Fluorescence quenching and spin-label electron spin resonance studies of stacking self-association in aqueous solutions of 2-aminopurine riboside and its 5'-mono- and -diphosphate

The autoassociation of 2-aminopurine riboside (rn²Pur) and its 5'-mono-(P-rn²Pur) and 5'-diphosphate (PP-rn²Pur) in neutral aqueous solutions was investigated using fluorescence quenching and ESR spin-label methods within the range 276–358 K. Respective equilibrium constants and thermodynamic functions were derived therefrom assuming two models of infinite autoassociation: (i) an isodesmic one (K₂ =K₃ = · K_p). and (ii) one in which K₂≠ K₃ = K₄ = · K_p. Comparative analysis of these data and that of the parent 2-aminopurine, obtained previously, allowed us to formulate the following conclusions: (1) the mechanism of autoassociation of rn²Pur varies with temperature in such a way that at T = 318 K the isodesmic model is fulfilled (K₂ = K_p); at higher temperatures $\text{K}{}_{\mathrm{p}}\text{K}{}_2$> 1. i.e., the process is cooperative, while at lower temperatures it becomes anticooperative (K_p/K₂ < I); (2) at 298 K the tendency to autoassociation decreases in the order; rn²Pur>P-rn²Pur>PP-rn²Pur; (3) rn²Pur forms highly packed complexes with the bases stacked and the ribofuranose residues interacting via hydrogen bonds or water bridges: (4) autoassociation of P-rn²Pur and PP-rn²Pur is mainly governed by stacking of the bases, while the ribose phosphate residues attain a trans configuration corresponding to the lowest electrostatic repulsion between charged phosphate groups: even at high ionic strength (I = 0.8). a positive electrostatic contribution to the free enthalpy of autoassociation is observed: (5) the two methods employed gave similar results for P-rn²Pur, but somewhat different ones for rn²Pur because the presence of the spin label (nitroxide stable radical) at the 2'(3')-OH group of the ribose residue prevents its interaction via hydrogen bonding with an unlabelcd one of an adjacent nucleoside.