-Carboxy-2',7'-dichlorofluorescein diacetate N-succinimidyl ester (cDCFDASE) Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C24H14O7CL2.C6H7NO4 |
| Molecular Weight | 642.39384 |
| Exact Mass | 641.049 |
| CAS # | 147265-60-9 |
| PubChem CID | 4351872 |
| Appearance | Typically exists as solid at room temperature |
| Melting Point | ≥220ºC(lit.) |
| LogP | 4.963 |
| Hydrogen Bond Donor Count | 0 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 43 |
| Complexity | 1170 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CC(ON1C(=O)CCC1=O)=O.CC(OC1=C(Cl)C=C2C3(C4=CC(Cl)=C(OC(=O)C)C=C4OC2=C1)OC(=O)C1=CC=CC=C31)=O |
| InChi Key | WNFQKOAKDVHNLA-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C29H17Cl2NO11/c1-12(33)39-23-10-21-17(8-19(23)30)29(18-9-20(31)24(40-13(2)34)11-22(18)41-21)16-4-3-14(7-15(16)28(38)42-29)27(37)43-32-25(35)5-6-26(32)36/h3-4,7-11H,5-6H2,1-2H3 |
| Chemical Name | (2,5-dioxopyrrolidin-1-yl) 3',6'-diacetyloxy-2',7'-dichloro-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate |
| Synonyms | Carboxy-DCFDA N-succinimidyl ester; 5(6)-Carboxy-2',7'-Dichlorofluorescein Diacetate Succinimidyl Ester; MFCD00037456; (2,5-dioxopyrrolidin-1-yl) 3',6'-diacetyloxy-2',7'-dichloro-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-carboxylate; DA-49766; |
| 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
| Targets | Fluorescent Dye |
| ln Vitro | The intracellular pH (pHin) of Leuconostoc mesenteroides subsp. mesenteroides 19D was evaluated by two different methods, fluorescent probe and ion-selective electrode. Two fluorescent probes 5 (and-6)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) and 5 (and-6)-carboxy-2',7'-dichlorofluorescein diacetate succinimidyl ester (cDCFDASE) were tested to evaluate the intracellular pH (pHin) of living cells at a medium pH (pHex) ranged from 5.0 to 6.5. Salicylic acid was used as a probe for the ion-selective electrode method. Cells kept 60-80% of cFDASE probe at all pHex values against 5-10% of cDCFDASE probe at pHex ≤ 6.0. The pHin values measured by the ion-selective electrode were higher by 0.1-0.6 pH units at pHex ranged from 5.0 to 6.5 than those determinated by fluorescent probe method. The possibility to study the intracellular pH at a wide external pH range using a single probe, and the simplicity of the material and experimental protocol may make the ion-selective electrode method most useful and easy to measure the intracellular pH of lactic acid bacteria compared with the other techniques like fluorescent probes [1]. |
| Enzyme Assay |
Bacteria Growth [1] L. mesenteroides subsp. mesenteroides 19D was obtained from the Institut National de Recherche Agronomique. The cells were grown overnight at 25 °C in a modified MRS broth, without fatty acids and acetate, and the cells were harvested in the exponential growth phase (OD575 nm = 0.7–0.8) to avoid the stress conditions that may lead to synthesize the multidrug efflux proteins. For fluorescent probe method, the cell preparation was carried out according to Breeuwer. Cells were washed twice in 50 mM N-2 hydroxyethyl piperazine-N-2-ethanesulfonic acid (HEPES) buffer (pH 8.0), incubated with 1 µM 5 (and-6)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) or 5 (and-6)-carboxy-2′,7′-dichlorofluorescein diacetate succinimidyl ester (cDCFDASE) fluorescent probes for 30 min at 30 °C. The cells were harvested by centrifugation, energized by incubation with glucose for 30 min at 30 °C in 50 mM potassium phosphate buffer. After washing by potassium phosphate buffer, cells were resuspended in 50 mM Tris-(hydroxymethyl)-aminomethane-2-(N-morpholino) ethanesulfonic acid (TRIS–MES) buffer at an appropriate pH, and placed on ice until use. For ion-selective method, the cells were washed twice in 100 mM potassium phosphate buffer (pH 7.0) and energized by incubation with 0.5 M glucose at 30 °C for 30 min. The cells were concentrated at 18 mg protein/mL in the potassium phosphate buffer at appropriate pH using a Beckman J2-HC centrifuge with a JA-10 rotor for 5 min and 3500×g, and cells were then placed on ice until use. |
| References |
[1]. Comparison Between Fluorescent Probe and Ion-Selective Electrode Methods for Intracellular pH Determination in Leuconostoc mesenteroides. Curr Microbiol. 2018 Nov;75(11):1493-1497. |
| Additional Infomation | In the present study, the intracellular pH of L. mesenteroides subsp. mesenteroides 19D was evaluated by two different methods, fluorescent probes and ion-selective electrode. These bacteria keep an alkaline pH inside of the cell by homeostasis system. For fluorescent probe method, L. mesenteroides subsp. mesenteroides 19D exhibited a low retention of cDCFDASE probe. Glaasker et al. reported that certain fluorescent probes were not suitable for application in bacteria because of their high rate of passive leakage from the cells. Calibration curves of fluorescent probes showed that the pHin determination at pHex inferior to 5.5 was not recommended for cFDASE probe because of the low correlation between the ratio R490/440 and the pH value. Similar results were reported when a correlation between pHin and R488nm/435nm ratio of both cFDASE and cDCFDASE was evaluated. The authors found that the ratio values varied between the two probes. Furthermore, the combined use of the two pH sensitive probes, i.e., cFDASE and cDCFDASE, to determine the pHin of cells over a wide range of pHex was previously studied by Ramos et al. for both Lactobacillus plantarum and Lactobacillus brevis and by Spilimbergo et al. for Listeria monocytogenes. The results of the latter two works supposed the necessity of using more than one fluorescent probe when the intracellular pH measurement of a bacterium at a wide pHex range is desired. For this reason, cDCFDASE probe which has a pKa lower than cFDASE was tested in our study (4.2 and 6.4, respectively), but this probe was slightly retained in the cells and consequently was not used afterwards. Results exhibited in Fig. 2 showed a good correlation between pHex and pHin values for pHex ranged from 5.0 to 6.5 determined by the both studied methods. Similar results were found when the pHin was evaluated for both L. plantarum and L. brevis using the two probes cFDASE and cDCFDASE [1]. |
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 | 1.5567 mL | 7.7834 mL | 15.5669 mL | |
| 5 mM | 0.3113 mL | 1.5567 mL | 3.1134 mL | |
| 10 mM | 0.1557 mL | 0.7783 mL | 1.5567 mL |