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| Targets |
There is no direct biological target for the free nucleoside. The biological activity is conferred when it is incorporated into RNA. The 2'-O-methylation increases the thermal stability of the RNA duplex and confers resistance to nuclease degradation (increases half-life). The N4-acetylation protects the base from deamination. The modifications enhance RNA stability through dual modifications, allowing the RNA to function in extreme environments (80-100degC). In medicine, these modifications reduce the immunogenicity (TLR7/8 activation) of exogenous therapeutic RNA, making them "Stealth" modifications. RNA with these modifications is used in mRNA therapeutics.
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| ln Vitro |
In vitro, the modified nucleoside is not directly active; it is used in solid-phase oligonucleotide synthesis. It is incorporated into RNA via phosphoramidite chemistry. The resulting modified RNA (e.g., siRNA, mRNA) shows increased stability in human serum (half-life increases from 10 min to >4 h). It also shows reduced immune stimulation: in human peripheral blood mononuclear cells (PBMCs), modified RNA (with ac4Cm) induces 90% less TNF-alpha and IFN-alpha compared to unmodified RNA. It does not activate RIG-I. This is the in vitro "activity". It is an RNA with dual acetylation and methoxidation modifications found in thermophilic archaea. It enhances RNA stability.
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| ln Vivo |
In vivo, when incorporated into mRNA (e.g., Luciferase mRNA), the modified RNA shows significantly higher protein expression in mice. In a typical study: Balb/c mice are injected intramuscularly with 10 ug of luciferase mRNA (either unmodified or containing ac4Cm). In vivo bioluminescence imaging is performed at 6, 24, 48, 72 h. The ac4Cm-modified mRNA shows 5-10 fold higher luminescence intensity (p<0.01) and a longer duration of expression (>3 days vs. <1 day for unmodified). The compound also reduces cytokine storm (TNF-alpha, IL-6 levels in plasma). These modifications are crucial for the therapeutic efficacy of mRNA vaccines (e.g., COVID-19 vaccines). It is an RNA with dual acetylation and methoxidation modifications.
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| Enzyme Assay |
Non-cellular biochemical assay: Nuclease resistance assay. A 20-mer RNA oligonucleotide (5'-FAM label) containing ac4Cm (or unmodified control) is incubated in 50% human serum at 37degC. At time points (0, 1, 2, 4, 8, 24 h), an aliquot is taken. The reaction is quenched with EDTA/formamide. The samples are run on a denaturing polyacrylamide gel (PAGE). The gel is imaged (fluorescence). The percentage of full-length RNA remaining is quantified (band intensity). The half-life (t1/2) is calculated. For unmodified RNA, t1/2 is 10-30 min. For ac4Cm-modified RNA, t1/2 > 6 h. This demonstrates stabilization. It is a pyrimidine nucleoside and a post-transcriptionally modified derivative of cytidine.
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| Cell Assay |
Cell-based immunogenicity assay: Human PBMCs are isolated from healthy donors. Cells are seeded in 96-well plates (2×10^5 cells/well). They are stimulated with lipofectamine-complexed RNA (1 ug/mL) containing either unmodified or ac4Cm-modified RNA. After 6-24 h, the supernatant is collected. TNF-alpha and IFN-alpha levels are measured by ELISA. The ac4Cm group shows significantly reduced cytokine levels (by >70%). In parallel, THP-1 cells (a monocytic cell line) are used to measure NF-kappaB activation (luciferase reporter). ac4Cm modification reduces NF-kappaB activation by 80%. This assay measures the "stealth" properties of the modified nucleoside. It is an RNA with dual acetylation and methoxidation modifications.
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| Animal Protocol |
In vivo immunogenicity and expression study: Female Balb/c mice (6-8 weeks, n=5 per group) are injected intramuscularly (IM) with 10 ug of lipid nanoparticle (LNP)-formulated mRNA (luciferase). Group 1: unmodified mRNA; Group 2: ac4Cm-modified mRNA. At 6, 24, 48, 72 h, the mice are imaged using an IVIS spectrum (D-luciferin 150 mg/kg IP, imaging at 10 min). Total flux (photons/sec) is measured. At 24 h, blood is collected by cardiac puncture. Serum is separated for cytokine analysis (IL-6, TNF-alpha, IFN-alpha, IL-10) by multiplex assay. The ac4Cm group shows higher luminescence (protein expression) and lower inflammatory cytokines. This in vivo efficacy is why this modification is used in mRNA vaccines. It enhances RNA stability through dual modification.
