Combinatorial Peptide and Nonpeptide Libraries

A Handbook

Edited by Günther Jung

VCH Weinheim • New York • Basel • Cambridge • Tokyo

Contents

Preface V List of Contributors XIX List of Abbreviations XXIII

1 Natural Peptide Libraries of Microbial and Mammalian Origin

Günther Jung

1.1 Introduction 1 1.2 Natural Peptide Libraries of Microbial Origin 2 1.2.1 Microbial Polypeptide Antibiotics by Multienzymatic Thiotemplate

Synthesis 2 1.2.2 Polypeptide Antibiotics by Ribosomal Precursor Protein Synthesis and

Posttranslational Modifications 4 1.2.3 Combinatorial Biosynthesis and Biological Diversity of Polyketids 8 1.3 Natural Peptide Libraries of Mammalian Origin 9 1.3.1 Self-Peptide Libraries Isolated from MHC-Class I Molecules 9 1.3.2 Self-Peptide Libraries Isolated from MHC-Class II Molecules 10 1.4 From Natural to Synthetic Peptide Libraries 12 1.4.1 Synthetic Methods and the Variety of Peptide and Oligomer

Libraries 12 1.4.2 Analysis of Synthetic Peptide Libraries 13 1.4.3 Selected Applications of Synthetic Peptide Libraries 14

References 15

2 Polymer Supported Organic Synthesis: A Review

Jörg S. Fruchtet and Günther Jung

2.1 Introduction 19 2.2 Solid-Phase Organic Synthesis and Analytics 20 2.2.1 Advantages of Solid-Phase Synthesis in Organic Reactions and Product

Work-Up 20 2.2.2 Supports and Anchors 22 2.2.3 Multiple, Parallel Syntheses 28 2.2.4 Analvtics and Monitorina of Solid-Phase Reactions 34

VIII Contents

2.3 Examples of Solid-Phase Syntheses of Small Molecules 36 2.3.1 Immobilization and Reactions with Hydroxy Compounds 36 2.3.1.1 Derivatization of Hydroxy Compounds by Mitsunobu Reaction 40 2.3.2 Immobilization and Derivatization of Aldehydes and Ketones 42 2.3.3 Immobilization and Derivatization of Dicarboxylic Acids and Their

Derivatives 44 2.3.4 Ring Closure Reactions 46 2.3.5 Heterocyclic Compounds: Benzodiazepines, Hydantoins and

Thiazolidines 49 2.3.6 Further Ring Closures on Solid Support 54 2.3.7 Palladium Catalyzed C-C Attachments 56 2.3.8 Further Reactions on Polymerie Support 59 2.4 Oligomer Synthesis 61 2.4.1 Peptoids 62 2.4.2 Oligocarbamates 64 2.4.3 Peptide-Nucleic Acids (PNA) 65 2.4.4 Oligoureas 67 2.5 Outlook 68

Acknowledgments 70 References 70

3 From Multiple Peptide Synthesis to Peptide Libraries

Annette G. Beck-Sickinger and Günther Jung

3.1 Introduction 79 3.2 Simultaneous Multiple Peptide Synthesis (SMPS) 80 3.2.1 Tea-Bag Synthesis 82 3.2.2 Cellulose as Support in Multiple Syntheses 84 3.2.3 Polystyrene-Grafted Polyethylene (PS-PE) Film, a New Resin? 85 3.2.4 Automated Multiple Peptide Synthesizers 85 3.2.5 Synthesis of Polymer-Bound Peptides 90 3.2.6 Spot Synthesis 93 3.2.7 Spatially Addressed Synthesis of Thousands of Peptides 93 3.2.8 Microstructured Peptide-Gold Electrode 94 3.2.9 Peptide Functionalized Surface by Electrochemical Polymerization 94 3.3 Peptide Libraries 94 3.3.1 Mixotopes 97 3.3.2 Mimotopes 98 3.3.3 Phage Libraries and Biopanning 98 3.3.4 Random Libraries 99 3.3.5 Modified Peptide Libraries 102 3.3.6 Identification of the Active ComDounds 103

