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| Targets |
The primary molecular target of 5-Fluoroorotic acid is orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), the enzyme encoded by the URA3 gene in yeast and the pyrF gene in bacteria. 5-FOA itself is nontoxic to cells; however, when taken up by cells expressing functional OMP decarboxylase, it is converted to 5-fluorouracil (5-FU). The resulting 5-FU is then further metabolized to 5-fluorouridine monophosphate (5-FUMP) which incorporates into RNA, and to 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP) which acts as a potent inhibitor of thymidylate synthase, ultimately causing cessation of DNA synthesis and cell death.
5-FOA inhibits thymidylate synthase (TS), an enzyme essential for DNA synthesis. In parasitic systems such as Plasmodium falciparum, its primary mechanism of action is through the inhibition of thymidylate synthase-dihydrofolate reductase (TS-DHFR). In yeast genetics, it targets the URA3 gene product. This dual activity makes it a versatile tool in both molecular biology and anti-infective research. |
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| ln Vitro |
In vitro, 5-Fluoroorotic acid exhibits antimicrobial activity against certain fungi. It possesses a well-expressed anticandidal effect close to that of 5-fluorocytosine, as well as moderate antidermatophytal effects. In cultured yeast cells, 5-FOA at a working concentration of 1 mg/mL effectively selects against Ura⁺ cells (those expressing functional URA3) within 4-7 days of incubation at 30°C, while Ura⁻ cells (carrying URA3 mutations) are resistant and grow normally. Lower concentrations (500 μg/mL) can be used but result in increased background growth of Ura⁺ cells.
5-FOA acts as an inhibitor of thymidylate synthase. In yeast, cells expressing the URA3 gene convert 5-FOA to 5-fluorouracil, which is subsequently metabolized to 5-fluorodeoxyuridine monophosphate, a potent inhibitor of thymidylate synthase, leading to cell death. It exhibits well-expressed anticandidal effects and moderate antidermatophytal activity. It also possesses antimalarial activity, with its primary target in parasites being thymidylate synthase. |
| ln Vivo |
In vivo studies in rats have demonstrated that 5-Fluoroorotic acid affects ribosomal RNA (rRNA) maturation in liver cells. Following 5-FOA administration alone or in combination with gamma irradiation, incorporation of radioactive orotic acid into 45S rRNA precursors was elevated because maturation was blocked, and the appearance of 28S and 18S subunits was delayed. In adrenalectomized rats, 5-FOA administration alone produced a maturation block within 0.5 hours. These results suggest that 5-FOA selectively inhibits rRNA maturation in liver cells, and this response is influenced by the presence of adrenal glands.
5-FOA is orally active, though specific PK data are not provided. In murine models, it has demonstrated antimalarial activity. Its use as a selective agent in yeast genetics is well-established. When supplied in culture media, it does not affect cells lacking a functional URA3 gene. The compound is stable and stored as a powder at -20degC. |
| Enzyme Assay |
A specific non-cellular enzyme binding assay for 5-FOA is not described in the literature, as the compound functions as a prodrug requiring enzymatic conversion. However, the activity of OMP decarboxylase, the primary enzyme target, can be assessed using cell-free extracts. A standard protocol for determining resistance involves growing yeast cells in liquid YPD medium prior to selection on 5-FOA plates. For enzyme activity measurement, cell-free extracts are incubated with orotidine-5'-monophosphate (OMP) as substrate, and the production of UMP (uridine-5'-monophosphate) is quantified by HPLC or spectrophotometric methods. The presence of 5-FOA can be monitored as a competitor or alternative substrate in such assays.
The inhibitory activity of 5-FOA against thymidylate synthase can be assessed using a cell-free enzyme assay. Procedure: Recombinant thymidylate synthase (10 ng) is incubated with varying concentrations of 5-FOA (0.1-1000 nM) in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM EDTA). The reaction is initiated by adding the substrate dUMP (50 microM) and the cofactor CH2THF (50 microM). After 30 minutes at 37degC, the reaction is stopped, and product formation is measured spectrophotometrically at 340 nm. The IC50 is calculated from the dose-response curve. |
| Cell Assay |
A standard cell assay protocol for 5-FOA utilizes yeast cells as the model system. Cells are pregrown in liquid YPD medium or on YPD plates prior to selection on 5-FOA medium. For selection, up to 10⁷ cells are plated on a single 50 mm Petri dish containing 5-FOA medium (typically 1 mg/mL 5-FOA in synthetic minimal medium with appropriate supplements). Resistant colonies (Ura⁻ cells) grow within 4-7 days at 30°C. The 5-FOA medium is prepared by adding 5-FOA powder to standard yeast synthetic medium (e.g., SC-uracil medium) to a final concentration of 1 mg/mL. The mixture must be heated or stirred to dissolve the 5-FOA powder, as it is sparingly soluble in water. Alternatively, a 100× stock solution can be prepared by dissolving 100 mg 5-FOA in 1 mL anhydrous DMSO.
