To Detect The Intracellular Redox State Reversible Design Synthesis And Biological Applications Of Near Infrared Fluorescent Probe

Redox homeostasis is extremely important to maintain the normal physiological functions withinthe organism’s cells .The redox state of the cells is primarily a consequence of the precisebalance between the level of reducing equivalents and reactive oxygen species (ROS) .Thethiols are one of the most important reducing substances within the organism cells .Thiols ,suchas glutathione(GSH)and cysteine(Cys),which are components of many proteins and simplemolecules, play an important role in the cellular antioxidant defense system. Numerous studiesindicate that many diseases are heavily dependent on the concentration levels of thiols in vivo.ROS plays an important role in intracellular signal transduction, on the other hand, unregulatedproduction of ROS results in potentially cytotoxic oxidative stress. Many diseases (e.g.Alzheimer’s disease) are linked with ROS damage as a result of an imbalance between radical-generating and radical-scavenging systems and thus harmful to cells, and many diseases areclosely-linked. The reversible oxidation biology of cells provides broad implications inphysiological and pathological fields. Therefore, the analytical methods that can be used to studythe dynamic redox chemistry in living cells are attractive and draw sustained attention. In thisregard, fluorescence detection or imaging with redox-responsive probes is a potentially powerfulapproach because of its merits of easy visualization, simple operation, and in vivoapplication.Fluorescence analytical methods for the advantage of intuitive, simple, and in situ, particularly combined with modern imaging techniques have been widely used in the field of bioanalytical.So development of new fluorescent probes for detection of intracellular redox state in dynamic, transient, reversible, is of great significance.Based on the thiols can specifically recognize the selenium-nitrogen bond of ebselen, andafter oxidation by H2O2the five-membered ring structure recovery. We have designed andsynthetized the novel reversible organoselenium fluorescent probes for reversible monitoring ofGSH and H2O2. We carried out two aspects of investigation:First, we report the synthesis and biological application of Cy-O-ebselen, a new near-infrared(NIR) organoselenium probe for selective detection of GSH and imaging redox cycles in livingcells. Our strategy is to take advantage of protective effect of selenide against GSH and theredox chemistry of selenium moiety to design a reversible fluorescent probe. Cy-O-ebselen features a reversible response to GSH and H2O2under physiological conditions, and exhibitsgood selectivity for GSH. Fluorescence imaging experiments by confocal microscopy show thatCy-O-ebselen can be applied to the imaging of multiple cycles of oxidative stress and reductiverepair HepG2 cells and it exhibits low toxicity, good membrane permeability. In addition,themaximum emission wavelengths are in near-infrared region, which could effectively avoidbackground fluorescence interference in biological samples.Therefore, this probe can provides anew approach and methods to research the physiological function of intracellular redoxregulation and signal transduction.Second, the near-infrared reversible Cy-O-ebselen is applied to detect the changes of redoxstate in HepG2 cells during apoptosis. By Western-blot analysis, nuclear staining, mitochondrialmembrane potential method for monitoring the signal indicators in this apoptotic process. Theexperimental results show that changes in redox state in HepG2 cells can indeed lead toapoptosis, and we probe can visual monitor this process. In addition, Cy-O-ebselen is applied toreal-time monitor the reversibility of the apoptosis in hepatoma cells and liver cells caused bythe redox environment changes. The results show that apoptosis can be reversed in hepatomacell, and normal liver cells is no such phenomenon.In short, the change in redox state caused bythe level of GSH levels in the process of apoptosis plays a crucial role in apoptosis, and the Cy-O-ebselen can provide a new tools to research the function of the changes of redox state insignal transduction and apoptosis .