Chemiluminescence of Cypridina Luciferin Analogs. Part 3. MCLA Chemiluminescence with Singlet Oxygen Generated by the Retro‐Diels‐Alder Reaction of a Naphthalene Endoperoxide

The chemiluminescence of the Cypridina luciferin analog, 2‐methyl‐6‐( p‐methoxyphenyl )‐3,7‐dihydroirnidazo[1,2‐a]pyrazin‐3‐one (MCLA), with O 2 ( 1 Δ g ) generated by the retro‐Diels‐Alder reaction of 3‐(4′‐methyI‐l'‐naphthyl)‐propionic acid endoperoxide was studied in an aqueous solution with pH 7.12 at 37°C. The retro‐Diels‐Alder reaction occurs with a first‐order rate constant of (4.16 ± 0.13) × 10 −4 /s to quantitatively yield O 2 ( 1 Δ g ) and 3‐(4′‐methyl‐l'‐naphthyl (‐propionic acid. MCLA consumed equimolar amounts of O 2 ( 1 Δ g ) with a second‐order rate constant (6.96 ± 0.27) × 10 8 /M/s to emit light in an aqueous solution with pH 7.12 at 37°C. The chemiluminescence spectrum was identified as the fluorescence spectrum of 2‐acetylamino‐5‐( p ‐methoxyphenyl)pyrazine (OMCLA), a major chemiluminescence reaction. Chemiluminescence spectra and product yields for MCLA reactions with O 2 1 Δ g , with O 2 ( 3 Σ − g ) and with superoxide anion radicals are identical, suggesting that all of these reactions occur via a common MCLA‐2‐hydrope‐roxide intermediate formed by a combination of MCLA radicals and superoxide anion radicals. We have established practical use of NEPO as an O 2 ( 1 Δ g ) source and MCLA as a biological probe for detecting O 2 ( 1 Δ g ).