# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 3 Results and Discussion  
The first step in this study involved measuring the chemiluminescent kinetic profiles of Coelenterazine, CLA-1, and CLA-2 in a protic solvent. Specifically, the measurements were first performed in methanol using potassium superoxide, which is a commonly used source of superoxide anion, to trigger the reaction [35; 36; 37; 39; 40]. The resulting kinetic profiles, which represent light emission as a function of time, can be seen in Figure 2 (when normalized). We can see that the chemiluminescent reaction proceeded with a flash-type profile, with a quick burst of light, followed by a rapid decay to basal levels. In fact, all of the studied compounds displayed qualitatively similar chemiluminescent profiles, with the emission of light finishing within the first minute. Nevertheless, the emission lifetime appeared to be shorter for CLA-1/2 than for native Coelenterazine. We attempted to measure chemiluminescence in deionized water but native Coelenterazine had too low a light emission to be measured. It should be mentioned that chemiluminescent reactions typically exhibit lower light-emission intensities in aqueous solution due to energy loss to water molecules [41; 42]. Indeed, previous studies have demonstrated that the chemiluminescence of Coelenterazine can be completely quenched in solvent mixtures with water contents as low as 20% [22]. However, it should be noted that this particular study was performed without the addition of superoxide anion as the triggering agent [22]. Studies have shown that the addition of acidic buffer can enhance the chemiluminescence of Coelenterazine and its analogs [43; 44; 45; 46]. To investigate this further, we attempted to measure the chemiluminescent emission in deionized water with the addition of acetate buffer pH 5.2 (1%). Remarkably, light emissions were then detected for all the compounds in this aqueous solution (Figure 2), and the results were similar to those observed in methanol. Interestingly, the peaks appeared to be more sharply defined in deionized water/acetate buffer. However, it should be noted that some of the observed features of the chemiluminescent kinetic profiles may not solely result from the intrinsic properties of the studied compounds. Instead, they may be attributed to the behavior of the superoxide anion, which can be quite unstable in protic solvents, particularly in aqueous solution due to its tendency for spontaneous disproportionation and strong solvation [47; 48]. Figure 2: Representative and normalized chemiluminescent emissions as a function of time for Coelenterazine, CLA-1, and CLA-2. The reactions were initiated by adding 5 mg of potassium superoxide to either methanol (**A**) or deionized water/1% acetate buffer pH 5.2 (**B**). Chemiluminescence measured in DMF/1% acetate buffer pH 5.2 (**C**). Final volume was 500 \(\mu\)L, with a concentration of 5 \(\mu\)M for all compounds. Finally, we measured the chemiluminescence of these compounds in DMF with the addition of acetate buffer pH 5.2 (1%) [35; 36; 43]. Previous studies have demonstrated that besides being triggered directly by superoxide anion, Coelenterazine and its derivatives can also emit light spontaneously in certain aprotic solvents such as DMF and dimethyl sulfoxide (DMSO) [43; 44; 45; 46; 49; 50]. In these reactions, the chemiluminescence signal is typically more stable than when triggered by superoxide anion in protic media [35; 36; 37]. Thus, by studying the chemiluminescence of these compounds in aprotic media, we can better determine their intrinsic properties, without potential interference from the instability of superoxide anion in protic media. Figure 2 presents the chemiluminescence kinetic profiles of Coelenterazine, CLA-1, and CLA-2 in DMF/acetate buffer pH 5.2 (1%). Although the chemiluminescence still exhibited a somewhat flash-type profile, the light emission persisted significantly longer compared to the reactions in both methanol and deionized water (Figure 2).