Chemistry of peptide synthesis

CHEMISTRY OF PEPTIDE SYNTHESIS

N. Leo Benoiton

University of Ottawa Ottawa, Ontario, Canada

Boca Raton London New York Singapore A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

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Published in 2006 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742

? 2006 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group

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International Standard Book Number-10: 1-57444-454-9 (Hardcover) International Standard Book Number-13: 978-1-57444-454-4 (Hardcover) Library of Congress Card Number 2005005753

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Library of Congress Cataloging-in-Publication Data

Benoiton, N. Leo. Chemistry of peptide synthesis / N. Leo Benoiton. p. ; cm. Includes bibliographical references. ISBN-13: 978-1-57444-454-4 (hardcover : alk. paper) ISBN-10: 1-57444-454-9 (hardcover : alk. paper) 1. Peptides--Synthesis. [DNLM: 1. Peptide Biosynthesis. QU 68 B456c 2005] I. Title.

QP552.P4B46 2005 612'.015756--dc22

2005005753

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Dedication

This book is dedicated to Rao Makineni, a unique member and benefactor of the peptide community.

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Preface

This book has emerged from courses that I taught to biochemistry students at the undergraduate and graduate levels, to persons with a limited knowledge of organic chemistry, to chemists with experience in other fields, and to peptide chemists. It assumes that the reader possesses a minimum knowledge of organic and amino-acid chemistry. It comprises 188 self-standing sections that include 207 figures written in clear language, with limited use of abbreviations. The focus is on understanding how and why reactions and phenomena occur. There are a few tables of illustrative data, but no tables of compounds or reaction conditions. The material is presented progressively, with some repetition, and then with amplification after the basics have been dealt with. The fundamentals of peptide synthesis, with an emphasis on the intermediates that are encountered in aminolysis reactions, are presented initially. The coupling of N-protected amino acids and N-protected peptides and their tendencies to isomerize are then addressed separately. This allows for easier comprehension of the issues of stereomutation and the applicability of coupling reactions. Protection of functional groups is introduced on the basis of the methods that are employed for removal of the protectors. A chapter is devoted to the question of stereomutation, which is now more complex, following the discovery that Nprotected amino acids can also give rise to oxazolones. Other chapters are devoted to solid-phase synthesis, side-chain protection and side reactions, amplification on coupling methods, and miscellaneous topics. Points to note are that esters that undergo aminolysis are referred to as activated esters, which is why they react, and not active esters, and that in two cases two abbreviations (Z and Cbz; HOObt and HODhbt) are used haphazardly for one entity because that is the reality of the peptide literature. An effort has been made to convey to the reader a notion of how the field of peptide chemistry has developed. To this end, the references are located at the end of each section and include the titles of articles. Most references have been selected on the basis of the main theme that the chapter addresses. When the relevance of a paper is not obvious from the title, a phrase has been inserted in parentheses. The titles of papers written in German and French have been translated. For obvious reasons the number of references had to be limited. I extend my apologies to anyone who considers his or her work to have been unjustifiably omitted. Some poetic license was exercised in the creation of the manuscript and the reaction schemes. Inclusion of all details and exceptions to statements would have made the whole too unruly.

I am greatly indebted to Dr. Brian Ridge of the School of Chemical and Biological Sciences of the University of Exeter, United Kingdom, for his critical review of the manuscript and for his suggestions that have been incorporated into the manuscript. I solely am responsible for the book's contents. I thank Professor John Coggins of the University of Glasgow for providing the references for Appendix 3,

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and I am grateful to anyone who might have provided me with information that appears in this book. I am grateful to the University of Ottawa for the office and library services that have been provided to me. I am indebted to Dr. Rao Makineni for generous support provided over the years. I thank the publishers for their patience during the long period when submission of the manuscript was overdue. And most important, I thank my wife Ljuba for her patience and support and express my sincere apologies for having deprived her of the company of her "retired" husband for a period much longer than had been planned.

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Table of Contents

Chapter 1 Fundamentals of Peptide Synthesis .....................................................1

1.1 Chemical and Stereochemical Nature of Amino Acids ..................................1 1.2 Ionic Nature of Amino Acids ..........................................................................2 1.3 Charged Groups in Peptides at Neutral pH.....................................................3 1.4 Side-Chain Effects in Other Amino Acids ......................................................4 1.5 General Approach to Protection and Amide-Bond Formation........................5 1.6 N-Acyl and Urethane-Forming N-Substituents ...............................................6 1.7 Amide-Bond Formation and the Side Reaction of

Oxazolone Formation.......................................................................................7 1.8 Oxazolone Formation and Nomenclature........................................................8 1.9 Coupling, 2-Alkyl-5(4H)-Oxazolone Formation and Generation of

