Miklos Bodanszky. Peptide Chemistry. A Practical Textbook. With 2 Figures and 5 Tables. Springer-Verlag Berlin Heidelberg New York London Paris Tokyo

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2 Miklos Bodanszky Peptide Chemistry A Practical Textbook With 2 Figures and 5 Tables Springer-Verlag Berlin Heidelberg New York London Paris Tokyo

3 Professor Dr. Miklos Bodanszky One Markham Road 1 E Princeton, NJ 08540, USA ISBN-13: e-isbn-13: DOl: / Library of Congress Cataloging-in-Publication Data Bodanszky, Miklos. Peptide chemistry. Bibliography: p. Includes index. 1. 'Peptides. 1. Title QD431.B ' ISBN-13: This work is subject to copyright. All rights reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or on other ways, and storage in data banks. Duplication of this publication or parts thereof is only permitted under the provisions of the German Copyright Law of September 9,1965, in its version of June 24,1985, and a copyright fee must always be paid. Violations fall under the prosecution act of the German Copyright Law. Springer-Verlag Berlin Heidelberg 1988 The use of registered names, trademarks, etc. in the 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. Product Liability: The publishers can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. Typesetting: Konrad Triltsch, Wiirzburg 2151/

4 To the memory of my brother Dr. S. Bodanszky

5 Preface Nature applied peptides for a great variety of specific functions. The specificity provided by the individual character of each amino acid is further ehanced by the combination of several amino acids into larger molecules. Peptides therefore can act as chemical messengers, neurotransmitters, as highly specific stimulators and inhibitors, regulating various life-processes. Entire classes of biologically active compounds, such as the opioid peptides or the gastrointestinal hormones emerged within short periods of time and it is unlikely that the rapid succession of discoveries of important new peptides would come to a sudden halt. In fact, our knowledge of the field is probably still in an early stage of development. Peptides also gained importance in our everyday life. A dipeptide derivative is used as sweetener, another to control high blood pressure. Peptides of moderate size, such as oxytocin or vasopressin have been available for medical purposes for many years and more recently peptide hormones of considerable chain-length, for instance calcitonin and secretin, are manufactured for therapy. These practical aspects alone might be considered as justification for teaching peptide chemistry in a special course. Yet, not less important is the experience that the chemistry of pep tides is treated as an orphan in textbooks both of organic chemistry and of biochemistry, a topic disposed of in a few pages. There are good reasons for this. While biosynthesis and metabolism of amino acids and biosynthesis and function of proteins are in the main line of biochemistry, peptide chemistry, and the synthesis of peptides particularly, are too strongly tied to organic chemistry to be treated in detail in biochemistry texts. A more than casual presentation of pep tides, however, would certainly transcend the limits of single-volume organic chemistry textbooks. Also, sequence determination lies outside the general methods of structure determination applied for the majority of organic compounds and synthesis of pep tides aims at compounds with molecular weights well beyond those encountered in the synthesis of other natural products, with the notable exception of nucleic acids. The ensuing problems, like simultaneous handling of numerous functional groups, the continued need for conservation of chiral purity and unusual modes of retrosynthetic analysis are particular to peptides. Peptide chemistry is indeed a discipline in itself with its own practical significance and intellectual challenge. It deserves to be taught in a special course.

6 VIII Preface The author of this small volume has taught Peptide Chemistry as a graduate course through many years. A suitable textbook was sorely missed both by students and instructor and the copious handouts were insufficient substitutes. This textbook was written with the assumption that its existence might stimulate the teaching of its subject. The size of the volume was limited by the desire to make it inexpensive, but the m a t e presented r i a ~ was intended to be adequate for a one-semester threecredit course. The first part, dealing with the determination of peptide structure, could be used separately in a short course while the second part, on synthesis, might support a one-semester two-credit course. It would give the author considerable satisfaction if instructors, prompted by the availability ofthis small textbook, would offer a course on Peptide Chemistry and if the students would find it helpful in their studies. The comments and suggestions by Professor G. T. Young (Oxford University) concerning my book Principles of Peptide Synthesis (Springer Verlag, 1984) were very helpful in writing the present volume and are gratefully acknowledged. I thank my wife Agnes for numerous corrections and improvements in the preparation of this manuscript. Princeton, May 1988 M. Bodanszky

