Molecular Basis for the P450-Catalyzed sp³ C–N Glycosidic Bond Formation in Staurosporine Biosynthesis

Abstract

Selective C(sp 3)–H bond activation and functionalization into a C–N bond has been one of the major challenges facing the pharmaceutical and fine chemical industries. Being the earliest example of C–N bond formation by cytochrome P450 enzyme, the formation mechanism of the sp 3 C5′–N12 glycosidic linkage in antitumor staurosporine (STA, 1) remains a long-standing unsolved issue. Herein, we present biochemical and mechanistic characterizations of P450 SpcN, which catalyzes the intramolecular sp 3 C5′–N12 glycosidic bond formation of holyrine A (2) to generate 3′-N-demethyl-4′-hydroxystaurosporine (6). X-ray crystal structure of SpcN in complex with 2 and QM/MM calculations revealed that the substrate undergoes a conformational switch from “distal” to “proximal” during the catalysis. The C(sp 3)–H amination is initiated by hydrogen atom abstraction from the substrate O8′–H8′ bond, which can serve as a radical relay catalyst to generate radicals at the indole N12 site of the substrate. Significantly, the resulting diradical species is highly unstable, thus leading to the facile C1′–O6′ cleavage to give an unprecedent zwitterionic intermediate that differs from the diradical mechanism recognized in other P450s. Our study sheds light on the molecular basis of intramolecular sp 3 C–N coupling by a natural P450, affording a potential biocatalyst to create nitrogen-containing heterocyclic bioactive compounds.