This grant in Organic and Macromolecular Chemistry provides support for the work of Professor Paul Schaap at Wayne State University. The work explores the properties of thermally stable dioxetanes which can be triggered by chemical reagents or enzymes to generate chemilumescence in organic solvents or in aqueous solution. This work will give significant new insights into chemiluminescent and bioluminescent processes. The results may also lead to the development of new methods for ultrasensitive biological assays including luminescent enzyme-like immunoassays and DNA probes. Various types of dioxetanes will be studied to provide an understanding of the structural requirements for efficient chemiexcitation. The mechanism for the formation of excited states will be further elucidated by an analysis of solvent effects and kinetic parameters. Chemical triggering will involve the synthesis of appropriately functionalized dioxetanes for use as chemiluminescent substrates with alkaline phosphatase, beta-galactosidase, chymotrypsin, aminopeptidase, and lipase. Preliminary studies have shown that binding of the dioxetane to the enzyme can result in enhanced chemiluminescence compared to chemical triggering in aqueous buffers. Enzyme kinetic studies will also be carried out to determine Michaelis constants and turnover numbers. Chemical and enzymatic triggering of dioxetanes will also be investigated in organized assemblies such as micelles and liposomes. Incorporation of the dioxetane and a flourescent co-micellar surfactant in micelles will afford efficient energy-transfer chemiluminescence. Experiments with reversed micelles will also be conducted.
