Solvolysis of 2’-deoxyribonucleosides and oligo-2’-deoxyribonucleotides in aqueous pyrrolidine or ammonia solutions

Abstract

To assess the feasibility of high-temperature aminolysis of deoxyribooligonucleotides containing rare bases as a method to determine their base sequence, the 2’-β-D-deoxyribosides of 5-bromouracil, 2-aminopurine, uracil, adenine, cytosine, 5-methylcytosine, hypoxanthine, N6-methyladenine, N4-ethylcytosine, and guanine were compared as to their rate of degradation in 0.5 M aqueous pyrrolidine at 110 °C, conditions used earlier in the analysis of oligonucleotides containing only the canonical bases. The reaction mixtures were analyzed by chromatography on Zorbax XDB-CN and UV absorption spectroscopy. The first-order rate constants for the nucleoside degradations decreased in the above order, spanning a wide range of reactivities. Some of these nucleosides were also tested in 0.5 M aqueous ammonia at 110 °C, giving similar first-order rate constants, except for 2’-deoxyguanosine, which is much more reactive with ammonia, due to the lower basicity of this reagent, leaving a larger proportion of the nucleoside in the non-ionized form, susceptible to nucleophilic attack at the base. Short oligothymidylates containing a single 2-aminopurine, adenine, guanine, or cytosine unit in central position were tested in pyrrolidinolysis, to determine the cleavage rates at these sites and the dependence of these cleavage rates on oligonucleotide length. A model decadeoxyribonucleotide containing all four canonical bases was also pyrrolidinolyzed, followed by ion-exchange chromatography, to deduce the nucleotide sequence from the resulting chromatographic profile.