SYNTHESIS OF AFFINITY LIGANDS AND RADIOACTIVE PROBES FOR ISOLATION AND STUDY OF MYOINOSITOL 1,4,5-TRISPHOSPHATE BINDING-PROTEINS

To synthesize an affinity matrix for isolation of D-myo-inositol 1,4,5-trisphosphate binding proteins, racemic 3-cyclohexene-l-carboxaldehyde was oxidized and converted to a mixture of trans-3,4-dihydroxycyclohexane-l-carboxylic acid methyl ester isomers, which was phosphorylated and separated into (±)-(lR,3R,4R)- and (±)-(lR,3S,4S)-trans-3,4-bis[(diphenoxyphosphoryl)oxy]cyclohexane-l-carboxylic acid methyl esters. Each of these racemic compounds was hydrogenolyzed and reacted with ethylenediamine to give a monoamide, N-(2-aminoethyl)-bis(phosphonyloxy)cyclohexane-l-carboxamide, that was coupled to cyanogen bromide activated Sepharose 4B to provide the desired affinity matrices. The intermediate trans-3,4-bis[(diphenoxyphosphoryl)oxy]cyclohexane-l-carboxylic acid methyl ester was also reduced with lithium borotritide to give the (hydroxy[3H]methyl)cyclohexane derivative, which was phosphorylated and hydrogenolyzed to yield trans-3,4-bis(phosphonyloxy)-l-[(phosphonyloxy)[3H]methyl]cyclohexane, a radiolabeled analogue of inositol 1,4,5-trisphosphate. The carboxamide was also coupled to 4-azidosalicylic acid, and the product was iodinated to provide a 125I-radiolabeled photoactivatable cross-linking derivative of cyclohexanediol bisphosphate. © 1990 American Chemical Society. All rights reserved.