Combinatorial Chemistry Synthesis, Analysis, Screening

Edited by Gunther Jung

@WILEY-VCH Weinheim . New York . Chichester . Toronto . Brisbane . Singapore

This Page Intentionally Left Blank

Combinatorial Chemistry

Edited by Giinther Jung

@WILEY-VCH

This Page Intentionally Left Blank

Combinatorial Chemistry Synthesis, Analysis, Screening

Edited by Gunther Jung

@WILEY-VCH Weinheim . New York . Chichester . Toronto . Brisbane . Singapore

Prof. Dr. Giinther Jung Institut fur Organische Chemie Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tiibingen Germany

This book was carefully produced. Nevertheless, authors, editor and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statement, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Reprint 2001

Library of Congress Card No: applied for

British Library Cataloguing-in-Publication Data: A catalogue rccord for this book is available from the British Library

Die Deutsche Bibliothek - CIP Einheitsaufnahme Ein Titeldatensatz fur diese Publikation ist bei der Deutschen Bibliothek erhiiltlich

0 WILEY-VCH Verlag GmbH, 0-69469 Weinheim (Federal Republic of Germany), 1999

Printed on acid-free and chlorine-free paper.

All right reserved (including those of translation in other languages). No part o f this book may be reproducted in any form - nor transmitted or translated into machine languagc without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law.

Composition: Datascan GmbH, D-64295 Darmstadt Printing: Strauss Offsetdruck, D-69509 Morlenbach Bookbinding: Osswald & Co. D-67433 Neustadt (WeinstraRe)

Preface

Since I edited Combinatorial Peptide and Nonpeptide Libraries - the first comprehen- sive book on the subject - in 1996, progress in the field of combinatorial chemistry has been truly dramatic. However, before I began to write this preface, I looked back and found the early work by no means outdated - as evidenced by the number of complimen- tary letters received from satisfied readers. A number of excellent books on combinatori- a1 chemistry have appeared since then, covering different aspects of the subject. How- ever, despite this earlier ‘boom’, I was encouraged to edit a second volume, and recog- nized that soon there will be very little overlap of Cornhinutorial Chemistry with the con- tents of other such books.

The past five years have seen the development and introduction of new and improved approaches to the combinatorial sciences, their practical applications having resulted in an almost ‘explosive’ increase in the number of experimental publications and new jour- nals devoted exclusively to these areas. Thus, a comprehensive book which emphasizes the state of the art of most of the methods and instrumentation of combinatorial chemis- try may be of value to those who may consider entering this exciting field. It is possible al- so that experts in combinatorial organic chemistry may find it interesting to examine more closely exciting new fields such as catalyst research, chemosensor development, new analytical tools, and the creation of biodiversity.

As a logical consequence of the demand for combinatorial chemists in industry, teach- ing the subject and related key technologies should be a major topic at our universities. It is hoped that some of the chapters of this book are sufficiently convincing as to encour- age future scientists not only to use combinatorial methods in their research, but also to offer training for their students or co-workers. Indeed, for some years, I have offered pub- lic courses in multiple methods in peptide chemistry, and presently my group teaches theoretical and practical methods in combinatorial organic chemistry.

As we approach the end of the 20th century, we observe two major trends in the drug discovery process: first, the production of large numbers of single compounds and com- plex libraries in small scale (microgram) quantities; and second, the production of prep- arative-scale amounts (mg) of single compounds with complete analytical characteriza- tion and defined purity criteria, and less complex mixtures. A t present, the nanoscale syn- thesis and on-line screening of 100000 compounds per day remains a dream for organic chemists - except perhaps for primitive compounds and rather simple biological targets. Nonetheless, such wishes may be realized for oligomers accessible by repetitive synthetic steps.

VI Preface

Comhinutoriul Chemistry begins with a general overview on recent advances in the subject, with expert surveys provided on selected solid-phase organic reactions and - for the first time - also on solution-phase combinatorial chemistry. These chapters are fol- lowed by a detailed survey of the broad research into multicomponent reactions.

The chapter on solid supports in the first book has now been supplemented by a com- plete listing of the various solid-phase anchors and linkers used in organic chemistry - in- formation which may be of particular value to newcomers in the field.

As a consequence of the progress and success of diverse oligomer libraries, experts in combinatorial synthetic oligomers, glycopeptide and oligosaccharide libraries, RNA- and DNA-aptamers have each contributed their news in these fields, while novel experi- mental examples of the use of templates in combinatorial chemistry for the solid-phase synthesis of multiple core structure libraries provide some insight to praxis-relevant work.

A complete chapter is devoted to novel combinatorial approaches to highly selective chemoreceptors designed for parallel analysis by sensor devices. An assay-oriented chap- ter on peptide libraries in T-cell-mediated immune response illustrates how libraries of extreme complexity can lead directly to single molecules with activities that may be

Results of combinatorial biosynthesis using gene clusters responsible for metabolites of potential pharmaceutical interest illustrate the state of the art in this field. Library de- sign and diversity analysis are of increasing importance, and the present programs and approaches are discussed, together with expert views and opinions. In particular, the chapter on a miniaturized ultra-high throughput screening system highlights the immedi- ate future in assay systems of this type.

Combinatorial approaches in materials sciences and solid-state catalysts are relatively new, and these are reviewed, together with expert contributions in the field.

Among the last five chapters, four are devoted to the important instrumentation used for analytical on- and off-bead controls. Whereas tagging concepts appear increasingly less interesting due to excessive time constraints and inherent problems introduced in the drug discovery process, a number of methods have proved suitable for routine single- bead analysis, including infra-red microscopy, separation techniques coupled to electro- spray-mass spectrometry, and ion-cyclotron resonance-FT-mass spectrometry to deter- mine the elemental composition of each component in a library. In addition, MAS-NMR has become a popular means of examining resin-bound intermediates during feasibility and optimization studies.

