COMPARISON OF THE SELF-ASSOCIATION PROPERTIES OF THE 5'-TRIPHOSPHATES OF INOSINE (ITP), GUANOSINE (GTP), AND ADENOSINE (ATP) - FURTHER EVIDENCE FOR IONIC INTERACTIONS IN THE HIGHLY STABLE DIMERIC [H2(ATP)]2(4-) STACK

The concentration dependence of the chemical shifts for the hydrogens H‐2, H‐8 and H‐1′ of ITP and for H‐8 and H‐1′ of GTP has been measured in D2O at 25°C under several degrees of protonation in the pD range 1.2–8.4. For reasons of comparison, inosine and guanosine have been included in the study. The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns > D(Ns)+/Ns in a 1:1 ratio > D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4−≲D(GTP)3− (K∼ 0.7 M−1) < D(GTP)3−/D2(GTP)2− in a 1:1 ratio (K∼ 2.9 M−1) > D2(GTP)2− > D3(GTP)− (K∼ 1.5 M−1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self‐association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3−/D2(ITP)2− are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3−/D2(GTP)2−; in all these pairs the N‐7 site is 50% protonated. Comparison of the association constants for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K∼ 3.3 M−1) > ITP4− (K∼ 0.4 M−1) and Guo (K∼ 8 M−1) > GTP4− (K∼ 0.8 M−1). In addition the series holds: Ado (K∼ 15 M−1) > Guo > Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4− (K∼ 1.3 M−1) < D(ATP)3− (2.1 M−1) < 1:1 ratio of D(ATP)3−/D2(ATP)2− (6 M−1) ≪ D2(ATP)2− (∼ 200 M−1) ≫ D3(ATP)− (K≲ 17 M−1). It is evident (a) that the self‐association tendency of the ATP species is larger and (b) that self‐association is most pronounced for D2(ATP)2− and not for the 1:1 ratio of D(ATP)3−/D2(ATP)2− [although the latter with a 50% protonation of the N‐1 site corresponds to that observed for Ado/D+ (Ado) (K= 6 M−1)]. The very large stability of the dimeric [H2(ATP)]4−2 stack has previously been attributed to the formation of intermolecular ion pairs (and hydrogen bonds) formed between the H+ (N‐1) site of one H2(ATP)2− and the γ‐P(OH)(O)−2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated. This interpretation is in perfect agreement with the properties observed now for H2(ITP)2− and H2(GTP)2−; both species show a low stacking tendency in accord with the fact that their neutral H(N‐1) site is not able to form the mentioned intermolecular ion pair with the (also present) γ‐P(OH)(O)−2 group, and the proton now located at the N‐7 site leads to repulsion of the purine moieties. The correctness of this interpretation is further confirmed by a study of the acid‐base properties of the purine residues in ITP, GTP and ATP in dependence on the concentration of the nucleoside 5′‐triphosphates (Corfù, N. A. & Sigel, H., unpublished results). The results of this study show that the acidity of the H+ (N‐7) site in H2(ITP)2− and H2(GTP)2− is enhanced upon stack formation (which leads to repulsion of the positively charged rings), while the acidity of the H+ (N‐1) site in H2(ATP)2− is reduced upon stack formation, because this site is also involved in ion pair and hydrogen bonding. Some consequences of these summarized results for biological systems are indicated. Copyright © 1990, Wiley Blackwell. All rights reserved