A phosphodiester analogue of the second messenger Ins(1,4,5)P3 has been synthesized and used to prepare a novel photoaffinity label and a selective bioaffinity matrix. A selectively protected inositol precursor was first converted by phosphite ester chemistry to an N-protected 1-O-(3-aminopropyl-l-phospho)-DL-myo-inositol and then phosphorylated to give a fully benzylated IP3 derivative. Hydrogenolysis gives the title compound, which was converted to a photolabile analogue and was immobilized on a polymeric resin. The analogues all competed with [3H] Ins(1,4,5)P3 for binding to purified IP3 receptors from rat brain. Reconstituted receptor liposomes showed calcium release when stimulated by the tethered IP3 materials. None of the new materials were substrates for the 5-phosphatase or the 3-kinase that normally act on Ins(1,4,5)P3. © 1991, American Chemical Society. All rights reserved.