Resorufin benzyl ether (BzRes) is a luciferase substrate that may be utilized to detect CYP3A4 enzyme activity. Resorufin benzyl ether can be used for ONOO- detection through the self-immolation mechanism after being modified with a boronic acid group with recognition function. Ex/Em=530-570 nm/590 nm.

Physicochemical Properties

Molecular Formula	C19H13NO3
Molecular Weight	303.311424970627
Exact Mass	303.09
CAS #	87687-02-3
PubChem CID	114982
Appearance	Typically exists as solid at room temperature
Density	1.26 g/cm3
Boiling Point	479.9ºC at 760 mmHg
LogP	3.871
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	23
Complexity	557
Defined Atom Stereocenter Count	0
SMILES	O=C1C=C2C(=NC3C(O2)=CC(OCC2C=CC=CC=2)=CC=3)C=C1
InChi Key	XNZRYTITWLGTJS-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H13NO3/c21-14-6-8-16-18(10-14)23-19-11-15(7-9-17(19)20-16)22-12-13-4-2-1-3-5-13/h1-11H,12H2
Chemical Name	7-phenylmethoxyphenoxazin-3-one
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	Assay for CYP3A4 activity[2]: 1. Get the reaction solution ready. a. The fluorescent substrate, Resorufin benzyl ether, has a concentration of 1 mM in the standard stock solution. 500 mL of dimethyl sulfoxide, 3.5 mL of acetonitrile, 1 mL of 2% w/v Poloxamer 188, and 5 mg of resorufin benzyl ether should all be dissolved. b. Make a new solution of the CYP3A4 enzyme. To create a 5 nM enzyme solution, dilute 5 mL of the 1 mM enzyme stock solution with 995 mL of buffer. 2. Find out what CYP3A4 activity is. In a 96-well plate, react. Fill each well with 1 mL of 1 mM Resorufin benzyl ether and 99 mL of buffer mix. Adjust to 5 mM as the final concentration. b. After transferring 100 mL of the 5 nM enzyme solution, incubate for 30 minutes at 37°C. C. Measure the enzyme activity using fluorescence detection at the excitation wavelength (lex=570 nm) and emission wavelength (lem=590 nm). d. variables influencing CYP3A4 activity measurement. phosphate and Tris-HCl buffers, for example; also, buffer concentrations (50–200 mM) and incubation times (0–50 min). Measuring the metabolic activity of CYP3A4 [3]: 1. At a final concentration of 5 pmol/well, add the CYP3A4 enzyme. 2. For every reaction, use 200 mM potassium phosphate buffer and 50 pM substrate. < br /> 3. Measure the metabolite fluorescence after 45 minutes of incubation with BzRes. 590 nm is the emission wavelength (Em) while 530 nm is the excitation wavelength (Ex). 4. Indicate the full strength extract concentration as 100% (diluted 1:4 in the final assay volume). 5. Double-do the 100% extract dilution at a ratio of 1:3. Determine the fluorescence values' standard deviation and mean.
ADME/Pharmacokinetics	Metabolism / Metabolites 7-Benzyloxyresorufin has known human metabolites that include Resorufin.
References	[1]. 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. [2]. Paper-based sol-gel thin films immobilized cytochrome P450 for enzyme activity measurement. Anal Chim Acta. 2020 Feb 15;1098:86-93. [3]. 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.

Solubility Data

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2970 mL	16.4848 mL	32.9696 mL
	5 mM	0.6594 mL	3.2970 mL	6.5939 mL
	10 mM	0.3297 mL	1.6485 mL	3.2970 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.