Combinatorial Peptide and Nonpeptide Libraries: A Handbook

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996 Combinatorial Pep...

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Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

Combinatorial Peptide and Nonpeptide Libraries A Handbook Edited by Gunther Jung

. Weinheim New York * Basel Cambridge * Tokyo

This 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%

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ISBN 3-527-29380-9

NE: Jung, Giinther [Hrsg.]

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 considered unprotected by law.

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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

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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 solidphase 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 progress 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 institutions 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

Preface V List of Contributors XIX List of Abbreviations XXIII 1

Natural Peptide Libraries of Microbial and Mammalian Origin Giinther Jung

1.1 1.2 I .2.1

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

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

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

2.1 2.2 2.2.1

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

2.2.2 2.2.3 3 3 4

Analvtirc and Mnnitnrino nf %lid-Phace Reartinnc

?A

VIII 2.3 2.3.1 2.3.1.1 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

Confents

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

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

IX

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

The Portioning-Mixing Method 111 Principles and Realization 111 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

4.3.1 4.3.1.1 4.3.1.2 4.3.1.3 4.3.1.4 4.4

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 5. I . 1 5.1.2 5.2 5.2.1 5.2.2 5.3 5.3.1

Introduction 139 Solid-Phase Peptide Synthesis 139 Peptide Libraries 140 Preparation of Synthetic Peptide Combinatorial Libraries 142 DCR Method 142 Coupling of Amino Acid Mixtures 143 Dual Positional SCLs 143 Use of SCLs 144

5 3.I . 1

Identificatinn nf Antipenic nettrminantc

I4A

X 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

Contents

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” 165 Conclusions 166 Acknowledgments 168 References 168

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

6.14 6.14.1 6.14.2

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

fi I d ?

MHr-Clnrr I Mnlwiik

6.11 6.12 6.13

191

Contents

XI

6.14.4 6.14.5 6.14.6 6.14.7 6.15

Releasable Assay Screening System 192 Posttranslational Modification such as Protein Phosphorylation 192 Small Organic Dye Molecule as a Target 193 Screening of Library of Libraries 194 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

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 Spectrometry 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

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

XI1

Contents

7.6.2 7.7

Positional Scanning of a Pentadecapeptide Epitope 237 Conclusions 241 References 241

8

Mass Spectrometric Analysis of Peptide Libraries J&rg W Metzger, Karl-Heinz Wiesmiiller, Stefan Kienle, Jente Briinjes and Giinther Jung

8.1 8.2 8.2.1

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 (MALDI-MS) 261 Tandem Mass Spectrometry (MS-MS) of Peptide Libraries and Diagnostic Ions 265 High Performance Liquid Chromatography-Mass Spectrometry (HPLCMS) of Peptide Libraries 274 Limits for Mass SpectrometricCharacterization 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

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

Contents

XI11

9

Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries Wieland Keilholz and Stefan StevanoviC

9.1 9.2

Introduction 287 Multiple Sequence Analysis as a Further Development of Edman Degradation 287 Applications 290 Natural Peptide Libraries: Ligand Motifs of MHC-I and MHC-I1 Molecules 290 Pool Sequencing of MHC-I Ligands 291 Pool Sequencing of MHC-I1 Ligands 293 Synthetic Peptide Libraries 296 References 301

9.3 9.3.1 9.3.1.1 9.3.1.2 9.3.2

10

Epitope Mapping with the Use of Peptide Libraries Stuart Rodda, Gordon Tribbick and Mario Geysen

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

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

lOA5

nnwnctream Prnrpccino nf Pin-PentiAe I ihrariec

10.1 10.1.1 10.1.2 10.1.3 10.1.4

21Q

XIV

10.4.6 10.5 10.5.1 10.5.2 10.5.3

11

Contents

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 ReferisRomanovskis

Introduction 327 Results and Discussion 328 1.2.1 Cyclic Pentapeptides 330 1.2.2 Cyclic Hexapeptides 336 Cyclic Heptapeptides 337 1.2.3 1.3 Summary 338 1.4 Materials and Methods 340 General Solid-Phase Peptide Synthesis Procedure 341 11.4.1 11.4.1 .I Synthesis of a Stylostatin Peptide Library with Six Sublibraries (6 x 256 Peptides) 341 Acknowledgments 346 References 346 11.1 1.2

12

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

12.1 12.2 12.3 12.4 12.5

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

12.6 12.7

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

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

14.7 14.7.1

Rerentnrc

307

XVI

Contents

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 14.9.1.1 Bromoacetylation of Rink-Amide-Resin and the N-Terminal Amine of an NSG Peptoid Chain 402 14.9.1.2 Displacement of the Bromide of Resin-Bound Bromoacetamides with Primary Amines 402 14.9.1.3 Cleavage of the Peptoid/Peptoid Mixture from the Solid Support 402 References 402 14.7.2 14.7.3 14.8 14.9 14.9.1

15

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

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

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 411 Current Solid-Phase l,4-Benzodiazepine Synthesis 411 Design of a Large 1,4-Benzodiazepine Library 413 Synthesis of an 11200 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 Amino Acid Fluoride Acvlation 420

15.12.2.2 15.12.3 15.12.3.1 15.12.3.2 15.12.3.3 15.12.4 15.12.4.1

Contents

15.12.4.2 15.12.4.3 15.12.4.4 15.12.4.5

Amino Acid Fmoc Cleavage and Benzodiazepine Cyclization 421 Benzodiazepine Alkylation 421 Cleavage from the Support 422 Analytical Evaluation of the 1,4-Benzodiazepine Library 422 Acknowledgments 423 References 423

16

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

16.1 16.2

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

16.3 16.4 16.5 16.6 16.7

17

Supports for Solid-Phase Organic Synthesis Martin Winter

17.1 17.2 17.2.1 17.2.2 17.2.3

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

17.3 17.4 17.5 17.6 17.7 17.8 17.9 17.10 17.10.1

XVIl

XVIII 17.10.2 17.10.3

18

18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8

Contents

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 Naturwissenschaftliches und Medizinisches lnstitut Abteilung Biochemie Eberhardstrane 29 D-72762 Reutlingen

Markus Herrmann Naturwissenschaftliches und Medizinisches Institut EberhardstraRe 29 D-72762 Reutlingen

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

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

Burkhard Fleckenstein lnstitut fur Organische Chemie Eberhard-Karls-UniversitatTubingen 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

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-UniversitatTubingen 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

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

XXI

Jorg W. Metzger Lehrstuhl fur Hydrochemie und Hydrobiologie 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

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

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

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

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

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

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

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

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

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

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

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-phenoxyaceticacid acetic anhydride acetylcholine receptor available chemicals directory acetic acid 5-(2-aminomethyL3,Sdimethoxy)-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-butoxycarbony1 benzotriazol-1-yI-oxy-tris(dimethy1amino)-phosphonium hexa fluorophosphate bromophenol blue biphenylisopropyloxycarbony 1 biphenylpropyloxycarbonyl 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

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

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

[D-Ah, MePh4, Gly-Ol']enkephalin dibenzylidene acetone 1,8-diazobicyclo[5.4.O]-undec-7-ene

EDC EGTA ELISA ES-MS ESI ESI-MS

N,"-dicyclohexylcarbodiimide

dichloromethane divide, couple, and recombine diethylamine diethylazodicarboxylate diethylaminoethyl 2,5-dihydroxybenzoic acid diisopropylazadicarboxylate diisobutylaluminium hydride N,"-diisopropylcarbodiimide diisopropylethylamine 4- dimethy lamino)-pyridine

dimethylformamide 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

FTIR

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

HATU

azabenzotriazolyl-NJV,",N'-tetramet

FAB FhuA Fm Fmoc

FT

hexafliinroahosnhate

hyluronium

List of Abbreviations

HBsAG HBTU

XXV

hepatitis B virus antigen 2-(1H-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate 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, 4-(4-hydroxymethyl-3-methoxy-phenyloxy)butyryl HMQC heteronuclear multiple quantum coherence HOBT/HOBt I-hydroxybenzotriazole HOBz 4-hydroxybenzyl HPLC high performance liquid chromatography HPDI 2-hydroxypropyl-dithio-2’-isobutyricacid high performance liquid chromatography-mass spectrometry HPLC-MS hydroxycrotonylamidomethyl HYCRAM ICR Ida ISMS

KG

ion cyclotron resonance iminodiacetic acid ionspray-mass spectroscopy

KLH

kieselguhr keyhole limpet hemocyanin

LDA

lithium diisopropylamide

mAb MALDI MALDI-MS MAP MAPS MAS MBHA MCPBA MECC MEM Met(0) MHC MOM MOPS

monoclonal antibody matrix-assisted laser desorption ionization matrix-assisted laser desorption ionization mass spectrometry multiple antigen peptide multiple antigen peptide system magic angle spinning

MPS

4-methylbenzhydr ylamine

3-chloroperbenzoic acid micellar electrokinetic chromatography methoxyethoxymethyl methionine-S-oxide major histocompatibility complex methoxymethyl 4-morpholinepropanesulfonic acid, monosized matrix polystyrene multiole oeotide svnthesis

XXVI

List of Abbreviations

MS MTT

mass spectrometry thiazolyl-blue-tetrazolium bromide

NCA NIMP NL NMF NMP NMR NOVC-CL NPEOC NSG

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

OD OspB

optical density outer surface protein B

PAL Pam,Cys 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,

5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valericacid tripalmitoyl-S-glyceryl-cysteine phosphate buffered saline plasma desorption poly(ethyleneglyco1) ylamide) poly(acrylamidopropyl-PEG-NJV-dimethylacr 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 polytetrafluoroethylene parathyroid hormone phenylthiohydantoin benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate bromo-tris-pyrrolidino-phosphoniumhexafluorophosphate Dyridine-S02 complex (oxidation reagent)

List of Abbreviations XXVII

RAM RaMPS RMM RP RP-HPLC

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

SAMBHA SASRIN SCAL SCL SEM SL SMPS SPCL SPOS SPPS

succinylamido-trimethoxybenzhydry 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

SWIFT

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

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

uv

ultraviolet

TAP TASC TASP TBDMS TBTU

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung OVCH Verlagsgesellschaft mbH, 1996

1 Natural Peptide Libraries of Microbial and Mammalian Origin Giinther Jung

1.1 Introduction The apparently unlimited microbial sources of novel natural products provide lead structures of highest molecular diversity and complexity. Moreover, screening for biologically active microbial metabolites will become established permanently as efficient tools become accessible, allowing the detection not only of novel activities but also the rapid elimination of reisolating, undesired toxicities or adverse nontarget activities. Many of the metabolites of microorganisms are isolated as mixtures of closely related molecules with varying substituent pattern on a common lead structure. Microheterogeneity is especially characteristicfor most peptide antibiotics. Two different principal biosynthetic pathways are used for the production of peptide antibiotics by fungi and bacteria as well: thiotemplate synthesis via multienzyme complexes [11 and ribosomal synthesis of precursors followed by post-translational enzymatic modifications [2]. Families of the largest known polypeptide antibiotics with about 20-40 amino acid residues in length and of shorter oligopeptide antibiotics are presented in the following section as examples of natural peptide libraries. The peptide transporters in bacterial cell walls can uptake and transport a large variety of di- and tripeptides including those containing nonnatural a-amino acids [3]. A complete dipeptide library can be taken up by a bacterial cell provided all components have the structural dipeptide backbone in common. These uptake systems for di- and tripeptides exist not only in bacteria but also microorganisms such as Streptomycetae, a single strain of which can produce a mixture of structurally related di- and tripeptides such as the amiclenomycins [4] which are active as biotin antimetabolites. Organic compound libraries are also found within most classes of natural products produced by plants [e. g., among terpenes, lipids, alkaloids, flavonoids, porphyrins]. A large variety of differently substituted parent compounds exists and these are often produced in a species-associated pattern. Within one type of natural library many structurally related compounds can exert similar functions in biological processes. Microbial metabolites are seldom synthesized as a single defined copy, with the exception of those products which occupy highly specialized key functions in all living organisms.

2

1 Natural Peptide Libraries of Microbial and Mammalian Origin

In recent years it has become more and more obvious that some of the most essential proteins involved in mammalian immune response are highly degenerate with respect to their specificity to act as peptide receptors. For example, a major histocompatibility complex molecule (MHC)-Class I can bind natural nonapeptide libraries consisting of thousands of different peptides from self-proteins. All these peptides have allele-specific sequence motifs in common [5, 6). Such a mixture of naturally selected oligopeptides was named a natural peptide library. Nature has developed the peptide library concept to distinguish between self and nonself during immune response mechanisms. It was found that MHC-Class I1 molecules, like the MHC-Class I molecules present peptide mixtures on the cell surface which also exhibit an allelespecific central core motif. However these peptides have different lengths from about 12 to 18 residues [7, 81. Furthermore, not only the antigen presenting MHC molecules bound to the outer cell membrane but also the protein processing proteasomes [9] and the peptide transporters (TAPS)[ 101 both localized in intracellular compartments have a degenerated specificity and therefore are able to recognize peptide libraries. Even the highly specific T-cell receptor recognizing the peptide-MHC complex or the antibodies are only to a very high degree, but not exclusively specific, for one peptide. Most peptide hormone receptors accept a large variety of related synthetic ligand analogs which we may call now a library or sublibrary. A similar situation is found for steroid receptors and other such systems. This diversity found in natural ligand-receptor interactions is the structural basis for the functioning of the modern approaches applied for the search for lead structures. Neither small and large molecule libraries and creation of chemical diversity was invented by chemists; nature has used the various library concepts since the beginning of Ive. In addition to combinatorial chemistry, combinatorial biosynthesis [l I] and biotransformations of organic molecules [12] will become the most important approaches towards creating molecular diversity exceeding that of natural diversity.

1.2 Natural Peptide Libraries of Microbial Origin 1.2.1 Microbial Polypeptide Antibiotics by Multienzymatic Thiotemplate Synthesis As a result of the low specificity of the nonribosomal biosynthesis via multienzyme

systems, 30 or more biologically active analogs of a cyclic or linear peptide antibiotic may be produced by a single strain [l]. A typical example is the steadily growing class of peptaibols (Fig. 1-1), e. g., alamethicins which are amphiphilic enzyme-resistant 20mer peptide antibiotics with a high content of the nonproteinogenic conforma-

1.2 Natural Peptide Libraries of Microbial Origin

a

tionally restricted a-aminoisobutyric acid (Aib), a N-terminal acetyl group and a Cterminal amino alcohol [13]. All form potential-dependent ion channels [14] in lipid bilayers membranes. They have mainly a-helical conformation, a proline bend and a high dipole moment. All natural amino acid exchanges (Fig. 1-1) occur with retention of these characteristic structural properties and therefore, there are few differences found in the biological activities of these variants. Designed, well-defined fully synthetic analogs exhibiting transmembrane ion-channel forming properties have been constructed [IS) based on the natural 3D-structural motif and on structureactivity relationships of a large number of natural and synthetic analogs. *Ac-Aib-Pro-Aib-Ala-Aib-Ala-Qla-Aib-Val-Aib-Gly-Leu-Aib-Pro-Va1-Aib-Aib-Q1u-Q1n-Ph~ol Aib Ala Aib Aib Val Val Qln

1

*Ac-Aib-Ala-Aib-Ala-Aib-Ala-Qln-~b-Aib-~b-Gly-~u-Aib-Pro-Val-Aib-Iva-Qla-Qla-Ph~l 2 IV. Aib LaU Aib '

Ac-Aib-Ala-Aib-Ala-Aib-Ala-Oln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Qln-Qln-Pheol

*Afi-Aib-Ala-Aib-Ala-Aib-Ala-Qln-Aib-Val-Aib-Gly-Aib-Aib-Pro-Va1-Aib-Aib-Qla-Qln-Phool Aib Leu

~-Aib-Pro-Aib-Ala-Aib-Aib-Qln-Aib-Leu-Aib-Gly-Aib-Aib-Pro-Val-Aib-Aib-~ln-Gln-&uol Ala Leu Ile Gly Rib Aib +Ac-~b-----Gly-Aib-Lu-Aib-Qln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Qlu-----Valol Ala Ala Ala Ala Ala Gly Ala Aib Qln Ac-Aib-----Ala-Ala-Aib-Aib-Qln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Ile-Q1n-Qln-Trpol

3 4 5

f

1

Figure 1-1. Alamethicin (1) is a well studied member of the large family of peptaibol polypep tide antibiotics [16], which are produced by fungal multi-enzyme complexes. Peptaibols arc helical amphiphilic polypeptides exhibiting potential-dependent ion channel formation [14! 151. The sequence alignment of some examples of micro-heterogeneous peptaibols produced by seven different microbial strains illustrates typical characteristics of a peptide library with fixed positions and variable positions: alamethicin 1, suzukacillin 2, gliodeliquescin 3! paracelsin 4, hypelcin 5, trichotoxin 6, and trichorzianin 7; Ac, acetyl; Aib, a-aminoisobutyric acid; h a , D-isovaline; Pheol, Leuol, Val01 and Trpol are amino acid alcohols.

The natural optimization process leading to a specific biological function is visualized by the production rate of a high number of analogs - a natural peptide library. In the case of peptaibols, those peptide-producing strains which survive generatf peptide antibiotics with optimal antimicrobial activity and enzyme resistance. Alamethicin is the most prominent member of the larger peptide family of pep taibols which are produced by various fungi. Similar membrane modifying proper. ties as found for alamethicin are exhibited by other helical and amphiphilic polypep tides such as melittin (insects) [17], magainins (frogs) and cecropins (insects) [18] and a number of polypeptides of mammalian origin. At micromolar concentrations alamethicin and other Deptaibols U91 which have a high content of Aib residues ex-

4

I Natural Peptide Libraries of Microbial and Mammalian Origin

hibit membrane lytic activity [19] against mammalian cells; similar effects are found for the bee venom constituent melittin, however this is built up solely from protein amino acids. On the other hand magainin analogs have been constructed which have no lytic properties [20] and which may be useful in therapeutics [21]. In principle, all of these medium-sized peptides could be easily prepared with great diversity in the form of synthetic libraries, and indeed, hundreds of analogs have been prepared already in attempts to optimize their properties. Among the many microbial metabolites elucidated in the laboratories various siderophores have also been found to exhibit microheterogeneity, and it was demonstrated that natural structural variety can be enlarged even further by excess feeding of various building blocks, e.g. nonnatural a,w-diamines in the directed fermentution of ferrioxamine (Fig. 1-2) producer strains [22, 231. The low substrate specificity of many microbial enzymes makes them very interesting tools to catalyze biotransformations in organic chemistry [12]. To mention one out of many examples, enzyme reactors have been proposed to produce chiral lactones from a long list of prochiral cyclic ketones [24].

1.2.2 Polypeptide Antibiotics by Ribosomal Precursor Protein Synthesis and Posttranslational Modifications Again a high natural variability, both in general structures and sequential analogs is found within the rapidly expanding classes of newly discovered peptidic compounds, which are biosynthesized from prepeptides via interesting posttranslational modifications. In contrast to the multienzyme complex-based antibiotic synthesis, here one natural strain usually produces one peptide antibiotic and not a mixture of analogs, because the precursor proteins are coded on a sequentially defined structural gene. %o prominent examples will be used to demonstrate the particularly pronounced structural diversity of natural peptide antibiotics originating from ribosomal synthesis and posttranslational enzymatic modifications: lantibiotics and microcins. The lantibiotics [2, 25) are conformationally constrained polypeptides with several thioether ring structures and a,/3-didehydroamino acids. The recently elucidated microcins [26,27] possess constraints in their backbone owing to various oxazole and thiazole ring systems. Using either the complete cellular biosynthesis system or the isolated peptide modifying enzymes, one can expect to produce large sets of conformationally constrained polypeptides. Indeed, by variation of the structural gene coding for the precursor proteins many variants of lantibiotics [2] and microcins [28] have already been produced in different microbial genetics laboratories. funtibiotics [2, 25) comprise a particularly broad variety of interesting biological activities. All lantibiotics, such as epidermin, nisin or pep5 contain a$-unsaturated amino acids formed from the dehvdration of Ser and Thr and thioether bridges of

1.2 Natural Peptide Libraries of Microbial Origin

5

11

C

D

E

2

-CHI-CH2-

-CH2*

*

*

*

OH OH

-CH2*

*

*

I

t

* *

* *

OH OH

-CH?-CH2-CHZ-CH?-

-CHZ-CH2-CH2-CH2-

-CH2-CH2-CH2-CH2-

-CH2-CH2-CH2-CHI-

* -CHz* -CHI-

OH OH H H

-CH?-CH2-0-

-CHI-0-0-

*

-0-0-0-

* * *

OH OH OH

Tc, -CH2-

-CH2-S-S-

* *

4-

*

t

-s-s-

OH OH OH

-CHI-

*

-C(O)- -NH-

Dcsfern-

A

B

-CH2-CH2-

oxamine

E Dz Xi

x2 X:, X4

Xs Xh

Et, El2 Et3

Te2

-CH2-

Te,

-S-

PlaJ

-CH2-

-

t t

*

*

OH

Figure 1-2. Structural formula of the siderophore desferrioxamine and some of its natural and nonnatural analogs obtained by directed fermentation 122, 231. Desferrioxamines belong to the large group of hydroxamate siderophores. *) denotes unoccupied positions; a) positinn 2''

rlnpc

nnt mitt in thic variant

6

I Natural Peptide Libraries of Microbial and Mammalian Origin

lanthionine and /I-methyl-lanthionine formed by the addition of thiol groups to the a,/I-didehydroamino acids. Novel enzymes are being isolated which catalyze dehydration, sulfide ring formation, hydrogenation [29], oxidative decarboxylation [30] and cleavage of a leader peptide. Some of these enzymes are currently being tested using peptide libraries to determine their substrate specificity. Natural variants and producer strains obtained by site directed mutagenesis have allowed the production of a large variety of analogs, e.g. of epidermin [31] and opened novel strategies for genetic engineering of large and conformationally restricted peptides containing unusual residues. Such peptides are of interest for both rational library approaches in drug design and optimization as well as random screening approaches. The lantibiotics are bacteriocins which can be subdivided in at least two major types (A and B) of natural polycyclic peptide libraries (Fig. 1-3) [2, 251. Type A lantibiotics have elongated, scew-like shapes with a high dipole moment, amphiphilic and basic properties, and inter alia they exhibit potential-dependent channel forming properties in lipid bilayer membranes as found for peptaibols (compare Section 2.1 .). Type B lantibiotics are more globular-shaped and compact. They exhibit diverse immunological and antimicrobial activities, and can act as enzyme inhibitors. The ring systems of the lantibiotics are highly constrained and experiments by genetic microbiologists using multiple site-directed mutagenesis shall reveal possible positions of exchange and the practical usefullness of this concept for preparing cyclopeptide libraries based on novel prepeptide-modifying enzymes. In any case such nonrandom libraries contain a very precious variety of sulfide ring structures and unsaturated amino acids which are barely (if at all) accessible by chemical synthesis. The structure of the microcins resisted elucidation for a long period of time. Recently, the structure of the polypeptide antibiotic microcin B17 [26] was determined, a 43 amino acids glycine-rich DNA gyrase inhibitor. During biosynthesis a

t

t

I

f

1.2 Natural Peptide Libraries of Microbial Origin

7

D

Duramycin

Cinnamycin

Duramycin B Duramycin C Ancovenin

X?

Lys

x’ X6

Gln Ah,‘

Arg Gln AhNY

Gln AI~IJ

x7

Phe

x In

Phe

X’*

Phe

Phe Phe Phe

XI3

Val OH

Val OH

R

Phe

Val Gln Dha:

Leu

Phe Leu

Phe Val

TrP Ser

OH

H

Figure 1-3. Examples of the structures of lantibiotics, lanthionine containing polypeptides produced by ribosomal synthesis of precursor proteins and posttranslational modification [2,25]. Lantibiotics are examples of conformationally restricted polycyclic polypeptides which can be sequentially varied by mutation of their structural gene to yield lantibiotic libraries. S p e A lantibiotics: a) gallidermin; b) pep 5 ; arrows indicate thioether bridges and 2,3-didehydroaminoacids. Type B lantibiotics: c) structural variety of the duramycins; x6 = AlaN is linked to the &-aminogroup of Lys” to form a lysinoalanine bridge; Ala-S-Ala = lanthionine; Ala-S- Abu = p-methyl-lanthionine; Asp(R) = 8-hydroxyaspartic acid; Dha = 2.3-didehydroalanine; Dhb = 2,3-didehydrobutyrine.

ribosomally synthesized precursor is enzymatically transformed via cyclization, dehydration, dehydrogenation and cleavage of the leader peptide into the mature and highly active microcin (Fig. 1-4). Microcin contains not less than 4 thiazole and 4 oxazole rings, which originate from Gly, Ser, Thr, and Cys residues within its backbone (261.

8

1 Natural Peptide Libraries of Microbial and Mammalian Origin

leader peptide (-26, -1) MELKASEFGWLSVDALKLSRQSPLG--

,.lavage

site

Figure 14. Biosynthesis and structure of the gyrase inhibitor microcin B17 (261. The mature,

highly active, glycine-rich 43mer peptide is produced as a 69mer precursor protein, which is posttranslationallymodified at 14 sequence positions (shaded) to give 8 thiazole and oxazole ring systems. Backbone modified microcin libraries are accessible by chemical synthesis and mutation of the structural gene.

As for the lantibiotics site-directed mutagenesis may be used to produce microcin libraries. However, such an approach and the isolation and purification of the products is very time consuming and the yields are very low. Therefore, a chemical synthesis protocol was recently elaborated for the construction of microcin libraries including various novel heterocyclic backbone modifications in addition to the common amino acid exchanges. The aminomethyl-oxazole(thiazo1e)carboxylic acids and the corresponding bicyclic building blocks with two linked 5-ring heterocycles (Fig. 1-4) are valuable new amino acids for various constructs. Thus, the synthesis of a variety of fully biologically active microcin analogs and nonactive compounds such as [all-thiazolel-microcin B17 are already been successful [32].

1.2.3 Combinatorial Biosynthesis and Biological Diversity of Polyketids The future prospects of combinatorial biosynthesis based on the extremely high biological diversity of polyketids were highlighted recently by J. Rohr (111. Polyketids are Droduced bv microornanisms. funni and Dlants and exhibit a Darticularlv high va-

1.3 Natural Peptide Libraries of Mammalian Origin

9

riety in both basic ring structures as well as variants. For many years, the polyketid producing Streptomyces strains have been a source of highly active antibiotics, cytostatic and immunosuppressiveagents. Gene technology of polyketid biosynthesis will eventually become a gold mine in the rapidly expanding field of exploiting biological diversity for drug discovery. In principle, it is possible to construct hybrid organisms which produce certain types of structural variants of polyketids based on a combination of modular arranged genes which code in gene clusters for the biosynthetic enzymes. Similar to the fatty acid synthetase complex, various acyl transferases, /I-keto synthetases, keto reductases, dehydratases, enoyl-reductases and cyclases catalyze the biosynthesis of, e. g. complex macrolide and anthracycline structures from CoA-thioester activated acetate, propionate, butyrate and amino acids. In addition, “postpolyketidic” modifications enlarge the diversity of the basic structures.

1.3 Natural Peptide Libraries of Mammalian Origin 1.3.1 Self-peptide Libraries Isolated from MHC-Class I Molecules Molecules of the major histocompatibility complex (MHC)-Class I and I1 are membrane-bound peptide receptors presenting their ligands to the corresponding T-cell receptors on the surface of cytotoxic T-lymphocytes (CTL) and T-helper cells. MHC molecules are normally occupied by a large number of sequentially related self-peptides processed from the self-protein pool of the cell (5-81. In addition, the peptides bound within the groove of the MHC molecule stabilize the conformation of the protein, thereby protecting it from being rapidly degraded by proteases. The self-peptide mixtures can be isolated and sequenced [33], as described in Chapter 9. For each MHC-Class I allele isolate, pool sequencing by multiple sequence analysis (331 of the natural peptide mixtures revealed a defined length of 9 (rarely 8 or 10) amino acids and 2-3 defined, so-called anchor positions occupied only by one amino acid (or structurally closely related amino acids) [5,6]. Moreover, the anchor amino acids fit exactly into allele-specific pockets of the MHC binding groove. Therefore these oligopeptide mixtures represent natuml peptide libmries consisting of thousands of peptides from intracellularly processed self-proteins. Foreign proteins expressed in virus-infected cells are equally processed intracellularly to present viral-derived peptides. The viral peptides fitting to the same allele-specific sequence motif may be recognized by the T cell receptor of cytotoxic T-lymphocytes which kill the infected cell (Fie. 1-51.

10

1 Natural Peptide Libraries of Microbial and Mammalian Origin The HLA-A1 motif (MHC class I)

1

2

3

.r

D E

S L

4

5

6

2

8

L

9

Y

Relative Dositions Anchor or auxiliary anchor residues Other prefemd residues

P G G G N V I Y I

Sequencesof isolated peptide ligands

Protein source

A I M Y L

Cyclin-like protein Proliferationcell nuclear antigen Ribosomal protein S16 Ets-1

L A I L L

D D E S D

F M P D P

K G R Y G

F H T F V

A M L K L Q I S L D

Y Y Y Y Y

Unknown

Reporled T-cell epitopes E A D P T G H S Y V S D G G P N L Y

MAGE I Influenza basic polymerase

Figure 1-5. Natural mammalian self-peptide library consisting of nona-peptides isolated from MHC class I molecules [S, 61. Amino acids printed in bold are anchoring within pockets of the peptide binding groove of the MHC molecule. The sequence motif of the natural mixture was determined by pool sequencing. The example shows a natural peptide library fitting to HLA molecule AI and the alignment of isolated individual peptide ligands and known CTL epitopes (K.Falk, 0.Rotzschke, M. Takiguchi, B. Grahovac, V. Gnau, S. Stevanovic, G. Jung, H . 4 . Rammensee, Zmmunogenefics1994, 40, 238-241).

1.3.2 Self-peptide Libraries Isolated from MHC-Class I1 Molecules Despite arguments of principle against its use for mixtures of peptides of different length, multiple sequence analysis [33] has recently been employed for pool sequencing of natural peptide libraries isolated from MHC-Class I1 molecules [7, 81. These self-peptide mixtures possess different chain lengths in the range of about 12- 18 amino acids. However, they also have defined sequence motifs within a core region consisting of anchor amino acids at defined relative positions. Processing of viral or bacterial proteins leads to the presentation of foreign peptides (T-helper epitopes) by MHC-Class I1 molecules which are then recognized by T-helper cells, thereby stimulating immune response by increased cytokine production (Fig. 1-6). Multiple sequence analysis of natural and synthetic peptide libraries can be carried out conveniently using sensitive, fully automated protein sequencers with standard programs. However, to evaluate the results of pool sequencing, several hints and rules should be followed carefully (331. Based on the lead motifs of natural peptide libraries svnthetic MHC blocker molecules and svnthetic DeDtide libraries can be con-

1.3 Natural Peptide Libraries of Mammalian Origin

11

structed to inhibit, e.g., autoreactivity of T-cells. Such “antagonists” of CTL and T-helper epitopes are a novel class of allele-specific immunosuppressors. The knowledge of the MHC-Class 1 and I1 restricted sequence motifs is also a prerequisite for the construction of fully synthetic peptide vaccines [34, 35) eliciting long-lasting Thelper and CTL responses. The HLA-DR17 motif (MHCclass 11)

-2-1

1

2 3

4

5

6

7

8

9

10

relative positions

P L I F Y M (V)

D

K R

L

(E)

Y Y L F

(Q) (N)

protein source

Sequences of isolated peptide ligands ~~

ISNQ ISNQ ISNQ VDT KPRA KQT YPD NIQ LLS lXDNXF

K V HG SLA

anchor or auxiliary anchor residues

~

~

L T L D S N T K Y FHKL L T L D S N T KYFHKLN L T L D S N T K Y F H K F L E D V K N L Y HSEA 1 V V D P V H G F MY I S P D Y R N M I F I M D P K E K D K V L I N D Q E V A R F D

apolipoprotein B 2877-93 apolipoproteinB 2877-94 apolipoproteinB 2877-92 a 1-antitrypsin 149-164 LDL-receptor 518-532 IgGZa, membrane domain no match no match

F V R D L L X S D G

transferrin receptor620-634 apolipoproteinB 1273-1290

N Q Y R AD1 R V K Y TLN

R A G K V R G

Q T PKV

human fau-1 protein 76-94

Alignment of previously reported T-helpher epitopes KT SKL KNP NWVRK GQTIEW MAHYNRVPALP

I A Y D I E Y D L F L D V F 1 D I F 1 D F P G D Y1

E E E T P P

E A R T A R Q L I I P N E A F R P Y

K A N S K F

I

R H

HSP 3-13 2nd 65KD 2- 13 HSP70 259-269 I MFFS H-aAChR310-327 T ENGEW H-yAChR 165-184 L H-yAChR 476-495

G ITEL

TT830-843

Figure 1-6. Natural self-peptide pool isolated from MHC class I1 molecules, which consists of peptides with a common core motif and different lengths [7,81. The example shows the natural peptide ligand motif of the HLA molecule DR17 associated with the disease Myasthenia gravis (G. Malcherek, K. Falk, 0.Rbtzschke, H.-G.Rammensee, S. Stevanovic, V. Gnau, G. Jung, A. Melms, Int. Immunol. 1993,10, 1229- 1237). Relative position 4 is occupied with the highly conserved anchor amino acid aspartic acid (D).All isolated individual self-DeDtide linands and Dreviouslv reDorted T-helmr edtoDes fit to this seauence motif.

12

I Natural Peptide Libraries of Microbial and Mammalian Origin

1.4 From Natural to Synthetic Peptide Libraries 1.4.1 Synthetic Methods and the Variety of Peptide and Oligomer Libraries In addition to the complete review in Chapter 3, this introductory section is intented to give only a brief overview of the most essential characteristics of various peptide libraries and guides to the literature and corresponding chapters of this book. Peptide librariesare mixtures of peptides consisting of hundreds (up to millions and more) of single components which have defined characteristics: equal length, defined amino acid positions (0positions) and mix positions (Xpositions) [36, 371. Peptides may consist of common L-amino acids, D-enantiomers, or unusual residues, and they may be N- and/or C-terminally blocked, or they can contain backbone modifying peptide mimetics, sugar or lipid containing building blocks. In addition, the peptides may be cyclized via disulfide, lactone or head-to-tail linkages (Chapter 11). Peptide libraries can be built-up of free linear peptides (Chapters 5 and 7). They can carry biotinyl or fluorescence labels, or they may be polymer-bound to plastic pins (Chapter lo), or resin beads (Chapters 4 and 6), cellulose (Chapter 13) or hydrophilic beads (Chapter 16). In addition to photolithographic techniques reviewed in (361, microstructured surfaces have been used recently to attach a variety of peptides, e.g., via self-assembling monolayers of thiol-modified peptides [38] or via surface attachment of peptides functionalized for electropolymerization (391. Both methods appear to be particularly promissing for the fast and reproducible peptide functionalization of surfaces using convenient and automated procedures for peptide library preparation (Chapter 7) and derivatization. A comprehensive review summarizes published work on simultaneous multiple peptide synthesis (SMPS) and applications [36]. All relevant literature up to 1991 is covered in this review (361 and in reviews updated until 1992 [37,41]. Further reviews appeared in 1994 [42-451, and Chapter 3 of this book should contain most of the relevant publications at least up to April 1995. The most practicable approaches leading to synthetic peptide libraries are described by experts in experimental details in this handbook. There are no limits in the trends observed in going from the natural biopolymers (peptides, sugars, and nucleic acid) to nonnatural oligomers. Peptoids (Chapter 14), peptide nucleic acids (PNA) [47], oligomers with repeating urea units [48] vinylogous peptides [49], pyrrolin-4-on oligomers [50], oligocarbamates [51], and many other oligomers [52] (Chapter 2) have been prepared. Furthermore, combinations of library concepts with tags for detection of active compounds called encoded combinatorial libraries have been proposed or realized [47, 53-56]. The review in Chapter 2 summarizes all important achievements in the organic chemistry on solid phase up to Julv 1995.

1.4 From Natural to Synthetic Peptide Libraries

13

The most useful libraries for lead structure searches are composed of mixtures of free peptides or free organic compounds of molecular masses below 1000 daltons, which can be screened in competition assays with high affinity ligands. The degree of complexity and purity of a peptide library is of importance for straightforward screening possibilities. Methods for the reproducible automated synthesis and reliable analysis of very large peptide pools up to 15mer peptides have been established (Chapter 7). Such extremely complex libraries of longer peptides are especially interesting tools, e.g., for MHC-Class I1 binding studies. We used a reproducible procedure of coupling premixed fluorenyl-methoxycarbonyl (Fmoc)-amino acids to simultaneously introduce different amino acids in any sequence position of peptides by applying only amounts equimolar to the resin loading (Chapter 7). Close to equimolar distribution for the amino acids in the mix positions was found by multiple sequence analysis [33] and electrospray-mass spectrometry (Chapter 8). Using this approach, there are no limits in the complexity of a library. Several authors in this book use the procedure called “split synthesis method” (also called “divide, couple and recombine synthesis”) originally developed by A. Furka (described in Chapter 4), which allows the synthesis of one defined peptide per resin bead. This method is of particularly high value for preparing libraries for assays on beads, selection of single active beads and sequencing of the one peptide per bead (Chapters 4 and 6). However, there are “natural” limits with respect to the kilogram amounts of resin in beads needed in case of a highly complex library (Chapters 4, 6, 16) in order to achieve the synthesis of all theoretically possible peptides.

1.4.2 Analysis of Synthetic Peptide Libraries About 50 possible side reactions are known to occur during common procedures of peptide synthesis. With modern techniques and equipment peptide chemists can prepare hundreds of peptides or peptide mixtures per day. Therefore, equally fast multiple methods of instrumental peptide analysis are necessary for high numbers of samples and especially for analyzing complex mixtures. It is obvious that simple amino acid analysis is only of limited value in characterizing a peptide library, however, it is better than nothing. At least for cyclopeptides an enantiomer analysis should be carried out. It is of utmost importance to warn beginners in this field against the use noncharacterized libraries and, moreover, unreproducible preparation of mixtures containing varying amounts of byproducts. The false opinion that purity is less important is often expressed using the arguement that one might be able also to eventually find active side products later in the iterative process. Multiple sequence analysis [33] by Edman degradation (described in Chapter 9) is restricted to N-terminally accessi-

14

I Natural Peptide Libraries of Microbial and Mammalian Origin

ble peptides. The much faster method, and indeed the method of choice is elec. trospray ionization mass spectrometry (ESI-MS or ES-MS) and ES-MS in combi. nation with HPLC, and tandem MS [57] (Chapter 8). Besides the convenient elec. trospray MS various other ionization techniques have been described for library ana. lyses [58, 591. ES-MS allows the analysis of solutions of free peptide libraries, N- and C-terminally blocked libraries, and libraries with unusual residues and even peptidomimetics. Mass discrimination effects due to differences in basicity, lipophilicity, etc., are normally negligible in ES-MS and from the mass spectrum of a peptide librarq an estimation of quantities is possible by comparison with the calculated intensities of the mass peaks using an especially developed computer program (Chapter 17). Mass spectrometry can detect side products even in mixtures of thousands of peptides [57, 601. Using HPLC-MS isobaric peptides may be separated or different pep tides eluting at the same time can be differentiated. For this purpose the most informative tool is online HPLC-MS/MS. The state-of-the-art of using MS for analyzing peptide libraries is described in Chapter 8 of this book.

1.4.3 Selected Applications of Synthetic Peptide Libraries The first successful and most straightforward applications of synthetic peptide li. braries in comparison with a high affinity ligand have been reported for epitope! mimotope mapping (Chapter 10). The sequence motifs of the MHC bound natural peptide libraries described in Chapter 9 can be determined successfully via assay: using synthetic peptide libraries as demonstrated in Chapter 12. In all selective protein-ligand systems relevant for immune response where natural self-peptide libraries have been found to be involved, e.g. MHC-Class I [5,6] and I1 [7, 81 molecules, proteasomes [9], peptide transporters [lo], and monoclonal antibodies, the use of synthetic peptide libraries was highly successful. Fine mapping of continuous epitopes of monoclonal antibodies using the pin enzyme-linked immuno assays (ELISA) [lo], or biotinylated peptides has revealed interesting results with respect to essential 01 nonreplacable amino acids within an epitope and the diversity allowed for other POsitions [61, 621. Experimental examples for the use of synthetic peptide libraries in competition assays with high affinity ligands illustrate convincingly the applicability of the library concept for complex octapeptide mixtures in MHC-Class I binding and cellular assays (Chapter 12). In addition successful binding studies of 15 mer-peptide and shorter peptide libraries on MHC-Class I1 molecules is reported (Chapter 7). In contrast to the many applications of libraries in epitope/antibody recognition, successful reports of the use of complex peptide libraries for finding novel high affinity ligands of hormone receptors are still somewhat rare. For example, whereas structure-activity relationships of neuropeptide Y analogs with respect to Y, and Y2

References

15

receptors revealed very informative assay data with a high number of single peptides prepared by multiple peptide synthesis [62], initial attempts to use complex libraries clearly demonstrated the limits of the library approach, at least for this particular example of a typical cellular radioligand competition assay. However, in opiod receptor assays, peptide libraries have been applied successfully to finding high affinity ligands (Chapter 6). In principle, the use of synthetic peptide libraries allows the unequivocal characterization of structural requirements for ligand-receptor interaction independently from the knowledge of the structure of the natural ligands. In reality, however, first unsuccessful attempts with, e.g., linear peptide libraries should not discourage one from proceeding further with either novel oligomer libraries or products from combinatorial chemistry. Very often, the limits of error of the assays are too large and small differences in the activities of sublibraries are not pronounced sufficiently. Further problems may concern the reproducibility of the chemical synthesis or the sample preparation of the libraries and the occurrence of synergistic effects. For example, and very typically, a sublibrary shows inhibitory activity in an enzyme assay and none of the less complex sublibraries, and single compounds prepared subsequently in the iterative process show higher activity. Expectations are extremely high, and one should soon find almost exclusively successful reports in the literature in this exploratory area of library research. Although critical contributions are not presently welcome, one has to admit that, in the search for a lead structure for inhibiting a particular interaction in a biochemical or biological target system many practical problems have to be overcome even with the use of the most sophisticated and straightforward approaches described in this book or in the forthcoming literature.

References [I] H. Kleinkauf, H.von Ddhren (Eds.), Biochemistry of Peptide Antibiofics, de Gruyter, Berlin, 1990. [2] G. Jung, H.-G. Sahl (Eds.), Nisin and Novel Luntibiotics, Escom, Leiden, 1991. (31 H. Diddens, H. Zilhner, E. Kraas, W. Gohring, G. Jung, Eur. J. Biochem. 1976, 66, 11-23. [4] A. Kern, U. Kabatek, G. Jung, R. G. Werner, M. Poetsch, H. Dhner, Liebigs Ann. Chem., 1985, 877-892. [S] K. Falk, 0. Rdtzschke, S. Stevanovic, G. Jung, H.-G.Rammensee, Nature 1991, 351, 290-296.

[a] 0. Rdtzschke, K. Falk, S. Stevanovic, B. Grahovac, M. J. Soloski, G. Jung, H A . Ram-

mensee, Nature 1993, 361, 642-644. (71 G. Malcherek, V. Gnau, S. Stevanovic, H.-G.Rammensee, G. Jung, A. Melms, J. Immunol. 1994, 153, 1141- 1149.

16

I Natural Peptide Libraries of Microbial and Mammalian Origin

[8] H. Max. T. Halder, M. Kalbus, V. Gnau, G. Jung, H. Kalbacher, Hum. Immunol. 1994 41, 39-45. 19) G. Niedermann, S. Butz, H.-G. Ihlenfeldt, R. Grimm, M. Lucchiari, H. Hoschutzky G. Jung, B. Maier, K. Eichmann, Immunity 1995, 2, 289-299. [lo] S. Uebel, T. H. Meyer, W. Kraas, S. Kienle, G. Jung, K.-H. Wiesmiiller, R. Tampe, J. Biol Chem. 1995, 31, 19512- 19516. 1111 M. Gerlitz, G. Udvarnoki, J. Rohr, Angew. Chem. 1995, 107, 1757-1761; Angew. Chem Int. Ed. E&. 1995, 34, 1617-1621. 1121 K. Faber, Biotransformations in Organic Chemistry, Springer, Berlin, 1992. 1131 H. Schmitt, G. Jung, Liebigs Ann. Chem. 1985, 321-344. 1141 G. Boheim, W. Hanke, G. Jung, Biophys. Struct. Mech. 1983, 9, 181-191. [lS] H. Vogel, L. Nilsson, R. Rigler, S. Meder, G. Boheim, W. Beck, H.-H. Kurth, G. Jung Eur. J. Biochem. 1993, 212, 305-313. [16] G. Jung, R.-P. Hummel, K.-P. Voges, C. Toniolo, G. Boheim, in: Protides of the Biologi. cal Fluids, (Ed.: H. Peeters), Vol. 35, Pergamon, Oxford, 1987, pp. 485-488. 1171 W. Hanke, C. Methfessel, H.4. Wilmsen, E. Katz, G. Jung, G. Boheim, Biochim. Biophys. A . 1983, 727, 108-114. 1181 H. G. Boman, Annu. Rev. Immunol. 1995, 13, 61-92. 1191 G. Irmscher, G. Jung, Eur. J. Biochem. 1977, 80, 165-174. [20] J. H. Cuervo, B. Rodriguez, R. A. Houghten, Pepf. Res. 1988, I , 81. [21] L. Jacob, M. Zasloff, in Antimicrobial Peptides, (Eds.: H. G. Boman, J. Marsh, J. Goode), Ciba Found. Symp., Wiley, Chichester, 1994, 187- 197. 1221 H. P. Fiedler, J. Meiwes, I. Werner, S. Konetschny-Rapp, G. Jung, J. Chromatogr. 1990, 513, 255 -262. 123) H. Drechsel, M. Tschierske, A. Thieken, G. Jung, H. Bhner, G. Winkelmann. J. Industrial Microbiol. 1995, 14, 105-112. I241 M. J. Taschner, D. J. Black, J. Am. Chem. Soc. 1988, 110, 6892; see also refs. in 1121, p. 189ff. 125) Reviews: a) G. Jung, Angew. Chem. 1991,103,1067-1084; Angew. Chem. Int. Ed. Engl. 1991.30, 1051-1068; b) H.-G. Sahl, R. W. Jack, G. Bierbaum, Eur. J. Biochem. 1995, 230,827-853; c) G. Jung in ref 121, p. 1-34; d) R. W. Jack, E Gdtz, G. Jung in Biotechnologu, 2nd Ed., Vol. 7, VCH, Weinheim, 1996, in print. 1261 A. Bayer, S. Freund, G. Nicholson, G. Jung, Angew. Chem. 1993, 105, 1410-1413; Angew. Chem. Int. Ed. Engl. 1993, 32, 1336-1339. [27] A. Bayer, S. Freund, G. Jung, Eur. J. Biochem. 1995, 234, 414-426. I281 P. Yorgey, J. Davignono, R. Kolter, Mol. Microbiol. 1993, 9, 897. (291 M. Skaugen, J. Nissen-Meyer, G. Jung, S. StevanoviC, K. Sletten, C. I. Mortvedt Abildgaard, I. F. Nes, J. Biol. Chem. 1994, 269, 27183-27185. (301 T. Kupke, C. Kempter, G. Jung, F. Gdtz, J. Biol. Chem. 1995, 270, 11282-11289. 131) B. Ottenwalder, T. Kupke, S. Brecht, V. Gnau, J. Metzger, G. Jung, F. Gdtz, Appl. Environm. Microbiol. 1995, 61, 3894-3903. 1321 G. Videnov, H.-G. Ihlenfeldt, A. Bayer, G. Jung, in Peptides 1994, Proc. 23rd Eur. Pept. Symp. (Ed.: H.L.S. Maia), 1995, Escom, Leiden, p. 351-352. [33] G. Jung, S. Stevanovic, Anal. Biochem. 1993, 212, 212-220. [341 K.-H. Wiesmiiller, G. Jung, G. Hess, Vaccine, 1989, 7, 29-33.

References

17

(351 K. Deres, H.-J. Schild, K.-H. Wiesmilller, G. Jung, H.-G. Rammensee, Nature, 1989,342, 561 -564. (361 G. Jung, A.G. Beck-Sickinger,Angew. Chem. 1992,104,375-391;Angew. Chem. Int. Ed. E&. 1992, 31, 367-383. (371 R.A. Houghten, TIG, 1993, 9, 235. (381 M. Knichel, P. Heiduschka, W. Beck, G. Jung, W. Gopel, Sensors and Actuators 1995, B28, 85-94. (391 P. Heiduschka, W. Beck, W. Kraas, G. Jung, W. Gopel, Polymer Preprints 1995, 36, 80-81. 1401 P. Heiduschka, W. Gopel, W. Beck, W. Kraas, S. Kienle, G. Jung, Chem. EUKJ., 1996, in print. (41) A.G. Beck-Sickinger, G. Jung, Pharmac. Acta Helv. 1993, 68, 3-20. 1421 E. R. Felder, Chimia 1994, 48, 531-541. (431 M. C. Desai, R. N. Zuckermann, W. H. Moos, Drug Development Research 1994, 33, 174. (441 M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E.M. Gordon, J. Med. Chem. 1994, 37, 1233-1251. 145) E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, M. A. Gallop, J. Med. Chem. 1994, 37, 1385- 1401. [46] R.J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel, D.V. Santi, F. E. Cohen, P. A. Bartlett, Proc. Natl. Acad. Sci. USA 1992,89,9367-9371. [47] J. N. Nielsen, S. Brenner, K.D. Janda, J. Am. Chem. Soc. 1993,115, 9812-9813 (and refs. cited therein). (481 K. Burgess, D. Scott Linthicum, H. Shin, Angew. Chem. 1995, 107, 975-977; Angew Chem. Int. Ed. Engl. 1995, 34, 907-909. [49] A. B. Smith, T. P. Keenan, R. C. Holcomb, P. A. Sprengeler, M. C. Guzman, J. L. Wood, P. J. Carroll, R. Hirschmann, J Am. Chem. Soc. 1992, 114, 10672-10674. [SO] M. Hagihara, N. J. Anthony, T. J. Stout, J. Clardy, S. L. Schreiber, J. Am. Chem. Soc. 1992, 114, 6568-6570. I511 C. Y. Cho, E. J. Moran, S. R. Cherry, J. C. Stephan, S. P.A. Fodor, C. L. Adams, A. Sundaram, J. W. Jacobs, P. G. Schultz, Science 1993. 261, 1303-1305. (521 R. S. Liskarnp, Angew. Chem. 1994, 106, 661-664; Angew. Chem. Int. Ed. Engl. 1994, 33, 633-636. (531 J. Kerr, C. Banville, R. N. Zuckermann, J. Am. Chem. Soc. 1993, 115, 2529-2531. [54] A. Borchert, W. C. Still, 1 Am. Chem. Soc. 1994, 116, 373-374. (551 J. J. Baldwin, J. L. Burbaum, I. Henderson, M.H. J. Ohlmeyer, J. Am. Chem. Soc. 1995, 117, 5588-5593. (561 M . Lebl, V. Krchnak, N. F. Sepetov, B. Seligmann, P. Strop, S. Felder, K. S. Lam, Biopolymers (Peptide Science) 1995, 37, 177- 198; and refs. cited. I571 J. W. Metzger, K.-H. Wiesmilller, V. Gnau, J. Brilnjes, G. Jung, Angew. Chem. 1993,105, 901-903; Angew. Chem. Int. Ed. Engl. 1993, 32, 894-896. (581 Y.-H. Chu, D. P. Kirby, B. L. Karger, J. Am. Chem. Soc. 1995, 117, 5419-5420. [59] B. J. Egner, G. J. Langley, M. Bradley, .l Org. Chem. 1995, 60, 2652-2653.

18

I Natural Pepride Libmries of Microbial and Mammalian Origin

1601 J. W. Metzger, C. Kempter, K.-H. Wiesmiiller, G. Jung, Anal. Biochem. 1994, 219,

261-277. 1611 E 0. Gomben, W. Werz, M. Schliiter, A. Bayer, R. G. Werner, W. Berthold, G. Jung, Biol. Chem. Hoppe-Seyler 1994, 375, 471-400. 1621 A. G. Beck-Sickinger.G. Juni. BioDolYmers @Wide Science) 1995. 37. 123- 142.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

2 Polymer Supported Organic Synthesis: A Review Jdrg S. Frtichtel and Giinther Jung

2.1 Introduction More than three decades ago, Merrifield (I] introduced the concept of solid phase synthesis for the first time. Subsequently automated solid phase syntheses of polypeptides (2, 3) oligonucleotides [S]and oligosaccharides (51 have been carried out with progressively increasing regularity. The introduction of synthesis robots for the simultaneous multiple synthesis of peptides (3b, 6) was the logical continuation of this development, followed by the systematic synthesis of peptide libraries (71. Compared with “classical” synthesis, the solid-phase synthesis of modified peptides, e.g., lipopeptides such as tripalmitoyl-S-glycerylcysteine peptides (81, glycopeptides (91 and cyclosporin (lo] can also be carried out more simply and rapidly. Even for less complicated peptides, several weeks are often required for the “classical” multistep synthesis in solution including isolation and purification while polymer-supported synthesis has considerably simplified these procedures by obviating the need for elaborate separation steps. However, its prerequisite is a quantitative yield in every step. Among the simple peptide derivatives, several peptide alcohols show interestingbiological activity (111, and some of them have already undergone clinical tests (e. g., DAGO 1121, Sandostatine (13)). The peptide aldehydes, which are obtained from alcohols, may be protease inhibitors (141. Such peptide alcohols and N-protected amino acid alcohols have so far been synthesized in solution (151. However, the methods proposed by Swistok el al. (16) and Neugebauer ef at. 117) also allow solid phase synthesis of C-terminal peptide alcohols. Recently, it has been demonstrated frequently often that complicated peptidomimetics and biologically active peptides can also be prepared by reactions carried out on solid supports. Until recently, solid phase synthesis of nonpeptide compounds, generally of nonpolymeric substances, has received less attention. However, the synthesis of DIVERSOMERM libturies by Hobbs DeWitt ef a/. (181 and other combinatorial organic synthesis has led to a renaissance of organic synthesis on solid support. The polymer-assisted reactions will be especially significant in future for the fast, simultaneous and multiple synthesisof many new compounds required in the search for lead structures and their optimization for preparation of novel pharmaceuticals. In addition to the repetitive methods of biopolymer synthesis, many other organic syntheses on solid supports, have been applied. In the following review a distinction is made between Solid Phase Peptide Synthesis (SPPS) and other types of Solid

20

2 Poryrner Supported Organic Synthesis: A Review

Phase Organic Synthesis, (SPOS). Those methods, by which classical organic synthesis on solid support have been carried out, and which show little resemblance to peptide, oligonucleotide and polysaccharide chemistry, will be emphasized. The introduced reactions are chosen especially from the point of view of their applicability for multiple synthesis of chemical and nonpeptidesligomer libraries. The aim of this review is to show that, in addition to the coupling reactions and protecting group cleavage in biopolymer synthesis, polymeric supports have also been applied to other organic reactions. The solid support-based reactions mentioned in this chapter are of fundamental significance to combinatorial chemistry, on which hope for efficient development of new lead structures must be based. In addition, synthesis on solid-phase supports further allows multiple and automated syntheses to be performed. Additional chapters on combinatorial organic chemistry for preparation of small molecules will appear later in this book.

2.2 Solid-Phase Organic Synthesis and Analytics 2.2.1 Advantages of Solid-Phase Synthesis in Organic Reacrions and Product Work-Up First, polymer-assisted synthesis must be differentiated from polymer-bound reagents and polymeric protecting groups. Reactions with polymer-bound reagents are one-step reactions in which the dissolved substrate is allowed to react with polymerbound reagents, mostly catalysts or enzymes (Fig. 2-1). A large number of support-bound reagents have been developed [19], which allow the performance of various chemical reactions on the support. Among others, solid supports have been used for fixing triphenylphosphine 1201, hydrogenation catalysts, oxidating and reducing agents and chiral co-reagentsor catalysts 1211. A series of these polymer-bound reagents are commercially available and facilitates the performance of reactions and especially the work up of reaction mixtures. Until now, most organic chemists appear to have failed to recognize the advantages of support-bound reagents and the opportunities offered by solid phase synthesis. This chapter will not focus primarily on reactions with polymeric reagents, but rather on those reactions in which the polymeric support functions as a protecting group for one of the many functional groups of the substrate, while another site on the substrate is being derivatized. These syntheses always consist of several consecutive reaction steps on the solid support, including linking of the substrate and cleavage of the reaction product (Fig. 2-2). Over three decades, this method has been investigated extensively and optimized in peptide chemistry, especially for carboxy, amino, thiol and hydroxy groups 122, 231 and the following advantages can be expected from this type of solid phase synthesis.

2.2 Solid-Phase Organic Synthesis and Analytics

-

21

-_re-

Tenu Gel

ICSb

+ Figure 2-1. (A) Schematic presentation of a one-step reaction with a polymer-supported reagent, a covalently linked hydrogenation catalyst [19-211. (B) Schematic presentation of an one-step reaction with polymer-supported substrate, a support-linker-anchor combination. PS/DVB: polystyrene-divinylbenzene copolymer (Table 2-1) as the solid support. PEG: polyethylene glycol as hydrophilic linker between the anchor and support. SCA: (Safety Catch Amide, Table 2-3) anchor for the covalent and reversible binding of the substrate. (a) Coupling of group X to the support, (b) removing of excess reagents, (c) derivatization of X with Y, cleavage of the final product.

Figure 2-2. Examples of multistep synthesis on a solid support: chiral 2-alkylcyclohexanone synthesized by enamine reaction according to Stork 1211. (a) 1. LDA, 2. alkyl iodides, (b) acidolysis.

22

2 Polymer Supported Organic Synthesis: A Review

(i) Considerably Simplified Reaction Procedure: Time-consumingpurification and isolation steps are eliminated by the covalent binding of the adduct and product to the support. Solutions of excess reagents are used, and the support-bound product is filtered off and washed. (ii) Thermodynamic and Kinetic Influence on the Reaction Course: Higher yields can be obtained by using a large excess of reagents. However, the conditions must also be chosen carefully so that no undesired side reactions, e.g., multisubstit utions take place. (iii) Possibility of Regenerating the Support: The polymeric support can be regenerated and reutilized for new syntheses if appropriate cleavage conditions and suitable anchor groups are chosen. Especially during industrial synthesis of small molecules involving a few reaction steps, the support should be chosen such that, after cleavage of the product, it can be regenerated in a few steps and used again. The price of support plays an unimportant role in the multistep synthesis of expensive products. Regeneratable supports include, amongst others, chloromethylated PS-DVB resin (Merrifield resin), chlorotrityl-PS-DVB resins, hydroxymethylated PS-DVB resins (two-step regeneration). (iv) Principle of High Dilution: In the case of supports with low loading ( <0.8 mmol/g), the undesired side-reactions, e. g. cross-linking and multiple couplings, can be suppressed by separation of the reactive sites (see Dieckmann cyclization, Section 3.6). (v) Influencing Thermodynamic and Kinetic Stability of Polymer-bound Reagents: For example, by utilization of the template effect in asymmetric synthesis. (vi) Possibility of Automation: An automated synthesis is a precondition for the multiple and parallel synthesis of individual products and compound libraries applying combinatorial chemistry.

2.2.2 Supports and Anchors

For the success of organic synthesis on solid supports, the correct choice of supports as well as their bound anchors (Fig. 2-1) is of utmost importance. At present, the choice of available linkers still imposes limits on the possible reaction sequence on the resin, since these are mostly optimized for biopolymer synthesis. In solution synthesis, extreme conditions often have to be applied to obtain satisfactory results. Synthesis at higher temperatures, the use of reactive components, or reactions under protecting gases, are the normal conditions and not the exceptions. In planning solid-phase synthesis, these factors must be taken into account, on one hand, by the choice of the support, on the other, by using suitable linkers. So far, no special supports and only a few anchors have been developed for solid phase organic svnthesis. However. the supports developed for DeDtide chemistrv can

2.2 Solid-Phase Organic Synthesis and Analytics

23

often be used for numerous organic syntheses (Table 2-1).The reaction course ultimately determines what kind of support can be used for which type of synthesis. Therefore, the choice of anchors is one of the most decisive factors for the success of proceeding reaction steps. Similar to the requirements for peptide chemistry, the anchor and any other required protecting groups must be stable in all of the reagents used during the synthesis (orthogonality principle). On the other hand, these groups must be removable under mild conditions, without damaging the final products. The anchors developed for peptide chemistry are generally either stable to bases or weak acids, but mostly they are only suitable for the immobilization of carboxylic acids [3c]. One exception is the 2-chlorotrityl-PS-DVB resin [36] (Table 2-2) on which all the nucleophilic substrates could theoretically be immobilized (Section 3.1). Unfortunately, this anchoring bond is cleavable by very weak acids such as acetic acid. The SCAL linker (Safety Catch Amide Linker) (Table 2-3), in oxidized form, is very stable against acids and bases, but after its reduction with (EtO),P(S)SH it can be cleaved with trifluoroacetic acid. However, using the SCAL linker, only carboxylic acids can be attached to the support to generate carboxamides upon cleavage. Additional safety catch linkers have already been investigated and reported (371. Several suitable and sparingly available anchors are listed in Tables 2-2and 2-3. For the reversible attachment of special functional groups, known anchors often have to be derivatized and optimized, or when necessary, completely new anchors must be developed. Some of the modified standard anchors with examples of their applications are presented in this chapter. In addition to the previously mentioned, for nucleophiles universally applicable, trityl or 2-chlorotrityl anchors, other anchor groups have been developed which allow the coupling of other functional groups. For example Ellman et al. [54] introduced an anchor group for immobilization of alcohols. The sodium salt of this anchor, (6-hydroxymethyl)-3,4-dihydro-2H-pyran,is covalently bound to chloromethylated Merrifield resin by a nucleophilic substitution reaction (Fig. 2-3). The alcohol is coupled to the support by electrophilic addition in the presence of pyridinium 4-toluenesulfonate ( P m )in dichloromethane (16h at 80"C). The resulting tetrahydropyranyl ether, a widespread protecting group for alcohols, is alkaline stable but can be cleaved by etherification or by 95% trifluoroacetic acid. Sucholeiki [55] coupled benzyl halides to a photo-labile a-mercapto-substituted phenylketone anchor (Fig. 2-4). Thus, the 2-methoxy-5-[2-[(2-nitrophenyl)dithio]1-oxopropy1))-phenylacetic acid anchor was protected as a disulfide and coupled to an aminofunctionalized resin, followed by reductive cleavage of the disulfide bond (a, b). Subsequently, various benzyl halides could be coupled to the free thiol group (c). The photochemical cleavage generates, depending on the substitution, a disulfide (for benzvl halides) or a toluene derivative (for o-arylbenzyl halides; d).

24

2 Polymer Supported Organic Synthesis: A Review

Table 2-1. Examples of supports used in organic synthesis Solid support

polystyrene-divinylbenzene copolymer, 1-5 To cross-linking

Remarks good swelling properties, swells up to fivefold of dry volume; limited thermostability (105- 130°C. dependent on solvent) by low cross-linking (1 To)

hexamethylenediamine- polyacryl resins and related polymers

polar resins; good swelling properties in polar solvents (water, DMF); no swelling in DCM.

poly(N-[2-(4-hydroxyphenyl)ethyI] acrylamide (core Q )

solid phase synthesis (SPPS) with high loading

poly(N-acrylyl-pyrrolidine) resins, PAP- and SPARE-polyamides resins

SPPS with high loading; swell in polar media (water, DMF and DCM)

polyethylene support functionalized with acrylic acid

used in the “PIN synthesis”; blocks with rods

kieselgur/polyamide (Pepsyn K)

pressure stable; used in continuous flow SPPS; low swelling properties due to the inorganic support; high rubbing down of the organic part by shaking

polyHipe, polystyrene/polydimethyl- continuous flow SPPS; high loading acrylamide copolymer capacity up to 5 mmol/g; high crosslinking of the polystyrene chains controlled pore glass (CPG)

pressure and thermostable; stable against reactive and aggressive reagents; low loading

polystyrene macro beads

up to 1 mm in diameter; 50 nmollbead loading; available with 3 different anchors

TentaGel. polyethylene glycolPS/DVB copolymers

polar resins; swell in water, methanol, acetonitrile, DMF and DCM; pressure stable; suitable for bioassays on resin

Ref.

2.2

Solid-Phase Organic Synthesis and Analytics

25

Table 2-2. Alkaline-stable anchors for substrate/support linking

Anchor

-8 / \

\ /

R

-

\

X

Cleavage conditions, reagents, mechanism, application .-

Ref.

R = H, Wang anchor, 95% TFA, 138, 391 acidolysis; immobilization of carboxylic acids; R = OMe, SASRIN anchor, 1070 TFA, acidolysis; immobilization of carboxylic acids

R = H, tritylchloride anchor, weak acid (AcOH), acidolysis, immobilization of nucleophilic compounds; R = Cl, 2-chlorotritylchloride anchor, very weak acids, 20% AcOHIDCM, acidolysis

[36]

0

OMe

PAM anchor HE TFMSA, acidolysis; I401 immobilization of carboxylic acids; hundredfold more stable than ester bond formed with chloromethylated Merrifield resin

X = OH,Rink acid anchor, 1411 AcOH/DCM, acidolysis; immobilization of carboxylic acids; X = NH-Fmoc Rink amide, TFA/DCM, acidolysis; immobilization of carboxylic acids yields amides

benzhydrylamine (BHA) anchor, TFMSA, acidolysis; immobilization of carboxylic acids generates amides

1421

Sieber amide anchor, 1% TFAIDCM, 1431 acidolysis; immobilization of carboxylic acids yields amides

26

2 Polymer Supported Organic Synthesis: A Review

Table 2-3. Acid-stabile anchor groups for substrateIsolid support linking Anchor

Cleavage conditions, reagents, mechanism, application

DBU, piperidine by 81441 elimination; suitable for peptide, oligonucleotide and oligosaccharide syntheses on aminomethylated solid supports; coupling of carboxylic acids

ti0

HOM

O

NaOH, saponification coupled to hydroxymethylated PSIDVB resins, coupling of alcohols; irreversible coupling of amines

H

NaOH, by P-elimination; [46] coupled to hydroxymethylated PSIDVB resins; immobilization of carboxylic acids

0

ii

HO-W

COOH

0 0

Bu4N +F‘ , coupled to 1471 aminomethylated PSIDVB resins; coupling of carboxylic acids; cumbersome synthesis of the linker

Bu4N+F’, as ref. [47]; coupling of carboxylic acids

[481

OH

J ‘yy ‘J N‘

NO,

Ref.

Polymer

nod:”

Hydmzine hydmte, by r491 hydrazinolysis; stable in 25 070 TFA; coupling of carboxylic acids

PdoH2, catalytic hydrogena- [SO] tion; coupling of carboxylic acids; HYCRAM supports

2.2 Solid-Phase Organic Synthesis and Analytics

21

Table 2-3. (continued) Anchor

Cleavage conditions, reagents, mechanism, application y

Ref.

(EtO),(PS)SH/TFA by reduc- [51) tive acidolysis; stable against acids and bases in the oxydized form; Safety Catch Amide Linkage (SCAL); immobilization of carboxylic acids

2

Pbotolysis (hv = 350 nm), RT., 72 h; stable in 50% TFA; labile in hydrazine hydrate; immobilization of carboxylic acids

152)

Photolysis (hv = 350 nm); labile in piperidine/DMF; up to 3% loss of peptide per Fmoc cleavage; somewhat more stable in piperidine/ DCM, up to 1% loss of peptide per Fmoc cleavage ; immobilization of carboxylic acid Photolysis (hv = 350 nm); (531 oxygen free atmosphere under inert gases; X = halogens, OH,NH2, immobilization of carboxylic acids

'&COOH

O"4'

+ CI-PSIDVB

a

Figure 2-3. Anchor used for immobilization of alcohols 154). The reaction of an alcohol with the anchor generates a tetrahydropyranyl ether, this common alcohol protecting group is basestable, but can be cleaved by acidolysis. (a) Dimethylacetamide, RT, 16 h, (b) 2 eq. PPTS, 5 eq. alcohol. 80°C. 16 h.

28

2 Polymer Supported Organic Synthesis: A Review

HOW I'SIDVB-PEG

-NH2

-

+

PS/DVB-PEG-N

I

4

I

1'

NpSSMpact

\CHI /

2

H,CO

H

H ,CO

1

/

H S J

a. h

-

JSR

PSIDVB-PEG-N HI 0

H,CO\ /

2

Figure 2-4. Photochemically cleavable linker for thiols [ U ] .Toluene derivatives or disulfides are obtained, depending on the reaction conditions.

2.2.3 Multiple, Parallel Syntheses Until a few years ago, new organic compounds were mostly synthesized individually and in succession, followed by testing their possible biological activities. In the last few years, the procedures in the search of new lead structures have become enormously more efficient. Progress in the fields of biotechnology, molecular biology, robot techniques and automation have led to the development of new screening assays, which are capable of testing a series of substances for receptor binding and biological activities in the cell test in a remarkably short time. As a consequence, the resources of compounds for testing in the pharmaceutical industry have been rapidly depleted. Therefore, the usual methods of synthesis are insuffcient to supply the demands for new compounds. In addition to the previously mentioned methods for automated synthesis of peptides, oligosaccharides and oligonucleotides, a new method has been described for the rapid and automated synthesis of a series of oligomeric and nonoligomeric compounds - combinatorial chemistry [56,59]. Combinatorial chemists no longer work with three-necked flasks, mixers, reflux condensers, dropping funnels and hot plates, but instead they try to use the new type of multiple reactors for the parallel and fully

2.2 Solid-Phase Organic Synthesis and Analyrics

29

automated simultaneous synthesis of a series of compounds. Solid-phase reactions are especially suitable for this type of synthesis, since the desired products remain bound to the support and can be separated from the excess reagents by filtration (Section 2.1). The starting materials (building blocks) should be versatile, easy to derivatize and accessible. For example, if by retrosynthesis a method is found which leads to the desired product, then by using multiple procedures a large series of structurally similar compounds can be prepared. Generally two different strategies are available for this purpose: (i) the multiple parallel synthesis of individual compounds. In this case, each time, one substance is prepared per reaction vessel. This method is specially suitable for rapid optimization of previously identified lead structures. (ii) the multiple parallel synthesis of so-called compound libraries, by which in each reaction vessel many different, but structurally similar, compounds are simultaneously synthesized. Compound libraries can be prepared by relatively simple techniques of “divide, couple and recombine” [75, 761 (Fig. 2-5). The resin-anchor-combination is allowed to react separately with the first components (A, -A3). At the end of the reaction, the resins are mixed thoroughly and the mixture is divided exactly into three portions for obtaining randomly distributed components. By subsequent reaction with the building blocks B,-B3, one obtains, in this example, nine different compounds after two reaction steps. Depending on the number of individual building blocks and synthesis steps, thousands of different compounds could be synthesized in each reaction vessel, which subsequently can be subjected to screening as polymer-bound products or as polymer-free libraries. In the course of development of combinatorial chemistry, reactions known from solution chemistry must be tested for their suitability for solid phase synthesis of nonpeptide and nonoligomeric compounds. In the last decade, in addition to the classical condensation used in peptide synthesis, a representative series of reaction types have been investigated for their applicability in solid-phase organic synthesis (SPOS)(Table 2-4). However, the published solid phase reactions and anchor-resin combinations are not sufficient for the preparation of a truly broad spectrum of compounds. The primary goal of SPOS must be the augmentation of currently available reactions, regardless of whether these reactions are suitable for the synthesis of single compounds or compound libraries. Having this intention, this chapter presents the known solid-phase reactions without differentiating between “combinatorial” and “non-combinatorial” solid-phase chemistry. The reproducible, i.e., automated synthesis of compound libraries, whether oligomers (e. g., peptides, oligonucleotides and biopolymer analogs) or small molecules (e.g., benzodiazepines) is a challenge; identification and structural elucidation of biologically active compounds from “combinatorial compound libraries” is another.

30

2 Polymer Supported Organic Synthesis: A Review

Q Q Q Recombine

Divide

Couple

1

Figure 2-5. Scheme of the preparation of nine different products by the “divide-couplerecombine” method [75, 761: (a) coupling, (b) mixing, (c) dividing. Table 24. Several of the so far known reactions on solid supports

Reaction type

Application examples in solid phase synthesis

Ref.

[2 + 21 cycloadditions

trapping of butadiene

[1031

[2+3] cycloadditions

isoxazolines, furans, modified peptoids.

[104, 108, 1471

acetal formation

immobilization of diols, aldehydes and ketones

[72, 82-85]

aldol condensation

derivatization of aldehydes, preparation of propanediols

[82-85, 1361

benzoin condensation

derivatization of aldehydes

183, 841

cyclocondensations

benzodiazepines, hydantoins, thiazolidines, &turn mimetics, DOrDhYrinS. Dhthalocvanines

[115-117, 119, 123, 125, 128, 1291

2.2 Solid-Phase Organic Synthesis and Analytics

31

Table 24. (continued)

Reaction type

Application examples in solid phase synthesis

Ref.

Dieckmann cyclization

cyclization of diesters

Diels-Alder reaction

derivatization of acrylic acid

electrophilic addition

addition of alcohols to olefins

Grignard Reaction

deri vat izat i on of aldehydes

Heck reaction

synthesis of disubstituted olefins

Henry reaction

in situ synthesis of nitrile oxides, see I2 + 31 cycloadditions

catalytic hydrogenation

preparation of pheromones and peptides, hydrogenation of olefins

[70, 71, 1371

Michael reaction

/3-mercapto-ketones, bicyclo[2.2.2.]octanes

[73, 1421

Mitsunobu reaction

aryl ethers, peptide phosponates, thioethers

[78-81]

nucleophilic aromatic substitution

quinolones

oxidation

synthesis of aldehydes and ketones

169-71, 73, 1231

Pausen Khand cycloaddition

cyclization of norbornadiene with pentynol

11061

photochemical cyclization

helicenes

U271

reactions with organometallic compounds

derivatization of aldehydes and aryl chlorides

(69-71, 72d, 89, 90,94)

reduction with complex reduction of carbonyl compounds, acids, hydrides or Sn compounds esters and nitro group

[72d, 83, 84, 89, 90, 1411

Soai reaction

reduction of carboxy group

[811

Stille reaction

biphenyl derivatives

[122, 1321

Stork reaction

substituted cyclohexanones

[211

reductive amination

quinolones

S181

Suzuki reaction

derivatives of phenylacetic acid

[I341

Wittig and Wittig- Horner reactions of aldehydes, pheromones and reactions 8-mercapto ketones

[70-73, 83, 841

32

2 Polymer Supported Organic Synthesis: A Review

Due to the new developments in the synthesis of nonpeptide compound libraries, heavy demands are put on analytics. Besides the problem of monitoring the solidphase synthesis mentioned in Section 2.4, the common chromatographic methods such as HPLC and TLC are not suitable for the analysis of the liberated complex substance libraries. A I3C NMR spectrum of a mixture of hundreds or more similar compounds is not completely interpretable and, like an IR spectrum, it is only suitable for group analysis. In this case, the typical parameters which determine the quality of a synthesis such as purity, yield, exact structure or defined stereochemical course of a reaction can no longer be determined. Recently, electrospray-MS was introduced as a method for the investigation of peptide libraries [57] and furnishes useful information for this class of compounds, with respect to the identity and purity of a library. The coupling of MS with HPLC, GC or MS-MS (581 allows the analysis of these mixtures. However, the use of these coupled techniques does not completely resolve the problem of an unequivocal characterization of mixtures, especially in the case of low molecular weight Ubraries. For the identification of active substances in a library, a series of proposals have been made, a few of which will be illustrated here. Various reporter systems have been proposed (tags, code molecules) [59] for the subsequent identification of active lead structures, aiming to label individually the components of a library in a parallel synthesis, thus making the identification easier. AAIAAzAA,

/

H,N-Lys

TG-SCAL- NH,

J

ii

TG-SCAL-N-L

~(Boc)

I y NHz .

H

A

AA,hA,AA,(Boc)

/

TG-SCAL-N-L TG-SCAL- N HI -L~s(Boc) Trp.NH,

I

/

TG-SCAL-N-L

I H

s

tTrP

t

-

A/

0

e, f

H

o&kfRz

c.d

AA,(Bw)

S

‘Trp

&h

O

AAIAA,(Boc)

i.

TG-SCAL-N-L

s

Trp

&A-,

R

0

Figure 2-6. Examples for the synthesis of a peptide encoded compound library according to Nicolaev et al. [61] (see text). (a) 1. Boc-Lys(Fmoc)-OH, DIC/HOBT, 2. piperidine/DMF (1 : l), (b) 1. Fmoc-Trp-OH, DICIHOBT, 2. piperidine/DMF (1 :I), (c) 1. divide, 2. bromoacetic acid, DIC, (d) 1. TFA, 2. encoding with Boc-amino acids (Boc-AA,), DIC, 3. mixing, (e) 1. dividing, Boc-AA,, DIC/HOBT, (f) RNH2, mixing, (g) dividing, coupling of carboxylic acids, DICIDMF, (h), Boc-AA3, DICIHOBT, (i) mixing, reductive acidolysis (TFA).

2.2 Solid-Phase Organic Synthesis and Analylics

3:

Kerr et al. [60] as well as Nicolaiev et al. [61] have used a peptide code for encoding their synthesized compounds. The coding principle, as used in the compound librarj prepared by Nicolaiev et al., should be illustrated briefly (Fig. 2-6). A nonpeptide compound library was prepared by acylation of the support with halo carboxylic acids, followed by a nucleophilic displacement and subsequent condensation with cyclic carboxylic acids. By using the standard protocol, the dipeptide Fmoc-TrpLys(Boc) was synthesized on a TentaGel support containing the SCAL anchor. Aftei removal of the Fmoc protection with piperidine, the compound library could be assembled on the free amino group of Trp using the divide-couple-recombine tech. nique [75,76). In a parallel synthesis, the &-aminogroup of Lys was deprotected with TFA and the code sequence was assembled of this site, resulting in each building block being encoded by an amino acid. The free library can be obtained by reduction of the anchor followed by cleavage with TFA. If an active substance is found by screening, the structure of the compound could be established by sequencing the peptide code. However, this method is very time-consuming and requires elaborate instrumentation, thus does not seem to be very useful. Further problems could appear by the coding structure itself, since on one hand, it must be worked with an orthogonal protection strategy, and on the other, the code peptides can participate in the subsequenl receptor recognition. Finally, the coding can also cause side reactions and give rise to false interpretations of the test results. Ohlmeyer et al. [62] encoded their compounds by a chlorophenol binary code which was bound to the resin by a photolabile anchor. In each synthesis, a maximum of 1070 of the available anchor position was used for the coding. The remaining binding sites served for the linking of the compound undergoing synthesis. In this way, the resins, and consequently the synthesized compounds, are provided with a sort of individual bar code. The code molecules of active polymer beads can be cleaved photolytically from the resin and are detected by gas chromatography. Due to the extreme sensitivity of this detection method, even the smallest amount of these aryl halides can be detected. A variation of this coding is proposed by Nestler et al. and Baldwin et al. [140) in which the covalent binding of the tag molecules is affected by a carbene insertion to the resin. The removal of the tag is achieved after oxidation with ammonium cernitrate. After original silylation of the molecules, the code sequence is analyzed by electron capture gas chromatography (EC-GC). Another, completely different, method has been proposed by Brenner and Lerner 1631. They inserted a binary code into oligonucleotide sequences (DNA tag), followed by detection with the sensitive analytical method, the polymerase chain reaction (PCR). The DNA tag of the isolated, active molecule can be amplified using PCR and the code can be decoded by DNA sequencing. Very small amounts of the code molecule can be identified and decoded by multiplication of the code sequence using PCR. However, due to the inherent chemical lability of oligonucleotides, the aDDlicabilitY of this method is severely limited.

34

2 Polymer Supported Organic Synthesis: A Review

2.2.4 Analytics and Monitoring of Solid-Phase Reactions Every step of a multistep synthesis on solid supports leads to a resin-bound intermediate whose characterization poses a serious problem for the solid phase chemist. The quantitation of loading (yield), the structure of the polymer-bound product and possible byproducts, all of which can more easily be investigated during solution-phase methods, cause difficulties in macromolecular systems. On the other hand, it must be emphasized that analysis of resin-bound peptides and peptidomimetics produced by multiple synthesis, has already reached a high standard. Quantitative determination of coupling yields, e. g., by rapid quantitative UV measurement and calculation of the amount of Fmoc cleaved, as well as direct sequencing on the support by ES-MS and Edman degradation is possible for individual peptides as well as for peptide libraries bound to a single bead. By contrast, little information is available on the methods siutable for a support analysis during solid phase organic synthesis, although this is the precondition for optimization of successive synthetic steps in combinatorial chemistry. In addition to the classical methods (i.e., 1. derivatization, 2. cleavage, 3. purification, 4. analysis by MS, IR and/or NMR), there are several methods which enable analysis of the polymer-bound compounds and give information on the type and completeness of reactions. (i) FT-IR and FT-Raman spectroscopy (ii) Solid state and gel-phase I3C NMR [64] as well as 'H and I3C correlation NMR (iii) High resolution 'H-MAS and MAS-CH correlation in gel-phase [65] (iv) Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) [66]-mass spectrometry (v) Elemental analysis (vi) Titration of reactive groups (-NH2, -COOH, ArOH and -SH) (vii) Gravimetric monitoring (viii) Photometric monitoring (-NH2 by photometric Fmoc determination). Due to the interference of the polymer backbone and low loadings, commonly used, analytical results obtained by the conventional methods such as IR and/or NMR are difficult to interpret and are often ambiguous. FT-IR measurements performed under definite measuring conditions furnish qualitative as well as quantitative data on the reaction. By difference measurements of the loaded and unloaded supports under identical conditions, absorption bands can be detected which otherwise are only seen as weak shoulders, especially in the finger print region. For example, the absorption band of H-C-CI stretching vibrations (1250 cm-') can serve as the basis for quantitation of coupling of carboxylic acids to a chloromethylated PS-DVB support. By calibrating measurements [72d,

2.2 Solid-Phase Organic Synthesis and Analytics

35

1241 on several loaded resins, a calibration curve can be constructed (log. absorption vs 070 chlorine) and the completeness of the reaction can be verified. The developments in the areas of solid state and gel phase NMR in the last years led to further suitable methods to follow support-bound reactions. The gel phase NMR spectroscopy [64] is a mixture of “normal” solution NMR and solid state NMR. For this purpose, a solid sample, e.g., PS-DVB resin is filled in an ordinary NMR tube and allowed to swell in a suitable solvent (CD2Cl2, CDCI,). After degassing the sample, it can be measured under the conditions of solution NMR. The ”C NMR spectra are dominated by the strong resonance signals of polymeric support containing weak signals of the polymer-bound substrate. Due to the strong line broadening, one-dimensional ‘H NMR spectra cannot be interpreted. High-resolution ‘H NMR spectra can be obtained by a combination of MAS (Magic Angle Spinning) [65], a technique normally used in the solid state NMR, and the already described gel phase NMR method. The combination of both methods drastically prevents the strong band broadening, even by a short measuring time, moreover, the signal to noise ratio improves considerably. However, the proton coupling can only be resolved when resins such as TentaGel are used. Egner, Langley and Bradley [66] succeeded in following chemical reactions by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDlTOF-MS). In this example, a series of peptides were synthesized on the commercially available acid-labile Rink amide-PS/DVB support. After every reaction step, 1- 100 polymer beads were removed and placed directly onto the sample holder. The synthesized compounds were then removed from the polymer by addition of acidic cleavage solutions. The polymer beads and liberated compounds were then embedded into a matrix (e.g., 2,5-dihydroxybenzoic acid) and analyzed by MALDI-TOF-MS. This analytical method was initially for applied to the extremely acid-labile Rink amide anchor (Table 2-2), but has subsequently been repeated with other acid-labile anchors such as MBHA-, trityl-, Wang-, aminotrityl- and hydroxytrityl- [66b]. In addition, the photolabile anchors, which can be cleaved by laser light, have been employed for this method. Other methods mentioned also enable quantitative analysis of polymer-bound compounds. In particular, elemental analysis of resins is often used, but is accompanied by difficulties. In general, the reason for inaccuracy and deviation of the results obtained is associated with the low loading of the substrates on the support. It is also very difficult to obtain information on the real availability of reactive or functional groups on the resin by such an analysis. C, H, N and halogen determinations often show poor reproducibility and fail to quantitate the small variations of their respective content. Several independent investigations on BrCH,(O)CO-trityl-PS-DVB resins [67] gave values between 0.9 and 1.8 mmol Br/g resin, with a possible maximal loading of 1.47 mmol/g. Furthermore, it could be shown that because of frequently occurring side reactions, e.g., cross-linking and multiple coupling of the substrate, the results obtained deviated severely from the real yields 172dl.

36

2 Polymer Supported Organic Synthesis: A Review

In spite of the great progresses made so far, the problem of analysis of supportbound compounds and of compound libraries is far from resolved. Further attempts must be made to either optimize the known methods, or to introduce new techniques for the direct monitoring of reactions. New techniques might include methods such as electrochemical sensors.

2.3 Examples of Solid-Phase Syntheses of Small Molecules A frequent problem of preparative chemistry is the selective protection of one of the two identical functional groups of a compound. Normally a mixture of di- , monoand unprotected products is obtained, which must be separated by elaborate procedures. In solution synthesis, this problem is often circumvented noneconomically by use of an excess of the bifunctional adduct. Consequently, predominantly the monoprotected derivative is obtained although one is confronted with the problem of its separation from the excess of nonreacted starting material. This problem of preparative isolation does not occur in the case of polymer-bound protecting groups, since one of the functional groups is always protected by immobilization on the support. Thus, further reactions can be selectively carried out on the unprotected group, and the excess reagents are removed by filtration. This reaction is an example of the so-called ‘~fishhook’’princip1e[68]. In particular, the group of Leznoff [69] has worked intensively on the selective blocking of one of many functional groups and the subsequent problems incurred.

2.3.1 Immobilization and Reactions with Hydroxy Compounds Extensive studies have been carried out using polymer-bound symmetrical diols to synthesize the insect sex attractants [70,71]. For this purpose, series of synthetic procedures have been developed, some of which are presented here. The heavily crosslinked polystyrene-DVB(2To) resin, carrying the acid-labile trityl anchor group, was employed as the support. Immobilization of diols on the support was affected by reacting the functionalized resin with a solution of an appropriate alcohol in pyridine (Fig. 2-7). Starting with this functionalized resin [69-711, the solid phase synthesis of pheromones was achieved by several different procedures. A polymer-bound sulfonate was prepared by reacting the immobilized diol with methanesulfonyl chloride which was then allowed to react with suitable alkynyllithium compounds to yield a protected alkynol (Fig. 2-7). When R = H, the molecule can be substituted by deprotonation with butyllithium and a subsequent reaction with alkyl halides. However, in contrast to the analogous reaction in solution, a large excess of BuLi must be used for deprotonation on a solid support. Subsequent hydrogenation of the triple bond with

p

2.3 Examples of Solid-Phase Syntheses of Small Molecules

PSIDVI3-Trt-

37

I oc6 / \

Polymer

\ /

I

Ib

PS/DVB-Trt/OW0\

H H

1

n

SO,Me

f, g. h

H H

Acoqi="R' AcoqL= Figure 2-7. Synthesis of several insect sex attractants. The polymer supported synthesis can be carried out by the so-called alkyne method as well as the two-step-alkyne method on trityl anchor [69-711. (a) Pyridine, (b) MeS02CI, (c) R-CECLi (R = H,alkyl, or aryl), inert gas, (d) BuLi, (e) RI-Br, (f) reduction with , e.g., 9-BBN, (g) HCI or acetic acid, (h) acylation, AC20.

H2/Pd-CaCO, produces a mixture of cis and trans alkenes, the stereoselective reduction to cis alkenes is achieved using 9-borabicyclo(3.3.l)nonane (9-BBN), or with disiamylborane. The synthesized alkene is then cleaved and the resin regenerated by hydrolysis with either HCI or acetic acid; the final product can then be reacted with acetic anhydride in solution. Further variations of the pheromone synthesis have also been investigated using Wittig and "reverse Wittig" reactions 169-71). The acid lability of the trityl anchor

38

2 Polymer Supported Organic Synthesis: A Review

requires basic to neutral conditions for oxidation of the polymer-bound alcohol to the corresponding aldehyde. In addition, precipitation of the oxidation reagent on the resin, either before or during the reaction, must be prevented. Oxidation can be achieved by CrC1202,pyridine and tert-butyl alcohol in dichloromethane (Fig. 2-8, left). The subsequent reaction with an alkyl triphenylphosphonium halide followed by the Wittig reaction yields the cis-alkene with a high stereoselectivity (68-91.5%, as determined by HPLC). In the so-called reverse Wittig reaction on solid polymers (Fig. 2-8, right) the phosphonium salt is prepared on the resin starting with polymer-bound alkyl halides

BuLi

n- I

n- I

1. BuLi

I . HCI 2. ACZO

2.

RzYo H

PSIDVB-Trl 1. HCI 2. Ac20

Ac0*R2

Figure 2-8. Preparation of pheromones by Wittig and Reverse Wittig reactions (71, 721 (left) on trityl resin. Because of the sluggish reaction of the mesitylated diol, the resin must be heated in the presence of triphenylphosphine at 100°C.Immobilized halides can also be used as well.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

39

or mesylated alcohols. This reaction requires very drastic conditions, since triphenylphosphine does not react with mesylated alcohol or with halides at room temperature. Preparation of the polymer-bound Wittig reagent is effected only after heating the resin with molten phosphine at 100°C for 24 h. It is not possible to link the less reactive aromatic diols to the trityl resin. But when instead of the trityl anchor, benzoic acid derivatives such as benzoyl chloride [71c] are used as the anchor, the aromatic diols can then be immobilized by esterification without problems. However, the base lability of this aryl ester bond enables only reactions which can be carried out in neutral to acidic media. Thus, for example, monomethyl ethers of resorcinol, hydroquinone and 7-hydroxy-2-naphthol can be obtained by reacting them with diazomethane; their monoalkyl ethers are prepared by their reaction with potassium tert-butylate and alkyl halides. The yields after cleavage, and based on the maximal loading of the support, ranged from 22% to 74%. These variations were due to the incomplete alkylation or cleavage reactions. The possibility of a simultaneous binding of both hydroxy groups to the resin has also been discussed. This reaction is not very probable, since the best results have been achieved with hydroquinone, although because of its para substitution it would have been the most suitable for a cross-linking coupling. Generally, by the evaluation of experimental results, the possibility of a cross-linking reaction involving bifunctional molecules cannot be ruled out. For immobilization of cis-diols on polymers, in addition to the trityl anchor, Frechet [72] has investigated the interesting support-anchor-combination, polystyrene- boric acid. This anchor possesses several very promising properties : (i) (ii) (iii) (iv)

moderate to high loading (2-3 mmol/g), coupling of diol as borane proceeds simply and with high yield, only cis-diols are bound, after cleavage of the reaction product, the resin can be used again without reactivity losses.

This anchor has been used for the selective blocking of the cis-hydroxy groups of carbohydrates. By employing this method, it has been possible to synthesize desoxysugars on solid supports which otherwise could not have been prepared. Based on the previous work of Leznoff et al. [69-711, a recent article has appeared describing a combinatorial method for the simultaneous synthesis of several different /3-mercapto ketones [73]. The resin-bound mercapto ketone is prepared on a trityl resin immobilized with lY4-butanediol,following it is oxidised and subjected to the Wittig-Horner reaction and Michael addition [74] of an aromatic thiol (Fig. 2-9). Cleavage from the resin was affected by acidolysis with formic acid. A series of nine different ketones could be obtained by the “divide, couple and recombine” [75, 76) technique using three different ylids as well as three different thiols.

40

2 Polymer Supported Organic Synthesis: A Review

PSIDVR-Tn

-0/OH

-

0

(R0)rP

=P

R '

PSIDVB-Trt-0

0

Figure 2-9. A set of nine different /?-mercapto-ketones could be prepared by a combination of Wifrig-Horner reaction and Michael addition 1731. (a) pyr*S03, DMSO,ET3N, RT,2 h, (b) thiophenol, NaOMe (cat.) THF,RT, 2d.

2.3.1.1 Derivatization of Hydroxy Compounds by Mitsunobu Reaction

Mitsunobu reaction [77] is an efficient and commonly used organic reaction for the synthesis of esters and aromatic ethers under configuration inversion. Therefore, Richter and Gadek [78] have chosen this reaction to immobilize N-Boc-L-tyrosine methyl ester on hydroxymethyl-PS/DVB resin via its phenolic hydroxy group. Their aim was to synthesize tail-to-head coupled cyclopeptides. Using N-methylmorpholine as the solvent and base for deprotonation of tyrosine and azodicarboxylic acid diethyl ester (DEAD)/triphenylphosphine as the Mitsunobu reagent, a maximum loading of 0.9 mmol/g resin (theoretical maximum loading = 1.08 mmol/g resin) was obtained. A modified Mitsunobu reaction has also been carried out by Campbell and Bermak [79] for the preparation of peptide phosphonates from peptides (Fig. 2-10). As transition state mimetics, peptide phosphonates show inhibitory activity against peptidases, esterases and metalloproteases. The described synthetic method is compatible with the Fmoc strategy of SPPS and enables the limitless incorporation of phosphonic acid groups into peptides. An a-0-Fmoc- hydroxycarboxylic acid is coupled to the peptide with HOBWHBTU, followed bv the removal of Fmoc eroun with nineridine/N-methvlnvrrolidinone.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

41

Figure 2-10. Peptide phosphonates can be prepared by a modified Mitsunobu reaction (791. The synthesis is compatible with the Fmoc-strategy. (a) HBTU, HOBT, DIPEA, NMP, (b) 30% piperidine/NMP (c) tri(4-~hlorophenyl)phosphine,DIAD, DIPEA, THF, (d) 5 Qo DBU(NMP), (e) Fmoc-amino acids, HOBT,HBTU, DIPEA, NMP, ( f ) thiophenol/triethylamineldioxane (1 :2 :2), (g) scavenger, TFA.

The above modified peptide was converted to the peptide phosphonate using a-[N-(4-nitrophenylethoxycarbonyl)amino]-alkylphosphonic-acidethyl ester under Mitsunobu conditions (azodicarboxylic acid diethyl ester, tri(4-ch1orophenyl)phosphine and NMP). The synthesis can be continued after cleavage of the 4-nitrophenylethoxy-carbonyl (NPEOC) protecting group under basic conditions (DBUINMP). Alkylphosphonic acids were obtained with couplings yields of >90% under conditions of the modified Mitsunobu reaction. The classical application of Mitsunobu reactions for the synthesis of phenol ethers has recently been used by Ran0 and Chapman [80] during polymer-bound synthesis (Fig. 2-11). Coupling of phenols to the polymer-bound alcohols, and of alcohols to the resin-bound phenols, was carried out for the synthesis of two model compounds. The optimal coupling reagents were found to be N,N',":N"'-tetramethylazodicarboxamide and tributyl-phosphine in THF/CH2C12. By using a fivefold excess of coupling reagents and 10 eq. of phenol (based on the maximal loading of the TentaGel support), after about 15 min of reaction time, yields of 72-99070 and purities in the range of 89 to 97% as judged by HPLC were obtained. Similarly, the reaction with polymer-bound phenols proceeded smoothly. Coupling reagents and alcohol were used in fivefold excess with reaction times of 45-60 min. The yields were 68-94%, with purities from 81 to 97%. The first steps for the synthesis of the new oligomers, oligosulfones and oligosulfoxides, have been described by Moran et al. [81]. The starting materials for these compounds were polymer-bound thioethers which were prepared in five steps. To follow the reaction course more accurately, the chromophore group 2-(4'-carboxy-

42

2 Polymer Supported Organic Synthesis: A Review

benzy1oxy)-ethanol was coupled to the Rink amide resin and the terminal hydroxy group was thioacetylated with thioacetic acid under Mitsunobu conditions (PPh3, DIAD and THF at OOC). Reduction of the thioacetate with NaBH4 generated the free thiol. The thiol was alkylated with (S)-2-bromopropionic acid and the carboxy group was subsequently reduced to an alcohol by using a slightly modified Soai reaction [82]. Further synthesis steps could then be carried out on the free hydroxy group to obtain polymer-bound thioethers which were oxidized to oligosulfones and oligosulfonates.

xH:r

TeniiGel- NH Teniael

-NH,

TentaCel c.d

O&OR

-NH

4

m e R . 0*o

-

R"

a -

0

-

6

R"

-

Figure 2-11. The classical Mitsunobu reaction for solid-phase synthesis of aryl ethers [80]. (a) 3-(4-hydroxyphenyl)propionic acid, EDF, 2 h, (b) ROH, TMAD, Bu3P, (c) 4-hydroxymethylbenzoic acid, EDC, DMF, 2 h, (d) phenol derivatives, TMAD, Bu3P, (e) 90% TFA/HzO.

i

2.3.2 Immobilization and Derivatization of Aldehydes and Ketones Immobilization of aldehydes and ketones on polymeric supports is achieved by the acetal (ketal) formation using resins functionalized with 1.2- or 1,3- diols. The respective 1,3-dioxolanes and 1,3-dioxanes thus formed, are base-stable but can be cleaved by strong acids. A series of these functionalized polymers has already been prepared by Leznoff et al. [83] (Fig. 2-12). To prevent a cross-linking between the resin molecules, the reaction has always been carried out in the presence of a large excess of dicarbonyl compounds and the acetal formation is catalyzed by m-benzene-disulfonic acid. Leznoff er al. (83a-83 c] have developed six methods for derivatization of the polymer-bound dialdehydes (Fig. 2-13). Moreover, analogous reactions can be carried out with ketals. By carrying out Grignard reactions on these solid supports, substituted olefins may be obtained [84]. Ren et al. [85] synthesized a 1,3-diol-functionalized polystyrene resin by aldol condensation and disproportionation (mixed Cannizzaro reaction) of a butyrylated PS-DVB with paraformaldehyde in the presence of NaOH. Aldehydes (especially

2.3 Examples of Solid-Phase Syntheses of Small Molecules

cHx

Polymer

43

PSIDVB-CHZ-0

O P I Nu’ ‘0-CH,

0

PSiDVB-CHI-

OH

,..”

Figure 2-12. Preparation of a modified chloromethyl-PS/DVBsupport for immobilization of aldehydes and ketones [83,84].The reversal of this reaction is used for linking of diols to the support.

PN-

0

0

)&a

H 0

-

H

Figure 2-13. Several organic reactions have been carried out by using immobilized aromatic aldehydes, e.g., oxime synthesis, Grignard reaction, selective reduction of carbonyl groups, benzoin condensation, aldol condensation and Wittig reaction (83, 841. (a) PhMgBr, (b) Na[AIH2(0CH20CH3),I, (c) PhCHO, NaCN, (d) NH20H, (e) acetophenone, (f) acidoIvsis.

44

2 Polymer Supported Organic Synthesis: A Review

terephthalaldehyde) have been immobilized on this resin and reactions with hydroxylamine, NaOH, Grignard and Wittig reagents have been carried out. Thus, p-formylbenzaldoxime, p-formylbenzyl formate, mono-p-formylstilbene, and mono-p-formyldiphenyl formate have been obtained with high yields.

2.3.3 Immobilization and Derivatization of Dicarboxylic Acids and Their Derivatives Monoester -monoamide compounds are normally prepared by the reaction of carboxylic acid monoester-monochloride with ammonia [86-871.However, synthesis of the corresponding precursors generally proceeds with yields lower than 50% [MI. Only in the reactions using anhydrides, yields of up to a maximum of 70% can be achieved. Higher yields have been obtained in solid phase synthesis of such compounds [89-901.As long as no strongly acidic conditions are required, hydroxymethylated PS-DVB resins and p-alkoxy resins [91](Wang resins) are reacted with the corresponding dicarboxylic acid chlorides (Fig. 2-14and 2-15).Subsequently, the polymer-bound carboxylic acids monoester-monochlorides are derivatized further (Table 2-5). P S D V B-CH,-OH

I. NaBH, 2. KICOI. MeOH

PSDVB-CHZ-0,

l-

O

c

3. CHIN2

I

I. PhCdCI 2. KICO,. MeOH

F'b#OMe

0

0

I. RMnl

Figure 2-14. Linking and derivatization of dicarboxylic acid dichlorides to hydroxymethylated PS/DVB sumorts 186. 871.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

45

1. VOCI, 2. CHzNz

3. KZCO,, MeOH

Me0 0

Figure 2-15. Oxidative diphenyl synthesis on polymer supports [69].

Table 2-5. Educts for solid-phase synthesis of dicarboxylic acid monoamides Dicarboxylic acid dichloride

Amine

isophthalic acid terephthalic acid succinic acid, succinic anhydride glutaric acid adipic acid sebacic acid

fert-butylamine fert-butylamine aniline, benzylamine, methylamine and ammonia

,.

. ,*

Based on the resin loading of 0.3-0.56 mmol/g, yields of 65-98% have been obtained. Several of the employed acid chlorides and amines are listed in Table 2-5. The resin-substrate bond is cleaved by alkaline saponification using potassium carbonate. Ketones can be prepared by reacting polymer-bound acyl chlorides with phenylcadmium chloride [92]. The selective reduction of acyl chlorides to primary alcohols is achieved with NaBH, without cleavage of the ester group [93]; alkylmanganese iodides [94] convert acyl chlorides to tert-alcohols. Some of these alcohols are precursors for the synthesis of 4,4-diphenyl-y-butyrolactones,which have known biological activity against pathogenic viruses [95]. Similar reactions have been carried out with a specially derivatized hydroxyethanesulfonyl-PS-DVB resin [72d]. This anchor combination possesses ester bonds with different reactivity, one of which is cleaved by /?-elimination. The remaining ethenesulfonyl moiety can be regenerated without reactivity losses and utilized for new synthpcpc

46

2 Polymer Supported Organic Synthesis: A Review

Camps er al. [96] and Patschorkin and Kraus [97] have succeeded in finding reac. tion conditions which allow monoalkylation and monoacylation of polymer-bounc carboxylic acids. Side reactions which are often observed in solution synthesis, suct as Claisen condensation, could be completely suppressed by using a low loadine (<0.2 mmollg).

2.3.4 Ring Closure Reactions In this Section, we wish to discuss those ring closure reactions which have formerlj been succesfully applied in solution phase synthesis of a number of organic molecules and subsequently have been adapted to use in solid phases. We will not propose to further discuss methods for the polymer-bound cyclization of peptides and pro. teins [98]. Due to the high regio- and stereoselectivity pericyclic reactions are ofter been applied for the synthesis of compounds [99]with defined structure [lo01 Therefore, several groups are engaged with the investigation of polymer-bounc cyclization reactions. Blazka and Harwood [I011 have prepared a resin-bound Diels- Alder product bj reacting polymer-bound maleimide with a cyclopentadiene. Nieuwstad and co-work. ers [lo21 have employed polymer-bound dienes for desulfurizing of fume gase: through cycloaddition with sulfur dioxide (Fig. 2-16). However, in both cases the syn. thesized products have not been cleaved from the polymer. Polymer

--p? \\

0

Polymer

Figure 2-16. Gas desulfurization through trapping of SO2 by the use of polymer-bound buta. dienes [IOZ].

Resin-bound dienes or dienophiles have also been used as “traps” for reactive intermediates [I031 (Fig. 2-17). These trapping reactions involving reactive polymers could also be interesting for combinatorial chemistry in solution, for example, for facilitating the work-up procedure and automation. The group of Leznoff (1041 investigated the reaction of substituted 1,3-butadiene! with polymer-bound dienophiles, in particular immobilized acrylic acid. A reaction with electron deficient dienophiles in solution synthesis yields a mixture of 3,4- and 3,5-disubstituted cyclohexanes, with the 3,4-disubstituted isomer as the major product [105]. The aim of these investigations has been to shift the reaction in favor 01 the 3,5-disubstituted product through a higher degree of steric hindrance caused bj the resin-bound components. Acrylic chloride was anchored to a hydroxymethylated

2.3 Examples of Solid-Phase Syntheses of Small Molecules

41

CHINH,

I

:

$

0

.

CH

Figure 2-17. Tko different resins have been combined to generate an interesting “trap” for the reactive intermediates [103]. The trap, maleimide, was immobilized on PS/DVB (2%) copolymer. The second support, a chlorosulfonated resin, was converted to the butadiene precursor by reacting with 5-amino-o-phenanthroline and cyclobutadienyl iron tricarbonyl. Through the oxidation of a suspension of these resins with metals or pyridin-1-oxide. after separation of resins and cleavage of methylamine, N,lv’-dimethylbicyclo[2.2.0.]hex-l-ene-4,5dicarboxylic amide is obtained.

PS-DVB resin as described in Section 3.3, and was then allowed to react with E-l-phenyl-ly3-butadiene, dissolved in hot, swell-promoting xylene (Fig. 2-18). The Diels- Alder product is removed from the polymer with alkali. In addition, the regio- and stereoselectivity of 1,3-dipolar cycloadditions of nitrile oxides to polymer-bound alkynes has been investigated [104] (Fig. 2-19, see also Section 4.1). The reactions of polymer-bound propynoic or phenylpropynoic acids with benzonitrile oxide, and the subsequent alkaline cleavage and esterification with acid methyl ester diazomethane, yielded exclusively 3-phenylisoxazole-5-carboxylic and 3,5-diphenylisoxazole-4-carboxylicacid methyl ester, respectively.

R ps-DvB%=-

0

A

R

F‘S-DVBCHZ00C

PS-DVBCH,OOC

R

-b

9 R

Fieure 2-18. Diels-Alder reaction of butadiene with Dolvmer-bound acrvlic acid 11041.

48

2 Polymer Supported Organic Synthesis: A Review

R=H I . ) Ph-CmN+-OPolymer

O'i:

0

2.) (n-Uu),NOH

3.) CHINt R=H.Ph

-&Ph

R

R = Ph " 0

Figure 2-19. 1,3-dipolar cycloaddition of nitrile oxides to resin-bound alkynoic acids (1041.

Although Leznoff et al. [lo41 could not conclusively demonstrate that regioselec. tivity can generally be influenced by carrying out resin-bound reactions, these experi. ments have shown that pericyclic reactions can successfully be carried out on poly mer supports. Thus, the stereoselectivity is at worst only minimally influenced. whereas the yields and purity are considerably improved. Nowadays, one can go back to the experiences made in the 1980's [ W ] . Schorc and Najdi [I061 reacted 4-pentyn-1-01 with excess norbornadiene by Pauson-Khand cycloaddition [I071 and, after hydrolysis, obtained the mono-cyclic product 3 with 69% yield (26% by solution synthesis) (Fig. 2-20); the bicyclic adduct 6 (20% yield, only traces are found in solution synthesis) could also be isolated by using norbornadiene in equimolar concentration. An analogously performed reaction with I-methyl-5-norbornen-2-onefurnished a 30 :70 mixture of isomers 4 and 5 (yield 99%, 10% in solution synthesis) (Fig. 2-21). The formation of 6 can be explained by assuming a cross-linking reaction involving norbornadiene. This has been confirmed in further experiments using 2 % crosslinked PS/DVB. The reactive groups of a higher cross-linked polymer are more isolated and, therefore, the cross-linking reaction is suppressed. In fact, the formation of 6 could be suppressed under analogous reaction conditions [106b].

3

Dicobalt octacarbonyl mediated Pausen-Khand cycloaddition [lM, 1071 (a) Co,COa, (b) norbornadienc 80-100°C. (c) n-BuNC1, KOH,H,O, S O T , 4 h.

Figure 2-20.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

49

0

Figure 2-21. Substances prepared by Pausen-Khand cycloaddition using different starting materials and reaction conditions.

In 1992 Beebe, Schore and Kurth [lo81 described solid phase synthesis of 2,5-disubstituted tetrahydrofurans by a “tandem” 1,3-dipolar cycloaddition of a support-bound nitrile oxide with lJ-hexadiene. Nitrile oxides were produced by reacting nitromethane with polymer-bound aldehydes under the conditions of Henry reaction. After protection of the hydroxy group as the trimethylsilyl ester and dehydration, a cyclization took place to yield a resin-bound isoxazoline. The subsequent electrophilic cyclization by addition of iodo monochloride (ICl) yielded 2,5-disubstituted dihydrofurans. The cis/truns ratio was 1 :2.1 (1 : 1.92 in solution synthesis) with a total yield of 17% based on a maximal loading of 0.65 mmol/g (in solution 18%) (Fig. 2-22). [3 + 21-cycloaddition reactions have also been carried out for derivatization of olefin- and alkyne-substituted peptoids [147] (Section 4.1).

2.3.5 Heterocyclic Compounds: Benzodiazepines, Hydantoins and Thiazolidines The benzodiazepine skeleton is a constituent of many bioavailable therapeutic agents. Thus, the CCK A and B receptor antagonists [109], opiate receptor ligands [110] (e.g., Valium), fibrinogen receptor antagonists [lll, 1121 (GP-IIbIIIa inhibitors), reverse transcriptase (RT) inhibitors [113] and HIV-TAT-RNA antagonists I1141 all Dosses a benzodiazeDine skeleton.

50

2 Polymer Supported Organic Synthesis: A Review

C

Polymer

d

Polymer

NO1

N=c I

Poylmer& -

-

-

Figure 2-22. Tandem 1,jl-dipolar cycloaddition for the preparation of substituted tetrahydro. furans (1081. (a) DMSO, NaHC03, 155"C, 6 h, (b) CH3N02, Et3N, THF, EtOH, 14 h (c) TMSCI, Et3N, (d) PhNCO, Et,N, PhH, 80°C, Wdienes, (e) ICI, CHzClz, -78"C, 1.5 h

Following the solid-phase synthesis of benzodiazepines by Camps, Castells and P [115]in 1974, Hobbs DeWitt et al. [116]have developed a method for the synthesi: of benzodiazepine and hydantoin based on the Fmoc strategy (Fig. 2-23).The syn. thesis of Camps et al. began with coupling of Boc-amino acids to hydroxy-PS-DVE resin using carbonyldiimidazole (CDI) as a coupling reagent (Fig. 2-23).Benzodia. zepines are obtained directly by cleavage of the Boc group, followed by the reactior with acetophenone, A further development of this reaction allows the simultaneous multiple synthesis of benzodiazepines or hydantoins, starting with the resin-bound Boc- or Fmocamino acids. Hobbs DeWitt et al. (1161 synthesized a series of 40 different ben. zodiazepines and hydantoins by reacting 5 differently loaded resins with 8 differenl benzophenones and isocyanates, respectively. Additionally, in a patent published in early 1994, Hobbs DeWitt et al. 11171 described the simultaneous multiple synthesis of a series of heterocyclic compounds starting with commercially available standard resins (hydroxy or chloromethylated PS-DVB) (Fig. 2-24). Recently, MacDonald and Ramage [118],in co-operation with Hobbs DeWitt and Hogan have described a procedure for the multiple parallel synthesis of quinolones (Fig. 2-25). Thus, 3-(3-alkyl-2,4,5-trifluorophenyl)-3-oxopropionic acid ethyl estei was coupled to commercially available Wang resin by transesterification (Table 2-2), followed by reaction with a series of amines to obtain enamines. These could be cyclized by a nucleophilic aromatic substitution. The quinolone skeleton could be subsequently enlarged by a further nucleophilic aromatic substitution. The final Droduct was cleaved from the anchor bv trifluoroacetic acid/DCM.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

1

YR H

R'

\

R'

o

0

--N

I'

R

2

Rn q R' -

51

'

=\ O

H

R

I

A

B

C

Figure 2-23. Solid phase synthesis of benzodiazepines and hydantoins. (a) Benzodiazepine synthesis according to Camps, Castells and Pi [l IS], synthesis of benzodiazepines according to Hobbs DeWitt 11161, (c) hydantoin synthesis according to Hobbs DeWitt 1116).

Ellman et al. [119] have described a slightly different procedure for benzodiazepine synthesis. The benzodiazepines have been synthesized on the resin by using three different building blocks. An aminobenzophenone derivative (carboxylic acid or alcohol) was attached to the polymer via an acid-labile anchor and subsequently reacted with a Fmoc-amino acid fluoride (Fig. 2-26). After deprotection, it was cyclized by addition of 5 % acetic acid. After deprotonation and alkylation with a series of alkyl halides, benzodiazepines could be cleaved from the resin by acids. In further investigations, in addition to the solid-phase synthesis of benzophenone derivatives [I201 (Fig. 2-27) a set of 192 benzodiazepines were simultaneously synthesized and their biological activity was determined. Thus, miniaturized syntheses were carried out on derivatized polypropylene pins [121J loaded with two different benzophenones. A block of 96 of these pins was consecutively immersed in microtiter plates filled with N-protected amino acid fluorides or alkyl halides (a definite compound in each cavity) and was allowed to react.

52

2 Polymer Supported Organic Synthesis: A Review

I2

II

10

Figure 2-24. Besides benzodiazepines and hydantoins, Hobbs DeWitt and co-workers [117] prepared a series of heterocyclic compounds, a few examples are shown here: 7 quinolone, X = N, CH2, 8 ketourethanes, 9 diketopiperazines,10 pyrimidindiones, 11 benzothiazolones, X = N, CH2, 12 spirosuccinimides.

PSIDVB~ OH

HN

F

& o El0

F

R'

-

-

a

b. c

PSIDVB7

F'SIDVBi

0

0

-

\ /

e. 1

0

no 0

Figure 2-25. Solid-phase synthesis of quinolones 1118). (a) toluene, DMAP, llO"C, 16 h, (b) (CH30)2CHN(CH3)2,THF, 25 "C, 18 h, (c) H2NR2, 25 "C, 72 h, (d) tetramethylguanidine VMG), DCM, 6 5 T , 18 h, (e) amine, NMP, llO"C, 4 h, (f) TFAIDCM = 2 : 3, 25"C, 1 h.

53

2.3 Examples of Solid-Phase Syntheses of Small Molecules 0

NH-Fmoc

Polymer Q N F R 2

Polymer QNH*

Q

-

a

RI

R'

1

Li /

Polymer

b,c

Polymer

d

Figure 2-26. Benzodiazepine synthesis according to Ellman et al. [119, 1201. (a) Frnocamino acid fluorides, (b) 50% piperidine/DMF, (c) 5 % HOAc/DMF, (d) alkyl iodides, DMF.

o Polymer -N

y

a

o

s

I Pd(0)

I

H

-

\ /

:mR

Me,% NO1

2. NaSH. Md)WH20 (4: I)

Figure 2-27. Synthesis of polymer-bound benzophenones 1119, 1201.

Recently Plunkett and Ellman [122] developed a further method for preparation of benzodiazepines by the Stille reaction (see also Section 3.7) using polymer-bound N-Bpoc protected (2-aminoaryl)-stannane with aromatic or aliphatic acyl halides. After removal of Bpoc, the free amine could be acylated and cyclized to benzodiazepine with 5% acetic acid. h t e k et al. [123] have published a method for the simple solid phase synthesis of thiazolidines by cyclocondensation of polymer-bound /I-mercaptoalkylamine with ketones or aldehydes (Fig. 2-28).

54

Polymer-0

2 Polymer Supported Orgunic Synrhesis: A Review

-s=

\-STrt R2

Po '0

Figure 2-28. Preparation of compounds with thiazolidine skeleton 1123). (a) 20% piperidinel

DMF, 2.5% TFA/DCM/BuiSiH3,(b) R'CHO, HOAc, (c) R'COOH, DIC, DCM, (d) MCPBA, DCM, (e) 0.5% NaOH.

The 8-mercaptoalkylamine moieties were introduced in the form of FmocCys(Trt)-OH and the Fmoc and Trt groups were consecutively removed. Subsequent reactions with aldehydes or ketones generated the polymer-bound thiazolidine skeleton which was acylated at the nitrogen atom through a condensation reaction with carboxylic acids in DMF. To enhance the acid-lability of the 5-membered ring, the anchor was first oxidized to the sulfoxide by 3-chloroperbenzoic acid (MCPBA). The thiazolidine synthesis was further investigated by employing a series of carbony1 and carboxy groups containing compounds; the yields were determined and compared with the analogous solution reactions. Since in this reaction a new asymmetric center is created and, thus, diastereomers are formed, the diastereomeric ratio was also determined. In comparison with the solution synthesis, the diastereoselectivity of the solid-phase reaction is on the average higher although with almost the same low yield. The yields varied between < 10 and 90% (in solution 66-92%) with dr values (dr = 100 x [(IR, 4R/(2R, 4R) + (2$4R)]) of 65 to >97% (in solution 29 to >97%).

2.3.6 Further Ring Closures on Solid Support The intramolecular ester condensation (Dieckmann condensation) of asymmetrically substituted esters normally leads to an isomeric mixture. In the solid phase procedure developed by Crowley and Rapoport, this ring closure reaction takes place regioselectively [124] (Fig. 2-29). An asymmetrically disubstituted diester has been synthesized by deprotonation of pimelic acid monomethyl ester and its reaction with chloromethylated PS;the product was finally cyclized by addition of a base. By using 13C labeling, it could be demonstrated that the condensation proceeds in two directions (Fig. 2-29). Hence,

2.3 Examples of Solid-Phase Syntheses of Small Molecules

x) 0-c

“’14

I

+

\’

0

a

5:

b

Figure 2-29. Polymer-bound Dieckmann cyclization. Product a remains immobilized on thc resin during the reaction whereas product b is liberated by the self-cleavage.The polymer-bound product can also be cleaved by saponification (1241.

a product is removed from the resin by “auto cleavage”, whereas the other remains immobilized on the resin. This product can be obtained by alkaline cleavage. By using the three building blocks, a-amino acids, bromocarboxylic acids and aminoalkylmercaptanes, Virgilio and Ellman [I251 (Fig. 2-30) carried out simulta neous syntheses of a series of 9- and 10-membered ring systems which supposedlq H Polymer-N~Hl

0

MOz

O

9

-

o:#.l HzNXNH 0

MOz

Figure 2-30. Preparation of nonpeptide 8-turn mimetics on polymer supports [125]. (a) abromocarboxylic acids, DIC, (b) 2-aminoethanol-tert-butylsulfide,(c) Fmoc-amino acids, HATU,(d) 50% piperidine/DMF, (e) symmetrical anhydride of an a-bromocarboxylic acid, (f)tributvbhosDhine. H,O. (n) tetramethvlnuanidine. (h) TFA/water/Me,S (18 : 1 :1).

56

2 Polymer Supported Organic Synthesis: A Review

imitate the 8-turn of proteins. The synthesis was carried out on polypropylene pins

or PEG/PS graft copolymers which allowed the preparation of mimetics possessing (R) as well as (S)configurations in the i + 1 or i + 2 positions of the 8-turn. What makes the reaction interesting is that the choice of commercially available building blocks already allows the simultaneous synthesis of a compound library containing more than 2000 components [I26]. The multiple step synthesis began with coupling of bromocarboxylic acid to the support and displacement of bromine by aminoalkylmercaptane, protected as a disulfide. The secondary amine formed was condensed with a Fmoc-amino acid and, after removal of the Fmoc group, was acylated again with bromocarboxylic acid. The disulfide bond was cleaved with tributylphosphine and, finally, the nucleophilic cyclization was affected by addition of IV,N'-tetramethylguanidine. Solid phase synthesis has not only been confirmed to the preparation of small ring systems, syntheses of helicenes [127], porphyrins [128] and phthalocyanines I1291 have also been reported. The synthesis of asymmetrically substituted tetraarylporphyrins on PS-DVB was for the first time reported in 1977. For this purpose, hydroxybenzaldehydes were bound to an acid-labile linker-resin combination and allowed to react with tolualdehyde, pyrrole and propionic acid at high temperature for 1 h. In addition to a soluble mixture of several porphyrins which were separable by extraction, a polymer-bound asymmetrically substituted porphyrin with 4-5'70 yield was also obtained. The product could be obtained by alkaline saponification in pure form. Recently asymmetricallysubstituted phthalocyanines were also synthesized by a similar procedure [129].

2.3.7 Palladium Catalyzed C-C Attachments In several recently published articles, the application of different reactions for C-C bond formation on a polymeric support has been described, which have already been carried out in solution synthesis. A common feature of this solid-phase reaction is the use of soluble palladium compounds as reaction catalysts. Countering from the results of the elegant experiments described below, further successful applications in this area of combinatorial chemistry can be expected. The Heck reaction [130] allows simple synthesis of disubstituted olefins. Reaction of aryl iodides or styrenes with olefins or aryl halides in the presence of Pd" acetate produced olefins with high purity and yields. As model compounds, Yu, Deshpande and Vyas [131] immobilized 4-vinyl- and 4-iodobenzoic acid on a Wang resin and reacted it with a series of substrates under Heck reaction conditions (Fig. 2-31, Table 2-6). With a few exceptions, such as ethyl propionate which did not react under the chosen conditions, olefins with high yields were obtained. Furthermore, immobilized 4-iodobenzoic acid has been used to investigate the Stille reaction 11321. Reaction of iodobenzoic acid with vinvl- or arvlstannanes in the

w

2.3 Examples of Solid-Phase Syntheses of Small Molecules

Polymer -0

a'd

0

-

/-

Polymer -0

0K J 1

c. d

5;

Ar

AI - C E C G C O O H

Figure 2-31. Palladium-catalyzed Heck reaction [130, 1311 for the preparation of substituted aromatics. The synthesized compounds could be cleaved from the resin by 90% TFA. (a) Aryl halides, Pd, (b) arenes or olefins, Pd, (c) aromatic alkynes, Pd, (d) 90% TFAIDCM. Table 2-6. Educts and reagents for the solid phase Heck reaction [131] Polymer

Reagent

Reaction conditions

Yield

1 1

iodobenzene 2-bromonaphthalene 2-bromothiophene 3-bromopyridine methyl 4-vinylbenzoate phenylacetylene ethyl acrylate

Pd(OAc)2, Bu~NCI,90"C, 16 h Pd2(dba)3. P(2-Tol),, 100°C, 20 h Pd,(dbab, P(2-Tol),, lOO"C, 20 h Pd2(dbah, P(~-ToI)~, 100°C, 20 h P ~ ( O A C )Bu4NCI, ~, W"C, 16 h Pd2(dbab, P(~-ToI)~, 100°C, 20 h Pd(OAc)l, BU~NCI,90"C, 16 h

81 64 76 87 90 90 91

1 1

2 2 2

la]Yields based on the loading of support with the substrate. (Bu = n-butyl, To1 = tolyl, dba = dibenzylidene acetone).

presence of palladium and As(Ph), generated substituted arenes or biphenyl derivatives (Fig. 2-32). In the above cases, yields from 85 to 92% were obtained, based on the loading with iodobenzoic acid. Backes and Ellman [134] have carried out solid-phase synthesis of a series of phenylacetic acid derivatives (Table 2-7) by enolate alkylation and the subsequent Suzuki reaction [133]. The syntheses were carried out by employing a rarely used "Safety-Catch" linker [37c]. Since, the latter was stable under the chosen reaction conditions (Fig. 2-33). R-Sa(R'),/

Pd

-

NMP

Figure 2-32. Polymer-supported Stillecross-coupling-reaction[132]. Substitutedaromatics are obtained by the reaction of organo tin compounds in the presence of Pd. The prepared aromatics are sdit off from the resin bv TFA.

58

2 Polymer Supported Orgonic Synthesis: A Review

Table 2-7. Educts and reagents for solid phase synthesis of phenylacetic acid derivatives I1341

R'

R2

Nucleophile

Yields [Yo]

H Me Me Bn Et Pr' Me Me H Me Me Me Me

(Me),CHCH2 (Me)2CHCH2 (Me)2CHCH2 (Me)2CHCH2 (Me),CHCH2 (Me),CHCH2 (Me)$HCH2 (Me)2CHCH2 Ph Ph 4-F3CPh 4-MeOPh 2,4-ClzPh

H20 H2O BnNH2 BnNH, BnNH2 BnNH2 piperidine aniline H2O BnNH2 BnNH2 BnNH2 BnNH,

100 96 96 98 92 91 96 0 93 95 87 88 88

Bn = benzyl.

R'

X = OH. NHU. NU,

Figure 2-33. Substituted phenylacetic acid derivatives can be prepared on the resin by a combination of an enolate alkylation and a Swuki reaction [133,134]. By employing this procedure, Backes and Ellman have prepared, among others, ibufenac, ibuprofene and felbinac (Table 2-7). (a) pentafluorophenyl pbromophenylacetate, DMAP. (b) LDA, THF, O"C, (c) alkyl halides, O"C, (d) alkyl9-BBN, or aryl boric acids, Pd(PPhb, Na2C03,THF, 65 "C, (e) diazomethane, (0hvdroxides or amines.

