Biocatalytic Synthesis of a Key Chiral Delamanid Precursor using an Engineered P450 Monooxygenase

We report the development of an engineered P450 monooxygenase that mediates a chemo- and stereo-selective alkene epoxidation to generate a key chiral precursor of the anti-tuberculosis drug delamanid. Screening of an in-house P450 monooxygenase panel led to the identification of a BM3 variant, containing five mutations, with activity for the target transformation. Over a single round of laboratory evolution and gene shuffling, three further beneficial mutations were introduced leading to an order of magnitude increase in the de-sired activity, with a total turnover number (TON) of >3000. This newly engineered enzyme generates a chiral epoxide intermediate from an alkene precursor in a single step with 98% e.e. and >97% conversion. Initial efforts to scale the biocatalytic transformation high-lights the potential of the engineered enzyme to provide a more efficient and sustainable route for the manufacture of delamanid.