Applied Biocatalysis E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 911

Veröffentlichungsjahr: 2020

Beschreibung

Provides clear and comprehensive coverage of recently developed applied biocatalysis for synthetic organic chemists with an emphasis to promote green chemistry in pharmaceutical and process chemistry This book aims to make biocatalysis more accessible to both academic and industrial synthetic organic chemists. It focuses on current topics within the applied industrial biocatalysis field and includes short but detailed experimental methods on timely novel biocatalytic transformations using new enzymes or new methodologies using known enzymes. The book also features reactions that are "expanding and making the enzyme toolbox available to chemists"--providing readers with comprehensive methodology and detailed key sourcing information of a wide range of enzymes. Chapters in Applied Biocatalysis: The Chemist's Enzyme Toolkit are organized by reaction type and feature a short introductory section describing the current state of the art for each example. Much of the book focuses on processes for which the enzymes are readily available so that organic chemists can synthesize appropriate quantities of chemicals with available materials in a standard chemical laboratory. Advanced methods are included to present examples of new enzymes that might encourage collaboration with suppliers or academic groups and that will educate chemists of rapidly expanding future possibilities. * Focuses on current topics within the applied industrial biocatalysis field * Offers experimental methods on novel biocatalytic transformations using new enzymes or new methodology using known enzymes * Covers the hot topics of enzyme and chemoenzymatic cascades and biocatalysis in flow * Edited by noted experts from both academia and industry with years of experience in the field of biocatalysis--particularly, the industrial applications of enzymes Written for synthetic organic chemists working in all industries but especially the pharmaceutical industry and for those in academia with an eye for biocatalysis, Applied Biocatalysis: The Chemist's Enzyme Toolkit will also benefit academic groups in chemistry and related sciences that are using enzymes for synthetic purposes, as well as those working in the area of enzymology and molecular biology.

Leseprobe

Table of Contents

Cover

Abbreviations

1 Directed Evolution of Enzymes Driving Innovation in API Manufacturing at GSK

1.1 Introduction

1.2 Drug Development Stages

1.3 Enzyme Panels

1.4 Enzyme Engineering

1.5 Case Studies

1.6 Outlook

Acknowledgements

References

2 Survey of Current Commercial Enzyme and Bioprocess Service Providers

Commercial Enzyme Suppliers/Distributors

Bioprocess Service Providers

Chemical Transformations of Selected Commercially Available Enzymes

Acknowledgements

Reference

3 Imine Reductases

3.1 Imine Reductase‐Catalysed Enantioselective Reductive Amination for the Preparation of a Key Intermediate to Lysine‐Specific Histone Demethylase 1 (LSD1) Inhibitor GSK2879552

References

3.2 Expanding the Collection of Imine Reductases Towards a Stereoselective Reductive Amination

References

3.3 Asymmetric Synthesis of the Key Intermediate of Dextromethorphan Catalysed by an Imine Reductase

References

3.4 Identification of Imine Reductases for Asymmetric Synthesis of 1‐Aryl‐tetrahydroisoquinolines

Acknowledgements

References

3.5 Preparation of Imine Reductases at 15 L Scale and Their Application in Asymmetric Piperazine Synthesis

References

3.6 Screening of Imine Reductases and Scale‐Up of an Oxidative Deamination of an Amine for Ketone Synthesis

4 Transaminases

4.1 A Practical Dynamic Kinetic Transamination for the Asymmetric Synthesis of the CGRP Receptor Antagonist Ubrogepant

References

4.2 Asymmetric Biosynthesis of L‐Phosphinothricin by Transaminase

References

4.3 Application of In Situ Product Crystallisation in the Amine Transaminase from Silicibacter pomeroyi‐Catalysed Synthesis of (S)‐1‐(3‐Methoxyphenyl)ethylamine

References

4.4 Enantioselective Synthesis of Industrially Relevant Amines Using an Immobilised ω‐Transaminase

References

4.5 Amination of Sugars Using Transaminases

References

4.6 Converting Aldoses into Valuable ω‐Amino Alcohols Using Amine Transaminases

References

5 Other Carbon–Nitrogen Bond‐Forming Biotransformations

5.1 Biocatalytic N‐Acylation of Anilines in Aqueous Media

References

5.2 Enantioselective Enzymatic Hydroaminations for the Production of Functionalised Aspartic Acids

References

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

References

5.4 Convenient Approach to the Biosynthesis of C2,C6‐Disubstituted Purine Nucleosides Using E. coli Purine Nucleoside Phosphorylase and Arsenolysis

References

5.5 Production of L‐ and D‐Phenylalanine Analogues Using Tailored Phenylalanine Ammonia‐Lyases

References

5.6 Asymmetric Reductive Amination of Ketones Catalysed by Amine Dehydrogenases

References

5.7 Utilisation of Adenylating Enzymes for the Formation of N‐Acyl Amides

References

6 Carbon–Carbon Bond Formation or Cleavage

6.1 An Improved Enzymatic Method for the Synthesis of (R)‐Phenylacetyl Carbinol

References

6.2 Tertiary Alcohol Formation Catalysed by a Rhamnulose‐1‐Phosphate Aldolase : Dendroketose‐1‐Phosphate Synthesis

References

6.3 Easy and Robust Synthesis of Substituted L‐Tryptophans with Tryptophan Synthase from Salmonella enterica

References

6.4 Biocatalytic Friedel–Crafts‐Type C‐Acylation

References

6.5 MenD‐Catalysed Synthesis of 6‐Cyano‐4‐Oxohexanoic Acid

References

6.6 Production of (R)‐2‐(3,5‐Dimethoxyphenyl)propanoic Acid Using an Aryl Malonate Decarboxylase from Bordetella bronchiseptica

References

7 Reductive Methods

7.1 Synthesis of Vibegron Enabled by a Ketoreductase Rationally Designed for High‐pH Dynamic Kinetic Reduction

References

7.2 Synthesis of a GPR40 Partial Agonist Through a Kinetically Controlled Dynamic Enzymatic Ketone Reduction

Reference

7.3 Lab‐Scale Synthesis of Eslicarbazepine

References

7.4 Direct Access to Aldehydes Using Commercially Available Carboxylic Acid Reductases

Acknowledgements

References

7.5 Preparation of Methyl (S)‐3‐Oxocyclohexanecarboxylate Using an Enoate Reductase

References

8 Oxidative Methods

8.1 Macrocyclic Baeyer–Villiger Monooxygenase Oxidation of Cyclopentadecanone on 1 L Scale

References

8.2 Regioselective Lactol Oxidation with O2 as Oxidant on 1 L Scale Using Alcohol Dehydrogenase and NAD(P)H Oxidase

References

8.3 Synthesis of (3R)‐4‐[2‐Chloro‐6‐[[(R)‐Methylsulfinyl]methyl]‐Pyrimidin‐4‐yl]‐3‐Methyl‐Morpholine Using BVMO‐P1‐D08

Reference

8.4 Oxidation of Vanillyl Alcohol to Vanillin with Molecular Oxygen Catalysed by Eugenol Oxidase on 1 L Scale

References

8.5 Synthesis of Syringaresinol from 2,6‐Dimethoxy‐4‐Allylphenol Using an Oxidase/Peroxidase Enzyme System

References

8.6 Biocatalytic Preparation of Vanillin Catalysed by Eugenol Oxidase

References

8.7 Vanillyl Alcohol Oxidase‐Catalysed Production of (R)‐1‐(4′‐Hydroxyphenyl)ethanol

References

8.8 Enzymatic Synthesis of Pinene‐Derived Lactones

References

8.9 Enzymatic Preparation of Halogenated Hydroxyquinolines

References

9 Hydrolytic and Dehydratase Enzymes

9.1 Synthesis of (S)‐3‐(4‐Chlorophenyl)‐4‐Cyanobutanoic Acid by a Mutant Nitrilase

References

9.2 Nitrilase‐Mediated Synthesis of a Hydroxyphenylacetic Acid Substrate via a Cyanohydrin Intermediate

References

9.3 Production of (R)‐2‐Butyl‐2‐Ethyloxirane Using an Epoxide Hydrolase from Agromyces mediolanus

References

9.4 Preparation of (S)‐1,2‐Dodecanediol by Lipase‐Catalysed Methanolysis of Racemic Bisbutyrate Followed by Selective Crystallisation

