SPRINGER LAB MANUAL

M. Bodanszky A. Bodanszky

The Practice of Peptide Synthesis Second, Revised Edition

Springer-Verlag Berlin Heidelberg New York London Paris Tokyo Hong Kong Barcelona Budapest

Professor Dr. MIKLOS BODANSZKY

One Markham Road Princeton, NJ 08540, USA

AGNES BODANSZKY (1925-1989)

The First edition was published as Vol. 21 of the series "Reactivity and Structure Concepts in Organic Chemistry" (3-540-13471-9)

ISBN-13:978-3-540-57505-4 DOl: 10.1007/978-3-642-85055-4

e-ISBN -13 :978-3-642-85055-4

Library of Congress Cataloging·in·Publication Data. Bodanszky, Miklos. The practice of peptide synthesis/ M. Bodanszky, A. Bodanszky. - 2nd, rev. ed. p. cm. - (Springer laboratory) Includes bibliographical references and indexes.ISBN-13:978-3-S40-S7S0S-4 I.Peptides- Synthesis. I. Bodanszky,A.(Agnes) II. Title. III. Series.QD431.B763 1994 547.T56-dc20 94-890

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,1965, in its current version, and permissions for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law.

CG; Springer-Verlag Berlin Heidelberg 1994

The use of general descriptive names, 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.

Typesetting: Macmillan India Ltd., Bangalore 51/3130 543 2 1 0 Printed on acid-free paper

To our daughter Eva

Preface

During the years 1980-81, as guests of the Deutsches Wollforschungsinstitut in Aachen, Germany, we were working on a small book entitled, "Principles of Peptide Synthesis". In the library of the Institute we noted that the volumes of Houben-Weyl's Handbuch der Organischen Chemie dealing with peptide synthesis were so much in use that they were ready to fall apart because the researchers of the Institute consulted them with amazing regularity. They were looking for references, but even more for experimental details which could be adapted to the particular problem they happened to face. In planning a new synthetic endeavor they tried to lean on the experience of others in analogous situations. This suggested to us that a smaller and hence more tractable book may be needed, a volume which can be kept on or near the bench to make examples of fundamental methods readily available in the laboratory. Such a collection could save numerous short trips to the library, a point particularly important where a library well equipped with the sources of the literature of peptide synthesis is not near at hand. Also, we thought that the envisaged book may be welcome by those who are more versed in English than in German. To the best of our knowledge no similar publication is available.

In our attempt to provide the peptide chemist with a collection of well established procedures we resisted the temptation to include novel, but untes­ted methods, although some of these are quite original and hence intellectually attractive. It seems, that in the practical execution of peptide synthesis most investigators stick to classical approaches. It might indeed be somewhat imprudent to add to the risks inherent in the synthesis of a long chain potentially hidden unknown factors of yet untried methods. In certain respects the synthesis of peptides is different from the synthesis of other organic compounds. The construction of a longer peptide chain involves the simulta­neous handling of numerous functional groups and this difficulty is often compounded by the high molecular weight of the target compounds. The ensuing technical problems, such as the lack of solubility in the commonly used solvents, the need to carry out bimolecular reactions at low concen­trations of the reactants and, last but not least, limitations in the analytical information available during synthesis, all warn the investigator to be cau­tious. Thus, most peptide chemists are conservative in their choice of ap­proach and probably rightly so.

Several examples selected for demonstration of well established methods result in compounds which are, by now, commercially available. Yet, the work

VIII Preface

of the peptide chemist may require materials which are not available from research supply houses, for instance derivatives of unusual amino acids. In such a case, the synthesis can follow the pattern used in the preparation of a trivial intermediate. Also, the methods described for small peptides are usually applicable, mutatis mutandis, in operations involving more complex materials as well. The examples assembled in this book should serve in preparative studies which are based on analogies. To render these examples more practical we followed the literature faithfully but not exactly. Amounts of starting materials were recalculated and expressed in molar ratios. The quantities of liquids are given both in weight and in volume. More impor­tantly, points which require comment are followed by footnotes and in these an attempt was made to put the extensive experience of the authors to good use. Hazards were pointed out to protect the uninitiated and pertinent references were added to assist those who wish to look deeper into the matter. We hope that our colleagues active in the field of peptide synthesis will consider "The practice of Peptide Synthesis" a worthwhile addition to the rich literature of peptide chemistry.

