Chemical Biology
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Chemical Biology
Edited by Stuart L. Schreiber, Tarun M. Kapoor, and Cunther Wess Volume I
Related Titles
Larijani, B., Woscholski, R., Rosser, C. A. (eds.)
Casteiger, I. (ed.)
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Kubinyi, H.,Muller, G . (eds.)
Chemogenomics in Drug Discovery
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A Medicinal Chemistry Perspective
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1807-2007 Knowledge for Generations Each generation has its unique needs and aspirations. When Charles Wiley first opened his small printing shop in lower Manhattan in 1807, it was a generation of boundless potential searching for an identity. And we were there, helping to define a new American literary tradition. Over half a century later, in the midst of the Second Industrial Revolution, it was a generation focused on building the future. Once again, we were there, supplying the critical scientific, technical, and engineering knowledge that helped frame the world. Throughout the 20th Century, and into the new millennium, nations began to reach out beyond their own borders and a new international community was born. Wiley was there, expanding its operations around the world to enable a global exchange of ideas, opinions, and know-how. For 200 years, Wiley has been an integral part of each generation’s journey, enabling the flow of information and understanding necessary to meet their needs and fulfill their aspirations. Today, bold new technologies are changing the way we live and learn. Wiley will be there, providing you the must-have knowledge you need to imagine new worlds, new possibilities, and new opportunities. Generations come and go, but you can always count on Wiley to provide you the knowledge you need, when and where you need it!
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Peter Booth Wiley Chairman of the Board
Chemical Biology From 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
Prof: Dr. Stuart L. Schreiber Howard Hughes Medical Institute Chemistry and Chemical Biology Harvard University Broad Institute o f Harvard and MIT Cambridge, MA 02142 USA
Prof: Dr. Tarun M. Kapoor Laboratory o f Chemistry and Cell Biology Rockefeller University 1230 York Ave. New York, NY 10021 USA
Prof: Dr. Ciinther Wess CSF - Forschungszentrum fur Umwelt und Gesundheit lngolstadter Landstr. 1 85764 Neuherberg Germany
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ISBN 978-3-527-31150-7
Iv
Preface
XV
List of Contributors
XVll
Volume 1 Part I
chemistry and Biology - Historical and Philosophical Aspects
1
Chemistry and Biology - Historical and PhilosophicalAspects
1.1 1.2 1.2.1 1.2.2 1.2.3 1.3 1.3.1 1.3.2 1.3.3
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
1.4 1.4.1 1.4.2 1.5 1.5.1 1.5.2 1.G
1.7 1.7.1
Gerhard Quinkert, Holger Wallmeier,Norbert Windhab,and Dietmar Reichert
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
3
vi
1
Contents
1.7.2
Two Lessons From the Wealth of Published Total Syntheses 55 Acknowledgments 58 References 59
Part II
Using Natural Products to Unravel Biological Mechanisms
2
Using Natural Products to Unravel Biological Mechanisms
2.1
Using Small Molecules to Unravel Biological Mechanisms Michael A. Lampson and Tarun M . Kapoor
2.1.1 2.1.2 2.1.3 2.1.4
2.2
2.2.1 2.2.2 2.2.3 2.2.4 2.2.5 2.2.6
3 3.1
3.1.1 3.1.2 3.1.3 3.1.4 3.1.5
71 71
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
96
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
Contents
3.2
Controlling Protein Function by Caged Compounds 140 Andrea Giordano, Sirus Zarbakhsh, and Carsten Schultz
3.2.1 3.2.2 3.2.3 3.2.4
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
3.2.5 3.2.6
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
4.1.1 4.1.2 4.1.3 4.1.4 4.1.5
4.2
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 191 199 Chemical Complementation: Bringing the Power of Genetics to Chemistry 199 Pamela Peralta-Yahya and Virginia W. Cornish
Controlling Protein-Protein Interactions
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 Outlook
227
1
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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
4.3
Protein Secondary Structure Mimetics as Modulators of Protein-Protein and Protein-Ligand Interactions 250 Hang Yinand Andrew D. Hamilton
4.3.1 4.3.2 4.3.3 4.3.4 4.3.5 4.3.6
5
5.1
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 250 Introduction 250 History and Development 251 General Considerations 253 Applications and Practical Examples Future Developments 264 Conclusion 265 Acknowledgments 2G5 References 265
228
255
271 Synthetic Expansion of the Central Dogma Masahiko Sisido Expanding the Genetic Code
271
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
5.1.7
Application of Nonnatural Mutagenesis - Fluorescence Labeling 289 Future Development and Conclusion 291 Acknowledgments 291 References 291
Part Ill
Engineering Control Over Protein Function Using Chemistry
6
Forward Chemical Genetics
5.1.6
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
299
StephenJ. Haggarty and Stuart L. Schreiber Outlook 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
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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
7.2
Chemical Biology and Enzymology: Protein Phosphorylation as a Casestudy 385 Philip A. Cole
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
8 8.1
8.1.1 8.1.2
366
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
415
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
Contents
8.1.3 8.1.4 8.1.5 8.1.6
8.2
8.2.1 8.2.2 8.2.3 8.2.4
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
Volume 2 Part IV
Controlling Protein- Protein Interactions
483 483
9
Diversity-orientedSynthesis
9.1
Diversity-oriented Synthesis Derek S. Tan
9.2
Combinatorial Biosynthesis of Polyketides and Nonribosomal Peptides 519 Nathan A. Schnarr and Chaitan Khosla
10
Synthesis of Large Biological Molecules
10.1
Expressed Protein Ligation 537 Matthew R. Pratt and Tom W. Muir
10.2
Chemical Synthesis of Proteins and Large Bioconjugates Philip Dawson
10.3
New Methods for Protein Bioconjugation Matthew B. Francis
11
Advances in Sugar Chemistry
11.1
537
567
593
635 The Search for Chemical Probes to Illuminate Carbohydrate Function 635 Laura L. Kiessling and Erin E. Carlson
1
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xii
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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 Ganesan Part V
Expandingthe Genetic Code
13
Chemical Informatics
13.1
Chemical Informatics Paul A. Clemons
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
723 723
Volume 3 Part VI
Forward Chemical Genetics
14
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 Hopkins
15
Target Families
15.1
The Target Family Approach Hans Peter Nestler
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
825
825
Contents
891
15.3
The Nuclear Receptor Superfamily and Drug Discovery John T. Moore, Jon L. Collins, and Kenneth H . Pearce
15.4
The GPCR - 7TM Receptor Target Family 933 Edgar Jacoby, Rochdi Bouhelal, Marc Gerspacher, and Klaus Seuwen
15.5
Drugs Targeting Protein-Protein Interactions Patrick Che'ne
16
Prediction of ADM ET Properties
Part VII
Reverse Chemical Genetics Revisited
17 17.1
1045 Systems Biology of the JAK-STATSignaling Pathway 1045 lens Timmer, Markus Kollrnann, and Ursula Klingmiiller
17.2
Modeling Intracellular Signal Transduction Processes Jason M. Haugh and Michael C. Weiger
18 18.1
Genome and Proteome Studies
18.2
Scanning the Proteome for Targets of Organic Small Molecules Using Bifunctional Receptor Ligands 1118 Nikolai Kley
Part Vlll
Tags and Probes for Chemical Biology
19
Chemical Biology - An Outlook
979
I003 UEfNorinder and Christel A. S. Bergstrom
Computational Methods and Modeling
1 061
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
Giinther Wess Index
1151
1143
I
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I
Preface Small 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 products”used 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 field’s 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 methodology Chemical 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
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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 Contributors Stephen R. Adarns Department o f Pharmacology University o f California, San Diego 310 George Palade Laboratories 0647 La Jolla, CA 92093-0647 USA
Elisabetta Bianchi lmmunoregulation Laboratory Department o f Immunology Institute Pasteur 25, rue du Dr. Roux 75724 Paris Cedex 15 France
Anke Arnold Ecole Polytechnique Federale de Lausanne (EPFL) Institute o f Chemical Sciences and Engineering 1011 Lausanne Switzerland
A h a Bora Division o f Biocomputing University o f New Mexico School o f Med, MSC11 6445 Albuquerque, N M 87131 USA
Christel A. S. Bergstrom AstraZeneca R&D Discovery Medicinal Chemistry 15185 Sodertalje Sweden
Rochdi Bouhelal Novartis Institutes for BioMedical Research Lichtstrasse 35 4056 Basel Switzerland
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
Rolf Breinbauer Institute 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
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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
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
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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 Johnsson Center 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
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
ofcontrjbutors
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
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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
Pamela Peralta-Yahya Department o f Chemistry Columbia University 3000 Broadway, MC 3167 New 'fork, NY10027-6948 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
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
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
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
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
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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 Yin
Sirus Zarbakhsh
Department o f Chemistry Yale University 225 Prospect St. New Haven, CT 06520-8107 USA
European Molecular Biology Laboratory Gene Expression Programme Meyerhofstr. 1 691 17 Heidelberg Germany
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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 Biology Edited 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 Aspects Gerhard Quinkert, Holger Wallmeier,Norbert Windhab,and Dietmar Reichert Dedicated to Profs. Helmut Schwarz and Utz-Hellmuth Felcht on the occasion of their respective GOth birthdays.
1.1 Prologue
The reductionistic attitude of philosophers [ 11has given way to the emergencebased 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 chemistry’s 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 and 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
4
I between moleculesthe crucial aid is the open sesame represented by the periodic 1 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 Semantics 1.2.1 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 structurally 1) 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].If it is taken to mean an attempt to construct a previously decided upon 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 Wohler’s 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 Wohler’s 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
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.
15
6
I endeavors with degradation products (aniline,anthranilic acid,isatin) obtained 7 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-P’eger synthesis (Scheme 1-1).It was envisaged that the artificial preparation of dyes from coal tar should become a source of national wealth. Baeyer’s Miinchen University laboratories and the two representatives of Germany’s flowering chemical
1
r
4
5
7
a
1
6
1
H 9
0
I
Scheme 1-2
colleagues.
Laboratory studies ofthe preparation of indigo 3 by A. (uon) Baeyer and his
1.2 Semantics 17
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
cis-2,3-dihydroxy2,3-dihydroindol
H
/ H 11
12
11
Tryptophanase
-
13
Naphthalenedioxygenase
12
+
13
1
- H2O
Air oxidation 3
-
Scheme 1-3 Formation of indigo 3 in a recombinant strain of E. coli.
2
8
I
1 Chemistry a n d Biology
Indol-3glycerolphosphate
Historical and Philosophical Aspects
- --
12
2
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 Corey’s 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
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 of organic synthesis. Woodward’scategorical 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 Woodward’scomment 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;
19
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 of molecular skeletons it appears expedient to switch to a handful of 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
-A
AB
BC
AC
ABD
I”
7 ABCD
\?AAD
N A Y D1 BD
6 further planning variants
CD
B
A
+
A B C D t C
4 further planning variants
4 D Scheme 1-5
Virtual 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
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 industry’s 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 Burton’s 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-component 7 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. Eschenrnoser’s 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 Kiliani’s 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 Van’t 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 cs c2
0C1
Glyceraldehyde
Eryihrose
/
$
Ribose
/
\
\
/
Xylose
\
/
\
LYXOSQ
HO
OH
Allose
Arabinose
OH
H
CH,OH
gl:$4
CH20H
CH20H
CH20H
CHzOH Altrose
H $
OH
CH20H
Glucose
CH>OH
Mannose
CH>OH OH CH,OH
CH70H
Gulose
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).