References

9.5 Biocatalytic Synthesis of n‐Octanenitrile Using an Aldoxime Dehydratase from Bacillus sp. OxB‐1

References

9.6 Access to (S)‐4‐Bromobutan‐2‐ol through Selective Dehalogenation of rac‐1,3‐Dibromobutane by Haloalkane Dehalogenase

Appendix

References

10 Glycosylation, Sulfation and Phosphorylation

10.1 Rutinosidase Synthesis of Glycosyl Esters of Aromatic Acids

References

10.2 Biocatalytic Synthesis of Kojibiose Using a Mutant Transglycosylase

References

10.3 Biocatalytic Synthesis of Nigerose Using a Mutant Transglycosylase

References

10.4 Easy Sulfation of Phenols by a Bacterial Arylsulfotransferase

References

10.5 Shikimate Kinase‐Catalysed Phosphorylations and Synthesis of Shikimic Acid 3‐Phosphate by AroL‐Catalysed Phosphorylation of Shikimic Acid

References

10.6 Kinase‐Catalysed Phosphorylations of Ketohexose Phosphates and LacC‐Catalysed Synthesis of D‐Tagatose‐1,6‐Diphosphate Lithium Salt

References

10.7 Kinase‐Catalysed Phosphorylations of Xylulose Substrates and Synthesis of Xylulose‐5‐Phosphate Enantiomers

References

10.8 Phosphoramidates by Kinase‐Catalysed Phosphorylation and Arginine Kinase‐Catalysed Synthesis of Nω‐Phospho‐L‐Arginine

References

11 Enzymatic Cascades

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

References

11.2 Asymmetric Synthesis of α‐Amino Acids through Formal Enantioselective Biocatalytic Amination of Carboxylic Acids

References

11.3 Enantioselective, Catalytic One‐Pot Synthesis of γ‐Butyrolactone‐Based Fragrances

References

11.4 Synthesis of Six out of Eight Carvo‐Lactone Stereoisomers via a Novel Concurrent Redox Cascade Starting from (R)‐and (S)‐Carvones

References

11.5 One‐Pot Biocatalytic Synthesis of D‐Tryptophan Derivatives from Substituted Indoles and L‐Serine

References

11.6 Escherichia coli Lysate Multienzyme Biocatalyst for the Synthesis of Uridine‐5′‐Triphosphate from Orotic Acid and Ribose

References

11.7 Aerobic Synthesis of Aromatic Nitriles from Alcohols and Ammonia Using Galactose Oxidase

References

11.8 Hydrogen‐Borrowing Conversion of Alcohols into Optically Active Primary Amines by Combination of Alcohol Dehydrogenases and Amine Dehydrogenases

References

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

References

12 Chemo‐Enzymatic Cascades

12.1 Synergistic Nitroreductase/Vanadium Catalysis for Chemoselective Nitroreductions

References

12.2 Chemo‐Enzymatic Synthesis of (S)‐1,2,3,4‐Tetrahydroisoquinoline Carboxylic Acids Using D‐Amino Acid Oxidase

References

12.3 Amine Oxidase‐Catalysed Deracemisation of (R,S)‐4‐Cl‐Benzhydrylamine into the (R)‐Enantiomer in the Presence of a Chemical Reductant

References

12.4 Asymmetric Synthesis of 1‐Phenylpropan‐2‐amine from Allylbenzene through a Sequential Strategy Involving a Wacker–Tsuji Oxidation and a Stereoselective Biotransamination

References

12.5 Chemoenzymatic Synthesis of (2S,3S)‐2‐Methylpyrrolidin‐3‐ol

References

13 Whole‐Cell Procedures

13.1 Semipreparative Biocatalytic Synthesis of (S)‐1‐Amino‐1‐(3′‐Pyridyl)methylphosphonic Acid

References

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

References

13.3 Clean Enzymatic Oxidation of 12α‐Hydroxysteroids to 12‐Oxo‐Derivatives Catalysed by Hydroxysteroid Dehydrogenase

References

13.4 Whole‐Cell Biocatalysis Using PmlABCDEF Monooxygenase and Its Mutants: A Versatile Toolkit for Selective Synthesis of Aromatic N‐Oxides

References

Index

End User License Agreement

List of Tables

Chapter 2

Table 2.1 Commercial enzyme suppliers/distributors.

Table 2.2 Bioprocess service providers.

Table 2.3 Chemical transformations of selected commercially available enzymes

Chapter 3

Table 3.1 Primers used for amplification.

Table 3.2 HPLC method.

Table 3.3 Retention times for HPLC analysis.

Table 3.4 Retention times of DHIQs and THIQs in HPLC analysis.

Table 3.5 HPLC conditions used for the chiral analysis of THIQ products.

Table 3.6 Conversion and enantioselectivity of selected imine reductases towa...

Table 3.7 HPLC conditions for analysis of conversion of amine substrate to ke...

Chapter 4

Table 4.1 GC method.

Table 4.2 Retention times for GC analysis.

Table 4.3 Further substrates.

Table 4.4 Conversion of monosaccharides (100 or 200 mM) [4] to the correspond...

Chapter 5

Table 5.1 Enantioselective synthesis of

N

‐heterocycloalkyl‐substituted L‐aspar...

Table 5.2 Retention times for HPLC analysis.

Table 5.3 HPLC methods for the monitoring of enzymatic reactions (determinati...

Table 5.4 HPLC methods for the determination of the enantiomeric excess (ee) ...

Table 5.5 Reaction time, conversion, isolation yield and enantiomeric excess ...

Table 5.6 Substrates accepted by at least one of the reported AmDHs.

Table 5.7 Enzyme sources.

Table 5.8 Extinction coefficient (ɛ) at λ of 280 nm and molecular weight of t...

Table 5.9 Panel of fatty acid and amine substrates used to screen the TamA‐ca...

Chapter 6

Table 6.1 Activated ketones used as RhuA electrophiles.

Table 6.2 Typical conversion to substituted tryptophans catalysed by tryptoph...

Table 6.3 Reaction buffer.

Table 6.4 Conditions for reversed‐phase chromatography.

Table 6.5 Chiral SFC method.

Chapter 8

Table 8.1 Gradient programme.

Table 8.2 GC method.

Table 8.3 Retention times for GC analysis.

Table 8.4 GC method.

Table 8.5 Retention times for GC analysis.

Table 8.6 Reaction components for PCR amplification of rdc2.

Table 8.7 PCR conditions for amplification of rdc2.

Chapter 9

Table 9.1 HPLC method.

Table 9.2 Retention times for HPLC analysis.

Table 9.3 TLC method.

Table 9.4 Retention factors for TLC analysis.

Table 9.5 HPLC conditions for assessing Nitrilase screening reactions.

Table 9.6 GC method.

Table 9.7 Retention times for GC analysis.

Table 9.8 Aliphatic nitrile synthesis using OxdB in 10 mL scale for different...

Chapter 10

Table 10.1 Eluent profile for HPAEC‐PAD analysis.

Table 10.2 Retention times for HPAEC‐PAD analysis.

Table 10.3 Retention times for HPAEC‐PAD analysis.

Table 10.4 Retention times for the sugar‐purification system.

Chapter 11

Table 11.1 GC‐MS programmes used for analysis of α‐hydroxy acids.

Table 11.2 Retention times of substrates and products during GC‐MS analysis.

Table 11.3 Chiral UV‐HPLC setup used for analysis of AAs.

Table 11.4 Retention times of substrates and products for HPLC analysis.

Table 11.5 Preparative synthesis of L‐amino acids (

1c–5c

).

Table 11.6 Enzyme expression strains and expression systems.

Table 11.7 GC method.

Table 11.8 One‐pot redox reaction of (+)‐carvone

1a

into the corresponding car...

Table 11.9 One‐pot redox reaction of (−)‐carvone

1b

into the corresponding car...

Table 11.10 Data and results from obtained product (−)‐(4S,7R)‐7‐methyl‐4‐(pr...

Table 11.11 Data and results from obtained product (−) ‐(3

R

,6

S

)‐3‐methyl‐6‐(p...

Table 11.12 Data and results from obtained product (−)‐(4

S

,7

S

)‐7‐methyl‐4‐(pr...

Table 11.13 Data and results from obtained product (−)‐(4

R

,7

R

)‐7‐methyl‐4‐(pr...

Table 11.14 Synthesis of D‐tryptophan derivatives D‐

2b–l

from substitut...

Table 11.15 Other substrates that can be synthesised via this procedure.

Table 11.16 HPLC gradient method.

Table 11.17 Retention times for HPLC analysis.

Table 11.18

GC method

.

