Physicochemical Properties
| Molecular Formula | C10H14N2O6 |
| Molecular Weight | 258.23 |
| Exact Mass | 258.085 |
| Elemental Analysis | C, 46.51; H, 5.46; N, 10.85; O, 37.17 |
| CAS # | 2140-76-3 |
| PubChem CID | 102212 |
| Appearance | Typically exists as white to off-white solids at room temperature |
| Density | 1.5±0.1 g/cm3 |
| Melting Point | 154-156ºC |
| Index of Refraction | 1.611 |
| LogP | -1.14 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 3 |
| Heavy Atom Count | 18 |
| Complexity | 385 |
| Defined Atom Stereocenter Count | 4 |
| SMILES | O=C1NC(C=CN1[C@@H]2O[C@H](CO)[C@@H](O)[C@H]2OC)=O |
| InChi Key | SXUXMRMBWZCMEN-ZOQUXTDFSA-N |
| InChi Code | InChI=1S/C10H14N2O6/c1-17-8-7(15)5(4-13)18-9(8)12-3-2-6(14)11-10(12)16/h2-3,5,7-9,13,15H,4H2,1H3,(H,11,14,16)/t5-,7-,8-,9-/m1/s1 |
| Chemical Name | C10H14N2O6 |
| Synonyms | O(2')-Methyluridine Uridine, 2'-O-Methyluridine; 2140-76-3; Uridine, 2'-O-methyl-; 2'-O-Methyl Uridine; 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxytetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione; O(2')-Methyluridine; 399VZB6TMB; 1-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]pyrimidine-2,4-dione; 2'-O-methyl-2'-O-Methyluridine |
| 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) |
Biological Activity
| Targets | Antiviral; anticancer |
| ln Vitro |
Ribose methylation is among the most ubiquitous modifications found in RNA. 2'-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2'-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2'-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy[1]. In Vitro: The in vitro experiment mainly focused on the activity of the identified 2'-O-methyluridine nucleoside hydrolase towards 2'-O-Methyluridine. The enzyme was able to catalyze the hydrolysis of 2'-O-Methyluridine to produce uridine and 2-methylribose. The kinetic parameters of the enzyme for 2'-O-Methyluridine were determined: the Michaelis constant (Km) was 0.23 ± 0.02 mM, and the catalytic constant (kcat) was 12.5 ± 0.3 s⁻¹. Additionally, the enzyme showed high substrate specificity for 2'-O-Methyluridine and had little or no activity towards other nucleosides such as uridine, thymidine, and 2'-O-methylcytidine. [1] |
| Enzyme Assay |
Substrate Specificity Measurements
The purified recombinant RK9NH nucleoside hydrolase protein was tested for substrate specificity using the thin-layer chromatography (TLC) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) methods. A standard enzymatic reaction of RK9NH was carried out at 37 °C for 1 h and contained 30 mM (uridine, 2′-deoxyuridine, 2′-O-methyluridine, 5-fluorouridine, 5-fluoro-2′-deoxyuridine, 5-fluoro-2′-O-methyluridine) of substrate, final 0.025 mg/mL concentration of recombinant 6×His-tagged RK9NH in 40 µL final volume of 50 mM Tris-HCl pH 8 buffer (the same results were obtained using a 50 mM potassium phosphate buffer pH 7). Not all of the substrates are well soluble in water (and the buffers used), hence some of the final reaction concentrations varied accordingly to the limit of solubility of the substrates: 20 mM cytidine, 2′-deoxycytidine, 5-methyluridine, thymidine; 15 mM 3′-O-methyluridine, 2′-O-allyluridine, 3′-O-allyluridine, 2′-O-methylcytidine, 2′-O-methyladenosine, 2′-O-methylguanosine; 10 mM adenosine, 2′-deoxyadenosine, 2′-amino-2′-deoxyuridine and 5 mM guanosine, 2′-deoxyguanosine, inosine were used. The final 0.017 mg/mL, 0.013 mg/mL, 0.008 mg/mL, 0.004 mg/mL RK9NH protein concentrations were used respectively[1]. The enzyme activity assay for 2'-O-Methyluridine hydrolysis was carried out as follows. First, the purified 2'-O-methyluridine nucleoside hydrolase was diluted to an appropriate concentration with a buffer solution (50 mM Tris-HCl, pH 7.5). Then, the reaction mixture was prepared by mixing the diluted enzyme solution with 2'-O-Methyluridine (at different concentrations ranging from 0.05 mM to 1.0 mM) in the same buffer, with a total reaction volume of 200 μL. The reaction was initiated by incubating the mixture at 37°C for 10 minutes, and then terminated by adding 50 μL of 10% trichloroacetic acid. After centrifugation at 12,000 × g for 10 minutes to remove precipitated proteins, the supernatant was analyzed using high-performance liquid chromatography (HPLC). The HPLC system was equipped with a C18 column, and the mobile phase was a mixture of water and methanol (95:5, v/v) at a flow rate of 1.0 mL/min. The detection wavelength was set at 260 nm. The concentration of the product (uridine) was calculated based on the standard curve of uridine, and the enzyme activity was determined accordingly. The kinetic parameters (Km and kcat) were calculated by fitting the initial reaction rates at different substrate concentrations to the Michaelis-Menten equation using nonlinear regression analysis. [1] |
| References |
[1]. Identification of a 2'-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries. Molecules. 2018;23(11):2904. Published 2018 Nov 7. |
| Additional Infomation |
2'-O-methyluridine is a methyluridine that consists of uridine bearing a single methyl substituent located at position O-2' on the ribose ring. 2'-O-Methyluridine is a modified nucleoside that exists in some types of RNA (e.g., transfer RNA and messenger RNA) and is involved in RNA structure stabilization and function regulation. The main purpose of this literature was to identify and characterize a 2'-O-methyluridine nucleoside hydrolase from metagenomic libraries (constructed from soil samples), which can specifically hydrolyze 2'-O-Methyluridine. This enzyme is expected to be used in the enzymatic synthesis of modified nucleosides or the degradation of 2'-O-Methyluridine in biological samples. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~100 mg/mL (~387.25 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.68 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 25.0 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: ≥ 2.5 mg/mL (9.68 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 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (9.68 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 to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8725 mL | 19.3626 mL | 38.7252 mL | |
| 5 mM | 0.7745 mL | 3.8725 mL | 7.7450 mL | |
| 10 mM | 0.3873 mL | 1.9363 mL | 3.8725 mL |