Novel Mg
2+ fluorescent molecular probes (KMG-20-AMand KMG-27-AM; where AM is an acetoxymethyl group)based on a coumarin possessing a charged
-diketonestructure were designed and synthesized. These fluorescent probes produced a red shift from 425 to 445 nm inthe absorption spectra after formation of a complex withMg
2+. The fluorescence spectra of these probes alsoshowed a red shift from 485 to 495 nm and an increasingfluorescence intensity after formation of a complex withMg
2+. The optimum experimental conditions were excitation wavelength of 445 nm and a monitored wavelengthof 500 nm, where these probes functioned as an indicatorshowing an image of increasing fluorescence in the presence of Mg
2+. These probes showed a "seesaw-type"fluorescent spectral change with the isosbestic point at480 nm due to the light excitation at 445 nm, whichindicates that ratiometry can be used for the measurement. The molecular probes formed a 1:1 complex withMg
2+ and the dissociation constant (
Kd) was 10.0 mM forKMG-20. The association constants of the probes withMg
2+ were ~3 times higher than that with Ca
2+, whichshowed that the selectivity of Mg
2+ versus Ca
2+ for theseprobes was over 200 times higher than that for commercially available Mg
2+ fluorescent molecular probessuch as mag-fura-2, Magnesium Green. As an applicationof these probes, intracellular fluorescent imaging of Mg
2+was demonstrated using a fluorescent microscope. Afterthe addition of KMG-20-AM and KMG-27-AM into PC12cells, a strong fluorescence was observed in the cytoplasmand a weak fluorescence in the nuclei region. Aftertreatment with a high-K
+ medium, the fluorescence intensity increased due to increasing intracellular Mg
2+. Thereal image of Mg
2+ release from the magnesium store wassuccessfully observed with these Mg
2+ fluorescent probes.