Selective C18-Methyl Oxidation of Sclareol via an Engineered P450 Enables the Synthesis of Spiromyrrhene B

While selective oxidation of unactivated C-H bonds has emerged as an effective tactic to reshape retrosynthetic strategies in the synthesis of natural products, the primary methyl C-H bonds remain inert and inaccessible to contemporary methods without preinstalled directing groups. Herein, we report a biocatalytic C-H oxidation strategy that leverages an engineered P450BM3 platform for the selective C18-methyl hydroxylation of sclareol. Through five rounds of directed evolution, variant Ox18M3-3 was developed, elevating the regioselectivity from 4% to >99% alongside a 2.2-fold increase in catalytic activity. The site-specific enzymatic hydroxylation facilitated the first synthesis of the complex heptacyclic tumor stemness inhibitor spiromyrrhene B in 10 steps (longest linear sequence) from sclareol, underscoring the benefits of merging precision biocatalysis with classical synthesis to streamline access to bioactive natural products.