title: Design and synthesis of Mg2+-selective fluoroionophores based on a coumarin derivative and application for Mg2+ measurement in a living cell.
abstract: 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 beta-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 (Kd) was 10.0 mM for KMG-20. The association constants of the probes with Mg2- were approximately 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.
authors.affiliation: Collaboration of Regional Entities for the Advancement of Technological Excellence, Kanagawa Academy of Science and Technology, Kawasaki, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Komatsu
authors.forename: Takafumi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Naohiko
authors.initials: N
authors.lastname: Saito
authors.forename: Sawa
authors.initials: S
authors.lastname: Araki
authors.forename: Daniel
authors.initials: D
authors.lastname: Citterio
authors.forename: Deaki
authors.initials: D
authors.lastname: Hisamoto
authors.forename: Yoshiichiro
authors.initials: Y
authors.lastname: Kitamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kubota
authors.forename: Jun
authors.initials: J
authors.lastname: Nakagawa
authors.forename: Kotaro
authors.initials: K
authors.lastname: Oka
authors.forename: Koji
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D003374:Coumarins / Q000737:chemistry*; D007476:Ionophores / Q000138:chemical synthesis / Q000737:chemistry*; D008274:Magnesium / Q000032:analysis*; D016716:PC12 Cells; D051381:Rats; D013050:Spectrometry, Fluorescence
chemical_list: D003374:Coumarins; D007476:Ionophores; C030123:coumarin; D008274:Magnesium