Preparation Of Coumarin Derivative Fluorescent Probes For Detection Of Hypobromic Acid And Their Applications In Animal Model Of Inflammation

As an important active oxygen substance in human body,HOBr plays a strong role in oxidation and antibacterial ability.It is considered to be an significant part of the human immune system.It has been reported that hypobromic acid in organism can be formed by the interaction of bromine anion(Br-),hydrogen peroxide(H2O2)and eosinophil peroxidase(EPO).Under normal physiological conditions,HOBr in the body fluctuates in a certain range.Once this limit is exceeded,a series of health problems will be triggered.Therefore,maintaining the dynamic equilibrium of hypobromic acid in organisms and detecting its content will contribute to better comprehend the occurrence and course of diseases.However,the content of HOBr in blood is extremely low,and the detection process is very easy to be interfered by the analog hypochlorous acid(HOCI).Consequently,higher demand are present for its detection method.Because of its advantages,fluorescent probes have been expanded to the study of biological samples.In the research progress of natural pharmaceutical chemistry,coumarin,as a common natural product,shows blue fluorescence under UV light.When the 7-position is replaced by hydroxyl,it will produce stronger fluorescence signal.In this paper,fluorescent small molecule probes specific for hypobromic acid are synthesized through simple design and logical reasoning,which lays an experimental foundation for preparing more fluorescent probes for detecting HOBr coumarin derivatives.The main research contents are as follows:In the first part,a fluorescent probe 1((Z)-N',7-dihydroxy-2-oxo-2H-chromene-3-carboximidamide)based on coumarin was constructed using 3-cyanoumbelliferone and hydroxylamine as raw materials.The characterization results show that the probe has great fluorescence quality,high fluorescence quantum yield,rapid and specific reaction with HOBr,strong light stability,fluorescence intensity can remain unchanged for more than 60 minutes.It is an ideal fluorescent small molecule probe for detecting HOBr.The results of animal experiments show that probe 1 can accurately monitor the change process of HOBr content in arthritis model mice induced by Freund's complete adjuvant,and this dynamic process is positively correlated with the level of eosinophils in mice and the degree of swelling of the right palm pad tissue,which shows that probe 1 can be used as an effective tool for early diagnosis of inflammation.However,although probe 1 has good biocompatibility,it can not penetrate the plasma membrane and enter the cell,and can not detect endogenous HOBr.Combined with the research progress of hypobromic acid fluorescent probes in recent years,it is essential to develop fluorescent probes for monitoring the changes of hypobromic acid content in cells and in vivo,so as to have a more thorough understanding and comprehension of the physiological and pathological functions of hypobromic acid.In the second part,on the basis of probe 1,the fluorescent probe 1-2((Z)-7-amino-N'-hydroxy-2-oxo-2H-chromene-3-carboximide)was synthesized through further optimization and structural modification.Through characterization of the probe,the experiment shows that there is a fine linear dependence between the probe and hypobromic acid,0-60?M(F=161.88+31.706[HOBr],R=0.992);60-200?M(F=1634.8+6.3933[HOBr],R=0.995)and the detection limit is 0.32?M.More importantly,probe 1-2 can enter the cell and realize the real-time monitoring of endogenous HOBr in the cell.The probe can be applied to the detection of EPO enzyme activity through the investigation of enzyme activity in vitro and at cell level.The detection results of inflammatory animal models show that probe 1-2 can also accurately monitor the change process of HOBr content in arthritis model mice and bacterial infection model,and can enter into tissue cells.It can directly reflect the content of endogenous HOBr and EPO enzyme through the fluorescence intensity of tissue sections,which can be used as a representative of fluorescence probe for detecting endogenous HOBr.In conclusion,according to the characteristics of chemical structure and optical properties of coumarin,two fluorescent probes for detecting HOBr were synthesized.Because two fluorescent probes have amidoxime groups on their molecular structure,the addition of HOBr can oxidize oxime structure to cyano group quickly,the process of flu-orescence quenching of fluorophores by oxime is inhibited to release fluorescence,thus realizing the purpose of detecting HOBr.The reaction mechanism provides the experimental foundation and scientific basis for further establishment of fluorescent probes for the test of HOBr.