Iron Chelators Designed to Study Aspects of Siderophore-Mediated Iron(III) Uptake

The investigation of the chemical characteristics of siderophores has been of interest for many years, for both the understanding of their fundamental chemistry and exploiting the design features of these natural compounds for novel applications. Within the present project, the properties of a biomimetic model of the tetradentate siderophore known as the enterobactin linear dimer was investigated, followed by those of the enterobactin linear dimer itself. Both compounds were chemically synthesised, with an overall yield of 52% in four steps for the biomimetic, and 26% in seven-step convergence synthesis for the linear dimer. Investigation of the coordination chemistry, showing both can form complexes with a ligand-to-metal ratios of 3:2 and 1:1 in solution. This was followed up by X-ray diffraction investigations of the ferric-complexes with the periplasmic binding protein CeuE. Both ferric-complexes bound to CeuE in a novel mode, with conserved tyrosine and histidine residues binding directly to the iron(III) centre to complete the octahedral coordination of the metal. Both complexes have a Λ metal centre configuration when bound to CeuE. The dissociation constants of CeuE with ferric-4-LICAM and ferric-linear dimer were determined to be 29.3 ± 11.7 nM and 8.4 ± 4.3 nM for the ferric-4-LICAM and ferric-linear dimer, respectively. In addition, selected applications of siderophores were investigated. Firstly, a synthetic route to a novel hexadentate siderophore mimic possessing a chemical linking group was developed, in an eight-step synthesis with an overall yield of 10.5%. Secondly, the natural hydroxamate siderophore ferricrocin was labelled with an Alexa Fluor® 488 fluorophore. Total internal reflection fluorescence microscopy was used to demonstrate that the resulting conjugate binds to E. coli cells that possess the ferricrocin outer membrane receptor protein FhuA.