Synthesis of 2‘-Modified Oligodeoxynucleotides via On-Column Conjugation

Oligodeoxynucleotides modified at the 2‘-position of 2‘-amino-2‘-deoxyuridine or uridine were prepared in high yield and purity using phosphoramidites 2 and 3, respectively. Oligodeoxynucleotide conjugates were prepared on the solid-phase synthesis support following selective unmasking of the nucleophile incorporated in these phosphoramidites. Synthesis of oligodeoxynucleotides modified at the 2‘-position of an internal nucleotide provides molecules that are complementary to those previously prepared via a similar approach using C5-substituted pyrimidines. The efficiency of functionalization of the 2‘-O-alkylamino-uridine derived from 3 in a protected oligodeoxynucleotide was less susceptible to steric hindrance than the 2‘-amino-2‘-deoxyuridine in the same polymeric substrate. However, the greater reactivity of the 2‘-O-alkylamine containing nucleotide gave rise to undesired acetamide formation resulting from nucleophilic attack on the 5‘-terminal acetate in capped failure sequences. This problem was overcome by using 2,2,2-trimethylacetyl anhydride as a capping agent during the automated synthesis cycles. Finally, the efficiency of the photochemical unmasking of the support bound alkylamine on a 1 μmole scale was improved by using two 20 min photolysis cycles, coupled with removing reaction byproducts between cycles.