographically detected axial ligand nor coordination of either DMB or His) has been observed in a corrinoid methyltransferase involved in acetyl-CoA synthesis.147 Clearly, as discussed for CblC above,129 the presence of a 5-coordinated methyl-Co(III)-center activates the cobalt-bound methyl group strongly for its heterolytic transfer to a nucleophilic acceptor. The histidine of the His-Asp-Ser-triad helps to position the protein-bound MeCbl in MetH for methyl group transfer.146 A significant thermodynamic role of the histidine coordination in the methyl transfer reactions of MetH is also indicated.146 Indeed, a significant thermodynamic trans-effect of the DMB-coordination in MeCbl on heterolytic methyl group transfer reactions has been observed in aqueous solution.67,68 The coordinating DMB-ligand stabilized MeCbl, opposing nucleophilic abstraction of the methyl group by roughly 4 kcal/mol.68 Furthermore, the His-Asp-Ser-triad appears to function as the “relay” for H+-uptake/ release accompanying the enzymatic methylation/demethylation cycles.148,149 It may, thus, fine-tune the bound corrinoid for enzyme catalysis: weakening of the axial (CodN)-bond not only activates the methyl group of MeCbl for abstraction by a nucleophile, but it also assists the re-reduction of adventitiously formed CblII to protein-bound CblI.

15.04.6.1.2

B12- and S-adenosylmethionine-dependent radical methyl transferases

The incorporation of intact methyl groups at un-activated and saturated carbon-positions in the course of the biosynthetic methylation of some antibiotics was a puzzling observation, incompatible with known methylation mechanisms, such as the one used by MetH, and suggesting a different biological methylation path.150 The identification of a class of the abundant enzymes with protein signatures of “radical” SAM-enzymes was consistent with a broad biosynthetic involvement of radicals.151 The more recently provided evidence for a large sub-class of B12-dependent “radical” SAM-enzymes has moved B12-dependent radical methyltransferases further into the focus.99,100,144 The remarkably direct, efficient and thermodynamically very favorable methylation of a carbon-radical by abstraction of the cobalt-bound methyl group of MeCbl has provided a first chemical model reaction.98 Indeed, methylation of radicals by methyl-Co(III)-corrins is now an accepted mechanism for biosynthetic methylation reactions at saturated carbon-positions, catalyzed by B12-dependent “radical” SAM-enzymes. So far, the case of the methyltransferase Fom3 that

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83

Fig. 11 Cbl-dependent methionine synthase (MetH) catalyses the coupled formation of methionine from homocysteine and demethylation of N5-methyltetrahydrofolate to tetrahydrofolate involving the protein bound Cbls MeCbl in a “base-off/His-on”-state and CblI (the imidazole ring symbolizes His759 of MetH). The heterolytic methyl group transfer occurs in a ping-pong mechanism via two nucleophilic substitution (SN2) steps. The resting state with MeCbl bound “base-off/His-on” is highlighted at the bottom left.

methylates 2-hydroxy-ethylphosphonate in the course of the biosynthesis of the antibiotic fosfomycin is the probably most thoroughly studied representative.99,101,152

15.04.6.2 Organometallic chemistry of enzymes dependent on coenzyme B12 The AdoCbl-dependent enzymes perform chemical transformations that are difficult to achieve by typical “organic reactions” and that rely on the chemistry of protein-bound organic radicals.18–20,70,71 The substrate radicals are generated (directly or indirectly) via an H-atom abstraction by a tightly controlled, protein-bound 50 -deoxy-50 -adenosyl radical.73,153 The 50 -deoxy-50 -adenosyl radical, in turn, originates form the homolysis of the (CodC)-bond of the “radical starter” AdoCbl.45 Hence, AdoCbl acts as a structurally highly sophisticated “pre-catalyst” (or catalyst precursor) in its mere function of a reversible source for the 50 -deoxy-50 -adenosyl radical.12,154 However, it has been a matter of further discussion to which degree the remaining Co(II)-corrin fragment CblII would also participate as a “conductor” in the AdoCbl-dependent radical enzymes, or remain a mere “spectator.”155–157 The question has remained puzzling, how the protein environment and the binding of the substrate actually induce (CodC)-bond homolysis of AdoCbl. The homolysis of the (CodC)-bond of the protein-bound AdoCbl is activated and accelerated by a factor of about 1012 to agree with the observed reaction rates of the coenzyme B12-dependent enzymes in comparison with the rate of thermal CodC bond homolysis of AdoCbl.45,77,158 The dramatic destabilization of the bound organometallic cofactor towards homolysis of the (CodC)-bond and its mechanism are, hence, much discussed features of the AdoCbl-dependent enzymes.17,47,133,159 There is no indication for a covalent restructuring of the bound cofactor (except for the formation of the “base-off/His-on”-form in the carbon skeleton mutases). Furthermore, a radical center could be stabilized only very weakly by interacting with the protein and with solvent molecules. A large number of studies have been dedicated to the question of “how the activation of AdoCbl towards homolysis of its CodC bond” could come actually about (see e.g. Ref. 20,45,155,160–162). Earlier investigations suggested a protein induced “butterfly” deformation of the corrin moiety as basis for the enigmatic activation of the bound AdoCbl toward homolysis of its (CodC)bond.45,80,81 However, the X-ray crystal analysis of CblII,47 the entire corrinoid fragment from CodC bond homolysis of AdoCbl, revealed its structure as very similar to the cobamide part of AdoCbl,38 providing no evidence for the relevance of a conformational deformation of the corrin moiety. Rather contrastingly, these observations suggested that the enigmatic protein-induced activation

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of the bound AdoCbl toward homolysis of its (CodC)-bond in AdoCbl-dependent enzymes, would largely come about by a strong binding of the largely separated homolysis fragments (without their further deformation).47 The existence of a binding interface (e.g. of an “adenosine-binding pocket” in some of these enzymes) which does not allow for unstrained binding of the organometallic moiety, helps to support this picture.135,163 In order to mimic such a hypothetical enzyme bound activated AdoCbl with a “stretched” CodC bond, the homologue of coenzyme B12, “homocoenzyme B12” (Cob-(50 -deoxy-50 -adenosylmethyl)-cob(III) alamin) was prepared.164 As was roughly expected, the crystallographically observed distance between the cobalt center and C50 of the homoadenosine moiety of “homocoenzyme B12” was increased to 2.99 A˚ . This would roughly be the distance found between the corrin-bound cobalt center and C50 in one of the two “activated” forms of coenzyme B12 in the crystal structure of the AdoCbl-dependent glutamate mutase.135 Homocoenzyme B12 has, indeed, been observed to strongly inhibit the AdoCbl-dependent enzyme dioldehydratase.165

15.04.6.2.1

Coenzyme B12-dependent isomerases

As was first revealed by X-ray crystallography, the B12-coenzyme AdoCbl was bound in “base-off/his-on” form in a bacterial AdoCbl-dependent methylmalonyl-CoA mutase, and at the interface between the two large domains of the enzyme,166 similar to the previously discovered situation (but involving bound MeCbl) in the methyltransferase MetH.134,167 The binding characteristics of the B12-cofactor AdoCbl, displayed in methylmalonyl-CoA mutase,166,168 turned out to be similar to the ones found subsequently in the known AdoCbl-dependent acyl-CoA mutases (such as isobutyryl-CoA mutase169), in glutamate mutase,135,159 and in other carbon-skeleton mutases. The two H-bonded “regulatory protein residues” Ser-Asp that is seen in the mutases may not be involved in proton-transfer steps, and it may conserve its structure largely during enzymatic turnover. Indeed, “electronic effects” of the axial “lower” ligand on the (CodC)-bond homolysis in AdoCbl are less important.93 However, a likely mechanistic or kinetic advantage for the carbon-skeleton mutases from their binding of AdoCbl in the “base-off/his-on” form could not be deduced. Whereas enzyme catalysis of carbon skeleton rearrangements appears to be an exclusive capacity of AdoCbl-dependent enzymes, isomerases of the “eliminase”-category may also function independent of B12-based catalysis.70,155 Interestingly, AdoCbl-dependent enzymes come in two structural classes with respect to the mode of binding of the AdoCbl cofactor. All the AdoCbl-dependent isomerases that are also classified as “eliminases,” as well as AdoCbl-dependent ribonucleotide reductase, bind the B12-coenzyme AdoCbl in the conventional “base-on” form.133 Fixed placement of the corrin moiety at the interfaces of the B12-binding and substrate-binding/activating domains of methylmalonyl-CoA mutase (Fig. 12) and other AdoCbl-dependent mutases appears to be of high significance. The proper substrate to product rearrangement steps of AdoCbl-dependent enzymatic rearrangements are accomplished by tightly protein-bound radicals that are controlled in their reaction space.12,73,153 A major function of the protein-part of the enzyme, clearly, is the activation of protein-bound AdoCbl.12,70,71,73,133,170 However, its additional crucial roles concern the spatial control of the proper enzyme reactions involving (reversibly generated) radical intermediates and the protection of its protein environment from non-specific radical chemistry, a function that was classified as “negative catalysis.”153 The AdoCbl-dependent enzymes are disproportionately distributed in living organisms. Only methylmalonyl-CoA mutase21 is required for a functioning metabolism of humans and other mammals.15 The other radical enzymes dependent upon AdoCbl (or an analogous AdoCba) as cofactor occur in bacteria and are further carbon skeleton mutases (ethylmalonyl-CoA-mutase137, glutamate mutase138, methylene glutarate mutase16 and isobutyryl-CoA-mutase139), diol dehydratase and glycerol dehydratase106, ethanolamine ammonia lyase106 and the two amino mutases, ornithine-4,5-aminomutase and D-lysine/L-blysine-5,6-aminomutase.70,71

15.04.6.2.2

Coenzyme B12-dependent ribonucleotide reductase

Ribonucleotide reductases (RNRs) convert the building blocks of RNA into corresponding 20 -desoxyribonucleotides, or “DNA,” and occur in all living organisms that require DNA.171 The known RNRs have in common the formation of a critical thiyl radical form of an internal Cys residue but differ by the initiation of the involved H-atom transfer.172 Among the three major classes of RNRs the class II RNRs depend upon AdoCbl as the initiator of the formation of the thiyl radical.19,171,173 The subsequent steps of the RNR-catalyzed radical processes adhere to a common basic path, but using either two properly placed Cys residues as the reducing agents (as depicted in Fig. 13 for the AdoCbl-dependent RNR) or formate. X-ray crystallography of the type-II RNR from Lactobacillus Leichmannii binding 5-adeninylpentyl-Cbl, an inhibitory analogue of coenzyme B12, indicated “base-on” binding of the AdoCbl cofactor,174 analogous with the situation in the AdoCbl-dependent isomerases that are also classified as “eliminases,” such as dioldehydratase.133 Indeed, while AdoCbl-dependent (or type-II) RNRs differ in the final enzymatic reduction step,19 they use a similar mechanism (in a formal sense) as the “eliminases” for the catalysis of the preceding isomerization via the vicinal exchange of a hydrogen atom and of a hydroxyl group, as, e.g. observed in the AdoCbl-dependent dioldehydratases.20

15.04.6.3 B12-dependent dehalogenases The ability of some anaerobic microorganisms to dehalogenate haloalkanes and haloaromatics reductively, requiring corrinoids as cofactors, is important for the environment of this globe.175–177 A variety of relevant enzymatic dehalogenation processes have been described, among them of chloroethenes178 and of chlorinated phenols.17 The anaerobic bacterium Sulfurospirillum multivorans harbors the membrane bound B12-dependent tetrachloroethene reductive dehalogenase that uses a reduced form of an unusual

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Fig. 12 Methylmalonyl-CoA mutase converts (R)-methylmalonyl-CoA to succinyl-CoA. (A) The 1,2-skeletal isomerization is induced by (CodC)-homolysis of enzyme-bound AdoCbl furnishing CblII and the 50 -deoxy-50 -adenosyl radical (Ado%). The proposed mechanism involves abstraction of an H-atom from (R)methylmalonyl-CoA by Ado%, leading to the 2-methyl-malon-20 -yl-CoA radical. The thioester moiety of this enzyme-bound radical undergoes a 1,2-migration (a carbon skeleton rearrangement) to the succin-30 -yl-CoA radical (bottom, right); H-atom abstraction from 50 -deoxyadenosine (Ado-H) converts the later radical into succinyl-CoA (bottom, left) and regenerates Ado%, ready for recombination with CblII. The sequence of steps of this reversible enzyme reaction is only shown in the “forward” direction. (B) The rearrangement is proposed to be intramolecular and to proceed via a cyclopropyloxyl structure, where the migrating thioester moiety is attached to two skeleton carbons.

Fig. 13 Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotide-phosphates (RNA) to deoxyribonucleotide-phosphates (DNA) (P stands for di- or tri-phosphate). The proposed mechanism in AdoCbl-dependent (ot type-II) RNR involves the generation of the protein thiyl radical via H-atom abstraction by a 50 deoxy-50 -adenosyl radical (Ado%, from homolysis of the (CodC)-bond of AdoCbl) and subsequent abstraction of an H-atom from the 30 -position of the ribonucleotide. Following the loss of water from the 20 -position an H-atom is abstracted by the latter from a protein-bound thiol, and a successive one-electron reduction furnishes the 20 -deoxy-30 -ribonucleotidyl radical. Back transfer of an H-atom from Ado-H provides the reduced 20 -deoxyribonucleotide and the Ado %-radical, ready for the regeneration of protein bound AdoCbl by recombination with CblII.

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“complete” corrinoid cofactor, isolated as nor-pseudovitamin B1239 in order to reduce tetrachloroethene to trichloroethene (first) and to cis-dichloroethene.178 Nor-pseudovitamin B12 was about fifty times more active than CNCbl in an in-vitro reduction of trichloroacetate.179 In aqueous solution nor-cobamides (and the related nor-Cbls) assemble to the B12 “base-on” forms with lesser preference than Cbls themselves,37 enabling redox reactions with nor-cobamides to occur at potentials less negative than those of the corresponding Cbl redox-couples.180 Recent studies with the structures of the reductase of Sulfurospirillum multivorans revealed the “base-off” Co(II)-form of the bound nor-pseudovitamin B12178 and suggested the reduction of the haloalkene to involve a long distance electron-transfer from the Co(I)-form of the enzyme.181 The structural studies with a dehalogenase from a nitrate reducing anaerobe involved in the respiratory chlorophenol dechlorination with the “base-off” form of CblII bound suggested an “innersphere” reduction by CblI68 of the halophenol via a formal halogen-atom transfer.182 Hence, according to these structural studies of B12-dependent enzymatic dehalpgenation reactions formation of an organometallic intermediate was not indicated, in contrast to what was considered earlier.183–185

15.04.7 Gene-regulatory roles of organometallic B12-derivatives 15.04.7.1 B12-riboswitches Around the turn of the century a range of puzzling and surprising observations indicated a role of coenzyme B12 in the direct genetic control of the bacterial B12 metabolism in the absence of a protein factor.186,187 Conserved elements in the 50 -untranslated region (50 -UTR) of the corresponding mRNAs proved to be essential for this type of gene regulation, feeding the hypothesis of RNA-mediated genetic control.188,189 Indeed, in 2002, Breaker and coworkers showed the direct binding of coenzyme B12 (AdoCbl) to E. coli btuB mRNA, thereby inducing a structural reorganization.190 This work spearheaded the discovery of the more broadly relevant class of the riboswitches that regulate gene expression via binding of a specific ligand to the 50 -UTR of mRNAs.190,191 In more recent years, besides the so called B12-riboswitches,192,193 a range of riboswitches sensing important cofactors and metabolites could be identified.194,195 Interestingly, the structure of the first riboswitch discovered, the E. coli btuB riboswitch, is still poorly characterized. Like other riboswitches, B12-riboswitches consist of a sensing aptamer domain that selectively recognizes the corresponding ligand, and an expression platform, which undergoes a structural switch upon ligand binding, thereby sequestering sequence elements crucial for further transcription or translation of the mRNA.196 To accommodate coenzyme B12, the largest known cofactor, the aptamer domain forms a highly complex binding pocket comprising 200 nucleotides. In-line probing experiments examining the 202nt aptamer domain of the E. coli btuB riboswitch revealed major structural changes at eight positions upon binding of coenzyme B12.190 In order to further address the question of the structural basis of the binding of AdoCbl and other Cbls the interaction of the E. coli btuB aptamer domain with various B12-derivatives was studied.66 These studies supported the view that AdoCbl was the preferred ligand of the E. coli btuB riboswitch190 and its large apical 50 -deoxy-50 -adenosyl group was essential for its high-affinity binding. In the meantime, the crystal structures of the aptamer region of another AdoCbl-binding riboswitch could be analyzed197 as well of two other Cbl riboswitches198 that indicate natural B12-riboswitches may exhibit structural means to prefer binding of other (organometallic) Cbls, such as methylcobalamin (MeCbl), over AdoCbl.199

15.04.7.2 Photo-regulation of gene expression by coenzyme B12 Photo-regulation of gene expression is based on photoreceptors that respond to light via light-sensitive chromophores, classic representatives using flavins, bilins, and retinal.200–202 These photo-receptors were recently joined by AdoCbl-dependent DNA-binding enzymes that make use of the light sensitive organometallic coenzyme B12 (AdoCbl) in an amazing photo-regulatory role of gene expression.203 This striking twist for AdoCbl from its known biological roles in radical enzymes is based on the efficient light-induced cleavage of the (CodC)-bond of AdoCbl (see above).84,85 As first revealed by the AdoCbl-based photoreceptor CarH of the bacterium Myxococcus xanthus203–205 that regulates biosynthesis of the photo-protecting carotenoids in this bacterium, the class of B12-based photo-regulators appear to be abundant in bacteria.205 Thus, AerR, relevant for the regulation of tetrapyrrole biosynthesis in the photosynthetic bacterium Rhodobacter capsulatus206 has joined the group the AdoCbl-dependent photo-regulators first revealed by the discovery of CarH.207 Combined crystallographic, mutational and mechanistic studies have largely clarified the question how the organometallic B12-cofactor AdoCbl could be repurposed to play a broadly relevant light-sensing gene-regulatory role in CarH.208–210 According to these investigations, AdoCbl is not directly involved in DNA binding and the mechanism of gene regulation by CarH relies on the modulation of the structure of the AdoCbl-binding protein through interaction with the B12-cofactor. Under low-light conditions CarH binds intact AdoCbl in a dimer-of-dimers-type tetramer that inhibits the transcription of genes coding for carotenoid biosynthesis. The exceptional heterolytic photolytic cleavage of the (CodC)-bond of AdoCbl attached to CarH leads to a bis-His-coordinated “base-off” Cbl and the unreactive 40 ,50 -anhydroadenosine (Fig. 14), rather than the Ado-radical produced by the photolysis of AdoCbl in solution.210 The restructuring of the CarH protein upon photolysis of AdoCbl weakens the intermolecular contacts in the DNA-binding CarH-tetramer, setting the stage for carotenoid biosynthesis.208 How the protein moiety “reprograms” the path of the light-triggered cleavage of the CodC bond to the here observed unique (“heterolytic”) mode211 is still a subject of thorough biochemical,212,213 photo-physical214,215 and computational216 investigations.

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Fig. 14 AdoCbl is bound “base-off/His-on” to the photoreceptor protein CarH and is cleaved by visible light with eventual formation of a histidine-bis-coordinated CblIII and 40 ,50 -anhydroadenosine, accompanied by a significant restructuring of the protein environment.

15.04.8 Organometallic cobalamins as antivitamins B12 Organometallic Cbls that are not tailored by CblC to CblII, thus resisting the subsequent cellular transformation to the B12-cofactors AdoCbl and MeCbl, have been conceived and classified as antivitamins B12.49,217 Such analogues of vitamin B12 would be designed to behave like a wolf in a sheep’s clothing by resembling vitamin B12 at the outside, but differing by the chemical character at their corrin-bound cobalt core.217 They would be poisonous for humans and other mammals, but are thought to be useful for inducing “functional” Cbl-deficiency in a highly controlled way in experiments with lab animals.218 So far, representatives of two classes of organometallic Cbls have been shown to be inert to tailoring by CblC and, hence, to represent potential antivitamins B12: (i) aryl-Cbls, which have a substitution and reduction inert CodCsp2 bond, with 4-ethylphenylcobalamin (EtPhCbl)49 and phenylcobalamin (PhCbl)50 as their first examples (Fig. 15). By the same criteria, (ii) suitably structured alkynyl-Cbls, such as 2-phenyl-ethynylcobalamin (PhEtyCbl),51 and difluorophenyl-ethynylcobalamin (F2PhEtyCbl)76 may also be potential antivitamins B12217 and such hydrolysis-, light- and heat-resistant arylalkynyl-Cbls have recently attracted broad interest in chemical biology.219–223 EtPhCbl and PhEtyCbl were observed to bind to the three important human B12-transporting proteins intrinsic factor, transcobalamin and haptocorrin,63 suggesting these aryl-Cbls to induce functional B12-deficiency in mammals when supplied orally.217 Indeed, when mice were treated with the EtPhCbl intravenously, this antivitamin B12, caused functional B12-deficiency in these animals with high efficacy.218 Typical Cbl-based (or other) antivitamins B12 may likewise be resistant to metabolic use by bacteria and archaea that depend upon B12, and they would, thus, also have the features of useful antibiotics.22,217,224–226

Fig. 15 Symbolic formulae of some antivitamins B12. (A) Cobalamin-based (left and center): the arylcobalamin EtPhCbl and the difluorophenylalkynyl-cobalamin F2PhEtyCbl are organo-Cbls with (CodCsp2)- and (CodCsp)-bonds, respectively. (B) Rhodibalamin-based (right): adenosyl-rhodibalamin (AdoRhbl), the Rh-analogue of AdoCbl.

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15.04.9 Metbalamins: Transition-metal analogues of cobalamin Suitably structured B12-mimics, in which the B12-specific cobalt-center is replaced by the homologous rhodium-ions, may have the features of antivitamins B12.217 As described recently, the organometallic Rh-analogue of AdoCbl, adenosyl-rhodibalamin (AdoRhbl), and AdoCbl have very similar structures.227 As expected,22,217 AdoRhbl (see Fig. 15) displayed the biological features of an antivitamin B12 in experiments with bacteria, as it very effectively inhibited a bacterial diol-dehydratase and the growth of Salmonella enterica.227 In the 1970s Koppenhagen and coworkers have already reported the preparation of rhodibalamins (Rhbls), among them AdoRhbl, which could only be partially characterized at that time,228 but behaved as a B12 antimetabolite in tests with microorganisms and human cell cultures.229 Since the cofactor activity of AdoCbl largely depends upon the homolytic cleavage of its CodC bond, the strength of the corresponding RhdC bond in the cofactor mimic AdoRhbl would be a critical factor for its behavior as antivitamin B12. So far the homolytic RhdC BDE of AdoRhbl has not been determined, but it probably exceeds the one of the corresponding CodC bond in AdoCbl. In a welcome contrast to the observations with the antivitamin B12 EtPhCbl and coenzyme B12 (AdoCbl), the organometallic bond of the related AdoRhbl also proofed to be stable under irradiation with sun light.227,230 Indeed, Rhbls, the Rh-analogues of the Cbls, appear to constitute a broader group of promising antivitamins B12, and a systematic and more direct chemical-biological methodology for the synthesis of Rhbls was developed, based on a newly bioengineered preparative route to the metal-free B12-ligand hydrogenobyric acid (Hby).231 The metal-free Hby (see Fig. 16) represents a remarkable helical ligand structure that acts on cobalt- and other transition metal ions as a “Procrustean Bed,” imposing unusual reactivity.231 Most importantly, Hby also is a rational starting material for the partial synthesis of hydrogenobalamin (Hbl),132 the complete metal-free ligand of the Cbls, and from there, of specific Metbls, an old dream and topical subject in the B12field.228,232,233 The semisynthetic Hbl has served as starting material for the synthesis of a range of Rhbls, among them chlororhodibalamin (ClRhbl) and methylrhodibalamin (MeRhbl),22,230,234 the Rh-analogues of chlorocobalamin (ClCbl) and of MeCbl, respectively (see Fig. 17). The crystal structures of the organometallic AdoRhbl and of the “inorganic” ClRhbl revealed these pairs of Rh(III)-corrins and Co(III)-corrins to be closely iso-structural and showed the slightly larger Rh(III)-ion to even fit slightly better into the natural corrin ligand of the Cbls than the “natural” Co(III)-ions.227,234 The crystallographic analysis of the two Rh(III)-corrins revealed a remarkable structural trans-influence of the axial ligands that is quantitatively similar to the corresponding phenomenon considered typical of their homologous Co(III)-corrins.12,235 The metal-free B12-ligands Hby and Hbl are starting materials not only for the syntheses of Rhbls, but also of other Metbls. So far, the synthesis and the detailed structural characterization of zincobalamin (Znbl),236 the Zn(II)-analogue of vitamin B12,237 and of the novel Ni(II)-analogue, nibalamin (Nibl)132 were achieved via this route (see Fig. 17). The redox-inactive penta-coordinate (“base-on”) Znbl has been proposed to constitutes a luminescent structural mimic236 of the penta-coordinate “base-on” Co(II)-cobalamin (CblII).47 The alternative tetra-coordinate diamagnetic “base-off” Ni(II)-corrin Nibl is a largely redox-inactive structural mimic132 of the highly reactive tetra-coordinate “base-off” CblII and of CblI.

Fig. 16 Partial synthesis of metbalamins (Metbl), transition metal analogues of vitamin B12, from biotechnologically produced hydrogenobyric acid (Hby) by the sequence metal incorporation and attachment of the B12-Nucleotide (e.g. Met(L) ¼ Zn(II) without axial ligand).

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Fig. 17 The partial synthesis of hydrogenobalamin (Hbl) from hydrogenobyric acid (Hby) provides a versatile platform for the direct preparation of rhodibalamins (Rhbls), such as chloro-Rhbl (R ¼ Cl, ClRhbl), methyl-Rhbl (R ¼ methyl, MeRhbl) and adenosyl-Rhbl (R ¼ 50 -desoxyadenosyl, AdoRhbl) as well as other metbalamins (Metbls), among them zincobalamin (Znbl) and nibalamin (Nibl).

15.04.10

Summary and outlook

The discovery of the organometallic nature of coenzyme B12 (AdoCbl) has opened the field of bio-organometallic chemistry. Indeed, Nature makes use of the capacities of the organometallic B12-derivatives in remarkable ways. Increasing insights into the B12-dependency of organisms from most kingdoms of life reveal ever increasing numbers of biological functions of the complex cobalt-corrinoids238–240 in often very specific and important ecological roles.241–243 The topic of B12-controlled natural gene expression also continues to generate amazing discoveries.210,212 A range of hardly rationalized effects of B12-deficiency in human and mammalian physiology240,244 call for new and better diagnostic tools in B12-medicine.217,245 Important contributions may come about by the application of antivitamins B1222 and of structurally related organometallic B12-based biological vectors,221,246 some of them anti-cancerous agents,222 in which the B12 carrier acts as a “Trojan Horse.”120,247 The organometallic B12-derivatives offer many opportunities to extend the range of chemical119,248 and biological catalysis,249 as well as to meet the quest for control of life processes106,250,251 with new biomedical applications.225,246,252 The B12-cofactors, when bound and controlled by proteins, are exceptional organometallic catalysts promoting and targeting cellular metabolism.154,253,254 In alternative functions they are structuring ligands of B12-binding nucleotides, e.g. in bacterial riboswitches.194 Hence, the various roles of B12-cofactors in living cells will continue to be “in the spotlight.”212,252,255 B12-derivatives also have a remarkable potential for the development of novel applications based on “purely” chemical (organometallic and analytical) research.120 Bio-structural, biochemical and chemical biological studies with organometallic B12-derivatives will, thus, continue to foster deeper insights into the growing number of roles of B12 in biology,7,24,126,256 and to fascinate the B12-fraternity and the research community in the neighboring fields with a rich bio-organometallic chemistry.

Acknowledgments I have enjoyed working with a group of dedicated and talented doctoral and post-doctoral coworkers, whose names are listed in the references. I am grateful also to many wonderfully collaborating colleagues for fruitful research interactions. Over the years our work in Innsbruck in the B12-field was supported by the European Commission and by generous and continuous support by the Austrian National Science Foundation (FWF), current project P-33059.

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15.05 Bioorganometallics: Artificial Metalloenzymes With Organometallic Moieties Michela M Pellizzoni and Andriy Lubskyy, Adolphe Merkle Institute, University Fribourg, Fribourg, Switzerland © 2022 Elsevier Ltd. All rights reserved.

15.05.1 Introduction 15.05.2 Artificial metalloenzymes based on the biotin-streptavidin technology 15.05.2.1 C–H activation 15.05.2.2 Suzuki cross-coupling 15.05.2.3 Transfer hydrogenation 15.05.2.4 Ring closing metathesis (RCM) 15.05.2.5 Hydroamination and hydroarylation 15.05.3 Artificial metalloenzymes based on human carbonic anhydrase 15.05.3.1 Imine transfer hydrogenation 15.05.3.2 Metathesis 15.05.4 Artificial metalloenzymes based on myoglobin 15.05.4.1 Carbene insertion and cyclopropanation 15.05.5 Artificial metalloenzymes based on thermophilic cytochrome P450 (CYP119) 15.05.5.1 Carbene insertion into C–H bond 15.05.5.2 Cyclopropanation 15.05.5.3 C–H amination 15.05.6 Artificial metalloenzymes based on POP scaffold 15.05.6.1 Si–H insertion 15.05.6.2 Cyclopropanation 15.05.7 Artificial metalloenzymes based on nitrobindin 15.05.7.1 Rh alkyne polymerization 15.05.7.2 Ru metathesis 15.05.7.3 C–H functionalization 15.05.7.4 Other reactions with nitrobindin scaffold Acknowledgment References Relevant Website

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15.05.1 Introduction The new millennium witnessed the rebirth of the research on artificial metalloenzymes envisioned in the late 70s. Since then, several abiotic reactions catalyzed by artificial metalloenzymes were reported1,2 such as hydrogenation,3 transfer hydrogenation,4 hydrogen production/decomposition,5 allylic substitution,6 cross-coupling,7 benzannulation,8 metathesis,9 cyclopropanation, polymerization,10 Diels-Alder reaction,11 Friedel-Crafts alkylation,12 as well as hydroformylation,13 hydroxylation,14 alcohol oxidation,15 fluorination,16 hydrolysis,17 NO reduction.18 An artificial metalloenzyme (ArM hereafter) results from the incorporation of a catalytically competent metal-cofactor within a biological protein scaffold able to catalyze abiotic reactions. Following pioneering reports by Whitesides19 and Kaiser,20 several groups started to investigate the potential of ArMs in combining and complementing the advantages of both homogeneous catalysis (wide range of reactivity, robustness and wide substrate scope) and enzymatic catalysis (high activity, selectivity and water tolerance).21 Importantly, the protein scaffold of ArMs can interact with metal cofactors, intermediates or substrates to promote the reaction with chemo-, regio- and stereoselectivity. The use of artificial metalloenzymes allows the evolution of the system by chemical and genetic optimization procedures. Chemical optimization allows exchanging and modifying both the metal center and the ligand. Most importantly, since this protein scaffold is genetically encoded, ArMs could be considered as a homogeneous catalyst endowed with genetic memory.2 This feature offers the opportunity to improve the catalytic performance of ArMs applying the Darwinian evolution scheme relying on directed evolution.22 Introduced by Arnold23 and Stemmer,24 this methodology has already had a strong impact on biotechnology and biocatalytic processes,25,26 and was recently applied for ArMs evolution.27,28 Different approaches have been investigated to create ArMs. Localization of an abiotic metal cofactor within a well-defined second coordination sphere environment provided by the biological scaffold can be pursued using (i) covalent, (ii) supramolecular, (iii) dative, or (iv) metal substitution strategies (Fig. 1). (i) Covalent anchoring is established by an irreversible reaction between a residue of the protein and the cofactor functionalized with a reactive moiety. Commonly, a cysteine residue29 or an unnatural amino acids bearing a terminal alkyne or azide30 are used. (ii) Supramolecular anchoring strategies exploit the low nanomolar affinity that proteins display for a small molecule.8,31,32

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Fig. 1 Anchoring strategies to embed the metal cofactor within the biomolecules. (A) Covalent, (B) supramolecular, (C) dative and (D) metal substitution. Color code: protein green, supramolecular anchor red, spacer and ligand blue, abiotic metal black. Nu, nucleophilic protein residues; E, electrophile; X, ligand coordinating moiety. Reprinted with permission from Schwizer, F.; Okamoto, Y.; Heinisch, T.; Gu, Y.; Pellizzoni, M. M.; Lebrun, V.; Reuter, R.; Köhler, V.; Lewis, J. C.; Ward, T. R. Artificial Metalloenzymes: Reaction Scope and Optimization Strategies. Chem. Rev. 2018, 118 (1), 142–231. Copyright (2018) American Chemical Society.

(iii) Dative anchoring is based on the coordination of a Lewis acid metal center to an electron-rich amino acid residue (e.g., His, Cys, Glu, Asp, Ser, etc.).13,33,34 (iv) Metal substitution exploits the reactivity of non-native metals combined with the binding pocket of natural metalloenzymes.35–38 An additional way to create new-to-nature enzymatic reactivity relies on an enzyme repurposing approach, which uses the natural metalloproteins without metal substitution (e.g., heme containing enzymes such as cytochrome P450 and myoglobin). Only the protein scaffold is engineered to promote abiotic reactions. Since these catalysts do not contain exogenous organometallic moieties, they are outside the scope of this chapter, but interested readers can find additional details on recent excellent publications reported in literature.39–41 The following sections will highlight the most prominent examples of ArMs-catalyzed reactions containing organometallic moieties.

15.05.2 Artificial metalloenzymes based on the biotin-streptavidin technology The use of biotin-streptavidin technology, is by far one of the most implemented strategies to create ArMs.42,43 The high affinity of biotin for streptavidin, ensures the quantitative incorporation of a biotinylated metal cofactor within the host protein via a supramolecular interaction. The geometry of the biotin-binding pocket is ideally suited to accommodate organometallic moieties, leaving enough space for substrate binding.44

15.05.2.1 C–H activation The rhodium-catalyzed benzannulation represents an outstanding example in the context of asymmetric C–H activation catalyzed by ArMs.8 Recently, the Cp RhL3-moiety emerged as a privileged catalyst for directed C–H activation.45 It was speculated that the introduction of a biotinylated rhodium Cp -moiety [Biot–Cp RhCl2]2 1 within streptavidin (Sav hereafter) may provide secondary coordination sphere interactions enabling an enantioselective C–H activation reaction. Since the reaction requires three coordination sites around RhCp to activate and couple both substrates, the introduction of an asymmetric ligand appeared difficult and in fact no stereoselective version of this reaction was reported. Knowing that the presence of an external base lowers the activation energy for the orthometallation step,46 the groups of Rovis and Ward screened the capability of [Biot–Cp RhCl2]2 1 to catalyze C–H functionalization reactions between pivaloyl-protected benzhydroxamic acid 2a and methyl acrylate 3a to afford dihydroisoquinolone 4a in MeOH/acetate buffer. While the free cofactor afforded the benzannulated products in 80% yield with a regioisomeric ratio (rr) of 4:1 and no enantioselectivity, the incorporation of the biotinylated rhodium cofactor 1 within Sav WT improved both the regio and the enantioselectivity of the reaction up to 9:1 rr and 50% ee with a lower conversion (46% yield) (Table 1, entries 1 vs 3). Thanks to the remarkable resistance of Sav against chaotropic agents and organic solvents,47 aqueous solutions containing 20% of methanol could be used without any noticeable denaturation of the protein. The same reactions were also performed substituting the buffer with pure water. In the absence of base, both the free cofactor and the assembled ArMs were virtually inactive, and no conversion was observed (Table 1, entries 2 and 4). Exploiting the easy manipulation of the secondary coordination sphere of Sav scaffolds and guided by docking studies, which highlighted the proximity of residues S112, N118 and K121 to the biotinylated metal center upon incorporation within Sav, basic residues (aspartate (D) or glutamate (E)) were introduced using side-directed mutagenesis. Gratifyingly, [Biot–Cp RhCl2]2 1
Sav K121D allowed for the success of the reaction in pure water to afford (R)-dihydroisoquinolone 4a in 89% yield, 15:1 rr and 56% ee (Table 1, entry 5).

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Table 1

Bioorganometallics: Artificial Metalloenzymes With Organometallic Moieties

Selected results for an artificial enantioselective benzannulase based on biotin-streptavidin technology.

Entry

Sav mutant

Buffer

Conv %

Regioisomeric ratio (rr)

ee

1 2 3 4 5 6 7

– – WT WT K121D N118K–K121E S112Y–K121E

Acetate buffer H2O Acetate buffer H2O H2O H2O H2O

80 adenine > cytosine > thymine.86 Rapid hydrolysis under neutral and alkaline conditions has limited corresponding studies on titanocene dichloride to acidic pH80,87 even though binding to long nucleic acids has been detected at physiological pH by inductively coupled plasma spectrometry.88 Coordination to phosphate was reported as the driving force of nucleotide binding but base-dependent differences in the affinities (guanine > thymine  adenine > cytosine) suggested additional interactions with the nucleobase moiety.

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Fig. 5 Examples of organometallic complexes with intercalating (22—27) and minor groove binding (28) ligands.

NMR studies have revealed the interaction of organotin compounds, such as diethyltin dichloride, dibutyltin dichloride and 11 (Fig. 2) with monomeric nucleosides and nucleotides to involve coordination to the sugar and phosphate oxygens, respectively.89–91 The relevance of these results to interactions with oligo- and polynucleotides, however, is not clear and for example in the case of 11 intercalation has been proposed instead. The results obtained on binding of metal arene complexes to model oligonucleotides by mass spectrometric and NMR spectroscopic92–97 as well as computational98,99 methods generally agree with the coordination patterns established for monomeric nucleosides and nucleotides. Within an oligonucleotide, however, the preferred binding site is determined not only by the identity of the coordinating nucleobase itself but also by its immediate environment59 and in some cases migration from a kinetic to a thermodynamic binding site has been observed.100 An interesting example is offered by G-quadruplex oligonucleotides where the preferred coordination site, guanine N7, is engaged in Hoogsteen hydrogen bonding. In such sequences, thymidine was found to be able to compete with guanine for coordination to Ru(II) arene complexes more effectively than in single-stranded oligonucleotides.92,101

15.07.2.2.3

Cross-linking

While cisplatin and its analogs act through cross-linking nucleobases (typically two guanines), this binding mode is less commonly encountered with mononuclear organometallic complexes, even when the metal center is coordinated to two readily exchangeable ligands.102 With Ru(II) and Os(II) arene complexes 29a and 29b bearing two chlorido ligands and one inert 1,3,5-triaza-7phosphatricyclo[3.3.1.1]decane (pta) ligand (Fig. 6), however, displacement of both of the chlorido ligands upon binding to single-stranded oligodeoxynucleotides was observed mass spectrometrically, consistent with intrastrand cross-linking.98 The Ru(II), but not the Os(II), complexes, also lost the arene ligand at lower stoichiometric ratios of the complex to the oligonucleotide. The phosphine ligand, on the other hand, appeared completely inert. Metallocene dichlorides have also been found to lose both of the chlorido ligands on binding to di- and oligonucleotides but, as discussed above, nucleobase and phosphate coordination probably compete in formation of the putative cross-links.78,79 Benzaldehyde thiosemicarbazone platina- and palladacycles 30a and 30b have been reported to form interhelical cross-links in plasmid DNA, a binding mode not observed with the classical platinum anticancer agents.103 The nature of this cross-linking remains unclear but, considering that the metallacycles were administered as tetranuclear complexes, coordination of different metal ions to different helices appears likely. Analogous multinuclear cross-linking between different DNA helices as well as between DNA and proteins has been firmly established with dinuclear Ru(II) arene complexes.104–107 Not surprisingly, many multinuclear organometallic (as well as coordinative) complexes show higher cytotoxicity than their mononuclear counterparts but in most cases the detailed target binding mode remains obscure.108–110

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Fig. 6 Examples of cross-linking organometallic complexes.

15.07.3 Organometallic and organometalloid oligonucleotides Compared to coordination complexes between natural or modified oligonucleotides and metal ions, examples of oligonucleotides featuring covalent bonds between carbon and metals or metalloids (mainly boron) are more scarce but no less diverse,111–113 even though the requirement for stability in aqueous solution limits the selection of metals. Organometallic and organometalloid oligonucleotides have been prepared as synthetic intermediates, affinity tags, electrochemical sensors and, more recently, to evaluate their potential as therapeutic agents. The synthetic methods used are as varied as the applications, ranging from direct metalation of oligonucleotides to enzymatic polymerization of metalated nucleoside triphosphates.

