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CHEMISTRYOF PEPTIDESYNTHESISN. Leo BenoitonUniversity of OttawaOttawa, Ontario, CanadaBoca Raton London New York SingaporeA CRC title, part of the Taylor & Francis imprint, a member of theTaylor & Francis Group, the academic division of T&F Informa plc. 2006 by Taylor & Francis Group, LLC

DK3236 Discl.fm Page 1 Wednesday, June 8, 2005 2:56 PMPublished in 2006 byCRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742 2006 by Taylor & Francis Group, LLCCRC Press is an imprint of Taylor & Francis GroupNo claim to original U.S. Government worksPrinted in the United States of America on acid-free paper10 9 8 7 6 5 4 3 2 1International 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 2005005753This book contains information obtained from authentic and highly regarded sources. Reprinted material isquoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable effortshave been made to publish reliable data and information, but the author and the publisher cannot assumeresponsibility for the validity of all materials or for the consequences of their use.No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, andrecording, or in any information storage or retrieval system, without written permission from the publishers.For permission to photocopy or use material electronically from this work, please access www.copyright.com(http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive,Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registrationfor a variety of users. For organizations that have been granted a photocopy license by the CCC, a separatesystem of payment has been arranged.Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used onlyfor identification and explanation without intent to infringe.Library of Congress Cataloging-in-Publication DataBenoiton, 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 2005612'.015756--dc222005005753Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.comTaylor & Francis Groupis the Academic Division of T&F Informa plc. 2006 by Taylor & Francis Group, LLCand the CRC Press Web site athttp://www.crcpress.com

DK3236 C000.fm Page iii Tuesday, June 28, 2005 8:32 AMDedicationThis book is dedicated to Rao Makineni, a unique memberand benefactor of the peptide community. 2006 by Taylor & Francis Group, LLC

DK3236 C000.fm Page v Tuesday, June 28, 2005 8:32 AMPrefaceThis book has emerged from courses that I taught to biochemistry students at theundergraduate and graduate levels, to persons with a limited knowledge of organicchemistry, to chemists with experience in other fields, and to peptide chemists. Itassumes that the reader possesses a minimum knowledge of organic and amino-acidchemistry. It comprises 188 self-standing sections that include 207 figures writtenin clear language, with limited use of abbreviations. The focus is on understandinghow and why reactions and phenomena occur. There are a few tables of illustrativedata, but no tables of compounds or reaction conditions. The material is presentedprogressively, with some repetition, and then with amplification after the basics havebeen dealt with. The fundamentals of peptide synthesis, with an emphasis on theintermediates that are encountered in aminolysis reactions, are presented initially.The coupling of Nα-protected amino acids and Nα-protected peptides and theirtendencies 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 areemployed for removal of the protectors. A chapter is devoted to the question ofstereomutation, which is now more complex, following the discovery that Nαprotected amino acids can also give rise to oxazolones. Other chapters are devotedto solid-phase synthesis, side-chain protection and side reactions, amplification oncoupling methods, and miscellaneous topics. Points to note are that esters thatundergo aminolysis are referred to as activated esters, which is why they react, andnot active esters, and that in two cases two abbreviations (Z and Cbz; HOObt andHODhbt) are used haphazardly for one entity because that is the reality of the peptideliterature. An effort has been made to convey to the reader a notion of how the fieldof peptide chemistry has developed. To this end, the references are located at theend of each section and include the titles of articles. Most references have beenselected on the basis of the main theme that the chapter addresses. When therelevance of a paper is not obvious from the title, a phrase has been inserted inparentheses. 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 myapologies 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 reactionschemes. Inclusion of all details and exceptions to statements would have made thewhole 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 reviewof the manuscript and for his suggestions that have been incorporated into themanuscript. I solely am responsible for the book’s contents. I thank Professor JohnCoggins of the University of Glasgow for providing the references for Appendix 3, 2006 by Taylor & Francis Group, LLC

DK3236 C000.fm Page vi Tuesday, June 28, 2005 8:32 AMand I am grateful to anyone who might have provided me with information thatappears in this book. I am grateful to the University of Ottawa for the office andlibrary services that have been provided to me. I am indebted to Dr. Rao Makinenifor generous support provided over the years. I thank the publishers for their patienceduring the long period when submission of the manuscript was overdue. And mostimportant, I thank my wife Ljuba for her patience and support and express mysincere apologies for having deprived her of the company of her “retired” husbandfor a period much longer than had been planned. 2006 by Taylor & Francis Group, LLC

