Doctoral Dissertations
Date of Award
8-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Chemistry
Major Professor
Joshua Baccile
Committee Members
Michael Best, Tessa Calhoun, Barry Bruce
Abstract
Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central, five-carbon precursors, to all isoprenoids. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is not possible as the negatively charged pyrophosphate renders these molecules membrane impermeant. In this dissertation, we present a chemical strategy for the isomer-specific delivery of IPP and DMAPP using self-immolative, ester-protected (SIE-IPP and SIE-DMAPP) analogs. These analogs mask the negative charge of the β-phosphate of IPP and DMAPP allowing passive diffusion of the protected analogues across membranes. Once inside the cell general esterase activity initiates cleavage of the SIEs, resulting in release of IPP and DMAPP. Using these analogs, we demonstrate effective intracellular delivery in U-87MG human glioblastoma cells, where SIE-IPP or SIE-DMAPP rescues growth during statin-induced inhibition of isoprenoid biosynthesis. We extend this approach to the Gram-positive bacterium Bacillus subtilis, where SIE-IPP and SIE-DMAPP similarly restore growth under inhibition of isoprenoid biosynthesis. Furthermore, we demonstrate that side products generated during the intracellular cleavage of the analogs are biologically inert compared to the released IPP and DMAPP, confirming that both rescue and toxicity arise specifically from the released isomers. We then extend this strategy to directly track isoprenoid biosynthesis by synthesizing a stable isotope-labeled analog of DMAPP (¹³C₃-SIE-DMAPP) to monitor its incorporation into native isoprenoids. Using this probe, we demonstrate that DMAPP-specific incorporation into menaquinone-7 (MK-7) depends on isopentenyl pyrophosphate isomerase (IPPI) activity, and that the ability of B. subtilis to grow solely with IPP or DMAPP is determined by IPPI expression. Finally, we apply ¹³C₃-SIE-DMAPP to reveal the spatial compartmentalization of isoprenoid biosynthesis during sporulation in B. subtilis, highlighting distinct isoprenoid biosynthesis between the mother cell and the endospore. Collectively, this work establishes a versatile chemical toolkit for the isomer-specific delivery and isotopic labeling of IPP and DMAPP in living cells. The creation of SIE-IPP and SIE-DMAPP facilitates the investigation of isomer-specific isoprenoid metabolism in native biological contexts, opening new avenues for exploring prenyl metabolism, terpene biosynthesis, and the regulatory mechanisms that control cellular isoprenoid homeostasis.
Recommended Citation
McBee, Dillon P., "Chemical tools for the independent interrogation of isopentenyl and dimethylallyl pyrophosphate metabolism. " PhD diss., University of Tennessee, 2025.
https://trace.tennessee.edu/utk_graddiss/12739
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