Cleveland, Ohio February, 1984

Preface to the Second Edition

MIKLOS BODANSZKY

AGNES BODANSZKY

Continued interest in The Practice of Peptide Synthesis prompted the publica­tion of this Second Edition. Revision of the First Edition was not limited to correction of errors. The book now contains several procedures, which have gained significance during recent years; for instance, additional blocking groups of the thiol function and novel coupling reagents. The author hopes that these changes enhance the usefulness of the volume for investigators active in the field of peptide synthesis.

Princeton, New Jersey May, 1994

M.B.

Contents

I Introduction . . . . 1

II Protecting Groups 7

1 Introduction of Amine Protecting Groups . 9 1.1 The p-Toluenesulfonyl Group . . . . . 9

p-Toluenesulfonyl-L-isoleucine . . . . . 9 1.2 Phthalyl (Phthaloyl, Pht) Amino Acids ..... . .. . 10

Phthalyl-L-Ieucine . . . . . . . . . . . . . . . . . . . . . . 10 1.3 The Benzyloxycarbonyl Group ....... . . . . . . . 11

Benzyloxycarbonyl-L-proline . . . . . . . 11 Benzyloxycarbonyl-L-Ieucine . . . . . . . . . . . . . . . . 11 Na-Benzyloxycarbonyl-L-arginine . . . . . . . . . . . . . 12 Benzyloxycarbonyl-L-aspartic Acid p-Benzyl Ester. . . 12

1.4 4-Methoxybenzyloxycarbonylamino Acids ....... 14 1.5 The tert-Butyloxycarbonyl (Boc) Group . . . . . . . . . 15

1.5.1 Introduction of the tert-Butyloxycarbonyl Group with 2-tert-Butyloxycarbonyloximino-2-phenylacetonitrile . . . . . . . . . . . . . . . . . . 15 tert-Butyloxycarbonyl-glycine . . . . . . . . . . . 16 tert-Butyloxycarbonyl-L-tryptophan . . . . . . . 16

1.5.2 Introduction of the tert-Butyloxycarbonyl Group with tert-Butyl Pyrocarbonate . . . .. . . .... 17

1.6 The Biphenylylisopropyloxycarbonyl (Bpoc) Group 18 Bpoc-L-Ieucine . . . . . . . . . . . . . . . . . . . . . . . . 18 Bpoc-L-Ieucine . . . . . . . . . . . . . . . . . . . . . . . . 19

1.7 The 9-Fluorenylmethyloxycarbonyl (Fmoc) Group. . . 20 9-Fluorenylmethyl Chlorocarbonate ..... 20 9-Fluorenylmethyloxycarbonyl-L-tryptophan . . . . . . 21

1.8 o-Nitrophenylsulfenylamino Acids .. . ... . ..... 21 1.9 2-Trimethylsilylethyloxycarbonylamino Acids. . . . . . 22

4-Nitrophenyl-2-Trimethylsilylethyl Carbonate . . . . . 22 2-Trimethylsilylethyloxycarbonyl-L-proline . . . . ... 23

1.10 Maleoylamino Acids and Maleoyl-Peptides . . . 24 N-Methoxycarbonylmaleimide .... . ... . ..... 24

X Contents

Maleoyl-glycine ........ 24 Maleoyl Peptides ....... 25

1.11 Triphenylmethyl-amino Acids 26 Trityl-L-Ieucine . . . . . . . . . 26 N -Trityl Amino Acids. . . . . . . . . . . . . . . . . . . . 27

2 Blocking of the IX-Carboxyl Group 28 2.1 Methyl Esters ........................ 28 2.2 Ethyl Esters ......................... 29

2.2.1 Esterification with the Help of Gaseous HCI . . . 29 L-Tyrosine Ethyl Ester . . . . . . . . . . . . . . . . 29

2.2.2 Esterification Catalyzed by p-Toluenesulfonic Acid 30 L-Methionine Ethyl Ester p-Toluenesulfonate. . . 30

2.3 Benzyl Esters. . . . . . . . . . . . . . . . . . . . . . . . . 30 Glycine Benzyl Ester p-Toluenesulfonate ........ 30 tert-Butyloxycarbonyl-L-asparagine Benzyl Ester. . . . 31 L-Glutamic Acid IX-Benzyl Ester. . 32 2.3.1 Polymeric Benzyl Esters . . . . . . . . 33

