A binuclear copper enzyme platform for enantioconvergent radical (pseudo)halogenation

Despite their intriguing native metalloenzymology, naturally occurring copper enzymes remain underexploited for new-to-nature biocatalytic reactions. Here we report the systematic investigation and reprogramming of natural copper enzymes to catalyse unnatural free-radical (pseudo)halogenation reactions with high enantioselectivity. Evaluation of Cu enzymes in the decarboxylative azidation of redox-active esters revealed activity across multiple copper enzyme families, with type III binuclear copper enzymes, particularly Bacillus megaterium tyrosinase (BmTyr), exhibiting superior activity and enantioselectivity towards both stabilized and unstabilized alkyl radicals after further engineering. The strong halide binding affinity of the binuclear copper system also enabled challenging enantioconvergent bromination, chlorination and isothiocyanation reactions that remained inaccessible to repurposed non-haem iron enzymes. Electron paramagnetic resonance and ultraviolet-visible spectroscopic analyses confirmed the coupled binuclear nature of wild-type and engineered bacterial tyrosinases and provided insight into the origin of their enhanced activity. Collectively, this study establishes binuclear copper enzymes as a powerful platform for new-to-nature stereoselective radical reactions, expanding the scope of metalloenzyme catalysis beyond mononuclear systems. The image explains the catalytic activity of the binuclear copper enzyme tyrosinase from Bacillus megaterium with mutations V218A and N205M. The enzyme contains two copper ions each coordinated by three histidine residues. The enzyme acts on a substrate with two chiral centers labeled R superscript L and R superscript S. On the left, the substrate with an azide group (N3) attached to the R superscript S center is shown as a reactant. On the right, the product has a substituent labeled X, which can be chlorine, bromine, or thiocyanate, attached to the R superscript S center. The enzyme catalyzes the substitution of the azide group by X at the R superscript S position.