Idose
Galactose
Talose
14
I considerations in the process. There is a tendency toward charge balancing 7 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 Dane’s 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. Woodward’s seminars set a new standard for natural products chemistry4).The other is Albert Eschenrn~ser~), the sole
P O
,-
Me
Me
Scheme 1-7 Analysis ofthe relative orientation o f Dane’s diene 14 and the complementary dienophile following Robinson’s 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
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 Dane’s 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
I
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 in I 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-Alder 6 ) 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.
8) The (S,S)-configurated Ti-TADDOLate [42] complex with four phenanthren-9-yl residues is used at -80°C in CH2C12: 65% chemical yield, 93% ee or 78% chemical yield, and 85% ee (2 or 0.2 equiv, respectively).
7) See [41] for the meaning of the term “chi-
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
\
&& C.r:"
Me0
25
\
Me0
\
26
27
a:R=Me b: R = Et
Me0
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.
1.4.1.1
The pre-Darwinian Era
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 Acade‘mie 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” [511. Cuvier represented the functionalist approach of 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’~). It was also evident that evolutionary thinking in biology could no longer be kept in its cage.
1.4.1.2
The Darwinian Era
In the narrow sense, the Darwinian era began with the publication of The Origin of Species in 1859 and ended at the beginning of the twentieth century with the rediscovery of Gregor Mendel’s 1866 Versuche iiber Pflanzen-Hybriden (Experiments in Plant Hybridization). Charles Darwin’s 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 of 12) See [SO] for the Cuuier-Geofioydebate before
and beyond the Academie.
13) See [52]: Discussions between Goethe and
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 Eigen’s paper on the role of “Self-organization of Matter and the Evolution of Biological Macromolecules” [58] Darwin’s 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, Eigen’s 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 conveyed 14) 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 of 7 Chemistry and Biology
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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 Eigen’s 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, biology’s evolutionary thinking has contributed on two occasions to enhance that science’s 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. Fischer’s 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 of candidates to the totality of&nctional tests. This is 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.
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
I
22
I early 1950s under the reproductive biologist Gregory G. Pincus at the Worcester I Chemistry and Biology
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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 former’s 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 Fischer’s static lock-and-keymetaphor [31a]ought to be replaced with a dynamic one. This was done by D. E. Koshland’s 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 Problems 123
HO
& 3,
Me0
32
a: R = M e b: R = Et
33
;fi
\ 35
a: R = Me b: R = Et
34
Me0
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-tetrahydronaphthalene (rac-34)with 2ethylcyclopentane-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 [77]. Compound 31b, incidentally, can be easily obtained starting from 47 [78]. Desogestrel 40 (Scheme 1-11) is a progestagen that is transformed in the intestinal mucosa and in the liver into the actual effective metabolite 3-ketogestrel. The bioavailability is around 75%. Desogestrel, obtained partially synthetically by chemists at Orgunon [79], displays minimal androgenic and estrogenic activity. The long pathway from the 19-norsteroid estr-4-ene-3,17-dione includes a microbiological hydroxylation of
39
40
41
Scheme 1-11 Cestagens of the Pill of later generations: (-)-gestodene 39, (-)-desogestrel40, and (-)-drospirenone 41.
1.4 Bringing Chemical Solutions to Biological Problems 125
.J-:3:1
&&
42
43
0
44
0
/
O A O E t
46
45
48
Scheme 1-12
47
49
Collection offormul ae relevant to syntheses of (-)-norgestrel 31b a nd
(-)-gestodene 39 in both cases via 47.
the steroid skeleton at C(11) and an intramolecular functionalization of C(18).
E. J . Corey et al. [80]reported a total synthesis (Scheme 1-13) beginning with the reduction product 50, easily accessible from 42'"'. Alkylation of the metallated enol derived from 52 with m-methoxyphenylethyl-iodide to afford the tricyclic P-keto ester 53, followed by cationic cyclization of this to furnish the steroid derivative 54, warrants particular attention. Corey and colleagues have recently published another total synthesis of 40 [82], beginning with an enantioselective Diels-Alder reaction between Dane's diene 14 and dienophile 61. An oxazaborolidinium salt (see Section 1.3.2.3)was used as an efficient catalyst (Scheme 1-14). Drospirenone 41 (Scheme 1-11),the latest of the exogenous gestagens, differs from its antecedents in some characteristic ways: 16) The bicyclic, chiral, non-racemic building block 42 represents a milestone in the his-
tory of organic chemistry. It is accessible in high chemical yield and enantiomeric
excess from the achiral triketone precursor through a proline-catalyzed, intramolecular aldol condensation (Hajos-Parrish-EderSauer- Wiechert reaction [76,81]).
26
I
7 Chemistry and Biology - Historical and Philosophical Aspects
54
55
56
59
58
57
60
Scheme 1-13 Collection offormulae relevant t o a synthesis of (-)-desogestrel40 opened by the asymmetric Hajos-Parrish-Eder-Sauer-Wiechert reaction.
61
63
62
26 b
64
38
65
Scheme 1-14 Collection o f formulae relevant t o a synthesis of (-)-desogestrel 40 opened by an asymmetric Diels-Alder reaction o f Dane’s diene 14 and dienophile 61.
I . 4 Bringing Chemical Solutions to Biological Problems
127
constitutionally, in that both angular positions are occupied by methyl groups whilst the tetracyclic steroid skeleton is endowed with three additional rings, and biologically, in that 41 is an unnatural gestagen that both acts as an aldosterone antagonist and at the same time displays pronounced antiestrogenic and antiandrogenic properties. With this combination of activities in one and the same dosage, drospirenone currently holds a leading position in hormonal contraception, although it requires a higher dosage than gestagens with an ethyl group at C( 13). The synthesis ofDrospirenone 41 (Scheme 1-15) [83]starts with the inexpensive androstenolone 66, which can be converted microbiologically (Colletotrichum h i ) into the 7a,lSa-dihydroxy derivative 67. A selective epimerization at C(7) proceeds by way of the acetalG8. Methylenation of the intermediate (C=C) bond appearing between C(15) and C(1G) is successfully accomplished with the aid of dimethylsulfoxonium methylide to provide 71, and that of the (C=C) bond between C(G) and C(7) through a Simmons-Smith reaction. The conversion of 76 into 41 can be carried out in a one-pot procedure, with a Pd-catalyzed hydrogenation being followed by a Ru-catalyzed oxidation and a hydrochloric acid-induced dehydration.
66
67
70
71
74
69
68
73
72
75
Scheme 1-15 Collection o f formulae relevant t o a synthesis of (-)-drospirenone 41 starting from the easily accessible androstenolone 66.
76
28
I
I Chemistry and Biology - Historical and Philosophical Aspects
Pinkus and Chang (Section 1.4.2.1.1),in their search for orally applicable contraceptives, had decided upon norethindrone after some 200 steroidal candidates had been examined one by one. Chemists at Schering AG had stumbled upon drospirenone after some 600 newly prepared molecules with antialdosterone activity had become available [84].It can be justifiably stated that the hardly ineffectual pharmaceutical industry had finished up in a Mind alley in its search for new active substances by using traditional strategies [85]. The rapidly progressing expansion of the world market, where new suppliers have arrived in great numbers (globalization), places serious decisions before the management of every multinational company [86] (see Section 1.3.1). These are not merely restricted to restructuring of portfolios of the products manufactured; they also do not exclude the reorganization of the entire company structure”). Under real pressure from financial analysts and resumptive pressure from shareholders, questions have also been directed toward the scientists involved: whether there might be new methods that could afford more rapid access to new active substances. The answer was not long in coming: with chirotechnologyI8)and the combinatorial acceleration of the preparation and screening of whole populations of molecular candidates, a new turn has been taken in the solution of biological problems through chemical methods.
1.4.2.2
Multicornponent Simultaneous Procedure
Darwinian evolution is kept in motion by a continual succession of newly arising variation and its modification by natural selection. The search for active substances proceeds through multiple-component simultaneous procedures, in which a restricted variant population is prepared on a microscale by a combinatorial strategy, to be subjected to the new form of selection, that is, collective screening. After a successfully applied unnatural selection of a particular variant with the desired properties, synthesis on a macroscale can take place. In Section 1.4.2.2.1 a static variation is going to be prepared and screened for anti-inflammatory 17) The consequences arising from reorganiza-
tion of the structure of a business may be guessed by careful market analysis. Most difficult to predict is the reaction of employees. If the creative people among them are not convinced by the new orientation, or have even been put off by the way in which it has been implemented, they may defect to the competition, thus doubly weakening their previous employer.
18) One of the main challenges of synthetic chemistry in the post-Woodwardian era (see Section 1.3.2.3) is to find routes that satisfy the demands of industrial applicability to enantiomerically pure compounds [37]. In 1992, various international journals (Financial Times, Neue Ziircher Zeitung, Science, and Chemical & Engineering News), as if coordinated by a global editor, touched on the phenomenon of chirality. C&EN even predicted that chirotechnology may progress in the future as biotechnology had grown in the past.
1.4 Bringing Chemical Solutions to Biological Problems
activity of individual variants that might be useful in controlling asthmatic inflammation19’. The worldwide incidence, morbidity, and mortality of allergic asthma are increasing. Asthma has become an epidemic, affecting 155 million individuals throughout the world. It is a complex disorder characterized by local and systemic allergic inflammation, mucus hypersecretion, and reversible airway obstruction [88].The pathogenesis of asthma reflects the activity of cytokines from T Hcells. ~ Without these cells there is no asthma. Animal models support important roles for the cytokines IL-4, IL-5, and the recent IL-13 [89].The latter is closely related to IL-4: they both bind to the same IL-4 receptor, to the a-chain of that receptor, particularly. The molecular biologist is interested in the molecular consequences of allergen binding to the T-cell receptor. Experimental investigations have revealed various signal-transduction pathways that link T-cell surface molecules with nuclear transcription events. A [Ca2+]-dependentroute has been discovered, emanating from the T-cell receptor, which can be blocked by natural products of fungi: cyclosporine A (CsA) and FK 506 (Scheme 1-16). Another signal-transducing pathway, independent of [Ca2+],emanates from the IL-2 receptor and controls translational events on ribosomes. It can be blocked by a third natural product, rapamycin, but not by CsA or FK 506. Two signaling pathways have been targeted for pharmacological treatment of unwanted immune responses. It is essential to realize that blocking signal transduction leading to regulated transcription or regulated translation, requires CsA or FK 506 on the one hand and rapamycin on the other to be more than an inhibitor of a cognate target protein: calcineurin in the former and fascilin related adhesive protein (FRAP) in the latter case. As a matter of fact, the fungi-derived ligands in each case act as a “molecular glue” that mediates the interactions of primary and secondary receptors, forming a ternary receptor-ligand-receptor complex. Calcineurin is blocked by CsA and by FK 506, but only, after the two ligands have been activated by each complex primary receptor, cyclophilin A and FK-506 binding protein 12 (FKBP 12), respectively. In a similar way, rapamycin, on forming a binary complex with the primary receptor FKBP 12, is promoted to block the secondary receptor called FRAP on ternary complex formation (Table 1-1). An antigen bound by the receptor of a T cell sets in motion a long cascade of signal carriers and subsequent proliferation of T cells. In allergic subjects, this signal cascade can be initiated by allergens, which are by themselves actually harmless, leading to undesired T-cell overproduction. For allergy sufferers, therefore, it is desirable to specifically interrupt or slow down transcriptional or translational signal cascades involved in T-cell production. Because FK 506, rapamycin, and CsA are effective immunosuppressants, they cannot be 19) Project of the G e m a n Federal Ministry of’
Education and Research (87a], initiated by A. Kleemann, K. Brune. G . Quinkert; fordetails see (631 and [87b]. Beginning: 1 July 1994.
