Combinatorial Peptide and Nonpeptide Libraries

A Handbook

Edited by Gunther Jung

VCH Weinheim New York * Basel Cambridge * Tokyo

This Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left Blank

Combinatorial Peptide and Nonpeptide Libraries

A Handbook

Edited by Gunther Jung

Further Reading from VCH:

Biotechnology Second Completely Revised Edition H.-J. Rehm et al. (Eds.) (12 Volumes)

Methods and Principles in Medicinal Chemistry R. Mannhold, P. Krogsgaard-Larsen, H. Timmerman (Eds.)

Vol. 1: H. Kubinyi QSAR: Hansch Analysis and Related Approaches

Vol. 2: H. van de Waterbeemd Chemometric Methods in Molecular Design

Vol. 3: H. van de Waterbeemd Advanced Computer-Assisted Techniques in Drug Discovery

Vol. 4: V. PliSka, B. Testa, H. van de Waterbeemd Lipophilicity in Drug Action and Toxicology

Vol. 5: H.-D. Htiltje, G. Folkers Molecular Modeling - Basic, Principles and Application

Antibiotics and Antiviral Compounds K. Krohn, H. A. Kirst, H. Maag (Eds.)

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1996

Distribution: VCH, P.O. Box 101161, D-69451 Weinheim (Federal Republic of Germany) Switzerland: VCH P. 0. Box, CH-4020 Basel (Switzerland) United Kingdom and Ireland: VCH (UK) Ltd., 8 Wellington Court, Cambridge CBI 1HZ USA and Canada: VCH. 333 7th Avenue, New York, NY lo001 (USA) Japan: VCH, Eikow Building. 10-9 Hongo 1-chome, Bunkyo-ku, Tokyo 113 (Japan)

ISBN 3-527-29380-9

Combinatorial Peptide and Nonpeptide Libraries

A Handbook

Edited by Gunther Jung

VCH Weinheim New York * Basel Cambridge * Tokyo

Prof. Dr. Giinther Jung Institut fiir Organische Chemie der Universitat Auf der Morgenstelle 18 D-72076 Tiibingen Federal Republic of Germany

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

Published jointly by VCH Verlagsgesellschaft mbH, Weinheim (Federal Republic of Germany) VCH Publishers, Inc., New York, NY (USA)

Editorial Directors: Dr. Ute Anton, Dr. Gudrun Walter Production Manager: Dip1.-Wirt.-lng. (FH) Bernd Riedel

Every effort has been made to trace the owners of copyrighted material; however, in some cases this has proved impossible. We take this opportunity to offer our apologies to any copyright holders whose rights we may have unwittingly infringed.

Library of Congress Card No. applied for.

A catalogue record for this book is available from the British Library.

Die Deutsche Bibliothek Cataloguing-in-Publication Data:

Combinatorial peptide and nonpeptide libraries : a handbook 1 ed. by Giinther Jung. - Weinheim ; New York ; Basel ; Cambridge ; Tokyo : VCH, 19%

NE: Jung, Giinther [Hrsg.] ISBN 3-527-29380-9

0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1996

Printed on acid-free and chlorine-free paper.

All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine-readable language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be con- sidered unprotected by law.

Composition: Filrnsatz Unger & Sommer GmbH, D-69469 Weinheim Printing: Strauss Offsetdruck. D-69509 Msrlenbach Bookbinding: J. Schaffer GmbH & Co. KG, D-67269 Griinstadt

Printed in the Federal Reuublic of Germany

This book describes the most practicable approaches to create combinatorial peptide libraries and compound libraries. These are valuable tools for basic research and discovery of new lead structures in pharmaceutical industry. Presently Combinatorial Chemistry is the most fascinating area in organic and bio-organic chemistry and extremely fast-developing. Originally developed and applied by relatively few peptide chemists, automated synthesizers for the multiple parallel synthesis of thousands of compound libraries have moved into general organic and medicinal chemistry. It has taken almost 30 years for most organic chemists to recognize the advantages of solid- phase synthesis for the assembly of new molecules with high molecular diversity. Today, there is no scepticism any more that most future lead structures and drugs will come from the enormous repertoire of small molecule libraries.

I am aware of the fact that, the day this book appears on the market, additional and perhaps even more sophisticated strategies to create chemical diversity and their applications will have been published. Nevertheless, the basic principles of planning, preparing, analyzing and testing peptide and compound libraries will remain valid.

The comprehensive book is addressed both to newcomers in this interdisciplinary field and to experts, who may benefit at least from the references of the work published to about mid-1995. Each chapter is kept in its own entirety for the sake of immediate access to topics of special interest, and therefore some repetition in the individual introductions could not be avoided.

The chapters covered begin with natural diversity, organic chemistry and multiple parallel peptide chemistry on solid supports and the one-bead -one-compound strategy. Particular emphasis is given to special analytical tools for the analysis of libraries. Some recent applications illustrate the successful use of scanning libraries both in solution and on supports for evaluating molecular details of ligand-receptor interactions. Leading experts of the early days describe their ideas and practical pro- gress on various exciting new methods revolutionary to traditional organic chemistry. The book also provides an up-to-date list of available resins for solid-phase chemistry and a glossary for beginners.