Contents IX

3.4 Conclusions 103 References 104

4 Chemical Synthesis of Peptide Libraries

Ar päd Furka

4.1 The Portioning-Mixing Method 111 4.1.1 Principles and Realization 111 4.1.2 Experimental Verification 113 4.1.3 The ELPLC Program 114 4.1.4 Simple Device for the Manual Synthesis of Peptide Libraries 116 4.1.5 Efficiency and Limitations 118 4.2 Composition of Peptide Libraries 120 4.2.1 Libraries and Sublibraries 120 4.2.2 First-Order Sublibraries 122 4.2.3 Second-Order Sublibraries 124 4.2.4 Higher Order Sublibraries 127 4.3 Potential Use of Partial Libraries in Screening: A Theoretical

Approach 128 4.3.1 The Domino Strategy 130 4.3.1.1 Determination of the Amino Acid Occurrence Library (Stage 1) 130 4.3.1.2 Determination of Positional Occurrence Library (Stage 2) 131 4.3.1.3 Determination of Active Sequences (Stage 3) 132 4.3.1.4 Generality of the Domino Strategy 135 4.4 Experimental Realization of the Portioning-Mixing Procedure 135

Acknowledgments 137 References 137

5 The Versatility of Nonsupport-Bound Combinatorial Libraries

Clemencia Pinilla, Jon Appel, Colette Dooley, Sylvie Blondelle, Jutta Eichler, Barbara Dörner, John Ostresh and Richard A. Houghten

5.1 Introduction 139 5.1.1 Solid-Phase Peptide Synthesis 139 5.1.2 Peptide Libraries 140 5.2 Preparation of Synthetic Peptide Combinatorial Libraries 142 5.2.1 DCR Method 142 5.2.2 Coupling of Amino Acid Mixtures 143 5.3 Dual Positional SCLs 143 5.3.1 Use of SCLs 144 5.3.1.1 Identification of Antieenic Determinants 144

X Contents

5.3.1.2 Identification of Opioid Peptides 150 5.3.1.3 Development of Antimicrobial Peptides 150 5.3.1.4 Development of Inhibitors of Melittins's Hemolytic Activity 153 5.3.1.5 Development of Enzyme Inhibitors 154 5.3.2 Use of all D-Amino Acid SCLs 155 5.4 SCLs Composed of Peptides Containing L-, D- and Unnatural Amino

Acids 155 5.5 Positional Scanning SCLs 157 5.5.1 Identification of Antigenic Determinants 159 5.5.2 Identification of Opioid Ligands 161 5.5.3 Identification of Inhibitors of Melittin's Hemolytic Activity 162 5.5.4 Decapeptide PS-SCL 162 5.5.5 D-Amino Acid PS-SCL 164 5.6 Modified Peptide "Libraries from Libraries" 165 5.7 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 Introduction 173 6.2 The Basic Concept of "One-Bead-One-Compound" 175 6.3 Synthesis of Random Peptide Library 176 6.4 Screening with an "On-Bead Binding Assay" 176 6.5 Screening with a "Releasable Assay" 177 6.6 Libraries of Organic Molecules 178 6.7 Scaffold Libraries 179 6.8 Structure Determination of Positive Reaction Compounds 179 6.9 Coding 182 6.10 Elimination of Possible Interaction of Target Macromolecule with

Coding Structure: Bead Shaving 183 6.11 Is It Necessary To Have Füll Representation in a Selectide

Library? 184 6.12 One-Bead-One-Motif Libraries ("Libraries of Libraries") 185 6.13 The Selectide Process Versus Other Combinatorial Library

Methodologies 185 6.14 Examples of Application 189 6.14.1 Anti-/?-Endorphin Monoclonal Antibody 189 6.14.2 Anti-Insulin Monoclonal Antibody 190 6.14.3 MHC-Class I Molecule 191

Contents XI

14.4 Releasable Assay Screening System 192 14.5 Posttranslational Modification such as Protein Phosphorylation 192 14.6 Small Organic Dye Molecule as a Target 193 14.7 Screening of Library of Libraries 194 15 Perspective 194

Acknowledgments 195 References 195

Peptide and Cyclopeptide Libraries: Automated Synthesis, Analysis and Receptor Binding Assays

Karl-Heinz Wiesmüller, Susanne Feiertag, Burkhard Fleckenstein, Stefan Kienle, Dieter Stoll, Markus Herrmann and Günther Jung

1 Introduction 203 2 Methods for the Generation of Peptide Libraries 204 2.1 Manually Synthesized Peptide Libraries 204 2.2 Automation to Ensure Reproducible, Simultaneous, Multiple Peptide

