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
2 Survey of Current Commercial Enzyme and Bioprocess Service Providers 27
2.1 Commercial Enzyme Suppliers/Distributors 28
2.2 Bioprocess Service Providers 92
2.3 Chemical Transformations of Selected Commercially Available Enzymes 103
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
3.2 Expanding the Collection of Immine Reductases Towards a Stereoselective Reductive Amination 138
3.3 Asymmetric Synthesis of the Key Intermediate of Dextromethorphan Catalysed by an Imine Reductase 143
3.4 Identification of Imine Reductases for Asymmetric Synthesis of 1-Aryl-Tetrahydroisoquinolines 148
3.5 Preparation of Imine Reductases at 15 L Scale and Their Application in Asymmetric Piperazine Synthesis 156
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
4.2 Asymmetric Biosynthesis of L-Phosphinothricin by Transaminase 168
4.3 Application of In Situ Product Crystallisation in the Amine Transaminase from Silicibacter pomeroyi-Catalysed Synthesis of (S)-1-(3-Methoxyphenyl)ethylamine 173
4.4 Enantioselective Synthesis of Industrially Relevant Amines Using an Immobilised ω-Transaminase 178
4.5 Amination of Sugars Using Transaminases 182
4.6 Converting Aldoses into Valuable ω-Amino Alcohols Using Amine Transaminases 187
5 Other Carbon–Nitrogen Bond-Forming Biotransformations 193
5.1 Biocatalytic N-Acylation of Anilines in Aqueous Media 193
5.2 Enantioselective Enzymatic Hydroaminations for the Production of Functionalised Aspartic Acids 196
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
5.4 Convenient Approach to the Biosynthesis of C2,C6-Disubstituted Purine Nucleosides Using E. coli Purine Nucleoside Phosphorylase and Arsenolysis 211
5.5 Production of L- and D-Phenylalanine Analogues Using Tailored Phenylalanine Ammonia-Lyases 215
5.6 Asymmetric Reductive Amination of Ketones Catalysed by Amine Dehydrogenases 221
5.7 Utilisation of Adenylating Enzymes for the Formation of N-Acyl Amides 231
6 Carbon–Carbon Bond Formation or Cleavage 237
6.1 Improved Enzymatic Method for the Synthesis of (R)-Phenylacetyl Carbinol 237
6.2 Tertiary Alcohol Formation Catalysed by a Rhamnulose-1-Phosphate Aldolase : Dendroketose-1-Phosphate Synthesis 241
6.3 Easy and Robust Synthesis of Substituted L-Tryptophans with Tryptophan Synthase from Salmonella enterica 247
6.4 Biocatalytic Friedel–Crafts-Type C-Acylation 250
6.5 MenD-Catalysed Synthesis of 6-Cyano-4-Oxohexanoic Acid 256
6.6 Production of (R)-2-(3,5-Dimethoxyphenyl)propanoic Acid Using an Aryl Malonate Decarboxylase from Bordetella bronchiseptica 259
7 Reductive Methods 263
7.1 Synthesis of Vibegron Enabled by a Ketoreductase Rationally Designed for High-pH Dynamic Kinetic Reduction 263
7.2 Synthesis of a GPR40 Partial Agonist Through a Kinetically Controlled Dynamic Enzymatic Ketone Reduction 265
7.3 Lab-Scale Synthesis of Eslicarbazepine 267
7.4 Direct Access to Aldehydes Using Commercially Available Carboxylic Acid Reductases 270
7.5 Preparation of Methyl (S)-3-Oxocyclohexanecarboxylate Using an Enoate Reductase 277
8 Oxidative Methods 281
8.1 Macrocyclic Baeyer–Villiger Monooxygenase Oxidation of Cyclopentadecanone on 1 L Scale 281
8.2 Regioselective Lactol Oxidation with O2 as Oxidant on 1 L Scale Using Alcohol Dehydrogenase and NAD(P)H Oxidase 286
8.3 Synthesis of (3R)-4-[2-Chloro-6-[[(R)-Methylsulphinyl]methyl]-Pyrimidin-4-yl]-3-Methyl-Morpholine Using BVMO-P1-D08 291
8.4 Oxidation of Vanillyl Alcohol to Vanillin with Molecular Oxygen Catalysed by Eugenol Oxidase on 1 L Scale 295
8.5 Synthesis of Syringaresinol from 2,6-Dimethoxy-4-Allylphenol Using an Oxidase/Peroxidase Enzyme System 301
8.6 Biocatalytic Preparation of Vanillin Catalysed by Eugenol Oxidase 308
