| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| Other Sizes |
| Targets |
The primary target of (R)-mchm5U is likely the mammalian tRNA methyltransferase ALKBH8 (alkylated DNA repair protein alkB homolog 8), which is responsible for the formation of the naturally occurring (S)-mchm5U (5-(S)-methoxycarbonylhydroxymethyluridine) in the wobble position of tRNAs. ALKBH8 is a multifunctional enzyme that first hydroxylates 5-methoxycarbonylmethyluridine (mcm5U) to form (S)-mchm5U, and then methylates it to form (S)-mchm5U. (R)-mchm5U is the unnatural diastereomer and may act as an inhibitor or a probe for studying the stereospecificity of ALKBH8 and other modifying enzymes. By binding to specific enzymes or receptors, (R)-mchm5U may modulate nucleic acid metabolism or signaling processes, thereby affecting cell proliferation, repair, or gene expression. However, its specific biological targets and molecular interactions are not fully characterized; it is primarily used as a reference standard for analytical chemistry and as a tool to study the stereochemical preferences of enzymes involved in tRNA modification.
|
|---|---|
| ln Vitro |
In vitro studies for (R)-mchm5U are typically analytical and enzymological. The compound is used as a reference standard for the quantification of (S)-mchm5U and its isomers in biological samples by LC-MS/MS. It is also used as a substrate or inhibitor to study the activity of the tRNA-modifying enzyme ALKBH8. In an in vitro ALKBH8 activity assay, recombinant human ALKBH8 is incubated with its natural substrate (mcm5U-containing RNA or the free nucleoside) in the presence of cofactors (Fe2+, alpha-ketoglutarate, ascorbate) and varying concentrations of (R)-mchm5U or (S)-mchm5U. The reaction products are analyzed by LC-MS/MS. (S)-mchm5U is the product of the hydroxylation reaction, while (R)-mchm5U may not be a substrate or may be a very poor substrate, and may competitively inhibit the hydroxylation of the natural substrate. The compound can also be tested as a substrate for other RNA-modifying enzymes, such as methyltransferases, to assess its ability to be further modified (e.g., methylation). The stability of (R)-mchm5U in vitro can be studied by incubating it in buffer or biological fluids (e.g., serum, cell lysates) at 37degC and measuring its concentration over time by LC-MS/MS. The half-life of the compound is determined. As a modified nucleoside, it may be more stable than unmodified uridine due to the presence of the bulky substituent. The compound's interaction with RNA can be studied by NMR or molecular dynamics simulations to understand how the stereochemistry of the modification affects RNA structure and dynamics.
|
| ln Vivo |
In vivo activity data for (R)-mchm5U is not available, as it is not a drug. It is a research tool primarily used in vitro. The compound could be administered to cells in culture (e.g., HEK293, HeLa) to study its effects on RNA modification and gene expression. For example, cells can be treated with (R)-mchm5U (1-100 uM) for 24-48 hours, and the levels of modified nucleosides in tRNA can be analyzed by LC-MS/MS to see if the unnatural isomer is incorporated into RNA or if it affects the levels of natural modifications. The effects on cell proliferation and viability can be assessed by MTT assay. However, no specific in vivo studies have been reported.
|
| Enzyme Assay |
Non-cell-based assays for (R)-mchm5U primarily involve analytical methods for its detection and quantification, as well as enzyme kinetics with ALKBH8. For LC-MS/MS analysis, a standard protocol uses a C18 reverse-phase column (e.g., 150 × 2.1 mm, 2.7 um) with a mobile phase of 0.1% formic acid in water (A) and acetonitrile (B). A gradient elution is typically employed. The mass spectrometer is operated in positive ion mode with multiple reaction monitoring (MRM) transitions for (R)-mchm5U (m/z 332.1 → 200.1, or other diagnostic transitions). Calibration curves are prepared using the purified compound. For ALKBH8 activity assay, recombinant ALKBH8 (0.1-1 ug) is incubated with 10 uM of the substrate (e.g., mcm5U-RNA or free nucleoside) in assay buffer (50 mM Tris-HCl pH 7.5, 2 mM FeSO4, 2 mM alpha-ketoglutarate, 2 mM ascorbate, 0.1 mM DTT) for 60 minutes at 37degC. (R)-mchm5U (0.1-100 uM) is added to the reaction mixture to test for inhibition. The reaction is stopped by adding 10% TCA, and the supernatant is analyzed by LC-MS/MS to quantify the product (S)-mchm5U. The IC50 for inhibition is calculated. For binding studies, surface plasmon resonance (SPR) can be used to measure the binding of (R)-mchm5U to ALKBH8. ALKBH8 is immobilized on a sensor chip, and varying concentrations of (R)-mchm5U (0.1-1000 uM) are injected. The KD is calculated from the steady-state binding response. The compound may also bind to RNA; this can be studied by UV melting experiments. A short RNA oligonucleotide (e.g., containing the anticodon loop) is synthesized with (R)-mchm5U at a specific position. The thermal stability (Tm) of the RNA is measured by monitoring the absorbance at 260 nm as the temperature is increased. The Tm of the modified RNA is compared to that of the unmodified or the natural (S)-isomer-containing RNA. The (R)-isomer may destabilize or stabilize the RNA structure differently than the (S)-isomer.
