A comparative study of supports for the synthesis of oligonucleotides without using ammonia

A comparative study of the cleavage efficiency of succinyl, phthaloyl, oxalyl, 2-(2-nitrophenyl)ethyl, 9-fluorenylmethyl, and 2nitrophenyl supports in 0.5M DBU solutions is described. A decrease in cleavage efficiency is observed when small oligonucleotides containing thymidine are linked to the supports. In these conditions oxalyl supports gave the best yields followed by 2-(2-nitrophenyl)ethyl and 9fluorenylmethyl supports. Solid-phase oligonucleotide synthesis relies on the use of solid supports in where the first nucleoside is covalently attached to the solid support. In almost all the cases, a 3’-succinate is attached to the support via an amide bond. At the end of the synthesis the oligonucleotide is released with concentrated ammonia together with the rest of the amino and phosphate protecting groups. Recently, the need for modified oligonucleotides carrying ammonia sensitive moieties has led to the development of linkers that could be cleaved under milder conditions such as oxalyl, o-nitrobenzyl and o-nitrophenylethyl linkers. The oxalyl linker is much more sensitive to bases than conventional nucleoside-succinyl supports, and it is cleaved using diluted solutions of primary and secondary amines. The o-nitrobenzyl linker is cleaved by photolysis and the onitrophenylethyl is cleaved by non-nucleophillic bases (1,8diazabicyclo[5.4.0]undec-7-ene, DBU). The utility pf these special linkers has been demonstrated during the preparation of oligonucleotides carrying ammonia sensitive molecules such as O-methyl-phosphotriesters, 5’-fatty ester conjugates, thymine C5-hydrate, O-alkylthymidine 2fluorohypoxanthine and 5-aza-2’-deoxycytidine. During the preparation of oligonucleotides using the onitrophenylethyl linker the presence of dimethoxytrityl (DMT) groups was still observed on DMT-oligonucleotide-supports after long DBU treatments (16 hrs with 0.5M DBU in pyridine). This result was surprosong knowing that DMT-nucleosides were released from o-nitrophenylethyl supports ysing DBU solutions in less than one hour. In the present communication we analyze the release of small oligonucleotides from supports having different linkers such as succinyl, phthaloyl, oxalyl, o-nitrophenylethyl (NPE), 9fluorenylmethyl (FM) and o-nitrobenzyl. Some of these supports have been described to be cleaved by DBU solutions but the cleavage was always measured on DMT-nucleoside supports. No data was available for oligonucleotides linked to the supports.