Empowering Precision C–H Functionalization: Advances in Peroxygenase Engineering

Abstract Selective C–H functionalization remains a central challenge in modern synthesis as it enables direct diversification of molecular scaffolds without pre‐functionalization. Peroxygenases, including both unspecific peroxygenases and P450 peroxygenases, offer a biocatalytic solution to this challenge, catalyzing oxyfunctionalization reactions in aqueous media under mild conditions with hydrogen peroxide (H 2 O 2 ) as the sole oxidant. In this review, we highlight recent advances in peroxygenase engineering over the past five years, with a particular focus on strategies that enhance heterologous expression, catalytic activity, and control of regio‐ and enantioselectivity. We also discuss protein engineering approaches that mitigate H 2 O 2 ‐induced inactivation and summarize efforts to repurpose NADPH‐dependent P450 enzymes into self‐sufficient peroxygenases, thereby expanding their catalytic repertoire. Furthermore, we examine the integration of peroxygenases with in situ H 2 O 2 generation systems by enzymatic, chemical, photocatalytic, and electrochemical methods to achieve balanced oxidant delivery and sustained turnover. Collectively, these developments have established peroxygenases as versatile and robust catalysts for selective C–H functionalization, opening new opportunities for their application in the synthesis of pharmaceuticals, fine chemicals, and agrochemicals.