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New Biocatalysts for Synthetically Useful Metabolites from Available Phenols

Howard, Daniel

[Thesis]. Manchester, UK: The University of Manchester; 2019.

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Abstract

Toluene dioxygenase (TDO) is a Rieske type non-haem enzyme found within the soil bacteria, Pseudomonas putida (P. putida) and is responsible for catalysing the enantioselective cis-dihydroxylation of aromatic substrates. This remarkable trait of the TDO enzyme has enabled its application in the preparation of over 400 novel cis-diol bioproducts using whole cell biocatalysis. Recently, a new and synthetically useful bioproduct, (4S,5S)-4,5-dihydroxy-3-iodocyclohex-2-en-1-one, has been identified from the TDO catalysed biotransformation of 3-iodophenol. The purpose of this project was to demonstrate the synthetic application of this new bioproduct in the synthesis of natural product analogues, which possess anti-proliferative activity against different cancer cell lines. This was achieved by developing methodology towards a key enone building block, which was successfully applied in the synthesis of an analogue of the natural product incarviditone. Furthermore, a robust computational docking model was developed using the Goldâ„¢ software, which showed significant correlation between the predicted docking outcome and the experimentally observed results for a series of monocyclic substrates. The docking model was also used to rationalise the major and minor binding modes of the 3-iodophenol substrate in the TDO enzyme active site. The project also sought to develop an alternative synthetic methodology using butan-1,2-diacetal (BDA) protected (-)-quinic acid, which would provide the same stereochemical outcome as the newly discovered bioproduct. This was achieved despite considerable synthetic challenges encountered when trying to control the diastereoselectivity of a key conjugate addition reaction. The antipodal compounds of the natural product analogues obtained using the 3-iodophenol bioproduct and BDA protected (-)-quinic acid were also synthesised using acetonide protected (-)-quinic acid. By reacting the enantiomers of a key hydroxyenone intermediate obtained using the two pathways, a novel heterodimerisation was reported to afford an analogue of the natural product, incarvilleatone. Finally, all the biological activities of the synthesised natural product analogues were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay: the results showed significant differences between the enantiomers of the incarviditone natural product analogues.