Synthesis And Application Of Glucuronidase Substrate

Foodborne pathogens are an important cause of foodborne diseases and a major hidden danger to food safety.Therefore,the rapid and effective detection of pathogenic bacteria is particularly important.Traditional detection methods(such as isolation culture and biochemical identification,etc.)have low specificity,low sensitivity,tedious and time-consuming operation for pathogenic bacteria detection,and cannot achieve rapid detection.In recent years,the synthesis and application of enzyme substrates have developed rapidly and have been widely used for microbial detection,but it is relatively expensive to use this method as a daily detection method.Therefore,it is urgent to develop a simple and easy synthetic process to reduce the production cost of enzyme substrates.6-Chloro-4-methylumbelliferyl-?-D-glucuronide has high application value in enzyme activity test.In this paper,the synthesis of 6-chloro-4-methylumbelliferyl-?-D-glucuronide was studied.The cheap 4-chloro-resorcinol was used as the starting material,the fluorophore was obtained through the modified Pechman condensation reaction,and the Koenigs-Knorr glycosylation reaction was used as the key step,then,the target enzyme substrate was obtained by removing the acetyl group and methyl uronate.In this experiment,a simple and practical synthesis scheme of 6-chloro-4-methylumbelliferyl-?-D-glucuronide was developed.At the same time,we modified the original fluorophore by introducing fluorine atom to the coumarin methyl group,then,designed and synthesized methyl(6-chloro-4-trifluoromethylumbelliferyl-?-Dglucuronide)uronate.The carboxyl group of traditional fluorescein can be replaced with a methyl group,and the specificity of a ?-glucuronidase substrate is higher than that of a ?-galactosidase substrate.In this paper,the synthesis of methyl(fluorescein-?-Dglucuronide)uronate was studied.Methyl(fluorescein-?-D-glucuronide)uronate was synthesized by bromination,Koenigs-Knorr glycosylation,and deacetylation with methyl(acetylated glucuronate)uronate as a starting material.Then,methyl(2,6-dichloro-4-acetylphenol-?-D-glucuronide)uronate was synthesized using the same method,and the p Ka of 2,6-dichloro-4-acetylphenol was tested.The experimental result of p Ka show that methyl(2,6-dichloro-4-acetylphenol-?-Dglucuronide)uronate is expected to replace 6-chloro-4-methylumbelliferyl-?-Dglucuronide to become a new substrate for enzyme activity testing.The red shift of fluorophores and a certain degree of Stokes shift are helpful for biological imaging research.In this paper,a malononitrile isoflavone substrate was synthesized by the extended design of the conjugate system,which is the basis for the subsequent biological activity testing.Methyl(2-(2-(4-hydroxystyryl)-4Hbenzopyran-4-methylene)malononitrile-?-D-glucuronide)uronate was synthesized by bromination reaction,Aldol reaction,introduction of malononitrile at 4 position,Koenigs-Knorr glycosylation reaction,Knoevenagel condensation reaction,and deacetylation with o-hydroxyacetophenone as a starting material.This paper evaluated the potentiality of 4-methylumbelliferyl-?-D-glucuronide(4-MUG),methyl(6-chloro-4-methylumbelliferyl-?-D-glucuronide)uronate(6-CMUG methyl uronate)and sodium(6-chloro-4-methylumbelliferyl-?-Dglucuronide)salt(6-CMUG sodium salt)for rapid detection of E.coli activity in water.It has been proven that the two synthetic substrates are expected to replace commercially available 4-MUG as new specific substrates for microbial activity testing by comparing the spectral and enzymatic properties of commercially available 4-MUG,synthetic 6-CMUG methyl uronate and 6-CMUG sodium salt.