Dual-Enzyme Relay Catalysis Enables Asymmetric Construction of Nonadjacent Stereocenters in Polycyclic Alcohols

Abstract The asymmetric construction of nonadjacent stereocenters in a single catalytic system remains a significant challenge due to the difficulty in controlling stereochemical relay between sequential transformations. Herein, we report a dual-enzyme relay catalytic system that integrates an artificial streptavidin-based copper metalloenzyme (Sav-Cu) with an engineered ketoreductase (KRED) for the one-pot synthesis of polycyclic alcohols bearing 1,3-non-adjacent stereocenters. In this relay process, the engineered SavDMase variant (S112A/K121D) catalyzes a non-natural decarboxylative Mannich reaction to establish the R-configured stereocenter in the enantioenriched ketone intermediate, which is then recognized by the engineered KREDAV mutant (S96A/Y190V) and reduced with high diastereo- and enantioselectivity to install the S-configured alcohol stereocenter, thereby furnishing the (R,S)-configured product as the dominant stereoisomer. The optimized system furnishes the products in up to 83% yield, 99% ee, and 96% de across a broad substrate scope, and is readily applicable to 2 mmol-scale synthesis. Structural analysis, docking, and molecular dynamics simulations support complementary roles of the two catalytic modules: SavDMase promotes productive organization of the Mannich step, while KREDAV stabilizes the reactive binding pose and constrains the hydride-transfer trajectory to enforce stereoselective reduction. This work demonstrates how artificial and natural enzymes can be integrated in relay catalysis to solve challenging stereochemical relay problems beyond the reach of individual biocatalysts.