2.3 Examples of Solid-Phase Syntheses of Small Molecules

59

The active ester pentafluorophenyl 4-bromophenylacetate was immobilized on the support in the presence of dimethylaminopyridine. By deprotonation with LDA, a trianion was obtained which was then alkylated with alkyl halides (Table 2-7). For the palladium-catalyzed Suzuki reaction, a series of either alkylboranes produced by in situ hydroboration of olefins, or arylboronic acids served as coupling components. After methylation of the sulfonamide nitrogen atom of the safety catch anchor, the reaction products can be cleaved by a nucleophile. The presented methods for C-C attachment are of particular significance for preparation of molecular diversity, since they allow the synthesis of novel skeletons.

2.3.8 Further Reactions on Polymeric Support A generally applicable method for the synthesis of ureas has been described by Hutchins and Chapman (1351 (see also Section 4.4). These compounds have been produced by reacting polymer-bound p-nitrophenylcarbamates with primary or secondary amines. The carbamates could, in their part, be obtained through the reaction of immobilized amines with p-nitrophenyl chloroformate. Thus, for example, Fmoc-Glu was anchored to the polymer via its y-carboxy group while the a-carboxy group was protected as an ally1 ester. After removal of Fmoc protection, the free amine was reacted with p-nitrophenyl chloroformate and several amines to obtain the corresponding substituted urea derivatives (Fig. 2-34). With the exception of pnitroaniline which did not react, all the amines employed yielded relatively pure products (90-98%, estimated by HPLC). It is also probable that this method can be extended for preparation of polymers (see Section 4.4). 0

a,b

PS/DVB-HMPB-0

FmocNH

0

PSDVB-HMPB-0 +

oLl 0-<"-"

O-rno' 0

0

h&LL

0

0-<"-"

N-R'

I

R'

-

PS/DVB-HMPB 0+

oLl 0-<"-" N--RI

I

R'

Figure 2-34. Preparation of polymer-bound ureas [135]. (a) 20% piperidine/DMF, (b) p nitrophenyl chloroformate,DIPEA, THF/CH2C12, 0.5 h, (c) R'R'NH, DMF,15 min., (d) 2% TFA/CH,CI,, 10 min.

60

2 Polymer Supported Organic Synthesis: A Review

Kurth et al. [136] described the three-step solid phase synthesis of a series of 1,3-propanediols (Fig. 2-35). The synthesis began with the coupling of carboxylic acids (acetic acid, 2-methoxyacetic acid and 3-phenylpropionic acid) to chloromethylated Merrifield resin. After their deprotonation with LDAITHF, they were allowed to undergo an aldol condensation reaction with a series of aromatic carbonyl compounds. In the last step, the immobilized P-hydroxy esters were reductively cleaved (DIBAL-H/toluene, O T ) and converted to 1,3-diols. A set of 27 diols have been prepared by using three different carboxylic acids and nine carbonyl compounds.

Figure 2-35. Polymer-bound synthesis of 1,3-propandiols 1136). (a) R1-C002-Na+, Bu,N+Br- (cat.), THF,A, (b) LDA, THF,(c) ZnCI,, THF,(d) aldehyde, ketone, (e) DIBALH, toluene.

As already mentioned at the beginning of this review, polymers have routinely been used for the anchoring of hydrogenation catalysts [19-211, while soluble palladium complexes have also been employed for cleavage of protecting groups and anchor bonds. Recently, the use of palladium catalysts for the asymmetric catalytic hydrogenation of polymer-bound substrates (olefins) has also been described [137]. The reaction has been employed for the preparation of a tripeptide, during which various hydrogenation catalysts and reaction conditions were investigated. The tripeptide was synthesized in solution and then coupled to Wang resin. It contained an a,@didehydroamino acid, which has been reduced enantioselectively. The best results have been achieved by the employment of catalysts prepared in sifu [R h(diPAMP)(NBD)JBF, [1381 and [Rh(Ph -CAPP)(NBD))-BF, [1391 in toluene/Zpropanol. The reaction yields range between 46% (in dioxane) and 100% (in toluene/2-propanol). Moreover, the diastereomeric ratios (R, S):(S,5') were determined using various catalysts and reaction conditions; for (Rh(diPAMP)(NBD))BF, the respective diastereomeric ratios of 30.9 :69.1 070 in dioxane and 6.2 :93.8% in toluene/2-propanol were obtained. For [Rh(Ph-CAPP)(NBD)]BF, ratios of 43.6 :69.1 070 in dioxane and 94.9 :5.1 070 in toluene/2-propanol have been found. A series of further compounds, prepared by combinatorial chemistry have recently been presented in the various congresses and symposia on this subject. A few of them. esDeciallv those reactions with interesting future Dotential should be men-

61

2.4 Oligomer Synthesis

tioned here. J. C. Chabal et al. (1401 have described an acylpiperidine and a benzopyran library as well as preparation of sulfonamides, Pavia [141] presented the synthesis of dibenzamide phenols by immobilization of 3-nitro-4-aminophenol on resin, followed by a selective reduction of the nitro group with Sn" chloride and acylation of the new amino group. A tandem Michael addition [142] of phenolates on polymer-bound acrylic acid has also been used to prepare substituted bicyclo[2.2.2.]octanes on Wang resin (Fig. 2-36). These have been removed from the polymer either by TFA/DCM or by reduction with DIBAL-H.

PSIDVB-Wang- OH

-

H O P R ' 0

PSPVB-Wang-0 ? O

hLR1

0

PS/DVB-Wang-0

a

R' 0

N-R'

Figure 2-36. Solid-phase synthesis of bicyclo[2.2,2]octanes(142). (a) R3R4NH, NaBH(OAch, ultrasonic, (b) TFAIDCM, (c) DIBAL-H.

2.4 Oligomer Synthesis Nowadays, the systematic screening of peptide libraries [3b, 7) is routinely and successfully applied in the search for lead structures for therapeutic applications. If biologically active peptides are found, these must often be converted to orally available low molecular weight drugs [143], This is generally achieved, at least in part by very difficult processes involving peptidomimetics.

62

2 Polymer Supported Organic Synthesis: A Review

In the previous Sections, two methods have already been described which help to shorten this search. Thus, benzodiazepine and hydantoin libraries consist of compounds which are much closer to the final biologically active desired agents than a peptide. An active compound found from such a library can be converted to more effective derivatives by a few additional steps. In addition to the peptide libraries, oligomer libraries from different building blocks also furnish interesting additional possibilities for the discovery of new biologically active compounds and/or lead structures, as shown below. In contrast to biopolymers, these oligomer libraries are enzymatically more stable and often show a far higher bioavailability.

2.4.1 Peptoids Peptoids are the compounds most related to peptides, because they possess N-substituted glycines (NSG) as the smallest building block (Fig. 2-37). Simon et al. [144] have reported the synthesis of peptoids by coupling of NSG building blocks, presynthesized in solution, according to a standard Fmoc protocol. Zuckermann [I451 and co-workers have synthesized NSGs directly on the polymeric support by the DIC mediated coupling of bromoacetic acid and subsequent reaction of the bromoacyl group with an amine. In addition to peptoids with “natural” side chains, commercial availability of a wide range of amines allows the syntheses of a series of “unnatural” NSGs and therefore, a large number of peptoid libraries. Principally, it is possible to prepare lo’* different hexamers by employing loo0 commercially available amines. Thus by screening of a tetrapeptoid library (4500 individual compounds) with side chains chosen according to required diversity criteria an a-1-adrenergic receptor antagonist has been found in a short time [146]. The possible variations are not only restricted to the choice of amines (side chains), since new classes of polymers can also be developed by varying the employed halocarboxylic acids. For example, a-alkyl or o-bromocarboxylic acids can be used to obtain N-and Ca-disubstituted peptoids or products with longer backbone chain. Pei and Moos [I471 have synthesized isoxazoline and isoxazole substituted peptoids (Fig. 2-38) by reacting nitrile oxides [104, 1081 with N-alkenyl- and N-alkynylglycines, respectively (Section 3.4). In these solid phase syntheses, nitrile oxides have been prepared in situ from nitro compounds, isocyanates and triethylamine, or by oxidation of oximes with sodium hypochlorite in the presence of Et,N. Nitrile oxides are usually very unstable and yield a series of side products under the conditions of [3 + 2) cycloaddition. Here, however, the byproducts were formed in the solution phase and could be removed by washing the support. Aside from two cases reported in this series [147], isoxazoline and isoxazole derivatives have been obtained with more than 80010 purity (as measured bv HPLCL

2.4 Oligomer Synthesis

63

We have carried out the automated synthesis of octa- and nonapeptoids on preparative scale in our laboratories. Using ligand-receptor assay, we could show, for example, the interesting binding of peptide/peptoid hybrids to the MHC-Class1 molecules [67] of the MHC-haplotype Kb in competition with the CTL epitope SIINFEKL [148]. The identity and purity of the peptoids were established by electrospray-MS, HPLC, and for the first time by automated sequencing [149]. The sequencing could be optimized for routine procedures and the products from the Edman degradation could be identified. For this purpose, the monomeric NSG has been synthesized on chlorotrityl-PS/DVB and converted to the corresponding phenylthiohydantoins (PTH-NSG). The PTH-NSGs were isolated, purified and characterized by MS and UV procedures. Afterwards, an HPLC standard has been constructed from PTH derivatives by which the degradation products of sequencing of peptoids and peptide-peptoid hybrids could be identified by co-elution characteristics.

I

PSIDVB-Rink NH,

PSIDVB-Rink NH

LLH

Figure 2-37. Synthesis of peptoids on Rink resins. Left: the monomeric method of Simon et al. [ M I , the required monomers are separately prepared by solution procedure. Right: the submonometer method of Zuckermann et al. [145]. N-substituted glycines can be prepared on resin by starting from bromoacetic acid and different amines. (a) N-Fmoc-N-alkylamino acids, (b) 50% piperidine/DMF, (c) bromoacetic acid, DIC/DMF, 2 x 30 min, (d) RNH,. DMSO, 2 h. le) reDetitive cowlina. tfl TFAIDCM.

64

2 Polymer Supported Organic Synthesis: A Review

7

0

hN<7p d

PSIDVB-Rink -NH

R1

0 0

0

1.b

R'

PSIDVB-Rmk-NH

d

0

7

0 PSIDVB-Rink -NHh N < N A R{

0

a b

;i 0

0

0

\

P S I D V B - R m k - N b N < N q

R'

0

Figure 2-38. Subsequent modification of N-alkenyl- and N-alkynylglycines by a 13 + 2) cycloaddition of in situ prepared nitrile oxides 11471. (a) Phenyl isocyanate, nitroalkyl or aryl compounds, triethylamine, toluene, loO°C, (b) 20% TFA/DCM. R' = alkyl, R2 = alkyl, pyridine, halogenes, R3 = methyl, butyl, hydroxymethyl, phenyl.

2.4.2 Oligocarbamates The solid phase synthesis of oligocarbamates offers another new source of potentially active oligomers in the search of lead structures. Compared with peptides, the oligocarbamate backbone consists of chiral ethylene units connected by relatively rigid carbamate units (Fig. 2-39). Like peptides, the C, atom could be substituted for different functional groups. Whereas in the first solid phase synthesis of oligocarbamate by Cho er al. [148, 1501 the C, atom has been left unsubstituted, further backbone modifications can be achieved by psubstitutions or by alkylation of the carbamoyl nitrogen atom. When compared with peptides, oligocarbamates are more hydrophobic and more stable against proteases such as trypsin and pepsin. Polymer-bound oligocarbamates are obtained by repetitive couplings of commercially available N-protected aminocarbonates (or prepared by the reduction of Nprotected amino acids pentafluorophenyl esters), to an aminofunctionalized resin (Fig. 2-39). Cleavage from the resin is carried out according to routine procedures in peptide chemistry. To demonstrate a microstructured synthesis of oligocarbamates, a series of 256 different oligocarbamates has been prepared using a photo-labile amino protecting group (nitroveratroyl chloroformate, NVOC-CI) in a light-induced parallel synthesis [ I S ] . The screening of this surface-bound library with an antioligocarbamate antibody, obtained by immunization (antigen AcXCKCFL?,showed the selective binding of antibody to this and to related sequences. It has been proposed that the seauence -FLc- is the dominant eDitoDe.

R

2.4 Oligomer Synthesis

65

Starting with commercially available amino acid alcohols and the Fmoc strategy, in our laboratories CO-substituted free oligocarbamates were prepared on a preparative scale by employing fully automated and considerably simpler procedures [152].

n

R'

PS-DVB, R , h K O A N , H

0

I PG

OMe

Figure 2-39. Solid-phasesynthesis of oligocarbamates [lSO]. (a) Deprotections: NVOC by hv

at 350 nm, Fmoc: 50% piperidine/DMF, (b) repetitive monomer coupling, (c) TFA/DCM.

2.4.3 Peptide- Nucleic Acids (PNA) Hybrid oligomers with the properties of both oligonucleotides and peptides have been described for the first time in 1991 and designated as peptide-nucleic acids (PNAs) [153]. Designed as DNA analogs, these PNA compounds can easily be varied by convenient procedures. It could be shown that the complete DNA backbone can be replaced by a polyamide (pseudo-peptide) structure without impairing the base specific hybridization. Thus, it is hoped that these compounds can find applications in antisense DNA therapy and in diagnostics. For the synthesis, the nucleo bases (thymine and cytosine) have firstly been N-substituted using bromoacetic acid followed by their conversion to pentafluorophenyl esters (OPfp) and the subsequent reaction with N-(N-Boc-aminoethy1)-glycine (Fig. 2-40). After a second esterification with pentafluorophenol, the monomers have been coupled in situ by DCC and according to a standard protocol. The PNA-DNA binding characteristics have been established by melting point determination (T,) of two complementary PNA-DNA strands (dA,,,-PNA- Tl0)as well as by NMR structural analysis [153e]. The homosequence hybrid obtained, form a triple helix with a melting point of T,[Alo-PNA-Tlo] = 73°C.

66

2 Polymer Supported Organic Synthesis: A Review

AN

+0

I

B

i MP c

I

L

B;

-

I

-

PNA Monomer

X

Jh Figure 2-40. Scheme of the peptide-nucleic acids (PNA) synthesis j153). (a) Methyl bromoacetate, K2C03, (b) NaOH/H20, 100°C, (c) PfpOH, DCC, DMF, (d) H-Orn(Boc)-OH, (e) coupling to the amide resin, (f) TFA, (8) repetitive monomer coupling, (h) HF.

Substitution of phosphoric acid diester by a diisopropylsilyl analog has led to a DNA-compatible DNA backbone modification. Solid-phase synthesis of this class of compounds has been thoroughly investigated by Saha et al. (1541 (Fig. 2-41). The oligonucleotide analogs have been obtained by repetitive coupling of 3'-O-diisopropylsilyl-nucleotide to the already loaded CPG-support. In addition to "all-silyl" analogs, diisopropylsilyl phosphonic acid diester hybrids (yields based on the maximal loading = 60%-8O%) have also been synthesized, and their binding to complementary DNA strands was investigated. By determining the melting temperature of the duplex-strands, which depend on the chain length and substitution degree, values have been obtained which range between 2 "C and 5 "C lower than those of the native DNA-duDlm.

67

2.4 Oligorner Synthesis

b. c

L

X

Figure 2-41. Synthesis of the Si-bridged oligonucleotide analogs [154]. (a) Imidazole, (b) Ac20, N-methylimidazole,(c) 3 070 trichloroacetic acid, (d) repetitive monomer coupling.

2.4.4 Oligoureas Enzyme inhibitors [I551 often contain a urea functional group as an important structural element. Recently Burgess et al. (1561 have reported the possibility of solid phase synthesis of oligoureas (Fig. 2-42). The starting point for their synthesis is the preparation of a phthaloyl protected diamine. This monomer has been prepared in solution by reducing Boc-amino acids followed by reaction with phthalimide under the conditions of the Mitsunobu reaction. By treating diamines with triphosgene [bis(trichloromethyl)carbonate] reactive isocyanates are obtained which can be reacted with polymer supports, functionahzed with amines or amino acids. After hydrazinolysis (cleavage of phthaloyl protecting group), the free amino group can be used for repetitive reaction steps. Using this solid-phase method, oligoureas have been prepared in preparative scales. The total

68

2 Polymer Supported Organic Synthesis: A Review

yield of a tetrameric test substance was 46%, corresponding to an average coupling yield of 86%. This synthetic route is not only suitable for preparation of oligourea, because of the virtue of hydrazine-labile phthaloyl protecting group, it is also suitable for the synthesis of peptidomimetics.

L

Figure 242. Solid-phase synthesis of oligoureas 11561. (a) 0.33 eq. (CC130)2C0,Et,N, DCM, (b) PSIDVB-Rink-NH2, DCM, (c) 60% hydrazine hydrate, DMF, 15 h, 2 5 T , (d) repeated monomer coupling and protecting group removal, (e) cleavage from the resin with TFA.

2.5 Outlook Without causing the problems inherent to the usual working procedure of the organic chemist, solid-phase synthesis can be carried out using a high excess of reagents. In addition to the possibility of obtaining higher yields, solid phase synthesis has the advantage of enabling complete automation of all the reaction steps as well as analytical monitoring. The required technologies for the automated biopolymer synthesis have been met almost completely in recent years. The excess reagents and all soluble byproducts can be removed by filtration and washing of the resin. The examples mentioned in this chapter demonstrate that almost all organic reactions can be carried out by solid-phase procedure. However, an intensive optimization and special adaptation will be required for achieving the present perfection of biopolymer synthesis. The methodological developments required for combinatorial chemistry cannot be achieved with the equipment available to organic chemists today, but rather it will be necessary to have new PC-controlled robots and reactors for the simultaneous and parallel synthesis of great numbers of compounds in 100 mg scale. Therefore, in future the chemistry development laboratory will in part resembles the present assay laboratories for biological screening. Three-necked flasks, reflux condensers, distillation and filtration apparatus will be replaced by minireactors with a large number of reaction vessels and sensors with fully auto-

2.5

Outlook

69

mated devices for adding and removal of reagents. New techniques for rapid heating with microwaves as well as simultaneous and efficient working up procedures for the products of multiple synthesis are being presently investigated. Even though many small companies have already been founded with the goal to synthesize extensive compound libraries for the pharmaceutical screening, the “combinatorial chemist” must urgently and extensively deal with the systematic reaction optimization for simultaneous preparation of single compounds, since as always the problems of realizing this vision are of experimental nature. In the case of biopolymer libraries and their analogs, as well as the products of combinatorial chemistry, the interest will be focused on unbound compounds and strategies without encoding procedures. The practical implementations of the label and tagging concepts in the already with byproducts loaded libraries will not easily be realized, and one will not achieve the desired and theoretically possible simplifications in the discovery of biologically active products. The concepts of direct linking of labels to investigating ligands have especially little prospect for finding general application. Great successes in fundamental biological research as well as medical diagnostics and therapy have become possible only after progresses made in the multiple solidphase peptide and DNA synthesis. Therefore, it can be assumed with certainty that future developments of solid-phase organic chemistry are not only revolutionary for chemical laboratories but also for large areas of allied sciences. Not only the pharmaceutical industry, but particularly the universities must also be intensively involved to train and familiarize the students with these new technologies. Now, where lie the limits of solid-phase organic chemistry, and in which direction might combinatorial chemistry develop in the coming years? Currently, there are very high expectations for what can be acheived by combinatorial chemistry; however, the future will show which of these are reasonable. In addition, the results of future screening programs should demonstrate the feasibility of using combinatorial chemistry for example in the search of novel lead compounds. Presently, automation of peptide and nucleic acid synthesis is possible; however, the technical part of combinatorial chemistry is still comparatively primitive and fully functioning automatic synthesizers are not commercially available. The future of automation offers two alternatives: either known chemistry must be adapted to suit present technology or the currently available synthesisers and other machines must be radically redesigned to suit new chemistries. Either way, a great deal of developmental work is still required. It is already apparent that it is pointless to synthesize many very large and complex compounds, since results using nature, in the form of biotechnology, are far superior. It is also worth combinatorial chemists considering whether the use of biotechnology may not be more suitable for the production of certain small compounds as well. Several examples of such strategies already exist, for example “combinatorial biosynthesis” 11571 or “biological diversity”.

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2 Polymer Supported Organic Synthesis: A Review

An expansion of the natural product pool is possible if, for example, microorganisms are enabled to produce new variants of, for example. a siderophore [158] or an antibiotic through feeding them with unnatural building blocks. These modest possibilities of the so-called directed fermentation remain far behind the enormous diversity, which, for example, is achievable via polyketide biosynthesis. Using different combinations of polyketide biosynthesis genes, unimaginable degrees of combinations can be achieved [I591 for the production of new molecules. Finally, it should be mentioned that, by the genetic manipulation of precursor structural genes, an array of variants and analogs of ribosomally synthesized polycyclic peptide antibiotics can be prepared. Thus, several new types of enzymes have been found which could effect, for example dehydration, cyclization, epimerization and oxidative decarboxylation in the post-translational modification of lantibiotics precursor proteins [160]. In contrast to the use of phage libraries, numerous conformationally restricted soluble active peptides could be prepared using the microbial biosynthesis systems of lantibiotics.

Acknowledgments We sincerely thank all of our colleagues who helped and supported us during preparation of this review chapter: Prof. Austel (Thomae), Dr Ch. Tsaklakidis and Dr R. Heck (Boehringer Mannheim), Dr R. Jack, Dr K.-H. Wiesmuller, Priv.-Doz. Dr J. Metzger, Dr J. Jauch, A. Fischer, H. Eickhoff, M. Winter, T. Redemann, W. Haap and S. Kapitza.

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(113) R. Pauwels, K. Andries, J. Desmyter, D.Schols, M. J. Kukla, H. J. Breslin, A. Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykants, K. Schellekens, M. A. C. Janssen. E. De Clercq, P. A. J. Jannsen, Nature, 1990, 343, 470. (1141 M.-C. Hsu, A. D. Schutt, M. Holly, L. W. Slice, M. J. Sherman, D.D. Richman, M. J. Potash, D. J. Volsky, Science, 1991, 254, 1799. [IlS] F. Camps, J. Castells, J. Pi, Ann. Quim., 1974, 70, 848. (1161 S. Hobbs DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. Reynolds Cody, M. R. Pavia, Proc. Natl. Acad. Sci. USA, 1993, 90, 6909. [I171 D. Reynolds Cody, S. H. Hobbs DeWitt, J. C. Hodges, B. D. Roth, M. C. Schroeder, C. J. Stankovic, W. H. Moos, M. R. Pavia. J. S. Kiely, PCT/US93/09666, 1993 (publiziert 1994), Warner-Lambart Company US, Ann Arbor, 143 pages. [I181 A. MacDonald, R. Ramage, S. Hobbs DeWitt, E. Hogan, Poster, Combinatorial Synthesis Symposium, July 1995, University Exeter, GroRbritannien. (119) (a) B. A. Bunin, M. J. Plunkett, J. A. Ellman, Proc. Natl. Acad. Sci. USA, 1994, 91, 4708; (b) B. A. Bunin, J. A. Ellman, J. Am. Chem. Soc., 1992, 114, 10997. (1201 (a) L. Weber, Nachr. Chem. Tech. Lab., 1994,42,698; (b) E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, M. A. Gallop, J. Med. Chem., 1994, 37, 1385; (c) J. A. Ellman, Lecture, Conference “‘ExploitingMolecularDiversityfor Drug Discovery”, San Diego, USA, Januar 1994. [I211 H. M. Geysen, S. J. Rodda, T. J. Mason, G. Tribbick, P. G. Schoofs, J. Immunol. Methods, 1987, 102, 259. (1221 M. J. Plunkett, J. A. Ellman, J. Am. Chem. Soc., 1995, 117, 3306. [I231 M. Patek, B. Drake, M. Lebl, Tetrahedron Lett., 1995, 36, 2227. (1241 (a) J. I. Crowley, H. Rapoport, J. Am. Chem. Soc., 1970, 92, 6363; J. I. Crowley, H. Rapoport, A Org. Chem., 1980,45, 3215; (b) solution phase synthesis: Y. Yamada, T. Ishii, M. Kimura, K. Hosaka, Tetrahedron Lett., 1981, 22, 1353. (1251 A. A. Virgilio, J. A. Ellman, J. Am. Chem. Soc., 1994, 116, 1180. [I261 A. A. Virgilio, J. A. Ellman, Lecture, Coderence on Exploiting Molecular Diversity, Small Molecule Libraries for Drug Discovery, 1995, La Jolla, California, USA. [127] J.-M. Vanest, M. Gorsane, V. Libert, J. Pecher, R. H. Martin, Chimia, 1975, 29, 345. (1281 (a) C. C. Leznoff, P. I. Svirskaya, Angew. Chem., 1978, 90,1001; Angew. Chem., In?. Ed. Engl., 1978, 17, 647; (b) R. K. Pandey, T. P. Forsyth, K. R. Gerzevske, J. J. Lin, K. M. Smith, Tetrahedron Lett., 1992 33, 5315. [I291 C. C. Leznoff, P. I. Svirskaya, B. Khouw, R. L. Cerny, P. Seymour, A. B. P. Lever, J. Org. Chem., 1991, 56, 82. [I301 R. E Heck, Org. React., 1982, 27, 345. [I311 K.-L. Yu, M. S. Deshpande, D. M. Vyas, Tetrahedron Lett., 1994, 48, 8919. [132] M. S. Desphande, Tetmhedron Lett., 1994, 31, 5613 (1331 A. Suzuki, Pure Appl. Chem., 1985, 57, 1749. (1341 B. J. Backes, J. A. Ellman, J. Am. Chem. Soc., 1994, 116, 11 171. 11351 S. M. Hutchins, K. T. Chapman, Tetrahedron Lett., 1994, 34, 4055. (1361 M. J. Kurth, L. A. Ahlberg Randall, C. Chen, C. Melander, R. B. Miller, J. Org. Chem., 1994, 59, 5862. [I371 I. Ojima, C.-Y. Tsai, Z . Zhang, Tetmhedron Lett., 1994, 35, 5785. [138] B. D. Vineyard, W. S. Knwoles, M.J. Sabacky, G. L. Bachmann, D. J. Weinkauff, J. Am. Chem. Soc., 1977, 99. 5946.

References

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[I391 1. Ojima, N. Yoda, Tetrahedron Lett., 1980,21, 1051. [140](a) J. C. Chabala, J. J. Baldwin, J. J. Burbaum, D. Chelsky, L. W. Dillard, I. Henderson, G. Li, M. H.J. Ohlmeyer, T. L. Randle, J. C. Reader, L. Rokosz, N. H. Sigal, Perspectives in Drug Discovery and Design, 1994,2,305;(b) J. C. Chabala, Lecture, Conference on Exploiting Molecular Diversitj Small Molecule Librariesfor Drug Discovery, 1995, La Jolla, California, USA; (c) H.P. Nestler, P. A. Bartlett, W. C. Still, J. Org. Chem. 1994,59, 5723;(d) J. J. Baldwin, J. J. Burbaum, I. Henderson, M. H. J. Ohlmeyer, J. Am. Chem. Soc., 1995, 117, 5588. [I411 M. R. Pavia, Lecture, Conference on Exploiting Molecular Diversity, Small Molecule Libraries for Drug Discovery, 1995, La Jolla, California, USA. [I421 S. V. Ley, D.M. Wynett, W.-J. Koot, Lecture, Combinatorial Synthesis Symposium, July 1995, University Exeter, Groflbritannien. 11431 Ubersichtsartikel: (a) R. A. Wiley, D. H. Rich, Med. Res. Rev., 1993,327;(b) G. R. Marshall, Tetrahedron, 1993,49,3547;(c) W. c. Ripka, D. V. de Lucca, A. c. Bach, R. S. Pottdorf, J. Blaney, Tetrahedron, 1993,49, 3593; (d) M. Chorev, M. Goodman, Acc. Chem. Res., 1993. 26, 266. (1441 R. J. Simon, R. S. Kania, R. N. Zuckermann. V. D. Huebner, D. A. Jewell, S. Banvill, S. Ng, C. Wang, S. Rosenberg, C. K. Marlowe, D. C. Epellmeyer, R. Tan, A. D. Fankel, D. V. Santi, F. E. Cohen, P. A. Bartlett, Proc. Natl. Acad. Sci. USA, 1992, 89,9367. [145]R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, W. H. Moos, J. Am. Chem. Soc., 1992, 114, 10646. I1461 R. Zuckermann, E. J. Martin, D. C. Spellmeyer, G. B. Stauber, K. R. Shoemakel; J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S.C. Banville, E. G. Brown, L. Wang, L. S. Richter, W. H. Moos, J. Med. Chem., 1994, 37, 2678. [147]Y. Pei, W. H. Moos, Tetrahedron Lett., 1994, 32, 5825. [148]K.-H. Wiesmuller, B. Teufel, R. Brock, J. Fruchtel, R. Warrass, G. Jung, P. Walden in Peptides, Chemistry, Structure and Biochemistry (Eds.: P. T. P. Kaumaya, R. S. Hodges), in print. [149]D. Stoll, J. S. Fruchtel, K.-H. Wiesmiiller, G. Jung, 2. Deutsches Peptidkolloquium, Absfracts, 1995, Tubingen, FRG. (1501 C. Y. Cho, E. J. M o m , S. R. Cherry, J. C. Stephans, S. P. A. Fodor, C. L. Adams, A. Sundaram, J. W. Jacobs, P. G. Schultz, Science, 1993, 261, 1303. (1511 S. P. A. Fodor, Science, 1991, 251, 767. I1521 R. Warrass, K.-H. Wiesmiiller, G. Jung, 1995,unpublished data. 11531 (a) P. E. Nielsen. M. Egholm, R. H. Berg, 0. Buchardt, Science, 1991, 254, 1497; (b) M. Egholm, 0. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S.K. Kim, B. Norden, P. E. Nielsen, Nature, 1993,365,566;(c) M. Egholm, 0. Buchardt, P. E. Nielsen, R. H. Berg, J. Am. Chem. Soc., 1992, 114, 1895; (d) M. Egholm, 0. Buchardt, P. E. Nielsen, R. H. Berg, Peptides 1992 Proc. 22"dEuc Pept. Symp. (Eds.: C. H. Schneider, A. N. Eberle) ESCOM, Leiden, 1993, S. 152; (e) S. C. Brown, S. A. Thomson, J. M. Veal, D. G. Davis, Science, 1994,265, 777. I1541 A. K. Saha, M. Sarsaro, C. Waychunas, D. Delecki, R. Kutny, P. Cavanaugh, A. Yawman, D. A. Upson, L. I. Kruse, J. Org. Chem., 1993, 58, 7827. [I551 P. Y. S. Lam, P. K. Jadahav, C. J. Eyermann, C. N. Hodge, Y. Ru, L. T. Bacheler, J. L. Meek, M. J. Otto, M. M. Rayner, Y. N. Wong, C.-H. Chang, P. C. Weber, D. A. Jackson. T. R. Sharpe, S. Erickson-Viitanen, Science, 1994, 263, 380.

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2 Polymer Supported Organic Synthesis: A Review

(1561 K. Burgess, D. S. Linthicum, H. Shin, Angew Chem., 1995, 107. 975; Angew. Chem., Int. Ed. Engl., 1995, 34, 907. (1571 (a) H. Decker, S. Haag; G. Udvarnoki, J. Rohr, Angew. Chem., 1995, 107, 1214; Angew. Chem., Int. Ed. Engl., 1995, 34, 1107; (b) R. A. Houghten, Methods, 1994, 6, 354. 1158) H. Drechsel, M. Tschierske, A. Thieken, G. Jung, H. Zihner, G. Winkelmann, J. Ind. Microbiol., 1995, 14, 105. [159] (a) C. R. Hutchinson, H. Decker, K. Maddari, S. L. Otten, L. Tang, Antonie van Leeuwenhoek, 1994, 64, 165; (b) H. Fu, S. Ebert-Khosla, D. A. H Hopwood, C. Khosla, 1 Am. Chem. Soc., 1994, 116, 4166. (160) (a) G. Jung, Angew. Chem., 1991, 103, 1067; Angew. Chem., Int. Ed. Engl., 1991, 30, 1051 ; (b) G. Jung, H.-G. Sahl, Nisin and Novel Antibiotics, ESCOM, Leiden, 1991. (c) M. Skaugen, J. Nissen-Meyer, G. Jung, S. Stevanovic, S. Kletten, C. I. Mortvedr Abildgaard, I. Nes, J. Biol. Chem., 1994,269, 27 183; (d) T. Kupke, C. Kempter, G. Jung, F. Getz, .l Biol. Chem., 1995,270, 11282. (e) N. Zimmermann, J. W. Metzger, G. Jung, Eur. J. Biochem., 1995, 228, 786; (f) A. Bayer, S. Freund, G. Jung, Eur. J. Biochem. 1995, 234, 414; (g) A. Bayer, S. Freund, G. Nicholson, G. Jung, Angew. Chem. 1993, 105, 1410; Angew. Chem. Int. Ed. Engl. 1993, 32, 1336.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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

3.1 Introduction Rapid developments in the biotechnology of new proteins, as well as advances in immunology and development of pharmaceuticals based on inhibitors and antagonists, have led to immense demands for novel synthetic peptides. Simultaneous preparation of 100-150 completely different peptides, having chain lengths of up to 20 amino acids, can nowadays be achieved using multiple methods. The afforded yields and qualities are high enough to permit reliable screenings in vivo and in virro for biological activities. Moreover, it is possible to optimize synthetic conditions, and to carry out comparative studies on the secondary structure and conformational mapping of proteins. Using special multiple methods, epitope mapping of larger peptides for the development of diagnostic tools and vaccines is achieved by employing hundreds of free or rod-bound peptides that are accessible for immunoassays. Multiple methods of peptide synthesis enable preparation of the so-called peptide libraries which could comprise hundreds of thousands of peptides, and by which new perspectives for the screening of lead structures will be opened. Peptide synthesis using a combination of photolabile protecting groups and photolithographic procedure enables assembling of peptide libraries on small plates for use in miniature immunoassays. Furthermore, lipopeptide-antigen conjugates allow preparation of both peptide specific and monoclonal antibodies as well as a complete screening of epitopes from B-, T-helper and T-killer cells. Applications in the areas of AIDS diagnosis, development of vaccines, and screening for hormone analogs demonstrate just some of the possibilities that have been opened by the advances in multiple peptide synthesis methods. At present, peptide chemistry is witnessing an especially rapid impetus. The numerous research possibilities using synthetic peptides for solving biological problems are increasingly better recognized. No other area of organic chemistry seems so intensively dependent on interdisciplinary co-operation as is peptide chemistry. The research fields of peptide chemistry include synthesis and analysis, isolation and structural determination, conformational investigations and molecular modelings. The project oriented studies are carried out in conjunction with research groups in pharmacology, physiology, immunology, biology and biophysics. More and more peptides with unusual amino acids, containing modified peptide bonds, having

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3 From Multiple Peptide Synthesis to Peptide Libraries

linker or spacer molecules and peptide mimetics, are being prepared. Highlights of the medicinal chemistry of agonists or antagonists of biologically active peptides and inhibitors have recently been summarized in [l, 2). Great progress has also been made in the immunology of the molecular recognition mechanisms, using both synthetic peptides and vaccines. Peptide chemistry is being confronted with new challenges at shorter and shorter intervals. There are greater demands for new strategies, faster syntheses, better coupling reagents and protecting groups, and especially for methods of simultaneous preparation, and analysis of very large numbers of peptides in a short time.

3.2 Simultaneous Multiple Peptide Synthesis (SMPS) To respond to the rapidly growing demands for a large number of peptides with completely different sequences, several groups have been developing different concepts for a multiple peptide synthesis since 1985. The term multiple peptides synthesis (MPS), also known as simultaneous multiple peptide synthesis (SMPS), denotes parallel synthesis of many peptides with various lengths and amino acid sequences. In principle, the various methods can be distinguished by the differences in polymeric support, number of possible peptides, and by the amount of products obtained. Typically, if more than 10 mg of product is obtained, not only the characterization but also a purification step can be carried out. Subsequent modification of the peptide then becomes possible. The development of synthesis strategies for a large number of peptides in parallel reviewed by [3] led to a new area in epitope mapping [4]. Characterization of the antigenic determinants of a protein with peptides, covering the whole sequence [5, 61 became possible not only for small proteins, but also for larger ones with more than 10oO residues. Different strategies were elaborated to achieve the goal of multiple peptide synthesis. The rapid synthesis of several up to a thousand peptides in parallel, each comprising a different sequence or a different C-terminus, was denoted by this term. Manual strategiescan be distinguished from automated or semiautomated ones. And methods that lead to polymer-bound peptides may be distinguished from those that can be used to obtain free ones. In addition to epitope screening, the multiple synthesis methods are applied in hormone and inhibitor research. For identification of the relevant side chains, every amino acid of a biologically active peptide can be substituted systematically, the chain length can be varied, and N- as well as C-termini can be modified. Whereas N-terminal modifications such as acetylation, biotinylation, or succinimidylation could be achieved by dividing the peptide resin, modifications within the peptide chain or at the C-terminus can only be achieved by multiple methods or by consecutive svntheses. Thus. in mulhle Debtide svnthesis. it is Dossible to

3.2 Simultaneous Multiple Peptide Synthesis (SMPS)

81

simultaneously prepare the same peptide bound to different anchors, and to cleave it to obtain peptide acid, peptide amide, alkylated amide, hydrazide, or a fully protected fragment. In the sequential mapping of a protein for immunological and biochemical investigations, it is often sufficient to have a few milligrams of peptide if overlapping sequences are used. The length of sequences and width of the raster then have to be adjusted to the types of investigation, and to determine the number of sequences which must be synthesized. Such fine scanning with the overlapping partial sequences is particularly applicable in the determination of immunoglobulin binding sites. Longer peptides are required for the investigation of immunogenicity of protein segments, however, with a less dense raster. The P6-protein of Haemophilus influenza type b, containing 134 amino acids, could be mapped immunologically using 28 peptides with a raster of 4 and a peptide segment containing 20 amino acids (Fig. 3-1) [7]. Here, we shall discuss the most advanced multiple synthesis methods according to the number and the amounts of peptides that are obtained, and with respect to their most frequent applications.

Haemophilus influenzae P6 protein faat

fleubility aoogerucity acrophll hydropath hydrophll cod Iurn

sheet hehx

LO

20

30

40

50

60

70

80

90

100

110

120

130

residue number

Figure 3-1. Epitope prediction of Haemophilus influenzae type b protein P6, consisting of 134 amino acids. Experimentally confirmed epitopes are marked by bars [I]; prediction according to the programme HYCON.

82

3 From Multiple Peptide Synthesis to Peptide Libraries

3.2.1 Tea-Bag Synthesis The first generation of multiple peptide synthesis in preparative amounts was created by inventing the tea-bag method [8]. This method is suitable for the preparation of more than 150 peptides in parallel using 100 mg up to several gram of resin. Separation of the polymeric support is achieved by sealing it in containers of polypropylene net (“tea-bags”) (Fig. 3-2). Each container will lead to a distinct peptide during the synthesis cycle. In a common synthesis protocol, 100 mg of polystyrene-divinylbenzene (1 070) are sealed into 3 x 4 cm bags; however, larger containers or different resins may be used as long as the size of the polymer fits with the pores of the net. Deprotection of the a-amino group and the washing steps are carried out together for all peptides in screw-capped polyethylene bottles, the size of which can be adjusted depending on the number of peptides and the washing volume. npically 3-4 ml solvent per container are required. For the coupling, the tea-bags are sorted according to the amino acids to be coupled and are reacted in parallel with the activated amino acids in separate bottles. A high excess and vigorous shaking is recommended in order to obtain complete reactions. After the removal of the solvent, the bags are washed twice separately and then reunified for washing and deprotection steps. In principle, the tea-bag method is not dependent on the protection or coupling strategy. Houghten (81 developed the method for the tert-butoxycarbonyl(Boc)benzyl strategy with diisopropylcarbodiimide activation. We employed the Fmoc/But strategy with tetramethyluronium tetrafluoroborate (TBTU) activation, which allows not only faster coupling cycles but also avoids halogenated solvents [9]. The tea-bag method has great advantages over other methods with respect to low costs and high flexibility. Since mainly it is performed manually, extra containers may be handled separately if, for example, nonproteinogenic amino acids are included in coupling cycles. Relatively large amounts of free peptides are obtained, which can be used for immunization, but also for structure-activity investigation or conformational analysis [101. Successful examples of epitope mapping by using the tea-bag approach were reported [ll] for the investigations of HIV gp 41 protein as well as by Van? Hoff et al. [I21 for the Dermatophagoidespteronyssimus house dust mite major allergen Der pII. Peptides, obtained by multiple synthesis were used as probes in studies of antibody-antigen interaction [lo]. In hormone research, scans of centrally truncated neuropeptide Y analogs are reported [13]. Conformational mapping has been performed [141 to clarify the secondary structure of thymidine kinire

3.2 Simultaneous Multiple Peptide Synthesis (SMPS) front

83

side

- polypropylene mesh -resin

, (b)

Deprotection

Washing steps

Sorting and Coupling

I

53----f-----label

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1

i l 1

poh/elhylene bonle piperidine in dimelhylformamide lea-bags

dimelhylformamide methanol

dislribulionof the lea-bags according lo Ihe amino acids lo be coupled coupling of Fmoc-amino acids wilh TBTU, HOBI. DIPEA

Washing steps dimelhylformamide methanol

Figure 3-2. (a) Schematic representation of a tea-bag, which is used in the multiple peptide

synthesis and synthesis of peptide libraries to separate the resins of the different peptides 01 sublibraries. (b) Scheme of the synthesis steps of the multiple peptide synthesis. Fmoc = 9-fluorenyl-methoxycarbonyl,TBTU = 0-(benzotriazole-1-y1)tetramethyluroniumtetrafluoro. borate. HOBt = I-hvdroxv-benzotriazole.DIPEA = diisoDrowlethylamine.

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3 From Multiple Peptide Synthesis to Peptide Libraries

3.2.2 Cellulose as Support in Multiple Syntheses Functionalized paper is also used as a polymeric support in multiple peptide synthesis. Paper strips (Whatman 540) loaded with 1-2 pmol protected amino acids were shown to be compatible with both Boc/Bzl and Fmoc/But strategy. Synthesized peptides can either be detached from the paper or, after removal of the side chain protection, they can remain attached to the paper and be employed for the antibody formation test by means of ELISA on paper (dot blot). Coupling cycles, applications and advantages are comparable to those of the tea-bag synthesis. Further advantages include the simple operation - cutting instead of sealing and filling (as in the teabags) - as well as lower costs through both the use of cheaper materials and low consumption of washing solvents. However, up to now, only very few groups gained experience in working with cellulose as a support for peptide synthesis. Moreover, low mechanical stability of the support imposes certain limitations. Frank et al. [15, 161 described simultaneous multiple peptide synthesis on labeled cellulose paper disks as solid supports. A peptide is synthesized on each disk using a manually operated multicolumn continuous flow synthesizer. For each coupling, the paper disks (1.55 cm in diameter, Whatman 3MM paper) must be sorted and tightly stacked in column reactors (1.5 x 10 cm glass tubes). As many as 100 disks can be reacted simultaneously with the same amino acid derivate in one reactor column, but several columns can be used in parallel. Therefore, this method is especially suitable for systematic sequence alteration. A computer program furnishes the correct compilation for a minimal number of cycles. The peptides can be employed either for immunological tests while on the cellulose support or, after detachment, as free peptides, e.g., for structure-activity relationship studies [17]. The allylic anchor group has been used for the parallel preparation of protected fragments of parathyroid hormone. A yield of only 8- 10 mg of raw peptide somewhat limits the application of the paper disks; the size of the disks cannot be varied indefinitely owing to the specificity of the column of the apparatus. As already described for other automatic methods, there is a limited flexibility with respect to the synthetic strategy and coupling reagents; savings in time and chemicals are, however, very great. Cotton, the purest form of cellulose, is mechanically much more stable than paper. Lebl et al. [18] have used this support previously for both solid phase synthesis, and in continuous flow peptide synthesis (CFPS) (181. Cotton pieces (3 cm in diameter) have been used for multiple synthesis of overlapping fragments of CRF, by which decapeptides with yields of 4-6 mg have been obtained.

3.2 Simultaneous Multiple Peptide Synthesis (SMPS)

85

3.2.3 Polystyrene-Crafted Polyethylene (PS-PE) Film, a New Resin? With the use of polystyrene-grafted polyethylene film (PS-PE) as a support, a further variation of multiple peptide synthesis has been accomplished 1191. In this procedure, styrene is polymerized on a polyethylene film using X-ray irradiation. Like the tea-bags, small pieces of film, 1.5 x 3 cm, can be washed and deblocked together, while couplings are carried out simultaneously in separate vessels. Because of the covalent bonding of polystyrene to polyethylene, there are no losses of the support, and it is also not necessary to prepare polypropylene bags by sealing. Peptide yields per gram of PS-PE film correspond to those of the tea-bag synthesis; therefore, in principle, all the uses described above are also applicable in this case. Structure-activity investigations of both melittin analogs and parathyroid hormone (PTH)have already been carried out using peptides prepared by this procedure.

3.2.4 Automated Multiple Peptide Synthesizers The tediousness of the tea-bag synthesis with the many repetitive steps has so far prevented the employment of the multiple peptide synthesis in many laboratories. During multiple synthesis as in common solid phase peptide synthesis, the deprotection, washing and coupling steps are repeated in every cycle; therefore, the multiple synthesis can be automated as well. In the second generation of multiple peptide synthesis, several synthesizers were developed and are now commercially available. Different principles were realized in order to obtain machines that handle several to many vessels in parallel. Modified pipetting robots were used successfully for automated multiple peptide synthesis [20, 211. Schnorrenberg 19Gerhardt, for example, modified the Tecan RSP 5052, a robot with two needles moving freely in x, yt and z direction, attached to two arms (SMPS, MultisynTech, Bochum; Advanced ChemTech GmbH, Frankfurt) (Fig. 3-3). Distribution of the solvents, the amino acids and the reagents is performed with these needles, which are controlled by a diluter system. The resin for the synthesis is placed either in tubes or in filters. For the tube system, the removal of the solvent is achieved with a second needle, which is covered with a platin net to avoid contamination of resin. If filters are used for reaction vessels, a pump system is employed and the removal of the solvents is achieved by suction. The second method is faster and allows a larger amount of resin to be handled, but fewer problems with sequences to synthesize were found for the first method (no methionine oxidation, no air gaps during the coupling). Depending on the size of the tubes, 48, 96 or 144 peptides can be synthesized in parallel. The multiple peptide synthesizer (Abimed AMS 422), developed [21] is based on a Gilson M222 laboratory robot. The one-arm apparatus is controlled by an extra personal computer, is equipped with six additional needles for the distribution of the

86

3 From Multiple Peptide Synthesis to Peptide Libmries removal of solvent or additional pipetling ann

dimethylfomamide reagents

(c)

piperidhe

reaction vessels

valve line to waste Figure 3-3. Schematic representation of a fully automated multiple peptide synthesizer based on the robot system TECAN RSP (a). Front view (b)and scheme of the reaction vessel unit (c).

3.2 Simultaneous Multiple Peptide Synthesis (SMPS)

87

reagents, and can be used for the synthesis of 48 peptides in parallel. The synthesis is performed in filter tubes, and the suction of the solvents is carried out by means of a vacuum pump and control valves. The parallel automated synthesis of eight peptides in the range of 5-400 pmol has been published [22]. The reaction vessels, reservoirs and amino acid derivatives have been supplied on eight tracks, each of which contains the complete set for the synthesis of one peptide The software can be varied with respect to different activation methods and the scale of the peptides to be synthesized. However, the number of peptides is limited to eight. So far, automated, robot-assisted multiple peptide synthesis is only possible with the Fmoc strategy [23]. Open vessels prohibit the use of trifluoroacetic acid in each cycle as required for the Boc strategy. Successful examples of robot synthesis for epitope mapping were reported from Beck-Sickinger et al. [7]. The antigenicity and immunogenicity of Haemophilus influenzae type b surface protein P6 was investigated by using 24 2Omer-lipopeptides and 45 12mer-lipopeptides. Mapping the influenza nucleoprotein was achieved by A successful using the automatic robot system SMPS 350 (Zinsser Analytics) [U]. complete talanine scan of the 36-amino acid neuropeptide Y has been reported previously [25] (Fig. 3-4), as well as the syntheses of conformationally restricted analogs [26, 27). Segments of adenylate kinase were synthesized, and secondary

rn L-A&scal

10.5 r 10.0

9.5 9.0

8.5 8.0

7.5 7.0

6.5

6.0

Jl

Y P S K P D N P G E D A P A E D L A R Y Y S A L R H Y l N L l T R Q R Y SOqWlCbofNPY

EPgore 34. Application of multiple peptide synthesis for the analysis of the binding site of neuropeptide Y. tAlanine and &amino acid scans revealed that the C-terminal amino acids are relevant for binding to the receptor. Affhty was investigated in a displacement assay at human neuroblastoma cell lines [25, 123).

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3 From Multiple Peptide Synthesk to Peptide Libraries

structure of the peptides revealed good agreement with the X-ray structure (281 (Fig. 3-5). Further applications of automated simultaneous synthesis of hundreds of peptides have been published recently which demonstrate the high advantages of the use of the robot systems: identification of a pentapeptide as monoclonal antibody binding site in the tissue-plasminogen activator (291, conformational and epitope mapping of the cytosolic linker region of the cardiac sodium channel protein (301, competitive peptide mapping for identification of receptor binding sites of the gating loop of the bacterial FhuA transport protein, preparation of overlapping biotinyl peptides for epitope mapping of hepatitis C virus (311 and for the OspB protein of the pathogen Borrelia burgdotferi (321 (Fig. 3-6) and peptides interacting with major histocompatibility complex Class 11 (331 and Class 1 molecules (see Section 3.4). In addition to the fully automated methods, several semiautomated ones are realized. Addition of reagents and solvent into 96 vessels was carried out simultaneously with a semiautomated machine [34, 351, which removes the liquid by reduced pressure and N, compensation. Wolfe [36] introduced the relatively inexpensive semiautomated RaMPS system for the parallel synthesis of 25 peptides by manual pipetting and parallel suction, and Krchnak et al. 137) used the continuous flow procedure at low pressure for the synthesis of ten peptides in parallel. The use of Fmoc as well as Boc strategy is described for the semiautomated methods. Instead of polystyrene, cellulose can be used as a polymer for peptide synthesis [38]. Semiautomated peptide synthesis using cotton disks [18] or paper disks [15] are

Sequence X-ray CO

151

11

160

12

170

13

180

190

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a

w

Figure 3-5. Sequence and comparison of secondary structure elements of adenylate kinase 1 as obtained by X-ray and circular dichroism. The segments of adenylate kinase are marked in gray and were obtained by automated multiple peptide synthesis. H = a-helix, S = &sheet, G = 31ahelix. T = reverse turn with H-bond, R = reverse turn [28].

3.2 Simultaneous Multiple Peptide Synthesk (SMPS)

89

I 0.6-

1

0.8

-

5

10

20

25

F 1

core1

N-terminal minimization

0.6 * 0.4

15

30

/ZZ-1 C-terminal minimizaIion

trapezoid minimization

.

0.2. 0-

B r l

I

Figore 3.6. Fine mapping of one epitope of the protein of hepatitis C virus type 1 using biotinylated 12mer peptides overlapping by nine amino acids on streptavidin-coated ELISA plates incubated with the serum of a HCV infected patient [31]. The core 1 decamer epitope is minimized to the nonamer core 1 m. The alanine scan of core 1 m emphasizes the importance of positions 1,3,4,5 and 7 for recognition by the patient’s antibodies, whereas positions 2,6,8 and 9 can be exchanged by alanine without loss of binding [w. Kraas, Thesis, University of Tobingen. 19961.

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3 From Multiple Peptide Synthesis to Peptide Libraries

reported. Whereas the paper disks can be tightly stacked in columns of commercially available continuous flow synthesizers, the cotton disks are washed by centrifugation, similar to the procedure used in laundry machines.

3.2.5 Synthesis of Polymer-Bound Peptides Geysen et al. [39] developed a concept for the synthesis of several hundred peptides on solid polymer sticks. The peptides remain bound to the carrier after the synthesis for the biological assays. Only the side chain protecting groups are removed. Aminofunctionalizedpolyethylenerods (4 mm diameter, 40 mm lengh) are employed as support. Of these so-called “pins” 96 are mounted on a block in 8 rows of 12 rods each (Fig. 3-7). The pins of this arrangement exactly fit into the wells of ELISA plates. Washing and deprotection are carried out in large baths, coupling is performed in ELISA plates stable towards DMF. The amount and distribution of the Fmoc-amino acids to be coupled is calculated by a computer program. Since the polyethylene rods cannot swell or shrink, washing steps have to be carried out thoroughly. In a new variant, detachable heads of the pins and not the rods themselves are functionalized. The blocks themselbes can be used several times. To obtain free peptides by the pin synthesis method, several methods are suggested. Chemical or enzymatic cleavage of additionally introduced C-terminal sequences (Asp-ProGly allows acidic cleavage, Lys-Gly allows tryptic cleavage [40],Lys-Pro enables diketopiperazine formation [41], as well as the cleavage with NH3 vapour [42] are reported). However, the amounts of free peptide are limited, since 80 to 300 nmol of peptides are synthesized per rod. This efficent method of synthesis of peptides on rods is used mainly in the enzyme linked immuno assays (ELISA). Many investigations were published on the successful epitope mapping of proteins, or identification of the binding sites of monoclonal antibodies or sera. The antibodies to be tested are distributed in ELISA plates, and the pin-bound peptides incubated. The corresponding specific antibodies in the sera bind preferably to the peptide rod having the sequence of the continuous B e l l epitope. Incubation of the antibody-peptide-pin complex with a second enzyme-linked anti-antibody and substrate indicates the pin that contains the binding peptide by the color reaction of the corresponding well of the plate. Typical applications are the mapping of large proteins by synthesizing overlapping tetra- to nonapeptides and testing of antiprotein sera or antibodies, minimization of epitopes on the level of oligopeptides or evaluation of the residues essential for recognition by alanine scans, introduction of spacer amino acids or full replacement analysis [43-451. Epitope fine mapping of neutralizing antibodies against the preS-protein of duck hepatitis B virus led to the identification of a sequence of 14 residues in which two (Gln, Tkp) amino acids were identified to be crucial by an alanine scan [46]. Anti-cvtochrome C antibodies were mamed bv usinn Din-bound and free Deotides

3.2 Simultaneous Multiple Peptide Synthesis (SMPS)

91

Fmoc-&alanine-hexamthylendiamine-spac8r polypropylene pin derhratiaedwith acrylic acid

-

10 50 nmol of covalently bound peptide

antiserum in microtiter plate Figure 3-7. Schematic representation of the pins (a) and the block containing % pins (b), After the synthesis of the rod-bound peptide, the side chain protecting groups are removed and the pins are tested by the pepscan method in an ELISA assay.

in a competitive ELISA [47]. Except for one epitope, the free peptides were able tc displace the pin-bound peptides. By mapping of mono- and polyclonal antibodies against the Torpedo acetylcholine receptor (AcCHR), three major epitopes on the Torpedo AcChR a-subunil were identified [48]. Since the monoclonal antibodies were used to investigate thc transmembrane orientation of the polmeptide chain, the knowledge of the epitopc

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3 From Multiple Peptide Synthesis to Peptide Libraries

is of strong interest. By mapping 11 monoclonal antibodies against Bcell differentiation (CD) marker CD 24, which is identical to the small cell lung cancer cluster w4 antigen, five antibodies were identified to react against the protein part of the glycoprotein. They all recognize the same sequence Leu- Ala-Pro, which is suggested to be localized in a turn region [49] (Fig. 3-8). Further successful investigations of antibody binding sites are reported from virus protein (VP1-protein) of the footand-mouth- disease virus [39], the capsular protein of the Epstein-Barr virus [43], which consists of 1381 amino acids, the hepatitis delta virus antigen [50], which was mapped by using 209 hexapeptides. Various proteins of HIV subtypes [51, 521 and parasite proteins [53] - as well as the plant proteins of potyvirus and TMV [54] were mapped using the pin technology. Whereas the investigation of monoclonal antibodies frequently can be achieved by routine protocols, some experience is necessary for the handling of human sera in order to suppress nonspecific reaction. Since the synthetic peptides may cover the sequence of the entire protein with only one amino acid shifted in each peptide, continuous B-cell epitopes are identified very precisely. In the case of monoclonal antibodies, the continuous binding sites are analyzed down to the amino acid level.

Relative

0.

N-termhdamino acid of the peptides

Figure 3-8. Epitope fine mapping of a monoclonal antibody raised against leukocyte workshop antigen CD24. Binding of the monoclonal antibody to hexa-, penta-, tetra- and tripeptides, which were prepared by the pin technology, was investigated and is shown by the bars. The smallest peptide that was recognized is the tripeptide LAP [49].

3.2 Simultaneous Multiple Peptide Synthesis (SMPS)

93

3.2.6 Spot Synthesis An inexpensive procedure for the preparation of polymer-bound peptides has recently been introduced [55, 16). Sheets of paper membranes are used as polymer. Droplets of liquid are applied onto the membrane and spread over a restricted circular area (spot). This so-called spot synthesis allows the parallel synthesis of about 50 peptides on a sheet of paper (55 x 105 mm) because a great number of distinct spots can be arranged, and each of these spots remains individually addressable. The steps of the peptide synthesis are performed by immersing the whole paper membrane into respective reagents or solvents. For the coupling, the amino acids are dispensed on the defined spots. Spically 5 to 10 pL are used. Miniaturization is suggested to accommodate the synthesis for 100 peptides/cm2 (16). The entire sheet was then immersed into appropriate solutions for washing, acetylation and deprotection. After the last step, an ethanol washing, the paper was dried. Coupling of the next amino acid was then carried out by distribution of the aliquots on the corresponding spots. This could be accomplished manually or by using a pipetting robot. The peptides were not removed from the paper under the cleavage conditions of the side chain deprotection (2OVo trifluoroacetic acid, 1Yo triisobutylsilane); therefore, the sheets can be directly employed for immunological tests. The peptide-bound paper sheet can be subsequently treated and tested in a manner similar to the membranes of the Western-blot. Frank et af. exemplified this by performing epitope analysis of the immunogenic region of cytomegalovirus (hCMV), using 49 peptides made simultaneously on paper sheets. If free peptides are to be prepared, a cleavage point will be introduced before the first amino acid. The spots are then cut out and the peptides are cleaved separately (see also Chapter 1.3).

33.7 Spatially Addressed Synthesis of Thousands of Peptides Even more peptides/cm2 can be prepared by the method of Fodor et af. [56]. This fascinatingtechnology combines the miniaturization of solid phase peptide synthesis with the process of photolithography. Thousands of oligopeptidesare synthesized at defiied sites by using aminoalkylated glass slides as solid support, and the photolabile nitroveratryloxycarbonyl amino protecting group. Irradiation and hence deprotection of a surface area of only 100 x 100 pm is possible by means of stencils. This so-called light-directed, spatially addressable, parallel chemical synthesis will allow the synthesis of 100 x 100 peptides per cm2, if a suitable choice and combination of stencils is used. Since the pattern of exposure to light through the photolithography mask determines the regions of deprotection and therefore coupling of the next residue, the identity of the peptide at each location is known. Detection and location of binding is camed out by incubation of the array with fluorescent-labeled antibodies, which can be identified in a fluorescence microscope. The precision of

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3 From Multiple Peptide Synthesis to Peptide Libraries

the method and the efficiency are impressive, and characterization of a HLA B7 pep tide sequence motif fitting to the major histocompatibility complex (MHC) Class 1 molecule was shown recently [57]. However, the establishment of this process as routine requires special and very expensive equipment. Future applications in biosensor research or as diagnostic chips can be assumed.

3.2.8 Microstructured Peptide-Gold Electrode Self-assembled peptide monolayers attached on gold surfaces can be prepared by modification of peptide or peptide libraries with a o-hydroxyundecanethiol linker. These peptide derivates can be applied together with nonderivatized o-hydroxyundecanethiol for adsorption on to microstructured gold surfaces according to selfassembling procedures [58, 591. Using synthetic, thiol modified peptides, e. g., the well-known epitope VPI-(135- 154) of foot-and-mouth disease virus, the specific binding of antibody to peptide recognition layers could be detected by impedance or capacitance measurements. The peptide-gold electrode system can be microstructured, and in addition it can be optimized as a flow-through system, and used as a multiple array detector (Fig. 3-9).

3.2.9 Peptide Functionallzed Surface by Electrochemical Polymerization The reproduceable and high density attachment onto surfaces of a high variety 01 different peptides and peptide libraries is a prerequisite for their use in biosensor and for lead structure search. Recently, a novel technique for the microstructured immobilization of antigenic peptides by electrochemical polymerization was reported [a]. Peptides were derivatized N-terminally with 3-hydroxy-phenylacetic acid, which serves as an ideal polymerizable group. In a one-step procedure, these polymerizable peptides can be directly attached via aromatic oxidation in the form of thin and stable films without further additives onto glass carbon or metal elm trodes. The application for the recognition of peptide functionalized platinum interdigital microstructures by specific antibodies has been demonstrated successfully (Fig. 3-10).

3.3 Peptide Libraries Peptide libraries, which consist of thousands of individual peptides, are synthesized as a mixture of amino acids or as separate peptide pools. They either remain bound to the polymer and can be tested in the bioassay (antibody binding assay) directly, or are cleaved from the bolvmer and used like single Debtides 13. 611.

3.3 Peptide Libmries

95

[M+3H]3+ .O ILRGOLQVLAQK

654.0

SH

400

600

800

1000

m/z

1200

1400

1600

epitope VP1-(135-154) of the FMDV (foot-and-

moulh-diease-virus)

gold layer

Figure 33. Peptide-functionalized gold surface coated by self-assembly of the thiol modified epitope W1-(135-154) of the foot-and-mouth disease virus for detection of antibodies of infected animals [58, 591. Succinylated bis-(w-hydroxyundecyl)-disulfide was coupled N-terminally and the reduced peptide (electrospray-MS, upper part) was obtained by reductive cleavage from the resin using thiol-scavengers. The surface functionalization (non-peptidic alkane thiol was added for dilution; schematic representation, lower part) and antibody binding can be demonstrated by ELISA, cyclovoltametry and impedance measurements (58,591.

96

e-

3 From Multiple Peptide Synthesis to Peptide Libraries

e'

extinction

mc

non-specific

antibody

6.7

1.74

8.7

87

pg/ml

pglml

pg/ml

pglml

Figure 3-10. Peptide-functionalized gold surfaces prepared by electropolymerisation of peptides carrying N-terminally 3-hydroxy-phenylacetic acid as a polymerizable phenolic moiety [60](schematic representation, upper part). Spacer molecules: Ahx, &-aminocaproic acid; Abu, 2-aminobutyric acid. The peptide represents the epitope VP1-(135-154) of the footand-mouth disease virus. The peptide functionalized surface is recognized by antiviral antibodies as shown by ELISA (lower part) and comparison of binding properties of surfaces made by applying either constant potential mode or cyclic mode.

3.3 Peptide Libraries

9;

For the synthesis of libraries, consisting of linear peptides, several methods have been developed and proved, but challenges still remain in the testing of libraries in biological assays, in the synthesis of modified libraries and in the identification of the active compounds of the library. From peptide pools and modified peptide libraries and other oligomer libraries such as peptoids, peptide nucleotide acids (PNAs)or polyurethanes, the aim is to build up pools consisting of peptoids and nonpeptide building blocks, which lead to a new field of organic chemistry, the socalled combinatorial chemistry (Review see Chapter 2). One branch is the attempt to perform organic synthesis on polymeric support, the other approach is the randomization of synthesis procedures in solution. In the following paragraphs, the strategies to obtain linear and modified peptide libaries will be described, and strategies with respect to characterization, testing and identification of active compounds will be discussed.

3.3.1 Mixotopes The first publications on parallel, nonseparated peptide synthesis appeared in 1990 [62]. Inspired by the “mixed” synthesis of oligonucleotides, mixtures of different amino acids have been activated and coupled at definite positions, instead of a single amino acid derived. The resulting analogs have been separated by preparative highperformance liquid chromatography (HPLC),analyzed and characterized; the parallel synthesis of six analogs has been described. If small amounts (<0.1- 1 mg) of analogs of peptide are required, it seems that this method is very promising. HPLC isolation of the components effects their purification at the same time. Problems are encountered with the analogs having very similar structure and containing by-products which cannot be separated simply be reversed phase R P HPLC. This phenomenon is mostly unpredictable, especially when several positions are substituted simultaneously, and in the case of larger peptides when their chromatographic behavior is influenced by the formation of secondary structure. Moreover, a structurally dependent, nonuniform coupling of different amino acids leads to dissimilar product distribution. Such an approach was applied for the use as synthetic immunogens [63]. High numbers of analogs (7.5 x 10’) of the V3-loop of HIV-gpl20 were synthesized, whereas the length of each of the peptides consisted of 22 to 25 residues. The substituted positions reflected the variable amino acids in different HIV isolates, whereas the conserved positions were maintained. These so-called mixotopes are therefore not accidental sequences, but analogs of the original peptide for the develoDment of a vaccine against more than one strain.

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3 From Multiple Peptide Synthesis to Peptide Libraries

3.3.2 Mimotopes To identify linear peptides that mimic discontinuous or conformational epitopes, the mimotope strategy was invented [45]. Hexa- or octapeptide mixtures are used, since an epitope comprises mostly 6-8 amino acids. The linear sequence DFLEKI was identified recently [64] as an epitope of the antimyohemerythrin monoclonal antibody by using a set of hexapeptide mixtures XX0,02XX: 0, and O2 are defined positions, the X-positions are mixtures of amino acids. Therefore, 400 peptide pools were required in the first synthesis, which was performed by the pin technology [39] and tested directly by Pepscan ELISA. Single amino acid substitution of the epitope by 19 other amino acids as well as multiple D-enantiomer substitution revealed the significance of each residue [64].However, it has to be stressed that the peptide, which is binding best is only mimicking the discontinuous epitope, and frequently it is impossible to correlate the found linear sequence with the original protein sequence.

3.3.3 Phage Libraries and Biopanning In the molecular biology laboratories of Scott and Smith (651, Devlin et al. [a] and Cwirla et al. [67], a completely different kind of epitope library has been developed. Fusion phages in E. coli are transformed through oligodesoxy-nucleotides (KKN),, or (KNN)6 - where K corresponds to a mixture of all four desoxynucleotides and N to a mixture of guanidine and thymidine. Every triplet KKN can code for all of the 20 amino acids but only for one stop codon. The multiple protein production takes place for example in the p-111 protein, which is coded by the phage and which is expressed at the tip of the virion. Alternatively, the extra sequence can be cloned into the p-VIII coat protein [68], which has several thousand copies at the surface of the phage body. Identification of the active peptide sequence is achieved again at the DNA level. A ligand that is to be mapped is used for affinity chromatography of the phages that display the binding peptides. For example, phages that bind to the biotin-labeled antibody are incubated on a steptavidin-coated plate The unbound phages are removed by washing, the bound ones separated from the antibody propagated in E. coli, and the sequences of the peptides displayed on the phage are identified by sequencing of the corresponding coding region of the viral DNA after PCR amplification. In addition, molecular biology techniques have been used to generate libraries of random peptides fused to the lac repressor protein, and the phage lamda receptor [69]. Recently Ginsberg et al. [70] successfully used a recombinant library for the epitope fine mapping of monoclonal antibodies against human Willebrand factor, and a monoclonal antibody against adenovirus was mapped with a hexapeptide librarv bv Arsenault and Weber 1711. Cull et al. 1721 used a similar aDDroach for the

3.3 Peptide Libraries

99

screening of a monoclonal antibody against dynorphin B with a dodecamer library, a decapeptide library was affinity selected with a monoclonal antibody specific for the V3 loop of the gp 120 protein of HIV 1 [73), and a biotin binding peptide was identified by Saggio and Laufer [74] using this approach. Further successful applications of phage peptide libraries were reported for the identification of an epitope on an antikeratin antibody [75], of an urokinase receptor antagonist [76], of a streptavidin binding peptide [77], of an integrin binding peptide [78], and of a peptidic ligand for a sugar-binding protein [79]. Conformationally constrained phage peptide libraries were constructed with hexapeptide mixtures flanked by cysteine residues. Whereas analogs with affinities to fibronogen in the nanomolar range were discovered by this approach, peptides which contain Ser, instead of Cys, bound 1000-fold less tightly (80).

3.3.4 Random Libraries To obtain random libraries, where all or some positions of the peptides consist of several amino acids, the methods of divide, couple and recombine [81, 821, portioning-mixing 183, 841 and split synthesis [85] were developed (Fig. 3-11). Problems of the presence of different percentages of various analogs, which are due to nonuniform couplings when mixtures of amino acids in molar excess are coupled, can be avoided by these strategies. The resin for the synthesis of the library is divided in portions for the coupling of the 20 (or more, if unnatural derivatives are used) amino acids. Uniform couplings can therefore be achieved, since the competition between slow and fast coupling amino acid derivatives no longer exists. After washing steps, the polymer is mixed again, washed, and the N-terminal protection group removed simultaneously for all peptides in one vessel. For the next coupling, the distribution of the polymer in each vessel is purely statistical. However, one has to consider that enough resin is used to obtain a ratio of at least three beads per peptide (Chapters 4 and 5). Lam et al. [85] established a library of oligopeptides, which remain bound to the polymeric support and were liberated only from their side-chain protecting groups. A standard solid-phase resin can be used [86, 871, and one bead only contains one defined sequence and its failure sequences. A suitable method for the screening of this type of polymer-bound library for antibody binding is the enzyme-linked immunoassay (ELBA). A monoclonal antibody, which was coupled to the enzyme alkaline phosphatase, was used. The beads, which contain the peptides, that interact with the monoclonal antibody become colored, and they can be removed from the dish by a micromanipulator under a low power dissecting microscope [MI. The peptides of such a bead can be characterized by a protein sequencer because 50 to 500 Dmol should be Dresent on each bead. A monoclonal antibody against &endor-

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3 From Multiple Peptide Synthesis to Peptide Libraries

Synthesis of 400 Sublibraties, 20 mg each

Synthesis on beads: polymer bound peptides assay on soluble receptors

Ala-Ala-X-X-Phe-X-X-Leu Ala-Cys-X-X-Phe-X-X-Leu Ala-Asp-X-X-Phe-X-X-Leu I I Trp-Trp-X-X-Phe-X-X-Leu

t

Identificationof the beads, , containing the active peptides

t

Bioassay: active sequence: Sublibrary: &&-X-X-Phe-X-X-Leu

Sequencingof polymer bound peptides

Synthesis of 20 mixtures:

Synthesis of 80 peptide mixtures:

Ser-Ile-Ala-X-Phe-X-X-Leu Ser-Ile-Cys-X-Phe-X-X-Leu

Ser-Ile-Ala-X-Phe-X-X-Leu

Ser-Ile-T;p-X-Phe-X-X-Leu

Ser-Ile-Trp-X-Phe-X-X-Leu

Bioassay, active sequence: Ser-ne-k-X-Phe-X-X-Leu

Ser-Ue-X-Ala-Phe-X-X-Leu

Synthesis of 20 mixtures:

Ser-Ue-X-Trp-Phe-X-X-Leu

Ser-lle-Ue-Ala-Phe-X-X-Leu Ser-Ile-Ile-Cys-Phe-X-X-Leu

Ser-Ile-X-X-Phe-Ala-X-Leu

Ser-Ile-ne-Tr-Phe-X-X-Leu

Ser-Ile-Cys-X-Phe-X-X-Leu

Ser-Ile-X-Cys-Phe-X-X-Leu

I

Ser-Ile-X-X-Phe-Cvs-X-Leu Ser-Ik-X-X-Phe-Trp-X-Leu

Bioassay, active sequence: Ser-ne-ne-A~-Phe-X-X-Leu

Ser-Ile-X-X-Phe-X-Cys-Leu

etc.

Ser-Ile-X-X-Phe-X-Trp-Leu

Each bioassay defmes one more position sequential approach

1-evitca( Ser-Ile-Ilr;-X-Phe-X-X-Leu

Ser-Ile-X-Bsn-Phe-X-X-Lei

Ser-Ile-X-X-Phe-~-X-Le~

Ser-Ile-X-X-Phe-X-Ala-Leu

Ser-Ile-X-X-Phe-X-&-Lei

active sequence can be deduced from the bioassays of the mixhues Ser-Ile-Ue-Asn-Phe-Glu-Lys-Leu

Figure 3-11. Scheme of peptide libraries and possible identification of active sequences exemplified by Kb-restricted cytotoxic T lymphocyte (CTL)-epitope ovalbumin 258-267. Recognition of the MHC-Class I peptide complexes on target cells by the receptor of the killer cells serves as bioassay. A: Position 5 (Phe) and 8 (Leu) are known as anchor residues. Construction of a library can be performed either by the method of Lam et al. 1851 (B) or by means of free sublibraries (C). 400 sublibraries have to be prepared, each consisting of two defined and four mixed (X)positions. After evaluation of the bioactive mixture identification can be achieved either sequentially (D)or by a combinational approach (E).

3.3 Peptide Libraries

101

phin was mapped using a pentapeptide random library and a limited octapeptide library. In addition to the native ligand YGGFL (ICs0 17.5 nM), the following peptides were identified: YGGFQ (ICso 15 nM), YGGFT (ICso 36.9 nM), YGGFA (ICso 32.9 nM) and YGAPM (ICso 52 nM) [86]. Although some low affinity ligands were also identified, the peptides fitted into the motif G(A/G)(L/W) for the octapeptide library and YG(A/G)(F/L/M/W) for the pentapeptide library. In a similar approach, D-amino acid containing ligands were tested [87] (Chapter 6). R. A. Houghten et al. [81] introduced the synthetic peptide combinatorial libraries (SPCLs), which consist of free peptides, and which in total exceeds 50 million hexapeptides. For an oligopeptide having a certain length, anchor amino acids, defined positions (0) and mixed positions (X)are distinguished. Anchor amino acids characterize the already known, essential positions of a sequence and are found in all peptides. The number of defined positions determines the number of peptide mixtures to be synthesized. For one defined O-position, 20 sublibraries are required, each containing one of the 20 amino acids at this position. Consequently two defined positions lead to 400 and three to 8000 peptide mixtures. In the case of 20amino acids and n mixed positions, each sublibrary consists of 20n peptides regardless of the length of the peptides (number of defined residues). If amino acids other than the proteinogenic ones are used, the number of resulting peptides increases rapidly. The remaining X-positions are mixed positions and contain all 20 amino acids (less for limited libraries, more, if nonproteinogenic residues are allowed). They are introduced by the system of divide, couple and recombine or by mixtures containing the amino acids in defined ratios, that were empirically determined to give almost equimolar incorporation of each amino acid during each coupling step [88] (Chapter 5). Houghten et al. [81], who used the tea-bag approach for the synthesis of the libraries, identified epitopes binding to monoclonal antibodies. In an iterative process, by using 364 sublibraries in the first synthesis (two fixed positions, 18 amino acids in each X position) four subsequent syntheses with 20 peptide mixtures each were required. Positional scanning libraries [3, 891, which reveal the active sequence by a combinatorial process, are described as well. Positional scanning with 300 hexapeptide sublibraries using ten representative amino acids for undefined positions (four per each sublibrary) and two defined positions next to each other O,0,O2O2O3O3 with O,O, representing the defined residues in the first 100 subin the second and 0303 in the third) was used to identify the binding libraries, 0202 diversity of a monoclonal antibody against Pseudomonus ueruginosu [go]. In addition to the identification of monoclonal antibody binding sites by competitive ELISAs, hexapeptide to decapeptide libraries were used to identify antibacterial an antifungal peptides, novel enzyme inhibitors, antivirals or hormone analogs [91,92]. Conformationally defined libraries were used to detect folding abilities of peptide chains [93]. Radiolabeled acceptor molecules [94] were shown to be helpful in the bioassay, whereas unspecificity can cause problems in routine affinity tests [95].

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3 From Multiple Peptide Synthesis to Peptide Libraries

In a similar approach using an automatic peptide synthesizer Stevanovid et al. [96] synthesized and characterized the sublibrary LNYRF-(T,I,E,S)-(N,K,Q)-(L,M,I,N) consisting of 48 analogs, which represent octapeptides with potential binding capacities to the groove of a defined major histocompatibility molecule Class 1. Such an octapeptide pool had been characterized by mass spectrometry and pool sequencing of the natural Kb-restricted MHC-bound peptide library (Chapter 9). High affinity ligands binding to a monoclonal antibody against HIV gp 120 protein were selected from a synthetic library, which consisted of 361 peptides [97]. The peptide mixtures were generated using a peptide library synthesizer, which is based on a Zymark robot system [98]. A fully automated machine to synthesize combinatorial peptide libraries and its use for the synthesis of FLAG peptides was reported recently [99].

3.3.5 Modified Peptide Libraries To overcome the limitation to the natural 20 amino acids, new trends are to develop modified peptide libraries. These are expected to solve some of the problems of low biostability, which is a general problem of peptide drugs, and to increase the number of lead structures through those modifications. Several groups use cyclic peptide libraries 1100, 101) to provide conformationally constrained analogs, and dimerization of oligopeptides is suggested by Zhang et al. [102]. N-terminally attached libraries were introduced by Holmes and Rybek [103]. The use of mixtures of D- and L- and nonproteinogenic amino acids does not require specific technical alterations in synthesis; however, the analysis of the active compound may be much more difficult, e.g., in cases where no Edman degradation is possible [W]. Approaches that face this problem are summarized in the next paragraph. Modifications of the resinbound library after the synthesis were shown for amide alkylation [lOS], polyalkylation [lo61 and thionation with Lawesson’s reagent [107]. Polymers built up by new monomers such as N-alkylated glycine residues (peptoids) [lo81 or iminodiacetic acid (1091 were investigated. Flegelova et al. [110] synthesized libraries consisting of Nacyl-N-alkyl-aminoacids by coupling and reduction of aldehydes to polymer-bound amino acids and further acylation by carboxylic acids. Further modifications include the use of scaffolds or templates for the generation of libraries, so even the structural features of the peptide chain are altered. Wallace et al. [lll] developed the multimeric peptide library approach, which uses lysine to branch the chain, similar to the multiple antigen peptide (MAP) concept. Using the TASP (template associated single protein) scaffold (1121, libraries may be displayed on the surface of the so-called TASC molecule (template associated side chains) by selective removal of side chains and coupling of randomized residues [113, 1141. A similar approach was shown by Eichler et al. [115] using linear or cyclic lysine tetrapeptides with randomly acetylated side chains. Many more attempts are cur-

3.4 Conclusions

103

rently under investigation to apply organic chemistry on polymeric support or to randomize organic chemistry in solution (Chapter 2).

3.3.6 Identification of the Active Compounds In the linear, soluble peptide libraries, the active sequence is found by an iterative process going from unspecific sublibraries to better characterized sublibraries and ending with single peptides or by positional scanning of sublibraries, and the identification of the peptides is finally performed by comparing the results. It is obvious that those approaches become difficult for modified libraries. Nonproteinogenic amino acids lead to an explosion of the required sublibraries and postsynthesis modification cannot be handled in a similar way. The use of specific subarrays [I161 or orthogonal sublibraries which are self-deciphering [117]reduce the number of required sublibraries. The partial cleavage approach [118]uses a polymer, where various functionalities are coupled to, and the peptide is released step by step and can be tested. For the detection of the sequence, the final polymer-bound peptide is subjected to Edman degradation. For nonsequencable oligomers, however, a new approach for decoding has to be developed. The so-called macro beads [119],which have selectively functionalized inner and outer surfaces of the beads, are a very useful and necessary tool of high capacity. Novel heterobifunctionalized polystyrene- polyethylene glycol resins have been prepared for the simultaneous synthesis of free and immobilized peptides, and biological activity has been detected by confocal microscopy [120].New encoding systems translate the stepwise build-up of a desired polymer either to a oligonucleotide sequence [121, 1221 or to peptide sequence [104]. After the coupling of each building block by a conventional strategy, either a di-, or tripeptide or a trinucleotide is coupled to the same bead by an orthogonal strategy. The one-bead-one-compound concept and the split-resin strategy have to be applied. After the identification of the bead that carries the active compound, either the DNA is amplified by PCR and sequenced, or the bead is subjected to Edman degradation and the coding peptide identified. A translation protocol leads to the active compound.

3.4 Conclusions Multiple peptide synthesis methods have been applied for the characterization of epitopes, for selection of receptor ligands and antimicrobial peptides, for the investigation of structure-activity relationships, for conformational mapping of proteins, as well as for methodological investigations. Peptide libraries are under investigation for the screening of novel peptidic lead compounds for drug development. Including the Droduct mixtures of combinatorial organic chemistry. an enor-

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3 From Multiple Peptide Synthesis to Peptide Libraries

mous number of molecules will be available for testing. However, strong efforts have t o be made with respect t o the analysis and characterization of active compounds a n d with respect t o reproducibility of synthesis of libraries. Multiple peptide synthesis methods undoubted will offer surprising new possibilities in many more areas in the future.

References [l] R. Hirschmann, Angew. Chem. Int. Ed. Engl. 1991, 30, 1279-1301. [2] A. Giannis, T. Kolter. Angew. Chem. 1993 105, 1303-1326. [3] G. Jung, A. G. Beck-Sickinger, Angew. Chem. Int. Ed. Engl. 1992, 31, 367-383. [4] A. G. Beck-Sickinger, G. Jung, Pharm. Acta Helv. 1993, 68, 3-20. [5] I. Callebaut, A. Burny. V. Krchnak, H. Gras-Masse, B. Wathelet, D. Portetelle, Virology, 1991, 195, 48-55. [6] D. Ou, P. Chong, Y. Choi, P. McVeigh, W. A. Jefferies, G. Koloitis, A. J. Tingle, S. Gillam, J. Virol., 1992. 66, 6788-6793. [7] A. G. Beck-Sickinger, H. Rothering, K.-H. Wiesmilller, F. Dorner, G. Jung, Biol. Chem. Hoppe-Seyler, 1994, 375, 173- 182. [8] R. A. Houghten, Proc. Natl. Acad. Sci. USA, 1985, 82, 5131-5135. [9] A. G. Beck-Sickinger, H. Dilrr, G. Jung, Pept. Res., 1991, 4, 88-94. [lo] R. A. Houghten, J. R. Appel, C. Pinilla, In Vaccines 88 (Ed.: H. Ginsberg, F. Brown, R. A. Lerner, R. M. Chanock), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, pp. 9-12. [ll] M. Saelberg, U. Ruden, L. 0. Magnius, E. Norrby, B. Wahren, Immunol. Lett, 1991,30, 59-68. [12] W. Van’t Hof, P. C. Driedijk, M. Van den Berg, A. G. Beck-Sickinger, G. Jung, R. C. Aalberse, Mol. Immunol., 1991, 28, 1225- 1232. [13] A. G. Beck-Sickinger, W. Gaida, G. Schnorrenberg, R. Lang, G. Jung, Int. J. Pept. Protein Res., 1990, 36, 522-530. [14] N. Zimmermann, A. G. Beck-Sickinger, G. Folkers, S. Krickl, I. Milllei-, G. Jung, Eur. J. Biochem. 1991, 200, 519-528. I151 R. Frank, R. Dbring, Tetrahedron, 1988, 44, 6031-6040. I161 R. Frank, Bioorg. Med. Chem. Lett., 1993, 3, 425-430. I171 T. Bbldicke, S. Kindt, E Maywald, G. Fitzlaff, M. Bbcher, R. Frank, T. Colin, Eur. J. Biochem., 1988, 175, 259-264. I181 M. Lebl, A. Stierandova, J. Eichler, M. Patek, V. Pokorny, J. Jehnicka, P. Murda, K. Zenisek, J. Kalousek, In: Innovation Perspect. Solid Phase Synth. Collect. Pap. (Ed.: R. Epton) 2nd Int. Symp. Intercept, Andover, 1992, pp. 251-257. [19] R. H. Berg, K. Almdal, W. Batsberg Pedersen, A. Holm, J. P. Tam, B. Merrifield, J. Am. Chem. Soc., 1989, 111, 8024-8026. [20] G. Schnorrenberg, H. Gebhardt, Tetmhedron, 1989, 45, 7759-7764. [21] H. Gausepohl, M. Kraft, C. Boulin, R. W. Frank, In: Innovation Perspect. Solid Phase Synth. Collect. Pan (Ed: R. Epton) Int. Symp. SPCC Birmingham, 1990, pp. 487-490.

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Perspect. Solid Phase Synth. Collect. Pap. (Ed. : R. Epton), 2nd Int. Symp. Intercept, Andover, 1992, pp. 445-448. (23) G. B. Fields, R. L. Noble, Int. J. Pept. Protein Res. 1990, 35, 161-214. [24] G. Schnorrenberg, K. H. Wiesmiiller, A. G. Beck-Sickinger, H. Drechsel, G. Jung, Peptides 1990, (Eds.: E. Giralt, D. Andreu), Proc. Eur. Pept. Symp. Escom, Leiden, 1991, pp. 202-203. [2S] A. G. Beck-Sickinger, H. A. Wieland, H. Wittneben, K. Willim, K. Rudolf, G. Jung, Eur. J. Biochem., 1994, 225, 947-958. (261 A. G. Beck-Sickinger, H. Durr, W. Gaida, G. Jung, Biochem. Soc. Pans., 1993, 20, 847-850. (27) A. G. Beck-Sickinger, E. Hoffmann, K. Paulini, H.4. ReiRig, K.-D. Willim, H. A. Wieland, G. Jung, Biochem. Soc. Trans., 1994, 22, 145-149. [28] A. G. Beck-Sickinger, M. Kiess, P. Kern, G. Folkers, Pharm. Actu Helv., 1995, 70, 33-41. [29] F. 0. Gombert, W. Werz, M. Schliiter, A. Bayer, R. G. Werner, W. Berthold, G. Jung, Biol. Chem. Hoppe-Seyler, 1994, 375, 471 -480. [30] W. Beck, G. Jung, W. G. Bessler, I. Benz, M. Kohlhardt, Biochim. Biophys. Acta, 1994, 1206, 263-271. (311 H. G. Ihlenfeldt, W. Kraas, C. Seidel, U. Wienhus, G. Jung, In: Innovationsand Perspectives in Solid Phase Synthesis, (Ed: R. Epton), Intercept. Ltd., Andover, 1995, pp. 555-558. [32] H. Otto, K.-H. Wiesmiiller, A. G. Beck-Sickinger, S. StevanoviC, G. Jung, In: Innovations and Perspectives in Solid Phase Synthesis, Intercept Ltd., Andover, 1994, 623-626. [33) G. Malcherek, V. Gnau, G. Jung, H.-G. Rammensee, A. Melms, J. Exp. Med. 1995, 181, 527- 536. [34] A. Holm, M. Meldal, In: Peptides 1988 (Eds.: G. Jung, E. Bayer), Proc. 20th. Eur. Pept. Symp. de Gruyter, Berlin, 1989, pp. 208-210. [35] M. Meldal, C. B. Holm, G.Bojesen, M. H. Jakobsen, A. Holm, Int. J. Pept. Protein Res. 1993, 41, 250-260. [36] H. Wolfe, RaMPS - Peptide Synthesis System. Dupont de Nemours Product Information 1987. (371 V. Krchnak, J. Vagner, Pept. Res. 1990, 3, 182-193. [38] J. Eichler, M. Bienert, A. Stierandova, M. Lebl, Pept. Res. 1991, 4, 296-307. [39] H. M. Geysen, R. H. Meloen, S. J. Barteling, Proc. Natl. Acad. Sci. USA 1984, 81, 3998-4002. [40]R. Van der Zee, W. Van Eden, R. H.Meloen, A. Noordzji, T. D. A. Van Embden, Eur. 1 Immunol. 1989, 19, 43-47. [41] A. M. Bray, N. J. Maeji, H. M. Geysen, Tetrahedron Lett. 1990, 31, 5811-5814. [42] A. M. Bray, N. J. Maeji, A. G. Jhingran, R. M. Valerio, Tetrahedron Lett. 1991,32,6132. 143) J. M. Middeldorp, R. H. Meloen, J. Viml. Methods 1988, 21, 147-159.

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Escom Verlag, Leiden. (581 J. Rickert, T. Weiss, W. Kraas, G. Jung, W. Gcipel, Biosens. Bioelectron., 1995, 11, 591-598. (591 M. Knichel, P. Heiduschka, W. Beck, G.Jung, W. Gclpel, Sensors and Actuators 828, 1995, 85-94. (601 P. Heiduschka, W. Gclpel, W. Beck, W. Kraas, S. Kienle, G. Jung, Chem. Eur. J. 1996,

in print.

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Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

4 Chemical Synthesis of Peptide Libraries Arpad Furka

4.1 The Portioning-Mixing Method 4.1.1 Principles and Realization With the intention of speeding up the process of drug discovery, in 1988 we introduced a radically new method for peptide synthesis 111. The portioning-mixing (PM) method makes it possible to synthesize millions of peptides easily in the form of multicomponent mixtures (or as they were called later “peptide libraries”). The procedure is based on Merrifield’s solid phase method [2]. The coupling cycle of this method is replaced by three simple operations: (i) dividing the solid support into equal portions before coupling; (ii) coupling a different amino acid to each portion; (iii) mixing the portions. The number of portions in (i) is determined by the number of amino acids one intents to vary in a certain position. One may choose this number to be the same (e.g. 20) in all positions or let it vary from position to position. In the general case, let the number of amino acids varied in the coupling position i be denoted by k i . The numbers of amino acids varied in the C-terminal and N-terminal positions are k, and k,,,respectively, where n is the residue number in the peptides to be synthesized. Synthesis is as follows. In the first cycle, the solid support is divided into k, equal portions, i.e. into as many portions as the number of amino acids intended to vary at the C-terminus of peptides. If a support-bound, or a free, library is prepared, an aminomethyl or a chloromethyl resin, respectively, is used. Then each portion of resin is coupled with one of the k, kinds of amino acids and the portions are mixed. If a free peptide mixture is prepared starting from protected aminoacyl resins, this first cycle can be replaced simply by mixing the aminoacyl resins in equimolar quantities (one may choose to conclude this and the subsequent cycles by the removal of the N-protecting groups but in this case this operation should be omitted after portioning in the next cycles). In the second cycle, the mixture is divided into k2 equal portions and, after the removal of the N-protecting groups, each of them is coupled with one of the k2 kinds of protected amino acids. Then the portions are mixed again.

112

4 Chemical Synthesis

of Peptide Libmries

...

The 3rd, 4th, and nth cycle is executed like the second one with the exception that the number of portions and that of the coupled amino acids, are k3,k4, and k,,,respectively. At the end of the last (nth) cycle the peptides are removed from the resin, also losing, as usual, their protecting groups. If a support-bound library is prepared, in this last operation only the protecting groups are removed. A schematic representation of the process is demonstrated in Figure 4-1. The three types of circles denote three different amino acids and P represents the polymer support. The divergent, parallel and convergent arrows show portioning, coupling and

P f

...

P

I

-P

P

01

ii

0-P

1 /

0-P

\ /

iii

-P

i

0-P

6-P e-P

0-P -P e-P

OI

0-P -P

e-P

01

ii

GO-P -P W - P P W-P 00- P

.o-

Figure 4-1. The portioning-mixing process. P: polymer support, the white, gray and black circles: different amino acids, divergent arrows: portioning, parallel arrows: coupling, convergent arrows: mixing.

4.1 The Portioning-Mixing Method

113

mixing, respectively. Formation of all possible sequences in this three monomertwo-cycle process is clearly traceable as well as the fast growing number of components. Formation of peptides in 1 to 1 molar ratio - which is very important in screening - is assured in the process described above by dividing the resin into equal portions. It is also important that execution of couplings on spatially separated portions makes it possible to achieve a complete conversion with every amino acid component. Finally, the alternating dividing and mixing operations make the method highly efficient.

4.1.2 Experimental Verification In order to verify experimentally that the expected peptides are really present in the final synthetic mixtures, simple libraries had to be synthesized. It was obvious at the time of our first experiments that identification of peptides in mixtures comprising thousands of components would have been (and still is) a hopeless job. Therefore, in the mixtures described in our earliest publications [l, 31, the maximum number of components was only 180. The main method used in the analysis of peptide mixtures was a computerassisted paper electrophoretic identification procedure developed for this purpose [4]. A simple synthetic mixture was prepared starting from A-resin. In both the second and third coupling position, E, F and K were varied, and the N-terminal position was unvaried and occupied by E in all peptides. The mixture of nine peptides was submitted to two-dimensional (PH 6.5 and pH 2) high voltage paper electrophoresis and then the electrophoretogram was compared to a computer-made predicted map (Fig. 4-2). The six experimental spots exactly matched those predicted. In accordance with prediction, isolation and sequencing of peptide from spots 1, 3 and 6 yielded sequences EEEA, EFFA and EKKA, respectively. For the remaining three spots, double occupancy was predicted: two EFEA and EEFA, four EKEA and EEKA, five EKFA and EFKA. These predictions were also confirmed by further experiments including HPLC separations. In addition to the expected sequences, the program also optionally generated the omission sequences. Their map or the superimposed maps were again compared to the experimental one in order to detect omission sequences. No such byproducts were found, however, in the above mixture. All experiments carried out with simple mixtures supported the expectations, and showed that the PM method worked. The predicted peptides were present in the mixtures without significant contamination by non correct sequences.

114

4 Chemical Synthesis of Peptide Libraries

No of peptides: 9 No of acidic p: 6 No of basic p: 1 Noof neutr p: 2 Noof spots : 6

pH 6.5 nob of GlyOMe: 100 pH 2 nobil of GlyOMe. 100 max mob of basic pep : 33 max mob of acidic p : -98 maxmobalpH2 : 98 DH 2 A I - )

assignementof spots: a circle size : c distance ‘ d turn to HPLC : I back to MENU 3 : b

I

print: P

Figure 4-2. Computer-predicted paper electrophoretic map of a synthetic mixture of nine tetrapeptides, made by ELPLC, a newer version of the original program.

4.1.3 The ELPIC Program Considering the unprecedentedly high number of peptides promised by the PM method, the use of computer assistance seemed to be inevitable. The first version of our computer program 14) could be used to calculate the expected as well as the defective sequences formed by varying up to 20 amino acids in up to 15 positions, to calculate their molecular weight and in addition their electric charge and relative electrophoretic mobility at pH 6.5 and pH 2. The new version of the program, named “ELPLC” [5], is an extension of the former one enabling the user to predict - in addition to the electrophoretic mobilities - HPLC retention times as well as separability by combination of paper electrophoresis and HPLC. The electrophoretic mobilities are calculated from the molecular weights and electric charges according to Offord [6]. The HPLC retention times of peptides are summed from the retention factors of the constituent amino acids. One can choose from seven sets of retention factors [7-111. The program can handle data of up to lo00 peptides (from dipeptides to pentadecapeptides). The input data are: number of residues in peptides, amino acids varied in different positions. electrobhoretic minration distances at DH 2 and DH 6.5 of GlvOMe used as

115

4.1 The Portioning- Mixing Method

reference substance (optional), and the desired set of retention factors. The program is written in Pascal and can be used on IBM-compatible PCs with a color EGA or VGA monitor. Hard copies of plots can be made with a Star LC24-10printer. The output data are: the number and sequences of peptides, the retention times, and the electrophoretic migration distances at both pH 2 and pH 6.5. These data are available in alphanumerical as well as in graphical form, and it is very easy to assign sequences to the graphical objects. Figures 4-3 and 4-4show the predicted twodimensional electrophoretic map and the calculated HPLC elution pattern of 18 tetrapeptides, respectively. The two solid circles on Fig. 4-3 - appearing as a doupH 6.5 nob of GlyOMe : pH 2 nobil of GlyOMe : max mob of basic pep : max mob of acidic p : maxmobatpH2 .

No of peptides: 18 No of acidic p: 6 No of basic p: 9 No of neutr p: 3 Noof spots : 18

100 100 80 -63 127

assignemenlof spots: circle size : distance : turn to HPLC . backtoMENU3 :

a c d I b

0 0 0 0

0

0 0

-

(-) pH 6.5

'rint: P

Figure 4-3. Predicted two-dimensional paper electrophoreticmap of a syntheticmixture of 18 tetrapeptides. Varied amino acids in coupling positions (starting from the C-terminus) are: 1 (R, D. L), 2(W, H), 3(G), and 4(E, K, F). The double solid circle belongs to EGWL and FGWD.

ble spot - belong to peptides EGWL and FGWD. As can be seen, an unsatisfactory electrophoretic separation paper is predicted. The considerable distance between the two broken vertical lines, however, indicating their position on the HPLC plot (Fig. 4-4)promises a better separation by HPLC. The ELPLC2 program is a modified version of ELPLC. The modification allows the input of individual (noncombinatorial) sequences, and separability can be predicted as in the case of mixtures.

116

4

Chemical Synthesis of Peptide Libraries PRINT : P

rlo of peptides: 18 3elentions

BACK TO MOBILITY PLOT : ANY OTHER KEY No of lines: 16 min : -29.20

Re1faktors: meek7 max : 36.30

Figure 4-4. Predicted HPLC plot of 18 tetrapeptides. The two vertical broken lines correspond to the two peptides occupying the double solid circle on the electrophoretic map.

4.1.4 Simple Device for the Manual Synthesis of Peptide Libraries Although the PM procedure is very simple, synthesis of peptide libraries requires parallel treatment of about 20 samples. In order to facilitate handling and mixing of samples in the manual procedure, a simple and cheap device has been constructed, the usefulness of which has already been verified in practice. The device is a vacuum chamber (Fig. 4-5 A) mounted on a shaker (Fig. 4-5 B). It is made up of a 55 cm long aluminum tube, b. The diameter and wall thickness are 8 cm and 0.5 cm, respectively. Both ends of the tube have removeable threaded-end caps, c and rubber packings, e. One of the end caps has an outlet, f which is connected to a water aspirator pump via a filter flask serving as a waste collector. On one side of the vacuum chamber there are 20 holes with rubber rings, d in them to hold the reaction vessels a strongly, and support a vacuum (it is advisable to apply a thin film of vacuum grease). TWO kinds of different-sized reaction vessels can be used as an alternative: screw-cap glass tubes with a glass frit at the bottom, or polypropylene syringe tubes with fitted-in polyethylene frit. The lower outlet part of the reaction vessels fits tightly into the rubber ring to provide a firm attachment. The aluminum tube can be rotated on its axis, so the reaction vessels can be fixed in any position between vertical and horizontal (Fig. 4-5B,1 and 2). A vertical arrangement is used in all operations except shaking, when a tilted position (Fig. 4-5 B, 3) is recommended. The mixer (Fig. 4-5 C) is a polyethylene vessel with a gas inlet tube mounted at the bottom. The eas enters the vessel through a Dolvethvlene frit. It is imDortant to ad-

4.1

L

The Portioning-Mixing Method

117

C

C

A

3

2

3

I I

I

B Figure 4-5. Device for manual synthesis. The vacuum chamber A, the vacuum chamber mounted on shaker B, and the mixing chamber C.

just the density of the solvent mixture used as diluent in mixing to that of the resin in order to avoid sedimentation of the beads during portioning. A 2: 1 mixture of dichloromethane (DCM) and dimethyl formamide (DMF) proved to be applicable in our experiments when polystyrene type resin (density 1.19 g/cm3) was used as solid support. Mixing is brought about by bubbling nitrogen gas through the suspension of resin beads. Portioning is carried out by pipetting the resin suspension into the reaction vessels.

118

4

Chemical Synthesis of Peptide Libraries

4.1.5 Efficiency and Limitations The number of n residue peptides (N,)formed in the PM synthesis depends on the number of amino acids (k) varied in the different (1, 2, ..-,n) positions. In the general case, this can be expressed by the following formula:

The number of coupling cycles (C,)one has to execute in the same synthesis is: C,, = k , + k2 + k3 + ... + k, The two formulae show the source of the high efficiency of the PM synthesis: the number of the synthetic steps can be calculated by summing the numbers of amino acids varied in different positions, while the number of peptides is given by the product of those same numbers. If the number of the varied amino acids is the same in every position (k):

N n =k" and C,,=n. k or N, = 20" and C,, = 20n

if the 20 common t a m i n o acids are varied in all positions (k = 20). The difference between the efficiency of the PM method (121 and that of the conventional one-by-one synthesis can best be characterized by comparing the number of coupling cycles needed in the synthesis of the same number of peptides. Table 4-1

Table 4-1. Number of coupling cycles in the synthesis of complete oligopeptide libraries

Number of coupling cycles PM One by one

Number of Residues

Peptides

2 3 4 5 6 7 8 9 10

4.00 x 8.00 x 1.60 x 3.20 x 6.40 x 1.28 x 2.56 x 5.12 x 1.02 x

~

~

~

~

~

~

~

Id

lo3

10'

lo6

10'

lo9 1O'O

10"

1013

~ ~~~

~

~

40 60 80 100 120 140 160 180 200

_

_

_

8.00 x 2.40 x 6.40 x 1.60 x 3.84 x 8.96 x 2.05 x 4.61 X 1.02 x

Id

104

los 10'

lo8 lo9 10"

loJ2 1014

119

4.1 The Portioning-Mixing Method

shows that the differences, particularly in the case of higher residue number peptides, are enormous. Another figure characterizing the efficiency of the PM method is the number of new substances formed in a single coupling cycle. As Table 4-2 shows, this figure is rapidly increasing from position to position. The synthesis of peptides has always been a laborious time-consuming job. The PM method makes it possible to synthesize millions of peptides in a couple of days and so it practically eliminates the demands of labour as limiting factors in the synthesis. In principle, even the synthesis of 10240 billion decapeptides seems to be an easily realizable task. One also has to consider, however, some practical details, e.g. the weight of reagents, the volume of solvents and reaction mixtures, etc. Let us consider now only the weight of the resin depending on the size of the beads. First, it must to be taken into account that the number of the synthesized peptides can not exceed the number of beads of resin. Moreover, for statistical reasons, the number of beads has to exceed the number of peptides by a factor of at least ten. Table 4-3 shows the weight of the starting resin to be used in the synthesis of

Table 4-2. Number of new peptides formed in a single coupling cycle in the synthesis of complete peptide libraries Number of new peptides

Coupling position

2.00 x 10 4.00 x Id 8.00 x lo3 1.60 x 10s 3.20 x Id Table 4-3. Dependence of the weight of the resin on the size of the beads in the synthesis of complete oligopeptide libraries Number of residues

2 3 4 5 6 7 8 9 10

Size of beads (mesh)

400

136.8 mg 2.7 mg 54.7 mg 1.1 g 21.9 g 437.6 g 8.7 kg 175.1 kg 3.5 t

200

1.1 mg 21.0 mg 420.1 mg 8.4 g 168.2 g 3.4 kg 67.3 kg 1.3 t 26.9 t

100

8.4 mg 168.2 mg 3.4 g 67.3 g 1.3 kg 26.9 kg 538.3 kg 10.8 t 215.3 t

50

60.8 mg 1.2 g 24.3 g 486.3 g 9.7 kg 194.5 kg 3.9 t 77.8 t 1.6 kt

120

4

Chemical Synthesis of Peptide Libraries

the complete libraries of oligopeptides ensuring ten beads for each kind of peptide. In our calculations, diameters of 0.0038, 0.0075, 0.015 and 0.029 cm were used for beads of 400, 200, 100 and 50 mesh, respectively, and the density was taken to be 1.19 g/cm3. Data of Table 4-3 clearly demonstrate that the quantity of starting resin - and, as a consequence, also that of the other materials - is so large in the synthesis of the higher member oligopeptides that no laboratory could be expected to manipulate with them. The synthesis of these libraries is, in practice, limited by the quantity of the reagents. These quantities could be reduced substantially by using finer resins. The use of finer resins seems to be advantageous for another reason: the statistical nature of the method. Both of the characteristic operations of the PM method (i.e. mixing and portioning), are governed by the laws of probability. As a consequence, as the number of peptides approaches the number of beads in a sample taken in the portioning operation, it becomes more and more uncertain whether all the peptides are represented in the sample. So, if a high number of peptides is prepared, it is advisable to use beads as fine as possible in order to keep a few order of magnitude difference between the number of beads and the number of peptides, even in the last portioning operation. The effect of statistics has to be taken into account even in sampling for screening experiments, if support-bound libraries are used. This can be demonstrated with a simple example. Let us suppose that we have a hypothetical pentapeptide library, in which each of the 3.2 million components is represented by five beads, and one-fifth of the whole mixture is removed as a sample. A simple calculation shows that the probability of finding all peptides represented by at least one bead in the sample is only 63%.

4.2 Composition of Peptide Libraries 4.2.1 Libraries and Sublibraries Since so many types of peptide libraries can be synthesized by the PM procedure, it seems worthwhile introducing some new definitions [13]. Thus, the name normal library may be suggested for mixtures formed by varying the same number of amino acids in all positions. If the number of amino acids differs in one or more positions, one can speak about general libraries. A special case of normal libraries, when the 20 common amino acids are varied in all coupling positions, is called standard /i&mry.Figure 4-6 demonstrates a standard pentapeptide library. The sequences of peptides can be deduced by drawing 3.2 million different lines on the Figure, connecting five amino acids, one in each coupling position. The normal libraries, derived from standard libraries by omission of one or several amino acids in all positions, are named omission libraries. Cysteine is often omitted. This can be indicated

4.2

Composition of Pepride Libraries

121

Figure 46. Standard pentapeptide library. The figures and lines denote coupling positions and sequences, respectively.

by a “minus C” termination (standard-C orjust -C library). A special combined library, containing a genetic series of standard libraries will be named a genetic library (e. g. the genetic pentapeptide library includes all standard libraries from dipeptides to pentapeptides). The use of noncommon amino acids in addition to the common ones can be indicated by the name extended library. Any kind of library - except genetic libraries can be divided into sublibraries. Sublibraries (SLs) are special partial libraries of parent libraries which are synthesized by coupling (without portioning) with a single amino acid in one or more position, while in the rest of positions the amino acids are varied as in the parent library. A nonvariedposition is occupied by the same amino acid in all peptides. SLs may be classified according to their order. The first-, second-, third-order etc SLs have one, two, three, etc. nonvaried positions, respectively. Omission sublibraries - like SLs - are also special partial libraries. They can be characterized by their one or more omission positions. In an omission position, all amino acids are varied except one. In the nonomission positions, on the other hand, the amino acids are varied as in the parent library. Depending on the number of omission positions, one can speak about first-, second-, third-order etc omission SLs. In the following discussions, if not stated otherwise, the term sub-library means SL of standard libraries, and mostly pentapeptide libraries and pentapeptide SLs are used as examples. Coupling positions (CPs) are numbered starting from the C-terminus to express the order of couplings during the synthesis. The amino acids are denoted by one-letter symbols.

-

122

4 Chemical Synthesis of Peptide Libraries

4.2.2 First-Order Sublibraries First-order SLs have a single nonvaried position. Figure 4-7 demonstrates a first. order pentapeptide SL. Coupling position No. 2 (CP2) is nonvaried, occupied bq glycine, while in the first, third, fourth and fifth positions all 20 amino acids art varied. A short symbol suggested for this type of SL shows the nonvaried position and the amino acid occupying it: 2E. An additional (optional) figure may be used to indicate the number of residues: 2E5. There are 20 different CP 2 type SLs: 2A, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2K, 2L, 2M, 2N, 2P, 24, 2R, 2S, 2T, 2V, 2W, 2Y A short notation for them might be 2X or 2x5. A first-order pentapeptide SL has 160000 components, and 81 coupling cycles are needed in its synthesis. It may be interesting to note that the number of those components in 2A5, for example, in which A occupies one, two, three, four or five positions is 130321, 27436, 2166, 76 and 1, respectively. The 1 to 1 mixture of the 20 different CP2 type first-order SLs is equivalent to the standard library (3.2 million components). The number of possible pentapeptide SLs, however, is more than 20, since there can be five different nonvaried positions, and 20 SLs can be assigned to each position. The 100 different first-order pentapeptide SLs are listed in Table 4-4. In general, the SLs belonging to a given residue number, and a given order is considered to form a kit. The total number of coupling cycles needed in the synthesis of the kit is 8100.

Figure! 4-7. First-order pentapeptide sublibrary 2GS. The black circle denotes the nonvaried amino acid G in coupling position No. 2. A11 seauence tines have to cross this amino acid.

4.2 Composition of Peptide Libraries

123

Table 4-4. The possible first-order pentapeptide sublibraries ~

1A 2A 3A 4A 5A

1C 2C 3C 4C 5C

IE IF IG IH 2E 2F 2G 2H 3E 3F 3G 3H 4E 4F 4G 4H 5D SE 5F 5G 5H

ID 2D 3D 4D

11 I K 21 2K 31 3K 41 4 K 51 5K

1L 2L 3L 4L 5L

1M 2M 3M 4M 5M

IN 2N 3N 4N 5N

1P 2P 3P 4P 5P

IR 2R 3R 4R 5Q 5R

1Q 24 34 44

1s 1T 1V IW 2 s 2T 2V 2W 3 s 3 1 3V 3W 4 s 4T 4V 4W 5s 5T 5V 5W

1Y 2Y 3Y 4Y 5Y

A remarkable mixture is obtained by mixing in 1 to 1 ratio the 5 SLs of any column of Table 4-4. Let us consider the mixture 1A + 2A + 3A + 4A + 5A (E A). All those - and only those - peptides are present in the mixture in which A occupies one or more positions. The number of components is 723901. This is less than 5 x 1600o0 = 800000, and this indicates that the 1 to 1 molar ratio of components is no longer maintained. The number of peptides in which 1, 2, 3, 4 and 5 positions are occupied by A is 651605, 68590, 3610, 95 and 1, respectively, and their molar ratio is 1 :2: 3 :4: 5, respectively. In total, 20 types of such positional mixtures of first-order sublibraries can be prepared :

Their short notation is X or X5. A peptide mixture comprising all those - and only those - peptides in which a certain amino acid (e.g. A) occupies one or more positions and, in addition, the molar ratio of the components is 1 to 1, can be prepared directly. Although the synthetic pathway seems somewhat complicated (therefore not described in detail here) the number of coupling cycles needed is reasonably low, only 158 for pentapeptides. For reasons described later, the name of such a partial library is an amino acid tester mixture (Fig. 4-8). A short name for a leucine tester may be an L tester or, if the number of residues are also indicated, an L5 tester. An amino acid tester kit comprises all 20 different amino acid tester mixtures: AS, C5, D5,E5, F5, G5, H5,15, K5, L5, M5,N5, P5,Q5,R5, S5, T5, V5, W5,Y5.

Their short notation may be X or X5. A first-order omission SL has one omission position. If E is omitted in position 2, for example, the suggested short symbol is -2E or -E5 Since only one amino acid is omitted in position 2, the number of components is 3.04 million. The number of coupling cycles in the synthesis is 99. There are 100 different first-order pentapeptide omission SLs. The 1 to 1 molar mixture of 2E5 and -2E5 SLs is equivalent to a standard librarv.

Figure 4-8. An amino acid tester pentapeptide mixture (LS). The black circles indicate the amino acid present in all peptides L. All sequence lines cross at least one such circle.

4.2.3 Second-Order Sublibraries The second-order SLs are characterized by two nonvaried positions. An example of such a pentapeptide SL is shown in Figure 4-9. All sequence lines have to cross both black circles, indicating the two amino acids A and L occupying the two nonvaried

5

Figure 4-9. A vicinal2.3 type second-order pentapeptide sublibrary 2G3L. All sequence lines cross both nonvaried amino acids (black circles).

2N31 2P31 2431 2R3I 2S3I 2T3I 2V3I 2W31 2Y31

2A3Y 2C3Y 2D3Y 2E3Y 2F3Y 2G3Y 2H3Y 2I3Y 2K3Y 2L3Y 2M3Y 2N3Y 2P3Y 2Q3Y 2R3Y 2S3Y 2T3Y 2V3Y 2W3Y 2Y3Y

2A3W 2C3W 2D3W 2E3W 2F3W 2G3W 2H3W 2I3W 2K3W 2L3W 2M3W 2N3W 2P3W 2Q3W 2R3W 2S3W 2T3W 2V3W 2W3W 2Y3W

2A3V 2C3V 2D3V 2E3V 2F3V 2G3V 2H3V 2I3V 2K3V 2L3V 2M3V 2N3V 2P3V 2Q3V 2R3V 2S3V 2T3V 2V3V 2W3V 2Y3V

2A3T 2C3T 2D3T 2E3T 2F3T 2G3T 2H3T 2I3T 2K3T 2L3T 2M3T 2N3T 2P3T 2Q3T 2R3T 2S3T 2T3T 2V3T 2W3T 2Y3T

2A3S 2C3S 2D3S 2E3S 2F3S 2G3S 2H3S 213s 2K3S 2L3S 2M3S 2N3S 2P3S 2Q3S 2R3S 2S3S 2T3S 2V3S 2W3S 2Y3S

2A3R 2C3R 2D3R 2E3R 2F3R 2G3R 2H3R 2I3R 2K3R 2L3R 2M3R 2N3R 2P3R 2Q3R 2R3R 2S3R 2T3R 2V3R 2W3R 2Y3R

2A3Q 2C3Q 2D3Q 2E3Q 2F3Q 2G3Q 2H3Q 213Q 2K3Q 2L3Q 2M3Q 2N3Q 2P3Q 2 4 3 4 2R3Q 2S3Q 2T3Q 2V3Q 2W3Q 2Y3Q

2A3P 2C3P 2D3P 2E3P 2F3P 2G3P 2H3P 2I3P 2K3P 2L3P 2M3P 2N3P 2P3P 2Q3P 2R3P 2S3P 2T3P 2V3P 2W3P 2Y3P

2A3N 2C3N 2D3N 2E3N 2F3N 2G3N 2H3N 213N 2K3N 2L3N 2M3N 2N3N 2P3N 2Q3N 2R3N 2S3N 2T3N 2V3N 2W3N 2Y3N

2A3M 2C3M 2D3M 2E3M 2F3M 2G3M 2H3M 213M 2K3M 2L3M 2M3M 2N3M 2P3M 2Q3M 2R3M 2S3M 2T3M 2V3M 2W3M 2Y3M

2A3L 2C3L 2D3L 2E3L 2F3L 2G3L 2H3L 2I3L 2K3L 2L3L 2M3L 2N3L 2P3L 2Q3L 2R3L 2S3L 2T3L 2V3L 2W3L 2Y3L

2A3K 2C3K 2D3K 2E3K 2F3K 2G3K 2H3K 213K 2K3K 2L3K 2M3K 2N3K 2P3K 2Q3K 2R3K 2S3K 2T3K 2V3K 2W3K 2Y3K

2A31 2C31 2D3I 2E3I 2F3I 2G3I 2H31 2131 2K3I 2L3I 2M3I

2A3H 2C3H 2D3H 2E3H 2F3H 2G3H 2H3H 2I3H 2K3H 2L3H 2M3H 2N3H 2P3H 2Q3H 2R3H 2S3H 2T3H 2V3H 2W3H 2Y3H

2A3G 2C3G 2D3G 2E3G 2F3G 2G3G 2H3G 213G 2K3G 2L3G 2M3G 2N3G 2P3G 2Q3G 2R3G 2S3G 2T3G 2V3G 2W3G 2Y3G

2A3F 2C3F 2D3F 2E3F 2F3F 2G3F 2H3F 2I3F 2K3F 2L3F 2M3F 2N3F 2P3F 2Q3F 2R3F 2S3F 2T3F 2V3F 2W3F 2Y3F

2A3E 2C3E 2D3E 2E3E 2F3E 2G3E 2H3E 2I3E 2K3E 2L3E 2M3E 2N3E 2P3E 2Q3E 2R3E 2S3E 2T3E 2V3E 2W3E 2Y3E

2A3D 2C3D 2D3D 2E3D 2F3D 2G3D 2H3D 213D 2K3D 2L3D 2M3D 2N3D 2P3D 2Q3D 2R3D 2S3D 2T3D 2V3D 2W3D 2Y3D

2A3C 2C3C 2D3C 2E3C 2F3C 2G3C 2H3C 213C 2K3C 2L3C 2M3C 2N3C 2P3C 2Q3C 2R3C 2S3C 2T3C 2V3C 2W3C 2Y3C

2A3A 2C3A 2D3A 2E3A 2F3A 2G3A 2H3A 213A 2K3A 2L3A 2M3A 2N3A 2P3A 2Q3A 2R3A 2S3A 2T3A 2V3A 2W3A 2Y3A

Table 4-5. The 400 different 2,3 type pentapeptide sublibraries

9

2

8 $ 5a

3

8

‘h

k

126

4

Chemical Synthesis of Peptide Libraries

positions 2 and 3, respectively. The symbol of this SL should be either 2A3L or 2A3L5. The number of such CP 2,3 type SLs is 400 (Table 4-5). Each SL has 800U components, and 62 coupling cycles are needed in the synthesis. To synthesize the 400 2,3 type SLs, 24800 coupling cycles have to be carried out. Shorter forms of description may be: 2A3X, 2C3X, 2D3X, 2E3X, 2F3X, 2G3X, 2H3X, 2I3X, 2K3X, 2L3X, 2M3X, 2N3X, 2P3X, 2Q3X, 2R3X, 2S3X, 2T3X, 2V3X, 2W3X, 2Y3X

where, e.g., 2A3X replaces the 20 SLs occupying the first column in Table 4-5, or: 2X3A, 2X3C, 2X3D, 2X3E, 2X3F, 2X3G, 2X3H, 2X31,2X3K, 2X3L, 2X3M, 2X3N, 2X3P, 2X3Q, 2X3R, 2X3S, 2X3T, 2X3V 2X3W, 2X3Y

where each element represents 20 SLs constituting a row in Table 4-5. The shortest form 2X3X may refer to the whole set of the 2,3 type SLs. The 1 to 1 mixture of the 400 2,3 type second-order SLs forms a standard library, and the mixture of 20 of them occupying the same row or the same column in Table 4-4 is equivalent to a first-order SL. The mixtures of 2A3X and 2X3A are equivalent to 2A and 3A, respectively. There are, however, several kinds of second-order SLs differing in their pairs of nonvaried positions. Figure 4-10, for example, demonstrates a 1,4 type second-order SL. The number of types of second-order pentapeptide SLs is ten (Table 4-6), so the total number of all possible second-order pentapeptide SLs is 4OO0, and 248000 coupling cycles are needed in their synthesis. One may call the types listed in the first row of Table 6 the vicinal type, and all the others disjunct type second-order SLs.

Figure 4-10. A disiunct 1.4 tvrx second-order DentaDeDtide sublibrary 114PS.

4.2

Composition of Peptide Libraries

127

Table 4-6. The types of second-order pentapeptide sublibraries

4.2.4 Higher Order Sublibraries The maximum order of SLs is limited by the number of residues in the peptides. The maximum order of a pentapeptide SL is fifth order. All positions in such a SL art nonvaried and, as a consequence, the SL has a single component: a pentapeptide. One can also speak about zero-order SL,which has no nonvaried coupling positions, and is identical with the parent library. The third-, fourth-, fifth-, sixth-order etc. SL: have three, four, five, six etc. nonvaried positions, respectively (i.e. the order of SLz is identical with the number of their nonvaried positions). The number of types of SLs (7) depends on both the order (r) and the numbei of residues (n),and can be expressed by a simple formula:

The calculated number of types is summarized in Table 4-7. The number of possible SLs S within a type, however, depends only on order, and is independent of both the type of SL and the number of residues.

s = 20' The numbers of zero-, first-, second-, third- and fourth-order standard SLs are 1, 20, 400, 8000 and 160000, respectively. Table 4-7. Dependence of the number of types of sublibraries on order (r) and the numbei of residues (n)

n\r

0

1

2

1 1 1 1

2 3 4 5 6 7 8

3 4 5 6 7 8 ~~

1 1 1 ~

~

~

~

2

~

3

1 3 6

1 4

10 15

10 20

21 28

35 56

4

5

6

1 6 21 56

7 28

7

8

1 8

1

1

5 15 35 70

1

128

4

Chemical Synthesis of Peptide Libraries

The number of components N within SLs, however, is again dependent on both the order and on the number of residues.

Examples are shown in Table 4-8. Table 4-8. Dependence of the number of components of sublibraries on order ( r ) and the number of residues (n) ~~

n\r

0

1

2

3

4

5

6

2 3 4 5 6 7 8

202 203 204 205 206 207 208

20 2d 203 204 205 206 207

1 20 202 203 204 205 206

1 20 2d 203 204 205

1 20 202 203 204

1 20 202 203

I 20 2d

7

8

1 20

I

4.3 Potential Use of Partial Libraries in Screening: A Theoretical Approach The partial libraries mentioned so far can be classified into four main groups: (i) (ii) (iii) (iv)

different order SLs; positional mixtures of first-order SLs; amino acid tester mixtures; and omission libraries and SLs.

The following discussions focus on the potential applicability of screening partial libraries belonging to groups (i) and (iii). Applications are not associated with any special screening method. It is considered advantageous, however, if the method provides a quantitative estimation of activity. It is assumed that, in general, not one but several active components are found in the library. These active components form a special partial library, the active library, which is the most important of all partial libraries, and at the same time its identification is the aim of screening. Testing with an amino acid tester mixture Since a certain amino acid (e.g. alanine) is present in every component of the amino acid tester mixture, a positive response in a screening experiment indicates that alanine also occurs in at least one of the active comDonents. On the other hand. a

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negative response is expected to show that alanine is not present in the active components. Amino acid tester mixtures in themselves are unsuitable for determination of the sequences of the active components. There is, however, a strange and unlikely exception: the active library comprises only a single component in which all positions are occupied by the same amino acid. Testing with a first-order sublibrary

As mentioned, it is a characteristic feature of first-order SLs that the only nonvaried position is occupied by the same amino acid in all peptides. For this reason the firstorder SLs can be used to detect the occurrence or absence of a particular amino acid in the nonvaried position of the active components. If, for example, a screening experiment is carried out with the 2L5 SL and the response is negative, it means that leucine does not occur in CP2 of the active pentapeptides. A positive response, on the other hand, indicates that leucine is present in the CP2 of at least some of the active components. Screening experiments carried out only with first-order SLs may - in two special cases demonstrated on Figure 4-11 - lead to the determination of the active sequences: if there is only one active peptide in the active library a, or if there are more active peptides but their sequences are the same except one position (b, c and d mean two, three and five such active peptides, respectively).

a

b

C

d

Figure 4-11. Nonoverlapping a and partially overlapping sequences differing in a single position b, c and d.

Testing witb a second-order sublibrary

Since two (identical or different) amino acids occupy the two nonvaried positions in all components of a second-order SL, these partial libraries can be used to detect, by screening experiments, the presence or absence of amino acid pairs occupying definite Positions within the active peptides. A positive response obtained with

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2A4L5, for example, indicates that there is at least one active peptide in which alanine and leucine occupy positions 2 and 4, respectively. A negative response, on the other hand, shows that there is no active peptide with such occupancy. The second-order SLs can be considered as tools which can be used like overlapping dominoes to determine the sequences of all components of the active library. The positive responses obtained, for example, with the following four vicinal secondorder pentapeptide SLs 5L4T, 4T3A, 3A2D, 2DlG define the sequence of an active pentapeptide as: LTADG if no partial overlapping occurs with sequences of other peptides (see also Fig. 4-16a). It could also be proved easily that any pair of active peptides having partially overlapping sequences are potentially distinguishable by testing with properly chosen second-order SLs. Despite the undeniably high screening potential embodied in second-order SLs, it is not suggested that the screening process should be started with them. Instead, a much more economical stepwise process is proposed, known as “the domino strategy”.

4.3.1 The Domino Strategy The proposed screening strategy was designed to allow the most economical determination of active sequences in three stages, gradually defining simpler and simpler partial libraries which, however, include all active sequences. It was also an important requirement that the design should eliminate the continuous need for synthesis during the screening process and open a way to the commercial availability of the kits used, at least in the first and second stage of screening, making it possible for biologists - lacking the skills of peptide chemists - the apply library technology. 4.3.1.1 Determination of the Amino Acid Occurrence Library (Stage 1)

By carrying out screening tests with all 20 components of the amino acid tester kit (see the black circles on Fig. 4-12), one can determine which amino acids occur as building blocks in the active peptides (marked with “ + ” on the Figure). Since absolutely no information is gained about their positions, it is the best to suppose their potential presence in every position (gray circles on the Figure). In this way a partial library, named an amino acid occurrence library, can be deduced. If, for example, the amino acids found are A, F, G, H, K, P, Y, the amino acid occurrence library is a normal library in which these seven amino acids are varied in all positions. If the number of found amino acids is less than 20 - as in this hypothetical case -

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A\IIHO ACID

-

OCCURRENCE LIBRARY

I

2

;

4

5

Figure 4-12. Determination of the amino acid occurrence library. Black circles: components

of the amino acid tester kit (+ sign mean positive response), gray circles: amino acids and positions defining the amino acid occurrence library.

the number of components in the amino acid occurrence library is less than that in the parent library. The number of components in the amino acid occurrence library of Fig. 4-12 is only 7’ = 16807 (instead of 3.2 million). 4.3.1.2 Determination of Positional Occurrence Library (Stage 2)

In this second stage of screening, one has to determine the position of amino acids occurring in the active peptides. It is possible to do this by avoiding Stage 1, and testing directly with the components of the first-order SL kit. In this case, however, all the components (100 in Table 4-4) have to be tested. It is more economical to take advantage of the information gained in the first stage, and so reduce the number of screening tests necessary. This, again, can be accomplished in two different ways. Synthesis and use of the first-order sublibrary kit belonging to the amino acid occurrence library

Once the amino acid occurrence library is determined, one can choose to synthesize the first-order SL kit belonging to this partial library. In the example shown in Fig. 4-12 the kit has only 35 components (instead of 100 in Table 4-4). Each element of this kit comprises 74 = 2401 peptides. The synthesis of an element needs 29 coupling cycles, and only 1015 coupling cycles are required to prepare the whole kit. Use of this kit requires fewer screening tests (only 35 in our example) as compared to the approach in which Stage 1 is omitted. Let us suppose that a positive respons

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is obtained with SLs marked with gray in Figure 4-13. These SLs show which aminc acids occur in different positions of the active peptides. At the same time they define a library named positional occurrence library. This library is a partial library of both the parent standard library and the amino acid occurrence library. It comprises all the active peptides and presumably the more or less inactive ones. The positional oc. currence library, exemplified in Figure 4-13, has eight components.

