7-Azido-4-methylcoumarin

7-Azido-4-methylcoumarin is a selective coumarin-based fluorescent probe for hydrogen sulfide (H₂S). In the presence of H₂S, the aromatic azido group of 7-Azido-4-methylcoumarin is selectively reduced to produce the fluorescently active 7-amino-4-methylcoumarin (AMC). 7-Azido-4-methylcoumarin binds to the coumarin/phenol-binding site of BSA, the aglycone-binding site of UGT1A6, and the substrate-binding site of SULT1A1, respectively. 7-Azido-4-methylcoumarin retains its fluorescent properties after covalent binding, acts as a fluorescent H₂S probe, and does not react with cysteine, homocysteine or glutathione (Ex/Em = 340/445 nm)^[1][2][3][4]. In Vitro:Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Dye Preparation
1.1 Preparation of Stock Solution
Prepare a 10-50 mM stock solution using anhydrous DMSO.
Note: It is recommended to aliquot the stock solution and store it at -20 °C or -80 °C, protected from light.
1.2 Preparation of Working Solution
Dilute the stock solution with pre-warmed, serum-free cell culture medium or PBS to prepare a 10-50 μM working solution.
Note: Please adjust the working solution concentration according to your specific requirements, and prepare it immediately before use.
2. Staining of Magnaporthe oryzae
2.1 Obtain Magnaporthe oryzae samples at various developmental stages.
2.2 Add 50 μM of the dye working solution, gently agitate to ensure the solution completely covers the cells, and incubate at room temperature for 30 minutes, protected from light.
2.3 Aspirate the dye working solution, wash with culture medium 2–3 times (5 minutes per wash), and observe using a fluorescence microscope (Ex/Em = 340/445 nm).


7-Azido-4-methylcoumarin (0.25-4.0 mM; 1 min pre-incubation, 0-30 min UV irradiation) irreversibly inactivates UGT1A6 from V79 cells expressing recombinant human liver UGT1A6 in a time- and concentration-dependent manner, with a K_d of 2.89 mM and k2 of 0.113 min^-1, decreasing the V_max of pNP glucuronidation without altering the enzyme's affinity for pNP^[1].
7-Azido-4-methylcoumarin (0.1-2 mM; 1-5 min pre-incubation, 20 min UV irradiation) specifically binds to the aglycon (substrate) binding site of UGT1A6 from V79 cells expressing recombinant human liver UGT1A6, as demonstrated by protective effects of aglycon site ligands and lack of interference with co-substrate site binding, and also binds specifically to phenol/coumarin sites on BSA^[1].
7-Azido-4-methylcoumarin (15.6 μM-2 mM; 30 min UV exposure) covalently labels purified human SULT1A1 in a concentration- and light-dependent manner^[2].
7-Azido-4-methylcoumarin (AzMC) (10 μM; 100 nM-100 μM; 1 mM-10 mM) functions as a selective fluorogenic probe for H₂S, producing a linear fluorescent signal across NaHS concentrations from 100 nM to 100 μM and showing no significant reactivity with cysteine, homocysteine, PLP, or AdoMet^[3].
7-Azido-4-methylcoumarin (50 μM; 30 min) reliably detects intracellular H₂S levels in Magnaporthe oryzae mycelium, appressoria, and conidia, revealing significantly higher H₂S levels in CSE1 overexpression mutants and H₂S level changes with NaHS or hypotaurine treatment^[4].