www.pdfgrip.com


Edited by
Andrew B. Hughes
Amino Acids, Peptides and
Proteins in Organic
Chemistry

www.pdfgrip.com


Further Reading
Drauz, K., Gröger, H., May, O. (eds.)

Lutz, S., Bornscheuer, U. T. (eds.)

Enzyme Catalysis in Organic
Synthesis

Protein Engineering Handbook

Third completely revised
and enlarged edition

2009

2 Volume Set
ISBN: 978-3-527-31850-6

3 Volumes

Sewald, N., Jakubke, H.-D.

2011
ISBN: 978-3-527-32547-4

Fessner, W.-D., Anthonsen, T.

Peptides: Chemistry
and Biology
2009

Modern Biocatalysis
Stereoselective and Environmentally
Friendly Reactions
2009

ISBN: 978-3-527-31867-4

Jakubke, H.-D., Sewald, N.

Peptides from A to Z

ISBN: 978-3-527-32071-4

A Concise Encyclopedia
Pignataro, B. (ed.)

2008


Ideas in Chemistry and
Molecular Sciences

ISBN: 978-3-527-31722-6

Royer, J. (ed.)

Advances in Synthetic Chemistry

Asymmetric Synthesis of
Nitrogen Heterocycles

2010
ISBN: 978-3-527-32539-9

2009

Reek, J. N. H., Otto, S.

ISBN: 978-3-527-32036-3

Dynamic Combinatorial
Chemistry

Hecht, S., Huc, I. (eds.)

2010

Foldamers


ISBN: 978-3-527-32122-3

Structure, Properties, and Applications
2007
ISBN: 978-3-527-31563-5

www.pdfgrip.com


Edited by
Andrew B. Hughes

Amino Acids, Peptides and Proteins
in Organic Chemistry
Volume 3 - Building Blocks, Catalysis and
Coupling Chemistry

www.pdfgrip.com


The Editor
Andrew B. Hughes
La Trobe University
Department of Chemistry
Victoria 3086
Australia

All books published by Wiley-VCH are carefully
produced. Nevertheless, authors, editors, and
publisher do not warrant the information contained

in these books, including this book, to be free of
errors. Readers are advised to keep in mind that
statements, data, illustrations, procedural details or
other items may inadvertently be inaccurate.
Library of Congress Card No.: applied for
British Library Cataloguing-in-Publication Data
A catalogue record for this book is available from the
British Library.
Bibliographic information published by
the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this
publication in the Deutsche Nationalbibliografie;
detailed bibliographic data are available on the
Internet at .
# 2011 WILEY-VCH Verlag & Co. KGaA,
Boschstr. 12, 69469 Weinheim, Germany
All rights reserved (including those of translation into
other languages). No part of this book may be
reproduced in any form – by photoprinting,
microfilm, or any other means – nor transmitted or
translated into a machine language without written
permission from the publishers. Registered names,
trademarks, etc. used in this book, even when not
specifically marked as such, are not to be considered
unprotected by law.
Composition Thomson Digital, Noida, India
Printing and Bookbinding betz-druck GmbH,
Darmstadt
Cover Design Schulz Grafik Design, Fgưnheim
Printed in the Federal Republic of Germany

Printed on acid-free paper
ISBN: 978-3-527-32102-5

www.pdfgrip.com


V

Contents
List of Contributors

XVII

Part One Amino Acids as Building Blocks
1
1.1
1.2
1.2.1
1.3
1.3.1
1.3.2
1.3.3
1.4
1.4.1
1.4.2
1.4.3
1.4.3.1
1.4.3.2
1.4.3.3
1.5

1.5.1

1

Amino Acid Biosynthesis 3
Emily J. Parker and Andrew J. Pratt
Introduction 3
Glutamate and Glutamine: Gateways to Amino Acid Biosynthesis 5
Case Study: GOGAT: GATs and Multifunctional Enzymes
in Amino Acid Biosynthesis 6
Other Amino Acids from Ubiquitous Metabolites: Pyridoxal
Phosphate-Dependent Routes to Aspartate, Alanine, and Glycine 8
Pyridoxal Phosphate: A Critical Cofactor of Amino Acid Metabolism 8
Case Study: Dual Substrate Specificity of Families
of Aminotransferase Enzymes 10
PLP and the Biosynthesis of Alanine and Glycine 15
Routes to Functionalized Three-Carbon Amino Acids: Serine,
Cysteine, and Selenocysteine 16
Serine Biosynthesis 16
Cysteine Biosynthesis 18
Case Study: Genome Information as a Starting Point for Uncovering
New Biosynthetic Pathways 19
Cysteine Biosynthesis in Mycobacterium Tuberculosis 19
Cysteine Biosynthesis in Archaea 20
RNA-Dependent Biosynthesis of Selenocysteine and Other
Amino Acids 21
Other Amino Acids from Aspartate and Glutamate: Asparagine
and Side Chain Functional Group Manipulation 22
Asparagine Biosynthesis 23


Amino Acids, Peptides and Proteins in Organic Chemistry.
Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes
Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-32102-5

www.pdfgrip.com


VI

Contents

1.6
1.6.1
1.6.2
1.6.2.1
1.6.2.2
1.6.2.3
1.6.2.4
1.6.2.5
1.6.3
1.7
1.7.1
1.7.2
1.7.3
1.7.4
1.7.4.1
1.7.4.2
1.7.4.3
1.8

1.8.1
1.8.2
1.8.3
1.8.3.1
1.8.3.2
1.8.4
1.9

2
2.1
2.2
2.2.1
2.2.2
2.2.3
2.2.4
2.2.5
2.2.6
2.3
2.3.1
2.3.2
2.3.3

Aspartate and Glutamate Families of Amino Acids 25
Overview 25
Aspartate Family Amino Acids: Threonine and Methionine 25
Case Study: Evolution of Leaving Group Specificity in Methionine
Biosynthesis 28
Threonine, Homocysteine, and PLP 30
Threonine Synthase 30
Methionine, Cysteine, and Cystathionine 32

