Biosynthetic Formation of the 6/7/5-Odyverdiene Diterpenes via a 6/10- <i>trans</i> -Eunicellanyl Intermediate

Odyverdienes are a rare class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons, and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skeletons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, S alkS from Streptomyces alkaliterrae, using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a trans -eunicellane named alkacellene, isolated from S alkS variants, and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling studies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of S alkS highlights the frequent formation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.