Practical Methods for Biocatalysis and Biotransformations 3

Biocatalysts are increasingly used by chemists engaged in fine chemical synthesis within both industry and academia. Today, there exists a huge choice of high-tech enzymes and whole cell biocatalysts, which add enormously to the repertoire of synthetic possibilities.

Practical Methods for Biocatalysis and Biotransformations 3 will be a companion book to Practical Methods for Biocatalysis and Biotransformations (2009) and Practical Methods for Biocatalysis and Biotransformations 2 (2012). Following the successful format of the two volumes, it will be a “how-to” guide focusing on commercially available enzymes and strains of microorganisms that are readily obtained from culture collections. The source of starting materials and reagents, hints, tips and safety advice (where appropriate) will be given to ensure, as far as possible, that the procedures are reproducible. Comparisons to alternative methodology will be given and relevant references to the primary literature will be cited.

Contents include:

• Biotransformation Process Technology
• Industrial Biooxidation
• Hydrolase catalysed hydrolysis/synthesis
• Reduction
• Oxidation
• Halogenation
• Transferase catalysed glycosylation, methylation, etc
• C-C bond formation
• Tandem Biocatalytic Reactions

Practical Methods for Biocatalysis and Biotransformations, Volume 3 is an essential collection of validated biocatalytic methods which will find a place on the bookshelves of synthetic organic chemists, pharmaceutical chemists, and process R&D chemists in industry and academia.

Dr John Whittall, Centre of Excellence for Biocatalysis, Biotransformations and Biocatalytic Manufacture (CoEBio3), Manchester Interdisciplinary Biocentre, University of Manchester, UK.

Dr Peter W Sutton, Senior Scientist, GlaxoSmithKline Research and Development Limited, UK.

Dr Wolfgang Kroutil, Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Austria.

List of Contributors ix

Abbreviations xxi

1 Considerations for the Application of Process Technologies in Laboratory- and Pilot-Scale Biocatalysis for Chemical Synthesis 1

1.1 Introduction 1

1.2 Process Intensification and Proposed Scale-Up Concept 2

1.3 Enabling Technologies 5

1.4 Enhancing Technologies 20

1.5 Conclusion 28

References 28

2 Cytochrome P450 (CYP) Progress in Biocatalysis for Synthetic Organic Chemistry 31

2.1 Introduction 31

2.2 CYP Development 32

2.3 Recent Developments 34

2.4 Conclusion 41

References 41

3 Use of Hydrolases and Related Enzymes for Synthesis 43

3.1 Continuous-Flow Reactor-Based Enzymatic Synthesis of Phosphorylated Compounds on a Large Scale 43

3.2 Deracemization of sec-Alcohols via Enantio-Convergent Hydrolysis of rac-Sulfate Esters 45

3.3 Dynamic Kinetic Resolution of a Primary Amine by an Efficient Bifunctional Pd-CALB Hybrid Catalyst. A Metalloenzyme Mimic for Enhanced Cooperative Catalysis 50

3.4 Highly Efficient DKR of Secondary 1-Phenylethanol Derivatives Using a Low-Cost Solid Super Acid as Racemization Catalyst 53

3.5 Identification of New Biocatalysts for the Enantioselective Conversion of Tertiary Alcohols 58

3.6 Enzyme-Catalyzed Hydrolysis of Bicycloheptane Diester to Monoester 60

3.7 Double Mutant Lipase with Enhanced Activity and Enantioselectivity for Bulky Secondary Alcohols 64

3.8 Stereoselective Synthesis of β-Amino Acids by Hydrolysis of an Aryl-Substituted Dihydropyrimidine by Hydantoinases 68

References 72

4 Non-Redox Lyases and Transferases for C-C, C-O, C-S, and C-N Bond Formation 75

4.1 Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrenes Employing Co-Factor-Independent Decarboxylases 75

4.2 Stetter Reactions Catalyzed by Thiamine Diphosphate-Dependent Enzymes 81

4.3 Asymmetric Michael-Type Additions of Acetaldehyde to Nitroolefins Catalyzed by 4-Oxalocrotonate Tautomerase (4-OT) Yielding Valuable γ-Nitroaldehydes 85

4.4 Michael-Type Addition of Aldehydes to β-Nitrostyrenes by Whole Cells of Escherichia coli Expressing 4-Oxalocrotonate Tautomerase (4-OT) 91

4.5 Norcoclaurine Synthases for the Biocatalytic Synthesis of Tetrahydroisoquinolines 95

4.6 Streptavidin-Based Artificial Metallo-Annulase for the Enantioselective Synthesis of Dihydroisoquinolones 101

4.7 Regiospecific Benzylation of Tryptophan and Derivatives Catalyzed by a Fungal Dimethylallyl Transferase 102

4.8 Enantioselective Michael Addition of Water Using Rhodococcus Rhodochrous ATCC 17895 106

4.9 Sulfation of Various Compounds by an Arylsulfotransferase from Desulfitobacterium hafniense and Synthesis of 17β-Estradiol-3-Sulfate 111

4.10 Asymmetric Synthesis of Cyclopropanes and Benzosultams via Enzyme-Catalyzed Carbenoid and Nitrenoid Transfer in E. coli Whole Cells 113