Methionine Synthase 33
Glutamate Family Amino Acids: Proline and Arginine 33
Biosynthesis of Aliphatic Amino Acids with Modified Carbon Skeletons:
Branched-Chain Amino Acids, Lysine, and Pyrrolysine 37
Overview 37
Valine and Isoleucine 37
Homologation of a-Keto Acids, and the Biosynthesis of Leucine
and a-Aminoadipic Acid 41
Biosynthesis of Lysine: A Special Case 44
Diaminopimelate Pathway to Lysine 44
a-Aminoadipic Acid Pathways to Lysine 45
Pyrrolysine 47
Biosynthesis of the Aromatic Amino Acids 49
Shikimate Pathway 49
Case Study: Alternative Synthesis of Dehydroquinate in Archaea 53
Biosynthesis of Tryptophan, Phenylalanine, and Tyrosine from
Chorismate 58
Tryptophan Biosynthesis 58
Phenylalanine and Tyrosine Biosynthesis 59
Histidine Biosynthesis 61
Conclusions 64
References 65
Heterocycles from Amino Acids 83
M. Isabel Calaza and Carlos Cativiela
Introduction 83
Heterocycles Generated by Intramolecular Cyclizations 83
a-Lactones and a-Lactams 83
Indolines 84
Aziridinecarboxylic Acids and Oxetanones 86
b-Lactams and Pyroglutamic Acid Derivatives 87

Amino Lactams and Amino Anhydrides 88
Azacycloalkanecarboxylic Acids 89
Heterocycles Generated by Intermolecular Cyclizations 89
Metal Complexes 89
a-Amino Acid N-Carboxyanhydrides and Hydantoins 90
Oxazolidinones and Imidazolidinones 91

www.pdfgrip.com


Contents

2.3.4
2.3.5
2.3.6
2.3.7
2.3.8
2.3.9
2.4
2.5
2.6
2.6.1
2.6.2
2.6.3
2.6.4
2.6.5
2.6.6
2.6.7
2.6.8
2.6.9

2.6.10
2.6.11
2.6.12
2.6.13
2.6.14
2.6.15

3
3.1
3.2
3.2.1
3.2.2

Oxazolones 93
Oxazinones and Morpholinones, Pyrazinones
and Diketopiperazines 94
Tetrahydroisoquinolines and b-Carbolines 96
Oxazo/Thiazolidinones, Oxazo/Thiazolidines,
and Oxazo/Thiazolines 97
Sulfamidates 101
Tetrahydropyrimidinones 102
Heterocycles Generated by Cycloadditions 102
Conclusions 104
Experimental Procedures 104
Synthesis of 1-tert-Butyl-3-phenylaziridinone (5) 104
Synthesis of Dimethyl (2S,3aR,8aS)-1,2,3,3a,8,8aHexahydropyrrolo[2,3-b]indole-1,2-dicarboxylate (Precursor of 10) 105
Synthesis of Benzyl (R)-1-Tritylaziridine-2-carboxylate (15) 105
Synthesis of (S)-N-tert-Butoxycarbonyl-3-aminooxetan-2-one (18) 106
Synthesis of (S)-1-(tert-Butyldimethylsilyl)-4-oxoazetidine2-carboxylic Acid (24) 106
Synthesis of 9H-Fluoren-9-ylmethyl (R)-Hexahydro-2-oxo-1H-azepin3-yl Carbamate (31) 106

Synthesis of Ethyl (S)-N-(tert-Butoxycarbonyl)-a(tert-butoxymethyl)proline Ester (36) 107
Synthesis of Proline N-Carboxyanhydride (49) 107
Synthesis of (2S,4S)-2-Ferrocenyl-3-pivaloyl-4-methyl-1,3oxazolidin-5-one (54b) 107
Synthesis of (6S)-6-Isopropyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2one (71) 108
Synthesis of (3S,6R)-6-Isopropyl-3-methyl-5-phenyl-1,2,3,6tetrahydro-2-pyrazinone (78) 108
Synthesis of (3S)-3,6-Dihydro-2,5-dimethoxy-3isopropylpyrazine (85) 109
Synthesis of (3S)-1,2,3,4-Tetrahydroisoquinoline3-carboxylic Acid (87) 110
Synthesis of Methyl (S)-N-tert-Butoxycarbonyl-2,2-dimethyloxazolidine4-carboxylate (109) 110
Synthesis of (2S,6S)-2-tert-Butyl-1-carbobenzoxy-4-oxopyrimidin-6carboxylic Acid (126) 111
References 111
Radical-Mediated Synthesis of a-Amino Acids and Peptides
Jan Deska
Introduction 115
Free Radical Reactions 115
Hydrogen Atom Transfer Reactions 116
Functional Group Transformations 121

www.pdfgrip.com

115

VII


VIII

Contents

3.3
3.3.1

3.3.2
3.3.3
3.3.4
3.4
3.5
3.6
3.6.1

3.6.2
3.6.3

3.6.4
3.6.5

4

4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.7.1
4.7.2
4.7.3
4.7.3.1
4.7.3.2
4.7.4
4.7.5