Hydroxymethyl-Polymer . . . . . . . . 33 tert-Butyloxycarbonyl-glycyl Resin. . 33 Benzyloxycarbonyl-L-isoleucyl-Polymer . . . . . . 34

2.4 4-Nitrobenzyl Esters .................... 34 L-Alanine-4-Nitrobenzyl Ester p-Toluenesulfonate . . . 34

2.5 4-Methoxybenzyl Esters of N"-Protected Amino Acids. . . 35 Benzyloxycarbonylglycine 4-Methoxybenzyl Ester . . . 35

2.6 Benzhydryl Esters (Diphenylmethyl Esters) . . . . . . . 36 2.7 Phthalimidomethyl Esters ............. 37 2.8 tert-Butyl Esters . . . . . . . . . . . . . . . . . . . . . . . 38

Addition of N'-Acylamino Acids to Isobutylene .. . . 38 L-Proline tert-Butyl Ester ................. 38 Transesterification with tert-Butyl Acetate. . . . . . . . 39 Esterification of Free Amino Acids through Acid Catalyzed Addition to Isobutylene .. . . . . . . . . . . 39 L-Tyrosine tert-Butyl Ester. . . . . . . . . 39

2.9 Phenacyl Esters ............... 40 Benzyloxycarbonylglycine Phenacyl Ester 40

2.10 2-Trimethylsilylethyl Esters ........ 41 2.11 4-Picolyl Esters ..................... 41

Benzyloxycarbonyl-L-phenylalanine 4-Picolyl Ester. . . 41 Benzyloxycarbonyl-L-valine 4-Picolyl Ester . . . . . . . 42

2.12 9-Fluorenylmethyl Esters. . . . . . . . . . 43 tert-Buty loxycarbon yl- L-phenylalanine 9-Fluorenylmethyl Ester .......... 43

2.13 2-Benzyloxyphenyl Esters ......... 44 L-Alanine 2-Benzyloxyphenyl Ester Hydrochloride. 44

Contents XI

2.14 Phenyl Esters. . . . . . . . . . . 45 2.15 Allyl Esters . . . . . . . . . . . . 45

3 Protection of Side Chain Functions. 47 3.1 Serine Ethers . . . . . . . . . . . 47

N -tert-Butyloxycarbonyl-O-benzyl-L-Serine Cyclohexylammonium Salt . 47

3.2 Ethers of Threonine . . . 48 O-tert-Butyl-L-threonine 48

3.3 Tyrosine Ethers ..... 50 O-Benzyl-L-tyrosine . . . 50

3.4 Acylation of the 8-Amino Group of Lysine 51 N'-Trifluoroacetyl-Iysine . . . . . . . . . . . 51 N'-p-Toluenesulfonyl-L-Iysine . . . . . . . . 52

3.5 Protection of the Guanidino Group of Arginine 53 Nitro-L-arginine . . . . . . . . . . . . . . . . . . . 53 N~-Benzyloxycarbonyl-NG-p-toluenesulfonyl-L-arginine 54 N"-Benzyloxycarbonyl-N°, NW-bis-(l-adamantyloxycarbonyl)-L-arginine . . . . . . . . . . . 56 N"- 4 -Methoxybenzyloxycarbonyl-NG -mesitylene-sulfonyl-L-arginine Cyclohexylammonium Salt . . . . . 57 NG -4 -Methoxy-2, 3,6-trimethylbenzenesulfonyl-L-arginine . . . . . . . . . . . . . . . . . . . . . . . . 57

3.6 Masking the Imidazole in Histidine ........ 59 3.6.1 Nim-p-Toluenesulfonyl-L-histidine ...... 59

N"-Benzyloxycarbonyl-N im -tosyl-L-histidine 59 3.6.2 N"-tert-Butyloxycarbonyl-Nn-benzyloxymethyl-L-

histidine . . . . . . . . . . . . . . . . . . . . . 60 3.6.3 Nim-Trityl-L-histidine . . . . . . . . . . . . . 62

3.7 Blocking of the Indole Nitrogen in Tryptophan. 62 Nim _ Formyl-L-tryptophan . . . . . . . . . . . . . 62

3.8 Protection of Side Chain Carboxyl Groups . . . 63 L-Aspartic Acid fJ-Benzyl Ester (fJ-Benzyl-L-aspartate) . 63 L-Glutamic Acid y-tert-Butyl Ester . . . . . . . . . . . . 64

