Improvement in the synthesis of oligonucleotides of extended length by modification of detritylation step.

4,4'-Dimethoxytrityl, (DMTr), is at present commonly used for the protection of the 5'-terminal hydroxy function in solid phase DNA or RNA synthesis. During the oligonucleotide synthesis the first step, prior to coupling with the appropriately protected phosphoroamidite monomer, is the removal of the 5'-protecting group, (DMTr, detritylation) by a suitable protic acid. Strong protic acids, such as benzene (1 —3) or toluene sulfonic acid (4), and trifluoro(5) or trichloroacetic (6-9) acid have been utilized as effective detritylating agents. However, their use is complicated by the modification of the protected purine base, 6N-benzoyl-2'-deoxyadenosine and, to a lesser extent, 2Nisobutyryl-2'-deoxyguanosine residues, during the deprotection step leading to depurination and chain cleavage in the final amide deprotection step in an ammonium hydroxide treatment. As an alternative, many Lewis acids, like A1C13, ZnCl2, SnCl4, and TiCl4, have been examined under various conditions as detritylating agents (10,11). There are also reports in the literature on usage of a Lewis acid, BF3 as methanol complex, for detritylation (12,13). Zinc bromide has also been used (3,4,10,11,14—19) as detritylating agent which does not cause significant depurination. However, the selective removal of Nacyl protecting group in nucleoside derivatives was observed by treatment with zinc bromide in the presence of alcohols (14) and the rate of trityl removal decreased rapidly as the DNA chain becomes longer (2,20) effectively limiting the length of sequence. As an alternative, dichloroacetic acid (DCA, pKa=1.5) was found to be a detritylating agent of choice (20), and which is at present widely used for the deprotection of the 5'-hydroxyl group prior to coupling the next nucleoside in automated DNA synthesis on a solid support.