The authors’ objective has been to concentrate on amino acids and pep- tides without detailed discussions of proteins, although the book gives all the essential background chemistry, including sequence determination, synthesis and spectroscopic methods, to allow the reader to appreciate protein behaviour at the molecular level. The approach is intended to encourage the reader to cross classical boundaries, such as in the later chapter on the biological roles of amino acids and the design of peptide- based drugs. For example, there is a section on enzyme-catalysed synthesis of peptides, an area often neglected in texts describing peptide synthesis. This modern text will be of value to advanced undergraduates, graduate students and research workers in the amino acid, peptide and protein field. Amino Acids and Peptides G. C.IBARRETT D. T. ELMORE The Pitt Building, Trumpington Street, Cambridge, United Kingdom The Edinburgh Building, Cambridge CB2 2RU, UK 40 West 20th Street, New York, NY 10011-4211, USA 477 Williamstown Road, Port Melbourne, VIC 3207, Australia Ruiz de Alarcón 13, 28014 Madrid, Spain Dock House, The Waterfront, Cape Town 8001, South Africa http://www.cambridge.org © Cambridge University Press 2004 First published in printed format 1998 ISBN 0-511-03952-2 eBook (netLibrary) ISBN 0-521-46292-4 hardback ISBN 0-521-46827-2 paperback Contents Forewordpagexiii 1Introduction1 1.1Sources and roles of amino acids and peptides1 1.2Definitions1 1.3‘Protein amino acids’, alias ‘the coded amino acids’3 1.4 Nomenclature for ‘the protein amino acids’, alias ‘the coded amino acids’7 1.5 Abbreviations for names of amino acids and the use of these abbreviations to give names to polypeptides7 1.6 Post-translational processing: modification of amino-acid residues within polypeptides11 1.7 Post-translational processing: in vivo cleavages of the amide backbone of polypeptides11 1.8 ‘Non-protein amino acids’, alias ‘non-proteinogenic amino acids’ or ‘non-coded amino acids’11 1.9Coded amino acids, non-natural amino acids and peptides in nutrition and food science and in human physiology13 1.10The geological and extra-terrestrial distribution of amino acids15 1.11Amino acids in archaeology and in forensic science15 1.12Roles for amino acids in chemistry and in the life sciences16 1.12.1Amino acids in chemistry16 1.12.2Amino acids in the life sciences16 1.13␤- and higher amino acids17 1.14References19 2Conformations ofamino acids and peptides20 2.1 Introduction: the main conformational features of amino acids and peptides20 vii Contents 2.2Configurational isomerism within the peptide bond20 2.3Dipeptides26 2.4Cyclic oligopeptides26 2.5Acyclic oligopeptides27 2.6Longer oligopeptides: primary, secondary and tertiary structure27 2.7Polypeptides and proteins: quaternary structure and aggregation28 2.8 Examples of conformational behaviour; ordered and disordered states and transitions between them29 2.8.1The main categories of polypeptide conformation29 2.8.1.1One extreme situation29 2.8.1.2The other extreme situation29 2.8.1.3The general case29 2.9Conformational transitions for amino acids and peptides30 2.10References31 3Physicochemical properties ofamino acids and peptides32 3.1Acid–base properties32 3.2Metal-binding properties of amino acids and peptides34 3.3 An introduction to the routine aspects and the specialised aspects of the spectra of amino acids and peptides35 3.4Infrared (IR) spectrometry36 3.5 General aspects of ultraviolet (UV) spectrometry, circular dichroism (CD) and UVfluorescence spectrometry37 3.6Circular dichroism38 3.7Nuclear magnetic resonance (NMR) spectroscopy41 3.8 Examples of assignments of structures to peptides from NMR spectra and other data43 3.9References46 4Reactions and analytical methods for amino acids and peptides48 Part 1Reactions of amino acids and peptides48 4.1Introduction48 4.2General survey48 4.2.1Pyrolysis of amino acids and peptides49 4.2.2Reactions of the amino group49 4.2.3Reactions of the carboxy group49 4.2.4Reactions involving both amino and carboxy groups51 4.3A more detailed survey of reactions of the amino group51 4.3.1N-Acylation51 4.3.2Reactions with aldehydes52 4.3.3N-Alkylation53 viii Contents 4.4A survey of reactions of the carboxy group53 4.4.1Esterification54 4.4.2Oxidative decarboxylation54 4.4.3Reduction54 4.4.4Halogenation55 4.4.5 Reactions involving amino and carboxy groups of ␣-amino acids and their N-acyl derivatives55 4.4.6 Reactions at the ␣-carbon atom and racemisation of ␣-amino acids55 4.4.7Reactions of the amide group in acylamino acids and peptides57 4.5Derivatisation of amino acids for analysis58 4.5.1 Preparation of N-acylamino acid esters and similar derivatives for analysis58 4.6References60 Part 2 Mass spectrometry in amino-acid and peptide analysis