Finally, Comhinutoriul Chemistry closes with a critical discussion of the various at- tempts to automate combinatorial synthesis, with a presentation of a newly developed workstation used to perform fully automated parallel synthesis, including all steps through work-up to sample preparation for screening modules.

I would like to close this Preface by expressing my gratitude to all authors and co-au- thors who spent their precious time in contributing to this work. I also acknowledge the present and former co-workers of my group who contributed novel experimental results.

I am very grateful to Mrs. Ursula Sanzenbacher for her help during the editorial work, and to Dr. Gudrun Walter and Dr. Peter Golitz from Wiley-VCH for their continuous interest on this edition.

up to 100000-fold higher than those of natural epitopes.

During the past few years - and in parallel with combinatorial chemistry - communica- tion via the Internet and the availability of published material from databases have emerged exponentially. However, I am confident that a broad readership may still - from time to time - enjoy reading a scientific book such as Combinatorial Chemistry.

Tiibingen, September 1999 Giinther Jung

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Contents

1.

1.1 1.2 1.3 1.3.1 1.3.1.1 1.3.1.2 1.3.1.3 1.3.2 1.3.2.1 1.3.2.2 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.2

1.4.2.1 1.4.2.2 1.5 1.5.1 1.5.2 1.6 1.6.1 1.6.2 1.7 1.8 1.9

Combinatorial Chemistry Dominikn Tiebes

Introduction 1 Principles of Combinatorial Chemistry 2 Methods and Techniques of Combinatorial Synthesis Synthetic Strategies Towards Combinatorial Libraries Split-Pool Synthesis Towards Combinatorial Libraries Parallel Synthesis Towards Combinatorial Libraries Reagent Mixture Synthesis Towards Combinatorial Libraries Synthetic Methodology for Organic Library Construction Solid-Phase Organic Synthesis 12 Synthesis in Solution and Liquid-Phase Synthesis Characterization of Combinatorial Libraries 14 Analytical Characterization 14 Analytical Characterization of Compound Mixtures Analytical Characterization of Single Substances Hit Identification in Combinatorial Libraries by High-Throughput Screening 15 Strategies for Libraries of Compound Mixtures Strategies for Libraries of Separate Single Compounds Automation and Data Processing 24 Synthesis Automation and Data Processing Automated Purification 25 Library Design and Diversity Assessment Diversity Assessment for Selection of Building Blocks or Compounds Iterative Optimization Methods 28 Economic Aspects 28 Acknowledgements 30 References 30

4 4 4

7 10

12

13

14 15

15 23

24

26 27

X Con tcw ts

2.

2.1 2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.2 2.2.3 2.2.4 2.3 2.4 2.4.1

2.4.1.1 2.4.1.2 2.4.1.3 2.4.1.4 2.4.1.5 2.4.1.6 2.4.1.7 2.4.1.8 2.4.2

2.4.2.1 2.4.2.2. 2.4.2.3 2.4.2.4 2.4.3 2.4.4 2.4.4.1 2.4.4.2 2.4.4.3 2.4.4.4 2.4.4.5 2.4.4.6 2.4.4.7 2.4.4.8 2.4.4.9 2.4.4.10 2.4.4.11 2.4.4.12 2.4.4.13

Survey of Solid-Phase Organic Reactions Susan E. Booth, C. Marijke Dree,f- Tromp, Petro H. H. Hermkens, Jos A.PA. de Man and H.C. J. Ottenheijm

Introduction 35 Observed Trends 36 The Synthetic Repertoire 36 Robust, Reliable Solid-Phase Reactions 36 Emerging Solid-Phase Reactions 36 Linkers and Cleavage Step 37 Reaction-Monitoring 37 Highlights 37 Conclusions 37 Reaction Tables 38 Substitution Nucleophilic and Electrophilic Type of Reaction: Amide Bond Formation and Related Reactions 38 Sulphonamide 39 (Thio)urea 39 Carbonate 40 Urethane 40 Guanidine 41 Imide 41 Amide 42 Lactam 42 Type of Reaction: Aromatic Substitution; Electrophilic Carbon-Carbon Bond Formation 42 Suzuki 43 Stille 43 Heck 44 Other 45 Type of Reaction: Aromatic Substitution; Nucleophilic (N-Arylation) 45 Type of Reaction: Cleavage 44 Cyclative Cleavage 46 Functional Group: None (traceless) 47 Functional Group: Halogens 48 Functional Group: Alkenes 48 Functional Group: Alcohols, Phenols 49 Functional Group: Primary Amine 49 Functional Group: sec-Amine SO Functional Group: tert-Amine SO Functional Group: Aldehyde/Ketone 51 Functional Group: Hydroxamic Acid 51 Functional Group: Amidine 52 Functional Group: Guanidine 52 Functional Group: sec Amideltert Amide/Sulfonamide 52

Con ten I S XI

2.4.5 2.4.6 2.4.6.1 2.4.6.2 2.4.6.3 2.4.7 2.4.8 2.4.8.1 2.4.8.2 2.4.8.3 2.4.8.4 2.4.9 2.4.9.1 2.4.9.2 2.4.9.3 2.4.10 2.4.1 1 2.4.11.1 2.4.11.2 2.4.1 1.3 2.4.11.4 2.4.12 2.4.12.1 2.4.12.2 2.4.12.3 2.4.12.4 2.4.12.5 2.4.13 2.4.13.1 2.4.13.2 2.4.13.3 2.4.13.4 2.4.13.5 2.4.13.6 2.4.14 2.4.14.1 2.4.14.2 2.4.14.4 2.4.14.4 2.4.14.5 2.5

