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

ABSTRACT Pentacyclic triterpenoids (PTs) feature a steroid‐like privileged scaffold, but late‑stage functionalization of their inert B‑ring C(sp 3 )─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 (IC 50 = 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.