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4-Methylumbelliferyl-beta-D-glucuronide (4-MUG) is an organic compound with the molecular formula C16H13NO7 and a molar mass of 333.27 g/mol for CAS 6160-80-1. It usually appears as a white to light yellow powder or crystal. It has good water solubility and can dissolve in water. The molecular structure of this compound contains a glucuronic acid group (GlcA) and a 4-methylumbelliferone (4-MeUmb) group. The glucuronic acid group is connected to the 4-methylumbelliferone group through glycosidic bonds. Characteristic spectral properties.
For example, under ultraviolet light, the compound exhibits strong fluorescence and can be used for its quantitative analysis. Its maximum excitation wavelength and maximum emission wavelength are 365nm and 448nm, respectively. It is a weak acid with a lower pKa value in water. As a substrate with fluorescent properties, it has broad application value in enzyme activity detection and screening. Its simple principle, convenient operation, high sensitivity, and quantifiability make it an important tool in multiple fields such as biomedical, chemical, and biological sciences.
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Chemical Formula |
C16H16O9 |
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Exact Mass |
352 |
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Molecular Weight |
352 |
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m/z |
352 (100.0%), 353 (17.3%), 354 (1.8%), 354 (1.4%) |
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Elemental Analysis |
C, 54.55; H, 4.58; O, 40.87 |
4-Methylumbelliferyl-beta-D-glucuronide (MUG) is an organic compound with unique fluorescent properties, with a molecular formula of C ₁₆ H ₁₆ O ₉ and a molecular weight of 352.29. This compound has demonstrated extensive application value in various fields such as biochemistry, molecular biology, environmental monitoring, and food testing.
Biochemistry and Molecular Biology Research
1. β - glucosidase (GUS) activity detection
MUG is a specific fluorescent substrate for β - glucosidase (GUS). Under the action of GUS, the β - glycosidic bond of MUG is hydrolyzed, releasing 4-methylumbelliferone (4-MU) with strong fluorescence. This reaction produces blue white fluorescence under ultraviolet light with an excitation wavelength of 362nm and an emission wavelength of 445nm, and its fluorescence intensity is proportional to the GUS activity. Therefore, by detecting the intensity of the fluorescence signal, the activity of GUS can be quantitatively determined.
Application example: In plant molecular genetics research, GUS genes are often used as reporter genes to analyze promoter activity or gene expression levels by detecting GUS activity. As a fluorescent substrate for GUS, MUG can sensitively reflect the expression of GUS genes, providing a powerful tool for gene function research.
Experimental conditions: Typically, the concentration of MUG in the reaction system is 156 μ M to 5mM, the reaction temperature is 37 ℃, and the reaction time is 30 minutes to several hours. After the reaction is complete, stop the reaction by adding carbonate termination buffer and observe the fluorescence signal under ultraviolet light.
2. Detection of genetically modified plants
Due to the natural lack of GUS activity in most plants, GUS genes are widely used in the screening and identification of transgenic plants. By co transforming GUS genes with target genes into plant cells and detecting GUS activity using MUG, transgenic positive plants can be quickly screened.
Application example: In the research of genetically modified crops, MUG detection method is used to verify the expression of exogenous genes in plants. By detecting GUS activity in tissues such as leaves, roots, or seeds, the genetic stability and expression efficiency of genetically modified crops can be evaluated.
Advantages: Compared with traditional PCR or Southern blot detection, MUG detection method has the advantages of simple operation, high sensitivity, and low cost, especially suitable for screening large-scale transgenic plants.
Environmental monitoring and food testing
1. Detection of Escherichia coli
Escherichia coli is one of the important indicator bacteria for water pollution. The MUG detection method is widely used for rapid detection of Escherichia coli in drinking water, surface water, and food. Escherichia coli can produce GUS enzyme, which hydrolyzes MUG to release fluorescent substance 4-MU, thereby achieving quantitative detection of E. coli.
Application example: In the monitoring of drinking water safety, the MUG detection method is used to quickly screen for E. coli contamination in water samples.
By inoculating the water sample into a medium containing 4-methylumbelliferyl-beta-d-glucuronide and observing the fluorescence signal after a period of cultivation, the presence of E. coli contamination in the water sample can be determined.
