Reactivity and Structure Concepts in Organic Chemistry Volume 16

Editors:

Klaus Hafner Charles W. Rees Barry M. Trost

Jean-Marie Lehn P. von Rague Schleyer Rudolf Zahradnik

M. Bodanszky

Principles of Peptide Synthesis

Springer-Verlag Berlin Heidelberg New York Tokyo 1984

Miklos Bodanszky

Department of Chemistry Case Western Reserve University Cleveland, OR 44106 USA

List of Editors

Professor Dr. Klaus Hafner Institut fUr Organische Chemie der TH Darmstadt Petersenstr. IS, D-6IOO Darmstadt

Professor Dr. Jean-Marie Lehn Institut de Chimie, Universite de Strasbourg I, rue Blaise Pascal, B.P. 296/R8, F-67008 Strasbourg-Cedex

Professor Dr. Charles W. Rees, F. R. S. Hofmann Professor of Organic Chemistry, Department of Chemistry Imperial College of Science and Technology South Kensington, London SW7 2A Y, England

Professor Dr. Paul v. Rague Schleyer Lehrstuhl fUr Organische Chemie der Universitat Erlangen-Nurnberg Henkestr. 42, D-8520 Erlangen

Professor Barry M. Trost Department of Chemistry, The University of Wisconsin HOI University Avenue, Madison, Wisconsin 53706, U.S.A.

Professor Dr. Rudolf Zahradnik Tschechoslowakische Akademie der Wissenschaften J.-HeyrovskY-Institut fUr Physik, Chemie und Elektrochemie Machova 7, 121 38 Praha 2, C.S.S.R.

ISBN-13: 978-3-642-96765-8 e-ISBN-13: 978-3-642-96763-4 001: 10.1007/978-3-642-96763-4

Library of Congress Cataloging in Publication Data Bodanszky, Miklos. Principles of peptide synthesis. (Reactivity and structure; v.I6). Includes bibliographical references and indexes. I. Peptide synthesis -Addresses, essays, lectures. I. Title. II. Series. QP552.P4B63 1984 547.7'56 83-4664

This work is subject to copyright. All rights are reserved, whether the whole part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under p 54 of the German Copy­right Law where copies are made for other than private use, a fee is payable to "Verwertungsgesellschaft Wort", Munich. © by Springer-Verlag Berlin Heidelberg 1984 Softcover reprint of the hardcover 1 st edition 1984 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

2152/3020-543210

To the memory of my parents

Preface

A look at the shelves of a major library awakens doubts in the author of this small volume about the importance of writing a new introduction to peptide synthesis. This rather narrow area of bio-organic chemistry already has received considerable attention. A whole series of books deals with the synthesis of peptides. Some of these are textbooks written to support lecture courses on peptide synthesis. Others try to help the beginner, otherwise well versed in organic chemistry, to embark on some experimental project that requires the construction of peptide chains. Not less useful are the monographs which were compiled to aid the adept practitioner and to provide him with references to the growing literature of a very active field. Is there any need for a new book on peptide synthesis? Should we add a new volume to an already impressive and certainly useful series? The answer is not obvious.

The author has already participated in two similar en­deavors. The first edition! of "Peptide Synthesis", with M. A. Ondetti as coauthor, was meant to serve as an introduction for the beginner. It was rather well received by researchers who joined the field of peptide chemistry and were looking for initiation. While working on the second edition2 with Drs. Klausner and Ondetti, we became painfully aware of the impossibility of the task. In the ten years elapsed between the two editions, the body of knowl­edge in peptide synthesis has grown to such dimensions that it could not be condensed into a small book. As a com­promise, many potentially important new developments were presented only as entries in one of the numerous tables. Also, the obvious lack of personal experience with several relatively recent procedures excluded the critical evaluation that was the strength of the first edition. A mere rendering of methods of protection, activation and coupling cannot help the uninitiated who has to chose between alternatives without the benefit of personal experience. The beginner was better served when the authors, based on their own experience, treated the material more subjectively. The

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Preface

explosive growth of the literature made, however, a brief and yet essentially complete and also critical account of peptide synthesis nigh impossible. The heroic efforts of E. Wiinsch and his associates, resulting in two impressive volumes,3 were indeed necessary to approach a much desired full presentation. Such an admirable completeness, however, could be achieved only at the expense of simplicity: the reader has to master the handling of the extensive material. Also, the ongoing enrichment of the field with novel proce­dures and reports of new experience gained with old methods must continuously reduce the completeness, the up-to­dateness and thereby one of the most important merits of any defmitive work.

