Abstract
NADH and NADPH are ubiquitous biological reducing agents essential for both respiration and biosynthesis. The discovery that increased pentose-phosphate pathway activity in cervical cancer cells leads to increased levels of NAD(P)H, emphasises the need for a sensitive detection system as an indication of cellular viability and vitality. The remit of this project was to design and synthesise a novel molecular sensor system whose emissive properties are "switched on" upon reduction by NAD(P)H. Research using the reducible, non-fluorescent dye, resazurin, has shown that, in the presence of a non-enzymic electron transfer agent phenazinium methosulphate (PMS)-NADH can effect reduction to the highly fluorescent dye resorufin. Mechanistic studies have shown that the reduction proceeds via a two-electron hydride transfer to the heterocyclic mediator, followed by a one electron transfer to the dye and disproportionation to furnish the final fluorescent product. It has been shown that direct reduction by NADH does not occur and that the reaction depends upon there being an electron transfer agent present. A new type of reagent for the detection of NAD(P)H has been synthesised, comprising a reducible heterocycle and a masked fluorophore. It has been shown that reduction of the precursor conjugate by NADH results in the release of a detectable fluorescent moiety methylumbelliferone. The synthesis of an analogous conjugate probe containing a known hindered dioxetane moiety is described. Prepared using a previously unreported route, the key vinyl ether intermediate is generated via a Wadsworth-Emmons reductive coupling of an alkoxy phosphonate to 2-adamantanone. Reduction by NADH and subsequent cleavage of a conjugate ether link generates an electron rich phenolate substituted dioxetane which is metastable, resulting in emission from the generated excited product. Work towards a dioxetane containing functionalised alkyl group for conjugation to a fluorophore is also outlined.