and in peptide-sequence determination61 4.7General considerations61 4.7.1Mass spectra of free amino acids61 4.7.2Mass spectra of free peptides62 4.7.3Negative-ion mass spectrometry65 4.8Examples of mass spectra of peptides65 4.8.1 Electron-impact mass spectra (EIMS) of peptide derivatives65 4.8.2Finer details of mass spectra of peptides68 4.8.3Difficulties and ambiguities69 4.9 The general status of mass spectrometry in peptide analysis69 4.9.1 Specific advantages of mass spectrometry in peptide sequencing70 4.10Early methodology: peptide derivatisation71 4.10.1N-Terminal acylation and C-terminal esterification71 4.10.2N-Acylation and N-alkylation of the peptide bond72 4.10.3Reduction of peptides to ‘polyamino-polyalcohols’72 4.11 Current methodology: sequencing by partial acid hydrolysis, followed by direct MS analysis of peptide hydrolysates72 4.11.1Current methodology: instrumental variations74 4.12Conclusions77 4.13References77 ix Contents Part 3 Chromatographic and related methods for the separation of mixtures of amino acids, mixtures of peptides and mixtures of amino acids and peptides78 4.14Separation of amino-acid and peptide mixtures78 4.14.1Separation principles78 4.15Partition chromatography; HPLC and GLC80 4.16Molecular exclusion chromatography (gel chromatography)80 4.17Electrophoretic separation and ion-exchange chromatography82 4.17.1Capillary zone electrophoresis (CZE)83 4.18Detection of separated amino acids and peptides83 4.18.1 Detection of amino acids and peptides separated by HPLC and by other liquid-based techniques84 4.18.2Detection of amino acids and peptides separated by GLC85 4.19Thin-layer chromatography (planar chromatography; HPTLC)86 4.20Quantitative amino-acid analysis86 4.21References87 Part 4Immunoassays for peptides87 4.22Radioimmunoassays87 4.23Enzyme-linked immunosorbent assays (ELISAs)88 4.24References90 Part 5Enzyme-based methods for amino acids90 4.25Biosensors90 4.26References90 5Determination ofthe primary structure ofpeptides and proteins91 5.1Introduction91 5.2Strategy92 5.3Cleavage of disulphide bonds96 5.4Identification of the N-terminus and stepwise degradation97 5.5Enzymic methods for determining N-terminal sequences105 5.6Identification of C-terminal sequences106 5.7Enzymic determination of C-terminal sequences107 5.8Selective chemical methods for cleaving peptide bonds107 5.9Selective enzymic methods for cleaving peptide bonds109 5.10Determination of the positions of disulphide bonds112 5.11 Location of post-translational modifications and prosthetic groups114 5.12Determination of the sequence of DNA117 5.13References118 x Contents 6Synthesis ofamino acids120 6.1General120 6.2Commercial and research uses for amino acids120 6.3Biosynthesis: isolation of amino acids from natural sources121 6.3.1Isolation of amino acids from proteins121 6.3.2 Biotechnological and industrial synthesis of coded amino acids121 6.4 Synthesis of amino acids starting from coded amino acids other than glycine122 6.5 General methods of synthesis of amino acids starting with a glycine derivative123 6.6Other general methods of amino acid synthesis123 6.7Resolution of -amino acids125 6.8Asymmetric synthesis of amino acids127 6.9References129 7Methods for the synthesis ofpeptides130 7.1Basic principles of peptide synthesis and strategy130 7.2Chemical synthesis and genetic engineering132 7.3Protection of ␣-amino groups134 7.4Protection of carboxy groups135 7.5Protection of functional side-chains138 7.5.1Protection of ␧-amino groups138 7.5.2Protection of thiol groups139 7.5.3Protection of hydroxy groups140 7.5.4Protection of the guanidino group of arginine141 7.5.5Protection of the imidazole ring of histidine142 7.5.6Protection of amide groups145 7.5.7Protection of the thioether side-chain of methionine145 7.5.8Protection of the indole ring of tryptophan146 7.6Deprotection procedures146 7.7Enantiomerisation during peptide synthesis146 7.8Methods for forming peptide bonds149 7.8.1The acyl azide method150 7.8.2The use of acid chlorides and acid fluorides151 7.8.3The use of acid anhydrides151 7.8.4The use of carbodiimides153 7.8.5The use of reactive esters153 7.8.6The use of phosphonium and isouronium derivatives155 7.9Solid-phase peptide synthesis (SPPS)156 7.10Soluble-handle techniques163 7.11Enzyme-catalysed peptide synthesis and partial synthesis164 xi Contents 7.12Cyclic peptides168 7.12.1Homodetic cyclic peptides168 7.12.2Heterodetic cyclic peptides170 7.13The formation of disulphide bonds170 7.14References172 7.14.1References cited in the text172 7.14.2References for background reading173 8Biological roles ofamino acids and peptides174 8.1Introduction174 8.2The role of amino acids in protein biosynthesis175 8.3Post-translational modification of protein structures178 8.4Conjugation of amino acids