Self‐association and protonation of adenosine 5′‐monophosphate in comparison with its 2′‐ and 3′‐analogues and tubercidin 5′‐monophosphate (7‐deaza‐AMP)

The concentration dependence of the chemical shifts of the protons H-2, H-8 and H-1' for 2'-, 3'- and 5'-AMP2- and of the protons H-2, H-7, H-8 and H-1' for tubercidin 5'-monophosphate (= 7-deaza-AMP2-; TuMP2-) has been measured in D2O at 27 degrees C to elucidate the self-association of the nucleoside monophosphates (NMPs). The results are consistent with the isodesmic model of indefinite non-cooperative stacking; the association constants for all four NMPs are very similar: K approximately 2 M-1. These 1H-NMR measurements and those on the dependence of the chemical shifts on the pD of the solutions indicate that the NMP2- species exist predominately in the anti conformation. Comparison of the shift data for 5'-TuMP and 5'-AMP shows that no hydrogen bonding between N-7 and -PO3H- occurs; hence, the previously observed and confirmed 'wrongway' chemical shift Martin, R. B. (1985) Acc. Chem. Res 18, 32 connected with the deprotonation of the -PO3H- group most probably results from the anisotropic properties of the phosphate group which is in the anti conformation close to N-7. From the dependence between the chemical shift and the pD of the solutions the acidity constants were calculated for the four protonated NMPs, and for adenosine and D-ribose 5'-monophosphate. The measurements also allow an estimation of the first acidity constant of H3(5'-AMP)+ (pKDD3(AMP) = 0.9 and pKHH3(AMP) = 0.4). The values for pKHH2(NMP) and pKHH(NMP) were also determined from potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO3; 25 degrees C). The agreement of the results obtained by the two methods is excellent. The position of the phosphate group at the ribose moiety and the presence of N-7 in the base moiety influence somewhat the acid-base properties of the mentioned NMPs. Measurements with 5'-AMP in 50% (v/v) aqueous dioxane show that lowering of the solvent polarity facilitates removal of the proton from the H+(N-1) site while the -PO2-3 group becomes more basic; this increases the pH range in which the monoprotonated H(5'-AMP)- species is stable and which is now also extended into the physiological pH region. Some consequences of this observation for biological systems are indicated.