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

Catalytic selective hydroxylation of unactivated aliphatic (sp(3)) C-H bonds without a directing group represents a formidable task for synthetic chemists. Through directed evolution of P450(BS beta) 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, C beta/C alpha > 20:1, > 99 % ee). The X-ray structure of the evolved variant, P450(BS beta)-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.

Automated summary

Selective hydroxylation of non-activated aliphatic C-H bonds was achieved through directed evolution of P450(BS beta) hydroxylase. The reaction showed high selectivity towards various aliphatic carboxylic acids with different chain lengths, functional groups, and aromatic moieties. The X-ray structure of the evolved enzyme variant provided insights into the regio- and enantioselectivity of the reaction.