Allan M. Cooke

Stereospecific recognition sites for [3H]inositol(1,4,5)-trisphosphate in particulate preparations of rat cerebellum

A very high density of stereospecific binding sites for inositol-(1,4,5)P3 have been identified in rat cerebellar membranes using [3H]inositol-(1,4,5)P3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (KD 23 nM). Incubations were carried out at 4 degrees and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [3H]-(1,4,5)P3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P3 was the only other phosphate to show relatively high affinity (KD 1500 nM). HPLC-pure Ins(1,3,4)P3 and Ins(1,3,4,5)P4 were substantially weaker and Ins(1,4)P2, Ins-2-P1, Ins-1-P1, Ins(1,2)-cyclic P1 and inositol were totally inactive at concentrations less than 50 microM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P3 can initiate the release of bound Ca2+.

Synthesis of DL-Myo-inositol 1,4,5-triphosphate

Abstract DL- Myo -inositol 1,4,5-trisphosphate has been synthesised from (±) 1,2,4-tri- O -benzyl- myo -inositol using a phosphite chemistry approach.

DL-Myo-inositol 1,4,5-trisphosphorothioate mobilizes intracellular calcium in Swiss 3T3 cells and Xenopus oocytes

The initial water-soluble product of receptor-stimulate polyphosphoinositide hydrolysis, D-myo-inositol 1,4,5-trisphosphate is now accepted as the second messenger that stimulates release of Ca2+ from intracellular pools. We report here the first examples of Ca2+ release by a novel phosphatase-resistant inositol trisphosphate analogue, DL-myo-inositol 1,4,5-trisphosphorothioate, in Swiss 3T3 cells and Xenopus oocytes. L-myo-inositol 1,4,5-trisphosphate was inactive in the latter system.

Myo-inositol(1,4,5)trisphosphorothioate binds to specific [3H]inositol(1,4,5)trisphosphate sites in rat cerebellum and is resistant to 5-phosphatase.

D-Myo-inositol(1,4,5)trisphosphorothioate, a synthetic analogue of inositol(1,4,5)trisphosphate was shown to bind with a relatively high affinity to specific sites on rat cerebellar membranes labelled with [3H]inositol(1,4,5)trisphosphate. Use of this binding assay has also established that unlike the trisphosphate, the trisphosphorothioate is completely resistant to a specific 5-phosphatase prepared from human erythrocytes. The ability of this novel analogue to release intracellular Ca2+ has already been reported and it offers considerable potential in the investigation of phosphoinositide-linked receptors.

myo-Inositol 1,4,5-trisphosphorothioate is a potent competitive inhibitor of human erythrocyte 5-phosphatase

The effect of the myo‐inositol 1,4,5‐trisphosphate (IP3) analogue, myo‐inositol 1,4,5‐trisphosphorothioate (IPS3) on the dephosphorylation of D‐5‐[32P]IP3 by the 5‐phosphatase from human erythrocyte membranes has been investigated. DL‐IPS3 was found to act as a competitive inhibitor with a K i of 6 μM, making it the most potent inhibitor currently available for this enzyme. L‐IP3 inhibited the enzyme with a K i of 124 μM and was more potent than D‐2,3‐diphosphoglycerate (K i 978 μM).

myo-Inositol 1,4,5-trisphosphorothioate: a novel analogue of a biological second messenger

DL-myo-Inositol 1,4,5-trisphosphorothioate, a novel analogue of the biologically active second messenger D-myo-inositol 1,4,5-trisphosphate, has been synthesised by use of phosphite chemistry.

Synthesis of (±)-myo-inositol 1,4,5-trisphosphate and the novel analogue (±)-myo-inositol 1,4-bisphosphate 5-phosphorothioate

Abstract Novel routes to myo -inositol 1,4,5-trisphosphate and a phosphorothioate analogue involving mixed P(V) and P(III) chemistry have been developed. Phosphorylation of 2,3,6-tri- O -benzyl- myo -inositol 1-[di-(2,2,2-trichloroethyl) phosphate] with bis(2,2,2-trichloroethyl) phosphorochloridate gave a mixture of the 1,4- and 1,5-bisphosphate derivatives from which the 1,4-bis[di-(2,2,2-trichloroethyl) phosphate] 9 crystallised. Phosphitylation of HO-5 in 9 followed by oxidation yielded the 1,4-bis[di-(2,2,2-trichloroethyl) phosphate] 5-[di-(2-cyanoethyl) phosphate] which was deblocked using sodium in liquid ammonia to give (±)- myo -inositol 1,4,5-trisphosphate. Phosphitylation of HO-5 in 9 followed by sulphoxidation generated the 1,4-bis[di-(2,2,2-trichloroethyl) phosphate] 5-[di-(2-cyanoethyl) thiophosphate] which was deblocked to give (±)- myo -inositol 1,4-bisphosphate 5-phosphorothioate. Removal of the 2,2,2-trichloroethyl group, using sodium in liquid ammonia, represents a new method for removing this protecting group.

Synthesis of myo-inositol 1,4-bisphosphate-5-phosphorothioate

Synthesis of myo-insoitol 1,4,5-triphosphate and myo-inositol 1,4,-bisphosphate-5-phosphorothioate, a phosphatase-resistant analogue of a biological second messenger, have been accomplished using a novel combination of PIII and PV chemistry and a new deprotection method for the 2,2,2-trichloroethyl group.

Phosphorothioate analogues of inositol phosphates

Abstract Novel analogues of the intracellular second messenger D-myo-inositol 1,4,5-trisphosphate, which possess phosphorothioate groups in place of phosphate groups have been synthesized. They exhibit unusual biological properties which will be of considerable application in understanding the phosphoinositide cycle.