2'-O-MethylpseudoUridine

2^'-O-methylpesudoUridine

The 2^'-O-methylated derivative of pesudoUridine has been observed in tRNAs of archaea and eukaryotes, in the 18+26S rRNA of eukaryotes, and in snRNAs in eukaryotes. In general, the 2^'-O-methylation of the ribose stabilizes the C3^'- endo conformation and makes the base pairing more thermodynamically stable in RNA. It can also disrupt the participation of the 2^'-OH group in tertiary interactions. The steric effect of the methyl group can also influence RNA−protein interactions. CD and NMR studies suggested that although Ψm preferred the syn conformation predominantly, the preference was less than that of Ψ. In contrast to most other 2^'-O-methylated nucleosides, Ψm was observed to slightly prefer the C2^'-endo conformation over the C3^'-endo conformation.

Synthesis of 2^'-O-methylpesudoUridine

2^'-O-methylpesudoUridine was first reported to be synthesized by using a stannous chloride catalyzed diazomethane reaction with the pesudoUridine ribonucleoside. The synthesis of the phosphoramidite derivative was later accomplished, when the 3^'- and 5^'- hydroxyl protected with the Markiewicz disiloxane reagent and the protection pivaloyloxymethyl groups on N-1 and N-3 of pesudouridine. This route required eight steps and several chromatographic separation processes.

A shorter albeit less selective method to produce 2^'-O-methylpesudoUridine has been reported, which avoids amino protection and through intermediate of dibutyltin oxide complex. The tin complex was formed and isolated by crystallization. Treatment with methyl iodide afforded a mixture of the 2^'' and 3^'-O-methylpesudoUridine isomers. After removing unreacted starting material, the mixture was reacted with dimethoxytiiphenylmethyl chloride and the product isomers were separated chromatographically and their identities were confirmed by TOCSY and NOESY NMR. The 2'-O-methylpesudoUridine was carried on to give phosphoramidite.

In short, the synthesis of 2^'-O-methylpesudoUridine and its phosphoramidite derivative is described which avoids the use of protecting groups on the nitrogen of amine. A binding study of oligonucleotides containing this modification suggest an increased binding affinity to RNA when compared to oligonucleotides incorporating 2^'-O-methyluridine. Study compared compare 2'-O-methylpesudoUridine to 2^'-O-methyluridine against RNA complement via melting temperature studies. The increase in melting temperature was dependent upon the number of methylation nucleosides incorporation and spacing. There was a positive effect which averaged 0.8^oC per 2^'-O-methylpesudoUridine over 2^'-O-methyluridine. This result supports further structure activity relationships of related bases and 2^' modifications.