Taxadiene Synthase-Catalyzed Cyclization of 6-Fluorogeranylgeranyl Diphosphate to 7-Fluoroverticillenes

The mechanism of the taxadiene synthase-catalyzed cyclization of (E,E,E)-geranylgeranyl
diphosphate (GGPP, 7) to taxadiene (5) is proposed to proceed through a verticillen-12-yl carbocation
intermediate (8) that undergoes an 11 → 7 proton transfer leading to formation of the C ring. The substrate
analogue 6-fluoroGGPP (17) was synthesized to elucidate the stereochemistry of the putative verticillenyl
intermediate. It was expected that the inductive electron-withdrawing effect of the fluoro substituent would
prevent the critical proton transfer to the Δ7 double bond and thereby derail the cyclization at the bicyclic
stage. Incubation of the fluoro analogue with recombinant taxadiene synthase yielded a mixture of three
major and two minor fluoro diterpenes according to GC/MS analyses. The three major products were
identified as the exocyclic, endocyclic, and 4(20)-methylene 7-fluoroverticillenes, i.e., Δ3,7,12 (18), Δ3,7,12, and
Δ4(20),7,11 isomers (22, 23, and 24) on the basis of 1H NMR analyses and comparisons with the parent
bicyclic diterpenes. The H1β, H11α (1S,11R) configurations at the bridgehead positions of 22 were
established by means of NOE experiments and CD spectra. The absolute configuration of (+)-verticillol
(4) was revised after the anomalous dispersion X-ray analysis of (+)-verticillol p-iodobenzoate. Of particular
note, all absolute configurations of verticillane diterpenes in the literature should be reversed. This work
affords compelling evidence supporting the H11α (11R) stereochemistry of the verticillen-12-yl+ ion
intermediate in the taxadiene synthase-catalyzed reaction and illustrates the capability of vinyl fluoro
analogues to intercept complex cyclization cascades.