Outstanding Marine Molecules [1st ed.] 3527334653, 9783527334650

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Stéphane La Barre is a senior research scientist at the Centre National de la Recherche Scientiique in France. He gained his MSc from Auckland University, New Zealand, and his PhD from James Cook University, Townsville, Australia, before joining CNRS in 1984. His multi-disciplinary career includes marine chemical ecology, natural products chemistry of terrestrial and marine organisms, and polymer chemistry. Dr. La Barre is currently the coordinator of the research cluster BioChiMar (Marine Biodiversity and Chemodiversity), and is investigating novel analytical tools to evaluate and predict environmental change affecting coral reef diversity, both biological and chemical.

Outstanding Marine Molecules

Emeritus professor at the University of Nantes, France, since 2003, Jean-Michel Kornprobst has a chemical engineering degree from Montpellier University and a PhD from the University of Lyon. After being assistant professor at the University of Paris 7 from 1970 to 1973, he became professor of organic chemistry at the University of Dakar, Senegal, where he worked on marine natural products before joining the University of Nantes in 1990. Professor Kornprobst has over 100 publications and three books to his name, and was responsible for two research programs on manapros in Doha, Qatar, and Jeddah, Saudi Arabia. He has recently been an invited professor at the universities of Louvain-la-Neuve, Belgium, Campinas, Brazil, and Blida, Algeria, and is currently an external member on the scientiic advisory board of the Marine Biotechnology Research Center in Québec, Canada.

La Barre . Kornprobst (Eds.)

Using a number of outstanding examples, this text introduces readers to the immense variety of marine natural compounds, the methodologies to characterize them, and the approaches to explore their industrial potential. Care is also taken to discuss the function and ecological context of the compounds. Meticulously produced and easy to read, this book serves students and professionals wishing to familiarize themselves with the ield, and is ideally suited as a course book for both industry and academia.

Outstanding Marine Molecules Chemistry, Biology, Analysis Edited by Stéphane La Barre and Jean-Michel Kornprobst

Edited by St
ephane La Barre and Jean-Michel Kornprobst Outstanding Marine Molecules

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Edited by St
ephane La Barre and Jean-Michel Kornprobst

Outstanding Marine Molecules Chemistry, Biology, Analysis

Editors St
ephane La Barre Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty can be created or extended by sales representatives or written sales materials. The Advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor authors shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. Library of Congress Card No.: applied for

and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Jean-Michel Kornprobst Institut Mer et Littoral B^atiment Isomer 2, rue de la Houssiniere 44322 Nantes BP 92208 Cedex 3 France Cover: Photo Ó Alain Diaz, Îles Glorieuses, Indian Ocean

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the Internet at . Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical, and Medical business with Blackwell Publishing. All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Print ISBN: 978-3-527-33465-0 ePDF ISBN: 978-3-527-68152-5 ePub ISBN: 978-3-527-68153-2 mobi ISBN: 978-3-527-68151-8 obook ISBN: 978-3-527-68150-1 Cover Design Typesetting

Formgeber, Mannheim, Germany Thomson Digital, Noida, India

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Markono Print Media Pte Ltd., Singapore

Printed on acid-free paper.

jV

Contents List of Contributors Foreword Preface

XIII

XIX XXI

Part One Outstanding Marine Molecules from a Chemical Point of View 1 1

1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.3.1 1.2.3.2 1.2.3.3 1.3 1.4 1.5 1.6

2

2.1 2.2 2.2.1 2.2.2 2.2.2.1 2.2.2.2 2.2.2.3

Marine Cyanotoxins Potentially Harmful to Human Health 3 M
elanie Rou
e, Muriel Gugger, Stjepko Golubic, Zouher Amzil, Romulo Ar
aoz, Jean Turquet, Mireille Chinain, and Dominique Laurent Introduction 3 Marine Cyanobacteria as Causative Agent of Ciguatera-Like Poisoning 4 Ciguatera Fish Poisoning 4 Ciguatera Shellfish Poisoning (CSP): A New Ecotoxicological Phenomenon 7 Ciguatera-Like Poisonings Involve Complex Mixtures of Cyanotoxins 7 Ciguatoxins and Homoanatoxin 7 Ciguatoxins and Saxitoxins 8 Ciguatoxins and Palytoxins 8 Marine Cyanobacteria: A Potential Risk for Swimmers 10 Microcystins Could also be Found in the Sea 12 Risk of Neurodegenerative Disease in the Sea 13 Conclusion and Future Prospects 13 Acknowledgments 16 References 16 Outstanding Marine Biotoxins: STX, TTX, and CTX 23 Philippe Amade, Mohamed Mehiri, and Richard J. Lewis Introduction 23 Saxitoxins (STXs) in Paralytic Shellfish Poisoning 24 Causes of Paralytic Shellfish Poisoning 24 Saxitoxins (STXs) 24 Chemical Aspects of the STXs 25 Detection of PSP Toxins 27 Poisoning Records 27

2.3 2.3.1 2.3.1.1 2.3.1.2 2.4 2.4.1 2.4.2 2.4.2.1 2.4.2.2 2.4.2.3 2.4.2.4 2.4.2.5 2.4.2.6 2.5

3

3.1 3.2 3.3 3.3.1 3.3.2 3.4 3.4.1 3.4.2 3.4.2.1 3.4.3

3.4.3.1 3.4.3.2

Tetrodotoxin (TTX) in Puffer Fish Poisoning (PFP) 28 Puffer Fish Poisoning (PFP) 28 Chemical Aspects of TTX 30 Detection of TTXs 32 Ciguatoxin (CTX) in Ciguatera Fish Poisoning (CFP) 33 Ciguatera Fish Poisoning (CFP) 33 Ciguatoxins 34 Chemical Aspects 35 Detection of CTX Toxins 36 Poisoning Records 37 Persistence and Recurrence of Symptoms 37 Fish Containing Ciguatoxins 37 Qualitative and Quantitative Methods for Toxins Detection 38 Conclusions 39 References 40 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs 45 Marieke Vansteelandt, Catherine Roullier, Elodie Blanchet, Yann Guitton, Yves-FranScois Pouchus, Nicolas Ruiz, and Olivier Grovel Introduction 45 Molecules Isolated from Marine-Derived Penicillium Species With Potent Cytotoxic Activity 46 Marine-Derived Cytotoxic Penicillium 46 Where Were Marine-Derived Penicillium Searched and Isolated? 46 Which Penicillium Species? 46 What are these Promising Molecules from Marine Penicillium? 57 Statistics 57 Focus on Interesting Molecules 59 Cytotoxic Alkaloids: The Example of Communesins 59 Cytotoxic Alkaloids/Diketopiperazine Compounds: Examples of Fructigenine A and Verticillin Derivatives 68 Fructigenine A (¼ Rugulosovin B ¼ Puberulin) 68 Verticillin A and Derivatives 68

VI

j

3.4.4 3.4.4.1 3.4.4.2 3.4.4.3 3.4.4.4 3.4.4.5 3.4.4.6 3.5

4

4.1 4.2 4.2.1 4.2.2 4.2.3 4.3 4.3.1 4.3.2 4.3.3 4.3.4 4.4 4.4.1 4.4.2 4.4.3 4.5

5

5.1 5.2

5.2.1 5.2.1.1 5.2.1.2 5.2.1.3 5.2.1.4 5.2.2 5.2.2.1

Contents

Cytotoxic Sesquiterpenes: Ligerin, a Chlorinated Sesquiterpene 72 Ligerin is Produced by a New Species of Penicillium 72 Isolation of Ligerin 72 The Chlorine Atom: The Originality of Ligerin’s Chemical Structure 74 The Many Structural Analogs of Ligerin 74 Ligerin Semisynthesis 75 Bioactivities 75 Conclusions 75 References 76 Astonishing Fungal Diversity in Deep-Sea Hydrothermal Ecosystems: An Untapped Resource of Biotechnological Potential? 85 Ga€etan Burgaud, Laurence Meslet-Cladiere, Georges Barbier, and Virginia P. Edgcomb Introduction 85 Deep-Sea Hydrothermal Vents as Life Habitats 85 Generation of Marine Hydrothermal Systems: A Story of Interactions 86 Different Vent-Fluid Compositions Shaping Different Ecological Niches 86 Hydrothermal Lifestyles At the Macro- and Microscopic Scale 87 The Five “W”s of Marine Fungi: Who? What? When? Where? Why? 89 Definition and Novel Concept 89 Patterns of Distribution 90 Ecological Roles 90 Origin of Marine Fungi 91 Fungi in Deep-Sea Hydrothermal Vents 91 Hydrothermal Vents as Life Oases for Fungi 92 Physiological Adaptations 92 Biotechnological Potential 93 Conclusions 94 Acknowledgments 94 References 94 Glycolipids from Marine Invertebrates 99 Gilles Barnathan, Aur
elie Couzinet-Mossion, and Ga€etane Wielgosz-Collin Introduction 99 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity 101 a-Glycopyranosylceramides 102 a-Monoglycosylceramides 102 a-Diglycosylceramides 102 a-Triglycosylceramides 109 a-Tetraglycosylceramides 109 b-Glycopyranosylceramides 109 b-Glycopyranosylceramides with Saturated, Mono-, and Diunsaturated Sphingoid Bases 109

5.2.2.2 5.2.3 5.2.3.1 5.2.3.2 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.1.3 5.3.2 5.3.3 5.4 5.4.1 5.4.2 5.4.3 5.5

6 6.1 6.2 6.3 6.4 6.5 6.6

b-Glycopyranosylceramides with Triunsaturated Sphingoid Bases 125 Biological and Pharmacological Properties of GSLs from Marine Invertebrates 127 Immunostimulating and Antitumor Properties of a-Galactosylceramides 127 Biological Activity of b-Glycosylceramides 128 Gangliosides 129 Occurrence and Structure 129 Inositolphosphoceramide Gangliosides 130 Lactosylceramide Gangliosides 131 Glucosylceramide Gangliosides 136 Biological Activity 143 Conclusion 145 Atypical Glycolipids 145 Occurrence and Structure 146 Biological Activity 152 Conclusion 155 General Conclusion 155 List of Abbreviations 155 References 155 Pigments of Living Fossil Crinoids 163 C
ecile Debitus and Jean-Michel Kornprobst The Discovery of Stalked Crinoids 163 Anthraquinonic Pigments of Stalked Crinoids 163 Axial Chirality of Gymnochromes and Hypochromines 165 Towards a Fungal Origin of Gymnochromes? 167 Biological Activities of Gymnochromes 168 Perspectives 168 References 169

Part Two Outstanding Marine Molecules from an Ecological Point of View 171 7

7.1 7.2 7.3 7.4 7.5 7.6 7.6.1 7.6.2 7.6.3

Bacterial Communication Systems 173 Tilmann Harder, Scott A. Rice, Sergey Dobretsov, Torsten Thomas, Alyssa Carr
e-Mlouka, Staffan Kjelleberg, Peter D. Steinberg, and Diane McDougald Coordination of Multicellular Behavior in Bacteria 173 The Repertoire of Chemical Signals 174 Molecular Mechanisms of QS 175 The Effective Range of QS-Regulated Processes 175 The Inhibition of QS: Quorum Quenching 176 Examples of Cross-Kingdom Signaling in the Marine Environment 179 Chemical Defense of the Red Seaweed Delisea pulchra 179 The Mutualistic Association of Vibrio fischeri with the Hawaiian Bobtail Squid 180 Exploitation of Bacterial QS During Settlement of Marine Spores and Invertebrate Larvae 182

Contents

7.7 7.8

“-Omic” Approaches to QS 182 Concluding Remarks 183 References 183

8

Domoic Acid 189 St
ephane La Barre, Stephen S. Bates, and Michael A. Quilliam Historical Background 190 Case Studies 192 Case Study #1: The 1987 Outbreak on Prince Edward Island 192 Case Study #2: The 1991 Bird Intoxication Event in California 193 Case Study #3: Massive Sea Lion Mortality in Just a Few Weeks 194 Chemistry 194 Physico-Chemical Properties 194 Structure Determination 194 The Kainic Acid Family 194 Nuclear Magnetic Resonance (NMR) Spectroscopy 195 Mass Spectrometry (MS) 196 UV spectroscopy (UV) 196 Extraction, Separation, Purification, and Detection of DA 197 Extraction and Cleanup 197 Separation and Purification 197 Detection, Quantification, and Monitoring in Food Samples 197 Immunological Method 198 Domoic Acid and Related Molecules 198 Synthesis 198 Biosynthesis 199 Labeled Precursor Investigations 199 Regulation of DA Production 200 Degradation 201 Photodegradation 201 Photo-oxidative Degradation 201 Bacterial and Enzymatic Degradation 201 DA-Producing Organisms 201 Red Algae 201 Diatoms 202 Molecular Basis of DA Acute and Chronic Poisoning 203 The Kainoids’ Mode of Action 203 Glutamate Receptors 204 Short- and Long-term Neurological Problems Associated with DA 207 Mammal Studies 207 Cures Against ASP 207 Understanding and Predicting Toxigenic Diatom Blooms (Macroscopic Scale) 207 Natural Factors that Enhance Bloom Formation and/or DA Production 209 Silicon 209

8.1 8.2 8.2.1 8.2.2 8.2.3 8.3 8.3.1 8.3.2 8.3.2.1 8.3.2.2 8.3.2.3 8.3.2.4 8.3.3 8.3.3.1 8.3.3.2 8.3.3.3 8.3.3.4 8.3.4 8.3.5 8.3.6 8.3.6.1 8.3.6.2 8.3.7 8.3.7.1 8.3.7.2 8.3.7.3 8.4 8.4.1 8.4.2 8.5 8.5.1 8.5.1.1 8.5.2 8.5.2.1 8.5.3 8.6 8.7 8.7.1

8.7.2 8.7.3 8.7.4 8.7.5 8.8 8.8.1 8.8.2 8.8.2.1 8.8.2.2 8.9

9 9.1 9.1.1

9.1.2 9.1.3 9.2 9.2.1 9.2.2 9.2.2.1 9.2.2.2 9.3 9.3.1 9.3.2 9.4

10 10.1 10.2 10.2.1 10.2.2 10.2.3 10.3 10.3.1 10.3.2

j VII

Phosphorus 209 Nitrogen 209 Iron 209 The Role of Bacteria in the Biosynthesis of DA by Toxigenic Diatoms 209 Functional Genomics of Diatoms 210 The Key to the Evolutionary Success of Diatoms 210 Genomics of DA Biosynthesis and Regulation Networks 210 Genomic Aspects 210 Transcriptomics of DA-Producing Diatoms 210 Conclusions 210 Acknowledgments 211 References 211 Algal Morpho-Inducers 217 Zofia Nehr and B
en
edicte Charrier Introduction 217 Marine Macroalgae: Different Evolutionary Histories Leading to Similar Morphologies 217 Macroalgal Morphologies and Adaptation 217 What Exactly does the Term “Algal MorphoInducer” Cover? 219 Morpho-Inducers of Animals and Land Plants Produced by Macroalgae 219 Algal Compounds as Morpho-Inducers of Animals 219 Algal Compounds as Morpho-Inducers of Land Plants: Phytohormones 219 Auxins 219 Cytokinin 220 Morpho-Inducers of Macroalgae 220 Are Macroalgal Phytohormones also MorphoInducers on Algae? 220 Morpho-Inducers of Macroalgae Produced by Bacteria 221 Conclusions 222 Acknowledgment 222 References 222 Halogenation and Vanadium Haloperoxidases 225 Jean-Baptiste Fournier and Catherine Leblanc Introduction 225 Biochemical Characterization of VanadiumDependent Haloperoxidases (VHPOs) 227 Occurrence of VHPO Activities in Living Organisms 227 Enzymatic Assays and Biochemical Properties 228 Biological Functions of VHPOs 229 Structural Characterization of VHPOs 230 Protein Sequences of VHPOs 230 Overall Quaternary Structures of VHPOs 231

VIII

j

10.3.3 10.3.4 10.3.5 10.4 10.4.1 10.4.2 10.4.3 10.4.4 10.4.5

Contents

Tertiary Structure of VHPOs 231 Active Site Structure of VHPOs 232 Fine Structure and Vanadate Coordination into the Active Site 232 Catalytic Cycle and Halide Specificity 234 Acid Phosphatases, “Cousins” of VHPOs 234 Inhibition of VHPOs 235 Reaction with Hydrogen Peroxide 236 Oxidation of Halides 236 Site-Directed Mutagenesis Studies and Catalytic Mechanisms 236 References 238

Part Three Outstanding Marine Molecules with Particular Biological Activities 243 11

11.1 11.2 11.2.1 11.2.2 11.2.3 11.2.4 11.3 11.3.1 11.3.2 11.3.3 11.3.4 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.5

12

12.1 12.2 12.3

Promising Marine Molecules in Pharmacology 245 Marie-Lise Bourguet-Kondracki and Jean-Michel Kornprobst Introduction 245 Promising Substances Isolated from Microorganisms 248 Salinosporamide A 248 Thiocoraline 249 Ammosamides 251 Largazole 252 Promising Substances Isolated from Macroalgae and Invertebrates 254 Griffithsin 254 PM-050489 and PM-060184; Two New Sponge Polyketides 254 Immucothel1 (Keyhole Limpet Hemocyanin; KLH) 255 Jorumycin (Zalypsis1) 255 Promising Substances Synthesized from Natural Models 255 Plitdidepsin from the Ascidian Aplidium albicans 255 Roscovitine (Seliciclib, CYC202): A Synthetic Analog of Natural Purines 255 DMXBA (GTS-21): A Synthetic Analog of Anabaseine 256 Bryologs: Synthetic Analogs of Bryostatins 258 Conclusion 259 References 259 Promises of the Unprecedented Aminosterol Squalamine 265 Marie-Lise Bourguet-Kondracki and Jean-Michel Brunel Introduction 265 Discovery of the Unprecedented Aminosterol Squalamine 265 Syntheses of Squalamine 268

12.4 12.4.1 12.4.2 12.4.3 12.4.4 12.5 12.6 12.6.1 12.6.2 12.7 12.7.1 12.7.2 12.8 12.9

13

13.1 13.2

13.3 13.4

13.5

13.6

13.7

13.8

14 14.1 14.1.1 14.1.2 14.1.3 14.2 14.2.1 14.2.2

Biological Activities 270 Antimicrobial Activities of Squalamine and Its Mimics 270 Antiangiogenic Activity of Squalamine 274 Antitumor Activity of Squalamine 274 Antiviral Activities 275 Mechanism of Antiangiogenic Activity of Squalamine 275 Preclinical Studies of Squalamine 276 Antitumor Therapy 276 Retinopathy 277 Clinical Studies of Squalamine 277 Human Cancers 277 Age-Related Macular Degeneration 278 Bioactive Potential of Trodusquemine, a Natural Squalamine Derivative 278 Conclusion 280 References 280 Marine Peptide Secondary Metabolites 285 Bernard Banaigs, Isabelle Bonnard, Anne Witczak, and Nicolas Inguimbert Introduction 285 Ribosomal- and Nonribosomal-Derived Peptides: A Virtually Unlimited Source of New Active Compounds 286 Laxaphycins and their Derivatives: Peptides Not So Easy to Synthesize 291 Dolastatins: From Deception to Hope Through Structural Modification Leading to Reduced Toxicity 294 Didemnins and Related Depsipeptides: How Perseverance Should Lead to Their Low-Cost Production 297 Kahalalide F: A Study in Chemical Ecology as a Starting Point for New Antitumoral Agent Discovery 299 Azole/Azoline-Containing Cyanobactins Isolated from Invertebrates: An Example of Nature’s Own Combinatorial Chemistry 304 Conclusion 310 Acknowledgments 311 References 311 Conotoxins and Other Conopeptides 319 Quentin Kaas and David J. Craik Background 319 Historical Interest in Cone Snails 319 Biology of Cone Snails 319 Cone Snail Venoms, their Conopeptides and Molecular Targets 320 Diversity of Conopeptides 321 Conopeptide Maturation and The Origin of Venom Diversity 321 Diversification at the Gene Level 321

Contents

14.2.3 14.2.4 14.2.4.1 14.2.4.2 14.2.4.3 14.3 14.3.1 14.3.2 14.4 14.4.1 14.4.2 14.5 14.6

15

15.1 15.1.1 15.1.2 15.2 15.2.1 15.2.2 15.2.2.1 15.2.2.2 15.2.3 15.2.3.1 15.2.3.2 15.2.4 15.2.4.1 15.2.4.2 15.2.4.3 15.2.5 15.2.6 15.2.6.1 15.2.6.2 15.2.7 15.2.7.1 15.2.7.2 15.2.8 15.3 15.3.1 15.3.1.1 15.3.1.2 15.3.1.3

Additional Diversity at the Protein Level 322 Nomenclature and Classification Schemes 323 Gene Superfamilies 323 Cysteine Frameworks 323 Pharmacological Families 323 Isolation Techniques 323 Transcriptomics-Based Conopeptide Discovery 324 Proteomics Studies of Conopeptides 324 Conopeptide Three-Dimensional Structures 325 Two-Disulfide Conotoxins 325 Tri-Disulfide Conotoxins 327 Conopeptide Pharmacological Activities 327 Outlook 328 Acknowledgments 328 References 328 Mycosporine-Like Amino Acids (MAAs) in Biological Photosystems 333 St
ephane La Barre, Catherine Roullier, and Jo€el Boustie Background 333 Life in Full Light and its Constraints 333 MAAs: To Protect and Serve, Occasionally to Defend 334 Chemistry 335 Physico-Chemical Characteristics of MAAs 335 MAAs and Related Molecules 335 MAAs in the Marine World 335 MAAs and Related Molecules in Lichens 335 Extraction, Separation, Purification, and Detection 335 Extraction, Separation, and Purification 335 Detection, Quantification, and Monitoring in Live Samples 339 Structure Determination 339 Ultraviolet (UV) Spectroscopy 339 Mass Spectrometry (MS) 339 Nuclear Magnetic Resonance (NMR) Spectroscopy 340 Synthesis 341 Biosynthesis: Labeled Precursor Investigations 341 The Shikimic Acid Pathway 341 The Pentose Phosphate Pathway 342 Regulation of MAA Production: Light and Nutrients 342 Light 342 Nutrients 343 Degradation 344 MAA-Producing Organisms 344 Chemical Protection Against Abiotic Stress 344 Symbiont-Assisted Metabolism 344 The “m
enage a trois” Solution 344 The Chemical Answer to an Exposed Mode of Life 345

15.3.1.4 15.4 15.4.1 15.4.2 15.4.3 15.4.4 15.4.5 15.4.6 15.5 15.6 15.6.1 15.6.2 15.7 15.A

16

16.1 16.2 16.3 16.4 16.4.1 16.4.2 16.4.3 16.4.4 16.4.5 16.5 16.6 16.6.1 16.6.2 16.7 16.7.1 16.7.2 16.7.2.1 16.8

j IX

Simple, Effective, and Ubiquitous: Why Change a Winning Recipe? 345 Hermatypic Corals: Living Under Tight Constraints 345 Coral Reefs are Monumental Bioconstructions 345 Corals are Highly Efficient Photosynthesizers 345 High Temperatures and UV Exposures Induce Oxidative Stress and Bleaching in Corals 346 The Chemical Acclimation of Scleractinian Corals to an Exposed Lifestyle 346 Biogenic Sources of MAAs in Scleractinian Corals 347 The Phylogenomics of MAAs in Scleractinian Corals 347 Lichenic Systems: Living in the Extremes 347 Modes of Action and Applications to Human Welfare 348 Skin Care and Cosmetics 349 Biotechnological Applications 349 Conclusions 349 Acknowledgments 349 Appendix 15A.1 Proton NMR data of Mycosporines and Mycosporine-like Amino Acids (MAAs) 350 Appendix 15A.2 Carbon thirteen data of Mycosporines and Mycosporine-like Amino Acids 354 References 357 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology 361 Franck Zal and Morgane Rousselot Introduction 361 Annelid Extracellular Hemoglobins 362 Architecture 364 Model of Quaternary Structures 366 Electron Microscopy 366 Estimation of Heme Number and Minimal Molecular Weight 367 Small-Angle Light Scattering 368 Low- and High-Pressure Liquid Chromatography and SDS–PAGE 369 Electrospray Ionization-Mass Spectrometry 369 Biotechnology Applications 370 Organ Preservation 370 Preservation Solutions 370 Hypothermic Continuous Reperfusion 371 Anemia 371 Hemoglobin Oxygen Carriers 372 Normovolemic Hemodilution 372 HEMOXYCarrier1 372 Conclusion 372 Acknowledgments 373 References 373

X

j

17

17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 17.10

Contents

Lamellarins: A Tribe of Bioactive Marine Natural Products 377 Christian Bailly Introduction 377 Lamellarins: Bioactive Marine Natural Products 378 Anticancer Activities of Lamellarins 379 Inhibition of Topoisomerase I by Lamellarins 380 Inhibition of Protein Kinases by Lamellarins 380 Lamellarin-induced Mitochondrial Perturbations 380 Antiviral Activity of Sulfated Lamellarins 382 Synthesis of Lamellarins 382 Non-Natural Lamellarin Analogs 383 Conclusion 384 References 384

Part Four New Trends in Analytical Methods 18 18.1 18.2 18.3 18.4 18.4.1 18.4.1.1 18.4.1.2 18.4.1.3 18.4.1.4 18.4.2 18.4.3 18.4.4 18.4.4.1 18.4.4.2 18.4.4.3 18.4.4.4 18.4.4.5 18.5 18.6 18.6.1 18.6.2 18.6.3

18.6.4 18.6.5 18.6.6 18.6.7

387

NMR to Elucidate Structures 389 Ga€elle Simon, Nelly Kervarec, and St
ephane C
erantola Introduction 389 NMR to Elucidate Structures 389 Sample Preparation 390 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei 393 1 H Spectra 393 Chemical Shift 394 Multiplicity 394 Integration 396 Special Features of Sample 396 13 C Spectra 400 2D Spectra 402 Other Nuclei Spectra 408 Isotopes with No NMR Properties 408 Isotopes (I ¼ 1/2) with 100% Abundance 408 Isotopes (I ¼ 1/2) with Low Abundance 411 Isotopes (I > 1/2) with Long T1-Values 415 Isotopes (I > 1/2) with Short T1-Values 415 Cryoprobes: Obtaining 1D and 2D Spectra Mainly in 1 H, 13 C 417 HRMAS NMR: Obtaining 1 H, 13 C, 31 P, 15 N 1D and 2D Spectra 417 Studies of Bacterial Strains from the Marine Deep 420 Differentiation Between Two Species 421 Effect of Exposure to Pollutants on Species Metabolism and Possible Pollutant Bioaccumulation 421 Application of 1H HRMAS NMR to Define Organ Cartography 423 Identification of Different Cultivable Marine Bacteria 424 Monitoring Quantitative Seasonal Variations of a Molecule 424 Understanding the Metabolism of a Species 425

18.7 18.8

19 19.1

20

20.1 20.2 20.2.1 20.2.2 20.2.2.1 20.2.2.2 20.2.2.3 20.2.3

20.2.3.1 20.2.3.2 20.3 20.3.1

20.3.1.1 20.3.1.2 20.3.1.3 20.3.2 20.3.2.1 20.3.2.2 20.3.2.3 20.3.2.4 20.3.2.5 20.3.3 20.3.3.1 20.3.3.2 20.3.3.3 20.3.3.4 20.4

CPMAS NMR: Obtaining all NMR Observable Nuclei Spectra 425 Conclusion 426 References 428 An Introduction to Omics 431 Jonas Coll
en and Catherine Boyen What are “Omics”? 431 References 434 Gene Mining for Environmental Studies and Applications: Examples from Marine Organisms 435 Simon M. Dittami and Thierry Tonon Introduction 435 Techniques 435 Sampling and Extraction: An Overview 435 Properties of Nucleic Acids 436 Genomic DNA 436 RNA 436 mRNA, rRNA, and rDNA 437 Recent Technological Advances in Molecular Biology and their Impact on Marine Biology 437 Sequencing Technology 437 Gene Expression Profiling 437 Current Applications 439 Development of Genomic and Transcriptomic Resources for Molecular Analysis of Organisms Under Environmental Threats: Application to Coral Physiology 439 Context 439 Selection of Coral Transcriptomics Studies in Relation to Climate Change 440 Concluding Remarks 443 Search for Genes Involved in Toxin Production within the Dinoflagellate Haystack 443 Context 443 Genes Involved in the Synthesis of Polyketide Dinotoxins 444 Molecular Bases of Dinoflagellate Saxitoxin Production 445 Influence of Abiotic and Biotic Factors on Dinotoxin Biosynthetic Pathways 446 Concluding Remarks 448 Molecular Biomonitoring of Marine Environments 448 Hierarchical Taxon-Specific and Function-Specific DNA Probes 448 Quantifying Biomass 449 Short and Mid-Term Monitoring of Marine Bacteria and Microalgae 450 Molecular Biomonitoring of Harmful Algae 450 Conclusions and Outlook 452 References 452

Contents

21

21.1 21.2 21.2.1 21.2.2 21.2.3 21.2.3.1 21.2.3.2 21.2.4 21.3 21.3.1 21.3.1.1 21.3.1.2 21.3.2 21.3.2.1 21.3.2.2 21.3.3 21.3.4 21.3.5 21.3.6 21.3.6.1 21.3.6.2 21.3.6.3 21.4

22

22.1 22.2 22.2.1 22.2.2

Proteomics and Metabolomics of Marine Organisms: Current Strategies and Knowledge 457 Fanny Gaillard and Philippe Potin Introduction 457 General Strategies for Proteomics and Peculiarities of the Marine Environment 458 Protein Extraction 458 Prefractionation 460 Quantification 460 Relative Quantification 460 Absolute Quantification 461 Direct Cell or Tissue Analysis 461 General Strategies for Metabolomics, and Peculiarities of the Marine Environment 461 Experimental Design and Sample Preparation for Metabolomics 462 Experimental Design 462 Sample Preparation 462 Analytical Tools for Metabolomics 464 Nuclear Magnetic Resonance (NMR) 464 Mass Spectrometry (MS) 464 Spectral Signal Processing in NMR and MS Metabolomics 465 Statistical Analysis 466 Challenges of Metabolite Identification 466 Current Applications of Marine Metabolomics 466 Health and Disease of Marine Organisms 466 Biodiversity and Chemometry 467 Signals in the Sea: Metabolomics and Marine Chemical Ecology 467 Conclusions 468 Acknowledgments 468 References 469 Genomics of the Biosynthesis of Natural Products: From Genes to Metabolites 473 Olivier Ploux and Annick M
ejean Introduction 473 Biosynthesis of PKs, NRPs and RiPPs: Basic Principles 474 The PKSs Polymerize Acetate Units 474 The NRPSs: A Biological Solid-Phase Peptide Synthesis 475

22.2.3 22.2.4 22.3

22.3.1 22.3.2 22.3.3 22.3.4 22.4

23

23.1 23.2 23.2.1 23.2.2 23.2.3 23.2.4 23.2.4.1 23.2.4.2 23.2.5 23.3 23.3.1 23.3.2 23.3.3 23.3.4 23.4

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Connecting Biosynthetic Genes to Natural Product Structure 475 The Diversity of RiPPs 476 Connecting Genes and Metabolites: Selected Examples of Aquatic Natural Product Biosynthesis 476 Curacins 477 Anatoxin-a and Homonatoxin-a 478 Microcystins 480 Cyanobactins 482 Conclusions and Perspectives 483 Abbreviations 483 References 484 High-Throughput Screening of Marine Resources 489 Arnaud Hochard, Luc Reininger, Sandrine Ruchaud, and St
ephane Bach Introduction 489 High-Throughput Screening and Drug Development 490 Screening Assay Development and Validation 490 Statistical Tools for Quality Assessment of HTS Assays 491 Choice of Screening Strategy 492 Data Analysis: From Hits to Leads 492 Hits 492 Leads 493 From HTS Assay to Market: The Drug Development Process 493 Examples of High-Throughput Screening 493 Chemical Libraries: The Fuel of HTS 493 Biochemical Assay: The Example of Protein Kinases 494 Protein–Protein Interactions (PPIs) 494 Cell-Based Assay: The Example of Bryostatins 495 Conclusions and Perspectives 495 List of Abbreviations 496 Acknowledgments 496 References 496 Index 499

j XIII

List of Contributors Ali Al-Mourabit Natural Product Chemistry Institute (ICSN) Department of Natural Products & Medicinal Chemistry (SNCM) Research Center of the CNRS at Gif sur Yvette Avenue de la terrasse 91190 Gif sur Yvette France [email protected]

St
ephane S. Bach Sorbonne Universit
es UPMC Univ Paris 06 USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected]

Philippe Amade Universit
e de Nice Sophia Antipolis Institut de Chimie de Nice, UMR 7272 CNRS, Facult
e des Sciences Parc Valrose 06108 Nice cedex 2 France [email protected]

and

Zouher Amzil IFREMER (Institut FrancS ais de Recherche pour l’Exploitation de la Mer) Laboratoire Phycotoxines Rue de l’Ile d’Yeu, BP21105 F-44311 Nantes cedex 3 France [email protected]
oz Romulo Ara Institut F
ed
eratif de Neurobiologie Alfred Fessard FR2118, Center de recherche CNRS de Gif-surYvette, Laboratoire de Neurobiologie et D
eveloppement UPR 3294 1 avenue de la Terrasse 91198 Gif sur Yvette Cedex France [email protected]

CNRS USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Christian Bailly Institut de Recherche Pierre Fabre Centre de Recherche et D
eveloppement 3 Avenue Hubert Curien - BP 13562 31035 Toulouse Cedex 1 France [email protected] Bernard Banaigs Universit
e de Perpignan via Domitia Laboratoire de chimie des biomol
ecules et de l’environnement, EA4215 52 avenue Paul Alduy 66860 Perpignan cedex France [email protected]

Georges Barbier Universit
e Europ
eenne de Bretagne, Universit
e de Brest, ESMISAB Laboratoire Universitaire de Biodiversit
e et Ecologie Microbienne (EA3882) IFR 148, Technopole Brest-Iroise 29280 Plouzan
e France [email protected] Gilles Barnathan Universit
e de Nantes Groupe Mer-Mol
ecules-Sant
e MMS/EA
CHIM – Lipides marins a 2160, Equipe activit
e biologique, Facult
e des Sciences pharmaceutiques et biologiques, Institut Universitaire Mer et Littoral FR3473 CNRS 9 rue Bias BP 53508 44035 Nantes France [email protected] Stephen S. Bates Fisheries and Oceans Canada Gulf Fisheries Centre P.O. Box 5030 Moncton New Brunswick E1C 9B6 Canada [email protected] Elodie Blanchet University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France and

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List of Contributors

Atlanthera, Atlantic Bone Screen F-44800 Saint Herblain Nantes France [email protected] Isabelle Bonnard Universit
e de Perpignan via Domitia Laboratoire de chimie des biomol
ecules et de l’environnement, EA4215 52 avenue Paul Alduy 66860 Perpignan cedex France [email protected] Marie-Lise Bourguet-Kondracki Mus
eum National d’Histoire Naturelle Mol
ecules de Communication et Adaptation des Micro-Organismes (MCAM) UMR 7245 CNRS/MNHN 57 rue Cuvier (CP 54) 75005 Paris France [email protected] Jo€el Boustie Universit
e de Rennes 1 Equipe PNSCM (Produits Naturels, Syntheses et Chimie M
edicinale), UMR CNRS 6226, Facult
e des Sciences Pharmaceutiques et Biologiques 2 Av. du Pr. L
eon Bernard 35043 Rennes Cedex France [email protected]

Jean-Michel Brunel Aix-Marseille Universit
e Centre de Recherche en Canc
erologie de Marseille (CRCM), CNRS, UMR7258; Institut Paoli Calmettes UM 105; Inserm, U1068 F-13009 Marseille France [email protected] Ga€etan Burgaud Universit
e Europ
eenne de Bretagne, Universit
e de Brest, ESMISAB Laboratoire Universitaire de Biodiversit
e et Ecologie Microbienne (EA3882) IFR 148, Technopole Brest-Iroise 29280 Plouzan
e France [email protected] Alyssa Carr
e-Mlouka National Museum of Natural History 75005 Paris France [email protected] St
ephane C
erantola Universit
e de Bretagne Occidentale Technological Platform of Nuclear Magnetic Resonance, Electron Paramagnetic Resonance and Mass Spectrometry 6, av. Victor Le Gorgeu, CS93837 29238 Brest Cedex 3 France [email protected]

Catherine Boyen Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected]

B
en
edicte Charrier Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected]

and

and

CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

Mireille Chinain Institut Louis Malard
e Laboratoire de recherche sur les Microalgues Toxiques BP30, 98713 Papeete Tahiti French Polynesia [email protected] Jonas Coll
en Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Aur
elie Couzinet-Mossion Universit
e de Nantes Groupe Mer-Mol
ecules-Sant
e MMS/EA
2160, Equipe CHIM – Lipides marins a activit
e biologique, Facult
e des Sciences pharmaceutiques et biologiques, Institut Universitaire Mer et Littoral FR3473 CNRS 9 rue Bias BP 53508 44035 Nantes France aurelie.couzinet-mossion@ univ-nantes.fr David J. Craik The University of Queensland Institute for Molecular Bioscience Brisbane QLD 4072 Australia [email protected]

List of Contributors

C
ecile Debitus Institut de Recherche pour le D
eveloppement UMR 241 BP 529, 98713 Papeete Polyn
esie FrancS aise [email protected] Simon M. Dittami Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] Sergey Dobretsov Sultan Qaboos University P. O. Box 50 Muscat 123 Oman [email protected] Virginia. P. Edgcomb Woods Hole Oceanographic Institution Geology and Geophysics Department Woods Hole MA 02543 USA [email protected] Jean-Baptiste Fournier Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected]

and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Fanny Gaillard Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] Stjepko Golubic Boston University Biological Science Center 5 Cummington Street Boston MA 02215 USA [email protected] Olivier Grovel University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected] Muriel Gugger Institut Pasteur, Collection des Cyanobacte
eries D
e´partement de Microbiologie 28 rue du Dr Roux 75015 Paris France [email protected]

j XV

Yann Guitton University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected] Tilmann Harder University of New South Wales Centre for Marine Bio-Innovation, School of Biological, Earth and Environmental Science Sydney Australia 2052 [email protected] Arnaud Hochard USR3151-CNRS Protein phosphorylation and human diseases, Kinase Inhibitor Specialized Screening facility (KISSf ) Station Biologique CNRS-UPMC Place Georges Teissier, CS 90074 29688 Roscoff Bretagne France [email protected] Nicolas Inguimbert Universit
e de Perpignan via Domitia Laboratoire de chimie des biomol
ecules et de l’environnement, EA4215 52 avenue Paul Alduy 66860 Perpignan cedex France [email protected] Quentin Kaas The University of Queensland Institute for Molecular Bioscience Brisbane QLD 4072 Australia [email protected] Nelly Kervarec Universit
e de Bretagne Occidentale Technological Platform of Nuclear Magnetic Resonance, Electron Paramagnetic Resonance and Mass Spectrometry 6, av. Victor Le Gorgeu, CS93837 29238 Brest Cedex 3 France [email protected]

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List of Contributors

Staffan Kjellberg University of New South Wales Centre for Marine Bio-Innovation, School of Biotechnology and Biomolecular Science Sydney Australia 2052 and Nanyang Technological University Singapore Centre on Environmental Life Sciences Engineering Singapore 639798 [email protected] Jean-Michel Kornprobst (Editor) Institut Mer et Littoral Bâtiment Isomer 2, rue de la Houssinière 44322 Nantes BP 92208 Cedex 3 France [email protected] St
ephane La Barre (Editor) Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] Dominique Laurent Institut de Recherche pour le D
eveloppement (IRD) Pharma-Dev UMR 152 BP529, 98713 Papeete Tahiti French Polynesia [email protected]

Catherine Leblanc Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] and

Annick M
ejean Chimie ParisTech, ENSCP Laboratoire Charles Friedel 11 rue Pierre et Marie Curie 75231 Paris Cedex 05 France and CNRS, UMR 7223 11 rue Pierre et Marie Curie 75231 Paris Cedex 05 France and

CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

Universit
e Paris Diderot 35 rue H
elene Brion 75205 Paris Cedex 13 France [email protected]

Richard J. Lewis University of Queensland Institute for Molecular Bioscience 306, Carmody Road St Lucia QLD 4072 Australia [email protected]

Laurence Meslet-Cladiere Universit
e Europ
eenne de Bretagne, Universit
e de Brest, ESMISAB Laboratoire Universitaire de Biodiversit
e et Ecologie Microbienne (EA3882) IFR 148, Technopole Brest-Iroise 29280 Plouzan
e France [email protected]

Diane McDougald University of New South Wales Centre for Marine Bio-Innovation, School of Biotechnology and Biomolecular Science Sydney Australia 2052 and Nanyang Technological University Advanced Environmental Biotechnology Centre, Nanyang Environment and Water Institute Singapore 639798 [email protected] Mohamed Mehiri Universit
e de Nice Sophia Antipolis Institut de Chimie de Nice, UMR 7272 CNRS, Facult
e des Sciences Parc Valrose 06108 Nice cedex 2 France [email protected]

Zofia Nehr Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

List of Contributors

Olivier Ploux Chimie ParisTech, ENSCP Laboratoire Charles Friedel 11 rue Pierre et Marie Curie 75231 Paris Cedex 05 France and CNRS, UMR 7223 11 rue Pierre et Marie Curie 75231 Paris Cedex 05 France [email protected] Philippe Potin Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] Yves-FrancS ois Pouchus University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected] Michael Quilliam National Research Council Canada Measurement Science and Standards 1411 Oxford Street Halifax Nova Scotia B3 H 3Z1 Canada [email protected]

Luc Reininger Sorbonne Universit
es UPMC Univ Paris 06 USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] and CNRS USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Scott A. Rice University of New South Wales Centre for Marine Bio-Innovation, School of Biotechnology and Biomolecular Science Sydney Australia 2052 and Nanyang Technological University Singapore Centre on Environmental Life Sciences Engineering Singapore 639798 [email protected] M
elanie Rou
e Research Scientist IRD-UMR 241 (EIO) Centre Polynésien de Recherche et de valorisation de la Biodiversité Insulaire B.P. 529, 98713 Papeete, Polynésie Française [email protected] Catherine Roullier University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected]

j XVII

Morgane Rousselot HEMARINA SA Biotechnop^ ole A
eropole Centre 29600 Morlaix France [email protected] Sandrine Ruchaud Sorbonne Universit
es UPMC Univ Paris 06 USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] and CNRS USR 3151 Protein Phosphorylation and Human Diseases Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France Nicolas Ruiz University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected] Ga€elle Simon Universit
e de Bretagne Occidentale Technological Platform of Nuclear Magnetic Resonance, Electron Paramagnetic Resonance and Mass Spectrometry 6, av. Victor Le Gorgeu, CS93837 29238 Brest Cedex 3 France [email protected]

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Peter D. Steinberg University of New South Wales Centre for Marine Bio-Innovation, School of Biological, Earth and Environmental Science Sydney Australia 2052 and Sydney Institute of Marine Science Mosman NSW Australia 2088 [email protected] Torsten Thomas University of New South Wales Centre for Marine Bio-Innovation, School of Biotechnology and Biomolecular Science Sydney Australia 2052 [email protected] Thierry Tonon Sorbonne Universit
es UPMC Univ Paris 06 UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France

and CNRS UMR 8227 Integrative Biology of Marine Models Station Biologique de Roscoff CS 90074 F-29688 Roscoff cedex France [email protected] Jean Turquet ARVAM CYROI, La Technopole 2, rue maxime Riviere 97490 Sainte Clotilde La R
eunion France [email protected] Marieke Vanstellandt University of Nantes Faculty of Pharmacy MMS, 9 rue Bias F-44000 Nantes Cedex 1 France [email protected]

Ga€etane Wielgosz-Collin Universit
e de Nantes Groupe Mer-Mol
ecules-Sant
e MMS/EA
2160, Equipe CHIM – Lipides marins a activit
e biologique, Facult
e des Sciences pharmaceutiques et biologiques, Institut Universitaire Mer et Littoral FR3473 CNRS 9 rue Bias BP 53508 44035 Nantes France [email protected] Anne Witzak Universit
e de Perpignan via Domitia Laboratoire de chimie des biomol
ecules et de l’environnement, EA4215 52 avenue Paul Alduy 66860 Perpignan cedex France [email protected] Franck Zal HEMARINA SA Biotechnop^ ole A
eropole Centre 29600 Morlaix France [email protected]

j XIX

Foreword Natural products (secondary metabolites) which once were focused on alkaloids and terpenes now cover an infinite molecular diversity, and are merging with primary metabolites through “omics” connections. Today, it is assumed that druggable molecules can also be a matter of bioinspired thinking through close and synergistic partnerships between chemists, biologists and chemical ecologists. Unfortunately, the discovery of new scientific concepts, novel analytical approaches or simply of state-of-the-art techniques tends to be overemphasized, overestimated or overpublicized, relegating essential questioning and basic concerns to the background. In the case of natural products, the isolation of new molecules is currently greatly hampered by this shift in focus, and we feel that the quest for new natural structures (i.e., sourcing) should be actively maintained in the face of pending climate- or humankind-driven habitat degradations and biodiversity destruction. The structural determination of natural new molecules is vital, given its considerable importance for any biological investigation, and includes an understanding of the ecosystems that function at the molecular level and the development of rational products for the treatment of diseases. While structural determination can be achieved more quickly by spectroscopic and crystallographic means, the acquisition of adequate funding for natural products projects is becoming increasingly difficult for both industrial and academic communities alike. The paradox is that the demand for new active molecules is now heralded as a major priority! Recent advances in organic chemistry and in metabolomics analyses, together with the advent of the postgenomic era, now

make it possible to envisage a critical role for natural products chemistry in chemical biology and in chemical ecology, with a timely integration into the multidisciplinary systems biology approach. But, can all of this be envisaged without knowing the structure of the molecule? The answer is definitely: no! In this book is presented a selection of marine molecules which have attracted the attention of a wide panel of reputed scientists worldwide, and especially within the national research network that I am proud to have managed for several years. To the series of comprehensive chapters on marine molecules, deemed outstanding for their interesting structures, their amazing bioactivities, or their environmental significance, critical and highly documented reviews of modern laboratory experimentation have been added. It is hoped that this contribution will provide inspiration to the generation of new scientists and motivate them to embrace a meaningful human healthoriented career, or to invent environmentally dedicated tools and approaches for the benefit of all.

Ali Al-Mourabit Director of BioChiMar Network Natural Product Chemistry Institute (ICSN) Department of Natural Products & Medicinal Chemistry (SNCM) Research Center of the CNRS at Gif sur Yvette France December 2013

j XXI

Preface Our original idea was to provide a series of comprehensive chapters, devoted to molecules that are either naturally produced or transformed by marine organisms, each having a recognized influence on human welfare, or having a significant impact on our chemosphere, and thus on depending life forms. The individual chapters would introduce the molecule(s) of interest in its/their historical or its environmental perspective, develop the analytical aspects (chemistry, structure–activity, synthesis), and finally mention the ecological significance and the pertaining biotechnological developments in the light of the existing literature. Graduate students would have access to essential information on a given molecule, all bundled up in a single chapter pointing out useful references for consultation on specific details. Likewise, teachers would be able to structure a complete lecture on a single topic, with references from which they can follow up a specific aspect. The immediate challenge we had to face was to select 20–25 molecules within the hundreds of eligible candidates. Our second challenge was to contact experts who were willing to spend some of their time and enthusiasm to join our project with at least one contribution. Not an easy task – as excellent textbooks, reviews, handbooks and communications have been published on marine natural products within the past few years. The choice of molecules by our authors naturally fell into three sections: (i) molecules deemed outstanding for their structural originality, their spectral characteristics, or their reactivity and its consequences on synthesis; (ii) molecules that are known to play an important role in isolated organisms or in whole ecosystems; and (iii) molecules that have attracted special interest in the quest for new drugs or new treatments. As the editorial project was being constructed, it was decided that a review of modern analytical approaches, using state-of-the art instrumentation would add a useful complement to the metabolite chapters. Thus, ultimately four Parts were proposed for the book, in which each chapter would be a stand-alone source of information and a useful starting point for someone willing to investigate. Part One includes six chapters, selected as an assortment based on biodiversity as representative criteria, given the editorial constraints. Cyanobacterial toxins represent a well-known problem in the treatment of freshwater for household and recreational uses, but the occurrence of cyanotoxins in maritime zones is not well documented, and a growing concern for isolated populations

which live off their natural resources on a daily basis. The first chapter is devoted to an overview of this subject, by a team of field specialists in association which pharmacologists and neurobiologists (Chapter 1). Highly efficient chemical defenses are produced by microbes or phytoplankton, and concentrated through the food chain, or result from functional interactions between sessile marine organisms and their dedicated microbiomes. In the second chapter are reviewed three major examples of seafood contaminants, which have puzzled generations of investigators and for which prevention remains essential. Some structures are extraordinarily complex, yet highly stable, with surprising bioactivities, as explained by the authors, chemists and pharmacologists who have longstanding experience in working with marine toxins (Chapter 2). The next two chapters deal with marine fungal metabolites, a recently explored source of novel molecules of pharmacological potential. After a review of the importance the genus Penicillium, both as marine fungi and as historical sources of drugs, the first “fungus” chapter expands on three examples of novel structures that have potential as anticancer agents, by leading researchers (Chapter 3). The following chapter explores the hitherto unsuspected source of bioactive drugs from fungi of deep-sea hydrothermal vents, and the biotechnological promises we can anticipate from this newly explored environment – a story told in association between benchtop scientists and field investigators (Chapter 4). The next chapter is devoted to glycoconjugates from marine invertebrates, an often underestimated source of original molecules endowed with bioactivities usually sought in other classes of so-called “secondary metabolites“ (Chapter 5). Part One ends with a very original chapter on molecules found in crinoids which were thought to be extinct since the Triassic– Jurassic extinction event . . . until the unexpected discovery of living representatives in the twentieth century (Chapter 6). Part Two of the book is devoted to metabolites that have no particular originality in terms of structure, but offer some benefits to the source organism, or act as “positive” communication signals between congeners, or between a host and its microbial associates. On the other hand, some of them have a clearly toxic effect on other taxa, and may be the cause of environmental concern. Leading scientists explore the bases of bacterial communication systems in the first chapter (Chapter 7). In the second chapter, the extraordinary story of the discovery of domoic acid is documented by two pioneers, Steve Bates and Mike Quilliam, and its ecological and pharmacological importance is further

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Preface

examined in the light of the most recent research (Chapter 8). The third chapter introduces us to algal morphoinducers, and to the resemblances and differences of cell differentiation and growth patterns between algae and terrestrial plants (Chapter 9). The fourth and last chapter of this “ecology” Part reviews halogenation processes in marine molecules, from molecular mechanisms involving haloperoxidases, to the biogeoclimatic consequences halogenated molecules have locally (Chapter 10). In Part Three, more emphasis is placed on the structure– activity and pharmacological applications in which some molecules have recently been involved, during screenings on targets of interest for major and diverse pathologies. The first chapter reviews recent “highlights,” some of which have interesting potential, mostly as inhibitors (Chapter 11). The second chapter provides a prime example of this multifunctionality, as the authors focus on squalamine, an aminosterol produced by dogfish, and which has revealed a wide array of potential therapeutic applications (Chapter 12). The third chapter reviews marine peptides which have been modified to acquire so-called secondary metabolite characteristics, and are actively studied for their potential as anticancer agents. A whole range of microbial and of metazoan examples are reviewed by authors from a group that has gained longstanding expertise in this class of molecules (Chapter 13). Conotoxin venoms and other conopeptides illustrate further the offensive–defensive specialization made by some carnivorous mollusks of these modified peptides, in a welldocumented text written by authorities on the subject (Chapter 14). Mycosporine-like amino acids (MAAs) are natural antioxidants and sunscreens used by diverse terrestrial and marine organisms or whole photosystems, enabling them to live totally exposed to solar radiations. The authors focus on MAAs produced by lichens and by reef corals, two models with very different lifestyles (Chapter 15). Next, in the pharmacology applications, is a chapter which relates a successful biotechnological adventure. The authors show how they adapted the hemoglobin produced by a lugworm, to an array of therapeutic applications, from firstaid to the optimal storage of organs prior to transplantation (Chapter 16). The closing chapter for this Part introduces lamellarins, a family of complex alkaloids that were originally produced by didemnid ascidians and which represent a fine example of structure–activity relationship, particularly in relation to sulfation patterns (Chapter 17).

Roscoff and Nantes January 2014

Lastly, Part Four provides a state-of-the art technical complement to whoever extracts, purifies, analyzes, mimics and valorizes marine natural products. The chapter on NMR is written by a team of spectroscopists who have developed a range of tools and approaches to cater for a wide variety of marine samples and address specific challenges posed by fellow chemists and biologists. Through multiple examples, the authors provide a rationale for the treatment of individual situations (Chapter 18). The next three chapters provide a comprehensive overview of “omics” – that is, molecular approaches that can be applied to single cells, organisms (systems biology approach), and to whole ecosystems, in order to study interaction dynamics. The range of analytical techniques (genomes, transcriptomes, proteomes, metabolomes) is explored by a panel of scientists who are leaders in their field, and whose research will undoubtedly revolutionize our examination of the ways in which organisms interact in the oceans (Chapters 19–21). Next, Chapter 22 is devoted to the biosynthesis of natural products, using genemining approaches, and is written by world experts in the subject. Finally, a team of young and enthusiastic investigators has devoted the closing chapter to the latest high-throughput screening methods which allow rapid responses to be obtained from a large number of minute samples of molecules exposed to specific molecular targets, especially those that directly control cell division cycles (Chapter 23). Finally, we wish to thank our fellow members of the French research cluster BioChiMar who responded very rapidly, spared some of their time, and shared their enthusiasm by writing chapters on some of their research or on favorite subjects, for the benefit of others. Marine natural products is indeed a treasure chest for ecologists to explore, for pharmacologists to investigate, and for humankind to preserve in anticipation of the unprecedented climatic changes that are forecast to occur during the next decades as a consequence of global warming. Undoubtedly, the latter topic will result in massive collapses in species diversity in fragile and complex ecosystems, and especially in areas subjected to direct human interference.

St
ephane La Barre Jean-Michel Kornprobst

j1

Part One Outstanding Marine Molecules from a Chemical Point of View

Ó Aquarium des Lagons, New Caledonia.

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

j3

1 Marine Cyanotoxins Potentially Harmful to Human Health M
elanie Rou
e, Muriel Gugger, Stjepko Golubic, Zouher Amzil, Romulo Ar
aoz, Jean Turquet, Mireille Chinain, and Dominique Laurent

Abstract

Many people around the world depend on the marine environment, for its nutritional, recreational, and general economic value. For many years, a notable increase has been observed in the number of cases of severe intoxication, through the consumption of contaminated seafood and through external exposure. While dinoflagellates and diatoms

1.1 Introduction

Many people around the world depend on the marine environment, for its nutritional, recreational, and general economic value. This is especially true in tropical regions, where the economy is highly dependent on seafood for subsistence, for the local export industry, and for tourism. Although fish and shellfish are an essential nutritional resource to the populations of islands and coastal regions, a notable increase in numbers of cases of severe poisoning related to the consumption of seafood during recent years has forced these populations to modify their eating habits. In French Polynesia, for example, the reduction of fishing in coral reefs and lagoons and the increased reliance on imported food instead, may have already contributed to the rising prevalence of chronic diseases such as diabetes, hypertension and cardiovascular diseases in indigenous Pacific populations (Chinain et al., 2010). Cyanobacteria (formerly “blue-green algae”) occupy a wide range of marine, freshwater, and terrestrial habitats. They are ubiquitous in marine ecosystems, where they play a major role in oxygen production and the fixation of atmospheric carbon and nitrogen. Cyanobacteria proliferating in marine environments represent an important source of structurally diverse bioactive secondary metabolites (Burja et al., 2001; Tan, 2007; Uzair et al., 2012), with over 800 compounds identified (Jones et al., 2010). However, compared to freshwater cyanobacteria,

are considered the main source of marine biotoxins, there is also growing evidence that certain groups of marine cyanobacteria are likely to produce various toxins with potential harmful effects on humans, especially in cases of massive proliferation. Some of the recent findings that support this hypothesis are summarized in this chapter.

relatively little attention has been paid to the toxicity of marine cyanobacteria as a human health hazard. Microalgae – in particular dinoflagellates but also diatoms (Fritz et al., 1992) – are considered to be the main source of marine biotoxins, particularly those that are biomagnified (i.e., accumulated and concentrated) along the food chain, and are at the origin of many human poisoning syndromes, such as Ciguatera Fish Poisoning, which is highly prevalent in the Pacific. The question is: Could marine cyanobacteria also have the ability to biosynthesize toxins? And if so, are they able to contaminate seafood such as to become potentially harmful to human health? Marine cyanobacteria are an enormous source of ever-increasing bioactive compounds that include cytotoxins, neurotoxins and dermatotoxins, among others (Shimizu, 2003; Nunnery, Mevers, and Gerwick, 2010). However, to date, no human mortality related to cyanobacterial marine toxins has been demonstrated. Some cyanobacterial isolates in freshwater (and brackish environments) are known to produce cyanotoxins and are a public health hazard when ingested with drinking water, leading to severe human or livestock poisonings (Falconer and Humpage, 2005; Funari and Testai, 2008). In addition, they can be fatal through hemodialysis (Azevedo et al., 2002), recreational exposure, or accumulation in food (Funari and Testai, 2008). There is recent evidence that marine cyanobacteria can indeed have harmful effects on humans through the consumption of contaminated seafood and through external exposure, especially in cases of massive proliferation (Figure 1.1).

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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1 Marine Cyanotoxins Potentially Harmful to Human Health

Figure 1.1 Headlines about the harmful effects of marine cyanobacteria on humans through the consumption of contaminated seafood (French Polynesia, New-Caledonia) and through external exposure (Mayotte). (a) Ó La D
ep^eche, no. 791, March 7th, 2010; (b) Ó Les Nouvelles Cal
edoniennes, November 11th, 2011; (c) Ó Cellule de l’Institut de Veille Sanitaire en R
egion Oc
ean Indien, BVS no. 9, February, 2012 (according to Lernout et al., 2012); (d) Ó M. Valo, Le Monde, August 20th, 2012.

1.2 Marine Cyanobacteria as Causative Agent of Ciguatera-Like Poisoning 1.2.1 Ciguatera Fish Poisoning

Ciguatera Fish Poisoning (CFP) is the most common marine foodborne disease, and is responsible for more cases of human poisonings than all other marine toxins combined (Fleming et al., 2006; EFSA, 2010). While CFP occurs primarily in tropical regions of the South Pacific Ocean, Indian Ocean and Caribbean Sea (Lewis, 2001), its incidence rates have recently been shown to be increasing in temperate regions (Aligizaki, Nikolaidis, and Fraga, 2008; Dickey and Plakas, 2010; Boada et al., 2010). CFP is classically known to result from the ingestion of tropical coral reef fish contaminated with ciguatoxins (CTXs) (Figure 1.2). CTXs are heat-stable polyethers mainly produced by microalgal benthic dinoflagellates belonging to the genus Gambierdiscus (Yasumoto et al., 1977;

Bagnis et al., 1980, Holmes et al., 1991) (Figure 1.3). These toxins are further transferred through the marine food web to herbivorous and then to carnivorous fish (Litaker et al., 2010). Human poisoning typically occurs after the consumption of herbivorous or carnivorous toxic fish (Randall, 1958; Bagnis et al., 1980). CTXs are potent activators of voltage-sensitive sodium channels (VSSCs), and cause an increase of the neuronal excitability and neurotransmitter release (Nicholson

H3C H

H H C HO 3 O

H

O

H

O

H H O

H

H OH

H HO

O

H

O H H OH H

O H H

Figure 1.2 Chemical structure of ciguatoxin P-CTX-3C.

O

H

CH3

H O H H3C

OH H O O CH3

1.2 Marine Cyanobacteria as Causative Agent of Ciguatera-Like Poisoning

Figure 1.3 Microphotograph of a Gambierdiscus sp. cell, the toxic dinoflagellate responsible for the production of ciguatoxins. Image Ó Institut Louis Malard
e.

and Lewis, 2006). Through the food web, significant biotransformations of Gambierdiscus-produced CTXs occur by oxidative changes, enhancing their potency (Mills, 1956, Yasumoto et al., 2000). Symptoms of CFP intoxication include a combination of more than 30 gastrointestinal, neurological and general medical disturbances (Bagnis, Kuberski, and Laugier, 1979; Gillepsie et al., 1986; Quod and Turquet, 1996); the most typical of these include temperature reversal sensations, paresthesia, pruritus, asthenia, and gastrointestinal disturbances. The severity of CFP symptoms depends on a combined influence of ingested dose, toxin profiles, and individual susceptibility. The realization that ciguatera-type fish and clam poisoning, intensified by an accumulation along the food chain, may be caused by benthic cyanobacteria rather than dinoflagellates came as recently as 2005 (Laurent et al., 2005). Between 2001 and 2005, the villagers of the tribe of Hun€et€e village in the island of Lifou (Loyalty Islands, New Caledonia) had observed that many cases of seafood poisoning that occurred following the consumption of giant clams or of grazing and molluskivorous fish, resembled the familiar CFP. However, the villagers were surprised by the severity of the symptoms, the elevated number of hospitalizations, and by the inefficiency of traditional remedies. On their initiative, the Institut de Recherche pour le D
eveloppement (IRD) conducted a thorough environmental survey of the affected area and found an outward-expanding degradation of the coral reef environment. No evidence of the presence of Gambierdiscus blooms was found; rather, large populations of benthic cyanobacteria of the genus Hydrocoleum K€ utzing were present (Laurent et al., 2005; Laurent et al., 2008; Laurent et al., 2012). Although early studies from many research groups had shown Gambierdiscus spp. and dinoflagellates to be the primary causative agents of CFP intoxications (Yasumoto et al., 1977; Bagnis et al., 1980), cyanobacteria were actually suspected first. Randall (1958) assumed that a benthic organism, most likely a blue-green alga, was the source of the toxin responsible for CFP. When, in 1964, a group of 33 people in

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Bora Bora Island (French Polynesia) were seriously poisoned following consumption of the giant clam Tridacna maxima (Tridacnidae), Bagnis (1967) emphasized the presence of bluegreen algae covering the giant clams in a limited area of the lagoon containing ciguateric fishes. This was also the first report of the implication of giant clams in ciguateric intoxications with a triple vasomotor, digestive and nervous syndrome that was in agreement with the typical syndromes of CFP. In the same year, based on the presence of the benthic cyanobacterium Lyngbya majuscula in the gut of a large number of poisonous fishes, Halstead (1967) hypothesized that these cyanobacteria might produce CTXs. During the 1990s, two experimental studies showed for the first time that a marine pelagic cyanobacterium, Trichodesmium erythraeum, could – just as Gambierdiscus dinoflagellates – be a potential source of toxins in CFP (Hahn and Capra, 1992; Endean et al., 1993). Hahn and Capra (1992) demonstrated typical signs of CFP intoxication in mice injected intraperitoneally with extracts from T. erythraeum and from mollusk samples collected during and shortly after the cyanobacterial bloom. Endean et al. (1993) demonstrated that the toxin profiles of extracts from T. erythraeum were similar to the corresponding fractions obtained from the flesh of the plankton-eating fish Scomberomorus commersoni (mullet, Mugilidae). Mullet are known to graze on Trichodesmium blooms, and are often implicated with CFP intoxications. This observation was later confirmed by the detection of CTXs-like compounds in Trichodesmium blooms collected in New Caledonian waters, where cases of CFP intoxications following the ingestion of mullets have been reported (Kerbrat et al., 2010). Planktonic Trichodesmium and benthic Hydrocoleum, recently observed in a toxic area of Lifou, New Caledonia, where inhabitants were intoxicated, are the most common bloom-forming filamentous cyanobacteria in tropical seas. They are morphologically similar, closely related with respect to 16 S rRNA gene (Abed et al., 2006), and are also both toxic. Plankton blooms of Trichodesmium are subject to drift by wind and currents to the coasts. Recently, an interrelationship between the disturbances of the reef ecosystem, the presence of benthic cyanobacterial blooms (Figure 1.4), and the occurrence of CFP-like incidents was observed in different regions of the South Pacific: New Caledonia (Lifou island), French Polynesia (Raivavae and Rurutu islands) and Republic of Vanuatu (Emao island) (Chinain et al., 2010; Laurent et al., 2012). Finally, Ehrenreich et al. (2005) documented that diverse marine and freshwater cyanobacteria possess the sequences of gene fragments from nonribosomal peptide synthetases (NRPS) and modular polyketide synthases (PKS). The construction of CTXs is achieved via the polyketide pathway and thus probably involves PKS; these results thus support the hypothesis that many cyanobacteria – just as the dinoflagellates – could be a potential source of CTXs, or CTX-like compounds. CTXs detection and quantification remains a difficult issue due to the wide range of congeners present in trace amounts in contaminated matrices. Presently, several detection methods are

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Figure 1.4 Benthic cyanobacterial blooms observed in Raivavae and Rurutu islands (French Polynesia), showing field aspects (left) and corresponding microscopic images of the organisms (right). (a) Marine benthic Anabaena sp. forming partially detached fibrous mats; (b) Mats of Aulosira schauinslandii Lemmermann 1905, a marine benthic heterocystous cyanobacterium first described from Hawaii; (c) Oscillatoria bonnemaisonii Crouan ex Gomont forms loose, bright red colonies which may fuse into contiguous covers; (d) Hydrocoleum cantharidosmum (Montagne) Gomont (genetically close to planktonic Trichodesmium), one of several toxic populations forming mats. Microscopic images Ó S. Golubic.

1.2 Marine Cyanobacteria as Causative Agent of Ciguatera-Like Poisoning

available with varying sensitivity and selectivity. These include the mouse bioassay (MBA), the receptor-binding assay (RBA), the cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), as well as various immunological assays (see Caillaud et al., 2010 for a review). The gene cluster encoding ciguatoxins is still unknown. However, the construction of these polyketides is achieved via the polyketide pathway and thus probably involves PKS with some additional functional segments (Lopez-Legentil et al., 2010). A hybrid NRPS and PKS gene was, for example, characterized from the toxic dinoflagellate Karenia brevis which produces brevetoxins – polyketides that show very strong structural homologies with ciguatoxins (Lopez-Legentil et al., 2010). According to the current EU legislation, fishery products containing biotoxins, such as ciguatoxins, are not to be introduced to the market (Paredes et al., 2011). Currently, the European Food Safety Authority (EFSA) could not characterize the risk associated with CTXs, because of the scarce data available (EFSA, 2010). However, the presence of CTX in fish from the Madeira Archipelago (Europe) was recently confirmed for the first time (Otero et al., 2010). In other parts of the world, there are only a few specific regulations for CTX, although some bans have been installed as public health measures, such as the prohibition of selling high-risk fish species coming from known toxic locations. These bans have been installed in American Samoa, Queensland, French Polynesia, Fiji, Hawaii, and Miami (Paredes et al., 2011).

1.2.2 Ciguatera Shellfish Poisoning (CSP): A New Ecotoxicological Phenomenon

Epidemiological studies conducted in New Caledonia (Lifou), French Polynesia (Raivavae) and the Republic of Vanuatu (Emao) have suggested a link between disturbances of the reef ecosystem, the development of oscillatoriacean cyanobacterial blooms, and an increase in ciguatera-like incidents following the consumption of giant clams from contaminated areas (Laurent et al., 2008; Kerbrat et al., 2010; Chinain et al., 2010; Laurent et al., 2012). As mentioned above, cyanobacteria and giant clams were implicated in 33 cases of human intoxications with two deaths as early as 1964 in Bora Bora Island, French Polynesia (Bagnis, 1967). Later, during the 1970s, an epidemiological survey of CFP conducted in the Gambier Islands (French Polynesia) established that 4% of all cases were in fact due to giant clam consumption (Bagnis, 1974). More recently, giant clams were implicated in several Ciguatera-like poisonings in the Cook Islands (Rongo and van Woesik, 2011). Cyanobacteria are known to respond with massive growth to increased nutrient flux accompanying eutrophication, pollution, and coastal construction. Many are able to fix nitrogen, which is a clear advantage in often nitrogen-limited oceans. Trichome fragmentation and the release of hormogonia is normal mode of reproduction in filamentous cyanobacteria. Thus, filter-feeding mollusks could easily become

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contaminated when cyanobacterial trichomes or hormogonia are released in the water column, especially during or following cyanobacterial blooms. Giant clams in conjunction with cyanobacterial growth and reproduction, thus provide a vector for the transfer of cyanotoxins to upper trophic levels, including humans. The term CSP, in relation to the term CFP, was proposed to designate this distinct ecotoxicological phenomenon (Laurent et al., 2012). Moreover, the symptomatology of CSP appears different from that of CFP, which features an elevated severity and rapid onset of certain symptoms, sometimes leading to paralysis. Further studies are required to chemically characterize the cyanobacterial compounds that may be implicated in ciguatera-like poisoning and to determine their mechanism of action. However, this could be explained by the fact that cyanobacteria as a group have the potential to produce a complex of toxic compounds leading to severe intoxications with multiple symptoms.

1.2.3 Ciguatera-Like Poisonings Involve Complex Mixtures of Cyanotoxins 1.2.3.1 Ciguatoxins and Homoanatoxin The severity of CSP intoxications following the consumption of giant clams in the tribe of Hun€et€e (Lifou, New Caledonia) could be explained by the co-occurrence in Hydrocoleum mats of CTX-like compounds (Laurent et al., 2008) and homoanatoxin-a (HANTX), a derivative of anatoxin-a (ANTX) (Figure 1.5). Indeed, HANTX was detected for the first time by applying gas chromatography–mass spectrometry (GC-MS) analyses to extracts of Hydrocoleum lyngbyaceum mats collected in the toxic area (Figure 1.4d), and also to extracts of giant clams collected in the vicinity during the cyanobacterial bloom (M
ejean et al., 2010). Both, ANTX and HANTX are bicyclic secondary amines usually produced by freshwater cyanobacteria belonging to the genera Microcystis, Oscillatoria, Planktothrix, Phormidium, Anabaena, Aphanizomenon, and Raphidiopsis. These alkaloids are potent neurotoxins that affect the muscular and neuronal nicotinic acetylcholine receptors (nAChRs) (Carmichael, Biggs, and Peterson, 1979; Spivak, Witkop, and Albuquerque, 1980). The alkaloids block the transmission of signal conduction at the level of the neuromuscular junction, causing staggering, muscle twitching, gasping, and rapid death by respiratory arrest in animals (Falconer, 2008). ANTX and HANTX produced by freshwater cyanobacteria Oscillatoria spp. and Phormidium favosum were involved in dog poisonings in France (Gugger

H2N

O R

Figure 1.5 Chemical structures of anatoxin-a (R ¼ CH3) and of homoanatoxin-a (R ¼ CH2–CH3).

8

1 Marine Cyanotoxins Potentially Harmful to Human Health

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et al., 2005; Cadel-Six et al., 2007), Scotland (Edwards et al., 1992), and New Zealand (Wood et al., 2007). Interestingly, cases of dog mortality similar to those conclusively associated with ANTX-producing freshwater cyanobacteria were reported in two Polynesian atolls (Fangatau and Pukarua) following the consumption of giant clams (M
ejean et al., 2010). Given the dangerous nature of these neurotoxins, the occurrence of ANTX or HANTX-producing-cyanobacteria in marine waters and their transmission along the food chain represent a serious potential health risk to human populations. ANTX and HANTX can be detected with GC-MS and ligandbinding assays (Araoz et al., 2005; Araoz et al., 2010). The 25 kblong biosynthetic gene cluster for ANTX and HANTX synthesis was described recently from benthic freshwater Oscillatoria sp. (Cadel-Six et al., 2009; M
ejean et al., 2009), while an homologous gene cluster of about 22 kb was recovered from a freshwater heterocystous Anabeana sp. (Rantala-Ylinen et al., 2011). The presence of these biosynthetic clusters could thus be investigated in other marine cyanobacteria to determine their potential to produce ANTX and HANTX. 1.2.3.2 Ciguatoxins and Saxitoxins Some cases of CSP were also reported in Raivavae (French Polynesia) (Laurent et al., 2012). As in other cases of intoxication following the consumption of giant clams, the symptoms, particularly neurological, were very severe. This could be explained by the co-occurrence of CTX-like compounds (Laurent et al., 2012) and Paralytic Shellfish Toxins (PSTs), known also as saxitoxin (STX) (Figure 1.6). Indeed, the presence of the sxtG gene fragment involved in the synthesis of saxitoxin was detected in two mats comprised of marine benthic filamentous cyanobacteria collected in the Rairua Bay (Raivavae, French Polynesia), an area known as toxic by the islanders, and suggested that these cyanobacteria are able to synthesize STX or its analogs (Villeneuve et al., 2012). Interestingly, STX was found previously in samples from the plankton bloom of the pelagic marine cyanobacterium Trichodesmium erythraeum collected off the Brazilian coast (ProenSc a, Tamanaha, and Fonseca, 2009). STX and its 57 analogs (Vale, 2010; Wiese et al., 2010) are alkaloids involved in the toxic syndrome known as Paralytic Shellfish Poisoning (PSP) (Etheridge, 2010). They selectively block the VSSC present in excitable cells such as muscle and nerve cells, and thereby inhibit the generation of correct action potentials in nerves and muscle fibers, leading to neuromuscular paralysis and death by respiratory arrest. Potentially produced by bloom-forming marine dinoflagellates such as Alexandrium species, Gymnodinium catenatum, and 21

H2N

O 17 H O H NH HN 1

+ H2N

N

+ NH2

NH OH OH

11

Figure 1.6 Chemical structure of saxitoxin.

Pyrodinium bahamense, as well as by freshwater or brackish cyanobacteria such as Lyngbya wollei, Rivularia sp., Aphanizomenon sp., Anabaena circinalis and Cylindrospermopsis raciborskii, PSTs can be transmitted and accumulated through tropical food webs (Llewellyn, Negri, and Robertson, 2006). Filter-feeding mollusks are the traditional vectors throughout aquatic food webs, but other potential vectors such as gastropods and crustaceans have also been involved in human cases of PSP (Deeds et al., 2008). Resistance to PSTs caused by the natural mutation of a single amino acid residue, which causes a 1000-fold decrease in affinity at the STX-binding site in the sodium channel pore of resistant, but not sensitive, clams (Mya arenaria), could be an important risk factor for human PSP (Bricelj et al., 2005). The mosaic structure of the gene cluster of 25 kb to 37 kb-long encoding saxitoxins was recently recovered from several freshwater cyanobacteria (Kellmann et al., 2008; Moustafa et al., 2009; Stucken et al., 2010; Mihali, Carmichael, and Neilan, 2011) and marine dinoflagellates (St€ uken et al., 2011; Hackett et al., 2012). St€ uken et al. (2011) demonstrated the presence of sxtA, the unique PKS gene involved in STX synthesis in the marine dinoflagellate Alexandrium sp., and showed very good agreement between the presence of sxtA and STX synthesis. Due to ethical issues, new analytical methods for the detection of PSTs have been developed as an alternative to the mouse bioassay, including a direct chemical analysis by liquid chromatography/fluorescence detection (LC/FD) (Oshima, 1995; Lawrence, Niedzwiadek, and Menard, 2005), immunoassay, in-vitro bioassay, and whole-animal invertebrate bioassay. Comparative laboratory calibration assays have indicated that the neuroblastoma assay shows a great potential for the detection of PSTs (Humpage et al., 2007). The established health guideline value for STXs in bivalve mussels is 0.8 mg kg 1 (expressed in STX toxicity equivalents) which is used as a regulatory reference value by Food Standards in Australia and New Zealand (FSANZ Food Standard 1.4.1) and the Victorian Shellfish Quality Assurance Program (EFSA, 2009b; Paredes et al., 2011). 1.2.3.3 Ciguatoxins and Palytoxins The ability to produce a complex of toxic compounds was also observed in blooms of pelagic cyanobacteria of the genus Trichodesmium collected in New Caledonian waters, in the toxic area of Lifou Island (Figure 1.7). Indeed, CTXs-like toxins combined with paralyzing toxins were detected (Kerbrat et al., 2010). The paralyzing toxins were further identified by LC-MS/ MS analyses as palytoxin (PLTX) (Figure 1.8) and one of its derivatives, 42-hydroxy-palytoxin (Kerbrat et al., 2011). PLTX and its analogs, such as ovatoxins, ostreocins, ostreotoxins and mascarenotoxins, constitute the family of palytoxins (Katikou, 2008), which were identified in marine organisms ranging from microorganisms to fishes (Vale and Ares, 2007; Wu, 2009; Ramos and Vasconcelos, 2010; Aligizaki et al., 2011; Amzil et al., 2012). PLTX is one of the most potent nonprotein compounds known to date, and exhibits a high toxicity in mammals (Katikou, 2008; Vale and Ares, 2007; Wu, 2009). One of the main actions of PLTX is to bind to the Naþ/Kþ-ATPase, converting the pump into an ion channel and causing Kþ efflux and Naþ influx, leading to

1.2 Marine Cyanobacteria as Causative Agent of Ciguatera-Like Poisoning

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Figure 1.7 (a) Lifou Island (Loyalty Islands, New Caledonia) in South Pacific Ocean (map Ó IRD); (b) Bloom of Trichodesmium erythraeum in Lifou Island (image Ó A.S. Kerbrat); (c) Microscopic view of Trichodesmium erythraeum colonies with trichomes typically oriented in parallel (image Ó A. Trottet); (d) Microscopic view of a trichome of Trichodesmium erythraeum Ehrenberg (scale bar 50 mm) (image Ó S. Golubic).

membrane depolarization (Vale and Ares, 2007). The osmotic imbalance that results from this flux of ions can be compared to CTX mechanisms, and could explain why PLTX has often been implicated in CFP (Kodama et al., 1989; Deeds and Schwartz, 2010). PLTX is also likely to play a role in clupeotoxism, a marine poisoning resulting from the ingestion of plankton-eating fish such as herrings and sardines (Clupeidae), anchovies (Engaulidae) or mullet (Mugillidae) in tropical regions (Randall, 2005; Deeds and Schwartz, 2010). This was postulated following the detection of PLTX and analogs in the remains of fish responsible for serious human intoxications (Kodama et al., 1989; Onuma et al., 1999; Taniyama et al., 2003). The symptoms appear abruptly, and include a metallic taste, digestive disorders, generalized paralysis, tachycardia, convulsions, and acute respiratory distress. Occasionally, this poisoning can be fatal (Deeds and Schwartz, 2010). Clupeotoxism is classically associated with blooms of the benthic dinoflagellate Ostreopsis, most

notably with the species O. siamensis and O. mascarenensis (Rhodes et al., 2002; Lenoir et al., 2004; Ramos and Vasconcelos, 2010). However, according to the latest findings, clupeotoxism could also be associated with blooms of pelagic cyanobacteria of the genus Trichodesmium (Kerbrat et al., 2011). The detection and quantification of PLTXs in biological samples can be conducted by biological and chemical analyses (LCMS/MS), though there is currently no officially approved method (Riob
o and Franco, 2011; Ciminiello et al., 2011; Amzil et al., 2012). The gene cluster encoding palytoxins is not yet known. Although PLTXs have become a global concern due to their toxic effects on animals and humans, there are currently no regulations, either in the European Union or in other parts of the world, concerning PLTXs in shellfish (Paredes et al., 2011). Recently, the EFSA established a low threshold value of 30 mg PLTX per kg shellfish flesh for the protection of public health (EFSA, 2009a).

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1 Marine Cyanotoxins Potentially Harmful to Human Health

OH

O OH

O

O

NH2

OH O HO

OH

OH

HO

OH OH

OH OH

Me

OH

OH OH OH

OH O HO

N H

O N H

Me

OH Me

HO OH

OH O

OH Me

Me

OH

O

OH OH

OH OH

HO

HO O

OH

O O

Me

HO

OH 42 HO HO

O

OH

OH

Me

OH OH

OH OH

OH

OH Figure 1.8 Chemical structure of palytoxin (from Riob
o and Franco, 2011).

1.3 Marine Cyanobacteria: A Potential Risk for Swimmers

All previous cases of intoxication presented in this chapter were due to the consumption of seafood contaminated with cyanotoxins; however, marine cyanobacteria can also be toxic for humans through external exposure. During the late 1950s, the marine benthic filamentous cyanobacterium Lyngbya majuscula was implicated as the cause of an epidemic of a previously unreported acute contact dermatitis. During July and August of 1958, 125 people were reported as suffering from dermatitis on the windward beaches on northeast Oahu, Hawaii (Grauer, 1959; Chu, 1959; Arnold, 1959), and this was attributed to exposure to L. majuscula. In 1968, 242 of 274 (88.3%) people bathing at Gushikawa Beach, Okinawa, developed acute dermatitis, with symptoms that included itching, rash, burning, blisters and deep desquamation, causing pain. L. majuscula was known to occur at this beach, and samples collected later were found to cause rashes and blistering on human skin (Hashimoto et al., 1976). The toxic compound extracted had almost identical properties to an uncharacterized toxin found by Moikeha et al. (1974), and was later shown to be a mixture of aplysiatoxin and debromoaplysiatoxin (Mynderse et al., 1977; Fujiki et al., 1981; Osborne, Webb, and Shaw, 2001) (Figure 1.9). These compounds were also isolated from a red alga Gracilaria coronopifolia involved in a human poisoning in Hawaii due to ingestion of the algae (Nagai, Yasumoto, and Hokama, 1996). More recently, in 2011, 86 persons frequenting one of the most popular beaches of Mayotte (Comoros, Southwestern of Indian Ocean) presented with respiratory and eye

irritations, while swimmers reported cutaneous irritations. According to the local sanitary report, these cases were caused by a large bloom of the benthic cyanobacterium L. majuscula, which covered the seafloor, while massive brownish green patches were deposited on the beach (Figure 1.10) (Lernout et al., 2012). Lyngbya majuscula presents a particularly remarkable complex toxinogenic potential. Widespread in tropical regions, it has been found to contain over 70 biologically active compounds, many of which have been shown to be highly toxic (Osborne, Webb, and Shaw, 2001). The proliferation of L. majuscula on Australian coasts is considered as a health hazard (Albert et al., 2005; Osborne, 2012), and consequently in some regions the beaches are closed to swimming to assure public safety (Department of Health, Northern Territory Government, Australia, 2011). Lyngbyatoxin A (Figure 1.11) is an ichthyotoxic cyclic depsipeptide that has been linked to at least one severe human poisoning (Cardellina et al., 1979). This compound is a potent phorbol-ester-type tumor promoter (Fujiki et al., 1985) and a highly inflammatory metabolite; it has also been suspected of

HO O O

O

O

OMe Br

O O

OH

OH

Figure 1.9 Chemical structure of aplysiatoxin.

1.3 Marine Cyanobacteria: A Potential Risk for Swimmers

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Figure 1.10 (a) Mayotte Island in SW Indian Ocean (map Ó ARVAM); (b) Beach covered by stranded mats of Lyngbya majuscula (Dillwyn) Harvey in April 2010 (image Ó K. Ballorain, see Lernout et al., 2012); (c) Hairy biofilms of L. majuscula rising from a seagrass bed of N’Gouja (Picture: Ó K. Ballorain, see Lernout et al., 2012); (d) Microphotograph of Lyngbya majuscula from N’Gouja beach (image Ó S. Golubic).

contributing to chelonitoxism, a human poisoning caused by the ingestion of meat from marine turtles. In fact, lyngbyatoxin A was identified by LC-MS in the meat of a turtle Chelonia mydas implicated in fatal intoxications (Yasumoto, 1998). Furthermore, the pathological effects of lyngbyatoxin A observed in mice are compatible with epidemiological data reported for marine turtle poisoning (Ito, Satake, and Yasumoto, 2002). Lyngbyatoxins B and C are irritant analogs isolated from L.

majuscula (Aimi et al., 1990). Additionally, lyngbyatoxins were shown to be accumulated by grazers feeding on L. majuscula (Capper et al., 2005). Other neurotoxic compounds that are strongly ichthyotoxic, such as antillatoxins (Orjala et al., 1995) (Figure 1.12), kalkitoxin (Wu et al., 2000) (Figure 1.13), and jamaïcamides (Edwards et al., 2004) (Figure 1.14), were also isolated from L. majuscula. Antillatoxin A is one of the most ichthyotoxic compounds to

CH3 H3C

CH3

H3C

H3C

O

H3C

HN CH2

N

NH

HO

Figure 1.11 Chemical structure of lyngbyatoxin A.

R

O OH

CH3 CH3 CH3 CH3 CH3

NH H3C N H H O NH O H CH3 O CH2 CH3

Figure 1.12 Chemical structures of antillatoxin A [R ¼ (CH3)2–CH2] and antillatoxin B [R ¼ Ph–CH2–CH2].

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1 Marine Cyanotoxins Potentially Harmful to Human Health

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H2C

S

CH3

CH3 CH3 N CH3

N CH3 CH3

O

O

D-Glu OH

O N

HN

NH Adda O

H3C

O

Cl

O

H

N H

N

R

L-Arg

O

HN

O

H3C H3C

O

H

N H

N

O

Cl

O

CH3

O Figure 1.14 Chemical structure of jamaïcamides A (R ¼ Br), B (R ¼ H), and C.

be isolated from marine cyanobacteria. Antillatoxin A and an N-methyl homophenylalanine analog, antillatoxin B, are, as CTXs, highly potent activators of VSSC (Li et al., 2001; Nogle, Okino, and Gerwick, 2001), whereas kalkitoxin is a potent inhibitor of VSSC (Lepage et al., 2005). Antillatoxin and kalkitoxin each induced neuronal cytotoxicity with a distinct temporal pattern; antillatoxin neurotoxicity was induced acutely, whereas kalkitoxin caused a delayed neurotoxic response (Berman, Gerwick, and Murray, 1999). Finally, jamaïcamides A, B and C are potent VSSC blockers, and exhibit cytotoxicity to human lung and on Neuro-2a neuroblastoma cells (Edwards et al., 2004). The genetic bases for producing those bioactive compounds from L. majuscula are largely known. A 11.3 kb-long gene cluster with a two-module NRPS was found to be required for the synthesis of lyngbyatoxins (Edwards and Gerwick, 2004). Similarly, a 58 kb-long gene cluster of NRPS and PKS is responsible for the synthesis of jamaïcamides in L. majuscula (Edwards et al., 2004). However, the genetic basis encoding the aplysiatoxin, antillatoxins and kalkitoxin remain to be identified.

1.4 Microcystins Could also be Found in the Sea

A newly recognized problem for the marine environment is the potential inflow of freshwater cyanotoxins to the ocean, eventually resulting in the deaths of marine species. Indeed, a recent study revealed deaths of sea otters from microcystin (MC) intoxication, which provided evidence implicating land-sea flow, most likely through trophic transfer via marine

D-ala O

HN H N

H N O

CH3

NH

O

O

O

Figure 1.13 Chemical structure of kalkitoxin.

H3C

Me-Dha

O

L-Leu

O OH D-meAsp

NH NH2

Figure 1.15 Chemical structure of microcystin-LR. D-Ala ¼ D-alanine; L-Leu ¼ L-Leucine; MeAsp ¼ D-erythro-b-methylaspartic acid; L-Arg ¼ L-arginine; Adda ¼ 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6dienic acid; D-Glu ¼ D-glutamate; Me-Dha ¼ N-methyldehydroalanine.

invertebrates (Miller et al., 2012). MCs from freshwater cyanobacteria are known to be transferred along the food chain (Ibelings et al., 2005; Smith and Haney, 2006), suggesting that animals and humans are at risk from microcystin poisoning when consuming aquatic products, including those harvested at the land–sea interface (Miller et al., 2012). The MCs belong to a family of cyclic hepatopeptides, which includes about 70 isoforms that vary by the degrees of methylation, hydroxylation and epimerization, and in peptide sequence (Figure 1.15). The MCs cause acute liver injury and are active tumor promoters via the inhibition of protein phosphatases 1 and 2A (Ohta et al., 1994). They may disrupt the actin filaments and intermediate filaments of the cytoskeleton, primarily in the hepatocytes and sinusoidal capillaries of the liver, leading to shrinkage and loss of cell-adhesion, and eventually lethal intrahepatic hemorrhage (Carmichael and Falconer, 1993; Carmichael, 1994). Exposure to environmentally stable MCs in food, drinking water, nutritional supplements and through medical dialysis can cause significant, and sometimes fatal, hepatotoxicity and possible tumor induction in humans and animals (Jochimsen et al., 1998; Humpage and Falconer, 1999; Gilroy et al., 2000; Carmichael et al., 2001; Azevedo et al., 2002; Lankoff et al., 2004). MCs have been isolated from different genera of freshwater cyanobacteria, such as Microcystis, Oscillatoria, Planktothrix, Anabaena, Nostoc, and Hapalosiphon, through it is also possible that marine cyanobacteria produce such toxins. Indeed, the identification of MCs by a Synechococcus from the slightly hypersaline Salton Sea, an inland body of water in California, indicates that these toxins may have a more common occurrence in water of elevated salinity (Carmichael and Li, 2006), including marine environments. Furthermore, the production of MCs by marine cyanobacteria belonging to the genera Pseudoanabaena, Phormidium and Spirulina isolated from Black Band Disease (BBD) and from sediment and water columns, was demonstrated according to HPLC, ELISA and protein phosphatase 2A (PP2A) data (Richardson et al., 2007; Gantar,

1.6 Conclusion and Future Prospects

Sekar, and Richardson, 2009). A positive result for the presence of NRPS and PKS gene clusters encoding MCs, previously related and found only in fresh and brackish water cyanobacteria (Nishizawa et al., 2000; Tillett et al., 2000; Dittmann et al., 2012), was also reported in one marine Leptolyngbya strain and one marine Oscillatoria strain from rocky beaches along the central Atlantic coast of Portugal (Frazao, Martins, and Vasconcelos, 2010), suggesting that these marine cyanobacteria could also produce MCs. Thereafter, by using UPLC-MS/MS, MCs were detected in cultured laboratory strains of BBD Oscillatoria and Geitlerinema from the Caribbean reefs (Stanic et al., 2011). MCs were also found in samples of Trichodesmium erythraeum from the Canary Islands Archipelago (Ramos et al., 2005) and the Brazilian coast (ProenSc a, Tamanaha, and Fonseca, 2009). More recently, the presence of MCs in water and their accumulation in mussels in the Amvrakikos Gulf (Greece) was associated with the presence of Synechococcus-Synechocystis cyanobacteria (Vareli et al., 2012). Produced by the similar biosynthesis pathway and structurally close to MCs, nodularin is a cyclic pentapeptide first isolated from the planktonic cyanobacterium Nodularia spumigena, which occurs in brackish waterbodies (Carmichael et al., 1988). Seven isoforms were isolated from Nodularia sp., N. harveyana, and from a Papua New Guinean sponge, Theonella swinhoei (Pearson et al., 2010). Like MCs, nodularin inhibits protein phosphatases 1 and 2A and can act as a liver tumor initiator and promoter (Ohta et al., 1994). Nodularin was involved in the death of animals by severe liver hemorrhage (Nehring, 1993). Moreover, it could be accumulated in shellfish and other seafood, which could become hepatotoxic and hazardous to human health (Mazur-Marzec et al., 2007; Falconer, Choice, and Hosja, 1992). The NRPS and PKS gene clusters encoding MCs and nodularins are related and found in fresh and brackish water cyanobacteria (Nishizawa et al., 2000; Tillett et al., 2000; Dittmann et al., 2012). The MCs have been the most thoroughly investigated cyanobacterial toxin group, and still represent the major toxin group under investigation. They rapidly became a global health concern and, at the present time, they are the only cyanobacterial toxin family internationally assessed as a health risk by the World Health Organization (WHO). This regulation actually concerns mainly freshwater habitats, but should be extended to marine environment in accordance with the latest emerging data that suggests a possible transfer of MCs along the marine food chain (Miller et al., 2012) and their potential production by marine cyanobacteria (Gantar, Sekar, and Richardson, 2009; Frazao, Martins, and Vasconcelos, 2010).

1.5 Risk of Neurodegenerative Disease in the Sea

A chapter describing marine cyanotoxins that are potentially hazardous to human health would not be complete without mention of b-N-methylamino-L-alanine (BMAA) (Figure 1.16), a

H2N

j 13

H O OH

HN CH3 Figure 1.16 Chemical structure of BMAA.

neurotoxic nonprotein amino acid that selectively injures motor neurons (Rao et al., 2006). BMAA is suspected of being involved in Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) and Alzheimer’s disease, although further investigations are necessary to confirm these suspicions. BMAA has been found in high concentrations in aquatic animals and in seafood in many areas of the world where cyanobacterial blooms occur (Ibelings and Chorus, 2007). In 2005, Cox et al. detected BMAA in diverse laboratory cultures from freshwater to marine cyanobacteria. Furthermore, BMAA may bioaccumulate in the marine food web, up to the level of sharks (Brand et al., 2010; Mondo et al., 2012). A regain of interest in BMAA was seen when cyanobacteria were found to produce this neurotoxic compound which was only found in Cycas circinalis palm seeds. The finding of BMAA in flying foxes (Pteropus mariannus mariannus, Pteropodidae) led Cox et al. (2003) to propose endophytic cyanobacteria as a primary source of BMAA biomagnification. BMAA was detected in endophytic cyanobacteria (Nostoc sp.) isolated from roots of the cycads (Banack and Cox, 2003). Flying foxes, that are salient components of the traditional Chamorro diet in Guam, Mariana Islands, accumulate BMAA by foraging on the seeds of Cycas micronesica (Cycadaceae). The Chamorro people are known to have high levels of ALS/PDC, and the link between BMAA and this neurodegenerative disease has been postulated (Cox, Banack, and Murch, 2003). Thereafter, BMAA was found in the brain tissues of Chamorros who died of ALS/PDC, but not in patients that died of unrelated causes (Murch, Cox, and Banack, 2004). The detection and quantification of BMAA in cyanobacteria could be subject to misidentifications using LC/MS/MS that detect derivatized and underivatized BMAA (Li et al., 2010). The genetic basis for the synthesis of BMAA is unknown to date; consequently, Bienfang et al. (2011) recommended that nuclear magnetic resonance (NMR) analysis could be used for the unequivocal identification of BMAA in biological samples.

1.6 Conclusion and Future Prospects

Many investigations remain to be conducted on marine cyanobacteria, their development, environmental responses, and toxinogenic potential. Recent findings have supported the hypothesis that benthic and planktonic blooms of marine cyanobacteria are able to produce various toxins and are one of the causes of human poisoning. However, the production of toxins by marine cyanobacteria needs to be unequivocally confirmed

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on pure cultures, because field-collected samples of marine cyanobacteria are often composed of a mixture of species, including prokaryotic or eukaryotic (diatoms, dinoflagellates) microorganisms, which might also contribute to the toxinogenicity of the sample. At present, only a small percentage of cyanobacteria have been successfully cultured, due largely to the difficulty of isolation in axenic culture. Based on experiences to date, it is important to note that seafood poisonings are not always due to a single toxin, or even a particular group of toxins, and that these toxins do not necessarily originate from a single species. Conversely, a single species of a given microorganism (dinoflagellate, diatom or cyanobacterium) is able to produce different toxic metabolites. Consequently, complex mixtures of toxins may originate from mixed blooms, as well as from blooms that are dominated by a particular toxic population. The heterogeneity of composition, distribution and destiny of toxins in marine environments are further complicated by specifically different feeding preferences of invertebrate and vertebrate grazers and filter-feeders, which consume these toxins and may concentrate them further along the food web. These observations highlight the fact that symptoms experienced during seafood poisoning incidents may well be the result of a synergistic action of a toxic mixture that is further modified by individual human responses to poisoning. Further analysis is needed to determine the routes of passage and concentration levels of toxins along various food chains and webs leading to human consumption. A thorough knowledge of the genetic architecture of biosynthetic gene clusters of cyanobacterial toxins could be a great help when assessing the health hazard posed by cyanobacteria. The acute effects of different toxins presented in this chapter have been relatively well studied; in contrast, potential chronic effects are still insufficiently documented. For example, some reports of CFP intoxications have suggested that neurologic and diffuse symptoms experienced by patients following the gastrointestinal troubles may persist for weeks, months, and even years after their onset (Ting, Brown, and Pearn, 1998; Pearn, 2001; Chateau-Degat et al., 2007). Although many outbreaks of ciguatera have been reported, epidemiologic studies on the severity and duration of ciguatera disease remain scarce (Lange, Snyder, and Fudala, 1992; Glaziou and Martin, 1993; Katz, Terrell-Perica, and Sasaki, 1993; Chateau-Degat et al., 2007). While recommendations and regulation policies regarding the management and risk of freshwater toxic cyanobacteria are already implemented in different countries (Australia, USA, Canada, France, Finland, the Netherlands, Germany, etc.), the corresponding problems associated with marine toxic cyanobacteria and their proliferation in marine environment are yet to be acknowledged by public authorities. In the absence of an accurate assessment of the extent of transfer of these cyanotoxins in the trophic chain and, hence, the level of toxic risk to humans, all fishery products should be regarded as a potential exposure source. In French Polynesia, for example, this risk involves not only all lagoonal fish but also several marine invertebrates such as giant clams and sea urchins, which are part of the diet of local populations.

This finding is particularly worrisome in countries and territories of the Pacific islands whose populations rely heavily on fish and other marine products as essential life-supporting resources. As a consequence of the high risk of ciguatera poisoning to which these communities are exposed on a daily basis, a thorough knowledge of nature and of fish species has allowed these populations to develop numerous traditional tests to recognize and monitor the suspected fish for toxicity (Banner et al., 1963). These “bioassays” include the repulsion of ants and flies, observing the discoloration of silver coins or copper wire, or looking for some physical characteristic of the flesh, such as rigor mortis or signs of hemorrhage (Lewis, 1983; Darius et al., 2013) (Figure 1.17). The most widespread test, however, remains the feeding of fish to cats and dogs (Lewis, 1983; Park, 1994). Although often mentioned in the literature, very few of these native tests have been examined scientifically for their effectiveness (Darius et al., 2013), and of those examined almost all have been rejected as invalid (Park, 1994). Complementary to these traditional detection tests, popular beliefs have also maintained the knowledge about several traditional remedies used to treat ciguatera (Kumar-Roin
e et al., 2011). Among the herbal remedy candidates evaluated both in in vivo and in vitro tests, the most popular one by far is a decoction prepared from the leaves of Heliotropium foertherianum (Boraginaceae) (Figure 1.18), for which an industrial development project is presently underway in French Polynesia (Kumar-Roin
e et al., 2010; Rossi et al., 2012). Apart from these traditional practices, several actions could be undertaken by the scientific community and health authorities that are suitable for the health monitoring and risk assessment associated with the contamination of seafood by cyanotoxins. A qualitative screening for the different cyanotoxins should be conducted in sites of economic interest (fishery and tourism). Given the great diversity of toxic metabolites potentially involved, one possible strategy would be the use of passive monitoring systems in high-risk sites. In particular, the use of passive Solid-Phase Adsorption Toxin Tracking (SPATT) devices, when coupled with appropriate analytical methods, can provide a highly sensitive and rapid monitoring of cyanobacterial compounds in waters. Several studies aimed at evaluating the use of various adsorbed materials have shown promising results, especially with regards to the monitoring of microcystins and anatoxin-a (MacKenzie, 2010). The other parameters to be surveyed in routine risk assessment programs include the visual surveillance of fishing sites for cyanobacterial proliferation. Although not all cyanobacterial blooms are toxic and constitute a danger to human health, in the absence of thorough toxicological data, the consumption of fish and invertebrates (clams, trochus, sea urchins, etc.) collected in areas with excessive growth of cyanobacteria (as planktonic or benthic blooms) should be considered a potential hazard. Therefore, it is strongly recommended that health-monitoring programs operating in the tropics also include the survey of cyanobacterial blooms, along with ciguatera-causing dinoflagellates. Such surveys should combine qualitative analyses to

1.6 Conclusion and Future Prospects

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Figure 1.17 Two traditional tests used by Polynesian populations to monitor toxicity in suspect ciguateric fish. (a) Rigor mortis test: if the fish looks flabby, it is regarded as toxic by the locals; (b) Bleeding test: if hemorrhagic signs are visible at the site of the tail incision, the fish is considered toxic (according to Darius et al., 2013) (images Ó Institut Louis Malard
e). Reused from Darius et al. 2013, with kind permission by Francis & Taylor.

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Figure 1.18 Heliotropium foertherianum (Boraginaceae), one of the traditional plant largely used by Polynesian populations to treat ciguatera (image Ó F. Rossi).

identify toxinogenic genera and species, and quantitative biomass assessments including studies of spatial and temporal heterogeneity of the blooms, as well as of the correlating environmental parameters (Sabart et al., 2010; Agha et al., 2012). The detection and quantification of toxins in the environment requires ad hoc extraction and purification protocols to ensure the complete extraction of all toxins, while minimizing

the coextraction of interference factors, such as cellular proteins or lipids that might affect toxicity tests with their potential matrix effects (Caillaud et al., 2010; Pawlowiez et al., 2013). The detection of toxins by LC-MS/MS analyses requires toxin standards, which are essential for the calibration of these methods. In conclusion, there is evidence that the frequency of harmful algal or cyanobacterial bloom events is increasing globally. The increase of coastal eutrophication, climate change and ocean acidification are stress factors that affect biological diversity in the oceans and likely contribute to the development of harmful cyanobacterial blooms, which are also hazardous to human health (Moore et al., 2008). Water management agencies, which have to date focused primarily on the reduction of nutrients (O’Neil et al., 2012), will need to incorporate the monitoring of qualitative and quantitative biological changes affecting aquatic ecosystems, which includes the formation of toxic microbial blooms and their effect on different levels of consumers, including humans.

Acknowledgments

Some of the results presented in this chapter were supported by the Agence Nationale de la Recherche, (ARISTOCYA program, ANR CES-2008), grants from the Country of French Polynesia and by the Agence de Sant
e de l’Oc
ean Indien and the Direction de l’environnement, de l’am
enagement et du logement of Mayotte island.

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dinoflagellate as a likely culprit of ciguatera. Bull. Jpn. Soc. Sci. Fish., 43 (8), 1021–1026. Yasumoto, T. (1998) Fish poisoning due to toxins of microalgal origins in the Pacific. Toxicon, 36 (11), 1515–1518. Yasumoto, T., Igarashi, T., Legrand, A.M., Cruchet, P., Chinain, M., Fujita, T., and Naoki, H. (2000) Structural elucidation of ciguatoxin congeners by fast-atom bombardment tandem mass spectroscopy. J. Am. Chem. Soc., 122, 4988–4989.

About the Authors M
elanie Rou
e, PhD, is a research scientist at the Institut de Recherche pour le D
eveloppement (Tahiti, French Polynesia) where she works on the isolation and characterization of marine biotoxins implicated in human intoxications. She received her PhD in microbial and chemical ecology from the Pierre et Marie Curie University (Paris, France). Her multidisciplinary doctoral research at the Museum National d’Histoire Naturelle (Paris, France) focused on the contribution of associated bacteria to the secondary metabolism of marine sponges. Muriel Gugger is the Head of the Laboratory Collection des Cyanobact
eries and the Curator of the PCC at Pasteur Institute since 2009. She obtained her PhD from the University Paris VII based on her studies in Prof. K. Sivonen’s group (Finland) on toxic cyanobacteria. During two postdoctoral positions (Luxembourg and France), she investigated the diversity of cyanobacteria, in particular the bloom-forming species and their cyanotoxins, notably on neurotoxin dog poisonings. Her main interests are the evolution of the cyanobacterial phylum, and the diversity of the genetic pathways involved in the synthesis of cyanotoxins and secondary metabolites in cyanobacteria. Stjepko Golubic is professor at the Boston University. His research concerns the relationship between microorganisms and mineral deposits. It is by nature ecological and interdisciplinary, involving aspects of paleontology, sedimentology, and geochemistry. His interests include the role of phototrophic microorganisms in carbonate deposition and dissolution, ecology of modern stromatolites, biokarst formation, and use of microborings as paleoenvironmental indicators. His special interests focus on the diversity of cyanobacteria in the ecological context, reconciling molecular and phenotypic characterizations. Research projects include: (a) biosedimentation of microbialite-forming and nitrogen-fixing marine benthic cyanobacteria in tropical lagoons and coral reefs; (b) bioerosion and biokarst formation by microbial euendoliths in conjunction with grazing in shallow marine environments; (c) the study of microbial fossils including microborings as trace fossils; (d) the role of phototrophic and organotrophic euendoliths in coral skeletons, as potential opportunistic diseases when reef corals are under stress; and (e) the formation of

carbonate tufa deposits under microbial influence in past and present freshwater systems. Zouher Amzil, PhD, is head of the Phycotoxin-laboratory at the IFREMER (French public institute for marine research). He spent most of his career working on natural toxins produced by phytoplanktonic species (micro-algae), their accumulation in seafood, and resulting human poisoning. He is involved in the development of analytical methods using mass spectrometry for the characterization of toxins and of their metabolites, in particular emerging toxins. Dr Amzil provides scientific and technical support for IFREMER’s national phytoplankton and phycotoxins monitoring network and other French national authorities. Romulo Ar
aoz obtained his PhD in Natural Sciences from Friedrich-Alexander Universit€at Erlangen-N€ urnberg (Germany) for his work on the mechanisms of tolerance to solar ultraviolet radiation in high-land freshwater cyanobacteria. During his postdoctoral training he worked on the mechanisms of resistance to vancomycin, on neurotoxic freshwater cyanobacteria, and on the postgenomic exploitation of Mycobacterium leprae. He is a Research Scientist at the Laboratory of Neurobiology and Development at the CNRS of Gif sur Yvette. He develops his research on the pharmacology of nicotinic acetylcholine receptors, the discovery and characterization of cholinergic neurotoxins, and bioassay development. Jean Turquet, PhD, is a research scientist at the laboratory of ARVAM (Agence pour la Recherche et la Valorization Marines, Reunion island, France). He obtained his PhD in 1997, from the University Paris VII, based on toxic tropical microalgae, involved in ciguatera and other seafood poisoning observed in the Indian Ocean. To date, he has been involved in management plans and research studies on seafood poisoning and associated tropical harmful algal blooms. He is head of the analytical unit at ARVAM. Mireille Chinain, PhD, is a research scientist at the Louis Malard
e Institute (Tahiti, French Polynesia), where she serves as head of the ciguatera research program. Current research in her laboratory focuses on the ecology, taxonomy and

22

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1 Marine Cyanotoxins Potentially Harmful to Human Health

systematics of the ciguatera-causing dinoflagellate Gambierdiscus. She also currently manages algal and toxin-based fieldmonitoring programs throughout French Polynesia for ciguatera risk assessment and management purposes. Dr Chinain is the recipient of the Tyge Christensen 2010 Award (International Phycological Society), the Albert S
ezary 2006 Award (Acad
emie Nationale de M
edecine, Paris, France) and the Tregouboff 2005 Award. Dominique Laurent, PhD, is an investigator at UMR152 Pharma-Dev, a joint research/academia unit involving IRD

(Institut de Recherche pour le D
eveloppement) and hosted by Paul Sabatier University (Toulouse, France). He spent most of his career in the South Pacific, working on natural products from terrestrial or marine fungi, marine invertebrates and marine microorganisms ranging from dinoflagellates to cyanobacteria. Now based in French Polynesia, Dr Laurent is currently investigating marine biotoxins and their neurotoxicity to humans. He is the coordinator of the Aristocya project (Toxic risk assessment linked to tropical benthic marine cyanobacteria blooms) which includes collaborators from seven major French research institute.

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX Philippe Amade, Mohamed Mehiri, and Richard J. Lewis

Abstract

Countless marine species, including invertebrates, are able to produce, accumulate and use a variety of toxins for predation and defense. Many of those marine toxins arise from toxic microscopic algae that are accumulated through the marine food-chain and can contaminate seafood to cause food poisoning, including various neurological and gastrointestinal illnesses in humans. The most important toxic syndromes associated with marine toxin contamination are paralytic

2.1 Introduction

Oceans provide a habitat for countless marine species, including invertebrates which are conspicuous inhabitants, often soft-bodied, relatively immobile, and lacking in obvious physical defenses. These marine animals frequently have evolved chemical defenses against predators and overgrowth by fouling organisms; typically, they are able to produce, accumulate and use a variety of toxins (zootoxins) for predation and defense purposes, including compounds that have been sequestered from either prey organisms or symbiotic microorganisms. These toxic animals are particularly abundant in the oceans compared to their terrestrial counterparts, and the diversity of toxins that they employ ranges from small molecules to highmolecular-weight proteins. Many such animals display unique chemical and biological features that make them useful research tools or molecular templates for the design of new drugs and pesticides (Fusetani and Kem, 2009). Marine toxins that contaminate seafood typically do not alter the look, smell or taste of the food, despite their potential to cause food poisoning. Many of the accumulated toxins arise from toxic microscopic algae that are accumulated through the marine food chain to cause various neurological and gastrointestinal illnesses in humans. Through well-implemented management practices, these poisoning events are relatively rare, especially in relation to the quantity of seafood consumed, although mild poisoning events are likely to go unreported.

shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), and the well-known pufferfish poisoning (PFP), with each related to marine toxins with specific chemical structures and biological properties. In this chapter, attention is focused on PSP, PFP, and CFP that are produced by the saxitoxins, tetrodotoxins and ciguatoxins, respectively.

The one exception is ciguatera, which is associated with the consumption of reef fish. The toxins involved are typically not affected by cooking, and there are no specific treatments other than managing its complications, of which dehydration – caused by diarrhea and vomiting – is the most common. About 40 of the 5000 species of marine phytoplankton identified to date, which primarily are dinoflagellates and diatoms, produce potent toxins (Park et al., 1999). These toxins accumulate in the shellfish tissues and are harmful to humans when contaminated bivalves are eaten. Other marine animals, including gastropods, crustaceans and fish, can also accumulate the toxins through the food chain and pose a threat to seafood consumers. Once contaminated, some shellfish depurate the toxins relatively quickly, whereas others (e.g., scallops) can retain the toxins for months and even years, particularly in the digestive glands and gonads. The occurrence of these phytoplankton and shellfish toxins is widespread in both temperate and tropical waters (Leftley and Hannah, 1998). On some occasions, the proliferation of certain species of planktonic algae produces “blooms” which discolor the water red, brown, or green, giving rise to the confusing term “red tide.” Blooms do not have to occur for shellfish to become toxic, however, and phytoplankton that are normally associated with toxins do not always produce toxins. Indeed, the conditions which trigger the occurrence of toxic phytoplankton species or the production of toxins are not well understood and cannot be predicted accurately. Different species producing different toxins may be present at the same time (Whittle and Gallacher, 2000).

