Hypoxanthine is a mutagenic purine base that most commonly arises from the oxidative deamination ofadenine. Damaged bases such as hypoxanthine are associated with carcinogenesis and cell death. Thisinevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA.Alkyladenine DNA glycosylase (AAG) is the enzyme responsible for excising hypoxanthine from DNAin humans. In an effort to understand the intrinsic properties of hypoxanthine, we examined the gas-phase acidity and proton affinity using quantum mechanical calculations and gas-phase mass spectrometricexperimental methods. In this work, we establish that the most acidic site of hypoxanthine has a gas-phase acidity of 332 ± 2 kcal mol
-1, which is more acidic than hydrochloric acid. We also bracket a lessacidic site of hypoxanthine at 368 ± 3 kcal mol
-1. We measure the proton affinity of the most basic siteof hypoxanthine to be 222 ± 3 kcal mol
-1. DFT calculations of these values are consistent with theexperimental data. We also use calculations to compare the acidic and basic properties of hypoxanthinewith those of the normal bases adenine and guanine. We find that the N9-H of hypoxanthine is moreacidic than that of adenine and guanine, pointing to a way that AAG could discriminate damaged basesfrom normal bases. We hypothesize that AAG may cleave certain damaged nucleobases as anions andthat the active site may take advantage of a nonpolar environment to favor deprotonated hypoxanthineas a leaving group versus deprotonated adenine or guanine. We also show that an alternate mechanisminvolving preprotonation of hypoxanthine is energetically less attractive, because the proton affinity ofhypoxanthine is less than that of adenine and guanine. Last, we compare the acidity in the gas phaseversus that in solution and find that a nonpolar environment enhances the differences in acidity amonghypoxanthine, adenine, and guanine.
