Evaluation of five imidazopyrazinone-type chemiluminescent superoxide probes and their application to the measurement of superoxide anion generated by Listeria monocytogenes

Superoxide-triggered chemiluminescence of five new imidazopyrazinone derivatives was investigated using the hypoxanthine-xanthine oxidase system as the source of superoxide anion. The results showed that they are highly sensitive and have favorable properties in measuring superoxide anion. With those new probes, the generation of superoxide anion from the bacteria Listeria monocytogenes was examined. The results confirmed the previous report that L. monocytogenes is an unusual organism that extracellularly and continuously generates a high level of superoxide anion in the presence of acetaldehyde. The data indicated that two of the probes, 3,7-dihydro-2-methyl-6-phenylethynylimidazo[2,2-a]pyrazin-3-one (4) and its methoxy derivative (5), are highly sensitive and useful in the measurements of superoxide anion and are clearly superior to 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3-one (MCLA), which has been generally considered the most sensitive superoxide probe in the past. When tested at a probe concentration of 3.3 mu M, the luminescence response and the signal-background ratio of compound 4 were 1.5 and 2.5 times those of MCLA, respectively, and the signal-background ratio of compound 5 was almost 15 times that of MCLA, though the luminescence response of this compound was slightly lower than that of MCLA. The low probe concentration used enhances the usefulness of probes in the measurements of superoxide in functioning biological systems. (C) 1998 Academic Press.