|
| Cell Assay |
For cell-based studies, cells (e.g., HEK293, HeLa, or cancer cell lines) are cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. Cells are seeded in 6-well plates (5×10^5 cells/well) and treated with (R)-mchm5U at concentrations of 1, 10, 50, 100 uM for 24-48 hours. Control cells receive DMSO or the natural (S)-isomer. After treatment, total RNA is extracted using TRIzol reagent, and the RNA is enzymatically digested to nucleosides (using nuclease P1 and alkaline phosphatase). The resulting nucleoside mixture is analyzed by LC-MS/MS to quantify the levels of (R)-mchm5U and other modified nucleosides in the RNA. The incorporation of (R)-mchm5U into RNA would be an indicator of its metabolic stability and utilization by the cellular RNA modification machinery. To assess the effect of (R)-mchm5U on cell proliferation, cells are seeded in 96-well plates (5×10^3 cells/well) and treated with the compound (0.1-500 uM) for 48-72 hours, and viability is measured by MTT or CellTiter-Glo. The compound is expected to have low cytotoxicity (IC50 > 100 uM). For gene expression analysis, cells are treated with (R)-mchm5U (50 uM, 48 hours), and RNA-seq is performed to identify changes in gene expression, particularly in pathways related to stress response, tRNA modification, and translation fidelity. qPCR can be used to validate the expression of specific genes. For studying the effect on translation, a dual-luciferase reporter assay with a construct containing a potential wobble codon (e.g., UUG or CUG) can be used to measure the efficiency and fidelity of codon recognition by the modified tRNA. However, these assays require the introduction of the modified nucleoside into tRNAs, which is not straightforward.
|
| Animal Protocol |
In vivo protocols for (R)-mchm5U are not standard. For pharmacokinetic studies, the compound could be administered to mice (e.g., 10-50 mg/kg, i.p. or i.v.), and blood and tissue samples would be collected at various time points for LC-MS/MS analysis.
|
| ADME/Pharmacokinetics |
The compound is a small polar molecule (MW 331) and may be rapidly cleared by the kidneys. Oral bioavailability would likely be low. However, since it is not a drug, such studies are not typically performed.
|
| Toxicity/Toxicokinetics |
No toxicity data is available for (R)-mchm5U. It is a modified nucleoside, and naturally occurring modified nucleosides are generally non-toxic. In cell viability assays, the compound is expected to have an IC50 > 100 uM. Standard safety precautions for handling (gloves, lab coat, safety goggles) should be followed. The compound is for research use only and is not for human use.
|
| References |
[1]. van den Born E, et al. ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA. Nat Commun. 2011 Feb 1;2:172.
|
| Additional Infomation |
(R)-mchm5U is a diastereomer of (S)-mchm5U, which is a naturally occurring modified uridine found in the wobble position of some tRNAs (e.g., tRNAArg, tRNAGlu). The compound is used as an analytical reference standard for the identification and quantification of these modifications in RNA and as a tool to study the stereospecificity of enzymes involved in tRNA modification (e.g., ALKBH8). It is not a drug and is not FDA-approved. It is a white to off-white solid powder, soluble in DMSO (e.g., 100 mg/mL) and water (limited). The product should be stored at -20degC, protected from light and moisture. In solution (DMSO), it should be stored at -80degC. This compound is a valuable tool for studying epitranscriptomics, RNA biology, and the function of tRNA modifications in gene expression.
|
| CAS # |
89665-84-9
|
|---|---|
| Related CAS # |
(S)-mchm5U;89665-83-8
|
| Appearance |
White to off-white solid powder
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
H2O: 125 mg/mL (376.21 mM)
|
|---|---|
| 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.) |
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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
