A Photoactivatable Free Mycolic Acid Probe to Investigate Mycobacteria–Host Interactions

Mycolic acids are long-chain, α-branched, β-hydroxylated fatty acid lipids that populate the outer mycomembrane of mycobacteria, including the pathogen <i>Mycobacterium tuberculosis</i>. Mycolic acids predominantly occur in the form of glycolipids, but nonglycosylated free mycolic acids (fMA), which are generated during mycomembrane remodeling, are major constituents of the <i>M. tuberculosis</i> biofilm extracellular matrix and promote host immune evasion during <i>M. tuberculosis</i> infection. However, our understanding of these processes is nascent, and there is limited information about the fMA-protein interactions involved. To facilitate such studies, we synthesized a fMA analogue probe (x-Alk-MA) containing a photo-cross-linking diazirine and a clickable alkyne to enable live-cell capture and analysis of protein interactors. The synthetic strategy featured asymmetric hydrogenation to establish the β-hydroxy group, diastereoselective alkylation to establish the α-branch, and late-stage modification to install the functional tags. In macrophages, x-Alk-MA recapitulated the cytokine response of native MA and selectively photolabeled TREM2, a host cell receptor for fMAs that suppresses macrophage activation and has been implicated in <i>M. tuberculosis</i> immune evasion. The synthetic strategy, chemical probes, and photolabeling methods disclosed herein should facilitate future studies aimed at understanding the roles of fMA in mycobacterial physiology and pathogenesis.