15.07.3.1 Synthesis of organometallic and organometalloid oligonucleotides 15.07.3.1.1

Electrophilic aromatic substitution

The relatively easy access to arylmercury compounds through electrophilic aromatic substitution with Hg(II) salts, known since the mid-1800s, makes mercury unique among the metals used in the synthesis of organometallic oligonucleotides.114,115 The reaction conditions required for the mercuration of unsubstituted carbocyclic arenes are harsh, typically involving refluxing in glacial acetic acid. The canonical nucleobases, however, are considerably more electron-rich, allowing high mercuration yields under conditions that nucleic acids withstand.116,117 For example cytosine and uracil C5, the most reactive sites of naturally occurring nucleic acids, are mercurated nearly quantitatively by incubation in 4 mM aqueous mercuric acetate at pH 6.0 and 50  C for 2 h (Scheme 1A and B). The purine bases are unreactive toward Hg(II) salts under these conditions but methylmercuration of guanine and hypoxanthine C8 by treatment with excess methylmercuric nitrate at comparable pH and temperature has been reported (Scheme 1C).118 Finally, the methyl substituent at C5 completely prevents mercuration of thymine bases. (A)

(B)

(C)

Scheme 1 Mercuration and methylmercuration of canonical nucleobases. Reagents and conditions: a) Hg(OAc)2, NaOAc, H2O, 50  C, 3 h; b) MeHgNO3, H2O, 50  C, 30 min.

With oligonucleotides and nucleic acids, the rate of electrophilic aromatic substitution by Hg(II) depends not only on the identity of the reacting nucleobase but also on the overall sequence. Homopolymers of cytidine and uridine, for example, react much more rapidly than naturally occurring heteropolymers. Owing to the denaturating conditions of a typical reaction mixture, dependence of the rate on secondary structure is generally not observed.117 Demercuration of a polymercurated nucleic acid by thiols, on the other hand, can be carried out under non-denaturing conditions and such experiments have revealed site-dependent reactivity patterns, in all likelihood attributable to secondary and tertiary structure. The different susceptibilities of different mercurated residues to demercuration have been exploited to prepare monomercurated tRNA through sequential mercuration and demercuration, although the single residue persisting after the thiol treatment was not identified.119

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Mercuration of nucleic acids through electrophilic aromatic substitution predates chemical oligonucleotide synthesis and was, hence, initially limited to natural nucleobases. Later, the versatility of this reaction has been demonstrated also with a number of artificial nucleoside and nucleotide analogs. The C7 of 7-deazaadenine, for example, exhibits similar reactivity toward Hg(II) as the C5 of cytosine and uracil (Scheme 2A).116 Electron-donating substituents on the aromatic ring serve not only to facilitate the reaction but also to direct it to the ortho and para positions. An appropriate combination of activating (such as amino and hydroxy) and blocking (such as methyl or the sugar moiety) substituents allows precise control over the site of mercuration, as exemplified by the artificial 3-fluoro-6-methylaniline C-nucleoside (Scheme 2B).120 Nucleobase analogs having more than one electron-rich carbon atom available, such as phenol121 or 6-phenyl-1H-carbazole,122 can be mercurated twice (Scheme 2C and D) although the second mercuration tends to be sluggish. Finally, in synthetic oligonucleotides the site of mercuration within the sequence can be controlled by replacing any cytosines and uracils at which reaction is not desired with 5-methylcytosines and thymines, respectively. (A)

(B)

(C)

(D)

Scheme 2 Mercuration of selected artificial nucleobases. Reagents and conditions: a) Hg(OAc)2, NaOAc, H2O, 50  C, 3 h; b) Hg(OAc)2, NaOAc, H2O, 55  C, 24 h; c) Hg(OAc)2, NaOAc, H2O, 70  C, 16 h; d) Hg(OAc)2, NaOAc, H2O, 55  C, 16 h.

15.07.3.1.2

Oxidative addition

Palladium-catalyzed cross-coupling reactions at C5 of a 5-iodouracil residue, notably Suzuki and Sonogashira couplings, are among the most popular methods for functionalization of oligonucleotides. With an appropriate choice of a catalyst, these reactions can even be carried out post-synthetically in aqueous solution.123–127 The first step of these reactions is oxidative addition of Pd(0) at C5 and in one case the resulting arylpalladium intermediate has been reported to be stable enough to be isolated chromatographically (Scheme 3A).128 In our hands, oligonucleotides incorporating 5-palladauracil modifications were found to decompose rapidly but a more stable product was obtained by oxidative addition of Pd(0) to a 2-iodobenzamide moiety (Scheme 3B).129 The greater stability could be understood if the product in question actually adopts a palladacyclic structure (see below) with the Pd(II) ion additionally coordinated to the amide oxygen or nitrogen. (A)

(B)

Scheme 3 Oxidative addition of Pd(0) to iodoaryl oligonucleotides. Reagents and conditions: a) Pd(OAc)2(2-aminopyrimidine-4,6-diol)2, 3-furanboronic acid pinacol ester, TRIS buffer (pH 8.5), H2O, 37  C, 4 h; b) Pd2(dba)3, H2O, MeCN, Ar atmosphere, 55  C, 18 h. In both cases, the products presented are those detected mass spectrometrically after RP-HPLC purification (the 3-hydroxypicolinate ligand in reaction A originates from the MALDI matrix). Possible coordination to oxygen is indicated by a dashed line.

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Cobalamins are probably the best-known examples of organometallic biomolecules and adenosylcobalamin remains the only organometallic nucleoside derivative identified in nature.130 The unique Co—C50 bond of adenosylcobalamin offers an elegant approach for oligonucleotide metalation and a few oligonucleotide analogs of adenosylcobalamin have indeed been described.131,132 The organometallic bond was formed post-synthetically by oxidative addition of Co(I)-cobalamin to a 50 bromo-20 ,50 -dideoxythymidine residue incorporated at the 50 -terminus of the oligonucleotide (Scheme 4). The brominated oligonucleotide was synthesized with a labile tert-butylphenoxyacetyl protection on the exocyclic amino groups, allowing relatively mild deprotection in a 10:1 mixture of concentrated aqueous ammonia and ethanol in 2 h at room temperature. Co(I)-cobalamin, in turn, was generated by electrochemical reduction of aquocobalamin chloride (31) in situ, a method originally described for the preparation of cobalamin dimers and rotaxanes.133 The resulting organocobalt oligonucleotides were stable at room temperature in darkness but decomposed back to aquocobalamin on exposure to light.

Scheme 4 Synthesis of oligonucleotide cobalamin conjugates through electrochemical reductive alkylation. Reagents and conditions: a) 50 bromo-oligodeoxynucleotide, tetrabutylammonium hexafluorophosphate, H2O, MeOH, −1.10 V vs. 0.1 N calomel electrode, N2 atmosphere, room temperature, 30 min. Substituents of the corrin ring have been omitted for clarity.

15.07.3.1.3

Ligand-directed cyclometalation

Cyclometalation was first described in the 1960s and has since become a popular method for the formation of carbon—metal s bonds under relatively mild conditions.134,135 Interestingly from the point of view of synthesis of organometallic oligonucleotides, the resulting metallacycles are also more stable than respective monodentate arylmetal compounds. The first cyclometalations were carried out between azoarenes and Ni(II),136 Pd(II) and Pt(II)137 and they still remain typical examples of this reaction, featuring initial coordination of the metal by a heteroatom and subsequent intramolecular activation of an otherwise inert CdH bond of an aromatic ring. Depending on the metal in question, the latter step may be considered either an electrophilic aromatic substitution or an oxidative addition. While ligand-directed cyclometalation has been reported with nearly all d-block transition metals, the selection usable for the synthesis of organometallic oligonucleotides is limited by the reaction conditions and the stability of the product in aqueous solution. Sufficiently stable metallacycles are obtained with gold, iridium, nickel, osmium, palladium, platinum, rhenium, rhodium and ruthenium, whereas cyclometalation under conditions compatible with the stability (if not always the solubility) of nucleic acids has so far only been demonstrated with gold, iridium, palladium, platinum, rhodium and ruthenium. 6-Arylpurine derivatives readily undergo cyclometalation with a number of metals of the platinum group.138 Depending on the metal ion and the purine derivative used, the N donor can be either N1 or N7 of the purine ring. Reaction of cyclopentadienyl complexes of Rh(III) and Ir(III) with 6-phenylpurine nucleosides and nucleotides (Scheme 5A), for example, yielded exclusively N1-coordinated products,139 whereas Pd(II) and Pt(II) favored N7-coordination with 6-furylpurine (Scheme 5B)140 and N1-coordination with 6-naphthylpurine (Scheme 5C)141 derivatives. All of these reactions were accomplished under conditions tolerated by nucleic acids and in the case of Rh(III) and Ir(III) even proven on dinucleotide models representing the shortest possible DNA sequence. It should be noted, however, that the phosphate groups of these model compounds were fully alkylated to allow the reaction to be carried out in a nonpolar organic solvent. Indeed, finding a solvent system suitable for both the cyclopentadienyl complexes as well as unprotected oligonucleotides might prove exceedingly difficult but on-support cyclometalation could be feasible if the oligonucleotide protecting groups and linker are designed to be cleavable under sufficiently mild conditions. Finally, cycloosmation of 6-phenylpurine and 4-phenylpyrimidine nucleosides was recently reported but the reaction conditions employed (refluxing in toluene) are not directly applicable with oligonucleotides.142

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(A)

(B)

(C)

Scheme 5 Cyclometalation of purine derivatives. Reagents and conditions: a) [MCl2Cp ]2, NaOAc, CH2Cl2, room temperature, 12—24 h; b) PdCl2, AgBF4, MeCN, 60  C, 18 h; c) PtCl2(cod), AgNO3, H2O, 60  C, 18 h; d) Pd(OAc)2, benzene, 60  C, 2 h.

Palladium is the metal most extensively studied in the field of cyclometalation chemistry and so far the only one with which cyclometalation of oligonucleotides has been demonstrated. Phenylpyridine,143 benzaldoxime144 and benzylamine145,146 residues have all been successfully cyclopalladated by treatment with lithium tetrachloropalladate in aqueous solution (Scheme 6). Limiting cyclopalladation to the desired site is straightforward as canonical nucleobases are unreactive under these conditions. Mass spectrometric analysis suggests that the m-chloro-bridged dimers initially formed dissociate during chromatographic purification of the cyclopalladated oligonucleotide products. (A)

(B)

(C)

Scheme 6 Cyclopalladation of modified oligonucleotides. Reagents and conditions: a) Li2PdCl4, NaOAc, MeOH, H2O, 55  C, 24 h; b) Li2PdCl4, NaOAc, H2O, 55  C, 40 h; c) Li2PdCl4, MeCN, H2O, 25  C, 16 h.

15.07.3.1.4

Post-synthetic conjugation in solution

While the methods described above provide access to a wide range of organometallic oligonucleotides, it is obvious that many potentially interesting metalation reactions require conditions incompatible with the stability or solubility of oligonucleotides. Such problems may be overcome through post-synthetic conjugation of the oligonucleotide with the organometallic moiety. Of the plethora of established conjugation reactions available, most have been successfully employed for the preparation of organometallic or organometalloid oligonucleotides.

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Arguably the very compound that established the field of modern organometallic chemistry, ferrocene is highly stable but none of the known synthetic procedures allow formation of a ferrocene moiety directly on a cyclopentadienyl-functionalized oligonucleotide.147 Ferrocene—oligonucleotide conjugates were, however, first prepared as early as 1994 by peptide coupling between an amino-functionalized oligonucleotide and an N-hydroxysuccinimide ester of ferrocenecarboxylic acid (32) in an aqueous buffer (Scheme 7A).148 The ferrocene structure places few limitations on the conjugation chemistry used and, in addition to peptide coupling,149–152 Michael addition between thiol-functionalized oligonucleotides and acrylamide- (33) or maleimidefunctionalized ferrocenes153 (Scheme 7B), oximation between aldehyde-functionalized oligonucleotides and aminooxyfunctionalized ferrocenes (34, Scheme 7C)154 and Cu(I)-catalyzed cycloaddition between alkyne-functionalized oligonucleotides and azido-functionalized ferrocenes (35, Scheme 7D)155 have all been used for preparation of ferrocene—oligonucleotide conjugates in aqueous solution. The latter approach has also been successfully applied to the related ferracarboranes (36, Scheme 7E) as well as other boron clusters, with the alkyne function tethered either to the base or the sugar moieties.156,157

(A)

(B)

(C)

(D)

(E)

Scheme 7 Post-synthetic conjugation of ferrocene to appropriately functionalized oligonucleotides. Reagents and conditions: a) NaHCO3 buffer (pH 9.0), DMSO, H2O, room temperature, overnight; b) phosphate buffer (pH 7.3), H2O, room temperature, overnight; c) ammonium acetate buffer (pH 4.6), H2O, room temperature, 6—8 h; d) CuSO4, sodium ascorbate, MeOH, H2O, room temperature, 1 h; e) CuSO4, sodium ascorbate, tris-(3-hydroxypropyltrazolylmethyl)-amine, DMSO, tBuOH, H2O, room temperature, 4 h. In scheme E, the white and black circles refer to boron and carbon atoms, respectively.

While in all of the above examples the oligonucleotide was modified with a dedicated reactive handle prior to conjugation, ferrocenes linked to a carbodiimide group (37) can also be conjugated directly with the imino nitrogen of guanine, uracil or thymine bases. Such an approach is simpler and readily applicable to naturally occurring DNA and RNA but, naturally, does not allow site-specific functionalization (Scheme 8A).158 Another example of ferrocene functionalization of unmodified DNA is the EDC-promoted condensation between a 50 -terminal phosphate group and an amino-functionalized ferrocene (38) in aqueous imidazole buffer, resulting in an N-alkyl phosphoramidate linkage (Scheme 8B).159,160

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(A)

(B)

Scheme 8 Conjugation of ferrocene to unmodified nucleic acids. Reagents and conditions: a) borate buffer (pH 8.5), DMSO, 37  C, 12 h; b) imidazole buffer (pH 6.8), EDC, H2O, 25  C, overnight.

Polyoxometalates are well-defined clusters consisting of several transition metal oxyanions linked together through shared oxygen atoms.161–163 While completely inorganic themselves, some polyoxometalates have been covalently tethered to oligonucleotides through SndC bonds, resulting in organometallic polyoxometalate—oligonucleotide conjugates. More specifically, activated monooxoacylated Keggin and Dawson heteropolytungstates, obtained by sequential transmetalation with carboxyethyltin trichloride164,165 and condensation to an intramolecular mixed anhydride,166 were allowed to react with amino-functionalized oligonucleotides to afford a peptide-bonded conjugate (Scheme 9).167 Curiously, Cu(I)-catalyzed cycloaddition between alkyne-functionalized oligonucleotides and azido-functionalized polyoxometalates was found to be low-yielding despite the success of this method in conjugation of polyoxometalates to smaller organic molecules.

Scheme 9 Post-synthetic conjugation of the Keggin-type heteropolytungstate {SiW11O39[Sn(CH2)2CO]}8− to an amino-functionalized oligonucleotide. Reagents and conditions: Et3N, DMSO, H2O, 40  C, 30 h. Conjugation of the Dawson-type heteropolytungstate {P2W17O61[Sn(CH2)2CO]}6− was accomplished under the same conditions.

The chemical versatility of boronic acids makes them attractive for functionalization of oligonucleotides but at the same time limits the methods applicable for introducing them – although perhaps not as much as feared initially.168,169 It is, hence, not surprising that the first successful chemical synthesis of boronic acid—modified oligonucleotides was achieved by post-synthetic conjugation exploiting strain-promoted alkyne—azide cycloaddition between a cyclooctyne-functionalized oligonucleotide and 4-(azidomethyl)benzeneboronic acid (39), the reaction of choice for the introduction of challenging conjugate groups (Scheme 10A).170 Peptide bond conjugation soon followed, however, and has since been used to introduce carboxylate-

(A)

(B)

(C)

Scheme 10 Post-synthetic conjugation of boronates to appropriately functionalized oligonucleotides. Reagents and conditions: a) H2O, room temperature, 30 min; b) EDC, DIPEA, DMF, H2O, room temperature, 24 h; c) TBTU, triethanolamine, carbonate buffer (pH 8.3), DMF, H2O, 60  C, 2 h.

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158

functionalized boronic acids, such as 40 and 41, to terminal (Scheme 10B) as well as intrachain (Scheme 10C) positions of amino-functionalized oligonucleotides.171,172 Peptide coupling of boronate carboxylic acids seems, however, to be rather particular about the coupling agent used and extensive screening may be required to find the optimal conditions.

15.07.3.1.5

On-support conjugation

Sometimes the desired organometallic moiety may be labile toward reagents used during the assembly of the oligonucleotide chain, such as oxidants or acids, but sufficiently stable under the nucleophilic or alkaline conditions used at the end of the synthesis for removal of the nucleobase and phosphate protecting groups and cleavage of the linker. In such cases it is advantageous to perform the conjugation on-support as the organometallic reactant can be used in large excess and washed off after completion of the reaction, eliminating the need for an additional chromatographic purification step. Ferrocene withstands not only the final nucleophilic treatment but also the acidic and oxidative treatments used in each coupling cycle, allowing on-support conjugation during as well as after chain assembly. For example, Sonogashira cross-coupling between the propargylated ferrocene derivative 42 and 8-bromoadenine or 5-iodouracil bases afforded the desired oligonucleotide—ferrocene conjugates in good yields (Scheme 11A).173 On-support conjugation to the internucleosidic linkages has also been achieved, through oxidative amination of an H-phosphonate linkage either directly with an amino-functionalized ferrocene (43, Scheme 11B) or with propargylamine, followed by Cu(I)-catalyzed alkyne—azide cycloaddition with an azido-functionalized ferrocene.174 A high density of ferrocene units could be incorporated by this method although the yields were generally better when a longer spacer between the oligonucleotide and the bulky ferrocene moiety was used. Alkylation of phosphite triesters, obtained as the immediate products of phosphoramidite coupling, by metal ethylene complexes offers another potential approach toward oligonucleotides with organometallic moieties conjugated to the internucleosideic linkages. In the case of (dicarbonyl) (
5-cyclopentadienyl)(
5-ethylene)iron, a “half-sandwhich” analog of ferrocene, this reaction afforded a protected dinucleotide in good yield but unfortunately failed when carried out on a solid-supported oligonucleotide.175 (A)

(B)

(C)

(D)

(E)

Scheme 11 On-support conjugation of organometallic and organometalloid moieties to oligonucleotides. Reagents and conditions: a) Pd(PPh3)4, CuI, Et3N, DMF; b) conventional oligonucleotide synthesis steps; c) NH3, H2O, 55  C, 16 h; d) CCl4, pyridine, room temperature, 30 min; e) NH3, H2O, 55  C, 5 h; f ) CuSO4, sodium ascorbate, tris(benzyltriazolylmethyl)amine, MeCN, H2O, 60  C, 60 min; g) NH3, H2O, 55  C, 5 h; h) CuSO4, sodium ascorbate, tris(benzyltriazolylmethyl)amine, dioxane, MeCN, H2O, MW 60  C, 2 h; i) DBU, MeCN, room temperature, 3 min; j) NH3, H2O, 37  C, 3 h; k) CH2Cl2, 25  C, 12 h; l) MeNH2, NH3, H2O, 65  C, 10 min. In scheme E, the exchangeable ligands on Pd(II) following cleavage and RP-HPLC purification could not be identified.

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159

While copper-free strain-promoted alkyne—azide cycloaddition yielded the first boronate—oligonucleotide conjugates,170 the original Cu(I)-catalyzed version of this reaction was long thought to be impractical for this purpose owing to the instability of boronic acids in the presence of copper salts.169 Recently, however, on-support conjugation of the pinacol ester of 4-ethynylbenzeneboronic acid (44) and the N-methyliminodiacetic anhydride of 4-(azidomethyl)benzeneboronic acid (45) with oligonucleotides bearing a 50 -terminal azido or an intrachain alkyne modification, respectively, has been demonstrated in high yield and purity (Scheme 11C and D).176 Protection of the boronic acid function appears to be crucial as attempts to introduce unprotected 4-ethynylphenylboronic acid in the same way resulted in quantitative oxidation of the phenylboronic acid to phenol. In many applications of organometallic oligonucleotide conjugates, such as labeling, the length or flexibility of the linker or the steric bulk of the linkage are not critical issues. In metal-mediated base pairing, however, the organometallic moiety has to fit within the base stack and this requirement rules out bulky linkages, such as the fused-ring triazoles resulting from strain-promoted alkyne—azide cycloaddition. Oxime is among the smallest linkages afforded by established conjugation methods, making oximation an attractive approach for the synthesis of nucleoside analogs featuring an organometallic base moiety, especially when the oxime nitrogen participates as a donor to form a metallacycle. The feasibility of this strategy has been demonstrated by on-support oximation of an aminooxy sugar residue with ortho-mercurated and -palladated benzaldehydes (46, Scheme 11E).177 The cleavage and deprotection conditions had to be optimized, however, to avoid demetalation.

15.07.3.1.6

Solid phase synthesis using organometallic and organometalloid building blocks

Introduction of organometallic moieties to oligonucleotides by phosphoramidite coupling during chain assembly would be preferable to post-synthetic metalation or conjugation as it eliminates the need for an extra purification step. Unfortunately, for most metals such an approach seems all but impossible. For example, the trivalent phosphorus atom of the phosphoramidite function itself readily coordinates many metal ions.178–181 With the notable exception of ferrocene and related structures, the acidic, alkaline and oxidative conditions used during oligonucleotide synthesis by the conventional phosphoramidite strategy would also present an overwhelming problem with most organometallic and organometalloid functions. That being said, innovative modifications to the standard synthesis strategy have enabled incorporation of organometallic and organometalloid phosphoramidite building blocks in a few select cases. Despite its tendency to oxidize during the iodine treatment following coupling, ferrocene can nevertheless be considered fairly compatible with conventional phosphoramidite strategy and numerous examples of ferrocene-containing phosphoramidite building blocks have been described since the first report in 1996.182 In addition to being introduced to the base (47—52),183–189 sugar (53 and 54)173,190 and phosphate (55)191 moieties of nucleoside phosphoramidites (Fig. 7), ferrocene has also been converted into non-nucleosidic phosphoramidite building blocks with various scaffolds192–196 (56—59, Fig. 8). The sugar phosphate backbone can even be partially replaced by a ferrocene backbone, with the cyclopentadienyl rings and the iron atoms formally replacing the sugar rings and the phosphodiester linkages, respectively (Fig. 9).197 Metallacarboranes are close structural analogs of metallocenes and, in the case of cobalt or iron as the metal center, have been proven to be compatible with oligonucleotide synthesis with some limitations.198 Phosphoramidite building blocks of thymidine (60)199 and 20 -deoxyguanosine (61)200 with a cobaltacarborane cluster and an H-phosphonate building block of 20 -deoxyadenosine (62)201–203 with a ferracarborane cluster linked to their base moieties (Fig. 10) have all been successfully incorporated into oligonucleotides albeit only as the final building block at the 50 -terminus. Furthermore, building block 60 was coupled manually rather than on the synthesizer. Capping after the final coupling was invariably omitted and, in the case of the phosphoramidite building blocks, oxidation was carried out by treatment with tert-butyl hydroperoxide rather than iodine. In other words, full compatibility of metallacarborane building blocks with standard oligonucleotide syntheses, including the potential to be incorporated in the middle of the chain, remains to be demonstrated. Carborane phosphoramidites (Fig. 11) can be employed in oligonucleotide synthesis without any modifications to the conventional strategy.204–206 In addition to carborane-bearing nucleoside phosphoramidites similar to the metallacarborane derivatives discussed above, Ortho-, meta- and para-carboranes have all been converted into bis(hydroxypropyl) derivatives that were successively dimethoxytritylated and phosphitylated to afford building blocks 63—65, suitable for backbone modification. Monofunctionalization of ortho-carborane, in turn, has afforded several glycol nucleic acid (GNA) type building blocks (66—68) placing the carborane moiety in a position analogous to that of the base moiety in nucleosides. Excellent coupling yields have been reported for all of these building blocks, even when several were incorporated consecutively. In fact, most of the oligomers synthesized contained a long sequence of carborane units and only a few, if any, natural nucleotides. In all cases, a short ammonolysis allowed recovery of the oligomers with the carborane residues in their original closo structure while a longer treatment resulted in quantitative conversion to the nido structure (Scheme 12).

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Fig. 7 Examples of ferrocene-containing nucleoside phosphoramidite building blocks.

Fig. 8 Examples of non-nucleosidic ferrocene phosphoramidite building blocks.

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161

Fig. 9 Comparison of an oligonucleotide having a part of its sugar phosphate backbone replaced by a ferrocene backbone with the respective canonical sequence.

Fig. 10 Examples of metallacarborane-containing nucleoside phosphoramidite and H-phosphonate building blocks. The white and black circles refer to boron and carbon atoms, respectively.

Fig. 11 Examples of carborane phosphoramidite building blocks. The white and black circles refer to boron and carbon atoms, respectively.

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Scheme 12 Choice of the cleavage conditions allows recovery of carborane oligomers with the carborane residues as the closo or nido structure. Reagents and conditions: a) NH3, H2O, room temperature, 5 min; b) NH3, H2O, 80  C, 30 min. The white and black circles refer to boron and carbon atoms, respectively.

Fig. 12 The 5-(E)-(2-tributylstannylvinyl)-20 -deoxyuridine phosphoramidite building block 69.

Outside the realm of metallocenes and metallacarboranes, 5-(E)-(2-tributylstannylvinyl)-20 -deoxyuridine remains the sole example of a true organometallic compound successfully incorporated into an oligonucleotide by the phosphoramidite strategy (Fig. 12).207 The building block (69) featured a base-labile 50 -fluorenylmethoxycarbonyl (Fmoc) protecting group instead of the conventional dimethoxytrityl (DMTr) protecting group to avoid acid-promoted destannylation during the deblocking step. Similarly to the metallacarborane phosphoramidite building blocks, 69 was coupled as the final building block of the sequence, capping was omitted and iodine was replaced by an organic peroxide (3-chloroperoxybenzoic acid) as the oxidant. Removal of the 50 -Fmoc protection was accomplished during the final ammonolysis step. Introduction of organoboron compounds by phosphoramidite coupling was first achieved with 1-(50 deoxythymidine)-methylboronic acid (70),208 and later also by a similar analog of uridine (71, Fig. 13).209,210 The boronic acid function of these building blocks was protected either as a pinacol ester or a mixed anhydride with N-methyliminodiacetic acid. The 20 -OH of the uridine building block 71 was protected with an unconventional pivaloyloxymethyl group, possibly to avoid nucleophilic attack of fluoride ion on boron during removal of the standard fluorolabile tert-butyldimethylsilyl (TBDMS) protection. The established synthesis protocols were followed with the exception that the capping step after incorporation of the boronate building block was omitted. The 50 -boronate phosphoramidites were always used as the final building block of the sequence but this limitation stems simply from the lack of a 50 -hydroxy group rather than lability of the boronate function. Indeed, recent studies have demonstrated incorporation of a thymidine phosphoramidites bearing an arylboronate group on the base moiety (72) in the middle of the sequence.211 The only deviation from standard phosphoramidite strategy was the use of the more labile tert-butylphenoxyacetyl groups to protect the exocyclic amino functions, allowing deprotection under mild conditions in methanolic potassium carbonate. Even this precaution may not be necessary, as evidenced by a very recent synthesis of oligonucleotides incorporating 5-dihydroxyboryluridine residues (73).212

Fig. 13 Examples of boronate-containing nucleoside phosphoramidite building blocks.

Oligonucleotide Complexes in Bioorganometallic Chemistry

15.07.3.1.7

163

Enzymatic polymerization

Enzymatic polymerization remains the method of choice for the preparation of longer nucleic acid sequences. Enzymatic polymerization can also be carried out under milder conditions than chemical oligonucleotide synthesis, providing access to organometallic oligonucleotides hardly available otherwise. The obvious limitation is that the substrates have to bear a sufficient resemblance to natural nucleoside-50 -monophosphates but a number of organometallic structures have been shown to meet this requirement. The first examples of enzymatic polymerization of organomercury nucleoside triphosphates were described in the same seminal paper as the mercuration of nucleoside derivatives by electrophilic aromatic substitution.116 This first study covered both deoxyriboand ribonucleotides as well as a range of enzymes, including E. coli DNA polymerase I, calf thymus terminal deoxinucleotidyl transferase, E. coli RNA polymerase, T7 RNA polymerase and avian myeloblastosis virus DNA polymerase. The polymerization was only found to proceed in the presence of excess thiol and different polymerases had different steric requirements for the thiol. For example, E. coli DNA polymerase I efficiently incorporated 5-mercuriuridine-50 -triphosphate in the presence of bulky thiols, such as 1-thioglucose, whereas polymerization by calf thymus terminal deoxinucleotidyl transferase was only supported by the two smallest thiols tested, namely methyl- and ethylmercaptan. These findings strongly suggest that the organomercuric nucleoside50 -triphosphates themselves are polymerase inhibitors whereas their thiol complexes are readily accepted as substrates, exhibiting nearly identical kinetics of incorporation as their unmetallated counterparts.116,213 Coordination of Hg(II) to a critical mercapto function within the polymerase enzyme appears a likely explanation for the observed inhibition. In any case, given the known susceptibility of organomercuric nucleotides and nucleic acids to thiol-promoted demercuration, the thiol concentration should always be carefully optimized.214 Fidelity of the incorporation of organomercuric nucleotides by DNA polymerase I was assessed using either poly(dG)•poly(dC) or poly[d(AT)] as a template.213 Incorporation of 5-mercuriuridine-50 -triphosphate on the former or 5-mercuricytidine-50 -triphoshpate on the latter template failed, suggesting that the fidelity of these nucleotides is not compromised on mercuration. In addition to template-directed polymerases, polymerization of organomercuric nucleotides has also been demonstrated with the template-independent E. coli polynucleotide phosphorylase, accepting 5-mercuriuridine-50 -diphosphate as a substrate and exhibiting a similar dependence on thiols as the other polymerases.215 Finally, polymerization of organomercuric nucleotides is not limited to purified enzymes but has also been accomplished in nuclei isolated from L cells and mouse myeloma cells216,217 and even in whole B. subtilis cells made permeable by treatment with toluene.218 Ferrocene-modified oligonucleotides are readily accessible by chemical synthesis but enzymatic polymerization of ferrocene-bearing nucleoside-50 -triphosphates also finds use in various bioanalytical applications, especially when long nucleic acid sequences are needed. The feasibility of this approach was first demonstrated by labeling of the 30 -end of an oligonucleotide with a 20 ,30 -dideoxyuridine-50 -triphosphate having a ferrocene tethered to its C5 (74a, Fig. 14).219 Incorporation of this chain-terminating nucleotide was catalyzed by the template-independent terminal deoxynucleotidyl transferase. Several examples of templated polymerization of ferrocene-functionalized nucleoside-50 -triphosphates (74 and 75) have also been reported, allowing multiple ferrocene units to be incorporated.220–223 For minimal interference with the action of the enzyme, modification of the

Fig. 14 Examples of ferrocene-containing nucleotides successfully incorporated by enzymatic polymerization.

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nucleotide substrates has been invariably carried out at C5 of pyrimidine or C7 of 7-deazapurine bases, placing the ferrocene moiety in the major groove of a double-helical nucleic acid. Even so, the structures obtained are not perfect substitutes for their parent nucleotides and generally require a careful selection of the optimal enzyme for a given task. The Klenow DNA polymerase fragment, for example, exhibits a lower fidelity than the DyNAzymeII DNA polymerase but performs better when two or more consecutive ferrocene-modified nucleoside-50 -triphosphates need to be incorporated.221 The approach described above for post-synthetic conjugation of Keggin and Dawson heteropolytungstates to amino-functionalized oligonucleotides has also been applied to preparation of respective derivatives of 7-deaza-20 -deoxyadenosine and -guanosine-50 -triphosphates (76a and 76 g, Fig. 15).224 Despite the considerable size of the polyoxometalate moiety, these modified nucleotides were substrates of polymerase chain reaction (PCR) by KAPA2G Robust DNA polymerase. With the less bulky Keggin derivatives up to 40% replacement of the corresponding natural nucleotides was tolerated whereas with the bulkier Dawson derivatives polymerization failed when more than 2% of the natural nucleotides were replaced. Enzymatic polymerization is the method that afforded the first boronate-modified oligonucleotides225 but has since been somewhat overshadowed by chemical synthesis. All of the substrates described in the literature are analogs of thymidine50 -triphosphate having an arylboronate group tethered to the C5 via a relatively long linker (77a—77f, Fig. 16).225–227 In addition to the standard primer extension experiments with the Klenow DNA polymerase fragment, nucleotides 77c—77f have also been proven compatible with PCR by Taq DNA polymerase.227 In contrast to chemical oligonucleotide synthesis, protection of the boronic acid function was not necessary.

15.07.3.2 Organometallic and organometalloid oligonucleotides as synthetic intermediates 15.07.3.2.1

Halodemercuration and halodestannylation

Haloarenes, including halonucleobases, can be prepared through a two-step process involving electrophilic aromatic substitution by Hg(II) and halodemercuration of the arylmercury intermediate by a halogen electrophile, such as elemental iodine or N-bromosuccinimide.115 While many halogenated nucleosides and their phosphoramidite building blocks are readily available commercially, successive mercuration and halodemercuration is still a useful method for labeling of nucleosides and, possibly, oligonucleotides with short-lived radioactive isotopes of iodine. The potential of this reaction was demonstrated on both nucleotides and nucleic acids shortly after the pioneering work on organomercury nucleotides (Scheme 13).228 Since then, iododemercuration has been developed into a facile and reliable method for the synthesis of 123I-, 125I- and 131I-labeled 5-iodo-20 -deoxyuridine.229,230 In this procedure, the combination of sodium iodide and 1,3,4,6-tetrachloro3a,6a-diphenylglucoluril (iodogen) has replaced elemental iodine as the iodine source.

Fig. 15 Examples of nucleotides bearing the Keggin-type heteropolytungstate {SiW11O39[Sn(CH2)2CO]}8− and incorporated by enzymatic polymerization. Respective derivatives of the Dawson-type heteropolytungstate {P2W17O61[Sn(CH2)2CO]}6− were also prepared and tested successfully.

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165

Fig. 16 Examples of boronate-containing nucleotides successfully incorporated by enzymatic polymerization.

Scheme 13 Radioiodolabeling of 20 -deoxyuridine by successive mercuration and iododemercuration. Reagents and conditions: a) Hg(OAc)2, H2O, 50  C, 3 h; b) NaCl, H2O, refrigerate, overnight; NaI , iodogen, H2O, 15 s. I stands for either 123I, 125I or 131I.

Halodestannylation has largely replaced halodemercuration as the method of choice for the radiohalide labeling of nucleosides and nucleotides.230–234 While the halodemetalation step is analogous for these two procedures, the initial metalation step differs considerably (Scheme 14). Unlike arylmercury compounds, aryltin compounds are not accessible through direct electrophilic aromatic substitution of the parent arene but are instead prepared by a Pd(0)-catalyzed reaction between the respective aryl halide and an organostannane. For this reason, halodemercuration may still be preferred in applications involving radiolabeling of naturally occurring nucleic acids. On the other hand, selective radioiodination of an oligonucleotide at a single arbitrarily predetermined site has to date only been achieved through iododestannylation of an organotin oligonucleotide.207

Scheme 14 Radioiodolabeling of 5-iodo-20 -deoxyuridine by successive stannylation and iododestannylation. Reagents and conditions: a) bis(tributyltin), Pd(PPh3)4, dioxane, N2 atmosphere, 105  C, overnight; b) NaI , H2O2, AcOH, EtOAc, room temperature, 15 s. I stands for 125I.

15.07.3.2.2

Palladium-catalyzed cross-coupling reactions

The seminal paper of 1968 described a Pd(II)-catalyzed reaction between organometallic compounds and alkenes that later became known as the Heck coupling.235 The key difference to the variant better known today is that the reactive organopalladium intermediate was obtained through transmetalation of the organometallic starting material with Pd(II), rather than oxidative addition of Pd(0) to an aryl halide. The original study largely focused on arylmercury compounds, which were deemed to be the preferred starting materials. This finding, combined with the easy accessibility of C5-mercurated pyrimidine nucleosides, makes the

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legacy Heck coupling a useful approach for functionalization of pyrimidine nucleosides even today (Scheme 15).236–238 The reaction works best with conjugated alkenes such as styrene or methyl acrylate, other alkenes typically giving mixtures of products. A wide variety of C5 substituents have been introduced by Heck coupling between 5-mercuripyrimidines and alkenes, including simple vinylic and allylic groups,239–244 catalytically active side chains,245,246 linkers for further functionalization,247–252 carbohydrates253–255 and even metal complexes.256,257 Finally, replacing the alkene starting material with an organic disulfide affords thioethers through a related reaction.258–260 Heck coupling employing organomercury starting materials can be carried out in hydroxylic solvents and appears, hence, applicable to the functionalization of organomercury oligonucleotides although such a study has not yet been forthcoming.

Scheme 15 Example of functionalization of 5-mercuripyrimidine bases through Heck coupling. Reagents and conditions: a) styrene, Li2PdCl4, MeOH, room temperature, 7 h.

In addition to Heck coupling, organomercury compounds also undergo a Pd(0)-catalyzed reaction with aryl halides, akin to the Negishi coupling of organozinc compounds (Scheme 16).261,262 In this reaction, transmetalation takes place between the organomercury starting material and an organopalladium intermediate obtained through oxidative addition of Pd(0) to the aryl halide. Examples of compounds synthesized by this reaction have so far been limited to fairly simple 5-aryl derivatives of 20 -deoxyuridine.

Scheme 16 Example of functionalization of 5-mercuripyrimidine bases through Negishi-type coupling. Reagents and conditions: a) iodobenzene, Pd(PPh3)4, THF, diglyme, inert atmosphere, 120  C, 3 h.

Suzuki coupling between boronic acids and aryl halides is a popular method for the functionalization of biomolecules, including nucleosides and oligonucleotides. In all such reactions described in the literature, the aryl halide (typically 5-iodouracil) is part of the oligonucleotide and the conjugate group is supplied as a boronic acid. Recent advances in the synthesis of boronate oligonucleotides,176,211,212 however, pave way for the opposite approach even if no examples are as yet forthcoming.

15.07.3.3 Reversible ligation of organoboron oligonucleotides Coordinate covalent bonding between a Lewis base and a Lewis acid offers an interesting approach for reversible ligation of nucleic acids. To be practical in a biological context, both the Lewis base and the Lewis acid should be bonded irreversibly to their respective nucleic acid strands. This concept has been proven using the vicinal diol of a ribonucleoside as the Lewis base and an alkylboronic acid as the Lewis acid (Scheme 17).263,264 Ligation of corresponding oligonucleotides proceeded smoothly under physiological conditions but only in the presence of the correct template sequence.208–210 Given an appropriate template, several short bifunctional oligonucleotides with 30 -ribose and 50 -boronic acid termini can be ligated to give one long sequence and even auto-templated self-assembly has been demonstrated.265,266 However, despite the superficial isostericity between the boronate linkage and the natural phosphate linkage, the resulting duplexes were considerably less stable than respective unmodified duplexes. Some of the destabilization was attributed to angle strain of the sp2-hybridized boron and considerable alleviation was indeed observed on conversion to the sp3-hybridized hydroxyboronate by increasing the pH from 7.5 to 9.5. The reversible ligation of boronic acid -functionalized oligonucleotides has been studied as a model for the origin of life and it is interesting to note in this regard the proposed role of boron coordination in the prebiotic synthesis of ribose.267

Scheme 17 Reversible ligation of oligonucleotides featuring 30 -ribose and 50 -boronate termini.

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15.07.3.4 Metal-mediated base pairing of organometallic oligonucleotides Formal replacement of one or more of the hydrogen bonds of a Watson—Crick (or Hoogsteen) base pair with a coordinate bond between a metal ion and a Lewis base gives rise to a metal-mediated base pair. The first examples were described between canonical nucleobases268–270 but they have since been supplemented by a growing number of artificial metal-mediated base pairs.271–278 Proposed and realized applications for metal-mediated base pairing include sensors and switches,279–283 molecular wires,284–287 nanoclusters,288,289 expansion of the genetic alphabet290–293 and high-affinity oligonucleotide probes294–296 and their detailed treatise would lie beyond the scope of this chapter. The desire to employ the latter in a biological context, however, has prompted research on metal-mediated base pairing of organometallic nucleobase analogs that would not dissociate even under metal-deficient conditions, such as those prevailing inside a cell.112

15.07.3.4.1

Hg(II)-mediated base pairing

Although 5-mercuripyrimidine nucleosides and nucleotides were first synthesized already in the 1970s117 and 5-mercuriuridine was reported to form N3-Hg(II)-C5-linked polymers in the 1980s,297 the potential of organomercuric nucleosides for Hg(II)-mediated base pairing was not investigated systematically at that time. In fact, it was not until 1996 that complexation of a relevant model compound, (1,3-dimethyluracil-5-yl)mercury(II) with simplified analogs of the canonical nucleosides was studied thoroughly by 1 H and 199Hg NMR.298 The methyl substituent of N3 of this model compound prevents Hg(II)-mediated self-aggregation, facilitating study of the desired mixed nucleobase complexes. The preferred binding sites were found to be largely the same as with methylmercury(II)1,10 and in the case of guanine, thymine and uracil Hg(II) coordination was accompanied by deprotonation of the donor atom (Fig. 17). Another two decades would pass before the first report on Hg(II)-mediated base pairing of an organomercury nucleobase within an oligonucleotide.299 The kinetic lability of Hg(II) complexes allows such systems to be studied by conventional UV melting experiments. Melting temperatures of short double-helical DNA oligonucleotides incorporating a central Hg(II)-mediated base pair between 5-mercuricytosine and guanine or thymine were found to be similar to the melting temperatures of fully matched canonical duplexes of equal length. Corresponding duplexes featuring 3-fluoro-2-mercuri-6-methylaniline as the organomercury nucleobase were even more stable, pairing with thymine and guanine again being favored.120 In other words, the most stable Hg(II)-mediated base pairs were formed with bases capable of serving as anionic ligands upon deprotonation of the donor atom (N3 and N1, respectively), consistent with the results obtained on Hg(II) salts, methylmercury(II)1 and (1,3-dimethyluracil-5yl)mercury(II).298 Hg(II)-mediated base pairing between two thymine residues within an oligonucleotide has been thoroughly studied by NMR,300,301 X-ray crystallography302 and computational methods303 and conclusively shown to involve coplanar orientation of the two thymine bases and deprotonation of both of the N3 donor atoms (Fig. 18A). The 5-mercuricytosine—thymine (Fig. 18B) and 3-fluoro-2-mercuri-6-methylaniline—thymine (Fig. 18C) base pairs in all likelihood share a similar structure. The Hg(II)-mediated base pair between the dinuclear 1,8-dimercury-6-phenyl-1H-carbazole and thymine, on the other hand, was predicted by DFT calculations to have a very different structure, with the two Hg(II) atoms coordinated to the two oxo substituents

Fig. 17 Complexes of (1,3-dimethyluracil-5-yl)mercury(II) with simplified analogs of the canonical nucleosides.

