Cysteine Fluorescent Probes Design And Their Bioimaging Applications

As the cellular sulfur metabolism center,cysteine?Cys?is the unique amino acid that containing the reduced thiol in the twenty natural amino acids.In animal cells,Cys is involved in a large number of life processes such as peptide synthesis and folding,redox balance control,cell signaling,detoxification and apoptosis,which maintained the splendid biological system.Generally,the concentration of Cys is exquisitely regulated in living body.Both elevated and reduced level of Cys in living body is closely related to a variety of major diseases such as cardiovascular and cerebrovascular diseases,neurodegenerative diseases and cancers.To value the concentration change of Cys in living cells and deepen its biological roles,we,in this paper,developed a series of Cys specific fluorescent probes based on the pH-regulated nucleophilic reaction activity gradient of Cys,homocysteine?Hcy?and glutathione?GSH?.Utilizing these probes,we realized the in situ lysosomal Cys specific imaging and the cellular Cys metabolism.The thiol nucleophilic reaction activities of the three biothiols in vivo and in vitro are studied meticulously.The pH-regulated Cys probes can be used for Cys dynamic imaging specifically which hold great potential in the pathological study of Cys.Specific research works are as follows:1.Incipiently,we synthesized two chromene derivates 2-1-CH₃ and2-1-Br to detect thiols in aqueous solution.Probe 2-1-CH₃ displayed supra specificity towards Cys with Turn-On fluorescent responses.In the spectroscopic study,we found that the rise of pH in the detection system would accelerate the reaction rate of the probe and Cys.Combined with the detection mechanism study,we thought that pH could regulate the nucleophilic addition reactivity of Cys during the Michael addition reaction between Cys and?,?-unsaturated ketone.2.Based on the pH regulated strategy,we further developed a coumarin derivate 3-1 to detect thiols with acryloly group as the reaction site.Indeed,the fluorescent responses of 3-1 toward Cys and Hcy were pH related.In pH 7.4 system,the probe could detect Cys specifically with Turn-On green fluorescent response.However,in pH 7.8 system,the firstly enhanced green fluorescent emission quenched subsequently while a new yellow fluorescent emission appeared.In the pH 7.8 system,the reaction between probe and Hcy displayed Turn-On green fluorescent response.Thus,in pH 7.8 system,the probe could detect Cys and Hcy distinctively through two emission channels.The detection mechanism was established to be the pH regulated continuous nucleophilic addition reaction between–SH and acryloly group and intramolecular nucleophilic substitution reaction between–NH₂ and ester group.The pH regulated fluorescent responses of probe toward thiols demonstrated that pH rise could enhance the nucleophilic reaction activities of both–SH and–NH₂in Cys.3.Instead of the widely used–SH initiated multi-stage reaction processes between thiols and probe to realize Cys special detection by unique fluorescent signal,we now utilize the–SH different nucleophilic reaction activities of Cys,Hcy,and GSH to result in exclusive reaction between Cys and probe in acidic system which can effectively avoid the probe consumption induced by other–SH contained species in coexist systems.Opportunely,lysosome with acidic internal environment builds up supra platform for the specific reaction.Cellular experiments demonstrated the specificity of the probe toward lysosomal Cys and the in situ lysosomal Cys detection process.Utilizing time dependent fluorescent imaging of the probe labeled A549 cells,we demonstrated that dexamethasone induced A549 apoptosis process was lysosomal Cys independent.These results may provide preliminary information about pathogenesis associated with Cys and lysosome.4.Fluorescent probes as non-invasive tools to visualize the metabolism of bio-molecules hold great potential to explore their physiological and pathological processes.As one of the intracellular Cys concentration control path,the catabolism of Cys could produce SO₂ with the catalysis of cysteinesulfinate decarboxylase and aspartate aminotransferase.To image this process,we developed a coumarin derivate based on the rational design of the dual recognition sites for Cys and its metabolite SO₂,respectively.The probe displayed distinct two channels turn-on fluorescent emission towards Cys and SO₂,which were successfully applied for both A549 cells and zebra fish imaging.Further,with reversible fluorescent responses towards Cys,the probe could image the enzymatic conversion of Cys to SO₂ in living A549 cells though ratiometric manner.The present work reported the first probe to image the endogenous generated SO₂ without incubation of the SO₂ donors.