title: Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties.
abstract: We report the synthesis and characterization of a fluorescent iron chelator (4), shown to be effective in inhibiting the growth of Mycobacterium avium in macrophages, together with the synthesis and characterization of two unsuccessful analogues selected to facilitate identification of the molecular properties responsible for the antimicrobial activity. Partition of the chelators in liposomes was investigated and the compounds were assessed with respect to uptake by macrophages, responsiveness to iron overload/iron deprivation and intracellular distribution by flow cytometry and confocal microscopy. The synthesis of the hexadentate chelators is based on a tetrahedral structure to which three bidentate 3-hydroxy-4-pyridinone chelating units are linked via amide bonds. The structure is synthetically versatile, allowing further addition of functional groups such as fluorophores. Here, we analyse the non-functionalized hexadentate unit (3) and the corresponding rhodamine B (4) and fluorescein (5) labelled chelators. The iron(III) stability constant was determined for 3 and the values log beta = 34.4 and pFe(3+) = 29.8 indicate an affinity for iron of the same order of magnitude as that of mycobacteria siderophores. Fluorescence properties in the presence of liposomes show that 4 strongly interacts with the lipid phase, whereas 5 does not. Such different behaviour may explain their distinct intracellular localization as revealed by confocal microscopy. The flow cytometry and confocal microscopy studies indicate that 4 is readily engulfed by macrophages and targeted to cytosol and vesicles of the endolysosomal continuum, whereas 5 is differentially distributed and only partially colocalizes with 4 after prolonged incubation. Differential distribution of the compounds is likely to account for their different efficacy against mycobacteria.
authors.affiliation: REQUIMTE, Departamento de Química, Faculdade de Ciências, Universidade do Porto, 4069-007, Porto, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Nunes
authors.forename: Maria
authors.initials: M
authors.lastname: Podinovskaia
authors.forename: Andreia
authors.initials: A
authors.lastname: Leite
authors.forename: Paula
authors.initials: P
authors.lastname: Gameiro
authors.forename: Tao
authors.initials: T
authors.lastname: Zhou
authors.forename: Yongmin
authors.initials: Y
authors.lastname: Ma
authors.forename: Xiaole
authors.initials: X
authors.lastname: Kong
authors.forename: Ulrich E
authors.initials: UE
authors.lastname: Schaible
authors.forename: Robert C
authors.initials: RC
authors.lastname: Hider
authors.forename: Maria
authors.initials: M
authors.lastname: Rangel
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D015195:Drug Design; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D042541:Intracellular Space / Q000378:metabolism*; D007501:Iron / Q000737:chemistry; D007502:Iron Chelating Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D000090463:Iron Deficiencies; D019190:Iron Overload / Q000473:pathology; D008081:Liposomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008968:Molecular Conformation; D009162:Mycobacterium avium / Q000187:drug effects; D011725:Pyridines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D007502:Iron Chelating Agents; D008081:Liposomes; D011725:Pyridines; D012235:Rhodamines; C506370:hexadentate 3-hydroxypyridin-4-one; D007501:Iron; C029773:rhodamine B; D019793:Fluorescein