Abbreviations xi
1 Directed Evolution of Enzymes Driving Innovation in API Manufacturing at GSK 1
1.1 Introduction 1
1.2 Drug Development Stages 3
1.3 Enzyme Panels 6
1.4 Enzyme Engineering 10
1.5 Case Studies 18
1.6 Outlook 22
Acknowledgements 23
References 23
2 Survey of Current Commercial Enzyme and Bioprocess Service Providers 27
Acknowledgements 133
References 133
3 Imine Reductases 135
3.1 Imine Reductase-Catalysed Enantioselective Reductive Amination for the Preparation of a Key Intermediate to Lysine-Specific Histone Demethylase 1 (LSD1) Inhibitor GSK2879552 135
References 138
3.2 Expanding the Collection of Immine Reductases Towards a Stereoselective Reductive Amination 138
References 143
3.3 Asymmetric Synthesis of the Key Intermediate of Dextromethorphan Catalysed by an Imine Reductase 143
References 148
3.4 Identification of Imine Reductases for Asymmetric Synthesis of 1-Aryl-Tetrahydroisoquinolines 148
Acknowledgements 155
References 155
3.5 Preparation of Imine Reductases at 15 L Scale and Their Application in Asymmetric Piperazine Synthesis 156
References 161
3.6 Screening of Imine Reductases and Scale-Up of an Oxidative Deamination of an Amine for Ketone Synthesis 162
4 Transaminases 165
4.1 A Practical Dynamic Kinetic Transamination for the Asymmetric Synthesis of the CGRP Receptor Antagonist Ubrogepant 165
References 167
4.2 Asymmetric Biosynthesis of L-Phosphinothricin by Transaminase 168
References 172
4.3 Application of In Situ Product Crystallisation in the Amine Transaminase from Silicibacter pomeroyi-Catalysed Synthesis of
(S)-1-(3-Methoxyphenyl)ethylamine 173
References 177
4.4 Enantioselective Synthesis of Industrially Relevant Amines Using an Immobilised -Transaminase 178
References 181
4.5 Amination of Sugars Using Transaminases 182
References 186
4.6 Converting Aldoses into Valuable -Amino Alcohols Using Amine Transaminases 187
References 190
5 Other Carbon-Nitrogen Bond-Forming Biotransformations 193
5.1 Biocatalytic N-Acylation of Anilines in Aqueous Media 193
References 195
5.2 Enantioselective Enzymatic Hydroaminations for the Production of Functionalised Aspartic Acids 196
References 203
5.3 Biocatalytic Asymmetric Aza-Michael Addition Reactions and Synthesis of L-Argininosuccinate by Argininosuccinate Lyase
ARG4-Catalysed Aza-Michael Addition of L-Arginine to Fumarate 204
References 210
5.4 Convenient Approach to the Biosynthesis of C2,C6-Disubstituted Purine Nucleosides Using E. coli Purine Nucleoside Phosphorylase and Arsenolysis 211
References 214
5.5 Production of L- and D-Phenylalanine Analogues Using Tailored Phenylalanine Ammonia-Lyases 215
References 220
5.6 Asymmetric Reductive Amination of Ketones Catalysed by Amine Dehydrogenases 221
References 230
5.7 Utilisation of Adenylating Enzymes for the Formation of N-Acyl Amides 231
References 236
6 Carbon-Carbon Bond Formation or Cleavage 237
6.1 Improved Enzymatic Method for the Synthesis of (R)-Phenylacetyl Carbinol 237
References 241
6.2 Tertiary Alcohol Formation Catalysed by a Rhamnulose-1-Phosphate Aldolase : Dendroketose-1-Phosphate Synthesis 241
References 246
6.3 Easy and Robust Synthesis of Substituted L-Tryptophans with Tryptophan Synthase from Salmonella enterica 247
