Construction Of Novel Rhodol Fluorescent Probes For Reactive Sulfur And Their Biological Application

Sulfur metabolism is essential for cell growth and survival.In biology,sulfur exists in a wide range of oxidation states,and almost all valences from+6 to-2 occur in normal cells.Almost all oxidation states of sulfur can be converted to each other,and the unique reactivity of these oxidation states is a major feature of sulfur biology.The lowest oxidation states of sulfur include cysteine（Cys）,homocysteine（Hcy）,glutathione（GSH）and hydrogen sulfide（H₂S）,and so on.They are an important reactive sulfur species（RSS）in living organisms.In recent years,research on the biological function of RSS has become a hot spot.RSS are important antioxidants and free radical scavengers in living organisms.H₂S is an important signaling molecule in living organisms.Biothiols can maintain the redox homeostasis of cells.Therefore,in order to further study the physiological functions of these reactive sulfur species,it is necessary to develop reliable detection methods to monitor and track them.Small-molecule fluorescent probes have attracted great attention from researchers due to their excellent performance such as high sensitivity,fast response,real-time monitoring,and low cost.In this paper,two organic small molecule fluorescent probes with novel structure for detecting active sulfur species（Cys and H₂S）were designed and synthesized successfully.The specific work contents are as follows:（1）In chapter 2,we developed a novel near-infrared fluorescent probe Cys-WR with a large Stokes shift（about 110 nm）,which is composed of Rhodol near-infrared fluorophore WR-4 and cromarate,of which cromarate is the response site of Cys.The probe can detect Cys in a phosphate buffer solution,which makes it suitable for biological imaging systems.Probe Cys-WR shows off-on response to Cys in a wide linear range of 0-100μM,and the color of the solution is observed from the dark pink to brown with the naked eye.The cytotoxicity study found that the probe Cys-WR has low toxicity to human Non-small-cell carcinoma cell A549.The results of cell and in vivo imaging showed that the probe Cys-WR could image Cys in cells and zebrafish in vitro and in vivo.The experimental results in this chapter prove that the probe Cys-WR has great potential for dynamic monitoring of Cys concentration in Cys-related diseases.（2）H₂S is both nucleophilic and reductive.In recent years,researchers have reported many H₂S probes based on the chemical properties of H₂S above.However,the biggest challenge in constructing H₂S probes is how to eliminate the potential interference of other biothiols（Cys,Hcy,and GSH）.To solve this problem,in Chapter 3,we designed and synthesized two Rhodol fluorescent probes SNARF-SSPy and SNARF-Se SPy for H₂S detection.Spectral experiment results show that the probe SNARF-Se SPy can show excellent anti-interference performance through the dual selenium-sulfur exchange reaction even in the presence of high concentration of thiols,while the probe SNARF-SSPy can’t detect H₂S under the same conditions.In addition,the probe SNARF-Se SPy can respond to H₂S quickly and with high sensitivity,and its detection limit is as low as 34 n M.Cytotoxicity studies found that the probe SNARF-Se SPy was very toxic to cells A549.Cell and in vivo imaging results indicate that the probe SNARF-Se SPy can image H₂S in cells and zebrafish in vivo/in vitro.The experimental results in this chapter prove that the probe SNARF-Se SPy can be used for the detection of H₂S in complex organisms,and has potential for the diagnosis of H₂S-related diseases.