Complete Resolution of the Microscopic Protonation Equilibria of D-myo-Inositol 1,2,6-Tris(phosphate) and Related Compounds by 31P NMR and Potentiometry

31 P NMR and potentiometric titration experiments allowed the complete microconstant system to be resolved for D-myo-inositol 1,2,6-tris(phosphate) [Ins(1,2,6)P 3 ] and DL-myo-inositol 1,2-bis(phosphate). For comparison, DL-myo-inositol 1 -mono(phosphate) and DL-myo-inositol 2-mono(phosphate) were also considered. The studies have been performed in 0.1 M tetraethylammonium perchlorate or tetrabutylammonium bromide solutions at 25 °C (media 1) and, in addition, for Ins(1,2,6)P 3 in a 0.2 M KCl medium at 37 °C (medium 2). The 31 P NMR titration curves of the polyphosphates indicate that the protonation process of a given phosphate group is complex and far from that of a simple monoester. The interactivity parameters derived from the microconstants support the conclusion that two cis phosphates interact less than two trans phosphates. The influence of the presence of K + on the microconstants determined in medium 2 can be explained by the competition between H + and K + for binding to the ligand. The distribution curves of the microspecies versus pH allow a direct determination of the protonation state of each phosphate group.