Fluorescent Probes for Hydrogen Peroxide Based on a Non‐Oxidative Mechanism

Abstract

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...

Redox regulation of calcium ion channels: Chemical and physiological aspects

“…Other commonly used fluorescent dyes are based on rhodamine, ethidine and the phenoxazine backbones, well known examples include Amplex Red and Amplex Ultra Red which are based on phenoxazine (resourfin). Commonly used chemiluminescent based indicators include lucigenin as a specific detector for O 2¯• while luminol has mostly been used as an indicator for general ROS production.Based on redox active substituents in the benzene ring and using the nucleophilic character of some reactive species, the development of a new generation of dyes with potentially improved characteristics has been described [31][32][33][34][35][36][37][38][39][40].Because of the quite complex chemistry of all of these dyes, great care has to be taken in designing biological experiments and in their interpretation. For example, there is evidence that DCF may produce secondary radicals induced by its own oxidation [41].…”

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells

“…To examine the effect of ERW on highly metastatic HT1080 cells, the amount of intracellular H 2 O 2 was determined using BES-H 2 O 2 , a fluorescent probe that more specifically detects intracellular H 2 O 2 than 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (Maeda et al 2004). HT1080 cells were cultured in control or ERWcontaining medium in the presence of 0, 20 or 40 lM H 2 O 2 for 24 h, and then the amount of intracellular H 2 O 2 was determined by an In Cell Analyzer.…”

ER membrane-localized oxidoreductase Ero1 is required for disulfide bond formation in the rice endosperm

“…A fluorescent probe, BES-H 2 O 2 , which is converted to the membrane-impermeant derivative by intracellular esterase, exhibits high selectivity for intracellular H 2 O 2 by H 2 O 2 -mediated hydrolytic deprotection of acyl derivative of fluorescein (21). BES-H 2 O 2 was used to determine whether H 2 O 2 is generated in the rER of the subaleurone cells.…”