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

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Table 2.1 Marine biotoxins and corresponding toxic syndromes.

Marine biotoxin Alkaloids

Polyethers

Toxic syndrome

Saxitoxin

STX

Gonyautoxins Tetrodotoxins

GTX TTXs

Brevetoxin

PbTx

Okadaic acid

OA

Dinophysistoxins Domoic acid

DTXs DA

Ciguatoxins Maitotoxin

CTXs MTX

Paralytic shellfish poisoning

PSP

Puffer fish poisoning Neurotoxic shellfish poisoning Diarrheic shellfish poisoning Amnesic shellfish poisoning Ciguatera poisoning

PSP PFP NSP DSP DSP ASP CFP CFP

The most important toxic syndromes associated with marine toxin contamination are paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), and the well-known pufferfish poisoning (PFP), each of which is related to marine toxins with specific chemical structures and biological properties. In this chapter, attention is focused on PSP, PFP and CFP that result from contact with the saxitoxins (STXs), tetrodotoxins (TTXs) and ciguatoxins (CTXs), respectively (see Table 2.1 and Figure 2.1).

2.2 Saxitoxins (STXs) in Paralytic Shellfish Poisoning 2.2.1 Causes of Paralytic Shellfish Poisoning

Paralytic shellfish poisoning (PSP) is caused by a red-brown dinoflagellate which, in large numbers, can cause a red streak in the ocean, referred to as “red tide.” PSP is usually associated with the consumption of filter-feeding shellfish such as mussels, cockles, clams, scallops, oysters, and also some crustaceans such as crabs and lobsters. Under red tide conditions, the dinoflagellate population increases to “bloom” levels, when optimal

R1HN

O

+ H2 N

7

N 10

R2 STX

NH2+

N H OH OH R3

12

2.2.2 Saxitoxins (STXs)

The STXs are the most potent agents of PSP, and are produced by a range of dinoflagellates, including species of the genera Gonyaulax, Alexandrium, Gymnodinium, and Pyrodinium. STXs are also produced by certain freshwater and brackish prokaryotic cyanobacteria belonging to the genera Anabaena, Cylindrospermopsis, Aphanizomenon, Planktothrix and Lyngbya, as well as calcareous red macroalgae and, more controversially, by marine bacteria (Leftley, 1998). The toxins are passed through the marine food web via vector organisms, which accumulate the toxins by feeding on paralytic shellfish toxins (PSTs), producing dinoflagellates without apparent harm to themselves (Gainey et al., 1988; Shumway, 1995). Eating contaminated shellfish causes PSP, the symptoms of which may occur within 2 h and last as long as 10 h after the consumption of a contaminated meal. The first symptoms include numbness or tingling in the face, arms, and legs, followed by headache, dizziness, nausea, and a loss of coordination. Severe PSP poisoning results in

OH

H HH N

O HN 1

nutrient and hydrographic conditions coincide, although the precise environmental conditions that cause red tides are not completely understood. The water becomes discolored red or brown due to the presence of dinoflagellate cells which number up to 20  106 cells per liter, typically dominated by one species of dinoflagellate. Some, but not all, red tides are toxic. In toxic red tides, the dinoflagellates produce one or more compounds that are toxic to other animals; subsequently, when the dinoflagellates are ingested by shellfish the chemicals accumulate in the shellfish tissues at levels sufficient to cause adverse effects in birds and marine animals, and in any humans that might ingest the latter. Often, the most prominent signs of intoxication are of a neurological nature. PSP is a distinctive, neurological illness caused by heat-stable and water-soluble tetrahydropurines (Leftley, 1998) known as paralytic shellfish toxins (PSTs). These toxins vary in potency and are present in toxic shellfish in different concentrations and combinations. They are also able to convert from one form to another as they pass through the food chain (Leftley, 1998; Kao, 1993), causing a change in toxicity and poisoning potential. All PSPs inhibit nerve action potential generation by blocking the movement of sodium ions across cell membranes.

HO HO HN HN

O

HO

O OH OH

N OH H

H

R

O

H

H H O

O O H H H OHH

TTX

Figure 2.1 Structures of saxitoxin (STX), tetrodotoxin (TTX), and ciguatoxin (CTX).

H HO O H H

CTX

H

H O O H H O H H

O H

H O H

OH H O O R2

2.2 Saxitoxins (STXs) in Paralytic Shellfish Poisoning

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Table 2.2 Structures of the saxitoxins.

R4 R1 +H2N

HHH N

N1

7

NH2+

N H 12 R5 10 OH R2 R3 N

Toxin

Rl

R2

R3

R4

Rs

Toxin

R1

R2

R3

R4

Rs

STX neoSTX GTX1 GTX2 GTX3 GTX4 GTX5 (B1) GTX5 (B2) C1 C2 C3 C4 dcSTX dcneoSTX

H OH OH H H OH H OH H H OH OH H OH

H H H H OSO3 OSO3 H H H OSO3 H OSO3 H H

H H OSO3 OSO3 H H H H OSO3 H OSO3 H H H

OCONH2 OCONH2 OCONH2 OCONH2 OCONH2 OCONH2 O3 O3 O3 O3 O3 O3 H H

OH OH OH OH OH OH OH OH OH OH OH OH OH OH

dcGTX1 dcGTX2 dcGTX3 dcGTX4 LWTX4 LWTX1 LWTX2 LWTX3 LWTX5 LWTX6 GC1 GC2 GC3

OH H H H H H H H H H H H H

H H OSO3 OSO3 H OSO3 OSO3 H H H H OSO3 H

OSO3 OSO3 H H H H H OSO3 H H OSO3 H H

H H H H H OCOCH3 OCOCH3 OCOCH3 OCOCH3 OCOCH3 OCOC6H4 OCOC6H4 OCOC6H4

OH OH OH OH OH H OH OH OH H OH OH OH

respiratory failure and death within 2–12 h (Van Egmond et al., 1993). Mussels, scallops and clams in PSP-prone areas pose the highest poisoning risk, unless monitored. However, PSP has also been reported following the consumption of crustacea, gastropods and fish, which are not routinely monitored. STX causes an inhibition of the voltage-gated sodium channel, which in turn results in a reduced action potential (Lagos and Andrinolo, 2000). Levels of STX causing human intoxification vary considerably due to differences in individual susceptibility, the methods of analysis, and verification of the quantity of material actually consumed (Whittle and Gallacher, 2000). 2.2.2.1 Chemical Aspects of the STXs The STXs are alkaloids that contain a tetrahydropurine nucleus with various substitutions, allowing many structural possibilities (see Table 2.2) that are related to the presence, or not, of sulfate and hydroxyl groups (R1 to R5), and of carbamoyl, acetate or hydroxybenzoate at R4 position. That is, STXs have no sulfate group, while gonyautoxins (GTXs) and C toxins have one or two sulfate groups, respectively. Likewise, dcSTXs have no carbamoyl group, while other toxins (LW and GC) were obtained from Lyngya wollei and Gymnodinium catenatum toxins, respectively (AWWA, 2007). The molecular weights of the STXs range between 241 and 491 Da. The less-toxic C toxins are transformed by slow hydrolysis in the much more toxic dcGTX. To date, a total of 57 PST analogs has been reported (Wiese et al., 2010). Pure STX was first isolated from the Alaskan butter clam, using weakly basic Amberlite IRC50 and alumina chromatography, and this source remains the best for STX (Schantz et al., 1975). However, the isolation procedure for STXs is not applicable to the less-basic PSP toxins found in shellfish, and a widely

used general procedure for this utilizes adsorption onto Bio-Gel P-2 or Sephadex G-15 (Oshima et al., 1977). The toxin-containing fraction eluted with a dilute acetic acid solution is then applied to a column of weakly acidic carboxylic resin, Bio-Rex-70 in acid form. The acetic acid gradient elution separates the nearly pure toxins in the reverse order of their net positive charge (LC-FLD in Figure 2.2). However, the separation of most

Figure 2.2 Analysis of the final CRM-STX-e by liquid chromatography with post-column oxidation and fluorescence detection (LC-FLD). Conditions: column ¼ Keystone Betabasic-C8, 250  4.6 mm i.d. at 37  C; flow ¼ 0.8 ml min 1; mobile phase ¼ 30 mM ammonium phosphate with 2 mM sodium heptane sulfonate, pH 7.1, and 4% acetonitrile; 10 ml injection; post-column oxidation using 5 mM periodic acid in 0.1 M sodium phosphate, pH 7.8, 0.4 ml min 1, reaction coil at 85  C, effluent acidified with 0.75 M nitric acid 0.4 ml min 1; detection ¼ fluorescence, excitation 330 nm, emission 390 nm. Retention times of related toxins are indicated: dcSTX ¼ decarbamoylsaxitoxin; NEO ¼ neosaxitoxin (CRM-STX-e, 2006).

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

Figure 2.3 Stereochemistry of dihydrosaxitoxins. Proton signal parameters of 270 MHz spectra at different pHs. The spectra were recorded in D2O at 296 K. Reprinted from Shimizu et al. 1981 with permission by the American Chemical Society.

analogs is not possible, even when using preparative thin-layer chromatography or a Bio-Gel P-2 resin. The chemistry of STX has been extensively studied (Shimizu, 1979, 1984; Bordner et al., 1975), with a tentative structure first proposed in 1971 (Wong et al., 1971), and the final structure of STX was later elucidated following X-ray crystallography of its pbromobenzenesulfonate salt (Schantz et al., 1975). STX has several striking structural features; its perhydropurine skeleton with an additional five-membered ring fused at the angular position is unprecedented. It also has a ketone hydrate at position 12, stabilized by two neighboring electron-withdrawing guanidinium groups; the ketone is also readily enolized to effect a rapid exchange of protons at position 11. The molecule has two pKa values of 11.5 and 8.1. Proton and carbon nuclear magnetic resonance (NMR) chemical shift studies conducted under different pH conditions (Figures 2.3 and 2.4) revealed that the latter pH value is associated with the imidazoline guanidinium group (Shimizu et al., 1981; Rogers and Rapoport, 1980). It is suggested that the abnormally low pKa for the guanidinium group is a result of the weak participation of N-7 in the guanidinium resonance, most likely due to the stereochemical strain imparted by the five-membered ring. The results of a high-resolution NMR study (Shimizu, 1984) suggested that, at physiological pH, STX exists as an equilibrium of three molecular species: a divalent cation; a monovalent cation of the hydrated form; and a monovalent cation in a keto form (see 13C NMR in Table 2.3). Saxitoxin is very stable in

acidic solution, and survives for long periods in dilute hydrochloric acid solutions, without degradation. However, the toxin is unstable under alkaline conditions, especially in the presence of oxygen, and undergoes facile oxidative degradation relatively rapidly to yield the highly fluorescent aromatized aminopurine derivatives (Ghazarossia et al., 1976), which can readily be measured for quantitative estimation.

Figure 2.4 1 HNMR spectrum of saxitoxin stock solution in D2O. Saxitoxin resonances used for quantitation are marked with asterisks (). Other peak identities: 1 ¼ CH3COOH; 2 ¼ CH3OH; 3 ¼ CH3CN; 4 ¼ 13C satellite peaks from CH3COOH. CH3OH and CH3CN are residues left from final column chromatography.

2.2 Saxitoxins (STXs) in Paralytic Shellfish Poisoning Table 2.3 13 C NMR chemical shifts in ppma) for saxitoxin hydrate and its keto form. Reprinted with permission from (Rogers and Rapoport, 1980). # (1980) American Chemical Society.

H2 N

O 13 HHH O N HN 6

8

2 +H2N

N 10

12

NH2+

N H OH OH

N C

STX

STX ketob)

C-11 C-10 C-6 C-5 C-13 C-4 C-12 C-2 C-8 C-14

33.37 43.31 53.46 57.45 63.57 82.84 99.00 156.43 158.28 159.30

33.35 41.63 53.86 60.37 64.89 84.90 156.56 164.73

a) Relative to internal dioxane, 67.4 ppm from Me4Si, at the pH attained upon solution. b) Keto form at pH 10.18.

2.2.2.2 Detection of PSP Toxins 2.2.2.2.1 Bioassays The mouse bioassay is the “Official Procedure” in use today as the primary analytical technique to support the majority of toxin-monitoring programs in shellfish for providing a single integrated response from all of the toxins (AOAC Int., 2000). 2.2.2.2.2 Sodium Channel Assays Saxitoxins bind to sodium channels in nerve cell membranes, preventing the influx of sodium and causing a subsequent depolarization of the membrane. A number of electrophysiological systems have been utilized for measuring the binding events, including the frog sciatic nerve (Strichartz, 1984), voltage clamp of single nerve cells (Frace et al., 1986), and blockade of sodium conductance through single-sodium channels isolated in lipid bilayers (Moczydlowski et al., 1984). These techniques are useful for determining the pharmacological properties of the toxins, but unsuitable for routine assay techniques. Cell cytotoxicity assays can be established that reflect the level of sodium channel blockade, and have the potential for use as rapid screening (Kogure et al., 1988). However, the speed and sensitivity of assays that measure the displacement of 3H-TTX from the brain membrane could replace the mouse bioassay as an integrated measure of PSP toxicity. Indeed, this method has recently been validated, confirming its potential (Van Dolah, 2012). 2.2.2.2.3 Immunoassays Several immunological assays for STXs have been developed (Johnson and Mulberry, 1966; Carlson et al., 1984), including an enzyme-linked immunosorbent assay (ELISA) that is sensitive at a level of 2–10 pg STX (Chu and

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Fan, 1985). As the toxicity of a shellfish extract is due to various toxins, the application of an immunoassay for accurate detection is very difficult. However, immunoassay methods can be very useful as rapid “field tests” for simply detecting the presence of PSP toxins. 2.2.2.2.4 Chemical Assays A colorimetric assay based on the reaction of STXs with picric acid was developed (Schantz et al., 1958), but is not sensitive and prone to interference as it is based on a reaction with 2,3-butanedione (Gershey et al., 1977). An alternative method, that is extremely sensitive and fairly specific for PSP toxins, involves a chemical assay for STX based on fluorescence of 8-amino-6-hydroxymethyl-2-iminopurine 3(2H)propionic acid, a hydrogen peroxide oxidation product of STX (Bates and Rapoport, 1975). The fluorometric assay is based on the degradation product formed by treatment with NaOH-H2O2 and is quite sensitive for STX derivatives, but is not suitable for neosaxitoxin derivatives. The main limitation is that other fluorescence products present in the crude extracts will interfere with the measurements. Chromatographic analyses have been also developed for PSP toxins, the most common being ion-pair liquid chromatography coupled with post-column oxidation and fluorescence detection (HPLC-ox-FLD). This is based on the rapid conversion of PSP toxins into fluorescent derivatives under alkaline oxidative conditions (Jaime et al., 2001), and provided an overall limit of detection of 89 mg STXdiHCL-eq per kilogram of shellfish meat (Sayfritz et al., 2008). HPLC methods may also be applicable to monitor marine toxins in shellfish (mussels, oysters, and scallops), including STX, neosaxitoxin (NEO), gonyautoxins (GTX1 and -4, together; and GTX2 and -3, together), and decarbamoyl saxitoxin (dcSTX B-1, C-1, and C-2, together; C-3 and C-4, together), but these remain to be validated. More comprehensive approaches based on high-pressure liquid chromatography with mass spectrometry detection (HPLC-MS/MS) have included a new analytical method based on a combination of hydrophilic interaction liquid chromatography and tandem mass spectrometry (HILIC-MS/MS). This allows a selective and sensitive detection and accurate quantitation of all STX-related compounds in a single 30 min analysis, with no need for further confirmatory analyses (Dell’Aversano et al., 2008; Sayfritz et al., 2008). 2.2.2.3 Poisoning Records One of the earliest recorded incidents of PSP occurred when Captain Vancouver landed in British Columbia in 1793. Tragedy struck when several of his crew suffered agonizing deaths due to paralysis and asphyxiation after eating shellfish taken from an area that Captain Vancouver named “Poison Cove.” Another report of PSP intoxication occurred in 1920 in California (USA), when at least six people died (Meyer et al., 1928). Until the 1970s, PSP toxins were only detected in European, North American and Japanese waters, but nowadays STXs are widely reported in countries outside the US and Europe, including Chile, South Africa and Australia (Pitcher et al., 2007; Krock et al., 2007; Robertson et al., 2004). In most countries,

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

monitoring programs have been established to protect the consumer; for example, the EU has established a permitted level of 800 mg saxitoxin 2HCl equivalents per kilogram of shellfish, while in 2009 the EFSA recommended a safe level as low as 75 mg saxitoxin 2HCl equivalents per kg in order to avoid exceeding the Acute Reference Dose (ARfD) (Alexander et al., 2009; Gerssen et al., 2010). In the USSR during the early 1980s, several fatal poisonings of this type were registered in the Kamchatka region when people ate mussels collected at the Avacha bay coast. The main toxin involved was identified as 11-hydroxysaxitoxin sulfate (Valeev et al., 1989). In early June 2000, an intense bloom (>7  105 cells l 1) of the dinoflagellate Alexandrium tamarense, a known producer of PSP toxins, occurred in southeast Nova Scotia (Canada). This was shown to be responsible for the enhanced mortality of farmed Atlantic salmon in aquaculture cages. LC-ox-FLD analyses of the plankton showed a range of PSP toxins to be present, with (in decreasing order of relative abundance) C2, GTX4, NEO, GTX5, GTX3, GTX1, STX, C1 and GTX2 being identified in the plankton. During the investigation of this event, samples of wild blue mussels (a mixture of Mytilus edulis and M. trossulus) were collected from the vicinity of the salmon cages. The mussel samples showed very high toxicity (up to 67 mg saxitoxin equivalents per kg tissue), using the AOAC mouse bioassay (Dell’Aversano et al., 2008). On 5th July 2002, fishermen harvesting sea urchins (Loxechinus albus) in the Patagonia Chilean fjords were intoxicated after eating the filter-feeder bivalve Aulacomya ater. After eating only between seven and nine ribbed mussels, two fishermen died at only 3–4 h. A forensic examination failed to show any pathological abnormalities except for the lungs, which were “crackling” to the touch and showed associated pulmonary congestion and edema. The toxic mussel sample was found to contain 8575 mg equivalents of STX per 100 g of shellfish meat, when measured with the mouse bioassay. By using a post-column derivatization HPLC method with fluorescent online detection, it was possible to measure the levels of individual PSP toxins in the gastric content, body fluids (urine, bile and cerebrospinal fluid) and tissue samples (liver, kidney, lung, stomach, spleen, heart, brain, adrenal glands, pancreas and thyroids glands). The PSP toxins in the gastric contents were STX and gonyautoxins (GTX4, GTX1, GTX5, GTX3 and GTX2), while those in the urine and bile were mainly neoSaxitoxin (neoSTX) and GTX4/

GTX1 epimers (both STX analogs with an hydroxyl group in the N1 of the tetrahydropurine nucleus). The fact that neoSTX was not present in the gastric contents indicated that oxidation of N1 in the STX tetrahydropurine nucleus had resulted in neoSTX, in a similar way that the GTX3/GTX2 epimers had been transformed to GTX4/GTX1 epimers. Hydrolysis of the carbamoyl group of STX to form the decarbomoyl analog, decarbamoylsaxitoxin, was also detected in the liver, kidney and lung. Together, these findings showed that the PSP toxins had undergone metabolic transformation during the 3–4 h period after the mussels had been eaten, during which the PSP toxins had mediated the enzymatic oxidation of N1 in the tetrahydropurine nucleus, producing neoSTX and GTX4/GTX1 epimers starting from STX and GTX3/GTX2 epimers, respectively. The study authors (García et al., 2004) concluded that, in humans, the PSP toxins had been metabolically transformed and excreted in the urine and feces, much like many xenobiotic compounds.

2.3 Tetrodotoxin (TTX) in Puffer Fish Poisoning (PFP) 2.3.1 Puffer Fish Poisoning (PFP)

Puffer fish poisoning (PFP) is caused by water-soluble tetrodotoxins (TTXs) which are most commonly found in the puffer fish (family Tetraodontidae) (Figure 2.5a–c). The toxins are concentrated in the liver, gonad, roe and skin of the fish, and PFP usually results from the consumption of incorrectly prepared puffer soup, fugu-chiri, or occasionally from raw puffer meat, sashimi fugu. While fugu-chiri is much more likely to cause death, sashimi fugu can be deliberately prepared to cause signs of mild intoxication, including light-headedness and numbness of the lips, and is often eaten for this reason. At higher doses, TTX causes dizziness and vomiting, followed by numbness and prickling over the body, rapid heart rate, decreased blood pressure, muscle paralysis and, potentially, respiratory failure. People who live longer than 24 h after TTX ingestion typically survive, although possibly after a coma lasting for several days. Puffer fish are not believed to produce toxins themselves, as fish kept in tanks or fish farms can be toxin-free. The toxicity of puffer fish species varies

Figure 2.5 The porcupine puffer fish, Diodon holocanthus, is widespread and can be found in the Atlantic, Pacific, and Indian Oceans. The spines will only become erect when the fish, stressed or excited, is puffed up. (a) With courtesy of Fouqui
e, (b) and (c) Ó Shutterstock.

2.3 Tetrodotoxin (TTX) in Puffer Fish Poisoning (PFP)

depending on the tissues or organs, the geography, the season of the year, and gender (Hashimoto and Kamiya, 1970). The female puffer fish is more poisonous than the male, as the ovaries tend to be much more poisonous than the testes (Goto et al., 1965). TTX is not restricted to puffer fish, and is widely distributed among various types of animal, including the California newt Tarichi torosa (Mosher et al., 1964), the goby Gobius criniger (Noguchi and Hashimoto, 1973), Atelopus frogs (Kim et al., 1975), the gastropod mollusks Charonia sauliae (Narita et al., 1981) and Babylonia japonica (Noguchi et al., 1981; Yasumoto et al., 1981), the xanthid crab Atergatis floridus (Noguchi et al., 1983), the blue-ringed octopus Octopus maculosus (Sheumack et al., 1978), Astropecten starfishes (Maruyama et al., 1984, 1985; Narita et al., 1987; Noguchi et al., 1982), the frog shell Tutufa lissostoma (Noguchi et al., 1984), and the small gastropod mollusks Zeuxis siquijorensis (Narita et al., 1984) and Niotha clathrata (Jeon et al., 1984; Hwang et al., 1992). As TTX is distributed in a variety of invertebrates and vertebrates, and there is a wide individual variation of toxin content, even among members of the same species, the origin of TTX was deduced to be a universal organism, such as a microbe. The reason why TTX-bearing animals struggle to accumulate TTX may be different, depending on the species. For example, Noguchi and colleagues (Noguchi et al., 2011) reported the isolation of a TTX-producing bacterium from the intestines of the crab Atergatis floridus which is known to produce the toxin (Saito et al., 2006). Another TTX-producing bacterium has also been found in a calcareous red alga, Jania sp. (Yasumoto et al., 1989). Since these early reports, TTX-producing bacteria have been isolated from various marine organisms, including a starfish, Astropecten polyacanthus (Narita et al., 1987), the blueringed octopus, O. maculosus (Simidu et al., 1987), and a horseshoe crab, Carcinoscorpius rotundicauda from Thailand (Kungsuwan et al., 1988) or Bangladesh (Tanu and Noguchi, 1999) that feeds mainly on mollusks, arthropods, and detritus (Chatterji and Parulekar, 1992). Bacteria which inhabit the marine sediments are again suspected of being the primary origin of TTX

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for this crab. The number of bacterial strains claimed to produce the toxin has expanded quickly, with most having been identified as members of the genus Vibrio, including Vibrio alginolyticus, which is widely distributed in seawater and marine animals of temperate and tropical latitudes (Narita et al., 1987). The most likely mechanism of toxification of TTX-bearing animals involves Vibrio fischeri, isolated from the xanthid crab Atergatis floridus, and V. alginolyticus, isolated from the puffer fish Fugu vermicularis, both of which produced TTX and anhydro-TTX (anh-TTX) (Jensen and Fenical, 1994; Noguchi et al., 1987; Sugita et al., 1987). Another TTX-producing bacterium has also been found in a calcareous red alga, Jania sp. TTX-producing bacteria have been isolated from various marine organisms, including the starfish A. polyacanthus and the blue-ringed octopus O. maculosus (Savage et al., 1977; Sheumack et al., 1984). During recent years the number of bacterial strains reported to produce TTX has been increasing (Simidu et al., 1987), and now includes Vibrio spp. (Noguchi et al., 1986), Pseudomonas spp. (Yasumoto et al., 1986), and actinomycetes (Do et al., 1991). In puffer fish, TTX may serve a defensive role against predators of the adults, and can be released following aggravation, potentially to deter consumption of the eggs. In other animals, TTX may be used to immobilize and capture prey by secretion or envenomation of the toxin, whether from the proboscis in ribbon worms or from the salivary glands in the blue-ringed octopus (Williams, 2010). Thus, two possible intoxication routes for TTX have evolved: orally as a poison arising from its bacterial production and transfer to higher animals through the food web; or as a venom produced by symbiotic bacteria. Because of its frequent involvement in fatal food poisoning, notably in Japan (see Table 2.4), TTX is one of the best studied marine neurotoxins. Its unique chemical structure and specific mode of action to block sodium channels of excitable membranes has made TTX an invaluable research tool for studying TTX-sensitive sodium channels. The TTX site on sodium

Table 2.4 Puffer poisoning incidents in Japan.a)

Year

1965 1970 1975 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989

No. of

Mortality (%)

Incidents

Patients

Deaths

106 46 52 46 30 26 18 23 30 22 35 26 31

152 73 75 90 46 33 34 39 41 38 52 46 45

88 33 30 15 12 8 6 6 9 6 4 5 5

57.9 45.2 40.0 16.7 26.0 24.2 17.6 15.4 21.9 15.8 7.7 10.9 11.1

Year

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

No. of

Mortality (%)

Incidents

Patients

Deaths

33 29 33 28 16 30 21 28 27 20 29 31 32

55 45 57 44 23 42 34 44 39 34 40 52 49

1 3 4 4 1 2 3 6 4 2 0 3 5

1.8 6.7 7.0 9.1 4.3 4.8 8.8 13.6 10.3 5.9 0 5.8 10.2

a) Data cited from Ministry of Health and Welfare in Japan MHW 1997. Reprinted from Hwang and Noguchi 2007 with kind permission by Elsevier.

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

Table 2.5 Severity of tetrodotoxin poisoning and associated symptoms (Hwang and Noguchi, 2007).

Table 2.6 Operating conditions of HPLC for the analysis of new toxins

TTXs.

Degree

Characteristic symptoms

HPLC system

D-7480

First

Neuromuscular symptoms (paresthesia of lips, tongue, and pharynx; taste disturbance; dizziness; headache; diaphoresis; pupillary constriction); gastrointestinal symptoms (salivation, hypersalivation, nausea, vomiting, hyperemesis, hematemesis, hypermotility, diarrhea, abdominal pain) Additional neuromuscular symptoms (advanced general paresthesia; paralysis of phalanges and extremities; pupillary dilatation, reflex changes) Increased neuromuscular symptoms (dysarthria; dysphagia, aphagia; lethargy; incoordination, ataxia; floating sensation; cranial nerve palsies; muscular fasciculations); cardiovascular/pulmonary symptoms (hypotension or hypertension; vasomotor blockade; cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, and atrioventricular node conduction abnormalities; cyanosis; pallor; dyspnea); dermatologic symptoms (exfoliative dermatitis, petechiae, blistering) Respiratory failure, impaired mental faculties, extreme hypotension, seizures, loss of deep tendon and spinal reflexes

Column (4.6  250 mm) Column temperature Mobile phase

Inertsil ODS-3

Second

Third

Fourth

channels (Site 1) is a high-affinity site (Kd ¼ 10 9 M) that overlaps the STX binding site. Due to its analgesic properties, TTX has even proved to be a promising new drug candidate in the field of pain management, and is currently in Phase III clinical trials for the treatment of cancer-related pain (Hwang and Noguchi, 2007). The specific toxicity of TTX is 5000–8000 MU mg 1, where 1 MU is defined as the amount of TTX that can kill a male ddY strain mouse of bodyweight 20 g in 30 min (this corresponds to ca. 0.2 mg TTX). The minimum lethal dose (MLD) of TTX for humans is estimated at approximately 10 000 MU (equivalent to 2 mg pure TTX). The minimum dose required to develop symptoms of TTX poisoning in humans has been estimated at 0.2 mg (approximately equivalent to the STX dose). Various TTX derivatives from pufferfish and other TTX-bearing organisms have been identified as a result of recent advances in the analysis of TTX. The type, severity and range of symptoms will depend on the amount of toxin ingested, and the age and health of the victim; the four main stages or degrees of tetrodotoxication are outlined in Table 2.5. The treatment of TTX intoxication is symptomatic, as there is no known antidote for the toxin (Buwer et al., 1981; Furman, 1967; Mosher et al., 1964). The maintenance of controlled ventilation (with an endotracheal tube) and hemodynamic control of the circulatory function associated with cardiac arrhythmias, will require the victim to be hospitalized. Gastric washing is indicated for an intoxication of less than 3 h, while anticholinesterase drugs can also be useful, with the first 24 h of care being most critical.

30  C 60 mM ammonium phosphate buffer (pH 5.0) containing 10 mM HAS and 2% CH3CN 4 M NaOH 0.8 ml min 1 110  C

Reagent Flow rate Reaction temperature Detection

Excitation 384 nm, emission 505 nm

Source: Tsuruda et al. (2001).

pharmacological characterization of TTX. In 1909, Tahara was able to partially purify the puffer toxin and named the active constituent TTX. Pure TTX has a molecular weight of 319 Da, and is poorly soluble in water and organic solvents. However, much like the PSP toxins, it is soluble and stable in acid aqueous solutions and unstable in alkaline solutions. TTX is heat-resistant and does not decomposed under standard cooking conditions; typically, it is a colorless crystalline compound with no clear melting point, but is black at temperatures above 200  C. Due to its novel structure and high density of functional groups, elucidation of the TTX structure proved challenging, although several groups each independently arrived at the same structure, namely octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a- dimethano-10aH-[l,3]dioxocino[6,Sd]pyrimidine-4,7,10,11,12-pentol (Goto et al., 1963a,b; Goto et al., 1965; Tsuda et al., 1964; Woodward, 1964; Woodward and Gougoutas, 1964). The absolute stereochemistry of TTX was determined unambiguously by the X-ray crystallographic analysis of a derivative (Furusaki et al., 1970). Despite being a small molecule, TTX has an extremely complex structure that is characterized by a dioxaadamantane skeleton, a guanidine residue at C2 that forms part of a hemiaminal at C4, and an orthoacid bridge at ClO. One remarkable feature of TTX is that the number of oxygen and nitrogen atoms equals the number of carbon atoms (Figure 2.6). The three nitrogen atoms of TTX are present in the molecule as a guanidine moiety; yet, despite the presence of this moiety the toxin was only weakly basic (pKa 8.5) and attempts to prepare crystalline salts failed. Treatment of the toxin with 0.2 N HCl yielded a crystalline, O-methyl-O,O0 -isopropylidene-tetrodotoxin hydrochloride monohydrate. About 1-2 g of crystalline TTX was obtained from 100 kg of puffer

OHO

2.3.1.1 Chemical Aspects of TTX On account of the many serious poisonings due to the consumption of puffer fish in Japan (see Table 2.6), many Japanese research groups have undertaken studies of the chemical and

Hitachi

HO HN +H2N

O

O OH OH

N OH H

Figure 2.6 Structure of tetrodotoxin (TTX).

2.3 Tetrodotoxin (TTX) in Puffer Fish Poisoning (PFP)

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Figure 2.9 1 H NMR spectrum of TTX. 5 mg of the ribbon worm toxin was dissolved in 0.5 ml of 1% CD3COOD in D2O and measured for 1H NMR spectrum using a JEOL JNM-500NMR spectrometer, with acetone as the internal standard (Asakawa et al., 2012). Figure 2.7 HPLC of authentic TTXs (left) and the Japanese newt (C. pyrrhogaster) toxin (right). (a) TTX; (b) 6-epi-TTX; (c) 4-epi-TTX; (d) 4,9-anhydro-6-epi-TTX; (e) 4,9-anhydro-TTX (Tsuruda et al., 2001).

ovaries, using Hirata’s procedure (Goto et al., 1964). TTX derivatives are found in puffer fish, newts, and a frog. The toxins can be detected using a highly sensitive TTX analyzer which separates them on a reverse-phase column and detects fluorescent products formed when the toxin is heated with sodium hydroxide solution (Yasumoto et al., 1982). The extraction and purification of natural TTX is complicated and depends on the availability of suitable animal sources; there is, however, an ongoing need to develop more cost-effective syntheses. The first total synthesis was reported by Kishi in 1972, and afforded TTX as a racemic mixture after 29 steps in 0.66% overall yield (Kishi et al., 1972a; Kishi et al., 1972b). The first asymmetric synthesis of TTX, which was reported in January 2003 (Ohyabu et al., 2003), relied on a Claisen rearrangement, a Shonogashira coupling, and an intramolecular carbamate–ester conjugate addition as the key steps. TTX was obtained in a 1.2% overall yield after 67 steps, although since 2003, in order to reduce the number of steps and increase the yield of the final compound, several total syntheses of TTX

Figure 2.10 Infrared (IR) spectrum of puffer toxin (Onoue et al., 1984). Although the spectrum in IR analysis is complex, it is a helpful tool for identification of TTX once purified. Reprinted from Onoue et al., 1984 with permission by the American Chemical Society.

have been proposed to provide an alternative, cost-effective and efficient synthesis of TTX and new analogs with useful biological and pharmacological properties (Hinman and Du Bois, 2003; Nishikawa et al., 2004; Koert, 2004; Sato et al., 2005). The HPLC operating conditions for the analysis of TTXs toxins are provided in Table 2.6 and Figure 2.7; the structural data of TTX are shown in Figures 2.8–2.10 (MS data, 1H NMR, and infrared spectra, respectively).

Figure 2.8 Mass spectra obtained in ESI mode (left), and negative (center) and positive (right) FAB mass spectra of TTX (Asakawa et al., 2012). TTX shows (M þ H)þ and (M þ H - H2O)þ ion peaks at m/z 320 and 302, respectively, in the positive mass spectrum, and an (M-H) peak at m/z 318 in the negative spectrum.

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2.3.1.2 Detection of TTXs TTX levels in pufferfish are normally estimated with the mouse bioassay. Following the intraperitoneal injection of mice with TTX-associated extracts, the mice show characteristic signs and symptoms, such as scratching of the shoulder/mouth with the hind limbs, weakness progressing to paralysis in the hind limbs, uncoordinated movement, shallowness of breathing, convulsion, and jumping preceding respiratory failure. The toxicity of an extract is expressed in terms of mouse units (MU) (for details, see Section 2.3.1). Historically, the mouse bioassay has been the most universally applied tool to determine toxicity levels in monitoring programs, but shows a low precision and alternative chemical methods have been developed (see Table 2.7). Thin-layer chromatography (TLC) is a useful technique when HPLC and other costly analytical systems are unavailable. Capillary isotachophoresis has proved to be very effective technique for the analyses of organic acids, carbohydrates, drugs, and amino acids, and offers a rapid and accurate detection for TTX (Shimada et al., 1983). The quantitative detection limit with this method was high (0.25 mg TTX), and the TTX content of contaminated puffer extracts could be assessed without any pretreatment. Among the assay methods employed, TLC, electrophoresis, LC, spectrophotometry and ELISA are not specific and lack sensitivity and precision at low concentrations. Both, HPLC-FID and LC-MS/GC-MS are sensitive techniques for the identification of TTX although, due to the complexity of sample matrices and insolubility of TTX in organic solvents, HPLC-FID and LCMS (or LC-MS/MS) are more preferable than GC-MS (Asakawa et al., 2012). MS spectrometry, in addition to high sensitivity and selectivity, can provide structural information that is useful for confirming toxin identity and identifying new toxins, but involves the use of expensive, high-maintenance instruments. Nevertheless, for the routine analysis of TTXs, HPLC-FID and LC-MS (LC-MS/MS) are expected to replace the conventional mouse bioassay.

A monoclonal TTX antibody to detect TTX with high sensitivity has been developed (Kawatsu et al., 1997), and the microdistribution of TTX in several tissues of puffer fish (Mahmud et al., 2003a; Mahmud et al., 2003b; Tanu et al., 2002) and newt (Tsuruda et al., 2002) were elucidated. However, an antibody to treat TTXpoisoned patients has still to be developed. A rapid and simple detection method for TTX in the urine and plasma of patients with puffer fish poisoning was developed using commercially prepacked solid-phase extraction (SPE) cartridges (C18 and weak cation-exchange columns), with subsequent analyses by HPLC with ultraviolet detection. The detection limit of the TTX standard and the TTX-spiked urine and plasma samples were all 10 ng ml 1, and the average TTX recovery in samples after SPE was 90% and 87%, respectively. Creatinine-adjusted urinary TTX levels obtained within the first 24 h of presentation appeared to correlate much better with the severity of poisoning than did urinary TTX concentrations, without adjusting for variations in concomitant creatinine excretion (Yu et al., 2010). A sensitive analytical method was developed for the quantitative determination of TTX in human postmortem hole blood, using hydrophilic interaction LC-tandem MS. The sample mixture was worked-up using a cation-exchange SPE cartridge after protein precipitation by methanol, and then separated with phosphorylcholine-hydrophilic interaction liquid chromatography (PC-HILIC). TTX was identified using tandem MS with positive electrospray, and the limit of detection (and quantification) was 0.32 ng ml 1 (Cho et al., 2012). Recently, HPLC coupled to electrospray ionization (ESI) mass spectrometry (CID-MS/MS) allowed the detection of TTX and its analogs for the first time in five different tissues (liver, gonads, gastrointestinal tract, muscle, skin) of six specimens of marine puffer fish, Lagocephalus sceleratus, from European waters (Aegean Sea, Greece). The detection limit of the LC-ESICID-MS/MS analysis was low (0.08 mg g 1), and two isomers of 5,6,11-trideoxy-TTX were detected in all specimens as the major TTX analogs, followed by 11-deoxy-TTX, 11-nor-TTX-6(S)-ol, and TTX. Among all puffer fish specimens the gonads,

Table 2.7 The features of detection methods for TTX.

Method

Detection system

Detection limit

Features

Bioassay HPLC TLC Electrophoresis Capillary isotachophoresis UV spectroscopy GC–MS IR spectrometry FABMS LC–MS ESI-TOF/MS NMR spectrometry Cytotoxicity test Immunoassay

Mice Fluoromonitor Fluorescent spot Fluorescent spot Potential stand Spectrometer Mass spectrometer IR spectrometer Mass spectrometer Mass spectrometer Mass spectrometer NMR spectrometer Microscopy ELISA

0.2 mg TTX 0.03 mg TTX 2 mg TTX 2 mg TTX 0.25 mg TTX Qualitative Qualitative Qualitative Qualitative 0.05 ng TTX Qualitative Qualitative 3 nmol l 1 2 ng ml 1

Death time Intensity Spot mobility Spot mobility Ion mobility Alkali decompose Alkali decompose + ion-monitoring Functional group Ion-monitoring Ion-monitoring Ion-monitoring Proton spectrum Cell death Monoclonal antibody

Source: Reprinted from Hwang and Noguchi 2007 with kind permission by Elsevier.

2.4 Ciguatoxin (CTX) in Ciguatera Fish Poisoning (CFP)

gastrointestinal tract and liver contained the highest toxin levels, while muscle and skin contained lower amounts. Toxin distribution among the tissues differed depending on the fish size, and the geographic area and season in which the fish were caught (Rodríguez et al., 2012).

2.4 Ciguatoxin (CTX) in Ciguatera Fish Poisoning (CFP) 2.4.1 Ciguatera Fish Poisoning (CFP)

Ciguatera fish poisoning (CFP) has for many centuries been known to be associated with the consumption of fish from coral reefs and inshore habitats in subtropical and tropical regions that have accumulated polyether toxins known as ciguatoxins (Figure 2.11). Islands of the Caribbean, Pacific, and Indian Oceans are particularly susceptible, but even within ciguatera-prone countries the occurrence of CFP is highly variable, both spatially and temporally.

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Traveling to an endemic area of the Caribbean is estimated to carry a 3% chance of acquiring CFP (Mines et al., 1997). In Europe, USA or Canada, travelers may present with CFP after visiting endemic areas, or it may occur after eating imported fish species. Worldwide, CFP is considered to be the most common type of poisoning from eating coral reef fish (Bagnis, 1993). Ciguatera not only endangers public health but also hampers local fisheries in tropical and subtropical regions of the world. It is estimated that approximately 10 000 to 50 000 people suffer annually from such poisoning (Lipp and Rose, 1997). CFP outbreaks are generally sporadic and unpredictable, but may be associated with reef disturbances (Bagnis, 1993). Positive correlations have been observed between the annual incidence of fish poisoning and local increases in sea surface temperature, in Pacific Island countries and territories that experience warming during El Ni~ no conditions (Hales et al., 2001). However, subsequent analyses have indicated that any relationship between ciguatera and climate change needs to take account of more complex associations between temperature and the abundance of Gambierdiscus, the production of the ciguatoxin precursors, and metabolism in fish (Figure 2.12) (Llewellyn, 2010).

Figure 2.11 The global distribution of ciguatera. Reprinted from Lewis 2001 with kind permission by Elsevier.

Figure 2.12 Annual cases rates in the South Pacific. Reprinted from Llewellyn, 2010 with kind permission by Elsevier.

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

Figure 2.13 Scanning electron microscopy image of Gambierdiscus.

2.4.2 Ciguatoxins

Ciguatoxins (CTXs) isolated from reef fishes accumulate through the marine food chain from precursors called gambiertoxins, that are elaborated by marine benthic dinoflagellates of the genus Gambierdiscus (Figure 2.13). These toxins are produced by the certain species of Gambiersdiscus and transferred to herbivorous fish and subsequently to carnivores through the marine food chain (Lewis and Holmes, 1993). The maitotoxins and a number of poorly defined toxins originating from various epiphytic and benthic dinoflagellate species of Ostreopsis, Prorocentrum and Amphidinium (Tindall and Morton, 1998) may also be involved, though there is no definitive evidence that these accumulate to toxic levels in fish. Over 400 species, including groupers, sea basses, snappers, sea perches, emperor fish, Spanish mackerel, jacks, trevallies, carangs, wrasses, surgeon fish, parrot fish, mullet, moray eel and barracuda, reportedly cause CFP (Bagnis, 1993) (Figure 2.14a–e). The prevalence of ciguatoxins in barracuda has resulted in its sale being banned in parts of Florida (Mines et al., 1997) and the Dominican Republic (Todd, 1993). The liver and viscera typically contain higher levels of the ciguatoxins, and larger fish can pose a higher risk. The term ciguatera was coined by Felipe Poey in 1866 to describe fish poisoning events originally associated with consumption of the marine snail, the Turbo Livona pica, known as “cigua” in Cuba, although the source of poisoning was probably misidentified at the time. Ciguatera was first reported in the Indian Ocean in 1601. In the South Pacific Ocean, the first noted case of ciguatera was in 1770 when the crew of the Fernandez de Quiros became intoxicated. In 1774, James Cook described a CFP in New Hebrides, while in French Polynesia the historic records of intoxicated fish date back to 1792.

CFP is characterized by variable combinations of over 30 gastrointestinal, neurological and cardiovascular symptoms. Pacific and Caribbean ciguatoxin-1 (P-CTX-1 and C-CTX-1, respectively) are regarded as the principal toxins responsible for human illness in the Pacific Ocean and Caribbean Sea, respectively. The symptoms vary by location and among individuals, appear within a few minutes to over 30 h, and include nausea, vomiting, diarrhea, cramps, excessive sweating, headache, and muscle aches. A feeling of burning and “pins and needles,” as well as weakness, itching, dizziness, unusual taste sensations, nightmares, and hallucinations are common. The symptoms usually last for one to four weeks, though some victims may suffer for months, especially if allergy-like reactions to certain foods develop. People eating similar portions of the same fish may ultimately suffer different symptoms or none at all, indicating a highly variable response, even to the same mix of ciguatoxins (Mines et al., 1997). The most commonly recognized symptom is a reversal of temperature perception, in which victims interpret cold as burning or “dry-ice” sensation (Bagnis, 1993). The molecular basis of this cold allodynia has recently been elucidated (Vetter et al., 2012). The effects of ciguatera are usually short-term and self-limited, and there is no antidote. In most cases, digestive symptoms subside within one to two days, cardiovascular within two to five days, and neuromotor and neurosensory in two to three weeks. In severe attacks, neurological symptoms may last years and recur intermittently, induced by the consumption of fish, shellfish, alcohol or nuts, and during periods of overwork and stress (Bagnis, 1993). Death may occur in 0.1% cases, but mortality can be as high as 20% in isolated outbreaks associated with the consumption of highly toxic fish. Fatalities have been attributed to direct cardiovascular depression, hypovolemic shock or respiratory paralysis. CFP may also be transmitted by sexual intercourse; in pregnant women it can result in premature labor or harm to the fetus, sometimes causing abortion (Mines et al., 1997). An intravenous infusion of mannitol may relieve symptoms if administered soon after intoxification (Palafox et al., 1988; Pearn et al., 1989), though a double-blind study conducted in a small cohort of moderately poisoned ciguatera sufferers failed to show any difference between mannitol and saline (Schnorf et al., 2002). An accurate differential diagnosis is critical before commencing any treatment, as the symptoms can be confused with organophosphate, palytoxin, botulism, puffer fish and shellfish poisoning, and potentially even chronic fatigue syndrome (Peam, 1997) if the onset coincides with the consumption of risk fish species.

Figure 2.14 Some popular ciguateric fishes. (a) Moray eel; (b) Trigger fish Ó Shutterstock; (c) Surgeonfish Ó Shutterstock; (d) Barracuda Ó Shutterstock; (e) Grouper.

2.4 Ciguatoxin (CTX) in Ciguatera Fish Poisoning (CFP)

delivered intraperitoneally (LD50 0.13 mg kg 1), its oral potency is less than that of the ciguatoxins, which have a similar potency irrespective of the route of administration (Lewis et al., 1991b). The structure of MTX was elucidated by Japanese and American groups, using state-of-the-art NMR spectroscopy, mass spectrometry and the synthesis of model compounds using material produced by cultured Gambierdiscus toxicus. Ciguatoxins arise from biotransformation in the fish of precursor gambiertoxins (Lewis and Holmes, 1993; Lehane and Lewis, 2000). In areas in the Pacific, the principal and most potent ciguatoxin is Pacific ciguatoxin-1 (P-CTX-1, mol. wt. 1112 Da), that likely arises from gambiertoxin-4B (GTX-4B). The main ciguatoxins in the Pacific (P-CTX-1, P-CTX-2, and P-CTX-3) are present in fish in different relative amounts (Lewis et al., 1991b; Lewis and Sellin, 1992; Lehane, 2000; Lehane and Lewis, 2000). The structures of more than 20 congeners of ciguatoxin have recently been elucidated (see Figure 2.15 and Table 2.8). Structural modifications were mainly seen in both termini of the toxin molecules, mostly associated with oxidations and ring openings (Naoki et al., 2001; Yasumoto et al., 2000) associated with acid-catalyzed spiro-isomerization (Lewis and Holmes, 1993). Caribbean CTX-1 (C-CTX-1) is less polar than P-CTX-1, and the structures of two Caribbean ciguatoxins (C-CTX-1 and C-CTX-2) were elucidated using NMR in 1998 (Lewis et al., 1998), as shown in Figure 2.16. Multiple forms of the C-CTX with minor mass differences and pathogenicity have been described (Pottier et al., 2002a), but the precursor of these ciguatoxins has not been identified in Gambierdiscus from this region. CTX-1 is the major toxin found in carnivorous fish, and poses a human health risk at levels above 0.1 mg kg 1 fish

2.4.2.1 Chemical Aspects Scheuer’s group at the University of Hawaii was the first to name ciguatoxin isolated from ciguateric moray eel (Scheuer et al., 1976). However, limited amounts of highly purified material and access to two-dimensional NMR prevented Scheuer and colleagues from determining its structure. Later, Yasumoto and coworkers (Murata et al., 1989) were the first to elucidate the structure of the major ciguatoxin obtained (0.35 mg) from the viscera of the moray eel Gymnothorax javanicus (125 kg) caught in Tahiti, with the structure of two related ciguatoxins named CTX-2 and CTX-3, determined a few years later (Lewis et al., 1991a; Lewis and Holmes, 1993). More recently, CTXs have been isolated from all major regions where ciguatera is endemic. The structurally distinct Caribbean (CCTXs) and Indian Ocean (I-CTXs) differ from the CTXs of the Pacific (P-CTXs) (Lewis et al., 1998; Hamilton et al., 2002), which explains the different poisoning syndromes observed between regions (Vernoux and Lewis, 1997). CTXs are lipophilic, heat- and acid-stable polyethers of molecular mass of 1100 Da that selectively open voltagesensitive sodium channels in excitable cells, including sensory neurons (Strachan et al., 1999; Vetter et al., 2012). At large doses, CTXs block the function of potassium channels, further impairing the conduction of nerve impulses (Birinyi-Strachan et al., 2005). In contrast, maitotoxin (MTX) is a water-soluble, bisulfated compound of molecular mass 3424 Da, which activates both voltage-sensitive and receptor operated calcium channels in the plasma membrane of cells. Although MTX is the most toxic and structurally complex of all known nonprotein toxins when

H

R1

H

O

H

O

H

H OH

H

O

H

H O

H HO O H H

P-CTX-3C

H

O H H

H HO

OH H O O

H O H

O H H

R2

P-CTX-1:

R1 = CHOH–CH2OH,

R2 = OH

P-CTX-3 (P-CTX-2):

R1 = CHOH–CH2OH,

R2 = H

P-CTX-4B (P-CTX-4A)

R1 = CH2 = CH;

R2 = H

H

H O O H H O H H

O H

O H H

O O H H H OH H

Paciic ciguatoxin-1:

HO

O

H

H O

HO

H H O

O H

H O H

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OH H O O

HO

HO

H

O

H

O

H

H

H

O

H

H HO

H O H

H

H O O H H O H H

O H

H O H

OH O H H O

O

O H HO

Caribbean ciguatoxin-1: C-CTX-1 (C-CTX-2)

Figure 2.15 Ciguatoxin (CTX) molecules. The energetically less favored epimers, P-CTX-2 (52-epi P-CTX-3), P-CTX-4A (52-epi P-CTX-4B) and C-CTX-2 (56-epi C-CTX-1) are indicated in parenthesis. 2,3-Dihydroxy P-CTX-3C and 51-hydroxy P-CTX-3C have also been isolated from Pacific fish (Lewis, 2001).

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

Table 2.8 Principal ciguatoxin congeners analyzed by HPLC-MS with their corresponding molecular ions masses [M þ H]þ (Caillaud et al., 2010).

Origins

Name

[M þ H]þ

Source

Pacific CTXs

P-CTX-1; CTX1B P-CTX-2; CTX2A2; 52-epi-54-deoxyCTX P-CTX-3; CTX2B2; 54-deoxyCTX 49-epi-CTX-3C; CTX-3B M-seco-CTX-3C CTX-3C 2,3-dihydroxy CTX-3C 51-hydroxy CTX-3C; CTX-2C1 CTX-4B; GT-4B 52-epi-CTX-4B; CTX-4A; GT-4A C-CTX-1 C-CTX-2, 56-epi-C-CTX-1 C-CTX-1127 C-CTX-1143 C-CTX-1157 C-CTX-1159 I-CTX-1 I-CTX-2 I-CTX-3 I-CTX-4

1111.6 1095.5 1095.5 1023.6 1041.6 1023.6 1057.6 1039.5 1061.6 1061.6 1141.6 1141.6 1127.6 1143.6 1157.6 1159.6 1141.6 1141.6 1157.6 1157.6

Carnivorous fish Carnivorous fish Carnivorous fish G. toxicus G. toxicus G. toxicus Carnivorous fish, G. toxicus Carnivorous fish Carnivorous fish, G. toxicus G. toxicus Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish Carnivorous fish

Caribbean CTXs

Indian CTXs

of silver coins or copper wire, or the repulsion of flies and ants, but all of these were rejected as invalid. Feeding tests to cat or mongoose are simple and relatively sensitive, but are cumbersome and nonquantitative, and are no longer routinely used. The mouse bioassay requires the purification of fish extracts as the mouse is not very sensitive to ciguatoxin. An alternative is the chicken, which is sufficiently sensitive to detect moderate to highly toxic fish (Vernoux et al., 1985). A mosquito bioassay correlates with the cat and mouse bioassay, but is neither robust nor easy to implement (Bagnis et al., 1987). Other bioassays have been developed but typically suffer from issues including insufficient sensitivity (brine shrimp), cost, and lack of specificity (guinea pig atrium). Recent studies have also focused on the

(Lewis et al., 1999; Pottier et al., 2002b, 2003). Various species of parrotfish have previously been reported to contain a toxin which is less polar than CTX-1, named scaritoxin. Judging from the reported chromatographic properties, scaritoxin seems to correspond to a mixture of CTX-4A and CTX-4B (De Fouw et al., 2001), suggesting a limited biotransformation in this species of herbivorous/corallivorous fish. 2.4.2.2 Detection of CTX Toxins Ciguatoxins are odorless, tasteless and generally undetectable by any simple test, and consequently bioassays have traditionally been used to monitor suspected fish. Many native tests for the toxicity of fish have been examined, including the discoloration

28.29

HO

136.0 132.57

67.01

HO

H HO

74.30 40.00 85.50

H O 78.50

27.19 45.99 46.59 73.13 83.69

O 80.90 H 36.38 H O H H

83.80 85.80 H 37.57 H H 34.48 H O 83.63 126.92 O 32.40 85.30 F 76.56 79.67 83.30 136.13 128.30 32.4 A 87.73 73.57 D O 81.86 80.43 74.01 78.00 128.20 H H O 132.50 131.19 O H H H 133.57 H OH 72.92 131.33

H

20.17

O H

43.90

81.51 84.45

74.78

OH 76.85

87.58

H O O

K

72.61

41.28

H O H

77.56

16.18

109.64

38.98 42.15

M

45.75

75.10 70.69

OH

13.92

OH Figure 2.16 13 C NMR assignments of CTX in C5D5N-D2O (20:1). Assigned on the basis of cross peaks in HMQC spectra. No clear peaks were observed at the corresponding chemical shift in the 13 C NMR (broadband decoupling) spectrum. Reprinted from Murata et al. 1992 with kind permission by Elsevier.

2.4 Ciguatoxin (CTX) in Ciguatera Fish Poisoning (CFP)

development of chemical methods, such as TLC and LC for the detection and quantification of ciguatera-related toxins. Alternative assays based on immunochemical technology have been developed and have shown promise for use in seafood safety monitoring programs. Immunochemical methods such as a radioimmunoassay (RIA) (Hokama et al., 1977), a competitive enzyme immunoassay (EIA) (Hokama et al., 1983, 1984; Tsumuraya et al., 2010), and a rapid enzyme immunoassay stick test (Hokama, 1985; Hokama et al., 1985b, 1987) have been developed for Pacific ciguatoxins, but these suffer from crossreactivity with other polyether compounds and the limited antibody supply. The presence of other family of ciguatoxins in the Caribbean and Indian Ocean has important implications for the detection of ciguateric fish, since antibodies raised against P-CTX-1 or P-CTX-1 fragments may not be suitable for detecting Caribbean ciguatoxins (Lewis et al., 1998). Recent studies have focused on the development of analytical methods, including LC/MS/MS for the detection and quantification of ciguatera-related toxins. By using a new rapid extraction procedure (Figure 2.17), this approach can detect levels of P-CTX (>0.1 ppb) sufficient to cause human intoxication (Lewis et al., 2009). Moreover, with recent developments in MS, sensitivity is likely to be improved a further 10-fold, allowing detection at the proposed regulatory limit of 0.01 ppb for P-CTX-1 (Dickey and Plakas, 2010). 2.4.2.3 Poisoning Records Currently, as many as 50 000 cases of CFP are reported annually on a worldwide basis, as the condition is endemic in tropical and

2 g minced fish, cooked in a 50 ml Falcon tube Homogenize in 8 ml methanol/hexane (3/1) Centrifuge, discard upper hexane layer Filter≈5.5 ml of methanol layer, add 2 ml H2O C 18 SPE cleanups (steps 1 – 4)

Condition with 4 ml H2O

Apply sample to SPE column

Wash with 6.5 ml 65% methanol

Elute with 8 ml 80% methanol

Add 4.2 ml 1 M NaCl + 6.7 ml chloroform Shake, centrifuge, discard upper aqueous layer Evaporate lower layer, redissolve in 4 ml chloroform Silica SPE cleanups (steps 1 – 4)

Condition with 4 ml Chloroform

Apply sample to SPE column

Wash with 4.5 ml chloroform

Elute with 8 ml 90% chloroform

Evaporate, redissolve in 200 ml 50% aqueous methanol LC/MS/MS analysis (20 ml)

Figure 2.17 CTX in fishes. Rapid extraction procedure for LC-MS/MS analysis. Reprinted from Lewis et al. 2009 with kind permission by Elsevier.

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subtropical regions of the Pacific Basin, Indian Ocean and Caribbean. Isolated outbreaks occur sporadically (but with increasing frequency) in temperate areas such as Europe and North America. The increase in travel between temperate countries and endemic areas, and the importation of susceptible fish has led to an encroachment of CFP into regions of the world where it has previously been rarely encountered (Ting and Brown, 2001). In the primary endemic areas, including the Caribbean and South Pacific Islands, the incidence is between 50 and 500 cases per 10 000 people (Perez et al., 2001). In the developed world, CFP poses a public health threat due to delayed or missed diagnosis. Without treatment, distinctive neurologic symptoms persist, occasionally being mistaken for multiple sclerosis. Constitutional symptoms may be misdiagnosed as chronic fatigue syndrome (Ting and Brown, 2001). It was supposed that the incidence figures were likely to represent only 10–20% of actual cases, with the extent of under-reporting likely to vary between countries and over time (De Fouw et al., 2001). 2.4.2.4 Persistence and Recurrence of Symptoms Neurological disturbances usually resolve within weeks of onset, although some symptoms may persist for months or even years. Symptoms such as pruritus, arthralgia and fatigue can also persist for months or years. The analysis of ciguatoxins in blood samples suggests that the toxin can be stored in adipose tissue, and that symptoms may recur during periods of stress, such as exercise, weight loss, or excessive alcohol consumption; sensitivity to alcohol may also persist for years after the first exposure (Lehane, 2000). The phenomenon of ciguatoxin sensitization has been observed after eating apparently nontoxic fish and unrelated foods including nuts, and many months or even years after an attack of CFP (De Fouw et al., 2001). Therefore, patients suffering from CFP are recommended to avoid these food products. Eating fish with low levels of toxins over several years in the absence of symptoms could eventually result in sensitization to the toxin. This most likely results from an accumulative effect of ciguatoxin in the host, possibly involving an immunological reaction. 2.4.2.5 Fish Containing Ciguatoxins Many families of reef fishes are involved in ciguatera globally. These include the herbivorous Acanthuridae and corallivorous Scaridae (parrotfish), which are considered key vectors in the transfer of ciguatoxins to larger carnivorous fish. Many more species of carnivorous fish are known to cause ciguatera than herbivorous species. These include Muraenidae (moray eels) and Lutjanidae (snappers such as red bass), which are notorious in the Pacific, Serranidae (groupers) including coral trout from the Great Barrier Reef, Epinephelidae, Lethrinidae, Scombridae (mackerel), Carrangidae (jacks) and Sphyraenidae (barracudas) (see Figure 2.14). The latter two families are a particular problem in the Caribbean (Crump et al., 1999; Lewis, 2001). More than 400 species of bony fish have been reported in the literature to have caused ciguatera poisoning. The larger carnivores such as moray eels, snappers, groupers, carrangs, Spanish

38

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

Table 2.9 Examples of fish associated with ciguatera (Farstad and Chow, 2001).

Lined surgeonfish Bonefish Gray triggerfish Gaucereye porgy Horse-eye jack Whitetip shark Humphead wrasse Heavybeak parrotfish Red grouper Moray eel (giant) Hogfish Red snapper

Acanthurus lineatus Albula vulpes Batistes carolinensis Calamus calamus Caranx latus Carcharinus longimanus Cheilinus undulatus Chlorurus gibbus Epinephelus morio Gymnothorax javanicus Lachnolaimus maximus Lutjanus bohar

mackerels, emperors, certain inshore tunas and barracuda are considered the highest risk and are likely to be the most toxic (IPCS, 1984). For example, along the southwest coast of Puerto Rico, the caught barracuda is involved in ciguatera poisoning. The head, viscera and flesh tissue components of 219 barracudas (528 tissue samples) were screened for their toxicity during the period March 1985 through May 1987. Subsequently, 29% of these fish yielded toxic preparations in at least one of their tissue components (De Fouw et al., 2001). In the continental United States, grouper, red snapper, jack and barracuda are the most commonly reported fish species associated with ciguatera poisoning (De Fouw et al., 2001). In Florida, the majority of these cases are associated with consumption of the great barracuda, with snapper, hogfish, jack, and grouper also involved (De Fouw et al., 2001). In Hawaii, jack, black snapper and surgeonfish are most frequently involved with ciguatera toxin (De Fouw et al., 2001). In the Mascareignes archipelago, 34 fish species have been identified to be involved in ciguatera poisoning, with larger predators such as grouper (Serranidae 53%, Carangidae 10%, Lethrinidae 15%) most often implicated. An incomplete list of fish species associated with ciguatera is reported in Table 2.9. 2.4.2.6 Qualitative and Quantitative Methods for Toxins Detection The mouse bioassay (MBA; see also Section 2.3.1) test is the official procedure for the analysis of shellfish for PSP and DSP, and these have been used effectively in monitoring programs worldwide for many years. This method is straightforward, simple, and provides a quick answer whether the material under analysis is toxic, or not. In the MBA test, each mouse weighing 20 g is injected (i.p.) with 1 ml of test solution of adjusted pH and toxicity. The time of death is measured, and the toxicity in MUs is found from the standard table and corrected by factor obtained from control mice injected with the standard saxitoxin dihydrochloride solution and expressed in micrograms equivalent of saxitoxin dihydrochloride. One MU is the amount of toxin needed to kill a 20 g mouse in 15 min, which is equivalent to 5, 48, and 18 ng of Pacific CTX-1, CTX-2 and CTX-3, respectively. However, these techniques have limitations in terms of specificity and accuracy, and the use of animals for such

Northern red snapper Tarpon Narrowhead gray mullet Yellowtail snapper Spotted coral grouper Blue parrotfish Spanish mackerel Lesser amberjack Great barracuda Chinamanfish Swordfish

Lutjanus campechanus Megalops atlanticus Mugil capurri Ocyurus chrysurus Plectropomus maculatus Scarus coeruleus Scomberomorus maculatus Seriola fasciata Sphyraena barracuda Symphorus nematophorus Xiphias gladius

purposes is becoming increasingly unacceptable, for ethical reasons. Furthermore, this method is expensive and provides little specific information on the nature of the toxins. The fly bioassay was developed to substitute the mouse assay (Bagnis et al., 1987). In this method, flies are temporarily immobilized at low temperature and injected with a minute quantity of test solution, using a microsyringe. However, as the method has not been officially recognized this has resulted in the development of alternative techniques such as cell culture assays, ELISAs, HPLC, CE-MS and LCMS, although none of these has yet been validated for monitoring purposes. Alternatives to in vivo testing were developed to detect ciguatoxins using sodium channel specific cytotoxicity (Manger et al., 1993, 1995) and sodium channel receptor binding in rat brain synaptosomal preparations (Lombet et al., 1987; Lewis et al., 1991a,b; Poli et al., 1997). These assays can detect subpicogram levels of ciguatoxins in fish extracts, and provide qualitative and quantitative estimates of toxicity. Both assays can be formatted for high sample throughput (Van Dolah et al., 1994). The assay provides a composite response to all sodium channel-specific toxins in an extract, and it cannot discriminate molecular species acting in the same manner (Dickey and Plakas, 2010). The receptor-binding assay is based on binding competition between a ciguatoxin standard or a sample with tritiated brevetoxin for the sodium channel receptor. The bound and unbound toxins are separated by centrifugation or filtration, and the amount of bound radiolabeled brevetoxin is measured by liquid scintillation counting. Samples are quantitated by comparison with a standard competition curve, generated by the addition of increasing concentrations of unlabeled ciguatoxin or brevetoxin to a mixture of receptor and tritiated brevetoxin. The ciguatoxin detection limits are in the picomolar range. One disadvantage when applying receptor assays to ciguatoxin and other algal toxins is that they are technically complex, and require the use of radiolabeled materials with their attendant supply issues and licensing requirements (Dickey and Plakas, 2010). The development of immunoassays for ciguatoxins in fish tissues appear to have the best potential for meeting the utilitarian requirements of speed, simplicity and accuracy in the measurement of toxicity relative to human susceptibility.

2.5 Conclusions

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Box 2.1: Ciguatera in the Caribbean On 24th February 1995, six U.S. soldiers serving with the Multinational Force in Haiti became ill after eating a locally caught fish identified as the greater amberjack Seriola dumerili. The victims presented with nausea, vomiting, watery diarrhea and abdominal cramps 5-8 h after consumption. Also present in some victims were numbness in the extremities or perioral region, bradycardia and scalp paresthesia. Patients were treated with i.v. hydration therapy and antiemetics. All recovered without sequelae over the course of one to three months. A portion of the cooked fish was obtained for analysis. A semipurified lipid extract was prepared according to standard methods and analyzed for the presence of Naþ

The initial development of immunoassays for ciguatera began with a radioimmunoassay format using a polyclonal antibody to a partially purified ciguatoxin preparation (Hokama et al., 1977). The later monoclonal antibody assays developed for ciguatera were also reported to react with abnormal lipids in the sera of chronic fatigue syndrome, chronic ciguatera fish poisoning, hepatitis B and cancer patients, which suggested a causal link (Hokama et al., 2003a,b, 2006, 2008). More recently reported developments of mouse and chicken antibodies specific to synthetic fragments of ciguatoxin were considered to produce reliable and accurate results in the screening of fish populations for ciguatoxins (Campora et al., 2008a,b). The application of MS played a critical role in the structure elucidation of most ciguatoxin congeners recovered in trace quantities from toxic fish and Gambierdiscus. By using as templates the fragmentation patterns of those ciguatoxins obtained in sufficient quantity for NMR structure elucidation, the structures of many congeners were deduced by a combination of FAB/MS/MS and synthetic conversions to known structures (Yasumoto et al., 2000). The application of MS to ciguatoxin detection in crude extracts at the sub-ppb levels required for public health relevance was first reported by Lewis et al. (1999), who described a method for gradient reversedphase HPLC/tandem MS (HPLC/MS/MS) of P-CTX-1 and CCTX-1 in spiked and naturally incurred crude extracts of fish. Levels equivalent to 0.04 ppb P-CTX-1 and 0.10 ppb C-CTX-1 were detectable and, by using P-CTX-1 as an internal standard, the analysis of fish extracts from the Caribbean Sea suggested an estimated risk level of >0.25 ppb C-CTX-1. A rapid extraction method (CREM) optimized for the HPLC/MS/MS analysis of ciguatoxins was recently reported (Lewis et al., 2009). It was suggested that, in the assessment of toxic fish for consumer protection, a two-part protocol comprising an in vitro mouse neuroblastoma cell assay to measure toxic potency and LC-MS/MS to confirm the molecular presence of ciguatoxins, might provide the most appropriate information for decisions of public health and economic importance (Dickey and Plakas, 2010) (see Box 2.1).

channel site 5 binding activity using a brevetoxin receptor binding assay. By this assay, the fish sample contained the equivalent of approximately 20 ng Caribbean ciguatoxin per gram of flesh. The presence of the major Caribbean ciguatoxin (C-CTX-1) was confirmed by liquid chromatographymass spectrometry. Using the receptor binding assay to monitor activity in TSK and PRP-1 column fractions, two minor toxins were detected in addition to C-CTX-1. One of these minor toxins was more polar, and the other less polar, than C-CTX-1. These data provide firm evidence that a family of C-CTX-1 is responsible for ciguatera in the Caribbean (Poli et al., 1997).

2.5 Conclusions

Many marine toxins accumulate in seafood to levels sufficient to cause human illness. The major seafood toxin classes include the saxitoxins, which are responsible for paralytic shellfish poisoning, the tetrodotoxins which are responsible for puffer fish poisoning, the ciguatoxins which are responsible for ciguatera fish poisoning, as well as the palytoxins which are responsible for a range of severe fish and shellfish intoxications. Recent progresses made in analytical chemistry, in particular with hyphenated techniques such as HPLC coupled to highly sensitive mass spectrometry, have greatly improved the isolation and identification of the specific toxins involved. Most seafood toxins have proven epidemiological links to human illness, and agreed regulatory levels have been fixed to minimize the risks associated with their exposure. These levels are currently measured in seafood, in a time- and location-specific context, using a range of methods that include the traditional mouse lethality assay, molecular interaction assays using the target receptor or high-affinity antibodies, and HPLC/MS methods that can identify the molecular species involved. As a consequence of their extreme oral potency, the ciguatoxins remain the only major class of seafood toxins where action levels are not formally agreed upon and commercial catches are not routinely monitored before consumption. Marine toxins have greatly contributed to the life sciences as biochemical or pharmacological tools. Both, STX and TTX have helped to define the structure and function of voltage-gated sodium channels. In contrast, toxins such as palytoxin and maitotoxin exhibit a wider range of physiological effects which are not yet fully understood. Dinoflagellates are the main producers of marine toxins, with biosynthetic pathways now beginning to be resolved at the molecular level. Today, seafood intoxications are only occasionally fatal due to the implementation of effective management strategies. However, research devoted to the development of effective monitoring methods are required to minimize public exposure to ciguatoxins and palytoxins.

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2 Outstanding Marine Biotoxins: STX, TTX, and CTX

About the Authors Philippe Amade, PhD, spent most of his career working for INSERM and leads a group working on Marine Natural Products Chemistry at Nice University (UMR7272 CNRS), France. Most of his works were dedicated to marine invertebrates: mainly sponges, algae (Caulerpa), and microalgae (Gambierdiscus); but also to ciguatera and folk remedies (three-yearprogram in New Caledonia). He organized expeditions in several coral reef regions of the word to collect marine invertebrates by scuba diving. Dr Amade has authored about 50 scientific publications and is a reviewer of several scientific journals. Mohamed Mehiri is assistant professor at the Chemistry Institute of Nice, France. After a post-doc experience at Lehight University under the supervision of Dr Steven Regen, he was hired at the University of Nice-Sophia Antipolis in 2008 in the

Marine Natural Products group with Dr Philippe Amade. His research interests encompass the isolation and structural elucidation of bioactive secondary metabolites from marine invertebrates, mostly sponges, and the synthesis of interesting chemical structures initially produced by selected sponges. Richard J. Lewis is a group leader and Professor at the Institute for Molecular Biosciences at the University of Queensland (UQ). After completing his PhD on ciguatera at UQ, he continued this research with the Queensland Department of Primary Industries before moving back to UQ to expand his research interests to include the discovery and molecular pharmacology of conotoxins. This research resulted in the discovery of two new classes of allosteric inhibitor peptides, including the x-conopeptides being developed for the treatment of pain by Xenome Ltd.

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs Marieke Vansteelandt, Catherine Roullier, Elodie Blanchet, Yann Guitton, Yves-FranSc ois Pouchus, Nicolas Ruiz, and Olivier Grovel

Abstract

Since the discovery of penicillin, Penicillium has become one of the most well-known genera of fungi for the discovery of bioactive compounds. The genus Penicillium is ubiquitous and widespread in all environments, and especially in marine coastal zones where it represents half of the fungal species present. In this chapter, attention is focused on the importance

3.1 Introduction

Over the past five decades, marine organisms have been increasingly investigated for new potential bioactive compounds (Blunt et al., 2011; Montaser and Luesch, 2011). Among this still largely unexplored resource, microorganisms – including fungi to a significant degree – appear to be prolific producers of secondary metabolites (Bhatnagar and Kim, 2010). Among these fungi, the genus Penicillium is one of the most widespread fungal genera in the marine environment. Since terrestrial Penicillia are known to produce numerous biologically active compounds,

of marine-derived Penicillium strains in the discovery of potential new antitumor drugs. A detailed review of potent cytotoxic compounds isolated from marine-derived Penicillium spp. is presented. Three examples are then provided to yield some insight into the chemistry of these compounds, and to help assess the chemical diversity that marine-derived Penicillium spp. are capable of producing.

their abundance in the marine environment represents a rich promising source of novel agents with potential activity (Kozlovskii, Zhelifonova, and Antipova, 2013). It is worth noting that so far, Penicillium species have already led to the isolation of some major clinical drugs. It is impossible, talking about Penicillium, not to cite penicillin, which has completely changed the physicians’ therapeutic armamentarium, with the advent of antibiotics and the eradication of several human diseases caused by pathogenic bacteria. The discovery of penicillin is attributed to Sir Alexander Fleming who, in 1929, described the effects of penicillin produced by Penicillium notatum on bacteria

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

(Fleming, 1929), and to Howard Florey and Ernst Chain who subsequently isolated the compound in 1939. In fact, mycophenolic acid – another fungal metabolite isolated from Penicillium brevicompactum in 1893 (Gosio and Ferrati, 1893) – is considered to be the first-described antibiotic of microbial origin. Mycophenolic acid presents a wide variety of activities, and is now mainly used in therapeutics for its immunosuppressive properties, in order to avoid rejection crises after organ transplant surgery. Last, but not least, a similar fungal metabolite was simultaneously isolated from Penicillium brevicompactum and Penicillium citrinum by two independent research groups in 1976 in UK and Japan, respectively (Brown et al., 1976; Endo, Kuroda, and Tsujita, 1976). This molecule, which was named either compactin or mevastatin, became the lead compound of the statins, a class of drugs that are potent antagonists of cholesterol biosynthesis and are used in the prevention of coronary diseases. Apart from these approved drugs, many other compounds have been isolated from Penicillium species and evaluated for their activities, particularly those of an antitumoral nature (Nicoletti et al., 2008). Today, cancer remains a major issue for human health and new anticancer therapies continue to be needed. With this point in mind, a review is provided here of the potential antitumor compounds that have been isolated from marine-derived Penicillium species. The details of only 135 compounds that have presented highly cytotoxic, cytostatic or antiproliferative properties on mammalian cells are provided here. The in-vivo toxicity data for some cases are included, but all data relating to toxicity on insects, on microorganisms such as viruses, bacteria or fungi, and also on plant cells have been discarded. Then, through three representative examples, this chapter gives some insight into the chemistry of such compounds and leads to assess the chemical diversity, which marine-derived Penicillium spp. are able to produce.

3.2 Molecules Isolated from Marine-Derived Penicillium Species With Potent Cytotoxic Activity

A review of the literature led to the construction of a table presenting all potent cytotoxic compounds isolated from marine-derived Penicillium species (Table 3.1). This table includes compounds that proved to be cytotoxic against mammalian cell lines and demonstrated a half-maximal inhibitory concentration (IC50) of less than 30 mM. Any compounds for which only moderate or low cytotoxicity were reported were not included; indeed, only those molecules with a high or potent activity were retained in order that attention could be focused on the most promising leads for anticancer drugs. In order to emphasize the impact of a marine environment on the production of new or even novel natural products, those compounds which were first isolated from a marine-derived Penicillium are printed in bold text.

3.3 Marine-Derived Cytotoxic Penicillium 3.3.1 Where Were Marine-Derived Penicillium Searched and Isolated?

Most articles relating to the isolation of cytotoxic molecules from marine strains of Penicillium described the nature of the samples from which the fungal strains were isolated. The origins were various (Figure 3.1); sea water did not appear to be a good feature for the isolation of cytotoxic Penicillium, as very few articles reported this environment as the source for strains of interest. In contrast, marine sediments – which corresponded to the origin of one-third of the strains studied – appeared to be favorable to the isolation of cytotoxic Penicillium strains. Together, the plant and animal origins represented half of the samples, thus highlighting the interest of these origins in the identification of cytotoxic compounds. 3.3.2 Which Penicillium Species?

The identification of fungi belonging to the genus Penicillium is difficult, even with the assistance of molecular biology, which explains why many articles describing the isolation of cytotoxic metabolites from marine-derived Penicillium strains do not provide the name of the species studied; typically, it is described as a simple “Penicillium sp.,” which is not informative. When considering only those identified species that produce cytotoxic compounds (Table 3.2), 26 were cited. Two of these, identified as P. purpurogenum and P. rugulosum, are now considered as belonging to the genus Talaromyces (teleomorphic forms of various Penicillium spp.). In 2011, Houbraken and Samson described a new phylogeny for the genus Penicillium which is now divided into two subgenera Aspergilloides and Penicillium and into 25 clades (sections) – 14 for Aspergilloides and 11 for Penicillium (Houbraken and Samson, 2011). The repartition of cited species between these subgenera and sections does not allow a peculiar section which could be particularly bound to the marine environment to be distinguished. In fact, some species such as P. citrinum, P. crustosum (¼ P. terrestre), P. expansum, P. chrysogenum and P. mononematosum seem to be widely spread among the marine environment, and have been isolated and studied by different research groups at different times.

Figure 3.1 Repartition of origins of cytotoxic marine-derived Penicillium strains.

3.4 What are these Promising Molecules from Marine Penicillium?

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Table 3.2 Identified species of marine-derived Penicillium described to produce cytotoxic metabolites.

Subgenus

Section

Species

Reference

Aspergilloides

Aspecrgilloides

P. thomii Maire P. flavidorsum ¼ P. glabrum Westling P. fellutanum Biourge P. bilaiae Chalab. P. obscurum ¼ P. corylophilum Dierckx P. janthinellum Biourge

Chen et al., 2007 Ren, Gu, and Cui, 2007 Shigemori et al., 1991 Capon et al., 2007 Gautschi et al., 2004 Smetanina et al., 2007

P. oxalicum Currie and Thom P. simplicissimum (Oudem.) Thom P. citrinum Thom

Liu et al., 2007; Sun et al., 2012a Pivkin et al., 2011 Tsuda et al., 2004; Sasaki et al., 2005; Chen et al., 2011; Khamthong et al., 2012; Yurchenko et al., 2013 Kossuga et al., 2012 Amagata, Minoura, and Numata, 1998 Xin et al., 2005; Song et al., 2012 Gao et al., 2011; Shang et al., 2012 Indriani, 2008 Liu et al., 2005a; Liu et al., 2005b; Chen et al., 2008; Li et al., 2011b Kerzaon et al., 2009; Lu et al., 2010; Wang et al., 2012 Li et al., 2011a Bringmann et al., 2003; Bringmann et al., 2005; Ma, H. et al., 2011; Ma, C. et al., 2011 Cui et al., 1996; Numata et al., 1991; Frisvad et al., 2004 Bringmann et al., 2004 Ma et al., 2012 He et al., 2005 Liu et al., 2012 Xin et al., 2005

Charlesii Sclerotoria Exilicaulis Lanatadivaricata

Citrina

Penicillium

Not attributed Genus Talaromyces

Fasciculata

P. paxilli Bainier P. waksmanii Zaleski P. aurantiogriseum Dierckx P. commune Raper and Thom P. polonicum Zaleski P. terrestre ¼ P. crustosom Thom

Penicillium Roquefortorum Chrysogena

P. expansum Link P. paneum Frisvad P. chrysogenum Thom

Brevicompacta Ramosa Canescentia Eladia

P. mononematosum Frisvad P. brevicompactum Dierckx P. raistrickii Smith P. janczewskii Zaleski P. sacculum Dale P. fructigenum Takeuchi P. purpurogenum ¼ Talaromyces purpurogenus (Stoll) Samson, Yilmaz, Frisvad and Seifert P. rugulosum ¼ Talaromyces rugulosus (Thom) Samson, Yilmaz, Frisvad and Seifert

These species could then be considered as being particularly acclimatized to the marine environment. 3.4 What are these Promising Molecules from Marine Penicillium?

Chai et al., 2011; Chai et al., 2012; Fang et al., 2012 Kozlovsky et al., 2001

against cancer cell lines and being isolated from marine-derived Penicillium species, they were mapped according to their chemical similarity. By using 49 basic molecular and topological descriptors such as LogP and the Wiener index, a principal component analysis (PCA) was performed without any filter (Figure 3.3).

3.4.1 Statistics

Among the different compounds that have been isolated from marine-derived Penicillium strains, and which present interesting bioactivities against mammalian cancer cell lines, many diverse classes corresponding to different biosynthetic pathways, are represented. As shown in Figure 3.2, the three major natural products classes that are observed are polyketides, alkaloids, and terpenes. This is not surprising as Penicillium are rich producers of this type of compound. It is worth noting that cytotoxic compounds are not exclusively found in one or two classes of natural products. However, one class failed to show a sufficiently high activity towards cancer cell lines and so was not included in the selection, namely peptides. In order to better assess the chemical diversity of the 135 molecules described in this chapter as having potent activity

Figure 3.2 Natural products class distribution of the potent antiproliferative compounds isolated from marine-derived Penicillium strains.

3.4 What are these Promising Molecules from Marine Penicillium?

It appeared that compounds from the same class grouped quite well together, such as steroids or diketopiperazines compounds. The first axis explains more than 40% of the variations between molecules, and seems to be strongly correlated to the molecular complexity, the number of rings, and the number of atoms. The second axis, which explains around 9% of chemical variation between molecules, seems to be partly related to LogP, as steroids group together and are opposed to hydroxylated compounds such as terrestrols. This graphical representation of the 135 compounds reviewed in this chapter, allows many other observations. First, many of the polyketides described are in fact monocyclic compounds. In contrast, among the alkaloids many structures appear to be quite complex, such as penochalasins or even verticillin analogs or shearinine E. With regards to penochalasins, two groups are represented as one group corresponds more specifically to penochalasins with an epoxide group. Similarly, although less obvious, terrestrols also seem to divide into two groups, which correspond to chlorinated and nonchlorinated molecules. Among polyketides, one group of compounds is rather far from the others, and corresponds to the very complex trichodimerol derivatives, which were first isolated from Trichoderma fungi. One interesting point concerns roquefortine F, meleagrin and fuctigenine A, which were mapped very close; this finding is relevant as the meleagrin group and the roquefortine group are biogenetically interrelated alkaloids (Figure 3.4). Effectively, roquefortine was reported to be the biosynthetic precursor of oxalin (methoxylated meleagrin) (Steyn and Vleggaar, 1983) and meleagrin (Reshetilova et al., 1995). More recently, Martin and coworkers also reported that a single gene cluster of Penicillium NH

NH N

N O

O H 3CO HN N

H 3CO HN N

N

N O

O

OCH 3

OH N

Meleagrin OCH3 H N

Oxalin

HN O N

NH H

O

Roquefortine C O N

O

H N

NH H

O

Fructigenine A Figure 3.4 Structures of meleagrin, oxalin, roquefortine C, and fructigenine A.

j 59

chrysogenum was involved in the biosynthesis of both roquefortine C and meleagrin (Garcia-Estrada et al., 2011). However, to the best of our knowledge, no studies have revealed any correlation between roquefortine and fructigenine biosynthesis, while their scaffold are really similar. The molecular diversity of the different bioactive compounds, as described here, appears to be quite important. Very different scaffolds are represented, and many of these were first discovered in a marine-derived Penicillium species, which highlights the importance of investigating the marine environment in order to identify more chemical diversity and more potential promising new drugs.

3.4.2 Focus on Interesting Molecules

Some of the molecules in Table 3.1 display very potent activity, and show much promise for potential antitumor drug development. Others show original chemistry, especially novel natural products with very unique carbon skeleton. Based on these criteria, some compounds from the selection of 135 were chosen to be thereafter reviewed in detail. 3.4.2.1 Cytotoxic Alkaloids: The Example of Communesins Among the potent cytotoxic metabolites isolated from Penicillium species, original heptacyclic scaffolds sharing indolic and quinoline moieties were found in the communesin alkaloids. These are very interesting molecules to discuss, with regards not only to their bioactivity but also to their chemical structure and biosynthesis. Indeed, this is a good example of Nature’s outstanding skill at producing complexity and originality in natural products, thereby trespassing the human imagination and ability for organic synthesis. 3.4.2.1.1 When Research Groups Isolate the Same Compound The story began in 1993, when a Japanese research group described active materials from a strain of Penicillium sp. which had been isolated from the marine alga Enteromorpha intestinalis. Two novel metabolites were identified as communesin A and B (Numata et al., 1993), both of which were reported to be cytotoxic against murine leukemia P388 cells with half-maximal effective dose (ED50) values of 7.7 mM and 0.9 mM, respectively. Many years later, two other research teams claimed they had in fact isolated those molecules at the same time. Effectively, in the Pfizer laboratories, structurally related compounds were identified during the same period and were named “commindolines,” in relation to their initially characterized producing organism P. commune and their indolic moiety. It appeared that the structures of the principal compounds, “commindolines” B and A, equated to that of communesins A and B, respectively (Wigley, 1996; Wigley, Mantle, and Perry, 2006). The actual producing organism of “commindolines” was further identified as being P. marinum (Wigley, Perry, and Mantle, 2008). Concurrently, it was reported that another study undertaken at the UK Central Science Laboratory in Slough had also led to the isolation of compounds that corresponded to communesins A and B from P. buchwaldii at

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

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H3C

O

CH3

O H N

H3C

N H3C

6

N

N H

Communesin B

O

H3C

CH3

O H N

H3C

N

6

N

O

H3C « Nomofungin »

Figure 3.5 Described molecular structures of communesin B and “nomofungin.”

the same time. Moreover, these compounds appeared to be toxic to brine shrimps (Scudamore and Hetmanski, personal communication in Wigley, Perry, and Mantle, 2008). The explanation as to why these research teams described, more than 10 years later, their isolation of the same compounds as Numata and colleagues, relates to a whole body of investigations carried out on the structural identification of these molecules since 2001, and the discovery of “nomofungin.” Indeed, Ratnayake and colleagues described in 2001 the isolation from an unidentified fungus of a new molecule, nomofungin. As the original strain of the fungus had been lost during successive subcultures, the name of the molecule then referred to “no more fungus” (Ratnayake et al., 2001). The nomofungin and communesin B structures were almost similar, differing by only one oxygen, which replaced a nitrogen atom (Figure 3.5). This original heptacyclic structure, which included two vicinal asymmetric carbons and one intracyclic oxygen atom, caught the attention of many synthetic chemists. Stoltz and coworkers were the first to be intrigued by the “nomofungin” structure as, contrary to communesin B, they could not propose a reasonable biogenetic pathway that would lead to this molecule. Moreover, the chemical shifts for the proton and carbon in position 6 were more in favor of a diaminal CH group. In a biomimetic approach towards communesin B, intermediates were obtained with the diaminal C-6 as a tetracyclic diamine residue. Subsequent NMR examinations of these compounds led to the suggestion that nomofungin and communesin were in fact the same molecule, and that communesin B was the appropriate structure (May, Zeidan, and Stoltz, 2003). Independently, other synthetic chemists had at the same time obtained a nomofungin analog as a hexacyclic N,O-acetal residue, and showed a serious discrepancy between chemical shifts for the CH group in position 6. They then confirmed that the structure of nomofungin was incorrect and that communesin B was the actual structure (Crawley and Funk, 2003). Subsequently to these studies, the report describing “nomofungin” was withdrawn from the literature and the structure of communesin B was validated as the most consistent. Despite this structural error – which can be explained by poor mass spectrometry data – studies on “nomofungin” allowed the absolute configuration of the molecule to be established. Cytotoxicity against different cell lines and potential activity on the microfilament network of mammalian cells, which had not been previously reported, were also highlighted (Ratnayake et al., 2001). Last but not least, six new communesins were isolated from different strains of Penicillium sp. First, in 2004, communesin B

was re-isolated from another Penicillium sp. from a Mediterranean sponge Axinella verrucosa, along with two new comunesins C and D (Jadulco et al., 2004), again revealing the importance of the marine environment in discovering these compounds. The three molecules were then further tested against different human leukemia cell lines, including U937, THP-1, NAMALWA, MOLT3 and SUP-B15, and showed activities with ED50-values ranging from 14 to 31 mM. Independently, while communesins B, C and D were being isolated, another research team isolated the same compound – namely communesin D – along with two new communesins, E and F, from a Penicillium expansum that had been obtained from a soil sample collected in Osaka (Hayashi, Matsumoto, and Akiyama, 2004); however, in the latter report the communesins D, E and F were named C, D and E. Four other reported communesins, named F to H, have been isolated from soil and from the arctic region, but were not reported as cytotoxic (Dalsgaard et al., 2005; Hayashi, Matsumoto, and Akiyama, 2004). The novelty and structural complexity of these indole alkaloids, and their activity on cancer cells, have generated intense interest from synthetic chemists, leading to the construction of hexacyclic substructures via different synthetic routes (Crawley and Funk, 2003; Crawley and Funk, 2006; Seo, Artman, and Weinreb, 2006; Yang et al., 2006). In 2007, a total synthesis of communesin F, requiring 23 steps with a yield of 3%, was finally performed (Yang et al., 2007). More recently, investigations on a marine Penicillium expansun strain allowed the identification of communesins A, B, D and E, along with seven new derivatives, which clearly displayed communesin-characteristic features as demonstrated with mass spectrometry experiments (Kerzaon et al., 2009). The MS data showed that communesins with their original skeleton may have more representatives than the currently recognized eight molecules, opening a route to the discovery of an extended structural diversity. 3.4.2.1.2 Production, Extraction, Separation, and Purification of Communesins To date, the communesins seem to be specific to Penicillium as they have only been isolated from this fungal genus, and communesins A and B are now considered as characteristic metabolites of the species P. expansum and P. marinum (Frisvad et al., 2004). It seems that their production is fairly widespread in the species P. expansum, and on different substrates. A study of 260 isolates of various terrestrial origin showed a constant production of communesins from this species.

3.4 What are these Promising Molecules from Marine Penicillium?

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Table 3.3 Description of the production and purification methods used for isolation of communesins.

Species

Origin

Culture medium

Culture conditions

Extraction solvent

Purification

Isolated communesins

References

Penicillium sp.

Alga Enteromorpha intestinalis

Liquid, artificial sea water, 2% glucose, 1% peptone, 0.5% yeast extract, pH 7.5

3 wk, 27  C

MeOH

A, B

Numata et al., 1993

Unidentified

Ficus microcarpa

Liquid: deionized water, potato dextrose extract

3 wk, r. t., dark

EtOAc

B

Ratnayake et al., 2001

P. expansum

Soil

Acetone extract of okara (insoluble residue of whole soybean)

A, B, D, E, F

Penicillium sp.

Sponge Axinella verrucosa

Hayashi, Matsumoto, and Akiyama, 2004 Jadulco et al., 2004

P. rivulum

Arctic

P. marinum

Soil

P. expansum

Marine sediment

Liquid: yeast extract (3 g l 1), malt extract (3 g l 1), peptone (5 g l 1), glucose (10 g l 1), and sea salt (24.4 g l 1), pH 7.2–7.4 Solid: covered by CYA: saccharose, Czapeck yeast, KH2PO4 Liquid: Soluble starch 7.5 g l 1, Trusoy flour 7.5 g l 1, NaCl 2 g l 1, yeast extract 0.05 g l 1, Pharmamedia 2 g l 1, pH 6.5 Solid: Dextrose Casein Agar prepared with sea water

Sephadex LH 20, Si chromatog., RP HPLC Repeated normal phase chromatog. Si column chromatogr, flash chromatog., RP HPLC Sephadex, Si column

EtOAc

3 wk, r. t., dark

EtOAc

19 d, 20  C, dark 4–6 d, 27  C, agitation

EtOAc

11 d, 27  C

P. buchwaldii

MeOH EtOAc

EtOAc/ CH2Cl2 (1 : 1)

HSCCC, RP HPLC RP HPLC

Vacuum liquid chromatog., RP HPLC

B, C, D

G, H A, B

Dalsgaard et al., 2005 Wigley, Mantle, and Perry, 2006

A, B, D, E, F 7 analogsa)

Kerzaon et al., 2009

A, B

not publishedb)

a) Detected using HRMS analysis. b) Personal communication from Wigley, Perry, and Mantle, 2008.

Fungal secondary metabolism is considered to be highly dependent on the conditions used to develop the microorganisms. The temperature, nature and composition of the substrate, and the solid or liquid culture medium are each important parameters that may influence the biosynthetic pathways implemented during the growth of microorganisms (Turner, 1971). Based on current knowledge, the production of communesins does not appear to need any specific requirements during culture, as the molecules were isolated from strains grown in various conditions (Table 3.3). However, the eight communesins have not all been isolated from the same strain, which suggests that culture conditions could influence the type of communesins to be produced, or that some specificity exists between species. As an example, communesins G and H were only isolated from P. rivulum, but this strain did not produce the other derivatives (Dalsgaard et al., 2005). In contrast, other molecules have been identified in association with communesin B, regardless of species (Hayashi, Matsumoto, and Akiyama, 2004; Jadulco et al., 2004; Numata et al., 1993). During studies on the biosynthesis of communesins A and B, a preliminary investigation into the growth and accumulation of communesins in submerged culture provided some insight into communesins production. Under the conditions used (27  C,

agitated liquid medium, pH 6.5), the production and accumulation of communesins A and B began after 20 h of incubation during the fungal growth phase, and continued until 200 h, despite growth having peaked after 70 h and then declined. The production curve perfectly matches the theoretical curve for biosynthesis of the secondary metabolites (Tortora, Funke, and Case, 2003). An independent analysis of the culture medium and mycelium also suggested that the excretion of communesin B into the medium would be disadvantaged compared to communesin A, because of its long side chain (R1) (Wigley, Mantle, and Perry, 2006). The extraction of communesins usually involves ethyl acetate (or occasionally methanol), which indicates that these molecules present an intermediate polarity. In order to purify communesins, a variety of common techniques were employed, including silica gel chromatography or reverse-phase HPLC. The latter method appears to be the most frequently used for the final purification of these compounds (Table 3.3). 3.4.2.1.3 Physico-Chemical Properties of Communesins As noted above, communesins consist of a heptacyclic skeleton that contains a heterocycle-fused indoline core. Other characteristics of these natural products include the vicinal quaternary centers at C-7 and C-8, and two diaminal linkages at C-6 and C-9 (Table 3.4).

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

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Table 3.4 Structures and chemical properties of communesins.

R1

O H

R3

10

N

11

1''

N 16

9

12a 7a

13

14a 15 1'

N

R2

1

2

8 8a

7 14

18

19

N H

=

3

5 6

4a

R1

R3

17

20

12

R2

4

Molecular weight (g mol 1)

Molecular formula

Communesin A

456.6

Communesin B

Compound

Chemical properties

References

Boiling point ( C)

UV (MeOH) lmax (nm)

C28H32N4O2

194–196

204; 250; 268

Numata et al., 1993

508.7

C32H36N4O2

152–154

204; 272

Numata et al., 1993 Nielsen and Smedsgaard, 2003

Communesin C

494.6

C31H34N4O2

ND

206; 271

Jadulco et al., 2004

Communesin D

522.6

C32H34N4O3

190–195

206; 267

Hayashi, Matsumoto, and Akiyama, 2004 Jadulco et al., 2004

Communesin E

442.6

C27H30N4O2

250

243

Hayashi, Matsumoto, and Akiyama, 2004

Communesin F

440.6

C28H34N2O

144–147

268

Hayashi, Matsumoto, and Akiyama, 2004

Communesin G

470.6

C29H34N4O2

162–166

208; 248; 268; 316

Dalsgaard et al., 2005

Communesin H

484.6

C30H36N4O2

143–147

208; 248; 268; 316

Dalsgaard et al., 2005

3.4 What are these Promising Molecules from Marine Penicillium?

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All together, the eight reported communesin compounds differ only by the acyl group at position 100 (R1 substituent), the 15-N group (R2 substituent), and by the presence or absence of an epoxide between C-21 and C-22 (R3 substituent). Communesin F is the only molecule to have a dimethyl-vinyl group instead of a dimethyl epoxide on R3. Mass Spectrometry (MS) Analysis by MS shows that communesins with a dimethyl-epoxide group in R3 display a characteristic fragment corresponding to the ionic species [M þ H 72]þ resulting from a loss of the dimethyl-epoxide (Figure 3.6). This fragmentation was first observed for communesins B, C and D when using an electrospray ionization (ESI) source (Jadulco et al., 2004). A thorough examination of the mass data produced for communesins A–E also highlighted a similar fragmentation by electronic impact mass spectrometry (EI-MS), with the loss of 71 units of mass (uma) (Hayashi, Matsumoto, and Akiyama, 2004; Jadulco et al., 2004). In 2009, a detailed interpretation of communesins MS/MS spectra provided a better understanding of their fragmentation patterns (Kerzaon et al., 2009). The latter authors confirmed the characteristic 72 uma loss observed by ESI-MS for the dimethyl epoxide-containing communesins, and further identified other characteristic fragments. A general fragmentation pattern and a predictive model for substituent determination were then proposed, as shown schematically in Figure 3.7. NMR Spectroscopy 1 H NMR spectroscopy on communesin compounds provides many signals, with seven protons in the aromatic zone, and four additional signals for communesin B, C and D, corresponding to the unsaturated chain on R1 (Table 3.5). Protons in positions 12 and 14 (shown as orange circles in Figure 3.8) are typically more shielded than the others, and appear quite clearly as doublets, their coupling constant being in the range 7.5–8. The signals for the other aromatic protons often overlap. Other remarkable signals are the singlets for the methyl groups in positions 10 (blue), 23, and 24 (red). Additionally, the

Figure 3.7 Simplified fragmentation pattern according to Kerzaon et al., 2009.

Inten.(x10,000,000) (a) 457.2620(1) fragmentation

2.0 1.0 110.0214(2)

0.0 100

174.1296

306.2652

200

385.2032(1)

300

487.2367(1)

400

500

600

700

800

900

m/z

Inten.(x10,000,000) 7.5

(b) 385.2042(1)

5.0

2.5

0.0 100

185.1077(1) 168.0826(1) 201.1019

150

200

240.1395(1)

250

400.2180 439.2547(1)

309.1392(1)

300

350

400

450

m/z

Figure 3.6 Characteristic ESI-mass spectrum of a communesin; example of communesin A. The MS spectrum of communesin A obtained in the positive mode (a) shows a major ion at m/z 457, which by fragmentation leads to different ions (b) including an ion at m/z 385. (These spectra were recorded on an ion trap-time of flight mass spectrometer apparatus).

66

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

O

O

CDCl3

N

N

1.0

2.34

0.8

CH3 CH3

CH3

1.39

N H

N

0.7

1.54

0.6

Ar-CH (doublets)

0.89

1.52 1.37 1.29 1.26

2.37 2.36 2.30 2.10 2.00 1.99 1.98

4.71

N-CH-N

4.59 4.10 4.08 3.92 3.90 3.89 3.88 3.47 3.45 3.38 3.35 3.03 3.01 2.89 2.87 2.82

0.1

7.02 7.02

0.2

N-CH-N 5.03

0.3

6.08 6.06 5.97 5.95

0.4

CH3

1.60

0.5

6.89 6.88 6.70 6.69

Normalized Intensity

2.85

0.9

0 1.01 0.97 2.91

7.0

6.5

0.99 0.88

6.0

0.94 0.94

5.5

5.0

0.90 0.890.93 1.10 1.02 0.98 1.03 2.61 1.06 3.82 1.04 0.95 3.23 3.12

4.5 4.0 3.5 Chemical Shift (ppm)

3.0

2.5

2.0

1.5

1.0

Figure 3.8 Characteristic 1H NMR spectrum of a communesin; example of communesin A (CDCl3, 500 MHz).

terminal methyl group on the R1 substituent (green) appears as a singlet around 2.35 ppm for communesins A, E and F, but forms a doublet around 1.85 ppm for communesins B, C and D, and a triplet around 1.00–1.20 ppm for communesins G and H. With regards to the two diaminal CH at positions 6 (brown) and 9 (purple), their chemical shifts are quite constant among communesins, namely around 4.70 ppm for H-6 and 5.00 ppm for H-9, with some differences for communesins D and E, where the chemical shifts are slightly deshielded (Figure 3.8). One interesting feature in communesins molecules is the occurrence of different rotamers, which have been described for communesins D and F. This occurs when the molecule adopts privileged configurations on rotatable bonds, where there should be free rotation. In the case of communesin D, this phenomenon was explained by the different privileged configurations that the R2 substituent can adopt, allowing 10 -H to correlate with 14-H by nuclear Overhauser effects (NOEs) in one case, and with 6-H in the other case. For communesin F, the R1 substituent was more incriminated with the observation of 200 -H NOEs correlations, either with 9-H or with 17-H. 3.4.2.1.4 Potential Activities Against Cancer Cells Communesins A and B were originally isolated during the screening of marine microorganisms for cytotoxic activity in P388 cell cultures (cells from a mouse lymphoma collected from Mus musculus) (Numata et al., 1993). Cell-based assays performed showed that these communesins have interesting cytotoxic activities, with communesin B being the most active among the different tests.

To date, no mechanism of action has been described for the cytotoxic activity of these molecules. However, the action of nomofungin (communesin B) on the cytoskeleton was assessed using indirect immunofluorescence microscopy on mammalian A-10 cells (rat smooth muscle cells). The results indicated a disruption of the cell microfilaments that could explain the cytotoxic activity observed in different cell lines (Ratnayake et al., 2001). 3.4.2.1.5 Biosynthesis Three main biosynthetic pathways have been proposed to create the hexacyclic structure. In a first report (Bugni et al., 2003), an oxidative coupling between a N-methylated aurantioclavine and an oxidized tryptamine derivative was envisioned, but this proposal was slightly modified at a later stage (see Figure 3.9a) (May and Stoltz, 2006). In this pathway, methylation of the indolic nitrogen atom would occur after construction of the hexacyclic structure, together with epoxidation and the acetylation of R1. In a second series of modeling studies, a new biosynthetic approach was proposed based on the biosynthesis of calycanthaceous alkaloids. The communesin skeleton would then be derived from the dimerization of two tryptamines (Figure 3.9b and c), but the N-methylation remained unclear. Subsequently, this was suggested by Wigley et al. (2006) to be an early step before cyclization, arising from S-adenosyl methionine (Figure 3.9b), before Siengalewicz et al. (2008) proposed an alternative sequence (Figure 3.9c). The use of radiolabeled compounds demonstrated the need for tryptophan, methionine, mevalonate, tryptamine and acetate in the biosynthesis of communesins A and B (Wigley et al., 2006). The addition of 6-fluorotryptophan in the culture medium of a

3.4 What are these Promising Molecules from Marine Penicillium?

CO2H

CO2H

NH2

j 67

NH2

NH2

NH2 N H

N H

N H

N H

dimerase

H N

H2N

+

N H

H3CHN

dimerase

H2N

N

N

O N

N

NHCH3

N

NH2

aurantioclavine methylase H N H NH O

HN

H H H N N

HN

N

N H

N

N H

N H

Me

H2N

N

prenylation acetylation [O] H

N

NH2

H N

N

O

R

H N

N H

NH2

O N

H N

H N

N H

methylation epoxydation acetylation

N Me

N H

N H

N H

prenylation O

R

H N

N Me

N

H N

N

N H

N H

N H

Me

H N

N Me

O N

N H

communesin F

(a)

(b)

(c)

Figure 3.9 Communesin biosynthetic pathways: the three hypotheses. (a) May and Stoltz (2006); Wigley, Perry, and Mantle (2008); (b) Wigley, Mantle, and Perry (2006); (c) Siengalewicz, Gaich, and Mulzer (2008).

68

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

Penicillium sp. led to the evidence of an incorporation of only one indole moiety; however, by using 13C-analogs it was shown that two tryptophans were necessary to form the skeleton. Furthermore, the selection of P. marinum mutants, which presented a deficient tryptophan decarboxylase activity, showed that tryptamine is not necessary for communesin production (Wigley, Perry, and Mantle, 2008). The results of these studies tended to confirm that the aurantioclavine pathway would be the most likely, and this proposal was supported by the combined observations of aurantioclavine and communesins A and B in P. marinum culture extracts. However, others suggested that a such common occurrence in all communesin-producing fungi would need to be established (Frisvad et al., 2004; Wigley, Perry, and Mantle, 2008). Nevertheless, details on the final steps leading to complete communesins have not been described in any model, notably of the final acylation and epoxidation of the putative dimethylvinyl precursors of communesins A and B (Figure 3.9a). 3.4.3 Cytotoxic Alkaloids/Diketopiperazine Compounds: Examples of Fructigenine A and Verticillin Derivatives

Among the cytotoxic metabolites isolated from Penicillium species, some share a common feature, namely a hybrid diketopiperazine-indolic scaffold as fructigenine A and verticillin analogs. 3.4.3.1 Fructigenine A (¼ Rugulosovin B ¼ Puberulin) Fructigenine A (Figure 3.10) was first described in 1989 from P. fructigenum Takeuchi (Arai et al., 1989). Subsequent studies led Russian research teams to isolate the same compound again, which they named after the corresponding producing strains, namely puberulin isolated from Penicillium puberulum (Solov’eva et al., 1992) and rugulosuvin B isolated from Penicillium rugulosum (Kozlovsky et al., 2001). This molecule was reported to inhibit mouse lymphoma L-5178y cell proliferation (Arai et al., 1989), and was later described also as being cytotoxic against mouse carcinoma cells tsFT210 (Xin et al., 2005), murine fibrosarcoma L929, as well as human cervical tumor HeLa cells and human erythroleukemia K562 cells, with moderate cytotoxicity (Chai et al., 2012; Kozlovsky et al., 2001). The first total synthesis of fructigenine A, which is a challenging structure, was achieved in 2010 by Kawasaki and coworkers, in 14 steps with 11.9% yield from the 1-acetylindolin3-one (Takiguchi et al., 2010). This should allow further biological evaluation of the compound. Fructigenine A seems to be quite ubiquitous among Penicillium species, as it was isolated from many different species such O N

O

H N

NH H

O

Figure 3.10 Structure of fructigenine A.

Cl H 3CO OH

N

HO O

O S

S

H 3CO

CH3

O

N

O S S

Gliotoxin

O

N NS

N CH3

CH3

HO

HO

O HN H

N

OH

Sporidesmin

OH

S

O

O

Epicorazine

Figure 3.11 Some representatives of epipolythiodioxopiperazines (ETPs).

as P. rugulosum (Kozlovsky et al., 2001), P. puberulum (Solov’eva et al., 1992), P. fructigenum (Arai et al., 1989), P. aurantiogriseum (Xin et al., 2005), P. purpurogenum (Bu, Cui, and Li, 2010; Chai et al., 2011; Chai et al., 2012), P. chrysogenum, and P. expansum (Kozlovskii et al., 2002), of either terrestrial or marine origin. 3.4.3.2 Verticillin A and Derivatives Turning to the sea, marine-derived Penicillium species led to the isolation of other similar compounds, two of which were very potent antitumor agents, namely 11,110 -dideoxyverticillin A and 110 -deoxyverticillin A (Son et al., 1999). These epidithiodioxopiperazine molecules, which were first isolated from a Penicillium sp. obtained from the Caribbean green alga Avrainvillea longicaulis, exhibited very potent cytotoxicity against human colon carcinoma cells HCT-116, with an IC50-value of about 45 nM. This reported IC50 corresponded to the highest cytotoxic or cytostatic activity observed in Table 3.1, which means that compounds isolated from marine-derived Penicillium strains have the best activity against cancer cell lines. The verticillins and their derivatives belong to the family of dimeric epipolythiodioxopiperazines (ETPs), among the larger class of ETPs, which are well known for their toxic fungal secondary metabolites such as gliotoxin or sporidesmin (Figure 3.11) (Gardiner, Waring, and Howlett, 2005). Among the dimeric ETPs, many verticillin compounds and structural analogs have been isolated, and all contain a polysulfide bridge in the molecule. Their simplified structures are displayed in Table 3.6. It is interesting to note that the presence of a disulfide bridge was often easily recognized by fast atom bombardment MS, with the reported loss of 64 uma. 3.4.3.2.1 Acquisition, Extraction and Purification of Verticillins and Analogs In contrast to the communesins, these fungal natural products have not been isolated only from Penicillium species. Rather, they appear to be quite ubiquitous, having been mainly isolated from species belonging to the Leptosphaeria, Chaetomium, Verticillium, Gliocladium and Penicillium genera (Table 3.7). 3.4.3.2.2 Bioactivities of Verticillins and Analogs These molecules were described for their highly cytotoxic activities, even before 11,110 -dideoxyverticillin A and 110 -deoxyverticillin A had been isolated. Effectively, verticillin A, which was initially isolated from a Verticillium sp. strain TM-759 in 1970, from the basidiocarp of Coltricia cinnamomea (Polystictus cinnamomeus), already displayed at that time a potent cytotoxicity against HeLa cells, with an ED50-value of 0.3 mM. Verticillin A was also reported as being

3.4 What are these Promising Molecules from Marine Penicillium?

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Table 3.6 Verticillin compounds and analogs: chemical description.

