Tracking Uptake and Metabolism of Xenometallomycins Using a Multi-Isotope Tagging Strategy

Synthetic and naturally occurring siderophores and their conjugates provide access to the bacterial cytoplasm via active membrane transport. Previously, we displaced iron with the radioactive isotope ⁶⁷Ga to quantify and track in vitro and in vivo uptake and distribution of siderophore Trojan Horse antibiotic conjugates. Here, we introduce a multi-isotope tagging strategy to individually elucidate the fate of metal cargo and the ligand construct with radioisotopes ⁶⁷Ga and ¹²⁴I. We synthesized gallium(III) model complexes of a ciprofloxacin-functionalized linear desferrichrome (Ga-D6) and deferoxamine (Ga-D7) incorporating an iodo-tyrosine linker to enable radiolabeling using the metal-binding (⁶⁷Ga) and the cargo-conjugation site (¹²⁴I). Radiochemical experiments with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa wt strains show that ⁶⁷Ga-D6/D7 and Ga-D6-¹²⁴I/D7-¹²⁴I have comparable uptake, indicating intact complex import and siderophore-mediated uptake. In naive mice, ⁶⁷Ga-D6/D7 and Ga-D6-¹²⁴I/D7-¹²⁴I demonstrate predominantly renal clearance; urine metabolite analysis indicates in vivo dissociation of Ga(III) is a likely mechanism of degradation for ⁶⁷Ga-D6/D7 when compared to ligand radiolabeled compounds, Ga-D6-¹²⁴I/D7-¹²⁴I, which remain >60% intact in urine. Cumulatively, this work demonstrates that a multi-isotope tagging strategy effectively elucidates the in vitro uptake, pharmacokinetics, and in vivo stability of xenometallomycins with modular chemical structures.