Evolved P450 Mutants for Streamlining Synthesis via Late-stage C-H Activation

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

The ability to effect selective hydroxylation of a specific C-H bond has profound implications for chemical synthesis and drug discovery. This proposal will further develop an ongoing collaborative effort to apply evolved cytochrome P450s in this important endeavour. We have shown that a panel of >100 unique bacterial P450 mutants developed within the Wong group converts a remarkably diverse set of organic substrates into (usually) mono-hydroxylated derivatives. Neutral lipophilic, polar, and charged molecules are oxidised, e.g. lidocaine, naproxen, and steroids such as testosterone. The general scheme for these oxidations underlies the enzymes' remarkable reactivity; reactions at 100mg substrate, routinely convert at catalyst loadings of 0.1 mol% or less. The only requirement is for a C-H bond to be physically accessible to the ferryl intermediate, and un-activated C-H bonds may therefore be oxidised. In contrast, chemical reagents show a complete preference for allylic and benzylic C-H bonds, and heteroatoms. P450-mediated oxidations are easy to conduct, require no special handling, employ mild conditions with no heavy metals and are tolerant of heteroatoms (including N and S), clean, sustainable and safe.
Our research within this area falls under three headings: i) Achieving 'difficult' oxidations; ii) Facilitating target synthesis; and iii) Medicinal chemistry applications. The initial object of this proposal concerns the second heading with application to the development of novel, selective herbicides. Viridiol, a furanosteroid secondary metabolite found in a handful of fungal strains, is potentially interesting as a new herbicide but there are concerns surrounding its mammalian toxicity that must be addressed. Viridiol has not yet been synthesised but the closely related antifungal kinase inhibitor viridin has been the subject of a few synthetic studies, including one total synthesis.
This molecule provides an ideal test-case to show how retrosynthetic considerations can be 're-programmed' based on a dual skeleton-building/late-stage oxidation strategy. Here, we will be free to construct the furanosteroid core by short skeleton-building sequences, carrying through minimal oxy-functionality thus dispensing with protecting group regimes. With the pentacyclic furanosteroid core prepared, research will begin to explore mutant P450 oxidation at both the activated (benzylic) and un-activated sites around the periphery, including the benzenoid ring-C. All the obtained viridiol analogues will be tested for herbicidal activity and in mammalian toxicity models to provide a structure-activity-relationship profile.
This project falls within the EPSRC Physical Sciences research area.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509711/1 01/10/2016 30/09/2021
1809776 Studentship EP/N509711/1 01/10/2016 30/06/2020 Victoria Alexandra Atkinson