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| 1mg |
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| Other Sizes |
| Targets |
The primary target of NHC-triphosphate tetraammonium is the viral RNA-dependent RNA polymerase (RdRp). This compound acts as a weak alternative substrate for the viral replication complex. When the viral RdRp incorporates NHC-triphosphate into the growing RNA chain instead of the natural cytidine triphosphate (CTP), it leads to the production of mutated viral genomes. This can happen through two primary mechanisms: non-obligate chain termination, where the added nucleotide prevents further extension, or viral error catastrophe (lethal mutagenesis), where the viral genome accumulates so many mutations that the resulting virus is no longer viable. This mechanism is distinct from most other antiviral drugs and provides a high barrier to resistance development. NHC-TP shows a high degree of selectivity for viral RdRps over host cellular DNA and RNA polymerases, contributing to its safety profile.
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| ln Vitro |
HCV replicon cells are treated with 10 μM 3H-labeled NHC in an intracellular metabolism test. After 1, 2, and 8 hours of incubation, intracellular nucleotide levels are measured. NHC is quickly transformed into the mono-, di-, and triphosphate forms; within 8 hours, NHC-TP can reach 71.12 pM[1]. In the absence of NHC-triphosphate tetraammonium (NHC-TP) (3-5 μM), full-length polymerization products result, and it may be a poor substitute substrate. Furthermore, in cell-free HCV NS5B polymerization events, an evident electrophoretic shift is seen, and the molecular weight of the polymerization product increases by 16 (one extra oxygen) upon insertion of NHC-TP rather than CTP[1]. NHC (10–50 μM; 4 h) or a McGuigan phosphoramidate prodrug of NHC are cultured in Huh-7 cells. LC-MS/MS is used to evaluate the intracellular levels of the primary chemicals and phosphorylated metabolites. While NHC-triphosphate tetraammonium continues to be the most prevalent metabolite, trace levels of NHC-monophosphate (MP) and NHC-diphosphate (DP) are also visible[2]. The metabolite NHC-triphosphate tetraammonium (NHC-TP) may act as a nonobligate chain terminator and directly target the viral polymerase. Through chain termination or mutagenesis, it inhibits early negative-strand RNA synthesis in a significant way. This can potentially obstruct the proper assembly of replicase complexes.
In an intracellular metabolism assay using HCV replicon cells treated with 10 uM 3H-labeled NHC, NHC-triphosphate (NHC-TP) reaches up to 71.12 pM after 8 hours, highlighting its efficient generation. In a cell-free HCV NS5B polymerase activity assay, NHC-TP (5-40 uM) acts as a weak alternative substrate. Its incorporation instead of CTP increases the molecular weight of the polymerization product by 16 Da (one extra oxygen) per event and results in a distinct electrophoretic mobility shift on a polyacrylamide gel. In Huh-7 cells incubated with NHC (10-50 uM for 4 hours), intracellular metabolism studies using LC-MS/MS have identified NHC-triphosphate as the most abundant phosphorylated metabolite, while only small amounts of the mono- and diphosphate are observed. |
| ln Vivo |
No specific in vivo data is available for this tetraammonium salt. The in vivo effects of the parent NHC prodrug (molnupiravir) are well-documented. In mouse models of influenza, SARS-CoV-2, and other RNA viruses, the prodrug has shown robust antiviral efficacy. Following oral administration, the prodrug is rapidly converted to NHC, which is then taken up by cells and phosphorylated to the active triphosphate metabolite (NHC-TP). NHC-TP is believed to directly target the viral polymerase and behave as a nonobligate chain terminator, playing a pivotal role in inhibiting early negative-strand RNA synthesis. This action interferes with the correct formation of the viral replicase complex. The tetraammonium salt is a research-grade tool for in vitro mechanistic studies and analytical chemistry.
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| Enzyme Assay |
Cell-free RNA polymerase assays are used to evaluate the direct inhibitory activity of NHC-triphosphate tetraammonium. The assay is performed in a 96-well format. Each well contains 20 uL of assay buffer (50 mM Tris-HCl pH 7.5, 10 mM NaCl, 2 mM MnCl2, 5 uM of a biotinylated RNA template-primer). Serial dilutions of NHC-triphosphate tetraammonium (0.1-100 uM) are mixed with a fixed concentration of CTP (e.g., 2 uM) and 100 uM each of ATP, GTP, and UTP. The reaction is initiated by the addition of 50 nM purified SARS-CoV-2 RdRp (nsp12-nsp7-nsp8 complex) and incubated at 30degC for 2 hours. The reaction is stopped with EDTA (final 25 mM). The reaction mixture is transferred to a Streptavidin-coated plate and incubated for 1 hour at room temperature. After washing, an anti-RNA antibody conjugated to HRP is added, followed by a chemiluminescent substrate. The signal is read on a luminometer. The IC50 is calculated by plotting the relative luminescence units (RLU) vs. the log concentration of the inhibitor. Incorporation of NHC-TP can also be confirmed by sequencing of the RNA products or by mass spectrometry.
