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| Other Sizes |
| Targets |
As a product of DNA methylation, 5-Methylcytosine itself is not a drug target but an epigenetic "mark." Its "reader" proteins, specifically the 5-methylcytosine binding domain (MBD) family of proteins, recognize and bind to 5-mC on DNA, subsequently recruiting other protein complexes (e.g., NuRD) to regulate chromatin structure and gene transcription. These "reader" proteins are considered potential therapeutic targets for treating diseases, including cancer.
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| ln Vitro |
As a component of DNA templates, 5-Methylcytosine influences in vitro transcription activity. Studies have shown that when oxidized derivatives of 5-mC, such as 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC), are located on the transcribed strand, they exhibit modest inhibitory effects on DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II. 5-Hydroxymethylcytosine (5-HmC) displayed a relatively milder blocking effect. Furthermore, oxidized derivatives of 5-mC can induce C to T transition mutations in bacterial cells at frequencies of 0.17%-1.12%.
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| ln Vivo |
The levels of 5-Methylcytosine are dynamically regulated in vivo. Research demonstrates that TET2-mediated oxidation of 5-mC plays a key regulatory role in CD8+ T cell memory differentiation in vivo following acute viral infection. Additionally, vitamin C enhances residual TET2 activity, increasing oxidized 5-methylcytosine (oxi-mC) formation and promoting active DNA demethylation via base-excision repair (BER), which slows leukemia progression.
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| Enzyme Assay |
A common non-cellular assay for measuring DNA methyltransferase (DNMT) activity is the LC-MS/MS-based method. The general protocol is as follows: 1. Reaction Setup: The reaction contains purified DNMT enzyme (e.g., DNMT1, DNMT3A/3B), a specific DNA substrate, the methyl donor S-adenosylmethionine (SAM), and the test compound. 2. Incubation: The mixture is incubated to allow the DNMT to catalyze the transfer of the methyl group from SAM to the C5 position of cytosine in DNA, forming 5-mC. 3. Sample Processing: After the reaction, the DNA is hydrolyzed into individual nucleosides. 4. LC-MS/MS Detection: The product 5-mC and unmodified cytosine (dC) are directly quantified by LC-MS/MS, and SAM and its byproduct S-adenosyl-L-homocysteine (SAH) can be measured simultaneously. This method is label-free, does not use radioactivity or antibodies, and has high sensitivity.
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| Cell Assay |
A classic cell-based protocol for analyzing changes in 5-Methylcytosine levels is the RNA Dot Blot. The procedure is as follows: 1. Cell Treatment: Culture target cells (e.g., Hep3B hepatoma or PC3 prostate cancer cells) to 80-90% confluency in plates and treat them with the test compound. 2. RNA Extraction and Quantification: After treatment, extract total RNA and quantify its concentration and purity. 3. RNA Denaturation: Denature equal amounts of RNA samples by heat. 4. Membrane Spotting and Crosslinking: Spot the denatured RNA samples directly onto a nylon or nitrocellulose membrane, then crosslink them to the membrane using UV light. 5. Immunodetection: Incubate the membrane with a primary antibody specific to 5-mC (e.g., D3S2Z rabbit mAb), followed by a labeled secondary antibody. 6. Imaging and Analysis: Visualize via chemiluminescence and quantify the spot signals using software like ImageJ.
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| Animal Protocol |
An exemplary in vivo protocol involves studying the role of TET2 in CD8+ T cell differentiation. Animal Model: T-cell specific Tet2 knockout (Tet2-cKO) mice and control mice are used. Experimental Procedure: 1. Viral Infection: Mice are infected with acute lymphocytic choriomeningitis virus (LCMV) to induce an immune response. 2. Cell Harvesting: Mice are euthanized at various time points post-infection (e.g., day 8, day 30), and T cells from the spleen and lymph nodes are isolated. 3. Phenotyping: The differentiation of CD8+ T cell memory subsets is analyzed by flow cytometry and compared between Tet2-cKO and control mice. 4. Molecular Analysis: DNA methylation and RNA sequencing are performed on the isolated T cells to analyze changes in 5-mC levels and downstream gene expression.
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| ADME/Pharmacokinetics |
Although 5-Methylcytosine itself is not commonly used as a drug, its pharmacokinetic properties can be analyzed using specific methods. Generally, 5-Methylcytosine in biological samples (e.g., plasma, tissues) can be analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) with a mobile phase containing acetonitrile and water; for mass-spectrometry compatibility, formic acid can be used instead of phosphoric acid. This liquid chromatography method is scalable and suitable for pharmacokinetic studies. Furthermore, as a water-soluble small molecule, the water solubility of 5-Methylcytosine may influence its bioavailability and distribution in the body.