Table 11.19

Retention times for GC analysis

.

Table 11.20 GC retention times and analysis methods for substrates, intermedi...

Table 11.21 GC retention times and analysis methods for derivatised amine ena...

Table 11.22 Biocatalytic synthesis of α‐chiral amines from alcohols using the...

Table 11.23 Enzyme sources.

Chapter 12

Table 12.1 Additive screening for reduction of NR‐14.

Table 12.2 Substrate scope of NR‐04, NR‐14, NR‐17 and NR‐24.

Table 12.3 Chemo‐enzymatic deracemisation of

rac

‐

2a–4a

employing FsDAAO....

Table 12.4 Retention times for LC analysis.

Table 12.5 GC method for the measurement of product ratios.

Table 12.6 Retention times for GC analysis using an Agilent HP‐1 or HP‐5 colu...

Table 12.7 GC method for the measurement of reaction conversion.

Table 12.8 Retention times for GC analysis using the Agilent HP‐1 column.

Table 12.9 HPLC method for the measurement of amine enantiomeric excess.

Table 12.10 Retention times for HPLC analysis using Chiralcel OD column.

Table 12.11 Sequential cascade process to transform allylbenzenes into optica...

Chapter 13

Table 13.1 GC method.

Table 13.2 Retention times for GC analysis.

Table 13.3

1

H‐NMR analysis of the (

S

)‐

O

‐acetylmandelate esters derivi

...

Table 13.4 Purification table for El12α‐HSDH.

Table 13.5 Bioconversion tables for Procedures 3 and 4. In both cases, a reac...

List of Illustrations

Chapter 1

Figure 1.1 A general drug discovery path overlapped with biocatalytic opport...

Scheme 1.1 Selected enzyme classes currently in GSK's collection.

Figure 1.2 Strategies for generating an enzyme panel.

Scheme 1.2 Reductive amination of ketones performed by imine reductases.

Scheme 1.3 Successful synthesis of alkylation products via enzymatic reducti...

Figure 1.3 Enzyme evolution and biocatalysis adoption.

Figure 1.4 Design–build–test cycle for an evolution round.

Scheme 1.4 Epoxide hydrolase (EH5)‐catalysed resolution of

rac

‐2‐butyl‐2‐eth...

Scheme 1.5 Synthesis of chiral synthons for the preparation of SeH inhibitor...

Scheme 1.6 Nelarabine formation catalysed by PNPase.

Scheme 1.7 Key chiral amine intermediate for the synthesis of GSK2879552.

Scheme 1.8 Modified conditions reflecting phosphate buffer synthesis of tert...

Chapter 3

Scheme 3.1 Imine reductase M3‐catalysed enantioselective reductive amination...

Scheme 3.2 Reductive amination target and a summary of the genome mining app...

Figure 3.1 Chiral analysis of the reductive amination catalysed by IR88‐2.

Scheme 3.3 Synthesis of dextromethorphan

1

from 1‐(4‐methoxybenzyl)‐3,4,5,6,...

Scheme 3.4 Asymmetric reduction of DHIQs by imine reductases.

Scheme 3.5 1‐Aryl DHIQ substrates.

Scheme 3.6 Imine reductase‐catalysed preparation of enantiopure (

R

)‐

N

‐methyl...

Scheme 3.7 Imine reductase‐mediated oxidative deamination.

Chapter 4

Scheme 4.1 Industrial‐scale production of tert‐butyl((5S,6R)‐6‐methyl‐2‐oxo‐...

Scheme 4.2 Enzymatic asymmetric synthesis of L‐PPT

2

from PPO

1

by Pf‐TA.

Scheme 4.3 Synthesis of IPA‐3DPPA salt (donor salt)

5

.

Scheme 4.4 ISPC‐based conversion of 3‐methoxyacetophenone

1

into (

S

)‐1‐(3‐me...

Scheme 4.5 Synthesis of optically pure (R)‐3‐amino‐1‐Boc‐piperidine

2

.

Scheme 4.6 Synthesis of (R)‐1‐(1‐naphthyl)ethylamine

4

.

Scheme 4.7 Synthesis of 1‐amino‐1‐deoxy‐D‐xylitol hydrochloride

1

.

Scheme 4.8 Synthesis of 2‐amino‐2‐deoxy‐D‐mannitol hydrochloride

2

.

Scheme 4.9 (a) Overall transformation and conversion of D‐deoxyribose to (2

R

Chapter 5

Scheme 5.1 Conversion of aniline derivatives to the corresponding acetanilid...

Scheme 5.2 (a) Natural reaction catalysed by EDDS lyase. (b) EDDS lyase‐cata...

Scheme 5.3 Selected enzymatic carbon–nitrogen bond‐forming Michael addition ...

Scheme 5.4 Argininosuccinate lyase ARG4‐catalysed aza‐Michael addition of L‐...

Scheme 5.5 Synthesis of 3'‐deoxyriboside 6‐methoxyguanine (6‐

O

‐Me‐Gua‐3’‐dR)...

Figure 5.4 1H‐NMR spectrum.

Scheme 5.6 Ammonia elimination reaction of p‐CH

3

‐phenylalanine rac‐

1a

and am...

Scheme 5.7 Biocatalytic amination using AmDHs with co‐factor recycling.

Figure 5.2 Comparison of the chemical and biocatalytic routes to the synthes...

Scheme 5.8 Preparative‐scale synthesis of the

N

‐C

12

‐L‐histidine amide using ...

Chapter 6

Scheme 6.1 Enzymatic reactions catalysed by PDC and AHAS enzymes (Reproduced...

Scheme 6.2 Enzymatic preparation of R‐PAC using CSU‐GST of E. coli AHAS I as...

Scheme 6.3 Preparation of dendroketose‐1‐phosphate

3

catalysed by rhamnulose...

Figure 6.2 NMR spectrum for dendroketose‐1‐phosphate

3

.

Scheme 6.4 Preparation of substituted L‐tryptophans.

Scheme 6.5 Conversion of resorcinol derivatives

1a

–

h

to the corresponding ac...

Scheme 6.6 MenD‐catalysed synthesis of 6‐cyano‐4‐oxohexanoic acid

3

.

Scheme 6.7 Asymmetric synthesis of (

R

)‐2‐(3,5‐dimethoxyphenyl)propanoic acid...

Chapter 7

Scheme 7.1 Ketoreductase (KRED) designed for activity at high pH and elevate...

Scheme 7.2 Scalable synthesis of a GPR40 partial agonist enabled by a dynami...

Scheme 7.3 Series of carbazepine APIs.

Scheme 7.4 Ketoreductase (KRED)‐mediated synthesis of

3

.

Scheme 7.5 Reduction of carboxylic acid

1

to aldehyde

2

with cell‐free recyc...

Scheme 7.6 Selected carboxylic acids.

Figure 7.1 Screening results of carboxylic acid synthons as substrates for a...

Scheme 7.7 Preparative‐scale reduction of racemic

1c

to aldehyde

2c

using cr...

Scheme 7.8 ERED‐207‐mediated reduction of methyl 3‐oxocyclohex‐1‐ene‐1‐carbo...

Chapter 8

Scheme 8.1 Baeyer–Villiger monooxygenase (BVMO)‐catalysed oxidation of cyclo...

Figure 8.1 1 L‐scale reactor setup used for the Baeyer–Villiger oxidation of...

Figure 8.2 Progress curve of cyclopentadecaone (CPD) oxidation to pentadecal...

Scheme 8.2 General synthesis route to δ‐lactones

8

via: 2‐deoxyribose‐5‐phos...

Scheme 8.3 Oxygen and NADP

+

oxidation of lactol

9

derived from DERA reac...

Figure 8.3 1 L‐scale reactor setup used for the oxidation of lactol

9

to hyd...

Figure 8.4 Progress curve of lactol

9

oxidation with ADH‐99 and NOX‐01 on 50...

Scheme 8.4 Synthesis of the chiral sulfoxide

2

using Codexis BVMO P1‐D08.

Scheme 8.5 Reactions catalysed by eugenol oxidase (EUGO): target reaction of...

Scheme 8.6 Oxidation of vanillyl alcohol to vanillin by molecular oxygen cat...

Figure 8.5 Left: 30 mL‐ and Right: 1 L‐scale reactor set‐up used for the oxi...

Figure 8.6 Example chromatogram of a reference solution.

Figure 8.7 Progress curve of vanillyl alcohol oxidation by eugenol oxidase (...