30
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1 Chemistry and Biology - Historical and Philosophical Aspects
\
FK 506
Rapamycin
-4
CsA Scheme 1-16
Natural immunosuppressants.
Table 1-1 Naturally occurring immunosuppressants (ligands) and their receptor complexes Ligand
Cyclosporine FK 506 Rapamycin
Primary receptor
Secondary receptor
Cyclophilin FKBP FKBP
Calcineurin Calcineurin FRAP
Binary complex Ternary complex
considered suitable for long-term treatment of allergic patients. The search is on for nonnatura120)ligands with a more specific action on the immune system. A collection of non-natural ligands - synthesized independently in various laboratories - has demonstrated an immense chemical production effort in search of specific modulators of the immune system with significantly reduced 20) V. Prelog [90]has underlined the viewthat nat-
ural products hold a worthwhile message. H. Waldrnann et al. [91] entertain the plausible
argument that “natural products are biologically validated starting points in structural space for compound library development”.
1.4 5r;nging Chemical Solutions to Biological Problems
molecular complexity. One can’t help wondering why the traditional method, making one compound at a time, analyzing it, and evaluating it biologically indubitably was applied by all synthetic groups involved. As the synthetic target structures aimed at are represented by isolated points scattered irregularly over a relatively small segment of structure space, a combinatorial approach furnishing a focused variation, whose members ought to be represented by a cluster of points in abstract structural space, would seem promising. 1.4.2.2.1 Preparation and Screening o f a Static Variation The combinatorial approach that was pursued in search of an antiasthma drug based on a split-and-mix strategy [92] as a practical use of the operational principle of parsimony was to get the most with the least; in this case, to get 343 different types of variants in only 21 reaction steps. Scheme 1-17 sketches
Scheme 1-17 Construction o f a binary-encoded [93]combinatorial variation using the split-and-mix protocol (resulting in an one-bead-one-variant state) and an
encoding-decoding alternation (resulting in a state with every bead carrying a single tripeptide sequence).
I
31
32
I how a biased variation of 343 members was obtained on resin-beads in three 7 Chemistry and Biology
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preparative rounds, each round allowing for the parallel attachment of one out of seven building blocks available. The complete set of monomeric building blocks used in the construction of the combinatorial variation of Scheme 1-17 is shown in Scheme 1-18.The aesthetic elegance of the combinatorial strategy reveals itself when compared with alternative strategies*’). The bead-bound substrate variation was screened for binding to a biological receptor (a fluorescence-conjugated immunophilin [87])by mixing a sample of the charged beads with a buffer containing the complementary protein. The beads that carry variants with affinity for the receptor are easily identified by visual inspection under a microscope with a fluorescent illuminator and removed with the aid of a (non-plastic) syringe. The sequence of each beadbound substrate variant has been determined indirectly but unambiguously by Clark Still’s encoding-decoding alternation [93].
Molecular encoding: During each step of the construction of a focused variation of tripeptides (see Scheme 1-17)tagging molecules are attached to the beads
Scheme 1-18 21 building blocks for the preparation o f t h e 343 tripeptides of Scheme 1-17 (building blocks 6,10, and 11 were used as racemates). 21) A divergent approach would require 399
+ +
(7’ 7’ 7 3 ) reaction steps, a serial approach even 1029 (73+ 7’t 7’) reaction steps to reach the same 343 variants [63, 871.
7.4 Bringing Chemical Solutions t o Biological Problems
that encode both the step number (one through 21) and the reagent (amino acid or acid chloride, respectively) used in that step. A combinatorial encoding of the 21 reaction steps requires altogether seven molecular tags (i.e., A, B, C; AB, AC, BC; ABC in one round). Molecular decoding: After screening the variation, the molecular tags22'can be cleaved photochemically from each of the selected beads and analyzed by gas chromatography [93].The specified on-bead selection test afforded a mixture of ruc-77 and rac-78 (Scheme 1-19). To explore its biological properties by various functional tests [94], a substantial amount had to be synthesized. Instead of going for 79 (Scheme 1-19)the more distant compound 80 (Scheme 1-20)was aimed at, by conventional synthesis technique. The cause for replacement oftarget structure 79 with 80 was accidental. While looking for linkers for solid-phase synthesis that can be cleaved enzymatically, the substitution took place. Substitution of the B-methoxyethylamino residue by the Z-protected lysine residue [87] led to higher biological activity in various functional tests. Compound 80, recently, [94] has been considered to be a promising candidate for the treatment of diseases accompanied by immunological inflammation. The combinatorial approach produces large variations of related molecules, which can be exploited by appropriate screening techniques. As far as the production ofthese variations and their screening are concerned, combinatorial chemistry reminds one of the immune system. In the immune system, antibodies recognize cognate antigens. Those antibody-producing cells that are effective against a particular type of invader molecules preferentially evolve from a huge population. If the invaders are pathogens or parasites, dynamic
6 OCH3
77
6 OCH3
78
OCH3 79
Scheme 1-19 On-bead molecules (rac-77 and roc-78) selected from the variation of Scheme 1-17. and the seeming target structure 79. 22) The molecular tags that were used are
composed of a series of electrophoric tags (halophenol derivatives) plus a photolabile linker [93].
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0
H
80
0
\
81 82
81 81 82+83
a)82
- - bl
CI
83 84
d)
85
+86
80
e)
a) 6 0 ~ ~aq0 NaOH, , dioxane, 90 % b) MeOH. SOClp, 98 % c ) 2-Chloro-1methylpyridiniumiodide, CH2Cl2.NEt3. 50 % d) MeOH. 2.5 N NaOH, 74 % e) 2-Chloro-1methylpyridiniumiodide, CH2Clp,NEt3. 86 %
Scheme 1-20 Collection of formulae relevant to a synthesis of the biologically active candidate 80.
coevolution between them and the host may occur. There is, however, a tremendous difference between a static variation and the immune system. While the processes of preparation and screening of a static variation were designed by chemists, what happens in immunology was not designed but rather evolved. The preparation of a dynamic variation (to be described in the following section) is somewhat in between the two extremes, though very much closer to the designer's end. 1.4.2.2.2
Preparation and Screening of a Dynamic Variationz3)
In the previous section, a well-known method was applied to a long-standing biological problem: the discovery of a new biologically active substance. With 23) For dynamic non-covalent chemistry see 1951.
1.4 Bringing Chemical Solutions to Biological Problems
the intention of finding such a substance displaying properties closest to a setup profile, a static molecular variation was prepared (on microscale) and screened (collectively) to afford a select variant qualifying as the candidate for subsequent synthesis (on macroscale). In this section, we present the selfassembly ofa variation ofthree sets ofconjugates from which an added receptor selects a number of effectors by molecular recognition. This selection works by way of the interactions of protein surfaces within the receptor-effector supermolecule, the knowledge of which ought to be helpful in drug design. The self-assembly to be introduced is based on three pyranosyl-RNA (p-RNA) [96] single strands (a, b, and c, Scheme 1-21) associating in a Watson-Crick-like manner, initially into binary and further on into ternary super molecule^^^). In
Scheme 1-21
Base-pairing dynamics of single strands a, b, and c.
24) Project of the G e m a n Federal Ministry of
Education and Research [97a];for details see [87b][97b]. Initiated by A. Eschenmoser, U.-H. Felcht, G. Quinkert [97c]. Beginning: 1 April 1995.
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I addition to the H bridges, intercatenary n,n-stackingeffects make a substantial I Chemistry and Biology - Historical and Philosophical Aspects
contribution to the stabilization of the resulting duplexes [9Ga, 9Gd]. In its current form, the self-assembly is based on three p-RNA single strands with 7 (a and b) or 14 (in the case of c) nucleobases. The two short strands are sequence complementary to the first seven or the last seven bases in the longer strand. The pairing gives rise eventually to water-soluble ternary complexes acb (Scheme 1-21). Strand c is involved in all the equilibria. Since strands a and b are unable to pair with one another and as they bind to non-overlapping regions of c, they do not compete with each other in binding to c. The unusual designation acb is used to reflect the dominant role of the longer strand c in complex formation. The following equilibria, with five independent equilibrium constants25), apply to the pairing of the complementary strands: ci
+ aj *aj
: ci,
Subscripts i,j , and k are used to distinguish various possible sequences displaying the required complementarity. Scheme 1-22 shows a network representation of the above set of equilibria. The nodes in the network correspond to the individual strands involved in the equilibria, while the lines represent their possible associations or dissociations. Along a given line, the concentrations of a single strand or of several strands vary between zero and the maximum disposable value. Each of the colored lines corresponds to a single strand, whilst black lines relate to more than one strand or to a binary complex. With the exceptions of a and b, which have only two connections each, all other nodes have at least three available connections, whilst the node for the ternary acb complex has as many as five. The network here results from the superposition of the synchronous formation from a, b, and c with the formation both from ac plus b and from cb plus a. 25) (1)and (2) form closed subsystems. As soon
as all three components are present, however, the full system of equilibria (1-5) is valid. Equilibrium (5) represents the synchronous formation of the ternary complex
out of the three single conjugates. Since this corresponds to third-order kinetics, a process of this type is significantly less probable than the purely bimolecular processes (1-4).
1.4 Bringing Chemical Solutions to Biological Problems
I '
I a
acb
I b
l
\
rh
C
Variation of [a] Variation of [b] ~
Scheme 1-22
Variation of [c] Network representation of equilibria (1)-(5)
In a three-dimensional representation, the strands and their complexes can be arranged as the vertices of a trigonal bipyramid, its edges corresponding to the equilibrium arrows from (l)-(S)26).Each state ofthe system is thus a point within the trigonal bipyramid. The stability of the complexes may be preserved when the pairing-capable strands a, b, and c are extended into sets of conjugates2'' A, B, and C (Scheme 1-23). Coupling with a series of oligopeptides transforms the pairing system (selfassembly system) with the three single strands a, b, and c into an exploring system (molecular recognizing system) with the three sets of conjugates A, B, and C. The equilibria (1)-(5) also apply to the conjugates, if the subscripts i, j, and k are used to denote the oligopeptides employed. For the resulting system there is a particular assignment of roles: the pairing system based on the p-RNA strands a, b, and c serves to bring the peptide regions into proximity with each other, thus supporting their joint function. The law of mass action applies here not only to the self-assembly but also to molecular recognition, ensuring that the full potential of the structural variation can be exploited. As effectors, the triple peptide combinations are capable of entering into specific interactions with a further component, a receptor R (Scheme 1-24).As a selector of complementary oligopeptide combinations, the receptor enables unnatural selection from the variation of conjugates. 26) I t should be pointed out that the transition
from ac to cb does not take place as a direct, single process, but should be regarded only as a conflation of processes ac cf a + c and cb c + b. The corresponding edge of the bipyramid thus - unlike the other edges - does not symbolize a single equilibrium. c)
27) For the conjugates the following p-RNA sequences have been used: a = {CGGGGGNJ. b = [NGAAGGG], and c = (CCCTCTNCC CCCG}. N is a tryptamine nucleoside [98],
which serves to attach the oligopeptides (discrete random variation of hexapeptides composed of the amino acids C, E, F, H , K , L, N, R, S, T, W).
