Mechanistic Aspects of Bioluminescence and Chemiluminescence(By a grant Reserch Institute for Integrated Science, Kanagawa University)

Bicyclic dioxetane bearing a (3-cyanoethenyl) phenyl group was found to be triggered by the Michael addition of a malonate anion with an accompanying emission of red light. 3-Adamantylidene-4-methoxy-1,2-dioxetanes bearing a 4-(3-hydroxy)phenyl substituted with a podand-type group, namely, 1,4,7-trioxaoctyl, 1,4,7,10-tetraoxaundecyl, or 1,4,7,10,13 -pentaoxa- tetradecyl were synthesized and their base-induced chemiluminescent decomposition was examined in an aqueous system. Optically active dioxetanes bearing a naphthylnaphthol moiety were also synthesized. They exhibited chemilumines" cence in molecular recognition

Mechanistic Study on Bioluminescence of Fireflies and Related Chemiluminescence

A dioxetane bearing a phenoxide anion decomposes with the accompanying emission of light, effectively by the intramolecular charge-transfer-induced chemiluminescence (CTICL) mechanism, by which the bioluminescence of fireflies has been believed to take place through an intermediary dioxetanone. In the course of our investigation of highly effective dioxetane-based chemilumine scent substrates, bicyclic dioxetanes bearing a 3-hydroxyphenyl moiety substituted with a 5-aryl, such as naphthalen-1-yl, carbazol-9-yl, indol-1-yl, and benzotriazol-1-yl, were synthesized. Base-induced decomposition of these dioxetanes displayed intense light, the maximum wavelength of which changed depending on the crown ether complex used as a base. As a further development, we realized four optically isomeric bicyclic dioxetanes bearing a 2-hydroxy-1, 1\u27-binaphthy1-4-y1 moiety. These isomeric dioxetanes were decomposed with the accompanying emission of light on treatment with three base systems. The chemiluminescence efficiencies of all isomers were practically the same for tetrabutylammonium fluoride (TBAF) in the DMSO system, whereas they split into two diastereomer-groups for 18-crown-6 ether complex of t-BuOK in the benzene-THF system. For the base system using t-BuOK complex of optically active crown ether, the chemiluminescence efficiencies split further into four. The shape of the chemiluminescence spectrum, maximum wavelength, and rate of decomposition were also different between each of the four stereoisomers

Benzothiazolylphenol–Substituted Ketoester is a Useful Fluorescent Probe for Detection of the Mitochondrion in Sea Urchin Sperm

One of the ketoesters derived from benzothiazolylphenol-substituted dioxetane,benzothiazolylphenol-substituted ketoester (TPKE), demonstrates fluorescence in a 0.1 MNaOH 1). In this study, the fluorescent staining of a living cell with TPKE was demonstratedby fluorescence microscopy. When sperm from two species of sea urchins—Pseudocentrotusdepressus and Anthocidaris crassispina—were used as biological materials, TPKE showed afluorescent signal in the midpiece that was composed of a single mitochondrion. The ratioof fluorescent signal intensity to background noise (S/N) was high in the sperm stained with1.0–5.0 μg/ml TPKE in normal artificial seawater (pH 8.0). The S/N ratio decreased inacidic seawater (pH 6.0); acidic conditions repress respiratory activity in sea urchin sperm.Moreover, in the presence of the respiratory chain inhibitor antimycin A and the uncouplercarbonyl cyanide p--trifluoromethoxyphenyl-hydrazone, the sperm showed faint or nofluorescence in normal artificial seawater (pH 8.0). Sea urchin sperm stained with TPKEafter fixation showed faint or no fluorescence. These results suggest that TPKE is apotential fluorescent probe of living sea urchin sperm mitochondria with high respiratoryactivities

Design and Synthesis of Dioxetane-Based Chemiluminescent Substrates with High Efficiency in Aqueous System(By a grant Research Institute for Integrated Science, Kanagawa University)

The chemiluminescent efficiency of hydroxyphenyl-substituted dioxetanes is well known to decrease significantly in an aqueous medium, though more than bioluminescence in an aprotic medium. We investigated here the effect of hydrogen bonding on singlet-chemiexcitation and fluorescence efficiency of the emitter produced for charge-transfer-induced chemiluminescence by the use of dioxetanes bearing a 3-hydroxyphenyl moiety substituted with a proton-donating group at the 4-position as a model substrate. Based on this investigation, four bicyclic dioxetanes bearing a 4-(benzoazol-2-yl)-3-hydroxyphenyl moiety were designed and synthesized. The thus-realized dioxetanes exhibited chemiluminescence with markedly high efficiency in aqueous medium as well as in aprotic medium

生物発光および関連化学発光の機構解明

ホタルやオワンクラゲなどに代表される生物の発光系において時、発光前駆体として高エネルギー物質であるジオキセタノン誘導体が関与しているらしいといわれている。しかしこの種のジオキセタノン誘導体からどのような仕組みで高効率の発光が起るか、その素過程、特に励起種生成の過程については今だよく分かっていない。一方、いわゆるCIEEL活性ジオキセタンは生物発光との関連だけでなく、光医学検査や生化学分析への応用面から、注目されている。このような背景の中で合成化学、有機化学の側面から生物発光および関連化学発光の機構解明を行おうとするのが本共同研究である。本年度において行なった研究の成果は次のようなものである