Practical Methods for Biocatalysis and Biotransformations 2

John Whittall, Manchester Interdisciplinary Biocentre, Manchester University, UK.

Peter W Sutton, GlaxoSmithKline Research and Development Limited, UK.

List of Contributors ix

Abbreviations xxiii

1 Biocatalysis in the Fine Chemical and Pharmaceutical Industries 1

1.1 Introduction 1

1.2 Biotrans Outsourcing – AstraZeneca 4

1.3 Biotrans Trends – Lonza 5

1.4 Biocatalysis in the Pharma Environment 9

1.5 Industrial Use of Hydrolases 24

1.6 Industrial Biooxidation and Reduction 32

1.7 Industrial Application of Transaminases – Cambrex 36

1.8 Biocatalyst Discovery and Improvement 38

1.9 From Pathway Engineering to Synthetic Biology 42

1.10 Prioritization of Future Biocatalysis and Synthetic Biology Needs 47

1.11 Concluding Remarks 52

2 Reductive Amination 61

2.1 o-Transaminases – Useful Biocatalysts for Chiral Amine Synthesis 61

2.2 Preparative Scale Production of a Bulky–Bulky Chiral Amine Using an Engineered Transaminase 64

2.3 Synthesis of Optically Pure Amines Employing o-Transaminases 69

2.4 A Fast, Sensitive Assay and Scale-Up of o-Transaminase Catalysed Reactions 74

2.5 Asymmetric Synthesis of L-3-Hydroxyadamantylglycine Using Branched Chain Aminotransferase 79

2.6 Asymmetric Reduction of Aryl Imines Using Candida parapsilosis ATCC 7330 83

3 Enoate Reductases for Reduction of Electron Deficient Alkenes 87

3.1 Asymmetric Bioreduction of Activated Alkenes Using Ene-Reductases from the Old Yellow Enzyme Family 87

3.2 Efficient Baker's Yeast Mediated Reduction with Substrate Feeding Product Removal (SFPR) Technology: Synthesis of (S)-2-Alkoxy-3-Aryl-1-Propanols 96

3.3 Asymmetric Reduction of (4S)-(+)-Carvone Catalyzed by Enoate Reductases (ERs) Expressed by Non-Conventional Yeast (NCY) Whole Cells 100

3.4 Preparation of Enantiomerically Pure Citronellal Enantiomers Using Alkene Reductases 104

3.5 Highly Enantiomeric Hydrogenation of C–C Double Bond of Methylated N-Phenyl and N-Phenylalkylmaleimides by Aspergillus fumigatus 108

4 Industrial Carbonyl Reduction 115

4.1 Bioreduction Using Immobilized Carbonyl Reductase Technology 116

4.2 Preparative Ketoreductase-Catalyzed Kinetic Resolution of a Racemic Aldehyde 118

4.3 Enzymatic Reduction of 2,6-dichloro-3-fluoro-acetophenone to Produce (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol 121

4.4 Preparative Scale Production of Poorly Soluble Chiral Alcohol Intermediate for Montelukast 124

5 Regio- and Stereoselective Hydroxylation 129

5.1 Engineering of an Amycolatopsis orientalis P450 Compactin Hydroxylase into a Pravastatin Synthase by Changing the Stereospecificity of the Enzyme 130

5.2 Recombinant Human Cytochrome P450 Enzymes Expressed in Escherichia coli as Whole Cell Biocatalysts: Preparative Synthesis of Oxidized Metabolites of an mGlu5 Receptor Antagonist 138

5.3 Alpha-Keto Biooxidation Using Cunninghamella echinulata (DSM 63356) 147

5.4 Aromatic Hydroxylation: Preparation of 3,4-Dihydroxyphenylacetic Acid 150

5.5 Regioselective Aromatic Hydroxylation of Quinaldine Using Living Pseudomonas putida Cells Containing Quinaldine 4-Oxidase 153

5.6 Regioselective Preparation of 5-Hydroxypropranolol with a Fungal Peroxygenase 158

5.7 Microbial Conversion of b-Myrcene to Geraniol by a Strain of Rhodococcus 159

6 Oxidation of Alcohols 163

6.1 Preparative Method for the Enzymatic Synthesis of 5-Ketogluconic Acid and its Isolation 163

6.2 Selective Enzymatic Oxidation of Atropisomeric Diaryl Ethers by Oxidation with Oxygen and Catalytic Galactose Oxidase M3–5 166

6.3 Kinetic Resolution of Chiral Secondary Alcohols by Oxidation with Oxygen and Catalytic Galactose Oxidase M3-5 169

6.4 ADH Catalyzed Oxidation of Sec-Alcohols Using Molecular Oxygen 172

6.5 Irreversible Non-Enantioselective Oxidation of Secondary Alcohols Using Sphingobium ADH and Chloroacetone as Oxidant 175

6.6 Chemoselective Oxidation of Primary Alcohols to Aldehydes 177

7 Selective Oxidation 181

7.1 Enantioselective Biocatalytic Oxidative Desymmetrization of Substituted Pyrrolidines 182

7.2 Large Scale Baeyer–Villiger Monooxygenase-Catalyzed Conversion of (R,S)-3-phenylbutan-2-one 186

7.3 Synthesis of Optically Active 3-Alkyl-3-,4-dihydroioscoumarins by Dynamic Kinetic Resolutions Catalyzed by a Baeyer–Villiger Monooxygenase 190