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Scheme 1-23 Equilibria between members ofthe three sets o f conjugates of types A, B, and C each with p-RNA moieties (gray) t o make self-assembly possible and oligopeptide moieties (green) t o allow molecular recognition.
The equilibria (1-5) described above now need to be supplemented, first to take account of the receptor itself, and second to allow for the receptor complexes with the various components of binary and ternary aggregates shown in Scheme 1-23: altogether eight molecular species are now involved. Scheme 1-25 shows the corresponding network of 8 nodes and 28 possible equilibria, each of the nodes having 7 connections. As in Scheme 1-22, green, red, and blue lines represent the possible binary equilibria, whilst black lines denote potential ternary and quaternary equilibria. In the interactions with a receptor, unlike in the case of the separate ternary complex, there are several types of substitution equilibria in which conjugates
1.4 Bringing Chemical Solutions to Biological Problems
Scheme 1-24 Sketch o f molecular recognition of a receptor (R) by a complementary effector (here by a discrete variant of type ACB).
are exchanged. There are three types of pure binary substitutions, and two higher order substitutions where one conjugate is substituted for two others at a time. Whether these simultaneous exchanges of several conjugates, as well as the higher order associations and dissociations are relevant, though, remains to be determined experimentally. The alternative of stepwise processes is available in any case. Topologically, the molecular species can be ordered into four levels of complexity28’(Scheme 1-25). On the simplest level is the free receptor R. The level above is represented by the binary complexes R:A, R B , and R C , the next level by the ternary complexes RAB, RAC, and RBC, whilst lastly the level of highest complexity is occupied by the quaternary complex R:ACB. Accordingly, the participating species can be arranged as vertices of a cube. All possible equilibria are now either edges, or face- or space-diagonals of the cube and the system is, by definition, described by a point inside the cube at any time. The cube-style representation shows, firstly, that pathways from one species to another are possible either via both edges and diagonals, or exclusively via 28) The free ternary complex and its subsystems
are found on these levels likewise and are continuously present over the full span of equilibria. For the sake of clarity, however, they are not explicitly taken into account here.
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Scheme 1-25 Network representation of all possible equilibria extending Scheme 1-24. The eight nodes are labeled by bold characters. All other intersections are
artifacts of the two-dimensional representation. For the sake o f clarity, faceand space-diagonals ofthe cube are not shown.
edges or diagonals. Secondly, it also demonstrates the high syntactic symmetry (equivalenceof the different types of interactions) of the system and underlines the exchangeability of receptor and effectors. To delineate pharmacological properties of members of the dynamic system shown in Scheme 1-25, data of an enzyme-binding experiment from a realtime biomolecular interaction analysis27)and data of an enzyme-inhibition experiment from a photometric assay30)have been correlated (Scheme 1-26). One can see that the strongest affinity (binding) does not give rise to the greatest activity (inhibition). Affinity is not proportional to activity. Species RAC shows the strongest affinity, whilst species RACB causes the greatest activity. Since species RCB has the weakest affinity, it is clear that B makes no cooperative contribution to affinity, but is important for effective activity. 29) The biotinylated conjugates (ACB, AC, BC, 30) The enzyme is mixed with its photolabeled or C) are captured by a sensor chip, whose substrate S. Upon cleavage by the enzyme,
surface is coated with immobilized streptavidin and which acts via surface plasmon resonance as a tool for enzyme (R) binding experiments.
the label is activated and fluorescence can be detected. In case ofinhibition by the effector, cleavage does not occur and fluorescence is not detected.
7.4 Bringing Chemical Solutions to Biological Problems
Obviously, there is no additivity of the individual conjugates’ contributions. From the quantitative point of view this corresponds to non-linear behavior. The influence on the enzymatic reaction has to be interpreted in terms of either competitive inhibition (ACB:R)31), uncompetitive inhibition (ACB:RS), mixed inhibition (ACB:R ACB:RS), or substrate capture by the conjugates. It should be noted that interactions of A, B, and C with the receptor may mutually influence one another in both cooperative or anticooperative fashion. Furthermore, the coordinating role that conjugate C is playing in self-assembly (Scheme 1-23) may be pushed into the background or may even be absent entirely while interacting with the receptor.
+
Scheme 1-26 Correlation diagram of affinity (binding) and activity (inhibition) for some nodes ofthe network of Scheme 1-25. Values for ACB are set to 100%. 31) Here, and in the other possibilities men-
tioned, ACB:R stands for any ofthe molecular species from Scheme 1-25 containing the receptor.
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1.4 Bringing Chemical Solutions to Biological Problems
For a screening experiment on enzyme inhibition (Scheme 1-27),a variation of conjugates of types A, B, and C was formatted spatially addressable using 16 microtiter plates. One out of 1308 different C conjugates was given each in a separate well, together with 1of 8 different A conjugates and 1 of 11 different B conjugates, as indicated on the margins. In 99 of the remaining wells, the single A or B conjugates were given as inactive blank controls. The last well was filled with solvent and buffer, only. To each of the various mixtures the enzyme used was added, together with its fluorescence-labeled substrate s. In each well, the enzyme could either select the substrate or the conjugates of Scheme 1-25. In the first case, the labeled substrate would be cleaved by the enzyme and fluorescence observed. In the second case, inhibition of the enzyme would occur and little or no fluorescence detected. The color coding in Scheme 1-27 indicates the degree of inhibitory activity found in each case. White and pale blue denote inactive substances, red and violet denote strong inhibitory effects. In a separate measurement, an ICs0 value of 23 nM was found for the strongest inhibitor (position A 8 / B l l on the plate in the fourth column, third row). Surprisingly, there are not only single point hits but also whole clusters of hits in which the participating conjugates display inhibitory activity. A closer inspection of, for example, all the wells in which conjugate A4 is present, reveals that the majority indeed shows activity, independently of the B and C conjugates added. This notwithstanding, not all 16 plates show the same distribution of active and inactive triplets, even though the A and B conjugates are the same in each plate. So, variation in the C conjugate significantly influences the activity of the A and B conjugates. This is especially apparent in the mixtures of A3 with B1 through B8 and of A2 with B1, B3, and B5 through B7 in the plate of the second column, third row. Only in the presence of a C conjugate do A and B conjugates contribute to the observed activity in this case. The law of mass action suggests to depart from the 1 : 1: 1 stoichiometry in the search for maximum activity. On changing the concentrations of individual conjugates, one shifts the molecular system parallel to edges or planes of the cube (Scheme 1-25).The statistical weights of the contributions of individual conjugates to the network of interactions are altered in the process. Scheme 1-28 shows the results of a pilot experiment in which the inhibitory activity was measured as a function of the concentrations of the A and B c o n j ~ g a t e s ~The ~ ) .results are displayed as a hypersurface for a constant concentration of conjugate C. The sigmoidal dose-activity relationship is clearly evident with regard to both A and B. The stoichiometric composition with [A] = [B] = [C] = 555 nM is represented by a point located on top of a ridge, separating a flat region of the hypersurface from a descending slope. Starting from the stoichiometric point, activity increases with the concentrations of A and B. The strongest inhibition value was found at the bottom of the slope 32) Results relate to the second strongest inhibitor found in the screening. In Scheme 1-27 it is to be found on the plate in
the third row and the second column with the conjugates A3/B1. The results presented in Scheme 1-26 refer to the same complex.
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Scheme 1-28 Three-dimensional (hypersurface) view ofenzyme-inhibition activity o f a combination ofthree conjugates, A, B, and C as a function of the concentrations o f conjugates A and B. The
stoichiometric composition [A] = [B] = [C] = 555 nM is close t o a ridge. Increasing the concentrations o f A and B enhances the activity.
with [A] = [B] = 5000 nM and [C] = 555 nM, where the properties of A and B have a 10 times greater statisticalweight than those of C33).From the foregoing discussion it can be directly inferred that the activity of a conjugate triplet is not connected to a single molecular species from Scheme 1-25. Given the dynamics of the supramolecular system described, one could go a step further and transgress the confinements of molecular constitution. It should be just as possible to use carbohydrates, steroids, terpenes or even nonbiogenic substance classes - dendrimers, for example - in place of the peptides. Through the addition of conjugates of different types of constitution, the transition from one type to another could be studied in a quasi-continuous way, opening up a further, new option for the determination of structure-activity relationships. The dynamics of the system allows it to adapt to changes in the environment. Adaptation here means that the balance between the interactions inside the 33) Comparing Scheme 1-28 with Scheme 1-26, one can see that the increase of activity on going from C to ACB, from CB to ACB, and from AC to ACB is consistent with the topology ofthe hypersurface in Scheme 1-28.
1.5 Bringing Biological Solutions to Chemical Problems
effector (between the individual conjugates) on the one hand and those between the effector and the receptor on the other hand, can change. Therefore, depending on the prevailing conditions, different molecular species may be responsible for the effects produced at the receptor. Particular combinations of members of the three sets described may be used to map the affinity profile of the receptor. In short: receptor profiling directly results from a thorough investigation of the dynamic system under discussion. It reveals the complementarity between the sites of the interacting surfaces of receptor and effectors and suggests the design for a specific, biologically active substance finally taking over from the analyzing effectors. Ultimately, the potential ofbiologicallyactive substances can only be assessed in actual biological systems by means of animal experiments (Scheme 1-29) and confirmed by subsequent clinical studies. En route to this, however, the dynamic system described here offers various options for the analysis and optimization of pharmacological parameters like affinity and activity. It is the heterobifunctional character of the dynamic system that allows the synthetic chemist to influence both intrinsic self-assembly as well as extrinsic molecular recognition in a controlled way. 1.5 Bringing Biological Solutions to Chemical Problems
1.5.1 Proteins 1991
Among the bio-macromolecules, proteins are distinguished all-round players. As fibrous proteins they are used for structural purposes. As enzymes they catalyze almost every chemical reaction in a cell with great power and high specificity. As gene regulators they control gene expression in development and evolution. As antibodies (immunoglobulins) they bind invading antigens. As motor proteins they convert chemical energy into kinetic energy. As transport proteins they mediate transmembrane movements of ions or metabolites. 1.5.1.1 A Look at Protein Structure and Generation from Different Angles The chemist fills the void in structure space left by the physicist who dislikes the integrated complexity of the molecular world. Even the chemist, for some time, had been treating his structure space rather unevenly. According to the Beilstein Doctrine341,macromolecules neglected by the organic chemist for a 34) Beilstein Handbook of
Organic Chemistry, an encyclopedia of known micromolecular carbon compounds, does not concern itself with macromolecular carbon compounds [17e].
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Scheme 1-29
Outlook: supramolecular network concept in pharmacology.
long time [17f],were finally taken up by the biochemist who could not afford to ignore bio-macromolecules like nuclear acids and proteins any longer. The bottom-up view of the biochemist eventually was complemented by the top-down attitude of the (molecular) biologist. Quite a few of those scientists who considered themselves molecular biologists entertained the idea [ 100aI that “other laws of physics’ might be discovered by studying the gene”. This search for the physical paradox [100b] remained an important element of the psychological infrastructure of the creators of molecular biology. As a matter of fact, the physicists among the new group were going to create a new approach to biology [loll.
