Azotochelin

Life on Earth depends on N2 -fixing microbes to make ammonia from atmospheric N2 gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N2 fixation was once believed to have evolved during the Archean-Proterozoic times using Fe as a cofactor. However, δ15 N values of paleo-ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo-oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral-associated trace metals to a model N2 -fixing bacterium Azotobacter vinelandii. N2 fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N2 fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N2 fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral-associated trace metals are bioavailable as cofactors of nitrogenases to support N2 fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox.

We report an electrochemical setup comprising a boron-doped diamond (BDD) working electrode for the electrochemical study of iron(III) catecholate siderophores. We demonstrate its successful application in the voltammetric investigation of iron(III) azotochelin, an iron complex of a bis(catecholate) siderophore. Cyclic voltammetry results, when complemented by UV-vis and native electrospray ionization-mass spectrometry (ESI-MS) characterization, reveal the formation of a coordinatively unsaturated tetracoordinate 1:1 complex of Fe:azotochelin (M1:L1) at neutral pH, contrary to iron(III) tetradentate siderophore complexes of other classes which favor the hexacoordinate environment of an M2:L3 species. A notable effect of pH and buffer composition on the reduction potential of iron(III) azotochelin is demonstrated. Lower pH values and buffers encompassing primary or secondary amines facilitate a positive potential shift of up to +290 mV and +250 mV vs Ag/AgCl 3 M NaCl, respectively. The study was extended to the investigation of the iron(III) complexes of hexadentate siderophores. For tris(catecholate) siderophores, enterobactin and protochelin, the reduction potentials were found to lie beyond the potential window accessible to the BDD electrode; however, we were successful in observing the electrochemical behavior of a tris(hydroxamate) siderophore, ferricrocin.

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation.