Studies on kinase inhibition and mechanisms of phosphoryl transfer.

Phosphoryl transfer from P1-0-ethyl-P1-S-methyl-thio[ P2-(Rp)-16 0,170,180] pyrophosphate to 2-0-benzyl-(S)-propan-1, 2-diol is shown to proceed with complete racemisation of configuration. Further evidence is presented to show that monomeric metaphosphate is a viable intermediate in dissociative phosphoryl transfer. Non-stereoselective reaction and epimerisation at the phosphorus centre of a 1,3,2-oxazaphospholidinone shows that alternative mechanisms to the "addition-pseudorotation-elimination" mechanism need to be considered and are reviewed. A novel strategy of irreversible inhibition of kinases is identified. The anticipated irreversible (suicide) inhibition of yeast hexokinase by ATP? SCH3 is not observed and is rationalised by stabilization of the precursor of the inhibiting species. The mechanism of decomposition of ATP? SCH3 is reviewed and evidence presented for the existence of a cyclo-diphosphate (dioxadiphosphetane) intermediate. Phosphofructokinase (pyrophosphate dependant) catalysed phosphoryl transfer is observed between inorganic thiopyrophosphate and fructose-6-phosphate. Methods of determining the stereochemical course of this enzyme are discussed. Choline kinase catalysed phosphoryl transfer is shown to proceed with inversion of configuration. The substrate specificity of choline kinase is also studied and a novel application for the stereochemical analysis of [?-160,170,180] ATP identified. A novel dithio-analogue of ATP is synthesized (ATP?S?S). Although exhibiting no substrate properties with yeast hexokinase ATP?S?S is shown to be an effective competitive inhibitor of this enzyme with Ki = 55 ?M. Analogues of ATP?S?S and inorganic dithiopyrophosphate are prepared by intramolecular cyclisation with diiodomethane. Intramolecular oxidation studies with inorganic dithiopyrophosphate show that disulphide formation could be applicable to ATP?S?S to generate nucleotide analogues