Chemodivergent C(sp)-H and C(sp)-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. 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 demonstrate engineered enzymes that effect a new-to-nature C-H alkylation with unparalleled selectivity: two complementary carbene C-H transferases derived from a cytochrome P450 from deliver an -cyanocarbene into the -amino C(sp)-H bonds or the -arene C(sp)-H bonds of -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(sp)-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 enzymes as C-H functionalization catalysts for divergent molecular derivatization.