Taxadiene synthase catalyzes the formation of taxadiene from GGPP, the universal building block of diterpenes. The cyclization of GGPP to taxadiene is generally thought to proceed through a series of monocyclic and bicyclic carbocation intermediates, all of which are mechanistically plausible but experimentally not isolable, and therefore, unobservable. To gain a better understanding of the mechanism of the cyclization of GGPP to taxadiene, a series of GGPP analogs were chemically synthesized and fed in vitro to taxadiene synthase (overexpressed in truncated form in E. coli). These analogs were designed to interrupt the cyclization cascade, such that the monocyclic and/or bicyclic carbocation intermediates could not react further and therefore would be quenched to give isolable and observable monocyclic or bicyclic hydrocarbon products. Four monocyclic hydrocarbon compounds were obtained from four reactions of different analogs, and the structure of each product was unambiguously solved by 1D and 2D NMR. These results support the intermediacy and the existence of the cembrenyl cation in the cyclization of GGPP to taxadiene, and indicate that modifications at the 10,11 double bond of GGPP are tolerable in the cyclization.

Taxadiene synthase catalyzes the formation of taxadiene from GGPP, the universal building block of diterpenes. The cyclization of GGPP to taxadiene is generally thought to proceed through a series of monocyclic and bicyclic carbocation intermediates, all of which are mechanistically plausible but experimentally not isolable, and therefore, unobservable. To gain a better understanding of the mechanism of the cyclization of GGPP to taxadiene, a series of GGPP analogs were chemically synthesized and fed in vitro to taxadiene synthase (overexpressed in truncated form in E. coli). These analogs were designed to interrupt the cyclization cascade, such that the monocyclic and/or bicyclic carbocation intermediates could not react further and therefore would be quenched to give isolable and observable monocyclic or bicyclic hydrocarbon products. Four monocyclic hydrocarbon compounds were obtained from four reactions of different analogs, and the structure of each product was unambiguously solved by 1D and 2D NMR. These results support the intermediacy and the existence of the cembrenyl cation in the cyclization of GGPP to taxadiene, and indicate that modifications at the 10,11 double bond of GGPP are tolerable in the cyclization.