4.7.5.1
4.7.5.2
4.7.5.3

Radical Addition to Imine Derivatives 124
Glyoxylate Imines as Radical Acceptors 125
Oximes and Hydrazones as Radical Acceptors 126
Nitrones as Radical Acceptors 129
Isocyanates as Radical Acceptors 130
Radical Conjugate Addition 130
Conclusions 135
Experimental Protocols 135
Preparation of ((1R,2S,5R)-5-methyl-2-(1-methyl-lphenylethyl)cyclohexyl 2-[(tert-butoxycarbonyl)amino]-4-methylpent-4-enoate) (7) 135
Synthesis of (2S)-3-{(1R,2S)-2-[(N-bis-Boc)amino]-1-cyclopropyl}-2benzyloxycarbonylamino-propionic Acid Methyl Ester (26) 136
Synthesis of (3aR,6S,7aS)-hexahydro-8,8-dimethyl-1-[(2R)-3,3dimethyl-1-oxo-2-(2,2-diphenylhydrazino)butyl]-3H-3a,6-methano-2,1benzisothiazole 2,2-dioxide (42) 136
Synthesis of N-(2,6-diphenyl-methylpiperidine-2carboxamide (59) 137
Synthesis of Methyl 2-(2-naphthylcarbonylamino)pentanoate
(80) 136
References 138
Synthesis of b-Lactams (Cephalosporins) by Bioconversion 143
José Luis Barredo, Marta Rodriguez- Sáiz, José Luis Adrio,
and Arnold L. Demain
Introduction 143
Biosynthetic Pathways of Cephalosporins and Penicillins 146
Production of 7-ACA by A. chrysogenum 147
Production of 7-ADCA by A. chrysogenum 149
Production of Penicillin G by A. chrysogenum 151
Production of Cephalosporins by P. chrysogenum 152
Conversion of Penicillin G and other Penicillins to DAOG
by Streptomyces clavuligerus 153

Expandase Proteins and Genes 153
Bioconversion of Penicillin G to DAOG 155
Broadening the Substrate Specificity of Expandase 155
Resting Cells 155
Cell-Free Extracts 156
Inactivation of Expandase during the Ring-Expansion Reaction 157
Further Improvements in the Bioconversion of Penicillin
G to DAOG 158
Stimulatory Effect of Growth in Ethanol 158
Use of Immobilized Cells 159
Elimination of Agitation and Addition of Water-Immiscible
Solvents 159

www.pdfgrip.com


Contents

4.7.5.4
4.7.5.5
4.8

Addition of Catalase 160
Recombinant S. clavuligerus Expandases
Conclusions 162
References 163

5

Structure and Reactivity of b-Lactams 169

Michael I. Page
Introduction 169
Structure 170
Reactivity 174
Hydrolysis 176
Base Hydrolysis 176
Acid Hydrolysis 178
Spontaneous Hydrolysis 180
Buffer-Catalyzed Hydrolysis 180
Metal Ion-Catalyzed Hydrolysis 180
Micelle-Catalyzed Hydrolysis of Penicillins 182
Cycloheptaamylose-Catalyzed Hydrolysis 184
Enzyme-Catalyzed Hydrolysis 185
Serine b-Lactamases 185
Metallo b-Lactamases 187
Aminolysis 191
Epimerization 195
References 195

5.1
5.2
5.3
5.4
5.4.1
5.4.2
5.4.3
5.4.4
5.4.5
5.4.6
5.4.7

5.4.8
5.4.8.1
5.4.8.2
5.5
5.6

160

Part Two Amino Acid Coupling Chemistry 201
6
6.1
6.2
6.2.1
6.2.1.1
6.2.1.2
6.2.1.3
6.2.1.4
6.2.1.5
6.2.1.6
6.2.2
6.2.2.1
6.2.2.2
6.2.2.3
6.2.2.4
6.2.2.5
6.2.3

Solution-Phase Peptide Synthesis 203
Yuko Tsuda and Yoshio Okada
Principle of Peptide Synthesis 203

Protection Procedures 205
Amino Group Protection 205
Z Group 205
Substituted Z and other Urethane-Type Protecting Groups 207
Boc Group 207
Tri Group 208
Fmoc Group 209
Other Representative Protecting Groups 211
Carboxyl Group Protection 212
Methyl Ester (-OMe) and Ethyl Ester (-OEt) 213
Benzyl Ester (-OBzl) 213
tBu Ester (-OtBu) 213
Phenacyl Ester (-OPac) 214
Hydrazides 214
Side-Chain Protection 215

www.pdfgrip.com

IX


X

Contents

6.2.3.1
6.2.3.2
6.2.3.3
6.2.3.4
6.2.3.5

6.2.3.6
6.2.3.7
6.2.3.8
6.2.3.9
6.3
6.3.1
6.3.1.1
6.3.1.2
6.3.1.3
6.3.1.4
6.3.2
6.3.2.1
6.3.2.2
6.3.2.3
6.4
6.4.1
6.4.2
6.4.3
6.4.4
6.4.5

e-Amino Function of Lys (d-Amino Function of Orn) 215
b-Mercapto Function of Cys 216
b- and c-Carboxyl Functions of Asp and Glu 217
Protecting Groups for the c-Carboxyl Function of Glu 219
d-Guanidino Function of Arg 219
Phenolic Hydroxy Function of Tyr 221
Aliphatic Hydroxyl Function of Ser and Thr 222
Imidazole Nitrogen of His 222
Indole Nitrogen of Trp 223

Chain Elongation Procedures 223
Methods of Activation in Stepwise Elongation 223
Carbodiimides 223
Mixed Anhydride Method 224
Active Esters 225
Phosphonium and Uronium Reagents 227
Methods of Activation in Segment Condensation 229
Azide Procedure 229
Carbodiimides in the Presence of Additives 230
Native Chemical Ligation 231
Final Deprotection Methods 232
Final Deprotection by Catalytic Hydrogenolysis 233
Final Deprotection by Sodium in Liquid Ammonia 233
Final Deprotection by TFA 233
Final Deprotection by HF 233
Final Deprotection by HSAB Procedure 234
References 234