Scheme 8.7 Conversion of 2,6‐dimethoxy‐4‐allylphenol

1

to syringaresinol

3

u...

Scheme 8.8 Oxidation of vanillyl alcohol (4‐(hydroxymethyl)‐2‐methoxyphenol)...

Scheme 8.9 VAO‐catalysed conversion of 4‐ethylphenol to (

R

)‐1‐(4′‐hydroxyphe...

Scheme 8.10 Conversion of (−)‐cis‐verbenone

2

to the normal pinene‐derived l...

Scheme 8.11

In vitro

and

in vivo

enzymatic chlorination of hydroxyquinolines...

Chapter 9

Scheme 9.1 Conversion of 3‐(4‐chlorophenyl) glutaronitrile

1

into optically ...

Scheme 9.2 Nitrilase‐mediated synthesis of hydroxyphenylacetic acid derivati...

Scheme 9.3 Epoxide hydrolase (EH) from

Agromyces mediolanus

catalysed enanti...

Scheme 9.4 Procedure for the separation of enantiopure (

S

)‐1,2‐dodecanediol ...

Scheme 9.5 Tosylation of product (

S

)‐

1

for HPLC analysis [6].

Scheme 9.6 Conversion of

n

‐octanaloxime

1

to

n

‐octanenitrile

2

using the ald...

Scheme 9.7 Biocatalytic sequential dehalogenation of

rac

‐1,3‐dibromobutane w...

Figure 9.1 GC‐FID chromatogram trace obtained on a chiral column with separa...

Chapter 10

Scheme 10.1 Glycosylation of p‐coumaric acid with rutinosidase [5].

Scheme 10.2 Transglucosylation of glucose by double‐mutant sucrose phosphory...

Scheme 10.3 Conversion of sucrose and glucose into kojibiose.

Figure 10.1 HPAEC‐PAD profile of purified kojibiose (note that the small pea...

Figures 10.2 (a)

1

H NMR and (b)

13

C NMR spectrum of purified kojibiose.

Scheme 10.4 Transglucosylation of glucose catalysed by BaSP‐YGQF. The disacc...

Scheme 10.5 Enzymatic Sulfation of

E

‐resveratrol by arylsulfotransferase (AS...

Scheme 10.6 Shikimate kinase‐catalysed phosphorylations of cyclic trihydroxy...

Scheme 10.7 Recombinant

E. coli

K12 shikimate kinase AroL‐catalysed phosphor...

Scheme 10.8 Selective kinase‐catalysed phosphorylations of D‐fructose phosph...

Scheme 10.9 Enzymatic phosphorylation of D‐tagatose‐6‐phosphate in the 1‐pos...

Scheme 10.10 Selection of xylulokinase‐catalysed phosphorylation reactions....

Scheme 10.11 Comparison of the chemical route (bottom) to D‐xylulose‐5‐phosp...

Scheme 10.12 Selected N‐phosphorylation reactions to phosphoramidates cataly...

Scheme 10.13 Arginine kinase‐catalysed N

ω

‐phosphorylation of L‐arginine...

Chapter 11

Scheme 11.1 Enzymatic oxidation–reduction cascade for conversion of

1

into

4

Scheme 11.2 One‐pot cascade for regio‐ and enantioselective transformation o...

Scheme 11.3 Chemoenzymatic synthesis of the quercus lactone

1

[3].

Scheme 11.4

Chemoenzymatic synthesis of lactone 6 [3,6]. Yields and selectiv

...

Scheme 11.5 Catalysis of (+)‐ and (−)‐carvone into six carvo‐lactone stereoi...

Scheme 11.6 One‐pot conversion of indoles

1

into D‐tryptophans D‐

2

.

Scheme 11.7

Synthesis of UTP

8

from orotic acid

4

and ribose

1

with

in situ

Figure 11.1 Workflow for cell lysate biocatalyst preparation and application...

Scheme 11.8 Biocatalytic cascade catalysed by GOx

M3–5

for the synthesi...

Scheme 11.9 Biotransformation of 1.2 mmol (151 mg) 2'‐fluorobenzyl alcohol

1

Figure 11.2 Chromatogram of preparative‐scale synthesis of

2

. solv, solvent;...

Scheme 11.10 Elucidated list of substrates accepted by GOx

M3–5

[6].

Scheme 11.11 One‐pot hydrogen‐borrowing amination of racemic alcohols into o...

Scheme 11.12

Homologation of phenylpropiolaldehyde

1

into phenylpentynol (

S

)

...

Scheme 11.13

Homologation of aldehydes

1

and

4

into alcohol derivatives (

S

)‐

...

Chapter 12

Scheme 12.1 General reaction scheme for the reduction of nitroaromatics

1

to...

Scheme 12.2 Reduction of 2‐methyl‐5‐nitropyridine.

Figure 12.1 Result of fed‐batch nitroreduction of 2‐methyl‐5‐nitropyridine....

Scheme 12.3 Chemo‐enzymatic derecemisation of racemic carboxyl‐substituted T...

Scheme 12.4 Overview of the amine oxidase‐catalysed deracemisation of

rac

‐

1

...

Figure 12.2 Gene sequence of the R283G variant. The mutated residue is under...

Figure 12.3 Gene sequence of the Y228L/R283G variant. The mutated residues a...

Figure 12.4 Gene sequence of the I230A/R283G variant. The mutated residues a...

Scheme 12.5 Transformation of allylbenzenes

1

into optically active 1‐arylpr...

Scheme 12.6 Wacker–Tsuji oxidation of allylbenzene.

Scheme 12.7

Biotransamination of 1‐phenylpropan‐2‐one using (

...

Scheme 12.8 Synthesis of 1‐phenylpropan‐2‐amine enantiomers from allylbenzen...

Scheme 12.9

Two‐step chemo‐enzymatic synthesis of iminocyclitols. (a) Aldola

...

Scheme 12.10

Two‐step chemoenzymatic synthesis of (2S,3S)‐2‐methylpyrrolidin

...

Chapter 13

Scheme 13.1

Preparation of racemic mixture of 1‐amino‐1‐(3′‐pyridyl)methylph

...

Scheme 13.2 Bioconversion synthesis of 1‐amino‐1‐(3′‐pyridyl)methylphosphoni...

Figure 13.1 Mycelia after immobilisation.

Figure 13.2 System for continuous‐flow column experiments.

Figure 13.3

31

P NMR spectra of 1‐amino‐1‐(3′‐pyridyl)methylphosphonic acid w...

Scheme 13.3 Lactobacillus

strain‐mediated biotransformation of oleic acid 1a

...

Scheme 13.4

Oxidation of 12α‐OH group of hydroxysteroids catalysed by 12α‐HS

...

Figure 13.4 (a) Elution profile of El12α‐HSDH. The black and grey lines corr...

Figure 13.5

Scheme of the flow reactor employed in the preparative biotransf

...

Figure 13.6

HPLC analyses of biocatalytic oxidation of CA 1a into 12‐oxo‐CDC

...

Scheme 13.5

Three different routes in the synthesis of aromatic

N

‐oxides uti

...

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Applied Biocatalysis

The Chemist's Enzyme Toolbox

Edited byJOHN WHITTALLUniversity of Manchester ManchesterUK

PETER W. SUTTONGlycoscience S.L. Barcelona ES

Copyright

This edition first published 2021

© 2021 John Wiley & Sons Ltd

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by law. Advice on how to obtain permission to reuse material from this title is available at http://www.wiley.com/go/permissions.

The right of John Whittall and Peter W. Sutton to be identified as the authors of the editorial material in this work has been asserted in accordance with law.

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Library of Congress Cataloging‐in‐Publication Data

Names: Whittall, John, editor. | Sutton, Peter (Peter W.), editor.

Title: Applied biocatalysis : the chemist's enzyme toolbox / edited by Dr

 John Whittall, Dr Peter W. Sutton.

Description: First edition. | Hoboken, NJ : Wiley, 2021. | Includes

 bibliographical references and index.

Identifiers: LCCN 2020015374 (print) | LCCN 2020015375 (ebook) | ISBN

 9781119487012 (cloth) | ISBN 9781119487029 (adobe pdf) | ISBN

 9781119487036 (epub)

Subjects: LCSH: Biocatalysis.

