Synthesis And Biological Application Of Fluorescent Probe For Detecting Seleno Alcohol In Living Cells

Selenium is one of the indispensable trace elements in human body, and plays an importantrole in many of the key of the body metabolism in the process. For example, Selenium canmaintain the body’s redox state, and can protect the body’s immune system against infringement,and can prevent the cancer, etc. Selenium plays an important physiological role in vivo, it ismainly because the form of selenocysteine(Sec) incorporating into a variety of selenoproteins(SePs). Selenocysteine is the21st amino acid, and is the most of selenols. It is similar tocysteine, both of them are important reducing substance, except that it has the selenium atomin place of the sulfur atom in Cys. Whereas, up to now, physiological functions of selenols andselenoproteins have been rarely reported due to the lack of an available in vitro and in vivomethod to assess the contents of Sec and SePs. It is mainly because the selenol is not verystable, and is more lively than thiol. Thus there is a clear need to identify different Se speciessuch as Sec in vivo in order to investigate their biological functions.The biological effect of selenium has a dual nature, both the anti-cancer effect and thecancer prevention. On the one hand, the deficiency of selenium can cause the abnormalreaction of the body, on the other hand, the high concentrations of selenium can cause toxicreaction. The nutritional supplement of selenium is mainly organic selenium and inorganicselenium. The inorganic selenium supplements are mainly sodium selenite, taken by oral orinjected to supply. After taking sodium selenite tablets in the body, most of the sodium selenitewere metabolized selenols, or converted to methyl selenide, or excreted through urine or sweat,etc. However, about the way in which sodium selenite plays the role of cancer prevention ortreatment is still without a clear research mechanism.Fluorescence detection is regarded to have both high sensitivity and high spatiotemporalresolution for visualizing biomolecules in vitro and in vivo, and is developed rapidly in recentyears. In consideration of the important physiological of selenium and anticancer mechanism ofconstant exploration, it is important to design a kind of method to detect selenols in living cells.Based on theses, we designed and synthesized a fluorescence probe to image selenol in livingcells, and applied the probe to biological systems. We carried out the following two aspects: Fistly, we found the2,1,3-benzoselenadiazole (BS) can react rapidly with selenocysteine(Sec), but not with cysteine in phosphate buffer solution (pH7.4). Then designed andsynthesized a new type of organic selenium fluorescence probe (HB) based on the nucleophilicaddition reaction between selenol and2,1,3-benzoselenadiazole (BS). We integrated BS moietyinto a hemicyanine dye because of its low toxicity and good solubility in water. At last, theexperimental data showed that fluorescent probe HB exhibited an obvious fluorescenceintensity enhancement upon reaction with selenol under the physiological conditions, and has agood linear and selectivity. Whereas the responses toward thiols and amino acid, reactiveoxygen species, reducing agents were negligible. The regression equation was F=104.9+14.7[Sec] μM with a linear coefficient of0.9942. The limit of detection was7.0nM (standarddeviation3.5%, n=11). And the probe has a good light stability, so it showed excellentperformance. We analysed the spectral characteristics of the fluorescence probe in physiologicalconditions.Secondly, we set up an appropriate biological model using the probe to the cellular leveland the living level. We first applied HB to visualize the intracellular Sec. The results revealthat the probe could monitor the dynamic changes of Sec level, and (CysSe)2could betransformed into Sec in living cells. Next imaging of Sec in living mice was carried out, whichdisplays that selenol level in vivo can be successfully assessed by the probe. The results showthat HB is a highly selective and sensitive probe for Sec in physiological conditions and is ableto monitor the changes of selenol level in living cells and in vivo. Furthermore, we haveobserved that selenol concentrations increase gradually in HepG2cells induced by Na2SeO3in atime-and dose-dependent manner, with accompanying tumor cell apoptosis, cell cycle arrestand depletion of mitochondrial membrane potential.