Type of Reaction: Condensation 53 Type of Reaction: Cycloaddition 54 [2+2] Cycloaddition 54 [3+2] Cycloaddition 54 [4+2] Cycloaddition 55 Type of Reaction: Grignard and Related Reactions Type of Reaction: Heterocycle Formation Nitrogen-Containing Heterocycles 56 Multiple Nitrogen-Containing Heterocycles 57 Oxygen-Containing Heterocycles 58 Sulphur-Containing Heterocycles 58 Type of Reaction: Michael Addition Immobilized Nucleophile 59 Thiol Addition 59 Amine Addition 59 Type of Reaction: Miscellaneous 60 Type of Reaction: Olefin Formation Wittig 61 Horner-Emmons 62 Cross-Metathesis 62 P-Elimination 62 Type of Reaction: Oxidation 62 Alcohol to Aldehyde/Ketone 62 Sulfide to Sulfoxide/Sulfone 63 Epoxidation 64 Ozonolysis 64 Other 64 Type of Reaction: Reduction 65 Aldehyde/Ketone to Alcohol 65 Nitro to Aniline 65 Azide to Amine 66 Amide to Amine 66 Reductive Alkylation/Amination 66 Other 67 Type of Reaction: Substitution 67 C-Alkylation (Aldol, Anion) 67 0-Alkylation (Mitsunobu, Anion) 68 N-Alkylation (Mitsunobu, Anion) 69 N-Alkylation (Sulfonamide, Amide) 69 S-Alkylation 70 References 70

55 55

58

61

XI1 Con tent.\

3.

3.1 3.2 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 3.3.11 3.3.12 3.3.13 3.4 3.4.1 3.4.2 3.4.3 3.4.4 3.4.5 3.5 3.6

4.

4.1 4.2 4.3

4.4 4.5 4.6

4.7 4.8 4.9 4.10 4.11

Solution-Phase Combinatorial Chemistry Volkhard Austel

Comparison with Solid-Phase Combinatorial Synthesis 77 Synthesis of Mixtures 79 Reactions Applied to Solution Phase Combinatorial Chemistry Acylation of Alcohols and Amines Sulfonation of Amines 86 Formation of Ureas, Thioureas and Carbamates Alkylation and Addition Reactions 86 Reductive Amination 88 Arylation of Amines 89 C-C Bond Formation via Condensation Reactions Pd-Catalyzed C-C Bond Formation 89 Hydrogenations and Reductions 93 Multicomponent Reactions 93 Cyclisation Reactions 95 Miscellaneous Reactions 95 Reaction Sequences 103 Purification 106 Solid-Phase-Bound Reagents 106 Solid-Phase Extraction 109 Liquid-Phase Extraction 114 Fluorous Synthesis 116 Synthesis on Soluble Polymers 117 Conclusions 119 References 11 9

80 81

86

89

Combinatorial Chemistry of Multicomponent Reactions Ivar Ugi, Alexander Diimling and Birgit Ehert

Classical and Modern Chemistry of Isocyanides and MCRs Early Studies and Concepts of MCR Chemistry Conceptual Differences between Conceptual Chemical Reactions andMCRs 127 The Different Types of MCRs The First Century of Isocyanide Chemistry Complementary Aspects of Natural Product Syntheses by Tandem-Domino Reactions and MCR Chemistry Modern Synthesis of the Isocyanides The Introduction of the New Isocyanide MCRs and their Libraries The Great Variability of the U-4CR The Usual and Unusual Peptide Chemistry Stereoselective U-4CRs and their Secondary Reactions

125 126

128 130

132 134

135 137

141 144

Contents XI11

4.12 4.13 Unions of MCRs and Related Reactions 153 4.14 The MCRs of More than Four Functional Groups 154 4.15 4.16 Progress in Combinatorial MCR Chemistry 160 4.17 Perspectives 161 4.18 Acknowledgements 162 4.19 References 162

The MCRs of Educts with Two or Three Functional Groups 149

New Unions of the U-4CR and Further Chemical Reaction 156

5. Solid-Phase Anchors in Organic Chemistry Ralf Warrass

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.8.1 5.8.2 5.8.3 5.8.4 5.8.5 5.9 5.10

Introduction 167 Acid-Labile Anchors 171 Anchors Cleaved by Nucleophiles 187 Photolysis-Labile Anchor 200 Allyl-Functionalized Anchors 204 Safety-Catch Anchor 207 Silicium-Anchor/Traceless-Anchor 212 Miscellaneous 21 6 Multifunctional Anchors 216 Anchors Cleaved by Reduction 217 Hydrogenolysis-Labile Anchors 218 Anchors Cleaved by Oxidation 219 Enzymatically Cleavable Anchors 219 Acknowledgements 220 References 221

6. The Use of Templates in Combinatorial Chemistry for the Solid Phase Synthesis of Multiple Core Structure Libraries Hartmut Richter, Axel Trautwein, Tilmann Walk and Giinther Jung

6.1 6.2 6.3 6.3.1 6.3.2 6.3.2.1 6.3.2.2 6.3.2.3 6.3.2.4 6.3.2.5

Introduction 229 Squaric Acid as Template 231 Templates Derived from the Baylis-Hillman Reaction Baylis-Hillman Reaction on the Solid Phase The Use of 3-Hydroxy-2-Methylidene Propionic Acids as Templates Michael Addition of Amines 235 Mitsunobu Reaction 236 1,3-Dipolar Cycloaddition with Nitrile oxides Synthesis of 2-Methylidene-P-Alanines 238 Synthesis of Pyrazolones 240

