Chemical Biology

Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Cunther WessVolume I

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Chemical BiologyFrom Small Molecules to Systems Biology and Drug Design Edited by Stuart 1. Schreiber, Tarun M. Kupoor,and Cunther Wess

.,CENTENNIAL

B I C I W T E N N I I L

WILEY-VCH Verlag CmbH & Co. KCaA

The Editors

All books published by Wiley-VCH are carefullyproduced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free o f errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Prof: Dr. Stuart L. SchreiberHoward Hughes Medical Institute Chemistry and Chemical Biology Harvard University Broad Institute o f Harvard and MIT Cambridge, MA 02142 USA

Library ofcongress Card No.: applied for British Library Cataloguingin-Publication DataA catalogue record for this book i s availablefrom the British Library.

Prof: Dr. Tarun M. KapoorLaboratory o f Chemistry and Cell Biology Rockefeller University 1230 York Ave. New York, NY 10021 USA

Bibliographic information published by the Deutsche NationalbibliothekThe Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet a t < http://dnb.d-nb.dez.

Prof: Dr. Ciinther WessCSF - Forschungszentrum fur Umwelt und Gesundheit lngolstadter Landstr. 1 85764 Neuherberg Germany

0 2007 WILEY-VCH Verlag CmbH & CoKCaA, Weinheim All rights reserved (including those o f translation into other languages). No part o f 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.

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ISBN 978-3-527-31150-7

Iv

Preface

XV XVll

List of Contributors

Volume 1Part I1

chemistry and Biology - Historical and Philosophical Aspects Chemistry and Biology - Historical and PhilosophicalAspects

Gerhard Quinkert, Holger Wallmeier,Norbert Windhab,and Dietmar Reichert

3

1.1 1.2 1.2.1 1.2.2 1.2.3 1.3 1.3.1 1.3.2 1.3.3 1.4 1.4.1 1.4.2 1.5 1.5.1 1.5.2 1.G

1.71.7.1

Prologue 3 Semantics 4 Synthesis - Genesis - Preparation 4 Synthetic Design - Synthetic Execution 8 Preparative Chemistry - Synthetic Chemistry 9 Bringing Chemical Solutions to Chemical Problems 10 The Present Situation 10 Historical Periods of Chemical Synthesis 12 Diels-Alder Reaction - Prototype of a Synthetically Useful Reaction IG Bringing Chemical Solutions to Biological Problems 18 The Role of Evolutionary Thinking in Shaping Biology 18 On the Sequence of Chemical Synthesis (Preparation) and Biological Analysis (Screening) 20 Bringing Biological Solutions to Chemical Problems 45 Proteins [99] 45 Antibodies 52 Bringing Biological Solutions to Biological Problems 53 EPILOGUE 54 The Fossil Fuel Dilemma of Present Chemical Industry 54

Chemical Biology. From Small Molecules to System Biology and Drug Design Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Cunther Wess Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31150-7

vi

1

Contents

1.7.2

Two Lessons From the Wealth of Published Total Syntheses 55 Acknowledgments 58 References 59Using Natural Products to Unravel Biological Mechanisms Using Natural Products to Unravel Biological Mechanisms

Part II 2

2.1

Using Small Molecules to Unravel Biological Mechanisms Michael A. Lampson and Tarun M . Kapoor

71 71

2.1.1 2.1.2 2.1.3 2.1.4

Outlook 71 Introduction 71 Use of Small Molecules to Link a Protein Target to a Cellular Phenotype 72 Small Molecules as Probes for Biological Processes 77 Conclusion 89 References 90 Using Natural Products to Unravel Cell Biology Jonathan D. Gough and Craig M . Crews Outlook 95 Introduction 95 Historical Development 95 General Considerations 96 Applications and Practical Examples Future Development 109 Conclusions 109 Acknowledgments 110 References 110 95

2.2

2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6

96

33.1

Engineering Control Over Protein Function Using Chemistry

115 Revealing Biological Specificityby Engineering Protein- Ligand Interactions 115 Matthew D. Simon and Kevan M. Shokat Outlook 115 Introduction 115 The Selection of Resistance Mutations to Small-moleculeAgents 116 Exploiting Sensitizing Mutations to Engineer Nucleotide Binding Pockets 126 Engineering the Ligand Selectivelyof Ion Channels 130 Conclusion 134 References 136

3.1.1 3.1.2 3.1.3 3.1.4 3.1.5

Contents

1

vii

3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6

Controlling Protein Function by Caged Compounds 140 Andrea Giordano, Sirus Zarbakhsh, and Carsten Schultz Introduction 140 Photoactivatable Groups and Their Applications 140 Caged Peptides and Proteins I S 0 Caged Proteins by Introduction of Photoactive Residues via Site Directed, Unnatural Amino Acid Mutagenesis 156 Small Caged Molecules Used to Control Protein Activity 159 Conclusions 168 References 168 Engineering Control Over Protein Function; Transcription Control by Small Molecules 174 j o h n T. Koh Outlook 174 Introduction 174 The Role of Ligand-dependent Transcriptional Regulators 175 Engineering New Ligand Specificities into NHRs 179 The Requirement of Functional Orthogonality 180 Overcoming Receptor Plasticity 180 Nuclear Receptor Engineering by Selection 183 Ligand-dependent Recombinases 184 Complementation/Rescue of Genetic Disease 186 De Novo Design of Ligand-binding Pockets 188 Light-activated Gene Expression from Small Molecules 189 References 191Controlling Protein-Protein Interactions

3.3

3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6 3.3.7 3.3.8 3.3.9 3.3.10

4 4.1

199 Chemical Complementation: Bringing the Power of Genetics to Chemistry 199 Pamela Peralta-Yahya and Virginia W. Cornish

4.1.1 4.1.2 4.1.3 4.1.4 4.1.5

Outlook 199 Introduction 199 History/Development 202 General Considerations 208 Applications 21 G Future Development 222 References 223 Controlling Protein- Protein Interactions Using Chemical Inducers and Disrupters of Dimerization 227 T i m Clackson Outlook227

4.2

viii

1

Contents

4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6

Introduction 227 Development of Chemical Dimerization Technology Dimerization Systems 229 Applications 237 Future Development 245 Conclusion 245 Acknowledgments 246 References 246

228

4.3

Protein Secondary Structure Mimetics as Modulators of Protein-Protein and Protein-Ligand Interactions 250 Hang Yinand Andrew D. Hamilton Outlook 250 Introduction 250 History and Development 251 General Considerations 253 Applications and Practical Examples Future Developments 264 Conclusion 265 Acknowledgments 2G5 References 265Expanding the Genetic Code

4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6

255

5

5.1

271 Synthetic Expansion of the Central Dogma Masahiko Sisido

271

5.1.1 5.1.2 5.1.2.2 5.1.2.3 5.1.2.4 5.1.2.5 5.1.3 5.1.3.2 5.1.3.3 5.1.4 5.1.4.2 5.1.4.3 5.1.5

Outlook 271 Introduction 272 Aminoacylation of tRNA with Nonnatural Amino Acids 274 Micelle-mediatedAminoacylation 275 Ribozyme-mediatedAminoacylation 276 PNA-assisted Aminoacylation 277 Directed Evolution of Existing aaRS/tRNA Pair to Accept Nonnatural Amino Acids 278 Other Biomolecules That Must Be Optimized for Nonnatural Amino Acids 281 Adaptability of EF-Tu to Aminoacyl-tRNAsCarrying a Wide Variety of Nonnatural Amino Acids 283 Adaptability of Ribosome to Wide Variety of Nonnatural Amino Acids 283 Expansion of the Genetic Codes 284 Four-base Codons 285 Synthetic Codons That Contain Nonnatural Nucleobases 286 In vivo Synthesis of Nonnatural Mutants 287

Contents

I

ix

5.1.6 5.1.7

Application of Nonnatural Mutagenesis - Fluorescence Labeling 289 Future Development and Conclusion 291 Acknowledgments 291 References 291Engineering Control Over Protein Function Using Chemistry Forward Chemical Genetics

Part Ill

6

StephenJ. Haggarty and Stuart L. SchreiberOutlook 299 Introduction 299 History/ Development 302 General Considerations 307 Small Molecules as a Means to Perturb Biological Systems Conditionally 307 Forward and Reverse Chemical Genetics 308 Phenotypic Assays for Forward Chemical-Genetic Screening 3 12 Nonheritable and Combinations of Perturbations 316 Multiparametric Considerations: Dose and Time 318 Sources of Phenotypic Variation: Genetic versus Chemical Diversity 318 The Target Identification Problem 329 Relationship between Network Connectivity and Discovery of Small-molecule Probes 323 Computational Framework for Forward Chemical Genetics: Legacy of Morgan and Sturtevant 325 Mapping of Chemical Space Using Forward Chemical Genetics 326 Dimensionality Reduction and Visualization of Chemical Space 330 Discrete Methods of Analysis of Forward Chemical-genetic Data 334 Applications and Practical Examples 336 Example 1: Mitosis and Spindle Assembly 336 Example 2: Protein Acetylation 338 Example 3: Chemical-genomic Profiling 340 Future Development 344 Conclusion 347 Acknowledgments 348 References 349

299

6.1 6.2 6.3 6.3.1 6.3.2 6.3.3 6.3.4 6.3.5 6.3.6 6.3.7 6.3.8 6.3.9 6.3.10 6.3.11 6.3.12 6.4 6.4.1 6.4.2 6.4.3 6.5 6.6

