PseudoUridine 5'-Triphosphate

PseudoUridine 5'-Triphosphate - CAS 1175-34-4

PseudoUridine 5'-Triphosphate is a modified nucleoside used for the polymerase-mediated synthesis of RNA molecules. The sodium salt form PseudoUridine 5'-Triphosphate Sodium (B2706-358098) is in stock.

Catalog Number
Molecular Weight
Molecular Formula
Pseudo-UTP; 5-[5-O-[Hydroxy[[hydroxy(phosphonooxy)phosphinyl]oxy]phosphinyl]-β-D-ribofuranosyl]-2,4(1H,3H)pyrimidinedione; 5-β-D-Ribofuranosyluracil 5'-Triphosphate; Pseudouridine Triphosphate
[[(2R,3S,4R,5S)-5-(2,4-dioxo-1H-pyrimidin-5-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate
Canonical SMILES
≥95% by HPLC
clear aqueous solution
Storage at -20°C

The feature of mRNA

It has been well known that ribonucleoside triphosphates (NTPs) are served as the building blocks for the synthesis of RNA molecules both in vitro and in vivo that mediated by polymerase. It is noteworthy that the utilization of messenger RNA (mRNA) provides a powerful tool for gene therapy applications in the field of cancer immunotherapy and stem cell-based biomedical research which is complimentary to plasmid DNA technique. The feature of mRNA is that it does not need to enter the nucleus and the chance of integration into the host genome is minimal, thus avoiding aberrant transcription and expression of oncogenes caused by insertional mutations.

The use of pseudoUridine-5'-triphosphate

However, the whole process greatly reduces expression efficiency and stimulates an immune toxicity in the host, which is influenced by the ubiquitously expressed nuclease and the immune stimulation. This disadvantage has been circumvented by using RNA modification that utilizes the naturally occurring modified nucleotides. There are several common modified ribonucleotides, including pseudoUridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate and N1-methylpseudoUridine-5'-triphosphate, which have been used in mRNA applications. In particular, these molecules have important therapeutic and diagnostic applications. It has been demonstrated in the literatures that modified mRNAs, such as pseudoUridine, substituting unmodified mRNAs has resulted in increased translational properties and stability with significantly reduced innate immune responses in human and other mammalian cells. Given the improved properties exhibited by pseudoUridine make such kind of modified mRNA serves as a promising tool for gene replacement and vaccination.

Synthesis of nucleoside triphosphates

For mRNA modification, successful molecular biology applications depend heavily on the quality and purity of the nucleoside triphosphates (NTPs). Therefore, giving method for efficient chemical synthesis of NTPs is of great importance. The most commonly used method for the synthesis of NTPs was developed by Ludwig, referred a one-pot strategy with three steps. The method involves first monophosphorylation of nucleoside to generate nucleoside dichlorophosphoridate intermediate, subsequent reaction with bis-(tri-n-butylammonium) pyrophosphate and hydrolysis of the resulting cyclic intermediate to afford the final nucleoside triphosphate. The commonly used phosphorylating agent is POCl3 and solvent is trialkylphosphate. This method does not require prior protection and subsequent deprotection of nucleobase and ribose moiety.

However, this method has serious disadvantages, including low yields, poor selectivity and purification challenges due to the presence of some by-products. Moreover, the utility of this method has not been extended to various nucleoside derivatives. Although several methods have been developed, which served alternative methods for the synthesis of nucleoside triphosphate such as nucleophilic reaction of nucleoside with triphosphate, the more reliable and promising method is still containing a three-step process starting from the corresponding nucleoside. The overall synthetic step involves monophosphorylation of nucleoside, followed by activation of 5'-monophosphate and subsequent reaction with pyrophosphate to produce the corresponding nucleoside triphosphate. But the three steps increased the difficulty of the synthesis, the final step is slow and the overall yields are moderate. In general, the phosphorylation of nucleoside is extremely challenging because of its purification problems, poor selectivity and low purity due to side products. To overcome these issues, Kore et al. developed an improved one pot method based on the Ludwig synthesis strategy through the choose of proper base, the optimization of phosphorous oxychloride and reaction time.

Synthesis of pseudoUridine-5'-triphosphate (ΨTP)

The procedure of pseudoUridine-5'-triphosphate (ΨTP) synthesis has been reported. Firstly, pseudoUridine solved in trimethylphosphate treating with phosphorous oxychloride to produce pseudoUridine-5'-monophosphate. Subsequently, adding tributylammonium pyrophosphate and tributylamine to the pseudoUridine-5'-monophosphate following reacted in solvent acetonitrile. Through the process of extraction, separation and purification, to afford desired product ΨTP. The synthesis method utilizes the ion-exchange chromatography column to purify product and finally to afford the sodium salt of ΨTP. In conclusion, this development of improved one-pot method can be used to generate modified nucleoside triphosphates, such as pseudoUridine-5'-triphosphate in moderate yields. Notably, the procedure utilizes a short experimental time for the reaction to be completed and simple purification processes to afford high purity product without inorganic impurity.

1. An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5'-Triphosphates
Muthian Shanmugasundaram, Annamalai Senthilvelan, Zejun Xiao, Anilkumar R Kore Nucleosides Nucleotides Nucleic Acids . 2016 Jul 2;35(7):356-62. doi: 10.1080/15257770.2016.1163382.
A simple, reliable, and an efficient "one-pot, three step" chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5'-triphosphate (5-MeCTP), pseudouridine-5'-triphosphate (pseudoUTP) and N(1)-methylpseudouridine-5'-triphosphate (N(1)-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).
2. Biosynthetic precursors of some modified nucleosides in the transfer ribonucleic acid of Mycoplasma mycoides var. capri
R T Walker J Bacteriol . 1971 Sep;107(3):618-22. doi: 10.1128/jb.107.3.618-622.1971.
The ribosomal and transfer ribonucleic acid (tRNA) from Mycoplasma mycoides var. capri, grown in a medium containing uridine-((14)C)-5'-triphosphate and cytidine-(5-(3)H)-5'-triphosphate, were isolated and separated. The uridine in both species of RNA was shown to contain (14)C and the cytidine to contain both (3)H and (14)C. Comparison of the labeling of 4-thiouridine and pseudouridine, obtained from an enzymatic digest of the RNA, indicates that their biosynthetic precursor is uridine, not cytidine. It is probable that ribothymidine and dihydrouridine have the same derivation.
3. Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts
Frauke Hausburg, Silke Na, Anna Skorska, Paula Müller, Robert David, Natalia Voronina, Gustav Steinhoff Cell Physiol Biochem . 2015;35(4):1360-71. doi: 10.1159/000373957.
Background:By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required.Methods:Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins.Results:Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies.Conclusion:The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

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