Structure

Compound

Molecular formula

Molecular weight (Da)

Verticillin A

C30H28N6O6S4

11,110 dideoxyverticillin A

Structure

Compound

Molecular formula

Molecular weight (Da)

696.8

Verticillin D

C32H32N6O8S4

756.9

C30H28N6O4S4

664.8

Verticillin E

C32H28N6O8S4

752.9

110 -deoxyverticillin A

C30H28N6O5S4

680.8

Verticillin F

C32H30N6O8S4

754.9

Verticillin B

C30H28N6O7S4

712.8

Verticillin G

C30H28N6O7S4

712.8

Verticillin C

C30H28N6O7S5

744.9

Verticillin H

C32H32N6O6S4

724.9

Sch 52901

C31H30N6O6S4

710.9

Gliocladine A

C30H28N6O6S5

728.9

Sch 52900

C31H30N6O7S4

726.9

Gliocladine B

C30H28N6O6S6

761.0

(continued )

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

Table 3.6 (Continued) Structure

Compound

Molecular formula

Molecular weight (Da)

Melanicidin II

C30H28N6O6S4

Melanicidin III

Compound

Molecular formula

Molecular weight (Da)

696.8

Leptosin A

C32H32N6O7S4

740.9

C30H28N6O7S4

712.8

Leptosin B

C32H32N6O7S5

773.0

Melanicidin IV

C30H28N6O8S4

728.8

Leptosin C

C32H32N6O7S6

805.0

Chaetocin

C30H28N6O6S4

696.8

active in vivo against Ehrlich ascites tumor, with a tumor index of 0.17 at a daily dose of 1 mg kg 1, and no deaths were observed (Katagiri et al., 1970). Subsequent studies also noted the inhibition of proto-oncogenic pathways (c-fos) at low concentrations (20– 500 nM) (Chu et al., 1995). More recently, verticillin A was found also to be cytotoxic for HepG2 cells, with an IC50 of 62 nM, partially through inducing apoptosis. The administration of 2 mg kg 1 per day of verticillin A to mice with the HepG2 tumor inhibited lesion growth in a dose-dependent manner (Liu et al., 2011). Another example involved leptosins isolated from a marine fungus (Leptosphaeria sp.), which showed potent cytotoxicity against cultured P388 cells, and more particularly leptosins A and C, which exhibited significant antitumor activity against sarcoma 180 ascites (Takahashi et al., 1994a; Takahashi et al., 1994b). This was further confirmed by experiments on leptosin C, which showed a strong inhibition of topoisomerase I activity, with IC50-values of 3–10 mM. Leptosin C was then reported to be a topoisomerase I catalytic inhibitor in vitro as well as in vivo, and to induce apoptosis in a number of human cancer cell lines (Yanagihara et al., 2005). 11,110 -Dideoxyverticillin A, after its first isolation from a marinederived Penicillium, was further investigated when subsequently isolated from the fungus Shiraia bambusicola, which is traditionally used in Chinese folk medicine (Table 3.7). The molecule’s cytotoxic activity was confirmed in vitro and in vivo, but an inhibition of angiogenesis was also reported in cultured tumor cell lines, as well as a reduction in the production of vascular endothelial growth factor (Chen et al., 2005a; Zhang et al., 2005). 11,110 -Dideoxyverticillin A also potently inhibited the proliferation of four human

Structure

breast tumor cell lines, with an average IC50 of 0.2 mM. In vivo, the compound exhibited a remarkable efficacy against mice sarcoma 180 and hepatoma 22 after daily intraperitoneal administration at 0.5 or 0.75 mg kg 1, with inhibition rates ranging from 45.0% to 72.4%. The molecular mechanisms implied in 11,110 -dideoxyverticillin A activity have been further investigated (Chen et al., 2005b), but some molecular links remain to be clarified. A total synthesis of this promising compound was performed in 2009, following an elegant biomimetic approach in 11 steps (Kim, Ashenhurst, and Movassaghi, 2009). Other related compounds from this class were also reported as having high cytotoxic activities. For example, Sch52900 was found to inhibit the growth of various tumor cell lines by 90% at concentrations ranging from 34 to 68 nM, but was less active on Colo 320 (IC50 680 nM) and HepG2 (IC50 1360 nM) cells. Sch52900 also appeared to induce differentiation and apoptosis in HL-60 cells by interfering with the signaling pathways leading to AP-1 activation downstream of MAP kinase, but the exact target remains to be elucidated (Erkel et al., 2002). Verticillin B and chaetocin each demonstrated cytotoxicity against Jurkat cells, with IC50-values less than 0.6 mM (Watts et al., 2010), while D displayed toxicity against L5178Y cells with an EC50value less than 0.13 mM (Ebrahim et al., 2012). Lastly, the newly discovered verticillin H was found to inhibit the growth of several cancer cell lines, with IC50-values ranging from 0.037 mM to 0.325 mM (Figueroa et al., 2012). It has been suggested that, although the verticillins are highly cytotoxic, with IC50-values in the low nanomolar range, they lack

3.4 What are these Promising Molecules from Marine Penicillium?

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Table 3.7 Production and purification methods used for isolation of verticillin-related compounds. Species

Origin

Verticillium sp. Coltricia TM-759 cinnamomea (fungus)

Culture medium

Culture Extraction conditions solvent

Purification

Isolated verticillins and related compound

References

Liquid: glucose (30 g l 1), peptone (20 g l 1), NaCl (5 g l 1), pH 6.8

Acetone 3 d, EtOAc 27  C Agitation 20 Lpm

Crystallization from pyridine/ acetone Si chromatography

A A, B, C

Katagiri et al., 1970 Minato, Matsumoto, and Katayama, 1973

MeOH/ CH2Cl2 (1:1)

Sephadex LH20, Si chromatography, RP HPLC

Leptosins A-I

Takahashi et al., 1994b Yanagihara et al., 2005

Si chromatography, RP HPLC, Sephadex LH20

A Sch .52900 Sch 52901

Chu et al., 1995

Leptosphaeria sp.

Sargassum tortile Liquid: glucose (2%), peptone (1%), yeast (alga) extract (0.5%), artificial seawater, pH 7.5

Gliocladium sp. SCF-1168

Dicot leaf litter (rainforest)

liquid: KH2PO4 (0.5%), peptone (0.5%), NaCl (0.5%), yeast extract (0.3%), cerelose (2%), soybean grits (0.5%),tap water, pH 7.0; neopeptone (1%), cerelose (4%), CaCO3 (0.4%), tap water, pH 7.0

Aspergillus flavus solid: rice soaked with H2O overnight sclerotium (fungus) Penicillium sp. Avrainvillea liquid: yeast extract (0.5%), peptone (0.5%), longicaulis (alga) glucose (1.0%), crab meal (0.2%), sea water (100%) Gliocladium Rotting plant liquid: corn meal medium: corn meal (1%), sp. strain 4-93 material glucose (1%), KH2PO4 (0.15%), KCl (0.05%), NaNO3 (0.05%), MgSO4  7 H2O (0.05%), pH 5.5 Gliocladium catenulatum

Gliocladium roseum

Submerged wood (freshwater)

Shiraia bamboo bambusicola Verticillium sp. Amanita flavorubescens (fungus) Bionectria soil byssicola Nectria inventa Marine sediment Bionectria ochroleuca Bionectriaceae

Sonneratia caseolaris (leaves)

solid: wheat

3 wk, 27  C

EtOAc 90 h, 24  C Agitation 6 d, 24  C, Agitation 55.0 lpm 45 d, 25  C

EtOAc

Si gel VLC, RP HPLC

D, E, F

Joshi, Gloer, and Wicklow, 1999

17 d, 27  C

MeOH/ CH2Cl2 (1 : 1)

Si VLC, HPLC

11,110 -dideoxy A, 110 -deoxy A,A

Son et al., 1999

MeOH/ 10 d, Acetone 22  C Agitation (1:1) 3 lpm

20 d 26  C

MeOH

Zhang et al., 2005

EtOAc

A

Liu et al., 2006 Liu et al., 2011

Acetone EtOAc

Si chromatography, RP HPLC

DG

Zheng et al., 2006 Zheng et al., 2007

XAD-16 Acetone

Partition, flash chromatography, HPLC, RP-HPLC Si chromatography, Sephadex LH20, RP HPLC

B chaetocin

Watts et al., 2010

D

Ebrahim et al., 2012

H

Figueroa et al., 2012

Not described

7 d, 28  C Agitation liquid: malt extract (1.5%), deionized water 21 d, r. t. Agitation

solid: rice, distilled water

EtOAc

solid

Dong et al., 2005

11,110 -dideoxy A

Ethanol

40 d, 22  C

Erkel et al., 2002

Partition, Si chromatography CCC, RP HPLC

Not described

liquid: glucose (2%), yeast extract (0.2%), peptone (0.5%), MgSO4 (0.05%), KH2PO4 (0.1%), pH 5.7

DIAION HP 21 Sch .52900 resin, Si chromatography, RP HPLC, Sephadex LH20 Si chromatography, Gliocladine A-E MPLC, RP HPLC, 110 -deoxy A, Si gel VLC, A Sephadex LH20 Sch .52900 Sch 52901

CH3CNMeOH

Lpm: liters of air per minute; VLC: Vacuum liquid chromatography; RP HPLC: Reverse-phase high-performance liquid chromatography; CCC: countercurrent chromatography.

the tumor specificity that is generally considered essential for further consideration as therapeutic agents (Son et al., 1999). However, when the effects of verticillin A on the growth of multiple types of tumor cell lines were compared to the effects on normal human colon epithelial cells CDD-841, verticillin A appeared to be between six- and 20-fold more active against tumor cells than normal cells. It was further concluded that verticillin A is a potent apoptosis sensitizer, and has the potential to be developed as a low-toxicity anticancer drug (Liu et al., 2011).

Interestingly, among the different alkaloid/diketopiperazine compounds that have been isolated from Penicillium species, and which have exhibited cytotoxic activity, it appears that dimers with disulfide bridges (such as verticillin A and its analogs) are the most active. Although the importance of the disulfide bridge for antibacterial activity has been described (Zheng et al., 2006), its role in cytotoxic activity remains unclear. As an example, Gliocladine C – which also contains the disulfide bridge in the diketopiperazine moiety of the molecule (Figure 3.12) – was less active than verticillin

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

Figure 3.12 Verticillin-related compound structures.

A as to date it has only shown activity against HepG2 cells, with an IC50-value of 19.6 mM (Ren and Yu, 2011). Verticillin A and its analogs are the only dimeric structures among the alkaloid/diketopiperazine described in this chapter for potent antitumor activity, and it is this dimeric feature that seems to play a role in their potency rather than the presence of a disulfide bridge in the molecule. In fact, another dimeric structure of this class without the disulfide moiety, WIN 64821, was isolated from P. expansum (Wang et al., 2012), but its cytotoxicity was not tested. Nonetheless, it proved to be a competitive antagonist of substance P, with a submicromolar potency for human neurokinin 1 and cholecystokinin B receptors (Barrow and Sedlock, 1994). 3.4.4 Cytotoxic Sesquiterpenes: Ligerin, a Chlorinated Sesquiterpene

Among the cytotoxic metabolites produced by Penicillium strains isolated from marine environments, 19 terpenes have been reported to exhibit activity against various cancer cell lines. Interestingly, only one terpenoid – ligerin – which is produced by a marine-derived species of Penicillium (Vansteelandt et al., 2013), includes a chlorine atom in its structure. This recently described sesquiterpene is an analog of fumagillin, a well-known secondary metabolite produced by different fungal genera, including Aspergillus and Penicillium. Ligerin is one of the most potent active terpenoids reported in this chapter, and is the only one to have been tested against osteosarcoma cell lines (IC50 ¼ 0.117 mM). This compound and its potential analogs are subject to a patent for their potential use in the treatment or prevention of bone tumors (Egorov et al., 2011).

Figure 3.13 Penicillium SP MMS351: the ligerin-producing strain.

3.4.4.1 Ligerin is Produced by a New Species of Penicillium Ligerin is a chlorinated sesquiterpene produced by an undescribed species of Penicillium designated as P. SP MMS351. The producing-strain was isolated in 1997, from a seawater sample gathered on the French Atlantic coast near the Loire river estuary (La Pr
ee, Loire Atlantique, France), and is stored in the laboratory fungal collection (MMS-Marine Fungal Collection, University of Nantes) under the reference number MMS351, as well as in the collection of Museum National d’Histoire Naturelle (MNHN) in Paris, France, with the code LCP.99.43.43. Its identification has been performed by sequencing the internal transcribed spacers (ITS) and beta-tubulin regions (GenBank accession number JN676192 for ITS and JN794530 for beta-tubulin sequence), and by a phenotypic approach (Prof. J. C. Frisvad, Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark). The strain has been identified as a new species of the genus Penicillium, belonging to the section Canescentia of the subgenus Penicillium (Figure 3.13).

3.4.4.2 Isolation of Ligerin This strain was selected after a screening for cytotoxicity against tumor cell lines, and more particularly against osteosarcoma cell lines. An ethyl acetate extract of this strain grown on YES medium exhibited activity against three cancer cell lines (POS1, AT6-1 and KB), without showing toxicity on a nontumor cell line (L929) when tested at the same concentration.

3.4 What are these Promising Molecules from Marine Penicillium?

Cl Cl20 7ab CH3 HO HO 6

5

13

1

4

3

O

2

18

O

H8

17

H2β

14

O 11

12

CH3

H19

19

O

8

10O

H20

15

OCH3 O O

9

CH3 16

O

j 73

H10

OH

H18

H11

OH

O

H3

H4 H16

H7

H7

H14

H1 H15 H15

OH

1.05 3.14 3.17 3.04 2.12 1.18

1.20

2.05

3.0

4.22

1.03

4.0

4.14

0.98

5.0

0.99

1

0.80

Figure 3.14

6.0

1.06

1.00

7.0

ppm (f1)

H5 H5

2.0

1.0

0.0

H NMR spectrum of ligerin (400 MHz, CDCl3).

In order to isolate the bioactive compound from the ethyl acetate extract, the P. SP MMS351 strain was grown on Yeast Extract Sucrose solid medium prepared with 20 g l 1 agar, 5 mg l 1 CuSO4, 10 mg l 1 ZnSO4, 0.5 g l 1 MgSO4, 20 g l 1 yeast extract, and 150 g l 1 sucrose, dissolved in sterilized natural seawater (salinity of 32.8 g l 1). Cultures were incubated at 27  C for 11 days before extraction with ethyl acetate. The crude extract was submitted to three successive bioassayguided fractionations, based on activity against osteosarcoma cell line (POS1 cells), and this led to 10 mg of ligerin, obtained as a colorless oil. The chemical structure of ligerin has been established by interpretation of spectroscopic data (UV, IR, HRESIMS), one- and two-dimensional NMR spectra (Figure 3.14) and X-ray analysis, enabling the determination of its absolute configuration. The analysis of the isotopic pattern of ligerin revealed the presence of a chlorine atom, with a 3 : 1 ratio for peak intensities of m/z 441 ([M þ Na]þ) and m/z 443 ([M þ 2 þ Na]þ), respectively. The elemental composition has been established as C20H31ClO7 (five degrees of unsaturation) by analysis of the HRESIMS spectrum with the observation of a sodium adduct [M þ Na]þ at m/z 441.16507 and its cluster ion [2 M þ Na]þ at m/z 859.34186. One- and two-dimensional NMR spectral analyses, and particularly HMBC and HSQC correlations, together with a comparison of the data with those in the literature, led to the establishment of a planar structure for ligerin, and

suggested that it was an analog of fumagillin. NOE experiments enabled the determination of the relative configuration, which was in agreement with that reported for fumagillin derivatives (Asami et al., 2006; Chu et al., 2001; Hal
asz et al., 2000). The three-dimensional structure of ligerin has been confirmed by X-ray analysis (Figure 3.15), and its absolute configuration determined as 1S,2S,3R,6R,13R,14R, which is the same as that previously established for fumagillol and fumagillin (Marui et al., 1992; Rodeschini et al., 2004).

Figure 3.15 ORTEP drawing of ligerin. Reprinted from Vansteelandt et al. 2013 with kind permission by the American Chemical Society. Ó (2013).

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3.4.4.3 The Chlorine Atom: The Originality of Ligerin’s Chemical Structure Despite the abundance of this atom in the marine environment, only eight compounds from marine-derived Penicillium strains have been described to include a chlorine atom in their chemical structures (e.g., AntiBase 2011; Laatsch, 2011). These are griseofulvin, 3-chloro-4-hydroxyphenylacetamide, and six monomer or dimer derivatives of gentisyl alcohol, all of which displayed cytotoxicity against cancer cell lines (Chen et al., 2008; Hiort, 2002; Petit et al., 2004; Zhang et al., 2007). Among the natural analogs of ligerin, chlovalicin – which has been reported to be produced by a terrestrial strain of Sporothrix sp. – is the only one to include a chlorine atom in its structure. Thus, ligerin is the first C-7-chlorinated analog to be produced by a marine-derived fungal strain. 3.4.4.4 The Many Structural Analogs of Ligerin As previously reported in this chapter, ligerin is an original chlorinated sesquiterpene analog of fumagillin, a natural secondary metabolite produced by various fungal genera such as Aspergillus sp. and Penicillium sp. Fumagillin was isolated for the first time in 1949 by Hanson and Elbe from a crude extract of an Aspergillus fumigatus strain (Elbe and Hanson, 1951; Hanson and Eble, 1949). Various natural analogs of fumagillin have been described, such as RK-95113 (Asami et al., 2006), Sch 528647 (Chu et al., 2001), as well as fumagiringillin produced by

A. fumigatus (Jiao et al., 2004), RK-805 produced by a strain of Neosartorya sp. (Asami et al., 2004), ovalicin produced by Pseudorotium ovalis and Sporothrix sp. (Bollinger, Sigg, and Weber, 1973; Hayashi et al., 1996; Sigg and Weber, 1968), chlovalicin, as well as two cyclized analogs of ovalicin produced by a strain of Sporothrix sp. (Hayashi et al., 1996; Takamatsu et al., 1996), and 5-demethylovalicin produced by Chrysosporium lucknowense (Son et al., 2002) (Figure 3.16). Despite sequencing of the A. fumigatus genome (Nierman et al., 2005), the complete biosynthetic pathway of fumagillin remains unknown. Based on feeding and isotope incorporation studies of ovalicin, a fumagillin-related compound produced by Pseudorotium ovalis, fumagillol and thus fumagillin were suggested to be derived from b-trans-bergamotene, also produced by A. fumigatus (Cane and Levin, 1976; Cane and King, 1976; Cane and Buchwald, 1977; Cane and McIlwaine, 1987). Recently, Lin et al. (2013) uncovered in A. fumigatus a b-transbergamotene synthase, the membrane-bound terpene cyclase responsible for the formation of b-trans-bergamotene from farnesyl-pyrophosphate. The early biosynthetic steps appear to have been identified, but the description of complete fumagillin biosynthesis remains in progress, especially with the study of the fumagillin biosynthetic gene cluster in A. fumigatus. Although several natural analogs of fumagillin have been reported, most analogs described to date are synthetic or semisynthetic derivatives of fumagillol. The first total synthesis of

Figure 3.16 Structures of the natural analogs (N) and important semisynthetic analogs of fumagillin and fumagillol, as reported in the literature.

3.5 Conclusions

CH3

O

CH3

CH3

CH3

O a) NaOH/H2O

CH3

O OCH3

O

O

OH O

CH3

O OCH3

Fumagillin

OH O O

b) DMAP/Py

O Cl HO

CH3

CH3 CH3

O

CH3

O

OCH3 O OH

O

CH3

O

OCH3 O O

CH3

c) LiCl-AcOH/THF

O

OH O

Ligerin

Figure 3.17 Semisynthesis of ligerin (DMAP: dimethylaminopyridine, Py: pyridine, THF: tetrahydrofuran).

racemic fumagillin was described in 1972 (Corey and Snider, 1972), and some 25 years later Kim et al. (1997) reported the first asymmetric synthesis of ( )-fumagillol. A review prepared in 2010 described the different synthetic strategies to access fumagillin derivatives (Yamaguchi and Hayashi, 2010). 3.4.4.5 Ligerin Semisynthesis As many synthetic derivatives of fumagillol have been described along with the synthetic methods used, the semisynthesis of ligerin has been performed to confirm the structure and to determine further the bioactivity evaluation of this compound. The studies were conducted in two steps from fumagillol, obtained after an alkaline hydrolysis of commercial fumagillin (Fumidil B1; Ceva sant
e animale), with an overall yield of 38%. The first step involved the esterification of fumagillol, adding a succinyl moiety to the hydroxyl at C-3, while the second step involved in opening the C6–C7 epoxide in order to introduce a chlorine atom (Figure 3.17) (Vansteelandt et al., 2013). 3.4.4.6 Bioactivities Fumagillin was first studied for is antimicrobial activity, and subsequently for veterinary use, especially in apiculture to treat the microsporidial parasite Nosema apis, which causes infections in honey bees (Kaltzelson and Jamieson, 1952). Unfortunately, the toxicity of the fumagillin molecule and, more especially, the potential genotoxic effects on humans and other vertebrates exposed through the consumption of contaminated honey, led to the withdrawal of fumagillin preparations such as Fumidil B1 in France and in the majority of EU member states (Stanimirovic et al., 2007). The antiangiogenic properties of fumagillin were investigated during the 1990s (Ingber et al., 1990), but because of the compound’s toxic effects many synthetic derivatives with lesser toxicity were prepared, leading to the publication of more than 200 patents. The molecular target of these compounds was identified as a cytosolic enzyme, methionine aminopeptidase-2 (MetAP2), which is

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responsible for the removal of methionine residues from newly synthetized polypeptides. Fumagillin and most of its structural analogs inhibit MetAP2 selectively and irreversibly through a covalent bond between the C-7 of the spiro-epoxide group and a nitrogen of the 231-histidine residue in the active site of the enzyme (Liu et al., 1998). Among the semisynthetic analogs, TNP470 (6-O-chloroacetylcarbamoyl-fumagillol; AGM-1470; Takeda & Abott) was the first to attract attention. This compound exhibited potent antiangiogenic and antitumor activities, and entered Phase II clinical trials for the treatment of pancreatic adenocarcinoma, but unfortunately its development was stopped due to its toxicity, and especially its neurotoxicity (Datta, 2009). Recently, another semisynthetic analog, CKD-732 [6-(4-dimethylaminoehoxy)cinnamoyl-fumagillol] entered Phase I clinical trials for the treatment of refractory solid cancer (Shin et al., 2010), and also in combination with capecitabine and oxaliplatine for the treatment of metastatic colorectal cancer in patients who had progressed on chemotherapy based on irinotecan (Shin et al., 2012). A third semisynthetic analog, PPI-2458 {[(3R,4S,5S,6R)-5methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan2-yl]carbamate} also entered Phase I clinical trials for the treatment of non-Hodgkin lymphoma and solid tumors (Arico-Muendel et al., 2013). These two compounds highlight the interest in this chemical family in the quest for new anticancer drugs. Ligerin has been isolated by using a bioguided fractionation based on the activity against an osteosarcoma cell line (POS1). In addition, its cytotoxicity activity has been evaluated against a panel of four cancer cell lines, including two osteosarcoma cell lines (KB, AT6-1, POS 1 and OSRGa), and one nontumor fibroblastic cell line (L929). Ligerin displayed antiproliferative activity against four of the five cell lines used (AT6-1, POS1, OSRGa, L929), with its more potent activity being observed against the POS1 cells (IC50 0.117 mM). The activities against AT6-1, OSRGa and L929 cell lines were weaker, and no IC50-values could be calculated. Rather, only a plateau of cell viability inhibition was observed (at respectively 20%, 22%, and 35%) for all concentrations tested over the range of 6 to 2400 nM.

3.5 Conclusions

The marine environment has emerged as an incredible resource for drug discovery, in which many fungi have developed ways not only of living and surviving but also occasionally producing surprising compounds with novel scaffolds and/or potent activities. Although these compounds may sometimes be highly toxic and threatening to human health following their consumption via intoxicated sea food, they also have the potential for use as drugs to prevent or treat several diseases. Despite the content of this chapter having been limited to potent cytotoxic fungal metabolites isolated only from marine-derived Penicillium

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species, more than 130 compounds have already been identified that can provide great promise in the development of new anticancer drugs. Fungi have the added advantage of being a sustainable resource, as most can be cultivated to produce unlimited quantities of compounds if the culture conditions are optimized, without disturbing the marine ecosystem. The molecules discussed at the end of the chapter appear very interesting with regards to their activity or chemical originality. However, in vitro-observed activities on cancer cell lines have their limits, and this is usually only the first step in the discovery of potential anticancer drugs. The mechanisms of action must first be elucidated and toxicity on normal human cells evaluated. Likewise, during the early stages of development from “hit” to

“lead” compound, the pharmacokinetic properties must be evaluated if dramatic failures in the drug “pipeline” are to be avoided. Notably, studies of chemically related compounds such as verticillins or fumagillin analogs appear useful for acquiring an understanding of the mechanism(s) of action and structure–activity relationship(s) of these materials. The final stage is the quest for less toxic bioactive compounds, using well-proven approaches of drug design and semisynthesis. Although the genus Penicillium has long been studied and has for many years provided a wide range of important pharmaceuticals, it will surely continue to surprise drug research communities with its ability to produce unprecedented and bioactive metabolites while inhabiting a marine environment.

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3 Impact of Marine-Derived Penicillium Species in the Discovery of New Potential Antitumor Drugs

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About the Authors Marieke Vansteelandt is a research scientist in the UMR 152 IRD-UPS “Pharma-Dev” unit (Medicinal Chemistry and Pharmacology for Developing Countries), and assistant professor in Pharmacognosy at the University Paul Sabatier (Toulouse III, France). She obtained her PharmD and PhD on marine natural product chemistry at the University of Nantes (Mer Molecules Sant
e research group), studying cytotoxic metabolites produced by marine-derived fungi. She then completed a postdoctoral fellowship at Nautilus Biosciences Canada Inc. (UPEI, Canada), where she worked on bioactive metabolites produced by marine microorganisms, utilizing a metabolomic approach. In 2013, she joined the staff of Prof. Nicolas Fabre at the University of Toulouse III to work within the PEPS team (Pharmacognosy, EthnoPharmacology and Pathologies from developing countries) in the field of natural product chemistry. Currently she is involved in a research project on plant-associated endophytic fungi.

Catherine Roullier is a research scientist in the group “Sea, Molecules and Health,” and assistant professor in Pharmacognosy at the University of Nantes, France. She completed her PhD in Chemistry at the University of Rennes in 2010 under Prof. Jo€el Boustie’s supervision. Her doctoral thesis mainly focused on a marine lichen and mycosporine-like compounds. In 2011, she began a research postdoctoral fellowship in Australia at Eskitis Institute in the Drug Design and Discovery group of Prof. Ron Quinn. She took part in the isolation and identification of bioactive compounds resulting from the high-throughput screening of terrestrial and marine organisms extracts against different therapeutic targets. In 2012, she joined the staff of Prof. Y. F. Pouchus at the University of Nantes, to work on the isolation of bioactive metabolites from marine-derived fungi. Elodie Blanchet is PhD student in Natural Product Chemistry at the University of Nantes. Her research is are carried out under the guidance of Prof. Yves-FranSc ois Pouchus and Dr

References

Olivier Grovel of the MMS “Sea, Molecules and Health” research group of the University of Nantes, France, and Dr Ronan Le Bot, CEO of Atlantic Bone Screen company. This leading Contract Research Organization is specialized in highquality preclinical evaluation services in the field of bone and joint diseases, and more particularly in bone diseases of tumor origin (www.atlantic-bone-screen.com). The thesis concerns the isolation and characterization of marine-derived Penicillium metabolites with anti-osteosarcoma activity. Yann Guitton is a Postdoctoral fellow in the group “Sea, Molecules and Health” at the University of Nantes, and in the Phycotoxin-laboratory at the IFREMER (French public institute for marine research), France. He completed his PhD in Plant Physiology at the University of Saint-Etienne in 2010 under Prof. Laurent Legendre’s supervision. His doctoral thesis mainly focused on terpenes production in the genus Lavandula, and on GC-MS data treatment bioinformatic approaches. In 2011, he began a research postdoctoral fellowship in Lille at the University of Lille in the SADV group of Prof. Hilbert. He took part in the development of a metabolomics core facility and developed R processing tools dedicated to mass spectrometry data treatment. In 2012, he joined the staff of Prof. Y. F. Pouchus at the University of Nantes to work on automated R tools for the fast dereplication of bioactive metabolites from marine-derived fungi. Yves Francois Pouchus is the MMS-team head at the Faculty of Pharmacy in Nantes, France. He is specialized in mycology and

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has been a pioneer in marine mycotoxinology. His research on biology and chemistry of marine fungi led to more than 60 publications and 80 communications. For that purpose, he has created a marine fungal collection in Nantes which is quite unique in France. He is member of various International Societies, and President of the Society of Botanists and Mycologists of French-speaking Faculties of Pharmacy. Nicolas Ruiz is from the research group “Sea, Molecules and Health” of the University of Nantes, France. He obtained his PhD from the University of Nantes in 2007. In 2008, he joined the staff of Prof. Y. F. Pouchus at the University of Nantes as assistant Professor in mycology to work on the isolation of bioactive metabolites from marine-derived fungi. His research interests have been concerned with the isolation and chemotaxonomy of marine-derived fungi, and the study of bioactive metabolites from marine marine-derived fungi and metabolomics. Olivier Grovel is a member of the MMS laboratory and head of the Pharmacognosy and Phytotherapy department at the University of Nantes, France. He obtained his Pharm D and PhD in marine natural products chemistry from University of Nantes before a two-year appointment at the University of Rouen, working on synthesis of fluorinated derivatives of plant alkaloids. He joined the marine fungi chemistry team of MMS in 2003, and has authored 16 scientific publications, one patent, and 44 communications; he has also received the National Academy of Pharmacy Award.

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4 Astonishing Fungal Diversity in Deep-Sea Hydrothermal Ecosystems: An Untapped Resource of Biotechnological Potential? Ga€etan Burgaud, Laurence Meslet-Cladiere, Georges Barbier, and Virginia P. Edgcomb

Abstract

Marine fungi have long been considered as exotic microorganisms that fascinate only a very small proportion of scientists. Ecologically important relationships between marine fungi from open oceans or coastal waters and other organisms have been clearly demonstrated. However, the diversity, ecological role(s) and biotechnological potential of fungal communities from deep-sea marine extreme environments such as hydrothermal vents are far from being resolved. Based on data from recent surveys, hydrothermal

vents have emerged as oases of life for fungi, with unexpected communities revealed by culture-independent and culturebased methods, in addition to newly described species that can adapt specifically to deep-sea conditions. As the natural product chemolibraries from marine fungi continue to expand rapidly, it can be hypothesized that an extensive exploration of fungi from extreme environments – and in particular from deep-sea hydrothermal vents – will cause the current catalog of natural products to be dramatically enriched with novel active biomolecules.

4.1 Introduction

4.2 Deep-Sea Hydrothermal Vents as Life Habitats

Marine fungi form an ecologically defined group of microorganisms that have colonized every type of environment in the oceans, from coastal waters to the deep biosphere. Although deep-sea extreme ecosystems, including deep sediments, cold seeps and sunken wood, have recently been explored for fungi, hydrothermal vents remain one of the best-studied environments for fungi. Hydrothermal vents are rich oases of life that belong to all three domains. Recent reports of fungi in the deep oceans have provided insights into their diversity, activity and ecological role, but further knowledge of the biotechnological potential of recently discovered fungal communities from “extreme” marine environments, in terms of their biological and pharmacological properties, is eagerly awaited. As natural product chemolibraries from marine fungi are rapidly expanding with newly reported structures, it can be hypothesized that the further exploration of fungi from extreme environments, and in particular of deep-sea hydrothermal vents, will cause a dramatic increase in the libraries of novel active biomolecules in the near future. These efforts will require expertise in marine microbiology, biology, chemistry and computational sciences in order to optimize screening strategies.

Many decades ago, the seafloor was characterized as being flat, uniform and abiotic, with an absence of light, low temperatures and high hydrostatic pressures being advanced as factors that would preclude life (Jorgensen and Boetius, 2007). During the Travaillier and Talisman expeditions (1882–1883), however, living bacteria were found in sediment and water samples collected at 5000 m depths (Jannasch and Taylor, 1984) while later, during the 1950s, the Danish Galathea expedition collected several samples from 10 000 m depth that contained bacteria (Zobell and Morita, 1959). Such unexpected observations clearly elicited a new perception of the seabed as an extreme, but dynamic, ecosystem that incorporated active microbial life. The importance of microorganisms in the deep-sea became extremely clear with the discovery of hydrothermal vents off the coast of the Galapagos in 1977. Scientists diving with the Autonomous Underwater Vehicle ALVIN serendipitously discovered rich benthic animal communities – that is, large clams, mussels and vestimentiferan worms, associated with chimneys that emanated black smoke composed of precipitated minerals (Corliss et al., 1979). This novel food chain was driven by

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by Stephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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chemosynthetic prokaryotic production, with chemolithoautotrophic microorganisms thriving as the primary producers. 4.2.1 Generation of Marine Hydrothermal Systems: A Story of Interactions

Hydrothermal venting occurs along the approximately 60 000 km-long open-ocean spreading centers that split the Earth’s crust much like the seams on a baseball. Sites of hydrothermal venting have been discovered along mid-ocean ridges, in back-arc basins, rifted arcs and at submerged islandarc volcanoes (Tivey, 2007) at low to high latitudes and depths (Figure 4.1). Hydrothermal vent generation results from the emissions of hot, reducing, metal-rich fluids, often chaperoning mineral deposits that shape chimney structures (Orcutt et al., 2011). Mineral deposits associated with the seafloor hydrothermal vent fluids result from different interactions: (i) the penetration of cold seawater into the fissured oceanic crust; (ii) increases in seawater thermal energy from the underlying magma; (iii) mineral leaching of the oceanic crust by hydrothermal fluids, that is, heated seawater becoming chemically reduced and metal-rich; and (iv) mixing of hydrothermal fluids with cold seawater after re-emergence at the seafloor, followed by mineral particle deposition. Such interactions modify the chemical composition of the oceanic crust, shape the hydrothermal chimneys, and deliver energy sources for chemosynthetic prokaryotes (members of the Bacteria and Archaea) which form the basis of a complex food chain at these sites. Hydrothermal fluids vent from the ocean crust in many different ways and exhibit a wide range of temperatures and

chemical compositions determined by the subsurface reaction conditions, which leads in turn to different types of chimney (Figure 4.2). Black and white smokers, with temperatures rising to 400  C or 100–300  C, respectively, are found directly above magma chambers (Martin et al., 2008) and represent a small fraction of the global hydrothermal heat flux (Schultz, Delaney, and McDuff, 1992). The black “smoke” is mainly composed of sulfide minerals and metal precipitates, while white “smoke” results from the precipitation of calcium, barium and silica minerals. Both lead to the formation of anhydrite (CaSO4). Offaxis vents are located several kilometers away from the spreading zone, and their alkaline vent fluids (pH 9–11) with temperatures of 40–91  C lead to the formation of carbonate chimneys (Ludwig et al., 2006). Diffuse flows – that is, the emission of diffuse low-temperature fluids (from a few degrees to 100  C) across a broad area of porous sulfide deposits – appear as a much more quantitative hydrothermal process based on chemical fluxes and habitat size (Schultz, Delaney, and McDuff, 1992). 4.2.2 Different Vent-Fluid Compositions Shaping Different Ecological Niches

Hydrothermal systems can be allegorized as natural geothermal power plants, with the oceanic crust as a reaction zone converting deep cold seawater into energy-rich fluids. Consequently, the final chemistry of hydrothermal fluids is correlated to the initial seawater composition, the oceanic crust composition leached by the fluid, and also the temperature and pressure conditions of the reaction zone (Tivey, 2007). Indeed, the pressure-temperature couple controls mineral dissolution and

Figure 4.1 Location of known hydrothermal sites with reported depth. This figure was generated using GeoMapAppÓ and the actualized InterRidge Vents Database http://www.interridge.org/irvents/ (Beaulieu, 2010).

4.2 Deep-Sea Hydrothermal Vents as Life Habitats

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Figure 4.2 (a,b) Photographs of (a) diffuse low-pH fluid at Mid-Atlantic Ridge Rainbow site and (b) black smoker with associated fauna and the NAUTILE arm sampler at Mid-Atlantic Ridge TAG site; (c,d) Photographs of alkaline vents from (c) Mid-Atlantic Ridge Lost City site with (d) microbial colonization module. The photographs were processed during the IFREMER-Victor/EXOMAR cruise (French Research Institute for Exploitation of the Sea).

precipitation reactions, and has a direct influence on fluid composition (German and Von Damm, 2003). Depth variation and associated physical parameters, along with different source rocks, will have a direct effect on the fluid composition. If vent fluids can be characterized as modified seawater without magnesium or sulfates, but with reduced species (H2S, H2 or CH4) and metals (Cu, Fe, Mn, Zn), then no two described vents will have exactly the same chemistries. However, patterns appear depending on the spreading rates of ridges – that is, slow-spreading Atlantic Ocean and fast-spreading Pacific Ocean ridges, or sediment-covered or – uncovered ridges (for specific fluid compositions of representative hydrothermal vents, see Orcutt et al., 2011). Finally, gradients are another interesting feature to consider, as strong gradients of temperature, pH and chemical composition offer a tremendous range of physical and chemical niches for many lifestyles. 4.2.3 Hydrothermal Lifestyles At the Macro- and Microscopic Scale

Hydrothermal vents have long been referred to as “oases of life,” based on the dense and unusual fauna that are clustered around chimneys relative to the surrounding, nonhydrothermal seafloor. Indeed, deep-sea vents represent one of the most productive ecosystems on Earth, with chemolithoautotrophic prokaryotes as the primary producers for macroeukaryotic

metazoan, the density of which may reach 50 kg m 2 (Prieur and Marteinsson, 1998). Clumped species distributions observed in concentric rings around vent orifices are mostly a result of temperature and toxicity of vent fluids (Minic, Serre, and Herve, 2006). Faunal endemism rates are high and are correlated with depth, as nonendemic penetrating species occur, but decrease with increasing hydrostatic pressures (Desbruyeres et al., 2000). Animals with symbionts are icons of hydrothermal life (Figure 4.3). Tubeworms, shrimps and mussels are distributed along a “hot-to-cold” gradient and have developed mutualistic relationships with the bacteria that convert chemical compounds from fluids into organic matter for the host. A few examples of these relationships are discussed here. Alvinella pompejana is a tubicolous worm that assembles in colonies around chimneys of the East Pacific Rise, and is one of the best-known animals described from hydrothermal vents (Bris and Gaill, 2007). A. pompejana is among the most thermotolerant metazoans on Earth, tolerating temperatures of 20–45  C at the tube opening and 40–80  C inside the tubes (Desbruyeres et al., 1998). A. pompejana hosts multiple species of episymbionts clustered in a hair-like biofilm on its dorsal surface. Metagenome analysis revealed a complex consortium of diverse bacteria involved in carbon dioxide fixation, sulfur oxidation and denitrification, as well as vitamin and amino acid biosynthetic pathways from which the host may benefit (Grzymski et al., 2008).

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Figure 4.3 (a,b) Symbiotic animals as icons for hydrothermal life with aggregates of Rimicaris exoculata shrimps from Mid-Atlantic Ridge Rainbow site; (c) Bathymodiolus azoricus mussels with covered by white filamentous bacterial mats (d); (e) Alvinella pompejana polychaetes colonizing tubes on black smoker chimneys along the East-Pacific Rise; (f) Riftia pachyptila vestimentiferan tubeworm from hydrothermal vents of the eastern Pacific. The photographs were processed during the IFREMER-Victor/EXOMAR (a–d), the IFREMER-Victor/PHARE (e), and the IFREMER-Nautile/BIG (f) oceanographic cruises.

The tubeworm Riftia pachyptila resides in lower-temperature fluids (ca. 25  C) that issue through seafloor openings or from the base of hydrothermal chimneys on the East Pacific rise (Luther et al., 2001). R. pachyptila has no mouth or digestive tract, and maintains an obligate nutritional association with chemolithoautotrophic symbionts housed in a specialized organ named the trophosome (Rodibart et al., 2008). The shrimp species Rimicaris exoculata dominates the faunal biomass at different sites along the Mid-Atlantic Ridge, and forms dense aggregates of thousands of individuals per square meter (Schmidt, Le Bris, and Gaill, 2008). Shrimps are found living close to active chimneys at temperatures between 13.2  5.5  C (Desbruyeres et al., 2001) and 8.7  2.3  C

(Zbinden et al., 2004). The niche of R. exoculata remains unclear, as no predator or scavengers were observed. Although its nutrition is not fully understood, it seems able to graze on the chimney walls (Segonzac, de Saint Laurent, and Casanova, 1993). Large apparently stable and specific chemoautotrophic symbiotic bacterial communities in the enlarged gill chamber (Petersen et al., 2010) and gut (Durand et al., 2010) appear to support the nutrition of these dense shrimp aggregates. R. exoculata appears to function much like a microbial interactions reactor, with mutualistic relationships between the shrimps and their autotrophic bacteria. These relationships appear to play a role in nutrition and/or detoxification processes, while syntrophic exchanges between sulfur-oxidizing and

4.3 The Five “W”s of Marine fungi: Who? What? When? Where? Why?

sulfur-reducing epibionts may increase the efficiency of metabolic pathways of the different partners (Hugler et al., 2011). Mussels of the Mytilidae family occur in hydrothermal vents around the world (von Cosel, 2002). The species Bathymodiolus azoricus is a major component of faunal communities at the shallower Mid-Atlantic ridge vent sites, and dwells at 6.0  3.9  C to 10.1  0.5  C (Desbruyeres et al., 2001). Symbioses between B. azoricus and sulfur-oxidizing and methane-oxidizing bacteria have been demonstrated (Cavanaugh et al., 1987; Duperron et al., 2006); this dual symbiosis allows the host to colonize sulfide- and/or methane-rich environments. Recently, B. azoricus was characterized as a mixotrophic organism, which obtains energy from both its symbionts and from filter-feeding, at a ratio that depends on the mussel size (Martins et al., 2008). The establishment of these endosymbionts may be a long process that is facilitated by filterfeeding at early development stages (small mussels), with an energetic-source switch appearing for only the larger mytilids. At the microscopic scale, dozens of bacteria and archaea have been isolated and fully characterized (Figure 4.4). Archaea at hydrothermal vents are mostly hyperthermophilic (with an optimal growth temperature above 80  C), and include methanogens, sulfur- or iron-reducers, and heterotrophs. Bacteria at vents can be either thermophiles (45–80  C) or mesophiles (15–45  C), with autotrophic to heterotrophic metabolisms. As observed for hydrothermal metazoa, strong physical and chemical gradients at the deep-sea vents allow for many different microbial lifestyles. Culture-independent methods now support this key point; the recent use of high-throughput sequencing coupled with qPCR has drawn patterns of microbial distribution depending on fluid chemistries, with hydrogen-rich fluids supporting methanogens and hydrogen-oxidizing thermophiles, and hydrogen-poor fluids supporting hyperthermophilic microaerophiles (Flores et al., 2011).

Figure 4.4 Description of new species of Archaea, Bacteria, and Eukarya, with their optimal growth temperature, isolated from deep-sea hydrothermal vents between 1983 and mid-2013.

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Hydrothermal vents are spectacular in their distribution, composition and biomass, and the discovery of life in these extreme environments has aroused a strong and longstanding research effort into the diversity, ecology and physiology of endemic macro and microorganisms that are inextricably linked to the unique fluid chemistries of these features. Yet, despite the many hundreds of multidisciplinary studies that have been conducted on deep-sea vents, knowledge of these ecosystems remains in its infancy, mostly because many of Earth’s spreading centers – and the unique biota they may harbor – have yet to be explored.

4.3 The Five “W”s of Marine fungi: Who? What? When? Where? Why?

Fungi in oceans form an ecologically defined group of microorganisms that have been intensively studied, starting with the first comprehensive study by Barghoorn and Linder in 1944. Important ecological roles have been clearly demonstrated in different types of marine ecosystem, yet few species of marine fungi have been listed to date. 4.3.1 Definition and Novel Concept

According to the universally accepted definition proposed by Kohlmeyer and Kohlmeyer (1979), fungi in the oceans are divided into two groups: obligate and facultative marine fungi. This dichotomic definition separates obligate marine fungi, which grow and sporulate only in a marine or estuarine habitat, from facultative marine fungi, which are from freshwater or terrestrial environments and are able to grow and possibly also sporulate in the marine environment. The definition of facultative marine fungi, as proposed by Kohlmeyer and Kohlmeyer, has not escaped debate, and a three-level classification has recently been proposed for categorizing marine fungi into groups of occurrence: (i) strict endemic, active marine fungi; (ii) ubiquitous, metabolically active marine fungi; and (iii) ubiquitous, passive fungi (Mahe et al., 2013). The motivation behind this revised definition was to provide an update, based on the huge recent advances in marine molecular mycology. Today, metagenomics and metatranscriptomics provide sufficient information to propose a definition based on function and activity. Interestingly, biotechnological studies are serendipitously providing ecological data to support this novel three-level classification concept. Facultative marine fungi synthesize a wide spectrum of secondary metabolites, and many of them differ from those of their terrestrial counterparts (Bhakuni and Rawat, 2010). The synthesis of original secondary metabolites from facultative marine fungi is evidence of metabolic activity and indicates that many of those fungi are active members of marine microbial consortia and not simply present as dormant spores in this ecosystem (Damare, Singh, and Raghukumar, 2012).

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Based on a quantitative view, marine fungal species represent a tiny fraction of global fungal diversity compared to terrestrial representatives. Indeed, recent reports have suggested that there are only 549 marine fungi, including 439 Ascomycota, 94 mitosporic fungi, and 16 Basidiomycota (Jones, 2011). Hence, only 0.74% of the fungal species described to date are derived from the oceans. Based on a recent minimum estimate of global fungal diversity, with 712 000 fungal species worldwide (Schmit and Mueller, 2007), this ratio would fall to 0.08%. These types of count are clearly biased due to: (i) a strong difference in sampling efforts between terrestrial and marine ecosystems; and (ii) a strict application of the dichotomic definition, leading to only the true marine fungi being considered and the facultative fungi omitted. On considering the newly proposed definition that centers on the whole active fungal community having an ecological role in marine environments, fungi are thought to represent a much more significant fraction in the marine environment with a great diversity of lineages and forms – that is, unicellular forms (yeasts and chytrids) as well as multicellular forms with filamentous hyphae. 4.3.2 Patterns of Distribution

The distribution of marine fungi is ruled by many biotic and abiotic factors, with some being more influential than others. Temperature, salinity and organic matter availability are the key elements that shape the distribution of marine fungal communities. Consequently, five temperature-determined biogeographical zones were proposed by Hughes (1974): temperate; subtropical; tropical; arctic; and Antarctic. Indeed, Spathulaspora antarcticum has only been isolated from the red algae Ballia callitricha in Antarctic seawaters, while Halosphaeria quadricornuta and Asteromyces cruciatus have only been recovered respectively in tropical and temperature seawaters (Kohlmeyer and Kohlmeyer, 1979). Digitatispora marina is another interesting candidate that fits this pattern, as it is a psychrotrophic fungi detected only in temperate waters during the winter. However, some ubiquitous marine fungi have also been identified, with Corollospora maritima, Halosphaeria appendiculata or Lignicola laevis each occurring in both tropical and temperate waters (Jones, 2000). Salinity is another important determinant of fungal distribution. Some fungal strains exhibit a capability to grow or to form fruiting bodies at specific concentrations of sea salts (Johnson and Sparrow, 1961), and recent results have suggested that salinity does indeed select for distinctive fungal communities (Mohamed and Martiny, 2011; Burgaud et al., 2013). Covarying factors may also have an important influence on fungal distribution; for example, organic matter availability appears fundamentally important as many marine fungi are decomposers of marine carbon substrates (Hyde et al., 1998). This factor is directly linked to the phytoplankton biomass in the overlying water column, and thus organic matter availability will correlate with periods of water column productivity. Large

fungal biomasses have often been seen to occur during seasonal peaks of production (Gutierrez et al., 2011). The distribution of marine fungi is also governed by interactions between factors that include temperature, salinity, organic matter availability, seasonality, and water depth. Each of these factors can be represented in a four-dimensional space with: (i) longitudinal dimension (salinity gradients, nutrient availability); (ii) latitudinal dimension (temperature); (iii) vertical dimension (hydrostatic pressure, nutrient availability); and (iv) time dimension (seasonality) with a consortium of complementary factors leading to a complex model of biogeographic distribution. 4.3.3 Ecological Roles

Marine fungi have been detected, by using culture-based and culture-independent methods, in a broad diversity of photosynthesis- and chemosynthesis-based ecosystems. In littoral regions the fungal species are mainly involved in wood decay, with an ability to cause soft rot and white rot attacks by the synthesis of extracellular cellulose-degrading cellulases and lignin-degrading laccases (Jones, 2011). Wood appeared as a suitable substrate for marine fungi in the oceanic biome; indeed, fungal sporocarps have been detected on wood fragments in the Vanuatu archipelago at depths of between 100 and 1200 m (Dupont et al., 2009). Marine fungi growing on algae are parasites, saprobes or endophytes, most of them being ascomycetes (Zuccaro et al., 2004; Zuccaro, Schulz, and Mitchell, 2003). The species richness of fungi growing on marine algae remains scant, with about 80 marine fungi isolated from specific algae classified in the four orders Dothideales, Halosphaeriales, Hypocreales and Lulworthiales (Zuccaro and Mitchell, 2005; Zuccaro, Schulz, and Mitchell, 2003). However, algae definitely represent a diversified substratum, distributed worldwide, for colonization by marine fungi. Marine planktonic ecosystems also contain a huge reservoir of life, with around 10 to 100 billion organisms per liter of oceanic water. Planktonic fungi, also referred to as mycoplankton, can form densities of 103 to 104 fungal cells per milliliter of seawater (Kubanek et al., 2003), and thus may play an important role as parasites or saprobes in the marine planktonic realm. Marine fungi may have a role in any type of sample in which they have been detected, such as marine plants, corals, mollusk shells, hydrozoan exoskeletons, annelid tubes and even in the deep-sea in extreme environments. Recently, fungal metabolic transcripts in deep marine sediments up to 159 m below the sea floor were reported, providing direct evidence for active fungal metabolism in the deep subsurface biosphere (Orsi et al., 2013). Fungi in deep sediments are clearly involved in carbohydrate, amino acid and lipid metabolism, indicating a potentially significant role in organic carbon turnover in sub-seafloor sediments. While some ecological roles can be easily determined using traditional culture-based approaches, some lend themselves

4.4 Fungi in Deep-Sea Hydrothermal Vents

more easily to DNA- or RNA-based molecular studies. Culturebased studies coupled with mRNA-based analyses using metatranscriptomics will certainly reveal interesting and possibly fundamental roles in marine biogeochemical cycles. 4.3.4 Origin of Marine Fungi

Much like the chicken-egg dilemma, an analogous question can be asked of marine fungi: “Which came first in the oceans? Marine fungi or terrestrial fungi?” A more accurate question would be to ask whether fungi have evolved in the oceans, or if their presence in this biome is a secondary adaptation after an initial terrestrial colonization. Many of the fungal isolates from marine ecosystems, and many of the fungal sequences detected using rDNA, are close relatives of terrestrial fungal species, and this leads to the hypothesis that marine fungi originated in terrestrial environments and then colonized the oceans. Several terrestrial-tomarine transitions have been detected using small and large subunit ribosomal RNA (SSU rRNA) -based phylogenetic analyses of Halosphaeriales, the major marine fungal group comprising one-fourth of the all marine fungal species, and also Lulworthiales (Campbell et al., 2005; Spatafora, VolkmannKohlmeyer, and Kohlmeyer, 1998). However, this picture of the evolution of marine fungi may be biased due to a relative undersampling of marine communities. Some answers to these questions may derive from further explorations of the deep, as many basal fungal lineages – that is, below the Dikarya radiation – have been detected in different deep extreme environments, such as cold seeps (Nagahama et al., 2011; Thaler, Van Dover, and Vilgalys, 2012), deep sediments (Nagano et al., 2010), or hydrothermal vents (Bass et al., 2007; Le Calvez et al., 2009). Such basal fungal lineages are useful for drawing new paradigms regarding the origin of marine fungi. The occurrence of ancient lineages in marine environments may indicate that fungi emerged and diversified in oceans, and more precisely in deep-sea extreme environments, and then colonized terrestrial environments (Le Calvez et al., 2009). Alternatively, a fungal emergence in freshwater has recently been proposed after the recovery of novel basal fungal sequences in freshwater ponds (Jones et al., 2011). Deep-branching fungal sequences were at a much higher frequency in freshwater than in marine environments (Richards et al., 2012). This new paradigm totally contradicts the previous version, by suggesting a terrestrial origin of marine fungi, although quantitative comparisons of the occurrences of freshwater and marine deep-branching sequences appear risky because of the different methods used. The high-throughput sequencing of genetic markers from several aquatic freshwater and marine ecosystems would most likely settle this dilemma with regards to the emergence of fungi. However, whether such results demonstrate an initial marine or freshwater emergence, much robust data still attest multiple marine-terrestrial transitions in both directions.

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4.4 Fungi in Deep-Sea Hydrothermal Vents

The present view of the composition of microbial communities at deep-sea hydrothermal vents was dramatically changed by the first SSU rDNA analyses to use specific primers targeting microeukaryotes. The detection of SSU rRNA gene sequences belonging to different eukaryotic lineages revealed complex communities, and allowed the formulation of hypotheses regarding ecological roles as grazers, detritivores, symbionts, or parasites (Edgcomb et al., 2002; Sauvadet, Gobet, and Guillou, 2010). One of the best-studied Pacific hydrothermal sites with regards to eukaryotes is the Guaymas Basin, in the Gulf of California. This site features vent plumes, seeps and anoxic sediments, each exhibiting a wide range of temperatures. Moreover, the sediments are covered in areas with surface-attached microbial mats of filamentous sulfur-oxidizing bacteria called Beggiatoa. Based on phylogenetic analyses of SSU rRNA gene sequences, some of the taxa detected appeared to have derived from novel, deep-branching taxa, while others represented novel, deep branches within well-described eukaryotic clades that included the stramenopiles, apicomplexa, dinoflagellates, ciliates, acantharea, and radiolaria (Edgcomb et al., 2002). In addition, SSU rRNA signatures of eukaryotes generally distributed in marine environments, such as certain fungi, radiolarian, acantharea, stramenopiles and ciliates, were detected (Edgcomb et al., 2002). A similarly diverse picture of protist communities (primarily diversity within alveolates) was obtained from hydrothermal sites in the Pacific, along the Mid-Atlantic Ridge at the Rainbow site, where abundant signatures of microbial eukaryotes were detected in fluid-seawater mixing regions, hydrothermal sediments, and on colonization devices deployed at these sites (Lopez-Garcia et al., 2003). However, some differences in the hydrothermal vent eukaryotic communities were observed between Atlantic and Pacific systems. For example, kinetoplastids, which appear to be abundant and diverse in Atlantic systems, were not detected in Pacific systems (Lopez-Garcia et al., 2003). Evidence of populations adapted to the anoxic, hydrothermal lifestyle comes from the recovery of sequences of taxa affiliated with known taxa from anaerobic sediments, such as the ciliates Metopus contortus and Trimyema compressum (e.g., Edgcomb et al., 2002). Ciliates (one group within the alveolates) appear to be common inhabitants of a wide range of extreme environments, including hydrothermal habitats (Coyne et al., 2013). They also live within complex protist communities found inside hydrothermal vent bivalves (Sauvadet, Gobet, and Guillou, 2010). The most abundant ciliate communities appear to be similar in different hydrothermal systems, although in the Guaymas Basin unique populations of ciliates were found to inhabit pigmented versus nonpigmented microbial mats and bare sediments (Coyne et al., 2013). The specific environmental factors driving these differences in observed diversity remain unknown. While it is difficult to interpret differences within molecular “snapshots” of SSU rRNA diversity from different hydrothermal environmental surveys due to methodological differences,

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fungal sequences were consistently retrieved in different studies (as discussed below). This opens a new era of research in marine mycology, with deep-sea hydrothermal vents as potential ecological niches for fungi, and potentially interesting “hotspots” for the discovery of novel taxa. 4.4.1 Hydrothermal Vents as Life Oases for Fungi

Using SSU rDNA clone library and Sanger-sequencing methods, the occurrence of fungal sequences in hydrothermal environments was assessed at different oceanic ridges (Bass et al., 2007; Edgcomb et al., 2002; Lopez-Garcia et al., 2003; LopezGarcia, Vereshchaka, and Moreira, 2007; Sauvadet, Gobet, and Guillou, 2010). Fungal sequences were retrieved from the nearsurface (first 2 cm) layers of sediments sampled at the Guaymas Basin hydrothermal vents (Gulf of California). No fungal sequences were detected at 3 cm depth in sediment cores, where the temperatures rose to 65  C (Edgcomb et al., 2002). Fungal sequences were also harvested from seawater, microcolonizers and sediment samples at Mid-Atlantic ridge hydrothermal sites (Bass et al., 2007; Lopez-Garcia et al., 2003; Lopez-Garcia, Vereshchaka, and Moreira, 2007). Finally, some fungal sequences were also retrieved from Pacific and Atlantic Ocean deep-sea bivalves Bathymodiolus azoricus and Calyptogena magnifica (Le Calvez et al., 2009; Sauvadet, Gobet, and Guillou, 2010). The sequences recovered provided information about fungal community structures in deep-sea hydrothermal vents that seems to be dominated by ascomycetes and basidiomycetes. This group, named “hydrothermal and/or anaerobic fungi” by Lopez-Garcia et al. (2007), appeared as the most consistently detected fungi in hydrothermal vents, but they were also detected in other deep-sea environments such as deep sediments, where ribosomal RNA detection suggests they are metabolically active (Edgcomb et al., 2011). Clone library surveys also revealed unique fungal sequences branching within the fungal radiation closest to chytrids. Culture-based analyses led to the isolation of many known, but also novel, yeast species (Burgaud et al., 2010; Burgaud et al., 2011; Gadanho and Sampaio, 2005; Nagahama, Hamamoto, and Horikoshi, 2006) and also many filamentous fungi (Burgaud et al., 2009). The only two cultured endemic fungal species from hydrothermal vents described to date have been yeasts belonging to higher fungi – one basidiomycete and one ascomycete – named Rhodotorula pacifica (Nagahama, Hamamoto, and Horikoshi, 2006) and Candida oceani (Burgaud et al., 2011), respectively. R. pacifica was isolated from sediment samples collected at a depth of 991 m at north-west Pacific Ocean, while C. oceani was isolated from two sites: (i) from water samples near Menez Gwen hydrothermal field at 825 m (Gadanho and Sampaio, 2005); and (ii) from deep-sea coral near the Rainbow hydrothermal vent at 2300 m depth (Burgaud et al., 2011). However, many novel fungal species have not yet been described. Fungal strains have also been isolated at Atlantic and Pacific oceanic ridges from sediment, seawater and endemic animal tissue samples. The culture conditions employed did

not allow for the isolation of chytrids or representatives of the “hydrothermal and/or anaerobic fungi”, as only ascomycetes and basidiomycetes were retrieved. Statistical distribution tests on fungal isolates have highlighted a strong heterogeneity of isolation frequency, depending on the sampling sites. Most of the strains were obtained from the Mid-Atlantic Ridge Rainbow site, a hydrothermal vent which displays unique characteristics, with high H2, CH4, CO and Fe concentrations and low H2S concentrations that may lower fluid toxicity and therefore improve conditions for fungal growth. Such distribution tests also led to the visualization of an aggregate distribution of fungi in deep-sea hydrothermal vents. This indicated that those fungi associated mostly with endemic animals were only retrieved from a few individuals; this, in turn, suggests that such fungi may be facultative parasites or opportunistic pathogens of deep-sea animals (Burgaud et al., 2009). The ecological roles of fungi in hydrothermal vents are far from resolved, but hints exist regarding parasitic or saprotrophic lifestyles. For example, the results of different studies have clearly indicated that fungi isolated from hydrothermal mussels are not mutualists but seem to be facultative parasites (Burgaud et al., 2009; Van Dover et al., 2007). The presence of chytrids was revealed using culture-independent methods, though no representative has been isolated to date. This could be explained by a strict parasitic lifestyle that is clearly an impediment to isolation. This hypothesis is congruent with observations of chytrids that are known to parasitize macroalgae (K€ upper et al., 2006). Saprotrophic lifestyles also seem to occur among fungi, since fungi are mostly retrieved from organic-rich microenvironments, such as the inner side of shrimp branchiostegites and the interior or exterior of hydrothermal vent mussels. 4.4.2 Physiological Adaptations

Recently, molecular studies have greatly enhanced the present knowledge of fungal communities dwelling at deep-sea hydrothermal vents, with hundreds of different fungal sequences having been retrieved. Although those molecular studies allow the identification and processing of phylogenetic placements of fungal signatures, SSU rDNA sequences offer no information regarding the physiology of the fungi behind the sequences. As stated by Bass et al. (2007), it is possible that the organisms represented by these sequences are specifically adapted to deepsea habitats, but this can only be confirmed with more detailed genomic and/or culture-based analyses. Culture collections of fungal isolates from deep-sea hydrothermal sites have been examined to identify individuals at the species level, and to assess their physiological adaptations to deep-sea conditions (Burgaud et al., 2010; Burgaud et al., 2009). The objective was to determine whether the fungal strains were: (i) inactive spores at deep-sea vents; (ii) active, even if the in-situ conditions are not optimal; or (iii) active and adapted to in-situ conditions. Temperature separated the filamentous fungal

4.4 Fungi in Deep-Sea Hydrothermal Vents

strains into two groups, namely psychrotrophs and mesophiles. Most of the filamentous fungal strains isolated were psychrotrophs, isolated from mussels or shrimps thriving at low temperatures in proximity to hydrothermal vents (Desbruyeres et al., 2001). Such psychrotrophic strains thus appeared active, and adapted to hydrothermal conditions at those locations near vents. Salinity was another structuring parameter for yeasts that separates halophiles from halotolerant and nonhalophilic taxa. One hydrothermal vent taxon described above, Candida oceani, was found to be a halophile (Burgaud et al., 2011). This seems to indicate that halophilic strains are endemic deep-sea marine fungi, while others may be ubiquitous but adapted taxa that occur at hydrothermal vents as a result of sedimentation or other natural phenomena such as the circulation of water masses. Future studies of growth rates at different elevated hydrostatic pressures will certainly add to these data on temperature and salinity. Indeed, hydrostatic pressure appeared as the most significant physical parameter in the cold abyss (Lauro and Bartlett, 2008), and will definitely determine whether some strains are able to live at great depth, or not. The fact that some terrestrial fungal representatives are able to adjust their membrane composition under pressure to tolerate high-hydrostatic pressure (Fernandes et al., 2004) indicates that the colonization of hydrothermal vents by terrestrial fungi is possible. It may also explain why so many sequences affiliated with known terrestrial representatives are found in deep-sea hydrothermal vents. Physiological analyses of fungal strains isolated from deep-sea hydrothermal vents are providing interesting information that will certainly be reinforced by future deep-sea fungal genome analyses. Full-genome sequencing of deep-sea fungal representatives would also be useful for biotechnological applications – that is, the screening of secondary metabolites using in-silico secondary metabolite pathway analyses.

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organisms, including plants and microorganisms. A large number of studies to investigate these pathways have been based on an analysis of the presence or absence of biosynthetic genes in the genome, such as polyketide synthase (PKS) or non-ribosomal peptide synthetase (NRPS) (Medema et al., 2011; Ziemert et al., 2012). In the absence of information from whole genomes, the presence of these genes can be detected by amplification using random priming (Amnuaykanjanasin et al., 2009; Amnuaykanjanasin et al., 2005), followed by sequencing and further phylogenetic analysis to establish whether the encoded enzymes are involved in the production of antimicrobial or anticancer compounds (Trindade-Silva et al., 2013). These analyses can be processed step-by-step, following a flow chart (Figure 4.5). A strong focus on marine fungi associated with sponges or corals was initiated to search for PKS or NRPS, and to estimate the biotechnological potential of different microorganisms (Thomas, Kavlekar, and LokaBharathi, 2010). Recently, an increasing number of studies have shown that marine fungi are an interesting source of secondary metabolites displaying interesting biological and pharmacological properties, with 690 structures having been elucidated between 2006 and mid-2010 (Rateb and Ebel, 2011). Different categories of bioactive metabolites were retrieved from those marine fungi with polyketides (40% of 690 structures), alkaloids (20%), peptides (15%), terpenoids (15%), prenylated polyketides

4.4.3 Biotechnological Potential

Oceans are the future for natural products research. Hundreds of interesting molecules have been found in marine habitats, and microbes – including Bacteria, Archaea, Fungi and other eukaryotes – are the main producers. Each year, several global ocean cruises are organized to identify new marine organisms and especially microbes, the most recent being the Tara Oceans expedition, a three-year expedition to better understand planktonic ecosystems from viruses and bacteria to metazoa. Microorganisms produce a variety of secondary metabolites, which could have bioactive properties, and many marine microbes have been shown to produce antibacterial, antifungal, antitumor or anti-allergic agents (Bhatnagar and Kim, 2012; Mayer et al., 2011; Mayer et al., 2009). Secondary metabolites can be divided into several chemical groups of terpenes, polyketides, peptides, and alkaloids (not including the proteins) (Mayer et al., 2011). The pathways responsible for the biosynthesis of these metabolites have been identified and characterized in several terrestrial

Figure 4.5 Flow chart depicting the methodology to use for a complete screening of fungal strains from isolation and culture to the development of a pharmaceutical product.

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(7%), shikimates (2%), and lipids (1%). Polyketides from marine fungi showed different types of activity, including: (i) anthelmintic compounds, such as nafuredin, from Aspergillus niger  isolated from sponges (Omura et al., 2001); (ii) anticancer compounds, such as chaetomugilins, leptosphaerone C or penicillenone isolated from Chaetomium globosum and Penicillium sp. isolated respectively from marine fishes and mangrove plants (Lin et al., 2008; Yamada, Muroga, and Tanaka, 2009); (iii) antimicrobial compounds with ascochyatin obtained from Ascochyta sp. isolated from a floating debris (Rateb and Ebel, 2011); and (iv) antiviral compounds, such as balticols obtained from an ascomycetous fungi isolated from driftwood (Shushni et al., 2009).

4.5 Conclusions

Fungi dwelling in deep-sea ecosystems, and especially in hydrothermal vents, appear to represent an interesting untapped resource with biotechnological potential. Indeed, some prokaryotes from hydrothermal habitats have led to significant developments in molecular biology, such as the discovery of the

proofreading DNA polymerase Pfu, from Pyrococcus furiosus (Lundberg et al., 1991). The possibility that marine fungi isolated from extreme environments may also produce thermostable proteins and novel metabolites makes them extremely interesting to current research groups. Coupled to the fact that the establishment of cultures in the laboratory is relatively easy (as most do not require partner organisms to grow, and their ecophysiological features are known), these organisms may hold the key to the identification, isolation and exploitation of molecules for future therapeutic use.

Acknowledgments

The authors thank Anne Godfroy, current director of LM2E (IFREMER-Brest), for providing the photographs of hydrothermal vents and associated fauna. All members of the GDR ECCHIS are also thanked for their fruitful discussions. The authors are grateful to ANR Deep-Oases and French Research Ministry for their financial support. This project was also supported by MaCuMBA funds from the European Community (Ref. FP7-KBBE-2012-6-311975) and Ocean Life Institute (WHOI) to VE (OLI-27071359).

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About the Authors Ga€etan Burgaud obtained his PhD degree in Marine Microbiology from European University of Brittany (France) for his work on fungal communities from hydrothermal vents. During his post-doctoral training, he worked on the characterization of marine subsurface fungi. He is now an associate professor at the European University of Brittany. He develops his research on: (i)

the characterization of fungal diversity in marine extreme environments using culture-based approaches; (ii) the taxonomic identification of novel fungal species; (iii) the high-throughput sequencing of fungal communities in the marine realm; and (iv) assessment of the biotechnological potential of such extreme eukaryotic microorganisms.

References

Laurence Meslet-Cladiere is an associate professor at LUBEM laboratory, European University of Brittany. Her subject is to elucidate the secondary metabolic pathways of fungi in cheese and in oceanic environments. She obtained her PhD degree in 2003, on Photorhabdus temperate, a Gram-negative entomopathogenic bacterium of the Enterobactericae. She tried to elucidate the virulent factors implicated in the killing of insect larvae. During three years, she worked on the DNA replication and repair in hyperthermophile Archaea, Pyrococcus abyssi. Between 2007 and 2009 she tried to create a method to perform homologous recombination in the Chlamydomonas reinhardtii nucleus and mutagenesis experiments, at the IBPC Institute, in Paris. Starting in 2010, at the Biological Institute of Roscoff, she worked on the biosynthesis pathway(s) of phlorotannins in brown macroalgae, by the integration of functional genomics and ecological/chemical methods. Georges Barbier is professor in microbial ecology at the Universite de Bretagne Occidentale (UBO), Ecole Superieure d’Ingenieurs en Agro-alimentaire de Bretagne atlantique (ESIAB), and head of the university laboratory of biodiversity and microbial ecology (LUBEM, EA3882). With 66 peerreviewed publications recorded in WOS, his research activity

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was devoted to microbial taxonomy and ecology, first at Ifremer, Brest center (1989–2002), mainly related to bacteria and archaea living at deep-sea vents, and then at UBO (2002now) concerning fungi of different niches, including those in the ocean. Virginia P. Edgcomb is a Research Specialist in the Department of Geology and Geophysics at the Woods Hole Oceanographic Institution (WHOI), Woods Hole, MA. She earned her PhD from the University of Delaware (Department of Biology) in 1997. As postdoctoral researcher, she spent three years at the Marine Biological Laboratory, where she was involved in studies of early eukaryotic evolution and of microbial diversity at hydrothermal vents, followed by two years at WHOI where she studied the tolerance of several marine prokaryotes to extreme conditions found at hydrothermal vents. Her current research interests include the diversity and evolution of protists, the microbial ecology of dysoxic and anoxic/sulfidic marine environments, investigations of the deep biosphere, and symbioses between protists and prokaryotes in extreme environments, including hypersaline anoxic basins, anoxic and sulfidic marine water column and sedimentary environments, and subsurface marine sediments.

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5 Glycolipids from Marine Invertebrates Gilles Barnathan, Aur
elie Couzinet-Mossion, and Ga€etane Wielgosz-Collin

Abstract

Glycolipids constitute a broad class of natural compounds belonging to the glycoconjugates. They can usually be subdivided into large groups comprising neutral glycosphingolipids, gangliosides, and various atypical glycolipid structures. Most glycolipids have been found in echinoderms (sea stars, sea cucumbers) and sponges, but the most surprising observation is the very large variety of

5.1 Introduction

Glycolipids (GLs) constitute a broad class of natural compounds belonging to the glycoconjugates. Depending on the nature of the lipid moiety, GLs can be usually subdivided into large groups comprising glycoglycerolipids, glycosphingolipids (GSLs), and various atypical GL structures. Glyceroglycolipids are more widely distributed among microbes and plants, and have only been found in sea urchins as sulfolipids. Marine invertebrates have been found to contain mainly GSLs. The GSLs are undoubtedly the most abundant and diverse class of GLs, occurring not only in marine invertebrates but also in animals, plants, and fungi. A variety of GSLs have been reported from marine invertebrates, with the majority being found in echinoderms and marine sponges. GSLs, in turn, form part of a larger family of sphingolipids (lipids possessing ceramide as their core structure such as sphingomyelin). Some GLs exhibit various biological activities such as antifungal, antitumor, immunomodulatory and nitric oxide (NO) release-inhibiting activities. Usually, GSLs are further subclassified as neutral (with no charged sugars or ionic groups), sulfated, or sialylated (having one or more sialic acid residues). They are built on a ceramide lipid moiety that consists of a long-chain amino-alcohol (sphingoid base) in amide linkage to a fatty acid. In this chapter, only

structures identified. In addition, new glycolipids may be encountered in different organisms. Some glycolipids exhibit various biological activities such as antitumor, immunomodulatory and nitric oxide release-inhibiting activities. In this chapter, the natural occurrence of glycolipids and their structure identification and biological properties are described, including details of immunostimulatory and antitumor activities.

neutral GSLs will be designated as such; other GSLs containing a sialic acid will generally be termed gangliosides. An initial comprehensive review included literature details up to 1982 (Kochetkov and Smirnova, 1987). The GLs determined were found mainly sphingolipids, but a number of various different structures have also been described; interestingly, no biological activities were reported in the initial review. In 1970, a large-scale study using thin-layer chromatography (TLC) demonstrated the occurrence of GLs in several phyla of marine invertebrates, sponges and echinoderms having the highest GL contents (Vaskovsky et al., 1970). The GLs were mainly cerebrosides, while ganglioside-like lipids were also observed in echinoderms. Species of marine invertebrates containing GLs are listed in Table 5.1. The status of the different species was controlled by applying the World Register of Marine Species (Appeltans et al., 2012). In this chapter, attention is focused on GLs from marine invertebrates that have been identified between 1970 and early 2013. Several areas of GLs will be examined in succession, based on their gross structures and natural occurrences; these will include neutral GSLs, glycosylceramides, gangliosides, and various atypical GLs. One part of the chapter will be devoted to the biological activities of these three classes of compounds. In addition to the natural sources, examples will be provided of the synthesis of some important GLs and related structures, with regards to their interesting bioactivities and the need to have sufficient quantities for further investigation.

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Table 5.1 Species of marine invertebrates containing glycolipids.

Phylum

Subphylum

Class

Trivial name

Species

Chordata

Tunicata

Ascidiacea

Tunicate

Cnidaria

—

Anthozoa

Soft coral

Microcosmus sulcatus (now accepted M. vulgarisa)) Phallusia fumigata Lobophytum crassum Lobophytum sp. Sarcophyton ehrenbergi

Sea anemone

Metridium senile

Echinodermata

Asterozoa

Asteroidea

Starfish

Acanthaster planci Allostichaster inaequalis Anasterias minuta Aphelasterias japonica Asterias amurensis Asterias amurensis versicolor Asterias rubens Asterina pectinifera (now accepted Patiria pectiniferaa)) Astropecten latespinosus Cosmasterias lurida Culcita novaeguineae Evasterias echinosoma Evasterias retifera Lethasterias fusca Linckia laevigata Luidia maculata Luidia quinaria bispinosa Narcissia canariensis Ophidiaster ophidiamus Oreaster reticulatus Pentaceraster regulus Protoreaster nodosus Stellaster equestris

Porifera

Ophiuroidea

Brittle star

Ophiocoma scolopendrina

Crinozoa

Crinoidea

Feather star

Comanthus japonica (now accepted Oxycomanthus japonicusa)) Comanthina schlegeli (now accepted Comaster schlegeliia))

Echinozoa

Echinoidea

Sea urchin

Anthocidaris crassispina (now accepted Heliocidaris crassispinaa)) Diadema setosum Echinarachnius parma Echinocardium cordatum Hemicentrotus pulcherrimus Strongylocentrotus intermedius Temnopleurus toreumaticus

Holothuroidea

Sea cucumber

Acaudina molpadioides Bohadschia argus Cucumaria echinata (now accepted Pseudocnus echinatusa)) Cucumaria japonica (now accepted Cucumaria frondosa japonicaa)) Cucumaria frondosa Holothuria leucospilota Holothuria pervicax Holothuria coronopertusa Pentacta australis Stichopus chloronotus Stichopus japonicus (now accepted Apostichopus japonicusa))

Demospongiae

Sponge

Agelas axisera Agelas conifera Agelas dispar Agelas clathrodes (synonymy Chalinopsis clathrodes) Agelas longissima Agelas mauritianus (mauritiana) Amphimedon viridis Amphimedon sp. Aplysinella rhax

—

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

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Axinella sp. Axinella corrugata Axinella damicornis Axinyssa djiferi Calyx sp. Caminus sphaeroconia Chondrilla nucula Chondropsis sp. Discodermia dissoluta (synonymy Desmahabana violacea) Ectyoplasia ferox Erylus placenta Erylus lendenfeldi Haliclona (Reniera) sp. Halichondria cylindrata Halichondria japonica Halichondria panicea Ircinia fasciculata Luffariella sp. Myrmekioderma dendyi (synonymy Raspaigella dendyi; Raspaigella tulearensis) Pachymatisma johnstonia Penares sollasi Plakortis simplex Phyllospongia foliascens (now accepted Carteriospongia foliascensa)) Pseudoceratina crassa (now accepted Aiolochroia crassaa)) Rhizochalina incrustata (now accepted Oceanapia incrustataa)) Siphonodictyon coralliphagum (synonymy Aka coralliphagum) Oceanapia phillipensi Oceanapia sp. Oceanapia ramsayi (synonymy Phloeodictyon ramsayi; Rhizochalina ramsayi) Stylissa flabelliformis Terpios sp. Trikentrion laeve a) The status of the species was controlled by the World Register of Marine Species (Appeltans et al., 2012).

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

Glycosphingolipids (GSLs, glycosylceramides) are an important class of membrane lipids, with a high structural diversity and a variety of biological functions. They occur in the cell membranes of organisms, from bacteria to human. A GSL contains a carbohydrate chain of one to several sugars linked to an aglycone (the ceramide), which is composed of a long-chain aminoalcohol (the sphingoid base) amide linked to a fatty acyl chain. Each sphingoid base and fatty acid (FA) is a mixture of various long alkyl chains (Figure 5.1).

If R ¼ R0 ¼ OH in Figure 5.1, the structure corresponds to amphimelibioside C, a major GSL from the sponge Amphimedon sp., whose the sugar chain 1-O-b-D-glucopyranosyl-(1 ! 6)a-D-galactopyranosyl is linked to the ceramide (2S,3S,4R,6E)-2(20 R)-2-hydroxydocosanoyl-2-amino-6-octadecene-1,3,4-triol (Emura et al., 2005). The structure of the reported GSLs was elucidated by using mass spectrometry (MS) and nuclear magnetic resonance (NMR), with the compounds often present as the peracetylated derivatives. Acid methanolysis allowed the detailed composition of the lipidic part of the molecule to be determined. In the GSLs of marine animals, the sphingoid base may be unsaturated and/or branched, while the fatty acyl chain may

Figure 5.1 Structure of representative glycosphingolipid (R ¼ H or OH/R0 ¼ H or OH).

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be a-hydroxylated. The first sugar linked to ceramide (inner sugar) in marine invertebrates is typically b-linked glucopyranose (GlcCer) or galactopyranose (GalCer). Among the GSLs, “cerebrosides” is the trivial name often used for the lipid-class monoglycosylceramides. Usually, the natural glycosylceramides contain a b-glycosyl linkage (Berg
e and Barnathan, 2005; Kornprobst, 2010). Glycolipids from marine sponges with immunomodulatory activities have been reviewed (Costantino, Fattorusso, and Mangoni, 2001a). Because of their very promising immunomodulatory and antitumor properties, a new class of GSLs has emerged, which have an a-galactosylceramide structure that is unprecedented among natural products. 5.2.1 a-Glycopyranosylceramides 5.2.1.1 a-Monoglycosylceramides The first reported examples of a-GSLs were probably amphicerebrosides E and F, which contained glucopyranosamine as the sugar moiety a-linked to the ceramide (Table 5.2). These were isolated from the sponge Amphimedon viridis, harvested in the Gulf of Eilat (Red Sea), and also contained GSLs with b-glucopyranosamine (Hirsch and Kashman, 1989). Another a-GSL with glucopyranose was isolated along with some b-glucopyranosylceramides during an investigation of the soft coral Sarcophyton ehrenbergi, collected near Taiwan (Cheng et al., 2009). The fatty acyl chain proved to be monounsaturated and 2-hydroxylated (20 R,30 E), while the long-chain base (LCB) was (4E,8E,10E) triunsaturated and 9-methyl-branched. This rare sphingoid type will be described for several b-glycosylceramides (see Section 5.2.2). The most interesting a-GSLs, in terms of biological activity, contain galactopyranose as the inner sugar. Indeed, an important discovery was made in 1993 (Natori, Koezuka, and Higa, 1993) when new GSLs named agelasphins were isolated from a lipophilic extract of the marine sponge Agelas mauritianus, collected near Okinawa, Japan, and determined as the first natural a-galactosylceramides (Table 5.2). The agelasphins, which were shown to be active substances during the course of screening of antitumor agents (Natori, Koezuka, and Higa, 1993; Natori et al., 1994), occur in the sponge as a mixture of homologs that differ in their composition of FAs and sphingoid bases. They were first isolated via an antitumor and immunostimulatory bioassayguided purification from an extract of A. mauritianus (Natori et al., 1994), during which three b-glucosylceramides were also identified. The absolute configurations of the agelasphins were obtained by mean of a total synthesis of the most efficient agelasphin, AGL-9b (Akimoto, Natori, and Morita, 1993; Natori et al., 1994). The fatty acyl chains proved to be saturated (C23 to C25), as were the long-chain bases (2S,3S,4R)-2-amino1,3,4-alkyltriols (normal, iso, and anteiso C16 to C19). Several additional sponge species of the Agelas genus were then studied for GSLs. These a-galactosylceramides were identified in lipid extracts of the sponges A. clathrodes (Costantino, Fattorusso, and Mangoni, 1995a), A. longissima (Cafieri et al.,

1995) and A. conifera, collected along the coasts of Little San Salvador Island (Costantino, Fattorusso, and Mangoni, 1995b). The three major fatty acyl chains and sphinganines for all other cited GSLs are listed in Table 5.2. In fact, four GSLs were isolated from the sponge A. clathrodes, each being a mixture of the homologs (Costantino, Fattorusso, and Mangoni, 1995a). One of the GSLs was a b-glucopyranosylceramide containing a branched triunsaturated sphingoid base and 2-hydroxy FAs (see Section 5.2.2), while another was an a-galactopyranosylceramide possessing C22:0–C24:0 acyl chains and C16:0–C21:0 sphinganines. 5.2.1.2 a-Diglycosylceramides In a first investigation of GSLs of the Caribbean sponge A. longissima, the sponge was shown to produce large amounts of a novel digalactoside named longiside, an a-digalactosylceramide in which the rare b-galactofuranose is (1 ! 4)-linked to the a-galactopyranose, which is itself linked to the ceramide (Cafieri et al., 1994). This was the first example of a marine natural diglycosylceramide. A reinvestigation of GSLs from A. longissima collected in the Caribbean Sea allowed the identification of new GSLs, each as a mixture of homologs (Cafieri et al., 1995). The composition in terms of FAs and sphingoid bases of the ceramide moiety was established by the chemical degradation of an aliquot of the GSL being investigated. Such an a-digalactosylceramide with the rare b-galactofuranose has been also identified in A. clathrodes (Costantino, Fattorusso, and Mangoni, 1995a), A. conifera (Costantino, Fattorusso, and Mangoni, 1995b), and A. axisera collected near Okinawa, Japan (Uchimura et al., 1997a). In addition, A. longissima and A. dispar contained a-digalactosylceramides where the first a-galactopyranose is (1 ! 2)linked to the a-galactopyranose, which is itself linked to the ceramide (Cafieri et al., 1995; Costantino et al., 1996). These specimens of A. longissima also contained small amounts of the already described a-mono-GSL. Details of the three major fatty acyl chains and sphinganines are listed in Table 5.2. This type of GSL was also observed in an unidentified Japanese sponge (Uchimura et al., 1997a). Other interesting new GSL molecules were isolated from A. conifera and A. longissima, where an a-glucopyranose is (1 ! 2)linked to the inner a-galactopyranose (Cafieri et al., 1995; Costantino, Fattorusso, and Mangoni, 1995b). Indeed, in addition to the three GSLs previously identified in A. clathrodes, the Caribbean sponge A. conifera contained a new a-GSL present as the major component of the GSL mixture (Costantino, Fattorusso, and Mangoni, 1995b). Such compounds also occurred in A. longissima (Cafieri et al., 1995), where the latter GSL was found as a mixture of homologs. Interestingly, the sugar moiety of the novel GSL was established as a-glucopyranosyl-(1 ! 2)a-galactopyranoside, the acid methanolysis of which allowed the composition of the lipidic part of the molecule to be determined. The 2-hydroxytetracosanoic acid accounted for 77% of the 2hydroxy FA mixture, while the sphingoid bases were C17:0–C21:0 (2S,3S,4R)-4-sphinganines (Costantino, Fattorusso, and Mangoni, 1995b).

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

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Table 5.2 Glycosphingolipids containing an a-glycosyl linkage.

Ceramide/GSL name

sarcoehrenoside A

Organism

Biological activity

Reference

Sponge Amphimedon viridis

n. r.

Hirsch and Kashman, 1989

Octocoral Sarcophyton ehrenbergi

No antibacterial Reduced iNOS protein expression Anti-inflammatory

Cheng et al., 2009

Sponge Agelas mauritianus

Antitumor Immunostimulatory

Natori, Koezuka, and Higa, 1993; Natori et al., 1994

Sponge Agelas clathrodes

n. r.

Costantino, Fattorusso, and Mangoni, 1995a

Sponge Agelas longissima

n. r.

Cafieri et al., 1995

(continued )

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Table 5.2 (Continued) Ceramide/GSL name

longisidec x ¼ 21 R ¼ -(CH2)11-CH3

three GSLs FA=LCB 2-OH-C22:0 =C16:0 2-OH-C22:0 =C16:0 2-OH-C22:0 =C18:0

Organism

Biological activity

Reference

Sponge Agelas conifera

n. r.

Costantino, Fattorusso, and Mangoni, 1995b

Sponge Agelas longissima

n. r.

Cafieri et al., 1994

Sponge Agelas longissima

n. r.

Cafieri et al., 1995

Sponge Agelas clathrodes

n. r.

Costantino, Fattorusso, and Mangoni, 1995a

Sponge Agelas conifera

n. r.

Costantino, Fattorusso, and Mangoni, 1995b

Sponge Agelas dispar

n. r.

Costantino et al., 1996

Sponge Agelas axisera

Immunostimulatory

Uchimura et al., 1997a

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

three GSL FA=LCB 2-OH-C22:0 =C16:0 2-OH-C24:0 =C16:0 2-OH-C24:0 =C18:0

not reported

Sponge Agelas longissima

n. r.

Cafieri et al., 1995

Sponge Agelas dispar

No immunostimulatory

Costantino et al., 1996

Unidentified sponge

Immunostimulatory

Uchimura et al., 1997a

Sponge Agelas conifera

n. r.

Costantino, Fattorusso, and Mangoni, 1995b

Sponge Agelas longissima

No immunostimulatory

Cafieri et al., 1995

Sponge Agelas clathrodes

n. r.

Costantino, Fattorusso, and Mangoni, 1995a

Sponge Agelas dispar

Stimulatory effect on lymphocyte proliferation

Costantino et al., 1996

j 105

(continued )

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5 Glycolipids from Marine Invertebrates

Table 5.2 (Continued) Ceramide/GSL name

Organism

Biological activity

Reference

Sponge Axinella damicornis

Immunostimulatory

Costantino et al., 2005

Sponge Agelas sp.

Anticancer

Pettit et al., 1999

Sponge Agelas dispar

Immunomodulatory

Costantino et al., 1996

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

Sponge Agelas longissima

n. r.

Cafieri et al., 1996

Sponge Axinella sp.

n. r.

Costantino et al., 1994a

Sponge Axinella sp.

n. r.

Costantino et al., 1994a

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(continued )

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Table 5.2 (Continued) Ceramide/GSL name

Organism

Biological activity

Reference

corrugoside

Sponge Axinella corrugata

Immunostimulatory

Costantino et al., 2008

two GSLs FA=LCB 2-OH-C24:0 =C18:0 2-OH-C26:0 =C18:0

Sponge Stylissa frabeliformis (¼ flabelliformis)

Immunostimulatory

Uchimura et al., 1997a

Sponge Agelas longissima

n. r.

Cafieri et al., 1996

Sponge Agelas clathrodes

n. r.

Costantino et al., 2004

Composition of fatty acyl chains and sphinganines are given for the three major GSL structures of each type (relative percentages in brackets) n. r. ¼ not reported; FA ¼ fatty acid; LCB ¼ long-chain base

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

Among the continuing investigations on sponge GSLs, the Italian group also examined the sponge A. dispar, collected along the coasts of Islands of the Bahamas (Costantino et al., 1996). Additional new a-diglycosylceramides were isolated from A. clathrodes and A. dispar, with 2-acetamido-2-deoxy-a-galactopyranosyl-(1 ! 3)-a-galactopyranose as the sugar moiety (Costantino, Fattorusso, and Mangoni, 1995a; Costantino et al., 1996). The sponge Axinella damicornis, collected along the coast of Sorrento, Italy, was studied for GLs, and a new a-GSL – damicoside – was identified with 2-acetamido-2-deoxy-a-galactopyranosyl-(1 ! 4)-a-galactopyranose as the sugar moiety (Costantino et al., 2005). This was a rare example of an a-galactopyranosylceramide being isolated from a sponge belonging to the genus Axinella. 5.2.1.3 a-Triglycosylceramides A human cancer cell line bioassay-guided investigation of the Western Pacific marine sponge Agelas sp. led to the isolation of a minor novel GSL, agelagalastatin, which contained an inner a-galactopyranoside linked to a digalactofuranosyl unit (Pettit et al., 1999). In a further investigation of A. dispar, Constantino and coworkers isolated a novel triglycosylceramide in which the inner a-galactopyranose is linked to another a-galactopyranose and to N-acetyl-b-galactopyranose (Costantino et al., 1996). The same GSL structure was identified in the more polar GL fractions from the methanolic extract of A. longissima, together with a new tetraglycosylated GSL (Cafieri et al., 1996). The occurrence of a-GSLs is not limited to the genus Agelas, as shown by their identification in the sponge Axinella sp. (Costantino et al., 1994a). The latter species, collected along the coasts of the Reunion Island, contained two new GSLs, named axiceramides A and B (Costantino et al., 1994a), which were the first examples of marine natural triglycopyranosylceramides. Interestingly, the two hexose units linked to the inner a-galactopyranose were also engaged in a-glycosidic linkages. Both axiceramides had N-acetyl-a-2-amino-2-deoxygalactopyranose linked at C-30 , and differed by the second sugar linked to the C-20 , a-glucopyranose or a-galactopyranose (Table 5.2). Interestingly, a close relationship of the genera Agelas and Axinella is generally accepted on the basis of similarities in chemical composition, and also for biological reasons (Bergquist, 1978). Additional evidence for this close relationship was mainly obtained by Chombard et al. (1997), using 28 S rRNA sequence data. Further support for this chemotaxonomic relationship emerged from an investigation of lipids of the sponge Axinella corrugata, collected along the coasts of Bahamas Islands, that led to the isolation of a new triglycosylated a-Gal-GSL along some known b-Glc-GSLs (Costantino et al., 2008). The ceramide portion of the molecule was composed of a trihydroxylated, saturated sphinganine and a 2-hydroxy FA residue. Thus, almost only sponges of the genera Agelas and Axinella were shown to contain a unique class of GSLs with a-galactose as the first sugar of the saccharide chain.

j 109

Surprisingly, two new a-triglycosylceramides were reported from the sponge Stylissa flabelliformis, collected in Okinawa, Japan (Uchimura et al., 1997a). This seemed to be a unique example of an a-Gal-GSL isolated from a sponge genus other than Agelas and Axinella. 5.2.1.4 a-Tetraglycosylceramides As indicated above, chemical analyses of the more polar GL fractions of A. longissima had led to the isolation of a new tetraglycosylated GSL (Cafieri et al., 1996). The terminal sugar of the sugar moiety is a-fucofuranose (1 ! 6)-linked to 2-acetamido-2-deoxygalactopyranose. A re-examination of GSLs from the Caribbean sponge A. clathrodes led to the isolation and characterization of a further a-Gal-GSL, clarhamnoside, a new tetraglycosylceramide which was the first a-Gal-GSL to have an unusual L-rhamnose in the sugar head (Costantino et al., 2004). As usual for sponge GSL, this new GSL was composed of a very complex mixture of homologs. Fatty acyl chains of the ceramide moiety were 2-hydroxy-saturated (C21:0–C25:0, including C23:0 at 42%), while the LCBs were saturated 4-hydroxysphinganines ranging from C16:0 to C20:0, whether branched or not. 5.2.2 b-Glycopyranosylceramides

b-Glycopyranosylceramides isolated from the lipids of marine invertebrates are undoubtedly the most widely encountered GSL structures. They contain one or more sugars, saturated or unsaturated ceramide moieties, with or without 2-hydroxylated fatty acyl chains. Nevertheless, several investigations of various marine invertebrates have been led to the identification of glycosylceramides, the ceramide part of which was either saturated or monounsaturated, mostly with only one sugar (mainly glucose). 5.2.2.1 b-Glycopyranosylceramides with Saturated, Mono-, and Diunsaturated Sphingoid Bases 5.2.2.1.1 b-Monoglycosylceramides The first detailed structural study of a marine GSL appeared to be conducted on the cerebroside present in relatively large amounts in the sea star Asterias rubens (Bj€ orkman, Karlsson, and Nilsson, 1972a; Bj€ orkman et al., 1972b). This contained glucose, 2-hydroxylated C16:0–C26:0 acyl chains, and C18 and C22 LCBs with one or two double bonds.

Ceramides of the Dihydrosphingosine Type Two series of new b-glucocerebrosides, temnosides, were identified in lipid extracts of the sea urchin Temnopleurus toreumaticus, collected from the India coasts (Babu, Bhandari, and Garg, 1997) (Table 5.3). Temnosides A and B had fatty acyl chains with or without a 2hydroxyl group, respectively. These were unique examples of cerebrosides containing 2-amino-1,3-eicosanediol (dihydrosphingosine type).

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Table 5.3 Glycosphingolipids containing a b-glycosyl linkage.

Ceramide/GSL Name

Organism

Biological activity

Reference

b-Monoglycopyranosylceramides with a saturated or mono- and diunsaturated sphingoid base

R1 ¼ OH; C16:0 -C26:0 fatty acids C17:0 -C19:0 sphingoid bases

halicerebroside A R1 ¼ OH x ¼ 19 R ¼ CH2 CH ¼ CHðCH2 Þ10 CH3

Sea urchin Temnopleurus toreumaticus

n. r.

Babu, Bhandari, and Garg, 1997

Sponge Chondrilla nucula

n. r.

Schmitz and McDonald, 1974

Starfish Asterina pectinifera

n. r.

Sugita, 1977

Starfish Acanthaster planci

n. r.

Kawano et al., 1988a

Starfish Asterina pectinifera

n. r.

Higuchi, Natori, and Komori, 1990a

Sponge Haliclona sp.

n. r.

Hirsch and Kashman, 1989

Starfish Astropecten latespinosus

n. r.

Higuchi, Kagoshima, and Komori, 1990b

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

j 111

Starfish Asterias amurensis versicolor

n. r.

Higuchi et al., 1991a

Tunicate Botrillus leachii

n. r.

Aiello, Fattorusso, and Menna, 1996

Starfish Stellaster equestris

n. r.

Higuchi et al., 1996

Starfish Pentaceraster regulus

Moderate wound-healing

Venkannababu, Bhandari, and Garg, 1997

Tunicate Cystodytes cf. dellichijei

Inactive against PLA2

Loukaci et al., 2000

Sea cucumber Holothuria pervicax

n. r.

Yamada et al., 2002

Starfish Luidia maculata

n. r.

Kawatake et al., 2002

Feather star Comanthus japonica

n. r.

Inagaki et al., 2004

Starfish Linckia laevigata

n. r.

Maruta et al., 2005

(continued )

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Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

Sponge Haliclona (Reniera) sp.

n. r.

Park et al., 2009

Sea cucumber Stichopus japonicus

n. r.

Kisa et al., 2005

Sea cucumber Cucumaria frondosa

n. r.

La et al., 2012

Tunicate Microcosmus sulcatus

n. r.

Aiello et al., 2003

Sponge Amphimedon viridis

n. r.

Hirsch and Kashman, 1989

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

j 113

Sponge Halichondria cylindrata

Antifungal Cytotoxic

Li, Matsunaga, and Fusetani, 1995

Sponge Oceanapia sp.

n. r.

Guzii et al., 2006

Sponge Amphimedon compressa

n. r.

Costantino et al., 2009

Starfish Acanthaster planci

n. r.

Kawano et al., 1988a

Sea cucumber Cucumaria echinata

n. r.

Higuchi et al., 1994a

Sea cucumber Pentacta australis

n. r.

Higuchi et al., 1994b

Starfish Luidia maculata

n. r.

Kawatake et al., 2002

(continued )

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5 Glycolipids from Marine Invertebrates

Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

Sea cucumber Cucumaria echinata

n. r.

Yamada et al., 1998a

Sea cucumber Holothuria coronopertusa

n. r.

Hue et al., 2001

Sea cucumber Holothuria pervicax

n. r.

Yamada et al., 2002

Sea cucumber Holothuria leucospilota

n. r.

Yamada et al., 2005a, 2005b

Starfish Asterias amurensis

Plant-growth promotion

Ishii, Okino, and Mino, 2006

Sea cucumber Stichopus japonicus

n. r.

Kisa et al., 2005

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

Sea cucumber Stichopus japonicus

n. r.

Kisa et al., 2005

Feather star Comanthus japonica

n. r.

Inagaki et al., 2004

Sea cucumber Acaudina molpadioides

Anti-fatty liver

Xu et al., 2011

Sea cucumber Cucumaria frondosa

n. r.

La et al., 2012

x ¼ 17

Sea anemone Metridium senile

n. r.

Karlsson, Leffler, and Samuelsson, 1979

x ¼ 17–19

Soft coral Cladellia sp.

n. r.

Dmitrenok et al., 2001

x ¼ 17

Sponge Ircinia fasciculata

n. r.

Zhang et al., 2005

x ¼ 16

Soft coral Lobophytum sp.

Non cytotoxic

Muralidhar et al., 2005

x ¼ 10, 14–16

Octocoral Sarcophyton ehrenbergi

No antibacterial Reduced iNOS protein expression

Cheng et al., 2009

j 115

(continued )

116

j

5 Glycolipids from Marine Invertebrates

Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

R1 ¼ -ðCH2 Þ11-13 -CH3

Starfish Asterias amurensis

n. r.

Irie, Kubo, and Hoshi, 1990

Sponge Agelas mauritianus

Immunostimulatory Antitumor

Natori et al., 1994

Sea star Ophidiaster ophidiamus

n. r.

Jin, Rinehart, and Jares-Erijman, 1994

Sponge Agelas clathrodes

n. r.

Costantino, Fattorusso, and Mangoni, 1995a

Sponge Agelas conifera

n. r.

Costantino, Fattorusso, and Mangoni, 1995b

Starfish Stellaster equestris

n. r.

Higuchi et al., 1996

Starfish Cosmasterias lurida

n. r.

Maier, Kuriss, and Seldes, 1998

Ascidian Phallusia fumigata

n. r.

Dur
an et al., 1998

Starfish Anasterias minuta

n. r.

Chludil, Seldes, and Maier, 2003

R2 ¼ H or CH3 and x ¼ 6 R2 ¼ CH3 and x ¼ 6 agelasphin-10 R1 ¼ -ðCH2 Þ21 -CH3 agelasphin-12 R1 ¼ -ðCH2 Þ22 -CH3 R2 ¼ CH3 and x ¼ 6 ophidiacerebrosides Aðx ¼ 17Þ; Bð18Þ; Cð19Þ; Dð20Þ; Eð21Þ R2 ¼ CH3 and x ¼ 6 R1 ¼ -ðCH2 Þ19 -CH3 ð19:7Þ ¼ -ðCH2 Þ20 -CH3 ð23:4Þ ¼ -ðCH2 Þ22 -CH3 ð7:1Þ R2 ¼ CH3 and x ¼ 6 R1 ¼ -ðCH2 Þ20 -CH3 ð10:9Þ ¼ -ðCH2 Þ21 -CH3 ð75:5Þ ¼ -ðCH2 Þ21 -CH3 ð56:9Þ R2 ¼ CH3 and x ¼ 6 S-1-3 R1 ¼ -ðCH2 Þ19 -CH3 S-1-4

¼ -ðCH2 Þ120 -CH3

S-1-5

¼ -ðCH2 Þ21 -CH3

R2 ¼ CH3 and x ¼ 6 GSL 3 R1 ¼ -ðCH2 Þ14 -CH3 GSL 4

¼ -ðCH2 Þ15 -CH3

GSL 6

¼ -ðCH2 Þ21 -CH3

R2 ¼ CH3 x ¼ 6 phallusides 1 R1 ¼ -ðCH2 Þ14 -CH3 2

¼ -ðCH2 Þ15 -CH3

3

¼ -ðCH2 Þ21 -CH3

4 R2 ¼ H R1 ¼ -ðCH2 Þa -CH3 a þ x ¼ 20 R2 ¼ CH3 x ¼ 6 GSL 1

R1 ¼ -ðCH2 Þ13 -CH3

GSL 2

¼ -ðCH2 Þ14 -CH3

GSL 3

¼ -ðCH2 Þ15 -CH3

GSL 4 R1 ¼ -ðCH2 Þ12 -CH ¼ CH-ðCH2 Þ7 -CH3 R2 ¼ H x ¼ 6 anasterioside A R1 ¼ -ðCH2 Þ11 -CH ¼ CH-ðCH2 Þ7 -CH3 GSL 5 R1 ¼ -ðCH2 Þ12 -CH ¼ CH-ðCH2 Þ7 -CH3

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

j 117

Starfish Allostichaster inaequalis

n. r.

Díaz de Vivar, Seldes, and Maier, 2002

Starfish Oreaster reticulus

Cytotoxic Proangiogenic Antiproliferative

Costantino et al., 2007

Octocoral Sarcophyton ehrenbergi

n. r.

Cheng et al., 2009

R1 ¼ -ðCH2 Þ14;18-20 -CH3 R2 ¼ H x ¼ 6 renierosides A m n d:b:

Sponge Haliclona (Reniera)

n. r.

Mansoor et al., 2007

Starfish Narcissia canariensis

Cytotoxic on human cancer cells

Farokhi et al., 2010

Sponge Halichondria japonica

n. r.

Hayashi, Nishimura, and Matsubara, 1991

Sponge Chondropsis sp.

Histidine decarboxylase inhibitor Hypotensive

Endo et al., 1986

Sponge Axinyssa djiferi

Antimalarial

Farokhi et al., 2013

R2 ¼ CH3

x¼6

GSL 1

R1 ¼ -ðCH2 Þ13 -CH3

GSL 2

¼ -ðCH2 Þ14 -CH3

GSL 3

¼ -ðCH2 Þ15 -CH3

GSL 5a

¼ -ðCH2 Þ19 -CH3

5b ¼ -ðCH2 Þ12 -CH ¼ CH-ðCH2 Þ7 R2 ¼ H

CH3

x¼6

GSL 4 R1 ¼ -ðCH2 Þ12 -CH ¼ CH-ðCH2 Þ7 -CH3 R2 ¼ H x ¼ 6

R2 ¼ CH3 x ¼ 6

oreacerebrosides

ophidiacerebrosides

A R1 ¼ -ðCH2 Þ19 -CH3 C R1 ¼ -ðCH2 Þ19 -CH3 B R1 ¼ -ðCH2 Þ20 -CH3 D R1 ¼ -ðCH2 Þ20 -CH3 C R1 ¼ -ðCH2 Þ21 -CH3 E R1 ¼ -ðCH2 Þ21 -CH3 R2 ¼ CH3

x¼8

A1

17

5

D21

A2

13

7

D17

A3 12 7 D16 R2 ¼ CH3 x ¼ 6 renierosides B m n d:b: B1

17

5

D21

B2

13

7

D17

B3

16

5

D20

R2 ¼ CH3 x ¼ 6 ophidiacerebrosides B R1 ¼ -ðCH2 Þ18 -CH3 C R1 ¼ -ðCH2 Þ19 -CH3 ð63%Þ D R1 ¼ -ðCH2 Þ20 -CH3

Major FAs 2-OH-C22:0 (64.2%), 2-OH-C23:0 (10.4), 2-OH-C24:0 (8.6) Major bases iso-C18:0 (39.5), n-C18:0 (22.6), anteiso-C19:0 (16.9)

(continued )

118

j

5 Glycolipids from Marine Invertebrates

Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

Starfish Stellaster equestris

n. r.

Higuchi et al., 1996

Starfish Culcita novaeguineae

n. r.

Inagaki, Nakata, and Higuchi, 2006

Starfish Protoreaster nodosus

n. r.

Pan et al., 2010

Sponge Halichondria panicea

n. r.

Nagle et al., 1992

Sea cucumber Bohadschia argus

n. r.

Ikeda et al., 2009

Sea star Oreaster reticulatus

Cytotoxic Anticancer cell proliferation

Costantino et al., 2007

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

fatty acid: anteiso-C17:0 sphingoid base: anteiso-C19:0

Sponge Plakortis simplex

Immunosuppressor

Costantino et al., 1997

Sponge Ectyoplasia ferox

n. r.

Costantino et al., 2000

Sponge Ectyoplasia ferox

n. r.

Costantino et al., 2003

Starfish Asterina pectinifera

n. r.

Sugita, 1977

Starfish Acanthaster planci

n. r.

Kawano et al., 1988b

Starfish Luidia maculata

n. r.

Inagaki et al., 2003

j 119

Glycosylceramides with two sugars or more

(continued )

120

j

5 Glycolipids from Marine Invertebrates

Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

Sponge Amphimedon sp.

n. r.

Emura et al., 2005

Starfish Luidia maculata

n. r.

Inagaki et al., 2003

Sponge Halichondria japonica

n. r.

Hayashi, Nishimura, and Matsubara, 1991

Sponge Spheciospongia vesparia

n. r.

Costantino et al., 2005

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

amphiceramide A S ¼ b-NAcGlcp

Sponge Spheciospongia vesparia

n. r.

Costantino et al., 2008

Sponge Aplysinella rhax

Nitric oxide release inhibitor

Borbone et al., 2001

Sponge Amphimedon compressa

n. r.

Costantino et al., 2009

Tunicate Microcosmus sulcatus

n. r.

Aiello et al., 2002

j 121

amphiceramide B S ¼ b-Galp amphimelibioside S ¼ a-Galp

(continued )

122

j

5 Glycolipids from Marine Invertebrates

Table 5.3 (Continued) Ceramide/GSL Name

Organism

Biological activity

Reference

Tunicate Microcosmus sulcatus

n. r.

Aiello et al., 2003

Sponge Terpios sp.

n. r.

Costantino et al., 2008

Sponge Terpios sp.

