Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O‐cyanoethyl‐N,N‐diisopropyl‐phosphoramidites of 5′‐O‐dimethoxytrityl (DMT) protected deoxyribonucleosides (dGib, dAbz, dCbz, T) were analyzed by HPLC‐MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile‐induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid‐catalyzed hydrolysis could also be reduced by addition of small amounts of base.