ACID-BASE PROPERTIES OF NUCLEOSIDES AND NUCLEOTIDES AS A FUNCTION OF CONCENTRATION - COMPARISON OF THE PROTON AFFINITY OF THE NUCLEIC BASE RESIDUES IN THE MONOMERIC AND SELF-ASSOCIATED, OLIGOMERIC 5'-TRIPHOSPHATES OF INOSINE (ITP), GUANOSINE (GTP), AND ADENOSINE (ATP)

The acid-base properties of the nucleic base residues of ITP, GTP, and ATP, and for comparison also as far as possible of the corresponding nucleosides, were studied in dependence on their concentration, i.e. on the effect of self-association. From the dependence between the H-1-NMR chemical shifts of H-2 (where applicable), H-8, and H-1', and the pD of the solutions, the acidity constants for the deprotonation of the D+(N-7) site in D2(ITP)2-, D2(GTP)2-, D(Ino)+, and D(Guo)+, and of the D+(N-1) site in D2(ATP)2- and D(Ado)+ were calculated. Chemical shift/pD profiles for a whole series of varying concentrations of the nucleic base derivatives (= N) were constructed, including those for infinite dilution (delta-o), which give the acidity constant for the monomeric N, and for infinitely concentrated solutions (delta-infinity), which give the acidity constant of an N in an infinitely long stack. The acidity constants determined from the delta-o/pD plots are in excellent agreement with the pK(a) values measured by potentiometric pH titrations of highly diluted solutions of N. The effects of self-association are striking: e.g. the pK(a) value of the D+(N-7) site in D2(GTP)2- is lowered by about 1 (as calculated from the delta-o/pD and delta infinity/pD profiles), while the pK(a) value of the D+(N-1) site in D2(ATP)2- is increased by approximately 0.3; i.e. in the first case deprotonation is facilitated and in the second it is inhibited. The increasing inhibition of the H+(N-1) deprotonation with an increasing ATP concentration is due to the high stability of the dimeric [H2(ATP)]2(4-) stack for which the intermolecular H+(N-1)/gamma-P(OH)(O)2- ion pairs between the two ATP molecules are crucial. In those cases where no other significant interaction but aromatic-ring stacking in the self-association process occurs, the release of protons from protonated nitrogen-ring sites is facilitated with increasing stacking; this holds not only for D2(GTP)2- as indicated above, but also for D2(ITP)2-, D(Ino)+, and D(Ado)+. The latter example especially suggests that the situation for the D2(ATP)2- system is exceptional. Some consequences of the considered acid-base properties for biological systems are indicated.