Chemoenzymatic Synthesis of (4R,7R)-Zingiberene Enables Construction of nor-Sesquiterpene Semiochemical Analogs.

The wild tomato sesquiterpene (4R,7R)-zingiberene (6a) acts as a potent repellent of the economically important agricultural pest, the silverleaf whitefly (Bemisia tabaci). While its biosynthesis is catalyzed by 7-epi-zingiberene synthase from the noncanonical terpene precursor (2Z,6Z)-farnesyl pyrophosphate (5a), the precise enzymatic mechanism has remained undetermined. Using deuterated substrates generated chemoenzymatically, the 1,6-cyclization mechanism was established to proceed via a 1,3-hydride shift from the C1 position of the initial bisabolyl carbocationic intermediate. Product diversity was expanded by identifying two single residue switches within the G1/2 helix that govern enzyme fidelity, thereby redirecting product distributions to alternative C6-cyclized zingiberenols as well as γ- and β-curcumene. Applying this chemoenzymatic synthesis with alternative allyl alcohols plus isoprenol enabled the production and structural diversification of two novel nor-sesquiterpene analogs (C14/C16), 13-nor-(4R,7R)-zingiberene (23) and 12-ethyl-13-nor-(4R,7R)-zingiberene (24). Notably, these syntheses were only possible because the tomato (2Z,6Z)-farnesyl pyrophosphate synthase overcomes conventional prenyltransferase substrate selectivity to catalyze chain extension via atypical secondary allyl carbocation intermediates. The catalytic promiscuity provides opportunities to engineer terpene products with diverse methyl-branching patterns, providing the platform to investigate the molecular basis of terpene semiochemical activity needed for the development of broad-spectrum repellents to sustainably manage insect pests in agriculture.