Syntheses of D-myo-inositol 1,4,5-trisphosphate affinity ligands

A mixture of 2,3,6-tri-O-benzoyl-4,5-di-O-benzyl-D-myo-inositol and 1,3,6-tri-O-benzoyl-4,5-di-O-benzyl-D-myo-inositol, obtained during our synthesis of D-myo-inositol 1,4,5-trisphosphate [C.E. Ballou and W. Tegge, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 94-98], was separated after tetrahydropyranylation of the free hydroxyl group in each. 2,3,6-Tri-O-benzoyl-4,5-di-O-benzyl-1-O- (tetrahydro-2-pyranyl)-D-myo-inositol was debenzylated and the two free hydroxyl groups were phosphorylated by a dibenzyl phosphoramidite procedure. The tetrahydropyranyl group was then removed, and phosphorylation at position 1 with benzyl 3-(benzyloxycarbonylamino)propyl di-N-isopropylphosphoramidite, followed by oxidation and deprotection, provided 1-[3-aminopropoxy(hydroxy)phosphinyl]-D-myo-inositol 4,5-bisphosphate. This compound was coupled to activated agarose to prepare an affinity matrix for the isolation of D-myo-inositol 1,4,5-trisphosphate-binding proteins, and it was coupled to 4-azido-2-hydroxybenzoic acid to give a product that was labeled with 125I to prepare a photoactivable derivatizing reagent. The new derivatives retain significant biological activity as assessed by their ability to stimulate the release of stored Ca2+ from the endoplasmic reticulum of permeabilized rat basophilic leukemia cells.