7 Table of Contents I. Introduction.... References and Additional Sources Part One: Structure Determination II. Amino Acid Analysis Additonal Sources III. Sequence Determination A. End Group Determination Determination of the N-terminal Residue Determination of the C-terminal Residue B. Sequential Degradation Hydrolysis Catalyzed by Exopeptidases Edman Degradation a) The Two-step Procedure b) The Three-step Procedure c) Automated Sequencing d) The Edman-Dansyl Process C. Sequence Determination with the Help of Mass Spectra D. Fragmentation of Peptides Hydrolysis with Specific Endopeptidases Chemical Methods of Cleavage Fission of Disulfides References and Additional Sources IV. Secondary and Tertiary Structure A. Architectural Features in Peptides The Peptide Bond Secondary Structures Tertiary Structure Quaternary Structure

8 X Table of Contents B. Methods for the Analysis of Conformation in Peptides Optical Rotatory Dispersion and Circular Dichroism Nuclear Magnetic Resonance (nmr) Spectroscopy X-Ray Crystallography C. Prediction of Conformation in Peptides References and Additional Sources Part Two: Peptide Synthesis V. Formation of the Peptide Bond A. The Acid Chloride Method B. The Acid Azide Method C. Anhydrides D. Active Esters E. Coupling Reagents F. Enzyme Catalyzed Bond Formation..., G. Non Conventional Methods of Peptide Bond Formation References and Additional Sources VI. Protection of Functional Groups A. Blocking of a-amino Groups B. Blocking of Side Chain Amino Groups C. Protection of the Carboxyl Group D. Protection of Hydroxyl Groups E. Blocking of the Sulfhydryl Group F. Masking the Thioether in Methionine G. Protection of the Guanidine Function in the Arginine Side Chain H. Masking the Imidazole in Histidine I. Indole Protection J. Carboxamide Blocking Groups References and Additional Sources VII. Undesired Reactions during Synthesis A. Base Catalyzed Ring Closure B. Acid Catalyzed Ring Closure C. Acyl Migration D. Alkylation E. Friedel-Crafts Reaction F. Saturation of Aromatic Nuclei

9 Table of Contents XI G. Reductive Cleavage of the Peptide Chain H. Oxidative Decomposition of the Indole in Tryptophan. 111 I. Side Reactions Caused by Steric Hindrance J. Prevention and Suppression of Side Reactions Additional Source VIII. Racemization A. Mechanism of Racemization B. Detection of Racemization C. Racemization Studies in Model Systems D. Prevention of Racemization References and Additional Source IX. Design of Schemes for Peptide Synthesis A. Segment Condensation B. Stepwise Chain-lengthening Starting with the N-terminal Residue C. Stepwise Chain-lengthening Starting with the C-terminal Residue D. Tactical Considerations E. Synthesis of Special Target Peptides Polyamino Acids and Sequential Polypeptides Disulfide Bridges Cyclic Peptides Peptides Containing Non-amino Acid Constituents Semisynthesis (partial Synthesis) References and Additional Sources X. Solid Phase Peptide Synthesis A. The Insoluble Polymeric Support and the Bond Linking the Peptide to the Resin B. Removal of Temporary Blocking Groups C. Coupling: Acylation of the Resin-bound Amine-component D. Separation of the Peptide from the Polymeric Support and Final Deprotection E. Some Problems Encountered in Solid-phase Peptide Synthesis Premature Chain-termination Deletion References and Additional Sources

10 XII Table of Contents XI. Methods of Facilitation A. Chains Attached to a Soluble Polymer (The Liquid Phase Method) B. The Picolyl Ester Method C. Rapid Synthesis Chain-building Without Isolation of Intermediates Chain-building Through Isolated Intermediates D. Synthesis "in situ" E. Simultaneous Synthesis of Peptide Analogs F. Insoluble Reagents Active Esters Polymeric Coupling Reagents Insoluble Cleaving Reagents References and Additional Sources XII. Analysis and Characterization of Synthetic Peptides A. Homogeneity Tests B. Amino Acid Analysis and Sequence Determination C. Elemental Analysis D. Measurement of Optical Activity E. Infrared and Ultraviolet Absorption Spectra F. Nuclear Magnetic Resonance Spectra G. Molecular-weight Determination Reference and Additional Sources Subject Index