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

Abstract

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 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 enzymes as C-H functionalization catalysts for divergent molecular derivatization.

Fig. 1 |. Reaction design.

a, Enzymatic C–H functionalization reactions can be highly selective and divergent; b, Previous work on abiological enzymatic C–H functionalization has mainly focused on modifying C(sp³)–H bonds; c, The goal of this study is to demonstrate that closely related C–H alkylases can enable divergent functionalization of arene C(sp²)–H bonds and nearby C(sp³)–H bonds; d, Model reaction for the initial activity discovery.

Fig. 2 |. Crystallographic studies of P411-PFA (PDB 8DSG).

a, Overall fold (white cartoon) and active site cavity of P411-PFA (left, purple mesh) compared to a closely related C–H aminase, P-4 A82L A78V F263L (PDB 5UCW) (right, orange mesh); b, An unusual backbone carbonyl flip is observed in the I helix (teal) of P411-PFA at residue position E267. The E267 carbonyl is indicated by a red arrow. Mutations on the I helix from wild type P450BM3 to P411-PFA are indicated as black residue labels. The AlphaFold2-predicted model of P411-PFA successfully templated the bulged helix, but (unsurprisingly) failed to capture the rearrangement of the I helix and carbonyl flip; c, Close-up view of the helical bulge which resides at the bottom of a water channel. The disruption of the I helix generates a partial dipole near the active site, which may make the local electronic environment more favorable for diazo binding and activation. Waters stabilizing the bulged helix in d, P411-PFA (white) and e, P411-PFA are overlaid on P-4 A82L A78V F263L (orange).

Fig. 3 |. Reaction discovery and directed evolution of a C(sp²)–H cyanomethylase.

Reaction conditions: anaerobic; 5 mM 1a or 1b; 48 mM 2 (9.63 equiv.); E. coli whole cell harboring P411 variants (OD₆₀₀ = 30) suspended in M9-N aqueous buffer (pH 7.4); 10% v/v EtOH (co-solvent); room temperature; 18–20 h. a, Initial activity screening revealed the formation of C(sp³)–H insertion product 3a and ortho-arene C–H alkylation product 4a; b, Locations of beneficial mutations are highlighted in the P411-PFA structure; c, Directed evolution of a highly selective arene C(sp²)–H cyanomethylase; d, Observed product distribution with 1b includes ring-expanded Buchner products. Basic pH suppresses Buchner product formation (5b, *inseparable mixture of tautomers).

Fig. 4 |. Substrate scope study.

a, Substrate scope of P411-PFA and P411-ACHF. Analytical yields of desired products were determined by using their GC calibration curves (See Supplementary Information, Section VII). Reaction conditions: anaerobic; 2.5 mM 1; 51.6 mM 2 (20.63 equiv.); E. coli whole cells harboring P411 variants (OD₆₀₀ = 30) M9-N aqueous buffer (pH 7.4); 10% v/v EtOH (co-solvent); room temperature; 18–20 h. Yields and total turnovers (TTNs) are reported as the averages of the triplicate experiments (See Supplementary Table 5); b, The arene C(sp²)–H functionalization activity and selectivity can be further optimized on individual target molecules; c, Chemodivergent, preparative-scale syntheses using P411-PFA and P411-ACHF. Reaction conditions: anaerobic; 5 mM 1d; 48 mM 2 (9.63 equiv.); E. coli whole cells harboring P411 variants (OD₆₀₀ = 30) M9-N aqueous buffer (pH 7.4); 10% v/v EtOH (co-solvent); room temperature; 18–20 h.