FIRST

ORDER

SUB-LIBRARY

C O M P O N E N T S TO BE T E S T E D

KIT

POSITIONAL OCCURRENCE LIBRARY

Figure 4-13. Determination of the positional occurrence library. The first-order SLs marked with gray are supposed to give positive responses in screening experiments.

The use of elements of a presynthesized first-order sublibrary kit

It may prove to be advantageous to synthesize, or even more convenient to buy, if commercially available, the first-order SL kit belonging to the standard library (Table 4-4), and to use its required elements (carrying the same symbols as described above) in the Stage 2 experiments. This approach may well be justified by the fact that, like an amino acid tester kit, the same first-order SL kit can be used in searching for different groups of bioactive peptides showing widely different biological effects. 4.3.1.3 Determination of Active Sequences (Stage 3)

The task to be solved in this stage is the determination of active sequences. This can be done by using second-order SLs like dominoes. Considering their very large number in a standard library (4OOO in the case of pentapeptides), one may probably find it more economical to synthesize the kit belonging to the positional occurrence library. The second-order SLs of the positional occurrence library shown on Figure 4-13 are the followina:

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133

Vicinals: 1A2G, 1P2G, 2G3H, 3H4K, 3H4P, 4K5F, 4K5Y, 4P5F and 4P5Y. Disjuncts: lA3H, 1A4K, 1A4P, IASF, IASY, 1P3H, 1P4K, 1P4P, lPSF, IPSY, 2G4K, 2G4P, 2GSF, 2G5Y, 3HSF, 3H5Y.

However, only those, which are printed in bold, are needed. For example, IA2G and 1P2G are not needed because one can be sure that both 1A and 1P are followed by G in CP 2. One-way and two-way divergent sequences

Looking at the positional occurrence library of Figure 4-13, one can see that the sequences are partially identical (overlapping in region CP 2 to CP 3). In general, the sequences of the positional occurrence library can be classified into three groups: (i) nonoverlapping sequences (Fig. 4-14 a and b); (ii) partially overlapping one-way divergent sequences (Fig. 4-14 c, d, and e). They have only one-way divergency points. This means that, if one goes through the sequences in one direction one finds only divergency points and, in the opposite direction, only convergency points; (iii) partially overlapping two-way divergent sequences (Fig. 4-15 a, b and c). In these sequences, divergency (convergency) points are found in both directions. The use of vicinal second-order sublibmries

It is advisable to start the screening experiments with the vicinal SLs. The dominolike application of the second-order SLs has already been demonstrated. No problem of any kind is expected in sequence determination if the active peptides do not have

a

b

C

d

e

Figure 4-14. Nonoverlapping a, b and one-way divergent partially overlapping sequences c, d, e. Black circles mark the divergency points.

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4 Chemical Synthesis of Peptide Libraries

1

2

3 4 5 a

C

b

Figure 4-15. Partially overlapping two-way divergent sequences. Black circles mark the divergency points.

(a)

Ib)

(c)

Figare 4-16. Domino-like use of secondader sublibraries in determination of adve sequences. Nonoverlapping a, one-way divergent b and two-way divergent sequences c.

identical sequence portions (see Fig. 4-16a). The one-way divergent partially overlapping sequences can also be determined easily using vicinal second-order SLs (Fig. 4-16b). One can conclude therefore that, if the positional occurrence library contains only nonoverlapping and/or one-way divergent active sequences, all these sequences can be determined by using only vicinal second-order SLs.

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135

The use of disjunct second-order sublibraries Disjunct type second-order SLs are needed in Stage 3 experiments only if the use of the vicinal ones reveals the presence of two-way divergent active sequences in the positional occurrence library. Even in this case all sequence lines can be traced by vicinal SLs. One uncertainty remains, however: how the sequences on the two sides of the convergency point (or section) are connected to each other. This problem can be solved completely by disjunct SLs. Their function is very simple: they bridge the two sequence parts already determined via the divergency point (Fig. 4-16c). Positive responses given by 2A4Q and 2E4V indicate that two sequences of the four possible ones are active (MQNAG and PVNEY). MQNEY and PVNAG are inactive sequences. No higher-order SLs are needed in sequence determinations.

The active library The identified active peptides together form the active library, which deserves a short mention. In contrast to all libraries mentioned so far, the active library, in most cases, is not a “combinatorial” library. Therefore, such a library could not be synthesized by the PM method. 4.3.1.4 Generality of the Domino Strategy

The SL structure of different nonpeptide oligomer libraries synthesized by the PM method, like that of the very promising peptoid libraries [14], is determined exclusively by the method of synthesis. In this respect they do not differ from peptides. For this reason the domino strategy, which is based solely on component partial libraries, is expected to be the best method for determining bioactive sequences in any of these “strange” libraries, since it is entirely independent of the current state of the art in sequence determinations of different categories of oligomers.

4.4 Experimental Realization of the Portioning-Mixing Procedure Practical application of the PM method is exemplified by the synthesis of a resinbound first-order (- C) pentapeptide sublibrary (1G5) by using manual synthesis carried out in the device described in Section 1.4. In order to prepare a resin carrying Gaba spacer, 5.0 g of aminomethyl resin (200-400mesh, 0.5 meq N/g) was coupled [15] with Boc-Gaba resulting in BocGaba-aminomethyl resin. Since the amino acids were not varied in the first coupling position, the whole amount of resin was coupled [15] with Boc-Gly, the resin was transferred into the mixinn chamber and. bv addition of a mixture of DCM and

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DMF (2: 1, v/v), the volume was made up to 100 ml. The suspension was thoroughly mixed by bubbling nitrogen through it. The suspension was then submitted to four consecutive coupling cycles each comprising the following operations. Portioning

Equal volumes (5 ml) of the slurry were pipetted into each of the 19 reaction vessels (volume 12 ml). The volume of the remaining 3-4 ml of suspension was made up again to 100 ml with the same solvent mixture, mixed, and 5 ml of this thin slurry was added to each of the 19 reaction vessels. The solvent was then removed by suction. Coupling

Each sample was submitted to deprotection by shaking twice with 5 ml DCM/trifluoroacetic acid (2 :1, v/v) then washed (MeOH, DCM, DCM, 5 ml each). In order to keep the reaction vessels in their places, the vacuum was maintained during all shaking operations. After deprotonation by shaking twice with 5 ml DCM/diisopropylethyl amine (9: 1, v/v) and washing (3 x 5 ml DCM), each sample was submitted to coupling with the active ester of one of the 19 common Boc-amino acids (Cys was omitted). Side chain protective groups were OBzl for Asp and Glu, Bzl for Ser, Thr and Q r , 2 for Lys, and Tos for Arg and His. The active esters (used in four times molar excess) were prepared in separate vessels from 0.8 mmol Boc derivatives dissolved in 5 ml DCM/DMF (3 : 1, v/v) - except in the case of Boc-Gln, Boc-Asn, Boc-Arg(Tos) and Boc-His(Tos), when the solvent was 5 ml DMF - by adding with stirring, N-hydroxybenzotriazole (HOBt) (135 mg; ca. 1 mmol) then diisopropyl carbodiimide (DIC) (0.124 ml; 0,s mmol). The freshly prepared solutions of active esters were transferred into the reaction vessels which were then shaken for 3 h at room temperature. After removal of excess reagents, the samples were washed with 3 x 5 ml of DMF, 3 x 5 ml of DCM and 5 ml of DCM/DMF (2: 1, v/v). Mixing

All samples were washed into the mixing chamber with portions of DCM/DMF (2: 1, v/v), then the volume was made up to 100 ml and mixed by bubbling in nitrogen for 15 min. After the mixing operation of the last coupling cycle, the solvent was filtered off by suction, then the side chain protecting groups of the formed resin-bound pentapeptides were removed by trifluoromethane-sulfonic acid [16]. The beads were then washed with 3 x 20 ml ether and 3 x 20 ml MeOH, and dried over phosphorus pentoxide.

References

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Acknowledgments The author thanks the Hungarian Scientific Research Foundation (OTKA T4192)for financial support and Dr J. A Durgo for constructing the manual synthesizer.

References 111 A. Furka, F. Sebestyen, M. Asgedom, G. Dibo, Abstr. 14th Int. Congr. Biochem. Prague, Czechoslovakia, 1988, Vol. 5, p. 47; Abstr. 10th Int. Symp. Med. Chem. Budapest, Hungary, 1988, p. 288. [2]R. B. Merrifield, 1 Am. Chem. Soc. 1963, 85, 2149-2154. [3] A. Furka, F. Sebestyen, M. Asgedom, G. Dibo, Int. J. Pept. Protein Res. 1991, 37, 487-493. [4]A. Furka, F. Sebestykn, J. Gulyas, “Computer made electrophoretic peptide maps” in Proc. 2nd Int. Conj Biochem. Separations, Keszthely, Hungary (Eds: J. Pick, J. Vajda), 1988, p. 35-42. [5]A. Furka, F. Sebestyen, G. Dibo “Computer program facilitating separation of peptides from mixtures prepared by the portioning-mixing method” in Peptides 1992(Eds.: C. H. Schneider, A. N. Eberle), ESCOM, Leiden, 1993, p. 463-464. [6]R. E. Offord, Nature (London), 1966, 211, 591-593. [7]J. L. Meek, Proc. Natl. Acad. Sci. USA, 1980, 77, 1632-1636. [8]C. A. Browne, H. P. J. Bennett, S. Solomon, Anal. Biochem., 1982, 124, 201-208. [9]K. Jinno, E. Tanigawa, Chromatographia, 1988,25, 613-617. [lo]D. Guo, C. T. Mant, A. K. Taneia, J. M. R. Parker, R. S. Hodges, J. Chromatogr. 1986, 359, 499-517. [ll]T. Sasagawa, T.Okuyama, D. C. Teller, J. Chromatogr. 1982, 240, 329-340. (121 F. Sebestyen, G. Dibh A. Kovacs, A Furka, Bioorg. Med. Chem. Lett. 1993.3, 413-418. [13] F. Sebestyen, G. Dibh A. Furka “Efficiency and limitations of the portioning-mixing peptide synthesis” in Peptides I992 (Eds.: C. H. Schneider, A. N. Eberle), ESCOM, Leiden, 1993, p. 63-64. [14]R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banwille, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D.Frankel, D. V. Santi, E E. Cohen, P. A. Bartlett, Proc. Natl. Acad. Sci. USA 1993,89,9367-9371; R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, W. H. Moos, J. Am. Chem. Soc. 1992, 114, 10646-10647. [IS] B. Gutte, R. B. Merrifield, J. Biol. Chem. 1974, 246, 1922-1941. [16]H. Yajima, N. Fujii, H. Ogawa and H . Kawatani, 1 Chem. Soc., Chem. Commun. 1974, 107-108.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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

5.1 Introduction Peptides are being recognized increasingly as broadly useful tools in all areas of biomedical research, and as effective immunodiagnostic and therapeutic agents. The process involved in the development of new diagnostically or therapeutically useful peptides, as with all pharmaceutical drug development, requires the synthesis and screening of hundreds to thousands of analogs of an original active sequence. Developments in recent years have made it possible for a modestly equipped laboratory to prepare, using a variety of approaches [l-41, thousands of peptides per year at a fraction of the cost per peptide of earlier methods [5]. Automated synthesizers, which can synthesize 24-96 peptides simultaneously, have also been described [6-81. In spite of these advances, the synthesis of the immense number of possible analogs of even short peptides (i. e. , there are 64 x lo6 L-amino acid substitution analogs of a hexapeptide) remains impractical. Over the past century, one of the primary goals of biochemical research has been to identify individual compounds from the immense diversity present in biological sources. While not even a scientific “dream” a decade ago, the recent explosion in new synthetic techniques, in particular the systematic preparation of extremely large numbers of compounds from which individual compounds can be readily identified, has made this goal a practical reality. The introduction of combinatorial libraries made up of tens to hundreds of millions of individual peptide sequences prepared by chemical approaches is at the forefront of a revolution in drug discovery and basic research. Such libraries offer a fundamental, practical advance in the study of interactions between peptides and their biochemical or pharmacological targets. 5.1.1 Solid-Phase Peptide Synthesis Merrifield’s approach to the solid phase synthesis of peptides, published in 1963 (51, can be considered the conceptual father of all of the recently presented synthetic chemical molecular diversity approaches [9- 111. Merrifield’s approach broke away

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5 The Versatility of Nonsupport-Bound Combinatorial Libraries

from traditional solution phase methods, which required that each intermediate step in a multistep synthetic process must be purified to homogeneity prior to its being used in the next step of the synthetic process. This conceptual breakthrough entailed the use of an insoluble polymeric resin support as a synthetic platform. This allowed large excesses of reagent and multiple washes to be used to remove excess reagent for the rapid synthesis of peptides. Secondarily, this process requires purification only of the resulting crude synthetic product. When coupled with the power of preparative RP-HPLC, which enables the separation and therefore purification of a desired compound from a complex mixture, solid phase synthesis enables individual peptides to be prepared much more rapidly and less expensively than the classical solution phase approaches. Furthermore, since indvidual reactions could be readily driven to 99% completion, not only were larger numbers of peptides available for research purposes, but many more became available that would have been obtainable only with great difficulty from classic solution phase approaches. Over the ensuing 20 years, developments in peptide chemistry, resin supports, amino acid protecting groups and coupling reagents steadily advanced the solid phase synthesis technology of peptides. Two approaches appeared in 1984 and 1985 that again greatly increased the number of individual peptides one could prepare in a given period of time: Geysen’s “pin” approach [l] and Houghten’s “tea-bag” approach (21. Both approaches capitalize on the fact that, in the solid phase synthesis of peptides, all of the washes, neutralizations, and deprotection steps are identical and therefore not sequence dependent, with only the addition of the individual protected amino acids to the growing immobilized peptide chain being specific to the given peptide’s sequence. The two approaches differ mainly by the nature of the solid support: multiple plastic pins versus multiple compartmentalized resin beads. Both of these approaches have been used to synthesize milligram to multigram quantities of literally tens of thousands of peptides per year.

5.1.2 Peptide Libraries The peptide libraries presented to date fall into three broad categories, the difference being the manner in which the sequences are synthesized and/or screened. The first category represents synthetic approaches, in which peptide mixtures are synthesized and cleaved from their solid support and assayed as free peptides. This category includes synthetic combinatorial libraries (SCLs, which are reviewed in this paper), as well as other approaches [12- 151. The second category includes synthetic approaches, in which peptide mixtures are synthesized and assayed while attached to either plastic pins [lo], resin beads [ll], or cotton [16]. The third category encomDasses the molecular biolow aDDroach. in which DeDtides or Droteins are Dresented

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141

on the surface of filamentous phasge particles or plasmids, as recently reviewed [17, 18).

In 1986, Geysen presented immobilized libraries made up of mixtures of millions of peptides on plastic pins, which were used to identify “mimotopes” or mimics of the epitopes recognized by antibodies [lo]. While conceptually sound, Geysen’s original approach has found limited application outside the field of immunochemistry, owing to the fact that many receptors of importance are immobilized and therefore cannot interact with extremely large pin-bound diversities of peptides, or any other immobilized ligand. Other support-bound peptide libraries that have since been reported are made up of very large numbers (i. e., millions) of individual peptides, each attached to a separate resin bead [ll], a cotton support [16, 191, the silica surface of a microchip (tens of thousands) (201, cellulose membranes (211. The effect of solid supports on the expected interactions between immobilized ligands and soluble receptors remains unclear. For instance, it has been shown that the interaction of a monoclonal antibody with cotton-bound libraries was much less effective than when the same libraries were cleaved from the cotton and used in solution [16]. In 1991, this laboratory presented soluble peptide libraries which, by their nature, could be used in virtually any assay system [9]. This is an important advantage over other procedures since a large number of molecular interactions involve membranebound (i. e., immobilized or insoluble) receptors located in specific tissues. Thus, one can consider receptors to be immobilized and constrained in a “solid phase”, and to be surrounded by a myriad of compounds available for interaction in the “mobile phase” of biological fluids. In order to illustrate the versatility and advantages of soluble synthetic combinatorial libraries (SCLs), the current paper reviews the published work by this laboratory on the preparation of SCLs made up of millions of peptides or nonpeptidic compounds, as well as their use in a variety of applications. The soluble peptide SCLs described, to date, range from three to ten amino acids in length, and the number of peptides per library varies from thousands to trillions. Libraries have been prepared in a variety of formats, and are composed either entirely of L-amino acid sequences, entirely of D-amino acid sequences, or of a mixture of L-, D- and unnatural amino acid sequences, as well as positional scanning hexa- and decapeptide libraries, which in many instances enable the overnight identification of individual compounds from tens of millions of sequences. Thus, using either an iterative selection and enhancement process or the positional scanning format, such soluble libraries have been successfully used for the identification of: novel antibacterials; new peptide agonists and antagonists to opioid receptors; new trypsin inhibitors; compounds that inhibit melittin’s hemolytic activity; and antigenic peptides recognized by monoclonal antibodies.

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5.2 Preparation of Synthetic Peptide Combinatorial Libraries The peptide mixtures making up the SCLs, as well as the peptide mixtures synthesized for the iterative steps, were prepared using methylbenzhydrylamine (MBHA) polystyrene resin and standard t-Boc chemistry in combination with simultaneous multiple peptide synthesis (SMPS) [2]. Peptide mixture resins were prepared either by a process termed divide, couple and recombine (DCR:[9, 11, 22]), or by using a mixture of amino acids that are incorporated simultaneously during the coupling procedure [23-251. The side chain protecting groups of the peptide mixtures were removed and the peptides cleaved from their respective resins using the low-high hydrogen fluoride method [26, 271. Peptide mixtures were extracted with water or dilute acetic acid and the solutions lyophilized. 5.2.1 DCR Method The DCR process is limited by its very nature. The physical process of dividing the resin pools, mixing them, and dividing them again leads to the generation of one peptide per resin bead. Since the distribution of peptides within mixtures is governed by the position distribution [28], inclusion of each peptide within a library of approximately 100OOO compounds is expected only if a large number of resin beads per peptide (>10) is used. An even larger number of resins beads (> 100) is needed if equimolarity (less than two-fold differences in concentration between the most and least prevalent peptide) is to be expected. For the practical generation of approximately equimolar mixtures, the DCR method is limited in the diversity of compounds possible. For example, even with a hexamer library using 20 defined amino acids at two positions and mixtures of 20 amino acids at the other four positions (400 pools of 160000 compounds), >0.3 g resin (200-400 mesh, = 5000000 beads/g) per pool (120 g for the library) is necessary to expect inclusion of each peptide in the library, while > 3 g resin per pool (1.2 kg for the library) is necessary to achieve approximate equimolarity within the mixture. The production of approximately equimolar libraries of diversities greater than lo6 using the DCR method is impractical. Briefly, 19 porous polypropylene packets, each containing 18.6 mmoles (20 g) of MBHA resin, were coupled to each of the protected N-a-t-Boc natural L-amino acids; cysteine was excluded from the mixture positions, since its presence would either require the continued presence of a reducing agent in the assay being used, or in the absence of a reducing agent one would expect the presence of difficult to define disulfide aggregates in the library. AI1 coupling reactions proceeded to completion (>99.5%) as assessed by Kaiser’s ninhydrin test [29] or Gisin’s picric acid procedure [30]. The resulting resins (typically 100-500 mg) from each packet were then combined and thoroughly mixed (there are approximately 4000 beads per mg

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143

of resin). This resin mixture was divided into 19 portions of equal weight, which were placed back into porous polypropylene packets, followed by N-a-t-Boc protecting group removal and neutralization. Each resin packet was then reacted once again with solutions of individual activated amino acids, and the resins mixed thoroughly to yield 361 (192) dipeptide combinations. The above two cycles were repeated twice more, yielding a final mixture (XXXX-resin) of 130321 protected tetrapeptide resins (194).

5.2.2 Coupling of Amino Acid Mixtures Mixture positions were incorporated by coupling mixtures of side chain-protected Boc-amino acids (L- or D-isomers) in a predetermined molar ratio, which compensates for the different coupling rates of the various amino acid derivatives. Amino acid analysis of the peptide mixtures showed approximately equimolar representation of each amino acid [23, 251. The advantages of this method over the DCR method are the ease of synthesis, as well as the ability to prepare complete libraries with more than five mixture positions. We have prepared decapeptide SCLs 1311 having nine mixture positions, which was successfully used for the identification of antigenic decapeptides. Furthermore, the defined positions in a library can be readily located in positions other than the N-terminal.

5.3 Dual Positional SCLs The two initial soluble SCLs [9,32] consisted of six-residue peptide sequences having either acetylated (Ac) or nonacetylated N-termini and amidated C-termini. The first two amino acids in each peptide chain were individually and specifically defined, while the last four amino acids consisted of approximately equimolar mixtures of 19 of the 20 natural L-amino acids. Cysteine was omitted from the mixture positions of the two SCLs, but included in the defined positions. These libraries can be generally represented by the formulas Ac-OIO2XXXX-NH2 and 0102XXXX-NH2 with O1and O2equal to AA, AC, AD, etc. through YV, YW, YY, for a total of 400 (2d) combinations, and each X position representing an equimolar mixture of the 19 amino acids. Four mixture positions result in a total of 130321 (194) combinations. Each of the 400 different peptide mixtures which make up each of these libraries thus consists of 130321 individual hexamers, which in total represent 52321400 peptides. Therefore, if one assumes that the average molecular weight of Ac-0,02XXXX-NH2 is 785, then a mixture of 130321 peptides at a total final concentration of 1.0 mg/ml yields a concentration of every peptide within each mixture of 7.67 nglml(9.8 m).Once the library of mixtures is screened in an assay, the

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remaining mixture positions are defined through an iterative selection process in order to identify active sequences.

5.3.1 Use of SCLs For the initial screening of the peptide library, typically each peptide mixture is assayed for activity at a concentration of 2.5 mg/ml for inhibition of antibody, receptor, or enzyme binding to its ligand, or inhibition of bacterial growth. The peptide mixtures found to exhibit significant inhibition are tested using serial dilutions to determine their IC,, values (i.e., in Ag- Ab studies, the concentration necessary to inhibit 50% of mAb binding to the antigen on the plate), which permits the selection of the most effective peptide mixture from the initial screening. An iterative process is then carried out in which the subsequent X positions of the peptide mixtures are individually defined with each of the 20 natural L-amino acids. This process involves ranking, selecting and reducing the number of peptide sequences while synthetically defining one more position at each step. It is sometimes necessary to move forward with several peptide mixtures that have similar activities upon screening the peptide library. Often, the difference between these peptide mixtures is due to a conservative substitution at the same position (i.e., a valine residue substituted for an isoleucine residue). If there are more mixtures with relatively good activity, then those peptide mixtures having amino acid side chains with significant chemical differences are pursued. For instance, both DVXXXX and DIXXXX would not be moved forward, but if DVXXXX and AKXXXX were both found to be effective, they would both be followed, since they are quite different in chemical character. Those cases not pursued initially from the screening or during the iterative process, however, can always be moved forward at a later date. 5.3.1.1 Identification of Antigenic Determinants

We have used competitive ELISA, in which an antigenic peptide or protein recognized by the target monoclonal antibody, is adsorbed to microtiter plates. Each of the 400 peptide mixtures of this SCL (Ac-O,O,XXXX-NH,) is screened for inhibition of antibody binding to the antigen on the microtiter plate. A number of monoclonal antibodies (mAb) have been screened in this manner, and the antigenic determinants recognized have been precisely identified. Table 5-1 illustrates four different peptide-antibody interactions that have been studied using the dual positional SCL. In the first application of the SCL approach for antigenic determinant mapping, a known peptide-mAb interaction was used as a model system [9]. This peptide-mAb interaction had been extensively characterized by ELISA using individual

5.3 Dual Positional SCLs

145

Table 5-1. Identification of antigenic peptides from dual positional SCLs ~~~

Peptide library

mAb

AC- DVPDYA- NH2

30

DVPDYA

Ac- DVPDYA- NH2

2

125-10F3 PYPNLS

AC-PYPNLP-NH2 Ac-PY PNLT-NH2 Ac-PYPNLS-NH2 Ac-Y PNLSS-NH2 Ac- YPNLSC- NH2 Ac-Y PYPNL-NHZ Ac-Y PYPNM-NH2

4 5 6 144

222-35C8 GRTTVFS

Ac- GRWTVF-NH2 Ac- GRYTVF- NH2 AC- GRTTVF- NH2 Ac- GRFTVF- NHZ Ac-GRHTVF- NHZ Ac-QEFH AG-NH2 Ac- RGTVFP-NH2

2 5 25 26 31 131 9

3E7

YGGFMT-NH2 YGGFMS-NH, YGGFME- NH?

1719

a

ICSo(nM) Ref.

DVPDYA

Ac-O,O~XXXX-NH~ 19B10

0,02XXXX-NH2

Known Individual antigenic peptides determinanta identified

YGGFMT

150

2 14

3 4 4

~.

Previously determined by omission or truncation analog analysis.

omission and substitution peptide analogs [33]. This prior knowledge of the specificity of the peptide-antibody interaction provided a clear model, which could be used to test the practical utility of the SCL approach. Upon screening each peptide mixture of the SCL (Ac-0,O2XXXX-NH2) and the subsequent iterations by competitive ELISA, a single peptide (Ac-DVPDYA-NH,) was found to effectively inhibit mAb 19B10 interacting with a 13-residue peptide (Ac-YPYDVPDYASLRSNH2 from the influenza virus hemagglutinin, HA1 : 98- 110). This sequence exactly matched the antigenic determinant sequence known to be recognized by this mAb. More recently, another mAb that was generated against the same peptide immunogen as mAb 19B10 [34] was also extensively characterized by competitive ELISA using substitution analogs of Ac-YPYDVPDYASLRS-NH2 [35], and by structural means using X-ray crystallography [36]. This mAb (1719) was screened against Ac-0,O2XXXX-NH2, which is made up of more than 52 million peptide

146

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

sequences [37]. Upon screening each of the 400 different peptide mixtures, the peptide mixture Ac-DVXXXX-NH, was found to have the greatest inhibition of antibody binding (ICso = 58000 nM). The iterative process, in which each subsequent position is identified by the preparation and testing of peptide mixtures, is illustrated in Fig. 5-1. Twenty new peptide mixtures were synthesized in which the third position of Ac-DVXXXX-NH, was defined with the 20 L-amino acids (Ac-DVOXXXNH,). The most effective inhibiting peptide mixture was Ac-DVPXXX-NH2, with an IC,, = 15000 nM. The iterative process was repeated in order to define the fourth position of Ac-DVPXXX-NH2 by synthesizing another set of 20 peptide mixtures (Ac-DVPOXX-NH2). Only one peptide mixture (Ac-DVPDXX-NH2, IC,, = 1100 nM) was found to have better activity than Ac-DVPXXX-NH, from the previous iterative step. The same degree of specificity was found when the fifth posi-

Ac- DVOXXX- NH, **7,

Ac- DVPOXX-NH, I

OD'

-

5.3 Dual Positional SCLs Ac-DVPDOX-

147

NH,

-.

Ac-DVPDYO-NH,

--

Figure 5-1. Iterative process for Ac-DVXXXX-NH2. Individual bars are labeled on the xaxis by the 20 amino acids used to define each subsequent position (represented as 0).The first bar in each graph represents the most active peptide mixture from the previous step. The y-axis represents the reciprocal of the ICJovalue of each peptide mixture.

tion of Ac-DVPDXX-NH, was defined, in that only one peptide mixture, AcDVPDYX-NH,, ICso = 40 nM, was found to be effective. This clearly illustrates the importance of the presence of specific peptide sequences in each peptide mixture throughout the iterative process. Upon defining the sixth and final position of the sequence, Ac-DVPDYA-NH,, with an ICso= 2 nM, was found to be the most effective peptide recognized by this monoclonal antibody. The sequence -DVPDYAexactly matches the antigenic determinant found in earlier studies to be recognized by this monoclonal antibody. The fine specificity found here for this peptide-an-

148

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

tibody interaction, in which the most specific amino acids were the two aspartic acid: and the tyrosine, as well as the replaceability of each position, are in good agreemeni with the mapping studies using complete sets of the substitution analogs of the anti. genic determinant [35] and the X-ray structure of a F(ab) of mAb 17/9 complexed with the nonapeptide (YDVPDYASL) (36, 381. In another example, the same peptide library was screened against a mAb, which was made against a synthetic peptide representing residues 703-732 of the oncogene. related protein from v-fes [32, 391. In this example, the antigenic determinani recognized by the mAb was previously characterized using single omission and substitution analogs [33,35]. Each of the 400 peptide mixtures was assayed to determine its ability to inhibit the interaction of mAb 125-10F3 with the antigenic peptide Ac-GASPYPNLSNQQT-NH, by competitive ELISA. An iterative process was car= ried out for Ac-PYXXXX-NH, (IC,, = 20 VM) and Ac-YPXXXX-NH2 (E5, 200 VM), which were found to be the most effective peptide mixtures. Thirteen of the final 20 peptides (Ac-PYPNLO-NH,), including the known antigenic determinani sequence (-PYPNLS-), had IC,, values within three-fold of the most effective peptide (Ac-PYPNLP-NH,, ICso = 4 IIM) [32]. Since no significant increase in activity was found, the sixth position of the antigenic determinant can be considered redundant, which was also found in previous mapping studies. In the case of Ac-YPXXXX-NH,, the second most effective peptide mixture from the initial screening of the SCL [39], it was initially believed that the activity of Ac-YPXXXX-NH, was due simply to the sequence homology that it has with the second and third positions of the known antigenic determinant, -PYPNLS-. We chose to pursue the identification of specific sequences from Ac-YPXXXX-NH, using the iterative process to test this hypothesis, as well as to accumulate a base of information about the utility of SCLs in such studies. Thus, the third position of AcYPXXXX-NH, was defined, resulting in 20 new peptide mixtures (Ac-YPOXXXNH,). Assayed by competitive ELISA, only Ac-YPNXXX-NH, (IC,, = 7329) and Ac-YPYXXX-NH, (IC,, = 12780) were found to inhibit antibody binding at a lower concentration than Ac-YPXXXX-NH,. Since these peptide mixtures were different in chemical composition and were found to have similar activities, the iterative process was carried out for both sequences. In the final iterative step, Ac-YPYPNL-NH2 (ICso = 2 nM) was found to inhibit antibody binding more than 100-fold better than Ac-YPNLSS-NH, (ICso= 144 nM). From the final results of the iterative process for both Ac-PYXXXX-NH, and Ac-YPXXXX-NH,, it is evident that Ac-PYXXXX-NH, was initially more effective, primarily because at least 20 peptides representing Ac-PYPNLO-NH2 within Ac-PYXXXX-NH, were equally effective in inhibiting antibody binding. In contrast, only one peptide, namely Ac-YPYPNL-NH,, which was found from the iterative process of Ac-YPXXXX-NH,, inhibited antibody binding at a similar concentration. The differences in inhibitory activity of the two peptide mixtures, Ac-PYXXXX-NH~ and Ac-YPXXXX-NH,, can be attributed to the number of

5.3 Dual Positional SCLs

149

high affinity peptide sequences (i.e., 20 vs. 1) within each peptide mixture. Also, whereas Ac-PYPNLS-NH, and Ac-YPYPNL-NH, were found to have similar in. hibitory activities, the 40 peptides representing Ac-YPNLSO-NH2 and AcYPNLPO-NH, were found to have 100-fold less activity, which indicates the im. portance of the presence of at least one additional residue at the amino terminal of the -YPNL- motif. The same SCL was screened for the inhibition of mAb 222-3928 binding to the antigenic peptide LHNNEAGRTTVFSC-NH, [40]. Three peptide mixtures, namely Ac-GRXXXX-NH,, Ac-RGXXXX-NH,, and Ac-QEXXXX-NH2, were found to have the greatest inhibition of antibody binding. Upon completion of the iterative selection and synthesis process, a number of peptide sequences were identified, including the antigenic determinant (Ac-GRTTVF-NH,), and are shown in Table 5-1. It should be noted that tyrosine and tryptophan substitutions of the threonine residue, for example Ac-GRYTVF-NH, and Ac-GRWTVF-NH, versus Ac-GRTTVF-NH,, resulted in peptides having nearly ten-fold greater activity. Also, two other sequences, Ac-RGTVFP-NH, and Ac-QEFHAG-NH,, were found to effectively inhibit antibody binding. MAb 3E7 [41], which was raised against &endorphin [42], was screened with a nonacetylated SCL having the same format (0,02XXXX-NH3 in order to identify the antigenic determinant, as well as to compare these results with those found by others investigating mAb 3E7 using different peptide library approaches [ll, 431. The 400 peptide mixtures of the nonacetylated SCL were assayed by competitive ELISA in a manner similar to the previous examples. Only peptide mixtures YGXXXX-NH, (ICs0 = 2900 nM) and YAXXXX-NH2 (ICs0 = loo00 nM) were found to cause significant inhibition of mAb 3E7 binding to &endorphin. An iterative process was carried out for YGXXXX-NH, in order to define the remaining positions of this peptide mixture. Upon completion of the iterative process for the most effective peptide mixture, YGGFMO-NH,, YGGFMT-NH, was found to have the greatest antibody inhibiting activity, even though only an eight-fold difference in activity was found for a majority of the defined peptides. The peptide YGGFMT-NH2 exactly matches the first six amino acids of b-endorphin. The precise identification of antigenic determinants proves the concept of the SCL approach for its use in mapping antigen-antibody interactions, and indicates its potential as well as its usefulness for the systematic study of the level of cross-reactivity of antibody recognition. It should also be noted that no prior information about the sequence of the antigen or antibody is required to carry out determinations of this kind.

150

5 The Versatility of Nonsupport-Bound Combinatorial Libraries

5.3.1.2 Identification of Opioid Peptides

An opioid assay was chosen to demonstrate the application of SCLs to receptor ligand identification. The enkephalins (i.e., YGGFM-OH and YGGFL-OH) were the first natural ligands found for the opioid receptors [44]. Since the enkephalins are not N-acetylated, a nonacetylated SCL was used in an initial test study. Each of the 400 different peptide mixtures of the SCL (0,02XXXX-NH2) was assayed to determine its ability to inhibit binding of [3H]-[~-Ala2,MePhe4,Gly-o15]enkephalin (DAMGO, specific for mu receptor) to membrane-bound receptors in crude rat brain homogenates. Following the initial screening, IC,, values were determined for each of the most effective peptide mixtures. YGXXXX-NH,, with an IC,, = 3452 nM, was found to be the most effective inhibiting peptide mixture. The remaining four mixture positions in YGXXXX-NH, were iteratively chosen [32,45a]. It was found that the first five residues of the most effective peptides, YGGFMO-NH2 and YGGFLO-NH,, matched the sequences of the naturally occurring methionineand leucine-enkephalins. n o other peptide mixtures, WWXXXX-NH2 and YPXXXX-NH,, showed activity, and their iterative process has been completed [45b]. In order to determine if new peptide sequences with opioid activity could be identified from peptide libraries, a sizing library (Ac-OXX-NH2 to Ac-OXXXXXXXNH2) made up of a series of individual, N-acetylated libraries ranging from three to eight residues in length was used to determine the optimal length associated with a potential N-acetylated peptide. None of the mixtures exhibited inhibitory activity until a length of six amino acids was reached. Mixtures containing peptides with more than five amino acids were highly active when arginine was in the defined position (461. Upon screening, the hexapeptide SCL Ac-O,O,XXXX-NH,, AcRWXXXX-NH, and Ac-RFXXXX-NH, were found to be the most active mixtures. The standard iterative enhancement process was carried out for Ac-RFXXXXNH,. The 20 defined peptides, termed acetalins (Ac-RFMWMO-NH,), were found to be potent opioid antagonists [47]. The activities of the individual peptides derived from iterations completed for both the acetylated and nonacetylated SCLs are shown in Table 5-2.

5.3.13 Development of Antimicrobial Peptides

The increasing occurrence of bacterial strains that are resistant to commonly used antibiotics has lead to a critical need for new antimicrobial agents. SCLs represent a rapid means to develop new antimicrobial leads for pharmaceutical research and drug discovery. This more complex application, which involves whole cells in suspension in biological-like fluids, further illustrates the strength of using soluble combinatorial libraries rather than support-bound libraries. Several microorganisms have

5.3 Dual Positional SCLs

151

Table 5-2. Use of a dual positional peptide SCL for the identification of p opioid receptor ligands Peptide library

Mixture iterated

OOXXXX-NH2

YGXXX-NH2

Most active peptides

YGGFMA-NH2 YGGFLA-NH2 WWXXXX-NH2 WWPKHG-NH2 YPXXXX-NH2 YPFGFR-NH2 AC-OOXXXX-NH2 AC-RFXXXX-NH2 Ac-RFMWMK-NH2 Ac- RFMWMT-NH2 Ac-RFMWMR-NH2 AC-RWXXXX-NH2 Ac-RWIGWR-NH2 Ac-FRXXXX-NH2 Ac-FRWGGM-NH2 A c - o ~ x x x x - N H ~ ~ Ac-rfxxxx-NH2 Ac-rfwink-NH2 a

ICsn (nM) Ref.

28 59 9 13 5 5 6 5 137 18

Lowercase letters indicate D-amino acids.

been used in these studies :gram-positive bacteria such as Staphylococcusaureus and Streptococcus sanguis, gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, and yeast such as Candida albicans.

Against Staphylococcus aureus The peptide mixtures of the hexamer SCL Ac-0,02XXXX-NH2 described earlier were screened for inhibition of S. aureus growth in a microdilution assay. While several peptide mixtures showed potent antimicrobial activities, two of the most active peptide mixtures (Ac-RRXXXX-NH2 [9, 481 and Ac-FRXXXX-NH2 [32]) were initially selected to carry out an iterative process as described above. A number of peptide mixtures were found to have potent activity at each iterative step. The three most active mixtures are shown in Table 5-3. The criteria of selection of the peptide mixtures to be followed in the first iterative process were: highest antimicrobial activity, low cytolytic activity against red blood cells, and differences in the chemical character of the amino acids selected at a given position. One can always initiate an iterative process from a second selection of the peptide mixtures at each iterative step following similar criteria. Thus, the most active individual hexapeptides of the three series against S.aureus identified through this process that have been reported to date are: Ac-RRWWCR-NH2 [9], Ac-RRWWRF-NH2 1481 and Ac-FRWLLF-NH, [32] (Table 5-4). The spectrum of activity of the AcRRWWCO-NH, series were further studied against S. sanguis, E. coli, Ps. aeruginosa and C.albicans 1491. These peptides exhibited a broad activity spectrum against these microorganisms (Table 5-4) and appeared to be bactericidal. It is noteworthy that the individual peptides derived from SCLs, while shorter in length, exhibit higher or equal activities to naturally occurring antimicrobial peptides (i.e., eramicidins. maeainins. cecrodns. defensins. etc. - activities reviewed in 1501).

152

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

Table 5-3. Iterative process for the identification of antistaphylococcal peptides

G o (Wml)

GO (vLg/ml)

Sequences Ac-RRXXXX-NH2

450

Sequences Ac- FRXXXX- NH2

Ac- RRWXXX- NH2 Ac-RRYXXX-NH2 Ac- RRRXXX-NH2

216 239 275

Ac-FRWXXX-NH2 Ac- FRLXXX-NHZ AC- FRFXXX - NH2

459 526 559

Ac- RRWWXX-NH2 Ac-RRWFXX-NH2 AC- RRWRXX-NH2

36 58 77

Ac-FRWWXX-NH2 Ac-FRW LXX-NH2 Ac- FRWMXX- NH2

67 111 135

Ac- FRWLCX- NHZ Ac-FRWLWX-NH2 Ac- FRWLLX-NH2

12 13 19

Ac-RRWWCX-NH2 Ac- RR WW WX-NH2 Ac-RRWWRX-NH2

8.7 9.9 9.9

1730

Table 5-4. Antimicrobial activities of hexapeptides derived from Ac-O1O2XXXX-NH2

Sequences

S. aureus

Ac-RRWWCR-NH2 Ac-RRWWCW-NH2 Ac-RRWWCV-NH,

3.4 4.1 4.9

S. sanguis 22

55 41

Ac-RRWWRR-NH2 Ac-RRWWRH-NH2 Ac-RRWWRW-NH2 Ac-RRWWRF-NH2

16 19 59 5

9 12

Ac-FRWLLF-NH2 Ac-FRWLLR -NH2 Ac- FRWLLW-NH2

G o (Clg/ml) E. coli

Ps. aeruginosu

C. albicans 100 151 103

7

28 30 37

5

39

11 13 14

5 6 5

685 54 78

Ac-FRWWHR-NH2 Ac-FRWWHK-NH2 Ac-FRWWHW-NH2

18 30 23

10 130 29

Ac-RRWCKR- NHz

-

Ac- RRWCKM- NH2

Ac-RRWCKH- NHz

-

-

-

67 71 70

Against Escherichia coli Ac-FRXXXX-NH, was also among the most active peptide mixtures against E. coli (ICso = I078 vg/ml) and was selected to carry out an iterative process [51]. Thus, based on the same criteria of selection described above, Ac-FRWXXX-NH, (IC5,, = 222 pg/ml), Ac-FRWWXX-NH2 (I& = 36 pg/ml) and Ac-FRWWHX-

5.3 Dual Positional SCLs

153

NH, (ICso = 52 pg/ml) were chosen in this process. From the Ac-FRWWHO-NH2 series, Ac-FRWWHR-NH, was found to be the most active peptide with an lCso = 13 vg/ml (Table 5-4). The hexapeptides derived from the mixture AcFRWWHO-NH, showed activities against the two gram-positive bacteria tested (Table 5-4). Against Candida albicans

In a similar manner, an iterative process from the peptide mixture Ac-RRXXXXNH2 was carried out to identify individual hexapeptides having activity against C. ulbicuns (ICso = 1704 pg/ml) (521. The peptide mixtures chosen at each iterative step were: Ac-RRWXXX-NH, (ICso = 405 pg/ml), Ac-RRWCXX-NH, (Ks0= 127 pg/ml), Ac-RRWWXX-NH2 (ICso = 170 pg/ml), Ac-RRWCKX-NH2 (ICso = 74 pg/ml), and Ac-RRWWRX-NH, (IC50= 109 pg/ml). Ac-RRWCKR-NH2 (ICso = 67 pg/ml) and Ac-RRWWRR-NH, (ICso = 28 pg/ml) exhibited the greatest activity of the two series (Table 5-4). None of the sequences described here have been reported or are part of known protein sequences (Registry Database produced by Chemical Abstracts Service). These examples further illustrate the strength of soluble SCLs to rapidly develop new leads for drug discovery.

5.3.1.4 Development of Inhibitors of Melittin’s Hemolytic Activity

SCLs can also be used to study the structure-activity relationships of a compound of interest. The following example involves the identification of peptides that inhibit the interactions with, and, in turn the lysis by, larger peptides to cell membranes. Thus, the same hexapeptide SCL was screened for inhibition of the hemolytic activity of melittin, a 26-residue peptide isolated from bee venom [53, 541. AcIVXXXX-NH2 showed the greatest inhibitory activity (ICso = 386 pg/ml) and was chosen to carry out the iterative process. A 3.9-fold improvement was observed upon defining the third position with isoleucine (Ac-IVIXXX-NH,, ICso= 97 pg/ml). Defining the fourth position with leucine or phenylalanine resulted in a 4.6- or 3-fold increase in activity, respectively (Ac-IVILXX-NH,, ICso = 21 pg/ml, and AcIVIFXX-NH2, ICso = 32 pg/ml). Smaller increases in activity were observed upon defining the fifth and sixth positions. Thus, Ac-IVILLX-NH, and Ac-IVIFDXNH2 had ICsOvalues of 13 and 32 pg/ml, respectively; and Ac-IVILLW-NH2 and Ac-IVIFDC-NH, showed the highest inhibitory activity of the two series (Eso =5 and 11 pg/ml, respectively). The hydrophobic character of these peptide inhibitors suggests a mechanism of inhibition through hydrophobic interactions between the peptides and melittin, which would prevent melittin from interacting with the cell membrane. Such an application can be extended to any general lytic assay that involves an initial binding and/or interaction of a comDound with a membrane.

I54

3

The Versatility of Nonsupport-Bound Combinatorial Libraries

5.3.1.5 Development of Enzyme Inhibitors

Enzyme inhibitors have a wide range of research and therapeutic uses. The use oi peptide libraries for the identification of peptide enzyme inhibitors was investigated using trypsin as the model enzyme. Trypsin was chosen for study because numerous polypeptide and protein inhibitors of trypsin from plant and animal sources are known, and range in length from 30 to several hundred amino acids. These inhibitors are highly specific, limited proteolysis substrates for their target enzymes (551. Their amino acid sequences contain at least one peptide bond called the reactive site (-P,-P;-), which specifically interacts with the active site of the cognate enzyme, and is identical to the potential cleavage site. Trypsin inhibitors are known to have lysine or arginine at the PI position. In contrast to normal substrates, the dissociation constant of an enzyme-inhibitor complex is extremely low, resulting in very slow hydrolysis. It was anticipated that short peptides, having a potential reactive site for a trypsin inhibitor in their sequence, would therefore be capable of inhibiting this enzyme. A specifically designed L-amino acid library of hexapeptide mixtures was synthesized on cotton carriers, cleaved in situ, and screened. This library was designed to represent all possible reactive sites for trypsin inhibitors with the PI residue (lysine or arginine) in every position of the sequence except the last [19]. The library was made up ten groups of peptide mixtures, represented by the formulas: AC-KOXXXX, AC-XKOXXX, AC-XXKOXX, AC-XXXKOX, AC-XXXXKO, ACROXXXX, Ac-XROXXX, Ac-XXROXX, Ac-XXXROX, and Ac-XXXXRO. Each of these groups consisted of 20 different peptide mixtures representing a total number of 2606420 individual peptides. In each peptide mixture, four positions (XI represent a mixture of 19 of the 20 genetically coded amino acids (cysteine excluded), whereas 0 represents those positions occupied by individual amino acids (A through Y). The mixtures were synthesized using cotton as solid support [4]. The results of the initial screening of the peptide library revealed that the peptide mixtures Ac-XKIXXX and Ac-XXKIXX were the most effective inhibitors of the By iteratively defining tryptic hydrolysis of Na-benzoyl-qbarginine-p-nitroanilide. the X positions adjacent to the lysine in Ac-XKIXXX, 20 new mixtures with three X positions (Ac-OKIXXX) were synthesized and screened. Ac-AKIXXX was found to be the strongest trypsin inhibitor. Upon defining the remaining positions in a similar manner, it was found that the most active inhibitor was Ac-AKIYRP-NH,, with an ICso of 46 pm (Table Ma). This sequence was chosen to serve as a motif in a new library of 20 dodecapeptide mixtures with five mixture positions, represented as Ac-XXXAKIYRPOXX. The iterative process for the most active mixture (Ac-XXXAKIYRPDXX) was carried out, and dodecapeptides two- to five-fold more effective than the hexapeptide were identified (Table 5-5 b).

5.4

SCLs Composed of Peptides Containing

L-, D-

155

and Unnatural Amino Acids

Table 5-5. Trypsin inhibitors derived from L-amino acid SCLs

Ac-AKIYRP-NH2 Ac-AKIYRE-NHz Ac-AKIYRD-NH2 Ac-AKIYRK-NH,

46 108 122

135

Dodecapeptides

G o (nM)

Ac-YYGAKIYRPDKM-NH, Ac-AYGAKIYRPDKM-NH2 Ac-SYGAKIYRPDKM-NH2 Ac-CYGAKIYRPDKM-NH2 Ac-DYGAKIYRPDKM-NH2 Ac-MYGAKIYRPDKM-NH2

10 20 20 21 22 22

5.3.2 Use of all D-Amino Acid SCLs An acetylated hexapeptide library composed entirely of D-amino acids (represented as Ac-o,02xxxx-NH2, was screened in the opioid receptor binding assay. The most active peptide found upon completion of the iterative process, Ac-rfwink-NH,, is an agonist with high selectivity for the1 receptor [56]. Furthermore, this peptide can produce analgesia in mice when administered i. c. v. or i. p. (i. c. v. = intracerebroventricularly, i. p. = intraperitoneally) [56]. While bearing some resemblance to the acetalins, this peptide was, in contrast, found to be a full agonist.

5.4 SCLs Composed of Peptides Containing L-, D- and Unnatural Amino Acids Since L-amino acid containing peptides may be susceptible to proteolytic breakdown and rarely have oral activity, combinatorial libraries composed of peptides containing a mixture of L-, D- and unnatural amino acids were generated. Such libraries expand the chemical diversity that can be screened for biological activity. Thus, two C-amidated tetrapeptide SCLs, one N-acetylated, the other not, were prepared having the first position defined with one of 58 amino acids (“U” - 20 L-, 19 D- and 19 unnatural amino acids, listed in Table 5-6) and the three remaining positions as a close to equimolar mixture of 56 of the same amino acids (“Z” - L- and D-cysteine were omitted). These libraries were synthesized using the DCR method in a manner similar to the hexapeptide libraries described above. Each SCL is composed of 58 peptide mixtures; each peptide mixture contains 175 616 (563) individual tetrapeptides, for a total of 10185728 (58 x 175616) tetrapeptides per SCL. When assayed at 2.5 mg/ml, each tetrapeptide within a mixture is present at 14.2 ng/ml (26 nM if one assumes an average molecular weight of 540). The two SCLs were assayed for inhibition of S. aureus [57] and E. coli [54]. (aFmoc-dys)ZZZ-NH, showed the highest activity in both assays (ICso= 44 and

I56

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

179 &ml, respectively). Following an iterative process, individual tetrapeptides having antimicrobial activities in a range similar to the L-hexapeptides described above were identified (Table 5-7). These tetrapeptides also showed a broad spectrum of acTable 5-6. Unnatural amino acids included in the two tetramer peptide SCLs Abbreviation used in the sequences

Protected amino acid used during the synthesis

Bala aABA gABA aAIB eAca 7aHa bAsp gGlu Cys[ACM] Elys aFmoc-dys MetOz Nle Nve Orn dOrn NOzF HYP Thiopro

Boc-8-alanine Boc-L-a-amino butyric acid Boc-L-y-amino butyric acid Boc-L-a-amino isobutyric acid Boc-L-&-aminocaproic acid Boc-7-amino heptanoic acid Boc-L-aspartic acid (a-Bzl) Boc-L-glutamic acid (a-Bzl) Boc-cysteine (acetaminomethyl) N-E- Boc-N-a-CBZ-L-lysine

N-E-Boc-N-a-Fmoc-L-lysine

Boc-L-methionine sulfone Boc-L-nor leucine Boc-L-norvaline N-a-Boc-N-6-CBZ-L-ornithine

N-6-Boc-N-a-CBZ-bornithine Boc-p-nitro-L-phenylalanine Boc-hydroxyproline (Bzl) Boc-L-thioproline

Table 5-7. Antimicrobial activities of tetrapeptides derived from (aFmoc-dys)ZZZ-NHz

Sequences

MIC (pg/ml)

S. aureus

E. coli

(aFmoc-dys)WfR-NHz (aFmoc-e1ys)Wfl-NHz (aFmoc-e1ys)Wf (Thiopro)-NHz

4- 8 3-4 4-8

31-62 62- 125 31-62

(aFmoc-dys)WKW- NH2 (aFmoc-e1ys)WKw- NHz (aFmoc-dys)WK (NO2F)-NH2

5-8 8- 16 8- 16

16-31 31-62 31-62

(aFmoc-dys)WYr-NH2 (aFmoc4ys)WY (aABA)-NHz

5-8

5-8

3 1-62 62- 125

(aFmoc-dys)cir-NH2 (aFmoc-e1ys)ciK-NHz (aFmoc-&lys)ci(dOrn)-NHz

4-8 8- 16 8- 16

31-62 62-125 31-62

5.5 Positional Scanning SCLs

157

tivities as determined against five different microorganisms (S. OUEUS, methicillin resistant S. aureus (MRSA), S.sanguis, E. coli, and C. albicans - [57]). These pep tides appeared to have activity by inhibiting cell growth and development rather than through cell lysis. The same two libraries were screened in the opioid receptor assay; the most active mixture found was YZZZ-NH, [58). Upon completing the iterations, the most active mixtures found were YmFA-NH,, YMFa-amino butyric acid-NH, and YmF(p-nitro)F-NH,. Peptides containing a similar YMF-motif at the N terminus have been described [59, 601.

5.5 Positional Scanning SCLs In order to shorten the time required for the identification of active peptide sequences from a mixture of millions of compounds, an approach, termed a positional scanning SCL (PS-SCL), was developed by this laboratory (231. Using peptides as an example, PS-SCLs are composed of individual positional SCLs in which a single position is defined with a single amino acid, while the remaining positions are composed of mixtures of amino acids. The defined position is “walked” through the entire sequence of the PS-SCL. Therefore, the number of positional SCLs is equal to the number of residues in each peptide of the PS-SCL. It should also be noted that each positional SCL, while addressing a single position of the sequence, represents the same collection of individual peptides. When used in concert, the data derived from each positional SCL yield information about the most important amino acids for every position. This library can be screened, and data obtained, in as little as a single assay. The information can also be used to synthesize individual peptides representing all possible combinations of the most active amino acids at each position. This serves to confirm the PS-SCL screening results, as well as to identify peptide sequences with the highest activities. The screening results also give information about the replacement of each position as well as its relative importance. The first PS-SCL prepared was made up of L-amino acid hexapeptides. This library consists of six separate positional SCLs, each composed of 20 different peptide mixtures having a single position defined with one of the 20 natural L-amino acids (represented as 0),with the remaining five positions composed of mixtures of 19 amino acids (represented as X;cysteine was omitted). The six positional SCLs differ only in the location of the defined position and can be represented as O,XXXXX, X02XXXX,XX03XXX,XXXO,XX, XXXXO,X, and XXXXXO, (120 peptide mixtures in total). Each peptide mixture represents approximately 2.5 million (1g5) individual sequences; each of the six positional peptide libraries contains over 50 million hemmers. The positional format has been used to prepare decapeptide PS-SCLs composed of more than loJ2sequences, which have been used successfullv for the studv of antinen-antibodv interactions 1311. as well as in

158

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

the identification of opioid sequences using a radioreceptor assay [61]. Also, an Nacetylated D-amino acid hexapeptide PS-SCL was prepared and used for the identification of trypsin inhibitors [62]. PS-SCLs can be used in virtually any assay for the rapid identification of active compounds. It should be noted that, in order to increase the rate of success upon screening a PS-SCL, assay conditions must be optimized to their maximum sensitivity, owing to the enormous number of compounds within each mixture. The results one obtains from screening a PS-SCL will vary depending on the nature of the interaction being studied. The investigator must decide, based on criteria such as percent inhibition, ICso value, chemical character, etc., which amino acids in each of the defined positions of the most active mixtures should be selected. The amino acids chosen at the specified positions are used to create a number of individual peptide sequences, which are synthesized and assayed to confirm the screening results and to identify the most active sequences. The advantage of the PS-SCL approach is that each position of the library i5 screened simultaneously, and the results found for each position enable the identification of individual, active compounds (peptides in the examples shown) withoul the iterative synthesis and selection steps that are necessary for the dual positional SCLs, or sequencing or decoding that are necessary for support-bound methods. The screening of PS-SCLs can be carried out in a single assay. If one or two amino acids are effective for each position, peptides representing the combinations of these amino acids may then be synthesized in order to confirm the screening results (i.e., 26 = 64 peptides). For those cases in which the screening profile yields three 01 more effective peptide mixtures at each position, the required number of individual peptides increases dramatically (i.e., 36 = 729 peptides), and at some juncture the synthesis of the combinations becomes impractical. As an alternative to the im. mediate synthesis of individual peptides, one can prepare a sublibrary in the same scanning format composed only of those amino acids found to be effective, or which fall below a suitable ICso cutoff. If three amino acids are chosen at each position, then a sublibrary could be prepared composed of six positional SCLs of three pep tide mixtures each, and each one is composed of 243 individual peptides. This increases the effective peptide concentration within each peptide mixture and can be expected to lead to much stronger signals. Furthermore, since the PS-SCL is composed of six separate positional SCLs, each one can be considered independent and self contained. Therefore, each positional SCL can be independently pursued using the iterative synthesis and selection process as described earlier 19, 32) and illustrated for the identification of the D-amino acid peptide inhibitors of trypsin. It should be noted that the PS-SCL approach does not guarantee the identification of the mosl active compounds present in the library in each assay. This fact, however, is inherenl to all combinatorial library approaches. The activity threshold of PS-SCLs is inherently lower than the SCL defined with two oositions due to the increased number of individual oeotides making UD each

5.5 Positional Scanning SCLs

159

peptide mixture (2476099 vs. 130321, respectively). At the same total peptide mixture concentration (i.e., 10 mg/ml), the concentration of each individual peptide is 19 times lower in the PS-SCL than it is in the dual positional SCL. In either peptide library, it is believed that the presence of analogs of the most active peptides will increase the “effective” concentrations of active peptides and elevate the overall signalto-noise ratio in any assay.

5.5.1 Identification of Antigenic Determinants As first presented, an L-amino acid hexapeptide PS-SCL, with an acetylated N-terminus and C-terminal amide, was used for the identification of two different antigenic determinants recognized by monoclonal antibodies (mAbs) [23]. The specificity of monoclonal antibodies 19B10 and 125-l0F3 have been studied using individual peptide analogs as well as SCLs. PS-SCLs were used to identify the most active amino acids at each position in a single assay (Table 5-8). This information is sufficient to locate the antigenic determinant in the immunogenic sequence; however, individual peptides representing all the possible combinations of the most active amino acids at each position were synthesized and their activities determined. In both peptide-mAb interactions, highly active individual compounds were identified. Also, their sequences were part of the immunogenic sequence. For the peptide-mAb interactions, the fact that all of the individual peptides had comparable affinities implies that the activities of the most active peptide mixtures in the screening of the PS-SCL are connected. This means that the individual peptides responsible for the activity of Ac-DXXXXX-NH2 are the same or similar to the individual peptides in Ac-XXXXYX-NH2. Monoclonal antibody 3E7, which was raised against /3-endorphin 1421, has been extensively studied using synthetic peptide analogs [20, 42) as well as a number of different peptide library approaches [ll, 43, 631. An N-acetylated L-amino acid PS-SCL was screened against mAb 3E7 by competitive ELISA [62]. No significant inhibition of mAb 3E7 binding to /3-endorphin was found for any of the 120 Nacetylated peptide mixtures (data not shown). This result agrees with the previous studies in which only peptide sequences having a free N-terminal tyrosine were recognized by mAb 3E7. ICs0 values were determined for the most active peptide mixtures from the screening of the nonacetylated L-amino acid PS-SCL. The most active amino acids are shown in Table 5-8. From the screening results alone, it can be seen that the antigenic determinant region of @endorphin recognized by mAb 3E7 is located at the first four residues of the N-terminus (YGGF). In order to confirm the screening results, individual peptides were synthesized which represent combinations of amino acids from the most active peptide mixtures at each position. This set of peptides was synthesized and assayed by competitive ELISA. A11 were found to have ICso values of 1-5 nM. The

G

A G

F

T

L M

F Y

A M

Q

D N P

E

16

1-4

2-430

YGGFMP-NH2 Y GGFMQ- NH2 YGAFLP- NH2

Ac- GESTFE- NH2 Ac-GDSTY E-NH2 Ac-GESTY E-NHZ

Y

S

S

P

8

3E7

S

H

D

G

134B29

E

Ac-STTSMM-NH2 Ac-SSTSMM-NH2 Ac-STTSAM-NH2

390- > 100000

16

T

H M

S

S T

12

L R Ac-PY PNLR-NHZ Ac-PY PNIL-NH2 Ac- PY PNLL-NH2

I L N 13-33000

N P 12

P

Y

P

123-10F3

Ac- DVPDYA- NH, Ac- DIPDYA-NH2 Ac-DVEDY A-NHZ

Three most active individual sequences

2-33

# of individual peptides Range of synthesized ICso nM

17/9

mAb

Most active amino acids Positions: 1 2 3 4 5 6

Table 5-8. Identification of antigenic peptides from hexapeptide PS-SCLs

2

1 1

2 2 2

170 873 2800

13 18 21

2 2 2

ICs0 (nM)

I621 I621 [62]

I731 1731 I731

1641

1641 I641

1231 I231 I231

(231 1231 1231

Ref.

-

$

il

5

f?

3

0

$

s

i3

s

r;

%r,

G

8

0

2

e

G

2

-3

cr

C

a

c

5.5 Positional Scanning SCLs

161

first five positions of four of the 16 peptides exactly match the five residues of the N-terminal amino acid sequence of /%endorphin recognized by mAb 3E7 [42]. The remaining 12 peptides contain conservative substitutions for the third (alanine substitution) and fifth positions (leucine substitution) of YGGFM. The fact that all of the individual peptides have comparable affinities demonstrates that the same peptide sequences are most likely responsible for the activity of the most active peptide mixtures at each position. In a final example, the acetylated version of the hexapeptide PS-SCL was screened against a monoclonal antibody that was raised against the surface antigen of Hepatitis B virus (HBsAg) [64a]. Sixteen individual peptides, representing the combinations of amino acids from the most effective peptide mixtures, were synthesized and assayed by competitive ELlSA to identify the most active sequences (Table 5-8). The most effective peptide (Ac-STTSMM-NH,, ICso = 170 nM) was nearly 10-fold less effective than HBsAg (ICJ0= 14 nM). The -STTS- motif is found in residues 114- 117 of HBsAg and probably represents the linear portion of a larger, possibly discontinuous, antigenic determinant. It should be noted that the proline residue of the peptide mixture Ac-XXXPXX-NH2, which was found to effectively inhibit antibody binding, was not part of any of the active individual peptides. An iterative process was carried out on this peptide mixture and resulted in the identification of a completely different, but equally active, peptide sequence (Ac-SVGPPH-NH2) when compared to the previous peptide [64b]. In contrast to the three previous examples, this example illustrates that the activities of the most active peptide mixture at a given position are not always connected to the activities of peptide mixtures with other positions defined. However, the activity of a peptide mixture does translate into the activity of an individual active peptide that can be identified through the iterative process. It should be noted that, if the peptide or protein sequence is known, in most cases the screening data alone will provide enough information to locate the antigenic determinant. For such cases, synthesis of individual peptides is necessary only to confirm the screening results and obtain more details on the specificity of the antigen-antibody interactions.

5.5.2 Identification of Opioid Ligands PS-SCLs have been used for the identification of specific opioid receptor ligands through the inhibition of p- and &specific opioid peptides binding to rat brain homogenates [24, 621. The use of PS-SCLs in radioreceptor assays illustrates their application in a more complex assay system. The nonacetylated L-amino acid PS-SCL was screened for inhibition of either 'H-DAMGO ( p selective) or 'HDPDPE (6 selective).

162

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

Combinations of the amino acids corresponding to the most active peptide mixtures at each position were synthesized for both receptor assays; 24 individual peptides were synthesized for the p receptor assay and 32 individual hexapeptides for the 6 receptor. The IC,, values obtained for the most active of these peptides are given in Table 5-9. The enkephalin sequences were found in both assay systems. The variability in peptide sequences for the 6 receptor suggests that at least two families of sequences are contributing to the activities seen in the library. The sequences of the four most active peptides (Table 5-9) reveal that glycine or histidine at the second position distinguish the two most active families of sequences.

5.5.3 Identification of Inhibitors of Melittin's Hemolytic Activity In a manner similar to the dual-position SCLs, the two PS-SCLs described above were assayed for inhibition of melittin's hemolytic activity [62]. The N-acetylated peptide mixtures generally showed higher activities than the nonacetylated mixtures (ICso values ranged from 0.5 to 4 mg/ml). It is noteworthy that, similar to the active peptide mixtures identified through the screening of the dual-position Ac-0,02XXXX-NH2 and iterative process [53, 541, the defined residues of the most active peptide mixtures of the N-acetylated PS-SCL were mostly hydrophobic residues [62,65]. In contrast, the defined residues of the most active peptide mixtures of the nonacetylated PS-SCL were acidic or hydrophilic, except for positions 4 and 6 [62]. TWO sets of individual peptides were generated representing all possible combinations of the most active amino acids selected following the screening of the two PS-SCLs (Table 5-10). As for the peptide mixtures, the N-acetylated individual peptides showed up to 100-fold higher activity than the most active nonacetylated peptide (Table 5-10). The N-acetylated peptides having a lysine at the sixth position showed little or no activity, while the corresponding peptides having a glutamic acid at this position showed high activity. This is probably due to the occurrence of electrostatic repulsion between the lysine-containing peptides and the positively charged residues of melittin (three lysines and two arginines). These results support the earlier hypothesis that the inhibitors act by interacting with melittin rather than with the cell membranes.

5.5.4 Decapeptide PS-SCL The positional scanning format was used to prepare a decapeptide PS-SCL, which is made up of ten positional peptide libraries, each one composed of 20 peptide mixtures having a single position defined and nine positions as mixtures. Each peptide mixture is made up of approximately 200 billion (2 x 10") individual sequences. Thus, each positional set of 20 peptide mixtures, as well as the entire peptide library,

~

'H ligand DAMGO

6: DPDPE

p:

Receptor

~~

Y H

Y

G F G L

G

M M W H

G F

F

F

L

F Y M L V

T

F R

Most active amino acids Positions: 1 2 3 4 5 6

32

24

# of individual peptides synthesized

45-20000

17- > 40000

Range of ICso nM

YGMFLV YGMHLV YGMMLV YHGWLV

YGGFMY YGGFMK YGGFYY YGGFLY

Most active individual sequences

Table 5-9. Use of a nonacetylated hexapeptide PS-SCL for the identification of p and 6 opioid receptor ligands

45 53 53 61

17 24 40 49

ICso

Ref.

164

5 The Versatility of Nonsupport-Bound Combinatorial Libraries

Table 5-10. Inhibitors

of melittin’s hemolytic activity

N-acet ylated PS - SCL Position : Amino acid:

F, I

Nonacetylated PS-SCL Position : Amino acid:

1

D,N

Sequences

Ic50 (pg/ml)

Sequences

G o (Wml)

Ac-FIIWCE-NH2 Ac-FIlWFE-NHZ Ac- FIIYCE- NH2 Ac-FIIY FE-NH, Ac- FQIWCE-NH2 Ac-FQI WFE-NH2 Ac- FQIYCE-NH2 Ac- FQIYFE-NH2 Ac- FIIYFK-NH2 Ac- FIIWFK-NH2

3.4 5.4 4.7 6.8 9.4 8.2 14 19 365 1298

Ac-IIIWCE-NH~ Ac-111 WFE-NH2 Ac-IIIYCE-NH2 Ac-IIIYFE-NH~ Ac-IQIWCE-NH~ Ac-IQIWFE-NH2 Ac- IQIYCE-NH2 Ac- IQIYFE-NH2 Ac- IQJYCK -NH2

4.5 5.4 12 4.9 5.6 21 4.8 17 1061

DDEITF-NH2

1201

all others

>2000

NDEIVI-NH2 NEEIVI-NH2 NDEIVF- NH2 NEEIVF-NH2

1

311 364 627 905

2 1.Q

3

4

5

6

I

W,Y

C,F

E,K

2

3

4

5

6

D,E

E

I

TY

F,I

is composed of approximately four trillion (4 x decapeptides. Despite the immense number of decapeptides, specific sequences having nanomolar affinities have been identified for four different antibodies [31, 661. 5.5.5 D-Amino Acid

PS-SCL

An N-acetylated hexapeptide D-amino acid PS-SCL was screened for trypsin inhibition (621. The initial premise was that peptides composed solely of D-amino acids would be stable towards proteolytic enzymatic degradation. The results of the screening did not provide information about highly specific residues in any of the positions. The combination of the most active amino acids would have generated 576 individual peptides. Alternatively, an iterative process was carried out to define the mixture positions of Ac-ryoxxx-NH,, since arginine and tyrosine were the most effective amino acids at positions 1 and 2, respectively. The activities of the individual peptides identified are shown in Table 5-11. Ac-ryrpwp-NH, (ICso= 62 VM) was identified as the D-amino acid hexapeptide with the strongest trypsin inhibitory activity. This peptide was shown to be stable towards tryptic hydrolysis. This example

5.6 Modified Peptide “Libraries from Libraries”

165

Table 5-11. Trypsin inhibitors derived from a D-amino acid PS-SCL

Sequence Ac- ryrpwp- NH2 Ac-ryrpww-NH2 Ac-ryrpwv-NH2 Ac-ryrpwc-NH, Ac-ryrpwt-NH2 Ac-ryrpwi-NH, Ac-ryrpwl-NH2 Ac-ryrpwf-NH, Ac-ryrpwr-NH, Ac-ryrpwy-NH, Ac-ryrpwa-NH, Ac-ryrpwm-NH2 Ac-ryrpws-NH2 Ac-ryrpwn-NH, Ac-ryrpwh- NH, Ac- ryrpwg- NH2 Ac-ryrpwq- NH2 Ac-ryrpwd-NH, Ac- ryrpwe- NH, Ac-ryrpwk-NH,

Icso

(W)

62 125 131 135 142 150 152 234 239 250 253 255 271 279 281 284 296 469 470 548

illustrates the fact that the positional SCLs making up a PS-SCL can be considered independent of each other, and can each be used as a starting point for an iterative synthesis and screening process as described for the dual positional SCLs [9].

5.6 Modified Peptide “Libraries from Libraries” The physico-chemical properties of peptides are strongly influenced by the amide bonds that make up peptide chains. We reasoned that if the character of the -CONH-moiety of peptides could be changed, then this would result in an overall change in the chemical property of these compounds. A range of possibilities included N-alkylation and/or reduction as modification. If such changes could be made to an entire library, a much wider range of chemical diversities would be possible. Through straightforward approaches, modified peptide libraries have been prepared which greatly extend the range and repertoire of chemical diversity [67-701. As a first example, a modified peptide library was prepared by the permethylation of the amides in an existing resin-bound hexapeptide PS-SCL, with the protected peptides remaining attached to the solid phase peptide support used in their syn-

166

5 The Versatility of Nonsupport-Bound Combinatorial Libraries

thesis [67-691. Following removal from the solid phase synthesis resin, libraries of this type, which lack the typical -CONH- amide bonds of peptides, can be used in solution with existing assay systems for the identification of active individual permethylated compounds. Such permethylated compounds are expected to have very different physical, chemical, and biological properties when contrasted with existing -CONH- based peptides. An illustration of the utility of the permethylated library is shown here, in which a permethylated PS-SCL was screened for antistaphylococcal activity [67, 681. Following cleavage from the solid support, it was used along with a standard microdilution assay to identify unique individual permethylated compounds with poten; antimicrobial activity against S. aureus. The permethylated mixtures having a hydrophobic residue or the permethylated form of histidine at the defined position showed the highest activity at each position. A set of 144 individual peptides representing all combinations of the most active residues was then generated and permethylated to be assayed for inhibition of S.aureus. The most active permethylated compounds from this set were pm[LFIFFF-NHJ, pm[FFIFFF-NH,], pm[FFFFFF-NH2] and pm[LFFFFF-NH2] (IC,,, = 6, 6, 7 and 10 pg/ml, respectively). These permethylated compounds showed similar activity against MRSA and S. sanguis, and no activity against the gram-negative bacteria E. coli and yeast C. albicans. Since phenylalanine was active at every position of the permethylated PS-SCL, a series of phenylalanine-containing peptides, which ranged in length from a single phenylalanine amide to its octapeptide form, was then synthesized and permethylated. This series was prepared in order to determine if the repetitive appearances of phenylalanine found at all six positions in the permethylated PS-SCL were due to an individual hexamer sequence, or to a frame-shifting fit by a shorter sequence (i. e., permethylated di-, tri-, tetraphenylalanine). The highest activity found (MIC = 2.5 pg/ml) was associated with the permethylated heptamer sequence.

5.7 Conclusions The range of studies in which SCLs have been used illustrates the broad utility of differently formated SCLs by their use in four separate biological assays representing differing levels of complexity. While the ELISA uses a soluble receptor (i.e., antibodies), the radio-receptor binding assay involves a more complex interaction, since the receptor is membrane bound. The studies involving the identification of an antigenic determinant by ELISA and of opioid peptides by a radio-receptor binding assay demonstrate the validity of the PS-SCL approach, since the active sequences expected to be present in the library were rapidly identified by the screening process. In the trypsin inhibition assay, which also involves soluble receptors (i.e., enzymes), no active seauences were known to be mesent within the librarv screened Drior to

5.7 Conclusions

167

the assay. This example shows not only that sequence information is not required for the screening data to be of value, but also that low affinity binding systems can be studied using the PS-SCL approach. The inhibition of melittin’s cytolytic activity and the development of antimicrobials are examples of assays with a higher level of complexity, which include whole cells as targets. Such applications have not previously been presented for support-bound libraries. In contrast to the support-bound methods, the SCL approach provides further information on the relative specificity of each residue within the peptide sequence. The versatility of PS-SCLs, as well as SCLs, is due to the fact that: (i) the compounds making up the libaries are not support bound, which allows each compound to interact freely in solution with relevant receptors; (ii) the compounds making up each mixture are present in approximately equimolar amounts; and (iii) each compound is present at a concentration expected to yield a detectable signal in most in vitro bioassays. The recent availability of immense libraries of chemically diverse compounds that can be used to identify highly active peptide and nonpeptide libraries is at the forefront of a revolution in basic research and drug discovery. In contrast to the expectations of “rational drug design”, which enables analogs to be designed based on the detailed understanding of molecular interactions, chemical library diversity enables the direct de novo discovery of new lead compounds, as well as the enhancement of the activity of existing compounds. Currently available libraries permit the identification of the most active sequences from hundreds of millions of peptides. We have now successfully utilized the concepts briefly reviewed here for the preparation and screening of organic, chemistry-based libraries using soluble combinatorial libraries as starting materials [67-701. For instance, the generation of libraries through chemical modifications using benzyl bromide, ally1 bromide, etc. have been successfully carried out [69], as have libraries of polyfunctional amines generated by the reduction of amide carbonyls to methylenes using peptide SCLs as starting materials [70]. Such organic chemical libraries can be readily prepared using known pharmacophores such as diazepams, piperazines, etc. Peptide libraries are also being constructed which are cyclic [71] or disulfide linked in nature [52]. In a manner similar to the generation of diverse planer or topographical landscapes, combinatorial libraries have been successfully inserted in structurally defined peptide sequences to generate conformationally defined libraries [52, 721. Such libraries have been used for the identification of new catalytic peptides [52] or to improve the biological activity of a peptide of interest [72]. These developments, as well as others not yet presented, will broaden the utility of libraries to enable the full repertoire of existing molecular architectures to be screened. It can be anticipated that libraries will assume an ever increasing role in drug discovery and basic research.

168

5

The Versatility of Nonsupport-Bound Combinatorial Libraries

Acknowledgments We thank Eileen Silva for her assistance in preparing this manuscript. This work was funded in part by Houghten Pharmaceuticals, Inc., San Diego, California.

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[21] A. Kramer, R. Volkmer-Engert, R. Malin, U. Reineke, J. Schneider-Mergener, Pept. Res. 1993, 6, 314-319. [22] A. Furka, E Sebestyen, M. Asgedom, G. Dibo, Int. J. Pept. Protein Res. 1991, 37, 487 -493. [23] C. Pinilla, J. R. Appel, P. Blanc, R. A. Houghten, Biotechniques 1992, 13, 901-905. 1241 C. T. Dooley, R. A. Houghten, Life Sci. 1993, 52, 1509-1517. [25] J. M. Ostresh, J. H. Winkle, V. T. Hamashin, R. A. Houghten, Biopolymers 1994, 34, 1681- 1689. (261 R. A. Houghten, M. K. Bray, S. T.De Graw, C. J. Kirby, Int. J Pept. Protein Res. 1986, 27, 673-678. (271 J. P. Tam,W. E Heath, R. B. Merrifield, J. Am. Chem. Soc. 1983, 105, 6442-6455. [28] M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon, J. Med. Chem. 1994, 37, 1233-1251. I291 E. T. Kaiser, R. L. Colescott, C. D. Blossinger, P. I. Cook, Anal. Biochem. 1970, 34, 595-598. [30] B. E Gisin, Anal. Chim. Acta 1972, 58, 248-249. [31] C. Pinilla, J. R. Appel, R. A. Houghten, Biochem. J. 1994, 301, 847-853. [32] R. A. Houghten, J. R. Appel, S. E. Blondelle, J. H. Cuervo, C. T. Dooley, C. Pinilla, Biotechniques 1992, 13, 412-421. [33] J. R. Appel, C. Pinilla, H. Niman, R. A. Houghten, J. Immunol. 1990, 144, 976-983. [34] I. A. Wilson, H. L. Niman, R. A. Houghten. A. R. Cherenson, M. L. Connolly, R. A. Lerner, Cell 1984, 37, 767-778. [35] C. Pinilla, J. R. Appel, R. A. Houghten, Mol. Immunol. 1993, 30, 577-585. (361 J. M. Rini, U. Schulze-Gahmen, I. A. Wilson, Science 1992, 255, 959-965. [37] C. Pinilla, J. R. Appel, R. A. Houghten, “Synthetic peptide libraries for research and

drug discovery” in Immunological Recognition of Peptides in Medicine and Biology (Eds. : N. Zegers, W. Boersma, E. Claassen), CRC Press, Boca Raton, 1995, 1- 14. 1381 U. Schulze-Gahmen, J. M. Rini, I. A. Wilson, J. Mol. Biol. 1993, 234, 1098-1118. [39] J. R. Appel, C. Pinilla, R. A. Houghten, Immunomethods 1992, I, 17-23. [40] C. Pinilla, J. R. Appel, S. Chendra, R. A. Houghten, “Determination of multiple binding specifities of a monoclonal antibody using a synthetic peptide combinatorial library” in Peptides: Proceedings of the 22nd European Peptide Symposium (Eds. : C. H. Schneider, A. N. Eberle), Escom, Leiden, 1993, p. 903. (411 C. Pinilla, J. R. Appel, R. A. Houghten, Gene 1993, 128, 71-76. [42] C. Gramsch, T. Meo, G.Riethmuller, A. Herz, J. Neurochem. 1983, 40, 1220-1226. I431 S. E. Cwirla, E. A. Peters, R. W. Barrett, W. J. Dower, Proc. Natl. Acad. Sci. USA 1990, 87, 6378-6382. [44]J. Hughes, T.W. Smith, H. W.Kosterlitz, L. A. Fothergill, B. A. Morgan, H. R. Morris, Nature 1975, 258, 577-579. [45a] R. A. Houghten, C. T. Dooley, BioMed. Chem. Lett. 1993, 3, 405-412. [45b] C. T. Dooley, R. A. Kaplan, N. N. Chung, P. W. Schiller, J. M. Bidlack, R. A. Houghten, Peptide Research, 1995, 8, 124- 137. [46] R. A. Houghten, J. R. Appel, S. E. Blondelle, J. H. Cuervo, C. T.Dooley, J. Eichler, C. Pinilla, “Optimal peptide length determination using synthetic peptide combinatorial libraries” in Peptides: Chemistry, Structure and Biology. Proceedings of the 13th American Peptide Symposium (Eds.: R. S. Hodges, J. A. Smith), Escom, Leiden, 1994, p. 971.

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5 The Versatility of Nonsupport-Bound Combinatorial Libraries

[47] C. T. Dooley, N. N. Chung, P. W. Schiller, R. A. Houghten, Proc. Natl. Acad. Sci. USA 1993, 90, 10811-10815. [48] R. A. Houghten, S. E. Blondelle, J. H. Cuervo, “Development of new antimicrobial agents using a synthetic peptide combinatorial library involving more than 34 million

hexamers” in Innovation and Perspectives in Solid Phase Synthesis - Peptides, Polypeptides and Oligonucleotides (Ed. : R. Epton), Solid Phase Conference Coordination, Oxford, UK, 1992, p. 237. [49] S. E. Blondelle, E. Takahashi, K. T. Dinh, R. A. Houghten, J. Appl. Bacteriof. 1995, 8, 39-46. [50] S. E. Blondelle,

R. A. Houghten, Ann. Rep. Med. Chem. 1992, 27, 159-168. [Sl] R. A. Houghten, K. T. Dinh, D. E. Burcin, S. E. Blondelle, “The systematic development of peptides having potent antimicrobial activity against E. coli through the use of synthetic peptide combinatorial libraries” in Techniques in Protein Chemistry IV (Ed.: R. H. Angeletti), Academic Press, Orlando, 1993, p. 249. [52] S. E. Blondelle, E. Perez-Pay& C. T. Dooley, C. Pinilla, R. A. Houghten, R n d s Anal. Chem. 1995, 14, 83-92. [53] S. E. Blondelle, L. R. Simpkins, R. A. Houghten, “Inhibition of melittin’s hemolytic activity using synthetic peptide combinatorial libraries” in Proceedings of the 22nd European Peptide Symposium (Eds.: C. H.Schneider, A. N. Eberle), Escom, Leiden, 1993, p. 761. [54] S. E. Bondelle, R. A. Houghten, “Membrane protecting sequences and new antimicrobial peptides identified through the screening of synthetic peptide combinatorial libraries” in Rchniques in Protein Chemistry V (Ed. : J. W. Crabb), Academic Press, Orlando, 1994, p. 509. [55] M. Jr. Laskowski, I. Kato, Ann. Rev. Biochem. 1980, 49, 593-626. [56] C . T. Dool9, N. N. Chung, B. C. Wilkes, P. W. Schiller, J. M. Bidlack, 0.W. Pasternak, R. A. Houghten, Science 1994, 266, 2019-2022. [57] S. E. Blondelle, E. Takahashi, P. A. Weber, R. A. Houghten, Antimicrob. Agents ChemotheE 1994, 38, 2280-2286. [58] C. T. Dooley, S. K. Hope, R. A. Houghten, “Identification of tetrameric opioid peptides from a combinatorial library composed of L-, D-and non-proteinogenic amino acids” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed.: H. L. S. Maia), Escom, Leiden, 1995, p. 805-806. 1591 L. H. Lazarus, S. Salvadori, V. Santagada, R. Tomatis, W. E. Wilson, J. Med. Chem. 1991, 34, 1350-1355. I601 S. Charpentier, S.Sagan, A. Delfour, P. Nicolas, Biochem. Biophys. Res. Commun. 1991, 179, 1161-1168. [61] R. A. Houghten, Gene 1993, 137, 7-11. [62] C. Pinilla, J. R. Appel, S. E. Blondelle, C. T. Dooley, J. Eichler, J. M. Ostresh, R. A. Houghten, Drug. Dev. Res. 1994, 33, 133-145. [63] R. W. Barrett, S. E. Cwirla, M. S. Ackerman, A. M. Olson, E. A. Peters, W. J. Dower, Anal. Biochem. 1992,204, 357-364. [64a] C. Pinilla, J. R. Appel, D. Milich, R. A. Houghten, “Identification of an antigenic determinant for the surface antigen of hepatitis B virus using peptide libraries” in Peptides: Chemistrx Structure and Biology, Proceedings of the 13th American Peptide Symposium (Eds.: R. S. Hodges, J. A. Smith), ESCOM, Leiden, 1994, p. 1016.

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[64b] C. Pinilla, J. Buencamino, R. A. Houghten, J. R. Appel, “Detailed studies of antibody

specificity using synthetic peptide combinatorial libraries”, in Vaccines 95: Molecular Approaches to the Control of Infectious Dkeases (Eds.: E Brown, R. M. Chanock, H. S. Ginsberg, R. A. Lerner), Cold Spring Harbor Laboratory Press, New York, 1995, pp. 1-6. [65] S. E. Blondelle, L. R. Simpkins, R. A. Houghten, “Inhibitors of cytolytic compounds identified using synthetic peptide libraries” in Innovation and Perspectives in Solid Phase Synthesk, Peptides, Proteins and Nucleic Acids, (Ed.: R. Epton), Mayflower Worldwide Ltd., Birmingham, U.K. 1994, p. 163- 168. 1661 J. R. Appel, J. Buencamino, R. A. Houghten, C. Pinilla, “Study of peptide-antibody interactions using a decapeptide positional scanning library” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed.: H. L. S. Maia), Escom, Leiden, 1995, p. 815-816. 1671 J. M. Ostresh. G. M. Husar, S. E. Blondelle, B. Dorner, P. A. Weber, R. A. Houghten, Proc. Natl. Acad. Sci. USA 1994, 91, 11138-11142. (681 R. A. Houghten, J. M. Ostresh, G. M.Husar, B. Dorner, S. E. Blondelle, “Libraries from libraries: the generation of peptidomimetic combinatorial diversities” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed.: H. L. S. Maia), Escom, Leiden, 1995, p. 459-460. [69] B. Dtirner, J. M. Ostresh, G. M. Husar, R. A. Houghten, “Extending the range of molecular diversity through amide alkylation of peptide libraries” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed. : H.L. S. Maia). Escom, Leiden, 1995, p. 463-464. [70] J. H. Cuervo, E Weitl, J. M. Ostresh, V. T. Hamashin, A. L. Hannah, R. A. Houghten, Polyalkylamine chemical combinatorial libraries in Peptides 94: Proceedings of the 23ml European Peptide Symposium (Ed.: H. L. S. Maia), Escom, Leiden, 1995, p. 465-466. [711 J. Eichler, A. W. Lucka, R. A. Houghen, “Cyclic peptide template synthetic combinatorial libraries” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed. : H. L. S. Maia), Escom, Leiden, 1995, p. 461-462. [72] S. E. Blondelle, E. Takahashi, R. A. Houghten, E. P&ez-Paya, Biochemical J. 1996,313, ’

141- 147. [73] C. Pinilla, J. Buencamino, R. A. Houghten, J.

R. Appel, “Detailed studies of antibody specificity using synthetic peptide combinatorial libraries” in Peptides 94: Proceedings of the 23rd European Peptide Symposium (Ed.: H. L. S. Maia), Escom, Leiden, 1995, p. 113-114. 1741 C. T. Doolev. S. H o w R. H. Houghten. Remlatorv Rwtides 1994. 54. 87-88.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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

6.1 Introduction Combinatorial peptide library methods not only offer great potential for facilitating the drug discovery process but also provide a powerful tool for basic research of various disciplines. Currently, there are four general approaches for preparing and screening huge random combinatorial peptide libraries: (i) the biologic peptide library method using filamentous phage (1-41, plasmids [5], or polysomes (61, (ii) the combinatorial library method with iterative processes on polyethylene pins (71, in solution (8-111, and on paper (“spot synthesis”) (12, 131, or libraries utilizing the “positional scanning” principle [14, 151, (iii) the combinatorial library method based on the “one-bead-one-compound’’ concept, or the Selectide process [16-301, and (iv) the combinatorial library method using affinity chromatography selection techniques (31, 321. In this chapter, we shall focus on the development and application of the Selectide process. This method uses the solid phase “split synthesis” method [8, 16, 331, to generate random peptide libraries such that each individual bead displays only one peptide entity [16] (see Fig. 6-1). The random library of millions of beads is then screened in parallel for a specific acceptor molecule (receptor, antibody, enzyme, virus, etc.). The beads that interact with the acceptor molecule are then isolated, and the structure of the peptide determined by microsequencing. Much of our earlier efforts concerned the development of peptide libraries using the “on-bead binding assay” screening method and the use of automatic Edman degradation by microsequencer as the sole method for structure determination. Over the last three to four years, several important developments have been made in the Selectide process. We are now able to synthesize chemical libraries with releasable linkers such that ligands are released into solution for screening with any existing solution-phase assay (34). Unnatural amino acids have been incorporated into our peptide libraries and specific ligands were isolated [35, 36). In addition to linear and constrained peptide libraries, combinatiorial nonpeptide libraries with small organic molecules as subunits have been synthesized and screened for the identification of nonpeptide ligands [37-431. A peptide coding scheme has been developed by us (441, as well as by others [26, 27, 45-48] for the nonpeptide libraries. Peptide and nonpeptide scaffoldings have been designed and

114

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

(a)

-@

Coupling Step

I

A A-@

n (9 dipeptides)

A AA-@ A G-@ AV*

m

G

V

G-@ (randomize. split) G

V-@

V

GA* G G-@ GV-@ (randomize. split)

VG-@ V G-@ V V-@

I

A

G

V

(27 tripeptides)

A A A -@ AAG-@ AAV-@

G A A -@ GAG-@ GAV-@

VAA-@ VAGVAV-@

AGA-@ A G G ~ AGV-@

GGAGGG-@ GGV-@

VGA-@ VGGV G V a

AVAAVO-@ AVV-@

GVA-@ GVG-@ GVV-@

VVA-@ VVG-

vvv-@

Figure 6-1. Scheme of split synthesis illustrated by an example of tripeptide library in which three amino acids were randomized in each step (from [la]).

built for the synthesis and screening of libraries [37-39, 41, 491. Strategies for the synthesis of resin beads with differential protecting groups on the surface and inside of the beads have also been developed [50]. Structure determination of small nonpeptide ligands by mass spectroscopy [a] and digital coding have been evaluated. The concept of “library of libraries” has been applied and proven on model targets (51, 521. More recently, using a novel screening method where the peptide library was radiolabeled with [y3*P]ATPand protein kinase, substrate motif for these important enzymes were determined [53,54]. We have been successful in identifving short linear DeDtideS (both D- and bamino acids) that interacted simifkantlv

6.2 The Basic Concept of “One-Bead-One-Compound”

175

with a small organic dye, indigo carmine (551. A fully automated random peptide library synthesizer has been built and used routinely in the synthesis of our standard peptide libraries [56]. We have also applied a dual color screening method to eliminate false positive beads resulting in the more efficient use of the microsequencer [57]. In this chapter, the concept, the basic method, as well as some of these newer developments will be explored. In addition, examples of how to apply Selectide process to various disciplines will be given. Technical details concerning the Selectide process have been recently reviewed (58, 591.

6.2 The Basic Concept of “One-Bead-One-Compound” Furka et al. [33, 60,61) first described the “split synthesis method” to overcome the differential coupling rates of various activated amino acids in the synthesis of peptide mixtures. The resin was first divided into several aliquots, and different activated amino acids were added into each resin aliquot. After coupling, the resin was mixed, washed, deprotected, and split into several aliquots again. The same process was repeated a few more times. The side chain protecting groups were then removed, the linker cleaved, and an equimolar mixture of solution peptides was ready for testing. The “split synthesis method” was conceived independently later by us and others (8, 161. We recognized that this synthetic method not only produced equimolar mixture of peptides but more importantly, since each resin bead encountered only one single activated amino acid at each coupling cycle and the coupling was driven to completion, each bead would have expressed only one peptide-entity [16, 171. By using a noncleavable linker, the deprotected peptide remained bead bound. We also noted that each 100 pm bead contained approximately 100 pmole of peptide, which was plenty of material for microsequencing. Using an enzyme-linked colorimetric assay similar to Western blot, we were able to identify beads that interacted with an anti-8-endorphin antibody and streptavidin. The color beads were then physically isolated for microsequencing [la]. Since synthetic chemistry is used in the synthesis of our libraries, in addition to eukaryotic amino acids, we may also use unnatural amino acids (35, 361 as well as small organic subunits resulting in a nonpeptide libmy [37, 40-431. This “one-bead-one-compound” concept offers numerous potential applications. Besides identifying binding ligands for acceptor molecules, or compounds that elicit a biological response, we can potentially screen for compounds with specific physical, chemical, photochemical, or electromagnetic properties. In addition, we may also screen for compounds with catalytic activity.

176

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

6.3 Synthesis of Random Peptide Library Details on the synthesis of a random peptide library have been described elsewhere [59].Standard Fmoc chemistry and split synthesis method (8, 16, 331 were used. The resin has to be compatible with solid-phase peptide synthetic methods (i.e., stable to base, trifluoroacetic acid, and organic solvents) used during the synthesis. In addition, it has to be compatible with the aqueous condition used in the screening process. The commercially available TentaGel S (polystyrene grafted with polyoxyethylene, Rapp Polymere, Germany) [62] or polydimethylacrylamide resin (Pepsyn Gel) first described by Sheppard’s group [63] have been found to be satisfactory. The resin (0.2-0.4 meq/g) is first divided into several aliquots, and a fourfold excess of specific Fmoc-amino acids are added into each aliquot of resin. The coupling reaction is initiated with the addition of either diisopropylcarbodiimide (DIC) or benzotriazol-l-yl-oxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate (BOP) and N-hydroxybenzotriazole (HOBt) with gentle mixing. In the case of DIC coupling, coupling reaction can be monitored by incorporating a trace of bromophenol blue into the reaction mixture (641, and the completion of the reaction is confirmed by the ninhydrin test [65]. On rare occasions, double or even triple coupling is needed to ensure completion of each coupling cycle. If couplings are not complete, deletion sequences will appear. After each coupling cycle, all the resin aliquots are mixed, washed, Nu-Fmoc group deprotected, the resin is washed, and divided into several aliquots for the next cycle of synthesis. The same process is repeated several times until the desired length of the library is achieved. The Nu-Fmoc group is deprotected by 20% piperidine in dimethylformamide (DMF) (v/v) and side chain protecting groups are removed by a mixture of trifluoroacetic acid/p-cresole/water/ thioanisole/ethanedithiol (82.5 :5 :5 :5 :2.5) [66].

6.4 Screening with an “On-Bead Binding Assay” The on-bead binding assay allows one to rapidly identify peptide beads that interact with an acceptor molecule. The acceptor needs to be tagged directly either with an enzyme [16,67], a fluorescent probe 1261, a radionuclide [24], or a chromophore [19]. Alternatively, a secondary reagent with the above tags can be used. For example, the primary acceptor molecule may be biotinylated, and the secondary reagent may then be streptavidin-alkaline phosphatase conjugate. The fully deprotected bead library is first washed extensively with double distilled water, then twice with phosphate buffer saline (PBS, 8 mM Na2HP04, 1.5 mM KH2P04, 137 mM NaC1, and 2.7 m~ KCl, pH 7.2) containing 0.1% gelatin (w/v) and 0.1 070 ’keen 20 (v/v). Other blockings agents such as bovine serum albumin or other nonionic detergents may also be used. The 0.28 M salt in the 2 x PBS prevents

6.5 Screening with a ‘Releasable Assay”

177

some of the nonspecific ionic interaction. The library is then incubated in the same buffer with, for example, the biotinylated acceptor molecule. After at least one hour, the library is washed and streptavidin-alkaline phosphatase is added. After incubating for one hour, the library is washed extensively, mixed with 5-bromo4-chloro-3-indolyl phosphate (BCIP), and plated in a dozen of polystyrene petri dishes (100 x 20 mm). Usually within one hour, the positive beads change to turquoise in color. The turquoise beads are then isolated for microsequencing. To eliminate false positive beads, the positive beads are decolorized with DMF and restainded in the presence of specific ligand. Alternatively, a dual color screening system may also be used [17, 571.

6.5 Screening with a “Releasable Assay” The “on-bead binding assay” is extremely rapid, and up to lo* beads can be screened readily. However, with this assay, one is not able to screen for biological activities where the soluble receptor is unavailable or when the assay requires that the ligand be in solution in order to elicit the biological response. This is true for many standard drug screening assays available in the pharmaceutical industry. To develop a solution phase screen and maintain the “one-bead-one-compound concept”, we modified the library and applied a dual releasable linker technology in a two-stage release and screening process (33, 58, 68, 691. Screening in solution requires equimolar amounts of test compound be released at each step. We selected two release methods which are independent and can be performed in distinct steps, intramolecular cyclization and hydrolysis, for construction of releasable libraries. Linkers utilizing intramolecular cyclization [70-721 have the disadvantage of leaving the diketopiperazine structure attached to the released peptide molecule. We have overcome this limitation by redesigning the cleavable linker [68], and later we developed a more convenient and substantially less expensive two-stage releasable linker [69] composed of the dipeptide motif Ida-Ida (Ida = iminodiacetic acid), which is particularly suitable for the construction of double cleavable linkers. The structure of Ida-Ida dipeptide-based linkers and the mechanisms of the staged release are shown in Fig. 6-2. For the screening, approximately 500 beads are first pipetted into each well of the 96-well filtration plate (Millipore). The double cleavable linker is first cleaved with the addition of neutral buffer into each well. After at least 4 hours, the peptide supernatants are suctioned into the 96-well receiving plate below. A solution-phase biologic assay is then performed, and the positive wells are identified. The amount of ligands released in each stage depends on the size as well as loading of each individual bead. From larger beads (e. g., 220 pm) up to 5 nmol can be released in each stage. This is equivalent to 50 NM when released into a volume of 100 ul. In certain

178

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

pon

Figure 6-2. Scheme of double release from iminodiacetic acid-based doubly cleavable linker. Reagents: a, trifluoroacetic acid; b, aqueous buffer pH 8.5; c, 0.1 To NaOH or gaseous ammonia.

specific applications, beads can also be immobilized on polyethylene sheets and/or agar [17, 231 and the ligand released for in situ biological assay.

6.6 Libraries of Organic Molecules Construction and screening of peptide and peptide-like libraries limit the structural search to a very limited conformational space. Expansion of this space is possible by using libraries of nonpeptide, or low molecular weight organic compounds. The potential for small organic libraries is enormous. Thousands of building blocks are readily available, eliminating the limitations imposed by the 50 or so commercially available amino acids. For instance, a small organic trimer with 100 possible subunits in each coupling step will generate a complexity of lo6 and the synthesis of such libraries is feasible using current synthetic methods. Ligands isolated from such libraries are more likely to be able to cross cellular membranes, resulting in a lead with characteristics closer to that of a therapeutic agent. An example of a ligand identified in a simple nonpeptide library constructed using subunits selected from readily available amino acids, aldehydes, and carboxylic acids is given in Fig. 6-3 (401.We have used alkylation and acylation reactions for the construction of some of these libraries [42, 44). The addition of amines to isocyanates [73], and Wittig reaction followed by Michael addition [21] have also been used by other researchers. ExDlosion of activities in this area resulted in the flood of Dublications. For recent

6.8 Structure Determination of Positive Reaction Compounds

p

179

h e

Figure 6-3.

Streptavidin ligand selected from small nonpeptide library.

reviews, see e.g. [74-761. The dynamic data base of papers in molecular diversity field can be found on Internet (771.

6.7 Scaffold Libraries An alternative to the use of various organic reactions for linking building blocks containing different functional groups, is the application of well-developed techniques for amide bond formation, using large numbers of commercially available mines and carboxylic acids [37-39, 41, 431. These buildings blocks can be attached to a great variety of “scaffolds”, ranging from linear and flexible structures allowing the exploration of a wide conformational space, to cyclic and rigid structures, mapping a smaller space, but potentially providing higher affinity ligands (Fig. 6-4). Figure 6-5 shows structure inhibiting enzymatic activity of thrombin as an example of ligands found in these libraries [78]. Inhibitory activity of the depicted compound was at the level of 4 WM, which is equivalent to that found in the primary peptide libraries.

6.8 Structure Determination of Positive Reaction Compounds Edman degradation is a standard method for the determination of peptide sequences. Sequencing of bead-bound peptides often requires modifications of the standard sequencing protocol. However, even optimized conditions will not allow separation of all amino acid derivatives, and in some cases, alternative methods, such as coding may become necessary. An average degradation cycle takes up to 40 minutes, making structure elucidation the rate limiting step in the screening process. However, not all positive compounds need to be sequenced individually. It may be advantageous to sequence multiple positive beads simultaneously to obtain an initial idea about structure-activity relationships [79] and to speed up the process signifi-

180

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

--.-

d

0

r

2

L

-E

a 0

f

a?

I

z

I

Figure 6-4. Structures of various "scaffold" libraries.

I

6.8 Structure Determination of Positive Reaction Compounds

0

181

0

Figure 6-5. Thrombin inhibitor identified in scaffold library based on Kemp’s triacid.

cantly. Figure 6-6 shows as an example the result of multiple sequencing of anti-&endorphin antibody ligands. The motif YG-F, is clearly visible. For peptide and nonpeptide molecules other than oligonucleotides, the only viable alternative for direct structure elucidation is mass spectroscopy. This method can use the exchange of labile hydrogenes in the peptide molecule to significantly improve the speed and precision of structure determination [80]. Diminishing the number of possible compositions allows the application of an algorithm beginning from the composition of a full-length peptide [U] rather than from small fragments (see, e.g., [74]). Knowledge of fragmentation pathways is important to the success of mass 6000 i

Y FSWG

Step 1

GQI E Step 2

GAYFVWI P D F Q Step 3

FLWPGAIYO Step 4

F I Y LQTAGVHSMENWDKRP Step 5

Figure 6-6. Result of multiple sequencing of 99 beads which expresses binding affinity to anti8-endorphin antibodies (from 1711).

182

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

spectroscopy in structure elucidation; however, even without this knowledge it has been possible to determine structure of hits form a small nonpeptidic library [40].

6.9 Coding Three main reasons for the introduction of coding techniques are: (i) structure identification of nonsequencable compounds; (ii) improvement of sensitivity; and (iii) increase of the speed and throughput of compound identification. The idea of encoding library compounds was derived from biological systems in which the composition of peptides/proteins is encoded by DNA. The phage technology, in which the structure of the peptide displayed on the phage surface is decoded by sequencing the phage DNA, takes advantage of this biological coding [I -41. Coding of peptide libraries by nucleic acids was suggested by Brenner and Lerner [45, 461, and further developed by others [26]. Recently, coding by halogenated molecules followed by gas chromatographical analysis was published [27, 471. This technique employs a “digital” code comprising separate sets of coding structures for each position of the library, with coding subunits within each set defining the identity of the specific compound subunit. A single step of analysis is needed to identify the test compound. A similar technique for structural elucidation was described using mass spectroscopy (28,811. In these cases, in each step of synthesis, a fraction of available amino groups (in the case of a peptide) is capped, and the “history” of synthesis is documented. Once a positive bead is isolated, the test compound is cleaved together with all capped intermediates. The resulting mixture is then injected into a mass spectrometer and the sequence of the test compound can be identified by reading the mass differences of individual molecules in the spectrum. Methods using peptide molecules for coding were first described for the structure determination of test compounds identified from peptide libraries with unnatural amino acids [17] and nonpeptide chemical libraries or small organic libraries [44]. In these approaches, each step of building the nonsequencable structure is accompanied by the attachment of one or several amino acids onto an independent point on the solid-phase particle resulting in a linear coding molecule. This “peptidic tag” can then be sequenced by the application of standard techniques (Edman degradation or mass spectroscopy), and the structure of the nonpeptide molecule recalled. Alternatively, the peptide tag can be built in such a way that it can be cleaved in one step of Edman degradation, followed by HPLC analysis as a “bar code” [78). Figure 6-7 illustrates the structure determination of a compound from the library with four randomizations coded by doublets of amino acid derivatives. N amino acids used as a doublet can code for a total of N x (N-1)/2 structures (e.g., 10 amino acids can code for 45 building blocks). To have more amino acids for coding. we have DreDared the set of side chain acvlated diaminocarboxvlic acids

6.10 Possible Interaction of Target Macromolecule with Coding Structure

183

(Propionyl + Eulyryl)

1

H-L”T codiq molecule

n

0.0 59

(H-Lys(CI2) + H.Glu)-Lyy7

4

Pr)

(H-lle + H-Val)-Lys-

1

(H-Sar + H-Asp(OBzl))-Lys-Polymer

Asp(0BzI)

-I -I

Figure 6-7. HPLC trace of bar code. Note that building block F is coded by a different amino acid doublet in position 1 (Lys(propiony1) Lys(Pr) and Lys(butyry1) Lys(Bu)) and 4 (Sar and Asp(OBz1)).

(Dap, Dab, Om, Lys). The HPLC retention times of these acid derivatives (in the form of phenylisothiohydantoins)do not overlap with those of natural amino acid derivatives.

6.10 E limination of Possible Interaction of Target II [acromolecule with Coding Structure: Bead Shaving Coding structure in on-bead screening may interfere with the interaction between the target molecule and the test compound. This problem can be addressed in at least four ways. One approach is to resynthesize both code and test structures from positive beads independently and determine their activity, Another is to use only a very small fraction of the material on the bead as the coding (- 1 to 5 Vo), or use a mixture of molecules for coding. This mixture, however, must provide unambiguous sequencing data. The fourth approach is to arrange the coding and screening structures in such a way that only the test compound is available for interaction with the target molecule. The interior of polyoxyethylene grafted polystyrene (TentaGel) beads is not available to macromolecular targets owing to the microporous structure of this polymer. This carrier can first be modified with a protected peptide sequence, which is then cleaved by a reagent that has access to the “surface” of the solid phase

184

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

support only, thus exposing a free amino group on the surface. This free amina group is subsequently protected with an orthogonally cleavable protecting group. In this way it is possible to synthesize the test compound exclusively on the bead surface and the coding structure exclusively in the bead interior. We have used enzymatic “shaving” to produce this selective surface modification, using chymotrypsin as the shaving agent and the sequence Boc-Ala-Gly-Val-Phe-Gly-PAla-Gly-TGas the substrate [50].

6.11 Is It Necessary To Have Full Representation in a Selectide Library? Assuming the resins are distributed equally in each coupling step during the “split synthesis”, the statistical distribution of individual species is based on Poisson distribution and the number of possible species actually synthesized is determined by the total number of beads present in the synthesis. For example, approximately 95 070 of all possible permutations will be present in a library if the number of beads used in the synthesis and screening equals three times the number of possible permutations. From our experience with peptide library screening, most of the biologic targets that we have studied have three to five contact residues necessary for specific molecular interaction. These contact residues can be separated by a variable number of non-essential residues. Because of this degeneracy, for most biological targets, a full representation of every species in a library is not needed for the determination of the general binding motif. For a motif with five contact residues, one would need a library of approximately lo7beads to ensure that 95 070 of all possible motifs (consisting of 20 eukaryotic amino acids) are included. For practical reasons, the upper limit for the synthesis and screening of an 80 pm bead library is probably los beads (approximately 30 g of resin). For routine screening, a lo7 bead library can easily be handled by one person in a day. However, this does not imply that the “best” ligand can always be isolated from a limited library. Except in simple cases where the high affinity ligand is a short linear epitope (e.g., opiate receptor ligands, anti-P-endorphin monoclonal antibody epitope, YG-F),often the binding affinity of the initial leads isolated from a primary library screen, particularly for a ligand with a “discontinuous epitope”, is relatively weak ( > I NM). To improve the binding affinity, one may use a sequential screening approach by synthesizing and screening a secondary library based on the primary motif. This can be repeated in several additional cycles, and perhaps with branching structures, until a higher affinity ligand is isolated. This sequential screening approach has been applied successfully to isolate high affinity ligands for an anti-insulin monoclonal antibody that recognizes a discontinuous eDitoDe C361.

6.13 The Seleclide Process Versus Other Combinatorial Library Methodologies

185

6.12 One-Bead-One-Motif Libraries (“Libraries of Libraries”) The number of expected positive beads in the library depends on the number of “critical residues” (or contact residues) in the peptide sequence, or critical pharmacophores in a nonpeptide structure, i.e., residues required for minimal observable binding. This number can be calculated according to the formula: n = (X)(Pf) [S/(A,)”C“‘] In this equation n is the number of expected positive hits, x i s the number of different motifs which exhibit binding, P f is the “placement” factor, i.e., number of possible placements of each motif in the peptide chain, S is the number of beads screened, A, is the number of amino acids (subunits) used for randomization, and ncri,is the number of critical residues. The number of positive hits depends on the number of beads tested, but it does not depend on the length of the library. Therefore, even a very incomplete library, e.g., a library of decapeptides, can provide a reasonable number of positive beads if only three or four residues in the sought-after peptide are critical for binding under the screening conditions used. The individual sequence identified in the peptide library with incomplete representation of all permutations is less valuable than information describing the motif responsible for binding or other biological activity. We have designed a library which addresses this question [51, 521. This library contains all possible motifs (e.g., all tripeptide motifs in a hexapeptide library) in the framework of a library of given length. At the same time, each individual bead contains a multiple of peptides (each with the same motif). In this arrangement all tripeptide motifs in up to a l5meric peptide can be synthesized and screened in a manageable library size (see Table 6-1). Synthesis of this library follows a simple scheme, given in Fig. 6-8. This library combines the principles of iterative library techniques [7-9, 831 with those of the onebead-one-compound approach; however, it results in the screening of all possible motifs at the same time, rather than in consecutive dipeptide motifs - which may be too short [84] or not appropriately localized in the peptide chain to identify the ligands. Each bead of the library of libraries represents a unique entity the structure of which is determined only after its biological relevance is established.

6.13 The Selectide Process Versus Other Combinatorial Library Methodologies As mentioned earlier, the four general combinatorial library methods are (i) biological libraries, (ii) iterative libraries, and (iii) one-bead-one-compound libraries, and (iv) affinity chromatography selection libraries. All these four methods have

186

6 Combinatorial Library Based on the One-Bead-One-CompoundConcept

-

Table 6-1. Numbers of beads (and weight of resin 130 wm bead size) required for testing full representation of peptides of various lenght using (a) one-bead-one-peptide approach and (b) library of libraries approach with tripeptide motif

Length

One bead-one peptide (a) Number of beads

3 4 5 6 7 8 9 10 11 12 13 14 15

8000 160000 3 200000 64000000 1280000000 25600000000 512000000000 10240000000000 204800000000000 40%000000000000 81920000000000000 1638000000000000000 32770000000000000000

Resin amount 8 mg 160 mg 3.2 g @g 1.28 kg 25.6 kg 512 kg 10.2 t 204t

Library of libraries (b) Nuber of beads Resin amount 8000 32000 80000 160000 280000

672000

8 mg 32 mg 80 mg 160 mg 280 mg 448 mg 672 mg

1320000 1760000 2288000 2912000 3640000

1.76 g 2.29 g 2.91 g 3.64 g

448000 960000

960 mg 1.32 g

both advantages and disadvantages. Comparisons between the four methods are summarized in 'Thble 6-2. The main advantage of the biological libraries is that long peptides can be produced with this method, thus allowing one to incorporate big scaffolds such as immunoglobulin folds [85] or scorpion toxin folds into the library. On the other hand, only relatively short peptides (e.g., up to 20-mers) can be produced by the synthetic methods. Another possible advantage of the biological library is that perhaps up to 109 molecules can easily be produced by the biological method. However, because it is a biological system, only the 20 bamino acids can be incorporated into the library. In contrast, the three latter synthetic methods allow one to incorporate tamino acids, D-amino acids, unnatural amino acids, small organic nonpeptide moieties, carbohydrates, lipids, nucleic acids, various secondary structures and synthetic scaffolds into the libraries. The major difference between the iterative process and the Selectide process is that the latter is addressable where each chemical species is physically separated, and the structure of the ligand is determined by chemical analysis of individual positive beads. In other words, similar to the biological library, the Selectide process uses a parallel approach where all the chemical species are screened concurrently in one step. The corollary is that all the multiple independent motifs present in the library can be discovered with one screen. This is particularly important for receptors that recognize a discontinuousepitope as multiple motifs are likely to be discovered. This will be illustrated by the anti-insulin monoclonal antibody system in the later section.

6.13 The Selecride Process Yersus Other Combinatorial Libmry Methodologies

Portioning

187

314

.. . Figure 6-8. Scheme of the synthesis of “library of libraries” with a tripeptide motif in a hexapeptide frame. The polymeric carrier is split in the ratio nR,:,n (numbers located on the lines connecting circles denote the fraction of carrier undergoing the specified operation) (nRI= number of remaining randomization steps; ,n = number of remaining steps in which a mixture of amino acids is to be coupled) based on the status nR/nM (number located in the shaded circles) (nR= number of randomizations performed on the carrier; nM= number mixtures to be coupled). At the beginning of the synthesis nR/nM = O/O, at the end nR/nM= 3/3. The lines pointing up designate a randomization step, the lies pointing down designate a mixture step. This scheme can be applied to any length of library and motif.

The iterative process, on the other hand, is a convergent approach, in which mixtures of compounds are synthesized and tested either on solid-phase or in solution in a multistep fashion. The result of the biological assay will determine the next synthetic step. This is straightforward if there is only one clearcut motif. However, if there were multiple motifs of similar activity, one has to decide which path to take for each of the subsequent steps. Even though remarkable results have been obtained with this method (see e.g., 111, 84, 86, 87]), this convergent approach may result in the possible omission of otherwise very important motifs. Since the standard iterative approach requires multiple synthetic and analysis steps which are time-consuming, a rapid positional scanning method has been developed [14, 151. In this method, each amino acid position is f i i d while the rest of the positions are randomized. With these standard mixtures (e.g., 120 mixtures for a hexapeptide), one can rapidly determine the binding motif. This method is very efficient for receptors with only one dominant linear binding motif. However, for receptors with multiple binding motifs, the results will be very complicated and likely uninterpretable.

188

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

Table 6-2. Comparison of combinatorial library methods

Biological

Iterative process

Selectide process

Affinity selection

109

106- 108

106- 108

106- 108

No

Yes

Yes

Yes

Small

Small

Small

Yes

Yes

Yes

Binding assay

Small to very large Simple disulfide or large scaffolds of proteins Yes

Yes

Yes

Solution assay Approach Structure determination

No Parallel Addressable, DNA sequencing

Yes (pin and spot) Yes Convergent Based on multistep synthetic and assay algorithm

Solution motif Biological bias

Multiple Yes

Often one No

Yes Parallel Addressable, Edman degradation, encoding mass spectroscopy Multiple No

No Parallel Summation of many possible solutions, Edman degradation Often one No

Number of species Unnatural amino acids or small organics Size of ligand Constrain structures

Although a solution-phase assay is preferred in many biologic assays, it also poses some important limitations. For instance, a signifcant number of ligands, particularly from the non-peptide libraries, may be insoluble or only very sparingly soluble, and their identification will be missed if a solution-phase assay is used. In this instance, an on-bead screening assay where the relatively insoluble ligand is coupled to a highly hydrophilic linker will be preferred. Often a solution-phase binding assay requires a radiolabeled-ligand as a tracer. In the case where no known ligand is available, the on-bead binding assay is preferred. Additionally, if a known ligand is available, the dual-color on-bead binding assay allows one to identify binders that bind to the ligand binding pocket or those that bind to the receptor surface outside the ligand binding pocket. As mentioned above, all the four combinatorial library methods have advantages and disadvantages. The choice of which method to use will depend on specific applications, expertise, and resources available in individual laboratories. Sometimes it may be advantageous to combine some of these methods together as they are comolementarv to each other.

6.14 Examples of Application

189

6.14 Examples of Application 6.14.1 Anti-P-Endorphin Monoclonal Antibody Using an on-bead binding assay with an anti-/?-endorphin monoclonal antibody (clone 3E7) that recognizes an N-terminal continuous epitope (YGGF),we were able to idenify ligands of various affinities from an all L-amino acid library [16, 34, 881. The general motif YG-F/W is readily identified. We have also screened several Damino acid containing libraries with the same antibody. The results are shown in Table 6-3. The “on-bead binding” screening process is rapid and usually takes less than a day. The rate limiting step is often the microsequencing. To speed up the structure determination step, we have pooled the positive beads (e.g., 30 beads) of some of our screen and subjected them to concurrent sequencing. The result is shown in Fig. 6-8, which demonstrates that with simple motifs and with limited critical residues that are relatively “in-frame”, the multiple sequencing method works very well. This is quite analogous to the positional scanning method described by Houghten et al. [14, IS]. However, if multiple motifs were significantly “off-frame”, both multiple sequencing or positional scanning methods will likely produce uninterpretable data. The multiple sequencing method not only works for short motifs but also for longer ligands with few “in-frame” critical residues. For example, anchor residues for the 9-mer of the MHC-Class I molecule binding peptides (see below) can readily be identified by multiple sequencing [89]. Table 6-3. Peptide ligands that interact specifically with anti-8-endorphin monoclonal antibody (clone 3-E7) ~~~~

Library

xxxxx

XXXXX

xxxxx XMXX

X = all

~

~

Ligands isolated YGGFA, YGGFT, YGGFQ, YGGFI, YGGFL YGGLS, YGGMV YGAFF, YGAFM, YGAFQ, YGAFT, YGALQ, YGALT, YGAWD YGNFF, YGVFA, YGVFI, YGQFV, YGVFE, YGVFQ YGFFQ, YGWFN, YGWWM, YGYWQ, YGWFH, YGWFQ, YGYWQ YGEAF, YGHAF, YGMGF, YGGGF, YGLGF, YGPGF YKGGF, YQGGF, YLGGF LYGGF, NYGGF, MYGGF, RYGLL YGGfM, YGAfW, YGAff YqGGF, YGAaF YGFGL, YGFGF, YGGGF IyGGF, YaNaW, YaQaW yGGFa, yGGFv wtGGy

18 L-amino acids plus glycine, no cysteine. x = all 18 D-amino acids plus glycine, no cysteine.

190

6 Combinatorial Library Based on the One-Bead-One-Compound Concept

6.14.2 Anti-Insulin Monoclonal Antibody Unlike anti-/?-endorphin monoclonal antibody, the anti-insulin monoclonal antibody recognizes a discontinuous epitope on an intact porcine or human insulin molecule. Multiple libraries have been screened systematically with this antibody. The results are summarized in Table 6-4. Immediately apparent is that multiple motifs can bind to this antibody. Importantly, the binding of these motifs to the monoclonal antibody was blocked by porcine or human insulin. When specific structures such as cyclic (disulfide bond) or type I1 /?-turns (D-proline) structures were incorporated into the library, some totally new motifs were detected. It is interesting that, although multiple motifs were identified for the L-amino acid libraries, only one motif was identified for the two a h - a m i n o acid libraries (6-mer and 8-mer). The binding affinities of most of the ligands identified during the primary screen were A sequential screening approach was then applied to one relatively low (> 10 p). of these motifs --W,-GF, identified from the primary screen (Table 6-5). Based on the motif of the primary screen, longer secondary libraries were synthesized and screened under higher stringency. This process was repeated several times until ligands of higer affinity were finally isolated. The binding affinity (1C5,,) of the best ligand identified (SKQDIWGRGF) was 0.3 PM, five fold weaker than that of insulin, the native ligand.

Table 6-4. Peptide motifs indentified from the primary screen on an anti-insulin monoclonal antibody (Clone AE9D6)

6.14 Examples of Application

191

Table 6-5. Optimization by sequential screening with secondary and tertiary libraries Library

Peptide motif

1"

xxxxxx

2" 3"

XXXWXXGF XXXXWKYGF XXQXIWGXGF

-W--GF - - -WKYGF, Q- I WG-GF NH-(G)WKYGF S(R/K)Q(D/A)I WG-GF

6.14.3 MHC-Class I Molecule Using papain-treated MHC-Class I molecules purified from the human lymphoblastoid JY cells, it was possible to rapidly identify the anchor residues for the binding peptides. The MHC-Class I molecule was first dissociated from its P2-microglobulin by 5~ potassium thiocyanate. Upon dialysis in the presence of excess P2-microglobulin and a random nonapeptide library, the MHC-Class I molecule reassociated on the peptide bead with the correct anchor residues. Detection was accomplished by probing with an enzyme-linked antinative MHC-Class I molecule antibody (clone W6/32). The results are shown in Table 6-6 [90]. In one experiment, several beads were pooled and microsequencing was performed on multiple beads (multiple screening) as described earlier. The anchored residues identified for A2 and B7 were determined to be FL/M L/I and -PR L/I/V, respectively. These results corrobrate closely with those reported in the literature [91-931.

______

Table 6-6. Peptide ligands that interact specifically with MHC-Class I molecules HLA-A2.1

HLA-B7

FLWEFPHDL FLWAIMHTE FLLPSFAPD FLWTLEGDV FMLGYDFYI FMLDWFPTI MMQDIDFY L MLWEGFTYI LLYDWDFGL

TPRFLNSPI APRVVQMPL LPRNVTFAV APRGGMY HV KPRGFVPMN RPRPVSHMW RPRGAYGDL LPFKRGGSL IPMGRRGGI

_____

192

6 Combinatorial Libmry Based on the One-Bead-One-Compound Concept

6.14.4 Releasable Assay Screening System As mentioned earlier, the releasable assay screening system, although significantly slower than the on-bead binding screen, offers an important alternative since solution phase assay can be used. The two-stage releasable assay screening method has been applied successfully in identifying ligands for anti-@-endorphinmonoclonal antibody (YGGF) and gpIIb-IIIa (CRGDC) (341.

6.14.5 Posttranslational Modification such as Protein Phosphorylation To determine the phosphorylation site of MP-dependent protein kinase, we have modified our “on-bead binding assay” by actually covalently radiolabeling the positive bead with [y”P]ATP and the enzyme. Random penta- or heptapeptide libraries were first mixed with [y 32P]ATP. The catalytic subunit of the CAMPdependent protein kinase was then added to initiate the phosphorylation reaction. After incubation, the library was washed thoroughly and immobilized on a glass plate with 1.5% low-gelling temperature agarose. The immobilized beads were then exposed to an X-ray film. Beads at the area corresponding to the dark spot on the autoradiograph were isolated, the agar redissolved by heating, diluted with additional agarose, and re-immobilized on the glass plate as described above. The single beads corresponding to the dark spots were then isolated for microsequencing. Figure 6-9 shows the autoradiograph of a primary screen. The result of such a screen

Figure 6-9. Autoradiograph of protein kinase screen.

6.14 Examples of Application

193

is shown in Table 6-7. The motif ,RR,S, identified from such a random screen is exactly identical to that reported in the literature [94]. More recently, we have applied the same method and have identified a novel peptide substrate for p W s K protein tyrosine kinase. We have shown that this peptide, YIYGSFK,is about sevenfold more efficient than the commonly used peptide substrate, cdc2(6-20) peptide, for tyrosine kinase [95]. Currently, work is being done on using this method to determine phosphorylation for other tyrosine kinases, many of which are without any known physiological substrate Table 6-7. Peptide substrate for CAMP dependent protein kinase Library

Peptide

Xxxxx

RRYSV SQRRFST YRRTSLV IIRRKSE

XXXxxxX

6.14.6 Small Organic Dye Molecule as a 'Igrget Using a small organic dye, indigo carmine (MW = 466.56 dalton) as a probe, an all Gamin0 acid heptapeptide and an all D-amino acid octapeptide library were screened. The beads that absorbed the dye and turned blue were isolated and microsequenced. The sequences obtained are summarized in Table 6-8 (551. All the sequences (both from the all G or all D-libraries) have the following motif: X(K/R)OOO(K/R)X where 0 = I, L,V, Y,F, or M,the relatively more hydrophobic amino acid. A series of three of these amino acids were flanked by two positively charged amino acids. This is not unexpected since indigo carmine, as shown in Fig. 6-10, is planar and has one sulfonic acid group at each end of the molecule. This simple experiment proves that small peptides that interact specifically with a small organic molecule can be isolated readily with this method. a b l e 6-8. Peptide ligands that interact specifically with indigo carmine Libraries

Peptide

XxMxXx

YKVVYKL, LTKLVLK, VTKIIFK klilkf, wlikmk ikivyrfr, akwkwvyr ykwyris, vkkmvikf

XXXXXX XXXXXXXX

194

6 Combinalorial Library Based on ihe One-Bead-One-Compound Concept

Figure 6-10. Chemical structure of indigo carmine.

6.14.7 Screening of Library of Libraries The library of tripeptide motifs in the framework of a linear hexapeptide composed of 19 natural amino acids (Cys excluded) was screened against anti-j3-endorphin and streptavidin. Peptides with the expected motifs YG-F and HPQ were identified as evidenced in Table 6-9 [51-521. The illustrated results were obtained using only a small sample of the library (300000 beads) for screening - demonstrating substantial savings in the consumption of reagents needed for both synthesis and screening, compared with a complete library of unique hexapeptides which would be represented on 64 million beads. Table 6-9. Ligands for streptavidin and anti-/3-endorphin antibody identified in library of libraries Streptavidin

Anti-&endorphin

XXXHPQ XXXHPM XXHPQX XWXHPX WXXHPX WXXXPQ

YGXFXX YXGFXX YGGXXX YGAXXX XGAFXX XGGFXX

6.15 Perspective Using a combinatorial library method based on the “one-bead-one-structure” concept and with an appropriate detection scheme, potentially one can isolate and identify a pure bead-bound compound with the desired physical, chemical, electrochemical, photochemical, biological, or enzymatic properties. Thus far, our efforts have been focused primarily on the discovery of compounds that bind specifically to various macromolecular targets such as antibodies, enzymes, MHC-Class I molecules, viral proteins and biologic receptors. The development of nonpeptide or small organic combinatorial libraries is particularly exciting as this will allow us to explore further diversity and at the same time discover drug leads that are smaller and more likely to be permeable to the cell membrane. There has been enormous in-

References

195

terest from both the pharmaceutical industry as well as academia in this area. Undoubtedly within the next five years, new coupling chemistries suitable for the library format will be developed. Since combinatorial library methods often generate a vast amount of data from each screen, it is particularly powerful when coupled with modern computational chemistry. We are currently beginning to explore the possible application of the Selectide process to other areas as well. The identification of small peptides that bind specifically to an organic dye [55] or the studies of ligands to artificial receptors (19, 961 are milestones, as they indicate that we should be able to identify specifc binding pairs for the design of self-assembled supramolecular structures. Additionally, one may also apply this method to identify efficient binders for specific toxic waste. As mentioned earlier, the technology also offers great potential for the design of novel catalyst or “artifical enzymes”. For example, one can envision that amino acids with cofactors such as FAD, NAD, or CoA can be attached to a side chain and incorporated into a random mono- or bicyclic peptide library with a constrained structure. By doing so, an individual peptide bead with a desired enzymatic activity may be isolated. In the area of material science and electronics, one can envision that with the appropriete detection scheme, new materials with the desired physicochemical or electronic properties can be isolated. For instance, from a combinatorial library of various chromophores strung together, one may be able to isolate a compound with efficient photoelectric properties. Likewise, materials with the desired conductance properties could also be isolated. It is evident from the above discussion that combinatorial library methodologies will allow us to rapidly explore a huge number of compounds concurrently. These have already proven to be extremely powerful tools for drug discovery as well as basic research. Undoubtedly in the next few years, we’ll see more laboratories using these techniques to solve problems across a wide range of disciplines.

Acknowledgments This work is supported by Selectide Corporation and NIH grants CA17094, CA57723, and CA13074. Kit S. Lam is a Scholar of the Leukemia Society of America.

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6 Combinatorial Library Based on the One-Bead-One-Compound Concept

[3] J. K. Scott, G. P. Smith, Searching for peptide ligands with an epitope library. Scienct 1990, 249, 386-390. [4] E Felici, L. Castagnoli, A. Musacchio, R. Japelli, G. Cesareni, Selection of antibod) ligands from a large library of oligopeptides expressed on a multivalent exposition vector. J. MO/.Biol. 1991, 222, 301-310. (51 M. G. Cull, J. E Miller, P. J. Schatz, Screening for receptor ligands using large librarie! of peptides linked to the C terminus of the lac repressor. Proc. Natl. Acad. Sci. USA 1992, 89, 1865-1869. [6] K. Kawasaki, Cell-free synthesis and isolation of novel genes and polypeptides. Interna. tional Patent Application WO 91105058 1991. [7] M. H. Geysen. H. M. Rodda, T. J. Mason, A priori delineation of a peptide whict mimics a discontinuous antigenic determinant. MO/.Immunol. 1986, 23, 709-715. (81 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, J. H. Cuervo, Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature (London) 1991, 354, (7 November), 84-86. [9] R. A. Owens, P. D. Gesellchen, B. J. Houchins, R. D. DiMarchi, The rapid identification of HIV protease inhibitors through the synthesis and screening of defined peptide mix. tures. Biochem. Biophys. Res. Commun. 1991, 181, 402-408. [lo] J. Blake, L. Litzi-Davis, Evaluation of peptide libraries: an iterative strategy to analyze the reactivity of peptide mixtures with antibodies. BioconjugafeChem. 1992,3,510-513. Ill] R. N. Zuckermann, E. J. Martin, D. C. Spellmeyer, G. B. Stauber, K. R. Shoemaker, J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S. C. Banville, E. G. Brown, L. Wang, L. S. Richter, W. H., Moos, Discovery of nanomolar ligands for 7-transmembrane G-protein-coupled receptors from a diverse N-(substitued)glycine peptoide library. J. Med. Chem. 1994, 37, 2678-2685. [12] R. Frank, “Spot-synthesis: an easy and flexible tool to study molecular recognition” in (Ed: R. Epton) Innovation and Perspectives in Solid Phase Synthesis, Vol. 3, Mayflower Worldwide Ltd. Birmingham 1994, pp. 509-512. [13] A. Kramer, R. Volkmer-Engert, R. Malin, U. Reineke, J. Schneider-Mergener, Simultaneous synthesis of peptide libraries on single resin and continuous cellulose mem brane supports: examples for the identification of protein, metal and DNA binding pep tide mixtures. Pept. Ra. 1993, 6, 314-319. [14] C. Pinilla, J. R. Appel, P. Blanc, R. A. Houghten, Rapid identification of high affinity peptide ligands using positional scanning synthetic peptide combinatorial libraries. BioTechniques 1992, 13, 901 -905. [15] C. Pinilla, J. R. Appel, R. A. Houghten, Investigation of antigen-antibody interactions using a soluble, non-support-bound synthetic dacapeptide library composed of four trillion (4 x sequences. Biochem. J. 1994, 301, 847-853. [la] K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, R. J. Knapp, A new type of synthetic peptide library for identifying ligand-binding activity. Nature (London) 1991, 354, (7 November), 82-84. [17] K. S. Lam, S. E.Salmon, V. J. Hruby, E. M. Hersh, Random bio-oligomer library, amethod of synthesis thereof and a method of use thereof. International Patent WO 91117823 1993. [I81 A. Borchardt, W. C. Still, Synthtic receptor for internal residues of a peptide chain. Highly selective binding of (L)X-(L)Pro-(L)X tripeptides. J. Am. Chem. Soc. 1994, 116, 7AA7-7AAR

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release of equimolar amounts of peptides from a polymeric carrier using orthogonal linkage-cleavage chemistry. Int. 1 Pept. Protein Res. 1993, 41, 201-203. 1691 P. Kocis, V. Krchnak, M. Lebl, Symmetrical structure allowing the selective multiple release of a defind quantity of peptide from a single bead of polymeric support. Tetrahedron Lutt. 1993, 34, 7251-7252. [70] N. J. Maeji, A. M. Bray, H. M. Geysen, Multi-pin peptide synthesis strategy for T cell determination analysis. L Immunol. Meth. 1990, 134, 23-33. [71] A. M. Bray, J. N. Maeji, R. M. Valerio, R. A. Campbell, M. H.Geysen, Direct cleavage of peptides from a solid support into aqueous buffer. Application in simultaneous multiple peptide synthesis. J. Org. Chem. 1991, 56, 6659-6666. [72] A. M. Bray, J. N. Maeji, A. G. Jhingran, R. M. Valerio, Gas phase cleavage of peptides from a solid support with ammonia vapour. Application in simultaneous multiple p e p tide synthesis. Tetrahederon Lett. 1991, 32, 6163-6166. [73) C. Y. Ch0.E. J. Moran, S.R. Cherry, J. C. Stephans, S.P. A. Fodor,C. L. Adams, A. Sundaram, J. W. Jacobs, P. G. Schultz. An unnatural biopolymer. Science 1993,261,1303- 1305. 1741 J. A. Ellman, L. A. Thompson, Synthesis and application of small molecule libraries. Chem. Rev. 1996, 96, 555-600. [75] M. Rinnova, M. Lebl, Molecular diversity and libraries of structures: Synthesis and screening. Collect. Czech. Chem. Commun. 1996, 61, 161-222. [76] N. K. Terrett, M. Gardner, D.W. Gordon, R. J. Kobylecki, J. Steele, Combinatorial synthesis: The design of compound libraries and their application to drug discovery. Tetrahedron 1995, 51, 8135-8173. [77] M. Lebl (1996), Compilation of papers in molecular diversity field, INTERNET World Wide Web address: http: //vesta.pd.com. [78] M. Lebl, V. Krchnak, N.E Sepetov, B. Seligmann, P. Strop, S. Felder, K. S. Lam, One-beadone-structure combinatorial libraries. Biopolymers (Peptide Science) 1995, 37, 177- 198. (791 M. Lebl, K. S. Lam, P. h i s , V. Krchnak, M. Patek, S. E. Salmon, V. J. Hruby, “Methods for building libraries of peptide structures and determination of consensus structures’’ in Peptides 1992 (Eds.: C. H. Schneider, A. N. Eberle), Proceedings of the 22nd European Peptide Symposium. ESCOM, Leiden, 1993, pp. 67-69. [SO] N. F. Sepetov, 0. L. Issakova, M. Lebl, K. Swiderek, D.C. Stahl, T. D. Lee, The use of hydrogen-deuterium exchange to facilitate peptide sequencing by electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom. 1993, 7, 58-62. [Sl] N. E Sepetov, 0. L. Issakova, V. Krchnak, M. Lebl, Peptide sequencing using mass spectrometry. USA Patent 5.470.753 (1995). [82] K. Biemann, In Mass Spectrometry of Biological Materials. (Eds.: C. N. McEwen, B. S. Larsen), Marcel Dekker, New York, 1990, pp. 3-(N). [83] M. H. Geysen, “Generation of diverse chemically synthesized peptide libraries’’ in Peptide Chemistry 1992 (Ed. : N. Yanaihara), Proceedings of the 2nd Japanese Symposium on Peptide Chemistry. ESCOM, Leiden, 1993, pp. 3-6.

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Combinatorial Peptide and Nonpeptide Libraries by. Gunther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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

7.1 Introduction The generation of molecular diversity and the combinatorial chemistry approach both have their origins in the laboratories of peptide chemists. Peptide mixtures and completely randomized peptide sequences (libraries) or combinations of defined positions and mixed positions (sublibraries) are widely used tools in the search for new lead compounds. These mixtures can be screened either in solution [l-41 or immobilized on solid phase supports [5-81. Methods originally developed for simultaneous multiple peptide synthesis (SMPS) have subsequently been adapted to the preparation of peptide libraries [9, lo]. The split resin method was used for the preparation of polymer-bound and soluble tetra- and hexapeptide libraries. This often applied procedure was designed to ensure equimolarity of single peptides in solution or to generate one peptide per resin bead [ll]. Several time and cost efficient methods have been described to couple premixed Fmoc- or Boc-amino acids for the synthesis of libraries. The “single resin approach” [6], the use of different molar ratios of Fmoc-amino acids [12) or Boc-amino acids [13, 141 and procedures for equimolar coupling of mixed amino acids have been developed for the generation of peptide mixtures. Studies of classical structure-activity relationships have been enhanced significantly by the parallel synthesis and screening of numerous compounds. Within this new field of combinatorial chemistry there are a lot of examples for successful applications of the peptide library approach, e. g., the determination of sequences for binding of SH2 domains [15] and SH3 domains [16] in investigations of the signal transduction pathway, or the identification of enzyme inhibitors [2, 121 or opioid peptides [3] in the search for new lead compounds. In addition, antigenic peptides [17] and MHC-Class I binding peptides [4] have been identified in immunochemistry and metal binding peptides [a], and peptides interacting with an organic dye [18] have also been found by screening of peptide libraries.

204

7 Peptide and Cyclopeptide Libraries

Several approaches have been established for the identification of active compounds from assays with libraries. The iterative selection and synthesis process [l, 3) or the positional scanning method (171 do not require special equipment and experience for structure elucidation. However, sequencing of active soluble peptides [IS] or of single peptides on beads [7], mass spectrometry of compounds from one bead [19] as well as decoding concepts like DNA tags [20], introduction and sequencing of coding peptide strands [21] and inert chemical tags for recording the reaction steps by gas chromatography [22] require experience in molecular biology and expensive, time consuming technologies for microanalysis. To enhance chemical diversity, peptide libraries were chemically modified: permethylated libraries [23], hybrids from peptide and nonpeptide components [24], libraries on cyclic templates [25] or molecular scaffolds [26] or libraries in multimeric form [27] have all been investigated. The incorporation of unusual amino acids [2], libraries of peptoids [28) or oligocarbamates [29], as well as the synthesis of organic compound mixtures [30], offer new possibilities in the search for lead structures by means of the solid phase organic chemistry which was originally initiated by Bruce Merrifield [3 11. This chapter describes practical aspects of the automated synthesis of peptide libraries from low to very high diversity, including cyclopeptide libraries. An example for the application of even pentadecapeptide libraries in biological screenings is presented as well as methods which have been elaborated and applied for the most reproducible and straightforward production of thousands of peptide libraries and sublibraries in the past few years.

7.2 Methods for the Generation of Peptide Libraries Solid-phase peptide synthesis on polymeric swellable beads is the method of choice for the synthesis of peptide libraries (Chapter 3). Among numerous carriers (Chapter 17) polystyrene or polystyrene-PEG copolymers are the most frequently used materials for resin beads. They offer the possibility to synthesize peptide libraries by the split resin approach (111 or by several “premix methods”. Polystyrene-PEG copolymers (Chapter 16) are biocompatible and swell in buffer solution. This offers the possibility to test polymer-bound peptides for their interaction with protein receptors.

7.2.1 Manually Synthesized Peptide Libraries The tea-bag method [32] and the multipin method [33] are widely distributed and accepted procedures for simultaneous multiple peptide synthesis (SMPS). Furka introduced the sblit resin method in 1988 f“Dortioninn-mixing techniaue” 1341) for the

7.2 Methods for the Generation of Peptide Libraries

205

synthesis of peptide mixtures (Table 7-1). This procedure has been used for the “onebead-one-peptide” concept (7J which was also adapted to the so-called “Selectide” technology [35]. The multipin method (Chapter 10) is restricted to the application of premixed amino acids as the carrier material cannot be divided. Table 7-1. Comparison of two principal methods for the synthesis of peptide libraries

Premix methods

Split resin method

Benefits :

Benefits :

-

-

no special equipment necessary

-

one peptide from one resin bead

-

can be used by most peptide laboratories equipped for automated synthesis of libraries loi4 different peptides from one resin bead thousands of sublibraries can be prepared within short time minimal amounts of solvents and chemicals are needed

- equimolar distribution of peptides in mixture can be expected

- standard synthesis procedures are applicable

Disadvantages :

Disadvantages :

-

-

upscaling difficult

- analysis of peptide pools requires specialists

time consuming should be carried out manually possible contact of laboratory workers with hazardous chemicals during resin handling

7.2.2 Automation to Ensure Reproducible, Simultaneous,

Multiple Peptide Synthesis Over the last two decades, a variety of techniques have been developed that permit automated and semiautomated peptide synthesis. The most experienced suppliers offer synthesizers which dissolve amino acids immediately before the peptide coupling reaction. These machines are equipped with mechanically shaken reaction vessels. In addition, couplings are carried out under oxygen exclusion conditions, and monitoring of the coupling and Fmoc deprotection steps are included. The run is stopped automatically when incomplete coupling or deprotection steps are monitored so that double couplings and resynthesis with modified chemistry are possible. Common automated peptide synthesizersthat combine all these technical, chemical and software prerequisites are commercially available, but they are not equipped for simultaneous multiple peptide synthesis, and are thus of restricted use for the synthesis of hundreds or thousands of sublibraries in combinatorial chemistry.

206

7 Peptide and Cyclopeptide Libraries

Pipetting robots for delivery of solvents, amino acids and coupling reagents to re. actors carrying resins have been used for highly parallel peptide synthesis. They art equipped with one way reactors made from polypropylene and with an especially de. signed needle [36,37) or with a multivalve block [38) for removal of all solubles from the reaction vessels. The parallel and fully automated synthesis of 98 overlapping pentadecapeptides for epitope mapping of influenza nucleoprotein in microcentrifuge tubes with polystyrene resin and Fmoc strategy demonstrated the practical application of fully automated simultaneous multiple peptide synthesis for the gen. eration of a large series of different peptides for subsequent biological screening [39]< This automated alternative to the tea-bag (Chapter 5 ) or multipin (Chapter 10) a p proaches was further improved in software and design and commercialized by twc European companies (Zinsser Analytic, Frankfurt; Abimed, Langenfeld). Both robots have also been applied for the fully automated synthesis of peptides on polyethylene pins [40] or clips [41]. A related robotic delivery system for the parallel synthesis of peptides has been constructed and was equipped for the automated synthesis of equimolar peptide mixtures by the split resin procedure [42,43]. It should be emphasized that the split resin approach is not applicable to highly complex libraries as the methodological borders are set by the amount of resins necessary in the “onebead-one-peptide” concept. Also, from the technical standpoint, the problem of the reproducible handling of multigram amounts of resins in automated split synthesis has not yet been completely solved. These large amounts of resin are necessary as the smallest component in a reactor that can be distributed is the single bead. Thus the number of peptides to be prepared is the same as the total number of beads (Table 7-2). A multiplication of this number by a statistic factor is recommended to overcome the inherent problem of missing sequences in the library prepared by split synthesis. Table 7-2. Amount of resin and yields calculated for the generation of a sublibrary OX7 synthesized by the split resin approach and the premix method (based on: bead size 50- 100 pm. 3000 beadslmg, average molecular mass of OX7: 1000 D, 0.5 mmol/g resin loading) Number of beads for 893871739 peptides (1g7) Amount of resin Total amount of peptides Amount of peptides necessary for the preparation of 10 ml test solution (1 mM concentration) Resin necessary for the preparation of 10 mg peptide mixture containing all 19’ different peptides Concentration of individual peptides in bioassay using a stock solution of 1 mM

Split resin approach

Premix methods

839871 739 -300g -150 g 10 mg

1 0.3 Pg 0.15 pg 10 mg

-3oog

20 mg

-1

-1 pu

PM

7.2 Methods for the Generation of Peptide Libraries

207

7.2.3 Peptide Diversity Determines Procedures for Synthesis and Bioassay So-called premix methods help to overcome difficulties connected with the split resin method. They facilitate the synthesis of highly complex soluble and polymer-bound libraries and sublibraries. It is obvious that the composition of the amino acid mixtures used for coupling and the molar excess to the reactive groups on the resin have a strong influence on the quality of the library. The different reaction constants of the protected amino acids as well as influences by the molarity of the reagents, the solvent, the types of coupling reagents, temperature, sequence length, the onset of secondary structures and the amino acid or amino acids on the resin (coupling to an 0 or X position) are reflected in the library. To overcome potential non-equal distributions of the different peptides in libraries, premix methods with equimolar or reaction rate-adjusted acylating amino acid mixtures are recommended for library synthesis (Table 7-3). Molecular diversity is not only limited by the methodological borders set for the split resin synthesis but also by the bioassays which accept only limited amounts and numbers of nonrelated peptides or byproducts. For example, the heterogeneity of a mixture of 104976 peptides from sublibraries containing four mixed positions was too high for re-evaluation of an epitope recognized by a monoclonal antibody against HIV Nef protein. This antibody was investigated for binding to a stepwise randomized hexapeptide epitope. Using a competition assay, significant binding effects were observed up to a heterogeneity of 5832 individual peptides per mixture [44].Octa- and hexapeptide mixtures were also responsible for the induction of nonspecific effects of the hormone neuropeptide Y (NPY) in a standard competition assay in the search for NPY analogs [44].It is obvious that the detailed concept for library screenings has also to be adapted to the biological target and to the detection methods used in the bioassay. In most of the receptor binding studies reported so far, a natural ligand has been already identified. For evaluation of the application of libraries in these bioassays, this ligand has to be randomized by stepwise introduction of random positions (Table 7-4). These randomizations can be carried out in the format for an iterative screening process, in the positional scanning format or in a format which allows the determination of the intercorrelation [4] of amino acid residues. The peptide length plays a critical role in receptor ligand studies, especially when the peptide binding groove of the receptor has interaction sites with the charged Nand C-terminal ends of the peptide ligands (Table 7-5). For example, there is no chance to identify a highly active ligand for MHC-Class I molecules from heptapeptide libraries, as MHC-Class I molecules bind preferably nonapeptides and for some few alleles octa- or decapeptides. One of the natural ligands for the MHC-Class I allele H-2Kb is the octapeptide RGYVYQGL. From pilot studies [U]we concluded that the Dositional scanning amroach 19. 171 and the testing of OX, sublibraries

0.8, capping 5.7

19

millions

AB1 430A

19

AB1 431A HPK, AAA, MS

5

HPLC,MS

1471

AAA

> 105

19

5

tea-bags

1141

AAA

1121

AAA

(2 h) DMF

(2 h) (*) NMP/DMSO/ DMF DIEA 6 18

(40min)

(12 h) DCM

324

single

single

double

*

0.8

Frnoc PyBOP Tenta Gel/ Cellulose

(1 h) DMF

(*I

161

AMS 422 HPLC,MS, CE

19, millions

19

1481

AB1 430A AAA

millions

19

single

DMF

1.o

141

Syro robot AAA, MS, Edman degradation

millions

19

(3 h) DMNDCM (1 :7)/DIEA

double

equimolar, equimolar but for R, 1 :50% excess 0.075 M

1.1

Fmoc Fmoc DCC/HOBt DIC/HOBt HMPA resin Wang/chlorotrityl resins

single

individual, individual, equimolar e.g., S:0.29; cg., G:0.20; I: 1.83 I :2.51 * * 0.3 M 0.13 M

10

Boc Fmoc DIC/HOBt DIC/HOBt MBHA resin Cotton

single

*

equimolar

1

BOC DCC/HOBt PAM resin

single

Reference I451 I461 * no information from the reference cited.

Degeneracy (X position) Degeneracy (peptides) Equipment Analysis

Number of couplings (time) Solvent

1.16

Molar excess of mixture of amino acids Molar ratio of amino acids (maximin) Concentration of amino acids

Boc DCC/HOBt PAM resin

individual, equimolar e.g., A: 1.02; V: 1.06 * 0.038 M

Boc DCC/HOBt PAM resin

Chemistry Coupling reagent Solid support

Table 7-3. Synthesis of soluble peptide mixtures using Boc- or Fmoc-amino acids (“premix methods”)

$

g!?

g

Q

$

L

c,

%

,5 fb ri

b

3

U

s

I4

7.2 Methods for the Generation of Peptide Libraries

209

Table 7-4. Randomization of an octapeptide ligand RGYVYQGL to validate MHC-Class I binding assay 141

Sequence

Number of peptides

Positional scan sequence

Number of peptides

RXXXXXXX RGXXXXXX RGYXXXXX RGYVXXXX RGYVYXXX RGYVYQXX RGYVYQGX RGWYQGL

893 87 1739 47045881 2476099 130321 6859 36 1 19 1

RXXXXXXX XGXXXXXX XXYXXXXX

893 87 1739 893 871 739 893 871 739 893 87 1 739 893 87 1739 893 87 1 739 893 871 739 893 87 1739

xxxvxxxx XXXXYXXX XXXXXQXX XXXXXXGX XXXXXXXL

Table 7-5. Examples for the investigation of the optimal length and different N- and C-terminal end groups of a peptide library ~~

Length

End groups

xxx xxxx xxxxx xxxxxx xxxxxxx xxxxxxxx

NH2-XXX-COOH NHz-XXX-CONH~ NH2-XXX-CHzOH Ac-XXX -COOH AC-XXX -CONH2 Ac-XXX -CHPOH

(Table 7-4) containing about 9 x 10' individual peptides would be highly effective [4, see also Chapter 121. Criteria for the heterogeneity of the sublibraries to be tested for MHC-Class I binding are influenced by the observation that a completely randomized X, peptide mixture, containing about 17 x lo9 individual peptides, can be titrated to the same saturation level as observed with a single peptide constituting the natural epitope which induces a signal at picomolar concentrations (Chapter 12). The concentration of the library required for half maximal stabilizing effects was only 180 times higher than the concentration of the natural octapeptide ligand SIINFEKL [4]. These findings prove that, in bioassays with peptide libraries, several or numerous individual components of the library will contribute to the overall activity of the mixture. Controversial discussions about libraries often arise by the fact that the concentration of an individual compound may be too low in comparison to the natural ligand. Calculations of the number of peptidedbead are important to numerically demonstrate the limitations in the split resin methodology and the advantage of the methodologically more complicated and, from the chemist's view sometimes questionable, premix methods (Table 7-3).

210

7 Peptide and Cyclopeptide Libraries

7.2.4 Coupling Reactions with Premixed Amino Acid Derivatives An important argument for the use of the split resin synthesis in comparison to premix methods is the necessity for the generation of equimolar peptide mixtures during synthesis. So far, there is no systematic study published about influences of workup procedures and purification steps on the composition of the peptide libraries. Discussions about the benefit of solubility supporting influences or precipitation supporting influences of individual components in complex peptide libraries are therefore mainly speculative. For example, hydrophobic peptides will not precipitate completely from ether as shown by sequence analysis of peptide mixtures or by mass spectrometric analysis of cyclopeptide libraries (see Section 5.2). Premixed Fmoc-amino acids or Boc-amino acids were used by several groups for the generation of peptide libraries. The procedures differ in the molar excess and in the molar ratio of individual amino acids used for couplings. Ostresh et al. 1141 as well as Eichler and Houghten [I21 recommend a heterogeneous composition adapted to coupling rates of amino acids whereas others use equimolar mixtures and standard coupling procedures [46-481 or equimolar mixtures and optimized couplings [4, 61. Complex peptide mixtures generated by the different premix methods were successfully screened for biological activity. These peptide mixtures can be prepared in mg amounts (e.g. 10 mg are sufficient for most assays) and were prepared with 20 mg of resin. To obtain identical heterogeneity, one would require at least 300 g of resin when the library has to be synthesized by the split resin method (Table 7-2). These 300 g of resin have to be increased by a reasonably high factor to avoid the loss of individual peptides.

7.2.5 Soluble and Polymer-Bound Libraries in One Run Bioassays carried out with resin-bound peptides are affected by unspecific binding to the polymer surface or to the tagging molecules sometimes used for the identification of nonpeptide compounds. For example, receptors such as MHC-Class I proteins strongly interact with the carboxy group of the C-terminal amino acid as well as with the amino terminus of peptide ligands [49,50] and optimal epitopes for these receptors will not be identified by screening polymer-bound peptides. Sequential release of picomolar quantities of peptides from single beads [51] or from cotton thread [52] helps to overcome disadvantages of immobilized ligands. Novel heterobifunctional resin beads [53] allow cleavage of the peptides from the interior (98%) while peptides on the polymer surface (2%) remain uncleaved. The surface-bound peptides or peptide sublibraries (K.-H. Wiesmuller, unpublished data) have been used as immobilized antigens for the screening for antibody binding sites in ELISA (Fig. 7-1). The resin is modified by a PEG surface which is biocompatible and reduces nonspecific antibody binding. Optical sections of the peptide resin

7.3 Analytical Control of Peptide Mixtures

211

FmoC

‘4

,CH: CH-CH o,\ C/ \CHI J-CH2-Cn,)R/ NC*0 LfCH2

Figure 7-1. Scheme of the structure of the heterobifunctionalized polystyrene- 1 To divinylbenzene resin (ADPV anchor on the inner surface, acid-stable PEG loo00 with marker amino acids on the outer surface) [53].

beads incubated with a tetramethylrhodamine labeled antibody have been carried out using a laser scanning confocal microscope [53]. As proteins will not penetrate such resin beads due to the exclusion limit of > about 10 kD, the ligands necessary for interaction will be recognized by soluble receptors specifically on the outer surface of the beads (Fig. 7-2). The inner surface is available for the synthesis of different compounds (e.g., peptide tags for sequencing) synthesized orthogonally to the surface molecules [54]. Whereas high loading on the interior is useful for unequivocal analyses by MS or Edman degradation, the outer surface has to be biocompatible and should mediate the contact to aqueous solvent and soluble protein receptors.

7.3 Analytical Control of Peptide Mixtures Pool sequencing of natural peptide libraries [55] was a breakthrough in the determination of MHC-Class I ligand motifs and in understanding of the presentation of self and nonself information hidden in the peptides recognized by killer cells [56] or T helper cells [57] during the cellular immune response. Multiple sequence analysis was also applied to the characterization of synthetic peptide libraries [58] (Chapter 9). Mass spectrometry proved to be valuable for the analysis of side products in mixtures of 48 octapeptides [59] and for the characterization of complex peptide mixtures [60] (Chapter 8). In addition, amino acid analysis as well as HP-LC and capillary electrophoresis have been used for partial characterization of peptide mixtures as shown in the followine.

212

7 Peptide and Cyclopeptide LibMries

a)

Figun 7-2. Laser scanning microscopy: Optical sections of five heterobifunctionalized pep.

tide resin beads (Fig. 74) incubated with a peptide-specific, fluorescence labeled antibody (a] and a series of optical sections in the zdirection (distance 2.5 pm) of a single resin bead (b), obtained on a laser scanning microscope (LSM 410, Carl Zeiss, Oberkochen). The spatial fluorescence distribution demonstrates the specific antigen-antibody interaction on the bead ciirfare

7.3 Analytical Control of Peptide Mixturn

213

7.3.1 Monitoring During Synthesis Online monitoring of the dibenzofulvene piperidine adduct is established for continuous flow and batch synthesizers [all but, like conductivity monitoring (621, is not sensitive enough for the quality control of library synthesis. Non-coupled amino groups on the resin can also be detected by ninhydrin assay [63], by bromophenol blue [64] or by chloroanil detection [65]. So far, there is no published example for the quantitative determination of noncoupled amino acids. We investigated the amount of residual amino acids in coupling cycles of automated synthesis of peptide libraries. They can be detected by amino acid analysis on condition that an activated amino acid mixture was used as a standard. This indirect determination of the coupling efficiency was carried out in five reactors during the synthesis of octapeptide libraries using the standard protocol for our premix method. The synthesis was carried out by a robot for simultaneous multiple peptide synthesis (see Section 4.1).

73.1.1 Method for Indirect Determination of the Coupling Yield by Amino Acid Analysis After completion of the first coupling cycle, using premixed Fmoc-amino acids, the coupling solution which was an equimolar mixture of 19 Fmoc-amino acids (no cysteine) prior to coupling, HOBt, DIC and DIEA in the solvent mixture DMF/DCM (1 :2) was filtered into microcentrifuge tubes, the resin was washed once with DMWDCM (1 :2). The filtrate was evaporated in vacuo and the residue was treated for 1 h with piperidine/DCM (1 :1) (0.2 ml) for removal of the Fmoc group and dried in vacuo. Side chain-protecting groups were cleaved with TFA and 5qo scavenger mixture bheno1:ethanedithiol: thioanisol (2 :2:1)] (0.2 ml) for 4 h. The solvent was evaporated in vacuo, the residue was dissolved in acetonitrile/H,O (1 :1) and the amount of noncoupled amino acids was determined by gas phase hydrolysis and amino acid analysis (AB1 477A, Applied Biosystems, Weiterstadt). The positive control was a mixture of 19 Fmoc-AA (1.5 mg, 3.5 pmol), HOBt (50 mg, 327 pmol), DIC (250 ml, 1635 pmol) and DIEA (280 pl, 1635 pmol) in DMF/ DCM (1 :2) (1.5 ml) which was treated as described above. The coupling rate of single amino acids in the equimolar amino acid mixture used for introduction of a mixed position to a randomized dipeptide resin was higher than 98%. Glycine and asparagine showed a slightly lower coupling efficiency. Therefore, almost 98% of the reactive sites on the XX-resin are acylated by amino acids in close to equimolar distribution after the first coupling cycle. The second coupling cycle ensures that there are no reactive sites left on the resin and will not significantly affect the combosition of the librarv.

214

7 Peptide and Cyclopeptide Libraries

7.3.2 Amino Acid Analysis, Capillary Electrophoresis and Mass Spectrometry of Peptide Libraries Deviations from equimolar distribution of peptides in highly complex peptide libraries can be estimated by comparison of the amount of defined residue(s) to the sum of amino acids occurring in X-positions. Repeating incomplete coupling will cause irregularities in the amino acid pattern. Deviations from the calculated amino acid distribution in peptides synthesized with our premix method are within the standard deviation of AAA (Fig. 7-3). Capillary electrophoresis (CE) extends analytical information obtained by high performance liquid chromatography (HP-E), and has been successfully used for the separation of amino acids, c h i d drugs, carbohydrates, oligonucleotides, peptides and proteins. Whilst separation by capillary zone electrophoresis (CZE) is based on differences in migration of components in an electric field (i.e., size and charge of the analyte molecules) micellar electrokinetic chromatography (MECC) represents a

*

*

A D E F G H I K L M P R S T V Y

A D E F G H I K L M P R S T V Y

A D E F G H I K L M P R S T V Y

A D E F G H I K L M P R S T V Y

Figure 7-3. Amino acid analysis for determination of the composition of peptide mixtures SIINFEM (a), XIINFEKX (b), SMXFEKL (c) and SIINXXXX (d). 0 :values predicted, : values observed, *: the Ile-Ile peptide bonds are only partially cleaved by gas-phase hydrolysis (6 M HCl, 160°C, 85 min). The mass spectrum (Fig. 7-6) proved the correct

distribution of Ile.

215

7.3 Analytical Control of Peptide Mixturn

much more efficient separation mechanism. As in CZE,analyte molecules are separated according to their size and charge. Additionally, there are hydrophobic interactions and coulomb interactions with the micelles of the buffer, which enable separation even of uncharged solutes. The chromatograms of an equimolar mixure of 19 Fmoc-amino acids containing all proteinogenic amino acids except for cysteine and of the sublibrary SIINXEKL, which contains 19 peptides in a close to equimolar ratio, are shown in Fig. 7-4 and 7-5. The Fmoc-amino acid mixture was separated into 18 peaks and the sublibrary SIINXEKL showed 13 distinct peaks. Single peaks were characterized by spiking the mixtures with single Fmoc-amino acids or individual synthetic octapeptides. Both chromatograms correspond in the order of elution of the Fmoc-amino acids and octapeptides, respectively. Those compounds containing Ala, Asp, Glu, Gly, His, Asn, Gln, Ser and Thr show short retention times, whilst those with Phe, Ile, Lys, Leu,

W Y

I.,

15

18

21

F

24

27 30 retention time (min)

33

36

39

42

Figure 74. MECC of an equimolar mixture of 19 Fmoc-amino acids containing all proteinogenic amino acids m p t for cysteine Peaks are assigned with the one-letter-code of the corresponding Fmoc-amino acids, impurities are assigned with an asterisk. Experimental conditions: capillary electrophoresis system AB1 270A-HT (Applied Biosystems, Weiterstadt, Germany), fused silica capillary, 72 cm (length to detector 50 cm) x 50 pm inner diameter, voltage 25 kV, UV detection at 200 nm, electrolyte system 50 mM Na2B4O7x 10 H20, 50 m~ SDS,adjusted to pH 9.5, 30°C.

216

7 Peptide and Cyclopeptide Libraries

I

K

4,\r

I --I--,-----10 15 20 25 30 35 retention time (min)

Figure 7-5. MECC of the sublibrary SIINXEKL containing 19 free octapeptides in close to equimolar ratio. Peaks are assigned with the one-letter-code of the corresponding amino acid at sequence position 5 of the individual peptides, impurities are assigned with an asterisk. Experimental conditions: see Fig. 7-4.

Arg and Trp elute after longer retention times and analytes with Met, Pro, Val and Tyr show different retention times for Fmoc-amino acids and octapeptides. The order of elution of the Fmoc-amino acids and the single peptides of the sublibrary SIINXEKL, respectively, depends on PI or charge, hydrophobic interactions with the inner parts of the micelles and coulomb interactions with the negatively charged surface of the micelles formed by sodium dodecylsulfate (SDS). Negatively charged analytes with high hydrophilicity are expected to show low retention times; positively charged analytes with low hydrophilicity will show high retention times. The size of the analyte molecules plays an important role, but it shall not be taken into account in the following discussion. The hydrophilicity and the PI of the amino acids and of the single peptides of the sublibrary SIINXEKL can be calculated (Tables 7-6 and 7-7). The Fmoc-amino acids Asp and Glu and the corresponding octapeptides SIINDEKL and SIINEEKL with high hydrophilicity and low PI show the shortest retention times. For derivatives and octapeptides containing Ala, Gly, His, 11%Leu, Met, Asn. Pro, Gln. Ser. Thr. Val and Tvr. the hvdroDhilicitv and DI are not Dromi-

7.3 Analytical Control of Peptide Mixtures

217

Table 7-6. Hydrophilicity of amino acids according to Hopp and Woods [66] and PI values [67]

Amino acid R, D. E, K S,N,Q,G,P,T,A,H M,V, I, L

Y, F

Hydrophilicity 3 0.3t00.5 -1 to -1.8

-2.3 to -2.5 - 3.4

W

Amino acid PI D 2.77 E 3.24 A, F, G, I, L, M, N, P, Q, R, V, 5.41 to6.30 w,y H 7.59 K 9.82 R 10.76

Table 7-7. Hydrophobicity of the individual octapeptides from the sublibrary SIINXEKL was calculated by using the uncorrected retention coefficients of Gou et al. [691 and PI values were calculated according to Skoog and Wichman [68]. Peptides are listed corresponding to the amino acid at sequence position 5 Peptides with

X in pos. 5

W, F, L, I M, V, Y P. A, H, R, T,Q,K, G

S, N, E, D

Hydrophobicity 23 to 25 20 to 21 15 to 17 13 to 15

Peptides with X in pos. 5 D

E

A,F,G,I,L,M,N,P,Q,

S, R, V, W, Y H

K R

PI 4.1 1 4.31 6.74 7.59 9.67 9.85

nent, which resulted in medium retention times. The highest retention times were observed for Arg, Phe, Lys and Trp containing compounds. For Phe and Trp medium PI values, but low hydrophilicity, for Lys and Arg high hydrophilicity but low PI values were calculated. Thus, good interactions with the micelles of the buffer and therefore high retention times were measured for octapeptides and Fmoc-derivatives containing Arg, Phe, Lys and Trp. In conclusion, MECC is superior to CZE in the separation of neutral molecules or analytes with similar charge and size because of the more efficient separation mechanism. Also, MECC offers additional information when compared to HPLC separations, and therefore it represents a useful tool for the separation of peptide mixtures. Electrospray mass spectrometry is a fast and efficient method for the determination of composition and purity of peptide mixtures (Chapter 8). For the optimization of the premix method established for the synthesis of octapeptide mixtures [4], we synthesized a series of 19- and 361-component mixtures. The mass spectra of SIINFEKX and XIINFEKX (Fig. 7-6) show a good correlation between calculated

218

7 Peptide and Cyclopeptide Libraries

925

950

975 m/z

1000

1025

900

950

1000

1050

1100

m/z

Figure 7-6. Electrospray mass spectra of the 19 component mixture SIINFEKX (a) and of the 361 compound mixture XIINFEKX (b) (upper part). The patterns of isobaric peptides were calculated by the program QMass (Chapter 18) (lower part). The mass distribution was approximated by counting the number of different peptides within one atomic mass unit.

and measured mass distributions. The mass patterns are influenced by machine parameters and by the loss of peptides due to side reactions during synthesis and work up procedure. Incomplete cleaveage of side chain protecting groups, incomplete precipitations as well as deviations from expected distribution caused by insoluble and adhesive or aggregate forming peptides and by multiple charged ions were also reflected in the spectra.

7.4 Pipet Robot for the Synthesis of Peptide Libraries n o different technologies were elaborated in recent years for multiple peptide synthesis which allow the parallel synthesis of up to 1152 individual peptides on standard resin in pmol amounts (701 or the parallel synthesis of loo00 spots on cellulose membranes (711 in one run (nmol amounts). Light-directed, spatially addressable parallel chemical synthesis was designed for the synthesis of minimal amounts of tens-of-thousands of defined peptides on a solid planar carrier [5]. In principle, these arrays are also applicable for the synthesis of libraries and sublibraries. In addition, microstructured peptide functionalization of surfaces using electropolymerizationof peptides has been achieved and shown to be of particular practical value, since the peptides can be characterized and purified before they are attached to the surface (for references, see Chapter 1). From the view of practical application, the library synthesizer is a pipet robot equipped with multivalve blocks containing microreaction vessels used for the parallel synthesis of hundreds of peptide sublibraries and libraries (Fig. 7-7). The equip-

7.4 Pipet Robot for the Synthesis of Peptide Libraries

hundreds of

several reagents

hundreds to thousands

educts

for coupling

of reactors with resin

219

a

Figure 7-7. Array for an optimized multiple peptide and peptide library synthesizer containing a nitrogen or argon inlet (-& ) a cooling or heating device ( -@), a multivalve block for removal of all solubles from reactors by filtration ( ), a shaker for mixing of resin ) and a controller for movements of arms and valve switching as well as empty sen( ' sors and optical sensors. The software is running on a standard PC,integrated in a network. The reaction vessels should be adressed individually, and chemfiles must be highly flexible.

:

ment of the robot includes racks for storage of up to 300 predissolved amino acids or amino acid mixtures, bottles for coupling and deprotection reagents, diluters for moving syringe pumps and waste and wash stations for the pipet needle. Bottles for storage of wash DMF,and a vacuum trap, are also integrated into the system. All chemicals and the reaction vessels are protected from moisture and oxygen and the resins should be mixed when reactors are loaded with more than 50 mg of resin beads.

220

7 Peptide and Cyclopeptide Libraries

7.4.1 Procedure for the Synthesis of Peptide Libraries by the “Premix Method” The experimental procedure for the premix method is characterized by extended coupling times, double couplings, high portions of DCM and open reactors. About 50% of DCM evaporates at room temperature during long lasting coupling steps, which results in concentration of remaining Fmoc-amino acids and reagents. Coupling is supported by the addition of DIEA after 3 h coupling time. For the synthesis of free peptides, we applied Fmoc-~-Pro-2-chlorotrityl-resinand Fmoc-L-amino acid-p-benzyloxybenzyl alcohol-polystyrene resins. Fmoc-L-amino acids were used with the following side chain protecting groups: ?er?.-butylethers for Ser, Thr, Tyr; ?er?.-butyl esters for Asp and Glu; trityl for Cys, His, Asn, Gln; tert.-butyloxycarbonyl for Lys and Trp; 2,2,5,7,8-pentamethyIchroman-6-sulfonyl (Pmc) for Arg. Fmoc-amino acids (0.5 M) were dissolved with HOBt (0.5 M) in DMF. The solutions were used for coupling of defined positions. Equimolarly premixed Fmocamino acids [0.075M in DMF: DCM (1 :7, v/v)] were distributed for coupling of mixed positions. Multiple peptide library synthesis was carried out on a robot (Syro, MultiSynTech, Bochum) equipped with one arm for the distribution of reagents and a second arm connected to a diluter with four syringes for the delivery of DMF during washing steps.

7.4.1.1 Experimental Procedure

A resin mixture was prepared that contains 19 Fmoc-L-amino acid resins (Cys was excluded) in equimolar amounts. Fmoc-amino acid resins and resin mixtures were distributed in 30 mg aliquots (15 pmol) to filter tubes (polypropylene pipet tips, 1 ml, glass wool filter), which were positioned in the format of a microtiter plate on valve blocks. Fmoc deprotections were carried out two times, 7 min each, with 50% piperidine in DMF (220 pl). After nine washing steps with DMF (300 pl) coupling reagent DIC [50 p1, 1.5 M in DMF: DCM (1 :2, v/v)] and Fmoc-amino acids and Fmoc-amino acid mixtures (200 pl) were distributed to the reaction vessels. Double couplings (3 h each) were carried out in open tubes. After 2 h coupling time DIEA (20 pl, 1 M in DMF: DCM (1 : 1, v/v)] was added. Coupling reagents were filtered off and the resins were washed three times with DMF. The peptide mixtures were cleaved from the resins and side chain deprotected with TFA: phenol :EDT: thioanisole (96 :2 :1 :2, v/w/v/v) (1 ml). The peptide solutions were filtered from the resins and washed with acetic acid (0.3 ml). The peptide mixtures were precipitated at -20°C by the addition of cold n-heptane :diethylether (1 :1. v/v) (5 m h The DreciDitates were washed twice bv sonification with n-heD-

7.5 Conformationally Constrained Peptide Libraries

221

tane: diethylether (1 :1, v/v) and were lyophilized from acetic acid :water :tert.-butyl alcohol (1 : 10: 50, v/v/v) yielding an average amount of 12 mg peptide library or sublibrary per reaction vessel.

7.5 Conformationally Constrained Peptide Libraries The generally poor oral availability and the enzymatic degradation of linear peptides built up by L-amino acids are the reason for their restricted application in drug development. The main interest in the field of combinatorial chemistry focuses at present on the development of chemically modified, peptidomimetic and nonpeptide libraries [72]. Cyclicpeptides have been reported to show increased stability to enzymatic degradation [73, 741 and the constrained flexibility by cyclization enhances peptide binding affinity to various acceptor molecules [75-771. Cyclopeptide libraries are therefore intriguing targets for drug discovery, having the advantage that leads identified by screening these libraries have properties desired for drug development (see also Chapter 11). Several methods of peptide cyclizalion on the solid support through lactam formation [78-841 or disulfide bridging [85] have been described which also provide the methods necessary for the synthesis of cyclic peptide libraries. Bradshaw et al. [86] synthesized a cyclic peptide library which contained disulfide bridged rings and rgmained covalently attached to resin beads for further screening experiments. The library comprised the sequence CXXXXXC and contained 19’ individual peptides. Fassina et al. [87] pursued a similar approach by synthesizing dimeric peptide libraries, starting from a Fmoc-Lys(8-Fmoc)-Gly -resin. After completion of randomized sequences, two cysteine residues were introduced at the N-termini and a disulfide bond was formed. Kramer et al. (881 describe the synthesis of structurally different types of combinatorial peptide libraries on continuous cellulose membrane supports. A combinatorial linear all G or all D-library XX0,02XX and two libraries conformationally constrained, either via a disulfide bridge between C- and N-terminal cysteines of CXX0,02XXC or an amide bond between the am i n o group of the N-terminus and the y-carboxy group of a C-terminal glutamic acid of XXX0,02XXE were screened with transforming growth factor-b, resulting in structurally different peptide mixtures mimicking natural ligand activity. Darlak et al. [89] report a procedure for the preparation of cyclopeptide mixtures involving head-to-tail peptide cyclization while attached to the resin via the side chain of a C-terminal aspartic acid. A technique of reversing the sense of the peptide sequence from N - C to C + N on solid support is described by Holmes et al. [NI.Thereby, following the synthesis of a library, the N-terminus is cyclized to the support and ultimately the bond an-

222

7 Peptide and Cyclopeptide Libraries

choring the C-terminus to the support is cleaved to generate the C-terminally exposed library. A different approach is pursued in the concept of cyclic peptide template libraries where a cyclic peptide template is generated prior to introducing the chemical diver. sity. Eichler et al. [25] synthesized a combinatorial library on a cyclic peptide template in a positional scanning format using solid-phase chemistry and four orthogonal protecting groups (Fmoc, Boc, Dde, OAII). The cyclic peptide template is composed of three lysine residues and one glutamic acid residue. The chemical diversity was introduced by on-resin acylation of the &-aminogroups of the lysine residues using 10 carboxylic acids in addition to the 20 proteinogenic amino acids. Sila and Mutter [91] also use in their TASP concept (template-assembled side chains) a cyclopeptide as a structural motif upon which amino acid residues are assembled by chemoselective ligation. During cyclizations on solid supports dimer formation has to be regarded as a possible side reaction and is dependent on resin loading [U]. Low resin loadings (0.1 -0.3 mmol/g) are recommended for on-resin cyclizations and help to avoid dimer formation. If larger amounts of cyclopeptides ( > I g) are to be synthesized, it may be advantageous to cyclize in solution where there is no limitation in the amount of peptide to be cyclized and costs for resin material can be reduced (10 g of resin with a loading of 0.2 mmol/g would be required for 1 g cyclopeptide with a moleculai mass of lo00 D and 50% yield after purification). The cyclization tendency of a linear precursor depends on the size of the ring ta be formed. Usually there are no difficulties during the cyclization of heptapeptides and longer peptides, whereas the ring closure within certain hexa- and pentapeptides is reported to be hindered. So far, it is generally accepted that the cyclization in these cases is enhanced by the presence of turn structure inducing amino acids such as glycine, proline or a D-amino acid [92]. On the other hand, successful cyclizations with all-L-hexapeptides which lack these amino acids have been reported [93]. Thus, cyclization tendency of hexapeptides seems to be strongly sequence dependent, and no predictions of side products can be made. Linear peptides containing amino acids that cause steric hindrance 01 cyclization suffer from long lasting activation of C-terminal carboxy groups and side reactions such as cyclodimerization, racemization of the C-terminal residue and chemical modifications are also increased. Cyclizations of protected peptides in solution are carried out at high dilutions M) to suppress dimer and multimer formation. Cyclization products have to be carefully analyzed for their content of cyclodimers, C-terminally epimerized cyclopeptides and modified linear peptides. Coupling reagents TBTU and HBTU [94] are reported to provide fast cyclizations [95], but may also lead to racemization levels which are comparable to those observed with DCC/HOBt (961. The newly developed uronium salt HAPyU [97] was found to be highly effective for cyclization with minimal content of diastereomeric cyclopeptide [98]. Furthermore, for cyclizations on the solid support, it has been re-

7.5 Conformationally Constrained Peptide Libraries

223

ported that HBTU can lead to N-terminally blocked, linear tetramethylguanidinium derivatives [99].

7.5.1 Synthesis of Cyclopeptides We describe here a general method for the partially automated, simultaneous multiple synthesis of head-to-tail cyclopeptides and cyclopeptide libraries (Fig. 7-8). Using this procedure, we have synthesized a series of 39 cyclohexapeptides c[RGDSPO] (with 0 = 19 L-amino acids and Gly and 19 D-amino acids) comprising the cell-adhesive motif of fibronectin (RGDSP). We applied the three different cyclization reagents TBTU [94],HATU [97]and PPA [I001and compared them with regard to cyclization efficiency, cyclodimerization and racemization.

7.5.1.1 Loading of 2-Chlorotritylcbloride Resin with Fmoc- Amino Acids

2-Chlorotritylchloride-polystyrene- 1 To-divinylbenzene-resin (2.0 g, loading with C1: I .4 mmol/g, Novabiochem, Liiufelfingen) was suspended in dichloromethane/dimethylformamide (1 : 1) (20 ml) and 39 equal portions (500 pl containing 50 mg resin, 0.07 mmol) were distributed to the reaction vessels of a multiple peptide synthesizer (Syro, Multi SynTech, Bochum). Resin loadings with C-terminal Fmocamino acids were performed in the individual reaction vessels. Stock solutions of 20 Fmoc-camino acids and 19 Fmoc-D-amino acids (0.7M in DMF) and DIEA (1 M in DMF) were prepared and stored at -20°C. Each reaction vessel containing 2-chlorotritylchloride resin (50 mg, 0.07 mmol) was equilibrated and washed with DMF. Fmoc-amino acids (110pl, 0.077 mmol) and DIEA (200 p1,0.193 mmol) were added and allowed to react at room temperature for 2 h. Methanol (50 pl) was distributed and, after reaction for 30 min, all solvents were sucked off and the resins were washed three times with DMF. Loadings of the resins wereO.5-0.6 mmol/g (depending on the Fmoc-amino acid) as measured by quantitative Fmoc determination: FmocAla-resin (0.63 mmol/g), Fmoc-D-Ala-resin (0.63 mmol/g), Fmoc- Arg-resin (0.39mmol/g), Fmoc-Gly-resin (0.59 mmol/g), Fmoc- Asp-resin (0.55 mmollg), Fmoc-Ser-resin (0.58 mmol/g), Fmoc-Pro-resin (0.58 mmol/g).

7.5.1.2 Synthesis of Linear Peptides

The linear peptides were synthesized by simultaneous multiple peptide synthesis using Fmoc/t Bu strategy and the analytically characterized Fmoc-amino acid2-chlorotrityl -polystyrene- 1 %-divinyl-benzene resins. Amino acid couplings were Derformed using a fivefold excess of Fmoc-amino acids (0.7M in DMF containing

224

7 Peptide and Cyclopeptide Libraries

0.7 M HOBt) and DIC activation. Fmoc groups were removed by treatment with piperidine/DMF (1 : 1) within 15 min. 7.5.1.3 Cleavage of Fully Side Chain-Protected Peptides from the Resins

Cleavage of each linear peptide from the resin was achieved by acetic acidlmethanol/dichloromethane (2 :2 :6) (1.5 ml) within 3 h. Resins were filtered off and washed with the cleavage mixture five times. The combined filtrates of each peptide were evaporated using a rotating vacuum concentrator (RVC, Christ, Osterode) and the peptides were lyophilized from tert.-butyl alcohol/water (4 : 1). The crude side chain-protected peptides were characterized by RP-HPLC (1021 and electrospray-

MS 11031.

7.5.1.4 Cyclization Reactions

The crude linear and side chain-protected peptides were cyclized in DMF at room M) using three different reagents. temperature (0.8 x Cyclization with TBTU/HOBt/DIEA

The crude linear peptides (0.005 mmol) were dissolved in DMF (6 ml). DIEA (4 eq.) and TBTU/HOBt (3 eq., 0.4 M in DMF) were added dropwise to the stirred peptide solutions. After 3 h, the solvents were removed in a vacuum concentrator, and the residual reaction products were dissolved in dichloromethane (3 ml). Organic phases were extracted by three washing steps with KHSO, (5% in water) and water. Solvents were removed in vacuo using the rotating vacuum concentrator. Cyclization with HATU/HOAt/DIEA

This method was carried out in a manner analogous to the TBTU cyclization except that HATU/HOAt were used for cyclization. Cyclization with PPA /DIEA

The crude linear peptides (0.005 mmol) were dissolved in DMF (6 ml). DIEA (12 eq.) and PPA (9 eq., 50Vo solution in DMF) were added dropwise to the peptide solutions. After 3 h the solvents were removed in a vacuum concentrator and the residues were dissolved in dichloromethane. The organic phases were washed three times with water and evaDorated in vacuo.

7.5 Conformationally Constrained Peptide Libraries

225

7.5.1.5 Cleavage of Side Chain-Protecting Groups

Side chain-protected cyclopeptides (0.005 mmol) were treated with cleavage mixture [TFA/thioanisole/thiocresol(95 :2.5 :2.5)] (250 pl) for 4 h at room temperature. Cyclopeptideswere precipitated from ether (3 ml) at -20°C (12 h). Precipitates were collected by centrifugation, washed twice with icecold ether and lyophilized from tert.-butyl alcohoVwater (4 : I). Crude cyclopeptides were analyzed by RP-HPLC [lo21 and electrospray-MS [103]. Analytical data are shown for a representative set of five cyclohexapeptides with a constant pentapeptide sequence c[RGDSPO] or c[RGDSPo], where 0 represents a L-amino acid and o a D-amino acid (Table 7-8). The purities of crude cyclohexapeptides, which were cyclized by three different cyclization reagents (TBTU, HATU, PPA), were determined by RP-HPLC at 214 nm [lo21 (Fig. 7-9) and proved to be better than 80% for nearly all 39 cyclopeptides, contains large and for all of the three reagents used for cyclization. ~[RGDSPV] amounts of side products. Peptides cyclized by TBTU and HATU show comparable purities. HATU yields cyclopeptides with marginally higher purity, whereas cyclopeptides made with PPA are less homogeneous, especially for c[RGDSPk] and c[RGDSPv]. Monitoring the degree of cyclodimerizationis carried out by RP-HPLC [I021 and electrospray-MS [103]. Distinguishing between cyclic monomers and cyclic dimers (or higher oligomers) by electrospray-MS is not trivial, since aggregation of monomers during the measurement can give rise to artifactual dimers. Real dimers may be detected as doubly charged molecules with an identical m/z-value to that of the cyclic monomer, but these cyclic dimers would show distances of 0.5 amu between the resolved isotopic peaks, whereas cyclic monomers show differences of 1 amu between the resolved isotopic peaks. An unambiguous distinction between cyclic monomers and dimers can only be made by on-line HPLC-MS where, after HPLC separation, the individual peaks can be scanned with high resolution to resolve the fine structure of the isotopic distribution.

Table 7-8. Retention times of five randomly selected cyclohexapeptides and percentage of the respective diastereomeric cyclohexapeptide as determined by RP-HPLC [lo21 ______

Sequence

Retention time [minl

c[RGDSPk] c[RGDSPG] c[RGDSPd] c[RGDSPC] ~[RGDSPV]

18.21 18.66 19.55 21.59 26.61

~

Cyclization reagent and 070 of diastereomer TBTU HATU PPA 4.1

0.0

3 .O

1.4 0.0 11.0

0.0 2.8 5.0

0.8 2.4 7.0

-

-

-

226

7 Peptide and Cyclopeptide Libraries

Fmoc-AA-COOH(1.1 eq) + CI-Trt(C1)-resin (1 q) Resin loading: I .4 mmoUg DlEA (2.75 eq),2 h Methanol (14 eq), 30 min DIC activation Fmoc-AAIHOBt (5 eq, positions defined) or Fmoc-AA-Mix/HOBt(1 eq,for positions mixed)

H2N-lpeptide mixture (protected)l-COO-Tr((Cl)-resin

Cleavaee:

acetic acid:methanol:dichloromethan (2:2:6)

+

%N{ peptide mixture (protected)l-C00

-

0.01 M in DMF TBTURIOBt (3 eq) D E A (4 eq)

cyclopeptide mixture

/-

95 95 TFA 5 95 thioanisol/thiocresol 3h

cyclopeptide mixture (side-chainsdeprotected) ~~

Figure 7-8. General scheme for the synthesis of cyclopeptides and cyclopeptide mixtures.

7.5 Conformationally Constrained Peptide Libraries

227

For all of the five cyclohexapeptides we found the correct masses for the cyclic monomers (Table 7-9) and 0-5% of the dimeric masses. These are most probably artifactual dimers, since in the RP-HPLC no significant peaks can be seen. The percentage of racemization of the C-terminal residue was determined by RP- HPLC (Table 7-8). All diastereomeric cyclopeptides were synthesized and used as standards for HP-LC analysis [102]. HATU as cyclization reagent showed the lowest percentages of C-terminal racemization, TBTU and PPA were similar with respect to their relative effects on racemization. For ~[RGDSPV], relatively high values for racemization were found. This can be put down to the fact that the peptide RGDSPv cyclizes slowly and therefore the C-terminal carboxy group persists for quite a long time in the activated state, leading to racemization and other side reactions. Linear peptide sequence, cyclization reagent and the C-terminal amino acid residue all have significant influence on the quality of the resulting product. In our study we obtained good purities for peptides that were cyclized with TBTU and HATU, where HATU had the best results for low racemization. PPA as cyclization reagent was found to be less effective resulting in more byproducts as determined by RP-HPLC. In addition, branched side chains on the C-terminal amino acid (e.g., valine) may cause steric hindrance and lead to incomplete cyclization. Table 73. Calculated and experimental molecular masses for 5 cyclohexapeptides as analyzed by ESI-MS [lo31

c[RGDSPC] cIRGDSPd] c[RGDSPG] c[RGDSPk) ~[RGDSPV]

615.68 627.62 569.58 640.65 611.66

616.0 628.5 570.5 641.5 612.0

616.0 628.5 570.5 641.5 612.0

616.0 628.5 570.0 641.5 612.0

7.5.2 Characterization of Cyclopeptide Sublibraries Linear hexapeptide libraries were synthesized using the standard protocol for automated peptide library synthesis (see Section 4). 2-Chlorotritylchloride resin was used to facilitate the cleavage of side chain-protected peptide libraries with 20% acetic acid in chloroform/methanol(3 : 1). Cyclization and work-up procedures are similar to those described for individual cyclopeptides (Fig. 7-8). Cyclopeptide libraries were characterized by amino acid analysis, nuclear magnetic resonance and, most important, by ESI mass spectrometry (see Chapter 8 by J. W.Metzaer et a/.). ESI mass snectra of cvclic Dentide libraries can be recorded

220

7 Peptide and Cyclopeptide Libraries

2400

I800 1200

600 0 min

0.0

15.0

30.0

45.0

60.0

75.0

0.0

15.0

30 0

45 0

600

75 0

3

0.0

15.0

30 0

45 0

60 0

, .o

Figure 7-9. RP chromatograms of five crude cyclohexapeptides cyclized by TBTU (top),

HATU (middle) and PPA (bottom). Cyclohexapeptides from lower line to upper line: c[RGDSPKI (11, c[RGDSPG] (2), c[RGDSPd] (3), c(RGDSPC1 (4), c[RGDSPv] (5).

229

7.5 Conformationally Constrained Peptide Libraries

automatically by the use of an autoinjector. Scanning with sufficient resolution facilitates the identification of all 18 individual cyclopeptides in a sublibrary c[HAGXHG] (with X = L-amino acids with the exception of Cys and Trp) (Fig. 7-10). The relative intensities of the detected signals indicate close to equimolar distribution of all 18 individual components. The two cyclopeptides c[HAGQHG] and c[HAGKHG] are isobaric and cannot be distinguished by the applied ESI techniques. The cyclopeptides c[HAGIHG], c[HAGNHG] and c[HAGDHG] can be resolved in separate signals with differences of 1 amu. Each monoisotopic mass (based on C = 12.00000) of the [M + HI+ ions is accompanied by a resolved '3C-isotopic peptide signal (marked by asterisks). An expanded view of the m/z range from 555 to 564 amu reveals the three cyclohexapeptides c[HAGPHG] ([M + H]zp = 557.22), c[HAGVHG] ([M + HI.& = 559.22) and c[HAGTHG] ([M+ HI;, = 561.29) and also their respective isotopes. This proves the absence of cyclodimers, which would be detected as doubly charged molecules

-3 G A

573.26

s

P

V

T

I, L

1574.25

N D

607.33

Q, K

j

E M H F R

Y

480

SO0

520

540

560

580 m/z

600

620

640

+ + 517.5 531.5 547.5 557.6 559.6 561.6 573.6 574.6 575.5 588.6 589.6 591.7 597.6 607.6 616.6 623.6

660

680

Figure 7-10. ESI mass spectrum of the 18-componentcyclohexapeptide mixture c[HAGXHG] (X = randomized position with 18 L-amino acids except for Cys and Trp) and calculated molecular masses of the [M+ H)+ ions. Mass peaks marked by asterisks correspond to isotopic peaks. The box shows the enlargement of the region of 555-564 amu, which includes the three cyclohexapeptides c(HAGPHG1, c(HAGVHG1and c(HAGTHG1and their respective isotows.

230

7 Bptide and Cyclopeptide Libmries

at the same m/z-value, but with differences of 0.5 amu between the isotopic peptide signals. Analysis of more complex peptide mixtures by ESI mass spectrometry is more complicated and comprises the assessment of characteristic mass distributions, which can serve as fingerprints for individual sublibraries. With QMass, a computer program designed in our group (see Chapter 19 by J. BrILnjes et al.), peak lists of all peptides present in a given peptide library, as well as the graphic depiction of the mass distribution are calculated very easily. It is also possible to simulate theoretical mass spectra by overlaying two different calculated mass distributions. This option is useful for the analysis of cyclopeptide mixtures, as the detection of incomplete cyclization provides important information. Complete cyclization of a linear hexapeptide library FAXXXG (with X = all amino acids excluding Cys and Trp) causes an 18 amu shift of the mass distribution to lower m/z values, whereas the characteristic peak pattern remains constant (Fig. 7-11). If each individual peptide of the 5832-component mixture is cyclized to a proportion of 50%, the resulting mass distribution is a 1 :1 overlay of the linear and the cyclic mass spectra, which can be simulated and plotted with the QMass program. The peak pattern in the overlay has changed slightly and new peak patterns can be observed (marked by lines). The vertical lines show that a peak maximum at 622 m u in the linear library, covers the right side of a broad peak in the theoretical overlay and that a peak maximum at 617 amu in the cyclic peptide library covers the left side of this broad peak. The theoretical simulation of a cyclopeptide library containing linear precursor peptides indicates that peak patterns are very sensitive to changes in the composition of a library. Peak families change their width, relative height and shape significantly when impurities are present in the synthetic library. From our experience with single peptides we concluded that during cyclization of a peptide library, there will be a broad pattern of completely and partially cyclized peptides. Modifications of the peak pattern due to these inconsistencies were not predictable and difficult to simulate. Further investigations on cyclization tendencies in relation to the sequence will give insight into this “hot topic” of conformational restrictions [93]. The calculated mass distribution in comparison to the experimental ESI spectrum is demonstrated for the cyclic peptide mixture c[FAXXXG] (X = randomized position containing all amino acids except Cys and ’ZLp) (Fig. 7-12). The mass distribution calculated for the masses of the [M + H]+ions of the 5832 peptides and the experimental mass spectrum show the same distinct peak groups, which are composed of isobaric peptide families. Deviations from the calculated mass distribution are observed in the experimental spectrum, especially at the lower m/z values (520-620 m u ) which are slightly underrepresented. The interpretation of these deviations is connected with the distribution of hydrophobic and charged peptides within the peak pattern. The theoretical and character-

7.5 CorlformationallyConstmined Peptide Libmries

231

mlz

Figure 7-11. Calculated mass distributions of the linear hexapeptide mixture FAXXXO (top) and the cyclic hexapeptide mixture c F A x x x o ] (middle). Linear and cyclic peptide mixtures show identical characteristic peak patterns which are shifted by 18 amu to lower m/z values. In the superimposition of the 5832 linear and the 5832 cyclopeptides (bottom)some peak groups have changed their width, relative height and shape (an example is marked by vertical lines).

istic XXX-distribution includes all possible combinations of the 18 amino acids in three positions XXX. QMass facilitates the identification of the peptides in each distinct peak group and the determination of the respective amino acids in the XXXcombination. We divided the amino acids of the X-position into two groups: the non-polar amino acids (A, D and E in the protonated form, F,G,I, L,M,N, P,Q, S, T, V, Y) and the positively charged amino acids (protonated H,K and R). All individual peptides from a given peak group were classified according to their composition of amino acids in the X-Dositions. The resulting four classifications were:

232

7 Peptide and CycfopeptideLibruries

- 620 m u

*520

I

0631.40 645.84

I(

673.61

“i”l ll*l

588.44

I

450

500

550

II I I

600

650

700

750

800

850

d Z

Figure 7-12. ESI mass spectrum of the 5832component cyclic hempeptide mixture c[FAXXXG] (X = 17 tamino acids and Gly except Cys and Rp) (top) and calculated mass +IHI+ ions (bottom). distribution of the &

(i) three nonpolar amino acids in the XXX-positions, (ii) two nonpolar and one charged amino acid in XXX, (iii) one nonpolar and two charged amino acids in XXX and (iv) three charged amino acids in XXX.This detailed analysis of the composition of each peak group shows that the nonpolar and hydrophobic peptides (mainly with three nonpolar amino acids in XXX) are found in the left part of the mass spectrum. The right part of the mass spectrum contains mostly two nonpolar amino acids in XXX and one and no nonpolar amino acid in XXX (Fig. 7-13)with the exception of the peak group at 663 m u , where mainly nonpolar amino acids are represented. Thus, from the mass spectrum it is obvious that representatives with 663 amu are missing. This loss of hydrophobic sequences in a cyclopeptide library was caused by the work-up procedure, especially the precipitation from etherh-heptane, where some

233

7.5 Codormationally Constmined Peptide Libmries 631.4 I

645.8

700

mlZ

cxpcrimeneal mlz values

Figure 7-13. ESI mass spectrum of c[FAXXXG] (top) and distribution of hydrophobic and charged peptides in the individual peak group (bottom). The amino acids of the X-position were assigned either to the group of non-polar amino acids (A, protonated D and E, F, G, G, I, L, M,N, P,Q S, T,V, Y) or to the group of positively charged amino acids (protonated H, K, R). The peptides in each group were classified according to their composition of amino acids in the X-position: three (m), two (m), one (D) and no (m)nonpolar amino acid in the XXX-positions. Peak groups at 574,588,602,616 and 663 amu contain mainly three nonpolar amino acids in the XXX-positions, peak groups at 631,645.655,657, 673,688 and 692 amu contain mainly two or less nonpolar amino acids in the XXX-positions.

234

7 Peptide and Cyclopeptide Libmries

cyclic peptides, in particular those containing hydrophobic amino acids in three Xpositions, remain soluble in the organic solvents. Pool sequencing of linear peptide libraries was performed both with peptides precipitated from cold ether and the peptides remaining in the ether supernatant. These two samples showed different amino acid composition in the X-positions. As expected peptides containing many hydrophobic residues were enriched in the ether supernatant (IOI]. The linear nonapeptide sublibrary XXXXEVXRX, prepared for studies of the interdependance in receptor binding of different amino acids by nonapeptide ligands, was analyzed by ESI-MS, and mass distribution patterns were compared with the theoretically expected mass distribution (Fig. 7-14). The loss of lipophilic peptides

I

loo0

.~

I100 mlz

1200

1300

1400

FSgure7-14. ESI mass spectrum of XXXXEVXRX (X= 19bamino acids and Gly) with 64OOOOOO components (bottom) and calculated mass distribution of the [M+H]+ ions (top).

7.6 Pentadecapeptide Libturies for Receptor Binding Studies

235

from the left flank of the mass pattern was not as striking as observed for 03x3 cyclopeptide sublibraries as discussed above. The built-in polarity mediated by the free N-terminal amino group and the additional amide bonds in XXXXEVXRX is obvious. These effects are presumably accompanied by a precipitating supporting effect of interacting peptide chains in highly complex linear nonapeptide mixtures. However, this assumption has yet to be proven experimentally. From our cyclization studies on hexapeptides and hexapeptide libraries we conclude that: (i) The cyclization tendency of linear side chain-protected hexapeptides is much higher than expected from previous studies. (ii) The use of H A W as a reagent for cyclizations is advantageous in comparison to PPA and TBTU in respect of racemization during cyclization. (iii) ESI-MS is valuable (or even essential) for the characterization of cyclopeptide libraries of both high and low complexity. (iv) 2-Chlorotritylchloride-resinis the best choice for the preparation of side chainprotected peptides. (v) Cyclization in solution yields crude cyclopeptides with purities of more than 80% with only a few exceptions. (vi) Precipitation from etherh-heptane is a critical step, which may lead to nonequimolar distribution of peptides in peptide libraries differing in polarities. (vii) Racemization of the residue activated for cyclization should be analyzed.

7.6 Pentadecapeptide Libraries for Receptor Binding Studies The examples of peptide libraries shown in this Section demonstrate that even libraries of the highest diversity and which include longer peptides can be synthesized by the automated premix procedure. These peptide mixtures are obtained in reproducible quality, and the biological results are convincing with respect to the diversity of the applied samples of peptide sublibraries. As shown in the following, we have chosen pentadecapeptidelibraries to screen for peptide ligands binding to HLA-Class I1 molecules. HLA-Class I1 proteins are cell-surface glycoproteins that take up peptides within the cell and present them on the cell surface. T-helper cells (CD4') are part of the cellular immune system and responsible for identification of and response to foreign antigens. They are involved in the formation of a ternary complex between HLAClass I1 protein and its bound antigenic peptide which is necessary for stimulation of the T-helper cell and essential for induction of an immune response. The understanding of the molecular interactions which determine peptide binding to HLA- or MHC-Class I and Class I1 molecules is of meat importance for prediction and identi-

236

7 Peptide and Cyclopeptide Libmries

fication of T-cell epitopes and, therefore, a prerequisite for the design of synthetic vaccines. The numerous different peptides able to bind to a distinct allele of a MHC protein can be considered as a natural peptide library (Chapter 1) [SS]. MHC-Class I binding peptides are 8-10 residues in length and are characterized by allele-specific binding motifs which exhibit strong preference for a few side chains of epitope amino acids at 2 or 3 positions in the sequence and tolerance for many side chains at the other positions (561. In contrast, MHC-Class I1 proteins bind longer peptides with no apparent restriction in length [104], showing also allele-specific motifs which have been more difficult to characterize. Binding of peptides to MHC-Class I1 proteins has been analyzed using methods such as M13 phage display libraries [lOS], substituted MHC-Class 11 binding peptides [lM] and pool sequencing of natural ligands, revealing allele-specific motifs [107]. Peptide binding to the MHC-Class I molecule H-2Kb was examined using soluble octapeptide sublibraries in the positional scanning format [4]. The effects of the individual amino acids in the different positions of octapeptides were specified, and it has been shown that the capacity of every single amino acid side chain to contribute to MHC-Class I binding is influenced by other amino acids in the sequence (Chapter 12). We determined Thelper epitopes within the nucleoprotein (NP)of the measles virus. This protein is the major target of antibodies first detectable after infection. First, 255 overlapping pentadecapeptide amides derived from NP were analyzed for their ability to bind to the human MHC-Class I1 protein HLA DRB1'0101 and secondly pentadecapeptide sublibraries were investigated to examine the contribution of the side chains of a newly defined epitope on DR1 binding. The problems due to the high diversity of these sublibraries, especially with respect to the protocols performed for the synthesis as well as to the binding assays, are discussed in Section 6.2.

7.6.1 Competition Assay for MHC-Class XI Binding Peptides A set of 255 pentadecapeptide amides, spanning the complete NP and overlapping by 13 residues, was screened in a competition assay for binding to the soluble HLA I1 molecule, DRBl'0101. MHC was isolated after lysis of the transformed homozygous B cell line WT-1OOBIS by affinity chromatography with the anti-HLA-DR monoclonal antibody L243, as described elsewhere [108]. MHC-associated self-peptides were released by adjusting to pH 2, and were separated from the solubilized protein by ultrafiltration [109]. NP-derived individual pentadecapeptide amides or peptide sublibraries were incubated with the soluble DRl to compete with the fluorescent labeled DR1 specific ligand AMCA-IM19-31 (AMCA-PLKAEIAQRLEDV)at 37 "C for 48 h (AMCA: 7-amino4methylcoumarin-3-aceticacid). The AMCA-labeled peptide was used in a concentration of 1.5 UM (5.8- and 11.5-fold molar excess

7.6 kntadecapeptide Libraries for Receptor Binding Studies

237

relative to DR1). As competitors, different individual peptide amides were added in a ten-fold molar excess relative to AMCA-IM19-31 and in a three-fold molar excess when using peptide sublibraries. MHC-Class I1 proteins with bound ligands (competitor peptides or AMCA-IM19-31) were separated from nonbound ligands by High Performance Size Exclusion Chromatography (HPSEC) on a Pharmacia Superdex75 HR 5/20 gel filtration column and the effluent was run through a Merck fluorescence spectrophotometer (350/450 nm) and a Merck UV detector (214 nm) set up in series. Competition was quantitated by calculating the ratio of the measured fluorescence- and UV-intensities of the MHC signal. An obvious interaction with DRB1’0101 was shown for 20 NP peptides (competition >60%). The peptide NP 87, QIWVLLAKAVTAPIYT, showed 97% competition and the most pronounced effect among all 255 peptides tested (Fig. 7-15). NP91 NP90 NP89 NP88

AVTAPDTAADSELRR AKAVTAPDTAADSEL LLAKAVTAPDTAADS WVLLAKAVTAPDTAA

NP86 NP85 NP84 NP83 NP82

LAQIWVLLAKAVTAP TILAQIWVLLAKAVT LGTILAQIWVLLAKA MILGTILAQIWVLLA FNMILGTILAQIWVL

wpai QI~VUARAVTAPDT

IM19-31 IM20-2 7

40 60 80 1 0 competition [%] Figure 7-15. Results of MHC-Class I1 binding assays of 10 pentadecapeptide amides overlap ping by 13 amino acids. NP 87 is the most potent competitor. Competition assays were performed with 1.5 p~ of the fluorescence labeled peptide ligand AMCA-IM19-31 and 0.26 plc of DR1. Different peptides were added as competitors in a tenfold molar excess relative to AMCA-IM19-31. IM19-31 and IM 20-27 representpositiveandnegativecontrols, respectively. 0

20

7.6.2 Positional Scanning of a Pentadecapeptide Epitope The contribution of every single amino acid in the sequence of NP 87 on binding to DR1 was investigated by means of l5mer peptide sublibraries. The diversity of peptide sublibraries increases with a growing number of X-positions as outlined in Table 7-10. Each of the pentadecapeptide sublibraries containing 14 X-positions and

238

7 Peptide and Cyclopeptide Libraries

Table 7-10. Molecular diversity of peptide sublibraries with a growing number of X positions. Calculation for i) 19 different amino acids in the X-positions (average molecular mass 110 D), ii) 4.0 pmol (7.2 mg) resin per reaction vessel, iii) 0.3 pg peptide mixture in the assay (0.18 nmol using l5mers with 14 X positions)

Number of X-positions 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

.

Theoretical number of peptides within the library

Copies of an individual sequence generated by premix synthesis

Copies of an individual sequence in the competition assay

15.2 x lo1* 799 x 1015 42.1 x loN5 2.21 x 1015 116 x 10l2 6.13 x 10l2 323 x lo9 16.0 x 1 4 893 x lob 47.1 x lob 2.48 x lob 130321 6859 361 19

0.16 3 57 1089 20691 393 129 7.45 x 106 143 x lob 2.7 x 14 51 x 1 4 973 x lo9 18.5 x 10l2 351 x 10l2 6.7 x lOIs 127 x lOIs

0.000007 0.00013 0.003 0.06

1.3 27 566 12000 263000 5.8 x lob 132 x lob 3.2 x 1 4 80 x 109 2.3 x loi2 86 x loL2

one defined 0-position contains the inconceivable number of 799 x 10” different pentadecapeptides. For our calculation, we assume that during the performed synthesis (4.0 pmol resin per reaction vessel) every sequence has been generated with equal probability. That means an individual peptide is generated in only three copies when using 7.2 mg of resin. For incubation with MHC-Class I1 proteins 0.18 nmol of such a sublibrary is added. Thus, there is a gap of 7700 peptides in the assay due to an incomplete representation of defined peptides in the sublibrary. Consequently, the question arises whether these numerical limitations of the complexity of synthetic peptide libraries sets the border for determination of MHC-Class I1 epitopes. Unequivocally, our experimental results using pentadecapeptide sublibraries containing 14 randomized positions and one defined position (0:one of the corresponding sequence position in NP 87) showed significant differences in their potency to compete with the fluorescence labeled ligand for MHC-Class I1 binding (Fig. 7-16). Hydrophobic amino acids (0= W,V, L, L, A) in the sequence positions 3 to 7 increase binding to DRB1’0101 and the same effects were found for A in position 9 and 12. These results correspond well to the natural binding motif of the HLA DRB1’0101 molecule (1101 and to the results from the crystal structure of the DR1 protein complexed with the influenza virus peptide HA306-318 t1111. Stern et al.

Z6 Rntadecapeptide Libmries for Receptor Binding Studies

239

Figan 7-16. Competition assay using OX14 sublibraries kfmed residues (0) are indicated in their sequence position (1 to 15). X represents the completely randomized pentadccapeptide library (XIS).Competition assays were performed with 1.5 pM of AMCA-IM19-31 (fluorescent ligand) and 0.13 pM of DR1 (MHC-Class I1 molecule). OXlosublibraries (competitors) were added in a threefold molar excess relative to AMCA-IM19-31.

[lll] showed several smaller cavities or pockets within the peptide-binding site of HLA-DR1, five of which accommodate side chains of the bound HA peptide. The largest and most hydrophobic pocket in the binding site has a strong preference for large hydrophobic side chains (W, Y, F, L, I) and appears to be the major determinant of peptide binding to DR1. In contrast to these sequence-specific interactions, the binding also includes sequence-independent contributions from interactions of MHC side chains with the main chain of the peptides. The peptides bind in an extended conformation using a core of 10 to 11 residues for specific interactions. The termini of the peptide can exceed both ends of the open MHC-Class I1 binding UOOVe.

The completely randomized XISlibrary shows a low but significant competition

(WO), which is surpassed by the sublibraries W3 (llvo), V4 (15%), L5 (27Vo), L6 (33%), A7 (l8%), A9 (25%) and A12 (13%). In agreement with the structural data from DR1 [lll], the residues W3, V4, L5 and L6 within the OXI4sublibraries fit to

240

7 Peptide and Cyclopeptide Libraries

the first, and most important, pocket. Residues A7, A9 and A12 support competition, probably by contacting the three smaller cavities in the binding groove. In con. trast, defined residues QI,12, VIO, TI1 and PI3 have a negative influence on biological activity of the library (below 8%). These side chains in the corresponding positions have to be considered as noncontributing or negative with regard to DRI binding. The binding affinities of pentadecapeptide sublibraries with a maximal number of 11 X positions were investigated by competition experiments (Table 7-11). Sublibraries 1 and 2 exhibit triplets of defined amino acids in the N-terminal region. The overall competition effect induced with these sublibraries seems to be defined by the residue with the highest competition in the OXlosublibraries. Thus, the competition of sublibrary 2 (360/0),is in the same range as observed for the X,LX, sublibrary (33%). One residue of the triplet VLL interacts with the first hydrophobic pocket in the binding groove and additional residues have supporting influences. In contrast, competition assays with sublibraries 3 and 4, containing triplets AVT and VTA with one supporting and two disturbing amino acids, show an intermediate (sublibrary 3) or no activity (sublibrary 4) when compared to the completely randomized XI,-library. Thus, binding is influenced by neighbouring residues. The competition-supporting effects of anchor amino acids will be partly compensated by disturbing flanking residues. Sublibraries 5 to 8 contain two triplets, where each triplet is spaced by 2 to 5 randomized positions. The competition clearly increases, especially for sublibraries 5 and 8 which carry the favorable triplet AVT in the C-terminal region. The highest competition effect was measured with sublibrary 8. This is explained by synergistic interactions of the two very favourable residues L6 and A9

Table 7-11. Binding assays with pentadecapeptide sublibraries containing several defined positions. Competition assays were performed with 1.5 wM of AMCA-IM19-31 (fluorescent ligand) and 0.13 FM of DRl (MHC-Class I1 molecule acting as peptide receptor). Peptide libraries (competitors) were added in a threefold molar excess relative to AMCA-IM19-3 1.

Pentadecapeptide sublibraries

Competition “701

1 IWVXXXXXXXXXXT 2 XXXVLLXXXXXXXXT 3 XXXXXXXXAVTXXXT 4 XXXXXXXXXVTAXXT

36.0 18.4

XIWVXXXXAVTXXXX XXXVLLXXXVTAXXX XIWVXXXXXVTAXXX XXXVLLXXAVTXXXX

30.4 40.2 21.7 56.1

9 XWLLAKAVTXXXX 10 QIWVLLAKAVTAPDT

72.9

5 6 7 8

14.6

8.5

94.5

References

241

(only present in sublibrary 8) with DRI. They mediate contacts to the first and second pocket within the binding groove of DRI proteins as described by Stern et al. [lll]. In peptide mixture 9, amino acids with an influence on the competition are combined to a decapeptide core. The high competition (73 To) clearly indicates the mutual dependence on the contribution of the amino acid side chains to DRI binding. This is also outlined by 95% competition for the pentadecapeptide NP 87 itself (Table 7-11). It should be emphasized that due to stoichiometry and the assay conditions only one out of 7700 peptides of a OX,, peptide sublibrary is accessible for MHC binding. Despite this fact, the OX,, peptide mixtures are useful tools in the search for new MHC-Class 11-binding peptide ligands.

7.7 Conclusions Peptide libraries are well-established tools in basic research and as a source for new lead structures. Several methods for their reproducible, effective and fully automated synthesis and analysis have been established. Peptide sublibraries must be optimized in length and their heterogeneity is dependent on the bioassay used for screening. Conformationally constrained peptide libraries are available in high yield and purity by a combination of automated solid phase synthesis and cyclization in solution. Methods for the characterization of linear and cyclic peptide libraries are well developed. Even extremely complex pentadecapeptide libraries are valuable for receptor binding studies and amino acid residues interacting with their respective binding pockets can be identified.

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1991 S. C. Story, J. V. Aldrich, Int. J. Peptide Protein Res. 1994,43, 292-296. [lo01 H. Wissmann, W. Kbnig, R. Geiger, in Peptides 1983 (Eds.: V. J. Hruby, D. H. Rich)! Pierce Chemical Co., Rockford, IL, USA, 1983,pp. 111-114. [loll J. Metzger, K.-H. Wiesmuller, S. StevanoviC, G. Jung, “Analytical methods for the char. acterization of synthetic peptide libraries” in Peptides 1992, Proc. 22nd Europ. Pept. Symp. (Eds.: C. H. Schneider, A. N. Eberle), Escom, Leiden, The Netherlands, 1992, pp. 481-482. 11021 Analytical HPLC was carried out on a Gynkotek system (Germering), consisting of an analytical pump M480, an autosampler GINA160 and a photodiode-array detectoi UVD 320-S. Separations were performed on a Nucleosil C-18 column 5 p, 2 x 250 mm (Grom, Ammerbuch) with a flow rate of 0.3 ml/min at room temperature, detection a1 214 nm. The solvent systems were: A: water/trifluoroacetic acid (100 :O.l), B: aceto. nitrile/trifluoroacetic acid (100:O.l) using a gradient from 0% B to 50% B within 60 min. All pairs of diastereomeric cyclohexapeptides were available and used as standards in HP-U=-analysis for the detection of C-terminally racemized cyclohexapeptides, Retention times of diastereomeric cyclohexapeptides: c(RGDSPK] (16.91 rnin), c[RGDSPD] (17.85 min), c(RGDSPc] (23.79 min), cIRGDSPV] (25.66 rnin). [lo31 Mass spectra were measured with a VG Quattro I1 (Fisons, Manchester) equipped with an ion spray source at atmospheric pressure (API-MS). Solutions of peptides in acetoni. trile/water (1 :1) were prepared (c = 0.1 mg/ml) and were introduced at a constant flow rate of 5 pl/min with a microliter syringe using a medical infusion pump (Harvard A p paratus, Southnatick). I1041 R. M. Chicz, R. G. Urban, W. S. Lane, J. C. Gorga, L. J. Stern, D. A. A. Vignali, J. L. Strominger, Nature 1992, 358, 764-768 I1051 J. Hammer, B. Takacs, E Sinigaglia, J. Exp. Med. 1992, 176, 1007-1013. I1061 C. M. Hill, A. Liu, K. W. Marshall, J. Mayer, B. Jorgensen, B. Yuan, R. M. Cubbon, E. A. Nichols, L. S. Wicker, J. B. Rothbard, J. Immunol. 1994, 152, 2890-2898. [lo71 K. Falk, 0.RC)tzschke, S. StevanoviC, G. Jung, H.-G. Rammensee, ImmunogeneticJ 1994,39,230-242. [lo81 H. Kropshofer, I. Bohlinger, H. Max, H. Kalbacher, Biochemistry 1991, 30, 9177. [lo91 H. Max, T. Halder, M. Kalbus, V. Gnau, G. Jung, H. Kalbacher, Human Immunol. 1994,41, 39-45. [110] J. Hammer, P. Valsasnini, K. Tolba, D. Bolin, J. Higelin, B. Takacs, E Sinigaglia, Celr 1993, 74, 197-203. (1111 L. J. Stern, J. H. Brown, T. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, D. C. Wilev. Nature 1994. 368. 215-221.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

8 Mass Spectrometric Analysis of Peptide Libraries Jorg W; Metzger, Karl-Heinz Wiesmiiller, Stefan Kienle, Jente Briinjes and Giinther Jung

8.1 Introduction Although simultaneous solid-phase peptide synthesis is an established and reliable method [I], it has to be considered that, without careful purification, crude synthetic peptides can contain considerable amounts of byproducts. Thus, identity and purity of crude peptides should be checked routinely by meaningful analytical techniques. In the case of combinatorial peptide libraries, besides the purity, a quantitative aspect also plays an essential role, i. e., it is of interest, whether or not all peptides are present in equimolar amounts. Deviations in equimolarity can result in wrong interpretation of the data obtained in biological assays during the iterative process of searching for a lead structure. The possibilities for analyzing soluble combinatorial peptide libraries with modern methods of mass spectrometry will be discussed. The examples, which were selected to demonstrate these powerful analytical methods, include crude peptide mixtures containing less than 100 peptides, but also peptide libraries consisting of several thousand components.

8.2 Results and Discussion 8.2.1 Analytical Techniques for the Characterization of Soluble Combinatorial Peptide Libraries For quality control of peptides synthesized on a solid support or in solution, standard analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), capillary electrophoresis (CE), mass spectrometry (MS), amino acid analysis (AAA), sequencing by Edman degradation and nuclear magnetic resonance (NMR) are commonly applied methods. In principle, each of these methods can be used for the analysis of mixtures. However, with the growing complexity of mixtures, interpretation of the analytical data gradually becomes more difficult and also less meaningful. HPLCand CE are of limited use for the characterization of peptide libraries containing more than 100 components. Online mass spectrometric detection of the com-

248

8 Mass Spectrometric Analysis of Peptide Libraries

pounds eluting from the HPLC column has a clear advantage over UV/VIS diode array detection, because it allows the differentiation of components even if they are not completely separated by RP-HPLC [2].A maximum amount of information is obtained if different detection methods (e.g., MS and UV/VIS) are used simulta. neously either in series or in parallel. Although it is impossible to distinguish individual components of mixtures con taining several thousand peptides by means of HPLC or CE, HPLC chromatograms or electropherograms (UV/VIS trace or total ion current chromatogram, TIC) can still be useful as characteristic ‘fingerprint’ spectra of the mixtures, because the components are separated into groups of peptides with similar polarity (HPLC) or electrophoretic mobility (CE). Because HPLC and CE are based on different separation principles, the combination of both techniques gives a more detailed view of the composition of a mixture. AAA of the total hydrolysate of a peptide library allows the determination of the percentage of each amino acid present in all peptides of the library. For libraries of general formula X,X2. .. X,, which contain an identical set of amino acids in each randomized position X i , all amino acids should be present in the hydrolysate in equimolar concentrations. As is commonly done, values should be corrected for losses by decomposition of amino acids during hydrolysis. If the kind and the number of residues in particular positions differ, an amino acid composition characteristic for the individual library is obtained. Thus, AAA can be used to reveal larger deviations in equimolarity. A computer program (e.g., program QMass, see below) facilitates the calculation of the expected amino acid compositions (especially of more complex libraries). Peptide pool sequencing by Edman degradation allows constant positions with one or only few residues to be distinguished from fully randomized positions [3]. A limitation, however, is that it can reveal neither modified peptides (e. g., peptides containing acid-labile side chain-protecting groups or piperidine-adducts) nor allow sequence determination of N-terminally blocked or cyclized peptides. Edman degradation and mass spectrometry are the most suitable analytical techniques for characterizing peptide libraries. Edman degradation, however, is relatively slow compared with mass spectrometry. Nuclear magnetic resonance. 2D NMR (e.g., heteronuclear multiple quantum coherence, HMQC) can also be used to characterize mixtures consisting of not too many components and having a sufficient purity [4]. The superimposition of cross-peaks in the 2D maps, however, may complicate any complete assignment and interpretation of the data. But in a similar way to the 2D HPLC-MS contour plots (see below) the 2D NMR spectra of even more complex libraries are useful as characteristic finnerwint mectra of the mixture.

8.2 Results and Discussion

249

8.2.2 Mass Spectrometry of Peptides Peptides are thermolabile molecules which decompose if heated to temperatures beyond the melting point. Thus, conventional ionization techniques like electron impact ionization cannot be used for molecular mass determination of underivatized peptides. ‘Soft’ ionization techniques are capable of producing molecular ions (usually in protonated or cationized form, e.g., as [M + HI+ or [M + Na]’) with high abundance. The most common soft ionization technique for peptide analysis in the 1980s was fast-atom-bombardment (FAB-MS) [5]. In the meantime two new exciting techniques, electrospray ionization (ESI)[6, 71 and matrix-assisted laser desorption ionization (MALDI) [8] are available as powerful techniques for determinating the relative molecular masses (RMM) of peptides and proteins. These techniques are characterized by a high sensitivity (low pmol to fmol range and below) and the capability to measure very high mass (i.e., far beyond 100 kDa). At present, these two ionization techniques constitute the mass spectrometric methods of choice for biopolymers (e.g., proteins, oligonucleotides and carbohydrates) and their non-natural analogs.

8.2.3 Electrospray Ionization (ESI) ESI is one of the softest ionization techniques known so far. During the electrospray process, ions preformed in solution by protonation or cationization are ejected from nanometer-sized droplets into the gas phase. Even highly polar and thermolabile analytes form protonated molecular ions with high abundance. Molecules with several protonation sites (in the case of peptides and proteins mainly the residues Arg, Lys or His) form multiply charged ions [M + nH]’+ . Therefore, the ESI mass spectrum of a protein or a oligonucleotide (the latter is usually measured in the negative ion mode) contains not one but a series of several molecular peaks with a (non symmetrical) bell-shaped intensity distribution [9]. Fragmentation during ESI is usually low. However, the gas-phase ion undergoes collisions in the transport region with air or with solvent molecules before it reaches the high vacuum region of the mass spectrometer. The collision energy, which influences the degree of fragmentation and the charge distribution of polyelectrolytes, depends to a large extent on the so-called ‘orifice’ or ‘skimmer voltage’. At low orifice voltages, high charge states and little fragmentation are observed. If one wants to enhance the intensity of singly charged ions or the degree of fragmentation (‘orifice’ or ‘skimmer’ or ‘cone fragmentation’), the orifice voltage has to be set to high values [lo].

250

8 Mass Spectrometric Analysis of Peptide Libraries

8.2.4 Peptide Families in Peptide Libraries Each peptide library defined by the constant and randomized positions has a characteristic mass distribution, which can serve as a fingerprint for the mixture. The total number of peptides of a peptide library is X" where X = number of residues in the mixed position and n = peptide length (Table 8-1). For simplification of synthesis, the residues cysteine (oxidation leads to crosslinks) and tryptophan (acid lability, tendency to chemical modifications) are sometimes avoided in library construction. If all the other protein amino acids are incorporated into the mixed positions, the number of peptides in such a chemically more stable library is 19" (Cys omitted) and 18" (Cys and Trp omitted). The lightest and the heaviest peptide(s) form the lower and upper mass limit within which all the other peptide masses are found [lo, 111. This mass range becomes greater with increasing peptide length. The 400 peptides of a complete dipeptide library X,X2containing all protein amino acids in the randomized positions, for example, have nominal (i.e. integer) masses between 132 and 390 (difference between lightest and heaviest peptide is 258 U), whereas the 1.28 billion peptides of the respective heptapeptide library are found between the mass of 417 and 1320 (mass difference 903 U). Peptides of the same nominal mass form an 'isobaric peptide family'. The total number of peptide families, the m/z value of the family and the number of peptides in each isobaric family are specific for a given peptide library.

Table 8-1. Number of compounds in peptide libraries of different length XI to X,, which arise by combination of i = 20 (e.g., all protein amino acids), 19 (e.g. Cys is missing) and 18 (Trp and a further amino acid e.g., Cys, are missing) amino acids in the different mixed positions X,. RMM are the masses of the peptides with the smallest and the largest mass (for i = 20: peptides G, and W,, and for i = 18: peptides G, and Y,,). Am (W,-G,) and Am (Y,-G,) are the respective mass differences between G, and the heaviest peptide W, or Y, of the mixture. i = Characteristics n = 1

2

3

4

8000 6859 5832

160000 3 200000 64000000 130321 2476099 47045881 104976 1889568 34012 224

5

6

7

of the library 2o Number of

:! peptides

20 19 18

400

361 324

1280000000 893 871739 612220032

132.0 189.1 246.1 390.2 576.2 762.3 344.1 507.2 670.3

303.1 948.4 833.3

360.1 1134.5 996.4

417.2 1320.6 1159.4

258.1 387.2 516.2 212.1 318.1 424.2

645.3 530.2

774.3 636.3

903.4 742.3

8.2 Results and Discussion

251

8.2.5 Calculation of Mass Distributions and Peak Clans The calculation of the mass distribution of even very small peptide libraries is tedious and time consuming if performed ‘by hand’. For more complex libraries, a computer program is absolutely necessary. With QMass, a UNIX-based program designed in the authors’ group, the masses of all peptides present in the library (up to several hundred billions), can be calculated very quickly. The program has a couple of additional features, such as the selection of charge states, an import function for experimental mass spectra, and analysis functions for byproducts and side reactions, which are very helpful for a rapid interpretation of ESI mass spectra. QMass is described in more detail by J. Briinjes et al. in Chapter 18 of this book. The mass distribution calculated for the masses of the [M + HI+ions of the 6859 peptides of the library XXINFXKL (X is a randomized position where all protein amino acids except Cys occur; cf. Table 8-1) shows distinct peak groups, which are composed of several isobaric families and for which we suggest the term peak ‘clans’ (Fig. 8-la).

8.2.6 Mass Spectrometry of Peptide Libraries Experimental determination of mass distributions requires mass spectrometric tech. niques which can cope with complex mixtures. Surface ionization techniques likt FAB, which are still commonly used for peptide analysis, tend to discriminate or even suppress the ionization of components [12]. Thus, there is a danger that the recorded mass spectrum of the mixture is not representative of the actual distribution of constituents in the sample. The same problems also arise if other ionization technique$ widely used for peptide analysis such as plasma desorption ionization (PD-MS)or matrix-assisted laser desorption ionization (see below) are applied.

8.2.7 Electrospray Mass Spectrometry

- A Potent Method

for the Characterization of Peptide Libraries ESI mass spectrometry has several advantages over other possible ionization tech-

niques, which makes it the method of choice for mass spectrometric analysis of pep tide libraries [lo, 11, 13-15]. The sample is introduced into the electrospray source in dissolved form, which greatly facilitates sample preparation and sample introduction into the mass spectrometer. Both preparation and introduction can be automated conveniently by the use of an autosampler (2, lo]. This allows the measurement of about one sample per minute so that hundreds of samples can be run automatically overnight.

252

a)

8 Mass Spectrometric Analysis of Pepride Libmries

992

1251

800

900

loo0

I200

Nominal Mass

Figore 8-1. a) Calculated mass distribution of the octapeptidemixture XXINFXKL (X = randomized position with X = all protein amino acids except Cys; theoretically 6859 peptides); the exact calculated monoisotopic masses of the FI+ HI+ions were rounded to the closest integer. b) ESI mass spectrum (orifice voltage: +lo0 V).; the masses of the lightest (GGINFGKL; m/z 805) and heaviest peptide (WWINFWKL; m/z 1193) of the rnixture are indicated by vertical dotted lines. The analyzing quackupole was scanned with a step sue of 0.1 mass units. Therefore, the spectrum looks more ‘spiky’ than the calculated mass distribution, which is based on integer masses.

8.2 Results and Discussion

25:

-

8.2.8 Relative Ion Intensities A Measure for the Number of Isobaric Peptides in Peptide Libraries? For mixture analysis by mass spectrometry it is desirable that the relative ion intensities in the spectrum reflect the actual composition of the mixture. For example, the peak intensities (or more precisely the peak areas) of the molecular ions in the spectrum of a binary mixture containing analyte A and B in a 1 : 1 ratio ideally should be identical. This is only true if the ionization of constituent A is not influenced by constituent B and vice versa, i.e., if discrimination effects are absent. Under these conditions, the mass spectrum should reflect the calculated mass distribution. The ion intensities for molecular ions of different peptides with the same mass should then be additive and sum up to the intensity of a mass peak of the respective isobaric family. If all components of a mixture are present in equimolar amounts, the relative intensities of mass peaks of isobaric families should be a measure of the number of isobaric peptides in these families. The absolute ion intensity observed in a mass spectrum chiefly depends on the concentration of the analyte. Within the dynamic range, ion intensity is proportional to concentration. For ESI, linearity between ion intensity and concentration is usually found at concentrations below 5 x M (161. At higher concentrations a saturation effect is observed leading to a deviation from linearity (in this case the number of analyte molecules exceeds the number of available charging species, e g., protons). So far, complete mass spectrometricsuppression of individual compounds of peptide libraries has never been observed, if their concentrations are within the dynamic range. The degree of mass spectrometric discrimination with ESI is usually low; however, the latter depends very much on the structural similarity or dissimilarity (hydrophilicity, polarity, basicity) of the individual components of the mixture During the last five years hundreds of synthetic peptide libraries have been compared and it has been found that the mass and intensity distributions observed in the ESI mass spectra of peptide libraries are very close to the expected calculated distribution (provided that they are pure and the sample solutions are not highly concentrated). This indicates that the majority of the peptides of a peptide library has very similar physical properties, leading to similar ionization yields in the ESI process. Each library, however, usually also contains a much smaller number of components, whose properties differ more significantly from each other, for example, arginine- in comparison to tryptophan-containing peptides. For such peptides more pronounced deviations from an ‘ideal’ behaviour may be observed. The similarity between calculated mass distribution and the experimental ESI spectrum is demonstrated for the peptide mixture XXINFXKL (X = randomized position containing all amino acids but Cys; Fig. 8-1 a/b). The relative intensities of the mass peaks in the ESI mass spectrum are almost proportional to the number of peptides belonging to each isobaric family. Figure 8-2a shows an expanded view of the

254

8

0’

Mass Spectrometric Anabsis of Peptide LibMries

.

942

945

947

950

952

955

950

952

955

mlZ

b)

80

70

4i60

-g 50 a $40

3

B

30 20 10 0

942

945

947

MaSS

Figure 8-2. a) Expanded region of the peak clan at m/z 949 in the ESI-MS of XXINFXKL (indicated by an arrow in Fig. 8-1 b). b) Calculated mass distribution for the different isobaric peptide families showing the number of isobaric peptides corresponding to each isobaric family of this peak clan (the natural isotope distribution was neglected in this calculation).

peak clan at m/z 949, which is indicated by an arrow in the full range ESI mass spectrum (Fig. 8-1 b). The ion intensities of the isobaric families in the different clans are the result of an addition of the ion intensities of the individual isobaric peptides of each family (calculated mass distribution for the peptide families of the clan at m/z 949 see Fig. 8-2b). For these peptides, discrimination effects seem to be low, and the ionization of the constituents does not seem to be very dependent on the total composition of the mixture. Such an additive effect can also be demonstrated by mixing two or more different libraries in defined concentrations. Figures 8-3a and b show the ESI mass spectra of

8.2 Results and Discussion

255

a) - l o o ]

c

1loo

F ~ r 8-3. e a) ESI-MS of the 48-component peptide mixtures ANYRY(T,I,E,S)(N,K,Q)(L,M, I,Wand b) SIWRF(T,I,E,S)(N,K,Q)(L,M,IY)(the three C-terminal positionsare randomized with three or four different residues, respectively). c) Superimposition of both spectra a) and b). d) ESI-MS of a 1 :1 mixture of ANYRY(T,I,E,S)(N,K,Q)(L,M,I,V)and SNPRF(T,I,E,Sl (N.K.OWL.M.1.V) (96 rtebtides).

256

8 Mass Spectmmetric Analysis of Peptide Libraries

two different peptide mixtures : ANYRY(S;r,I,E)(N,K,Q)(V,I,L,M)and SNPRF(S,T,I,E) (N,K,Q)(V,I,L,M),each consisting of 48 octapeptides in 16 different isobaric families

(in the formulas, residues occurring in the mixed positions are given in brackets) [lo, 141. The m/z range of the [M+ HI+ ions of these peptides is m/z 920-1008, and 986- 1074, respectively. In Fig. 8-3c these two spectra are superimposed (i. e displayed in one graph). Figure 8-3d shows the ESI mass spectrum of a 1 : 1mixture of these two 48-component mixtures. The intensity distribution in the superimposed spectra of Fig. 8-3c and in the spectrum of the mixture are very similar (Fig. 8-3d). There is almost no interference between the individual peptides of this particular mixture during ionization. To elucidate quantitative or semiquantitative aspects of ESI mass spectrometry of mixtures, ionization yields and relative ion intensities have to be studied systematically. This is not an easy task, because it is tedious or impractical to prepare ideal peptide mixtures containing hundreds, thousands or even millions of peptides with defined concentrations. Mixtures having less than ten components were investigated [lo]. Essentially, these studies showed that the ionization of the majority of components is not influenced much by all other components. The most striking deviation of calculated and measured ion intensities is observed when the peptides vary significantly in hydrophobicity or charge, as, for example, oligoarginine and oligotryptophan; also peptides of different lengths are frequently ionized with different efficiency leading to deviations up to 50% from the ‘expected’ ratios. Peptides of a library with strikingly different properties are in the minority. In peptide libraries containing large numbers of constituents the majority of the components have a similar ‘average’ basicity, hydrophobicity, etc., and are ionized equally efficiently. If a few peptides of the mixture have a higher or a lower ionization yield, the overall intensity pattern essentially is not changed much. Therefore, differences in experimental and theoretical intensity distributions very often indicate a deviation from equimolar concentrations. To estimate the relative composition of the mixture on the basis of intensity distribution, it is important to consider all the ions which are related to the molecule of interest. These are, in particular, all possible multiply charged ions [M + nH]”+, which are formed under the chosen conditions, adduct ions (eg., [M + Na]’), fragment ions and isotopic ions (eg., the I3C ion). A ‘nonideal’ relative ion intensity distribution is often caused by the fact that the total ion intensity corresponding to a particular molecule arises from all these ion species. If fragment ions or multiply charged ions are present in the spectrum, the ion intensities in the [M+ HI+ region do not give a representative view of the relative composition either. Fragmentation and formation of multiply charged ions should be avoided by choosing a suitable ‘orifice’ or (‘skimmer’) voltage. This optimal voltage has to be determined experimentally. However, this is often difficult to achieve. Under condi-

8.2 Results and Discussion

257

tions which suppress the formation of multiply charged ions, the degree of fragmentation is high, whereas conditions which suppress fragmentation favor the formation of multiply charged ions. The ESI mass spectrum of the 6859 component mixture XXINFXKL (see above] was recorded at a high orifice voltage of 140 V (Fig. 8-4). Although no doubly charged ions are observed, fragment ions are formed under these 'harsher' electrospray conditions. These are found in the low m/z region of the spectrum. All 6859 peptides of this mixture have an identical C-terminal part KL and a common inna part with the sequence INF. Fragmentation of the peptides in the transport region of the ESI source therefore leads to high abundant fragment ions INF at m/z 375 (plus further fragment ions thereof, cg., the internal fragment ion IN at m/z 228) and Y{ at m/z 260 (nomenclature of fragment ions see [17]). Mass peaks corresponding to fragment ions, which contain randomized positions, have lower intensities due to the lower percentage of peptides involved (Fig. 8-4). If the program for calculation of the mass distribution does not consider the natural isotope distribution, the simulated spectrum is simplified and might differ slightly from the experimental spectrum owing to this inaccuracy. The "C ions of an isobaric peptide family with a molecular mass Menhance the 12Cintensity of the

L

INF

3:

578

rj

593

-m

707

1193 I

69 1

lo00

t

Figure 84. ESI mass spectrum of the octapeptide mixture XXINFXKL (X= randomized position with X = all protein amino acids except Cys; theoretically 6859 peptides) recorded at a high orifice voltage of + I 4 0 V, a condition which favors fragmentation. The boxes mark the regions of the [M + HI+ions and of the fragment ions Yt-Yd, (the numbers at the upper left and right comer of each box are the m/z values of the lightest and heaviest ion of the reswctive s d e s ) .

250

8 Mass Spectrometric Analysis of Peptide Libraries

ions of the isobaric peptide family with the mass M + 1. The monoisotopic masses (based on C = 12.00000) of the [M+ H]+ ions of the two peptides SIINQEKL and SIINEEKL (m/z 945.5 and 946.5, respectively) differ by only one mass unit. Figure 8-5 shows the calculated natural isotope distribution of protonated molecular 945.5

947.5

945

946

941

948

949

950

Figure 8-5. Calculated isotope distribution of the monoisotopic [M+ HI+ ions of a 1 :1 mixture of octapeptides SIINQEKL (C41H74N11014; m/z 944.5) and SIINEEKL (C41H+\, OI5;m/z 945.5) based on the assumption that the ion intensities are additive. Table 8-2. Possible reasons for the deviation from linearity between peak intensity (area) and number of isobaric peptides in peptide libraries

a) Peptides are present in equimolar amounts: 0 ion yields of individual peptides differ from each other 0 ion intensities are distributed to several charge states 0 fragmentation 0 clustering b) Peptides are not present in equimolar amounts: 0 solubility problems 0 aliquots of resin were not equal (split synthesis) 0 differences in reaction rates (mixed Fmoc-AA approach) 0 formation of byproducts - truncated sequences (coupling problems) uncleaved side chain-protecting groups (cleavage conditions) - chemical modifications (oxidation, alkylation, acylation, etc.)

-

8.2 Results and Dkcussion

259

ions for a 1 : 1 mixture of these two peptides. The calculation is based on the assump tion of an additive behaviour of the ion intensities. If the algorithm for the calculation of the mass distribution neglects this natural isotope distribution, only two peaks with identical intensities at m h 945.5 and 946.5 are obtained. Some major reasons for a deviation from the ‘ideal’ relative intensity distribution are summarized in Table 8-2.

8.2.9 Experimental Conditions for Recording ESI Mass Spectra of Peptide Libraries For ESI-MS analysis, the peptide libraries have to be dissolved in a suitable solvent, usually a mixture of methanol or acetonitrile and water. Lowering the pH of the sample by addition of formic or acetic acid (ca. 1 - 10010) favors the ionization process by enhancing the degree of protonation of the peptides in solution. Above all, the choice of the solvent system is dependent very much on the solubility of the components of the library. Mass peaks of peptides with a tendency to precipitate in solvents commonly used for ESI-MS might be missing in the spectrum, regardless of a successful synthesis. If solubility problems arise, the addition of trifluoroethanol, hemfluoropropanol or trifluoroacetic acid can be advantageous. The analyte concentration has to be within the dynamic range. The upper concentration limit of the linear range of a peptide is determined by the concentration chi of this single peptide, rather than by the total peptide concentration c (n is the number of peptides of the mixture) [lo]. Thus, solutions with total peptide concentrations higher than commonly used for ESI-MS are suitable (ca. 1-2 mg/ml, if there are no solubility problems). Within the dynamic range, the relative intensity distribution is independent of the total concentration of the peptide library [lo]. The ESI detection limit for a peptide with a conventional ESI source (flow rate of 2-5 pllrnin, fused silica capillary of 100 pm ID) is usually ca. 200 fmol. At this sensitivity, ESI is capable of detecting individual peptides of a library (assuming an average relative molecular mass of lo00 and a total peptide concentration of 2 mg/ml), if it does not contain more than loo00 different peptides. However, since the ion intensities of many isobaric peptides sum up to the observed intensity of the mass peak of the isobaric family, it is possible to obtain spectra of mixtures containing many more peptides (millions or even billions). Apart from the composition and the pH of the solvent, the most important parameters are the orifice/skimmer voltage of the ESI source. By influencing the collision energies of the analyte ions in the transport region of the ion source, the applied voltage effects fragmentation and charge distribution. The formation of multiply charged ions and fragment ions can complicate the interpretation of ESI mass spectra.

260

8 Mass Spectromelric Analysis of Peptide Libmries

8.2.10 Mass Resolution and Accuracy of Mass Determination The shape of the mass distribution or rather the diagram ‘number of peptides versus mass’ changes slightly depending on the value the masses were rounded to after mass calculation, i.e., if the masses were rounded to integers, 0.5 or 0.1. Usually the mass distributions of peptide libraries calculated with mass intervals of 1 or 0.5 show clearer and more distinct clans than those calculated with a higher accuracy of 0.1. The same effect is observed if the ESI mass spectrum is recorded with a step size of I or 0.1 U, or at higher or lower mass resolution. In general, the ESI mass spectrum and the theoretical calculation should be based on the same accuracy. The theoretical mass distribution and ESI mass spectrum differ slightly if the program for calculation does not consider the natural isotope distribution of the molecular ions.

8.2.11 Mass Analyzers Peptides having masses which exceed the upper mass range of a quadrupole mass analyzer can be measured only if they form multiply charged ions. The doubly charged ion [M+ 2HJ2+of a peptide with the mass of 4000, for example, gives a mass peak at m/z 2001. For the comparison of relative ion intensities, it is advantageous if only a single ion species (e.g., singly charged ions) is formed. By adjusting the orifice/skimmer voltage, the formation of ions other than singly charged ones can, in principle, be suppressed (see above). For larger molecules, the analyzer should therefore have an increased m/z range (most commercially available mass spectrometers can be equipped optionally with quadrupole analyzers with a m/z range up to 4OOO). Most electrospray mass spectrometers are equipped with quadrupole mass analyzers. These are relatively easy to operate and are more compatible with the high voltages of the ESI source than, for example, sector instruments. Quadrupole mass analyzers usually have a resolving power m/Am of no better than ca. 2000. During the past few years there have been efforts to combine ESI with other analyzers: with sector instruments, time-of-flight analyzers (TOF) or hybrid systems (any combination of these analyzers). These analyzers differ in ease of operation, resolution power and in mass range available. TOF analyzers, which are part of most commercial laser-desorption ionization mass spectrometers, in principle, have no upper mass limit. Mass determination is based on the time it takes for an ion to reach the detector after being formed in the ion source. The mass resolution of TOF analyzers has improved greatly over the last few years (resolving powers between 5000 and 10000 have been demonstrated). Therefore, a combination of ESI with a TOF is highly promising.

8.2 Results and Discussion

261

8.2.12 Fourier Transform Ion Cyclotron Resonance ESI Mass Spectrometry A very exciting and relatively new field is the combination of ESI with Fourier transform ion cyclotron resonance mass spectrometers (ESI-FT-ICR-MS). Ions introduced from the ESI source are trapped for extended time periods, owing to the combination of magnetic and electric fields. In contrast to other mass spectrometric methods, both high sensitivity and high resolution can be obtained simultaneously

WI. The high resolution capabilities of ESI -FT-ICR-MS allow differentiation of components with very similar mass. Figure 8-4a shows the ESI-Fourier transform mass spectrum of SIINXEKL (X = 19 protein amino acids, no cysteine). Supposing all peptides were present in equimolar amounts, and the ionization yields for all components were exactly the same, one would expect a relative ion intensity of 100% for the isobaric peptide families with Ile/Leu and Gln/Lys substitution and 50% for all other peptides. The relative peak intensities observed in the spectrum suggest that the peptides with X = S,V, T, N, E, H and R are present in almost equimolar quantities (relative intensities 40-60%), whereas the other peptides are under- (X = G, A, D, M, Y, W) or over-represented (X = P, F). The high intensity of the mass peak corresponding to the F-substituted peptide is due to the presence of two isobaric peptides SIINFEKL and SIINM(0)EKL (a peptide with an S-oxidized methione residue) with a nominal mass of 964. No mass peak for the nonoxidized peptide SIINMEKL (nominal mass 948) is found, which indicates that the methionine residue is completely oxidized. The presence of these two isobaric peptides can be shown clearly by HPLC-MS (see below), because they have quite different retention times. Using SWIFT (stored waveform inverse Fourier transform), a technique which allows the selective ejection of high intensity ions or low mass background ions from the FT-ICR cell to permit observation of weaker ions [19], the presence of these two peptides with very similar mass (their mass difference is only 0.033 U) was proved (Fig. 8-6b). Applying the same technique, it was also possible to separate the Q-substituted from the K-substituted peptide (the mass difference of these two peptides is 0.036 U). Rvo clearly separated mass peaks of the peptides SIINKEKL and SIINQEKL with relative intensities of almost 50% are observed in the FT-ICR-MS (Fig. 8-6c).

8.2.13 Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) Apart from ESI, the most important ionization technique for the analysis of biopolymers is matrix-assisted laser desorption ionization [8]. It is a highly sensitive

262

8 Mass Spectrometric Analysis of Peptide Libraries

Figure 8-6. a) ESI-Fourier transform mass spectrum of SIINXEKL (X = 19 protein aminc

acids, no cysteine); b) High-resolution mass separation of SIINFEKL and SIINM(0)EKL (mass difference 0.033 U; m/Am 50000) and c) of SIINKEKL and SIINQEKL (mass differ. ence 0.036 U; m/Am 6OOOO) using the SWIFT techniaue.

8.2 Results and Discussion

263

technique, which allows mass determination of molecules of several hundred thousand Daltons. For MALDI-MS, the peptide is dissolved in a suitable solvent (e.g., methanollwater) and mixed with the same volume of a UV-absorbing matrix (e.g. , 2,5-dihydroxybenzoic acid, Dhb). The sample is then dried on a stainless steel target and ionized by short laser pulses. The possibilities of MALDI for the analysis of peptide libraries were investigated. Figure 8-7a shows the positive-ion MALDI mass spectrum of the 48-component ocThe mass peaks of all expected tapeptide mixture ANYRY(S,T,I,E)(N,K,Q)(V,I,L,M). peptides ([M + HI+ m/z 986.5-1075.5) are present in the spectrum. The intensity distribution of the mass peaks is similar (though not completely identical) as it is found in the ESI mass spectrum (Fig. 8-7 b). However, usually a more pronounced discrimination effect for particular compounds is found in MALDI (see below). Figure 8-7c shows the negative ion mass spectrum of the same octapeptide mixture containing phosphotyrosine in sequence position 5 (m/z 1066.5- 1154.5). Figure 8-8 shows the MALDI-TOF mass spectrum of the 19-component octapeptide mixture SIINXEKL (see FT-MS investigation above). When compared with the ESI mass spectrum, it turns out that the major mass signals correspond to peptides with more hydrophobic residues X = Trp, Phe, Ile/Leu, but also to peptides having charged residues arginine, lysine and histidine in the X position; the relative intensities of these ions differ slightly from laser shot to laser shot, so that in other spectra of the same sample the signal for the peptide with X = W became more intense than for the arginine peptide (data not shown). In principle, two effects can be responsible for this observation. First, components can be separated spatially on the target during sample preparation. This could lead to an accumulation of particular components either in the crystalline or amorphous regions on the target. In the amorphous regions (i.e. where no co-crystals of analyte and matrix are formed), ion yields are usually zero or very low, so that low peak intensities result for these components. Second, some components might be discriminated mass spectrometricallyor totally suppressed. Such effects are common for surface ionization techniques and they make it difficult or impossible to reproduce the mass spectrum exactly with respect to signal intensities. The MALDI spectra obtained do not necessarily represent the actual distribution of the peptides in the mixture, which is the major disadvantage of MALDI TOF-MS in the analysis of compound libraries. Similar problems arise when FAB-MS or PD-MS is used. In comparison to ESI, MALDI-TOF has some advantages: (i) when the masses of the peptides exceed the mass range of the quadrupole analyzer, and (ii) when the molecules tend to fragment in the ion source. Lipopeptides are a typical example. were elongated N-terThe peptides of the mixture ANYRY(S,TJ,E)(N,K,Q)(V,I,L,M) minally with the lipohexapeptide Pam,Cys-SKKKK [20] (Pam,Cys = N-palmitoyl-S[2,3-bis(palmitoyloxy)propyl]-cysteinyl) affording a 48-component lipotetradecapeptide mixture. Electrospray MS is well suited for the analysis of such lipopeptides [211.

8 Mass Spectrometric Analysis of Peptide Libraries

264

lo00

980

960

I020

1040

1060

1080

1040

1060

1080

lah

b)

1013%

too

g

-

7s

br

o

B

r?

1

2s

0

-

E !

0.10

960

lo00

980

1020 nlz

.

1051)

1070

1090

1110

1130

1151)

I170

Figure 8-7. a) Positive ion MALDI-TOF mass spectrum and b) ESI mass spectrum ol ANYRY(SyT,I,E)(NyKyQ)(V,I,L,M) (m/z 986.5- 1075.5). c) Negative ion MALDI mass spec. trum of the same octapeptide mixture containing phosphotyrosine in position 5 ( m h 1066.5- 1154.5) (matrix 2.5-dihydroxybenzoic acid).

8.2 Results and Discussion

265

R

W

850

900

950

loo0

1050

1100

1150

Figure 8-8. MALDI-TOF mass spectrum of SIINXEKL (same sample as used for FT-MS; cf. Fig. 8-6). The spectrum does not reflect the actual composition of the mixture. The most intense mass peaks observed in the spectrum are peptides with hydrophobic residues (W, Y, F, I/L) or positively charged residues (R, K, H) in the variable X-position (matrix: 2,5-dihydroxybenzoicacid).

However, the masses of the singly charged [M + HI+ions (m/z 2480-2568) are too high to be measurable by the triple-quadrupole mass spectrometer used in this study (a Sciex API 111). Although all components form doubly charged ions by ESI, isobaric families differing only a little in mass can be distinguished more easily in the [M+ HI+ region. Therefore, a MALDI spectrum was recorded (Fig. 8-9). This allowed the identity of the mixture to be unequivocally confirmed. MALDI has a higher sensitivity than ESI and is less sensitive to contaminations by nonvolatile salts, buffers or detergents. Both advantages, however, are not relevant for the analysis of synthetic peptide libraries; usually, these can be obtained in mg amounts and in sufficient purity.

8.2.14 Tandem Mass Spectrometry (MS-MS) of Peptide Libraries and Diagnostic Ions Tandem mass spectrometry (MS-MS) can be used to obtain structural information on individual peptides or byproducts in peptide libraries [lo]. In a typical tandem MS experiment (a daughter ion scan), the ion of interest is selected by a first mass analyzer (e.g. a quadrupole analyzer). The selected ion enters a collision cell (a Rfonly quadrupol) and collides with the collision gas (e.g., argon) atoms, which causes fragmentation of the ion. The product spectrum is then recorded with a second ana-

8 Mass Spectrometric Analysis of Peptide Libraries

266

1620.8

-b

0.08

v)

I

-P-

.-af

0.06

0.04

0.02

2470

2520

2570

mh

Figure 8-9. a) Positive ion MALDI-TOF mass spectrum of the lipopeptide mixture

Pam,Cys-SKKKK- ANYRY(S,TI,E)(N,K,Q)(L,M,IY) (m/z 2480.3-2568.4; the mass peak at m/z 1620.8 corresponds to bombesin, which was used for calibration). b) Expanded view of the [M + HIC region (matrix 2’5-dihydroxybenzoic acid).

lyzer (this configuration of three quadrupoles in a series is called a ‘triple-quadrupole mass spectrometer’). The fragment ions formed are specific for the precursor ion and can be used for the elucidation of the primary structure of a peptide. Parent ion and neutral loss scans, two further tandem mass spectrometric techniques, are helpful for the identification of common structural features of molecules in a mixture. Therefore, they are of particular interest for the analysis of peptide lihmriec.

8.2 Results and Discussion

267

Crude peptides usually contain varying amounts of byproducts, which are formed during solid-phase peptide synthesis or cleavage of the peptides from the resin. For example, truncated sequences or modified peptides might be present. Common chemical modifications include 0-acylation by trifluoroacetic acid, oxidation of methionine-containing peptides, alkylation by reactive species formed during cleavageldeprotection (e.g., tert-butylation). In addition, crude peptides can be contaminated by low-molecular mass compounds (e.g., scavengers) or salts. The synthesis of a defined peptide library is usually more complex than that of a single peptide; therefore, peptide libraries should be checked even more carefully for the presence of byproducts than single crude peptides. The ESI mass spectrum of peptide libraries often directly shows whether byproducts are present or not. They are indicated by a second ‘population’ of mass peaks shifted to higher or lower mass than the masses expected for the unmodified peptides of the library. n p i c a l mass shifts caused by incomplete coupling of a particular residue, by incomplete removal of side chain protecting groups or by chemical modification are shown in Table 8-3. Table 8-3. Determination of byproducts by ESI-MS based on the observed mass difference

Am between [M+ HI+ of unmodified peptide and byproduct (negative Am: deletion peptides with one residue missing; positive Am: chemically modified peptide). Am - 186 - 163 - 156 - 147 - 137

- 131

- 129 - 128 - 128 - 115

-114 -113 -113 - I03 - 101 -99 - 97 - 87 - 71 - 57

Assignment (deletion) W,Trp Y,Tyr R, Arg F, Phe H,His M, Met E, Glu

Q,Gln K, LYS

4 ASP N, Asn L, Leu I, Ile

c, CYS T, Thr V, Val P, Pro S, Ser A, Ala G, G b

Am

16 42 56 66 + 96 + 222 + 242 + 266

+ + + +

Assignment (modification) oxidation acetyl tert-butyl

piperidine amide trifluoroacetyl 9-fluorenylmethoxycarbonyl trityl

2,2,5,7,8-pentamethylchromane-

6-sulfonyl

268

8 Mass Spectrometric Analysis of Peptide Libraries

Crude peptides, which contain side chain protected residues (structures of common protecting groups are shown in Fig. 8-10), can be identified collectively by parent ion or neutral loss scans, if !be precursor ion (usually a [M+ HI+ or [M + 2H]*+ ion of the respective modified peptide) is capable of forming 'diagnostic', fragment ions by collision-induced dissociation. Some of these MS-MS techniques are demonstrated below for the 19-component mixture SIINFEKX (X is a randomized position containing all protein amino acids except cysteine).

-- N H - p

-

-0-

P 2

Ser(tBu)

Fmoc

Ser(Tfa) Figure 8-10. Structural formulas of chemically modified residues or residues containing side chain-protecting groups (tBu, rerr-butyl; Pmc, 2,2,5,7,8-pentamethyIchrornan-dsulfonyl; Trt, trityl; Tfa, trifluoroacetyl; Met(O), methionine-S-oxide).Preferential cleavage sites in the CID experiment and scanning modes for collective identification of protecting groups by tandem mass spectrometry are indicated (P = parent ion scan: NL = neutral loss scan).