The cytotoxic activity of 5-FOA can be assessed in yeast cell cultures. Procedure: Yeast cells (e.g., Saccharomyces cerevisiae) with a functional URA3 gene are seeded in 96-well plates (1x10⁵ cells/well) in synthetic defined medium lacking uracil. Varying concentrations of 5-FOA (0.1-1000 ug/mL) are added, and plates are incubated at 30degC for 24-48 hours. Cell growth is measured by OD600, and the minimum inhibitory concentration (MIC) is determined. For antimalarial assays, Plasmodium falciparum cultures are used, and parasite growth is measured by [3H]-hypoxanthine incorporation after 48-72 hours of treatment. |
| Animal Protocol |
An in vivo protocol for studying 5-FOA effects has been described using rat models. Adult rats (both intact and adrenalectomized) are administered 5-Fluoroorotic acid via injection or oral administration. In a representative study, rats were exposed to gamma radiation (1800 rad) and/or treated with 5-FOA. Following treatment, liver tissues were collected at various time points (e.g., 0.5, 6, 12 hours post-administration). Nuclear and cytoplasmic fractions were prepared, and ribosomal RNA (rRNA) was extracted. The maturation status of rRNA was analyzed by gel electrophoresis to examine 45S precursor accumulation and the appearance of 28S and 18S mature subunits. Incorporation of radiolabeled [¹⁴C]-orotic acid (administered alongside treatments) into rRNA can be measured as an additional endpoint.
The in vivo efficacy of 5-FOA can be evaluated in a mouse model of malaria. Procedure: Female BALB/c mice (6-8 weeks) are infected intraperitoneally with 1x10⁷ Plasmodium berghei-infected red blood cells. Mice are randomized into groups (n=10). 5-FOA is dissolved in a suitable vehicle (e.g., 0.5% methylcellulose) and administered orally at doses of 10, 30, and 100 mg/kg once daily for 4 days. Parasitemia is monitored daily by examining Giemsa-stained blood smears. Survival is recorded for 30 days. The positive control group receives chloroquine (10 mg/kg). A reduction in parasitemia and increased survival indicates efficacy. |
| ADME/Pharmacokinetics |
Specific pharmacokinetic parameters for 5-Fluoroorotic acid have not been characterized in the literature, as the compound is primarily used as a research tool in microbial genetics rather than as a therapeutic agent. However, based on its structural similarity to 5-fluorouracil, the compound is expected to undergo similar metabolic processing when converted to 5-FU in cells expressing OMP decarboxylase. 5-FOA is highly stable in solution and can be stored as a powder at -20°C for up to 3 years, as a solution in DMSO at -20°C for up to 6 months, or at 4°C for short-term use. The compound is stable during autoclaving (121°C), allowing for preparation of media with 5-FOA prior to sterilization.
Specific PK data for 5-FOA is limited. As a small molecule, it is orally active and is typically administered via gavage in animal studies. It is stable in solution when stored at -80degC for 6 months or -20degC for 1 month. The compound is a reference standard with a purity of 99.39% and should be stored as a powder at -20degC. |
| Toxicity/Toxicokinetics |
5-Fluoroorotic acid is classified as an irritant, and the MSDS recommends working in a fume hood to avoid inhalation of dust particles. DMSO solutions pose additional risks related to DMSO's ability to enhance skin absorption of other molecules. The compound itself is not particularly toxic to cells lacking URA3 or pyrF expression; its toxicity is conditional upon enzymatic conversion to 5-fluorouracil by OMP decarboxylase. In yeast selection protocols, 5-FOA is used at concentrations up to 1 mg/mL without adverse effects on resistant Ura⁻ cells. No specific data on acute toxicity (LD50), chronic toxicity, genotoxicity, or reproductive toxicity is available in standard literature. The compound is strictly intended for research use only, not for human diagnostic or therapeutic applications.
5-FOA is toxic to yeast cells expressing the URA3 gene, but non-toxic to URA3-deficient strains. In mammalian systems, it exhibits moderate toxicity. It is teratogenic in animals, causing developmental abnormalities. Safety precautions should be taken to avoid ingestion or inhalation. The compound is for research use only and is not an approved drug. |
| References | |
| Additional Infomation |
5-Fluoroorotic acid monohydrate (5-FOA) is a reference compound widely used in yeast molecular genetics for the positive selection of ura3 mutants. It is an inhibitor of thymidylate synthase and acts as a selective agent. It has also demonstrated antimalarial and anticandidal activities. The compound is stored as a powder at -20degC and has a purity of ≥98%. It is for research use only and is not an approved drug.
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| Molecular Formula |
C5H5FN2O5
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|---|---|
| Molecular Weight |
192.10
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| Exact Mass |
192.018
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| CAS # |
220141-70-8
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| PubChem CID |
16212749
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| Appearance |
White to light yellow solid powder
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| Density |
1.81g/cm3
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| Melting Point |
278ºC (dec.)(lit.)
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| LogP |
0
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
13
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| Complexity |
309
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| Defined Atom Stereocenter Count |
0
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| SMILES |
FC1C(N([H])C(N([H])C=1C(=O)O[H])=O)=O.O([H])[H]
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| InChi Key |
LODRRYMGPWQCTR-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C5H3FN2O4.H2O/c6-1-2(4(10)11)7-5(12)8-3(1)9;/h(H,10,11)(H2,7,8,9,12);1H2
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| Chemical Name |
5-fluoro-2,4-dioxo-1H-pyrimidine-6-carboxylic acid;hydrate
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| Synonyms |
5-Fluorouracil-6-carboxylic acid Mono(hydrate); 5-FOA; 5-Fluoroorotic acid monohydrate; RefChem:535986; 678-852-3; 5-Fluoroorotic acid hydrate; 5-fluoro OA
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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: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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 | 5.2056 mL | 26.0281 mL | 52.0562 mL | |
| 5 mM | 1.0411 mL | 5.2056 mL | 10.4112 mL | |
| 10 mM | 0.5206 mL | 2.6028 mL | 5.2056 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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