I am very grateful to all authors and co-authors, who contributed so much of their expertise. My acknowledgments also include many of the co-workers of my group, Mrs Ursula Becker-Sanzenbacher, Ralph Jack, and colleagues from industry and in- stitutions who helped in preparing, improving, and proof-reading the manuscripts. Dr Peter Golitz, Dr Gudrun Walter and Dr Ute Anton from VCH were the driving forces in initiatine and accomDanvine the Droeress of this work.

VI Preface

Finally, I should like to encourage all researchers in the field of combinatorial chemistry to communicate to me their published or unpublished new results because the next edition is already in preparation. I hope that the book will find acceptance among the readers, and that it will serve as a source for new ideas while reading.

Tiibingen, October 1995 Giinther Junn

Contents

1

1.1 1.2 I .2.1

1.2.2

1.2.3 1.3 1.3.1 1.3.2 1.4 1.4.1

1.4.2 1.4.3

2

2.1 2.2 2.2.1

2.2.2 2.2.3 3 3 4

Preface V List of Contributors XIX List of Abbreviations XXIII

Natural Peptide Libraries of Microbial and Mammalian Origin Giinther Jung

Introduction 1 Natural Peptide Libraries of Microbial Origin 2 Microbial Polypeptide Antibiotics by Multientymatic Thiotemplate Synthesis 2 Polypeptide Antibiotics by Ribosomal Precursor Protein Synthesis and Posttranslational Modifications 4 Combinatorial Biosynthesis and Biological Diversity of Polyketids 8 Natural Peptide Libraries of Mammalian Origin 9 Self-peptide Libraries Isolated from MHC-Class I Molecules 9 Self-peptide Libraries Isolated from MHC-Class I1 Molecules 10 From Natural to Synthetic Peptide Libraries 12 Synthetic Methods and the Variety of Peptide and Oligomer Libraries 12 Analysis of Synthetic Peptide Libraries 13 Selected Applications of Synthetic Peptide Libraries 14 References 15

Polymer Supported Organic Synthesis: A Review J&g S. Friichtel and Giinther Jung

Introduction 19 Solid-Phase Organic Synthesis and Analytics 20 Advantages of Solid-Phase Synthesis in Organic Reactions and Product Work-Up 20 Supports and Anchors 22 Multiple, Parallel Syntheses 28 Analvtirc and Mnnitnrino nf %lid-Phace Reartinnc ?A

VIII Confents

2.3 Examples of Solid-Phase Syntheses of Small Molecules 36 2.3.1 Immobilization and Reactions with Hydroxy Compounds 36 2.3.1.1 Derivatization of Hydroxy Compounds by Mitsunobu Reaction 40 2.3.2 2.3.3

2.3.4 2.3.5

2.3.6 2.3.7 2.3.8 2.4 2.4.1 2.4.2 2.4.3 2.4.4 2.5

3

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.6

Immobilization and Derivatization of Aldehydes and Ketones 42 Immobilization and Derivatization of Dicarboxylic Acids and Their Derivatives 44 Ring Closure Reactions 46 Heterocyclic Compounds: Benzodiazepines, Hydantoins and Thiazolidines 49 Further Ring Closures on Solid Support 54 Palladium Catalyzed C-C Attachments 56 Further Reactions on Polymeric Support 59 Oligomer Synthesis 61 Peptoids 62 Oligocarbamates 64 Peptide-Nucleic Acids (PNA) 65 Oligoureas 67 Outlook 68 Acknowledgments 70 References 70

From Multiple Peptide Synthesis to Peptide Libraries Annette G. Beck-Sickinger and Giinther Jung

Introduction 79 Simultaneous Multiple Peptide Synthesis (SMPS) 80 Tea-Bag Synthesis 82 Cellulose as Support in Multiple Syntheses 84 Polystyrene-Grafted Polyethylene (PS-PE) Film, a New Resin? 85 Automated Multiple Peptide Synthesizers 85 Synthesis of Polymer-Bound Peptides 90 Spot Synthesis 93 Spatially Addressed Synthesis of Thousands of Peptides 93 Microstructured Peptide-Gold Electrode 94 Peptide Functionalized Surface by Electrochemical Polymerization 94 Peptide Libraries 94 Mixotopes 97 Mimotopes 98 Phage Libraries and Biopanning 98 Random Libraries 99 Modified Peptide Libraries 102 Identification of the Active ComDounds 103

Contents I X

3.4 Conclusions 103 References 104

4 Chemical Synthesis of Peptide Libraries Arpad Furka

4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.2 4.2.1 4.2.2 4.2.3 4.2.4 4.3

4.3.1 4.3.1.1 4.3.1.2 4.3.1.3 4.3.1.4 4.4

The Portioning-Mixing Method 11 1 Principles and Realization 11 1 Experimental Verification 113 The ELPLC Program 114 Simple Device for the Manual Synthesis of Peptide Libraries 116 Efficiency and Limitations 118 Composition of Peptide Libraries 120 Libraries and Sublibraries 120 First-Order Sublibraries 122 Second-Order Sublibraries 124 Higher Order Sublibraries 127 Potential Use of Partial Libraries in Screening: A Theoretical Approach 128 The Domino Strategy 130 Determination of the Amino Acid Occurrence Library (Stage 1) 130 Determination of Positional Occurrence Library (Stage 2) 131 Determination of Active Sequences (Stage 3) 132 Generality of the Domino Strategy 135 Experimental Realization of the Portioning-Mixing Procedure 135 Acknowledgments 137 References 137