Classification: LCC TP248.65.E59 A677 2021 (print) | LCC TP248.65.E59

 (ebook) | DDC 660.6/34–dc23

LC record available at https://lccn.loc.gov/2020015374

LC ebook record available at https://lccn.loc.gov/2020015375

Cover Design: Wiley

Cover Images: (Background) © Angelatriks/Shutterstock,

(Inset diagram) Courtesy of Gheorghe‐Doru Roiban

Abbreviations

a/a

Area/area

AA

Amino acid

AADH

Amino acid dehydrogenase

ABTS

2,2′‐Azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid

ABS

Amide bond synthetase

ACN

Acetonitrile (Also MECN)

AcOEt

Ethyl acetate (Also EtOAc)

ADH

Alcohol dehydrogenase (Also KRED)

ADP

Adenosine diphosphate

AEAA

(

S

)‐

N

‐(2‐Aminoethyl)aspartic acid

AHAS

Acetohydroxyacid synthase

AHTC

Anhydrotetracycline

AMDase

Aryl malonate decarboxylase

AmDH

Amine dehydrogenase

AP

Area percent

API

Active pharmaceutical ingredient

ArgK

Arginine kinase

ArgK‐LP

ArgK from

Limulus Polyphemus

AroL

E. coli

K12 shikimate kinase

AST

Arylsulfotransferase

ATA

Amine transaminase (Also TA)

ATase

Acetyl transferase

atm

Atmosphere (pressure)

ATP

Adenosine triphosphate

BCA

Bicinchoninic acid assay

BCL

Agarose beads crosslinked

BA

Benzaldehyde

BaSP

Sucrose phosphorylase from

Bifidobacterium adolescentis

Bis‐tris

2‐[Bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol

BL21

Competent

Escherichia coli

BL21

BME

β‐Mercaptoethanol

BMGH

Buffered minimal glycerol medium

BMGY

Buffered glycerol‐complex medium

BMMH

Buffered minimal methanol medium

BMMY

Buffered methanol‐complex medium

BnONH

2

O

‐Benzylhydroxylamine

Boc

t‐Butoxycarbonyl

BSA

Bovine serum albumin

BSM

Basal salt medium

BVMO

Baeyer–Villiger monooxygenase

BVO

Baeyer–Villiger oxidation

c

Conversion

CA

Cholic acid

CAD

Charged aerosol detection

CALB

Candida antarctica

lipase B

CAR

Carboxylic acid reductase

Cbz‐OSu

N (Benzyloxycarbonyloxy)succinimide

CDCA

Chenodeoxycholic acid

CDW

Cell dry weight

CDMO

Cyclododecanone monooxygenase

CFE

Cell free extract

c.f.u

Colony‐forming unit

CHMO

Baeyer–Villiger monooxygenase (cyclohexanone monoxygenase)

CLR

Controlled laboratory reactor

CoG

Cost of goods

CP

Citrate phosphate (buffer)

CPD

Cyclopentadecanone

CPME

Cyclopentyl methyl ether

CSU

Catalytic subunit

CSU‐GST

Catalytic subunit – glutathione S‐transferase tag

CV

Column volume

DAAO

D‐Amino acid oxidase

DAAT

D‐Amino acid transferase

DAD

Diode‐array detection

DAPG

2,4‐Diacetylphloroglucinol

DCC

Dicyclohexylcarbodiimide

DCM

Dichloromethane

ddH

2

O

Double distilled water

DEA

Diethyl amine

DEAE

Diethylaminoethanol (group in ion‐exchange resin)

DERA

2‐Deoxyribose‐5‐phosphate aldolase

dH

2

O

Distilled water

DHA

Dihydroxyacetone

DHAK

Dihydroxyacetone kinase

DHAP

Dihydroxyacetone phosphate

DHIQ

Dihydroisoquinoline

dI

Deoxyinosine

DI

Deionised

DIBAL‐H

Diisobutyl aluminium hydride

DK(R)

Dynamic kinetic (resolution)

DMAP

4‐Dimethylaminopyridine

DMF

Dimethylformamide

DMSO

Dimethyl sulfoxide

DNA

Deoxyribonucleic acid

DoE

Design of experiments

DSP

Downstream processing

DPPA

3,3‐Diphenylpropionic acid

DTT

Dithiothreitol

E

Enantioselectivity

EDDS

Ethylenediamine‐N,N′‐disuccinic acid

EDTA

Ethylenediaminetetraacetic acid

ee

Enantiomeric excess

EH

Epoxide hydrolase

ELSD

Evaporative light scattering detector

ER

Ene‐reductase (often same as ERED)

ERED

Enoate reductase (often same as ER)

Et

2

O

Diethyl ether

EtOAc

Ethyl acetate (Also AcOEt)

EUGO

Eugenol oxidase

EV

Expansion vessel

EWG

Electron‐withdrawing group

FA

Formic acid

FAD

Flavin adenine dinucleotide

FADH

2

Flavin adenine dinucleotide reduced form

FDH

Formate dehydrogenase

F&F

Flavour and Fragrance

FID

Flame ionisation detector

FMN

Flavin mononucleotide

FMO

Flavin monooxygenase enzyme

Fre

Flavin reductase

FSA

D‐Fructose‐6‐phosphate aldolase

Fs

DAAO

D‐Amino acid oxidase from

Fusarium solani

FTIR

Fourier‐transform infrared spectroscopy

g

Gram (× g is centrifuge unit)

G6P

Glucose‐6‐phosphate

G6PDH

Glucose‐6‐phosphate dehydrogenase

GC

Gas chromatography

GC‐FID

Gas chromatography/flame ionisation detection

GC‐MS

Gas chromatography/mass spectrometry

GDH

Glucose dehydrogenase

GHMP

Galactokinase, homoserine kinase, mevalonate kinase

GMP

Good manufacturing practice

Gox

Galactose oxidase

GPDH

α‐Glycerophosphate dehydrogenase

GPR40

G‐protein‐coupled receptor 40

GRAS

Generally recognised as safe

GST

Glutathione S‐transferase

GTP

Guanosine‐5′‐triphosphate

hr

hour

HA

Hydroxyacid

HEPES

4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid

HEWT

Amine transaminase from

Halomonas elongata

DSM 2581

HFA

Hydroxy fatty acid

HIC‐DH

Hydroxyisocaproate dehydrogenase

HMM

Hidden Markov model

HMU

Worldwide PDB entry aminotransferase from

Silicibacter pomeroyi

HPAEC

High‐performance anion exchange chromatography

HPLC

High‐performance liquid chromatography

HPLC‐DAD

High‐performance liquid chromatography with diode‐array detection

HPLC‐PAD

High‐performance liquid chromatography with photodiode‐array detection

HPLC‐RI

High performance liquid chromatography with refractive index

HRMS

High‐resolution mass spectrometry

HRP

Horse radish peroxidase

HSDH

Hydroxysteroid dehydrogenases

HTP

High throughput

HWE

Horner–Wadsworth–Emmons reaction

iBAT

Ileal bile acid transport

IBX

o‐Iodoxybenzoic acid

ID

Internal diameter

IMAC

Immobilised metal affinity chromatography

IMB

Immobilised

IP

Intellectual property

IPA

Isopropyl alcohol

IPA

Isopropyl amine

IPAc

Isopropyl acetate

IPEA

Isopropenyl acetate

IPTG

Isopropyl β‐D‐1‐thiogalactopyranoside

IR

Imine reductase

IRED

Imine reductase

IS

Internal standard

ISM

Iterative saturation mutagenesis

ISPC

In situ

product crystallisation

ISPR

In situ

product removal

kDA

Kilodalton

KHK

Ketohexokinase

KIRED

Ketimine reductase

KPB

Potassium phosphate buffer

KPi

Potassium phosphate buffer

KR

Kinetic resolution (Also ADH)

KRED

Ketoreductase

LAAD

L‐Amino acid deaminase

LacC

D‐Tagatose 6‐phosphate kinase

LB

Lysogenic broth (also known as Luria–Bertani medium)

LC‐MS

Liquid chromatography/mass spectrometry

LDH

Lactate dehydrogenase

LE‐AmDH

Lysine amine dehydrogenase

LSD

Lysine specific histone demethylase

MALDI

Matrix‐assisted laser desorption/ionisation

MAO‐N

Monoamine oxidase from

Aspergillus niger

MAP

Methoxyacetophenone

MeCN

Acetonitrile (Also ACN)