DK3236 C000.fm Page vii Tuesday, June 28, 2005 8:32 AMTable of ContentsChapter 151.161.171.181.191.201.211.22Chemical and Stereochemical Nature of Amino Acids .1Ionic Nature of Amino Acids .2Charged Groups in Peptides at Neutral pH.3Side-Chain Effects in Other Amino Acids .4General Approach to Protection and Amide-Bond Formation.5N-Acyl and Urethane-Forming N-Substituents .6Amide-Bond Formation and the Side Reaction ofOxazolone Formation.7Oxazolone Formation and Nomenclature.8Coupling, 2-Alkyl-5(4H)-Oxazolone Formation and Generation ofDiastereoisomers from Activated Peptides .9Coupling of N-Alkoxycarbonylamino Acids without Generation ofDiastereoisomers: Chirally Stable 2-Alkoxy-5(4H)-Oxazolones.10Effects of the Nature of the Substituents on the Amino andCarboxyl Groups of the Residues That Are Coupled toProduce a Peptide.11Introduction to Carbodiimides and Substituted Ureas .12Carbodiimide-Mediated Reactions of N-Alkoxycarbonylamino Acids .12Carbodiimide-Mediated Reactions of N-Acylamino Acids and Peptides.13Preformed Symmetrical Anhydrides of N-Alkoxycarbonylamino Acids.14Purified Symmetrical Anhydrides of N-Alkoxycarbonylamino AcidsObtained Using a Soluble Carbodiimide.15Purified 2-Alkyl-5(4H)-Oxazolones from N-Acylamino andN-Protected Glycylamino Acids .162-Alkoxy-5(4H)-Oxazolones as Intermediates in Reactions ofN-Alkoxycarbonylamino Acids.17Revision of the Central Tenet of Peptide Synthesis.18Strategies for the Synthesis of Enantiomerically Pure Peptides.19Abbreviated Designations of Substituted Amino Acids and Peptides .20Literature on Peptide Synthesis .21Chapter 22.12.22.3Fundamentals of Peptide Synthesis .1Methods for the Formation of Peptide Bonds.25Coupling Reagents and Methods and Activated Forms .25Peptide-Bond Formation from Carbodiimide-Mediated Reactions ofN-Alkoxycarbonylamino Acids.26Factors Affecting the Course of Events in Carbodiimide-MediatedReactions of N-Alkoxycarbonylamino Acids .28 2006 by Taylor & Francis Group, LLC

DK3236 C000.fm Page viii Tuesday, June 28, 2005 8:32 172.182.192.202.212.22Intermediates and Their Fate in Carbodiimide-Mediated Reactions ofN-Alkoxycarbonylamino Acids.29Peptide-Bond Formation from Preformed Symmetrical Anhydrides ofN-Alkoxycarbonylamino Acids.30Peptide-Bond Formation from Mixed Anhydrides ofN-Alkoxycarbonylamino Acids.32Alkyl Chloroformates and Their Nomenclature.34Purified Mixed Anhydrides of N-Alkoxycarbonylamino Acids andTheir Decomposition to 2-Alkoxy-5(4H)-Oxazolones.34Peptide-Bond Formation from Activated Esters ofN-Alkoxycarbonylamino Acids.36Anchimeric Assistance in the Aminolysis of Activated Esters .38On the Role of Additives as Auxiliary Nucleophiles:Generation of Activated Esters .391-Hydroxybenzotriazole as an Additive That Suppresses N-AcylureaFormation by Protonation of the O-Acylisourea.40Peptide-Bond Formation from Azides ofN-Alkoxycarbonylamino Acids.41Peptide-Bond Formation from Chlorides ofN-Alkoxycarbonylamino Acids:N-9-Fluorenylmethoxycarbonylamino-Acid Chlorides .43Peptide-Bond Formation from ated Reactions ofN-Alkoxycarbonylamino Acids.44Coupling Reagents Composed of an Additive Linked to aCharged Atom Bearing Dialkylamino Substituents and aNonnucleophilic Counter-Ion.45Peptide-Bond Formation from mHexafluorophosphate-Mediated Reactions ofN-Alkoxycarbonylamino Acids.46Peptide-Bond Formation roniumHexafluorophosphate- and Tetrafluoroborate-MediatedReactions of N-Alkoxycarbonylamino Acids .48Pyrrolidino Instead of Dimethylamino Substituents for theEnvironmental Acceptability of Phosphonium and CarbeniumSalt-Based Reagents.50Intermediates and Their Fate in Benzotriazol-1-ylOxyphosphonium and Carbenium Salt-Mediated Reactions .511-Hydroxybenzotriazole as Additive in Couplings ofN-Alkoxycarbonylamino Acids Effected by Phosphonium andUronium Salt-Based Reagents .53Some Tertiary Amines Used as Bases in Peptide Synthesis.54 2006 by Taylor & Francis Group, LLC