7

Solid-Phase Peptide Synthesis: Historical Aspects 253
Garland R. Marshall
Introduction 253
Selection of Compatible Synthetic Components 253
Racemization and Stepwise Peptide Assembly 257
Optimization of Synthetic Components 258
Foreshadowing of the Nobel Prize 258
Automation of SPPS 260
Impact of New Protecting Groups and Resin Linkages 261
Solid-Phase Organic Chemistry 262

Early Applications of SPPS to Small Proteins 263
Side-Reactions and Sequence-Dependent Problems 264
Rapid Expansion of Usage Leading to the Nobel Prize 265
From the Nobel Prize Forward to Combinatorial
Chemistry 267
Protein Synthesis and Peptide Ligation 268
Conclusions 269
References 270

7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
7.10
7.11
7.12
7.13
7.14

www.pdfgrip.com


Contents

8

8.1
8.1.1
8.1.2
8.1.3
8.2
8.2.1
8.2.1.1
8.2.1.2
8.2.1.3
8.2.2
8.2.3
8.2.4
8.2.5
8.3
8.4
8.4.1
8.4.2
8.4.3
8.4.4
8.4.5
8.4.6
8.5
8.5.1
8.5.2
8.5.2.1
8.5.2.2
8.5.2.3
8.5.3
8.5.4
8.5.5

8.6

9

9.1
9.1.1
9.1.2
9.1.3
9.1.4
9.1.5

Linkers for Solid-Phase Peptide Synthesis 273
Miroslav Soural, Jan Hlavá4c, and Viktor Krch4nák
Introduction 273
Immobilization Strategies 275
Overview of Linker Types 276
Selection of a Linker 277
Immobilization via Carboxyl Group 279
Esters 280
Hydroxy Linkers for Preparation of Resin-Bound Esters 281
Electrophilic Linkers for Preparation of Resin-Bound Esters 282
Cleavage from the Resin 282
Amides 288
Hydrazides 291
Oximes 291
Thioesters 292
Immobilization via Amino Group 294
Backbone Immobilization 296
Benzaldehyde-Based Linkers 298
Indole Aldehyde Linkers 299

Naphthalene Aldehyde Linkers (NALs) 299
Thiophene Aldehyde Linkers (T-BALs) 300
Safety-Catch Aldehyde Linkers 300
Photolabile Aldehyde Linker (PhoB) 300
Immobilization via Amino Acid Side-Chain 300
Carboxyl Group 301
Amino and Other Nitrogen-Containing Groups 302
Lys 302
His 302
Arg 303
Hydroxy Group 303
Sulfanyl Group 304
Aromatic Ring 305
Conclusions 306
References 306
Orthogonal Protecting Groups and Side-Reactions in Fmoc/tBu
Solid-Phase Peptide Synthesis 313
Stefano Carganico and Anna Maria Papini
Orthogonal Protecting Groups in Fmoc/tBu Solid-Phase Peptide
Synthesis 313
Arg 313
Asn and Gln 315
Asp and Glu 316
Cys 318
His 323

www.pdfgrip.com

XI



XII

Contents

9.1.6
9.1.7
9.1.8
9.1.9
9.1.10
9.1.11
9.2
9.2.1
9.2.2
9.2.3
9.2.4
9.2.5
9.2.6
9.2.7
9.2.8
9.2.9
9.2.10
9.2.11

10

10.1
10.2
10.2.1
10.2.2

10.2.2.1
10.2.2.2
10.2.2.3
10.2.3
10.3
10.3.1
10.3.2
10.3.3
10.3.3.1
10.3.3.2
10.3.3.3
10.3.3.4
10.3.3.5
10.3.3.6
10.3.3.7
10.3.3.8

Lys 324
Met 327
Ser and Thr 327
Trp 328
Tyr 329
Conclusions 330
Side-Reactions in Fmoc/tBu Solid-Phase Peptide Synthesis 330
Imidazole Ring-Mediated Racemization of Chiral a-Carbon 332
Hydroxyl-Mediated O ! N Acyl Transfer 332
Met Oxidation to Methionyl Sulfoxide 334
Dehydration of Asn and Gln Amide Side-Chain 334
Aspartimide Formation 336
Formation of Diketopiperazines 337

Side-Reactions Affecting Protected Cys 338
Deletion Peptides, Truncated Sequences, and Multiple
Additions 338
Uronium/Guanidinium Salts-Induced Guanidino
Capping 340
Arg Cyclization and Arg Conversion into Orn 341
Conclusions 342
References 343
Fmoc Methodology: Cleavage from the Resin
and Final Deprotection 349
Fernando Albericio, Judit Tulla-Puche, and Steven A. Kates
Introduction 349
‘‘Low’’ TFA-Labile Resins 351
Cleavage 351
Choice of Resin for the Preparation of Peptide Acids 352
CTC Resin 353
SASRIN Resin 355
Bromide Resin 356
Final Deprotection 356
‘‘High’’ TFA-Labile Resins 356
Cleavage 357
Final Deprotection of Protected Peptides in Solution 359
Side-Reactions 360
Linker/Resin 360
Trp and Tyr Modification 361
Sulfur-Containing Residues: Cys and Met 362
Ser and Thr, N ! O Migration 363
Asp and Asn 363
Arg 364
N-Alkylamino Acids 365

Work-Up 366

www.pdfgrip.com


Contents

10.4

Final Remarks 366
References 366

11

Strategy in Solid-Phase Peptide Synthesis 371
Kleomenis Barlos and Knut Adermann
Synthetic Strategies Utilizing Solid-Phase Peptide Synthesis
Methods 371
Solid Support: Resins and Linkers 373
Developing the Synthetic Strategy: Selection of the Protecting
Group Scheme 374
Resin Loading 376
SBS Peptide Chain Elongation: Coupling and Activation 377
Piperazine Formation 378
Solid-Phase Synthesis of Protected Peptide Segments 379
Fragment Condensation Approach: Convergent and Hybrid
Syntheses 379
Cleavage from the Resin and Global Peptide Deprotection 382
Disulfide Bond-Containing Peptides 384
Native Chemical Ligation (NCL) 386