X I

Contents

7

7.1

Reverse Chemical Genetics Revisited 355 Reverse Chemical Genetics - An Important Strategy for the Study of Protein Function in Chemical Biology and Drug Discovery 355 Rolf Breinbauer, Alexander Hillisch, and Herbert Waldmann

7.1.1 7.1.2 7.1.3 7.1.4 7.1.5 7.1.6

Introduction 355 History/Development 356 General Considerations 361 Applications and Practical Examples Future Developments 376 Conclusion 379 Acknowledgments 380 References 380

366

7.2

Chemical Biology and Enzymology: Protein Phosphorylation as a Casestudy 385 Philip A. Cole Outlook 385 Overview 385 The Enzymology of Posttranslational Modifications of Proteins 387 References 401 Chemical Strategies for Activity-based Proteomics NadimJessani and Benjamin F. Cravatt Outlook 403 Introduction 403 History/Development 404 General Considerations 407 Applications and Practical Examples Future Development 421 Conclusions 422 Acknowledgments 423 References 423 403

7.2.1 7.2.2

7.3

7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.3.6

415

8 8.1

Tags and Probes for Chemical Biology

427 The Biarsenical-tetracysteine Protein Tag: Chemistry and Biological Applications 427 Stephen R. Adams Outlook 427 Introduction 427 History and Design Concepts of the Tetracysteine-biarsenical System 429

8.1.1 8.1.2

Contents

1

xi

8.1.3 8.1.4 8.1.5 8.1.6

General Considerations 430 Practical Applications of the Biarsenical-tetracysteine System 439 Future Developments and Applications 453 Conclusions 454 Acknowledgments 454 References 454 Chemical Approaches to Exploit Fusion Proteins for Functional Studies 458 Anke Arnold, India SielaJ NilsJohnsson, and Kailohnsson Outlook 458 Introduction 458 General Considerations 459 Applications and Practical Examples 463 Conclusions and Future Developments 476 Acknowledgments 477 References 477

8.2

8.2.1 8.2.2 8.2.3 8.2.4

Volume 2Part IV Controlling Protein- Protein Interactions Diversity-orientedSynthesis

9

9.1 9.2

Diversity-oriented Synthesis Derek S. Tan

483 483

Combinatorial Biosynthesis of Polyketides and Nonribosomal Peptides 519 Nathan A. Schnarr and Chaitan KhoslaSynthesis of Large Biological Molecules

10

537

10.1

Expressed Protein Ligation 537 Matthew R. Pratt and Tom W. Muir Chemical Synthesis of Proteins and Large Bioconjugates Philip Dawson New Methods for Protein Bioconjugation Matthew B. FrancisAdvances in Sugar Chemistry

10.2 10.311

567

593

11.1

635 The Search for Chemical Probes to Illuminate Carbohydrate Function 635 Laura L. Kiessling and Erin E. Carlson

xii

I

Contents

11.2

Chemical Glycomics as Basis for Drug Discovery Daniel B. Werz and Peter H. Seeberger

668

12

The Bicyclic Depsipeptide Family of Histone Deacetylase Inhibitors 693

Paul A. Townsend, Simon]. Crabb, Sean M. Davidson, Peter W. M. Johnson, Graham Packham, and Arasu GanesanPart V13

Expandingthe Genetic Code Chemical Informatics

13.1

Chemical Informatics Paul A. Clemons

723 723

13.2

WOMBAT and WOMBAT-PK Bioactivity Databases for Lead and Drug Discovery 760 Marius Olah, Ramona Rad, Liliana Ostopovici, Alina Bora, Nicoleta Hadaruga, Dan Hadaruga, Ramona Moldovan, Adriana Fulias, Maria Mracec, and Tudor 1. Oprea

Volume 3Part VI14

Forward Chemical Genetics Chemical Biology and Drug Discovery

14.1

789 Managerial Challenges in Implementing Chemical Biology Platforms 789 Frank L. Douglas

14.2

The Molecular Basis of Predicting Druggability 804 Bissan Al-Lazikani, Anna Gaulton, Gaia Paolini, Jerry Lanfar, John Overington, and Andrew HopkinsTarget Families

15

825

15.1

The Target Family Approach Hans Peter Nestler

825

15.2

Chemical Biology of Kinases Studied by NMR Spectroscopy 852 Marco Betz, Martin Vogtherr, Ulrich Schieborr, Bettina Elshorst, Susanne Grimrne, Barbara Pescatore, Thomas Langer, Krishna Saxena, and Harald Schwalbe

Contents

I

xiii

15.3

The Nuclear Receptor Superfamily and Drug Discovery John T. Moore, Jon L. Collins, and Kenneth H . Pearce

891

15.4

The GPCR - 7TM Receptor Target Family 933 Edgar Jacoby, Rochdi Bouhelal, Marc Gerspacher, and Klaus Seuwen Drugs Targeting Protein-Protein Interactions Patrick Che'nePrediction of ADM ET Properties

15.5

979

16

I003 UEfNorinder and Christel A. S. Bergstrom

Part VII

Reverse Chemical Genetics Revisited

17 17.1

1045 Systems Biology of the JAK-STATSignaling Pathway 1045 lens Timmer, Markus Kollrnann, and Ursula KlingmiillerComputational Methods and Modeling

17.2

Modeling Intracellular Signal Transduction Processes Jason M. Haugh and Michael C. WeigerGenome and Proteome Studies

1 061

18 18.1

1083 Genome-wide Gene Expression Analysis: Practical Considerations and Application to the Analysis of T-cell Subsets in Inflammatory Diseases 1083 Lars Rogge and Elisabetta Bianchi

18.2

Scanning the Proteome for Targets of Organic Small Molecules Using Bifunctional Receptor Ligands 1118 Nikolai KleyTags and Probes for Chemical Biology Chemical Biology - An Outlook

Part Vlll19

1143

Giinther WessIndex1151

I

xv

PrefaceSmall molecules are at the heart of chemical biology. The contributions in this book reveal the many ways in which chemical biologists studies of small molecules in the context of living systems are transforming science and society. Macromolecules are the basis of heritable information flow in living systems. This is evident in the Central Dogma of biology, where heritable information is replicated via DNA and flows from DNA to RNA to proteins. Small molecules are the basis for dynamic information flow in living systems. They constitute the hormones and neurotransmitters, many intra- and intercellular signaling molecules, the defensive and offensive natural productsused in information flow between organisms, among many others. They are the basis for memory and cognition, sensing and signaling, and, of course, for many of the most effective therapeutic agents. One dominant theme in many of the chapters concerns small molecules and small-molecule screening. Together, these have dramatically affected lifescience research in recent years. Many of the contributors to Chemical Biology themselves both provided new tools for understanding living systems and affected smoother transitions from biology to medicine. The chapters they have provided offer riveting examples of the fields impact on life science. The range of approaches and the creativity that fueled these projects are truly inspiring. After a period of widely recognized advances by geneticists and molecular and disease biologists, chemists and chemical biologists are returning to a position of prominence in the consciousness of the larger scientific community. The trend towards small molecules and small-molecule screening has resulted in an urgent need for advances in synthetic planning and methodology. Synthesis routes are needed for candidate small molecules and for improved versions of candidates identified in biological discovery efforts. Several contributors give hints to the question: How do we synthesize candidate structures most effectively poised for optimization? They note that planning and performing multi-step syntheses of natural products in the past resulted in the recognition and, often, resolution of gaps in synthetic methodology. The synergistic relationship between organic synthesis planning and methodologyChemical Biology. From Small Molecules to System Biology and Drug Design. Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Giinther Wess Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31150-7

xvi

1

Preface

is even more profound as synthetic organic chemists tackle the new challenges noted above. The objects of synthesis planning, no longer limited by the biochemical transformations used by cells in synthesizing naturally occurring small molecules, require radically new strategies and methodologies. Several contributors help us answer a related question that also influences synthetic plannig: What are the structural features of small, organic molecules most likely to yield specific modulation of disease-relevant functions? They note that the ability to assess the performance of these compounds, and to compare their performance to other small molecules such as commercially available or naturally occurring ones, is possible through public small-molecule screening efforts and public small-molecule databases (e.g., WOMBAT, PubChem, ChemBank). These developments are reminiscent of the early stage of genomics research, where visionary scientists recognized the need to create a culture of open data sharing and to develop public data repositories (e.g., GenBank) and analysis environments (e.g., Ensembl, UCSC Genome Browser). Sometimes the line between small and macromolecules is blurred. Oligosaccharides are often presented as a third class of macromolecules, yet several contributions here reveal arguably greater similarities of carbohydrates to small-molecule terpenes than to nucleic acids and proteins, both in terms of their biosynthesis and cellular functions. Oligosaccharides are shown to be synthesized by glycosyl transferases (analogous to isopentenyl pyrophosphate transferases used in terpene biosynthesis) and, like the terpenes, are subject to tailoring enzymes. Transferase enzymes are used to attach oligosaccharides and terpenes to proteins, where they serve key functions (e.g., glycoproteins, farnesylated Ras). Chemical biologists have illuminated and manipulated oligosaccharides and the unquestionable member of the macromolecule family, the proteins, with great aplomb. Several of our contributors are pioneers in the revolution of protein chemistry and protein engineering, and their chapters provide clear testimony to the consequences of these advances to life science. Finally, in examing the similarities of and synergies between chemical biology and systems biology, several of our contributors have perhaps offered a glimpse into the future of these fields.Stuart L. Schreiber, Cambridge Tarun M. Kapoor, New York Gunther Wess, Neuherberg