(A)

(D)

(B)

(C)

(E)

Fig. 18 Structure of the Hg(II)-mediated thymine—thymine base pair (A), compared with proposed structures of 5-mercuricytosine—thymine (B), 3-fluoro-2-mercuri-6-methylaniline—thymine (C) and 1,8-dimercury-6-phenyl-1H-carbazole—thymine base pairs (D) and the thymine—2,6-dimercuriphenol— thymine base triple (E).

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of the thymine base, rather than the endocyclic N3 (Fig. 18D).122 In line with previous results on coordinative dinuclear Hg(II)-mediated base pairs,304 but in contrast with their Ag(I)-mediated counterparts,305–311 the additional metal bridge did not offer additional stabilization compared to the mononuclear 5-mercurycytosine—thymine base pair. Hg(II)-mediated base pairing has also been studied in the context of triplex-forming oligonucleotides but with mixed results.312 In most cases, covalent C5-mercuration of a terminal cytosine or uracil residue of a homothymine triplex-forming oligonucleotide actually decreased the affinity for homoadenine•homothymine target duplexes, presumably through competing intramolecular Hg(II)-mediated base pairing with one of the thymine bases.312 With A•T or T•A as the target base pair, increase of both Hoogsteen and the Watson—Crick melting temperatures of the triplex was observed but could not be unambiguously attributed to Hg(II)-mediated base pairing. Modification of the homopurine strand has been more successful, with a central dinuclear 2,6-dimercuriphenol C-nucleoside greatly increasing both the Hoogsteen and the Watson—Crick melting temperatures of a homothymine homoadenine•homothymine triplex through formation of dinuclear Hg(II)-mediated base triples.121 The favored partner was, again, thymine (Fig. 18E).

15.07.3.4.2

Pd(II)-mediated base pairing

In contrast to Hg(II)-mediated base pairing, studies on Pd(II)-mediated base pairing within oligonucleotides are complicated by the relatively slow ligand exchange of Pd(II). Accordingly, UV melting profiles of duplexes incorporating Pd(II)-mediated base pairs often exhibit several overlapping transitions and considerable hysteresis between the denaturation and renaturation curves.129,143 The most convincing evidence on stabilization by Pd(II) coordination has been obtained on short duplexes with the putative Pd(II)-mediated base pairs at terminal positions.145,313,314 Unambiguous demonstration of a stabilizing Pd(II)-mediated base pair in the middle of a double helix has been much more difficult but a recent study has shown that destabilization by an intrachain Pd(II)-mediated base pair can be considerably alleviated by making the organopalladium nucleoside analog more flexible.144 All of these results suggest that while the square planar coordination geometry of Pd(II) in principle lends itself to metal-mediated base pairing within a double helix, finding a structure that works in practice is more challenging that with metals preferring linear coordination geometry, such as Hg(II) and Ag(I).

15.07.3.5 Organometallic nucleobases as affinity tags One of the earliest successful applications of covalently metalated residues in nucleic acids was as affinity tags, harnessing the very high stability of Hg(II)—thiol complexes. Mercuration of as few as one out of 200 bases was found to be sufficient for quantitative retention of a mercurated polynucleotide on thiol-functionalized agarose beads.213 After the impurities had been washed out, the mercurated polynucleotide could be recovered by adding 2-mercaptoethanol to the elution buffer. In combination with enzymatic incorporation of organomercury nucleoside-50 -triphosphates, affinity chromatography on thiol-functionalized agarose, controlled-pore glass (CPG) or cellulose has been used for isolation of a specific nucleic acid from a mixture of cellular nucleic acids.315–317 5-Mercuricytidine residues were incorporated either selectively at both 30 -termini of a double helix or randomly throughout the sequence. Consistent with the previous results, terminally mercurated double helices up to 510 base pairs long were retained quantitatively and beyond this point binding decreased with increasing size. Larger numbers of randomly distributed 5-mercuricytidine residues, on the other hand, allowed quantitative retention irrespective of the size of the nucleic acid. In addition to purification on thiol-functionalized solid phases, the high affinity of thiols for mercurated nucleobases has been exploited for sequence-specific visualization of cellular nucleic acids.318–320 Mercurated nucleic acid probes were first allowed to hybridize with metaphase chromosomes or interphase nuclei, after which a thiol-functionalized hapten ligand (trinitrophenyl, biotinyl or fluorescyl) was introduced. In contrast to the purification experiments discussed above, the mercurated probes were obtained by treatment of the appropriate nucleic acid with mercuric acetate. The fluorescyl-labeled sequences could be detected directly whereas with the trinitrophenyl- and biotinyl-labeled ones further immunochemical amplification was required.

15.07.3.6 Organomercury nucleotides as isomorphous heavy atom derivatives in X-ray crystallography X-ray crystallographic determination of RNA tertiary structures using the multiwavelength anomalous dispersion (MAD) method321 relies on the incorporation of isomorphous heavy atom derivatives of nucleotides in the sequence to be studied. 5-Mercuripyrimidine nucleotides show potential in this application owing to their structural similarity to canonical nucleotides and the favorable absorption characteristics of mercury.322 It should be pointed out, however, that while bulky substituents at pyrimidine C5 are accommodated with relative ease within the major groove of DNA, the major groove of RNA is considerably narrower, making it advisable to limit mercuration near the ends of double-helical regions. Such site-selectivity could be achieved for example through ligation of a short mercurated oligonucleotide with a large unmodified RNA.

15.07.3.7 Sensor and imaging applications The programmable sequence recognition properties, complemented by the essentially limitless possibilities of developing high-affinity aptamers, make nucleic acids an attractive scaffold for various types of sensors. Functionalization with metals and metalloids widens the scope of such applications even further. In principle, the role of an organometallic or an organometalloid

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moiety in an oligonucleotide-based sensor could be either as a passive label facilitating detection at low concentration or as an active component in target recognition. Examples of both kinds can be found in the literature.

15.07.3.7.1

Electrochemical labeling of hybridization probes

Hybridization probes designed for the detection of very small amounts of a target nucleic acid traditionally bear either a radioactive or a fluorescent label to allow highly sensitive detection. Redox labeling was introduced in the 1980s as a quick and affordable alternative that avoids the use of hazardous radioactive materials.323,324 While a number of redox active compounds, including aniline and nitrobenzene,325 azidobenzene,326 anthraquinone,327 methoxyphenol and dihydrobenzofuran,328 benzofurazane,329 polyoxometalates,167,224,330 metallacarboranes331 and osmium complexes,332–334 have been investigated for their potential for electrochemical labeling of nucleic acids, ferrocene148 remains the gold standard. A single ferrocene unit allows reliable detection of femtomole quantities of the labeled oligonucleotide.148,335 When the oligonucleotide is in solution and the ferrocene moiety is tethered to the end of the sequence through a flexible linker, the electrochemical response is independent of secondary structure, making quantification straightforward.336 Ferrocene units forming a part of the backbone, on the other hand, can respond to changes in the secondary structure of the oligonucleotide (Scheme 18A).337–339 (A)

Fc

Fc

Fc Fc

Fc

(B)

Fc Au

Au

(C)

Fc

Fc Au

Au

Scheme 18 Various strategies for making the redox properties of ferrocene labels responsive to oligonucleotide conformation. “Fc” denotes ferrocene units with different scaffolds.

Proximity to an electrode dramatically increases the oxidation current of a ferrocene label. Accordingly, the signal can be made highly sensitive to the electron transfer tunneling distance between the label and the electrode and, hence, changes in the oligonucleotide conformation by covalently tethering the labeled oligonucleotide to the electrode. The most common example of such a conformational change is the hybridization of the loop region of a hairpin oligonucleotide with a complementary single-stranded oligonucleotide (Scheme 18B)340–342 but triple helix formation has been utilized as well.343 As a variation of this strategy, the ferrocene-labeled oligonucleotide can also be immobilized to the electrode through hybridization with a complementary sequence (Scheme 18C).344,345 Different bases may be tagged with ferrocene derivatives exhibiting different oxidation potentials, allowing quantification of their relative abundancies in an unknown target sequence by primer extension using the respective nucleoside-50 -triphosphates.346 Finally, ferrocene can also act as a quencher when brought to close proximity of an electrochemiluminescent label, such as ruthenium(II) bipyridine complexes, analogous to a Förster resonance energy transfer (FRET) pair.347,348 Such quenching and the electrochemical signal of the ferrocene label itself have been used in conjunction with each other in highly sensitive dual-channel hybridization probes.

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15.07.3.7.2

Detection of single-nucleotide polymorphisms

The role of organometallic and organometalloid nucleotides and oligonucleotides in detection of single-nucleotide polymorphisms (SNPs) depends on the strategy used. In methods that are based on canonical Watson—Crick base pairing with the variable nucleobase, such as various types of hybridization assays,349–352 single-base extension,353 or PCR,354 the organometallic moiety typically acts as a passive label, as discussed above. Ferrocene355–357 and polyoxometalates330 have both been employed in this role. Multiple signaling can be achieved by different labeling of different hybridization probes or nucleotides, as exemplified by ferrocenes with varying oxidation potentials.346,356 Many fluorescence-based hybridization probes make use of Förster resonance energy transfer (FRET) between a fluorophore and a quencher and similar strategies have been described also with electrochemical organometallic labels. For example, quenching of the electrochemical signal of ferrocene upon inclusion within b-cyclodextrin has been exploited in a binary hybridization probe for SNP genotyping (Scheme 19).358

B

B Scheme 19 A binary hybridization probe consisting of ferrocene and b-cyclodextrin oligonucleotide conjugates. The electrochemical signal of ferrocene is quenched upon inclusion within b-cyclodextrin. Hybridization affinity of the ferrocene-labeled oligonucleotide is sensitive to identity of the polymorphic base B.

Approaches where the organometallic moiety interacts directly with the polymorphic base to produce a base-specific signal are less common than those where the organometallic moiety is employed as a passive label but some interesting examples have nevertheless been described. For example the redox properties of a ferrocene label, when placed opposite to a base within a double-helical oligonucleotide, were found to depend on the identity of the “base pairing partner” although the nature of the interaction remained obscure.192 In an optimal case reliable discrimination between the four canonical nucleobases could be achieved but the results were complicated by the fact that bases flanking the ferrocene label also influenced the signal. Nevertheless, direct coupling between a redox label and a nucleobase appears a promising method for detection of single-nucleotide polymorphisms once the factors affecting the readout are better understood. Besides superior affinity, metal-mediated base pairing can also exhibit improved base discrimination compared to canonical Watson—Crick base pairing. The UV melting temperatures of short duplexes featuring a central Hg(II)-mediated base pair between the organometallic nucleobase analog 3-fluoro-2-mercuri-6-methylaniline and any of the natural nucleobases, for example, were all separated by a margin of at least 7  C, allowing reliable identification of the natural base pairing partner (Fig. 19).120 The 19F NMR spectra of all duplexes were also clearly different, providing an independent method to validate the results of the UV melting studies. In contrast, while the canonical Watson—Crick base pairs are much more stable than any of the mispairs, the stability differences between the various mispairs are generally too small and unpredictable for reliable identification. The superior discrimination by 3-fluoro-2-mercuri-6-methylaniline has recently been exploited in a FRET-based molecular beacon probe by placing this organometallic nucleobase in the middle of the loop region.359 At an appropriate temperature, the fluorescence emission intensity of this probe depended on the identity of the polymorphic base in the target sequence in the same way as the UV melting temperature of short duplexes (thymine > guanine > cytosine > adenine), allowing robust discrimination.

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Fig. 19 Comparison of melting temperatures of short double-helical oligonucleotides pairing either 3-fluoro-2-mercuri-6-methylaniline (F-Hg), cytosine (C) or thymine (T) with either adenine (dense hash), cytosine (white), guanine (sparse hash) or thymine (black).

The interesting ability of oligonucleotides incorporating a boronate linkage to stabilize the complementary strand within a duplex against nuclease degradation has been harnessed in a binary hybridization probe for SNP detection.360 Two short oligonucleotides, bearing 30 - ribose and 50 -boronic acid termini, were allowed to hybridize with a longer sequence containing the polymorphic base, after which snake venom phosphodiesterase (SVPD) and SYBR green I dye were added (Scheme 20). With a fully matched target sequence, the short oligonucleotides were ligated through a boronate linkage, yielding a nuclease-resistant double helix detectable by SYBR green staining. With a mismatched target, on the other hand, ligation did not take place and the target was left vulnerable to SVPD digestion to give short single-stranded fragments poorly stained by SYBR green I.

Scheme 20 A binary hybridization probe based on ligation of oligonucleotides with ribose and boronic acid termini on a fully matched template and the resistance of the resulting duplex to enzymatic degradation.

15.07.3.7.3

Biomolecule sensors

Aptamer oligonucleotides can be selected by directed evolution to bind virtually any molecule with high affinity and selectivity.361–366 Incorporation of labels responsive to the change in the aptamer conformation upon binding its target converts an aptamer into a sensor.367–375 For example, the aforementioned dependence of the electrochemical properties of ferrocene on its distance from the electrode and thus on the conformation of the intervening oligonucleotide sequence has been harnessed to develop an aptamer-based sensor for cocaine.376 A previously optimized aptamer sequence377–379 was immobilized on a gold electrode through its 50 -terminus and functionalized with ferrocene at its 30 -terminus. Binding of cocaine induces a change in the secondary structure of the aptamer, bringing the ferrocene moiety close to the electrode surface which, in turn, leads to increased oxidation current. When applicable, splitting the aptamer into two fragments which are joined upon binding the target may lead to improved sensitivity and lower background current.380 The aptamer and the labeled oligonucleotide can also be two separate sequences, as exemplified by a recently reported sensor for insulin.345 In the absence of insulin, the aptamer oligonucleotide forms part of a double-helical stem, keeping the ferrocene label away from the electrode surface. On binding insulin, the aptamer strand dissociates, leaving the ferrocene label tethered to the electrode by a relatively flexible single-stranded oligonucleotide, allowing closer contact with the electrode surface. Target binding can also be designed to mask a recognition site for a restriction enzyme.381

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In the absence of the target molecule, the labeled aptamer sequence is promptly cleaved off the electrode by the restriction enzyme, resulting in loss of signal. In the presence of the target, on the other hand, the aptamer folds into a conformation that resists cleavage and signal persists. The advantage of this approach is greatly reduced background signal. Finally, release of the labeled oligonucleotide from the electrode and subsequent decrease in the signal can also be affected by interaction with the target.382 The versatility of aptamers makes the strategies discussed above, or variations thereof, applicable to practically any target, proven examples including adenosine,383–386 theophylline,152,387 adenosine triphosphate,388–391 cocaine,392 codeine,393 thrombin,394,395 platelet-derived growth factor396–398 and lysozyme.399,400 Molecules that catalyze the cleavage of nucleic acids form a special class of targets for oligonucleotide-based sensors. In such cases, detection can be based on irreversible sequence-specific cleavage of the labeled oligonucleotide by the target, rather than affinity of an aptamer oligonucleotide for the target. Detection of femtomolar concentrations of bleomycin, for example, has been achieved with a sensor featuring a ferrocene-labeled hairpin oligonucleotide immobilized on a gold electrode coated with an electrochemiluminescent film of tris(2,20 -bipyridyl)ruthenium(II)—gold nanoparticle (Ru—GNP) complexes.348 In the absence of bleomycin, the hairpin oligonucleotide places the ferrocene moiety close to the electrode surface, resulting in quenching of the Ru— GNP electrochemiluminescence. Sequence-specific cleavage of the oligonucleotide in the presence of Fe(II) releases the ferrocene-containing fragment and restores the electrochemiluminescence signal. A large variety of carbohydrate sensors have been developed based on the propensity of boronic acid to form cyclic boronate esters with cis-1,2- and 1,3-diols.401–403 In nature, interactions between carbohydrates and other biomolecules are often multivalent and nucleic acids have been studied as a potential scaffold to simulate such interactions in a synthetic sensor.171 Accordingly, the ability of oligonucleotides functionalized with two benzeneboronic acid groups at either the 30 - or the 50 -terminus to bind various sugars was assessed both as single strands and as a double helix placing four benzeneboronic acid groups in close proximity. Cellulose paper chromatography in the absence of other sugars revealed significant retention of only the tetraboronic acid duplex, attributed to interaction of the boronic acid functions with the cis-diols present in cellulose. Of the sugars tested, galactose, maltose, trehalose and both a- and b-lactose were able to displace cellulose and thus restore mobility of the tetraboronate duplex but only at a high concentration. In addition to underlining the importance of multivalency, the results also serve as a proof of concept for selective recognition of carbohydrates by boronic acid oligonucleotide sensors.

15.07.3.7.4

Cation sensors

Organometallic oligonucleotide sensors for small cations are analogous to the electrochemical biomolecule sensors discussed above except that the change in the oligonucleotide conformation is induced by protonation or metal coordination, rather than the interaction between an aptamer and its target. Folding of a cytosine-rich sequence into an i-motif under acidic conditions is an example of such a conformational change and it has been used to bring a ferrocene label close to the electrode surface, resulting in increased oxidation current.404 A linear dependence between pH and oxidation current was observed over a pH range of 5.6—7.1. Similarly, the K+-promoted folding of a guanine-rich sequence into a G-quadruplex has been employed in potassium sensors, both to bring the ferrocene label close to the electrode405 as well as to push it away.406 The intermolecular parallel G-quadruplex proposed in the latter case exhibited higher selectivity to K+ than the chair-form intramolecular G-quadruplex proposed in the former case.

15.07.3.7.5

Intracellular imaging

The ability of 50 -boronic acid-modified oligonucleotides to inhibit ribonuclease H407 (RNase H) and the susceptibility of the boronic acid function to oxidation specifically by peroxynitrite have been harnessed to develop an oligonucleotide sensor for intracellular imaging of peroxynitrite.408 The sensor consisted of a 50 -boronic acid-modified 12-mer DNA oligonucleotide hybridized with a 21-mer RNA oligonucleotide, leaving a 9-mer 30 overhang. The 30 -terminus of the DNA oligonucleotide bore a quencher and the 50 -terminus of the RNA oligonucleotide a fluorophore, allowing the dissociation of the two strands to be detected by FRET imaging. No fluorescence was observed when untreated RAW264.7 murine macrophages, deficient in peroxynitrite, were transfected with the oligonucleotide sensor, indicating that the double-helical stem remained intact. Upon addition of external peroxynitrite or stimulation with interferon-g, known to induce production of peroxynitrite, increase of fluorescence was observed, consistent with oxidation of the boronic acid to an alcohol and subsequent RNase H catalyzed cleavage of the RNA strand (Scheme 21).

Scheme 21 Intracellular imaging of peroxynitrite based on oxidation of a terminal boronic acid function and subsequent activation of RNase H.

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15.07.3.8 Toward therapeutic applications While myriad small molecule organometallic compounds have been studied for their potential as therapeutic agents,22–35 the field of therapeutic organometallic oligonucleotides is still in its infancy. However, the few examples found in the literature nicely illustrate how organometallic modifications could be used to tackle some of the greatest challenges of oligonucleotide therapeutics,409–412 including delivery, stability and affinity. In an unrelated approach, the phosphate backbone of nucleic acids has been repurposed as a scaffold for construction of well-defined boron-rich oligomers for boron neutron capture therapy (BNCT).413–415

15.07.3.8.1

Organometallic oligonucleotides and drug delivery

DNA nanoparticles have received great attention for their potential as a drug delivery platform.416–437 Compared to short oligonucleotides, DNA nanoparticles exhibit improved cellular uptake and, in the case of larger nanoparticles, prolonged biological half-life owing to reduced renal clearance. DNA nanoflowers, so termed because of the petal-like structures protruding from their surface, are a special class of DNA nanoparticles, assembled by liquid crystallization of long DNA strands,438 in contrast to the more common DNA origami nanoparticles obtained by selective hybridization of several short DNA strands. The long DNA building blocks are synthesized by rolling circle replication (RCR) and the template can be designed for incorporation of various functional sequences, such as aptamers and drug loading sites. The enzyme used for RCR, F29 DNA polymerase, exhibits similar tolerance to artificial substrates as many other polymerases, as exemplified by the successful incorporation of 20 -deoxyuridine-50 -triphosphate carrying a Cy5 fluorescent label at C5. More extensive modifications can be introduced by using corresponding chemically synthesized oligonucleotides as primers or hybridizing them with complementary sequences within the DNA nanoflower. The latter approach was employed for the preparation of ferrocene-functionalized DNA nanoflowers.196 Adjusting the concentration of the ferrocene oligonucleotide provided a means for controlling the size of the DNA nanoflowers, with higher concentrations leading to decreased size and more condensed morphology. The ferrocene moieties also made the DNA nanoflowers susceptible to self-degradation by Fenton’s reaction in the presence of hydrogen peroxide. The power of combining the strategies outlined above was demonstrated by doxorubicin-carrying DNA nanoflowers incorporating an aptamer sequence for protein tyrosine kinase 7 (PTK7) and hybridized with ferrocene oligonucleotides.196 The aptamer moieties allowed selective recognition and internalization of the DNA nanoflowers into Michigan Cancer Foundation-7 (MCF-7) cancer cells overexpressing PTK7. Furthermore, at the relatively high hydrogen peroxide concentration prevailing within these cancer cells, the DNA nanoflowers readily degraded and released their doxorubicin cargo. Finally, in vivo experiments in mice confirmed the ability of the ferrocene-functionalized DNA nanoflowers to reduce distribution of doxorubicin to healthy organs while at the same time improving its therapeutic efficiency. Cobalamins are indispensable vitamins synthesized only by some prokaryotes439 and acquired by mammalian cells through specific B12 transporters.440–442 In addition to its natural cargo, the B12 transport system can be exploited for the delivery of artificial cobalamin derivatives with various conjugate groups, such as peptides,443–445 toxins446 and radioactive447–449 and fluorescent450 labels. Cobalamin appears an attractive delivery vector also for oligonucleotides given that one of the naturally occurring cobalamins (adenosylcobalamin) is actually a nucleoside derivative. This possibility has been explored with oligonucleotides ranging from 18 to 39 nucleotides in length, bonded to the cobalt center through C50 of a 50 -terminal thymidine residue.131,132 Of the three key proteins of the human B12 transport system, transcobalamin (TC) was found to bind efficiently with all of the oligonucleotide conjugates tested, suggesting the possibility of delivery to cobalamin-accumulating cells through parenteral administration. Intrinsic factor (IF) and haptocorrin (HC), on the other hand, exhibited much weaker binding, attributed to both steric crowding and the polyanionic charge of the oligonucleotide. The inefficient binding to IF and HC makes oral administration difficult but a less bulky linker between the oligonucleotide and cobalamin moieties might alleviate this problem. Cobalamin conjugation shows, hence, promise as a novel delivery vector for therapeutic oligonucleotides.

15.07.3.8.2

Biostability of organometallic and organometalloid oligonucleotides

Reports on the stability of organometallic and organometalloid oligonucleotides against nucleases are scarce but in most cases increased resistance has been observed. A 50 -boronic acid modification, for example, conferred improved stability against calf spleen (CSPD) and snake venom (SVPD) phosphodiesterases, exonuclease III, lambda nuclease and RNase H but not against exonuclease I. Interestingly, the stabilization was not limited to the modified strand itself but, in the case of double-helical oligonucleotides, extended also to the complementary strand.360,407,408 A free boronic acid function was crucial for the RNase H resistance whereas also an internucleosidic boronate linkage provided protection against CSPD, SVPD, exonuclease III and lambda nuclease, suggesting a different mechanism of stabilization. The difference in the stability of an RNA oligonucleotide against RNase H degradation when hybridized with a DNA oligonucleotide featuring either a 50 -boronic acid terminus or an internucleosidic boronate linkage offers a means to control the activity of this key enzyme of antisense strategy. An RNA oligonucleotide hybridized with a 50 -boronic acid-modified DNA oligonucleotide resisted degradation by RNase H but the activity could be switched back on by supplying an additional DNA oligonucleotide complementary to the downstream sequence of the RNA strand and featuring a 30 -ribose terminus. Esterification with the 30 -terminal ribose masked the 50 -boronic acid function and restored RNase H activity.407 Nuclease stability of oligonucleotides incorporating organomercury nucleobases has not been studied systematically but digestion with nuclease P1 was found to be sluggish and failed to proceed beyond dimer stage.121,122 This retardation could stem from Hg(II) coordination to a catalytically important mercapto group, as discussed above in the context of polymerase enzymes.

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Incorporation of carborane moieties makes oligonucleotides resistant to both 30 - and 50 -exonucleases, such as SVPD and CSPD.451–453 On the other hand, the ability of DNA oligonucleotide to induce RNase H catalyzed cleavage of a complementary RNA strand is not compromised by carborane functionalization.452 Steric shielding by the bulky carborane moiety has been proposed as a reason behind the stability toward exonucleases. Consistent with this proposal, carborane modification near the 50 - or 30 -terminus has been shown to specifically inhibit digestion by 50 - or 30 -exonucleases, respectively.206 In striking contrast to the increased nuclease stability of carborane-modified oligonucleotides, a recent study has revealed decreased stability toward SVPD upon incorporation of a ferracarborane complex.454 Facilitation of the cleavage was attributed to high affinity of the ferracarborane moiety for the enzyme (Kd ¼ 20 nM), promoting association of the oligonucleotide and the enzyme. The ferracarborane complex itself also exhibited a fair affinity for the respective unmodified oligonucleotide (Kd ¼ 5 mM) and, at sufficiently high concentration, could facilitate degradation by SVPD even when not covalently tethered to the oligonucleotide.

15.07.3.8.3

Affinity and selectivity of organometallic and organometalloid oligonucleotides for intracellular targets

The potential of metal coordination to promote hybridization of a therapeutic oligonucleotide with its intended target sequence was studied throughout the 1990s and 2000s on DNA and PNA oligonucleotides platinated at N3 of pyrimidine or N7 of purine bases or at a dedicated artificial Pt(II) coordination site.455–457 Sequence-specific interstrand cross-linking was demonstrated with both single-458–460 and double-stranded461 oligonucleotide targets but few follow-up studies with biological systems462 have been forthcoming. Recently, however, the idea was revisited by assessing the ability of organopalladium phosphorothioate oligonucleotides to induce splice-switching in human cervical cancer (HeLa Luc/705), human osteosarcoma (U-2 OS_705) and human liver (HuH7_705) cell lines expressing an aberrant version of the luciferase reporter gene.146 Restoration of correct splicing on treatment with the organopalladium oligonucleotide complementary to the aberrant splice site was observed in all three cell lines, with no marked toxicity. In the HeLa Luc/705 cell line, the splice-switching was modestly more efficient with the organopalladium oligonucleotide than with its unmodified counterpart, especially on naked delivery. The improved splice-switching activity could not, however, be unambiguously attributed to improved hybridization affinity, as the duplex stabilization observed with short oligonucleotides having a phosphate backbone145 was not reproduced with longer oligonucleotides having a phosphorothioate backbone.146 Nevertheless, the concept of therapeutic organometallic oligonucleotides appears sound although a better understanding of the origin of the observed modest enhancement in splice-switching ability is required. Carborane-modified oligonucleotides have been studied for their potential as small interfering RNAs (siRNAs) and, more recently, as antisense oligonucleotides (ASOs). The ability of the siRNAs to silence the gene coding for beta-site APP cleaving enzyme 1 (BACE1) in HeLa cells was essentially unchanged upon introduction of a carborane moiety to either the sense or the antisense strand or both.463 Some of the single-stranded carborane- or ferracarborane- modified antisense oligonucleotides, in turn, were more efficient in silencing the gene coding for epidermal growth factor receptor (EGFR) than their unmodified counterparts despite their slightly lower hybridization affinity.156,464 The origin of the silencing activity remains obscure but, at least in the case of the ferracarboranes, generation of reactive oxygen species may be involved. On the other hand, the dependence of silencing activity on the number and position of the carborane or ferracarborane moieties on the ASO paralleled that previously observed for the susceptibility of the complementary RNA strand to cleavage by RNase H,156,452 suggesting that activation of RNase H plays a key role.

15.07.3.8.4

Boron neutron capture therapy

Phosphodiester-linked oligomeric nido-carboranes have been studied as 10B-containing payload of immunoproteins for tumor-specific delivery in BNCT.465 Up to 90 10B atoms per immunoprotein molecule have been introduced in this way and, importantly, the resulting material was well-defined and homogeneous. Compared to the parent immunoprotein, the carborane immunoconjugates exhibited similar biological half-lives in mice but somewhat more pronounced accumulation in the liver and kidney. The carborane oligomers themselves were not taken up by mammalian cells but when introduced to the cytoplasm by microinjection they accumulated in the nucleus.466 The initial results on phosphodiester-linked carborane oligomers were encouraging but, 20 years later, their application in BNCT is yet to be demonstrated. The carborane- and metallacarborane-modified ASOs discussed above could also serve as carriers of 10B to cancer cells, especially when multiple boron clusters are incorporated.156,157 The combination of antisense activity against a gene overexpressed in cancer (such as the EGFR gene) and high 10B payload makes such dual-action oligonucleotides an interesting new class of therapeutic agents. However, while the antisense activity of carborane- and metallacarborane-modified oligonucleotides has been proven, the applicability to BNCT has not been tested yet.

15.07.4 Summary and outlook Complex formation between oligonucleotides and simple organometallics, such as methylmercury or arylmercury compounds or dimethyltin dichloride, takes place through coordination to the same donor atoms within nucleosides and nucleotides as with the respective metal salts. With organometallic compounds featuring large p-expansive ligands, non-coordinating interactions, notably intercalation, may become important or even dominant. The most extensively studied organometallic compounds in the context of binding to nucleic acids are those of the platinum group metals and gold and these compounds appear to exhibit a general

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preference for coordinating to guanine N7. Metallocene dichlorides, in turn, can coordinate to either the nucleobases or to the phosphate backbone depending on the hardness of the metal ion but the relevance of these interactions to the observed biological effect is not clear. Chemical synthesis of oligonucleotides bearing organometallic or organometalloid moieties is complicated by the limited stability of such moieties under the reaction conditions of the conventional phosphoramidite approach. Accordingly, most organometallic and organometalloid oligonucleotides described in the literature have been prepared by post-synthetic methods, such as direct metalation or conjugation in solution or on solid support. Efforts to develop strategies for the solid-phase synthesis of organometallic and organometalloid oligonucleotides are, however, underway and the range of structures that can be introduced in this way is continuously increasing. Finally, enzymatic polymerization provides access to longer oligonucleotides and in some cases can be used to circumvent the stability limitations of chemical synthesis. Surprisingly bulky modifications, such as polyoxometalates, can be introduced as long as they are placed in the major groove. Historically, covalently mercurated pyrimidine nucleobases have been used as affinity tags on nucleic acids but this method has largely been replaced by more modern approaches that do not require the use of heavy metals. Organomercury and organotin nucleosides have been employed as synthetic intermediates, notably in radioiodolabeling, and extension of these reactions to oligonucleotides seems feasible. Ligation of oligonucleotides bearing boronate and ribose termini and metal-mediated base pairing of organometallic nucleobase surrogates both exploit Lewis acid—base interactions for the formation of strong but reversible bonds, either along or across the double helix. Both approaches could find use in bio- as well as nanotechnological applications. The most common contemporary application of organometallic oligonucleotides is, however, the use of ferrocene-labeled oligonucleotides in various types of electrochemical sensors. Small molecular organometallic compounds have been extensively studied to discover new anticancer agents and recently the therapeutic potential of organometallic oligonucleotides has also started to attract attention. Organometallic modifications have been studied in the context of some of the main challenges faced by therapeutic oligonucleotides, including delivery, stability and affinity and the initial results have been encouraging.

Acknowledgments Funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 721613, from the Academy of Finland (decisions 286478 and 294008), from Finnish Cultural Foundation and from Turku University Foundation is gratefully acknowledged.

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15.08 Organometallic Receptors and Conjugates With Biomolecules in Bioorganometallic Chemistry Benjamin Neuditschkoa,b, Bernhard K Kepplerb, Christopher Gernera,c, and Samuel M Meier-Menchesa,b,c, aDepartment of Analytical Chemistry, University of Vienna, Vienna, Austria; bInstitute of Inorganic Chemistry, University of Vienna, Vienna, Austria; c Joint Metabolome Facility, University of Vienna and Medical University of Vienna, Vienna, Austria © 2022 Elsevier Ltd. All rights reserved.

15.08.1 15.08.2 15.08.2.1 15.08.2.2 15.08.3 15.08.3.1 15.08.3.2 15.08.3.3 15.08.4 15.08.4.1 15.08.4.1.1 15.08.4.1.2 15.08.4.1.3 15.08.4.1.4 15.08.4.1.5 15.08.4.1.6 15.08.4.1.7 15.08.4.2 15.08.4.2.1 15.08.4.2.2 15.08.4.2.3 15.08.4.2.4 15.08.4.2.5 15.08.5 References

Introduction Analytical techniques to assess the accumulation and distribution of organometallics In vitro In vivo Approaches to study organometallic-protein interactions Metalloproteomics Protein target identification Metabolomics and multi-omics approaches Organometallic conjugates with biomolecules Strategies to synthesize organometallic-peptidic conjugates Amide coupling Direct metalation of amino acids Sonogashira coupling Alkyne-azide coupling Maleimide-thiol coupling NHC coupling Miscellaneous Targeting strategies Blood Membrane receptors Cell penetrating peptides Subcellular targeting Miscellaneous Conclusions

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15.08.1 Introduction Metal complexes significantly improved the treatment of cancer patients. Especially, the platinum derivatives cisplatin, carboplatin and oxaliplatin are still successfully applied in the clinics for certain cancer types, including testicular, ovarian, head and neck, as well as colorectal cancer and represent a cornerstone of anticancer chemotherapy. Using cisplatin and carboplatin chemotherapy, the 10-year survival of patients presenting with testicular cancer increased to 95%.1 Oxaliplatin has recently been used in adjuvant FOLFOX4-regime to treat colorectal cancer,2 which is a combination treatment of this metal-based anticancer agent with folic acid and 5-fluorouracil. The mode of action of platinum anticancer agents typically relies on the formation of cytotoxic DNA-lesions, which lead to apoptosis of the cancer cells.3 Arsenic trioxide is another example of a clinically relevant metalloid anticancer agent and used in combination with all-trans retinoic acid to treat acute promyelocytic leukemia (APL).4–6 In contrast to platinum therapy, treatment with arsenic trioxide is performed at low doses over long time periods and does not represent a cytotoxic chemotherapy. The combination of arsenic trioxide with all-trans retinoic acid relieves the differentiation blockade7 in APL cancer cells, which leads to their differentiation into functional neutrophils and activates their physiological apoptosis program. Neutrophils are generally short-lived8 and therefore, primed for apoptosis as soon as they terminally differentiate. This combination therapy cures >90% of APL cancer patients, which is achieved by an efficient targeting of the PML-RARa fusion protein, the characteristic oncogenic driver of APL.7 Arsenic trioxide targets the PML moiety, while all-trans retinoic acid activates the RARa transcription factor. Encouraged by the clinical success of platinum(II) derivatives and arsenic trioxide, metals in medicine are intensively investigated and the related drug discovery programs cover many different transition metals, including titanium, gold, ruthenium, iron, iridium, rhodium, osmium, vanadium and copper to name a few.9,10 Next to coordination complexes, organometallic anticancer agents form a promising class of metals in medicine. Although several coordination metal compounds entered clinical trials, only a few organometallic drug candidates achieved such a state of development. Dichloridobis(Z5-cyclopentadienyl)titanium(IV) (titanocene dichloride, Chart 1) was among the first organometallics reported to display anticancer properties in vivo in 1979 by Köpf and Köpf-Maier.11 The structural similarity of the cis-dichlorometal moiety

Comprehensive Organometallic Chemistry IV

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Organometallic Receptors and Conjugates With Biomolecules in Bioorganometallic Chemistry

Chart 1 Chemical structures of clinically approved platinum and arsenic coordination complexes and experimentally investigated organometallic drug candidates. Ferroquine has finished clinical phase II trials as a promising treatment against Plasmodium falciparum (NCT02497612 and NCT03660839).

compared to cisplatin was believed to be responsible for such an effect. Titanocenes are cytotoxic agents, which induce apoptosis in cancer cells,12 possibly via coordinating to phosphate groups of nucleotides in DNA and inducing strand-breaks.13 It was shown that titanocene dichloride exhibits a complex solution chemistry.13,14 The chlorido ligands hydrolyze rapidly forming aquo-, hydroxo- and m-hydroxo-species, amongst others, eventually hydrolyzing the Cp-rings. The mixed aquo-hydroxo species seems to be the major product in unbuffered aqueous solution. Titanocene dichloride was investigated up to clinical phase II trials in patients with metastatic breast cancer15 and advanced renal-cell carcinoma,16 but showed no partial or complete responses. Thus, efficacy was low, but toxicity was generally acceptable. More recently, dichloridobis(Z5-(p-methoxybenzyl)cyclopentadienyl)titanium(IV) (titanocene Y) was identified as a promising second generation titanocene as it showed 100-fold higher cytotoxicity.17 Additionally, it showed superior tumor-inhibition in a renal cancer model in vivo compared to cisplatin18 and additionally, a clear tumor-inhibition in freshly explanted human breast cancer cells.19 The higher in vitro potency of titanocene Y compared to titanocene dichloride might translate into an improved efficacy in the clinical setting. Titanocene Y is subjected to a similar solution chemistry as compared to titanocene dichloride, but seems to induce apoptosis by chelating to the phosphodiester bond in DNA without inducing DNA backbone cleavage.14 Molybdenocenes20–22 are based on the same structural motif as titanocenes and extend the medicinal investigations of metallocenes. Several molybdenocenes were recently reported to exhibit promising antiproliferative activity in vitro and possessing distinct chemical properties in comparison to titanocene.23 Ferrocene represents a further metallocene motif, which is successfully incorporated into drug candidates and these were extensively investigated.24 Notable contributions to the use of ferrocenes for medicinal applications came from the lab of Jaouen and Vessières.25 In contrast to titanocenes and molybdenocenes, the ferrocene moiety is a pure sandwich compound and does not display labile chlorido ligands. It benefits from a reversible redox potential between FeII/FeIII, which is in the physiologically accessible range of 0.4 V.26 This redox activity was shown to increase reactive oxygen species (ROS) in cancer cells, which correlated to some degree with their cytotoxicity in a series of ferrocifen complexes. Ferrocifens are ferrocene-containing tamoxifen-analogs, in which a phenyl was substituted by ferrocene.25 Interestingly, a two electron-two proton reduction may lead to the formation of quinone methides that exert specific reactivities in cells, e.g., inhibition of thioredoxin reductase by nucleophilic addition to selenolate27 and this may be implicated in their mode of action. Despite their promising properties, these ferrocene-derived anticancer drug candidates did not progress to clinical trials so far. However, a ferrocene-containing derivative of chloroquine (i.e., ferroquine) was found to have superior antimalarial activity even in chloroquine resistant strains of Plasmodium falciparum and Plasmodium vivax.28 Ferroquine is investigated in a clinical phase II trial in patients with uncomplicated Plasmodium falciparum malaria in a combination treatment with artefenomel (NCT02497612 and NCT03660839). Results of these trials were received, but not yet published. Already in 1965, Rosenberg et al. reported the filament-forming effects in E. coli for [RuIII(NH3)4Cl(OH)]Cl, similarly to cisplatin.29 Thus, research into this compound class has grown steadily and was extensively reviewed.30–36 Initially, research focused on ruthenium(III) coordination complexes because of their synthetic availability and presuming a similar mode of action. These efforts resulted in the development of two lead structures, namely sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (NKP-1339, BOLD-100) and 1H-imidazoium trans-[tetrachloro(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)] (NAMI-A).33 The latter recently completed phase I/II clinical trials to treat non-small cell lung cancer patients in combination with gemcitabine, but the combination was less effective than gemcitabine alone and some adverse effects occurred emphasizing so far unrecognized off-targets.37,38 In contrast, BOLD-100 was shown to be safe39 and is currently undergoing a phase Ib clinical trial (NCT04421820).

