HIRATA Kazumasa Osaka University, Graduate School of Pharmaceutical Sciences, Associate Professor, 大学院・薬学研究科, 助教授 (30199062)
ITOH Norio Osaka University, Graduate School of Pharmaceutical Sciences, Lecturer, 大学院・薬学研究科, 講師 (60176352)
|Budget Amount *help
¥10,200,000 (Direct Cost : ¥10,200,000)
Fiscal Year 2005 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 2004 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 2003 : ¥6,900,000 (Direct Cost : ¥6,900,000)
(1) Hydrogen Peroxide (H_2O_2) Probe : Based on the proposed working hypothesis, we examined the reactivity of various types of benzenesulfonyl fluoresceins toward H_2O_2, and found that 3'-(pentafluorobenzenesulfonyl) 2',7'-difluorofluorescein (1) works as a specific H_2O_2 fluorescent probe. In addition, it was demonstrated that the acetyl derivative of 1 allows fluorometric analysis of H_2O_2 generated inside a green algae Chlamydomonas reinhardtii and human Jurkat T cells by stimulation with Cu^<2+> and butyric acid, respectively. The study on developing these probes, we have been able to propose a novel and general strategy for probe design.
(2) Superoxide (O_2^<-・>) Probe : Based on the proposed strategy, we succeeded in developing 3',5'-bis(2,4-dinitrobenzenesulfonyl) 2',4',5',7'-tetra-fluorofluorescein (2) as a novel O_2^<-・> probe. A further study allowed demonstrating that 3'-(4,5-dimethoxy-2-nitrobenzenesulfonyl) 2',4',5',7'-tetrafluorofluorescein (3) as a more practical O_2^
<-・> probe than 2. By loading cells with the acetoxymethyl derivative of 3, flowcytometry and fluorescent microscopy were able to measure O_2^<-・> generated inside human Jurkat T cells stimulated with butyric acid.
(3) Thiol Probe : Based on the proposed strategy, we succeeded in developing 3'-(2,4-dinitrobenzenesulfonyl) 2',7'-dimethylfluorofluorescein (4) as a novel thiol probe. High miscibility of 4 to aqueous media tolerated development of highly sensitive fluorometric assay of cholinesterases useful as a screening method of acetylcholinesterase specific inhibitors.
(4) Selenol Probe : It was demonstrated that at pH 5.8, 4 provides no fluorescent responses toward thiols, while bringing about strong fluorescent augmentation on reaction with selenol, namely, that 4 works as a first selenol fluorescent probe. Thus, the fluorometry with 3 works as a sensitive quantification method for selenocysteine generated from selenocystine by reaction with dithiothreitol, and as an estimating tool for selenocysteine residues in selenoproteins. Less