Understanding pantothenate synthetase: mechanism, inhibition and monitoring of enzymatic reactions

The research described herein is directed towards understanding the enzyme pantothenate synthetase; including monitoring of the enzymatic reaction with fluorescent small-molecule chemosensors and the development of nucleoside-based enzyme inhibitors. Chapters 2 to 4 detail the design, synthesis and evaluation of fluorescent pyrophosphate chemosensors. In Chapter 2, a previously described naphthalene diimide chemosensor 50 was prepared and evaluated. The naphthalene diimide chemosensor 50 demonstrated good selectivity for pyrophosphate when compared with other biologically relevant anions. However, when evaluated in an enzymatic assay it was not sufficiently robust to be applied to real-time monitoring. Chapter 3 details the preparation of two novel anthracene-based chemosensors 61 and 62. These two anthracene-based chemosensors 61 and 62 utilised chelation enhanced fluorescence (CHEF) to generate a high fluorescent output. However, their utility was limited by a lack of selectivity, resulting in difficulties resolving pyrophosphate from structurally related adenosine mono phosphate, diphosphate and triphosphate (AMP, ADP, ATP). Chapter 4 describes the development of two novel core-substituted naphthalene diimide chemosensors 92 and 93, aiming to combine the good selectivity for PPi of the first generation NDI chemosensor with the excellent quantum yields of the second generation anthracene chemosensors. Chemosensors 92 and 93 demonstrated interesting photophysical properties. However, limited solubility in aqueous solution prevented their application to the monitoring of enzymatic reactions. Chapter 5 details the preparation, isolation and purification of the enzyme pantothenate sythetase from E. coli. Furthermore, work toward the preparation of nucleoside-based inhibitors of pantothante synthetase is also described. While the initial route to access the target compounds was unsuccessful, preliminary studies on a revised strategy addressed the shortcomings of the initial approach. Chapter 6 describes the subsequent application of anthracene-based chemosensors 61 and 62 to the detection of zinc in aqueous systems. Chemosensors 61 and 62 provided accurate detection of zinc at environmentally relevant concentrations in the presence of competing analytes. However, they were unable to detect any zinc present in real environmental samples.