Production of epoxydammaranes by the enzymatic reactions of (3R)- and (3S)-2,3-squalene diols and those of 2,3:22,23-dioxidosqualenes with recombinant squalene cyclase and the mechanistic insight into the polycyclization reactions.

The enzymatic cyclizations of (3R)- and (3S)-2,3-squalene diols by squalene cyclase afforded bicyclic compounds and epoxydamamranes in a ca. 3 : 2 ratio. Formation of the epoxydammarane scaffold indicates that a 6/6/6/5-fused tetracyclic cation is involved as the intermediate in the polycyclization reaction. 2,3:22,23-Dioxidosqualenes also afforded an epoxydammarane skeleton, i.e., 3alpha- or 3beta-hydroxyepoxydammaranes, but the amount of bicyclic compounds produced was markedly lower than that of the squalene diols, indicating that the larger steric bulk of the diols had a more significant influence on the polycyclization pathway than the smaller bulk of the expoxide. All the epoxydammaranes had 17R,20R stereochemistry except for one product, demonstrating that these analogs were folded into an all-chair conformation in the reaction cavity. The mechanistic insight into the observed stereochemical specificities indicated that the organized all-chair conformation is rigidly constricted by squalene cyclase and, thus, free conformational change is not allowed inside the reaction cavity; a small rotation of the hydroxyl group or the epoxide toward the intermediary cation gave a high yield of the enzymatic products, while a large rotation led to a low yield of the product. The stereochemistries of the generated epoxydammaranes are opposite to those from natural sources, and thus almost all of the enzymatic products described here are novel.