Use of ribonucleosides as protecting groups in synthesis of polynucleotides with phosphorylated terminals

Abstract

Two new protected 5''-ribonucleotides, 2'',3''-O-bis(4,4''-dimethoxytrityl)uridine 5''-(4-chlorophenyl phosphate) and 2'',3''-O-(methoxymethylene)uridine 5''-(4-chlorophenyl phosphate), form the basis of a chemical procedure for phosphorylating the 5''-ends of DNA fragments synthesized by the phosphotriester approach. Condensation of either of these mononucleotide units with the free 5''-hydroxyl of an otherwise fully protected oligomer results in high-yield formation of a 5''-5''-triester linkage. Subsequently, the terminal ribonucleotide of the deprotected product rU5''-5''d(N-Nn-N) can be cleaved by periodate oxidation of its 2'',3''-cis-hydroxyl system followed .beta.-elimination, leaving its phosphate attached to the 5''-hydroxyl group of the oligodeoxyribonucleotide. This procedure, together with a tactic employing a 2'',3''-O-acylribonucleotide residue at the 3''-terminus of the chain, allows the synthesis of oligomers possessing monophosphate groups at either end or both ends. Furthermore, oligonucleotide intermediates possessing a 5''-5''-linked uridine terminal are shown to have a special application as acceptors in RNA ligase reactions, where the presence of the ribonucleoside cap on the 5''-phosphate limits ligation specifically to the 3''-ends of the oligomers. Removal of the uridine residues to expose free 5''-phosphates would then enable the products to participate as donors in further elongation reactions.