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

Preparation of 2'-OMe-Bz-m 6 A-CE phosphoramidite. 59-(4,49-Dimethoxytrityl)-6-N-benzoyl-6-N-methyl-adenosine,29-O-methyl-39-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (2'-OMe-Bz-m 6 A-CE phosphoramidite) was prepared as follows: tetrabutylammonium bromide (260 mg, 807 mmol) and aq. NaOH (1M, 7 ml) were added to a stirring solution of 2'-OMe-Bz-A-CE phosphoramidite (Link Technologies, 708 mg, 797 mmol) and iodomethane (200 ml, 3.21 mmol) in dichloromethane (7 ml). After vigorous stirring for 30 min, ether and water were added and the resulting layers were separated. The aqueous layer was extracted with ether, the combined organic extracts were dried over Na 2 SO 4 and the volatiles were removed in vacuo. 2'-OMe-Bz-m 6 A-CE phosphoramidite was readily purified by column chromatography (1:1 petrol/etherR9:1 ether/methanol) affording 2'-OMe-Bz-m 6 A-CE phosphoramidite (403 mg, 56%, white foam, 1:1 mixture of diastereoisomers) and 2'-OMe-Bz-m 1 A-CE phosphoramidite (182 mg, 25%, white foam, 1:1 mixture of diastereoisomers). Data for 2'-OMe-Bz-m 6 A-CE phosphoramidite and for 2'-OMe-Bz-m 1 A-CE phosphoramidite are presented in Supplementary Table S1. online.