Design,synthesis And Application Of Multifunctional Probe For Protein S-sulfenylation

Reactive oxygen species(ROS),which is formed upon incomplete reduction of O2,plays a role of "antiheroes" in both physiology and pathology.An appropriate amount of reactive oxygen species can interact with biological molecules to trigger a series of signal transduction processes and exhibit different physiological functions.High concentrations ROS can cause protein peroxidation.DNA damage and lipid peroxidation to cause oxidative stress.The continuous increase of ROS is also closely related to many diseases,such as atherosclerosis,hypertension,diabetes,and further into stroke or cancer.As a post-translational modification,Protein S-sulfenylation is mainly occurred by ROS oxidizing protein cysteine sulfhydryl.As a metastable intermediate,Protein S-sulfenylation can regulate protein function and maintain redox balance,including a variety of signal transduction and cellular processes.And these cellular processes are also closely related to the pathology of many major diseases and drug design.Because of the fate of protein S-sulfenylation in a dynamic and short-lived state,most of the existed tools for protein S-sulfenylation are biotin affinity probes and fluorophore tethered(not fluorescent responded)probes with single function,which hindered our understanding of this post-translational modification spatially and temporally.Therefore,we are committed to developing a new orthogonal reaction between sulfenic acid and hydrazine derivatives.Based on this reaction,a new tool was designed and synthesized to study protein S-sulfenylation in living cells and tissues spatially and temporally.The main research contents are as follows:1.Due to the electrophilicity of sulfur atom in sulfenic acid,a series of hydrazine nucleophilic reagents(1a-8a)with different substituents were designed and selected to react with sulfenic acid,and the best model substrate(5a)was selected according to the reaction effect.The model substrate(8a)containing fluorescent group was determined according to the best model substrate(5a)and the reaction product with sulfenic acid.The reaction mechanism was deduced and verified based on the reaction products,at the same time,the model substrate(8a)can react with sulfenic acid and release fluorescence.The model substrate(8a)was used to study the selectivity.Finally,it was confirmed that 8a had better chemical selectivity under TCEP conditions,and it could respond quickly to the sulfenic acid and release fluorescence at the same time.2.Based on the model substrate(8a),a multifunctional fluorescent probe SA-HYD was designed and synthesized.It was verified that the probe SA-HYD responded to the small molecular sulfenic acid in solution.The fluorescence intensity of SA-HYD showed a good linear relationship with the concentration of sulfenic acid.It could specifically react with sulfenic acid with good selectivity.According to the kinetic data,it was found that the probe SA-HYD could respond quickly sulfenic acid.3.The "OFF-ON" labeling effects of probe SA-HYD on HSA,BSA and β-lactoglobulin were studied,including the effects of H2O2 concentration and oxidation time on protein S-sulfenylation.Then,the reaction sites and labeling sites of β-lactoglobulin sulfenic acid were identified and characterized by mass spectrometry.The distance between the labeled sites and reaction sites,as well as the four cysteines in β-lactoglobulin cavity to Val43.Val41,and Tyr42 were calculated.The response mechanism of probe SA-HYD with protein S-sulfenylation were described.The probe SA-HYD was used to study the response and labeling of protein S-sulfenylation in HeLa cells under three different conditions of H2O2,TCEP and NAC,and on this basis,GAPDH-SOH was verified.4.Flow cytometry and laser confocal were used to analye and image the protein sulfenic acid in living cells quantitatively,including H2O2 stimulation.TCEP/NAC inhibition,and subcellular distribution of protein S-sulfenylation in HeLa using co-stained with nuclear dyes and mitochondrial dyes.5.Meanwhile,the probe SA-HYD was used to stain prostate cancer cells PC-3 and DU 145 by LPA stimulation,in order to study the signal transduction of LPA/H2O2 in prostate cancer cells.Subsequently,the probe SA-HYD was used to study the oxidating rate and degree of protein cysteine sulfhydryl under different concentrations of H2O2.And the mechanism of protein cysteine sulfhydryl to protein S-sulfenylation was explained.Finally,the model probe 8a was used to analyze the content of protein sulfenic acid/formaldehyde in different cells,and the model probe 8a was used to study the content of protein sulfenic acid/formaldehyde in Arabidopsis roots at different growth stages.