5 The Versatility of Nonsupport-Bound Combinatorial Libraries Clemencia Pinilla, Jon Appel, Colette Dooley, Sylvie Blondelle, Jutta Eichlec Barbara DOrnec John Ostresh and Richard A. Houghten

5.1 Introduction 139 5. I . 1 Solid-Phase Peptide Synthesis 139 5.1.2 Peptide Libraries 140 5.2 Preparation of Synthetic Peptide Combinatorial Libraries 142 5.2.1 DCR Method 142 5.2.2 Coupling of Amino Acid Mixtures 143 5.3 Dual Positional SCLs 143 5.3.1 Use of SCLs 144 5 3 . I . 1 Identificatinn nf Antipenic nettrminantc I 4 A

X Contents

5.3.1.2 5.3.1.3 5.3.1.4 5.3.1.5 5.3.2 5.4

5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5 5.6 5.7

Identification of Opioid Peptides 150 Development of Antimicrobial Peptides 150 Development of Inhibitors of Melittins’s Hemolytic Activity 153 Development of Enzyme Inhibitors 154 Use of all D-AminO Acid SCLs 155 SCLs Composed of Peptides Containing I-, D- and Unnatural Amino Acids 155 Positional Scanning SCLs 157 Identification of Antigenic Determinants 159 Identification of Opioid Ligands 161 Identification of Inhibitors of Melittin’s Hemolytic Activity 162 Decapeptide PS-SCL 162 D-Amino Acid PS-SCL 164 Modified Peptide “Libraries from Libraries” Conclusions 166 Acknowledgments 168 References 168

165

6 Combinatorial Library Based on the One-Bead-One-Compound Concept Kit S. Lam and Michal Lebl

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10

6.11

6.12 6.13

6.14 6.14.1 6.14.2 fi I d ?

Introduction 173 The Basic Concept of “One-Bead-One-Compound” 175 Synthesis of Random Peptide Library 176 Screening with an “On-Bead Binding Assay” 176 Screening with a “Releasable Assay” 177 Libraries of Organic Molecules 178 Scaffold Libraries 179 Structure Determination of Positive Reaction Compounds 179 Coding 182 Elimination of Possible Interaction of Target Macromolecule with Coding Structure: Bead Shaving 183 Is It Necessary To Have Full Representation in a Selectide Library? 184 One-Bead-One-Motif Libraries (“Libraries of Libraries”) 185 The Selectide Process Versus Other Combinatorial Library Methodologies 185 Examples of Application 189 Anti-p-Endorphin Monoclonal Antibody 189 Anti-Insulin Monoclonal Antibody 190 MHr-Clnrr I Mnlwiik 191

Contents XI

6.14.4 Releasable Assay Screening System 192 6.14.5 Posttranslational Modification such as Protein Phosphorylation 192 6.14.6 Small Organic Dye Molecule as a Target 193 6.14.7 Screening of Library of Libraries 194 6.15 Perspective 194

Acknowledgments 195 References 195

7 Peptide and Cyclopeptide Libraries: Automated Synthesis, Analysis and Receptor Binding Assays Karl-Heinz Wiesmiiller, Susanne Feiertag, Burkhard Fleckenstein, Stefan Kienle, Dieter Stoll, Markus Herrmann and Giinther Jung

7.1 7.2 7.2.1 7.2.2

7.2.3

7.2.4 7.2.5 7.3 7.3.1 7.3.1.1

7.3.2

7.4 7.4.1

7.4.1.1 7.5 7.5.1 7.5.1 .I 7.5.1.2 7.5.1.3 7.5.1.4 7.5.1.5 7.5.2 7.6 7.6.1

Introduction 203 Methods for the Generation of Peptide Libraries 204 Manually Synthesized Peptide Libraries 204 Automation to Ensure Reproducible, Simultaneous, Multiple Peptide Synthesis 205 Peptide Diversity Determines Procedures for Synthesis and Bioassay 207 Coupling Reactions with Premixed Amino Acid Derivatives 210 Soluble and Polymer-Bound Libraries in One Run 210 Analytical Control of Peptide Mixtures 211 Monitoring During Synthesis 213 Method for Indirect Determination of the Coupling Yield by Amino Acid Analysis 213 Amino Acid Analysis, Capillary Electrophoresis and Mass Spectro- metry of Peptide Libraries 214 Pipet Robot for the Synthesis of Peptide Libraries 218 Procedure for the Synthesis of Peptide Libraries by the “Premix Method” 220 Experimental Procedure 220 Conformationally Constrained Peptide Libraries 221 Synthesis of Cyclopeptides 223 Loading of 2-Chlorotritylchloride Resin with Fmoc-Amino Acids 223 Synthesis of Linear Peptides 223 Cleavage of Fully Side Chain-Protected Peptides from the Resins 224 Cyclization Reactions 224 Cleavage of Side Chain-Protecting Groups 225 Characterization of Cyclopeptide Sublibraries 227 Pentadecapeptide Libraries for Receptor Binding Studies 235 Cornnetition Assav for MHC-Class 11 Bindine Peatides 236

