Syntheses Of Fluorescent Probes Based On Coumarin Derivatives And Their Applications In Biological Imaging

Fluorescent probes are widely used in biological imaging research due to their advantages of strong selectivity,high sensitivity,simple operation and non-invasive real-time monitoring.Coumarin compounds are a kind of secondary metabolites widely distributed in the plant kingdom.The basic structure of benzopyranes has no fluorescence characteristics.However,when 7 or 3,4 position is replaced by some substituent,can produce strong fluorescent signal,and has the high fluorescence quantum yield,large Stokes shift,light chemical stability and so on.So it is an ideal fluorescent sensing molecule and has been widely used in the field of biological im-aging.In this thesis,three coumarin derivatives with organelle targeting were synthe-sized by chemical reaction method,which lays and accumulates experimental founda-tion for the preparation of rich coumarin fluorescent probes and cell imaging observa-tion of disease diagnosis and treatment.The details are as follows:Based on the ICT(intramolecular charge transfer)mechanism,a coumarin de-rivative fluorescent probe was designed and synthesized.We named it as(8-(diethylamino)-2-thioxo-2,3-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one,the first probe 2-1)was synthesized by 4-(diethylamino)salicylic aldehyde and 4,6-dihydroxy-2-mercaptosine.The experimental results showed that the probe could rapidly and specifically bind to the GABAA receptor sites on the cell membrane,and could conduct real-time fluorescence imaging of a variety of living cell membranes for a long time.Compared with commercial dye Dil,this fluorescent probe had a simple synthesis method,strong light stability,low cytotoxicity,after incubation with cells,it could target the cell membrane in a short time(about lmin)and stabilize for a long time(0-3h).It could carry out wash-free treatment for cell imaging,making it an ideal fluorescent probe targeting cell membranes.In addition,this paper had con-ducted a preliminary study on the application of this fluorescent probe in the screen-ing of GABAA receptor drugs.The results showed that this fluorescent probe had no specificity to other receptors on the cell membrane,and could be used as an effective tool for the screening of GABAA receptor drugs,which was worthy of further re-search.In the second part,chemical synthesis method was adopted based on the TICT(twisted intramolecular charge transfer)mechanism.We obtained two kinds of struc-ture similar to coumarin derivatives fluorescent probe and named them as(E)-2-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-3-methylbenzo[d]thiazol-3-iumiodide(probe3-1)and(E)-2-(2-(7-(diethylamino)-2-oxo-4-(thiophen-2-yl)-2H-chro men-3-yl)vinyl)-3-methylbenzo[d]thiazol-3-ium iodide(probe 3-2).The experimental results showed that due to the differences in structure of the two probes,the binding of the two fluorescent probes to DNA and RNA was significantly different.The re-sults of molecular docking showed that the introduction of thiophene ring at 4 site made the three-dimensional molecular structure of probe 3-2 tend to be curved,while the single-stranded structure of RNA was easier to fold and bend than the dou-ble-stranded structure of DNA,making the probe 3-2 had better specific binding abil-ity with RNA.The results of cell experiments showed that both fluorescent probes had low toxicity to cells,and the confocal fluorescence showed that they had good correlation coefficients compared with commercial dyes(the overlap coefficients were 0.91 and 0.95,respectively),indicating that both fluorescent probes could target mitochondria well.Due to the positive and reverse the existence of double bond structure,two kinds of fluorescent probes to the mitochondrial membrane potential and viscosity had a specific response,and these two features were significant differ-ence in normal cells and cancer cells,so could be used to distinguish between normal and cancer cells,so as to realize the difference between the normal tissue and tumor tissue and used for the diagnosis of cancer,which was a kind of small molecule fluo-rescent probe with great potential.In summary,according to the characteristics of coumarin chemical structure and its optical characteristics,this paper used chemical synthesis methods to prepare three kinds of small molecule fluorescent probes with organelle targeting,which broadens the practical application of these compounds in fluorescence imaging and lays the experimental and scientific foundation for the development and application of cou-marin derivative fluorescence probes.