Exploring bacterial cytochrome P450s for selective activation of aliphatic C-H bonds in pentacyclic triterpenoids

Selective activation of aliphatic C-H bonds in polycyclic terpenoids offers a potent strategy for exploring diverse chemical space in drug design, yet challenges persist in achieving site- and diastereoselectivity, especially with increasing structural complexity. Here we report a bioinformatics-driven terpene-P450 discovery strategy to develop bacterial cytochrome P450s for selective activation of aliphatic C-H bonds in structurally intricate pentacyclic triterpenoids (PTs). The identified ApPT demonstrated elegant diastereoselectivity and broad substrate tolerance, facilitating a chemo-enzymatic platform to explore the chemical space of PTs at previously inaccessible sites. Protein crystallization and computational analysis reveal the mechanism of the preliminary C-H bond activation selectivity of ApPT towards various PTs, particularly an example of enzymatic C7-to-C15 relay oxidation mediated by 1,5-hydrogen atom transfer. This work offers ApPT as a valuable biocatalyst to explore the chemical space of PTs via aliphatic C-H bond activation, demonstrating the advantage of our biocatalyst-discovery strategy for the late-stage diversification of polycyclic terpenoids.