1H-NMR comparative studies of dinucleoside monophosphates. Influence of different factors on the conformational equilibria of nucleoside and dinucleoside monophosphates in aqueous solution

Abstract

In order to obtain information about different factors which exert a great influence on the conformational equilibria of mono-, di- and oligonucleotides, the average conformation of four heterodinucleoside monophosphates, ApC, CpA, ApU and UpA, was studied by ¹H-NMR at 250 MHz and compared with that of GpC, CpG, GpU, UpG and the corresponding nucleosides and nucleotides. The ratio of $\text{N}\text{S}$ conformers and the rotamer distribution of the exocyclic group were evaluated from proton-proton coupling constants, and the orientation of the base about the glycosidic bond from proton relaxation involving selective deuteration. The influence of intra- and intermolecular interactions on proton chemical shifts and conformational equilibria has been carefully studied from temperature and concentration effects. The results show that intermolecular base-base stacking does not modify the average conformation of dinucleoside monophosphates, while the intramolecular interaction (between residues of the same molecule) favours the N conformer, the gg rotamer and the anti conformation. The large upfield shift of H_5′ (lower field resonances) compared with H_5″ (higher field resonances) and the large variation of the N proportion with increasing temperature suggest that H_3′ is statistically closer to H_5′ than H_5″. There is a correlation between temperature, the gg rotamer and the variation in the relative chemical shifts between H_5′ and H_5″. These conclusions are in agreement with those of our previous work on GMPs using pH and temperature effects. T₁ relaxation studies indicate a large predominance of the anti conformation for both residues of ApC and ApU; these two compounds also exhibit a very high degree of association. For the naturally occurring ribonucleoside derivatives in aqueous solution, the base chemical structure, the position of the phosphate group, the temperature and pH are among the most important factors which can modify the conformation equilibria of molecules: the N conformation is generally more favoured in pyrimidine than in purine derivatives. The anti proportion is higher in 5′- than in 2′- or 3′-mononucleotides. However, the influence due to the position of the phosphate group is not determined in dinucleoside monophosphates. The temperature effect is negligible on the conformation of monomers but is remarkably large for the dimers: it consists of reducing the N and gg proportions which are preponderant in the dimers at room temperature. The pH effect is particularly large for the G residue, the conformation of which changes upon pr tonation at N⁷ (pK ≈ 2.3); the N conformer is favoured at acidic pH.