XI1 Contents

7.6.2 7.7

8

8.1 8.2 8.2.1

8.2.2 8.2.3 8.2.4 8.2.5 8.2.6 8.2.7

8.2.8

8.2.9

8.2.10 8.2.1 1 8.2.12

8.2.13

8.2.14

8.2.15

8.2.16 8.3 8.3.1 8.3.2 8.3.3 8.3.4 8.4

Positional Scanning of a Pentadecapeptide Epitope 237 Conclusions 241 References 241

Mass Spectrometric Analysis of Peptide Libraries

J&rg W Metzger, Karl-Heinz Wiesmiiller, Stefan Kienle, Jente Briinjes and Giinther Jung

Introduction 247 Results and Discussion 247 Analytical Techniques for the Characterization of Soluble Combinatorial Peptide Libraries 247 Mass Spectrometry of Peptides 249 Electrospray Ionization (ESI) 249 Peptide Families in Peptide Libraries 250 Calculation of Mass Distributions and Peak Clans 251 Mass Spectrometry of Peptide Libraries 251 Electrospray Mass Spectrometry - A Potent Method for the Characterization of Peptide Libraries 251 Relative Ion Intensities - A Measure for the Number of Isobaric Peptides in Peptide Libraries? 253 Experimental Conditions for Recording ESI Mass Spectra of Peptide Libraries 259 Mass Resolution and Accuracy of Mass Determination 260 Mass Analyzers 260 Fourier Transform Ion Cyclotron Resonance ESI Mass Spectrometry 261 Matrix Assisted Laser Desorption Ionization Mass Spectrometry

Tandem Mass Spectrometry (MS-MS) of Peptide Libraries and Diagnostic Ions 265 High Performance Liquid Chromatography-Mass Spectrometry (HPLC- MS) of Peptide Libraries 274 Limits for Mass Spectrometric Characterization of Peptide Libraries 281 Materials and Methods 281 Peptide Synthesis 281 Mass Spectrometry 282 Narrow-Bore RP-HPLC 283 Calculation of the Mass Distribution with QMass 283 Summary 283 Acknowledgment 284 References 284

(MALDI-MS) 261

Contents XI11

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

Wieland Keilholz and Stefan StevanoviC

9.1 Introduction 287 9.2 Multiple Sequence Analysis as a Further Development of Edman

Degradation 287 9.3 Applications 290 9.3.1 Natural Peptide Libraries: Ligand Motifs of MHC-I and MHC-I1

Molecules 290 9.3.1.1 Pool Sequencing of MHC-I Ligands 291 9.3.1.2 Pool Sequencing of MHC-I1 Ligands 293 9.3.2 Synthetic Peptide Libraries 296

References 301

10 Epitope Mapping with the Use of Peptide Libraries

Stuart Rodda, Gordon Tribbick and Mario Geysen

10.1 10.1.1 10.1.2 10.1.3 10.1.4

10.2 10.2.1 10.2.2 10.2.3 10.2.4 10.3 10.3.1 10.3.1.1 10.3.1.2 10.3.2 10.3.2.1 10.3.2.2 10.4 10.4.1 10.4.2 10.4.3 10.4.4 l O A 5

Introduction 303 Definition of Epitope 303 Brief History of Antibody-Defined Epitope Mapping 304 History of T-cell Epitope Mapping 305 Comparison between Linear Epitope Scanning and the Combinatorial Library Approach 305 Synthetic Peptides for Epitope Mapping 306 Difficulty of Predicting Epitopes 306 Nature of the Screening Task 306 “Format” of Peptides for Epitope Mapping 307 Peptide Purity and Characterization 309 Validity Testing of Peptide Assay Results 310 Testing the Relevance of Peptide Binding Data 310 Antibody Binding 310 Major Histocompatibility Complex (MHC) Binding 311 Testing the Relevance of Bioactivity Data 312 Bioactivity of Antibody-Defined Linear Peptide Epitopes 312 Bioactivity of T-cell Epitopes 313 Peptide Libraries from Pins 313 Types of Library: Strategies 313 Methods of Synthesis of Peptide Libraries on Pins 316 Strategies for Maximizing the Usefulness of Pins 317 Approaches to Amino Acid Mixtures 318 nnwnctream Prnrpccino nf Pin-PentiAe I ihrariec 21Q

XIV Contents

10.4.6 10.5

10.5.1 10.5.2 10.5.3

11

11.1 1.2 1.2.1 1.2.2 1.2.3 1.3 1.4 11.4.1 11.4.1 .I

12

12.1 12.2 12.3 12.4 12.5

12.6 12.7

Screening Methods Applicable to Pin Peptides 319 Comparison of Methods of Peptide Library Generation for Epitope Mapping 320 Systems Using Spatially Stable Matrices 320 Systems Producing “Loose” Solid-Phase Peptides 321 Systems Producing Cleaved Peptides 322 References 322

Cyclic Peptide Libraries: Recent Developments Arno E Spatola and Referis Romanovskis

Introduction 327 Results and Discussion 328 Cyclic Pentapeptides 330 Cyclic Hexapeptides 336 Cyclic Heptapeptides 337 Summary 338 Materials and Methods 340 General Solid-Phase Peptide Synthesis Procedure 341 Synthesis of a Stylostatin Peptide Library with Six Sublibraries (6 x 256 Peptides) 341 Acknowledgments 346 References 346

Random Peptide Libraries as Tools in Basic and Applied Immunology Keiko Udaka, Karl-Heinz Wiesmiiller, Stefan Kienle, Susanne Feiertag, Giinther Jung and Peter Walden

Introduction 349 Peptide Binding to MHC Molecules 350 Synthetic Random Peptide Libraries 351 Peptide Selection by MHC Molecules 352 Interdependence of the Contribution of Individual Amino Acids to Peptide-MHC Interaction 356 T-cell Epitopes Defined with Peptide Libraries 358 Conclusions 361 References 361

Contenrs XV

13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique: Imaging Peptide Sequence and Shape Space Ronald Frank, Stefan Hoffmann, Michael KieA Heike Lahmann, Werner Tegge, Christian Behn and Heinrich Gausepohl

13.1 13.2 13.2.1 13.2.2 13.2.3 13.2.4 13.3 13.3.1 13.3.1.1 13.3.1.2 13.3.1.3 13.3.2 13.3.3 13.4 13.4.1 13.4.2 13.4.3 13.4.4

Introduction 363 General Technical Aspects of SPOT Synthesis 365 Instrumental 365 Peptide Synthesis on Spots 366 Peptide Library Synthesis 369 Library Design 370 Applications of Peptide Libraries on Spots 372 Solid-Phase Ligand Binding Assay 372 Positional Scanning Libraries 375 Iterative Library Search (Mimotope Approach) 376 Dual-Positional Scanning 378 Enzymatic Transformations of Peptide Libraries 379 Other Applications and Future Developments 382 Methods 383 Peptide Library Assembly 383 Side Chain Deprotection 383 Ligand Binding Assay on SPOTS Membranes 384 Enzymatic Phosphorylation 384 References 385

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concept Lutz S. Richtec David C. Spellmeyec Eric J. Martin, Gianine M. Figliozzi and Ronald N. Zuckermann

14.1 14.2 14.3 14.4 14.5 14.6

14.7

14.7.1

Introduction 387 Criteria and Goals for the Generation of Molecular Diversity 387 The Peptoid Approach 389 Synthesis of NSG Peptoids 391 Automated Synthesis of Equimolar Peptoid Mixtures 394 Rational Approaches for Library Design and the Generation of Structural Diversity 3% Peptoid Ligands with Nanomolar Affinity for Adrenergic and Opiate Receptors 397 Design of a Biased Library for 7-Ikansmembrane/G-Protein Coupled Rerentnrc 307

XVI Contents

14.7.2 14.7.3 14.8 14.9 14.9.1

14.9.1.1

14.9.1.2

14.9.1.3

15

15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 15.9 15.10 15.1 1 15.12 15.12.1 15.12. I .1 15.12.1.2 15.12.2 15. 12.2.1

15.12.2.2 15.12.3 15.12.3.1

15.12.3.2 15.12.3.3 15.12.4

Identification of Peptoid Ligands with Nanomolar Affinity 398 Discussion 399 Summary 401 Experimental Procedures 402 Standard Protocol for the Synthesis of NSG Peptoids with C-Terminal Amides Using the Submonomer Method 402 Bromoacetylation of Rink-Amide-Resin and the N-Terminal Amine of an NSG Peptoid Chain 402 Displacement of the Bromide of Resin-Bound Bromoacetamides with Primary Amines 402 Cleavage of the Peptoid/Peptoid Mixture from the Solid Support 402 References 402

Synthesis and Evaluation of Three l&Benzodiazepine Libraries Barry A. Bunin, Matthew J. Plunkett and Jonathan A. Ellman

Introduction 405 Synthesis Criteria for a Benzodiazepine Library 406 Chiron Mimotopes (Geysen) Pin Apparatus 406 Solid-Phase 1,4-Benzodiazepine Synthesis 407 First Generation 1,4-Benzodiazepine Library 410 Second Generation 1,4-Benzodiazepine Library 41 1 Current Solid-Phase l,4-Benzodiazepine Synthesis 41 1 Design of a Large 1,4-Benzodiazepine Library 413 Synthesis of an 11 200 Member l&Benzodiazepine Library 415 Alternate Strategies for Benzodiazepine-Based Diversity 417 Conclusion 418 Experimental Section 418 Reagents and General Methods 418 Fmoc Deprotection of Aminomethyl Solid Support (Pins) 419 2-Aminobenzophenone 419 Method A 419 Coupling Fmoc-Protected 2-Aminobenzophenones (1) to Pins to Give 2 419 Fmoc Cleavage 419 Method B 420 Coupling Aminoaryl Stannane Cyanomethyl Ester to Pins to Give 7 420 Stille Coupling Reactions 420 Bpoc Cleavage 420 Benzodiazepine Synthesis from 2-Aminoarylketones 420

15.12.4.1 Amino Acid Fluoride Acvlation 420

Contents XVIl

15.12.4.2 Amino Acid Fmoc Cleavage and Benzodiazepine Cyclization 421 15.12.4.3 Benzodiazepine Alkylation 421 15.12.4.4 Cleavage from the Support 422 15.12.4.5 Analytical Evaluation of the 1,4-Benzodiazepine Library 422

Acknowledgments 423 References 423

16

16.1 16.2

16.3 16.4

16.5 16.6 16.7

17

17.1 17.2 17.2.1 17.2.2 17.2.3

17.3 17.4 17.5 17.6 17.7 17.8 17.9 17.10 17.10.1

PEG Grafted Polystyrene Tentacle Polymers: Physico-Chemical Properties and Application in Chemical Synthesis Wolfgang Rapp

Introduction 425 Physico-Chemical Properties of Polystyrene-Poly(ethyleneglyco1)- Tentacle Polymers 427 Peptide Synthesis 436 Monosized Tentacle Microspheres for Screening and High Speed Peptide Synthesis 438 TentaGel Peptide Conjugates in Immunization 442 Oligonucleotide Synthesis 445 Macrobeads as Polymeric Microreactors : Peptide Libraries and Combinatorial Chemistry 446 References 458

Supports for Solid-Phase Organic Synthesis Martin Winter

Introduction 465 Polystyrene Supports 468 Polystyrene Base Resins 468 Acid-Labile Polystyrene Resins 472 Base-Labile, Photo-Labile and Nucleophilic Cleavable Polystyrene Resins 481 TentaGel Resins 484 PolyHIPE Resins 489 PEGA Resins 491 Kieselguhr-Polyamide Supports (“Pepsyn K”) 492 Controlled-Pore Supports (CPG, CPC) 495 Other Silicate Supports 498 Miscellaneous Support Components 499 Appendix 502 Conversion Table (mesh - Darticle size. mm) 502

XVIII Contents

17.10.2 17.10.3

18

18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8

Addresses of Suppliers 502 Index of Solid Supports 505 References 509

QMass: A Computer Program for the Analysis of Mass Spectra of Peptide Libraries Jente Briinjes, JOrg U! Metzger and Giinther Jung

Introduction 511 Concepts of QMass 511 Library Concepts of QMass 513 Calculations 514 System Tools, Shell Scripts and Automated Analysis 516 Visualization and Alternative Setup of Calculation Options 518 System Requirements and Limitations 519 Summary 519 References 520

Glossary 521 Index 533

List of Contributors

Jon Appel Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Annette G. Beck-Sickinger Departement Pharmazie ETH Ziirich Winterthurer Strasse 190 CH-8057 Zurich

Christian Behn ABIMED - Analysen-Technik GmbH RaiffeisenstraRe 3 D-40764 Langenfeld

Sylvie Blondelle Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Jente Brilnjes Institut fiir Organische Chemie Eberhard-Karls-Universiat Tiibingen Auf der Morgenstelle 18 D-72076 Tiibineen

Barry A. Bunin Department of Chemistry University of California, Berkeley Berkeley, CA 94720 USA

Barbara Diirner Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Colette Dooley Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Jutta Eichler Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Jonathan A. Ellman Department of Chemistry University of California, Berkeley Berkeley, CA 94720 USA

XX List of Contributors

Susanne Feiertag Markus Herrmann Naturwissenschaftliches und Naturwissenschaftliches und Medizinisches lnstitut Medizinisches Institut Abteilung Biochemie EberhardstraRe 29 Eberhardstrane 29 D-72762 Reutlingen D-72762 Reutlingen

Gianine M. Figliozzi Chiron Corporation 4560 Horton Street Emeryville, CA 94608 USA

Burkhard Fleckenstein lnstitut fur Organische Chemie Eberhard-Karls-Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen

Ronald Frank GBF - Gesellschaft fur Biotechnologische Forschung Mascheroder Weg I D-38124 Braunschweig

Jorg Steffen Fruchtel Institut fur Organische Chemie Eberhard-Karls-Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen

Arpad Furka Advanced ChemTech 5609 Fernvalley Road Louisville, KY 40228 USA

Heinrich Gausepohl ABIMED - Analysen-Technik GmbH RaiffeisenstraRe 3 D-40764 Laneenfeld

Stefan Hoffmann GBF - Gesellschaft fiir Biotechnologische Forschung Mascheroder Weg 1 D-38124 Braunschweig

Richard A. Houghten Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Giinther Jung Institut fur Organische Chemie Eberhard-Karls-Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen and Naturwissenschaftliches und Medizinisches Institut Abteilung Biochemie EberhardstraRe 29 D-72762 Reutlingen

Wieland Keilholz Deutsches Krebsforschungszentrum Abteilung Tumorvirus-Immunologie 0620 Im Neuenheimer Feld 242 D-69120 Heidelbere

List of Contributors XXI

Stefan Kienle Institut fur Organische Chemie Eberhard-Karts-Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen

Michael KieR GBF - Gesellschaft fur Biotechnologische Forschung Mascheroder Weg 1 D-38124 Braunschweig

Heike Lahmann GBF - Gesellschaft fur Biotechnologische Forschung Mascheroder Weg 1 D-38 124 Braunschweig

Kit S. Lam Arizona Cancer Center and Department of Medicine University of Arizona College of Medizine Tucson, AZ 85724 USA

Michal Lebl Selectide Corporation 4580 East Hanley Boulevard lbcson, AZ 85737 USA