Potent inhibitor of NO release

Costantino et al., 2010

Composition of ceramide moieties are given for the major GSL structures of each type (relative percentages in brackets) n. r. ¼ not reported

Ceramides of the Phytosphingosine Type Indeed, the most encountered GSLs contain 2-amino-1,3,4-trihydroxylated longchain bases (phytosphingosine type). A pioneering study of lipids from the sponge Chondrilla nucula allowed the isolation of a saturated b-glucosylceramide as a mixture of homologous GSLs (Schmitz and McDonald, 1974). The starfish Asterina pectinifera, collected in the Japan Sea, contained various GSLs including mono- and dihexosides (Sugita, 1977). Two series of b-monoglucosylceramides were identified in this starfish, possessing saturated long-chain phytosphingosines and fatty acyl chains with or without a 2-hydroxyl group. Some years later, six new ceramide monohexosides (b-glucopyranose) were isolated

from the lipid fraction of the starfish Acanthaster planci, including the saturated acanthacerebrosides A and B, and the monounsaturated acanthacerebroside C (Kawano et al., 1988a). Acanthacerebrosides A and B contained ceramides with 2hydroxylated C24:0 acid and C16 and C22 trihydroxylated LCBs, respectively. The latter starfish has been reinvestigated, and this led to the identification of the known ceramide monohexoside, acanthacerebroside B, obtained in a pure state from the GSL mixture of another starfish, Asterina pectinifera (Higuchi, Natori, and Komori, 1990a). Table 5.3 shows three other representative GSLs isolated in this study. Almost 10% of a dichloromethane– methanol extract of the sponge Haliclona sp. from the Red Sea

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

was composed of a single GSL, named halicerebroside A (Hirsch and Kashman, 1989). This b-glucosylceramide contained a 2-hydroxy C22 acyl chain and a C18 D6-phytosphingosine. Investigation of the less-polar fraction of a chloroform– methanol extract of the starfish Astropecten latespinosus allowed the isolation of three new cerebrosides, astrocerebrosides A, B and C, together with the known acanthacerebroside A (Higuchi, Kagoshima, and Komori, 1990b). Six new cerebrosides, named asteriacerebrosides A–F, were isolated from the starfish Asterias amurensis versicolor, besides two known cerebrosides, astrocerebroside A and acanthacerebrosides C that had been obtained earlier from other starfishes (Higuchi et al., 1991a). The structures of astrocerebrosides D, E and F, which have a monounsaturated ceramide moiety, are depicted in Table 5.3. The ceramide part of astrocerebroside F contains 2-hydroxy-15-tetracosenoic acid and a saturated phytosphingosine. A cerebroside mixture was obtained from the water-insoluble lipid fraction of an n-butanol extract of the ascidian Botrillus leachii from the Gulf of Venice (Aiello, Fattorusso, and Menna, 1996). This was identified as the peracetate derivative and was a mixture of homologs, and appeared to be the first GSL to be isolated from a tunicate. A study of the less-polar lipid fraction of the starfish Stellaster equestris led to the identification of eight new cerebrosides, glucosides or galactosides with saturated or unsaturated ceramides (Higuchi et al., 1996). Two of these were isolated and characterized as homogeneous compounds, termed S-2a-3 and S-2a-11. Reguloside A is a new cerebroside isolated from the starfish Pentaceraster regulus found along the Indian coast (Venkannababu, Bhandari, and Garg, 1997); two minor homologous regulosides B and C were also identified. A mixture of homologous saturated glycosylceramides was isolated from the tunicate Cystodytes cf. dellechiajei collected in Tunisian waters (Loukaci et al., 2000). In Japan, Higuchi’s group isolated ten glucocerebrosides, HPC-3-A to HPC-3 J, from a lipid extract of the sea cucumber Holothuria pervicax (Yamada et al., 2002). The GSLs were a mixture of iso and anteiso isomers. Two new glucocerebrosides, luidiacerebrosides A and B, were isolated from the Japanese starfish Luidia maculata, along with the known CE-2b and CE-3-2 isolated from the sea cucumber Cucumaria echinata, and astrocerebroside B and acanthacerebroside B isolated from the starfish Astropecten latespinosus (Higuchi, Kagoshima, and Komori, 1990b) and Acanthaster planci (Kawano et al., 1988a), respectively (Kawatake et al., 2002). Following the report on the isolation and structure of gangliosides from the polar lipid fraction of the feather star Comanthus japonica (class Crinoidea) (Arao, Inagaki, and Higuchi, 2001), Inagaki and colleagues examined the less-polar lipid fraction and four glucocerebrosides (Inagaki et al., 2004). Two of these, JCer-3 and JCer-4, had saturated ceramide moieties, while the others had diunsaturated ceramides (JCer-1 and JCer-2). The compounds JCer-2 and JCer-4 were unprecedented. JCer-3 has also been reported from a sea cucumber (Hue et al., 2001). Interestingly, JCer-3 had a 2-hydroxy fatty acyl chain, while JCer4 was unique in being the first type of cerebroside from echinoderms to be composed of a phytosphingosine and a nonhydroxylated fatty acyl chain.

j 123

In their continuing intense quest for biologically active GSLs, Higuchi and coworkers isolated a series of glucocerebrosides that possessed saturated ceramide moieties from the less-polar fraction of a chloroform–methanol extract of the starfish Linckia laevigata (Maruta et al., 2005). Only two of these glucocerebrosides have been isolated as homogeneous GSLs, giving a single FA methyl ester upon acid methanolysis. The first compound was a new glucocerebroside, linckiacerebroside A, while the second was reported from the starfish Stellaster equestris (Higuchi et al., 1996) (Table 5.3). Three other “pseudo” GSLs were isolated as mixtures of homologous compounds in S. equestris. Fourteen phytosphingosine-type cerebrosides were isolated from the sponge Haliclona (Reniera) sp., collected off the Korean coasts (Park et al., 2009). Nine of these, GSLs 1–9, contained saturated acyl chains and were new compounds; the exception was GSL-4, known as renieroside C4, which had already been reported from the starfish Linckia laevigata (Maruta et al., 2005). The phytosphingosine-type glucocerebroside “molecular species,” SJC-3, has been isolated from the less-polar lipid fraction of a chloroform–methanol extract of the sea cucumber Stichopus japonicus (Kisa et al., 2005). “Molecular species” generally refers to a set of GSL molecules presenting the same sugar moiety but several LCBs and FAs. Nevertheless, in some cases pure compounds (single sphingoid base and FA) have been isolated from the molecular species. The four other GSL molecular species were of the typical sphingosine type, with nonhydroxylated and hydroxylated fatty acyl moieties. SJC-4 and SJC-5 were new glucocerebroside molecular species with unique sphingosine bases. Among the new glucocerebrosides recently isolated from the sea cucumber Cucumaria frondosa, the new GSL CF-3-1 has a ceramide part with a monounsaturated FA and a phytosphinganine-type base (La et al., 2012). A new b-glucopyranosylceramide with a phytosphingosine type and dihydroxy FAs was isolated from the tunicate Microcosmus sulcatus (Aiello et al., 2003). This was a mixture of homologous of b-glucopyranosides of phytosphingosine-type ceramides that were the first natural GSLs of which the ceramide contained a 2,3-dihydroxy FA (Aiello et al., 2003). 2,3Dihydroxy FAs, which until now have been reported only as constituents of ceramides of plants and fungi, could be derived from the diet (Aiello et al., 2003). b-N-Glucosaminyl and b-N-Acetylglucosaminylceramides The sponge Amphimedon viridis, which has already been cited for b-N-glucosaminylceramides, also contained b-N-glucosaminylceramides, amphicerebrosides B, C and D, with a 2-hydroxy fatty acyl chain (Hirsch and Kashman, 1989). During the course of screening for bioactive metabolites from the Japanese marine sponge Halichondria cylindrata, ten new N-acetylglucosaminylceramides, named halicylindrosides, have been isolated having phytosphingosine-type bases and nonhydroxylated fatty acyl chains (halicylindrosides A1–A4) or 2-hydroxylated (halicylindrosides B1–B6) (Li, Matsunaga, and Fusetani, 1995). Two cerebrosides were isolated from the sponge Oceanapia sp., and their chemical structures determined (Guzii et al., 2006). They proved to be new modifications of N-acetylglucosamine-containing

124

j

5 Glycolipids from Marine Invertebrates

GSLs, and contained n-, iso- and anteiso-phytosphingosines, and C24:0–C28:0 2-hydroxy FAs. The GL composition of the Caribbean sponge Amphimedon compressa was examined by Costantino and colleagues, who showed it to contain two novel diglycosyl GSLs and a new molecular species of acetamidoglucosylceramide (Costantino et al., 2009). Ceramides of the Sphingosine Type Three new glucocerebrosides with a sphingosine-type base, acanthacerebrosides D, E and F, were isolated from the starfish Acanthaster planci (Kawano et al., 1988a). Three new sphingosine-type glucocerebrosides, CE-2b, CE-2c and CE-2d, were obtained as pure compounds from a cerebroside molecular species, CE-2, from the less-polar lipid fraction of the Japanese sea cucumber Cucumaria echinata (Higuchi et al., 1994a). Ceramide moieties of these GSLs have a D4-sphingosine base and a 2-hydroxy fatty acyl chain. Chemical investigations of the GLs of the sea cucumber Pentacta australis led to the isolation of new glucocerebrosides PA-O-1 and PA-O-5, which also had ceramides with a D4-sphingosine base and a nonhydroxylated acyl chain (Higuchi et al., 1994b). The less-polar fraction of a chloroform–methanol extract of the Japanese starfish Luidia maculata yielded two complex mixtures of homologous glucocerebrosides, which were separated (Kawatake et al., 2002). One of these, LMC-2, led to the isolation of ten GSLs, each showing a single quasi-molecular ion peak. Only five of the latter GSLs (GSL-2, GSL-3, GSL-4, GSL-5, and GSL-6) gave a single FA upon acid methanolysis and, consequently, were regarded as homogeneous cerebrosides. Compounds GSL-2 and GSL-6 are new cerebrosides named luidiacerebrosides A and B. Compounds GSL-3 and GSL-4 have been found to be identical to astrocerebroside B (Higuchi, Kagoshima, and Komori, 1990b) and to acanthacerebroside B (Kawano et al., 1988a), isolated from the starfish Astropecten latespinosus. Compound GSL-5 has been found to be identical to CE-3-2 isolated from the sea cucumber Cucumaria echinata (Yamada et al., 1998a). The other GSL fraction from L. maculata, LMC-1, led to the isolation of 11 fractions, including six that revealed a single quasi-molecular ion peak. Only one fraction was identified as a pure compound, GSL-1 (Table 5.3), which has been found identical to the compound CE-2b isolated from the sea cucumber Cucumaria echinata (Higuchi et al., 1994a). In addition to the GSLs with saturated ceramide moieties (Higuchi et al., 1994a), the sea cucumber Cucumaria echinata was shown to contain ceramides with diunsaturated sphingoid bases (Yamada et al., 1998a). A mixture of 18 glucocerebrosides was isolated from lipids of the sea cucumber Holothuria coronopertusa, collected in New Caledonia (Hue et al., 2001). The structures of these compounds were established using a combination of liquid-secondary ion mass spectrometry (LSIMS) as the ionization mode, with highenergy tandem mass spectrometry (MS/MS). This was an interesting approach to analysis for the structure elucidation of unresolved mixtures of cerebrosides. The Japanese sea cucumber Holothuria pervicax was shown to contain GSLs, the ceramides of which were of the sphingosine type or phytosphingosine type bases that were iso- and anteiso-branched

(Yamada et al., 2002). The new sphingosine-type glucocerebroside HLC-2-A with an anteiso-type side chain of the long-chain base moiety was isolated from the whole bodies of the Japanese sea cucumber Holothuria leucospilota (Yamada et al., 2005a). This compound has been isolated from its parent glucocerebroside molecular species HLC-2 composed of iso and anteiso isomers. The absolute configuration of the branched methyl group in the anteiso-type side chain moiety on the long-chain base of HLC-2A was determined chemically (Yamada et al., 2005b). The new glucocerebroside, asteriacerebroside G, together with two known cerebrosides, asteriacerebrosides A and B, were isolated from lipophilic fractions of the whole bodies of the Northern Pacific (coasts of Hokkaido) starfish Asterias amurensis L€ utken (Ishii, Okino, and Mino, 2006). Among the GSL molecular species isolated from the sea cucumber Stichopus japonicus, SJC-1 and SJC-2 were typical sphingosine-type glucocerebrosides with nonhydroxylated and hydroxylated fatty acyl moieties (Kisa et al., 2005). The molecular species SJC-4 and SJC-5 are also sphingosine-type glucocerebrosides with hydroxylated fatty acyl moieties, although they are new glucocerebroside molecular species with unique sphingosine bases in that they contain additional double bond and hydroxy functionalities. Three glucocerebrosides, JCer-1–JCer-3, have been isolated from their parent molecular species from the less-polar fractions of a lipid extract of the Japanese feather star Comanthus japonica (Inagaki et al., 2004). These are of the sphingosine type with 2-hydroxy fatty acyl chains. A novel glucocerebroside was isolated from the sea cucumber Acaudina molpadioides (Xu et al., 2011). The sphingosine-type base is iso- or anteiso-branched, and the 2-hydroxy acyl chain may be monounsaturated. Three molecular GSL species, CF-3-1, CF-3-2 and CF-3-3, were isolated from the Arctic sea cucumber Cucumaria frondosa (La et al., 2012). The latter two species were obtained as pure compounds for the first time. These GSLs were shown to contain the same 2hydroxy D5-C24:1 acid and anteiso-branched or diunsaturated sphingoid bases. b-Glucocerebrosides with Diunsaturated Branched Sphingoid Bases A new cerebroside was isolated from the sea anemone Metridium senile (Karlsson, Leffler, and Samuelsson, 1979). This was a b-glucopyranosylceramide with various 2-hydroxy FAs (mainly C16:0 and C20:0), and the major base, diunsaturated and 9-methyl branched, was a novel base. A glucocerebroside was isolated from the soft coral Cladellia sp. collected on the seaboard of the Andaman Islands (Indian Ocean) (Dmitrenok et al., 2001). This contained 2-hydroxy FAs and its base was the same as above, 2-amino-9-methyloctadeca-4E,8E-diene-1,3-diol. The same sphingosine type was identified again in the ceramide part of an isolated pure GSL, ircicerebroside, from the sponge Ircinia fasciculata collected from Wei-zhou Island, China (Zhang et al., 2005). Ircicerebroside was present in the GSL mixture of M. senile. The octocoral Sarcophyton eherenbergi, collected in Taiwan waters, and already cited for containing a new a-glucocerebroside with a triunsaturated long-chain base, also contained a new b-glucocerebroside. Its ceramide part possess the same diunsaturated base type as M. senile and I. fasciculata (Karlsson, Leffler,

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

and Samuelsson, 1979; Zhang et al., 2005) (Table 5.3). Among several metabolites isolated from the soft coral Lobophytum sp., collected in the Indian Ocean, a pure unprecedented glucocerebroside was identified bearing the same diunsaturated sphingoid base as in the latter reports, and a saturated 2-hydroxy FA (Muralidhar et al., 2005). In addition to the a-glucocerebroside, the ceramide of which was composed of a triunsaturated branched sphingoid base, the octocoral Sarcophyton ehrenbergi harvested off the coasts of Taiwan contains b-glucocerebroside molecular species including a new compound (Cheng et al., 2009). This a further example of a 9-methyl-4E,8E-sphingadiene-type cerebroside. 5.2.2.2 b-Glycopyranosylceramides with Triunsaturated Sphingoid Bases A new b-glucosylceramide was isolated as a mixture of related compounds from the spermatozoa of the starfish Asterias amurensis, collected in Japanese waters (Irie, Kubo, and Hoshi, 1990). The ceramide moieties of the these GSLs contain 2-hydroxylated C14–C25 FAs, most of which were saturated and unbranched. Long-chain bases consisted of dihydroxy (C18:2, C18:3, C19:3, and C22:2), and trihydroxy (C22:1) types. Two of the triunsaturated bases (C18:3 and C19:3) were identified as (4E,8E,10E)-2-amino4,8,10-octadecatriene-1,3-diol and (4E,8E,10E)-2-amino-9methyl-4,8,10-octadecatriene-1,3-diol, which is a novel base. Some Agelas sponges, reported above to contain a-GSLs, also contained b-glucocerebrosides, agelasphin-10 and -12 (Natori et al., 1994). Thus, agelasphin-10 and -12 are b-glucocerebrosides in which the ceramides are triunsaturated and identical to those identified previously (Irie, Kubo, and Hoshi, 1990). Five unprecedented bioactive b-glucocerebrosides, named ophidiacerebrosides A to E, have been purified from the large sea star Ophidiaster ophidiamus, collected in waters off the Balearic Islands, Spain (Jin, Rinehart, and Jares-Erijman, 1994). These GSLs have the same sphingosine part containing a methyl branch and a conjugated diene, and differ only in the chain lengths of the 2-hydroxy fatty acyl chain. The major component, ophidiacerebroside C, underwent extensive chemical investigation. The latter GSL, together with the other ophidiacerebrosides, were present in the GSL mixtures previously studied (Irie, Kubo, and Hoshi, 1990), but no individual glucosylceramide was isolated. b-Glucocerebrosides possessing this type of sphingosine and mainly the 2-hydroxy C24:0 FA were also identified from sponges such as A. clathrodes (Costantino, Fattorusso, and Mangoni, 1995a) and A. conifera (Costantino, Fattorusso, and Mangoni, 1995b). The acetone-insoluble part of the less-polar lipid fraction from the starfish Stellaster equestris, collected from the East China Sea, was investigated (Higuchi et al., 1996) and this led to the isolation of five new saturated and three known unsaturated cerebrosides. The latter compounds were glucocerebrosides S-1-3, S-1-4 and S-1-5 from the starfish Stellaster equestris (Higuchi et al., 1995a), and contained the same triunsaturated sphingosine type and various 2-hydroxy FAs. They were shown to be identical to ophidiacerebrosides C, D and E from the starfish Ophidiaster ophidiamus (Jin, Rinehart, and Jares-Erijman, 1994), while compound S-1-5 was the same as

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agelasphin-10 found in the sponge Agelas mauritianus (Natori et al., 1994). Two new glucosylceramides, GSL-1 and GSL-2 (see Table 5.3), together with the known ophidiacerebroside E, were isolated from the starfish Cosmasterias lurida, collected in cold waters off the Patagonian coast of Argentina (Maier, Kuriss, and Seldes, 1998). The new GSLs were b-glucopyranosylceramides with the same long-chain base and 2-hydroxylated C17:0 and C18:0 FA chains. Glucosphingolipids also occur in tunicates, as reported for Phallusia fumigata collected off the southern coast of Cadiz, Spain (Dur
an et al., 1998). Four new glucocerebrosides were identified in this ascidian, namely phallusides 1 to 4, which had also the same sphingosine type and different 2-hydroxy FAs. The very common starfish, Anasterias minuta, collected along the Patagonian coast, was shown to contain seven known related GSLs and the new polyunsaturated glucosylceramide, anasterocerebroside A, obtained from the dichloromethane–methanol extract (Chludil, Seldes, and Maier, 2003). They contained a branched or nonbranched sphingoid base, and the 2-hydroxy FAs may be monounsaturated. Glucosylceramides GSL-3 to -7 were methanolized and gave a single FA methyl ester by GC-MS (Table 5.3). GSL-3 was identified as phalluside-1, previously isolated from the ascidian Phallusia fumigata (Dur
an et al., 1998), whereas GSL-4 and GSL-5 are also known compounds previously isolated from Cosmasterias lurida (Maier, Kuriss, and Seldes, 1998). GSL-6 was identified as ophidiacerebroside C, previously isolated from Ophidiaster ophidiamus (Jin, Rinehart, and Jares-Erijman, 1994), while GSL-7 has been isolated and characterized as a pure compound for the first time. GSL-7 has been isolated previously as a mixture of related glucosylceramides from the Patagonian starfishes Cosmasterias lurida (Maier, Kuriss, and Seldes, 1998) and Allostichaster inaequalis (Díaz de Vivar, Seldes, and Maier, 2002). Chemical investigations of the sea star Oreaster reticulus, collected along the coasts of Grand Bahama Island, led to the isolation of nine new GLs named oreacerebrosides A–I, along with the known ophidiacerebrosides C–E (Costantino et al., 2007). Among these, oreacerebrosides A–C are glucocerebrosides containing a 4E,8E,10E-trienoic sphingoid base. The same triunsaturated sphingosine type, found as a part of the ceramide of an a-glucocerebroside, from the octocoral Sarcophyton ehrenbergi, also composed the ceramide moiety of a new b-glucocerebroside (Cheng et al., 2009). Cerebrosides possessing polyunsaturated ceramide moieties were reported from a sponge Haliclona (Reniera) sp. harvested from Korean waters (Mansoor et al., 2007). A first series, renierosides A1–A5, contained a 4E,8E,10E-trienoic sphingoid base, and three of these had 2-hydroxy monounsaturated fatty acyl chains. The second series, renierosides B1–B3, contained a 9-methyl-4E,8E,10E-trienoic sphingoid base, and their 2hydroxy acyl chains were monounsaturated. The sphingoid base may be branched or not, and the 2-hydroxyl chain may be unsaturated. In addition to the new immunostimulatory b-glucocerebrosides, corrugoside, the sponge Axinella corrugata was shown to contain GSLs bearing unusual endoperoxide and allylic hydroperoxide functionalities on the sphinganine chain (not shown in Table 5.3) (Costantino et al., 2008).

126

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5 Glycolipids from Marine Invertebrates

The starfish Narcissia canariensis, harvested from the coasts off Dakar, Senegal, was investigated for GLs (Farokhi et al., 2010). Three homologous GLs were isolated from a glycolipidic fraction; these glucocerebrosides contained a b-glucopyranoside as the sugar head, a 9-methyl-branched 4,8,10-triunsaturated long-chain amino-alcohol as the sphingoid base, and amide-linked 2-hydroxy FA chains. The major GSL (63%) had an amide-linked 2-hydroxydocosanoic acid chain, and was identified as ophidiacerebroside C, first isolated from the starfish Ophidiaster ophidiamus (Jin, Rinehart, and Jares-Erijman, 1994). The two minor components differed by one more or one less methylene group, and corresponded to ophidiacerebrosides B and D. 5.2.2.2.1 b-Monogalactosylceramides In addition to a new digalactoside ceramide, a ceramide monogalactoside was isolated from the GSL fraction from the sponge Halichondra japonica (Hayashi, Nishimura, and Matsubara, 1991). This accounted for 81% of the fraction (ceramide monoglucoside at 19%). The ceramide moiety had a very similar composition to the di-GSL (Table 5.3). Among pharmacologically active substances from southern Pacific marine invertebrates, new homologous bioactive galactosyl monohexosides were isolated from the Australian sponge Chondropsis sp. (Endo et al., 1986). The absolute stereochemistry of the new GSLs was further determined by asymmetrical synthesis (Honda et al., 1991). These galactocerebrosides contained 2-hydroxytetracosanoic acid and C17- and C18-phytosphingosines bearing a trans-D6 double bond and an iso-type terminal in the long chain. The sponge Axinyssa djiferi, collected on mangrove tree roots in Senegal, was studied for GL fractions (Farokhi et al., 2013). A mixture containing new GSLs, named axidjiferoside-A (60.8%), -B (22.4%) and –C (16.8%), accounted for 2.16% of the total lipids. These were homologous b-galactopyranosylceramides with phytosphingosine bearing a trans-D6 double bond (2-amino-(6E)-octadec-6-en-1,3,4-triol). Among the new cerebrosides isolated from the starfish Stellaster equestris, three homogeneous galactosides having saturated ceramides, S-2b-2, S-2b-4 and S-2b-16 were purified (Higuchi et al., 1996). A mixture of related galactocerebrosides, namely molecular species CNC-2, was isolated from the starfish Culcita novaeguineae collected near Okinawa, Japan (Inagaki, Nakata, and Higuchi, 2006). Recently, 16 new b-galactopyranosides with saturated ceramides and 2-hydroxy FAs were isolated from a chloroform–methanol extract of pyloric ceaca dissected from the starfish Protoreaster nodosus as three series of homologous GSLs possessing normal, iso, and anteiso sphingosine chains (Pan et al., 2010). Among these, only four GSLs were isolated as pure compounds (see Table 5.3), the others being mixtures of homologs. In addition to the glucocerebrosides identified in lipids of the sea cucumber Bohadschia argus, a new galactosylceramide, BAC4-4a, was obtained from its parent molecular species (Ikeda et al., 2009). This contained a 2-hydroxy D15(Z)-C24:1 acid, and normal and iso-saturated sphingoid bases. Oreacerebrosides A–C were shown to have a b-glucopyranoside as the sugar residue, as found in ophidiacerebrosides C–E and in all known compounds of this type; in contrast, oreacerebrosides D–I were

the first examples of b-galactosylceramides containing this unusual triunsaturated sphingoid base (Costantino et al., 2007). Two unique galactocerebrosides, plakosides A and B, were isolated from the sponge Plakortis simplex, collected along the coasts of Little San Salvador Islands (Costantino et al., 1997). These were considered as belonging to a new class of prenylated GSLs. Indeed, the galactopyranose residues were alkylated at O-2 by a 3,3-dimethylallyl group. In addition, the ceramide moiety contained cyclopropane in the fatty acyl chain and in the sphingoid base. Two further new plakosides, C and D, were isolated from the sponge Ectyoplasia ferox collected along the coast of Grand Bahama Island (Costantino et al., 2000). These authors indicated that the presence of similar compounds in taxonomically distant sponges may raise doubts on the actual organism responsible for their biosynthesis. In these cases a bacterial or, more generally, a dietary origin is often hypothesized. More recently, ectyoceramide, isolated from Ectyoplasia ferox, proved to be a rare example of a b-galactofuranosylceramide (Costantino et al., 2003). 5.2.2.2.2 b-Glycocerebrosides with Two Sugars or More The investigation of polar lipids from the starfish Asterina pectinifera allowed the isolation of not only ganglioside and monohexoside ceramide (as shown elsewhere in this chapter) but also lactosylceramide (Sugita, 1977). The ceramide moiety contained long-chain 2-hydroxy FAs and normal and iso-branched phytosphingosines. Two new ceramide lactosides, acanthalactosides A and B, were isolated from the starfish Acanthaster planci (Kawano et al., 1988a). The ceramide moiety was composed mainly of 4-hydroxy-iso-octadecasphinganine and 2-hydroxydocosanoic acid. Five new ceramide dihexosides, amphimelibiosides B–F (with C as the major constituent), were individually isolated from a Japanese marine sponge Amphimedon sp. (Emura et al., 2005). An inseparable mixture of two combinations of alkyl chains in the ceramide moiety was also obtained as amphimelibioside A. Amphimelibiosides have an a-galactopyranose as the second sugar, and this carbohydrate moiety is well known as melibiose. Four new ceramides, luidialactosides A–D, were isolated from the Japanese starfish Luidi maculata (Inagaki et al., 2003). Luidialactoside A was seen to possess a sphingosine-type ceramide, while the others had phytosphingosine-type bases with a D9 double bond in the case of luidialactoside C. The main GSL of the sponge Halichondria japonica was identified as a ceramide digalactoside having an a-Galp-(1 ! 4)-b-Galp as sugar unit, and a ceramide moiety mainly composed of 2-hydroxydocosanoic acid and 4-hydroxy-iso-octadecasphinganine (Hayashi, Nishimura, and Matsubara, 1991). The sponge Spheciospongia vesparia, collected in the Caribbean Sea (Bahamas Islands), was shown to produce a new diglycosylated GSL, vesparioside A, which was the first example of a diglycosylceramide with a pentose sugar residue (b-arabinopyranoside) (Costantino et al., 2005). A reinvestigation of the GSL composition of S. vesparia revealed the presence of a new vesparioside B, a new furanose-rich hexaglycosylated GSL with a very complex structure (Costantino et al., 2008).

5.2 Glycosphingolipids from Marine Invertebrates: Occurrence, Characterization, and Biological Activity

Several new GSLs were isolated from the choloroform-soluble part of the methanol extract of the freeze-dried sponge Aplysinella rhax, collected in the shallow waters of New Caledonia (Borbone et al., 2001). A single FA and a single LCB were detected only from the GSLs 4, 8 and 12, which were thus considered as pure compounds; the others were mixtures of isomers that differed by the ceramide various combinations. Investigations of glycolipids from the Caribbean sponge Amphimedon compressa led to the isolation of two novel GSLs, amphiceramides A and B, which possessed the unusual D6phytosphingosine, and the known amphimelibioside C reported by Emura et al. (2005) (Costantino et al., 2009). These compounds had the same ceramide moiety, but differed by the second sugar linked to the inner glucopyranose. A new triglycosylceramide, sulcaceramide, was isolated from the Mediterranean tunicate Microcosmus sulcatus and shown to contain an unprecedented fucosylated carbohydrate moiety (Aiello et al., 2002). In addition to the monoglucocerebroside mentioned above, this tunicate was found to contain two other new glucocerebrosides possessing 2,3-hydroxy fatty acyl chains in their ceramide (Aiello et al., 2003), one of them having the same sugar moiety than sulcaceramide. The new diglycosylceramide, terpioside 1a, was isolated from the marine sponge Terpios sp., collected from the tropical waters of Key Largo, Florida. Terpioside 1a is a diglycosylated GSL, and the first example of a natural GSL having an L-fucofuranose unit (Costantino et al., 2008). Interestingly, iso and anteiso FAs each comprised more than 50% of the total FA residues. A recent reinvestigation of the GSL composition of the sponge Terpios sp., collected in Florida waters, revealed the presence of an additional, unique GSL, terpioside B, which is a pentaglycosylated GSL characterized by the presence of two terminal a-L-fucofuranose units (Costantino et al., 2010). 5.2.3 Biological and Pharmacological Properties of GSLs from Marine Invertebrates

Interestingly, the first review on marine GLs reported no biological activity for compounds reported up to 1982 (Kochetkov and Smirnova, 1987). A b-galactocerebroside isolated from a sponge, Chondropsis sp., was shown to significantly inhibit histidine decarboxylase, and also to have hypotensive activity in anesthetized rats (Endo et al., 1986) (Table 5.3). Histidine decarboxylase is known to synthetize histamine in mammals, a major mediator involved in various physiological and pathological conditions. The active cerebroside was then

Figure 5.2 Structure of the synthetic KRN 7000.

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prepared by organic synthesis (Honda et al., 1991). Some years later, no biological activity was identified for the amphiceramides from the sponge Amphimedon viridis, including a-glycosylceramides, other than a low antifungal activity for their peracetylated derivatives (Hirsch and Kashman, 1989). Biological activity for b-glycosylceramides has been mainly reported since the late 1990s. The most important GSLs, in terms of biological activity, are undoubtedly the a-galactosylceramides. 5.2.3.1 Immunostimulating and Antitumor Properties of aGalactosylceramides Agelasphins, a-galactosylceramides, were isolated by antitumor and immunostimulatory bioassay-guided fractionation from an extract of the marine sponge Agelas mauritianus (Natori, Koezuka, and Higa, 1993; Natori et al., 1994). These GSLs, which had no precedent in marine natural products, were evaluated by an antitumor assay in which mice were implanted intraperitoneally with B16 cells, and also by an in-vitro mixed leukocyte reaction (MLR) assay. All of the GSLs examined showed a high activity, especially those with the a-galactosyl linkage, with tumor growth inhibition (T/C) ratios of between 160% and 190%. These a-GSLs also showed a low toxicity in rats (after intravenous injection). When the most active GSL, agelasphin-9b, was synthesized, it demonstrated the same antitumor activity and stimulatory effects as the natural compound (Akimoto, Natori, and Morita, 1993). This was the first report of immunomostimulatory activity by GSLs, and their marked antitumoral properties were considered due to an activation of the immune system (Costantino, Fattorusso, and Mangoni, 2001a). Natural a-GSLs are potent ligands of the major histocompatibility complex (MHC) class I-like CD1d protein, which is present on the surface of antigen-presenting cells (APCs), and are capable of activating, both in vitro and in vivo, a specialized population of T cells, known as natural killer (NK) cells. The latter play an important role in the regulation of immunity during infection, tumor growth, and autoimmune diseases (Godfrey and Berzins, 2007). The synthesis of various GSLs that differ only by the anomeric linkage led to the realization that a-GSLs are more potent than b-GSLs in terms of their antitumor activity (Morita et al., 1995a; Uchimura et al., 1997b). Subsequently, studies of the structure– activity relationship of various related synthetic GSLs led to the creation of a chemically optimized variant of the naturally occurring agelasphins, named KRN 7000 (Figure 5.2) (Banchet-Cadeddu et al., 2011; Morita et al., 1995b). KRN 7000 quickly became the prototypical antigen for studies of NK T-cell stimulation, and a suitable candidate for clinical use as an anticancer agent (Giaccone et al., 2002; Kikuchi et al., 2001).

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KRN 7000 has shown great potential in the treatment of several diseases, including cancer, malaria, and hepatitis B, and may also assist in fending off certain bacterial infections and suppressing autoimmune disorders (Wu, Fujio, and Wong, 2008). Some years later, a human cell line bioassay-directed investigation of the sponge Agelas sp. from Papua New Guinea led to the isolation of a new digalactofuranosyl ceramide, agelagalastatin, which inhibited the growth of cancer cells (Pettit et al., 1999). With the aim of understanding the relationships between the structural elements and the immunomodulatory and anticancer activities, various GSL analogs were rapidly synthetized (Morita et al., 1995a; Motoki et al., 1995). The importance of the lengths of the lipidic moieties, and of the three hydroxyl groups of the sphingoid base, has been investigated. Agelasphin analogs were assayed that presented the galactose OH groups at positions 2, 3 or 6 as glycosylated, or modified in other ways. Those compounds with a free 2-OH on the sugar directly linked to the ceramide moiety exhibited immunostimulatory activity (Costantino et al., 1996). The results obtained indicated that glycosylation or any other substitution at position 2 would cancel the immunomodulatory activity of the GSL, while glycosylation at position 6 had no effect; the effect of modifying the 3-OH group was less clear. The new a-galacto-GSL damicoside from the sponge Axinella damicornis, which possessed a glycosylated galactose 4-OH group, exhibited a stimulatory activity comparable to that of agelasphin, thus demonstrating that a free 4-OH group is not essential for the immunostimulatory activity (Costantino et al., 2005). Corrugoside has been verified as a new agonist of murine NK T cells and as a new immunostimulatory a-galacto-GSL, though its potency was much lower than that of a-GalCer (Costantino et al., 2008). 5.2.3.2 Biological Activity of b-Glycosylceramides The biological activity of the b-glycosylceramides is much less well documented than that of the a-glycosylceramides. Indeed, biological studies on isolated b-GSLs have rarely been conducted.

Cytotoxicity, Immunomodulatory, and Antitumor Activities of b-Glycosylceramides A dichloromethane–methanol extract of the sponge Haliclona sp., containing 10% of the new single GSL halicerebroside A, exhibited a mild antitumor activity against P388 leukemia cells (Hirsch and Kashman, 1989) (Table 5.3). Halicylindrosides isolated from the sponge Halichondria cylindrata were also cytotoxic against P388 murine leukemia cells (Li, Matsunaga, and Fusetani, 1995), while cerebrosides of the sea cucumber Cucumaria echinata showed lethality towards brine shrimps (Yamada et al., 1998a). Indeed, based on the results of brine shrimp lethality assays, new cerebrosides were isolated from an extract of the marine sponge Haliclona (Reniera) sp., but failed to demonstrate cytotoxicity towards a panel of five human solid tumor cell lines (Mansoor et al., 2007). The five newly identified GSLs, ophidiacerebrosides A–E, isolated from the sea star Ophidiaster ophidiamus, and including the major component ophidiacerebroside C (40%), showed

strong cytotoxicity against L1210 murine leukemia cells in vitro, exhibiting an up to 96% inhibition of L1210 cell growth at a concentration of 2 mg ml 1 (Jin, Rinehart, and Jares-Erijman, 1994). Recently, a mixture of ophidiacerebrosides C (63%), and B and D, isolated from the starfish Narcissia canariensis, displayed an interesting cytotoxic activity on various adherent human cancerous cell lines, with IC50-values in the range of 10 to 30 mM on multiple myeloma, colorectal adenocarcinoma, epidermoid carcinoma, and glioblastoma multiforme (Farokhi et al., 2010). GSLs from the sea star Oreaster reticulatus were tested for their cytotoxic activity on rat glioma C6 cells by evaluating cell growth, and shown to be mildly effective (Costantino et al., 2007), with glucosylceramides being more active than galactosylceramides. Oreacerebroside A and ophidiacerebroside E (gluco-GSL) showed the greatest effects. Glycosphingolipids are also known to modulate the signaling of angiogenic growth factors, including vascular endothelial growth factor (VEGF). One major point of interest would be to identify compounds capable of blocking the formation of new blood vessels, whether in chronic ischemia, heart failure or cancer therapy. In this respect, oreacerebroside I (galacto-GSL), but not the corresponding ophidiacerebroside E (gluco-GSL), improved the biological response to VEGF. Galacto-GSL was shown to exert a proangiogenic activity, and also to increase VEGFinduced human endothelial cell proliferation (Costantino et al., 2007). The presence of a prenylated galactose in the structure of the unique plakosides A and B, from the sponge Plakortis simplex, is of major importance for their bioactivity. In a T-cell proliferation assay, both plakosides inhibited lymphocyte proliferation following stimulation with concanavalin A, but this appeared not to be related to any cytotoxic effect (Costantino, Fattorusso, and Mangoni, 2001a). The plakosides were strongly immunosuppressive on activated T cells, and provided a useful model to improve the current understanding of the structural requirements for the immunomodulatory activity of glycosphingolipids (Costantino et al., 1997). Notably, the immunological behavior of plakosides differs from that of other bioactive GSLs, in that they are not only immunostimulatory agents but also effective immunosuppressors. Interestingly, the new galactosylceramides, axidjiferosides A and B, obtained from the African sponge Axinissa djiferi, showed a significant antimalarial activity with only a low cytotoxicity against various human cancer cell lines (Farokhi et al., 2013). Antifungal and Antibacterial Activities of b-Glycosylceramides The heptaacetyl amphicerebrosides B–D (with b-glucosamine), and amphicerebrosides E and F (a-glucosamine), from the sponge Amphimedon viridis, exhibited low antifungal activities against Candida albicans (Hirsch and Kashman, 1989) (Table 5.3). Halicylindrosides isolated from the sponge Halichondria cylindrata were moderately antifungal against Mortierella remanniana (Li, Matsunaga, and Fusetani, 1995). None of the GSLs isolated from the Chinese octocoral Sarcophyton ehrenbergi, including the new a-glycosylceramide, exhibited any antibacterial activity (Cheng et al., 2009).

5.3 Gangliosides

Anti-Fatty Liver Activity of b-Glucosylceramides The anti-fatty liver activity of the new GSL AMC-2 was studied in rats in which fatty liver had been induced by treatment with orotic acid (Xu et al., 2011). Subsequently, AMC-2 was shown to cause significant reductions in levels of hepatic triglycerides and total cholesterol and, as a consequence, would ameliorate nonalcoholic fatty liver disease. Activity as Nitric Oxide Release Inhibitors A mixture of the new fucosylated GSLs from the sponge Aplysinella rhax exhibited an effective inhibitory activity on lipopolysaccharide (LPS)-induced NO2 release by J774 A.1 macrophages (Borbone et al., 2001). In another study, the production of NO2 (a stable metabolite of NO) was measured as a parameter of macrophage activation and inducible NO-synthase (iNOS) induction. iNOS is regulated by inflammatory mediators (LPS, cytokines), and the excessive production of NO by iNOS has been implicated in the pathogenesis of the inflammatory response. These results were confirmed by a subsequent synthetic study (Hada et al., 2007). Most recently, terpioside B, a new difucosyl-GSL from the sponge Terpios sp., was shown to be a more potent inhibitor against LPS-induced NO release than were previously reported GSLs, such as terpioside A and monoglucosylceramide (Costantino et al., 2010). Terpioside B has been shown to possess an anti-inflammatory activity that is related to its ability to prevent the production of proinflammatory mediators such as nitrite. These results confirm those of previous studies (Borbone et al., 2001; Hada et al., 2007). The activity is retained even in analogs where the ceramide is replaced by a greatly simplified aglycone; this suggests that the nonpolar part of the molecule is not important, although activity is lost when an analog does not contain fucose, confirming the importance of this sugar. The in vitro anti-inflammatory activity of GSLs from Sarcophyton ehrenbergi has been monitored using LPS-stimulated cells. The stimulation of RAW 264.7 macrophages with LPS resulted in an upregulation of the proinflammatory iNOS and cyclo-oxygenase (COX)-2 proteins. The GSL with 2-hydroxy-C16:0 FA and diunsaturated sphingoid base reduced the levels of iNOS to 20.3  6.8%, and of COX-2 to 64.3  8.6%, in comparison to those of control groups (Cheng et al., 2009). The other isolated new GSLs, including the a-glycosylceramide, reduced iNOS protein expression (by up to 55.6%), but did not inhibit COX-2 protein expression. Miscellaneous Activities Among the new homologous regulosides A–C, obtained from the Indian starfish Pentaceraster regulus, reguloside A showed a moderate wound-healing activity (Venkannababu, Bhandari, and Garg, 1997). The new asteriacerebroside G, and the known asteriacerebrosides A and B, promoted plant growth in sprouts of Brassica campestris (Ishii, Okino, and Mino, 2006). The new galactosylceramides, axidjiferosides A and B (axidjiferoside A at 60%), obtained from the African sponge Axinyssa djiferi, showed a significant antimalarial activity, with an IC50value of 0.53  0.2 mM (Farokhi et al., 2013). Interestingly, these galactocerebrosides provided a good antiplasmodial activity, with low cytotoxicity against various human cancer cell lines and no significant antitrypanosomal and antileishmanial activities.

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Gangliosides are unusual glycosphingolipids, in that they are isolated from the water-soluble lipid fraction of a chloroform– methanol extract. They were first named by Ernst Klenk in 1942, after their isolation from ganglion cells of brain, and initially were described as “ . . . glycosphingolipids containing glucose and sialic acid” (Hoshi and Nagai, 1975). The two sialic residues found in echinoderms are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc) (Figure 5.3), as is the case in many other species except for humans, where the latter compound is typically undetectable in normal tissues (Kohla and Schauer, 2005). Investigations into gangliosides isolated from marine invertebrates started during the 1960s (Eldredge, Read, and Cutting, 1963), since when they have been described almost solely in echinoderms. Although one study reported their assumed presence in mollusca (squid and Pacific octopus) (Saito, Kitamura, and Sugiyama, 2001), the structures were not provided in detail. In other invertebrates, sialic acid was shown to be replaced by glucuronic acid (Kolter, 2012). Kochetkov and coworkers were among the first to examine gangliosides from marine invertebrates, especially sea urchins (Kochetkov, Smirnova, and Glukhoded, 1978; Shashkov et al., 1986; Smirnova, Chekareva, and Kochetkov, 1978; Smirnova, Chekareva, and Kochetkov, 1980). As marine GLs have been reviewed previously (Fattorusso and Mangoni, 1997), the aim of this section will be to update the present data on echinoderm gangliosides. 5.3.1 Occurrence and Structure

Until now, about 95 “molecular species” analyzed from 27 species of echinoderms (Table 5.1) have been described. As indicated previously for neutral GSLs (see Section 5.2.2), the term “molecular species” generally refers to a set of molecules that present the same sugar moiety; however, several LCBs and FAs, as well as some other molecules (single base and FA) have also been isolated. In contrast to mammalian gangliosides, no specific nomenclature has yet been established to assign names to gangliosides obtained from echinoderms (Kolter, 2012). The highest variability among different ganglioside species described in marine organisms relates to the carbohydrate sequence, including sialic acid. Consequently, in this section gangliosides will be presented by considering the linkage between the ceramide and

HO O

OH OH

COOH O

HN

HO

NeuAc

OH

HO HO O

OH OH

COOH O

HN

OH

HO

NeuGc

Figure 5.3 N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc): sialic residues of gangliosides from marine invertebrates.

130

5 Glycolipids from Marine Invertebrates

j

O HN S

(i)

HO HO

OH O O P O OH

O

HO

R

OH OH S O

R' (ii)

HO

(iii)

S

O

OH O

O

OH

HN

OH

O

O

OH O HO

O

OH

O

R

HN

O

R'

(iv)

HO HO

O OH

O

9

O

OH OH

COOH O

O HN

R'

O S

R

HN

MeO HO

O

HO

NH O

HO HO

HO

OH

O O P O OH

20,22

OH

Figure 5.5 CPJ2 isolated from C. japonica (Arao, Inagaki, and Higuchi, 2001). R

R'

Figure 5.4 Different linkages between ceramide and sugar moieties in gangliosides from echinoderms with fatty acid (R) and sphingoid base (R0 ). (i) inositolphosphoceramide gangliosides; (ii) lactosylceramide type; (iii) galactosylceramide type; (iv) glucosylceramide type.

possible). Biological activity is specified when available, and will be discussed in Section 5.3.2, while the ceramide moieties will be discussed for each table, and sugar moieties for all gangliosides in Section 5.3.1.5.

carbohydrate moieties, leading to four groups: (i) the glycosyl inositolphosphoceramide core; (ii) linkage by C-3 of galactose in a lactose moiety (derived from lactosylceramide); (iii) linkage by C-3 of galactose (derived from galactosylceramide); and (iv) linkage by C-6 of glucose (derived from glucosylceramide) (Figure 5.4). Among the previous groups, gangliosides will be classified by the most common part between all of the molecular species, which is in fact the ceramide moiety of either phytosphingosine or sphingosine type. Sphingoid bases and FAs described in Tables 5.4 to 5.11 represent respectively more than 10% of the analyzed mixture (the major components are indicated when

5.3.1.1 Inositolphosphoceramide Gangliosides The inositolphosphoceramide gangliosides refer to particular gangliosides described for the first time in 2001 in the feather star Comanthus japonica, collected at Koinoura, Japan (Arao, Inagaki, and Higuchi, 2001). Since then, they have been identified only in another feather star, Comanthina schlegeli, collected at Hedo Cape, Okinawa, Japan (Inagaki et al., 2007a) (Table 5.4). Those compounds present a 15 carbon chain length sphingosine base with saturated or unsaturated FAs. The sugar moiety is composed of phosphoinositol linked to one, two, or three methylated NeuGc (sialic residues) in C. japonica, and to one NeuAc in C. schlegeli (Figure 5.5). Glycosyl inositolphos-

Table 5.4 Glycosyl-inositolphosphoceramide-type gangliosides isolated from feather stars, with R and S representing fatty acids and sugar according to the model.

Fatty acid (R)

Sugar (S)

Name

Feather star

Biological activity

Reference

9-O-Me-a-NeuGc-(2 ! 3)-

CJP2

Comanthus japonica

Arao, Inagaki, and Higuchi, 2001  Arao et al., 2004

9-O-Me-a-NeuGc-(2 ! 11)-9-O-Me-a-NeuGc-(2 ! 3)-

CJP3

potentiate neuritogenesis of NGFa) (PC12 cells)

9-O-Me-a-NeuGc-(2 ! 11)-9-O-Me-a-NeuGc-(2 ! 11)9-O-Me-a-NeuGc-(2 ! 3)-

CJP4

9-O-Me-a-NeuAc-(2 ! 3)-

CSP2

Comanthina schlegeli

n. r.b)

Inagaki et al., 2007b

9-O-Me-a-NeuGc-(2 ! 11)-9-O-Me-a-NeuGc-(2 ! 3)-

CJP3

Comanthus japonica

potentiate neuritogenesis of NGF (PC12 cells)

Arao, Inagaki, and Higuchi, 2001  Arao et al., 2004

a) NGF ¼ nerve growth factor. b) n. r. ¼ not reported.



Arao et al., 2004

5.3 Gangliosides

j 131

Table 5.5 Gangliosides isolated from starfishes containing carbohydrate moiety linked to one phytosphingosine base (R0 ) by C3 of galactose in a lactose moiety.

R0

Fatty acid (R)

Sugar (S)

Name

Starfishes

Biological activity

Reference

b-Galf-(1 ! 3)-a-Galp(1 ! 4)-a-NeuAc-(2 ! 3)-

AGa) A, B, C (AG 2)

Acanthaster planci

neuritogenic activity (PC12 cells)

Kawano, Higuchi, and Komori, 1990 Miyamoto et al., 1997  Kaneko et al., 2007

AG F, G

n. r.b)

Miyamoto et al., 1997

b-Galf-(1 ! 3)-a-Galp(1 ! 3)-a-Galp-(1 ! 4)a-NeuAc-(2 ! 3)-

AG D, E (AG 3)

potentiate neuritogenesis of NGF (PC12 cells)

Kawano, Higuchi, and Komori, 1990 Miyamoto et al., 1997  Kaneko et al., 2007

b-Fucf-(1 ! 4)-a-Galp(1 ! 4)-a-NeuAc-(2 ! 3)-

AG I, J

n. r.

Miyamoto et al., 2000

1-a-Araf-(1 ! 3)-[a-Araf(1 ! 3)-a-Galp-(1 ! 6)]a-Galp-(1 ! 4)-a-NeuAc(2 ! 3)-

Asterinaganglioside A

Asterina pectinifera

n. r.

Higuchi et al., 1991b

b-Galf-(1 ! 3)-a-Galp(1 ! 4)-a-NeuAc-(2 ! 3)-

AGa) H

A. planci

n. r.

Miyamoto et al., 1997

a) AG ¼ acanthaganglioside. b) n. r. ¼ not reported.

phoceramide core gangliosides seem to be particular to the subphylum Crinozoa, class of Crinoidea.

5.3.1.2 Lactosylceramide Gangliosides Gangliosides deriving from lactosylceramide (b-Galp-(1 ! 4)b-Glcp) seem to be specific to starfishes, since all starfishes studied – except for Protoreaster nodosus (which will be discussed later) – present this linkage either with phytosphingosine (Tables 5.5 and 5.6) or sphingosine bases (Table 5.7). Moreover, a ganglioside deriving from lactosylceramide has been observed in a sea cucumber, Stichopus (¼ Apostichopus), provided by Bizen-Kaisan Co., Ltd (Kaneko et al., 2003). Ganglioside molecular species differ depending on the chain length of the sphingoid base (from 16 to 22 carbons),

and whether the bases are branched (iso or anteiso) or not, and saturated or not. The major FA from starfishes is always 2-hydroxylated and saturated with a chain length varying from 16 to 24 carbons, except for three molecular species described in Luidia maculata and Evasterias echinosoma (gathered respectively in Nha Trang Bay at the Vietnam coast and in Possjet Bay of the Sea of Japan in September), which present unsubstituted FAs (Smirnova, 2000a, 2000b). Recently, three particular gangliosides, named PNG-1 (Figure 5.6), PNG-2A and PNG-2B, were isolated from the pyloric caeca of the starfish Protoreaster nodosus collected at Katsuren, on the east coast of Okinawa in June 2009 (Pan et al., 2012). The fact that this was the first time that gangliosides deriving from galactosylceramide had been described in the literature

132

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Table 5.6 Gangliosides containing carbohydrate moiety linked to phytosphingosine bases (R0 ) by C3 of galactose in a lactose moiety (mixture of fatty

acids and bases).

R0

Fatty acid (R)

Sugar (S)

Name

Organism (starfish unless otherwise stated)

Biological activity

Reference

8-O-Me-a-NeuGc-(2 ! 3)-

A

Aphelasterias japonica

n. r.a)

Smirnova, Kochetkov, and Sadovskaya, 1987

22

Luidia maculata

b-Galp-(1 ! 4)-8-O-Me-NeuAc(2 ! 3)-

—

Luidia quinaria bispinosa

b-Galf-(1 ! 4)-a-NeuAc-(2 ! 3)-

1

A. planci

IDENTICAL SUGAR MOIETY

(major)

þ n ¼ 19

þ n ¼ 11, 13, 14

(major)

Kawatake et al., 2002

(major)

SATURATED BASES

n. r.

Smirnova and Kochetkov, 1985 Smirnova, 1990

(major) 2

(major) (3) þ n ¼ 14

(major) þ n ¼ 12, 14, 16 (1) þ n ¼ 11, 12, 14 (2)

(major) þ n ¼ 21

(major) þ n ¼ 12, 15 (1) þ n ¼ 11, 12 (2)

8-O-Me-a-NeuAc-(2 ! 11)a-NeuGc-(2 ! 3)-

LLG-3a)

8-O-Me-a-NeuGc-(2 ! 11)a-NeuGc-(2 ! 11)-a-NeuGc(2 ! 3)-

LLG-5(2)

a-NeuAc-(2 ! 8)-a-NeuAc(2 ! 3)-

LMG-4

8-O-Me-a-NeuGc-(2 ! 3)b-GalNAc-(1 ! 3)-

1

8-O-Me-a-NeuGc-(2 ! 3)[a-NeuAc-(2 ! 6)]-b-GalNAc(1 ! 3)

2

(3)

Linckia laevigata

potentiate neuritogenesis of NGF (PC12 cells)

Inagaki, Isobe, and Higuchi, 1999  Inagaki et al., 2005

L. maculata

Kawatake et al., 2004 Evasterias retifera

n. r.

Smirnova, 2003

5.3 Gangliosides

(major) n ¼ 10, 11, 12, 13

j 133

a-NeuAc-(2 ! 3)-

LMG-2

L. maculata

potentiate neuritogenesis of NGF (PC12 cells)

Kawatake et al., 1999

8-O-Me-a-NeuGc-(2 ! 3)And 11-O-Me-a-NeuGc(2 ! 3)-

GAA-6

Asterias amurensis versicolor

n. r.

Higuchi et al., 1993

8-O-Me-a-NeuGc-(2 ! 3)-[8-OMe-a-NeuGc-(2 ! 6)]-b-GalNAc(1 ! 3)-

GAA-7

a-Galp-(1 ! 4)-a-NeuAc-(2 ! 3)-

LG-1

a-Arap-(1 ! 4)-a-Galp-(1 ! 4)a-NeuAc-(2 ! 3)-

LG-2

a-NeuAc-(2 ! 4)-a-NeuAc(2 ! 3)-

—

Lethasterias fusca

n. r.

Smirnova, Glukhoded, and Kochetkov, 1986

8-O-Me-a-NeuGc-(2 ! 11)a-NeuGc-(2 ! 3)-

B1

A. japonica

n. r.

8-O-Me-a-NeuGc-(2 ! 11)-[8-O-

B2

Smirnova, Kochetkov, and Sadovskaya, 1987

Sea cucumber Stichopus japonicus

potentiate neuritogenesis of NGF (PC12 cells)

Kaneko et al., 2003

(major) n ¼ 20, 21

UNSATURATED

(major) n ¼ 13, 15, 19, 20, 22

neuritogenic and growthinhibitory activity towards the mouse Higuchi neuroblastoma et al., 1993 cell line  Kaneko potentiate et al., 2007 neuritogenesis of NGF (PC12 cells)

BRANCHED

(major) þ n ¼ 9, 11

(major) þ n ¼ 10, 12

(major) þ n ¼ 11, 13, 14

(major)

Astropecten latespinosus No for Antitumor activity against murine lymphoma Antitumor activity against murine lymphoma

Higuchi et al., 1995a Higuchi et al., 1995a, 1995b

þ n ¼ 20, 21

(major)

þ n ¼ 19 (B1) Me-NeuGc-(2 ! 3)]þ n ¼ 11, 20, 21(B2)

a-NeuAc-(2 ! 3)-[a-NeuAcSJG-2 (2 ! 4)]-b-Galp-(1 ! 8)-a-NeuAc(2 ! 3)-b-GalNAc-(1 ! 3)-

þ n ¼ 14, 18, 20

þ n ¼ 9, 11 (continued )

134

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Table 5.6 (Continued) R0

Fatty acid (R)

Sugar (S)

Name

Organism (starfish unless otherwise stated)

Biological activity

Reference

A. pectinifera

n. r.

Sugita, 1979a

SATURATED AND BRANCHED

(major) þ n ¼ 11 (all) þ n ¼ 9 (GP-1a, 1b, GP-2) þ n ¼ 12

(major) þ n ¼ 20, 21 (all) þ n ¼ 17, 18 (GP1a, 1b, GP-2)

(major) n ¼ 11, 14, 15

a) n. r. ¼ not reported.

b-Arap-(1 ! 6)-b-Galp(1 ! 4)-NeuGc-(2 ! 3)-

Ganglioside 2 Kochetkov and Smirnova, 1983 Glukhoded, Smirnova, and Kochetkov, 1990

(1 ! 4)-NeuGc-(2 ! 3)b-Galp-(1 ! 4)-

þ n ¼ 9(ganglioside 1, 2, 3, ) þ n ¼ 11 ( )

(major)

Sugita, 1979b

(1 ! 4)-8-O-Me-NeuGc(2 ! 3)-

Araf-(1 ! 3)-a-Galp-(1 ! 4)- — 8-O-Me-NeuAc-(2 ! 3)-Galp(1 ! 3)-Galp-(1 ! 4)-NeuAc(2 ! 3)— a-Arap-(1 ! 3)-b-Galp-

þ n ¼ 12,13 (GP-1a, 1b, GP-2) þ n ¼ 10 ( )

þ n ¼ 11

b-Araf-(1 ! 6)-b-Galp-(1 ! 4) Ganglioside 3 [b-Galp(1 ! 8)]-NeuGc(2 ! 3)Ganglioside 1 b-Arap-(1 ! 6)-b-Galp-

a-Arap-(1 ! 3)-b-Galp(1 ! 4)-8-O-Me-NeuGc(2 ! 3)-b-Galp-(1 ! 4)-

—

a-Araf-(1 ! 3)-a-Galp(1 ! 4)-a-NeuAc-(2 ! 3)-

GP-1a

a-Araf-(1 ! 3)-[a-Araf(1 ! 4)]-a-Galp-(1 ! 4)a-NeuAc-(2 ! 3)-

GP-1b

a-Araf-(1 ! 3)-[a-Araf(1 ! 3)-a-Galp-(1 ! 6)]a-Galp-(1 ! 4)-a-NeuAc(2 ! 3)-

GP-2 Sialoglycolipid Ia)

Support survival of cultured cerebral cortex cells

a-Araf-(1 ! 3)-a-Galp(1 ! 4)-a-NeuAc-(2 ! 6)b-Galf-(1 ! 3)-[a-Araf(1 ! 4)]-a-Galp-(1 ! 4)a-NeuAc-(2 ! 3)-

GP-3

potentiate Higuchi neuritogenesis of et al., 2006 NGF (PC12 cells)

a-NeuAc-(2 ! 9)-a-NeuAc(2 ! 3)-b-GalNAc-(1 ! 3)-

—

E. retifera

n. r.

Kochetkov, Smirnova, and Glukhoded, 1982

a-NeuGc-(2 ! 3)-

LLG-1

A. pectinifera

n. r.

Inagaki et al., 2009

Higuchi et al., 1991b

(major)

Higuchi et al., 1991b 1 Smirnova and Kochetkov, 1980

þ n ¼ 11, 12

(major) n ¼ 13, 14, 20, 21

5.3 Gangliosides

j 135

Table 5.7 Gangliosides containing carbohydrate moiety linked to sphingosine base (R0 ) by C3 of galactose in a lactose moiety in starfishes.

R0

Fatty acid (R)

Not determined

Sugar (S)

Name

Starfish

Biological activity

Reference

8-O-Me-a-NeuAc-(2 ! 3)-

LMG-3

L. laeviga

n. r.a)

Kawatake et al., 2002

8-O-Me-a-NeuGc-(2 ! 3)-

—

L. maculata

Smirnova, 2000b

8-O-Me-NeuAc-(2 ! 3)-[NeuAc-(2 ! 6)]GalNAc-(1 ! 3)-

3

Evasterias echinosoma

Smirnova, 2000a

8-O-Me-NeuAc-(2 ! 3)-[NeuAc-(2 ! 6)]GalNFor-(1 ! 3)-

4

a) n. r. ¼ not reported.

Table 5.8 Gangliosides isolated from the starfish Protoreaster nodosus containing carbohydrate moiety linked to phytosphingosine base (R0 ) by C3 of

galactose.

R0

Fatty acid (R)

a) n. r. ¼ not reported.

Sugar (S)

Name

Biological activity

Reference

8-O-Me-a-NeuAc-(2 ! 3)-

PNG-1

n. r.a)

Pan et al., 2012

b-Galf-(1 ! 3)-a-Galp-(1 ! 4)-8-O-Me-a NeuAc -(2 ! 3)-

PNG2A

b-Galf-(1 ! 3)-a-Galp-(1 ! 9)-a-NeuAc-(2 ! 3)-

PNG-2B

136

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5 Glycolipids from Marine Invertebrates

O HN OMe HO HO AcHN

HOOC O HO

HO O

OH O

R OH

O

OH

R' OH

Figure 5.6 PNG-1 isolated from Protoreaster nodosus (Pan et al., 2012).

may have been due to the fact that authors had analyzed the pyloric caeca and not the whole body of the starfish. Details of the phytosphingosine base and FAs of PNG-1, PNG-2A and PNG-2B are listed in Table 5.8. The fatty acids, as in other starfishes, are 2-hydroxylated, and the phytosphingosine base is saturated or iso-branched. NeuAc, whether 8-O-methylated or not, is the only sialic residue analyzed. 5.3.1.3 Glucosylceramide Gangliosides The last group of gangliosides, derived from glucosylceramide (Tables 5.9–5.11), has only been described in the echinoderm subphylum Echinozoa and in the brittle star studied. Ganglioside molecular species differ depending on the chain length of the saturated sphingoid base (from 16 to 18 carbons), and whether the bases are branched (iso or anteiso) or not. The major FAs are 2-hydroxylated or unsubstituted, contrary to starfishes, and saturated or monounsaturated with a chain length varying from 15 to 25 carbons. Four neutral sugars – namely, arabinose, fucose, galactose and glucose – form the sugar moieties of gangliosides in either the pyranose or furanose form, except for glucose, which is only described in the pyranose form. Fucose and galactose in the furanose form, and arabinose in both forms (furanose or pyranose), have only been described in starfishes. In fact, Araf have been described in gangliosides isolated from Asterina pectinifera (Higuchi et al., 1991b, 2006; Kochetkov and Smirnova, 1983), some specimens of which were collected from Wakasa Bay (Japan Sea) (Sugita, 1979a). Arap was described in the same starfish (Glukhoded, Smirnova, and Kochetkov, 1990; Sugita, 1979b), and has also been isolated from Astropecten latespinosus (Higuchi et al., 1995a, 1995b) (Tables 5.5 and 5.6). Fucose was only described in sea cucumbers (namely, Cucumaria echinata collected in 1997 from the Sea of Genkai, Japan) (Kisa et al., 2006a), from Holothuria pervicax collected in 1994 at Tsuyazaki, Fukuoka, Japan (Yamada et al., 1998b, 2000), and from Holothuria leucospilota collected at Ushibuka, Kumamoto, Japan in 1997 (Yamada et al., 2001) (Table 5.9). This sugar could be 4-acetylated, or not, and arabinose and fucose were always in the terminal position. Galactose in the furanose form has only been observed in starfishes belonging to the superorder Valvatacea and the order Valvatida, since terminal galactose-furanose is only observed in Acanthaster planci (Tables 5.5 and 5.6) (Kawano, Higuchi, and Komori, 1990; Miyamoto et al., 1997; Smirnova, 1990) and

Protoreaster nodosus (Table 5.8), while inner galactose-furanose has been described in Asterina pectinifera (¼ Patiria pectinifera) (Table 5.6; GP3) (Higuchi et al., 2006). Moreover, N-acetylgalactosamine (GalNac) and N-formylgalactosamine (GalNFor) (Figure 5.7) are only described in gangliosides deriving from lactosylceramide. GalNAc has been identified in three starfishes belonging to the same phylogenetic family of Asteriidae (1 and 2 (Smirnova, 2003); GAA-7; 3 (Smirnova, 2000b) and in the sea cucumber (Stichopus japonicas) (SJG-2). Meanwhile, GalNFor has been described in one of the starfishes (Asteriida 4; Smirnova, 2000b). Gangliosides from echinoderms are mostly monosialogangliosides with NeuAc as the sialic acid, although disialo- up to tetrasialogangliosides (Hp-s5) have also been described. Multisialogangliosides contain either NeuGc or NeuAc, or both. Naturally occurring sialic acids are assumed to be the D-form, and this configuration has been proven for nine gangliosides isolated from Cucumaria echinata (Kisa et al., 2007). Although sialic acids are mainly terminal, in some cases they may locate within the oligosaccharide moieties, which represents a huge difference from vertebrate species (Kohla and Schauer, 2005). In total, 50 sialic acids have been described in literature, with a huge diversity resulting on the one hand from the different a linkages that can be formed with adjacent sugars, and on the other hand from a variety of natural modifications (Schauer, 2004). Only some of natural modifications have been described in gangliosides from echinoderms. In fact, 8-O-methylation has only been observed in starfishes (Figure 5.8), in contrast to the 9-Omethylation observed in the case of inositol-gangliosides (feather stars) and in one ganglioside, DSG-A, isolated from the sea urchin Diadema setosum collected from the sea coast of Kagoshima, Japan in 1999 (Yamada et al., 2008). Until now, the O-methylation of sialic acids in gangliosides has only been described in echinoderms. Both, 8-O- and 4-O-sulfated sialic acids have been analyzed in sea cucumbers, sea urchins and the only brittle star described, Ophiocoma scolopendrina, collected in 1998 at Cape Zanpa, Okinawa, Japan (Inagaki et al., 2001) (Figure 5.9), but never in starfishes. All of the linkages formed by sialic acids in gangliosides are summarized in Table 5.12. The linkage between C-2 and C-11 of NeuGc, such as in LLG-5 from Linckia laevigata collected in 1995 at Okinawa (Japan) (Inagaki, Isobe, and Higuchi, 1999) (Figure 5.10), seems specific to nonmammalian gangliosides (Kolter, 2012). In gangliosides deriving from lactosylceramide, the terminal unit is either a neutral sugar (galactose, fucose, arabinose) or a sialic residue (NeuAc or NeuGc, methylated or not); this is in contrast to gangliosides deriving from glucosylceramide, where the terminal unit is principally sialic acid, NeuAc or NeuGc, neither modified, methylated, nor sulfated. The longest glycan moieties, which contain eight sugar units and are branched (GP-3) or not (Kochetkov and Smirnova,

5.3 Gangliosides

j 137

Table 5.9 Gangliosides with phytosphingosine base (R0 ) linked to ceramide by C6 of glucose.

R0

Fatty acid (R)

Sugar (S)

Name Organism

Activity

Reference

8-[OSO3 ]-a-NeuGc-(2 ! 6)-

III

Sea urchin Echinocardium cordatum

n. r.a)

Kochetkov, Smirnova, and Chekareva, 1976

T1

Sea urchin Anthocidaris crassispina Now: Heliocidaris crassispina

CG11 21b

Sea cucumber Cucumaria echinata

potentiate neuritogenesis of NGF (PC12 cells)1b

Yamada et al., 1998a 1b Kisa et al., 2006a

II

Sea urchin Echinarachnius parma

n. r.

Smirnova, Chekareva, and Kochetkov, 1980

1

Sea urchin Diadema setosum

potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 2008

IDENTICAL SUGAR MOIETY

þ n ¼ 11 (III)

(major) Kubo et al., 1990

(major)

(1)

1

þ n ¼ 15, 20 þ n ¼ 20

(1b)

a-NeuAc-(2 ! 6)-

(major) þ n ¼ 12,17,19

(major) 2

(major) 5

(major) (continued )

138

5 Glycolipids from Marine Invertebrates

j

Table 5.9 (Continued) R0

Fatty acid (R)

Sugar (S)

a-NeuGc-(2 ! 6)-

Name Organism

Activity

Reference

Hp-s1 Sea urchin, Hemicentrotus pulcherrimus

n. r.

Ijuin et al., 1996

II

Sea urchin Strongylocentrotus intermedius

Kochetkov, Smirnova, and Glukhoded, 1978

M5

Sea urchin A. crassispina

Kubo et al., 1990

HLG1

Sea cucumber H. leucospilota C. echinata

potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 2001  Kisa et al., 2006b

SJG-1

Sea cucumber S. japonicus1 C. echinata2

potentiate neuritogenesis of NGF (PC12 cells)

1 Kaneko et al., 1999 2 Kisa et al., 2006a

OSG0

Brittle star Ophiocoma scolopendrina

potentiate neuritogenesis of NGF (PC12 cells)

Inagaki et al., 2001

(major) þ n ¼ 17,19

(major) xþy ¼ 17, 18

(major)1 (1)

þ n ¼ 11, 12 (1)

(major) þ n ¼ 21 (1 and 2) (1) þ n ¼ 18, 20 (2) þ n ¼ 16, 17, 18, 19 (1)

(2)

(major)2 þ 1

(major) n ¼ 18, 20, 21, 22

þ n ¼ 12

5.3 Gangliosides

j 139

SATURATED BASES

Sea urchin S. intermedius

NeuGc-(2 ! 6)-Glcp-(1 ! 8)NeuGc-(2 ! 6)-

G-1

8-[OSO3 ]-NeuGc-(2 ! 6)Glcp-(1 ! 8)-NeuGc-(2 ! 6)-

G-2

a-NeuGc-(2 ! 4)-a-NeuGc(2 ! 6)-

I

Sea urchin E. cordatum

9-O-Me-a-NeuAc-(2 ! 6)-

DSGA

Sea urchin D. setosum

a-NeuAc-(2 ! 8)-a-NeuAc(2 ! 6)-

Hp-s2 Sea urchin

a-NeuAc-(2 ! 8)-a-NeuAc(2 ! 8)-a-NeuAc-(2 ! 6)-

Hp-s3

8-[OSO3 ]-NeuAc-(2 ! 6)

Hp-s4

a-NeuAc-(2 ! 8)-a-NeuAc(2 ! 8)-a-NeuAc-(2 ! 8)a-NeuAc-(2 ! 6)-

Hp-s5

8-[OSO3 ]-NeuAc-(2 ! 8)a-NeuAc-(2 ! 6)-

Hp-s6

a-Fucp-(1 ! 11)-a-NeuGc(2 ! 6)-

CEG51

NeuGc-(2 ! 11)-NeuGc(2 ! 4)-NeuAc-(2 ! 6)-

CEG92

n. r.

Prokazova et al., 1981

Smirnova, Chekareva, and Kochetkov, 1978

þ n ¼ 14 (I) potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 2008

n. r.

Ijuin et al., 1996

potentiate neuritogenesis of NGF (PC12 cells)

1

(major)

H. pulcherrimus

BRANCHED BASES

Sea cucumber C. echinata

2

Kisa et al., 2006a Kisa et al., 2006b

(major) (1) (2)

þ n ¼ 15, 17, 20, 21 þ n ¼ 15, 17, 21

(2)

(continued )

140

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5 Glycolipids from Marine Invertebrates

Table 5.9 (Continued) R0

Fatty acid (R)

Sugar (S)

Name Organism

a-Fucp-(1 ! 8)-a-NeuGc(2 ! 4)-a-NeuAc-(2 ! 6)-

HPG1

a-NeuGc-(2 ! 4)-a-NeuAc(2 ! 6)-

HPG3

4-[OSO3 ]-a-NeuAc-(2 ! 6)-

HPG8 HPG7

Activity

Reference

potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 1998b

DIFFERENT BRANCHED BASES

n ¼ 10, 11

þ n ¼ 19, 20

a-Fucp-(1 ! 4)-a-NeuAc(2 ! 11)-a-NeuGc-(2 ! 4)a-NeuAc-(2 ! 6)-

Sea cucumber Holothuria pervicax

Yamada et al., 2000

Sea cucumber H. leucospilota

a-Fucp-(1 ! 11)-a-NeuGc(2 ! 4)-a-NeuAc-(2 ! 6)-

HLG3

8-[OSO3 ]-a-NeuAc-(2 ! 6)-

OSG- Brittle star 11 Ophiocoma 42 scolopendrina

a-NeuGc-(2 ! 8)-a-NeuAc(2 ! 6)-

OSG21

Yamada et al., 2001

SATURATED AND BRANCHED BASES

(major OSG-1, 2) þ n ¼ 12 (OSG-1)

(major OSG-1) n ¼ 15, 19, 21

(major 4)

(major) þ n ¼ 15, 20, 21

a) n. r. ¼ not reported.

potentiate neuritogenesis of NGF (PC12 cells)

1 Inagaki et al., 2001 2 Yamada et al., 2008

5.3 Gangliosides

j 141

Table 5.10 Gangliosides with sphingosine base (R0 ) linked to ceramide by C6 of glucose.

R0

Fatty acid (R)

Sugar (S)

Name

Organism

Activity

Reference

a-NeuAc-(2 ! 6)-

Compound 1

n. r.a) Sea urchin A. crassispina

Hoshi and Nagai, 1975

a-NeuAc-(2 ! 8)-a-NeuAc(2 ! 6)-

Compound 5

a-NeuGc-(2 ! 6)-

—

Sea cucumber Cucumaria japonica

Chekareva, Smirnova, and Kochetkov, 1991

a-NeuGc-(2 ! 4)-a-NeuAc(2 ! 6)-

HLG-2

Sea cucumber Holothuria leucospilota

a-Fucp-(1 ! 11)-a-NeuGc(2 ! 6)-

SCG-3

Sea cucumber S. chloronotus

Yamada et al., 2003

CEG-4

Sea cucumber C. echinata

Kisa et al., 2006a

ONE BASE SATURATED

þ n ¼ 13, 15 ( )

(major) þ n ¼ 15

BASE BRANCHED

(major)

potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 2001

(SCG-3 þ HLG-2)

(major) þ n ¼ 20, 21

m ¼ 14, 18, 21

(major) þ n ¼ 14, 18, 20, 22 (continued )

142

j

5 Glycolipids from Marine Invertebrates

Table 5.10 (Continued) R0

Fatty acid (R)

Sugar (S)

Name

Organism

Activity

Reference

a-Fucp-(1 ! 11)-a-NeuGc(2 ! 4)-a-NeuAc-(2 ! 6)-

CEG-6

Kisa et al., 2006b

4-O-Ac-a-Fucp-(1 ! 11)a-NeuGc-(2 ! 6)-

CEG-3

Kisa et al., 2006a

NeuGc-(2 ! 11)-NeuGc(2 ! 4)-NeuAc-(2 ! 6)-

CEG-8

Kisa et al., 2006b

8-[OSO3 ]-a-NeuAc-(2 ! 6)-

SCG-2

(major) þ n ¼ 16, 21, 22

(major) þ n ¼ 15, 20, 21

(major) þ n ¼ 15, 17, 19 Sea cucumber Stichopus chloronotus

Yamada et al., 2003

a) n. r. ¼ not reported.

1983), have been described in the starfish Asterina pectinifera (¼ Patiria pectinifera). Moreover, starfishes and the sea cucumber Stichopus (¼ Apostichopus) appear to be the only echinoderms capable of generating gangliosides with a branched carbohydrate moiety, the ramification being whether with

neutral sugars (arabinose and/or galactose) or sialic acid residues (NeuAc or 8-O-Me-NeuGc). A branched group could be carried by galactose or galactosamine, and rarely by the sialic residue, as in Asterina pectinifera (¼ Patiria pectinifera) (Ganglioside 3 and GP1b).

Table 5.11 Gangliosides with sphingosine (R0 ) and phytosphingosine base linked to ceramide by C6 of glucose.

R0

Fatty acid (R)

Sphingosine þ Phytosphingosine þ

þ n ¼ 20, 21

m ¼ 20, 21

Sugar (S)

Name

Organism

Biological activity

Reference

a-NeuGc-(2 ! 6)-

SCG-1

Sea cucumber S. chloronotus

potentiate neuritogenesis of NGF (PC12 cells)

Yamada et al., 2003

5.3 Gangliosides

OH OH

OH OH

O

HO

NH

OH

O

HO

HO

NH H

O

(i)

OH

O O

(ii)

Figure 5.7 Structures of (i) N-acetylgalactosamine and (ii) Nformylgalactosamine.

O

8

OMe OH

HN

COOH OHOH O O O

HO

OH O HO

NH OH

19

OH O

O 13

OH

O

OSO3OH

COOH NH O

HN

HO HO HO

O

15

OH O

O 9

OH

OH

Figure 5.9 Structure of SCG-2 (S. chloronotus) with 8-O-sulfation on sialic acid (Yamada et al., 2003).

O HO

8

j 143

OH

Figure 5.8 Structure of LMG-3 (L. maculata) with 8-O-methylation on sialic acid (Kawatake et al., 2002).

5.3.2 Biological Activity

The biological activity of a small number of the gangliosides isolated from marine echinoderms has been investigated since 1991 by groups at Kyushu University, Japan, and summarized in previous reviews (Higuchi et al., 2007; Kaneko et al., 2007). Initially, the effect of GP-2 on the survival of cultured cortex cells of rat fetuses was monitored (Table 5.6), and showed a more potent activity at 1 mg ml 1 than did the mammalian ganglioside, GM1 (Higuchi et al., 1991b). Subsequently, the neuritogenic and growth-inhibitory activities of GAA-7 (Table 5.6), AG C and AG E (Table 5.5) towards a mouse

neuroblastoma cell line (Neuro 2a) were monitored (Higuchi et al., 1993). Both, AG C and AG E showed a slight neuritogenic activity at 1 mM, but this was inhibited in the presence of fetal calf serum (FCS). GAA-7 was slightly more active than AG C and AG E, and its activity was less sensitive to FCS. Moreover, GAA-7 was the only ganglioside to reveal a weak growth-inhibitory activity towards the Neuro 2a cell line. Two neutral GSLs – acanthacerebroside A and acanthalactoside A (Kawano et al., 1988b) – were also monitored but neither showed any activity. The 8-O-Me and/or GalNac sialic residues appeared to be related to GAA-7 activity. When, in 1995, LG-1 and LG-2 were tested for antitumor activity, using murine lymphoma L1210 cells and human epidermoid carcinoma KB cells (Higuchi et al., 1995a), none of the gangliosides showed any activity against KB cells, but the IC50-value for LG-2 against L1210 was 17 mg ml 1. Since 1995, all biological tests reported for gangliosides from echinoderms have involved neuritogenic activity towards the rat pheochromocytoma cell line PC12. The gangliosides failed to show any activity in the absence of nerve growth factor (NGF), and consequently they were thought to potentiate the neuritogenetic activity of NGF. The effects of gangliosides from echinoderms were compared to those of a mammalian ganglioside GM1, which is known to enhance

Table 5.12 Diversity in sialic acids resulting from the alpha linkage between Cx of NeuAc and NeuGc with other sugars by carbon y (number in brackets).

a linkage by (Cx)

NeuAc

C2 (Terminal)

Inositol (3), NeuGc (11), galactose (3), GalNac (3), NeuAc (8, 4), glucose (6) GalNAc et GalNFor (3, 6), NeuAc (9) NeuGc (4) Inner Galactose (3), Glucose (6) Galactose (6), GalNac (3) Inositol (3), NeuGc (11), NeuAc (4)

C2 C4

Fucose (1), NeuAc (2) C8 NeuAc (2), NeuGc (2) C11

NeuGc

–

galactose (1), NeuGc (2) – Galactose (1) Glucose (1), fucose (1) NeuAc (2), NeuGc (2), Fucose (1)

144

5 Glycolipids from Marine Invertebrates

j

O NH OH

OH HO OH COOH OH O O COOH H OH OH 2 N O H O O O O OH O 11 N HO HO HO OH O OH 11 O

HO

HOOC H3CO HO HO

2

19

OH OH 12

OH

O OH

HN O OH

Figure 5.10 2,11-linked sialic acid in LLG-5 (Inagaki et al., 2005).

neurologic recovery (Geisler, Dorsey, and Coleman, 1991) and whose structure was confirmed as b-Gal-(1 ! 3)-b-GalNac(1 ! 4)-[a-NeuAc-(2 ! 3)]-b-Gal-(1 ! 4)-b-Glc-(1 ! 0 )-ceramide. The results of these biological assays are summarized in Table 5.13. One important finding was that, unlike marine gangliosides, the neutral GSLs showed no activity towards the rat pheochromocytoma cell line PC12. Unfortunately, it may be difficult to compare results for gangliosides, as NGF and GM1 have not always been tested at the same concentrations. Indeed, when their concentrations have been similar, the results obtained were different. Nevertheless, when differences between the proportions of neurite-bearing cells with ganglioside þ NGF, or with NGF alone, were calculated, five gangliosides – SJG-2, CEG-3, LLG-3, GAA-7 and LLG-5 – seemed to be more potent than the others. Moreover, when this difference was calculated between gangliosides from echinoderms and GM1, the glycosyl-inositolphosphoceramide-type gangliosides appeared to be the most active, though the five noted above were also among the eight most active. When considering glycosyl-inositolphosphoceramide-type gangliosides, the biological activity seems not to be linked to the sialic residue, since CJP1 – which does not contain sialic residue – shows the same activity as CJP2, CJP3 and CJP4 that contain, respectively, one, two or three sialic residue(s). Biological activity may also be due to the presence of an “inositolphospho” group. Among SJG-2, CEG-3, LLG-3, GAA-7 and LLG-5, the last three have a terminal 8-O-Me sialic acid. Moreover, CEG-3 with a terminal 4-O-Ac-a-Fucp is more potent than CEG-6, which presents a terminal a-Fucp. SJG-2 presents a b-GalNac function and two terminal sialic residues. Of particular note here is that SJG-1 isolated from C. echinata presents the same activity as GM1, in contrast to SJG-1 isolated from S. japonicas, which is less potent than GM1; these molecular species differ by their ceramide moiety.

In order to better understand the relationship between the structure and biological activity of the gangliosides obtained from echinoderms, many attempts have been made since 1994 to synthesize these compounds. Although gangliosides M5, isolated from the sea urchin Anthocidaris crassispina, were subsequently synthesized, the compound obtained did not present the same FA as its natural counterpart (Yamamoto et al., 1994). Synthesis of the pentasaccharide moiety of AG-2 isolated from the starfish Acanthaster planci was reported in 2009 (Hanashima et al., 2009a), but no information is yet available concerning the total synthesis or biological assay. Also during 2009, the first total synthesis was reported for HLG-2 isolated from the sea cucumber Holothuria leucospilota (Iwayama et al., 2009) but, again, the biological assays of these synthesized compounds remain unreported. Synthesis of the sugar moiety of HPG-1 and HPG-7, isolated from the sea cucumber Holothuria pervicax, has also been reported, and the total synthesis and biological assays are currently under way (Iwayama et al., 2011; Shimizu et al., 2011). Recently, the pentasaccharide moiety of GAA-7, isolated from the starfish Asterias amurensis versicolor, has been synthesized (Tamai et al., 2012) and, according to the authors, the complete synthesis is currently also under way. In some cases, the total synthesis of compounds has been monitored in parallel with testing for biological activity. Three syntheses have been described for the main compound of LLG-3, isolated from the starfish Linckia laevigata (Hanashima et al., 2009b; Rich and Withers, 2012; Tamai et al., 2011a, 2011b); one of the synthesized compounds appeared to demonstrate neurogenetic activity towards the rat pheochromocytoma cell line PC12 (Tamai et al., 2011a). An analog of Hp-s1 isolated from the sea urchin Hemicentrotus pulcherrimus (Tsai et al., 2012) was also synthesized via a chemoselective activation glycosylation, but differed from Hp-s1 by the length of the sphingosine base (one carbon less) and the FA, which was saturated. This analog was found to present a slight

5.4 Atypical Glycolipids

j 145

Table 5.13 Biological activity of gangliosides.

Gangliosides

Concentration of gangliosides

SCG-1 SCG-2 SCG-3

3.3 mg ml

DSG-A 2 4 5 1

10 mg ml

CJP2 CJP3 CJP4 CJP1b)

10 mM

SJG-1 (C. echinata) CG-1 CEG-3 CEG-4 CEG-5 CEG-6 CEG-8 CEG-9 HLG-3 SJG-2 LLG-3 GAA-7 LLG-5 AG-2 (AG A þ Bþ C) HLG-2 LMG-4 AG-3 (AG D þ AG E) HLG-1 HLG-3 GP-3 SJG-1 (S japonicus) LMG-2 Neutral GSLc)

1

1

% neurite bearing cells with NGF alone

% neurite bearing cells with GM1

Reference

34 24 24

Not reported (5 ng mL 1)

22 (3.3 mg mL 1)

Yamada et al., 2003

41 34 30 26 25

19 (5 ng mL 1)

25 (10 mg mL 1)

Yamada et al., 2008

50 49 49 48

17 (3 ng mL 1)

20 (10 mM)

Arao et al., 2004

39

7.5 (5 ng mL 1)

35.6 (10 mM)

Kisa et al., 2006a, 2006b

21  2 (5 ng mL 1)

47  2,(5) (10 mM)

Kaneko et al., 2007

% neurite bearing cells with NGFa)

43 51 34 36 43 40 35 42 65  8 63  6 61  2 59  6 50  4 48  3 48  2 46  3 45  3 45  4 38  2 35  4 33  5 19  4

a) NGF ¼ Nerve growth factor. b) CJP1 did not contain sialic acid. c) b-Gal-(1 ! 4)-b-Glc-(1 ! 10 )-ceramide from the starfish Luidia maculata (Inagaki et al., 2003).

neuritogenic activity towards the human neuroblastoma cell line SH-SY5Y, in the absence of NGF. 5.3.3 Conclusion

To conclude, gangliosides obtained from marine invertebrates have invariably been derived from echinoderms, and have almost always a weak biological activity, with the exception of LG-1. Moreover, modification of the sugar units appears to enhance biological activity towards the rat

pheochromocytoma cell line PC12. Investigations are still required, however, to understand the relationship between structure and activity, using either synthesized compounds or analogs, or both.

5.4 Atypical Glycolipids

The glycoglycerolipids (GGLs) are ubiquitous metabolites in algae, having been mainly isolated from sponges, though some

146

j

5 Glycolipids from Marine Invertebrates

reports exist describing their isolation from marine invertebrates. Some of these compounds are linked to the glycerol molecule via an ether bond, while others have a five-membered cyclitol instead of a sugar moiety. Many of them have proved to be glycosides of a very long-chain alcohol derived from FAs. In containing a b-glycoside moiety, the two-headed GLs represent a group of unprecedented bis-a,v-amino alcohol derivatives, known as “hydrid sphingolipids.” 5.4.1 Occurrence and Structure

The first known examples of GGLs were a galactolipid and a GL with a sulfonic acid function (Table 5.14), isolated from the sponge Phyllospongia foliascens. The R1 and R2 groups are acyl groups with 14 and 16 carbon atoms, respectively (Kikuchi et al., 1982). A galactolipid of similar structure was isolated from the Okinawan sponge Pseudoceratina sp. (Meguro, Namikoshi, and Kobayashi, 2002), while three sulfonoquinovosyl-glycerides were isolated from the sea urchins, Strongylocentrotus intermedius (Sahara et al., 1997) and Anthocidaris crassispina (Kitagawa, Hamamoto, and Kobayashi, 1979). The fatty acyl chains consist mainly of myristic and palmitic acids, as well as some saturated and monounsaturated FAs (18:0; 20:0; 14:1; 16:1; 18:1; and 20:1). Unusual ether GLs of glycerol have been found in the class of Demospongia (Table 5.15). One notable feature is the presence of an O-alkyl rather than an O-acyl chain at position 3 of the glycerol unit. The sponge

Trikentrion laeve, harvested in Senegal waters, was the source of trikentroside, a 1-O-alkyl-sn-glyceryl-glycoside, the glycosylated part of which contained only b-xylose, and the fatty part an unsaturated linear chain of 24 carbon atoms (Costantino et al., 1993a). Other diglycosylglycerols, notably myrmekiosides A and B, were obtained from a Japanese species of the genus Myrmekioderma (Aoki et al., 1999); these showed the same glycosylated part, with two glucoses on carbon 2 of glycerol and a xylose on carbon 3, and differed only in the fatty acyl chain linked to carbon 1. In addition, new myrmekiosides C, D and E have been isolated from Myrmekioderma dendyi, harvested in Vanuatu (Farokhi et al., 2012; Genin et al., 2004); these contained a glycerol backbone linked to xylose and N-acetylglucosamine, and an alkyl long-chain with a terminal alcohol group. A series of similar etheroside compounds, with one sugar, was isolated from soft corals as early as 1988. Thus, two species of the genus Sinularia have yielded an a-fucoside (Anjaneyulu, Subba Rao, and Radhika, 2000), while an undetermined species of the genus Cladiella, harvested in the Andaman and Nicobar Islands, yielded a b-arabinoside (Anjaneyulu, Subba Rao, and Radhika, 2001). Both compounds contained a glycerol ether and a fatty chain with 18 carbon atoms. One compound isolated from Sinularia in 2000, proved to be a stereoisomer of lochmodoside, isolated from Sinularia lochmodes (Long, Lin, and Lian, 1988), where the sugar is b-connected to allopyranose, and sarsoliside from Sarcophyton solidum (Zhang and Long, 1995). Another GL in which b-arabinose is linked on carbon 2 (2R) to glycerol

Table 5.14 Glycoacylglycerides.

Name

Species

S

R2

R1

Biological activity

Reference

M-5

Phyllospongia foliascens Sponge

b-Galp

Cis 8hexadecenoic acid 14:0; 16:0

Cis 8hexadecenoic acid 14:0; 16:0

Anti-inflammatory

Kikuchi et al., 1982

14:0, 15:0, 16:0, 16:1, 18:1

14:0, 15:0, 16:0, 16:1, 18:1

Stimulators of tubulin polymerization

Meguro, Namikoshi, and Kobayashi, 2002

Cis 8hexadecenoic acid 16:0

Cis 8hexadecenoic acid 16:0

Anti-complement

Kikuchi et al., 1982

Kitagawa, Hamamoto, and Kobayashi, 1979

Pseudoceratina sp. Sponge M-6

Phyllospongia foliascens Sponge

Anti-1

Anthocidaris crassispina

14:0, 16:0

H

n. r.

4

Sea urchin

14:0, 16:0

14:0, 16:0

n. r.

A-4

Strongylocentrotus intermedius

14:0, 16:0

14:0, 16:0

No activity against human lung cancer cells

A-5

Sea urchin

16:0

H

Cytotoxicity against human lung cancer cells (A549)

a-sulfonoquinovosyl-p

Myristoyl (14:0), Pentadecanoyl (15:0), Palmitoyl (16:0), Palmitoleoyl (16:1), Oleoyl (18:1).

Sahara et al., 1997

5.4 Atypical Glycolipids

ethers was isolated from the Formosan species Lobophytum crassum (Chao et al., 2007). The verongid sponge Pseudoceratina crassa, harvested in the Caribbean, contains the crasserides with a cyclitol (cyclopentanepentol) (Costantino, Fattorusso, and Mangoni, 1993b; Costantino, Fattorusso, and Mangoni, 1994b) (Table 5.16). These are the first – and, until now, the only – natural compounds containing a five-membered cyclitol. In

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contrast, the six-membered cyclitol, inositol, is very common and also found in some phospholipids. In 2002, Costantino and coworkers showed that several species of demosponges contained crasserides (in the majority) and isocrasserides, in which one of the glycerol ester functions is attributed to a cyclitol (Costantino et al., 2002). Crasserides have been isolated from the sponges Verongula gigantea, Aplysina fistularis fulva, Aplysina cauliformis, Neofibularia nolitangere,

Table 5.15 Mono-O-alkyl-glycosylglycerols.

Species and Name

S1 or R1

S2 or R2

Sponges

b-Xylp

b-Xylp

S3

R0

Biological activity

Reference

17-24:1

Cytotoxicity against human lung cancer cells (NSCLC-N6)

Costantino et al., 1993a; Farokhi et al., 2012

Reversing tumor cell morphology of rastransformed cells

Aoki et al., 1999

Cytotoxicity on THP1 cells

Genin et al., 2004

Cytotoxicity against human lung cancer cells (NSCLC-N6 and A549) for acetylated Myrmekioside E

Farokhi et al., 2012

Trikentrion laeve Trikentroside Myrmekioderma sp.

b-Glcp

(2 ! 1)b-Glcp

Myrmekioside A Myrmekioside B

16:0 10 Me 16:0 b-2-N-Ac– glucosamine

Myrmekioderma dendyi Myrmekioside C Myrmekioside D

17OH 17:0 br 17OH 17:0

Myrmekioderma dendyi Myrmekioside E

9 Me 18OH 18:0

Soft corals

b-Arap

16:0; 18:0

Cytotoxicity on Hep G2, Hep 3B, MDAMB-231, Ca9-22 cells

Chao et al., 2007

H

18:0

n. r.

Anjaneyulu, Subba Rao, and Radhika, 2001

Lobophytum crassum

OH or OAc

Cladiella sp.

b-Arap

Sinularia gravis

a-Fucoside

Anjaneyulu, Subba Rao, and Radhika, 2000; Dmitrenok et al., 2003

Sinularia lochmodes Lochmodoside

b-Allop

Long, Lin, and Lian, 1988

Sarcophyton solidum Sarsoliside

pentofuranoside

Zhang and Long, 1995

Sinularia sp. Sinularia grandilobata

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Table 5.16 Cyclitol glycolipid.

Fatty acid

R1

Species of sponges/Reference

Biological activity for Keruffarides and Crasserides

Luffariella sp.

Plakortis simplex

Agelas clathrodes

Siphonodictyon coralliphagum

Pseudoceratina crassa

Pseudoceratina sp.

Ishibashi, Zeng, and Kobayashi, 1993

Costantino et al., 2002

Costantino et al., 2002, 2006

Costantino et al., 2002

Costantino, Fattorusso, and Mangoni, 1993b, Costantino, Fattorusso, and Mangoni, 1994b

Meguro, Namikoshi, and Kobayashi, 2002

keruffarides

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Crasserides

Stimulators of nerve growth factors synthesis Anti-feedant on fish

9 Me-14:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Crasserides

Anti-feedant on fish

10 Me-14:0

Isocrasserides

Iso/ Crasserides

Iso/Crasserides

12 Me-14:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Crasserides

Anti-feedant on fish

13 Me-14:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Crasserides

Anti-feedant on fish

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

9 Me-15:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

n.r

10 Me-15:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Anti-feedant on fish

14 Me-15:0

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

Iso/ Crasserides

Iso/ Crasserides

Iso/Crasserides

14:0

15:0

16:0

keruffarides

keruffarides

10 Me-16:0 17:0

keruffarides

n.r

keruffarides

Crasserides

Stimulators of nerve growth factors synthesis Antimitotic

Anti-feedant on fish keruffarides

Stimulators of nerve growth factors synthesis Antimitotic

Crasserides Stimulators of NGF synthesis

5.4 Atypical Glycolipids

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Table 5.17 Simple ether glycolipid.

Species of sponges

S1 to S8

R1 and R2

Biological Activity

Reference

Plakortis simplex simplexides

S1¼ b-Galp S2 ¼ (4 ! 1)a-Glcp

n-C16H33; iso-C16H33; n-C17H35; anteiso-C17H35; n-C18H37

Immunosuppressive

Costantino et al., 1999

Plakortis simplex plaxylosides

S1 ¼ b-Xylp S2 ¼ (4 ! 1)b-Xylp

Plakortis simplex plaxylosides

S1 ¼ b-Xylp S2 to S6 ¼ (4 ! 1)b-Xylp

Name

Biemna sp., and Xestospongia sp. (Costantino, Fattorusso, and Mangoni, 1994b; Ishibashi, Zeng, and Kobayashi, 1993). An analog, keruffaride, has been isolated from Japanese Luffariella species (Ishibashi, Zeng, and Kobayashi, 1993). Many compounds are composed of a glycosyl moiety (one or several saccharide units) linked to the hydroxyl group of fatty alcohol (Tables 5.17–5.20). The most recent studies on these compounds have been performed since 2006. The GLs were isolated from the Caribbean sponge Agelas clathrodes, and the derivatives characterized by the presence of a,b-ethylenic alcohols with a highly branched long-chain (C32 and C33) bound to a b-glucopyranose. Clathrosides and isoclathrosides were seen to differ only in the stereochemistry of the double bond of the aglycone moiety (Costantino et al., 2006). Plakopolyprenoside and plaxyloside are b-xylosides with a polyprenic chain of seven isoprene units. The simplexides form a series of glycosides involving a diholoside a-glucoseb-galactose in a series of long-chain alcohols. The latter derivatives were isolated from Plakortis simplex, harvested in the Caribbean (Costantino et al., 1999, 2000, 2001b). Caminosides A–D were isolated from Caminus sphaeroconia, harvested in the waters of the Dominican Republic (Table 5.18). These are original glycosides formed from the same aglycone and a variously substituted tetrasaccharide in which a rare sugar, 6-deoxytalose, exists as well as a quinovose (Linington et al., 2002, 2006). The species Pachymastisma johnstonia, harvested from the Isle of Man (Irish Sea) belongs to the same family Geodiidae as Caminus spaeroconia, and was

Costantino et al., 2000

n. r.

Costantino et al., 2001b

the source of a new GL which was formed by the same aglycone and comprised two galactoses and six variously acetylated glucoses. The aglycone is the amide formed from 14-keto-23-hydroxy-octacosanoic acid and N-methylglycine (Warabi et al., 2004). The group of glycosides isolated from Erylus placenta and E. lendenfeldi are characterized by a tri- or tetrasaccharide linked to a ketodihydroxy FA and a diamine (Table 5.19). The sugars were identified as b-galactopyranose, b-glucopyranose, a-arabinopyranose, and b-xylopyranose (Fusetani et al., 1993; Goobes et al., 1996; Sata et al., 1994). A series of glycosides in which the aglycone is a substituted tetramic acid has been isolated from sponges of the genera Ancorina and Penares (Table 5.20). The first of these, ancorinoside A, was isolated from an undetermined species of the genus Ancorina. With regards to the GL from Ancorina species (Ohta, Ohta, and Ikegami, 1997), the glycoside part is a diholoside formed from galacturonic acid and glucose. Ancorinosides B–D are ancorinoside A homologs with variation in the sugars and fatty acyl chain; these three derivatives were isolated from the marine sponge Penares sollasi, harvested in Japan (Fujita et al., 2001). The two-headed sphingolipids linked to b-galactose found in sponges formed a new series that had rhizochalin as the representative component (Table 5.21). Calyoxide differed by the fact that it is a sphingolipid without any FA linkage to the 2amino group, and is derived from the sponge Calyx sp., a member of the same sponge family from which the rhizochalin compounds were isolated (Zhou et al., 2001). The first member

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Table 5.18 Simple ether glycolipid.

Species of sponges

S1 to S8

R1 and R2

Biological activity

Reference

Antibacterial

Linington et al., 2002

Name Caminus sphaeroconia caminoside A

S1 ¼ b-Glcp S2 ¼ (2 ! 1)-3-OBu-4OAc-b-Glcp S3 ¼ (2 ! 1)b-quinovose S8 ¼ (5 ! 1)-6deoxytalose

Caminus sphaeroconia caminoside B

S1 ¼ b-Glcp S2 ¼ (2 ! 1)-3-OBu-4OBu-b-Glcp S3 ¼ (2 ! 1)b-quinovose S8 ¼ (5 ! 1)-6deoxytalose

Caminus sphaeroconia caminoside C

S1 ¼ b-Glcp S2 ¼ (2 ! 1)-3-OBu-4OAc-b-Glcp S3 ¼ (2 ! 1)3-OBub-quinovose S8 ¼ (5 ! 1)-6deoxytalose

Caminus sphaeroconia caminoside D

S1 ¼ b-Glcp S2 ¼ (2 ! 1)-3-OBu-4OBu-b-Glcp S3 ¼ (2 ! 1)3-OBub-quinovose S8 ¼ (5 ! 1)-6deoxytalose

Pachymatisma johnstonia pachymoside

S1 ¼ b-Glcp S2 ¼ (4 ! 1)-b-Glcp S3 to S4 ¼ (3 ! 1)b-Glcp S7 and S8 ¼ (2 ! 1)b-Galp

of the rhizochalin series was described in 1989 in the marine sponge Rhizochalina incrusta, and in 2008 from the species Oceanapia ramsayi (Bensemhoun et al., 2008; Molinski, Makarieva, and Stonik, 2000). Isorhizochaline is an epimer of rhizochalin, with an erythro configuration at the glycosylated 2-amino3-alkanol a-terminus. Another parent molecule, oceanapiside, was later described in the sponge Oceanapia phillipensis, and

Linington et al., 2006

Warabi et al., 2004

contains a b-glucose group instead of a b-galactose group, as in rhizochalin (Nicholas et al., 1999). Some analogs were described as the 2-ethylcarbamate of rhizochaline (rhizochalin A) or containing an oxymethyl group (rhizochalin D) (Makarieva et al., 2005, 2009). Discoside is obtained from the sponge Discodermia dissoluta, harvested in the Bahamas (Figure 5.11), and is composed of a

152

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Table 5.20 Simple ether glycolipid.

Sponge species

Name

S1 to S8

Ancorina sp.

ancorinoside A

Penares sollasi

ancorinoside D

R1 and R2

Biological activity

Reference

S1 ¼ bGlcUp S2 ¼ (4 ! 1)b-Galp

Inhibits blastulation of starfish embryos

Ohta, Ohta, and Ikegami, 1997

S1 ¼ bGalUp S2 ¼ (4 ! 1)b-Glcp

Inhibitors of matrix metalloproteinases

Fujita et al., 2001

ancorinoside C ancorinoside B

S1 ¼ bGlcUp S2 ¼ (4 ! 1)b-Galp

4,6-O-diacyl mannose attached to the 2-hydroxyl group of a myoinositol unit. It is an analog of phosphatidylinositol a-mannoside (Barbieri et al., 2005). 5.4.2 Biological Activity

Erylusamine and its derivatives are cytotoxic antagonists of the interleukin-6 receptor, and could be used as anti-inflammatory agents. The galactolipid M-5 also showed anti-inflammatory activity, while a similar galactolipid to M-5 showed stimulatory activities on microtubule polymerization, much

Figure 5.11 Discosides (Barbieri et al., 2005).

like taxol. The sulfonoglycodiacylglyceride, M-6, showed resistant activity against the complement-fixation reaction. When the antitumor effects of A-4 and A-5 were evaluated in vivo in nude mice bearing solid tumors of a human lung adenocarcinoma cell line A-549, A-5 suppressed tumor growth significantly but A-4 had no effect. All mono-O-alkyl-glycosylglycerols demonstrate cytotoxicity on cancer cells. In addition, trikentroside and the acetylated myrmekioside E were shown to inhibit the proliferation of the human non-small cell lung cancer cell line NSCLC-N6, whereas myrmekiosides C and D acted against THP1 cells (Farokhi et al., 2012; Genin et al., 2004). The myrmekiosides A and B each

References

altered the tumor cell morphology of H-ras-transformed NIH3T3 fibroblasts to that of parental NIH3T3 cells (Aoki et al., 1999). A compound isolated from Lobophytum crassum inhibited the proliferation of Hep-G2, Hep-3B, MDA-MB-231, and Ca9-22 cells (Chao et al., 2007). Ancorinoside A has been shown to arrest the embryonic development of starfish at the blastulation stage, while ancorinosides B–D are inhibitors of matrix metalloproteinase (MMP), the enzyme implicated especially in tumor progression. The crasserides and isocrasserides are thought to play the role of natural feeding deterrents (Costantino, Fattorusso, and Mangoni, 1993b), as these unique compounds were found to exhibit a three- to fourfold stimulation in NGF synthesis in cultured astroglial cells. It should be noted that enhancers of NGF synthesis are considered as potential drugs for peripheral or central nerve disorders. The simplexides have been shown to strongly inhibit the proliferation of activated T cells by a noncytotoxic mechanism. Although the clathrosides and isoclathrosides are similar to simplexides, they have no significant immunomodulatory activity (Costantino et al., 2006). Caminosides demonstrate antibacterial activity against the pathogenic forms of Escherichia coli, which possess a specific type III secretion system by which toxic proteins are excreted directly into the host cell cytosol; caminosides inhibit this excretion. The rhizochalin family compound has antimicrobial activity (antibacterial against Staphylococcus aureus, antifungal against the pathogenic fluconazole-resistant yeast Candida glabrata) as well as cytotoxic properties (leukemia HL-60 cell and Ehrlich carcinoma cell) (Kalinin et al., 2012). Rhizochalin and some analogs (rhizochalins A and C) from the same animal were also shown to be anticarcinogenic and proapoptotic agents (Fedorov et al., 2009). 5.4.3 Conclusion

These atypical GLs were isolated mainly from demospongia, and from some cnidarians and echinoderms. Two-thirds of atypical GLs possess a monosaccharide, with rare sugars such as quinovose, 6-deoxytalose, as well as many more common saccharides (glucose, galactose, xylose and arabinose) being found. Atypical glycosides obtained from sponges demonstrate mainly antibacterial and antitumor activities.

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5.5 General Conclusion

During the past 25 years, the GSLs of marine invertebrates have undergone increasing chemical investigations, such that their structures have been revealed as unique and often highly complex, notably following the recognition of immunomodulatory and antitumor properties in the case of a-galactosylceramides. Glycolipids are known to be widely distributed in marine invertebrates, and unique and fascinating structures of these compounds have been identified. Consequently, echinoderms and sponges, as well as other classes of marine invertebrates, should be investigated for the presence of glycolipids. In addition, it is vital that a number of identified compounds of interest are investigated for their biological effects; indeed, further investigations in this area may lead to new active compounds that might be used to treat health disorders in humans.

List of Abbreviations

Ara FA(s) Fuc f Gal GalNAc GalNFor GGL(s) Glc GL(s) GSL(s) iNOS LCB LPS p Me NeuAc NeuGc NGF Rha VEGF

arabinose fatty acid(s) fucose furanose galactose N-acetylgalactosamine N-formylgalactosamine glycoglycerolipid(s) glucose glycolipid(s) glycosphingolipid(s) inducible nitric oxide synthase long-chain base lipopolysaccharide pyranose methyl N-acetylneuraminic acid N-glycolylneuraminic acid nerve growth factor rhamnose vascular endothelial growth factor

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activities of mono- or diglycosylated a-galactosylceramides. Bioorg. Med. Chem., 5, 1447–1452. Uchimura, A., Shimizu, T., Morita, M., Ueno, H., Motoki, K., Fukushima, H., Natori, T., and Koezuka, Y. (1997b) Immunostimulatory activities of monoglycosylated a-dpyranosylceramides. Bioorg. Med. Chem., 5, 2245–2249. Vaskovsky, V.E., Kostetsky, E.Y., Svetaschev, V.I., Zhukova, I.G., and Smirnova, G.P. (1970) Glycolipids of marine invertebrates. Comp. Biochem. Physiol., 34, 163–177. Venkannababu, U., Bhandari, S.P.S., and Garg, H.S. (1997) Regulosides A–C: glycosphingolipids from the starfish Pentaceraster regulus. Liebigs Ann., 1997, 1245–1247. Warabi, K., Zimmerman, W.T., Shen, J., Gauthier, A., Robertson, M., Finlay, B.B., Van Soest, R., and Andersen, R.J. (2004) Pachymoside A – A novel glycolipid isolated from the marine sponge Pachymatisma johnstonia. Can. J. Chem., 82, 102–112. Wu, D., Fujio, M., and Wong, C.H. (2008) Glycolipids as immunostimulating agents. Bioorg. Med. Chem., 16, 1073–1083. Xu, J., Wang, Y.-M., Feng, T.-Y., Zhang, B., Sugawara, T., and Xue, C.-H. (2011) Isolation and anti-fatty liver activity of a novel cerebroside from the sea cucumber Acaudina molpadioides. Biosci. Biotechnol. Biochem., 75, 1466–1471. Yamada, K., Hara, E., Miyamoto, T., Higuchi, R., Isobe, R., and Honda, S. (1998a) Constituents of Holothuroidea. 6. Isolation and structure of biologically active glycosphingolipids from the sea cucumber Cucumaria echinata. Eur. J. Org. Chem., 1998, 371–378. Yamada, K., Harada, Y., Nagaregawa, Y., Miyamoto, T., Isobe, R., and Higuchi, R. (1998b) Constituents of Holothuroidea. 7. Isolation and structure of biologically active gangliosides from the sea cucumber Holothuria pervicax. Eur. J. Org. Chem., 1998, 2519–2525. Yamada, K., Harada, Y., Miyamoto, T., Isobe, R., and Higuchi, R. (2000) Constituents of Holothuroidea. 9. Isolation and structure of a new ganglioside molecular species from the sea cucumber Holothuria pervicax. Chem. Pharm. Bull. (Tokyo), 48, 157–159. Yamada, K., Matsubara, R., Kaneko, M., Miyamoto, T., and Higuchi, R. (2001) Constituents of Holothuroidea. 10. Isolation and structure of a biologically active

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ganglioside molecular species from the sea cucumber Holothuria leucospilota. Chem. Pharm. Bull. (Tokyo), 49, 447–452. Yamada, K., Sasaki, K., Harada, Y., Isobe, R., and Higuchi, R. (2002) Constituents of Holothuroidea. 12. Isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax. Chem. Pharm. Bull. (Tokyo), 50, 1467–1470. Yamada, K., Hamada, A., Kisa, F., Miyamoto, T., and Higuchi, R. (2003) Constituents of Holothuroidea. 13. Structure of neuritogenic active ganglioside molecular species from the sea cucumber Stichopus chloronotus. Chem. Pharm. Bull. (Tokyo), 51, 46–52. Yamada, K., Wada, N., Onaka, H., Matsubara, R., Isobe, R., Inagaki, M., and Higuchi, R. (2005a) Constituents of Holothuroidea. 15. Isolation of Ante-iso type regioisomer on long chain base moiety of glucocerebroside from the sea cucumber Holothuria leucospilota. Chem. Pharm. Bull. (Tokyo), 53, 788–791. Yamada, K., Onaka, H., Tanaka, M., Inagaki, M., and Higuchi, R. (2005b) Constituents of Holothuroidea. 16. Determination of absolute configuration of the branched methyl group in Ante-iso type side chain moiety on long chain base of glucocerebroside from the sea cucumber Holothuria leucospilota. Chem. Pharm. Bull. (Tokyo), 53, 1333–1334. Yamada, K., Tanabe, K., Miyamoto, T., Kusumoto, T., Inagaki, M., and Higuchi, R. (2008) Isolation and structure of a monomethylated ganglioside possessing neuritogenic activity from the ovary of the sea urchin Diadema setosum. Chem. Pharm. Bull. (Tokyo), 56, 734–737. Yamamoto, T., Teshima, T., Saitoh, U., Hoshi, M., and Shiba, T. (1994) Synthesis of ganglioside M5 from sea urchin egg. Tetrahedron Lett., 35, 2701–2704. Zhang, M. and Long, K. (1995) A new batyl alcohol glycoside from Sarcophyton solidum of the South China Sea. Tianran Chanwu Yanjiu Yu Kaifa, 7, 12–15. Zhang, G.-W., Ma, X.-Q., Zhang, C.-X., Su, J.-Y., Ye, W.-C., Zhang, X.-Q., Yao, X.-S., and Zeng, L.-M. (2005) Two new ceramides from the marine sponge Ircinia fasciculata. Helv. Chim. Acta, 88, 885–890. Zhou, B.-N., Mattern, M.P., Johnson, R.K., and Kingston, D.G.I. (2001) Structure and stereochemistry of a novel bioactive sphingolipid from a Calix sp. Tetrahedron, 57, 9549–9554.

About the Authors Gilles Barnathan is now emeritus professor at the University of Nantes, France. He received his first PhD from the University of Paris VI in organic synthesis of new potentially antitumor Cnucleosides in the Laboratory of Therapeutic Chemistry at the Pasteur Institute, Paris. He then worked as associate professor

at several overseas foreign universities and joined the University of Nantes, College of Pharmacy, where he received a second PhD for a work on lipids of marine sponges. As the head of a team on marine lipids with biological activity, one part of the research group “Sea, Molecules and Health,” he pursued

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his activities in marine lipidology with a main topic on antitumor and antiparasitic glycolipids until he retired in September 2013. He has authored about 50 journal articles and two book chapters, and recently has been guest editor of special issues on marine lipids in Marine Drugs. He has been a member of the scientific committee of the French Society for Studies and Research in Lipidomics (2004–2012). Aur
elie Couzinet-Mossion is a chemical engineer and an assistant professor in the department of marine chemistry at the research team MMS (Mer Mol
ecules Sant
e) in Nantes, France. Having obtained her academic degrees from Toulouse University in analytical chemistry, she began her career

working on tea infusion before changing to marine lipids research. She is in charge of lipid analyses, using liquid chromatography. Ga€etane Wielgosz-Collin is a pharmacist and assistant professor in the department of marine chemistry at the research team MMS (Mer Mol
ecules Sant
e) in Nantes, France. Having obtained her academic degrees from Nantes University in pharmaco-chemistry, she spent most of her career working in marine lipids research. Dr Ga€etane Wielgosz-Collin has authored scientific publications, especially on biologically active marine molecules. She is also a member of the Conseil National des Universit
es of France.

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6 Pigments of Living Fossil Crinoids C
ecile Debitus and Jean-Michel Kornprobst

Abstract

Stalked crinoids were, for a long time, known as fossils found in different quarries, and also from ocean depths since the great expeditions of the eighteenth century. The chemical analyses of fossils and also of living organisms led to the discovery of new

bioactive pigments, and also confirmed that the core structures of these pigments had been preserved during the fossilization process. The existence of these quinoid pigments also leads to several interesting questions related to their stereochemistry and the photoactivation of their bioactivity.

With kind permission by the Office des Postes et T
el
ecommunications de la Nouvelle Cal
edonie.

6.1 The Discovery of Stalked Crinoids

The existence of stalked crinoids was first proposed by A.H. Clark during the early twentieth century, while taking part in a scientific expedition aboard Albatross off the coast of the Philippines. Historically, the first stalked crinoid – the pentacrine Cenocrinus asterius – was described by Guettard in 1761, while details of the first description of stalked crinoid biodiversity in the deep ocean were published by Carpenter in 1884, after the famous expedition of the HMS Challenger, between 1873 and 1876 (Ameziane and Roux, 1997). These species have long been considered as “living fossils,” because only fossilized species were previously known from the Jurassic and Cretaceous eras (Clark, 1910, 1912, 1923; Oji, 1985). Subsequently, Proisocrinus ruberrimus and Naumachocrinus hawaiiensis were rediscovered off Okinawa at a depth of 1800 m (Oji and Kitazawa, 2008). In addition, during the early 1980s two new species – Diplocrinus alternicirrus and, again, Proisocrinus ruberrimus – were sampled in the central Pacific off the coast of Tahiti by dredging at depths of between 980 and 1135 m (Roux, 1980).

During the Chalcal 2 expedition in 1986, the new species Gymnocrinus richeri1) was discovered at 520 m (Bourseau, AmezianeCominardi, and Roux, 1987), and the exceptional characteristics and abundance of this species in this area led the Institut de Recherche pour le D
eveloppement (IRD; formerly ORSTOM) in New Caledonia to issue a commemorative stamp. Today, approximately 100 stalked crinoids species have been sampled at between 60 m and abyssal depths, and have been described in the literature (Am
eziane Cominardi, 1991; Roux, Messing, and Am
eziane, 2002). Some examples of these “fossil” crinoids are shown in Figure 6.1.

6.2 Anthraquinonic Pigments of Stalked Crinoids

Until now, three types of anthraquinonic pigment have been characterized: (i) fringelites2) and hypericin have been identified 1) The species name was dedicated to the project leader Dr Bertrand

Richer de Forges. 2) The name « fringelite » comes from the Swiss village of Fringeli, where

these fossils were discovered.

