Synthesis, stability, and protonation studies of a self-complementary dodecamer containing the modified nucleoside 2deoxyzebularine

The nucleoside 2ʹdeoxyzebularine (K) was incorporated into the self-complementary dodecamer 5ʹCGTACGKGTACG-3ʹ by solid-phase 2-cyanoethylphosphoramidite chemistry using dimethoxytrityl (DMT) as the 5ʹhydroxyl protecting group. Standard synthesis cycles using trichloroacetic acid and short ammonia treatment (50°C for 30 min) were found to be the optimal conditions to obtain the desired dodecamer with minimum acid and basic degradation of the acid- and base-sensitive 2-pyrimidinone residue. The protonation equilibria of the K nucleoside and of the dodecamer at 37°C were studied by means of spectroscopically monitored titrations. For the K nucleoside, a pKa value of 3.13 (plus-minus) 0.09 was obtained. For the dodecamer, four acid-base species were found in the pH range 2-12, with pKa values of 9.60 (plus-minus) 0.07, 4.46 (plus-minus) 0.16, and 2.87 (plus-minus) 0.19. Melting experiments were carried out to confirm the proposed acid-base concentration profiles. Finally, kinetic experiments were also carried out at several pH values to evaluate the stability of the K nucleoside and of the dodecamer. An increased stability was shown by the K nucleoside when incorporated into the dodecamer. Multivariate methods based on both hard- and soft-modeling were applied for the analysis of spectroscopic data, allowing the estimation of concentration profiles and pure spectra.