Diastereoisomers from Activated Peptides ......................................................9 1.10 Coupling of N-Alkoxycarbonylamino Acids without Generation of

Diastereoisomers: Chirally Stable 2-Alkoxy-5(4H)-Oxazolones..................10 1.11 Effects of the Nature of the Substituents on the Amino and

Carboxyl Groups of the Residues That Are Coupled to Produce a Peptide...........................................................................................11 1.12 Introduction to Carbodiimides and Substituted Ureas ..................................12 1.13 Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids ........12 1.14 Carbodiimide-Mediated Reactions of N-Acylamino Acids and Peptides.....13 1.15 Preformed Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids......14 1.16 Purified Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids Obtained Using a Soluble Carbodiimide.......................................................15 1.17 Purified 2-Alkyl-5(4H)-Oxazolones from N-Acylamino and N-Protected Glycylamino Acids ....................................................................16 1.18 2-Alkoxy-5(4H)-Oxazolones as Intermediates in Reactions of N-Alkoxycarbonylamino Acids......................................................................17 1.19 Revision of the Central Tenet of Peptide Synthesis......................................18 1.20 Strategies for the Synthesis of Enantiomerically Pure Peptides...................19 1.21 Abbreviated Designations of Substituted Amino Acids and Peptides ..........20 1.22 Literature on Peptide Synthesis .....................................................................21

Chapter 2 Methods for the Formation of Peptide Bonds...................................25

2.1 Coupling Reagents and Methods and Activated Forms ................................25 2.2 Peptide-Bond Formation from Carbodiimide-Mediated Reactions of

N-Alkoxycarbonylamino Acids......................................................................26 2.3 Factors Affecting the Course of Events in Carbodiimide-Mediated

Reactions of N-Alkoxycarbonylamino Acids ................................................28

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2.4 Intermediates and Their Fate in Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids......................................................................29

2.5 Peptide-Bond Formation from Preformed Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids......................................................................30

2.6 Peptide-Bond Formation from Mixed Anhydrides of N-Alkoxycarbonylamino Acids......................................................................32

2.7 Alkyl Chloroformates and Their Nomenclature............................................34 2.8 Purified Mixed Anhydrides of N-Alkoxycarbonylamino Acids and

Their Decomposition to 2-Alkoxy-5(4H)-Oxazolones..................................34 2.9 Peptide-Bond Formation from Activated Esters of

N-Alkoxycarbonylamino Acids......................................................................36 2.10 Anchimeric Assistance in the Aminolysis of Activated Esters .....................38 2.11 On the Role of Additives as Auxiliary Nucleophiles:

Generation of Activated Esters ......................................................................39 2.12 1-Hydroxybenzotriazole as an Additive That Suppresses N-Acylurea

Formation by Protonation of the O-Acylisourea...........................................40 2.13 Peptide-Bond Formation from Azides of

N-Alkoxycarbonylamino Acids......................................................................41 2.14 Peptide-Bond Formation from Chlorides of

N-Alkoxycarbonylamino Acids: N-9-Fluorenylmethoxycarbonylamino-Acid Chlorides .................................43 2.15 Peptide-Bond Formation from 1-Ethoxycarbonyl-2-Ethoxy1,2-Dihydroquinoline-Mediated Reactions of N-Alkoxycarbonylamino Acids......................................................................44 2.16 Coupling Reagents Composed of an Additive Linked to a Charged Atom Bearing Dialkylamino Substituents and a Nonnucleophilic Counter-Ion.........................................................................45 2.17 Peptide-Bond Formation from Benzotriazol-1-ylOxy-tris(Dimethylamino)Phosphonium Hexafluorophosphate-Mediated Reactions of N-Alkoxycarbonylamino Acids......................................................................46 2.18 Peptide-Bond Formation from O-Benzotriazol-1-yl-N,N,N,N-Tetramethyluronium Hexafluorophosphate- and Tetrafluoroborate-Mediated Reactions of N-Alkoxycarbonylamino Acids ................................................48 2.19 Pyrrolidino Instead of Dimethylamino Substituents for the Environmental Acceptability of Phosphonium and Carbenium Salt-Based Reagents.......................................................................................50 2.20 Intermediates and Their Fate in Benzotriazol-1-ylOxyphosphonium and Carbenium Salt-Mediated Reactions ........................51 2.21 1-Hydroxybenzotriazole as Additive in Couplings of N-Alkoxycarbonylamino Acids Effected by Phosphonium and Uronium Salt-Based Reagents .......................................................................53 2.22 Some Tertiary Amines Used as Bases in Peptide Synthesis.........................54

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