A fluorescent probe for rapid detection of thiols and imaging of thiols reducing repair and H₂O₂oxidative stress cycles in living cells

Abstract

A diselenide containing fluorescent probe based on a fluorescein scaffold for thiols was developed. The fluorescent probe exhibited rapid response, high selectivity and reversibility. Confocal fluorescence microscopy was used to visualize the redox changes mediated by thiols and reactive oxygen species in living HeLa cells.Intracellular thiols play an essential role in maintaining redox homeostasis by regulating the redox status between reduced free thiols and oxidized disulfides.1 Glutathione (GSH), which has been identified as the most abundant non-protein thiol, is considered to be the main player in combating oxidative stress and regulating the redox environment of the internal cellular compartments. Further development of intracellular thiol detection methods is necessary to understand the complicated roles of thiols in redox biology and elucidate the pathogenesis of various disorders in living systems. Compared with other approaches, fluorescence imaging is less invasive, more sensitive and more convenient. 11Numerous fluorescent probes for thiols based on Michael addition, aldehyde cyclization, fluorophore deprotection, and thiol-disulfide exchange protocols have been reported. 2,12 However, very few of these probes react with thiols rapidly enough to achieve real-time detection in vivo. And most of these probes can only respond to thiols statically. Research has shown that fluorescent probes which are capable of responding to thiols and ROS reversibly offer better chances of visualizing redox signaling, stress, and thiols repair. Real-time methods for measuring thiols in vivo based on redoxsensitive green fluorescent protein have been reported. 1,14 However, such techniques require gene manipulation while redox-responsive probes based on small molecules are scarce. 13,15Glutathione peroxidase (GPx) is a selenoenzyme that protects various organisms from oxidative damage by catalyzing ROS reduction in the presence of GSH. 16 To clarify the catalytic mechanism of GPx and develop antioxidant drugs, researchers have synthesized many organoselenium compounds as GPx mimics, in which monoselenides, diselenides, and ebselen analogs are the major ones. 16,17 Steinmann et al. 18 recently reported that the cleavage of diselenide bonds by thiols is 5 orders of magnitude faster than that of disulfide bonds. Building on these results, we designed and synthesized a diselenide-based fluorescent probe (FSeSeF, Scheme 1) for the rapid detection of thiols and redox changes mediated by thiols and ROS in living cells. FSeSeF is a weakly fluorescent molecule that contains a diselenide bond as a fast, reversible recognition center for thiols and two fluoresceins as signal reporters. Once FSeSeF is cleaved by GSH, both the selenenyl sulfide FSeSG and the Scheme 1 Structure of FSeSeF and the nucleophilic attack by GSH at Se-Se bond with the generation of selenenyl sulfide FSeSG and selenol FSeH.

A reversible fluorescent probe for detecting hypochloric acid in living cells and animals: utilizing a novel strategy for effectively modulating the fluorescence of selenide and selenoxide

“…11 Until now, even though several fluorescent probes for HOCl 12 have been reported, few reversible fluorescent probes for visualizing the redox changes mediated by HOCl and antioxidants have been developed. 13 Here we present such a reversible fluorescent probe for HOCl.…”

A ratiometric fluorescent probe for cysteine and its application in living cells

“…The results revealed that probe DPCA was able to be applied to complicated conditions with negligible interference from these species ( Figure S4). DPCA displayed good selectivity toward Cys, which outweighs numerous reported probes [60][61][62].…”

An arylboronate-based fluorescent probe for selective and sensitive detection of peroxynitrite and its applications for fluorescence imaging in living cells

“…Finally, condensation of S1 and 2-benzothiazoleacetonitrile in the presence of piperidine provided probe 1 with the yield of 80%. [38,39]. These results suggest that probe 1 holds the big potential for real-time detection of ONOO  in biological systems.…”