DEPROTECTION OF METHYLPHOSPHONATE OLIGONUCLEOTIDES USING A NOVEL ONE-POT PROCEDURE

Deprotection of methylphosphonate oligonucleotides with ethylenediamin e was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N-2-ibu-O6-D PC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC w as found to transaminate at the C4 position when treated with ethylene diamine. A similar displacement reaction with ethylenediamine was obse rved at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser exte nt of N2-ibu-dG. Side reactions were not observed when oligonucleotide s containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediami ne. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief trea tment with dilute ammonia for 30 minutes followed by addition of ethyl enediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to sto p the reaction and prepare the crude product for chromatographic purif ication. This method was used to successfully deprotect a series of ol igonucleotides at the 1, 100, and 150 mumole scales. These deprotectio n results were compared to a commonly used two-step method and found t o be superior in yield of product by as much as 250%.