Acinetobacter baumannii, Pseudomonas aeruginosa, and carbapenem-resistant Enterobacteriaceae are emerging multidrug-resistant Gram-negative bacterial pathogens. With increasing frequency, they often prove untreatable or treatable only with toxic antimicrobials. Therefore, the CDC now categorizes such organisms in their top antibiotic resistance threat level. New anti-infective strategies are urgently needed. Apramycin is an aminocyclitol aminoglycoside that has impressive activity spectrum against these pathogens. Further, in contrast to plazomycin, it retains activity against strains expressing widely circulating ribosomal methylases that for example are often found in the alarmingly multidrug-resistant, NDM-1 carbapenemase strains. Importantly, apramycin also does not appear to have significant nephrotoxicity and ototoxicity side effects that have limited clinical use of other aminoglycosides. We also found that apramycin showed rapid bactericidal killing, high selectivity for prokaryotic ribosomes, and efficacy against A. baumannii in a murine thigh infection model. Based on such compelling properties, we propose to pursue medicinal chemistry based optimization of the apramycin scaffold in two specific aims: The first goal is to perform structure-activity relationship and structure-pharmacokinetic relationship studies to identify derivatives with enhanced antimicrobial potency while maintaining selectivity. In the past, medicinal chemistry evaluation of the apramycin scaffold made use of semi- synthetic approaches based on modification of the existing apramycin natural product. These approaches necessarily placed significant constraints on chemical space available for exploration. Here, we hypothesize that use of modern, total synthetic glycochemistry approaches will allow us to optimize and develop compelling therapeutic leads with activity against Acinetobacter and other resistant Gram-negative organisms. The second proof-of-principle goal will be to characterize the ability of apramycin analogues to treat XDR Acinetobacter infection in a murine thigh infection model. Experiments will also address whole animal toxicities and iterate back into the medicinal chemistry optimization plan with the long-term goal of developing safe and effective therapy. The near-term goal of this two year, exploratory R21 proposal is to address potential of total synthetic glycochemistry approaches to identify tractable, potent analogues worthy of further exploration.