January 2007

List of ContributorsStephen R. Adarns Department o f Pharmacology University o f California, San Diego 310 George Palade Laboratories 0647 La Jolla, CA 92093-0647 USA Anke Arnold Ecole Polytechnique Federale de Lausanne (EPFL) Institute o f Chemical Sciences and Engineering 1011 Lausanne Switzerland Christel A. S. Bergstrom AstraZeneca R&D Discovery Medicinal Chemistry 15185 Sodertalje Sweden Marco Betz Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany Elisabetta Bianchi lmmunoregulation Laboratory Department o f Immunology Institute Pasteur 25, rue du Dr. Roux 75724 Paris Cedex 15 France A h a Bora Division o f Biocomputing University o f New Mexico School o f Med, MSC11 6445 Albuquerque, N M 87131 USA Rochdi Bouhelal Novartis Institutes for BioMedical Research Lichtstrasse 35 4056 Basel Switzerland

Rolf BreinbauerInstitute o f Organic Chemistry University o f Leipzig Johannisallee 29 041 03 Leipzig Germany

Erin E. Carkon Department o f Chemistry University o f Wisconsin 1101 University Avenue Madison, WI 53706 USA

Chemical Biology. From Small Molecules to System Biology and Drug Design. Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31150-7

xviii

1

List

ofContributors

Patrick Chene Oncology Research Novartis Institutes for Biomedical Research 4002 Basel Switzerland Tim Clackson ARIAD Pharmaceuticals, Inc. 26 Landsdowne Street Cambridge, MA 021 39-4234 USA Paul A. Clemons Chemical Biology Broad Institute o f Harvard & MIT 7 Cambridge Center Cambridge Center, MA 02142 USA Philip A. Cole Department o f Pharmacology Johns Hopkins School o f Medicine 725 N. Wolfe St. Baltimore, MD 21 205 USA Jon L. Collins Discovery Research. GlaxoSmithKline Discovery Research Research Triangle Park, NC 27709 USA Virginia W. Cornish Department o f Chemistry Columbia University 3000 Broadway, MC 31 67 New York, NY 10027-6948 USA Simon J. Crabb School o f Chemistry University o f Southampton Highfield Southampton SO1 7 1 BJ United Kingdom

Craig M. Crews Yale University School o f Medicine 333 Cedar Street New Haven, CT 06510 USA Benjamin F. Cravatt Neuro-Psychiatric Disorder Institute The Skaggs Institute for Chemical Biology The Scripps Research Institute BCC 159 10550 North Torrey Pines Rd. La Jolla, CA 92037 USA Sean M. Davidson The Hatter Cardiovascular Institute 67 Chenies Mews University College Hospital London WC1 E 6DB United Kingdom Philip Dawson Department o f Cell Biology and Chemistry The Scripps Research Institute 10550 N. Torrey Pines Road La Jolla, CA 92037 USA Frank L. Douglas Aventis Pharma lndustriepark Hochst 65926 Frankfurt Germany Bettina Elshorst Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany

List ofcontributors

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xix

Matthew B. Francis Department o f Chemistry University of California, Berkeley Berkeley, CA 94720-1460 USA Adriana Fulias Division of Biocomputing University o f New Mexico School of Med, MS C l l 6445 Albuquerque, N M 87131 USA Arasu Canesan School of Chemistry University o f Southampton Highfield Southampton SO1 7 1BJ United Kingdom Anna Caulton Pfizer Global Research and Development Pfizer Ltd. Sandwich, Kent, CT13 9NJ United Kingdom Marc Cerspacher Novartis Institutes for BioMedical Research Klybeckstrasse 141 4057 Basel Switzerland Andrea Giordano European Molecular Biology Laboratory Gene Expression Programme Meyerhofstr. 1 691 17 Heidelberg Germany

Jonathan D. Cough Yale University Department of Molecular, Cellular, and Developmental Biology Kline Biology Tower 442 New Haven, CT 06520-8103 USA Susanne Crimme Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany Dan Hadaruga Division of Biocomputing University of New Mexico School of Medicine, MS C l l 6445 Albuquerque, N M 87131 USA Nicoleta Hadaruga Division of Biocomputing University of New Mexico School o f Med, MS C l l 6445 Albuquerque, N M 87131 USA Stephen J. Haggarty Broad Institute of Harvard and MIT 320 Bent Street Cambridge, MA 02141 USA Andrew D. Hamilton Department of Chemistry Yale University 225 Prospect St. New Haven, CT 06520-8107 USA

xx

I

List ofcontributors

JasonM. Haugh Department o f Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905 USA Alexander Hillisch Bayer Healthcare AG PH-GDD-EURC-CR Aprather Weg 18a 42096 Wupperta! Germany Andrew Hopkins Pfizer Global Research and Development Pfizer Ltd. Sandwich, Kent, CT13 9NJ United Kingdom Edgar Jacoby Novartis Institute for Biomedical Research Lichtstrasse 35 4056 Basel Switzerland Nadim Jessani Department of Cell Biology Celera 180 Kimball Way South San Francisco, CA 94080 USA Kai Johnsson Ecole Polytechnique Federale de Lausanne (EPFL) Institute o f Chemical Sciences and Engineering 1011 Lausanne Switzerland

Nils JohnssonCenter for Molecular Biology o f Inflam mat io n Institute o f Medical Biochemistry University o f Muenster Von-Esmarch-Str. 56. 48149 Muenster Germany

Peter W. M. Johnson School o f Chemistry University of Southampton Highfield Southampton SO17 1BJ United Kingdom Tarun M. Kapoor Laboratory of Chemistry and Cell Biology Rockefeller University Flexner Hall 1230 York Ave. New York, NY 10021 USA Laura L. Kiessling Department o f Chemistry University o f Wisconsin 1101 University Avenue Madison, WI 53706 USA Nikolai Kley CPC Biotech, Inc. 610 Lincoln Street Waltham, MA 02451 USA Chaitan Khosla Department o f Chemistry Stanford U n iversi ty 381 North South Mall Stanford, CA 94305 USA

List

ofcontrjbutors

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xxi

Ursula Klingmiiller German Cancer Research Center (DKFZ) Im Neuenheimer Feld 280 69120 Heidelberg Germany John T. Koh Department o f Chemistry and Biochemistry University o f Delaware Newark, DE 19716 USA Markus Kollmann Physics Institute Hermann-Herder-Str. 3 79104 Freiburg Germany Michael A. Lampson Laboratory o f Chemistry and Cell Biology Rockefeller University Flexner Hall 1230 York Ave. New York, NY 10021 USA Jerry Lanfear Pfizer Global Research and Development Pfizer Ltd. Sandwich, Kent, CT13 9NJ United Kingdom Thomas Langer Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany

Bissan Al-Lazikani lnpharmatica Ltd. 60 Charlotte Street London, W1T 2NU United Kingdom Ramona Moldovan Division o f Biocomputing University o f New Mexico School o f Med, M S C l l 6445 Albuquerque, N M 87131 USA JohnT. Moore Discovery Research GlaxoSmithKline Discovery Research Research Triangle Park, NC 27709 USA Maria Mracec Division o f Biocomputing University o f New Mexico School o f Med, M S C l l 6445 Albuquerque, N M 87131 USA Tom W. Muir The Rockefeller University 1230 York Avenue New York, NY 10021 USA Hans Peter Nestler Sanofi aventis Combinatorial Technologies Center 1580 East Hanley Blvd. Tucson, AZ 85737 USA Ulf Norinder AstraZeneca R&D Discovery Medicinal Chemistry 15185 Sodertalje Sweden

xxii

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Marius Olah Division o f Biocomputing University o f New Mexico School o f Med, M SC l l 6445 Albuquerque, N M 87131 USA Tudor 1. Oprea Division o f Biocomputing University o f New Mexico School o f Med, MS C l l 6445 Albuquerque, N M 87131 USA Liliana Ostopovici Division o f Biocomputing University o f New Mexico School o f Med, M SC l l 6445 Albuquerque, N M 87131 USA John Overington lnpharmatica Ltd. 60 Charlotte Street London, W1T 2NU United Kingdom Graham Packham School o f Chemistry University o f Southampton Highfield Southampton SO1 7 1BJ United Kingdom Gaia Paolini Pfizer Global Research and Developme nt Pfizer Ltd. Sandwich, Kent, CT13 9NJ United Kingdom Kenneth H. Pearce Gene Exp. and Protein Chem. GIaxoSmith Kline Discovery Research Research Triangle Park, NC 27709 USA

Pamela Peralta-Yahya Department o f Chemistry Columbia University 3000 Broadway, MC 3167 New 'fork, NY10027-6948 USA Barbara Pescatore Center for Biomolecular Magnetic Resonance Institute of Organic Chemistry and Chemical Biology Johann Wolfgang CoetheUniversity Frankfurt Max-von-Laue-Str.7 60439 Frankfurt Germany Matthew R. Pratt Laboratory of Synthetic Protein Chemistry The Rockefeller University New York, NY 10021 USA Ramona Rad Division o f Biocomputing University o f New Mexico School of Med, MS C l l 6445 Albuquerque, N M 87131 USA Dietmar Reichert Degussa AG Exclusive Synthesis & Catalysis Rodenbacher Chausssee 4 63457 Hanau Germany Lars Rogge lmmunoregulation Laboratory Department of Immunology Institute Pasteur 25, rue du Dr. Roux 75724 Paris Cedex 15 France

List ofcontributors

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Cerhard Quinkert lnstitut fur Organische Chemie und Chemische Biology Johann Wolfgang Goethe Universitat Marie-Curie-Str. 11 60439 Frankfurt Germany Krishna Saxena Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany Ulrich Schieborr Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany Nathan A. Schnarr Department o f Chemistry Stanford University 381 North South Mall Stanford, CA 94305 USA Harald Schwalbe Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str. 7 60439 Frankfurt Germany

