Intracellular Small Molecule Thiol Compounds Detected Near-infrared Fluorescence Probes

There are a variety of small thiol compounds that are indispensable in theorganisms. These small thiol compounds, mainly including cysteine (Cys),homocysteine (Hcy), and glutathione (GSH), varied in their concentration andphysiological functions. Cys, as the only natural amino acid that possesses freesulfydryl, is important for the protein synthesis. Hcy is mainly originated from themetabolic transformation of methionine. GSH is in the highest levels (1-10mmol/L)of non-protein thiol compounds in cells which plays important roles in redox balancemaintenance, exogenous toxins elimination, signal transduction and gene regulation.The abnormal levels of those small thiol compounds usually induce many diseases,such as slow growth, edema, cardio vascular disorder, Alzheimer’s disease, andcarcinogenesis, etc. Therefore, it’s of great significance to explore the production,distribution and level fluctuation of small thiol compounds for better understanding oftheir impacts on cell viability and physiological and pathological conditions of livingorganism.Probe-based fluorescent imaging has been effective tools for studies onphysiological and pathological functions of thiols through direct and dynamicobservation of their production and distribution at the cellular level. Fluorescentprobes for small thiol compounds developed in the recent years fall into three groupsaccording to their analytical targets, including probes specific for one single thiol,probes for two thiols, and probes for the total thiols in the cells. As can be seen, mostfluorescent probes that specific for one single thiol fluoresce in the visible region.Moreover, few near-infrared (NIR) probes for differentiating two or more thiols arereported.Inspired by this, in this dissertation, a series of NIR fluorescent probes that specificfor one single thiol and two-thiol differentiation are synthesized and screened. Theprobes combine heptamethyl cyanine (Cy.7.Cl) as the NIR fluorophore with variousbenzene derivatives, such as phenylamine, phenol, phenthiol, nitrophenol (o-, m-, p-),2,4-dinitrofluorobenzene and4-nitrothiophenol as the leaving groups. Based on the difference in nucleophilicity of thiol towards Cy.7.Cl median carbon, and theeletrophilicity of Cy.7.Cl median carbon that modulated by the various leaving groups,as well as the steric-hinerance effect and the different molecular structure of thiolcompound, GSH, Cys, and Hcy can be specificly detected. After investigation on thespectra properties and reaction dynamics, two type of probes have been screened andselected:(1) probes specific for GSH, including Cy-2-NO2and Cy-Ph-Ph;(2) probesspecific for Cys/GSH, namely Cy-3-NO2.The recognition mechanism of the selectedprobes for GSH and Cys/GSH were studied by MS and LC-HRMS methods. Inaddition, the probes were applied to the in vitro imaging of thiols in living cells. Theresearch results shown here not only provide new NIR probes for the biological thiols,but afford useful information about the interpretation of the nucleophilicsubstitution mechanism of biological thiols.