3.9 Protection of the Carboxamide Function ........ 65 Introduction of the 4,4'-Dimethoxybenzhydryl Group. 65

3.10 Blocking the Sulfhydryl Group in Cysteine 66 S-Benzyl-L-cysteine ...... 66 S-Ethy1carbamoyl-L-cysteine . . . . . . . . . 67 S-Acetamidomethyl-L-cysteine . . . . . . . . 68 S-Triphenylmethyl-L-cysteine (S-Trityl-L-cysteine). 68 N -tert-Butyloxycarbonyl-S-(3-nitro-2-pyridinesulfenyl-L-cysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 S-tert-Butyl-L-cysteine hydrochloride. . . . 71

3.11 Protection of the Thioether in Methionine ....... 72

XII Contents

L-Methionine Sulfoxide 72 N -tert-Butyloxycarbonyl-S-methyl-L-methionine p-Nitrophenyl Ester p-Toluenesulfonate . . . . . . . . . 72

III Activation and Coupling. . . . . . 75

1 The Acid Chloride Procedure 77 p-Toluenesulfonyl-L-isoleucyl-glycine Ethyl Ester. 77 p-Toluenesulfonyl-L-isoleucyl-L-glutaminyl-L-asparagine. . . 77

2 The Azide Process . . . . . . . . . . . . . . . . . . . 79 2.1 Peptide Hydrazides Through Hydrazinolysis

of Peptide Esters . . . . . . . . . . . . . . . . . 79 N -Benzyloxycarbonyl-S-benzyl-L-cysteinyl-L-seryl-L-histidine Hydrazide. . . . . . . . . . . . . . . . . . . . . 79

2.2 Preparation of Peptide Hydrazides from Carboxylic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 tert -Butyloxycarbonyl-/3- benzyl-L-aspartyl-L-Ieucyl- L-N"-benzyloxycarbonyl-Iysine Hydrazide .. . . . . . . . 80

2.3 Protected Hydrazides . . . . . . . . . . . . . . 81 N -Benzyloxycarbonyl-L-prolyl-glycyl-N' -tert-butyloxycarbonyl-hydrazine. . . . . . . . . . . 81 N -Benzy loxycarbonyl-glycyl-glycyl-N' -tert -butyloxy-carbonyl-hydrazine. . . . . . . . . . . . . . . . . . . . . 81 Glycine Benzyloxycarbonylhydrazide Trifluoroacetate. 82

2.4 Conversion of Hydrazides to Azides and Coupling with Azides. . . . . . . . . . . . . . . . . . 83 2.4.1 Generation of the Azide with the Aid

of Sodium Nitrite and Coupling . . . . . . . . 83 Benzyloxycarbonyl-L-seryl-L-tyrosyl-L-seryl-L-me thionyl-}'-benzyl-L-glutamic Acid . . . . . . 83

2.4.2 Conversion of a Hydrazide to the Azide with the Aid of Butyl Nitrite and Acylation with the Azide "In Situ" . . . . . . . . . . . . . . . . . . . . . . . . 84 Benzyloxycarbonyl-L-tyrosyl-L-isoleucyl-L­glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-L-Ieucyl-glycinamide. . . . . . . . . . . . . . 84

2.4.3 Conversion of Protected Amino Acid or Peptide Hydrazides to the Azides Followed by Acylation with the Isolated Azide. . . . . . . . . . . . . . . . 85

3 Symmetrical Anhydrides. . . . . . . . . . . . . . . . . . . . . . 87 tert-Butyloxycarbonyl-L-phenylalanine Anhydride 87

4 Mixed Anhydrides ........................ 89

Contents XIII

4.1 The Isovaleric Acid Mixed Anhydride Method . . . . . 89 Benzyloxycarbonyl-L-leucyl-glycine Ethyl Ester. . . . . 89

4.2 Trimethylacetic Acid (Pivalic Acid) Mixed Anhydrides. 90 Benzyloxycarbonyl-rx-methylalanyl-rx-methylalanine Methyl Ester . . . . . . . . . . . . . . . . . . . . 90 N~-Benzyloxycarbonyl-Ne-p-toluenesulfonyl-L-

lysylglycine Ethyl Ester . . . . . . . . . . . . . . 91 4.3 The Ethyl Carbonate Mixed Anhydride Method 92