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| Cell Assay |
To study the intracellular pharmacology of NHC, a common protocol uses Huh-7 or Calu-3 cells. Cells are seeded in 6-well plates at 5×10⁵ cells per well and allowed to grow for 24 hours. The cells are then treated with the parent compound, NHC (e.g., 0.1-100 uM) or the prodrug, for 2-24 hours. At the end of the incubation, cells are washed twice with ice-cold PBS. Intracellular metabolites are extracted by adding 1 mL of ice-cold 80% methanol containing an internal standard (e.g., a stable isotope-labeled analog of NHC-TP). The cells are scraped off the plate and the mixture is incubated at -80degC for 1 hour. The lysate is then centrifuged at 15,000 g for 15 minutes at 4degC. The supernatant is transferred to a clean tube and dried under a gentle stream of nitrogen. The dried residue is reconstituted in water:methanol (1:1 v/v) and analyzed by LC-MS/MS. NHC-triphosphate tetraammonium is used as the calibration standard. The MS/MS is operated in negative ion MRM mode, monitoring transitions specific to the triphosphate moiety. The data is used to calculate the concentration of the active triphosphate inside the cells.
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| Animal Protocol |
Although the charged triphosphate is not used in vivo, the parent prodrug molnupiravir is dosed orally in animals. For example, to evaluate antiviral efficacy, BALB/c mice are infected intranasally with mouse-adapted SARS-CoV-2 (MA10) or influenza A virus. At 4 hours post-infection (Day 0), mice are treated with the prodrug (e.g., 250 mg/kg) by oral gavage twice daily (BID) for 5 days. Body weight is measured daily. On day 2 or day 5 post-infection, some animals are euthanized, and lungs and nasal turbinates are collected. These tissues are homogenized, and viral RNA is extracted for qRT-PCR to determine viral load. To quantify the active metabolite, lung homogenates can be processed for LC-MS/MS analysis. The tetraammonium salt of NHC-TP is used as the analytical standard to generate a calibration curve. The concentration of the active metabolite in the lung tissue is correlated with the reduction in viral load to determine the efficacy of the treatment.
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| ADME/Pharmacokinetics |
The tetraammonium salt form of NHC-triphosphate has a molecular formula of C9H28N7O15P3 and a molecular weight of 567.28 g/mol. The lyophilized powder should be stored at -80degC, protected from light and stored under an inert atmosphere like nitrogen to maximize stability. It is highly soluble in water (≥200 mg/mL, 352.56 mM). Solutions should be stored at -80degC for up to 6 months and at -20degC for up to 1 month. The tetraammonium salt is specifically chosen to maximize aqueous solubility and stability, making it ideal for preparing concentrated stock solutions for biochemical assays. The compound is membrane-impermeable, so it is used as a standard or in cell-free systems. The CAS number for the free acid is 39023-73-9.
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| Toxicity/Toxicokinetics |
This product is for research use only and is not intended for clinical use. No specific toxicity data is available for the tetraammonium salt form. Ammonium salts can be irritating to the eyes, respiratory system, and skin. Therefore, standard laboratory safety precautions, including the use of personal protective equipment (PPE) such as gloves, lab coats, and safety glasses, should be strictly observed when handling the compound. Avoid generating dust. The active metabolite NHC-triphosphate is a ribonucleotide analog; while it has a high selectivity for viral polymerases, there may be theoretical risks of host cell toxicity at very high concentrations. The product is for non-human research only.
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| References | |
| Additional Infomation |
This compound is the active antiviral metabolite of the drug molnupiravir (EIDD-2801), which has been clinically approved for the treatment of COVID-19. The tetraammonium salt is the preferred salt form for many research applications due to its enhanced solubility and stability compared to the tetrasodium salt or free acid. It is a weak alternative substrate for viral RNA-dependent RNA polymerase, which acts as a non-obligate chain terminator and causes lethal mutagenesis. This compound is widely used in virology research to study the replication of RNA viruses such as influenza, SARS-CoV-2, RSV, and HCV. Researchers use it to screen for viral resistance, to characterize novel viral polymerases, and to understand the molecular mechanism of mutagenesis. It is also used in drug development to evaluate the potential of new nucleoside analogs.
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| Molecular Formula |
C9H28N7O15P3
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| Molecular Weight |
567.28
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| Related CAS # |
NHC-triphosphate;34973-27-8;NHC-triphosphate tetrasodium
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| Appearance |
White to off-white 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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). 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) |
H2O :~200 mg/mL (~352.56 mM)
DMSO :~170 mg/mL (~299.68 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 4.25 mg/mL (7.49 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 42.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 4.25 mg/mL (7.49 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 42.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: 100 mg/mL (176.28 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 | 1.7628 mL | 8.8140 mL | 17.6280 mL | |
| 5 mM | 0.3526 mL | 1.7628 mL | 3.5256 mL | |
| 10 mM | 0.1763 mL | 0.8814 mL | 1.7628 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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