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| 5mg |
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| Targets |
Human Endogenous Metabolite
(6S)-Tetrahydrofolic acid targets folate-requiring enzymes including thymidylate synthase (TS), serine hydroxymethyltransferase (SHMT), and dihydrofolate reductase (DHFR). As the active cofactor form, it donates one-carbon units for the synthesis of thymidylate, purines, and methionine. The (6S) diastereomer is 1000-fold more active than the (6R) form in promoting fluorodeoxyuridylate binding to thymidylate synthase and 600-fold more active as a growth factor for Saccharomyces cerevisiae. |
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| ln Vitro |
In vitro, (6S)-Tetrahydrofolic acid exhibits potent cofactor activity in folate-dependent enzyme assays. It promotes the binding of fluorodeoxyuridylate (FdUMP) to thymidylate synthase, forming a stable ternary complex with the enzyme and inhibiting thymidylate synthesis. In cell-free systems, it serves as the preferred substrate for SHMT in the conversion of serine to glycine. Its stability in aqueous buffer (pH 7.4, 37degC) is limited, with a half-life of 2-4 hours, necessitating careful handling with reducing agents such as DTT or beta-mercaptoethanol to prevent oxidation.
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| ln Vivo |
In vivo, (6S)-Tetrahydrofolic acid is rapidly cleared from circulation, with a half-life of less than 30 minutes in rat serum due to enzymatic degradation and renal excretion. It is transported into cells via folate receptors and reduced folate carriers. As an endogenous metabolite, it does not produce pharmacological effects when administered exogenously, but it can rescue cells from folate deficiency in dietary restriction models. In animal studies, it is primarily used as a nutritional supplement or as a control compound in anti-folate drug investigations.
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| Enzyme Assay |
The cofactor activity of (6S)-Tetrahydrofolic acid for thymidylate synthase is assessed in a cell-free system using purified recombinant TS enzyme. Reaction buffer (50 mM Tris-HCl pH 7.5, 25 mM MgCl2, 1 mM EDTA) contains 50 uM FdUMP, 100 uM (6S)-THF, and 10 nM TS. After 30 min at 25degC, the ternary complex is stabilized by glutaraldehyde crosslinking and analyzed by SDS-PAGE. The amount of complex correlates with TS inhibition and is quantified by densitometry. The (6R) isomer is used as a negative control to demonstrate stereospecificity.
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| Cell Assay |
Cellular activity can be assessed in folate-deficient cell culture medium. Cells (e.g., CHO or HeLa) are cultured in folate-free RPMI supplemented with dialyzed serum and varying concentrations of (6S)-THF (0.1-100 nM). After 72 h, cell proliferation is measured by MTT assay or by [3H]-thymidine incorporation. The (6S) isomer supports growth at 600-fold lower concentrations than the (6R) isomer. Enzyme activity in lysates is measured by SHMT or TS assays using radiolabeled substrates.
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| Animal Protocol |
Animal studies involving (6S)-Tetrahydrofolic acid are typically conducted in rats to study folate metabolism. Folate-deficient diets are used to induce deficiency, followed by oral or intraperitoneal administration of (6S)-THF (0.1-5 mg/kg/day). Blood samples are collected at multiple time points for analysis of homocysteine, methionine, and folate levels by LC-MS/MS. Tissues (liver, kidney) are harvested for analysis of folate polyglutamates and one-carbon metabolites. The compound restores folate pools and corrects hyperhomocysteinemia in deficient animals.
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| ADME/Pharmacokinetics |
(6S)-Tetrahydrofolic acid is a water-soluble compound with limited stability in aqueous solution, requiring reducing agents (5-10 mM DTT or beta-mercaptoethanol) to prevent oxidation. Its half-life in buffer (pH 7.4, 37degC) is 2-4 hours, and in rat serum it is <30 minutes. It is rapidly cleared from circulation via renal excretion of intact molecule and folate-binding protein-mediated uptake. For storage, powder should be kept at -20degC under inert atmosphere; solutions should be prepared fresh for each experiment to avoid oxidative degradation.
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| Toxicity/Toxicokinetics |
(6S)-Tetrahydrofolic acid has low intrinsic toxicity as an endogenous metabolite. The oral LD₅0 is estimated to be >2000 mg/kg in rodents. At high pharmacological doses, it may cause minor gastrointestinal disturbance. Potential for skin and eye irritation is low. It is not classified as a carcinogen by IARC or NTP. Standard laboratory precautions should be used when handling the powder: wear gloves, goggles, and a lab coat. It is not for human therapeutic use.
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| References | |
| Additional Infomation |
(6S)-Tetrahydrofolic acid is not an approved drug but is used as a research tool in folate metabolism studies, anti-cancer drug mechanism investigations (particularly regarding methotrexate and 5-fluorouracil), and one-carbon metabolism research. It serves as the physiologically relevant cofactor standard for enzyme assays. The marked stereospecificity between (6S) and (6R) diastereomers makes it invaluable for studying folate-dependent enzyme mechanisms and folate antagonist development.
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| Molecular Formula |
C19H23N7O6
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| Molecular Weight |
445.43
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| Exact Mass |
445.171
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| CAS # |
71963-69-4
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| Appearance |
Typically exists as solid at room temperature
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| Synonyms |
(6S)-5,6,7,8-Tetrahydrofolic acid; (6S)-Tetrahydrofolic acid
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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 |
| 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) |
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
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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 | 2.2450 mL | 11.2251 mL | 22.4502 mL | |
| 5 mM | 0.4490 mL | 2.2450 mL | 4.4900 mL | |
| 10 mM | 0.2245 mL | 1.1225 mL | 2.2450 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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