Molecular Basis\nfor the P450-Catalyzed <i>sp</i><sup>3</sup> C–N\nGlycosidic Bond Formation in Staurosporine\nBiosynthesis

Selective C(<i>sp</i><sup>3</sup>)–H\nbond activation\nand functionalization into a C–N bond has been one of the major\nchallenges facing the pharmaceutical and fine chemical industries.\nBeing the earliest example of C–N bond formation by cytochrome\nP450 enzyme, the formation mechanism of the <i>sp</i><sup>3</sup> C5′–N12 glycosidic linkage in antitumor staurosporine\n(STA, <b>1</b>) remains a long-standing unsolved issue. Herein,\nwe present biochemical and mechanistic characterizations of P450 SpcN,\nwhich catalyzes the intramolecular <i>sp</i><sup>3</sup> C5′–N12 glycosidic bond formation of holyrine A (<b>2</b>) to generate 3′-<i>N</i>-demethyl-4′-hydroxystaurosporine\n(<b>6</b>). X-ray crystal structure of SpcN in complex with <b>2</b> and QM/MM calculations revealed that the substrate undergoes\na conformational switch from “distal” to “proximal”\nduring the catalysis. The C(<i>sp</i><sup>3</sup>)–H\namination is initiated by hydrogen atom abstraction from the substrate\nO8′–H8′ bond, which can serve as a radical relay\ncatalyst to generate radicals at the indole N12 site of the substrate.\nSignificantly, the resulting diradical species is highly unstable,\nthus leading to the facile C1′–O6′ cleavage to\ngive an unprecedent zwitterionic intermediate that differs from the\ndiradical mechanism recognized in other P450s. Our study sheds light\non the molecular basis of intramolecular <i>sp</i><sup>3</sup> C–N coupling by a natural P450, affording a potential biocatalyst\nto create nitrogen-containing heterocyclic bioactive compounds.