title: Design of a practical fluorescent probe for superoxide based on protection-deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups.
abstract: A strategy for designing probes based on protection-deprotection chemistry involving fluoresceins and their benzenesulfonyl (BES) derivatives has led to the development of a much more practical superoxide (O(2) (-.)) probe than the previously reported bis(2,4-dinitro-BES) tetrafluorofluorescein (6 a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6 a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7 j) as the optimal reagent. A microtiter plate assay with BESSo showed a tenfold improved detection limit for O(2) (-.) compared with such an assay based on 6 a. BESSo showed markedly better specificity for O(2) (-.) than for GSH or other reactive oxygen species, and this specificity was significantly higher than that of Fe(2+) and some reducing enzymes. These features have resulted in the development of an assay based on BESSo that is capable of providing more unambiguous results for O(2) (-.) release from neutrophils, with or without stimulation by phorbol myristate acetate, as compared with an assay based on 6 a. Intracellular generation of O(2) (-.) in human Jurkat T cells stimulated by butyric acid has been measured by using flow cytometry and fluorescence microscopy utilizing the acetoxymethyl derivative of BESSo.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. h-maeda@phs.osaka-u.ac.jp
authors.forename: Hatsuo
authors.initials: H
authors.lastname: Maeda
authors.forename: Kayoko
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Iho
authors.initials: I
authors.lastname: Kohno
authors.forename: Leila
authors.initials: L
authors.lastname: Hafsi
authors.forename: Norio
authors.initials: N
authors.lastname: Itoh
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Nakagawa
authors.forename: Naoko
authors.initials: N
authors.lastname: Kanagawa
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Uno
mesh_terms: D002478:Cells, Cultured; D002625:Chemistry, Organic / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D013451:Sulfonic Acids / Q000737:chemistry; D013481:Superoxides / Q000032:analysis* / Q000737:chemistry*; D013601:T-Lymphocytes; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D013451:Sulfonic Acids; D013481:Superoxides; D013755:Tetradecanoylphorbol Acetate