Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 13, Issue 7, Pages 1946-1954Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.200600522
Keywords
fluoresceins; fluorescence spectroscopy; fluorescent probes; molecular design; superoxide
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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 (6a). Examination of various BES derivatives, developed from the starting point of the prototype probe 6a, yielded 4,5-dimethoxy-2-nitro-BES tetrafluorofluorescein (BESSo; 7j) 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 6a. 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 Fe2+ 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 6a. 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.
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