Synthesis 205 2.3 Peptide Diversity Determines Procedures for Synthesis and

Bioassay 207 2.4 Coupling Reactions with Premixed Amino Acid Derivatives 210 2.5 Soluble and Polymer-Bound Libraries in One Run 210 3 Analytical Control of Peptide Mixtures 211 3.1 Monitoring During Synthesis 213 3.1.1 Method for Indirect Determination of the Coupling Yield by Amino

Acid Analysis 213 3.2 Amino Acid Analysis, Capillary Electrophoresis and Mass Spectro-

metry of Peptide Libraries 214 4 Pipet Robot for the Synthesis of Peptide Libraries 218 4.1 Procedure for the Synthesis of Peptide Libraries by the "Premix

Method" 220 4.1.1 Experimental Procedure 220 5 Conformationally Constrained Peptide Libraries 221 5.1 Synthesis of Cyclopeptides 223 5.1.1 Loading of 2-Chlorotritylchloride Resin with Fmoc-Amino Acids 223 5.1.2 Synthesis of Linear Peptides 223 5.1.3 Cleavage of Fully Side Chain-Protected Peptides from the Resins 224 5.1.4 Cyclization Reactions 224 5.1.5 Cleavage of Side Chain-Protecting Groups 225 5.2 Characterization of Cyclopeptide Sublibraries 227 6 Pentadecapeptide Libraries for Receptor Binding Studies 235 6.1 ComDetition Assav for MHC-Class II Bindina PeDtides 236

XII Contents

7.6.2 Positional Scanning of a Pentadecapeptide Epitope 237 7.7 Conclusions 241

References 241

8 Mass Spectrometric Analysis of Peptide Libraries

Jörg W. Metzger, Karl-Heinz Wiesmüller, Stefan Kienle, Jente Brünjes and Günther Jung

8.1 Introduction 247 8.2 Results and Discussion 247 8.2.1 Analytical Techniques for the Characterization of Soluble

Combinatorial Peptide Libraries 247 8.2.2 Mass Spectrometry of Peptides 249 8.2.3 Electrospray Ionization (ESI) 249 8.2.4 Peptide Families in Peptide Libraries 250 8.2.5 Calculation of Mass Distributions and Peak Clans 251 8.2.6 Mass Spectrometry of Peptide Libraries 251 8.2.7 Electrospray Mass Spectrometry — A Potent Method for the

Characterization of Peptide Libraries 251 8.2.8 Relative Ion Intensities — A Measure for the Number of Isobaric

Peptides in Peptide Libraries? 253 8.2.9 Experimental Conditions for Recording ESI Mass Spectra of Peptide

Libraries 259 8.2.10 Mass Resolution and Accuracy of Mass Determination 260 8.2.11 Mass Analyzers 260 8.2.12 Fourier Transform Ion Cyclotron Resonance ESI Mass

Spectrometry 261 8.2.13 Matrix Assisted Laser Desorption Ionization Mass Spectrometry

(MALDI-MS) 261 8.2.14 Tandem Mass Spectrometry (MS-MS) of Peptide Libraries and

Diagnostic Ions 265 8.2.15 High Performance Liquid Chromatography-Mass Spectrometry (HPLC-

MS) of Peptide Libraries 274 8.2.16 Limits for Mass Spectrometric Characterization of Peptide Libraries 281 8.3 Materials and Methods 281 8.3.1 Peptide Synthesis 281 8.3.2 Mass Spectrometry 282 8.3.3 Narrow-Bore RP-HPLC 283 8.3.4 Calculation of the Mass Distribution with QMass 283 8.4 Summary 283

Acknowledgment 284 References 284

Contents XIII

9 Multiple Sequence Analysis of Natural and Synthetic Peptide Libraries

Wieland Keilholz and Stefan Stevanovic

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

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

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

References 301

10 Epitope Mapping with the Use of Peptide Libraries

Stuart Rodda, Gordon Tribbick and Mario Geysen

10.1 Introduction 303 10.1.1 Definition of Epitope 303 10.1.2 Brief History of Antibody-Defined Epitope Mapping 304 10.1.3 History of T-Cell Epitope Mapping 305 10.1.4 Comparison between Linear Epitope Scanning and the Combinatorial

Library Approach 305 10.2 Synthetic Peptides for Epitope Mapping 306 10.2.1 Difficulty of Predicting Epitopes 306 10.2.2 Nature of the Screening Task 306 10.2.3 "Format" of Peptides for Epitope Mapping 307 10.2.4 Peptide Purity and Characterization 309 10.3 Validity Testing of Peptide Assay Results 310 10.3.1 Testing the Relevance of Peptide Binding Data 310 10.3.1.1 Antibody Binding 310 10.3.1.2 Major Histocompatibility Complex (MHC) Binding 311 10.3.2 Testing the Relevance of Bioactivity Data 312 10.3.2.1 Bioactivity of Antibody-Defined Linear Peptide Epitopes 312 10.3.2.2 Bioactivity of T-Cell Epitopes 313 10.4 Peptide Libraries from Pins 313 10.4.1 Types of Library: Strategies 313 10.4.2 Methods of Synthesis of Peptide Libraries on Pins 316 10.4.3 Strategies for Maximizing the Usefulness of Pins 317 10.4.4 Approaches to Amino Acid Mixtures 318 10.4.5 Downstream Processina of Pin-PeDtide Libraries 318

Contents

10.4.6 Screening Methods Applicable to Pin Peptides 319 10.5 Comparison of Methods of Peptide Library Generation for Epitope

Mapping 320 10.5.1 Systems Using Spatially Stable Matrices 320 10.5.2 Systems Producing "Loose" Solid-Phase Peptides 321 10.5.3 Systems Producing Cleaved Peptides 322

References 322

11 Cyclic Peptide Libraries: Recent Developments

Arno F. Spatola and Peteris Romanovskis

11.1 Introduction 327 11.2 Results and Discussion 328 11.2.1 Cyclic Pentapeptides 330 11.2.2 Cyclic Hexapeptides 336 11.2.3 Cyclic Heptapeptides 337 11.3 Summary 338 11.4 Materials and Methods 340 11.4.1 General Solid-Phase Peptide Synthesis Procedure 341 11.4.1.1 Synthesis of a Stylostatin Peptide Library with Six Sublibraries

(6 X 256 Peptides) 341 Acknowledgments 346 References 346

12 Random Peptide Libraries as Tools in Basic and Applied Immunology

Keiko Udaka, Karl-Heinz Wiesmüller, Stefan Kienle, Susanne Feiertag, Günther Jung and Peter Waiden

12.1 Introduction 349 12.2 Peptide Binding to MHC Molecules 350 12.3 Synthetic Random Peptide Libraries 351 12.4 Peptide Selection by MHC Molecules 352 12.5 Interdependence of the Contribution of Individual Amino Acids to

Peptide-MHC Interaction 356 12.6 T-Cell Epitopes Defined with Peptide Libraries 358 12.7 Conclusions 361

References 361

Contents XV

13 Combinatorial Synthesis on Membrane Supports by the SPOT Technique: Imaging Peptide Sequence and Shape Space

Ronald Frank, Stefan Hoffmann, Michael Kieß, Heike Lahmann, Werner Tegge, Christian Behn and Heinrich Gausepohl

13.1 Introduction 363 13.2 General Technical Aspects of SPOT Synthesis 365 13.2.1 Instrumental 365 13.2.2 Peptide Synthesis on Spots 366 13.2.3 Peptide Library Synthesis 369 13.2.4 Library Design 370 13.3 Applications of Peptide Libraries on Spots 372 13.3.1 Solid-Phase Ligand Binding Assay 372 13.3.1.1 Positional Scanning Libraries 375 13.3.1.2 Iterative Library Search (Mimotope Approach) 376 13.3.1.3 Dual-Positional Scanning 378 13.3.2 Enzymatic Transformations of Peptide Libraries 379 13.3.3 Other Applications and Future Developments 382 13.4 Methods 383 13.4.1 Peptide Library Assembly 383 13.4.2 Side Chain Deprotection 383 13.4.3 Ligand Binding Assay on SPOTs Membranes 384 13.4.4 Enzymatic Phosphorylation 384

References 385

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 387 14.2 Criteria and Goals for the Generation of Molecular Diversity 387 14.3 The Peptoid Approach 389 14.4 Synthesis of NSG Peptoids 391 14.5 Automated Synthesis of Equimolar Peptoid Mixtures 394 14.6 Rational Approaches for Library Design and the Generation of