Benzyloxycarbonyl-L-leucyl-glycine Ethyl Ester. 92 4.4 Isobutylcarbonic Acid Mixed Anhydrides . . . . 93

Benzyloxycarbonyl-glycyl-L-phenylalanyl-glycine Ethyl Ester. . . . . . . . . . . . . . . . . 93

4.5 Coupling with the Aid of o-Phenylene Phosphorochloridite . . . . . . . . . . . 94 Preparation of N -Benzyloxycarbonyl-S-benzyl-L-cysteinyl­L-tyrosyl-L-isoleucyl-L-glutaminyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-L-leucyl-glycine Amide . . . . . . . . . 94

5 Preparation of Active Esters. . . . . . . . . . . . . . . . . . . . . 96 5.1 Cyanomethyl Esters . . . . . . . . . . . . . . . . . . . . . . 96

N-Benzyloxycarbonyl-S-benzyl-L-cysteine Cyanomethyl Ester 96 5.2 Preparation of p-Nitrophenyl Esters . . . . . . . . . . . 97

Benzyloxycarbonyl-L-phenylalanine p-Nitrophenyl Ester 97 5.3 o-Nitrophenyl Esters. . . . . . . . . . . . . . . . . . . . . 98

tert-Butyloxycarbonyl-glycine o-Nitrophenyl Ester. . . 98 5.4 2,4-Dinitrophenyl Esters. . . . . . . . . . . . . . . . . . 99

N -Benzyloxycarbonyl-L-threonine 2,4-Dinitrophenyl Ester. . . . . . . . . . . . . . 99

5.5 2,4,5-Trichlorophenyl Esters . . . . . . . . . . 100 5.6 Pentachlorophenyl Esters. . . . . . . . . . . 101

Benzyloxycarbonyl-glycyl-L-phenylalanine Pentachlorophenyl Esters. . . . . . . . . . . 101

5.7 Pentafluorophenyl Esters. . . . . . . . . . . 102 Benzyloxycarbonyl-glycyl-L-phenylalanine Pentafluorophenyl Ester. . . . . . . . . . . . 102 tert-Butyloxycarbonylamino Acid Pentafluorophenyl Esters. . . . . . . . . . . . . . . . . . . . . . . . . . . 103

5.8 N-Hydroxyphthalimide Esters. . . . . . . . . . . . . . 103 5.9 N-Hydroxysuccinimide Esters ............. " 104

tert-Butyloxycarbonyl-L-alanine N-Hydroxysuccinimide Ester. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 104

5.10 1-Hydroxypiperidine Esters ............... " 105 Benzyloxycarbonyl-L-Ieucine 1-Hydroxypiperidine Ester 105

5.11 Esters of 5-Chloro-8-Hydroxy-Quinoline. . . . . . . .. 106

XIV Contents

6 Coupling with Active Esters . . . . . . . . . . . 108 6.1 Coupling in Organic Solvents . . . . . . . 108

6.1.1 Acylation with Cyanomethyl Esters. 108 N -Benzyloxycarbonyl-S-benzyl-L-cysteinyl-L-tyrosine . . . . . . . . . . . . . . . . . . . . . . 108

6.1.2 Chain Lengthening with p-Nitrophenyl Esters. 109 Benzyloxycarbonyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-L-Ieucyl-glycinamide . . . . . .. 109

6.1.3 Acylation with N-Hydroxysuccinimide Esters .. , 110 tert-Butyloxycarbonyl-glycyl-L-phenylalanyl-glycine Ethyl Ester. . . . . . . . . . . . . . . . . 110 Benzyloxycarbonyl-L-phenylalanyl-L-tyrosine Ethyl Ester . . . . . . . . . . . . . . . . . . . . . 111

6.1.4 Coupling with 2,4-Dinitrophenyl Esters . . . . .. 112 Benzyloxycarbonyl-L-threonyl-L-alanyl-L-alanyl-L-alanine Ethyl Ester . . . . . . . . . . . . . . . .. 112

6.2 Coupling in Organic-Aqueous Media. . . . . . . . . ., 113 6.2.1 Acylation with p-Nitrophenyl Esters ....... , 113

Benzyloxycarbonyl-L-phenylalanyl-glycyl-L-proline. . . . . . . . . . . . . . . . . . . . . . . . .. 113