Stuart L. Schreiber Howard Hughes Medical Institute Department o f Chemistry and Chemical Biology Harvard University Broad Institute o f Harvard and M I T Cambridge, MA 02142 USA Carsten Schultz European Molecular Biology Laboratory Gene Expression Programme Meyerhofstr. 1 691 17 Heidelberg Germany Peter H. Seeberger Laboratory for Organic Chemistry Swiss Federal Institute o f Technology Zurich ETH-Honggerberg HCI F315 Wolfgang- Pa u Ii-Str. 10 8093 Zurich Switzerland Klaus Seuwen Novartis Institutes for BioMedical Research Lichtstrasse 35 4056 Basel Switzerland Kevan M. Shokat Department o f Cellular and Molecular Pharmacology UC San Francisco 600 16th Street, Box 2280 San Francisco, CA 90143-2280 USA hdia Sielaff Ecole Polytechnique Federale de Lausanne (EPFL) Institute o f Chemical Sciences and Engineering 1011 Lausanne Switzerland

xxiv

I

List ofcontributors

Matthew D. Simon Department o f Cellular and Molecular Pharmacology UC San Francisco 600 16th Street, Box 2280 San Francisco, CA 90143-2280 USA Masahiko Sisido Department o f Bioscience and Biotechnology Okayama University 3-1-1 Tsushimanaka Okayama 700-8530 Japan Derek S. Tan Laboratory of Chemistry and Chemical and Chemical Genetic Sloan-Kettering Cancer Center 1275 York Ave. RRL 1317 New York, NY 10021 USA lens Timmer Physics Institute Hermann-Herder-Str. 3 79104 Freiburg Germany Paul A. Townsend School o f Chemistry University o f Southampton Highfield Southampton SO1 7 1BJ United Kingdom Martin Vogtherr Center for Biomolecular Magnetic Resonance Institute o f Organic Chemistry and Chemical Biology Johann Wolfgang GoetheUniversity Frankfurt Max-von-Laue-Str.7 60439 Frankfurt Germany

Herbert Waldmann MPI of Molecular Physiology University of Dortmund Otto-Hahn-Str. 11 44227 Dortmund Germany Holger Wallmeier Aventis Pharma Deutschland GmbH Research &Technologies lndustriepark Hochst, K801 65926 Frankfurt am Main Germany Michael C. Weiger Department o f Chemical and Biomolecular Engineering North Carolina State University Raleigh, NC 27695-7905 USA Daniel B. Werz Laboratory for Organic Chemistry Swiss Federal Institute o f Technology Zurich ETH-Honggerberg HCI F315, Wolfgang-Pauli-Str. 10 8093 Zurich Switzerland Ciinther Wess GSF - Forschungszentrum fur Umwelt und Gesundheit Ingolstadter Landstr. 1 85764 Neuherberg Germany Norbert Windhab Degussa AG CREAVIS Rodenbacher Chausssee 4 63457 Hanau Germany

List ofContributors

Hang YinDepartment o f Chemistry Yale University 225 Prospect St. New Haven, CT 06520-8107 USA

Sirus ZarbakhshEuropean Molecular Biology Laboratory Gene Expression Programme Meyerhofstr. 1 691 17 Heidelberg Germany

I

xxv

PART I Introduction

Chemical Biology. From Small Molecules to System Biology and Drug Design. Edited bv Stuart L. Schreiber. Tamn M. Kauoor. and Gunther Wess Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31150-7

Chemical BiologyEdited by Stuart L. Schreiber, Tarun M. Kupoor,and Gunther Wess CoDvriaht 0 2007 WILEY-VCH Verlaa CmbH & Co KCaA. Weinheim

13

1 Chemistry and Biology - Historical and Philosophical AspectsGerhard Quinkert, Holger Wallmeier,Norbert Windhab,and Dietmar ReichertDedicated to Profs. Helmut Schwarz and Utz-Hellmuth Felcht on the occasion of their respective GOth birthdays.

1.1 Prologue

The reductionistic attitude of philosophers [ 1 has given way to the emergence1 based thinking [2] of biologists. In place of the view that phenomena occurring at a higher level in a complex system [3] with hierarchically structured levels of organization can also be described by rules and in terms of concepts already verified at a lower level, it has come to be accepted that some of these rules or concepts may be altered or even gained in the transition from lower to higher level. This applies even in the case of the structural and functional basic unit of all biological systems: the living cell. The living cell is a protected region in which diverse ensembles of molecules interact with one another in a harmony achieved through self-assembly [4]. The reality of the cell, with its overlapping functional networks [S] (for regulation of metabolism, signal transduction, or gene expression, for example) can serve as a model. The question of the hierarchical organization of such networks arises. Top-down analysis proceeds in the direction of decreasing complexity of the biological systems, a cell, a tissue, or even an organism, step by step all the way down to the level of molecules underlying their intra- and intermolecular interactions. From chemistrys molecules and supermolecules bottom-up synthesis starts in the direction of increasing complexity to reach the totality of the cell and its higher organizations emerging through modular motifs and supramodular functional units [6]. Bottom-upsynthesis and top-down analysis are signposts for changes in complexity in emergent systems, lending themselves not only to narrative representation of what is, but also to reflective conjecture on why something is as it is. The interdisciplinary union of the worlds of chemistry and of biology has to begin with the different entry points to the two disciplines. In the world of chemistry, for material atoms and its associated interactions within andChemical Biology. From Small Molecules to System Biology and Drug Design. Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Gunther Wess Copyright 0 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-31150-7

4

I between moleculesthe crucial aid is the open sesame represented by the periodic1 Chemistry and Biology - Historical and Philosophical Aspects

system of the chemical elements. In the world of biology, the fundamental information flow and the associated ascent from the biochemical network of metabolism to the biological network of genetic information transfer can be deciphered by the Rosetta Stone that is the genetic code. Fundamental to this is the understanding that in biology - as in cosmology'), but wholly different in chemistry (and physics) - earlier historical events influence future developments. It is a characteristic of historical events that they may have been played out completely differently under other circumstances. In such cases, it is reasonable to ask why questions. Why did Darwinian evolution eventually come to entrust its further fate to the chemistries of two polymer types, nucleic acids and proteins, and their later collaboration in a ribosome? Why did the dice fall in favor of a genetic code with triplet character? Why did protein genesis satisfy itself with the 20 canonical amino acids? For a transdisciplinary perspective it is worth addressing such cases in which the emergence of chemistry (or, more precisely, biochemistry) into biology (or, more precisely, molecular biology) signifies a tipping point. This came about with the appearance of macromolecules possessing the aptitude to store and distribute information and to translate it into catalytic function [gal. It became manifest as awareness grew of the double-faceted nature of protein synthesis: as an enzymatic chain of chemical reaction steps in biochemical space and as a genetic information transfer process in molecular biological space 191. This essay deals with the structures and functions of material things produced by chemical or biological means. While the products obtained in both routes are comparable, if not identical, the production facilities differ substantially.As facilities of human design, they happen to be formed by machines in the laboratory or in the factory;as facilities of Darwinian evolution, they start to exist in generative supermolecules of the living world. Having distinguished the generation of natural products by supramolecular facilities built up by self-assemblyof complementary molecules from the production of materials in man-made facilities, it seems appropriate to add a brief excursion into semantics.1.2 Semantics1.2.1 Synthesis - Genesis - Preparation

By a chemical reaction, whether it takes place in a laboratory, in a factory, or in a living cell, an educt is converted into a product. If the product is structurally1) The developments of stars and galaxies offer

no analog to Darwinian evolution by natural selection, of course [7].

1.2 Semantics

more complex than the related educt, the conversion is called a construction (in biochemistry: an anabolic pathway). In contrast, the conversion is called a degradation (in biochemistry: a catabolic pathway), if the product is less complex than the related educt. According to another classification, one may distinguish between synthesis, genesis, and preparation. While execution follows a subtle plan in the first and instructions of a naturally selected program in the second case, tinkering takes place in the last instance. That such a differentiation may prove useful to the keen mind of a synthetic chemist is demonstrated by the example of the natural dye, indigo. While its first offspring is often popularly held to be urea, synthetic chemistry actually began in the last quarter of the nineteenth century, with the production of artificial indigo [lo]. This dissent can be resolved if consensus is reached on what should be understood by the term synthesis in organic chemistry [ll]. it is taken to mean an attempt to construct a previously decided upon If target molecule with a known structure from a suitable starting molecule (or molecules) according to some plan [12],the choice has to be for indigo. Urea, in contrast, was discovered by chance as an isomerization product of ammonium cyanate by Wohler [13]in 1828, and was not in any way prepared intentionally [14].This qualification, however, does not mean that the urea synthesis can be discounted as inconsequential. On the contrary, Friedrich Wohlers production of artificial urea from hydrogen cyanate and ammonia in 1828 was a key discovery for the dawning chemical sciences, and researchers at the everadvancing frontiers of the science have to this day venerated the narrative connection between Wohlers urea synthesis and their own new findings and future perspectives. What historians like to unmask as a benign legend [14] serves scientists as a rhetorical shorthand and metaphorical paraphrase. In the industrially used Heurnann-Pfleger synthesis, N-phenylglycine 1, readily accessible from aniline, is transformed through indoxyl2 into indigo 3 in a targeted fashion (Scheme 1-1). This process represents the culmination of a development first set in motion in the laboratories of the Munchen University under Adolf Baeyer. Baeyer had begun his efforts to prepare indigo in the laboratory at a time (before 1883) when the constitution of indigo was not even known [lG],starting his

15

1

2

3

Scheme 1-1 Industrial production o f indigo 3 by the Heurnann-Pfleger synthesis [15]: from 1 via 2 t o 3.

