PseudoUridine 5'-Triphosphate

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.