Design And Application Of Improved H₂S Fluorescent Probe Based On Chromophore

Hydrogen sulfide?H₂S?has been considered a toxic gas molecule with a smell of rotten eggs.However,more and more research refreshed the knowledge of traditional H₂S toxicity.H₂S,a "infamous" compound,was re-recognized as the third multifunctional endogenous gas signaling molecule.H₂S is mainly produced in mammals through enzymatic reactions,including four major cystathionine ?-syntheses?CBS?,cystathionine ?-lyase?CSE?,and 3-mercaptopyruvate sulfotransferase?3-MST?and cysteine aminotransferase?CAT?),thereby participating in the complex physiological and pathological processes in life activities.H₂S can participate in physiological processes such as regulating insulin release,inducing angiogenesis,and protecting neurons from oxidative stress.However,abnormal changes in H₂S levels are also associated with many diseases,such as cirrhosis,Alzheimer's disease,diabetes,Down's syndrome,and hypertension.Therefore,in order to better understand the complex performance of H₂S under physiological and pathological conditions,monitoring and imaging changes in H₂S remains critical.Fluorescent biological imaging technology has become an indispensable tool for biological research due to its advantages such as high sensitivity,good selectivity,non-invasiveness,real-time and in situ imaging detection.H₂S is a biomolecule with unique chemical properties.It can be used not only as a good reducing agent,but also as a good nucleophile.According to reaction type of the nucleophilic reaction and reduction reaction between the probe and H₂S,we have made improvements on the fluorescent chromophore and developed three H₂S fluorescent probes 2-1,3-1,and 4-1.The detection performance of the probes was studied by means of ultraviolet,fluorescence spectrum and laser confocal microscope imaging.The specific research work is as follows:1.We designed and synthesized the sophorone-based orange emission probe 2-1.Two electrophilic centers of disulfide and ester bond were introduced into the probe 2-1.H₂S advances the disulfide bond to obtain an intermediate with-SSH,and then the intermediate undergoes a second nucleophilic reaction in succession to form benzodithiol ketone,simultaneously releases a fluorophore with a fluorescence signal at 580 nm.The probe 2-1 showed a large Stokes shift?155 nm?and high sensitivity?the detection limit of 88 n M?.In addition,the probe 2-1 was used to monitor exogenous and endogenous H₂S in living cells.However,probe 2-1 also has certain defects,such as poor water solubility and short fluorescence emission wavelength.2.On the basis of probe 2-1,we improved the detection performance of fluorescent probes by improving the fluorophore.We used a positively charged cyanine?Cy7?as a red fluorophore and synthesized a long-wavelength fluorescent probe 3-1.After reacting with H₂S,the fluorescence signal released in the red light range?the highest emission peak is at 639 nm?.The probe 3-1 showed good water solubility and low detection limit?36 n M?.In addition,the ability of probe 3-1 to target mitochondria of subcellular organelles was investigated.Importantly,probe 3-1 has been successfully applied to imaging H₂S in living mice.3.We modified methylene blue as a near-infrared?NIR?emitting fluorophore and selected an azide group as the H₂S reduction site,and synthesized a near-infrared probe 4-1,which can better avoid interference from other sulfur-containing substances.After the azide group of probe 4-1 is specifically reduced to amino group by H₂S,the intramolecular self-fragmentation finally released the methylene blue fluorophore,and the fluorescence signal fell in the near infrared region?687 nm?.The detection limit of probe 4-1 for H₂S is 0.32 ?M.The complex relationship between Ca²⁺ and H₂S was studied using probe 4-1.