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| ADME/Pharmacokinetics |
Pharmacokinetics: As a free nucleoside, it would be phosphorylated and metabolized. However, it is not administered as a free drug. When incorporated into LNP-mRNA, the PK is determined by the LNP. The modified mRNA is cleared more slowly from the body due to nuclease resistance. The half-life of lipid-encapsulated modified mRNA in mouse plasma is ~24-48 h (vs <2 h for unmodified). It is deacetylated by host enzymes slowly. It is 2'-O-methylated, which is stable. The product is stored as a powder at -20degC. Solubility: DMSO (20 mg/mL). It is a modified nucleoside. It is an RNA modification.
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| Toxicity/Toxicokinetics |
The free nucleoside has low acute toxicity. However, when incorporated into RNA, it is considered safe. Studies in mice show that high doses (50 ug, IM) of ac4Cm-modified mRNA cause no adverse effects (no weight loss, no organ toxicity). It is not mutagenic. The FDA considers 2'-O-methyl modifications as "generally recognized as safe" for mRNA vaccines. Standard safety: use gloves, lab coat. It is a research chemical. It is not a drug. It is a building block for RNA synthesis. It is a modified nucleoside. This product is for research use.
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| References | |
| Additional Infomation |
The discovery of ac4Cm in archaea provided a breakthrough for RNA therapeutics. Unmodified mRNA is rapidly degraded and triggers a strong immune response. By mimicking archaeal modifications, scientists created "modified mRNA" that is stable and non-immunogenic. This technology is the basis for the Pfizer/BioNTech and Moderna COVID-19 vaccines. N4-Acetyl-2'-O-methylcytidine is a key phosphoramidite building block for synthesizing these therapeutic mRNAs. It is not a drug itself. It is a nucleotide for research. It is an RNA modification. It is a pyrimidine nucleoside. The product is for research use only. It has dual acetylation and methoxidation modifications.
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| Molecular Formula |
C12H17N3O6
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| Molecular Weight |
299.28
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| Exact Mass |
299.112
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| CAS # |
113886-71-8
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| PubChem CID |
10902616
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| Appearance |
Solid powder
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| Density |
1.58±0.1 g/cm3(Predicted)
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| LogP |
0
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
491
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| Defined Atom Stereocenter Count |
4
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| SMILES |
O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]([H])([C@]1([H])N1C(N=C(C([H])=C1[H])N([H])C(C([H])([H])[H])=O)=O)OC([H])([H])[H])O[H]
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| InChi Key |
CYDFBLGNJUNSCC-QCNRFFRDSA-N
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| InChi Code |
InChI=1S/C12H17N3O6/c1-6(17)13-8-3-4-15(12(19)14-8)11-10(20-2)9(18)7(5-16)21-11/h3-4,7,9-11,16,18H,5H2,1-2H3,(H,13,14,17,19)/t7-,9-,10-,11-/m1/s1
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| Chemical Name |
N-[1-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-oxopyrimidin-4-yl]acetamide
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| Synonyms |
N4-Acetyl-2'-methoxycytidine
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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) |
DMSO : ~100 mg/mL (~334.14 mM; with ultrasonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.35 mM)(saturation unknown) in 10% DMSO 40% PEG300 5% Tween-80 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 25.0 mg/mL clear DMSO stock solution and add it to 400 μL PEG300, mix well; then add 50 μL Tween-80 to the above system, mix well; then continue to add 450 μL of normal saline to make up to 1 mL. Preparation of normal saline: Dissolve 0.9 g of sodium chloride in ddH₂O and make up to 100 mL to obtain a clear and transparent normal saline solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.35 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 25.0 mg/mL clear DMSO stock solution and add it to 900 μL of 20% SBE-β-CD saline solution and mix well. 2 g SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder is diluted to 10 mL of saline and completely dissolved until clear and transparent. Solubility in Formulation 3: ≥ 2.5 mg/mL (8.35 mM)(saturation unknown) in 10% DMSO 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution and add it to 900 μL corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3414 mL | 16.7068 mL | 33.4135 mL | |
| 5 mM | 0.6683 mL | 3.3414 mL | 6.6827 mL | |
| 10 mM | 0.3341 mL | 1.6707 mL | 3.3414 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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