Chemodivergent C(sp3)–H and C(sp2)–H Cyanomethylation Using Engineered Carbene Transferases

The ubiquity of C–H bonds presents an attractive opportunity to elaborate and build complexity in organic molecules. Methods for selective functionalization, however, often must differentiate among multiple chemically similar and, in some cases indistinguishable, C–H bonds within the same molecule. An advantage of enzymes is that they can be finely tuned using directed evolution to achieve control over divergent C–H functionalization pathways. Here, we present engineered enzymes that effect a new-to-nature C–H alkylation (C–H carbene insertion) with unparalleled selectivity: two complementary carbene C–H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an α-cyanocarbene into the α-amino C(sp3)–H bonds or the ortho-arene C(sp2)–H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold (nine mutations, less than 2% of the sequence) were needed to adjust the enzyme’s control over the site-selectivity of cyanomethylation. The X-ray crystal structure of the selective C(sp3)–H alkylase, P411-PFA, reveals an unprecedented helical disruption which alters the shape and electrostatics in the enzyme active site. Overall, this work demonstrates the advantages of using enzymes as C–H functionalization catalysts for divergent molecular derivatization.