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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6 Pigments of Living Fossil Crinoids

Stalked fossilized crinoid Angulocrinus polydactylus

Proisocrinus ruberrimus (« Moulin Rouge »)

(Courtesy of Michel Roux)

(Courtesy of Michel Roux)

Gymnocrinus richeri (Photos Delphine Brabant)

Gymnocrinus richeri

Hyocrinus biscoitoi

Endoxocrinus parrae

(Courtesy of Michel Roux)

(Courtesy of Michel Roux)

(Courtesy of Michel Roux)

Figure 6.1 Examples of present and fossilized stalked crinoids.

6.3 Axial Chirality of Gymnochromes and Hypochromines

OH

O

OH

OH O

HO HO

OH

OH O

HO HO

OH

O

OH

Demethylhypericin C29H14O8 OH O

HO HO

OH O

OH OH

OH

O

O

OH

OH

O

Fringelite E C28H12O9 OH

OH

O

O

OH

O

OH

OH O

OH

OH

OH

O

Fringelite F C28H12O8 OH

R1 R2

OH

O

HO HO

OH

HO HO

OH

OH

OH

OH

OH

OH

Pseudohypericin C30H16O9

HO HO

Fringelite D C28H12O10 OH

O

Hypericin C30H16O8

OH

OH

HO HO

OH

j 165

Homologous hypericinoids R1, R2 = alkyl M ranging from C31H18O8 to C36H28O8

OH

Fringelite H C28H12O6 Figure 6.2 Examples of fringelites and related compounds.

from Liliocrinus munsterianus, an upper Jurassic crinoid, and also from the middle Triassic Carnallicrinus carnalli; (ii) proisocrinins A–F were isolated from Proisocrinus ruberrimus; and (iii) gymnochromes A–F from Gymnocrinus richeri and Holopus rangii. Fringelites, hypericin and gymnochromes, which possess the same hydrocarbon skeleton of the phenanthroperylene quinone type, were discovered during the early 1960s, but their structure was not determined definitively until the early twenty-first century (Blumer, 1960, 1962a, 1962b; O’Malley, Ausich, and Chin, 2008; Wolkenstein, Gross, and Sch€ oler, 2006, Wolkenstein et al., 2008). All of these structures can be considered as dimers derived from a radical coupling of the proisocrinins identified in 2009 in Proisocrinus ruberrimus. The proisocrinins and gymnochromes display the same special brominated and sulfated patterns. Similar structures, but without bromine atoms, have long been known in presentday crinoids (Kornprobst, 2014), and some of these structures are presented in Figures 6.2–6.4. Before it isolation from fossilized crinoids, hypericin was identified in 1942 when extracted from the herb plant

Hypericum perforatum L. (known as St John’s wort), the extract having been used since ancient times to treat depression.3) This antidepressant activity of hypericin was considered due to its ability to inhibit the reuptake of neurotransmitters such as dopamine and serotonin (Falk, 1999).

6.3 Axial Chirality of Gymnochromes and Hypochromines

The proximity of the substituents at positions 3 and 4, and also at positions 10 and 11, results in a twisting of the molecule; this is clearly visible on the circular dichroism spectra of gymnochromes and isogymnochromes, which present opposite helical configurations (axial chirality), with a left-handed configuration (M) for gymnochromes and a right-handed configuration (P) for isogymnochromes (De Riccardis et al., 1991). The equilibrium between both forms is very slow, even at 160  C, and also leads to 3) Dioscoride was advocated for its antidepressant action about 2000 years

ago.

166

6 Pigments of Living Fossil Crinoids

j OH

O

OH

OH O Br

R1 HO 11 HO 10

OH

3

(S)

4

(R)

R2

Br OH

O

OH

OH

HO HO

OSO3

Na

(R)

Br O

OSO3 Na

OH

O

OH Br

Br

Br OH

OSO3

Na

OH OH

HO HO

O

OSO3 Na

OH

Isogymnochrome D Gymnocrinus richeri De Riccardis et al., 1991

OH Br

Na

OH

Br

Gymnochrome D Gymnocrinus richeri De Riccardis et al., 1991 OH O

O

OSO3

HO HO

(R)

OH

Br

Gymnochrome C Gymnocrinus richeri De Riccardis et al., 1991

OH

Br

(S)

OH

Br

OH (S)

Br

Gymnochrome A (R1 = R2 = Br) Gymnochrome B (R1 = H, R2 = Br or R1 = Br, R2 = H) Gymnocrinus richeri De Riccardis et al., 1991 OH O

Br

HO HO

OH

Br

OH

Br

O

OH Br

Br

OH

HO HO Br OH

O

OH

OH

OH

Gymnochrome E Holopus rangii Wangun et al., 2010 OH O

Br

Br O

OH

OH

Gymnochrome F Holopus rangii Wangun et al., 2010

OH Br

R1

OH

HO HO R2

Br OH

O

OH

OH

Isogymnochrome B (R1 = H, R2 = Br or R1 = Br, R2 = H) Holopus rangii Wangun et al., 2010 Figure 6.3 Structures of gymnochromes.

a partial degradation in the case of gymnochrome B and isogymnochrome B, as well as a conformational change of the side chains. The two compounds are both configurational helices, one left-handed and the other right-handed (Figure 6.5).

A helical configuration of the P form has been discovered more recently for hypochromines A and B, when isolated from the marine fungi Hypocrea vinosa from a sand sample taken at Okinawa, Japan (Ohkawa et al., 2010). This helical arrangement

6.4 Towards a Fungal Origin of Gymnochromes?

OMe O

OH

Br

OMe O R2

OSO3 Na

HO

j 167

OH

Br

R2

OSO3 Na

HO Br

O

R1

Proisocrinin A (R1 = R2 = Br) Proisocrinin B (R1 = Br, R2 = H) Proisocrinin C (R1 = H, R2 = Br) Proisocrinus ruberrimus Wolkenstein et al., 2009 OMe O

O

Br

R1

Proisocrinin D (R1 = R2 = Br) Proisocrinin E (R1 = Br, R2 = H) Proisocrinin F (R1 = H, R2 = Br) Proisocrinus ruberrimus Wolkenstein et al., 2009 OMe O

OH

OH

Na

Na

O3SO

MeO

OH O

OSO3 O

O

Comantherin-O-sulfate Comantheria perplexa Kent et al., 1970

O

Comatula pectinata Ridout and Sutherland, 1981

Figure 6.4 Structures of proisocrinins and similar sulfated anthraquinones from crinoids.

Figure 6.5 Circular dichroism spectra of gymnochrome B (_._), gymnochrome D (___), and isogymnochrome D(__).

could explain the very high optical rotation values of both compounds (þ410 and þ340 for hypochromines A and B, respectively), while the low rigidity of the carbon skeleton may explain why there is such a high helical conformation. OH

OH O

OH

OH O

OH HO HO

O O

HO HO

O O

O OH

OH

O

Hypochromine A

O OH

OH

O

Hypochromine B

6.4 Towards a Fungal Origin of Gymnochromes?

The discovery of halogenated and nonhalogenated anthraquinones has been described in two reports made in 2012, using extracts of marine fungi that had been isolated from marine sediments and subsequently cultured. The species Streptomyces spinoverrucosus has been isolated from a coastal sediment taken at Trinity Bay, Galveston, Texas, to produce the two nonhalogenated compounds, galvaquinones A and B. However, the strain of Aspergillus sp. SCIO F063, isolated from a deep-sea sediment (1451 m) of the South China Sea was found to contain chlorinated anthraquinones. The culture of this latter strain in a medium containing sodium bromide led to an isolation of the

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6 Pigments of Living Fossil Crinoids

j

corresponding brominated compounds (Hu, Martinez, and MacMillan, 2012; Huang et al., 2012). All of these halogenated anthraquinones are derivatives of averantin, a biosynthetic precursor of aflatoxin B1, isolated from a mutant strain of Aspergillus parasiticus (Bennett et al., 1980).

OH O

OH

O

O

Galvaquinone A

OH

O

OH O

OH

O

OH

B, were enhanced by light, with gymnochrome B in particular showing the best activity under these conditions. The lateral chains also appear increase both antiviral and virucidal activities, independent of any photoactivation.

O

Galvaquinone B

OH OH (S)

HO

OH

Averantin

O OH

O

OH O

OH OR2

Cl

OH

Cl

R1O

OH O

R1 = H, R2 = H R1 = H, R2 = Me R1 = H, R2 = n-Bu R1 = Me, R2 = H R1 = Me, R2 = Me

RO R = H, R = Me,

OH O

The structural analogy between chlorinated derivatives of averantin and gymnochromes allows the consideration of a fungal origin for these derivatives, as well as for most of anthraquinone pigments of present crinoids (Kornprobst, 2014).

6.5 Biological Activities of Gymnochromes

Dengue fever is a viral disease transmitted by the mosquito Aedes aegypti in intertropical areas. Both, the hemorrhagic form and an alternative form that provokes a shock syndrome associated with a reduction in blood pressure and subsequent myocardial dysfunction, are responsible for many deaths. The gymnochromes B and D, and also isogymnochrome D, each demonstrated a strong in vitro antiviral activity at concentrations as low as 1 nM, and showed no signs of cytotoxicity towards the host cells. The most active agents were sulfated gymnochrome D and isogymnochrome D. A similar antiviral activity was also demonstrated for hypericin and tetrabromohypericin (Falk and Schmitzberger, 1993; Laille, Gerald, and Debitus, 1998; Laurent et al., 2005). The virucidal and antiviral activities of three compounds, namely hypericin, tetrabromohypericin and gymnochrome

The activity of gymnochrome A was evaluated on myocardial tissue; typically, gymnochrome A acts as an oxidant of the heart membrane, inducing intracellular changes. As for other cellular activities, the photoactivation of gymnochrome A seems also to play a role in enhancing its activity (Sauviat et al., 2001). Gymnochrome B presents a similar activity as hypericin on herpes simplex virus, with an MIC100 of 10 acyl carbons N-(2-phenylethyl)- isobutyramide (1) N-(2-phenylethyl)- isobutyramide (2) Bromoalkaloids (3, 4) Diketopiperazines Cyclo(L-Pro-L-Tyr) (5) Cyclo(L-Phe-L-Pro) (6) Cyclo(L-Pro-L-Val) (7)

Unknown mode of action Manoalide (8), Manoalide monoacetate (9), Secomanoalide (10) Demethoxy encecalin (11) Hymenialdisin (12) Tumonoic acid F (13) Malyngamide C (14), 8-epimalyngamide C (15) Lymbyoic acid (16) Malyngolide (17) Diketopiperazines Proteoanemonin (18)

of AHL signal molecules in vitro (Fuqua and Greenberg, 2002; McClean et al., 1997). There are many reports of QQ compounds produced in a diverse range of marine organisms (see Table 7.1, Figure 7.2). This may be a good reflection of the coevolution of pathogens and hosts, where the hosts evolve a novel chemistry to reduce the pathogenicity of the bacteria. For example, 23% of extracts of 284 marine organisms from the Great Barrier Reef inhibited the LuxR-based reporter strain C. violaceum (Skindersoe et al., 2008). Malyngamide-C and 8epimalyngamide-C, present in extracts of the cyanobacterium Lyngbya majuscula, inhibited the QS of LasR-based reporter strains (Kwan et al., 2010). Extracts of cyanobacterial species from Florida (USA), Belize and Oman were also assessed for their ability to inhibit the QS of C. violaceum CV017 (Dobretsov et al., 2010). The strongest QS inhibition was observed in extracts of Symploca hydnoides and L. majuscula from South Florida, and the inhibitor produced by L. majuscula was identified as malyngolide (Dobretsov et al., 2010). This QQ compound inhibited responses of the LasR reporters and Las

Dong et al., 2002

QS-dependent production of elastase by P. aeruginosa PAO1. It was proposed that malyngolide inhibits bacterial QS by repressing the expression of lasR; this mode of action was also proposed for lyngbyoic acid isolated from L. majuscula (Kwan et al., 2011) and could be one way of inhibiting bacterial QS systems. In a recent screen for bacterial QS inhibitors, Marinobacter sp. SK-3 isolated from a hypersaline mat was shown to produce four related diketopiperazines with different QQ activities (Abed et al., 2013). While cyclo(L-Pro-L-Phe), cyclo (L-Pro-L-Leu), and cyclo(L-Pro-L-iso-Leu) inhibited QS-dependent luminescence of the reporter strain E. coli pSB401, only cyclo(L-Pro-L-Phe) and cyclo(L-Pro-L-iso-Leu) inhibited violacein production by C. violaceum CV017. Cyclo(L-Pro-DPhe), the epimer of cyclo(L-Pro-L-Phe), did not have any QQ properties. It is interesting that some QS inhibitors, such as malyngolide, display antibiotic properties. In this respect, it has been proposed recently that antibiotics can be considered as either

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signaling or signaling-disruptive molecules (Yim, Wang, and Davies, 2007). For example, these compounds are produced at specific phases of growth and cause significant changes in the transcriptional activity of nearby neighbors that is not solely related to their metabolism and detoxification. It has also been recently reported that antibiotics affect community behavior, as manifested by a reduction in biofilm formation, and interference with QS signaling (Nalca et al., 2006).

Furthermore, both antibiotics and signal molecules are toxic at high concentrations (Kaufmann et al., 2005; Martinelli et al., 2004). The same considerations of mobility and signal attenuation described above for QS-signals apply also to QQ compounds, at least in theory. However, this subject has not yet been addressed in the scientific literature, and many questions and speculations regarding an “arms race” of chemical signals and their antagonists

7.6 Examples of Cross-Kingdom Signaling in the Marine Environment

operating in complex natural environments remain to be resolved in the future.

R3

7.6.1 Chemical Defense of the Red Seaweed Delisea pulchra

A cross-kingdom communication strategy is used by the seaweed, Delisea pulchra, to defend itself from fouling and disease (Maximilien et al., 1998). Natural populations of D. pulchra on the east coast of Australia undergo seasonal bleaching (Campbell et al., 2011), characterized by white patches of algal fronds that have lost the typical red pigmentation. This bleaching phenomenon is prevalent in summer and correlates strongly with elevated seawater temperatures. The direct consequences of bleaching include reductions in growth and fecundity of the seaweed. Seaweeds affected by bleaching characteristically have reduced concentrations of halogenated furanones (Campbell et al., 2011). These compounds (Figure 7.3) are structurally similar to AHL QS signals (De Nys et al., 1993; Manefield et al., 1999; Pettus, Wing, and Sims, 1977), and antagonize the bacterial AHL receptor (Manefield et al., 2000). This results in inhibition of bacterial cell-to-cell communication, with important consequences for the algal host. To determine if algal bleaching is a temperature-mediated bacterial infection that can be prevented by QQ, defended (furanone-containing) and undefended (furanone-depleted) sporelings of D. pulchra were exposed to water temperatures representing low and high summer temperatures, and to high and low bacterial abundances (Campbell et al., 2011). This experiment revealed that sporelings maintained in natural seawater containing ambient bacterioplankton bleached more frequently and severely than conspecifics maintained in sterile

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7.6 Examples of Cross-Kingdom Signaling in the Marine Environment

The range of effects of bacterial QS are incredibly broad, including the production of exoenzymes for the scavenging of nutrients, mediating defensive responses, biofilm formation and regulating the phenotypes of symbionts (McDougald, Rice, and Kjelleberg, 2007). The coevolution of bacteria and higher eukaryotes in the marine environment has therefore likely resulted in numerous examples of QS and antagonistic QSregulated processes in marine organisms. Given the widespread role of QS in the formation of bacterial biofilms, and thus possible pathogenicity and disease, several groups have proposed that potential hosts should have evolved compounds that specifically interfere with QS and thereby defend themselves from colonization and infection by bacteria (Alagely et al., 2011; Bauer and Teplitski, 2001; McDougald, Rice, and Kjelleberg, 2007; Steinberg et al., 2011). These interactions, which occur across the boundary of kingdoms, have been coined inter- or cross-kingdom signaling or communication (Hughes and Sperandio, 2008).

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Figure 7.3 Halogenated furanones produced by the red alga Delisea pulchra.

seawater. Moreover, bleaching was more severe in sporelings lacking halogenated furanones than in defended seaweed (Campbell et al., 2011). These results were consistent with the premise that bleaching is the result of a bacterial infection. A large number of epiphytic bacteria from the surface of D. pulchra have been screened in vitro for their capability to induce bleaching similar to that observed in the field. The bacterial strain Nautella sp. R11 (formally Ruegeria sp. R11) is to date the best-studied bacterial pathogen capable of infecting D. pulchra (Case et al., 2011; Fernandes et al., 2011). Nautella sp. R11 colonizes and subsequently forms pronounced biofilms on chemically undefended sporelings of D. pulchra. Subsequent to colonization and at elevated temperatures, Nautella sp. R11 has been seen to penetrate the algal tissue and invade individual algal cells (Case et al., 2011), which coincides with localized bleaching of the thallus. While details of the virulence mechanisms expressed by Nautella sp. R11 are yet to be elucidated, genome analysis of this strain suggests a role for the QSdependent regulation of virulence genes (Fernandes et al., 2011). Such regulation is not uncommon among bacterial pathogens, where QS signals are used to control concerted bacterial traits, such as virulence gene expression (Atkinson and Williams, 2009; von Bodman, Bauer, and Coplin, 2003). In this particular case, a direct effect of elevated temperature on virulence and pathogenicity of Nautella sp. R11 has yet to be determined; however, a positive correlation between the expression of virulence genes and temperature has been observed in a broad range of other bacterial pathogens (Konkel and Tilly, 2000).

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Figure 7.4 A conceptual model of the causes and ecological consequences of bleaching in Delisea pulchra. Environmental stressors cause a decrease in concentrations of halogenated furanones, which facilitates a shift in microbial communities associated with the algal surface; abiotic conditions may also induce or increase bacterial virulence. Pathogens colonize and infect D. pulchra and cause algal bleaching. The bleached algae are smaller and less fecund than healthy conspecifics, and are also more likely to be consumed and used as a host by locally abundant herbivores. With kind permission by the Royal Zoological Society of NSW, Mosman, Australia.

The QS antagonistic mode of action of halogenated furanones of D. pulchra against pathogenic bacteria is a classic example of how eukaryotic organisms can successfully exploit prokaryotic communication strategies (Figure 7.4). The case study of D. pulchra demonstrates, in a predictable pattern, how at the ecological level of entire populations environmental stress interferes with the production of QS antagonists, which in turn allows bacterial pathogens to invade the algal host causing downstream processes that ultimately result in disease (Harder et al., 2012). While bacterial resistance to natural furanones has not been detected in the marine environment, some strains of P. aeruginosa isolated from pediatric patients were shown to be resistant to C-30, a furanone derivative that has been used extensively in laboratory studies on QQ (Garcia-Contreras et al., 2013). Furthermore, when P. aeruginosa was grown in the laboratory under conditions where growth was dependent on QS, as degradation of the sole provided nutrient was under QS regulation, resistance to C-30 occurred and was shown to be due to mutations that increase the efflux of C-30 (Maeda et al., 2012). Thus, resistance to these natural compounds may occur in the environment, although the selection pressure to evolve resistance is much less than that for development of antibiotic resistance, where the selection pressure is high. Examples of eukaryotic hosts that, like D. pulchra, produce metabolites that interfere with bacterial QS are not limited to the

marine environment, and other examples include fungal, plant and herbal sources (Bjarnsholt et al., 2005; Rasmussen et al., 2005). In particular, extracts from garlic have exceptional QQ activity and reduce virulence in a nematode infection model (Rasmussen et al., 2005). The roots of Medicago truncatula have multiple QS-active compounds (Gao et al., 2003). Even fungi, which may be commensals on plants, have been shown to produce QS-inhibiting compounds (Rasmussen et al., 2005). Thus, it is clear that QS antagonists have evolved in a broad range of hosts that interact in Nature with bacteria. 7.6.2 The Mutualistic Association of Vibrio fischeri with the Hawaiian Bobtail Squid

As discussed above, QS systems are often found in bacterial species that associate with other organisms, either as a pathogen or symbiont, and control the expression of genes involved in colonization and virulence in the case of pathogens. V. fischeri is found free-living in seawater and in the light organs of various squid and fish, and is a symbiont of the light organ of the Hawaiian bobtail squid Euprymna scolopes. This symbiosis serves as a model host/microbe system for understanding inter-kingdom interactions (Busetti and Gilmore, 2010; McFall-Ngai et al., 2012; Ruby, 1996; Ruby and Lee, 1998). In V. fischeri, the expression of bioluminescence is a QS-regulated phenotype that is necessary for the symbiosis to occur.

7.6 Examples of Cross-Kingdom Signaling in the Marine Environment

Shortly after hatching, the crypts of the juvenile squid are colonized by V. fischeri from the seawater. After 12 h of colonization, a persistent state is reached with a reduction in symbiont growth rate, loss of flagella, and induction of bioluminescence. The light that is emitted by the symbiont acts an antipredation tactic by providing counterillumination. V. fischeri mutants that do not produce light are eventually lost from the crypts, and it is now known that the host tissues can perceive the light produced by the symbiont (Tong et al., 2009; Visick et al., 2000). Between 90% and 95% of the symbiont population is expelled from the light organ at dawn each day; the remaining cells then proliferate throughout the day and by nightfall have reached a high cell density such that QS-regulated bioluminescence is again induced. Bioluminescence is generated when the luxICDABEG operon is induced. Luciferase is comprised of LuxA and LuxB, which converts reduced flavin mononucleotide (FMNH2), O2 and an aliphatic aldehyde to FMN, water and an aliphatic acid (Hastings and Nealson, 1977). LuxC, LuxD, LuxE and LuxG generate and recycle the substrates. V. fischeri has two ASL QS systems, the LuxIR and the AinSR systems, which operate sequentially to regulate luminescence and colonization of the host (Gilson, Kuo, and Dunlap, 1995; Lupp et al., 2003; Visick et al., 2000). At high cell densities, the autoinducer N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) produced by LuxI reaches a threshold concentration, which results in binding to its cognate receptor LuxR to activate transcription of the lux operon, causing luminescence. Genes involved in late colonization of the inner crypts of the light organ are also induced. The AinSR system induces luminescence at cell densities that precede activation of the LuxIR system (Lupp and Ruby, 2004). AinS produces the autoinducer N-octanoyl-L-homoserine lactone (C8-HSL) that is bound by the transcriptional regulator AinR. The AinSR QS system acts to: (i) derepress the lux operon by inhibiting LuxO; and (ii) induce low levels of lux induction by the binding of C8-HSL to LuxR. This allows a temporal regulation of various early and late colonization genes (Antunes et al., 2007; Lupp and Ruby, 2005). V. fischeri also possesses the AI-2

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QS system, which is coordinately regulated with the AinSR system through LuxO phosphorelay and acts to delay the induction of luminescence at low cell density (Kuo, Callahan, and Dunlap, 1996). There is evidence that luminescence acts to reduce the exposure of the symbiont to host-derived reactive oxygen species found in the light organ, since the reaction consumes oxygen (Bose et al., 2007; Bose, Rosenberg, and Stabb, 2008; Visick et al., 2000). This may explain why dark strains of V. fischeri are eliminated from the light organ, as they are more sensitive to the oxidative environment than wild-type strains. Under reducing conditions, luminescence is repressed by the ArcAB twocomponent regulatory system (Bose et al., 2007), but once 3-oxoC6-HSL has initiated the positive autoinduction feedback ArcA cannot reverse the induction state (Septer and Stabb, 2012). Thus, the redox state of the environment is an important environmental factor regulating lux expression. AinSR QS is necessary for the efficient early colonization of the squid host, E. scolopes, as microarray analysis has demonstrated that the regulation of phenotypes is important for colonization, including chemotaxis and flagellar motility, but through a second receptor protein, LitR (Lupp and Ruby, 2005). AinS regulates genes that are important for colonization, such as the acetate switch through LitR but independently of LuxR (Studer, Mandel, and Ruby, 2008). The ability to uptake and utilize acetate is important for the persistence of V. fischeri in the squid light organ. AinSR also regulates a number of phenotypes independently of LitR (Fidopiastis et al., 2002; Lupp and Ruby, 2005). The relationship between V. fischeri as a bacterial symbiont of the light organ of the Hawaiian bobtail squid is reminiscent of another symbiotic bacterial relationship with a cephalopod (Figure 7.5). Nautiloid cephalopods are marine invertebrates sheltered by an external chambered shell filled with nitrogen gas. These organisms are considered living fossils, being the descendants of cephalopods that roamed the oceans during the Cambrian (over 500 Myr ago). In Nautilus macromphalus, the excretion of waste products is operated by the pericardial

Figure 7.5 Examples of transphyletic cooperation between bacteria and a cephalopod host. (a) The Hawaiian bobtailed squid, Euprymna scolopes, which establishes a symbiosis with Vibrio fischeri within its light organ. Image reproduced with permission of M.J. McFall-Ngai; (b) Nautilus macromphalus, a cephalopod sheltering specific bacterial phylotypes involved in nitrogen metabolism. Photograph courtesy of L.R. Berger.

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appendages formed from numerous contractile finger-like villi (Schipp et al., 1985). Both, electron microscopy and fluorescence in-situ hybridization experiments have shown that only two Nautilus-specific bacterial phylotypes were present in these appendages (Pernice et al., 2007). Despite the fact that ammonia is the main nitrogenous waste product of Nautilus, the measured excretion rates are relatively low. This suggests that the bacterial symbionts are involved in the transformation of ammonia to nitrogen, which is trapped in the chambers of the Nautilus shell and serves as a lifting gas for buoyancy. The two symbionts have been affiliated to widespread bacterial lineages in aquatic ecosystems, b-proteobacteria distantly related to Nitrosomonadaceae, an ammonia-oxidizing lineage; and spirochaetes (Pernice et al., 2007). While it is currently unknown if the establishment of a symbiotic relationship between the Nautilus host and these two bacteria is controlled by QS signals, the striking analogy to the squid model presents an interesting scientific challenge to future QS studies in this particular system. 7.6.3 Exploitation of Bacterial QS During Settlement of Marine Spores and Invertebrate Larvae

Many benthic marine organisms have a biphasic life cycle that includes a pelagic larva that is microscopic and morphologically distinct from the adult form. In most cases, the transition from pelagic larvae to benthic juvenile requires contact with an inductive environmental cue (Hadfield, 2011). The ability to discriminate and respond to signals associated with benthic substrata ensures that larvae settle in a habitat that is suitable for juvenile growth and survival. The location and selection of appropriate settlement sites is rarely random, and is instead guided by a range of positive and negative cues associated with the habitat (Grassle, Butman, and Mills, 1992; Pawlik, 1992; Wieczorek and Todd, 1998). Among the cues that trigger the pelagobenthic transition of marine invertebrates, settlement signals associated with natural habitat sources have received considerable attention (Hadfield, 2011; Qian et al., 2007). For example, conspecific and sympatric species and epibiotic or epilithic microbial biofilms in the habitat have been identified as sources of biomolecular settlement cues for a broad range of marine invertebrates. Both, surface-attached and water-borne attractants from microbial biofilms have been demonstrated as larval settlement cues (Hadfield, 2011; Wieczorek and Todd, 1998), but very few have identified the actual chemical signals and/or mode of action behind these triggers (Chung et al., 2010; Harder et al., 2002; Harder and Qian, 1999; Huang, Callahan, and Hadfield, 2012; Tebben et al., 2011). The role of bacterial QS signals as facilitators of eukaryote larval settlement has been demonstrated in a seminal study by Joint et al. (Joint et al., 2002). These authors showed that diffusible AHL signal molecules produced by many bacterial biofilms were detected and exploited by the planktonic phase of a marine eukaryotic organism, the green seaweed Enteromorpha.

Three experimental approaches were used to provide evidence of the role of AHLs as settlement cues; Vibrio anguillarum mutants defective in AHL production; Escherichia coli strains expressing AHL synthases from recombinant plasmids; and synthetic AHLs. While wild-type V. anguillarum biofilms strongly enhanced zoospore settlement compared to controls, no density-dependent stimulation of attachment was observed with a vanM mutant that does not produce C6-HSL and 3hydroxy-C6-HSL and is also deficient for 3-oxo-C10-HSL (Milton et al., 2001). Zoospore attachment was stimulated by another mutant (vanI), which produces C6-HSL and 3-hydroxy-C6-HSL but not 3-oxo-C10-HSL. The involvement of all three AHLs in spore settlement was confirmed, as the vanIM double mutant which is AHL-negative failed to stimulate zoospore attachment. These results indicated that Enteromorpha zoospores sensed and responded to AHL produced by V. anguillarum. It was shown that the spores exhibited reduced chemokinesis in the presence of high concentrations AHLs (i.e., bacterial biofilms), which decreased their swimming speed and indirectly increased spore settlement (Wheeler et al., 2006). The settlement response of barnacle larvae to bacterial biofilms has been intensively studied, and laboratory settlement assays have revealed that cyprid larvae distinguish between biofilms with different community compositions and prefer to settle on biofilms obtained from their adult habitats (Lau et al., 2005). In an investigation of the role of AHL signal molecules on the settlement of cyprid larvae of Balanus improvisus (Tait and Havenhand, 2013), a comparison was made between the settlement response of cyprids to AHLs produced by biofilms of the marine bacteria V. anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 with the response to AHLdeficient variants of these three isolates. All three AHL-producing strains significantly increased cyprid settlement in comparison to non-AHL-producing biofilms, which suggested that cyprid settlement was either mediated directly by an AHL signal, or indirectly by another QS-regulated bacterial cue (Tait and Havenhand, 2013).

7.7 “-Omic” Approaches to QS

The vast majority of what has been learned about QS – namely regulation, the phenotypes controlled and even interactions with specific hosts – has been achieved by using laboratorybased experiments with pure cultures of QS organisms. This approach has been essential to define the mechanisms of QS. Thus, while the general mechanisms of QS are quite well understood, the occurrence and distribution of QS in Nature is far less explored. For example, while the diel cycle of AHLs associated with microbial mats has been defined, it remains to be determined which organisms in the community are QSactive and the specific behaviors that are QS-controlled. Similarly, QS has been shown to drive ammonia oxidation in a complex community, but the mechanisms remain unclear (De Clippeleir et al., 2011).

References

One of the major bottlenecks of QS investigations at the community level or in natural environments is the difficulty in culturing the vast majority of microorganisms (Rappe and Giovannoni, 2003). Recent advances in sequencing technologies and mass spectroscopy, as well as bioinformatics, have generated the potential to provide insights into the role and function of QS systems at the whole community level by identifying and quantifying not only DNA and mRNA but also metabolites. These methodological approaches have now advanced to the point where it is possible to sequence proteins or identify macromolecules in complex mixtures, without the need to separate out individual nucleic acids, proteins, or metabolites. Because the techniques can be used to investigate complex samples, they are collectively called “-omics,” where transcriptomics is the sequencing of total mRNA transcripts, proteomics is the sequencing of total proteins present, and so on. For example, metagenome screens have identified putative lactonases from uncultured soil bacteria, suggesting that the community may be naturally exposed to QS organisms and that they may compete with such bacteria (Riaz et al., 2008; Schipper et al., 2009). Recently, when meta-omics approaches were applied to study community-level QS, using a water-water sludge community in a laboratory bioreactor, the concentration of specific AHLs was observed to correlate with the function of the community, for both nutrient removal as well as dense biofilm formation (unpublished data). Community analyses using the “-omics” approach have shown a strong correlation to AHL production, where the identified species were related to known AHL producers, suggesting which bacteria might be responsible for QS and the overall behavior, nutrient removal and biofilm formation, that was QS-controlled. Similarly, it was possible to identify

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AHL synthase and receptor genes, as well as the genes encoding QQ enzymes. Thus, it seems clear that both QS and QQ are relevant at the community level, affecting community function. Although there is still a long way to go to achieve the same levels of details observed for QS studies of single-species populations, it is now possible to investigate QS in highly diverse, complex communities.

7.8 Concluding Remarks

The discovery that bacteria produce and respond to extracellular signals as a means of controlling gene expression at the population level has changed not only the present understanding of gene regulation in bacteria, but also the view that bacteria act as autonomous, individual units. Instead, they appear to be able to coordinate behaviors in order to adapt to their environment. The mechanisms for communication are beginning to be unraveled, with several QS mechanisms now well defined, and new mechanisms continuing to be discovered. While much has been learned about QS in the laboratory, the next steps must move into the natural environments where QS occurs to define the genes, behaviors, and organisms involved. This is particularly important because such bacteria do not exist as individual populations, but rather as complex, multispecies consortia, and it is clear that QS plays important roles in the interaction of microorganisms in these complex communities. Thus, studies on interspecies and cross-kingdom signaling in situ will be particularly exciting avenues of research to better understand the connections between organisms sharing the same habitats.

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formation in a coral pathogen Serratia marcescens. ISME J., 5, 1609–1620. Antunes, L.C.M., Schaefer, A.L., Ferreira, R.B. R., Qin, N., Stevens, A.M., Ruby, E.G., and Greenberg, E.P. (2007) Transcriptome analysis of the Vibrio fischeri LuxR-LuxI regulon. J. Bacteriol., 189, 8387–8391. Atkinson, S. and Williams, P. (2009) Quorum sensing and social networking in the microbial world. J. R. Soc. Interface, 6, 959– 978. Bassler, B.L., Wright, M., Showalter, R.E., and Silverman, M.R. (1993) Intercellular signaling in Vibrio harveyi – sequence and function of genes regulating expression of lumininescence. Mol. Microbiol., 9, 773–786. Bauer, W.D. and Teplitski, M. (2001) Can plants manipulate bacterial quorum sensing? Aust. J. Plant Physiol., 28, 913–921. Bjarnsholt, T., Jensen, P.O., Rasmussen, T.B., Christophersen, L., Calum, H., Hentzer, M., Hougen, H.P., Rygaard, J., Moser, C., Eberl, L. et al. (2005) Garlic blocks quorum sensing and promotes rapid clearing of pulmonary

Pseudomonas aeruginosa infections. Microbiology, 151, 3873–3880. Bose, J.L., Kim, U., Bartkowski, W., Gunsalus, R. P., Overley, A.M., Lyell, N.L., Visick, K.L., and Stabb, E.V. (2007) Bioluminescence in Vibrio fischeri is controlled by the redox-responsive regulator ArcA. Mol. Microbiol., 65, 538–553. Bose, J.L., Rosenberg, C.S., and Stabb, E.V. (2008) Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture. Arch. Microbiol., 190, 169–183. Busetti, A. and Gilmore, B.F. (2010) Marinederived bacteria; a source of quorum sensing inhibitors? J. Pharm. Pharmacol., 62, 1390– 1391. Campbell, A.H., Harder, T., Nielsen, S., Kjelleberg, S., and Steinberg, P.D. (2011) Climate change and disease: bleaching of a chemically defended seaweed. Glob. Change Biol., 17, 2958–2970. Case, R.J., Longford, S.R., Campbell, A.H., Low, A., Tujula, N., Steinberg, P.D., and Kjelleberg, S. (2011) Temperature induced bacterial

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About the Authors Tilmann Harder graduated from the University of Oldenburg, Germany, in chemistry. During a postdoc at the Hong Kong University of Science and Technology, he worked on the identification of chemical and biological inducers involved in the pelago-benthic transition of marine organisms. In 2002, he was appointed Junior Professor at the Institute of Chemistry and Biology of the Marine Environment (Germany). He moved to Australia in 2008, where he currently serves as Deputy Director of the Centre for Marine Bio-Innovation (CMB), University of New South Wales. In this role he leads and manages the research cluster of Marine Chemical Ecology and Host-Microbe Interactions at the CMB. He is on the Editorial Board of PLoS One, Frontiers in Marine Ecosystem Ecology, and Journal of Chemical Ecology.

Scott A. Rice is Associate Professor at The Centre for Marine Bio-Innovation UNSW Australia and the Singapore Center on Environmental Life Sciences Engineering NTU Singapore. He has developed a research program around the themes of biofilm development and quorum sensing and, in particular, has made important contributions to understand the role of quorum sensing in biofilm development as well as dispersal, crosstalk between signaling systems, and biotechnology applications of quorum sensing inhibitors for biofilm control. His interests and current projects include the role of bacteriophage in biofilm development and infection, as well as intracellular signals such as nitric oxide and environmental cues, for example, nutrient limitations that induce biofilm dispersal. He is also developing polymicrobial models of

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biofilm development to better relate the laboratory studies of community assembly to the natural consortia of microorganisms. Sergey Dobretsov is an Associate Professor at the Sultan Qaboos University, Oman. His research interests are wide, and encompass marine chemical ecology, biofouling and its prevention, extremophiles, biofilms and quorum sensing. He has authored more than 60 peer-reviewed articles, five book chapters, and is a coinventor of four international patents. He has been an Alexander von Humboldt Scholar (Germany) and a recipient of George E. Burch Fellowship in Theoretical Medicine and Affiliated Sciences at the Smithsonian Institution (USA). He is on the Editorial Boards of Marine Ecology Progress Series and Biofouling journals. Torsten Thomas holds an MSc from the University in Bonn and a PhD from the University of New South Wales (UNSW). From 2001–2004 he worked as a Senior Scientist at the biotech company Nucleics Pty Ltd. In 2005, he joined the Center for Marine Bio-Innovation (CMB) at UNSW, and received fellowships from the American Australian Association and the Australian Research Council. He was appointed Senior Lecturer (2009) and Associate Professor (2012) at the School of Biotechnology and Biomolecular Sciences at UNSW. In 2010, he joined the Executive Team of the CMB, and is a current member of the National Scientific Advisory Committee of the Australian Society of Microbiology. His current research covers bacteria-sponge symbiosis, the microbial conversion of coal to methane, the functional diversity and redundancy of marine communities, antibiotics production and resistance in the marine environment and the genomics of evolving, bacterial populations. Alyssa Carr
e-Mlouka is an Assistant Professor at the National Museum of Natural History (MNHN) in Paris, France. She obtained her PhD on hepatotoxic cyanobacteria at the Pasteur Institute in Paris, and then worked as a teaching assistant at the University of Versailles. She currently studies the diversity of active molecules produced by microorganisms from salty ecosystems, focusing on halophilic archaea and sponge-associated bacteria. She teaches microbiology and biochemistry at the MNHN and the University Denis Diderot-Paris 7, and is a member of the French Society for Microbiology. Staffan Kjelleberg is a Professor and Director of the Singapore Centre on Environmental Life Sciences Engineering NTU

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Singapore, and Co-Director of the Centre for Marine BioInnovation, UNSW, Australia. He is internationally renowned for his research into bacterial biofilm biology, chemically mediated interactions used by bacteria and higher organisms, and harnessing/controlling biofilms for engineering and public health applications. His research programs are aimed at understanding the role of complex microbial communities in the urban water cycle, as well as in marine coastal ecosystems, using complementary top-down meta-omics/systems biology and bottom-up biofilm mechanism approaches. Commonalities in biofilm biology underpin his broad-based translational research and applications. Peter D. Steinberg is Director and CEO of the Sydney Institute for Marine Science on Sydney Harbour, as well as Professor of Biology and Director of the Centre for Marine Bio-Innovation at UNSW, and Co-Director of the Advanced Environmental Biotechnology Centre at Nanyang Technological University, Singapore. He has authored some 140 international papers in a diversity of biological fields, and is an inventor on eight patents. He has been a Fulbright Scholar, a Queen Elizabeth II Fellow, and CEO of a publically listed biotechnology company focusing on the development of novel antibacterials and antifoulants from marine organisms. He is on the Editorial Boards of leading scientific journals such as Ecology and Marine Ecology Progress Series. His research interests include marine chemical ecology, seaweed ecology, marine herbivory, diseases of marine organisms, bacterial biofilm biology and ecology, environmental biotechnology, biofouling, and novel antifouling technologies. Diane McDougald is a program leader and Deputy Director at the Centre for Marine Bio-Innovation, as well as a cluster leader for the Marine Health and Biotechnology Cluster in the Advanced Environmental Biotechnology Centre (AEBC), Nanyang Technological University, Singapore. She has made significant contributions to the fields of Vibrio biology, bacterial adaptation to stress and mechanisms of molecular control of these responses, cell-to-cell communication, biofilm formation and interactions of bacteria with higher eukaryotes. Projects supervised by Dr McDougald include the investigation of the interactions of bacteria and heterotrophic protists, and how the evolution of predation defenses drives the evolution of pathogenicity. She serves on the scientific committee of the Association of Vibrio Biologists, of which she was a founding member, and on the organizing committee of the international conferences Vibrios in the Environment 2010 and Vibrios 2005, 2007, 2009, and 2011.

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8 Domoic Acid St
ephane La Barre, Stephen S. Bates, and Michael A. Quilliam

Abstract

Domoic acid (DA) was of no special scientific interest until a series of case studies revealed its role as the major marine neurotoxin causing amnesic shellfish poisoning (ASP). The analysis, toxicology, synthesis and degradation of the highly polar amino acid DA and its kainoid congeners are discussed in this chapter. Although DA is structurally simple and ubiquitous in contaminated food samples, it was not simple to prove that it was the causative agent of ASP in humans and of DA poisoning in carnivorous birds and mammals. Furthermore, its detection and the prevention of ASP requires regular monitoring of seafood using rapid and accurate analyses. The main producers of DA are certain seasonally blooming diatoms of the genus Pseudo-nitzschia, major components of coastal phytoplankton. Here, details are provided

of the species most likely to be involved in food-poisoning episodes, together with a brief account of the molecular mechanisms that underlie DA toxicity, which cause symptoms of acute and chronic neurotoxicity. DA may attain critically toxic levels within two major food chains involving benthic filter-feeders (e.g., mussels) or planktivorous fish (e.g., anchovies). Preventive measures must be complemented by risk assessments of seasonal toxigenic blooms, especially in nutrient-enriched coastal areas. The major chemical and biotic factors that influence diatom bloom formation and toxigenicity are outlined. Genomics of DA production allow the development of novel molecular tools to better understand DA biosynthesis at the gene level, and the evolutionary significance of DA as a metabolite with primary and secondary characteristics.

Box 8.1: Domoic Acid {2S-[2a,3b,4b(1Z,3E,5R)]}-2-Carboxy-4-(5-carboxy-1-methyl-1, 3-hexadienyl)-3-pyrrolidineacetic acid (IUPAC) Isolated from the red alga Chondria armata from Japan (Takemoto and Daigo, 1958) and Alsidium corallinum from the Mediterranean Sea (Impellizzeri et al., 1975). Domoic acid is also produced by at least 14 of the over 38 species of the pennate diatom genus Pseudo-nitzschia, as well as some strains the of pennate diatom Nitzschia navis-varingica (q.v. Lelong et al., 2012; Trainer et al., 2012) Elemental formula: C15H21NO6 MW: 311.33 CAS RN: 14277-97-5

HOOC

BRN: 5768789 Colorless crystal needles, highly water soluble

HOOC

COOH N H

Used traditionally in Japan as an anthelmintic agent (Daigo, 1959a, 1959b, 1959c) and insecticide (Maeda et al., 1984; Maeda et al., 1987a). Synonyms: 2a-carboxy-4b-(5-carboxy-1-methyl-1,3b-hexadienyl)-3-pyrrolidineacetic acid, (2S,3S,4S)-2-carboxy-4-[(1Z, 3E,5R)-5-carboxy-1-methyl-1,3-hexadienyl]-3-pyrrolidineacetic acid; (3S,4S)-4-[(2Z,4E,6R)-6-carboxyhepta-2,4-dien-2-yl]-3(carboxymethyl)-L-proline. Caution: Toxic if ingested, inhaled, or in skin contact. The use of dust mask type N95 (US), eyeshields and gloves is required within a well-ventilated space. May cause rapid gastrointestinal and neurological disturbances (amnesic shellfish poisoning syndrome) as acute symptoms; causes brain/CNS long-term functional impairments and structural damages on chronic exposure.

Commercially available as an analytical and pharmacological tool; cost ca. D 200 per milligram.

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA. Ó 2014 Her Majesty the Queen in Right of Canada, reproduced with the permission of the National Research Council Canada 2014.

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8.1 Historical Background

The story of this relatively simple molecule is unusual in many respects, its significance having gradually unfolded since its presence in the red alga Chondria armata was first discovered in 1958 (Takemoto and Daigo, 1958). Domoic acid (DA, see Box 8.1) (from domoi, the vernacular name of C. armata in Japan) is the active ingredient of this seaweed, which has been used traditionally on the island of Tokinoshima to treat ringworm infestations, and this may have prompted the initial chemical investigations. DA resembled another molecule, kainic acid, which had been identified a few years earlier (in 1953) from another red alga, Digenea simplex, and used as an anthelminthic in Japan since the ninth century to cure infants of roundworm infection (Higa and Kuniyoshi, 2000). Domoi was also used for insect control by the inhabitants of Yakushima Island, when it was noticed that flies landing on these algae became intoxicated and died (Maeda et al., 1984). DA was identified as the active ingredient in 1958 (Daigo, 1959a, 1959b, 1959c), and its insecticidal properties, along with those of the isodomoic acids A, B and C (Table 8.1), were further studied (Maeda et al., 1984, 1986, 1987b). Although a total synthesis of the molecule was completed in 1982 (Ohfune and Tomita, 1982), DA was relatively unheard of outside Japan at the time because its neurotoxic effects after oral intake were not apparent at prescribed levels. The global significance of DA emerged gradually (Trainer, Hickey, and Bates, 2008; Trainer et al., 2012; Lelong et al., 2012). Initially, a single massive seafood intoxication in 1987, originating at Prince Edward Island in eastern Canada, had caused several deaths and severe complications in well over 100 people who had consumed blue mussels (Mytilus edulis) (Perl et al., 1990; Teitelbaum et al., 1990; as described in Case study #1). Subsequently, a new term – amnesic shellfish poisoning (ASP) – was coined to account for the disorientation and memory deficiencies observed in many individuals; these were accompanied by gastrointestinal effects, followed some time later by epileptic seizures in at least one patient, and death in four others. Investigations promptly led to DA being designated as the causative agent (Quilliam and Wright, 1989), and the pennate diatom Nitzschia pungens forma multiseries (now known as Pseudo-nitzschia multiseries) as the source of the toxin, after having examined the shellfish flesh and gut contents, and isolating the diatom in culture (Bates et al., 1989; see Case study #1). This was the first time that a biotoxin had been shown to be produced by a diatom, and the monitoring of shellfish beds has been undertaken consistently since then. Safety measures were immediately implemented, which forbade the sale or harvesting of molluskan shellfish when the DA content of the edible flesh exceeded 20 mg g 1 fresh weight (Wekell et al., 2002). The first verified case of vertebrate animal DA intoxication occurred in 1991, in Monterey Bay, California, when brown pelicans (Pelecanus occidentalis) and Brandt’s cormorants (Phalacrocorax penicillatus) died after having eaten anchovies contaminated by DA from another producer, Pseudo-nitzschia australis (Fritz et al., 1992; Work et al., 1993; as described in Case study #2). High levels of DA contamination were also

reported in crabs, razor clams and mussels at many other sites in the USA (Bates, Garrison, and Horner, 1998; Trainer et al., 2012). In 1998, epizootics affecting sea lions (Zalophus californianus) were attributed to DA accumulation in planktivorous fish that had consumed toxigenic P. australis (Scholin et al., 2000; as described in Case study #3). In addition to the documented acute toxicity syndromes, repeated exposure to sublethal doses of DA was found to be responsible for epileptic seizures observed over the following decade among sea lion populations. With the annual increase in toxigenic blooms along the California coast, DA is now established as a prominent environmental neurotoxin (Trainer, Hickey, and Bates, 2008), with acute and long-term neurological effects on wildlife that feeds on intoxicated fish and invertebrates (Bejarano et al., 2008). The full environmental significance of recurrent blooms of toxigenic Pseudo-nitzschia diatoms has shifted progressively from isolated risk zones in eastern Canada and the Pacific coast of the USA to a worldwide concern, such that DA monitoring has become an emerging necessity in some temperate Asian, European and South American localities. For example, P. australis, which originally was described only from the southern hemisphere, was later identified on the west coast of California, and more recently in Europe (Lelong et al., 2012). The global transport of exogenous plankton in ships’ ballast water could be held partly responsible for this expansion. Moreover, these problems can be expected to worsen with increased global warming, as this may allow certain toxigenic species to proliferate in new locations. Increased levels of carbon dioxide, which accompany ocean acidification and global warming, will increase Pseudo-nitzschia toxicity (Sun et al., 2011; Tatters, Fu, and Hutchins, 2012). The experimental and natural iron enrichment of oceanic waters may also stimulate plankton productivity and reduce carbon dioxide levels, but this correlates positively with the occurrence of toxigenic Pseudo-nitzschia blooms (Silver et al., 2010; Trick et al., 2010); the environmental role of DA as a siderophore (metalcapturing molecule) is still debated, however (Lelong et al., 2012). Blooms of toxic Pseudo-nitzschia tend to occur in high-productivity areas, and occasionally in association with waters impacted by urban and farm discharges, which provide abundant nitrogen (e.g., nitrate, ammonium) for growth. Urea can be used as a primary nitrogen source by these diatoms, and this clearly enhances the production of DA in P. australis (Howard et al., 2007), though this may not always be the case for other Pseudonitzschia species (Auro and Cochlan, 2013). The enrichment of coastal waters exacerbates the recurrence of harmful algal bloom (HAB) episodes in North America, and this has now become a major issue (Anderson et al., 2008; Heisler et al., 2008). A newly described DA-producing diatom is Nitzschia navis-varingica, isolated from shrimp farms in Viet Nam (Lundholm and Moestrup, 2000). This diatom is found over a large latitudinal range in Asia, where it thrives in brackish waters (Kotaki et al., 2004; Thoha et al., 2012) and has become a major concern to local shrimp farmers. The full toxicological significance of DA has taken years to investigate, with numerous neurophysiological studies having been carried out in laboratory animals, including vertebrates (fish to mammals) and invertebrates (insects, crustaceans,

8.1 Historical Background

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Table 8.1 The domoic acid family. From left to right: Domoic acid with its isoforms A–F, its 50 - epimer and its two lactone derivatives, as defined by the side chain at position 4. The black dot represents the common pyrollidineacetic moiety; Original biological source in which the molecule was first identified; Reported bioactivities; Novel syntheses.

Domoic acid

50 -epi-Domoic acid (DA diastereoisomer)

First isolation Chondria armata (Takemoto and Daigo, 1958); Alsidium corallium (Impellizzeri et al., 1975); 14 Pseudo nitzschia species (see Lelong et al., 2012), Mytilus edulis (Wright et al., 1988) First isolation Mytilus edulis (Walter, Falk, and Wright, 1994)

Bioactivity Potent insecticide (Maeda et al., 1987a) Very potent ASP

Total synthesis (Ohfune and Tomita, 1982)

Bioactivity Potent ASP

DA heat-degradation product

Isodomoic acid A (DA geometric isomer)

First Isolation Chondria armata (Maeda et al., 1986)

Bioactivity Potent insecticide (Japanese thesis) Weak ASP

Isodomoic acid B (DA geometric isomer)

First isolation Chondria armata (Maeda et al., 1986)

Bioactivity Potent insecticide Weak ASP

Total synthesis (Lemiere et al., 2011)

Isodomoic acid C (DA geometric isomer)

First isolation Chondria armata (Maeda et al., 1986)

Bioactivity Potent insecticide Weak ASP

Total synthesis (Clayden, Knowles, and Baldwin, 2005b)

Isodomoic acid D (DA geometric isomer) Isodomoic acid E (DA geometric isomer)

First isolation Chondria armata (Maeda et al., 1985); Mytilus edulis (Wright et al., 1990) First isolation Mytilus edulis (Wright et al., 1990)

Total synthesis (Lemiere et al., 2011)

Isodomoic acid F (DA geometric isomer)

First isolation Mytilus edulis (Wright et al., 1990)

Total synthesis (Lemiere et al., 2011)

Isodomoic acid G (DA geometric isomer)

First isolation Chondria armata (Zaman et al., 1997)

Isodomoic acid H (DA geometric isomer)

First isolation Chondria armata (Zaman et al., 1997)

Total synthesis (Ni et al., 2003, 2009; Denmark, Liu, and Muhuni, 2009, 2011) Total synthesis (Ni et al., 2009; Denmark, Liu, and Muhuni, 2009, 2011)

Domoilactone A (DA analog)

First isolation Chondria armata (Maeda et al., 1987b)

Domoilactone B (DA analog)

First isolation Chondria armata (Maeda et al., 1987b)

mollusks). These studies have been supplemented by postmortem investigations on the brain and central nervous system (CNS) of humans with a history of ASP. DA intoxication is dosedependent with regards to acute symptoms, while long-term (chronic) exposure can result in a cumulative impairment of function. As blooms of toxic Pseudo-nitzschia tend to occur naturally in high-productivity areas, and occasionally in association with waters impacted by urban discharges, residents are

facing a higher risk of chronic intoxication (with onset after up to 20 years) by consuming contaminated seafood on a regular basis, even if the detected levels of DA are deemed acceptable (Lefebvre and Robertson, 2010). Indeed, DA is present in many animal species (in addition to mussels) that are consumed by humans, including recreational fish, anchovies, razor clams, Dungeness crabs, king scallops, squid, and cuttlefish (Trainer et al., 2012).

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8.2 Case Studies 8.2.1 Case Study #1: The 1987 Outbreak on Prince Edward Island

Prior to 1987, the only biotoxins of concern to Canada, and much of North America, were those such as saxitoxins produced by dinoflagellates of the genus Alexandrium that caused paralytic shellfish poisoning (PSP). This syndrome usually occurred during the summer months, when the stratified water column produced conditions that were conducive to the proliferation of these dinoflagellates. It was therefore a great surprise when very sick individuals exhibiting similar symptoms began arriving at hospitals in New Brunswick and Quebec, starting on 22 November 1987 (a chronology of events is given in Anderson et al., 2001). On 29 November, epidemiologists from Health and Welfare Canada

(HWC) determined that all of the patients had consumed blue mussels (Mytilus edulis) from eastern Prince Edward Island, eastern Canada. Tests for PSP toxins, trace metal contamination and the usual bacterial or viral agents proved negative, while water samples taken through holes drilled in the ice in early December, in Cardigan Bay, Prince Edward Island, showed an absence of toxic dinoflagellates, but this was not surprising given the time of year. The deaths of at least four elderly individuals and the sickness of over 100 others (Perl et al., 1990; Teitelbaum et al., 1990) led to an immediate closure for harvesting of all shellfish, including mussels, clams, quahogs and scallops, on 11 December. This was devastating to the aquaculture and wild shellfish industries, especially just prior to the lucrative Christmas season, and consequently the story made national headlines and great pressure was applied to the Canadian government to resolve the problem. Hence, a major effort was mounted to identify the toxin in the contaminated mussels.

Figure 8.1 Flow chart showing the original extraction and separation procedure used to identify the toxic fraction from mussel flesh in the 1987 intoxication event on Prince Edward Island, Canada. HPLC coupled with diode array detection (DAD) used the 242 nm UV peak of domoic acid for quantification. Active fractions are indicated by red arrows; the final extracts obtained by HPLC and by HVPE (high-voltage paper electrophoresis) were crosschecked to confirm activity. (Adapted with permission from Quilliam and Wright. Ó (1989) American Chemical Society.)

8.2 Case Studies

On 12 December, the National Research Council of Canada (NRC), in Halifax, Nova Scotia, assembled a team of 40 chemists and biologists to tackle the problem. Other scientists from Fisheries and Oceans Canada (DFO) and the Atlantic Veterinary College of the University of Prince Edward Island (Charlottetown), joined in the efforts. A (mouse) bioassay-directed strategy (Figure 8.1) traced the toxicity to a water-soluble fraction of the mussels (Quilliam and Wright, 1989), and chemical methods that included column chromatography, high-voltage paper electrophoresis, HPLC with ultraviolet diode array detection (DAD) and NMR spectrometry were used to analyze the toxic fraction. After an unprecedented 104 h period of detective work, the culprit toxin was identified as DA, an amino acid that had already been isolated in the 1950s from the red seaweed Chondria armata (see above). The identification was at first treated with disbelief, because this was the same compound used in Japan to treat children infested with intestinal worms! However, in the case of the Canadian illnesses and deaths, an order of magnitude higher dose of DA was estimated (290 mg) than was ever given for anthelmintic treatments (20 mg) (Trainer, Hickey, and Bates, 2008). Furthermore, those affected in 1987 were elderly and had preconditions, such as renal dysfunction and compromised blood–brain barriers, which made them more vulnerable than the children. These findings were later reinforced by several studies that showed an agedependent DA toxicity in mice and rats (Ramsdell, 2007). The lessons learned from this 1987 incident were that unexpected biotoxins could be discovered in novel biological sources, and that monitoring efforts must be strengthened. Finally, analytical methods, such as LC-MS/MS (see below),

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must be used to maintain vigilance against such incidents. As a consequence, several other biotoxins, including spirolide toxins, pectenotoxins, yessotoxins and azaspiracids, which are also found elsewhere in the world, have since been discovered in Canadian waters.

8.2.2 Case Study #2: The 1991 Bird Intoxication Event in California

A bloom of the pennate diatom Pseudo-nitzschia australis, which occurred in early September 1991 at Monterey Bay, California, coincided with an episode of mortality in brown pelicans (Pelicanus occidentalis) and Brandt’s cormorants (Phalacrocorax penicillatus). High levels of DA, the ASP toxin, were recorded in the plankton samples (Fritz et al., 1992; Work et al., 1993). Furthermore, high levels of DA, as well as numerous remnants of P. australis frustules, were found in the stomach contents of the affected birds and in the visceral contents of local anchovies, a major food source of the seabirds. This was the first confirmed report of DA poisoning since the 1987 outbreak on Prince Edward Island (see Case study #1), and was also the first evidence of a herbivorous fish acting as a vector for this toxin. Interestingly, currently available data indicate that DA-producing algal blooms do not cause fish kills or neuroexcitotoxic behavior in fish (Lefebvre et al., 2012). A little known fact is that, at the time, a large shipment of the highly toxic anchovies was heading for another country but was recalled a short while later, possibly saving many lives.

Box 8.2: The Birds: A Suspense Story Generations of thriller lovers have seen Alfred Hitchcock’s The Birds since its original screening in 1963. The screenplay, written by Evan Hunter, borrowed the title of Daphn
e Du Maurier’s 1952 short story The Birds, about angry birds attacking humans and invading small English towns. This was developed into a suspense story which started from trivia and gradually climaxed into full horror. Hitchcock’s scenario, however, drew its inspiration from an 18 August 1961 report in a local Californian newspaper, The Santa Cruz Sentinel, titled “Seabird invasion hits coastal homes” (http://www.santacruzpl.org/history/articles/183/): “A massive flight of sooty shearwaters, fresh from a feat of anchovies, collided with shoreside structures from Pleasure Point to Rio del Mar during the night. Residents, especially in the Pleasure Point and Capitola area were awakened about 3 a.m. today by the rain of birds, slamming against their homes. Dead and stunned seabirds littered the streets and roads in the foggy, early dawn. Startled by the invasion, residents rushed out on their lawns with flashlights, then

rushed back inside, as the birds flew toward their light. ( . . . /) When the light of day made the area visible, residents found the streets covered with birds. The birds disgorged bits of fish and fish skeletons over the streets and lawns and housetops, leaving an overpowering fishy stench.” It was not until a similar episode in 1991, involving brown pelicans and cormorants in Monterey Bay, California (see Case study #2), that what became “Hitch’s secret” was finally resolved. Carefully preserved samples of the gut contents of zooplankton collected in July–August 1961, from Monterey Bay, California, were shown to contain Pseudo-nitzschia species (Bargu et al., 2012). In the 1991 incident, high DA concentrations were found in the food regurgitates of the birds, together with Pseudo-nitzschia diatom frustules (Fritz et al., 1992; Work et al., 1993). The similarities between events in 1961 and the DA-induced poisoning of 1991, suggested that toxic Pseudonitzschia were probably responsible for the odd behavior and death of sooty shearwaters in August 1961.

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8.2.3 Case Study #3: Massive Sea Lion Mortality in Just a Few Weeks

In the spring of 1998, hundreds of sea lions (Zalophus californianus) were found stranded along the central Californian coast; many of these were already dead, while others displayed severe neuropathological symptoms. Investigations showed that this event had coincided with seasonal “flash” blooms of Pseudo-nitzschia australis, a newly discovered producer of DA, which causes ASP. The identified trophic link was northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax) that feed on phytoplankton in the nutrient-rich waters, and accumulate DA to toxic levels during P. australis blooms. Indeed, the toxin was found both in extracts of these fish and in the body fluids of dead sea lions (Scholin et al., 2000). An examination of the stomach contents of the fish showed frustules of P. australis to be present, and blooms of this species co-occurred during sea lion mortality events, whereas blooms of the nontoxic P. pseudodelicatissima corresponded with a fall in DA levels in the flesh of these fish. Furthermore, histopathological sections of the anterior hippocampal region of the sea lion brain (see Section 8.5) revealed DA-induced lesions typically found in other post-mortem autopsies. As was the case with seabirds, earlier observations of similar but unresolved cases of marine mammal intoxication events could then be linked to deadly blooms of toxigenic diatoms. Thus, other sea lion, fur seal, dolphin and cetacean mortalities reported from Mexican (Baja California peninsula) and Californian coasts could be documented in connection with potentially toxigenic Pseudo-nitzschia blooms (e.g., Ochoa et al., 1998). There is now a growing concern that repeated seasonal blooms of toxigenic Pseudo-nitzschia species may expose marine wildlife to sublethal doses of DA that will trigger pathological symptoms years later. Although water-soluble DA is not bioaccumulated (excess DA is rapidly eliminated by kidneys), chronic exposure to DA may cause irreversible damage to the brain and CNS by repeated fixation to glutamate receptors (see Section 8.5). For example, Goldstein et al. (2008) have predicted that chronic DA toxicosis will induce epileptic fits that can be qualified as novel symptomatology, in contrast to the acute syndromes described above. Young female sea lions are known to be under high risk due to chronic exposure to DA, with effects on their embryos and subsequent early life stages; this adds to poisoning by industrial discharges of other neurotoxins (e.g., DDT) at localities where human influence has already been highly detrimental (Ramsdell, 2010). It is now necessary to reassess the health risks to marine wildlife and humans in light of predictive anthropogenetic and biogeoclimatic factors that may synergistically lead to the emergence of high-risk DA-producing diatom blooms.

8.3 Chemistry 8.3.1 Physico-Chemical Properties

Domoic acid is highly water-soluble (8 mg ml 1) but much less soluble in methanol (0.6 mg ml 1). The pure compound is most often described as colorless crystal needles, with a density of 1.273 g cm 3. DA is a moderately thermostable molecule (it resists cooking conditions), and is photodegradable upon sunlight and UV exposure. Acidic conditions (pH downfield

H (d ppm)

H splitting

J (H, H) Hz

Position

13

1 2 7 4 5

1.27 1.81 2.50 2.76 3.05

d s dd dd dddd

50 -CH3 10 -CH3 6b 6a 3

18.6 23.5 35.4

qdd qdd bt

44.6

bd

10 9 8 6 11 12 13 14

3.30 3.49 3.71 3.84 3.98 5.78 6.13 6.35

dq dd dd ddd d dd d dd

(50 , 60 ) 7,1 None (3, 6b) 9.1 (3, 6a) 5.8 (2, 3) 8.1 (3, 4) 8.4 (3, 6a) 5.8 (3, 6b) 9.1 (40 , 50 ) 7.8 (50 , 60 ) 7.1 (40 , 5a) 7,3 (5a, 5b) 12.2 (4, 5b) 7.9 (5a, 5b)- 12.2 (4, 5a) 7.3 (4, 5b) 7.9 (2, 3) 8.1 (30 -40 ) 14.9 (40 -50 ) 7.8 (20 , 30 ) 11.1 (20 , 30 ) 11.1 (30 -40 ) 14.9

44.9 49.1

bd bt

42.7 67.1 135.2 132.8 128.6 133.8 177.5 174.9 181.9

bd bd bd bd dd bm dt dd m

50 5a 5b 4 2 40 20 30 10 7 2-COOH 50 -COOH

C (d ppm)

13

C splitting

Attached H type allylic methyl vinylic methyl vicinal to COOH vicinal to COOH methine methine beta to N beta to N methine methine ethylenic H ethylenic H ethylenic H none none none none

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Figure 8.3 Typical product ion mass spectrum of domoic acid in positive ion mode. Peaks in red correspond to species resulting from fragmentation pattern in Figure 8.4. (Personal data provided by Dr Michael Quilliam.)

8.3.2.3 Mass Spectrometry (MS) A typical product ion mass spectrum of the DA molecule is represented in Figure 8.3. The protonated molecule [M þ H]þ appears at m/z 312, with major peaks at m/z 266 and 220 corresponding to successive decarboxylation losses of HCOOH (46). Other peaks correspond to eliminations of H2O (18), HCN (27) and CO (28) (Figure 8.4). One low-mass ion at m/z 74 can be attributed to the ion [CO2H CH NH2]þ, which is characteristic of protonated amino acids (Thibault et al., 1989). The peaks with red labels in Figure 8.3 correspond to ions in the fragmentation pathway of Figure 8.4. 8.3.2.4 UV spectroscopy (UV) The UV spectrum as scanned from 200 to 300 nm consists of a single intense band (signal onset at 275 nm) with a maximum

at 242 nm (Figure 8.5), corresponding to an a,b-unsaturated carboxylic acid in the side chain. The absorbance peak is pHsensitive, the 242 nm maximum corresponding to an approximately neutral pH. Falk, Walter, and Wiseman (1989) determined the lmax and emax profiles of DA at different protonation stages in order to facilitate the interpretation of UV spectra commonly used in the detection of the toxin in seafood samples analyzed using liquid chromatography (see Wright et al., 1988). At first glance, the outstanding feature of DA is its unusual ionization properties due to the presence of the aforementioned carboxyl and amino groups, which leads to five potential charge states of the molecule. At physiological pH, the prevailing form of DA is deprotonated at all three carboxyl groups and protonated at the amino group, leading to a net charge of 2. This has

Figure 8.4 Characteristic fragmentation pathway of domoic acid in positive ion mode. The peaks correspond to red labeled species in Figure 8.3. (After Thibault et al., 1989.)

8.3 Chemistry

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Figure 8.5 Ultraviolet spectrum of domoic acid using diode-array spectrometer (UV-DAD). There is a small shift in the lmax (wavelength for maximum absorbance) according to the pH of the solution. (Personal data provided by Dr Michael Quilliam.)

important implications for the neurotoxicity of DA when administered to animals (Ramsdell, 2007). DA bears a hexadienoic C4 side chain, which confers its unique toxigenic character with respect to its kainate/glutamate analogs. The presence of two conjugated double bonds in the C4 side chain, and the geometry of these moieties, are directly related to the interaction of DA at the glutamate receptor and to the toxicity of the molecule (Swanson and Sakai, 2009). The presence of these stable dienes enables DA to absorb UV light, which at neutral pH gives an emission maximum of 242 nm, and this is used in one method to quantify DA by liquid chromatography (Quilliam et al., 1989a).

8.3.3 Extraction, Separation, Purification, and Detection of DA 8.3.3.1 Extraction and Cleanup Most of the extraction procedures use mollusk flesh as the starting material, whether to obtain the pure compound or to perform routine toxin identification and quantitation from samples. Aqueous methanol (1 : 1) or hot water may be used as solvent under homogenizing conditions to achieve an efficient extraction of DA from tissue samples (Quilliam et al., 1989a; Quilliam, Xie, and Hardstaff, 1995). In preparative isolation procedures, pigments and other molecules of medium polarities may be removed from crude extracts by evaporating the methanol and partitioning the extract with a nonmiscible solvent, such as dichloromethane. A selective sample cleanup of aqueous methanol extracts, based on solid-phase extraction (SPE) with a strong anion-exchange column, is used widely as a cleanup for analytical purposes (Quilliam, Xie, and Hardstaff, 1995; He et al., 2010). 8.3.3.2 Separation and Purification The high polarity of DA is due to the presence of three carboxyl groups and one imino group with pKa values of 1.85, 4.47, 4.75, and 10.60, respectively. Thus, DA may exist in five different charge states, from 3 to þ1 depending on the pH, and this can

affect its retention in different chromatographic systems. For example, an acidic mobile phase is required for reversed-phase chromatography in order to suppress ionization of the carboxyl groups which, in their anionic form, will lead to a poor retention as well as an adverse interaction with residual silanol functions (Quilliam et al., 1989a; Quilliam, Xie, and Hardstaff, 1995). Both, capillary electrophoresis (CE) and capillary electrochromatography have proved to be useful alternatives to HPLC for DA monitoring (Zhao, Thibault, and Quilliam, 1997; He et al., 2010); in this case, a basic running buffer is used to ensure that DA is in an anionic form for maximum electrophoretic mobility. 8.3.3.3 Detection, Quantification, and Monitoring in Food Samples The method of choice for detecting and quantifying DA in samples is liquid chromatography combined with UV spectrophotometry (using a diode array detector; DAD) at a fixed wavelength of 242 nm to detect DA specifically, or with full spectrum scanning (e.g., between 220 and 360 nm) for a better confirmation of identity. Reversed-phase thin-layer chromatography with DA visualized by a ninhydrin spray has been proposed for those laboratories lacking HPLC equipment (Quilliam, Thomas, and Wright, 1998). LC-MS has proven to be a very sensitive and selective method for DA (Quilliam et al., 1989b; Quilliam, Xie, and Hardstaff, 1995). Routine monitoring in shellfish tissue requires the detection of DA at concentrations well below safe limits for human consumption, and protocols must be simple to operate and reliable regarding quantitation. This subject is reviewed by Quilliam (2003). When quantifying DA in complex extracts, careful attention is required regarding the selection of mobile and stationary phases, and also to the column temperature, in order to achieve an adequate separation of DA from the many other natural compounds with chromophores present in the sample. One such example is tryptophan and its oxidation products, which can have similar retention times to that of DA (Quilliam et al., 1989a). If the instrument used is fitted with a DAD, the compounds can be easily distinguished by their full UV spectra.

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A strong anion-exchange SPE cleanup prior to HPLC provides a much higher degree of selectivity (Quilliam, Xie, and Hardstaff, 1995). This cleanup is quite remarkable; when aqueous methanol extracts are loaded, only very strongly acidic compounds such as DA will be retained, while weaker amino acids such as tryptophan are washed away; the DA can then be eluted with acidic water (pH 3) or with 1 M NaCl in water. The limit of detection (LOD) for DA when using this method is 20 ng g 1, after sample cleanup. Mafra et al. (2009) developed a highsensitivity HPLC-UV method for detecting trace levels of DA in seawater and phytoplankton cells, using a large-volume injection with an ion-pairing agent and gradient elution; a LOD for DA of 42 pg ml 1 was achieved in this way. As a variant to classical reversed-phased HPLC columns packed with fine particles, Regueiro et al. (2011) proposed a simple and efficient method for the routine determination of DA from shellfish samples. Using graphitized nonporous carbon as a dispersive solid-phase extraction (dSPE) cleanup sorbent for the dispersive extraction of the shellfish flesh homogenates (concentrated at 50 mg ml 1), the authors were able to separate the centrifuged supernatant using a monolithic silica stationary phase (high flow rate and high exchange surface). This helped to reduce the backpressure buildup and decrease the runtime versus efficiency of the HPLC separation process. With acetonitrile/water gradient as the mobile phase under acidic conditions, it was possible to separate the sample and detect DA and its congeners (UV at 242 nm) in about 3 min, which was three- to fourfold faster than with conventional HPLC procedures, without reducing the performance of the column between runs. A very selective and sensitive detection of DA can be achieved by using electrospray ionization (ESI) LC-MS, especially in the selected reaction monitoring mode where transitions from the [M þ H]þ ion to specific product ions (as in Figures 8.3 and 8.4) are measured. Triple quadrupole or ion-trap MS systems can be used to produce such data (Quilliam et al., 1989b; Furey et al., 2001; Mafra et al., 2009), using reversed-phase HPLC columns eluted with an acidified acetonitrile–water gradient from 5 : 95 to 40 : 60. Mafra et al. (2009) demonstrated the suitability of the large-volume injection method with MS detection for the trace level detection of DA in seawater and phytoplankton cells; in this case, an LOD for DA of 15 pg ml 1 could be achieved. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS is suitable for the routine detection of higher-level DA samples (Paz, Riob
o, and Franco, 2011). Although the DA signal is hampered by matrix noise and limited observable fragmentation patterns (193, 248, 266), the technique has been used in the efficient diagnosis of DA-related toxicosis of sea lion samples (Neely et al., 2012). Other protocols aimed at detecting DA diluted in seawater and in phytoplankton samples include derivatization followed by HPLC with fluorescence detection (reviewed by Riob
o, L
opez, and Franco, 2011). 9-Fluorenylmethylchloroformate (FMOC-Cl) is known to react rapidly with DA, and its application to seawater samples gave an LOD for DA of 15 pg ml 1 (Pocklington et al., 1990). Other derivatizing reagents that have been used successfully on phytoplankton and seawater samples after

preconcentration with SPE include 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). 8.3.3.4 Immunological Method Recently, enzyme-linked immunosorbent assays (ELISAs) have been widely used for toxin detection because they are relatively cheap, rapid, and simple in operation. Garthwaite et al. (1998) developed an antibody to detect DA in shellfish and seawater extracts via an indirect competitive enzyme-linked immunosorbent assay (cELISA) with a LOD for DA of 3% (dry weight) DA (Laycock, de Freitas, and Wright, 1989), some strains of reputedly toxigenic species may not always test positive for DA (Lelong et al., 2012). The same is true for certain strains of Nitzschia navis-varingica, the other DA-producing species (e.g., Romero et al., 2012). Thus, investigations of the environmental and epigenetic factors that regulate DAproduction are necessary, in addition to classical taxonomy, for characterizing Pseudo-nitzschia and Nitzschia blooms as potentially harmful.

Following the 1987 event, other DA-producing species were documented in connection with: (i) the mortalities of marine birds and mammals; (ii) the contamination of finfish consumed by humans or other marine organisms; (iii) shallow-water benthic shrimp (by toxic Nitzschia navis-varingica), crabs and soft-bodied worms; and (iv) deeper benthic food webs caused by the rapid transport of particulate DA to depths in excess of 800 m (Sekula-Wood et al., 2009). Low, chronic doses of DA have yet-to-be-understood negative impacts on human and marine populations, and therefore DA has broad ramifications not only for human health but also for ecosystem health. A detailed account of which Pseudo-nitzschia species was involved with each toxic episode (Lelong et al., 2012), and maps showing the location of each species and where the harm had occurred (Trainer, Hickey, and Bates, 2008; Trainer et al., 2012) are available elsewhere. Most Pseudo-nitzschia species form chains with overlapping tips (Figure 8.10), which distinguishes them from Nitzschia species that are solitary cells. These chains are of variable length in a natural situation (up to hundreds of cells), but in culture they tend to be only a few cells long, and can become singlecelled when they are depleted of nutrients for growth. Diatoms secrete a cell wall, a frustule, which is made from amorphous silica (hydrated silicon dioxide). The biogenic source of the silica is mostly silicic acid Si(OH)4, the availability of which is critical for successful diatom bloom formation. Indeed, diatoms are regarded as important silicon sinks and

Figure 8.10 Live toxigenic Pseudo-nitzschia species under light microscopy. (a) P. australis chain, side (girdle) view, showing the overlapping see-through frustules revealing the plastid pigments; sampled during a toxic bloom (Cabrillo Beach, CA, USA; 9 March 2011); (b) P. multiseries cultivated strain from eastern Canada, showing a cell in the early stage of division; (c) P. australis under epifluorescence microcopy. The nucleus appears bright green and chlorophyll appears red; (d) P. australis chain, top (valve) view. Panels (c) and (d) are taken from the same source as panel (a). (All images reproduced with kind permission of Karie Holtermann.)

8.5 Molecular Basis of DA Acute and Chronic Poisoning

primary producers of organic carbon in the oceans. Photosynthesis removes volatile carbon, which was originally in the form of carbon dioxide in the atmosphere. When diatoms die, they sink to the oceans’ depths where the carbon remains, in some cases long-term in the form of fossil fuel. Diatoms thus not only mediate a major greenhouse gas but also produce almost half of the world’s oxygen. Diatom frustules are made up of two halves (hence the name “diatom,” which is Greek meaning “cut in two”), that are referred to as valves. The larger valve fits over the rim of the smaller one, somewhat like the lid of a Petri dish, and the two are held together by girdle bands. The general shape of the long Pseudo-nitzschia frustule (whether the sides of the frustule are symmetrical, straight or curved), the cell width (whether >3 mm or 200% for doses of 6–12 mg kg 1) was first detected in 1969, but the minute levels present in the sponge (10 4%) and the complexity of its structure required an intense input from several research teams for

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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N

N O

HOH2C

O NH2

N

HO

O

HOH2C

N

HO

OH

NH2

N N

CKGKGAKCSRLMYDCCTGSCRSGKC

OH

Cytarabine, Ara-C, 2 Cytosar-U® (FDA 1969) Anticancer

Vidarabine, Ara-A, 1 Vira-A® (FDA 1976) Antiviral

ω-conotoxin MVIIA, 3 Ziconotide, Prialt® (FDA 2004) Anticancer O O

O O

Omega-3 acid ethyl esters, 4 Lorvaza® (FDA 2004) Antitriglyceride

OH

HO

H3N NH

MeO O AcO O Me

OMe

HO S H H

O Me S O O

Me

N

O

O O

O

Me

H O

O H O O

O

N O O

OH Eribulin mesylate, 6 Halaven® (FDA 2010, EMEA 2011) Anticancer

Trabectedin, ET-743, 5 Yondelis® (EMEA 2007) Anticancer cAC10

S

O N

O

O O

H N

N H

O

O

O

N

O O

HN O

NH2

Brentuximab vedotin, 7 Adcetris® (FDA 2011) Anticancer OSO3-

OH O

O

OH

O

O O

O

OSO3-

n iota-Carrageenan, 8 Carragelose® (FDA 2013) Antiviral Figure 11.1 Commercially available molecules of marine origin.

N

N

O

N H

H N

O

H N

O O

OH

11.1 Introduction

almost two decades before its identification in 1990 (Wright et al., 1990, Rinehart et al., 1990). The first total synthesis in 1996 gave a disastrous yield (0.75%) that precluded any further industrial development (Corey, Gin, and Kania, 1996). Subsequently, aquaculture (which was subject to random variation) and semi-syntheses were developed to obtain the quantities of this remarkable antitumor agent required to perform clinical trials. Today, ecteinascidin is produced by the Spanish pharmaceutical company PharmaMar via a semi-synthesis of 27 steps from cyanosafracin obtained by the fermentation of Pseudomonas fluorescens (Cuevas et al., 2000). ET-743 has proved to be a DNA alkylating agent with a particularly original activity, acting at the minor groove of DNA with selectivity for guanine residues (Mendola, 2000). In 2007, this antineoplastic alkaloid, with its unique mechanism of action, was approved by the European Commission for the treatment of soft sarcomas, and is marketed as Yondelis1 by Pharmamar in Europe and by Johnson & Johnson/OrthoBiotech in the United States; in 2009, availability was extended to the Japanese market. A structurally related ecteinascidin analog named lurbinectedin (PM01183) is a new synthetic alkaloid produced by Pharmamar and currently in Phase II clinical trials. This compound targets the minor DNA groove, and has shown activity and in vivo synergy with cisplatin, when used in the treatment of orthotopic cisplatin-sensitive and cisplatin-resistant patient-derived preclinical tumor models (Vidal et al., 2012). The recent success of marine natural products is illustrated by the synthetic anticancer compound eribulin mesylate (6), a cyclic polyether linked to a macrolactone. The story began in 1985 with the discovery of norhalichondrin-A in the Japanese sponge Halichondria okadai and analogs, all of which were highly cytotoxic (Uemura et al., 1985). An additional 10 halichondrins and similar molecules were subsequently isolated from other genera of Halichondrida, such as Axinella and Phakellia, and of Poecilosclerida, such as Lissodendoryx and Raspailia (Kornprobst, 2014, vol. 2, Chapter 19). Interest in these molecules derives from their in vitro and in vivo cytotoxicity; their IC50 values range from 10 9 to 10 10 M, and from their T/C ratio, which is approximately 300% for 60 human tumor cell lines. Halichondrins are antitumor agents of the spindle poison type, acting by binding to tubulin, much like the periwinkle (Vinca minor) alkaloids vinblastine and vincristine (Bai et al., 1991). Halichondrin B revealed exceptional antitumor properties at nanomolar dose levels, but although it was found in other types of sponge (e.g., Axinella spp. and Phakellia carteri), its bioavailability was insufficient for preclinical studies. Although the synthesis was proposed in 1992, the 90 steps involved and complexity of the molecule caused the chemical production to be impractical. However, the serendipitous discovery of a new source, a sponge collected in deep waters off New Zealand Lissodendoryx n. sp. by the team of Professors Blunt and Munro, allowed the exploration of harvest trawling in parallel with aquaculture. Thus, the combined efforts of chemists, biologists and ecologists yielded 310 mg halichondrin B, which was sufficient to start preclinical trials.

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Studies on structural modulations have led to many synthetic analogs, including eribulin (E7389 and ER-086526) and eribulin mesylate (HalavenTM; ER-076349, 7). These induce apoptosis by binding to the ends of microtubules, Eribulin mesylate is currently in Phase II clinical trials for lymphoma U937 (Towle et al., 2001; Kuznetsov et al., 2004; Jordan et al., 2005). A review has been provided of all aspects of halichondrins, particularly their production (Hart et al., 2000). Developed by Eisai Co., eribulin mesylate (6) entered Phase III clinical trials in 2009 and received FDA agreement on 15 November 2010, and EMEA agreement on 17 March 2011, for the treatment of metastatic breast cancer (Alday and Correia, 2009; Twelves et al., 2010; Smith, J.A. et al., 2010) under the trade name HalavenTM, and has since been approved by several countries worldwide. The second recent success of marine natural products is the anticancer agent brentuximab vedotin (7), a dolastatin derivative that took advantages of the progress of antibody-based immunotherapy. Dolastatins are a family of linear peptides isolated so far only from one species of mollusk anaspidae Dolabella auricularia, collected in the Indian Ocean and the Pacific Ocean. This mollusk is also associated with only two species of cyanobacteria, Lyngbya majuscula and Symploca hydnoides. Dolastatins revealed high cytotoxicity by inhibition of the polymerization of tubulin into microtubules, with IC50values generally ranging between 50 and 1 nM. Their presence in the mollusk is very low, from 10 6 to 10 7 % of the wet weight of the animal. Almost 30 dolastatins were identified and characterized by the presence of one or more nonribosomal amino acids and/or of thiazole derivatives. Dolastatin10 (9) is the most active derivative of the series (Figure 11.2). It was postulated that dolastatin-10 and its analogs were produced by these three organisms, separated or in combination (Luesch et al., 2002). Dolastatin-10 entered Phase II clinical trials in 2002 for various types of cancer (Vaishampayan et al., 2000; VerdierPinard et al., 2000; Von Mehren et al., 2004), but its development was withdrawn in 2005 for insufficient activity on advanced breast cancer (Perez et al., 2005). Soblidotin (Auristatin PE, TZT-1027; 10), another very promising synthetic pentapeptide analog of dolastatin-10, developed by Aska Pharmaceuticals, was also recently discontinued in Phase III of clinical trials for advanced or metastatic soft tissue sarcoma and locally advanced or metastatic nonsmall-cell lung cancer. Similarly, tasidotin (ILX-651; 11) and synthadotin, two highly cytotoxic linear synthetic depsipeptides (Ray et al., 2007; Bai et al., 2009), analogs of dolastatin-15 (12) (Pettit et al., 1993) in development for various types of cancer by Genzyme Corporation, were discontinued when in Phase II clinical trials. Despite this lack of success, brentuximab vedotin (7), an antibody–drug conjugate that used a derivative of the microtubule inhibitor agent dolastatin-10, was approved in August 2011 for the treatment of Hodgkin and systemic anaplastic large cell lymphoma (Katz et al., 2011). Brentuximab vedotin (7) is a dolastatin-10 derivative monomethylauristatin E, that is

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H Me

H

H

N (S) N H Me O

(S)

N Me

(S)

O

H

H

H

N

(S)

OMe O

OMe O

N H

Dolastatin-10, 9

N

(S)

S

H Me

Me

Me

H N Me

O

N H

H N Me

O

N H

H N Me

O

N H

N Me

O

H

H

H

OMe

H

H

N O Me

O

H

H

N O Me

N

O

N

OMe O

O

H

N

O

N

H O

Soblidotin, 10

N H

H H N

Tasidotin, 11

O

N

H

H

H (S)

O O

OMe

Dolastatin-15, 12

N O O

Figure 11.2 Soblidotin, Tasidotin and Dolastatins-10 and -15.

linked to an antibody that targets CD30, a cell membrane protein present on the surface of Hodgkin’s lymphoma cells, via a valine-citrulline linker. The linker system was designed to be stable in the bloodstream but to release monomethylauristatin upper internalization into CD30-expressing tumor cells, resulting in target cell death. Similarly, two other auristatin-based antibody–drug conjugates, namely SGN-LVI1 and ASG-22ME, targeting LIV-1 and Nectin-4, respectively, that are expressed in several types of solid tumor, are in Phase I clinical trials. Finally, antiviral iota-carrageenan (8) was approved in 2013 by the US FDA. Carrageenans are sulfated galactose polymers, derived from red seaweeds of the genera Eucheuma and Chondrus, and are commonly used in the food industry for their gelling and emulsifying properties. They are also used in the pharmaceutical industry thanks to their “Generally Recognized as Safe” (GRAS) status from the FDA. The recent antiviral properties of the copolymer iota-carrageenan (Eccles et al., 2010), especially against human rhinoviruses (HRVs), recently led to a commercially available nasal spray for the prophylaxis and therapy of respiratory infections, produced by Marinomed Biotechnologie GmbH. Although chemical synthesis remains the preferred route for obtaining naturally occurring substances in sufficient quantities, aquaculture, semi-synthesis, synthesis and the development of structural analogs – the properties of which can be better controlled – represent alternatives that can overcome the failures of total synthesis. Further investigations into the origins

of these molecules, obtained from sessile marine organisms, led to the discovery of microbial communities that have often emerged as being the “real” producers of metabolites of interest. Consequently, new avenues of research have opened in order to overcome problem of only minute quantities of these marine natural products being available. Highly promising investigations with associated microbes suggest that, over the past 10 years, progress in the cultivation of microorganisms has led to considerable developments in the study of marine bacteria and fungi. The aim of this chapter is to introduce some lead compounds with potent pharmacological activities that have been isolated from marine microorganisms, algae and marine invertebrates (see Table 11.1). Attention is also paid to those compounds which have been produced synthetically when large-scale extraction was not feasible, or when synthesis was not economically viable.

11.2 Promising Substances Isolated from Microorganisms 11.2.1 Salinosporamide A

Collected in 1989 from a shallow sediment (1 m) of the Bahamas mangrove (Chub Cay), culture of the strain CNB-392 led to the isolation of salinosporamide A (13), which showed significant

11.2 Promising Substances Isolated from Microorganisms

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Table 11.1 Marine molecules in development as promising drugs.

Compound Salinosporamide A (Marizomib) Griffithsin Plitidepsin (Aplidin1) Jorumycin (Zalypsis1, PM00104) Bryostatin 1 PM-060184 Lurbinectedin PM01183 DMXBA (GTS-21) SGN-LIV1A ASG-22 M

Origin

Therapeutic target

Current status

Microorganisms Actinomycete Salinispora tropica Algae Red alga Griffithsia sp. Invertebrates Ascidian Aplidium albicans

Nereus Pharmaceuticals

Cancer

Phase I

Microbicide Trials Network PharmaMar

HIV-1 viruses

Phase I

Cancer

Phase II/III

Mollusk Jorunna funebris

PharmaMar

Cancer

Phase II

Bryozoan Bugula neritina Sponge Lithoplocamia lithistoides Synthetic, bioinspired Analog of ecteinascidin Ascidian Ecteinascidia turbinata Analog of anabaseine Worm Paranemertes peregrina Analog of dolastatin-10 Ascidian Dolabella auricularia Analog of dolastatin-10 Ascidian Dolabella auricularia

National Cancer Institute PharmaMar

Cancer Cancer

Phase I/II Phase I

PharmaMar

Cancer

Phase II

Taiho Pharmaceuticals

Schizophrenia

Phase II

Seattle Genetics

Cancer

Phase I

Seattle Genetics

Cancer

Phase I

in vitro cytotoxicity against the HCT-116 cell line (colon cancer) with an IC50-value of 11 ng ml 1, as well as against the panel of 60 cell lines from the National Cancer Institute (NCI) with a GI50 value1) less than 10 nM (Feling et al., 2003). The strain CNB-392 was identified as Salinispora tropica, an actinomycete of the family Micromonosporaceae of the former genus Salinospora. Developed by the Nereus Pharmaceuticals company, salinosporamide A (Marizomib; NPI-0052) is a potent inhibitor of the 20 S proteasome, currently in Phase I of clinical trials for various types of multiple myeloma (Fenical et al., 2009). Furthermore, salinosporamide A, previously recognized as an inducer of apoptosis in lymphocytes for patients with chronic lymphocytic leukemia with an activity higher than that of bortezomib (14), was marketed under the tradename Velcade by Millennium Pharmaceuticals Corporation (Ruiz et al., 2006). In 2005, culture of the same strain CNB-392 furnished seven new derivatives (15–21) related to salinosporamide-A, including salinosporamides B (15) and C (16) (Williams et al., 2005). In 2007, culture of a new strain of Salinispora tropica NPS000465 from Cross Harbor, Bahamas, yielded seven additional new derivatives salinosporamides D–J (22–28). It has been shown that, by replacing sea salt with sodium bromide or sodium fluoride in the culture medium, the bromosalinosporamide (29) or fluorosalinosporamide (30) would be produced (Reed et al., 2007). In 2009, a large-scale cultivation of the initial strain NPI0052 led to the isolation of antiprotealide, a hybrid of salinosporamide A, and omuralide (31), a product of the transformation of lactacystin (32) that had been isolated in 1991 from a terrestrial Streptomyces sp., used as a reference inhibitor to the 1) The GI50 is the concentration of drug that causes 50% reduction of the

proliferation of cancer cells.

Company

20 S proteasome subunit (Omura et al., 1991). The production of salinosporamide A varies with the considered strain of S. tropica, ranging from 87 to 277 mg l 1 for strains CNB476 and NPS21184, respectively (Manam et al., 2009). The biosyntheses of salinosporamide A (Liu et al., 2009) and its derivatives, especially of fluorosalinosporamide (30), have been widely investigated (Reddy, Saravanan, and Corey, 2004; Endo and Danishefsky, 2005, Mulholland, Pattenden, and Walters, 2008; Nett et al., 2009; Eust
aquio, O’Hagan, and Moore, 2010, Ling, Potts, and Macherla, 2010, Nguyen et al., 2010). Figure 11.3 shows the above-mentioned molecules, while two reviews on salinosporamide A and the salinosporamides were produced by Lam et al. (2009) and Gulder and Moore (2010), respectively. Several studies of pharmacomodulation have shown that substituents at positions 2 and 5 lead to a considerable modulation of antiproteasome activity. The most significant reported results are listed in Table 11.2 (Feling et al., 2003; Nett et al., 2009; Gulder and Moore, 2010). The total synthesis of (–)-salinosporamide A was achieved in 14 steps, with 19% overall yield, from 4-pentenoic acid (Satoh, Yokoshima, and Fukuyama, 2011). 11.2.2 Thiocoraline

The cyclic thiodepsipeptide thiocoraline (33) was isolated from the marine bacterium Micromonospora marina-13-L-092 ACM2, obtained from a soft coral harvested off the coast of Mozambique in the Indian Ocean (Perez Baz et al., 1997). Thiocoraline revealed a significant antibacterial activity against the Grampositive bacteria Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus, with minimum inhibitory concentration

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O H

OH 5 O

H N O

H

H

O N

2

H N

N H

O

OH B OH

H

H N O

H O

H

CH2Cl Salinosporamide A, 13

H

H H N

O

O OH CO2Me OH

H N

H H

H N

O OH CO2Me

O

17

OH H

Cl

19

H

20

21

H

H H N

O

OH O

O

OH

O

O

H H N

O

H N

H H N

OH

Cl Salinosporamide I, 27

H

OH O

O

O

O

H N O

O

O

OH O

Salinosporamide F, 24, R = CH2CH2Cl Salinosporamide G, 25, R = Me Salinosporamide H, 26, R = Et

Salinosporamide D, 22, R = Me Salinosporamide E, 23, R = n.Pr

H N

O

R

O

O

O OH

Cl

18

R

H H N

O Cl

O

H N H

Cl

H N

OH

Cl Salinosporamide C, 16

Salinosporamide B, 15

Bortezomib, 14

H

N

O

O

N

OH O

OH HO2C O S OH

X

Cl Salinosporamide J, 28

Bromosalinosporamide, X = Br, 29 Fluorosalinosporamide, X = F, 30

Omuralide, 31

Lactacystin, 32

Figure 11.3 Salinosporamide A and related compounds.

Table 11.2 Inhibition of rabbit 20 S proteosomal chymotrypsin-like activity.

R H N O

H

H OH O O

H

R

IC50 (nM)

H

H

1.9 ± 0.2

2.2 ± 0.1

9.3 ± 1.6

27.5 ± 3.5

CH2Cl

H H N O R

H

OH O

R

C2H4OTs

C2H4Cl

C2H4Br

O

IC50 (nM)

2.4 ± 0.4

2.6 ± 0.2

2.6 ± 0.4

C2H4I

C2H4ODs

C2H4OMs

2.8 ± 0.5

3.0 ± 0.5

4.3 ± 0.8

O N H

11.2 Promising Substances Isolated from Microorganisms

Thiocoraline (3 33)

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HQA

QA

OH N,S-Me2Cys O H D-Cys Me N S (R) S (S) O N O NH Me O (R) S N-MeCys Gly O O N Me Me N

N

N-MeCys

O N,S-Me2Cys

Me

S

(R)

S

N Me S

(S)

O

N D-Cys H HO

C48H56N10O12S6 HQA: 3-Hydroxyquinaldic acid

N O

D-Ser

O

L-N-MeVal

O

O

NH Me S L-Ala O Me N O

N O

(S)

N-MeCys

N Me L-Ala

(S)

Me S HN Me L-N-MeVal N O O

OO

(R)

H N

N-MeCys

Gly

HN

N

N

O HQA

OO

N D-Ser H

N N QA

C51H64N12O12S2 QA: Quinoxalic acid

Figure 11.4 Thiocoraline and Echinomycin.

(MIC) values less than 0.03 mg ml 1, but without any activity (MIC > 10 mg ml 1) against Gram-negative bacteria such as Escherichia coli, Klebsiella aeruginosa or Pseudomonas aeruginosa. The cytotoxic activity of thiocoraline was considerable, with IC50-values 0.01 mg ml 1 against the murine leukemia cell line P388, the non-small-cell lung cancer line A549, the colorectal adenocarcinoma HT-29, and the melanoma MEL-28 (Romero et al., 1997). This strong cytotoxic activity is a result of the bis-intercalation character of thiocoraline and its interaction with a polymerase DNA (Erba et al., 1999; Brandon et al., 2004; Negri et al., 2007). Developed by the Spanish company PharmaMar, thiocoraline is currently undergoing preclinical testing as an anticancer agent. Thiocoraline (33) is a dimer of a tetrapeptide linked to a 3hydroxyquinaldic acid (3-HQA). The tetrapeptide is formed of four amino acids Cys-Gly-NMeCys-N, S-Me2Cys, where a disulfide bridge connects both NMe-cysteines of D configuration. Such a dimeric depsipetide is similar to those of echinomycin (34) (quinomycine), which has two serines of D configuration and two NMe valines of L configuration. Used as internal standard for the determination of biological fluids, echinomycin (34), is a potent inhibitor of nucleic acid synthesis; the mechanism of action is likely similar to that of thiocoraline (Sparidans et al., 1999; Yin et al., 2003). Echinomycin was isolated from the bacterium Streptomyces echinatus isolated from soil sediment from Cuenza, Angola (Corbaz et al., 1957). The structural analogies between thiocoraline (33) and echinomycin (34) are summarized in Figure 11.4. Five derivatives related to thiocoraline – 120 -sulfoxythiocoraline (35), 220 -deoxythiocoraline (36), and thiochondrillines A–C (37–39) – were recently isolated along with thiocoraline from the culture of a bacterium of the genus Verrucosispora (strain WMMA107), associated with the Caribbean sponge Chondrilla caribensis f. caribensis (Wyche et al., 2011, Hu et al., 2011). Furthermore, structural analogs azathiocoraline (40), oxathiocoraline (41), and NMe-azathiocoraline (42) have been synthesized (Tulla-Puche et al., 2009). Most of these compounds are

highly cytotoxic, though less so than thiocoraline. Data relating to thiocoraline and analogs are summarized in Figure 11.5 and Table 11.3. Several syntheses of thiocoraline and its analog BE-22179 have been reported, especially by Boger and Ichikawa (2000, 2001), and the biosynthesis of thiocoraline was established in 2006 (Lombo et al., 2006). Recently, strategies for the synthesis of thiocoraline–triostin hybrids have been developed, and this has led to the design of new potent anticancer agents that could act as DNA bisintercalators (Tulla-Puche et al., 2013). 11.2.3 Ammosamides

The ammosamides A and B were isolated from a culture of a bacterium of the genus Streptomyces (strain CNR-698) collected from a deep sediment (1618 m) of Little San Salvador, Bahamas in 20032). A bioguided extraction (HCT-116) allowed the isolation of both compounds, which differed only by the presence of Table 11.3 In vitro cytotoxic activities of thiocoraline and analogs against

A-549 cell line. Compound 33 36 35 39 37/38

EC50 (mM) 9.5  10 0.13 1.26 2.86 > 10

3

Compound

Gl50 (mM)

42 33 41 40

3.39  10 5.57  10 0.31 0.37

3 3

Data from Tulla-Puche et al., 2009 and Wyche et al., 2011. The EC50 is the concentration of the molecule that gives half-maximal response. The GI50 is the concentration of drug that causes 50% reduction of the proliferation of cancer cells.

2) The authors state that the culture does not require seawater (Hughes

et al., 2009a).

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11 Promising Marine Molecules in Pharmacology

j

22

N

OH H 11 N O

O S 1'

N

R12'

O

N

O

NH Me O S O O N Me Me N S O HN OO N Me 1 S S Me N 12 H 22' O R22'

N

O

O

S

Me

N

N

O Me

S

Me

N

O

O

Me

N

S

N

S

Me

S NH

Me

3

O

O

N Me

N

X=S X = NH X=O X = NMe

OH H N O

O

Me 1

Me

N

NH

O

Me 3

O S

S

Me

N

S

X

Thiocoraline (3 33) 40) Azathiocoraline (4 Oxathiocoraline (4 41) 42) N-MeAzathiocoraline (4

OH H N O

NH

O

NH Me O S O O N Me Me N S O HN OO N Me S S Me N H O HO

Thiocoraline (3 33) R12' = SMe, R22' = OH 35) R12' = SMe, R22' = H 12'-Sulfoxythiocoraline (3 36) R12' = SOMe, R22' = OH 22'-Deoxythiocoraline (3

OH H N

OH H N

12'

O

Me 1

Me

O

S

N 3

Me

O

Me

O S

N

O 37) Thiochondrilline A (3 (amide C3 trans)

Me 1

O

Me

O Thiochondrilline B (3 38) (amide C3 cis)

Thiochondrilline C (3 39) (amide C3 cis)

Figure 11.5 Thiocoraline-related compounds.

oxygen in ammosamide A (43) or by a sulfur atom in ammosamide B (44) (Hughes et al., 2009a)3). These two compounds belong to the pyrroloquinoline family, of which several members are known from sponges and ascidians (Kornprobst, 2014). Nevertheless, ammosamide A is the first known example of a pyrrolo[4,3,2-de]quinoline cycle with a thio-c-lactam. In 2012, the bacterium Streptomyces variabilis (strain ANS-020) isolated from a sediment of Sweetings Cay, Bahamas, yielded ammosamide D (46), which may arise from opening of the pyrrole ring of ammosamide B (Pan et al., 2012). An analysis of cytotoxic fractions of the culture of S. variabilis recently led to the isolation of ammosamide E (47), and the incorporation of tryptophan in order to increase the production of ammosamide E. In addition, the introduction of tryptophan in the culture medium allowed the isolation of ammosamide F (48). The addition of a series of aliphatic and aromatic amines in the culture medium indicates that they are incorporated into the biosynthetic pathway, leading to ammosamides G–P (49–52) (Pan et al., 2013). Some of these ammosamides are presented in Figure 11.6. Ammosamides A and B are highly cytotoxic against the HCT116 cancer cell line (colon), with IC50-values of 0.32 mM for both

compounds. The preparation of a fluorescent probe with ammosamide B showed that the ammosamides act on the cytoskeleton by interaction with myosin and microtubule depolymerization (Hughes et al., 2009b; Fenical et al., 2013)4). Ammosamide D (46) is only moderately cytotoxic against the pancreatic cancer cell line MIA PaCa-2, with an IC50-value of 3.2 mM (Pan et al., 2012). In contrast, most analogs are potent inhibitors of quinone reductase 2, with IC50-values in the nanomolar range (Reddy et al., 2012). Several total syntheses of ammosamides A and B were reported in 2010 (Hughes and Fenical, 2010; Reddy, Banerjee, and Cushman, 2010; Wu et al., 2010; Zurwerra, Wullschleger, and Altmann, 2010), and patented in 2011. It is likely that the ammosamides will quickly enter the preclinical phases of their development.

3) Ammosamide C (45) was also isolated from the culture of CNR-698

4) The probe was prepared from the ammosamide B, which is more

strain, but little information is available on this compound.

11.2.4 Largazole

Largazole (53) is a cyclic depsipeptide isolated from a cyanobacterium of the genus Symploca, harvested in Key Largo, Florida, in 2008. This compound exhibited a highly proliferative

stable than ammosamide A.

11.2 Promising Substances Isolated from Microorganisms CONH2

CONH2

CONH2

N

N H2 N

S

Cl NH2

N Me

NH2

N Me

N Me

NH2

O Ammosamide D, 46

Ammosamide C, 45

CONH2 N

N H2 N

NH

Cl NH2

N Me

H2N

CO2H

N N Me

Cl NH2

Ammosamide E, 47

NH2

Cl NH2

N Me

N H2 N

N N Me

Cl NH2

Ammosamide L, 50

Cl

CONH2

N H2 N

N

N Me

Ammosamide G, 49

CONH2

N

N

Cl

Ammosamide F, 48

CONH2

H2 N

HN Me O

Cl

CONH2

N H2 N

O

H2N

Cl

Ammosamide B, 44

CONH2

N

H2 N

O

Cl

Ammosamide A, 43

CONH2

N

H2N

j 253

N

Cl NH2

Ammosamide M, 51

N Me

NO2 N H

N

Ammosamide P, 52

Figure 11.6 Ammosamide A and related compounds.

inhibition of histone deacetylase class (HDAC) 1 (Taori, Paul, and Luesch, 2008; Seiser and Cramer, 2009; Cole et al., 2011). These zinc-dependent enzymes remove the acetyl groups of e-N-acetyllysines, promoting interaction between histones and DNA. The regulation of transcription genes is controlled by the acetylation and deacetylation of histones; histone deacetylase inhibitors increase the interaction between histones and DNA, O Val

7

NH

O

S

N

O

O

S

O

N

S

N H

17

O

O HN

S

N

O

HS H N

Reduction

O N H

O HN

HS NH

O

S

N H

NH O

O

Largazole thiol, 54

N H S

O

HS

O H N

N

Hydrolysis

Largazole, 53

O

S NH

(S)

O

and inhibit the transcription genes of cytostatic agents that block tumor cell proliferation in vitro and induce apoptosis in vivo (Bowers et al., 2008; Bhansali et al., 2011, Cole et al., 2011). The hydrolysis of largazole produces largazole-thiol (54), which chelates with the zinc of class I HDACs; this mode of action is similar to that of FK228 (55) which, by reduction, led to a dithiol (Figure 11.7). Many structural analogs of largazole have

O

FK228, 55 Figure 11.7 Largazole, FK228, and other HDAC inhibitors.

O

O

reduced FK228, 56

O

254

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11 Promising Marine Molecules in Pharmacology

been synthesized and tested against HDACs 1, 2, 3, and 6, and also against different cell lines of human cancers (Seiser, Kamena, and Cramer, 2008; Bowers et al., 2009a; Chen et al., 2009; Souto et al., 2010; Hong and Luesch, 2012). Although largazole has not yet entered clinical trials, it is considered to be a very promising treatment for colon cancer (Liu et al., 2010). Following its discovery in 2008, several total syntheses of largazole have been described, and studies of structure–activity relationships with structural analogs have established the importance of a methyl group at position 7 of the macrocycle, as well as the terminal aliphatic chain (Ghosh and Kulkarni, 2008; Ying et al., 2008, Bowers et al., 2009b; Xiao et al., 2010; Zeng et al., 2010; Ungermannova et al., 2012).

11.3 Promising Substances Isolated from Macroalgae and Invertebrates 11.3.1 Griffithsin

The aqueous extract of a New Zealand species of the genus Griffithsia (Ceramiaceae) collected at 10 m depth from two different sites (Chatham Island and Fiordland) in 2003 led to the isolation of griffithsin (GRFT, 57), a lectin that binds in a monosaccharide-dependent manner to the glycoprotein envelopes gp41, gp120, gp160 of the HIV-1 virus (Mori et al., 2005; Moulaei et al., 2010). This protein, of molecular weight 12 770 Da, contains 121 amino acids and is specific for mannose and glucose. The amino acid at position 31 has not yet been identified, but its replacement by alanine in recombinant protein 58 does not affect the antiviral activity (Giomarelli et al., 2006), and griffithsin does not contain any cysteine (Figure 11.8). The considerable anti-HIV activity of griffithsin, with an EC50value of 43 pM, originates from the presence of an unusual domain-swapped dimeric structure with three mannosebinding sites on each monomer (mGRFT), as established by

1

31

X-ray analysis. This latter approach has shown that griffithsin “ . . . can be described as a domain-swapped dimer, although it is not clear whether a corresponding monomeric form does (or even could) exist” (Zi
olkowska et al., 2006, 2007; Zeitlin, Pauly, and Whaley, 2009). The potent anti-HIV activity of griffithsin, in the picomolar range, led to its production by extraction from Nicotiana benthamiana transduced with a tobacco mosaic virus vector expressing GFRT. Thus, 60 g of pure recombinant griffithsin could be produced in a 500 m2 greenhouse (O’Keefe et al., 2009). This protein, both recombinant and native, is currently being tested as a vaginal microbiocide in order to prevent infection through sexual transmission (Emau et al., 2007). Its potent antiviral activity against hepatitis C, HIV-1 and coronaviruses, such as the virus that causes SARS (severe acute respiratory syndrome) and, more recently, against herpes simplex virus type 2 (HSV-2), has also been explored (O’Keefe et al., 2010; Meuleman et al., 2011, Alexandre et al., 2012; F
erir et al., 2012a; F
erir, Palmer, and Schols, 2012b, Nixon et al., 2013). Recently patented, griffithsin is currently in preclinical phase for its antiviral activity in partnership with the NCI. (Many details of the antiviral activity of griffithsin are provided in the thesis of Christopher L. Barton, at the University of Louisville, 2011; also available online at: http://digital.library.louisville.edu/utils/getfile/collection/etd/id/2179/filename/4976.pdf ). 11.3.2 PM-050489 and PM-060184; Two New Sponge Polyketides

PM-050489 (59) and its dechlorinated analog PM-060184 (60) represent a new type of marine polyketide isolated from the Madagascan sponge Lithoplocamia lithistoides (Figure 11.9). Both compounds exhibited antimitotic activities in human tumor cell lines at subnanomolar concentrations, and showed potent antimitotic activity by a distinct biochemical mechanism of interaction with tubulin (Pera et al., 2013). Their total syntheses allow their production on a multigram scale, thus overcoming the

40

SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDXIIIDGVHHG 80

GSGGNLSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGR

121

RFGPYGGSGGSANTLSNVKVIQINGSAGDYLDSLDIYYEQY Native griffithsin, 57 (the amino acid 31 is still unknown)

1

SLTHRKFGGSGGSPFSGLSSIAVRSGSYLDAIIIDGVHHGGSGGN LSPTFTFGSGEYISNMTIRSGDYIDNISFETNMGRRFG PY GGSGGSANTLSNVKVIQINGSAGDYLDSLDIYYEQY Recombinant griffithsin, 58 (with an alanine at position 31); in bold the carbohydrate binding domains.

Figure 11.8 Native and recombinant griffithsin.

11.4 Promising Substances Synthesized from Natural Models MeO O

NH2 O

O

O N H

O

O HN X

X = Cl, PM-050489 59 X = H, PM-060184 60 Figure 11.9 PM-050489 and PM-060184 isolated from Lithoplocamia lithistoides.

supply issue that often limits the development of marine natural products (Martin et al., 2013). PM-060184 has entered Phase I clinical trials in France, Spain, and the United States of America. 11.3.3 Immucothel1 (Keyhole Limpet Hemocyanin; KLH)

Along the coasts of California and Mexico lives a large archaeogastropod,5)Megathura crenulata, the giant patella or giant keyhole limpet. The hemocyanin of this mollusk (keyhole limpet hemocyanin; KLH) has been known since 1970 as an immunostimulant and as a vector for the transport of small molecules (e.g., lipids, peptides, hormones and oligonucleotides). KLH also displayed anticancer activity by immunostimulation. Easily extractable from the living organism without killing it, this protein (immunocyanin) is marketed under the name Immucothel1 by the German pharmaceutical company Biosyn Arzneimittel.6) Hemocyanin levels in the hemolymph are not correlated with the mass of the animal and average 5.45 g l 1, but with significant seasonal variation (Senozan and Briggs, 1989). KLH is used as a tool in immunobiology and has entered Phase III of clinical trials for the treatment of superficial bladder cancer (JurincicWinkler et al., 1995, 1996; Lamm, 2003). KLH also reduces significantly the growth in vitro of several human cancer cell lines: breast (ZR75-1), pancreas (PANC-l and MIA-PaCa), prostate (DU145) and esophagus (SEG-1 and BIC-l) (Riggs et al., 2002; McFadden et al., 2003). A general review of the biomedical applications of KLH, containing almost 180 references, was produced by Harris and Markl, 1999. KLH contains a unique oligosaccharide pattern with (Gal-b16-Man)-motifs (Kurokawa et al., 2002). This respiratory pigment contains copper in the cuprous state; the reversible fixation of molecular oxygen produces absorbance at 340 nm, which is responsible for the blue color of hemocyanin. The Cu/protein ratio is of the order of 2  10 3. The protein is in fact a mixture of two isoforms, KLH1 and KLH2, which are separable by preparative electrophoresis. Each of these isoforms is formed from 10 units of approximately 400 kDa, eight of them being functional units and containing two copper atoms (Swerdlow et al., 1996; Gebauer, Harris, and Markl, 2002). The production 5) On average a dozen centimetres. 6) The average price is around D 100 for 25 mg (ammonium sulfate).

j 255

of KLH by raising shellfish has shown that the percentages of the isoforms depend on the diet (Oakes et al., 2004). Despite the interest shown by the KLH, this latter is not a synthesizable molecule, and is produced via shellfish farming in California and France (Beninger et al., 2001). This type of production is similar to that of “Limulus Lysate Amoebocyte” (LAL; Pyrochrome 1), which is used as a biological tool for the detection of endotoxin, and is prepared from blood of the ancestral arthropod Limulus polyphemus, better known as the Horseshoe crab (Walls, Berkson, and Smith, 2002). 11.3.4 Jorumycin (Zalypsis1)

Jorumycin (61) is a tetrahydroisoquinoline alkaloid which has some structural analogy with renieramycin E (62; isolated from a sponge of the genus Reniera), saframycin C (63; bacterial origin), and with ecteinascidin 743 (ET-743; 5), a powerful antitumor agent isolated from the colonial ascidian Ecteinascidia turbinata (Figure 11.10). Isolated from the nudibranch Jorunna funebris harvested in India, jorumycin permeates the mantle and mucus of the animal and is probably a defense allomone (Fontana et al., 2000). Although a less powerful antitumor agent than ecteinascidin 743, jorumycin has an IC50-value of 12.5 ng ml 1 for human cancer lines A549 (lung), HT-29 (colon), and MEL-28 (melanoma). Developed by Pharmamar, jorumycin was patented in 2001.