233 233

234

237

XIV Con ten t.s

6.3.2.6 6.3.3 6.4 6.4.1 6.4.2 6.4.3

6.4.4 6.4.5 6.4.6 6.5 6.5.1 6.5.2 6.5.3 6.5.4 63.5 6.5.6 6.5.7 6.6 6.7

Synthesis of 2-Diethoxy-Phosphorylmethyl Acrylic Acids 242 Alkylation of 2-Arylsulfonylaminomethyl Acrylic Acids 243 5-(2-Bromoacetyl)Pyrroles as Templates 244 Synthesis of Polymer-Bound 5-(2-Bromoacetyl)pyrroles 244 Synthesis of .5-(2-Aminoacetyl)pyrroles 246 Synthesis of Thiazolpyrroles, Aminothiazolylpyrroles and Selenazolylpyrroles 246 Synthesis of Imidazol[l,2-a]Pyri(mi)dylpyrroles 247 Synthesis of Benzofurylpyrrolylketones 248 Examples 248 The Use of Enones as Templates Synthesis of Polymer-Bound Enones 250 Michael Addition of Aryl Thiolates Synthesis of Pyridines and Pyrido[2,3-d]pyrimidines Synthesis of Pyridones 252 Synthesis of Pyrrolidines 252 Synthesis of Pyrimidines and Pyrimidones Synthesis of Pyrazoles 254 Conclusions 255 References 255

249

250 2.51

253

7. Combinatorial Synthetic Oligomers Alherto Bianco

7.1 7.2 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.3 7.4 7.5 7.6 7.6.1 7.6.2 7.6.3 7.6.4 7.7 7.7.1 7.7.2 7.7.3 7.7.4

Introduction 257 Peptoids 258 Submonomer Approach to Peptoid Synthesis Monomer Approach to Peptoid Synthesis Peptomers 260 Peralkylated Peptides 261 P-Peptoids 262 Oligocarbamates 263 Sulfonopeptides and Vinylogous-Sulfonamidopeptides 264 Poly-N-Acylated Aniines 266 Oligoureas 268 Linear Ureas 268 Thioureas 270 Oligocycloureas and Cyclothioureas 270 Ureapeptoids 271 Heterocyclic Ring-Containing Oligomers 271 Poly-N-Methyl-Pyrroles and Imidazoles 271 Thiazole and Oxazole Ring-Containing Peptides Polyisoxazol i nes 273 Oligothiophenes 273

258 259

272

Contenrs XV

7.7.5 7.7.6 7.8 7.8.1 7.8.2 7.8.3 7.8.4 7.8.5 7.8.6 7.8.7 7.9 7.9.1 7.9.2 7.9.3 7.9.4 7.9.5 7.9.6 7.10 7.11 7.12

8.

8.1 8.2 8.3 8.4 8.5 8.5.1 8.5.2 8.5.3

8.6 8.6 8.5 8.9 8.10 8.11

8.12 8.13 8.14 8.15

Oligotetrahydrofurans 273 Pyrrolinone-Containing Oligomers 274 Other Examples of Synthetic Oligomers Vinylogous Polypeptides 275 Retro-inverso Pseudopeptides 276 Azatides and Azapeptides 277 Polyketides 278 P-Polypeptides 278 a,a-Tetrasubstituted Amino Acids-Containing Peptides 279 Peptide Nucleic Acids 280 Single Position Peptide Bond Modifications Thioamide Pseudopeptides 281 Reduced-Amide Bond Peptides 282 N-Hydroxyamide Bond-Containing Peptides 282 Hydroxyethylamine Peptide Bond Isosteres 283 Methylene Ether Isosteres 284 Phosphono- and Phosphinopeptides 285 Summary 286 Acknowledgements 286 References 287

275

281

Glycopeptide and Oligosaccharide Libraries Phizedria M. St. Hilaire and Morton Meldal

Parallel Arrays versus Libraries of Compounds Proteins Recognising Carbohydrates 291 The Carbohydrate Ligands 293 Supports for Solid-Phase Libraries 296 Analytical Tools for Oligosaccharide and Glycopeptide Libraries Tagging Techniques in Oligosaccharide Libraries Analysis by Mass Spectrometry 298 Structural Analysis of Compounds Linked to Single Beads

Introduction to Array and Library Synthesis of Oligosaccharides Parallel Synthesis of Oligosaccharide Arrays Synthesis of Oligosaccharide Libraries 302 Glycopeptide Templates as Oligosaccharide Mimetics Parallel Synthesis of Glycopeptide Arrays Preparation and AnaIysis of Solid-Phase Clycopeptide Template Libraries 31 0 Screening of a Solid-Phase Glycopeptide Library 3 12 Conclusions 31 3 Acknowledgements 313 References 314

291

298 298

by MAS-NMR 300 301

301

305 307

XVI Contents

9.

9.1 9.2 9.3

9.4 9.5 9.6 9.7

10.

10.1 10.2 10.3 10.4 10.5

10.5.1 10.5.2

10.6 10.7

11.

11.1 11.1.1 11.1.2 11.1.3 11.1.4 11.1.4.1 11.1.4.2 11.1.5 11.2.5.1 11.1.5.2

RNA and DNA Aptamers Michael Fumulok

Introduction 319 Aptamers for Small Molecules 320 Functional Aptamers for Proteins and their Application in Biotechnology, Molecular Medicine, and Diagnostics 324 Functional Aptamers In Vivo 327 Conclusions 330 Acknowledgements 331 References 331

Combinatorial Approaches to Molecular Receptors for Chemosensors Dietmar Leipert, Jiirgen Muck, Rolf Tiinnemann and Giinther Jirng

Introduction 335 Supramolecular Recognition Sites 336 Macrocyclic Peptides 339 Combinatorial Receptor Libraries 340 Cyclopeptides as Supramolecular Recognition Sites for Chemosensors 342 Quartz Microbalance Measurements in the Liquid Phase Reflectometric Interference Spectroscopy (RIfS) in the Liquid Phase 346 Outlook 3.51 References 351