References 250
6.4 Biocatalytic Friedel-Crafts-Type C-Acylation 250
References 255
6.5 MenD-Catalysed Synthesis of 6-Cyano-4-Oxohexanoic Acid 256
References 258
6.6 Production of (R)-2-(3,5-Dimethoxyphenyl)propanoic Acid Using an Aryl Malonate Decarboxylase from Bordetella bronchiseptica 259
References 261
7 Reductive Methods 263
7.1 Synthesis of Vibegron Enabled by a Ketoreductase Rationally Designed for High-pH Dynamic Kinetic Reduction 263
References 265
7.2 Synthesis of a GPR40 Partial Agonist Through a Kinetically Controlled Dynamic Enzymatic Ketone Reduction 265
References 267
7.3 Lab-Scale Synthesis of Eslicarbazepine 267
References 270
7.4 Direct Access to Aldehydes Using Commercially Available Carboxylic Acid Reductases 270
Acknowledgements 276
References 276
7.5 Preparation of Methyl (S)-3-Oxocyclohexanecarboxylate Using an Enoate Reductase 277
References 279
8 Oxidative Methods 281
8.1 Macrocyclic Baeyer-Villiger Monooxygenase Oxidation of Cyclopentadecanone on 1 L Scale 281
References 286
8.2 Regioselective Lactol Oxidation with O2 as Oxidant on 1 L Scale Using Alcohol Dehydrogenase and NAD(P)H Oxidase 286
References 291
8.3 Synthesis of (3R)-4-[2-Chloro-6-[[(R)-Methylsulphinyl]methyl]-Pyrimidin-4-yl]-3-Methyl-Morpholine Using BVMO-P1-D08 291
References 295
8.4 Oxidation of Vanillyl Alcohol to Vanillin with Molecular Oxygen Catalysed by Eugenol Oxidase on 1 L Scale 295
References 301
8.5 Synthesis of Syringaresinol from 2,6-Dimethoxy-4-Allylphenol Using an Oxidase/Peroxidase Enzyme System 301
References 307
8.6 Biocatalytic Preparation of Vanillin Catalysed by Eugenol Oxidase 308
References 311
8.7 Vanillyl Alcohol Oxidase-Catalysed Production of (R)-1-(4'-Hydroxyphenyl) Ethanol 312
References 319
8.8 Enzymatic Synthesis of Pinene-Derived Lactones 319
References 325
8.9 Enzymatic Preparation of Halogenated Hydroxyquinolines 326
References 331
9 Hydrolytic and Dehydratase Enzymes 333
9.1 Synthesis of (S)-3-(4-Chlorophenyl)-4-Cyanobutanoic Acid by a Mutant Nitrilase 333
References 337
9.2 Nitrilase-Mediated Synthesis of a Hydroxyphenylacetic Acid Substrate via a Cyanohydrin Intermediate 337
References 339
9.3 Production of (R)-2-Butyl-2-Ethyloxirane Using an Epoxide Hydrolase from Agromyces mediolanus 339
References 343
9.4 Preparation of (S)-1,2-Dodecanediol by Lipase-Catalysed Methanolysis of Racemic Bisbutyrate Followed by Selective Crystallisation 344
References 349
9.5 Biocatalytic Synthesis of n-Octanenitrile Using an Aldoxime Dehydratase from Bacillus sp. OxB-1 349
References 353
9.6 Access to (S)-4-Bromobutan-2-ol through Selective Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase 354
Appendix 360
References 361
10 Glycosylation, Sulphation and Phosphorylation 363
10.1 Rutinosidase Synthesis of Glycosyl Esters of Aromatic Acids 363
References 368
10.2 Biocatalytic Synthesis of Kojibiose Using a Mutant Transglycosylase 369
References 376
10.3 Biocatalytic Synthesis of Nigerose Using a Mutant Transglycosylase 377
References 381
10.4 Easy Sulphation of Phenols by a Bacterial Arylsulphotransferase 381
References 386