1.5 Bringing Biological Solutions to Chemical Problems
1.5.1.1.1
The Chemist’s Look (1021
The HofFneister-Fischer Theory of Protein Structure was made public in 1902 [103, 1041. Accordingly, proteins consist of polypeptide chains in which the individual a-amino acids are linked to one another through amide (peptide) bonds formed between the COOH group of one amino acid and the NH2 group of the next amino acid. The structure of proteins, Linus Pauling has demonstrated, some time later, how deep knowledge of chemistry can lead to general rules [105]. The nature of the strong peptide bond, the role of weak hydrogen bonding, and the importance of complementarity [lo61 were such rules used in model building: one of Pauling’s methods to work out the structure of bio-macromolecules. Stepwise protein synthesis normally requires [ 1071 protection of the amino group of the first amino acid and the carboxy group of the next amino acid; activation of the carboxy group of the amino acid carrying the protected amino group to form a peptide bond; and finally, removal of the protecting groups. Polypeptide synthesis on insoluble polymer supports was pioneered by R. B. Merriield [108].This method could be automated and has facilitated protein synthesis enormously [ 1091. Chemical ligation of even unprotected peptide segments has recently been reported [IlO]. To summarize: systematic variation of structure with the aim of developing peptides for therapeutic use gives the synthetic chemist a good excuse for chemical synthesis. a-Amino acids, obtained from natural sources or from the synthetic chemist’s laboratory, play a trailblazing role in the gradual growth of chemical biology. For the synthetic protein chemist they are the obvious building blocks, for the teaching chemical generalist they are ideal demonstration objects with an unmistakable structural profile: two unlike functional groups and - with the exception of glycine - at least one stereogenic center within the smallest possible space. Nearly 50 years were to pass from Emil Fischer’s view that synthetic chemistry should contribute to the solution of biological problems [30] to Du Vigneaud’s synthesis of the neuropeptide oxytocin [ 1111. Preparative stumbling blocks in the selective protection and/or activation of functional groups as well as in the effective separation of complex reaction products, first had to be cleared from the path. Methodological progress toward the achievement of automated solid-phase synthesis, with or even without utilization of protecting group technology, finally made peptide synthesis more or less a routine matter. Sophisticated methods have been developed to ligate smaller peptide segments together to make larger peptides. As far as larger proteins are concerned, the chemist’s ability to control their structure (and functions) specifically is still in its infancy.
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The Biochemist’s Look [112]
In his study of endergonic protein genesis,3s)the biochemist is driven by the desire to understand how the energy barrier from the amino acids to the peptide is overcome [113]. Paul C. Zamecnik, Mahlon Hoagland, and their colleagues developed and used a cell-free system for the in uitro study of the mechanistic details of protein genesis [114]. By the use of radioactive amino acids, it could be shown that, in an initial step, enzymatic activation of the one amino acid out of 20 induced by the hydrolysis of ATP took place following the reaction: Amino acid + ATP
Enzyme,
AMP-amino acid residue:enzyme pyrophosphate
+
The resulting adenylated amino acid appears to be tightly bound to its specific enzyme, the corresponding aminoacyl-tRNA synthetase. without leaving its enzyme, the former, in a consecutive step, reacts with a low-molecular-weight RNA (called soluble RNA = sRNA, later more logically known as transfer RNA = tRNA) to afford an aminoacyl-tRNA [115,116]. AMP-amino acid residue:enzyme tRNA
+
GTP Amino acid residue-tRNA +
+ AMP + enzyme
This transacylation furnishes conjugates that structurally bridge the gap between amino acids and their ordered arrangement in proteins.
1.5.1.1.3
The Molecular Biologist’s Look [117]
Aminoacyl-tRNAs not only bridged the gap between activated amino acids and their ordered arrangement in proteins but they also, rather dramatically, brought together the experimental biochemist and the theoretical molecular biologist [113, 1181. The biochemist, beyond biogenesis, takes a lively interest in flow of matter and energy during metabolism. The molecular biologist takes additional interest in the flow of genetic information during gene expression on the one-way road: D N A + RNA + Protein. M. Hoagland [115] and P. C. Zamecnik [116]with their sRNAs acted as the experimental biochemists while Francis Crick, by offering his adaptor hypothesis [119], figured as the theoretical biologist. Several years, before sRNAs were discovered, Crick had already proposed 20 types of adaptor-RNAmolecules, which could line up along an unspecified template-RNA, and each bind to a particular amino acid. In his own words: “one would require twenty adaptors, one for each amino acid, and separate enzymes would be needed to join each adaptor to its cognate amino 35) We distinguish in this essay products of
protein synthesis which were designed by man from products of protein genesis which were produced by evolution.
1.5 Bringing Biological Solutions to Chemical Problems
acid. Thus one is lead to suppose that after the activating step, discovered by Hoagland and described earlier (vide supra), some other more specific step is needed before the amino acid can reach the template”. Which template? Several observations had excluded rRNAs from being candidates for acting as templates. A cell, for example, could make a new type of protein without making a new type of ribosome. The template-RNA was finally disinterred as a class of unstable intermediates, self-explanatorilycalled messenger-RNAs ( ~ R N A s ) ~When ~ ) . J . D. Watson informed the scientific community “About the Involvement of RNA in the Synthesis of Protein” [117a]he could begin with the sentence: “The ordered interaction of the three classes of RNA controls the assembly of amino acids into protein”. Now essential details in brief: protein genesis (translation) is the central event in molecular biology. It takes place in the incredibly complex machinery3’) of the ribosome [124], where the syntactic structure of ribonucleic acids is translated into the syntactic structure of proteins. During the translation process, the information contained in a triplet codon of mRNA is decrypted by an anticodon of a tRNA molecule, according to the instructions of the genetic code. The genetic code is an abstract scheme for the redundant correlation of 64 “words” (nucleoside triplets) in the language of nucleic acids with 20 “words” (canonical amino acids) in the language of proteins. The synthetic chemist accepts the limitation on the number of amino acid building blocks as the price for his readymade use of the ribosomal protein generating system. The undisputed leading actors in the translation process at the stage of information transfer from ribonucleic acids to proteins are aminoacyl-tRNAs [ 1251. These are conjugates made up of proportions of both biopolymer types (language systems), produced through esterification of an amino acid with a tRNA. A particular tRNA with its anticodon corresponding to a specific amino acid is covalently coupled (esterified) with precisely this amino acid. The esterification takes place through the help of an enzyme (an aminoacyl-tRNA synthetase) capable of specifically recognizing and coupling that particular tRNA and its cognate amino acid [126].Whilst the self-assembly of mRNA and tRNA during translation is due to codon-anticodon interaction, based on Watson-Crick 36) Messenger-RNAs were the last of the RNA trio engaged in protein genesis, to be detected [120]. A further type of RNA has been discovered as a widespread, universal tool in biology for gene regulation by means of antisense-like interactions [121]. It is called inductive RNA (RNAi) and is produced from double stranded RNA in a cascade of enzymatic processes by a set of specific RNAses. Several regulatory pathways involving RNAi are known in many eukaryotes, including plants and mammals. RNAi is used extensively as a tool for research and its therapeutic potential is getting more and more obvious [122].
37) In an urgent appeal, we are certainly going to
follow henceforth, Carl Woese [123] requests to stop looking at an organism as a molecular machine. The machine metaphor, according to his view, overlooks much of what biology is. To understand living systems in any deep sense, “we must come to see them not materialistically, as machines, but as stable complex, dynamic organization”.
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I pairing of complementary nucleobases, the mutual recognition of a tRNA and its cognate synthetase during aminoacyl-tRNA formation is due to molecular shape complementarity.
1.5.1.2 1.5.1 2.1
The Genetic Code [127] Cracking the Genetic Code
The genetic code was cracked in the early 19GOs, beginning with investigations by Marshall Nirenberg and Heinrich Matthaei by using a cell-free E. coli system. The N I H researchers, in an inaugural experiment demonstrated that the homopolymer polyuridylic acid coded for the nonnatural protein polyphenylalanine [ 1281. Clearly, the natural system of protein genesis would translate any appropriate message, natural or artificial, into a polypeptide chain, natural or artificial [116]. 1.5.1.2.2
Expanding the Genetic Code
By Natural Selection
The genetic code has the potential for 64 (=43) triplet codons, 61 of which redundantly specify the 20 canonical amino acids. The methionine-specifying triple code AUG may take on the role of a starting signal at the beginning of protein synthesis: it thus has a double function. Three triplet codes in a mRNA - UAA (ochre), UGA (opal), and UAG (amber) - known as nonsense codons, specify no amino acids; that is, there are no tRNAs with complementary anticodons for these codons. As a consequence, translation breaks off here. The nonsense codons are also, therefore, termed stop signals (termination codons). Broader roles in protein genesis, however, have also been established for two of these three stop signals in recent years. In E. coli (and also in a whole range of other organisms) the UGA codon may be redefined to perform one of two different functions: either it may function as a stop codon and thus end the elongation of the protein chain under construction, or further growth of the polypeptide chain may carry on with incorporation of selenocysteine [129],not a member of the standard set of canonical amino acids. Which of the two instructions is followed by the translation system is dictated by the secondary and tertiary structure of the mRNA to be decrypted (and possibly by protein factors). Similarly, structural alterations in mRNA are able to modify the programming of the UAG codon: once more, a codon that continues a translation in progress, in this case through the incorporation of pyrrolysine [130], is produced from a stop codon. The genetic code is thus naturally expanded from the standard set. Instead of the original 20 amino acids, 22 amino acids specified by mRNA sequences are currently recognized. Further as yet unrecognized extensions of the genetic code through natural selection cannot be excluded. Why no sense codon has (yet) been found to be doubly
1.5 Bringing Biological Solutions to Chemical Problems
coded, is unclear. The discovery that the genetic code, as a result of natural selection, already has more than 20 amino acid building blocks for protein genesis in store, poses the question of whether the genetic code might also be expandable by design; that is, whether amino acids not specified by the genetic code in their original version might be introducible into a polypeptide chain by translation. By Design [131]
Peter G. Schultz, a leading protagonist of the movement to consider biology an engineering discipline, is aiming at the construction of new proteins and, eventually of new organisms with enhanced properties. Two alternatives for site-specific in vivo incorporation into proteins, of amino acids not specified by the genetic code in their original version, have been designed to achieve that goal: systematic reassignment of three-base nonsense codons or use of supersized codons. The addition of a non-canonical amino acid to the genetic code requires - in the first case - additional components of the protein producing system: a noncanonical amino acid, an exogenous tRNA/aminoacyl-tRNA synthetase pair, and an unique codon that specifies the amino acid of interest. Orthogonality between the exogenous translational components (Scheme 1-30) and their endogenous opposite numbers is the key feature of this approach. With the effect that the codon for the noncanonical amino acid should not encode a canonical amino acid; that the new tRNA or the cognate aminoacyl-tRNA synthetase should not cross-react with any endogenous tRNA/synthetase pair; and that the new synthetase should recognize only the noncanonical and not any of the canonical amino acids.
