Synthesis of affinity ligands and radioactive probes for isolation and study of myo-inositol 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-1-carboxaldehyde was oxidized and converted to a mixture of trans-3,4-di-hydroxycyclohexane-1-carboxylic acid methyl ester isomers, which was phosphorylated and separated into (+-)-(1R,3R,4R)- and (+-)-(1R,3S,4S)-trans-3,4-bis[(diphenoxyphosphoryl)oxy]cyclohex an e-1- 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-1-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-1-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)-1-[(phosphonyloxy)[3H]methyl]cy clohexane, 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.
Jina
A N
AN
Department of Biochemistry, University of California, Berkeley 94720.
Ralph
J
J
Ballou
C E
CE
D015220:Calcium Channels; D002240:Carbohydrate Sequence; D002352:Carrier Proteins; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D015544:Inositol 1,4,5-Trisphosphate / Q000302:isolation & purification*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear
D015220:Calcium Channels; D002352:Carrier Proteins; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate