Selective purification of catecholate, hydroxamate and ?-hydroxycarboxylate siderophores with titanium dioxide affinity chromatography

Siderophores, high affinity iron chelators, play a key role in the uptake of iron by microorganisms and regulate many biological functions. Siderophores are categorized by their chelating group, e.g., catecholates, hydrox-amates, alpha-hydroxycarboxylates. Natural concentrations of siderophores are often either too low or sample matrices are too complex for direct analysis by, e.g., liquid chromatography - mass spectrometry. Therefore, both concentration and purification are prerequisite for reliable analyses. However, a chromatographic technique that is selective for all siderophore classes and affords high levels of purification is lacking. We developed a titanium dioxide affinity chromatography (TDAC) solid-phase extraction (SPE) that affords the selective purification of these siderophore classes from complex sample matrices with recoveries up to 82%. The one-step purification removed most non-ligand sample 'contaminants', therefore, affording the straightforward identification of siderophore peaks in base peak chromatograms. As a proof of concept, the bioinformatic processing, der-eplication of known features and selection of significant features in the TDAC eluates afforded a fast identifi-cation of six novel siderophores (woodybactines) from bacterial supernatants. We propose TDAC SPE as a fast and cost-effective methodology to screen for known or discover novel siderophores in natural samples in com-bination with untargeted bioinformatic processing by, e.g., XCMS. The method is scalable and yielded large amounts of highly purified siderophores from bacterial culture supernatants, providing an effective quantitative sample clean-up for, e.g., NMR structure elucidation.