11.4 Promising Substances Synthesized from Natural Models 11.4.1 Plitidepsin from the Ascidian Aplidium albicans

Plitidepsin (64) is a cyclic depsipeptide identified from the Mediterranean marine ascidian Aplidium albicans. This compound is a synthetic derivative of didemnin B (65), discovered in 1981 (Rinehart et al., 1981) from the ascidian Trididemnum solidum. Didemnin B was the first marine natural product that entered in clinical trials, but was withdrawn in 1995 (Figure 11.11). Plitidepsin (64) showed similar cytotoxic activity in the nanomolar range as didemnin B, but without its side effects. Plitidepsin (Aplidin1) is a PharmaMar’s compound and is currently in Phase II clinical trials for solid and hematological malignant neoplasias such as T-cell lymphoma, and in Phase III clinical trials for multiple myeloma. Plitidepsin was approved in 2003 in the EU as an orphan drug for the treatment of acute lymphoblastic leukemia. 11.4.2 Roscovitine (Seliciclib, CYC202): A Synthetic Analog of Natural Purines

The study of the cell cycle regulation of starfish oocytes led to the development of inhibitors of cyclin-dependent kinases (CDKs)

256

11 Promising Marine Molecules in Pharmacology

j

OMe O

H

O

N

MeO

OH

O

O H

O

H O

OH

Jorumycin 61

NH O

O

O

O Me

N

MeO

H NH

O H N

N O

H

O Me

N MeO

H O

H

O Me

N

OMe

OMe

O H

Renieramycin E 62

OH O

Saframycin C 63

Figure 11.10 Jorumycin and natural analogs.

O

N

O

NH

O

O

O O

Me O

O

O H N

N

O N H

MeO

OH

O N

NH

O

N H

MeO

OH

NMe O

NMe O O

N

O

O

O N

O

Me O

O O

Dehydrodidemnin B, 64 Plitidepsin, Aplidin

OH

O H N

N

O

O

Didemnin B, 65

Figure 11.11 Structures of Plitidepsin and Didemnin B.

(Meijer and Pondaven, 1988; Meijer et al., 1989), which belong to the group of 2,6,9-trisubstituted purines (66, 67). (R)-Roscovitine (68), a powerful inhibitor of CDK1, CDK2 and CDK5 kinases, revealed remarkably antimitotic effects on a wide variety of culture tumor cells, leading to cell cycle arrest, and forcing cells into apoptotic cell death (Azevedo et al., 1997; 2002; Fisher et al., 2003; Hahntow et al., 2004; Meijer et al., 1997, 2006; Taylor et al., 2004, Rossi et al., 2006). Chemical synthesis of (R)-roscovitine was obtained by a simple three-step procedure, starting from commercially 2,6-dichloropurine (Havlícek et al., 1997; Wang et al., 2001) as presented in a detailed review of roscovitine (Meijer and Raymond, 2003). Today, (R)-roscovitine (Seliciclib, CYC202; 68), is developed by Cyclacel Pharmaceuticals (Phase II trials) for the treatment of lung and nasopharyngeal cancers. (R)-Roscovitine also showed potent long-term effects in a murine model of polycystic kidney syndrome (Bukanov et al., 2006), a genetic disease that affects 12.5 million people worldwide and has so far no effective available treatment. The efficacy of (R)-roscovitine to treat this disease has been recently established, showing effective inhibition cell cycle, proliferation and apoptosis (Bukanov et al., 2012). Currently,

(R)-roscovitine is in Phase I clinical trials for the treatment of glomerulonephritis. (R)-Roscovitine also showed protective effects against neuronal cell death. Furthermore, the leucettines that possess a 2aminoimidazole-4-one scaffold, such as leucettamine B (69) isolated from the marine sponge Leucetta microraphis (Kong and Faulkner, 1993), are kinase inhibitors, that could be drug candidates to treat Alzheimer’s disease and also to reduce Down’s syndrome (trisomy 21). In addition, a new generation of molecules related to roscovitine, the aftins (e.g., 70), that have the ability to increase the production of amyloid (Ab)42 peptide, could become a new molecular tool for studying one hallmark of Alzheimer’s disease (Hochard et al., 2013). Patented by the Company ManRos Therapeutics, these compounds are studied for their potent prevention in the Alzheimer’s disease (see Figure 11.12). 11.4.3 DMXBA (GTS-21): A Synthetic Analog of Anabaseine

Anabaseine (71) is a nicotinic alkaloid produced and used by the carnivorous nemertean worms Paranemertes peregrina in order to

11.4 Promising Substances Synthesized from Natural Models

NH

NH N

N

N H

N

HN

NH N

N N

N Me

HN

N

OH

Olomucine, 67 (7 µM)

(R)-Roscovitine, 68 (0.45 µM)

Me O

N N

N

N

N

(R)

O Me N

N

N

OH Isopentenyl adenine, 66 (55 µM)

j 257

O HN

H2N

N

N

(R)

OH An Aftin, 70

Leucettamine B, 69

(values into brackets represent IC50 for CDK1/cyclin B)

Figure 11.12 Roscovitine and some other purines inhibiting cyclin-dependent kinases.

paralyze their prey (Kem, 1971; Kem, Scott, and Duncan, 1976). Studies on structural modulations led to the synthesis of 3-(2,4dimethoxybenzylidene)-anabaseine (DMXBA, GTS-21; 72) which is an agonist of 7a-acetylcholine nicotinic receptors and is less toxic than nicotine (73; Figure 11.13). Developed by Taiho Pharmaceutical Company, DMXBA is in stages I/II of clinical trials for the treatment of Alzheimer’s disease (Meyer et al., 1997, 1998,

Azuma et al., 1999a, 1999b; Kem, 1997, 2000, Machu et al., 2001; Uteshev, Meyer, and Papke, 2003; Kem et al., 2006; Stokes et al., 2004; Bourguet-Kondracki and Kornprobst, 2005; Chen et al., 2010; Zawieja, Kornprobst, and M
etais, 2012). Three hydroxylated derivatives of DMXBA (74–76) revealed similar properties of DMXBA, and are being studied for the treatment of neurological diseases (Kem et al., 2004) (Figure 11.13).

OMe

MeO H N N

N N

Anabaseine, 71

DMXBA (GTS-21), 72

N

Me

Nicotine, 73

R2

R1 H N

R1 = OH, R2 = OMe, 2-Hydroxybenzylidene anabaseine (2-OH-MBA), 74 R1 = OMe, R2 = OH, 4-Hydroxybenzylidene anabaseine (4-OH-MBA), 75 R1 = R2 = OH, 2,4-Dihydroxybenzylidene anabaseine (2,4-DiOH-BA), 76

N Figure 11.13 Anabaseine, DMXBA, and related compounds.

258

11 Promising Marine Molecules in Pharmacology

j

percentage obtained varying from 10 6 to 10 4 % of the fresh weight of the colony. This leads to considerable problems in obtaining sufficient quantities for structural analysis and in-vivo testing. Bryostatin 1 has several types of activity encompassing immunostimulation, inhibition of cell growth, induction of cell differentiation, and synergy with other substances to increase their cytotoxic activity against certain tumors. Bryostatin 1 acts synergistically with Taxol and Ara-C with respect to effectiveness against human leukemia U937 and human myelocytic leukemia HL-60 cells, doubling in both cases the number of cells in apoptosis (Mutter and Wills, 2000). Although its mode of action is not yet known, bryostatin 1 has properties that appear to result from interaction with protein kinase C and its isoenzymes, stimulating the activity of the latter. Studies of structure–activity relationships have shown that the interaction sites of bryostatin 1 with PKC include carbons 1 and 19, and oxygen at position 26. These results led to the preparation of bryostatin 1 analogs called bryologues, in which the environment of the three sites mentioned above is the same, with the possibility of modulating the substituent at C-7 on ring A, as illustrated below (Wender et al., 1998a,b,c, 1999; Wender and Hinkle, 2000; Wender and Lippa, 2000). These analogues

11.4.4 Bryologues: Synthetic Analogs of Bryostatins

Discovered in 1982 in the species Bugula neritina, bryostatins are cyclic polyethers with a unique carbon skeleton that display exceptional antitumor activity (Pettit et al., 1982). Bryostatin-1 (77), currently the most active compound of the series, is in Phase II clinical trials for various types of cancer (Hale et al., 2002; Clamp et al., 2002; Madhusudan et al., 2003). The 20 bryostatins currently known all have the same 26-member macrolactone skeleton of bryopyran. Variation occurs at carbon 7 on the A ring, and at carbons 19 and 20 on the C ring; a hydroxyl group at position 19 is present in most bryostatins. The species containing bryostatins were found near the bryozoan Bugula neritina, which suggests that bryostatins are produced by associated microorganisms and then transferred to the zoarium of B. neritina. These associated microorganisms, which cannot yet be cultured, are regarded as c-proteobacteria and are generally referred to as Candidatus Endobugula neritina and Candidatus Endobugula sertula (Haygood and Davidson, 1998; Davidson and Haygood, 1999; Davidson et al., 2001; Hale et al., 2002; Trindade-Silva et al., 2010). The quantities of bryostatins that can be isolated are extremely small, the average HO MeO2C

B

O

H

H

O

O

7

A

O

O

OH HO O

O

C

H CO2Me

H

C7H15

O

H

C7H15

26

H

26

O R

OH O

CO2Me

Bryostatin-1 analogues, 83-85 R = Me, C13H27, Ph Wender & Hinkle, 2000

H HO

H

O

O

O OH OH O

1

O 26

OAc

CO2Me

Bryostatin-1 analogues, 86-87 (R1 = H R2 = H, OBz ) Wender & Lippa, 2000 Figure 11.14 Bryostatin-1 and some bryologues.

R

OH OH O

CO2Me

n.Pr

Merle 21 (R = C6H5), 88 Merle 22 (R = C7H15), 89 Merle 23 (R = (CH=CH)2C3H7), 90 Keck et al., 2009a, b

O O OH

O

OH O

O

O O

O

R2 O

O

7

O

19

1

19

Bryostatin-1 analogues, 79-82 (R1 = H, OH, R2 = OH, Ac) Wender et al., 1998a, b, c

O

O

O

OH O

CO2Me

R1

OH HO O

OH

R2

H 7

O

O

O

O

OH

26

Bryostatin-1, 77

O

O

1

19

O

O

H

O

OH O

1

O 20 n.Pr

O R1

O

19

H

OAc

O

CO2Me

Merle 28, 91 Keck et al., 2009a,b

References

79-91 interact strongly with PKC and inhibit the growth of some human cancer cell lines. It has also been shown on synthetic analogs that “. . . the C30 carbomethoxy group is not essential to obtain bryostatin-like biological responses and the structure in the C7 -C9 region of the A-ring is critical in conferring bryostatin-like biological responses” (Keck et al., 2009a,b). The structures of bryostatin-1 and some bryologues are presented in Figure 11.14.

11.5 Conclusion

These selected marine promising molecules illustrate the optimism of scientists in the search for new marine drugs. Indeed, the marine field continues more than ever to provide exciting molecules with varied potencies and tremendous biological diversities. In addition to the exploration of macroalgae and marine invertebrates such as sponges, mollusks and tunicates,

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investigations of the marine microorganisms associated with these organisms have enabled studies of the oceanic microbial diversity as a new drug discovery. Most of these hopeful marine molecules have displayed new strategies to treat diverse cancers, with new mechanisms of action as illustrated by the inhibitor of the 20 S proteasome, salinosporamide A, the inhibitor histone of deacetylase, largazole, or the inhibitor of cyclin-dependent kinase, roscovitine. The identification of new targets further supports the promising treatments to fight asthma, HIV, neurological conditions and Alzheimer’s disease or melanoma. If the supply of these unique biologically active chemicals remains the thorny issue of marine natural products, some solutions may now be provided. A synergistic combination of advances in sampling techniques, structural determination and target identification, as well as chemical syntheses, the fermentation of culturable microorganisms, molecular biology tools and genomic studies are relevant solutions to solve this major challenge. These innovative methodologies and advanced techniques will determine the future success of marine natural products.

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About the Authors Marie-Lise Bourguet-Kondracki is a Director of Research at the CNRS in France. She spent most of her career at the Mus
eum National d’Histoire Naturelle in Paris, focusing her research activity on the isolation, structure elucidation and structure– activity relationships of bioactive natural products from marine sponges. She now leads a research group involved in pluridisciplinary studies combining microscopic, cultural, molecular biology and chemical approaches in order to understand the role of the associated bacteria within the complex ecosystem bacteria-sponges. She was the chairperson of the 4th European Conference on Marine Natural Products in 2005 in Paris. Jean-Michel Kornprobst is emeritus professor at the University of Nantes, France. He is an engineer in chemistry (Montpellier) and received his PhD at the University of Lyon in 1969. Assistant-professor at the University Paris 7 from 1970 to

1973, he became professor of organic chemistry at the University of Dakar, Senegal and worked on marine natural products from 1974 to 1990, and created the scuba diving school Oceanium de Dakar in 1984. He was the organizer of the 6th International Symposium on Marine Natural Products held in Dakar, Senegal, in July 1989. He joined the University of Nantes in 1990 and became emeritus professor in 2003. He was responsible for two research programs on manapros at the University of Doha, Qatar, and Jeddah, Saudi Arabia. He has authored more than 100 publications and three books. He has recently been an invited professor at the Universities of Louvain-la-Neuve, Belgium, Campinas, Brazil, and Blida, Algeria and is currently an external member of the scientific advisory board of the Marine Biotechnology Research Center (MRBC) of Rimouski, Qu
ebec, Canada.

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12 Promises of the Unprecedented Aminosterol Squalamine Marie-Lise Bourguet-Kondracki and Jean-Michel Brunel

Abstract

Squalamine, a water-soluble cationic steroid that was originally isolated during the 1990s from the dogfish Squalus acanthias, has begun to attract attention due to its significant antimicrobial activities against fungi, Gram-positive and Gram-negative bacteria. Additionally, this unusual sulfated

12.1 Introduction

Life goes through transformations resulting from adaptive processes in order to perpetuate itself in an ever-changing environment, and in the face of biotic pressures. Genomes include all the information that is essential for self-subsistence and the reproduction of each living entity. Dynamic interactions, whether positive (functional) or negative (defense, competition), are however necessary for the overall fitness of each individual, both with microbiota and macrobiota. Throughout historical times, humans have been able to make sensible use of what the environment could offer to improve their livelihood and health. Infections have always been a threat to all creatures, and scientists have long been intrigued by the ways in which living things defend themselves against invasion from the thousands of different microbes in the environment. In recent years, the pace of investigation has quickened, with attention focused on discovering bioactive substance from plants, insects, animals and, especially, from marine organisms. Indeed, the oceans – which comprise 70.8% of the Earth’s surface – represent an important pipeline for the discovery of new drugs that has been so far little studied. Such exploration awaited technological refinements, and began in 1943 with the invention of scuba diving by the French engineer Emile Gagnan, in collaboration with the Commandant Jacques-Yves Cousteau. Today, more than 275 000 marine animals have been identified, and this exceptional marine biodiversity has afforded an incredible chemodiversity with more than 24 000 marine natural molecules. Some of these have displayed significant pharmacological activities in the anticancer, antibiotic, anti-inflammatory, immunomodulatory and analgesia fields. The first marine chemical studies appeared in 1950, with the discovery of analogs of nucleosides called spongosides by

aminosterol could be a very promising leader of a new class of drugs in the antiangiogenic, anticancer and antiviral fields. In this chapter, a current overview is provided of all the potentialities of this remarkable aminosteroid and its mimics in human disease treatment.

Bergman and coworkers, where the classical sugar ribose was replaced by an arabinose. These studies were rapidly followed by the discovery of large amounts of prostaglandins that industrial companies had labored to synthesize. The subsequent explosion of marine natural products during the late 1970s evolved into three areas: the search of new biologically active compounds; the study of toxins; and, more recently, chemical ecology. Some marine invertebrates such as sponges, corals and ascidians, which are filter-feeders with a specific mode of life as they live fixed on a substrate, have been particularly well studied. Other marine animals have also provided a special interest due to their potential in numerous therapeutic fields, as illustrated by the discovery of squalamine. This was later described in 1993, following its isolation from the tissues of a deep-water small omnivorous shark that grew to 0.8–1.3 m in length (Ebert et al., 2010) (Figure 12.1) and was well-known for being sold with other spiny dogfishes in the fish and chip shops of England. Squalamine was reported as a powerful killer of a variety of bacteria, fungi, viruses and parasites (Moore et al., 1993). Whilst it was too soon to appreciate exactly how squalamine could be used, it was rapidly speculated that it might serve as a powerful weapon in the arsenal of antibiotics, not only because it was new but also because it might be effective against infectious diseases, as the causative microbes became more resistant to existing drugs.

12.2 Discovery of the Unprecedented Aminosterol Squalamine

The squalamine story began in late July 1989, when Dr M. Zasloff was invited to speak about magainin antibiotic peptides at Mount Desert Island Marine Biological Laboratory

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Figure 12.1 Squalus acanthias. Illustration courtesy of Andy Murch, Elasmodiver.

at Salisbury Cove, where a main focus of research was on the physiology of dogfish sharks. An amazing point was how the shark pups escaped infection despite being constantly exposed to sea water during their two years of gestation when their mothers flushed their Fallopian tubes. Another point was that sharks seldom became infected after surgery; thus, the immune systems of sharks and humans must differ significantly, with the shark system appearing much less plastic and less responsive than the human one, yet the sharks were resistant. Four years later, a new compound (see Figures 12.2–12.4 for mass and 1 H and 13 C NMR spectra of squalamine, respectively) that did not belong to any former known class of chemicals, was isolated using a procedure of purification involving organic extraction, size-exclusion chromatography, and reverse-phase and cation-exchange HPLC. This compound (1) was called

Figure 12.2 Fast atom bombardment mass spectroscopy of squalamine lactate, positive-ion mode.

squalamine, because it has been derived in part from Squalus acanthias, the Latin name of the dogfish shark in which it was first found, and from its spermidine moiety. The structure of 1 was confirmed by two-dimensional NMR and fast atom bombardment mass spectroscopy as a water-soluble cationic aminosterol, characterized by the condensation of an anionic bile salt intermediate with a polyamine spermidine (Moore et al., 1993). In 1995, a stereoselective synthesis of squalamine dessulfate allowed the precise configuration at C-24 of squalamine to be determined as 3b-N-1-{N[3-(4-aminobutyl)]-1,3diaminopropane}-7 a,(24R)-dihydroxy-5a- cholestane, 24-sulfate (Moriarty et al., 1995). Although the liver and gallbladder were the richest sources of squalamine in the shark, it could also be detected in many other tissues. This gave rise to speculation that it might provide the fish with its principal defense against infection, and also explain the shark’s reputation of being particularly resistant to infection. Seven additional structurally related aminosterol compounds (2–8) were mainly isolated from the liver and gallbladder of the shark (Figure 12.5). These all possessed a relatively invariant cholestane skeleton with a trans AB ring junction, a spermidine or spermine attached equatorially at C3, and a steroidal side chain that may be sulfated. Some members of this family of aminosterols exhibited a broad spectrum of antimicrobial activity, similar to that of squalamine. The two most abundant natural amphiphilic steroids, squalamine 1 and trodusquemine 2, are 7-hydroxylated, -24 sulfated cholestanes conjugated to spermidine or spermine at C-3, respectively (Rao et al., 2000). Later, squalamine was also isolated within the circulating white blood cells of the sea lamprey Petromyzon marinus (Yun and Li, 2007). Consequently, various authors postulated that squalamine had an important role in the innate immunity as a defense substance against microbial invasion.

12.2 Discovery of the Unprecedented Aminosterol Squalamine

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Figure 12.3 Proton NMR spectrum of squalamine lactate in D2O.

Trodusquemine 2 (MSI-1436) was also of particular interest due to its capacity to suppress appetite, which resulted in a significant weight loss in mammals (Zasloff et al., 2001), and to its antidiabetic properties in genetically obese mice. Both activities highlighted a great promise in antiobesity and diabetes therapy.

Figure 12.4 Carbon NMR spectrum of squalamine lactate in D2O.

In this chapter, details of squalamine and its synthetic derivatives will be provided, ranging from the discovery of this unique steroid structure to its remarkable and highly potent antimicrobial, antiangiogenic, anticancer, and antiviral activities.

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NH2

H N

OSO3H

NH2

OSO3H

HN

HN N H

N H

OH Squalamine 1

H

NH2

NH2

O

HN OH

H 3

NH2

O

OSO3H

NH2

H 4

O

HN

OH

H 5

OH

NH2

OH

HN N H

OSO3H N H

OH

NH2

O

HN

S N H

OH Trodusquemine 2

H

N H

OH

H 6

OH

OSO3H OH

NH2

OSO3H

HN

HN N H

H 7

OH

N H

H 8

OH

Figure 12.5 Structures of the naturally aminosterols isolated from Squalus acanthias.

12.3 Syntheses of Squalamine

The synthesis of squalamine emerged as a crucial challenge for organic chemists, as it provided the ability to obtain the compound without killing any sharks. The first synthesis to confirm, unambiguously, the proposed structure for squalamine was performed in 1994 by Moriarty et al., starting from 3bacetoxy-5-cholenic acid (9) in a 17-step sequence and a 0.3% overall yield that was epimeric in C-24 (Scheme 12.1) (Moriarty et al., 1994). Thus, introduction of the 7a-hydroxyl group was obtained via an allylic oxidation, followed by a subsequent hydrogenation of the 5-6 double bond by the K-selectride stereoselective reduction of the ketone group. One year later, the same group reported the first totally stereoselective synthesis of natural (24R) squalamine and its non-natural (24S) epimer in a 20-step synthesis from stigmasterol (Moriarty et al., 1995). In this case, the key step was the lateral chain coupling in C-22 using (2R)-16 or (2S)-1,2-epoxy-3methylbutane 17 (Scheme 12.2).

Stigmasterol 13 has been also used in a 15-step stereoselective synthesis (Jones et al., 1998) involving derivative 20 as the key intermediate for the preparation of squalamine 1 (Scheme 12.3) (Rao et al., 1997). In 2000, Kinney et al. reported an 11-step preparation of squalamine 1 from a microbial metabolite 26 available from 3-keto-23,24-bisnorchol-4-en-22-ol (Scheme 12.4), in which the 7a-hydroxylation of the 3-keto-23,24-bisnorchol-4-en-22-ol 25 was realized by the Diplodia gossypina fungus (Kinney et al., 2000). More recently, Zhou et al. described a stereoselective construction of the squalamine side chain by using methyl-3-keto5a-chenodeoxycholanate 33 as a starting material, and an improved Sharpless catalytic asymmetric dihydroxylation as the key step (Scheme 12.5) (Zhou et al., 2001). In this case, squalamine 1 was obtained in 19% overall yield (14 steps) and with a diastereomeric purity of about 100%. In this synthesis, the key step consisted of a totally stereoselective introduction of the 24R, 25-dihydroxy moiety on the lateral chain of derivative 34, with subsequent formation of derivative 39 in 31% yield.

12.3 Syntheses of Squalamine

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COOH OTBDMS

OTBDMS i-vi

AcO

AcO

vii-xi

O

H2N

10

9

OH

NC OTBDMS HN

I

N Ts

xii Squalamine 1

11

OH

xiii-xvii

12 CN

N Ts

Scheme 12.1 Conditions: (i) (COCl)2, CH2Cl2, reflux, 2 h (100%); (ii) (CH3)2CHCdBr, C6H6, rt, 1 h (60%); (iii) Ca(BH4)2, THF, rt, 5 h (80%); (iv) TBDMSCl, Imidazole, CH2Cl2, rt, 16 h (90%); (v) Cr(CO)6, t-BuOOH, CH3CN,reflux, 12 h (46%); (vi) Li, liq. NH3, Et2O, 78  C, 10 min (81%); (vii) K-selectride, THF, 50  C, 5 h (80%); (viii) NaCN, MeOH, reflux, 8 h (88%); (ix) (t-BuO)3Al, Cyclohexanone, Toluene, 110  C, 20 h (59%); (x) C6H5CH2O-NH2.HCl, C5H5N, C2H5OH, reflux, 16 h (97%); (xi) LiAlH4, Et2O, reflux, 16 h (98%); (xii) K2CO3, CH3CN, reflux, 20 h; (xiii) C6H5CH2OCOCl, NaOH, THF, 0  C to rt, 4 h (70%); (xiv) Na, liq. NH3, THF, 78  C to rt, 18 h (91%); (xv) LiAlH4, Et2O, reflux, 6 h (93%); (xvi) HCl, EtOH, rt, 3 h (98%); (xvii) C5H5N.SO3, C5H5N, 75  C, 2 h (10%).

Otherwise, it was found by Okumura et al. (2003) that desmosterol 41, a natural sterol available in wool filtrates, was a substrate of choice for the synthesis of squalamine due to its peculiar lateral chain that permitted the introduction of a sulfate moiety in a few steps via a Sharpless catalytic asymmetric

dihydroxylation; this was followed by a subsequent regioselective elimination of the hydroxyl group in C-25 (Scheme 12.6). In 2005, Zhang et al. reported a new efficient and stereoselective synthesis of squalamine 1 from methylchenodeoxycholanate 46 (Scheme 12.7) in nine steps, with a 14% overall

CHO

iv-vii

i-iii

HO

SO2Ph

15 OMe

14 OMe 13

OAc OH

xi-xv

H

AcO

HO

18a

16 O

(24R)-1

OAc

19a

OAc

OH

or xvi-xix

(24S)- epi 1

viii-x

H O

xi-xv

17

HO

AcO

OAc

19b 18b

Scheme 12.2 Conditions: (i) TsCl, Pyridine, 25  C, 14 h; (ii) MeOH, KOAc, reflux, 4 h; (iii) O3, MeOH, 78  C; (iv) NaBH4, MeOH, 25  C; (v) CH3SO2Cl, Et3N, CH2Cl2, 0  C, 2 h; (vi) NaI, Me2CO, reflux, 17 h; (vii) PhSO2Na, DMF, 25  C, 32 h; (viii) n-BuLi, Epoxide 16 or 17, 78  C, 2 h; (ix) Li, NH3, 78  C, 30 min; (x) TsOH, Dioxane-H2O (7:3), 80  C, 1 h; (xi) Ac2O, Pyridine, 25  C, 14 h; (xii) CrO3, DMP, CH2Cl2, 20  C, 24 h; (xiii) Li, NH3, 78  C, 10 min; (xiv) KB[CH(CH3)C2H5]3H, THF, 50  C, 6 h; (xv) Ac2O, DMAP, CH2Cl2, 25  C, 14 h; (xvi) NaCN, MeOH, 25  C, 48 h; (xvii) CrO3, H2SO4, H2O; (xviii) tBocNH (CH2)4N(tBoc)(CH2)3NH2, NaBH3CN, 25  C, 14 h; (xix) HCl, MeOH, 14 h.

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12 Promises of the Unprecedented Aminosterol Squalamine

j

OH

10 steps

iii

i, ii

O O

HO

OSO3H

13

O

20

O

O

O

21 O

Ph

Ph

OSO3H

OSO3H

v

iv +

O

Squalamine 1

NH2

N H

CN

HN

OH

24

OH

23

22

CN

N H

Scheme 12.3 Conditions: (i) Amberlist 15, Acetone, 20  C; (ii) SO3.Py, Pyridine, 80  C; (iii) KOH, MeOH, reflux; (iv) Trimethylorthoformiate, NaBH4, MeOH, 78  C; (v) PtO2, EtOH, H2 (40 psi).

yield (Zhang et al., 2005). Derivative 46 was first selectively oxidized, leading to the formation of 7a-hydroxy-3-one 47, followed by protection of the remaining hydroxyl group to afford 7a-methoxymethylether 48 in 91% yield. A subsequent dehydrogenation and reduction (in the presence of Li/NH3) of the generated double bond led to the formation of derivative 50 in 73% yield. The introduction of an isopropyl group by reacting on the aldehyde group afforded the expected key intermediate 39 in 84% yield and 99% diastereomeric excess, which could then lead to the preparation of squalamine 1 using well-known procedures.

OH

12.4.1 Antimicrobial Activities of Squalamine and Its Mimics

The unique steroid skeleton of squalamine revealed remarkable antibacterial properties, similar to that of ampicillin, with activity on both Gram-positive and Gram-negative bacterial strains. Typical minimum inhibitory concentrations (MICs) ranged from 1 to 8 mg ml 1, depending on the strain. Furthermore,

OH

45%

OH

O

O

OH

O

26

25

OH

ii

i

D. gossypina

O

12.4 Biological Activities

28

OH

27

OH

O

O

H

OH

O

iii

iv O

v

O O

OH

O O

29

30

OH

O

OH

31

OSO3H

vi-vii

Squalamine 1 2 steps O

H

OH

32

Scheme 12.4 Conditions: (i) Li, NH3, THF; (ii) TMSCl, Ethylene glycol; (iii) NaOCl, TEMPO, NaBr, CH2Cl2; (iv) (EtO)2P(O)CH2C(O)CH(CH3)2, t-BuONa, THF; (v) (R)-MeCBS, BH3.THF, THF-Toluene; (vi) Et3N, Toluene, 10% Pt/C, H2 (50 psi); (vii) p-TsOH, Water, Acetone; (viii) SO3.Py (1.05 equiv.), Pyridine.

12.4 Biological Activities

j 271

OH COOMe OH

i-iv

vi

v O

O

33

O OMOM

O

OH

OMOM

O

34

35

OAc

OAc

OH

OH

viii

vii O

O O

36

OMOM

O O

OMOM

OMOM

O

37

38

OH H N

ix

+ O

OH

40

39

NH2

N

boc

Squalamine 1

boc

2 steps

Scheme 12.5 Conditions: (i) CH3OCH2OCH3, P2O5, CHCl3, 20  C; (ii) Ethylene glycol, PTSA, Benzene, reflux; (iii) LiAlH4, THF, 20  C; (iv) (COCl)2, DMSO, Et3N, CH2Cl2, 78  C; BuLi, Ph3PþCH(CH3)2I , THF, 20  C; (v) (DHQD)2PHAL, K2OsO2(OH)4, K3Fe(CN)6, K2CO3, CH3SO2NH2, tert-butanolmethyl tert-butyl ether - H2O (2.5:3:2.5), 20  C; (vi) Ac2O, Pyridine, 20  C; (vii) CH3SO2Cl, DMAP, Et3N, CH2Cl2, 0–20  C; (viii) NH2OH, EtOAc, DMF, 95  C, then KOH, MeOH, reflux; (ix) PPTS, tert-BuOH, reflux.

unlike ampicillin, squalamine also displayed antifungal activity and caused an osmotic lysis of protozoa (Moore et al., 1993). This novel aminosterol, due to its broad-spectrum antibiotic activity, quickly attracted intense interest and consequently, in parallel to the synthesis and evaluation of the bioactivities of squalamine, intensive syntheses of the analogs were developed. This allowed the effects of modifying the steroid nucleus to be explored, and highlighted the importance of polyamine substitution as well as the presence of the sulfate group in diverse activities, especially in field of antimicrobials (Figure 12.6). With the emergence of multidrug-resistant bacteria and the consequent urgent need for new antimicrobial agents, squalamine

mimics have attracted considerable attention. Unlike squalamine, the mimics were very simple to prepare, in few steps with high yields, and from readily available and inexpensive starting materials. The first squalamine mimic to be designed was the molecule 52, the synthesis of which was guided by its structural and functional resemblance to the amphiphilic antibiotic, amphotericin B. Synthesized in three steps, the new analog 52 possessed the spermine chain attached onto the steroid side chain, and the sulfate group at the C-3 position. Although, the structure of mimic 52 was unable to adopt a macrocyclic conformation similar to that of amphotericin B, this compound revealed a slightly better activity than squalamine against Pseudomonas aeruginosa, and a reduced hemolytic potential. However, it was not efficient against OH OH

ii

i

41

HO

9 steps

42

AcO

43

AcO

Desmosterol OSO3K

OH

N3

iii

39 O

OH

O

44

H N HCl

45 NH2 HCl

Squalamine 1

OH

Scheme 12.6 Conditions: (i) Ac2O, Pyridine, 20  C; (ii) (DHQD)2PHAL, K2OsO2(OH)4, K3Fe(CN)6, K2CO3, CH3SO2NH2, tert-BuOH, tert-butylmethylether, H2O, 20  C; (iii) SO3.Pyr, Pyridine, 80  C.

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12 Promises of the Unprecedented Aminosterol Squalamine

j

O

O O

i

HO

H

O

OH

46

47

O

OMOM

H

48

OH

H

v

iv

O

OMOM

ii

O

O

O

O

iii

OH

H

O

O O

OMOM

H

49

vi

O

50

OMOM

H

51 OH

Squalamine 1 O

H

OH

39

Scheme 12.7 Conditions: (i) Ag2CO3 on Celite, Toluene, reflux; (ii) MOMCl, i-Pr2NEt, cat. NaI, CH2Cl2, reflux; (iii) 2 equiv. IBX, 30 mol% TFA, DMSO, rt, 24 h; (iv) Li, NH3, THF, 78  C, for 1 h and quenched with anhydrous NH4Cl; (v) 20 mol% (N,N)-di-n-butylamino-1-phenylpropane-1-ol, 2.2 equiv. i-Pr2Zn, Toluene, 0  C, 4 h; (vi) PPTS, tert-BuOH, reflux.

a large panel of bacterial strains such as Escherichia coli, Staphylococcus aureus, Proteus vulgaris, Serratia marcescens, and the yeast Candida albicans (Sadownik et al., 1995). A wide variety of bile acid analogs was later synthesized, starting from hyodeoxycholic acid, with the aim of evaluating the effects of different polyamine substitutions with different stereochemistry at the C-3 position, as well as the presence of a methyl ester on the side chain and the stereochemistry modification of hydroxyl substitution of the B ring. From a series of analogs, structure-activity relationships were generated which showed that changing the identity of the polyamine, especially when replacing the spermidine by spermine, had little effect upon the antimicrobial activity. However, it did reveal a different selectivity of the agents against different species of microorganisms, with the 3b-analogs being slightly more effective than the corresponding 3a-analogs. The methyl esters were more potent antimicrobial agents than the free acids against S. aureus, and the hydroxyl substituent proved to be non-essential in the antimicrobial activity. The most potent analog, the 3b-ethylene diamine methyl ester 53, displayed an antimicrobial activity similar to that of squalamine but was less effective against E. coli and P. aeruginosa, but more active against C.albicans (Joneset al., 1996). Other squalamine mimics were synthesized in a two- or threestep procedure, revealing the importance of hydrophobicity of the sterol backbone and the length and cationic charge of the side chain in antimicrobial activity. The analog 54a revealed bactericidal activities against E. coli, P. aeruginosa, and S. aureus in a dosedependent manner. Interestingly, subinhibitory concentrations of this analog markedly enhanced the antimicrobial activity of

rifampin against Gram-negative rods, which suggested that the compound might disrupt the outer membrane of Gram-negative strains. However, problems triggered by the hemolytic activity as well as interactions with serum proteins of these bile acid analogs limited their use as systemic agents. Rather, it was postulated that such compounds might serve better as topically applied antimicrobial agents (Kikuchi et al., 1997). Another squalamine analog 55, with a sulfate group at C-22, produced from the inexpensive 22-hydroxy-23,24-bisnorchola-4-en3-one, showed weaker activity than squalamine, indicating that a shorter side chain could maintain the activity (Kim et al., 2000). In the meantime, it was shown by others that whereas squalamine was not ionophoric, compound 56 acted as a proton ionophore and did not possess any lytic activity. Taken together, these results suggested that either multiple mechanisms for the antimicrobial activity of aminosterols existed, depending on the aminosterol structure, or that a possibly unrelated common mechanism was in operation that remained to be discovered (Selinsky et al., 2000). In a study designed to investigate the antibacterial properties of cationic steroid antibiotics, mimics of squalamine and of polymyxin were examined. Squalamine 1 and its mimics 54b and 56 were able to adopt a facially amphiphilic morphology in the presence of membranes; moreover, they revealed a rapid bactericidal activity and an ability to permeabilize the outer membrane of Gram-negative strains (Savage et al., 2002). Starting from bile acid-based aminosterols, and synthesized in good yields from C-3b-oxiranes as the key intermediates, the squalamine mimics 57 and 58 which had extra amino groups revealed activities comparable to that of gentamicin (IC50

12.4 Biological Activities

H O N

H N

HN

j 273

O O CH3

NH2 H3N HO3SO

OH

H

53

OH

H3N

O N H

OSO3-

N HH N

H2N HO

N H2

52

NH2

54a: 5 H 54b: 5 H

H

N H2

OH

H

55

O

+

H2+ N

H2N

OH

H O N

NH3

HO

R

H

56

O3SO

OCH3

57: R = H 58: R = OH

NH

H2N

HO

H

N H

HO

NH2

N H

HN 59

HO

N H

N H

NH2

N H

HO

61a: 7 61b: 7

HO

N H

NH2

NH2

N H

60

NH2 62

63

Figure 12.6 Squalamine mimics.

5.14 mg ml 1 for 57 and 4.12 mg ml 1 for 58, compared to 4.46 mg ml 1 for gentamicin) against S. aureus, and good antifungal activity (although less than fluconazole). These findings underlined the importance of the presence of an extra amino group, and suggested that more potent compounds could be created by increasing the chain length (Aher et al., 2009).

Several squalamine-related polyaminosterols (59–61b) were synthesized in six to eight steps using cholesterol as an inexpensive starting material. The purpose of these investigations was to introduce spermidine into the B steroidal ring, and the compounds produced revealed similar antibacterial and antifungal activities to squalamine, as well as cytotoxic effects

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on a human non-small-cell bronchopulmonary carcinoma line (NSCLC-N6). The analogous 7a-and 7b-polyaminocholesterols revealed an activity against Gram-negative bacteria, while 7apolyaminocholestanol and 6a-polyaminocholestanol were both inactive. The stereochemistry of the spermidinyl moiety on the B ring demonstrated a small effect on the antimicrobial activity (Choucair et al., 2004). Evaluation of the antimicrobial activities of these different squalamine mimics led to the conclusion that: (i) the precise structure of the polyamine was not important in terms of activity; (ii) the sulfate group could be replaced by a carboxylate or a hydroxyl group (or even removed); and (iii) the structure of the rigid hydrophobic unit could also be varied. Nonetheless, squalamine remained the most effective antimicrobial agent and an ideal lead compound for exploring the antimicrobial potencies of these compounds. Clearly, by targeting membrane integrity, by preserving its antimicrobial activity towards Gram-negative bacteria among multidrug-resistant clinical isolates, and by being insensitive to efflux resistance mechanisms, squalamine was proving to be the ideal candidate to overcome the thorny problem of multidrug-resistant Gram-negative bacteria and the associated nosocomial diseases (Salmi et al., 2008). Relevant results were described on the evaluation of squalamine and of two mimics (62 and 63) against two well-known aminosterol antibiotics, colistin and amoxicillin, to treat multidrug-resistant bacteria. The comparison was made notably with clinical isolates of P. aeruginosa and S. aureus strains, which are the most common pathogens in cystic fibrosis lung infections (Alhanout et al., 2009). Based on the results of these studies, it was postulated that squalamine could be administered locally, in the form of an aerosol. Squalamine was also shown to display two different mechanisms of action, according to the nature of the bacteria: (i) it could disrupt the outer membranes of Gramnegative bacteria via a detergent-like mechanism of action; or (ii) it could depolarize the bacterial membranes of Gram-positive bacteria, with subsequent possible use as a disinfectant and/or a detergent (Alhanout, Rolain, and Brunel, 2010a; Alhanout et al., 2010b). The suitability of squalamine in an aerosol formulation to treat cystic fibrosis was demonstrated (Alhanout et al., 2011). Moreover, in order to overcome bacterial resistance the chemosensitizing effect of squalamine was evaluated, when it was realized that the ability to increase internal antibiotic levels to treat resistant strains would require smaller amounts of antibiotics to be administered (Lavigne et al., 2010). Squalamine was also proposed as a potent alternative to skin and nasal antisepsis before surgery (Djouhri-Bouktab et al., 2011a). More recently, a comparable efficacy of aerosol squalamine and colistin to reduce the lung bacterial load and pulmonary lesions was demonstrated in a rat model of chronic P. aeruginosa infection, and revealed a promising therapeutic treatment strategy (Hraiech et al., 2012). Soluble squalamine tablets could also be developed for quickacting, easily used home nebulizers for cystic fibrosis patients (Djouhri-Bouktab et al., 2011b). Squalamine also revealed a significant fungicidal activity due to its capability to disrupt the outer membrane of yeasts, and could

be used against superficial dermatophyte infections (Coulibaly et al., 2012). Recently, the affinity of mycobacteria to cholesterol was exploited due to the desperate need for new tuberculosis drugs. Squalamine and its derivatives are very promising as antitubercular agents as they have been proved to act at specific sites in each organism (Walker and Houston, 2013). Antiparasitic activities were also investigated from a series of simplified analogs of squalamine synthesized in order to evaluate their activity against Trypanosoma brucei and T. cruzi, the causative agents of African trypanosomiasis and Chagas disease, respectively, and against the causative agent of visceral leishmaniasis Leishmania donovani. Several compounds showed in vitro activity, especially against T. brucei and L. donovani, but only minimal effect on T. cruzi. In these investigations the spermine compounds appeared slightly more active, while the presence of a sulfate group decreased the antiparasitic activities (Khabnadideh et al., 2000). 12.4.2 Antiangiogenic Activity of Squalamine

In addition its impressive antimicrobial activities, squalamine was found to cause changes in vascular endothelial cell shape, and also reported to possess significant antiangiogenic activity in models of lung, breast, brain, and ovarian cancer (Sills et al., 1998; Teicher et al., 1998). In the shark, squalamine is found primarily at sites of bile synthesis such as the liver and gallbladder, though the aminosterol compound also occurs in smaller amounts in the spleen, testes, stomach, gills, and intestine. The widespread distribution of squalamine among shark tissues indicates the importance of its biologic role in this marine specimen. Squalamine has been shown to be an angiostatic steroid by virtue of its inhibition of growth of vascular endothelial cells in culture. This activity was observed in the chick embryo chorioallantoic membrane assay and a rabbit corneal micropocket assay, as well as growth inhibition of gliomas and lung cancers in vivo (Sills et al., 1998, Teicher et al., 1998 and Williams et al., 2001). One particularity of squalamine as an antiangiogenic agent in development is that it inhibits endothelial cell proliferation and migration induced by a wide variety of growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) (Williams et al., 2001; Herbst et al., 2003). This broad antiangiogenic activity of squalamine may result from its inhibition of surface sodium-proton exchangers and other downstream signaling pathways in endothelial cells. 12.4.3 Antitumor Activity of Squalamine

Squalamine, which has no direct cytotoxic or antiproliferative effects on any tumor cell lines, revealed antitumor activity in multiple animal models due to its angiogenesis effect by preventing the neovascularization of the tumor and its migration. Indeed, avascular tumors are incapable of growth and have little metastatic potential. It is for this reason that squalamine activity in tumor

12.5 Mechanism of Antiangiogenic Activity of Squalamine

treatment and other diseases characterized by neovascularization in humans was investigated in both monotherapy and polytherapy. The formation of an adequate blood supply and tumor angiogenesis have been reported as determinants for the progressive growth and spread of many solid tumors, and this may have prognostic significance in several human cancers (Alvarez et al., 1999). As avascular tumors exhibit only limited growth and tumor aggression, and metastatic potential commonly correlates with tumor vascularity, therapy directed towards the vasculature of a solid tumor is now being regarded as an important tool in cancer treatment (Ferrara et al., 1993; Folkman and Ingber, 1992a; Folkman and Shing, 1992b). VEGF is a major regulator of physiological and pathological angiogenesis (Keck et al., 1989; Leung et al., 1989), and its activity is mediated by binding to receptor tyrosine kinases to activate downstream signaling enzymes, including MAP kinase. This, in turn, regulates gene expression and also specific endothelial cell responses such as proliferation, migration, and apoptosis (Cuenda and Rousseau, 2007; Rousseau et al., 2000; Soker et al., 1996). Several studies have revealed the important role of VEGF in the development of many cancers (Alvarez et al., 1999; Kurebayashi et al., 1999; Paley et al., 1997). In contrast, growth factor pathways, such as those which depend on epidermal growth factor (EGF) and its receptor (known as HER-2) appear to upregulate VEGF production in solid tumors (Li, Williams, and Pietras, 2002; Petit et al., 1997). Since the EGF and HER family receptors are activated (Gilmour et al., 2001) and/or overexpressed in significant numbers of human cancers (Slamon et al., 1987; Slamon et al., 1989; Wong et al., 1995), these growth factor receptor pathways may play a role in promoting the further growth of human malignancy by increasing VEGF-dependent tumor angiogenesis. 12.4.4 Antiviral Activities

Investigations of the antiviral activities of squalamine were recently described by Zasloff and coworkers, who demonstrated a broad spectrum of activity towards human viral pathogens both in vitro and in vivo, including RNA- and DNA-enveloped viruses (Zasloff et al., 2011). The ability of squalamine to cause the displacement of membrane-anchored proteins renders the host cells less effective in supporting viral replication. Therefore, by targeting the host membranes and by modifying the electrostatic interactions of cellular membranes onto which it binds, squalamine could modify the infectivity of the tissues. Moreover, due to its safe profile in humans, the therapeutic potential of squalamine remains to be examined in detail. 12.5 Mechanism of Antiangiogenic Activity of Squalamine

One proposed antiangiogenic mechanism of action of squalamine involves an inhibition of the mammalian brush-border Naþ/Hþ exchanger isoform, NHE3. The Naþ/Hþ exchanger is a transport protein that is known to regulate changes in cell volume or shape (Akhter et al., 1999). Using transfected PS120 fibroblasts,

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the investigators showed the inhibitory effects of squalamine on Naþ/Hþ exchanger activity to be both time- and concentrationdependent, and also reversible. The inhibition of Naþ/Hþ exchange was confirmed by independently monitoring 22 Na-uptake by cells. The effect of squalamine was specific for NHE3, as it had no effect on NHE1, NHE2, Naþ-dependent þ D-glucose uptake, or Na -independent D-glucose uptake. Confirmation of the non-cytotoxicity of squalamine was achieved by monitoring the release of lactate dehydrogenase (LDH), which was found to be 150-fold specificity over the highly homologous phosphatase TCPTP. Values normalized as percent of maximum response on a 0–100% scale; (b) Increased IRS-1 phosphorylation following a single dose of trodusquemine (10 mg kg 1, i.p.). Insulin injected via hepatic portal vein, livers collected 2 min later; (c) Trodusquemine binds to dopamine (DAT) and norepinephrine (NET) transporters and inhibits neurotransmitter (DA, NE) uptake in the cells.

explained by an inhibition of the projection of NPY/AGRP on the PVN. With regards to the mechanism of action, it has been postulated that trodusquemine selectively inhibits protein tyrosine phosphatase 1B (PTP1B), a key enzyme regulating insulin and leptin signaling (Figure 12.11). Thus, trodusquemine significantly enhanced the insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-beta and STAT3 (both of which are direct targets of PTP1B) in HepG2 cells in vitro and/or hypothalamic tissue in vivo. These data establish trodusquemine as an effective central and peripheral PTP1B inhibitor, with the potential to elicit noncachectic fat-specific weight loss and improve insulin and leptin levels. Recently, the Ohr Pharmaceutical Inc. company has explored the high PTP1B inhibitory activity of trodusquemine, which is currently undergoing preclinical trials for undisclosed indications.

12.9 Conclusion

Squalamine is an unprecedented aminosterol, characterized by a polyamine spermidine moiety and a sulfate group. Originally isolated from the dogfish Squalus acanthias, squalamine is now produced via a chemical synthesis route (Jones et al., 1998).

The broad-spectrum antimicrobial activity of squalamine, encompassing antifungal activity and antibacterial power against Gram-positive and Gram-negative strains (including multidrugresistant strains), has been explored. Varied formulations have been proposed for its use for quick-acting and easily used home nebulizer disinfection, especially for cystic fibrosis therapy, as an ointment for S. aureus nasal and skin decolonization, or in topical applications against superficial dermatophyte infections. The discovery of an antiviral activity of squalamine has also opened new avenues for its therapeutic development. In addition, although without antiproliferative activity on human cancer cell lines, squalamine has been widely investigated as an antitumor agent, based on its antiangiogenic activity. Moreover, its potential development as a therapeutic agent in the future has been reinforced by improvements in its antitumor activity when coadministered with carboplatin or cisplatin. The next few years should bring about significant results in the treatment of ovarian cancer and age-related macular degeneration, for which squalamine is currently in advanced clinical trials. Another natural derivative, trodusquemine, has revealed its potential to combat against obesity, and may also be linked with the therapy of diabetes. Currently, shark liver extracts are available via the internet, under the trademark SqualamaxTM; these contain “highly concentrated” natural squalamine as a remarkable dietary supplement. Clearly, this promising aminosterol or other related aminosterols will continue to astonish us!

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About the Authors Marie-Lise Bourguet-Kondracki is a Director of Research at the CNRS in France. She spent most of her career at the Mus
eum National d’Histoire Naturelle in Paris, focusing her research activity on the isolation, structure elucidation and structure–activity relationships of bioactive natural products from marine sponges. She now leads a research group involved in pluridisciplinary studies combining microscopic, cultural, molecular biology and chemical approaches in order to understand the role of the associated bacteria within the complex ecosystem bacteria–sponges. She was the chairperson of the fourth European Conference on Marine Natural Products in 2005 in Paris.

Jean Michel Brunel obtained his PhD in organic chemistry in 1994 in Marseille in the field of organophosphorus and asymmetric synthesis. He spent two years (postdoctoral position) in Kagan group, working on the catalytic asymmetric oxidation of sulfides, and then integrated the CNRS in 1997. Between 1997 and 2001, he worked on the development of new chiral organophosphorus catalysts and their application in asymmetric synthesis. Since 2002, he has been implicated in the synthesis of biologically active compounds, and in January 2012 he joined the Center de Recherche en Canc
erologie de Marseille with a new project team gathering chemists and biologists, where their mission is to design specific inhibitors modulating the effects of proteins, first in vitro and, ultimately, in animal models.

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13 Marine Peptide Secondary Metabolites Bernard Banaigs, Isabelle Bonnard, Anne Witczak, and Nicolas Inguimbert

Abstract

A large proportion of the marine natural products actually under clinical investigation are peptide secondary metabolites with unusual amino acids residues. This growing family of compounds can be produced via two major pathways, ribosomal and nonribosomal. In this chapter are presented and illustrated, with a few famous examples, ribosomal- and nonribosomal-derived peptides. Attention is focused on five families of biologically active marine peptides of diverse origin, ranging from the oldest living

13.1 Introduction

Marine organisms have, for five decades, been recognized as a rich source of potential drugs. The isolated marine natural products (MNPs) are mostly secondary metabolites that are defined as compounds not necessary for the growth and development of the producer but involved in their defense, survival, or even in interspecies communication phenomena. The term MNPs seems to have been formalized in 1971 (Sims et al., 1971), and until 2013 was widely used, as accounted for by the growing number of publications which use it as a keyword. A simple search using this keyword in the ISIweb of knowledge attested to the continuous and ever-increasing interest in MNPs from 1974, and retrieved over 7000 publications in the peer-reviewed literature (Figure 13.1). The FDA approval in 1969 of cytarabine (Ara-C) and vidarabine (Ara-A) for anticancer and antiviral therapies is often reported as the cornerstone of the launching of MNPs. Two striking features of MNPs are their production as minute quantities, and their dilution in the environment when emitted by their hosts. These characteristics, which could be considered as drawbacks, are counterbalanced by an hydrophobic character and, more importantly, by their high biological activities that prompted research groups to divert these activities with the aim of using them as drugs or as templates in the search for new medicines. Therefore, many hundreds of MNPs have entered preclinical trials and clinical trials. However, when considering that MarinLit, a database dedicated to MNPs (MarinLit database,

cell of the world, the cyanobacteria, to more sophisticated organisms such as tunicates or mollusks. The discussed peptides illustrate differently the structural features and diversity found in marine-derived peptides; laxaphycins as an example of true peptides; dolastatins, didemnins and kahalalides are discussed in connection with their clinical trials; and azole/azoline-containing cyanobactins to exemplify the extraordinary chemical diversity within a single organism. Information about their biological properties and mechanisms of action is discussed when available.

Department of Chemistry, University of Canterbury: http:// www.chem.canterbury.ac.nz/marinlit/marinlit.shtml), inventoried approximately 22 000 compounds originating from 6000 species derived from 2200 genera appertaining to 39 phyla, and that only three compounds have reached the market over these five decades, it should be pointed out that the quest for a “magic bullet” originating from the sea is somehow disappointing (Liu, 2012). From a different point of view, if this is compared to a classical drug discovery process based on the high-throughput screening of rationally designed compounds, or of in-house chemical libraries, then the number of MNP hits is relatively high and competes advantageously with the success rate of the previous methods (Gerwick and Moore, 2012). Furthermore, MNPs, as did natural products from terrestrial sources, cover a wider chemical area than that achievable by generating a focused synthetic drug library. This allows research teams working in the field of MNPs to highlight their chemical diversity and to speak of combinatorial biosynthesis performed by Mother Nature (Welker and Von D€ ohren, 2006). This chemical diversity arises from the biological and habitat diversities found in the oceans that cover 70% of the Earth’s surface and remain largely unexplored. MNPs produced by bacteria (Burja et al., 2001) and invertebrates (Leal et al., 2012) encompass peptides, polyketides, alkaloids, terpenes, and polyphenols. This structural diversity allows the MNPs to act on various biological targets implicated in cancers, malaria, HIV or infectious diseases; however, in most cases the MNPs were developed for their antineoplastic activities (Russo, Nastrucci, and Cesario, 2011; Nastrucci, Cesario, and Russo, 2012; Russo and Cesario,

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by Stephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

13 Marine Peptide Secondary Metabolites

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of compounds that can be produced via two major pathways, namely ribosomal and nonribosomal.

2011

2007

2003

1999

1995

1991

1987

1983

1979

1975

700 600 500 400 300 200 100 0 1971

publication number

286

year

Figure 13.1 Numbers of publications using Marine Natural Products as a keyword (ISIweb search) between 1970 and 2012.

2012). Three MNPs (Figure 13.2) – ziconotide a peptide, ecteinascidin 743 an alkaloid, and eribulin mesylate, a macrocyclic analog of halichondrin B – gained FDA approval between 2004 and 2010 (Liu, 2012; Montaser and Luesch, 2011). It should be emphasized that one-third of the 18 MNPs currently under clinical investigation are peptide secondary metabolites with “unnatural” amino acids residues. The decision was taken to focus this chapter on five families of biologically active marine peptides of diverse origin, ranging from the oldest living cell of the world, the cyanobacteria, to more sophisticated organisms such as tunicates or mollusks. The discussed peptides illustrate differently the structural features and diversity found in ribosomal- and nonribosomalderived marine peptides. They are classified in order of ascending molecular complexity, and will be presented in the following order: laxaphycins, as an example of true peptides; dolastatins, didemnins and kahalalides, to be discussed in connection with their clinical trials; and azole/azoline-containing cyanobactins to exemplify the extraordinary chemical diversity within a single organism. However, it was first thought necessary to present and illustrate, with a few famous examples, this growing family

13.2 Ribosomal- and Nonribosomal-Derived Peptides: A Virtually Unlimited Source of New Active Compounds

The large structural diversity encountered in ribosomal- and nonribosomal-derived peptides is not solely due to the huge number of possible combinations of the 20 common proteinogenic L-amino acids, but rather arises from the high contents of nonproteinogenic amino acids resulting from: i) Post-translational modifications mostly performed by enzymes, leading to ribosomally synthesized and posttranslationally modified peptides (RiPPs). ii) A synthesis of nonribosomal peptides (NRPs) by nonribosomal peptide synthetase (NRPS). iii) A mixed process in which both polyketide synthase (PKS) and NRPS intervene. The NRPs can indeed contain, together with the 20 common acids, some of the D series and both types could be N-methylated, N-formylated, glycosylated, acylated, halogenated orhydroxylated.Withinthesenonproteinogenicaminoacidsshould also be include b-, c-, or v-amino acids. Unlike NRPs, RiPPs cannot explore amino acids beyond the canonical 20 proteinogenic amino acids, which limits their structural diversity to some degree (McIntosh, Donia, and Schmidt, 2009). The cyclization of amino acids against the peptide backbone is often performed, resulting in oxazoline and thiazoline rings or, after oxidation, in oxazole or thiazole rings. Dehydration on serine results in dehydroalanine (Figure 13.3). As a result, the extensive modifications led in turn to restricted conformational flexibility that allowed a better fitting to biological L-amino

Figure 13.2 Ziconotide (SNX-111, Prialt), ecteinascidin 743 (trabectin, Yondelis) and eribulin mesylate (Halaven), three marine natural products approved for use as drugs in USA and/or Europe during the past ten years.

13.2 Ribosomal- and Nonribosomal-Derived Peptides: A Virtually Unlimited Source of New Active Compounds

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Figure 13.3 Some unusual and rare constituents of ribosomal and nonribosomal derived peptides.

targets, and also to enhanced metabolic and chemical stabilities. Moreover, RiPPs and NRPs are two diverse families of peptide secondary metabolites with a broad range of biological activities and pharmacological properties that are used in the context of human and animal health. Some of these peptides are currently in

commercial use, such as the antibiotics actinomycin, bacitracin, daptomycin, vancomycin, tyrocidine, gramicidin or zwittermicin A, the antifungal caspofungin, the cytostatics epothilone or bleomycin, the immunosuppressant cyclosporine A, and the siderophores enterobactin or myxochelin A (Figure 13.4).

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Figure 13.4 Natural modified peptides in commercial use. Cyclosporine is an immunosuppressant drug used to prevent the rejection of organ transplants. Actinomycin D, an antibiotic that shows anticancer activity, is used in the treatment of a variety of cancers under the trade name Cosmegen. Tyrocidine is an antibiotic. Caspofungin is a member of a new class of antifungals. Bleomycin is an anticancer drug used to treat head and neck cancers.

Historically, the first nonribosomal pathways for cyanobacterial peptides were described in 2000 (Tillett et al., 2000; Rouhiainen et al., 2000). The NRPSs are large multimodular and multidomain enzymes (Schwarzer, Finking, and Marahiel, 2003), and although the genetic potential for nonribosomal synthesis seems to be common to prokaryotes and eukaryotes, NRPs are apparently produced by bacteria and fungi (Tiburzi, Visca, and Imperi, 2007). Of the 678 marine cyanobacterial natural products reported in the literature, the majority is polyketide synthase-non-ribosomal peptide synthetase-derived (Malloy et al., 2012). As one of the most structurally diverse natural products, NRPs contain not only nonproteogenic amino acids or aryl acids from amino acid metabolism, but also fatty acids or polyketide-derived units. These have often a cyclic and/or

branched structure. The depsipeptides subfamily is characterized by the replacement of one, or more, amide bond by an ester linkage. NRPSs are also responsible for the biosynthesis of the tetrahydroisoquinoline family of alkaloids, which includes saframycin, naphthyridinomycin, and ecteinascidin (Peng et al., 2012). The term ribosomally synthesized and post-translationally modified peptide (RiPP) was recently defined in a very complete review that provides an overview of the structures of, and the biosynthetic processes leading to, this large group of natural products (Arnison et al., 2013). In this review, the authors included a recommendation for a universal nomenclature in the field of RiPPs. Peptide secondary metabolites can also represent a serious threat to the aquatic environment and to human health. The

13.2 Ribosomal- and Nonribosomal-Derived Peptides: A Virtually Unlimited Source of New Active Compounds

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Figure 13.5 Toxic peptide secondary metabolites isolated from cyanobacteria. The LD50 of microcystin-LR, the most common isoform of microcystins family, and nodularin is 50 mg kg 1 in mice.

most frequently found cyanotoxins in fresh and brackish water are the cyclic peptide toxins, microcystins and nodularin (Figure 13.5). These are hepatotoxic compounds that specifically inhibit the activity of the protein phosphatases PP1 and PP2A. The microcystins (more than 75 isoforms exist) are monocyclic heptapeptides produced by multiple genera of cyanobacteria (Microcystis, Anabaena, Oscillatoria, Planktothrix, Chroococcus or Nostoc). For some species there is a cosmopolitan distribution, whereas the nodularins are pentapeptides most commonly isolated from the filamentous cyanobacterium Nodularia spumigena, a species that forms toxic blooms in brackish and estuarine environments (Pearson et al., 2010). Microcystins and nodularins contain a c-branched methyl aspartic acid (MeAsp) and the unique b-amino acid 3-amino-9-methoxy2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) (Rinehart et al., 1988). Since 1980, when two of the first peptide secondary metabolites, ulicyclamide and ulithiacyclamide, were first discovered in marine organisms (Ireland and Scheuer, 1980), many RiPPs and/or NRPs have been discovered on an almost weekly basis. Some examples of these are shown in Figure 13.6. Apratoxin A (Luesch et al., 2001a), isolated from the marine cyanobacterium Lyngbya majuscula, possesses an intricate structure composed of a polyketide section fused with a modified pentapeptide, and shows an extremely promising profile of selective cytotoxicity towards cancer cell growth. The total synthesis of apratoxin A and analogs was achieved by different groups (Chen and Forsyth, 2004; Doi et al., 2006; Ma et al., 2006; Gilles, Martinez, and Cavelier, 2009; Robertson, Wengryniuk, and Coltart, 2012). Apratoxin A prevents the cotranslational translocation of proteins destined for the secretory pathway (Liu, Law, and Luesch, 2009). The putative biosynthetic apratoxin A gene cluster was reported using a single-cell genome amplification (Grindberg et al., 2011). Largazole (Hong and Luesch, 2012) was isolated from a marine cyanobacterium of the genus Symploca. This depsipeptide contains a 4-methylthiazoline fused to a thiazole ring and an octanoic thioester side chain, and is another novel chemical scaffold that may preferentially target cancer cells over normal cells; consequently, largazole is a potentially valuable cancer chemotherapeutic. Largazole potently inhibits class I histone

deacetylases (HDACs), and is a novel class of ubiquitin E1 inhibitor (Ungermannova et al., 2012). Due to the intriguing structure and the potential as selective anticancer drug candidate, a great deal of attention has been focused on the synthesis of largazole and its analogs (Li et al., 2011). Lyngbyastatins 4–10 are among the most potent natural elastase inhibitors (IC50-values in the nanomolar range), and were isolated from another marine cyanobacterium Lyngbya confervoides (Taori et al., 2007). Lyngbyastatins 4–10 are analogs of dolastatin-13 (Pettit et al., 1989a), somamides A–B (Nogle, Williamson, and Gerwick, 2001), scyptolins A–B (Matern et al., 2001), and also of the new symplostatins 5–10 (Salvador et al., 2013) and stigonemapeptin (Kang, Krunic, and Orjala, 2012). All of these compounds, isolated from diverse cyanobacteria, share a relatively conserved 19-membered cyclic hexadepsipeptide containing the unique 3-amino-6-hydroxy-2-piperidone (Ahp) unit and a variable (in both length and composition) pendant side chain. The crystal structure of an elastase, in complex with its inhibitor scyptolin A, was first described in 2003 (Matern et al., 2003). More recently, in order to evaluate the therapeutic potential of this Ahp containing cyclodepsipeptides, structure– activity relationship (SAR) studies and X-ray cocrystal structure analyses were conducted, using a representative family member, symplostatin 5 (Salvador et al., 2013). Hoiamide A is a novel cyclodepsipeptide isolated from an assemblage of the marine cyanobacteria Lyngbya majuscula and Phormidium gracile, collected in Papua New Guinea (Pereira et al., 2009). Hoiamide A, a complex metabolite with three highly modified regions (peptidic, triheterocyclic and polyketide), illustrates the intricate biosynthetic machinery responsible for the production of cyanobacterial compounds. Hoiamide A is a partial agonist of site 2 on the voltage-gated sodium channel, making it a structurally novel lead compound for drugs capable of promoting neuronal growth and plasticity. Polytheonamide B, isolated from the Japanese marine sponge Theonella swinhoei (Hamada et al., 2005), is the largest nonribosomal peptide known to date, and perhaps one of the most wellstudied marine NRPs. Solution structure (Hamada et al., 2010) and synthesis (Inoue et al., 2010) of this unique NRP were achieved five years later. This highly cytotoxic compound is a transmembrane channel-forming peptide (Matsuoka et al., 2011).

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Figure 13.6 Intriguing peptide secondary metabolites from marine organisms. Polytheonamide: The D-chirality and L-chirality of amino acids are indicated by red and blue structures, respectively.

A number of reviews on lipopeptides, depsipeptides, nonribosomal peptides or post-ribosomal-modified peptides have been produced over the past ten years. The different structural features and physico-chemical properties of three families of Bacillus lipopeptides, surfactins, iturins and fengycins, effective surface- and membrane-active amphiphilic biomolecules, were

reviewed (Ongena and Jacques, 2008). Lipopeptide antibiotics represent an old class of antibiotics, which includes polymyxins but also new entries, such as the recently approved daptomycin. This class of antibiotics was reviewed (Pirri et al., 2009), focusing on their therapeutic applications and placing particular emphasis on the chemical modifications introduced to improve

13.3 Laxaphycins and their Derivatives: Peptides Not So Easy to Synthesize

their activity. Another review (Raaijmakers et al., 2010) provides a detailed summary of the versatile functions of lipopeptides in the biology of Pseudomonas and Bacillus species, and highlights their role in competitive interactions with coexisting organisms. This review provides also an update on lipopeptide detection and discovery as well as on novel regulatory mechanisms and genes involved in lipopeptide biosynthesis in the two bacterial genera. A new family of lipopeptides produced by Bacillus thuringiensis, the kurstakins, was discovered in 2000 and considered as a biomarker of this species. A mini-review gathers all the relevant published information about these promising bioactive molecules (Bechet et al., 2012). Recent biochemical and structural studies on several NRPS assembly lines have been reviewed (Marahiel, 2009). Structural biology has provided significant insights into the complex chemistry and macromolecular organization of NRPSs. In addition, novel pathways are continually described, expanding the knowledge of known biosynthetic chemistry (Condurso and Bruner, 2012). Fungal PKS–NRPS hybrids manufacture a wide range of structurally diverse secondary metabolites that play an eminent role in the environment, as molecular tools and leads for therapeutic development (Boettger and Hertweck, 2013). The synthesis of bioactive cyclodepsipeptides was also recently reviewed (Stolze and Kaiser, 2013). Highlights of bioactive modified peptides from marine sources have been reported in several reviews (Aneiros and Garateix, 2004; Romanova et al., 2011). Modified peptides from particular types of organism were detailed in reviews on marine sponges (Matsunaga and Fusetani, 2003; Andavan and LemmensGruber, 2010) or marine cyanobacteria from the genus Lyngbya (Liu and Rein, 2010). Reviews on more specific types of biological activities, including antifungal (Fusetani, 2010), antitumor (Zheng et al., 2011) and anticancer (Suarez-Jimenez, BurgosHernandez, and Ezquerra-Brauer, 2012) peptides have been produced in recent years. One of the most active groups in the chemistry, chemical ecology and biological chemistry of marine lipopeptides – that of Gerwick – reviewed several mechanistic transformations identified in marine cyanobacterial biosynthetic pathways, with an emphasis on modular PKS/NRPS gene clusters (Jones et al., 2010).

13.3 Laxaphycins and their Derivatives: Peptides Not So Easy to Synthesize

Laxaphycins and their congeners are cyclic lipopeptides isolated from cyanobacteria, photosynthetic prokaryotes that produce a wide range of secondary metabolites (Burja et al., 2001; Liu and Rein, 2010; Tan, 2007; Moore, 1996). For a rapid overview, laxaphycins can be classified as two types (Figure 13.7):  Laxaphycin A-type peptides, which are cyclic undecapeptides in which the sequence shows a segregation between hydrophobic and hydrophilic residues.

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 Laxaphycin B-type peptides, which are cyclic dodecapeptides in which hydrophobic and hydrophilic residues are alternated. Both types contain a rare b-amino acid with an aliphatic side chain ranging from six (Aoc) to eight (Ade) carbons, and alternating L- and D-amino acid sequences. For example, starting from Ade towards the N-terminal part of laxaphycin B, the consecutive stereochemistry of the alpha or beta carbons constituting the macrocycle are as follows: R-S-R-S-R-S-S-R-S-S-R-S, and this is similar for laxaphycin A and others cyclic lipopeptides such as the recently described cyclodysidins (Abdelmohsen et al., 2012). These peptides were first discovered in 1992 by Frankm€ olle, when they were extracted from Anabaena laxa, a freshwater species isolated from a sample collected off the Manoa Campus at Hawaii University (Frankm€ olle et al., 1992a). Laxaphycins A and B are in a 3 : 2 ratio in the crude extract, and constitute the major products of a mixture of related peptides. Shortly thereafter, hormothamnin A – which only differs from laxaphycin A by the geometry of the double bond, respectively Z and E, of the dehydroaminobutyric acid (Dhb) unit – was isolated from Hormothamnion enteromorphoides, whereas no laxaphycin B-type peptide seems to have been detected (Gerwick et al., 1989; Gerwick et al., 1992). Later, the structure of laxaphycins A and B were fully elucidated. Two other congeners, B2 and B3, having different degrees of hydroxylation, were also isolated from Anabaena torulosa harvested in French Polynesia (Bonnard et al., 1997; Bonnard et al., 2007). This peptide family broadened over the years with the successive isolation and structure elucidation of lobocyclamide A from Lyngbya confervoides containing serine (Ser2), tyrosine (Tyr6) in place of the homoserine (Hse2) and phenylalanine (Phe6) found in laxaphycin A, while conserving the same E geometry of Dhb3 (MacMillan et al., 2002). In this series, the equivalent to laxaphycin B-type peptides are lobocyclamides B and C, which differ only by the stereochemistry of the 3-hydroxyleucines (Hleu). The two Hleu in lobocyclamides B and C share the same configuration while they are diastereoisomers in laxaphycin B. Another difference is the presence of the rare 4-hydroxythreonine having the same configuration as its 3-hydroxyasparagine counterpart found in laxaphycin B. The last exemplars are the lyngbyacyclamides A and B, isolated from Lynbya sp. collected at Ishigaki Island in Japan (Maru, Ohno, and Uemura, 2010), and the scytocyclamides A, B and C, extracted from the cultivated fresh water Scytonema hofmanni PCC7110. Lyngbyacyclamide A contains a Pro10 replaced in the B form by (2 S,4R)-Hyp10 and are [(2SHse4), (3R-Phe)] laxaphycin B3 analog, but contrary to the previous studies the stereochemistry of nonproteinogenic amino acids was not determined. Scytocyclamide A is a laxaphycin A analog in which S-Hse2 is mutated to S-Gln2, while scytocyclamides B and C correspond respectively to laxaphycin D and (Leu5)-laxaphycin D (Grewe, 2005). Finally, laxaphycins, hormothamnin, lobocyclamides, lyngbyacyclamides and scytocyclamides are ubiquitous peptides with minimal changes in the primary sequences, or subtle

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Table 13.1 Laxaphycin derivatives, their collection sites and bioactivity.

Cyanobacterium species

Collection site

Isolated peptide

Biological activity

Reference

Laxaphycin A derivatives Anabaena laxa

Hawaii

Laxaphycin A

No reported activity

Frankm€olle et al., 1992a Frankm€olle et al., 1992b

French Polynesia Puerto Rico

Laxaphycin A

No reported activity

Hormothamnin A

Gerwick et al., 1992 Gerwick et al., 1989 MacMillan et al., 2002

Anabaena torulosa

Lobocyclamide A

Scytonema hofmanni

Cay lobos Bahamas Cultured

Antimicrobial and antifungal: nonactive Cytotoxic and moderately ichthyotoxic Modest antifungal activity

Scytocyclamide A

Cytotoxic

Grewe, 2005

Laxaphycin B derivatives Anabaena laxa

Hawaii

Laxaphycin B

French Polynesia Cay Lobos Bahamas Ishigaki Island Japan Cultured

Laxaphycins B, B2 B3

Antifungal: moderate Cytotoxic Cytotoxic

Lobocyclamides B, C

Modest antifungal activity

Frankm€olle et al., 1992a Frankm€olle et al., 1992b Bonnard et al., 1997 Bonnard et al., 2007 MacMillan et al., 2002

Lyngbyacyclamides A, B

Cytotoxic on B16 mouse melanoma cells (IC50 0.7 mM)

Scytocyclamides B, C

Cytotoxic

Hormothamnion enteromorphoides Lyngbya confervoides

Anabaena torulosa Lyngbya confervoides Lyngbya sp.

Scytonema hofmanni

variations in the stereochemistry. These observed variations in stereochemistry can be due to differences in their biosynthetic pathway, but could also result from a misinterpretation of the HPLC analysis of Marfey’s derivatives, or because of typing errors in the original article. Hormothamnin A is described, in the text of the article (Gerwick et al., 1992), containing the sequence D-Leu-L-Ile-D-allo-Ile-L-Leu, whereas in the figure the sequence is represented as D-Leu-D-Ile-D-allo-Ile-L-Leu. A comparable typing error is also noted for lobocyclamide B, in which all of the natural amino acids are described to be of the natural L configuration in the text (MacMillan et al., 2002), whereas Leu and Glu are represented in the figure in the D configuration. An error is also suspected in the proposed structure of laxaphycin B (Bonnard et al., 2007). The two Hleu residues are described as (2S,3S)-Hleu3 and (2R,3S)Hleu5, whereas they are both of (2R,3S) configuration in lobocyclamide B (MacMillan et al., 2002). Such confusing information will add difficulties for the chemist involved in their synthesis. These peptides have been selected after a long evolutionary history by different species of cyanobacteria of freshwater and marine environments (Table 13.1) appertaining to three families, namely the Nostocaceae, Oscillatoriaceae and Scytonemataceae, and are believed to confer on them an ecological advantage. In consequence, it is understandable that the biological activities of these NMPs were examined. Secondary metabolites issuing from cyanobacteria cover a wide range of bioactivities, ranging from antibiotic to anticancer and antiviral, to cite but a few. In brief (Table 13.1), all members

Maru et al., 2010 Maru, Ohno, and Uemura, 2010 Grewe, 2005

of the laxaphycin A-type family do not shown any antimicrobial or antifungal activity, in contrast to the laxaphycin B-type members which present a moderated effect that is synergistically enhanced when the two compounds are combined (Frankm€ olle et al., 1992b). The result is even more contrasted with regards to their cytotoxicity; indeed, among the laxaphycin A series only hormothamnin (the Z-Dhb isomer of laxaphycin A) exerts a cytotoxic effect in the micromolar range (IC50 value between 0.13 and 0.72 mM) (Gerwick et al., 1989). The laxaphycin B-type family shows a cytotoxic effect on different cell lines that is comparable to those of hormathamnin A. Therefore, much effort was undertaken to decipher the molecular mechanism that is implicated in their action. All studies reported to date have highlighted a cellular lysis occurring in a nonspecific manner, whereas immunofluorescence experiments performed on HeLa cells using antibodies raised against alpha-tubulin have shown that scytocyclamide B induces the depolymerization of alpha-tubulin (Grewe, 2005), a phenomenon commonly observed for many cyanobacterial MNPs. The MNP content of cyanobacteria was not examined solely for its value in human health; rather, it also ignited the interest of research groups investigating the chemical ecology of cyanobacteria (Le~ao et al., 2012). Cyanobacteria appeared on Earth billions of years ago, in an unfriendly environment that was exposed to ultraviolet irradiation and saturated with carbon dioxide. Today they can be found in very diverse habitats, from terrestrial to aquatic (freshwater and marine) environments, in extreme latitudes, and growing as endosymbionts or

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planktonic cells. Under these conditions, which often are very competitive, it is not surprising that cyanobacteria have developed chemicals to protect themselves against UV, competitors, or predators. It has been observed that algal bloom cycles are provoked by increases in the carbon dioxide concentration in both air and water, along with convenient lighting, feeding, and also iron sources. Consequently, the cyanobacteria act as a “sea curer” by removing carbon dioxide and thus participating in the sea homeostasis (Smetacek et al., 2012). The ecological role of laxaphycins remains especially unclear, with conflicting results having been reported. For example, when lyngyacyclamides or hormothamnin A were tested on the brine shrimp Artemia, they failed to show any activity at doses of 70 mM, whereas scytocyclamides A and B were lethal in less than 2 h for the freshwater crustacean Thamnocephalus platyrus, with LD50-values of 18 mM and 3.7 mM, respectively (Maru, Ohno, and Uemura, 2010; Grewe, 2005). Therefore, the commonly proposed use of these peptides as chemical defenses could not be confirmed. Although these peptides do not have an exceptional bioactivity, they still raise the question of why cyanobacteria from different localizations produce peptides that contain so little variation in their sequences, if this were not to confer an ecological advantage. Alternatively is it because they act on an asyet unidentified biological target? As hypothesized already for the heptapeptide cyanobacterial toxin microcystins (see Figure 13.5), these peptides should have evolved from a common precursor and also have common biological actions and/or ecological roles (Rantala et al., 2004). The synthesis of these compounds will enable the establishment of SARs and allow a better comprehension of their action mechanisms, along with the possibility of exploring their ecological role(s). One much-evoked explanation of their mechanism relies on pore formation within the cell membrane, and again this hypothesis was tested and partially demonstrated by J. C. Grewe, who showed that model membranes containing cholesterol were permeable after treatment with scytocyclamide B. In order to clarify this point, one possibility might be to check if the peptides are able to promote the formation of supramolecular entities by a self-assembly process, since this has already been demonstrated for the lipodepsipeptide pseudodesmin A (Sinnaeve et al., 2009).

Figure 13.8 Heterogeneity in the dolastatin family.

Due to their great potential as producers of cytotoxic drugs, it is hoped in the future to cultivate cyanobacteria under favorable conditions for the production of secondary metabolites (Burja et al., 2002). Nevertheless, as noted above, their synthesis remains an alternative to their expression that will allow investigations into their SARs. Some research groups involved in synthesizing precursor amino acids have paved the way to a total synthesis of laxaphycins and their analogs (MacMillan and Molinski, 2002; Boyaud et al., 2012; Boyaud, Viguier, and Inguimbert, 2013a). Very recently, when the first total synthesis of laxaphycin B was accomplished, this led to a structural revision of its stereochemistry. Together with the total synthesis of lyngbyacyclamide A, this also led to a stereochemical determination of the undetermined amino acids (Boyaud et al., 2013b).

13.4 Dolastatins: From Deception to Hope Through Structural Modification Leading to Reduced Toxicity

The following example of dolastatins involves one of the most successful groups of MNPs. Indeed, it was not only the synthesis of this class of compound that was performed; rather, the SARs of these compounds were studied in great detail, which in turn allowed a reduction of their toxicity and their clinical evaluation, which culminated in one of the derivatives receiving FDA approval. Compounds isolated from the mollusk Dolabella auricularia are grouped under the same generic name of dolastatin; consequently, a high structural heterogeneity is observed in this family that incorporates linear and cyclic peptides (dolastatin 13) (Pettit et al., 1989a), peptides containing the thiazole and oxazole heterocycles (dolastatin E) (Nakamura et al., 1995), and also macrolides (dolastatin19) (Pettit et al., 2004) (Figure 13.8). The variety and structural heterogeneity grouped under the dolastatin generic name is amazing; nonetheless, it has now been ascertained that some of these molecules are not produced by the mollusk itself, but rather are ingested and concentrated in the organism, as their diet consists largely of cyanobacteria (Luesch et al., 2002a).

13.4 Dolastatins: From Deception to Hope Through Structural Modification Leading to Reduced Toxicity

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Figure 13.9 Structure of dolastatins 10 and 15, and symplostatin-1.

Among the compounds isolated from the sea hare, the most remarkable with respect to their biological activities are the linear peptides dolastatin 10 and 15 (Pettit et al., 1987b; Pettit et al., 1989b; Pettit et al., 1989c) (Figure 13.9). Whilst these compounds were first identified in the sea hare Dolabella auricularia, dolastatin 10 and its symploplastin 1 analogs were shortly thereafter isolated from the marine cyanobacterium Symploca (Luesch et al., 2001b). To date, up to 15 different linear or cyclic dolastatins have been isolated, and the group has been widened by the isolation of the comparable compounds symploplastin 3 (Luesch et al., 2002b) and malevamide D (Horgen et al., 2002). As the synthesis and antineoplastic activities of these compounds have been reviewed (Poncet, 1999), the proposal here is to briefly showcase the final developments in improving their compounds’ structures with regards to their use as potential drugs. As is often the case, structural originality and high biological potencies do not guarantee an easy transfer to clinical trials, and only dolastatin 10 has demonstrated subnanomolar activities during in vitro assays and thus proved amenable to clinical trials. This compound acts on tubulin by binding to it on a site close to the Vinca alkaloid site. Unfortunately, Phase II clinical trials with dolastatin 10 were disrupted for reason of insufficient anticancer activity (Perez

Figure 13.10 Structure of C-terminal-modified dolastatins 10 and 15.

et al., 2005) and induced side effects, notably of neuropathy. Following such disappointment, intense efforts were undertaken to manage these side effects through making structural modifications of the natural scaffolds. For this, the molecules were simplified at their C terminus, leading respectively to TZT-1027 derived from dolastatin 10, and ILX-651 related to dolastatin 15; these two modifications were subsequently launched into clinical trials for the treatment of cancer (Figure 13.10). This type of modification resulted from classic SAR studies (Pettit et al., 1998; Ogawa et al., 2001), in which the thiazolo moiety of the dolaphenine was replaced by a phenylalanine methyl ester, leading for example to auristin PHE (Figure 13.10). Similar types of modification were carried out, thereby extending the auristatin family (Shnyder et al., 2007). It should be noted that similar modifications were also applied to malevamide D (Horgen et al., 2002), where dolaphenine was replaced by the more simple 3-phenyl-1,2 propanediol and, consequently, the amide bond by an ester. These compounds retained the same mechanism of action as has been reported for dolastatin 10, and hence were active at the low nanomolar range via tubulin depolymerization (Horti et al., 2008; Tan, 2010). Their potential binding site was deduced as being adjacent to the exchangeable GTP site present on b-tubulin, according to molecular modeling

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13 Marine Peptide Secondary Metabolites

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Dolastatin 10 continues to attract research interest, and efforts are being made for its use as a template in the development of new compounds with reduced toxicity and comparable or more potent biological activities. Very recently, a proposal to replace dolaproline (Figure 13.11) with a sugar amino acid, thus conserving bioactivity, was shown to preserve the antiproliferative effect on cancer cells by dampening the microtubule dynamics (Gajula et al., 2013). The delivery of drugs to their targets can be impaired in many ways, including poor water-solubility so that a therapeutic dose is difficult to achieve (Savjani, Gajjar, and Savjani, 2012), rapid excretion, and/or side effects due to actions on either nontarget receptors or healthy organs. Dolastatin derivatives do not constitute an exception to these basic rules, and those seeking to improve this situation have taken a variety of options. For example, auristatin E was recently modified on its phenylethylamide portion by the introduction of a phosphate moiety; this resulted in a water-soluble form that conserved the strong anticancer potential (Pettit, Hogan, and Toms, 2011). Further improvements of dolostatin resulted in the development of hybrid structures in which a dolastatin derivative was coupled to a specific ligand of a receptor overexpressed on tumoral cells (Figure 13.12). This ligand may be an antibody or a human serum albumin carrier of a homing tumor peptide. In the latter case, the peptide included the RGD sequence that recognizes the avb3-integrin receptor expressed on endothelial cells that are present at the tumor level but are underexpressed on quiescent endothelial cells or healthy tissues. At nanomolar concentrations, the drug conjugate was able to kill the endothelial cells, and shown to accumulate in the mouse C26 tumor, without any notable dispersion into other organs (e.g., spleen, liver, kidneys) (Temming et al., 2006). At the same time, a comparable strategy was developed using a monoclonal antibody (mAb), raised against the transmembrane cytokine receptor CD30, tethered to monomethyl auristrin E by a spacer (Figure 13.12). The development of this antibody–drug conjugate (ADC) (Gerber, Koehn, and Abraham, 2013) was supported by the conjunction of favorable factors that

Figure 13.11 Structure of dolaproline-modified dolastatin 10.

(Mitra and Sept, 2004) and photoaffinity labeling studies (Bai et al., 2004). Furthermore, other studies had established that TZT1027 (soblidotin) would also act as an antivascular agent (Natsume et al., 2003); indeed, this compound had been shown to destroy pre-existing blood vessels, causing tumor hemorrhage which, in turn, led to necrosis of the lesion by starvation of nutrients and oxygen. Thus, compounds which combined cytotoxic and antivascular effects with a reduced toxicity would have a higher therapeutic potential than would dolastatin 10. In fact, during in vivo studies soblidotin was shown to be superior to the classical anticancer agents of cisplatin, vincristine, doxetaxel, and combretastatin A4 (Natsume et al., 2003; Watanabe, Natsume, and Kobayashi, 2007a). This effect was even more pronounced on tumors that expressed the vascular endothelial growth factor (VEGF), which promotes the creation of blood vessels from the pre-existing vasculature, leading to the angiogenesis required for growth and metastasis of the primary solid tumor. TZT-1027 was shown to exert an antiangiogenic effect at lower concentrations, but to become antivascular at higher concentrations by causing damage to the vascular endothelial cells (Watanabe et al., 2007b). A combination of TZT-1027 with a mitogen activated protein kinase (MEK) inhibitor induced a significant regression of a xenografted tumor, highlighting the potential benefits of such drug association (Watanabe et al., 2010). These favorable results have resulted in TZT-1027 reaching Phase II clinical trials stage for different types of cancer (Patel et al., 2006; Riely et al., 2007).

Targeting head D F G

K R

Albumin or

O

mAB S

H N

O O N O

H N

N H

O

O N H Self immolative spacer part

HN O

O

N O

O

H N

N

N O

O

O

O

Monomethyl auristatin E (MMAE)

NH2

Peptidic fragment subject to proteolysis Spacer

Figure 13.12 Simplified structures of RGD-albumin-MMAE conjugates, and of the antibody–drug conjugate Adecetris.

OH

13.5 Didemnins and Related Depsipeptides: How Perseverance Should Lead to Their Low-Cost Production

included the most potent antimitotic drug ever described, a pretested mAb, together with a well- defined and controlled spacer technology (Doronina et al., 2006). Due to its peptidic nature, auristin could be linked to the spacer at the C-terminal or N-terminal regions, and both option were envisioned (Doronina et al., 2008). In this case, the mAb serves as a carrier, allowing identification of the specific CD30 antigens located at the tumor cell surface. Once internalized in the cell, the linker is cleaved by a protease, thus ensuring a correct delivery of the drug in the vicinity of its receptor. This approach is also believed to reduce the toxicity, due to the lower concentration of the administered drug, and also to reduce its ability to interact with healthy tissues that are deprived of the CD30 antigens. These ADCs constitute the most recent development of dolastatin derivatives, and acquired FDA approval in August 2011, under the name of Adcetris, for the treatment of Hodgkin lymphoma and systemic anaplastic large-cell lymphoma (Senter and Sievers, 2012). This accomplishment should pave the way to extend ADCs to others mAbs with different specificities, and thus to a broadened scope of their application. In conclusion, it has taken a long time from the discovery of dolastatin as a potential drug to its marketing as an ADC, and this provides great encouragement for those working in the field of MNPs. This highlights the need for not only subventional multidisciplinary research but also the maintenance of strong links between academic research and the pharmaceutical industry.

13.5 Didemnins and Related Depsipeptides: How Perseverance Should Lead to Their Low-Cost Production

Didemnin B (Rinehart et al., 1981) occupies a particular place in the world of MNPs, being the first marine natural product to

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enter clinical trials against cancer in the US (Chun et al., 1986). Unfortunately, however, its development was halted because of important adverse side effects. However, based on Mother Nature’s inventiveness and a prodigious capacity to perform combinatorial chemistry, the closely related compounds of tamandarine (Vervoort et al., 2000) and dehydrodidemnin B were isolated from different species of tunicates (Figure 13.13). The latter compound was also synthesized as part of SAR studies on didemnins (Sakai et al., 1996), and showed a reduced toxicity and an enhanced activity in comparison to didemnin B. Thus, it was re-launched in clinical trials under the name of aplidine (plitidepsin) and acquired orphan drug status in the EU for the treatment of multiple myeloma in 2004 (Cooper and Yao, 2012). Didemnins are cyclic depsipeptides – that is, the peptide contains at least one ester bond in place of an amide link. Didemnins contain two ester bonds; the first bond ensured the linkage of two of the most striking units inserted into the macrocycle, the isostatin (Ist) or norstatin (Nst) units, with the a-(a-hydroxyisovaleryl)-propionic acid (Hip), while in tamandarine the last residue was replaced by the shorter and simpler a-hydroxyisovaleric acid (Hiv). A threonine residue allows not only closure of the macrocycle through an ester bond with the N,O-dimethyl-tyrosine residue, but also the presence of a tail of amino acids inserted onto its amino function. This feature is commonly found in numerous socalled “lariat-type” cyclic depsipeptides of marine origin, such as the families of lyngbyastatins (Kwan et al., 2009), the kahalalide F (which will be discussed below), hassallidin A (which contains a mannose branched on a threonine; Neuhof et al., 2005), and the remarkable almost fully N-methylated coibamide A (Medina et al., 2008), to mention but a few of the most outstanding compounds (Figure 13.14). Despite their resemblances, these peptides share different bioactivities. Lyngbyastatin derivatives are serine protease

Figure 13.13 Didemnins and tamadarines derivatives numbered according to their official nomenclature.

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Figure 13.14 Structure of the lariat-type cyclic depsipeptides kempopeptin A (lyngbyastatin family), kahalalide F, hassallidin A, and coibamide A.

inhibitors that have been cocrystallized with their targets (Salvador et al., 2013; Matern et al., 2003), while didemnin and its derivatives have a multifaceted mechanism of action leading to the abrogation of proteins, DNA, and RNA syntheses, a reduction of VEGF secretion and of its receptor expression (VEGFR-1) by acting on several targets that have not yet been identified (with the exception of elongation factor-1 alpha), ornithine decarboxylase, or the FK-506 apoptotic pathway. Although these interactions have been confirmed, it has been pointed out that these targets, when considered alone, could not account for all of the observed biological effects. The fact that most biological targets of MNPs remain unidentified constitutes a major bottleneck in the comprehension of their mechanism of action, and delays their optimization on a rational basis. Therefore, research effort has been turned towards conventional SAR studies based on the threedimensional structure of the compounds, by speculating which residues are essential for optimal bioactivity and, conversely, by modifying the natural scaffold in order to verify their implication in target recognition. As a forerunner of

MNPs in clinical trials, didemnins have attracted the interest of numerous laboratories that have synthesized dozen of analogs, thus shedding light on their SARs. In briefly summarizing the main results of SAR studies on didemnin B relatives, the tail of amino acids containing N-methyl-D leucine7 is essential for its activity, but certain modifications could be made. Notably, the tail could serve as a point of insertion of a fluorescent label (coumarin or fluorescein), for tags in in vivo studies (benzophenone, or biotin), or of tritium atoms for pharmacokinetics studies. More recently, a folate conjugate was synthesized for the potential treatment of inflammatory diseases (Henne et al., 2012). The macrocycle itself is less prone to modification because of difficulties in its synthesis, and all modifications of the isostatin region induce a drop of activities. Finally, the N,O-dimethyl-L-tyrosine can be replaced by isosteric amino acids only as long as they remain N-methylated. The main results of these studies were reported in two exhaustive reviews that covered the total synthesis, SAR studies, ecological role and clinical trials of didemnins (Vera and Joullie, 2002; Lee et al., 2012). It is notable that the SAR of

13.6 Kahalalide F: A Study in Chemical Ecology as a Starting Point for New Antitumoral Agent Discovery

didemnin can easily be transposed to tamandarine, proving that both molecules have the same biological targets (Lee et al., 2012). This point was reinforced by the fact that, in solution, the two molecules adopt a similar conformation (Vervoort et al., 2000). Surprisingly, none of the modifications performed during SAR studies of the didemnins has provided a new compound that reached the clinical trial stage. Thus, after three decades of continuous effort, although one didemnin derivative has reached its goal by being marketed and clinical trials actively pursued, it was clear that in the conclusion of the last review on didemnin (Lee et al., 2012) there was a hint of skepticism concerning the future of derivatives as novel therapeutics, mainly due to their high costs of synthesis. One solution to producing these compounds would rely on microorganisms, and such an approach is already being used to produce the synthon cyanosafracin B, through fermentation of the bacterium Pseudomonas fluorescens, which permits the hemisynthesis of yondelis (Cuevas and Francesch, 2009). Although in this case the solution was provided by a terrestrial strain, the possible use of marine bacteria should not be discounted, even if they are not always cultivable. Indeed, recent studies have confirm the seminal views of Rinehart and coworkers, who suspected didemnin to be produced from a symbiotic bacterium (Sings and Rinehart, 1996; Schmidt et al., 2012). Hence, didemnin B and nordidemnin B were shown to be the primary metabolites of more complex didemnins produced by different Tistrela strains, even if the association between the ascidian and the microbe has not been yet proved (Tsukimoto et al., 2011; Xu et al., 2012). Nevertheless, the peptide could be produced from these bacteria at a lower cost and under environment-friendly conditions; a genetic modification of the producer could later lead to the production of aplidin. Overall, these results underscore the need to screen the microbial contents of collected marine invertebrates, and perhaps also to focus on marine bacteria as potential drug producers.

13.6 Kahalalide F: A Study in Chemical Ecology as a Starting Point for New Antitumoral Agent Discovery

Kahalalides are lipopeptides originally isolated from the Hawaiian sacoglossan mollusk Elysia rufescens by Mark T. Hamman and Paul J. Scheuer in 1993 (Hamann and Scheuer, 1993). Sacoglossans (Mollusca, Opisthobranchia) are one of few groups of specialized herbivores in the marine environment. These sea slugs feed suctorially on the cell sap of siphonous green algae and of some macroalgae. Since the discovery of “chloroplast retention” in Elysia atroviridis (Kawaguti and Yamasu, 1965), sacoglossan sea slugs have become famous for their ability to extract and incorporate functional chloroplasts from algal food organisms, mainly Ulvophycae, into their gut cells. In some sacoglossan species the sequestered chloroplasts, called kleptoplasts, retain photosynthetic activity for a few days to several months (Marín and Ros, 2004), enabling them to survive during periods of food insecurity

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(Maeda et al., 2012). It has been observed that the photosynthetic products of kleptoplasts (e.g., sugars and amino acids) are transferred to and used by sea slugs when they are starved (Trench, Trench, and Muscatine, 1972). However, the ecological role of kleptoplasts under natural conditions, when food is abundant and nutritional yields from kleptoplasts are lower than from food digestion, remains unclear. Sacoglossan sea slugs also famously steal deterrent substances from their diet algae, which constitutes a second “loot” qualified of kleptochemistry (Clark, Jensen, and Stirts, 1990). These mollusks prefer to live on and eat chemically defended seaweeds; they are able to accumulate metabolites through suction of the algae (Clark, 1992), and produce chemical exudation by selective concentration or by the in vivo transformation of major algal metabolites (Cimino and Ghiselin, 1998). Finally, sacoglossan sea slugs belonging to the genus Elysia, which is widely distributed pan-tropically, have developed numerous diet-derived defense mechanisms and are able to either accumulate sesquiterpenoids (caulerpenyne), diterpenes (udoteal) (Cimino and Ghiselin, 1998), and depsipeptides (kahalalides) (Becerro et al., 2001), to modify such molecules (thuridillins, halimedatrial tetracetate), and to biosynthesize de novo polypropionates (elysione, polypropionatederived c-pyrones) (Gavagnin et al., 1994). Hamman and Scheuer, in their pioneer studies in Hawaii, observed that E. rufescens fed on the green alga Bryopsis sp. However, instead of diterpenes, they then isolated a series of depsipeptides among which kahalalide F appeared to be the most outstanding congener. In another Hawaiian sacoglossan species, Elysia degeneri, that was reported to feed on calcareous green algae Udotea sp., diterpene aldehydes with feeding-deterrent properties were reported (Paul, 1992). Some years later, Scheuer’s group in Hawaii isolated kahalalides (polypropionates have not been found) from E. rufescens and E. ornata, both of which graze on Bryopsis sp. (Horgen et al., 2000). E. grandifolia is an Indian species which the algal diet is B. plumosa, and from which kahalalides have been isolated (Ashour et al., 2006). New kahalalide derivatives were also characterized directly in the green algae Bryopsis sp. (Kan et al., 1999) and B. pennata (Gao et al., 2009). To date, 24 kahalalides including 19 cyclic peptides, kahalalide A (KA), norKA, kahalalides B–F, isokahalalide F, 5-OHKF, K, kahalalides O–Q, R1, S1, R2, S2, W, Y, and five linear peptides, kahalalides G, H, J, V, X, ranging from a C31 tripeptide to a C77 tridecapeptide, have been isolated from the mollusks E. rufescens, E. ornata, and E. grandifolia, or from the green alga Bryopsis sp. (Table 13.2). As is the case for dolastatins, the kahalalide family is very heterogeneous, and includes both cyclic and linear peptides, ranging from three to 13 amino acid residues. Common to all kahalalides is a fatty acid unit conjugated to the N terminus; this fatty acid unit can be either:  A 3-hydroxy-methylocta- or decanoic acid. In this case the cyclization, when it occurs, is formed via an ester linkage involving the C terminus and the 3-hydroxy function of the fatty acid (Figure 13.15),

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Table 13.2 Kahalalides: the organisms from which they are isolated, and their biological activities.

Cyclic

Kahalalide

Species

Biological activities

References

A

Elysia rufescens Bryopsis sp. Bryopsis pennata Elysia rufescens Bryopsis sp. Elysia rufescens, E. grandifolia (Malvan) Elysia rufescens, E. grandifolia (Malvan) Elysia rufescens Elysia rufescens, E. grandifolia (Malvan) Bryopsis pennata, B. plumosa (Malvan) Bryopsis pennata Bryopsis pennata Elysia rufescens, Bryopsis sp. Elysia ornata Bryopsis sp. Bryopsis sp. Bryopsis sp. Elysia grandifolia (Mannar) Elysia grandifolia (Mannar) Elysia grandifolia (Malvan) Elysia grandifolia (Malvan) Elysia rufescens Elysia rufescens Bryopsis pennata Elysia rufescens, E. grandifolia (Malvan) Bryopsis sp. (Hawaii) Elysia rufescens Elysia rufescens Elysia rufescens Elysia rufescens

Modest antimalarial activity

Hamann et al., 1996

Y1 receptor ligand

Gao et al., 2009 Hamann et al., 1996

norKA B C D E F isoKF 5-OHKF K O P Q R1 S1 R2 S2 W Y Linear

G H J V X

Antiviral activity against HSV II Cytotoxic, antiviral Cytotoxic Moderate cytotoxicity No activity No activity

Hamann et al., 1996 Hamann et al., 1996 Hamann et al., 1996 Hamann and Scheuer, 1993 Gao et al., 2009 Gao et al., 2009 Gao et al., 2009 Kan et al., 1999 Horgen et al., 2000

No significant activity No significant activity Cytotoxic Low cytotoxicity Not tested Not tested

Dmitrenok et al., 2006 Dmitrenok et al., 2006 Ashour et al., 2006 Ashour et al., 2006 Tilvi and Naik, 2007 Tilvi and Naik, 2007 Rao et al., 2008 Rao et al., 2008

No activity

Hamann et al., 1996

No activity No activity

Goetz, Nakao, and Scheuer, 1997 Goetz, Nakao, and Scheuer, 1997 Rao et al., 2008 Rao et al., 2008



All Elysia rufescens, E. ornata, Bryopsis pennata and Bryopsis sp. have been collected in O’ahu Island, Hawaii; Elysia gandifolia and Bryopsis plumosa have been collected in Malvan, west coast if Indian or ashore in the Gulf of Mannar, India.

 A butanoic (eventually a methylbutanoic), methylhexanoic or octanoic acid. The cyclization, when it occurs, is formed via an ester linkage in a head-to-side chain manner between the C terminus and a b-hydroxylated amino acid, a threonine and, in one case, a serine unit (Figure 13.16). The most important peptides of this series are kahalalide F (KF) and its analogs, which were licensed to PharmaMar by the University of Hawaii during the 1990s and the subjects of diverse patent applications for the treatment of tumors and viral infections in mammals (Jimeno et al., 2002) and the treatment of psoriasis (Izquierdo Delso, 2004). In 2000, KF entered Phase I clinical trials in Europe for the treatment of androgen-independent prostate cancer (Rawat et al., 2006). The activity of KF was then investigated in Phase II clinical studies with patients having liver, non-small-cell lung cancer (Provencio et al., 2009) and melanoma. KF has also been tested in Phase II clinical trials for severe psoriasis (Gao and Hamann, 2011). IsoKF (elisidepsin, PM02734; Irvalec), the 4S-methylhexanoic isomer of KF, currently obtained via synthetic process, has been developed by PharmaMar in a Phase II clinical study (Serova

et al., 2013). All are 13-amino acid peptides with a 6-amino acid residue depsicycle and a 7-amino acid residue side chain ending with a fatty acid moiety. As with the lyngbyastatins, which are cyclic depsipeptides isolated from cyanobacteria belonging to the genus Lyngbya, relatives of KF contain the Z-dehydrobutyric acid (Z-Dhb) residue, which is commonly encountered in cyanobacterial peptides, but are devoid of the N-methylated amino acid residues that are characteristic of prokaryotic secondary metabolites (Figure 13.17). The analogs of KF exhibit only minor changes in their amino acid sequences and are the result of isosteric replacement, whereby D-allo-Ile is replaced by valine in KR1, or ornithine by lysine in KS2. The fatty acid is also the site of slight modifications that include hydroxylation found in 5-OHKF, or the isomeric position of the methyl group, as in isoKF (elisidepsin, PM02734; Irvalec). In most cases, when the stereochemistries are known, they are conserved at the Ca and Cb atoms, which not only led to the suspicion of a common biosynthetic pathway for these peptides but also highlighted the importance of preserving their bioactivity. Among the many kahalalides tested for their biological properties the most outstanding have been

13.6 Kahalalide F: A Study in Chemical Ecology as a Starting Point for New Antitumoral Agent Discovery

Figure 13.15 Kahalalides with a 3-hydroxy C9 and C11 fatty acid (indicated by red) on the N terminus.

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Figure 13.16 Kahalalides with a C4, C5, or C7 fatty acid (indicated by red) on the N terminus.

Figure 13.17 Kahalalide F and its analogs. The N-terminal fatty acid is indicated in red, and the non-natural amino acid Z-Dhb in blue.

13.6 Kahalalide F: A Study in Chemical Ecology as a Starting Point for New Antitumoral Agent Discovery

isoKF, 5-OHKF, KR1 and, of course KF, and all have shown significant activities. All acyclic peptides of the kahalalides family, such as KG – the counterpart of KF – lack activity. The gross structure of KF, as described initially by Scheuer’s group in 1993, was reported to contain three D-Val and two L-Val, along with one D-allo-Thr and one L-Thr, leading to the existence of multiple possible stereoisomers. The determination of an absolute configuration of the molecule has represented a serious challenge due to its complexity, particularly with regards to the correct positions in the sequence of the three D-Val and two L-Val units, together with the D-allo and L-Thr isomers. A first report on KF stereochemistry (Goetz, Yoshida, and Scheuer, 1999) assigned the configurations D and L to Val10 and Val11, respectively, and configurations D-allo- and L- to Thr6 and Thr12, respectively. Further investigations carried out independently by Rinehart and coworkers revaluated the stereochemistry of KF by proposing to switch the absolute configuration of the two valines (Bonnard, Manzanares, and Rinehart, 2003). The two compounds were synthesized by the group of Albericio and Giralt, and compared to natural KF using NMR spectroscopy, chromatographic analyses and activity assays, and confirming without any doubt the correctness of this last proposition (L opezMacia et al., 2001). This achievement permitted the launch of SAR studies in order to offer new kahalalide analogs with improved pharmacological properties, and also to identify the importance of a structural framework on the biological activity of this series of peptides. No less than 143 new KF analogs have been obtained by the groups of Hamann (Shilabin et al., 2007) and Albericio (Jimenez et al., 2008). To resume these studies, the three parts of KF – the depsipeptide ring, the side chain, and the aliphatic moiety – are considered separately. First, as acyclic KG was found to be inactive, it would appear that the ring is essential for biological activity, and that the non-natural amino acids, (Z)-Dhb, D-alloThr, along with the alternated L/D configurations of the a-carbon, have a key role in maintaining the rigidity and enzymatic stability of the ring, and thus are crucial for activity. The introduction of any residue that increases the hydrophobicity at the Phe position will increase activity. The peptide tail is rich in aliphatic residues, including a D-allo-Ile, three Val (two D and one L) and a D-Pro, and also contains two polar residues, one Thr and one Orn. The elimination of any residue and the reversion of any configuration – except that of Val13 in the peptide tail – will induce a loss of activity. The importance of chirality at the a-carbon atoms suggests that this part of the peptide can adopt or induce some sort of folding and/or interactions with another molecule (Jimenez et al., 2008). Some amino acid residues in the tail play a more important role than others; D-Pro and the configuration of Val10 and Val11, as previously observed when the total stereochemistry of KF was determined, are essential, although the hydroxy group of the Thr is not necessary for antitumoral activity. Orn, which is the only charged amino acid at physiological pH, appears to be unnecessary and almost undesirable for activity (Jimenez et al., 2008). Orn can be replaced by Lys, in synthetic analogs and in the natural product KS2, without losing activity, and

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analogs resulting in coupling of the amino group of Orn to an aliphatic group are the most active compounds of the series. Finally, the broad range of active compounds modified on Orn suggests that this residue is not involved in any recognition interface (Jimenez et al., 2008). The side chain of the peptide ends with a fatty acid moiety that is also crucial for activity. Compounds with no N-terminal fatty acid or short aliphatic group have lower activities than KF. Any introduction of polar groups capable of generating hydrogen bonds will decrease the activity. This observation for synthetic analogs was made previously for natural compounds 5-OHKF and KS1. Finally, synthesis of the KF enantiomer devoid of biological activity is a clue that KF acts via a specific recognition mechanism (Jimenez et al., 2008). As noted by Albericio and Hamann, KF has a more defined structure than expected, and is highly sensitive to stereotypical changes that affect the chirality of a-carbon atoms of the residue. Overall, this SAR study led to the conclusion that “Nature does things right,” because if some synthesized analogs have a higher activity than the natural KF or isoKF, then none of these synthetic compounds will be developed in clinical studies. However, they do provide the possibility of generating tools to study the biological activities of kahalalide. Kahalalide F analogs conjugated with gold nanoparticles (NPs) have been located subcellularly at lysosome-like bodies (Hosta et al., 2009). Furthermore, isoKF modified with a fluorophore coupled to the amino function of ornithine indicated that isoKF could insert into the cell membrane via a selfassociation process that, in turn, altered the normal architecture and function of the membrane (presumably by pore formation), leading to a lytic process that resulted in cell death (MolinaGuijarro et al., 2011; Shilabin and Hamann, 2011). Multiple targets involved in the action of KF have been identified, many of which are associated with the hydrophobic nature of the compounds as lysosomes or plasma membrane. KF has been found to interact with the epidermal growth factor receptor (ErbB) family, which promotes cell proliferation and opposes apoptosis. The Erb family receptors play an important role in the pathogenesis and progression of certain aggressive cancers. The exposure of breast, vulval and hepatic human tumor cell lines to KF induced a downregulation of protein expression levels of ErbB3 (Janmaat et al., 2005). Detailed information on the biological activities, mechanism of action and clinical status of KF is provided in an excellent review (Gao and Hamann, 2011). Concerning the biosynthetic origin of kahalalides, it should be emphasized that extensive epiphyte growth, including that dinoflagellates, cyanobacteria and diatoms, was observed in association with Bryopsis (Kan et al., 1999). The structural similarities between kahalalides and cyclic depsipeptides of cyanobacterial origin suggest that kahalalides may have an ancient evolutionary origin within cyanobacteria. This hypothesis would be in agreement with an involvement of the PKS and NRPS pathways in the biosynthesis of such peptides that bear typical structural carbon architectures of prokaryotes. Hamann and Hill described the production of KF by a Vibrio associated with both the algae Bryopsis sp. and Elysia rufescens (Hill et al.,

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2007). The hypothesis of Hamann’s group was that E. rufescens acquires the Vibrio from the surface of Bryopsis, and maintains these microbes as symbionts. As genes involved in the biosynthetic process and comprising nucleotide sequence information are not yet accessible, a compelling knowledge of the origin of kahalalides is currently unavailable. The isolation of kahalalides had demonstrated variability in the production of the peptides. KB was first reported in high yield from E. rufescens, after which only trace amounts were identified from recent collections. KK, which has been found in Bryopsis sp., was later isolated in a collection of E. rufescens (Kan et al., 1999). These findings suggest that the production of kahalalides could potentially be from a microbial association with the mollusk and the algae, and that this relationship could continuously evolve in response to changes in the environment and predation.

