Development And Utilization Of Fluorogens For Direct Profiling Of Cystathionine β/γ-Lyase Activity

In mammals and nonmammals,cystathionine lyase,as key enzymes involved in the pathways of transsulfuration and reverse transsulfuration,regulates a varity of physiological and pathological processes and plays an indispensable role in many diseases.Therefore,it is of great significance to study the changes of cystathionine lyase activity in organisms for the evaluation and treatment of related diseases.Although many current studies have discussed the changes of hydrogen sulfide/cystathionine lyase system in various diseases,the detection of cystathionine lyase in chemical biology is a major challenge.The traditional chemical colorimetry and methylene blue method can not be applied in biological system due to their limitations.Fluorescent probe is conside to be a promising tool for biological application owing to its high sensitivity,simple operation and low toxicity.However,the existing fluorescent probes of cystathionine lyase are ultilized to indicate the cystathionine lyase expression through the content of H2S.Regulated by multiple factors in complex biological environment,the results presented by H2S are inaccurate and unreliable to some content.At present,there are no reports about fluorescent probes for direct dectcting of cystathionine lyase in biological systems.Based on this,we first attempted to design and develop a novel fluorescent probe with effective and precise,in direct monitoring cystathionine lyase in complex biological samples.The research content of this paper are as follows:1.Based on the slectivity of cystathionine β-lyase（CBL）for catalytical cracking cystathionine in vivo,a novel fluorescent probe（CBLP）was constructed by utilizing 1,8-naphthalimide（NI）as scaffold,cysteine as recognition group,and a carbamate ethyl sulfide group as connector.The structure of CBLP was characterized by 1H NMR,13C NMR and high-resolution mass spectrometry,and the variation of UV-vis,fluorescent spectrum and liquid mass spectrum illustrate the feasibility of this design strategy.CBLP can specifically recongnize without the influence of other active substances,and display an excellent linear response to CBL.Besides,the results of cell experiment also indicate that CBLP has good biocompatibility.Thus,CBLP was applied to analyze the expression levels of CBL in the roots of wild-ype arabidopsis thaliana and two mutants（cbl knockout:SALK₀14740C,overexpressed:OE-CBL）.The results maintained that CBLP dipalyed high sensitivity to the changes of CBL and revealed the relationship between plant growth and CBL content.2.Considering the catalytical principle of cystathionine y-lyase（CSE）,we designed and synthesized a new and activatable fluorescent probe CSEP to detect the CSE directly in living organisms.According to the density functional theory,we found that the probe has remarkable intramolecular charge transfer（ICT）characteristics.The emission wavelength of CSEP is 470 nm,which redshifted to 540nm after CSEP reacts with CSE due to the ability of CSE to specifically cleave C-S bond of CSE and subsequently thiol-induced NH-COO-β-S decomposes and eventually releases product（NI-NH2）fluorescent signals.The probe has the advantages of high selectivity,low detection limit and low cytotoxicity.Employing this probe,the CSE in HCC-LM3 cells and zebrafish liver and abdomen have been successfully labeled,and the fluorescence intensity at 540nm rapidly decreased indicating the variety of CSE activity after appending inhibitors.Additionally,CSEP can successfully distinguished the changes of CSE content in liver tissue frozen sections of sepsis rats induced by sham and cecal ligation puncture（CLP）due to the high sensitivity of CSEP,revealing the up-regulation of CSE level in liver tissue of sepsis rats,which was verified by western blot and immunohistochemical analysis.In brief,the development of CBLP/CSEP promotes the research of cystathionine lyase activity.This design tactic not only realizes the visual analysis of cystathionine activity in complex bioprocess and the semi quantitative profiling of cystathionine lyase in heterobiological samples,but also provides the theoretical basis and reference for the further development of accurate and efficient methods for the direct detection of cystathionine lyase.