3-hydroxybenzene 1,2,4-trisphosphate, a novel second messenger mimic and unusal substrate for type-I myo-inositol 1,4,5-trisphosphate 5-phosophatase:: Synthesis and physicochemistry

3-Hydroxybenzene 1,2,4-trisphosphate 4 is a new myo-inositol 7,4,5-trisphosphate analogue based on the core structure of benzene 1,2,4-trisphosphate 2 with on additional hydroxyl group at position-3, and is the first noninositol based compound to be a substrate for inositol 1,4,5-trisphosphate 5-phosphatase. In physicochemical studies on 2, when three equivalents of protons were added, the P-31 NMR spectrum displayed monophasic behaviour in which phosphate-1 and phosphate-2 behaved independently in most of the studied pH range. For compound 4, phosphate-2 and phosphate-4 interacted with the 3-OH group, which does not titrate at physiological pH, displaying complex biphasic behaviour which demonstrated co-operativity between these groups. Phosphate-1 and phosphate-2 strongly interacted with each other and phosphote-4 experienced repulsion because of the interaction of the 3-OH group. Benzene 1,2,4-trisphosphate 2 is resistant to inositol 1,4,5-trisphosphate type I 5-phosphotase catolysed dephosphorylation. However surprisingly, 3-hydroxybenzene 1,2,4-trisphosphate 4 was dephosphorylated by this 5-phosphatose to give the symmetrical 2,3-dihydroxybenzene 1,4-bisphosphate 16. The extra hydroxyl group is shown to form a hydrogen bond with the vicinal phosphate groups at -15 degrees C, and H-1 NMR titration of the ring and hydroxyl protons in 4 shows the OH proton to be strongly stabilized as soon as the phosphate groups ore deprotonated. The effect of the phenolic 3-OH group in compound 4 confirms a critical role for the 6-OH group of the natural messenger in the dephosphorylation mechanism that persists even in radically modified analogues.