SPPS of Peptides Modified at their C-Terminus 388
Side-Chain-Modified Peptides 390
Cyclic Peptides 392
Large-Scale Solid-Phase Synthesis 394
Conclusions 395
References 396

11.1
11.2
11.3
11.4
11.5
11.6
11.7
11.8
11.9
11.10
11.11
11.12
11.13
11.14
11.15
11.16

12
12.1
12.2
12.2.1
12.2.1.1
12.2.2

12.3
12.3.1
12.3.2
12.4
12.4.1
12.4.2
12.4.3
12.4.4

Peptide-Coupling Reagents 407
Ayman El- Faham and Fernando Albericio
Introduction 407
Carbodiimides 409
General Procedure for Coupling Using Carbodiimide and HOXt;
Solution Phase 413
General Procedure for Solid-Phase Coupling via Carbodiimide
Activation 414
Loading of Wang Resin Using Carbodiimide 415
Phosphonium Salts 416
Preparation of Phosphonium Salts 418
General Method for the Synthesis of Phosphonium Salts 420
Aminium/Uronium Salts 420
Stability of Onium Salts 425
General Procedure for the Preparation of Chloroformamidinium
Salts 426
Synthesis of Aminium/Uronium Salts 427
General Procedure for Coupling Using Onium Salts
(Phosphonium and Uronium) in Solution Phase 427

www.pdfgrip.com


XIII


XIV

Contents

12.4.5
12.4.6

12.5
12.5.1
12.5.2
12.5.3
12.6
12.6.1
12.7
12.7.1
12.8
12.9

13

13.1
13.2
13.2.1
13.2.2
13.2.3
13.2.4

13.2.5
13.3
13.3.1
13.3.1.1
13.3.1.2
13.3.2
13.3.2.1
13.3.2.2
13.3.2.3
13.3.2.4
13.4
13.4.1
13.4.1.1
13.5

General Procedure for Coupling Reaction in Solid-Phase Using
Onium Salts (Phosphonium and Uronium) 427
General Procedures for Coupling Reaction in Solid-Phase Using
Onium Salts (Phosphonium and Uronium) Boc-, Fmoc-Amino
Acids via Phosphonium and Uronium Salts 427
Fluoroformamidinium Coupling Reagents 429
General Method for the Synthesis of Fluoroformamidinium
Salts 431
Solution- and Solid-Phase Couplings via TFFH 432
General Method for Solid-Phase Coupling via TFFH 432
Organophosphorus Reagents 432
General Method for Synthesis of the Diphenylphosphoryl
Derivatives 435
Triazine Coupling Reagents 435
Formation of the Peptide Bond Using DMTMM (128) 437

Mukaiyama’s Reagent 437
Conclusions 438
References 439
Chemoselective Peptide Ligation: A Privileged Tool for
Protein Synthesis 445
Christian P.R. Hackenberger, Jeffrey W. Bode, and Dirk Schwarzer
Introduction 445
Chemoselective Peptide Ligations Following a Capture/
Rearrangement Strategy 449
Basic Concepts and Early Experiments 449
NCL 452
Protein Semisynthesis with NCL 454
Protein Semisynthesis with Expressed Protein Ligation 456
Protein Trans-Splicing 457
Chemical Transformations for Cys-Free Ligations in Peptides
and Proteins 460
Chemical Modification of NCL Products 460
Desulfurization 463
Alkylation and Thioalkylation Protocols 464
Auxiliary Methods 466
(Oxy-)Ethanethiol Auxiliary 467
Photoremovable Na-1-Aryl-2-Mercaptoethyl Auxiliary 468
4,5,6-Trimethoxy-2-Mercaptobenzylamine Auxiliary 468
Sugar-Assisted Glycopeptide Ligations 469
Other Chemoselective Capture Strategies 471
Traceless Staudinger Ligation 471
Imine Ligations with Subsequent Pseudo-Pro Formation 473
Peptide Ligations by Chemoselective Amide-Bond-Forming
Reactions 474


www.pdfgrip.com


Contents

13.5.1
13.5.2
13.5.3
13.6
13.6.1
13.6.2
13.6.2.1

Thio Acid/Azide Amidation 475
Thio Acid/N-Arylsulfonamide Ligations 475
Chemoselective Decarboxylative Amide Ligation 477
Strategies for the Ligation of Multiple Fragments 479
Synthetic Erythropoietin 480
Convergent Strategies for Multiple Fragment Ligations 480
Ubiquitylated Histone Proteins 484
References 486

14

Automation of Peptide Synthesis 495
Carlo Di Bello, Andrea Bagno, and Monica Dettin
Introduction 495
SPPS: From Mechanization to Automation 497
Deprotection Step: Monitoring and Control 500
Coupling Step: Monitoring and Control 505

Integrated Deprotection and Coupling Control 509
References 514

14.1
14.2
14.3
14.4
14.5

15

15.1
15.2
15.3
15.3.1
15.3.1.1
15.3.1.2
15.3.2
15.3.2.1
15.3.2.2
15.3.2.3
15.3.2.4
15.3.2.5
15.3.2.6
15.3.2.7
15.3.2.8
15.3.2.9
15.4
15.5
15.6

