Redirecting Regioselectivity in Cytochrome P450 ApPT for B-Ring Functionalization of Pentacyclic Triterpenoids.

Pentacyclic triterpenoids (PTs) feature a steroid-like privileged scaffold, but late‑stage functionalization of their inert B‑ring C(sp3)─H bonds remains a formidable synthetic challenge due to the absence of directing groups and the consequent poor regio- and stereoselectivity. Here we report a semi-rationally engineered P450 variant, MB4, that enables highly selective C7β hydroxylation of 18β-glycyrrhetinic acid, achieving C-H activation on the B-ring with > 99% regioselectivity. Protein crystallography and computational studies revealed that the reshaped active pocket of MB4 disrupts the native 1,5-hydrogen atom transfer (1,5-HAT) relay oxidation, thereby redirecting the regioselectivity exclusively to the B-ring C7 position. The synthetic utility of MB4 was demonstrated through substrate scope investigation, large-scale preparation, and a chemoenzymatic synthesis platform that generated a library of previously inaccessible B-ring-modified PT derivatives. Biological evaluation showed that several C7-acylated derivatives exhibited antiproliferative activity (IC50 = 0.31-5.04 µM) by triggering caspase-3-dependent apoptosis and inducing G2/M-phase arrest. This work provides a promising blueprint for reprogramming P450 selectivity for late-stage C-H functionalization of polycyclic natural products.