A completely autonomous bacterium with a 21 amino acid genetic code was engineered. The bacterium can generate p-aminophenylalanine from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon (1321. As the restriction of non-coding triplet codons limits the number of noncanonical amino acids, the question arises as to whether or not expansion of the genetic code by use of a supersized codon and cognate tRNA with an expanded anticodon loop might be possible. A study Exploring the Limits of Codon and Anticodon Size [133] reveals that the E. coli ribosome is capable of using codons of three to five nucleobases. The tRNAs that decode these codons are most efficient with a Watson-Crick complementary anticodon containing two additional nucleotides on either side of the normal-sized anticodon in the loop. An orthogonal synthetase/tRNA pair was designed and constructed, which site-specifically incorporates a noncanonical amino acid (L-homoglutamin) into proteins of E. coli in response to the four-base codon AGGA [134].
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Scheme 1-30 Incorporation of (a) canonical (yellow) and (b) noncanonical (red) amino acids into proteins in vivo.
1.5.2 Antibodies
The ribosomal system is not the only evolutionary accomplishment the synthetic chemist might use in pursuit of his ends. The immune system offers an example of how a biological solution can successfully be brought to exploit antibodies as enzymatic catalysts. As far as their functions are concerned, enzymes and antibodies normally are quite different. Enzymes have been selected for the transition state of a catalyzed reaction over millions of years [105].Antibodies have been selected for their affinity for the immunogen over a period ofweeks [135].Ifthe immunogen were a transition state analogue, the resulting antibodies should catalyze the appropriate reaction. Richard A. Lemer and Peter G. Schultz with their respective colleagues have designed molecules
1. I; Bringing Biological Solutions to Biological Problems
that could be used to guide the process of clonal expansion and somatic mutation to generate catalytic antibodies for a variety of reactions [136].Rather than going into details here, we refer to the authoritative book on catalytic antibodies 11371. The various articles ofthat book make for interesting reading: for the synthetic chemist who wants to design new catalysts as well as for the molecular biologist who wants to gain structural insight into antibody evolution.
1.6 Bringing Biological Solutions to Biological Problems
The composition of this essay followed the matrix
chemical problems
biological problems
Biological answers to biological questions are, of course, given by Nature directly. Man may use the complex systems of Nature with the aim to correct a fault (as, e.g., was done by Robert Edwards and Patrick Steptoe [ 1381 in reproductive medicine). Reproductive medicine cannot be discussed disregarding bioethical aspects [ 1391. The present authors are not competent to meet the bioethical requirements. For this reason, reproductive medicine is not further commented on. Up to now synthetic chemistry has been the dominant part of our reflection. Now synthetic biology comes in to meet the requirements of the sophisticated observer who wants to be informed about the newest development. At any rate, the fundamental question, WHAT IS LIFE? comes up. Under this title, two essays have been published; one by Erwin Schrodinger [140] in 1944 and the other by J . B. S. Haldane [141] in 1949. While the former focused on the physical aspect of the living cell, the latter considered life essentially as a pattern of chemical processes. A very pragmatic point of view was formulated in 1994 by Antonio Lazcano 11421 with the statement: “Life is like music, you can describe it, but not define.” In a state-of-the-art survey, Biology and the Future o f M a n 11431, of the US National Academy of Sciences, the chances to realize the dream of a man-made cell were pondered. The conclusion reached was: “Those who are hopeful about synthesizing a cell in the foreseeable future have every reason to retain their optimism.” However, they should be warned against false claims. Synthesis of life is one such false claim. Living things (i.e., a cell) can be synthesized but not life itself, and that is what people really mean when they are talking about synthesizing life. A question that keeps busy scientists in chemistry as well as in biology is about where the line separating inanimate from animate matter can be
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life’s origin in terms of molecular evolution [144]. Recently, sequencing of the human and other complete genomes has shed some new light on this field. The question of what the minimal set of genes would be necessary for a living organism can be put more concisely in the context of what is now called synthetic biology [145]. Both approaches, the top-down way of deactivating more and more genes of an existing species [146]and the bottomup way of assembling genes to build an organism with a fully synthetic genome [147],have not yet reached the goal to explain the transition from the inanimate to the animate world. On the one hand, results obtained through different methods to identify the minimal set of genes that constitute a living organism point to roughly 250 genes [148]. On the other hand, none of the synthetic constructs obtained so far covers the central functionality of life, self-construction, metabolism, adaptation, self-repair, reproduction, and evolution [149]. Nonetheless, the bottom-up route has turned into an engineering approach to synthetic biology [150].The strategy is to combine predefined DNA modules, so-called bio-bricks that can be combined to bio-circuits, designed to be implementations of biological functions [ 1511. In that sense, synthetic biology is seen as the successor of molecular cloning, in particular, with respect to safety issues.
1.7 EPI LOCUE
To round offthis essay, we point to two issues gaining more and more emphasis in chemistry. One thing is the problem of shared use of the limited sources of energy and raw materials. The other thing is the concept of a total synthesis, in particular for complex natural substances. Both topics underline that organic chemistry is far from being pure routine applying a comprehensive toolbox to solve any problem in synthesis [ 1521. Medical therapeutics, agrochemicals, and high-performance materials must be provided by organic chemistry to fulfill global needs. 1.7.1 The Fossil Fuel Dilemma o f Present Chemical Industry
For chemical industry, the interdependence of energy source and raw material supply is typical. This double function of fossil fuel to act as a source of raw material supply as well as an energy source will have to be terminated in a not-too-distant future [153]. Being the main source of raw material, fossil fuel should be maintained as long as possible for the chemical industry. A final way out to disentangle energy requirement and raw material supply
I would be to find new sources for one field or the other. Nuclear energy, 1.7 EPlLOCUE
despite political moves to dispense with nuclear power, could play a role as an alternative to fossil fuel. With petroleum supplies dwindling, there is increasing interest in selective methods for transforming other carbon feedstocks into hydrocarbons suitable for transportation fuel. The reductive oligomerization of CO and H l to produce hydrocarbons (specificallyn-alkanes) with highly controlled molecular weight (Fischer-Tropsch process [154]) from the vast reserve of coal, natural gas, oil, or biomass is one such process that was developed in the 1920s. The Goldman-Brookhart process (tandem alkane dehydrogenation-olefin metathesis [155]) is of a similar kind, but of recent origin.
1.7.2 Two Lessons From the Wealth o f Published Total Syntheses
The final proof of the structure of a natural product after the latter has also been synthesized in the chemist’s lab was, for a long time, common procedure [156]. In a few cases, disagreement raised a few eyebrows. This was the case for patchouli alcohol and for a molecule called hexacyclinol [157]. Quinine is an example of the difficulties associated with the notion of a total synthesis. Shouts [35, 37,1581 and murmurs [llb,159] have been expressed to comment on the wealth of total syntheses of natural products performed in the second half of the twentieth century.
1.7.2.1
Synthetic Lesson from Patchouli Alcohol: The Trouble with “the Last Structural Proof’ [160]
The peculiar case of patchouli alcohol (87) (Scheme 1-31) was told and commentated by Jack D. Dunitz [IbOa]. Following W. H. Perkin’s jun. advice [I561 to perform, as a final proof of structure a total synthesis of a natural product 87 was synthesized [IGOc]. The synthetic product proved to be identical to sesquiterpene whose structure had been derived from the results of a long series of chemical experiments lasting more than 50 years and apparently confirmed in 1961 by total synthesis [IGOc]. In spite of this, X-ray structure determination [IbOa] revealed that the accepted structure of patchouli alcohol was wrong. A careful reinvestigation showed that during chemical degradation as well as during synthesis a rearrangement of the molecular skeleton had taken place. The first reaction step of the chemical degradation (acetate pyrolysis affording patchoulene 88) and the last reaction step of the chemical synthesis (hydrolysis of the epoxide 89 obtained from 88) were accompanied by a rearrangement proceeding in precisely the reverse direction of the rearrangement in the other case. Taking this
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Degradation
a7
t
87
Synthesis
88
i 89
(b) Scheme 1-31
Synthesis and degradation of Patchouli alcohol.
finding into consideration, a new synthetic approach furnished 87 without any difficulty [lGOd].
1.7.2.2
Synthetic Lesson From Quinine 90: The Trouble with Formal Total Syntheses [161a]
In the period between 1918 and 2001, a series of publications appeared that changed the claim of the total synthesis of 90 (Scheme 1-32) as a fact into a myth. It started with a paper of Rabe and Kindler in 1918 [lGlb]on the partial synthesis of 90 from quinitoxine (91),via quininone (92) (Scheme 1-32a).91 is a relais compound to 90, since it can easily be made from 90. In 1944 and 1945, Woodward and Doring published two papers [lGle]where they linked the partial synthesis of Rabe and Kindler to their own synthesis of 91 (Scheme 1-32b), taking the combination as a total synthesis of 90. Not being convinced of the view of Woodward and Doring, Stork published a new total synthesis of 90
1.7 EPILOGUE
92
HOP
Me
N
A
57
9-epf-quinine
90
quinidine
HO
I
HO F
9-epr-quinidine
MeN
, Ac
- qN, 0
Ac
Me mixture of stereoisomers
isoquinoline-7dl
OMe
91
Scheme 1-32 Synthesis of 90. (a) The Robe-Kindler partial synthesis of 90 I161 b]. (b) The Woodward-Diin'nglRabe-Kindler formal total synthesis of 90 [161e]. (c) The Stork total synthesis of 90 [161fl.
90
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Historical and Philosophical Aspects
+
.POTBDPS
J.-+.OTBS
oAf= OTBDPS
94
Scheme 1-32
(Continued)
in 2001 [Iblfl. He started from the Taniguchi lactone (94) and proceeded via desoxyquinine (95) (Scheme 1-32c).According to Stork, a distinction between a real total synthesis and a formal one is necessary. Accordingly, the work of Woodward and Doring is an example of a formal total synthesis.
Acknowledgments
Our own investigations on multicomponent simultaneous procedures were supported by the German Ministry of Education and Research and carried out by a team ofpostdoctoral fellows. In addition to these colleagues whose names are mentioned in the references, Susanne Feiertag, Stefan Kienle, Stefan Raddatz, Jochen Muller-lbeler,Jochen Muth, Christoph Brucher, Heike BehrensdorJ; Andreas Kappel, and Marc Pignot have contributed to our understanding of dynamic variations. Oliver Boden took care of the equipment for the electronic version of the manuscript. We are indebted to n e o d o r a Ruppenthal for patient and skillful secretarial help. The greater part of this essay has been translated from German into English by Dr. Andrew Beard. We are grateful to the mentioned persons for their assistance and to the indicated institution for its generous support. Last but not least, we would like to emphasize that it was Albert Eschenmoser's idea to use p-RNA or analogs for selecting appropriate candidates from a self-assembly of a dynamic variation.
References 159 References 1.
2.
3.
4.
5.
6.
7.
8.
9.
