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The Development of Novel Biocatalytic Routes for the Synthesis of Enantiomerically-Pure Chiral Amines

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The Development of Novel Biocatalytic Routes for the Synthesis of Enantiomerically-Pure Chiral Amines The development of novel biocatalytic routes for the synthesis of enantiomerically-pure chiral amines A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Science and Engineering 2017 Shahed Hussain School of Chemistry Contents Chapter 1: Introduction ................................................................................................. 11 1.1 Chirality and chiral amines .................................................................................... 11 1.2 Strategies for the production of chiral amines ........................................................ 12 1.2.1 Resolution ..................................................................................................... 12 1.2.2 Asymmetric synthesis .................................................................................... 15 1.2.3 Chemical imine reduction as a means to access chiral amines .......................... 15 1.3 Background on the evolution of biocatalysis .......................................................... 21 1.4 Reduction reactions – imine reduction ................................................................... 22 1.4.1 Artificial metalloenzymes for the reduction of imines ....................................... 22 1.4.2 Imine reductases ........................................................................................... 23 1.5 Reduction reactions – carboxylic acid reduction ..................................................... 33 1.6 Biocatalytic asymmetric synthesis - ω-transaminases ............................................. 34 1.7 Biocatalytic asymmetric synthesis – phenylalanine ammonia lyase .......................... 38 1.8 Biocatalytic asymmetric synthesis - amine dehydrogenase ...................................... 40 1.9 Deracemisation of racemic amines ........................................................................ 40 1.9.1 Chemical deracemisation methods .................................................................. 40 1.9.2 Amine oxidases for the biocatalytic deracemisation of amines .......................... 42 1.10 Research project aims ........................................................................................ 45 Chapter 2: Characterisation of the (R)-imine reductase from Streptomyces sp. GF3587 and comparison of the enzyme with the (S)-imine reductase from Streptomyces sp. GF3546 ... 46 2.1. Overview ............................................................................................................ 46 2.1.1 Brief background on imine reductases as biocatalysts ...................................... 46 2.1.2 Aims ............................................................................................................. 46 2.2 Characterisation of the (R)-imine reductase from Streptomyces sp. GF3587 ............ 47 2.2.1 Previously reported substrate scope and synthesis of screening compounds ..... 47 2.2.2 Biotransformation results with (R)-IRED and characterisation of the enzyme substrate scope ..................................................................................................... 53 2.2.3 Comparison of (R)-IRED substrate scope with (S)-IRED ................................... 59 2.2.4 Establishing substrate-enzyme kinetic parameters ........................................... 62 2.2.5 Enzyme mechanistic study – mutagenesis of the (R)-imine reductase ............... 64 2.3 Conclusions and outlook ....................................................................................... 68 Chapter 3: One-pot enzymatic cascade reactions combining imine reductase (IRED) with amine transaminase (ATA) and carboxylic acid reductase (CAR) enzymes ......................... 73 3.1 Overview of enzymatic cascade reactions .............................................................. 73 3.1.1 Existing strategies for enzymatic cascade reactions ......................................... 73 3.1.2 Aims ............................................................................................................. 76 3.2 ω-transaminase/imine reductase cascade (ATA/IRED cascade) ............................... 78 2 3.2.1 Principle and synthesis of screening compounds .............................................. 78 3.2.2 One-pot two-step enzymatic cascade for the production of 2,6-disubstituted piperidines............................................................................................................. 80 3.2.3 One-pot two-step enzymatic cascade for the production of 2,5-disubstituted pyrrolidines ........................................................................................................... 88 3.3 Carboxylic acid reductase/ω-transaminase/imine reductase cascade ....................... 89 3.3.1 Principle and synthesis of screening compounds .............................................. 89 3.3.2 One-pot multi-enzyme cascade for the production of 3-phenylmorpholine 19 and 3-phenylthiomorpholine 22 ..................................................................................... 90 3.4 Conclusions and outlook ....................................................................................... 93 Chapter 4: Amine oxidase-catalysed redox deracemisation reactions of 2-substituted cyclic amines, tetrahydroquinolines and indolines ..................................................................... 97 4.1 Overview of deracemisation strategies ...................................................................... 97 4.1.1 Existing strategies for enzymatic deracemisation of amines .............................. 97 4.1.2 Aims ............................................................................................................. 98 4.2 Amine oxidase/imine reductase catalysed deracemisation of 2-substituted cyclic amines ...................................................................................................................... 99 4.2.1 Optimisation of the biocatalytic deracemisation of amines with 6-HDNO and (R)- IRED ..................................................................................................................... 99 4.2.2 Deracemisation of racemic cyclic amine 2-phenylpiperidine 6c with 6-HDNO/(R)- IRED cascade ...................................................................................................... 101 4.3 Amine oxidase-catalysed deracemisation of tetrahydroquinolines .......................... 104 4.3.1 Current strategies for the deracemisation of tetrahydroquinolines .................. 104 4.3.2 Synthesis of tetrahydroquinoline compounds ................................................. 105 4.3.3 Establishing an autoinduction protocol for the expression and growth of CHAO 108 4.3.4 Biocatalytic deracemisation/kinetic resolution of tetrahydroquinolines – with AO/IRED combination .......................................................................................... 115 4.4 Deracemisation of indolines ................................................................................ 117 4.4.1 Brief background on chiral indolines and synthesis of indolines ...................... 117 4.4.2 Kinetic resolution of indolines ....................................................................... 119 4.5 Conclusions and outlook ..................................................................................... 128 Chapter 5: (R)-IRED-catalysed bioreduction reaction in continuous flow ......................... 133 5.1 Brief introduction to reactions in flow and current strategies ................................. 133 5.2 Imine reductase reaction in flow using the Freactor modular flow reactor units – principle .................................................................................................................. 135 5.3 (R)-imine reductase reaction in flow – results ...................................................... 138 5.4 Conclusions and further work ............................................................................. 140 Chapter 6: Conclusions and outlook.............................................................................. 142 6.1 Conclusions ....................................................................................................... 142 3 6.2 Outlook ............................................................................................................. 143 Chapter 7: Methods and experimental .......................................................................... 146 7.1 Materials, equipment and general analysis procedures ......................................... 146 7.1.1 Chemicals ................................................................................................... 146 7.1.2 Equipment and general analysis procedures .................................................. 146 7.2 Media, buffers and stock solutions ...................................................................... 147 7.2.1 Growth media ............................................................................................. 147 7.2.2 Buffers ....................................................................................................... 149 7.2.3 Antibiotics and IPTG stock solutions ............................................................. 149 7.3 Synthesis of substrates and analytical standards .................................................. 150 7.3.1 Synthesis of 2-substituted cyclic imines and amines ....................................... 150 7.3.2 Synthesis of 2-substituted
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