Analytical studies of 'mixed sequence' oligodeoxyribonucleotides synthesized by competitive coupling of either methyl- or beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, including 2'-deoxyinosine

Abstract

High-performance liquid chromatography (HPLC) and 1H/31P nuclear magnetic resonance (NMR) spectroscopy were used to measure the molar ratio of oligodeoxyribonucleotide products in mixtures obtained with automated DNA synthesizers that employed competitive coupling of either standard methyl- or newer beta-cyanoethyl-N,N-diisopropylamino phosphoramidite reagents, which include deoxyinosine. Mixtures of these reagents when used as freshly prepared solutions afforded ratios of products that indicated negligibly small differences among the rates of the various competitive coupling reactions. However, studies of reagent stability in solution revealed that both types of the N-isobutyryl deoxyguanosine reagent decompose faster than their corresponding dA, dC, and dT phosphoramidites, which led to significantly lower proportions of dG-containing sequences. This problem was attenuated for the beta-cyanoethyl reagents due to their slower rate of decomposition.