Metabolic Labeling and Visualization of Isoprenoids Using Alkyne‐Modified DMAPP Analogs in <i>Bacillus subtilis</i>

Abstract

ABSTRACT Isoprenoids are a chemically and functionally diverse class of metabolites that underlie essential bacterial physiology including respiration and cell envelope biogenesis. Chemical interrogation of isoprenoid metabolism remains limited because the two central five‐carbon (C 5 ) precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are highly polar and cell‐impermeant, rendering exogenous addition of them or functional analogs intractable. Here, we report the development of cell‐permeant alkynylated DMAPP precursors ( 1‐3 ) that enable DMAPP‐specific bioorthogonal labeling of isoprenoids in Bacillus subtilis . Growth rescue, toxicity profiling, and downstream incorporation analyses showed that alkyne geometry and substitution pattern strongly influence the metabolic suitability of DMAPP surrogates. We found that a directly substituted Z ‐alkynyl DMAPP analog was the only biosynthetically productive surrogate tested. Metabolic incorporation of this probe resulted in regiospecific labeling of membrane‐embedded isoprenoids, including menaquinone‐7 (MK‐7), bactoprenol, and tetraprenyl‐β‐curcumene. Pathway inhibition and supplementation assays demonstrate that the labeling efficiency of DMAPP surrogates is governed by competition with endogenous DMAPP and IPP. Lastly, post‐incorporation CuAAC derivatization enabled direct visualization of isoprenoid localization and targeted mass‐spectrometric profiling of labeled prenyl metabolites. These results establish a general chemical platform for bioorthogonal tagging of C 5 metabolism in B. subtilis .