15.7
15.8
15.9

Peptide Purification by Reversed-Phase Chromatography 519
Ulrike Kusebauch, Joshua McBee, Julie Bletz, Richard J. Simpson,
and Robert L. Moritz
RP-HPLC of Peptides 519
Peptide properties 520
Chromatographic Principles 520
Choice of Mobile Phase 520
Mobile-Phase Aqueous Buffer pH 520
Organic Solvent 522
Stationary Phase 523
Surface Bonding 523
Pore Diameter 523
Particle Size 524
Ultra-High-Pressure Liquid Chromatography 525
Synthetic Polymer Packings 525
Monolithic Stationary Phase 525
Packed Bed (Column) Length 526
Gradient Effect 527
Temperature 527
Prediction of Peptide Retention Times 528
Advantages of Reduced Scale 531
Two-Dimensional Chromatographic Methods 532
Peptide Analysis in Complex Biological Matrices 533
Standard Methods for Peptide Separations for Analysis by
Hyphenated Techniques 534
Emerging Methods for Peptide Separations for Analysis by

Hyphenated Techniques 534

www.pdfgrip.com

XV


XVI

Contents

15.10

Practical use of RP-HPLC for Purifying Peptides (Analytical
and Preparative Scale) 539
15.10.1
Simple Protocol for Successful RP-HPLC 540
15.10.1.1 Buffer Preparation 540
15.10.1.2 HPLC Chromatographic System 542
15.10.1.3 Test Sample 542
References 544
16
16.1
16.2
16.3
16.4
16.5
16.5.1
16.5.2
16.5.3

16.5.4
16.5.5
16.5.6
16.5.7
16.5.8
16.6
16.7

Difficult Peptides 549
M. Ter^esa Machini Miranda, Cleber W. Liria, and Cesar Remuzgo
Importance of Peptide Synthesis 549
Methods for Peptide Synthesis 550
Chemical Peptide Synthesis 551
‘‘Difficult Peptide Sequences’’ 554
Means to Overcome Peptide Aggregation in SPPS 556
In Situ Neutralization 556
Solvents for Peptide Chain Assembly 557
Type and Substitution Degree of Resins for Peptide Chain
Assembly 557
Use of Chaotropic Salts During Peptide Chain Assembly 558
Use of Amide Backbone Protection 558
The Use of Pseudo-Prolines 560
O-Acyl Isopeptide Approach 561
Use of Elevated Temperatures 562
Monitoring the Synthesis of a ‘‘Difficult Peptide’’ 562
Conclusions 564
References 564
Index

571


www.pdfgrip.com


XVII

List of Contributors
Knut Adermann
Pharis Biotec GmbH
Feodor-Lynen-Strasse 31
30625 Hannover
Germany

University of Barcelona
Department of Organic Chemistry
Martí i Franqués 1–11
08028 Barcelona
Spain

José Luis Adrio
Neuron BPh
Avda. de la Innovación 1, Edificio BIC
Parque Tecnológico de ciencias de la
Salud
18100 Armilla
Granada
Spain

Andrea Bagno
University of Padova

Department of Chemical Process
Engineering
Via Marzolo 9
35131 Padova
Italy

Fernando Albericio
Institute for Research in Biomedicine
Barcelona Science Park
Baldiri Reixac 10
08028 Barcelona
Spain
and
Networking Centre on Bioengineering,
Biomaterials and Nanomedicine
(CIBER-BBN)
Barcelona Science Park
Baldiri Reixac 10
08028 Barcelona
Spain

Kleomenis Barlos
University of Patras
Department of Chemistry
Rion-Patras
Greece
Josộ Luis Barredo
IỵD Biologia
Antibiúticos S.A.
Avda. Antibióticos, 59–61

24009 León
Spain
Julie Bletz
Institute for Systems Biology
1441 North 34th Street
Seattle, WA 98103-8904
USA

Amino Acids, Peptides and Proteins in Organic Chemistry.
Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes
Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-32102-5

www.pdfgrip.com


XVIII

List of Contributors

Jeffrey W. Bode
Eidgenössische Technische
Hochschule Zürich
Laboratorium für Organische Chemie
Wolfgang Pauli Strasse 10
8093 Zürich
Switzerland
M. Isabel Calaza
Universidad de Zaragoza – CSIC
Instituto de Ciencia de Materiales

de Aragón
Departamento de Qmica Orgánica
50009 Zaragoza
Spain
Stefano Carganico
University of Firenze
Polo Scientifico e Tecnologico
Laboratory of Peptide and Protein
Chemistry and Biology
Via della Lastruccia 13
50019 Sesto Fiorentino
Italy
PRES University of Cergy-Pontoise
Laboratoire SOSCO-UMR 8123
5 mail Gay-Lussac, Neuville sur Oise
95031 Cergy-Pontoise
France
Carlos Cativiela
Universidad de Zaragoza – CSIC
Instituto de Ciencia de Materiales
de Aragón
Departamento de Qmica Orgánica
50009 Zaragoza
Spain
Arnold L. Demain
Drew University
RISE
HS-330
Madison, NJ 07940
USA


Jan Deska
Stockholm University
Arrhenius Laboratory
Department of Organic Chemistry
106 91 Stockholm
Sweden
Monica Dettin
University of Padova
Department of Chemical Process
Engineering
Via Marzolo 9
35131 Padova
Italy
Carlo Di Bello
University of Padova
Department of Chemical Process
Engineering
Via Marzolo 9
35131 Padova
Italy
Ayman El-Faham
King Saud University
College of Science
Department of Chemistry
PO Box 2455
1451 Riyadh
Kingdom of Saudi Arabia
Alexandria University
Faculty of Science

Department of Chemistry
Horria Street, PO Box 246, Ibrahimia
21321 Alexandria
Egypt
Institute for Research in Biomedicine
Barcelona Science Park
Baldiri Reixac 10
08028 Barcelona
Spain

www.pdfgrip.com


List of Contributors

Christian P.R. Hackenberger
Freie Universität Berlin
Institut für Chemie und Biochemie
Takustrasse 3
14195 Berlin
Germany
Jan Hlavá4c
Palacky University
Department of Organic Chemistry
Trida 17, Listopadu 12
771 46 Olomouc
Czech Republic
Steven A. Kates
Ischemix
63 Great Road