MenD

2‐Succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate synthase

MeOH

Methanol

MES

2‐(

N

‐Morpholino)ethanesulfonic acid

MeTHF

2‐Methyl tetrahydrofuran

MDH

Malate dehydrogenase

MFC

Mass flow controller

min

Minute

MOPS

4‐Morpholinepropanesulfonic acid

MPLC

Medium‐pressure liquid chromatography

m.pt

Melting point

MRS

de Man, Rogosa and Sharpe broth

MS

Mass spectroscopy

mS.cm

−1

Millisiemens per centimeter

MTBE

Methyl

tert

‐butyl ether

MWCO

Molecular weight cut‐off

m/z

Mass‐to‐charge ratio

NA

Nutrient agar

NAC

N

‐Acetyl‐L‐cysteine

NAD

+

β‐Nicotinamide adenine dinucleotide

NADH

β‐Nicotinamide adenine dinucleotide, reduced form

NADPH

β‐Nicotinamide adenine dinucleotide 2′‐phosphate, reduced form

NADP

+

β‐Nicotinamide adenine dinucleotide 2′‐phosphate

NH

3

·BH

3

Ammonia‐borane complex

Ni‐NTA

Nickel‐nitrilotriacetic acid

nm

Nanometre

NMAADH

N

‐Methylamino acid dehydrogenase

NMP

Nucleoside monophosphate

NMR

Nuclear magnetic resonance spectroscopy

NOX

NAD(P)H oxidase

NP

Nitrophenol

NP

Normal phase (chromatography)

NPS

Nitrophenyl sulfate

NR

Nitroreductase

nr

Nonredundant

NTP

Nucleoside triphosphate

OD

Optical density

Omd

Orotidine‐5′‐monophosphate decarboxylase

OMP

Orotidine‐5′‐monophosphate

OPA

o‐Phthalaldehyde

Opt

Orotate phosphoribosyl transferase

OYE

Old yellow enzyme

OUR

Oxygen uptake rate

P

ortho

‐Phosphate

P450

Cytochrome P450

P5CR

Δ

1

‐Pyrroline‐5‐carboxylate reductase

PA

Phenyl acetate

PAD

Pulsed amperometric detection

PAGE

Polyacrylamide gel electrophoresis

PAL

Phenylalanine ammonia lyase

PAPS

Adenosine‐3′‐phospho‐5′‐phosphosulfate

PBS

Phosphate buffered saline

PCR

Polymerase chain reaction

PD

Potato‐dextrose

PDa

Potato dextrose agar

PDA

Photodiode array

PDb

Potato dextrose broth

PDB

Protein data bank

PDC

Pyruvate decarboxylase

PDL

Pentadecanolide

PE

Petroleum ether

PEP

Phosphoenolpyruvic acid

PFAM

Protein family

Pf‐TA

Transaminase from

Pseudomonas fluorescens

Phe‐DH

Phenylalanine dehydrogenase

PK

Pyruvate kinase

Pk

Porcine kidney

PLIF

Protein–ligand interaction fingerprint

PLP

Pyridoxal 5′‐phosphate

PMP

Pyridoxamine 5‐phosphate

PMSF

Phenylmethylsulfonyl fluoride

PNPase

Purine nucleoside phosphorylase

polyP

Polyphosphate

PP

Pyrophosphate

PpATaseCH

Acylase from

Pseudomonas protegens

PPB

Potassium phosphate buffer

PPK

Polyphosphate kinase

PPO

4‐(Hydroxy(methyl)phosphoryl)‐2‐oxobutanoic acid

Pps

PRPP synthetase

PPT

Phosphinothricin

PRPP

Phosphoribosyl pyrophosphate

PTM

Pichia

trace metal salt supplement

PTV

Programmed temperature vaporisation

PWM

Position weight matrix

PYR

Pyruvate

QbD

Quality by design

rac

Racemic

R&D

Research and Development

RCF

Relative centrifugal force

Rdc2

Halogenase from

Pochonia chlamydosporia

Rf

Retention factor

RhuA

Rhamnulose‐1‐phosphate aldolase

ROH

Generic alcohol

ROP

Ring‐opening polymerisation

RP

Reverse phase

R

‐PAC

(

R

)‐Phenylacetyl carbinol

rpm

Revolutions per minute

RSU

Regulatory subunit

rt

Room temperature

Rt

Retention time (also t

R

)

SASA

Solvent accessible surface area

SDS

Sodium dodecyl sulfate

SDS‐PAGE

SDS–polyacrylamide gels

sec

Seconds

SeH

Soluble epoxide hydrolase

SFC

Supercritical fluid chromatography

SLM

Supported liquid membrane

sLpm

Standard litre per minute

SOC

Super optimal broth with catabolite repression

STRAP

Structure based sequences alignment program

TA

Transaminase

TA‐CV

ξ‐Transaminase from

Chromobacterium violaceum

TATP

Tri‐acetone‐triperoxide

TB

Terrific broth

TBAP

Tetrabutylammonium phosphate

TBDAc

2,3‐Di‐

O

‐acetyl‐6‐

O

‐t‐butyldimethylsilyl)‐β‐cyclodextrin

TEA

Triethylamine

TEoA

Triethanolamine

TEV

Tobacco etch virus

TFA

Trifluoroacetic acid

ThDP

Thiamine diphosphate

THIQ

Tetrahydroisoquinoline

TLC

Thin layer chromatography

TMS

Tetramethyl silane

TMSCHN

2

Trimethylsilyl diazomethane

TOF

Time‐of‐flight

TPI

Triosephosphate Isomerase

t

R

Retention time (also Rt)

Tris

Tris(hydroxymethyl)aminomethane

TrpS

Tryptophan synthase

TY

Tryptone yeast extract broth

U

Units

UAB

Universitat Autònoma de Barcelona

UDCA

Ursodeoxycholic acid

UFA

Unsaturated fatty acid

UHPLC

Ultra‐high‐performance liquid chromatography

UPLC

Ultra‐performance liquid chromatography

UTP

Uridine‐5′‐triphosphate

UV

Ultraviolet

UV‐Vis

Ultraviolet/visible

VAO

Vanillyl alcohol oxidase

v/v

Volume/volume

vvm

Gas volume flow per unit of liquid volume per minute (vessel volume per minute)

wcp

Wet cell pellet

wcw

Wet cell weight

wrt

With respect to

wt

Wild type

w/v

Weight/volume

w/w

Weight/weight

x g

Centrifugal force (relative centrifugal force more precise than rpm)

YNB

Yeast nitrogen base

YPD

Yeast extract peptone dextrose medium

YT

Yeast extract tryptone

ZmPDC

Zymomonas mobilis

pyruvate decarboxylase

1Directed Evolution of Enzymes Driving Innovation in API Manufacturing at GSK

Jonathan Latham1, Anne A. Ollis2, Chris MacDermaid3, Katherine Honicker2, Douglas Fuerst2, and Gheorghe‐Doru Roiban*1

1Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline Medicines Research Centre, Stevenage, Hertfordshire, UK

2Synthetic Biochemistry, Medicinal Science & Technology, GlaxoSmithKline, Collegeville, PA, USA

3Molecular Design, Computational and Modelling Sciences, GlaxoSmithKline, Collegeville, PA, USA

1.1 Introduction

Biocatalysis has had a significant impact on the synthesis of active pharmaceutical ingredients (APIs) in recent years. The main driver for this is the ability to harness the regio‐ and stereoselectivity of enzymes to improve the efficiency of synthetic routes. For example, enzymes can offer direct access to enantiopure products, where traditional organic synthesis would require either resolution or the use of auxiliary groups [1], whilst enzymes applied in manufacture have improved syntheses or generated molecules that would otherwise be either impossible or impractical to synthesise. Other factors supporting the adoption of enzymes in the synthesis of APIs include:

Reduced manufacturing costs:

The selectivity of biocatalytic processes often results in fewer overall processing and purification steps, reducing labour and material requirements.

Environmental sustainability:

Traditional organic solvents and reagents raise environmental concerns. Enzymes often function in aqueous solutions using natural biochemical co‐factors as reagents, providing environmentally friendly and operationally safe alternatives to traditional chemical transformations.

Sustainable supply:

Enzymes fulfil sustainability principals [

2

]. They are produced from microbial fermentation – a renewable feedstock requiring simple sugars to grow – and are themselves biodegradable.

Simplified drug manufacturing:

The increasing complexity of APIs and the need to implement new products has ramped up the pressure to find new synthetic strategies to simplify the way drugs are made. Additionally, GlaxoSmithKline's (GSK) focus on making medicines broadly accessible across the globe requires economical manufacture of APIs and contributes to the drive for simplification of manufacturing processes.