8.2 Results and Discussion

269

The ESI mass spectrum of this mixture (Fig. 8-11) contains a wealth of information on the composition and presence of byproducts. Primarily, it shows all [M + H]+ions at the expected m/z values for the individual peptides. The mass peak at m/z 850 labelled with a closed circle, however, corresponds to a truncated sequence. In the ESI mass spectrum of Fig. 8-11, the relative intensities for the peptides with X = G, A, S, V, D, E, F are similar and are in relatively good agreement with the ‘ideal’ relative intensity (dashed line in Fig. 8-11). Also in good agreement with the ideal relative intensity is the intensity of the mass peak for the isobaric family containing the two peptides with X = I and L,which therefore has double intensity. By contrast, the peptides with X = P and Y are slightly under-represented. The peptides with a positively charged residue (X = K, R and H) in the randomized position are clearly under-represented. For X = Q and K one mass peak with double intensity is expected. However, only a mass peak with half the intensity expected is observed. The reason for this is that, despite the high orifice voltage of + 120 V, these peptides form abundant doubly charged ions, which reduces the ion intensities of the singly charged ions. The mass peaks expected for the peptides with X = Met (m/z 981.5) and X = Trp (m/z 1036.5) are missing almost completely from the spectrum. By HPLC-MS it could be shown that the methionine residue is oxidized. The [M+ H]+-ion of the Met(0)-peptide forms an isobaric family together with the protonated molecular ion of the Phe-peptide. In principle, the ion intensity should be higher for this mass ................., ..... ...... ......,,,

2

f

B 1 %

3

U.

P

tz

800

900

lo00 mlZ

1100

1200

Figure 8-11. ESI-MS of SIINFEKX consisting of 19 peptides with all possible protein amino acids except cysteine in the mixed position X. The letter above the mass peaks denotes the residue in the X position of the peptide (one letter code). Mass peaks marked by an asterisk correspond to additional trifluoroacetylated peptides (Am = 96 U). The mass peak marked by a closed circle corresponds to a truncated peptide.

210

8 Mass Spectrometric Analysis of Peptide Libraries

peak. Either the Phe or the Met(0) peptide or both are not present in equimolar amounts (provided that they have identical ionization yields). It turned out, that besides Met Trp also underwent chemical modification. Trp was found to be alkylated in the 2-position by a 4-hydroxybenzyl (HOBz) moiety, which arises from the Wang-anchor. This side reaction, which shifts the peptide mass by 106 U to higher m/z values, was described previously by Riniker and Kamber [22]. The corresponding mass peak of this byproduct has a relative low intensity. Presumably, owing to a relatively high solubilty of this hydrophobic peptide in ether (ether precipitation was used for purification), most of the modified peptide was lost. The ESI mass spectrum shows a second 'population' of mass peaks shifted to higher mass and with a similar intensity distribution like the expected [M + HI+ions (marked by asterisks in Fig. 8-11). The mass difference between the most intense mass peak of the [M+ HI+-ions, which corresponds to the peptide with X = I, L, and the most intense mass peak of the byproducts, is 96 U. Using MS-MS and HPLC-MS, these byproducts can be identified as peptides, which are trifluoroacetylated at the hydroxy-group of the N-terminal serine residue. Daughter ion spectra of the ions at m/z 964 ([M + HI+ of the unmodified peptides SIINFEK(1,L)) and m/z 1060 ([M + HI + of the respective O-trifluoroacetylated peptides) were recorded (Fig. 8-12). Some fragment ions are present in both ; and Y;). However, there are ions characteristic either for the modspectra (e.g., Y

[M+H]+

410

1060

9

75

$

50

v

4

2

25 n "

200

400

600

mh

800

loo0

Figure 8-12. Daughter ion mass spectra of the [M+ HI+ ions of the isobaric families a) SIINFEK(1,L) (m/z 964) and b) the Ser-0-trifluoroacetylatedpeptides S(Tfa)IINFEK(I,L) (m/z 1060).

8.2 Results and Discussion

271

ified or the unmodified peptides. The unmodified peptides, for example, form abundant B, fragment ions (m/z 201), whereas an abundant B, fragment ion (m/z 410) is the result of preferential cleavage of the amide bond between Ile and Asn of the Tfa-modified peptides. These 'diagnostic' ions can be used as the basis for precursor (parent) ion scans to reveal the masses of all unmodified and all modified peptides in the mixture [lo]. Figure 8-13a shows a parent ion mass spectrum (parent ions of m/z 201). This spectrum reveals the masses of all those peptides which are capable of cleaving off IS963

I

A

)'

50.

'Oo-

HPLC-MS

I W(Tfa/HOBz) 25 850

900

950

lo00

1050

mb

1100

1150

1200

1250

1300

Figure 8-13. a) Parent ions of m/z 201 (=the fragment ion B2) revealing the masses of all un. modified peptides of the 19-peptide mixture SIINFEKX (the mass peaks are assigned with the one-letter code indicating the X-position for the corresponding peptide@); HOB2 = 4-hy droxybenzyl). b) Parent ions of m/z 410 (=the fragment ion B3) revealing all peptides of the mixture S(Tfa)IINFEKX containing an 0-trifluoroacetylated serine residue. The spectrum was ob. tained by loop injection. c) Parent ion spectrum spectrum (parent ions of m/z 410) obtained by averaging 84 scans oi an finline

UP1 P - M C - M C

nin

272

8 Mass Spectrometric Analysis of Peptide Libraries

the N-terminal dipeptide Ser-Ile in the tandem experiment. All unmodified peptides, a truncated peptide (m/z 850; labelled with -X)and the W-modified peptide (labelled with W(H0Bz)) are registered in this scan mode. If the same experiment is performed with the fragment ion at m/z 410 (Fig. 8-13 b), an almost identical spectrum is obtained, however, shifted by 96 U to higher mass. This spectrum contains only the masses of all 0-trifluoroacetylated peptides of the mixture (note that the truncated and the W-modified peptide are also trifluoroacetylated, i. e. , they have two modifications each). The unmodified peptides are not registered. Parent ion scans can also be performed online after separation on a RP-HPLC column (see below). Fig. 8-13c shows the parent ion spectrum (parents of m/z 410) obtained by HPLCMS-MS and averaging all scans recorded during the elution of the peptides. Tandem mass spectrometry is not restricted to small libraries and can be used also for mixtures containing many thousand components. The ESI-MS of the mixture XXINFXKL (Fig. 8-4) contains several diagnostic ions, e. g. the internal fragment ion INF and the C-terminal fragment ion Y;, which can be used for identification. Parent ion scans of m/z 375 (= INF) and m/z 260 (= YT) were performed (Fig. 8-14a/b). The parent ion spectra obtained look very similar to each other and contain the [M+ HI+ mass peaks of the expected peptides of the mixture. Based on common structural features, the identity of libraries can be confirmed very easily by tandem mass spectrometry. A scan mode suitable for collective identification of a particular kind of byproduct follows from its daughter ion spectrum. In this spectrum, ions have to be found which are characteristic for the respective modification and which correspond to

800

loo0

mh

I100

1200

I300

Figure 8-14. ESI-MS-MSof XXINFXKL (cf. Fig. 8-4). Parent ions of a) m/z 375 (=internal fragment ion INF)and b) parent ions of m/z 260 (=Y;).

8.2 Results and Discussion

273

common structural features of the byproduct. If the protecting group or parts of it form a stable fragment ion, parent ion scans can be used for collective identification. If the protecting group or parts of it are cleaved from the peptides as neutral species, neutral loss scans can be used (Fig. 8-10). Parent ion scans can be used, for example, for collective identification of fluorenylmethoxycarbonyl-protectedpeptides (parent ions of m/z 179) or tritylated peptides (side chain protecting group of Asn; parent ions of m/z 243). Scanning the neutral loss of 56 U reveals all peptides containing a tert-butyl group, because isobutene (=56 U) is cleaved off very easily from the precursor ion by collision-induced dissociation. This is demonstrated for a (nonequimolar) mixture of ANPDF(S,T,I,E)(N,K,Q)(L,M,I,V) (m/z877-965) and SNYRY(S,T,I,E)(N,K,Q)(L,M, 1,V) (m/z893-981). The ESI mass spectrum of this mixture, which theoretically consists of 96 components, shows all expected mass peaks (Fig. 8-15a). Additionally, the mixture contains tert-butylated peptides. They, however, can hardly be identified in the ESI mass spectrum, because the mass range for the [M + HI+ ions of tertbutylated products (m/z 934- 1038) overlaps with the mass range of the respective unmodified peptides (m/z 877-981). With the help of a neutral loss scan of 56 U, it is possible to prove the presence of tert-butylated peptides (Fig. 8-15 b).

"

800

lo00 d Z

Figure 8-15. a) ESI-MS of a (nonequimolar) mixture of ANPDF(T,I,E,S)(N,K,Q)(L,M,IY) (m/z 877-965) and SNYRY(T,I,E,S)(N,K,Q)(L,M,IY)(m/z 893-981), which should theoretically be composed of 96 components. b) Neutral loss of 56 U revealing the presence of terf-butylated peptides (mass range for the IM + HI+ ions of these byproducts are m/z 934- 1022 and 950- 1038, respectively).

274

8 Mass Spectrometric Analysis of Peptide Libraries

Some modifications like 0-trifluoroacetylation, oxidation of Met, or N-acetylation, do not give rise to fragment ions indicative for the modification itself; such byproduct ions, however, may form mass-shifted B or Y”-fragment ions series (see above), which can be used for collective tandem mass spectrometric identification.

8.2.15 High Performance Liquid Chromatography-Mass Spectrometry

(HPLC-MS) of Peptide Libraries As sample solutions are introduced into the ion source and most of the eluent is already removed outside of the vacuum system, ESI is the ideal interface for combining high performance liquid chromatography (HPLC) with the mass spectrometer. Coupling is achieved simply by connecting the fused-silica capillary, which is part of the ESI interface, to the outlet of the HPLC column. The components are separated by chromatography and reach the ion source at different times, where they are ionized in succession at atmospheric pressure. During HPLC-MS, a so-called reconstructed total ion current chromatogram (TIC) is stored on the hard disk of a computer. The TIC is a display of the total ion current (i. e., all ions within a particular mass window, which reach the detector) versus the retention time and scan number. At any point in the TIC, the corresponding mass spectrum can be obtained. Reversed-phase columns (100 x 2 mm i.D.) with flow rates of ca. 200 pl/min are a good choice for the HPLC-MS analysis of synthetic peptides. The HPLC pump used should be pulse free and should guarantee reproducible and reliable gradients; syringe-based pumps are suitable, for example. An additional UV detector in series or in parallel with the mass spectrometer is recommended for simultaneous detection. Also, the combination with an automated fraction collector is helpful, especially if additional information on particular fractions is required. Fractions could, for example, be analyzed additionally by tandem MS and Edman degradation. If a nebulizer-assisted electrospray (=ion spray) [23] source is used, flow rates of 200 pl/min can be introduced directly into the source. However, the sensitivity is

qeccioll valve (5 CJ -pk

bop)

Figure 8-16. Experimental setup for HPLC - electrospray MS.

8.2 Results and Discussion

275

higher at lower flow rates, so that an eluent splitter can be useful. In addition, the condensation of eluent at the interface plate, which after longer operation times can lead to ‘puddles’ of solvent at the bottom of the source, can be avoided by a splitter T. A typical setup for HPLC-MS is shown in Figure 8-16. HPLC-MS gives a very detailed view of the composition of peptide libraries [2, 141. Especially in the case of smaller peptide libraries (not more than about of 100 components), it is possible to differentiate between isobaric components. The analysis of the results of a HPLC-MS experiment is facilitated very much by displaying the mass-analyzed chromatogram in a two-dimensional fashion, in which the m/z values are plotted against the retention time. Whereas from the TIC (and also the UV trace) it can only be concluded that the components are not completely separated, this 2D contour plot reveals the number and also the masses of co-eluting components. Figure 8-17 shows the TIC (upper part) and the contour plot (lower part) of an online HPLC-MS separation of the 48-component octapeptide mixture LNYRF (S,T,I,E) (N,K,Q)(V,L,I,M) [lo, 141. This mixture consists of 16 isobaric families (the number of peptides and the respective m/z values of each isobaric family 1- 16 are given in the capture of Fig. 8-17). In the TIC and the 2D contour-plot, five regions I-V can be defined, which are more or less separated from each other and correspond to peptides with similar retention time. The horizontal lines in the contour plot of Fig. 8-17 connect mass spots of peptides of the same isobaric family. Missing or additional components of smaller libraries can be identified very easily with the help of a contour plot. In the isobaric family 4,which theoretically consists of eight peptides, at least one peptide is obviously missing, other peptides seem to be under-represented. Most of the 16 peptides with Lys in sequence position 7 have mass spots with relatively low intensity due to the formation of doubly charged ions. For example, the intensities of the two peptides LNYRFIQM and LNYRFIKM in isobaric family 14 or LNYRFEQM and LNYRFEKM in isobaric family 16 differ significantly from each other (in both cases the more hydrophilic Lys-peptide elutes before the Gln-peptide). In addition to providing the possibility of distinguishing isobaric compounds, HPLC-MS is also very helpful for revealing byproducts. Very often a// components of a peptide library are modified chemically (e.g., tert-butylated) to about the same degree. In the ESI mass spectrum, such a modification can be recognized by the presence of a second population of mass peaks shifted to higher mass (in the case of the formation of deletion peptides to lower mass). In RP-HPLC, the relative order of elution for members of the unmodified peptides is usually the same as for members of the modified peptides. The contour plot therefore shows two almost identical patterns of mass spots, which are shifted to higher mass and longer or shorter elution times (depending on hydrophilicity of the modified peptides in comparison to the unmodified peptides).

276

I

8 Mass Spectrometric Analysis of Peptide Libmries

I

250 14.1

260

14.7

210 15.3

I

I

I

I

280 290 15.9 16.4 S a r f b (min) ~ ~

I I 300

17,O

I I

I

310 17,6

320

-

I

18,l

Figure 8-17. HPLC-MS of the 48 component octapeptide mixture LNYRF(S,T,I,E)(N,K,Q) (L,M,IY) (MS: m/z 1000-1120, step size 0.2, dwell time 0.8 ms; HPLC: Nucleosil C-18, 100 x 2 mm, 5 pm, 200 pl/min, 5-20070 B in 20 min; A 0.1% aqueous Tfa; B 0.1% Tfa in acetonitrile). Above: Reconstructed total ion current chromatogram. Below: 2D contour plot showing five separate regions I-V containing the isotopic [M + HI+ mass signals of the different peptides. Horizontal lines connect mass spots corresponding to peptides of the same isobaric family (labelled 1 to 16). The m/z values and the three C-terminal residues of the peptides of each isobaric families are: 1 = 1013 (1 x ;SNV); 2 = 1027 (5 x ; SNI, TNV, SNL, SQV, SKV); 3 = 1039 (1 x ;INV), 4 = 1041 (8 x ;TNL, SQI, TQV, TNI, SQL, TKV, SKI, SKL); 5 = 1044 (1 x ;SNM); 6 = 1053 (4 x ;IQV, INL, INI, IKV); 7 = 1055 (5 x ; ENV, TQI, TQL, TKL, TKI); 8 = 1058 (3 x ;TNM, SQM, SKM); 9 = 1067 (4 x ; IQI, IQL, ; ENL, EQV, EKV); 11 = 1071 (1 X ; INM); 12 = 1073 IKL, IKI); 10= 1069 ( 4 ~ ENI, ( 2 x = TQM, TKM); 13 = 1083 ( 4 ;~EQI, EQL, EKI, EKL); 14 = 1085 ( 2 ;~IQM, IKM); I5 = 1086 (1 x ; ENM); 16 = 1101 ( 2 ;~EQM, EKM).

8.2 Results and Discussion

271

In the HPLC-MS contour plot of the crude peptide mixture SIINFEKX (X = all protein amino acids but Cys; Fig. 8-18), for example, region I contains the expected mass spots for the [M+ H]+ions of the peptides, whereas region 11, which is shifted in mass by 96 U, corresponds to peptides which contain 0-trifluoroacetylated 288115.2

13417.1

I

I \I

101

0,o

5,3

20 1

10,6 Scad'ime (min)

307116.2

365119.3

301

15.9

421122.3 .I

I

40 1

21.2

Figure8-18. HPLC-MS of the 19 component peptide mixture SIINXEKL (MS: m/z 800-1200, step size 0.2, dwell time 0.8 ms; HPLC: Nucleosil C-18, 100 x 2 mm, 5 pm, 40-70Vo B in 25 min; A 0.1% aqueous Tfa; B 0.1% Tfa in methanol). Above: Reconstructed total ion current chromatogram. Below: 2D contour plot showing the region of the [M + HI+ ions of the unmodified peptides (I), of the 0-trifluoroacetylated peptides (11) and a truncated peptide (111). In region I and I1 instead of the mass for the peptide with X = Met the one for the respective oxidized peptide containing X = Met(0) is found.

270

8

M m Spectrometric Analysis of Peptide Libraries

Ser residues. The patterns of I and I1 are almost identical. Three mass spots on the left-hand side of pattern I, however, are missing in 11; they correspond to the peptides with X = Arg, His and Lys, which have a high tendency to form doubly charged ions. The ion intensities are therefore distributed to both charge states, which leads to very low intensities of the [M+ HI+ ions. A truncated peptide as byproduct can be recognized easily in region 111.

Figure 8-19. Expanded view of region I of the ZD-contour plot displayed in Fig. 8-11. The ‘massspots’ are labelled with the three letter code of the respective residue in the X position of SIINXEKL. Isobaric peptides with X = Lys and Gln, as well as with X = Ile and Leu are clearly separated. As a result of oxidation, a peptide with X = Met(0) is observed, which has the same nominal mass as the Deotide containing Phe in the X-Dosition.

8.2 Results and Discussion

279

A closer look at region I (Fig. 8-19) shows that the expected 19 different mass spots are present, but that the peptide with X = Met is missing. Instead of this peptide, a second peptide with the same mass as the Phe-substituted peptide occurs. It turned out that this is a peptide containing oxidized Met. The residue Met(0) has a mass of 131 plus 16 = 147, which is identical with the mass of the Phe residue. The order of elution observed is in good agreement with the differences in hydrophilicity/hydrophobicity of the peptides. Thus, HPLC-MS provides a convenient and fast way to study retention times of peptides systematically. The contour plot also shows that the isobaric peptides present in the mixture (X= Lys/Gln and Ile/Leu) were successfully separated by the chosen chromatographic conditions. For libraries containing more than several thousand peptides, HPLC-MS is of only limited use. The reconstructed total ion chromatogram obtained by HPLC-MS of the 6859 octapeptides of XXINFXKL (cf. Fig. 8-1) shows a broad ‘peak’ without distinct features (Fig. 8-20). Mass spectra corresponding to single scans should contain several FI+ H]+ ions, which, however, are difficult to identify owing to a low signal to noise ratio (the concentration of individual peptides registered in only one scan is very low). Mass spectra with better signal/noise ration can be obtained by averaging several scans in different regions (Fig. 8-21 a/b). An interesting finding is that the mass and intensity distribution is very similar in a spectrum obtained by direct infusion of the mixture into the ESI source (Fig. 8-21 c) and in a spectrum obtained by averaging all scans during the elution of the peptides (Fig. 8-21 d). The spectrum obtained by direct injection looks a bit more

7

I

0.0

--

101 7.1

201 14.1 scanmm (mia)

301 213

Figure 8-24). HPLC-MS of XXINFXKL. Reconstructed total ion chromatogram (Nucleosil C-18. 5 um, 40-70% in 25 min; A 0.1% TFA in water; B = 0.1% TFA in methanol).

8 Mass Spectrometric Analysis of Peptide Libraries

280

a)

Average of Scans 120-I42

"-

800

800

C)

900

94R 963

LOO0 mlz

I 100

I200

loo0

1100

I200

1 100

1200

mlz

Average of Scans 65-390

mlz

d)

direct iniection

" 800

90

loo0 ml2

Figure!8-21. ESI mass spectra of XXINFXKL obtained: a) by averaging scans 120- 142 (corresponding to more hydrophilic peptides), b) by averaging scans 222-247 (corresponding to more hydrophobic peptides), c) by averaging scans 65-390 (=sum of all scans recorded during elution of all peptides of the mixture) and d) by introducing the mixture into the ESI interface without separation.

8.3 Materials and Methods

281

spiky, which is the result of the higher mass spectrometric resolution chosen for this experiment than for HPLC-MS. At a total peptide concentration of 1-2 mg/ml (which is much higher than the dynamic range concentration of single peptides), the ionization of a particular component is obviously not affected much by all other peptides which are present during ionization. Using standard conditions for HPLC and MS, the obtained elution patterns are quite reproducible. It is therefore possible to evaluate the quality of libraries or to differentiate libaries by means of contour plots (‘fingerprint spectra’). In principle, the interpretation and comparison of different contour plots can be done in a very similar way to that commonly used for the analysis of 2D electrophoresis gels of proteins.

8.2.16 Limits for Mass Spectrometric Characterization of Peptide Libraries There are limits for application of ESI mass spectrometry for the analysis of peptide libraries. Libraries containing over a million to several billions of peptides usually have mass distributions which approach a continuum. Thus, the ESI mass spectrum shows one broad mass ‘peak’ without distinct and characteristic peak patterns, which are desired for an unambiguous identification and characterization. For mass spectrometric analysis of such libraries, one has to reduce the complexity of the library. This can be done in two ways. One possibility is to collect resin samples after each step of the synthesis and to analyze the less complex mixture obtained after cleavage from the resin. In addition to mass spectrometric analysis, it is recommended to check the samples with multiple sequence analysis [3]. A second possibility is to perform MS-MS or HPLC-MS-MS experiments. Parent ion or neutral loss scans allow the complexity of the mixture to be reduced by filtering out all peptides with particular features (e.g., all peptides having the C-terminal dipeptide KL; see 8.2.14).

8.3 Materials and Methods 8.3.1 Peptide Synthesis Synthetic peptide mixtures were preparec by solid phase peptide synthesis using Fmoc-L-amino acid-p-benzyloxyalcohol-polystereneresins loaded with a single amino acid, or equimolar mixtures of 19 different resins with all the protein amino acids except cysteine. A robot system controlled by an optimized software for multiple peptide synthesis (MultiSynTech, Bochum, Germany) was employed. The residues at the constant positions were introduced with a fivefold molar excess (with re-

282

8 Mass Spectrometric Analysis of Peptide Libraries

spect to loading of the resin) of single activated Fmoc-amino acids. The mixed positions X were coupled using equimolar mixtures of activated Fmoc-amino acids equimolar with the coupling sites on the resins or using the divide-couple-recombine procedure [24]. Lipopeptides were prepared as described previously [20]. For the preparation of the phosphopeptides, Fmoc- phosphotyrosine dimethylester was used as the building block. For activation, the dicyclohexylcarbodiimide/l-hydroxybenzotriazole method was used. Extended coupling times, double coupling, an initially high content of dichloromethane, and open vessels to allow evaporation of dichloromethane and thereby concentration of the reagents in the course of the coupling cycles, supported the generation of close to equimolar distribution of the peptides in the libraries. Cleavage of the peptides from the resins and side chain deprotection was achieved with trifluoroacetic acid/phenol/ethanedithiol/thioanisole(96 :2 : 1 : 1 ; v :v :v :v). The resins were filtered off, and peptides were precipitated by adding cold n-heptaneldiethylether (1 : 1 ; v :v) washed and lyophilized from acetic acid/water/terl butylalcohol (1 : 10 :50; v :v :v). In some cases the solution obtained after cleavage of the peptide mixtures from the resin was evaporated to dryness at 40T, to enhance the degree of byproduct formation. More details of the automated synthesis of peptide libraries are given by Wiesmuller et al. in Chapter 7 of this book.

8.3.2 Mass Spectrometry Positive ion electrospray mass spectra and tandem mass spectra were recorded on a API 111 Taga 6OOO equipped with an ion spray source (Sciex, Thornhill, Toronto, Canada). The clusters of CsI were used to calibrate both quadrupoles Q1 and 4 3 . Approximately ten spectra were accumulated by scanning at unit resolution with a step size of 0.1 Da and a dwell time of 4 ms. For the H P K - M S experiments, a step size of 0.3 Da and a dwell time of 1 ms were chosen. The lyophilized samples were dissolved in acetonitrile/l Vo aqueous formic acid (1 : 1; ca. 1 pg/ml). The solution was introduced into the ion spray source at a constant flow rate of 5 pl/min with a microliter syringe using a medical infusion pump (Harvard Apparatus, Southnatick, USA). For rapid analysis of the peptide mixtures, 5 pl of a 1 m M solution (acetonitrileAVo aqueous formic acid) were injected on to a carrier stream (methanol/l Vo aqueous formic acid; 80 pl/min) at intervals of 1 min by an autosampler (Gilson Abimed, Model 131). Argon was used as collision gas for collision-induced dissociation in the tandem MS experiments. The orifice voltage was set between +50 and +140 V. ESI-FT-ICR-MS was performed with a Extrel-Millipore 6.2 T superconducting magnet and an Odyssey data system that acquires 1 M data points (Waters-Extrel, Madison. Wisconsin. USA).

8.4 Summury

283

The MALDI mass spectra were recorded with a Bruker MALDI mass spectrometer using a nitrogen laser and 2,5-dihydroxybenzoic acid as matrix.

8.3.3 Narrow-Bore RP-HPLC For online HPLC-MS and HPLC-MS-MS, an Applied Biosystems AB1 140A HPLC system and a narrow-bore column (Nuclesosil C-18, 2 x 100 mm; 5 pm; Grom, Herrenberg, Germany) equipped with a precolumn (2 x 10 mm; same filling) at a flow rate of 200 pl/min were used. A linear gradient was used (solvent A: 0.1 070 trifluoroacetic acid in water; solvent B: 0.1 070 trifluoroacetic acid in methanol). The flow was split so that 40 pl/min entered the ESI interface. 5 pl of a solution of the peptide mixture (ca. 0.2 mg/ml) were injected with a injection valve (Rheodyne 7125) equipped with a 5 pl-loop.

8.3.4 Calculation of the Mass Distribution with QMass The mass and intensity distribution of the peptide mixtures were calculated with the computer program QMass. This program is described in detail by Brunjes et al. in Chapter 18 of this book.

8.4 Summary Although the synthesis protocols of peptide and nonpeptide libraries very often look straightforward and are based on established and proven procedures, the success of the synthesis should never be taken for granted. More than once it has been observed that, in a parallel synthesis, one or other mixture of the batch for some reason did not have the expected composition, whereas the majority of the samples were satisfactory. For an effective optimization of the synthesis, a careful characterization of the resultant product mixture is indispensable, and biological screening should not be performed with only poorly defined mixtures. Existing analytical methods are well suited for this task. In particular, modern mass spectrometric techniques (ESI, MS-MS and HPLC- or CE-MS) give a wealth of information on composition and purity in very short time. ESI mass spectra of all peptide libraries synthesized are routinely recorded. This can preferentially be done overnight with the help of an autoinjector. To save time it may be sufficient only to spot-check the analytical data to get an idea about the quality of the synthesis. A detailed examination of the data can be undertaken after-

284

8 Mass Spectrometric Analysis of Peptide Libraries

wards, including only those samples which attract attention during the biological testing. Due to the large number of samples, a perfect control over sample logistics during the synthesis, product characterization (i.e., evaluation, handling and processing of analytical data) and the biological testing step are prerequisites for successful exploitation of molecular diversity. Development of appropriate computer software is therefore of the utmost importance. Automation of all these steps as far as possible is necessary in order to take advantage of the high speed and output of combinatorial synthesis.

Acknowledgment This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 323) and the Fonds der Chemischen Industrie. We thank Dr. B. Winger (Waters-Extrel, Madison, WI, USA) for the FT-ICR-MS measurements, and Dr. F. J. Meyer-Posner (Bruker-Franzen Analytik, Bremen, Germany) for recording the MALDI-TOF mass spectra.

References [l] A. Beck-Sickinger, G. Jung, Angew Chem. Int. Ed. Engl. 1992, 31, 367-486. [2J J. W. Metzger, C. Eckerskorn “Electrospray mass spectrometry” in Microchamcterization of proteins, (Eds.: R. Kellner, F. Lottspeich, H. E. Meyer), VCH, Weinheim, 1994, pp. 167-188. 131 S. Stevanovik, G. Jung, Anal. Biochem. 1993, 212, 212-220. 141 J. A. Boutin, P. Henning, S. Bertin, P.-H. Lambert, J.-P. Volland, J.-L. Fauchere “Robotic synthesis of peptide libraries: analysis, pitfalls and success’’ in Abstmcts of the 14th International Symposium on the Separation and Analysis of Proteins, Peptides, and Polynucleotides, Heidelberg, 2-4 November, 1994, p. 34 (abstract no. 614). [S] M. Barber, R. S. Bordoli, R. D.Sedgwick, A. N. Tyler, J. Chem. Soc., Chem. Commun. 1981, 325-327. [6] M. L. Aleksandrov, L. N. Gall, V. N. Krasnov, V. I. Nikolaev, V. A. Pavlenk, V. A. Shkurov, Dokl. Akad. Nauk. SSSR 1984, 277, 379-383. [7]M. Yamashita, J. B. Fenn, J. Phys. Chem. 1984, 88, 4451-4459. (81 M. Karas, F. Hillenkamp, Anal. Chem. 1988, 60, 2299-2301. [9] M. Mann, C. K. Meng, J. B. Fenn, Anal. Chem. 1989, 61, 1702-1708. [lo] J. W. Metzger, C. Kempter, K.-H. Wiesmuller, G. Jung, Anal. Biochem. 1994, 219, 261 -277. [ll] J. W. Metzger, S. StevanoviC, J. Brtinjes, K.-H. Wiesmtiller, G. Jung, Methods: a Companion to Methods in Enzymology 1994, 6, 425-431.

References

28:

[12] M. R. Clench, G. V. Garner, D. B. Gordon, M. Barber, Biomed. Mass Spectrom. 1985, 12, 355-357. [13] S. Stevanovid, K.-H. Wiesmuller, J. W. Metzger, A. Beck-Sickinger, G. Jung, Bioorg. Med. Chem. Lett. 1993, 3, 431-436. (141 J. W. Metzger, K.-H. Wiesmuller, V. Gnau, J. Brunjes, G.Jung, Angew. Chem. 1993, 105, 901-903; Angew. Chem. Int. Ed. Engl. 1993, 32, 894-896. [l5] J. W. Metzger, K.-H. Wiesmiiller, S. Stevanovit, G. Jung, “Analytical methods for the characterization of synthetic peptide libraries” in :Peptides 1992, (Eds. : C. H. Schneider, A. N. Eberle), Escom, Leiden, 1993, pp. 481 -482. (161 L. Tang, P. Kebarle, Anal. Chem. 1991, 63, 2709-2715. (171 P. Roepstorff, J. Fohlman, Biomed. Mass Specrrom. 1984, 11, 601. (181 B. Asamoto (Ed.), “FT-ICRIMS: Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry“, VCH, Weinheim, 1991. (191 A. G.Marshall, T. C. Wang, T. L. Ricca, J. Am. Chem. Soc. 1985, 107, 7893-7897. [20] J. Metzger, K.-H. Wiesmiiller, R. Schaude, W. G.Bessler, G. Jung, Inr. J. Pept. Protein Res. 1991, 37, 46-57. (211 J. W. Metzger, W. Beck, G.Jung, Angew. Chem. 1992,104, 235-237. Angew. Chem. Int. Ed. Engl. 1992, 31, 226-228. [22] B. Riniker, B. Kamber “Byproducts of Trp-peptides synthesized on a p-benzyloxybenzylalcohol polysterene resin” in Peptides 1988, (Eds. :G.Jung, E. Bayer), Walter de Gruyter, Berlin, New York, 1989, pp. 115-117. [23] A. R. Bruins, T. R. Covey, J. D. Henion, Anal. Chem. 1987, 59, 2642-2646. (241 R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, J. H. Cuervo, Nature 1991, 354, 84-86.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries Wieland Keilholz and Stefan Stevanovic'

9.1 Introduction Biological processes often rely on the interaction between biologically active molecules such as peptides and proteins. In this context, the binding of a peptide or a defined sequence out of a protein as a ligand to a receptor plays an important role. Processes based on these interactions are signal transduction, enhancement of transcription by factors binding to DNA, antigen-antibody interactions and self-nonself discrimination by ap receptors of Tlymphocytes in the immune system. To understand these processes completely, it is obvious that one has to investigate the interactions on the molecular level. A new approach for conducting these investigations uses binding studies of a given receptor molecule with a peptide library containing millions of different peptide sequences [I]. In general, there are two different methods of building up a peptide library: on the genetic level using phages as library creators [2] and on the chemical level using simultaneous multiple peptide synthesis by applying Merrifield's solid phase method [31. For the application of synthetic peptide libraries to biological systems, it is of enormous importance to characterize the product and make sure that it fulfils the requirements. This chapter describes multiple sequence analysis based on Edman degradation as a method of peptide and protein sequencing for the characterization of either synthetic or natural peptide libraries.

9.2 Multiple Sequence Analysis as a Further Development of Edman Degradation The goal of the chemical reaction steps developed by Pehr Edman [4] was to find out the amino acid sequence of a given peptide or protein by stepwise removal of derivatized amino acids from the N-terminal end of the molecule. Therefore, in most cases it was not necessary to know the exact amount of peptide present. In the case of multiple sequences analyzed by Edman degradation, however, it is necessary to work with high reproducibility and reliability in quantification. Thus, characteriza-

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

288

tion of the sources of inaccuracy in quantification is one of the basic features of multiple sequence analysis [5]. In sequence analysis, three phenomena affecting exact quantification have to be considered : (i) washout and partial hydrolysis causing loss of the sample; (ii) different recoveries of PTH-amino acids;

(iii) lag (or memory) effect.

In addition, two further terms have to be defined, namely signal and background. Washout:Sample loss occurs by detachment of protein during washing and extraction steps of automated Edman degradation and by partial hydrolysis of peptide bonds due to TFA treatment in every degradation cycle. It is calculated as repetitive yield, which is the part of a sample remaining after one degradation cycle, given in percentages. This repetitive yield is usually determined from amino acids occurring twice or more in one sequence. In multiple sequence analysis of peptide libraries, the pmol PTH-AA

\ 100

\ '\

\ \

\'. \

\

10

\

7

M

Q

I

F

V

K

T

L

T

G

K

T

I

T

L

Figure 9-1. Different recoveries of FTH-amino acids in sequence analysis, as shown by the

analysis of ubiquitin. The columns represent the amount of FTH-amino acid detected, the black line indicates the amounts expected according to a repetitive yield of 89%.

9.2 Multiple Sequence Analysis as a Further Development of Edman Degradation

289

calculation of the repetitive yield is based on the sum of all amino acids present in one position. Recoveries: All amino acids containing functional groups in their side chain are detected in amounts lower than expected. This is seen in Figure 9-1, which shows a sequencing experiment with a repetitive yield of 89%, represented by the black line, while the hatched bars indicate the amount of PTH-amino acids detected. To quantitate the recoveries for every amino acid, correction factors have been introduced which were derived out of a series of sequencing experiments (Table 9-1). These correction factors are sequencer dependent and may alter even after exchanging the HPLC column responsible for the separation of PTH-amino acids. Therefore, they have to be determined in periods of several months (51. Lag-effect: Tailing of PTH-amino acids to the following cycle, caused by inefficient extraction of the ATZ-amino acid out of the reaction chamber and/or inefficient coupling/cleavage reactions. Signal: PTH-amino acids that belong to a peptide sequence or are regarded as part of a motif. The criteria for signal definition are different in every experiment but in general defined as 1.5 to 2-fold increase of PTH-amino acid compared to the previous cycle.

Table 9-1. Recoveries of amino acids detected as FTH-derivatives after automated Edmam degradation AA

Recovery (070)

A

90.75 71.58 57.26 97.47 83.12 43.60 97.71 78.88 96.17 70.40 63.69 85.41 73.24 63.27 26.86 38.98 96.26 21.20 76.18

D E F G H I

K L M N P

Q

R S T

v

W

Y

f

f f f f f f f f f f f f

*

f f f f f f

Dev. 11.46 10.59 10.99 8.13 7.69 5.78 9.16 13.69 9.22 6.94 8.70 8.01 8.44 20.28 4.62 5.59 7.55 2.94 9.15

290

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

Background: While in traditional sequence analysis, background is defined as every signal not belonging to the sequence, in multiple sequence analysis it has to be defined as every nonsignal value. Multiple sequence analysis can be carried out in every protein sequencer, whether samples are attached covalently or in the adsorptive mode. With multiple sequence analysis, even resin-bound synthetic peptide libraries can be characterized after every coupling step during synthesis, thus representing the only analytical method capable of analyzing resin-bound samples [6). The steps of sample fixation and Edman chemistry used routinely are applicable to multiple sequences, too; only the calculation of results is more sophisticated, including preview analysis and correction of recoveries.

9.3 Applications 9.3.1 Natural Peptide Libraries: Ligand Motifs of MHC-I and MHC-I1 Molecules The function of MHC molecules localized on the cell surface, is to present pathogen-

or tumor-derived peptides to T-cell receptors [7]. Every individual expresses two HLA-A, HLA-B and HLA-C alleles, respectively, and therefore only up to six different types of MHC-I molecules are present on the cell surface. Rvo opposite requests have to be fulfilled by every MHC molecule. On the one hand, it has to be specific in its choice of ligands, one the other hand, it has to be able to bind a large number of varying ligands. This is achieved by the following specificity rules which are alike for MHC-I and -11 molecules and summarized as allele-specific ligand motifs. For MHC-I, peptides are of a defined length (eight or nine residues; exceptions have been observed) containing usually two anchor residues in their sequence, which can only be occupied by few, closely related amino acids. Typically the C-termina1 amino acid of an MHC-I ligand represents one of these anchor residues, another one is often situated in position two or three [S]. For MHC-11, ligands consist of 12-25 residues carrying 3-4 anchor residues inside the peptide chain, depending upon the MHC allele. Neither the C-nor the N-terminal part of the peptide is used as an anchor; ligands may hang out of the binding groove at both ends [9]. Thus, MHC molecules carry a large number of different peptides, which are uniform only at a few positions. These peptides can be extracted by acid treatment [lo], and they represent a natural peptide library with allele-specific motifs [l].

9.3 Applications

291

9.3.1.1 Pool Sequencing of MHC-I Ligands

When sequencing a peptide pool of MHC-I ligands, every anchor position is characterized by only a few signals of closely related PTH-amino acids. Table 9-2 shows the multiple sequence analysis of HLA-B*0701 ligands (self-peptides). Signals were split into two categories: 3-fold increase of the amount of PTH-amino acids (bold), and 1.5-fold increase (underlined). The first anchor residue is easy to determine as Pro in position 2, which shows an 8-fold increase referring to position 1. In cycle 3 many PTH-amino acids show an increase in amount compared to cycle two. This is due to the strong signal of Pro in cycle 2; no other amino acids are allowed in this position of HLA-B*0701 ligands which leads to a general decrease of all other amino acids. Therefore, it is advisable to compare cycle 3 to cycle 1, which reveals only a few real signals belonging to Asn, Asp and Arg. In cycles 4 to 8, several changes of the amino acid pattern are observed which mostly lie under the threshold of signal definition. Some signals, e. g., Phe and Thr in cycle 6, are probably not of great relevance for the binding motif owing to the different nature of these two amino acids. In position 7, however, a preference of hydrophobic amino acids can be seen clearly, as manifested by the Val and Ile signals. The extraordinarily high amount of Gln is probably caused by the addition of Gln to the medium used for cell culture, and is regarded as an artefact. Cycle 9 shows two signals of 1.5 and 1.9 fold increase, namely Phe and Leu which can be assigned to the binding motif because of their closely related nature and the strength of the signals appearing in this late cycle. Since cycles 10 and 11 show a strong decrease of all PTH-amino acids detected, the two signals in cycle 9 represent the C-terminus. As a result, this experiment determines the binding motif of self-peptides obtained from the HLA-B*0701 peptide receptor as Pro in position 2 and Leu/Phe at the Cterminus, which in this case is position 9. For the interpretation of sequencing data derived out of a MHC-I self-peptide pool, two rules can be set. (i) In cycles of anchor residues, the background of most other PTH-amino acids is very low, leading to insignificant increases in the following cycle. (ii) Due to sample loss, the quality of signals decreases with the number of cycles performed. Therefore, C-terminal anchor residues sometimes do not even reach the threshold set for signal definition. Compared to the drastic decrease of all other amino acids, however, those C-terminal anchor amino acids can be identified readily.

F Phe

123.6 186.0 103.1 50.0 45.8 127.3 70.9 11.9 29.8 85.5 34.4 21.5 28.8 66.7 49.0 17.4 35.0 68.0 42.4 16.6 35.8 50.6 45.5 25.3 42.1 71.7 86.4 15.5 27.0 64.7 61.4 15.3 23.0 43.1 115.5 23.7 13.3 25.5 67.0 13.4 8.6 13.8 38.6 7.4

L

Leu

1 2 3 4 5 6 7 8 9 10 11

Val

V

I Ile

Cycle

42.1 7.3 36.8 14.4 10.7 12.7 13.5 12.1 12.0 7.5 4.4

M Met

381.2 152.6 215.4 119.2 99.4 68.3 71.4 80.4 49.0 26.6 16.3

A Ala

T Thr

S Ser

Tyr

Y

185.1 114.6 245.5 56.3 131.1 47.6 106.1 24.0 143.8 41.3 98.8 36.4 202.5 62.0 77.1 24.3 167.9 54.4 75.3 20.6 164.0 87.4 66.2 11.8 112.7 76.9 62.5 13.2 77.9 75.5 57.1 11.0 50.7 43.7 30.2 8.2 37.0 31.1 17.9 5.8 28.8 18.7 10.4 4.2

G Gly

H His

66.4 12.9 9.9 177.2 16.7 135.7 14.3 168.4 17.6 64.5 11.0 37.4 11.8 18.8 11.8 10.9 11.0 7.6 11.7 5.7 9.2

546.0

P Pro 772.7 476.3 378.2 197.6 112.7 46.2 44.3 39.8 21.1 12.5 7.3

Gln

Q

20.6 23.0 16.1 8.7 5.4 3.0

28.4 28.6 30.4

20.1 11.6

148.4 97.8 109.4 103.4 68.0 33.6 31.3 34.5 20.4 13.8 7.9

13.7 9.5 30.1 36.8 39.4 23.3 21.0 18.6 14.3 9.9 7.6

32.1 18.5 12.9 9.4 1.3 0.5 0.7

51.6 58.9

74.1 24.6

N E D K Asn Glu Asp Lys

98.8 31.7 128.3 58.3 69.0 60.8 43.9 34.9 24.6 17.9 13.1

R Arg

Table 9-2. Multiple sequence analysis of peptides eluted from HLA-B*0701 molecules. Amino acids are ordered by decreasing hydropathy. Shown are the picomol amounts of every PTH-amino acid detected during 11 cycles of Edman degradation (cycle 1, N-terminus)

e

Y

2

h

9.3 Applications

293

9.3.1.2 Pool Sequencing of MHC-I1 Ligands Multiple sequence analysis of MHC-I1 pools is even more complicated compared to MHC-I pools owing to the different lengths of the ligands. Fortunately, in the majority of ligands, the distance from the N-terminus to the first anchor differs only slightly, and therefore in the sequence data anchor residues appear as signal clusters, split into two or three neighboring positions. This phenomenon makes it possible to locate the exact position of anchor residues and is helpful in identifying individual sequences present in the peptide pool which appear as single signals. Figure 9-2shows a sequencing experiment of a MHC-I1 (DRB1*0405)extracted self-peptide pool. The course of 18 different FTH-amino acids (Cys and Trp omitted) over the cycles of degradation is shown. This kind of graphics is easier to survey than a table containing all values of FTH-amino acids and was therefore chosen for this chapter. The most striking signal appears in position 2 and is caused by Pro. Since this high Pro signal in position 2 has been observed in many class 11-extracted peptide pools, it is not part of the ligand motif of DRB1*0405,but probably a feature of processing [9]. Some FTH-amino acids show no signals at all, but their amount is steadily decreasing which is best seen for Leu but as well for His, Ala and Lys, respectively. Weak increases in their profile may be caused by individual sequences or unstable background, but can not be seen as part of the ligand motif. Other FTH-amino acids show the clustered signals which are expected and it is possible to obtain a consensus motif out of these data. For example, Phe and Q r have strong signals in position 4/5 which reflect the first anchor position, defined as relative position 1 of the ligand motif. Another striking cluster of signals is in cycle 718, where the hydrophobic amino acids Met and Val have a maximum. This represents the second anchor position at relative position 4 of the peptide motif. A third anchor can be found in cycle 9/10(relative position 6), which is occupied by Gly, Ser, Thr and Asn. Finally, there is a fourth anchor position detected in cycle 12/13 (relative position 9). This position is mainly occupied by the negatively charged amino acids Asp and Glu. After having determined the natural ligand motif of DRB*0405 molecules, it is possible to align all the individual peptides isolated and analyzed (Table 9-3).Out of 17 ligands characterized, five carry the first anchor amino acid in position 4 relative to the N-terminus, six have it in position 5. Thus, the majority of individual ligands contributes to the cluster in position 4/5 of the peptide pool, while three of the ligands in Table 9-3possess the first anchor residue in position 3, one in position 6, and two in position 8, respectively. The danger which lies in the presence of dominant individual ligands in a peptide pool has to be mentioned. There is, for example, a strong signal cluster of Ile in position 617 which lies exactly between the relative positions 1 and 4. When examining only the pool data, this signal cluster might be interpreted as an anchor position. But in contrast, it is an example of how dominant individual peptides may bias the pat-

294

9 MultipIe Sequence Analysis of Natural and Synrheric Peptide Libmries

9.3 Applications

2007

295

50T

ao 60 40 20

0 50 40

30 20 10

0

Figure 9-2. Multiple sequence analysis of MHC 11 (DRBl"0405) - extracted peptides. Shown are the pmol amounts of FTH-amino acids. Left, cycle 1 (N-terminus). Note the different scales. Anchor residues amear as signal clusters, e.g. Phe and '@r in positions 4 and 5.

A

C

I

V

T

H A V

E E

A P A

P P T Y

P E R R R F F

P L A A A A R R R D D H L L K N D

Q K R R R E E E E H H Y L L R T K

1

Y I Y Y Y F F F F Y Y Y Y Y V F I

I D Q Q Q K K K K V V A Y Y T K Q

2

3

A I W W W L L L L V V V T T I I L

V I V V V S S S S V V A E E M L I

4

H P R R R K K K K G G V F F P D N

5

V N C C C V V V V A A V T T K S N

6

V P N N N W W W W Q Q K P P D W M

7

P Q P P P R R R R R R K T T I R L

8

9

D E D D D D D D D D D D E E Q D D

Q R S N S S N S S N S Q H Q Q H Q A A T D F K D K D E Y

K

-

MIF (32-45) Hsp 90-beta (68-81) PGSG (1-19) PGSG (4-19) PGSG (5-19) Transferrin receptor (173 186) Transferrin receptor (173- 185) Transferrin receptor (174-186) Transferrin receptor (1 74- 185) Transferrin receptor (397-41 1) Transferrin receptor (398-41 1) similar to transferrin P2-microglobulin(83-96) P2-microglobulin(84-98) Histone H3 (110-124) ras-related protein RAB-7 (rat) (86-98) Phosphoglycerate kinase (216-228)

Source

Peptide sequences are aligned according to the first anchor position, anchor residues are printed in boldface. MIF. macrophage migration inhibitory factor; PGSG, platelet proteoglycan core protein.

P

Y

K K Q Q

Table 9-3. Individual ligands extracted from HLA-DRB1*0405 molecules

29

y:

0

U

h

9.3 Applications

297

tern of a pool sequencing experiment. In this case, the peptide MIF 32-45 as part of the pool was present in a high amount as well as HSP90 68-81. These peptides were the only ligands analyzed containing Ile at position 6 or 7 (relative position 3 or 4, see Table 9-3). Also, other prominent signals not appearing as clusters, like His9, Ala’ and Glu2 were caused by these two individual peptides. This example shows that multiple sequence analysis is a powerful tool to determine ligand motifs of MHC-I1 molecules, but the results have to be confirmed by sequence analysis of individual peptides to avoid misinterpretations.

9.3.2 Synthetic Peptide Libraries To define the quality of a synthetic peptide library, the application of multiple sequence analysis offers several advantages compared to other analytical methods. Multiple sequence analysis reveals coupling efficiencies per coupling cycle as well as a quantitative evaluation of every amino acid in each position. The preview effect as a special feature of synthetic peptides undergoing Edman degradation has to be quantified, owing to its correlation to coupling efficiency. A small amount of a PTH-amino acid appears in position n-1, although expected in position n. This value is reversed proportionally to the coupling efficiency of the cycle n-1. By using correction factors, one is able to state the amounts of the different amino acids in every variable position. A peptide library is well suited for use in biological systems, only if the distribution of amino acids in X-positions is approximately equimolar. The analysis of the synthetic peptide library SF, which is a library of 19 = 361 different octapeptides LSRXEXDL, is shown as an example. Synthesis was carried out with an equimolar mixture of Fmoc-amino acids, and therefore 19 different amino acids should be present at the X-positions (Cys was omitted). Three sequencing experiments have been performed to investigate alternative coupling conditions during synthesis. (i) SF1 was synthesized using DIC/HOBt as activating reagents and analyzed after synthesis without cleavage from the resin. (ii) SF2 was synthesized the same way but was removed from the resin before analysis. (iii) SF3 was synthesized using TBTU/HOBt for activation and was also analyzed after cleavage. Figure 9-3 shows corrected data of PTH-amino acids seen in every degradation cycle of SF2. X-positions are calculated as the sum of the amount of every amino acid detected. The course of samde loss can be sdit into three Dhases:

1 1IIL

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

290

pmol PTH-AA

1600 1400 1200 1000

800 600 400 200 0

T

L

S

R

X

E

X

D

L

Figure 9-3. Repetitive yield plot of the synthetic peptide library SF2. All pmol amounts are corrected in respect of recoveries, in X-positions the sum of all 19 PTH-amino acids was used for the calculation. It fits well with a repetitive yield of 80% between cycles 2 and 6.

Initialitation: The second position yields a higher total amount of PTH-amino acids than the first position. This feature is always seen when a sequencing experiment is done without running a precycle to include conditioning of the filter on which the sample is adsorbed.

deviation

[%I

100

-50

-100

A D E F G H I K L U N P O R R T V W Y

9.3 Applications

b)

deviation

299

['/I

I

I

I

-50 -

- l o o ~ , , , ~ l , l l l , , l , , l l l l , ,, ,, l, , , ,

1 1 , 1 1 1 1 , , /

A D E F G H I K L M N P Q R S T V W Y C)

deviation

[%I

100 -

50 -

I

0

rn

II

I

I

-50 -

-'0°

1 A D E F G H I K L M N P Q R S T V W Y

Figure 9-4. Analysis of the equimolar distribution of amino acids in position 4 of SFl-SF3. The deviations (To) from the calculated average of all amino acids are shown. a. SFl, b. SF2, c. SF3 (see text, page 301).

Repetitive Yield: Due to the contrast decrease of PTH-amino acids in cycles 2-6, the total amount of PTH-amino acids in every X-position was used to determine the repetitive yield of 80%. C-terminal part: A very strong decrease is detected between cycle 7 and 8. Since amino acid 8 is the C-terminus, the peptides analyzed in cycle 7 consist of only two residues and therefore are more easily washed out. It is even more difficult to analyze

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9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

the C-terminal amino acid (position 8) which, as a single free amino acid, is very weakly attached to the glass fiber filter. For the characterization of synthetic peptide libraries, the most important question is the equimolar distribution of peptides. Multiple sequence analysis can answer this question by analyzing the equimolar distribution of amino acids in variable a)

deviation [%] I

I

100

50

0

A D E F G H I K L M N P Q R S T V W Y

HSFI a S F 2

b)

deviation [%I

100

50

0

I

I

1

-loo A D E F G H I K L M N P Q R S T V W Y HSFl a S F 3

Comparison of the equimolar distribution of amino acids in position 4 of a) SF1 and SF2,and b) SFl and SF3. Deviations (To) from the average of all amino acids are shown

Figure 9-5.

References

301

positions. Figures 9-4a-c show the deviations in percent (referring to the average of all amino acids) analyzed in the XI-position for each of the different peptide libraries SF1-3. Figure 9-5a shows the same data of SFl and SF2 within one diagram. The profiles are very similar, showing that there is no difference in quality of the data obtained from multiple sequence analysis carried out either directly from the resin or after cleavage. Furthermore, it is pointed out that side chain protecting groups have no influence on sequencing. This is expected, because the acid-labile protecting groups usually used in Fmoc chemistry, will be removed under the conditions of automated Edman chemistry. In Figure 9-5 b, data of SFl and SF3 for the XI-position are compared. It can be seen that the deviations of the PTH amino acids of SF3 are smaller than those of SFI. Therefore, it can be concluded that TBTU/HOBt as activating reagents lead to a more uniform distribution of amino acids in this case. Not only does multiple sequence analysis represent a powerful tool in controlling the synthesis of peptide libraries, it can also reveal changes in the composition after the library has passed a biological assay. For instance, binding motifs of phosphorylated peptides interacting with SH2 domains have been determined in this way [12].

References [I] R. A. Houghten, lknds Genet. 1993,9 (7), 235-239. [2]J. K. Scott, G. P. Smith, Science 1990, 249, 386-390. [3] R. A. Houghten, Proc. Nufl. Acud. Sci. USA 1985,84, 5131-5135. [4]P. Edman, Acta Chem. Scand. 1950, 4, 283-293. [5] S. StevanoviC, G. Jung, Anal. Biochem. 1993, 212, 212-220. [6]J. W. Metzger, S. StevanoviC, J. Brllnjes, K.-H. Wiesmuller, G. Jung, Methods, A companion of Methods in Enzymology, 6, 425-432. [7]R. M. Zinkernagel, P. C. Doherty, Nature 1974, 248, 701-702. [8]H. G. Rammensee, K. Falk, 0. Rotzschke, Annu. Rev. Immunol. 1993,11, 213-244. [9]K. Falk, 0.Rtitzschke, S. StevanoviC G. Jung, H. G. Rammensee, Immunogenefics 1994, 39, 230-242. [lo]0.Rotzschke, K. Falk, Immunol. Toduy 1991, 12, 447-455. (11) 0.RGtzschke, K. Falk, H. Schild, M. Norda, H.G. Rammensee, K. Deres, J. Metzger, S. StevanoviC, K.-H. Wiesmllller and G. Jung, in &prides, Chemistry and Biology (Eds.: J. A. Smith and J. Rivier), Proceedings, 12th American Peptide Symposium 1992,Escom Science, Leiden, pp. 832-834. [12]Z. Songyang et al., Cell 1993, 72, 767-778.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

10 Epitope Mapping with the Use of Peptide Libraries Stuart Rodda, Gordon Tribbick and Mario Geysen

10.1 Introduction 10.1.1 Definition of Epitope The term epitope was introduced to help with the nomenclature describing the binding site on the surface of an antigen molecule where the antibody is in contact with antigen [l]. As understanding of the immune system grew, it was realized that there were two repertoires of specific antigen receptors: antibodies, as found in membranebound form on the surface of B lymphocytes (B cells); and T-cell receptors (TCR), the antigen receptors found on T-lymphocytes (T cells). As applied to antibodydefined antigens, “epitope” can still mean the entire surface area of the antigen which is rendered inaccessible to water as a result of interaction with atoms of the antibody, or it can be used to refer to just that part of the antigen which is necessary and sufficient for antibody binding. As applied to TCR-defined antigens, “epitope” can again mean the entitv area defined on the protein surface recognized by interaction with the TCR. This area is partly one of the self molecules, Class I or Class I1 MHC, and partly a peptide fragment of the protein antigen molecule [2]. Alternatively, the term T-cell epitope can be used to refer just to the antigenic peptide required for binding by a particular TCR, with the understanding that the epitope does not bind at all to the TCR unless presented on self MHC. Concepts of antibody-defined epitope identity have been enhanced in recent years through X-ray crystallography of several antigen-antibody complexes [3-51. Likewise, crystallography of MHC-peptide complexes [6, 71 clarified the manner in which the epitope was created by a combination of the requirements for MHC binding and recognition of the MHC-peptide complex by a TCR selected in the thymus. Study of antibody-defined eptitopes by noncrystallographic methods identifies residues or sequences which are critical to the energy and specificity of binding. These critical residues are universally few in number, and thus comprise only a small portion of the total area of the interface revealed by crystallography. For TCRdefined epitopes, very clear data is obtained with synthetic peptides, and thus there is a tendency to refer to the peptide alone as the epitope. In this review, the term “epitope” will be taken to mean the structure as defined by the technique used to study the antigenlreceptor pair, and thus may encompass

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10 Epitope Mapping with the Use of Peptide Libraries

the entire range from a linear peptide a few amino acid residues in length, to a surface involving up to 22 or more residues of the protein antigen. Discussion will focus on protein antigens, but it should always be borne in mind that carbohydrate, whether as glycosylation of a protein or as a polysaccharide antigen, may also be important in defining particular epitopes. While the term “epitope” was devised for immunology, the concept applies equally well to any protein-protein or even hapten-protein interaction, and thus it is common to refer to an “epitope” when protein subunits or hormone-receptor interactions are being studied. In this chapter we will not be dealing with epitopes in this broader sense, but many of the techniques and studies described in this review would find ready extension to other fields, so use of the term epitope here should not be considered as limiting the applicability of the methods or concepts only to receptors in the immune system.

10.1.2 Brief History of Antibody-Defined Epitope Mapping Several reviews have covered the history of antibody-defined epitope mapping in detail [6, 91. In brief, early work with myoglobin and lysozyme developed the concepts of linear (continuous) versus assembled (discontinuous) epitopes [lo]. These early studies promoted the idea of a multipronged approach to gathering information about epitopes: chemical modification of amino acid side chains of protein antigens, study of the effects of natural sequence variation, cleavage of proteins at different points to create families of peptides, and final confirmation of binding sites using synthetic peptides. Later studies, particularly following the availability of monoclonal antibodies, were at variance with some of these conclusions [8, 91, without invalidating the concepts of continuous versus discontinuous epitopes. In the early 1980s, the practicality of epitope mapping by use of synthetic peptides advanced rapidly, when Geysen and colleagues [ll, 121 devised the Multipin method of parallel peptide synthesis and testing. Other methods of multiple peptide synthesis emerged shortly thereafter [13, 141 and allowed thorough exploration of the continuous epitopes of many antigens [15- 181. The method of synthesis of linear peptides homologous with the sequence of the antigen is limited to the identification of continuous epitopes; more imaginative strategies are needed if discontinuous epitopes are to be found by the synthetic peptide methodology (see below). The ability to express cloned genes to yield native protein antigens, and to make large numbers of mutants with single, directed amino acid changes, opened the way for epitope mapping by comparison of antibody binding to a series of designed whole protein homologs. This is beautifully illustrated in a study of hGH mutants with a set of monoclonal antibodies [19]. While this is perhaps the most “elegant” and uneauivocal wav of definine what antibodies “see” on the surface of a Drotein antinen.

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305

it does not lead directly to information usable for design of a synthetic antigen for diagnostic or immunization purposes. A synthetic peptide with specificity for a particular antibody or group of antibodies can be located by searching through a library of peptides. Such a specific peptide can act as a surrogate for the original antigen. The term “mimotope” was coined to describe this type of specific antibody ligand [20], to indicate that the molecule is a mimic of the native epitope rather than being the epitope itself, as no assumptions are made about whether the epitope is “continuous” or “discontinuous”.

10.1.3 History of T-cell Epitope Mapping Schwarz [21] summarized the work which led to the establishment of the nature of T cell epitopes. The essentially peptide, rather than protein, nature of T-cell epitopes, makes the use of peptides highly effective for their location. Current methods available to generate the target epitope peptides include enzymic or chemical fragmentation of the protein antigen [22], expressed gene fragments generated by restriction enzyme digestion [23], or PCR using synthetic oligonucleotide primers [24]. Synthetic peptides of 15 to 30 residues [25-271 and short peptides synthesized using the Multipin peptide system [28-351 are a highly effective tool for the mapping of epitopes in antigens with known primary sequence.

10.1.4 Comparison between Linear Epitope Scanning and the Combinatorial Library Approach An alternative approach to the identification of specific ligands is the peptide library, or “mimotope” approach. In principle, the aim is to identify a specific ligand for an antibody or TCR without prior knowledge of the epitope. This can include the epitopes of protein antigens for which the sequence is not known, or even nonprotein epitopes [36]. A library of peptides is synthesized, where each member of each set in the library has one or more defined characteristics, such as a particular amino acid at a particular position in the sequence. The ability to identify a peptide which will be an acceptable surrogate for the epitope depends on several factors. Among the most critical are the size (total number of peptide combinations) of the library; the character (peptide length, amino acid set size, degree of degeneracy) of the library; and the discrimination power (sensitivity and specificity) of the test to which the library is applied. Without doubt, a set of overlapping peptides of a protein antigen represent a far more powerful tool than a combinatorial library for location of an epitope where there is an expectation that the epitope will be a linear homolog of the protein. In other cases, the library approach may be the only avenue available.

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10 Epitope Mapping with the Use of Peptide Libraries

10.2 Synthetic Peptides for Epitope Mapping 10.2.1 Difficulty of Predicting Epitopes Many attempts have been made to develop efficient predictive algorithms for both antibody-defined [37] and T cell [38, 391 epitopes. The predictors of antibodydefined epitopes have had a range of theoretical bases, such as the likely exposure of hydrophilic sequences at the surface of a protein antigen [40]. A flaw in the testing of many of these algorithms was the small and biased database of well-defined epitopes from which they were derived and on which they were tested. Despite claims that methods such as the Hopp method “works”, a proper statistical evaluation (371 does not give any of the methods any better than a 60% success rate. The only thorough study of a highly defined protein antigen which minimized the distorting effects of self-tolerance, affecting many common model antigens such as myoglobin, immunoglobulins, albumin and cytochromes [16] did not support the usefulness of the available prediction methods. Likewise, for T cell epitopes, the earlier prediction methods were based on models built on sketchy data [38, 391 and failed to usefully predict major epitopes [27], whereas the discovery of MHC allotype-dependent “motifs” which are necessary, but not sufficient, for Class I [41] and Class I1 [42] peptide presentation, allows a more confident prediction of potential epitopes for each MHC allotype (see Chapters 9 and 12).

10.2.2 Nature of the Screening Task Epitope mapping, through use of synthetic peptides, may be seen as either a “screening” process, where no assumptions are made about the peptides likely or desirable to be found as epitopes; or as a test of a hypothesis such as a predictive method. When the process is a screening one, it may be desirable but not critical to be certain that all possible instances of peptide binding or bioactivity are being detected. The “Law of Diminishing Returns” probably applies: 90% of all epitopes may be detected with an initial experiment and, to detect the last 10% of all possible epitopes, the effort may have to be increased manyfold. In most instances, a judgement of the likely costlbenefit ratio will end up deciding the question of how much effort is expended. The costlbenefit ratio can only be assessed as the experiment progresses; before the experiment is undertaken it has to be estimated based on experience and subjective factors (beliefs). This is highly relevant to the criteria for design of peptide sets and the required target quantity, standard of purity, and characterization. For instance, the discovery of a number of cytotoxic T-cell epitopes occurred because of the presence of “contaminating” truncated peptides in an otherwise acceptably pure peptide preparation. Use of ultrapure peptide in this case may have led to the “correct” result, namely, no epitope would have been detected.

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Ideally, screening is done in a way which maximizes the likelihood of finding those epitopes which exist (i.e., to minimize false negatives) but minimizes the likelihood of observing false positives (apparent epitopes which cannot be confirmed, see below). It seems that the best way to optimize these conditions is to make sets of peptides with built-in “redundancy”. Thus, it is unwise to rely on a single long peptide homologous with a sequence as a reagent to test the presence of an epitope within that sequence. It seems better to make several overlapping peptides, perhaps with different endings or different lengths/offsets, to ensure that potentially bioactive or binding peptides will be present. If, on the other hand, the task is one of testing a prediction or hypothesis, it may be critical that the peptide is of a particular length or of a particularly high level of purity and characterization. The consequences of “false positives” due to contaminants are much more serious in this case, for example, the reinforcing of an incorrect hypothesis. An example is where one is aiming to demonstrate that a predicted peptide is (or is not) acting as an epitope for a particular monoclonal antibody (MAb) or T-cell clone.

10.2.3 “Format” of Peptides for Epitope Mapping The seemingly simple matter of “format” of peptide used can have far-reaching effects on the outcome of an epitope mapping study. By format, is meant whether the peptide is in solution or on a solid phase; if on a solid phase, the character of the solid phase including method of attachment and physicochemical properties of the solid phase; and if in solution, aspects of the peptide sequence including the endcapping groups and whether or not the peptide has a “handle” by which it can be manipulated. When the epitope mapping experiment relates to antibody-defined epitopes, the readout is frequently a simple binding assay. In this case there is a choice between solid and solution phase peptides, with the solid phase route frequently being used for its simplicity and economy. In contrast, for epitope studies on the TCR, solution phase peptides are universally used, as the interaction being studied is a ternary complex between MHC molecules, peptide, and the TCR. One study with binding of solid phase peptides to MHC molecules showed that the binding had little discernible specificity [43], whereas binding of solution-phase peptide to solid-phase MHC [44]or to cell-bound MHC [45] has high specificity and parallels the binding of naturally processed peptides. Specificity of binding to peptide on a solid phase has long been debated and no true consensus has emerged. This is probably because of the large variety of solid phases and methods for attaching peptides to those solid phases, giving many opportunities for the peptide-antibody interaction to be perturbed or for artifacts to be

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10 Epitope Mapping with the Use of Peptide Libraries

Before conclusions are drawn about reactivity of a peptide on a solid phase, thert should be evidence that the peptide is indeed present. Some of the earliest work usec peptides covalently coupled on hydrophilic gels [46] in an effort to quantitate tht antibody specific to an epitope. It is common to bring peptides in contact with native plastics [47, 481 on the assumption they will bind to the plastic, and will be in an orientation which makes the linear sequence accessible to antibodies. For greatei reliability of capture, they can be coupled to proteins before adsorption of the pro. tein to plastic [49]. This method also has the advantage that the peptide is presented in the microenvironment of a protein surface. Biotinylated peptides can be reliablj captured by avidin or streptavidin which has been precoated onto plastic, [50] effectively creating a bound peptide-protein conjugate of each peptide. Testing of pep tides covalently coupled to the solid phase on which they were made, such as conventional synthesis resins [51], paper [52], or graft polymers (121 greatly simplifies the handling and testing of the peptide, and in the latter case allowed the simultaneous testing and re-use of thousands of peptides. In each case, it is important to establish that the solid phase does not significantly perturb the peptide-antibody interaction. Factors other than the nature of the solid surface which affect specificity include the peptide density [53], ligand valency [IgM vs IgG], and the technique adopted to block “nonspecific” binding [54]. In the case of solution-phase assays, aspects of format which are important in epitope mapping include the length of the peptides, especially insofar as long peptides may adopt a preferred structure, slowing the binding kinetics for a ligand which has a requirement for an alternative structure. Thus, shorter peptides are to be preferred on kinetic grounds. Identity of the N- and C-terminal groups on the peptide is also important. Presence of charge due to an unblocked N- or C-terminus can prevent recognition of what would otherwise be a linear epitope. This problem can be avoided by acetylation of the N-terminus and by making the C-terminus an amide, or by making the peptide longer so that the charge is “spaced” away from the amino acid residues being recognized by the ligand. A good example of this is the effect that end groups have on the “minimal” epitope recognised by a T-cell clone [31, 551 or by polyclonal T-cells [29, 561. Effectively, less of the native peptide sequence is required for T-cell recognition if the peptide is flanked with additional bland residues like glycine or beta-alanine, or is N-terminally acetylated or C-terminally amidated. This fact can be put to use in designing sets of peptides to cover a protein sequence with the minimal number of overlapping shorter peptides. For example, if the required length of a T-cell epitope were to be taken as 13 residues, a protein of 900 residues could be covered with approximately 300 synthetic peptides if l5mers offset along the sequence by three residues were used. If the required epitope length were only 10 residues instead of 13, the same sequence could be covered with only 150 peptides by offsetting along the sequence by 6 residues. This shorter epitope length requirement can be achieved with N-terminal acetylation and a constant C-terminal betaalanine diketopiperazine tail, equivalent to three amino acid residues 129, 35, 561.

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10.2.4 Peptide Purity and Characterization While solid phase synthesis of peptides has been a tremendously useful tool in biological research, one of its problems is the “accumulation” of impurities arising from incomplete or side reactions at each step in synthesis, as no purification is normally carried out until the synthesis is completed. Thus, it is unrealistic to expect synthetic peptides to be of “analytical” purity, even after a purification process has been applied, since impurities with similar chemical or physical properties can copurify with the target peptide. In every case, a decision has to be made as to the appropriate purity level required for the project in hand. This decision will affect the choice of methodology used for the synthesis, purification, and characterization phases of peptide preparation. Generally speaking, “unpurified” or “as synthesized” peptides are only suitable for initial investigative or speculative phases of a project. The presence of the target peptide in reasonable yield is assumed; it is also assumed that the presence of impurities will not invalidate the assay results obtained. For antibody epitope mapping, the information contained in the specificity of the antibody for its antigen helps overcome the risk of spurious positive results; as does the fact that binding between antibody and peptide is linearly rather than exponentially related to peptide concentration, so that minor contaminants are unlikely to dominate the binding interaction. The great advantage of screening with unpurified peptides is to obtain indicative information more rapidly and at lower cost than would be required if highly purified peptide had to be used for the same purpose. Results obtained in screening with uncharacterized peptides should be confirmed in more detail with purified or characterized peptides to ensure the absence of serious artifact. In work which uses relatively few peptides, it may be more efficient to use peptide which has been characterized or purified, even if only to a low degree, e.g., >70% of the peptidic material being the target peptide, as measured by a reliable method such as mass spectrometry. Peptide of higher purity (>%To, >99%) can then be used to show that the activity being measured is an inseparable property of the target peptide. As a cautionary point, evidence for purity should not be based solely on a single method such as RP-HPLC, since impurities can co-purify. Similarly, for reliable evidence of identity, several analytical methods may have to be used unless a powerful technique like ion spray mass spectrometry is employed. Even with the most thorough routine testing of a peptide, it may be necessary to synthesize twice, using different chemistries, to confirm that the target peptide made by both methods has the same DroDerties and has not been modified in subtle wavs.

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10 Epitope Mapping with the Use of Peptide Libraries

10.3 Validity Testing of Peptide Assay Results 10.3.1 Testing the Relevance of Peptide Binding Data 10.3.1.1 Antibody Binding

The mere existence of measurable binding by an antibody is not sufficient for classification of a peptide as an epitope. Similar qualification applies to positives in other direct binding assays with peptides. One school of thought holds that linear peptide epitopes do not exist in native antigens [57, 581 but that peptide-binding antibodies exist as a consequence of a response to denatured antigen. Certainly, the proportion of monoclonal antibodies specific for native antigen which are able to bind to one or more peptides of the protein is small [59], but there are many instances in the literature of linear epitopes defined with MAbs which recognize native antigens [60]. When dealing with polyclonal sera, the number of clonal antibody specificities is sufficiently large that some which are able to bind both native antigen and linear peptide are to be expected; however, tests to confirm this are mandatory (see below). As mentioned above, evidence is first needed that the binding measured is attributable to the target peptide, free of artifact arising from impurities. There are several methods for checking the antigen- or antibody-specificityof peptide binding. First, a set of sera including appropriately chosen control sera which are expected to be negative for binding to the peptide, are tested. High binding by prebleed (unimmunized or uninfected) animal sera is presumptive evidence that the binding data obtained with immune sera is nonspecific, i.e., due to antibodies other than those under study [12, 52, 611. We have observed peptides which bind antibodies from a variety of sera, unrelated to the antigen of origin of the peptides under test [62]. Solid phase peptides which are combinations of three residues chosen from the set of large amino acids, particularly W (Trp), Q(G1n) and M (Met), can have high binding to particular antibodies, with some specificity as revealed by the partial dependence for binding on the L-, D-optical isomerism of the residues in question [62] [Geysen et al., unpublished observations]. Specificity can also be tested with binding by control peptides, such as peptides with the same amino acid composition but a significantly different sequence, e.g. the reverse sequence. Alternatively, control peptides can be chosen at random or from parts of the same protein where there is a similar amino acid composition but no commonly observed antibody binding. A more powerful test of specificity is the competition binding test. Peptide-binding antibody populations which are directed against native antigen can be identified by showing that reaction of antibodies with native antigen prior to exposure to the peptide reduces peptide binding [ll]. It is desirable but not essential to remove the immune comolexes formed between the antibodies and native antieen. orior to

10.3

validity Rsting of Peptide Assay Results

31 1

testing the competed antibody preparation for binding on peptides. Excess antigen should be used. In a polyclonal serum, the concentration of antibodies directed to a particular epitope is rarely known, and the “concentration” of the epitope itself may be difficult to determine, especially for a particulate or impure antigen preparation, so it is difficult to determine absolutely when the antigen is in excess. In such cases, a titration of antigen dose should show that the reduction in peptide binding is a function of the concentration of competing antigen. One underlying assumption in this type of competition test is that the affinity of the antibodies for the native antigen is higher than for the peptide, a reasonable assumption when one considers that a short linear peptide is unlikely to contain all elements contributing to the binding of an antibody to a protein antigen. A less obvious factor in the outcome of a competition test is the role of peptide concentration, particularly when the peptide is on a solid phase. For example, when a peptide is on the grafted surface of a plastic pin, the peptide concentration can be very high (millimolar). While this is good for ensuring high sensitivity of detection of peptide-binding antibodies, it can have two undesirable effects. It can result in detection of low affinity antibodies. When the affinity of the antibody for the peptide is high, it can also make the concentration of native antigen, required for effective competition, very high indeed [43]. Additionally, high peptide surface density on a solid phase gives a high probability of multivalent binding (through both arms of IgG or multiple sites on IgM) which can magnify the apparent affinity of antibodies for the peptide. Some of these factors can be avoided by using solution-phase peptide as the competitor in a binding test on native antigen. Due to the presence in polyclonal sera of antibody populations unrelated to a specific peptide, this method is only feasible for monoclonal antibodies or epitope-specific fractions of polyclonal sera. A further method of value in testing the relevance of peptide-binding antibodies is the elution test. After an initial step of binding antibodies on a peptide which is captive on a solid phase, the complex is washed free of unbound material and a nonspecific method for dissociation of the peptide-antibody complex is applied, e.g., low or high pH; chaotropic agents [63]. Released antibody is then available for retesting in a binding or functional assay involving the native antigen, e.g., a neuAn advantage of this method over competition tests is that the tralization assay [MI. final test on the peptide-specific antibody population can be more relevant to a biological function. Possible problems with the method include difficulty of eluting the higher-affinity antibodies and denaturation of antibodies due to harshness of the elution conditions.

10.3.1.2 Major Histocompatibility Complex (MHC) Binding

A different situation applies to the study of the specificity of peptide binding to MHC molecules. Binding of peptide to a MHC molecule is a necessary but not suffi-

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cient condition for the peptide to be recognized by a TCR. The specificity of MHC binding has been largely elucidated by sequencing of peptide extracted from purified MHC (651 but can also be studied by allowing MHC molecules to select from a peptide library, whether a random library or a library created by selection of sequences from the antigen of interest [66]. In this case, it appears essential for the peptide to be in solution phase [43]; the issue of valency does not arise as there is a single binding site in each MHC heterodimer, although we cannot ignore the possibility that noncovalent polymers of MHC molecules may function in vivo [67]; and the only alternative antigen which could be used in competition tests is another peptide known to be presented by that MHC allotype. However, since peptides homologous with a known epitope can be effective antagonists [68], screening of a library for simple binding could be an effective way of identifying potential therapeutic peptides.

10.3.2 Testing the Relevance of Bioactivity Data In principle, one would expect the bioactivity of a peptide to be less subject to artifact, and more likely to reflect the in vivo situation, than an in vitro binding assay, if only because the mechanisms for the ultimate production of the biological effect are more complex and possibly multistep. Thus, tests for specificity in bioactivity assays are frequently limited to testing of analogs as part of a structure-activity study. One would expect that some analogs will be inactive in the assay, and these can be taken as “negative controls”. This is not, however, proof that the biological activity is being brought about by the mechanism on which the peptide selection or design was predicated. For example, a pentapeptide from human IgE was developed as an anti-allergy drug [69]. The peptide sequence was chosen as a blocker of the uptake of IgE by the epsilon FcR on mast cells, but studies failed to support this as the mechanism of action. Similarly, a peptide from the human immunodeficiency virus gp 160 sequence was found which was able to prevent the binding of virus to its cellular receptor, the CD4 molecule. Study of fractions of the peptide from purification runs showed that the major activity resided in a peptide fraction which was side chain protected, and the target peptide was devoid of activity, showing that the theoretical basis for the bioactivity was not supported [70]. 10.3.2.1 Bioactivity of Antibody-Defined Linear Peptide Epitopes

The major biological test usually applied to linear antibody-defined peptide epitopes is to see if they will induce the formation of new antibodies with specificity similar to those produced in response to the whole antigen. In the early 1980s, it was widely claimed that most accessible peptide sequences would induce the formation of such antibodies [71, 721. Such antipeptide antibodies have been very useful as a research

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tool, such as for protein antigen tracing. However, the assessment of the antibodies generally included detection of antibodies to denatured antigen, leaving open the question of the bioactivity of the antibodies against native antigens. Despite limited successes in highly defined homologous challenge systems [73,74], peptide vaccines have not generally been shown to be effective in inducing protective antibodies. This may be due to lack of “structural” information in the peptide immunogen, or due to steric factors preventing high affinity binding of antipeptide antibodies to the same sequence in the native protein. 10.3.2.2 Bioactivity of T-cell Epitopes

Because natural T-cell epitopes are short linear peptides, they are readily characterized and ultilized in bioassays. The bioactivity of peptides as T-cell epitopes can be very clearcut, as with an epitope recognized by a stable, characterized T-cell clone [31, 331 or it can be uncertain, as with peptides recognized by freshly collected polyclonal T-cells [35, 751. A major unanswered question in studies on T-cell epitopes as recognized by polyclonal T-cells is: to what extent can the recognition of a peptide of one antigen by T-cells be attributed to cross-reactivity with peptides from other antigens [76]? The other antigens in question may be completely unrelated antigens, or may be from other organisms (viruses etc.) sharing sequence homology.

10.4 Peptide Libraries from Pins In this discussion, a peptide library is taken to be any collection of peptides used in a screening exercise. It may be a highly structured set, such as the set of all overlapping peptides of a given length homologous with the sequence of a protein antigen of interest, or it may be a “random” set made by mixing amino acid solutions prior to the coupling reaction. Thus, each peptide preparation could consist of one reasonably pure peptide, or of a mixture of tens to millions of peptides.

10.4.1 Types of Library: Strategies The strategies for linear epitope mapping with Multipin-peptides, whether for antibody- or T cell-defined linear epitopes, have been well spelt out by Geysen et al. [12]. Briefly, it is feasible to test all possible overlapping peptides of a given (sufficient) length homologous with a protein sequence. In such a case, the offset of the N-terminal residue of each peptide from the preceding and following peptide is one residue; the overlap is therefore n-1 residues, where n = the chosen peptide length. Such a “scan” through a protein can reveal all linear epitopes if the peptides are long

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enough, provided that the peptides are not so long that secondary structure forma. tion interferes with transition of the solution-phase conformation to the ultimate bound conformation of the peptide, whether bound to an antibody or MHC mole cule. While the early work on antibody-defined epitopes was done with hexa- or oc. tapeptides, we have found that use of peptides up to 15 residues in length is satisfac tory for this purpose (unpublished results). When using longer peptides, it is alsc feasible to use an offset of greater than one residue. Thus, as mentioned above (Sec. tion 2.3), the combination of peptide length and offset should be chosen to ensure that no sequences shorter than the expected maximum length epitope are missed. Foi example, to ensure that all octapeptide sequences are represented, one could makf decapeptides offset by 3, 12mers offset by 5, 15mers offset by 8, etc. We have termed the use of multiple sets of peptides, each of different length, a “Window” analysis [12]. The additional information gained by this method over that from a single pep. tide length is the more accurate definition of the boundaries of an epitope [15, 331, This approach may give the minimal active peptide immediately, or may allow such a peptide to be located by synthesis of one or a very few peptides, perhaps a smaller “Window” set, deduced to contain the key elements of recognition by the antibody, MHC molecule, etc. After first identifying the minimal effective length of an epitope, so that there are few or no residues at the N- or C-terminal end which make no contribution to the binding of (e.g.) antibody, it is practical to study the role of each residue systematically with another peptide library consisting of a set of analogs. The simplest is a set of single-residue substitutions, known as a replacement set [12]. Substitution with each of the alternative genetically coded amino acids gives a detailed picture of the requirements for binding (77). A less detailed but still very informative picture can be obtained with a small set of replacements which presents a diverse set of functional group properties, i. e., lysine (positive charge), leucine (hydrophobic), glutamate (negative charge), phenylalanine (aromatic) and glycine (no side chain). It is clear that, even within “continuous” epitopes, there are residues whose side chains play no part in the binding energy of the peptide-antibody ligand interaction [77]; these have been called “spacer” residues by the author, while others have redefined these epitopes as discontinuous, negating the otherwise useful classification of pep tide epitopes into two types [17]. A completely different approach is the Mimotope strategy, based on “random” peptide libraries. The element of randomness referred to is usually that at each coupling cycle, more than one amino acid is incorporated, producing a peptide mixture in which the sequence of any particular molecule is randomly determined, with the exception of one or more “defined” positions in the sequence, at which only a single amino acid was coupled (20). The output of such a synthesis is a set of pins (or a set of soluble peptides) in which the peptides are mainly random but have predetermined residues in certain positions within the sequence. Thus, stronger binding to one peptide preparation rather than others tells us something about the

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preferred binding peptide sequence for that protein, whether antibody, receptor, etc. Through a process of resynthesis of ever more defined peptides, a peptide (now termed a “mimotope”, or mimic of an epitope) with the “optimal” binding affinity can be determined without the requirement for sequence information from the antigen. Any combination of peptide design from fully randomized to fully specified is possible. For example, it is feasible to make peptides corresponding to a known linear epitope, with substitution of a mixture of amino acids at each position in turn. In an experiment to determine the dependence of binding on the degree of randomness introduced by such a synthesis, it was seen that specific binding by a MAb decreased with each substitution until the substitution of five out of six residues left no significant specific signal (Fig. 10-1). The same approach of using a mixture rather than a single amino acid could be used in a simplified replacement analysis to see the dependence of binding on the nature of the residue in each position in the epitope. In the course of studies of the binding of antibodies to peptide libraries on pins, we observed that antibodies would bind strongly to pins with a high density of certain dipeptides (781. Further exploration of this phenomenon showed that it was

a

Yw

401

r

1

I

I

I

I

I

0

1

2

3

4

5

6

Number of Residues Replaced

Figure 10-1.Effect of replacing residues in a monoclonal antibody-defined epitope DFLEKI with mixtures. Hexapeptides were synthesized on pins where, rather than always using a single pure amino acid at each position, a mixture of all 20 genetically coded amino acids was used in one, two, or more positions. For each “level” of replacement (1, 2, 3. .residues replaced), the mean and range (not SD) of the ELISA values is shown, relative to the single peptide DFLEKI (= 100%) and the complete random hexapeptide mixture (=O%). There are 6 single replacements, 15 double replacements, 20 triple, I5 quadruple and 6 quintuple replacements. (H. M. Geysen, T. J. Mason, personal communication)

.

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I0 Epitope Mapping with the Use of Peptide Libraries

possible to use a dipeptide as the starting point for building up a binding peptide (mimotope) with specificity for a particular MAb [79]. This approach to mimotope delineation, dubbed the “incremental” approach in contrast to the “decoding” ap. proach described in the preceding paragraph, has not been very successful due to the rather nonspecific starting point (dipeptides) [62] and the frequent lack of any incremental binding energy with increase in peptide length (unpublished observations).

10.4.2 Methods of Synthesis of Peptide Libraries on Pins The success of the pin method of peptide synthesis is dependent on the properties of the graft polymers used to attach peptide to the inert plastic of the pin. Polyethylene pins are gamma irradiated in the presence of vinyl monomers such as acrylic acid, resulting in the formation of a covalently bound, saturated polymer. The functional groups on the polymer are determined by the choice of monomer; polymers can be more or less hydrophilic and their solvation by the organic solvents used in peptide synthesis is likewise controlled by the choice of monomer. The original polyacrylic acid polymer [Ill was compatible both with DMF and water, allowing testing of peptide in aqueous solution on the same polymer on which it had been synthesized. Later developments [SO, 811 created a range of polymers for different applications, including polymers which are highly efficient for conventional peptide synthesis, and can be cleaved in either aqueous or nonaqueous solvents. Choice of amino acid protection scheme for synthesis is not critical, but we have switched from the earlier Boc chemistry [Ill to Fmoc chemistry [S2] because of the milder conditions of the latter, especially for side chain deprotection. Use of pins for synthesis of peptides with a wide variety of choices of C-terminal endings necessitated the development or application of new linkers, including the diketopiperazine-forminglinker [28]; esters cleavable to free acids or methylamides [83]; and Rink amide-forming handles [Sl]. The DKP-forming linker was particularly useful for cleavage of peptides directly into aqueous solution at pH 7, free of residual scavengers, etc., and ready for use in biological assays [28]. Table 10-1. Comparison of solid phase and solution phase libraries for epitope mapping

Characteristic

Solid Phase

Solution phase

Amount of peptide Peptide characterization Re-use possible Ease of handling Reproducibility of successive tests Versatility Control of “dose”

Small: 1 - 100 nmol Difficult Yes Simple, rapid Questionable Binding only Restricted

Large: 1 - 100 Bmol Unrestricted No Time consuming High Many types of assay Unlimited

10.4 Peptide Libraries from Pins

311

Thus, peptide libraries can be made with equal convenience as permanently bound to the “solid” (gel) phase, or as cleaved (solution phase) peptides. The relative merits of these have been discussed briefly above; further comparisons are given in Table 10-1. While the original pins were limited to about 100 nmol peptide per pin, later designs with scaled-up surface area can be used to make up to 50 micromole per pin while retaining the convenience of the microtiter plate format, with 96 pins per holder.

10.4.3 Strategies for Maximizing the Usefulness of Pins The simplest way of making a peptide library on pins is to make one pinlone peptide. This strategy works well when doing a scan through a protein sequence. However, in some cases the screening test is more difficult, costly or longer than the process of making peptides, and it is worthwhile pooling a sample of each peptide for the initial assay [56]. For any pool yielding a positive result, the identity of the peptide(s) responsible is easily determined by testing the individual peptides comprising the pool. This pooling process only has an advantage, compared with testing all peptides individually, if there are some “negative” pools; the more negative pools there are, the greater the advantage. The next level of complexity in making libraries on pins is to synthesize mixtures of peptides on a single pin. It is not feasible to use with pins the “divide, couple, recombine” approach which is used in resin synthesis of libraries [84). In practice, an effective way to produce mixed peptides on pins is to couple mixtures of activated amino acids. The total amount of amino acid added is slightly greater than the substitution level of the pin. This minimizes unequal coupling due to the different reaction rates of the different amino acids. The coupling time can be extended to promote complete coupling, followed if necessary by a recoupling (without deprotection), with an identical mixture, to drive the reaction to completion. The ability to handle large numbers of pins simultaneously, using equipment designed for microtiter work, allows the number of individual mixtures handled to be high, reducing the complexity of each mixture. For example, to test 200000 peptides, it is feasible to handle 2000 pins, each pin producing a mixture consisting of 100 peptides, rather than using (say) 400 resin samples to produce 400 mixtures, each consisting of 500 peptides. In this example, the lower complexity of each mixture from pins means that the concentration of each peptide is high, and there is less uncertainty about the interpretation of the results; “decoding” or “deconvolution” to a single binding or bioactive Deptide also is faster and takes less effort.

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I0 Epitope Mapping with the Use of Peptide Libraries

10.4.4 Approaches to Amino Acid Mixtures In the initial library work, mixtures of all 20 genetically coded amino acids were used [20]. The chemical and physical properties of amino acids are of such variety that it is (a) reasonable to exclude some of them because of unwanted side reactions (Cys, Met), and (b) possible to treat them as groups in order to better control coupling rates and conditions. For example, it makes sense to couple a mixture of Val, Leu and Ile but not to mix these with Asn and Gln. It is reasonable to include, with grouped amino acids, nonnatural amino acids with similar physico-chemical properties, if this does not result in an unmanageably large set of peptides. The interpretation of data on mixtures of similar peptides is also more straightforward than on mixtures of dissimilar peptides. Naturally, the larger the number of separate groupings of amino acids, the larger is the overall size of the library in terms of numbers of discrete mixtures which have to be handled, so it can be impractical to subdivide into too many groups. A new concept in random libraries is based on sets of peptides made with reassorted groupings of the same starting set of amino acids. From the change in binding pattern observed as the groupings are altered, it is possible to deduce which amino acids in which positions make the major contribution to binding (A. Bray, personal communication). The advantage of this approach is that resynthesis of several sets of peptides of progressively decreasing complexity (“decoding” or “deconvolution”) may not be necessary; the sequence of the best-binding peptides can be deduced from the primary data.

10.4.5 Downstream Processing of Pin-Peptide Libraries As with any library of compounds, creative ideas are needed to ensure that the materials being tested are “as designed” and are present at the expected concentration. Since many of the components of the library are new compounds with unknown properties, the handling methods have to try to cope with all possibilities including poor solubility, instability, etc. Thus, to ensure that selective solubility does not bias the composition of the library too much, it is important to minimize the number of handling or purification steps, and to redissolve peptides in solvents which have the greatest likelihood of taking all components into solution. Preferred solvents are DMSO for its broad solubilization properties and acetonitrile for its low toxicity, although DMF can also be used. Once solubilized, the master library solutions are stored at - 20 “C or colder. Working solutions can be prepared and frozen (cg., in microtiter plates) ready for thawing just before an assay. Characterization of individual peptides making up a systematic library set is straightforward. The usual methods of chromatography and amino acid analysis can confirm homogeneity and composition. A recent technological advance in mass

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spectroscopy, ion spray MS,allows the identity and purity of a peptide to be rapidly checked [85]. In contrast, characterization of random libraries is difficult. Because random libraries are complex mixtures, any gross analysis will not detect the absence of individual compounds, but may indicate general failure, e.g., failure to couple a particular amino acid. The technique of semiquantitative ion spray MS can be applied to compare the predicted profile of the m/z spectrum of the sample with the observed profile (T.Mason, personal communication; see also Chapter 8).

10.4.6 Screening Methods Applicable to Pin Peptides The original method developed for pin peptides was the direct binding test. Being able to test peptides on the same solid support on which they had been synthesized produced great savings in time and effort of handling the peptides themselves. Through use of computer programs for guiding the synthesis and testing, the data was rapidly acquired and analyzed without the need for transcribing or manual calculation. Removal of the bound ligand (antibody) by use of warm detergent (SDS), reducing agent (mercaptoethanol), and sonic agitation, allowed the peptides to be retested many times. Conditions were adopted which protect the peptides from rapid breakdown; e.g., any long incubation steps are carried out in the cold, and the sonic cleaning of the pins keeps them free of microbial contamination. At the end of each use, the pin peptides are dewatered with warm methanol and after air drying can be stored desiccated in the cold for months. ELISAs on pin-bound peptides show a linear relationship between antibody concentration and OD reading over the linear reading range of a microplate reader. This conveniently allows estimates to be made of the relative concentration of particular peptide-specific antibodies in fractionated sera [12, 861. As mentioned above, specificity tests such as the competition test and the elution test can also be done with permanently pin-bound peptides. The direct binding test on pin-peptides has some disadvantages. It is difficult to characterize the peptide on the pin, or to make a meaningful estimate of the peptide quantity. On occasions, antibodies will bind so strongly to pin-peptides that disruption is not completely effective, necessitating additional control tests to demonstrate that pins are free of antibody before the next test is carried out. Pin-peptides which have been cleaved into physiological solutions, such as those synthesized on pins prepared with the DKP-forming cleavable linker [28], can be used directly in bioassays such as T-cell stimulation assays [35]. Similarly, sets of pinpeptides which have been made with a constant capture handle such as a biotin group can be tested in ELISA or RIA [50]. The solid phase in such tests will present the peptide at a lower surface density than found on a pin, and they are thus of lower sensitivity, but are consequently less subject to artifact. We have recently developed

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I0 Epitope Mapping with the Use of Peptide Libraries

a monoclonal antibody specific for an N-terminal dipeptide (N-acetyl-Cys-beta. amino-alanine), which allows capture of any peptide with this N-terminal structure. Peptides made with this N-terminal sequence can thus be captured by this MAt for presentation to a second antiserum, or the Cys residue can be used for covalenl coupling to a protein, gel, or other macromolecular entity. Synthesis of peptide set: without this precaution of incorporation of a handle can lead to difficulty in using the peptide for measurement of binding in a solid phase method since peptides maj not adsorb directly on a plastic surface [87] and for small peptides, the process 01 binding to a solid phase can mask the antigenic determinants. In many instances, sets of pin-peptides for testing in bioassays cannot include tag ging sequences, because the modifications would prevent their biological effect. In such cases, pin-peptides can be made with C-terminal acid or amide groups rathe1 than the more convenient DKP group. After side chain deprotection and cleavage, the peptides can be handled as sets, still in the same layout as they were during synthesis, employing common laboratory equipment designed for efficient handling of multiple samples in microtiter format. Provided the assay is amenable to being carried out in microtiter trays, handling time is minimized and there is a reduced chance of mistakes being made.

10.5 Comparison of Methods of Peptide Library Generation for Epitope Mapping Peptide library generation requires multiple peptide synthesis. Here we will only consider the methods for chemical synthesis, leaving biosynthetic methods for others to cover. All modern, efficient methods of multiple peptide synthesis use the solid phase concept. The approaches differ mainly in consequence of the physical nature of the solid phase used. Factors which are important are: the quality of the peptides, the amount which can be made, the ease of physical handling of the solid phase, the ease of recovery of the peptide from the solid phase at the end of synthesis, the cost, and the adaptability of the method to variations in chemistry, peptide design etc. Each method has its particular strengths and weaknesses, which are discussed below.

10.5.1 Systems Using Spatially Stable Matrices Peptides permanently attached to the solid phase on which they were synthesized can either be located at a predetermined position on a spatially stable matrix or can be “loose”. Stable matrices include the Multipin system, synthesis as spots on paper [14], synthesis on the surface of a glass slide [88], and synthesis on grafted plastic dates (MiniDeDscan 1891). These svstems have the advantage that the identitv of the

10.5 Comparison of Methods of Peptide Library Generationfor Epitope Mapping

321

peptide at each location on the solid phase is determined at the outset and remains fixed during the life of the peptide, simplifying all aspects of handling and recording. They all suffer from the limitation of being suitable only for binding studies, and thus not useful for T-cell epitope mapping. There is uncertainty about peptide quantity and purity, as well as the often unknown influence of the solid support on the specificity and strength of binding of macromolecules to the peptide. While qualitative ELISA results from these techniques may be comparable, there is always the need to apply a quantitative cutoff in discriminating “significant” binding from “background” binding. For this purpose, a method in which the data are machine read and expressed as a number (optical density) is clearly preferable to one in which the intensity is judged subjectively or quantitated in a nonlinear fashion. Similarly, if results of ELISAs run in parallel (as in a competition test) need to be compared, the availability of quantitative ELISA optical density data allows a statistical test for significance of differences between test samples and the appropriate controls, whereas only gross differences can be deduced from the subjective reading of color in ELISA. Re-use of peptides is practical with pins, plastic plates and glass slides but may present problems with spots on paper due to instability of the paper. Pins and plates allow quantitation, while spots are qualitative [90]. The “Spots” has a slight advantage in simplicity, while the technique of synthesis on glass slides is “high-tech” and consequently not widely available. Both Spots and microplates have the benefit of low reagent usage. The glass slide technique employs unusual and incompletely understood chemistries. A drawback of spots or plates is that the reaction zone has a diffuse edge which may compromise peptide quality and ELISA quantitation. An advantage of pins is that they allow individual peptides to be tested without testing all in the set, or if necessary they can be re-sorted to create subsets for particular assays. The graft polymers available on pins now allow synthesis of peptides of quality as high as or higher than the best resin synthesis [91].

10.5.2 Systems Producing “L~ose”Solid-Phase Peptides These differ from the previous category in having no physical connection between the solid phases bearing different peptides. Peptides can be made on paper or cotton disks in a way which ensures that the whole reactive area is used [14, 921 such as by placing disks in a tea-bag [93]. Since each disk can be used once and discarded, problems associated with re-use are avoided. A gain in sensitivity for IgE measurement is possible but some of the mass handling advantages of rigidly located peptides are lost. Synthesis of random peptide libraries on resin has been used in combination with direct binding to the peptide-resin beads to identify de novo binding peptides for

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10 Epitope Mapping with the Use of Peptide Libmries

monoclonal antibodies [92]. These are presumably subject to the same problems of nonspecificity found previously with resin [94] and with pins (Section 3 above) and should similarly be checked by competition, elution and then by tests involving solution-phase peptide [12].

10.5.3 Systems Producing Cleaved Peptides This category includes all types of multiple peptide synthesis on resin, such as teabags, RAMPS, and peptide synthesis machines (951. It includes pins with a cleavable linker, both the 1 pmol and 5 pmol scale [91]. While several of these systems are computer assisted or controlled, the pin system also keeps the peptides in their 8 x 12 array and they can be handled in this form during and after cleavage. Multiple peptide synthesis machines are very expensive and a large synthesis program is necessary to justify their use. Speed of synthesis slows as the number of peptides being made increases. After synthesis, deprotection and handling can become a bottleneck, as it is with tea-bags and RAMPS also. In contrast, the number of peptides being made on pins can be doubled, etc., by handling two blocks of 96 pins instead of one, or four instead of two, without overloading the time of one researcher. The scale of synthesis on multiple synthesizers is up to about 50 RmoI, whereas commercially available pins can give up to 5 pmol per pin. This latter quantity is sufficient for most initial screens, such as antibody epitope mapping [96], T-cell epitope mapping [35, 561, or hormone activity bioassay [97, 981, but if larger amounts are needed, a multiple synthesizer is the most attractive option. In summary, there is now a broad choice of methods for epitope mapping with synthetic peptides, and for searching for specific peptide ligands of antibodies. The choice appropriate to a particular project depends on the resources available to the researcher, the scale of the project, and the researcher’s knowledge of peptide chemistry. The Multipin system is attractive for rapid initial screening on solid phase or in solution phase, or small-scale work; spots, paper disks or Minipepscan may be more suitable for low budget semiquantitative work, while multiple peptide synthesis machines satisfy the well-funded researcher’s need for large amounts of large numbers of peptides.

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S. J. Rodda, P. C. Ho, P. Csurhes, H. Dunckley, A. Saul, H. M. Geysen. C. M. Rzepczyk, J. Immunol. 1991, 147, 2507-2513. [33] S. R. Burrows, S. J. Rodda, A. Suhrbier, H. M. Geysen, D. J. Moss, Eur. J. Immunol. (321 A. Suhrbier,

1992, 22, 191- 195. [34] S. J. Rodda, M. Benstead, H. M. Geysen, “Exhaustive Helper T Cell Determinant Mapp

ing of Influenza Type A Antigens with Synthetic Peptides and Human PBMC”, in Op tions for the Control of Influenza II (Ed. Hannoun C. et al.), Excerpta Medica, Amster. dam, 1993, pp. 237-243. [35] J. C. Reece, H. M. Geysen, S. J. Rodda, J. Immunol. 1993, 151, 1-10. [36] H.M. Geysen, R. MacFarlan, S. J. Rodda, G. Tribbick, T. J. Mason, P. G. Schoofs, “Pep tides which Mimic Carbohydrate Antigens” in Towards Better Carbohydrate Vaccna (Eds.: R. Bell, G. Torrigiani) Wiley, Chichester, 1987, pp. 103-118. [37] J. L. Pellequer, E. Westhof, M. H. V. van Regenmortel, Methods Enzymol. 1991, 2038, 176-201. [38] J. B. Rothbard, W. R. Taylor, EMBO J. 1988, 7, 93-100. (39) C. DeLisi, J. A. Berzofsky, Proc. Natl. Acad. Sci. USA 1985, 82, 7048. [40]T. P. Hopp, J. Immunol. Methods 1986, 88, 1-18. [41] K. Falk, 0. Rtitzschke, S. Stefanovik, G. Jung, H.-G. Rammensee, Nature, 1991, 351, 290-296. (42) J. H. Brown, T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger, D.C. Wiley, Nature 1993, 364, 33-39. (43) B. P. Chen, J. Rothbard, P. Parham, J. Exp. Med. 1990, 172, 931-936.

[44]K. Matsui, J. J. Boniface, P. A. Reay, H. Schild, B. Fazekas de St Groth, M. M. Davis, Science 1991, 254, 1788-1791. [45] R. Busch, J. B. Rothbard, J. Immunol. Methods 1990, 134, 1-22. [46]S. S. lhining, M. Z . Atassi, J. Immunol. Methods 1979, 30, 139-151. [47] J. A. Tainer, E. D. Getzoff, H.Alexander, R. A. Houghten, A. J. Olson, R. A. Lerner, W. A. Hendrickson, Nature, 1984, 312, 127-134. (481 H. J. Geerligs, W. J. Weijer, W. Bloemhoff, G. W. Welling, S. Welling-Wester, J. Immunol. Methods 1988,106, 239-244. (491 J. P. Briand, S. Muller, M. H. V. Van Regenmortel, 1 Immunol. Methods 1985, 78, 59-69. [50] A. J. Weiner, H. M. Geysen, C. Christopherson, J. E. Hall, T. J. Mason, G. Saracco,

F. Bonino, K. Crawford, C. D. Marion, K. A. Crawford, M. Brunetto, P. J. Barr, T. Miyamura, J. McHutchinson, M. Houghton, Proc. Natl. Acad. Sci. USA 1992, 89,

3468-3472. [51] J. A. Smith, J. G. R. Hurrell, S. J. Leach, Immunochemistry 1977, 14, 565-568. I521 W. van t’Hof, M. van den Berg, R. C. Aalberse, J. Immunol. Methods 1993, 161, 177- 186. [53] H. S. Marsden, A. M. Owsianka, S. Graham, G. W. McJxan, C. A. Robertson, J. H. Subak-Sharpe, J. Immunol. Methods 1992, 147, 65-72. [54] R. E Vogt (Jr.), D. L. Phillips, L. 0. Henderson, W. Whitfield, E W. Spierto, 1 Immunol. Methods 1987, 101, 43-50. I551 P. M. Allen, G. R. Matsueda, S. Adams, J. Freeman, R. W. Roof, L. Lambert, E. R. Unanue. Int. Immunol. 1989. 1. 141-150.

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1561 J. C. Reece, D. L. McGregor, H. M. Geysen, S. J. Rodda, J. Immunol. Merhods 1994 172, 241- 254. 1571 R. Jemmerson, Proc. Natl. Acad. Sci. USA 1987, 84, 9180-9184. [58] W. G. Laver, G. M. Air, R. G. Webster, S. J. Smith-Gill, S. J., Cell 1990, 61, 553. [59] B. D. Spangler, 1 Immunol. 1991, 146, 1591-1595. (60)M. E. Duncan, M. E. McAleese, N.A. Booth, W. T.Melvin, J. E. Fothergill, J. Immunol. Methods, 1992, 151, 227. (611 R. Savoca, C. Schwab, H. R. Bosshard, J. Immunol. Methods 1991, 141, 245-252. (621 F. v. Bockstaele, Math. Comput. Modelling 1992, 16, 161-178. 1631 G. R. Pullen, M. G. Fitzgerald, C. S. Hosking, J. Immunol. Methods 1986, 86, 83. [64)G. Tribbick, B. Triantafyllou, R. Lauricella, S. J. Rodda, T. J. Mason, H. M. Geysen. J. Immunol. Methods 1991, 139, 155-166. 165) D. F. Hunt, R. A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A. L. Cox, E., V. H. Engelhard, Science 1992, 255, 1261-1266. [66]J. A. Lenstra, J. H. F. Erkens, J. G. A. Langeveld, W. P. A. Posthumus, R. H. Meloen, E Gebauer, I. Correa, L. Enjuanes, K. K. Stanley, J. Immunol. Methods 1992, 152, 149- 157. [67] J. H. Brown, T. S. Jardetzky, J. C. Gorga, L. J. Stern, R. G. Urban, J. L. Strominger, D. C. Wiley, Nature 1993, 364, 33-39. [68] M. T. De Magistris, J. Alexander, M. Coggeshall, A. Altman, E C. A. Gaeta, H. M. Grey, A. Sette, Cell, 1992, 68, 625-634. 1691 R. N. Hamburger, Immunology 1979, 38, 781-787. (701 J. D. Lifson, K. M. Hwang, P. L. Nara, B. Fraser, M. Padgett, N. M. Dunlop, L. E. Eiden, Science 1988, 241, 712-715. 171) N. Green, H. Alexander, A. Olson, S. Alexander, T. M. Shinnick, J. G. Sutcliffe, R. A. Lerner, Cell 1982, 28, 477-487. [72] H. M. Geysen, S. J. Barteling, R. H. Meloen, Proc. Nutl. Acad. Sci. USA, 1985, 82, 178-182. 1731 M. J. Francis, C. M. Fry, D. J. Rowlands, F. Brown, J. L. Bittle, R. A. Houghten, R. A. Lerner, J Gen. Y i d . 1985, 66, 2347-2354. [74] D. M. A. Evans, P. D. Minor, G. S. Schild, Nature 1983, 304, 459-462. 1751 D.A. Mutch, J. R. Underwood, H. M. Geysen, S. J. Rodda, J. Acquired Immune Deficiency Syndromes 1994, 7, 879-890. I761 V. Bhardwaj, V. Kumar, H. M. Geysen, E. E. Sercarz, 1 Immunol. 1993,151, 5000-5010. (771 H. M. Geysen, T. J. Mason, S. J. Rodda, J. Mol. Recog. 1988, I , 32-41. 1781 H. M. Geysen, Immunol. Today 1985, 6, 364-369. (79) G. Tribbick, A. B. Edmundson, T. J. Mason, H. M. Geysen, Mol. Immunol. 1989, 26, 625 -635. [SO] N. J. Maeji, R. M. Valerio, A. M. Bray, R. A. Campbell, H. M. Geysen, Reactive Polymers 1994, 22, 203-212. [81] R. M. Valerio, A. M. Bray, N. J. Maeji, Int. 1 Pept. Protein Res. 1994, 44, 158-165. I821 A. M. Bray, R. M. Valerio, A. J. DiPasquale, J. Greig, N. J. Maeji, J. Pept. Sci. 1995, I , 80-87. 1831 A. M. Bray, A. G. Jhingran, R. M. Valerio, N. J. Maeji, J. Org. Chem. 1994, 59, ~

I

O

~

-

Y

~

326

10 Epitope Mapping with the Use of Peptide Libraries

(84) R. A. Houghten, J. R. Appel, S. E. Blondelle, J. H. Cuervo, C. T. Dooley, C. Pinilla, Bic Techniques, 1992, 13, 412-421. (851 C. Dass, J. J. Kusznierz, D. M. Desiderio, S. A. Jarvis, B. N. Green, J. Am. Soc. Mass, Spectrom. 1991, 2, 149-156. 1861 D. D.Shukla, G. Tribbick, T. J. Mason, D. R. Hewish, H. M. Geysen, C. W. Ward, Proc. Nail. Acad. Sci. USA 1989, 86, 8192-8196. (871 H. J. Geerligs, W. J. Weijer, W. Bloemhoff, G. W. Welling, S. Welling-Wester, J. Im. munol. Methods 1988, 104, 239-244. I881 S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 1991, 251, 767-773. [89) W. C. Puijk, G. C. Ligtvoet, R. H. Meloen, World Patent WO 93109872, 1993. (901 R. Frank, S. Guler, S. Krause, W. Lindenmaier. Peptides 1990, Proc. 21st. Eur. Pept. Symp. (Eds.: E. Giralt, D. Andreu) 1990, pp. 151-152. [91] N. J. Maeji, A. M. Bray, R. M. Valerio, W. Wang, Pept. Res. 1995,8, 1-6. (921 J. Eichler, M. Bienert, A. Stierandova, M. Lebl, Pept. Res. 1991,4, 296-307. I931 W. van’t Hof, M. van den Berg, R. C. Aalberse, J. Immunol. Methods 1993,161,177-186. (941 P. Shi, J. P. Riehm, P. E. E. Todd, S. J. Leach, Mol. Immunol. 1984,21, 489-496. [95) G. Jung, A. G. Beck-Sickinger, Angew. Chem. 1992,31, 367-486. I961 J. R. Underwood, G. A. Cartwright, A. M. McCall, G. Tribbick, M. H. Geysen, M. T. W. Hearn, J. Autoimmunity 1994, 7, 291-320. [97] M. A. Seldon, M. Karabatsas, A. M. Bray, N. J. Maeji, H. M. Geysen, Immunomethods 1992, I , 25-31. [98] J.-X. Wang, A. M. Bray, A. J. DiPasquale, N. J. Maeji, H. M. Geysen, Int. J. Pept. Protein Res. 1993. 42. 384-391.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

11 Cyclic Peptide Libraries: Recent Developments Arno I? Spatola and Peteris Romanovskis

11.1 Introduction Cyclic peptides represent attractive lead candidates for new drug discovery. Cyclization lowers conformational flexibility and removes exopeptidase vulnerable sites when head-to-tail amide rings are formed. Cyclic peptide libraries are therefore intriguing targets as interest in a broad range of diverse structure for peptide combinatorial libraries increases [l]. But as molecular diversity broadens beyond linear peptides to new organic molecules [2, 31, where years of optimized solid phase synthesis methods are no longer the norm, providing complex mixtures with high degrees of structural fidelity will be far more difficult. Using modified Merrifield chemistries [4], it is now possible to synthesize linear peptides of up to 100 amino acids and more in acceptable yields [ 5 ] . But peptide chemists have often struggled to prepare cyclic peptides, even of modest ring size, and with yields of SO%, assuming purity of 97% or greater. When we began our work directed toward the preparation of cyclic peptide libraries, it was clear that considerable effort would have to be expended in perfecting the synthetic methods and in characterizing the resultant products. Accordingly, we selected a synthetic strategy based primarily on the Boc method of synthesis and have incorporated optimized side chain protecting groups and potent condensing and cyclization reagents in order to minimize the synthetic variables. Additionally, the synthetic targets were primarily based on medium size rings with known structural motifs, turn inducing structures, and conservative side chain modifications. Finally, as we demonstrate below, we have kept the number of individual components in the cyclic libraries small enough such that, in most cases, individual structures and possible side products are more readily detected and identified. Our initial synthetic objectives were cyclic pentapeptides based on a recently described naturally occurring endothelin antagonist. But other libraries with five, six and seven amino acids have been prepared, and fall into two major groups: (i) cyclic peptides based on known biologically active sequences such as the endothelin antagonists, cyclic penta- or hexapeptides containing the cell adhesion motif, Arg-Gly- Asp, and an anti-leukemic heptapeptide (stylostatin) [6] (Fig. 11-1); (ii) cyclic peptides and cyclic pseudopeptides with no known biological actions but chosen for their potentials for new drug discovery, metal binding properties, or to explore fundamental parameters such as sequence dependence on cyclization.

328

11 Cyclic Peptide Libraries: Recent Developments

cyclic Pentapeplides: Endothelin Antagonists

D-Leu

Cell Adhesion

Cyclic Heptapeplides: (stybst8tin) Cell GmWh Inhibition

Figure 11-1. Authentic stylostatin (Www = Ile, Xxx = Ala, and Yyy = Leu) 161.

11.2 Results and Discussion Our general synthetic approach is shown in Scheme 11-1. Attachment of amino acids to a solid support through their side chain is not new. Isied et al. [7],Trzeciak and Bannwarth [8] and Sklyarov et al. [9]have prepared cyclic peptides in this fashion, and Rovero and coworkers reported the preparation of a head-to-tail Asp-linked peptide with resin-bound cyclization in 1991 [lo].More recently, Kates et al. reported the preparation of head-to-tail cyclic peptides using an Fmoc-based approach and orthogonal ally1 protection (111.Brugghe and coworkers recently reported the synthesis of 7-14 residue cyclic peptides using Fmoc chemistry and a 8-aspartyl side chain resin linkage [12].Various groups (13-151 have described other modes of side chainside chain amide or disulfide cyclization using resin-attached peptides. But when we began our work [16, 171, the only reports of cyclic peptide libraries consisted of disulfide bridged rings. These were formed via phage-based linear peptides containing pairs of cysteine residues [MI. In preparing our first cyclic peptide mixture, we chose an analog of the cyclic pentapeptide endothelin antagonist, BQ-123 [19].The synthesis began with the L-aspar-

11.2 Results and Discussion

A

Boc-Asp-OFm + HOC&H I CH2C02H

FmooAA,,-*Asp--OFm I CH~CO~CH~C&-R~S~~

D

Condensation

Boc-Asp-OFm I CHzCOzCH2QHrResin

(Mitsunobu;

(@3P;DEAD)

C

329

B1

1 ) deprotected (50% TFA) 2) BOC-AAZ...~.~; BOP. HOB1

Boc-Aaa*-Asp-OFm I CH2CO2CH2QHrResin

ir$;i

removal of FmocandOFm (20% piperidine)

i

H-AAn---AsFOH I CH~C&CI+~C&-RS~~

-

(Bop or HATU)

I

CH2C02CH&H4-Resin cleavage from resin (anhydrous HF)

Evaluation of Asp for DJ. content

G

9 I

hydrolpis (6N HCI)

cH2c02H

Scheme 11-1. General synthetic approach used for preparation of cyclic peptide mixtures. Letters (A-G) identify separate steps as potential racemization or epimerization loci.

tyl residue Boc-L- Asp-OFm, linked to a hydroxymethylated polystyrene resin through the side chain. The one-bead/one (cyclic) peptide approach [20, 21J was adopted, and we reasoned that this would permit us to monitor such factors as sequence dependence on cyclization more readily. As with other examples of combinatorial libraries, we based our work on the pioneering efforts of others, and especially of Geysen, who first described the pin method for multiple peptide synthesis (221, and Houghten for his development of the tea-bag method which has been extended to the preparation of literally millions of linear peptide mixtures [231. As is evident in the sections below, cyclic mixtures provide unique problems. An additional level of orthogonality is required to produce defined cyclic products while retaining side chain functional groups protected. Traditional protein sequencing methods are no longer available. Furthermore, distinguishing between cyclic monomers and higher oligomers is not trivial, especially since some forms of mass spectrometry such as electrospray ionization normally result in multiple charges, while other MS techniques may result in aggregation that can give rise to artifactual dimers [25]. These are often difficult to distinguish from authentic dimers. Finally, the ease

330

I1 Cyclic Peptide Libraries: Recent Developments

of cyclization depends on ring size, amino acid sequence, amino acid configuration and additional factors that pertain to linear peptide couplings such as condensing agents, solvents, temperature, and additives 126).

11.2.1 Cyclic Pentapeptides Our first synthetic cyclic libraries consisted of the two sequences CYCIO(X~X-D-L~UVal-D-Pro-Asp), where Xxx = Ala, Phe, Tyr, Trp, and cyclo(Xxx-D-Leu-YyyD-Pro-Asp), where Xxx = Phe, Tyr, Trp, Val and Y = Gly, Ala, Val, Leu (Table 11-1, entries 1 and 2). These are retro-inverso analogs of BQ-123. In the smaller mixture, it was possible to individually separate all four components and to verify their structures by comparison with an authentic individual synthetic product (Fig. 11-2a). Using FAB-MS analyis of the mixture, we also verified that a set of four by products at +85 units higher mass were formed in the synthesis (Fig. 11-2b). These were assumed to be linear piperidides and such products have also been reported by Kates and coworkers in a closely related synthetic procedure [I I]. In later preparations, using unusually stringent wash procedures, we were able to remove residual piperidine and these products are no longer observed as major contaminants. A greater concern was uncovered when we undertook a detailed analysis of amino acid chirality using Marfey’s reagent (271. Liquid chromatography (LC)analysis had suggested the existence of minor shoulders to most major peaks, possibly due to diastereotopic contamination. When the individual cyclic peptides were hydrolyzed, the amino acids derivatized with Marfey’s reagent, and the resulting products analyzed by reversed phase-high performance liquid chromatography (RP-HPLC), the only amino acid to show significant evidence of racemization was L-aspartic acid. At first we assumed that epimerization of the C-terminal side chain linked acidic moiety was occurring during the cyclization step. Detailed LC-MS analysis (Fig. 11-3) indicated that at least three of the four products were accompanied by a minor second peak of identical molecular weight. But the differences in percentage of D-ASPfound using Marfey’s reagent with the cyclic peptide and the percentage found when the linear peptide precursor was identically treated led us to undertake a more detailed stepwise analysis of the cause of epimerization. First, it is known that aspartic acid is partially racemized upon acid hydrolysis. When samples of L-ASPand D-ASPwere treated with 6~ HCl/lIO°C hydrolysis conditions for 24 hours, the amount of racemization found was 6.3% and 3.2%. respectively (Table 11-2). When isolated pure endothelin antagonist analogs were submitted for hydrolysis, subsequent derivatization and HPLC analysis, the results were: 3.4% D-ASPfrom cyclo(Trp-D-Leu-Val-D-Pro- Asp) and 5.4% D-ASPfrom cyclo(PheD-kU-Val-D-PrO-ASp). According to Peter el al. [28], the percentage of Asp epimer formed during 6~ HCl hydrolysis under conventional conditions (24 h, 1lOOC) was

11.2 Results and Discussion

331

Table 11-1. Several examples of cyclic peptide libraries and their characteristics

No. Library I 2

Variations

C(XXX-D-LeU-VaI-D-PrO-ASp-)

Xxx = Ala, Phe, Tyr,

(endothelin)

TrP

c(Xxx-D-Leu- Yyy-D-Pro- Asp-)

(endothelin)

3

c(Aaa- Bbb- Ccc- Ddd- Asp-) (RGD; others)

4

c(Ala-Gln-Phe-Ccc-Ddd- Asp-) (RGD; others) c(D-Ala- Gln- Phe- Ccc- DddAsp-) (RGD; others)

Xxx = Phe, Tyr, Trp,

9

4

5

16

5

Nal Yyy = Gly, Ala, Val, Leu Aaa = Ala, Phe, S r , 1296 Ile, His, Gly Bbb = Lys, Ala, Thr, Phe, Glu, Pro Ccc = Arg, Gly, Trp, Phe, Met, Gln Ddd = Gly, Tyr, Val, Leu, Ser, Glu

Ccc = Arg, Gly, Trp, Phe 6 c(Pro-Gln-Phe-Ccc-Ddd- Asp-) Ddd = Gly, Tyr, Val, Leu (RGD; others) 7 c(D-Pro-Gln-Phe-Ccc- DddAsp-) (RGD;others) 8 c(Tyr - Aaa- Bbb- Ccc- Pro- Ddd Am-) (stylostatin analogs) 5

Number of Amino acids Compounds

c(D-Tyr- Aaa-Bbb-Ccc-Pro-

5

16

6

16

6

16

6

16

6

256

7

256

7

256

7

256

7

256

7

256

7

4

5

Ddd- Asp-) (stylostatin analogs) 10 c(Ser - Aaa- Bbb- Ccc- Pro- Ddd -

Asp-) (stylostatin and analogs)

c(DSer-Aaa-Bbb-Ccc-ProDdd- Asp-) (stylostatin analogs) 12 c(Thr- Aaa- Bbb- Ccc- Pro- DddAsp-) (stylostatin analogs) 13 c(D-Thr-Aaa-Bbb-Ccc-ProDdd- Asp-) (stylostatin analogs)

Aaa = Leu, Phe, Trp, Ala Bbb = Ala, D-Ala, Phe, D-Phe CCC = 11% Ala, DAla, Val Ddd = Phe, D-Phe, Ala, D-Ah

11

14

C(Xxx-D-LeU-VaI-D-PrO-ASp-)

X x x = Ala, Phe, Tyr, Trp

332

b7

I1 Cyclic Peptide Libraries: Recent Developments Ala

740

(a) 100-

(2a) LC (Retention time)

Phe

Tm

TYr

$ C

em 9

50-

>

3

965

1036

847

684 rrfk

FABMS

-

800

1000

I . .

1400

.

time (seconds)

Phe

Ala

572.3

496.2

450

I , ,

1200

500

611.3 Trp

gI

TYr 588.

550

I

600

(2b)FABMS

Phe (+85)

657.4

I

650

Tyr (+85)

673.4

700

750

di!

Figure 11-2. HPLC (a) and FAB-MS (b) characterization of ~ClO(Val-D-PrO-ASp-XxxD-L~u)(crude mixture), where Xxx = Ala, Phe, Qr*Trp, showing presence of linear piperidide byproducts (M + H + 85) in the FAB-MS spectrum (b)

II.2 Results and Discussion

333 E+07

1.658

Phe

Tyr

100

-

50-

100 50

Trp

739

--

ion chromatogram)

E+07 1.083

1463

1562 2 peaks (920 + 929 sec): M+H = 61 1 1167 1297 11% 1260 1360 1446

--

1.494

I

10050,

100 50

1229 1281

2 peaks (788 + 803 sec): M+H = 588 €+07

I

Ric (ran*bt&

1204

m 6 I9 3 p l

545

I1

111

I

I

1

1111

I

E+07 1.463

I

E+07 5.902

6

1 600I

;I I I I I

relative time

Figure 11-3. Detailed LC-MS analysis of a cyclic pentapeptide mixture furnishes data regarding diastereomer presence in three of four components

2.5-3.0%. Other authors have reported 4.3% D-ASPpresent in the amino acid attached to the resin after coupling by diisopropylcarbodiimide in the presence of pyridine, and 5.7% D-ASPwhen coupling was done in the presence of dimethylaminopyridine (DMAP) [29]. Examination of our synthetic Scheme 11-1 suggests at least seven major points where epimerization or racemization can take place, and undoubtedly all procedures are vulnerable to some extent. By a process of elimination, we were able to conclude that the major amount of aspartic acid epimerization was occurring during solid phase chain elongation, and probably through activation of a prematurely deprotected C-terminal carboxylate. Bednarek and coworkers had shown that the fluorenylmethyl ester may be cleaved during routine steps of solid phase synthesis or modest exposure to base (301. Presumably, such side reaction could be avoided by using a preactivated amino acid rather than adding a condensing agent at each step. In a later section, we provide evidence that active esters do provide one solution to this particular problem. Our next more ambitious library resulted in a total of 16 cyclic peptides (endothelin antagonist analogs; Table 11-1, entry 2) with a more varied structure selection. Again, this library component retained a single proline residue and a mixture of D- and L-amino acids. Cyclizations were effected using either benzotriazol-l-yloxy-tris-(dimethylamino)-phosphonium-he~fluorophosphate/~-hydroxy-benzotriazole (BOPIHOBt) or the newly described aza-equivalent of HBTU and the related aza-additive HOAt [31]. Using either reagent group, cyclizations were judged com-

3 34

I1 Cyclic Peptide Libraries: Recent Developments

Table 11-2. Selected early results on percentage D vs. and peptides using Marfey's reagent

Coupling agent BOP BOP HATU BOP BOP BOP HATU BOP HATU HATU HATU HATU

a

L

aspartic acid content of amino acids

Object hydrolyzed c(Trp-D-Leu-Val-D-Pro-Asp)a c(Trp-D-Leu-Val-D-Pro-Asp)b Boc-L-As~(P)-OH c(Trp-D-Leu- vat - D-Pro-D - Asu)' c(Trp-D-Leu- Val-D-Pro-D- &U)' Boc- D-As~(P)-OH C(Xxx-D-LeU-Val-D-PrO-ASp) C(XxX-D-LeU-Yyy-D-PrO-ASp)

c(Aaa- Bbb -Ccc-Ddd -Asp) c(Aaa- Bbb-Ccc- Ddd- Asp) H-Pro-Gln-Phe-Ccc-Ddd-Asp(P)-OH c(A1a-Gln-Phe-Ccc-Ddd- Asp) c(D-Ala-Gln-Phe-Ccc-Ddd- Asp)

c(Pro- Gln- Phe- Ccc- Ddd - Asp) c(D-Pro-Gln - Phe-Ccc -Ddd -Asp) L-ASP D-ASP

ASP Yo L

YO D

74.6 74.8 99.2 26.2 33.5 3.6 82.8 61.4 54.0 62.3 72.9 64.8 66.9 61.6 67.1 93.7 3.2

25.4 25.2 0.8 73.8 66.5 96.4 17.2 38.6 46.0 37.7 27.1 35.2 33.1 32.9 32.9 6.3 96.8

NMF (N-methylformamide) as solvent. DMF as solvent. Asu = aminosuccinamidyl (peptide was a reaction byproduct).

plete in under one hour, as monitored by the standard ninhydrin test [32]. Overall yields were in excess of 70070, and the products gave expected amino acid ratios, excepting the customary low value observed for Trp. Upon analysis by FAB-MS, only about two-thirds of the peaks were heavily represented and the ratios were not unity. Closer examination revealed a remarkable periodicity based on an over-representation of the most hydrophobic residues (Fig. 11-4). Although this effect, sometimes termed ion suppression, has often been seen in FAB-MS [25], we believe this observation using cyclic peptide libraries is the first instance of its application to a synthetic macrocycle mixture, and should prove valuable for the further identification of mass spectral biases when more universally applied. We also investigated the utility of matrix-assisted laser desorption ionization as an alternative to FAB-MS to overcome this problem. However, when this technique was applied to a similar sized library but made up of analogs with the cell adhesion sequence, Arg-Gly-Asp, we found that a bias now favored hydrophilic amino acids and in particular, arginine containing sequences. In any case, the combination of quantitative amino acid analysis results and various forms of mass spectrometry was

11.2 Results and Discussion

loo

1

335

FL 586

I

XL 636

629.3

N 5721 I

I

VL 602

50

500

540

580

620

Figure 11-4. FAB-MS analysis of a 16-component cyclic pentapeptide (cyclo(Yyy-D-ProASP-XM-D-L~U), where Xxx = Phe, Vr, Ttp, Nal and Yyy = Gly, Ala, Val, Leu, suggests bias of more hydrophobic constituents by the FAB technique. L = Leu V = Val A = Ala G = Gly X = Nal F = Phe Y = Tyr W = Trp

able to confirm that the libraries were structurally intact and that all the expected components were present with little evidence of major by-product contamination, epimerization products excepted. We also found little evidence of dimer contamination often reported in the literature [24], although some small quantities of oligomers at the calculated masses were observed (Fig. 11-5). We have concluded that the presence of these, especially dimeric by-products is most likely a consequence of peptide length, solvent effects, and the relative nature of the peptides to aggregate prior to cyclization, leading to intermolecular reactions. The effect of solvents on this problem have been described in detail by Rothe and coworkers, who reported how changing solvents could lead to dominant fractions of monomers, dimers, trimers or tetramers [33].