6

I endeavors with degradation products (aniline,anthranilic acid,isatin) obtained7 Chemistry and Biology - Historical and Philosophical Aspects

by the application of one of the usual degradative methods (alkali melt, effect of oxidizing agents) to the naturally occurring dyestuff. These degradation products were treated with an extraordinarily broad range of chemicals in a form of intuitive combinatorial process, to examine whether the resulting products would contain 3. In this way, Baeyer and Emmerling succeeded in transforming isatin 10 into 3 in 1870.The preparation of 10 (from phenylacetic acid4: 1878)was however too elaborate to becomrnerciallyviable (Scheme 1-2). As long as the constitution of a target molecule is unknown, the above definition of a synthesis is inadmissible. The sequence of reactions depicted in Scheme 1-2, however, characterizes a venture that serves for the preparation of indigo. Two other pathways that afforded indigo in the laboratory were also not industrially viable. A. von Baeyer encouraged BASF and Farbwerke Hoechst to undertake a systematic search for an industrial synthesis of artijicial indigo (the constitution of which had meanwhile been established) in competition with one another. This was finally achieved in a strategicallyclear and tactically flexible manner through the already mentioned Heumann-Peger synthesis (Scheme 1-1).It was envisaged that the artificial preparation of dyes from coal tar should become a source of national wealth. Baeyers Miinchen University laboratories and the two representatives of Germanys flowering chemical

r

1

4

5

1H

6

1

7

a0

9

I

Scheme 1-2

Laboratory studies ofthe preparation of indigo 3 by A. (uon) Baeyer and his

colleagues.

1.2 Semantics17

industry had exchanged ideas and experiences in a previously unknown scale and had thus passed the test for a collaboration in partnership. In 1905, Adolf von Baeyer was awarded the Nobel Prize for Chemistry for his contribution to the development of organic chemistry and the chemical industry. It has thus been demonstrated that the example of indigo is suitable for conceptual differentiation between molecule construction according to a plan (synthesis) and one without a plan (preparation). It can also provide an illustration, based on the different character of the synthetic steps involved, of differentiation between chemical and biological synthesis steps within the overall indigo syntheses. Chemical synthesis steps [ 17a] can be understood to include transformations achieved not only through the use of reagents or catalysts prepared by chemists but also those in which enzymes, antibodies, or even dead cells are used. Synthesis steps in which the synthetic capabilities of living cells, either possessing their original genomes or new recornbinant variants, are deployed in a targeted manner, are classified as a part of biological synthesis [17a]. Indigo was synthesized biologically in 1983 (Scheme 1-3) [18]. Biological indigo synthesis made use of an Escherichia coli strain with a recornbinant genome, being capable of converting aromatic hydrocarbons in general into cis-l,2-dihydrodiols and, in particular, indole (obtained from tryptophan 11 with the aid of tryptophanase) into cis-2,3-dihydroxy-2,3dihydroindol13. The recombinant E. coli strain was augmented with the genes expressing naphthalene dioxygenase from Pseudomonas putida. The initially produced oxidation product spontaneously loses water, and the resulting indoxyl 2 is converted by aerial oxidation into 3, which can be taken up into organic solvents.

&NH2

H

cis-2,3-dihydroxy2,3-dihydroindol

/H11 12 13

11

Tryptophanase

-

12

Naphthalenedioxygenase

+

13

1

- H2O

Air oxidation3 2

Scheme 1-3 Formation of indigo 3 in a recombinant strain of E. coli.

8

I

1 Chemistry a n d Biology

Indol-3glycerolphosphate

- --

Historical and Philosophical Aspects 2

12

3

Scheme 1-4 On the formation of indigo 3.

After the discussion on the biological synthesis of indigo with the aid of a recombinant E. coli strain, one question still remaining relates to the programmed genesis of indigo precursors in plants. Plants cultivated for indigo production contain 2, stabilized by glycosylation (e.g., as indican = indoxyl B-D-glucoside or as isatan B = indoxyl 5-ketogluconate) [19]. Indoxyl on its part is produced from indole 3-glycerinephosphate [20] (Scheme 1-4) and that in turn by the chorismate pathway. This essay deals not only with preparation (intuitive) and synthesis (planned) but also with genesis (programmed). Such (genetically and somatically regulated) programs have arisen through Darwinian evolution. A plan for a synthesis is devised by a synthetic chemist as designer and enacted by the synthetic chemist as molecule maker. How is a synthesis planned?

1.2.2 Synthetic Design - Synthetic Execution

Unlike the bottom-up-oriented execution of a synthesis, involving real molecules, the designing of a synthesis is a top-down event using virtual structuresZ).Design begins with the target structure and moves through a greater or lesser number of intermediate structures to the starting structure, with the complexity generally decreasing. The starting structure is worthy of that name, once it can reasonably be said to represent a comfortably accessible starting molecule for the carrying out of the synthesis. E. J . Corey coined some terms for top-down-oriented synthesis design which intended to highlight the fact that retrosynthetic structure analysis and synthetic building up of the molecule are concurrent processes. Whilst bottom-up synthesis takes place with molecules and in synthetic steps through the deployment of suitable synthetic building blocks, from the appropriate starting molecule to the resulting target molecule, top-down retrosynthesis operates with structures and in transformation steps through the identification of appropriate retron structure elements, from the particular target structure to the resulting starting structure. Some of Coreys achievements through his endeavors in the logic ofsynthesis [21] include: the fact that organic synthesis can be taught [22] even where it is not actively practiced;2) Differentiation between abstract structures

and concrete molecules will also pay for itself in other circumstances.

1.2 Semantics

19

the availability of computer-aided synthesis planning [23]as a procedure to generate a population of synthesis plans from which the synthetic chemist can select the best one to use; and his being awarded the 1990 Nobel Prize for Chemistry for development and methodology of organic synthesis. Twenty-five years earlier, R. B. Woodward had been awarded the Chemistry Nobel Prize for his outstanding achievements in the art o organic synthesis. f Woodwardscategorical imperative [12] - Synthesismust always be carried out by plan - rapidly became the sign of the coming generation of natural products synthesis chemists. His qualifying statement in the following sentence can easily go unremarked: The synthetic frontier can be defined only in terms of the degree to which realistic planning is possible. This is probably the reason for Woodwardscomment at the end ofhis essay on the total synthesis of chlorophyll [24a].At the beginning there was detailed synthetic planning. The degree to which our plans proved realizable is very gratifying, but laboratory discoveries and knowledge obtained from observation and experimentation contributed at least as much to the advancement of our studies. We learned and found out much that would previously not have been knowable or at best would have been only approximately imaginable. Elsewhere he sounds the Leitmotif of natural products synthesis [24b]: In our time many organic chemists address themselves explicitly to mechanistic and theoretical problems - and make outstanding contributions in so doing - it should not be forgotten that questions too self-consciouslyasked of Nature may well receive subconsciously determined answers - answers which only with difficulty contain more than was presupposed in the questions. It is important to keep open the avenues for innovation and surprise.

1.2.3 Preparative Chemistry - Synthetic Chemistry

The terms preparative chemistry and synthetic chemistry are often used synonymously. We wish to draw some distinction between them: in preparative chemistry we see a rich fund of knowledge from which the synthetic chemist can draw, gained from work on chemical reactions. The preparative chemist is concerned with broadly aimed investigations geared toward the discovery of chemical reactions and the development and improvement of already known ones. A chemical reaction may qualify as mature [17a] if it is capable of transforming a starting compound of not too restricted substrate specificity in a predictable manner: under easily maintainable reaction conditions; as far as possible with the use of substoichiometric proportions of effective catalysts;

10

I

I Chemistry and Biology - Historical and Philosophical Aspects

without restriction to a particular scale; with high chemical yield; and with high regio- and stereospecificity into an envisaged product. There is now such an extensive available reservoir of preparatively useful reactions of this level of comprehensiveness that for the construction o molecular skeletons it appears expedient to switch to a handful of f trusted reactions in the first instance [25]. In the introduction, modijication, and elimination offinctional groups, the a priori restriction on only a few methods is already becoming more difficult. Organic synthesis presupposes a substantial body of knowledge, usually developed through bottom-up strategies ofthe structures and reactivities oforganic molecules. In education, though, it is important to begin concurrently practicing top-down approaches based on this knowledge and its extension and further enrichment, as early as possible. As example speaks louder than a long discussion of principles: to demonstrate the problem-solving potential of synthetic chemistry, it would be useful to identify a molecule that has served for a long time, commanding undiminished interest both in the past and in the present, as a sought-after target molecule for a solid synthetic pathway. One such molecule is estrone. If a particular target structure has been decided upon, it is appropriate to select a particular synthetic pathway from the multitude ofvirtual ones identifiable by combinatorial analysis (Scheme 1-5).In the process, it usually remains open whether the whole set of alternative synthetic pathways for the particular decision is evaluated or intuitively only a part of it is considered.

