Biocatalytic Enantioselective β‐Hydroxylation of Unactivated C−H Bonds in Aliphatic Carboxylic Acids

Catalytic selective hydroxylation of unactivated aliphatic (sp3) C−H bonds without a directing group represents a formidable task for synthetic chemists. Through directed evolution of P450BSβ hydroxylase, we realize oxyfunctionalization of unactivated C−H bonds in a broad spectrum of aliphatic carboxylic acids with varied chain lengths, functional groups and (hetero‐)aromatic moieties in a highly chemo‐, regio‐ and enantioselective fashion (>30 examples, Cβ/Cα>20 : 1, >99 % ee). The X‐ray structure of the evolved variant, P450BSβ‐L78I/Q85H/G290I, in complex with palmitic acid well rationalizes the experimentally observed regio‐ and enantioselectivity, and also reveals a reduced catalytic pocket volume that accounts for the increased reactivity with smaller substrates. This work showcases the potential of employing a biocatalyst to enable a chemical transformation that is particularly challenging by chemical methods. Directed evolution rendered P450BSβ capable of β‐hydroxylating unactivated C−H bonds in aliphatic carboxylic acids with broad substrate scope and excellent chemo‐, regio‐, and enantioselectivity. The crystal structure of the evolved variant rationalizes the improved reactivity and selectivity. This study demonstrates the potential of exploring biocatalysts to fulfill reactions that are otherwise elusive with chemical strategies.