Amino Acids, Peptides and Proteins in Organic Chemistry Volume 3

Amino Acids, Peptides and Proteins in Organic Chemistry
 
Author:
Andrew B. Hughes
Publisher: Wiley-VCH
ISBN No: 978-3-527-32102-5
Release at: 2011
Pages: 595
Edition:
Volume 3 Building Blocks, Catalysis and Coupling Chemistry
File Size: 6 MB
File Type: pdf
Language: English



Description of Amino Acids, Peptides and Proteins in Organic Chemistry


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 the 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. The ribosomally encoded a-amino acids described here ultimately derive from a-keto acids by a process corresponding to reductive amination.

Content of Amino Acids, Peptides and Proteins in Organic Chemistry



Part One Amino Acids as Building Blocks 1

1 Amino Acid Biosynthesis 3
Emily J. Parker and Andrew J. Pratt
1.1 Introduction 3
1.2 Glutamate and Glutamine: Gateways to Amino Acid Biosynthesis 5
1.2.1 Case Study: GOGAT: GATs and Multifunctional Enzymes
in Amino Acid Biosynthesis 6
1.3 Other Amino Acids from Ubiquitous Metabolites: Pyridoxal
Phosphate-Dependent Routes to Aspartate, Alanine, and Glycine 8
1.3.1 Pyridoxal Phosphate: A Critical Cofactor of Amino Acid Metabolism 8
1.3.2 Case Study: Dual Substrate Specificity of Families
of Aminotransferase Enzymes 10
1.3.3 PLP and the Biosynthesis of Alanine and Glycine 15
1.4 Routes to Functionalized Three-Carbon Amino Acids: Serine,
Cysteine, and Selenocysteine 16
1.4.1 Serine Biosynthesis 16
1.4.2 Cysteine Biosynthesis 18
1.4.3 Case Study: Genome Information as a Starting Point for Uncovering
New Biosynthetic Pathways 19
1.4.3.1 Cysteine Biosynthesis in Mycobacterium Tuberculosis 19
1.4.3.2 Cysteine Biosynthesis in Archaea 20
1.4.3.3 RNA-Dependent Biosynthesis of Selenocysteine and Other
Amino Acids 21
1.5 Other Amino Acids from Aspartate and Glutamate: Asparagine
and Side Chain Functional Group Manipulation 22
1.5.1 Asparagine Biosynthesis 23
1.6 Aspartate and Glutamate Families of Amino Acids 25
1.6.1 Overview 25
1.6.2 Aspartate Family Amino Acids: Threonine and Methionine 25
1.6.2.1 Case Study: Evolution of Leaving Group Specificity in Methionine
Biosynthesis 28
1.6.2.2 Threonine, Homocysteine, and PLP 30
1.6.2.3 Threonine Synthase 30
1.6.2.4 Methionine, Cysteine, and Cystathionine 32
1.6.2.5 Methionine Synthase 33
1.6.3 Glutamate Family Amino Acids: Proline and Arginine 33
1.7 Biosynthesis of Aliphatic Amino Acids with Modified Carbon Skeletons:
Branched-Chain Amino Acids, Lysine, and Pyrrolysine 37
1.7.1 Overview 37
1.7.2 Valine and Isoleucine 37
1.7.3 Homologation of a-Keto Acids, and the Biosynthesis of Leucine
and a-Aminoadipic Acid 41
1.7.4 Biosynthesis of Lysine: A Special Case 44
1.7.4.1 Diaminopimelate Pathway to Lysine 44
1.7.4.2 a-Aminoadipic Acid Pathways to Lysine 45
1.7.4.3 Pyrrolysine 47
1.8 Biosynthesis of the Aromatic Amino Acids 49
