Synthesis Of 5-membered Heterocycles Containing Nitrogen And Its Application In Sulfhydryl Protein Subsulfonation Fluorescence Labeling

Five-membered nitrogen-containing heterocycles are important in the field of drug design and synthesis,because of excellent biological performance.less toxicity and high efficiency,and therefore become the hot research target of many researchers.In the field of medicine,nitrogen-containing heterocyclic compounds have been proved to have excellent medical effects in diseases treating,such as the basic structure of antipyretic and pain relieving antipyrine,which is pyrazole ring.Although five-membered nitrogen-containing heterocyclic compounds have relatively mature synthesis methods and applications,it is still necessary to develop more efficient and environmentally friendly methods to construct five-membered nitrogen-containing heterocyclic compounds,and further explore its potential in more biological fields.Cysteine hyposulphonic acid is one of the important products of sulfhydryl protein oxidation products and is necessary to regulating cell signal transduction and protein function.At present,the methods for detecting sulfhydryl protein and oxidation process are mainly divided into indirect method and direct method.Among them,the design concept of the indirect method is generally to first block the reduced thiol group with thiol specific reagents,reduce the oxidized thiol group,and then perform specific detection.However,this method has significant limitations,such as the variability of protein thiol groups during processing and the inability to achieve in situ detection in vivo,which limits the development and application of the indirect method.The general strategy of the direct method is to develop small molecular probes for in situ detection of sulfhydryl protein and its oxidation products.However,cysteine disulfonic acid is an extremely unstable intermediate product,which is difficult to detect in real time,and there is no effective strategy to detect disulfonic acid on the market at present,which largely limits the development and application of cysteine disulfonic acid.To further solve the above problem,we have explored the new strategy for the synthesis of1,2,3-triazole,two important branches of five-membered nitrogen-containing heterocycles,and the new strategy for the detection of protein subsulfonation using tetrazolium small molecular fluorescence probes.The specific work is as follows:(1)A novel multicomponent labeling reaction for the synthesis of nitrogen-containing five membered heterocycles catalyzed by bimetalsWe developed a three-component one-pot coupling strategy between alkynyl-terminated,phenol-substituted alkenylazide and trifluoromethyl-substituted 1,3-dicarbonyl compounds under mild conditions,and synthesized a series of multifunctional 1,2,3-triazole derivatives.First of all,we determined the best reaction conditions for the coupling reaction,that is,under the catalysis of metal Pd and Cu,and using triethylamine as the basic catalyst,the reaction was carried out in argon at 60 ℃ for two hours.Secondly,we explored the universality of the reaction substrate,and the results showed that the reaction substrate was highly applicable.Finally,we also explored the reaction mechanism and speculated the possible reaction mechanism.The construction of this method further enriches the new strategy for the synthesis of five membered nitrogen-containing heterocyclic compounds,namely 1,2,3-triazole.(2)Tetrazolium small molecular probe for detection of protein subsulfonation fluorescence labelingWe have designed a small molecule fluorescence probe Tet SOH using tetrazole derivatives as the probe matrix and norbornene as the recognition group.Firstly,the probe Tet SOH norbornene part is labeled with protein sulfonic acid,and then irradiated with ultraviolet light.The probe tetrazole undergoes a halo reaction with the olefin part to achieve fluorescence activation.The research results indicate that the probe Tet SOH can recognize BSA SOH in a short period of time,and complete its own photocyclization under illumination within 1 minute,with significantly enhanced fluorescence.Finally,we successfully used the probe Tet SOH to perform fluorescence imaging of sulfonic acid in smooth muscle cells A7r5 under oxidative stress conditions.This strategy provides a fast and efficient new tool for labeling protein subsulfonation.