342

Peptide Libraries in T-Cell-Mediated Immune Response Burkhard Fleckenstein, Giinther Jung and Karl-Heinz Wiesmiiller

Introduction 355 Generation and Presentation of Antigens Combinatorial Peptide Libraries 356 Peptide Libraries for the Investigation of TAP 356 MHC Class I and Class I1 Molecules Peptide Ligands of MHC Class I Molecules 358 Peptide Ligands of MHC Class I1 Molecules T-cell Response 360 Recognition of MHC Class I-Bound Peptides by TCR Recognition of MHC Class 11-Bound Peptides by TCR

355

357

359

360 361

11.2 11.2.1 11.2.2 11.2.3 11.2.4 11.3 11.3.1 11.3.2 11.3.2.1 11.3.2.2

11.3.2.3

11.3.2.4 11.3.2.5 11.3.2.6 11.3.2.7 11.3.2.8 11.4 11.5 11.6

12.

12.1 12.2 12.3 12.3.1 12.3.2 12.3.3 12.3.4 12.3.5 12.3.5.1 12.3.5.2 12.3.5.3 12.4 12.4.1

12.4.2 12.4.3 12.5

Methods 362 Synthesis of Peptides and Peptide Libraries Analytical Characterization of Peptide Libraries HPSEC-Competition Assay 363 Isolation of HLA Class I1 Molecules Results and Discussion 366 Optimal Length of Peptides for MHC Class I1 Interaction Activity Pattern Describing DRl/Peptide Interaction Tolerance to Amino Acid Variations in a HLA Class I1 Ligand Competition of Peptides Deduced from the Activity Pattern of the Undecapeptide Library 371 Predictions of HLA Class 11-Ligands and T-cell Epitopes by the Algorithm, Actipat 371 Peptide Libraries and the Antigen Recognition of CD4' T Cells T-cell Response to Completely Randomized Peptide Libraries Complete Dissection of the Epitope for TCC 5G7 Evaluation of TCR Contact Positions Superagonists and Prediction of Epitopes Conclusions 376 Acknowledgements 376 References 376

362 363

363

366 366

370

372 373

374 375

375

Combinatorial Biosynthesis of Microbial Metabolites Andreas Bechthold and Jos& Antonio Salns Fernurzdez

Introduction 381 Cloning of Biosynthetic Gene Clusters New Drugs by Genetic Engineering 382 New Drugs by Targeted Gene Disruption New Drugs by Expression of Single Genes New Drugs by Expression of Gene Clusters New Drugs by Variation of the Starter Units New Drugs by Recombinant Assembly of Enzymatic Subunits Polyketide Synthases 391 Peptide Synthases 399 Proteins Involved in Deoxysugar Biosynthesis Future Perspectives 403 Use of Genes Involved in the Biosynthesis of Oligosaccharide Antibiotics 403 Use of Genes from Other Sources - New Compounds from Earth Changing the Substrate Specificity of an Enzyme References 404

381

384 385 388 390

390

402

404 404

XVIII Contents

13.

13.1 13.2 13.3 13.4 13.4.1 13.4.2 13.4.3 13.4.4 13.5 13.5.1 13.5.2 13.5.3 13.6 13.6.1 13.6.2 13.6.3 13.6.4 13.7 13.7.1 13.7.2 13.7.3 13.8 13.9 13.10

14.

14.1 14.1.1 14.1.2 14.1.3 14.1.4 14.2 14.2.1 14.2.2 14.2.3 14.2.4

Design and Diversity Analysis of Compound Libraries for Lead Discovery Hans Matter and Matthias Rarey

Introduction 409 Concepts and Issues for Combinatorial Library Design The Similarity Principle 412 Molecular Descriptors and Selection Techniques 413 Two-Dimensional Fingerprints 413 Pharmacophore Definition Triplets 414 Other Descriptors 414 Compound Selection Techniques 415 Selected Examples of New Approaches to Molecular Similarity 416 Affinity Fingerprints 417 Feature Trees 418 Automated Structural Superposition of Fragments Descriptor Validation Studies 421 2D Versus 3D Descriptors for Global Diversity 421 Random Versus Rational Design for Global Diversity 3D Pharmacophore Definition Triplets Versus 2D Fingerprints 424 Local Similarity -The Radius of Similarity Designing Combinatorial Libraries 428 Strategy 428 Practical Issues 429 Analysis of Diverse Libraries 432 Conclusions 433 Acknowledgement 435 References 435

410

420

422

426

How to Scale-upwhile Scaling Down: EVOscreenTM, a Miniaturized Ultra High Throughput Screening System Rodney Turner, Sylvia Sterrer, Karl-Heinz Wiesmiiller and Franz-Josef Meyer-Almes

Introduction to Drug Discovery and Screening A Rationale for New Methods in Drug Discovery 441 Chemistry, Biology and Technology 442 Sources of Compounds for HTS Demands on HTS 444 Combinatorial Chemistry 445 Design and Production of Chemical Diversity Microreactor Systems 446 Procedures for the Synthesis of Combinatorial Compounds Solution-Phase Combinatorial Chemistry 447

441

444

445

446

14.2.5 14.3 14.3.1 14.3.2 14.3.2.1 14.3.2.2 14.3.2.3 14.3.2.4 14.3.3 14.3.3.1 14.3.3.2 14.3.4 14.3.5 14.4 14.4.1

