O- and N-linked TMM derivatives for selective labeling of mycolate-containing components of the mycomembrane. (A) O-linked TMM derivatives (modified with an unnatural R group) mimic the mycoloyl donor function of TMM and are proposed to undergo periplasmic mycoloyltransferase-catalyzed transfer of their unnatural acyl chain onto mycoloyl acceptors, producing labeled TDM, AGM, proteins, and potentially other mycolate-containing products. (B) N-linked TMM derivatives (modified with an unnatural R group) mimic the mycoloyl acceptor function of TMM and are proposed to undergo periplasmic mycoloyltransferase-catalyzed mycoloylation of their free 6-position hydroxyl group, producing only a labeled version of TDM. Enz, enzyme with mycoloyltransferase activity (e.g., Ag85). (C and D) Structures of published O-linked (C) and N-linked (D) TMM derivatives bearing bioorthogonal functional groups, fluorophores, and photo-cross-linking tags.