For synthetic applications, a higher substrate loading than previously employed (1 mM at pH 7) would be highly desirable to reduce solvent use and facilitate purification. Based on the pKa of 2-Se-uracil (1) (7.18 at RT) we hypothesized that a slightly higher reaction pH would facilitate dissolution of 1 and 2-Se-thymine (2) as their corresponding anions. Since it was not available from the literature, we determined the pKa of 2 through analysis of its UV absorption spectra at different pH values and found a similar value (7.49 ± 0.01, Figure ) to that of 1. Hence, compared to the natural nucleobases, the Se-containing analogues were more acidic. Next, we investigated the solubility of 1 and 2 at pH values below and above their pKa to see if a pH shift would allow higher substrate loading. At pH 7, 1 was not soluble at 10 mM, as indicated by the presence of precipitate (Figure ). In contrast, full dissolution was observed at pH 9, confirming that a higher reaction pH can increase the solubility of the free nucleobase through deprotonation and solvation of the corresponding salt. Nonetheless, this effect was modest, as only 2 was fully soluble at 20 mM at pH 9 and RT.