University College London UCL Biocatalysis Using Plant and Metagenomic Enzymes for Organic Synthesis Sophie Alice Newgas Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) 2018 [1] [2] Declaration I, Sophie Alice Newgas, 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. Signed: Dated: [3] Abstract Biocatalysts provide an excellent alternative to traditional organic chemistry strategies, with advantages such as mild reaction conditions and high enantio- and stereoselectivities. The use of metagenomics has enabled new enzymes to be sourced with high sequence diversity. At UCL a metagenomics strategy has been developed for enzyme discovery, in which the library generated is annotated and searched for desired enzyme sequences. In this PhD, a metagenomic approach was used to retrieve 37 short chain reductase/dehydrogenases (SDRs) from an oral environment metagenome. Eight enzymes displayed activity towards cyclohexanone and their substrate selectivities were investigated. Four of the SDRs displayed activity to the Wieland-Miescher ketone (WMK), a motif found in several pharmaceutically relevant compounds. SDR- 17 displayed high conversions and stereoselectivities and was co-expressed with the co-factor recycling enzyme glucose-6-phosphate dehydrogenase. This system was then successfully used to reduce (R)-WMK on a preparative scale reaction in 89% isolated yield and >99% e.e.. In further studies using reductases, the substrate specificities of two ketoreductases known as tropinone reductase I and II (TRI and TRII respectively) from the plant D. stramonium and MecgoR from E. coca were investigated. These studies expanded on reported substrate activities with these enzymes in the literature. A selection of symmetric and asymmetric tropinone analogues were synthesised, towards which MecgoR and TRI showed high activities, providing a strategy to access novel alcohols. Furthermore, sixteen ketoreductases were selected from a drain metagenome based on their sequence similarity of over 24% to MecgoR. They were annotated as aldo/keto reductases (ARKs) and five were successfully expressed in E. coli. Interestingly, the novel enzyme AKR-3 displayed activities toward aromatic ketones and aldehydes such as 2-indanone, phenylacetaldehyde and benzaldehyde. [4] Transaminases (TAms) from the enzyme library toolbox at UCL were also tested with tropinone analogues and related cyclic compounds, several of which showed good activities. [5] Impact Statement This PhD project explores on the use of biocatalysis as an environmentally friendly alternative to the use of traditional chemical catalysts. The use of enzymes to perform chemical reactions is a sustainable alternative because the method results in low energy consumption and waste production. The reactions can be highly efficient with excellent selectivity, and the use of enzymes is both renewable and may be less toxic than alternatives. Because of this, there has been a recent increase in interest in this sustainable topic. The use of enzymes to perform organic chemical reactions relies on the availability of enzymes which accept the targeted molecule of interest. Searching for novel enzymes is critical both inside academia, as well as in industry, for the sustainable production of pharmaceuticals and fine chemicals. This thesis addresses this issue by mining a metagenomic database to discover enzymes. From the identified enzymes, several were identified as performing useful reactions with a selection of substrates. Highlighting the industrial applicability of this work, one of these enzymes was scaled up to a preparative scale with a particularly useful molecule, the Wieland Miescher Ketone, which is a motif found in a wide selection of pharmaceutical drugs. A co-factor recycling system was employed to achieve excellent conversions and stereoselectivities. This research also utilises plant enzymes to efficiently perform biocatalytic reactions, with a focus on the use of the bicyclic natural product tropinone as a substrate. The tropinone structure is a motif that is found in a wide selection of pharmaceutical and recreational drugs and the tropinone alkaloid family is known for its anticholinergic and chemotherapeutic properties, with applications ranging from treatments for Parkinson’s disease to intestinal tract problems. Plant enzymes were employed to perform reactions on a selection of embellished tropinone substrates and accomplished high conversions. With a focus on the interface between biology and chemistry, a broad range of techniques were utilised including organic synthesis, molecular biology, analytical [6] chemistry and biochemical approaches. This research has been presented at multiple conferences, both