Chemodivergent C(sp3)-H and C(sp2)-H cyanomethylation using engineered carbene transferases

The ubiquity of C-H bonds presents an opportunity to efficiently elaborate and build complexity in organic molecules. Methods for selective functionalization, however, must differentiate among multiple, chemically similar C-H bonds: enzymes are attractive because they can be finely tuned using directed evolution to achieve divergent reaction outcomes. Here we present engineered enzymes that effect a new-to-nature C-H alkylation (C-H carbene insertion) with distinct selectivities: cytochrome P450-based carbene transferases deliver an alpha-cyanocarbene either into the alpha-amino C(sp(3))-H bonds or the ortho-arene C(sp(2))-H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold were needed to adjust the enzyme's chemoselectivity. Structural studies of the C(sp(3))-H alkylase reveal an active-site helical disruption, which alter the structure and electrostatics of the substrate-binding pocket compared to the native enzyme. Overall, this work demonstrates advantages of using highly tuneable enzymes as C-H functionalization catalysts for divergent molecular derivatization.