Design, synthesis and properties of novel iron(III)-specific fluorescent probes

Bidentate chelators such as hydroxypyridinones and hydroxypyranones are highly iron selective. The synthesis of two novel fluorescent probes N-[2-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (CP600) and N-[(3-hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (CP610) is reported. The method involves coupling the bidentate ligands, 3-hydroxypyridin-4-one and 3-hydroxypyran-4-one, with the well-characterised fluorescent probe methoxycoumarin. Fluorescence emission of both probes at 380 nm is readily quenched by Fe(3+). The fluorescence was quenched to a greater extent by Fe(3+) than by Mn(2+), Co(2+), Zn(2+), Ca(2+), Mg(2+), Na(+) and K(+) and to approximately the same extent as Cu(2+). Comparison of the fluorescence-quenching ability by a range of metal ions on CP600 and CP610 and the hexadentate chelator, calcein, under in-vitro conditions, demonstrated advantages of the two novel fluorescent probes with respect to both iron(III) sensitivity and selectivity. Chelation of iron(III) by CP600 and CP610 leads to the formation of a complex with a metal-to-ligand ratio of 1:3. Fluorescence is quenched on formation of such complexes. These probes possess a molecular weight less than 400 and thus they are predicted to permeate biological membranes by passive diffusion, and have potential for reporting intracellular organelle labile iron levels.