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.