Design and synthesis of Mg²⁺-selective fluoroionophores based on a coumarin derivative and application for Mg²⁺ measurement in a living cell

Novel Mg²⁺ fluorescent molecular probes (KMG-20-AM and KMG-27-AM; where AM is an acetoxymethyl group) based on a coumarin possessing a charged β-diketone structure were designed and synthesized. These fluorescent probes produced a red shift from 425 to 445 nm in the absorption spectra after formation of a complex with Mg²⁺. The fluorescence spectra of these probes also showed a red shift from 485 to 495 nm and an increasing fluorescence intensity after formation of a complex with Mg²⁺. The optimum experimental conditions were excitation wavelength of 445 nm and a monitored wavelength of 500 nm, where these probes functioned as an indicator showing an image of increasing fluorescence in the presence of Mg²⁺. These probes showed a "seesaw-type" fluorescent spectral change with the isosbestic point at 480 nm due to the light excitation at 445 nm, which indicates that ratiometry can be used for the measurement. The molecular probes formed a 1:1 complex with Mg²⁺ and the dissociation constant (K_d) was 10.0 mM for KMG-20. The association constants of the probes with Mg²⁺ were ∼3 times higher than that with Ca²⁺, which showed that the selectivity of Mg²⁺ versus Ca²⁺ for these probes was over 200 times higher than that for commercially available Mg²⁺ fluorescent molecular probes such as mag-fura-2, Magnesium Green. As an application of these probes, intracellular fluorescent imaging of Mg²⁺ was demonstrated using a fluorescent microscope. After the addition of KMG-20-AM and KMG-27-AM into PC12 cells, a strong fluorescence was observed in the cytoplasm and a weak fluorescence in the nuclei region. After treatment with a high-K⁺ medium, the fluorescence intensity increased due to increasing intracellular Mg²⁺. The real image of Mg²⁺ release from the magnesium store was successfully observed with these Mg²⁺ fluorescent probes.