8.7 Vanillyl Alcohol Oxidase-Catalysed Production of (R)-1-(4′-Hydroxyphenyl) Ethanol 312
8.8 Enzymatic Synthesis of Pinene-Derived Lactones 319
8.9 Enzymatic Preparation of Halogenated Hydroxyquinolines 326
9 Hydrolytic and Dehydratase Enzymes 333
9.1 Synthesis of (S)-3-(4-Chlorophenyl)-4-Cyanobutanoic Acid by a Mutant Nitrilase 333
9.2 Nitrilase-Mediated Synthesis of a Hydroxyphenylacetic Acid Substrate via a Cyanohydrin Intermediate 337
9.3 Production of (R)-2-Butyl-2-Ethyloxirane Using an Epoxide Hydrolase from Agromyces mediolanus 339
9.4 Preparation of (S)-1,2-Dodecanediol by Lipase-Catalysed Methanolysis of Racemic Bisbutyrate Followed by Selective Crystallisation 344
9.5 Biocatalytic Synthesis of n-Octanenitrile Using an Aldoxime Dehydratase from Bacillus sp. OxB-1 349
9.6 Access to (S)-4-Bromobutan-2-ol through Selective Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase 354
10 Glycosylation, Sulphation and Phosphorylation 363
10.1 Rutinosidase Synthesis of Glycosyl Esters of Aromatic Acids 363
10.2 Biocatalytic Synthesis of Kojibiose Using a Mutant Transglycosylase 369
10.3 Biocatalytic Synthesis of Nigerose Using a Mutant Transglycosylase 377
10.4 Easy Sulphation of Phenols by a Bacterial Arylsulphotransferase 381
10.5 Shikimate Kinase-Catalysed Phosphorylations and Synthesis of Shikimic Acid 3-Phosphate by AroL-Catalysed Phosphorylation of Shikimic Acid 386
10.6 Kinase-Catalysed Phosphorylations of Ketohexose Phosphates and LacC-Catalysed Synthesis of D-Tagatose 1,6-Diphosphate Lithium Salt 393
10.7 Kinase-Catalysed Phosphorylations of Xylulose Substrates and Synthesis of Xylulose-5-Phosphate Enantiomers 397
10.8 Phosphoramidates by Kinase-Catalysed Phosphorylation and Arginine Kinase-Catalysed Synthesis of Nω-Phospho-L-Arginine 401
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
11.2 Asymmetric Synthesis of α-Amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids 413
11.3 Enantioselective, Catalytic One-Pot Synthesis of γ-Butyrolactone-Based Fragrances 420
11.4 Synthesis of Six out of Eight Carvo-Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)-and (S)-Carvones 426
11.5 One-Pot Biocatalytic Synthesis of D-Tryptophan Derivatives from Substituted Indoles and L-Serine 435
11.6 Escherichia coli Lysate Multienzyme Biocatalyst for the Synthesis of Uridine-5’-Triphosphate from Orotic Acid 4 and Ribose 1 441
11.7 Aerobic Synthesis of Aromatic Nitriles from Alcohols and Ammonia Using Galactose Oxidase 449
11.8 Hydrogen-Borrowing Conversion of Alcohols into Optically Active Primary Amines by Combination of Alcohol Dehydrogenases and Amine Dehydrogenases 455
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
12 Chemo-Enzymatic Cascades 475
12.1 Synergistic Nitroreductase/Vanadium Catalysis for Chemoselective Nitroreductions 475
12.2 Chemo-Enzymatic Synthesis of (S)-1,2,3,4-Tetrahydroisoquinoline Carboxylic Acids Using D-Amino Acid Oxidase 482
12.3 Amine Oxidase-Catalysed Deracemisation of (R,S)-4-Cl-Benzhydrylamine into the (R)-Enantiomer in the Presence of a Chemical Reductant 488
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
12.5 Chemoenzymatic Synthesis of (2S,3S)-2-Methylpyrrolidin-3-Ol 504
13 Whole-Cell Procedures 509
13.1 Semipreparative Biocatalytic Synthesis of (S)-1-Amino-1-(3’-Pyridyl)methylphosphonic Acid 509
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
13.3 Clean Enzymatic Oxidation of 12α-Hydroxysteroids to 12-Oxo-Derivatives Catalysed by Hydroxysteroid Dehydrogenase 521
13.4 Whole-Cell Biocatalysis Using PmlABCDEF Monooxygenase and Its Mutants: A Versatile Toolkit for Selective Synthesis of Aromatic N-Oxides 528
Index 535
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