14.4.1.1 14.4.2 14.4.3 14.5 14.6 14.7

Limitations on Combinatorial Design Strategies Screening Technology 448 The Ideal Read-out Technology: Confocal Fluorescence Examples of Specific Fluorescence Detection Technologies Fluorescence Correlation Spectroscopy (FCS) 450 Fluorescence Resonance Energy Transfer (FRET) Fluorescence Polarization (FP) 45 1 Fluorescence Imaging 451 EVOscreen ' 451 Liquid Handling 452 ScannerIPicker 453 Blurring the Lines Between Primary, Secondary and Tertiary Screening Using Primary Cells 455 Practical Application 45.5 Association Kinetics of Gene Product Fragments Derived from E. coli P-Galactosidase 455 Association Kinetics 457 Comparison of Native (EA), and Indicated (EA), FCS Adaption 459 Summary 459 Acknowledgements 460 References 460

448

448 450

450

454

459

15. High Throughput Experimentation in Catalysis Ferdi Schiith, Christiun Hofjiniunn, Anke Wolf; Stephan Schimk, Wolfram Stichert and Armin Brenner

15.1 15.2 15.2.1 15.2.2 15.3 15.4 15.5 15.6 15.7

Introduction 463 General Considerations 464 Motivation for and Problems of HTE Approaches in Catalysis Strategies for Library Design and Testing Approaches to Synthesis 469 Approaches to Testing 471 Conclusions 476 Acknowledgements 477 References 477

464 467

16. FT-Infrared Spectroscopy and IR-Microscopy for On Bead Analysis of Compound Libraries Holger Bandel, Wolfgang Haap and Giinther Jimg

16.1 Introduction 479 16.2 Analytical Methods Using FT-IR Spectroscopy 479

xx Contents

16.2.1 16.2.2 16.2.3 16.2.3.1 16.2.3.2

16.2.3.3

16.2.4 16.2.4.1

16.2.4.2 16.2.4.3 16.3 16.4

KBr Pellet Method 479 ATR-Spectroscopy 482 FT-IR Microscopy 484 Single Bead Reaction Monitoring 484 Examination of the Interaction between Resin-Bound Reactive Groups via IR-Microscopy 485 FT-IR Mapping: A New Tool for Spatially Resolved Characterization of Polymer-Bound Combinatorial Compound Libraries with IR-Microscopy 490 Other IR-Spectroscopy Methods 496 DRIFTS (Diffuse Reflectance Infrared Fourier Transform) Spectroscopy 496 Photoacoustic Spectroscopy 497 FT Raman Spectroscopy 497 Conclusions 497 References 498

17. Mass Spectrometric Analysis of Combinatorially Generated Compounds and Libraries Roderich Sujhuth, Axel Trautwein, Hurtmut Richter, Grueme Nicholson and Giinther Jung

17.1 17.2

17.2.1 17.2.2

17.2.2.1 17.2.3 17.2.4 17.2.4.1 17.2.4.2 17.2.5 17.2.6 17.2.6.1 17.2.6.2 17.2.6.3 17.3 17.3.1 17.3.1.1 17.3.1.2 17.3.1.3 17.3.1.4

Introduction 499 Significance of Mass Spectrometry in the Analytical Concept of Combinatorial Chemistry 499 Decisional Pathway for Combinatorial Synthesis and Analysis Mass Spectrometric Techniques for Combinatorial Compound Analysis 502 Ionization Techniques 503 Analysis of Peptide- and Oligonucleotide-Libraries SO4 Analysis of Non-Oligomeric Compounds and Libraries 505 Direct Analysis of Non-Oligomeric Compound Libraries Coupling of Separation Techniques with Mass Spectrometry “On-Bead’’ versus “Off-Bead’’ Analysis SO6 High-Throughput Systems for Sample Analysis and Purification High-Throughput Analysis SO6 High-Throughput Sample Purification using MS-Detection Automated Software-Aided Spectra Evaluation SO8 Application of ES-MS in Combinatorial Chemistry Analysis ES-MS-Analysis of Single Compounds SO9 Influence of Resin Types on the MS-Analysis Influence of Reagents on the MS-Analysis SO9 Influence of Fragmentation on the MS-Analysis High-Throughput Analysis 51 1

499

505 505

506

SO7

SO8

509

51 I

Conretits XXI

17.3.2 17.3.2.1 17.3.2.2 17.4 17.5 17.5.1 17.5.2 17.6 17.7 17.8

ES-MS-Analysis of Non-Oligomeric Libraries ES-MS-Analysis of a Pyrrol-Library 514 MS-Analysis of an Isoxazoline-Library 523 Conclusions 528 Materials and Methods 528 Electrospray-Mass-Spectrometry 528 GC-EI Mass Spectrometry 529 Acknowledgements 529 Appendix 529 References 531

5 14

18. High Resolution Magic Angle Spinning (MAS) NMR Spectroscopy for On-Bead Analysis of Solid-Phase Synthesis Ralf Warruss and Guy Lippens

18.1 Introduction 533 18.2 Methods Used for On-Bead NMR Analysis 533 18.3

18.4 Use of Protonated Solvent in HR MAS NMR 539 18.5 Summary and Perspectives 540 18.6 References 542

Sample Preparations, Observations and Quantifications by HR MAS NMR 535

19. Automated Combinatorial Chemistry Martin Winter, KarLHeinz Wiesmiiller and Giinther Jiing

19.1 19.2 19.3 19.4

19.4.1 19.4.2 19.4.3 19.4.4 19.5

19.6 19.7 19.8

Introduction 543 Parallel Synthesis of Individual Compounds Productivity in the Research Laboratory Automation Concepts: How Multicomponent Systems are Organized 545 General Considerations 545 Decentralized Automation Systems 549 Central-Controlled, Function-Oriented Multicomponcnt Systems 550 Central-Automated, Sample-Oriented Multicomponent Systems 551 Collateral Technologies for High-Throughput Purification and Analysis 556 Summary and Prospects 557 Acknowledgements 558 References 558

544 545

20.