Quality:

Regulatory pressures from government agencies to maintain high API quality standards whilst reducing carbon emissions drive the use of biocatalysis to harness the high regio‐, chemo‐ and stereoselectivity offered.

Accessibility:

Technology advances such as next‐generation sequencing and directed evolution have simplified biocatalysis adoption.

Early attempts to embrace biocatalysis relied on identification of native microorganisms and enzymes capable of catalysing the desired transformation with exquisite selectivity under the required process conditions. This represented a significant barrier, as an enzyme's natural sensitivity and substrate specificity often were not compatible with manufacturing process conditions, where solvent concentration, temperature and pH, for example, are often out of a typical physiological range. Early protein engineering attempts to overcome these challenges largely involved generating structure‐guided, rational mutations to an enzyme's primary sequence through site‐directed mutagenesis.

Advances in the directed evolution of proteins, however, have facilitated, greatly accelerated and enabled the wider‐scale implementation of biocatalysis by providing an accessible means of producing fit‐for‐purpose enzymes and increasing the overall speed of enzyme engineering [3]. Directed evolution can be used to tailor multiple enzyme properties that historically challenged the uptake of biocatalysis, rapidly alleviating problems with properties such as activity, specificity, expression, thermostability and tolerance to process conditions. This ability to engineer biocatalytic enzymes has been a boon to all fields of chemical manufacture, including pharmaceuticals.

Despite all the technological advances and positive trends, adoption of industrial‐scale biocatalytic processes has been generally slower than expected. This is mainly due to the fact that small‐molecule drug manufacturing processes are far from simple and efficient; development of a small‐molecule screening hit to a commercial product takes around 10–15 years [4]. Recently, in GSK, there has been a focus on adopting biocatalysis by first intent, rather than as a second‐ or third‐generation process. This aids in the reduction of costs associated with filing a new process post‐approval, as well as reducing the resource requirement over the lifecycle of the product [5]. As such, within GSK, we employ a unified technology platform to deliver our portfolio [6].

In addition to flow chemistry (continuous primary) and chemical catalysis, biocatalysis was identified as step‐changer technology that would boost GSK's ability to manufacture APIs. To accelerate internal biocatalysis and enzyme engineering capability, in 2014, GSK in‐licensed the CodeEvolver® platform from Codexis, a California, USA‐based biotechnology company. Internal investment in a team of scientists with expertise in biocatalysis, directed evolution, molecular biology, sequencing and computational chemistry provided dedicated support and operation of this platform. In addition, specialised theoretical and practical biocatalysis courses and workshops were organised for medicinal, organic and process chemists, to increase awareness of biocatalysis and directed evolution for synthetic chemistry applications. This training enabled scientists to identify potential enzymatic opportunities in chemistry routes and creen available biotransformations.

This chapter highlights how the directed evolution of biocatalysts has delivered impact for GSK, and contextualises this in the API manufacturing and drug development environment, enabling understanding of the timelines required to deliver a robust and manufacturable biocatalytic process. The chapter also provides examples of success stories in implementing directed evolution at GSK, and discusses the hurdles currently associated with embedding biocatalysis and how the process may be further accelerated.

1.2 Drug Development Stages

To understand how biocatalysis fits into the pipeline, knowledge of the drug development process is required. The journey from small‐molecule screening to commercialisation of a medicine starts with target selection and validation (Figure 1.1). During this process, scientists gather evidence to support the role of a target (e.g. an enzyme in a biochemical pathway or a receptor) in a given disease, and the potential therapeutic benefit of modulating its function. The second stage is lead discovery, where the scientists seek molecules capable of interacting with the target (hits), which can then be used as a starting template for further optimisation. These hits are derivatised into synthetic small substrates called lead molecules, which interact with the target and have additional qualities that give the team confidence that they can be optimised to deliver a medicine (e.g. favourable target binding strength and selectivity). This process, from target selection to identification of lead molecules, can take between 4 and 24 months.

During the lead discovery and optimisation stages, the attrition rate is very high, with most compounds being discarded because of poor biochemical or biophysical properties. The focus, therefore, is on the quick delivery of a large number of diverse compounds using whichever chemistry works – making the adoption of biocatalysis more difficult at this stage. As only small quantities of compound are required (mgs), techniques like chromatography and chiral resolution are considered acceptable, meaning that the selectivity and process advantages offered by biocatalysis are less likely to be harnessed. In the identification of compounds with the desired pharmacological properties, however, biocatalysis can still be a powerful tool at this stage, as it enables the synthesis of drug entities inaccessible by other chemistries.

Once several pre‐candidate molecules have been selected, medicinal chemists can begin to focus their effort on identifying biocatalysts that may provide a more efficient synthetic step. Quick read‐outs and fast delivery are required, meaning that enzyme hits (those identified as capable of performing the desired chemistry) need to be easily scaled to deliver grams of material and allow delivery of product for further toxicological studies. Reactions utilising non‐process‐ready biocatalysts (having low activity, poor stability or suboptimal selectivity) do not pose a significant problem at this stage, provided that the biotransformation affords product that meets the minimal quality criteria (e.g. chiral purity), since the quantity of product that must be prepared is small. Due to the relatively small scale, several of the downsides of non‐process‐ready enzymes can be easily mitigated (e.g. purification to meet quality requirements or centrifugation to deal with high enzyme loadings), affording some flexibility around the biocatalyst properties. However, if the catalyst presents challenges at this small scale (e.g. very low activity that cannot be reproduced on gram scale, or poor selectivity) that would require engineering to overcome, it is unlikely that the enzymatic step will be pursued as the high attrition of compounds means the resource commitment to enzyme evolution is difficult to justify. One solution to this problem is to increase the quality of the enzyme panels (collections of enzymes from various transformation classes that are initially screened for desired biotransformation), and therefore the likelihood of success on scale‐up, by expanding the number and diversity of enzymes within the panels – either by acquisition of new enzymes or through panel expansion with engineered enzyme variants.

Figure 1.1 A general drug discovery path overlapped with biocatalytic opportunities.

Once the few promising candidates have been selected, the drug journey continues with preclinical evaluation, where compounds are assessed for toxicity and efficacy using a combination of in vitro and in vivo animal models. With this data in hand, a decision is made as to whether or not a compound will progress to phase 1 clinical trials – also called ‘commit to first time in human’. During phase 1, which typically takes between 12 and 18 months, batches of API are prepared for later dosing. The challenges that occur during API preparation can have a knock‐on effect on clinical trials, causing them to slow or halt if drug supply is inadequate. Keeping these trials on time is key to timely assessment of drug candidates and ultimately to the delivery of approved medicines to patients. At this point, an ideal process for delivery of API is not required, provided that product quality is maintained. For assets in phase 1, the route employed by the medicinal chemistry team is usually scaled up to provide API for toxicology studies. Although these processes are usually not suitable or scalable for commercial‐scale manufacturing, the time‐critical nature of compound delivery and the small quantities required mean significant time isn't usually invested in process development at this stage.

Whilst API is being supplied using a non‐ideal manufacturing process, route scouting activities are undertaken concurrently to identify more appropriate long‐term syntheses. This is often where biocatalysis opportunities are identified and screened. At this point, the hits from enzyme panel screening must provide a significant advantage over the previous chemistry and provide API compound on a reasonable scale. Enzyme engineering becomes feasible at this stage and can play an important part in assessing a route's feasibility, impacting route scouting and selection.

As development progresses and the asset heads towards phase 2, delivery of API for clinical studies must occur concurrent with other process development activities. These include:

Discovery of new routes of synthesis and chemistries to facilitate this (route scouting)

Optimisation and understanding of the chemical process

Thorough understanding of parameters impacting drug substance and intermediates quality

Consideration of supply chain security for the process – that is, availability of starting materials and reagents on the required scale

Transfer of the process to a full‐scale manufacturing plant

Preparation of regulatory documentation to support file and launch

If found to be effective during phase 1, an asset reaches phase 2 trials – a process that can take 2–3 years before commitment to medicine development. In addition to drug production for future trials, it is also important that scale‐up process strategy is considered during this time. By now, the number of candidates has been reduced and route improvements driven by introducing biocatalysis have been linked directly with API production. The biocatalyst has been optimised to deliver a manufacturable process, but there is still room for further improvement and fine tuning if certain criteria (such as cost of goods) have not yet been met.

