Design of a practical fluorescent probe for superoxide based on protection - Deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups
Design of a practical fluorescent probe for superoxide based on protection - Deprotection chemistry of fluoresceins with benzenesulfonyl protecting groups
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DOI：
10.1002/chem.200600522
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Uno, Tadayuki
中科院分区：
文献类型：
--
作者：
Maeda, Hatsuo;Yamamoto, Kayoko;Uno, Tadayuki
基于荧光素及其苯磺酰基（BES）衍生物的保护-去保护化学设计探针的策略导致了更实用的超氧化物（O-2（-.））探针比以前报道的双（2，4-二硝基-BES）荧光素（6a）。从原型探针6a的起始点开发的各种BES衍生物的检查产生了作为最佳试剂的4，5-二甲氧基-2-硝基-BES荧光素（BESSO; 7 j）。用BESSO进行的微量滴定板测定显示O-2（-）的检测限提高了10倍。与基于6a的这种测定相比。BESSo对O-2（-.）对GSH等活性氧的特异性高于Fe ~（2+）和某些还原酶。这些特征导致了基于BESSO的测定的开发，该测定能够提供更明确的O-2（-）结果。与基于6a的测定相比，在有或没有佛波醇肉豆蔻酸乙酸酯刺激的情况下，从中性粒细胞释放。细胞内产生O-2（-.）在丁酸刺激的人Jurkat T细胞中，通过使用BESSo的乙酰氧基甲基衍生物，使用流式细胞术和荧光显微镜测量。
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.