10.5 Shikimate Kinase-Catalysed Phosphorylations and Synthesis of Shikimic Acid 3-Phosphate by AroL-Catalysed Phosphorylation of Shikimic Acid 386
References 393
10.6 Kinase-Catalysed Phosphorylations of Ketohexose Phosphates and LacC-Catalysed Synthesis of D-Tagatose 1,6-Diphosphate Lithium Salt 393
References 396
10.7 Kinase-Catalysed Phosphorylations of Xylulose Substrates and Synthesis of Xylulose-5-Phosphate Enantiomers 397
References 401
10.8 Phosphoramidates by Kinase-Catalysed Phosphorylation and Arginine Kinase-Catalysed Synthesis of N -Phospho-L-Arginine 401
References 407
11 Enzymatic Cascades 409
11.1 Redox-Neutral Ketoreductase and Imine Reductase Enzymatic Cascade for the Preparation of a Key Intermediate of the Lysine-Specific Histone Demethylase 1 (LSD1) Inhibitor GSK2879552 409
References 413
11.2 Asymmetric Synthesis of -Amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids 413
References 420
11.3 Enantioselective, Catalytic One-Pot Synthesis of -Butyrolactone-Based Fragrances 420
References 426
11.4 Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)-and (S)-Carvones 426
References 434
11.5 One-Pot Biocatalytic Synthesis of D-Tryptophan Derivatives from Substituted Indoles and L-Serine 435
References 441
11.6 Escherichia coli Lysate Multienzyme Biocatalyst for the Synthesis of Uridine-5'-Triphosphate from Orotic Acid 4 and Ribose 1 441
References 449
11.7 Aerobic Synthesis of Aromatic Nitriles from Alcohols and Ammonia Using Galactose Oxidase 449
References 455
11.8 Hydrogen-Borrowing Conversion of Alcohols into Optically Active Primary Amines by Combination of Alcohol Dehydrogenases and Amine Dehydrogenases 455
References 468
11.9 Ene-Reductase-Mediated Reduction of C=C Double Bonds in the Presence of Conjugated C C Triple Bonds: Synthesis of (S)-2-Methyl-5-Phenylpent-4-Yn-1-Ol 468
References 474
12 Chemo-Enzymatic Cascades 475
12.1 Synergistic Nitroreductase/Vanadium Catalysis for Chemoselective Nitroreductions 475
References 481
12.2 Chemo-Enzymatic Synthesis of (S)-1,2,3,4-Tetrahydroisoquinoline Carboxylic Acids Using D-Amino Acid Oxidase 482
References 487
12.3 Amine Oxidase-Catalysed Deracemisation of (R,S)-4-Cl-Benzhydrylamine into the (R)-Enantiomer in the Presence of a Chemical Reductant 488
References 496
12.4 Asymmetric Synthesis of 1-Phenylpropan-2-Amine from Allylbenzene through a Sequential Strategy Involving a Wacker-Tsuji Oxidation and a Stereoselective Biotransamination 497
References 504
12.5 Chemoenzymatic Synthesis of (2S,3S)-2-Methylpyrrolidin-3-Ol 504
References 508
13 Whole-Cell Procedures 509
13.1 Semipreparative Biocatalytic Synthesis of (S)-1-Amino-1-(3'-Pyridyl)methylphosphonic Acid 509
References 514
13.2 Practical and User-Friendly Procedure for the Regio- and Stereoselective Hydration of Oleic, Linoleic and Linolenic Acids, Using Probiotic Lactobacillus Strains as Whole-Cell Biocatalysts 515
References 520
13.3 Clean Enzymatic Oxidation of 12 -Hydroxysteroids to 12-Oxo-Derivatives Catalysed by Hydroxysteroid Dehydrogenase 521
References 527
13.4 Whole-Cell Biocatalysis Using PmlABCDEF Monooxygenase and Its Mutants: A Versatile Toolkit for Selective Synthesis of Aromatic N-Oxides 528
References 533
Index 535