4.11 Biocatalytic Production of Novel Glycolipids 118

4.12 Enzymatic Synthesis of 8-Aza- and 8-Aza-7-Deazapurine 2´-Deoxyribonucleosides 124

4.13 Phenylalanine Ammonia Lyase-Catalyzed Asymmetric Hydroamination for the Synthesis of L-Amino Acids 128

References 130

5 Oxidations 135

5.1 Semi-Preparative-Scale Drug Metabolite Synthesis with Human Flavin Monooxygenases 135

5.2 Biobased Synthesis of Industrially Relevant Nitriles by Selective Oxidative Decarboxylation of Amino Acids by Vanadium Chloroperoxidase 139

5.3 Terminal Oxygenation of Fatty Acids by a CYP153A Fusion Construct Heterologously Expressed in E. coli 142

5.4 Enantioselective Oxidative C-C Bond Formation in Isoquinoline Alkaloids Employing the Berberine Bridge Enzyme 144

5.5 Oxidation of Aldehydes Using Alcohol Dehydrogenases 148

5.6 MAO-Catalyzed Deracemization of Racemic Amines for the Synthesis of Pharmaceutical Building Blocks 150

5.7 Synthesis of (S)-Amines by Chemo-Enzymatic Deracemization Using an (R)-Selective Amine Oxidase 153

5.8 Selective Oxidation of Diols into Lactones under Aerobic Conditions Using a Laccase-TEMPO Catalytic System in Aqueous Medium 156

References 160

6 Reductions 163

6.1 Tetrahydroxynaphthalene Reductase: Broad Substrate Range of an NADPH-Dependent Oxidoreductase Involved in Reductive Asymmetric Naphthol Dearomatization 163

6.2 Chemoenzymatic Synthesis of Diastereo- and Enantiomerically Pure 2,6-Disubstituted Piperidines via Regioselective Monoamination of 1,5-Diketones 167

6.3 Asymmetric Amination of Ketones Employing ω-TAs in Organic Solvents 171

6.4 Stereoselective Synthesis of (R)-Profen Derivatives by the Enoate Reductase YqjM 176

6.5 Productivity Improvement of the Bioreduction of α,β-Unsaturated Aldehydes by Coupling of the In Situ Substrate Feeding Product Removal (SFPR) Strategy with Isolated Enzymes 181

6.6 Reduction of Imines by Recombinant Whole-Cell E. coli Biocatalysts Expressing Imine Reductases (IREDs) 186

References 191

7 Halogenation and Dehalogenation 193

7.1 Site-Directed Mutagenesis Changes the Regioselectivity of the Tryptophan 7-Halogenase PrnA 193

7.2 Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution 197

7.3 Enzymatic Production of Chlorothymol and its Derivatives by Halogenation of the Phenolic Monoterpenes Thymol and Carvacrol with Chloroperoxidase 201

7.4 Halogenation of Non-Activated Fatty Acyl Groups by a Trifunctional Non-Heme Fe(II)-Dependent Halogenase 204

References 211

8 Cascade Reactions 213

8.1 Synthetic Cascades via a Combination of Artificial Metalloenzymes with Monoamine Oxidases (MAO-Ns) 213

8.2 Amination of Primary Alcohols via a Redox-Neutral Biocascade 215

8.3 Biocatalytic Synthesis of a Diketobornane as a Building Block for Bifunctional Camphor Derivatives 218

8.4 Three Enzyme-Catalyzed Redox Cascade for the Production of a Carvo-Lactone 222

8.5 Preparation of Homoallylic Alcohols via a Chemoenzymatic One-Pot Oxidation-Allylation Cascade 226

8.6 Cascade Biotransformations via Enantioselective Reduction, Oxidation, and Hydrolysis: Preparation of (R)-δ-Lactones from 2-Alkylidenecyclopentanones 230

8.7 One-Pot Tandem Enzymatic Reactions for Efficient Biocatalytic Synthesis of D-Fructose-6-Phosphate and Analogs 232

8.8 Efficient One-Pot Tandem Biocatalytic Process for a Valuable Phosphorylated C8 D-Ketose: D-Glycero-D-Altro-2-Octulose 8-Phosphate 239

8.9 Chemoenzymatic Synthesis of (S)-1,2,3,4-Tetrahydroisoquinoline-3-Carboxylic Acid by PAL-Mediated Amination and Pictet-Spengler Cyclization 243

8.10 ω-TA/MAO Cascade for the Regio- and Stereoselective Synthesis of Chiral 2,5-Disubstituted Pyrrolidines 246

References 249

9 Biocatalysis for Industrial Process Development 253

9.1 Efficient Synthesis of (S)-1-(5-Fluoropyrimidin-2-yl)ethylamine Hydrochloride Salt Using an ω-Transaminase Biocatalyst in a Two-Phase System 253

9.2 Preparative-scale Production of a Chiral, Bicyclic Proline Analog Intermediate for Boceprevir 257

9.3 Focused Carbonyl Reductase Screening for Rapid Gram Supply of Highly Enantioenriched Secondary Alcohol Libraries 260

9.4 A Rapid, Inexpensive and Colorimetric High-throughput Assay Format for Screening Commercial Ketoreductase Panels, Providing Indication of Substrate Scope, Co-factor Specificity and Enantioselectivity 266

9.5 Stereoselective Production of (R)-3-quinuclidinol Using Recombinant Escherichia coli Whole Cells Overexpressing 3-Quinuclidinone Reductase and a Co-factor Regeneration System 273

9.6 Preparation of N-Boc-D-Serine Using a Coupled D-Acylase/Racemase Enzyme System 275

9.7 Scale-up of a Biocatalytic Oxidase in a Dynamically Mixed Tubular Flow Reactor 279

References 282

Index 285

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