Catalytic selective hydroxylation of unactivated aliphatic (sp 3 ) CÀ H bonds without a directing group represents a formidable task for synthetic chemists. Through directed evolution of P450 BSβ hydroxylase, we realize oxyfunctionalization of unactivated CÀ 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, P450 BSβ -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.

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Catalytic selective hydroxylation of unactivated aliphatic (sp 3 ) CÀ H bonds without a directing group represents a formidable task for synthetic chemists. Through directed evolution of P450 BSβ hydroxylase, we realize oxyfunctionalization of unactivated CÀ 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, P450 BSβ -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.