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Chemoenzymatic Synthesis of Novel, Structurally Diverse Compounds

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Chemoenzymatic Synthesis of Novel, Structurally Diverse Compounds Chemoenzymatic synthesis of novel, structurally diverse compounds A Thesis Submitted for the Degree of Doctor of Philosophy to the University of London Lydia G Coward Department of Biochemical Engineering University College London 1 I, Lydia Grace Coward confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 For my fantastic family. 3 Abstract The use of Diels Alder cycloaddition chemistries to access a diverse range of useful cyclic structures is well established throughout literature. However, the value of the product may be enhanced further still by linking this reaction with subsequent (biocatalytic) steps to create novel, structurally demanding, optically pure compounds. This project investigates the linking of Diels Alder (DA) chemistry to the enzyme, transketolase (TK) as a model integration pathway of a chemical syntheses and a biological transformation. The two-step process aims to provide a framework to synthesise small structurally diverse compounds with high enantiomeric excess. The demand for optically pure compounds is becoming a necessity due to the adverse affects frequently introduced by racemic compounds and the cost implications of the material possessing often only 50% active compound. Recombinant wild type Eschericha coli transketolase (EC 2.2.1.1) (WT-TK) was overexpressed in E. coli for the biocatalytic step of this two step synthesis. A substrate walking approach whereby a range of sequentially linked cyclic aldehydes, were applied to wild type transketolase and potential activity detected. Transketolase mutants, previously constructed based on information derived from the structural position within the active site of the dimeric enzyme were subsequently screened for activity with the cycloadduct of the Diels Alder reaction as aldehyde acceptor substrate for TK. Following identification, selection, culturing and sequencing of variants indicating enhanced activity towards the novel, bulky, hydrophobic, cyclic aldehyde the enantioselectivity and absolute stereochemistry of the product were determined. Activity displayed by wild type transketolase indicated an 18,000 fold activity improvement. Michaelis- Menten kinetic parameters were subsequently determined to have an apparent Km -1 -1 of 69.9 mM , kcat of 17.5 s and a vmax of 0.07 mM.min and preliminary process compatibility including product and substrate inhibition issues were highlighted. 4 Acknowledgements I would like to thank my supervisors, Dr. Paul Dalby, Dr. Helen Hailes and Professor John Woodley for their guidance throughout my project. The interesting multi-disciplinary discussions provided both motivation and inspiration for the topics. A big thank you is also due to Dr. Mark Smith, James Galman and Dr. Kirsty Smithies for the invaluable advice, assistance and kindness in all aspects of molecular chemistry, organic chemistry and analytical chemistry. The support received from the chemistry department throughout my entire project was very much appreciated. I am also very grateful to everyone within the Biochemical Engineering department at UCL, in particular those within the BiCE programme for the very useful advice and constructive discussions. I would like to thank Adam Stonier, Sandro Matosevic, Nina Remtulla and James Galman not only for help with IT quandaries and practical assistance but on a personal level, making this journey an enjoyable one. Finally, I would like to thank my family for their love, support and encouragement always. And to my lovely husband, Ritchie Crago whom without his support and understanding, this would have been impossible. 5 Abbreviations ABT 2-amino-1,3,4-butanetriol ACN Acetonitirile AmpR Ampicillin resistant AP Acetophenone BSA Bovine serum albumin CO 2 Carbon dioxide DA Diels Alder reaction DCHCA 3,4-dimethyl-3-cyclohexene-1-carboxaldehyde DCDHP 1-(3',4'-dimethylcyclohex-3'-enyl)-1,3-dihydroxypropan-2-one DHP 1,3-dihydroxypentan-2-one DNA Deoxynucleic acid DOT Dissolved oxygen tension (%) E.C. Enzyme commission E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid L-ery L-erythrulose EtOAc Ethyl acetate GA Glycolaldehyde GC gas chromatography His-tag Histidine-tag HPA Hydroxypyruvic acid HPLC High Performance Liquid Chromatography IPTG Isopropyl-β-D-thiogalactosidase ISPR in situ product removal kDa kilo dalton Km Michaelis constant kcat Enzyme turnover rate LB Luria -Bertani Li-HPA Lithium hydroxypyruvate MBA Methylbenzylamine MeOH Methanol mg milligrams mL millilitre MgCl Magnesium chloride 6 MPTA 2-methoxy-2-(trifluoromethyl) phenylacetic acid NaOH Sodium Hydroxide Ni-NTA Nickel – Nitriloacetic acid OD Optical Density PA Propanal PCR Polymerase chain reaction PLP Pyridoxal phosphate PMP Pyridoxamine phosphate PMA Phosphomolybdic Acid RO Reverse osmosis rpm revolutions per minute [S] Substrate concentration SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis SFPR substrate feed product removal SRW Standard round well TAm Transaminase TEMED Tetramethylethylenediamine TFA Trifluoroacetic Acid TLC Thin Layer Chromatography THF Tetrahydrofuran TK Transketolase TPP Thiamine Pyrophosphate TPTC 2,3,5-tripheyltetrazolium chloride Tris-HCl Tris(hydroxymethyl)aminomethane-hydrochloric acid UCL University College of London UV Ultraviolet v/v volume by volume vmax Maximal velocity WT Wild type X5P xylulose-5-phosphate µmax Maximal specific growth rate µL microlitre µg microgram 7 Contents Abstract ........................................................................................................................ 4 Acknowledgements ...................................................................................................... 5 Abbreviations ............................................................................................................... 6 Contents ........................................................................................................................ 8 List of figures ............................................................................................................. 15 List of Tables .............................................................................................................. 18 List of schemes ........................................................................................................... 18 1 Introduction ....................................................................................................... 19 1.1 Chemoenzymatic synthesis: Integrating chemistry and biology ................. 19 1.2 Chirality ....................................................................................................... 24 1.2.1 Optically pure pharmaceuticals ........................................................... 25 1.2.2 Enantioselectivity ................................................................................ 29 1.3 Carbon-carbon bond forming ...................................................................... 29 1.4 Biocatalysis ................................................................................................. 30 1.4.1 Advantages and disadvantages of biocatalysis .................................... 31 1.4.2 Ideal biocatalysts ................................................................................. 33 1.4.3 Biocatalysts in Industry ....................................................................... 34 1.4.4 Transketolase (TK) .............................................................................. 35 1.4.4.1 Synthetic potential of transketolase .................................................. 37 1.4.4.2 TK function In vivo .......................................................................... 38 1.4.4.3 Transketolase mechanism ................................................................. 38 1.4.4.3.1 Thiamine pyrophosphate (TPP) ................................................. 41 1.4.4.4 Sources of TK ................................................................................... 41 1.4.4.5 TK production .................................................................................. 42 1.4.4.5.1 TK purification .......................................................................... 42 1.4.5 Enzyme modification .......................................................................... 44 1.4.5.1 Enzyme evolution ............................................................................. 44 1.4.5.2 Directed evolution ............................................................................ 45 1.4.6 TK biotransformation .......................................................................... 47 1.4.6.1 Reactants and products ..................................................................... 47 8 1.4.7 TK stereoselectivity ............................................................................. 49 1.5 Diels Alder reaction ..................................................................................... 51 1.5.1 Diels Alder reaction substrates ............................................................ 51 1.5.2 DA cycloadducts ................................................................................. 52 1.5.3 Solvents and catalysts for Diels Alder reaction ................................... 53 1.6 Enzyme kinetics .......................................................................................... 55 1.6.1 Detection methods ..............................................................................
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