6.2.2 Acylation with N-Hydroxysuccinimide Esters. .. 114 Benzyloxycarbonyl-glycyl-L-proline . 114 Benzyloxycarbonyl-L-prolyl-glycine . 114

6.3 Catalysis of Active Ester Reactions . . . . 115 6.3.1 Catalysis with Imidazole . . . . . . . 115

Benzyloxycarbonyl-L-prolyl-L-tyrosine Methyl Ester. . . . . . . . . . . . . . . . . . . . . . . 115

6.3.2 Catalysis with 1-Hydroxybenzotriazole. . . . . 116 Benzyloxycarbonyl-L-Ieucyl-L-valinamide. . . . 116

6.3.3 Catalysis with 3-hydroxy-3,4-dihydro-quinazoline-4-one .......................... 116 Benzyloxycarbonyl-L-isoleucyl-glycinamide .... 116

7 Peptide Bond Formation with the Aid of Coupling Reagents 118 7.1 The Dicyclohexylcarbodiimide (DCC, DCCI) Method. 118

7.1.1 Coupling with DCC. . . . . . . . . . . . . . . . . 118 N -Phthaloyl-L-threonyl-L-phenylalanine Methyl Ester . . . . . . . . . . . . . . . . . . . . . . . . . . 118

7.1.2 Coupling with Carbodiimides in the Presence of 1-Hydroxybenzotriazole . . . . . . . . . . . . . 119 tert-Butyloxycarbonyl-L-Ieucyl-L-phenylalanine Methyl Ester . . . . . . . . . . . . . . . . . . . . . 119

7.1.3 Coupling with Dicyclohexylcarbodiimide in the Presence of N-Hydroxysuccinimide . . . . 120 Phthalyl-L-phenylalanyl-L-valyl-L-glutaminyl-L-

Contents XV

tryptophyl-L-leucyl-L-methionyl-L-asparaginyl-O-tert-butyl-L-threonine tert-Butyl Ester. . . . . 120 N -Benzyloxycarbonyl-O-tert-butyl-L-tyrosyl-L-leucyl-fi-tert-butyl-L-aspartic Acid Methyl Ester. 121

7.2 Coupling with 1-Isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ) . . . . . . . . . . . . . . .. 121 Benzyloxycarbonyl-L-alanyl-L-tyrosine Methyl Ester.. 121

7.3 The Carbonyldiimidazole Method. . . . . . . . . 122 Benzyloxycarbonyl-glycyl-L-tyrosine Ethyl Ester 122

7.4 Coupling with N -Ethyl-5-phenylisoxazolium-3'-sulfonate. . . . . . . . . . . . . . . . . . . . . . . . 123 N", Ne-Dibenzyloxycarbonyl-L-lysyl-glycine Ethyl Ester 123

7.5 Coupling with 1-Benzotriazolyl-tri-dimethylamino-phosphonium Hexafluorophosphate (BOP-Reagent) 124

7.6 O-Benzotriazolyl-tetramethyluronium Hexafluorophosphate . . . . . . . . . . . . . . . . . . 125 Preparation of the HBTU-Reagent ....... 125 Coupling with HBTU . . . . . . . . . . . . . . . . . . .. 125

IV Removal of Protecting Groups. 127

1 Hydrogenation . . . . . . . . 129 1.1 Hydrogenolysis of Benzyl Esters, Benzyl Ethers

and of the Benzyloxycarbonyl Group. . . . . . . 129 L-Prolyl-L-leucyl-glycinamide ........... 129

1.2 Catalytic Hydrogenation of Methionine Containing Pep tides . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Benzyloxycarbonyl-glycyl-L-tryptophyl-L-methionyl-fi-tert-butyl-L-aspartyl-L-phenylalanine Amide. . . 131

1.3 Transfer Hydrogenation with l,4-Cyclohexadiene as Hydrogen Donor. . . . . . . . . . . . . . . . . 133 Ne-tert-Butyloxycarbonyl-L-lysyl-O-tert-butyl-L-threonine Methyl Ester . . . . . . . . . . 133

2 Reduction with Sodium in Liquid Ammonia. . . . . . 135 L-Glutaminyl-L-asparagine . . . . . . . . . . . . . . . . 135

3 Removal of the Phthalyl (Phthaloyl) Group by Hydrazinolysis 137 Glycyl-glycine. . . . . . . . . . . . . . . . . . . . . . .. 137