1.3 Bringing Chemical Solutions to Chemical Problems

1.3.1 The Present Situation

At the beginning of the twenty-first century chemistry finds itself in the middle of a phase of reorientation. In the chemical industry there is a clear trend toward specialization and concentration. It cannot be ignored that traditional organizational structures can be altered appreciably by investment and disinvestment decisions, the maxim being away from the broadly diversified chemical concern of yesterday toward the megacorporation of tomorrow, with its focus on a few core competences. Measures adopted in established organizations are disposition of particular branches, horizontal fusion of adjoining core activities, and vertical integration of new high-tech ventures. In the chemical sciences, progressive integration with chemical biology and also with nanotechnology is underway. Self-organization of molecules and modules into supramolecular and supramodular functional units plays a prominent role in both fields of development, as is clear from research and

1.3 Bringing Chemical Solutions to Chemical Problems

I

-A

AC

ABD

7 ABCD

AB

BC

N A Y D 1BDCD A A B C D t C

\?AAD+B

6 further planning variants

4 further planning variants

4DVirtual synthetic pathways toward the steroid skeleton with rings A, 6, C, and D. Top row: stepwise conversion of a ring A (B,C, or D)-building block into the ABCD system; middle row: expansion in a

Scheme 1-5

single step of an AB (AC, AD, BC, BD, or CD)-building block into the ABCD system; bottom row: expansion in a single step of an A (B,C, or D)-building block into the ABCD system.

teaching in the top academic institutions. That this has been possible is due to the development of physical methods without the aid of which it would be impossible even to establish the existence or presence of systems with particular properties. The core competence of chemistry, though, remains the provision of new molecules through synthesis, a mission equally valid for synthetic chemists in both industrial and academic environments. Both can point to great successes in the past. Nonetheless, synthesis finds itself in a dilemma. Academic synthetic chemists tended to give the highest priority to the elegance of the design of a synthesis, and this veneration was passed on to their students. For industrys molecular engineers, the expediency with which the synthesis could be carried out held center stage: a concept which new graduates did not have to come to terms with until their entry into their industrial careers. Meanwhile, the constructive tension between elegance and efficiency was usurped by the dream of the perfect reaction and the ideal synthesis. The perfect reaction can be summarized in Derek Burtons utopian view: 100%yield, 100%stereoselectivity [25a]. B. M. Trost [25b]seeks to advance toward the ideal through observance of atom-economy, and M. Beller [25c]

12

I through transformation of multiple-component educts into single-component7 Chemistry and Biology - Historical and Philosophical Aspects

products. The ideal synthesis conforms to the prescription of K. B. Sharpless [26]: rather than being concerned with the innumerable synthetic methods in the textbooks one should assemble a handful of perfect reactions that may be used again and again by synthetic chemists in the many-step construction of a molecular framework. A solution to this dilemma lies in a radical new orientation, as the synthetic chemist begins to take on a role in chemistry similar to those long played by the medical doctor in biology or the engineer in physics [27]. In this way, the synthetic chemist provides assistance to the fundamental scientist as a practicing technologist for mutual benefit and being capable of demonstrating that, and in what way, fundamental chemical knowledge may be applied in a targeted fashion to problem solving in synthesis. There is still the matter of future target molecules for the synthetic chemist. The times are gone when it was sufficient to synthesize a target molecule just because it had not yet been synthesized in another laboratory. The accent of interest in chemistry has shifted. There are two reasons for this: one is that the structure space of supramolecular chemistry, unlike that of molecular chemistry, is in many regions only thinly populated and awaits selective filling. The attention of chemists has therefore moved from molecular structure to molecular function [28]. Molecules that combine themselves into supramolecular functional units attract particular attention from synthetic chemists. A. Eschenrnosers vision [29] of creating synthetically accessible supramolecular systems that will spontaneously assemble and may even be capable of reproducing themselves, thus representing the first artificial models of living systems, is heading in this direction, although far into the future.1.3.2 Historical Periods of Chemical Synthesis

From a distance, scientific and technological advancements look like a continuous stream, contributed to by many activists. On closer inspection, though, discontinuities due to outstanding contributions by individuals are unmistakable. If the development of chemical synthesis is reviewed, it is possible informally to identify three phases, following on from one another in the sense that a later phase is characterized by a greater degree of selectivity than the earlier, with which it partially overlaps. It is easy to make out prominent protagonists for each of the three phases. The example of the female sex hormone estrone serves well to demonstrate how the synthetic chemist has succeeded in meeting growing demands for selectivity.

1.3.2.1

The pre-Woodwardian Era

The first phase of chemical synthesis, ending at about the beginning of the Second World War, might be termed the pre-Woodwardian era.

1.3 Bringing Chemical Solutions t o Chemical Problems

The pre- Woodwardian era largely concerned itself with the collection and classification of synthetic tools: chemical reactions suited to broad application to the constitutional construction of molecular skeletons (including Kilianis chain-extension of aldoses, reactions of the aldol type, and cycloadditions of the Diels-Alder type). The pre- Woodwardian era is dominated by two synthetic chemists: Emil Fischer and Robert Robinson. Emil Fischer was emphasizing the importance of synthetic chemistry in biology as early as 1907 [30]. He was probably the first to make productive use of the three-dimensional structures of organic molecules, in the interpretation of isomerism phenomena in carbohydrates with the aid of the Vant Ho$ and Le Be1 tetrahedron model (cf. family tree of aldoses in Scheme I-G),and in the explanation of the action of an enzyme on a substrate, which assumes that the complementarily fitting surfaces of the mutually dependent partners are noncovalently bound for a little while to one another (shape complementarity) [31]. Robert Robinson looked for suitable reactions with the aid of which constitutional modifications in a pathway to, for example, a steroid synthesis might be achieved. He was probably the first to employ mechanistic

! c 7 csGlyceraldehyde

0 C1

c2

Eryihrose

CH20H

$HCH,OHAllose

/

Ribose

\

OH CHzOHAltrose

Scheme 1-6 The family tree o f aldoses derived f r o m

(+)-glyceraldehyde. The Fischer projections of the corresponding aldaric acids are, variously, chiral and asymmetrical (C,), chiral and symmetrical (C?), o r achiral and symmetrical (G).

gl:$4CH20H

CH20H

/

Arabinose

\

/

Xylose

\

/

LYXOSQ

\

OH

HO

OH

H $

CH>OH OH CH,OHIdose Galactose Talose

CH20H

CH>OH

CH70H

Glucose

Mannose

Gulose

14

I considerations in the process. There is a tendency toward charge balancing7 Chemistry and Biology - Historical and Philosophical Aspects

between anionoid and cationoid atom groups [32] through space and through the bonds lying between them (charge complementarity). Robinson used a transparent accounting system (curly arrows) to illustrate the direction of charge displacement (Scheme 1-7). Case Study Estrone: Elisabeth Danes attempts to produce estrone 24 (Scheme 1-8)synthetically [33], beginning with a Diels-Alder reaction that might formally give rise to two regioisomeric adduct components, ended in disappointment: whilst no adduct at all was obtained from an attempted reaction between the Dane diene 1 4 and the monoketonic dienophile 15a, the reaction between 14 and the biketonic dienophile 19a resulted in a mixture of rac-20a and rac-2la, in which rac-20a, with the steroidal molecular skeleton, was present only as a minor component. It is thus no surprise that the Dane strategy was consigned to the files, at the end of the 1930s.

1.3.2.2

The Woodwardian Era

In the second phase of organic synthesis, which could reasonably be termed the Woodwardian era, beginning in 1937, chemical reactions characterized by diastereoselection in the construction of a molecular skeleton found favor. Here as well, two synthetic chemists tower over all their contemporaries: one, naturally, is R. 3. Woodward, who advanced the intellectualization of organic synthesis like no one else. Woodwards seminars set a new standard for natural products chemistry4).The other is Albert Eschenrn~ser~), sole the

P O

Me

,-

Me

Scheme 1-7 Analysis ofthe relative orientation o f Danes diene 14 and the complementary dienophile following Robinsons way.3) Woodward graduated as a Doctor of Philosophy in 1937, after submission of his dissertation at M I T (Cambridge, Mass.) (341.4) I have no doubt that they ( Woodwards seminars

at ETH Zurich)played a major role in stimulating my ownpredilectioizforand enthrallment with the synthesis of complex natural products; A. E.: in 1351.

5) See the concise Preface in [36a].

1.3 Bringing Chemical Solutions to Chemical Problems

I

15

14

15a: R =M e 15b: R = Et

16a: R =M e 16b: R = Et

17a: R = Me 17b: R = Et

18a: R = Me 18b: R = Et

19a: R = Me 19b: R = Et

20a: R = Me 20b: R = Et

21a: R =M e 21b:RZEt

22a: R = Me 22b: R = Et

23

24

Scheme 1-8 Collections o f formulae relevant to Danes concept o f a steroid synthesis following the AB D + ABCD aufbau principle.

+

recipient of the privilege of a collaborative competition with Woodwurd [35]. To master the demands of stereoselection it is necessary to know the mechanism of the reaction used and its stereostructural consequences. In particular, knowledge of a mechanism demands the capability to gauge the diastereomorphic transition states of rival parallel reactions (see Scheme 36 in [37]).A necessary prerequisite for the acceptance of proposed ideas is that they should be able to predict the sense of chirality of the main product components, accurately. Case Study (f)-Estrone (ruc-24): In 1991, [33c] the presumed dead Dane strategy was resurrected by the use of Lewis acids as mediators. Compound 1 4 does in fact react with 15a between 0 C and room temperature in CH2Cl2 - to provide a mixture of (mainly) ruc-16a and (as a minor product) ruc-17a - as soon as Et2AlCl is added [33d]. In the presence of TiC14 in CHzCl2 at -80 C an 89% yield of ruc-18a is obtained.

