7-Amino-4-methylcoumarin

7-Amino-4-methylcoumarin belongs to the coumarin class, can be isolated from the endophytic fungus Xylaria sp. and has a broad spectrum of antibacterial activity. 7-Amino-4-methylcoumarin is also commonly used as an important laser dye that emits in the blue region, capable of analyzing glycoprotein monosaccharides and N-linked oligosaccharides, and is also utilized in tissue pathology analysis, enzyme activity measurement, and copper ion detection. The excitation wavelength and emission wavelength are 351 nm and 430 nm, respectively.^[1][2][3][4] In Vitro:7-Amino-4-methylcoumarin for Tissue Pathology Analysis^[2]
(1) Original solution preparation: Dissolve 7-Amino-4-methylcoumarin in 10 mg/mL DMSO and store it at room temperature in the dark.
(2) Working solution preparation: Dissolve 25 μL of the 7-Amino-4-methylcoumarin stock solution and 2.5 mg of 2-pyridine borane in 50 mL of 1 mg/mL citric acid solution.
(3) Dewax and hydrate the slices first, and then oxidize them in 0.5% (w/v) periodic acid solution for 10 minutes.
(4) After rinsing with distilled water, stain the slices with the 7-Amino-4-methylcoumarin working solution for 10 minutes.
(5) Rinse again with distilled water, then counterstain the slices with Mayer's hematoxylin for 1 minute.
(6) Wash the slices under slightly warm (about 40°C) tap water for 5 minutes, then immerse them in a solution of Eosin Y diluted with 95% ethanol in a 3:1 ratio for 2 minutes.
(7) After rinsing with distilled water, dehydrate and clear the slices, then mount them with the non-fluorescent, anhydrous mounting medium Entellan New.
(8) Use an Axio Observer inverted microscope illuminated with LED (385 nm UV fluorescence), employing a 96 H & E filter set with BP390/40, BS420, and BP450/40 for bright field morphological analysis and UV excitation fluorescence signal analysis.
7-Amino-4-methylcoumarin for Enzyme Activity Detection^[3]
(1) Cultivate red fungi and related actinomycetes on glucose yeast extract agar at 25°C for five days.
(2) Dissolve 5 mg of the enzyme to be tested in 200 μL N,N-dimethylformamide to make a 2×10^-3 M solution. Dilute it to 10 mL with 0.05 M Tris buffer, adjusting the pH to 7.0. The substrate solution can be used immediately without sterilization or stored at -20°C for up to 3 months.
(3) Generate fluorescent 7-Amino-4-methylcoumarin from the conjugated substrate through enzyme-catalyzed hydrolysis. At an excitation wavelength of 380 nm, close to the strong 366 nm line of a mercury arc lamp, there is strong fluorescence emission at 460 nm, falling into the blue region.
(4) Lightly draw a 20 mm diameter circle with a pencil on Whatman No. 3 filter paper (about 90 mm) placed inside a standard 100 mm petri dish lid.
(5) Transfer the cultured red fungal and related actinomycetes inoculum into the circular area on the filter paper using a wooden toothpick. Inoculate 8-10 spots in each petri dish, pressing them into the paper, and then add a drop of substrate using an automatic pipette.
(6) Use the bottom of the petri dish as a lid, and incubate the reactants at 37°C for 10 minutes.
(7) Record a positive reaction when a strong light blue fluorescence is observed under UV light.
7-Amino-4-methylcoumarin for Cu²⁺ Detection^[4]
(1) Dissolve 7-Amino-4-methylcoumarin and o-phenylenediamine (OPD) in 0.2M Tris-HCl buffer at pH 7.4, with concentrations of 50 μM and 50 mM respectively, adjusting the pH to 7-9.
(2) Prepare a copper sulfate storage solution by dilution to obtain different concentrations ranging from 0-300 μM.
(3) Mix 100 μL of the different concentrations of Cu²⁺ solution with AMC (20 μL, 50 μM), OPD (50 μL, 50 mM), and Tris-HCl (830 μL, 0.2 M, pH 7.4) thoroughly at 37°C for 2 minutes, then incubate the mixture in a water bath at 37°C for 2 hours. Record the changes in the fluorescence intensity ratios (F557/F438) using an F-4500 fluorescence spectrophotometer at an excitation wavelength of 360 nm.
(4) Maintain 20 μL of 50 μM AMC, 50 μL of 50 mM OPD, and 830 μL of 0.2 M Tris-HCl buffer (pH = 7.4) in a 2 mL centrifuge tube in a 37°C water bath for 2 minutes; then add 100 μL of different metal ions to the reaction solution and react at 37°C for 2 hours. Record the changes in the fluorescence intensity ratios using an F-4500 fluorescence spectrophotometer at an excitation wavelength of 360 nm.