N3-MethylpseudoUridine

N3-MethylpseudoUridine

3-MethylpseudoUridine (m³Ψ) was first identified in a ribosomal RNA in 1996 although its synthesis has been previously reported in 1982. Although the N3 position of this nucleoside is incapable of forming hydrogen bonds, it can still interact with adenine through the N1 position if it adopts the syn conformation. There is little idea why 3-methylpseudoUridine is so strongly conserved at position 1915 in helix 69 in prokaryotic ribosomal RNA. Mutation of m³Ψ at the 1915 position has been found to impair translation fidelity and subunit association.

Helix 69 of Escherichia coli 23S rRNA

The site of protein synthesis in the ribosome is comprised of a set of specific structural motifs of the large and small subunit rRNAs, known as 23S and 16S rRNA in Escherichia coli. A large number of nucleotides in this region are modified, including methylation and pseudouridylation. Helix 69 of domain IV, or residues 1906-1924 in 23S rRNA, is centered at the heart of the ribosome machinery. More specifically, the minor groove of helix 69 interacts with the minor groove of the D stem of the P-site tRNA, and the 1915 loop region interacts with the D stem of the A-site tRNA. Helix 69 is likely to undergo conformational changes during protein synthesis as it contacts with mobile tRNAs.

The helix 69 of Escherichia coli 23S rRNA consists of pseudoUridine (Ψ), 3-methylpseudoUridine and standard phosphoramidites. This rRNA fragment contains a total of 19 nucleotides, with two Ψ^'s at positions 1911 and 1917 and a single m³Ψ at position 1915. Selective introduction of a methyl group at the N3 position of pseudoUridine at site 1915 results in a slight enhancement in the thermodynamic stability of the RNA hairpin compared to pseudoUridine. RNA hairpin structure does not undergo any substantial changes after methyl modified RNA, which revealed by one-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs.Synthesis of 3-methylpseudoUridine

In nature, pseudoUridines are synthesized through isomerization of uridines by one of many specific pseudoUridine synthases. Methylations of pseudoUridines are hypothesized to occur via the action of specific RNA methyltransferases. Although the specific modification is considerably more challenging to the organic method, the chemical synthesis of the nature modified nucleside 3-methylpseudoUridine has been reported.

The synthesis method involved an N1 protection-deprotection strategy in which the N3 position can then be selectively methylated. Firstly, the 3^'- and 5^'- hydroxyl groups of pseudoUridine are simultaneous protected using dichlorotetraisopropyldisiloxane. Then, 2^'- hydroxyl group is protected via treating with trimethylsilyl chloride (TMSCl). After protection of the ribose hydroxyl groups, the N1 position was selectively protected by reaction with pivaloylmethyl chloride (POMCl). The TMS group can be removed selectively in the presence of the N1 protective group POM under mild conditions. Subsequently, N1 methylation of pseudoUridine is completed by the reaction with N,N-dimethylformamide dimethyl acetal (DMF-DMA). Treating with p-toluene- sulfonic acid (pTSA) followed by methanolic ammonia successively to remove the protection groups of 2^'-O-TMS and N1-POM. Finally, the 3^',5^'-O-TIPDS-protected (TIPDS ) 1,1,3,3-tetraisopropyldisiloxanediyl) 3-methylpseudoUridine can be obtained.