P450-Catalyzed Intramolecular C–S Bond Formation: Mechanistic Dichotomy Directed by Cyclization

While biocatalysis excels at controlling reaction selectivity, the detailed mechanisms by which heme enzymes govern C–S cyclization patterns remain largely uncharted. Herein, the potential energy landscape for P450-catalyzed intramolecular C–S bond formation in brassinin is mapped by using DFT calculations on cluster models. The computations predict a mechanistic divergence dictated by the mode of substrate activation. The epoxidation pathway is proposed to proceed via a rate-determining epoxide ring-opening step to yield a zwitterionic intermediate, favoring the construction of a six-membered thiazine scaffold. In contrast, the hydrogen abstraction pathway─characterized by a stepwise hydrogen atom transfer and proton transfer─is shown to generate a radical intermediate that would steer cyclization toward a five-membered thiazolidine ring. Notably, the radical pathway is predicted to be kinetically favored with a lower overall activation barrier. These findings offer a theoretical framework for understanding the competition between zwitterionic and radical manifolds, providing a rationale for the bioengineering of P450 enzymes toward programmable heterocycle synthesis.