Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids

Abstract

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2'',3''-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2'',3''-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) and G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloroacetate, lactate and cytosine (N-3) were obtained from 1H NMR line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: at neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7-9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. Protonation of the nucleobase probably effects a qualitative increase in the ability of the amino protons to form H-bonds: from very poor in the neutral base to normal in the conjugate acid.