Bio-orthogonal Catalysts for the Activation of Fluoroquinolone Prodrugs within Bacterial Cells

Iron is an essential metal in biology. Siderophores are low molecular-weight compounds excreted by bacteria to bind and retrieve iron. These systems have been exploited by bacteria for the design of antibacterial-siderophore conjugates called sideromycins. Disguised as useful iron sources these conjugates are internalised and release the active component to kill the competing bacteria.

This project investigates siderophore-conjugated bio-orthogonal catalysts for the activation of prodrugs within bacteria. The selected transformation for activation was the deprotection of an allyl ester-protected carboxylic acid of the fluoroquinolone antibacterial moxifloxacin performed by an organometallic ruthenium complex. As kinetics assays for prodrug activation identified catalyst decomposition by molecular oxygen, enteric pathogens were targeted due to the low oxygen atmosphere at parts of the gastrointestinal tract. Antibacterial activity assays showed the synthesised moxifloxacin prodrug was 300-fold less toxic than its active form against gut-relevant bacteria E. coli K12 (BW25113) grown under micro-aerobic (2% O2) and iron-limited conditions to mimic the environment of the intestines. Subsequent bacterial uptake studies suggested that the prodrug is internalised.

A variety of siderophores with different denticities were covalently attached to the catalyst core via glycine linkage to the hydroxyquinoline ligand, such as bidentate catechols, azotochelin, desferrioxamine and pyranone. Positively, prodrug activation kinetics were not significantly impeded by these catalyst decorations. The catechol and azotochelin conjugates improved the growth of iron-starved E. coli 2- fold and 9-fold respectively, implying their facilitation of iron-uptake. Moreover, co- addition experiments for each conjugate with prodrug at their upper, non-toxic concentrations gave antibacterial activity attributed to moxifloxacin formation. The growth recovery of a siderophore biosynthesis deficient strain of E. coli K12 (BW25113) suggested that the catechol and azotochelin conjugates act as weak siderophores. Prodrug incubation experiments mirrored these results, as the catechol conjugate gave the most significantly diminished bacterial growth (~40%) after their prior loading with prodrug.