1.8.1 Shikimate Pathway 49
1.8.2 Case Study: Alternative Synthesis of Dehydroquinate in Archaea 53
1.8.3 Biosynthesis of Tryptophan, Phenylalanine, and Tyrosine from
Chorismate 58
1.8.3.1 Tryptophan Biosynthesis 58
1.8.3.2 Phenylalanine and Tyrosine Biosynthesis 59
1.8.4 Histidine Biosynthesis 61
1.9 Conclusions 64
References 65
2 Heterocycles from Amino Acids 83
M. Isabel Calaza and Carlos Cativiela
2.1 Introduction 83
2.2 Heterocycles Generated by Intramolecular Cyclizations 83
2.2.1 a-Lactones and a-Lactams 83
2.2.2 Indolines 84
2.2.3 Aziridinecarboxylic Acids and Oxetanones 86
2.2.4 b-Lactams and Pyroglutamic Acid Derivatives 87
2.2.5 Amino Lactams and Amino Anhydrides 88
2.2.6 Azacycloalkanecarboxylic Acids 89
2.3 Heterocycles Generated by Intermolecular Cyclizations 89
2.3.1 Metal Complexes 89
2.3.2 a-Amino Acid N-Carboxyanhydrides and Hydantoins 90
2.3.3 Oxazolidinones and Imidazolidinones 91
2.3.4 Oxazolones 93
2.3.5 Oxazinones and Morpholinones, Pyrazinones
and Diketopiperazines 94
2.3.6 Tetrahydroisoquinolines and b-Carbolines 96
2.3.7 Oxazo/Thiazolidinones, Oxazo/Thiazolidines,
and Oxazo/Thiazolines 97
2.3.8 Sulfamidates 101
2.3.9 Tetrahydropyrimidinones 102
2.4 Heterocycles Generated by Cycloadditions 102
2.5 Conclusions 104
2.6 Experimental Procedures 104
2.6.1 Synthesis of 1-tert-Butyl-3-phenylaziridinone (5) 104
2.6.2 Synthesis of Dimethyl (2S,3aR,8aS)-1,2,3,3a,8,8a-
Hexahydropyrrolo[2,3-b]indole-1,2-dicarboxylate (Precursor of 10) 105
2.6.3 Synthesis of Benzyl (R)-1-Tritylaziridine-2-carboxylate (15) 105
2.6.4 Synthesis of (S)-N-tert-Butoxycarbonyl-3-aminooxetan-2-one (18) 106
2.6.5 Synthesis of (S)-1-(tert-Butyldimethylsilyl)-4-oxoazetidine-
2-carboxylic Acid (24) 106
2.6.6 Synthesis of 9H-Fluoren-9-ylmethyl (R)-Hexahydro-2-oxo-1H-azepin-
3-yl Carbamate (31) 106
2.6.7 Synthesis of Ethyl (S)-N-(tert-Butoxycarbonyl)-a-
(tert-butoxymethyl)proline Ester (36) 107
2.6.8 Synthesis of Proline N-Carboxyanhydride (49) 107
2.6.9 Synthesis of (2S,4S)-2-Ferrocenyl-3-pivaloyl-4-methyl-1,3-
oxazolidin-5-one (54b) 107
2.6.10 Synthesis of (6S)-6-Isopropyl-5-phenyl-3,6-dihydro-2H-1,4-oxazin-2-
one (71) 108
2.6.11 Synthesis of (3S,6R)-6-Isopropyl-3-methyl-5-phenyl-1,2,3,6-
tetrahydro-2-pyrazinone (78) 108
2.6.12 Synthesis of (3S)-3,6-Dihydro-2,5-dimethoxy-3-
isopropylpyrazine (85) 109
2.6.13 Synthesis of (3S)-1,2,3,4-Tetrahydroisoquinoline-
3-carboxylic Acid (87) 110
2.6.14 Synthesis of Methyl (S)-N-tert-Butoxycarbonyl-2,2-dimethyloxazolidine-
4-carboxylate (109) 110
2.6.15 Synthesis of (2S,6S)-2-tert-Butyl-1-carbobenzoxy-4-oxopyrimidin-6-
carboxylic Acid (126) 111
References 111
3 Radical-Mediated Synthesis of a-Amino Acids and Peptides 115
Jan Deska
3.1 Introduction 115
3.2 Free Radical Reactions 115
3.2.1 Hydrogen Atom Transfer Reactions 116
3.2.2 Functional Group Transformations 121
3.3 Radical Addition to Imine Derivatives 124
