Determination of horserdish peroxidase concentration using the chemiluminescence of Cypridina luciferin analogue, 2 methyl-6-(p-methyoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one

The chemiluminescence of the Cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) was observed at 462nm in the presence of horseradish peroxidase (HRP) and the total spectrum of light emitted was found to depend linearly on HRP concentration. Methods for the determination of HRP concentration using the chemiluminescence was investigated. HRP could be detected in the range from 100 pmol/L to 100nmol/L under the optimum condition, H₂O₂ (10mmol/L) and MCLA (10μmol/L) at pH 5.8.     

Chemiluminescence probe with Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, for estimating the ability of human granulocytes to generate O2-

The Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), in Hanks' balanced salt solution, emitted a weak luminescence which was not affected by superoxide dismutase or catalase and was not augmented by resting human granulocytes. In contrast, activated granulocytes caused a dramatic increase in the luminescence of CLA. The light emission by CLA in the presence of activated granulocytes was inhibited by superoxide dismutase, but not by catalase or benzoate. Azide at 0.5 mM did not inhibit light emission significantly. These results indicate that O2-, rather than H2O2, HO., singlet oxygen, or HOCl, was the agent responsible for eliciting the chemiluminescence of CLA. Moreover, the intensity of light emission by CLA correlated with the rate of production of O2- either by activated neutrophils or by the xanthine oxidase reaction.     

A chemiluminescent probe with a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one, specific and sensitive for O2- production in phagocytizing macrophages

When a Cypridina luciferin analog (the title compound) was added to a macrophage suspension in Hank's balanced salt solution (control), the system emitted a weak, but detectable light, which was not altered in the presence of superoxide dismutase. The same system, however, emitted a much stronger light, just after the addition of a trigger, opsonized zymosan. The luminescence was suppressed to the control level in the presence of superoxide dismutase, while it was only slightly influenced, if at all, by NaN3, a scavenger of singlet oxygen and an inhibitor of myeloperoxidase. Some other results obtained also indicate the participation of O2- in the luciferin analog-dependent luminescence in macrophages during phagocytosis.     

Enhanced chemiluminescence of 6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one by attachment of a cyclomaltooligosaccharide (cyclodextrin). Attachment of cyclomaltononaose (delta-cyclodextrin)

2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one (MCLA) is an oxygen-induced chemiluminescent compound. It has been shown that the chemiluminescence can be enhanced by forming a cyclomaltooligosaccharide (cyclodextrin)-bound MCLA, and therefore, in continuation of the survey of the types of cyclodextrins, in this study, MCLA was attached to the secondary hydroxyl face of delta-cyclodextrin, which consists of nine D-glucose units. Although the oxygen-induced chemiluminescence efficiency of delta-cyclodextrin-bound MCLA in a pH 8.0 aqueous phosphate buffer was 12 times greater than that of MCLA, the efficiency was markedly lower than that of gamma-cyclodextrin-bound MCLA, which exhibited the highest chemiluminescence efficiency in the previous investigation. Although fluorescence efficiency and light-emitter formation efficiency for delta-cyclodextrin-bound MCLA were similar to those for gamma-cyclodextrin-bound MCLA, singlet-excited state formation efficiency for delta-cyclodextrin-bound MCLA was lower than that for gamma-cyclodextrin-bound MCLA. This study distinctly indicated the optimum cyclodextrin for construction of greatly luminescent cyclodextrin-bound MCLA is gamma-cyclodextrin.     

The reaction mechanism for the high quantum yield of Cypridina (Vargula) bioluminescence supported by the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (Cypridina luciferin analogues).

To establish the reaction mechanism of the high-quantum-yield bioluminescence in Cypridina (Vargula), we investigated the chemiluminescence of 6-aryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones (1H) as Cypridina luciferin analogues in DMSO-1,1,3,3-tetramethylguanidine and in diglyme-acetate buffer. We found that the chemiluminescence of 1H with an electron-donating aryl group, such as a 4-(dimethylamino)phenyl, 3-indolyl or 3-(1-methyl)indolyl group, gave a high quantum yield (Phi(CL)) in diglyme-acetate buffer. This indicates that the reaction mechanism producing this high Phi(CL) involves the chemiexcitation of a neutral dioxetanone intermediate possessing an electron-donating aryl group to the singlet excited state of neutral acetamidopyrazine (the light emitter). In addition, we investigated the fluorescence of acetamidopyrazines and performed DFT calculations for neutral dioxetanones and the transition states (TS) of the dioxetanone's decomposition. The results made it clear that the electron-donating aryl group gives the TS and the singlet-excited acetamidopyrazine (S(1)) a strong intramolecular charge transfer (ICT) character, and their similar ICT character leads to the ICT TS --> S(1) route in the charge transfer-induced luminescence (CTIL) mechanism for efficient chemiexcitation. The reaction mechanism of the chemiluminescence of 1H can explain the highly efficient chemiexcitation of Cypridina bioluminescence.     

Sulfur-containing compounds quench 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one chemiluminescence: Discrimination between true antioxidants and quenchers using xanthine oxidase

The probe 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA) is widely used for studying the superoxide anion production and the efficiency of antioxidants in biological systems. Here we report that a number of sulfur-containing compounds applied in biochemical and cytological studies are able to suppress MCLA-derived chemiluminescence (MDCL) independent of their capability to scavenge superoxide anion. The most effective MDCL quenchers appeared to be the substances with thiocarbamoyl and thiocarbonyl groups coupled to cyclic molecules and several thiol- and disulfide-containing compounds. The analysis of MDCL kinetics in a xanthine oxidase system allows one to rapidly discriminate between true antioxidants and the quenchers of chemiluminescence.