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The mode of action of BOLD-100 is still debated, but seems to involve down-regulation of the glucose-regulated protein of 78 kDa (GRP78) and endoplasmic reticulum stress,40–43 probably mediated by direct interference with ribosomal constituents.44 Ruthenium(III) compounds are prodrugs, which require a reduction step, accompanied by ligand exchange reaction of the labile chlorido ligands in order to be activated and to exert their biological effects. This process was recognized as the “activation by reduction” hypothesis.45 The realization that ruthenium(II) is the active form of the otherwise largely substitution-inert ruthenium(III) complexes spurred efforts to directly prepare stable ruthenium(II) derivatives. Especially, N-heterocycles seemed to stabilize the lower oxidation state and the first series of stable ruthenium(II) was prepared by Reedijk et al., which were found to display and intriguing isomer-dependent cytotoxicity.46 Around 2001, Dyson et al.47 and Sadler et al.48 independently reported on organometallic ruthenium derivatives characterized by an Z6-arene ligand. The introduction of this class of “half-sandwich pianostool” complexes led to extensive investigations into organometallic ruthenium complexes as anticancer agents.33 Since ruthenium and osmium are in the same group of d-elements, interest in organometallic osmium congeners also grew. In a global attempt to construct in vitro structure-activity relationships, it turned out that the rate of hydrolysis, the charge of the organometallic compound or the pKa of the aqua-complex did not correlate with cytotoxicity in ovarian cancer lines.33 In contrast, the metal exerted a somewhat ambiguous effect on cytotoxic activity, while the use of kinetically inert chelating ligands was clearly favorable toward an improved cytotoxic potency. Loss of the chelating ligand was also associated with deactivation of organoruthenium compounds.49 While some examples of presumably inert and cytotoxic organometallics are known,50 ruthenium(arene) derivatives are generally prodrugs that require only a hydrolysis step for their activation and they exhibit intriguing binding preferences for their potential targets. In crystal soaking experiments with the nucleosome core particle, [RuCl2(Z6-p-cymene)(1,3,5-triaza-7-phosphaadamantane)] (RAPTA-C) and [RuCl(Z6-p-cymene)(1,2-ethylenediamine)]PF6 (RAED-C) were found to display distinct binding preferences (Chart 1, Fig. 1).51 While RAED-C coordinated to guanines of DNA, RAPTA-C was coordinated to surface exposed nucleophilic amino acids of the histone octamer. This exciting finding underlined the capacity of this class of anticancer metallodrugs to be fine-tuned with respect to their potential molecular interaction partners and eventually, with respect to modes of action. Thus, this compound class can be chemically tuned to target either DNA or proteins. Next to crystal-soaking experiments, a mass spectrometry-based approach was reported to delineate the protein vs DNA binding preference of organometallics.52,53 Evidence for the selective protein-targeting capability of organoruthenium derivatives was obtained by [chlorido(Z6-p-cymene)(N-fluorophenyl-2-pyridinecarbothioamide)ruthenium(II)] chloride (plecstatin-1).54,55 Plecstatin-1 was shown to interact with plectin, which is a cytolinker and scaffold protein, and consequently modulates the cytoarchitecture of cancer cells thereby affecting the migration and invasion of cancer cells.54 A structurally similar organo-osmium derivative [(Z6-p-cymene)Os(phenylazopyridineNMe2)I]+ (FY26) was found to be a highly potent cytotoxic agent in vitro probably based on redox modulation56 and rewiring of cancer metabolic processes.57 Even an organometallic platinum(II) bis-N-heterocyclic carbene (NHC) complex, [(bis-NHC)Pt(bt)] PF6, where Hbt ¼ 1-(3-hydroxybenzo-[b]thiophen-2-yl)ethenone (Chart 1), was proven to engage with a protein target as the main mode of action, namely asparagine synthetase.58 Recent developments of the class of half-sandwich piano-stool organometallics of different metals also extended into catalysis in cells.59,60

Fig. 1 Upon activation by hydrolysis, the ruthenium(arene) derivatives RAED-C and RAPTA-C exhibit distinct binding preferences for oligonucleotides and histone amino acid sequences, respectively. This supports the possibility to fine-tune their binding preferences by the choice of the ligand set for ruthenium(arene) derivatives. Crystal structures were obtained from the RCSB protein data bank with identifiers 4kgc (left) and 3mnn (right). The organometallic moieties are depicted in dark space-fill models with arrows.

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Since the clinical approval of auranofin to treat rheumatoid arthritis, gold-based anticancer agents also witnessed considerable progress in recent years.61–63 The gold(I) coordination compound auranofin is currently being repurposed for cancer treatment, e.g., ovarian cancer (NCT03456700).64,65 Gold(III) coordination complexes were found to target the proteasome,66 aquaporins67 and may induce ROS stress. Recent developments of organometallic, cyclometalated gold(III) complexes pointed toward a selective inhibition of the zinc finger domain of PARP-1, which detects DNA strand breaks.68 Moreover, cyclometalated gold(III) derivatives were found useful for cysteine arylation reactions.69,70 Finally, NHCs were used to stabilize gold(I) and several organometallic gold(I) NHCs and bis-NHCs were investigated as anticancer agents.61,71,72 Gold NHC complexes were found to be potent inhibitors of thioredoxin reductase,73–75 while the class of gold bis-NHC organometallics based on xanthine-ligands emerged as promising telomeric G-quadruplex stabilizers,72 which are potentially affecting the replicative immortality of cancer cells.76 Research efforts about therapeutically relevant organometallics are aimed at translating these compounds to the clinic, which is a considerable task.77 The drug candidate subsequently also needs to be successful in the clinical setting. Thus, it may be worthwhile to concisely reconsider the key milestones of preclinical and clinical investigations. According to the guideline S9 of the international council of harmonization (ICH) the preclinical investigations aim at identifying the pharmacological properties and the toxicological profile of a drug candidate.78 Additionally, a safe initial dose needs to be established for first human exposure. Clinical trials then rigorously study the drug action in humans with respect to safety and efficacy.79 The drug candidate must be safe, i.e. not causing extensive adverse effects, and be effective with respect to the clinical endpoints. In the context of cancer therapy, the most commonly accepted endpoints are overall survival, progression-free survival or disease-free survival.80 Past clinical investigations of organometallic compounds showed either adverse effects or lacked efficacy (vide infra). Detailed knowledge about the molecular mode of action of a drug, including targets and off-targets may thus aid in delineating safety aspects from preclinical to clinical settings. Furthermore, the selective accumulation of drug candidates at specific sites of interest may increase drug efficacy. Recent evidence emerged that representatives of this compound class can be exquisitely selective with respect to their targets, which contrasts the longstanding paradigm of organometallics being toxic, unspecific and poorly controllable. Thus, this chapter covers recent developments to characterize in detail the target preference (i.e., biological receptors) and modes of action of organometallic drug candidates after discussing analytical approaches to study their accumulation and distribution. Moreover, bioconjugation strategies are highlighted that enable the targeted accumulation of the organometallic payload at specific (sub-)cellular locations.

15.08.2 Analytical techniques to assess the accumulation and distribution of organometallics 15.08.2.1 In vitro Inductively coupled plasma mass spectrometry (ICP-MS) is a well-suited element-specific technique to study the cellular accumulation and distribution of organometallic therapeutics.81 ICP-MS uses an inductively coupled plasma to atomize and ionize the elements in a sample. It creates mostly singly-charged atoms, whose m/z-ratio is determined by the mass analyzer and can be used for a variety of metallomics investigations.82 Since most metals used in medicinal inorganic drug discovery (e.g., Pt, Ag, Ru or Os etc.) are not endogenous or only found in trace amounts in living cells, the abundance of these metals are indications of drug exposure. In a typical cellular accumulation experiment, cells are treated with the organometallic of interest at a given concentration and time period, after which they are harvested and digested with strong and often oxidizing acids. The amount of metal is then determined by ICP-MS. Usually, the results are normalized to cell number or protein content in order to account for potential reduction of proliferation or induction of apoptosis depending on the treatment. This approach is now widely applied to study the accumulation of medicinally relevant metals in vitro33,81,82 in time- or concentration-dependent manners.71,83 Of note, Ballesta et al. combined this method with mathematical modeling to investigate the time-dependent uptake mechanism of the organo-osmium compound FY26 in human ovarian cancer cells (A2780).84 Over the course of 72 h, they determined the osmium concentration in the cells treated at IC50 values. They could show a consistent accumulation of Os within the first 24 h followed by a gradual decrease within the next 48 h. Especially the decrease of Os was investigated more closely. When removing the drug from the medium after 24 h the reduction of intracellular Os was very similar to the original experiment. A further investigation of temperature dependency showed that with lower temperatures the uptake was considerably slower. With this information they formulated two hypotheses: The “enhanced efflux” model suggested an enhanced excretion of the drug, while the “reduced uptake” model suggested a reduced cellular accumulation. The calculations showed that the “reduced uptake” model better resembled the experimental data and led them to conclude that FY26 probably affected its own uptake after passing a certain drug concentration threshold inside the cells. The kinetic models of the drug uptake allowed valuable insights into the uptake process and its inhibition, which may lead to important considerations concerning possible resistance mechanisms.84 It may be noted that in contrast to most other transition metals, Os is sensitive toward oxidative digestion and prone to form volatile OsO4 under these conditions, which may impact on the accuracy of the analysis.85 This may be circumvented by using an appropriate stabilizing solution under reductive conditions.86,87 Besides studying accumulation, the intracellular distribution of organometallics reveals additional information about the fate of the drug candidate. This is particularly relevant when a drug candidate shows target selectivity for a biomolecule that is expressed only in specific intracellular compartments. For example, a drug targeting a specific nuclear protein or DNA ought to accumulate in

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the nucleus. In principle, ICP-MS can be used to study the intracellular distribution of organometallics in vitro either via subcellular fractionation protocols or by subcellular fractionation followed by chromatographic separation (e.g., size-exclusion chromatography).88,89 Of note, the interaction between the drug and the target ought to be sufficiently strong to reduce potential ligand scrambling during sample preparation or separation. The former approach can be used to quantify the metal content in subcellular fractions, such as organelles, cytoplasm or nucleus.88 For example, it was shown that RAPTA-T predominantly accumulated in the particulate fraction in A2780 cells, while it accumulated more strongly in the cytosolic and nuclear fractions in the cisplatin-resistant cell line A2780cisR. Tian et al. reported the subcellular distribution of different auranofin-related complexes between the nuclear, cytosolic, mitochondrial, and membrane fraction containing endoplasmic reticulum, among which was a gold NHC derivative.83 They witnessed a much slower accumulation of the gold NHC organometallic with respect to the other gold complexes. This latter approach involves chromatography as an additional dimension of information and may reveal the binding specificity of organometallics to proteins residing in the respective subcellular fractions.88 It was shown that RAPTA-T favored binding to large proteins in the nuclear fraction of A2780 cancer cells, while in the cisplatin-resistant A2780cisR, the binding pattern shifted to favor low mass proteins. Next to ICP-MS techniques, atomic absorption spectroscopy (AAS) was also used to determine the cellular uptake of organometallics.90 Recent developments in laser ablation (LA) ICP-MS with respect to improved resolution and speed of acquisition enabled studying the distribution of organometallics in 3D cell culture models, i.e. tumor spheroids.81 Here, plecstatin-1 was shown to distribute specifically to the periphery of HT-29 and HCT116 tumor spheroids.91 Nano-scale secondary ion mass spectrometry (NanoSIMS) may also be used to map the subcellular distribution of elements with unparalleled resolution.92,93 Platinum-94 and gold-based compounds95 can be detected with high sensitivity using this method, while this is more challenging for (organometallic) ruthenium derivatives.96 Finally, X-ray fluorescence microscopy (XFM)90 measures element-specific X-ray fluorescence emissions that are produced from core-shell transitions.97 XFM benefits from sufficient resolution and allows to study the cellular accumulation and intracellular distribution of metal-based drugs98 and organometallics, including tamoxifen-derivatives,99 half-sandwich ruthenium and osmium NHCs,100 FY26,101 as well as the rhenium carbonyl compound fac-[Re(CO)3(dmphen) (para-iodobenzeneisonitrile)]+, where dmphen ¼ 2,9-dimethyl-1,10-phenanthroline (I-TRIP).102 The latter allowed to study the co-localization of Re and iodide intracellularly and suggested that the complex remained largely intact upon entering the cells. Finally, an organometallic Re-carbonyl moiety was used as a multimodal probe to study the colocalization with manganese(II) superoxide dismutase mimics by XFM.103

15.08.2.2 In vivo Only a handful of therapeutically relevant organometallics have been investigated in vivo to date (cf. introduction), with most of the recent examples being either organometallic metal(arene)33 or rhenium carbonyl derivatives.104–106 However, next to the main read-outs of tumor-inhibition or survival, pharmacokinetic aspects are of relevance and might be considered as part of the experimental design. In fact, the in vivo distribution of the sandwich ruthenocene-melittin bioconjugate was investigated.107 After intraperitoneal administration, the bioconjugate distributed mainly to the liver, but also to the kidney and spleen as observed by ICP-MS analysis. Furthermore, investigations of blood of treated mice by ICP-MS give valuable insight into the distribution of organometals in this biofluid. While oxaliplatin and other platinum(IV) drugs tend to accumulate in blood cells108,109 plecstatin-1 and its osmium-analog featured higher abundances in the serum fraction.89 Depending on the route of administration, this may give an indication about the bioavailability of the administered organometallic. Additional analysis of the serum fraction by SEC-ICP-MS showed that plecstatin-1 and its osmium derivative were quickly bound to the serum album/transferrin fraction as well as to immunoglobulins in treated mice and in human plasma ex vivo. The distribution of these organometallic drug candidates among different organs was further investigated by ICP-MS in tumor-bearing mice treated with a single dose.87 Tissues of liver, kidney, lung, muscle and tumor were excised from the treated mice and split into two pieces: one was used to determine the total amount of Ru and Os, while the other was cryo-sectioned into 20 mm thick slices to investigate the distribution of the organometallics in the organs by LA-ICP-MS. The drug candidates seemed to accumulate in the kidney and liver. Muscle tissue did not reveal any significant accumulation. LA-ICP-MS imaging of the cryo-cut tissue slices revealed the spatial distribution of Ru and Os. Plecstatin-1 and the Os analog showed homogenous distribution in the liver, muscle and tumor. Only in the kidney the metal concentration was higher in the cortex compared to the medulla, which was also previously found for other metal-based drugs.110 Mass spectrometry for imaging of metallodrugs currently develops considerably into a direction that may allow investigations into structurally complex and heterogenous solid tumor tissues in the near future.81 Elucidating the coordination environment of metal-based therapeutics upon administration in vivo is of particular interest. To date, only a few reports were published among which was a case study about BOLD-100 in a SW480 mouse model.111 In this study, X-ray near-edge absorption spectroscopy (XANES) of tissue samples was performed and revealed that ruthenium was present in +III oxidation state, while at least one of the four chlorido ligands remained coordinated. X-ray absorption spectroscopy holds great promise in elucidating coordination environments of organometallics under relevant biological conditions.112,113

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15.08.3 Approaches to study organometallic-protein interactions Elucidating the molecular mechanism of action of metal-based therapeutics, including organometallics, is a major step in their early development, especially for emerging candidates (e.g., anti-migratory therapeutics).114 A detailed insight into the molecular mechanism of action may also be useful to guide patient selection and stratification strategies in clinical trials. The transcriptome and the proteome can provide comprehensive insight into the perturbation of cellular systems upon treatment with drug candidates. Gene expression analysis at the level of mRNA is highly sensitive, while the analysis of the proteome can include post-translational modifications and other mechanisms beyond translational control. In general, the correlation between the two levels is not clear-cut.115 Gene expression analysis has been employed on several occasions to investigate organometallic anticancer agents,57,116 and also in conjunction with cytotoxicity data of the NCI-60 screen.117,118

15.08.3.1 Metalloproteomics About 90% of the known drug targets are proteins.119 Consequently, proteomics represents a powerful tool to investigate organometallic drug candidates in the cellular context.120 Importantly, besides the analysis of drug mode of action, proteomics also allows the identification of potential targets.120 The investigation of the entirety of proteins in a biological system that are correlated or influenced by metal(-loid)s is termed metalloproteomics.121 This originally included approaches to study metalloproteins and their changes in time and space,122 but also includes the analysis of metal-binding proteins, metalloproteins or the effects or targets of metal-based drugs on the proteome121 and represents the intersection of proteomics and metallomics.82 Lately, the study of molecular mechanisms of organometallic drug candidates has been intensified due to the improvements in MS-based proteomics techniques.120,121,123–125 Proteomic response profiling uses a comparative approach to gather information about drug effects and mechanisms of action in vitro. Ultimately, it is aimed to compare the abundances of identified proteins in untreated cells to treated cells in order to obtain pathway information about the drug-induced perturbation. The experimental workflow typically involves a suitable cell culture model that is sensitive or resistant toward the drug of interest, meaning the cells induce a biological or morphological change at relatively low concentrations (Fig. 2). The concentration that inhibits cell growth by 50% (IC50) is determined by a cell viability assay prior to proteomic response profiling. Concentrations are usually chosen to be below IC50 concentrations. This assures that the cellular response to the drugs can be characterized and cell death signals (e.g., by apoptosis) are not overrepresented in the proteomic data. In order to be able to calculate significant protein expression changes,

Fig. 2 The figure displays the workflows for either gel-based (A) or gel-free (B) proteomic approaches. (A) Gel-based approaches include protein separation according to 2D gel electrophoresis. Protein spots are individually digested and analyzed by peptide mass fingerprinting. (B) Protein samples are digested into peptide mixtures using gel-free approaches, which are subsequently analyzed by nHPLC-MS/MS. Bioinformatic analysis then allows for protein identification and quantification.

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at least three biological replicates of each condition are carried out. After a given incubation time the cells are lysed and processed for proteomic analysis. While the analysis of whole cell lysates is the fastest method, the cells can also be fractioned (vide supra). Fractionation into cytoplasmic (CYT) and nuclear fraction (NE) can be achieved by cell lysis with isotonic buffers and simple successive centrifugation steps.123 Sample preparation procedures can also be adapted to purify other cellular compartments, including membrane fractions, mitochondria and endoplasmic reticulum (ER) largely using density gradients and ultracentrifugation.83 More extensive fractionation reduces the complexity of the sample and typically allows for a higher number of protein identifications, but increases acquisition time. Fractionation procedures also increase the time and complexity of sample preparations, which might increase the experimental variation and thus, reduce the number of significantly regulated proteins if not conducted carefully enough. The proteomic analysis can then be performed according to gel-based or gel-free approaches, both of which typically include peptide analysis by MS.120,121,125 The former relies on 2D-electrophoretic separation of the protein fraction of a sample (Fig. 2A). Individual protein spots are then tryptically digested and analyzed by peptide mass fingerprinting to identify the protein.125 Changes in gene expression are obtained by comparing the intensity of the protein spots under differential conditions, which can be used for pathway analysis, amongst others. Gel-based approaches have been especially applied to study a number of organometallic gold derivatives, including [(bipydmb–H)Au(OH)][PF6], where bipydmb ¼ 6-(1,1-dimethylbenzyl)-2,20 -bipyridine) (Aubipyc), as well as the gold NHC complexes Au(NHC1)Cl and [Au(NHC1)2]PF6, where NHC1 ¼ 1-butyl-3-methyl-imidazole2-ylidene.125 For this purpose, dedicated software for protein quantification was employed and the comparison of the three organometallic compounds revealed very distinct drug effects in A2780 cancer cells, including impairment of glucose metabolism for all three gold derivatives. Using gel-free proteomic analysis, protein fractions are tryptically digested and the produced peptide mixtures are separated by liquid chromatography (LC) and directly analyzed by high-resolution MS instruments (Fig. 2B).123 MS-based data acquisition consists of full mass scans, which identify peptides according to their accurate mass and associated tandem mass spectra (MS/MS). The MS/MS spectra reveal information about the peptide sequence.126 Nano-flow high performance liquid chromatography (nHPLC) coupled to MS further improves the sensitivity of the analysis.127 Bioinformatic tools are then used to search the mass spectra for information about the peptide sequences and annotate the identified tandem mass spectra to protein sequences resulting in intensity values (full scan) of all the detected peptides in the chromatographic run, which are further compiled to protein abundance. Bioinformatic calculations are absolutely required since already in 2017 one proteomic nHPLC-MS/MS run acquired five mass spectra per second amounting to 30’000 MS/MS spectra per run, which converge to roughly 5’000 proteins.123 Today, the latest mass spectrometers achieve theoretically up to 100 MS/MS per second.128 Differential analysis of untreated and treated conditions allows for calculating significantly regulated proteins and for further pathway analysis, for example by gene enrichment analysis using gene ontology (GO). This approach is called label-free quantification (LFQ) if the proteomic analysis does not involve any labeling of peptides with stable isotopes. The different quantification strategies are beyond the scope of this chapter and are expertly discussed elsewhere.126,129 The following paragraphs focus on different metalloproteomic techniques to study organometallic therapeutics using gel-free methods. Multidimensional protein identification technology (MudPIT) was one of the initial MS based methods used for proteomic response profiling. In 2007, it was used to investigate the protein interactions of [(Z6-p-cymene)RuCl2(DMSO)] in E. coli.130 The authors were even able to identify peptides that formed stable complexes with Ru, probably by multi-dentate binding to amino acids. This was facilitated by the isotopic pattern of the metal. The identified binding sites gave indications about the interactions of the metal ion of the organometallic complex in the biological system. The ruthenated cold-shock protein scpC and DNA helicase dinG indicated them as direct effectors in the antiproliferative effect of the drug. Further analysis of the protein abundances upon treatment showed the upregulation of stress-regulated proteins such as ppiD, osmY and sucC. Another study investigating the organometallic RAPTA-T in A2780 cells was able to show 25 significantly up- and down-regulated proteins upon treatment.88 Especially, the down-regulation of 14 histones indicated an activation of DNA damage signaling apparatus and the down-regulation of vimentin was correlated to the known antimetastatic effect of the drug candidate. Although MudPIT showed promise in the analysis of therapeutically relevant organometallics, it was not further pursued. A comparison to other, already established drug mechanisms would allow in principle a gauge for potential targets. Functional identification of targets by expression proteomics (FITExP) as an application of this principle was used to investigate RAPTA-T and an ethacrynic acid-derivative, named RAPTA-EA.131 This approach was able to confirm the anti-metastatic and anti-tumorigenic effects of RAPTA-T as well as a regulatory effect of RAPTA-EA on oxidative stress. While this technique does not allow an assignment of a specific drug target per se, it gives valuable insight into the ongoing proteomic changes that occur during the treatment. Besides the specific cellular effects of organometallics that can be identified with this method there are recurring mechanisms that seem to accompany many treatments even beyond metal-based drugs. For example, the induction of oxidative stress is often observed. Upregulation of heme oxygenase 1 (HMOX1), a detoxifying enzyme triggered upon oxidative stress through activation of the Nrf2-Keap1 pathway is often observed using proteomics.71,123,131–133 While an independent verification of oxidative stress in vitro through fluorescence microscopy (e.g., MitoSOX) or metabolomic measurements (e.g., glutathione) is well advised, the regulation of HMOX1 can give first insight into a possible oxidative mechanism accompanying drug effects of organometallics. Furthermore, the regulation of HMOX1 may also serve as a biological proxy for whether a drug of interest would accumulate in cells. A study involving LFQ proteomics and the organometallic Au(I) bis-NHC complex [Au(9-methylcaffeine-8-ylidene)2]+ (AuTMX2) was recently reported by us using the sensitive ovarian cancer cell line A2780.71 AuTMX2 has been previously shown to be a selective and potent stabilizer of telomeric G-quadruplexes.76,134,135 Cellular accumulation studies using ICP-MS were employed to confirm that Au distributed to the nucleus and was bound to DNA in living cells. Analysis of the telomere length in

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A2780 cancer cells in a concentration-dependent manner suggested a trend of telomere length reduction upon treating the cells with AuTMX2, suggesting that AuTMX2 may indeed represent a potent stabilizer of telomeric G4 in the cellular context if sufficient intracellular concentrations can be obtained. Moreover, the drug candidate was further shown to perturb telomere-associated proteins upon treatment in A2780 cells further supporting telomeric targeting. In fact, LFQ-based proteomic analysis suggested that AuTMX2 acted via multiple pathways, including telomeric perturbations, actin-related proteins, stress-response and the proteasome. The induction of reactive oxygen species (ROS) by 20 ,70 -dichlorofluorescin diacetate (DCFDA) supported oxidative stress upon treatment with AuTMX2. Moreover, several stress response proteins that are target genes of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2, NFE2L2) were significantly upregulated. Also, intracellular glutathione levels and the ratio between its reduced and oxidized form were elevated as signs of intracellular oxidative stress. Finally, the formation of actin stress bundles using fluorescence microscopy connected the Nrf2-stress response to morphological changes in the cancer cells. Overall, the proteomic response profiling underlined the telomere-targeting capability of AuTMX2 and indicated unexpected additional drug effects, especially relating to the Nrf2-stress response and actin cytoskeleton.

15.08.3.2 Protein target identification While proteomic response profiling gives valuable information about the cellular response to the treatment with a therapeutically relevant organometallic, it can hardly reveal the actual protein target of the organometallic drug candidates. Several mass spectrometry-based approaches have been recently established and successfully used to identify potential targets of organometallics in vitro.120,121 Research in this direction was initiated among others by a study in 2001, that evaluated the protein targets of cisplatin in a bacterial model.136 Therein, E. coli were treated with cisplatin and the extracted protein fraction was separated by 1D gel electrophoresis. Then, the gel lanes were analyzed by LA-ICP-MS with respect to their platinum content and the area with the highest platinum concentration was processed for peptide mass fingerprinting. Outer Membrane Protein A (OmpA) was identified as the most abundant protein and indicated this protein as one of the major interaction partners of cisplatin in this system. Sun and co-workers expanded this methodology to capillary electrophoresis coupled to ICP-MS to identify protein targets of bismuth subcitrate (CBS). CBS is an anti-ulcer agent used in the therapy against Helicobacter pylori.137 Cell lysates of H. pylori treated with CBS were separated by electrophoresis and the eluate was split in two fractions. One was used for online ICP-MS analysis while the other was fractionated and collected. The latter fractions were then subjected to MALDI-TOF-MS peptide mass fingerprinting to identify the proteins eluting in conjunction with high Bi abundance. This allowed the identification of seven proteins with significant amounts of Bi, among which were the subunits of urease UreA and UreB. Bismuth antimicrobial therapeutics were recently shown by the same group to be potent metallo-b-lactamase inhibitors.138 The binding preferences of RAPTA-C toward human serum albumin (HSA) and human transferrin (hTf ) was also investigated in comparison to cisplatin in a model system.139 The analysis revealed that RAPTA-C only binds to HSA while cisplatin is found bound to both serum proteins. Target identification based on metalloproteomic techniques represents the latest addition to the analytical arsenal for investigating organometallics. Target identification experiments in this context typically rely on affinity purification strategies, which are also termed drug pull-downs or chemical proteomics.54,120,140 Pull-down experiments often require to chemically modify the drug of interest to allow for immobilization. The modification often includes a linker and a functional moiety for selective conjugation to the solid support. Of course, this modification should only minimally alter the biological activity of the parent drug, and several derivatives may be prepared by attaching the linker at different positions and by using different linker types. Polyethylene glycol (PEG) is a commonly used linker due to its flexibility, stability and hydrophilicity. Prior knowledge about the speciation141 of the organometallic drug candidate is valuable, but the modified drug destined for immobilization might require additional verification in terms of biological properties and stability, especially in comparison to the parent drug. A recurring criticism of pull-down experiments is the unknown impact of non-selective interactions that might occur from hydrophobic interactions or from pulling down protein complexes. In fact, this led to the design of experimental setups that probe the target spectrum of a drug of interest under differential conditions.142 For this purpose, the cell lysates used for the pull-down experiment processed differentially (Fig. 3A). Half of the samples are pre-treated with the parent compound before the pull-down step, while the other half is used without pre-treatment (normal). The binding sites of the drug of interest are saturated in the pre-treated condition and consequently, proteins featuring such a binding site will not be able to interact with the immobilized drug. Consequently, the pull-down of this pre-treated condition gives rise to a collection of purely non-specific binding partners. The normal condition allows for the selective and the non-selective interactions to occur. The two conditions are then independently analyzed by LFQ proteomics. The collection of selective interaction partners can be obtained by subtracting the pre-treated from the normal condition, which can be ranked according to extent of enrichment and significance. Up to a thousand proteins may be identified in the pull-down experiments in such an approach. Drug pull-downs have been performed for the organoruthenium derivatives RAPTA-C143 and plecstatin-1.54,140 Importantly, the pull-down experiments might return a considerable number of potential interaction partners, which may hamper subsequent validation attempts. The decision process might be facilitated by integrating drug pull-down experiments with proteomic response profiling in a “target-response profile” (Fig. 3A). While the pull-down reveals potential binding partners, the response profiling shows the cellular response to the drug treatment. If a target is a true target, then its modulation will perturb proteins, which are functionally connected to it. Constructing a target-response network44,54 thus causally links potential targets to their observed perturbations in the cellular system on the level of the proteome. The proof-of-concept for such an approach was shown for plecstatin-1.54,140 In this case, the organometallic was modified at the arene with a PEG linker and biotin for performing the

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Fig. 3 Target identification of therapeutically relevant organometallics can be performed by target-response profiling (A). Combining pull-down experiments with proteomic response profiling causally links potential binding partners to cellular perturbations. The target spectra of plecstatin-2 and Ru-OH-1 are shown in (B) depicting the fold-change of enrichment (y-axis) together with the associated significance (x-axis), the intensity as the bubble size and the color representing the specific binding probability according to the CRAPome database. Exchanging the hydrogen bond acceptor (–F) to a hydrogen bond donor (–OH) drastically reduced the selectivity for plectin and altered the entire target spectrum. Adapted from Meier-Menches, S.M.; Zappe, K.; Bileck, A.; Kreutz, D.; Tahir, A.; Cichna-Markl, M.; Gerner, C., Metallomics, 2019, 11(1), 118–127, an open access article distributed under terms of the Creative Commons CC BY license. (C) Chemical structure of the cyclometalated gold(III) NHC photoaffinity probe.

pull-down on streptavidin-coated beads (Fig. 3A). Furthermore, the binding specificity of each potential binding partner was calculated according to the CRAPome database,144 which is a collection of known unspecific binding partners. Together with the fold-change of enrichment and the significance of the enrichment (p-value), the entire target spectrum of plecstatin-2 was obtained (Fig. 3B). Strikingly, it showed an unexpected target selectivity of this Ru organometallic mainly to two protein targets from a whole

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cell lysate, including plectin (PLEC) and outer dense fiber protein 2 (ODF2). The target-response network suggested a stronger influence on plectin and only minor changes attributable to targeting ODF2. Plectin targeting was then validated in a plectin knock-out model in keratinocytes and its anti-invasive properties were confirmed in 3D tumor spheroids.54 Most organometallic therapeutics undergo modifications in biological systems either through active metabolism or ligand exchange reactions. Ligand exchange reactions are required to activate organometallic prodrugs, but complicate the target identification process since the target specificity of the prodrug can be significantly different from the activated or metabolized drug. Ligand exchange reactions are subjected to kinetic rate constants and can thus be controlled in time. The effect of prodrug activation was investigated for plecstatin-2, which requires a hydrolysis step of one chloride ligand to be activated.140 By choosing appropriate incubation periods during the pull-down experiments, the target spectra of the prodrug and the activated drug were obtained, respectively. It was revealed that prodrug displayed low selectivity for plectin and generally a lower specificity. In contrast, the activated organometallic featured an exquisite selectivity for plectin. This finding suggested that the interaction of plecstatin-2 with plectin must be mediated by a coordination bond in addition to electrostatic and secondary interactions. After incubating for 19 h plecstatin-2 lost this specificity completely, which was correlated to the possibility of arene loss and thus, deactivation of the probe. Further investigations showed that the hydrogen bond acceptor (–F) in para-position of the phenyl ring is a major contributor to the target specificity. Exchanging the fluoride with a hydrogen donor (–OH) nearly completely abolished the selectivity for plectin (Fig. 3B).140 These investigations underline that insight into the speciation of organometallics prior to extensive pull-down experiments is relevant for choosing the correct time point and capturing the target spectrum of the organometallic species of interest. The organometallic-receptor interactions can also be comprehensively investigated by photoaffinity probes.145 For this purpose, the organometallic is combined with a photoaffinity linker and a reporter tag. Photoreactive moieties include phenylazides, diazirines and benzophenones.145,146 In contrast to the pull-down experiments with biotin-streptavidin, photoaffinity probes can be administered not only to cell lysates, but also to living cell cultures due to the formation of a covalent bond between probe and receptor. The probe is typically activated by irradiation with UV light to create highly reactive intermediates, which subsequently covalently bind to the nearest available reaction partner. Ideally, the photoaffinity group is stable in the dark and under biological conditions and once irradiated the covalent bonds formed are stable throughout the complete sample preparation process. The probe-receptor conjugate may be pulled-down from the cell lysate by the biotin-streptavidin approach. Alternatively, fluorescent tags may allow to identify the labeled proteins upon separation by 2D-gel electrophoresis.121,146,147 Another popular method uses click chemistry to couple an alkyne group to an azide-linked reporter tag.146 Then, the proteins are identified by mass spectrometry either through peptide mass fingerprinting or shotgun proteomics. Fung et al. investigated a cyclometalated gold(III) complex (I) containing a NHC ligand by a photoactivity protocol.147 By modifying the carbene with a diazirine (Fig. 3C) or benzophenone photoaffinity unit and a clickable alkyne reporter tag, the resulting cyclometalated gold(III) was used for target identification. Thereby, the alkyne was coupled to a fluorescent dye, which allowed to scan the target selectivity of the cyclometallated gold(III) in a 2D gel. This revealed a multi-targeted nature of the organometallic, which was found to target HSP60 and vimentin amongst others, and these targets were further validated. The temperature at which a protein precipitates is characteristic for each protein and can change significantly upon ligand binding. This principle is employed in thermal proteome profiling (TPP) to identify protein-ligand interactions in living cells.148,149 Treated cells are lysed and split into several aliquots (Fig. 4). Each aliquot is heated to a different temperature typically in the range of 37–67  C.148 A given temperature will cause certain proteins to denature, aggregate and precipitate, which are removed by centrifugation. The remaining soluble proteins are digested and analyzed by LC-MS. To parallelize the workflow the peptides/ proteins of the different temperature samples can be labeled with stable isotopes using tandem mass tags (TMT).149 This thermal profiling generates melting curves in the MS/MS spectra of each detected peptide/protein. Drug-target interactions may stabilize or destabilize against denaturation leading to upward or down-ward shifts of the melting curves, respectively. Recently, He et al. investigated a platinum(II) bis-NHC complex by TPP and were able to show significantly higher thermal stability of asparagine synthetase (ASNS), ferrochelatase, and glutathione S-transferase Mu 1.58 For ASNS even a dose-dependent stabilization at 54  C could be observed. The protein target was further validated on a metabolic level. Specifically, the asparagine level was significantly decreased to about 50% upon treatment with [(bis-NHC)Pt(bt)]PF6 (Chart 1). The treated cells showed strong correlations with the exogenous asparagine levels that were supplemented to the cells under culture conditions. While low asparagine levels decreased cell viability, elevated levels were able to restore cell viability. Furthermore, the N-terminus of ASNS contains an enzymatically relevant cysteine and the Pt(II) complex was shown to directly interact with this amino acid. Upon exchange with alanine the organometallic moiety did not bind to the peptide, as observed by mass spectrometry.

15.08.3.3 Metabolomics and multi-omics approaches Proteins form an emerging target class of investigational organometallic therapeutics, but the drug effects are not limited to those, as exemplarily evidenced for [(bis-NHC)Pt(bt)]PF6.58 Even for cisplatin, a classical DNA-targeted agent, a correlation of fatty-acid metabolism and resistance was recently observed.150 These examples underline the importance of metabolomic investigations for elucidating molecular mechanisms of metal-based drugs and organometallics. The general high variability and fast fluxes of metabolites may entail certain methodical pitfalls.151 Only few publications are available that explore the impact of organometallic therapeutics (and even coordination compounds) on the metabolome. We found interest in the method developed by Rusz et al. to identify and partially quantify 58 metabolites in a 3D tumor cell culture model. They investigated the effect of treatment with the

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Fig. 4 Thermal profiling of the proteome assesses the solubility of proteins depending on their state of denaturation under differential conditions, e.g. cells are treated with a drug of interest. Aliquots of the sample are heated to different degrees creating a thermal gradient of protein denaturation and thus, precipitation. Interaction of a drug of interest with a target may stabilize or destabilize this target, thus affecting the melting curve.

coordination compounds BOLD-100 and oxaliplatin and were able to detect significant metabolomic alterations that went in line with expected molecular mechanisms.152 The same group reported a comprehensive investigation of the metabolome of oxaliplatin resistant and sensitive variants of HCT116.153 Not only did the sensitive and resistant cell lines differ significantly in their metabolite profile with respect to energy metabolism, sulfur metabolism, fatty acid metabolism and polyamines but also in their response to oxaliplatin treatment. Wang et al. performed an untargeted gas chromatography MS (GC-MS) metabolomics analysis to investigate two Re(I) tricarbonyl complexes in a HeLa cell culture model.154 They were able to identify 219 metabolites and study the differential responses of the cells to the treatments. KEGG pathway analysis revealed amino acid-related pathways and glutathione metabolism as their main changes triggered by the organometallic compound. They further confirmed altered GSH levels using a commercial kit, which supported the induction by oxidative stress. To the best of our knowledge, combinations of multiple post-genomic analysis strategies (i.e., multi-omics) were not reported to date to investigate the mechanism of action of investigative organometallic therapeutics. Methodological advances may pave the way for such experimental designs and help elucidate the biological effects of those organometallics for which distinct modes of action remain elusive. Such approaches could adapt the recently published multi-omics approach to determine part of the mode of action of the ruthenium coordination compound BOLD-100.44 Here, a combination of proteomic target-response profiling and transcriptomic analysis revealed the ribosomal proteins RPL10 and RPL24, but also the transcription factor GTF2I as potential interaction partners (Fig. 5) and confirmed a strong influence on ribosomal constituents. Transmission electron microscopy (TEM) further validated the disturbance of ribosomes through the visible formation of polyribosomes in HCT116 cells treated with BOLD-100.

15.08.4 Organometallic conjugates with biomolecules The conjugation of metals to biomolecules is performed with the aim to combine the beneficial properties of both entities. The metals may invoke unprecedented effects with respect to therapy and diagnosis9,10,33 or as structural linkers,155,156 while the biomolecules exhibit distinct biological functions, which can be harnessed for biomedical applications, e.g. targeting, recognition or even therapeutic effects.157 The combination of the organometallic complex with the biomolecule may form a bioconjugate with novel properties, which are not obtained by the individual entities. Synthetic approaches to prepare metal-bioconjugates have been intensively investigated during the last two decades. Indeed, many useful strategies to conjugate organometallics to peptides, proteins or antibodies were developed and expertly reviewed.157–160 This subchapter covers organometallics conjugated to peptides, proteins or antibodies, as these biomolecules execute the targeting function most prominently, which can be capitalized on for accumulating an organometallic payload at specific systemic sites or tissues, cell types or subcellular compartments. An overview of the most common synthetic strategies is presented before discussing biological studies. Metal-biomolecule conjugates with DNA or peptide nucleic acids (PNAs)161,162 or their use in catalysis158 are comprehensively covered elsewhere.

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Fig. 5 The design of the multi-omics approach to elucidate the protein binding partners of the BOLD-100 included proteomic target-response profiling and transcriptome profiling, in addition to transmission electron microscopy. A similar concept might be applied to organometallic drug candidates with elusive modes of action. Adapted from Neuditschko, B.; Legin, A.A.; Baier, D.; Schintlmeister, A.; Reipert, S.; Wagner, M.; Keppler, B.K.; Berger, W.; Meier-Menches, S.M.; Gerner, C.; Angew. Chem. Int. Ed. 2021, 60(10), 5063-5068., an open access article distributed under terms of the Creative Commons CC BY license.