13.7 Azole/Azoline-Containing Cyanobactins Isolated from Invertebrates: An Example of Nature’s Own Combinatorial Chemistry

As noted above, cyanobacteria have been shown as a prolific source of novel bioactive natural products, with recent studies having centered attention on a group of small cyclic peptides, the cyanobactins. Cyanobactins are N-to-C low-molecular-weight macrocyclic peptides that belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The major structural and biosynthetic categories of RiPPs was first summarized in 2009 (McIntosh, Donia, and Schmidt, 2009); more recently, however, a comprehensive review was assembled by 65 authors from various countries under the coordination of Wilfred van der Donk (Arnison et al., 2013), detailing up-todate knowledge of the biosynthesis of the more than 20 distinct classes of RiPPs. The review also proposed a recommended

nomenclature for the biosynthesis of this rapidly growing class of natural products. The specific subclass of cyanobactins has been the subject of several recent reviews focused on chemical diversity (Sivonen et al., 2010; Houssen and Jaspars, 2010), metal-binding properties (Bertram and Pattenden, 2007), strategies used for the identification (Velasquez and Van der Donk, 2011), and biosynthetic mechanisms (Donia, Ravel, and Schmidt, 2008a; Schmidt and Donia, 2009; Katoh et al., 2011). A comprehensive review was also the subject of a book chapter by one of the leading groups in the field (Donia and Schmidt, 2010). Hence, in this section the decision was taken to focus only on azole- and azoline-containing cyanobactins isolated from Prochloroncontaining marine invertebrates, with the aim of introducing these structurally and functionally diverse metabolites. Prochloron spp. are uncultivated cyanobacteria commonly found as symbiotic partners of tropical didemnid ascidians. At an early stage they were suspected of being the producers of cyanobactins which, originally, were defined as containing oxazolines or thiazolines, or their oxidized derivatives oxazoles and thiazoles acids, alternating with “natural” amino acids. This definition was recently broadened to include cyclic peptides that consist solely of proteinogenic amino acids (Sivonen et al., 2010). The heterocyclization of threonines (Thr), serines (Ser) and cysteines (Cys) is a common post-translational modification in cyanobactin biosynthesis, as well as disulfide bridge formation or prenylation or geranylation. Heterocyclized Cys, Ser and Thr residues have been found in many different RiPPs, and are also known from NRPs. These heterocycles are formed when the side-chain thiol or hydroxy of Cys, Ser or Thr attacks the carbonyl carbon of the adjacent amino acid, yielding thiazoline (Tzl), oxazoline (Ozl) or methyloxazoline (mOzl), respectively. Further oxidation of the a–b linkage yields thiazole (Tzn), oxazole (Ozn), or methyloxazole (mOzn) (McIntosh, Donia, and Schmidt, 2009) (Figure 13.18).

Figure 13.18 Heterocyclization and subsequent oxidation of cysteine, threonine and serine to form thiazole/thiazoline (Tzl/Tzn) and (methyl)oxazole/ (methyl)oxazoline [(m)Ozl/(m)Ozn]. The sequence Val-Cys-Thr-Ala-Ser is hypothetical, but the heterocyclized and oxidized final portion is contained in leucamide A (Kehraus et al., 2002).

13.7 Azole/Azoline-Containing Cyanobactins Isolated from Invertebrates: An Example of Nature’s Own Combinatorial Chemistry

Almost all heterocyclized cyanobactins found in marine invertebrates are isolated from the three didemnid ascidians Lissoclinum patella, Lissoclinum bistratum, and Didemnum molle. These tropical species are Prochoron-containing ascidians, and it was speculated at an early stage that these cyanobacterial symbionts were the producers of cyanobactins. A few examples exist of azole- and azoline-containing cyanobactins in sponges and mollusks. In the case of sponges, it is likely that the cyanobactins are also synthetized by cyanobacterial symbionts. However, in the case of mollusks it is probably a dietary strategy, as they consume either cyanobacteria or cyanobacteria-containing ascidians or sponges. Various studies have shown that some mollusks actively select sponges or sponge zones with high concentrations of cyanobacteria (Becerro et al., 2003). The structure of ulicyclamide and ulithiacyclamide was elucidated in 1980 by Ireland and Scheuer (Ireland and Scheuer, 1980), since when more than 70 azole/azoline-containing cyanobactins have been described from ascidians, sponges, or

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mollusks. These are structurally diverse, and range in size from six to eight amino acids with varying numbers and combinations of oxazoles, oxazolines, thiazoles, and thiazolines. Azole/azoline-containing cyanobactins from marine invertebrates can be classified in three groups: hexapeptides, heptapeptides, and octapeptides. More than half of the members of the cyclohexapeptides (18-atom macrocycle) group belong to a series of analogs based on the bistratamide platform (Figure 13.19). These are isolated mainly from Lissoclinum bistratum, and also from Didemnum molle and from the gastropod Dolabella auricularia. Among this group are also compounds that include generally only one azole/azoline heterocycle (Figure 13.20) isolated from the ascidians Didemnum molle comoramides A-B (Rudi et al., 1998), mollamides B–C (Donia et al., 2008b), E–F (Lu et al., 2012), hexamollamide (Teruya, Sasaki and Suenaga, 2008), and Lissoclinum patella patellins 1-2 (Caroll et al., 1996), and from the gastropod mollusk Pleurobranchus forskalii keenamide A (Wesson and Hamann, 1996).

Figure 13.19 Structural variation within bistratamide-related cyclohexapeptides. Tzl ¼ thiazole; Tzn ¼ thiazoline; Ozl ¼ oxazole; Ozn ¼ oxazoline; mOzl ¼ methyloxazole; mOzn ¼ methyloxazoline.

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Figure 13.20 Structural variation within non-bistratamide-related cyclohexapeptides. Organisms in which compounds were isolated are indicated in brackets. Tzl ¼ thiazole; Tzn ¼ thiazoline.

A high proportion of cycloheptapeptides (21-atom macrocycle) belongs to a series of analogs based on the lissoclinamide platform (Figure 13.21), isolated from Lissoclinum patella. This group is also composed of compounds including one, two, or three azole/azoline heterocycles in the peptide backbone (Figure 13.22). They are isolated from the ascidians Didemnum molle cyclodidemnamide (Norley and Pattenden, 1998), cyclodidemnamide B (Arrault et al., 2002), mayotamides A–B (Rudi et al., 1998), leucamide A (Kehraus et al., 2002), mollamide (Carroll et al., 1994), and Lissoclinum sp. trunkamide A (Wipf and Uto, 2000) or from the sponge Haliclona nigra haligramides A-B (Rashid et al., 2000), Ircinia dendroides waiakeamide (Mau et al., 1996) and the mollusks Dolabella auricularia dolastatin 3 (Pettit et al., 1987a) and Hexabranchus sanguineaus sanguinamide A (Dalisay et al.,

2009). Ceratospongamide (Tan et al., 2000) was isolated from the marine red alga Ceratodictyon spongiosum containing the symbiotic sponge Sigmadocia symbiotica. The third group, the cyanobactins containing eight amino acids (24-atom macrocycle), consists of molecules based on the patellamide platform. This platform is characterized by the presence of two thiazole and two oxazoline heterocycles distributed symmetrically in the peptide backbone (Figure 13.23). This group is also composed of compounds that can be considered as precursors of the patellamide platform (Figure 13.24), such as patellamide G (Fu et al., 1998), prepatellamide A (Fu, Su, and Zeng, 2000), or ulithiacyclamides F–G (Fu et al., 1998). Tawicyclamides A–B (Mcdonald et al., 1992), patellins 3–6 (Carroll et al., 1996), sanguinamide B (Dalisay et al., 2009) and haliclonamides A–B (Guan et al., 2001) complement this third

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Figure 13.21 Structural variation within lissoclinamide-related cycloheptapeptides. Tzl ¼ thiazole, Tzn ¼ thiazoline.

group, and represent different families of cyclic octapeptides possessing thiazole and/or thiazoline but lacking either the oxazoline ring or the symmetrical distribution characteristic of cyclic peptides from L. patella. With few exceptions, all of the azole- or azoline-containing cyclooctapeptides are isolated from Lissoclinum patella, which makes this species and its obligate symbiont an extraordinary source of chemical diversity. Azole- and azoline-containing cyanobactins isolated from Prochloron-containing marine invertebrates are characterized by an alternating sequence of five-membered heterocycles and hydrophobic amino acid residues. Although methods using nuclear Overhauser effect (NOE)-restrained molecular dynamics (Morris et al., 2000), an advanced Marfey’s method (Fujii et al., 2002) or structure elucidation at the nanomolar scale (Dalisay et al., 2009) were developed, the structure of many of these compounds had to be either confirmed (Pettit et al., 1987a; Wipf and Fritch, 1996; Arrault et al., 2002) or revised after total

synthesis (see for example, Hamada, Shibata, and Shioiri, 1985a; Hamada, Shibata, and Shioiri, 1985b; Boden and Pattenden, 1994; Norley and Pattenden, 1998; Wipf and Uto, 2000; Boden and Pattenden, 2000). The presence of five-membered heterocycle residues, often symmetrically distributed, limits the conformational flexibility of the macrocycle. Depending on the size of the macrocycle (whether 18-, 21-, or 24-atom), these highly backbone-modified cyclopeptides may adopt molecular “triangle,” “square,” and “twisted eight” conformations in solution and the solid state (Schmitz et al., 1989; Ishida et al., 1992; Mcdonald et al., 1992; Ireland et al., 1982). The presence of five-membered heterocycles residues promotes also conformational exchanges. Cis,cisand trans,trans-ceratospongamides (Figure 13.22) are stable conformational isomers (Tan et al., 2000). The conformational variation of ascidiacyclamide (Figure 13.23) was studied with X-ray diffraction (Ishida et al., 1992). It has been suggested that conformational changes are necessary in order to provide the

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Figure 13.22 Structural variation within non lissoclinamide-related cycloheptapeptides. Organisms in which compounds were isolated are indicated in brackets.

optimum binding site for divalent metals (Morris and Jaspars, 2000). The topology of the compounds based on lissoclinamide (Figure 13.21) and patellamide (Figure 13.23) platforms has long led to speculation that metal binding may be a role of cyanobactins in Nature, and experimental evidence in favor of metal binding has indeed been achieved in several instances (Bertram and Pattenden, 2007; Morris and Jaspars, 2000). Some of these molecules have shown a preferential binding to Cu(II)

and Zn(II) (Freeman et al., 1998), while others such as haliclonamides (Figure 13.24) were shown to bind specifically to Fe(III) and Cr(III) (Guan et al., 2001). In the latter case, it was shown that the concentration of iron in Haliclona sp. sponge tissue was 100-fold higher than that in sea water, and 20- to 100-fold higher than in various types of other sponge tissues collected in the same area. It was concluded that iron accumulation in the sponge Haliclona sp. tissue may be related to the production of peptides haliclonamides A and B. However, although

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Figure 13.23 Structural variation within patellamide-related cyclooctapeptides isolated from Lissoclinum patella.

azole- and azoline-containing cyanobactins have shown significant biological activities, the relationship between metal-binding properties and activities has not been established. Conformational studies, metal-binding properties and biological activities from Lissoclinum patella were reviewed by Houssen and Jaspars (Houssen and Jaspars, 2010). As has been seen, azole- and azoline-containing cyanobactins exhibit considerable diversity in their chemical compositions.

This chemical diversity explains the activities shown by these peptides in a number of biological screenings. As is often the case, most of the interest in cyanobactins as potential new drugs has focused on anticancer activity. It has been observed that slight modifications in the chemical structure of some compounds (e.g., lissoclinamides 4 versus 5, ulithiacyclamides A versus B) resulted in significant differences in biological activity (Hawkins et al., 1990).

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Figure 13.24 Structural variation within non-patellamide-related cyclooctapeptides. Organisms in which compounds were isolated are indicated in brackets.

Until very recently, essentially nothing was known of cyanobactin biosynthesis, and only after cloning and sequencing of the genetic elements required for cyanobactin biosynthesis was their biosynthetic origin deduced. Donia and coworkers showed that Prochloron spp. could generate a diverse library of patellamides using small, hypervariable cassettes within a conserved genetic background. Each symbiont strain contained a single pathway, and mixtures of symbionts within ascidians led to the accumulation of chemical libraries (Donia et al., 2006). These complex natural peptides were biosynthesized by extensive posttranslational modification of ribosomally synthesized precursor peptides (McIntosh, Donia, and Schmidt, 2009). The biosynthetic genes for cyanobactin production have been described in distantly related cyanobacteria such as Prochloron, Trichodesmium, Microcystis, Nostoc, Lyngbya, and Anabaena (Sivonen et al., 2010). Recent advances have included heterologous production of patellamide D and ascidiacyclamide (Long et al., 2005) or the prenylated antitumor preclinical candidate trunkamide (Donia, Ravel, and Schmidt, 2008a), metagenome mining for engineering cyanobactin pathways (Schmidt and Donia, 2009), biochemical characterization of ribosomal peptide biosynthetic pathways (Koehnke et al., 2012; Leikoski et al., 2012; Agarwal

et al., 2012), or the rational synthesis of complex, new molecules over multiple-step biosynthetic pathways using genetic engineering (Tianero et al., 2012).

13.8 Conclusion

Peptides are often regarded as simple molecules, and their synthesis as an easy task. The discovery during the past decade of hundreds of different NRPs and RiPPs from marine organisms, and often from a single genus (e.g., Lyngbya, Anabaena, Lissoclinum, Dolabella, Elysia), illustrates the potential of these organisms, or of their symbionts or their diet sources, to execute their own combinatorial biosynthesis and structure optimization. The examples presented above underline not only the chemical variety of MNPs but also the difficulties faced by chemists when engaged in their synthesis. It is notable that MNPs, after having been studied by classical approaches comprising sample collection, product isolation, structure determination and bioassay, will be investigated in the near future using modern

References

techniques of biology that will encompass molecular biology and bioinformatics, with the aim to shed light on their biosynthesis, and to further exploit these tools for their production without exhausting natural resources (Leal et al., 2012; Kalaitzis, Lauro, and Neilan, 2009; Lane and Moore, 2011; Johnston, Ibrahim, and Magarvey, 2012). This could be completed by hemi-syntheses using bacterial strains of terrestrial origin, as is the case for the production of Yondelis (Cuevas and Francesch, 2009). Furthermore, the identification and structural elucidation of these compounds could be aided by applying powerful techniques such as capillary probe and microcryoprobe NMR (Molinski, 2010). It is noteworthy that marine natural peptides include interesting features such as a high content of N-methylated, D series and nonribosomal amino acids, along with cyclization-inducing conformation constraints that confer on them their exquisite bioactivity and enhanced stability.

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It is evident that most of the macroscopic or microscopic species which inhabit the oceans remain unknown, and that the exploitation of these resources can provide the therapeutic armamentarium of tomorrow. In particular, when considering microbial diversity, many compounds originally isolated from marine animals are in fact produced by microbial symbionts (didemnin) or are accumulated during the course of feeding by grazers (dolastatin). In addition, microorganisms may assist in the semisynthesis of these materials (e.g., Yondelis).

Acknowledgments

These studies on laxaphycines have been funded by ANR (ANRF2010FBLANF1533F02) and by la Ligue Nationale Contre le Cancer (LNCC).

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the marine mollusk Pleurobranchus forskalii. J. Nat. Prod., 59, 629–631. Williams, D., Moore, R., and Paul, V. (1989) The structure of ulithiacyclamide-B – antitumor evaluation of cyclic-peptides and macrolides from Lissoclinum patella. J. Nat. Prod., 52, 732–739. Wipf, P. and Fritch, P.C. (1996) Total synthesis and assignment of configuration of lissoclinamide 7. J. Am. Chem. Soc., 118, 12358–12367. Wipf, P. and Uto, Y. (2000) Total synthesis and revision of stereochemistry of the marine metabolite trunkamide A. J. Org. Chem., 65, 1037–1049. Xu, Y., Kersten, R.D., Nam, S.-J., Lu, L., Al-Suwailem, A.M., Zheng, H., Fenical, W., Dorrestein, P.C., Moore, B.S., and Qian, P.-Y. (2012) Bacterial biosynthesis and maturation of the didemnin anti-cancer agents. J. Am. Chem. Soc., 134, 8625–8632. Zabriskie, T., Klocke, J., Ireland, C., Marcus, A., Molinski, T., Faulkner, D., Xu, C., and Clardy, J. (1986) Jaspamide, a modified peptide from a Jaspis sponge, with insecticidal and antifungal activity. J. Am. Chem. Soc., 108, 3123–3124. Zainuddin, E.N., Jansen, R., Nimtz, M., Wray, V., Preisitsch, M., Lalk, M., and Mundt, S. (2009) Lyngbyazothrins A D, antimicrobial cyclic undecapeptides from the cultured cyanobacterium Lyngbya sp. J. Nat. Prod., 72, 1373–1378. Zampella, A., D’Auria, M.V., Paloma, L.G., Casapullo, A., Minale, L., Debitus, C., and Henin, Y. (1996) Callipeltin A, an anti-HIV cyclic depsipeptide from the New Caledonian lithistida sponge Callipelta sp. J. Am. Chem. Soc., 118, 6202–6209. Zheng, L.-H., Wang, Y.-J., Sheng, J., Wang, F., Zheng, Y., Lin, X.-K., and Sun, M. (2011) Antitumor peptides from marine organisms. Mar. Drugs, 9, 1840–1859.

About the Authors Bernard Banaigs is currently a Research Scientist employed by the French National Institute of Health and Medical Research (INSERM) at the “Chimie des Biomolecules et de l’Environnement” laboratory, University of Perpignan, France. He received his PhD in Organic Chemistry in 1983 from the University of Montpellier, France, and joined the University of Perpignan in 1986. He received the Habilitation qualification (HDR) in 2001. Bernard’s research interests include marine natural products chemistry, specifically focused on chemical ecology and medicinal chemistry. He is the author and coauthor of over 70 research papers and patents. Isabelle Bonnard obtained her academic degrees and PhD (in 1996) from the University of Perpignan after a specialization in marine chemistry at the University of Brittany. In continuing

her thesis on the isolation and structural determination of marine cyclic lipopeptides from cyanobacterial origins, she joined the team of Prof. Kenneth Rinehart, University of Illinois at Urbana-Champaign (USA), for a postdoctoral period (1997–1998), working on new saponins from holothuries and studying the configurational structure of marine depsipeptides. She was appointed lecturer in 1998 at the University of La Reunion, developing marine natural products isolation and structural elucidation from diverse marine organisms of the Mascareign area, and then moved in 2004 to the “Chimie des Biomolecules et de l’Environnement” laboratory, University of Perpignan, focusing her research interest on chemical ecology. Anne Witczak obtained her PhD at the university of Lille 1 in 1996 (studies and synthesis of potential topoisomerase 2

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inhibitors), and was appointed assistant professor in 1999 at the University of Perpignan, France. She then joined the group of Bernard Banaigs in the “Chimie des Biomolecules et de l’Environnement” laboratory, and focused her interest on peptides from marine sources. She is currently Vice President of Student Life and Culture at the University of Perpignan. Nicolas Inguimbert obtained his PhD at the university of Montpellier 2, and then joined the INSERM Laboratory of

Prof. B.P. Roques, where he specialized in medicinal chemistry and was appointed assistant professor in 2000. After having developed metallopeptidase inhibitors, he switched to peptide synthesis and studied protein–protein interactions with the aim of developing antagonists of the vascular endothelial growth factor receptor. He recently joined the university of Perpignan as professor, and focused his interests on peptides from marine sources. He has authored 30 publications and two patents.

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14 Conotoxins and Other Conopeptides Quentin Kaas and David J. Craik

Abstract

Conopeptides are a large family of peptide toxins produced by marine cone snails. They act with high potency and exquisite specificity on a range of ion channels and transporters of the nervous system, making them valuable drug leads and important molecular probes in neurophysiological studies. Most conopeptides are small, ranging from 10 to 30 amino acid residues, but some contain up to about 90 amino acids. They display a large chemical diversity because they have very diverse sequences and a large number of post-translational modifications. Disulfide-rich conopeptides are commonly referred to as

14.1 Background 14.1.1 Historical Interest in Cone Snails

Cone snails are marine carnivorous gastropods that have been linked to human history since ancient times because their beautiful shells are valuable objects that have been used as ornaments, or even as investments (Terlau and Olivera, 2004). One of the most striking examples of their worth comes from the late eighteenth century, when one shell from Conus gloriamaris was auctioned at a price three-times higher than the masterpiece painting of Vermeer, Woman in Blue Reading a Letter (Conniff, 2009; Olivera et al., 1990). Besides the appealing delicate patterns of their shells, cone snails are notoriously dangerous animals, and several fatalities resulting from their stings have been reported (Cox, 1884; Macgillivray, 1860; Nagami, 2005; Terlau and Olivera, 2004; Yoshiba, 1984). Current scientific interest in cone snails mainly focuses on understanding their biology and evolutionary history, as well as using their venoms for pharmaceutical applications. 14.1.2 Biology of Cone Snails

Cone snails live mainly in tropical seas and oceans, but also are found in some temperate waters. They are especially prevalent in the Indo Pacific region, and particularly abundant in South-

conotoxins, and have particularly diverse structures and a broad range of molecular targets. One conotoxin, MVIIA (also named Prialt1 or ziconotide), is an N-type calcium channel blocker that is used clinically as an analgesic to treat neuropathic pain. Other conopeptides have also attracted considerable interest for their potential pharmaceutical applications. In this chapter, the marine cone snails and their venoms are introduced and the chemical diversity of conopeptides is described, along with the techniques used to unravel this diversity. The three-dimensional structures of conopeptides are discussed and linked to their pharmacological activities.

East Asian seas. Of the more than 550 species belonging to the Conus genus (Duda, Kohn, and Matheny, 2009b; R€ ockel, Kom, and Kohn, 1995), about 50 are known to prey on fish, whereas most feed on worms or molluscs.1) The diversity of targets and the difference in pace between the fast-moving prey and the slow cone snails are possibly at the origin of their peculiar hunting strategy, which relies on chemically complex venoms and a specialized toxin delivery apparatus (Kohn, Saunders, and Wiener, 1960) (Figure 14.1). Cone snails have a proboscis capped by a spear-shaped radular tooth that acts as a syringe to inject venoms into targeted animals (Olivera, 2002). Offensive and defensive behaviors can differ widely between cone snail species; for example, some species can fire teeth in rapid succession, or diffuse numbing substances into the surrounding water, or extend their mouth to engulf entire schools of fish before stinging them one by one from inside their mouth (Olivera, 2002). There is substantial academic interest in studying the evolution and biology of these intriguing animals, but even greater interest has been associated with the venomic compounds and their pharmaceutical applications (Adams et al., 1999; Craik and Adams, 2007; Endean, Parish, and Gyr, 1974; Livett et al., 2006; Olivera and Cruz, 2001; Terlau and Olivera, 2004; Twede et al., 1) The Conus Biodiversity Web site (http://biology.burke.washington.

edu/conus/) is a valuable resource on cone snail species. Another important Internet resource is The Cone Snail (www.theconesnail. com), which is maintained by the laboratory of Baldomero Olivera, a pioneer in conopeptide discovery.

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by Stephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Figure 14.1 Cone snail envenomation apparatus. (a) Cone snails, such as Conus adamsonii shown here, have a siphon with which they detect prey or predators. In the snail’s mouth is a proboscis from which radular teeth are thrust, like harpoons, to tether the prey (Olivera, 2002); (b) Contraction of the venom bulb pushes the venom produced in the venom duct into the proboscis and through the radular tooth into the prey; (c) Radular teeth, here from Conus striatus, have a harpoon shape and act as hypodermic needles to inject venoms in the targeted animal. The image of Conus adamsonii was taken by Xavier Curvat, and is used with his permission. Courtesy of Xavier Curvat. The image of the cone snail radular tooth was kindly provided by Sebastien Dutertre. Courtesy of Sebastien Dutertre.

2009; Vincler and McIntosh, 2007). This interest derives from the pharmaceutical relevance of their molecular targets. 14.1.3 Cone Snail Venoms, their Conopeptides and Molecular Targets

Conopeptides are gene-encoded peptides that are typically 10 to 30 amino acids in size and are heavily post-translationally modified (Gray et al., 1981; Kaas, Westermann, and Craik, 2010; Olivera and Cruz, 2001) (Figure 14.2). The most common post-translational modification is the formation of disulfide crosslinks, which is characteristic of the conotoxin subgroup of conopeptides. Each cone snail species expresses between 200 and 2000 different conopeptides, and current estimates of the total number of different conopeptides vary between thousands

and hundreds of thousands of peptides (Davis, Jones, and Lewis, 2009; Olivera, 2002; Olivera and Cruz, 2001). The uncertainty in their upper estimate arises from recent studies that have discovered large intraspecies and even intraspecimen diversity (Davis, Jones, and Lewis, 2009). Most conopeptides tested so far act on ion channels of the nervous systems (Olivera and Teichert, 2007). The high degree of conservation of these targets in animals explains why conopeptides are active in humans, despite humans not being part of the cone snails’ diet. Every animal with a nervous system expresses a wide range of ion channel subtypes, which have different expression patterns throughout the nervous system and are involved in different functions (Bagal et al., 2013). Conopeptides display unparalleled potency and specificity for these receptor subtypes, and act with exquisite specificity and sometimes concerted activity during prey capture (Terlau and Olivera, 2004). For example, some conopeptides trigger maximum stimulation of the central nervous system (CNS), while others cause tetanic spasms or inhibit neuromuscular transmission, causing paralysis. Some conopeptides have also been shown to inhibit CNS functions, resulting in a rapid sedation of the prey before other toxins cause longer-term tetany (Terlau and Olivera, 2004). The expression patterns of ion channel subtypes differ between species, and their stimulation or inhibition therefore have different effects in fish and mammals. Conopeptides have been the subject of a vast number of studies. They represent a large natural library of compounds that are specific for receptor subtypes, and are commonly used in neurophysiological studies to understand the role of receptor subtypes in nervous system functions or diseases (Dutton and Craik, 2001; Lewis, 2009; Olivera and Cruz, 2001). Fundamental biological studies have also been carried out to understand how cone snail venoms mature (Dobson et al., 2012; Safavi-Hemami et al., 2011a), how the environment influences conopeptide expression (Duda and Lee, 2009; Duda and Palumbi, 2004; Duda et al., 2009a), and the genetic events at the origin of conopeptide diversity (Biggs et al., 2010; Chang and Duda, 2012; Duda and Palumbi, 2000; Puillandre et al., 2012). Conopeptides also have outstanding potential as drugs or drug leads as they are the result of millions of years of natural optimization of both specificity and potency; combining both properties is a major challenge faced in drug design programs (Teichert and Olivera, 2010). One conopeptide, MVIIA isolated from the venom of Conus magus (Olivera et al., 1985a) and commercialized under the name Prialt, is an FDA-approved analgesic drug used to treat neuropathic pain (Miljanich, 2004). It is currently the only clinically available conopeptide-based drug, although several others are currently being investigated for the treatment of neuropathic pain, epilepsy, cardiac infarction, and neurological diseases (Lewis et al., 2012). In this chapter a broad overview is provided of the bustling activity in conopeptide research. Several recent reviews have provided in-depth details on specific topics, including conopeptide sequence and function diversity (Olivera and Teichert, 2007), three-dimensional structures (Daly and Craik, 2009; Marx, Daly, and Craik, 2006), chemical modifications (Craik

14.2 Diversity of Conopeptides

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Figure 14.2 Sequences and structures of four conopeptides isolated from different cone snails. The shells of the cone snails from which the peptides were extracted are shown on the left and the solution three-dimensional structures on the right. The disulfide bonds are in orange, and the post-translational modifications in red. C-terminal amidations are denoted by (nh2), and c-carboxyglutamates are noted using c. Molecular targets are indicated below the sequences. The backbone conformations are represented in blue cartoon structures, and the disulfide bonds are in yellow sticks. The three-dimensional structures of these peptides are well defined except for conantokin-G, which mainly adopts an a-helical structure, the termini of which are disordered (Skjaerbaek et al., 1997). Alternative models of the family of conantokin-G structures are shown as red lines. The images of cone shells were taken by David Touitou. Photos of Conidae: Courtesy of David Touitou.

and Adams, 2007), and therapeutic applications (Halai and Craik, 2009; Lewis, 2009; Lewis and Garcia, 2003; Lewis et al., 2012; Livett, Gayler, and Khalil, 2004).

14.2 Diversity of Conopeptides

Conopeptides were initially discovered by direct studies of the peptidic contents of venoms, but the advent of molecular biology technologies has dramatically accelerated their characterization via studies of the encoding nucleic acids (Duda and Palumbi, 1999; Woodward et al., 1990). More recently, second-generation sequencing methods have been used to unravel the genomes and transcriptomes of cone snails, further increasing the pace at which the conopeptide universe is being mapped (Hu et al., 2011). The ConoServer database (www.conoserver.org) was created to keep track of conopeptide sequences and three-dimensional structures (Kaas et al., 2008, 2012). As of June 2013, ConoServer catalogs information on 4806 conopeptide sequences (1832 mature peptides, 1875 protein precursors, 412 synthetic peptides and 1287 patented sequences) and 156 three-dimensional structures from 100 species of cone snails. 14.2.1 Conopeptide Maturation and The Origin of Venom Diversity

As illustrated in Figure 14.3, conopeptide genes are transcribed into precursor mRNAs, which are then translated into protein precursors. These precursors are directed to the endoplasmic

reticulum and probably transit through the Golgi apparatus, where the toxins are matured by being excised from the precursors as well as post-translationally modified. The approximate 1800 conopeptides that have been at least partly characterized represent only 1–2% of the natural diversity. Nevertheless, the already known peptides display an extremely high level of chemical diversity, both in terms of amino acid sequence and post-translational modifications. The diversity of conopeptides therefore arises both from diversity at the genetic level (i.e., the encoded amino acid sequences) and at the protein level (i.e., the post-translational modifications). 14.2.2 Diversification at the Gene Level

The conopeptide gene family is undergoing amongst the most rapid evolution in the animal kingdom, with the genes continuously and extensively duplicating under positive selection pressure (Conticello et al., 2001; Duda and Palumbi, 1999; Puillandre, Watkins, and Olivera, 2010; Puillandre et al., 2012). The large number of pseudogenes observed in the genome of one cone snail also indicates that conopeptide genes have a highly accelerated turnover (Puillandre, Watkins, and Olivera, 2010). Other genetic mechanisms, including allelic variations (Duda and Lee, 2009; Duda and Palumbi, 2000) and genetic recombinations (Gilly et al., 2011), might also contribute to the overall diversity of the conopeptide genetic pool. An observation of geographic gene variability between individuals belonging to the same species suggests that ecological adaptation to different preys is the main

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Figure 14.3 Illustration of conopeptide synthesis and maturation in venom duct cells. (a) Conopeptides are the products of a multiple gene family and diversified through differential post-translational modifications; (b) Examples of these post-translational modifications are shown for the MrIA precursor, whose major product is conotoxin MrIA, an inhibitor of the norepinephrine transporter. The modifications include proteolytic cleavages as well as modification of the side chains, including the hydroxylation of proline residues (4-hydroxyproline residues are denoted by a red “O”), the deamination of asparagine residues and the hydroxylation of methionine residues (hydroxylated methionine residues are denoted by “m”).

evolutionary pressure driving conopeptide diversification (Duda et al., 2009a). 14.2.3 Additional Diversity at the Protein Level

During their journey though the cell secretory machinery, conopeptide precursors are cleaved by a range of proteases (Olivera, 2002; Woodward et al., 1990), including signal peptide peptidases, proprotein convertases, and exopeptidases. The chemical space of conopeptides is also dramatically increased during this maturation by an unusually large range of posttranslational modifications of the peptide side chains and termini (Craig, Bandyopadhyay, and Olivera, 1999a; Kaas, Westermann, and Craik, 2010). Some 14 different types of modification have been identified (Table 14.1), the most frequent being the formation of disulfide bonds, C-terminal amidations, the hydroxylation of prolines, and the c-carboxylation of glutamates. Some of these modifications have been shown to be important for improving specificity towards molecular targets or improving folding rates (Lopez-Vera et al., 2008). The formation of disulfide bonds between pairs of cysteine residues creates crosslinks, and is a major contributor to the stability of conopeptide three-dimensional structures (these are further detailed in Section 14.4). Conopeptides originating from the same gene can display different degrees of post-translational modification in the venom (Elliger et al., 2011). This differential post-translational

modification is an important mechanism contributing to venom diversity because of the combinatorial possibilities for peptides that display multiple modifications. Additionally, a significant number of partially truncated forms of conopeptides have been isolated in venoms, and some of these have different activities from the nontruncated conopeptides (Dutertre et al., 2013).

Table 14.1 List of post-translational modifications occurring naturally in

conopeptides. The formation of disulfide bonds, which is the most common modification, is not included in this list. Post-translational modification

No. of wild-type conopeptidesa)

4-Hydroxyproline 5-Hydroxylysine Bromotryptophan C-terminal amidation D-leucine D-phenylalanine D-tryptophan c-Carboxylic glutamic acid c-Hydroxy-D-valine Glycosylated serine Glycosylated threonine Oxomethionine Pyroglutamic acid Sulfotyrosine

110 1 19 170 4 6 8 58 2 2 3 5 16 6

a) 378 conopeptides have been recorded in ConoServer to have been at least partially characterized at the protein level.

14.3 Isolation Techniques

Therefore, peptide truncation might be functionally important and generate another level of conopeptide diversity. On average, each conopeptide gene results in about 10 chemically different conopeptides that differ in the chemistry of their side chains as well as their sequence length. This 1 : 10 ratio between genes and peptides broadly agrees with the number of 50 to 100 transcripts discovered in transcriptomics projects2), compared to the 500 to 2000 peptides identified by high-precision mass spectrometry.3) 14.2.4 Nomenclature and Classification Schemes

A standard nomenclature and several subclassification schemes have been used to describe conopeptides (Gray, Olivera, and Cruz, 1988; Kaas, Westermann, and Craik, 2010; Walker et al., 1999). Disulfide-poor and large conopeptides are divided into several classes, mostly on the basis of their sequence similarities. They are usually named by combining their class name with one or two letters describing the species (e.g., contryphanM from Conus marmoreus), and additionally a number when several peptides of the same class belong to the same species (e.g., contulakin-Lt1 from Conus litteratus). Conotoxin standard names consist of one or two letters indicating the cone snail organism, a Roman number categorizing the arrangement of cysteines along its amino acid sequence, and a letter denoting the order of discovery (Gray, Olivera, and Cruz, 1988; Walker et al., 1999). The pattern of cysteine residues along the amino acid sequence in the precursor is commonly referred to as the cysteine framework. As an example of a standard conotoxin name, AuIB is the second conotoxin (letter B of the alphabet) with a cysteine framework I extracted from Conus aulicus (Au). Some other nomenclatures have been devised, especially in data generated with new discovery technologies, but peptides for which the activities have been extensively studied are usually named using the standard naming scheme. Three classification schemes are commonly used to characterize conotoxins: “gene superfamilies” describe the sequence relationships between precursors; “cysteine frameworks” distinguish the arrangements of cysteines in the mature toxin region of the precursors; and “pharmacological families” correspond to the molecular targets and types of activity of the peptides. 14.2.4.1 Gene Superfamilies Gene superfamilies are based on the high degree of sequence similarity between endoplasmic reticulum signal peptides of related precursors (Kaas, Westermann, and Craik, 2010; 2) Recent transcriptomics projects discovered 37 conopeptide transcripts

in Conus geographus (Hu et al., 2012), 53 in Conus consors (Terrat et al., 2012), 82 in Conus pulicarius (Lluisma et al., 2012), and 105 in Conus marmoreus (Dutertre et al., 2013). 3) Recent proteomics projects discovered 419 peptide masses in Conus consor, 455 in Conus novaehollandiae (Safavi-Hemami et al., 2011b), 650 in Conus victoria (Safavi-Hemami et al., 2011b), 845 in Conus imperialis (Davis et al., 2009), 1147 in Conus marmoreus (Davis et al., 2009) and 2428 in Conus textile (Davis et al., 2009).

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Woodward et al., 1990). The distinct sequence separation between different precursor signal peptides indicates that conotoxins might have diverged from a limited number of genes (Chang and Duda, 2012; Duda and Palumbi, 1999). Gene superfamilies are usually noted by using a single letter and, in some cases, an appended number, for example, the O1 gene superfamily. The most studied – and probably the most naturally populated – gene superfamilies are A, M, O1, and T (Dutertre et al., 2013; Kaas, Westermann, and Craik, 2010). The gene superfamily classification was recently extended to include two class of disulfide-poor conopeptides, the conantokins (B1 gene superfamily) and the contulakins (C gene superfamily) (Puillandre et al., 2012). 14.2.4.2 Cysteine Frameworks Cysteine frameworks describe the arrangement of cysteine residues along a mature conotoxin sequence in the precursor (Kaas, Westermann, and Craik, 2010). The formation of disulfide bonds between the side chains of two cysteine residues generally contributes to protein stabilization, and a description of the disulfide bonds is therefore important. Unfortunately, the determination of disulfide bond connectivities is difficult, and those of only a few conotoxins have been determined experimentally. In contrast, the description of a sequence pattern of cysteine residues along a sequence is readily accessible for all peptide sequences, and therefore all conotoxins can be classified using cysteine frameworks. A given cysteine framework usually defines peptides with only a limited number of disulfide connectivities, which correspond to a limited number of folds (see Section 14.4). Currently, 25 cysteine frameworks have been reported; these are denoted by Roman numerals, and the most common are cysteine frameworks I, III, V, and VI/VII. 14.2.4.3 Pharmacological Families Pharmacological families describe the molecular targets of conotoxins and also the type of biological activity, whether agonist or antagonist (Kaas, Westermann, and Craik, 2010). Pharmacological families are usually designated using a Greek letter. Most disulfide-poor conopeptide classes have distinct activities and therefore do not require a pharmacological classification; for example, the conantokins are the only conopeptides that act on NMDA glutamate receptors. Conopeptide activities are further discussed in Section 14.5.

14.3 Isolation Techniques

At an early stage of conopeptide discovery, the peptides were directly isolated from venoms by chromatographic fractionation, guided by their effects upon injection in animals, most often mice (Olivera and Cruz, 2001). The first conopeptides were discovered at a snail’s pace by using this approach, which was typically followed by Edman degradation sequencing, also generally a slow process. With progress in molecular biology technologies, as well as the finding that large groups of

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conopeptide precursors have similar sequence regions (i.e., they are classified in gene superfamilies), the sequence of entire sets of conopeptides began to be discovered using polymerase chain reaction (PCR) and expressed sequence tag libraries (Duda and Palumbi, 1999). More recently, second-generation sequencing has offered the possibility of studying entire transcriptomes and genomes of cone snails, thus facilitating the discovery of conopeptides outside the already known gene superfamilies (Hu et al., 2011). Despite being considerably more efficient, conopeptide identification from nucleotide sequences cannot completely replace direct characterization at the peptide level, partly because of the poor ability to predict post-translational modifications. Today, proteomics using mass spectrometry is the preferred method for identifying conopeptides in venoms and then sequencing them (Bhatia et al., 2012; Davis, Jones, and Lewis, 2009; Gowd et al., 2008; Jakubowski, Kelley, and Sweedler, 2006; Tayo et al., 2010). Recent studies have used the complementarity between transcriptomics and proteomics to produce comprehensive lists of conopeptides in a given cone snail venom (Dutertre et al., 2013). 14.3.1 Transcriptomics-Based Conopeptide Discovery

The classical method of identifying conopeptide transcripts by cDNA cloning and PCR/RACE (Rapid Amplification of cDNA Ends) amplification makes use of primers that complement regions of the conserved signal peptide, 50 -untranslated region, or 30 -untranslated region. The sequences of the amplified transcripts are then typically determined by Sanger sequencing. A few gene superfamilies, including A (Puillandre, Watkins, and Olivera, 2010; Santos et al., 2004), I1 (Jimenez et al., 2003), I2 (Kauferstein et al., 2004; Liu et al., 2009), O1 (Conticello et al., 2001; Duda and Palumbi, 1999; Kauferstein, Melaun, and Mebs, 2005; Luo et al., 2006), O2 (Conticello et al., 2001), T (Conticello et al., 2001; Walker et al., 1999) and M (Conticello et al., 2001; Corpuz et al., 2005; Han et al., 2006; Wang et al., 2008), have been extensively studied using this approach. Recent second generation sequencing transcriptomics studies were all carried out using the 454 GS-FLX Titanium technology, generating 200 000–900 000 reads with an average read length of 200–400 bp (Dutertre et al., 2013; Hu et al., 2011, 2012; Lluisma et al., 2012; Terrat et al., 2012). With an average size of 68 amino acid residues in their coding regions, most conopeptide precursors could be directly discovered in the sequence reads, without requiring the assembly of contigs. Without access to a reference genome, contigs (ultimately corresponding to transcripts) must be assembled de novo; however, this is a difficult task that potentially is susceptible to generating erroneous information. The 454 technology suffers from a significant rate of sequencing errors, about 1% of incorrect base calls, and several corroborating reads are required to support each base of a newly discovered sequence (Gilles et al., 2011). The number of reads supporting each nucleotide base is referred to as the “sequencing depth,” which is linked to the level of expression of each

transcript. Besides the expression level, other technical factors – such as the differential fragmentation propensity of different nucleotide sequences – can also influence the sequencing depth. Transcripts with low sequencing depths (typically below 10 reads) should ideally be verified using alternative techniques, such as a classical combination of PCR amplification and Sanger sequencing. As mentioned above, mature conopeptides cannot be accurately predicted from their transcript sequences alone, and transcriptomics analyses can be complemented by proteomics studies aimed at identifying the corresponding mature conopeptides in the venom (Dutertre et al., 2013; Violette et al., 2012). 14.3.2 Proteomics Studies of Conopeptides

In general, the aims of modern proteomics studies of cone snail venoms have been either to globally evaluate their complexity and/or to sequence new conopeptides (Davis, Jones, and Lewis, 2009; Dutertre et al., 2013; Violette et al., 2012). Most of these studies have been carried out by first fractionating the diluted venom using gel filtration and/or reversed-phase high-performance liquid chromatography (HPLC), followed by an analysis of the fractions using mass spectrometry. A typical spectrum highlighting the complexity of cone snail venom is shown in Figure 14.4. Ionization is often carried out either by matrixassisted laser desorption ionization (MALDI) or by electrospray ionization (ESI), and the masses of produced ions are commonly evaluated using a series of time-of-flight (TOF) analyses. Advances in TOF analyses have been instrumental in unraveling the full diversity of conopeptide venoms, which were previously largely underestimated (Davis, Jones, and Lewis, 2009). The

Figure 14.4 Typical ion current trace of a cone snail venom. This particular trace was obtained from a liquid-chromatography-massspectroscopy (LC-MS) analysis of the injected venom of Conus marmoreus. Conopeptides corresponding to some of the fractions are identified on the trace. Reproduced with permission from Dutertre et al., 2013; Ó 2013, American Chemical Society. This research was originally published in “Molecular & Cellular Proteomics”

14.4 Conopeptide Three-Dimensional Structures

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sequences of conopeptides can now be determined using tandem MS/MS sequencing of reduced-akylated peptides, followed by either complete sequence assignment or searching in a database of known proteins, often carried using the Protein PilotTM software with the ParagonTM database search engine. A series of tools available on the ConoServer Web site has been designed to seamlessly help in the comparison of transcriptomics and proteomics data (Dutertre et al., 2013; Kaas et al., 2012). The transcript sequences are first analyzed, and a list of masses corresponding to differential post-translational modifications of the predicted mature conopeptides is then generated. This list is then matched with corresponding reconstructed mass spectra from proteomics analyses. Proteomics studies benefit from transcriptomics studies by helping to identify interesting precursor ions and by resolving sequencing problems, such as the ambiguity between isobaric leucine and isoleucine residues. Conversely, transcriptomics studies benefit from proteomics studies by validating mature peptide sequences and determining any post-translational modifications for each peptide. However, the connectivity between disulfide bonds is an important type of post-translational modification that cannot be fully determined using mass spectrometry. In some cases, disulfide connectivities can be determined by analyzing the mass of nonreduced conotoxin fragments (Dy et al., 2006; M€oller et al., 2005), but often more labor-intensive methods must be used such as the chromatographic coelution of wild-type peptides and synthetic disulfide isomers (Han et al., 2006; Hidaka et al., 1990), or even directly by structural characterization of the peptides either by X-ray crystallography (Hu et al., 1997) or nuclear magnetic resonance (NMR) spectroscopy (Hill, Alewood, and Craik, 2000; Kang, Jois, and Kini, 2006).

14.4 Conopeptide Three-Dimensional Structures

Most three-dimensional structures of conopeptides have been determined using NMR rather than X-ray crystallography (Kaas et al., 2012), principally because conopeptides are difficult to crystallize, whereas their relatively small size and good solubility are ideal for NMR structure determination. Moreover, the majority of conopeptides are small and can be studied using homonuclear NMR experiments; they do not need expensive and timeconsuming isotope labeling, which is often the case for NMR studies of larger proteins. A typical total correlation spectroscopy (TOCSY) NMR spectrum of a conotoxin is shown in Figure 14.5. The 107 currently available three-dimensional structures of wild-type conopeptides show that these peptides can adopt vastly different folds, which are often correlated within a given cysteine framework or disulfide bond connectivity. Most conotoxins adopt very well-defined and stable conformations, whereas some disulfide-poor conopeptides are disordered in solution (Kindahl et al., 2002). Conotoxins displaying two or three disulfide bonds have been the most extensively structurally characterized, and some representative structures are shown in Figure 14.6.

Figure 14.5 Two-dimensional 1H NMR spectra of conopeptide BtIIIA from (a) TOCSY and (b) NOESY experiments. Only the fingerprint regions are shown in the NOESY spectrum. Reproduced from Akcan et al., 2013 with kind permission by Elsevier.

14.4.1 Two-Disulfide Conotoxins

Conotoxins displaying cysteine framework I adopt conserved compact structures corresponding to disulfide connectivity {1– 3, 2–4}.4) They inhibit nicotinic acetylcholine receptors and are referred to as a-conotoxins (Millard, Daly, and Craik, 2004; Muttenthaler, Akondi, and Alewood, 2011), but some have also been found to act via the GABAB receptor to modulate calcium channels (Adams, Callaghan, and Berecki, 2012; Callaghan and Adams, 2010; Daly et al., 2011; Klimis et al., 2011; Nevin et al., 2007). A few cysteine framework I conotoxins have been crystallized in complex with structural homologs of the extracellular domain of nicotinic acetylcholine receptors called acetylcholine-binding proteins (AChBPs) (Dutertre et al., 2007). 4) Disulfide connectivities are noted using consecutive numbering of the

hemi-cystines along the mature conopeptide sequence.

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Figure 14.6 Selected sequences and structures of conopeptides with two (conotoxins GI, ImI, MII, PeIA and MrIA), or three disulfide bonds (conotoxins TIIIA, BtIIIA, PIIIE, MrIIIE, MVIIA and TxVIA). Disulfide bonds are shown in orange in the sequences and structures, and the hemi-cystines are numbered in orange on the structures. The disulfide connectivity is represented by orange lines in the sequences, and an asterisk ( ) indicates C-terminal amidation; “O” indicates 4-hydroxyproline residues. The conotoxins are grouped by cysteine frameworks, the cysteine pattern of which are indicated between squared brackets. Framework I peptides are classically subclassified according to the number of amino acid residues in the first and second loops, for example, 3/5 for conotoxin GI. Similarly, framework III conotoxins are subclassified according to the number of amino acid residues between the fourth and fifth hemicystines (third loop). For example, TIIIA belongs to the M-4 branch because it has four residues in its third loop. The disulfide connectivities are indicated between curly brackets for the conotoxins with three disulfide bonds. Three different connectivities displayed by wild-type conotoxins with a cysteine framework III have been structurally studied, including {1-5, 2-4, 3-6} (corresponding to the M-1 branch), {1-6, 2-4, 3-5} (M-2 branch) and {1-4, 2-5, 3-6} (M-4 and M-5 branches). The known subtype specificities of some of the conotoxins are indicated, as well as their typical IC50 values. The names of the molecular targets have been abbreviated, including nicotinic acetylcholine receptors (nAChR), gamma-aminobutyric acid receptor B (GABAB), voltagegated sodium channels (NaV) and voltage-gated calcium channels (CaV). Some conotoxins, including PeIA, have several molecular targets. Some conotoxins displaying the same cysteine framework have different molecular targets; for example, TIIIA and PIIIE display a M-4 branch cysteine framework III but inhibit NaV and nAChR, respectively; MVIIA (Prialt) and TxVIA display cysteine framework VI/VII and are specific for CaV and NaV, respectively. Conversely, some conotoxins with different cysteine frameworks can be specific for the same molecular target; for example, GI and PIIIE act on muscle-type nAChRs. The protein databank, biological magnetic resonance bank or ConoServer identifiers of the represented structures are indicated in gray.

Unfortunately, no nicotinic acetylcholine receptor has yet been successfully crystallized, and the AChBPs – which are also targeted by conotoxins – are conveniently used as surrogates in structural studies. The structures of complexes between the AChBPs and conotoxins provide important information that has been used to interpret their affinity and specificity for nicotinic acetylcholine receptor subtypes. The cysteine pattern of cysteine framework X is similar to that of cysteine framework I, and differs by the requirement to

have only two residues between the third and fourth cysteine residues (Balaji et al., 2000). Despite this similarity, conotoxins with cysteine framework X have been found to have a different disulfide connectivity {1–4, 2–3}, that is, the so-called “ribbon connectivity,” and a distinct fold to cysteine framework I conotoxins. The four conotoxins with a framework X that have been pharmacologically characterized display different activity to a-conotoxins and act specifically on norepinephrine transporters.

14.5 Conopeptide Pharmacological Activities 14.4.2 Tri-Disulfide Conotoxins

Most conotoxins with three disulfide bonds – that is, with six cysteines in their cysteine framework pattern – display cysteine frameworks III or VI/VII, which have been studied in 24 and 18 NMR solution structures, respectively. All structures of cysteine framework VI/VII conotoxins display a cystine knot motif, with disulfide connectivity {1–3, 2–4, 3–6}. In the cystine knot motif, two disulfide bonds and their interconnecting backbone segments form a “cystine ring” that is penetrated by a third disulfide bond. This structural element is generally associated with a high conformational stability and is found in a wide array of unrelated proteins in all kingdoms of life. In contrast to cysteine framework VI/VII, cysteine framework III leads to three different disulfide connectivities, each of which is associated with distinct folds. Most of the structurally studied conotoxins with a cysteine framework III have the same disulfide connectivity as cysteine framework VI/VII conotoxins – that is {1– 3, 2–4, 3–6} – but do not form a cystine knot. Interestingly, their fold resembles that of framework I conotoxins, and these peptides have also been shown to inhibit nicotinic acetylcholine receptors, similarly to framework I conotoxins (Hopkins et al., 1995). Nevertheless, most of the pharmacologically tested framework III conotoxins are specific blockers of voltage-gated sodium channels (m-conotoxins) (Heinemann and Leipold, 2007). Structural studies of conopeptides have revealed that some pharmacological activities are associated with particular folds, even if some conopeptides with the same fold can bind to different molecular targets (Figure 14.6). Structural studies are therefore important to understand how conopeptides achieve exquisite specificity for their molecular targets, which is the main motivation for the intense interest in them.

14.5 Conopeptide Pharmacological Activities

Although interest in conopeptides has arisen primarily from their potent biological activities, only 10% of known conopeptides have been tested for their pharmacological activity, most likely because of the cost and difficulty of discovering precise molecular targets. The preliminary characterization of conopeptides is often carried out by direct injection into animals, frequently by intracranial injection in mice (Clark, Olivera, and Cruz, 1981; Olivera and Cruz, 2001). The animals usually display distinctive symptoms for the different conopeptides, and some of the peptides have been nicknamed accordingly (Olivera and Cruz, 2001). For example, GVIA was coined the “shaker peptide” as it caused tremors in mice (Olivera et al., 1984) which are now known to be caused by an inhibition of calcium channels. This peptide is one of the most widely used conopeptides in neuroscience (Terlau and Olivera, 2004). The “sleeper peptides” (e.g., the disulfide-poor conopeptide conantokin-G) plunge mice into a state of mental torpor (Olivera et al., 1985b) due to their activity on NMDA glutamate receptors (Donevan and McCabe, 2000). Some other peptides

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have been given colorful names, including the “moonwalker peptides” (conolysins) that cause mice to adopt a “popstar’s signature dance maneuver” (Biggs et al., 2007), the “fin-popping peptide” (PVIIA), the “lock-jaw peptide” (PVIA) or the “King Kong peptides” (e.g., TxVIA), which causes injected lobsters to adopt an “exaggerated dominant stance,” probably due to agonist activity on sodium channels (Hillyard et al., 1989). The analgesic activity of some wild-type and modified conopeptides are of particular interest for pharmaceutical applications (Carstens et al., 2011). Several have been tested in animals for analgesia, most commonly in rats, using a range of assays that includes mechanical paw withdrawal threshold (Berecki et al., 2010; Ekberg et al., 2006; Klimis et al., 2011; Nevin et al., 2007; Satkunanathan et al., 2005), the hot-plate test (Lee et al., 2010; Malmberg and Yaksh, 1995; Sun et al., 2011), and nerve ligation/ compression surgery (Adams, Callaghan, and Berecki, 2012; Berecki et al., 2010; Klimis et al., 2011). Behavioral effects associated with treatments have also been monitored, for example by motor performance in rotating drums (Klimis et al., 2011). A precise characterization of the type of molecular target usually involves the patch–clamp electrophysiological recording of depolarization–repolarization currents of rat dorsal root ganglion (DRG) neurons. On the basis of competition with known specific blockers and inhibitor compounds, the molecular targets of conopeptides can be better described. For example, tetrodotoxin is a potent blocker of some voltage-gated sodium channel subtypes, which are commonly classified as either tetrodotoxin-resistant or tetrodotoxin-sensitive. Similarly, competition with a-bungarotoxin is classically used to test if conopeptides bind to the muscle type or to the a7 subtype nicotinic acetylcholine receptors (Ellison, McIntosh, and Olivera, 2003). The determination of selectivity at the receptor subtype level is commonly carried out using two-electrode patch–clamp current recording from Xenopus laevis oocytes, or more rarely human embryonic kidney (HEK) cells (Leipold et al., 2011), that heterologously express specific ion channel subtypes. A fluorescentbased assay known as FLIPR1 has also been used to measure the activity of conopeptides on ion channels (Tranberg et al., 2012; Vetter et al., 2012). Based on the dose–response curves, the halfmaximum inhibitory concentration (IC50) can be computed, and in turn the inhibitory constant Ki can be inferred. The direct determination of Ki without any assumption of mechanism has been measured for some conopeptides using radioligand-binding studies (Lewis et al., 2007; Schroeder et al., 2008). Conopeptides have been found to target numerous types of ligand-gated and voltage-gated ion channels, as well as transporters and other Gprotein-coupled receptors of the nervous system, as illustrated in Table 14.2 (Kaas, Westermann, and Craik, 2010; Teichert and Olivera, 2010). The most widely studied pharmacological family of conotoxins is the a-conotoxins, which specifically inhibits nicotinic acetylcholine receptor subtypes; other intensively studied pharmacological families target various voltage-gated calcium channels, including voltage-gated calcium channels (antagonized by v-conotoxins), voltage-gated sodium channels (antagonized by m-conotoxins and agonized by d-conotoxins) and voltage-gated potassium channels (antagonized by k-conotoxins).

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Table 14.2 Conopeptide pharmacological activities.

Familya)

Definition

Representative toxin(s)

Reference

a c d e i k m r s t x v Conantokins Contulakins Conopressins

Nicotinic acetylcholine receptors (nAChR) Neuronal pacemaker cation currents (inward cation current) Voltage-gated sodium channels (agonist, delay inactivation) Presynaptic calcium channels or G protein-coupled presynaptic receptors Voltage-gated sodium channels (agonist, no delayed inactivation) Voltage-gated potassium channels (blocker) Voltage-gated sodium channels (antagonist, blocker) a1-adrenoceptors (GPCR) Serotonin-gated ion channels (GPCR) Somatostatin receptor Neuronal norepinephrine transporters Voltage-gated calcium channels (blocker) NMDA glutatamate receptors Neurotensin receptors Vasopressin receptors

GI PnVIIA, TxVIIA TxVIA TxVA RXIA PVIIA GIIIA TIA GVIIIA CnVA MrIA, CMrVIA GVIA Conantokin-G Contulakin-G Conopressin-S

Gray et al., 1981 Fainzilber et al., 1998 Fainzilber et al., 1991 Rigby et al., 1999 Buczek et al., 2007 Scanlon et al., 1997; Terlau et al., 1996 Cruz et al., 1985 Sharpe et al., 2001 England et al., 1998 Petrel et al., 2013 Sharpe et al., 2001 Kerr and Yoshikami, 1984 Donevan and McCabe, 2000 Craig et al., 1999b Cruz et al., 1987

a) The listed families correspond to those that have been documented in the ConoServer Web site. Other families have been described in the literature, including kM, kA, mO, aA and y, but their definition does not correspond to the strict convention of describing a receptor target and a mode of action (Kaas et al., 2010). Consequently, these conopeptides have been grouped within the families described in this table; for example, the aA conotoxins are described as a conotoxins.

14.6 Outlook

As noted in this chapter, considerable efforts have been dedicated to the discovery and characterization of conopeptides over the past three decades, prompted by the outstanding properties and applications of these compounds. Starting in the early 1980s, this ongoing process has constantly relied on cutting-edge analytical techniques, and the most recently developed nucleotide sequencing and proteomics technologies are now able to provide access to the entire conopeptide contents of cone snail venoms – information which previously was out of reach. Other aspects of conopeptide characterization – and, unfortunately, perhaps the most interesting – seem still to be lagging behind because they are highly time-consuming and costly. Although the determination of conopeptide three-dimensional structures is still a very slow process, the approximately 100 experimental structures of wild type conopeptides that are available have revealed certain conserved structural features which can be used to generate tentative models for most conopeptides. These modeled structures could, eventually, also be used to help resolve the current bottleneck in conopeptide discovery, namely the elucidation of their pharmacological activity.

With scientific interest in conopeptides ever-growing, an attempt has been made in this chapter to outline the most exciting aspects of their research. Molecular biologists studying the evolution of conopeptides are continuing to identify more similarities between the mechanisms that generate the molecular diversity of conopeptides and those of animal immune systems, thus drawing an interesting comparison between attack and defense molecules. Today, pharmacologists are using newly discovered conopeptides to help understand how ion channels function at the molecular level, while neuroscientists use them in their quest to understand more broadly how cognitive functions are achieved through the different ion channel subtypes. Finally, chemists are using conopeptides as a source of inspiration in drug design through a better understanding of how conopeptides achieve receptor subtype specificity.

Acknowledgments

The studies on conotoxins, conducted in the authors’ laboratory, are supported by an Australian Research Council grant (DP1093115). D.J.C. is an NHMRC Professorial Fellow (APP1026501).

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of neuronal pacemaker cation currents. Biochemistry, 37, 1470–1477. Gilles, A., Meglecz, E., Pech, N., Ferreira, S., Malausa, T., and Martin, J.-F. (2011) Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics, 12, 245. Gilly, W.F., Richmond, T.A., Duda, T.F. Jr, Elliger, C., Lebaric, Z., Schulz, J., Bingham, J.P., and Sweedler, J.V. (2011) A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus. J. Exp. Biol., 214, 147–161. Gowd, K.H., Dewan, K.K., Iengar, P., Krishnan, K.S., and Balaram, P. (2008) Probing peptide libraries from Conus achatinus using mass spectrometry and cDNA sequencing: identification of d and v-conotoxins. J. Mass Spectrom., 43, 791–805. Gray, W.R., Luque, A., Olivera, B.M., Barrett, J., and Cruz, L.J. (1981) Peptide toxins from Conus geographus venom. J. Biol. Chem., 256, 4734–4740. Gray, W.R., Olivera, B.M., and Cruz, L.J. (1988) Peptide toxins from venomous Conus snails. Annu. Rev. Biochem., 57, 665–700. Halai, R. and Craik, D.J. (2009) Conotoxins: natural product drug leads. Nat. Prod. Rep., 26, 526–536. Han, Y.-H., Wang, Q., Jiang, H., Liu, L., Xiao, C., Yuan, D.-D., Shao, X.-X., Dai, Q.-Y., Cheng, J.-S., and Chi, C.-W. (2006) Characterization of novel M-superfamily conotoxins with new disulfide linkage. FEBS J., 273, 4972–4982. Heinemann, S.H. and Leipold, E. (2007) Conotoxins of the O-superfamily affecting voltage-gated sodium channels. Cell. Mol. Life Sci., 64, 1329–1340. Hidaka, Y., Sato, K., Nakamura, H., Kobayashi, J., Ohizumi, Y., and Shimonishi, Y. (1990) Disulfide pairings in geographutoxin I, a peptide neurotoxin from Conus geographus. FEBS Lett., 264, 29–32. Hill, J.M., Alewood, P.F., and Craik, D.J. (2000) Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 2. Three-dimensional solution structure. Eur. J. Biochem., 267, 4649– 4657. Hillyard, D.R., Olivera, B.M., Woodward, S., Corpuz, G.P., Gray, W.R., Ramilo, C.A., and Cruz, L.J. (1989) A molluscivorous Conus toxin: conserved frameworks in conotoxins. Biochemistry, 28, 358–361. Hopkins, C., Grilley, M., Miller, C., Shon, K.J., Cruz, L.J., Gray, W.R., Dykert, J., Rivier, J., Yoshikami, D., and Olivera, B.M. (1995) A new family of Conus peptides targeted to the nicotinic acetylcholine receptor. J. Biol. Chem., 270, 22361–22367. Hu, H., Bandyopadhyay, P.K., Olivera, B.M., and Yandell, M. (2011) Characterization of the Conus bullatus genome and its venom-duct transcriptome. BMC Genomics, 12, 60. Hu, H., Bandyopadhyay, P.K., Olivera, B.M., and Yandell, M. (2012) Elucidation of the molecular envenomation strategy of the cone snail Conus geographus through transcriptome

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About the Authors Quentin Kaas is a researcher at the Institute of Molecular Bioscience at the Institute for Molecular Bioscience (IMB), University of Queensland, Australia. He graduated with a PhD from the University of Montpellier and subsequently started his scientific career at the IMB. A major area of his research is the study of plant and animal toxins and their development as drugs. He leads a popular information system, called ConoServer, that specializes on Cone snail toxins, and has authored a number of articles and reviews on these toxins.

David J. Craik is a group leader and Professor of Biomolecular Structure at the Institute for Molecular Bioscience, University of Queensland. He obtained his PhD from La Trobe University, Melbourne, Australia in 1981, followed by two years’ postdoctoral experience in the USA. He is the author of over 450 scientific publications and the recipient of numerous awards, including the Ralph F. Hirschmann Award for Peptide Chemistry from the American Chemical Society. He is a board member of nine international journals, including The Journal of Biological Chemistry.

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15 Mycosporine-Like Amino Acids (MAAs) in Biological Photosystems St
ephane La Barre, Catherine Roullier, and Jo€el Boustie

Abstract

Mycosporine-like amino acids (MAAs) represent a suite of small molecules which have unique ultraviolet-absorbing capacities, based on their common cyclohexenone or cyclohexenimine conjugated arrangements. MAAs also have strong antioxidant properties, and participate in the osmotic equilibrium of the numerous and diverse marine organisms that produce them, especially in photosymbiotic partnerships. In this chapter a historical introduction is provided to the discovery of MAAs, together with details of their primary and secondary functions. Details are also provided of isolation and spectral information, illustrated by pertinent examples,

R1

O OCH3

HO HO

HO

NH R2

R cyclohexenone ring where R = glycine or taurine

OCH3

R3O

NH

N

cyclohexenimine ring (most MAAs) where R1= amino acid residue, R2 = H or CH3 or CH2COOR3 = H or SO3-

15.1 Background 15.1.1 Life in Full Light and its Constraints

Life probably appeared in the depths of a primitive ocean some 3.5 billion years ago, supposedly in the absence of light and under anoxic conditions. Cyanobacteria were the first organisms to use oxygenic photosynthesis, which deeply modified the

and aspects of biosynthesis and the modulating environmental factors involved in the production and degradation of MAAs. Two contrasting biological models models are described: lichens, which are mostly terrestrial and extremophiles; and shallow-water tropical reef corals, which are marine and stenotolerant; these have one point in common, namely a permanent exposure to direct sunlight. Finally, brief details are provided of the medical, cosmetological and biotechnological applications of these primary metabolites with secondary roles, that were most likely instrumental in the adaptation of early cyanobacterial life forms to surface conditions. Useful practical information for structural chemists is included in an Appendix.

Water-soluble and photostable Low-MW compounds (usually less than 400 Da) No proven toxicity Strong anti-UV absorption, especially between l ¼ 310– 340 nm, with molar absorptivities (e) at around 40 000 l mol 1 cm 1. Widely known as natural sunscreens, they also act as osmolytes and/or antioxidants with secondary metabolite characteristics that are mostly, but not exclusively, aquatic.

biogeochemistry of the primitive ocean. Later, the development of an atmosphere created the conditions for ever-more complex life forms to appear, evolve, and colonize new habitats under constantly changing environmental conditions. Today, oxygen redox chemistry and photon energy are indispensable to all aquatic and terrestrial life forms that have not receded to a chemolithotrophic mode of subsistence. This was made possible by the development of biochemical pathways that integrated molecular oxygen, using novel enzymatic reactions, and also by acquiring protection against oxygen toxicity and exposure to harmful solar radiations. The

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Box 15.1: Useful and Harmful Solar Radiations Ultraviolet radiation (UVR) is probably the single most influential abiotic factor that has shaped the evolution and ecology of the biosphere (Banaszak and Lesser, 2009). Under prebiotic conditions, exposure to unfiltered solar radiations (UVA, UVB and UVC) would have led rapidly to DNA damage and the death of exposed organisms. The gradual O2 enrichment of the atmosphere by cyanobacteria who invented photosynthesis and molecular sunscreens (most probably MAAs) led to the development of new life forms, both autotrophic and heterotrophic, but it was not until atmospheric O2 photooxidation into ozone had created an efficient antiradiation shield that terrestrial life was made possible. Today, the ozone photoprotective layer is being depleted, leaving “holes” above large tropical zones that are now under progressive risk of overexposure to harmful shortwave UVB radiations. Shallow-water (less than 20 m) coral reef

protective strategies employed include enzymes for DNA repair and against aggressive oxygenated and radicalar species, UV avoidance behavior, and the biosynthesis of small molecules acting as natural sunscreens. Sessile and planktonic organisms that live while exposed to sunlight have developed chemical protections that help them to absorb harmful and genotoxic ultraviolet radiation (UVR), especially in the 310–360 nm (more-penetrating) UVA range (see Box 15.1). In addition, oxidative stress due to the production of reactive oxygen species (ROS) can be controlled by the production of antioxidants. 15.1.2 MAAs: To Protect and Serve, Occasionally to Defend

Mycosporine-like amino acids (MAAs) are synthesized (or acquired) by these organisms in response to both challenges. Indeed, MAAs are known to be the strongest UVA-absorbing compounds (in the 320–365 nm range) in Nature, as well as having strong antioxidant properties and occasionally acting as osmoprotectants (Oren and Gunde-Cimerman, 2007). Interestingly, the existence of UV-protecting molecules had been known for some time before their exact chemical nature was revealed. For example, Wittenburg (1960) in the gas gland of an epipelagic jellyfish, Tsujino and Saito (1961) in red algae, and Shibata (1969) in aqueous coral extracts, had each reported the existence of molecules capable of strongly absorbing UV radiations in the 310–360 nm region, but without characterization. Independently, other research groups working on fungi reproduction mechanisms noticed the production of unidentified substances in the mycelia of several fungi (e.g., Pyronema omphalodes, Alternaria chrysanthemi, and Ascochyta pisi), when sporulation was induced by UV light (Leach, 1965). Molecules having a UV absorption maximum at 310 nm were notionally designated P-310. However, no correlation was made at the time

communities are at particular risk, especially in areas of high water transparency and under clear skies. Cryptic and softbodied coral-associated biota cannot withstand even accidental exposure to such conditions. The indirect effects of UVR is the generation of reactive oxygen species (ROS), including radical species,viatheexcitationofforexample,aromaticintermediates and the downstream production of highly reactive hydroxyl radicals in an iron-catalyzed Fenton reaction. ROS generate oxidative stress, with adverse effects on coral polyps and their symbiotic zooxanthellae, causing the bleaching phenomenon. Perhaps the most dramatic situation has to be faced by lichens, which thrive only on bare rock surfaces, in latitudes that are inhospitable (e.g., exposure to high-UVR regimes, temperature extremes, desiccation and almost no nutrients) to any other macroscopic life form, yet they also break longevity records!

between those studies and previously reported marine organisms observations. Consequently, fungi continued to be explored, and UV-absorbing compounds similar to P310 continued to be observed. Only several years later was the structure of the first P310 finally elucidated, after its isolation from the basidiomycete Stereum hirsutum; the compound became known mycosporine I, as its biosynthesis was largely observed in fungi and thought to be related to sporulation and reproduction mechanisms (Favre-Bonvin, Arpin, and Brevard, 1976). Subsequently, Ito and Hirata (1977) established the structure of the first MAA, namely mycosporine-glycine, from the zoanthid Palythoa tuberculosa, and this was followed by other similar molecules (Hirata et al., 1979). From 1977 onwards, this type of molecule began to be more largely isolated and identified from many organisms, such as cyanobacteria, algae, phytoplankton and even animals, mostly using reversed-phase high-performance liquid chromatography (Nakamura, Kobayashi, and Hirata, 1982). Many of these molecules were isolated from corals that live symbiotically with dinoflagellates, but also from mollusks and fish. As a result the term “mycosporine-like amino-acid” or MAA emerged, reserving “mycosporine” for molecules that had been isolated exclusively from fungi. The phylogenomics of the MAAs is still in progress as routes to MAAs appear to be multiple (Balskus and Walsh, 2010). In marine photosymbiotic systems such as reef corals, both the zooxanthellae photosymbionts and the coral host are genetically capable of producing MAAs (Shinzato et al., 2011), involving gene transfer from microbiont to host (Rosic, 2012). In addition, some MAAs can be acquired directly from food sources by the coral hosts, though this adds to the complexity of the biosynthetic origin of MAAs found in these organisms (Rosic and Dove, 2011). Hundreds of taxonomically diverse marine, freshwater and terrestrial organisms have the capacity to synthesize, accumulate and metabolize MAAs in order to address

15.2 Chemistry

the direct and indirect damaging effects of UVR (Carreto and Carignan, 2011). To date, at least 21 MAAs have been described from various marine sources, two with the cyclohexenone ring bearing one amino acid substituent, and 19 with the cyclohexenimine ring bearing up to three substituents (Figure 15.1). The quest is not over, however, as aplysiapalythines A, B and C (Kicklighter et al., 2011) were recently discovered in the sea hare Aplysia, which acquires them from the diet and subsequently uses them as alarm cues in defensive secretions (opaline). In the realm of lichens, new structures are continually being found (Nguyen et al., 2013) one example being mycosporine hydroxy-glutamicol from the lichen Nephroma laevigatum (Roullier et al., 2011). Altogether, up to 40 mycosporines and derivatives have been described from both terrestrial and marine sources, some bearing functional groups or being covalently linked with saccharidic units. Fungal mycosporines are closely related to marine mycosporines, and include mycosporine-2, mycosporine-alanine, mycosporine-glutamine, mycosporine-glutamicol, mycosporine-glutaminol, mycosporine-glutamic acid, mycosporine-glutaminol glucoside, mycosporine-serinol, and normycosporineglutamine (Sinha, Singh, and Hader, 2007). Interestingly, all ten fungal structures feature a maximum absorbance at a wavelength of 310 nm; this is in contrast to MAAs, some of which have a lmax of 360 nm. The accumulation of MAAs in the conidia is reported to increase the tolerance of the fungi against adverse environmental conditions, among other roles in their developmental biology, but this is beyond the scope of this chapter. Suffice to mention is the interesting parallel that Klisch and H€ader (2008) established between MAAs and toxins in their overlapping phyletic distributions, and possibly in their similar biogenetic pathways, namely the putative involvement of non-ribosomal polyketide synthase (NRPSs) in both, albeit with totally distinct and complementary functions: the MAAs are there to protect, while toxins are there to defend.

15.2 Chemistry 15.2.1 Physico-Chemical Characteristics of MAAs

The MAAs are low-molecular-weight (most are under 400 Da), generally colorless, water-soluble compounds that are also resistant to thermodegradation and photodegradation under environmental conditions. Yet, they are differentially susceptible to acidic and oxygenating conditions. The truly outstanding characteristic, however, is their high UV-absorbing capability, with a unique strong peak in the 305–360 nm (lmax) range (harmful UVA and shortwave UVB radiations) with molecular absorptivities (e) of about 40 000 l mol 1 cm 1. This makes them the strongest UVA-absorbing compounds in Nature, and they are also effective against shortwave UVB radiations. The physico-chemical properties of individual MAAs have been reviewed in detail (Carreto and Carignan, 2011).

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Together, these features are essential in the roles of MAAs as photoprotectants in the outermost tissue layers of marine metazoans, and also as antioxidants and osmoprotectants in cell or body fluids, or in external mucus layers. Especially surprising is the ease by which the MAAs can be ferried from one biological compartment to another. 15.2.2 MAAs and Related Molecules 15.2.2.1 MAAs in the Marine World Their high water solubility enables MAAs to be easily dispersed in the cytoplasm of microalgae and diatoms, and also to be rapidly accumulated in the superficial sun-exposed cell layers of numerous marine invertebrates. Some organisms acquire the MAAs directly from food by assimilation, without transformation (like free amino acids); examples include echinoderms such as starfish, sea-cucumbers and sea-urchins (for a review, see Dunlap and Shick, 1998). Cnidarians and ascidians acquire their MAAprecursors from their photomicrobionts (cyanobacteria, prochloron or dinoflagellates); as most are colorless this allows “filtered” sunlight to reach the photosynthetic zooxanthallae that are sequestered in the endodermal layers of the diploblastic photosymbionts. The presence of a suite of 10 MAAs in Stylophora pistillata (Shick et al., 1999) is most likely due to a need for photoprotection over an extended range of potentially harmful wavelengths, especially in photosymbiotic systems which may have different and specific sensitivities to sun radiations. This corresponds to MAA production being stimulated primarily by UV radiation of different wavelength ranges (Klish and Hader, 2008). 15.2.2.2 MAAs and Related Molecules in Lichens Lichens associate with different partners, typically a saprotroph (fungus) and a phototroph (unicellular green alga or cyanobacterium, sometimes both), where the eubacterial consortia play an important role in nutrient cycling. Not surprisingly, cyanolichens (which comprise 10% of all lichen species) have been shown to possess both mycosporines and MAAs, and possibly also some unique structures (Figure 15.2), according to the environmentally “favored” partner – that is, to its prevailing metabolic expression. 15.2.3 Extraction, Separation, Purification, and Detection 15.2.3.1 Extraction, Separation, and Purification As the semi-purification of mycosporines from other polar compounds is crucial, the protocol must be optimized, taking into account the possible hydrolysis and conversion of some of these compounds. Glycosylated mycosporines can be hydrolyzed under acidic conditions (0.5 M HCl, 100  C), and conversion from an aliphatic amino acid chain to a cyclized amide form is likely to occur on open-column resins in the Hþ form used for separation. Cleavage of the amino acid moiety is readily achieved by alkaline hydrolysis (Pittet et al., 1983).

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OXO-MYCOSPORINES O 2

R

OCH3 NH

HO HO

Absorbance (UVA-UVB)λmax in nm Extinction coefficient ε in l.mol-1.cm-1 Molecular weight in Da Molecular formula

1

R 2

C8H11NO4

R

DATA

SOURCE

REFERENCES

310 28100 245 C10H15NO2

Collema sp. Peltula spp. Gonohymenia sp. Lichina pygmaea

310 25516 261 C11H19NO6

Collema cristatum Lichina pygmaea Peltigera horizontalis

Torres et al. 2004 Roullier et al. 2009

310

Gnomonia leptostyla Degelia plumbea

Fayret et al. 1981 Roullier et al. 2011

Nephroma laevigatum

Roullier et al. 2011

1

R

Mycosporine glycine H

COOH

Büdel et al. 1997

de la Coba et al. 2009

Mycosporine serinol H OH OH

Mycosporine glutamicol H OH

303 C13H21NO7

COOH

Mycosporine hydroxy-glutamicol H OH

310 319 C13H21NO8

HO COOH

Collemine A 310 Collema cristatum 34000 496 C19H32N2O13

HO OH O HO HO HO

Torres et al. 2004

O NH O

HO

H

Figure 15.2 Mycosporine-like compounds more typically found in lichens.

The usual protocols start by extracting the crude material with water, alcohols (ethanol, methanol) and mixtures thereof (water/ methanol (75/25)  0.1% acetic acid), performed on fresh or freeze-dried samples with great variations in the temperature range (from cool to boiling). However, in order to prevent

artifact generation or hydrolysis, it is recommended that temperatures higher than 45  C are avoided. Two methods are available to perform a simultaneous extraction and characterization (by HPLC) of mycosporines and MAAs:

15.2 Chemistry

Cyanobacteria/Phytoplankton/ Macroalgae Gr€ oniger et al., 2000 Homogenization 2.5 h in MeOH 20%, 45  C Centrifugation þ lyophilization/ evaporation of the supernatant Redissolve the residue in 100% MeOH 2–3 min Centrifugation Supernatant evaporated to dryness, 45  C Redissolve the residue in 0.2% acetic acid/double-distilled water Filtrate passed through a 0.2 mm pore size

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Cyanobacteria/Fungi/Red algae Volkmann and Gorbushina (2006), (according to Whitehead et al., 2001a) 100 mg moist sample/Eppendorf Lyophilization Pulverization in liquid nitrogen 20 mg crushed, suspended in 1 ml water (þ0.2% acetic acid, þ0.5% MeOH) Vortex, 4  C, 12 h Centrifugation 14 000  g, 5 min Filtrate passed through a 0.2 mm pore size

The HPLC quantification of mycosporine-serinol from the lichen Lichina pygmea was performed following a variety of protocols, including varying parameters such as the percentage of methanol in water, the temperature (4  C or 40  C), and the presence or absence of mechanical crushing of the raw material. The mycosporine ratio was about 2–3% of the extracted polar compounds (50 mg raw material per ml solvent, 3 h). For this lichen, water was found to be the best extraction solvent and, somewhat surprisingly, the greatest quantity of mycosporines to be extracted (0.26 mg) was achieved with pure water at þ4  C, without crushing the lichen material. Metabolite screening on cyanolichens revealed the complexity of the extracts, which occasionally was too high to allow any conclusive characterization of the detected compounds. In most reports, the crude extract is semi-purified by chromatographic techniques involving gel permeation, activated charcoal and/or ion-exchange resins or precipitation techniques. Three different methods, including sugar precipitation, gel permeation and ionexchange chromatography have been compared. The relative efficiency of these methods was assessed on 50 mg of two cyanolichens, Lichina pygmaea and Peltigera horizontalis, for which a thin-layer chromatography migration had suggested mycosporine-serinol to be present (Figure 15.3). In this case, the cation-exchange resin Dowex 50W-X8 (Hþ form) was confirmed as the most efficient stationary phase for concentrating the mycosporines (elution was achieved with a solution of 200 mg ml 1 NaCl after the cleaning of nonadsorbed compounds). After evaporating to dryness, salt removal was achieved by the solubilization of mycosporines in ethanol. Ultimately, the mycosporine proportion of the purified extract was about 20%, compared to 2.5% in the crude extract. 15.2.3.2 Detection, Quantification, and Monitoring in Live Samples Because of the lack of commercially available MAA standards, much of the characterization has been accomplished using the distinctive nature of their absorption spectra. Rezanka et al. (2004) have proposed an approach adapted to the aquatic

Figure 15.3 Influence of different purification techniques on the content of mycosporine serinol from L. pygmaea and P. horizontalis extracts.

environment, based on liquid chromatography (LC) coupled with electron ionization mass spectrometry (ESI-MS-MS), which allows all known MAAs to be distinguished based on individual retention times, wavelength maxima and molar masses. A further advantage is that the fragmentation patterns of selected ions can be examined. 15.2.4 Structure Determination 15.2.4.1 Ultraviolet (UV) Spectroscopy As mentioned above, one of the most remarkable characteristic of mycosporines and MAAs is the presence of a unique strong absorption peak in the UV region (Figures 15.1 and 15.2) (Arpin, Curt, and Favre-Bonvin, 1979). The lmax values range from 310 to 360 nm, and are correlated to high molar extinction coefficients (i.e., e ¼ 25 000–60 000 l mol 1 cm 1; Bandaranayake, 1998), which explains their potential role in photoprotection. Based on this specific UV profile (symmetrical peak centered on 310 nm for aminocyclohexenone rings, 320 nm for noraminocyclohexenone rings, and 320–360 nm for iminocycloheximines), most can be detected and characterized as MAAs through diode-array detection (DAD), following separation on HP-TLC plates or HPLC/UPLC columns in order to perform the required spectroscopic analysis (Figure 15.4). The characterization and quantification of MAAs is sometimes performed using reverse-phased columns with a TFA/ ammonium mobile phase to enhance the polarity separation of MAA mixtures, and UV-DAD (Cardozo et al., 2008) (Box 15.2). 15.2.4.2 Mass Spectrometry (MS) Early mass spectroscopy studies on MAAs only enabled estimations of their molecular mass to be made. Recently, however, several MS techniques have been applied to the structural elucidation of MAAs (Whitehead and Hedges, 2003), especially since the analysis of the fragmentation patterns has become more reliable. For example, by using ESI-MS on

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Figure 15.4 Characteristic UV absorption spectrum of a mycosporine; example of, mycosporine serinol isolated from the marine lichen Lichina pygmaea.

positive mode, Cardozo et al. (2008) proposed a complete analysis of the fragmentation pattern of palythine, palythinol and asterina MAAs. In general, MSn with accurate HR-mass measurement enable known MAAs recognition and an anticipation of structures. However, the identification of new derivatives can only be ascertained through the isolation of suitable quantities. 15.2.4.3 Nuclear Magnetic Resonance (NMR) Spectroscopy NMR remains the method of choice for the scalar determination of MAA structures. As previously described, mycosporines and MAAs present a rather well-conserved scaffold, which consists of a cyclohexenone or cyclohexenimine ring that is substituted with amino alcohols or amino acids (Figures 15.1 and 15.2). Their

NMR spectroscopic data are then comparable and show characteristic patterns (as indicated in Appendices 15A.1 and 15A.2). For example: i) For 1 H NMR data, the hydroxymethyl group at C-5 usually appears around 3.60 ppm as a singlet (ranging from 3.50 to 4.04 ppm), together with the methoxyl group at C-2, which is usually represented by another singlet around the same region (3.55–3.93 ppm). In the same way, the four protons on C-4 and C-6 are often noted on 1 H NMR spectra as four doublets, with a geminal coupling constant around 17 Hz. In many cases, the authors did not assign these. ii) The 13 C NMR spectroscopic data are very well conserved among mycosporines and MAAs. One clear difference

Box 15.2: Bathochromic Shift The degree of electron delocalization varies with the nature of the different substituents on the oxygen, or on the nitrogen borne by the C-1 carbon. The greater it is, the higher the

wavelength of the maximum absorbance peak (lmax). This partly explains the lmax differences observed between different MAAs (Figure 15.5).

Figure 15.5 UV absorption and bathochromic effect observed in mycosporine-like amino acids.

15.2 Chemistry

Figure 15.6

1

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H NMR spectrum of mycosporine hydroxyl-glutamicol (500 MHz, D2O).

between oxomycosporines and iminomycosporines is the chemical shift of C-1, which is more around 180 ppm for oxo-mycosporines and 160 ppm for imino-mycosporines. C-4 and C-6 are usually located very close, with chemical shifts around 30–40 ppm. The proton (Figure 15.6) and the 13 C NMR (Figure 15.7) data from mycosporine hydroxy-glutamicol are presented as an example of NMR patterns typical of mycosporines (Roullier et al., 2011). 15.2.5 Synthesis

Early MAA syntheses are reviewed by Bandaranayake (1998). The exceptional efficiency of MAAs as sunscreens has prompted synthetic chemists to not only reproduce key natural structures but also devise synthetic analogs with enhanced activities, without any loss of molecular stability for their intended applications. Recent patents have been filed in relation to extraction methodology and formulation, more than to structural novelties. Today, most efforts are directed towards the production of MAAs by genetically modified microorganisms. 15.2.6 Biosynthesis: Labeled Precursor Investigations 15.2.6.1 The Shikimic Acid Pathway The common precursor to MAAs is 4-deoxygadusol (4-DG), as evidenced from genome-mining and biochemical investigations

on referenced strains of cyanobacteria. These serve as models to other MAA-producing marine organisms, as gene transfer (LTG) between cyanobacteria and host have occurred – and still occur – as a common evo-devo (evolutionary and developmental) feature. Multiple investigations have demonstrated the role of the shikimic acid pathway in the biosynthesis of 4-DG, a precursor common to both fungal mycosporines and cyanobacterial MAAs. In fungi, evidence was initially derived from the metabolic incorporation of a 14 C-labeled 3-dehydroquinate intermediate (DHQ) by the fungal parasite Trichothecium roseum, which subsequently transformed DHQ into structurally related mycosporines (Favre-Bonvin et al., 1987). Radiolabeling experiments on the cyanobacterium Chlorogloeopsis sp. PCC 6912 later confirmed that the shikimic acid pathway is involved in MAA biosynthesis, through the incorporation of 14 C-labeled pyruvate, an early and obligate precursor of this pathway, and its selective transformation into radiolabeled MAAs (Portwich and GarciaPichel, 2003). In the same study, it was also shown that the polyketide pathway was not involved, as 14 C-labeled acetate was not traceable in the MAA cytoplasmic pool of metabolites. Additional evidence was obtained via the use of exogenous tyrosine (a shikimic acid pathway repressor), while the use of glyphosate (Roundup1) as a shikimic acid pathway inhibitor was shown to block MAA biosynthesis in the cyanobacterium Nostoc commune (Sinha et al., 2003), and also in the scleractinian coral Stylophora pistillata (Shick et al., 1999). The suite of MAAs that are synthesized from 4-DG (itself a strong antioxidant) as a common precursor, is initiated with mycosporine-glycine (inclusion of glycine residue at C-3) as a

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Figure 15.7

15 Mycosporine-Like Amino Acids (MAAs) in Biological Photosystems

13

C NMR spectrum of mycosporine hydroxyl-glutamicol (125 MHz, D2O).

primary product, which is itself transformed into secondary structures through chemical and/or biochemical conversions into secondary products (Singh et al., 2008; Rosic and Dove, 2011). Through gene-mining and bioinformatics studies, Singh et al. (2010a) have determined the gene system which is responsible for the expression of the putative enzymes producing a 4DG precursor of shinorine in Anabaena variabilis strain PCC 7937 (Figure 15.8). In this figure the enzymes are indicated in red, the putative genes loci from the gene cluster which express these enzymes are indicated in black, and databank accession numbers in blue (see Singh et al., 2010a for details).

produced in a convergent manner as the general MAA precursor, via two different biosynthetic pathways (Spence et al., 2012). Gene-mining investigations in the cyanobacterial model strain Anabaena variabilis ATCC 29413 showed that four enzymes are involved in the synthesis of the specific MAA shinorine: (i) a dehydroquinase synthase homolog (DHQS); (ii) an O-methyl-transferase (O-MT); (iii) an ATP grasp ligase; and (iv) an NRPS-like enzyme. Cloning of the entire gene cluster in Escherichia coli led to the production of shinorine. The production of the intermediate 4-deoxygadusol by DHQS and O-MT could be reproduced in vitro (Balskus and Walsh, 2010).

15.2.6.2 The Pentose Phosphate Pathway Shinorine is a secondary product of mycosporine-glycine in which a serine residue is linked at the imino site of the cycle. Its biosynthesis in Anabaena variabilis (ATCC 29413) – via the intermediates 4-DG and shinorine – was recently proved to proceed via an alternative route to that of the shikimic acid pathway (Figure 15.9). Instead of DHQ (a branchpoint intermediate in the shikimic acid pathway), 2-epi-5-epi-valiolone (EV, a product of the pentose phosphate pathway) was found to be the precursor of 4-DG in this cyanobacterium. Besides structural similarities between the two precursors EV and DHQ, the enzymes involved in their conversion into 4-DG – respectively EV synthase (EVS) and DHQ synthase (DHQS) – have strikingly similar three-dimensional structures, and both belong to the same superfamily of enzymes. Genome mining studies have shown that the EVS gene is present only in cyanobacterial strains that produce MAAs. It appears that 4-DG can be

15.2.7 Regulation of MAA Production: Light and Nutrients 15.2.7.1 Light Exposure to UVB radiation has a definite positive effect on the production of MAAs in a variety of micro- and macro-organisms. W€angberg, Persson, and Karlson (1997) have shown that cultures of the dinoflagellate Heterocapsa triquetra exposed to artificial UVB, when compared to growth in absence of UVB, showed an increase in MAA production, in synchrony with cell cycle, and changes in the absorbance spectra during UVB exposure indicated that the composition of the MAAs varied accordingly. Sinha et al. (2001) showed that the cyanobacterium Scytonema sp. increased its MAA production during the light phases of its circadian cycle. Torres et al. (2007) found that Acropora cervicornis (stagshorn coral), when transplanted from 20 m to 1 m depth, reacted rapidly to the increase in UVR levels

15.2 Chemistry

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Figure 15.8 Proposed biosynthetic pathway of shinorine in the cyanobacterium Anabaena variabilis.

by a general 40-fold increase in MAA production, including both polyp hosts and photosymbionts. In the latter case, activation of the shikimic acid pathway (itself dependent on photosynthesis) with the production of MAA precursors is expectedly dependent on the amount of sunlight available. Shick et al. (1999) found that the bleaching process (loss of zooxanthellae) of UVBexposed Stylophora pistillata corals did not prevent MAA accumulation by the remaining microbionts.

15.2.7.2 Nutrients The effects of nutrient limitation on MAA production is less clear. Nitrogen limitation may lead to a quantitative decrease in MAA production (Karsten, Lembcke, and Schumann, 2007), while a sulfur deficiency may affect the qualitative bioconversion of a primary MAA to a secondary MAA (Singh et al., 2010b).

Figure 15.9 Proposed alternative biosynthetic pathway of shinorine in the cyanobacterium Anabaena variabilis. The enzymes are indicated in red, the putative genes loci from the gene cluster which express these enzymes are indicated in black, and databank accession numbers in blue. See Singh et al., 2010a for details.

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15.2.8 Degradation

The photoprotective role of MAAs has been demonstrated for all study models, from cyanobacteria to photosymbiotic systems, against UVA (315–400 nm) and UVB (280–315 nm) radiations. Since MAAs are considered to be highly photostable, very few studies have examined the molecular degradations aspects. Photosensitization by an external agent is required for indirect and temperature-dependent photodegradation. This was confirmed by Bernillon et al. (1990), who degraded mycosporine-glutamine (an oxo-MAA) into aminocyclohexenone and 2-hydroxyglutaric acid, in presence of flavin, oxygen and light. Later, Whitehead and Hedges (2005) confirmed this with imino-MAAs by testing different inducers (riboflavin, rose Bengal, natural seawater) on palythine, shinorine and porphyra-334, three early products of the putative MAA biosynthesis suite proposed by Sinha and H€ader (2008), and in the presence of light and oxygen. In the ocean, MAAs are resistant to photodegradation, a process that requires the presence of photoreactive solutes that have the potential to create radicals necessary to initiate the photooxidation process.

using them as associates). The first category includes heterotrophic consumers, ranging from browsing herbivores to carnivorous predators, while the second category lacks true mesodermderived organs and performs basic metabolic functions with help from microbionts that live in close association with them. This includes the synthesis of structural and energy-yielding primary compounds, and the various catalytic processes that enable their transformation into secondary metabolites through environmentally mediated regulatory networks.

15.3.1 Chemical Protection Against Abiotic Stress

15.3.1.1 Symbiont-Assisted Metabolism Many reef corals and sponges shelter dinoflagellates and/or cyanobacteria and exploit an alternative energy source from photosynthates produced during light hours, in addition to being night-time heterotrophs. Nutrient cycling in such photosystems is achieved with bacterial and archaeal functional consortia. Photosymbioses between a eukaryotic host and its dedicated protistan and/or bacterial microbionts represent elaborate adaptive processes that allow whole living systems to function as a biological entity, termed the holobiont (see Box 15.3). Some specialized herbivores transiently sequester plastids or even whole dinoflagellates in diverticula as part of their digestion process, and become “solar-powered” or, more precisely, “hybrid-powered.” This kleptoplasty is regarded by some as part of an evolutionary process towards acquiring a fully integrated organelle if the genes coding for essential photosynthesis proteins are acquired by horizontal gene transfer (e.g., Rumpho et al., 2008).

Aquatic life forms that derive their metabolic energy directly from capturing and transforming sunlight by photosynthesis are the primary producers of the organic matter upon which all food chains are based. In the marine world, these autotrophs range from cyanobacteria to phytoplankton (Whitehead et al., 2001b) and to large macrophytic algae, and are present from superficial oceanic waters worldwide to small tide pools. When sharing the same habitats, other life forms depend on the “goods and services” of these primary producers, whether by predation (i.e., eating and digesting them) or by symbiosis (i.e.,

 trois” Solution 15.3.1.2 The “m
enage a Symbiotic photosystems have also colonized terrestrial habitats, some to the point of becoming extremophiles, in a totally different direction to that taken by chemoautotrophydependent inhabitants of hydrothermal vents in oceanic depths. Lichens are able to colonize and even monopolize arid and extremely inhospitable habitats, possibly to escape predation pressures and access new resources without competition by contending life forms. Lichens are a composite cluster resulting from a three-way symbiotic association

15.3 MAA-Producing Organisms

Box 15.3: The Holobiont, the Hologenome, the Metagenome The original and primary definition of the term holobiont (one host and its dedicated symbiont) by Mindell (1992), was later expanded by Rohwer et al. (2002) and Rosenberg et al. (2007) to include the functional microbiome of corals. From a functional genomics perspective, Rosenberg and Zilber-Rosenberg (2008) coinedthetermhologenome,whichdefinesthesumofthegenetic information of the host and its symbiotic microorganisms, the host being any nucleated organism from dinoflagellate to human. In practice, the metagenome obtained by environmental (or clinical) microbial sampling of the host’s sphere may not coincide with the hologenome. The former may include neutral and pathological microbiota, whereas the latter refers to functional microflora (at least under no-stress conditions).

In evolutionary terms, a major challenge of modern environmental genomics is to study precisely how the environment influences the holobiont’s performance. Systems biology thus encompasses the transcriptomic responses of a model holobiont facing specific stresses, and their downstream proteomic and metabolic consequences on the host organisms, and the metagenomic diversity reduction and composition shifts of their microbiomes. Knowledge of the host’s genome is always advantageous, in order to correlate the observed changes to specific genes or gene clusters. Some holobionts are stenotolerant – that is, they are not tolerant to environmental fluctuations which would allow more resistant competitors to thrive and displace them.

15.4 Hermatypic Corals: Living Under Tight Constraints

between a photobiont (green alga or cyanobacteria), a mycobiont (fungus), and a dedicated bacterial pool (microbiont) which helps maintain the stability of this assemblage (Grube et al., 2009). The fungal partner supplies water and minerals as well as mechanical protection, while the green alga and/or cyanobacteria provides photosynthates; in some cases the fixation of atmospheric nitrogen occurs via specialized cyanobacterial structures, the cephalodia. 15.3.1.3 The Chemical Answer to an Exposed Mode of Life Some of the challenges to which sessile, slow-moving or freefloating marine life forms exposed to sunlight must to face include: (i) protection against genotoxic radiations; (ii) oxidative stress; and (iii) a maintenance of osmotic balance under fluctuating physico-chemical environmental parameters. The main survival strategy that soft-bodied or unprotected organisms frequently employ is chemical, whether toxins for protection and defense against biotic threats or sunscreens against harmful radiations and oxidative stress (i.e., abiotic damages). The cooccurrence of these two lines of defense may also be explained by features that many share in common: (i) they are formed by amino acid building blocks for toxic cyclic peptides and mycosporin-like amino acids; and (ii) their possible transmission along food chains and reuse by secondary consumers after further transformation (Klisch and H€ader, 2008). However, of all the MAAs known to date none has shown any sign of toxicity, and their biosynthesis is not linked to the regulation of the same genes as defense compounds. Lichens are geared to live in extreme conditions that do not fluctuate as rapidly as shallow-water marine organisms, and are much less exposed to the intense predation pressures typical of biodiverse ecosystems, such as tropical coral reefs. This is reflected in their chemical secondary repertoire, which is mainly composed of UV-absorbing compounds, whether of polyphenolic nature (depsides and depsidones) or oxo-carbonyl MAAs, depending on the photosymbiont type (algal for the former, cyanobacterial for the latter). In addition, the fungal “host” contributes fungal mycosporines of its own (Roullier et al., 2011), with some unique glycoconjugated examples (Torres et al., 2004). 15.3.1.4 Simple, Effective, and Ubiquitous: Why Change a Winning Recipe? The wide distribution – both phyletic (some 380 marine species identified to date) and geographic (from tropical to polar regions) – of MAAs is a measure of their longstanding evolutionary success as sunscreens and antioxidants. This is partly explained by their exceptional UVB and short-wave UVA absorbance characteristics, partly by their solubility (i.e., their homogenous cytoplasmic distribution within single cells and superficial localization of exposed tissues in multicellular organisms), in addition to being chemically “simple” (not requiring complex and metabolically expensive biosynthesis). General reviews on this topic have been produced by Shick and Dunlap (2002), and more recently by Careto and Carignan (2011); the multifaceted roles of MAAs are discussed by Bandaranayake (1998) and Oren and Gunde-Cimerman (2007).

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In addition to being typically found in cyanobacteria, MAAs are present in most microalgae and in light-exposed phytoplankton (Jeffrey et al., 1999; Llewellyn and Airs, 2010). Diatoms combine the optical anti-UV protection afforded by a siliceous skeleton with the chemical protection of MAAs (Ingalls et al., 2010). Bloom-forming dinoflagellates are able to respond rapidly to changing light regimes by regulating the amount and composition of these molecules. The earlier discovered and greatest source of MAAs is in the Rhodophyta (red algae), both in terms of structural diversity and concentration (Karsten et al., 1998), and more so than in the green algae. This is in contrast to the Phaeophyta (brown algae), which rely on phototannins to achieve equivalent anti-UV (Karentz, 2001), antioxidant, and antiradicalar roles (Karsten et al., 1998; Pavia and Toth, 2000; La Barre et al., 2004).

15.4 Hermatypic Corals: Living Under Tight Constraints 15.4.1 Coral Reefs are Monumental Bioconstructions

Coral reefs occupy 0.1% of the projected surface of the world’s oceans, but are confined to sunlit and warm waters in tropical and subtropical latitudes. Coral reef ecosystems shelter an estimated 30% of the total observable marine species diversity, typically organized into complex assemblages around limestone scaffolds made of coral skeletons and debris cemented by coralline algae. Hermatypic (shallow-water, reef-forming) corals require sunlight to gather the photosynthetic energy necessary to achieve biomineralization; that is, the accretion of calcium carbonate from dissolved carbonates and cementation into a most often colonial skeleton. In these diploblastic invertebrates (without mesoderm), the crossmembrane trafficking of ions (mineral and organic) is intense between the immediate hydrosphere and their tissues. Reef-building corals provide the limestone scaffold inside and around which innumerable communities of marine life forms coexist. Biodiversity and trophic chains are maintained if nutrient cycling is optimal, and without interference at any of the trophic levels (Rohwer, 2010). For example, the nutrient enrichment of oligotrophic waters induces drastic metagenomic shifts from diverse and functional microbiomes to reduced and pathogen-dominated forms (Mouchka, Hewson, and Harvell, 2010). However, climatic changes are likely to modify the degradation scenario even more than direct human interference, with global warming and increased exposure to harmful solar radiations being the most significant features, along with ocean acidification (La Barre, 2013). 15.4.2 Corals are Highly Efficient Photosynthesizers

The symbiosis between scleractinian corals and their indwelling zooxanthellae microalgae (Symbiodinium) is an ancient feature

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Figure 15.10 Chemical photoprotection of the reef coral holobiont. Coral colonies can be either branched (1a) or massive (1b), and are made of polyps (2a) which are often highly colored in daylight due to phycobilin pigments of the tiny zooxanthellae microalgal symbionts. Fluorescent GFP-like proteins glow under UV light in many coral species (2b). Under stress, the zooxanthellae (4) leave the endodermal layer of the polyp host tissues (3a), causing discoloration. Even when bleached, polyps are still partly protected by the MAAs concentrated in the epidermal tissue layer (3b).

that has required modifications on behalf of both parties to become a light-harvesting system, (Stambler and Dubinsky, 2005), up to sixfold as efficient as that of land plants (Enríquez, Eugenio, and Iglesias-Prieto, 2005), and involving a combination of interfacial and skeletal photon-scattering systems (Reef, Kaniewska, and Hoegh-Guldberg, 2009). 15.4.3 High Temperatures and UV Exposures Induce Oxidative Stress and Bleaching in Corals

Corals are sensitive to seawater temperature elevations above 32  C (Berkelmans and Willis, 1999), with some species more sensitive than others (Barshis et al., 2013). They are also sensitive to excess exposure to photosynthesis active radiation (PAR) (Banaszak and Lesser, 2009), and to any ensuing photooxidative stress (Lesser, 2006) involving physiological and genotoxic damage. A particularly crucial phase of corals undergoing temperature-induced bleaching is the accelerating negative effects of short-wave UVA and UVB on exposed tissues. Some corals produce green fluorescent protein (GFP)-like materials that afford some protection by quenching excess high-energy PAR and re-emitting it as fluorescent light (Catala-Stucki, 1959). Coral skeletons also have an intrinsic yellow luminescence that helps to dampen high-energy PAR while increasing its harvesting efficiency (Reef, Kaniewska, and Hoegh-Guldberg, 2009). However, MAAs are by and large the most efficient protection against the combined effects of temperature, PAR and oxidative stress (Figure 15.10).

15.4.4 The Chemical Acclimation of Scleractinian Corals to an Exposed Lifestyle

While characterizing the highly colorful pigments of several Acroporid and Pocilloporid corals from the surface waters of the Great Barrier Reef in Queensland, Shibata (1969) was surprised to find elevated concentrations of highly UV-absorbing compounds at and around 320 nm, as well as in a cyanobacterium in the vicinity of these corals. Collectively termed S-320, these compounds were subsequently found in massive Porites corals in concentration varying inversely with depth, thereby accrediting their role as anti-UV screens (Maragos, 1972). It was later shown that the production of S-320 in Pocilloporid corals is directly linked to the level and duration of exposure to shortwave UVB radiations (Jokiel and York, 1982). The first S-320 characterization came from the incidental discovery of mycosporineglycine from the zoanthid coelenterate Palythoa tuberculosa (Ito and Hirata, 1977), before a whole suite of similar molecules was described from various algal and invertebrate sources, revealing MAAs as a widespread class of compounds mainly involved in protection against the radicalar and oxidative damages caused by excess exposure to solar radiations. While the types and concentrations of MAA may differ among coral reef invertebrates, their range in hermatypic (photosymbiotic) corals and of giant clams is quite large (encompassing 15 structures in all, included in Figure 15.1), and seems consistently related to the presence of clades of Symbiodinium microalgal symbionts in these hosts. The single model species

15.5 Lichenic Systems: Living in the Extremes

Stylophora pistillata can possess up to 12 MAA structures. Various observations reported in Shick and Dunlap (2002) indicate that the MAAs are concentrated in the ectodermal cell layers of coral tissues, rather than in the endodermal layers where the microalgae are sequestered. Also, directly exposed regions (e.g., apical and central zones of the colonies) concentrate more MAAs than the shaded or lateral regions, which again suggests a photoadaptive relocalization from the biogenic site. In branched corals, the co-occurrence of higher concentrations of both MAAs and colorful phycobilin pigments at actively growing apical zones may afford a more general photo- and thermo-protective cover against potentially genotoxic and heat stress conditions. The relocation and accumulation to exposed tissue surfaces can be regarded as temporarily advantageous to coral polyps during bleaching episodes, in the absence of the symbiotic algae which produce these compounds. 15.4.5 Biogenic Sources of MAAs in Scleractinian Corals

In hermatypic (shallow-water and photosymbiotic) scleractinian corals, the microalgal symbionts generate much of the chemical photoprotection for the whole colony, except for the fluorescent GFP-like pigments introduced earlier, which are synthesized by the cnidarian host. This includes those MAAs which are transferred from the dinoflagellates to the host, and the colorful phycobilin pigments which are lost during bleaching episodes. However, some coral genomes possess the genes that would allow them to synthesize UV-absorbing compounds by themselves (Shinzato et al., 2011), and this is a possible further adaptation to extended bleaching periods. Finally, some MAA complements can be acquired by food and transformed, which may explain why a single coral species such as the model Stylophora pistillata may possess up to 15 MAA structures, while no more than five different MAA structures can be isolated from Symbiodinium microalgae freshly isolated from a coral holobiont, or from the culture medium (see Rosic and Dove, 2011). Apart from exposure-dependent, ectodermal accumulation and distribution and inversely depth-dependent considerations of the presence of MAAs in coral holobionts, the regulation of their biosynthesis and their expressed repertoire responds to rapid, as well as seasonal, fluctuations in PAR and UVB levels (see Shick and Dunlap, 2002). 15.4.6 The Phylogenomics of MAAs in Scleractinian Corals

The shikimic acid pathway is a major feature of the metabolisms of plants and microorganisms, but not of animals. However, the possibility of gene transfer involved in the shikimate pathway between symbiotic dinoflagellates and/or bacterial symbionts and the coral host is now seriously considered (Rosic and Dove, 2011). Indeed, in photosymbiotic systems such as reef-building coral holobionts, the complete MAA biosynthesis is predicted to be a “shared metabolic adaptation” of symbiosis, with the required biochemical intermediates from the shikimic acid pathway

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emanating from the dinoflagellate partner and final steps of the biosynthesis occurring in the host (Starcevic et al., 2010). Therefore, gene-mining approaches must take the coral host and its transient microbionts compartments into consideration. Singh et al. (2010a) have undertaken a bioinformatics approach on several strains of cyanobacteria, and hypothesized that genes YP_324358 (coding for DHQ synthase) and YP_324357 (coding for O-methyl transferase) are transferred from a cyanobacterial donor to dinoflagellates and finally to a metazoan via lateral gene transfer (LTG) events. A biochemical and phylogenetic study of the genes involved in the biosynthesis of MAAs in symbiotic dinoflagellates was recently undertaken (Rosic, 2012). This was in response, first, to the growing concern for thinning of the high-altitude ozone layer that acts as a protective shield against harmful solar radiations, and which is especially critical in the Great Barrier Reef provinces in Australia. A second concern was the need to produce protective and harmless skincare agents (e.g., Cardozo et al., 2007; de la Coba et al., 2009b) through biomimetic approaches. The putative MAA biosynthetic genes from symbiotic dinoflagellates were found to show a monophyletic clustering with their algal and bacterial homologs, confirming their microbial origin. The cluster of the four genes of the shikimic acid pathway involved in shinorine biosynthesis in cyanobacteria are found not only in the zooxanthellae (Balskus and Walsh, 2010), but also in cnidarians, which suggests that some metazoans may synthesize the UV-absorbing compounds by themselves (Shinzato et al., 2011). However, the precise origin and the regulation transcriptomics of MAAs in coral holobionts will be undertaken in several model species (see Chapter 20), and should be investigated within the context of early stress responses of whole coral holobionts in the face of climatic changes, and integrated into evaluation studies of the fitness of impacted communities (La Barre, 2013).

15.5 Lichenic Systems: Living in the Extremes

Some specialized fungi (mainly Ascomycota) have the ability to combine with algae and/or cyanobacteria, and this results in self-supporting symbiotic associations with extremophilic capabilities. The lichenous lifestyle is maintained by about 18 800 known species (Feuerer and Hawksworth, 2007), most of which are microscopic lichens. Whatever the shape of the thallus, most visible lichens expose their vegetative parts at the substrate surface, enabling the photobiont to harvest energy from solar radiation. For at least 600 million years, lichens have developed highly extremotolerant systems which allow them to withstand warm deserts, polar or alpine areas as pioneer organisms. In these preferred colder and humid habitats, a number of the lichens are cryptoendolithic and are hidden in rocks, while the thalli are associated with a variety of other organisms (e.g., black fungi, bacteria). Some alpine epilithic lichens, when taken as models of complex living organisms, have survived long-term (up to 1.5 years) exposures to cosmic radiation on the outer surface of the International Space Station (Onofri et al., 2012).

