Investigation of the acid-base and electromigration properties of 5‐azacytosine derivatives using capillary electrophoresis and density functional theory calculations

- Thermodynamic acidity constants of antiviral cidofovir analogs were determined by CE.
- PKa values calculated by DFT were in a good agreement with those determined by CE.
- Effective charges of 5-azacytosine derivatives at selected pH values were estimated.
- Effective and ionic mobilities of 5-azacytosine derivatives were determined.
- N3 atom of triazine ring was found to be preferably protonated by DFT calculations.

Capillary electrophoresis (CE) and quantum mechanical density functional theory (DFT) were applied to the investigation of the acid-base and electromigration properties of important compounds: newly synthesized derivatives of 5-azacytosine - analogs of efficient antiviral drug cidofovir. These compounds exhibit a strong antiviral activity and they are considered as potential new antiviral agents. For their characterization and application, it is necessary to know their acid-base properties, particularly the acidity constants (pKa) of their ionogenic groups (the basic N3 atom of the triazine ring and the acidic phosphonic acid group in the alkyl chain). First, the mixed acidity constants (pKamix) of these ionogenic groups and the ionic mobilities of these compounds were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities. Effective mobilities were measured by CE in a series of background electrolytes in a wide pH range (2.0-10.5), at constant ionic strength (25 mM) and constant temperature (25 °C). Subsequently, the pKamix values were recalculated to thermodynamic pKa values using the Debye-Hückel theory. The thermodynamic pKa value of the NH+ moiety at the N3 atom of the triazine ring was found to be in the range 2.82-3.30, whereas the pKa of the hydrogenphosphonate group reached values from 7.19 to 7.47, depending on the structure of the analyzed compounds. These experimentally determined pKa values were in good agreement with those calculated by quantum mechanical DFT. In addition, DFT calculations revealed that from the four nitrogen atoms in the 5‐azacytosine moiety, the N3 atom of the triazine ring is preferentially protonated. Effective charges of analyzed compounds ranged from zero or close-to-zero values at pH 2 to −2 elementary charges at pH ≥ 9. Ionic mobilities were in the range (−16.7 to −19.1) × 10−9 m2 V−1 s−1 for univalent anions and in the interval (−26.9 to −30.3) × 10−9 m2 V−1 s−1 for divalent anions.