Pathogenic bacteria scavenge ferric iron from the host for survival and proliferation using small-molecular chelators, siderophores. Here, we introduced solid-phase synthesis platform for the rapid and scalable synthesis of synthetic siderophores inspired by the siderophore protochelin, an enterobactin analogue. Four synthetic protochelin analogues were constructed and evaluated for their ability to be recognized as substrates for the transmembrane uptake machinery of Escherichia coli. The constructs KK-COOH, KO-NH2, KO-COOH and OK-COOH, were probed for their ability to restore growth in E. coli Dent and internalized in E. coli wt as well as transporter mutants E. coli ΔFepA and E. coli ΔFepC using time dependent quantitation of the uptake of 67Ga-labeled analogues. Our results suggest that the novel protochelin-analogues are internalized selectively by catechol-recognizing Fep transmembrane transporter family selective for Enterobactin (Ent), with likely additional uptake via the less selective Cir or Fiu system. Upon substitution of the trivalent metal ions Fe(III) and Ga(III) for 45Ti, radiochemical bacterial uptake was significant diminished by an order of magnitude, highlighting the role of complex charge for siderophore uptake. In vivo experiments investigating the distribution and metabolic stability in naïve mice, the lead [68Ga][Ga(KO-NH2)]3- clears renally and remains ≥ 95% intact. Further evaluation of [68Ga][Ga(KO-NH2)]3- in a soft tissue infection model demonstrated selective, enhanced uptake in the E. coli infected muscle (1.28 % ID/g), while inflamed and naïve muscle tissue uptake remained low (0.42 and 0.35 % ID/g). Determination of concentration-dependent growth-inhibition of protochelin-based siderophores with Ga(III) and Ti(IV) revealed that absence of metal-ion induced toxicity at tested concentrations. A corresponding siderophore antibiotic conjugates (SACs) [Ga(KO-NH2 CIP)]3-, constructed using solid-phase synthesis approach, exhibited MIC98 of 7.5 mM, whereas the corresponding apo, Fe(III) and Ti(IV) complexes MIC98 of 15 mM, 30 mM, 30 mM respectively. Taken together, our studies qualify [Ga(KO-NH2)]3- and [Ga(KO-NH2 CIP)]3- complexes of interest for further investigation as diagnostic and treatment tools for bacterial infections.