7-Amino-4-methylcoumarin  (Synonyms: Coumarin 120; AMC)

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]

Guidelines (The following is our recommended protocol. This protocol only provides guidance and should be modified according to your specific needs).
7-Amino-4-methylcoumarin for histopathological analysis^[2]
(1) Stock solution preparation: Dissolve 7-Amino-4-methylcoumarin in DMSO at a concentration of 10 mg/mL and store at room temperature in the dark.
(2) Working solution preparation: Dissolve 25 μL of 7-Amino-4-methylcoumarin stock solution and 2.5 mg of 2-pyridine borane in 50 mL of 1 mg/mL aqueous citric acid solution.
(3) Tissue sections are first deparaffinized and hydrated, then oxidized in 0.5% (w/v) periodic acid aqueous solution for 10 minutes.
(4) After rinsing with distilled water, stain the sections with 7-Amino-4-methylcoumarin working solution for 10 minutes.
(5) After rinsing again with distilled water, counterstain the sections with Mayer’s hematoxylin for 1 minute.
(6) Wash the sections under lukewarm tap water (approximately 40°C) for 5 minutes, then immerse in a solution of eosin Y diluted 3:1 with 95% ethanol for 2 minutes.
(7) After rinsing with distilled water, dehydrate and clear the sections, then mount with the non-fluorescent anhydrous mounting medium Entellan New.
(8) Use an inverted microscope equipped with LED illumination (385 nm UV fluorescence), with a 96 H & E filter set BP390/40, BS420, and BP450/40 for bright-field morphological analysis and UV-excited fluorescence signal analysis.
7-Amino-4-methylcoumarin for enzyme activity assay^[3]
(1) Culture Rhodococcus 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 prepare a 2×10^-3 M solution. Dilute to 10 mL with 0.05 M Tris buffer and adjust pH to 7.0. The substrate solution can be used immediately without sterilization or stored at -20°C for up to 3 months.
(3) Fluorescent 7-Amino-4-methylcoumarin is generated from conjugated substrates through enzymatic hydrolysis. Excitation occurs at 380 nm, close to the strong 366 nm spectral line of a mercury arc lamp, with strong fluorescence emission at 460 nm in the blue region.
(4) Lightly draw 20 mm diameter circles with a pencil on Whatman No. 3 filter paper (approximately 90 mm) placed inside the lid of a standard 100 mm Petri dish.
(5) Use wooden toothpicks to transfer inocula of five-day cultured Rhodococcus and related actinomycetes into the circled areas on the filter paper. Inoculate 8-10 spots per dish and rub firmly into the paper, then add one drop of substrate using an automatic pipette.
(6) Use the bottom of the Petri dish as a lid and incubate the reaction mixture at 37°C for 10 minutes.
(7) Record a positive reaction when strong light-blue fluorescence is observed under a UV lamp.
7-Amino-4-methylcoumarin for Cu²⁺ detection^[4]
(1) Dissolve 7-Amino-4-methylcoumarin and o-phenylenediamine (OPD) in 0.2 M Tris-HCl buffer at pH 7.4 to final concentrations of 50 μM and 50 mM, respectively, and adjust the pH to 7-9.
(2) Prepare a copper sulfate stock solution and dilute to obtain concentrations ranging from 0-300 μM.
(3) Mix 100 μL of 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 37°C water bath for 2 hours. Record the fluorescence intensity ratio change (F557/F438) of the ratiometric sensor using a fluorescence spectrophotometer at an excitation wavelength of 360 nm.
(4) Keep 20 μL 50 μM AMC, 50 μL 50 mM OPD, and 830 μL 0.2 M Tris-HCl buffer (pH = 7.4) in a 2 mL centrifuge tube at 37°C in a 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 fluorescence intensity ratio change of the ratiometric sensor using an F-4500 fluorescence spectrophotometer at an excitation wavelength of 360 nm.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

175.19

C₁₀H₉NO₂

26093-31-2

Solid

White to off-white

O=C1C=C(C)C2=CC=C(N)C=C2O1

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Żamojć K, et al. Fluorescence quenching of 7-amino-4-methylcoumarin by different TEMPO derivatives. Spectrochim Acta A Mol Biomol Spectrosc. 2015 Feb 5;136 Pt C:1875-80.  [Content Brief]

  [2]. Hiroshi Takase, et al. 7-Amino-4-methylcoumarin as a fluorescent substitute for Schiff's reagent: a new method that can be combined with hemalum and eosin staining on the same tissue section. Biotech Histochem. 2023 Jan;98(1):54-61.  [Content Brief]

  [3]. M. Goodfellow, et al. Characterisation of rhodococci using peptide hydrolase substrates based on 7-amino-4-methylcoumarin. FEMS Microbiology Letters,1987 Nov; 44 (3): 349-355.

  [4]. Xiaoyan Sun, wt al. A highly sensitive and selective ratiometric sensing platform based on 7-amino-4-methylcoumarin for naked-eye visual fluorescence sensing of Cu2. Spectrochim Acta A Mol Biomol Spectrosc. 2022 Feb 15;267(Pt 2):120627.  [Content Brief]