336

11 Cyclic Pepride Libraries: Recent Developments 757.3

100

50

0 600

800

1000

1200

1400

1600

1800

2000

FABMS: University of Nebraska

Figure 11-5. Analysis of a cyclic hexapeptide with 16 components cyclo(D-Pro-Gh-PheXxx-Yyy-Asp) shows minimal dimer content (Note dimer region above 1100 enhanced by IS x). FAB-MS: University of Nebraska

11.2.2 Cyclic Hexapeptides A set of libraries based on the cyclic hexapeptides c(Xxx-Yyy-Zzz- Arg-Gly-Asp) and additional analogs where Arg and Gly were also substituted as shown in Table 11-1, entries 4-7. These mixtures were again prepared using a one-bead-one(cyc1ic)peptide approach, resulting in a total of 14 separate condensation reactions. The linear peptides were separated at the pentapeptide stage in order to ascertain the effect of N-terminal chirality on ring closure. Next, the N-terminal residues added individually were D-Ah, L-Ala, D-Pro, and L-Pro. The ease of cyclization was qualitatively assessed by ninhydrin monitoring over time, while the integrity of the products was established by amino acid analysis, FAB-MS, and, in some cases, LC-MSanalysis. In general, the amount of background noise in the FAB-MS spectra appeared to be somewhat greater for the L-Ala mixtures than for the D-Ah, and for the L-Pro compared to the D-Pro mixture (Fig. 11-6). But, all libraries appeared to contain the expected components and the FAB-MS results did not provide evidence for unusual dimer formation. We concluded that the cyclic hexapeptide libraries formed in relatively clean fashion, but again with the exception that Marfey’s analysis gave the exbected high values for D-ASOcontent (Table 11-2).

11.2 Results and Discussion 100

331

.OE5

90

.8E5

80

.6E5

70

.4E5

60

.2E5

50

.8E4

40

.7E4

30

.5E4

20

.3E4

10

.2E4

0

.OEO .OE5 D-Pro

.8E5

RG

.8E4

GV

N

GL

I

17.7E4

,ld.:e

.5E4

3.3E4 .2E4

800

600

700

.OEO

Figure 11-6. FAB-MS spectrum of Idpeptidecyclic hexapeptide library cyclo(L-Pro(orD-Pro)Gln-Phe-Xxx-Yyy-Asp), where Xxx = Arg, Gly, Trp, Phe; Yyy = Gly, Tyr, Val, Leu; showing lower background noise for D-Pro sublibrary

11.2.3 Cyclic Heptapeptides In 1992, Pettit and coworkers described the isolation of a relatively potent cyclic heptapeptide known as stylostatin from an Indo-Pacific sponge [6].This peptide, named stylostatin, showed an ED,, of 0.8 mg/ml in a P388 lymphocytic leukemia assay. The structure of stylostatin itself is cvcloCeu-Ala-Ile-Pro-Phe-Asn-Ser).

338

11 Cyclic Peptide Libraries: Recent Developments

We undertook the synthesis of a library based on stylostatin with several objectives in mind. These included: (i) preparation of a side chain-linked, asparagine-containing, cyclic peptide; (ii) investigation of an active ester (pentafluorophenyl ester) mode of coupling to reduce or eliminate C-terminal epimerization during chain elongation; and (iii) further investigation of the consequence of a D-amino acid at the N-terminus of an otherwise all-L peptide sequence to correlate with product quality. Again, this synthesis was based on the split resin technique of Furka [20] and Lam et al. (211, and necessitated 23 separate coupling steps, as well as six additional cyclization reactions and six treatments with anhydrous hydrogen fluoride to liberate the cyclic sublibrary mixtures. As shown in Table 11-1, entries 8-13, each sublibrary mixture consisted of 256 components or a total of 1536 stylostatin analogs in the total library. Table 11-3 contains the percentage D-aspartic acid found upon analysis of each of the six sublibraries using Marfey's reagent and RP-HPLC with UV detection for quantification. It is apparent, from these results, that activation with preformed active esters virtually eliminated the epimerization problem, except for the L-nr entry, and generally exonerates the cyclization step as a significant cause of epimerization. At this time, we have no adequate explanation for the tyrosine result. Analysis of larger libraries through identification of individual components is more difficult. But, our preliminary mass spectral analysis is fully consistent with the amino acid results which verify that the expected residues are present in their calculated ratios. Detailed analysis of the stylostatin libraries as well as their activities in standard anti-tumor and anti-AIDS assays will be presented elsewhere. Table 11-3. Pentafluorophenyl active ester peptide elongation reduces aspartyl epimerization of cyclic libraries in nearly all cases

No. 8 9 10 11 12 13

Peptide library on polymer

L-ASP(To)

D-ASP(To)

L-'r'-styIostatin D-Tyr'-stylostatin L-Ser '-stylostatin D-Ser -stylostatin L-Thr '-stylostatin D-Thr'-stylostatin

83.98 97.27 93.89 98.03 96.21 96.73

16.02 2.73 6.11 1.97 3.79 3.27

'

11.3 Summary We have demonstrated that the preparation of cyclic peptide libraries is feasible, albeit not without complications. The ring sizes we have selected are modest, to preclude problems of strain on the one hand or entropy at the other extreme. A

II.3 Summary

339

useful program has been developed [34] that simplifies the calculation of molecular weight ratios and compositions for any defined cyclic peptide library (Fig. 11-7). It is evident that most naturally occurring cyclic peptides contain one or more proline Or glycine residues to facilitate turn features. Problems of oligomerization are so far manageable; if dimers or higher multiples are formed, these should not unduly interfere in identifying positive bioassay hits, provided that monomeric structures are present and identifiable. It is likely that many, very useful, library combinations of cyclic peptides can be formed even if they are biased toward structural features that would make their synthesis more facile. Our studies have suggested that newly optimized synthesis and cyclization procedures can provide high quality cyclic peptide mixtures with unusually rapid cyclization rates. We find evidence that the reported tendency for all L-sequences (not containing proline) to resist monomer cyclization without epimerization may be overcome. Theoretical predictions [35] and experimental reports [36-391 have offered sometimes conflicting evidence regarding this point. Our current working hypothesis is based on an enhanced facility toward cyclization of linear sequences as a consequence of the side chain attachment combined with the previously exploited advan-

1

350

I

I

450

I

I

I

I

I

450 650 Molecularweight of peptide

I

750

Figure 11-7. Statistical analysis of a cyclic pentapeptide library reveals molecular weight patterns. 1296 cyclic pentapeptides (Table 11-1, No. 3) Example: MW = 521 (N= 5 ) Gly + Glu + Gly + TYr + Asp Tyr + Glu + Gly + Gly + Asp Phe + Ala + Met + Gly + Asp Ala + Phe + Met + Gly + Asp Ala + Thr + Phe + Ser + ASD

340

I1 CycficPeptide Libraries: Recent Developments

tages of the “pseudo-dilution” effect [40]when carrying out resin-bound cyclization. We are continuing our efforts to answer these fundamental questions with appropriately designed experiments even as we expand our efforts to produce a wide range of peptide, pseudopeptide [41], and peptidomimetic structures based on the macrocyclic library concept.

11.4 Materials and Methods p-Methylbenzhydrylamine hydrochloride (MBHA) resin (0.36 meq/g) was obtained from Peninsula Laboratories, Inc. Reagent grade methylene chloride was refluxed for 0.5 h over phosphorus pentoxide and freshly distilled. DMF was distilled from ninhydrin and stored over molecular sieves. Boc- and Fmoc-amino acids were purchased from Bachem, Bioscience Inc. and Bachem, California. Pentafluorophenyl esters of Boc- and Fmoc-protected amino acids were obtained according to 142, 431. TLC analysis was performed on silica gel 60-F254 precoated aluminum sheets (Merck, Darmstadt), development with CHCI,/MeOH/AcOH (90: 8 :2). Spots were visualized by fluorescence, iodine visualization, and by spraying with ninhydrin reagent. Reversed phase HPLC analysis was performed on Shimadzu LC-600 system using a Zorbax C18 (4.6 x 25 cm) Protein Plus column, a Hitachi 655A variable wavelength UV monitor, and Shimadzu GR3A chromatopack integrator. A buffer: 0.05% TFA in water, B buffer: 0.05% TFA in acetonitrile. Amino acid analysis: hydrolysis of samples was performed using 6 M HCI-propionic acid (1 : 1) for amino acids and peptides attached to the resin in the presence of 1000 nM norleucine as internal reference standard or by 6 M HCI for all other samples in sealed ampules (in vacuo) at 105-115°C for 21 h. Amino acid analysis was carried out on a Applied Biosystems automated system using PTH derivatives. Marfey’s test was carried out on peptide hydrolysates according to [27]. HPLC analysis of the diastereoisomers was performed with a linear gradient from 10% to 30% B buffer over a period of 25 min, flow rate 1 ml/min, and with scanning of the effluent at 340 nm. Identification of the diastereomer peaks corresponding to either L- or D-aspartic acid (in case of each sample) was done in a separate run by coinjection with internal reference standard. Determination of the amount of -0Fm ester on the resin was done spectrophotometrically accordingly to [29). FAB-MS analysis of the peptides and mixtures was performed by the Midwest Center for Mass Spectrometry (University of Nebraska-Lincoln) with partial support by the National Science Foundation, Biology division (Grant No. DIR9017262).

11.4 Materials and Methods

341

11.4.1 General Solid-Phase Peptide Synthesis Procedure The Merrifield method of solid-phase synthesis was followed using the general methods described below except that side chain attachment of the first amino acid using Boc-Asp-OFm was used to generate Asp (hydroxymethyl resin) or Asn ( p methylbenzhydrylamine resin) cyclic peptides after resin bound cyclization. For Bocdeprotection, a solution of TFA in CH2C12with anisole (50: 45: 5) was used. For the synthesis of libraries of the cyclic peptides, the Merrifield method was used according to the principle of “one bead-one peptide” [21] or split resin [20] procedure. General syntheses were run on an automated synthesizer SYNTHOR 2000AT (Peptides International, Inc.). In the manual regime, the split resin was coupled in separate reaction vials. A typical synthetic procedure for a Boc-amino acid coupling cycle was: Bocdeprotection 1 x TFA/CH2C12/anisole (5 min), 1 x TFA/CH2C12/anisole (25 min); 6 x CH2C12(1 min); 2 x DMF (1 min); 2 x 10% DIPEAIDMF; 2 x 0.5 M INCE (ethyl isonitrosocyanoacetate), used according to [44]/DMF (1 min); 2 x DMF (1 min); coupling: pentafluorophenyl esters of Boc- or Fmoc-protected amino acids (4 equiv) in DMF in the presence of BPB (bromophenol blue, according to [45,46]; washing: 3 x DMF (1 min); 6 x CH2C12(1 min), followed by the Kaiser ninhydrin test. After Fmoc-amino acid coupling: 3 x DMF (1 min), 3 x 20% Pip/DMF (10 min), 3 x DMF (1 rnin), 6 x CH2CI2 (1 min), 2 x DMF (1 min); 2 x 10% DIPEA/DMF (1 min), 3 x DMF (1 min). Cyclization: coupling reagent (4 equiv), HOBt or HOAt (4 equiv), DIPEA (8 equiv) in DMF; washings after the cyclization: 3 x DMF (1 min); 3 x 10% DIPEAIDMF (1 min); 2 x DMF (1 min); 6 x CH2C12(1 min); 3 x MeOH (1 min); 2 x Et20 (1 min); 1 x hexane (1 min). Cyclic peptides and the mixtures or libraries of cyclic peptides were cleaved from the resin and deprotected by the action of liquid HF in the presence of anisole and MeSEt as scavengers (20°C, 60 min) [47]. After the removal of anhydrous HF, the resin was washed with Et20, filtered, dried and extracted with 50To acetic acid. The extracts were diluted with water to 25% and freeze-dried. 11.4.1.1 Synthesis of a Stylostatin Peptide Library with Six Sublibraries (6 x 256 Peptides)

Boc-Asp- (methylbenzhydrylamine (MBHA)- resin)- OFm

To a suspension of 5.0 g (1.8 mmol) of MBHA-HCl resin in 30 ml dimethylformamide (DMF) was added 0.31 (1.8 mmol) DIPEA followed by a solution of 1.48 g (3.6 mmol) Boc-Asp-OFm, 1.59 g (3.6 mmol) BOP, 0.55 g (3.6 mmol) HOBt*H20 and 1.23 ml (7.2 mmol) DIPEA in 10 ml DMF. After stirring the reaction mixture

342

I1 Cyclic Pepride Libraries: Recent Developments

for 24 h at room temperature, the resin was filtered and washed with 2 x 20 ml DME 3 x 20 ml CH2CI2 and dried over KOH/P205. Yield 5.674 g, theory 5.642 g. 0.317 mmol/g. Spectrophotometric determination of -0Fm ester on the resin provided 0.331 mmol/g (103.8 To substitution). Quantitative amino acid analysis suggested a lower loading or 0.184 mmol/g (58% substitution). The higher substitution amouni was utilized for purposes of calculating reagent excess. Stylostatin library (stylostatin is cyclo(Leu -Ala - I le- Pro-Phe -Asn -Ser))

Boc-Asp(MBHA-resin)-OFm (5.1 g) corresponding to 1.62 mM of the amino acid bound was swollen in CH2C12in an hour-glass reaction vessel and treated with TFA/CH,CI,. After deprotection and liberation from the TFA salt (DIPEA wash), the resin was split into four equal parts with four separate reaction vessels (vials). Each portion was acylated with the corresponding Boc-protected amino acid pentafluorophenyl ester (Boc-Xxx -0Pfp) : (i) (ii) (iii) (iv)

0.69 g (1.62 mM) Boc-Phe-OPfp; 0.69 g (1.62 mM) Boc-D-Phe-OPfp; 0.575 g (1.62 mM) Boc-Ala-OPfp; 0.575 g (1.62 mM) Boc-D-Aia-OPfp.

Each active ester was dissolved in 5 ml of DMF, and 1-3 drops of a 1070 solution of BPB in ethanol was added to the solution for each resin portion. As the reaction proceeded, the color of the suspension turned from blue to yellow; usually the acylation was complete within 5-15 min depending on the amino acid. A parallel Kaiser test [32] was used to confirm that the acylation was complete. After the reactions, the resin portions were recombined, washed, and then Bocdeprotected and TFA liberated according to the protocol. Second cycle

After Boc-deprotection, the TFA-liberated resin was acylated with Boc-Pro-OPfp: 2.47 g (6.48 mM) Boc-Pro-OPfp were dissolved in 15 ml DMF and added to the resin in the presence of 1 drop of 1070 solution of BPB in EtOH. The indicator suggested that, within 20 min, the coupling reaction was finished. After the reaction was completed, the resin was washed and treated with acid followed by a base neutralization. Third cycle

After the Boc-deprotectionand base treatment, TFA salt, the resin was split into four equal parts in four separate reaction vessels. Each part of the resin was acylated with the corresbonding -0Pfo esters:

11.4 Materials and Methods

(i) (ii) (iii) (iv)

343

0.643 g (1.62 mM) Boc-Ile-OPfp; 0.575 g (1.62 mM) Boc-Ala-OPfp; 0.575 g (1.62 mM) Boc-D-Ala-OPfp; 0.621 g (1.62 mM) Boc-Val-OPfp.

Each one of the active esters was dissolved in 5 ml DMF and added to the separate resin portion with occasional stirring. According to the BPB indicator, complete coupling of the Boc-Ile required 60 min and was the longest coupling; L-Boc-Val was the second longest. After all reactions were judged complete, the resins were mixed, washed, Boc-deprotected and liberated from TFA salt in preparation for the next coupling cycle.

Fourth cycle The Boc-deprotected and TFA-liberated resin was split into four equal parts in four separate reaction vessels. Each part of the resin was acylated with one of the following -0Pfp esters: (i) (ii) (iii) (iv)

0.575 0.575 0.698 0.698

g (1.62 mM) Boc-Ala-OPfp; g (1.62 mM) Boc-D-Ala-OPfp; (1.62 mM) Boc-Phe-OPfp; (1.62 mM) Boc-D-Phe-OPfp.

Each one of the active esters was dissolved in 5 ml DMF and added to the separate resin portion with occasional stirring. After the reactions were completed, the resin portions were mixed, washed, Boc-deprotected and liberated from the TFA salt according to the protocol for the next coupling cycle. Fifth cycle

The Boc-deprotected and TFA-liberated resin was split into four equal parts in four separate reaction vessels. Each part of the resin was acylated with the corresponding -0Pfp ester: (i) (ii) (iii) (iv)

0.643 g (1.62 mM) Boc-Leu-OPfp; 0.698 g (1.62 mM) Boc-Phe-OPfp; 0.807 (1.62 m ~ Boc-Trp(For)-OPfp; ) 0.575 (1.62 m ~ Boc-Ala-OPfp. )

Each one of the active esters was dissolved in 5 ml DMF and added to the separate resin portion with occasional stirring. After the reactions were completed, the resin was mixed, washed, Boc-deprotected and liberated from the TFA salt according to the Drotocol for the next coudine cvcle.

344

I1 Cyclic Peptide Libraries: Recent Developments

Sixth cycle The Boc-deprotected and TFA liberated resin was split into six equal parts into six separate reaction vessels. Each portion was reacted with the corresponding acylating agent: (i) (ii) (iii) (iv) (v) (vi)

0.442 g (1.08 mM) Fmoc-Ser(tBu)-N-carboxy-anhydride; 0.593 g (1.08 mM) Fmoc-D-Ser(tBu)-OPfp; 0.608 g (1.08 mM) Fmoc-Thr(tBu)-OPfp; 0.608 g (1.08 mM) Fmoc-D-Thr(tBu)-OPfp; 0.675 (1.08 mM) Fmoc-lYr(tBu)-OPfp; 0.675 (1.08 mM) Fmoc-DTyr(tBu)-OPfp.

Each one of the activated compounds was dissolved in 5 ml DMF and added to tht separate resin portion under occasional stirring. BPB indicated the presence of com plete coupling in all reaction vessels within 60 min. After the reaction, each portion of the resin received separate treatment. Each por. tion was washed, Fmoc- and -0Fm- deprotected with of 20% piperidine in DMF (20 min) and each of the six side chain residue linked deprotected linear heptapeptidt mixtures (256 components) was independently submitted to cyclization. Cyclization:(L-Tyr '-stylostat in library) After Fmoc- and -0Fm deprotection, the resin was washed in 3 x 20 ml DME 5 x 20 ml CH2CI2, 2 x 20 ml DMF, 2 x 20 10% DIPEA/DMF, 3 x 20 ml DME The solution of 0.411 g (1.08 mM) HATU, 0.147 g (1.08 mM) HOAt, 0.37 mL (2.16 mM) DIPEA in 4 ml DMF was added to the above prepared resin and allowed to run for 2 h with occasional stirring. After 1 h a Kaiser test was negative, indicating the cyclization reaction was complete. The polymer material was filtered and washed with 3 x 20 ml DMF, 3 x 20 ml10Yo DIPEA/DMF, 2 x 20 ml DMF, 6 x 20 ml CH2C12, 3 x 20 ml MeOH, 3 x 20 ml Et20, 3 x 20 ml hexane and dried. Y = 0.877 g. The resin (0.614 g, 70% of the material obtained) was treated with 7 ml liquid hydrogen fluoride in the presence of 0.6 ml anisole and 0.2 ml methyl ethyl sulfide for 1 h at m. After the evaporation of HF the resin was washed with 3 x 30 ml Et,O, dried and extracted with 2 x 20 ml 50% AcOH, extracts diluted with 40 ml H 2 0 and freeze dried. Y = 0.111 g. The five remaining portions were treated in an identical fashion. D-Pr '-stylostatin library

(i) Yield of the peptide resin after cyclization on the resin: Y = 0.938 g. (ii) Yield of the cvcloDebtide librarv after cleavafze and demotection: Y = 0.1438 E.

345

11.4 Materials and Methods

L-Ser’-stylostatin library (i) Yield of the peptide resin after cyclization on the resin: Y = 0.950 g. (ii) Yield of the cyclopeptide library after cleavage and deprotection: Y = 0.130 g.

0-Set-‘-stylostatin library (i) Yield of the peptide resin after cyclization on the resin: Y = 1.023 g. (ii) Yield of the cyclopeptide library after cleavage and deprotection: Y = 0.152 g.

L-Thr’-stylostatinlibrary

(i) Yield of the peptide resin after cyclization on the resin: Y = 0.946 g. (ii) Yield of the cyclopeptide library after cleavage and deprotection: Y = 0.129 g. D-Thr’-stylostatin library

(i) Yield of the peptide resin after cyclization on the resin: Y = 1.035 g. (ii) Yield of the cyclopeptide library after cleavage and deprotection: Y = 0.132 g. The amino acid ratios obtained for the stylostatin sublibraries are listed in Table 11-4; in each case, column A represents the resin-bound cyclic peptides while column B reflects amino acid ratios of the deprotected cleaved products. In aggregate, the ratios are reasonable for these complex cyclic peptide mixtures. Table 11-4. Stylostatin-based cyclopeptide libraries: results of amino acid analysis for resinbound (A) and released (B) cyclic peptide mixtures AA

SL

L-SerI A B

D-SerIA B

L-Thr’A B

D-Thr’A B

4.3 3.9 5.2 3.9 4.3 3.8 4.4 4.1 5.4 4.8 5.0 4.0 ASP - - - - 0.1 3.0 1.1 3.3 - - - Ser Leu 1.2 1.0 0.7 1.0 0.7 1.0 0.8 1.0 1.0 1.0 1.0 1.0 5.1 Phe 7.0 5.5 7.6 6.0 3.6 5.1 7.8 9.3 5.8 5.8 + 6.8 7.3 7.7 6.8 6.0 6.8 6.9 7.0 7.2 8.4 7.9 6.8 Ala Ile 1.0 0.9 1.0 1.0 1.0 0.6 1.0 1.0 1.0 0.8 1.0 0.7 0.7 0.8 0.6 Val 0.6 0.9 0.8 1.0 0.8 0.5 0.9 0.9 + 4.7 5.0 5.2 4.8 4.3 4.3 5.0 4.6 4.9 5.4 5.4 4.5 Pro 3.0 3.6 3.6 3.7 TYr 3.5 4.3 3.6 3.5 Thr n.d. n.d. n.d. n.d. n.d. n.d. TrP A = resin-bound; B = HF cleaved and deprotected. (+) = amino acid present but quantitation unreliable; n.d. = not determined. AA = amino acid. SL = sublibrarv (varies according to Dosition 1 substituent).

- - - - - -

-

Theory 4 4 1

5 7 1 1

4 4 4 1

346

11 Cyclic Peptide Libraries: Recent Developments

Acknowledgments This work was supported by NIH GM33376. Mass spectral determinations were performed by P. Andrews a n d coworkers at the University of Michigan, R. Cerny and coworkers at the University of Nebraska, and by J. Ftagsdale a t Finnigan-MAT.

References [l] M. A. Gallop, R. W. Barrett, W. J. Dower, S.P. A. Fodor, E. M. Gordon, A Med. Chem., 1994, 37, 1233-1251. [2]G. Jung, A. D. Beck-Sickinger, Angew. Chem. Int. Ed. Engl. 1992, 31, 367-486. [3]E. M.Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, M.A. Gallop, J. Med. Chem. 1994, 37, 1385-1401. (4)R. B. Merrifield, Science, 1986, 232, 341-347. 151 M. SchnBlzer, S. B. H. Kent, Science 1992, 250, 221-224. (61 G. Pettit, J. K. Srirangam, D. L. Herald, K. L. Erickson, D. L. Doubek, J. M.Schmidt, L. P. Tackett, G. M. Bakus, A Org. Chem., 1992, 57, 7217-7220. [7]S. S. Isied, C. G. Kuehn, J. M. Lyon, R. B. Merrifield, A Am. Chem. Soc. 1982, 104, 2632-2634. [8]A. Trzeciak, W. Bannwarth, Telmhedron Lett., 1992, 33, 4557-4560. [9]L. Y. Sklyarov, I. V. Shashkova, A Gen. Chem. USSR (in Russian), 1969,39, 2778-2783. [lo] P. Rovero, L. Quartara, G. Fabbri, Pfrahedmn Lett., 1991, 32, 2639-2642. [ll] S. A. Kates, N. A. Sol&C. R. Johnson, D. Hudson, G. Barany, F. Albericio, Tetrahedron Lett., 1993, 34, 1549-1552. (121 H. F. Brugghe, H. A. M. Timmermans, L. M. A. Van Unen, G. Jan Ten Hove, G. Van De Werken, T. Poolman, P. Hoogerhout, Int. A Pept. Protein Res., 1994, 43, 166-172. I131 P. W. Schiller, T. M. D. Nguyen, J. Miller, Int. A Pept. Protein Res., 1985,25, 171-177. [14]A. M. Felix, C. T. Wang, E. P. Heimer, A. Fournier, Int. A Pept. Protein Res., 1988, 31, 231 -238. [15]M. Lebl, V. J. Hruby, Tetrahedron Lett., 1984, 25, 2067. [16]K. Darlak, P. Romanovskis, A. E Spatola, in Peptides: Chemistry, Structure, and Biology, (Eds.: R. S. Hodges and J. A. Smith), ESCOM, Leiden, 1994, pp. 981-983. [17]A. E Spatola, J. J. Chen, I. Romanovska, P. Romanovskis, J. J. Wen, Peptides 1994, Proceedings of the European Peptide Symposium, 1994, 96-97. (181 K. T. O’Neil, R. H. Hoess, S. A. Jackson, N. S. Ramachandran, S. A. Mousa. W. E DeGrado, Proteins, 1992, 14, 509-515. (191 M. Ihara, K. Noguchi, T. Saeki, T. Fukurode, S. Tsuchido, S. Kimura, T. Jukami, K. Ishikawa, M. Nishibe, M. Yano, Life Sci., 1992, 50, 247. [20)A. Furka, F. Sebestyen, M. A. Asgedom, G. Dibo, Int. J. Pept. Protein Res., 1991, 37, 481-493. (211 K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, R. J. Knapp, Nature, London 1991.354, 82-84.

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[22] H. M. Geysen, R. H. Meloen, S. J. Barteling, Proc. Nut. Acad. Sci. USA, 1984, 81, 3998-4002. [23] R. A. Houghten, Proc. Nut. Acad. Sci. USA, 1985, 5131-5135. (24) J. S. McMurray, Tetrahedron Lett., 1991, 32, 7679-7682. [25] P. J. Andrews, J. Boyd, R. 0. Loo, R. Zhao, C. Q. Zhu, K. Grant, S. Williams, Techniques in Protein Chem. (Ed.: V. J. Crabbe), Academic Press, 1994, pp. 485-492. (26) S. A. Kates, N. A. Sole, F. Albericio, G. Barany, Peptides: Design, Synthesis and Biological Activity, (Eds. : C. Basava and G. M. Anantharamaiah), Birkhauser (Boston), 1994, pp. 39-58. (271 Marfey, P. Carlsberg Res. Commun., 1984, 591 -596. [28] A. Peter, G. Laus, D. Tourwe, E. Gerlo, G. Van Binst, Pept. Res., 1993, 5 , 48-52. [29] J. Green, K. Bradley, Etrahedron, 1993, 49, 4141-4146. (301 M. 1. Bednarek, M. Bodanszky, Int. 1 Pept. Protein Res., 1983, 21, 196-201. [31] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397-4398. [32] E. Kaiser, R. Colescott, C. Bossinger, P. Cook, Anal. Biochem., 1970, 34, 595-598. [33) M. Rothe, M. Lohmuller, W. Fischer, W. Taiber, U. Breuksch, Solid Phase Synthesis, (Ed.: R. Epton), SPCC (UK) Ltd., Birmingham, 1991, 551-558. [34] R. Sakamuri, A. E Spatola, 1995, in preparation. (351 M. J. Mutter, J. Am. Chem. Soc., 1977, 99, 8307-8314. (361 V. T. Ivanov, V. V. Shilin, Y. Bernet, Y. A. Ovchinnikov, J. Gen. Chem. USSR (Engl.), 1971, 41, 2341. [37] H. Kessler, B. Kutscher, Liebigs Ann. Chem., 1986, 39, 869-892. I381 G. H. Heavner, T. Audhya, D. Doyle, P. S. Tjoeng, G. Goldstein, Int. J. Pept. Protein Res., 1991, 37, 198-209. (391 S. E Brady, S. L. Varga, R. M. Freidinger, D.A. Schwenk, M. Mendlowski, F. W. Holly, D. E Veber, J. Org. Chem., 1979, 44, 3101-3105. (40) A. Patchornik, M. Fridkin, E. Katchalski in The Chemistry of Polypeptides, (Ed.: P. G. Katsoyannis), Plenum, New York, 1973, pp. 315-333. [41] A. E Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII, (Ed.: B. Weinstein), Marcel Dekker, NY, 1983, pp. 267-357. I421 L. Kisfaludy, J. E. Roberts, R. H. Johnson, J. Org. Chem., 1970, 35, 3563-3564. [43] L. Kisfaludy, M. Low, 0. Nyeki, T. Szirtes, I. Schoen, Liebigs Ann. Chem., 1973, 1421- 1429. 1441 M. Itoh, Bull. Chem. Soc. Jpn. 1973, 46, 2219-2221. (45) V. Krchnak, J. Vagner, M. Lebl, Int. J. Pept. Protein Res., 1988, 32, 415-416. I461 V. Krchnak, J. Vagner, P. Safar, M. Lebl, Coll. Czech. Chem. Commun., 1988, 53, 2542-2548. 1471 S. Sakakibara in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, (Ed.: B. Weinstein). Vol. I. Marcel Dekker. New York. 1971. DD. 51-85.

Combinatorial Peptide and Nonpeptide Libraries by. Gunther Jung 0 V C H Verlagsgesellschaft mbH, 1996

12 Random Peptide Libraries as Tools in Basic and Applied Immunology Keiko Udaka, Karl-Heinz WiesmuIIec Stefan KienIe, Susanne Feiertag, Gunther Jung and Peter Walden

12.1 Introduction T lymphocytes play central parts in induction and regulation of immune responses and in execution of immunological effector functions. Specific immunity against infectious agents and tumors is dependent on these cells, and they are believed to contribute to healing of injuries. On the other hand, escape from the strict specificity control of the immune system can lead to autoaggression and autoimmune diseases. Antigen recognition by T-lymphocytes is controlled by two gene complexes that code for T-cell receptors (TCR) and major histocompatibility complex (MHC) molecules [I]. MHC molecules bind antigenic peptides and present them at the surfaces of cells. The adducts of peptides and MHC molecules are the ligands for the TCR (Fig. 12-1).

cy#otoxic T wphoqvte

&Net cell or ant/gen presenting cell Figure 12-1. Molecular basis of MHC restricted antigen recognition by T-lymphocytes.

350

12 Random Peptide Libraries as Tools in Basic and Applied Immunology

Helper and cytotoxic T-lymphocytes (CTL) require different MHC molecules for antigen presentation. Helper T cells which express CD4 coreceptors recognize peptides bound to MHC Class I1 (MHC-11) molecules. CD8-expressingCTL respond to peptides presented by MHC Class I (MHC-I) molecules. Thus, the specificity of Tcell responses is governed by two binding events: first, selection and binding of suitable peptides by MHC molecules, and second, recognition of peptide-MHC complexes by complementary TCR. Elucidation of the rules that control these two steps is prerequisite for understanding specificity of T-cell-mediated immunity in general and the basis for rational approaches to vaccine development or immunotherapy of cancer and autoimmune diseases.

12.2 Peptide Binding to MHC Molecules MHC-I molecules are heterodimers composed of polymorphic MHC-encoded heavy chains of about 45 kDa and noncovalently associated, nonpolymorphic light chains, ~2-microglobulin,of about 12 kDa which are encoded by a gene outside of the MHC. MHC-I1 molecules consist of two polymorphic, MHC-encoded polypeptides of about 30 kDa each [2]. The current understanding of the rules for peptide selection by MHC molecules is derived from sequencing of peptides and natural peptide libraries eluted from MHC proteins [3, 41, from analysis of the effect of mutations in sequences of known T-cell epitopes on peptide binding to MHC molecules and on T-cell responses [5, 61, and from crystal structure analyses of defined peptide-MHC complexes [7-91. MHC I-binding peptides are mostly octa- or nonapeptides and exhibit allele-specific motifs as determined by pool sequencing of natural peptide libraries [3]. The motif-determining amino acids were interpreted as anchor residues. Crystal structure analysis has confirmed this concept. A peptide binding cleft was identified which is framed by two a helices on top of a P-pleated sheet and which is stabilized from beneath by j?2-microglobulin [7, 101. Specific pockets inside the peptide-binding groove were identified that accommodate the anchor amino acids. The orientation of the peptide is determined by conserved side chains of the MHC-I protein that compensate the N- and C-terminal charges of the peptide. The analysis of natural peptide libraries that were eluted from MHC molecules has provided valuable information on the molecular basis of antigen recognition by T cells. However, these natural libraries are a selected population of peptides generated by the antigen processing machinery of the cells [ll]. Proteasomes generate suitable peptides by proteolytic degradation of protein precursors [12, 131, ABC transporters deliver the peptides to the site of MHC biosynthesis in the endoplasmic reticulum [14] and molecular chaperonins assist in the assembly of the trimeric complexes of MHC heavy and light chains, and peptides [IS]. These processes introduce biases that preclude a complete protein chemical understanding of the relationship of an-

12.3 Synthetic Random Peptide Libraries

351

tigenic peptides and MHC molecules. Moreover, development of synthetic vaccines requires active epitopes. Identification of natural epitopes is extremely time consuming and laborious, and is often obscured by low abundance of the peptides or their weak association with MHC molecules. We employed random peptide libraries to overcome these restrictions. Applicability and limitations of peptide libraries were tested using the MHC-I molecule H-2Kb and a CTL clone that recognizes its antigen, the OVA-derived epitope SIINFEKL, together with H-2Kb [16, 17).

12.3 Synthetic Random Peptide Libraries Random octapeptide libraries with varying degree of complexity were synthesized with premixed amino acid derivatives and comprise randomly composed peptides in close to equimolar representation [18, 191. Randomized sequence positions in peptide libraries used in our studies contain 19 amino acids, all common proteinogenic amino acids except for cysteine. The complexity of these libraries ranges from mixtures of 19 different peptides in 0 7 X (0, defined sequence positions; X, randomized sequence positions) to 19* in X8. OX, sublibraries with one defined and seven randomized positions (Fig. 12-2) containing 1g7 peptides were prepared to investigate the effect of every amino acid in every sequence position of octapeptides on the behavior of the peptide ligands [20, 211. A complete set consists of 152 (19 x 8) OX, peptide libraries. The advantages of this analytical tool for studies on receptor ligand systems are, first, that the effect of every amino acid is specified by the combination of singly defined with randomized sequence positions and, second, that the entire structural variability of ligands can be investigated with a limited number of reagents. 0 7 X peptide libraries, on the other hand, allow the effect of loss of definition in individual sequence positions to be investigated. With OzX, and O,Xz libraries, interdependence of the effects of different amino acids was studied. The reproducible synthesis, equimolar or close to equimolar composition and the availability of analytical techniques for the control of these qualities are prerequisite for the use of random peptide libraries in biological systems and for the reliability of results obtained from experiments with these libraries. The details of the synthesis are described elsewhere [22]. Briefly, a solid phase peptide synthesis protocol was developed that uses q-fluorenyl-methoxycarbonyl (Fmoc)-L-amino acid-p-benzyloxybenzylalcohol-polyst yrene resins loaded with single amino acids, or equimolar mixtures of 19 resins for randomized carboxy-terminal positions. Couplings were performed with premixed Fmoc-amino acids equimolar to the coupling sites on the resins for randomized positions or with a fivefold excess of single Fmoc-amino acids for defined positions. The dicyclohexylcarbodiimide/ I-hydroxybenzotriazole method was used. Equimolar representation of the amino acids was achieved bv extended coudine times. double couding. initially high con-

352

12 Random Peptide Libraries as Tools in Basic and Applied Immunology

L and OX,

octapeptide libraries

P1 P2 P3 P4 P5 P6 P7 P8

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Figure 12-2. Design of X8 and OX, octapeptide libraries. 0, defined positions in peptide libraries; X, randomized positions containing A, D, E, F, G, H,I, K, L, M, N, P, Q,R, S, T, V, W,Y.

tent of the solvent dichloromethane, and open reaction vessels to allow evaporation of the solvent and thereby concentration of the reagents during the course of the coupling cycles. After cleaving the peptides from the resins, and side chain deprotection with trifluoroacetic acid and a mixture of scavengers, resins were filtered off and peptides were precipitated by adding cold n-heptane :diethyl-ether and lyophilized from acetic acid :water :tert-butylalcohol. Peptide libraries obtained this way were used directly in bioassays. The qualities of such preparations were controlled by amino acid analysis, pool sequencing [23],electrospray mass spectrometry (241 and, in the case of some less complex sublibraries, by HPLC-MS. Deviations from the projected equimolar representation of the amino acids in randomized positions were within the error limits of these analytical procedures and maximally 3%.

12.4 Peptide Selection by MHC Molecules The assays for peptide-MHC binding on cell surfaces make use of genetic defects in the antigen processing machinery found in some tumor cells which have escaped immune surveillance. These cells have deleted the genes for the endoplasmic peptide

12.4 Peptide Selection by MHC Molecules

35:

transporters and, as consequence of peptide deficiency in the endoplasmic reticulum. show reduced MHC expression on their surfaces [25]. MHC expression can bt restored by adding synthetic peptides to the cell cultures or by reducing the culturt temperature to 26°C [26]. The dependence of the thermal stability of MHC-I molecules on peptides can be used to assess the efficiency of peptide binding to these molecules. In the stabilization assays [22,27], outlined in Fig. 12-3, peptide-deficieni cells precultured at 26 "Cto allow accumulation of peptide-receptive MHC molecules were incubated with test peptides for 30 min at room temperature, exposed to 37 "C for 45 min to induce denaturation of "empty" MHC molecules. The remaining conformationally intact MHC-I molecules were quantitated by fluorescence flow cytometry. In extensive studies reported elsewhere, X, and all OX, libraries were tested for their capacity to bind to, and stabilize, the mouse MHC-I molecule

- 1

\-

peptides A

37 "C I S min

FlTC bb.(.d

Quantitation by flow cytometry Fieure 12-3. MHC stabilization assay.

354

I2 Random Peptide Libraries as Tools in Basic and Applied Immunology

H-2Kb [20]. All these libraries contain peptides that are effective in stabilization assays; however, half-maximal stabilization efficiency was different for the different libraries depending on the defined amino acids. By comparing the concentrations of OX7 sublibraries required for half-maximal MHC expression with the corresponding concentration of the X, library, stabilization indices were defined, and stabilizing or destabilizing amino acids identified for the individual sequence positions of octapeptides (221. The eight sequence positions differ greatly in their tolerance to amino acid variation (Fig. 12-4). Taking the chemical properties of stabilizing amino acids into consideration, constitutional, volumetrical and steric constraints were identified that govern preferences for particular amino acids in the individual sequence positions. Concordant with the crucial role of peptides for the conformational integrity of MHC molecules, amino acid preferences found are reminiscent of rules that govern tolerance to amino acid variations in defined sequence positions of globular proteins in general. Hydrophobic amino acids were preferred in most positions. The only exception is the penultimate position which requires hydrophilic side chains. Positions 4 and 6 can accommodate hydrophilic as well as hydrophobic amino acids. In addition to these constitutional constraints, volumetric constraints were found for some sequence positions. These more constrained positions correspond to anchor positions which had been defined on the basis of pool sequencing of natural peptide libraries extracted from immunoaffinity-isolated MHC molecules [3]. These positions allow only a few amino acids. With stabilization assays that use OX7 peptide libraries, anchor amino acids were found for the positions 3, 5 and 8 of H-2Kb, confirming the interpretation of results obtained with natural peptide libraries. Based on the stabilization analyses, amino acids at positions 1, 2, 3, 5 and 8 were classified as MHC contact sites and amino acids at positions 4, 6 and 7 as TCR contact sites (Fig. 12-4 and [22]). This classification applies to the majority of H-2Kb-binding peptides. In the case of some epitopes, however, side chains at position 1 are exposed to the TCR. Even though in all sequence positions the majority of the amino acids are destabilizing when compared with X8, it needs to be stressed that all amino acids are permitted in all positions. Moreover, no position is neutral, i.e., every amino acid in the sequence has a positive or a negative effect on the efficiency of peptide binding to MHC molecules. Stabilization experiments with the eight OX7 peptide libraries that scan the sequence of the OVA epitope SIINFEKL give a clear example for these concepts (Fig. 12-5). The MHC contact amino acids, serine in position 1, isoleucine in position 2 and 3, phenylalanine in position 5 and leucine in position 8 are stabilizing the peptide-MHC complexes. Asparagine in position 4 and glutamate in position 6 are destabilizing. They d o not contribute to the stability of the complex, but as shown later are important for interaction with the TCR. The slightly stabilizing effect of lysine in position 7 is presumably due to steric constraints that act on the conformation of peptides in the binding groove. The side chain of amino acids in position 7

12.4 Peptide Selection by MHC Molecules

355

w

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pointing out of the MHC molecule towards the f cell receptor P4

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burried insMe the peptide binding groove of the MHC molecule

Figure 12-4. Contribution of the individual sequence positions of octapeptides to the stability of the peptide-MHC complex. Upper panel: The average of the absolute values of the stabilization indices of all the amino acids are plotted for the eight sequence positions of octapeptides (221. These values are measures for the tolerance to amino acid variations in the indicated positions. In case of absolute tolerance, all amino acids would be accepted equally well and the concentrations required for half-maximal MHC stabilization would be the same for all OX, sublibraries and the completely random X8 peptide library. The resulting stabilization indices would be 1, their logarithms would be 0 and the average of the absolute values of these log SI would also be 0. Deviations of these average values from 0 are indicative of restricted tolerance to amino acid variations which may be due to biases in favor of, or against, particular amino acids (201. Lower panel: Side chain orientations in an average H-2Kb-binding peptide. The positions 5 and 8 bear the MHC allele-specific motif amino acids.

356

I2 Random Peptide Libraries as Tools in Basic and Applied Immunology

SXXXXXXX

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Figure 12-5. Contribution of every one of the eight amino acids of the CTL epitope SIINFEKL to the stability of the peptide-H-2Kb complex as measured with OX, peptide sublibraries (19’ peptides each) and peptide transporter-deficient RMA-Scells 1221.

is forced out of the groove and has contact with the surrounding water. Hydrophilic amino acids are favored in this position.

12.5 Interdependence of the Contribution of Individual Amino Acids to Peptide-MHC Interaction Randomizing single positions in SIINFEKL results in decreased peptide binding to H-2Kb (Fig. 12-6). This is true also for positions 4 and 6 which in SIINFEKL carry the destabilizing amino acids asparagine and glutamate. The specific sequence context in a defined epitope can alter the properties of individual amino acids in an epitope. This interdependence is particularly noticeable when 02X6and 06X2peptide libaries are compared (Fig. 12-7). As expected from the length of the peptides, XIINmKL SXInmKL

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1

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0

Figure 12-6. Loss of stability of the peptide-MHC complexes caused by randomizing single position in the CTL epitope SIINFEKL. In these assays 07Xpeptide libraries (19 peptides each) were used 1221.

12.5 Contribution of Individual Amino Acids to Peptide-MHC Interaction

357 i

k

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Figure 12-7. Mutual dependence of the contribution of individual amino acids to the properties of MHC ligands. a-h: Loss of the MHC-stabilizing power of 06Xzpeptide libraries with randomized positions in the CTL epitope SIINFEKL. Each library contains 361 peptides. i-p: Complementary stabilization experiments done with OzX6peptide libraries which each contain 196 different DeDtides 1221.

358

I2 Random Peptide Libraries as Tools in Basic and Applied Immunology

and the extent of surface contact between peptide and MHC molecule, no amino acid is independent from the amino acids occupying other positions [22]. This interdependence of the effects of the individual amino acids in the peptide sequence precludes an exact prediction of the binding properties. However, the stabilization indices defined in experiments with OX, peptide libraries provide a guideline for the identification of MHC-binding peptides, and might help to optimize the properties of CTL epitopes and potential synthetic vaccines.

12.6 T-cell Epitopes Defined with Peptide Libraries Binding of the antigenic peptides to MHC molecules generates the ligands for the antigen receptors of T-cells and is prerequisite for T-cell-mediated immune responses. However, whether a peptide is indeed a T-cell epitope is decided by T-cells. Thus, selection of T-cell epitopes is determined not only by MHC molecules but also influenced by processes that shape the T-cell repertoire. Identification of T-cell epitopes requires analysis of T-cell responses and can not rely on the knowledge of the peptide-binding properties of MHC molecules alone. Peptide recognition by CTL is easily analyzed in cell-mediated lympholysis assays (Fig. 12-8). CTL are incubated together with 51-chromium loaded target cells and test peptides or peptide libraries. The amount of radioactivity released from lysed target cells is taken as measure for the efficiency of peptide-induced cytolysis and

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Figure 12-8. Cell-mediated lympholysis assay.

12.6 T-cell Epitopes Defined with Peptide Libraries

359

thereby as a measure for the efficiency of the test peptide as epitope for the CTL clone used. The responses of the SIINFEKLspecific, H-2Kb-restricted CTL clone 4G3 to X8 and the eight OX7 peptide libraries that define the SIINFEKL sequence reveal the contribution of every amino acid to peptide recognition by this CTL clone (Fig. 12-9). Substantial improvement in target cell sensitization was observed when defined amino acids were introduced into the positions 1 , 2 , 4 , 6 and 7 of octapeptide libraries. Most pronounced is this effect for serine in position 1. The strong anchor amino acids, isoleucine, phenylalanine and leucine in positions 3, 5 and 8, respectively, on the other hand, cause drastic reduction of the power of the libraries to trigger the CTL responses. Using these three peptide sublibraries, cytolysis could not be titrated up to the 50% level. Strong suppression of the CTL responses is most likely due to an increased competition by peptides that bind to the MHC molecule but which are not recognized by the CTLs. Competition could be tested directly by adding a peptide library, LSXXYXDL, to SIINFEKL that contains the strong anchor amino acids tyrosine and leucine in the positions 5 and 8 but in the positions 1, 2 and 7 amino acids that do not allow recognition by 4G3 (Fig. 12-10). Background level of the cytolytic response is obtained with a 5 million-fold excess of competitor. OX7 peptide libraries with one defined anchor amino acid contain a much higher excess of MHC binding peptides over a single defined epitope. The reverse experiment with 0 7 X peptide libraries in which single sequence positions in SIINFEKL are randomized shows an decreased efficiency of all eight libraries when compared with SIINFEKL (Fig. 12-11). The strongest effects are seen when the anchor positions 5 and 8 are randomized. Efficient binding to H-2Kb is important for induction of peptide-induced responses of 4G3. However, reduction of CTL responses to OX, peptide libraries with strong anchor amino acids is not generally observed. Analysis of the capacity of X, and the complete set of I52 OX7 peptide libraries to

IlU(xxXX

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peptide concentration required for half-maximalcytoiysis [ g l l ]

Figure 12-9. Response of the SIINFEKLspecific, H-2Kb-restrictedCTL clone 4G3 to highly complex random OX7 octapeptide libraries [221.

I2 Random Peptide Libraries as Tools in Basic and Applied Immunology

360

sensitize target cells for cytolysis by a whole panel of CTL clones reveals Tcell-specific patterns of responses [28]. These patterns allow deduction of synthetic T-cell epitopes which are effective in concentrations comparable to natural epitopes. Identification of effective anchor residues was possible for some epitopes. In other

molar excess of competitor peptide ( LSXXWDL )

Figure 12-10. Inhibition of the SIINFEKGinduced cytolysis by the peptide sublibrary LSXXYXDL that lacks the appropriate TCR contact residues for the CTL clone 4G3 used in these assays.

SIINFEKL XIINFERL SXItWEIu SIXNFEIu SIIXFlGlcL SIIWXEKL s11mKL

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Figure 12-11. Randomizing single positions in the CTL epitope SIINFEKL causes reduced efficiencv of tareet cell sensitization.

References

361

cases, however, active epitopes were obtained only when canonical anchor amino acids were combined with the non-anchor amino acids found by positional scanning with OX7 peptide libraries. For all CTL clones tested, construction of a variety of active peptides was possible indicating degeneracy of antigen recognition by T-cells. The degree of degeneracy varied with the T-cell clone tested. Some T-cells allowed amino acid variations only in restricted positions of their cognate peptide. Other T-cell clones recognize completely unrelated epitopes, which often do not even conform to the MHC allelespecific peptide motifs. Degeneracy of peptide recognition by CTL is also indicated by the results shown in Fig. 12-9. Half-maximal responses of clone 4G3 were obtained with a few hundred fM of SIINFEKL. In comparison, the concentrations of X8 or OX7 peptide libraries required for the same degree of cytolysis is orders of magnitudes lower than expected from the representation of SIINFEKL in these libraries. Also natural T-cell epitopes were identified which are potent inducers of cellular responses but which are very inefficient in binding to the presenting MHC molecule [29, 301. Thus, efficient binding to the MHC molecules and strict adherence to the canonical anchor position motifs are important, but not in all cases essential for efficient T-cell epitopes. For many CTL clones, or even with bulk culture CTLs, synthetic epitopes can be deduced either from results obtained with synthetic peptide libraries directly or with peptide library scans in combination with information on the allele-specific peptide motif for the particular MHC molecule of interest.

12.7 Conclusions Rational design of reagents that are effective in induction or modulation of Tcell-mediated immunity requires knowledge of rules that control peptide selection by MHC molecules and recognition of MHC-peptide complexes by TCR. We have described a methodology that allows both levels to be elucidated. The critical features of epitopes can be identified directly by positional scanning with OX, or similar random peptide libraries, or indirectly by following an iterative approach. Peptides can be designed and optimized to meet the requirements for T-cell vaccines to induce active immunity or TCR antagonists to cause anergy in T-cells involved in aberrant immune responses.

References [I] L. D. Barber, P. Parham, Ann. Rev. Cell Biol. 1993, 9, 163-206. 121 J. Klein, Natural History of rhe Major Histocompatibility Complex, John Wiley & Sons, New York, 1986.

362

I2 Random Peptide Libraries as Tools in Basic and Applied Immunofogy

[3]K. Falk, 0.Rotzschke, S. StevanoviC, G. Jung, H.-G. Rammensee, Nuture (London) 1991, 35f. 290-296. (41 D. F. Hunt, R. A. Henderson, J. Shabnowitz, K. Sakaguchi, H. Michel, N.Selilir, A. Cox E. Appella, V. H. Engelhard, Science 1992, 255, 1261- 1263. [S)W. Chen, J. McCluskey, S. Rodda, E R. Carbone, A Exp. Med. 1990, 177, 869-873. [6]J. Ruppert, H. M. Grey, A. Sette, R. T. Kubo, J. Sidney, E. Celis, Cell 1993,74,929-937 [7]D. H. Fremont, M. Matsumura, E. A. Stura, P. A. Peterson, I. A. Wilson, Science 1992 257, 919-927. (81 M. Matsumura, D. H. Fremont, P. A. Peterson, I. A. Wilson, Science 1992,257,927-934 [9]D. R. Madden, D. N. Garboczi. D. C. Wiley, Ceff1993, 75, 693-708. [lo] P. J. Bjorkmann, M. A. Saper, B. Samraoui, W. S. Bennet, J. Strominger, D. C. Wiley Nature (London), 1987a, 329, 506-512. [ll] R. N. Germain, D. H. Margulies, Ann. Rev. Immunof. 1993, JJ, 403-450. [I21 J. Driscoll, M. G. Brown, D. Finley, J. J. Monaco, Nature (London) 1993,365,262-264 (131 M. Gaczynska, K. Rock, T. Spies, A. L. Goldberg, Proc. Natf.Acad. Sci. USA 1994,9f 9213-9217. (141 F. Momburg, J. Roelse, J. C. Howard, G. W. Butcher, G. C. Hammerling, J. J. Neefjes. Nature (London) 1994, 367, 648-651. (151 H.Udono, P. K. Srivastava, J. Exp. Med. 1993, 178, 1391-13%. [I61 P. Walden, H. N. Eisen, Proc. Natf. Acad. Sci. USA 1990, 87, 9015-9019. 1171 0.RBtzschke, K. Falk, S. StevanoviC, G. Jung, P. Walden, H.-G. Rammensee, Eur. J Immunof. 1991, 21, 2891-2894. [I81 G. Jung, A. G. Beck-Sickinger, Angew. Chem. Int. Ed. Engf. 1992, 31, 367-383. [I91 C. T. Dooley, R. A. Houghten, Life Sci. 1993, 52, 1509-1517. (201 K. Udaka, K.-H. Wiesmiiller, S. Kienle, G. Jung, P. Walden, A Biof. Chem. 1995, 270, 24130-24134. (211 L. Pridzun, K.-H. Wiesmiiller, S. Kienle, G. Jung, P. Walden, Eur. A Biochem. 1996,236, 249-253. [221 K. Udaka, K.-H. Wiesmuller, S. Kienle, G. Jung, P. Walden, A Exp. Med. 1995, 181, 2097-2108. [23]S. StevanoviC, G. Jung, Anal. Biochem. 1993, 212, 212-220. (241 J. W. Metzger, C. Kempter, K.-H. Wiesmflller, G. Jung, Anal. Biochem. 1994, 219, 261 -277. (25)A. Townsend, C. Ohlen, J. Bastin, H.-G. Ljunggren, L. Foster, K. Urre, Nature (London] 1989, 340, 443-448. I261 H.-G. Ljunggren, N. J. Stam, C. Ohlen, J. J. Neefjes, P. HBglund, M. T. Heemels, J. Bastin, T. N. M.Schumacher, A. Townsend, K. Karre, H. L. Ploegh, Nature (London] 1990,346,476-480. [27]T. N. M. Schumacher, M.T. Heemels, J. J. Neefjes, W. M. Kast, C. J. M.Melief, H. L. Ploegh, Ceff1990, 62,563-567. 128) B. Gundlach, K.-H. Wiesmuller, S. Kienle, G. Jung, P. Walden, A Immunof. 1996, J56, 3645-3651. [291 K. Udaka, T. J. Tsomides, H. N. Eisen, Ceff1992, 69,989-998. [30]E. Sadovnikova, X. Zhu, S. M. Collins, J. Zhou, K. Vousden, L. Crawford, P. Beverley, H. J. Strauss. Int. Immunof. 1994. 6. 289-296.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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 Introduction SPOT synthesis [I, 21 is a facile and very flexible technique for the simultaneous parallel chemical synthesis on membrane supports. The principle of the technique is to dispense small droplets of liquid onto a predefined array of positions on a porous membrane where the droplets get absorbed and form individual separate spots (Fig. 13-1). Using a solvent of low volatility containing appropriate reagents, such a spot can form a reactor for chemical conversions involving reactive functions anchored to the membrane matrix, e. g., conventional solid phase synthesis. A great number of distinct spots can be arranged on a larger membrane sheet and each of these is individually addressable by manual or automated delivery of the respective

Figure 13-1. Schematic cartoon showing the principle of SPOT synthesis.

364

‘

13 Combinatorial Synthesis on Membrane Supports by the SPOT nchnique

reagent solutions. The volume dispensed and the absorptive capacity of the membrane determine the spot size. According to the specific functionality of the matrix, the spot size correlates with the particular scale of the synthesis. The spot size also controls the minimal distance between spot positions and thereby the maximum density of the array. Synthetic steps common to all spot reactors are carried out by washing the whole membrane with respective reagents and solvents. Based on our many years of experience with membranes as solid supports for simultaneous synthesis of oligonucleotides and peptides [3, 41, SPOT synthesis was developed initially as an easy and economic alternative to the multiple parallel synthesis on arrays of pins (multipin synthesis) (51. Other types of reactors arranged into array formats have also been described such as tubes [a], wells [7],columns (81, and dots on membranes [9] (e.g., PILOT) for the multiple preparation of repertoirs or combinatorial libraries of peptides and other compounds [lo]. Furthermore, simultaneous synthesis techniques utilizing a checkerboard type arrangement of microsquares on a suitably functionalized flat surface have been developed. For the chemical coupling with nucleotide or amino acid monomers, arrays of squares are addressed either by light-induced terminal deblocking [ll] or by physical separation [12]. Because straight lines of squares are linked via a mask and react simultaneously with one type of monomer per coupling cycle, the spatial arrangement of the sequences is the result of a combinatorial process dictated by the movement of the mask. In particular, laser light-directed synthesis reaches an exceptional high density of distinct sites (>4OOOO per mm2). So far, SPOT synthesis has been elaborated for the chemistry of amide bond formation (peptide synthesis) following the conventional FmochBu-protection scheme on membranes made of pure cellulose (cotton linters) chromatography or filtration paper. Compared to other array-directed synthesis techniques, SPOT synthesisis particularly flexible with respect to numbers and scales that can be accomplished. The arrays are freely selectable to fit the individual needs of the experiment by variation of paper quality, thickness, specific anchor loading, and spot size. Recent technical advancements of the SPOT technique [13] now allow the semiautomated preparation of arrays including several thousands of peptide sequences (Fig. 13-2). These peptide arrays can be applied either directly or after cleavage of peptide products from individual cutout spots in binding or activity assays with, eg., biological acceptor molecules (antibodies, receptors, enzymes, etc) to the rapid search of active sequences [2, 131. The adaptation of the ‘‘mixed coupling’’ approach to selected randomization of positions in a peptide sequence, thereby generating arrays of defied peptide pools [13-151, and the use of digital image analysis devices for recording positional activity patterns has expanded the scope of this technique considerably. The entire sequence and shape space of combinatorial peptide libraries can be displayed in a detailed two-dimensional scene by a variety of different pooling strategies. In combination with suitable assay systems, this is being used in the (I Driori delineation of biologically active sequences, as well as in the analysis of se-

13.2 Geneml Echnical Aspects of SPOT Synthesis

36:

Figme 13-2. A: Automated performance of SPOT synthesis utilizing an array of 2000 spots on an 8 x 12 cm paper sheet. B: Recognition pattern of mouse monoclonal antibody 1D3 on an array of 2OOO peptide pools of a dual-positional scanning library experiment (see also Fig. 13-10).

quence fitness landscapes, thus experimentally verifying theoretical models and predictions of search strategies [la].

13.2 General Technical Aspects of SPOT Synthesis 13.2.1 Instrumental The initial standard array used for Sp(Tr synthesis was adapted to the microtitex plate format with 8 x 12 spots and manual pipetting of volumes between 0.5 to 2pl was adequate as well as reliable enough (Table 13-2) [l, 21. However, a substantial in-

366

13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

crease in spot number is only practically feasable by utilizing an automated pipetting workstation. We have modified a Gilson model XL222 sample changer plus 401 dilutor to provide a platform for fixing membranes up to 25 x 30 cm in size [13]. The instrument is controlled by a PC and a very flexible MS-Windowsm compatible software that includes a sequence editor and an array formator. The formator allows to define several (currently up to ten) independent arrays, two of these are used for the rack positions of reagent and stock solutions. Thus, different arrays may be simultaneously synthesized utilizing up to 48 reservoirs for preactivated amino acid derivatives. Special steel/Teflon needles were developed for an accurate dispensing of volumes down to a few nanoliters. In combination with appropriately selected paper qualities, synthesis arrays can now be miniaturized to, e.g., 25 spots per cm2 (Fig. 13-2; Table 13-2). This amounts to about 15000 synthesis sites on the working platform of the instrument. The instrument so far carries out all of the pipetting work; other steps of a synthesis cycle such as washing and deblocking are carried out as in the manual procedure [2]. A fully automated process in under development.

13.2.2 Peptide Synthesis on Spots Arrays of spots providing suitable anchor functions for peptide assembly on cellulose membranes are easily generated by, e.g., a two-step procedure including first, the preparation of “amino” paper by esterification of an N-Fmoc protected amino acid to the whole sheet followed by Fmoc-cleavage and secondly, spotwise coupling of another Fmoc-amino acid or suitable linker compound (Table 13-1). During the second derivatization step, the array of spot reactors is generated, and all residual amino functions in-between spots are blocked by acetylation. Particularly this array formation step requires a very accurate pipetting. During peptide assembly, slightly larger volumes are dispensed and the spots then formed slightly exceed those initially formed in order to avoid incomplete couplings at the edges. Some standard array configurations used by us are given in Table 13-2. Any linker chemistry used in conventional Fmoc SPPS [17] can be applied to SPOT synthesis for the construction of linear, modified, cyclic or branched peptide structures. A selection of those anchors and linkers being used with SPOT synthesis is given in Table 13-1. Particularly in the preparation of solution phase peptides, the work-up procedure for the removal of toxic chemicals from the final deprotection step includes inherently the risk of losing individual peptides due to their very different physicochemical properties. More important, the evident screening with soluble peptide libraries relies on complete pools. The synthesis and handling of large numbers of such peptide preparations is greatly facilitated by the use of orthogonal safety-catch linkers as forwarded by Bray et al. (18, 19). These allow the cleavage of protecting groups and removal of chemicals, while the peptides remain attached to ~ bv the final direct release into an aaueous buffer solution. the solid S U D D O ~followed

13.2 Genera( Technical Aspects of SPOT Synthesis

367

Table 13-1. Orthogonal safety-catch linkages and anchors used with SPOT-synthesis ~

~

~~

Entry

Anchor/linker”

Peptide product

Reference

I

peptide-PAla-PAla-COO-resin

Immobilized via C-terminus

[I, 21

~ CI o o - r e S i n Soluble, C-terminally modified CO with diketopiperazine

2

[181

Soluble, C-terminal acid

3

Soluble, C-terminal amide

4

peptide5 f2

peptide-NH

CO-PAla-resin

PI

Soluble, C-terminal amide (optional release after biological assay)

Pi1

Immobilized, cyclic

r221

700-TBDMS 6

a

n = 0 (R = H,CH3), 1 (R = H);m = I, 2; R’= methyl, isopropyl, rert-butyl, terr-butyl-dimethylsilyl (TBDMS), trimethylsilylethoxymethyl(SEM), methoxymethyl (MOM), methoxyethoxymethy1 (MEM); R2= ethyl, isopropyl, rerr-butyl, trityl; after selective removal of TBDMS and cyclization

Two new types of safety-catch linkages compatible with Fmoc SPPS were recently developed by us. The peptide acid linkage (Table 13-1, entry 3) exploits the intramolecular hydrolysis of an ester bond catalyzed by the intermediately protected imidazole side chain of a substituted a-hydroxy-acetic acid [20]. The final acidic deprotection simultaneously removes the Boc group from the linker, but the catalytic effect is still blocked by protonation of the imidazole ring. Toxic chemicals are removed by simple acidic washing steps and the final release of the peptide acids proceeds at neutral to slightly basic PH.The DeDtide amide linkages (Table 1, entries 4

368

I3 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

Table 13-2. Standard SPOT array configurations (each one fitted to a sheet of the size of a microtiter plate, 8 x 12 cm)

Format

Membrane Anchorb Type” pmol/cm*

8x12=96 540 7 x 10=70 Chrl 17 x25 =425 540 4 0 50=2000 ~ 50 a

0.2-0.4 0.4-0.6 0.2-0.4 0.2-0.4

Spotted volumeC

Spot size

Positional distance

Synthesis scaled

0.5/0.7 pl 1.0/1.5 pl 0.1/0.15 p1 0.03/0.05 p1

7 mm 8 mm 3 mm

9 mm 10 mm 4 mm 2 mm

25 nmol 50 nmol 6 nmol 1 nmol

I mm

Chromatography paper products from Whatman, Maidstone UK. Typical derivatization with first P-alanine. Volumes are given for array generation steplpeptide assembly step. Mean values.

and 5 ) exploit the spontaneous decomposition of an intermediately protected, acid stable a-hydroxy- or a-thiol-glycine intermediate [21]. The protecting group on linker type “4” is simultaneously cleaved with the side chain protection, whereas linker type “5” is acid stable, and the peptide array may be applied first in a biological screen and later deprotected with 5 % HgC12 in 30% acetic acid. The final release of the peptide amides proceeds at neutral to near neutral pH to leave glyoxalic acid bound to the solid support. These linkers work both on conventional polymeric resins and spots on cellulose membranes (Fig. 13-3). Cellulose membranes are dried in vocuo after the last acid wash and may be stored. The individual peptide preparations are released by punching out the corresponding spots and simply placing them into an aqueous buffer. As outlined in the Introduction, a spot on a cellulose membrane serves as an open reactor entirely made up from derivatized solid support. When the dispensed volumes are taken up, the ratio of liquid to support material adjusts by itself to a constant level all over the membrane and is typical for a given material. The stoichiometry of the coupling reactions can thus be quite accurately preadjusted as the amount of reactive functions per reactor volume is known from the area specific functionalization of the membrane. There is no void volume to be filled in a spot reactor and rate-limiting diffusion is minimal; high concentrations of reactants can be applied to drive coupling reactions rapidly to completion, yet avoiding unnecessary high excesses. Because the solvent within the spots is slowly evaporating, the concentration is even increasing during the coupling time. Then, additional aliquots of solution may be spotted onto the same positions without enlargening the spots and risking overlap with their neighbors. Free amino functions on the spots are routinely stained with bromophenol blue [23] prior to the coupling reactions. This allows the monitoring of proper performance of all synthesis steps such as correct dispensing, quantitative coupling and capping, and effective removal of piperidine from the Fmoc-deblocking steps.

13.2 General Technical Aspects of SPOT Synthesis

A Q)

H

H-DRWIICLNKC-OH

1

- 100

60-

369

1173.2

. 0.2

-2. s: 4

- 0.1 10

I

1

20

I

I

40

0-

lo00

I

1

elution time (min)

B

I

BCHSPCVRYRF-NHz

I

60-

-loo

- U

- 0.2

8

--

- 2 % Z i 2 30-

- 0.1 10

20

I

I

30 I

elution time (do)

1200

1

1

1400

1

masslcharge

e 451

1

40 I

0-

R -3 g 3 2. -9

1175.3

-

lo00 1200 1400 1 1 1 1 1

masslcharge

Figure 13-3. Reversed phase C18-HPLC chromatograms and MALDI-TOF spectra of two crude soluble peptide preparations synthesized on (A) conventional TentaGel-S-amine resin utilizing anchor 3 of Table 13-1 with n = 0 and R = CH3 or (B)on a spot of a cellulose membrane utilizing MOM-protected anchor 4 of Table 13-1 ; HPLC eluent was water/acetonitrile containing 0.1 To trifluoroacetic acid; MALDI-TOF matrix was sinapinic acid.

13.2.3 Peptide Library Synthesis For the introduction of randomized positions within a peptide sequence assembled on a spot, the only way is coupling with amino acid mixtures. Similar to Kramer et al. [15], we investigated the coupling with equimolar mixtures, applying these at a subequimolar ratio with respect to available amino functions on the spots. This is to allow all activated derivatives to react quantitatively during the first step and then

370

I3 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

to bring coupling to completion by successively repeating the treatment. We therefore combine equal aliquots of the usual 0.3 M stock solutions and dilute this mixture to result in an 0.4 to 0.6 molar ratio over amino functions according to the chosen spot scale and volume (Table 13-2).Coupling is then repeated up to four times. A quantitative reaction as monitored by the change of blue spots to yellow is observed during the third or fourth repeat. In a real case study, a series of such peptide pools prepared on spots were analyzed for amino acid representation at randomized positions by amino acid analysis after cleavage from the spots. Data in Table 13-3 clearly indicate that coupling with subequimolar mixtures yield a sufficiently homogeneous representation of amino acid residues even for critical amino acids such as Ile or Val at mixed positions in the peptide chain. These data confirm those reported by Kramer et al. [I51 obtained on a PEG-polystyrene support (TentaGel S RAM). Using this coupling procedure, any position can easily be randomized without special considerations or increase in technical effort. Table 13-3. Amino acid analysis of a series of random peptide pools

X Xt X3 X4 Xj Xr

D

E

2.3 2.1 2.1 2.2 2.2

2.1

1.7

2.0 1.8 1.8 1.9

1.7

S

G

H

R

A

P

Y

V

I

1.3

1.2 1.3 1.2 1.2 1.3 1.0

0.4 0.6 0.8 0.6 0.8 0.6

0.7 0.7 0.6 0.6 0.6 0.6

1.1 1.1 1.0 1.0 1.1 1.0

0.6 0.9 1.1 1.1 1.1 1.1

1.2 1.1 1.0 1.1 1.1 1.0

0.7 0.8 0.9 0.8 0.9 0.9

0.6 0.8 0.8 0.8 0.9 0.9

1.1 0.9 1.0 0.9 0.7

L

F

K

1.0 1.0 1.0 1.0 1.0 1.0

1.1 1.1 0.9 1.0 1.1 1.1

1.0 0.9 0.9 0.9

0.9 1.0

Peptide pools were cleaved from individual punched out cellulose spots by treatment with 1 M triethylamine in water at room temperature overnight and eluted by repeated washing with 0.1 M triethylammonium acetate (pH 7.5) in the presence of 20% ethanol. The combined eluates were evaporated to dryness and then subjected to acid hydrolysis and amino acid analysis. X2= X-X;X3= X-X-X;X4= X-X-X-X;etc. Amino acids in one-letter code; D = D + N, E = E + Q,cysteine(Acm) was not determined, threonine coeluted with P-alanine from the anchor, methionine and tryptophan were oxidized and destroyed to various degrees during the whole procedure. Loss of peptide components that strongly adsorb to cellulose cannot be excluded. Amounts given are relative to leucine.

13.2.4 Library Design For the deconvolution of individual sequences by activity screening of random pools, concepts of analysing strategic sets of sublibraries are applied as forwarded by Geysen et al. [24] and Furka et al. [25]. Meanwhile, a variety of pooling strategies have been reported which are strongly influenced by the technical features of the respective synthesis method used. These strategies are also theoretically treated by, e.g., Kauffman et al. [16]concerning their efficacy in finding local activity peaks by

13.2 General Technical Aspects of SPOT Synthesis

371

walking through a rugged fitness landscape of the shapes of combinatorial sequences. We will describe some typical examples to demonstrate the flexibility in testing and developing search strategies for library analysis offered by SPOT synthesis. The identity of each pool is given exactly by its site of synthesis, the spot coordinates. Special codes to describe the pool compositions are: 0 = unvaried position in a particular screen occupied by single amino acid residues; 1,2,3.. . = positions systematically varied by single amino acid residues in a particular screen; X = position occupied by a set of (e.g., all 20 L-) amino acid residues. (a) Iterative search starting with one or more defined positions (mimotope approach) 1241, e.g., x-x-1-2-x-x 400 pools first screen x-1-03-04-2-x 400 pools second screen l-O2-O3-O4-O5-2 400 POOIS third screen (b) Positional scanning with single fixed positions (PS-SPCLs) [26, 271, e.g., 1 -x-x-x-x-x 20 pools x-1-x-x-x-x 20 pools x-x- 1-x-x-x 20 pools x-x-x-I-x-x 20 pools x-x-x-x-1 -x 20 pools x-x-x-x-x- 1 20 pools with only 120 pools of hexapeptides, acceptor preferences for certain amino acid residues at all positions are obtained (consensus sequence); sequences of individual active compounds have to be identified by synthesizing and testing all possible combinations of hits from this “positional scanning library” screen. (c) Dual-positional scanning [I31

1-2-x-x-x-x x- 1-2-x-x-x x-x-1-2-x-x x-x-x-1-2-x x-x-x-x-1-2

400 pools 400 pools 400 pools 400 pools 400 pools with 2000 pools of hexapeptides, acceptor preference for certain dipeptide combinations at all positions are obtained; from matching overlaps of these, sequence connectivities can be directly delineated. The interpretation of signal patterns on spot peptide arrays should consider the following:

(i) on a standard 540 paper with initial 0.2 pmol/cm2 loading (Table 13-2) about 1 nmol peptide per mm2 is presented; particularly in binding assays, multivalent acceptor molecules can be trapped via more than one ligand peptide and weak affinities are enhanced:

N (ii) with increasing number of X-positions the densities of individual peptide sequences per spot decrease corresponding to the complexity of the pool; if X = 20, a “4X” pool with 16OOOO components displays only about 5 fmol of individual peptides. The dynamic range of the assay system will certainly not cover more than two orders of magnitude. Thus, pools containing a larger fraction of active sequences (e.g., sequences having degenerate positions) will preferentially show up. Additionally, binding via more than one ligand peptide becomes less significant. (iii) a signal results from the cumulative activity of all sequences in the respective pool; the same signal intensity may reflect a few high-affinity or many low-affinity compounds.

13.3 Applications of Peptide Libraries on Spots 133.1 Solid-Phase Ligaad Binding Assay The detection of acceptor molecules bound to the peptides that are immobilized via, e.g., #Ala-#Ala anchors on the spots after incubation of the whole membrane or parts thereof can be achieved by a variety of labels (radioisotopes, fluorescent dyes or enzymes). The majority of experiments were carried out with enzyme-conjugated acceptors or secondary antibodies (4.3). Conjugates with alkaline phosphatase performe superior to the initially employed #-galactosidaseconjugates [2] as less unspecific signals were observed. Signal development through hydrolysis of 6-bromo-5-chloro-3-indolyl phosphate (BCIP) and instant oxidation by thiazolylblue-tetmolium bromide (MTT) yields a bludviolet precipitate on positive spots (Fig. 13-4a). Signal intensity is linear over a wide range of enzyme concentrations and time intervals (Fig. 13-5a). This was proven by spectrophotometric quantitation of the dye eluted from individual spots after incubating different amounts of enzyme adsorbed to the cellulose paper by serial dilution and spotting the concentrated stock of enzyme conjugate. Rapid quantitative evaluation of signals on a spot membrane is thus feasible by utilizing a digital recording laser densitometer which reads the optical densities (OD) of dye distributed over the entire membrane. The particular instrument used by us, the pdi image analysis system model DNA 35, has a maximum resolution of 64 x 64 pm2. The software option “Diversity One” allows to fit an array of squares over the array of spots and gives “optical density” values for these squares. These ODs do not correspond linearly to the actual amounts of dye on the spots. However, all data from several calibration experiments fitted exactly to a quadratic equation represented as: dye-units = (0D/0.145)2. “Dye-units” are the real optical absorbancies (A,,x 100) of the dye eluted from individual spots and measured photometri-

13.3 Applications of Peptide Libraries on Spots

B

373

SPOTslib Analysis

Binding of Slreptavkiin-AP to Ac-XX12.W I C D E ~ C O l K L M N ? Q R 8 l ’ V W V A

I

P

f

f.

0

;

S

G

i

Y

t

:

o

r

i

a

1 D

0

I I(

1.

;

5 m

e

* : 2

0 h

t ?

: I

V ll

v

A C D E ~ C ~ I K L Y N I Q U S T V W V

Position1 U a

m

a

e

a

m

m

u

t

*

munia

Figure 134. A: Recognition pattern of the streptavidin-alkaline phosphatase conjugate on an array of 400 immobilized hexapeptide pools Ac-X-X- 1 -2-X-X- with systematic variation of the central two positions. B: Spectral diagram display of a. showing the correlation of quantified signals to the amino acid residues at positions 1 and 2; C* = Cys (Acm).

cally in solution at A, (Fig. 13-5b). Therefore, results from spot analyses can be accurately presented in column type or spectral type diagrams of signal intensities expressed in dye units (Fig. 13-4b). After development, quantitation and documentation of binding results, membranes can be regenerated by a “stripping procedure” (4.3) that washes off dyes and bound proteins. Thus, membranes are reusable several (>20) times.

314

A

13 Combinatorial Synthesis on Membrane Supports by the SPOT Rchnique OD440

0

20

40

60

80

100

80

100

time (min)

B

OD Scanner

0

20

1

40

60

Dye Units (OD 440 x 100)

Figure 13-5. A: Amount of dye formed (optical density of the eluate in dimethylformamide) versus time of color development by alkaline phosphatase labeled spots on a Whatman 540 sheet generated with 1 pl each of alkaline phosphatase conjugate (1 mglml) after indicated dilution. B: Calibration of spot intensities from the pdi scanner to the actual amount of dye (dye units).

375

13.3 Applications of Peptide Libmries on Spots

W Omodel acceptor proteins were used to evaluate the methods: streptavidin, applied as conjugate with alkaline phosphatase (strepavidin-AP); a wealth of reference data from studies with phage display [28] and synthetic combinatorial peptide libraries are available [29], which have revealed a preferential recognition of peptide ligands carrying the - HPQ/M- motif; specificity of binding can be easily proven by competition with the high affinity ligand biotin (K,= 2.5 x 10” M). the mouse monoclonal antibody 1D3, directed against the tubulin tyrosine ligase from pig [30] and an alkaline phosphatase conjugated goat antimouse secondary antibody; data on conventional epitope scanning and analog analysis are given in Fig. 13-6; the smallest epitope peptide recognized with high affinity is AcNYGKYE- and tolerates a broad range of single substitutions, tyrosine(2) and glycine(3) being the most critical residues; some smaller penta- and tetrapeptides are also bound with weaker affinity.

A

SPOTsbAna

Binding of mouse

Is

B

l h W

lher

her

ha

8ma

8mer

7ma

7mer

6mer

6ma

5mer

5ma

4mer

4ma

SPOTsalogueAnalysis Binding of mouse mAb 103

NHCIQKEYIKNYGKYLIGN~Y1

nZpig Sequencepos. 242 to 263 4

U

b

mvi*

an

a

a

4

N

Y

N

Y a

G

K

Y

E

G

K

Y

E

parentseguerrce 40

Wvi*

a

a

m

Figure 13-6. Peptide recognition data for mouse monoclonal antibody 1D3.A: An array of overlapping peptide fragments with indicated length and offset of one amino acid residue was prepared spanning the epitope region of pig tubulin tyrosin ligase (TTLpig); C = Cys(Acm). Signals from each spot are displayed relative to the N-terminal amino acid residue of the respective peptide sequence. B: Systematic single replacement analysis for the hapeptide Ac-NYGKYE-;C+= Cy~(Acm). 133.1.1 Positional Scanning Libraries

The most simple and less synthetic effort requiring type of library approach is the Dositional scanning described by Dooley et al. 1271 which has been also our initial

13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

376

approach to evaluate peptide libraries on spots [13]. A hexapeptide library of this type with only 120 pools (2.4) can be accommodated on a spot array of not more than 3 x 2.5 cm in size (e.g., a stamp). To determine simultaneously the minimal peptide size required for binding, we prepared a series of scanning libraries from two to six residues, altogether 400 pools on a 17 x 25 format (Table 13-2). The streptavidin- A P recognition pattern on this positional scanning library is shown in Fig. 13-7a (no signal at all was observed when the conjugate was saturated with 1 mM biotin; not shown). The most prominent signals that appeared already a few minutes after starting signal development correlate to proline at the third and second last positions in tri- to hexapeptides. In a systematic analysis of residues around a proline we found the same preferences as described in the literature, namely -HPQ-and -HPM-giving the strongest signals (Fig. 13-7b). This approach, however, did not give significant results for mAb 1D3.Apparently, pools of a minimum six residues long ligand peptide with more than four X-positions seem to be too complex for this type of solid phase binding assay (the same was observed by J. Eichler, pers. comm.).

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13.3.1.2 Iterative Library Search (Mimotope Approach) Iterative search starts with a general set of sublibraries having one or more positions of fixed residues within the peptide chain (2.4). From a synthetic point of view,

13.3 Applications of Peptide Libraries on Spots

377

400 pools with two positions systematically varied by all 20 amino acid residues are currently most convenient. However, fixing three positions which results in up to 8000 pools is technically feasible and under development. The hexapeptide library Ac-X-X-I -2-X-X- was first screened with streptavidin- A P (Fig. 13-4);no signals were obtained in the presence of 1 mM biotin. The motif-HP- gave the strongest signal followed by -DW-. A number of other weak signals were detected which include amino acid residues W, Y, F, H, and G. All these appeared also in Fig. 13-7b. The -DW- motif was then chosen for a second iterative step to search for the two neighboring positions (Fig. 13-8). Glycine and proline are clearly selected for at position I and tolerate almost any residue at position 2, -G-D-W-LA- and P-D-W-N/I- showing the highest affinities. SPOTslib Analysis

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One clear dominant signal is obtained when screening hexapeptide library Ac-XX-1-2-X-X- with mAb lD3: the motif -GK-(Fig. 13-9a).This is in perfect agreement with residues at the same positions in the natural epitope peptide. No signal was obtained from the secondary antibody alone. The second iterative step (Fig. 13-9b)revealed tyrosines and with less affinity phenylalanines at positions 1 or 2, then tolerating a series of other residues at the complementary positions. This again fits very well with the features of the natural epitope peptide (Fig. 13-6). Finally, several selections from the second search data of both acceptor molecules could be followed for a third iterative step.

I3 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

318

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13.3.1.3 Dual-Positional Scanning

Both approaches described above require the subsequent synthesis of new peptide arrays after analysis of the first general library to deconvolute individual active sequences. To achieve a direct access to sequence information, we realised a scanning strategy which uses two fixed neighboring positions, thus each systematic dipeptide variation thereof amounts to 400 pools, altogether 2000 for a complete hexapeptide scan (2.4). Thereby, information from dipeptide motifs is obtained, and those overlapping in their positions are likely to have a sequence connectivity. Exploiting the high density arrays of SPOT synthesis sites (Fig. 13-2), this type of library could also be accommodated on the same size of a microtiter plate sheet utilizing an array of 40 x 50 spots (Table 13-2), thus avoiding a massive increase in physical library size. The feasibility of this new search strategy is demonstrated by the pool recognition image obtained with mAb 1D3 (Fig. 13-2b). Signal patterns from four of the five sets of the dual-positional sublibraries are given in Fig. 13-10. Indeed, the strongest signals from each set, -NY-, -YG-, -GK-, and -KY-, perfectly match to give the natural epitope sequence -NYGKY -. The fifth set gave no significant signals over background for the last two residues. Most probably, these positions are quite degenerate (Fig. 13-6 and 13-9b) and specificity is determined primarily by the four preceding positions which would result in a very low abundance of such peptides in these pools.

13.3 Applications of Peptide Libraries on Spots

379

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Figure 13-10. Delineation of hexapeptide Ligands for mouse monoclonal antibody ID3 from a dual-positional scanning library; quantitative evaluation of the recognition pattern shown in Fig. 13-2b. Signals from each full set of 400 dipeptide combinations are displayed in separate graphs. Results from the Ac-X-X-X-X-1-2pools are not shown because no significant signals were obtained; C* = Cys(Acm).

13.3.2 Enzymatic Transformations of Peptide Libraries To address the question of whether spot peptide arrays on paper can be applied to the screening of not only binding affinities but also the sequence dependence of enzymatic transformations, we investigated the phosphorylation by the catalytic subunit of AMP-deDendent Drotein kinase as a model system. This enzymatic reac-

380

13 Combinatorial Synthesis on Membtune Supports by the SPOT nchnique

tion has several features that made it seem particularly well suited for this purpose: (i) protein phosphorylation is among the most important posttranscriptional modifications; (ii) it is well known from investigations with soluble peptides that the enzyme displays a specificity that is strongly dependent on the sequence around the site of transformation (the phosphorylation), so that relatively short peptides would be sufficient for the investigation; (iii) the extent of the transformation can easily and very sensitively be monitored and quantified by using the [''Plphosphate label; (iv) the specificity of the enzyme is well known and documented [34] and these data are an excellent lead for the performance of our approach. The last point turned out to be particularly important during the development of the assay conditions. We found that the incubation buffers must contain at least 100 m~ salt to avoid unspecific ionic trapping of ATP by basic amino acids. Also, the paper membrane had to be washed with solutions containing high amounts of salt (e.g. 1 M NaCl) after the phosphorylation reaction. 75 m~ phosphoric acid that is used for the washing of phosphocellulose paper in assays with soluble peptides is not sufficient. We followed the iterative approach (see 2.4) of constructing libraries consisting of pools with two defined amino acid residues each. The best amino acid combination that was obtained with a particular array was used throughout in the next one. The fist array had the structure Ac-X-X-X-1-2-X-X-X. Incubation with the kinase in the presence of [y-'*P]ATP gave a phosphorylation pattern where 1 and 2 are basic amino acid residues (Fig. 13-11 and Fig. 13-12a). The pool containing arginine

Figure 13-11. PhosphorImager scan of the peptide array Ac-X-X-X-1-2-X-X-X-after phosphorylation by cAMP-dependent protein b a s e ; C*= Cys(Acm).

381

13.3 Applications of &pride Libraries on Spots

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at both positions gave particularly high activity. This is in agreement with investigahas tions of soluble peptides, where the general motif -R-X-X-R-R/K-X-S-Xbeen identified for this kinase. The second array had the general structure Ac-XX-X-R-R-1-2-X. In this case, the pools with serine or threonine at postion 2 worked best (Fig. 13-12b). The discrimination of the enzyme at position 1 is much weaker. In a third array, the Ac-X-1-2-R-R-A-S-X library was constructed. Here we found that the kinase accepted almost any combination equally well, with a slight preference for Glu at position 2 (Fig. 13-12c). For the last scan in this series the array Ac-1-A-E-R-R-A-S-2 was used (Fig. 13-12d). Here we found that arginine at

382

13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

position 1 together with isoleucine at position 2 worked best, which again is in good agreement with investigations based on soluble peptides [32]. A comparison of the phosphorylation efficiency of sequences that were synthesized in five to ten copies each revealed a scattering of the values with a deviation of up to 30%. About 10% can be attributed to fluctuations in peptide density per spot [unpublished results]. In cases where small differences between several sublibraries are observed, a re-evaluation with such an array where the pools of interest are presented severalfold seems advisable before descisions on the structure of the next sublibraries are being made. We also found that the specificity at a certain position is influenced by the amino acids around it [33], so that the resulting “optimal sequence” that has been determined by such an iterative approach may depend on the search strategy employed and may not necessarily be the best possible one (2.4). In conclusion, this approach seems to be well suited for the determination of substrate motifs of protein kinases. It can be expected that many other enzymatic transformations can be investigated with the SPOT method as well.

13.3.3 Other Applications and Future Developments More applications of peptide library screening on spots described in the literature inion affinities, double-stranded clude the search for, e.g., Ni2+,Ag” ,and 99mT~03+ DNA binding peptides as well as peptide ligands for other protein acceptors such as transforming growth factor-beta (TGFP) [15, 22). With the recent technical and chemical improvements, SPOT synthesis is an easy tool to study molecular recognition phenomena in general thereby including modern aproaches to the generation and screening of complex peptide libraries. Several ten thousands of peptides or pools can be prepared and assayed in a few days, which offers a high flexibility in the development and testing of novel general and acceptor specific combinatorial search strategies. Peptide libraries are much more than just new resources for the identification of biologically active compounds, which satisfy the selection parameters of a given experiment. Beyond that, the recognition pattern (binding affinity or substrate transformation) by an acceptor molecule allows a complete statistic and thermodynamic description of all possible chemical ligand interactions for the building blocks in the set employed. Without presupposing an understanding of these interactions, important molecular details can be derived a priori (“irrational” molecular design, Sydney Brenner). Obviously, recognition patterns on arrays of peptides and peptide pools are characteristic properties of the acceptor molecule itself. This feature is being exploited by us in the development of new analytic and diagnostic assay systems. Synthesis scale is in the nmol range and, thus, also ng to pg of acceptor molecules can be trapped on spots. This should be sufficient for the isolation and identification of unknown acceptor molecules from, e. g., biological fluids or extracts.

13.4 Methods

383

13.4 Methods Derivatization of paper membranes and peptide assembly was essentially as described [2]. Details on array configurations used are given in Table 13-2. All spotting operations were carried out with the Model ASP 222 Synthesizer from ABIMED Analysen-Technik GmbH (Langenfeld, Germany). Instrumental details are provided in the manual.

13.4.1 Peptide Library Assembly In situ prepared N-hydroxybenzotriazole (HOBt) esters of Fmoc-amino acid derivatives in N-methylpyrrolidinone (NMP) were used throughout for coupling reactions. Side chain protection was Cys(Acm), Asp(0t Bu), Glu(0t Bu), His(Trt), Lys(Boc), Asn(Trt), Gln(Trt), Arg(Pmc), Ser(t Bu), Thr(t Bu), Trp(Boc), and Tyr(t Bu). Stock solutions of 0.2 M Fmoc-amino acid derivative plus 0.3 M HOBt in NMP were prepared and stored at -70°C. According to the total volumes of each derivative needed for a complete synthesis, stock solutions were diluted 1 :4 (v/v) with NMP divided into aliquots required for each elongation cycle. An additional part is used to mix amino acid derivatives for X-couplings. Equal parts were combined, diluted as above and devided again into aliquots required for each elongation cycle. All these solutions were stored at -70°C. One set of solutions was activated prior to use in the respective coupling cycle by addition of diisopropyl carbodiimide (DIC) to a final concentration of 0.07 M and was left at room temperature for 0.5 h. Spotting was repeated up to four times until the blue color of the spots changed to yellow. After the final cycle, peptides were N-terminally acetylated as described [2].

13.4.2 Side Chain Deprotection (Must be performed under a hood! Trifluoroacetic acid (TFA) is very aggressive!) 20 ml of a deprotection mix containing 50% TFA, 3% triisobutylsilane, 2 % water and 45% dichloromethane was used per treatment of one membrane. The dryed paper was placed in a tightly closed Teflon reaction trough containing the deprotection mix and agitated for 2 h. After one hour, the solution is replaced with a fresh equal portion. The membrane was then washed each time for 10 min with 20 ml of dichloromethane (four times), dimethylformamide (three times), 1 M acetic acid in water (three times), and methanol or ethanol (three times). The sheet was then dried with cold air and stored at -20°C or processed further as below.

384

13 Combinatorial Synthesis on Membrane Supports by the SPOT Dchnique

13.4.3 Ligand Binding Assay on SPOTS Membranes All incubations were carried out in buffers at pH 7.0! At higher pH the ester linkage to the cellulose membrane is slowly hydrolyzed and peptides are gradually lost. Major modification to the protocol described [2] was the use of alkaline phosphatase(AP) conjugated acceptor molecules. This made the following few changes necessary: color development was carried out in CBS-buffer (10 mM sodium citrate pH 7.0, 8 g/l sodium chloride, 0.2 g/l potassium chloride) and 6-bromo-5-chloro3-indolyl /3-D-galactoside (BCIG) is replaced by 6-bromo-5-chloro-3-indolyl phosphate (BCIP, toluidine salt, Sigma Chemical Co., St Louis, USA) of the same concentration. Prior to color development, the membrane is washed twice with CBSbuffer. During color development the membrane is not agitated! Alkaline phosphatase is active enough at pH 7. The reaction is stopped by washing with phosphate buffered saline (PBS). Membranes were blocked against unspecific protein adsorption by incubation overnight with membrane blocking buffer containing 2 ml blocking buffer concentrate (Genosys Biotechnologies, The Woodlands, Texas, USA), 8 ml T-TBS buffer pH 8, and 0.5 g saccharose, final pH is 7! For the ex. amples described in the text, mouse monoclonal antibody 1D3 (1.3 mg/ml, kindlq donated by J. Wehland, GBF) was applied at a 1 :100 dilution in membrane blocking buffer and was detected by the successive incubation with a 1 :500 dilution in membrane blocking buffer of alkaline phosphatase conjugated goat antimouse IgG (1 mg/ml, affinity pure, Fc,-fragment specific) from DIANOVA (Hamburg, Germany). Steptavidin conjugated with alkaline phosphatase (1 mg/ml) was also from DIANOVA and applied at a 1 :100 dilution in membrane blocking buffer. A developed membrane was placed wet on a glass plate, covered with plastic wrap and scanned with the image analysis instrument (Model DNA 35, pdi, Huntington Station, New York, USA) in the reflecting light mode. Quantification of spot signals after background subtraction with the “Diversity One” software gave tabulated OD data which were then further processed with the programs EXCELTMor COREL DRAWTM(spectral diagrams). During signal documentation and quantification, the membrane must not dry out to avoid denaturation and irreversible sticking of proteins to the paper. Stripping off dye and proteins for reuse of the membrane was as described [2).

13.4.4 Enzymatic Phosphorylation The paper with the peptide array is wetted with a few ml of ethanol, washed with 50 ml of buffer A (50 mM MOPS, 200 mM NaCL, 1 mM magnesium acetate, 0.4 mM EGTA and 1 mg/ml BSA, pH 6.9) and kept overnight in 200 ml of this buffer. After removing the buffer, 8 ml of fresh buffer are added and the mixture is preincubated at 30°C. 100 ul of 10 mM ATP. 100 uCi CV-~*PIATP and the catalvtic subunit of

References

385

CAMP-dependent protein kinase at a final concentration of 200 ng/ml are added. The paper is incubated for 10 min at 30°C with slight aggitation. The buffer is decanted off and the paper is washed repeatedly (at least ten times) with 100 ml each time of 1 M NaCl. 100 ml of a 8 M solution of guanidinium hydrochloride containing 1% SDS and 0.5% mercaptoethanol are added and the paper is treated for 1 h ar 40°C in an ultrasonic bath to remove background. The paper is washed several times with water and ethanol and dried. Radioactivity is determined with the PhosphorImagerTMsystem (Molecular Dynamics). Radiation times of the screen are in the range of a few hours. Quantitative evaluation of the measurements are carried ou1 with the program ImageQuantTMby placing equally sized circles that are slightly smaller than the smallest spot onto every position of the array.

References [I] R. Frank, S. Giiler, S. Krause, W. Lindenmaier, Peptides 1990 (Eds.: E. Giralt, D. Andreu), ESCOM Leiden, 1991, p. 151. (21 R. Frank, Tetruhedron 1992, 48, 9217-9232. 131 R. Frank, W. Heikens, G. Heisterberg-Moutses, H. Blticker, Nucl. Acids Res. 1983, 11,

4365-4377. [4]R. Frank, R. Dtiring, Tetmhedron 1988, 44, 6031-6040. 151 H. M. Geysen, R. H. Meloen, S. J. Barteling, Proc. Natl. Acud. Sci. USA 1984, 81, 3998-4002. (6)G. Schnorrenberg, H. Gerhard, Tetrahedron 1989, 45, 7759-7764. [7]A. Holm, M. Meldal, Peptides 1988 (Eds. : G. Jung, E. Bayer), de Gruyter, Berlin, 1989, p. 208. [8]H. Gausepohl, M. Kraft, C. Boulin, R. W.Frank, Solid Phase Synthesis (Ed.: R. Epton), SPCC, Birmingham, UK. 1990, p. 487. (91 J. A. Buettner, D. Hudson, C. R. Johnson, M. J. Ross, K. Shoemaker, Solid Phase Synthesis (Ed.: R. Epton) Mayflower Worldwide, Birmingham, UK, 1994, p. 169. [lo] G. Jung, A. G. Beck-Sickinger, Angew. Chem. 1992, 104, 375-391. [Ill S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 1991 251, 767-773. (121 U. Maskos, E. M. Southern, Nucl. Acids Res. 1992, 20, 1679-1684. [I31 R. Frank, Solid Phase Synthesis (Ed.: R. Epton) Mayflower Worldwide, Birmingham UK, 1994, p. 509. 114) S. Adler, R. Frank, A. Lanzavecchia, S. Weiss, FEBS Letters 1994, 352, 167-170. [IS] A. Kramer, R. Volkmer-Engert, R. Malin, U. Reineke, J. Schneider-Mergener, Pept. Res 1993, 6, 314-319. [16]S. A. Kauffman, W. G. Macready, J. Theor. Biol. 1994, in press. [I71 G. B. Fields, R. L. Noble, Int. J. Peptide Protein Res. 1990, 35, 161-214. (181 A. M. Bray, N. J. Maeji, H. M. Geysen, Tetrahedron Lett. 1990, 31, 5811-5814. 1191 A. M. Bray, N. J. Maeji, A. J. Jhingran, R. Valerio, Tetrahedron Lett. 1991, 32 hlhl-hl(iti

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13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique

[20] S. Hoffmann and R. Frank, Tetrahedron Lett. 1994, 35, 7763-7766. [21] S. Hoffmann, R. Frank, in preparation. [22] A. Kramer, A. Schuster, U. Reineke, R. Malin, R. Volkmer-Engert, C. Landgraf, J. Schneider-Mergener. Methods (Comp. Meth. Enzymol.) 1994, 6, 912-921. [23] V. Krchnak, J. Vagner, P. Safar, M. Lebl, Collect. Czech. Chem. Commun. 1988, 53, 2542. [24] H. M. Geysen, S. J. Rodda, T. J. Mason, MO/.Immunol. 1986, 23, 709-715. 1251 A. Furka, F. Sebestyen, E. CLmpian, “Peptide sub-Library kits” in Solid Phase Synthesis (Ed.: R. Epton), Mayflower Worldwide Ltd., Birmingham, UK, 1994, p. 385. [26] C. Pinilla, J. R. Appel, P. Blanc, R. A. Houghten, BioTechniques 1992, 13, 901-905. [27] C. T. Dooley, R. A. Houghten, Life Sci. 1993, 52, 1509-1517. 1281 J. J. Devlin, L. C. Panganiban, P. E. Devlin, Science 1990, 249, 404-406. [29] K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmiersky, R. 1. Knapp, Nature (London) 1991, 354, 82-84. [30] K. Ersfeld, J. Wehland, U. Plessmann, H. Dodemont, V. Gerke, K. Weber, J Cell Biol. 1993, 120, 725-732. (311 B. E. Kemp, R. B. Pearson, Trends Biochem. Sci. 1990, 15, 342-346. I321 J. D. Scott, M. B. Glaccum, E. H. Fischer, E. G. Krebs, Proc. Nutl. Acad. Sci. USA 1986, 86, 1613- 1616. I331 W. Tegge, R. Frank, F. Hofmann, W. R. G. Dostmann, Biochemistry 1995, 34, 10569- 10577.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

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

14.1 Introduction Historically, the most successful approach for t a x discovery of lead structures for drug development has been the broad screening of natural products. Lead compounds for pharmaceuticals have been obtained from plants, marine organisms, fungi or other microorganisms. In addition, the screening of collections of organic compounds or the modification of bioactive peptides have been used for the generation of drug candidates [l, 21. Over the past few years, automated and combinatorial methods for the rapid generation of diverse mixtures (“libraries”) of peptides, nucleic acids and organic molecules have been developed [3, 41. Highly diverse sets of large collections of biooligomers or organic compounds should have an increased probability to comprise a few molecules that bind to a specific pharmaceutical target like an enzyme or a receptor. The approach used here for the generation of molecular diversity [5] is based on the fully automated, high-through synthesis of N-substituted glycine (NSG) peptoid libraries. Equimolar mixtures of nonnatural oligomers are generated with precise control over the composition of the synthesized libraries. The diverse peptoid mixtures are screened in the solution phase to obtain binding data that are as accurate as possible.

14.2 Criteria and Goals for the Generation of Molecular Diversity To generate a large number of different compounds in a short period of time, automation of the synthetic process is highly desirable. A modular approach for the generation of molecular diversity limits the number of the required synthetic manipulations and thus greatly simplifies automation. Consequently, it was decided to take advantage of a simple, high-yielding linking chemistry for the assembly of oligomeric structures.

388

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concept

The building blocks for the intended modular approach should have a wide variety of structurally and functionally diverse side chains. Structural redundancy within components of the library can be avoided by rational selection of the incorporated monomers. The use of readily available building blocks for the oligomers would overcome the costly and time-consuming process of synthesizing large amounts of monomers prior to assembly of the libraries. Polymer-supported chemistry is particularly attractive for the generation of diverse mixtures with equimolar concentrations of the individual components. In contrast to solution phase chemistry, the attachment of the substrate to a matrix allows for the easy separation of the substrate and ensures that: (a) all reagents can be washed away after each synthesis step is completed. This allows the use of excess reagents or even the repetition of each reaction step to drive each reaction to completion. Consequently, equimolarity can be achieved despite some differences in the reactivity of the individual substrates and reagents; (b) the substrate molecules are spatially separated from one another (“pseudodilution”) [6]. Consequently, intermolecular side reactions are suppressed efficiently, resulting in more homogeneous reaction products. Moreover, reagents with multiple reaction sites react more selectively with a polymer-bound substrate (e.g., the reaction of a primary amine, a symmetrical diamine, etc., with a resin-bound electrophile often provides a single reaction product, as compared to a more complex reaction mixture for the same reaction using an electrophile in solution). (c) automation can be developed for almost all manipulations; (d) by recombining and splitting the resin after each reaction step, combinatorial methods can be used for the generation of a large number of compounds. A suitable resin splitting scheme would allow for the synthesis of equimolar mixtures with precise control over the composition of the libraries. Mixture equimolarity greatly simplifies the evaluation of binding data [7]because all assays will reflect normalized affinities of the tested pools. When synthetic libraries are used for the discovery of biologically active compounds, all initial assay data are generated from pools of compounds rather than single molecules. Therefore, the molecular diversity approach to drug discovery requires the retrogressive identification of the bioactive components. This has been accomplished (a) by affinity selection (7, 81, (b) by iterative resynthesis of successively smaller subpools [9], (c) by attaching a tag that codes for the synthesis of the molecule [lo- 13) or (d) by having the molecules attached to a matrix and using the location (“address”) on the matrix [14] or microsequencing [15] for their identification. The use of a linker that attaches the potential ligand to a matrix or tag, however, holds the risk of altering the structural characteristics of the individual molecules. Iterative resynthesis may be more labor-intensive than a decoding process, but allows for the identification of bioactive, structurally unmodified components from complex, diverse mixtures.

14.3

The Peptoid Approach

385

As a consequence of the availability of optimized protocols for solid phase peptide and nucleotide synthesis, all the initial work on synthetic molecular diversity was accomplished with peptide libraries [16].In general, however, peptides suffer from poor oral bioavailability and rapid metabolic inactivation [ 171. Therefore, the design of unnatural analogs of peptides [18]that possess increased plasma stability, better absorption characteristics and can be used for controlled oligomerization appeared particularly promising.

14.3 The Peptoid Approach Peptoids are nonnatural oligomers that contain N-substituted glycines (NSGs) as their structural motif [19].Peptoid oligomers are achiral and possess comparable spacing of the side chains (and the amide bonds) as their natural analogs (Fig. 14-1). In addition, the nature of the peptoid backbone is very similar to the nature of the peptide backbone. However, peptoids are devoid of amide protons, which decreases their overall polarity and should increase their oral bioavailability. Since they contain only tertiary amide bonds, they are not subject to degradation by common proteases [20].

altered conformation no H-bond donor Figure 14-1. General structure of an NSG pentapeptoid. The arrows indicate some major differences of NSG peptoids as compared to peptides.

Since this novel class of molecules is based on an oligomeric glycine backbone, peptoids were expected to have a similar conformational profile as glycine. The unsubstitued a-carbon should allow almost unhindered rotation about the phi and psi angles. In addition, both cis- and trans- conformers of the amide bond should be accessible at room temperature. However, the substituent on the nitrogen is expected to confer different structural properties on peptoids that might limit their flexibility. Figure 14-2 shows the Ramachandran plots for a common model of a peptide (Ac-Ala-NHMe) as compared to a common model of a peptoid (Ac-Sar-NMe,) [21].These maps were generated using the SM2-AM1semiempirical method with an implicit solvent term [22,231. Figure 14-2a (left) shows the traditional Ramachan-

390

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concept

dran plot [24] for Ac-Ala-NHMe. As expected, the beta-sheet region in the upper left-hand corner of the map is seen as a large area of conformations within 2 kcal/mol of the minimum. The right-handed alpha helix (phi = 100, psi = 60)is only 0.5 kcal/mol higher in energy than the beta-sheet region. The minimum corresponding to the C7-axial conformation (phi = +60, psi = -60) is approximately 2 kcal/mol higher in energy than the other two minima. Figure 14-2b (right) shows the Ramachandran plot for the trans-amide conformer of Ac-Sar-NMe,. Since the NSG peptoid backbone contains no stereocenter, there is an axis of symmetry in this plot. The minima for peptoids are clearly in a different region of the map than are the minima for peptides. The lowest energy conformation for Ac-Sar-NMe, corresponds to a phi angle = k90" and a psi angle that is 180". This is also true for the conformer with a cis-amide bond (data not shown). A second minimum, about 2 kcal/mol higher in energy, is found with phi and psi angles of approximately ( - 120, + 90) and ( + 120, - 90). A third minimum at ( - 60, -60) is 3 kcal/mol higher in energy.

Ac-Ala-NHMe

Ac-Sar-NMeo

0 0 0 0 0 0 0 0 0 0 0 0 0 (Qln"cycp'9c? c9oa)"cyV)Q)

7 7 7

F F F

Phi (C-N-Ca-C)

.

.,

.*

-,

Phi (C-NCa-C)

Figure 14-2. Ramachandran plots of Ac-Ma-NHMe (left)and the trans-amideconformerof Ac-Sar-NMe2 (right).

It is striking that the energy minima are so well defined. This shows that peptoids possess a different conformational profile than glycine. The different minimum energy positions suggest that peptoids sample a variety of backbone conformations that are inaccessible to peptides. The side chains are expected to be presented in entirely different manners as well. A direct comparison of the structural features of peptides and peptoids is shown in Fig. 14-3. The oligomers are aligned in a way that maintains the relative orientation of the amide-oxygen atoms and the side chains.

14.4 Synthesis of NSG Peproids

391

N-terminus

O

R

0

peptides

C-terminus O

0

R

peptoids

Figure 14-3. Sequence alignment of peptides and peptoids. To maintain the relative orientation of the amide-oxygen atoms and the side chains, the direction of the peptoid chain had to be reversed.

14.4 Synthesis of NSG Peptoids The initial approach for the synthesis of NSG peptoids was analogous to conventional solid phase peptide synthesis and based on the condensation of N-Fmoc-protected, N-substituted glycines [19]. Using the standard Fmoc protocol and PyBOP or PyBroP activation of the monomers, oligomeric peptoids were assembled in good yields and high purity. The NSG monomers had to be synthesized prior to assembly of the libraries and were obtained by alkylation of primary amines with electrophiles (e.g., haloacetic acids and acrylamides) or by reductive amination of primary amines with aldehydes (e.g., glyoxylic acid) [19].

H\N-@

I

R

* DIC, DMF

N

I

R

Figure 14-4. Synthesis of NSG peptoids using the submonomer method.

392

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepi

In a recent synthetic advance, it was possible to improve the efficiency of the synthesis for a large variety of NSG peptoid oligomers. Regarding NSG peptoids as alternating copolymers of primary amines and acetate units, a protocol was developed that allows for the assembly of peptoids from the readily available building blocks (“submonomers”) bromoacetic acid and primary amines [25]. As shown in Fig. 14-4,NSG peptoid oligomers can be synthesized using acylation reactions and S,Z-reactions in an alternating fashion, without the need to synthesize N-Fmocprotected monomers.

(4

?L--J

9 “-qqw2

47

0

9

~

0

20 Time (min)

40

0

20 Time (min)

40

0

20 Time (min)

(b)

(4

9&+* ! ! %

40

I . ,

0

20

,

;~

~

40

Time (min) Figure 14-5. HPLCchromatograms of four crude peptoid pentamers; each sample gave the expected peak in the ESI mass spectrum (data not shown). Prior to incorporation into a peptoid library. each amine was tested for its ability to incorporate into a Dentamer.

14.4 Synthesis of NSG Peproids

393

The submonomer approach requires the protection of side chain functionalities like carboxyl-, hydroxyl-, amino- and thiol-groups; phenols can be used without protection. Using optimized reaction conditions, it was possible to assemble homoand heteropentamers in greater than 80% purity from a wide variety of commercially available amines (Fig. 14-5). Moreover, a 24-mer (N-methoxyethylglycine-N-benzylglycine),,, was obtained in 60% yield (crude material) and in > 70% purity [26], thus further demonstrating the efficiency of the method (Fig. 14-6).

Time (min)

1055.7

1583.4

L 1400

lo00

m/z Figure 14-6. HPLC-chromatogram and ESI mass spectrum of a crude NSG-peptoid 24-mer N-methoxyethylglycine-N-benzylglycine),2.The mass spectrum shows the peaks corresponding to the doubly and triply charged species.

In summary, the submonomer protocol allows for the efficient synthesis of equimolar mixtures of NSG peptoid oligomers with a wide variety of side chain functionalities. The high-yielding, reproducible linking chemistry, combined with the structural and functional diversity of the monomers that can be incorporated, renders the peptoid approach ideally suited for the automated generation of molecular diversity.

394

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepi

14.5 Automated Synthesis of Equimolar Peptoid Mixtures The high-throughput synthesis of diverse peptoid libraries is performed by a robotic workstation of our own design [27]. The apparatus is capable of performing all the required synthetic and resin-splitting manipulations. The key features of the instrument have been described in detail [27] ; some of the most significant characteristics can be summarized as follows. (i) The workstation consists of a Zymate XP robot that is interfaced with a Macintosh computer [28]. The robotic arm delivers solvents and reagents from pressurized lines into a 6 x 6 array of reaction vessels (Fig. 14-7). (ii) The individual reactions are performed on polystyrene beads with acid-labile linkers. The resin is placed into 36 1.5 x 10 cm fritted glass reaction vessels. Bubbling with argon is used to ensure proper agitation of the reagents and the resin particles. Moreover, the argon provides a blanket that shields the reaction mixture from water and oxygen. (iii) The reaction vessels are mounted on a custom-designed aluminum block that can be heated to 100°C. Reagents and solvents are added through the open top of the reaction vessels. Argon delivery to, and solvent removal from, the reaction vessels is accomplished by applying vacuum or argon pressure to the bottom of the reaction Multi-spigot \

Extraction Station

Waste

Pressurized Solvent Lines

Figure 14-7. Schematic view of the automated workstation for the synthesis of equimolar peptoid mixtures and the integrated cleavage/deprotection station.

14.5 Automated Synthesis of Equimolar Peptoid Mixtures

395

vessels. The latter operations are controlled by three-way Teflon solenoid valves that are interfaced with the Macintosh computer. (iv) The distribution and recombining of the solid support (“resin splitting”) is performed after generating a free-flowing, isopycnic slurry of the resin in 1,2-dichloroethane/DMF. Resin splitting that allows for precise control over the composition of the libraries is accomplished using the scheme summarized in Fig. 14-8. Syringes are used to measure equal volumes of the slurry into the reaction vessels so that equimolar amounts of resin are transferred. By repeating each of the resin transfer steps three times, virtually qunatitative transfer of the resin particles is achieved.

-

Distribute into

1

..................

2

To each separate portion: (1) acylate with bromoacetic acid (2) displace with primary amhe

............ bn

Figure 14-8. Resin-splitting scheme for the generation of equimolar peptoid mixtures.

After completion of the synthesis, the peptoid libraries are detached from the solid support using standard, trifluoroacetic acid-based cleavage protocols. An automated cleavage station has been developed that greatly increases the library throughput [29]. Prior to assay, manual lyophilization of the crude samples is required.

396

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concept

14.6 Rational Approaches for Library Design and the Generation of Structural Diversity More than 13000 primary amines are listed in the Available Chemicals Database [30]. Over loo0 of these are inexpensive (priced less than SS.OO/g) and suitable for submonomer synthesis. In a set of peptoid trimers, more than lo9 different combinations can be generated with these inexpensive amines alone. If the synthesis of tetramers or modifications of the C- and N-termini are also taken into account, the number of possible combinations greatly exceeds that which can be handled by automated synthesis or automated binding assays. To limit the library synthesis to a reasonable number for assays and deconvolution, some a priori selections have to be made. As a consequence for the conformational flexibility of the backbone, an NSG peptoid library will be capable of accessing a variety of conformational states. Therefore, the individual side chains can be arranged in many different orientations. However, structural and functional redundancy of NSG peptoid libraries can be kept to a minimum by maximizing diversity among the monomeric building blocks. In addition, this strategy should suppress the occurence of “false positives” in the assays that could arise from the combined effects of several structurally related ligands (with only moderate affinity for each individual ligand). Maximally dissimilar building blocks are used for the synthesis of NSG peptoid libraries that are intended for random screening. Due to the conformational flexibility around the a-carbon atoms, and the accessibility of both cis- and trans-amide bonds, even a peptoid library consisting of a limited number of dissimilar building blocks should be able to cover a significant sector of a conformational and functional continuum. For these reasons, random screening of peptoid libraries should have a good chance of success even if the number of the incorporated, diverse monomers is limited. Following a semirational, target-oriented approach, libraries can be directed towards specific enzymes or receptors. This can be accomplished by including side chains containing functional units, e.g., transition-state analogs for the generation of lead structures for enzyme inhibitors. Alternatively, monomers that contain structural units which are known to commonly occur in ligands for certain classes of receptors can be used to bias the library. Maximally dissimilar monomers are also incorporated into these libraries to present the biased elements in many different geometries and chemical environments. Computational tools are used to determine and measure similarity and dissimilarity between monomers by computing a variety of structural and functional properties including lipophilicity, shape, branching and “atom layer properties” (roughly speaking, the number of bonds separating any functionality from the closest atom of the backbone) 1311. Each building block is remesented by a vector

14.7 Peptoid tigands with Nanomolar Affinity

397

of 16 numerical descriptors that summarize these structural and functional characteristics. Sets of monomers that are highly similar to a known pharmacophore, or to one another, are obtained by simply rank ordering every member of the pool by the Euclidean distance from a reference structure. Maximally dissimilar compounds are found with D-optimal design [32]. The algorithm chooses subsets of points from a larger pool that are well spread out and largely orthogonal in property space, i.e., that are maximally dissimilar. Any number and combination of monomers can initially be included (to serve as starting points) in the set. Subsequently, additional points that best fill out the set to a specified size will be selected by the optimizer. In summary, strategies and methods have been developed for selecting monomer subsets for peptoid library synthesis. Using computational tools, we maximize dissimilarity within the monomeric building blocks, thereby increasing the structural diversity and minimizing the structural overlap in our libraries. The methods and tools described here can also be used in biased libraries, combining approaches that rely on intuition, experience and on the incorporation of known pharmacophores with computational strategies for the generation of molecular diversity.

14.7 Peptoid Ligands with Nanomolar Affinity for Adrenergic and Opiate Receptors 14.7.1 Design of a Biased Library for 7-Traosmembrane/G-Protein Coupled Receptors A substantial amount of therapeutically relevant drugs bind to 7-transmembrane/

G-protein coupled receptors [33]. Since amino acid sequences of this class of receptors are highly homologous (341, ligands to this receptor family frequently share common pharmacophores. An analysis of structural elements of known drugs revealed that most of the ligands contained at least one hydroxyl or phenol group as hydrogen donor and one hydrophobic, aromatic group [35]. For the synthesis of a biased peptoid library, three sets of building blocks were selected from commercially available amines [35]. Set “A” was composed of 4 aromatic, hydrophobic amines, set “0”of 3 amines with side chains containing hydroxyl or phenol groups, and set “D”was designed to comprise 17 maximally diverse amines (Table 14-1). In addition, the N-termini of the NSG peptoids were either left as a free amine or capped as an acetamide or cyclohexylurea; the C-terminus of all compounds was fixed as a primary amide. The complete library contained 3672 peptoid trimers (18 combinatorial mixtures of 3 x 4 x 17 = 204 compounds). Moreover, all possible dimers were included into the library.

398

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepr

Table 14-1. Each of the 18 peptoid pools was made from all combinations drawn from the six permutations of three sets of amines, times three different N-terminal endings. Thus, each trimer contained at least one hydroxylic and one aromatic group.

N-Terminal Capping Groups

HF

Ha&

Free Amine

Acetamido

Cyciohexyiureido

Hydroxyl Set of Sidechains (0) -CH2CH2CH2CH20H

Aromatic Set of Sidechains (A) . .

Diverse Set of Sidechains (D) I

14.7.2 Identification of Peptoid Ligands with Naaomolar Affinity The equimolar NSG peptoid mixtures were screened for their ability to competitively inhibit bindinn of hieh-affinitv radiolkands to 7-transmembrane/G-~roteincouded

14.7 Peptoid Ligands with Nanomolar Affinity

399

receptors. Assays were performed in aqueous solution containing 0.1 to 1070 DMSO at a concentration of 100 nM for each individual compound (351. All 18 pools were tested for inhibition of [3H]-prazosin binding (361 to an a , adrenergic receptor preparation. The pool that showed greatest inhibition of [3H]prazosin binding was subjected to the subsequent cycle of deconvolution. In the third round of deconvolution, CHIR 2279 (Fig. 14-9) was resynthesized in pure form and showed competitive inhibition of ['HI-prazisin binding with a Ki = 5 f 3 nM. CHIR 2276 and CHIR 2283, two closely related peptoids, also inhibited [3H]-prazosin binding at nanomolar concentrations (Ki = 310 nM for CHIR 2276 and Ki = 140 n M for CHIR 2283; structures not shown) [35]. Similarly, the 18 NSG peptoid pools were assayed for inhibition of [3H]-DAMG0 @-specific) binding [37] to opiate receptors. After deconvolution and individual resynthesis of the three most potent compounds [35], CHIR 4531 (Fig. 14-9) was shown to inhibit [3H]-DAMG0binding with a Ki = 6 2 nM. Two related peptoids, CHIR 4537 and CHIR 4534, had Ki values of 31 nM and 46 nM (structures not shown).

*

Q

OH

6H

CHIR 2279 CHlR 453 1 Figure 14-9. Structures of CHIR 2279 and CHIR 4531, two nanomolar ligands for 7-transmembrane/G-protein coupled receptors.

14.7.3 Discussion Using a relatively small, biased library of NSG peptoid trimers, it was possible to identify novel, low-molecular weight ligands with high affinity for two pharmaceutically relevant receptors. Although an analysis of common pharmacophores for 7-transmembrane/G-protein coupled receptors served as the starting point for library design, the newly discovered ligands represent an entirely novel class of biologically active molecules. The peptoid trimers CHIR 2279 and CHIR 4531 are

400

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepi

capable of competively inhibiting binding of endogenous ligands to their receptors, despite their complete lack of chiral elements. Both CHIR 2279 and epinephrinehorpinephrine, the endogenous ligands for the a,-adrenergic receptor, contain a substituted tyramine derivative; however, the hydroxyl group of CHIR 2279 apparently is not required for biological acitvity, as evidenced by the high affinity of its deshydroxy analog (Ki = 4 nM). Other than that, CHIR 2279 shares few structural elements with either epinephrinehorepinephrine or prazosin (Fig. 14-10). CHIR 4531 inhibits binding of the natural ligands morphine and Met-enkephalin to the p-opiate receptor. Although some structural elements, like the substituted tyramine or a hydrophobic ring, are present in CHIR 4531 and the two natural ligands (Fig. 14-10), the different backbone, the lack of chirality and the nature of the aromatic side chains of CHIR 4531 clearly distinguish the peptoid lead from the natural ligands. The discovery of novel, high affinity ligands for 7-transmembrane/G-protein coupled receptors is the result of a multidisciplinary effort. Key factors that con-

n

OH NHp

epinephrine : R = CH3 norepinephrine: R = H

prazosin

(b)

Met-enkephalin morphine Figure 1410. Structures of known ligands for the a,-adrenergic receptor and the p-opiate receptor.

14.8 Summary

401

tributed to success were a general, highly reproducible protocol for the synthesis of nonnatural oligomers with a large variety of structurally and functionally different side chains; the availability of suitable automation for synthesis and deconvolution of equimolar peptoid mixtures; and efficient strategies for library design under consideration of the conformational characteristics of NSG peptoids.

14.8 Summary Since the concept of molecular diversity was introduced into pharmaceutical research, the research field has quickly expanded. Initially, the generation of diverse libraries was accomplished with biomolecules like peptides and nucleic acids, but more and more examples of libraries of non-natural oligomers and organic molecules have been described [38]. The ultimate goal of pharmaceutical research is the discovery of small molecule drug leads and drug canditates. To realize this goal, the structural diversity of the libraries is maximized. Computational tools have been developed that allow for the minimization of structural and functional redundancy of the building blocks that are incorporated. These methods can be applied to “diversify” libraries for random screening and to reduce overlap around constant units in biased libraries. In addition, synergistic effects of similar ligands (with only moderate affinity for each of the ligands) are kept to a minimum. Suitable automation allows for the high-throughput synthesis of equimolar peptoid mixtures with precise control over the composition of the libraries. A highyielding, reproducible protocol has been developed for the synthesis of diverse, unnatural oligomers from inexpensive, readily available building blocks. To obtain binding data that are as accurate as possible, all the libraries are screened in solution phase. Iterative resynthesis of successively smaller subpools is used for the identification of bioactive components. Like library production, the deconvolution process is greatly accelerated by automation. By using this approach for lead identification, the covalent attachment of molecules to a matrix or a tag which could structurally interfere with the binding to the pharmaceutical target is avoided. The strategies summarized here have been applied both to the synthesis of random mixtures and to the synthesis of libraries that are focused towards specific pharmaceutical targets. The design and synthesis of an NSG peptoid library which was biased towards 7-transmembrane/G-protein coupled receptors resulted in the discovery of novel, low-molecular weight ligands with nanomolar affinity for the a,-adrenergic and the p-opiate receptor. Since achiral, low-molecular weight peptoid trimers have the ability to competively inhibit binding of potent, endogenous ligands, the peptoid approach holds enormous potential for the discovery of novel lead structures for drug development.

402

14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepi

14.9 Experimental Procedures 14.9.1 Standard Protocol for the Synthesis of NSC Peptoids with C-terminal Amides Using the Submonomer Method [40] 14.9.1.1 Bromoacetylation of Rink-Amide-Resin and the N-terminal Amine of an NSG Peptoid Chain

After standard deprotection of N-Fmoc-Rink amide resin [39] with 20% piperidine in DMF, the resin (96 mg, 0.52 mmol/g) was extensively washed with DMF (5 x 1 ml) and drained. A 0.6 M solution of bromoacetic acid in DMF (830 pl) and subsequently a 3.2 M solution of diisopropylcarbodiimide in DMF (200 pl) was added, and the resin was agitated with argon. The reaction mixture was agitated at room temperature for 30 minutes, washed with DMF (3 x 1 ml), and the reaction was repeated once under identical conditions. 14.9.1.2 Displacement of the Bromide of Resin-Bound Bromoacetamides with Primary Amines

The resin-bound bromoacetamide was washed with DMSO (3 x 1 ml), and 1.0 ml of 2.0 M solution of the primary amine in DMSO was added. For amines with poor solubility in DMSO such as tyramine, a 1.0 M solution of the amine in DMSO was used instead. The reaction mixture was agitated with argon for 2 h at room temperature. Subsequently, the resin was washed with DMF (3 x 1 ml). 14.9.1.3 Cleavage of the Peptoid/Peptoid Mixture from the Solid Support

After all synthetic manipulations were completed, the resin was washed with DMF (3 x 1 ml), dichloromethane (3 x 1 ml) and dried. 95% aqueous TFA (5.0 ml) was added to the resin particles, and the mixture was stirred for 20 min at room temperature. The solid support was removed by filtration and washed with water (2 x 2.5 ml). The crude samples were lyophilized, followed by relyophilization from glacial acetic acid.