3.3.1 Glyoxylate Imines as Radical Acceptors 125
3.3.2 Oximes and Hydrazones as Radical Acceptors 126
3.3.3 Nitrones as Radical Acceptors 129
3.3.4 Isocyanates as Radical Acceptors 130
3.4 Radical Conjugate Addition 130
3.5 Conclusions 135
3.6 Experimental Protocols 135
3.6.1 Preparation of ((1R,2S,5R)-5-methyl-2-(1-methyl-l-
phenylethyl)cyclohexyl 2-[(tert-butoxycarbonyl)amino]-4-methyl-
pent-4-enoate) (7) 135
3.6.2 Synthesis of (2S)-3-{(1R,2S)-2-[(N-bis-Boc)amino]-1-cyclopropyl}-2-
benzyloxycarbonylamino-propionic Acid Methyl Ester (26) 136
3.6.3 Synthesis of (3aR,6S,7aS)-hexahydro-8,8-dimethyl-1-[(2R)-3,3-
dimethyl-1-oxo-2-(2,2-diphenylhydrazino)butyl]-3H-3a,6-methano-2,1-
benzisothiazole 2,2-dioxide (42) 136
3.6.4 Synthesis of N-(2,6-diphenyl-methylpiperidine-2-
carboxamide (59) 137
3.6.5 Synthesis of Methyl 2-(2-naphthylcarbonylamino)pentanoate
(80) 136
References 138
4 Synthesis of b-Lactams (Cephalosporins) by Bioconversion 143
José Luis Barredo, Marta Rodriguez- Sáiz, José Luis Adrio,
and Arnold L. Demain
4.1 Introduction 143
4.2 Biosynthetic Pathways of Cephalosporins and Penicillins 146
4.3 Production of 7-ACA by A. chrysogenum 147
4.4 Production of 7-ADCA by A. chrysogenum 149
4.5 Production of Penicillin G by A. chrysogenum 151
4.6 Production of Cephalosporins by P. chrysogenum 152
4.7 Conversion of Penicillin G and other Penicillins to DAOG
by Streptomyces clavuligerus 153
4.7.1 Expandase Proteins and Genes 153
4.7.2 Bioconversion of Penicillin G to DAOG 155
4.7.3 Broadening the Substrate Specificity of Expandase 155
4.7.3.1 Resting Cells 155
4.7.3.2 Cell-Free Extracts 156
4.7.4 Inactivation of Expandase during the Ring-Expansion Reaction 157
4.7.5 Further Improvements in the Bioconversion of Penicillin
G to DAOG 158
4.7.5.1 Stimulatory Effect of Growth in Ethanol 158
4.7.5.2 Use of Immobilized Cells 159
4.7.5.3 Elimination of Agitation and Addition of Water-Immiscible
Solvents 159
4.7.5.4 Addition of Catalase 160
4.7.5.5 Recombinant S. clavuligerus Expandases 160
4.8 Conclusions 162
References 163
5 Structure and Reactivity of b-Lactams 169
Michael I. Page
5.1 Introduction 169
5.2 Structure 170
5.3 Reactivity 174
5.4 Hydrolysis 176
5.4.1 Base Hydrolysis 176
5.4.2 Acid Hydrolysis 178
5.4.3 Spontaneous Hydrolysis 180
5.4.4 Buffer-Catalyzed Hydrolysis 180
5.4.5 Metal Ion-Catalyzed Hydrolysis 180
5.4.6 Micelle-Catalyzed Hydrolysis of Penicillins 182
5.4.7 Cycloheptaamylose-Catalyzed Hydrolysis 184
5.4.8 Enzyme-Catalyzed Hydrolysis 185
5.4.8.1 Serine b-Lactamases 185
5.4.8.2 Metallo b-Lactamases 187
5.5 Aminolysis 191
5.6 Epimerization 195
References 195

Part Two Amino Acid Coupling Chemistry 201

6 Solution-Phase Peptide Synthesis 203
Yuko Tsuda and Yoshio Okada
6.1 Principle of Peptide Synthesis 203
6.2 Protection Procedures 205
6.2.1 Amino Group Protection 205
6.2.1.1 Z Group 205