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| Toxicity/Toxicokinetics |
5-Methylcytosine possesses potential mutagenicity. A primary toxicological risk stems from its spontaneous deamination. While deamination of cytosine forms uracil, which is recognized and repaired by DNA repair systems, deamination of 5-methylcytosine forms thymine, leading to a G/T mismatch. If not repaired properly, this can cause C to T transition mutations, which is considered a major reason for the high mutability of CpG islands. Furthermore, oxidized derivatives of 5-mC can also induce C to T transition mutations in bacterial cells, albeit at low frequencies (0.17%-1.12%). Aberrant levels and distribution patterns of 5-mC are strongly associated with the development of various diseases, particularly cancer.
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| References | |
| Additional Infomation |
5-Methylcytosine is a pyrimidine derivative with a methyl group at the 5-position. It is a human metabolite, belonging to the pyrimidine class of compounds, and is also a methylcytosine. Functionally, it is related to cytosine. 5-Methylcytosine is a metabolite found or produced in Escherichia coli (K12 strain, MG1655 strain). It has also been reported to exist in Luffa acutangula, Luffa aegyptiaca, and other organisms with relevant data. 5-Methylcytosine is the methylated form of cytosine, primarily found in cytosine-phosphate-guanine (CpG) islands produced by DNA methyltransferases, and may regulate gene expression. Similar to cytosine, DNA sequences containing 5-methylcytosine (5-mC) can replicate error-free, and 5-mC can pair with guanine in double-stranded DNA. However, DNA sequences with locally higher concentrations of 5-mC may exhibit lower transcriptional activity than regions with higher proportions of unmodified cytosine. 5-mC is a methylated nucleotide base found in eukaryotic DNA. In animals, cytosine methylation to form 5-methylcytosine primarily occurs in the palindromic sequence CpG. In plants, the methylated sequence is CpNpGp, where N can be any base.
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| Molecular Formula |
C5H7N3O
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|---|---|
| Molecular Weight |
125.131
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| Exact Mass |
125.058
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| CAS # |
554-01-8
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| Related CAS # |
5-Methylcytosine-d4;1219795-15-9
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| PubChem CID |
65040
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
417.3ºC at 760 mmHg
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| Melting Point |
>270ºC (>518ºCF)
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| Flash Point |
206.2ºC
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| Index of Refraction |
1.651
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| LogP |
-1.12
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
9
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| Complexity |
204
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
LRSASMSXMSNRBT-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C5H7N3O/c1-3-2-7-5(9)8-4(3)6/h2H,1H3,(H3,6,7,8,9)
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| Chemical Name |
4-amino-5-methylpyrimidin-2(1H)-one
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| Synonyms |
5-Methylcytosine 5Methylcytosine5 Methylcytosine 5 mC 5mC 5-mC
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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) |
H2O : ~7.14 mg/mL (~57.06 mM)
DMSO : ~5 mg/mL (~39.96 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 0.83 mg/mL (6.63 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 0.83 mg/mL (6.63 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 8.3 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 0.83 mg/mL (6.63 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 6.67 mg/mL (53.30 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 7.9917 mL | 39.9584 mL | 79.9169 mL | |
| 5 mM | 1.5983 mL | 7.9917 mL | 15.9834 mL | |
| 10 mM | 0.7992 mL | 3.9958 mL | 7.9917 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03526666 | COMPLETED | Other: Peripheral blood collection | AML Acute Myeloid Leukemia CMML Chronic Myelomonocytic Leukemia |
Van Andel Research Institute | 2017-11-01 | |
| NCT05318989 | COMPLETED | Genitourinary Agents | Żelazna Medical Centre, LLC | 2021-02-08 | ||
| NCT01687400 | COMPLETEDWITH RESULTS | Drug: decitabine | Leukemia, Myeloid, Acute Myelodysplastic Syndromes |
Washington University School of Medicine | 2013-02-12 | Phase 2 |
| NCT05380999 | NOT YET RECRUITING | Diagnostic Test: Methylation tests of nucleic acids extracted from mature red blood cells (RBCs) |
Lung Cancer Methylation Red Blood Cells |
The Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School | 2022-05 | |
| NCT02297009 | TERMINATED | Procedure: Buccal swab | DNA Methylation | Universitaire Ziekenhuizen KU Leuven | 2014-12 |
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