20.1 20.2 20.2.1 20.2.1.1 20.2.1.2 20.2.1.3 20.2.2 20.2.2.1 20..2.2.2 20.2.2.3 20.2.2.4 20.3 20.3.1 20.3.1.1 20.3.1.2 20.3.1.3 20.3.2 20.3.2.1 20.3.2.2 20.4 20.5

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) for Analysis of Compound Libraries Tilman B. Walk, Axel Trautwein, Holger Bundel and Giinther Jung

Introduction 561 Physical Basis of FT-ICR Mass Spectrometry Instrument 562 Supercconducting Magnet 562 Vacuum System 563 Analyzer Cell 563 Detection Principle 564 Cyclotron Motion 564 Signal Generation 565 Mass Accuracy 567 MS”-Experiment 567 FT-lCR-MS Analysis 567 Combinatorial Compound Collections 567 Synthesis of Pyrrole Amide Collections Automated Direct Measurement 569 MicroLC/ES-FT-ICR-MS-Coupling 573 Single Bead Analysis 575 Synthesis of Pyrrolidine Libraries 575 Result of Single Bead Analysis Materials and Methods 578 References 579

562

568

577

Colour Plates 583

Index 589

List of Abbreviations

A-3CR Aha Ac ACN ACP ADME ADPV Alloc AM AMCA ANP APCI ATP

BAL BAP

BBTO BEMP

ATR-IR

BB-4CR

BHA BINAP Bn, Benz Boc BOP Bz CAD CALD CAN CBP Cbz CD CDI CE

Asinger - three component reaction 2-aminobenzoic acid acetyl acetonitrile acyl carrier protein absorption, distribution, metabolism, excretion 5-(4-aminomethyl-3,5-dimethoxy-phenoxy)-valeric acid allyloxycarbon yl aminomethyl 7-amino-4-methyl-coumarin-3-acetic acid 3-amino-3-(2-nitrophenyl) propionic acid atmospheric pressure chemical ionization adenosine triphosphate attenuated total reflection infrared spectroscopy backbone amide linker borane/pyridine complex Berg-Bucherer - four component reaction bis-( tributy1)tinoxide 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro- 1,3,2-diazaphos phorine benzyhydrylamine 2,2’-bis(dipheny1phosphino)-1 ,l’-binaphthyl benzyl tert .-butyloxycarbonyl benzotriazolyloxy-tris(dimethy1amino)phosphonium hexafluorophosphate benzoyl collision activated dissociation computer assisted library design ceric ammonium nitrate carbohydrate binding proteins benzyloxycarbony 1 circular dichroism N,N-carbon yldiimidazol capillary electrophoresis

XXIV

CHC CHO Chx CID CID CLEAR CLF CLIP CMPP CoA Cod CPG CRD CTL cHex

CZE Da DABCO DBE DBU DCI DCM Dde DDQ DEAD DEBS DH DIAD DIBAH DIBAL DIC DIEA DMAE DMAP DMF DMSO DMTST DNA DRIFTS DSB DVB EA EBV

CY

List of Ahhreviutions

a-cyano-4-hydroxy-cinnamic acid Chinese hamster ovary (cells) cyclohexyl collision induced decay collision induced decomposition cross-linked ethoxylate acrylate resin chain length factor protein class IT-associated invariant chain peptide chloromethyl phenyl pentyl polystyrene coenzyme A cycloactadien yl controlled pore glass carbohydrate recognition domain cytotoxic T lymphocytes cyclohexyl cyclohexyl capillary zone electrophoresis dalton 1,4-diazabicyclo[2.2.2]octane double bond equivalent 1,8-diazabicyclo[5.4.0]undec-7-ene desorption chemical ionization dichloromethane 1-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethy~ dichlorodicy ano-p-benzochinon diethylazodicarboxylate 6-deoxyerythronolide B synthase dehydratase diisopropyl azodicarboxylate diisobutylaluminium hydride diisobutylaluminium hydride diisoprop ylcarbodiimide diisopropylethylamine dimethy laminoethanol 4-N,N-dimethylaminopyridine N,N-dimethylformamide dimethylsulfoxide dimethyl(methylthio)sulfonium-trifluoroinethylsulfonat~ desoxyribonucleic acid diffuse reflectance infrared Fourier transform spectroscopy 4-(2,5-dimethyl-4-methylsulfinylphenyl)-4-hydroxybutyric acid divinylbenzene enzyme acceptor Epstein-Barr virus

ED enzyme donor

EDC EI ELISA ER ER ESI

Et FAB FCS FITC FMN Fmoc FP FRET FT

Fuc GalNAc GC GlcNAc HFIP HHR HIV hKGF HLA HMBA HMPB

HMQC HOBT HPLC HPSEC HQN HTE HTS HR iPr IR

ICR IRMDP kDa KHMDS KR

ES-MS

FT-IR

HMP-PS

ICR-MS

1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride electron impact enzyme-linked immunosorbent assay endoplasmatic reticulum enoyl reductase electrospray ionization electrospray-mass spectrometry ethyl fast atom bombardment fluorescence correlation spectroscopy iluoresceinisothiocyanate flavinemononucleotide fluorenylmethyloxycarbon yl fluorescence polarization fluorescence resonance energy transfer Fourier transform Fourier transform infrared spectroscopy L-fucose N-acetyl-D-galactosamine gas chromatography N-acetyl-D-glucosamine hexafluoroisopropanol hammer head ribozyme human immunodeficiency virus human keratinocyte growth factor human leukocyte antigen (MHC) 4-hydroxymethylbenzoic acid 4-hydroxymethyl-3-methoxyphenoxybutyric acid p-benzyloxybenzyl alcohol polystyrene heteronuclear correlation quantum coherence h ydrox ybenzotriazole high performance liquid chromatography high-performance size-exclusion chromatography h ydrox y-quinuclidine high-throughput experimentation high-throughput screening high resolution isopropyl infrared ion cyclotron resonance mass spectrometry ion cyclotron resonance multiphoton infrared photodissociation kilodalton potassium bis(trimethylsily1)amide ketoreductase

