Oligoribonucleotide Synthesis III. Synthesis of Trinucleotides Using a Stepwise Phosphotriester Method

A convenient general synthesis for protected di- and triribonucleotides of predetermined sequence is described. Starting with a 5′-p-methoxytrityl-2′-O-tetrahydropyranyl-ribonucleoside, the nucleoside unit destined to become the 5′-terminal of the oligomer, 2′-O-tetrahydropyranyl-nucleosides are coupled together by β,β,β-trichloroethyl 3′–5′-phosphotriester linkages. A two-step process is used for each coupling, (a) the terminal 3′-hydroxyl group is phosphorylated using β,β,β-trichloroethylphosphate activated by triisopropylbenzenesulfonyl chloride (TPS) and (b) re-activation of the newly inserted diphosphate grouping by TPS is followed by condensation with the 5′-hydroxyl of the incoming nucleoside derivative. These phosphotriester derivatives can be isolated and purified in good yield by silica gel column chromatography in methanol – methylene chloride. The fidelity of 3′–5′ linkage is checked. The p.m.r. spectra can indicate the nature and ratio of the bases in these protected oligomers. Deblocking conditions to give free trinucleoside diphosphates are described.