Inframolecular studies of the protonation of adenophostin A: Comparison with 1-D-myo-inositol 1,4,5-trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the D-myoinositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic proton ation process of this compound by perfoming P-31 NMR, H-1 NMR, and potentio metric titration experiments. The logarithms of the first to the fourth ste pwise protonation constants are, respectively, log K-1 = 8.48, log K-2 = 6. 20, log K-3 = 4.96, and log K-4 = 3.80. The latter constant refers to the p rotonation equilibrium involving the N1 adenine nitrogen. From the microcon stants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophos tin A at near physiological pH is very similar to those of inositol 1,4,5-t risphosphate, indicating that differences in phosphate charge cannot accoun t for the high potency of this molecule. The analysis of the H-1 chemical s hifts vs pH provided complementary conformational information. In particula r, a slight "wrongway shift" of H1" can be related to the protonation of P2 , thus indicating a short H1"-P2 distance. Our results are in line with a r ecently published model in which, however, a certain degree of constraint w ould beep the ribose 2'-phosphate moiety close to the glucose ring phosphat es. (C) 1999 Academic Press.