Inframolecular protonation process of 6-modified myo-inositol 1,4,5-tris(phosphates): substitution effects on the cooperativity between the phosphategroups

The protonation process of the individual functional groups of four analogu es of(+/-)-myo-inositol 1,4,5-tris(phosphate) [Ins(1,4,5)P-3], modified at position 6, were studied by P-31- and H-1-NMR titration experiments in orde r to gain insight into the reasons for the particular importance of OH6 for the binding of Ins(1,4,5)P-3 to its receptor. The fluorinated derivative 1 2) was studied to evaluate the H-bond donor or acceptor ability of OH6. Com pounds 3 and 4 should explain the presence and the configuration of OH6, re spectively, whereas the amino analogue (5) was considered to delineate the effect of a positively charged group of about the same size as OH6 on the v icinal phosphates P1 and P5. The P-31-NMR curves look alike for compounds 2 -4 and differ from that of Ins(1,4,5)P-3, as the initial downfield shift of P1 upon protonation is no longer observed. In the H-1-NMR titration curves , the wrongway shift found for H2 of Ins(1,4,5)P-3 is observed for H6 in co mpounds 3 and 4. In addition, both phosphorus and proton resonances of comp ound 5 are influenced by the protonation of the neighbouring NN, group. By considering the protonation constants it is shown that the log K-1 values d ecrease in the order 6-F-Ins(1,4,5)P-3 > 6-deoxy-Ins(1,4,5)P-3 > Ins(1,4,5) P-3 > epi-Ins(1,4,5)P-3 > 6-NH2-Ins(1,4,5)P-3, indicating that the substitu tion effect is the consequence of the lipophilicity of the substituents, th e basicity order following the order of the hydrophobic a constants. Consid eration of the microprotonation constants, calculated for all the studied c ompounds, leads to the conclusion that hydration effects and hydrogen bond donor ability of the equatorial OH6 hydroxyl should mainly account for the differences observed between compounds 2-4 and Ins(1,4,5)P-3. The amino der ivative 5 remains in a zwitterionic form over almost the entire pH range st udied (2.5 < pH ( 11.5). Through the presence of an intramolecular hydrogen -bonded ion pair interaction the basicity of all three phosphates is decrea sed whereas the basicity of the amine increases. The strength of the hydrog en-bonded ion pair appears to be of the same order of magnitude as that of the recently published monoammonium phosphate complexes.