15.08.4.1 Strategies to synthesize organometallic-peptidic conjugates Since the late 1980s,163 the synthesis of metal-biomolecule conjugates has steadily expanded from the initial amide-based ligation in amino acids and peptides to regio- and chemoselective reactions on antibodies.157,159,160 The synthesis of metal-peptide bioconjugates was enabled by advances in solid-phase peptide synthesis (SPPS).164 SPPS typically involves the N-terminal coupling of amino acids to the growing peptide using suitable protecting group chemistries, which remains coupled to the C-terminus on a resin. In general, the metal moiety can be conjugated to the peptide on the resin and subsequently be cleaved using tailored peptide cleavage conditions. When using the fluorenylmethoxycarbonyl protecting group-based SPPS (Fmoc-SPPS), the obtained peptides can contain a C-terminal amide or carboxylic acid using a Rink or Wang resin, respectively. Alternatively, the coupling step may be performed after cleavage of the peptide in solution when dealing with sensitive metals. Solution chemistry plays a major role in the synthesis of metal-protein bioconjugates, including metal-antibody conjugates.157,160 Importantly, the obtained metal-peptide and metal-protein conjugates are finally purified by chromatographic methods and thus, the metal fragments are required to display sufficient stability or kinetic inertness with respect to ligand exchange reactions in order to be biologically evaluated. The metal-containing payloads can be conjugated either directly or indirectly via suitable linkers.157,159 Metal coordination typically involves multi-dentate or face-capping coordination motifs, while organometallic gold(I) derivatives are examples of successful metalations using mono-dentate binding modes. The linker can be chosen according to the desired physicochemical properties (e.g., polarity)165,166 or display functional properties (e.g., photorelease).167 It may be ligated to the biomolecule exploiting orthogonal reactivities, but typically involves amide, maleimide or Sonogashira coupling reactions (vide infra).157 Since 2008,164 a number of novel organometallic synthons were reported157,159 beyond metallocenes, which led to an increased focus on organometallic-peptide and -protein bioconjugates. Essentially, the organometallic synthons contain functional groups or labile ligands in order to allow the conjugation to the peptidic moiety. Notable examples beyond metallocenes include half-sandwich organometallics based on metal(arenes), metal(Cps), where Cp ¼ cyclopentadienyl) or half-sandwich aqua complexes. A plethora of different conjugation strategies emerged that allow the selective metalation of the N-terminus and more recently, the C-terminus.159 Additionally, modifications were reported on the peptide backbone or amino acid side chains. The latter may include natural (e.g., Cys, Lys, etc.) or synthetic non-natural amino acids (e.g., iodophenylalanine, propargylglycine, thiazolylalanine, etc.). The most common conjugation strategies of organometallics to peptides and proteins reported since 2007 are discussed in more detail in the following paragraphs.

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Amide coupling

Amidation remains among the most widely reported metalation strategies for organometallic-peptide bioconjugates, probably due to the fact that SPPS relies on the selective formation of amide bonds to construct the peptide backbone. Direct N-terminal amidation was reported amongst others for Cp-based metallocene mono-carboxylic acids (M ¼ Co, Fe) before 2010 (Chart 2, 1).168,169 The synthetic strategy included the possibility to tether a fluorescent probe to a differentially deprotected lysine. A ferrocene moiety was also N-terminally conjugated by amidation via an indirect approach including a hexyl linker (Chart 2, 2). For this purpose, the ferrocene monocarboxylic acid was reacted with 6-aminohexanoic acid before conjugation to the peptide. Intriguingly, this linker led to an increase in cytotoxic potency compared to the directly conjugated ferrocene.165 In contrast, the linker type of N-terminally conjugated CpMn(CO)3 moieties, known as cymantrenes, did not have a significant effect on cytotoxic potency of the conjugates (Chart 2, 3).170 Here, two cymantrene moieties were also conjugated to the peptide, but only the introduction of a cathepsin B cleavage site next to the organometallic payload increased the cytotoxicity. Boc-protected 10 -aminoferrocene-1-carboxylic acid represents one of the few organometallics that can be efficiently incorporated into the peptide backbone.171 A similar building block was incorporated into the backbone of a cell-penetrating peptide, which did not influence the biological activity of the peptide (Chart 2, 4).172 The osmium(arene) complex [(6-bip)Os(4-CO2-picolinate)Cl], where bip ¼ biphenyl, was similarly conjugated N-terminally using standard SPPS techniques (Chart 2, 5).166,173 Of note, the organoosmium complex was sufficiently stable to withstand the cleavage conditions of 2 h in 95% trifluoroacetic acid (TFA).173 Ruthenium(arene)-containing conjugates were also prepared by incorporating amidation reactions. Interestingly, the linker consisted of a pyridyl-containing poly-ethylene glycol backbone that was used to coordinate the ruthenium(arene) moiety to the peptide (Chart 2, 6). Irradiation with visible light led to clavage of the Ru–pyridine bond and liberation of the organometallic fragment.174 Several different organometallic metal(arene) moieties were successfully conjugated using a similar approach involving imidazole or a chelating ethylenediamine.166 N,N-Bis(quinolinoyl) Re(I) tricarbonyls containing a free carboxylic acid were employed to generate organometallic-peptide conjugates with photosensitizing properties by N-terminal amidation (Chart 2, 7).175 Indeed, the Re(I) tricarbonyls were able to generate singlet oxygen.

15.08.4.1.2

Direct metalation of amino acids

Incorporation of the unnatural amino acid propargylglycine into the peptide backbone during SPPS allowed to synthesize organometal-peptide conjugates involving Co2(CO)8176 (Chart 3, 8) or organometallic tungsten177 (Chart 3, 9A–B). In the former,

Chart 2 Organometallic-peptide conjugation strategies via amide bond formation.

Chart 3 Organometallic-peptide/antibody conjugation strategies via direct metalation of amino acids. The crystal structure of the antibody was obtained from the RCSB protein data bank with identifier 1igy.

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the cobalt-alkyne conjugations were performed after peptide cleavage from the resin. In the latter case, the tungsten-alkyne bond was sufficiently stable to employ Fmoc-protected tungsten-propargylglycine as a building block for preparing N-terminal and internal bioconjugates by SPPS. The selectivity of cobalt carbonyls for terminal alkynes was used to successfully synthesize bimetallic bioconjugates that also included amide coupled ruthenocenes.178 Zobi and Spingler prepared a lysozyme-bound facRe(CO)3 and found that the chiral protein environment would influence the accessible chemistry of the organometallic fragment compared.179 A gold(I) NHC organometallic moiety was used to directly tether gold(I) to HSA and to a Trastuzumab mutant (ThiomAb LC-V205C), the latter featuring two free cysteines per light chain (Chart 3, 10).180 Conjugation was achieved by ligand exchange of the chlorido-containing parent compound, which was previously established.181 Although this direct metalation benefits from a relatively facile synthetic approach, the antibody-gold conjugates were not found to feature a significantly increased cytotoxicity compared to the parent gold(I) NHC organometallic complex. Arene metalation represents a special form of direct metalation in the synthesis of organometallic bioconjugates. This selective conjugation strategy involves the natural aromatic amino acids Phe, Tyr or Trp. Their aromatic rings can engage in p-bonding with suitable metals. This approach was successfully demonstrated for ruthenium(Cp) moieties that formed conjugates with Phe in secretin182 (Chart 3, 11) or Trp in melittin (Chart 3, 12).107 Ruthenation of Trp of melittin reduces the biological activity of the peptide suggesting that the aromatic Trp is crucial for the interaction with its receptor. Interestingly, arene metalation will occur even in the presence of other nucleophilic amino acids, including His and Lys. In contrast, rhodium(Cp ), where Cp ¼ pentamethyl-cyclopentadienyl, was used to successfully metalate the arene of Tyr in octreotide (Chart 3, 13). Octreotide contains Phe, Tyr and Trp amino acids. Rh(Cp ) selectively formed sandwich complexes with Tyr and to some extent with Trp.183

15.08.4.1.3

Sonogashira coupling

Incorporation of 4-iodophenylalanine into the peptide backbone allows the regioselective conjugation of an organometal featuring a terminal alkyne. These were realized for ferrocene184 (Chart 4, 14) and manganese tricarbonyl185 (Chart 4, 15) organometallics. The Pd/Cu-catalyzed Sonogashira coupling reactions are typically performed after cleavage of the peptide from the resin and require to work in an inert atmosphere and use degassed and dried solvents. Additionally, N-terminal amines are ideally acetylated, while the coupling tolerates the presence of carboxylic acids or phenols, as present in tyrosine.184 The Sonogahira coupling to N-terminal iodoarenes was equally successful.185

15.08.4.1.4

Alkyne-azide coupling

The Cu(I)-catalyzed azide–alkyne coupling (CuAAC) generates selectively 1,4-substituted triazoles and due to its robust nature is often described as “click chemistry.” The CuAAC may be carried out during the SPPS, in contrast to the Sonogashira coupling, but can also be performed in solution after cleavage. This regioselective conjugation reaction requires the introduction of either a terminal alkyne or azide on the peptide. Typically, propargylglycine is used for internal modification, while 4-pentynoic acid may be used for N-terminal labeling.186 Organometallic fragments containing an azide moiety are then required for successful CuAAC coupling reactions. Azdiomethyl-ruthenocene was reported in one of the initial publications in this context (Chart 5, 16).186 The most favorable reaction conditions were obtained by performing the CuAAC with CuI as catalyst in DMF, while remains of Cu may be efficiently removed by using diethyldithiocarbamate.187 More recently, the development of silyl-based linkers enable the C-terminal conjugation of alkyne-containing peptides with methylazido-ferrocene moieties by CuAAC.188 In improved version of a sterically less demanding silyl-based alkyne linker was subsequently reported and used for C-terminal conjugations of organometallic payloads.189 The conjugation of a ruthenium(arene) moiety was achieved by using an, O,O-chelating kojic azide derivative (Chart 5, 17).187 The ligand was conjugated using CuAAC during SPPS at the N-terminus and metalation was performed after peptide cleavage. The resulting organometallic-peptide conjugate showed antiproliferative activity. The inverse CuAAC was reported for an ethynylpyridine-containing Re(I) tricarbonyl moiety, which was successfully conjugated to an azide-containing lipopeptide (Chart 5, 18).190 In this case, the azide was incorporated as the lysine-derivative amino-6-azidohexanoic acid. An intriguing reactivity was reported for Au(I)-azido complexes,191 which induced triazole formation by a [3 + 2] cycloaddition (Chart 5, 19). This copper-free reaction is characterized by a rearrangement of the Au-nitrogen to an organometallic Au-carbon bond upon triazole formation. The advantage of this conjugation strategy is that it can be performed completely on solid support by incorporating a 4-iodophenylalanine, which is converted to into a 4-ethynylphenylalanine.191 The Au(I)-azido-mediated [2 + 3] cycloaddition reaction was also employed to generate inert organometallic Au(I)-containing moieties, which were successfully conjugated to antibodies (Chart 5, 20).192 As recently reported by Contel, Lewis and co-workers, this approach led to Trastuzumab–Au (I) conjugates, which featured an improved cytotoxicity compared to the unmetalated Trastuzumab. This approach employs an

Chart 4 Organometallic-peptide conjugation strategies via Sonogashira coupling.

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Chart 5 Organometallic-peptide conjugation strategies via alkyne-azide coupling. The crystal structure of the antibody was obtained from the RCSB protein data bank with identifier 1igy.

indirect conjugation strategy by using a pendant activated carboxylic acid. Thus, the organometallic Au(I) was conjugated favorably to surface-exposed lysine residues by amidation (s. above) resulting in an approximate drug-to-antibody ratio of 3.192

15.08.4.1.5

Maleimide-thiol coupling

The spontaneous addition of maleimide to thiol represents a regioselective conjugation strategy and found initial applications in the preparation of metal-HSA conjugates because HSA contains CYS34 as the single surface-exposed cysteine that is not engaged in disulfide bridges. Hence, a maleimide-derivatized arene was used as a precursor to synthesize several ruthenium(arene) moieties and subsequently conjugated to HSA (Chart 6, 21).193 Similarly, maleimide was incorporated into the ligand scaffold of an N-substituted pyridine carbothioamide, which forms stable S,N-chelates with metal(arenes).194 These maleimide-containing organometallics were shown to quantitatively react with thiols in model studies. An N,N-bis(quinolinoyl) Re(I) tricarbonyl moiety containing a pendant photo-labile protecting group was conjugated N-terminally to peptides (Chart 6, 22).195 Upon low dose irradiation, the photo-labile o-nitrophenyl linker released the intact organometallic moiety, which was found to increase the cytotoxic potency. An analogous concept was applied to study ROS-induction by the photo-release of ferrocene units from peptides (Chart 6, 23).167

15.08.4.1.6

NHC coupling

NHCs provide a rich ligand scaffold for air- and water-stable organometallics with particular physicochemical properties, which were initially harnessed for catalysis.158 It was also realized that these properties render NHC-metals promising scaffolds for selective biomolecule conjugation strategies,196 which generated catalytically active bioconjugates based on palladium(II) (Chart 7, 24).197,198 The synthesis of these conjugates was performed by incorporating imidazoles and pending pyridines. Subsequently,

Chart 6 Organometallic-peptide/protein conjugation strategies via maleimide-thiol coupling. The crystal structure of human serum albumin (HSA) was obtained from the RCSB protein data bank with identifier 4s1y.

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Chart 7 Organometallic-peptide conjugation strategies via N,N- or N,S-heterocyclic carbene (NHC) coupling.

deprotonation and metalation were carried out on solid support. Of note, the catalytic activity of the NHC-Pd(II) bioconjugate with respect to Sonogashira and Suzuki cross-coupling reactions was assessed in the resin-bound state and provided high yields.197 More recently, NHC-metal complexes emerged as promising scaffolds for medicinal purposes.199,200 NHC gold(I) and gold(II) amino acid conjugates were obtained by transmetallation from a NHC Ag(I) precursor (Chart 7, 25).181 The carbene was obtained by incorporation of an imidazole moiety via nucleophilic attack of bromoacetamide-derived amino acid. These NHC gold-amino acid bioconjugates showed medium cytotoxicity. A dimethylated histidine-analog was used in a further study to generate NHC Rh bioconjugates.201 Incorporation of Met into the peptide three positions upstream led to the unexpected cyclization of the peptide backbone by coordination of the thioether moiety to the metal generating one of the few examples of published organometallic macrocycles (Chart 7, 26). An interesting alternative approach to carbene-metal bioconjugates was presented by the formation of a synthetic thiazolylalanine amino acid, which was N-terminally coupled to dipeptides under SPPS conditions.202 The thiazolyl-moiety mimics the imidazole of histidine and upon deprotonation, can be used to conjugate organometals. The corresponding N,S-heterocyclic carbene-metal tripeptides were successfully synthesized with Ru(arene) and Rh(Cp ) metal synthons (Chart 7, 27A).202 This strategy was extended to Au(I) conjugates, which were found to exhibit potent cytotoxicity even in the multidrug-resistant cell line A549 (Chart 7, 27B).203

15.08.4.1.7

Miscellaneous

Symmetrical alkyne-bridged bis(peptides) were synthesized that offer an internal alkyne for conjugation. Such systems were used prepare organometallic tungsten(II)-peptide bioconjugates (Chart 8, 28).204 Together with the octahedral coordination geometry, the employed chelating ligands based on dithiocarbamates result in diastereomeric tungsten atoms. Simultaneously N- and C-terminally alkyne-modified peptides were used in a similar approach to generate peptide metallacycles.205 These organometallicpeptide bioconjugates were not biologically investigated. Schatzschneider and co-workers reported the N-terminal modification by aminoxy acetic acid, which allowed the conjugation of an aldehyde-containing Mo tetracarbonyl via formation of an oxime (Chart 8, 29).206 The conjugate was stable and was shown to exhibit favorable properties as a photo-activatable CO-releasing molecule (PhotoCORM). In a similar direction, lysine residues on HSA were modified with a hydrazine linker by amidation, followed by treating with an aldehyde-containing ruthenium(arene) moiety (Chart 8, 30).207 Mass spectrometry-analysis revealed a mass shift of approximately 1900 Da suggesting three to four metalated lysines on HSA. An increase in cytotoxic potency of the metal-HSA

Chart 8 Miscellaneous organometallic-peptide/protein conjugation strategies. The crystal structure of HSA was obtained from the RCSB protein data bank with identifier 4s1y.

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conjugate was observed compared to the unmetallated precursor or the organometal. Additionally, the hydrazone bond is generally stable under biological conditions and represents a regioselective conjugation chemistry. However, the application of hydrazones may be limited due to their tendency to hydrolyze at low pH.

15.08.4.2 Targeting strategies By means of the biological properties of the biomolecule, organometal-bioconjugates can be used to target specific compartments or favor their retention therein. Beneficial properties include the retention in the blood stream, or accumulation at the target site. Targeting cells or cell types may be achieved due to the expression of specific membrane receptors. Subcellular targeting may also be achieved by the conjugation to peptides, because distinct peptide sequences serve as intracellular sorting signals.208 Known natural examples include the nuclear localizing sequence (NLS) and the mitochondrial localizing sequence (MLS), which are peptide sequences typically found at the N-terminus of proteins while the peroxisomal targeting sequence (PTS-1) and the signal for retention in the endoplasmic reticulum are found at the C-terminus of proteins. The signaling code is mainly dictated by the spatial arrangement of charged and apolar amino acids.168 Obviously, organometallic moieties can be charged depending on the oxidation state of the metal and the ligand types, which in turn may modulate the recognition of the sorting signal by the receptor, when conjugated to such a peptide sequence. The following paragraphs focus on the biological effects that were achieved by forming organometallic bioconjugates with peptides, proteins and antibodies. In several cases, the organometallic bioconjugate exhibited properties distinct from their individual organometallic and biomolecule components.

15.08.4.2.1

Blood

Organometallic conjugates to peptides or serum proteins can be employed to increase their plasma life-time, as well as for accumulation in tumor tissues by traversing leaky blood vessels.209 Serum protein binding by coordination compounds was shown to be responsible for their relatively long plasma half-lives during clinical studies.33 Moreover, drug-HSA conjugates may be internalized210 by proliferating cancer cells due to their nitrogen dependency for anabolism, leading to an accumulation of the drug. Thus, the conjugation to serum proteins (e.g., HSA) infers properties distinct from antibodies, which tend to bind to membrane-bound receptors at the cell surface. Investigations with organometallic-protein bioconjugates are scarce. A RAPTAtype-HSA bioconjugate showed increased cytotoxicity compared to the parent compound in in vitro tests.207 This was achieved by conjugating the organometallic moiety via a hydrazone linker to lysine moieties. A more direct approach involved maleimide-thiol conjugation to Cys34 of HSA,193 the single surface-exposed cysteine not involved in disulfide bridges. Although this was achieved for ruthenium(arene), only a maleimide-containing platinum(IV) coordination compound was investigated in vivo and showed promising tumor inhibition.211

15.08.4.2.2

Membrane receptors

Peptides may serve as targeting vectors to membrane receptors. It is known that several membrane receptors are overexpressed on malignant cells that may be specific to the disease. For example, octreotide is a more potent synthetic analog of somatostatin and binds to the G protein–coupled somatostatin receptor, which inhibit proliferation upon stimulation174 by suppressing the release of growth hormones.212 The disulfide bridge in the cyclic octreotide was in some cases replaced by a dicarba-analog in order to improve the metabolic stability of the peptide.166 Octreotide was N-terminally modified with a linker to variably conjugate metal(arenes), where metal ¼ ruthenium or osmium,166,174 as well as a cyclometalated iridium(III).213 Alternatively, octreotide was directly metalated at Phe with Rh(Cp ), which selectively forms a sandwich complex.183 The latter was investigated in MCF7 and HT29 cancer cell lines, which express somatostatin receptors. Metalation of Phe retained the low micromolar inhibitory concentration of the organometallic bioconjugate with respect to free octreotide, suggesting that metalation at this position does not interfere with binding to the receptor.183 Of the metal(arene) bioconjugates, only the ruthenium(arene) moiety containing triphenylphosphine exhibited modest cytotoxicity in the somatostatin subtype-2-expressing MCF7 and DU-145 cancer cell lines.166 Interestingly, the cytotoxic potency seemed to correlate with the expression levels of the somatostatin subtype-2 receptor. Moreover, the internalization of a fluorescein-labeled octreotide (i.e., without the organometallic payload) into MCF7 cells was confirmed by fluorescence microscopy. The cellular accumulation of the cytotoxic ruthenium(arene)-octreotide bioconjugate was determined by ICP-MS, which yielded 143  17 pmol Ru/106 cells. This concentration was approximately two-fold higher compared to the unconjugated ruthenium(arene) precursor, which accumulated to 68  2 pmol Ru/106 cells.166 The cellular accumulation of the cyclometalated iridium-octreotide bioconjugate was also confirmed by ICP-MS analysis.213 Intracellular accumulation in cytoplasmic vesicles was also observed by fluorescence microscopy due to the luminescent properties of the iridium complex. For a fluorescently-labeled ferrocene-octreotide co-localization studies by fluorescence microscopy revealed accumulation in endosomal vesicles and finally deposition in lysosomes.214 The tripeptide RGD and NGR amino acid motifs allow the selective accumulation at the tumor site by interacting with integrins and aminopeptidase-N receptors of cancer cells.215 Biological investigations into a ruthenium(arene)-RGD bioconjugate were not reported.166 Due to the fact that there are several heterodimeric subtypes of integrins, the cyclic RGDfK-motif was found to exhibit favorable selectivity for integrin avb3.216 A cyclometalated Pt(II)-RGDfK bioconjugate was reported to have reduced cytotoxic potency compared to the parent Pt(II) compound. No correlation between integrin avb3 expression and cytotoxic activity was observed. However, the organometallic bioconjugate displayed anti-angiogenic properties in line with RGDfK targeting integrins.217 Very recently, a number of ferrocene-RGD bioconjugates were reported with containing linkers with different lipophilicity and

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variable RGD-motifs.218 Conjugating ferrocene to these peptides was carried out with the aim to increase ROS specifically at the tumor site. The authors identified ferrocene conjugated via a non-polar hexyl linker to the cyclic FRGDLAFp(NMe)K to be selective for integrin avb6, which is up-regulated in various tumor types, e.g. colorectal cancer or oral squamous carcinoma.219 The bioconjugate was more cytotoxic compared to the unconjugated organometallic and induced intracellular ROS and DNA damage. In a separate study, a ferrocene-RGD bioconjugate was also shown to possess increased cellular accumulation and cytotoxicity in the presence of a hexyl linker.165 Bombesin binds to G-protein coupled receptors on gastrin cells, which may also be found overexpressed on tumor cells. A Re(I) tricarbonyl conjugated via a photolabile linker to bombesin was shown to exert medium to low cytotoxic activity in several cell lines, without a clear-cut correlation concerning irradiation.195 Targeting of the epithelial growth factor receptor (EGFR) implicated in cancer proliferation was also accomplished by short peptide sequences containing LARLLT, although for a coordination Pt(IV) compound.220 Although not related to cancer, antimicrobial peptides (AMPs) are cationic and broad-spectrum antibiotics.221 They exert their mode of action by targeting negatively-charged bacterial membranes and exhibit biological activity on their own. Defensins are known biological AMPs, while promising synthetic AMPs were reported containing the sequence WRWRW, i.e. aromatic and positively-charged.222 In an elegant structure-activity guided approach,223 it was found that N-terminal conjugation to ferrocene or ruthenocene222 would increase the antibacterial activity, which was further enhanced by inverting the natural L- to the non-natural 224 Indeed, the ruthenocene-WRWRW bioconjugate was shown by transmission electron microscopy to D-stereo-conformation. accumulate in bacterial envelopes.225 Antibody-drug conjugates (ADC) are used for targeting and therapeutic purposes,160,226,227 but only a few ADCs exist in which the organometallic moiety acts as the connecting element. For example, an organometallic gold(I)-trastuzumab conjugate was used to target HER2-positive breast cancer cells.192 The bioconjugate exhibited a slightly lower binding affinity to HER2 compared to trastuzumab alone. However, it showed low micromolar cytotoxicity in HER2-expressing cancer cells and some selectivity over non-cancer cells, while trastuzumab alone was inactive. An interesting alternative organometallic conjugation was reported by the formation of a half-sandwich Rh(III) conjugate on Tyr in the presence of boronate displaying an ortho-carboxamide,228 similarly to the mentioned Rh sandwich compounds,183 which may be extended to biological investigations in the future. An intriguing point was highlighted by means of a ferrocene-neurotensin conjugates. It was noted that electron-poor ferrocenes would exhibit moderate cytotoxicity, while conjugated to neurotensin the cytotoxic properties were lost.229 Neurotensin binds to G-protein coupled membrane receptors that may be subsequently internalized by endocytosis for degradation in lysosomes.230 It was hypothesized that this pathway would lead to the inactivation of the ferrocene-containing bioconjugates. Indeed, endosomal entrapment was supported by fluorescence microscopy as a possible route of inactivation using a fluorescent derivative of the organometallic bioconjugate.

15.08.4.2.3

Cell penetrating peptides

Peptides are not only suitable ligands for membrane receptors, but may also serve as vectors to cross the cell membranes. This concept of cell-penetrating peptides (CPPs) was first proven by means of the transcription and transactivation (TAT) sequence 47–57 (e.g., YGRKKRRQRRR) of human immunodeficiency virus 1 (HIV-1),231 which is responsible for membrane translocation. In contrast to peptides that target membrane receptors and are internalized via endocytosis, CPPs are believed to be taken up by several possible mechanisms.232 Amongst other, they may cross the membrane without specific transporters or receptors, which makes them attractive carrier molecules.233 TAT is most probably taken up in endosomes by lipid raft micropinocytosis.234 An organometallic Re(I) tricarbonyl-TAT bioconjugate was prepared, which additionally featured a myristoylated N-terminus.190 The luminescent nature of the Re complex allowed the direct investigation of cellular accumulation by fluorescence microscopy. Indeed, the bioconjugate showed enhanced uptake with respect to the unconjugated Re complex to the cytoplasm of HeLa cells, in addition to some potential endosomal hotspots. As a side note, a ferrocene-containing cyclic peptide was proven to inactivate HIV-1 cell infection by interacting with the virions gp120 receptor.235 The C-terminal acid sequence of the antimicrobial peptide CAMP cathelicidin (CAP18) showed some similarity to the TAT sequence and a short 16 amino acid version termed sC18 (e.g., GLRKRLRKFRNKIKEK) was indeed found to be a CPP.236 sC18 is believed to be taken up by the endosomal pathway similarly to TAT, but a direct uptake is also thought to be possible.237 Schatzschneider, Neundorf and co-workers have investigated several cymantrene moieties with different linker types conjugated to sC18.170,238 Indeed, the organometallic bioconjugates were taken up in dot-shaped vesicles around the nucleus, which suggested endosomal pathway.170 Eventually, sC18 seems to accumulate in lysosomes. Interestingly, removal of a keto group from the linker directed the organometallic conjugates from endosomal vesicles into the nucleoli in MCF7 cancer cells, irrespective of the metal (e.g., manganese or rhenium).239 This strongly suggested that the linker types may modulate release from endosomal entrapment. Synthetic repetitive poly-cationic peptides are also suitable CPPs (e.g., R9F2K or R8).240,241 A ferrocene-bioconjugate was shown to be internalized in dot-shaped vesicles, similarly to cymantrene-sC18, which colocalized to some degree with lysosomes.241 Moreover, the organometallic bioconjugate was more cytotoxic with respect to its individual components. Sadler and co-workers conjugated an organometallic Os(arene) via a picolinate ligand to R8.173 The organometallic R8 bioconjugate was more effectively taken up by cells compared to the R5 and R1 analogs. A ferrocene moiety was conjugated into the backbone of hCT(9-32), which represents a CPP of the peptide hormone calcitonin.172 The conjugate accumulated efficiently in endosomal vesicles in HeLa cells.

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Subcellular targeting

Subcellular localization is complicated by the fact that the bioconjugate is required to cross the plasma membrane or escape endosomes in order to accumulate at a subcellular location. Endosomal entrapment needs to be considered if membrane receptors are targeted, which are subsequently internalized for degradation, intracellular protein sorting is generally dictated by short peptide sequences at the N- or C-terminus of a nascent protein.242 By conjugating a positively-charged cobaltocenium-fragment to the nuclear localizing sequence (NLS) consisting of (K)PKKKRKV, Metzler-Nolte and co-workers were able to efficiently escape endosomes and accumulate in the nucleus.168 Without the cobaltocenium, the fluorescently-labeled NLS would remain in the endosomes. The ferrocene-conjugate would also accumulate in the nucleus, but be retained to some degree in the endosomes.169 Conjugating a Re(I) tricarbonyl to a short NLS consisting of KKKR via a hexyl linker led to the accumulation of the bioconjugate preferentially in the nucleoli of the investigated cells.175 Two-membrane crossing properties were also achieved for synthetic peptides containing FxRFxK or FxRFxKFxRFxK, with Fx ¼ Phe or cyclohexylalanine, that target the mitochondria in cells.243 The octapeptide led to a two-fold increased uptake compared to the tetrapeptide. Based on this concept, organometallic gold(I)-FRFK bioconjugates were prepared and investigated for their biological activity.191 Although the subcellular targeting was not directly investigated, these bioconjugates were quite cytotoxic, induced ROS and depolarized the mitochondrial membrane potential.

15.08.4.2.5

Miscellaneous

Peptides also serve as hormones or signaling molecules, such as angiotensin, which increases blood pressure or [Leu5]-enkephalin, which is a neuropeptide involved in nociception by binding to opioid receptors. Besides their biological function, these peptides often served as a model system for synthetic proof-of-principle studies of metal-peptide bioconjugation.159 However, the permeation of a ferrocene-[Leu5]-enkephalin conjugate was indeed investigated in a model of the blood-brain-barrier (BBB).244 Incorporation of the neutral ferrocene increased the lipophilicity of the peptide and led to an increased cellular accumulation.

15.08.5 Conclusions The ultimate aim of drug discovery is to develop effective therapeutic agents for conditions with unmet medical need. Inorganic drug discovery provides scaffolds, which are inaccessible to classical medicinal chemistry and this complementary chemical space has the potential to generate therapeutic agents for so far elusive targets and exploring novel mechanisms of action. For example, the organoruthenium plecstatin was recently validated as a first-in-class modulator of plectin with direct implications on the invasiveness of cancer cells.54,140 So far, inorganic drug discovery has focused strongly on establishing synthetic procedures with sporadic biological explorations on a limited number of lead structures. With the rise of therapeutic mechanisms beyond the induction of apoptosis, the elucidation of these novel mechanisms of action mediated by potentially novel targets is of central importance to move organometallic drug candidates forward to the clinical setting. Therefore, the field might benefit from an increased focus on translational aspects. Detailed insight into the mechanism of action might crucially inform patient selection and stratification. Moreover, the low efficacy of the already tested clinical candidates might be improved by conjugation to targeting agents, which enhance the accumulation at the target site. The last decade has seen substantial progress in both the elucidation of targets (including mechanisms of action) and bioconjugation strategies. On the one hand, comprehensive omics-approaches were established for the analysis of drug targets and effects, culminating in the characterization of mechanisms of action. Especially, metalloproteomics emerged as a valuable tool for elucidating cellular targets and mechanisms of action of organometallic drug candidates. Based on the observed response and target profiles, it may additionally be possible to determine off-targets and resistance mechanisms. Such a mechanism-driven approach might also lead to more efficient animal testing. So far, metalloproteomics was mainly used for in vitro testing, but has the potential to be expanded to validate the proposed target engagements and mechanisms of action in vivo. The omics-methods for the comprehensive analysis of mechanisms of action have just emerged and promise to be useful for characterizing potential structural synergisms of organometallic-bioconjugates with respect to their biological effects. Bioconjugation strategies have also undergone significant advances during recent years. Versatile synthetic approaches were established to successfully conjugate organometallic payloads with high specificity to peptides, proteins and antibodies. Nonetheless, it is expected that additional chemo- and regioselective reactions will be discovered that enable the efficient conjugation of organometallic payloads under mild conditions to biomolecules, especially proteins and antibodies. The production of antibodies with non-natural amino acids may further allow the generation of novel site-specific antibody-drug conjugates.245 While bioconjugation strategies for targeting or antimicrobial strategies were realized on one level, combining systemic targeting with subcellular localization by sequential peptide sequences may be conceptually possible. This dual-targeting may necessitate the incorporation of enzymatically-cleavable linkers.226 Ideally, the first targeting moiety would accumulate the construct at the target site and be internalized by endocytosis. Upon transport to lysosomes cathepsin substrates (e.g., GFLG) might release the second targeting moiety, which may ultimately accumulate the payload at a given subcellular location. Further insight into the selective release of organometallic-bioconjugates from endosomes might be necessary to fully exploit such a double-targeting approach, especially since endosomal entrapment and subsequent degradation in lysosomes seems to contribute to the deactivation of the bioconjugates.

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Metalation can be considered as a synthetic form of post-translational modification (PTM) of peptides, proteins or antibodies that may confer biological effects that are only accessible by the organometallic bioconjugate. The structural determinants for the biological activity of the organometal-bioconjugate remain often unclear. In the case of antimicrobial peptides, the biological effect was clearly improved by the organometallic fragment. Thus, the organometal-bioconjugate may exhibit novel properties, which are not the simple sum of its organometallic payload and biomolecule. A rational exploration of the potentially synergistic effects of organometal-bioconjugates in terms of PTMs is expected in the near future. These efforts will consolidate organometallic therapeutics, including bioconjugates, as a promising element in inorganic drug discovery and development.

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K.; Kim, C.; Seo, C. H.; Park, Y. J. Microbiol. 2017, 55 (1), 1–12. Albada, H. B.; Chiriac, A. I.; Wenzel, M.; Penkova, M.; Bandow, J. E.; Sahl, H. G.; Metzler-Nolte, N. Beilstein J. Org. Chem. 2012, 8, 1753–1764. Albada, B.; Metzler-Nolte, N. Acc. Chem. Res. 2017, 50 (10), 2510–2518. Albada, H. B.; Prochnow, P.; Bobersky, S.; Bandow, J. E.; Metzler-Nolte, N. Chem. Sci. 2014, 5 (11), 4453–4459. Wenzel, M.; Chiriac, A. I.; Otto, A.; Zweytick, D.; May, C.; Schumacher, C.; Gust, R.; Albada, H. B.; Penkova, M.; Kramer, U.; et al. Proc. Natl. Acad. Sci. 2014, 111 (14), E1409–E1418. Bargh, J. D.; Isidro-Llobet, A.; Parker, J. S.; Spring, D. R. Chem. Soc. Rev. 2019, 48 (16), 4361–4374. Boros, E.; Holland, J. P. J. Labelled Compd. Radiopharm. 2018, 61 (9), 652–671. Ohata, J.; Miller, M. K.; Mountain, C. M.; Vohidov, F.; Ball, Z. T. Angew. Chem. Int. Ed. 2018, 57 (11), 2827–2830. Maschke, M.; Grohmann, J.; Nierhaus, C.; Lieb, M.; Metzler-Nolte, N. ChemBioChem 2015, 16 (9), 1333–1342. 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15.09 Organometallic Chemistry of Anticancer Ruthenium and Osmium Complexes Artem Osypenkoa, Adnan Ashrafb, Valentyn Pozhydaieva, Maria V Babakc, and Muhammad Hanifb, aInstitut de Science et d’Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, Strasbourg, France; bSchool of Chemical Sciences, University of Auckland, Auckland, New Zealand; cDepartment of Chemistry, Drug Discovery Lab, City University of Hong Kong, Hong Kong, People’s Republic of China © 2022 Elsevier Ltd. All rights reserved.

15.09.1 Introduction 15.09.2 Early discoveries and design of bioactive organometallic compounds 15.09.3 Sandwich metal complexes—ruthenocenes and osmocenes 15.09.4 Half-sandwich metal(II)-arene complexes of ruthenium and osmium 15.09.4.1 RAPTA inspired half-sandwich complexes 15.09.4.2 RAED-inspired organoruthenium and -osmium complexes 15.09.4.3 N-heterocyclic carbene (NHC) complexes 15.09.4.4 Cyclometalated Ru(II) and Os(II) arene complexes 15.09.4.5 Bioconjugates of half-sandwich organoruthenium and osmium complexes 15.09.5 Multinuclear Ru and Os organometallics 15.09.6 Cytotoxic organometallic clusters of Ru and Os 15.09.7 Conclusions Acknowledgments References

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15.09.1 Introduction The serendipitous discovery of anticancer properties of cisplatin has brought a revolution to the field of metal-based drugs. Both the careful design of ligands and the choice of the metal ion play an important role in the biological properties and mode of action of novel metallodrugs. Each metal ion offers unique physicochemical properties such as redox potentials, and ligand exchange kinetics to fine-tune the bioactivity of the complexes.1–4 Among several non-platinum metal ions investigated across the periodic table, complexes of ruthenium, gold, titanium, gallium and gadolinium have shown promising anticancer potential in advanced stages of preclinical development.1–9 Despite the fact that bioactive organometallic compounds occur in nature, such as vitamin B12 and metal carbonyls, the vast majority of research in metallodrugs discovery has been focused on the design of coordination complexes. Exploring the medicinal applications of organometallic compounds, with at least one metal-carbon bond, is still a relatively under-investigated area compared to the extensive literature focusing on the medicinal properties of coordination complexes. The stigma about organometallic anticancer complexes probably stems from the general view that organometallic compounds are generally too air and moisture-sensitive to be suitable for medicinal applications. However, in recent years there has been a shift in paradigm. Currently, virtually all classes of organometallic compounds such as metal carbonyls, arenes, carbenes and metallocenes, have been tested for a diverse range of biological properties including antimicrobial, anti-parasitic and anticancer activity in in vitro and in vivo assays and demonstrated promising therapeutic potential.10 It has been clearly shown that hydrolytic stability, reactivity and biological properties of organometallic compounds can be controlled by careful design of the ligand system. These organometallic compounds have demonstrated modes of action that are different from platinum-based anticancer drugs. The diverse modes of action range from redox modulation to targeting a specific protein or enzyme to induce antitumor immune responses in cancer cells.5–8,10,11 This article covers the latest advances in the design, chemistry and properties of anticancer organometallic compounds of ruthenium and osmium. We discussed their anticancer properties, modes of action and biomolecular interactions with cellular targets.

15.09.2 Early discoveries and design of bioactive organometallic compounds The first organometallic drug used in humans was the arsenic-based compound salvarsan developed in the early 20th century by Paul Ehrlich to treat syphilis.12 Subsequently, in the 1980s, anticancer properties of the organogermanium compounds spirogermanium and germanium-132 were explored. Spirogermanium demonstrated promising in vitro cytotoxicity in cancer cell lines.13 However, it showed limited efficacy and was neurotoxic when evaluated in human clinical trials.14 Germanium-132 showed anticancer activity in a wide range of cancer cell lines, possibly via stimulation of the immune system.15,16 At the same time, when the clinical potential of organogermanium compounds was investigated, Köpf and Köpf-Maier discovered the antitumor properties of titanocene dichlorido [Ti(Cp)2Cl2] (Cp ¼
5-C5H5; Fig. 1).17 The design concept of titanocene containing cis-dichlorido coordination motif was inspired by the structure of cisplatin; however, their mode of action was drastically different. While cisplatin is known to interact with DNA through the N7 of guanine, titanocene dichloride reacted

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Fig. 1 Schematic structures of titanocene, hydroxytamoxifen, metallocifen analogs and their quinone methides.

with DNA’s phosphate backbone.18 Titanocene dichlorido reached human clinical trials, but was further discontinued due to limited efficacy and low stability under physiological conditions.19 Promising early results of titanocene led to significant interest in the design of bioorganometallic compounds of other metals. Among them, Fe, Ru and Os complexes have demonstrated promising anticancer properties and some of them have reached advanced preclinical testing.5,6,20,21 Ru and Os offer unique features such as slower ligand exchange kinetics and the ability for stronger p-back-donation from lower oxidation states, all of which are important in the design of metallodrugs. Therefore, the biological properties of Ru and Os organometallic complexes have been extensively investigated in recent years. A diverse range of ligand systems and wide range of synthetic approaches have yielded structurally diverse library of compounds, which have been investigated for their chemical and biological properties.5–7,22–27 In the sections below, we describe leading examples of anticancer organoruthenium and -osmium complexes.