(a) F.J. Ayla, T. Dobzhansky, (Eds.), Studies in the Philosophy of Biology-Reduction and Related Problems, Macmillan, London, 1974; (b) J. Cornwell, (Ed.),Nature’s Imagination-The Frontiers ofscientijjc Vision, Oxford University Press, Oxford, 1995; (c) G.R. Bock, J.A. Goode (Eds.),Novartis Foundation Symposium 213, The Limits of Reductionism in Biology, John Wiley and Sons, Chichester, 1998; (d) F. Crick, The Astonishing Hypothesis (Introduction), Simon & Schuster, New York, 1995. A. Stephan, Emergenz-Von der Unvorhersagbarkeit zur Selbstorganisation, Dresden University Press, Dresden, 1999. (a) Several authors, Special section on complex structures, Science 1999, 284,79; (b) T. Vicsek, The bigger picture, Nature 2002, 418, 131; (c) J.M. Ottino, Engineering complex systems, Nature 2004, 427, 399. (a) Z.N. Oltvai, A.-L. Barabasi, Life’s complexity pyramid, Science 2002, 298, 763; (b) L.H. Hartwell, 7.7. Hopfield, S. Leibler, A.W. Murray, From molecular to modular biology, Nature 1999, 402, c47. Several authors, Special section on networks in biology, Science 2003, 301, 1863. Several authors, Special section on systems biology, Science 2002, 295, 1661. M. Rees, Our Cosmic Habitat, Weidenfeld & Nicolson, London, 2001. M. Eigen, R. Winkler-Oswatitsch, Steps Towards Lqe; (a) Part 11, Chapter 4; (b) Part 111, Oxford University Press, Oxford, 1992. (a) H.-J. Rheinberger, Toward History of Epistemic Things-Synthesizing Proteins in the Test Tube, Stanford University Press, Stanford, 1997; (b) H.-J. Rheinberger, A history of protein biosynthesis and ribosome research, in Protein Synthesis and
10.
11.
12.
13.
14.
15.
16.
17.
18.
Ribosome Structure, Eds.: K.H. Nierhaus, D.N. Wilson, Wiley-VCH, Weinheim, 2004. (a) M. Seefelder, Indigo-Kultur, Wissenschaft und Technik, 2nd ed., ecomed Verlagsgesellschaft. Landsberg, 1994; (b) W. Wetzel, Natunvissenschaften und Chemische Industrie in Deutschland, Franz Steiner Verlag, Stuttgart, 1991; (c)W. Abelshauser, (Ed.), Die BASF- Eine Unternehmensgeschichte, Verlag C.H. Beck, Munchen, 2002; (d) E. Baumler, Ein Jahrhundert Chemie (zum 1OOjahrigen Jubilium der Farbwerke Hoechst AG), Dusseldorf, 1963; (e) E. Steingruber, Indigo and indigo colorants, Ullmann’s Encyclopedia ofhdustrial Chemistry, 5th ed., Vol A14, Verlag Chemie, Weinheim. (a) C.A. Russell, Role of synthesis in organic chemistry, Ambix 1987, 34, 169; (b) J.W. Cornforth, The trouble with synthesis, Aust. /. Chem. 1993, 46, 157. R.B. Woodward, in Perspectives in Organic Chemistry, Ed.: A. Todd, Interscience Publishers, New York, 1956, p. 155. F. Wohler, Uber kunstliche Bildung des Harnstoffs, Ann. Phys. Chem. 1828, 12, 253. J. Weyer, 150 Jahre Harnstoffsynthese, Nachr. Chem. Tech. Lab. 1978, 26, 564. C Voigt, Immer eine Idee besser-Forscher und Erfinder der Degussa, Degussa AG, Frankfurt am Main, 1998. A. von Baeyer, Zur Geschichte der Indigo-synthese, Ber. Dtsch. Chem. Ges. 1900, 33, LI, (Sonderheft). G. Quinkert, E. Egert, C. Griesinger, Aspects oforganic Chemistry, Verlag Helvetica Chimica Acta, Basel, 1996; (a) p. 2; (b) p. 55; (c) Fig. 5.4; (d) Section 10.2.6; (e) p. 5 and p. 79; Section 7.5. B.D. Ensley, B.J. Ratzkin, T.D. Osslund, M.1. Simon, L.P. Wackett, D.T. Gibson,
(4
60
I
1 Chemistry and Biology
19.
20.
21.
22. 23.
24.
25.
-
Historical and Philosophical Aspects
Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo, Science 1983, 222, 167. (a) Zhi-Qiang X, M.H. Zenk, Biosynthesis of indigo precursors in higher plants, Phytochemistry 1992, 31, 2695; (b) H. Marcinek, W. Weyler, B. Deus-Neumann, M.H. Zenk, Indoxyl-UDPG-glucosyltransferase from baphicacanthus cusia, Phytochemistry 2000, 53, 201. T. Maugard, E. Enaud, P. Choisy, M.D. Legoy, Identification ofan indigo precursor from leaves of isatis tinctoria (Woad), Phytochemistry 2001, 58,897. (a) E.J. Corey, M. Ohno, R.B. Mitra, P.A. Vatakencherry, Total synthesis of longifolene, J . Am. Chem. SOC. 1964, 86,478; (b) E.J. Corey, General methods for the construction of complex molecules, Pure Appl. Chem. 1967, 14, 19; (c) E.J. Corey, Xue-Min Cheng, The Logic of Chemical Synthesis,Wiley, New York, 1989; (d) E.J. Corey, The Logic of Chemical Synthesis, Nobel Lectures Chemistry 1981-1990, World Scientific, Singapore, 1992, p. 686. S. Warren, Desigrting Organic Syntheses,Wiley, Chichester, 1978. (a) E.J. Corey, W. Todd Wipke, Computer-assisted design of complex organic syntheses, Science 1969, 166, 178; (b) E.J. Corey, Computer-assisted analysis of complex synthetic problems, Q. Rev. 1971, 25, 455; (c) E.J. Corey, A.K. Long, S.D. Rubenstein, Computer-assisted analysis in organic synthesis, Science 1985, 228, 408. (a) R.B. Woodward, Totalsynthese des chlorophylls, Angew. Chem. 1960, 72, 651; (b) R.B. Woodward, Fundamental studies in the chemistry of macrocyclic systems related to chlorophyll, Ind. Chim. Belg. 1962, 11, 1293. (a) D.H.R. Barton, The invention of reactions useful for the synthesis of specifically fluorinated natural
26.
27.
28.
29.
30.
31.
32.
33.
products, Pure Appl. Chem. 1977, 49, 1241; (b) B.M. Trost, Atom economy-A challenge for organic synthesis, Angew. Chem., Int. Ed. Engl. 1995, 34, 259; (c) J.F. Hartwig, Raising the bar for the “Perfect Reaction”, Science 2002, 297, 1653. H.C. Kolb, M.G. Finn, K.B. Sharpless, Click chemistry: Diverse chemical function from a few good reactions, Angew. Chem., Int. Ed. Engl. 2001, 40, 2004. A. Eschenmoser, in Neuorientierung der Chemie-Mode oder mehr? Podiumsdiskussion,Aventis Deutschland, Frankfurt am Main, 2002. G.S. Hammond, Restructuring of chemistry and chemical curricula, Pure Appl. Chem. 1970, 22, 3. A. Eschenmoser, Various comments made on organic synthesis and life sciences, in Chemical SynthesisGnosis to Prognosis, Eds.: C. Chatgillaloglu, V. Snieckus, Kluwer Academic Publishers, Dordrecht, 1996. E. Fischer, Synthetical chemistry in its relation to biology, 1.Chem. SOC. 1907,1749. (a) E. Fischer, Bedeutung der stereochemischen resultate fur die physiologie, Ber. Dtsch. Chem. Ges. 1894, 27, 3228; (b) D.E. Koshland, Jr, The key-lock-theoryand the induced-fit-theory,Angew. Chem., rnt. Ed. Engl. 1995, 33, 2375. A. Todd, J.W. Cornforth, Robert Robinson, Biographical Memoirs of the Fellows of the Royal Society, 1976, 22, 415. (a) E. Dane, Synthesen in der Reihe der Steroide, Angew. Chem. 1939, 52, 655; (b) G. Singh, Structure of Dane’s adduct,]. Am. Chem. SOC.1956, 78, 6109; (c) G. Quinkert, M. Del Grosso, A. Bucher, J.W. Bats, G. Durner, E. Dane’s route to estrone revisited, Tetrahedron Lett. 1991, 32, 3357; (d) G. Quinkert, M. Del Grosso, A. Doring, W. Doring, R.I. Schenkel, M. Bauch, G.T. Dambacher, J.W. Bats, G. Zimmermann, G. Durner, Total synthesis with a chirogenic
References I 6 1
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
opening move demonstrated on steroids with estrone or 18a-Homoestrone skeleton, Helv. Chim. Acta 1995, 78, 1345. R.B. Woodward, Experiments on the synthesis of estrone, 1.Am. Chem. SOC.1940, 62, 1478. A. Eschenmoser, RBW, Vitamin B12, and the Harvard-ETH Collaboration, in Robert Bums Woodward, Eds.: O.T. Benfey, P.J.T. Morris, Chemical Heritage Foundation, Philadelphia, 2001. G. Quinkert, M.V. Kisakurek, From Molecular Structure Towards Biology, Verlag Helvetica Chimica Acta, Zurich, 2001, (a) p. VII; (b) Section 3.2.1. (a) G. Quinkert, H. Stark, Stereoselective synthesis of enantiomerically pure natural products-estrone as example, Angew. Chem., lnt. Ed. Engl. 1983, 22, 637; (b) B. List, J.W. Yang, The organic approach to asymmetric catalysis, Science 2006, 313 1584. (a) K.B. Sharpless, Searching for new reactivity, Nobel Lecture Chemistry 2001; (b) S.Y. KO, A.W.M. Lee, S. Masamune, L.A. Reed, 111, K.B. Sharpless, F.J. Walker, Total synthesis of the L-Hexoses, Tetrahedron 1990, 46, 245. R. Noyori, Asymmetric catalysis: Science and opportunity, Nobel Lecture Chemistry 2001. E.J. Corey, Catalytic enantioselective Diels- Alder reactions: Methods, mechanistic fundamentals, pathways, and applications, Angew. Chem., Int. Ed. Engl. 2002, 41, 1650. S. Drenkard, J. Ferris, A. Eschenmoser, Chemie von a-Amonitrilen, Helv. Chim. Acta 1990, 73, 1373. (a) D. Seebach, A.K. Beck, A. Heckel, TADDOL and its derivatives-our dream of universal chiral auxiliaries, in From Molecular Structure Towards Biology, Verlag Helvetica Chimica Acta, Zurich, 2001; (b) K. Narasaka, Chiral lewis acids in catalytic asymmetric reactions, Synthesis 1990, 1. S.B. Tsogoeva, G. Durner, M. Bolte, M.W. Gobel, A C2-Chiral Bis(amidinium) catalyst for a
44.
45.
46.
47.
48.
49.
50.
51.
52.
