Reactive oxygen species (ROS) such as superoxide (O 2 ÀC ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (HOC) are important mediators of pathological processes in various diseases.[1] Detection by fluorescent probes is one of the most useful methods for evaluating the roles of ROS in pathological processes. 2',7'-Dichlorofluorescin (DCFH) and its diacetyl derivative (DCFH-DA) [2] have been widely used as fluorescent probes for measuring cell-derived H 2 O 2 , [3] but these compounds suffer from the major drawback that they are poorly selective toward H 2 O 2 . Researchers have demonstrated that oxidation of DCFH to dichlorofluorescein is also induced by peroxidase [4] and other hemoproteins [5] as well as by hydroperoxides in the presence of peroxidase, [6] nitric oxide, [7] and peroxynitrite. [8] Therefore, the fluorescent response based on the oxidation of DCFH provides an index, not for cell-derived H 2 O 2 , but for the total oxidants present in biological systems. This limitation stems from its mechanism of fluorescence, which is based on oxidation. Dihydro derivatives of fluorescent compounds such as dihydrorhodamine 123 [3c,g] and N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) [9] have been shown to function as probes for detecting H 2 O 2 . However, their mechanism of action is similar to that of DCFH, which implies that low selectivity toward H 2 O 2 is a shortcoming that must be accepted when utilizing these probes. In fact, dihydrorhodamine 123 was shown to react with various ROS, [3c, 7b] and although Amplex Red seems to have high selectivity toward H 2 O 2 , peroxidase is essential for its fluorescence, similar to the case of DCFH. Thus, developing probes for H 2 O 2 based on a non-oxidative fluorescence mechanism, which would allow the highly specific and peroxidase-independent detection of H 2 O 2 under the complicated oxidative circumstances found in biological systems, is a worthwhile goal.Recently, we found that perhydrolysis of acyl resorufins is a useful reaction that acts as a fluorescent indicator for H 2 O 2 assays.[10] The method is based on simple deprotection, not on oxidation, thus allowing acyl derivatives of fluorescent compounds such as resorufin and fluorescein to work as probes for detecting cell-derived H 2 O 2 with higher selectively than that provided by DCFH and its analogues. Unfortunately, the competition between perhydrolysis and hydrolysis of acyl resorufins and fluoresceins in biological systems was not altered in a manner favorable towards H 2 O 2 -based deacylation.We thus designed pentafluorobenzenesulfonyl fluoresceins (1 a-c, Scheme 1) as selective fluorescent probes for H 2 O 2 but would eliminate, or at least significantly reduce, competition from hydrolysis reactions of the acetyl derivatives. These compounds were chosen for the following reasons: sulfonates are more stable to hydrolysis than are esters; fluoresceins have high fluorescence quantum yields in aqueous solution; and the pentafluorobenzene ring enhances the reactivity of the sulfonates tow...

Fluorometric detection of O2-* is performed based on desulfonylation of 3 to the corresponding fluoresceins 4 through nucleophilic substitution, and this fluorescing process is quite specific toward O2-* over H2O2, t-BuOOH, NaOCl, 1O2, HO*, NO*, and ONOO-. Furthermore, effects of glutathione, cytochrome P450 reductase/NADPH, and diaphorase/NADH are relatively small on the fluorescing process of probe 3 with X = Y = F, which is useful to detect O2-* released from neutrophils stimulated by phorbol myristate acetate with satisfactory sensitivity.

Reactive oxygen species (ROS) such as superoxide (O 2 ÀC ), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (HOC) are important mediators of pathological processes in various diseases.[1] Detection by fluorescent probes is one of the most useful methods for evaluating the roles of ROS in pathological processes. 2',7'-Dichlorofluorescin (DCFH) and its diacetyl derivative (DCFH-DA) [2] have been widely used as fluorescent probes for measuring cell-derived H 2 O 2 , [3] but these compounds suffer from the major drawback that they are poorly selective toward H 2 O 2 . Researchers have demonstrated that oxidation of DCFH to dichlorofluorescein is also induced by peroxidase [4] and other hemoproteins [5] as well as by hydroperoxides in the presence of peroxidase, [6] nitric oxide, [7] and peroxynitrite. [8] Therefore, the fluorescent response based on the oxidation of DCFH provides an index, not for cell-derived H 2 O 2 , but for the total oxidants present in biological systems. This limitation stems from its mechanism of fluorescence, which is based on oxidation. Dihydro derivatives of fluorescent compounds such as dihydrorhodamine 123 [3c,g] and N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) [9] have been shown to function as probes for detecting H 2 O 2 . However, their mechanism of action is similar to that of DCFH, which implies that low selectivity toward H 2 O 2 is a shortcoming that must be accepted when utilizing these probes. In fact, dihydrorhodamine 123 was shown to react with various ROS, [3c, 7b] and although Amplex Red seems to have high selectivity toward H 2 O 2 , peroxidase is essential for its fluorescence, similar to the case of DCFH. Thus, developing probes for H 2 O 2 based on a non-oxidative fluorescence mechanism, which would allow the highly specific and peroxidase-independent detection of H 2 O 2 under the complicated oxidative circumstances found in biological systems, is a worthwhile goal.Recently, we found that perhydrolysis of acyl resorufins is a useful reaction that acts as a fluorescent indicator for H 2 O 2 assays.[10] The method is based on simple deprotection, not on oxidation, thus allowing acyl derivatives of fluorescent compounds such as resorufin and fluorescein to work as probes for detecting cell-derived H 2 O 2 with higher selectively than that provided by DCFH and its analogues. Unfortunately, the competition between perhydrolysis and hydrolysis of acyl resorufins and fluoresceins in biological systems was not altered in a manner favorable towards H 2 O 2 -based deacylation.We thus designed pentafluorobenzenesulfonyl fluoresceins (1 a-c, Scheme 1) as selective fluorescent probes for H 2 O 2 but would eliminate, or at least significantly reduce, competition from hydrolysis reactions of the acetyl derivatives. These compounds were chosen for the following reasons: sulfonates are more stable to hydrolysis than are esters; fluoresceins have high fluorescence quantum yields in aqueous solution; and the pentafluorobenzene ring enhances the reactivity of the sulfonates tow...