4 Acidolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 138 4.1 Hydrobromic Acid in Acetic Acid. . . . . . . . . . . .. 138

Glycyl-L-phenylalanine Benzyl Ester Hydrobromide .. 138 S-Benzyl-L-cysteinyl-L-prolyl-L-leucyl-glycinamide 138

4.2 Hydrobromic Acid in Trifluoroacetic Acid. . 140 cx-Melanotropin. . . . . . . . . . . . . . . . . . . . . 140

XVI Contents

4.3 Hydrochloric Acid in Acetic Acid. 141 Nitro-L-arginyl-copolymer . . . . . 141

4.4 Triftuoroacetic Acid . . . . . . . . . 142 L-Methionyl-L-glutaminyl-L-histid yl- L-phenylalanyl-L-arginyl-L-tryptophyl-glycinamide Acetate. 142

4.5 Removal of Protecting Groups with Triftuoroacetic Acid-Thioanisole. 144 Porcine Vasoactive Intestinal Peptide. 144

4.6 Liquid Hydrogen Fluoride 145 Oxytocin. . . . . . . . . . . . . . . . . . 145

5 Hydrolysis. . . . . . . . . . . . . . . . . . . . 148 5.1 Base Catalyzed Hydrolysis of Alkyl Esters

(Saponification with Alkali). . . . . . . . . . 148 N -Benzyloxycarbonyl-S-benzyl-L-cysteinyl-L-tyrosine. 148

5.2 Acid Catalyzed Hydrolysis . . . 149 Phthalyl-L-phenylalanyl-glycine . . . . . . . . . . . . . 149

5.3 Enzyme Catalyzed Hydrolysis . . . . . . . . . . . . . . 150 5.3.1 Enzymatic Removal of the Phenylacetyl Group

from the e-Amino Group of Lysine Residues. . . 150 Partial Deprotection of a Dipeptide Derivative. 150 Desamino Lysine Vasopressin . . . . 150

5.3.2 Chymotrypsin Catalyzed Hydrolysis of Alkyl Esters . . . . . . . . . . . . . 151 L-Prolyl-L-phenylalanine . . . . . . . 151 Benzyloxycarbonyl-L-valyl-L-tyrosine. 152

6 Base Catalyzed Elimination. . . . . . . . . . . . . . 153 6.1 Removal of the 9-Fluorenylmethyloxycarbonyl (Fmoc)

Group. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 153 9-Fluorenylmethyloxycarbonyl-L-asparaginyl-S-benzyl-L-cysteinyl-L-prolyl-L-tryptophyl-glycinamide . . . . .. 153

6.2 Cleavage of 9-Fluorenylmethyl Esters. . 155 L-Leucyl-L-phenylalanine . . . . . . . . . . . . . . . . .. 155

7 Nucleophilic Displacement . . . . . . . . . . . . . . . . . . . 156 7.1 Removal of the o-Nitrobenzenesulfonyl (Nps) Group.. 156

N'-tert-butyloxycarbonyl-L-lysyl-L-leucyl-L-pheny lalan yl-N' -tert -buty loxycar bon yl-L-lysy 1-N' -tert-butyloxycarbonyl-L-lysine. . . . . . . . . . . . . . . . .. 156

7.2 Cleavage of 2-Trimethylsilylethyl Esters with Fluorides 157 N-Benzyloxycarbonyl-O-tert-butyl-L-threonine . . . .. 157

8 Iodolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 Removal of the S-Trityl Group by Iodolysis with Concomitant Oxidation to the Disulfide. . . . . . . . . . . 159

9 Reduction of Methionine Sulfoxide Containing Peptides. 161 L- Prolyl-L-tyrosyl-N'-tosyl-L-lysyl-L-methionine. . . . . . . 161

Contents XVII

V Special Procedures. . . . . 163

1 Ammonolysis of Esters. 165 1.1 Conversion of a Peptide Ethyl Ester

to the Corresponding Amide. . . . . . 165 Benzyloxycarbonyl-L-prolyl-L-Ieucyl-glycinamide . . .. 165

