Chemo-, Regio-, and Stereoselective Oxidation of the Monocyclic Diterpenoid β-Cembrenediol by P450 BM3

Late-stage oxyfunctionalization of terpenoid scaffolds has been recognized as a powerful tool in the synthesis of complex molecules such as natural products to achieve their efficient diversification. Selective C–H oxidation of such hydrocarbon scaffolds remains challenging for chemical catalysts because of their insufficient regio- and stereoselectivity. To achieve this goal, cytochrome P450 monooxygenases are often used as biocatalysts. Here, we demonstrate the successful P450-catalyzed chemo-, regio-, and stereoselective oxidation of the tobacco cembranoid β-cembrenediol. This 14-membered macrocycle possesses a broad range of biological activities including antitumor-promoting and neuroprotective effects and carries seven potential sites for allylic hydroxylation as well as three epoxidation sites. On the basis of first-sphere active site mutagenesis, we generated in a few rounds a P450 BM3 minimal library and screened for β-cembrenediol oxidation activity. Several P450 BM3 variants were evolved, enabling the regioselective hydroxylation of the neighboring positions C-9 (100% regioselectivity and a diastereomeric ratio of 89:11 in the case of the F87A/I263L mutant) and C-10 (97% regioselectivity and a diastereomeric ratio of 74:26 in the case of the L75A/V78A/F87G mutant) of β-cembrenediol.