Structural Diversity 396 14.7 Peptoid Ligands with Nanomolar Affinity for Adrenergic and Opiate

Receptors 397 14.7.1 Design of a Biased Library for 7-Transmembrane/G-Protein Coupled

Recentors 397

XVI Contents

14.7.2 Identification of Peptoid Ligands with Nanomolar Affinity 398 14.7.3 Discussion 399 14.8 Summary 401 14.9 Experimental Procedures 402 14.9.1 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 TV-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

15 Synthesis and Evaluation of Three 1,4-Benzodiazepine Libraries

Barry A. Bunin, Matthew J. Plunkett and Jonathan A. Ellman

15.1 Introduction 405 15.2 Synthesis Criteria for a Benzodiazepine Library 406 15.3 Chiron Mimotopes (Geysen) Pin Apparatus 406 15.4 Solid-Phase 1,4-Benzodiazepine Synthesis 407 15.5 First Generation 1,4-Benzodiazepine Library 410 15.6 Second Generation 1,4-Benzodiazepine Library 411 15.7 Current Solid-Phase 1,4-Benzodiazepine Synthesis 411 15.8 Design of a Large 1,4-Benzodiazepine Library 413 15.9 Synthesis of an 11200 Member 1,4-Benzodiazepine Library 415 15.10 Alternate Strategies for Benzodiazepine-Based Diversity 417 15.11 Conclusion 418 15.12 Experimental Section 418 15.12.1 Reagents and General Methods 418 15.12.1.1 Fmoc Deprotection of Aminomethyl Solid Support (Pins) 419 15.12.1.2 2-Aminobenzophenone 419 15.12.2 Method A 419 15.12.2.1 Coupling Fmoc-Protected 2-Aminobenzophenones (1) to Pins

to Give 2 419 15.12.2.2 Fmoc Cleavage 419 15.12.3 Method B 420 15.12.3.1 Coupling Aminoaryl Stannane Cyanomethyl Ester to Pins

to Give 7 420 15.12.3.2 Stille Coupling Reactions 420 15.12.3.3 Bpoc Cleavage 420 15.12.4 Benzodiazepine Synthesis from 2-Aminoarylketones 420 15.12.4.1 Amino Acid Fluoride Acvlation 420

Contents XVII

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

Acknowledgments 423 References 423

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

Wolfgang Rapp

16.1 Introduction 425 16.2 Physico-Chemical Properties of Polystyrene-Poly(ethyleneglycol)-

Tentacle Polymers 427 16.3 Peptide Synthesis 436 16.4 Monosized Tentacle Microspheres for Screening and High Speed

Peptide Synthesis 438 16.5 TentaGel Peptide Conjugates in Immunization 442 16.6 Oligonucleotide Synthesis 445 16.7 Macrobeads as Polymerie Microreactors: Peptide Libraries and

Combinatorial Chemistry 446 References 458

17 Supports for Solid-Phase Organic Synthesis

Martin Winter

17.1 Introduction 465 17.2 Polystyrene Supports 468 17.2.1 Polystyrene Base Resins 468 17.2.2 Acid-Labile Polystyrene Resins 472 17.2.3 Base-Labile, Photo-Labile and Nucleophilic Cleavable Polystyrene

Resins 481 17.3 TentaGel Resins 484 17.4 PolyHIPE Resins 489 17.5 PEGA Resins 491 17.6 Kieselguhr-Polyamide Supports ("Pepsyn K") 492 17.7 Controlled-Pore Supports (CPG, CPC) 495 17.8 Other Silicate Supports 498 17.9 Miscellaneous Support Components 499 17.10 Appendix 502 17.10.1 Conversion Table tmesh — Darticle size. mm) 502

Contents

17.10.2 Addresses of Suppliers 502 17.10.3 Index of Solid Supports 505

References 509

18 QMass: A Computer Program for the Analysis of Mass Spectra of Peptide Libraries

Jente Brünjes, Jörg W. Metzger and Günther Jung

18.1 Introduction 511 18.2 Concepts of QMass 511 18.3 Library Concepts of QMass 513 18.4 Calculations 514 18.5 System Tools, Shell Scripts and Automated Analysis 516 18.6 Visualization and Alternative Setup of Calculation Options 518 18.7 System Requirements and Limitations 519 18.8 Summary 519

References 520

Glossary 521 Index 533