Unspecific Peroxygenase Catalyzed Benzylic C-H Functionalization of diN-Heterocycles and Its Application

While unspecific peroxygenases (UPOs) have been reported to mediate benzylic oxidation of both nitrogen-free and one nitrogen-containing aromatic heterocyclic substrates, the benzylic C-H functionalization of two-nitrogen (diN) heterocycles is rarely reported. Such diN-heterocycles serve as key building blocks for many high-value compounds, including food additives and first-line clinical drugs. Here, we report the selective benzylic (sp³ and sp²) C-H activation of aromatic diN-heterocycles catalyzed by recombinant Agrocybe aegerita UPO (rAaeUPO-Pada-I-H). We further develop a mild and efficient biocatalytic route for the synthesis of 2-acetylpyrazine (1b, 88.5% yield) via a one-pot, two-step cascade that combines rAaeUPO-catalyzed regioselective hydroxylation with alcohol dehydrogenase (ADH) and an economical NAD⁺/NADH recycling system. This work has achieved the benzylic oxidation functionalization of aromatic diN-heterocycles, thereby broadening the substrate scope of UPOs in the applications of heterocyclic chemistry. The developed system not only provides a green route to 2-acetylpyrazine as an alternative for industrial production, but also, owing to its extendability to other valuable diN-heterocyclic substrates, points toward a promising direction for advancing green biocatalysis in fine chemical and pharmaceutical manufacturing.