The pKa of 1 and 2 was determined by analysis of their UV/Vis absorption spectra between pH 4 and pH 10 at RT. In a total volume of 10 mL, the nucleobases were dissolved to a concentration of 100 µM in 50 mM MOPS buffer (initially pH 9). Desired pH values were adjusted with HCl and NaOH and samples of 200 µL were transferred to a UV/Vis-transparent 96-well plate to record the UV absorption spectra in the range of 250 to 350 nm in steps of 1 nm. The spectra were analyzed via spectral unmixing as previously described using a fully protonated (pH 4) and a fully deprotonated (pH 10) spectrum as substrate and product reference for the deprotonation reaction, as spectra obtained near these pH values displayed identical shape (indicating no further reaction). For the determination of the pKa, the experimental data were fitted to equation (1), whereby the pH was set as the input variable, α as the dependent variable and pKa as the parameter to fit. = + = 10 1 + 10