Interrogation of the metabolic fate of five-carbon isoprenoids by isomer-specific labeling of DMAPP and IPP

Isoprenoid biosynthesis is an essential pathway across all domains of life, biosynthesis of many critical primary metabolites, including crucial membrane components and nuclear hormones. In mammals, isoprenoids derive from the mevalonate (MVA) pathway beginning with acetyl-CoA, whereas many bacteria utilize the methyl-erythritol phosphate (MEP) pathway.1-3 These two essential routes converge at two structurally related five-carbon (C5) metabolites, dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP). The MEP pathway synthesizes both DMAPP and IPP concurrently, while the MVA pathway produces IPP exclusively, that is then isomerized into DMAPP. DMAPP and IPP are the simplest C5 isoprenoids and the universal precursors of all higher-order isoprenoids.4 IPP and DMAPP is interconverted by the enzyme isopentenyl diphosphate isomerase (IDI), which is responsible for maintaining the proper cellular balance.5,6 Despite their importance for cellular function, few chemical or biological tools interrogate the metabolic roles of each isomer separately, and little is known about their independent cellular activities or ultimate metabolic fates beyond incorporation into higher-order isoprenoids. Progress has been limited by the membrane-impermeant pyrophosphate due to the negatively charged β-phosphate, which blocks exogenous delivery of either isomer or analogs. To overcome this barrier, our lab developed cell-permeant, isomer-selective DMAPP and IPP probes in which the β-phosphate is masked by self-immolating esters (SIEs). After passive uptake, endogenous esterases trigger traceless release of the native isomer inside cells. Herein, we uncover the effort taken towards the synthesis of pyrophosphate probe analogs, and the preliminary results of their delivery into cellular system.