XXVI List of Ahbreviurions

KS ketosynthase Lau lauric acid LC liquid chromatography LCAA-CPG long-chain alkyl amino controlled pore glass LC-MS L D M-3CR MALDI MAMP Man

MBHA MBP mCPBA MCPS MCR MCSL Me

MAS-NMR

liquid chromatography-mass spectrometry laser desorption Mannich - three component reaction matrix assisted laser ionization alpha-methoxy-phenyl (4-methoxy-benzhydryl) mannose magic angle spinning-nuclear magnetic resonance 4-methylbenzhydrylamine myelin basic protein meta-chloroperoxobenzoic acid multiple column peptide synthesis multicomponent reaction multiple core structure libraries methyl

Me-Du-Phos 1,2-bis(2,5-dimethylphospholano)benzene MHC MOBHA MPR Ms MS MsCL MTBD MTP Mtr Nasyl NBA

NBHA NeuAc NIS NMR NOE NOESY NPE Npss NSG NBS-Cl P P-3CR PAAs

Nbb-PS

major histocompatibility complex 4-met hoxybenzhydrylamine mannose-phosphate receptor mesyl, methanesulfonyl mass spectrometry methanesulfon ylchloride 7-methyl-l,5,7-triazabicyclo[4,4,0]elec-5-ene microtiter plate 4-methoxy-2,3,6-trime thyphenyl-sulfonyl naphthylsulfonyl 4-aminomethyl-3-nitrobenzoic acid aminobenzhydryl-polystyrene ortho-nitrobenzylhydrylamino (2-nitrobenzhydrylamine) N-acetyl-neuraminic acid N-iodosuccinimide nuclear magnetic resonance nuclear Overhauser effect nuclear Overhauser effect spectroscopy 4-hydroxyethyl-3-nitro-benzoic acid 2-methoxy-5-(2-(2-nitrophenyl)dithio-l -oxyproplphenylacetic ac N-substituted oligoglycine o-nitrobenzenesulfonyl chloride partition coefficient Passerini - three component reaction poly-N-acylated amines

PAL

PAM PBS PCA PCR PDC PDTs PEG PEGA

PET

Ph PhFl Resin Pip PK PKSs PNAs PNP POEPOP POEPS

PPa PS PS-DVB PSD

PyBroP QSAR R RCM relSI REMPI Rev RIfS

RRE

SAR SASRIN SDS-PAGE SI S-Le SMSI SPOC

PEG-PS

P fP

PS-PEG

RP-HPLC

S3CR

peptide amide linker: 5-[4[9-fluorenylmethoxycarbonylaminomethyl-]-3,5- dimethoxyphenoxy]valeric acid phenylacetamidomethyl phosphate buffered saline principal component analysis polymerase chain reaction pyridinium dichromate pharmacophor definition triplets polyethyleneglycol polyethylene glycol-polyacrylamide co-polymer polyethylene glycol-polystyrene photo-induced electron transfer effect pentafluorophen yl phenyl 9-phenylfluorene-9-yl-PS/DVB piperidine pharmacokinetics polyketide synthases peptide nucleic acids para-nitrophen yl polyoxyethylene-epichlorohydrine derivative polyoxyethylene-cross-linked-oligostyrene phenylpropionic acid polystyrene polystyrene-divinylbenzene copolymer post-source decay polystyrene-polyethyleneglycol graft polymer bromo-tris-pyrrolidino-phosphonium-hexafluorophosphate quantitative structure-activity relationship alkyl ring closure metathesis relative stimulation index laser-induced resonant multiphoton ionization reverse transcriptase (HIV) reflectometric interference spectroscopy revers phase-high performance liquid chromatography Rev-responsive element Strecker - three component reaction structure-activity relationship super acid sensitive resin sodium dodecylsulfate-polyacrylamide gel electrophoresis stimulation index sialyl - Lewis string metal support interaction solid-phase organic chemistry

SPOCC SPOS SPPS ssRNA TAR RNA TAP TBAF Tbf tBu TCC TCR TE TEA Tf TFA TFE TFMSA THF THP TIC TIPS TIS TLC TMS TMOF TMSOTf Tn TOCSY TOF TPAP Ts U-4CR uHTS uv UVDD VEGF VLSIPS VPF WHIM Z

polyoxyethylene-oxetane derivative solid phase organic synthesis solid phase peptide synthesis single stranded ribonucleic acid trans-activation response region transporter associated with antigen processing tetrabutylammonium fluoride tetrabenzo[a,c,g,i]fluorene tert.-butyl CD4+ T-cell clones T-cell receptor t hioesterase triethyl amine trifluoromethylsulfonyl trifluoroacetic acid trifluoroethanol trifluoromethanesulfonic acid tetrahydrofurane tetrahydropyranyl total ion current triisopropylsilane (TIS) triisopropylsilane (TIPS) thin layer chromatography trimethylsilyl trimethylsilyl trifluoromethanesulfonate trimethylsilyl trifluoromethanesulfonate tags’for labeling total correlation spectroscopy time-of-flight thrombin receptor activating peptide tosyl, para-toluenesulfonyl Ugi - four component reaction ultra high-throughput screening ultra-violet ultra-violet photodissociation vascular endothelial growth factor very large-scale immobilized polymer synthesis vascular permeability factor weighted holistic invariant molecular (indices) benzyloxycarbon yl

Further special abbreviations of linkers and resins are found in Chapter 5.