Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. 'Hits' generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate/product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.

The research carried out within this thesis was aimed at the development and implementation of a biocatalytic route towards Ticagrelor, a platelet-aggregation inhibitor. A bio-retrosynthetic consideration of the target compound yielded different possible strategies, which were analysed in terms of enantioselectivity and efficiency. The ultimate goal was to generate a biocatalyst specifically tailored to the starting material to yield the target compound in high optical purity and conversion. Different approaches to the chemoenzymatic generation of the cyclopropyl subunit (cf figure) in enantiomerically pure form were proposed and tested. The lipase from Thermomyces lanuginosus proved to be the most selective and active enzyme tested and was used as a model enzyme, initially yielding an E of 76 at a conversion of 50% after 48h. Through both reaction engineering and rational protein design approaches the time to attain 50% conversion could be reduced to 24 h while the enantioselectivity of the process increased to 100. Moreover, in a rational protein design approach different residues in the lid of the lipase were identified through analysis of the resolved crystal structures and subsequently mutated in order to investigate the influence of these residues on the overall performance of the lipase towards the target biotransformation. Mutations on Asn88 resulted in the inactivation of the enzyme while an Asp57Asn mutation resulted in a more active enzyme. Ultimately, this research has contributed to making the synthetic route towards Ticagrelor more environmentally sustainable, diminishing the need for the use of toxic, unsustainable and sterically demanding auxiliaries, as well as the amount of waste produced. The principles of green chemistry have been applied to the case studied. The synthetic route towards a key fragment of Ticagrelor has been significantly shortened using a biotransformation with an enzyme that can be recycled and employed in catalytic quantities.