Maynard, MA 01754
USA
Viktor Krchñák
University of Notre Dame
Department of Chemistry and
Biochemistry
251 Nieuwland Science Center
Notre Dame, IN 46556
USA
Ulrike Kusebauch
Institute for Systems Biology
1441 North 34th Street
Seattle, WA 98103-8904
USA
Cleber W. Liria
University of São Paulo
Institute of Chemistry
Department of Biochemistry
Peptide Chemistry Laboratory
Av. Prof. Lineu Prestes, 748
05508-900 São Paulo
Brazil

Garland R. Marshall
Washington University
Center for Computational Biology
Departments of Biochemistry and
Molecular Biophysics and Biomedical
Engineering
700 S. Euclid Avenue

St. Louis, MO 63110
USA
Joshua McBee
Institute for Systems Biology
1441 North 34th Street
Seattle, WA 98103-8904
USA
Maria Terêsa Machini Miranda
University of São Paulo
Institute of Chemistry
Department of Biochemistry
Peptide Chemistry Laboratory
Av. Prof. Lineu Prestes, 748
05508-900 São Paulo
Brazil
Robert L. Moritz
Institute for Systems Biology
1441 North 34th Street
Seattle, WA 98103-8904
USA
Yoshio Okada
Kobe Gakuin University
Faculty of Pharmaceutical Sciences
Arise 518, Ikawadani-cho, Nishi-ku
651-2180 Kobe
Japan
Michael I. Page
University of Huddersfield
Department of Chemical and Biological
Sciences

Queensgate
Huddersfield HD1 3DH
UK

www.pdfgrip.com

XIX


XX

List of Contributors

Anna Maria Papini
University of Firenze
Polo Scientifico e Tecnologico
Laboratory of Peptide and Protein
Chemistry and Biology
Via della Lastruccia 13
50019 Sesto Fiorentino
Italy

Dirk Schwarzer
Leibniz-Institut für Molekulare
Pharmakologie (FMP)
Chemical Biology Section
Robert-Rössle-Strasse 10
13125 Berlin
Germany


PRES University of Cergy-Pontoise
Laboratoire SOSCO-UMR 8123
5 mail Gay-Lussac, Neuville sur Oise
95031 Cergy-Pontoise
France

Richard J. Simpson
Ludwig Institute For Cancer Research
Joint Proteomics Laboratory
Royal Melbourne Hospital
Parkville, Victoria 3050
Australia

Emily J. Parker
University of Canterbury
Department of Chemistry
PO Box 4800
Christchurch
New Zealand

Miroslav Soural
Palacky University
Department of Organic Chemistry
Trida 17, Listopadu 12
771 46 Olomouc
Czech Republic

Andrew J. Pratt
University of Canterbury
Department of Chemistry

PO Box 4800
Christchurch
New Zealand

Yuko Tsuda
Kobe Gakuin University
Faculty of Pharmaceutical Sciences
Minatojima 1-1-3, Chuo-ku
650-8586 Kobe
Japan

Cesar Remuzgo
University of São Paulo
Institute of Chemistry
Department of Biochemistry
Peptide Chemistry Laboratory
Av. Prof. Lineu Prestes, 748
05508-900 São Paulo
Brazil

Judit Tulla-Puche
Institute for Research in Biomedicine
Barcelona Science Park
Baldiri Reixac 10
08028 Barcelona
Spain

Marta Rodriguez-Sáiz
Antibióticos S.A.
Avda. Antibióticos, 59–61

24009 León
Spain

Networking Centre on Bioengineering,
Biomaterials and Nanomedicine
(CIBER-BBN)
Barcelona Science Park
Baldiri Reixac 10
08028 Barcelona
Spain

and

www.pdfgrip.com


Part One
Amino Acids as Building Blocks

Amino Acids, Peptides and Proteins in Organic Chemistry.
Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes
Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-32102-5

www.pdfgrip.com


j3

1

Amino Acid Biosynthesis
Emily J. Parker and Andrew J. Pratt

1.1
Introduction

The ribosomal synthesis of proteins utilizes a family of 20 a-amino acids that are
universally coded by the translation machinery; in addition, two further a-amino
acids, selenocysteine and pyrrolysine, are now believed to be incorporated into
proteins via ribosomal synthesis in some organisms. More than 300 other amino acid
residues have been identified in proteins, but most are of restricted distribution and
produced via post-translational modification of the ubiquitous protein amino
acids [1]. The ribosomally encoded a-amino acids described here ultimately derive
from a-keto acids by a process corresponding to reductive amination. The most
important biosynthetic distinction relates to whether appropriate carbon skeletons
are pre-existing in basic metabolism or whether they have to be synthesized de novo
and this division underpins the structure of this chapter.
There are a small number of a-keto acids ubiquitously found in core metabolism,
notably pyruvate (and a related 3-phosphoglycerate derivative from glycolysis),
together with two components of the tricarboxylic acid cycle (TCA), oxaloacetate
and a-ketoglutarate (a-KG). These building blocks ultimately provide the carbon
skeletons for unbranched a-amino acids of three, four, and five carbons, respectively.
a-Amino acids with shorter (glycine) or longer (lysine and pyrrolysine) straight
chains are made by alternative pathways depending on the available raw materials.
The strategic challenge for the biosynthesis of most straight-chain amino acids
centers around two issues: how is the a-amino function introduced into the carbon
skeleton and what functional group manipulations are required to generate the
diversity of side-chain functionality required for the protein function?
The core family of straight-chain amino acids does not provide all the functionality
required for proteins. a-Amino acids with branched side-chains are used for two

