Conformational and inframolecular studies of the protonation of adenophostin analogues lacking the adenine moiety

Four adenophostin analogues lacking the adenine moiety were subjected to 31P- and 1H-NMR titrations in order to determine the acid-base behaviour of the individual ionisable groups of the molecules and the complex interplay of intramolecular interactions resulting from the protonation process. For the two trisphosphorylated compounds, the curve pattern of the phosphorus nuclei corresponds to the superimposition of the titration curves of a monophosphorylated polyol and a polyol carrying two vicinal phosphates, suggesting that the two phosphate moieties behave independently. Also, the general shape of 1H-NMR titration curves of the studied compounds is very close to that of adenophostin A, indicating that the adenine moiety does not specifically interact with the phosphorylated sugar moieties. The curves show, however, that both trisphosphorylated compounds adopt slightly different preferential conformations which could contribute to explain the difference in their affinity for Ins(1,4,5)P3 receptor. Their macroscopic as well as the microscopic protonation constants are higher than those of adenophostin A, indicating that the adenine moiety plays a base-weakening effect on the phosphate groups. Further analysis of the microscopic protonation constants confirms that the compound whose conformation is the closest to that of adenophostin A also shows the highest biological activity. The two bisphosphorylated analogues studied behave very similarly, suggesting that the deletion of the hydroxymethyl group on the pentafuranosyl ring only weakly influences the protonation process of the phosphate groups that bear the glucopyranose moiety.