7.4 Oxidative Cleavage of the B-Ring of (+)-Catechin 193

7.5 18O-Isotopic Labeling in the Meta-Dioxygenase Cleavage of (þ)-Catechin B-Ring 196

7.6 Biocatalytic Cleavage of Alkenes with Oxygen and Trametes hirsuta G FCC047 199

8 Industrial Hydrolases and Related Enzymes 203

8.1 Dynamic Kinetic Resolution of a-Halo Esters with Hydrolytic Enzymes and Sec-amine Bases 203

8.2 Kinetic Resolution of an Amino Ester Using Supported Mucor miehei Lipase (Lipozyme RM IM) 207

8.3 Large Scale Synthesis of (S)-Allysine Ethylene Acetal via Amino Acylase Resolution 212

8.4 Pilot-Scale Synthesis of (1R,2S,4S)-7-Oxabicyclo[2.2.1] heptan-2-exo-carboxylic Acid 214

8.5 A Selective Lipase-Catalyzed Mono-Acetylation of a Diol Suitable for a Telescoped Synthetic Process 217

8.6 A Protease-Mediated Hydrolytic Kinetic Resolution of an Atropisomeric Ester Operating Within an Unusually Narrow pH Window 220

8.7 Asymmetric Synthesis of Quaternary Amino Acids from Simple Bis Nitriles Using a Dual Nitrile Hydratase/Amidase Biocatalyzed Reaction 223

8.8 Development of an Improved Immobilized CAL-B for the Enzymatic Resolution of a Key Intermediate to Odanacatib 227

9 Transferases for Alkylation, Glycosylation and Phosphorylation 231

9.1 Industrial Production of Caffeic Acid-a-D-O-Glucoside 232

9.2 Enzymatic Synthesis of 5-Methyluridine by Transglycosylation of Guanosine and Thymine 235

9.3 Preparation and Use of Sucrose Phosphorylase as Cross-Linked Enzyme Aggregate (CLEA) 240

9.4 Enzymatic Synthesis of Phosphorylated Carbohydrates and Alcohols 244

9.5 Biocatalyzed Synthesis of Chiral O-Phosphorylated Derivative of 2-Hydroxy-2-phenylethanephosphonate 247

9.6 High Activity b-Galactosidase Preparation for Diastereoselective Synthesis of (R)-(1-Phenylethyl)-b-D-Galactopyranoside by Reverse Hydrolysis 250

9.7 Stereospecific Synthesis of Aszonalenins by Using Two Recombinant Prenyltransferases 254

9.8 Enzymatic Friedel–Crafts Alkylation Catalyzed by S-Adenosyl-L-methionine Dependent Methyl Transferase 258

10 C–C Bond Formation and Decarboxylation 263

10.1 Enzymatic, Stereoselective Synthesis of (S)-Norcoclaurine 264

10.2 Preparation of Non-Natural Tyrosine Derivatives from Pyruvate and Phenol Derivatives 267

10.3 Enzymatic a-Decarboxylation of L-Glutamic Acid in the Production of Biobased Chemicals 269

10.4 Asymmetric Decarboxylation of Arylmalonates and Racemization of Profens by Arylmalonate Decarboxylase and its Variants 274

10.5 Improved Enzymatic Preparation of 1-Deoxy-D-xylulose 5-Phosphate 280

10.6 On the Use of 2-Methyltetrahydrofuran (2-MeTHF) as Bio-Based (Co-) Solvent in Biotransformations 284

10.7 The Lipase-Catalyzed Asymmetric Michael Addition of Thienyl Nitroolefin to Acetylacetone 291

11 Halogenation/Dehalogenation/Heteroatom Oxidation 297

11.1 Preparation of Halogenated Molecules by a Fungal Flavin-Dependent Halogenase Heterologously Expressed in Escherichia coli 299

11.2 Preparation of Optically Pure Haloalkanes and Alcohols by Kinetic Resolution Using Haloalkane Dehalogenases 301

11.3 Preparation of Enantiopure Sulfoxides by Enantioselective Oxidation with Whole Cells of Rhodococcus sp. ECU0066 307

11.4 Kinetic Resolution of an Insecticidal Dithiophosphate by Chloroperoxidase Catalyzed Oxidation of the Thiophosphoryl Group 310

12 Tandem and Sequential Multi-Enzymatic Syntheses 313

12.1 Production of Isorhamnetin 3-O-Glucoside in Escherichia coli Using Engineered Glycosyltransferase 313

12.2 Multienzymatic Preparation of (-)-3-(Oxiran-2-yl)Benzoic Acid 317

12.3 Enzymatic Synthesis of Carbohydrates from Dihydroxyacetone and Aldehydes by a One Pot Enzyme Cascade Reaction 321

12.4 Aldolase Based Multi-Enzyme System for Carbon–Carbon Bond Formation 323

12.5 Tandem Biocatalytic Process for the Kinetic Resolution of b-Phenylalanine and its Analogs 331

12.6 A Chemoenzymatic Synthesis of a Deoxy Sugar Ester of N-Boc-Protected L-Tyrosine 335

12.7 Electrochemical Systems for the Recovery of Succinic Acid from Fermentations 339

Appendix 347

Index 355

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