Construction And Application Of Fluorescent Molecular Probes For Detecting Biothiol

BackgroundIntracellular bio-thiols,such as glutathione?GSH?,cysteine?Cys?and homocysteine?Hcy?,because of their similar structures,play a key role in maintaining control of redox homeostasis and the higher structure of proteins.For example,Abnormal Cys level can cause edema,etc.On the other hand,elevated plasma Hcy is a risk factor for alzheimer's disease,cardiovascular disease and osteoporosis.And GSH is closely related to leukopenia,cancer and HIV infection.Therefore,the important biological role of bio-thiols has aroused great interest in the development of useful chemical tools to detect bio-thiols.PurposeA series of fluorescent probes were designed and synthesized with anthracene and naphthalene as fluorescent groups.Cys,GSH and Hcy were specifically detected from 20amino acids?cysteine?Cys?,arginine?Arg?and homocysteine?Hcy?,proline?Pro?,glutathione?GSH?,lysine?Lys?and glutamate?Glu?and glutamine?Gln?,serine?Ser?,aspartic acid?Asp?,phenylalanine?Phe?,leucine?Leu?,isoleucine?Ile?,glycine?Gly?,valine?Val?,methionine?Met?and histidine?His?,alanine?Ala?,tryptophan?Trp?,threonine?Thr??by a series of in vitro tests including UV-vis,fluorescence,electrochemistry and circular dichroism?CD?,and the mechanism of action was explored.Combined with the GSH content,recovery rate and cytotoxicity test,the practical fluorescent probes with fast detection,good selectivity,high sensitivity,and low toxicity were screened,which provided a basis for the further detection of biothiols.Methods1.Construction of fluorescent probes:AProbes were prepared by chemical methods such as nucleophilic addition and coordination,and characterized by high resolution mass spectrometry?HRMS?,nuclear magnetic resonance spectroscopy?¹H NMR?and other methods.2.In vitro thiol detection:Through UV-vis,fluorescence,electrochemistry,CD,etc.,the absorption and emission spectra,limit of the detection,time response,cyclic voltammetry curve changes,structure changes and fluorescence life changes of the interaction between amino acids and probes were explored.The non-linear least square curve fitting was used to calculate the binding constants of the host and the guest,and electrochemical,CD,IR and other methods are used to analyze and verify the possible mechanism of the host and guest.3.Application of drugs:The concentration standard curve of the interaction between the probe and GSH was measured,and the content of GSH in atopolam was obtained by fitting with the linear regression equation,and then the recovery test was carried out.The experimental results were compared with the pharmacopoeia to study the practical application value of the probes.4.Cytotoxicity evaluation:The toxicity of fluorescent probes to HepG2 cells for 24 h was measured by MTT method,so as to screen out the probes with low toxicity and appropriate drug concentration.Results1.The 9 compounds?HNANA,ABSA-1,ABSA-2,APA-1,APA-2,APA-3,APA-4,APA-5 and APA-6?were synthesized successfully,characterized by ¹H NMR and HRMS.2.In vitro detection method was used to find that 6 fluorescent probes(HNANA-Cu²⁺,ABSA-1-Cu²⁺,ABSA-2-Cu²⁺,APA-1-Cu²⁺,APA-2-Cu²⁺and APA-3-Cu²⁺)had specific detection for Cys,GSH and Hcy.The binding constant can reach 10^6-11,and the detection limit can be as low as 0.95?mol·L^-1.After the addition of biothiols,it was found that the UV-vis spectra and IR spectra of the HNANA-Cu²⁺and ABSA-Cu²⁺series probes were restored,while the spectra of APA-1-Cu²⁺,APA-2-Cu²⁺and APA-3-Cu²⁺probes has changed.3.When the probe was applied to atopolam,the detection percentage of GSH by 6probes reached above 99%and the recovery rate reached above 98%.4.MTT assay showed that the probe had little cytotoxicity to HepG2 cells in the range of 0-150?g·mL^-1.Conclusion1.The 9 kinds of fluorescent probes were successfully prepared.2.The 6 kinds of probes were screened for their high selectivity and sensitivity to Cys,GSH and Hcy.The possible mechanism of action between the host and the guest was divided into two types:HNANA-Cu²⁺,ABSA-1-Cu²⁺,ABSA-2-Cu²⁺were reduced to ABSA-1,ABSA-2,HNANA,and Cu²⁺was released to form a complex with biothiol;the reaction between the probes(APA-1-Cu²⁺,APA-2-Cu²⁺,APA-3-Cu²⁺)and the biothiol was a complex addition reaction,and the added biothiol was directly complexed with Cu²⁺in the probes.3.When the probes were applied to atamolam,the detection percentage and recovery rate of GSH reached the requirements of the Chinese pharmacopoeia.4.The probes showed little toxicity to HepG2 cells and can be tested in the next step.