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| Targets |
B12 -processing enzyme CblC[1]
The primary target is the human B12-processing enzyme CblC (also known as MMACHC). F2PhEtyCbl binds to CblC with high affinity (Kd = 130 nM). It is processed by CblC (cleaved) and stabilizes a ternary complex with the co-substrate glutathione (GSH), acting as a potent B12 antivitamin that interferes with the normal intracellular trafficking and function of cobalamin. |
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| ln Vitro |
In vitro, F2PhEtyCbl binds to human B12-processing enzyme CblC with high affinity (KD = 130 nM). It is cleaved by CblC and forms a stable ternary complex with the co-substrate glutathione (GSH). By interfering with normal B12 processing, it acts as an antivitamin, disrupting cobalamin-dependent enzymatic pathways in cells (e.g., methionine synthase and methylmalonyl-CoA mutase).
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| Enzyme Assay |
A non-cellular binding assay is performed using surface plasmon resonance (SPR) or fluorescence polarization. Recombinant human CblC protein is immobilized on a sensor chip. F2PhEtyCbl is flowed over at increasing concentrations (0-1000 nM), and the association and dissociation rates are monitored. The dissociation constant (Kd) is calculated from the binding curves, with a reported Kd of 130 nM, indicating high-affinity binding. Cellular uptake can be measured using a chemiluminescence-based B12 assay.
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| Cell Assay |
Cellular assays measure the antivitamin activity of F2PhEtyCbl on B12-dependent enzymes. Human cell lines (e.g., HEK293 or fibroblasts) are cultured in B12-deficient medium, then supplemented with physiological concentrations of vitamin B12 (e.g., 1-10 nM) in the presence or absence of F2PhEtyCbl (0.1-10 uM). The activity of B12-dependent enzymes (e.g., methylmalonyl-CoA mutase or methionine synthase) is measured by LC-MS/MS detection of their metabolites (methylmalonic acid and homocysteine, respectively). An increase in these metabolites indicates functional B12 deficiency due to the antivitamin.
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| Animal Protocol |
In vivo experiments are typically conducted in zebrafish or mouse models. For zebrafish, embryos are placed in water containing F2PhEtyCbl (1-100 uM) for 48-96 hours. The embryos are observed for developmental abnormalities related to B12 deficiency (e.g., neural tube defects, growth retardation). For mice, the compound is administered intraperitoneally or intravenously at doses of 1-10 mg/kg, and tissues are harvested to measure B12-dependent enzyme activities and metabolite levels. As a click chemistry reagent, it can be conjugated to fluorescent tags for imaging studies.
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| ADME/Pharmacokinetics |
F2PhEtyCbl has a molecular weight of 1466.45 (C70H91CoF2N13O14P). As a cobalamin derivative, it shares the general pharmacokinetic properties of vitamin B12. It is expected to be absorbed via the intrinsic factor-mediated pathway in the gut if administered orally, but intravenous or intraperitoneal administration is more typical for research. It is soluble in water and organic solvents due to the corrin ring structure. The fluorine substitution does not drastically alter its bioavailability.
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| Toxicity/Toxicokinetics |
Toxicity data for F2PhEtyCbl is not well-documented, but as a B12 antivitamin, it is expected to induce a state of functional B12 deficiency. Chronic exposure could lead to metabolic disturbances such as elevated methylmalonic acid and homocysteine, which are neurotoxic. The compound is for research use only, not for therapeutic applications. At research doses in zebrafish (1-100 uM), it is tolerated without acute mortality.
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| References |
[1]. Ruetz M, et al. Antivitamin B12 Inhibition of the Human B12 -Processing Enzyme CblC: Crystal Structure of an Inactive Ternary Complex with Glutathione as the Cosubstrate. Angew Chem Int Ed Engl. 2017 Jun 19;56(26):7387-7392.
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| Additional Infomation |
F2PhEtyCbl is a click chemistry reagent containing an alkyne group, enabling copper-catalyzed azide-alkyne cycloaddition (CuAAc) with molecules containing azide groups. This allows for its conjugation to fluorescent probes or affinity tags for imaging and proteomics studies. It was developed as a tool to probe the B12 processing pathway and study inborn errors of cobalamin metabolism (e.g., cblC deficiency). It has no approved therapeutic status.
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| Molecular Formula |
C70H91COF2N13O14P
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| Molecular Weight |
1466.45
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| CAS # |
2101750-19-8
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| Appearance |
Brown to red solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 100 mg/mL (68.19 mM)
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| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.6819 mL | 3.4096 mL | 6.8192 mL | |
| 5 mM | 0.1364 mL | 0.6819 mL | 1.3638 mL | |
| 10 mM | 0.0682 mL | 0.3410 mL | 0.6819 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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