Practical Methods for Biocatalysis and Biotransformations 2

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

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 2 is a "how-to" guide that focuses on the practical applications of enzymes and strains of microorganisms that are readily obtained or derived from culture collections. The sources of starting materials and reagents, hints, tips and safety advice (where appropriate) are given to ensure, as far as possible, that the procedures are reproducible. Comparisons to alternative methodology are given and relevant references to the primary literature are cited. This second volume - which can be used on its own or in combination with the first volume - concentrates on new applications and new enzyme families reported since the first volume. Contents include:
* introduction to recent developments and future needs in biocatalysts and synthetic biology in industry
* reductive amination
* enoate reductases for reduction of electron deficient alkenes
* industrial carbonyl reduction
* regio- and stereo- selective hydroxylation
* oxidation of alcohols
* selective oxidation
* industrial hydrolases and related enzymes
* transferases for alkylation, glycosylation and phosphorylation
* C-C bond formation and decarboxylation
* halogenation/dehalogenation/heteroatom oxidation
* tandem and sequential multi-enzymatic syntheses
 
Practical Methods for Biocatalysis and Biotransformations 2 is an essential collection of biocatalytic methods for chemical synthesis which will find a place on the bookshelves of synthetic organic chemists, pharmaceutical chemists, and process R&D chemists in industry and academia.

Biocatalysts are increasingly used by chemists engaged in finechemical synthesis within both industry and academia. Today, thereexists a huge choice of high-tech enzymes and whole cellbiocatalysts, which add enormously to the repertoire of syntheticpossibilities.

List of Contributors ix

Abbreviations xxiii

1 Biocatalysis in the Fine Chemical and PharmaceuticalIndustries 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 - Cambrex36

1.8 Biocatalyst Discovery and Improvement 38

1.9 From Pathway Engineering to Synthetic Biology 42

1.10 Prioritization of Future Biocatalysis and Synthetic BiologyNeeds 47

1.11 Concluding Remarks 52

2 Reductive Amination 61

2.1 o-Transaminases - Useful Biocatalysts for Chiral AmineSynthesis 61

2.2 Preparative Scale Production of a Bulky-Bulky ChiralAmine Using an Engineered Transaminase 64

2.3 Synthesis of Optically Pure Amines Employing o-Transaminases69

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

2.5 Asymmetric Synthesis of L-3-Hydroxyadamantylglycine UsingBranched Chain Aminotransferase 79

2.6 Asymmetric Reduction of Aryl Imines Using Candidaparapsilosis ATCC 7330 83

3 Enoate Reductases for Reduction of Electron DeficientAlkenes 87

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

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

3.3 Asymmetric Reduction of (4S)-(+)-Carvone Catalyzed by EnoateReductases (ERs) Expressed by Non-Conventional Yeast (NCY) WholeCells 100

3.4 Preparation of Enantiomerically Pure Citronellal EnantiomersUsing Alkene Reductases 104

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

4 Industrial Carbonyl Reduction 115

4.1 Bioreduction Using Immobilized Carbonyl Reductase Technology116

4.2 Preparative Ketoreductase-Catalyzed Kinetic Resolution of aRacemic Aldehyde 118

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

4.4 Preparative Scale Production of Poorly Soluble ChiralAlcohol Intermediate for Montelukast 124

5 Regio- and Stereoselective Hydroxylation 129

5.1 Engineering of an Amycolatopsis orientalis P450Compactin Hydroxylase into a Pravastatin Synthase by Changing theStereospecificity of the Enzyme 130

5.2 Recombinant Human Cytochrome P450 Enzymes Expressed in Escherichia coli as Whole Cell Biocatalysts: PreparativeSynthesis of Oxidized Metabolites of an mGlu5 Receptor Antagonist138

5.3 Alpha-Keto Biooxidation Using Cunninghamella echinulata (DSM63356) 147

5.4 Aromatic Hydroxylation: Preparation of3,4-Dihydroxyphenylacetic Acid 150

5.5 Regioselective Aromatic Hydroxylation of Quinaldine UsingLiving Pseudomonas putida Cells Containing Quinaldine4-Oxidase 153

5.6 Regioselective Preparation of 5-Hydroxypropranolol with aFungal 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 of5-Ketogluconic Acid and its Isolation 163

6.2 Selective Enzymatic Oxidation of Atropisomeric Diaryl Ethersby Oxidation with Oxygen and Catalytic Galactose Oxidase M3-5166

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

6.4 ADH Catalyzed Oxidation of Sec-Alcohols Using MolecularOxygen 172

6.5 Irreversible Non-Enantioselective Oxidation of SecondaryAlcohols Using Sphingobium ADH and Chloroacetone as Oxidant175

6.6 Chemoselective Oxidation of Primary Alcohols to Aldehydes177

7 Selective Oxidation 181

7.1 Enantioselective Biocatalytic Oxidative Desymmetrization ofSubstituted Pyrrolidines 182

7.2 Large Scale Baeyer-Villiger Monooxygenase-CatalyzedConversion of (R,S)-3-phenylbuta

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