Engineered enzymes form carbon-carbon and carbon-nitrogen bonds with high selectivity

Scientists at the Manchester Institute of Biotechnology have developed a new class of engineered enzymes capable of forming carbon-carbon and carbon-nitrogen bonds with high selectivity. The research, published in Nature Catalysis, addresses a long-standing challenge in chemistry: creating biological catalysts that can selectively construct complex molecular architectures used in pharmaceuticals and advanced materials.

The team, including Dr. Zachary Birch-Price and Professor Anthony Green, engineered proteins containing a non-natural catalytic amino acid to create enzymes termed allylic transferases. These enzymes generate highly reactive imidazolium intermediates that can be intercepted by various carbon- and nitrogen-containing molecules, enabling a broad range of bond-forming reactions from a single catalytic strategy.

Using directed evolution, the researchers improved enzyme performance significantly. One variant, ASB1.3, achieved more than 99% conversion in several reactions while delivering products with high stereochemical purity, and produced a target compound with 98% conversion at preparative scale. A second variant, ASA1.5, enabled formation of molecules containing all-carbon quaternary stereocenters, structures that are challenging to synthesize selectively, also achieving 98% conversion at preparative scale.

The engineered enzymes demonstrated versatility across a broad range of reaction partners, including substituted furans, indoles, pyrroles, cyanoesters, diketones, ketoesters, anilines, and isatins. Across the reactions examined, the enzymes generated a single major product with no observable byproducts other than the released leaving group used for monitoring.

Structural analysis revealed that a para-nitrophenol group released during the reaction remains in the enzyme active site and helps orient incoming reactants, contributing to stereoselective bond formation. This mechanistic insight helps explain how the enzymes achieve their high selectivity.

Professor Green noted that biocatalysis has transformed many chemical reactions but important areas remain difficult to access, and this work shows artificial enzymes can be engineered to perform a wide variety of bond-forming reactions with adaptability to different reaction partners. Dr. Birch-Price added that combining enzyme engineering with non-natural catalytic chemistry created a family of bond-forming enzymes new to the biocatalytic repertoire.

While further development is needed before wider application, the study expands the range of chemical transformations available through biocatalysis and highlights how engineered protein catalysts can provide new ways to access molecular structures difficult to produce with established small-molecule catalysts.