Abstract
Symplectin, which is one of the few photoproteins, forms covalent bonds with the dehydrocoelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cite first, and then moves to the active cite Cys-390 for the luminescence. In the first part of this dissertation, we developed an efficient synthetic route to yield keto-acetal (2-6a, 2-20b, and 2-20c) and keto-sulfone (2-24b and 2-24c) as the equivalent of enol-aldehyde, which was the key segment toward coelenterazine (CL) and its analogs as luminescent molecules. The improved synthesis of coelenterazine and its analogs employed an advanced condensation with the aminopyrazines using various keto-acetal and keto-sulfone segments, which resulted in much higher yields to give the final imidazopyrazinone heterocycles than the previous method using enol-aldehyde. In the second part, we focus on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4'-methoxyphenyl)- or 8-(2'-naphthyl)-group and 2-(2’,4’-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions. In the third part, the cysteine adducts 2-43b were treated to aequorin as the model of DCL in symplectin. This result showed us that the intramolecular peroxide oxidation happened on cysteine residue and lead decrease of bioluminescence. The intramolecular peroxide oxidation combined with the previous study lead us to determinate the absolute configuration of C2' position in cysteine adducts.