1.2 Ammonolysis of Active Esters . . . . . . . 166 Benzyloxycarbonyl-L-asparagine amide. . . . 166

2 Transesterification . . . . . . . . . . . . . . . . . . . 167 2.1 Cleavage of the Ester Bond Between Peptide

and Polymeric Support by Transesterification Followed by Hydrolysis. . . . . . . . . . . . . 167 Benzyloxycarbonyl-L-alanyl-L-phenylalanine. 167

3 Cyclization. . . . . . . . . . . . . . . . . . . . 169 3.1 Cyclization Through the Formation

of a Disulfide Bond . . . . . . . . . . . 169 Desamino-oxytocin. . . . . . . . . . . . 169

3.2 Ring Closure Through an Amide Bond. 170 Ditosyl-gramicidin S. . . . . . . 170 Desthiomalformin . . . . . . . . 171

4 Polycondensation. . . . . . . . . . . . 173 4.1 N -Carboxy anhydrides . . . . . . 173

N"-Benzyloxycarbonyl-N~-carboxy-L-Iysine Anhydride. 173 4.2 Poly-N"-benzyloxycarbonyl-L-Iysine............ 174 4.3 Synthesis of Sequential Peptides by Polycondensation. 175

Poly (L-Iysyl-L-alanyl-L-alanine) Trifluoroacetate . . .. 175 5 Partial Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . 178

Human Insulin. . . . . . . . . . . . . . . . . . . . . . . 178 Zinc Free Insulin. . . . . . . . . . . . . . . . . . . . . . 178 NA1 , NB1 -Di-tert-butyloxycarbonyl-B(23-30)-desocta-peptide Insulin, (Boch-DOL . . . . . . . . . . 179 Human Insulin Through Condensation with Dicyclohexylcarbodiimide. . . . . . . . . . . . 179 Human Insulin via Enzyme-Catalyzed Coupling 179

VI Models for the Study of Racemization 181

1 Benzoyl-Ieucyl-glycine Ethyl Ester. 183 2 The Anderson-Callahan Test. . . . 184

Benzyloxycarbonyl-glycyl-L-phenylalanyl-glycine Ethyl Ester 184 3 Acylation with Acetyl-L-isoleucine. 186

Acetyl-L-isoleucine . . . . . . . . . . . . . . . . . . . . . . . .. 186

XVIII Contents

Acetyl-L-isoleucyl-glycine Ethyl Ester and Acetyl-D-alloisoleucyl-glycine Ethyl Ester .......... .

4 The lzumiya-Muraoka Model ............ . Glycyl-alanyl-leucine. . . . . . . . . . ........ .

5 Synthesis and Enantioselective Enzymic Hydrolysis of Tetraalanine . . . . . . . . . . . . . . . . ..... .

186 189 189

191

VII Reagents for Peptide Synthesis . . . . . . . . . . . . 193

1 tert-Butyl Azidoformate . . . . 195 tert-Butyl Phenyl Carbonate. 195 tert-Butyl Carbazate . . . . . . 195 tert-Butyl Azidoformate . . . . 196

2 1-Adamantyl Chlorocarbonate . 197 3 1-Isobutyloxycarbonyl-2-isobutyloxy-1,2-dihydroquinoline

(IIDQ) . . . . . .. ...................... 198 4 Diphenyl Phosphorazidate (Diphenylphosphoryl Azide,

DPPA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 5 3-H ydroxy-3,4-dihydroq uinazoline-4-one

(4-Hydroxyquinazoline 3-oxide) . . . . . . 200 2-Aminobenzhydroxamic Acid. . . . . . . 200 3-Hydroxy-3,4-dihydroquinazoline-4-one. 200

6 1-Hydroxybenzotriazole. . . . . . . . . . . 201 7 Ethyl 2-hydroximino-2-cyanoacetate . .. .......... 202 8 1-Guanyl-3,5-dimethyl-pyrazole Nitrate ........... " 203

VIII Appendix . . . . . . . . . . . . . . . . . . . . . . .

1 Conversion of Dicyc1ohexylammonium Salts of Protected Amino Acids to the Free Acids. N -0-Nitrobenzenesulfonyl-O-tert-butyl-L-threonine N -hydroxysuccinimide Ester ... .

2 Preparation ~f Analytical Samples ....... . 2.1 Elemental Analysis. . . ....... . 2.2 Amino Acid Analysis 2.3 NMR Spectra. . ...

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

205

207

207 208 208 208 210

211