nationally and internationally, and will be used by other academics working in the discipline. The research presented in this thesis overcomes challenges associated with reduction and transamination reactions by utilising biocatalysis and contributes to the development of sustainable processes within organic chemistry. [7] Acknowledgements I am very grateful to my fantastic supervisor Professor Helen Hailes for all her guidance, support and teaching. She has helped me every step of the way during my PhD, and has been so encouraging. I also would like to thank Professor John Ward for all his help and ideas and also being an excellent supervisor. Both Helen and John have created a wonderful lab group and I couldn’t have asked better supervisors. I also would like to thank other who have helped me with this work, including Jack Jeffries, Maria Bawn, Damien Baud, Dragana Dobrijevic, Fiona Truscott, Nadine Ladkau, Fabiana Subrizi, and Dani Mendez-Sanchez. They have taught me so much day to day in the lab and have provided so much advice. My fellow PhD students including Leona, Alice, Rachael, and Harriet have been fantastic friends, and have supported me during this PhD. I couldn’t have done it without you guys. My whole lab group has been amazing, so I want to express my gratitude to everyone in the group. In addition, all the PhD students and PDRAs in John’s group have also been amazing, and have taught me so much. I also thank all the other friends and family from outside UCL who have been so important to me over that last few years. My parents, John and Celia, and my brothers, Adam and David, have helped me an indescribable amount and I am so grateful. My housemates Inez and Amy have also put up with me for three years and have been there for all the highs and lows. I’m also so grateful to my RSY friends, as well as all my school friends for their support and continuous encouragement. My friends from both Warwick University and those that I have made whilst at UCL have also been made these last three years unforgettable. [8] Abbreviations 2,5-DMTHF Dimethoxy tetrahydrofuran AcOH Acetic acid ACP Acyl-carrier-protein ADH Alcohol dehydrogenase AKR Aldo/keto reductase Aq. Aqueous BLAST Basic Local Alignment Search Tool bp Base pairs BVMO Baeyer-Villager Monooxygenases °C Degrees centigrade CBT Carbomethoxytropinone CCL Clarified cell lysate CDCl3 Deuterated chloroform CH2Cl2 Dichloromethane CHCl3 Chloroform Contig Contiguous sequences d.e. Diastereomeric excess Da Dalton DMAP 4-(Dimethylamino)pyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide e.e. Enantiomeric excess EC Enzyme Commission Et Ethyl Et2O Diethyl ether Et3N Triethylamine EtOAC Ethyl acetate EtOH Ethanol FID Flame ionisation detector [9] G6P Glucose-6-phosphate G6PDH Glucose-6-phosphate dehydrogenase GC Gas Chromatography GDH Glucose dehydrogenase h Hour(s) His-tag Hexahistidine tag HPK Hajos-Parrish ketone HPLC High Performance Liquid Chromatography HSD Hydroxysteroid dehydrogenase Hz Hertz IDABO 8,8- Dimethyl-3-oxo-8-azonia-bicyclo[3.2.1]octane iodide iPrNH2 Isopropylamine iPrOH Isopropanol or 2-propanol KRED Ketoreductase LB Lysogeny broth LC-MS Liquid chromotography mass spectrometry LDA Lithium diisopropylamide LEH Limonine-1,2-epoxide hydrolases L-Orn L-Ornithine MADO (1R,6S)-10-methyl-10-azabicyclo[4.3.1]decan-8-one MBA Methylbenzylamine Me Methyl MeOH Methanol mins Minute(s) mol Mole(s) Mp Melting point MS Mass spectroscopy Mw Molecular weight n.d. Not determined n/a Not applicable NAD(P)+ Nicotinamide adenine dinucleotide (phosphate) [10] NAD(P)H Reduced nicotinamide adenine dinucleotide (phosphate) n-BuLi n-Butyllithium NCBI National Centre of Biotechnology Information NMR Nuclear magnetic resonance ORF Open reading frame PCR Polymerase chain reaction PDB Protein Data Bank Pet. Ether Petroleum ether Pi Phosphate buffer PLE Porcine liver esterase PLP Pyridoxal 5'-phosphate PMP Pyridoxamine 5' phosphate Ppm Parts per million Pr Propyl R Generic alkyl group Rf Retention factor Rt Retention time RT Room temperature s Second(s) Sat. Saturated SDR Short chain dehydrogenase/reductase TAm Transaminase TB Terrific broth TBON 8-Thiabicyclo[3.2.1.]-octan-3-one TFA Trifluoroacetic acid THF Tetrahydrofuran TK Transketolase TLC Thin layer chromatography TMS Tetramethylsilane TR Tropinone reductase TRI Tropinone reductase 1 [11] TRII Tropinone reductase 2 TRL Tropinone reductase-like SDR UV Ultra violet WMK Wieland-Miescher ketone [12] Table of Contents Declaration ................................................................................................................... 3 Abstract .......................................................................................................................