Eric J. Martin Chiron Corporation 4560 Horton Street Emeryville, CA 94608 USA

Jorg W. Metzger Lehrstuhl fur Hydrochemie und H ydrobiologie Universitat Stuttgart Bandtiile 2 D-70569 Stuttgart

John Ostresh Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Clemencia Pinilla Torrey Pines Institute for Molecular Studies and Houghten Pharmaceuticals, Inc. 3550 General Atomics Court San Diego, CA 92121 USA

Matthew J. Plunkett Department of Chemistry University of California, Berkeley Berkeley, CA 94720 USA

Wolfgang Rapp Rapp Polymere GmbH Ernst-Simon-StraRe 9 D-72072 Tubingen

Lutz S. Richter Chiron Corporation 4560 Horton Street Emeryville, CA 94608 USA

XXII List of Confributors

Peter Romanovskis Department of Chemistry University of Louisville Louisville, KY 40292 USA

Stuart Rodda Chiron Mimotopes Peptide Sys. I1 Duerdin Street Clayton, Victoria 3168 Australia

Arno E Spatola Deptartment of Chemistry University of Louisville Louisville, KY 40292 USA

David C. Spellmeyer Chiron Corporation 4560 Horton Street Emeryville, CA 94608 USA

Stefan StevanoviC Institut fur Zellbiologie Abteilung Immunologie Eberhard-Karls-Universitat TIibingen Auf der Morgenstelle 15 D-72076 Tubingen

Dieter Stoll Naturwissenschaftliches und Medizinisches Institut Abteilung Biochemie Eberhardstrde 29 D-72762 Reutlingen

Werner Tegge GBF - Gesellschaft fur Biotechnologische Forschung Mascheroder Weg 1 D-38 I24 Braunschweig

Keiko Udaka Max-Planck-Institut fur Biologie Abteilung Immungenetik CorrenstraRe 42 D-72076 Tubingen

Peter Walden Dermatologische und Poliklinik Universitatsklinikum CharitC Humboldt-Universitat Berlin SchumannstraRe 20121 D-10117 Berlin

Karl-Heinz Wiesmiiller Naturwissenschaftliches und Medizinisches Institut Abteilung Biochemie EberhardstraRe 29 D-72762 Reutlingen

Martin Winter Institut fur Organische Chemie Eberhard-Karls-Universitat Tiibingen Auf der Morgenstelle 18 D-72076 Tubingen

Ronald N. Zuckermann Chiron Corporation 4560 Horton Street Emeryville, CA 94608 USA

List of Abbreviations

AAA AC AcZO AcCHR ACD AcOH ADPV Aib AM AMP AP

9-BBN BCIG BCIP BHA Bn, Bzl Boc BOP

BPB BPOC BPM BSA Bu BuLi

CBS CCK CD CDI CE CF CFPS c1z

amino acid analysis Chydroxymethyl-3-methoxy-phenoxyacetic acid acetic anhydride acetylcholine receptor available chemicals directory acetic acid 5-(2-aminomet hyL3,Sdimet hoxy)-phenoxy)valeryl a-aminoisobutyric acid aminomethyl aminopropyl alkaline phosphatase

9-borabicyclo[ 3.3. I] nonane 6-bromo-5-chloro-3-indolyl-/3-~-galactoside 6-bromo-5-chloro-3-indolyl phosphate benzhydrylamine benzyl tert-butoxycarbony 1 benzotriazol-1 -yI-oxy-tris(dimethy1amino)-phosphonium hexa fluorophosphate bromophenol blue biphen ylisopropylox ycarbon y 1 bipheny lpropy loxycarbonyl bovine serum albumine n-butyl n-butyllithium

citrate buffered saline cholecystokinin cell differentiation carbonyldiimidazole capillary electrophoresis continuous flow continuous flow peptide synthesis 2-chlorbenzvloxvcarbonvl

XXIV List of Abbreviations

CML CP CPC CPG CRF CTL

DAMGO dba DBU DCC DCM DCR DEA DEAD DEAE Dhb DIAD DIBALH DIC DIPEA DMAP DMF Dmt DPDPE DPTU DVB

EDC EGTA ELISA

ESI ES-MS

ESI-MS

FAB FhuA Fm Fmoc FT FTIR

HATU

carboxymethyl coupling positions controlled pore ceramics controlled pore glass corticotropin releasing factor cytotoxic T-lymphocyte

[D-Ah, MePh4, Gly-Ol ']enkephalin dibenzylidene acetone 1,8-diazobicyclo[5.4.O]-undec-7-ene N,"-dicyclohexylcarbodiimide dichloromethane divide, couple, and recombine diethylamine diethylazodicarboxy late diethylaminoethyl 2,5-dihydroxybenzoic acid diisopropylazadicarbox ylate diisobutylaluminium hydride N,"-diisopropylcarbodiimide diisopropylethylamine 4- dimethy lamino)-pyridine dimethyl formamide dimethoxytrityl [D-Pen', ~-Pen']enkephalin diphenylthiourea divinylbenzene