6.2.1.2 Substituted Z and other Urethane-Type Protecting Groups 207
6.2.1.3 Boc Group 207
6.2.1.4 Tri Group 208
6.2.1.5 Fmoc Group 209
6.2.1.6 Other Representative Protecting Groups 211
6.2.2 Carboxyl Group Protection 212
6.2.2.1 Methyl Ester (-OMe) and Ethyl Ester (-OEt) 213
6.2.2.2 Benzyl Ester (-OBzl) 213
6.2.2.3 tBu Ester (-OtBu) 213
6.2.2.4 Phenacyl Ester (-OPac) 214
6.2.2.5 Hydrazides 214
6.2.3 Side-Chain Protection 215
6.2.3.1 e-Amino Function of Lys (d-Amino Function of Orn) 215
6.2.3.2 b-Mercapto Function of Cys 216
6.2.3.3 b- and c-Carboxyl Functions of Asp and Glu 217
6.2.3.4 Protecting Groups for the c-Carboxyl Function of Glu 219
6.2.3.5 d-Guanidino Function of Arg 219
6.2.3.6 Phenolic Hydroxy Function of Tyr 221
6.2.3.7 Aliphatic Hydroxyl Function of Ser and Thr 222
6.2.3.8 Imidazole Nitrogen of His 222
6.2.3.9 Indole Nitrogen of Trp 223
6.3 Chain Elongation Procedures 223
6.3.1 Methods of Activation in Stepwise Elongation 223
6.3.1.1 Carbodiimides 223
6.3.1.2 Mixed Anhydride Method 224
6.3.1.3 Active Esters 225
6.3.1.4 Phosphonium and Uronium Reagents 227
6.3.2 Methods of Activation in Segment Condensation 229
6.3.2.1 Azide Procedure 229
6.3.2.2 Carbodiimides in the Presence of Additives 230
6.3.2.3 Native Chemical Ligation 231
6.4 Final Deprotection Methods 232
6.4.1 Final Deprotection by Catalytic Hydrogenolysis 233
6.4.2 Final Deprotection by Sodium in Liquid Ammonia 233
6.4.3 Final Deprotection by TFA 233
6.4.4 Final Deprotection by HF 233
6.4.5 Final Deprotection by HSAB Procedure 234
References 234
7 Solid-Phase Peptide Synthesis: Historical Aspects 253
Garland R. Marshall
7.1 Introduction 253
7.2 Selection of Compatible Synthetic Components 253
7.3 Racemization and Stepwise Peptide Assembly 257
7.4 Optimization of Synthetic Components 258
7.5 Foreshadowing of the Nobel Prize 258
7.6 Automation of SPPS 260
7.7 Impact of New Protecting Groups and Resin Linkages 261
7.8 Solid-Phase Organic Chemistry 262
7.9 Early Applications of SPPS to Small Proteins 263
7.10 Side-Reactions and Sequence-Dependent Problems 264
7.11 Rapid Expansion of Usage Leading to the Nobel Prize 265
7.12 From the Nobel Prize Forward to Combinatorial
Chemistry 267
7.13 Protein Synthesis and Peptide Ligation 268
7.14 Conclusions 269
References 270
8 Linkers for Solid-Phase Peptide Synthesis 273
Miroslav Soural, Jan Hlavác, and Viktor Krch 4 nák 4
8.1 Introduction 273
8.1.1 Immobilization Strategies 275
8.1.2 Overview of Linker Types 276
8.1.3 Selection of a Linker 277
8.2 Immobilization via Carboxyl Group 279
8.2.1 Esters 280
8.2.1.1 Hydroxy Linkers for Preparation of Resin-Bound Esters 281
8.2.1.2 Electrophilic Linkers for Preparation of Resin-Bound Esters 282
8.2.1.3 Cleavage from the Resin 282
8.2.2 Amides 288
8.2.3 Hydrazides 291
8.2.4 Oximes 291
8.2.5 Thioesters 292
8.3 Immobilization via Amino Group 294
8.4 Backbone Immobilization 296
8.4.1 Benzaldehyde-Based Linkers 298
8.4.2 Indole Aldehyde Linkers 299
8.4.3 Naphthalene Aldehyde Linkers (NALs) 299
8.4.4 Thiophene Aldehyde Linkers (T-BALs) 300
8.4.5 Safety-Catch Aldehyde Linkers 300
8.4.6 Photolabile Aldehyde Linker (PhoB) 300
8.5 Immobilization via Amino Acid Side-Chain 300
8.5.1 Carboxyl Group 301
8.5.2 Amino and Other Nitrogen-Containing Groups 302
8.5.2.1 Lys 302
8.5.2.2 His 302
8.5.2.3 Arg 303
8.5.3 Hydroxy Group 303
8.5.4 Sulfanyl Group 304
8.5.5 Aromatic Ring 305
8.6 Conclusions 306
References 306
9 Orthogonal Protecting Groups and Side-Reactions in Fmoc/tBu
Solid-Phase Peptide Synthesis 313
Stefano Carganico and Anna Maria Papini
9.1 Orthogonal Protecting Groups in Fmoc/tBu Solid-Phase Peptide
Synthesis 313
9.1.1 Arg 313
9.1.2 Asn and Gln 315
9.1.3 Asp and Glu 316
9.1.4 Cys 318
9.1.5 His 323
9.1.6 Lys 324
9.1.7 Met 327
9.1.8 Ser and Thr 327
9.1.9 Trp 328
9.1.10 Tyr 329
9.1.11 Conclusions 330
9.2 Side-Reactions in Fmoc/tBu Solid-Phase Peptide Synthesis 330
9.2.1 Imidazole Ring-Mediated Racemization of Chiral a-Carbon 332
9.2.2 Hydroxyl-Mediated O ! N Acyl Transfer 332
9.2.3 Met Oxidation to Methionyl Sulfoxide 334
9.2.4 Dehydration of Asn and Gln Amide Side-Chain 334
9.2.5 Aspartimide Formation 336
9.2.6 Formation of Diketopiperazines 337
9.2.7 Side-Reactions Affecting Protected Cys 338
9.2.8 Deletion Peptides, Truncated Sequences, and Multiple
Additions 338
9.2.9 Uronium/Guanidinium Salts-Induced Guanidino
Capping 340
9.2.10 Arg Cyclization and Arg Conversion into Orn 341
9.2.11 Conclusions 342
References 343
10 Fmoc Methodology: Cleavage from the Resin
and Final Deprotection 349
Fernando Albericio, Judit Tulla-Puche, and Steven A. Kates
10.1 Introduction 349
10.2 ‘‘Low’’ TFA-Labile Resins 351
10.2.1 Cleavage 351
10.2.2 Choice of Resin for the Preparation of Peptide Acids 352
10.2.2.1 CTC Resin 353
10.2.2.2 SASRIN Resin 355
10.2.2.3 Bromide Resin 356
10.2.3 Final Deprotection 356
10.3 ‘‘High’’ TFA-Labile Resins 356
10.3.1 Cleavage 357
10.3.2 Final Deprotection of Protected Peptides in Solution 359
10.3.3 Side-Reactions 360
10.3.3.1 Linker/Resin 360
10.3.3.2 Trp and Tyr Modification 361
10.3.3.3 Sulfur-Containing Residues: Cys and Met 362
10.3.3.4 Ser and Thr, N ! O Migration 363
10.3.3.5 Asp and Asn 363
10.3.3.6 Arg 364
10.3.3.7 N-Alkylamino Acids 365
10.3.3.8 Work-Up 366
10.4 Final Remarks 366
References 366
11 Strategy in Solid-Phase Peptide Synthesis 371
Kleomenis Barlos and Knut Adermann
11.1 Synthetic Strategies Utilizing Solid-Phase Peptide Synthesis
Methods 371
11.2 Solid Support: Resins and Linkers 373
11.3 Developing the Synthetic Strategy: Selection of the Protecting
Group Scheme 374
11.4 Resin Loading 376
11.5 SBS Peptide Chain Elongation: Coupling and Activation 377
11.6 Piperazine Formation 378
11.7 Solid-Phase Synthesis of Protected Peptide Segments 379
