Directed Evolution of a Fe(II)- and α-Ketoglutarate-Dependent Dioxygenase for Site-Selective Azidation of Unactivated Aliphatic C-H Bonds

Fe(II)- and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions. Achieving high chemoselectivity for functionalization using non-native functional groups remains rare, however, particularly for non-native substrates. The current study shows that directed evolution can be used to engineer variants of an engineered dioxygenase, SadX, that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved using variants with improved azidation yield and selectivity on a probe substrate as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.