Design,Synthesis And Properties Of Coumarin-based Fluorescent Probes

Biothiols,as basic biologically active sulfur species,play important roles in many key physiological processes.Intracellular biothiols play key roles in various physiological activities such as protein synthesis,cellular metabolism,signal transduction,and regulation of oxidative stress.Abnormal cellular levels of biothiols are closely related to many human diseases,so it is particularly important to realize real-time quantitative monitoring of biothiols in humans.Since biothiols such as cysteine(Cys),homocysteine(Hcy),and glutathione(GSH)all contain sulfhydryl groups and are structurally similar,it is a huge challenge to distinguish them.The synthesis and properties of biological thiol probes are of great significance.Coumarin has excellent biocompatibility,optical properties and stability.In this paper,four new fluorescent probes were designed and synthesized using coumarin as the parent fluorophore.The performance is systematically studied,and the specific contents are as follows:1)The probe(2E,4Z)-2-(benzo[d]thiazol-2-yl)-5-chloro-5-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)penta-2,4-dienenitrile(BCD)was synthesized by combining coumarin with benzothiazole-acetonitrile and heating under reflux in dichloromethane for 12 h.The probe has four reaction sites,and according to the different binding mechanisms between the probe and biothiols and hydrazine,it shows fluorescence turn-on,which is used to distinguish Cys,Hcy,GSH and hydrazine.After adding Cys,the fluorescence intensity of probe BCD at 502 nm increased by 760 times;after adding Hcy,the fluorescence intensity of BCD at 479 nm increased by 8 times;after adding GSH,the fluorescence intensity of probe BCD at 476 nm increased by 6 After adding hydrazine,the fluorescence intensity of probe BCD at 458 nm increased 637 times.The effect of pH on the fluorescence properties of BCD and the selectivity of the probe BCD show that the probe is very stable in the pH range of 1.55～11.77,and the probe has high specificity for biological thiols and hydrazine,so the probe BCD is expected to be promising.It will become an effective tool for the detection of biological thiols in cells and organisms or the detection of hydrazine in actual samples in the future.2)The probe 2-((1E,3Z)-4-chloro-4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)buta-1,3-dien-1-yl)-1,1,3-trimethyl-1 H-benzo[e]indol-3-ium(CTI)was synthesized by combining coumarin with methyl-hemicyanine and heating under reflux with acetic acid as a catalyst in absolute ethanol for 5 h.The maximum absorption wavelength of the probe CTI was at 592 nm,and the blue-shifted to about 530 nm after the addition of biothiol,and the change was obvious.It took 40 min,60 min and 80 min for the probe CTI to react completely with Cys,Hcy and GSH,respectively.Probe CTI reacted quickly with HSO3and S2-,which took 30 min and 10 min,respectively.Adding biological thiol or HSO3-,S2-,the fluorescence intensity of probe CTI only increased by 2～3 times.The effect of pH on the fluorescence properties of CTI shows that the probe is suitable for the detection of biological thiols or HSO3-and S2-in the environment of pH 6-8.Selectivity studies show that the probe CTI has a high specificity for biological thiols and HSO3-,S2-,which will be beneficial to the detection of biological thiols or HSO3-,S2-in actual samples by CTI.3)Using coumarin as the parent fluorophore,combined with carboxylated hemicyanine fluorophore,the probe 2-((1E,3Z)-4-chloro-4-(7-(diethylamino)-2-oxo-2Hchromen-3-yl)buta-1,3-dien-1-yl)-3-(6-ethoxy-6-oxohexyl)-1,1-dimethyl-1 H-benzo[e]indol3-ium(CDI-1)was synthesized using anhydrous ethanol as the solvent and trifluoroacetic acid as the catalyst;the probe 3-(5-carboxypentyl)-2-((lE,3Z)-4-chloro-4-(7(diethylamino)-2-oxo-2H-chromen-3-yl)buta-1,3-dien-1-yl)-1,1-dimethyl-1 H-benzo[e]indol-3-ium(CDI-2)was synthesized using acetic acid as the solvent and catalyst.The maximum absorption wavelength of the probe CDI-1 is at 609 nm.After adding Cys/Hcy,the blue-shift is about 530 nm,and the change is very obvious.However,when GSH is added,the maximum absorption wavelength of the solution is blue-shifted to 573 nm.To distinguish Cys/Hcy from GSH.After the probe CDI-1 reacted with Cys/Hcy,the fluorescence intensity of the solution was enhanced by about 2 times,while it was enhanced by 3 times after the reaction with GSH.Cys/Hcy and GSH can also be distinguished according to this phenomenon.The probe CDI-1 can react completely with Cys within 30 min,and it takes 80 min to react with Hcy or GSH.The probe CDI-1 reacts quickly with HSO3-and S2-,which can be completed within 20 min.The effect of pH on the fluorescence properties of CDI-1 shows that the probe is suitable for the detection of biological thiols or HSO3-and S2-in neutral or weak alkaline environments.The probe CDI-1 has higher specificity for biological thiols and HSO3-and S2-.Probe CDI-2 can react completely with Cys,Hcy and GSH in about 20 min,40 min and 22 min,respectively,while it reacts quickly with HSO3-and S2-,which only takes about 10 min.Therefore,the probes CDI-1 and CDI-2 have certain reference functions for the detection of biological thiols,HSO3-and S2-.