title: Design and synthesis of highly sensitive and selective fluorescein-derived magnesium fluorescent probes and application to intracellular 3D Mg2+ imaging.
abstract: The role of intracellular magnesium ions is of high interest in the fields of pharmacology and cellular biology. To accomplish the dynamic and three-dimensional imaging of intracellular Mg2+, there is a strong desire for the development of optimized Mg2+ fluorescent probes. In this paper we describe the design, synthesis, and cellular application of the three novel Mg2+ fluorescent probes KMG-101, -103, and -104. The compounds of this series feature a charged beta-diketone as a binding site specific for Mg2+ and a fluorescein residue as the fluorophore that can be excited with an Ar+ laser such as is widely used in confocal scanning microscopy. This molecular design leads to an intensive off-on-type fluorescent response toward Mg2+ ions. The two fluorescent probes KMG-103 and -104 showed suitable dissociation constants (Kd,Mg2+ = 2 mM) and nearly a 10-fold fluorescence enhancement over the intracellular magnesium ion concentration range (0.1-6 mM), allowing high-contrast, sensitive, and selective Mg2+ measurements. For intracellular applications, the membrane-permeable probe KMG-104AM was synthesized and successfully incorporated into PC12 cells. Upon application of the mitochondria uncoupler FCCP to the probe-incorporated cells, the resulting increase in the free magnesium ion concentration could be followed over time. By using a confocal microscope, the intracellular 3D magnesium ion concentration distributions were satisfactorily observed.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Komatsu
authors.forename: Naoko
authors.initials: N
authors.lastname: Iwasawa
authors.forename: Daniel
authors.initials: D
authors.lastname: Citterio
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kubota
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tokuno
authors.forename: Yoshiichiro
authors.initials: Y
authors.lastname: Kitamura
authors.forename: Kotaro
authors.initials: K
authors.lastname: Oka
authors.forename: Koji
authors.initials: K
authors.lastname: Suzuki
mesh_terms: D000818:Animals; D005452:Fluoresceins / Q000138:chemical synthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D008274:Magnesium / Q000032:analysis* / Q000737:chemistry; D018613:Microscopy, Confocal; D016716:PC12 Cells; D051381:Rats; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D008274:Magnesium