There is probably no entirely satisfactory solution for this dilemma. A new book that reflects the rapid growth of knowledge in peptide synthesis cannot be more than a snapshot, outdated within a few years. On the other hand, just because of the rich harvest of new procedures, an introductory monograph becomes more and more necessary. It seemed, therefore, not unreasonable to write, as an experiment, instead of a third edition of "Peptide Synthesis" rather a small volume on the general principles that govern the development of methodology. Such general lines or fundamental thoughts - if they can be discerned - could help in the understanding and perhaps even in the critical evaluation of alternative procedures. This emphasis on principles should also absolve the author from an obligation toward attempts for exhaustive literature references or a complete account of all methods known in peptide synthesis. Perhaps by concentrating on the ideas behind the procedures, he can produce an intellectually more satisfactory and there­fore more readable book.

Somewhat disappointingly, peptide synthesis resulted in only a few compounds which contribute to the armament of modem medicine. Not too many peptides are produced on an industrial scale. At the same time, the number of known, biologically-active peptides increases almost ex­ponentially and many important research tools can be obtained through synthesis in the laboratory. This requires the participation of numerous well informed, well trained, competent peptide chemists. It is unlikely that the revolution brought to this area by the production of peptides' via recombinant DNA would s~riously alter this picture in the near future. Thus, a sizeable number of new colleagues might join the field of peptide synthesis. The author hopes that they will find this volume, "Principles of Peptide Synthesis", helpful during their initiation.

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Preface

I. Bodanszky,M., Ondetti, M. A.: Peptide Synthesis, New York: Wiley-Interscience 1966.

2. Bodanszky, M., Klausner, Y. S., Ondetti, M. A.: Peptide Synthesis (2nd Edition), New York: Wiley-Interscience 1976.

3. Wiinsch, E., ed., Houben-Weyl, Methoden der Organischen Chemie, Vol. 15, Parts I and II, Stuttgart: Thieme 1974.

Cleveland/Ohio, August 1983 Miklos Bodanszky

IX

Acknowledgements

The author expresses his gratitude to his wife for her help in the search of the literature. He thanks Dr. John C. Tolle for reading and correcting the manuscript, to Ms. Elsie Hart and Ms. Roberta Sladeck for preparing the typescript and drawing the figures. Part of this book was written in 1981 when the author, as a recipient of the A. v. Humboldt Award, was the guest of the Deutsches Wollforschungs­institut in Aachen, Germany. His work was both encouraged and helpful by the Director of the Institute, Professor Dr. Helmut Zahn.

XI

Table of Contents

I. Introduction . J References of Chapter I . 7

II. Activation and Coupling . 9

A. Activation . 9 B. Coupling. 14 C. Coupling Methods 16

1. The azide procedure 16 2. Anhydrides 21 3. Active esters . 28 4. Coupling Reagents . 36 5. Auxiliary Nucleophiles 44 6. Enzyme-catalyzed Formation

of the Peptide Bond . . . . 49 7. A Comparison Between Various

Coupling Methods 51 References of Chapter II 52

III. Reversible Blocking of Amino and Carboxyl Groups . 59

A. General Aspects 59 1. The Need for Protecting Groups 59 1. Minimal Versus Global Protection 60 3. Easily Removable Protecting Groups

and Methods Used for Their Removal. 61 a. Reduction and Oxidation . 62 b. Acidolysis-Carbocation Formation 63 c. Proton Abstraction

(Carbanion Formation). 65 d. Nucleophilic Displacement 66 e. Photolysis 67 f. Enzyme-Catalyzed Hydrolysis. 68

B. Protection of the Carboxyl Group. 69 1. Benzyl Esters and Substituted

Benzyl Esters 70

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Table of Contents

2. Methyl Esters and Substituted Methyl Esters 73

3. Ethyl Esters and Substituted Ethyl Esters . 76

4. tert. Butyl Esters and Related Compounds. 78

5. Aryl Esters 79 6. Hydrazides 80

C. Protection of the Amino Group. 82 1. Alkyl and Alkylidene Protecting Groups. 82 2. Protection by Acylation . 85 3. Protection of the Amino Group

in the Form of Urethanes . 90 a. Urethane-type Protecting Groups 90 b. Introduction of Urethane-type