References [I] R. Hirschmann, Medicinal chemistry in the golgen age of biology: Lessons from steroid and peptide research. Angew. Chem. Int. Ed. Engl. 1991, 30, 1278-1301. [2] A. A. Patchett, Excursion in drug discovery. J. Med. Chem. 1993, 36, 2051-2058. [3] M. A. Gallop, R. W. Barrett, W.J. Dower, S. P. A. Fodor, E. M.Gordon, Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J. Med. Chem. 1994. 37. 1233-1251.

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[4) E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, M. A. Gallop, Applications of combinatiorial technologies to drug discovery. 2. Combinatorial organic synthesis, library screening strategies, and future directions. J. Med. Chem. 1994, 37, 1385- 1401. (51 W. H. Moos, G. D. Green, M. R. Pavia, Recent advances in the generation of molecular diversity. Annu. Rep. Med. Chem. 1993, 28, 315-324. [6] S. A. Kates, N. A. Sole, C. R. Johnson, D. Hudson, G. Barany, F. A. Albericio, Novel, convenient, three-dimensional orthogonal strategy for solid-phase synthesis of cyclic peptides. Tetrahedron Lett. 1993, 34, 1549- 1552 and references cited therein. [7] R. N. Zuckermann, J. M. Kerr, M. A. Siani, S. C. Banville, D. V. Santi, ldentification of highest-affinity ligands by affinity selection from equimolar peptide mixtures generated by robotic synthesis. Proc. Natl. Acad. Sci. USA 1992, 89, 4505-4509. (8) J. M. Kerr, S.C. Banville, R. N. Zuckermann, Identification of antibody mimotopes containing non-natural amino acids by recombinant and synthetic peptide library affinity selection methods. Bioorg. Med. Chem. Lett. 1993, 3, 463-468. [9] R. A. Houghten, C. Pinilla, S. E. Blondelle, J. R. Appel, C. T. Dooley, J. H. Cuervo, Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature (London) 1991, 354, 84-86. [lo] S. Brenner, R. A. Lerner, Encoded combinatorial chemistry. Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383. Ill] J. M. Kerr, S. C. Banville, R. N. Zuckermann, Encoded combinatorial peptide libraries containing non-natural amino acids. .l Am. Chem. Soc. 1993, 115, 2529-2531. (121 M. C. Needles, D. G. Jones, E. H. Tate, G. L. Heinkel, L. M. Kochersperger, W. J. Barrett, M. A. Gallop, Generation and screening of an oligonucleotide-encoded synthetic peptide library. Proc. Natl. Acad. Sci. USA 1993, 90, 10700-10704. [13] 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. USA 1993, 90, 10922-10926. [14] S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Light-directed, spatially addressable parallel chemical synthesis. Science 1991, 251, 767-773. [I51 K. Lam, S. Salmon, E. Hersh, V. Hruby, W. Kazmiersky, R. A. Knapp, A new type of synthetic peptide library for identifying ligand-binding activity. Nature (London) 1991, 354,8244. [I61 G. Jung, A. G. Beck-Sickinger, Multiple peptide synthesis methods and their applications. Angew. Chem. In?. Ed. Engl. 1992, 31, 367-486. [17] J. J. Plattner, D.W. Norbeck, “Obstacles to drug developement from peptide leads” in Drug Discovery khnologies (Eds.: C. R. Clark, W. H. Moos) Elllis Horwood Limited: Chichester, England, 1990, pp. 92- 126. (181 R. N. Zuckermann, The chemical synthesis of peptidomimetic libraries. Curr. Opin. Struct. Biol. 1993, 3, 580-584. (191 R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewel], S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, A. D. Frankel, D. V. Santi, F. E.Cohen, P. A. Bartlett, Peptoids: A modular approach to drug discovery. Proc. Natl. Acad. Sci. USA 1992, 89, 9367-9371. [20] S. M. Miller, R. J. Simon, S. Ng, R. N. Zuckermann, W. H. Moos, Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid and N-substituted nlvcine DeDtide and DeDtoid olinomers. Drug Dev. Res. 1995. 35. 20-32.

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14 Automated Synthesis of Nonnatural Oligomer Libraries: The Peptoid Concepi

[21] D. C. Spellmeyer, unpublished results. [22] C. J. Cramer, D. G. Truhlar, General parameterized SCF model for free energies of solvation in aqueous solution. J. Am. Chem. Soc. 1991, 113, 8305, 9901 (E). [23] C. J. Cramer, D. G. Truhlar, AMI-SM2 and PM3-SM3 Parameterized solvation models for free energies in aqueous solution. J. Computer Aided Mol. Design 1992, 6,629-666. [26) G. N. Ramachandran, V. Sasisekharan, Adv. Prot. Chem. 1968, 23, 283-438. (251 R. N. Zuckermann, J. M. Kerr, S. B. H. Kent, W. H. Moos, Efficient method for the preparation of peptoids [oligo(N-substituted glycines)] by submonomer solid-phase synthesis. J. Am. Chem. Soc. 1992, 114, 10646-10647. [26] R. N. Zuckermann. D. A. Goff, Synthesis of (N-substituted) glycine polymers of defined sequence and length. ACS Polymer Preprint 1994, 35, 975-976. [27] R. N. Zuckermann, J. M. Kerr, M. A. Siani, S. C. Banville, Design, construction and application of a fully automated equimolar peptide mixture synthesizer. Int. J. Pept. Protein Res. 1992, 40, 497-506. (281 R. N. Zuckermann, M. A. Siani, S. C. Banville, Control of the zymate robot with an external computer: construction of a multiple peptide synthesizer. Lab. Robotics Automation 1992, 4, 183-192. [29] R. N. Zuckermann, S. C. Banville, Automated peptide-resin deprotection/cleavage by a robotic workstation Pept. Res. 1992, 5 , 169-174. [30) Available Chemicals Directory 93.1, Molecular Design Limited, San Leandro, CA, 1993. [31] E. J. Martin, J. M. Blaney, M. A. Siani, D. C. Spellmeyer, A. K. Wong, W. H. Moos, Measuring Diversity: experimental design of combinatorial libraries for drug discovery. J. Med. Chem. 1995, 38, 1431-1436. [32] V. V. Federov, Theory of Optimal Experiments, Academic Press: New York, 1972. [33] M. N. Potenza, G. F. Graminski, M. R. Lerner, A method for evaluating the effects of ligands upon G-protein-coupled receptors using a recombinant melanophore-based bioassay. Anal. Biochem. 1992, 206, 315-322. [34) W. C. Probst, L. A. Snyder, D. I. Schuster, J. Brosius, S. C. Sealfon, Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 1992, 11, 1-20. (351 R. N. Zuckermann, E.J. Martin, D. C. Spellmeyer, G. B. Stauber, K. R. Shoemaker, J. M. Kerr, G. M. Figliozzi, D. A. Goff, M. A. Siani, R. J. Simon, S. C. Banville, E. G. Brown, L. Wang, L. S. Richter, W. H. Moos, Discovery of nanomolar ligands for 7-transmembrane G-protein coupled receptors from a diverse (N-substituted)glycine peptoid library. J. Med. Chem. 1994,37, 2678-2685. [36] P. B. M. W. M. Timmermans, E K. Ali, H.Y. Kwa, A. M. C. Schoop, E P. SlothorstGrisdijk, P. A. v. Zwieten, Identical antagonist selectivity of central and peripheral alphal-adrenoceptors. Mol. Pharmacol. 1981, 20, 295-301. (371 M. G. C. Gillan, H.W. Kosterliu, Spectrum of the mu-, delta-, and kappa-binding sites in homogenates of rat brain. Br. J. Pharm. 1982, 77, 461-469. [38] see refs. 3-5 and references cited therein. [39] H. Rink, Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Dtrahedron Lett. 1987, 28. 3787-3790. [40)G. M. Figliozzi, R. Goldsmith, S. C. Ng, S. C. Banville, R. N. Zuckermann, Synthesis of N-Substituted glycine peptoid libraries. Methods in Enzymolo~y.1996,267,431-447.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries Barry A. Bunin, Matthew J. Plunkett and Jonathan A. Ellman

15.1 Introduction One of the initial steps in the development of therapeutic agents is the identification of lead compounds that bind to the receptor or enzyme target of interest. Many analogs of the lead compounds are then synthesized to define the key recognition elements for maximal activity. In general, many compounds must be evaluated in both the lead identification and optimization steps. Recently, the demand for compounds for drug discovery efforts has increased dramatically. This is due in large part to recent technological advances in screening procedures for many therapeutic targets that allow for the rapid and efficient evaluation of thousands to millions of compounds. To address this demand, very powerful chemical and biological methods have been developed for the generation of large combinatorial libraries of peptides and oligonucleotides that are then screened against a receptor or enzyme to identify the high affinity ligands or potent inhibitors, respectively [I]. While these studies have clearly demonstrated the power of combinatorial synthesis and screening strategies, peptides and oligonucleotides generally have poor oral activities and rapid in vivo clearing times, which limit their utility as bioavailable therapeutic agents. As a result of such limitations, the synthesis and screening of libraries of nonbiological oligomers [2, 31, and most recently nonpolymeric organic compounds 14, 51, have rapidly become the focus of intensive research efforts. Derivatives of 1,Cbenzodiazepines have widespread biological activities, and are one of the most important classes of bioavailable therapeutic agents [6]. Benzodiazepine derivatives have been reported that act as anxiolytic, anticonvulsant, and antihypnotic agents [8], selective cholecystokinin (CCK) receptor subtype A or B antagonists 171, K-selective opioid antagonists [8], platelet activating factor antagonists [9],HIV trans-activator Tat antagonists [lo], GPIIbIIIa inhibitors 1111, reverse transcriptase inhibitors 1121, and ras farnesyl transferase inhibitors [13). Because of the broad biological activity and desirable pharmacokinetics of 1,4-benzodiazepine derivatives, we have developed general and expedient solid-phase synthesis methods for this class of molecules. Here, we describe the methods used for the design and simultaneous synthesis of three benzodiazepine libraries that incorporate a wide variety of chemical functionality.

406

I5 Synthesis and Evaluation of Three 1,4-BentodiazepineLibraries

15.2 Synthesis Criteria for a Benzodiazepine Library In the construction and evaluation of a library of 1,4-benzodiazepinederivatives, we felt that several criteria should be met. (i) The benzodiazepine derivatives should be synthesized on a solid support because the solid support strategy allows for facilc isolation of polymer-bound reaction products from reagent mixtures. This enable: one to drive reactions to completion by the use of excess reagents. (ii) The variable components, or building blocks, used for the synthesis of a benzodiazepine librarj should be readily synthesized or (ideally) commercially available. This greatly ex pedites the process of library synthesis, since time is not consumed in the repetitive synthesis of different building block derivatives. (iii) After synthesis of the compounds is complete, the compounds should be removed from the support so that the compounds can be assayed in solution because the solid support may complicate or interfere with receptor binding to the support-bound small molecule. (iv) Initially, in the construction of the library, the compounds should be synthesized in a spatially separate fashion to enable rigorous chemical and biological characterization of the library. In contrast to solid-phase peptide and oligonucleotide synthesis, general methods for the solid-phase synthesis of organic compounds have, until recently, seen limited development 114, IS]. When new solid-phase synthesis methods are employed, the chemical integrity and relative yields of library members can readily be determined when compounds are spatially separate. Also, by maintaining the compounds spatially separated, biological evaluation often provides detailed structure-versus-activity data. (v) The construction of a library of organic compounds that relies upon techniques already developed for high throughput screening procedures avoids the development of new instrumentation.

15.3 Chiron Mimotopes (Geysen) Pin Apparatus In constructing the libraries, we have employed the Chiron Mimotopes pin apparatus, originally developed by Geysen for peptide epitope mapping 116, 171. In this apparatus, 96 polyethylene pins are placed into a supporting block so that each pin fits into a separate well of a 96-well microtiter plate. The pins are prederivatized with aminoalkyl groups, providing sites for substrate attachment, and each well of the microtiter plate serves as a distinct reaction vessel for performing chemical reactions. Currently, pin loading levels that range from 100 nmol to 50 pmol of material per pin are available. Even 100 nmol of material is sufficient for multiple biological assays, as well as for analytical evaluation of the purity and chemical integrity of the individual compounds. While our libraries synthesized to date have been made using the Geysen pin apparatus, our development of solid-phase synthesis methods has generally been performed on crosslinked aminomethvl Dolvstvrene resin. which allows for vield deter-

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis

407

minations based on mass balance. Because optimization has been performed on gelform resin, our synthetic methods may readily be adapted to a library synthesized with a split-and-mix approach initially developed by Furka [18] and subsequently expanded by many others 1191.

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis Because of the broad biological activity and favorable pharmacokinetic properties of the 1,4-benzodiazepines, we set out to develop general methods for the synthesis of libraries of this class of molecules [20]. The first step in the synthesis of an organic compound library is to develop general reaction conditions that allow a variety of different functionality to be incorporated in high yield. The 1,6benzodiazepine derivatives were initially constructed from three components: 2-aminobenzophenones, amino acids, and alkylating agents [21]. Employing solution chemistry, substituted 2-N-Fmoc-aminobenzophenonesare coupled to the acid cleavable 4-hydromethylphenoxyacetic acid (HMP)linker. As shown in Scheme 15-1, the linker may be attached through a hydroxyl or carboxyl group located on either aromatic ring of the 2-aminobenzophenone. The linker derivatized aminobenzophenones 1 are then coupled to the solid support by standard amide bond-forming methods.

la

2a

lb

2b

Scheme 15-1.

Synthesis of benzodiazepine derivatives on solid support (Scheme 15-2) is initiated by removal of the Fmoc protecting group from 2 by treatment with piperidine in DMF. An a-N-Fmoc amino acid is then coupled to the resulting unprotected 2-aminobenzophenone. Standard activation methods for solid-phase peptide synthesis were not successful for this coupling step due to the poor basicity and nucleophilicity of 2-aminobenzo~henones. However, the activated a-N-Fmoc amino acid fluorides

408

15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

developed by Carpino [22]couple efficiently to provide the amide products 3 even for electron deficient 2-aminobenzophenone derivatives. The Fmoc protecting group is then removed using piperidine in DMF, and the resulting free amine is treated with 5 % acetic acid n NMP or DMF at 60°C to provide the benzodiazepine derivatives 4 that incorporate two of the three components for introducing diversity. NHFmoc

1. piperidine c

2. 5% AcOH. NMP

2.

NHFmoc

2

3 0 Li‘

TFA

4

5

6

Scheme 15-2.

Alkylation of the anilide of 4 provides the fully derivatized 1,Cbenzodiazepines 5. In order to ensure complete reaction on solid support, excess reagent is generally

employed. We therefore employ either lithiated 5-phenylmethyl-2-oxazolidinoneor lithiated acetanilide as the base since they are basic enough to completely deprotonate the anilide of 4, but will not deprotonate other functionality that may be present in the benzodiazepine structure such as amide, carbamate, or ester functionality. Treatment with the volatile acid cleavage cocktail trifluoroacetic acid/dimethyl sulfide/H20 (85 : 10 :5 ) then affords the benzodiazepine products 6, which after chromatography are obtained in high yield (85- 100%) based on the support-bound starting material 2. Finally, no racemization (c1 TO) of selected derivatives was detected as determined by chiral HPLC. Structures of representative compounds prepared by this route are shown in Fig. 15-1.

15.4 Solid-Phase 1,4-Benzodiazepine Synthesis

OH

409

OH

Figure 15-1. Structures of representative 1.4-benzodiazepinederivatives prepared by the route of Scheme 15-2. The average yield for the derivatives shown is 90'70, based upon the initial 2-aminobenzo~henoneloading level of the resin.

I5 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

410

15.5 First Generation 1,4-Benzodiazepine Library With the development of general solid-phase strategy for benzodiazepine synthesis, we set out to synthesize and evaluate a benzodiazepine library in order to evaluate the spatially separate synthesis of these compounds [23]. Using the Chiron Mimotopes pin support, a library of 192 compounds was assembled using ail combinations of two 2-aminobenzophenones, twelve amino acids, and eight alkylating agents, with a variety of functionality being displayed (Fig. 15-2). The chemical integrity and yield of many of the compounds in the library were determined by two analytical methods. For 28 of the structurally diverse benzodiazepine derivatives, FAB mass spectrometry confirmed the structure of the compound corresponding to the major peak (in almost all cases the only peak) observed

8^0-&

2-Aminobenzophenones

H2N

0

OH

\

H2N

0

HO \

CI

Amino Acids

H

H

Alkylating Agents

'-

/I

\I

-1

H' Figure 15-2. Building blocks used in the synthesis of first generation benzodiazepine library

(192 spatially separate compounds).

15.7 Current Solid-Phase 1,4-Bentodiazepine Synthesis

411

by HPLC. Yields were also determined for 20 derivatives, where each of the 2-aminobenzophenones, amino acids and alkylating agents was incorporated in at least one of the derivatives. This was accomplished by addition of a stock solution containing fluorenone as an internal standard followed by HPLC analysis. An 86% average yield for the benzodiazepine derivatives was observed as calculated from the experimentally determined extinction coefficients of the selected derivatives. The spatially separate library of benzodiazepines was screened to identify ligands to the cholecystokinin A receptor using a competitive radioligand binding assay. Detailed structure versus activity information was obtained for this receptor target. The data provided by screening the library was confirmed by synthesizing a number of the derivatives on large scale followed by purification and IC,, determinations. The most potent compound (K5,= 0.08 p ~ was ) synthesized from 2-amino-4-hydroxybenzophenone,D-tryptophan, and ethyl iodide. In addition, comparison of our structure-activity data with that obtained by Merck researchers for a series of structurally related benzodiazepines showed close correlation.

15.6 Second Generation 1,4=BenzodiazepineLibrary With the successful synthesis and evaluation of the initial 192 member benzodiazepine library, the same strategy was used to synthesize a larger library for screening against a variety of targets. This second generation library was synthesized from three 2-aminobenzophenones, 35 amino acids, and 16 alkylating agents, providing 1680 1,4-benzodiazepine derivatives (Fig. 15-3). With this library we have identified inhibitors of pp60c”R:tyrosine kinase [24] and ligands that block an autoimmune DNA-antibody interaction [25] implicated in systemic lupus erythematosus. These results suggest that a modestly sized library based upon an appropriate template can often be sufficient to identify ligands or inhibitors.

15.7 Current Solid-Phase 1,4-Benzodiazepine Synthesis The original benzodiazepine synthesis sequence was based upon the combination of three different building block sets: a 2-aminobenzophenone, an Fmoc amino acid fluoride, and an alkylating agent. While many alkylating agents are commercially available, and N-Fmoc amino acid fluorides can be prepared in a single step without purification from the corresponding N-Fmoc amino acids, few appropriately functionalized 2-aminobenzophenones are readily accessible. To increase the diversity of 1,4-benzodiazepine-2-onesavailable through solid-phase synthesis, we utilized the Stille coupling reaction to synthesize a variety of 2-aminoaryl ketones on solid support. The Stille reaction is particularly appealing for this purpose since it proceeds

412

15 Synthesis and Evaluation of Three 1,4-BenzodiazepineLibraries

2-Aminobentophenones

Hm OH

CI

H2N

0

JyQ ’ ’

CI OH

OH

Amino Acids (Both EnantiomersIncorporated)

Alkylating Agents H*

/I

W

I

W‘

‘-

&er

Figure 15-3. Building blocks used in the synthesis of second generation benzodiazepine library (1680 sDatially separate comDounds).

15.8 Design of a Large 1,4-BentodiazepineLibrary

411

under mild conditions, is tolerant of a wide range of functionality, and well over 300 structurally diverse and chemically compatible acid chloride building blocks are commercially available. The support-bound 2-(4-biphenylyl)isopropyloxycarbonyl (Bpoc) protected aminoarylstannane 7 (Scheme 15-3) is prepared in six steps from commercially available material. Coupling to support is again accomplished employing the HMP linker. Stille coupling can be carried out with a range of different acid chlorides and the catalyst Pd2(dba)3.CHC13. Because excess acid chloride is employed in the Stille coupling step, diisopropylethylamine and K2CO3 are added as acid scavengers to minimize protodestannylation. The Bpoc group is cleaved by brief treatment with 3% TFA in CH2C12,and the resulting support-bound 2-aminoaryl ketone 8 is then incorporated directly into 1,4-benzodiazepine derivatives using the previously described synthesis sequence 1261.

6 7

9

Scheme 15-3.

Using this strategy, numerous acid chlorides were employed to prepare supportbound 2-aminoaryl ketones 8 that were further incorporated into 1,Cbenzodiazepines 9, including aromatic acid chlorides that are electron rich, electron poor, alkyl substituted, polyaromatic, heterocyclic, and orthosubstituted, and aliphatic acid chlorides that can be sterically hindered. The desired benzodiazepines were isolated after the eight-step synthesis sequence in > 85% purity by 'H NMR analysis of the crude products. Yields of purified benzodiazepine products varied from 52 070 to 82% (Table 15-2) based on the initial aminomethyl loading of the polystyrene resin used. The structures of represantative compounds are shown in Fig. 15-4. The full characterization and mass balance based yields of the products obtained demonstrate that a wide variety of functional groups are compatible with this solid-phase synthesis methodology.

15.8 Design of a Large 1,4-Benzodiazepine Library With a versatile procedure for the solid-phase synthesis of 2-aminobenzophenones and 2-aminoacetophenones in hand, we designed the synthesis of a large library of structurallv diverse 1.4-benzodiaze~inederivatives. Since this librarv is to be evalu-

414

I5 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

Meal$ OMe

0

Figure 15-4. Structures of representative 1.4-benzodiazepine derivatives prepared by the Stille coupling route of Scheme 15-3. The average yield for the derivatives shown is 70V0,based upon the initial aminomethyl substitution level of the resin.

ated against a number of different therapeutic targets, we wanted to display a wide range of chemical diversity about the rigid benzodiazepine scaffold, thereby maximizing the possibility of finding lead compounds for each of these targets. A search of the Available Chemicals Directory (ACD) [27] for chemically compatible building

15.9 Synthesis of an 11 200 Member 1,4-Benzodiazepine Library

41

blocks shows that over 300 acid chlorides, 80 Fmoc-protected amino acids, and 800 alkylating agents can be purchased. If all of these components were included, the library would contain well over 19 million compounds! The component that are available and compatible do not realistically limit the ultimate size of a benzodiazepine library. Because our previous experience has shown that a library of less than 2000 benzodiazepine derivatives is sufficient to find ligands for several different medicinal targets, we decided to set the size of the third generation library at approximately IOOOO compounds. We chose to generate a 1,Cbenzodiazepine library containing 11 200 derivatives, which would be prepared from 20 acid chlorides, 35 amino acids [28], and 16 alkylating agents (Fig. 15-5). These numbers were chosen in part so that compounds in the library would divide neatly and rationally into the wells of a microtiter plate. Natural and unnatural amino acids containing amines, amides, carboxylic acids, alcohols, phenols, thiophenes and indoles were included. Alkylating agents containing a range of aromatic and aliphatic groups, as well as alkylating agents with hydrogen bond donors and acceptors were incorporated. The acid chlorides were selected with assistance from a structural similarity procedure developed by Steven Muskal at MDL Information Systems [29]. A list of over 500 commercially available acid chlorides was pared to approximately 350 based solely on the predicted chemical compatibility of each acid chloride with the benzodiazepine synthesis sequence. The acid chlorides were then grouped; structurally similar derivatives were placed into the same bin and structurally different acid chlorides were in separate bins. From the resulting 45 bins [30], 20 diverse acid chlorides were chosen for inclusion in the library. Generally, only the least substituted acid chloride was chosen from a particular bin. The components for each of the three building blocks were selected based on commercial availabilitiy and maximal structural diversity, to generate an 1,4-benzodiazepine library displaying a wide range of chemical functionality.

15.9 Synthesis of an 11200 Member 1,4-Benzodiazepine Library With a diverse set of building blocks in hand, we proceeded to synthesize the third generation benzodiazepine library [31]. This benzodiazepine library includes derivatives with indole, phenol, ether, cyclohexyl, heterocyclic, polyaromatic, halogen, thiophene, furan, cyano, carboxylic acid, amine, amide, and hydroxyl functional groups. The development of reliable solid-phase synthtic methods is the rate determining step in library synthesis. The actual construction of this library of 11 200 compounds took two graduate students about one month each, and could be faster with automation. The majority of the building blocks, or structurally similar building blocks like ethyl- and hexyl-alkylating agents, were incorporated into fully characterized

416

I5 Synthesis and Evaluation of Three 44-Benzodiazepine Libraries

Acid Chlorides 0 CI /

I

,'N

CN CI&OMe

CI

0

Amino Acids

Alkylating Agents H+

/I

V I

/GBr

-1

o^'

NC-1

Figure 15-5. Set of structurally diverse reagents used for the synthesis of the third generatior 1,4-benzodiazepinelibrary (11 200 compounds).

15.10 Alternate Strategies for Benzodiazepine-Based Diversity

411

1,4-benzodiazepinessynthesized on large scale. Assuming an untested building block will be compatible with a combinatiorial synthesis is risky. For example, the 1,4-benzodiazepines derived from cyclopropyl carbonyl chloride, with structural homology to the 1,4-benzodiazepines derived from cyclohexyl carbonyl chloride (previously synthesized and characterized on beads) [16], contained side products in addition to the expected structure. Approximately 100 nmol of racemic 1,4-benzodiazepinewas obtained in each well after removal of the cleavage cocktail. Ten aliquots of this library were made from a single synthesis, to be used for evaluation against a range of therapeutic targets by a number of industrial and academic collaborators. Also, a statistically significant portion of the current library of 11200 derivatives has been analyzed by mass spectrometry to confirm the presence of the expected derivatives (see note added in proof after section 15.12.4.5).

15.10 Alternate Strategies for Benzodiazepine-Based Diversity An alternate display of functionality is possible by use of the 1,4-benzodiazepine2,5-dione structure. Shown on the left of Fig. 15-6 is a 1,4-benzodiazepine-2-one, discussed in this chapter. The 1,4-benzodiazepine-2,5-dionescaffolding is shown on the right.

Figure 15-6. Alternate benzodiazepine-based diversity. A 1,4-benzodiazepine-2,5-dioneis

shown on the right.

We have recently reported [32] a general solid-phase method for the synthesis of this class of compounds, and the construction of a library of these compounds will be reported in due course. We have also developed a silicon-based method for linkage of our 1,4-benzodiazepine derivatives [33], shown in Fig. 15-7. Upon cleavage, this strategy leaves behind no trace of the linking functionality. We have also determined that substitution of silicon for germanium increases the lability of the linker so that

Finure 15-7. A silicon-based linker for traceless solid-Dhase svnthesis.

418

IS Synthesis and Evaluation of Three I,4-BenzodiazepineLibraries

cleavage by trifluoroacetic acid is facile. These linkage methods may prove useful for the construction of libraries of aromatic compounds where no memory of the solidphase synthesis is desired.

15.11 Conclusion The results reported in this chapter show that the parallel, multistep, solid-phase synthesis of organic molecules is an expedient strategy for the generation of combinatorial libraries that incorporate a variety of sensitive chemical functionality. In addition, a library of only a few thousand compounds can sometimes be sufficient to identify lead compounds for further modification and improvement. We have applied the principles and methods outlined in this chapter to the synthesis of a number of other therapeutically important classes of organic compounds including the prostaglandins 1341, arylacetic acids [35], and steroid derivatives 1361, and to designed recognition elements including P-turn mimetics [37] and aspartic acid protease inhibitors [38].

15.12 Experimental Section 15.12.1 Reagents and General Methods Fmoc-protected amine-derivatized pins were supplied by Chiron Mimotopes (Victoria, Australia). Fmoc-protected amino acids (including side-chain preprotected derivatives) [23] and 4hydroxymethylphenoxyacetic acid were purchased from Nova Biochem (San Diego, CA) or Bachem Bioscience Inc (King of Prussia, PA). All other reagents and solvents were purchased from Aldrich (Milwaukee, WI). Chemical synthesis was performed in chemically resistant polypropylene deep well microtiter plates purchased from Beckmann (Fullerton, CA), catalog No. 267006. In working with sets of pins that are not in a pin block, a peptide flask (Safe-Lab, catalog number M2570) is often useful for filtration and rinsing operations. (A peptide flask is a glass cylinder with a fine frit at the bottom and a three-way valve beneath the frit. Solvents may be forced through by nitrogen pressure at the top, and reaction mixtures may be gently agitated by bubbling in nitrogen from the bottom.) When working with sets of pin blocks, the polypropylene lids from micropipette tip boxes are convenient, chemically stable reservoirs for rinses and deprotection reactions. A set of printed documentation for the library synthesis was prepared in advance. This is used as a checklist during the construction of the library, and is a very important part of the overall process.

15.12 Experimenlal Section

4 1S

15.12.1.1 Fmoc Deprotection of Aminomethyl Solid Support (Pins)

Fmoc-protected amines on either 1.4 pmol (“small”) or 5 pmol (“large”) pins (Chiron Mimotopes Ltd.) are deprotected with 20% piperidine in DMF (20 min). The pins are rinsed with DMF (4 x ), methanol (4 x ), and dried under vacuum.

15.12.1.2 2-Aminobenzophenone

As noted in the text, the support-bound 2-aminobenzophenone derivatives 8 may be prepared by two different routes. Method A :The N-Fmoc-2-aminobenzophenones 1 are prepared via a solution 2-aminobenzophenone synthesis, coupling to the HMP linker allyl ester, Fmoc protection of the aniline, allyl deprotection to provide the free acid, coupling to the solid support, and Fmoc deprotection [21,23]. Merhod B: The Stille coupling route involves the synthesis of a Bpoc-protected 2-aminoarylstannane, Mitsunobu coupling to a preactivated HMP linker, acylation onto the solid support, Stille coupling, and Bpoc deprotection [26, 311. Below we describe general solidphase methods for 1,4-benzodiazepine library synthesis by either route using the Chiron Mimotopes pins.

15.12.2 Method A 15.12.2.1 Coupling Fmoc-Protected 2-Aminobenzophenones (1) to Pins to Give 2

Deprotected pins are added to a round-bottomed flask with DMF (0.15 ml/pin) that contains the phenoxyacetic acid 1 (0.05 M), I-hydroxybenzotriazole (0.055 M), and diisopropylcarbodiimide (0.055 M). The acylation is allowed to proceed for 12 h. The pins are rinsed with DMF (3 x), methanol (3 x), and air dried.

15.12.2.2 Fmoc Cleavage

The Fmoc protecting group of the 2-aminobenzophenones 2 is removed by treatment of the pins with 20% piperidine in DMF (20 min). The pins are rinsed with DMF, methanol (3 x), and air dried. (After this step, synthesis by this route continues in the “Amino acid fluoride acylation” section 15.12.4.1.)

420

15 Synthesis and Evaluation of Three I,4-Bentodiazepine Libraries

15.12.3 Method B 15.12.3.1 Coupling Aminoaryl Stannane Cyanomethyl Ester to Pins to Give 7

To an oven-dried Schlenk flask under nitrogen is added the active ester (5 mole equiv.), 4-dimethylaminopyridine (5 mole equiv.), diisopropylethylamine (8 mole equiv.), N-methylpyrrolidinone (minimal volume), and the deprotected pins. The reaction mixture is heated at 65°C for 12 h to give support-bound stannane 7. The pins are transferred to a peptide flask and rinsed with ethyl acetate (3 x ) and CH2Cl2(3 x), and are then dried under vacuum. Unreacted stannane is recovered by extraction of the ethyl acetate washes with 0.2 M citric acid (3 x ) and brine, concentration of the organic layer, and column chromatography. Multigram quantities of starting material may routinely synthesized and in general about half the material can be recovered after acylation of the solid support.

15.12.3.2 Stille Coupling Reactions

The Stille coupling reactions are performed separately for each acid chloride. To a Schlenk flask under nitrogen is added the pins, K2C03 (0.20 g/mmol support bound stannane), Pd2dba3-CHC13(1.0 equiv.), THF (100 ml/mmol stannane), and diisopropylethylamine (4.0 equiv.). The mixture is stirred for 3 min, at which point 20 equiv. of the appropriate acid chloride is added slowly and the reaction mixture is stirred for 1 h at room temperature. The pins are then transferred to a large reaction flask and rinsed with CH2C12( x 5), KCN/DMSO (to remove residual Pd), H 2 0 ( x 3), and methanol ( x 3). The protected 2-aminobenzophenonesand 2-aminoacetophenones may be stored at - 20 "C. 15.12.3.3 Bpoc Cleavage

The Bpoc protecting group by treating the pins with 1070 TFA/CH2C12(5 min), rinsing with CH2C12(2x), and repeating the sequence once. The pins are rinsed with CH2C12(5 x ) and methanol (3 x ) to give 8, and the pins are dried under vacuum. Every pin in a given flask now has the same 2-aminoarylketone attached to it.

15.12.4 Benzodiazepine Synthesis from 2-Aminoarylketones 15.12.4.1 Amino Acid Fluoride Acylation

(Synthesis by either route is identical from this point on.) The pins may be subdivided into vials for the acylation reaction, where the number of vials needed is

15.12 Experimental Section

42 I

equal to the number of amino acids times the number of acid chlorides or 2-aminobenzophenones used in the library construction. (Alternatively, at this step the pins may also be placed into the pin blocks so that all subsequent steps are performed in deep-well microtiter plates. We have found that reagents are conserved when reactions are performed in vials due to the smaller solvent volume needed per pin.) To each vial is added 0.2 ml/pin of a CH2C12solution that contains 0.2 M of the appropriate Fmoc protected amino acid fluoride [22] and 0.2 M of 2,6-dir-butyl-4-methylpyridine, providing the corresponding anilide. The coupling reactions are allowed to continue for three days to ensure complete coupling of the most hindered amino acid derivatives (valine and isoleucine). The pins are rinsed with CH,C12 (3 x), MeOH (3 x), and air dried. In order to obtain high yields in this coupling step, the Fmoc amino acid fluorides should be prepared with cyanuric fluoride, and the workup should include extraction with 1 M sodium bicarbonate (3 x ) and 1 M sodium bisulfate (3 x ) to remove any cynuric fluoride byproducts, with no further purification necessary.

15.12.4.2 Amino Acid Fmoc Cleavage and Benzodiazepine Cyclization

At this point each vial should contain one pin for each alkylating agent, with the total number of vials equal to the product of the number of amino acids and the number of 2-aminobenzophenones or acid chlorides. The pins are transferred to a 96-well microtiter plate pin block for the cyclization, alkylation and cleavage steps. The Fmoc protecting group is removed by treatment of the pin blocks with 20% piperidine in DMF (20 min). The pin blocks are rinsed with DMF, MeOH (3 x ), and air dried. The pin blocks are immersed in 5 % acetic acid in DMF or NMP at 65 "C for 12 h to provide the cyclic product 4. The pins are rinsed with DMF (2 x), MeOH (2 x ), T H F (2 x ), and air-dried.

15.12.43 Benzodiazepine Alkylation

After THF and DMSO rinses, the pin blocks are immersed in a 1 : 1 (v/v) solution of 0.12 M solution of lithiated 5-phenylmethyl-2-oxazolidinonein 10% DMF in THF/DMSO and sonicated for one hour. For the sonication, the pin blocks are placed in plastic ziplock bags to maintain dryness (although we have found that 2 070 water does not adversely affect the alkylation step). The pin blocks are removed from the bags and are then immersed, without rinsing, in a 0.40 M solution of alkylating agent in DMF (prepared immediately before alkylation) and sonicated for an additional three hours (again in a ziplock bag), to provide the fully functionalized, support-bound derivatives 5. The pin blocks are removed from the ziplock bag and rinsed with DMF, DMF/H,O, MeOH (air dried), and CHzClz.

422

15 Synthesis and Evaluation of Three 1,4-Bentodiazepine Libraries

15.12.4.4 Cleavage from the Support The derivatives 5 are cleaved from the support by placing the pin blocks into microtiter plates that contain 0.25 ml/well of a solution of 85 : 10 : 5 trifluoroacetic acid/Me2S/H20 for 24 h. For benzodiazepine derivatives incorporating tryptophan, 85 :5 :5 :5 trifluoroacetic acid/dimethylsulfide/H20/1,2-ethanedithiolis employed as the cleavage cocktail to prevent oxidative decomposition of the indole ring [39]. The cleavage cocktail is then removed with a Jouan RC1O.10 concentrator equipped with a microtiter-plate rotor to provide the free 1,Cbenzodiazepine derivatives 6, spatially separated in the individual wells of the microtiter plate. 15.12.4.5 Analytical Evaluation of the 1,4-Benzodiazepine Library Evaluation of the 1Q-benzodiazepine derivatives is accomplished by reverse phase HP-LC analysis using a Rainin C,*column and a 15-100Vo gradient of methanol in water buffered with 0.1 Yo trifluoroacetic acid with UV detection at 350 nm. The compound corresponding to the major peak (usually the only peak) can be isolated and submitted for mass spectrometric analysis to verify the structure of the benzodiazepine derivative. In addition, yields for synthesis on pin supports can be determined by addition of a stock solution of fluorenone in DMF followed by reversephase HPLC analysis to determine the relative peak area of the lY4-benzodiazepine derivative to the fluorenone standard. The quantity of material produced per pin is then calculated from the extinction coefficients of the derivatives that were determined on material prepared on a large scale. Alternately, for the synthesis of lY4-benzodiazepineson pins producing 1.4 or 5 pmol per pin, yields can be determined by addition of an aliquot of p-xylene as an internal proton NMR standard followed by peak area integration. Finally, characterization of the unpurified benzodiazepine products may be performed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) using a-cyano-4-hydroxycinnamicacid as matrix. Note Added in Proof A subset of the second generation library was analyzed by HPLC as described for the first generation library, and yields were found to range from 6 1 4 7 % (average 72%). In addition, 48 of the compounds (randomly selected, incorporating each of the building block derivatives at least twice) were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) using a-cyano-4-hydroxycinnamic acid as the matrix. For 46 of the derivatives the expected molecular ion was found. For one of the undetected derivatives the hydrolytically unstable alkylating agent cyanomethylbromide was used, and the expected unalkylated derivative was found. For the other compound, no product at all was found by HPLC, although

References

423

good yields were seen for all other library members analyzed that shared at least one building block. This suggests that an aliquotting error to loss of the compound. The 11 200 compound benzodiazepine library was also analyzed for purity, yield, and molecular ions. 'H-NMR spectroscopy of five compounds from the library showed the expected benzodiazepine as the major product in each case. Twenty derivatives were analyzed by reverse phase HPLC with UV detection at 315 nm for benzodiazepines derived from aromatic acid chlorides and 285 nm for those derived from aliphatic acid chlorides. One major product (with retention time identical to authentic material prepared on large scale) was observed in all cases, although the loading was lower than expected. To further confirm that the expected compounds had been synthesized, many of the resulting compounds were analyzed by MALDIMS. The expected peak was found for 67 of 72 randomly selected derivatives, where each building block derivative was analyzed at least twice.

Acknowledgments Separation of the acid chlorides into bins was performed by Steven Muskal (MDL Information Systems, Inc.) and is greatly appreciated. The technical expertise provided by Andrew Bray (Chiron Mimotopes) is gratefully acknowledged. This work was supported by the NIH, the NSF, the Arnold and Mabel Beckman Foundation, and the Burroughs Wellcome Fund. Chiron, Affymax, Tularik, Eli Lilly, and Hoffman La Roche are also gratefully acknowledged for their support.

References [I] M.A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M.Gordon, J. Med. Chem. 1994, 37, 1233. [2] R. J. Simon, R. S. Kania, R. N. Zuckermann, V. D. Huebner, D. A. Jewell, S. Banville, S. Ng, L. Wang, S. Rosenberg, C. K. Marlowe, D. C. Spellmeyer, R. Tan, Proc. Natl. Acad. Sci. USA 1992, 89, 9367. 131 C. Y. Cho, E. J. Moran, S. Cherry, J. Stephans, S. P. A. Fodor, C. Adams, A. Sundaram, J. W. Jacobs, P. G. Schultz. Science 1993, 261, 1303. [4] E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, M.A. Gallop, J. Med. Chem. 1994, 37, 1385. (51 L. A. Thompson, J. A. Ellman, Chem. Rev. 1996, 96, 555. [6] L. H. Sternbach, J. Med. Chem. 1979, 22, 1. [7] M. G. Bock, R. M. Dipardo, B. E. Evans, K. E. Rittle, W. L. Whitter, D. F. Veber, P. S. Anderson, R. M . Freidinger, J. Med. Chem. 1989, 32, 13. [8] D. Rimer, H. H. Buscher, R. C. Hill, R. Maurer, T. J. Petcher, H. Zeugner, W. Benson, E. Finner. W.Milkowski. P. W. Thies. Nature (London) 1982. 298. 759.

424

15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

[9] E. Kornecki, Y. H. Ehrlich, R. H. Lenox, Science 1984, 226, 1454. [lo] M.-C. Hsu, A. D. Schutt, M. Hooly, L. W. Slice, M. 1. Sherman, D. D. Richman M. J. Potash, D. J. Volsky, Science 1991, 254, 1799. [ll] W. E. Bondinell, J. F. Callahan, W. E Huffman, R. M. Keenan, T. W.-F. Ku, K. A Newlander, 1993 International Patent Application WO 93/00095. [12) R. Pauwels, K. Andries, J. Desmyter, D. Schols, M. J. Kukla, H. J. Breslin, A Raeymaeckers, J. Van Gelder, R. Woestenborghs, J. Heykants, K. Schellekens, M. A. C Janssen, E. D. Clercq, P. A.J. Janssen, Nature (London) 1990, 343, 470. [13] G. L. James, J. L. Goldstein, M. S. Brown, T. E. Rawson, T. C. Somers, R. S. McDowell C. W.Crowley, B. K. Lucas, A. D. Levinson, J. C. Marsters, Jr., Science 1993,260, 1937 I141 C. C. Leznoff, Acc. Chem. Res. 1978, 11, 327. [15] X. Beebe, N. E. Schore, M. J. Kurth, J. Am. Chem. Soc. 1992, 114, 10061. [16] H. M. Geysen, S. J. Rodda, T. J. Mason, G. Tribbick, P. G. Schoofs, J. Immunol. Methods 1987, 102, 259. [17] R. M. Valerio, A. M. Bray, N. M. Maeji, Int. J. Pept. Protein Res. 1994, 44, 158. [18] A. Furka, E Sebestyen, M. Asgedom, G. Dibo, Int. J. Pept. Protein Res. 1991, 37,487. I191 M. A. Gallop, R. W.Barrett, W. J. Dower, S. P. A. Fodor, J. Med. Chem. 1994,37, 1233. [20] A different approach has been reported by researchers at Parke-Davis: S. H. DeWitt, J. S. Kiely, C. J. Stankovic, M. C. Schroeder, D. M. Reynolds Cody, M. R. Pavia, Proc. Natl. Acad. Sci. USA 1993, 90,6909. (211 B. A. Bunin, J. A. Ellman, J. Am. Chem. Soc. 1992, 114, 11997. [221 L. A. Carpin6, S. Satat-Aalaee, H.G. Chao, R. H. DeSelms, J. Am. Chem. Soc. 1990, 112, 9651. (231 B. A. Bunin, M. J. Plunkett, J. A. Ellman, Proc. Nutl. Acad. Sci USA 1994, 91, 4708. (241 R. A. Buddie, V. Levin, M. D. Anderson Cancer Institute, Houston, TX, unpublished results, 1995. [25) G. Glick, University of Michigan, unpublished results, 1995. [26] M. J. Plunkett, J. A. Ellman, J. Am. Chem. Soc. 1995, 117, 3306. I271 MDL Information Systems, Inc San Leandro, CA. (28) The L- and D-amino acids were pooled together. 129) S. Muskal, MDL Information Systems, Inc, unpublished results, 1995. I301 The number of bins increases as the minimum similarity value for same-bin placement approaches unity. [31] B. A. Bunin, M. J. Plunkett, J. A. Ellman, Methods Enzymol. 1996, 267, in press. I321 C. G. Boojamra, K. M. Burow, J. A. Ellman, J. Org. Chem. 1995, 60, 5742. (331 M. J. Plunkett, J. A. Ellman, J. Org. Chem. 1995, 60, 6006. [34] L. A. Thompson, J. A. Ellman, unpublished results. I351 B. J. Backes, J. A. Ellman, J. Am. Chem. Soc. 1994, 116, 11 171. [36] 1. C. Choong, J. A. Ellman, unpublished results. [37] A. A. Virgilio, J. A. Ellman, J. Am. Chem. Soc. 1994, 116, 11580. [38] E. K. Kick, J. A. Ellman, J. Med. Chem. 1995, 38, 1427. I391 G. B. Fields, R. L. Noble, Int. J. Pept. Protein Res. 1990, 35, 161.

Combinatorial Peptide and Nonpeptide Libraries by. Gunther Jung OVCH Verlagsgesellschaft mbH, 1996

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

16.1 Introduction Since the introduction of solid phase peptide synthesis (SPPS) on low crosslinked polystyrene by R. B. Merrifield in 1962 [I -41, this method has been developed and applied in many other fields, e. g. oligonucleotide synthesis [5-71, catalysis [8- 161, biotechnology for immobilization of enzymes [17-281 and for polymeric reagents [29-341. A new, very rapidly growing field is the use of solid phase chemistry in combinatorial chemistry and libraries. The expanding fields of applications demand a need for new polymeric supports. In the early days of solid phase chemistry, 2 % crosslinked polystyrene supports were used almost exclusively while, nowadays, the 1% crosslinked polystyrene resin is the most used resin. Besides the classical support (1 To crosslinked chloromethylated polystyrene), originally introduced by R. B. Merrifield, a large variety of other polymeric supports have been developed and are in use (see also Chapter 17). From the chemical and physicochemical view, the solid supports can be classified into three main groups: (i) gelatinous polymer supports; (ii) macroporous supports and composite supports ; (iii) surface modified polymeric carriers. Group 1 comprises the well-known 1 Yo crosslinked polystyrene resins and the crosslinked polyacrylamides introduced by Sheppard [35], as well as the ultrahigh loaded Core Q-resins [36], which are also polyacrylamide-type resins. Typical capacities of these resins are in the range of 0.1-1.5 mmol/g, core Q up to 5 mmol/g, however, this high loaded resin has never become very important. All these gelatinous resins are available in beaded form, having a typical particle size distribution of 200-400 mesh (73-37 vm) or 100-200 mesh (150-75 pm). The described resins are low, crosslinked supports and have to swell up in the reaction solvent in order to built up a polymeric network accessible to the reactants. It is important to notice that, on gelatinous resins, up to 99% of the reactive sides are located inside the bead and only 0.1 to 1 % are on the outer surface. The network

426

16 PEG Grsfed Polystyrene lbtacle Polymers

is very soft and flexible. Therefore, the use of the swollen beads is restricted to batch processes and cannot be used in continuous column processes. In contrast to the gelatinous resins, macroporous supports of group 2 consist of a very rigid, nonswellable backbone. The backbone could be either kieselguhr, glass (CPG-glass) or highly crosslinked polystyrene. In contrast to the swellable resins the macroporousresins, show a permanent porosity and no swelling is necessary. Dependent on the manufacturing process, the supports have a beaded shape (CPC-glass, polystyrene) or an irregular shape like kieselguhr and polystyrene (Polyhipe). Composite supports consist of such a macroporous rigid skeleton which is either kieselguhr (Pepsyn resin) or polystyrene (Polyhipe) [37, 381. Low, crosslinked polyacrylamideis then polymerized within the pores. The structure of the composite resins is illustrated in Fig. 16-1. This resins are pressure stable but mechanically very fragile, and their use is strictly restricted to continuous flow processes. Again, the synthesis takes place in the gelatinous polyacrylamide part of the resin. npical capacities are in the range 0.1-0.5 mmol/g. In all polymer supported reactions the polymeric support represents the reaction space where the chemical reaction takes place. Using polymer beads for synthesis the total polymeric reaction space is divided into small, individual reaction compartments. Therefore, resin parameters like crosslinking, swelling properties, mass transport, phase transition, bead size and particle size distribution have to be taken in account, as each individual bead represents the reaction space. The resin type which combines the advantages of gelatinous resins (group 1) and macroporousresins (group 2) in addition of the advantages of the liquid phase chemPolystyrene Matrix or Kiselgur

Pores

Figure 16-1. Scheme of macroporous composite resins with Kieselgur or polystyrene ctelptnn

16.2 Properties of Polystyrene- Poly(ethyleneglyco1)-Tentacle Polymets

427

istry is the polystyrene-poly(ethyleneglyco1)-graft (PEG) copolymer (TentaGel-resins [39-441). This resin belongs to group 3 of the resins characterized above. The liquid phase method [45-49) was developed as an alternative approach to the solid phase method of Merrifield. In general, soluble and functionalized PEG of molecular masses between 3000-20000 D are used in the liquid phase method. PEG is very compatible with many solvents and with peptides and proteins. To assemble a peptide or nucleotide, the couplings are carried out in homogeneous solution on the polymer as shown by Mutter and Bayer [50]. Kinetic investigations have shown that the coupling rates are of the same order as low molecular mass amino acid esters [51]. Diffusion phenomena play no role in this technique, which is a major advantage over the solid phase technique. PEG often solubilizes even insoluble substrates. One practical advantage of the peptide-PEG conjugates is their easy use for instrumental analysis, e. g. for the recording of circular dichroism spectra for conformational analysis of the growing peptide chain. Instead of simple filtration used in solid phase chemistry, membrane filtration or crystallization is applied to separate the polymer bound product from the low molecular mass molecules. However, the main disadvantages of liquid phase technique are the time-consuming work-up operations like membrane filtration or crystallization and the impossibility of automation. To benefit from the advantages of both the liquid phase and the solid phase techniques, we have grafted poly(ethyleneglyco1) chains on to low crosslinked polystyrene beads.

16.2 Physico-Chemical Properties of

Polystyrene-Poly(ethyleneglyco1)-Tentacle Polymers

The simplest immobilization procedure is to couple PEG via one of its terminal hydroxy groups to chloromethylated polystyrene according to the classical ether synthesis [52,53], or to use other bifunctional PEGs for coupling on to the solid support [54]. However, when long chain PEGS (>800 D) are used, the yields of this approach are unsatisfactory. Moreover, the homo-bifunctional PEGs react to a large extent at both ends to give cyclic poly(ethyleneglyco1s) and additional crosslinking. Consequently, the number of free functions will be reduced with concomitant lowering of the capacity of the graft copolymer. We have found that, by means of anionic graft copolymerization setting up the PEG step by step directly on the matrix, PEG chains of molecular masses up to 20 k~ have been immobilized on functionalized crosslinked polystyrenes [55-601. Graft copolymers with PEG chains of about 3000 D proved to be optimal with respect to kinetic rates, mobility, swelling and resin capacity. These graft copolymers are pressure stable and can be used in batch processes as well as under continuous flow conditions. Figure 16-2 illustrates the chemical architecture of the tentacle polymer. The copolymer contains about 60-7070

428

16 PEG Gmfted Polystyrene Rntacle Pobmers

Figure 16-2.Architecture of the polystyrene-poly(ethyleneglyco1)-tentacle resin. In the swollen state the polymeric network is formed. The POEtentacles and the functional groups at the end of the spacers are completely solvated.

PEG (w/w).Therefore, the properties of these polymers are highly dominated by the properties of PEG and no longer by the polystyrene matrix. This is especially shown by the very consistent and, from the solvent, nearly independent swelling volumes of the graft copolymer in comparison to 1Vo crosslinked polystyrene. Table 16-1 summarizes the swelling volumes of TentaGel resins in comparison to polystyrene. The dry volume of TentaGel is approximately 1.7 ml/g, and that for polystyrene 1.6 d / g . For measuring the swelling volume, 1 g of resin was swollen in the solvent for 24 hours. The PEG-graft copolymer swells in all solvents which dissolve PEG and, on the other hand, swelling is negligible in solvents which do not dissolve PEG, e.g. aliphatic hydro-carbons and diethylether. This broad range of usable good swelling solvents allows the use of TentaGel resins in almost every solvent system. Thus, after a reaction, e.g. in methylene chloride, one can use a solvent gradient, e.g. from a b l e 16-1: Swelling volume of polystyrene, crosslinked with 1 Qo DVB (dry volume: 1.6 d / g ) and TentaGel resin (dry volume: 1.7 d / g ) Solvent

Polystyrene 1% DVB

TentaGel resins

Water

MeOH EtOH CH2C12 Toluene DMF

MeCN

THF Dioxane Ether

-

1.6

1.68

8.3

8.5

5.6

3.2

8.8

7.8

4.1

4.25

4.25

2.1

5.1

5.3

5.4

5.1

5.8

6.2

1.9

16.2 Properties of Polystyrene- Poly(ethyleneglycor)-l&ntaclePolymers

429

methylenc chloride via tetrahydrofurane to aqueous systems, and then back from this hydrophilic system via a gradient to pure organic solvent systems, e.g. from water via tetrahydrofurane or ethanol to toluene. The reactive sites which are located at the end of the spacer-arms behave kinetically as though they were in solution. This could be demonstrated by kinetic measurements where the coupling constants of the active ester Boc-Gly-ONp to both low molecular mass compounds and graftcopolymer are of the same order of magnitude [61]. Because of the flexibility and the good solvation properties of the PEG tentacles, polymer reactions could be performed on the solid support under quasihomogeneous conditions. The I3CNMR relaxation time measurements (7i)also indicate the high flexibility of the PEG-spacers and the reactive sites at the end of the spacer. Figure 16-3 illustrates the two extreme conditions when the resin is not swollen (Fig. 16-3 a) or completely swollen (Fig. 16-3 b). In the nonswollen or uncompletely swollen state of the resin, the NMR signals of the PEG spacers are rather broad as known from solid state NMR studies. When the resin is swollen, the PEG tentacles are well solvated and highly flexible, which results in high values. The NMR signals show a very narrow linewidth which is comparable to that of small molecules. This physicochemical property of the resin allows the use of I3CNMR and M A S 'HNMR spectroscopic techniques for investigations of the resin, for analysis of resin-bound molecules and for resin functionality [60-631. For example, palmitic acid was coupled to

PEG PS

solvation gelphase

PEG PS b

precipitation solid state

a)

ppm 90

70

50

PPm 90

70

50

Figure 163. Top: Tentacle resin in shrunken and swollen condition. The bottom line shows the corresponding gelphase 13C N M R of the PEG-tentacles: in swollen state, the tentacles show high 7i values (highly flexible, well solvated) resulting in sharp 13Cresonances, whereas in the nonswollen state the I3C resonances show broadening.

430

16 PEG Gnlfled Polystyrene Tentacle Polymers

the aminofunctionalized resin, and the resin was measured by I3C N M R spectroscopy under various conditions. Figure 16-4 shows the "C N M R spectrum of a sample of resin (100 mg), which is swollen in CDCI, and measured in a 5 mm tube (Fig. 16-4a). Because of the high flexibility and good solvation of the PEG spacer and the attached palmitic acid, rather sharp signals were recorded for the carbon resonances, and a good signal to noise ratio is obtained. The same investigation was done using water as a solvent. Again, the PEG spacer shows a sharp resonance signal, whereas the signals of the palmitic acid are rather broad. This is readily explained since PEG is well solvated in chloroform and in water, whereas the palmitic acid is only well solvated in chloroform and not in aqueous systems. This results in sharp signals for chloroform (highly flexible, good solvated) and broad signals for the water system (not solvated, restricted mobility). As a result of these investigations, it is necessary to take care of both the resin and the attached molecule and to choose a good solvent for each of them so as to have them solvated and to obtain informative NMR spectra. n b l e 16-2 summarizes the NMR chemical shifts of some TentaGel derivatives. Look ef 01. [62] have used I3C enriched molecules as an internal standard on the TentaGel resin to follow the chemical shift of the I3C enriched centers during a synthesis sequence on the resin. This technique allows fast I3C NMR analysis to be used for monitoring gel phase reactions. Fitch et al. (631 have used the magic angle spinning technique (MAS) to obtain 'H N M R spectra from the gel phase resin, for analysis of the functionalities and for molecules immobilized on the TentaGel matrix. All these NMR techniques allow analytical information of resin-bound molecules and analytical data to be obtained during a reaction sequence directly on the resin. As mentioned earlier, particle size and particle size distributions are of major importance, and have to be taken into account during organic chemistry on solid supports. In all polymer supported reactions there must be a mass transport of the activated compound from the surrounding solvent to the reactive sites which are located inside the resin bead. The driving force for mass transport is diffusion, and diffusion is dependent on pathlength. Figure 16-5 correlates the size fraction (5a) to the corresponding mass fraction ( 5 b). Assuming a theoretical size distribution in the range 20-60 pm, where 80% of the beads were represented in the main fraction, 10% fraction with smaller beads, 10% fraction of larger beads, and the 10% size fraction of the larger beads representing 29% of the total mass of all three fractions. Supposing there is an equal distribution of the reactive groups on the mass fraction, this 10% size fraction of larger beads also carries 29% of the reactive groups. Assuming that the kinetic rate is much faster than the diffusion rate, and that diffusion rate is dependent on particle size, the largest beads control the overall reaction time. Only monosized beads show a homogeneous distribution of reactive sites within all beads. Using monosized beads, there are identical reaction conditions on each bead at all times. Besides diffusion, sorption properties also influence the overall reaction rates.

16.2 Properties of Porusty~ne-Poru(ethyienegruco~-~ntacie Po&mers

43 I

2 1 1 6 15' 14' 1 3 ' s 4' 3 2' 1' PS POE 0- CHiCHrNH CO -CH&Ha (CHa)o-CWH&HSHs

CDCI,

a)

16'

n

2 1 18' 15' 14' 1 3 5 ' 4' 3' 2' 1' PS PO€ 0- CHrCHrNH CO CHrCHa (CHa)o-CHrCtk-CWb

2do

rQ

do

do

do

lb

PPM

8b

$0

do

Figure 16-4. Gelphase "C N M R of TentaGel immobilized palmitic acid. a) CDC13 as solvent, b) D20as solvent

2b

I

0

432

16 PEG Grafted Polystyrene Entacle Polymers

Table 16-2: Chemical shifts of TentaGel derivatives in gelphase I3C NMR

X CH2-X [CH,-CH2-0],, N-CH, N-(CH# Boc

61.9 71.0

Br

SH

NH2

N-(CHJ)2

30.9 70.9

31.6

42.5 71.1

59.3 71 46.1

70.8

BOC-NH N-(CH# CHZCOO 64.2 70.4

65.3 71.3 54.1

28.2

a) particle size distribution

100%

90%

60%

b) mass fraction

100% 84% 70%

52%

- 1 15.5%

0.59

Figure 16-5. Correlation of particle size fractions with the corresponding mass fractions of DolYdisDersed and monosized beads.

16.2 Properties of Polystyrene-Poly(ethyleneglyco1)-Tentacle Polymers

432

Sorption is dependent on crosslinking, swelling properties and polarity of the resin. The degree of sorption of reagents in the bead is rather important, and influence5 the kinetic rates to a great extent. To drive the reaction to completion, normally a large excess of reagents in the fluid medium is used. However, for the effective reaction rates (64-671 the concentration of reactants in the beads is decisive and is expressed by the partition coefficient k (Table 16-3). A sorption respective partition coefficient k > 1 indicates higher concentration inside the bead than that in the surrounding liquid medium. With increased PEG con. tent on the graftcopolymer, PS contents (w/w) are lowered, and the partition coefficient is increased. Table 16-3 shows the partition coefficient of polystyrenes in comparison to PS PEG-graft copolymers having different PEG molecular masses, resulting in different ratios of PEG to PS. The sorption and partition coefficients of Boc-Gly-ONP active esters were measured by standard procedures, developed by Hori et al. [66, 671. A comparison of sorption curves of the dye Oracett blue 2 R on polystyrene - 1 % divinyl benzene beads (44 pm particle size) and on PS-PEG graft copolymers is shown in Fig. 16-6. For the grafting process of PEG (molecular mass of 3000 D) to polystyrene, the same PS matrix was used. The diameter of the beads increases from 44 pm to approximately 80 pm and the graft copolymer now consists of 75% PEG. Although the particle size is doubled compared to the polystyrene matrix originally used, sorption increases by a factor of 4 accompanied by an increase of the sorption capacity. If monosized 7.5 pm TentaGel particles are used, the sorption rate increases dramatically (curve 3), and even higher crosslinked monosized microparticles show higher sorption rates compared to polystyrene. Setting up a chemical library or peptide library by the “one bead-one compound” approach [68, 691 it is essential to know the number of beads which are available in 1 g of resin as well as the capacity of single beads. Table 16-4 summarizes some particle sizes and correlates them with the corresponding capacity of single beads. The calculations are based on a typical loading of TentaCel beads which is in the range Table 16-3: Partition coefficients of Boc-Gly-ONp active ester for polystyrene and TentaGel beads in acetonitrile solution (25 “C) Polymer

BOC-Gly - PS BOC-Gly- PEG -PS Boc-GIY -PEG-PS BOC-Gly-MOPS* Boc-Gly- PEG- MOPS * Boc-Gly- PEG - MOPS *

*

Monosized matrix.

Crosslinking of PS matrix [To DVB]

Molecular mass of PEG

PS matrix substitution

[Dl

I0701

-

13.5 13.5 13.5 13.0 13.0 13.0

1 1 1 1

194 3300

1

194

1

2000

-

Partition coefficient k Ikg

L-7

1.11 1.30 1.57 1.14 1.37 1.58

434

16 PEG Grafted Polystyrene Tentacle Polymers

20 TcntaCct,

$as Lm

MOPS TcntaCcl, 0 7.5 #rn

-

2

5

0 time [min]

20 lime [rnin]

Figure 16-6. Sorption curves for oracet blue 2 dye in acetonitrile (1.3 x mol I-', 25°C) Polymer/solvent I :120 (w/w), Curve 1 represents 1To crosslinked polystyrene, having a mean particle size of 44 ym, whereas curve 2 shows the corresponding PS-PEGgraft copolymer. Curve 3, 4 and 5 compare monosized graftcopolymers with different degree of crosslinking.

Table 16-4: Correlation of particle size, number of beads per gram resin and capacity per single bead. Calculation of single bead capacity is based on a capacity of 0.25-0.3 mmol/g resin; functional group 0-CH2CH2NH2

Resin

size [ym]

TentaGel S NH2 TentaGel S NH2 TentaGel S NH2 TentaGel S NH2 TentaGel M NH2 TentaGel M NH2 TentaGel M NH2

750 ym 500 ym 130 ym 88 ym 35 ym 20 ym 10 ym

Beads/g 4.62 x 1.5 x 8.87 x 2.86 x 4.55 x 2.4 x 1.95 x

103 104 10s

lo6 107

10' 109

Loading/bead 65 nmol 19 nmol 280-330 pmol 80- 100 pmol 5.5 pmol 1.0 pmol 0.13 pmol

0.25-0.3 mmol/g. For example, a hemmer peptide library, containing all 20 natural amino acids, ends up in 206= 64OOOOOO individual peptides. Therefore, a minimum of 64 million beads are necessary. For 64 million beads there is a need for 30 mg of 10 ym resin, 1.5 g of 35 ym resin, 25 g of 88 ym resin and about 10 kg of 750 ym beads. For analytical characterization, at least 5 pmol of resin-bound peptide are needed for sequencing on a bead [70]. To estimate the optimum resin quantity for the library which can be handled economically, one has to take into account the different bead sizes and bead capacities. Table 16-5 summarizes some available tentacle derivatives for use in peptide synthesis, libraries and combinatorial chemistry.

16.2 Properties of Polystyrene- Poly(ethyleneglyco1)-Tentacle Polymers

435

Table 16-5. Functionalized and activated commercially available graftcopolymer resins for

use in organic synthesis, peptide and oligonucleotide synthesis Resin

Functionality

TentaGel S OH TentaGel S Br

-0-CH2 -CHz-OH

TentaGel S NH2 TentaGel S COOH TentaGel S SH

-

0- CH2 -CH2

-0-CH2 -CH2

- Br - NH2

wNH-CO-CH2 -CH2 -COOH -0 -CH2 -CH2 -SH

3

0

TentaGel S COOSu

-NH-CO-CH2-CH2-CO-O-N

0

TentaGel S CHO TentaGel S NHNHBoc

-

-

CH2 - CH2 - CHO

TentaGel S AC

TentaGel S Trt

TentaGel S PHB

TentaGel S HMB

TentaGel S AM

TentaGel S RAM

-

NH - CO - CH2 - CHI CO - NH - NH - BOC

- 0 o C H 2 - 0 H

436

16 PEG Grafted Polystyme Tentacle Polymers

16.3 Peptide Synthesis Physico-chemical properties like mechanical stability, pressure stability and equal swelling volumes in different solvents as well as high kinetic rates [60, 611, allow the use of the TentaGel resins in all commercial synthesizers (e. g. continuous flow (CF) [59], vortexing, shaking, gas bubbling or multiple synthesizers). Our investigations show that the overall reaction rate is influenced by the method of mixing (and therefore the synthesizer). There is no difference in kinetic rates by shaking, gas bubbling or even no mixing [71], whereas vortexing (ABI 431, 433) and flow-through systems (MilliGen 9050) have impact on the overall kinetic rates. Vortexing and optimized flow-through systems show remarkably higher coupling rates compared to other systems. We have investigated continuous flow systems in detail. Coupling rates are strongly influenced by the flow rate of the reagent under CF conditions and therefore adjustment of flow rate is an important factor for successful synthesis in CF systems (Fig. 16-7). We have increased the flow rate up to 20 ml/min during coupling. Acylation of 1 g of resin was performed then within 6- 12 min (dependent on activation) instead of the usually applied reaction time of 30-60 min. An efficient coupling and derivatization protocol was developed and adjusted to the MilliGen 9050 synthesizer. By using this high flow protocol, the synthesis time is 3-4 times less than at "normal" flow rates at 5 ml/min [72]. The combination of high mass transport into the TentaGel resins [66], high kinetic rates and optimized conditions allow synthesis conditions to be used where diketopiperazine formation on the dipeptide step can be prevented. The hexapeptide KHPRPP was synthesized applying a standard protocol on a MilliGen 9050 synthesizer (7 min Fmoc deprotection, 12 min DMF wash, 30 min acylation).

a' 3

8 U

s

/

'

/

/ , /

0

w >

/

80-

70-1

60

____ . . .

,

"

.

'

I

--

ZOmllmin l S m l Imin 10mllmin Smllmin

Figure 16-7. Dependence of coupling time on flow rate in CF-peptide synthesis.

16.3 Peptide Synrhesis

437

Approximately 50% of the peptide was lost during Fmoc deprotection of Pro' by diketopiperazine formation. By reducing the Fmoc deprotection time to 2.5 min, there is no remarkable loss of the peptide by diketopiperazine formation [73]. As indicated previously and shown in Table 16-1, TentaGel resins are compatible with many solvents, either aqueous or pure organic systems. There are currently two methods available to obtain 0-phosphorylated peptides: either by global phosphorylation on the resin or by the use of preformed building blocks (74, 751. Recently, we have described an alternative way where we utilize the compatibility of TentaGel resins to aqueous and organic solvents [76]. The partial sequence RLMAGDAYAAHAG of the tyrosine kinase was synthesized by Fmoc strategy and DIC/HOBt or BOP activation was used. Tyrosine at position 8 was attached via DIC/HOBt activation without 0-protection and immediately phosphorylated. The phosphorylation was carried out with bis(benzy1oxy)-(diisopropyl-amino)phosphine in the presence of tetrazole (7 fold excess, 10 min) followed by oxidation with iodine in a water/THF/lutidine solution. The synthesis was continued after phosphorylation and finally the phosphorylated peptide was biotinylated. Biotin was introduced by N"-biotinyl-aminocaproic acid-N-hydroxysuccinimide or stepwise via Fmoc-aminocaproic acid attached to the resin-bound peptide, followed by biotinylation. Biotin was activated by CDI, HBTU or BOP and coupled at 50°C in NMP. CDI activation is the method of choice, whereas the N-hydroxysuccinimide activation was the least effective but most expensive one [77]. Figure 16-8 shows the results of the synthesis. Scaling up the synthesis to 10 g of resin (2.2 mmol) yields the peptide in quantities of 3.6 g with an overall purity of 78% compared to 80% purity for the small-scale

K * .~,: HN

I

I4

8

12

i

16

20

NH

9

-NH&,

24

A

750

Arg-Lys-Met-Ala-Gly-Asp-Ala-Tyr-Ala-Ala-Hls.Ala. I

0

O=P-OH OH

loo0

1250

1500

1750

d Z

Figure 16-8. HPLC and electrosDray-MS of Durified biotinvlated tyrosine kinase DeDtide.

438

I6 PEG Grafted Polystyrene Tentacle Polymers

synthesis (Fig. 16-9). Optimized protocols and high kinetic rates reduce the synthesis time for the up-scale synthesis to 14.5 h [78].

sy Figure 16-9. HPLC of crude peptide RLMAGDAYAAHAG. CF-synthesis with a) 500 mg resin (0.1 mmol) and b) 10 g resin (2.2 mmol).

16.4 Monosized Tentacle Microspheres for Screening and High Speed Peptide Synthesis Because of the heterogeneous nature of the reaction in solid phase peptide synthesis, resin parameters like polarity, particle size and solvation, mass transport and diffusion are of prime importance in all polymer supported reactions [79]. The driving force for mass transport is diffusion, and diffusion is dependent on path length. All solid supports which are normally used in solid phase chemistry show a more or less broad particle size distribution. Particle sizes of polystyrene, polyacrylamide and TentaGel resins are in the range 50-200 pm, kieselguhr/polyamide and Polyhipe in the range 500-1000 pm. Therefore, mass transport and reaction rates are individual parameters for each particle, whereas the overall reaction time is controlled by the largest bead (801. To overcome these problems, we have developed small monosized 15 and 25 pm tentacle beads [81]. The monosized nature of the beads divides the total reaction space (represented by all beads) into identical small reaction compartments which are all of equal size. The monosized nature and the uniform architecture allow the reaction conditions to be optimized to a great extent because of identical reaction conditions for each individual bead. In contrast to 90 pm particles, where

16.4 Tentacle Microspheresfor Screening and High Speed Peptide Synthesis

439

the release of the Fmoc group and the washout was finished within 2.5 min, the time can be reduced to 80 s by using 25 ym particles at 45 “C. This time corresponds to the kinetic rate of the Fmoc deprotection. There is no longer any contribution of the mass transport to the overall reaction rate. For peptide synthesis, the following conditions are applied: for the ABI 431 A and 433 synthesizers: 200 mg resin, 180 s acylation, 2 x 2.5 min Fmoc deprotection; and for the continuous flow MilliGen 9050 synthesizer: 200 mg resin, 180 s acylation, 120 s Fmoc deprotection, 60 s washout, 45”C, total cycle time: 7 min 37 s. These short cycle times allow even larger peptides to be synthesized and screened within hours. In combination with modern analytical methods like HPLC/MS and control by on line monitoring, all synthesis and analytical data are available within shortest time. The nonapeptide oxytocine was synthesized within 80 min and the 31-peptide Pendorphine was built up and synthesized within 4.55 h [79, 811. By using the described synthesis conditions, so-called difficult sequences have been investigated (e. g. a partial sequence of the gp 41 [79]) very intensively and it was found that solvent mixtures, increased reaction temperatures, and agents which delay the onset of stable conformations, are the conditions of choice. Bayer and Goldammer [82] reported on the use of ethylene carbonate for destroying peptide secondary structure, and the use of DMF/NMP/CH2C12 containing 1 To Triton X-100 has been reported to be a very potent solvent system for prevention of peptide secondary structure and solubilizing the peptide [79, 80, 831. Triton X-100acts as a nonionic detergent. Due to the surfactant properties of Triton X-100, the formation of secondary structure of the polymer-bound peptide and its aggregation are prevented and the peptide remains solubilized (Fig. 16-10). Finally, a “magic mixture” was created for keeping the peptide solubilized and for prevention of secondary structure formation during synthesis 184). The “magic mixture” consists of DMF/NMP/CH2C12/1To Triton X-100 and 1.5-2.5 M ethylene carbonate (washing and coupling) and DMF/NMP/20%Pip/lTo Triton for Fmoc cleavage. The reaction temperature was raised to 42°C.

Figure 16-10. Prevention of peptide secondary structure by Triton X-100.

440

16 PEG Grafted Polystyrene Tenracle Polymers

We have screened the complete C-terminus of the histone H1 c-(176-220)-peptide:

KVKTPQPKKAAKSPAKAKAPKPKAAKPKSGKPKVTKAKKAAPKKK. The pre-

view synthesis of this 45mer peptide was completed within 7 h 15 min [84]. Breakdowns for the coupling efficiency and Fmoc deprotection are recognized in the regions 209-205 and 176- 183. This phenomenon is caused by bad solvation or peptide conformations on the resin.This finding was very unexpected because, up to now, the C-terminal sequences of the histones are known to be random. For further investigation and synthesis optimization, the HI c sequence 176-220 was cut into two overlapping sequences: 176-200: 196-220:

KVKTPQPKKAAKSPAKAKAPKPKAA KPKAAKPKSGKPKVTKAKKAAPKKK

The most dramatic decrease in the Fmoc deprotection yield was recognized for K 206: only 54% using TBTU activation and 57% with Fmoc NCAs (Fig. 16-11). IR spectra of the resin-bound peptide (196-220) show shifts for the amide I and amide I1 bands which indicate @-sheetand helical conformations. Therefore, it is reasonable that a change in the activation method did result in a negligible change of the yield because the reactive sites are not accessible. To overcome this problem, the solvent system was changed from DMF to a mixture of DMF/NMP/CH,CI, containing 1 Vo of Triton, a nonionic surfactant. The yield increased to 69%, and pro9

Fmoc cleavage

lW 80

-

60

-

40

-

20

-

86

TBTU DMf

NCA OMf

TBTU DMFINMPIDCM Triton

TBTU OMF/NMP/DCM Triton extended coupling

93

TBTU 'magic mixture'

Figure 16-11. Screening of the partial sequence 196-220 of histone Hlc KPKAAPKSGK PKVTKAKKAAPKKK: Coupling yields of K 206 dependent on activation method and struc-

ture destroying reagents.

16.4

Tentacle Microspheres for Screening and High Speed Peptide Synthesis

441

longed coupling reactions in the region 206-209 increased the yield to 86%. Due to the surfactant properties of Triton, the peptide conformations on the resin are destroyed. The total coupling and deprotection yield for K 206 rised to 93% when the “magic mixture” was used. The crude peptide was recovered with 90% purity. This effect of the “magic mixture” was confirmed by synthesizing several other difficult sequences successfully. These results encouraged us to synthesize the complete HI c sequence 176-220, again applying such optimized conditions. Figure 16-12 shows the HPLC and electrospray MS of the crude completely protected peptide. Due to the still incomplete coupling of K 206, we find the deletion peptide [-Lys(Boc)] corresponding to the mass signal 1711.5 [M + 4HI4+ and a second failure sequence 1693 [M + 4HI4+ where the dipeptide Lys(Boc)-Ala is missing. CD investigations of both the free peptide and the completely protected HI c-peptide confirmed the peptide conformations as expected: each of the two peptides adopted helical structures. From these experiments, it is obvious that the synthesis of longer conformational peptide libraries may suffer from serious problems: for example, the systematic introduction of X positions along a transmembrane segment of a membrane protein cannot be recommended without extensive optimization of the synthesis conditions. Although the use of solventldetergent mixtures as discussed above did improve the

[M + 5H] 5+1

[M + 6H]

1.5

6+

1180.0

[M + 4H) 4+ 1768.5

- Lvs

1000

1711.5 1

I

1

I

I

2000

IW

I

Figure 16-12. HPLC and electrospray-MS of completely protected histon H1 c sequence 176-220. Molecular mass of the protected peptide: 7096 D. HPLC was performed on a RPPl column, macroporous high crosslinked PS/DVB, 6 pm particle size, 125 x 4.6 mm, linear gradient: A: water, B: MeCN, 5B%-95B%, 55 min.

442

16 PEG Grufted Polystyrene Tentacle Polymers

yields considerably in the synthesis of all six transmembrane helices of the sh-potassium channel of Drosophilu melunoguster, the conditions could not be optimized to give a satisfying general protocol [85].

16.5 TentaGel Peptide Conjugates in Immunization Small antigenic molecules (haptens) such as peptides cannot initiate an immune response unless they are covalently bound to a carrier molecule. Commonly, they are hooked to carrier proteins providing also T-helper epitopes, e. g. BSA or KLH. The preparation of such immunoconjugates involves several additional steps besides synthesizing the peptide antigen, e.g. cleavage of the peptide from the resin, recovery of the peptide and coupling to the carrier protein. Very often, the poor solubility of the peptides causes problems in immobilization of the peptide to the carrier protein. In most cases the immobilization is statistical. On the other hand, unspecific antibodies are induced against the carrier protein. Several attempts were made to use synthetic carriers like polystyrene or polyacrylamide for synthesis and induction of antibodies. Time consuming cleavage, work-up and recoupling steps are not necessary [86-90]. Besides the advantage of synthetic carriers (easy handling, no unspecific interactions with the matrix, etc.) there are undoubtedly some drawbacks which have to be taken in account. Because of the heterogeneous nature, polymeric carriers often show incompatibility or toxicity in vivo or, in general, adverse effects are consequences of chemical and physical heterogeneities on the surface of the carrier systems. With the TentaGel resin, this adverse effect can be avoided because of the following: (i) chemically and physically homogeneous and uniform surface structure; (ii) increased hydrophilicity and chemical inertness ; (iii) identical links between all hapten groups and the polymer matrix; (iv) uniformly linked hapten molecule structures; (v) maximally exposed hapten structures due to the long PEG spacers and therefore optimal contact with the immunocompetent cells. Peptide fragments from the noncatalytic Zn2+ binding loop of liver alcohol dehydrogenase isoenzymes and of N-and C-terminal parts of histone H1 were synthesized on the resin and used as resin-bound epitope conjugates for immunization [91]. Free peptide, Eupergit-bound peptide and TentaGel-hooked peptides are used in in vifroand in in vivo immunization experiments. As a result of this investigation, it seemed that TentaGel-bound epitopes stimulate the growth of more clones in comparison to free peptide or Eupergit-bound peptide. Immunization with TentaGel in vivo shows no adverse effects in contrast to Eupergit. By using a special designed resin with an acid-labile matrix/PEG linkage for immunization procedures, moderate acid treatment (90-95'70 TFA, 1-3 h) deprotects

16.5 RntaGel Peptide Conjugates in Immunization

443

the peptide, but the peptide remains bound to the resin, whereas harsh acid treatment (95%-99% TFA, 1070 trimethylsilyl-bromide, 6-8 h) deprotects the peptide and cleaves off the C-terminal PEG-modified peptide. PEG solubilizes even insoluble peptides and therefore allows investigations of poor soluble peptides in solution. It has to be pointed out that PEG modification of proteins suppresses the immune response, whereas linear PEG peptides can induce antibody production. For comparison of immunogenicity we have synthesized the epitope 115-131 of the Huemophilus infruenzueP6 protein. Balbhmice were immunized by the following conjugates: PEG-epitope, TentaGel-epitope, Pam,Cys-epitope and BSA epitope. Serum samples were used for detection of antibodies in an ELISA. All conjugates are able to induce antibodies which recognize isolated P6 [92]. Except for the BSA conjugate, all synthetic immunogens show no nonspecific reactions. As the antibodies are directed specifically against the peptide and not against the resin, the TentaGel peptide resin can be used simultaneously for immunization and as a highly selective affinity column for antibody separation [93]. P14L, a 14-amino acid residue peptide from the C-terminus of mouse-p-catenin was synthesized by Fmoc strategy as a resin immobilized MAP system. Rabbits were immunized and those antibodies recognizing p-catenin specifically in uvomorulin-catenin complexes were separated from the serum by affinity chromatography using the TentaGel MAP peptide column. 50 mg on the TentaGel MAP peptide column recovers 60-80 pg of purified antibodies. Pam,Cys-peptides [94] and PEG-peptides are systems which can induce an antibody response without resp. with adjuvant. PEG-Pam,Cys itself is a new adjuvant system which shows several advantages in comparison with others, e. g. Titermax, Vegetan, Freund’s adjuvant 1951. A new immunization complex was created by the combination of PEG-peptide, PEG-Pam3Cys and Pam,Cys-peptide. Within the new immunization complex, the adjuvant system is integrated and covalently bound to the immunogenic part (Fig. 16-13). The lipopeptide-PEG-conjugate is very easy to synthesize by automated multiple simultaneous peptide synthesis, and ready for use directly after synthesis. No additional conjugation and adjuvant are necessary. The immunogenic potency of the Pam,Cys-PEG-peptide conjugate by foot pad immunization of Balbk mice was tested without additional adjuvant, and stimulation of T-cells was demonstrated in a proliferation assay (data not shown). For analytical control of the synthesized PEG-lipopeptide, electrospray-MS can be applied because chromatographic procedures are not suitable (Fig. 16-14). A series of peaks due to the molecular mass distribution of the PEG were found. The difference of each peak is 44 mu corresponding to one ethylene oxide unit. The theoretical mass of 6050 Da was detected, corresponding to the lipopeptide (2659 Da) and the mean mass of PEG (77 monomer units, 3391 Da). The synthesized conjugates therefore consist of a constant lipopeptide part and a variable PEG part [961.

444

16 PEG Grafted Polystyrene Tentacle Polymers

-

NHBM

POE

-

NHF-

1. P a m l C y r 2. p e p t i d e

sy n t h e s{ s

% peptide

T F A cloavnge

T F A depmlection

Figure 16-13. Synthesis scheme of PEG-Pam$ys-epitope

lipopeptide conjugate 1961.

0.20 0.18 0.16 0.14 0.12

0.10 0.08 0.06 0.04 1,

.

.

,

, . .

5300

.

. 5800

I

6300

-

-

.

.

,

6800

'

.

.

.

m/Z

Figure 16-14. Electrospray-MS of PEG-Pam,Cys-epitope lipopeptide conjugate. Epitope

sequence of the peptide from measles F protein: LLGILESRGIKARIT. Theoretical mass: 6050.8 D. found molecular mass: 6050

D.

16.6 OligonucleotideSynthesis

445

16.6 Oligonucleotide Synthesis In oligonucleotide chemistry, controlled pore glass (CPG) is the preferred solid sup. port [97]which typically shows a loading of 20-40 pmollg. With the antisense concept, relatively large quantities (multiple gram scale) of expensive synthetic oligonucleotides are required for clinical trials, diagnostic assays and physicochemical studies. Low crosslinked polystyrene which is used successfully in peptide synthe sis, shows very poor results in oligonucleotide synthesis [98-1001.The coupling efficiencies on polystyrene and TentaGel supports are compared: the coupling yield drops down on polystyrene after five cycles to less than 20%. The overall yield 01 oligonucleotide on polystyrene was 11 9'0whereas TentaGel gives a total yield of 87% [loll. High loaded CPG loses efficiency and shows mechanical instability during vortexing or vigorous mixing conditions [102-1051.Sinha et a/. [lo61recently have described a high-loaded CPG support (90-110 pmol/g) for synthesis of oligonucleotides up to the 1 mmol scale. Another drawback of CPC-glass is that, during cleavage by heating with NH,, a remarkable amount of the support is destroyed and precipitates with the product. TentaGel does not show the problems mentioned above, since the resin is stable against basic and acid treatment. Several oligonucleotides were synthesized by standard conditions for small scale oligonucleotide synthesis (0.2 pmol scale). For small scale synthesis, less than lmg of TentaGel resin is used [107-1101.Due to the high loading of the resin, standard small scale cartridges can be used for medium scale synthesis. Bleicher [I1 I]describes the synthesis of several antisense oligonucleotides and thioates as well as the modified synthesis protocols for 1 and 2 pmol scale on TentaGel on an ABI synthesizer. To increase the cell passage of the oligonucleotide, several PEG-modified nucleotides were synthesized. It is interesting to notice that the PEG-modified double strands show only 1-3 "C lower melting temperature compared to the unmodified double strand. Simple strands as well as the hybridized double strands are analyzed by micellar electrokinetic chromatography (MECC) and ESI-MS [101,1121. Medium-scale synthesis of 30-40 pmol of oligonucleotide in 10 pmol standard cartridges was described by Weiler et al. [113].They use also phosphoramidite chemistry, but in combination with P-eliminating protecting groups. This technique allows the oligonucleotide to be deprotected completely on the resin and the protecting groups to be removed simply by washing and filtration. The crude products have high purity, and time-consuming work-up can be avoided. Large-scale synthesis up to 1 mmol of oligonucleotidesand thioates was described by Andrus et al. [114].The oligos up to 24 mers are synthesized either by phosphoramidite or phosphodiester method on the large-scale ABI synthesizer 3902. For medium-scale synthesis (25-200 pmol) 6 equivalent excess of phosphoramidite nucleotides were used, whereas only 3-4 eq. were used in the case of 400-lo00 pmol synthesis. As an activator, the more acidic 5-ethylthio-l-H-tetrazole was used instead of 5-H-tetrazolc resulting in higher yields and purity of the oligonucleotide product.

446

16 PEG Grafted Polystyrene Tentacle Polymers

To minimize the depurination, 5 % dichloroacetic acid was used instead of the more acidic trichloroacetic acid. Up to 3.6 g of oligonucleotide were synthesized with an overall purity of 70% (Fig. 16-15). Recently, Andrus et a/. [115] have reported synthesizing PNAs on the resin and using the PNA-modified TentaGel resin as an affinity column for selective separation of oligonucleotide targets.

Figure 16-15. MicroGel capillary electrophoresis analysis of crude oligonucleotides a) 2lmer phosphothioate 5‘ GCG TCA CAG TCT GAT TTC GAC 3‘, 600 pm scale, 2.188 g yield, 15.3 min retention time, b) 18mer DNA S’TCA CAG TCT GAT CTC GAC 3’, 1 mmol scale, 3.66 g yield, 13.7 min retention time.

16.7 Macrobeads as Polymeric Microreactors: Peptide Libraries and Combinatorial Chemistry The synthesis of peptide libraries, and use of combinatorial chemistry methods for the generation of molecular diversity, require resins that are compatible with many organic reaction conditions as well as very polar and even aqueous solvent systems. Aqueous buffer systems are used for resin bound biological assays and screening. Based on the “divide, couple and recombine” concept for generating libraries, introduced by Furka [68] and further developed by Lam [69], a number of peptide libraries have been synthesized and screened on TentaGel beads of various particle sizes [116-1331. Whereas in peptide chemistry only a restricted number of solvents are in use (DMF, DCM, NMP), organic chemistry requires the whole range of solvents. With the development of combinatorial organic chemistry on solid supports, TentaGel resins are used extensively in this approach for screening reactions and creating small molecule nonpeptide libraries as well as tagging and encoding techniques [38,63,64, 134-1521. Fenniri el al. (1361 described a technique for a encoded cassette for the highly sensitive detection of the success of chemical reactions on TentaGel. They have svnthesized a DeDtide on the resin bv Fmoc stratem followed by DNA synthesis.

16.7 Macrobeads as Polymeric Microreactors

447

Enzymatic treatment in aqueous solution cleaved off the peptide-DNA complex which was then exposed to PCR.Virgilio and Ellman [I351 described the synthesis of P-turn mimetics on solid supports which involved a reduction of a disulfide bond in aqueous solvent mixtures. A clean reduction without side reactions was only found by using TentaGel supports. These two selected examples may demonstrate the importance of resin compatibility to different synthesis conditions. Nevertheless many applications suffer from the restricted amount of substance which is available on one single bead. Several attempts were made to overcome this disadvantage, either by increasing the degree of substitution on the bead, or by, e. g. lysine branching [121].Both attempts suffer from extremely slow reaction rates, incomplete reactions, intermolecular interactions within one bead and slow release from the support. Our goal was to increase the amount of substance per bead by increasing theparticle size and not the concentration of substance within the bead. Based on polystyrene-polyethylene glycol graft copolymers (TentaGel), macrobeads with unusually high particle sizes of 400-800 pm were developed. This increase of particle size raises the capacity/bead by a factor of 100 to lo00 to the nanomolar range, whereas the beads used so far have diameters of 90-130 pm and 50-200 pmol capacity. The yield obtained from one of these smaller beads by sequential cleavage is not sufficient for unequivocal analytical investigations and simultaneous application to bioassays. Dependent on particle sizes, capacities of 10-100 nmol/bead have been detected by quantitative Fmoc determination from single macrobeads. Each bead is characterized by measuring its size and capacity (Table 16-6).As the reaction volume and concentration of each single bead is now defined, the beads can be used as polymeric microreactors. The use of polymeric microreactors as new tools in combinatorial chemistry, bioassays and structural characterization of diverse molecules is described here. In many bioassays, high molecular weight biopolymers are used for screening a library. Due to the gelatinous nature of TentaGel resins, biopolymers (enzymes, receptors, etc.) cannot penetrate the resin and interact only with the outer surface of the bead. 98-99% of the reactive sites are located inside the bead and only 1-2% are accessible on the outer surface for interaction with large molecules. Vagner et al. (1221have described a “enzymatic shaving” method to modify the bead surface. We Table 16-6: Capacity of selected macrobeads, measured by UV absorption of Fmoc cleavage product bead size [km] expected [nmol] found [nmol] volume [nl] conc. [nmol]

458 459 494 547 14 14 18 24 21 20 25 26 50 50 63 86 0.42 0.42 0.39 0.3

564 590 675 777 860 26 30 45 68 93 32 38 50 61 93 94 101 161 245 332 0.34 0.36 0.31 0.25 0.28

448

16 PEG Grafted Polystyrene Tentacle Polymers

have developed a chemical method to distinguish between outer surface of the beac and the internal reaction volume. The reactive sides on the surface and within tht beads are protected orthogonally by Fmoc and Boc. The ortogonal protecting group: were cleaved selectively by using either polymeric acids or bases followed by modifi. cation of the reactive sides inside the beads with two orthogonal handles: acid-labile handle AC (4-hydroxymethyl-3-methoxy-phenoxyacetic acid) or RAM (Rink amide,p[(R,S)-a-(9H-fluoren-9-yl)-methoxyformamido]-2,4dimethoxy benzylJ-phenoxy-acetic acid) and the base labilep-hydroxymethylbenzoicacid (HMB) to create a multifunctional microreactor (Fig. 16-16). Dependent on particle size, the accessible sides on the surface are in the range 0.1 - 1 nmol. This amount of reactivesides allows screening with bioassays, sequencing,mass spectrometryor HPLC investigations. We have sequenced peptides on a single 30 pm microparticle, and peptides attached only on the outer surface of 90 pm and 640 pm particles (Table 16-7). A reasonable signalhoise ratio was

Figure 16-16. Scheme of a trifunctional microreactor. Table 16-7: Peptide sequence data from one single bead of 30 pm diameter in comparison to surface attached peptides of modified 90 and 640 pm particles

Bead parameter Sequence found [pmol] Bead parameter Sequence found [pmoll Bead parameter Sequence found [pmol]

Size [pm]

Beadslg

Total capacity

Surface capacity

30

72 x

3.8 pmol

40-80 fmol

7.59

K 2.13

2.37

G 2.76

Size [pm]

Beadslg

Total capacity

Surface capacity

90

2.8 x

80- 100 pmol

1-2 pmol

v

P

E

lo6 lo6

Q

F

2.2

Y 3.1

Size [pm]

Beadslg

Total capacity

Surface capacity

640

6200

40 nmol

400-800 pmol

4.0

P 591.9

G 667.1

Y

632.3

2.7

F 558.1

S

A

K 1.2

N 0.3

0.48

1.17

K N 268.8 106.8

16.7 Macrobeads as Polymeric Microreactors

445

obtained with a minimum amount of 2-4 pmol peptidelbead [70]. This means thal the whole amount of peptide on a 30 pm bead is neccessary for analysis and identification, whereas on particles of >90 pm the peptide content of the surface is sufficient. The capacity per bead and the number of beads per gram resin are strongly dependenl on the bead size. Figure 16-17correlates particle size, capacity per bead and the number of beads per gram. The amount of 10-90 nmol substance inside the 400-800 pm macrobeads allow multible sequential release from one single bead. The released compounds from 90 or 130 pm particles typically yield 1-3 p~ solutions (100-300 pmol/ 100 pl) compared to 1-5 mM solutions (2-10 nmoV200 pl) obtained by 5 sequential releasing steps on 400-800 pm beads. Thus assay sensitivity increases by a factor of 100- 1OOo. Based on the described polymer, three peptide libraries are synthesized on trifunctional macrobeads: acid-labile amide handle R A M and base-labile HMB are located inside the bead, and aminofunctions are on the surface [96]. Subsequent synthesis of the libraries XXYFKN, PXXFKN, PEXFKN by the “divide, couple and recombine” technique [68,69] results in a trifunctional peptide resin with acid-cleavable peptides, base-cleavablepeptides and noncleavable peptides on the outer surface. TFA treatment deprotects the peptides and creates a soluble peptide library which was screened by a solution assay. 60 beads of the polymer-bound hexapeptide amide library PEXFKN-NH, were investigated by ELISA and fluorescence assays. Peptide beads were filled in filter tubes (1 -8 per vessel) and, after washing steps, they were incubated with the Mab SN 20 which binds to the HIV 1-Nef protein epitope PEYFKN. Staining of antigen beads was

I>c.,adF/e Figure 16-17. Correlation of Darticle size, caDacity and number of beads/g resin.

450

16 PEG Grqfted Polystyene nntacle Polymers

achieved by peroxidase conjugated antimouse antibody and enzyme substrate tetramethylbenzidine.Blue colored beads were selected manually by a pair of tweezers. Basic treatment of the selected bead released the peptide from HMB linker. The soluble peptide from each bead was then characterized and identified by HPLC, MS and AAA. Five beads were selected and the following sequences identified by sequencing of the surface bound peptide: 3 x PEEFKN-NH2, 1 x PEGFKN-NH2 and 1 x PEYFKNNH2. The amount of peptide immobilized on the surface of a single macrobead is also sufficient for a quantitative ELISA. Mab SN 20 incubated beads XXYFKN were investigated for interaction with a tetramethylrhodaminelabeled anti mouse antibody (Fig. 16-18). ELISA positive beads

YH-NKFYXX

Figure 16-18. Screening of the library XXYFKN bound to macrobeads. a) incubation with Mab SN 20 antibody, b) rhodamin labeling with anti antibody, c) selection.

were detected by confocal laser microscopy, and beads showing surface fluorescence were selected. For positive control, 90 pm AcPEYFKN-beads were added (Fig. 16-19). The peptide sequence was determined following the same procedure as shown above (Fig. 16-20). Competition of peptides mixtures with the biotinylated antigen N-a-biotinyl-N-a-Ac-PEYFKN-Ahx-Ahx-Lys for binding to the Mab SN 20 was achieved with soluble peptide mixtures Ac-XXYFKN-NH2, Ac-PXXFKN-NH2 and AcPEXFKN-NH2. Streptavidin coated ELISA plates were incubated with increasing concentrations of acetylated hexapeptide amide mixtures and dose dependent (0.5 pg-0.5 mg) competition was obtained pig. 16-21). Both the solution assay and the resin bound ELISA indicate that is not essential for binding as expected up to now. For investigations of peptide/oligonucleotide interactions, the C-terminal sequence KSGKPKVXlX2A(XI = K,L,G,Y,F and X2 = R,L,G,E,F) of the histone H1 c was synthesized as a sublibrary [1531. The sublibrary was constructed on trifunctional macro-

m-3

16.7 Macrobeads 4s Fblymeric Microreactors

451

Figure 16-19. Detection of positive beads (400-800pm) by confocal laser microscopy of fluorescence labeled library. The small 90 pm PEYFKN-resin beads were used as positive control.

Sequence

P

0

Y

F

K

N

pmol

591.9

667.1

632.3

558.1

268.8

106.8

452

16 PEG Gmfted Polystymne lhtacle Polymers

Figure 16-21. Competition assay of the soluble library XXYFKN with Ac-PEYFKN-bio. tinylated peptide for binding to Mab SNU) antibody in a biotinlstreptavidm assay.

beads (NH2on the surface, AC and HMB inside) as described above Acid treatmenl deprotects the peptides and cleaves off approximately 50% of the peptide This procedure results in a soluble and a resin bound peptide library within one synthesis run. In natural systems,.histones interact with the DNA. To screen the oligonucleotide interaction with the modified histon sequences, 25 beads of the polymer-bound pep tide library were incubated with the fluorescence-labeledoligonucleotide5’-CAC TCG TTA G-FZ3’. To differentiate between the strength of the interaction, single beads were incubated with increasing concentrations of aqueous NaCl. Negative beads 01 beads with reduced intensity are selected. Release of the peptide by basic treatment, followed by HPLC and MS analysis, identifies the peptides. As a result, Phe decreases the interaction, whereas all sequences having show strong interaction. Hydrophobic amino acids show weak to medium interactions (Table 16-8). In combinatorialchemistry there are, in principle, two distinct approaches for construction of libraries: construction of the library by the split and recombine procedure as described above, or the spatially addressable technique where the compounds a r e synthesized individually by parallel and often semiautomated synthesis techniques. Reaction screeningand optimizationwere also performed in dozens of individual parallel reactions. The reduction in reaction volume to the size of one single bead allows the amount of required solvents to be minimized as well as the amount of expensive building blocks to the nmol range [153]. Even when applying a 100-foldexcess of building block, several hundered reactions can be carried out with one mmol of building block. Combinatorial chemistry methods were applied for the synthesis of a set of hvdantoins on single beads in nlass cadlaries (Fin. 16-22. Table 16-9). The formation

16.7 Macrobeads as Polymeric Microreactors

453

Table 16-8: Decreasing stability of the fluorescence-labeled resin-bound KsGKPKVXIX2AHCACTCGTTAG-FZ3' complex with increasing concentrations of NaCl solution: w = weak, m = medium, s = strong

Histone sequence KSGKPKVXXA KSGKPKVTKA KSGKPKVRKA KSGKPKVRLA KSGKPKVRGA KSGKPKVRYA KSGKPKVRFA KSGKPKVLKA KSGKPKVLLA KSGKPKVLGA KSGKPKVLYA KSGKPKVLFA KSGKPKVGKA KSGKPKVGLA KSGKPKVGGA KSGKPKVGYA KSGKPKVGFA KSGKPKVEKA KSGKPKVELA KSGKPKVEGA KSGKPKVEYA KSGKPKVEFA KSGKPKVFKA KSGKPKVFLA KSGKPKVFGA KSGKPKVFYA KSGKPKVFFA

Fluorescence after wash with 0.2 M NaCl solution

W

M

xx xxxx xx

S

Fluorescence after wash with 0.5 M NaCl solution

W

xx xx xxxx xx xx xx xx

3z

xx xx xx xxxx xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx

M

xx xx

xx

xx xx xx xx

S

Fluorescence after wash with 1 M NaCl solution

W

M

S

xx xx xx xx xx xx xx xx xx xx xx xx xx xx xx 3z xx xx xx xx xx xxxx xx xx xx xx xxxx xx xx xx xx xx xx xx

xx xx xx xx xx xx xx xx xx xx

454

16 PEG Gnlfted hlystymne nntacle Polymers

Figure 16-22. Single microreactor in a glass capillary of 2.6 mm diameter, bottom: sinter glass filter, top: open, solvent volume: 25 kl.

lbble 16-9: Hydmtoins kOCHRNHCONR' synthesized on TentaGel microreactors. 'l)pical reaction conditions: 1-4 single beads/capillary, Fmoc-amino acid attached to the acid labile AC handle, 20 pl 1 M isocyanate solution, reaction time 3 h, no protecting gas. Cleavage and coodensation: 6 M HCl in water, 100°C, 45 min, 4 M HCI in tetrahydrofurane, KT, 120 min Compound

R

R'

Hydantoin 1 Hydantoin 2 Hydantoin 3 Hydantoin 4 Hydantoin 5 Hydantoin 6 Hydantoin 7 Hydantoin 8 Hydantoin 10

phenyl phenyl phenyl H H H isopropyl isopropyl isopmpyl

phenyl ProPYl phenylethyl phenyl ProPYl phenylethyl phenyl ProPYl phenylethyl

16.7 Macrobeab as Polymeric Microreactors

455

of each resin-bound compound was controlled during synthesis and identified on each single bead by gelphase 'H NMR (Fig. 16-23). After release and cyclization of the hydantoins from one single bead, the compound was analyzed and characterized by HPLC and G U M S (Fig. 16-24). One of the key steps in all combinatorial aproaches is chemical analysis and structural identification of "hits" within a library. Identification by indirect methods like tagging or encoding has been proposed. An alternative way may be the direct identification of the ligand on the resin by the combination of different spectroscopic methods, ag. gelphase proton MAS NMR on single beads as shown in Fig. 16-23, FT-IR spectroscopy on single beads [141] as well as gelphase "C NMR spectroscopy. A "C enriched tripeptide was synthesized on macrobeads, and one bead was analyzed by 13CNMR spectroscopy. The modified 13C microprobe containing one bead is illustrated in Fig. 16-25a and Fig. 16-25b shows the resulting NMR spectrum.

Figure 16-23. Gelphase MAS 'H N M R of one single bead. Bruker NMR spectrometer A6-300,rotor 4 mm (modified), spinning frequence: 2000 Hz, solvent CDCIP, scans: 256.

Figure 16-24.

-..

:10

0:20

030

0.

0.269

0.538

0.808

.on 1

2.423

-.-A

w-91w-ia 175188

I"

200

__I.

141

lZ0

HPLC and GC/MS of crude hydantoins. TOR:M+ :260 D. bottom: M+ :232 D (phthalat impurities from sealings).

005

I

phthalat

2.692

j

1.243-

0.628

JL I

-

-

1.657

2.071

2.485-

3.728

3.314 2.899 -

4.142 -I

266

16.7 Macrobeads as lblyrneric Microwactors

457

Figure 16-25s. Single bead in a 13C NMR microprobe. The bead was adjusted by Teflon plugs.

I

-

I

lB0

.

160 I

*

1

uo

'

l20 I

"

.

mo

"

(ppn3

'

"

80

I

60

40

.

I

20

.

Figure 16-25b. Gelphase "C NMR of a single 740 pm Boc-Val-Ala-bead ("C enriched danine (CH3) and vane (CO) marked by *. Bruker 600 MHz, scans: 5920, solvent CDC13, tema 298 K.

458

16 PEG Grqfted polystyme Tentacle polymers

References (11 R. B. Memfield, Fed. Pmc., Fed. A m SOC. Exp. Biol 1962,21, 412. (21 R. B. Merrifield, A Am. Chem. Soc. 1963,85, 2149. [3]G. R. Marshall, R. B. Merrifield in Biochemical Aspectsof Reactions on Solid Supports, Academic Press, NY;1971. [4]G. Bamy, R. B. Merrifield, The Peptides, Academic Press, NY; 1980, 2. [5]R. L. Letsinger, J. L. Finnan, G. A. Heavner,W. B. Lunsford, A A m Chem SOC.1975, 97, 3278. [a]E. Uhlmann, A. Peyman, Chem. Rev. 1990, 90, 543. [7]J. W.Engels, E. Uhlmann, Angew. Chem. 1989,101,733;Angew. Chem. Znt. M.Engl. 1989, 28, 716. [8]R. H. Grubbs, L. C. Kroll, 1 Am. Chem SOC. 1971, 93, 3062. [9]C. U. Pittmann Jr., L. R. Smith, A Am. Chem. SOC. 1975, 97, 1749. [lo]C. U. Pittmann Jr., L. R. Smith, R. M. Hanes, J Am. Chem. Soc. 1975, 97, 742. [ll] R. H.Grubbs, Chem. Txhnol. 1977, 512. (121 Y. Chaurin, D. Commerenc, F. Dawens, Pmg. polym. Sci. 1977, 5, 95. (131 V. A. Kubanov, V. J. Smetanyuk, Makmmol Chem. Phys., Suppl. 1981, 5, 121. [l4]G. Manecke, W.Storck, Angew.Chem. 1978,90,691;Angew. Chem. Znt. Ed. Engl. 1978, 17, 657. [I51 C. U. Pittmann Jr., Comprehensive Organometallic Chemistry (Ed.: 0. Wilkiwn), Pergmon Press, Oxford, New York, Toronto, Sydney, Paris, Frankfurt, 1982, Vol. 8, p. 553ff. (161 M. Kaneko, E. Tsuchida, Macmmol. Rev. 1981, 16, 397. (171 H. D. Orth, W. Brllmmer, Angew. Chem. 1972, 84, 319;Angew. Chem Znt. Ed. Engl. 1972, 11, 249. [I81 H.H. Weetall, Chem. Ztg. 1973, 97, 611. 1191 G. Manecke, Chimia 1974, 28, 467. I201 J. Chibata in Zmmobilized Enzymes,Wiley, New York, 1978, pp. 9-142 and references therein. 1211 G. Manecke, E. Ehrenthal, J. Schlllnsen, Chamcterization of ZmmobilizedBiocatalysts (Ed. : K. Buchholz) Schon u. Wetzel GmbH, Frankfurt, 1979, pp. 49-109 and references therein. [221 B. P. Scharma, L. F. Baily, R. A. Messing, Angew.Chem 1982,94,836;Angew. Chem. Int. Ed Engl. 1982, 21, 837. 1231 L. Goldstein, G. Manecke, Applied Biochemistry and Bioengineering @is.: L. S. Wingard Jr., E. Katchalski-Kutzir, L. Goldstein), Academic Press, New York, San F m Ciscq London, 1976, VoI. 1, pp. 23-110 and references therein. [241 John F. Kennedy in Solid Phase Biochemistry, Analytical and SyntheticAspects (Ed.: William H. Scouten), John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, 1983,pp. 257-361 and references therein. [251 K. Podla, Biotechnology Vortrag Hauptversammlung MNU, 74th 1983, 21 ff. (261A. Rosevear, J Chem Ipchnol. Biotechnol, Biotechnol. 1984, 348(3), 127. [271 A. S. Attiyat, G. D. Christian, Am. Biotechnol. Lab., 2(2) 1984, 8,10, 12-16. 1281 T. M. Fyles, C. C. Lenzoff, Can. A Chem 1975,54, 935.

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460

16 PEG Grafted Polystyrene Tentacle Polymers

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462

16 PEG Grafted Polystyrene Tentacle Polymers

(1061 S. Frey, N. D. Sinha, Solid Phase Synthesis of Peptides, Proteins, Nucleic Acids, Biological and Biomedical Applications, Collected Papers, Third International Symposium, 1993, Oxford, UK, (Ed. : R. Epton) Mayflower Worldwide Limited, Birmingham 1994, pp. 9-20. [107)G. Griibler, H.Straubinger, W. Reinig, H. Echner, M. Geiger, W. Voelter in Solid Phase Synthesis of Peptides, Proteins, Nucleic Acids, Biological and Biomedical Applications, Collected Papers, Third International Symposium, 1993, Oxford, UK,(Ed. : R. Epton) Mayflower Worldwide Limited, Birmingham, 1994, pp. 9-20. (1081 H.Gao, B. L. Gaffney, R. A. Jones, Tetrahedron Lett. 1991, 32, 5477-5480. (lOS]P. Wright, D. Lloyd, W. Rapp, A. Andrus, Tetrahedron Lett. 1993, 34, 3373-3376. (llO] E. Bayer, Angew. Chem. 1991, 103, 117;Angew. Chem. Int. Ed. Engl. 1991, 30, 113. (111) C. Bleicher, Thesis, University of Tubingen, 1995. (1121 E. Bayer, K. Bleicher, M. Maier, 2. Narurforsch. 1995, 506, 1096-1100. 11131 J. Weiler, T. Maier, W. Pfleiderer, Solid Phase Synthesis of Peptides, Proteins, Nucleic Acids, Biological and Biomedical Applications, Collected Papers, Third International Symposium, 1993, Oxford, UK, (Ed.: R. Epton) Mayflower Worldwide Limited, Birmingham, 1994, pp. 9-20. (1141 D.Tsou, P. Wright, D. Lloyd, A. Andreus, Solid Phase Synthesis of Peptides, Proteins, NucleicAcids, Biological and BiomedicalApplications, Collected Papers, Third International Symposium, 1993, Oxford, UK, (Ed. : R. Epton) Mayflower Worldwide Limited, Birmingham, 1994, pp. 9-20. (1151 A. Andrus, presented at the 4th International Symposium on Solid Phase Synthesis, Edinburgh, 1995. (1161 M. Lebl, V. Krchiiak, N. F. Sepetov, B. Seligmann, S. Felder, Biopolymers (Peptide Science), (Ed.: J. Wiley & Sons), 1995, 177-198. (117)S. E. Salmon, K. S. Lam, M. Lebl, A. Kandola, P. S. Khattri, S. Wade, M. Patek, P. Kocis, V. Krchiiak, D. Thorpe, S. Felder, Proc. Natl. Acad. Sci. USA (Chemistry) 1993, 90, 11708-11712. (1181 M. Lebl, V. Krchiiak, S. E. Salmon, K. S. Lam, Methods: A Companion to Methods in Enzymology 1994, 6, 381-387. (1191 K. S. Lam, Z. Zhao, S. Wade, V. Krchiilk, M. Lebl, Drug DevelopmentResearch 1994, 33, 157-160. 11201 V. Nikolaiev, A. Stierandova, V. Krchiiak, B. Seligmann, K. S. Lam, S. E. Salmon, M. Lebl, Peptide Res. 1993, 6, 161- 170. (1211 M. Lebl, M. Patek, P. Kocis, V. Krchiiak, V. Hruby, S. E. Salmon, K. S. Lam, Inl. J. Pept. Protein Res. 1993, 41, 201-203. (1221 J. Vagner, V. KrchiiBk, N. F. Sepetov, P.Strop, K. S. Lam, G. Barany, M. Lebl, Innovation and Perspectives in Solid Phase Synthesis and Related Technologies, Collected Papers, Third Int. Symp. Oxford, England, (Ed.: R. Epton) 1993, p. 347. [123]M. Lebl, V. Krchiiak, N. F. Sepetov, V. Nikolaiev, A. Stierandova, P. Safar, B. Seligmann, P. Strop, K. S. Lam,S. E. Salmon, Innovation and Perspectives in Solid Phase Synthesisand Related Technologies, Collected Papers, Third International Symposium, Odord, England, (Ed.: R. Epton) 1993, p. 233. (1241 M. Lebl, V. Krchiiak, P. Safar, A. Stierandovl, N. F. Sepetov, P. Kocis, K. S. Lam, Techniques in Protein Chemistry t: 1994, 541 548.

-

References

463

[I251 V. Nikolaiev, A. Stierandova, V. Krchiiak. B. Seligmann, K. S. Lam, S. E. Salmon, M . Lebl, Pept. R ~ s 1993, . 6, 161-170. [I261 M. Lebl, M. Patek, P. Kocis, V. KrchAak, V. Hruby, S. E. Salmon, K. S. Lam, Int. J. Pept. Protein Res. 1993, 41, 201 -203. [I271 P. Kocis. V. Krchiiak, M. Lebl, Tetrahedron Lett. 1993, 34. 7251-7252. [128]S. E. Salmon, K. S. Lam, M. Lebl, A. Kandola, P. S. Khattri, S. Wade, M. Patek, P. Kocis, V. Krchiiak, D. Thorpe, S. Felder, Proc. Natl. Acad. Sci. USA (Chemistry) 1993, 90, 11708-11712. [I291 M. Torneiro, W. C. Still, J. Am. Chem. SOC.1995, 117, 5887-5888. [130]H. Wennemers, W. C. Still, Tetrahedron Lett. 1994, 35, 6413-6416. [I311 R. S. Youngquist, G. R. Fuentes, M. P. Lacey, T. Keough, J. Am. Chem. SOC.1995,117, 3900-3906. [I321 H.Wenschuh, M. Beyermann, S. Rothemund, L. A. Carpino, M. Bienert, Tetrahedron Lett. 1995, 36, 1247-1250. [133]R. S. Youngquist, G. R. Fuentes, M. P. Lacey, T. Keough, J. Am. Chem. SOC.1995, 117, 3900-3906. [I341 J. J. Baldwin, J. J. Burbaum, I. Henderson, M.H. J. Ohlmeyer, J. Am. Chem. SOC.1995, 117, 5589. (135)A. A. Virgilo, J. A. Ellman, J. Am. Chem. SOC.1994, 116, 11580-11581. [I361 H. FeMiri, K. D. Janda, R. A. Lerner, Proc. Natl. Acad. Sci. USA (Chemistry) 1995, 92, 2278-2282. [137]F. W. Forman, I. Sucholeiki, J. Org.Chem. 1995, 60, 523-528. [138]G. C. Look, M. M. Murphy, D. A. Campbell, M. A. Gallop, Tetrahedron Lett. 1995, 36, 2937-3940. [139)T. A. Rano, K. T. Chapman, Tetrahedron Lett. 1995, 36, 3789-3792. (140)V. Krchiihk, 2. Flegelovi, A. S. Weichsel, M . Lebl, Tetrahedron Lett. 1995, 36, 61936196. [141] B. Yan, G. Kumaravel, H. Anjaria, A. Wu, R. C. Petter, C. F. Jewel1 Jr., J. R. Wareing, J. Org. Chem. 1995, 60, 5736-5738. [I421 J. R. Hauske. P. Dorff, Tetrahedron Lett. 1995, 36, 1589-1592. [143]M. Patek, B. Drake, M. Lebl, Tetrahedron Lett. 1995, 36, 2227-2230. I1441 I. Sucholeiki, Tetrahedron Lett. 1994, 35, 7307-7310. [145)M. Patek, B. Drake, M. Lebl, Tetrahedron Lett. 1994, 35, 9169-9172. [I461 J. Nielsen, S. Brenner, K. D. Janda, Proceedings of the 14th American Peptide Symposium, (Eds.: P. T. P. Kaumaya, R. S. Hodges) Columbus, Ohio, 1995, pp. 92-93. [147]F. W. Forman, I. Sucholeiki, J. Org.Chem. 1995, 60, 523-528. (1481 J. J. Baldwin, J. J. Burbaum, I. Henderson, M.H. J. Ohlmeyer, J. Am. Chem. SOC.1995, 117, 5588-5589. I1491 D.A. Campbell, J. C. Bermak, T. S. Burkoth, D.V. Patel, J. Am. Chem. SOC.1995,117, 5381-5382. [I501 M. M. Murphy, J. R. Schullek, E. M. Gordon, M. A. Gallop, J. Am. Chem. SOC. 1995, 117, 7029-7030. [I511 K. C. Nicolaou, X. Xiao, Z. Parandoosh, A. Senyei, M . P. Nova, Angew. Chem. 1995, 107. 2476-2479: Anaew. Chem. Int. Ed. End. 1995. 34, 2289-2291.

464

16 PEG Grafted Polystyrene Tentacle Polymers

[152] J. J. Burbaurn, M. H. J. Ohlmeyer, J. C. Reader, I. Henderson, L. W. Dillard, G. Li,

T.L. Randle, N. H. Sigal, D. Chelsky, J. J. Baldwin, Proc. Natl. Acud. Sci. USA (Phar. malogy) 1995, 92, 6027-6031.

[153] W. Rapp, M. Maier, G. Schlotterbeck, M. Pursch, K. Albert, E. Bayer, Peptides,

Chemistry and Biology, Proceedings of the 14th American Peptide Symposium, (Eds. : P. T. P. Kaurnaya, R. S. Hodges), Columbus, Ohio 1995.

Combinatorial Peptide and Nonpeptide Libraries by. Gunther Jung 0 VCH Verlagsgesellschaft mbH, 1996

17 Supports for Solid-Phase Organic Synthesis Martin Winter

17.1 Introduction Peptide synthesis on a solid polymeric support, introduced by Merrifield in the early 1960s [l], has led to a substantial progress in the synthesis of biopolymers such as peptides [2]and oligonucleotides. The great advantage of solid phase peptide synthesis (SPPS) is the highly simplified procedure for purification of the product during the repeated steps of the synthesis. In the ideal case, all employed soluble reagents can be removed completely and conveniently by filtration and washing the solid resin, whereas the product remains covalently bound to the solid support. After completion of a synthesis, the synthesized final product is split off from the solid support by using a cleaving reagent, followed by its isolation from the cleavage solution. The bond between the growing product and the carrier has to be stable towards all reagents used during the synthesis, but quantitatively must be cleavable at the end of the synthesis without damaging the final product. Consequently, direct attachment of the substrate to the polymeric support normally is not considered. Therefore, at first the polymeric support must be modified by introducing a cleavable linkage between the substrate and the support (normally an ester bond). The polymeric support used by Merrifield [3,4]was a cross-linked polystyrene-divinylbenzene (DVB) (2070)containing the reactive benzyl chloride functional group, introduced into the resin by chloromethylation. This so-called “Merrifield resin” could then be coupled to the first amino-protected amino acid via its carboxy group. The resulting ester bond is highly stable towards the coupling and deprotecting reagents used during the peptide synthesis, but it is cleaved at the end of the synthesis with HF or trifluoromethanesulfonic acid (TFMSA). Therefore, it fulfils one of the most important criteria of SPPS, namely the orthogonality principle. The first starting point to improving this resin was to vary its extent of cross-linking by increasing or reducing the divinylbenzeneconcentration in the polymerization mixture. Higher degrees of cross-linking (more than cu. 5 070) yield more stable resins but they swell less and consequently possess lower loading capacity. On the contrary, lower degrees of cross-linking enhance the swelling properties and loading capacity and yield gel phases in the presence of suitable solvents, but reduce the mechanical stability of the polymer beads. The best compromise between these two possibilities is achieved by cross-linking the polymer with 1% DVB [3,41. The accessibility of internal surface of the support for the substrate plays a decisive role. For the loading capacity of a polymer to reach an appreciable extent,

466

I7 Supports for Solid-Phase Organic Synthesis

the reaction solutions largely must penetrate the internal surface of the beads. Tc achieve this, the polymer must be solvated properly by the used solvents and the resir must swell sufficiently. In the case of nonpolar polystyrene-DVB resin these re quirements are fulfilled by dichloromethane (DCM) and dimethylformamidt (DMF). In these cases the resin swells enough (ca. 3-6 ml/g), so loading capacitie! between 0.5- 1.4 mmol/g can be obtained. In contrary to the batch synthesis in which the reagents and washing solvents art added to the polymer at one stage, the usual polystyrene-DVB resins are not suitabk for continuous flow procedures in which the support is packed in a column and the reaction and washing solutions flow through the resin continuously. The pores between the polymer beads necessary for the flow are closed rapidly by the swollen resin which leads to high pressures and to the collapse of the gel phase. Attempts to increase the mechanical stability of this resin by coating polystyrene onto glass beads was not successful, owing to the rubbing down of the resin and the low loading capacity [5, 61. Finally, a successful experiment was the use of kieselguhr as support matrix into which polydimethylacrylamide was polymerized [7]; the resulting material possessed the mechanical stability of kieselguhr and the chemical properties of polyacrylamide. These supports are in use until today, although they are surpassed in their continuous flow properties by other fully organic supports (TentaGel, PolyHIPE). The TentaGel, a polyethyleneglycol-polystyrene/DVB graft copolymer, developed by Bayer and Rapp [6,8], as well as PolyHIPE [9], a copolymer consisting of porous and highly cross-linked polystyrene/DVB and PEG/polyacrylamide, introduced by Small and Sherrington, are sufficiently pressure stable. By using these copolymers, no problems are encountered regarding the loss of polymer from the support matrix and the low loading capacity associated with the kieselguhr-polyacrylamide. A second approach to introducing mechanically and thermally stable supports was the use of inorganic supports. Glass or silica supports are characterized by thermal stability, but they have limited loading capacities. These materials (Controlled PoreGlass, Spherisorb, etc.) are normally employed for the solid phase synthesis of oligonucleotides, as long as only a small amount of product is needed [lo]. In contrast to organic supports, they cannot swell and therefore have to be appropriately chosen by adapting the pore sizes to the size of the product molecule. With the introduction of milder synthesis strategies for SPPS (Bpoc, Fmoc, etc. instead of Boc for the amino protection) ill] it has become possible to introduce special linkers for the attachment of substrates to the supports, and consequently to carry out cleavage reactions under milder conditions. Among the first new linkers was the so-cdled “Wang” Iinker [12] which could be cleaved by 50% TFA, thus enabling the synthesis of side-chain protected peptide fragments. Other linkers such as the Rink [13] linker allow the synthesis of peptide amides. At present a wide range of linker systems on different supports is commercially available which can be cleaved under different conditions and bv various reaeents.

17.1 Introduction

461

Among them are highly acid-sensitive groups such as the trityl [14]or the Sasrin system [15],base-labile systems such as HMBA [16] or PPOA [17],palladiumcatalyzed cleavable systems such as HYCRAM (181and photolabile systems such as Brominated Wang and Brominated PPOA. In certain systems, such as oxime support 1191, the cleavage is effected by a nucleophilic attack to produce C-terminally modified peptides by a simple method. Meanwhile, even the cleavage of fully protected peptide fragments is possible without great difficulty. To prevent the premature cleavage of the substrates from the supports under extreme synthesis conditions, the so-called “Safety-catch-Linker” [20]was developed which must be rendered cleavable in a preceding step prior to the final cleavage. In combinatorial chemistry [21],the supports and linkers must meet special requirements since, in this case drastic reaction conditions are often necessary. There has been great activity in this area recently, so the need for suitable solid phases has given rise to growing commercial offers of such materials. To make organic solid phase synthesis a success, it is of the utmost importance to be able to choose the most suitable support. The present summary is intended to facilitate this goal by presenting and comparing a large variety of commercially available supports and their properties, classified according to the sort of the support and the cleavage conditions of the linkers. In the first section the traditional polystyrene supports are treated for which the largest number of linker systems is already established. In the following sections, first the other organic supports such as TentaGel [8,221 or PolyHIPE [9]will be mentioned, secondly the organic resins polymerized on inorganic supports (Pepsyn K) [7],and finally the fully inorganic solid supports (CPG, CPC, etc.) are presented. The various derivatized supports are first classified according to their cleavage conditions (acidic, basic, etc.) and secondly, they are arranged according to the structural similarity of their linker derivatization. To give a short introduction to the field of yet less established supports and linker systems in solid phase organic synthesis, some special support components described in literature are shown in Section 9. In the glossary at the end of this Chapter an alphabetic list of the mentioned solid phases is presented. The given price ranges may help to estimate the financial expense incurred by use of the selected supports. They refer to the approximate prices for 25 g packages drawn from the suppliers 1994 price lists. Where physical data of a polymeric support is not furnished by the supplier, this is characterized by “ - ”. up to Price ranges I 100 DM (70 US$) (per 25 g): I1 250 DM (170 US$) 111 500 DM (350US$) IV lo00 DM (7OOUS$) V 2500 DM (1700 US$) VI > 2500 DM (1700 US$)

468

17 Supports for Solid-Phase Organic Synthesis

17.2 Polystyrene Supports Polystyrene supports employed for chemical syntheses are normally cross-linked with 1070 divinylbenzene. This degree of cross-linking offers the best compromise between mechanical stability and swelling properties [3, 41. The higher cross-linked polystyrenes are mechanically and thermally more stable; however, their degrees of swelling and loading capacity are noticeably lower. If otherwise not mentioned, all the polystyrene supports considered are cross-linked with 1070 DVB. Although functionalization with linker molecules can affect the physical properties of the resin in certain cases, in the first approximation the properties of the unmodified resins and the resin-linker systems can be assumed to be identical. Polystyrene supports generally show good swelling properties and high loading capacity, but possess disadvantages in regard to the mechanical and thermal stability; they are therefore not suitable for continuous flow procedures and reaction temperatures above 100“C. The typical values for swelling factors are between 4 and 8 ml solvent/g resin for solvents which solvate polystyrene adequately (dichloromethane, dimethylformamide, etc.), whereas the polystyrene resin shrinks strongly in less suitable solvents such as methanol or water and loses its gelatinous character. Thus, on one hand, there are advantages to washing the excess reagents; on the other, there are limitations on the choice of solvents for the polymer-supported reactions, since only in the gel form and in the swollen state all the reactive sites are accessible. Moreover, the interior section of the polymer beads always takes up those components in which polystyrene is well solvated. The reactants not compatible with polystyrene are always found in lower concentration within the beads than in the surrounding solvent. Polystyrenes cross-linked with more than 5 070 DVB show complicated behavior. Depending on the preparation conditions, these “microporous” resins possess several kinds of varyingly large pores which arise as spaces from aggregation (a) of ca. 10 nm polymer nuclei to the so-called microspheres, (b) of ca. 100 nm microspheres to larger aggregates and (c) of large aggregates to agglomerates of several micrometers in size. Depending on the solvents and the properties of the materials, these different domains are not equally accessible which renders the treatment of these resins difficult. Therefore, commercially available polystyrene resins for chemical synthesis are mostly low cross-linked (1% DVB). 17.2.1 Polystyrene Base Resins Polystyrene itself does not possess functional groups for the attachment of substrates (generally carboxylic acid derivatives). Therefore, the polystyrene support has to be chemically modified prior to its use in solid-phase organic synthesis [S]. This functionalization is done mostlv bv chloromethvlation of the nonactivated Dhenvl

17.2 Polystyrene Supports

469

residues which leads to Merrifield’s famous chloromethyl polystyrene. The reactive benzylic chloride functional groups of this resin can be substituted easily by carboxylates to form stable ester bonds. Other nucleophiles like amines or alcohol may also be bound to the resin although their postsynthetic cleavage is much more difficult or even impassible. Further treatment of the chloromethyl polystyrene results in the formation of other polystyrene base resins like aminomethyl and hydroxymethyl polystyrene. The conversion of these resins into the corresponding esters or amides of carboxylic acids is effected by treatment with activated carboxylic acid derivatives. Some of the polystyrene resins can also be purchased as monodisperse beads in different sizes for their use in automatized sorting protocols [23].