Diels-Alder reaction constituting the key step of the quinkert-dane estrone synthesis, Eur. J . Org. Chem. 2003, 1661, and earlier papers. Qi-Ying Hu, P.D. Rege, E.J. Corey, Simple, catalytic enantioselective syntheses of estrone and desogestrel, 1.A m . Chem. Soc. 2004, 126,5984. (a) G. Quinkert, Five Decades of Steroid Synthesis, Vorlesungsreihe Schering, Berlin, 1988, Heft 19; (b) G. Quinkert, M. Del Grosso, Progress in the Diels-Alder reaction means progress in steroid synthesis, in Stereoselective Synthesis, Eds.: E. Ottow, K. Schollkopf, B.G. Schulz, Springer Verlag, Berlin, 1993, S. 109. K. Nicolaou, S.A. Snyder, T. Montagnon, G.E. Vassilikogiannakis, The Diels-Alder reaction in total synthesis, Angew. Chem., Int. Ed. Engl. 2002, 41, 1668. (a) M.B. Groen, F.J. Zeelen, Steroid total synthesis, Red. Trav. Chim. Pays-Bas 1986, 105,465; (b) F.J. Zeelen, Steroid total synthesis, Nat. Prod. Rep. 1994, 607. G . Quinkert, W.-D. Weber, U. Schwartz, H. Stark, H. Baier, G. Durner, Hochselektive totalsynthese von 19-Nor-Steroiden mit photochemischer Schlusselreaktion: Racemische zielverbindungen, Liebigs Ann. Chem. 1981, 2335. G . Quinkert, U. Schwartz, H. Stark, W.-D. Weber, F. Adam, H. Baier, G. Frank, G. Durner, Asymmetrische totalsynthese von 19-Nor-Steroiden mit photochemischer Schlusselreaktion: Enantiomerenreine zielverbindungen, Liebigs Ann. Chem. 1992,1999. T.A. Appel, The Cuvier-Geoffrey Debate, Oxford University Press, New York, 1987. M. Ruse, Evolution, in 7’he Oxford Companion to Philosophy, Ed.: T. Honderich, Oxford University Press, Oxford, 1995. J.P. Eckermann, Gespriiche mit Goethe in den LetztenJahren Seines Lebens, C. Michel, H. Grtiters (Hrsg.), Deutscher Klassiker Verlag, Frankfurt am Main, 1999.
62
I
I Chemistry and Biology - Historical and Philosophical Aspects 53. 54.
55.
56.
57.
58.
59.
60.
61.
62. 63.
64.
65.
66.
J. Browne, Charles Darwin, Vol. 11, A.A. Knopf, New York, 2002. E.A. Carlson, Mendel’s Legacy, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2004. W. Johannsen, Elemente der Exakten Erblichkeitslehre, G. Fischer, Jena, 1909. F.M. Burnet, Evolution made visible, in The Evolution ofLiving Organisms, Ed.: G.W. Leeper, Melbourne University Press, Melbourne, 1962. M. Eigen, Viren als modelle der molekularen evolution, Paul- Ehrlich-Ludwig Darmstadter Award Lecture, Frankfurt am Main, March 14th 1992. (a) M. Eigen, Self-organization of Matter and the Evolution of Biological Macromolecules, Naturwissenschaften 1971, 58,465; (b) Der Code des Lebens, 3 SAT, 26.04.2006, DVD, ZDF, 2006; (c) M. Eigen, From Strange Simplicity to Complex Familiarity, in preparation. G. Strunk, T. Ederhof, Machines for automated evolution experiments in vitro based on the serial-transfer concept, Biophys. Chem. 1997, 66, 193. E. Mayr, What Evolution is, Weidenfeld & Nicolson, London, 2002. I. Rechenberg, Evolutionsstrategie ‘94, frommann-holzboog, Stuttgart-Bad Cannstatt, 1994. J. Maynard Smith, Concept ofprotein space, Nature 1979, 280,445. G. Quinkert, H. Bang, D. Reichert, Variation and selection, Helv. Chim. Acta 1996, 79, 1260. W.B. Provine, Sewall Wright and Evolutionary Biology, The University of Chicago Press, Chicago, 1986. D.L. Hull, History of evolutionary thought, in Encyclopedia of Evolution, Vol. I, Ed.: M. Pagel, Oxford University Press, Oxford, 2002. (a) S.C. Gilbert, J.M. Opitz, R.A. Raff, Resynthesizing evolutionary and developmental biology, Dev. Biol. 1996, 173, 357; (b) J.S. Robert, Embryology, Epigenesis, and
67. 68.
69.
70.
71.
72.
73.
74.
Evolution- Taking Development Seriously, Cambridge University Press, Cambridge, 2004; (c) C.R. Woese, A new biology for a new century, Microbiol. Mol. Biol. Rev. 2004, 173, 68; (d) K.M. Weiss, The phenogenetic logic of life, Nut. Rev. Genet. 2005, 6, 36. J.-M. Lehn, Supramolecular Chemistry, VCH, Weinheim, 1995. P. Ehrlich, Partial cell functions, Nobel Lecture Physiology or Medicine 1908. (a) B. Asbell, The Pill, Random House, New York, 1995; L.V. Marks, Sexual Chemistry, Yale University Press, New Haven, 2001; C. Djerassi, This Man’s Pill, Oxford University Press, Oxford, 2001; (b) G. Pincus, Control of contraception by hormonal steroids, Science 1966, 153, 493. (a) A. Brzozowsky, A.C.W. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, 0. Engstrom, L. Ohman, G.L. Greene, J.-A. Gustafsson, M. Carlquist, Molecular basis of agonism and antagonism in the oestrogen receptor, Nature 1997, 389, 753; (b) E.-E. Beaulieu, Contragestion and other clinical applications of RU 486, an antiprogesterone at the receptor, Science 1989, 245, 1351. C. Djerassi, L. Miramontes, G. Rosenkranz, F. Sondheimer, Synthesis of 19-Nor-17aethynyltestosterone and 19-Nor-17a-methyltestosterone, J . A m . Chem. SOC.1954, 76,4092. G. Quinkert, Hans Herloff Inhoffen in His Times, Eur. J. Org. Chem. 2004,3727. C. Rufer, H. Kosmol, E. Schroder, K. Kiesslich, H. Gibian, Totalsynthese von optisch aktiven 13-Ethyl-gonan-Derivaten, Liebigs Ann. Chem. 1967, 702,141. (a) I.V. Torgov, Progress in the total synthesis of steroids, Pure Appl. Chem. 1963, 6,525; (b) C.H. Kuo, D. Taub, N.L. Wendler, Mechanism of the coupling reaction of a vinyl carbinol with a B-Diketone, J. Org. Chem. 1968,33,3126.
References I 6 3 75.
76.
77.
78.
79.
80.
81. 82.
83.
84.
H. Smith. et al., 13fi-Alkylgona1,3,5(10)-trienes, 13fi-Alkylgon-4en-3-ones, and related compounds, /. Chem. Soc. (London), 1964,4472. (a) U. Eder, G. Sauer, R. Wiechert, Neuartige asymmetrische cyclisierung zu optisch aktiven Steroid-CD-Teilstticken, Angew. Chem., Int. Ed. Engl. 1971, 10. 496; (b) Z.G. Hajos, D.R. Parrish, Asymmetric synthesis of bicyclic intermediates of natural product chemistry,/. Org. Chem. 1974, 39, 1615. H. Hofmeister, K. Annen, H. Laurent, K. Petzoldt, R. Wiechert, Syntheses of gestodene, Drug Res. 1986, 36, 781. G. Sauer, U. Eder, G. Haffer, G. Neef, R. Wiechert, Synthesis of D-Norgestrel, Angew. Chem., Int. Ed. Engl. 1975, 14, 417. M.J. van den Heuvel, C.W. van Bokhoren, H.P. de Jongh, F.J. Zeelen, A partial synthesis of desogestrel based upon intramolecular oxidation of an Recl. 1Ifi-hydroxy-19-norsteroid. Trav. Chim. Pays-Bas 1988, 107, 331. E.J. Corey, A.X. Huang, A short enantioselective total synthesis of the third-generation oral contraceptive desogestrel, /. Am. Chem. Soc. 1999, 121, 710. B. List, Proline-catalyzed asymmetric reactions, Tetrahedron 2002, 58, 5573. Qi-Ying Hu, P.D. Rege, E.J. Corey, Simple, catalytic enantioselective syntheses of estrone and desogestrel, 1.Am. Chem. Soc. 2004, 126,5984. (a) H. Laurent, D. Bittler, H. Hofmeister, K. Nickisch, R. Nickolson, K. Petzoldt, R. Wiechert, Synthesis and activities of anti-aldosterones, J . Steroid Biochem. Mof. Biof.1983, 19, 771; (b) W. Elger, S. Beier, K. Pollow, R. Garfield, S.Q. Shi, A. Hillisch, Conception and pharmacodynamic profile of drospirenone, Steroids 2003, 68, 891. R. Wiechert, in Schering 1971- 1993, S. 149, Schering AG, Berlin, 2005.
85.
86.
87.
88.
89.
90.
91.
92.
(a) G. Quinkert, in High-Tech-Das neue Gesicht der Arzneimitte(forschung, H.1. Dengler, S . Meuer (Hgb.), G . Fischer, Stuttgart, 1995; (b) Several authors in: Special Issue of Science on Drug Discovery 2005, 309, 721-735. F. Aftalion, A History ofthe International Chemical Industry, 2nd. ed., Chemical Heritage Press, Philadelphia, 2001. (a) G. Quinkert, D. Reichert, H.-G. Schaible, B. Cezanne, Final Report of the BMBF Project No. 0310792, Projekttrager Jiilich, 2000; (b) G. Quinkert, Kombinatorische Chemie-ein Paradigmenwechsel in der Chemischen Synthese, Verh. Ges. Dtsch. Naturforscher u. Arzte, 120. Vers., Hirzel Verlag, Stuttgart, 1999; (c) H . 4 . Schaible, Kombinatorische Synthese codierter Verbindungsbibliotheken und Selektion immunsuppressiver Verbindungen, Dissertation, University of Frankfurt am Main, 1997. (a) W.W. Busse, R.F. Lemanske, Asthma, N. Engl. /. Med. 2001, 344, 350; (b) Several authors in: Nature 1999, B l , 402. M. Wills-Karp, J. Luyimbazi, X. Xu, B. Schofield, T.Y. Neben, C.L. Karp, D.D. Donaldson, Interleukin-13: central mediator of allergic asthma, Science 1998, 282, 2258. V. Prelog, Gedanken nach 118 Semestern Chemiestudium, in Chemie und Geseflschaft, Ed.: G . Boche, Wissenschaftl Verlagsges, Stuttgart, 1984, p. 57. D. Brohm, S. Metzger, A. Bhargava, 0. Muller, F. Lieb, H. Waldmann, Natural products are biologically validated starting points in structural space for compound library development, Angew. Chem., Int. Ed. Engl. 2002, 41, 307. (a) A. Furka, F. Sebestyen, M. Asgedom, G. Dibo, General method for rapid synthesis of multicomponent peptide mixtures, Int. /. Pep. Protein Res. 1991, 37, 487; (b) K.S. Lam, S.E. Salmon,
64
I
I Chemistry and Biology
93.
94.
95.
96.
97.
-
Historical and Philosophical Aspects
E.M. Hersh,V.J. Ruby, W.M. Kazmierski, R.J. Knapp, A new type of peptide library for identifying ligand-binding activity, Nature 1991, 354, 82. (a) M.H.J. Ohlmeyer, R.N. Swanson, L.W. Dillard, J.C. Reader, G. Asouline, R. Kobayashi, M. Wigler, W.C. Still, Complex synthetic chemical libraries indexed with molecular tags, Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10922; (b) H.P. Nestler, P.A. Bartlett, W.C. Still, A general method for molecular tagging of encoded combinatorial chemistry libraries, I. Org. Chem. 1994,59,4723. A. Pahl, M. Zhang, K. Torok, H. Kuss, U. Friedrich, Z. Magyar, J. Szekely, I