Dibenzylfluorescein (DBF) is a fluorescent probe (Fluoresecent dye) and a specific substrate for cytochrome P450 (CYP) isoforms such as CYP3A4, CYP2C8, CYP2C9, CYP2C19 and aromatase (CYP19). Dibenzylfluorescein is typically used with Km values around 0.87-1.9 µM (Ex=485nm, Em=535nm). Dibenzylfluorescein is used to detect changes in CYP catalytic activity caused by drugs or diseases.

Physicochemical Properties

Molecular Formula	C34H24O5
Molecular Weight	512.551369667053
Exact Mass	512.162
CAS #	97744-44-0
PubChem CID	371436
Appearance	Yellow to orange solid powder
LogP	7.5
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	8
Heavy Atom Count	39
Complexity	980
Defined Atom Stereocenter Count	0
SMILES	C1=CC=C(C=C1)COC2=CC3=C(C=C2)C(=C4C=CC(=O)C=C4O3)C5=CC=CC=C5C(=O)OCC6=CC=CC=C6
InChi Key	YZJGKSLPSGPFEV-UHFFFAOYSA-N
InChi Code	InChI=1S/C34H24O5/c35-25-15-17-29-31(19-25)39-32-20-26(37-21-23-9-3-1-4-10-23)16-18-30(32)33(29)27-13-7-8-14-28(27)34(36)38-22-24-11-5-2-6-12-24/h1-20H,21-22H2
Chemical Name	benzyl 2-(3-oxo-6-phenylmethoxyxanthen-9-yl)benzoate
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	Experimental procedure for the metabolism of dibenzylfluorescein, catalyzed by P450 and affected by alkali [3]: Reaction process: P450 dealkylates dibenzylfluorescein to produce fluorescein benzyl ester, which, in the presence of sodium hydroxide, can hydrolyze into fluorescein. Dibenzylfluorescein is also broken down into fluorescein benzyl ether by adding 2 M sodium hydroxide. 1. This 150 μL reaction system contains the following components for analyzing CYP2C19 enzyme activity and inhibition: 0.1 M Tris-HCl buffer (pH 7.4), 10 μM Dibenzylfluorescein, 15 pmol CYP2C19 enzyme, and 50 μL NADPH regeneration system. 1.13 mM NADP, 12.5 mM isocitrate, 56.33 mM KCl, 187.5 mM Tris-HCl, pH 7.4, 12.5 mM MgCl2, 0.0125 mM MnCl2, and 0.075 U/ml isocitrate dehydrogenase make up the NADPH regeneration system. 2. Incubate samples for 30 to 60 minutes at 37°C. After centrifuging the reaction and quickly chilling it to 4°C, the supernatant was subjected to LC-MS analysis. 3. Pure Dibenzylfluorescein, Fluorescein Benzyl Ester, Fluorescein Benzyl Ether, and Fluorescein (10 μM) were used for analysis, both in the presence and absence of 2 M NaOH.
ADME/Pharmacokinetics	Metabolism / Metabolites Dibenzylfluorescein has known human metabolites that include Fluorescein benzyl ester and 2-[6-(benzyloxy)-3-oxo-3H-xanthen-9-yl]benzoic acid.
References	[1]. Substrate-dependent modulation of CYP3A4 catalytic activity: Analysis of 27 test compounds with four fluorometric substrates. Drug Metabolism and Disposition 28(12), 1440-1448 (2000). [2]. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab. Dispos. 32(7), 699-706 (2004). [3]. Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics. Drug Metabolism and Disposition 39(3), 412-418 (2011). [4]. Altered human CYP3A4 activity caused by Antley-Bixler syndrome-related variants of NADPH-cytochrome P450 oxidoreductase measured in a robust in vitro system. Drug Metabolism and Disposition 40(4), 754-760 (2012).

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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	1.9510 mL	9.7551 mL	19.5103 mL
	5 mM	0.3902 mL	1.9510 mL	3.9021 mL
	10 mM	0.1951 mL	0.9755 mL	1.9510 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.