11.8 Fragment Condensation Approach: Convergent and Hybrid
Syntheses 379
11.9 Cleavage from the Resin and Global Peptide Deprotection 382
11.10 Disulfide Bond-Containing Peptides 384
11.11 Native Chemical Ligation (NCL) 386
11.12 SPPS of Peptides Modified at their C-Terminus 388
11.13 Side-Chain-Modified Peptides 390
11.14 Cyclic Peptides 392
11.15 Large-Scale Solid-Phase Synthesis 394
11.16 Conclusions 395
References 396
12 Peptide-Coupling Reagents 407
Ayman El- Faham and Fernando Albericio
12.1 Introduction 407
12.2 Carbodiimides 409
12.2.1 General Procedure for Coupling Using Carbodiimide and HOXt;
Solution Phase 413
12.2.1.1 General Procedure for Solid-Phase Coupling via Carbodiimide
Activation 414
12.2.2 Loading of Wang Resin Using Carbodiimide 415
12.3 Phosphonium Salts 416
12.3.1 Preparation of Phosphonium Salts 418
12.3.2 General Method for the Synthesis of Phosphonium Salts 420
12.4 Aminium/Uronium Salts 420
12.4.1 Stability of Onium Salts 425
12.4.2 General Procedure for the Preparation of Chloroformamidinium
Salts 426
12.4.3 Synthesis of Aminium/Uronium Salts 427
12.4.4 General Procedure for Coupling Using Onium Salts
(Phosphonium and Uronium) in Solution Phase 427
12.4.5 General Procedure for Coupling Reaction in Solid-Phase Using
Onium Salts (Phosphonium and Uronium) 427
12.4.6 General Procedures for Coupling Reaction in Solid-Phase Using
Onium Salts (Phosphonium and Uronium) Boc-, Fmoc-Amino
Acids via Phosphonium and Uronium Salts 427
12.5 Fluoroformamidinium Coupling Reagents 429
12.5.1 General Method for the Synthesis of Fluoroformamidinium
Salts 431
12.5.2 Solution- and Solid-Phase Couplings via TFFH 432
12.5.3 General Method for Solid-Phase Coupling via TFFH 432
12.6 Organophosphorus Reagents 432
12.6.1 General Method for Synthesis of the Diphenylphosphoryl
Derivatives 435
12.7 Triazine Coupling Reagents 435
12.7.1 Formation of the Peptide Bond Using DMTMM (128) 437
12.8 Mukaiyamas Reagent 437
12.9 Conclusions 438
References 439
13 Chemoselective Peptide Ligation: A Privileged Tool for
Protein Synthesis 445
Christian P.R. Hackenberger, Jeffrey W. Bode, and Dirk Schwarzer
13.1 Introduction 445
13.2 Chemoselective Peptide Ligations Following a Capture/
Rearrangement Strategy 449
13.2.1 Basic Concepts and Early Experiments 449
13.2.2 NCL 452
13.2.3 Protein Semisynthesis with NCL 454
13.2.4 Protein Semisynthesis with Expressed Protein Ligation 456
13.2.5 Protein Trans-Splicing 457
13.3 Chemical Transformations for Cys-Free Ligations in Peptides
and Proteins 460
13.3.1 Chemical Modification of NCL Products 460
13.3.1.1 Desulfurization 463
13.3.1.2 Alkylation and Thioalkylation Protocols 464
13.3.2 Auxiliary Methods 466
13.3.2.1 (Oxy-)Ethanethiol Auxiliary 467
13.3.2.2 Photoremovable Na-1-Aryl-2-Mercaptoethyl Auxiliary 468
13.3.2.3 4,5,6-Trimethoxy-2-Mercaptobenzylamine Auxiliary 468
13.3.2.4 Sugar-Assisted Glycopeptide Ligations 469