-

Aminomethyl PS 1241

Aminomethylated polystyrene

Cleavage: Properties: base resin, can be acylated easily Supplier Adv. ChemTc Bachem AG Novabiochem Peptides Int. Propeptide Rapp Polymere Richelieu Sigma Saxon

Mesh 200-400 100-200 200-400 200-400 200-400 100-200 100-200 200-400 200-400 100-200 100-200 200-400 200-400 100-200

mmol/g

Price range

-

11-111

0.5- 1.2 0.1-0.5 0.3-1.5 0.1-0.3

-

1.o- 1.2 1.o- 1.2 0.4/0.6/0.8 0.5-1.5 0.6- 1 0.6- 1

470

I7 Supports for Solid-Phase Organic Synthesis

Hydroxymethyl- PS I251

Hydroxymethylated polystyrene OH

very strong acid (HF, TFMSA) base resin, can be acetylated easily

Cleavage: Properties:

Supplier Adv. ChemTc. Bachem AG Peptides Int. Saxon Sigma

Merrifield resin I261

Chloromethylated polystyrene

CI

Cleavage: Properties:

Mesh

-

200-400 200-400 200-400 100-200 200-400

mmol/g

-

Price range 11-111

0.6-1 0.6-1

-

HF or TFMSA base resin for substrate anchoring by nucleophilic substitution

Supplier Adv. ChemTc.

Aldrich 2VoDVB: Bachem AG Novabiochem

Peptides Int. 2% DVB: Propeptide Rapp Polymere Sigma 2VoDVB: Saxon

Mesh 100-200 100-200 200-400 200-400 200-400 200-400 200-400 200-400 200-400 100-200 100-200 200-400 200-400 100-200 100-200 200-400 200-400 200-400 200-400 100-200

mmol/g 0.4-0.7 1.O- 1.4 0.4-0.7 1.O- 1.4 1 1 ca. 1 0.6-0.8 1.2-1.6 0.6-0.8 0.3- 1.5 0.3-1.5

-

1.0-1.3 1.O- 1.3 0.9- 1.5 0.4-0.9

ca. 1 0.6-1.2 0.6-1.2

Price range I

I

17.2 Polystyrene Supports

Aminoethyl-PS I231

-

Cleavage: Properties: aminofunctionalized base resin without benzylic properties

Aminoethylated polystyrene

I

’

~

“Polystyrene A NH,” “Polystyrene M NH,” (monosized particles for automatized sorting) Hydroxyethyl-PS I231

Hydroxyethylated polystyrene

“Polystyrene A OH”

“Polystyrene M OH” (monosized particles for automatized sorting)

47 1

Supplier Mesh Rapp Polymere 100-200 100-200 200-400

mmol/g 0.4-0.6 0.9- 1.3

Price range IV

See above

Particle size Rapp Polymere 5/10/20 pm 0.7-1

Cleavage: Properties:

V

basic hydrolysis hydroxyfunctionalized base resin without benzylic properties

Supplier Rapp Polymere

Mesh

mmol/g

Price range

100-200 100-200 200-400

0.4-0.6 0.9- 1.3

111

See above

Particle size Rapp Polymere

5/10/20 pm 0.7- 1

V

Bromoetbyl-PS I231

Bromoethylated polystyrene

“Polystyrene A Br” “Polystyrene M Br” (monosized particles for automatized sorting)

Properties:

base resin for substrate anchoring by nucleophilic substitution (no benzylic properties)

Supplier Rapp Polymere

Rapp Polymere

Mesh

mmol/g

100-200 100-200 200-400

0.4-0.6 0.9-1.3

See above Particle size 5/10/20 um 0.7-1

Price range 111-IV

V

472

I7 Supports for Solid-Phase Organic Synthesis

17.2.2 Acid-Labile Polystyrene Resins The chemical modification of the afore-mentioned polystyrene b se supp rts by acid-labile linkers results in resins from which bound substrates can be cleaved by simple treatment with an acidic cleavage solution, after having finished the solid phase synthesis protocol. By adequate choice of the linker system, the acid strength required for cleavage can be adapted to the needs of the planned synthesis. The cleavage conditions given in the list refer to the ester or amide bond between substrate (carboxylic acid component) and resin-linker-system (alcohol or amino component). Wang resin 112)

p-Benzyloxybenzylalcohol-PS

Cleavage: strong acid (e.g., 95% TFA) Properties: standard resin for peptide synthesis using Fmoc strategy

Supplier Adv. ChemTc. Bachem AG Novabiochem Peptides Int. Sigma Saxon

Mesh

mmol/g

Price range

100-200 200-400 200-400 200-400 100-200 100-200

-

11-111

-

200-400 100-200 Rapp Polymere 200-400 100-200 10/20/30

ca. 1 0.6-0.9 0.6-0.8

-

0.3-0.5 0.5-0.7 0.6-1 0.6-1 1-1.3 1-1.3 pm 0.7- 1

473

17.2 Polystyrene Supports

Polyslyrene A PHB/M PHB I231 p-Alkoxybenzylalcohol, based on hydroxyethyl- PS

Cleavage: strong acid (e.g., 95% TFA) Properties: resin for peptide synthesis using Fmoc strategy

Supplier “Polystyrene A PHB”

“Polystyrene M PHB” (monosized particles for automatized sorting)

SASRIN [IS]

2-Methoxy-4-alkoxybenzylalcoholPS

(SASRIN = super acid sensitive resin)

Mesh

Rapp 100-200 Polymere 100-200

200-400

rnmol/g

0.4-0.6 0.9- 1.3 see above

particle size

Rapp 5/10/20pm 0.7-1 Poly mere

Price range IV

V

Cleavage: weak acid (e. g., 0.5 Vo TFA) Properties: resin for synthesis and release of fully protected peptides

Supplier Bachem AG Saxon

Mesh

mmol/g

200-400

Price range

0.9

v

200-400

0.6-1.0

PAM [27, 281 p-(H ydroxymethy1)phenylacetamidomethyl-PS

Properties: resin for particularly stable anchoring (100 times as stable as Merrifield anchoring). Suitable for Boc synthesis of long peptides.

0

Supplier

Mesh

mmol/g

Adv. ChemTc.

-

-

Price range VI

474

17 Supports for Solid-Phase Organic Synthesis

Rink-Acid (131

Cleavage: weak acid (e.8.. AcOH-DCM) Properties: resin for synthesis and release of p-(2,4-Dimethoxyphenylhydroxymethyl)fully protected peptides phenoxymethyl- PS

8 \ /

0%

0

Rink-Amide (131

p-(2,4-Dimethoxyphenyl(Fmoc-amino) methyl)- phenoxymethyl-PS

~~~~

Supplier

Mesh

Novabio- 100-200 chem Saxon

mmol/g

Price range

0.4-0.8

v

-

~

~~~~

1

Cleavage: strong acid (TFA) Properties: release of the carboxylic amide on cleavage

Supplier

Mesh

Adv. ChemTc. Novabio- 100-200 chem Peptides Int. Sigma Saxon

-

mmol/g

0.3-0.6

0.4-0.5

-

Price range IV-v

17.2 Polystyrene Supports R A M (“Knorr resin”)

415

~

Cleavage: strong acid (TFA) Properties: release of the related carboxylic amide on cleavage. Also available as monosized “Polystyrene Microspheres” (Polystyrene M)

p-(2,4-Dimet hoxyphenyl(Fmoc-amino)methyl)phenoxyacetyl functionalized aminomethyl (“Knorr”) and aminoethyl polystyrene (A, M)

- -. . \

/

I

\

mmol/g

Price range

-

V

_A*.

ChemTc. Bachem 200-400 AG Rapp 200-400 Polymere 100-200 ABI Rapp 200-400 Polymere 200-400 100-200 particle size Rapp 5/10/20 pm Polymere

“Polystyrene A RAM”

“Polystyrene M RAM”

0.5

0.7-0.8

0.6 0.4-0.6

0.9- 1.3

See above 0.7-1.0

V

Moaometboxy-alanyl-RAM I291

p-(CMethoxyphenyl(Fmoc-amino)methyl)- phenoxyacetamido - alanyl- aminomet hylpolystyrene FmocNH

Cleavage: Properties:

Supplier Bachem AG ~~~~

strong acid (TFA) release of the carboxylic amide on cleavage Mesh 200-400

mmol/g 0.5

Price range V

476

17 Supports for Solid-Phase Organic Synthesis

Rink-Amide AM 1131

p-(2,4-Dimethyoxyphenyl(Fmoc-amino)methyl)phenoxy-acetamido-norleucyl-amidomethyl- polystyrene

Cleavage: Properties:

strong acid (TFA) release of the carboxylic amide on cleavage

Supplier Novabiochem

SAMBHA 1301

4-(2,4-Dimethoxyphenyl(amino)methyl3-methoxy-phenylaminocarbonylpropionylamidomethyl- PS

Sieber Amide 1311

-A

I Cleavage:

Properties:

Cleavage: Properties:

6-Fmoc-amino-xanthen3-yloxymethyl -polystyrene

FmocNH/_7 I Supplier A

mmol/g 0.4-0.6

Price range

v

strong acid (TFA) release of the carboxylic amide on cleavage

I

1% TFA in DCM release of the related carboxylic amide on cleavage. Resin for the synthesis of fully protected peptide amides by cleavage under mildly acidic conditions

I Novabiochem I

Mesh 200-400

Mesh

100-200

mmol/g 0.3-0.6

Price range

V

I

471

17.2 Polystyrene Supports

PAL 1321

Cleavage: strong acid (TFA) 5-(4-Aminomethyl-3,5-dimethoxyphenoxy) Properties: release of the carboxylic amide on cleavage valeryl- oxymethyl- polystyrene Supplier

Mesh

;ice range

YoVg

Saxon

PAL-MBHA I331

7%

PAL linker on 4-Methylbenzhydrylamine- polystyrene

__

~

Cleavage: Properties:

Supplier Adv. Chemtc.

ADPV 1231

5-(2-Aminomethyl-3,5-dimethoxyphenoxy) valeryl- amidomethyl- polystyrene

H2NCH,

OCH,

~

~~

strong acid (TFA) release of the related carboxylic amide on cleavage Improved resin swelling due to the additional methyl substituent at the benzhydryl anchor

Mesh

-

mmol/g

-

Price range V

Cleavage: strong acid (TFA) Properties: release of the carboxylic amide on cleavage

Supplier

Mesh

mmol/g

RaPP Poly mere

200-400

0.7-0.8

Price range

V

1

418

17 Supports for Solid-Phase Organic Synthesis

BHA I341 Benzhydrylamine- polystyrene

Cleavage: Properties:

very strong acid (TFMSA) on cleavage release of the related carboxylic amide. In contrast to the aminomethyl polystyrene resins cleavage by HF does not proceed quantitatively

Supplier Adv. ChemTc.

Mesh

2% DVB: Bachem AG Novabiochem Peptides Int.

see above

Sigma (2% D) Saxon Richelieu

MBHA I351 4-Methylbenzhydrylaminepolystyrene

Cleavage: Properties:

200-400 100-200 200-400 100-200 200-400 100-200 80- 180 200-400 100-200 100-200

mmol/g

-

Price range 11-111

-

0.6-0.9 0.3- 1.5

0.3-0.5

-

0.4-0.8 0.4-0.8 0.4/0.8/1.2

very strong acid (HF, TFMSA) on cleavage release of the related carboxylic amide. In contrast to the BHA 1 resin (see above) HF treatment causes I quantitative cleavage I I

, Supplier Adv. ChemTc.

Mesh

mmol/g

200-400 100-200

-

2% DVB: see above Bachem AG Novabiochem 200-400 100-200 100-200 200-400 Peptides Int. 100-200 2% DVB: 100-200 Sigma 200-400 2% DVB: 50-200 Richelieu 100-200

-

0.4-0.6 0.4-0.6 0.8- 1.2 0.3-1.5 0.3- 1.5 0.3-1.5 1 0.5-0.7 0.4/0.8/1.2

Price range 11-111

17.2 Polystyrene Supports

479

HMPB-BHA, -MBHA 1361 444-H ydroxymethyl-3-methox yphenyloxy)butyryl-( (4-methylIbenzhyd ry1)amide- polystyrene R

R=H.CH,

I

Properties:

acid-labile resin with good swelling properties for the synthesis of protected peptides

Supplier Mesh Novabiochem 100-200 HMPB-MBHA: 100-200

mmol/g

Price range

0.2-0.5

V

0.3-0.6

Rink-Arnide-MBHA I131

p-(2,4-Dimethoxyphenyl(Fmoc-amino)methyl-phenoxyacetylnorleucyI -(4-methylbenzhydryl -amide)- polystyrene

Cleavage: Properties:

strong acid (TFA) chemical properties like the Rink Amide resin, but better swelling properties due to the MBHA-PS support

Supplier Novabiochem

Mesh 100-200

mmol/g 0.3-0.6

Price range V

480

I7 Supports for Solid-Phase Organic Synthesis

Trityl Resin I371

Chlorodiphenylmethylbenzoyl-

Cleavage: Properties:

amidomethyl- PS

Supplier PepChem

weak acid (AcOH) resin for the synthesis of side chain protected peptides by the Fmoc strategy. The bulky trityl group prohibits diketopiperazine formation in peptide synthesis. A wide range of substrates can be attached to the reactive trityl handle by nucleophilic substitution without activation (no danger of racemization)

Mesh 200-400

mmol/g 0.9

Price range V

Cblorotrityl-Resin (371 2-Chlorotritylchloride-polystyrene

CI

Properties: enhanced acid lability compared to the

Supplier Novabiochem

Tricyclic Amide Linker (on BHA-PS) 1381 5-Fmoc-amino- 10,ll-dihydro-5Hdibenzo[a,d]-cycloheptenyl- 2-oxyacetylNle-4-methylbenzhydrylamide- PS

l e v O 0

Mesh 200-400

mmol/g 1.1-1.6

Price range IV-v

Cleavage: mild acid (1 070 TFA) Properties: especially suitable for the synthesis of peptide amides with C-terminal Phe, 5 r or Ile positions. Because of the mild cleavage conditions protected peptides can be obtained

Supplier

Mesh

mmol/g

Bachem AG

200-400 0.5

Price

48 1

17.2 Polystyrene Supports

17.2.3 Base-Labile, Photo-Labile and Nucleophilic Cleavable Polystyrene Resins Basic cleavage conditions are much less established in solid phase organic synthesis than acidic ones because of the frequent use of base-catalyzed synthesis steps in organic chemistry. However, the functional principle of the few base-labile linkersystems already commercially available is mostly based on the sensitivity of the substrate-resin ester bond against nucleophilic attack. According to the kind of nucleophilic cleaving agent used, the substrate is detached as the corresponding carboxylic acid derivative. Typical cleaving agents together with their corresponding products are: Cleaving agent Product ammonia -methanol amide hydrazine- DMF hydrazide aqueous NaOH acid methanol -triet hylamine methyl ester sodium borohydride alcohol The application of such linker systems involves considerable restrictions for solid phase syntheses, so they are only used in special cases. HMB Ill]

Cleavage:

nucleophilic attack

p-(Hydroxymethyl)benzoyloxymethyl- PS

Polystyrene A HMB I231

Supplier

Mesh

mmol/g

Price range

Bachem AG

200-400

-

111

Cleavage:

nucleophilic attack

p-(Hydroxymethy1)benzoyloxyethyl - PS

Supplier

Mesh

mmol/g

200-400 100-200 100-200

0.4-0.6 0.9- 1.3 0.4-0.6

Price range

482

17 Supports for Solid-Phase Organic Synrhesis

HMBA-MBHA 1161

Cleavage:

p-Hydroxymethylbenzoyl-(bmethylbenzhydry1)amide- polystyrene

CH,OH

Ammonia-MeOH, hydrazine-DMF, NaOH-water, methanol-triethylamine, sodium borohydride-ethanol, etc. Properties: side chain protecting groups may react with the cleaving reagent and should therefore be removed prior to cleavage. Good swelling properties due to the MBHA support

Supplier range Novabiochem

Brominated Wang resin I391

Cleavage:

p-(2-Bromo-2-methylphenacyl)oxymethyl- polystyrene

6f

photolysis by irradiation at 350 nm (5-25 h), hydrazine sensitve Properties: acid stable ( i l l x t towards 50% TFA) Suitable for synthesis of fully protected peptides No side reactions known during photolysis

Supplier range Novabiochem

200-400

0.5-1.2

17.2 Polystyrene Supporrs

483

Brominated PPOA Ill, 401

p-(2-Bromopropionyl)phenoxyacetamidomethyl-PS

Cleavage:

nucleophilic attack : NaOH-dioxane- water,

methanol-triethylamine-dioxane, hydrazine

hydrate-methanol, etc. Photolysis by irradiation at 350 nm (10 h) yields 80% of the fully protected peptide.

Supplier: Novabiochem

Mesh 200-400

mmol/g 0.5-1.2

Price range V

Oxime Resin [191

p-(4-Nitrobenzophenonoxime)polystyrene

Properties:

suitable for the synthesis of side chain protected peptide fragments, cyclopeptides, and for fragment condensation

Supplier Novobiochem

HYCRAM 1181

Cleavage:

Hydroxycrotonylamidomethy1polystyrene

Properties:

"w:UP;;;

CH*-!

Propeptide

Mesh 200-400

mmol/g 0.2- 1.0

Price range V

palladium(0)triphenylphosphine and morpholine or dimedone orthogonality to all standard protecting group strategies Mesh

-

mmol/g 0.3-0.6

-

Price range

IV

484

17 Supports for Solid-Phase Organic Synthesis

17.3 TentaGel Resins Polyethyleneglycols (PEGS) are more polar compounds than polystyrene and are therefore more compatible with polar substrates like peptides. They are soluble in most of the common solvents and are well established in organic synthesis. Solid supports made from PEG-grafted polystyrene show similar behavior concerning solvent and substrate compatibility, and are suitable for various synthetic purposes. The swelling volumes of those so-called “TentaGel” resins (which consist of ca. 70% PEG)are rather good (about 4-6 ml/g) for a wide range of solvents including water, methanol, acetonitrile, dichloromethane and dimethylformamide 16, 81. Only ether and alkanes lead to destruction of the gel phase, and the polymer forms shrunken, hardly solvated particles. Compared to polystyrene supports, the good solvation and swelling properties also lead to increased accessibility of the TentaGel polymer especially for polar reagents. On the other hand, sometimes it may be difficult to remove water quantitatively from the resin after a synthesis step, which was carried out in aqueous medium, because of the hygroscopic properties of PEG (411. The pressure stability of TentaGel resins is also increased remarkably in comparison to polystyrene supports, so TentaGel resins are used frequently in continuous flow syntheses. The loading capacities of TentaGel resins (ca. 0.1-0.4 mmol/g) are lower compared to those of polystyrene resins, but still higher than those of other continuous flow resins. For these reasons, TentaGel resins are standard supports for the production of high amounts of nucleic acid fragments (e. g. primer for molecular biology purposes) in large-scale oligonucleotide synthesis. The spherical TentaGel supports (in size distribution or monodispersed) also offer the possibility to synthesize beads with different functionalized inner and outer surface. Such heterobifunctional resins can be used for the synthesis of encoded substance libraries [23, 421. In the meantime, most of the linker systems known from polystyrene resins are also available on TentaGel support. Chemical properties of the described TentaGel resins are approximately the same as those specified for the related linker systems on polystyrene support and will therefore not be explained further. Cleavage conditions and compatibilities closely correspond to the characteristics of the respective polystyrene resins. Physical properties such as swelling behaviour and pressure resistance have heen descrihed here.

17.3

TentaGel Resins

485

TentaCel S Functionalized ethyl-TG

Particle size: 90 microns (ca. 170 mesh) 130 microns (ca. 110 mesh) mmol/g: 0.2-0.3 Suppliers: Bachem Bioscience Inc. Fluka Peptides Int. Rapp Polymere Saxon

Functionalization Hydroxyethyl-TG (S OH) Bromoethyl-TG (S Br) Aminoethyl-TG (S NH2) Carboxyethyl-TG (S COOH) Ethanethiol-TG (S SH) 3-Propionaldehyde-TG ( S CHO)

N-Succinimidyloxycarbonylethyl-TG(S COOSu) N-Boc-N'-propionylhydrazide-TG (S NHNHBoc) 2-Methoxy -4-alkoxybenzylalcohoI-TG

Cleavage

-

Price range IV

V

1-95% TFA

(cf. SASRIN) (S AC)

p(4-Hydroxymethy1)phenoxyethyl-TG (cf. Wang)

95 Vo TFA

IV

(S PHB)

5-(4-Aminomethyl-3,5-dimethoxyphenoxy)valeryl- 25- 95 Vo TFA

oxyethyl-TG (cf. PAL) (S AM)

p-(2,4-Dimethoxyphenyl(Fmoc-amino)methyl)phenoxy-acetamidoethyl-TG (cf. aminomethyl-

60-95'70 TFA

RAM) (S RAM) 1-Hydroxytrit yl-4-carbamidoethyl-TG (cf. tntyl-PS) (S Trt)

50% AcOH

p-Hydroxymethylbenzoylamidoethyl-TG(S HMB)

basic, nucleophilic ~~~~~~

V

486

17 Supports for Solid-Phase Organic Synthesis

TentaGel R

Particle size: 90 microns (ca. 170 mesh) Properties: improved swelling properties, decreased pressure stability; recommended for the synthesis of long peptides mmol/g: 0.18-0.22 Supplier: Rapp Polymere

Functionalization Aminoethyl-TentaGel (R NH2) p-Alkoxybenzylalcohol-TG (cf. Wang) (R PHB)

Cleavage

p-(2,4-Dimet hoxyphenyl(Fmoc-amino)methyl)phenoxy-acetamidoethyl-TG (R RAM)

60-95V0 TFA

TeotaCel microspheres

Functionalization Hydroxyethyl-TG (M OH) Bromoethyl-TG (M Br) Aminoethyl-TG (M NH2)

50-95 Vo TFA

Price range

IV V

Particle sizes: 10/20/30 microns monodisperse support for synthesis of Properties: substance libraries, automatic sorting, ultrafast synthesis [42c] suitable for bioassays on polymerbound substances. mmol/g: 0.2-0.3 Supplier : Rapp Polymere

p-Alkoxybenzylalcohol-TG (cf. Wang) (M PHB) p-(2,4-Dimet hoxyphenyl(Fmoc-amino)methyl)phenoxy-acetamidoethyl-TG (M RAM)

Cleavage

50-95 Qo TFA 60-95 QO TFA

Price range

V

17.3

TentaGel B TenIaCel

- bifunctional

Particle size: Properties: mmol/g: Supplier :

TentuGel Resins

487

90 microns (cu. 170 mesh) 130 microns (cu. 110 mesh) beads with different functionalities on inner and outer surface 0.2-0.3 Rapp Polymere

0

Innerlouter functionality Fmoc- AminoethyVBoc- Aminoethyl Boc- AminoethyVFmoc- Aminoethyl Fmoc- Rink - Linker/Fmoc- Aminoethyl H ydroxymethylbenzoyl- LinkedFmoc- Aminoethyl

TentaGel N

Price range

VI

Particle size: 90 microns (cu. 170 mesh) Properties: suitable for oligonucleotide synthesis mmol/g: 0.17-0.22 Supplier : Rapp Polymere

Functionalization Hydroxyethyl-TG (N OH) Aminoethyl-TG (N NH2)

Cleavage

-

A, C, G or T on Succinyl-TG

0

Novasyn lG

NH,

V

N = A, C. T, 0

Cleavage: Properties:

Aminoethyl-TentaGel

Price range IV

standard resin for TentaGel-solid-phase synthesis. Good swelling properties even in

Supplier Novabiochem

Particle size mmol/g 90 km 0.2-0.3

Price range IV

488

17 Supports for Solid-Phase Organic Synthesis

Novasyn TGA p-(Hydroxymethy1)phenoxyacetamidoethyl -TG CH,OH

Cleavage: Properties:

Supplier Novabiochem

95% TFA

Wang linker on acetyl-TentaGelsupport. Suitable for continuous flow syntheses with high flow rates and comparatively high loading

Particle size 90 wm

mmol/g 0.1-0.3

Price range V

Novasyn TGR

Cleavage: 95% TFA Properties: Rink-linker on acetyl -TentaGel -support. Unprotected amino group

Supplier Novobiochem

Particle size 90 wm

mmol/g 0.2-0.3

Price range V

17.4 PolyHIPE Resins

489

17.4 PolyHIPE Resins PolyHIPE (HIPE = high internal phase emulsion) [9] consists of a highly branched, highly macroporous polystyrene-DVB matrix which is grafted by polydimethylacrylamide. The polystyrene matrix (90% porosity) is responsible for the mechanical stability which is required for continuous-flow synthesis supports. It replaces the inorganic (and irregular) kieselguhr matrix of Pepsyn K [7], and is covalently bound to the polyamide material which is the actual solid-phase synthesis polymer in both resins. The polarity of the polyamide support facilitates efficient peptide synthesis and the use of a wide range of solvents. In comparison with Pepsyn K, PolyHIPE is characterized by high loading capacity, compatibility to a wide range of solvents and better anchoring of the graft polymer onto the matrix. In the literature loading capacities of up to 5 mmoles per gram are described [9].

PolyHIPE SU 2OOO

Cleavage: Properties: base resin with aminofunctionality Very high loading

Aminoethyl- PolyHIPE

Supplier 1Alexis

Aminosyn P2000 NH,

Aminofunctionalized PolyHIPE resin

Supplier Alexis

Novasyn P500

Sarcosine methyl ester functionalized PolyHIPE

Mesh

mmol/g

-

2

Mesh

-

mmol/g 2

Price

Price range

-

Cleavage: Properties: PolyHIPE base support with sarcosine methyl ester functionality. Readily convertable into amino function by ethylenediamine

Supplier range Novabiochem

1

4%)

I7 Supports for Solid-Phase Otganic Synthesis

Novasyn PA500

p-(Hydroxy)phenoxyacetyl linker, attached to the sarcosine-PolyHIPE base resin via norleucine and ethylenediamine

OH

Cleavage: 95% TFA Properties: acid-labile Wang linker system on high loaded PolyHIPE support

Supplier Novabiochem

Mesh

-

mmol/g 0.3-0.6

Price range

V

Novasyn PR500

p-(2,4-Dimethoxyphenyl(Fmoc-amino)methyl)-phenoxyacetyl-norleucyl-ethylenediaminePolyHIPE

95% TFA Cleavage: Properties: acid cleavable Rink Amide linker on PolyHIPE support. On cleavage, the corresponding carboxylic amide is obtained

Supplier Novabiochem

Mesh 200-400

mmol/g 0.125

Price range 111

17.5 PEGA Resins

491

17.5 PEGA Resins PEGA (Acrylamidopropyl-PEG-N,N-dimethylacrylamide) is a relatively new material which is especially suited for the synthesis of polar compounds [3]. It consists of a highly branched polymer network giving sufficient stability for continuous-flow syntheses which is nevertheless able to swell in polar solvents making the synthesis of long peptides possible. Furthermore, it does not contain chromophores, and therefore allows spectrophotometric monitoring of reactions within the resin itself. The material shows weak tendency of adherence towards glass and metal, but not towards plastics. The swelling volumes in dichloromethane, alcohols and water are about 6 ml/g, in DMF up to 8 ml/g.

PEGA

Cleavage:

-

I

PEGA base support _ _ -polymer with amino functionality

Supplier

I Saxon Wang-PEGA

p(Hydroxymethy1)phenoxy- PEGA

Mesh

-

mmol/g

-

Price range IV

Cleavage: 95% TFA Properties: acid cleavable Wang linker system on PEGA support

492

17 Supports for Solid-Phase Organic Synthesis

Rink-glycyl- PEGA

p-(2,4-Dimethoxyphenyl(Fmoc-amino)methyl)phenoxyacetyl-glycyl-PEGA

FP

Q,’ ‘

Cleavage: Properties:

Supplier

95% TFA

acid cleavable Rink Amide linker system, via glycyl spacer attached to the PEGA support. On cleavage the carboxamide is obtained

Mesh

mmol/g

-

-

Price range

IV

17.6 Kieselguhr-Polyamide Supports (“Pepsyn K”) The first successful approach in continuous-flow SPPS utilized a polydimethylacrylamide synthesis resin which had been polymerized inside a kieselguhr matrix. The resulting, so-called, “Pepsyn K” support shows suitable chemical properties for the synthesis of polar compounds belonging to the polar dimethyl acrylamide polymer, and complete mechanical flow stability due to the rigid inorganic kieselguhr skeleton [7].Pepsyn K is a continuous-flow-suited derivative of the polydimethylacrylamide resin “Pepsyn” which had been especially designed for peptide synthesis (cf. Section 17.9) [43]. Because of the undefined pore sizes of kieselguhr, the accessibility of anchoring positions within the synthesis polymer is limited. So, low levels of substitution (about 0.05 to 0.2 mmoles/g) and poor product yields are often observed. Physical abrasion of the polymer and the formation of fines from the fragile kieselguhr matrix may also lower the success of a continuous flow synthesis using Pepsyn K resins. To overcome these problems, other supports with a rigid matrix have been developed for continuous flow svnthesis 191 (PolvHIPE. TentaGel).

17.6 Kieselguhr-Polyamide Supports (“Pepsyn K’) Novasyn K125

Sarcosine methyl ester functionalized polyacrylamide polymer in kieselguhr matrix

Cleavage: Properties:

Pepsyn K base support with sarcosine methyl ester functionality. Readily convertable into amino function by ethylenediamine

Mesh 0

-

Novabiochem

Novasyn KD125

Ethylenediamine functionalized K125

493

mmol/g 0.125

Price range IV

Cleavage: Properties: Pepsyn K base resin for acylation by carboxylic acid derivatives, e.g., for peptide synthesis without cleavage of the final product

Supplier

Price range

Novabiochem

0.05 -0.1 3

Novasyn KA125

p-(Hydroxymethyl)phenoxyacetylnorleucyl-derivatized KD125

Cleavage: Properties:

95% TFA Wang linker system on Novasyn KDl25 Pepsyn K support

Supplier Novabiochem

Mesh

mmol/g

-

0.05-0.1

Price range IV

494

I7 Supports for Solid-Phase Organic Synthesis

Novasyn KR125 p-(2,4-Dimethoxyphenyl(Fmoc-amino)methyl)-phenoxyacetyl-norleucyl-derivatizedKD125

Cleavage: Properties:

Supplier Novabiochem

95% TFA Rink linker system on Novasyn KD125 support. On cleavage the amide is obtained Mesh

mmol/g

Price range

-

0.07-0.13

V

Novasyn K HMPB 125 4-(4-Hydroxymethyl-3-methoxyphenoxy)butyryl-norleucyl-derivatized K D 125

r m

Cleavage: 1 Vo TFA Properties: extremely acid-labile SASRIN linker system on Novasyn KD125 support for continuous flow syntheses of protected peptide fragments Supplier Novabiochem

Mesh

-

mmol/g 0.05-0.1

Price range V

17.7 Controlled-Pore Supports (CPG, CPC)

495

Novasyn KB

p-(Hydroxymethy1)benzoyl-norleucyl-derivatized KD125 (cf. HMBA)

Cleavage: basic/nucleophilic Properties: HMBA linker system on Novasyn KD125 Pepsyn K support Supplier Novabiochem

Mesh

-

mmol/g

-

Price range V

17.7 Controlled-Pore Supports (CPG,CPC) Organic polymers have limitations in temperature and pressure stability on principle. Inorganic supports typically d o not show such limitations within the ranges of chemical synthesis. Such materials often have been developed for chromatography purposes and subsequently utilized as synthesis supports, for which controlled pore glass (CPG)is a typical example. CPG and CPC (controlled pore ceramics) possess large inner surfaces with welldefined accessibilities controlled by the generation of particular pore sizes during the manufacturing process. Because of their special properties, CP supports are predestined to be used in continuous flow synthesizers. Loading capacities of about 0.2 mmol/g render CP supports a good alternative for other continous flow resins. In olinonucleotide svnthesis. CPG has even become a standard resin.

496

17 Supports for Solid-Phase Organic Synrhesis

CPG 10 No derivatization

Suppliers: Fluka. Sigma Price range IV Pore size (A)

75 120 170 240 350 500 700 1000 1400 2000

Mesh

Or Mesh

Or mesh

80- 120 80- 120 80-120 80- 120 80-120 80- 120 80- 120 80- 120 80- 120 80- 120

120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200

200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400

Mesh

Or mesh

120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200 120-200

200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400 200-400

Mesh

Or mesh

120-200 120-200

200-400

Glyceryl-CPG Glycerine coated CPG Pore size (A) 75 120 170

'240 350 500 700 1000 1400

2000 3000

DEA-CPG Diethylamino-derivatized, weakly basic ion exchanger

Supplier : Fluka Price range VI

I Pore size (A)

170 1240

I

17.7 Controlled-Pore Supports (CPG, CPC)

CML-CPG Carboxvmethvl-derivatized. weaklv acidic ion exchanger

Supplier:

I Price range I

Fluka

VI

Pore size (A)

Mesh

170 240

AMP-CPG Aminopropyl- CPG, developed for affinity chromatography, also suitable for the attachment of carboxylic acid derivatives (e.g., SPPS)

spherical controlled pore-glass varticles

Aminosiloxane-, hydroxysiloxane- or epoxysiloxane functionalized TRISOPERL in the above mentioned pore and particle sizes

0.1/0.2/0.04 0.1/0.2/0.04 0.1/0.2/0.04

120-200 120-200 200-400

200-400 200-400 200-400

Supplier: Schuller Price range 111 Pore size (A) 10 20 30 40 50 60 80 100 120

TRISOPERL N, L fuactiooalized

120-200 200-400

Supplier: Fluka Price range VI

1400

TRISOPERL N, L

497

qm4

Particle size (microns)

140 120 110 100 80 60 40 30 20

(50- 1001100- 200 200-500)

Supplier: Schuller Price range 111

498

17 Supports for Solid-Phase Organic Synthesis

TRISOPOR N, L nonspherical controlled poreglass particles

Supplier: Schuller Price range I1 Pore size (A) 20 30 40 50 60 80 100

120 TRISOPOR N, L, functionalized

qm/g

Particle size (microns)

140 120

(50-100/100-200 200- 500/500- 800)

110

100

80 60 40 30

Supplier: Schuller Price range I1

Aminosiloxane-, hydroxysiloxane- or epoxysiloxane functionalized TRISOPOR in the above mentioned pore and particle sizes

CPC- Silica-carrier Controlled pore-ceramics

CPC-SC -Silane-coated Controlled pore-ceramics,

Supplier: FI LIka

Pore size (A) 375

Mesh 30-45

Price range I

Supplier: Fluka

Pore size (A) 375

Mesh 30-45

Price range I

3-aminopropyltriethox ysilane-

derivatized

17.8 Other Silicate Supports Micropil A NHz Amino-functionalized spherical silica particles with high porosity

~~

Supplier: Alexis

~

mmol/g ca. 0.3

~

~

Particle size 7-20 pm

~

Price range

I

17.9 Miscellaneous Support Components

499

Spherisorb GC Monodispersed

NH2

monodisperse amino-

Supplier: Alexis

mmol/g

Part.grtiBe

Price range

-

120 wm

-

17.9 Miscellaneous Support Components The aforementioned supports represent the major part of solid phase organic synthesis supports, but there is a wide range of further compounds which have been used as support components for special applications. Some examples are described below. Support components means linker molecules as well as support polymers or linker-polymer combinations. Pepsyn Pepsyn is a polydimethylacrylamidesupport which has been especially designed for the synthesis of polar substrates, particularly for peptide synthesis [43]. The conception that good yields in solid phase peptide synthesis require approximately the same polarity of both substrate and support led to the invention of this support. Due to its polyamidic backbone Pepsyn meets this requirement quite well. Nevertheless, Pepsyn could not overcome the dominance of polystyrene supports, not even in peptide synthesis. Only kieselguhr-polydimethylacrylamide attained some importance as a flow stable support in continuous-flow synthesis. Micropins/Macrocrowns The Micropin/Macrocrown system consists of plastic-sticks arranged on blocks and fitting in microtiter plate formate, which can be equipped with pins or crowns made of synthesis resins. By using ELISA reagent containments, the syntheses can be easily carried out by plunging the resin plates into the reagent or washing plates. The pins are available in different sizes, polymers and loading capacities, so multiple syntheses on a greater scale are feasible. Some of the polymers described for organic syntheses using the Micropin system are polystyrene, polyacrylic acid and polymethacrylate [44](Chiron Mimotopes). CellulosdCotton Supports Like many other supports, cellulose or cotton supports have been developed initially for chromatography and later on applied in solid phase synthesis. Its compatibility to polar and apolar solvents (even to water) is used in chromatography of highly polar compounds and is also advantageous for organic syntheses. Besides underivatized cellulose, DEAE-(diethylaminoethyl-) cellulose is the most widemread cellulose derivative 1451.

500

17 Supports for Solid-Phase Organic Synthesis

Membrane Supports Grafted membranes are a new form of solid supports for organic synthesis. In general, the membranes consist of polypropylene which is grafted by a synthesis resin like hydroxypropylacrylate [46, 471. The unusual membrane shape makes applications like affinity chromatography, epitope mapping or covalent sequence analysis easier to manage. Such membranes are flow stable and therefore suited for continuous-flow processes, and the synthesis resin is characterized by a highly controlled porosity and its large inner surface. So loading capacities of up to 0.1 mmole/g are achieved. Fractogel/Fractogel EMD Fractogel is a polymer made from methyl methacrylate and vinyl alcohol, and has been developed for chromatography purposes. It is compatible to the most organic solvents, possesses well-defined porosity and is stable in a range of pH1 to pH14, up to a temperature of 100°C and up to a pressure of about 7 atmospheres. Furthermore, the spherical shape of Fractogel beads makes it well suitable especially for continuous-flow syntheses. Loading capacities amount from 0.01 to 0.125 mmole/g. In Fractogel EMD, the polymer is grafted by polyglycine tentacles bearing different functional groups like imino diacetic acid, azlactones, sulfur compounds or epoxides [48] (E. Merck). Multiple Antigen Peptide System (MAPS) MAPS supports are especially designed for the effective production of antibodies. They consist of branched oligolysine dendrimers bound to conventional resin-linker systems. By synthesizing peptides starting at the ends of the oligolysine branches, unique, bulky bundles of peptides are obtained, which can be injected into animals to elicit immune response and antibody production. Mostly, the oligolysine-bound peptides are injected after cleavage from the resin and side chain deprotection, but it is also reported that the antigens can be injected as resin-bound conjugates, after side chain deprotection only (Novabiochem, Rapp Polymere). Macrobeads Macrobeads offer the possibility to synthesize resin-bound substance libraries following the one-bead-one-compound principle by split-and-combine synthesis protocols or by individual synthesis on one bead. Typical bead sizes are 400 to 750 microns. In bioassays, biological active substances are usually indicated as colored beads which can be selected manually. The amount of substance bound to the inner surface of the resin beads is sufficient for detailed analytical characterization (HPLC, MS, peptide-sequencing, amino acid analysis, NMR, etc.) of the biological active compounds [42]. Following these techniques, lead structures can be quickly determined by a minimum of experimental expense ( R a ~ pPolymere).

17.9 Miscellaneous Support Components

501

Heterobifunctional Polystyrene/Polystyrene-PEG resin Recently, the preparation of a resin has been described (491, which is heterobifunctionalized by two different linker molecules and large outer-surface bound PEG spacers. This functionalization is of particular use for the parallel synthesis of free and immobilized peptides on the same resin bead. For example, the soluble peptides cleaved from the interior of the resin, which contains more than 98% of the total amount of peptides, can be screened in immunological assays, whereas immobilized peptides on the highly biocompatible PEG resin surface can be directly screened for antibody binding. In contrast to the Macrobead approach described before, this resin offers a comparatively high loading capacity due to the pure polystyrene-DVB interior, which renders it a valuable tool also for simultaneous multiple synthesis of resin-bound and soluble peptide libraries for random screening. Polystyrene PTFE Polystyrene grafted polytetrafluoroethylene (PTFE) supports have recently been used successfully for the synthesis of long oligonucleotide chains. The chemically inert PTFE support is responsible for the high mechanical stability, whereas the polystyrene synthesis resin allows the use of a wide range of linker systems and a relatively high loading capacity (about 0.16 mmolelg). The hydrophobic PTFE-polystyrene support is not compatible to very polar solvents [50]. ExpansinlHPDI-Expansin Expansin is an aminofunctionalized polyacrylic resin with loading capacities of about 0.4 mmoles per gram. Recent investigations used Expansin resins as support acid) which polymer for the new HPDI linker (2-hydroxypropyl-dithio-2’-isobutyric possesses two different, chemically orthogonal cleaving sites: a disulfide bond and an ester bond. Each site is cleaved under different cleaving conditions without having an influence on the standard Fmoc peptide synthesis protocol [51].

Safety-Catch Amide Linker (SCAL) A safer connection between substrate and support is guaranteed by so-called SafetyCatch linkers of which SCAL [52) is the most widely known. The functional principle of Safety-Catch linkers is the non-cleavable attachment of the substrate to the linker and a chemical modification of the linker molecule prior to cleavage in order to obtain a cleavable substrate-linker bond.

502

17 Supports for Solid-Phase Organic Synthesis

In the SCAL system, the cleavability of this bond is achieved by reduction of the sulfoxide groups. Treatment of the reduced linker-substrate system with 50% TFA affords the free substrate in the shape of its carboxylic amide. COOH

FmocNH -0

s" AH3

CH3

17.10 Appendix 17.10.1 Conversion Table (mesh Mesh 2.5 3 5 8 10 14 18 20 30 40

- particle size, mm)

mm 8 6.73 4 2.38 2 1.41

1 0.84 0.59 0.42

Mesh 50 60 80 100

140 I70 200 270 325 400

17.10.2 Addresses of Suppliers Advanced ChemTech 5609 Fern Valley Road, Louisville, KY 40228-1075 Phone: (502) 969-oooO Fax: (502) 968- lo00 Advanced ChemTech GmbH Graf-Vollrath-Weg 4, D-60489 Frankfurt Phone:/Fax: (069) 7891 06-60 Aldrich-Chemie GmbH Postfach 1120, Riedstr. 2, D-89552 Steinheim Phone: (073291 97-21 1 1

mm 0.3 0.25 0.18 0.149 0.105 0.088 0.074 0.053

0.044

0.037

17.10 Appendix

Alexis Deutschland GmbH Gief3ener Str. 12, D-35305 Grilnberg Phone: (06401) 900770 Fax: (06401) 3823 Bachem AG Hauptstr. 144, CH-4416 Bubendorf Phone: (061) 9312333 Fax: (061) 9312549 Bachem Bioscience Inc. 3700 Horizon Drive, King of Prussia, PA 19406, USA Phone: 610/2390300, Fax: 610/2390800 CoshiSoft/PeptiSearch PO Box 68212, Tuscon, AZ 85737 Fax: (602) 7429252 EMC, ECHAZ microcollections Eberhardstr. 29, D-72762 Reutlingen Fluka Chemie AG Industriestr. 25, CH-9470 Buchs Phone: (081) 7552511 Fax: (081) 7565449 E. Merck Postfach 4119, D-6100 Darmstadt 1 Phone: (061 51) 720 Fax: (061 51) 72-2000 Novabiochem GmbH Postfach 1167, D-65796 Bad SodenITs Phone: (01 30) 6931 Fax: (06196) 62361 PepChem Goldammer & Clausen Im Winkelrain 73, D-72076 Tiibingen Phone:/Fax: (07071) 600393 Peptides International, Inc. PO Box 24658, Louisville, KY 40224 Phone: 1 (502) 266-8787 Fax: 1 (502) 267-1329 Propeptide Reuterweg 47, D-6000 FrankfurtIMain 1 Phone: (069) 729751 Fax: (069) 7240836 Rapp Polymere GmbH Ernst-Simon-Str. 9, D-72072 Tiibingen Phone: (0049) 7071-7631 57 Fax: (0049) 7071-7631 58

503

504

17 Supports for Solid-Phase Organic Synthesis

Richelieu Biotechnologies Inc. 11 177, Hamon, Montreal, Canada H3M 3E4 Phone: (541) 335-5934 Fax: (541) 339-1407 Saxon Biochemicals GmbH Feodor-Lynen-Str. 14, D-30625 Hannover 61 Phone: (05 11) 546080 Fax: (05 11) 5460888 Schuller GmbH Trtibach 2, D-96523 Steinach Phone: (036762) 360 Fax: (036762) 36298 Sigma-Aldrich Vertriebs-GmbH Postfach, D-82039 Deisenhofen Phone: (0130) 51 55 Fax: (089) 61301-405

17.10 Appendix

505

17.10.3 Index of Solid Supports

Name

Cleavage

PAL, PAL-MBHA. TentaGel S AM Aminomethyl-PS, TentaGels NHZ, PolyHIPE-SU 2000, Aminosyn P2000 NH2, PEGA, NovaSyn KD125, AMP-CPG, Trisoperl, Trisopor, CPC-SCSilane-Coated, Micropil A-NH2 Spherisorb GC

,, Aminosyn P2000 NH2 AMP -CPG BHA- polystyrene Brominated PPOA polystyrene Brominated Wang polystyrene Bromoethyl- polystyrene Cellulose Chlorotrityl- polystyrene CML-CPG CPC- SC-silane-coated CPC silica-carrier CPG 10 DEA-CPG Expansin Fractogel Fractogel EMD Glyceryl-CPG HMBA-MBHA- polystyrene

,,

., 3, N

B

MBHA-PS Brominated Wang PS Brominated PPOA PS Bromoethyl-TentaGels Trityl-PS, TentaGel S Trt Carboxyethyl -TentaGel Aminoethyl-PS and analogs

B, N

HMB-PS, Novasyn KB, TentaGel HMB

506

17 Supports for Solid-Phase Organic Synthesis

HMB - polystyrene HMPB-BHA-polystyrene HMPB- MBHA-polystyrene HPDI -Expansin HYCRAM-polystyrene Hydroxyethyl- polystyrene Hydroxymethyl- polystyrene Macrobeads Macrocrowns MAPS MBHA-polystyrene Membrane supports Merrifield Micropil A NH2 Micropins Monomethoxy -Alanyl- RAMpolystyrene Novasyn K HMPB 125 Novasyn K125 Novasyn KA125 Novasyn KB Novasyn KD125

S S S Pd-catal B

C

Hydroxymethyl -PS, Tentagels OH, Trisoperl jS

Y*

;S

5s Aminoethyl-PS and analogs Macrocrowns S S

S R N

Novasyn KR125

S

Novasyn P500 Novasyn PA500 Novasyn PR500

S S

Novasyn TG

S

Novasyn TGA Novasyn TGR Oxime-polystyrene PAL- MBHA-polystyrene

HMBA-MBHA-PS, Novasyn KB, TentaCel HMB SASRIN and analogs SASRIN and analogs

s

Rink-Amide-PS and analogs SASRIN and analogs Novasyn P500 (PolyHIPE) Wang-PS and analogs HMB-PS cf. Aminoethyl-PS, PolyHIPE SU2000 Rink-Amide-PS and analogs, Novasyn PR500, Novasyn TGR Novasyn K125 (Pepsyn K) Wang-PS and analogs Rink-Amide + analogs, Novasyn KR125, -TGR Aminoethyl-Tentagel, Aminoethyl-PS, etc. Wang-PS and analogs Rink- Amide + analogs, Novasyn KR125, -PR500

N S

-

PAL, ADPV

17.10 Appendix

507

PAL- polystyrene

S

PAM -polystyrene PEGA Pepsyn Pepsyn K PolyHlPE PolyHlPE SU 2000

SS

Polystyrene A PHB/M PHBpolystyrene Polystyrene- PTFE RAM (“Knorr”)-polystyrene Rink - Acid- polystyrene Rink - Amide AM - polystyrene Rink -Amide-MBHA-polystyren

Aminoethyl-PS and analogs, Novasyn KD125 S

Wang-PS and analogs

S

Rink- Amide-PS and analogs

S

S S

Rink-Amide-polystyrene

S

Rink -gl ycyl -PEGA Safety-Catch-Amide- Linker

S

SAMBHA- polystyrene SASRIN- polystyrene

S

SCAL Sieber- Amide- polystyrene Spherisorb GC Monodispersed NH, TentaGel B Boc/Fmoc TentaGel B Fmoc/Boc TentaGel B HMBIFmoc TentaGel B RAM/Fmoc TentaGel M Br

PAL-MBHA, ADPV, TentaGel S AM H ydroxymethyl- PS

S S

31 31 S

,, 9,

Rink-Amide and analogs, BHA-PS, MBHA-PS Monomethoxy - Alanyl- RAM, Novasyn KR125, -PR5OO, -n?R, RAM-PS (Knorr), Rink-Acid, Rink-Amide AM, Rink -Amide-MBHA, RinkGlycyl-PEGA, Sieber- AmidePS, TentaGels RAM, Tricyclich i d e - B H A - P S , SAMBHA Rink-Amide-PS and analogs Rink-Amide-PS and analogs, BHA, MBHA R i n k - h i d e and analogs HMPB-(M)BHA-PS, Novasyn E HMPB cf. Safety-Catch-Amide-Linker Rink-Amide and analogs Aminoethyl-PS and analogs Aminoethyl-PS and analogs

,* HMB Rink-Amide and analogs Bromoethyl- PS ~~~~

508

17 Supports for Solid-Phase Organic Synthesis ~

TentaGel M NH2 TentaGel M OH TentaGel M PHB TentaGel M RAM TentaGel MAP TentaGel N NH2 TentaGel N OH TentaGel R NH2 TentaGel R PHB TentaGel R RAM TentaGel S AC TentaGel S AM TentaGel S Br TentaGel S CHO TentaGel S COOH TentaGel S COOSu TentaGel S HMB TentaGel S NH2 TentaGel S NHNHBoc TentaGel S O H TentaGel S PHB TentaGel S RAM TentaGel S SH TentaGel S Trt Tricyclic- Amide-BHApolystyrene Trisoperl L Trisoperl N Trisopor L Trisopor N TrityI -polystyrene Wang-PEGA Wang- polystyrene

S S

S S

S S

Aminoethyl- PS H ydrox yethyl- PS Wang-PS and analogs Aminomethyl - RAM-PS and analogs cf. MAPS Aminoethyl- PS Hyd roxyet hy 1-PS Aminoethyl-PS and analogs Wang-PS and analogs Aminomethyl-RAM and analogs SASRIN, HMPB-(M)BHA, Novasyn K HMPB PAL, ADPV Bromoethyl -PS CML-CPG HMBA-MBHA-PS, HMB-PS, Novasyn KB Aminoethyl-PS and analogs

S S

S S

S S S

Hydroxyethyl-PS and analogs Wang-PS and analogs Aminomethyl-RAM, RinkAmide-PS + analogs Tritylchloride - PS, Trityl- PS Rink- Amide, Sieber- Amide and analogs CPG CPG CPG CPG Tentagel S Trt, chlorotrityl-PS Wang-PS and analogs Novasyn KA125, -PASOO, - m A , polystyrene A-PHB/M PHB, TentaGels PHB

P B N

S

ss

photolytic cleavage cleavage under basic conditions cleavage by nucleophilic attack cleavage under acidic conditions cleavage by very strong acids (TFMSA)

References [l] R. B. Merrifield, J. Am. Chem. SOC. 1963,85, 2149. [2]G. Jung, A. G. Beck-Sickinger, Angew. Chem. 1992,104, 375;Angew. Chem., Int. Ed. Engl. 1992, 31, 367. (31 M. Meldal, Tetmhedron Lett. 1992,33, 3077. [4]A. Guyot in Syntheses and Separations using Functional Polymers (Eds. :D. C. Sherrington, P. Hodge), John Wiley & Sons, New York 1988. (51 H.Gausepohl, PhD Thesis, University of Tubingen, Tiibingen 1990. [6]E. Bayer, Angew. Chem. 1991, 103, 117. [7]E. Atherton, R. C. Sheppard, A. J. Rosevar, J. Chem. SOC.,Chem. Commun. 1981,1151. [8]a) E. Bayer, W. Rapp, DE-A 3714258,(1988).b) E. Bayer, W. Rapp, Chem. Pept. Protein 1986,3, 3. 191 P. W. Small, D. C. Sherrington, J. Chem. Soc, Chem. Commun. 1989, 1589. [lo]a) M. D. Matteucci, M. H. Caruthers, Tetrahedron Lett. 1980,21,719;J. Am. Chem. SOC. 1981, 103,3188. b) G. R. Gougle, M. J. Bunden, P. T. Gilham, Tetrahedron Lett. 1981, 22, 4177. [Ill E. Atherton, C. J. Logan, R. C. Sheppard, J. Chem. SOC., Perkin Tmns. 11981, 538. [I21 S. S. Wang, J. Am. Chem. SOC. 1972, 95, 1328. 1131 H. Rink, Tetrahedron Lett. 1987, 28, 3787. [14]K. Barlos, D. Gatos, S. Kapolos, G. Papaphotiou, W. Schafer, Y. Wenquing, Tetrahedron Lett. 1989,30, 3947. [IS]M. Mergler, R. Tanner, J. Gosteli, P. Grogg, Tetrahedron Lett. 1988,29, 4005. (161 R. C. Sheppard, B. J. Williams, Int. J. Pept. Protein Res. 1982,20, 451. [17]D. Bellof, M. Mutter, Chimia 1985,39, 10. [l8]H. Kunz, B. Dombo, Angew. Chem., Int. Ed. Engl. 1988,27, 710. [19]W. F. DeGrado, E. T. Kaiser, J. Org. Chem. 1980,45, 1295. (20)M. PAtek, M. Lebl, Tetrahedron Lett. 1991,32, 3891. [21]a) M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon, J. Med. Chem. 1994,37,1233.b)E. M.Gordon, R. W. Barrett, W.J. Dower, S.P. A. Fodor, M. A. Gallop, J. Med. Chem. 1994.37, 1385. c) L. Weber, Nachr. Chem. Techn. Lab. 1994,42,698. 1221 a) E. Bayer, Int. J. Pept. Protein Res. 1985,25,178.b) W. Rapp, PhD Thesis, Universitiit Tubingen, Tubingen 1985. c) E. Bayer, W. Rapp in Peptide Chemistry (Eds.: T. Shiba, S. Sakakibara) 1988.

510

17 Supports for Solid-Phase Organic Synthesis

[23] Rapp Polymere, Produktkatalog 1994, Tubingen (1994). [24] A. R. Mitchell, S. B. H. Kent, J. Org. Chem. 1978, 43, 2845. [25] W. M. McKenzie, D. C. Sherrington, J. Chem. Soc., Chem. Commun. 1978, 541. [26] G. Barany, R. B. Merrifield in The Peptides. Vol. 2, 1979, 1. [27] B. Gutte, R. B. Merrifield, J. Biol. Chem. 1971, 246, 1922. [28] A. R. Mitchell, J. Am. Chem. Soc. 1976, 98, 7357. [29] W. Stuber, J. Knolle, G. Breipohl, Int. J. Pept. Protein Res. 1989, 34, 215. [30] B. Penke, L. Nyerges in Peptides 1988 (Eds.: G. Jung, E. Bayer) W. de Gruyter, Berlin 1989, 142. [31] P. Sieber, Tetrahedron Lett. 1987, 28, 2107. [32] F. Albericio, N. Kneib-Cordonier, L. Gera, R. P. Hammer, D. Hudson, G. Barany in Peptides-Chemistry and Biology (Ed.: G. R. Marshall), ESCOM, Leiden 1988, 159. [33] F. Albericio, N. Kneib-Cordonier, S. Biancalana, L. Gera, R. I. Masada, D. Hudson, G. Barany, J. Org. Chem. 1990, 55, 3730. [34] a) J. P. Tam, J. Org. Chem. 1985,50, 5291 ; Tetrahedron Lett. 1981,22, 2854. b) A Hirao, J. Chem. Soc., Chem. Commun. 1983, 25. [35] a) G. R. Matsueda, J. M. Stewart, Peptides 1981, 2, 45. b) K. Channabasavaiah, J. M. Stewart, Biochem. Biophys. Res. Commun. 1981, 86, 45. [36] A. FICrrsheimer, B. Riniker in Peptides 1990 (Eds.: E. Giralt, D. Andreu), ESCOM, Leiden 1991, 131. [37] K. Barlos, D. Gatos, J. Kallitis, G. Papaphotiou, Y. Wenquing, W. SchBfer, Tetrahedron Lett. 1989, 30, 3943. [38] R. Ramage, S. L. Irving, C. McInnes, Tefrahedron Lett. 1993, 34, 6599. [39] S. S. Wang, J. Org. Chem. 1976, 41, 3258. I401 N. A. Abraham, Tetrahedron Lett. 1991, 32, 577. (411 J. Weiler, W. Rapp, personal communication. [42] a) W. Rapp, K. H. Wiesmuller, B. Fleckenstein, V. Gnau, G. Jung in Peptides 1994 (Ed.: H. L. S. Maia), ESCOM, Leiden 1995. b) V. Nikolaiev, A. Stierandova, V. Krchfiik, B. Seligmann, K. S. Lam, S. E. Salmon, M. Lebl, Pept. Res. 1993, 6, 161. c) E. Erb, K. D. Janda, S. Brenner, Proc. Natl. Acad. Sci. USA 1994,91, 11422. d) W. Rapp in Peptides 1992 (Eds.: C. H. Scheich, A. N. Eberle), ESCOM, Leiden 1993, 25. [43] R. Arshady, E. Atherton, D. L. J. Clive, R. C. Sheppard, J. Org. Chem., Perkin Trans. I 1981, 529. 1441 R. M. Valerio, A. M. Bray, N. J. Maeji, Int. Pept. Protein Res. 1994, 44, 158. 1451 J. Eichler in Solid Phase Synthesis (Ed. : R. Epton), Mayflower Worldwide, Oxford 1994, 227. J. Eichler, A. Bienert, A. Stierandova, M. Lebl, Peptide Res. 1991, 4, 296. I461 S. B. Daniels, M. S. Bernatowicz, J. M. Coull, H. KBster, Tetrahedron Lett. 1989, 30, 4345. [47] R. Fitzpatrick, P. Goddard, R. Stankowski, J. Coull in Solid Phase Synthesis (Ed.: R. Epton), Mayflower Worldwide, Oxford 1994, 157. [48] M. P. Reddy, M. A. Michael, F. Farooqui, N. S. Girgis, Tetrahedron Lett. 1994,35, 5771. [49] B. Fleckenstein, K. H. Wiesmiiller, M. Brich, G. Jung, Lett. Pept. Sci. 1994, I , 117. [50] E. Birch-Hirschfeld, 2. F6ldes-Papp, H. H. Guhrs, H. Seliger, Nucl. Acids Res. 1994,22, 1760. [51] J. Brugidou, J. Mery, Pept. Res. 1994, 7, 40. [521 M. Pitek, M. Lebl, Tetrahedron Lett. 1991, 32, 3891.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

18 QMass: A Computer Program for the Analysis of Mass Spectra of Peptide Libraries Jente Brunjes, Jorg K Metzger and Giinther Jung

18.1 Introduction Synthetic peptide libraries offer a wide range of defined structural diversity allowing rapid screening for biologically active lead compounds [l -61. The characterization of peptide combinatorial libraries using conventional analytical techniques like HPLC is difficult and not very informative. The newly developed method of pool sequencing has proved to be a potent method to characterize natural and synthetic peptide libraries [7,8]. In recent investigationsit has been shown that ESI mass spectrometry, tandem mass spectrometry and high-performance liquid chromatography-mass spectrometry (HPLC-MS) are also well suited for quality control and determination of byproducts and other impurities in peptide libraries [8- 101. These possibilities are based on the finding that the ESI mass spectrum of a peptide library reflects the mass distribution of the peptides regarding both the m/z values of the mass peaks and their relative intensities. Because a ‘manual’ calculation of the mass distribution of even small peptide mixtures remains a time-consuming and errorproned task, the computer program QMass has been developed, which calculates the masses of expected peptides, byproducts and mass distributions of peptide libraries.

18.2 Concepts of QMass QMass is a command line driven, input-output oriented program for UNIX-like operating systems, written in a simple style of the C programming language. It uses a smaller number of UNIX system tools and shell features. Mechanisms, which automate analyses schemes are based on shell scripts, which are included in the distribution. Calculation results are given as plain ASCII files. The graphical user interface is kept apart from the calculating kernel. Therefore, QMass can be compiled on a wide range of computing platforms. Own applications can be easily written, and calculations can be run as batches on clients. QMass can not resolve combinatorial problems, but it performs calculations, which can be adjusted to the complexity of the library under investigation. For example, it might be useful to calculate all masses corresponding to modifications, deletions, insertions and combinations thereof for the singly, doubly and triply charged

512

18 A Computer Pmgmm for the Analysis of Mars Spectra of Peptide Libmries

ions of a single peptide simultaneously. Although this amount of information can be handled for a single peptide, this would not be true even for a 18 or 100 component library. In such cases, it would be more reasonable to look first for common modifications and, if no assignment is possible, to search for less probable byproducts in a selected mass range. Therefore, QMass offers the possibility to activate calculation options in a stepwise manner and shell scripts can be used to define these steps. QMass consists of some additional auxiliary programs, which perform additional calculations (the programs “cross”, “iso” and “deuterate”, see below) or support automated analyses based on shell scripts (the programs “range” and “compare”). The functionality of these programs is shortly described in Table 18-1, their relations to QMass are depicted in Fig. 18-1.

mble 18-1. Description of QMass, supplementary programs and UNIX system tools

Programs and Description system tools qmass Main program. Calculates mass distributions of expected and modified peptides (with sequence information) and mass distributions for complex libraries (without sequence information). Uses command line options to calculate different kinds of modifications and degrees of complexity. Writes output to stdout or into files (qmass output files) using output redirection. compare Uses experimental data (mass/intensity lists) and searches for corresponding masses in any qmass output fiies. Reports number of assigned mass peaks, not assigned mass peaks or gives masslintensity lists. Values can be used for an initial investigation of numerous sublibraries, mass/intensity lists for iterative searches of increasing complexity. Uses experimental data (mass/intensity lists), searches for corresponding range masses in any qmass output files and combines both of them for inspection. cross Calculates masses of crosslinked peptides for each qmass output entry and reports possible mass combinations for a selected mass range. is0 Calculates for each monoisotopic qmass output entry the natural isotopic distribution. Optionally restricted to a number of signals, the signal of lowest mass or the signal of maximum intensity. plot-iso Prepares is0 output fiies for plotting. UNIX system tools:

sort, wc, head, Sorting of qmass output files, number of calculated entries, mass range tail, grep(s) of a calculation result, searches for any meaningful identifier (eg., kind of modification, deletions in a defiied position, types of backbone fragmentation, possible sequences which tend to fragment).

18.3 Libmry Concept of QMass

5 13

Figure 18-1. Program structure of QMass. For complex libraries, the kernel (qmass) calculates mass distributions. For less complex libraries, the calculation provide the complete information of actual mass, actual sequence, original sequence, original mass, the type of a modification, the location of a modification and the ion charge. Additional programs can be used to automate calculations and analyses or to perform further calculations.

183 Library Concept of QMass Within QMass a peptide (Fig. 18-2) is defined as a linear or cyclic chain of building blocks and each building block can suffer from mass changes (covalent modifications or not cleaved protecting groups in a chemical sense). Each chain can suffer from insertions (duplications), deletions, successivetruncation and crosslinks to further components of the library. Backbone fragmentation and isotope distributions can be calculated.

514

I8 A Computer Program for the Analysis of Mass Spectra of Peptide Libraries

Figure 18-2. Library concept of QMass. Peptides are defined as linear (or cyclic chains) of building blocks, which might have deletions (D), insertions (I) truncation (T) crosslinks (C), noncovalent modifications or C/N terminal modifications (implicitely defined). Building blocks can suffer from modifications (uncleaved or cleaved protecting groups (P)and other chemical modifications (M)). Fragmentations occur during ionization (F), and the natural isotope distribution (iso) influences the mass distribution.

Building blocks and modifications can be defined by the user. N- or C-terminal modifications can be implicitly defined as additional residues, covalent and noncovalent byproducts as N-or C-terminal residues with zero mass, but defined mass change. For visualization, approximated isotope distributions can be calculated (using Poisson approximation and discrete convolution).

18.4 Calculations For libraries of high complexity, the program calculates the expected mass distributions (optionally monoisotopic, average mass or including the isotope distributions) for singly, doubly and triply charged ions. The results can be directly compared with the experimental spectra (Fig. 18-3). Distributions of modified peptides can be defined implicitly by residue or library definition (for example, distributions of peptides with deletions or modifications in invariant positions). For small-sized libraries, QMass reports sequences as well. In this case command line options can be used to select the kind of calculation. These options include peptides, which suffer from one modification (single deletions, insertions, side reactions, not cleaved protection groups and mass signals, which arise from backbone fragmentation) or combinations of modifications (e. g. deleted peptides, which are further modified in one position, or fragments of peptides with one insertion). Two modifications per peptide are the actual limit of the Drogram. There are only four exceptions. QMass also

18.4 Calculations

5 15

140 120

-

c 100

pE

80

-+ 6 0 840 20

Figure 18-3. ESI-MS spectrum of an octapeptide mixture, consisting of 5832 different peptides (above) in comparison to the calculated mass distribution (below). The experimental spectrum was recorded using a step size of 1 u. The calculated masses were sampled using a window size of 1 u.

Table 18-2. Calculation options of the qmass kernel Calculations of qmass

Additional remarks

Expected sequences

Modifications can include uncleaved protecting groups or chemical modifications. Calculations depend on the modification file actually used.

Single deletions Single modifications Single insertions Backbone fragments Double deletions Double insertions Double modifications Modifications of deleted peptides Modifications of insertions Fragments of deleted peptides Fragments of insertions Fragments of modified peptides Truncated peptides For complex libraries, only mass distributions will be given (using a window size of optionally 0.1, 0.5 and 1.0~)

Terminal modifications can be defined using a dummy residue of zero mass, but defined modification. The same is appropriate for noncovalent modifications. All calculations can be done for singly to triply charged peptides.

Linear or cyclic peptides. The results of each calculation can be restricted to a limited mass range (saves disk space) Optionally for each calculation, only the mass range will be reported. ~~~~

516

18 A Computer Program for the Analysis of Mass Spectra of Peptide Libraries

calculates the masses of successively truncated peptides, the programs ‘cross’ and ‘iso’ calculate crosslinks and isotope distributions for any QMass output file and the program ‘deuterate’ calculates peptide masses after deuteration. To support the analysis of observed mass signals, QMass optionally also reports only mass ranges for each type of modification. An overview of calculations and command line options is given in Table 18-2.

18.5 System Tools, Shell Scripts and Automated Analysis Because the output files of QMass are ASCII-based, each program, which can work on textual output, can be used to reduce calculation results further to a manageable size. For example, the use of a standard modification file for not cleaved protecting groups would result in a complete calculation of all events of this kind. With respect to tandem mass spectrometry, the user might be interested in a defined uncleaved protecting group. In this case a simple UNIX command (‘grep’, ‘fgrep’, etc.) would extract the required information from the QMass result file. For an MS-analyst, it might also be of interest, to get an overview of peptide sequences, which tend to fragment. In this case QMass could be used to calculate the masses of all backbone-fragment ions for a defined library. The usage of the same ‘grep’-command would segregate the desired information for display, plotting and comparison to experimental data. The simplest reason of the usage of shell-scripts is to avoid typing long command line options. Implicitly, they can be used to define analysis schemes for a given degree of library complexity. But the most important reason for their usage is to automate analysis (Table 18-3). For example, the script of Table 18-3a would calculate (and optionally plot) 100-200 sublibraries automatically. Different command line options of QMass would allow the mass ranges of all modifications to be calculated and results to be stored in a reportfile, using output redirection, for each sublibrary. The programs ‘compare’ and ‘range’ are related to this concept. ‘Compare’ compares an experimental list of mass signals to each QMass output file. Optionally, it reports the number of assigned peaks, not assigned peaks or produces an output file of not assigned mass signals, which can be recursively read by ‘compare’ itself. Therefore it becomes possible, to get initial hints, which of some hundred sublibraries are possibly affected by synthetic problems (Table 18-3 b). Alternatively, observed mass signals can be compared with the masses of expected peptides, and only for those mass signals that have not been assigned, further searches for possible byproducts can be performed. The program ‘range‘, in its simplest use, searches for QMass entries in a selected mass range. Using the same command in a shell script (Table 18-3c) would generate a list where experimental mass signals and possible byproducts are combined. Sample scripts as well as a small tutorial will be included in the final distribution.

18.5 System Too[s, Shell Scripts and Automated Analysis

5 I7

Table 18-3. Examples of UNIX csh-shell scripts, which can be used to calculate a large number of sublibraries without intervention (a), to compare experimental mass signals with calculated masses automatically (b) and to merge experimental mass/intensity files with calculation results (c)

Shell script examples

Remarks

a:

foreach name (*.lib) qmass -Eas.tab mod.tab $name > tmp sort -n $name.exp end

This script calculates for each library definition with extension ‘.lib’ (up to some hundred) the masses of expected peptides. Sorted results for each library will be stored in separate files with extension ‘.exp’.

b:

rm report

foreach name (*.lib) (qmass -Eas.tab mod.tab $name > tmp) >>& report compare -n -w 1.0 $name.x tmp >>report end

This script calculates for each library definition with extension ‘.lib’ the masses of expected peptides. Some statistics and the used files will be summarized in a report file. The calculated masses will be compared with experimental peaks, stored in corresponding files (expected as ‘.lib.x’). The number of assigned peaks (within 1 . 0 ~accuracy) will be combined with this information in a file named ‘report’.

C:

foreach name (‘cat 1ib.x’) low = $name - 0.5 high = $name + 0.5 echo $name >> report range low high qmass-file >> report

This script would search for each entry of an experimental masshtensity listing corresponding masses (within 1 . 0 ~ )and combines experimental values and calculated peptides. In practice a slightly modified version could be used by simply typing:

end compare qmass- file experimental- file

518

18 A Computer Pmgram for the Analysis of Mass SNctm of Peptide Libraries

18.6 Visualization and Alternative Setup of Calculation Options There is a large range of public domain programs (available via ftp), which can be compiled for commonly used workstations and which can be used to visualize and to plot calculation results. For example, some input files are provided for the program ‘gnuplot’ (GNU). Because one principle of QMass is to calculate subsets of expected and modified peptides, a simple X11-based viewer was programmed, which is based on the Tcl/Tkcommand language. This viewer is capable of reading in up to ten QMass output files, experimental peak lists and external digitized spectra. It allows a direct comparison of different calculation results with experimental data (Fig. 18-4). The same system can be used for an interactive setup of calculation options to avoid the usage of command line options.

Figore 184. TcVTk based viewer, which allows one to visualize up to ten calculated subsets of QMass (grey) in comparison to an experimental spectrum, imported as mashtensity values (black).

18.8 Summary

519

18.7 System Requirements and Limitations UNIX based systems are required because QMass uses a small number of system tools, shell features and requires long filenames. Required features of shell scripts are accepted by tcsh, ksh, bash, and csh, at least. QMass is written in a simple form of the C-programming language, therefore recompiling for commonly used workstations should not be a problem. The same should be true for the Tclflk-based visualizer and the interactive setup of calculation options. This system was compiled for Linux- and IRIX-based operating systems, further systems are reported in the Tcl/Tk distribution. Speed is not critical for workstation, i586 and i486 (Linux) based personal computers, but it should be realized that the calculation of the masses of all backbone fragments of a 16OOOO component library might give some problems with required diskspace. In such cases, it would be more appropriate to calculate only distributions, not sequences. Peptide length is actually restricted to 30 positions. Each variable position can be occupied by 30 different residues. QMass calculates monoisotopic masses for sequence-based results; the calculation of isotopes, which contribute to intensity distributions (at least in less complex libraries) is performed by the program ‘iso’. In short, ‘iso’ calculates distributions based on the most two common isotopes of the elements C, H,N, 0,S, P and Cl at the moment (using Poisson-approximation),and performs a discrete convolution of these individual distributions. ’Ibo options are available for comparing different degrees of accuracy with respect to speed. Because ‘iso’ works on result files of QMass, it can be replaced by alternative methods, if redy required. The accuracy of calculated masses is limited to 2 digits; therefore isotope contributions are calculated in multiples of l u (no high resolutions modus).

18.8 Summary QMass is a UNIX-based program for quality control of synthetic peptide libraries using mass spectrometry. Basically, QMass calculated the masses of expected peptides, masses of modified peptides (both sequence based) and mass distributions. Because the analysis of complex peptide libraries has to deal mainly with the number of peptides, depth of analysis and experimental design, the user can choose the amount of information provided by QMass 1111. Using implicit features of UNIXlike system environments, analysis schemes can be flexibly defined and applied to numerous sublibraries nearly automatically. If required, results of calculations can be compared interactively to experimental data. The information which can be obtained by such analysis can be used to optimize synthesis strategies and library concttiiction.

520

18 A Computer W m r n for rht Analpis of .Was w

m of

LIbmrkr

[I] S R A. Fodor, 1. L. R#d, M. C. Pirrunr), L. slym, A. T. Lu, R War, scknrv 1991,

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121 K. S Lua, S E. sllmon, E. M. Hcnh, V. J. Hruby, W. M. Kumknky, R. J. K n r p ~ Notun f-) 1991* 354, 82-84. 131 R. A. H@tcn, C. PiniUr, S E. Blondelk, 1. R. Appd, C. T. Dooky, 1. H. Cwnq NOlUn (-1 1991,354, 84-86. 141 G Jun& A. 0 Beck-Skkinlcl, An-. Clkm. fnr. Ed EngL 1992.31, 367-383. IS] A. G Beck-SkkinCn,G Junk plbnnoc Ac14 Hrh. 1993, 68, 3-20. 161 R. N. altemmn, 1. M. Ken, M. A. Siani, S C. Buwilk,D. V. -ti, Fmc. Nut4 Aco& sci. USA lm, a,4505-4u19. G J u ~ &And. Bloclknc. 199& 2l2*212-220. S 181 I W. MculCr, S S W W I W ~J. K.-H. W k d k G ~ J u ~ &M U M 1% 6, 43-431. 191 J. W. Mew-* K.-H. WkrmOlkr, V. Gnru, J. Bronjer,G Jun& An-. Clkn. Inr. Ed -4 1993,32*8944%. 110) J. W. M W C. kmpcCr, K.-H. WkrmQuer, G JAnd Bioclkm. 1 219, 261-277. [Ill The proyrmQMssis rnilrbk fnnn ECHAZ minocdlcetkn, (fw rddt#s see UU 01 supplkn, p sO2f.l.

Combinatorial Peptide and Nonpeptide Libraries by. Gunther Jung 0 VCH Verlagsgesellschaft mbH, 1996

Glossary

Acceptor

An acceptor (or ligate) is usually a biomacromolecule (antibody, enzyme, receptor) used for screening of ligands (epitopes, substrates, inhibitors, blockers) out of a peptide or compound library (+). Acceptors may be labeled for detection via fluorescence, bioluminescence or enzyme-linked immunosorbent assay (ELISA +). They can be used for screening free ligands in solution or polymer-bound ligands, e.g. bead pools (4).

Affinity

Measure for the strength of binding between, e. g. an antigenic determinant (epitope, +) and the binding site of an antibody (paratope).

Agretope

Part of an antigen (+) or antigenic fragment which interacts with the groove of cg. a MHC molecule (+) (“desetope”).

Allele

Variance of a defined gene locus within one species.

Allogen

Allogenic variation refers to genetic differences within one species.

Anchor

The anchor molecule provides the decisive chemical bond between the bead and the compound to be synthesized. First, the anchoring of an educt in SPPS must be stable during multistep reactions and secondly the final product must be cleaved under nondestructive conditions. Very often the term linker (+) is used simultaneously. Between the linker (anchor) and the bead structure, a spacer (+) moiety may be introduced to provide a distance between the reactive centers and the support (anchor-spacer combination). There are a large variety of anchors which exhibit chemical properties to allow orthogonal strategies of synthesis.

Anchor residue

Amino acid in the sequence of a ligand, that is essential for mediating contact to the receptor.

522

Glossary

Antagonist

Molecule which interacts specifically with a defined re. ceptor molecule but which prevents signal transduction. Hormone antagonists are often peptidomimetics derivec from rationally designed or randomly screened modified hormones (4).

Antigen

Any substance which induces a specific immune response or which interacts with components of an existing i m mune response.

Antigen presentation

Antigen fragments (from antigen processing, +) arc presented on the cell surface of molecules of the majoi histocompatibility complex (MHC-class 1 and 11, +), Only peptides which fit to the binding pockets within the MHC groove are good ligands. They all have a structural motif in common, which is allele-specific (+) for each MHC type.

Antigen processing

Enzymatic transformation of an antigen (-+) to a form which is recognizable by lymphocytes, e. g. processing of proteins by proteasomes and recognition of proteolytic fragments (peptides bound to MHC) by the T-cell receptor (see antigen presentation).

Aptamer

Aptamers are oligonucleotide ligands fitting to a target. Aptamers are selected from highly complex pools of potentially binding polymers.

Bead

A bead is the solid support (insoluble carrier) for immobilization of an educt through a multistep organic synthesis. Beads are polymer particles which can be functionalized in order to attach the educts via linkers (-1 and anchors (4) for solid phase synthesis. Supports may consist of polystyrene-divinylbenzene (swellable Merrifield resin), glass (nonswellable CPG), polyamides, polystyrene grafted with polyethylene glycol (PEG) and cellulose (paper, cotton). Monosized beads and macrobeads (up to 800 pm) are of advantage in the one-bead-onecompound approach (+ portioning/mixing procedure).

Bead pools

Libraries consisting of single beads (+), each of which carries only one defined compound can be synthesized by the one-bead-one-comDound amroach ( 4 Dortion-

Glossary

523

ing/mixing procedure). These libraries are usually tested as pools of beads for interaction with a labeled receptor (e. g. fluorescent labeled antibody or enzyme). Those single beads which interact, as shown by fluorescence, are selected and the ligands on the beads are identified by direct sequence analysis from the beads or by MS analysis after cleavage or via tagging (+) concepts. The assays with bead pools may also be done after release of the ligands to be tested. The size of the bead pools and the number of beads to be tested also depends on the degree of automation developed for high throughput. Biotransformation

The use of biocatalysts (isolated enzymes or whole cells) for the transformation of nonnatural synthetic organic compounds. An example of an in vivo biotransformation leading to a metabolite is the oxidation of benzene to phenol and the conjugation to phenyl-/h-glucuronide.

Chemical diversity

Term used to differentiate organic compound libraries from biopolymer libraries and natural libraries (+) (natural diversity).

Combinatorial chemistry Branch of pharmaceutical chemistry which deals with the development of strategies and methods for the automated production of compound libraries suitable for the search of low-molecular mass drug leads in high-throughput assays. The new developments originated from simultaneous multiple peptide synthesis, peptidomimetics and peptide libraries. Combinatorial compound Compound mixtures which have a core or basic libraries library (CCL) structure in common and are varied by introducing different elements in different positions. Combinatorial libraries are used for screening lead compounds for therapeutics and diagnostics (compound library, -). Combinatorics

Special discipline of mathematics dealing with the calculation of the number of possibilities to combine a definite number of single items. Fourth basic problem of combinatorics: the number of combinations A of kth order of n different, infinitely repetitive elements with respect to their arrangement is nk, and irrespective of their arrangement is A (n,k) = (n + k - l/k).

524

Glossary

Compound library

Mixture of organic compounds which possess common structural motifs imposed by synthetic strategies, methods and types of educts used in combinatorial chemistry.

Conformational epitopes Linear epitopes (4) which are recognized by antibody only in a folded structure, e.g. a-helix or particular turn conformation. Scans with systematic patterns of a-helix inducing residues and loop scans using disulfide or @-lactam bridges may occasionally locate such epitopes. Consensus sequence

A consensus sequence is derived by comparing a high

number of different ligands selected from a library. The consensus sequence contains most of the elements (or structurally very similar elements) essential for recognition. Consensus sequences of, cg. viral proteins and genes are constructed by alignment of the sequences of different subtypes and variants. Degeneracy Directed library

The degeneracy of a library or sublibrary (+) is given by the maximum number of potential ligands. Combinatorial library which is designed for SPOS or SMPS of scaffolds displaying various, e.g. message and address pharmacophors “directed” to specific receptor binding sites.

Diversity

Libraries with maximal element variability are so-called high diversity libraries. These libraries include a high number of different elements (building blocks) within a short sequence in contrast to low diversity libraries which have longer sequences and a smaller number of different elements. For example a tripeptide library XXX with 20 different elements includes 203 = 8000 different compounds whereas there are only 56 = 3125 different compounds in a hexapeptide XXXXXX built up using 5 different elements.

Diversity of libraries

Large libraries: lo3-lo8 and more components; small libraries: 10- lo00 individual molecules. Smaller libraries can be generated by multiple simultaneous SPOS as individual entities. Large libraries require several steps of deconvolution and selection.

Glossary

525

DiversomerTMlibrary

This expression relates to compound libraries (nonpeptide and nonoligomer libraries), which possess a common core. This core structure (e. g. benzodiazepin) is maintained and produced in the multiple parallel synthesis of analogs (diversomers) using always the same synthetic principles but analogous educts, which carry the same reactive centers (e. g. aldehydes, 1,3-dipolar compounds). Diversomer libraries can be constructed using all the various strategies of combinatorial chemistry and automated synthesizers.

Divide, Couple and Recombine

see portioning/mixing procedure

ELISA

Enzyme-linked immunoadsorbent assay is a fast and inexpensive screening assay which can be linked (adapted) to almost any receptor binding assay of interest; mostly used assay for epitope mapping.

Encoded combinatorial libraries

The use of tags has been proposed and procedures were elaborated for identifying active beads (+) in a screening process for those cases in which sequencing or mass spectrometry are not applicable to the direct analysis of the positive bead (+). Tag molecules are marker molecules (labels) which are attached in parallel with the ligand syntheses on the same bead; (- portioning/mixing procedure). Time-consuming, complicated orthogonal techniques must be applied to attach such tag sequences, which should be present in very low amounts (less than 1%) on the beads and should not themselves interact with the receptor. Bitmap tags are nonsequential tags.

Epitope

Defined determinant of an antigen (+) which binds to the paratope (+) of an antibody. An epitope may be continuous, conformational or discontinuous. Continuous (linear) epitopes are easily screened and exactly defined by using overlapping peptides synthesized by SMPS or screened from peptide libraries.

FACS analysis

Fluorescence-activated cell sorting (FACS) is a fast method for screening either fluorescence-labeled free libraries for interactions with whole cells or beads carrv-

526

Glossary

ing polymer-bound libraries for binding of a fluores. cence-labeled antibody (acceptor). Lead structure

A lead compound (or simply “lead”) is screened via a bioassay or competition assay using, e. g. a radioligandl receptor system, for inhibiting a hormone activity by antagonism (+). The sources for lead structures are natural or synthetic compound libraries. It is anticipated that combinatorial chemistry may lead immediately to morf precious low molecular mass leads, which do not need long developments for a useful drug.

Libraries from libraries

Expanded library approach which uses complex libraries as educts of scaffolds (+) to generate diversity.

Library

Defined mixture of synthetic organic compounds which is characterized by a common structural element, e.g. a backbone of defined length, or cyclic template, or core structure, which is varied systematically in certain positions (+ positional scanning) using methods of combinatorial chemistry.

Light-directed synthesis

Library technology combining photolithography and solid phase synthesis on functionalized flat glass surfaces. Photolabile temporary protecting groups are used and, for cleavage, a photolithography mask. The position of each compound on the surface is known (light-directed spatially addressable parallel synthesis).

Linker

The term linker is used simultaneously for anchor (+I molecules which provide a orthogonally cleavable bond between educt and the solid support. Furthermore, in order to prevent steric hindrance and crosslinking side reactions, spacers such as s-aminocaproic acid or the hydrophilic polyethyleneglycol (PEG) are introduced between linker and solid support (linker-spacer combinations). Anchors and spacers may be combined in one molecule, e. g. PAL (= ADPV) anchor.

Major histocompatibility MHC molecules constitute the centers of molecular immunological recognition of self and nonself-peptides. complex These peptide binding proteins present self-peptides and foreign DeDtides of defined seauence motifs (+) on the

Glossary

527

surface of cells. Foreign peptide-MHC-class I complexes are recognized by the T cell receptor (TCR) of cytotoxic T lymphocytes and foreign peptide-MHC-class I1 complexes stimulate TCR of T-helper cells.

Mimotope

Diccontinuous epitopes (+) which can not or can hardly be defined by overlapping peptides may be imitated by low-molecular mass mimics consisting of peptidic or nonpeptidic structures screened from libraries.

Mixotopes

Mixotopes consist of a mixture of different epitopes (+) fitting to the motifs of a highly variable region of a viral protein (Gras-Masse and Tartar). Immunization with mixotopes may create a population of antibodies possessing a higher chance of crossreactivity with a new viral variant.

Molecular evolution

Applied molecular evolution makes use of multiple recursive cycles of iterativer DNA mutations, selection and amplification to mimick and accelerate natural evolutionary design. Viruses and cells are used as vehicles and factories to create diversity and new molecules or even organisms. In vitro evolution (protein evolution) is a proven tool to discover active 3D structures, e. g. a novel antibiotic resistant /3-lactamase. Functional optimization of biomolecules is one aim of combinatorial biology (evolutionary biotechnology).

Monitoring

Test for completeness of deprotection or coupling during SPPS either by automated chemical or electrochemical analysis or spectroscopically using a color reaction (ninhydrin, Fmoc cleavage product by UV) and/or via automated conductivity monitoring.

Monoclonal antibody

Monospecific antibodies which are produced by transformed cell lines (clones) and used mainly in diagnostics.

Motif of a library

Natural or synthetic libraries fitting to a particular recep tor molecule exhibit common structural features for all components, e. g. helicity and amphiphilicity, a proline bend, or conserved amino acids in defined positions.

528

Glossary

Multicomponent reactions The concept is based on Ugi's four component reactior of the isocyanides (1959). MCR isonitrile chemistry re. (MCR) presents the first milestone in combinatorial organic syn. thesis. As early as 1971 Ugi described the first solutior library comprising 2560000 products. Multipin technology

Multiple simultaneous peptide synthesis

First simultaneous multiple solid phase synthesis origin. ally developed for peptide synthesis, also applicable foi peptide library and solid phase organic synthesis (SPOS: below 90"C.Based on grafted polypropylene pins arrang ed in a convenient 8x12 format fitting to 96-well microtiter tray. The pin blocks simplify synthesis of hundreds to thousands of compounds, since no filtration steps are required. A variety of anchor-linker systems also allows compound cleavage from the pins supports.

SMPS can be carried out using teabags (4). pin synthesis-technology (+), and with great advantages full) automated multiple synthesizersbased on pipetting roboi systems. Using small-scale synthesis (5 mg of resin per reactor vessel), up to 1200 compounds or peptides can be prepared simultaneously. The same approaches or instrumentation can be used for the simultaneous preparation of libraries.

Mutation

Replacement, deletion or insertion of building blocks in defined positions of polymers or biopolymers by using chemical synthesis or site-directed mutagenesis; leading to single or point mutations; multiple mutations of plasmids; phage libraries (4).

Natural libraries

In order to maintain target specificity and biological activity, natural product libraries exhibit common structural motifs (+), for example, the same heterocycle (e. g. actinomycins) or the same peptide sequence motif (e. g. MHC peptide ligands), whereas other parts of the molecule may be highly variable. Natural evolution is based on natural compound and peptide libraries.

Nonrationsl drug finding Process of selecting ligands as potential leads from a library without knowing structural information about the receDtor (tarnet).

Glossary

525

Paratope

Part of an antibody molecule which is in contact with the antigenic determinant (+ epitope) of an existing immune response.

Peptoids

Special class of peptide-related oligomers which possess the backbone of N-substituted glycines resistant to pep tidases.

Phage libraries

Phage surface proteins can be easily and highly varied within certain regions using gene technological methods. Bacteriophage libraries consist of up to 10'' phage clones, each displaying one defined surface protein with different sequence and thus different affinity to a defined receptor molecule used for ligand screening. Those phage clones displaying receptor binding properties are separated, and the amino acid sequence of interest is derived from the DNA sequence in the phage genome. Polymerase chain reaction (PCR) techniques are used for this purpose.

Pharmacodynamics

Properties of a drug with respect to biological activitj and bioavailability, absorption, distribution, metabolism and excretion.

Pharmacophore

Pharmacologically active compound which can be a useful drug, or which may be developed into a drug.

Photolabile linker

Anchor-linker combination with a photosensitive functionality, e. g. chromophor based on the o-nitrobenzyl system cleavable by photolysis at 365 nm.

Pin technology

Based on the ideas of Geysen and Meloen, small polymei pins are functionalized at their surface and used for parallel synthesis. The pins are arranged on a block in the format of 96-well microtiter plate. Coupling steps are car. ried out individually for each pin in each well and, fol washing and deprotection, all pins are treated simulta. neously (+ multipin technology).

Portioning/Mixing procedure

(= split synthesis, = divide, couple, recombine (DCR] method). The approach allows the simultaneous synthesis of one defined compound on one particular bead (one bead-one seauence svnthesisl. For DeDtide librart

530

Glossary

synthesis, the complete bead pool (+) is distributec (divided) by portioning into separate reaction vessels, ir which individual amino acids are coupled separately tc completion (In combinatorial organic synthesis only ow defined reactant is allowed to react with the polymer. bound educt placed in one of the multiple reaction vessels). After reaction the resin portions are mixed (recombined), washed (etc.), and again, before the nexl synthetic step, divided into portions to be reacted with individual reagents. The portioning/mixing method en. sures almost equal representation of all building blocks regardless of their reactivity (see also mixed synthesis method). The limitation of the method is the single bead, which cannot be divided. Thus, the number of beads defines the heterogeneity of the bead pool.

Positional scanning

Variation of a library by introducing mix positions (X) containing different elements in one position after the other along a biopolymer sequence or by introducing defined positions (0)containing only one element.

Random pool of fragments

Nonspecific cleavages of a macromolecule, e. g. polymer or biopolymer, may lead to random pools of fragments potentially binding to receptors.

Randomized library

Random combinations of building blocks at every position of a core structure or peptide.

Randomized position

Single position in a peptide sequence or on a template or core structure which contains all building blocks.

Rational design

Process of planning and designing ligands based upon the knowledge of the 3D structure of a binder or/and its receptor.

Sublibraries

Libraries of lower complexity than a fully randomized library; e.g. each sublibrary possesses at least one constant region and variable regions in defined positions of a template or a cyclic or linear oligomer. In the iterative process for lead structure selection sublibraries of lower complexity are tested in comparison to that library of higher complexity defined in the previous round as the best one. Repetitive cycles of assays and syntheses of

Glossary

531

sublibraries of lower complexity lead ultimately to a defined compound with e. g. the best antagonistic properties. There are no tags (+) and no analytical characterizations necessary in this strategy provided all synthetic steps are highly reproducible. Scaffold

Structural motif or template (e. g. cyclic polyol, cyclopeptides, multifunctionalized heterocycle, or even part of a protein such as a a-helix/turn/@sheet fold). Used for creation of molecular diversity via multimeric derivatizations, attachments and structurally compatible insertions.

Selector/Selectand

A selector is usually a low-molecular mass organic mole-

cule which specifically interacts with one defined selectand, e.g. a cage-like selector such as cyclodextrin interacts only with one enantiomer of dansylated phenylalanine, thus allowing chiral discrimination. Calixarenes have been applied as selectors for specific recognition of common structural elements of individual components in a peptide library. Small molecules libraries Compound libraries (+) consisting of compounds with molecular masses preferably below about 600 D, which are, e. g. peptidomimeticsor diversomers. Diversity library approaches for accelerated and direct drug discovery aim at the following properties of drug leads: selective, potent, orally available, biostable, bioavailable, safe, oneday therapy. The most successful therapeutics are typically low-molecular weight compounds below 600 D. Spacer polymers

Spacer molecules are introduced between the insoluble support and the anchor (linker) group. Spacers are oligomethylene carrying units (e. g. via c-aminocaproic acid) or the hydrophilic polyoxyethylene (POE or PEG) which introduce quasisolubilization and better accessibility of the reactive centers.

Split synthesis

see Portioning/mixing procedure.

Synergism

Interaction with cooperative intensifying effects.

Synthetic vaccine

Synthetic vaccines are chemically synthesized immunogens which induce either antigen-specific antibody,

532

Glossary

T-helper and killer cell response or all responses at once (complete vaccine). Such vaccines are constructed on the basis of defined B-cell, T-helper and CTL (cytotoxic T cell) epitopes (+) and an adjuvant (e. g. tripalmitoylS-glyceryl-cysteine, Pam,Cys). Different formulations of antigens and adjuvants can be used such as the MAP constructs (multiple antigen peptide).

Tags, Tagging

see Encoded combinatorial libraries.

Tes bags

In the Houghten approach of SMPS tea bags (“T-bags”) consisting of polypropylene net are used as reaction chambers for defined populations of beads (+). Resin beads are divided into equal or different portions (depending on the desired amounts of products) and packed into the numbered (coded) bags and sealed. The resin beads in the tea bags are accessible to reagents and swellable in solvents used for usual protocols of peptide synthesis. The bags are sorted prior to the coupling step and coupled in separated vessels, followed by combining them in one vessel for washing and N-terminal deprotection steps. Variable positions can be introduced, and libraries are also easily prepared.

Target

Biomolecule (cellular receptor or synthetic molecule) used in screening assays to select a ligand from a library.

Template

see Scaffold

Unrandomization

During the iterative process of lead structure search, a more complex library is unrandomized in the next round to less complex sublibraries, which contain more defined elements, e.g. in the sequential positions of a peptide. Multiple unrandomization steps in more than one position may be advisable occasionally to obtain faster access to interesting leads.

Combinatorial Peptide and Nonpeptide Libraries by. Giinther Jung 0 VCH Verlagsgesellschaft mbH, 1996

Index

acceptor 521 acetal formation 30 acetal(keta1) formation 42 acetylcholine receptor 91 activity threshold of PS-SCLs 158 acylpiperidine 61 adduct ions 256 adenovirus 98 adenylate kinase I ADPV 477 a,-adrenergic receptor 399f. affinity 521 affinity chromatography selection 173 affinity of antibody 3 1 1 agretope 521 alamethicin 3 alcohol dehydrogenase isoenzymes 442 aldol condensation 30,42f. cis-alkenes 37 N-alkenyl-glycines 62,64 akylating agents 407 alkylation of primary amines 391 2-alkylcyclohexanone 21 alkylphosphonicacids 41 alkynol 36 N-alkynyl-glycines 62,64 alkynyllithium compounds 36 allele 521 allele-specific motifs 235 allogen 521 amidosuccinamidylpeptide 334 amino acid analysis 213,247 amino acid fluorides 407 D-amino acid SCLs 155, 164 amino acid tester mixture 123 amino acids 407 aminobenzophenone 5 1,407,419 a~~~in~ethyl-PS 471 a-aminoisobutyric acid 3 7-amino-4-methylcoumarin-3-aceticacid 236 aminomethyl-PS 469 5-amino-0-phenanthroline 47 analysis by Edman degradation 287 ff.

analysis of benzodiazepins 422 analysis of libraries 21 1 analysis of mixtures 247ff. analysis of peptide libraries 5 1 1 ff. anchor amino acids 240 anchor groups (table) 25 anchor safety-catch 367 anchor residue 521 anchor residues 290 anchors 22,25ff., 521 ff. - benzylhydrylamine 25 - Rink acid anchor 25 - SASRINanchor 25 - tritylanchor 25 - Wanganchor 25 animalsera 310 antagonist 522 antiallergic drug 3 12 antibody 207 antibody binding 3 IOff. antibody inhibiting activity 149 anti-/&endorphin monoclonal antibody 189 antigen 522 antigen presentation 522 antigen processing 522 antigen-antibodyinteraction 149,212 antigenic determinants 144,159 anti-insulin monoclonal antibody 1% antikeratin antibody 99 antimicrobial peptides 150% antipeptide antibodies 3 12 antisense oligonucleotides 445 applications of synthetic peptide libraries 14 aptamer 522 arenes 57

-

configuration 368 density 372 directed synthesis 368 Ofspots 365 artifical enzymes 195 ASCII files 5 1 1 aspartic acid racemization 330 aspartyl epimerization 338

534

Index

aspartyl racemization 338 assay sensitivity 449 autoimmune diseases 349 ff. automated pipetting 365 automated and semiautomated peptide synthesis 205 automated multiple peptide synthesizers 85 automated premix procedure 235 automated workstation 394 autoradiograph of beads I92 auxiliary programs 512 available chemicals database 396 backbone fragmentation 51 3 backbone fragments 5 19 backbone modifications 8 background 290 bacteriocins 6 B-cell epitopes 92 bead 522 bead pools 522 bead shaving 183

benzhydrylamine-polystyrene 478

benzodiazepine 53 ff. - design 413 - library 410,405,411 - synthesis 407,415 - skeleton 51 benzodiazepine 49,53 f., 405

1,4-benzodiazepine-2,5-dione 4 I7 benzoin condensation 30,43 benzophenone derivatives 5 1 benzopyran library 61 BHA 478 - anchor 25 2-chlorotrityl anchor 25 biased peptoid library 397 bicyclo[2.2.2]octanes 61 binding assay solid phase 372, 384 bioactivity data 3 12 biopanning 98 biosynthesis via multienzyme systems 2 biotinylation 437 biotransformation 4,523 biphenyl derivatives 57 N-(N-Boc-aminoethy1)-glycine 65 9-borabicyclo[3.3. Ilnonane 37 Bpoc 413 brominated PPOA 483 brominated Wane resin 482

bromoacetic acid 392 bromoacetylation 402 bromoethyl-PS 47 1 bromophenol blue 368 tert-butylatedpeptides 267,273 byproduct analysis 247 ff. byproducts 267,267,275,51 I byproducts in libraries 265

I3CNMR relaxation time 429 CAMP-dependent protein kinase 192 candida albicans 153 Cannizzaroreaction 42 capacity of single beads 433 capacity per bead 447 capillary electrophoresis 247 capillary zone electrophoresis 2 14 catalytic hydrogenation 3 1,60 CCK A and B receptor antagonists 49 CE 247 cecropins 3 cellulose 499 cellulose as support 84 cellulose membrane 368 cellulose paper disks 84 chemical diversity 523 chemical modifications 267,268, 514 chiron mimotopes (Geysen) pin apparatus 406 chlorophenol binary code 33 2-chlorotritylchlorideresin 227,235 chlorotrityl-PS-DVB resin 23 chlorotritylresin 220,480 cholecystokinin (CCK) 405 cholecystokinin receptor 41 1 cleavable dipeptide linker 177 cleavage mixture for protected peptides 224 cleavage/deprotection station 394 code molecules 33 code peptides 32,33 coding 182ff. collision-induced dissociation 268,273 combinatorial biosynthesis 8,69 combinatorial chemistry 28,523 combinatorial compound library 523 combinatorial libraries 139ff. combinatorial library methodologies 185 combinatorial synthesis on membrane supports 363f. comhinatorics

523

Index

competition assay 207,239 competition assay, MHC-class [I 236 competition binding test 310 competition experiments 240 completely randomized XI, 209 composite supports 425 composition of peptide libraries 120K compound libraries 29,524 computational tools 396 computer program Qmass 5 I 1 ff. confocal laser microscopy 450 conformational epitopes 524 conformational mapping 82 confonnational restrictions 230 consensus sequence 524 continuous flow 466,484,492,495 contour-plot 275 control sera 3 10 controlled pore glass (CPG) 24,445 controlled-pore supports 495 convergent approach 187 conversion table mesh-mm 502 copolymer 466 core Q-resins 425 correction factors 289 cotton as solid support 84 cotton supports 499 coupling rate 2 13 CPC 495 CPG 495 C-programming language 5 19 critical residues 185 cross-references 505 tT. CTLepitopes 9 cyclic pentapeptides 330 cyclic peptide libraries 327K cyclic peptide template 222 cyclic peptides 221 ff. - disulfide bridging 221 - lactam formation 221 cyclization in solution 222,235 cyclizationreagents 223,224 cyclizationtendency 235 cyclizations 333 cyclizationson solid supports 222 [2+2] cycloadditions 30,47 [2+3] cycloadditions 30 cyclobutadienyl iron tricarbonyl 47 cyclocondensation 6,30 cyclocondensation to thiazolidines 53

535

cyclodimerization 222,225 cycloheptapeptides 337 cyclohexanes 46 cyclohexapeptide, scheme of synthesis 228 cyclohexapeptides 223 ff., 336 cyclohexapeptides, synthesis 223 ff. cyclopeptide library 227ff. cyclization tendency 222 cytolysis 361 cytomegalovirus 93 cytotoxic T-lymphocytes 9, 349 daughter ion scan 265 DCRmethod 142 decoding concepts 204 decoding of libraries 103 deconvolution degeneracy 524 degeneracy of peptide recognition 361 deletions 5 I 1 delineation 379 dendrimers 500 depurination 446 derivatization of dicarboxylic acid 44 desfemoxamine 5 determination of the coupling efficiency 213 device for manual synthesis 1 17 diagnostic ions 265,27 1 diastereomeric cyclopeptides 227 dicarboxylic acid monoamides 45 dicarboxylic acids 44 2,3-didehydroaminoacids 7 Dieckmann condensation 54,55 Dieckmann cyclization 3 1 Diels-Alder product 47 Diels-Alder reaction 3 I, 47 diffision in resins 430 digital code 182 digitized spectra 5 18 23-dihydroxybenzoicacid 263 3’-O-diisopropylsilyI-nucleotide 66 diketopiperazine forming linker 3 I6 diketopiperazine release linker 177 dimethylacrylamide 491 cis-diols on polymers 39 diphenyl synthesis 45 1,3-dipolar cycloaddition 47 directed fermentation 4 directed library 524 discontinuous epitope 184, 190

536

Index

discrimination effects 254 diversity 524 diversity of benzodiazepine libraries 417 diversity of libraries 524 diversomerTMlibrary 525 divide, couple and recombine 30,529 divinylbenzene crosslinker 465 DNA analogs 65 DNA gyrase inhibitor 8 DNAtag 33 domino strategy 130 doubly charged ions 260,265 downstream processing of pin-peptide libraries 318 drug finding 528 duramycins 7 dynamic range 253,259 dynorphin B 99 Edman degradation 63,211,274,287 electrochemical polymerization 94,96 electron impact ionization 249 electronics 195 electrophilic addition 31 electrophoretic mobilities 1 14 electrophoretogram 1 13 electropolymerisation of peptides 96 electrospray ionization 249 electrospray mass spectrometry 217 electrospray mass spectrum of peptide mixtures 218,231 ff. elemental analysis of resins 35 ELISA 525 ELISAassay 89 - on pin-bound peptides 3 19ff. enamine reaction 21 encoded combinatorial libraries 525 /?-endorphin 99,149 /?-endorphin antibodies 181 endothelin antagonist 328 enkephalins 150 enolate alkylation 57f. enzyme inhibitors 154 enzyme-linked immuno assays 90 enzyme-linked colorimetric assay 175 epidermin 4 epimerization 330 epimerized cyclopeptides 222 epitope 207,525 epitope fine mapping 89,92

epitope mapping 8 I , 303 ff. epitope screening 375 Epstein-Barr virus 92 equimolarity 256,282 equimolarity of library components 247 Escherichia coli 152

ESI 249

ESI mass spectrometry 227,263,511 ethylene carbonate 439 expansion 50I FA0 249,251 FAB mass spectrometry 334,410 FACS analysis 525 fast atom bombardment 249 filamentous phage 173 fingerprint spectra 248,281 fitness landscape 365,371 flow-through systems 436 9-fluorenylmethoxycarbonyl 267,273 fluorenylmethylester 333 fluorescence assays 449 fluorescence labeled ligand 236 fluorescent dye 372 fluoride activation 407 Fmoc-amino acid fluoride 5 1 foot-and-mouth disease virus 92,95,96 Fourier transform ion cyclotron resonance mass spectrometers 261 Fractogel 500 fragment ions 256,270 fragmentation 249,259,265 FT-ICR 282 FT-IR measurements 34

/3-galactosidase 372 gallidermin 7 gas desulfiuization 46 gelphaseNMR 35 gel phases 465 gelatinous polymer supports 425 gel phase NMR 455,457 gene clusters 9 genetic engineering 6 glossary 521 ff. graftcopolymer 466 graft copolymerization 427 graftpolymers 321 Grignard reaction 3 1,42,43 gyrase inhibitor 8

Index Haemophilus influenza type b 8 I , 87 Haemophilus influenzae 443 halogenated molecules I82 Heck reaction 3 1,57 helicenes 56 Henry reaction 31,49 hepatitis B virus 90 hepatitis C virus type I, epitopes of 89 hepatitis delta virus 92 heterobifunctional resin 210,21I, 484,501 heterobifunctionalized polystyrene-polyethylene glycol resins 103 heterocyclic compounds 49,52 heteronuclear multiple quantum coherence

248 hexamethylenediamine-polyacrylresins 24 high-performance size exclusion chromatography 237 high speed peptide synthesis 438 high-performance liquid chromatography 247 high-performance liquid chromatographymass spectrometry 5 1 1 high-throughput synthesis 394 histone HI c peptide 440 HIV gp120 protein 102 HIVsubtypes 92 HIV I -Nef protein epitope 449 HIV-TAT-RNA antagonists 49 HLA I1 molecule, DRB 1'0 101 236 HMB 481 HMBA-MBHA 482 HMPB-BHA, -MBHA 479 HMQC 248 HPDI-expansin 501 HPLC retention times I 14 HPLC-MS 269,270,274,511 HPLC-MS-MS 282 hybrid oligomers 65 HYCRAM 483 HYCRAM supports 26 hydantoins 49,50,5 1 hydantoins on microreactors 454 hydrogenation catalysts 60 hydrophobic peptides 280 hydrophobic pocket in MHC-class JI groove 239 hydroxamate siderophores 5 4-hydroxybenzyl byproducts 270 hydroxyethane-sulfonyl-PS-DVBresin 45

537

hydroxyethyl-PS 47 I hydroxymethyl-PS 470 (6-hydroxymethyl)-3,4-dihydro-2H-pyran 23 identification of active sequences 100 image analysis 372,384ff. iminodiacetic acid based linker 178 immobilization of alcohols 27 immobilization of aldehydes and ketones 42f. immobilized aromatic aldehydes 43 immunization 442 immunogenicity 8 I impurities 51 1 indigo carmine 193 influenza nucleoprotein 87 influenza virus hemagglutinin 145 influenzavirus peptide HA306- 3 18 238 inhibition of [3H]-prazosin binding 399 inhibition ofS. aumus 166 inhibitors of melittin's hemolytic activity

153, 162

inorganic supports 466 insect sex attractants 36 insertions 51 1 integrin binding peptide 99 ion channel formation 3 ionspray 274 ionization yield 253 IRIX 519 isobaric peptide family 250,253,257,275 isobaric peptides 218,279 isotope distribution 260,513,519 isotopes 519 isotopic ions 256 isoxazole 62 isoxazoline 62 iterative approach 187 iterative process 13, 147,173, 185 iterative resynthesis 388 kieselguhr 425 K kieselguhr support matrix 466 kieselguhr-polyamidesupports 492 kieselguhr/polyamide 24 killer cells 21 1 Knorrresin 475 lag-effect 289 lanthionine 7

538

Index

lantibiotics 4ff., 70 laser densitometer 372 laser desorption 369 laser-desorption ionization 26 I laser light-directed synthesis 364 laser scanning microscopy 2 12 lead structure 525 leukemia assay 337 libraries from libraries 165,525 libraries of libraries 185, 187 library 525 ff. - combinatorial on spots 363 library design 396 ff: ligand motifs 290 light-directed synthesis 526 linker 466,526 ff. - safety-catch 367 linker for benzodiapepsin synthesis 417 linux 519 lipopeptide-PEG-conjugate 443 lipopeptides 263,282 liquid phase method 427 loading of resins 223 lupus etythematosus 4 1 1 lympholysis assays 358 lysinoalanine 7 macro beads 103,446,500 macrocrowns 499 macroporous supports 425 magainins 3 magic angle spinning 35,430 ft: magic mixture 439 major histocompatibilitycomplex 526 major histocompatibilitycomplex (MHC) molecules 349 ff. MALDI 249,26 1,263,282 MALDI-TOF MS 369 manual synthesis of peptide libraries 1 I6 Madey’s reagent 330 Marfey’s test 340 mass analyzers 260 mass distribution 253,260,281,511,514 mass patterns 2 I8 mass spectrometric suppression 253 mass spectrometry 247 ff. mass transport 438 material science 195 matrix-assisted laser desorption ionization 249,251,261,334

matrix-assisted laser-desorption ionization time-of-flight mass spectrometry 35,369 MBHA 478 MBHAresin 340 melittin 3 membrane support 363,500 /?-mercapto ketones 39 Merrifield resin 470 metabolites, microbial I 2-methoxy-5-[2-[(2-nitrophenyl)dithio]- 1-oxopropyll-phenylaceticacid anchor 23 p-methylbenzhydrylaminehydrochloride /?-methyl-lanthionine 7 MHC blocker molecules 10 MHC molecule 290,3 1 I, 350 MHC-class I molecule 191 MHC-class I peptide ligands 207,209,357 MHC-class II binding assays 237 MHC-class I1 binding groove 239,240 MHC-class 11 molecule 10 MHC contact sites 354 MHC-I ligands 291 MHC-I1 ligands 293 micellar electrokinetic chromatography (MECC) 2 14,445 Michael addition 39f. Michael reaction 3 1 microcins 8 microheterogeneity 1 microparticles 433 micropins 499 microporous resins 468 microspheres 468 microsquares microstructured peptide-gold electrode 94 microstructured synthesis of oligocarbamates

64

microtiter plate 406 mimotope 305,376,527 mimotope strategy 98,3 14 miniaturization of solid-phase peptide synthesis 93 minipepscan 320 Mitsunobu reaction 3 1,40 f., 67 mixed position 2 13 mixotopes 97,527 modifications 51 1 modified peptide libraries 102, 165 modular approach 387 molecular evolution 527

Index monitoring 213,527 - bromophenol blue 2 13 - chloroanil detection 213 - conductivity monitoring 21 3

- ninhydrin assay 21 3 - of solid-phase reactions 34 monoclonal antibody 527 monoclonal antibody binding 88 monodisperse beads 469 monoisotopic masses 5 19 monomethoxy-alanyl-R4M 475 motifs of libraries 185,527 multicomponent mixtures 11 1 multicomponent reactions 528 multipin peptides 313 multipin peptide system 304 multipin technology 528 multiple antigen peptide (MAP) concept 102 multiple antigen peptide system 500 multiple, parallel synthesis 28 multiple peptide synthesis 320 ff. multiple peptide synthesis methods 80 ff. multiple peptide synthesizer 85 ff. multiple sequence analysis 13,21 I, 287 multiple sequencing 181 multiple simultaneous peptide synthesis 528 multiply charged ions 249,256,260 mutation 528 Myasthenia gmvis 1 1 natural libraries 528 natural peptide libraries 1 ff. nebulizer-assisted electrospray 274 neuropeptide Y 14,207 neuropeptide Y,binding site of 87 neutral loss scans 266,273 nihile oxides 49,62 nitroveratroyl chloroformate 64 nonapeptide sublibrary 234 nucleophilic aromatic substitution 3 1 octapeptide libraries 351 olefin synthesis 56 oligocarbamates 12,64f. oligomer libraries 387 oligomers 12 oligomer synthesis 61 oligonucleotideanalogs 66 f. oligonucleotides 364 oligonucleotidesynthesis 445

539

oligosulfones 41 oligosulfoxides 41 oligoureas 67,68 f. omission libraries 121 on-bead binding assay 176 one-bead-one-compound 175 opiate receptors 399,400 opiod receptor assays 15 opioid antagonists 150 opioid ligands 161 opioid peptides 150 opioid receptor ligands 15 1 optical sections 212 organometallic compounds 3 1 organ0 tin compounds 57 orifice 249 orthogonality principle 23 oxazole ring systems 4 oxidation 31 oxidation of Met 274,278,278 oxime resin 483 oxime synthesis 43 oxytocine 439 PAL 477 palladium catalysts 60 palladium catalyzed C-C attachments 56ff. palladium-catalyzed Heck reaction 57 PAL-MBHA 477 PAM 473 - anchor 25 Pam3Cys-epitope 443 Pam3Cys-PEG-peptide conjugate 443 paper electrophoretic identification 1 13 paper electrophoretic map 1 14, 1 15 parallel approach 186 parallel synthesis 2 18 paratope 529 parention 266 parent ion mass spectrum 271 parent ion scans 271,273 partial libraries 128 partition coefficient 433 Pauson-Khand cycloaddition 3 I , 48,49 PD-MS 25 I , 263 peakclans 251 PEGA resins 491 PEG-Pam3Cys adjuvant system 443 PEGsurface 210 pentadecapeptidelibraries 235

540

Index

pentadecapeptide sublibraries 237 pentafluorophenylesters 338 2,2,5,7,8-pentamethyIchromane-6-sulfonyl

267

pepscan method 90ff pePSyn 499 pepsynK 492 peptaibols 2f. peptide alcohols 19 peptide aldehydes I9 peptide amide 367 peptide analysis 13 peptide antibiotics 1 peptide binding to MHC 350 peptide coating for ELISA 308 peptide diversity calculation 207ff.,238 peptide dual positional scanning 378 peptide encoded compound library 32 peptide families 230 peptide fotmat for mapping 307ff. peptide-functionalized gold surface 95 peptide hnctionalized surface 94,95 peptide identity 247 peptide libraries 94,203 - activecompounds 204 - amino acid composition 248 - analysis 253 - applications 203 - characterization 247 - chemically modified 204 - frompins 313 peptide libraries for epitope mapping 316 peptide libraries in immunology 349fF. peptide libraries of microbial origin 2ff. peptide libraries on pins 3 17 peptide library synthesizer scheme 218 ff. peptide library, 32P-labeled 173 peptide ligands 241 peptide motifs 361 peptide nucleic acids 12 peptide-nucleic acids (PNA) 65 f. peptide-peptoid hybrids 63 peptide phosphonates 40f. peptide positional scanning 375 peptide purity 247 peptide purity 309 peptide recognition by CTL 358K peptide selection by MHC molecules 352 peptide tag 182 peptide transporters 14

peptide vaccines 1 1 peptoid concept 387 peptoid libraries 62 peptoid mixtures 398 peptoid oligomers 389ff. peptoids 62f., 529 pericyclic reactions 48 permethylation 165 phage libraries 98,529 pharmacodynamics 229 pharmacophore 529 phenol ethers 41 phenylacetic acid derivatives 57,58 phenylthiohydantoins 63 pheromones 36,38 phosphatase - alkaline 374,384 phosphopeptides 282 phosphoramidite chemistry 445 phosphorylation 437 phosphorylation patterns 379ff. photochemical cyclization 3 1 photochemically cleavable linker 28 photolabile linker 329 photolithography 93 phorolysis 27 phthaloyl protecting group 68 PILOT 364 pin block 91 pin method 3 I6ff. pins 418 pins with a cleavable linker 322 pin synthesis method 90ff. pin technology 9Off., 529 piperidides 330 pipet robot 218 pipetting robots 85 plasmids 173 PNA-modified TentaGel 446 Poisson distribution 184 polyacrylamide-type resins 425 polyacrylic acid polymer 3 16 polydimethylacrylamide 466,489,492,499 polyethylene glycol-PSDVB copolymers 24 polyethylene pins 3 16 polyethylene rods 90 polyethylene support 24 polyethyleneglycoles 427,485 poly Hipe, polystyrene/polydimethylacrylamide copolymer 24

Index

PolyHlPE resins 489 fT. polyketide biosynthesis 70 polyketids 8ff. polymer beads 465 polymerase chain reaction 33 polymer-bound acrylic acid 47 polymer-bound acyl chlorides 45 polymer-bound benzophenones 53 polymer-bound butadienes 47 polymer-bound carboxylic acids monoestermonochlorides 44 polymer-bound cyclization 46 polymer-bound Dieckmann cyclization 55 polymer-bound dienophiles 46 polymer-bound p-nitrophenylcarbamates 59 polymer-bound peptides, synthesis of 90ff. polymer-bound reagent 20 polymer-bound ureas 59 polymer supported organic synthesis 19 polymeric microreactors 446 poly(N-acrylyl-pyrrolidine)resins 24 poly(N-[2-[4-hydrophenyl)ethyl]acrylamide (coreQ) 24 polymeric supports 425 polymer-supported chemistry 388 polymer-supported reagent 21 polymer-supported substrate 21 polypeptide antibiotics 4 ff. polypropylene packets 142 polystyrene 426 ff. - cross-linking 465 - loading capacity 465 - macroporous 489 - PEG-grafied 484 - macrobeads 24 - swelling properties 465 polystyrene resins - acid-labile 472 - base-labile 481 - nucleophilic cleavable 481 - photo-labile 481 polystyrene-divinylbene copolymer 24 polystyrene-grafted polyethylene film 85 polystyrene-poly(ethyleneglyco1)-graft (PEG) copolymer 427 polystyrene supports 468ff. pool sequencing 21 1,234f. porphyrins 56 portioning-mixing method 1 1 1, 112

541

portioningmixing procedure 529 positional scanning 101,204,207,530 positional scanning format 222 positional scanning libraries 371 Positional scanning SCLs 157 posttranslational modifications 4,7,192 precursor protein 4,8 prediction methods 306 prediction of epitopes 81,306 premix method, procedure 205,206,220 premixed Fmoc-amino acids 220 preview effect 296 primary amines 392 I ,3-propanediols 60 protein - cAMPdependent 379 - kinase 173,380 - phosphorylation 192,379 protocol for the synthesis of NSG peptoids 402 Pseudomonas aeruginusa I0 1 PTFE support 501 pthalocyanines 56 pyrrolin-4-on oligomers 12 QMass 248,251,511 QMass, description 5 12 quadrupole analyzer 263,265 quadrupole mass analyzer 260 quality control 5 1 I fT. quantitative analysis 247 quantitative Fmoc determination 223 quinolones 50,52 racemization 227,235,330 radioisotope 380 radioreceptor assays 161 RAM 475 Ramachandran plots 390 random libraries 99 randompool 370 random pool of fragments 530 random position 371 randomized library 530 randomized position 530 rational design 530 receptor ligand identification 150 recognition pattern 373 reconstructed total ion current chromatogram 214

542

Index

recoveries of PTH amino acids 289 reduction of carbonyl groups 43 reduction of the nitro group 61 reduction to cis-alkenes 37 reduction with complex hydrides 31 reductive amination 3 I , 391 relative ion intensities 253,260 releasable assay 177, 192 release in solution 367 repetitive yield 288 reporter systems 32 resin-bound Diels-Alder product 46 resin-bound dienes 46 resin-bound isoxazoline 49 resins 465ff. resin splitting 395 resolving power 260 retention factors 1 14 retro-inverso analogs 330 reverse transcriptase (RT) inhibitors 49 reverse Wittig reaction 38 reversed phase chromatography, of cyclohexapeptides 226 ring closure reaction 46,54 Rink-acid 474 Rink-amide 474 Rink-amide AM 476 Rink-amide-MBHA 479 Rink-glycyl-PEGA 491 robot synthesis 87,394 safety catch amide linkage (SCAL) 27 safety-catch amide linker 23,501 safety-catch linkages 367 SAMBHA 476 SASRM 473 scaffold 102,531 scaffold libraries 179 screening 176, 177 screening methods 3 19 screening of epitopes 306 screening strategy I30 search strategy, iterative 378 secondary library 184 selectide process 173, 185 selector/selectand 53 1 self-assembled peptide monolayers 94 self-assembled supramolecular structures 195 self-assembly 95

self-peptide libraries 9 ff. self-peptide ligands 10, 11 self-peptidepool 291 semiautomated methods of parallel synthesis 88 sequence alignement 3 sequencing of peptide beads I79 shell scripts 5 12,519 side chain-protected peptides 224 side chain-protecting groups 248,273 siderophores 4,5 Sieber amide 476 Sieber amide anchor 25 signal 289 ff. - definition 289 - intensity 373 - pattern 376 silica supports 466 silicate supports 498 silicon-based linker 417 simultaneous multiple peptide synthesis

80ff.

single beads in glass capillaries 454 site-directed mutagenesis 6 skimmer voltage 249 small cell lung cancer 92 small molecule library 531 Soai reaction 31 software 384,511 ff. solid state NMR 35 solid supports, index of 505 solid supports, list of 465 K solid-phaseorganic synthesis 29 solubility 259 solution-phase assay 188 spacer polymers 53 1 specificity rules 290 spiking of mixtures 215 split resin approach 206 split resin method 205,206 split synthesis 529 split synthesis method 174lT. SPOT 363ff. spot synthesis 93,363 stabilizationassays 353,354 stabilizationexperiments with MHC 354 stabilization indices 354 Sfuphylococcusuureus I5 1 Stille coupling reaction 41 1 Stille cross-coupling-reaction 57

Index Stille reaction 31, 53,56 stored waveform inverse Fourier transform 26 1 Stork reaction 3 1 streptavidin binding peptide 99 streptavidin ligand 179 streptavidin-alkalinephosphatase 376 stylostatin 337 stylostatin library synthesis 341 ff. stylostatin-basedcyclopeptide libraries 328 sublibraries 120,530 submonomer approach 393 submonomer method 391,402 %substituted glycines NSGs 389 substrate motif 382 sugar-binding protein 99 suppliers of resins, adrenes 502 ff. support-bound reagents 20 support-linker-anchorcombination 2 1 - internal surface 454 supports 22,24 supports, list of 465 ff. surface functionalization 95,96 surface modified polymeric carriers 425 Suzuki reaction 3 1,57 f. swelling of resins 428 SWIFT 261 synergism 531 synthesis of NSG peptoids 391 synthesis of peptide libraries 204 ff. - one-bead-one-peptide 205 - premixmethods 204 - split resin approach 204 synthesis strategies for peptides 79 ff. synthetic vaccine 531 tag molecules 33 tags, tagging 525 tandem mass spectrometry 265 ff., 272,511 tandem Michael addition 61 tandem 1,3-dipolar cycloaddition 50 target 532 TASP concept 222 T-cell clones 360 T-cell epitopes 313,358 TcI/Tk language 518 teabags 532 tea-bag method 82,83 template 102,531 tentacle microspheres 438

543

tentacle polymer 427 TentaGel 24 TentaGel resins 484 ff. TentaGel-bound epitopes 442 TentaGel-resins 427 testing with sublibraries 128ff. tetrahydrofurans 49,50 f. tetrahydropyranyl ether 23,27,27 T-helper cell epitopes 235 T-helper cells 9,211 T-helper epitopes 1 1 thiazole ring systems 4 thiazolidine 54 thiazolidines 49,54 thioether bridges 4 thiol modified epitope 95 thiotemplate synthesis 2 thrombin inhibitor 181 tissue-plasminogen activator 88 titration of antigen 31 1 TOF analyzers 260 total hydrolysate 248 toxic waste 195 transformation enzymatic 379 transforming growth factor-p (TGFP) 382 7-transmembrane/G-protein coupled receptors 397 ticyclic a i d e linker (on BHA-PS) 480 0-trifluoroacetylatedpeptides 270,271 triphenylphosphine 20 tiphosgene 67 triple-quadrupolemass spectrometer 265 f. TRISOPERL 497 Triton X-100 439 tityl 267 trityl resin 480 truncated peptides 5 16 trypsin inhibitors 154f. tubulin tyrosine ligase 375 p-turn mimetics 55 tyrosine kinase 193,41 1 ubiquitin 288 UNIX 516 UNIX system tools 5 1 1 unrandomization 532 urea derivatives 59 urokinase receptor antagonist 99 variability of ligands 35 1 V3-l00p of HIV-gpl20 97,99

544

Index

Wang resin 472 Wang-PEGA 49 1 washout 288 Willebrand factor 98 Wittie. 31

Wittig and reverse Wittig reaction 38 Wittig reaction 37,38,43 Wittig-Homer reaction 3 1, 39 f. workup procedure for libraries 210