In phase 3, extensive work is undertaken to identify the logistics surrounding the distribution of clinical supply to the investigator sites and to develop a robust commercial, end‐to‐end supply chain to ensure continuity of both launch and long‐term drug supply to patients. By this time, in addition to optimisation of enzymes through engineering and biocatalytic process development, a fermentation process for enzyme supply and the manufacturing chain must be established.

Throughout the drug development phases, the aim is to generate a process that is safe, operable and ultimately efficient. In this context, the decision to engineer an enzyme for use in an API manufacturing route is complex, due to the time and resource commitment required for protein engineering. It is often the case that either wild‐type or panel enzymes will satisfy the requirements of early, small‐scale manufacturing campaigns of nascent assets. However, once an asset is significantly advanced along the drug development pipeline, it becomes increasingly difficult for enzymes to provide a manufacturable process for larger‐scale clinical supply campaigns. This is where directed evolution has a large impact. As strategies and technologies develop to expedite the evolution process, it becomes increasingly possible to adopt directed evolution earlier in the drug development cycle. In the next section, we discuss how recent developments have helped with this aim, and what future work is required to fully realise this vision.

1.3 Enzyme Panels

GSK has a significant number of panels, produced both internally and acquired from external sources, which are continuously enhanced through evolution and addition of new enzyme classes (Scheme 1.1).

A portfolio analysis reveals which transformations account for the most frequently used chemistries within GSK [6]. Heteroatom alkylation and arylations, together with aromatic heterocycle formations, make up approximately 40% of the portfolio. Functional group interconversion, C‐C bond formation and reductions, oxidations and protections are other types of transformations frequently encountered. Focusing on enzymatic alternatives for these transformations would have the most impact.

New enzyme panels are assembled using a diverse set of enzymes which have been identified through previously demonstrated activities or using predictive tools (Figure 1.2). In some instances (e.g. lipases), a significant number of enzymes are already commercially available, and therefore these panels comprise mostly enzymes from a commercial source. Although it is usually trivial to acquire larger quantities of commercial enzymes to facilitate scale‐up of any hits for a particular transformation, commercial enzymes can carry intellectual property (IP) restrictions around their use for commercial‐scale manufacture and can cause supply‐chain concerns as most are single‐source. Additionally, it is not usually possible to evolve a commercially‐available enzyme without licensing agreements with the source company.

Scheme 1.1 Selected enzyme classes currently in GSK's collection.

Figure 1.2 Strategies for generating an enzyme panel.

For most other enzyme classes, there are very few, if any, commercially‐available enzymes available to screen. Although significant efforts are underway to change the situation, this often means that enzymes of the desired class must be identified by other means. Panels are often assembled by acquisition of the genes encoding wild‐type enzymes reported to catalyse the reaction class of interest in the literature. In some cases, mutational studies have also been conducted, and variants of these enzymes may also be acquired.

Bioinformatic tools are the key to the assembly of an enzyme panel, allowing identification of additional putative enzymes from sequence databases based on similarity to known enzymes of the desired class. The goal of a search is straightforward: to identify naturally occurring enzymes that perform the same transformations but maximise their sequence and structural diversity in hopes of maximising the substrate scope, as well as the pliability of the enzymes to be evolved.

As with hit expansion for small molecules, enzymes identified in the literature are often used as seeds for subsequent similarity searching against large annotated sequence datasets, including those from Interpro [7], Uniprot [8], NCBI, PDB [9], CATH [10] and some metagenomics collections [11]. In a standard approach, homologous sequences are identified and clustered, and each cluster's functional annotations are examined. Homologous protein structures are often included in the clustering step as they help in the identification of the relevant clusters from which to sample. Selecting exemplars from the various clusters is often the most challenging part of the process. In a typical scenario, candidates are prioritised based on the availability of experimental annotations, starting with those with experimental data or structures or coming from extremophile organisms. In the absence of additional data, the remaining exemplars are chosen from a diverse set of clades that maximise coverage of each cluster's diversity. The HH‐Suite toolset [12], maintained by the Soeding group, provides great tools for identifying homologues, as well as Mmseqs2 [11] and CLANS [13] for clustering. Phylogenetic reconstruction can be performed using MEGA [14] or ETE3 [15] and their corresponding tree‐reconstruction and evolutionary‐analysis workflows.

One way to improve this approach is to select for enzymes that exhibit similar active sites, and thereby maintain activity and selectivity, by developing ‘fingerprints’ or protein‐based pharmacophores, sometimes referred to as PLIFs, which describe the residue composition and positionality within the active site and the interactions with bound ligands [16]. Once a fingerprint is created, it can be used to identify clusters of sequences that exhibit similar active site makeups, which can then be prioritised for acquisition. The goal of this approach is often to acquire enzymes that minimally perturb the active site but which sample diversity throughout the remainder of the enzyme. The method can also be extended to sample diversity at specific positions within the active site. The success of this approach hinges on the availability of experimentally determined structures from the structural families of interest. The approach also assumes that the active site can be unambiguously identified.

Beyond the acquisition of wild‐type enzymes, it is also possible to leverage variants produced in previous enzyme evolution campaigns to enrich a panel. As these evolved enzymes are likely to have generally improved properties (e.g. activity, stability and expression) compared to wild‐type enzymes, they can often provide hits more amenable to immediate scale‐up. These enzymes can be the result of in‐house evolution, in‐licensing of other companies' enzyme panels or from collaborations.

As these panels are used for the initial assessment of a biocatalytic transformation, it is important to maintain a significant amount of sequence diversity within them, in order to increase the likelihood that enzymes with the desired activity and selectivity can be identified for a wide range of substrates.

One example is the GSK IRED panel, where 85 IREDs were assembled from different sources [17]. These wild‐type variants were tested under industrially relevant conditions using equimolar loadings of amine. Screening a diverse set of substrates, selected to cover a broad chemical space, showed most enzymes were capable of driving reactions to completion (Scheme 1.2). Enzyme‐dependent stereoselectivity was observed for many products and successful scale‐up was performed for several enzymes and substrates.

Reductive amination of keto acids represents another area where we focused our efforts and successfully generated a small collection of enzymes that are very promising for the synthesis of N‐alkylated amino acids [18,19]. Enzyme such as N‐methylamino acid dehydrogenases (NMAADHs, EC 1.5.1.1 and EC 1.5.1.21), ketimine reductases (KIREDs, EC 1.5.1.25) and Δ1‐pyrroline‐5‐carboxylate reductases (P5CR, EC 1.5.1.2) have been shown to deliver highly enantioselective alkylated products, further extending the scope of these substrates (Scheme 1.3).

Following several successful in‐house enzyme evolution campaigns, we have also constructed enzyme panels incorporating the best variants produced therein. One of the drivers for this practice is that, by creating panels of robust enzymes with diverse activities, it becomes more likely that a panel can be used directly without the need for further enzyme engineering – greatly increasing the speed at which the biocatalytic process can be implemented, and therefore increasing uptake across the portfolio.

Scheme 1.2 Reductive amination of ketones performed by imine reductases.

Scheme 1.3 Successful synthesis of alkylation products via enzymatic reductive amination of keto acids.

GSK is also collaborating with various academics in the field with the purpose of continuously expanding its portfolio of enzymes (e.g. halogenases, nitration enzymes, unspecific peroxygenases, etc).

1.4 Enzyme Engineering

With high‐quality enzyme panels in hand, it becomes possible to develop a manufacturable process for an API using the hit from panel screening without the need for further enzyme optimisation by either engineering or evolution. The definition of a manufacturable process changes throughout the drug development cycle and becomes more stringent as the asset matures and the quantity of API required increases – making it more likely that engineering of a panel enzyme will be necessary to meet these needs. The specific process parameters required for assets at each stage of development are complex, but the following list summarises some key aspects that must be considered before committing to the evolution of an enzyme:

Product quality:

This is always a key factor in considering any manufacturing process. Depending upon the quantity of product required, more laborious purification steps (e.g. chromatography) may be considered in order to supply a compound in a timely manner. For large‐scale synthesis of later‐stage assets, processes capable of delivering the required purity directly are required, and enzyme engineering is often employed to achieve this.

Enzyme supply:

For small‐scale synthesis of early‐stage assets, it is likely that laboratory‐scale fermentation can provide sufficient quantities of enzyme for a reaction – even if activity is low and high enzyme loading is required. For large‐scale synthesis of later‐stage assets, enzyme loading needs to be reduced as low as is practicable for biocatalyst supply to be economical, and enzyme engineering is often employed to improve enzyme activity and reduce the loading of enzyme required.

Physical properties: