Peptide Synthesis Protocols

Methods in Molecular Biology John M. Walker, SERIES EDITOR

35. Peptide Synthesis Protocols, edited by Michael W. Pennington and Ben M. Dunn, 1994

34. Immunocytochemical Methods and Protocols, edited by Lorette C. Javois, 1994

33. In Si tu Hybridization Protocols, edited by K. H. Andy Choo, 1994 32. Basic Protein and Peptide Protocols, edited by John M. Walker, 1994 31. Protocols for Gene Analysis, edited byAdrian J. Harwood, 1994 30. DNA-Protein Interactions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by

Peter G. Isaac, 1994 27. Biomembrane Protocols: II. Architecture and Function, edited by

John M. Graham and Joan .4. Higgins, 1994 26. Protocols for Oligonucleotide Conjugates, edited by Sudhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by Hugh G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Techniques,

edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1994

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993 20. Protocols for Oligonucleotides and Analogs, edited by

Sudhir Agrawal, 1993 19. Biomembrane Protocols: I. Isolation and Analysis, edited by

John M. Graham and Joan A. Higgins, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Christopher Jones,

Barbara Mulloy, and Adrian H. Thomas, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCR Protocols, edited by Bruce A. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsell,

1993 13. Protocols in Molecular Neurobiology, edited by Alan Longstaf f

and Patricia Revest, 1992 12. Pulsed-Field Gel Electrophoresis, edited by Margit Burmeister

and Levy Ulanovsky, 1992 11. Practical Protein Chromatography, edited by Andrew Kenney

and Susan Fowell, 1992 10. Immunochemical Protocols, edited by Margaret M. Manson, 1992 9. Protocols in Human Molecular Genetics, edited by

Christopher G. Mathew, 1991 8. Practical Molecular Virology, edited by Mary K. L. Collins, 1991

Earlier volumes are stil l available. Contact Humana for details,

Peptide Synthesis Protocols

Edited by

M i c h a e l W. P e n n i n g t o n Bachem Bioscience, King of Prussia, PA

and

Ben M. D u n n University of Florida, Gainesville, FL

Humana Press ~ Totowa, New Jersey

© 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All fights reserved.

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permis- sion from the Publisher.

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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1

Library of Congress Cataloging in Publication Data

Peptide synthesis protocols / edited by Michael W. Pennington and Ben M. Dunn p. cm.--(Methods in molecular biology; 35)

Companion v. to : Peptide analysis protocols. Includes index. ISBN 0-89603-273-6 1. Peptides--Synthesis--Laboratory manuals. I. Pennington, Michael W. II. Dunn, Ben M.

IlL Peptide analysis protocols IV. Series: Methods in molecular biology (Totowa, NJ); 35 [DNLM: 1. Peptides--chemical synthesis. Wl ME9616J v.35 1994 / QU 68 P424 1994]

QP552.P4P458 1994 574.19'296---dc20 DNLM/DLC 94-26326 for Library of Congress CIP

Contents of the companion volume:

Peptide Analysis Protocols (Note: Cross references to these chapters will include the notation PAP)

Preface Companion Volume Contents Contributors

CH. 1.

CH. 2.

CH. 3.

CH. 4.

CH. 5.

CH. 6.

CH. 7.

CH. 8.

CH. 9.

CH. 10.

CH. 11.

Gel-Filtration Chromotography, Daniel M. Bollag

Ion-Exchange Chromatography, Daniel M. Bollag

Reversed-Phase HPLC: Analytical Procedure, Udo Nirenberg

Reversed-Phase High-Performance Liquid Chromatography: A Semipreparative Methodology,

