Abstract
Mycolic acids are long-chain, α-branched, β-hydroxylated fatty acid lipids that populate the outer mycomembrane of mycobacteria, including the pathogen Mycobacterium tuberculosis. 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 M. tuberculosis biofilm extracellular matrix and promote host immune evasion during M. tuberculosis 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 M. tuberculosis 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.