Resorufin benzyl ether  (Synonyms: BzRes; 7-Benzyloxyresorufin; 7-Benzyloxyphenoxazone)

Resorufin benzyl ether (BzRes), a fluorogenic enzyme substrate, can be used to detect CYP3A4 enzyme activity. Resorufin benzyl ether modified with a recognizing moiety boronate, can be used for ONOO^- detection via a self-immolation mechanism. Ex/Em=530-570 nm/590 nm^[1][2][3].

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
Measurement of CYP3A4 activity^[2]: Prepare the reaction solution.
a. The standard solution contains 1 mM Resorufin benzyl ether, which is used as the fluorescent substrate. Dissolve 5 mg of Resorufin benzyl ether in a mixture of 1 mL of 2% w/v Poloxamer 188 (HY-D1005A), 500 mL Dimethyl sulfoxide (HY-Y0320), and 3.5 mL acetonitrile.
b. Freshly prepared CYP3A4 enzyme solution. Mix 5 mL of 1 mM enzyme stock solution with 995 mL of buffer to obtain a 5 nM enzyme solution.
2. Measurement of CYP3A4 activity a. Perform the reaction in a 96-well plate. Add 99 mL of buffer mixture and 1 mL of 1 mM Resorufin benzyl ether to each well. Adjust the final concentration to 5 mM.
b. Before incubation at 37℃ for 30 minutes, transfer 100 mL of 5 nM enzyme solution.
c. Enzyme activity was determined under fluorescence detection at excitation wavelength (lex = 570 nm) and emission wavelength (lem = 590 nm).
d. Factors affecting the determination of CYP3A4 activity. Such as buffer solutions (phosphate, Tris-HCl buffer) and buffer concentrations (50-200 mM), as well as incubation time (0-50 min).

Metabolic Activity Assay of CYP3A4^[3] : Add the CYP3A4 enzyme to a final concentration of 5 pmol per well.
2. For each reaction, 50 pM of substrate and 200 mM phosphate buffer solution were used. < br / > 3. Before measuring the fluorescence of metabolites, incubate them with BzRes for 45 minutes. The excitation wavelength (Ex) is 530 nm and the emission wavelength (Em) is 590 nm.
4. The concentration of the full-strength extract is specified as 100% (diluted 1:4 in the final measurement quantity).
5. Dilute the 100% extract in a 1:3 ratio twice. Calculate the mean and standard deviation of the fluorescence values.

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

303.31

C₁₉H₁₃NO₃

87687-02-3

O=C1C=CC2=NC3=C(C=C(OCC4=CC=CC=C4)C=C3)OC2=C1

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Ji X, et al. Regulating the activity of boronate moiety to construct fluorescent probes for the detection of ONOO-in vitro and in vivo. Anal Methods. 2022 Dec 15;14(48):5027-5033.  [Content Brief]

  [2]. Nuchtavorn N, et al. Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement. Anal Chim Acta. 2020 Feb 15;1098:86-93.  [Content Brief]

  [3]. Yale SH, et al. Analysis of the inhibitory potential of Ginkgo biloba, Echinacea purpurea, and Serenoa repens on the metabolic activity of cytochrome P450 3A4, 2D6, and 2C9. J Altern Complement Med. 2005 Jun;11(3):433-9.  [Content Brief]