Empowering Precision C-H Functionalization: Advances in Peroxygenase Engineering

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 (H₂O₂) 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 H₂O₂-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 H₂O₂ 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.