One-Pot, Two-Step Hydroxylation of the Macrocyclic Diterpenoid beta-Cembrenediol Catalyzed by P450 BM3 Mutants

摘要

Cytochrome P450 monooxygenases (P450s) are involved in the biosynthesis of a wide range of bioactive secondary metabolites. They often introduce several oxy functionalities at different positions of a substrate through multiple steps and produce a range of oxidized derivatives. Herein, we describe a one-pot two-step hydroxylation of the diterpenoid beta-cembrenediol isolated from the plant Nicotiana tabacum. This 14-membered macrocycle shows neuroprotective effects and is, along with its oxidized derivatives, of pharmaceutical interest. Sequential hydroxylations catalyzed by the regioselective P450 BM3 mutants F87A/I263L and V78A/F87G yielded the epimeric (9S, 10R/S)-beta-cembrenetetraols with a diastereomeric ratio of 48: 52. The replacement of the mutant V78A/F87G with L75A/V78A/F87G in the second step improves the diastereomeric ratio up to 10: 90. Absolute configurations of the newly introduced hydroxy groups were determined by quantum-mechanical calculations of NMR spectra.