Evolved P450 mutants as general oxidation catalysts for target synthesis via C-H activation

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This project falls within the EPSRC research themes: Healthcare Technologies (related: synthetic biology, synthetic organic chemistry); Physical Sciences (related: catalysis, synthetic biology, synthetic chemistry); Manufacturing the Future (related: manufacturing technologies).This project will concentrate on the synthesis of a range of fragments and natural products via late stage oxidation using a library of mutant cytochrome P450 enzymes. This approach, by introducing oxy-functionality at the end of the synthesis, allows the focus to be on the skeleton-building process, which alleviates the need for protecting groups and makes the route more efficient and environmentally friendly. The use of P450s to effect asymmetric oxidation will be explored, and the use of these enzymes on a range of subtrates will help to build a profile of reactivity, moving these mutants closer to application as general oxidation catalysts.Late stage functionalisation has emerged as an efficient method of chemical synthesis. It allows the synthetic route to focus on skeleton-building without the need for a protecting group strategy. Hydroxylation is of great interest with the field of late stage functionalisation due to the ubiquity of oxy-functionality in both natural products and drug molecules.Recent work on late stage hydroxylation has tended to use chemical catalysts to achieve these transformations which, while practicable, can be expensive and difficult to dispose of. This project will develop a different catalytic method, one which utilises a library of mutant P450 (mP450) enzymes. These enzymes can be evolved and tailored so the desired reactivity can be developed, including activity complementary to current chemical oxidation methods.Cytochrome P450BM3 (CYP102A1) belongs to the P450 cytochrome (CYP) superfamily of haemproteins, found in almost every organism as oxidase enzymes. Their ubiquity in Nature has led to extensive study into their structure and mechanism of action; as a result, attention has turned to their use as catalysts for chemical synthesis. P450s catalyse the oxidation of a wide range of organic substrates and their signature transformation is the hydroxylation of C-H bonds. Using only molecular oxygen and NADPH, alcohol products are formed via C-H activation within the enzyme active site. The nature of the active site makes it possible to access un-activated positions and gives the opportunity for asymmetric hydroxylation.Work towards ferruginol, an abietane diterpene, will continue, with the key mP450 oxidation being the conversion of interest. The carbon skeleton will be screened against a panel of P450 mutants targeting an enzyme that carries out the necessary aromatic oxidation. The oxidation will be attempted on both the racemic mixture and the separate enantiomers in order to test for kinetic resolution. With a mP450 in hand which carries out the correct oxidation, other abietane natural products could be targeted to expand the scope of the enzyme. In addition, the diterpene eleutherobin and taxol derivative 'taxadienone' will also be targeted.