Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2–C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is com...