1.3.2.3

The post-Woodwordian Era

Characteristic of the third phase of organic synthesis, which would logically be termed the post- Woodwurdian era, is that the constitutional construction of a molecular framework is now concerned not only with the problem of diastereoselection but also with the more demanding problem of

16

I enantioselection [37]. Certain chemical reactions serving as key stages inI Chemistry and Biology - Historical and Phi/osophical Aspects

multistep syntheses have been developed to perfection through the preparation of tailor-made catalysts by Barry Sharpless6) (38a],R. NoyoVi [39]and E. J. Corey [40],setting the standard for the further development of organic synthesis. Case Study: (+)-Estrone 24. The Dane-style estrone synthesis provides a classic example of stereoselective access to an envisaged target molecule. The Diels-Alder reactions between 14 and 15a or 19a are chirogenic reaction steps or, put another way, the enantioselective access to the Diels-Alder adducts can already be set at this stage. This requires, for example, the participation of a nonracemic Lewis acid with the right sense of chirality. In the presence of a Ti-TADDOLate [42], cycloadduct 20a was thus obtained from the Dane diene 14 and the bidentate dienophile 19a and was further transformed via 23 into (+)-estrone 24*1 [33d]. Before leaving estrone, a synthetic model for oral contraceptives, as synthetic biologicals (vide infia), it should be pointed out that each historical period of chemical synthesis can be correlated with a characteristic synthetic level amenable to conscious perception [37]. The resurrection [33c] of the Dane strategy for estrone prompted synthetic chemists working on the design of metal-free, chirality-transferring catalysts to use the chirogenic opening step as a selection assay. In this context, acceleration of adduct formation and changes in the ratios of the resulting regioisomers are encouraging signs that enantioselection, which may be finished off here by recrystallization if necessary, may be anticipated [33d]. M. W. Gobel and coworkers [43] and E. J. Corey and coworkers [44]have reported on the application of amidinium catalysts and oxazaborolidinium catalysts, respectively,for the enantioselective treatment of the Dane diene 14 with 19a or with acyclic dienophile~~).

1.3.3 Diels-Alder Reaction - Prototype of a Synthetically Useful Reaction

The Diels-Alder reaction occupies a cherished place in the hearts of organic synthetic chemists, not only in the synthesis of steroids [45]but far and wide in the synthesis of structurally complex natural products [46].The Diels-Alder6 ) Thebottomline in Scheme 1-6shows the eight aldohexoses ofnatural origin; they all belong to the D-series. Their L-configured enantiomers have been synthesized by use of the abiotic Sharpless catalyst (38bj.7) See [41] for the meaning of the term chi-

8) The (S,S)-configurated Ti-TADDOLate [42] complex with four phenanthren-9-yl residues is used at -80C in CH2C12: 65% chemical yield, 93% ee or 78% chemical yield, and 85% ee (2 or 0.2 equiv, respectively). 9) With cyclic dienophiles, rings C and D in the cycloadduct are joined in cis fashion. With acyclic dienophiles containing E-configured C=C bonds, an adduct in which the atom groups necessary for construction ofthe D ring are oriented, trans is produced; see Chapter 3 in [33d].

rogenic reaction step and the usefulness of its application.

1.3 Bringing Chemical Solutions to Chemical Problems

reaction comes closest to meeting the stipulations of K. B. Sharpless [26] and B. M. Trost [25b] set out in Section 1.3.1. It only remains to comment that, besides diverse instances of intermolecular examples, the intramolecular version1o'of a Diels-Alder reaction was not left neglected in the synthesis of estrone and its derivatives. Scheme 1-9 summarizes the construction of a steroid framework by the A D + AD + [AD]* -+ ABCD aufiau principle"'. [AD]* 25a is a photoenol generated i n situ, and reacts under meticulously determined conditions [48] by cycloaddition and subsequent dehydration to provide the estrone derivatives 2Ga and 27a. The mixture of regioisomeric styryl derivatives can be reduced to give 24 after temporary protection of the 17-keto group. The photoenol 25a is produced by regioselective electronic excitation of the Michael adduct 28a with light having wavelengths of >340nm. The Michael adduct is accessible by treatment of the chiral enolate anion 30a with the achiral acceptor 29 [49]. The strength (the trans fusion of rings C and D is directly accessible) and weakness (there is still no solution to the problem of substitution of the multistep procedure that delivers diastereoselection for a shorter route proceeding in tandem with enantioselection) of the photochemical synthesis of 24 have already been commented upon [36b].

+

I&[Me0

\

Me0

25

&& C.r:"\Me0

\

26

27

Me0

a:R=Me b: R = Et

20

29

30

Scheme 1-9 Collection offormulae relevant to a steroid synthesis following an A D + AD + [AD]* + ABCD aufbau principle.

+

10) For further examples see the section "Intramolecular DielT-Alder Reactions" in Ref. [47].

11) Optimization of the reaction conditions was carried out in the racemic series 1481. See 1491

for the synthesis ofthe enantiomerically pure target compounds.

18

I

I Chemistry and Biology - Historical and Philosophical Aspects

1.4 Bringing Chemical Solutions to Biological Problems 1.4.1 The Role o f Evolutionary Thinking in Shaping Biology

Biology is such a hugely diversified field that a historical guide hardly helps as an aid to orientation. Given this, it might then be reasonable to consciously pick out some particular partial aspect, as Theodosius Dobzhansky did in his famous statement Nothing in Biology makes Sense except in the Light of Evolution. With evolutionary biology as a compass, it is not hard to discern three historical periods.The pre-Darwinian Era

1.4.1.1

One prominent event in the pre-Darwinian era is the Cuvier-Geofioy debate (concerning the primacy of anatomical structure over anatomical function or vice versa) before the Academie des Sceances in Paris in the spring of 18301*). Its immediate focus involved opposed viewpoints in comparative anatomy, while indirectly it represented endeavors to turn the static Chain of Being into an ever-moving escalator [51 . Cuvier represented the functionalist approach of 1 the designer: Formfollows Function. Geofioy Saint-Hilaire expanded the theme and took the structuralist standpoint of the evolutionist: Functionfollows Form. The public argument was unable to settle the difference between the two adversaries, though it became clear that fundamental scientific discussions would in future no longer take place in a neutral en~ironment~). was It also evident that evolutionary thinking in biology could no longer be kept in its cage.The Darwinian Era

1.4.1.2

In the narrow sense, the Darwinian era began with the publication of The Origin o Species in 1859 and ended at the beginning of the twentieth century f with the rediscovery of Gregor Mendels 1866 Versuche iiber Pflanzen-Hybriden (Experiments in Plant Hybridization). Charles Darwins book The Origin of Species by Means of Natural Selection could be read as one long argument. It supported the claims of science to understand the world in its own terms. Animals and plants are not the product of special design or special creation. Natural selection was not self-evident in nature, nor was it the kind of theory in which one could say, Look here and see. Darwin had no crucial experiment that conclusively demonstrated evolution in action. His whole concept of natural selection rested on analogy, an analogy between selective processes taking place under either artijcial or natural conditions [53]. A series of12) See [SO] for the Cuuier-Geofioydebate before 13) See [52]: Discussions between Goethe and

and beyond the Academie.

Eckerrnann of the 2nd August 1830.

1.4 Bringing Chemical Solutions to Biological Problems

questions was left open; that of whether in the union of two gametes into a zygote a mixture of the genes involved took place (blending inheritance), occupied a key position. It could only be answered after: Gregor Mendel [54]had set out statistical rules for the passing on of particular hereditary characteristics from generation to generation, which are useful for discussion on the complex relationships in questions of heredity, and Wilhelm]ohannsen [55] had coined the terms phenotype and genotype, which made it possible to distinguish between a statistically apparent type (the phenotype) of observable properties and the corresponding genetic make-up (the genotype) of an organism. The distinction between genotype and phenotype facilitated the separation between genetics and embryology. It is clear from this separation that the differentiation between genetic and environmental causes in embryology and the wider discipline of developmental biology is something to talk about.

1.4.1.3

The post-Darwinian Era

The post-Darwinianera saw the vision of Darwinian evolution through natural selection being accepted as a reality. Since then, evolution has been observed in action in many living organisms and also in innumerable viruses [56, 571. Through Manfied Eigens paper on the role of Self-organization of Matter and the Evolution of Biological Macromolecules [58] Darwins ideas have been placed on firm physical foundations and have been tested by in vitro evolution experiments [59]. The Darwinian view of evolution has prompted biologists to think in terms of dynamic populations while considering a species [60].To avoid misunderstandings among nonbiologists, Eigen introduced the term quasispecies. Because of mutability, self-replicating systems are always ensembles of mutants and are not, in any circumstances, single species made up of uniform individuals. To indicate quantitative proportional relationships between quasispecies and their mutants, Eigens evolutionary model uses a multidimensional representation (sequence space). In a nucleic acid space [61] (protein space [62]14)), each nucleic acid (protein) sequence is represented in the sequence space by a point and each change in the sequence by a vector. If the points in a sequence space are assigned specific scalar fitness values, a fitness landscape is obtained. The metaphor of a fitness landscape (adaptive landscape) was introduced into evolutionary biology in 1932 by Sewall Wright [64] and was afterwards used abundantly, if with a certain breadth of interpretation, by theoretical biologist^^^). The picture conveyed14) See [63]:Footnote 10. 15) R. A. Fisher, /. B. S. Haldane, and S. Wright

count as mathematical biologists; their publications were understood only by some of

their professional colleagues. T. Dobzhansky, G . G . Simpson, and E. Mayr successfully interpreted the mathematically formulated theorems [65].

