To demonstrate the cyclization and hydride transfers, we synthesized 1,1-D2-GGPP (Fig. , Supplementary Figure ) and incubated it with PcTS1. The deuterated product, compound 4, was isolated, purified, and characterized using GC-MS and NMR techniques. GC-MS analysis revealed the molecular formula of compound 4 as C20H30D2 (m/z 274.3), indicating an increase of two Da compared to the unlabeled compound 3, which corresponds to the replacement of two hydrogen atoms with deuteriums in 1,1-D2-GGPP. NMR comparison between compounds 3 and 4 showed that both share the same elisabethatriene core structure. The absence of correlations for H-6 to C-6 and H-11 to C-11 in the HSQC spectra of compound 4 (Fig. ) indicated the presence of deuterium at the C-6 and C-11 positions. This was confirmed by the lack of the 1 H NMR signals for H-6 and H-11 found in compound 3, and the signal for H-17 in 4 exhibits a singlet instead of a doublet in 3. Additionally, the carbon signals for C-6 and C-11 in compound 4 showed a decrease in intensity, consistent with deuterium isotope effects , confirming the presence of deuteriums at the C-6 and C-compound 3. These findings collectively support the proposed cyclization cascades and confirm the occurrence of a 1,3-hydride shift following the 1,10-cyclization and two 1,2-hydride shifts after the 1,6-ring closure in pathway I, or two 1,2-hydride shifts following the 1,6-cyclization and a 1,3-hydride shift after the 1,10-ring closure in pathway II.