The chemical nature of the 2′-substituent in the pentose-sugar dictates the pseudoaromatic character of the nucleobase (pKa) in DNA/RNA

Abstract

We here show that the pK_a (error limit: 0.01 to 0.03 pK_a unit) of a nucleobase in a nucleotide can be modulated by the chemical nature of the 2′-substituent at the sugar moiety. This has been evidenced by the measurement of nucleobase pK_a in 47 different model nucleoside 3′,5′-bis- and 3′-mono-ethylphosphates. The fact that the electronic character of each of the 2′-substituents (Fig. 1) alters the chemical shift of the H2′ sugar proton, and also alters the pK_a of the nucleobase in the nucleotides has been evidenced by a correlation plot of pK_a of N3 of pyrimidine (T/C/U) or pK_a of N7 of 9-guaninyl with the corresponding δH2′ chemical shifts at the neutral pH, which shows linear correlation with high Pearson′s correlation coefficients (R = 0.85–0.97). That this modulation of the pK_a of the nucleobase by a 2′-substituent is a through-bond as well as through-space effect has been proven by ab initio determined pK_a estimation. Interestingly, experimental pK_as of nucleobases from NMR titration and the calculated pK_as (by ab initio calculations utilizing closed shell HF 6-31G** basis set) are linearly correlated with R = 0.98. It has also been observed that the difference of ground and protonated/de-protonated HOMO orbital energies (ΔHOMO, a.u.) for the nucleobases (A/G/C/T/U) are well correlated with their pK_as in different 2′-substituted 3′,5′-bis-ethylphosphate analogs suggesting that only the orbital energy of HOMO can be successfully used to predict the modulation of the chemical reactivity of the nucleobase by the 2′-substituent. It has also been demonstrated that pK_a values of nucleobases in 3′,5′-bis-ethylphosphates (Table 1) are well correlated with the change in dipole moment for the respective nucleobases after protonation or de-protonation. This work thus unambiguously shows that alteration of the thermodynamic stability (T_m) of the donor–acceptor complexes [], as found with various 2′-modified duplexes in the antisense, siRNA or in triplexes by many workers in the field, is a result of alteration of the pseudoaromatic character of the nucleobases engineered by alteration of the chemical nature of the 2′-substitution.