Experimental method: Common MUG detection media include Crystal Violet Neutral Red Bile Salt-4-Methylumbelliferone - β - D-Glucoside Agar (VRBA-MUG), etc. These culture media achieve selective detection of Escherichia coli by inhibiting the growth of non target bacteria while promoting the proliferation of E. coli and the expression of GUS enzyme.
2. Microbial contamination detection in food
In addition to water quality monitoring, the MUG detection method is also used for detecting microbial contamination in food. For example, in dairy products, meat, and seafood, contamination by Escherichia coli and other microorganisms that can produce GUS enzymes can be quickly screened using the MUG detection method.
Application example: In the production process of dairy products, the MUG detection method is used to monitor the contamination of Escherichia coli in raw milk and finished milk.
Through regular sampling and testing, microbial contamination can be detected and controlled in a timely manner, ensuring the quality and safety of dairy products.
Advantages: Compared with traditional cultivation methods, MUG detection method has the advantages of fast detection speed, high sensitivity, and easy operation. It can screen a large number of samples in a short period of time, improve detection efficiency, and reduce detection costs.
Drug development and enzymatic research
1. Drug screening and evaluation
In the process of drug development, the MUG detection method is used to screen compounds with GUS inhibitory activity. By detecting the inhibitory effect of compounds on GUS activity, their efficacy and safety as potential drugs can be evaluated.
Application example: In the development of anti-tumor drugs, high expression of GUS enzyme is closely related to the occurrence and development of certain tumors.
Therefore, searching for compounds with GUS inhibitory activity has become one of the important directions in the development of anti-tumor drugs. The MUG detection method provides a sensitive and reliable detection method for drug screening in this field.
Experimental design: In drug screening experiments, MUG is usually incubated with GUS enzyme and the test compound, and the inhibitory effect of the compound on GUS activity is evaluated by detecting changes in the intensity of the fluorescence signal. By optimizing experimental conditions, the sensitivity and accuracy of screening can be improved.
2. Enzymatic research and enzyme kinetics analysis
The MUG detection method is also used in enzymatic research and enzyme kinetics analysis. By detecting the hydrolysis rate of MUS enzyme on MUG under different conditions, the catalytic mechanism, substrate specificity, and enzyme kinetic parameters of the enzyme can be studied.
Application example: In enzymatic research, the MUG detection method is used to determine the optimal reaction temperature, pH value, and ionic strength of GUS enzyme.
By changing the reaction conditions and detecting changes in the fluorescence signal, the optimal reaction conditions for the enzyme can be determined and its catalytic mechanism can be studied.
Experimental method: In enzyme kinetics analysis, different concentrations of MUG are usually used as substrates, and the enzyme kinetics curve is plotted by detecting the relationship between reaction rate and substrate concentration. By fitting the curve and calculating relevant parameters (such as Km, Vmax, etc.), the catalytic efficiency and substrate specificity of the enzyme can be evaluated.
The laboratory synthesis method of 4-Methylumbelliferyl-beta-D-glucuronide includes the following steps:
Select appropriate protective groups to protect glucuronic acid and avoid unnecessary side reactions in subsequent reactions. Common protective groups include ester groups, ether groups, etc. Choose methyl ester as the protective group to convert glucuronic acid into methyl ester glucuronic acid. This reaction uses methanol and carboxylic anhydride as reactants, with sulfuric acid as the catalyst.
The reaction equation is as follows:
(CH3)2C(OH)-CH2-COOH + H2SO4 → (CH3)2C(O)-CH2-COOH + CH3OH
Coupling reaction between 4-methylumbelliferone and glucuronic acid with protective groups. This step usually requires the use of coupling agents such as DCC, EDC, etc. to facilitate the reaction. The reaction conditions need to be controlled appropriately to ensure the purity and yield of the product. This reaction uses DCC as a coupling agent.
