Isoprenoids comprise one of the largest and most structurally diverse classes of natural products. Despite this remarkable chemical and biological diversity, every isoprenoid originates from the same two five-carbon building blocks: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Many aspects of the cellular fate of IPP and DMAPP, including their utilization, localization, biological activity, and metabolic regulation, remain poorly understood. The main focus of the Baccile Research Group is to integrate chemical tool development, analytical chemistry, and experimental biology into a platform for studying five-carbon metabolism. We develop cell-permeant probes that provide direct experimental access to IPP and DMAPP in living systems for exogenous metabolite delivery, stable-isotope tracing, and bioorthogonal labeling. Our chemical tools are paired with high-resolution mass spectrometry, metabolomics, and fluorescence microscopy to quantify metabolic flux, identify downstream metabolites of IPP and DMAPP, and visualize isoprenoid metabolism. The Baccile research program is distinguished by the integration of chemical tool development and mechanistic biology, using analytical workflows to guide both probe development and biological questions surrounding five-carbon metabolism.
Development of chemical tools for investigating five-carbon metabolism in diverse organisms
Direct investigation of five-carbon metabolism has historically been limited by a fundamental technical barrier: the central metabolites IPP and DMAPP are highly charged pyrophosphates that do not readily cross biological membranes. Our initial objective was to overcome this technical barrier through chemical probe development to provide direct access to IPP and DMAPP individually in living systems. Our first major advance was the development of self-immolative ester (SIE)-protected analogs of IPP and DMAPP that mask the β-phosphate charge, enabling cellular uptake and intracellular release of the native metabolites following esterase-mediated activation. This study (ChemBioChem, 2023, 24, e20220512) established the first general strategy for exogenous delivery of IPP and DMAPP to living cells and demonstrated restoration of isoprenoid biosynthesis following inhibition of the mevalonate pathway in U-87MG glioblastoma cancer cells. Using these compounds, we demonstrated that the sensitivity of cancer cells to farnesyl pyrophosphate synthase inhibition depends specifically on IPP, whereas DMAPP has no detectable effect. We next extended this platform to Gram-positive bacteria, where differences in membrane composition and esterase specificity required optimization of the delivery strategy. This study (ChemBioChem, 2024, 25, e20240064) provided the first direct comparison of IPP and DMAPP utilization in bacterial cells and established broadly applicable chemical tools for manipulating five-carbon metabolism across phylogenetically diverse organisms.
Targeted discovery and characterization of metabolites derived from five-carbon metabolism
Despite decades of research into isoprenoid biosynthesis, the full diversity of metabolites derived from IPP and DMAPP remains unknown because most analytical methods monitor only well-characterized pathway intermediates or end products. A major objective of my laboratory is to directly identify, trace, and characterize metabolites derived from five-carbon metabolism. Our initial efforts focused on developing cell-permeant stable-isotope probes capable of directly tracing IPP- and DMAPP-derived metabolites. By integrating isotope labeling with high-resolution mass spectrometry, these probes enable detection of metabolic flux through five-carbon metabolism. Application of this platform to B. subtilis revealed an unexpected level of metabolic organization during sporulation (J. Am. Chem. Soc. 2025, 147, 23, 19777-19787). These findings established that five-carbon metabolism is spatially organized throughout bacterial differentiation.
Beyond the biological discovery, these studies established stable-isotope tracing as a general platform for resolving metabolic organization and identifying new metabolites derived from IPP and DMAPP in living systems. My laboratory is now extending stable-isotope tracing to additional classes of metabolites. The first application of this expanded strategy focuses on prenylated RNA, a class of post-transcriptional modifications whose biological function remains poorly understood. By combining isomer-specific stable-isotope tracing with high-resolution metabolomics, my laboratory is pursuing previously unrecognized metabolites derived from IPP and DMAPP, quantifying their biosynthesis under changing physiological conditions, and defining biological functions across both bacterial and mammalian systems.
A reverse metabolomics approach to discovery of prenylated metabolites
Public mass spectrometry data repositories have massively expanded the scope of structure discovery and visibility of uncharted chemical space, allowing for community-driven efforts to assign chemical structures to previously detected but uncharacterized small molecules. We are currently taking advantage of these advancements through reverse metabolomics to interrogate public datasets and reveal new isoprenoid chemistry that has been previously overlooked. Using this strategy, we constructed a 240-member reference library spanning C5, C10, C15, and C20-prenylated metabolites and identified 38 prenylated small molecules across public datasets, including 26 structures that, to our knowledge, have not been previously reported. These finding demonstrate that small molecule prenylation is a chemically organized and broadly distributed metabolic transformation that extends across microorganisms, plants, animals, and host-associated microbiomes. These discoveries provide immediate targets for identifying the responsible prenyltransferases, defining the underlying biosynthetic pathways, and determining biological functions of these newly recognized metabolite classes.
Bioorthogonal visualization and enrichment in five-carbon metabolism
Although membrane-associated isoprenoids play indispensable roles in bacterial physiology, their spatial organization and dynamic regulation have remained relatively inaccessible because existing imaging methods rely on nonspecific lipid stains or fatty-acid probes. Building upon our cell-permeant probe platform, my laboratory developed the first bioorthogonal strategy for selectively labeling downstream isoprenoids through metabolic incorporation of alkynylated DMAPP analogs in living B. subtilis. Using this optimized probe, we demonstrated selective metabolic incorporation into endogenous membrane-associated isoprenoids, including menaquinone-7, bactoprenol, and tetraprenyl-β-curcumene, which were first verified by targeted LC-HRMS and subsequently visualized in intact bacterial cells following click-mediated fluorescent labeling. Fluorescence localized predominantly to the cell periphery and division septa, consistent with regions of active peptidoglycan biosynthesis.
Current efforts are extending this platform to investigate how bacterial isoprenoid metabolism is dynamically reorganized during cell-wall stress. Preliminary studies with vancomycin reveal striking temporal changes in bioorthogonal labeling that accompany inhibition of peptidoglycan biosynthesis, suggesting substantial redistribution of downstream isoprenoid metabolites during antibiotic treatment. Ultimately, we hope to establish how bacterial isoprenoid metabolism responds to antibiotic induced stress and define the relationship between membrane-associated isoprenoid remodeling and regulation of the central five-carbon network.