Construction Of Fluorescent Probe For Detection Of β-gal And Mercury Ion Based On ESIPT Mechanism And Its Application

Part Ⅰ Detection of β-gal and its inhibitors by fluorescence probe based on ESIPT mechanism Objective:1.A series of β-galactosidase(β-gal)recognition probes based on the Excited State Intramolecular Proton Transfer(ESIPT)mechanism were designed and synthesized by taking full advantage of the small molecular size,strong group modifiability and easy preparation of 3-hydroxyphthalamide fluorescence skeletons and the reaction characteristics between the probes and β-galactosidase were explored.In order to obtain more efficient and suitable substrate probe,the structure-activity relationship between probe structure and reactivity was discussed.2.The probe was used to detect β-galactosidase activity in common foods,and the effectiveness of the probe was tested through practical application.3.An inhibitor screening system based on fluorescence analysis was constructed by using the best fluorescent probe obtained from screening.To discover new β-galactosidase inhibitors from active monomers of traditional Chinese medicine.Methods:1.Synthesis of 3-hydroxyphthalamide fluorescence molecules: The probe derivatives were prepared by liquid phase organic synthesis.The key lacamidation reaction conditions were as follows: 3-hydroxyphthalic anhydride and corresponding amino compounds were dissolved in glacial acetic acid,the reaction solution was heated to 110℃ for acylation,and the reaction was monitored by TLC.The obtained intermediate compounds and final probe products were purified by silica gel column chromatography.The structure of the compounds was characterized by nuclear magnetic resonance spectroscopy(NMR)and high resolution mass spectrometry(HRMS).2.Synthesis of β-galactosidase substrate probe: tetraacetyl-α-D bromogalactose was alkylated with the phenol hydroxyl group on the fluorescence molecule of 3-hydroxyphthalamide.The basic conditions were as follows: Anhydrous acetonitrile(ACN)was used as the reaction solvent,and the reaction was monitored by TLC.The product was separated and purified by silica gel column and characterized by NMR and HRMS.3.Assay of the reaction between fluorescent probe and β-galactosidase: Substrate probe and β-galactosidase were mixed in PBS buffer(p H7.4)and the fluorescence intensity was recorded at 37℃ using a Multi Function Measuring Instrument.4.Select mature fruits in the season take appropriate amount of pulp,extract the supernatant after grinding and mix the supernatant with the probe solution for reaction.The fluorescence changes of the reaction system were recorded and the enzyme content in fruit was obtained by comparing with the standard curve.5.Dozens of interesting Chinese medicine monomers were selected and added into the above enzyme reaction system,and the fluorescence changes were recorded by Multi Function Measuring Instrument.The group without inhibitor was set as the control group.If necessary,half inhibitory concentration IC50 of the selected monomer for enzyme activity was calculated.Results:1.14 β-galactosidase probes with 3-hydroxyphthalamide as fluorescence groups were synthesized and characterized by NMR and HRMS.2.The results showed that the reaction rate of probe C11(containing benzyl groups)with the enzyme was better than other probes.It was found that 22 kinds of active monomers of traditional Chinese medicine,such as α-crocin,myricetin and kaferol,had certain inhibitory effects on the enzyme by using C11 for inhibitor screening.3.In the detection of fruit samples,the results of C11 probe in the recovery experiment are accurate and reliable,and has good practical detection performance.Conclusion:1.By analyzing the reactions of 14 probes with different substituents with β-galactosidase and combining with molecular docking,it is suggested that β-galactosidase may be inclined to recognize substrates with larger hydrophobic groups such as C11(containing benzyl groups).However,due to the limited number of probe molecules,it is impossible to discuss the effect of probe charge properties on the reaction,so the correlation structure-activity analysis is still preliminary and one-sided.2.The synthesized C11 probe has better detection sensitivity,and compared with the commercial probe(4-MU)and other probes,the 3-hydroxyphthalimide molecules in this paper are easier to achieve derivatization for probe structure expansion and enzyme structure activity analysis.The probe and enzyme structure activity analysis can guide the design and synthesis of inhibitors to a certain extent.Part Ⅱ Detection of mercury ions by fluorescent probe using ESIPT mechanism and its biological application Objective:1.Based on the ESIPT mechanism of 3-hydroxyphthalamide fluorescence molecules and the desulphurization reaction of mercuric ion to thiooxy heterocyclic ring,a new mercuric ion detection probe was constructed.The sensitivity,selectivity,anti-interference and the influence of p H on the detection were investigated..2.The probe was used to detect mercury ions in actual samples(such as Chinese medicinal materials),and the detection ability of the fluorescence probe method was investigated.3.A new fluorescent probe was designed and prepared to realize fluorescent labeling of mercury-ion binding proteins.Methods:1.Synthesis of mercury ion probe: small molecule organic synthesis method was used to prepare the probe.The compounds were isolated and purified by silica gel column chromatography,and the intermediates and end products were characterized by NMR and HRMS.2.Mercury ion detection: The detection performance of the probe was tested in the PBS buffer system,and the change of fluorescence intensity of the probe was recorded using a multifunctional enzyme label.The selectivity and anti-interference were investigated by adding other interferors.3.Detection in actual samples: detection of mercury ions in several commercially purchased Chinese medicinal materials,and adding standard concentration of mercury ions into Chinese medicinal materials and then detection,to obtain the recovery rate and other information,to investigate the reliability of the method.Results:1.Three probes(D1,D2,D3)for mercury ion detection were obtained by organic synthesis,and their structures were correctly characterized by NMR and LC-MS.Among them,D1 probe could realize fluorescence detection of mercury ion in aqueous solution,and showed good selectivity and anti-interference ability.2.In the Chinese medicinal materials tested was no found mercury ion by D1.3.A compound A16(probe intermediate)was obtained,which could be covalently labeled with protein.On this basis,the mercury-ion probe design was carried out,and fluorescence labeling of mercury-ion binding proteins could be realized theoretically.However,due to time issues,the relevant design has not been experimentally verified.Conclusion:1.Mercury-ion fluorescence probes can be designed by using the ESIPT mechanism of3-hydroxyphthalamide fluorescence molecules,and the related probes have high mercuryion selectivity and anti-interference ability.However,the reaction kinetics of probe D1 indicated that the probe reacted slowly with mercury ions,and the detection needed a relatively long time.2.D1 probe can realize the detection of mercury ions in complex samples such as Chinese herbal extracts.Further application and realization of fluorescent labeling of mercury ion binding proteins still need more in-depth work.