(CH3)2C(O)-CH2-COOH + NH2-CH=CH-COOH + DCC + C6H5N3O → (CH3)2C(O)-CH2-CO-NH-CH=CH-COOH + H2O + NH4OH
(CH3)2C(O)-CH2-CO-NH-CH=CH-COOH + NH2(CH3) → (CH3)2C(O)-CH2-CO-NH-CH=CH-CO-NH(CH3) + H2O
(CH3)2C(O)-CH2-CO-NH-CH=CH-CO-NH(CH3) + HBr → (CH3)2C(O)-CH2-CO-NH-CH=CH-CO-Br(CH3) + HBr
(CH3)2C(O)-CH2-CO-NH-CH=CH-CO-Br(CH3) → (CH3)2C(O)-CH2-CO[C16H8-(NH-)-6]-Br(CH3) + NaCl + NaBr
By using a mixture of sodium methanol and sodium ethanol as a catalyst, methyl glucuronic acid methyl ester is removed from 4-Methyllumbelliferyl beta D-glucuronide. The reaction equation is as follows:
(CH3)2C(O)-CH2-CO[C16H8-(NH-)-6]-Br(CH3) + CH3ONa + C2H5ONa → (CH3)2C(O)-CH2-CO[C16H8-(NH-)-6] + 2NaBr + 2CH3OH + C2H5OH
The crude product is purified by column chromatography and recrystallization to obtain high-purity 4-Methyllumbelliferol-beta-D-glucuronide. The specific process is as follows:
Dissolve the crude product in an appropriate amount of solvent and separate it by column chromatography. Select appropriate adsorbents and eluents, wash off impurities and unreacted raw materials in sequence to obtain pure products. Recrystallization is the process of dissolving pure products in an appropriate amount of solvent and obtaining pure products by cooling and crystallization. Recrystallization can remove a small amount of unreacted raw materials and by-products, further improving the purity of the product.
4-MUG is a glycoside derivative of 4-methylammonium rivarone (4-MU), which is an important member of coumarin compounds.
Coumarin was first isolated from beans by French chemist Nicolas Jean Baptiste Gaston Gibb and its basic structure was determined.
German chemists Wilhelm Henry Perkin and others began to systematically study the synthesis methods of coumarin derivatives, laying the foundation for the development of subsequent fluorescent probes.
With the development of organic synthetic chemistry, scientists discovered that coumarin derivatives (such as 7-hydroxycoumarin) could emit strong fluorescence under ultraviolet light.
R. Ansch ü tz and H. W. Kohlrausch synthesized various derivatives of umbrella shaped drugs and studied their fluorescence properties. These studies have laid a crucial chemical foundation for the subsequent development of 4-MUG.
With the development of glycochemistry, scientists began to study glycosylation reactions by combining fluorescent molecules (such as 4-MU) with glycosides to explore their applications in biological systems.
W. Klyne and R. D. Guthrie reported for the first time the synthesis of β -4-MU glycoside derivatives, including 4-methyl beta-1,3-glucoside (4-MUGic).
With the discovery of β - glucosidase (GUS) in mammalian tissues and bacteria, scientists began searching for specific fluorescent substrates.
J.L. Strominger and H.M. Kalka synthesized 4-methyl lubricating benzoyl - β - D-glucuronic acid ester (4-MUG) while studying bacterial glycosidases, and found that the compound could be efficiently hydrolyzed by GUS, releasing highly fluorescent 4-MU. This discovery established the key role of 4-MUG in enzyme detection.
4-MUG began to be used for clinical diagnosis, especially in the detection of liver and gallbladder diseases and bacterial infections. Due to the high expression of GUS in bacteria such as E. coli and its presence in mammalian tissues such as liver, 4-MUG has been developed as a sensitive fluorescent probe for detecting enzyme activity in urine and blood.
Molecular biology technology developed rapidly, and scientists needed an intuitive and sensitive reporter gene to monitor gene expression.
In 1987, Richard A. Jefferson (then a researcher at the University of Cambridge) discovered that the gusA gene (encoding β - glucuronidase) in Escherichia coli could serve as an ideal reporter gene for studying T-DNA transfer in Agrobacterium. Prove that GUS naturally does not exist in most plants and bacteria, with low background interference. Research has found that 4-MUG can generate high fluorescence signals under the action of GUS, with sensitivity far exceeding traditional X-Gluc (5-bromo-4-chloro-3-indole-β - D-glucoside) staining method.
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