13.4 Other Chemoselective Capture Strategies 471
13.4.1 Traceless Staudinger Ligation 471
13.4.1.1 Imine Ligations with Subsequent Pseudo-Pro Formation 473
13.5 Peptide Ligations by Chemoselective Amide-Bond-Forming
Reactions 474
13.5.1 Thio Acid/Azide Amidation 475
13.5.2 Thio Acid/N-Arylsulfonamide Ligations 475
13.5.3 Chemoselective Decarboxylative Amide Ligation 477
13.6 Strategies for the Ligation of Multiple Fragments 479
13.6.1 Synthetic Erythropoietin 480
13.6.2 Convergent Strategies for Multiple Fragment Ligations 480
13.6.2.1 Ubiquitylated Histone Proteins 484
References 486
14 Automation of Peptide Synthesis 495
Carlo Di Bello, Andrea Bagno, and Monica Dettin
14.1 Introduction 495
14.2 SPPS: From Mechanization to Automation 497
14.3 Deprotection Step: Monitoring and Control 500
14.4 Coupling Step: Monitoring and Control 505
14.5 Integrated Deprotection and Coupling Control 509
References 514
15 Peptide Purification by Reversed-Phase Chromatography 519
Ulrike Kusebauch, Joshua McBee, Julie Bletz, Richard J. Simpson,
and Robert L. Moritz
15.1 RP-HPLC of Peptides 519
15.2 Peptide properties 520
15.3 Chromatographic Principles 520
15.3.1 Choice of Mobile Phase 520
15.3.1.1 Mobile-Phase Aqueous Buffer pH 520
15.3.1.2 Organic Solvent 522
15.3.2 Stationary Phase 523
15.3.2.1 Surface Bonding 523
15.3.2.2 Pore Diameter 523
15.3.2.3 Particle Size 524
15.3.2.4 Ultra-High-Pressure Liquid Chromatography 525
15.3.2.5 Synthetic Polymer Packings 525
15.3.2.6 Monolithic Stationary Phase 525
15.3.2.7 Packed Bed (Column) Length 526
15.3.2.8 Gradient Effect 527
15.3.2.9 Temperature 527
15.4 Prediction of Peptide Retention Times 528
15.5 Advantages of Reduced Scale 531
15.6 Two-Dimensional Chromatographic Methods 532
15.7 Peptide Analysis in Complex Biological Matrices 533
15.8 Standard Methods for Peptide Separations for Analysis by
Hyphenated Techniques 534
15.9 Emerging Methods for Peptide Separations for Analysis by
Hyphenated Techniques 534
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 Difficult Peptides 549
M. Ter^esa Machini Miranda, Cleber W. Liria, and Cesar Remuzgo
16.1 Importance of Peptide Synthesis 549
16.2 Methods for Peptide Synthesis 550
16.3 Chemical Peptide Synthesis 551
16.4 ‘‘Difficult Peptide Sequences’’ 554
16.5 Means to Overcome Peptide Aggregation in SPPS 556
16.5.1 In Situ Neutralization 556
16.5.2 Solvents for Peptide Chain Assembly 557
16.5.3 Type and Substitution Degree of Resins for Peptide Chain
Assembly 557
16.5.4 Use of Chaotropic Salts During Peptide Chain Assembly 558
16.5.5 Use of Amide Backbone Protection 558
16.5.6 The Use of Pseudo-Prolines 560
16.5.7 O-Acyl Isopeptide Approach 561
16.5.8 Use of Elevated Temperatures 562
16.6 Monitoring the Synthesis of a ‘‘Difficult Peptide’’ 562
16.7 Conclusions 564
References 564
Index 571

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