15.09.3 Sandwich metal complexes—ruthenocenes and osmocenes The interest in Ru and Os complexes with a sandwich structure has started after the discovery of ferrocifen 3a (Fig. 1), a ferrocene functionized with anticancer drug Tamoxifen.6,20,21,28 The ferrocenyl moiety, where Fe(II) atom is “sandwiched” between two cyclopentadienyl moieties, did not only improve the lipophilicity of the complex to cross cell membranes but also contributed to the electron transfer capacity of the ferrocifens which enable the oxidation through a series of redox processes. In comparison to Tamoxifen, 3a demonstrated superior anticancer properties against resistant breast cancers.6,20,21,28 Following the success of ferrocifen, metallocifens of Ru 3b and Os 3c were prepared and their anticancer properties were investigated. The complexes were synthesized using the McMurry cross-coupling reaction of the two corresponding ketones, but yields were lower compared to 3a. In addition, the synthesis of Ru and Os complexes required a stronger acylating agent such as AlCl3.6,20,21,29,30 Regardless of the nature of metal ion, metallocifens 3a–3c were more lipophilic than Tamoxifen. Incorporation of metallocene moiety into the tamoxifen pharmacophore resulted in strong tumor-inhibiting properties against both hormone-independent (MDA-MB-231) and hormone-dependent (MCF-7) breast cancer cells compared to the Tamoxifen per se. The Os derivative showed cytotoxicity in a similar range to that of Fe analog but the ruthenocene was least cytotoxic.31,32 The lower activity of ruthenocene was attributed to its inability to undergo a redox process identical to its Fe counterpart. All three metallocenes showed strong inhibition of mitochondrial thioredoxin reductase, and induction of mitochondrial dysfunction and cell death in Jurkat cells.31,32 In addition, the intracellular distribution of osmocenyl-tamoxifen 3c in breast cancer MDA–MB–231 cells was studied using synchrotron radiation X-ray fluorescence.20 The high-resolution imaging demonstrated the accumulation of 3c in the endomembrane system. This may be due to electrostatic interactions between the membranes and protonated amino nitrogen chain of 3c at physiological pH. These studies provided important clues in understanding the cellular behavior of the Os complex 3c.6,20,21 Chemical oxidation of 3a–3c using Ag2O as oxidant led to the formation of the corresponding quinone methides, 4a–4c at different rates.29 The rates of formation for the osmium-based quinone methides such as 4c were significantly lower than their iron counterparts. This follows the trend in redox potentials of the metallocenes (0.47, 1.03, and 0.83 V for Fe, Ru, and Os, respectively), which indicates that ferrocifen was easier to oxidize compared to its Ru and Os analogs. However, the enzymatic oxidation showed that oxidation of Os complexes stopped at the carbenium ions, which are precursors to the formation of the quinone methides in the case of Fe and Ru analogs. These stable osmocenyl carbocations were isolated and characterized using UV-Vis and mass spectrometric methods.30

15.09.4 Half-sandwich metal(II)-arene complexes of ruthenium and osmium In half-sandwich complexes, one of the aromatic parts of metallocenes is replaced by mono or bidentate ligands. Due to their molecular shape, these are also called “piano stool” complexes (Fig. 2). The “piano stool” metal complexes consist of: (i) a wide variety of Z6-coordinated arene ligands such as benzene, toluene, biphenyl, p-cymene, (ii) a halido ligand including chlorido,

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Fig. 2 Archetypical structures of half-sandwich compounds with monodentate and bidentate ligands where M is Ru or Os.

bromido or iodido, and (iii) mono- or bidentate ligand(s).33–38 It has been shown that each component plays an essential role in the anticancer properties of the compound. The halido ligands undergo ligand exchange reactions with water and ultimately react with biological nucleophiles such as DNA or proteins. The arene moiety maintains the stability of the Ru(II) or Os(II) species as well as provides hydrophobicity for effective penetration through cell membranes. The half-sandwich ruthenium and osmium complexes have shown antitumor, anti-metastatic and antiangiogenic properties both in vitro and in vivo.1 One of the first studies reporting the half-sandwich [RuII(Z6-arene)(nicotinamide)Cl2] complex, which demonstrated low cytotoxicity (>100 mM).39,40 Since then, numerous other derivatives have been prepared and tested for biological properties. In particular, RAPTA-C ([Ru(
6-p-cymene)(PTA)Cl2] PTA ¼ 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane) and RAED-C ([Ru(
6-p-cymene)Ru(en)Cl]+, en ¼ ethylenediamine) were the most extensively studied lead compounds.10,41 A general synthetic route (Fig. 3) towards such complexes is based on reacting hydrate of metal(III) halide (usually chloride) with cyclohexadiene (usually obtained via a Birch reduction of arene) or cyclopentadienyl derivatives. The resulting halido bridged bimetallic precursors are subsequently reacted with a range of mono- or bidentate ligands, yielding a wide variety of half-sandwich complexes (Fig. 3). Arene exchange can also be used when diene precursor is unavailable. The half-sandwich scaffold provides an excellent platform to fine-tune the biological properties of complexes by structural variations at (i) the aromatic part; (ii) ligands; (iii) both aromatic component and ligands; and (iv) post-synthetic modification of aromatic part or/and ligands of the prepared complexes. To this end, several approaches to functionalize half-sandwich complexes have been explored. These include conjugation with drugs with known bioactivity; molecules with high affinity to protein targets (either non-covalent or covalent); compounds with stimuli-responsive properties (such as the pH, redox, photo or thermoresponsive); and compounds exhibiting enhanced permeability and retention (EPR) properties (such as polymers, dendrimers, and nanoparticles).42–48

15.09.4.1 RAPTA inspired half-sandwich complexes Half-sandwich organometallic complexes of PTA ligand are among the most studied compounds of this class of anticancer agents. These complexes with general formula [Ru(Z6-arene)(PTA)X2] (with X being Cl−, Br−, I−, and other anions) are so-called RAPTA complexes—Ruthenium Arene PTA. RAPTA complexes were shown to have high potency against metastatic tumors in vivo, while they are often being much less toxic to primary tumors in vitro studies.3 The first report on RAPTA-C (5, Fig. 4) showed promising results in pH-dependent DNA damage, suggesting possible control over its selectivity against cancer cells, which are known to have a more acidic pH.49 Further, in vitro studies revealed that RAPTA compounds 5–14 (Fig. 4) display relatively modest cytotoxic activity against mammary adenocarcinoma (TS/A) cells but were nontoxic towards nontumorigenic cells. In vivo studies for antimetastatic activity in CBA mice with mammary carcinoma (MCa) indicated that RAPTA complexes induced significant potency in reducing the number and weight of metastases. Although the effective concentration was relatively high, RAPTA-C was well tolerated by mice making these compounds suitable drug candidates to treat metastatic cancer.50 The osmium analog [Os(
6-p-cymene)Cl2(pta)] 6 (Fig. 4) and cationic derivative [Ru(
6-p-cymene)Cl2(pta-Me+)] 7 of RAPTA-C showed low to moderate tumor-inhibiting properties. The former showed selectivity towards cancer cells, whereas the cationic analog 7 was indiscriminately cytotoxic to both healthy and diseased cells.51–53 Studies on the structural variation of aromatic part and PTA helped to establish structure-activity relationships for RAPTA complexes. The modification of the aromatic fragment could be used to fine-tune the hydrophobicity of RAPTA compounds and possible conjugation with other bioactive molecules. The IC50 values for RAPTA complexes against TS/A cells were in range >300 to 66 mM. The compound 14 being the most active, while the toxicity against normal human HBL-100 cells was over 300 mM.50 RAPTA complexes with hydrogen bonding groups (such as alcohol, amines, or ammonium salts) on the aromatic part were less cytotoxic and less selective towards cancer cells than RAPTA-C. This was related to their lower cellular uptake and dissociation of hydrophilic arene part, resulting, probably, in the deactivation of the complex.52

(C)

Fig. 3 The general synthetic route towards piano-stool complexes.

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Fig. 4 Chemical structures of RAPATA-type half-sandwich compounds.

On the other hand, PTA ligand modification had a more dramatic influence on RAPTA behavior (Fig. 4). For instance, PTA methylation in 7 increased the toxicity and rendered the compound nonselective towards cancer, hence more toxic for normal cells.50 Ru complexes 8 with DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) were much less cytotoxic than their PTA analogs for both cancer and normal cells.52 The mechanism of action of RAPTA compounds was first believed to be related to DNA binding. However, more evidence suggests now that RAPTA mainly targets various proteins. For example, RAPTA-C weakly inhibits thioredoxin reductase and cathepsin B, the enzymes that take part in the cellular response to oxidative stress.54 Thus, RAPTA induces an increase in reactive oxygen species (ROS) concentration in cancer cells.54 5 and 9 have demonstrated promising antimetastatic and antiangiogenic effects both in vitro and in vivo.55,56 The treatments using relatively low doses (0.2 mg kg−1, i.v.) of RAPTA-C significantly reduced the growth of primary tumors in preclinical models for ovarian and colorectal carcinomas (Fig. 5C). In addition, RAPTA-C inhibited microvessel density in both models (Fig. 5A and B), further supporting its antiangiogenic properties. Pharmacokinetics studies revealed that RAPTA-C was rapidly cleared from the organs and the blood via excretion by the kidneys, suggesting translational potential of RAPTA-C to the clinic.56 Triphenylphosphine (PPh3) is one of the most widely used ligands in coordination complexes, which are widely used as catalysts in fine organic synthesis and industrial processes. The influence of PPh3 on the antitumor activity of Ru complexes was demonstrated on a series of compounds with pyridine and PPh3 ligands (Fig. 6). Cytotoxicity of the compounds was studied against HL60 leukemia tumor cell line. The half-sandwich Ru complexes with small pyridine ligands are often practically nontoxic (IC50 > 200 mM for complex 15).47 However, the presence of PPh3 significantly increased anticancer potency (low micromolar range) of complexes 16–19, which was comparable to cisplatin. Besides, it was also shown that these complexes were able to bind DNA and distort its secondary structure. While 15 was covalently linked to DNA, complexes with PPh3 could also intercalate between DNA base pairs, which might explain their increased cytotoxicity.48 The importance of hydrophilic/hydrophobic balance was highlighted in a study of a series of compounds featuring ruthenium complexes with PTA and PPh3 as co-ligands (20 and 21). Cytotoxicity studies against TS/A mammary adenocarcinoma cell line demonstrated that only complex 20 was moderately active (IC50 ¼ 124 mM) while 5 (RAPTA-C), and 21 did not show any cytotoxicity. These results correlated well with the ruthenium uptake, which was three times higher for 20 compared to 21. Notably, the presence of PPh3 yielded less selective complexes, as shown by the cytotoxic effect on nontumorigenic cell line HBL-100, which suggests a different mechanism of action in comparison with RAPTA-type complexes that were nontoxic for this cell line.49 Modification of phosphine ligand structure can also influence the biological activity of half-sandwich drug candidates, and some promising work has been done in this direction.50 For example, cytotoxicity of Ru complexes increased when larger, more hydrophobic phosphine ligands (such as 22 and 23) were used (Fig. 7). While ligand 22 was nontoxic for H460 carcinoma cells, its conjugation with a ruthenium core into complex ([Ru(Z6-p-cymene)(Z1–22)Cl2]) demonstrated increased cytotoxicity in a low micromolar range. When phosphine oxide was replaced by a diphosphine ligand, the toxicity of Ru complex was even comparable to cisplatin.51 Incorporation of pyrene moiety into phosphine ligand 23 resulted in a series of highly potent ruthenium complexes with the cytotoxicity against lung adenocarcinoma (A549), melanoma (A375), colorectal adenocarcinoma (SW620), and breast carcinoma (MCF7) human cell lines with IC50 from 2 to 36 mM. All tested compounds demonstrated low selectivity and were also toxic for

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Fig. 5 Representative immunohistochemical staining images (A) of the endothelial cell marker CD31 (brown) showing reduced microvessel density per mm2 in tumors treated with RAPTA-C normalized to the tumor surface area and provided as a % of the control (B) and Ki-67 positive nuclei (blue); growth curve of A2780 tumors grafted on the CAM showing tumor volume with respect to treatment day (C). Adopted from Ref. Weiss, A.; Berndsen, R. H.; Dubois, M.; Müller, C.; Schibli, R.; Griffioen, A. W.; Dyson, P. J.; Nowak-Sliwinska, P. Chem. Sci. 2014, 5, 4742–4748, published by the Royal Society of Chemistry.

Fig. 6 Ru complexes with PPh3 and pyridine derivatives.

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Fig. 7 Examples of triarylphosphine ligands utilized in Ru and Os cytotoxic complexes.

nontumorigenic epithelial breast cells (MCF10A). The most potent complex being [Ru(Z6-Me-benzoate)(23)Cl2]. Substitution of methyl groups in 23 by bulkier phenyl groups and replacing the methyl benzoate by p-cymene lowered cytotoxic activity of derived Ru complexes. Surprisingly, although pyrene is known to be an efficient intercalating agent, the mechanism of action of Ru complexes with pyrene-substituted phosphine ligands was not related to DNA targeting.52 Like phosphine ligands, phosphite derivatives of carbohydrates (3,5,6-bicyclophosphite-a-D-glucofuranoside) also became of interest as ligands for half-sandwich organometallic compounds due to their excellent solubility in water. A series of complexes of ruthenium and osmium with sugar phosphites have been developed and studied to fine-tune the hydrophilicity of the resulting complexes (Fig. 8).57–59 Cytotoxicity of these compounds against various human cancer and nontumorigenic cell lines was studied, with the highest activity detected against CH1 ovarian carcinoma (IC50 29–153 mM).57,58 Osmium complexes exhibited comparable cytotoxicity (IC50 50–113 mM).59 Ru complex 28 with sugar ligands functionalized with 5-fluorouracil or other substituents such uracil, or thymine were only modestly cytotoxic against human ovarian cancer cells, suggesting that the significant modifications of introducing a sugar ligand might alter anticancer properties of the half-sandwich Ru complex.60 Several conjugates with other molecules with sugar ligands have also been reported (Fig. 8). For instance, the Ru complex decorated with maleimide via an aromatic moiety was reported for cysteine ligation. In vitro studies against human ovarian carcinoma (CH1), colon (SW480), and non-small cell lung cancer (A549) cells showed a significant increase in cytotoxicity of Ru-maleimide conjugate 29 (IC50 15, 13 and 92 mM respectively) in comparison with RAPTA-C (IC50 65, 170 and >640 mM respectively), highlighting the effect of possible immobilization of complex on protein surface for improved anticancer activity.47 Arene decoration with fluorescent anthracene moiety was also beneficial for cytotoxic properties of the complex 30, which although was moderately active (IC50 values in the range of 40–200 mM), demonstrated two-fold increase in toxicity in comparison with RAPTA-C against several cancer cell lines.46 Incorporation of anticancer DNA alkylating agent chlorambucil into the aromatic part also significantly increased the cytotoxicity of resulting Ru complex compared to RAPTA-C, which was practically inactive. Moreover, the activity of the conjugate was also higher than the activity of chlorambucil alone (IC50 9 vs. 25 mM respectively against cisplatin-resistant ovarian cancer cells A2780cisR).61 The hydrolytic behavior of Ru and Os complexes was studied in detail in an aqueous medium by several spectroscopic techniques, including 1H and 31P{1H} NMR (Fig. 9). Initially, one chloride was substituted by a water molecule, resulting in the

Fig. 8 Ru and Os complexes 24–27 with sugar-derived phosphites, Ru complex 28 showing derivatization of sugar phosphite ligands with 5-fluorouracil and Ru phosphite containing arene ligand conjugated with maleimide 29, and anthracene 30.

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Fig. 9 Hydrolytic pathway of half-sandwich 24 (Ru) and 25 (Os) complexes of sugar-phosphates (left); 31P{1H} NMR spectra for the hydrolysis of 24 and 25 in D2O up to 72 h (right). Peak assignments for unhydrolyzed complex (24 or 25), monoaqua complex, products obtained by cleavage of the PdO bond; dimeric species. Adopted from Ref. Hanif, M.; Nazarov, A. A.; Hartinger, C. G.; Kandioller, W.; Jakupec, M. A.; Arion, V. B.; Dyson, P. J.; Keppler, B. K. Dalton Trans. 2010, 39, 7345–7352, published by the Royal Society of Chemistry.

formation of two diastereomeric complexes (b). This step can be reversed by adding 10-fold excess of NaCl. Next, hydrolysis of one of the phosphoester bonds leads to diastereomers c and ultimately leading to the formation of bis-aqua complex d. For 24, the formation of dimers with the general formula [Ru2(Z6-p-cymene)2(sugarHyd)2 ]3+ (X ¼ Cl−, OH−) was observed after 24 h of incubation. The addition of AgNO3 to a fresh solution of 24 resulted in the rapid formation of d, suggesting that such form will probably be present in a biological medium. The rate of hydrolysis for Os complex 25 was very slow compared to its Ru counterpart 24 (Fig. 9). Within the first 24 h, more than 50% of Ru complex 24 was hydrolyzed, whereas only 5% hydrolysis was observed for its Os analog 25.59 Replacing the chlorido ligands with bidentate carboxylato ligands significantly suppressed the hydrolysis both at the metal center and cleavage of PdO bond. However, this rendered the complexes non-cytotoxic, which indicate that the presence of the labile group is needed for interactions with cellular targets.59

15.09.4.2 RAED-inspired organoruthenium and -osmium complexes Sadler and co-workers developed half-sandwich Ru and Os complexes featuring a p-bonded arene, a halido ligand, and a 1,2-ethylenediamine or related bidentate ligands, yielding a class of so-called RAED complexes.33–36 The example of RAED half-sandwich complex is Ru[(
6-biphenyl)(ethylenediamine)Cl]PF6 31 (also known as RM175). RM175 and its osmium analog [(
6- biphenyl)Os(en)Cl]PF6 32 (Fig. 10) displayed promising anti-proliferative activity with IC50 values in the low micromolar range against human cancer cells.35,36 The halido ligands were shown to undergo ligand exchange reactions with water and ultimately with biological nucleophiles such as DNA. The hydrolysis rate and reactivity with biological molecules could be optimized by ligand choice. Generally, the rate of hydrolysis decreased depending on the halido ligand in the following order Cl  Br >I. In addition, the stronger electron-donating ability of the p-coordinated arene led to an increase in hydrolysis rate: Os(II) arene chloride complexes hydrolyzed up to 100 slower compared to their Ru(II) counterparts. The Os(II)-arene aqua complexes were also more acidic than their Ru(II) analogs.33–36 The nature of bidentate ligands has a strong influence on the hydrolysis, reactivity and biological properties of the complexes. While neutral N,N-donor 1,2-ethylenediamine provided essential H bonds to facilitate the interaction with DNA, which was suggested as cellular target for these complexes,33,34 its replacement with anionic O,O-bidentate ligands such as acac and hydroxyl pyr(id)ones resulted in complexes (see examples 33 and 34; Fig. 10) with remarkably fast hydrolysis rates and low anticancer activity. The low cytotoxicity of complexes with O,O-ligands was attributed to their low stability in an aqueous solution. The fast aquation of the complexes led to the cleavage of O,O-ligands and the formation of inert hydroxy-bridged dimers [(
6-arene)2M2(mOH)]+.62 On the contrary, M(II)-arene complexes 35 and 36 featuring anionic N,O-chelating picolinato ligands were significantly more stable in aqueous media and showed potent cytotoxicity. The cytotoxicity of OsII complex 36 was similar to that of carboplatin in human ovarian cancer cells.63–65 To enhance the cellular uptake, the OsII(picolinato) complex 36 was conjugated to cell-penetrating peptides (CPPs) such as polyarginine with adjustable chain lengths. The OsII(picolinato) conjugate bearing octaarginine resulted in significantly enhanced cell uptake and DNA binding, compared to the monoarginine analog; however, the latter was more cytotoxic.66 To exploit the overexpressed receptors of somatostatin on membranes of many tumor cells, a derivative of potent somatostatin agonist octreotide was incorporated into the OsII(picolinato) pharmacophore of 36. The conjugation of OsII(picolinato) with CPPs resulted in improved biological properties of the peptides. However, the bioconjugates were less cytotoxic compared to the parent OsII(picolinato).67

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Fig. 10 Ru and Os complexes with N,N-, O,O- and N,O-chelating ligands.

Additionally, bidentate azopyridine (azpy) derivatives were used to prepare [(
6-p-cymene)M(azpy)I]PF6 complexes 37 (M ¼ Ru) and 38 (M ¼ Os). It was shown that the strong back-donation of electrons from the metal center to azpy ligands has a pronounced effect on the reactivity and bioactivity of the complexes. Systematic structural variations were carried out to establish a structure-activity relationship. It was found that the anticancer properties were remarkably enhanced by replacing the p-cymene with biphenyl and the chloride ligand with iodide ligand. The presence of electron-donating substituents (e.g., OH or NMe2) on the phenyl ring or electron-withdrawing groups (e.g., F, Cl, Br or I) on the pyridine ring render metal complexes more cytotoxic compared to their unsubstituted analogs. The azo functionality was suggested to be involved in redox processes by interacting with glutathione. The complexes were highly stable and hydrolytically inert towards halide/aqua exchange reactions in the aqueous solution.68,69 The cytotoxicity of OsII(arene) complexes containing azpy ligands was in the nanomolar range against human colon, prostate, breast, lung, ovarian, and bladder cancer cells.68 The lead complex [Os(
6-p-cymene)(Azpy-NMe2)I]PF6 (FY026) 38 (Fig. 10) was at least 10-fold more potent than cisplatin. When used in combination with L-buthionine-sulfoximine, the anticancer activity of 38 was significantly improved in A2780 and A549 cancer cells. In in vivo studies using xenografted mice 38 demonstrated delayed growth of HCT-116 human colon cancer with no off-target toxicity.70,71 Os(II) arene complexes 39 and 40 containing iminopyridine as neutral N,N0 -donors were similarly cytotoxic as Os(II) azpy complexes. They underwent hydrolysis in aqueous conditions and showed adduct formation with the nucleobase guanine. These Os(II) complexes possibly exerted their cytotoxic effects by interfering with the redox signaling pathways in cancer cells.72 They were capable of oxidizing NADH to NAD+ in the aqueous solution via the formation of an Os-H intermediate. The Os(II) iminopyridine complexes 40 (Fig. 9) induced ROS production in A549 lung cancer cells. The Os(II) iodido complexes showed more potency and high selectivity for cancer cells. These were not cross-resistant with cisplatin or oxaliplatin.73 Using combinatorial chemistry and high-throughput screening, Ang and co-workers evaluated the anti-proliferative activity of a library of 442 Ru(II)iminopyridine complexes against ovarian (A2780, A2780cisR), breast (MCF7), and colorectal (HCT116, SW480) cancer cells. The lead compounds were hydrolytically stable and, in particular, [(Z6-1,3,5-triisopropylbenzene)RuCl(4methoxy-N-(2-quinolinylmethylene)aniline)]Cl showed excellent cytotoxicity in a low micromolar concentration range. It was suggested that this compound type might exert their cytotoxic effects independent of the p53 tumor suppressor gene.74 The biological properties of Ru(II) and Os(II) complexes of N,N0 -chelating ligands based on bioactive molecules have been investigated by several groups. The cytotoxicity and inhibition of cyclin-dependent kinases (CDKs) of organometallic Ru(II) and

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Os(II) complexes 41–46 derived from paullones and indolocarbazole have been reported by Keppler and co-workers.75–79 The Os(II) arene complexes showed significantly enhanced cytotoxicity compared to uncoordinated ligands.75 Other coordination motifs such as iminopyridine were incorporated into the paullone scaffold and in vitro cytotoxicity of the resulting organometallic complexes are reported in the micromolar range against A549, SW480 and CH1 cancer cells.76,77 The intraperitoneal and oral administration of Os(II) complexes demonstrated a significant reduction in the tumor in the murine colon cancer model CT-26. The compounds were well tolerated by the tested mice.78 The use of known kinase inhibitor staurosporine resulted in Ru(II) complexes 47 (also known as DW12) and its isostructural Os(II) complex 48, that showed nanomolar inhibition of kinase GSK-3b (glycogen synthase kinase-3).80 The metal center was essential in maintaining rigid 3D structures of molecules in the active site of the enzyme.81 A series of half-sandwich Ru(II) and Os(II) complexes (49 and 50, Fig. 11) has been prepared using bioactive 2-pyridinecarbothioamides (PCAs) with different groups substituted at the para-position of the phenyl ring.82,83 The nature of substituents at the phenyl ring influences the cytotoxicity of the PCAs and their corresponding metal complexes. The structure-activity relationship studies showed that the lipophilic PCAs with smaller substituents such as F, Cl, or Me and their organometallic complexes were more cytotoxic with IC50 values in the low micromolar range.83,84 The coordination of PCAs to Ru and Os metal centers as S,N-donor system led to remarkably stable compounds in aqueous conditions and in 60 mM hydrochloric acid (conditions resembling the stomach pH), allowing the use of the complexes for oral administration. The complexes underwent water/chloride ligand exchange reactions in aqueous conditions and the stable aqua complexes react with biomolecules. The X-ray diffraction structures of organometallic compounds with the nucleosome core particle revealed binding to histidine residues of the histone proteins.82 Subsequently, the proteomic-based target-response profiling investigation with an immobilized arene-functionalized derivative of 49 revealed that the PCA-organoruthenium complexes were highly selective towards cytoskeleton protein plectin.85,86 Plectin is a critical signaling scaffold protein that maintains the cytoskeletal architecture of the cell. When administered orally, 49 was well tolerated and demonstrated higher effectiveness against invasive melanoma (B16) compared to colon (CT-26) cancer in mouse syngeneic tumor models.85 Studies using laser ablation inductively coupled plasma mass spectrometry of organs of mice bearing a CT-26 tumor showed a homogenous distribution of higher concentrations of Ru in the liver and kidney compared to other organs.87 49 penetrated more deeply into the organs compared to an osmium analog.87 Size-exclusion chromatography inductively coupled plasma mass spectrometry experiments on the mice blood and serum showed that Ru complex bound to blood proteins transferrin, HSA, and immunoglobulins. In contrast, Os analog exhibited more selectivity for the ovary and the central nervous system.87

Fig. 11 (A) Chemical structures of plecstatin-1 (49) and it’s Os analog 50; (B) HMGB-1 release measured by ELISA from tumor spheroids treated with 49 (200 mM) for 72 h; (C) Representative confocal microscopy image of HCT-116 spheroids showing HMGB-1 translocation and enrichment in the cytoplasm. Adapted from Ref. Wernitznig, D.; Meier-Menches, S. M.; Cseh, K.; Theiner, S.; Wenisch, D.; Schweikert, A.; Jakupec, M. A.; Koellensperger, G.; Wernitznig, A.; Sommergruber, W.; Keppler, B. K. Metallomics 2020, 12, 2121–2133, published by the Royal Society of Chemistry.

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HCT-116 tumor spheroids treated with 49 demonstrated a collapse of the plectin network and a rearrangement of other cytoskeletal proteins. 49 was more cytotoxic in HCT-15, HCT-29, and HCT-116 monolayers with IC50 values of 32, 24, and 18 mM, respectively. However, it turned out to be inactive (IC50 >200 mM) in the multicellular tumor spheroids (3D).88 49 showed disruption of the cytoskeleton of tumor spheroids as a result of oxidative stress and immunogenic cell death (Fig. 11), providing evidence for the further clinical potential of this class of compounds.88

15.09.4.3 N-heterocyclic carbene (NHC) complexes N-alkylated imidazolium salts 51 are commonly employed in the preparation of NHC compounds and reacted with Ag2O to yield labile silver(I) NHC complexes 52 (Fig. 12). The latter are often used for the safe storage of ligands e.g., to avoid carbene dimerization and as precursors for facile carbene transfer. They afford the corresponding Ru-NHC compounds 53 via transmetallation with the dichloro(arene)ruthenium (II) dimer (Fig. 12). By virtue of their strong s-donor ability, NHC’s are ideal candidates for synthesizing moisture and air-stable compounds i.e., complexes, which can persist long enough in the human bloodstream and cell cytoplasm. This property may be harnessed by using these complexes as anticancer agents by tethering a biologically active molecule to the NHC moiety via an enzyme cleavable linker. In vitro studies showed excellent anticancer effects of a naphthalimide-conjugated Ru-NHC complex 54 (Fig. 13) to HT-29 cells (colon carcinoma).89 These effects were ascribed to the propensity of a planar naphthalimide fragment for intercalation between DNA strands. Another Ru-NHC half-sandwich complex 55 by conjugating 17-a-ethynyl testosterone to NHC via a disulfide bridge with subsequent attachment to Ru center (Fig. 13).90 This complex demonstrated a higher survival rate of mice with breast cancer xenografts upon a 44-day long treatment (5 doses spaced 4 days apart, 2 mmol kg−1, i.v.). Owing to the overexpression of hormone steroid receptors in tumor cells, the use of testosterone as a carrier vector facilitated the drug uptake by cancerous cells. Glutathione, which is present in the intracellular environment at high concentrations, quickly reduced the SdS bond, liberating the ruthenium complex for further action. Another study revealed that the use of a carboranyl-tethered NHC ligand 56 induced an 18-fold increase of cytotoxicity against A2780 cell lines. It also turned out to be efficient against cisplatin-resistant cells. Moreover, the carboranyl complex 56 can be potentially used in boron neutron capture therapy. Many complexes bearing aryl substituents on NHC moieties such as 57 and 58 have been investigated as well.91,92 Most of them displayed moderate to excellent cytotoxic activity against a wide range of cancer cell lines, which may be due to the presence of large hydrophobic aromatic surfaces and hence improved lipophilicity of these compounds. Although the precise mode of action of these

Fig. 12 The typical route to synthesis of Ru(II) and Os(II) NHC complexes.

Fig. 13 Representative examples of Ru-carbene and Os-carbene complexes with a high cytotoxic activity.

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NHC complexes is unknown, Ru-NHC half-sandwich complexes are believed to interfere with the DNA replication and are rather akin to cisplatin and inhibit certain enzymes such as thioredoxin reductase or cathepsin B. Recent studies provided some insight into the mechanism of protein inhibition by Ru-NHC complexes.93 X-ray structures of an adduct formed between the complex 59 (Fig. 13) and the hen egg-white lysozyme (HEWL) revealed that the cymene ligand was displaced by the His15 and Arg14 residues in one binding site and by Lys33 in the second one while residual water molecules completed the rest of octahedral coordination sphere. Interestingly, the NHC ligand remained tightly bound to the metal. Due to its strong s-donor ability, NHC was supposed to exert some kind of a trans effect and weaken the metal-cymene p-bonding. EPR studies pointed out the oxidation of the metal center to Ru(III), presumably due to the absence of stabilization of the lower oxidation state (II) brought about by the arene ligand. The high cytotoxic activity of a series of Ru (II) and Os(II) mesoionic carbene complexes 60 and 61 (Fig. 13) have been reported as well.94 High-resolution ESI-MS studies aimed at probing Ru complex-protein interactions with a model protein Ubiquitin (Ub) suggested the presence of a 60-Ub adduct, albeit at much lower concentrations than RAPTA-C complexes can attain when reacting with Ub, with the NHC ligand still bound to the metal. The Os complex 61 did not form any adduct at all; hence, the observed cytotoxicity may involve different cellular targets. These findings demonstrated the versatility of the mechanisms underlying the anticancer activity of Ru-NHC half-sandwich complexes.

15.09.4.4 Cyclometalated Ru(II) and Os(II) arene complexes Pfeffer and co-workers prepared a large library of cycloruthenated and cycloosmated complexes through a CdH activation reaction of N-donor ligands, which acted as C,N-, C,N,N- or C,N,C-chelators towards the Ru or Os center.95–97 Several of the compounds demonstrated potent anticancer activity similar or superior to cisplatin. Ru complex 62 (Fig. 14) showed inhibition of tumor growth in mice without any side effects on kidney, liver or the neural sensory system of the mice. Detailed modes of action revealed that the Ru complex did not cause DNA damage but induced the formation of ROS in cancer cells. Cancer cells treated with Ru complex showed activation of several genes such as Bip, XBP1, PDI, and CHOP, which are relevant to the endoplasmic reticulum stress pathway. Notably, the cytotoxicity of the Ru complex was significantly reduced when CHOP was silenced by RNA interference. The anticancer properties were dependent on the lipophilicity and inertness of the complexes towards ligand exchange reactions before cellular uptake. For example, the most lipophilic and kinetically inert Os complexes were the most cytotoxic. This is in contrast to triosmium organometallic clusters, where the presence of labile ligand was vital for their cytotoxic effects.98 Similar findings have been reported for
2-allene anticancer osmacycle 64 (Fig. 14) by Meggers and co-workers. The osmacycle showed remarkable stability and inertness towards ligand substitution reactions. This was attributed to the ability of Os to make simultaneously p and s bonds with an allene moiety and a carbon atom of a terminal double bond, respectively. The treatment of Burkitt-like lymphoma cells with osmacycle demonstrated the activation of caspases-9 and -3 and the reduction of the mitochondrial membrane potential.99 Another class of C,N-cyclometalated half-sandwich Ru(II)100 65 and Os(II)101 66 complexes (Fig. 14) containing the benzimidazole derivatives was developed by Ruiz and co-workers. The complexes showed potent anticancer activity with IC50 values in the low micromolar range in a panel of cancer cell lines including the cisplatin-resistant A2780cisR cells. Ru complexes increased caspase-3 activity in A2780 cells, indicative of apoptosis, and showed inhibition of angiogenesis in the human umbilical vein

Fig. 14 Representative examples of cyclometaled Ru and Os complexes.

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endothelial cell line.100 Os complexes demonstrated reduction of the NAD+ coenzyme, a decrease in the levels of intracellular ROS, and inhibition of tubulin polymerization.101 Similarly, a series of cyclometalated Ru(II) 67 and Os(II) 68 bearing 1,2,3-triazole-derived ligands demonstrated antiproliferative properties in the human tumor cell lines. The dissolution of the cyclometalates in DMSO-containing aqueous solution led to the formation of stable DMSO adducts. Detailed investigation using DMSO vs DMF as solvent as well as testing of isolated DMSO adducts demonstrated that DMSO coordination did no impact anticancer properties. This implied that in cell culture media the DMSO adduct formation may not be pronounced. Modification of the lipophilicity of the ligand has led to the increased cytotoxicity of the cyclometalates. Both Ru and Os complexes demonstrated effects on cell cycle distribution; however, Os complexes were more potent compared to their Ru counterparts.102

15.09.4.5 Bioconjugates of half-sandwich organoruthenium and osmium complexes Organoruthenium and -osmium complexes have been conjugated with a variety of bioactive agents. These include nonsteroid antiinflammatory drugs (NSAIDs) such as diclofenac or indomethacin,44 ethacrynic acid that per se potentiate anticancer activity,45,103–105 as well as cytotoxic drugs including cisplatin,106,107 and coumarin.108 It has been noticed that the combination of organometallic and organic drugs often leads to increased activity unseen for either component due to either additive or synergistic effects. For instance, NSAIDs can potentiate the activity of some anticancer drugs and also exhibit modest antitumor activity themselves. Thus, functionalization of Ru and Os organometallic complexes with NSAIDs was investigated to uncover the possible synergistic action. In this context, a number of such complexes has been synthesized with diclofenac (Dic) 69 and indomethacin (Indo) 70. First, ligands were obtained by modification of the NSAID via the available carboxylic acid function by esterification with pyridine derivative. Then, the ligands were reacted with RuII or OsII dimers to afford corresponding half-sandwich complexes 69 and 70 (Fig. 15). All the compounds were tested against human ovarian carcinoma cell lines A2780 and A2780cisR and showed cytotoxicity in the range of IC50 20–50 mM and 60–110 mM, respectively. Diclofenac derivative was more potent than indomethacin one against both cell lines, while 69b complex was more potent against cisplatin-resistant cell line A2780cisR.44 Another class of non-carboxylate NSAIDs such as meloxicam and piroxicam have demonstrated anticancer properties. These drugs offer multiple donor atoms for coordination to metal ions. As a result, half-sandwich Ru(II) and Os(II) complexes were prepared. The meloxicam and piroxicam either can act as mono-, or bidentate ligands depending on the reaction conditions such as pH and the solvent used in the reaction.109,110 The limited stability was observed for complexes with monodentate coordination of oxicam in DMSO. The oxicams underwent ligand exchange with DMSO very quickly, making these compounds unsuitable for biological studies where DMSO is used for dissolving compounds. In contrast, complexes with bidentate coordination of oxicams demonstrated significantly higher stability and were investigated for their in vitro cytotoxicity. The complexes exhibited low to moderate in vitro cytotoxicity. The meloxicam complexes were the most cytotoxic and their cytotoxicity was similar or superior to that of investigational drug NKP1339/IT-139 and NAMI-A.109 Ethacrynic acid (EA), a well-known diuretic and also a potent inhibitor of glutathione S-transferase (GST), was shown to enhance the anticancer activity of cisplatin,111 was also exploited for modification of half-sandwich Ru and Os compounds 71–74.

Fig. 15 Ru and Os half-sandwich conjugates with NSAIDs and ethacrynic acid.

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As grafting of EA via a carboxyl group did not change its bioactivity, this mode of functionalization was used to prepare imidazole and pyridine ligands to obtain metal complexes (Fig. 15). For instance, complex 71 showed activity against the cisplatin-sensitive A2780 (IC50 10 mM), and the cisplatin-resistant A2780cisR (IC50 13 mM) human ovarian carcinoma cell lines in comparison with EA (IC50 around 55 mM) and Ru-imidazole complex without EA attached (which did not demonstrate any anticancer activity).105 Other complexes also showed anticancer activity within 8–40 mM concentration range against cisplatin-sensitive and cisplatinresistant human ovarian cancer cells. In all cases, EA-conjugated complexes were more active than either EA or nonconjugated complexes, suggesting their synergistic action.103,104 Similar to the complexes with N-donor ligands, the P-donor ligands have also been employed as a platform for bioconjugation. For instance, Ru complex conjugates with acetylsalicylic acid, ibuprofen, diclofenac, indomethacin, ethacrynic and valproic acid via functionalization of (4-hydroxyphenyl)diphenylphosphine ligand have been synthesized. All the conjugates exhibited cytotoxic activity against human ovarian carcinoma (A2780) and cisplatin-resistant human ovarian carcinoma (A2780CisR) with IC50 as low as 9–10 mM for valproic acid and 12–14 mM for ibuprofen conjugates.112 Diclofenac and indomethacin conjugates of Os complex also demonstrated cytotoxicity in the range of IC50 4–75 mM.44 Various conjugates with RAPTA have also been obtained with promising activities, including ethacrynic acid,48 anthracene,46 chlorambucil, naphtalimide113. The latter conjugate, for instance, demonstrated a noticeable increase in cytotoxicity against cisplatin-sensitive (A2780) and -resistant (A2780cisR) human ovarian carcinoma with IC50 as low as 2 mM (in comparison with RAPTA-C with IC50 230 and 270 mM, and similar naphthalimide ligand alone, IC50 18 and 26 mM respectively). Though, the overall toxicity of such intercalating conjugates against normal cells is also high (IC50 ¼ 6.6 mM). Another way to improve the efficacy of anticancer drugs and reduce the collateral damage of healthy tissues is a smart delivery of drugs to target tissues. One of the approaches consists of in situ functionalization of human serum albumin (HAS), which can get into cancer cells via an EPR mechanism and thus deliver the drug inside a tumor.47,84,114 With this in mind, several Ru and Os complexes 75 and 76 with pyridine and indazole ligand bearing maleimide function have been prepared and tested (Fig. 16). The maleimide functionalized complexes 75 and 76 demonstrated fast reactions with biological thiols such as cysteine, confirming the possibility of their conjugation to HSA and thus possible enhanced delivery into a tumor. Anticancer activity of the complexes was evaluated in vitro against human colorectal carcinoma (HCT116), non-small cell lung carcinoma (NCI-H460), and cervical carcinoma (SiHa) cells, showing activity in the range of IC50 from 8 to 35 mM for complexes with pyridine ligand and from 11 to 164 mM for complexes with indazole ligand; while osmium complexes were generally more active. It was also noticed that chlorido complexes were less active than the more hydrolytically stable complexes with iodido or oxalato ligands.47,84,114 The molecular recognition process was also used to target tumors over healthy tissue selectively. It is known that cancer cells often overexpress specific receptors at their surfaces to bind useful molecules, such as vitamins, efficiently. In this case, conjugating an anticancer drug to a vitamin molecule could increase treatment selectivity and efficiency. By following this approach, Ru half-sandwich complexes 77 with biotinylated indazole and pyridine ligands with various linkers in between were prepared and tested against colon adenocarcinoma (COLO205), colon carcinoma (HCT116), and colon adenocarcinoma (SW620) cells, the latter having high expression of sodium-dependent multivitamin transporter (SMVT) protein (Fig. 16). Complexes with pyridine ligands were more toxic than the complexes with indazole, but no conclusive evidence could be seen on the spacer’s influence between the coordinating part and biotin. For instance, indazole complexes were almost inactive against COLO205 cell line, while Ru pyridine complexes showed activity in a range of 5–17 mM. All the complexes were more active against cell line with a high level of SMVT proteins (IC50 of 2–4 mM for pyridine and 14–29 mM for indazole complexes), suggesting that the molecular recognition of biotin by SMVT drives the increase in the anticancer activity of studied complexes.115

Fig. 16 Half-sandwich complexes of Ru and Os conjugated with maleimide and biotin moiety.

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15.09.5 Multinuclear Ru and Os organometallics Combination of more than one metal offer opportunities to develop novel chemotherapeutics with unique biological properties and modes of action due to specific interactions with cellular targets. The trinuclear Pt complex BBR3464 reached human clinical trial and has shown DNA binding mode distinct from cisplatin and other Pt-based drugs.27 Inspired by the BBR3464, significant attention was devoted to the design of bi- or multinuclear organometallic compounds. In many examples, the combination of more than one metal centers significantly increased the cytotoxicity of compounds compared to their mononuclear counterparts, suggesting strong synergic action between different metals and highlighting the potential of this approach.116–125 It was shown recently that although RAPTA-T (9) was not highly toxic itself, it was modulating the cytotoxicity of a platinum drug, which was inactive alone against chemo-resistant mesothelioma. It has been suggested that RAPTA-T was responsible for an increase in vascular density around the tumor, which allowed the access of cytotoxic platinum drug to cancer cells.126 Also, RAPTA-T displayed a distinct enhancement of auranofin cytotoxicity (gold-containing antiarthritic drug with sub-micromolar activity) when used together, via an allosteric mechanism. X-ray crystal structure of nucleosome core particle (NCP) revealed that two molecules of RAPTA-T were present in so-called “acidic patch” on the histone H2A-H2B dimer. Remarkably, auranofin could not bind NCP even at 1 mM concentration, but when RAPTA-T was present, an adduct of NCP with both auranofin and RAPTA-T was obtained, strongly suggesting the allosteric effect of the latter.127 These studies have demonstrated that combining the two or more metal centers in one drug may have result in overall improved or entirely new pharmaceutically properties. This multivalency approach was applied to increase the activity of RAPTA by linking two ruthenium metal centers 78 together via linkers of various lengths and nature (Fig. 17). Multivalency ensures higher binding constants of RAPTA to the histone binding site and increased cytotoxicity of compounds against HeLa cells with IC50 from 1893 mM (RAPTA-C) to 29.5 mM for C10 linked RAPTA (78c), comparable to cisplatin (35.7 mM).116 Using the same approach, isostructural homo- OsII-OsII and heterodinuclear RuII-OsII organometallics were prepared. Both homo- and heterodinucelar complexes demonstrated anticancer properties superior than cisplatin and mononuclear RAPTA compounds. For example, the homonuclear RuII-RuII and heteronuclear RuII-OsII complexes showed IC50 values of 1.6 and 1.3 mM compared to >50 mM for cisplatin in the human colorectal adenocarcinoma (HT-29) cells. Unlike the RAPTA-C which significantly impacts the cell-cycle distribution of cancer cells, the dinuclear compounds showed only minor effects on cell-cycle phases, confirming that they exert their biological effects in a manner atypical to mononuclear drugs.42,45 Similarly, heterodinuclear complex 79 showed promising results in terms of cytotoxicity against cisplatin sensitive (A2780) and cisplatin-resistant (A2780cisR) human ovarian carcinoma with IC50 of 6.9 and 8.5 mM, respectively.128 Recently, water-soluble bimetallic anticancer drug candidates have been prepared by conjugation of Ru half-sandwich compounds with octahedral platinum (IV) prodrug (Fig. 17).107 First, a Pt-pyridine ligand was synthesized from N-hydroxysuccinimide ester of 3-pyridinepropionic acid and platinum (IV) complex (c,c,t-[Pt(NH3)2Cl2(OH)2]). A set of bimetallic complexes 80a–80c were obtained by reacting Pt-pyridine ligand with Ru arene dimers. Apparently, such a combination resulted in several essential improvements due to the synergic action of both metals. First, the Ru-Pt bimetallic compounds 80 have a higher solubility in water in comparison with cisplatin. After solubilization, rapid changes in 1H NMR suggested the exchange of chlorides for water molecules next to the Ru atom. The spectrum did not change for up to 4 days suggesting good stability of formed aqua complexes.