purposes; the primary need is related to protein structural issues. Proteins fold into
well-defined three-dimensional shapes by virtue of their amphipathic nature: a
significant fraction of the amino acid side-chains are of low polarity and the
hydrophobic effect drives the formation of ordered structures in which these
side-chains are buried away from water. In contrast to the straight-chain amino
Amino Acids, Peptides and Proteins in Organic Chemistry.
Vol.3 – Building Blocks, Catalysis and Coupling Chemistry. Edited by Andrew B. Hughes
Copyright Ó 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
ISBN: 978-3-527-32102-5

www.pdfgrip.com


j 1 Amino Acid Biosynthesis

4

acids, the hydrophobic residues have large nonpolar surface areas by virtue of their
branched hydrocarbon side-chains. The other role of branched amino acids is to
provide two useful functional groups: an imidazole (histidine) and a phenol (tyrosine)
that exploit aromatic functional group chemistry.
This chapter provides an overview of amino acid biosynthesis from a chemical
perspective and focuses on recent developments in the field. It highlights a few
overarching themes, including the following:
i) The chemical logic of the biosynthetic pathways that underpin amino acid biosynthesis. This chemical foundation is critical because of the evolutionary mechanisms that have shaped these pathways. In particular, the way in which gene
duplication and functional divergence (via mutation and selection) can generate
new substrate specificity and enzyme activities from existing catalysts [2].
ii) The contemporary use of modern multidisciplinary methodology, including
chemistry, enzymology, and genomics, to characterize new biosynthetic
pathways.

iii) Potential practical implications of understanding the diverse metabolism of
amino acid biosynthesis, especially medicinal and agrichemical applications.
iv) The higher-level molecular architectures that control the fate of metabolites,
especially the channeling of metabolites between active sites for efficient
utilization of reactive intermediates.
Box 1.1: Nitrogen and Redox in Amino Acid Biosynthesis

Ammonia is toxic and the levels of ammonia available for the biosynthesis of
amino acids in most biochemical situations is low. There are a limited number of
entry points of ammonia into amino acid biosynthesis, notably related to glutamate and glutamine. Once incorporated into key amino acids, nitrogen is
transferred between metabolites either directly or via in situ liberation of ammonia
by a multifunctional complex incorporating the target biosynthetic enzyme. The
main source of in situ generated ammonia for biosynthesis is the hydrolysis of
glutamine by glutaminases. De novo biosynthesis of amino acids, like element
fixation pathways in general, is primarily reductive in nature. This may reflect the
origins of these pathways in an anaerobic world more than 3 billion years ago.

Box 1.2: The Study of Biosynthetic Enzymes and Pathways

The source of an enzyme for biochemical study has important implications. Most
core metabolism has been elaborated by studying a small number of organisms
that were chosen for a variety of reasons, including availability, ease of manipulation, ethical concerns, scientific characterization, and so on. These exemplar
organisms include the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, the plant Arabidopsis thaliana, and the rat as a typical mammal. Much of the
detailed characterization of amino acid biosynthesis commenced with studies on
these organisms. With the rise of genetic engineering techniques, biosynthetic

www.pdfgrip.com


1.2 Glutamate and Glutamine: Gateways to Amino Acid Biosynthesis


enzymes from a wide variety of sources are available for scientific investigation,
and there has been increasing emphasis on working with enzymes and pathways
from alternative organisms.
Metabolic diversity is greatest among prokaryotes. One fundamental change in
the underlying microbiology that has affected our understanding of pathway
diversity has been the appreciation of the deep biochemical distinctions between
what are now recognized to be two fundamental domains of prokaryotes:
eubacteria and Archaea [3]. The former bacteria include those well known to be
associated with disease and fermentation processes; while the latter include many
methanogens and extremophiles (prokaryotes that grow in extreme conditions,
such as hyperthermophiles that grow at temperatures above 60  C or halophiles
that grow in high ionic strength environments). Bioinformatics approaches are
complementing conventional enzymological studies in identifying and characterizing interesting alternative biosynthetic pathways [4]. The greater understanding of microbial and biosynthetic diversity is presenting exciting opportunities for
novel discoveries in biosynthesis.
Much of the focus of biosynthetic enzymology now focuses on enzymes from
pathogens and hyperthermophiles. The focus on the study of enzymes from
pathogens is predicated on the possibility that inhibitors of such enzymes may be
useful as pesticides and therapeutic agents. Since humans have access to many
amino acids in their food, they have lost the ability to make “dietary essential”
amino acids that typically require extended dedicated biosynthetic pathways [5].
The biosynthetic enzymes of the corresponding pathways are essential for many
pathogens and plants, but not for humans; hence, selective inhibitors of these
biosynthetic enzymes are potentially nontoxic to humans, but toxic to undesirable
organisms. Enzymes from hyperthermophilic organisms, produced by genetic
engineering, are scrutinized mainly because of their ease of structural characterization. These enzymes retain their native structures at temperatures that
denature most other proteins, including those of the host organism. These
proteins are of high thermal stability and simple heat treatment can be used to
effect high levels of purification of the desired protein.


1.2
Glutamate and Glutamine: Gateways to Amino Acid Biosynthesis

Glutamate and the corresponding amide derivative, glutamine, are critical metabolites in amino acid metabolism. The biochemistry of these two amino acids also
illustrates the distinct chemistry associated with the a-amino and side-chain functional groups, each of which is exploited in the biosynthesis of other amino acids.
These amino acids derive from ammonia and a-KG. Glutamate dehydrogenase
(GDH) interconverts a-KG and glutamate (Figure 1.1) [6]. Although glutamate is
formed in this way by reductive amination, this enzyme is generally not dedicated to
biosynthesis; the reverse reaction, an oxidative deamination to regenerate a-KG, is

www.pdfgrip.com

j5