N-ethyl-N'-(3-dimethylaminopropyl)carbodiim~de ethyleneglycol-bis(amino-ether)-NJV,","-tetmacetic acid enzyme linked immuno assays, enzyme linked immunosorbant assay electrospray mass spectrometry electrospray ionization electrospray ionization mass spectrometry

fast atom bombardment ferric hydroxamate uptake (transport protein A) fluorenylmethyl 9-fluorenylmethoxycarbonyl Fourier transform Fourier-transform infrared spectroscopy

azabenzotriazolyl-NJV,",N'-tetramet hyluronium hexafliinroahosnhate

List of Abbreviations XXV

HBsAG hepatitis B virus antigen HBTU 2-( 1H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium

hCMV human cytomegalovirus HIPE high internal phase emulsion HMB 4-hydroxymethylbenzoic acid, p-(hydroxymethy1)benzoyloxy-methyl HMP hydroxymethylphenoxyacetic acid HMPA p-hydroxymethylbenzamide HMPB 4-(4-hydroxymethyl-3-methoxy)phenoxybutyric acid,

HMQC heteronuclear multiple quantum coherence HOBT/HOBt I-hydroxybenzotriazole HOBz 4-hydroxybenzyl HPLC high performance liquid chromatography HPDI 2-hydroxypropyl-dithio-2’-isobutyric acid HPLC-MS HYCRAM hydroxycrotonylamidomethyl

hexa fluorophosphate

4-(4-hydroxymethyl-3-methoxy-phenyloxy)butyryl

high performance liquid chromatography-mass spectrometry

ICR ion cyclotron resonance Ida iminodiacetic acid ISMS ionspray-mass spectroscopy

KG kieselguhr KLH keyhole limpet hemocyanin

LDA lithium diisopropylamide

mAb MALDI

MAP MAPS MAS MBHA MCPBA MECC MEM Met(0) MHC MOM MOPS

MALDI-MS

MPS

monoclonal antibody matrix-assisted laser desorption ionization matrix-assisted laser desorption ionization mass spectrometry multiple antigen peptide multiple antigen peptide system magic angle spinning 4-methylbenzhydr ylamine 3-chloroperbenzoic acid micellar electrokinetic chromatography methoxyet hoxymet hyl met hionine-S-oxide major histocompatibility complex methoxymethyl 4-morpholinepropanesulfonic acid, monosized matrix polystyrene multiole oeotide svnthesis

XXVI List of Abbreviations

MS MTT

NCA NIMP NL NMF NMP NMR

NPEOC NSG

OD OspB

PAL

PBS PD PEG PEGA Pepsyn Gel PfP PHB Pht PM Pmc PNA PPOA PPTS PS PS-PE PS-SCL PSM PTFE PTH PTH PyBOP

PyBroP DYr *so,

NOVC-CL

Pam,Cys

mass spectrometry thiazolyl-blue-tetrazolium bromide

N-carboxyan hydride N-met hylpyrollidone neutral loss N-methylformamide N-methylpyrrolidinone nuclear magnetic resonance nitroveratroyl chloroformate p-nit rophenylethyloxycarbonyl N-substituted glycine

optical density outer surface protein B

5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid tripalmitoyl-S-glyceryl-cysteine phosphate buffered saline plasma desorption poly(ethyleneglyco1) pol y(acr ylamidopropyl-PEG-NJV-dimethylacr ylamide) polydimethylacrylamide resin pentafluorophenyl ester p-alkoxybenzyl alcohol (Wang) phthaloyl portioning-mixing methods 2,2,5,7,8-pentamethylchroman-6-sulfonyl peptide-nucleic acid p-(2-bromopropionyl)phenoxy-acetic acid pyridinium p-toluenesulfonate polystyrene polystyrene-grafted polyethylene film positional scanning synthetic combinatorial library p-( hydroxymethy1)phenyl-acetamidomethyl pol ytetrafluoroethylene parathyroid hormone phenylthiohydantoin benzotriazole- 1 -yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate bromo-tris-pyrrolidino-phosphonium hexafluorophosphate Dyridine-S02 complex (oxidation reagent)

List of Abbreviations XXVII

RAM RaMPS RMM RP RP-HPLC

SAMBHA SASRIN SCAL SCL SEM SL SMPS SPCL SPOS SPPS SWIFT

TAP TASC TASP TBDMS TBTU

TCR TentaGel S TFA Tfa TFMSA TG TGF R TIC TMAD TMG TMSCl TOF To1 trt/Trt TTL

uv

Rink amide rapid multiple peptide synthesizer relative molecular mass reversed phase reversed-phase high performance liquid chromatography

succinylamido-trimethoxybenzhydr y lamine super acid sensitive resin safety catch amide linker synthetic combinatorial library trimethylsilylethoxymethyl sublibrary simultaneous multiple peptide synthesis synthetic peptide combinatorial library solid-phase organic synthesis solid-phase peptide synthesis stored waveform inverse Fourier transform

peptide transporter template associated side chain template associated single protein tert-but yl-dimet h ylsil yl 2-(1H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate T-cell receptor polystyrene-grafted with polyoxyethylene trifluoroacetic acid tri fluoroacet yl trifluoromethanesulfonic acid TentaGelTM, polystyrene-polyoxyethylene copolymer transforming growth factor I3 total ion current chromatogram NflJV”-tetramethylazocarboxamide tetramethylguanidine trimethylsilyl chloride time-o f- flight tolyl trityl tubulin tyrosine ligase

ultraviolet

This Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left BlankThis Page Intentionally Left Blank