20

I by the metaphor is that of an evolving population subject to exclusion of7 Chemistry and Biology-

Historical and Philosophical Aspects

unfit mutants making uphill progress until a local peak is reached. For the evolutionary process in the high-dimensional sequence space, local peaks in the vicinity may readily be reached by small jumps, without the need to traverse the valleys between them, and a continuous sequence of small jumps to reach a global summit is a realistic prospect. To use Eigens own words: Because of frequent criss-crossing of paths in multidimensional sequence space, by virtue of its inherent non-linear mechanism which gives the appearance of goal-directednessthe process of evolution is steered in the direction of optimal value peak [8b]. In brief, biological evolution uses two processes: genetic mutation (as a means of generating random diversity) and natural selection (as a means to optimize the peak-jumping technique) in the environmentally shaped fitness landscape. Through the removal of subdisciplinary barriers, biologys evolutionary thinking has contributed on two occasions to enhance that sciences voice in the choir of the natural sciences. In the 1940s and 1950s, a union of Darwinian and Mendelian perspectives took place in Modern Synthesis [65], whilst at the turn of the twentieth to the twenty-first century a union of developmental and evolutionary biology into evolutionary developmental biology (Evo-Devo) is taking place before our eyes in the New Synthesis [66].

1.4.2 O n the Sequence of Chemical Synthesis (Preparation) and Biological Analysis (Screening)

In an ideal starting situation for the synthetic chemist the structure of the target molecule is already given. In the real world of the search for active substances, the matter of whether a target molecule is to be synthesized is determined by its presumed profile of properties. If a management decision is made in favor of a target molecule to be synthesized, the synthetic chemist then looks for a way to relate molecular function back to molecular structure. This is based on the supposition that a functional unit should contain at least two structurally complementary molecules non-covalently bound to one another in a supermolecule. The idea of supermolecules as supramolecular functional units, nowadays preached and systematically further developed most conspicuously by Jean-Marie Lehn [67], goes back directly to Emil Fischer [31], who introduced the instructive lock-and-key metaphor as early as 1894. Fischers metaphor, as the tip of the submerged model of molecular recognition, traces the function of a supermolecule back to structural interactions between its complementary constituents. Through this, the complementarity between substrate and enzyme was to become the basis of enzymology. Paul Ehrlich seized on the lock-and-key metaphor in his 1908 Nobel lecture [68], and the goal of chemotherapeutic endeavor thereafter came to be regarded as the activation or deactivation of a receptor through noncovalent binding of a

7.4 Bringing Chemical Solutions to Biological Problems

complementary effective substance. Structural complementarity of effector and receptor accordingly represents the fundamentals of chemotherapy, similar to the way in which complementarity of antigen and antibody is regarded as central to immunology. The goal of synthesizing a target molecule with particular properties can be achieved with the aid of two problem-solving processes based on different principles. In one problem-solving process, illustrated by the image of the key and its lock, the maxim is to m o d i h a designed target structure little by little until the corresponding target molecule has the very properties of interest. It involves an iterative procedure, usually of several rounds, based on trial and error. It is trivial to note that the screening can take place only after the synthesis. In the other problem-solvingprocess, which can be illustrated by the image ofan assortment of keys, hopefully containing the key that will be complementary to a given lock, the maxim is to develop a parallel structured search method, with the aid of which the matching key will befound, without it being necessary to subject the whole ensemble o candidates to the totality of&nctional tests. This is f a procedure based on the principle of trial and selection. Since a distinction has been drawn between synthesis and preparation (Section 1.2.1),some spin doctoring should come as no surprise. After preparation is performed on a microscale, screening will follow before the synthesis on a macroscale. For the time being, we should come back to the traditional search for a biological, with a very particular function.

I

1.4.2.1

Single-componentConsecutive Procedure

In traditional single-component consecutive procedures, the synthetic chemist each time focuses on a structure (a molecule) from a series of successive candidates. The example of the total synthesis of estrone in Sections 1.3.2 and 1.3.3 demonstrates the adaptation of synthetic goals to the state of the art in organic synthetics. The case studies described there have academic value that should not be underestimated, though for industrial synthetic practices they are not directly relevant because estrone will in general be commercially more advantageously accessible through partial synthesis than through total synthesis. In the search for an ovulation inhibitor outlined below, however, total synthesis plays a commercially acceptable role, since partial synthesis drops out as a serious contender from the second generation of inhibitors to be discovered in future.1.4.2.1.1 Oral Contraceptives

Thanks to initiatives instigated by Margaret Sanger, probably the highestprofile campaigner worldwide for family planning, a project geared toward the development of an orally administrable contraceptive was initiated in the

22

I early 1950s under the reproductive biologist Gregory G. Pincus at the WorcesterI Chemistry and Biology-

Historical and Philosophical Aspects

Foundation for Experimental Biological Research [69a]. It was known that progesterone established and maintained pregnancy as an endogenous gestagen and so was able to act as a contraceptive. As progesterone was not suited for oral application, a systematic search for the steroidal structure space was carried out for an exogenous gestagen [69b] that - orally administered - would bind to the progesterone receptor, hereby initiating a series of molecular events culminating in the induction or repression of a certain set of target genes. Binding of a gestagen to the progesterone receptor is necessary but not sufficient for the formers playing an active role as an agonist in reproductive biology. This became clear as soon as an antigestagen like R LJ 486 [70] was found, which bound to the progesterone receptor, but - unlike an agonist - was unable to trigger the gestagenic response. As it turned out, there is no known parameter of effector binding that can predict differential agonistic or antagonistic activity of a steroid. If a metaphorical statement can ever reveal how things are, Emil Fischers static lock-and-keymetaphor [31a]ought to be replaced with a dynamic one. This was done by D. E. Koshlands induced-jit concept [31b],which readily produced the self-explanatory hand-and-glove metaphor. Binding of a given effector will bring about a conformational change of the receptor that is favorable for catalytic activity of the formed supermolecule. G. G . Pincus and M . C. Chang investigated a diverse range of variants of about 200 steroids [69b], which were in most cases not naturally occurring compounds but products that had accrued in countless laboratories as a result of arduous individual studies on their biological functions. They found that combinations of a gestagenic and an estrogenic 19-nor-steroid exhibited the desired effects. These findings from animal experiments (rabbit and rat) were also confirmed in humans, in almost militarily planned (Pincus) clinical studies (by the gynaecologists I. Rock and C. R. Garcia). In the early 19GOs, a combination pill made up of norethindrone (prepared by C. Djerassi at Syntex in 1951 [71]) and 17w-ethynylestradiol (prepared by H . H . Inhofen at Schering AG in 1938 [72]) reached the market as the firstgeneration pill.Members of the First Generation

Norethindrone 31a, the gestagenic component in the combination pill, is smoothly accessible from estrone-methylether by partial synthesis [71]. The reaction sequence begins with a dearomatization (Birch reduction) and ends with an ethynylation (Scheme 1-10), necessary for the oral applicability. Technical production of estrone 24 (or estradiol) from inexpensive steroids such as diosgenin or cholesterol by partial synthesis is also feasible. Pyrolytic aromatization (Inhofen at Schering A G ) assists the transition from the steroid to the 19-nor-steroid class (such as from androsta-1,4-dien-17~-01-3-one 32 to estradiol33 [72]).

1.4 Bringing Chemical Solutions to Biological Problems123

HO

&3,a: R = M e b: R = EtMe0

32

33

;fi35a: R = Me b: R = EtMe0

\34

Me0

37

38

Scheme 1-10 Collection o f formulae relevant to Trogov's concept o f a steroid synthesis following the AB D + ABD + ABCD aufbau principle.

+

Members of the Second Generation

Here the gestagen (-)-norethindrone 31a has been supplanted by (-)norgestrel 31b. The difference between the two molecular structures, minor in itself, still has far-reaching consequences for biological action and synthetic accessibility. The presence of the ethyl group in place of the methyl group at C( 13) slows down the compound's metabolism, thereby increasing bioavailability and also ordaining that total synthesis now has to take the place of partial synthesis. This begins (Scheme 1-10)with the condensation of (~)-l-vinyl-l-hydroxy-G-methoxy-l,2,3,4-tetrahydronaphthalenewith 2(rac-34) ethylcyclopentane-l,3-dione (35b) [73]. The resulting seco-dione 3Gb, with a meso configuration, can be reduced microbiologically to one of four stereoisomers: the microorganism used (Saccharornycesuvarurn) approaches the surface of the five-membered ring differentially from one of the two diastereotopic half-spaces and selectively attacks only one of the two enantiotopic carbonyl groups [74b]. The reduction product 37b can be stereoselectively converted into (-)-38b (as reported by V. Torgov [74a]) and finally ( H . Smith [75])into (-)-norgestrel 31b.

24

I

I Chemistry and Biology - Historical and Philosophical Aspects

Members of Later Generations

The search for unnatural gestagens with improved properties by the trial and error approach continues. Oral applicability (through ethynylation at C(17)) and at low dosages (thanks to slow metabolism because of the ethyl group at C(13)) have already been achieved. A new, exogenous gestagen therefore has prospects of being favored over already known preparations only if it distinguishes itself in at least one of the three following aspects: through a higher binding specificity to the complementary receptor (i.e., biological); through more economically advantageous accessibility (i.e., chemical);and/or through some advantage arising from patent law (i.e., commercial). What this means in detail should become clear through illustration with later-generation gestagens. Gestoden 39 (Scheme 1-11) has the lowest ovulation inhibitory dose of all gestagens known to date. It displays both antiestrogenic and antimineralcorticoidal activity. A lower affinity to the androgen receptor is not sufficient to produce measurable anabolic androgenic effects. The pathway to 39 passes through compound 47 (Scheme 1-12) [7G] and after microbiological introduction of an 0 function at C(15) (with the aid of Penicilliurn ruistuickii), on through the stations 48 (R = H or Ac) and 49