Determination of the tautomeric equilibrium of Ψ-uridine in the basic solution

The pD-dependent C-13 shift and (3)J(CH) of Psi-uridine have shown that the pK(a) of its N1 and N3 are 9.6 and 9.3, respectively. For the monoanionic Psi-uridine, the N1-anion population (64%) is favored over the N3-anion (36%), suggesting a larger preference of the population for N1-Hg2+ complex for Psi-uridine over its N3-Hg2+ complex in the neutral pH, which has also been proven by the similarity of the pD-dependent titration curve of Psi-uridine and its N3-methyl-Psi-uridine. Finally, ab initio molecular orbital calculations with a 6-31G** basis set (Gaussian 94) have been used to explain why the N1-anion tautomer of Psi-uridine is thermodynamically more stable than the N3-anion tautomer of Psi-uridine, which cannot be corroborated by the results of the calculations at 6-31+G** and 6-31++G** levels. The fact that N1 of Psi-uridine (pK(a) 9.3) is more acidic than N3 (pK(a) 9.6) implies that the N1-H will compete favorably for any potential hydrogen bonding with a complementary nucleotide over the N3-H.