Michael E. Byrnes Applications of Strong Cation-Exchange (SCX)-HPLC

in Synthetic Peptide Analysis, Dan L. Crimmins

Principles and Practice of Peptide Analysis with Capillary Zone Electrophoresis,

Thomas E. Wheat Fast Atom Bombardment Mass Spectrometric Characterization

of Peptides, P. R. Das and B. N. Pramanik

Sequence Analysis of Peptide Resins from Boc/Benzyl Solid-Phase Synthesis,

Jan Pohl NMR Spectroscopy of Peptides and Proteins,

Mark G. Hinds and Raymond S. Norton Techniques for Conjugation of Synthetic Peptides

to Carrier Molecules, J. Mark Carter

Epitope Prediction Methods, J. Mark Carter

ix

P r e f a c e Peptide synthesis has emerged as one of the most powerful tools in

biochemical, pharmacological, immunological, and biophysical labo- ratories. Recent improvements include general solid-phase methodol- ogy, new protecting groups, and automated equipment. These advances have allowed the facile synthesis of increasingly more complex pep- tides. Many of these new and improved methods for the synthesis of peptides and peptide-related substances have been reported in various publications, but never compiled in a convenient handbook. Like other volumes in this series, Peptide Synthesis Protocols concentrates on the practical aspects of these procedures, providing the researcher with detailed descriptions and helpful tips about potential problems. This volume is not intended to serve as a basic guide to standard Merrifield- type solid-phase strategy, but rather to provide the researcher with some of the most recent applications in the field of peptide science. A com- panion volume, Peptide Analysis Protocols, will detail methodology for the charaterization of new synthetic peptides.

Development of new methods and applications has continued actively even as this volume was in preparation. Owing to the number of contributors to this volume, it was necessary to establish a cutoff for publication purposes. We feel that all of the protocols presented are timely and up-to-date. Several promising new strategies, such as allyloxycarbonyl-based syntheses, were being developed at the time this volume was in the editing stages and will be included in future editions.

The editors would like to thank all of the contributing authors and their colleagues for their many helpful suggestions regarding new applications and methods. Special appreciation must be given to R. B. Merrifield, whose original conceptualization and implementation of the solid-phase approach to peptide synthesis has now been extended to the preparation of peptide libraries, oligonucleotides, and organic mol- ecule libraries. With these new applications, we are certain that the next decade will be a truly exciting time for the peptide chemist.

Michael W. Pennington Ben M. Dunn

/3

C o n t e n t s

Preface ............................................................................................................................ v Companion Volume Contents ..................................................................................... ix Contributors ................................................................................................................. xi

CH. 1.

CH. 2.

CH. 3.

CH. 4.

CH. 5.

CH. 6.

CH. 7.

CH. 8.

Ca. 9.

CH. 10.

CH. 11.

Procedures to Improve Difficult Couplings, Michael W. Pennington and Michael E. Byrnes .................................. 1

Methods for Removing the Fmoc Group, Gregg B. Fields ...................................................................................... 17

Solvents for Solid-Phase Peptide Synthesis, Cynthia G. Fields and Gregg B. Fields ............................................... 29

HF Cleavage and Deprotection Procedures for Peptides Synthesized Using a Boc/Bzl Strategy,

Michael W. Pennington ........................................................................ 41 Acid Cleavage/Deprotection in Fmoc/tBu Solid-Phase Peptide Synthesis,

Fritz Dick ............................................................................................... 63 Bromoacetylated Synthetic Peptides: Starting Materials for Cyclic

Peptides, Peptomers, and Peptide Conjugates, Frank A. Robey ...................................................................................... 73

Formation of Disulfide Bonds in Synthetic Peptides and Proteins, David Andreu, Fernando Albericio, Ndria A. Sol~,

Mark C. Munson, Marc Ferrer, and George Barany ............... 91 Site-Specific Chemical Modification Procedures,

Michael W. Pennington ...................................................................... 171 Synthesis of Phosphopeptides Containing O-Phosphoserine

and O-Phosphothreonine, Anatol Arendt and Paul A. Hargrave ................................................ 187

Solid-Phase Synthesis of Phosphorylated Tyr-Peptides by "Phosphite Triester" Phosphorylation,

Michael W. Pennington ...................................................................... 195 Design of Novel Synthetic Peptides Including Cyclic Conformationally

and Topographically Constrained Analogs, Victor J. Hruby and G. Gregg Bonner .............................................. 201

vii

viii Contents

CH. 12.

