Addition of E⁻_aq and H Atoms to Hypoxanthine and Inosine and the Reactions of α-hydroxyalkyl Radicals with Purines. A Pulse Radiolysis and Product Analysis Study

Abstract

The reactions of hydrated electrons e⁻_aq with hypoxanthine and inosine were followed using pulse radiolysis methods. In a neutral solution the electron adduct of inosine is immediately protonated at the heteroatoms of the purine ring by water (k ≫ 2·5 × 10⁶s⁻¹) to give In(NO-H)·. These NO-protonated intermediates have a single absorption maximum at 300 nm. In basic solution the protonation of the electron adduct of inosine by water leads to other intermediate products with an absorption maximum at 350 nm. These intermediates are believed to be in C-protonated electron adducts of inosine (In(NO-H)·). In (NO-H)· and In(C-H)· differ strongly in their ability to reduce C-nitroacetophenone (PNAP). In(NO-H)· are strong reductants and reduce PNAP quantitively to PNAP·⁻. Based on the pH dependence of PNAP·⁻ yields, two types of tautomers of In(C-H)· could be distinguished. One of the tautomers can reduce PNAP, albeit with slower rate than In(NO-H)·, the other tautomer has no reducing properties. The latter is the one with the higher pK_a and therefore thermodynamically more stable. The absorption spectrum of the intermediates produced in the reaction of e⁻_aq with hypoxanthine at neutral pH is very similar to that of In(NO-H)· with a maximum at 300 nm. However, no build-up at 350 nm was observed in basic solution as in the case of the electron adduct of inosine. The reaction of H atoms with inosine produces in basic solution intermediate radicals with the same absorption spectrum as the C-protonated electron adducts of inosine. It is suggested that both the reactions of e⁻_aq and H· with inosine in basic solution produce the same radical, namely the H-adduct of inosine (In(C-H)) with the highest pK_a. α-Hydroxyalkyl radicals were found to react very slowly with purine bases and nucleosides in neutral to basic solutions. In acidic solution their reactivity increases and a number of rate constants were determined by pulse radiolysis measurements at pH 0·4. The intermediates from the reaction of 2-hydroxy-2-propyl radicals with inosine could be observed pulse spectrometrically in neutral and in basic solutions. In basic solution this reaction leads to intermediates with the same absorption maximum at 350 nm as that of the H-adduct of inosine. Furthermore, the yield of acetone was found to increase strongly in basic pH. It is suggested that the reaction of inosine with 2-hydroxy-2-propyl radicals leads to the formation of adducts which rapidly eliminate acetone in alkaline solution to give In(C-H). The addition product, 8-(2′-hydroxy-2′-propyl)adenine (G = 1·9 × 10⁻⁷ mol J⁻¹), was obtained in the reaction of 2-hydroxy-2-propyl radicals with adenine under steady-state γ-radiolysis conditions.