Kupyaphores─Self-Assembling Diisocyanolipopeptide Zn<sup>II</sup> Ionophores in <i>Mycobacterium tuberculosis</i> Zn<sup>II</sup>/Cu<sup>I/II</sup> Homeostasis and Antibacterial Effects

Mycobacterium tuberculosis ( Mtb ), the leading cause of infectious disease mortality from a single pathogen, requires essential metal ions to establish infection and persist in the host. Kupyaphores, a suite of recently identified amphiphilic diisocyanolipopeptides, were reported to assist with Zn II acquisition to support a multitude of Zn II -dependent metalloenzymes critical for Mtb 's survival and pathogenicity. However, compared to well-studied Fe III acquisition systems in Mtb , the mechanisms for Zn II acquisition and homeostasis remain virtually unexplored. Herein, we reveal them as novel metal ionophores in Mtb 's metal-fluctuating lipidic niche. A concise modular scalable synthesis was developed to assess the critical features required for activity. Synthetic kupyaphores were structurally and functionally validated, respectively, via LCMS and chemical complementation of kupyaphore-deficient (Δ rv0101 ) Mtb . MS, NMR, and IR evidence demonstrated that kupyaphores complex Zn II as a bidentate ligand. Fluorescence competition data indicated Zn II /Cu I/II binding capabilities, by which Mtb entraps excessive metals within o/w-type micelles against host-induced metal intoxication. The inhibition against Gram-positive Staphylococcus aureus and the low human toxicity imply the potential as a novel antibacterial scaffold. Collectively, this work provides insight into the Zn II /Cu I/II homeostasis of Mtb and a chemical basis for the development of mechanistic tools, therapeutic conjugates against Mtb , and antibiotics.