CH. 13.

CH. 14.

CH. 15.

Solid-Phase Synthesis of Peptides Containing the CH2NH Reduced Bond Surrogate,

Michael W. Pennington ...................................................................... 241 Approaches to the Asymmetric Synthesis of Unusual Amino Acids,

Victor J. Hruby and Xinhua Qian .................................................... 249 Synthesis of Fully Protected Peptide Fragments,

Monika Mergler ................................................................................... 287 Peptide Synthesis via Fragment Condensation,

Rol f Nyfeler .......................................................................................... 303

Index .......................................................................................................................... 317

x C o m p a n i o n V o l u m e C o n t e n t s

CH. 12.

CH. 13.

Cn. 14.

Cn. 15.

CH. 16.

CH. 17.

Index

Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure,

J. Mark Carter Analysis of Proteinase Specificity by Studies of Peptide Substrates:

The Use of UV and Fluorescence Spectroscopy to Quantitate Rates of Enzymatic Cleavage,

Ben M. Dunn, Paula E. Scarborough, Ruth Davenport, and Wieslaw Swietnicki

Synthesis of Recombinant Peptides, Gino Van Heeke, Jay S. Stout, and Fred W. Wagner

De Novo Design of Proteins: Template-Assembled Synthetic Proteins (TASP),

Gabrielle Tuchscherer, Verena Steiner, Karl-Heinz Altmann, and Manfred Mutter

Chemical Synthesis of the Aspartic Proteinase from Human Immunodeficiency Virus (HIV),

Paul D. Hoeprich, Jr. Multiple and Combinatorial Peptide Synthesis: Chemical Development

and Biological Applications, Philip C. Andrews, Daniele M. Leonard, Wayne L. Cody,

and Tomi K. Sawyer

C o n t r i b u t o r s

FERNANDO ALBERICIO * Department of Organic Chemistry, University of Barcelona, Barcelona, Spain; MiUipore Corporation, Bedford, MA; Department of Chemistry, University of Minnesota, Minneapolis, MN

DAVID ANDREU * Department of Organic Chemistry, University of Barcelona, Barcelona, Spain

ANATOL ARENDT * Department of Ophthalmology, University of Florida School of Medicine, Gainesville, FL

GEORGE BARANY * Department of Chemistry, University of Minnesota, Minneapolis, MN

G. GREGG BONNER * Department of Chemistry, University of Arizona, Tucson, AZ

MICHAEL E. BYRNES * Bachem Bioscience, King of Prussia, PA FRITZ DICK * Bachem Feinchemikalien AG, Bubendo~ Switzerland MARC FERRER * Department of Chemistry, University of Minnesota,

Minneapolis, MN CYNTHIA G. FIELDS "Depar tmen t of Laboratory Medicine

and Pathology, The Biomedical Engineering Center, University of Minnesota, Minneapolis, MN

GREGG B. FIELDS * Department of Laboratory Medicine and Pathology, The Biomedical Engineering Center, University of Minnesota, Minneapolis, MN

PAUL A. HARGRAVE * Department of Biochemistry and Molecular Biology, University of Florida School of Medicine, GainesviUe, FL

VICTOR J. HRUBV * Department of Chemistry, University of Arizona, Tucson, AZ

MONIKA MERGLER " B a c h e m Feinchemikalien AG, Bubendor~, Switzerland

xi

xii Contribzztors

MARK C. MUNSON ° Department of Chemistry. University of Minnesota, Minneapolis, MN. Present Address: Amgen Inc., Boulder, CO

ROLF NYFELER ° Bachem Feinchemikalien AG, Bubendorf, Switzerland MICHAEL W. PENNINGTON • Bachem Bioscience, King of Prussia, PA XINHUA QIAN ° Department of Chemistry, University of Arizona,

Tucson, AZ FRANK A. ROBEY • Peptide and Immunohistochemistry Unit,

Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD

NI/IRIA A. SOLd " Department of Chemistry, University of Minnesota, Minneapolis, MN. Present Address: Millipore Corporation, Bedford, MA