Avoidance of Secondary Carbocations, Unusual Deprotonation, and Nonstatistical Dynamic Effects in the Cyclization Mechanism of Tetraisoquinane

The complexity and versatility of terpene cyclization reactions contribute to the wide variety of functions and properties that terpenoid compounds exhibit in nature. The management of reactive carbocations over the course of the reaction to ultimately arrive at a particular carbon fragment connectivity and stereochemistry is no small feat. Bacteria possess a variety of TSs that generate diverse polycyclic terpene skeletons; however, terpenoids in myxobacteria are especially rare. Here, we report the first mechanistic study of tetraisoquinene biosynthesis from TiqS, a diterpene synthase from <i>Melittangium boletus</i>. To understand formation of the unique 5/5/5/5-fused tetraisoquinane skeleton, we used the isolation and structural elucidation of nine minor metabolites, site-directed mutagenesis, stable isotope labeling experiments, and quantum chemical calculations to propose and support its mechanism. This study reveals a new mechanism of diterpene cyclization, expands our understanding of terpenoid biosynthesis, and enables the discovery of novel natural products in myxobacteria.