A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA

Kruse, Susanne; Zhong, Silin; Bodi, Zsuzsanna; Button, James; Alcocer, Marcos J. C.; Hayes, Christopher J.; Fray, Rupert

In order to get quick access to the required m 6 Am phosphoramidite building block for solid-phase oligoribonucleotide synthesis, we performed a selective one-step methylation of the commercially available 2'-OMe-Bz-A-CE phosphoramidite. Due to its high reactivity and sensitivity towards acids and bases, Aritomo's relatively mild phase transfer catalysis (PTC) method was used 30 . Under these conditions (iodomethane, sodium hydroxide, tetrabutylammonium bromide) the N 6 -methylated product was obtained as the major product in 56% yield, with the N 1 -methylated isomer being formed as a byproduct in 25% yield. Due to their different Rf values, the N 6 -and N 1 -methylated products were easily separated via column chromatography, and the purified m 6 Am phosphoramidite was used directly for RNA synthesis.