Protecting Groups . 94 c. Removal of Urethane-type

Protecting Groups . 98 4. Protecting Groups Derived

from Sulfur and Phosphorus . 102 References of Chapter III . 107

IV. Semipermanent Protection of Side Chain Functions. 119

A. Carboxyl Groups of Aspartyl and Glutamyl Residues 119

B. Side Chain Amino Groups of Lysine and Ornithine 122

C. Hydroxyl Groups in Serine, Threonine and Tyrosine . 126

D. The Sulfhydryl Group in Cysteine. 130 E. The Guanidino Group of Arginine 137 F. Imidazole in Histidine . 142 G. The Thioether in Methionine . 146 H. The Indole Nitrogen in Tryptophan . 148 I. The Carboxamide Groups in Asparagine

and Glutamine . 149 References of Chapter IV . 151

V. Side Reactions in Peptide Synthesis . 158

A. Side Reactions Initiated by Proton Abstraction. 158 1. Racemization 159

a. Mechanisms of Racemization 159 b. Models for the Study of Racemization 163

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Table of Contents

c. Detection of Racemization. (Examination of Synthetic Peptides for the Presence of Diastereo-isomers. . . . . . . . . . . 166

d. Conservation of Chiral Purity.. 167 2. Undesired Cyc1ization. . . . . .. 173 3. O-Acylation . . . . . . . . . .. 177

B. Side Reactions Initiated by Protonation 178 1. Racemization . . . . 178 2. Undesired Cyclization. . . . . . 178 3. Alkylation. . . . . . . . . . . 179 4. Chain Fragmentation. . . . . . 181

C. Side Reactions Due to Overactivation 182 D. Side Reactions Related to Individual

Amino Acid Residues. . . . . . . 184 References of Chapter V. . . . . . . . 195

VI. Tactics and Strategy in Peptide Synthesis 202 A. Tactics. . . . . . . . . . . . . . 202

1. Combinations of Protecting Groups. 262 2. Final Deprotection . . . . . . .. 205

B. Strategies . . . . . . . . . . . .. 208 1. Segment Condensation . . . . .. 208 2. Stepwise Synthesis Starting with the

N-terminal Residue (N-+C Strategy). 210 3. Stepwise Synthesis Starting with the

C-terminal Residue (C-+N Strategy). . 211 C. Disulfide Bridges . . . . . . 214 D. Synthesis of Cyclic Peptides . 217

1. Homodetic Cyc10peptides . 217 2. Heterodetic Cyc10peptides . 223

E. Sequential Polypeptides . . . 225 F. Partial Synthesis (Semisynthesis) 226 References of Chapter VI. . 228

VIT. Techniques for the Facilitation of Peptide Synthesis . . . . . . . . . 233 A. Solid Phase Peptide Synthesis (SPPS) 233

1. The Insoluble Support . . . 235 2. The Bond Between Peptide

and Polymer. . . . . . . . 236 3. Protection and Deprotection . 238 4. Methods of Coupling in SPPS 239 5. Separation of the Completed Peptide

Chain from the Polymeric Support 240 6. Problems in Solid Phase Peptide

Synthesis . . . . . . . ... . .. 244

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Table of Contents

B. Synthesis in Solution . . . . . . . .. 246 1. Peptides Attached to Soluble Polymers 246 2. The "handle" Method. . . 248 3. Synthesis "in situ" . . . . . 249 4. Synthesis without Isolation

of Intermediates . . 250 References of Chapter VII . . . 251

vm. Recent Developments and Perspectives. 255

A. Formation of the Peptide Bond. 255 B. Protection . . . . . . . 259

1. Carboxyl Groups. . . . . 259 2. Temporary Protection

of Amino Groups . . . . 261 3. Semipermanent Protection

of Side Chain Functions. . 261 4. Removal of Protecting Groups . 263

C. Side Reactions . . . . . . . 264 D. Solid Phase Peptide Synthesis. 265 E. Catalysis. . . . . . . 266 References of Chapter VIII 266

Author Index . .- 269

Subject Index . . 286

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