An Evolved Artificial Radical Cyclase Enables the Construction of Bicyclic Terpenoid Scaffolds via an H-Atom Transfer Pathway

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalyzed hydrogen-atom-transfer (M-HAT) reaction offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Herein, we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyzed a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co-cofactor was confirmed by crystallography. A time-course experiment revealed a cascade reaction catalyzed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibited tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.