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A variety of compound classes is associated with these longliving and slow-growing organisms, and the abundance of some of these protective compounds is frequently correlated to environmental exposure (Heber et al., 2000). Their resistance to extreme temperatures, desiccation and UV radiation stress is sustained by various biochemical processes and the synthesis of protective molecules, but above all to their capacity to survive in the stage of anhydrobiosis (Beckett, Kranner, and Minibayeva, 2008). Lichens must cope with drastic and rapid balances of environmental conditions, often counterbalancing reactive oxygen species (ROS) production and deleterious UV lights (Bart
ak et al., 2004). This is well illustrated in polar, alpine or intertidal ecosystems, were lichens frequently constitute the highest represented flora. Chlorolichens are known to produce major phenol metabolites, and most of them have UV-absorbing properties; the cyanolichens are generally devoid of these compounds, although some species accumulate more polar materials, including mycosporines. Until now, these compounds have been reported in a very limited number of lichens having a cyanobacterial photobiont (Roullier et al., 2011). The first convincing report was made by B€ udel et al. (1997) from cyanolichens, most of which were black melanized lichens, standing on highly sun-exposed rocks in tropical and subtropical regions from various continents. Except for one of the species studied, near-UV-B absorbing MAAs cooccurred with the near-UVA range-absorbing scytonemin. The MAA concentration was found to be about 0.1–0.8% dry weight (concentrations were higher in Peltula euploca collected from shaded habitats), and mycosporine-glycine was characterized as the major compound. In contrast to scytonemin, which has been proven to be produced by cyanobacteria, the production of MAAs is not clearly defined, as both partners could contribute to the process. The glycosylated collemin A, isolated and identified from the gel-hydrated Collema cristatum, was also recognized as being produced by a culture of the mycobiont exhibiting a characteristic UV 311 nm-absorbing shape (Torres et al., 2004) (see Box 15.4). From the marine lichen, Lichina pygmea, which formed dark cushions in the intertidal zone and contained a Calothrix cyanobacterium, the antioxidant and photoprotective properties

were reported as related to mycosporine-glycine, characterized on the basis of HPLC behavior with a standard source supposed to contain this compound (de la Coba et al., 2009a). After isolation via a dual-mode Centrifugal Partition Chromatography experiment (Roullier et al., 2009), mycosporine-serinol was fully identified as the major MAA from Lichina pygmea gathered from the Brittany coast. In a screening carried out on 15 lichen species, mycosporineserinol and mycosporine-glutamicol were identified for the first time in five cyanolichens, while six unknown mycosporine-like compounds were characterized in five other cyanolichens from various habitats, some of which were mostly found in shaded and humid habitats (Roullier et al., 2011). Thus, it is questionable MAAs are produced as a response to UV only. While optimizing the extraction process of mycosporines, the highest quantity of mycosporine-serinol (ca. 0.6% dry weight) was obtained with a 3 h period of passive diffusion in 4  C distilled water from the uncrushed thallus of Lichina pygmea. Thus, a dynamic production and diffusion of these highly soluble compounds in an aqueous environment should be considered as they are mixed with other hydrophilic compounds such as polyols, sugars, and amino acids; hence, a suitable protocol (vide supra) must be specifically applied to recognize and isolate these MAAs. Following such a procedure, about 8 mg of the newly described mycosporine-hydroxyglutamicol was isolated from 3 g of the squamulose cyanolichen Nephroma laevigatum (Roullier et al., 2011).

15.6 Modes of Action and Applications to Human Welfare

Oxidative stress refers to a situation where ROS such as hydrogen peroxide and oxygen-derived free radicals are produced and initiate a chain reaction, with resulting damage to the cellular systems. ROS are produced when photosystems absorb more energy than can either be transferred by the electron transport chain to an electron acceptor, or dissipated as heat. Corals and other fixed invertebrate photosystems usually combine pigments, sunscreens and proteins that optimize protection in a species-specific fashion, and these molecules have an interest-

Box 15.4: Lichens: Looking into the Future As lichens are composed of organisms that possibly are producing their own mycosporines or MAAs, they represent an interesting challenge for investigating mycosporine biogenesis and distribution within their thalli. So far, cyanobacteria seem to be involved in the presence of such compounds in lichens, as they can absorb atmospheric nitrogen and incorporate it into their metabolism. However, all of the mycosporines described to date in lichens have the characteristic carbonyl moiety of fungal mycosporines. The fungal partner generally constitutes more

than 80% of the lichen biomass, and it will be especially interesting to investigate the production and distribution of these compounds in tripartite lichens, where the cyanobacteria are confined to specific tissues termed cephalodia. Preliminary results obtained with LC-HRMS analyses of the cephalodia compared to the whole thallus have suggested the presence of 330 nm-absorbing iminomycosporines in the cephalodia, while 310 nm-absorbing oxomycosporines are typically found in the whole thallus (Roullier et al., 2011).

15.7 Conclusions

ing biotechnological potential. Large-scale microalgal cultivation represents an important source of food additives – for example, carotenoids, vitamins C and E, unsaturated lipids and many others – the production of which can be boosted by an appropriate combination of stressors (Skjanes, Rebours, and Lindblad (2013). The production of MAAs in microscopic green algae (Sinha, Singh, and Hader, 2007) and marine organisms is directly linked to exposure to short-wave UVR (in terms of both intensity and duration), and inversely correlated with nitrogen levels in some investigations (Karsten et al., 2007). 15.6.1 Skin Care and Cosmetics

Thus, MAAs and synthetic derivatives have inspired applications as skin care products (Bandaranayake, 1998), corresponding to a strong demand in the cosmetics market for chemically stable, potent, and broad-spectrum molecules. As noted by Bhatia et al. (2011), many earlier formulations contained carcinogenic agents, some with estrogen-like effects, or generated harmful radical species. The sun-protection factor (SPF) is defined as the ratio of the time of UV exposure necessary to produce minimally detectable erythema in sunscreen-protected skin, to that of the time taken to produce the same effect for unprotected skin (Bhatia et al., 2010). Although useful, the SPF is insufficient to evaluate the carcinogenic potential of a substance, and must be complemented by more specific tests. As an example, Helioguard 3651 is a commercial formulation based on the mycosporine-like amino acids shinorine and Porphyra334 from the red alga Porphyra umbilicalis; these compounds are encapsulated into liposomes in order to increase their uptake by the skin.

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15.6.2 Biotechnological Applications

Nonmedical applications of MAAs include photostabilizing additives in plastics, paint, and varnish (Torres et al., 2006; Sampedro, 2011). A typical example is that of automobile paints, which are subjected to intense and prolonged exposures to sunlight.

15.7 Conclusions

Mycosporine-like amino acids and their natural analogs are lifeessential molecules for life forms that live directly exposed to sunlight, especially if they are fixed on a substratum. As well as being efficient sunscreens, most MAAs are also efficient antioxidants and heat dissipators, and may also contribute to the osmotic balance under ionic stress. In this way, the MAAs achieve both primary and secondary roles in the metabolism of the organism or the holobiont that produces them. Whilst the true origin of these molecules remains uncertain, their future is intricately linked with the survival of essential taxonomic groups, both marine and terrestrial.

Acknowledgments

The authors wish to thank Dr Marylene Chollet-Krugler for her continuing support, and Prof. Sophie Tomasi for her general interest and relevant discussions.

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and scytonemin, in Marine Chemical Ecology (eds J.B. McClintock and J. Baker), CRC Press, Boca Raton, FL, USA, pp. 481–520. Karsten, U., Sawall, T., Hanelt, D., Bischof, K., Figueroa, F.L., Flores-Moya, A., and Wiencke, C. (1998) An inventory of UV-absorbing mycosporine like amino acids in macroalgae from polar to warm temperate regions. Botanica Marina, 41, 443–453. Karsten, U., Lembcke, S., and Schumann, R. (2007) The effects of ultraviolet radiation on photosynthetic performance, growth and sunscreen compounds in aeroterrestrial biofilm algae isolated from building facades. Planta, 225, 991–1000. Kedar, L., Kashman, Y., and Oren, A. (2002) Mycosporine-2-glycine is the major mycosporine-like amino acid in a unicellular cyanobacterium (Euhalothece sp.) isolated from a gypsum crust in a hypersaline saltern pond. FEMS Microbiol. Lett., 208, 233–237. Kicklighter, C.E., Kamio, M., Nguyen, L., Germann, M.W., and Derby, C.D. (2011) Mycosporine-like amino acids are multifunctional molecules in sea hares and their marine community. Proc. Natl Acad. Sci. USA, 108 (28), 11494–11499. Klisch, M. and H€ader, D.-P. (2002) Wavelength dependence of mycosporine-like amino acid synthesis in Gyrodinium dorsum. J. Photochem. Photobiol. B, 66, 60–66. Klisch, M. and H€ader, D.-P. (2008) Mycosporinelike amino acids and marine toxins – the common and the different. Mar. Drugs, 6, 147–163. Kobayashi, J., Nakamura, N., and Hirata, Y. (1981) Isolation and structure of a UVabsorbing substance 337 from the ascidian Halocynthia roretzi. Tetrahedron Lett., 22 (321), 3001–3002. La Barre, S., Weinberger, F., Kervarec, N., and Potin, P. (2004) Monitoring defensive responses in macroalgae – limitations and perspectives. Pharmacogn. Rev., 3, 371–379. La Barre, S. (2013) Novel tools for the evaluation of the health status of coral reef ecosystems and for the prediction of their biodiversity in the face of climatic changes, in Topics in Oceanography (ed. E. Zambianchi), Intech, Ch. 5, pp. 127–155. ISBN 980-953-307-949-8. Leach, C.M. (1965) Ultraviolet-absorbing substances associated with light-induced sporulation in fungi. Can. J. Bot., 43, 185–200. Lemoyne, F., Bernillon, J., Favre-Bonvin, J., Bouillant, M.L., and Arpin, N. (1985) Occurrence and characteristics of amino alcohols and cyclohexenone. Components of fungal mycosporines. Z. Naturforsch. [C], 40, 612–616. Lesser, M.P. (2006) Oxidative stress in marine environments: Biochemistry and physiological ecology. Annu. Rev. Physiol., 68, 253–278. Llewellyn, C.A. and Airs, R.L. (2010) Distribution and abundance of MAAs in 33

species of microalgae across 13 Classes. Mar. Drugs, 8, 1273–1291. Lunel, M.C., Arpin, N., and Favre-Bonvin, J. (1980) Structure of normycosporin glutamine, a new compound isolated from Pyronema omphalodes [Bull ex Fr.] Fuckel. Tetrahedron Lett., 21, 4715–4716. Maragos, J.E. (1972) A study of the ecology of Hawaiian reef corals. PhD thesis, University of Hawaii, Honolulu. Mindell, D.P. (1992) Phylogenetic consequences of symbioses: Eukaria and Eubacteria are not monophyletic taxa. Biosystems, 27 (1), 53–62. Mouchka, M.E., Hewson, I., and Harvell, D. (2010) Coral-associated bacterial assemblages: current knowledge and the potential for climate-driven impacts. Integr. Comp. Biol., 50 (4), 662–674. Nakamura, H., Kobayashi, J., and Hirata, Y. (1981) Isolation and structure of a 330 nm UVabsorbing substance, asterina-330 from the starfish Asterina pectinifera. Chem. Lett., 28, 1413–1414. Nakamura, H., Kobayashi, J., and Hirata, Y. (1982) Separation of mycosporine-like amino acids in marine organisms using reversedphase high-performance liquid chromatography. J. Chromatogr. A, 250, 113–118. Nguyen, K.-H., Chollet-Krugler, M., Gouault, N., and Tomasi, S. (2013) UV-protectant metabolites from lichens and their symbiotic partners. Nat. Prod. Rep., 30, 1490–1508. Onofri, S., de la Torre, R., de Vera, J.-P., Ott, S., Zucconi, L., Selbmann, L., Scalzi, G., Venkateswaran, K.J., Rabbow, E., Sabchez, F.J., Homeck, I., and Homek, G. (2012) Survival of rock-colonizing organisms: after 1.5 years in outer space. Astrobiology, 12 (5), 508–516. Oren, A. and Gunde-Cimerman, N. (2007) Mycosporines and mycosporine-like amino acids: UV protectants or multipurpose secondary metabolites? FEMS Microbiol. Lett., 269, 1–10. Pavia, H. and Toth, G. (2000) Inducible chemical resistance in the brown seaweed Ascophyllum nodosum. Ecology, 81, 3212–3225. Pittet, J.L., Bouillant, M.L., Bernillon, J., Arpin, N., and Favre-Bonvin, J. (1983) The presence of reduced-glutamine mycosporines, new molecules, in several Deuteromycetes. Tetrahedron Lett., 24 (1), 65–68. Plack, P.A., Fraser, N.W., Grant, P.T., Middleton, C., Mitchell, A.I., and Thomson, R.H. (1981) Gadusol, an enolic derivative of cyclohexane1,3-dione present in the roes of cod and other marine fish. J. Biochem., 199, 741–747. Portwich, A. and Garcia-Pichel, F. (2003) Biosynthetic pathway of mycosporines (mycosporine-like amino acids) in the cyanobacterium Chlorogloeopsis sp. strain PCC 6912. Phycologia, 2003 (42), 384–392. Reef, R., Kaniewska, P., and Hoegh-Guldberg, O. (2009) Coral skeletons defend against ultraviolet radiation. PLoS ONE, 4 (11), e7995.

Rezanka, T., Temina, M., Tolstikov, V.M., and Dembitsky, V.M. (2004) Natural microbial UV radiation filters – mycosporine-like amino acids. Folia Microbiol., 49, 339–352. Rosenberg, E., Koren, O., Reshef, L., Efrony, R., and Zilber-Rosenberg, I. (2007) The role of microorganisms in coral health, disease and evolution. Nat. Rev. Microbiol., 5, 355–362. Rosenberg, E. and Zilber-Rosenberg, I. (2011) Symbiosis and development. Birth Defects Res. (Part C), 93, 56–66. Rohwer, F. (2010) Coral Reefs in the Microbial Seas, Plaid Press, USA, 201 pp. ISBN 978-09827012-0-1. Rohwer, F., Seguritan, V., Azam, F., and Knowlton, N. (2002) Diversity and distribution of coral associated bacteria. Mar. Ecol. Prog. Ser., 243, 1–10. Rosic, N.N. and Dove, S. (2011) Mycosporinelike amino acids from coral dinoflagellates. Appl. Environ. Microbiol., 77 (24), 8478– 8486. Rosic, N.N. (2012) Phylogenetic analysis of genes involved in mycosporine-like amino acid biosynthesis in symbiotic dinoflagellates. Appl. Microbiol. Biotechnol., 94, 29–37. Roullier, C., Chollet-Krugler, M., PferschyWenzig, E.-M., Maillard, A., Rechberger, G.N., Legouin-Gargadennec, B., Bauer, R., and Boustie, J. (2011) Characterization and identification of mycosporines-like compounds in cyanolichens. Isolation of mycosporine hydroxyglutamicol from Nephroma laevigatum Ach. Phytochemistry, 72, 1348–1357. Roullier, C., Chollet-Krugler, M., Bernard, A., and Boustie, J. (2009) Multiple dual-mode centrifugal partition chromatography as an efficient method for the purification of a mycosporine from a crude methanolic extract of Lichina pygmaea. J. Chromatogr. B, 877, 2067–2073. Rumpho, M.E., Worful, J.M., Lee, J., Kannan, K., Tyler, M.S., Bhattacharya, D., Moustafa, A., and Manhart, J.R. (2008) Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proc. Natl Acad. Sci. USA, 105, 17867–17871. Sampedro, D. (2011) Computational exploration of natural sunscreens. Phys. Chem. Chem. Phys., 13, 5584–5586. Sekikawa, I., Kubota, C., Hiraoki, T., and Tsujino, I. (1986) Isolation and structure of a 357 nm UV-absorbing substance, usujirene, from the red algae Palmaria palmata (L.) O. Kuntze. Jap. J. Phycol., 34, 185–188. Shibata, K. (1969) Pigments and a UVabsorbing substance in corals and a bluegreen alga living in the Great Barrier Reef. Plant Cell Physiol., 10, 325–335. Shick, J.M., Romaine-Lioud, S., Ferrier-Pages, C., and Gattuso, J.-P. (1999) Ultraviolet-B radiation stimulated shikimate pathwaydependent accumulation of mycosporine amino-acids in the coral Stylophora pistillata despite decrease in its population of symbiotic dinoflagellates. Limnol. Oceanogr., 44, 1667.

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Volkmann, M. and Gorbushina, A.A. (2006) A broadly applicable method for extraction and characterization of mycosporines and mycosporine-like amino acids of terrestrial, marine and freshwater origin. Microbiol. Lett., 255, 286–295. Volkmann, M., Gorbushina, A.A., Kedar, L., and Oren, A. (2006) Structure of euhalothece-362, a novel red-shifted mycosporine-like amino acid, from a halophilic cyanobacterium (Euhalothece sp.). FEMS Microbiol. Lett., 258, 50–54. W€angberg, S.-A., Persson, A., and Karlson, B. (1997) Effects of UV-B radiation on synthesis of mycosporine-like amino acid and growth in Heterocapsa triquetra (Dinophyceae). J. Photochem. Photobiol. B, 37, 141–146. Whitehead, K., Karentz, D., and Hedges, J.I. (2001a) Mycosporine-like amino acids (MAAs) in phytoplankton, a herbivorous pteropod (Limacina helicina), and its pteropod predator (Clione antarctica) in McMurdo Bay. Antarctica Mar. Biol., 139, 1013–1019. Whitehead, K., Gorbushina, A.A., and Hedges, J.I. (2001b) Mycosporines in the environment: their analysis and implications. American Chemical Society, Annual Meeting, April 2001, San Diego, USA. Whitehead, K. and Hedges, J.I. (2003) Electrospray ionization tandem mass spectrometric and electron impact mass spectrometric characterization of mycosporine-like amino acids. Rapid Commun. Mass Spectrom., 17, 2133–2138. Whitehead, K. and Hedges, J.I. (2005) Photodegradation and photosensitization of mycosporine-like amino acids. J. Photochem. Photobiol. B, 80, 115–121. Wittenburg, J.B. (1960) The source of carbon monoxide in the float of the Portuguese manof-war Physalia physalis L. J. Exp. Biol., 37, 698–705. Wu Won, J.J., Rideout, J.A., and Chalker, B.E. (1995) Isolation and structure of a novel mycosporine-like amino acid from the reefbuilding corals Pocillopora damicornis and Stylophora pistillata. Tetrahedron Lett., 36 (29), 5255–5256. Wu Won, J.J., Chalker, B.E., and Rideout, J.A. (1997) Two new UV-absorbing compounds from Stylophora pistillata: sulfate esters of mycosporine-like amino acids. Tetrahedron Lett., 38 (14), 2525–2526. Young, H. and Patterson, V.J. (1982) A UVprotective compound from Glomerella cingulata. A mycosporine. Phytochemistry, 21, 1075–1077.

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About the Authors St
ephane La Barre is a senior research scientist at the French Centre National de la Recherche Scientifique. He obtained his MSc Degree from Auckland University, New Zealand, and his PhD at James Cook University, Townsville, Australia, before entering CNRS in 1984. He spent two years (1990–1991) as a research scholar at University of California San Diego, working on synthetic peptides with the late Murray Goodman, and on marine natural products with the late John Faulkner. His multidisciplinary career includes marine chemical ecology, natural products chemistry of terrestrial and marine organisms and polymer chemistry. St
ephane La Barre is currently the coordinator of the research cluster BioChiMar (Marine Biodiversity and Chemodiversity), and is promoting research on new analytical tools to evaluate and predict environmental changes on coral reefs diversity, both biological and chemical. Catherine Roullier is a research scientist in the group “Sea, Molecules and Health,” and assistant professor in Pharmacognosy at the University of Nantes, France. She completed her PhD in Chemistry at the University of Rennes1 in 2010 under Dr Chollet/Prof. Jo€el Boustie’s supervision. Her doctoral thesis

was mainly focused on a marine lichen and mycosporine-like compounds. In 2011, she began a research postdoctoral fellowship in Australia at Eskitis Institute in the Drug Design and Discovery group of Prof. Ron Quinn. She took part in the isolation and identification of bioactive compounds resulting from the high-throughput screening of terrestrial and marine organisms extracts against different therapeutic targets. In 2012, she joined the staff of Prof. Y. F. Pouchus at the University of Nantes to work on the isolation of bioactive metabolites from marine-derived fungi. Jo€el Boustie is Professor of Pharmacognosy and Head of the PNSCM-team (Natural Products-Synthesis-Medicinal Chemistry, UMR CNRS 6226, ISCR), teaching pharmacognosy and mycology at the Faculty of pharmacy of Rennes 1. His research in phytochemistry is focused on bioactive compounds from lichens. He initiated in Rennes the chemical study of lichens, and is in charge of the Des Abbayes lichen herbarium. As President of the Association Francophone pour l’Enseignement et la Recherche en Pharmacognosie, he co-organized in Athens 2008 the World Congress on Natural Product Research.

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology Franck Zal and Morgane Rousselot

Abstract

This chapter highlights the diversity of invertebrate hemoglobin structures, and gathers together up of 100 years of research data. Among invertebrates, annelid hemoglobin structures have been the most widely studied, probably due to their typical symmetric shapes and sizes that allow direct observations to be made using transmission electron microscopy. Hemoglobin seems to be present in all phyla, which suggests a very ancient origin. Using the hemoglobin of the marine annelid Arenicola marina, the French biotechnology startup HEMARINA is developing medical

16.1 Introduction

All aerobic cells require oxygen and nutrients to fuel their energetic and growth requirements. They find these elements in their environment for biomolecule synthesis, and discard the degradation products of their metabolism. However, multicellular organisms are not directly in contact with the external environment where oxygen is available, and in this context two physical mechanisms exist for the transportation of the respiratory gases (oxygen and carbon dioxide), namely diffusion and convection. Diffusion mechanisms are effective only over small distances (millimeters or less), and so the process plays an important role only in unicellular organisms. If the distance between the external and internal media is important, however, then a convection process will be substituted in place of diffusion. Yet, these two processes may intervene at either the same time or separately during the circulation and respiration processes. The primary function of the circulation process involves the requirements for oxygen and nutrients, as well as the elimination of carbon dioxide and metabolic waste. Consequently, the circulating fluids establish important ties between distant specialized cellular populations and the external environment via the intermediate of the exchange organs. In this context, blood has a key position as it contains specialized molecules known as respiratory pigments. These exist either intracellularly or extracellularly, and are able to bind oxygen in a reversible fashion, which increases considerably the ability of the blood to carry these gases.

products. The first generations of blood substitutes (hemoglobin oxygen carriers) were manufactured using intracellular hemoglobin (human and bovine) in order to function outside the red blood cells. However, HEMARINA technologies are based on a natural extracellular hemoglobin. This circulatory pigment, which is present in the blood vessels of A. marina, has evolved over a million years and is able to function extracellularly. Currently, two main products are under development at HEMARINA, namely HEMO2life1 (for organ preservation) and HEMOXYCarrier1 (as a universal oxygen carrier).

During evolution, three different categories of oxygen carriers were selected, each of which retained a characteristic color (Table 16.1). Of these pigments, hemoglobin (Hb), with a typical red color, is the most familiar and is present in almost all phyla, whereas chlorocruorin, which is very similar to the Hb, is characterized by a green color and exists in four families of polychete annelids. Hemocyanin, a blue pigment, is currently present in certain mollusks and arthropods, while hemerythrin, which is pink in color occurs more sporadically among the animal kingdom (Figure 16.1). The heme-containing pigments are the most common among life kingdom as they are found in 33% of zoological classes (Toulmond, 1992); yet, surprisingly, these pigments are also present in plants (Landsmann et al., 1986; Bogusz et al., 1988; Fuchsman, 1992). Although the molecules are characterized by an important diversity of molecular weight and structure (Royer, 1992; Terwilliger, 1992), the active site is always similar and is constituted by a protoporphyrinic tetrapyrrolic ring (Mr ¼ 616.5 Da) that has an iron atom at the center (Fe2þ) (Figure 16.2). This Fe–protoporphyrin complex constitutes the heme molecule. The same active site is also found in other enzymes, such as cytochromes, catalases and peroxidases. In the case of hemoglobin the iron atom is only bound by an amino acid from the polypeptide chain, the proximal histidine. The complex constituted by the active site and the polypeptide chain forms the basic functional unit that is able to bind oxygen only in a reversible fashion. Two categories of heme-containing groups exist, but in both cases the heme molecule is a chelate of iron associated with

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Table 16.1 Classification and localization of the different respiratory pigment across the animal and plant kingdoms.

Pigments (localization)

Phylum

Mr (Da)

Active sites

Na)

Intracellular Hemerythrin

Without heme Sipunculida Brachiopoda Annelida Priapula

40–110  103

2 Fe atoms

2

Arthropoda Mollusca

4  105 to 3  106 3  106 to 9  106

2 Cu atoms

6–48 70–160

65  103

1 Fe atom

4

17–130  103

1 Fe atom

1–8

1 Fe atom 1 Fe atom 1 Fe atom 1 Fe atom

1 1 1 1 1

Extracellular Hemocyanin

Intracellular Hemoglobin stricto sensu Hemoglobin of invertebrates dissolved in their body fluids

Hemoglobin cytoplasmic Myoglobin Hemoglobin Leghemoglobin Neurhemoglobin Extracellular Hemoglobins

Erythrocruorin

Chlorocruorin

With heme Vertebrate Mollusca Echinodermata Annelida Echiuria Vertebrate Invertebrate Bacteria Leguminous plants Annelida Platyhelminthes Nematyhelminthes Nemerta Mollusca Arthropoda Annelida Vestimentifera Pogonophora Annelida

17  103 to 12  106 4  105 To 4  106 3  106 to 4  106

1 to >8

>8 >8

a) N ¼ no. of active sites capable of binding an oxygen molecule.

a protoporphyrin IX which is termed ferroprotoheme and contains two vinyl groups (CH CH2); this complex is the most common (Falk, 1975), but in ferrochlorocruoroporphyrin or chlorocruoroheme the vinyl group is replaced by a formyl group (CH¼O) (Fischer and Seemann, 1936) (Figure 16.2). The latter group is present only in the chlorocruorins. It is possible to distinguish between five different categories of globin chains (for a review, see Vinogradov et al., 1993):  Single-domain chains are the most common in the animal kingdom. They comprise about 145 amino acids and have an active site, and their existence is documented as three different Hbs, namely intracellular, extracellular, or cytoplasmic. These chains can associate to create monomeric, dimeric, tetrameric or polymeric Hbs.  Truncated single-domain chains that contain about 116–121 amino acids.  Chimeric single-domain chains have Mr  40 kDa, are cytoplasmic, and are characterized by an N-terminal region that is able tobind a heme group and a C-terminal with several functions.

 The chimeric bi-domain chains (40 kDa) are created by the covalent association of two domain chains, one of which bears a heme group.  Linear chimeric multidomain chains created by the covalent association of several globin domains. The main aim of this chapter is to describe the invertebrate hemoglobins known to date, and briefly to review the extracellular Hbs obtained from annelids. The details also provided of two therapeutical applications from Arenicola marina Hb.

16.2 Annelid Extracellular Hemoglobins

With numerous reviews having been produced on annelid extracellular hemoglobins (EHbs) and chlorocruorins, the interest of the international “invertebrate hemoglobinist” community was directed towards this remarkable family of molecules (Mangum, 1976a; Mangum, 1976b; Antonini and Chiancone,

16.2 Annelid Extracellular Hemoglobins

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Figure 16.1 Occurrence of the respiratory pigments among the major phyla and classes (Toulmond and Truchot, 1993). The color dots indicate the type of respiratory pigments eventually present. Red ¼ hemoglobins; green ¼ chlorocruorins; purple ¼ hemerythrins; blue ¼ hemocyanins. The absence of a dot means that no respiratory pigment has yet been detected. However, the presence of dot does not mean that all of the group’s species possess the respiratory pigment mentioned. With kind permission by La Recherche.

1977; Weber, 1978; Chung and Ellerton, 1979a; Garlick, 1980; Vinogradov et al., 1980c; Vinogradov, Kapp, and Ohtsuki, 1982; Vinogradov, 1985a; Vinogradov, 1985b; Gotoh and Suzuki, 1990; Riggs, 1990; Terwilliger, 1992; Lamy et al., 1996). The EHbs are present in the three annelids classes, namely polychetes, oligochetes, and achetes. The molecules are very large biopolymers with high molecular weights in the range of 3000– 4000 kDa, and corresponding sedimentation coefficients of 54–61 S, respectively. Each molecule consists of an assembly of about 200 polypeptide chains that belong to between six and eight different types that are usually grouped into two categories:  Functional chains, which possess an active site that is able to bind oxygen reversibly, and correspond to globin chains with Mr-values of about 15 and 18 kDa.

 Linker chains, which possess very few or no heme groups and play an important role in the assembly of one-twelfth of the molecule. These chains have Mr-values of between 22 and 27 kDa. Some authors have revealed the importance of glycans on the assembly of EHbs from Perinerreis aibuhitensis, and have termed this mechanism “carbohydrate gluing” (Ebina et al., 1995; Matsubara et al., 1996; Yamaki et al., 1996). However, this mechanism cannot be universal as not all of these molecules are glycosylated (e.g., Arenicola marina EHb; Zal et al., 1997a, 1997b). The EHbs of annelid are likewise characterized by an acidic isoelectric point and low heme and iron contents, about two-thirds of that observed for other hemoglobins, and corresponding to the presence of one mole of heme per 20000– 27000 Da of protein. This result indicated that not all of the

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

j

COO–

COO–

(a)

CH2

CH2 CH2 HC

C C C

C

C C

C

C H

C

CH3

C

H3 C

C

C

C

C CH

N

N

CH2

C

Fe C

C

C

C CH3

H2C

C H

CH2

CH2

H C

C

C C

N

C

N

C

N

C C

N C

C H

CH3

CH

Fe C

C

C

HC

C

C

CH2

CH2

CH2

N

N

HC H2C

H C

COO–

COO–

(b)

C

C CH3 C C O

CH3

H

H

Figure 16.2 Heme structure (Toulmond and Truchot, 1993). (a) Ferroprotoporphyrin or ferroprotoheme (porphyrin IX) corresponding to the hemoglobin oxygen-binding site; (b) Ferrochlorocruoroporphyrin, corresponding to the chlorocruorin oxygen-binding site. One of the vinyl groups (CH CH2) of molecule (a) takes over from a formyl group (CH O). With kind permission by La Recherche.

polypeptide chains possessed a heme group (Vinogradov, Kapp, and Ohtsuki, 1982; Vinogradov, 1985a; Vinogradov, 1985b).

16.3 Architecture

Interest in the global structure of these molecules first emerged during the 1960s, some 30 years after the molecular weights of the pigments had been determined. The initial studies involved observations using transmission electron microscopy (TEM), and the first micrographs were obtained from Arenicola marina (Roche, Bessis, and Thiery, 1960a; Roche, Bessis, and Thiery, 1960b). The

images revealed hexagonal structures with diameters of 22–26 nm, where each molecule was constituted by two superimposed hexagons with a height of 11–17 nm (Levin, 1963; Roche, 1965). This structure was typically referred to as a hexagonal-bilayer (HBL). Each hexagon was composed of an assembly of six elements with a water-drop shape (Kapp and Crewe, 1984; Van Bruggen and Weber, 1974), and were also referred to as having a hollow globular structure (HGS) (De Haas et al., 1996a, 1996b, 1996c, 1996d) or one-twelfth. The molecule was built up by 12 of these subunits, each of molecular weight ca. 250 kDa, being association one with another. Subsequently, these observations were extended to other molecules belonging to other annelid species, and revealed compounds of similar dimensions (Table 16.2).

Table 16.2 Dimension of several extracellular hemoglobins measured from TEM images.

Species

High (nm)

Polycheta Nephthyidae Nephtys incisa Nereidae Tylorrynchus heterochaetus

19.8  1.2

Perinereis aibuhitensis Arenicolidae Arenicola marina

Abarenicola affinis Opheliidae Euzonus mucronata Ophelia bicornis Travisia japonica Eophilia tellinii Eunicidae Eunice aphroditois

a (nm)a)

18.2 19.2  1.2 20.0  1.8

7.5  0.8

16.0  0.8 16.0 14.8 19.7 17.0

25.5  0.8 24.0 20 27.5 25.5

17.0 17.5 16.0 19.0

25.5 26.0 23.5 28.0

17.9  0.3

26.3  0.3

b (nm)b)

Stainingc)

References

31.6  1.1

AU

Messerschmidt et al., 1983

28.4 30.1  0.8 29.4  0.8 30.0 30.4 28.4 30.0

27.5

Suzuki, Kapp, and Gotoh, 1988 Kapp et al., 1982 Matsubara et al., 1996 PT PT PT AU PT

Levin, 1963 Roche, 1965 Breton-Gorius, 1963 Toulmond et al., 1990 Chung and Ellerton, 1982

MA, PT AU PT AU

Terwilliger et al., 1977 Mezzasalma et al., 1985 Fushitani et al., 1982 Cejka et al., 1989

AU

Bannister, Bannister, and Anastasi, 1976

16.3 Architecture Cirratulidae Cirriformia sp. Terrebelidae Pista pacifica Thelepus crispus Ampharetidae Amphitrite ornata

17.0

24.0

AU

Van Gelderen, Shlom, and Vinogradov, 1981

17.0  0.3 17.0  0.3

25.0  0.5 25.0  0.5

MA, PT MA, PT

Terwilliger and Koppenheffer, 1973 Terwilliger and Koppenheffer, 1973

10.5

21.8 29.0

PT AU

Mangum, 1976 Chiancone et al., 1980

30.4

MA, PT AU

Terwilliger and Terwilliger, 1984 Toulmond et al., 1990

AU

St€ockel, Mayer, and Keller, 1973 Kapp and Crewe, 1984

Alvinellidae Alvinella pompejana 19.7

24.5 27.0

21.1

16.0 31.2

Oligocheta Tubificidae Tubifex tubifex Lumbricidae Lumbricus terrestris

Eisenia fetida

Limnodrilus gotoi Maoridrilus montanus Megascolecidae Pheretima communis Pheretima hilgendorfi Acheta Hirudidae Haemopis sanguisuga

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20.0 16.0  0.7 16.0 23.7 20.0  0.9 21.5 17.5 16.5 14.0 16.0  0.7

30.0 26.5  0.0 26.5

16.7 16.7

15.2  1.4

26.0

AU AU PT PT AU PT

Levin, 1963 Levin, 1963 Roche, 1965 Ben Shaul, 1974 Kapp et al., 1982 Kapp and Crewe, 1984 Frossard, 1982 Frossard, 1982 Ochiai and Enoki, 1981 Yamagishi et al., 1966 Ellerton, Bearman, and Loong, 1987

26.0 26.0

PT PT

Ochiai and Enoki, 1979 Ochiai and Enoki, 1979

24.4  2

AU

Wood, Mosby, and Robinson, 1976

28.2  0.8 31.3 26.0 26.0 25.0 22  1.2 25.0

37.0 29.9  0.8

AU PT PT AU

a) Distance between two parallel sides. b) Maximal molecule diameter. c) AU ¼ uranyl acetate; PT ¼phosphotungstate, MA ¼ ammonium molybdate.

The hexagon center is generally devoid of subunits and was constituted by a hole of about 7–8 nm diameter; however, some annelid EHbs were shown to possess a central subunit, such as Oenone fulgida (Van Bruggen and Weber, 1974), Nepthys hombergii (Wells and Dales, 1976), Nepthys incisa (Wells and Dales, 1976; Messerschmidt et al., 1983; Vinogradov and Kapp, 1983), Ophelia bicornis (Ghiretti-Magaldi et al., 1985; Mezzasalma et al., 1985; Cejka et al., 1991, 1992), Maoridrilus montanus (Ellerton et al., 1987), Glossoscolex paulistus (El Idrissi Slitine, Torriani, and Vachette, 1990), Eophila tellinii (Cejka et al., 1989), Euzonus

mucronata (Terwilliger et al., 1977b), and Arenicola marina (Zal et al., 1997a, 1997b) (Figure 16.3). The existence of a central subunit suggested that this was similar or equivalent to another one-twelfth of the native molecule, although to date no central subunit has been isolated and/or characterized. GhirettiMagaldi et al. (1985) also postulated that the subunit for Ophelia bicornis would differ from the other one-twelfth, while Messerschmidt et al. (1983) proposed that the central subunit for Nephtys incisa was too small to correspond to one-twelfth of the native molecule.

Figure 16.3 Three-dimensional cryomicroscopic structure of Arenicola marina EHb. Ludovic Jouan, 2003.

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

The use of TEM revealed a symmetric structure, and consequently several quaternary structure models were proposed. However, in order to construct an accurate model, knowledge is required of three parameters: (i) the molecular weight of the biopolymer in its native condition; (ii) the number and relative amounts of subunits and polypeptide chains that constituted the molecule; and (iii) the accurate molecular weight of each component. Access to these parameters was gradually achieved due to the emergence of new technologies such as mass spectrometry and multiangle light-scattering, which were first applied by one of the present chapter authors (F.Z.). A sixfold axis of symmetry was identified by using X-ray  diffraction with a resolution of 6 A (Royer and Hendrickson, 1988), although the existence of minor components was suggested without this symmetry. The same observation was made several years later by Boekema and van Heel (1989), when using  electron microscopy with a resolution of 20 A. An analysis using small-angle X-ray scattering of Lumbricus terrestris EHb – the most extensively studied molecule – revealed the presence of a central subunit that was not visible with TEM (Pilz, Schwarz, and Vinogradov, 1980), in contrast to Tylorrynchus heterochaetus where the subunit was visible (Pilz et al., 1988). The same conclusion was drawn in the case of Arenicola marina and Glossoscolex paulistus EHbs (Wilhelm, Pilz, and Vinogradov, 1980; El Idrissi Slitine, Torriani, and Vachette, 1990). A threedimensional reconstruction of Lumbricus EHb confirmed these results when using scanning transmission electron microscopy (STEM), and suggested that the mass could correspond to onetwelfth at the molecule center (Crewe, Crewe, and Kapp, 1984). Alternatively, Vinogradov et al. (1986) suggested that the mass could correspond to the linker chains. The same observation was made via a three-dimensional reconstruction of the molecule by cryomicroscopy (Schatz et al., 1995; Taveau et al., 1999), but these authors suggested that only immunolabeling could confirm the exact nature of the central subunit. The major conclusions deduced by Schatz et al. (1995) were the presence of a sixfold axis of symmetry, in agreement with Royer and Hendrickson (1988), and a threefold axis of symmetry at the

one-twelfth level. The latter level of symmetry was also confirmed by Martin et al. (1996a) after crystallization and observa tion of the subunit at 2.9 A resolution.

16.4 Model of Quaternary Structures

Molecular weight determinations using the primary sequence permitted the realization of several models of quaternary structure for these EHbs. However, several approaches were employed to understand the different component assemblages inside the biopolymers (Table 16.3). 16.4.1 Electron Microscopy

Based on TEM images which showed molecular symmetry, Rossi-Fanelli et al. (1970) proposed a model for Lumbricus terrestris EHb (Figure 16.4). This model was inspired by the

Figure 16.4 First quaternary structure model proposed for Lumbricus terrestris extracellular haemoglobin. Reused from Rossi-Fanelli et al., 1970 with kind permission from Elsevier.

Table 16.3 Main structural models proposed in the literature for the extracellular hemoglobins of annelids and Vestimentifera.

Species

No. of globin chains

No. of linker chains

Methods

Assembly for the one-twelftha)

References

Tylorrynchus heterochaetus Tylorrynchus heterochaetus Perinereis aibuhitensis Arenicola marina Lumbricus terrestris Lumbricus terrestris Lumbricus terrestris Macrobdella decora Alvinella pompejana Alvinella pompejana Riftia pachyptila

192 144 192 156 192 144 144 144 144 120 144

24 36 24 42 24 36 36 42 60 72 36

HPLC ESI-MS HPLC ESI-MS HPLC HPLC ESI-MS ESI-MS ESI-MS ESI-MS ESI-MS

(T þ M)4 þ L2 (T þ M)3 þ L3 (T þ M)4 þ L2 (T þ M9) þ L3.5 (T þ M)4 þ L2 (T þ M)3 þ L3 (T þ M)3 þ L3 (D þ M2)3 þ L3.5 (T þ M)3 þ L5 (T þ M2)2 þ L6 (D þ M)4 þ L3

Suzuki et al., 1990a Green et al., 1995 Matsubara et al., 1996 Zal et al., 1997a Ownby et al., 1993 Vinogradov et al., 1991 Martin et al., 1996b Weber et al., 1995 Zal et al., 1997b Zal et al., 1997b Zal et al., 1996a

a) M ¼ monomeric globin chain; D ¼ covalent dimer of globin chains; T ¼covalent trimer of globin chains; L ¼ linkers chains.

16.4 Model of Quaternary Structures

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Figure 16.5 Proposed model for Lumbricus terrestris extracellular hemoglobin using transmission electron microscopy. (a) Face view; (b) Internal bracelet constituted by D1 (central ring) and D2 (external ring) subunits, where the twelfths of the molecule were fixed. With Courtesy by J.C. Taveau, 1991.

findings of Guerritore et al. (1965), who were first to establish a structure of chlorocruorine from Spirographis. In this model, the polymer was constituted by an HBL structure with a maximum diameter of 26.5 nm, where each hexagon was composed of six identical triangular subunits (A in Figure 16.4), located at each corner. Each of the subunits was built from three smaller subunits (B in Figure 16.4), assembled in a triangle. The molecular weights were estimated as 270 kDa for A and 92 kDa for B. The model proposed for the Lumbricus terrestris EHb by Vinogradov et al. (1986) is known as the “bracelet model,” and is constituted by the assembly of monomers M and trimers T with Mr-values of 16 and 50 kDa, respectively. The cohesion of this structure was produced by a central ring constituted by two other dimeric subunits that were devoid of heme, D1 and D2, with Mr-values of 31 and 37 kDa, respectively. The assembly of these subunits was modeled by Taveau (1991), based on TEM images (Figure 16.5). 16.4.2 Estimation of Heme Number and Minimal Molecular Weight

The minimal molecular weight of the native EHb, using either the heme or the number of iron atoms, revealed a value of about one mole of heme per 27 000 Da of protein. This value was higher than for the smaller subunit found on EHb, determined either by electrophoresis or by using the primary sequences. These observations confirmed that not all of the polypeptide chains possessed a heme group, in contrast to vertebrate Hbs. The mean mass of 27 000 Da corresponded to one heme for about 1.5–2 globin chains. Waxman (1971) suggested a model for the EHb of Arenicola cristata in which two polypeptide chains of the three would be able to bind a heme group. Some years later, Hendrickson and Royer (1986) proposed a unique model for EHb molecules, based on similar aspects of TEM images. In the latter model the molecule was proposed to have been built by the assembly of three different chains, of which two (a and b) would contain a heme group, in agreement with the findings of Waxman

Figure 16.6 Proposed model for all annelid hexagonal-bilayer hemoglobin. (a) Trion constituted by three polypeptide chains, two with the similar tertiary structure of the myoglobin (a and b) and one chain different (c); (b) Hexatrion built by the association of six trions; (c,d) Localization of these chains on the whole hexagonal-bilayer hemoglobin (c, profile view; d, face view). Reused from (Hendrickson, 1983) with kind permission from Elsevier.

(1971). The molecule would be characterized by the same folding as Mb but with the third (c) chain being different. The three chains would be associated in trimer or “trion” fashion, with six trions associated to a “hexatrion,” which itself would be associated by 12 to create the EHb molecule (Figure 16.6). This model described a biopolymer with Mr 3880 kDa, constructed from an association of 216 chains of which 144 would possess heme. The deficiency in the stoichiometry of one mole of heme per polypeptide chain proved to be a major result, and allowed an understanding of the quaternary structure of these huge molecules. However, the scientific community found this result very difficult to accept, and both Antonini and Chiancone (1977) and Garlick (1980) concluded that these results may have been due to purification artifacts. Subsequently, the absence of stoichiometry was admitted without real proof, although Fushitani et al. (1982a) rejected this hypothesis using high-performance liquid chromatography (HPLC). In fact, these authors showed that the polychete EHb from Travisia japonica was assembled from five components with Mr-values ranging between 14 and 18 kDa, each of which contained one heme, although the minimal molecular weight was about 23 000 Da (Fushitani et al., 1982a; Fushitani et al., 1982b; Fushitani, Morimoto, and Ochi, 1983) (Table 16.4). When Suzuki et al. (1983) were able to isolate (by HPLC) only six heme-components from the EHb of Perinereis brevicirris, their result was in agreement with that of Fushitani et al. (1982a). Subsequently, all of these results were challenged, and following the determination of the primary sequences of three globin chains involved in the trimer subunit of Lumbricus EHb, Fushitani and Riggs (1988) recalculated the heme

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

Table 16.4 Minimum molecular weight (Da) obtained by iron or heme content, or by electrophoresis (SDS–PAGE).

Species

Polycheta Nephtys incise Tylorrhynchus heterochaetus Perinereis cultifera Arenicola marina Arenicola cristata Abarenicola affinis Abarenicola pacifica Euzonus mucronata Travisia japonica Travisia fetida Eunice aphroditois Marphysa sanguinea Pista pacifica Thelepus crispus Alvinella pompejana Oligochaeta Tubifex tubifex Glossoscolex paulistus Lumbricus terrestris Maoridrilus montanus Eisenia fetida Pheretima communissima Acheta Haemopis grandis Haemopis sanguisuga Dina dubia ND ¼ not determined.

Minimal Mr Fe or Heme

SDS–PAGE

22 400 26 500 23 700 23 613 26 100 23 525 24 700 23 370 23 000 23 400 26 700 27 589 24 454 23 400

11 000 12 000 13 000 14 000 13 000 13 700 15 800 12 500 14 000 14 600 14 600 14 000 15 000 15 000 13 900

21 000 25 250 24 500 23 900 24 700 25 430

13 000 ND 12 400 13 500 14 800 14 500

24 000 24 800 21 200

13 000 12 600 12 000

percentage. Their results suggested that all three chains possessed heme and a proportion of about 32.5 kDa of protein without heme per one-twelfth of the molecule that remained (Fushitani and Riggs, 1988). Using the HPLC data, Gotoh and Suzuki (1990) proposed a quaternary model for Tylorrynchus heterochaetus EHb which, according to these results, would consist of an assembly of four globin chains. This model described a molecule built by assembling 192 globin chains. Later, Suzuki et al. (1990a) sequenced two linker chains without heme from the Tylorrynchus EHb, and confirmed that not all polypeptide chains in EHb would contain a heme group, thus rejecting in facto their previous model. 16.4.3 Small-Angle Light Scattering

The model proposed by El Idrissi Slitine (1991) for Arenicola marina was obtained by the assembly of 186 basic spherical  units of 21 A radius. This radius was calculated as being the distance between the next two spherical units, and was inferior to the units’ own diameter. This model was based on three different levels: (i) a “peripheral structure” made from 14 elemental spheres set out in three stairs (4/6/4); (ii) a “central structure” formed by six spheres arranged in this way (1/4/1); and (iii) a “contact structure” constituted by two spheres, which occupied the free space between the peripheral and central structures (Figure 16.7). This model described a molecule built by the assembly of 12 “peripheral structures” linked together by 12 “contact structures”; this assemblage surrounded a protein material, the density of which could not be attributed exactly to a complete one-twelfth (El Idrissi Slitine, Torriani, and Vachette, 1990). This model described

Figure 16.7 Model proposed for the extracellular hemoglobin from Arenicola marina, based on small-angle X-ray scattering data. P ¼ peripheral structure; C ¼ central structure; L ¼ contact structure. Courtesy of Fouzia El Idrissi Slitine, 1991.

16.4 Model of Quaternary Structures

a molecule of 168 chains, of which 144 globin chains and 24 linker chains were devoid of heme. 16.4.4 Low- and High-Pressure Liquid Chromatography and SDS–PAGE

To date, the details of two major quaternary models for Lumbricus terrestris EHb have been reported. For both models, each twelfth is constituted only by the globin chain, and the twelfths were tied together by the intermediate of linker chains that possessed no or little heme. The first of these models was proposed by Vinogradov et al. (1986), and described a molecule assembled from 12 subunits (“dodecamers”) and constituted 12 globin chains (Vinogradov et al., 1991; Sharma et al., 1996). The molecule was composed of 144 globin chains, associated with 36 linker chains. Although, the names of the different components have changed with time, at this point the first nomenclature will be used. For Vinogradov’s model, a monomer M (i.e., d) with a Mr of 16 750 Da possessing a heme group was associated with a trimer subunit T (i.e., a, b, c) with a Mr of about 50 000 Da; together, this complex constituted a tetramer. The trimer complex constituted the covalent assembly of three globin chains with Mr about 16 000 Da, and hence each twelfth or dodecamer was constituted by the assembly of three tetramers (3  (M þ T)). These twelfths were tied together in the native molecule by two different monomers D1 and D2 of Mr about 31 and 32 kDa, respectively. D1 and D2 were suspected of being devoid of heme, and to have a protein bracelet at the molecule centers. The bracelet model was based on TEM images as well as dissociation–reassociation experiments (Kapp et al., 1984, 1987; Mainwaring et al., 1986; Vinogradov, 1986; Vinogradov et al., 1986), and contained 200 polypeptide chains with a

global Mr of 3800 kDa. In addition, the model could explain the heterogeneity of the results observed with SDS–PAGE, as the molecule could be cleaved randomly at different levels (Figure 16.8). The second model proposed for Lumbricus terrestris was built by assembling 192 globin chains (i.e., a, b, c, d) and 24 linker chains (i.e., three majors L1, L2 and L3 and one minor L4) (Fushitani and Riggs, 1988; Ownby et al., 1993; Zhu et al., 1996). For this model, each twelfth would be constituted by a dimer of tetramer [(a,b,c,d)2] that would be able to dimerize to create a tetramer of [(a,b,c,d)4]. The molecule would be constituted in the following way: [(a,b,c,d)4,L2]  12, where a, b, c and d corresponded to globin chains, and L to linker chains. This conceptual model would have a Mr of 3800 kDa, as for the bracelet model (Fushitani and Riggs, 1988; Riggs, 1990). 16.4.5 Electrospray Ionization-Mass Spectrometry

A more accurate technique, electrospray ionization-mass spectrometry (ESI-MS), was successfully applied to some HBL Hbs, allowing the determination of their complete polypeptide chain composition. The EHbs examined were from Macrobdella decora (Weber et al., 1995), Tylorrynchus heterochaetus (Green et al., 1995), Lumbricus terrestris (Martin et al., 1996b), Riftia pachyptila (Zal et al., 1996a), and Arenicola marina (Zal et al., 1997a, 1997b). The models of these molecules, obtained by ESI-MS, were compared to those obtained with HPLC in Table 16.3. The model obtained for Arenicola marina EHb HBL, using ESIMS data, is shown in Figure 16.9. The one-twelfth protomer would be constituted by the following assembly [(3a1)(3a2)2T], where T corresponds to either T3, T4, or T5. T1 and T2 were probably located at the molecule center, as noted in previous studies.

(a)

(b)

T3 T5

T4 T T

T4

T5 T3

Figure 16.8 Cleavage possibility of the extracellular hemoglobin from Lumbricus terrestris, according to the bracelet model proposed by Vinogradov et al. (1986). This scheme explains the heterogeneity of SDS– PAGE results. (a) Face view (T ¼ trimer; M ¼ monomer; D1 (V) and D2 (VI) ¼ linker chains; (b) Profile view. V and VI correspond with the first nomenclature used to name these chains. Courtesy of S. Vinogradov.

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a2 a2 a1 a1 a1 T a2 a2 a2 a2

Figure 16.9 Model of Arenicola marina HBL Hb, adapted from Waxman (1971). (a) Drawing of typical HBL Hb, top view. Each one-twelfth is constituted by one trimer (T2, T3 and T4), as noted by Waxman (1971), with nine monomeric chains. The two trimers (T), for example, T1 and T1 or T1 and T2 at the center of the HBL structure, are represented as viewed on the TEM image; (b) Detail of a one-twelfth, showing the arrangement of monomeric chains (Zal et al., 1997a).

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16 Extracellular Hemoglobins from Annelids, and their Potential Use in Biotechnology

16.5 Biotechnology Applications

16.6 Organ Preservation

As noted briefly above, many research groups worldwide have investigated the annelid EHb, demonstrating the great interest in this molecule among the international scientific community. Among these groups, the more active in the field in France was led by Prof. Andr
e Toulmond and was located initially in Paris on the University Paris VI campus (Pierre-etMarie-Curie University). In 1993, this laboratory moved to the Marine Biological Station Roscoff (Finistere, France), and Prof. Toulmond became the Director of this very old and famous center (founded in 1872 by Prof. Henri de LacazeDuthiers, at the Sorbonne University), between 1993 and 2003. Franck Zal, a PhD student of Andr
e Toulmond, began to work on Arenicola marina in 1993, and described in detail the structure of several EHbs. Zal discovered that the Hb molecule of Arenicola marina (HbAm) had all the characteristics of a “universal oxygen carrier” that medics had been seeking for several decades for therapeutic applications. HbAm has exceptional properties:

In 2005, almost 93 000 organ transplants were conducted worldwide, and the number of transplants is growing each year by 5%. For example, 25 000 transplants were conducted in the US alone in 2003. In France, the annual number of transplants is constantly growing, having risen from 3115 in 1998 to 4946 in 2011. During the latter year about 300 people died in France due to a lack of transplants, whilst in 2012 some 10 400 patients were awaiting transplant. The success of organ transplantation has resulted in a rapid increase in the discrepancy between the number of patients waiting to be transplanted and the number of transplants available. The available solutions include splitting an organ between several receivers (e.g., liver), xenotransplantation (using organs from animals, mostly cardiac valves or islets of Langerhans from pigs), or the culture of organs from stem cells. Unfortunately, as these solutions are only at the exploratory stages, a much better solution might be to extend the duration of organ preservation after collection from a cadaver, by using hypothermic continuous reperfusion so as to minimize organ loss. The main objective of organ preservation is to extend the period of ex vivo viability of an organ, allowing for its transfer from the removal center to the transplant center. Under classical static organ conservation conditions, the conservation times possible before ischemia sets in differ widely and depend on the organ concerned. For example, the kidney, heart, liver, lungs and pancreas are very sensitive to cold ischemia. The limited life span of transplants is due to deleterious effects induced by interruption of the blood circulation (ischemia) which, at 37  C, causes a rapid cellular necrosis of the removed organ. Ischemia can be retarded by refrigerating the organ, as a decrease in temperature causes a decline in cellular energy requirements (the O2 requirement at 5  C is only 5% of that at 37  C). Ischemia in hypothermia (cold ischemia) starts as soon as the washed organ is refrigerated, and continues until it is reperfused in the recipient. Cooling does not completely stop cellular metabolism, but does slow down the speed of enzymatic reactions and delays cellular death. However, the use of cold ischemia is time-limited, as the absence of a sufficient supply of O2 causes a metabolic shift towards anaerobic glycolysis. This leads to the generation of lactic acid with cellular acidification from the rupture of lysosomal membranes and cellular destruction by the liberated proteolytic enzymes. The results is an accumulation of various toxins, including cytotoxic free radicals, that are passed to the receiver during organ reperfusion.

 It is naturally extracellular and polymerized.  Its molecular weight is 50-fold that of human Hb.  It has functional O2-binding and -liberating properties similar to those of human Hb (HbA inside the red blood cell).  Each HbAm molecule can bind 156 molecules of oxygen, compared to four in the case of human HbA  It has naturally antioxidative properties. Historically, and before this discovery, two approaches had been investigated to develop universal oxygen carriers: (i) a chemical method using perfluorocarbons; and (ii) biological methods using human or bovine Hb (Ketcham and Cairns, 1999; Gulati, Barve, and Sen, 1999; Winslow, 2000. In March 2007, Dr Franck Zal and Dr Morgane Rousselot created the French biotechnology company, HEMARINA, in order to develop and promote the molecule HbAm, which was seen to possess all of the characteristics necessary to become the leading third-generation blood substitute. In addition to the data outlined above, the principal characteristics of HbAm are that:  The functional properties are totally independent of secondary molecules, such as 2,3-DPG.  The molecule functions without any chemical modification, and with no additional treatment.  The molecule has a functional capability at temperatures between 4  C and >30  C, which is a major and essential advantage.  The absence of any vasoconstrictor effects is in contrast to all other products developed to date and referred to as hemoglobin oxygen carriers (HBOCs) (Tsai et al., 2012). Although several major applications for HbAm have already been identified, attention here will be focused on only two, namely organ preservation pending transplantation and the development of an innovative oxygen carrier.

16.6.1 Preservation Solutions

The deleterious effects of cold ischemia can be attenuated through the use of preservation solutions, which allow the reduction of cellular edema by maintaining the extracellular osmotic pressure, thus preventing intracellular acidosis and reducing reperfusion injuries due to free radical accumulation.

16.7 Anemia

Some years ago, the ANSM (Agence National de S
ecurit
e du M
edicament) questioned the efficiency of organ preservation solutions. 16.6.2 Hypothermic Continuous Reperfusion

In general, following its removal a transplant can be conserved by simple immersion in a preservation solution at 4  C. This static technique is simple and inexpensive; however, due to the urgency of the situation related to lack of organs and to the degree of ischemia of organs currently being transplanted, a hypothermic continuous reperfusion technique has been developed. This dynamic technique, which is presently being applied to kidney transplants, requires sophisticated and expensive equipment. Currently, two main dynamic systems have been developed, the performances of which are similar. Unfortunately, the use of perfusion equipment is limited to a small number of centers in Europe, and the preliminary results do not indicate a significantly better performance than static methods. Because it is functional in the natural environment at low temperature, HbAm was manufactured by HEMARINA as the product HEMO2life1 which, when added to several clinical storage media, insured oxygenation of the organs. The recovery of organ grafts maintained in media with HEMO2life1 and then transplanted into pig models was shown to be excellent (Figure 16.10). HEMO2life1 is currently at the final stage of registration, with efficiency having already been demonstrated for kidney, lung, and heart. HEMO2life1 also possesses naturally an intrinsic superoxide dismutase (SOD) activity on HbAm, which allows it to protect the cells against oxidative damage caused by an accumulation of free radicals liberated during cold ischemia. HEMO2life1 could also increase the efficiency of dynamic systems for the conservation of kidneys, and probably also for other organs with shorter preservation times (e.g., heart, liver, pancreas, lung).

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16.7 Anemia

According to a World Health Organization reports, one in ten people admitted to hospital requires a blood transfusion. The majority of these cases are anemia, due to a decrease in Hb levels following the loss or reduced production of red blood cells (RBCs). A loss of RBCs may be due to hemorrhage during a trauma (accidents, injuries, burns) or during surgery (coronary artery bypass, organ transplants, hip replacement, etc.). Anemia can also be caused by hemolytic anemia (e.g., drepanocytosis). A reduced RBC production may occur in cases of global medullary insufficiency and bone marrow cancer, as well as being a side effect of radiotherapy or chemotherapy. The aim of transfusing RBC concentrates is to maintain or to restore a sufficient level of Hb. Typically, 50% of all RBCs are transfused to patients aged >65 years, and demographics show that, in developed countries, this age category will double over the next 30 years. Certain types of surgery that are becoming increasingly common require an essential number of RBCs (e.g., up to 100 RBC units for one liver transplantation). Moreover, these trends are also observed in emerging countries (India, China, Brazil), and it is agreed that worldwide RBC demands to treat anemia will continue to increase. As an example, in 2009 the number of RBCs transfused was about 93 million units, which represents an increase of 33% compared to 2003. Moreover, this situation is not expected to improve with demands continuing to increase, especially when associated with a stagnation or even regression in donations. Today, the exclusion criteria for donors are expanding due to emergent risks relating to transfusions (Nile virus, severe acute respiratory syndrome, prions, chikungunya virus, etc.), and this is causing a regression in the number of regular donors. Experts in the field maintain that the shortage in blood products worldwide is 100 million liters per year, and these requirements are constantly increasing.

Figure 16.10 Kidney function following reperfusion. Porcine kidneys were cold-flushed and preserved for 24 h with UW or HTK supplemented with M101 at a concentration of 0 g l 1 (UW and HTK groups) or 5 g l 1 (UW þ M101 and HTK þ M101 groups). Follow-up was performed on the day before transplantation (D-1) and after transplantation (at 1 h ¼ R60; from day 1 to day 14 ¼ D1 to D14; and 1 month ¼ M1). Values shown are mean  SEM., p 50%) and versatile approaches (Fujikawa et al., 2006; Korotaev et al., 2008, 2010). More than 15 total syntheses of lamellarins have been reported by many groups. In one of the most concise syntheses, the two pentacyclic lamellarins D and H were both prepared via seven steps, while the simpler, nonfused lamellarin R was prepared via five steps with a total yield of 53% (Li et al., 2011).

Many marine-derived compounds are endowed with antiviral activities, including certain lamellarins (Yasuhara-Bell and Lu, 2010). In 1999, Reddy et al. showed that lamellarin a 20-sulfate was able to inhibit the integrase activity of the HIV-1 virus, interfering both with the terminal cleavage activity of the enzyme and the strand transfer activity (IC50-values of 16 and 22 mM, respectively). Lamellarin a 20-sulfate also restricted growth of the HIV-1 virus in cell culture, with an IC50-value of 8 mM (Reddy et al., 1999). More recently, the synthesis of sulfated and nonsulfated derivatives of lamellarin a has been successfully achieved, enabling the definition of antiviral structure–activity relationships. Interestingly, the sulfated derivatives proved to be active, whereas the nonsulfated analog was totally inactive; a ring-opened 20-sulfated analog also proved to be inactive. Lamellarin a 20-sulfate, lamellarin 13-sulfate and lamellarin 13,20-disulfate were all equally active. The key role of the sulfate group was previously established with another natural analog, lamellarin-f, for which the 20-sulfate derivative was active against HIV-1 integrase, but not the nonsulfated analog (Ridley et al., 2002). However, the sulfated derivatives of lamellarin a showed a very limited cell uptake, in line with their

RO RO

OR

RO R

NO2 O

N OR

CF3

or

+

MeO

RO CF3

CF3

MeO

NO2 O

O

N

OR

OR

MeO N

O

Figure 17.5 Example of a synthetic scheme leading to lamellarin derivatives.

CF3

OMe

17.9 Non-Natural Lamellarin Analogs

 Pyrrolo[2,1-a]isoquinolines, represented by the open-chain derivative (compound 2 in Figure 17.6), an intermediate in the synthesis of the bioactive lamellarin H (You et al., 2006). More recently, the same authors also described the synthesis of the corresponding 5,6-dihydro pyrrolo [2,1-a]isoquinolines (Liao et al., 2011).  Hybrid structures of lamellarin D and combretastatin A4, such as the dihydropyrroloisoquinoline derivative (compound 3 in Figure 17.6), which proved to be significantly cytotoxic towards a panel of tumor cell lines (Shen et al., 2010). The potential targets of these hybrid, topoisomerase I and/or tubulin, are not known.  Chromeno[3,4-b]indoles. In this series, a few highly potent inhibitors of the kinase DYRK1A were identified when a hydroxyl group was present at the C-2 position. Molecular modeling suggested that, in this case, the compounds would bind to the ATP active site of the kinase. In contrast, substitution at the C-3 and C-10 positions afforded a bis-hydroxylated chromenoindole derivative (compound 4 in Figure 17.6) which acted as a topoisomerase I inhibitor and exhibited a significant cytotoxic potential. However, these two activities are apparently not linked, and another target may be responsible for the cytotoxic action (Neagoie et al., 2012).  Polymeric forms of lamellarin D in order to increase the water-solubility of the molecules, with structures incorporating polyethylene glycol (PEG) ester moieties or in the form of PEG-based dendrimers (Pla et al., 2009a). The same authors also described bioconjugates of lamellarin D, including a peptidic nuclear localization signal (Pro-Pro-Lys-Lys-Lys-ArgLys-Val-OH) to favor the accumulation of the drug in cell

17.9 Non-Natural Lamellarin Analogs

A great variety of unnatural lamellarins with either a saturated or unsaturated D-ring have been reported, including:  Simplified structures lacking the aryl group perpendicular to the pentacyclic core. The synthesis of various 1-dearyllamellarin D derivatives was proposed, but their bioactivity – if any – was not reported (Ohta et al., 2009).  Isolamellarin (Figure 17.6), an isomeric analog of lamellarin G trimethyl ether, was obtained through a copper(I)-mediated and microwave-assisted coupling process (Thasana et al., 2007). Its bioactivity has not been reported.  The amino derivative of lamellarin D designated PM031379 (Figure 17.6) which was shown to induce the nuclear translocation of the AIF in the non-small-cell lung cancer cell line U1810, unlike the parent product lamellarin D (Gallego et al., 2008). This synthetic analog, designed by PharmaMar (Madrid, Spain), is a potent proapoptotic agent that triggers mitochondrial permeability transition via the generation of reactive oxygen species and an upregulation of the AIF (Gallego et al., 2008).  Oxazine derivatives, such as a compound possessing a planar 30 4-dihydro-2H-[1,3]oxazine core (compound 1 in Figure 17.6). This diazaindeno[2,1-b]phenanthrenone derivative, designed on the basis of molecular modeling of the LamD-topoI complex, is about 100-fold less cytotoxic than Lam-D, but it maintains a capacity – albeit reduced – to inhibit topoisomerase I (Cananzi et al., 2011).

MeO MeO MeO

H2N

OMe

HO MeO

O

O O

MeO

MeO

O MeO

N

O HO

MeO N

MeO

Me

OH

O-Pr

OPr O

HO

OMe

N

HO

MeO

N H MeO MeO

Compound 2

O

N

Compound 1

PM031379

MeO MeO

N MeO

HO Isolamellarin

OH

MeO

OH

OMe N

OMe

O

HO

Compound 3

Figure 17.6 Structures of selected synthetic lamellarin derivatives.

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Compound 4

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17 Lamellarins: A Tribe of Bioactive Marine Natural Products

nuclei. This approach was relatively promising: a peptide– lamellarin D conjugate proved to be over threefold more cytotoxic than the parent compound against three human tumor cell lines (Pla et al., 2009b). PEG-containing polymeric micelles can also be used as nanocarriers to facilitate the delivery of lamellarins (Pungkham et al., 2011). The above-described lamellarins are selected examples, and many other derivatives have been reported (Ishibashi et al., 2002; Boonya-Udtayan et al., 2010; Chittchang et al., 2010). 17.10 Conclusion

Lamellarins represent a valuable source of bioactive metabolites, and have inspired medicinal chemists to design and synthesize novel drugs endowed with reinforced anticancer and/or antiviral properties. The prominent member of the family, lamellarin D, possesses a wide range of important biological and pharmacological properties, including anticancer activities. Lamellarin D provides a robust pharmacological tool to study topoisomerase I inhibition, as well as the perturbation of mitochondrial metabolism. Due to functional defects in apoptosis signaling molecules, or deficient activation of apoptosis pathways, certain forms of cancer are particularly sensitive to lamellarin D, and there are ongoing studies to elaborate more efficient derivatives and drug candidates. Intensive studies on the syntheses of lamellarin derivatives should open many possibilities for the creation of novel structures and innovative functionalized compounds. The contribution of lamellarin D to drug discovery must be put into prospective due to its synthetic availability and relative

ease of modification, high potential applications, and complex pharmacology which is now relatively well understood. In an attempt to combat the increasing numbers of chemoresistant cancers, marine natural products such as lamellarins could serve as a potential source of novel drugs. Today, however, the challenge remains of using a mitochondria-targeted drug to treat cancers with a high degree of efficacy and safety. Future directions for using lamellarin-derived compounds as anticancer drugs include both a better characterization of the selective action of the compounds toward malignant cells versus normal cells, and the possibility of delivering the molecules to tumor sites in a more precise fashion. Approaches that involve conjugating a lamellarin to a cancer cell delivery factor are essential to increase drug potency towards tumor cells and reduce nonspecific cytotoxicity. Targeted cell delivery through cell-surface receptors seems important to extend the therapeutic index of these molecules, and along these lines a variety of nanoparticle-based platforms may be considered to promote the targeting of natural products to cancer cells, notably for the efficient delivery of lamellarin D to localized tumor sites. Modern cancer chemotherapy faces the major challenge of delivering chemotherapeutic drugs exclusively to tumor cells, while sparing normal proliferating cells (Sakhrani and Padh, 2013). Yet, opportunities exist to exploit lamellarins as anticancer drugs by profiting from the dual action at nuclear (topoisomerase I inhibition) and mitochondrial levels. Although, since the first isolation of lamellarins from a small sea snail tremendous progress has been made, it will take a long time before the findings from this fundamental research can be transformed into medical benefits, capturing the true value of this fascinating family of marine natural products.

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About the Author Christian Bailly is Director of Research at the Pierre Fabre Research Institute (IRPF, Toulouse, France). He received his PhD in life sciences in 1989 (University of Lille, France) for work on the synthesis and biological study of novel DNAbinding anticancer agents. He then joined the Department of Pharmacology, University of Cambridge (UK), for a postdoctoral period and held a research associate position (1990–1995) on the team of Prof. Michael J. Waring (University of Cambridge), studying drug–nucleic acid interactions. He joined the National Institute of Health and Medical Research (INSERM U-524, Lille, France), as Director of

Research, working for eight years on the mechanism of action of topoisomerase I and II inhibitors and DNA recognition. In 2001, he received the INSERM prize for Therapeutic Research, and in 2003 moved to the Pierre Fabre Research Institute as Director of Oncology Research, managing two research centers in immunology (CIPF, 2004–2010) and experimental oncology (2003–2010). He is now responsible for the research activities (drug discovery) of the IRPF. His research interests extend over a wide range of biologically active molecules, mostly anticancer agents, such as monoclonal antibodies and natural products.

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Part Four New Trends in Analytical Methods

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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18 NMR to Elucidate Structures Ga€elle Simon, Nelly Kervarec, and St
ephane C
erantola

Abstract

Nuclear magnetic resonance NMR has evolved appreciably during the past 50 years to become an astounding tool for molecular characterization. Recent progress has enabled NMR to enter the field of biology and has facilitated the identification of molecules. Indeed, the nondestructive NMR tool makes possible, thanks to its different one-dimensional (1D) and two-dimensional (2D) sequences on many nuclei (e.g., 1H, 13C, 31P, 15N), the complete characterization of increasingly smaller amounts of material. Moreover, the

18.1 Introduction

Nuclear magnetic resonance (NMR) spectroscopy was discovered in 1945 by Bloch and Purcell, who received in turn the Nobel Prize for Physics in 1952. Initially, NMR was used only in physics but, following the discovery of the chemical shift, the technique quickly became a very important analytical tool in chemistry. Indeed, NMR is a powerful and theoretically complex analytical technique which uses radiofrequency radiation to induce transitions between different nuclear spin states of samples in a magnetic field. However, in order to use the NMR tool to its full extent, it is important to have knowledge of mathematics, physics, chemistry, and biology. Over the past 50 years, NMR spectroscopy has undergone great developments, with multinuclear studies becoming possible with very-high-field operations (currently up to 1000 MHz for proton frequency). Applications of new pulse series also appeared, while new technologies of multidimensional spectra have been developed, high-resolution studies on solids have expanded, and a branch discipline of magnetic resonance imaging (MRI) has emerged. Consequently, the NMR technique became the dominant technique for determining the structure of organic compounds, and entered the field of biology. Based on its continuously increasing importance in modern chemistry, biochemistry and medicine, two additional Nobel Prizes for NMR followed in 1991 (for Richard Ernst) and in 2002 (for Kurt W€ uthrich) (Table 18.1). Although NMR spectroscopy can be used for quantitative measurements, it is mainly used for determining the structures

development of high-resolution magic angle spinning (HRMAS) probes has allowed direct analysis, without prior extraction, on living tissues (plant fragments, muscles or organs, bacterial pellet) to indicate the global content and ratio of molecules of the tissue(s), and thus permit differentiation between species. It is also possible to control the synthesis of compounds of interest according to various environmental or experimental conditions (e.g., pH, salinity, season, species, pollutant).

of molecules along with other spectroscopic methods (e.g., infrared, MAS). Of all the spectroscopic tools, NMR is the only one for which a complete analysis and interpretation of the entire spectrum is normally expected. Although larger amounts of sample are needed than for mass spectroscopy, NMR is nondestructive, and with modern instruments good data may be obtained from samples weighing less than 1 mg. Interest in NMR spectroscopy for structural characterization results from the fact that the atoms of a molecule, when placed in magnetic field, possess slightly different resonance frequencies depending on their chemical environment. Furthermore, splitting of the spectral peaks arises due to interactions between different nuclei, which provides information about the proximity of different atoms in a molecule and allows a structural identification of the molecules to be obtained. In this chapter, the main possibilities of studies that the NMR tool offers are presented, illustrated by chosen examples for marine environments. Moreover, in order that NMR can be used successfully as an effective analytical tool, the basics of NMR that allow interpretation of the spectra obtained in common studies are also outlined.

18.2 NMR to Elucidate Structures

NMR is based on the physical properties of nuclei, such as spin, isotopic abundance and frequency of the observation. Spin (I) is a quantic number that allows the orientation of nuclei in a

Outstanding Marine Molecules: Chemistry, Biology, Analysis, First Edition. Edited by St
ephane La Barre and Jean-Michel Kornprobst. Ó 2014 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2014 by Wiley-VCH Verlag GmbH & Co. KGaA.

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Table 18.3 Resonance frequency according to magnetic field.

Table 18.1 Typical applications of modern NMR.

Chemical structure analysis Synthetic organic chemistry (often together with other spectroscopic method) Natural product chemistry (identification of unknown compounds) Structural studies of soluble biomacromolecules Oligosaccharides Solid state NMR (ssNMR) Analysis of solids like metals, silicates, soils and clays, polymers . . .

Study of dynamic processes Reaction/binding kinetics Chemical/ conformational exchange Membrane protein structures Drug design Structure Activity Relationship (SAR) Magnetic Resonance Imaging (MRI)

magnetic field, and its value varies according to the nuclei. When the spin is “null,” the nucleus cannot be oriented in a magnetic field, and so cannot be observed in NMR (e.g.,12 C or 16 O). Natural abundance and gyromagnetic ratio are also other important NMR factors (Table 18.2). When a suitable electromagnetic wave is applied to a nucleus, an energetic transition from the energy base level to a higher energy level occurs. When the excited nucleus relaxes, energy is emitted at a wavelength corresponding to radiofrequencies. This emission is digitalized by the spectrometer and leads, after Fourier transformation, to an NMR spectrum of the concerned nucleus (Table 18.3). Today, NMR is now widely recognized as the most useful and powerful method for the structural elucidation of natural compounds, although a common drawback when studying such compounds is the limited amount of sample available. The relatively low inherent sensitivity of NMR, compared to mass spectrometry, for example, caused its use to be confined for many years to chemists for the identification of organic

Table 18.2 Properties of some nuclei.

Isotope

Spin (I)

Natural abundance

Relative sensitivity at constant field

1

1/2 1 3/2 1/2 1 1/2 5/2 1/2 1/2 1/2 3/2

99.98 1.56  10 2 81.17 1.108 99.635 0.365 3.7  10 2 100 4.70 100 100

1.000 9.64  10 0.165 1.59  10 1.01  10 1.04  10 2.91  10 0.834 7.85  10 6.64  10 2.51  10

H H 11 B 13 C 14 N 15 N 17 O 19 F 29 Si 31 P 75 As 2

3

2 3 3 2

2 2 2

Magnetic field (T)

Nuclei

Frequency (MHz)

7.04 T

1

300 75 400 100 500 125

H C 1 H 13 C 1 H 13 C 13

9.39 T 11.74 T

compounds. However, technical improvements of the technique, such as the introduction of cryomagnets with high magnetic field strengths (up to 23.5 Tesla, i.e., 1 GHz), cryogenically cooled probes, microprobes, and high-resolution magic angle spinning (HRMAS) probes, as well as the development of two-dimensional (2D) pulse sequences, allowed drastic reductions to be made in the quantities of material required for analyses. This, in turn, provided biochemists and biologists with a clear access to the study of natural products, using the NMR tool. Faced with a crude natural sample, NMR can be used as an initial step of an investigation before applying separation techniques such as gas chromatography (GC), high-performance liquid chromatography (HPLC) and ion-exchange chromatography, each of which would require prior knowledge of the nature of the molecules being examined. Subsequently, NMR can also be used to follow the stages of purification and, finally, for structural identification of the biomolecules of interest. Some of the practical aspects of NMR, with particular reference to marine samples, are discussed below.

18.3 Sample Preparation

In classical NMR, the sample must be solubilized prior to analysis. In order to avoid occurrence of additional protons signals which may overlap signals of interest, deuterated solvents (see Table 18.4) must be used as deuterium is invisible in a spectrometer tuned to, for example, protons or carbon. Moreover, deuterated solvents allow the magnetic field to be locked, which prevents it from shifting during the analysis, so as to improve signal resolution and avoid possible signal degradation. Unlike other spectroscopic methods, sample concentration and quality are very important factors in NMR. Indeed, dilute samples may lead to difficulties in obtaining NMR spectra, or may necessitate important scan accumulations and thus time-consuming analyses. In contrast, concentrated samples may result in broader lines due to increased solution viscosities and an altered resolution due to difficult shimming.

18.3 Sample Preparation

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Table 18.4 Properties of some deuterated NMR solvents.

Solvent

Bp ( C)

Residual 1 H signal (d in ppm)

Residual 13 C signal (d in ppm)

Acetone-d6 Acetonitrile-d3 Benzene-d6 Chloroform-d Cyclohexane-d12 Dichloromethane-d2 Dimethylsulfoxide-d6 Dimethylformamide-d7 Methanol-d4 Nitromethane-d3 Pyridine-d5 Tetrahydrofuran-d8 Toluene-d8

56 82 80 61 81 40 189 153 65 101 115 66 111

2.04 1.93 7.15 7.26 1.38 5.32 2.49 2.74, 2.91, 8.01 3.30, 4.78 4.33 7.19, 7.55, 8.71 1.73, 3.58 2.09, 6.98, 7.00, 7.09

206.0, 29.8 118.2, 1.3 128.0 77.0 26.4 53.8 39.5 30.1, 35.2, 162.7 49.0 62.8 123.5, 135.5, 149.9 25.3, 67.4 20.4, 125.2, 128.0, 128.9, 137.6

The quantity of material required varies from sample to sample, and depends on the nature of the studied nucleus and on the NMR experiment(s) to be performed. For example, with regular 5 mm tubes, a good range for 1 H acquisition is between 1–10 mg of a pure molecule according to its molecular weight. In the case of mixtures of molecules, the amount of material introduced must be adjusted. In the case of limited quantities of material, the concentration can be increased by reducing the solvent volume. This can be achieved by using specific tubes such as 3 mm or Shigemi1

Figure 18.1 NMR tubes.

tubes; these have a bottom section composed of solid glass with an interspace that accommodates the sample, thus limiting the solution volume. In this case, resolution is not perturbed because glass is inert in magnetic fields. By using these particular tubes and the same quantity of initial product, it is possible to increase the solution concentration almost twofold compared to the 5 mm tubes. The only drawback in this case is the need for the studied compound(s) to be very soluble, in order to obtain a homogeneous solution and thus good-quality NMR spectra (Figure 18.1).

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Figure 18.2 500 MHz 1 H NMR spectra of asparagine in D2O; ns ¼ 16.

Care must be taken for NMR sample preparation: 





Solid samples must not be scraped with a metallic spatula. In order to avoid the occurrence of paramagnetic particles in the sample, which would degrade the spectrum resolution, a plastic or glass spatula must be used. Samples must be filtered before analysis to remove any suspended particles; otherwise, the presence of solids will prevent magnetic field homogeneity, leading to a broadening of the NMR signals. The volume of a sample contained in a 5 mm NMR tube must be ca. 700 ml (4 cm high in the NMR tube). This



criterion is especially important in variable-temperature experiments. If the NMR tube is overloaded, temperature gradients will appear over the length of the sample, leading to a degraded resolution (Figure 18.2). Some biological samples undergo microbial degradation in aqueous solutions; to overcome this sodium azide can be added as biocide, except for 15 N NMR experiments.

To succeed in a NMR analysis, spectrometer probe must be adapted to the sample (Figure 18.3).

Figure 18.3 Different approaches to analyzing a sample by NMR: soluble sample.

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei 18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

Two types of conventional probe are available; some with direct detection (DUAL, BBO, QNP) and others with inverse detection (TXO, TXI, TBI). In the latter case the 1 H channel is closer to the receiver. Direct probehead conception aims at favoring 13 C detection, whereas the inverse probehead conception improves 1 H sensibility and is preferred for 2D analysis (Figure 18.4). Even if the observation of 1 H and 13 C can be made on multiprobeheads, it is necessary to emphasize that the main purpose

of broadband probes (such as TBI) is to allow the observation of a wide range of nuclei. Hence, 1D and 2D studies are possible on many nuclei, but only if their spin characteristics and abundances are compatible (Figure 18.5). All of these probes allow samples to be analyzes at temperatures ranging from 100  C to 100  C. 18.4.1 1 H Spectra

Each 1 H of a molecule, according to its electronic environment, appears on a spectrum as a peak characterized by its chemical shift, its multiplicity, and its integration.

Figure 18.4 Examples of conventional “liquid” probeheads.

Classical probes (TXI, TXO, TBI…)

1

H spectra

13

C spectra

Figure 18.5 Common NMR analyses.

2D spectra

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Other nuclei spectra

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Figure 18.6 1 H NMR table of samples in CDCl3 solution. The d scale is relative to TMS at d ¼ 0.

18.4.1.1 Chemical Shift The chemical shift, designated by the symbol d and expressed in units of parts per millions, is shown on the bottom axis of the spectra according to a reference compound, such as tetramethylsilane (TMS) or trimethylsilyl propionate (TSP), chosen because it produces a single sharp NMR signal that does not interfere with the resonances usually observed for organic compounds (Figure 18.6). 18.4.1.2 Multiplicity The signal multiplicity of a given nucleus can be predicted by the 2nI þ 1 rule, where n is the number of neighboring spincoupled nuclei and I is the spin of the considered nucleus. For example, if a proton has two neighboring spin-coupled protons, the observed signal is a triplet (2  2  1/2 þ 1 ¼ 3). The line intensity ratio of splitting pattern is given by Pascal’s triangle numbers. Thus, a doublet has 1 : 1 of equal intensities, a triplet has an intensity ratio of 1 : 2 : 1, a quartet 1 : 3 : 3 : 1, and so on (Figure 18.7). The coupling constant, denoted J, corresponds to values (in Hz) between two peaks of the same signal. They are denoted 1 2 3 J; J; J; and so on, according to the number of bonds between the two nuclei concerned. They correspond to the spatial geometry of the molecule, in which interatomic distances vary according to constraints, distortions, angles between atoms, configuration. The coupling constant value is linked directly to the stereochemistry (Table 18.5).

Figure 18.7

1

H NMR signal multiplicity.

On the ethanol molecule (as quoted below), the coupling constants between 1 H methyl and 1 H methylene are of the order of 7–8 Hz. The coupling constant between these two groups of protons is measurable on the two signals of the respective groups. 1 H, 13 C, 19 F and 31 P are spin 1/2 nuclei. Spin-coupling interactions may be of either the homo- or heteronuclear

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

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Table 18.5 Some typical 1 H–1 H coupling constants.

Structural type

J(Hz)

Structural type

J(Hz)

6 to 8 12 to 18

5 to 7

7 to 12

2 to 12 (depends on the dihedral angle and the nature of X and Y)

0.5 to 3

0.5 to 3

3 to 11 (depends on the dihedral angle)

2 to 3

type; for example, spin-coupled 19 F and 1 H nuclei will, respectively, appear as doublets with the same coupling constant. Coupling can become complex when stereochemistry implies forced or even forbidden movements. The thermodynamics conformation of molecule dictates the proximity between nuclei and atomic distortions. For example, protons H2 of malate are diastereotopic; that is, they magnetically different, and are coupled together such that each one couples with H3, yet the distances between H3 and H2a and between H3 and H2b must be different (Figure 18.8).

ortho 6 to 8 meta 1 to 3 para 0 to 1

Every expected signal should have, according to Pascal’s triangle, an intensity of 1/1/1/1, but undergoes spatial constraints which deform the heights of the lines; this is referred to the “roof” effect. For a molecule, the coupling constants retain the same value, whatever the recording conditions. 18.4.1.2.1 Magnetic Field Effect By increasing the magnetic field, the signal-to-noise ratio is improved and the multiplicity of closed signals is easier to observe (Figure 18.9).

Figure 18.8 (a) Theoretical 1 H NMR multiplicity of the CH2 aspartate signal; (b) 500 MHz 1 H NMR spectra of aspartate in D2O: multiplicity of the CH2.

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4.2

4.0

(a)

(b)

Figure 18.9

1

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

2.0

1.8

1.6 ppm

H NMR spectra of proline in D2O. (a) On a 500 MHz spectrometer; (b) On a 300 MHz spectrometer.

18.4.1.2.2 Solvent Effect Molecules are more or less sensitive to polarity, and their electronic environment can be modified or disrupted according to the solvent used. So, multiplets which overlap in a solvent can be easily observed in another solvent. This is called the “solvent effect,” and is often used to distinguish aromatics 1 H, for example (Figure 18.10).

because 90% H2O/10% D2O is used as the solvent. Most spectrometers are equipped with an analog converter, which allows a signal intensity of 1 to be observed when the intensity of the biggest peak is 64 000. This is why it may be important to use water suppression to improve small signal detection (Figure 18.12).

18.4.1.3 Integration If the multiplets of a spectrum are well separated and do not overlap with other peaks, the number of each type of proton of a molecule is given by the integration of each signal. However, where multiplets overlap the individual signal cannot be integrated, and the overall integral of the signal group must be considered. If a precise value of integration is required, the relaxation delay must be fivefold the longitudinal relaxation time (T1) of the signals of interest.

18.4.1.4.2 Saline Samples Extracts of marine tissues, albeit of animal or plant origin, contain salt in variable quantities. As a consequence, the length of the 90 pulse, which is a key parameter for NMR observations, is considerably increased to the point of preventing any signal detection. This is particularly true for cryoprobes with regular 5 mm tubes. There are, nevertheless, several possibilities to obtain spectra of saline samples, including: (i) previous desalination of the sample; (ii) reducing the sample concentration; or (iii) replacement with 3 mm NMR tubes to reduce the sample volume analyzed and thus the quantity of salt.

18.4.1.4 Special Features of Sample Sample preparation and acquisition parameters depend on the characteristics of the sample (Figure 18.11).

18.4.1.4.3 Samples Containing Exchangeable Protons In many samples, OH, NH and SH protons can be recognized from their characteristic chemical shifts or broadened appearance. Chemical shifts of OH, NH and SH protons vary over a wide range, depending on the experimental conditions. The shifts are very strongly affected by hydrogen bonding, with large downfield shifts of H-bonded groups compared to free OH, NH, or SH groups.

18.4.1.4.1 Samples with a High Water Content Water suppression allows the elimination of unwanted solvent resonances and the conservation of all others. This approach is commonly used when studying biological molecules where the water signal often dominates all others, because the sample quantity is low or

(b)

(a)

8.8

8.6

8.4

8.2

8.0

7.8

7.6

7.4

7.2

7.0

6.8

6.6

Figure 18.10 500 MHz 1 H spectra of pyridine. (a) In benzene-d6; (b) In CDCl3.

6.4

6.2 ppm

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

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Special features of the sample

Contains a lot of water

Saline

Contains exchangeab le protons (OH, NH, SH…type)

pH sensitive

Viscous

Unstable

Has to be measured (dosing)

Figure 18.11 Special features of NMR sample.

Figure 18.12 500 MHz 1 H NMR spectra of a D2O extract of abalone Haliotis tuberculata, ns ¼ 64; (a) Without presaturation of water signal; (b) With presaturation of water signal.

For example, in dilute solution of alcohols in non-hydrogenbonding solvents (CCl4, CDCl3, C6D6), the OH signal generally appears at 1–2 ppm, but at higher concentrations the signal moves downfield. Indeed, the OH signal of ethanol appears at 1.0 ppm in a 0.5% solution in CCl4, whereas it is observed at 5.13 ppm in the pure liquid (Bovey and Mirau, 1996). Under ideal conditions, exchangeable protons can show sharp signals with full coupling to neighboring protons even at room temperature, as in the spectrum of neat ethanol (Figure 18.13). However, under normal conditions used for NMR spectra acquisition, no coupling is observed between the hydroxyl, amine or thiol hydrogen and hydrogens on the carbon atom to which it is attached. A typical alcohol undergoes intermolecular proton exchange at a rate of 105 protons per second, which is too fast for NMR observation. Only the average environment is observed, which results in broadening or complete loss of coupling to neighboring protons. Such an exchange can also broaden or average the signals of multiple OH, NH or SH groups in the sample, if more than one is present. Rates of exchange will depend on the temperature, solvent, concentration, and the presence of acidic and basic impurities. In CDCl3, the presence of acidic impurities resulting from solvent

Figure 18.13 60 MHz 1 H NMR spectra of ethanol at various concentrations (Bovey and Mirau, 1996).

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Figure 18.14 500 MHz 1 H NMR spectra of isethionic acid isolated from red alga Grateloupia turuturu. (a) In D2O; (b) In DMSO-d6.

decomposition often leads to a rapid acid-catalyzed exchange between exchangeable protons. In contrast, solvents such as dimethylsulfoxide (DMSO) and acetone form strong hydrogen bonds to exchangeable protons. This has the effect of slowing down the intermolecular proton exchanges, usually leading to discrete OH, NH or SH signals with observable coupling to nearby protons. In order to observe coupling, the experiment temperature can also be reduced. Hence, the exchange rate of the hydroxyl, amine or thiol protons will be reduced, and coupling with other protons in molecule can then be observed (Figure 18.14). When compounds with OH, NH or SH protons are placed in protonated solvents (D2O, MeOD, etc.), these hydrogens often exchange spontaneously with deuterium; however, because of this deuterium exchange the signal of these protons “disappears” from the spectrum. This can be useful in assigning peaks in the NMR spectrum. Indeed, to identify the labile protons, the “D2O shake” can be used, which results in the disappearance of all OH, NH, and SH signals.

Deuterium exchange works best if the used solvent is water immiscible and denser than water (CDCl3, CD2Cl2, CCl4), because the DOH formed will be in the drop of water floating at the top of the sample, where it could not be detected. In watermiscible solvents (e.g., acetone, DMSO, acetonitrile, pyridine, THF), labile proton signals are largely converted to OD, ND and SD, but the DOH formed remains in solution and will be detected in the water region. 18.4.1.4.4 pH-Sensitive Sample Samples, especially tissue extracts, contain pH-sensitive metabolites (amino acids and other classical compounds of metabolism) that are characterized by chemical groups acting as donors or receivers of protons. Consequently, they are particularly sensitive to

cellular pH variations and thus represent good proxies of this parameter (Figure 18.15). This parameter tends to complicate NMR spectra interpretations, because chemical shifts and signal shape can be greatly modified according to pH conditions. 18.4.1.4.5 Viscous Samples At room temperature, the NMR spectra of viscous samples (e.g., polymers, polysaccharides) most often present large signals due to a poor resolution. Initially, the sample can be diluted in order to improve resolution; however, if the resolution is not improved or if the limit of detection is reached, the temperature of the sample can be increased. Finally, by increasing the rotating speed of chemical bonds higher than the NMR speed detection, better resolved signals can be observed (Figure 18.16). Clearly, the probe temperature must not come close to the boiling point of the solvent used. 18.4.1.4.6 Unstable Samples An unstable sample can be of biological origin (plant or animal) or a chemical product that is likely to degrade in time. There are two possible ways to treat the sample. First, especially if the analysis is likely to take a long time (e.g., 2D characterization), the spectra should be recorded at low temperature, perhaps down to 100  C. The second solution is to quickly analyze the sample at room temperature by making use of a cryoprobe. It has already been noted that sodium azide (NaN3) can also be used to stabilize biological samples. 18.4.1.4.7 Sample to Quantify Sample NMR quantification depends directly on peak integrations. In order to correctly quantify a compound in a sample, each peak area must truly represent the proton number of signals. Hence, for a proton acquisition the delay between the impulsions is generally set at least to 10 s to allow all proton relaxations and to avoid T1 influence. The most common way to quantify a sample is to “spike” the sample with an internal reference

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

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Figure 18.15 500 MHz 1 H NMR spectra of aspartic acid solubilized in D2O at different pH-values (same concentration, ns ¼ 16).

product of known concentration, or to use an external reference.  Internal reference: A compound of known concentration (close enough to that of the analyte) is added to the sample. The reference compound must: (i) be cosoluble with the sample; (ii) not react in any way with the sample; (iii) have chemical shifts that do not interfere with those of the sample; (iv) have T1 value(s) close (or inferior) to those of the sample; and (v) not be volatile or hygroscopic. However, internal standards rarely comply with all of the above criteria.

Figure 18.17 NMR tube with capillary tube.

 External reference: A reference compound is placed in a capillary tube, which is inserted into the NMR tube (Figure 18.17). This avoids problems of volatility, cosolubility and chemical interactions, but other problems appear including:

(b)

(a) 5.8

5.6

5.4

5.2

5.0

4.8

4.6

4.4

4.2

4.0

3.8

3.6

3.4

3.2

ppm

Figure 18.16 500 MHz 1 H NMR spectra of a polysaccharide in D2O at two different temperatures. (a) At 298 K; (b) At 333 K.

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Figure 18.18 500 MHz 1 H NMR spectrum. Dosing of phloroglucinol within an extract of brown alga Cystoseira tamariscifolia. The internal reference used was TSP. Courtesy of C. Jegou, 2011.

(i) the available volume for the sample is smaller, which reduces the signal-to-noise ratio; and (ii) the magnetic field homogeneity is not the same in the capillary and in the NMR tube. These two facts lead to an inferior precision and reproducibility compared to using an internal reference. When the acquisition has ended, the spectrum is carefully integrated and integral(s) of the molecule to dose and integral(s) of the compound of known concentration are compared to calculate the concentration of the studied molecule. The results are quickly obtained with a precision close to that obtained by HPLC post-column peak integration (Figure 18.18). This is a simple dosing method which requires the operator to optimize parameters such as baseline stability, correct selection of integration areas, correct phasing of spectrum, and digital resolution.

Figure 18.19

13

This method cannot be used when the signals overlap.

18.4.2 13 C Spectra 13

C is much less sensitive than 1 H, but has a much larger chemical shift range. The principle is the same as for 1 H NMR, whereby each nonequivalent 13 C gives a different signal (Figure 18.19). Because of the low natural abundance of 13C (1.108%), it is very unlikely to find two 13 C atoms close to each other in the same molecule, and consequently 13 C–13 C spin coupling is not observed. There is, however, heteronuclear coupling between

C NMR table of samples in CDCl3 solution. The d scale is relative to TMS at d ¼ 0.

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

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Figure 18.20 500 MHz 13 C NMR spectrum of isovaleric acid (one of the main acids entering the lipidic composition of triacylglycerides and wax esters of cetacean blubber).

13

C carbons and the 1 H bonded according to the (n þ 1) rule. Carbon–proton coupling constants of 100–250 Hz are common, which means that there is often a significant overlap between signals, which makes splitting patterns very difficult to determine (Figure 18.20). For clarity, broadband decoupling is used. In a hydrogendecoupled mode, the sample is irradiated two different ways: one frequency to excite all 13 C nuclei, and a broad spectrum of frequencies that compels all hydrogens in the molecule to undergo rapid transitions between their nuclear spin states. This greatly simplifies the spectrum and makes it less crowded because all of the carbon signals are singlets (Figure 18.21).

Unlike the 1 H-NMR spectrum, 13 C-NMR spectrum integration cannot be used to determine the number of carbons of each signal, because the signals for some types of carbon are weaker than for other types, especially quaternary carbons. This is due to long relaxation times of 13 C: a few seconds for a methyl (2–4 s) and up to hundreds of seconds for a carbonyl (generally about 200 s for an ester carbonyl) and to delay between scans generally of only a few seconds.

In hydrogen-decoupled mode, information on spin–spin coupling between 13 C and attached 1 H is lost. The Distortionless Enhancement by Polarization Transfer (DEPT) method is an instrumental mode that provides a way to acquire this information. DEPT is an NMR technique that is used to distinguish between 13 C signals for CH3, CH2, CH, and quaternary carbons.

DEPT methods use a complex series of pulses in both 1 H and C ranges:

13

 a 45 pulse: CH, CH2 and CH3 signals are positive.  a 90 pulse: only CH carbons are observed.  a 135 pulse: CH, CH3 signals are positive, whereas CH2 signals are negative. Quaternary carbons produce no signal. When used as an alternative to the DEPT experiments, Jmod (J-modulated spin-echo) sequences provide spectra in which the quaternary (C) and methylene (CH2) signals have opposite phases to those of methine (CH) and methyl (CH3). However, quaternary signals remain often rather weak (Figure 18.22). The J-mod sequence is normally used because, with one experiment, all of the 13 C chemical shifts of the sample can be obtained and CH, CH3 can be discriminated from CH2 and quaternary.

Care must be taken with this sequence because, according to the 1 H–13 C coupling constants in molecules, a signal that should appear positive may appear negative, and vice-versa. In case of doubt it may be necessary to determine the 1 H–13 C couplings constant of the considered signal by means of an 1 H-undecoupled 13 C spectrum.

Although only about 1 of 100 carbon atoms in a naturally occurring organic molecule is the 13 C isotope, carbon analyses can be enhanced by the 13 C labeling of molecules. For

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm Figure 18.21 500 MHz 1 H-decoupling 13 C NMR spectrum of isovaleric acid.

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Figure 18.22 500 MHz 13 C NMR series of spectrum of isovaleric acid. Comparison of 1 H-decoupled 13 C, DEPT45, DEPT90, DEPT135 and J-mod NMR experiments.

example, in biochemical studies it is possible to label one or more carbons in a small precursor molecule and then to trace the presence of the 13 C label through a metabolic pathway, all the way to a larger biomolecule product (see Box 18.1).

Box 18.1:

13

18.4.3 2D Spectra

Although 1D NMR experiments do not allow detailed structural analyses to be conducted, this can be accomplished by means of

C-NMR in isotopic labeling study (Bondu et al., 2009)

The aim of this research was to study the impact of the salt stress on the photosynthetic carbon flux used in the synthesis of low-molecular-weight carbohydrates (digeneaside and floridoside). This was investigated by 13 C and 1 H NMR spectroscopy in samples of the red seaweed, Solieria chordalis, incubated at different salinities of 22, 34, and 50 practical salinity units (psu). The carbohydrates were labeled, by pulse-chase, with the stable isotope 13 C from

NaH13 CO3 . In vivo NMR analyses carried out with a cryogenic probe optimized for 13 C detection were performed directly on the living algal tissues to evidence the labeling of the carbohydrates with neither preliminary extraction nor purification step(s). The isotopic enrichment of each compound was determined by high-resolution 1 H and 13 C NMR spectroscopy (Figure 18.23). These analyses evidenced different orientations of the flux of the

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

photosynthetic carbon in the algae according to the salt stress. At normal and low salinities, the photosynthetic carbon flux was responsible of 70% and 67% of the floridoside synthetized during the pulse period, respectively,

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whereas it was only of 30% in the thalli exposed to the high salinity, which means the biosynthesis of high amounts of floridoside from an endogenous source, leading to the osmotic regulation.

Figure 18.23 In vivo 500 MHz 13 C NMR proton-decoupled spectra obtained from S. chordalis prior to incubation in 13 C-enriched medium (lower spectrum) and after 24 h incubation in hypo-osmotic medium containing 2.5 mM NaH13 CO3 (upper spectrum). The letters F and D on the spectra refer to the resonances of carbons of floridoside and digeneaside, respectively.

2D NMR experiments. 2D experiments are a series of 1D experiments collected with different mixing times, and can be divided into two types: homonuclear; and heteronuclear. Each type can provide either through-bond (COSY, HMQC, HMBC, TOCSY) or through-space (NOESY, ROESY) coupling information. On modern spectrometers, a 90 pulse width proton is the key parameter to run a full series of 2D experiments. COSY, HMQC/HSQC and HMBC represents the front-line 2D experiments of molecular identification:  COSY (COrrelation SpectroscopY) is used to identify nuclei that share a scalar (J) coupling (through-bond correlation). The presence of off-diagonal peaks (cross-peaks) in the spectrum directly correlates coupled nuclei. 1 H–1 H COSY

correlations appear between neighboring protons (usually up to four bonds) (Figure 18.24). This sequence is most often used to analyze coupling relationships between protons, but may be used to correlate any high-abundance homonuclear spins, for example, 31 P, 19 F, and 11 B.  DQF-COSY (Double-Quantum Filtered Correlation Spectroscopy) is a variation of the standard 2D COSY experiment, used for identifying scalar (J) couplings between protons. In practice, it gives the same information as COSY, but appears “cleaner.” Indeed, double-quantum filter aids the analysis of crowded spectra, reduces cross-peak overlap, and allows potentially informative fine-structure within cross-peaks to be studied in detail. Filtering also suppresses all singlets in the spectrum (along the diagonal). If 13 C had a higher isotopic abundance, then 13 C–13 C COSY would be possible. An

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Figure 18.24 500 MHz COSY 1 H/1 H of floridoside isolated from red algae Grateloupia turuturu.

example of an unusual 13 C–13 C COSY on 13 C-enriched floridoside is shown in (Figure 18.25).  HMQC (Heteronuclear Multiple-Quantum Correlation) is used to correlate directly bonded X-proton nuclei. This

technique is based on proton detection, and has very high sensitivity (HMQC 1 H–13 C can even be quicker to acquire than a 1D carbon spectrum). HMQC 1 H–13 C gives proton assignments onto their directly attached carbons, and is very useful when proton multiplets overlap. It also provides a convenient way of identifying diastereotopic geminal protons (which are sometimes difficult to distinguish unambiguously, even in COSY) because only these two protons will produce correlations to the same carbon (Figure 18.26).  HMBC (Heteronuclear Multiple-Bond Correlation) is a 2D pulse sequence (closely related to HMQC, its 1-bond analog), used to identify long-range couplings between protons and X. It allows to correlate X-nucleus shifts that are typically two to three bonds away from a proton (2 JH X , 3 JH X and sometimes 4 JH X in extended conjuguated systems). The experiment utilizes proton detection, and so has good sensitivity. HMBC 1 H–13 C is an extremely powerful tool for grouping together intramolecular and/or intermolecular spin systems (Figure 18.27). Additional useful 2D NMR experiments include:

Figure 18.25 500 MHz two-dimensional 13 C–13 C COSY spectrum of floridoside obtained from red alga extract of G. doryphora incubated for 24 h in 52%-NaCl enriched with 2.5 mM NaH13 CO3 . Assignments of floridoside carbons are indicated on the 13 C projection (Simon-Colin et al., 2004).

 HSQC (Heteronuclear Single Quantum Correlation) permits a 2D proton-detected heteronuclear chemical shift correlation map to be obtained between directly bonded 1 H and X-heteronuclei (commonly, 13 C and 15 N), which provides the same information as the closely related HMQC. The difference between these two techniques is that, during the evolution time of an HMQC, both proton and X magnetization (e.g., X ¼ 13 C) are allowed to evolve, whereas in an HSQC only X magnetization is allowed to evolve. This means that an HMQC is affected by homonuclear proton J coupling during the evolution period, but

18.4 Conventional “Liquid” Probes: Obtaining 1D and 2D Spectra of all NMR-Observable Nuclei

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Figure 18.26 500 MHz HMQC 1 H/13 C of floridoside isolated from red algae Grateloupia turuturu.

Figure 18.27 500 MHz HMBC 1 H/13 C of floridoside isolated from red algae Grateloupia turuturu.

Figure 18.28 Comparison of 1 H/13 C HMQC and HSQC of menthol at 7.05 Tesla (http://u-of-o-nmr-facility.blogspot.fr/2009/01/hmqc-vs-hsqc .html).

an HSQC is not affected as there is no proton magnetization during the evolution time. Consequently, HSQC gives a slightly better resolution, whereas the resonances are broadened by homonuclear proton couplings in HMQC (Figure 18.28). If a high 13 C resolution is required, then HSQC is recommended; however, if a high 13 C resolution is not essential then HMQC is sufficient. Moreover, HSQC provides a factor of two of improvement in sensitivity for methine protons and amide nitrogen.  TOCSY (TOtal Correlated SpectroscopY), also known as HOHAHA (HOmonuclear HArtmann HAhn), is a 2D homonuclear correlation experiment used to analyze scalar (J) coupling networks between protons. It has a similar appearance to the 2D COSY spectrum, and is useful for dividing the proton signals into groups or coupling networks, especially when the multiplets overlap (have very similar chemical shifts), or there is extensive second-order coupling. A TOCSY spectrum yields

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Figure 18.29 500 MHz TOCSY 1 H/1 H of floridoside isolated from red algae Grateloupia turuturu.

through bond correlations via spin–spin coupling, and shows correlation among spins that are not directly coupled but exist within the same spin system. Correlation intensities are not related to the number of bonds connecting the protons; that is, a five-bond correlation may or may not be stronger than a threebond correlation. TOCSY is normally used for the analysis of large molecules (peptides, proteins, oligosaccharides and polysaccharides) composed of discrete subunits (spin systems) such as amino acids or glycosyl units (Figure 18.29).  NOESY (Nuclear Overhauser Effect SpectroscopY) is a 2D method used to establish through space correlations; that is,

to determine which signals arise from protons that are close to each other in space, even if they are not bonded. It is useful for the conformational organization of molecules and for observations of adjacent spin systems. The spectra have a layout similar to COSY, but crosspeaks here indicate contacts between the correlated protons. NOESY is not applicable to intermediate-sized molecules (Figure 18.30). When molecules are biologically active, it is often important to determine their conformation in order to understand their biological activity (see Box 18.2).

Figure 18.30 500 MHz NOESY 1 H/1 H of floridoside isolated from red algae Grateloupia turuturu.

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Box 18.2: Conformation explain biological activity of meridianins In meridianins, rotation is possible around the axis C3–C40 . NOESY NMR spectra of these molecules show the favorite conformation (Figure 18.31). Meridianins are CKIs (cyclin-dependent kinase inhibitor), whose inhibitory activity is due to their association with CDK or complex

Figure 18.31 500 MHz NOESY NMR spectrum of meridianin G from the tunicate Aplidium meridianum (Simon et al., 2007).

 ROESY (Rotating-frame NOE SpectroscopY) is a 2D experiment that measures NOEs in the “rotating-frame,” and is useful for determining which signals arise from protons that are spatially close to each other, even if they are not bonded, particularly for mid-sized molecules (1000–3000 Da) that have close-to-zero conventional NOEs. A ROESY spectrum provides information on through-space correlations via spin–spin relaxation, and also

CDK/cyclin. The CKIs are often ATP-competitive and function by substitution on the ATP fixation site. These chemical inhibitors, which block cellular proliferation by inactivation of CDK/cyclins, are potential antitumoral agents (Figure 18.32).

Figure 18.32 Modeling of meridianins A, D, F, and G in the ATP fixation site of CDK1/cyclin B.

detects chemical and conformational exchange. It has a similar overall appearance to COSY, but crosspeaks here indicate ROEs between the correlated spins. A ROESY spectrum can show interfering from TOCSY transfers (between J-coupled spins), and requires careful analysis (Figure 18.33).  DOSY (Diffusion-Ordered SpectroscopY) is a pseudo-2D NMR experiment which presents chemical shifts along the

Figure 18.33 500 MHz ROESY 1 H/1 H of floridoside isolated from red algae Grateloupia turuturu.

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Figure 18.34 (Top) Series of 1D 1H NMR diffusion experiments for a mixture containing adenosine triphosphate, sodium dodecyl sulfate, 4,4-dimethyl-asilapentane-1-sulfonic acid and HOD – (Bottom) the corresponding DOSY spectrum (Source: Bruker)

detected F2 axis and diffusion coefficients of the solutes along the other F1 axis. A series of 1D NMR diffusion spectra are acquired as a function of the gradient strength involving that signal intensity of compounds decreases as a function of the gradient strength. All signals corresponding to the same molecular species decay at the same rate. The variation of signal intensity according to gradient intensity is exponential. These data are transformed in a 2D DOSY map on which the horizontal axis encodes the chemical shift of the nucleus observed (generally 1 H) and the vertical dimension encodes the diffusion coefficients. In the ideal case of non-overlapping component lines and no chemical exchange, the 2D DOSY peaks align themselves along horizontal lines, each corresponding to one sample molecule (Figure 18.34). DOSY NMR provides a way to separate the different compounds in a mixture based on the differing translation diffusion coefficients of each chemical species in solution. It can be used to investigate molecular size or shape, complexation phenomena, binding, aggregation and physical properties of the surrounding environment such as viscosity, and temperature. 18.4.4 Other Nuclei Spectra

In order to complete a structural analysis of unknown compounds by NMR, it can be necessary to use other nuclei. The natural

abundance of some nuclei allows them to be observed directly (1D), for example, 31 P or 19 F. However, in many cases, when the isotopic abundance is weak or there is intense quadrupole moment, only an indirect detection using HMQC or HMBC experiments is possible (Figure 18.35). At this point, attention is focused on isotopes that are likely to be found in marine compounds. 18.4.4.1 Isotopes with No NMR Properties Isotopes with a null spin number have no magnetic properties, and the only effect on neighboring nuclei is the isotope shift. 18.4.4.2 Isotopes (I ¼ 1/2) with 100% Abundance These isotopes have a high NMR sensitivity. The spectra present sharp lines and full homonuclear and heteronuclear couplings are observable. 18.4.4.2.1 31 P NMR Phosphorus occurs predominantly as the isotope 31 P, which has a nuclear spin value of 1/2 and is therefore easily observable in NMR. 31 P NMR has been in use since the development of multinuclear, high-field Fourier-transform instruments during the late 1970s. The 31 P NMR method is widely used, with both 1D and multidimensional techniques, in such diverse areas as organic and inorganic molecular structure characterization, the analysis of biological fluids, determination of intracellular pH, noninvasive studies of intact tissues and organs, and quantitative assays of industrial products (Twyman, 2005).

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All isotopes

I=0

No NMR properties

I = 1/2

≈100% abundance 1

12

C, 16O, 32S,…

19

31

Low abundance

89

H, F, P, Y, Rh,…

103

I > 1/2

13

77

15

29

C, N, Si, Se, 109Ag, 113Cd, 119 Sn, 207Pb,…

Long T1 values 2

6

H, Li,

133

Cs,…

Short T1 values 7

Li, 11B, 14N, 17O, S, 35Cl, 39K,…

33

Figure 18.35 NMR properties of isotopes.

Figure 18.36 500MHz 31P NMR spectrum of a sea anemone (Meneses et al. 1988).

Typically, a 1D 31 P NMR experiment is much less sensitive than 1 H, but more sensitive than 13 C. 31 P is a medium-sensitivity nucleus that yields well-resolved signals and has a wide chemical shift range (about 250 to 250 ppm), depending primarily on the oxidation state and coordination number of the phosphorus atoms present. Chemical shifts also depend on the pH, concentration, solvent effects, and the electronegativity of any substituents. In general, phosphorus chemical shifts are referenced to an external standard, 85% phosphoric acid (H3PO4) (Figure 18.36; Table 18.6). 31 P NMR is usually acquired with 1 H decoupling, which means that spin–spin couplings are seldom observed; this greatly simplifies the spectrum and makes it less crowded. 1 J 31P 1H are typically 150 to 700 Hz, 2 J 31P 1H are 0.5 to 20 Hz, 3 J 31P 1H are 10 to 30 Hz, and 4 J 31P 1H