Selective oxidations of cyperenoic acid by slightly reshaping the binding pocket of cytochrome P450 BM3

The selective hydroxylation of unactivated C-H bonds in complex natural products represents a formidable challenge in synthetic organic chemistry. Cyperenoic acid, a sesquiterpenoid isolated from Croton crassifolius, possesses an antiangiogenic-promoting effect. Its C7- and C9-hydroxylated products can significantly inhibit the release of vascular endothelial growth factor (VEGF). To prepare these hydroxylated products, cytochrome P450 BM3 monooxygenase was chosen as a catalyst for the hydroxylation of cyperenoic acid. A simple and fast strategy, slightly reshaping the binding pocket of P450 BM3, was described to expedite the development of highly regio- and stereoselective P450 catalysts. P450 BM3 was evolved through one or two generations of mutations, and a highly enriched mutant library that contained fewer than 30 variants was then constructed. The obtained P450 BM3 variants achieved selective hydroxylation at positionsC7 (94% selectivity in the case of the F87A/A330W/F331L mutant) and C9 (90% regioselectivity and 100% stereoselectivity in the case of the L75V/F87A/T88F/A330W mutant) of cyperenoic acid and were also used to prepare the desired hydroxylated products at a preparative scale with high isolated yields.