Selective Oxidations of Cyperenoic Acid by Slightly Reshaping the Binding Pocket of Cytochrome P450 BM3

Abstract

AbstractThe 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 C9α‐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 positions C7 (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.