Synthesis of novel phosphorothioate and pyrophosphate analysis of inositol phosphates.
thesisposted on 19.11.2015, 08:46 by Nicholas Jason. Noble
A novel synthesis of cyclic pyrophosphates was developed. The vicinal bisphosphorothioate, l-phenyl-ethane-l,2-bisphosphorothioate, was synthesised using phosphorus (III) chemistry. This model compound was used to investigate the formation of the cyclic 7-membered pyrophosphate; N-bromosuccinimide was found to be the reagent of choice, giving the cyclic pyrophosphate in moderate yield. Activation of the bisphosphorothioate moiety by S-methylation, increased the yield of the pyrophosphate, as id reducing the conformational mobility of the vicinal bisphosphorothioate. Isotopic labelling experiments were used to investigate the mechanism of the reaction. However, it was concluded that the reaction proceeded via a complex combination of reaction pathways. The synthesis of the bacterial metabolite cyclic-2,3-bisphosphoglycerate was attempted using the new method, unfortunately, cyclic-2,3-bisphosphoglycerate was not isolated. In order to prepare pyrophosphate analogues of myo-inositol-1,4,5-trisphosphate, it was necessary to prepare compounds such as myo-Inositol-1-phosphate-4,5- bisphosphorothioate and 6-deoxy-D-myo-inositol-l,4,5-trisphosphorothioate. Inositol-l,4-bisphosphate-5-phosphorothioate was synthesised from a precursor, the structure of which was determined by 2-dimensional NMR spectroscopy. myo-Inositol- 1-phosphate-4,5-bisphosphorothioate and myo-inositol-1,4-bisphosphate-5- phosphorothioate were found to be potent inhibitors of the inositol-5-phosphatase enzyme, whereas 6-deoxy-D-wyo-inositol-l,4,5-trisphosphorothioate was found to be a weak inhibitor of the inositol-5-phosphatase enzyme. Initially, the synthesis of an inositol pyrophosphate was attempted where phosphate scrambling, a potential side reaction, could not compete with the formation of the pyrophosphate. To this end, the desulphurisation of 6-deoxy-2,3-cyclohexylidene-D- myo-inositol-l,4,5-trisphosphorothioate was attempted. The reaction gave the 6-deoxy- 2,3-cyclohexylidene-D-myo-inositol-l-phosphate-4,5-pyrophosphate in good yield. myo-Inositol-1-phosphate-4,5-bisphosphorothioate was prepared from wyo-inositol and was treated with N-bromosuccinimide. The result of the reaction was to give the target molecule myo-inositol-1-phosphate-4,5-pyrophosphate again in high yield. Unfortunately, the pyrophosphate was found not to be active at releasing Ca2+ nor active towards the phosphoinositide cycle enzymes.