Fig. 17 Representative examples of hetero- and homo-bimetallic half-sandwich compounds of Ru.

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Organometallic Chemistry of Anticancer Ruthenium and Osmium Complexes

Strikingly, all the bimetallic complexes 80 demonstrateed sub-micromolar and nanomolar cytotoxicity against multiple tested human cancer cell lines, including cisplatin-resistant A2780cisR and A549cisR cells (with 107-fold increase comparing to cisplatin). At the same time, their general toxicity against noncancerous skin fibroblasts (Hs27) was comparable, and against normal lung fibroblast cells (MRC-5) was even lower than that of cisplatin.107 Thus, a combination of Pt (IV) prodrug with Ru (II) half-sandwich complexes could be a game-changer in the field of organometallic anticancer therapies. Keppler and co-workers reported di- and trinuclear metal-arene complexes containing 3-hydroxy-4-pyridones with varying length of aliphatic spacers.129–132 The cytotoxicity of dinuclear complexes was dependent on the length of the aliphatic linker between the two 3-hydroxy-4-pyridone units. This may be related to the lipophilicity of the compounds. The most cytotoxic compound 81 (Fig. 17) was the most lipophilic with IC50 in the sub-mM range against a wide range of cancer cells, and no cross-resistance to oxoplatin was observed.11,133–135 In contrast to Pt drugs, dinuclear Ru(II) complexes demonstrated cross-linking of DNA duplexes and DNA with proteins.132 Dinuclear Ru(II) complexes (Fig. 17) were more potent compared to structurally related mononuclear and trinuclear analogs. The nature of monodentate halido ligands as well as p-bound arene did not significantly influence the antiproliferative properties.130 Os(II) dinuclear complex was less cytotoxic compared to its Ru(II) analog. However, swapping Ru(II) with Rh(III) and Ir(III) yielded dinuclear complexes with similar potency and DNA damaging ability.136 More recently, dinuclear Ru(II) and Os(II) complexes containing 3-hydroxy-4-thiopyridone were isolated in polar protic solvents. The sulfur of 3-hydroxy-4-thiopyridone acting as bridging ligand between two M(arene) fragments.137 The doubly charged dinuclear complexes showed high aqueous solubility and stability, and were potent antitumor agents in A549, SW480, and CH1/PA-1 cell lines. A similar trend in cytotoxicity was observed in HCT-116 tumor spheroids.137 On the way to “smart medicine,” the use of stimuli-responsive drugs is of primary interest. Such an approach was also used with respect to organoruthenium and osmium compounds. For instance, thermoresponsive Ru half-sandwich complexes were obtained when using pyridine ligand with grafted perfluoroalkyl chains (Fig. 18).138,139 Their anticancer activity was tested against various cancer cell lines, including human ovarian carcinoma (A2780) and human endothelial (ECRF24) cells. Strikingly, cytotoxicity of Ru complex 82a increased dramatically from almost negligible at physiological temperature (37  C) to high against all the studied cancer cell lines at mild hyperthermia (41  C) with IC50 values from 5 to 132 mM. The activity of other Ru and Os complexes with different perfluoroalkyl or alkyl chains was also assessed at 37  C and showed promising results.140,141 Activity of 82a complex was also studied in vivo against human adenocarcinoma (LS174T) implanted into mice. Application of both hyperthermia and injection of 82a (3 doses spaced 4 days apart, 12.5 mg kg−1, 300 mL, i.p.) led to up to 90% inhibition of tumor growth compared to the control group. Combined action was also more potent than the use of hyperthermia or 82a alone.142 In a follow-up study, it was shown that a slight modification of perfluoroalkyl chain from 10 to 8 carbon atoms did not alter antitumor activity but significantly altered the mode of action (antiangiogenic mechanism for C10 chain, and both anticancer and antiangiogenic mechanisms for C8 chain).143 Conjugation of Ru complexes with pyridine and imidazole ligand with pendant ferrocene (Fc) as a redox-active moiety also displayed promising results (Fig. 18).122,144,145 Their anticancer activity was assessed in vitro against human colon adenocarcinoma (HT29) and showed IC50 values in the range of 20–58 mM for all the complexes (82–86). It was found that the cytotoxicity of these

Fig. 18 Structures of pyridine ligand functionalized with C10 perfluoroalkyl chain; imidazole and pyridine ligands bearing ferrocene moiety; and half-sandwich complexes with tetrapyridyl porphyrin.

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compounds correlated with the redox potential of both metal centers (Fc+/0 and RuIII/II)—the lower the redox potential—the higher the cytotoxicity. The suggested mechanism implies Fc-catalyzed generation of ROS inside the cells, decreasing their viability. The complexes also demonstrated antibacterial and antioxidant activity.145 ROS can also be generated using photochemistry, and tetraphenyl porphyrins are known photoactive compounds in the field of photodynamic therapy. The combination of organometallic Ru and Os complexes with photodynamic therapy into one molecule delivered some promising results (Fig. 18).146,147 First, tetrakis Ru and Os adducts with tetrapyridyl-porphyrin (87a and 87b) were tested in dark in vitro against human Me300 melanoma cells and showed moderate cytotoxicity (IC50  50 mM). Although 87b was more active in the dark, 87a showed 90% cytotoxic efficiency upon exposure to light, while 87b was only 20% active.148 One of the interesting approaches to increase the effectiveness of a drug is conjugation with the compounds exhibiting the EPR effect, such as polymers. In this light, dendrimers are of particular interest as they are monodisperse, which is favorable for analysis and controlled delivery. They can also be easily functionalized in a homogenous manner with multiple groups attached to one dendrimer molecule. Thus, a set of carbosilane dendrimers (88–90) with pendant half-sandwich Ru complexes has been synthesized and tested against cervical HeLa, breast MCF-7, colon HT-29, and triple-negative breast MDA-MB-231 cancer cells (Fig. 19). Three dendrimer generations displayed cytotoxicity against all the tested cell lines. However, G0 dendrimer (88) was much less active (IC50 26–53 mM) than G1, 89 (IC50 2.5–4.9 mM) and G2 dendrimers, 90 (IC50 3.4–9.1 mM). Slightly higher activity of G1 vs. G2 can be explained by the higher solubility of the smaller dendrimer.

15.09.6 Cytotoxic organometallic clusters of Ru and Os Organometallic ruthenium and osmium clusters have demonstrated promising anticancer properties.23,27,149 A series of triruthenium-carbonyl clusters based on glucose-modified bicyclophosphite ligands showed cytostatic, and cytotoxic activity. The ruthenium clusters were found stable under aqueous conditions. The anticancer properties can be fine-tuned by increasing the number of bicyclophosphite, which enhances the lipophilicity and cellular uptake of the clusters in cancer cells. The organoruthenium clusters 91–93 (Fig. 20A) showed antiangiogenic activity in the in vivo chicken chorioallantoic membrane model (Fig. 20B and C).149 Similarly, antiproliferative properties of triosmium carbonyl clusters with the general formula Os3(CO)12-n(L)n (L ¼ nitriles, acetonitrile, maltol, triphenyl phosphine, etc.) were investigated against estrogen receptor (ER)-dependent MCF-7 and ER-independent MDA-MB-231 breast cancer cell lines.24,25,98 The osmium clusters were more potent with IC50 values in the low micromolar range against an ER-independent cell line as compared to an ER-dependent cell line. The triosmium clusters with labile ligands such as acetonitrile or maltol were more potent compared to those bearing a non-labile phosphine ligand.98 In the modes of action studies, the Os clusters showed induction of DNA fragmentations, chromatin condensation, disruption of the microtubules morphology, caspase inhibition and an elevated level of p53. Also, the interactions of Os clusters with intra- and extracellular sulfhydryl moieties were observed, which is relevant to their tubulin disruption ability.25

15.09.7 Conclusions Organoruthenium and -osmium compounds offer a versatile platform allowing fine-tuning of the biological properties towards the discovery of new therapeutic agents. During the last decade, bioactive organometallic compounds of Ru and Os have attracted considerable attention. Significant advances have been made in the field of organoruthenium anticancer agents, while organoosmium compounds picked up momentum relatively recently. Several compound classes have clearly demonstrated potential for biological applications. The individual components of organometallics dictate not only the biological properties but also the physicochemical properties such as stability in the aqueous environment and solubility. The most widely investigated organometallic compounds of ruthenium and osmium are metallocenes, half-sandwich complexes, cyclometaleted, metalloclusters and multinuclear organometallics. The lead compounds demonstrated potent bioactivities and diverse mechanisms of action. The inactive half-sandwich complexes with O,O-chelating ligands are typically unstable possibly due to the formation of inert dimeric species. The stable and anticancer active organometallic complexes with N,N-and N,O-bidentate ligands preferentially target DNA.

Fig. 19 Carbosilane dendrimers with pendant Ru half-sandwich complexes coordinated via pyridine ligands.

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Fig. 20 (A) Chemical structures of trinuclear Ru carbonyl clusters derived from sugar-phosphates, (B) Angiographic images visualized by FITC-dextran fluorescence angiography of the developmental CAM (EDD 9) treated with 0.9% NaCl (control) and 91 and 93 (150 mM/day), (C) Quantification of digital analysis of the fluorescence angiography images: number of branching points (mm2), scale bar 200 mm. Red arrows indicate the avascular zones. Adopted from Ref. Nazarov, A. A.; Baquie, M.; Nowak-Sliwinska, P.; Zava, O.; van Beijnum, J. R.; Groessl, M.; Chisholm, D. M.; Ahmadi, Z.; McIndoe, J. S.; Griffioen, A. W.; van den Bergh, H.; Dyson, P. J. Sci. Rep. 2013, 3, 1485, published by the Springer Nature.

Kinetically inert organometallics of azopyridine as N,N-bidentate ligands induced cytotoxic effects through intracellular redox chemistry. Whereas MII(arene) (M ¼ Ru or Os) scaffold coordinated to S,N-chelating ligands demonstrated exceptionally high stability and high selectivity for plectin. The data from a vast diversity of the structural motifs confirmed that the choice of ligand system profoundly influences the anticancer properties and modes of action of organoruthenium and -osmium compounds. As expected for 3rd row element, organoosmium compounds generally exhibited higher stability and inertness towards hydrolysis and/or ligand substitution reactions. In some compounds, the metal center remains an innocent spectator; Ru and Os play a structural role by maintaining the three-dimensional rigid structure essential for binding with a biotarget in DW12 and its Os analog. In many other compound classes, Ru and Os participate in binding with biological targets such as DNA, proteins or enzymes. When comparing ruthenium over osmium among different classes of bioactive compounds, no clear lead emerges. In some cases, organoruthenium were significantly more active while in others, organoosmium demonstrated superior biological properties. In addition, in some examples, the properties of organometallic compounds seemed independent of the nature of the metal center. To conclude, the interest in the field of bioactive organometallic compounds of ruthenium and osmium has been continuously growing. These compounds exert their biological effects by interfering with diverse cellular pathways. We believe that a number of compounds with interesting biological properties will be uncovered in the future.

Acknowledgments We thank the Health Research Council of New Zealand (Sir Charles Hercus Fellowship to MH), and the University of Auckland for financial support. The work described in this paper was partially supported by a grant from the City University of Hong Kong (Project No. 7200682 and No. 9610518).

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15.10

Organometallic Chemistry of Drugs Based on Technetium and Rhenium

Roger Alberto, Department of Chemistry, University of Zurich, Zurich, Switzerland © 2022 Elsevier Ltd. All rights reserved.

15.10.1 Introduction 15.10.1.1 General aspects about technetium and rhenium drugs 15.10.1.2 Properties of technetium in molecular imaging 15.10.1.3 Rhenium in radiotherapy and in “cold” drugs 15.10.2 Technetium imaging agents 15.10.2.1 Cancer targeting 15.10.2.2 Labelled peptides and proteins 15.10.2.3 Alzheimer’s disease and b-amyloid targeting with 99mTc 15.10.2.4 Cell nucleus targeting and Auger electrons 15.10.2.5 Labelled nanoparticles – Multi modality imaging 15.10.3 Small molecules with rhenium and 99mTc homologs 15.10.3.1 De novo matched-pair complexes with Re and 99mTc 15.10.3.2 Homologs of Re and 99mTc with pendent pharmacophores or substrates 15.10.3.3 Pharmacomimetics with integrated complexes 15.10.4 Miscellaneous 15.10.5 Concluding remarks Acknowledgments References

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15.10.1 Introduction 15.10.1.1 General aspects about technetium and rhenium drugs The radioisotope 99mTc is the workhorse of nuclear medicine and in molecular imaging since many decades, but this position is slowly adopted by competing or complementary imaging modalities such as positron emission tomography (PET), optical imaging, magnetic resonance imaging (MRI) and other non-invasive methods. Reasons for this are manifold; technical aspects such as higher image resolution, availability and shortages of 99Mo/99mTc generators1–3 and an industrial focus on PET is one, the more reliable retention of receptor affinities by labelling inhibitors with single atom substitutions such as 18F another. The conjugation of a 99mTc complex (or any other complex) represents generally a major structural and pharmacological impact on the lead structure, and new ways and concepts have to be sought for re-introducing 99mTc compounds in the imaging field as versatile drugs. Furthermore, as reported in another chapter of this book series, the comparably demanding chemistry of technetium further affects incentives to label biomolecules with 99mTc. Its chemistry covers at least eight oxidation states, many of which are accessible in water. This complexity is in stark contrast to the comparably simple coordination chemistry of radionuclides of the “3+ family,” comprising e.g. Gd3+, 67Ga3+, 153Sm3+, a full series of Tb3+ radioisotopes and others.4–6 For this family, well-established bifunctional ligands with known chemistry are available and in which redox chemistry does not play a role. These features render labelling a routine process, albeit the impact on bioactivity may be substantial since variations of the ligands are not easily achieved and chemically limited. For the preparation of 99mTc-based imaging agents, several cores or building blocks have been reported, among them the [99mTc]O]3+, [99mTc^N]2+ and the fac-[99mTc (CO)3]+ cores.7 Efforts to turn these cores into suitable molecular imaging agents or radiopharmaceuticals focus essentially on them. Over the past years, the “carbonyl fac-[99mTc(CO)3]+ core” was clearly at the center of interest, with one compound advancing through different clinical phases. Currently, this radiopharmaceutical is market introduced in Europe for prostate cancer imaging (vide infra). The situation with 99mTc is peculiar, as compared to its higher homolog rhenium. Due to the short half-life time of 99mTc (t1/2 ¼ 6 h), all preparations have to be effectuated on site, i.e. in the clinics, whereas rhenium complexes can be prepared like routine pharmaceuticals. This notion is relativized by alternative strategies, taking new approaches for 99mTc preparations into account. The radioisotope 99mTc is currently obtained from 99Mo/99mTc generators. New strategies have to be found for safeguarding future demands, since reactors producing 99Mo are shutting down. One of these ways is the direct preparation of 99mTc in the form of [99mTcO4]− from cyclotrons by e.g. proton irradiation of molybdenum.8–10 Such methods can ensure the availability of 99m Tc for the currently used radiopharmaceuticals but it does not support the development of new ones since research in this direction relies on the onsite availability of 99mTc. Still and as obvious from the following sections, 99mTc chemistry is a very active field. However, to regain its importance, new concepts for applying 99mTc have to be developed and implemented with the support of the “imaging industry.” One of these new concepts is the combination with radioactive rhenium in the form of the radioisotopes 186Re or 188Re. Alternatively, cold rhenium complexes as reviewed in Section 15.10.3 may act as congeners of 99mTc imaging agents. In this respect, the original “matched-pair” idea, rhenium for therapy and technetium for diagnosis with homologous complexes, has seen a certain revival. The matched-pair concept 188Re/99mTc is however affected by the limited availability of 188W/188Re generators. The current

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situation with high-flux reactors will hardly improve to cover the demand for 188Re, in case corresponding radiopharmaceuticals will be developed. Although 186Re has superior decay properties over 188Re as a therapeutic radioisotope, its specific activity as obtained from neutron irradiation of isotope enriched 185Re is too low. Both drawbacks could be overcome by a new strategy. In this respect, investigations toward “cold” rhenium-based complexes as medicinal inorganic drugs are gaining momentum (Scheme 1). In case such a “traditional” medicinal inorganic drug is discovered, it could be spiked with one of the radioisotopes mentioned above to follow its in vivo behavior.

Scheme 1 The different concepts for organometallic 99mTc and rhenium chemistry in the life sciences: De novo imaging agents with no biological structures (A), Receptor targeting 99mTc complexes for exclusive imaging purposes, little or no therapeutic activity expected (B), de novo rhenium complexes with distinct cytotoxic effects and luminescence properties for which the 99mTc homolog is accessible (C), 99mTc complex with cytotoxic tag for imaging and its rhenium homolog for therapy, the metal complexes as such are non-toxic (D) and the integrated approach, mimicking a lead structure which analogs are accessible for rhenium and 99m Tc (E). C-E have a theranostic option. The compounds will be referenced in the corresponding subsections.

Medicinal inorganic chemistry is a rapidly growing field. As reviewed in the subsequent sections, the vast majority of these investigations is based on complexes with the fac-[Re(CO)3]+ core. A few other oxidation states of rhenium complexes punctually complement ReI. In contrast to platinum, ruthenium, gold and other late transition metal elements, rhenium complexes have hardly been considered for therapeutic purposes. Only the advent of the 99mTc complex fac-[99mTc(OH2)3(CO)3]+ may have triggered research with cold rhenium homologs. Chemically spoken, the advent of rhenium might well be due to the H2O ligands bound to 99m Tc(I) or Re(I), which can easily be exchanged with numerous ligand classes. The known photoactivity of some diimine complexes of the fac-[Re(CO)3]+ fragment may be another reason. When searching the Web of Science® for the keywords “rhenium” and “cytotoxicity,” one can find three quotations for the 1990s, 38 for 2001–2009 and 188 for 2010–2020. This mirrors the exponentially growing role of cold rhenium complexes in medicinal inorganic chemistry very well. Rhenium has clearly an underestimated potential in this respect, as pointed out by Gasser et al.11 Based on this new matched-pair concept, cold rhenium complexes for therapy and hot 99mTc homologs for imaging might provide 99mTc with a new role for resuming its importance in diagnostic nuclear medicine and in theranostics.12 The following sections are divided according to these different aspects and considerations. Specialized reviews for the subchapters have been published, e.g. for the anti-cancer activities of rhenium complexes.13,14 The combination of rhenium and technetium (radio)pharmaceuticals15–17 and the chemistry and radiopharmacy of 99m Tc alone are the topics of others,18–22 as well as the speciation and toxicity of rhenium drugs.23,24 The combination of rhenium with other imaging modalities25–27 and 99mTc complexes for the targeting of special dysfunctions,28,29 for theranostics, multimodal

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imaging agents30 and others,12 mentioned at the corresponding subsections have been summarized as well but generally not with a focus on organometallic compounds.31,32 For 99mTc, excellent and comprehensive reviews appeared over the last decade,33,34 thus, this chapter will cover for 99mTc essentially those in context of the matched-pair concept over the last 1–2 decades, whereas for rhenium, earlier findings are reviewed where relevant for current developments. Scheme 1 gives a synopsis of the different concepts for rhenium and technetium applications in medicinal inorganic chemistry, together with references to the corresponding subchapters.

15.10.1.2 Properties of technetium in molecular imaging Two radioimaging modalities are core in today’s nuclear medicine applications, namely positron emission tomography (PET) and single photon emission computed tomography (SPECT), 99mTc being the most prominent representative for the latter application. 99m Tc is available from the 99Mo/99mTc generator, which is a convenient source, and one of the reason why this radionuclide has gained its importance and status it currently has. Its decay properties match the demands from routine clinical application and imaging instrumentations and finally yet importantly, 99mTc is cheap. Its prominent role can be extracted from an excellent compendium, summarizing not only 99mTc but also other radionuclides in nuclear medicine in the US over the past 50 years. Although the analysis focuses on the US, the situation would be similar if worldwide application is taken into consideration.35 From this analysis, it becomes obvious that 99mTc is carrying the field of nuclear medicine imaging, although this might change in the future. Furthermore, the availability of generators is jeopardized by the shutdown of some of the important reactors and facilities currently producing 99Mo as the parent to 99mTc.1,3 Alternative methods are being developed, all bringing advantages such as large GBq amounts of produced 99mTc along but also disadvantages such as the on-site, immediate availability of this radionuclide.9,36,37 Despite the favorable properties of 99mTc, there are drawbacks different from its availability but affecting its current role. Without going into detail on the advantage of PET, this imaging modality provides generally a better resolution and allows microdosimetry. With respect to PET radionuclides (apart from 18F and 11C), prominent representatives are from elements, which are most stable in their 3 + oxidation state and do thus not require any redox chemistry for the preparation of radiopharmaceuticals. Radionuclides in PET are 68Ga3+, various terbium isotopes,38 but also 111In3+ (SPECT) and others. In contrast, the preparation of 99mTc radiopharmaceuticals or molecular imaging agents may demand a complex series of reactions. These include the reduction of [99mTcO4]−, unless the target compound remains in the +VII oxidation state, which is rare,39–41 and concomitant chelation by a strong ligands accompanied by e.g. their deprotonation. Especially the redox processes are difficult to control since intermediate oxidations states may be thermodynamic sinks in which the 99mTc is trapped. This leads to undesired side products. As for regular drugs, the purities of administered radiopharmaceuticals must be >99%. If side products are present, imaging may lead to false interpretations or to decreased target to non-target ratios. Side products or starting materials such as [99mTcO4]− may accumulate in critical organs such as the thyroid, which leads to substantial doses in non-target organs. These features prohibit obviously applications in humans when side products are present. Whereas coordination of radionuclides of the 3+ family to multidentate chelators is a fast process, often performed at room temperature, reduction and coordination in 99mTc chemistry often demands elevated temperatures. Taking these conditions together reveals the difficulty of discovering and developing new radiopharmaceutical 99mTc complexes but at the same time offers numerous attractive opportunities for research and applications. Whereas reported ligands for 68Ga3+ are limited to a comparably small number of donor atoms, the coordination sphere around 99mTc is highly flexible and oxidation state dependent. This allows a case-dependent ligand design, adapted to the target and the biological environment in which it exists. Complex charges and lipophilicities can be altered, which leads straight into organometallic chemistry, particularly distinct in the low oxidation state of technetium. The +I state is well explored since the stabilities of complexes are of kinetic rather than of thermodynamic origin. Robustness was the key to the development of new radiopharmaceuticals and ultimately induced the field of rhenium complexes for therapeutic purposes.42,43 The chemistry with e.g. 68 Ga starts to make steps into the direction of more ligand flexibility but the chemical nature of this element still makes the design challenging since kinetic robustness is not an consequence of the electronics of the central atom but rather a mechanistic one of ligands with very high denticities.44–46 Although not largely developed, efforts to prepare the PET nuclide 94mTc for imaging purposes have been performed in the past.47,48 A few targeting molecules have been labelled with 94mTc49–51 but its difficult production in isotopically pure form, the relatively short half-live t1/2 ¼ 52.0 min and the high decay energies b+max ¼ 2.44 MeV (70.2%) as well as g-rays with energies of 871 (94.2%), 1522 (4.5%) and 1868 keV (5.7%) respectively, hamper a routine study of new radiopharmaceuticals.52,53 Today, 99mTc is mainly used in the development of rhenium-based, cytotoxic drugs comprising the fac-[M(CO)3]+ core. As outlined in Section 15.10.2, labelled targeting molecules still play a certain role, encouraged by the successful introduction of the 99mTc labelled prostate imaging agent MIP-1404.54–56 For the labelling of biovectors, other cores are applied as well, mainly the [99mTc]O]3+, the [O] 99mTc]O]+ and the [99mTc^N]2+ cores but reports about their applications are minor as compared to the carbonyl core. Relevant strategies and concepts of imaging with 99mTc are shown in Scheme 1A and B.

15.10.1.3 Rhenium in radiotherapy and in “cold” drugs Rhenium has two radioisotopes which are useful for targeted radionuclide therapy, namely 186Re with a half-live t1/2 ¼ 3.8d, b−max ¼ 1.07 MeV and a suitable g-emission with an energy of 137 keV, and 188Re with a shorter half-live t1/2 ¼ 17 h, b−max ¼ 2.1 MeV associated with a g-emission of 155 keV. In the spirit of the matched-pair concept, the preparation of homologous

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99m

Tc and 188Re radiopharmaceuticals enables combining radionuclide therapy (186/188Re) with imaging (99mTc). The g-emission at 155 keV allows for following 188Re alone, albeit its contribution with 15% of the decays is at the border of detection, especially at later time points.6 The radionuclide 188Re is, similar to 99mTc, available from the 188W/188Re generator. Such a generator is “nice to have” and the half-live of 188W with t1/2 ¼ 69d comparably long and convenient. However, it is produced by a 2n,2g reaction from enriched 186W which requires high flux reactors.57 It has been noted that the neutron flux at a suitable reactor should be higher than 1015 n s−1 cm−1, otherwise the amount of tungsten 188W to be loaded on the generator becomes too high or the elution volume too large. Currently, there are only two of such reactors operational, namely in Russia at the Reactor Institute for Atomic Research and at the High Flux Isotope Reactor at Oak Ridge, USA. This is insufficient for providing enough 188W for a broader research community or routine applications. Still, many research reports on 188Re labelled targeting radiopharmaceuticals corroborate the importance of radionuclide therapy, as discussed later in this chapter. There are reports about the preparation of 188Re with accelerators but these approaches generally suffer from a concomitant formation of other, undesired rhenium radioisotopes. It is evident that routine clinical application cannot be based on a mixture of radionuclides and the original generator is therefore the best source for 188Re as an important radionuclide.58 The other appropriate radionuclide, 186Re, has conceptually superior properties for radionuclide therapy. It has a longer half-live, consistent to pharmacokinetics of e.g. antibodies, together with well-suited decay properties. The common method of neutron irradiation of 185Re targets does however not yield 186Re in sufficiently high specific activity. From normal research reactors, the achievable specific activities are too low to allow for receptor targeting, i.e. molecular imaging or therapy, since too many cold rhenium atoms are accompanying 186Re. A way around neutrons is the production with charged particles from accelerators. Indeed, numerous studies focus on this way of preparation but none has led to a routine production so far.59–61 To make neutron produced 186Re in its carrier added form still a useful radioisotope, application in medicinal inorganic chemistry is a prospective concept. As mentioned in the introductory section and detailed in the following, cold rhenium complexes with potential therapeutic properties have emerged as complements to the core transition metals such as platinum or ruthenium. Instead of relying exclusively on radionuclide therapy with e.g. 188Re for which chemotoxicity is negligible due to their minute quantities, a combination of both, radio- and cytotoxicity for non-carrier free 186Re is an option. Even if the radiotoxic effect is minor, non-invasive imaging of the g-emission of 186Re could allow following treatment, thereby enabling therapy prognosis of the efficacy of the treatment. This strategy is similar to the theranostic concept with homologous 99mTc and cold rhenium compounds. Along this line, 186Re complexes play a role at the interface between exclusive radionuclide therapy with 188Re and non-radioactive chemotherapy with rhenium complexes. The latest developments in inorganic drugs are a growing focus on rhenium-based compounds without direct involvement of radionuclides. The fac-[Re(CO)3]+ core and its complexes in particular offer opportunities as cyto- and phototoxic drugs any many promising approaches have been published and been reviewed in part.14,43,62,63 Most of these complexes are also feasible for 99mTc which assets a theranostic option. In addition to these small molecule drugs, a more recent and widely neglected opportunity are rhenium-cluster compounds. Clusters in general are a field, which has found little attention in medicinal inorganic chemistry. Octahedral Re6-clusters have found some interest due to their properties as high-Z X-ray contrast agents, their characteristic of sensitizing and producing singlet oxygen 1O2 for photodynamic therapy. The incorporation of rhenium with a high atomic number (Z ¼ 75), opens the additional opportunity to utilize the cluster-compounds also as radiosensitizers. Synergistic effects, enhancing the therapeutic efficacy can thus be expected in combination with radiotherapy.64,65 Extending atom-precise clusters to nanoparticles, rhenium has also been used to decorate gold nanoparticles for delivering large cargos of cytotoxic compounds to cancer cells. Nanoparticles in combination with cytotoxic rhenium complexes are a growing field as well and lead into dual modality imaging or to theranostics.66 Thus, rhenium in the field of nuclear medicine and medicinal inorganic chemistry has many options, most of which are not yet explored to a sufficient extent. These options range from a multitude of small molecules and targeting agents to dual-modality imaging, clusters and nanoparticles. In combination with 99mTc, they can contribute to almost any field of diagnostic and therapeutic medicine. The matched-pair concept is in this respect equal or even superior to other combinations of isotopes.

15.10.2 Technetium imaging agents 15.10.2.1 Cancer targeting De novo 99mTc organometallic complexes or coordination compounds are not discussed in detail here since they have been reviewed many times in the past (see above). Some small molecules are discussed in the context of rhenium-based-drugs.67,68 In the development of imaging agents, the targeting of cancer is the most import application of 99mTc-based molecular imaging agents. These compounds are complexes bound to targeting vectors such as proteins or peptides, may carry a moiety of pharmaceutical activity or are de novo compounds but structurally adapted to some characteristic properties for the target. This section will review cancer targeting with 99mTc probes different from these classical motives and not treated in the other sections. A representative example in this field is the prostate cancer-imaging agent MIP-1404, developed by the companies Molecular Insight Pharmaceuticals and later by Progenics.69 The structure of MIP-1404 is shown in Scheme 2. The prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer, and small-molecule radiopharmaceuticals targeting PSMA are very well suited to detect the location and extent of disease. The molecular PSMA target, not expressed in normal human prostate epithelium, allows the detection of metastatic prostate cancer as well as primary tumors. It is upregulated in prostate cancer, including metastatic disease.71,72

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Scheme 2 The prostate imaging agent “MIP-1404,” [99mTc]Tc-MIP-1404, developed by Molecular Insight Products and Progenics is based on the [99mTc(CO)3]+ core.54,69,70

MIP-1404, trade name Trofolastat®, underwent phase IdIII clinical trials.73–76 The development of fac-[99mTc]Tc-MIP-1404 is a representative example about how to discover and develop a new small-molecule radiopharmaceutical.55 Whereas the target and the targeting vector was given, linkers and chelators had to be altered extensively, until a bioconjugate was found, displaying the desired low- to sub-nanomolar affinity for its antigen. fac-[99mTc]Tc-MIP-1404 was also used to assess the success of prostate cancer treatment. The concordance between SPECT and biochemical response was 75%, significantly better than between SPECT and CT. This implies a possible role of [99mTc]Tc-MIP-1404 as an imaging tool for monitoring treatment of metastatic prostate cancer.77 In a more recent study, the potential of [99mTc]Tc-MIP-1404 was evaluated in patients with primary prostate tumor and metastasis. Since PET is not available at many places worldwide, it was of interest to what level SPECT with [99mTc]Tc-MIP-1404 would be useful. It was found that at low PSA levels of 100 mM (HeLa)

56

Artemisinin hybrid

Pyrazole hybrid

Peptide hybrid

Sugar hybrid

Fig. 9 Ferrocene-functionalized derivative of NAMI-A.

Ferrocenyl-containing rhodium(I) complexes were also reported to target large-scale necrosis, confirmed by morphological analysis with fluorescence microscopy.61 The use of ferrocene-containing heterometallic complexes for photoactivated metallochemotherapy has come to prominence.62 With a ferrocenyl group tethered to another transition metal complex, under irradiation the ferrocenyl entity acts as an antenna by providing reactive, singlet oxygen species through a Fenton-type pathway.63 The 1 O2 interacts with DNA and proteins resulting in apoptosis in cancer cells. Ferrocenyl groups have also been incorporated into L-amino acids, producing increased toxicity against HeLa cells after irradiation with 400–700 nm light in comparison with studies conducted in the dark.64

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15.11.3.5 Multinuclear, macromolecular ferrocenyl compounds A strategy often used to enhance the biological properties of metal-based compounds is “multinuclearity”, allowing larger multimetallic constructs to access different properties not exhibited by analogous mononuclear complexes. This strategy is often employed to target tumors selectively, preserve healthy cells and reduce side effects. This often involves combining several metal entities with a compound to create a multinuclear, macromolecular platform, intended for multimodal targeting. This follows a suite of multinuclear platinum compounds evaluated for anticancer properties and a trinuclear complex having undergone clinical evaluation.65 A number of multinuclear complexes based on polymeric and dendritic scaffolds (Fig. 10), have been evaluated as potential anticancer agents, with alternate redox behavior and altered reactivity towards biological targets compared to mononuclear compounds.59d,66 In particular, ferrocene has been conjugated to various polymeric scaffolds with the design focus of carrier-drug conjugates to act as prodrugs and enhancing the therapeutic efficacy through increased drug availability in the target site.67 Neuse et al. conjugated ferrocene to polyaspartamide, water-soluble carrier polymers.68 These complexes were active against both colon and cervical cancer cell lines (IC50 ¼ 0.2–2 mg mL−1). The synergy between metals and dendrimers has also allowed for the development of novel functionalised metal-containing macromolecules, leading to a promising field of chemistry. Various metallodendrimers topologies exist, allowing for the incorporation of metals within the core, interstitial sites or at the periphery of the dendrimer.69 Most of the examples of metallodendrimers furnished with ferrocenyl entities are those found on the periphery of the dendrimer. Heterometallic ferrocenyl-derived metallodendrimers were prepared and led to an enhancement of anticancer activity when ferrocene was introduced into the periphery of the dendrimer.69a,70 de la Mata et al. prepared water-soluble ammonium-terminated carbosilane dendrons with ferrocene entities at the focal point (core).71 These compounds were effective as broad spectrum antibacterial agents and highly selective in both Gram-positive bacteria (Staphylococcus) and Gram-negative bacteria (Escherichia coli) over mammalian cells.

15.11.4 Organometallic ferrocene compounds against neglected tropical diseases The World Health Organization (WHO) describes neglected tropical diseases (NTDs) as a diverse group of infectious medical conditions endemic in tropical and subtropical regions that predominantly affect populations of low socio-economic status with limited access to adequate sanitation and clean water, and living in close proximity to infectious disease carriers, livestock and domestic animals.72 Despite the development of new technologies and cutting-edge advances in modern medicine, the control and eradication of NTDs is still out of sight, with more than 1 billion people living in 149 countries in developing economies affected yearly. Currently, 20 afflictions are classified as NTDs by WHO in its categorization of diseases. These diseases not only place a great economic burden on afflicted countries, but also threaten lives of infected individuals and can cause severe disability and death if left untreated. Because NTDs almost exclusively affect impoverished communities of low socio-economic and political standing, calls for public health awareness and efforts to curb these diseases often falls on deaf ears. Even more concerning, the pharmaceutical industry is reluctant to fund projects directed towards developing innovative drugs for treatment of NTDs on account of low economic incentives. To that end, currently available treatment options for a vast majority of NTDs are old drugs or existing drugs of other indications that have been repurposed for NTDs. Not a single innovative drug (i.e., new chemical entity) has been clinically approved for NTDs in the past 20 years, though a few have managed to enter the early stages of clinical trials. As a result, there are increasing fears in the medical community concerning high chances of clinical resistance development by NTD pathogens to currently employed drugs. Compounds containing transition metals are increasingly gaining attention as potential agents for NTDs owing to their diverse biological activities and peculiar modes of action, which may provide solutions to concerns about clinical resistance development by NTD pathogens to existing drugs. This area is extensively covered in literature with very encouraging results. Regarding iron-based organometallic complexes, ferrocenyl compounds derived from various bioactive chemical scaffolds have been heavily studied for antiproliferative effects against causative agents of well-known NTDs, such as human sleeping sickness (trypanosomiasis), leishmaniasis and schistosomiasis. In this section, we will highlight the pathology of these diseases and the biological activity of ferrocenyl compounds showing inhibitory activity against the responsible pathogens. Due to the expansive nature of this field, only a selection of representative research accounts available in literature will be used to emphasize the role of ferrocene-containing compounds as potential anti-NTD agents.

Fig. 10 Structures of typical ferrocenyl-containing polymers and dendrimers.

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15.11.4.1 Trypanosomiasis: Human African trypanosomiasis and Chagas disease Human sleeping sickness, also known as trypanosomiasis, is categorized into two types: Human African sleeping sickness (or human African trypanosomiasis (HAT)) and American trypanosomiasis (Chagas disease). Human African sleeping sickness is caused by species of the protozoan, Trypanosoma brucei brucei, further classified into subspecies T. brucei gambiense and T. rhodesiense, that are transmitted through bites by the tsetse fly vector, while Trypanosoma cruzi is responsible for Chagas disease spread via contact with fecal waste of kissing bugs.73 Both parasites require two hosts to complete their life cycles, a vector and a human host. The life cycle of T. brucei brucei species begins when the parasitic trypomastigotes are injected into the human bloodstream through a tsetse fly bite followed by proliferation and invasion of different cell types and other human bodily fluids, particularly of the lymphatic and central nervous systems.73 It is at this stage that symptomatic manifestation of the disease takes root, characterized by headaches, weakness and fatigue at 1–3 weeks after the infection.74 Acute symptoms are associated with disturbances in normal functioning of the central nervous system and include personality changes, confusion, impaired mobility and abnormal sleeping patterns with excessive daytime sleepiness. The life cycle is completed when the vector ingests trypomastigote-infected blood, satisfying the requirement of a second host where trypomastigotes mature and collect in the salivary glands of the vector until the next bite. The life cycle of the Chagas disease causal agent, T. cruzi, progresses similarly to that of T. brucei brucei with the only difference being the mode of transmission. Infection with T. cruzi occurs when trypomastigotes in the fecal matter of infected kissing bugs invade cells of exposed tissues (bite wounds, broken skin, mucous membranes or soft skin of the eye) and multiply and spread to other cells in the body, leading to clinical manifestation of the disease. Consumption of infected food products by humans can also lead to infection with Chagas disease. While there are several medication options for treatment of HAT shown in Fig. 11, nifurtimox and benznidazole are the only currently approved clinical drugs for Chagas disease. These drugs are effective at controlling and treating the diseases and can be administered to alleviate both early and advanced symptoms. However, the drawbacks caused by acute human toxicity severely limit the therapeutic utility of these drugs and exacerbate the devastating effects of trypanosomiasis. This is typified by the arsenic-containing melarsoprol (trade name: arsobal) used for effective treatment of advanced cases of HAT with side effects comparable to arsenic poisoning, including convulsions, loss of consciousness and encephalopathy that can be life-threatening. Initiatives by non-profit organizations, such as the open-access Pathogen Box from the Medicines for Malaria Venture (MMV) and Drugs for Neglected Disease Initiative (DNDi) have paved the way in the quest for novel bioactive compounds with improved therapeutic value and reduced toxicity to humans, providing opportunities for medicinal chemists and pharmacologists to explore the antitrypanosomal chemical space at expeditious rates.75 Organometallic iron derivatives of existing clinical antitrypanosomal drugs and other chemical scaffolds, as well as structures from the MMV Pathogen Box, containing the ferrocene unit have been extensively studied in the literature as potential trypanocidal agents.

Fig. 11 Chemical structures of clinical drugs for treatment of HAT (4.1a-f) and Chagas disease (4.1f-g).

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15.11.4.1.1

Trypanocidal ferrocenyl compounds derived from clinical antitrypanosomal drug scaffolds

In 2011, the research group of Klahn and associates reported the first ferrocenyl derivatives of an antitrypanosomal drug, nifurtimox (4.1f), used to treat both HAT and Chagas disease, by replacing the thiomorpholine unit with ferrocene whilst retaining the 5-nitrofurane pharmacophore (Fig. 12).76 Cyrhetrenyl analogs, such as 4.2b, were also included in the study to probe the electronic effects of displacing the electron-donating ferrocene unit with an electron-withdrawing metallofragment, cyrhetrene. Both classes of compounds displayed in vitro trypanocidal effects against the Tulahuen strain of T. cruzi epimastigotes in the mid-micromolar range (43.6–100.2 mM) with the ferrocenyl derivatives generally possessing slightly improved, albeit comparable, potencies than their cyrhetrenyl counterparts. However, for compounds 4.2a-b with electronic communication between the organometallic fragments and pharmacophore via a conjugated imine linker, the electron-withdrawing cyrhetrene unit was found to be favorable for antitrypanosomal activity as the cyrhetrenyl derivative 4.2b (IC50 ¼ 43.6  0.9 mM) was twice as potent as the ferrocenyl analog (IC50 ¼ 100.2  3.8 mM). This was be attributed to the strong electron-withdrawing character of cyrhetrene and thus its ability to facilitate bio-reduction of NO2 to form NO2− radicals that can degrade cellular membranes and disrupt crucial biological processes in trypanosomal cells, leading to therapeutic effects. This hypothesis was confirmed in a follow-up study in which ferrocenyl and cyrhetrenyl units were connected to the 5-nitrofurane or 5-nitrothiophene pharmacophoric motifs to produce organometallic Schiff bases 4.2a-c and 4.2e-g active in the

Fig. 12 Antichagastic ferrocenyl and cyrhetrenyl derivatives of nifurtimox.

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ranges 0.4–18.3 mM and 12.72–87.7 mM, respectively, against trypomastigote and epimastigotes forms of the Dm28c T. cruzi strain.77 Again, antichagastic activity of the electron-communicating cyrhetrenyl compounds, 4.2b and 4.2f, were more potent than their ferrocenyl counterparts, reinforcing the favorable electron-withdrawing effects of cyrhetrene for antitrypanosomal activity owing to better promotion of NO2− radical production. Furthermore, the sulfur atom in the 5-nitrothiophene pharmacophore was found to be more desirable for antitrypanosomal activity than oxygen (5-nitrofurane) as a consequence of its lower electronegativity and, consequently, better stabilization of generated NO2− radicals due to the presence of empty d-orbitals. Most noteworthy, a majority of these compounds exhibited superior activity to the clinical antitrypanosomal drug, nifurtimox (4.1f), on both forms of the T. cruzi strain. Remarkably, inhibitory effects of ferrocenyl bases were still better than the cyrhetrenyl analogs for the series with no electron-communication between the metallofragments and pharmacophores. The beneficial and pharmacological significance of incorporating ferrocene to known drug scaffolds is further illustrated by the activities of ferrocenyl compounds, which are superior to the parental drug, nifurtimox. Recently reported ferrocenyl and cyrhetrenyl azine derivatives (e.g., 4.2h-k) for which similar observations concerning the influence of electrochemical character of the compounds on biological activity were made are also worth mentioning.78 Ferrocenyl and benzyl diamines (4.3a-g and 4.3f-j) resembling the skeletal scaffold of pentamidine (4.1d) were assembled by Velásquez and colleagues and investigated for trypanocidal effects on T. brucei and T. cruzi strains (Fig. 13).79 A majority of the synthesized compounds were more active than the control drugs, pentamidine (4.1d) and benznidazole (4.1g), with ferrocenyl diamines 4.3a and 4.3b emerging as the most promising candidates against all three tested strains. Selectivity indices of the compounds evaluated on a hepatoma cell line (HepG2) were comparable to those of control drugs, highlighting minimal human toxicity profile of the compounds. The impressive antitrypanosomal potencies were tentatively attributed to the pharmacological properties of ferrocene, such as high lipophilicity and reversible redox character, which enables formation of the toxic ferrocenium ion and concomitant generation of reactive oxygen species (ROS) that are lethal to trypanosomal cells. This is supported by the fact that all the ferrocenyl amines were many times more active than their respective organic counterparts 4.3f-j (IC50 values in the mid-micromolar range) against all three tested strains. Collectively, these studies demonstrate the strategy of incorporating the ferrocene unit into organic antitrypanosomal drug scaffolds not only as a practical avenue to improve their biological performance but also offers opportunities to introduce novel mechanisms of action to the resulting organometallic compound.

15.11.4.1.2

Trypanocidal ferrocenyl compounds inspired by bioactive scaffolds contained in the MMV pathogen box

The Medicines for Malaria Venture (MMV) is one of the pioneering initiatives that has driven substantial developments to accelerate the drug pipeline for combating tropical diseases. The MMV pathogen box, containing more than 400 bioactive chemical entities as drug leads for clinical investigation, has inspired bioorganometallic and medicinal chemists in the field of drug discovery to conceptualize innovative chemotypes with potential biological activity against the causal agents of tropical diseases by incorporating metallofragments into similar bioactive scaffolds. Similarly, ferrocene has been appended to scaffolds structurally related to candidates in the MMV pathogen box to fashion novel compounds possessing antitrypanosomal activity.

Fig. 13 Ferrocenyl diamines based on the structural architecture of pentamidine showing inhibitory activity against T. brucei and T. cruzi strains.

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Fig. 14 Antitrypanosomal ferrocene-benzimidazole hybrid compounds with chemical motifs similar to MMV pathogen box candidates.

Biot and co-workers conjugated ferrocene to the benzimidazole scaffold, a prominent moiety in the MMV box, to generate antitrypanosomal ferrocenyl hybrids tested in vitro on a T. b. gambiense strain as well as the T. b. brucei Swiss mouse model in vivo (Fig. 14).80 The choice of benzimidazole scaffold was inspired by the multitude of biological properties that are well-documented in literature for this moiety, ranging from antiprotozoal to anticancer activities.81 Particularly, benzimidazolyl compounds like MMV676445 and MMV020081 from the MMV pathogen box are potent antiparasitic agents. Ferrocenyl benzimidazoles were assembled by grafting the ferrocene unit to position C2 of the benzimidazole motif, resulting in hybrid compounds comprising a single benzimidazole unit or two units appended to both the top and bottom ferrocenyl cyclopentadiene rings (Fig. 14). The tested compounds displayed varying in vitro antitrypanosomal potencies between 6.80  1.52 and >100 mM, with compound 4.4b boasting the most effective activity. Conjugation of a second benzimidazole unit presented no beneficial trypanocidal effects as this series was equipotent with the mono-benzimidazole variants, while fluorination at position C5 was generally favorable for activity. The presence of the basic dimethylaminomethyl (CH2NMe2) side chain on the ferrocene unit, a key structural feature in ferroquine, proved detrimental for the activity of compounds 4.5a-b. When taken for in vivo assessment, the most promising compound cleared T. b. brucei parasitemia to 36024  3100 parasites per microliter in the blood of infected mice following a 100 mmol kg−1 compound dosage, with no observed toxicity to the mice 4 days post treatment. However, this efficacy was deemed too modest for further investigation. Noteworthy, the antimalarial drug ferroquine and its 4-NMe analog were also surveyed in vitro for trypanocidal effects and IC50 activities of 0.13  0.04 and 1.02  0.08 mM, respectively, were recorded. Although ferroquine displayed impressive in vitro activity as well as in vivo efficacy (0 parasitemia at day 3) on the models, acute toxicity to the mice was observed at the highest tested dosage (100 mmol kg−1) on day 4 after drug administration. Propositions regarding mechanistic considerations of the compounds revealed that the attained activities were independent of the redox profile of the compounds (attributed to the ferrocene unit) as indicated by the in vitro antitrypanosomal activity of a non-redox active ruthenium congener, ruthenoquine (IC50 ¼ 0.10  0.05 mM), which was analogous to that of ferroquine, suggesting a mode of action that does not involve redox activation processes.82 A series of ferrocenyl benzoxazines were synthesized and evaluated for antiproliferative effects on nagana T. b. brucei 427 trypomastigotes in vitro.83 Motivation for the investigation of this class of compounds was, in part spurred, by the vast biological activity of benzoxazines, which are related analogs of well-known bioactive a-aminocresols and also claim eminence in the MMV pathogen box.83 In particular, the intramolecular hydrogen bond between the phenolic hydroxyl and amine groups in a-aminocresols is known to be of critical importance in the biological performance of this compound class. Introduction of a “pseudo-hydrogen bond” by linkage of these two groups via a methylene bridge, yielding 1,3-benzoxazines, has been demonstrated to be equally effective at mimicking the pharmacological role of the intramolecular hydrogen bond in biological applications.84 In fact, 1,3-benzoxazine derivatives are considered as pro-drugs that transform into active a-aminocresol analogs in vivo to elicit biological activity, shielding the labile OH and NH group from reacting with other biomolecules in biological systems.85 On this

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premise, the authors assembled ferrocenyl 1,3-benzoxazines, structurally related to MMV candidates like MMV003356 and MMV892646, and realized their trypanocidal IC50 potencies. Compounds endowed with the basic ferrocenyl CH2NMe2 moiety were more effective than their non-basic derivatives (IC50 ¼ 10.1–38.6 mM), exhibiting activities in the sub- and low micromolar range (IC50 ¼ 0.15–5.4 mM). Removal of the methylene linkage of compound 4.7d (IC50 ¼ 38.6 mM) to produce a-aminocresol 4.7l (IC50 ¼ 6.6 mM) magnified the antitrypanosomal activity by more than fivefold, proving the biological significance of the intramolecular hydrogen bond for activity. Preliminary studies to explain the plausible mechanism of action of these compounds revealed DNA interaction involving minor groove binding and consequent damage to DNA as likely pathways. The effect of the ferrocene unit in the binding interactions was elucidated by means of computational docking simulations employing a B-DNA structure as the receptor, which suggested non-covalent associations as the dominant stabilizing forces (Fig. 15).

15.11.4.1.3 Heteronuclear ferrocenyl antitrypanosomal compounds targeting NADH-fumarate reductase, epimastigote necrosis, DNA interaction and oxidative stress The approach of combining multiple structurally and mechanistically distinct molecular frameworks into a single molecule in order to modulate the biological activity of the resulting compound, creates diversity in both activity and mechanism imparted by the individual entities. Perspectives and trends in contemporary drug discovery purport this as a practical strategy for forging bioactive agents capable of eluding the mechanism associated with the development of clinical resistance by pathogens. This has been demonstrated in antitrypanosomal research wherein ferrocene-containing ligands have been chelated with other metallic centers of biological prominence (such as Pd and Pt) to produce complexes endowed with the therapeutic benefits of both metallofragments. The research groups of Gambino and Pérez-Díaz applied this approach and explored the antichagastic efficacy of Pd and Pt complexes obtained as hexafluorophosphate (PF6) salts chelating pyridine-2-thiol-1-oxide (mpo) and 1,10 -bis (diphenylphosphino)ferrocene (dppf ) bidentate ligands on T. cruzi parasites in several studies (Fig. 16).86 In the first cohort two complexes, [Pt(mpo)(dppf )][PF6] (4.8a) and [Pd(mpo)(dppf )][PF6] (4.8b), exhibited trypanocidal activities of 0.64  0.03 and 0.28  0.01 mM, respectively, with no acute toxicity towards normal mammalian Vero epithelial cells (selectivity indices  18).86a

Fig. 15 Ferrocenyl 1,3-benzoxazines with antitrypanosomal activity.

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Fig. 16 Heterobimetallic Pt(II) and Pd (II) ferrocenyl complexes active against T. cruzi strains.

Remarkably, the activities of both complexes were 10–20 times more superior to the reference drug, nifurtimox (4.1f). Most noteworthy, the observed reduced mammalian toxicity of the complexes was attributed to the presence of the ferrocenyl moiety since the non-ferrocenyl [Pd(mpo)2] and [Pt(mpo)2] equivalents from a previous study displayed significantly inferior selectivity indices (SI: 4.9 and >10, respectively) on mammalian macrophages.87 This shows incorporation of the ferrocene unit was successful in modulating the biological activity of the complexes. Both compounds show similar electrochemical behavior; however, discrepancies in their observed biological performance exclude the pharmacological significance of redox effects, like ROS production, as a probable mechanistic modality, pointing to other alternative modes of action. Indeed, inhibition of T. cruzi NADH-fumarate reductase was found to be a likely mechanism of action and the obtained data were consistent with observed trypanocidal potencies when mechanistic investigations were conducted. Additional studies to further elaborate the antitrypanosomal profile of the complexes on the CL Brener T. cruzi strain, employing the promising lead [Pt(mpo)(dppf )][PF6] (4.8a), revealed the mechanism of action proceeds via epimastigote necrosis mediated by mitochondrial membrane potential depolarization and interaction with macromolecules, i.e., nucleic acids (DNA and RNA) and proteins (soluble and insoluble forms).86b High compound accumulation rates of 75% in T. cruzi cells were also reported after 24 h of incubation with the parasitic culture. The highly lipophilic character of ferrocene could be a contributing factor for this observation. The complex was also found to induce morphological changes to the parasites by eliminating or shortening the parasitic flagella, leading to complete loss of mobility. Compound concentrations as low as 5  IC50 significantly reduced the initiation and progression of T. cruzi infection in mammalian Vero cells. These findings clearly warrant in vivo assessment and further clinical development of this ferrocene-based Pt complex as antichagastic clinical candidate. Similar derivatives containing a N^O-coordinated quinoline scaffold (4.9a-e) in lieu of the pyridine-2-thiol-1-oxide ligand have been recently reported for antitrypanosomal potential with comparable activities to complexes [Pd(mpo)2] and [Pt(mpo)2] in the sub-micromolar range (IC50: 0.14–0.93 mM) on 427 T. b. brucei parasites and high selectivity indices assayed in J774 murine macrophages (Fig. 16).86c Of note, these quinolinyl complexes also exhibited better anticancer activity than the clinical anticancer drug, cisplatin, against A2780 ovarian carcinoma cells in addition to trypanocidal effects. Most importantly, this new class of compounds was experimentally shown to produce ROS intracellularly, a property attributable to the redox-active ferrocene unit, as a probable mechanism of action along with DNA intercalation. Bimetallic ferrocenyl nickel(II) and palladium(II) dithiocarbazato complexes inhibiting the biochemical functioning of T. cruzi old yellow enzyme (TcOYE) with comparable activities to benznidazole (4.1g),88 as well as anti-T. b. brucei N-heterocyclic carbene gold(I) complexes targeting tubulin polymerization are some of the most recent advances in the current trends exploiting heteronuclear ferrocenes in trypanosomiasis research.89

15.11.4.1.4

Ferrocifens and heterocyclic ferrocenyl compounds as trypanocidal agents

Considering the exceptional medicinal properties of ferrocene, several heterocyclic compounds featuring the iron metallofragment have been extensively explored as potential antitrypanocidal agents. These compounds provide means to modulating the activity and imparting new therapeutic attributes to the resulting compounds as highlighted in the preceding sections. In this sub-section we highlight the antitrypanosomal activity of heterocyclic ferrocene derivatives with selected, representative examples explored in literature. Ferrocifens are considered the trailblazing ferrocene conjugates, incorporating a known bioactive drug scaffold, tamoxifen, and are consistently successful as drug leads in anticancer research. Ferrocifens 4.10a-b were investigated for inhibitory activity against the growth of T. cruzi epimastigotes (Y strain) and compound 4.10a emerged as an effective inhibitor (IC50 ¼ 62.2 mM) although the presence of ferrocene did not necessarily offer beneficial antichagastic effects on the tested strain (Fig. 17).90 The limited

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Fig. 17 Ferrocifens and heterocyclic ferrocenyl derivatives showing inhibitory activity against Trypanosoma strains.

biological activity of ferrocifen 4.10b could have been an effect of poor solubility in aqueous assay media, owing to the bulky, highly hydrophobic ester chain (C15H23). These compounds also demonstrated plasmocidal effects against the drug-resistant P. falciparum strain (W2 clone) below 3 mM. Derivatives of a thiazolidine-2,4-dione scaffold (e.g., 4.10c) famed for their antidiabetic properties,91 were constructed by grafting ferrocene at C5 of the heterocycle core. These compounds displayed antiproliferative effects against 427 T. b. brucei trypomastigotes and negligible toxicity towards mammalian HeLa cells, indicating high selectivity index of the compounds (Fig. 17).92 Compound 4.10c was the most potent from the investigated series (IC50 ¼ 1.94 mM). Ferrocene-adenine conjugates evaluated as potential antitrypanosomal agents by Kowalski et al. exhibited moderate half-maximal inhibitory growth values (GI50: 18.2  5.5 (4.11a) and 20.5  5.3 mM (4.11b)) that were 3-times superior to the cymantrenyl variant (GI50: 59.6  11.1 mM).93 This could be substantiated by the enhanced lipophilicity of these compounds and higher redox potentials due the ferrocene unit, alluding to their capacity to readily generate ROS in biological media via redox processes as a possible mechanistic modality.

15.11.4.2 Ferrocenyl compounds as potential agents against leishmaniasis Leishmaniasis is a collection of diseases caused by Leishmania parasites that are transmitted to humans by sandflies. Like the Trypanosoma pathogens, Leishmania species require two hosts to complete its life cycle. Leishmaniasis is classified into three common types depending on the affected part of the body: cutaneous, mucocutaneous and visceral leishmaniasis. The latter in this classification is the deadliest form of the disease and can lead to death if it goes untreated for a long time. The severity of visceral leishmaniasis is compounded by the fact that it is increasingly emerging as a serious co-infection of HIV and further compromises the immune system of infected individuals, making way for more fatal HIV-associated opportunistic diseases like tuberculosis and pneumonia to take root.94 Current front-line medicines for treatment of leishmaniasis include pentavalent antimonials, antibiotics (amphotericin B and paromomycin), miltefosine and pentamidine (Fig. 18).95 As with antitrypanosomal drugs, the effectiveness of these treatments is limited by side effects and development of clinical resistance.96

15.11.4.2.1

Heterobimetallic ferrocenyl antimonials possessing anti-leishmanial activity by targeting DNA interaction

Ferrocene has been appended to the pentavalent antimonial scaffold, a mainstay in anti-leishmanial treatment, to generate novel heteronuclear metallic complexes with high DNA binding affinity as potential agents inhibiting the growth of Leishmania species (Fig. 18).97 The anti-leishmanial activity of the complexes was established on Leishmania tropica KWH23 promastigotes and amastigotes, and their potencies were significantly superior to that of the reference antimonial drug, glucantime. SAR analysis revealed that the methyl substituent at position C4 of the aryl rings was favorable for leishmanicidal effects of the complexes. The compounds were also studied for cell membrane permeability employing the SYTOX™ green nucleic acid stain membrane permeability model, and they displayed high permeability effects.98 DNA interaction was proposed as a possible mechanism of

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Fig. 18 Frontline clinical drugs used for treatment of leishmaniasis.

Fig. 19 Ferrocenyl antimonials as potential antileishmaniasis agents.

action and the observed electrochemical shifts in the cyclic voltammograms in DNA titration experiments with tool compounds 4.12d and 4.13a indicated attractive electrostatic interactions as the predominant forces involved in the interactions. This could be attributed to the redox activity of the iron center in ferrocene as similar conclusions were drawn in an earlier study for similar ferrocene-antimonial conjugates.99 Indeed, several ferrocenyl compounds display high affinity for DNA in which electrostatic forces play a crucial role.100 This study further proves that introduction of ferrocene into known drug frameworks is a pragmatic strategy to enhance therapeutic value of the resulting compounds (Fig. 19).

15.11.4.2.2

Ferrocenyl quinolines inhibiting the growth of Leishmania parasites

Conjugation of ferrocene to the quinoline scaffold of antimalarial drugs, primaquine and chloroquine, yielded new anti-leishmanial ferrocenyl compounds that halted the growth of L. infantum and L. donovani parasites in in vitro bioassays, respectively (Figs. 20 and 21).101 In all studies, the compounds were non-toxic to mammalian cells as assessed on murine splenocytes and macrophages. The ferrocene unit was appended to the 8-NH2 position of the primaquine nucleus via either peptide

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Fig. 20 Alkyl and peptidomimetic ferrocene-primaquine conjugate compounds active against L. infantum promastigotes and amastigotes.

Fig. 21 Ferrocenyl triazole-quinoline conjugates inhibiting the growth of L. donovani promastigotes and amastigotes.

linkers or alkyl chains. The alkylated derivatives were generally more potent than the analogs bearing peptide linkers against late stage L. infantum promastigotes, possibly due to better lipophilic character (Fig. 20).101a Although both classes of compounds were less efficacious than the reference drugs, miltefosine and sitamaquine, compounds 4.14c and 4.15b emerged as the most effective in the series against L. infantum promastigotes and intracellular amastigotes, respectively, with activity rivaling that of the reference drugs exerting 97% parasite reduction in infected murine macrophages. What is also important to note here is that the antimalarial drug primaquine was potent on both types of investigated Leishmania parasites, producing a potential antileishmanial treatment which accentuates the emerging drug-repurposing strategy as an affordable avenue in contemporary drug discovery to alleviate costs of the clinical development pipeline.102

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Ferrocenyl quinolines reported by Adhikari and coworkers, based on the chloroquine scaffold, inhibited the proliferation of L. donovani promastigotes and amastigotes by promoting cell arrest and inducing cell apoptosis mediated by intracellular generation of nitric oxide (NO).101b,c The activity of the compounds was demonstrated to be associated with their ability to generate intracellular NO, a crucial mechanistic modality of anti-leishmanial agents.103 Interpretation of these data suggests that these ferrocenyl compounds likely act a NO synthase activators.

15.11.4.2.3

Quinazoline- and benzimidazole-based derivatives of ferrocene active against leishmaniasis

Quinazolines and benzimidazoles are eminent heterocycles in medicinal chemistry with a myriad of attractive biological properties. Consequently, these have been hybridized with organometallic entities like ferrocene to modulate their biological activity. Antileishmanial ferrocenyl quinazoline and benzimidazole derivatives have been reported by the different research groups of Hernández-Luis, Chauhan and Hanlon (Fig. 22).104 In the first study assessing compounds 4.18a-d, ferrocenyl quinazoline 4.18a emerged as the most active against the tested L. mexicana promastigotes, inducing 100% parasite growth reduction, through disruption of the folate metabolic cycle by inhibiting the function of parasitic pteridine reductase as well as dihydrofolate reductase.104a The activity of this compound was at least 15-times higher than that of organic derivatives. Comparing the potency of 4.18a (IC50 ¼ 0.93  0.1 mM) to its congener 4.18b (IC50 > 100 mM) shows that acetylation of the pyrimidinyl NH2 groups at C2 and C4 positions is detrimental for activity. Dihydroquinazoline compounds incorporating structural motifs present in bioactive natural products were assessed on L. donovani promastigotes and amastigotes along with their organic variants by Chauhan and co-workers.104b The derivatives containing ferrocene 4.19a-b exhibited low and mid-micromolar activities and were generally more potent than their organic counterparts, e.g., 4.19c, with no toxicity to mammalian J-774A.1 and Vero cells. These observations were reflected in the in vivo studies using golden hamster model. Mechanistic insights in murine macrophages with selected organic analogs suggest the compounds may act as activators of human immune response by promoting production of protective cytokines, as well as intracellular generation of nitric oxide, which are effective mechanistic pathways for clearance of the parasite from the body. Hanlon reported the first account of saltable ferrocene-based aryl- and chalcone-benzimidazole hybrids showing antiproliferative effects on L1 L. infantum strains.104c The methyl iodide salt forms of the compounds (e.g., 4.20a-b) were more effective than their neutral versions (4.20c-d), possibly due to better solubility in aqueous media, commanding activities as low as 0.50 mM (Fig. 22). Spacing of the aryl rings with an unsaturated ethene chain, as represented by 4.20d, at position C2 of benzimidazole enhanced the activity of the compounds.

Fig. 22 Quinazoline and benzimidazole derivatives with antileishmanial activity.

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15.11.5 Antiviral ferrocene-containing compounds Viral infections are among the most deadliest and prevalent ailments of the 21st century. In the face of the recent coronavirus disease (COVID-19) pandemic, caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2), and the highly contagious Ebola virus outbreak, infections of viral origin have raised grave concerns in the medical community regarding their spread and containment. This is amplified by their rapid transmission and low curable rates owing to their tenacity. Vaccination campaigns that boost the immune system to allow the body to fend off incoming infections have proven fruitful in preventing the spread of viral malignancies, however; devising and manufacturing a new vaccine is a complicated and strenuous process, limited by regulations, slow and limited approval rates. Although suffering from the same drawbacks, the use of chemotherapeutics remains essential to the treatment of viral diseases and for alleviating some of the symptoms resulting from these infections. To stay ahead of the virus’s ability to develop mechanisms that promote development of resistant clinical strains, bioorganometallic chemists have exploited the exceptional medicinal qualities of organometallic complexes, like ferrocene, to fashion novel bioactive chemotypes with innovative modes of action as potential antiviral agents. To that end, several research groups have studied metallodrugs for inhibitory effects against proliferation of several pathogenic viruses, like human immunodeficiency virus (HIV), hepatitis and herpes.105 In the following sections, we highlight these antiviral ferrocenyl compounds using representative studies documented in literature.

15.11.5.1 Ferrocene-containing compounds as anti-HIV compounds Human immunodeficiency viruses, a group of retroviruses HIV-1 and HIV-2, invade and destroy cells of the immune system (e.g., lymphocytes and microphages) that are important for the normal defense mechanism of the human body to fight incoming infections.106 Progression of the infection leads to a compromised immune system that results in AIDS, a condition whereby severe opportunistic diseases take a hold of the human body at rapid speeds leading to severe illnesses that can result in death.106a Although efforts towards containing the transmission and mortality rates of HIV have been successful, the search for a cure or vaccine remains elusive. Currently, HIV is managed by employing a cocktail of anti-retroviral drugs (known as highly active antiretroviral therapy (HAART)) that block the function of crucial biomolecules at different phases of the virus’ life cycle to reduce its burden.107 The HAART cocktail consists of drugs inhibiting HIV integrase, protease and nucleoside/non-nucleoside retroviral proteins, which are actively investigated in drug discovery for devising new anti-HIV candidates.107 Metal-based compounds, including ferrocenyl derivatives of various bioactive organic scaffolds, have been reported in literature as potential anti-HIV agents and inhibitors of these therapeutic targets and other essential viral biomolecules.

15.11.5.1.1

Ferrocene-peptides inactivate HIV-1 by targeting viral proteins

Different research groups have pursued peptides and peptidomimetics as probable anti-HIV agents owing to their innate capacity to interact with biological systems, such as enzymes and nucleic acids, due to better molecular and structural recognition by biological receptors as they can mimic proteins. Ferrocene is touted for its vast medicinal attributes, such as reverse redox activity, high lipophilicity and chemical robustness. It has been incorporated to these chemical entities to fine-tune and improve the therapeutic profile of the resulting organometallic peptides and impart new pharmacological benefits. Chaiken and co-workers have disclosed a number of ferrocenyl peptides active against HIV-1 by targeting the envelope spike gp120 glycoprotein (HIV-1 Env gp120) located on the surface of the virus (Fig. 23).108 This protein serves as the site of recognition and interaction for the invasion of human cells by HIV.109 By blocking the binding of gp120 proteins, the peptide can exclude the HIV pathogen from interacting with the recognition receptors on the surface of targeted cells of the immune system in the human host by acting as competitive antagonist binders. The first triazolyl ferrocene-peptide (HGN-156) reported in 2009 showed high binding affinity for all three types of HIV-1 Env gp120 proteins tested at multiple sites of interaction commanding target dissociation constants as low as 9 nM, which were at least 3-times better than those of the phenyl peptide (HGN-105).108a Similarly, peptide HGN-156 inhibited the infection of P4-CCR5 MAGI cells (immune system cells) by the HIV-1 strain with an IC50 value of 96 nM that was vastly superior to that of HGN-105 (IC50 ¼ 1.43 mM) with no indication of toxicity to normal cells. A second cohort of ferrocenyl peptidomimetics bearing indole (UM-24) and benzothiophene (KR-41 and KR-42) moieties in the internal peptide chain similarly displayed significant affinities for HIV-1 Env gp120 and inhibitory activity against the tested HIV-1 virus.108b Indolyl peptide UM-24 emerged as the most promising from the study with a dissociation constant of 21.1  1.4 nM and IC50 value of 6.7  1 mM. Another series of ferrocenyl peptides (A-C) binding to two distinct vicinal hydrophobic pockets of the HIV-1 Env gp120 exerted potent binding affinities and antiviral activities against the tested strain.108c Other examples of ferrocenyl peptides such as D-E have with anti-HIV activity have also been reported and most of them show better activity than their organic counterparts.110

15.11.5.1.2

Ferrocenyl anti-HIV compounds inhibiting HIV-1 integrase

Integrases are essential proteins in the pathology of HIV.111 After host cell invasion, HIV integrases incorporate the viral genomic information (DNA) into the chromosomal DNA of the host, setting in motion the mechanism by which the virus overrides the DNA replication machinery of the infected host to make more copies of itself.111 Integration of viral DNA into human genomic material is a multistep process, of which cleavage of two nucleotides of the viral DNA at 30 -ends (cleavage step) and its subsequent insertion to

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Fig. 23 Anti-HIV ferrocenyl peptides as antagonist inhibitors of HIV-1 Env gp120.

the host DNA (insertion step) are the most crucial.111 Due to its critical role in viral replication, HIV integrase is a therapeutic target of many clinical antiretrovirals in the HAART cocktail, e.g., raltegravir, by blocking either of the aforementioned integration processes.111,112 Considering the significance of integrase, some research groups in bioorganometallic chemistry have incorporated ferrocene into bioactive chemical scaffolds, some of which are known integrase inhibitors, to generate novel antiretroviral agents targeting this enzyme.113 Hillard and colleagues incorporated ferrocene to phenolic chalcone and difluoridoborate scaffolds via a chalcone linker to produce new hybrid compounds showing inhibitory activity against HIV-1 integrase (Fig. 24).113 This study was inspired by a previously reported anti-HIV integrase and antiretroviral activities of a ferrocenyl b-ketoacid (5.1a) with impressive biological performance in in vitro experiments.114 The compounds presented integrase inhibitory potencies in the low and mid-micromolar range, with the difluoroborates (5.2a-j) exerting higher activities as previously observed for similar non-ferrocenyl compounds in other studies. Both classes of compounds generally acted on the strand integration step with higher selectivity vis-à-vis the cleavage step. Also, no acute toxicity was observed for the compounds when assessed in vitro on normal endothelial cells. Most noteworthy, these ferrocenyl compounds were more effective than those of other organic diborates and chalcones reported in the literature.115 Notwithstanding, the presence of ferrocene alone was not found to be sufficient for anti-integrase activity.

15.11.5.1.3

Bimetallic ferrocene-based gold(I) complexes as anti-HIV compounds

Application of bioactive frameworks combining distinct metallic centers is a popular approach in the design of metallodrug agents and diversifying the therapeutic attributes of resulting complexes as each metal brings its unique coordination chemistry and biological properties. In HIV, this is typified by heteronuclear ferrocenyl gold (I) complexes with antiretroviral activity. Elmroth and colleagues reported a P^P-coordinated ferrocenyl bisphosphine gold(I) complex (5.3a) inhibiting replication of CM9 HIV-1 strains with a IC50 potency of 2.0  1.4 mM, in the same range as the control antiretroviral AZT (1.3  0.3 mM), with tolerable toxicity on human T-lymphoid cells (CEM-SS cell line) (Fig. 25).116 Though slightly less effective, the antiretroviral efficacy of this complex was comparable to that of its ruthenium conger (IC50 ¼ 1.5  0.5 mM). Encouraging antimalarial activity on the multidrug resistant P. falciparum strain was also noted for this complex as anticancer efficacy on the cancerous HeLa cell line. Unfortunately, complex 5.3b was unstable in aqueous media. Another similarly coordinated gold(I) complex containing a quinoline derivative (5.3c) was demonstrated to halt the replication of an HIV-1 ZM53 strain in TZM-bl cells expressing immune markers CD4, CCR5 and CXCR4 with no host toxicity (Fig. 25).117 Further investigation to elaborate the biological profile of this complex revealed cytostatic activity characterized by cell arrest of the G2/M cycle in TZM-bl cells. The cytostatic agent roscovitine is known to induce this process similarly through a mechanism involving cyclin-dependent kinase inhibition. Interpretation of this finding suggests the complex 5.3c effects G2/M cell cycle arrest in a similar fashion.

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Fig. 24 Design of ferrocenyl phenolic chalcones and their difluoroborate analogs as HIV-1 integrase inhibitors.

Fig. 25 Antiretroviral ferrocenyl gold(I) complexes.

15.11.5.2 Ferrocenyl complexes active against strains of herpes and hepatitis viruses Antiviral activity of bioactive compounds containing the ferrocene unit that exhibit inhibitory activity towards strains of viral infections other than HIV such as herpes and hepatitis viruses is documented in literature.53,118 This sub-section highlights the antiviral activity of ferrocenyl compounds against the aforementioned viral infections with relevant and recently reported examples.

15.11.5.2.1

Artemisinin- and betulin-ferrocene hybrids active against herpes virus

Tsogoeva and co-workers employed the molecular hybridization strategy to introduce ferrocene to the chemical scaffold of an antimalarial drug, dihydroartemisinin, and betulin for antiviral and antimalarial evaluation (Fig. 26).53,118a Out of the eight investigated artemisinin-ferrocene hybrids in the first series, compounds 5.4a-c commanded the highest in vitro antiviral efficacies (IC50 ¼ 0.16  0.06 mM (5.4a), 0.46  0.05 mM (5.4b), 0.11  0.11 mM (5.4c)) in infected human fibroblasts with the herpes virus, cytomegalovirus.53 Remarkably, these compounds were superior to the reference antiviral drug, ganciclovir, by more than 10-fold on average, and this was the first study to demonstrate antiviral potency of artemisinin-based ferrocenyl derivatives. Encouraged by these impressive results, the authors appended ferrocene to the structural framework of betulin or betulinic acid, both with established antiviral activities,119 to generate novel antiviral betulin-ferrocene hybrids.118a The compounds showed

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Fig. 26 Antiviral ferrocene hybrid compounds based on artemisinin (5.4a-c), and betulin/betulinic acid (5.4d) scaffolds.

comparable, albeit slightly lower, activities against replication of the cytomegalovirus between 2.46  0.85 mM and 10.75  0.51 mM IC50 values. Notably, hybrid 5.4d (IC50 ¼ 2.46  0.85 mM) was slightly more effective than betulinic acid (IC50 ¼ 3.62  1.21 mM) in the biological assays.

15.11.5.2.2

Ferrocene-based inhibitors of hepatitis C virus

Wiles and co-workers incorporated the ferrocene moiety into the structural core of a clinically approved drug, daclatasvir (5.5a), used to treat hepatitis C infections, by replacing the internal biphenyl unit (Fig. 27).118b Daclatasvir acts by blocking the function of the hepatitis C virus nonstructural 5A protein (HCV-NS5A) implicated in DNA replication of the virus and other critical biochemical processes in the virus life cycle.120 The inhibitory effects of the synthesized compounds were realized on the H77 (GT-1a replicon) and Con-1 (GT-1b replicon) hepatitis C virus strains. All the tested compounds exhibited activities in the sub- and low micromolar range, with the most active candidates exerting potencies of picomolar EC50 values, and no detectable toxicity was noted in healthy human liver cells (Huh-7). The effect of ferrocene was found to significantly enhance the activity of some organic analogs, although cases where this was not necessarily the case were also recorded. Compound 5.5b emerged as the lead (EC50 ¼ 15 pM (H77) and 2 pM (Con-1)) in the series and was taken further to examine its pharmacokinetic profile by studying its interactions with key metabolic human cytochrome P450 isozymes and stability in gastric fluid, rat plasma as well as human, monkey and rat liver microsomal preparations, which was found to be favorable. The rat in vivo model results revealed low clearance rates of compound 5.5b, suggesting the compound might be suitable for formulation as a convenient once-daily drug regimen. Ferroquine (FQ) has also been reported to be an inhibitor of HCV.121 FQ inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. FQ also inhibited HCV RNA replication at a higher concentration. Combinations of FQ with interferon (IFN), or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity.

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Fig. 27 Representative examples of ferrocenyl derivatives of daclatasvir (5.5a) active against hepatitis C virus.

15.11.6 Ferrocene-containing compounds as antitubercular agents The bacterium Mycobacterium tuberculosis (M. tuberculosis, MBT) is the pathogen responsible for causing the infectious disease tuberculosis (TB) that affects the lungs. According to WHO, TB is among the top 10 causes of death worldwide, and infected around 10 million people in 2019 with 1.5 million succumbing to the disease.122 It is one of the most critical opportunistic diseases of infection in people suffering from the advanced form of HIV, i.e., AIDS.123 The treatment of TB is a difficult process that requires the use of a drug cocktail of four (4) antibiotics taken over a course of 6 months. Failure to adhere to the prescribed TB treatment regimen results in the development of drug resistant MBT strains responsible for more serious forms of the disease, i.e., multiple drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB).124 Though preventable and curable, treatment of drug resistant MDR- and XDR-TB is more complicated.124 Approved clinical antibiotic agents result in development of resistance by bacterial pathogens, of which MBT bacteria are among the top contributors.125 Considering the promising benefits of introducing metallofragments to drug molecules as an emerging practical avenue for fashioning bioactive compounds with potential to evade or delay the process of clinical resistance, ferrocene, being an organometallic complex of significant therapeutic value, has been incorporated to several pharmacological scaffolds for anti-TB evaluation. There are various ferrocene compounds with inhibitory activity against other bacterial as well as fungal infections.12a,126,127 Here, we highlight the antimicrobial properties of ferrocene derivatives using TB as the archetypal bacterial infection.

15.11.6.1 Isatin- and uracil-ferrocene hybrids as anti-TB agents Presented with the diverse biological spectrum of isatin, the research groups of Kumar and Biot investigated a series of triazole-tethered ferrocenyl derivatives (6.2a-h, 6.3a-h, 6.5a-h and 6.6a-h) of this natural product modified at N1 position with varying substituents on C5 of the scaffold (Fig. 28).128 Generally, all the investigated compounds showed in vitro antitubercular

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Fig. 28 Ferrocenyl isatin and uracil derivatives showing antitubercular activity.

activities against the screened M. tuberculosis strains (mc26230 and mc27000) at minimal inhibitory concentrations (MIC) ranging from 105 to >463 mM, albeit less potent than those of reference clinical antibiotics, isoniazid (MIC ¼ 2.92 mM), rifampicin (MIC ¼ 0.12 mM), cephalexin (MIC ¼ 72–144 mM) and ethionamide (MIC ¼ 90.36 mM). Notwithstanding, a majority of ferrocenyl hybrids in both studies possessed higher efficacies than their respective non-ferrocenyl azido-analogs, e.g., MIC >213 (6.1a) vs. MIC >385 (6.3a). This observation could be explained by the favorable therapeutic benefits, such as high lipophilicity, of ferrocene imparted to the new hybrids. The presence of ferrocene could have also introduced a new mechanistic modality owing to its biological redox profile (for ROS generation) as suggested by the improved antitubercular performance of hybrids endowed with this motif. Continuing in their research efforts towards ferrocenyl antitubercular compounds, the researchers pursued uracil-ferrocene hybrids prepared similarly to the isatin variants.129 Potent growth inhibitory activities between MIC 26.67  1.67 and 35.00  7.64 mM were attained for compounds 6.6d-f whereas the rest of the hybrids in the series exerted potencies of no less than 50 mM. Inclusion of the chalcone moiety to the ferrocene spacer (6.7a-h) and of bromine at position C5 of the uracil motif were, respectively, found to be detrimental and favorable for anti-TB activity. Notably, these compounds presented favorable selectivity profile when assessed for general toxicity effects as none killed human HeLa cells at 50 mM concentration. b-Lactam-ferrocene hybrids have also been reported by the same groups, though these are ineffective against the M. tuberculosis pathogens.130

15.11.6.2 Bimetallic heteronuclear ferrocene complexes with anti-TB potency Bimetallic ferrocenyl complexes of different organic structural cores containing metallic centers other than iron have been studied in literature for inhibitory activity against M. tuberculosis strains, following a growing trend in antituberculosis drug discovery.

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Fig. 29 Heterometallic ferrocenyl complexes with antitubercular activity.

Fig. 30 Heterometallic ferrocenyl complexes with antitubercular activity.

Smith and co-workers reported a series of N^O-chelated isoniazid-ferrocene hybrids (6.8a-c) containing organometallic half-sandwich complexes with platinum-group metal centers (iridium, rhodium and ruthenium), along with uncomplexed monometallic ferrocene-isoniazid and -pyrazine hybrids (e.g., 6.9a-b), that inhibit the growth of the H37Rv M. tuberculosis strain in a glycerol-dependent manner (Fig. 29).131 The complexes, in conjunction with the uncomplexed monometallic ferrocenyl ligands (e.g., 6.9a), displayed MIC activities below 1 mM, and were superior to their pyrazine counterparts (e.g., 6.9b) by more than 47-fold. Compared to the parental antitubercular drug, isoniazid (6.9) (MIC 4 > 5, whereas they scarcely inhibited glutathione reductase.37,38 A comparative bond dissociation energy (BDE) study of the mentioned compounds suggested that the stabilities of the AudL bonds vary in the order chlorido-Au (3)