# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 3 Results and Discussion
Given these findings, it is important to investigate the factors that affect the chemiluminescence of these compounds in different media and explain the observed differences in light emission. To this end, we assessed the relative light-emission efficiency of the three compounds in the three reaction media. We then calculated the ratios between the light-emission maxima of the chemiluminescent reaction of each compound in different media using water/acetate buffer as the reference (\(\frac{Methanol\ or\ DMF/Acetate\ Buffer}{Water/Acetate\ Buffer}\)). The results are presented in Table 2.  
CLA-1 and CLA-2 exhibited similar behavior, with higher light-emission intensities observed in water/acetate buffer than in methanol. This could be partly attributed to the addition of acidic buffer in the former scenario [43; 44; 45; 46]. In contrast, when switching from protic to aprotic media, the light-emission intensity increased by approximately 2-2.5 times. However, the results for native Coelenterazine showed a markedly different trend (Table 2). Chemiluminescence was more intense in methanol than in water/acetate buffer by a factor of about 9, which was opposite to the behavior observed for CLA-1 and CLA-2. More importantly, the light-emission intensity maximum of Coelenterazine in DMF/acetate buffer was about 1.31 \(\times\) 10\({}^{3}\) times higher than that in water/acetate buffer, whereas for the two CLA analogs, the increase was only of 2.00-2.50 \(\times\) 10\({}^{0}\). So, the results suggest that although the chemiluminescence of CLA-1/CLA-2 is relatively unaffected by the reaction media, the light emission of Coelenterazine is significantly reduced in water compared to other solvents. Therefore, while the observed outcome is indeed an enhanced emission by CLA-1/CLA-2, it appears to be less of an enhancement and more of an inhibition of the chemiluminescence of Coelenterazine in aqueous solution, which barely affects CLA compounds. As mentioned earlier, chemiluminescent systems typically exhibit lower light-emission intensities in aqueous solution due to the energy loss to water molecules [41; 42]. Hence, the reduction in emission intensity for Coelenterazine in water is not unexpected. What may be surprising is that CLA-1/2, as with other similar CLA derivatives [35; 36; 37], are relatively unaffected by this quenching effect. Thus, our findings point out the relative insensitivity of CLA-1/CLA-2 to changes in media as the reason behind the enhanced emission in water, in contrast to the sensitivity of Coelenterazine to solvent effects. It is worth noting that we recently conducted a study on the Coelenteramide versions of both CLA-1 and CLA-2 [38]. Unlike their chemiluminescent reactions, the fluorescence \begin{table}
\begin{tabular}{l l l} \hline
**Compound** & \(\frac{Methanol}{Water\ |Acetate\ Buffer}\) & \(\frac{DMF1 Acetate\ Buffer}{Water\ |Acetate\ Buffer}\) \\ \hline Coelenterazine & \(8.98\times 10^{0}\pm 3.56\times 10^{0}\) & \(1.31\times 10^{3}\pm 3.16\times 10^{2}\) \\ \hline CLA-1 & \(6.19\times 10^{-1}\pm 2.44\times 10^{-1}\) & \(2.50\times 10^{0}\pm 5.07\times 10^{-1}\) \\ \hline CLA-2 & \(1.93\times 10^{-1}\pm 8.22\times 10^{-2}\) & \(2.00\times 10^{0}\pm 4.36\times 10^{-1}\) \\ \hline \end{tabular}
\end{table} 
Table 2: Measured ratios between the obtained light-emission maxima of the chemiluminescence reactions of Coelenterazine, CLA-1, and CLA-2 in different media with deionized water/1% acetate buffer pH 5.2 as a reference. Measurements were performed in either methanol, deionized water/1% acetate buffer pH 5.2, or DMF/1% acetate buffer pH 5.2. When in protic solvents, reactions were triggered by the addition of 5 mg of potassium superoxide. Final volume was 500 \(\mu\)L, with a concentration of 5 \(\mu\)M for all compounds. quantum yields of their Coelenteramide versions were indeed relevantly affected by the solvent. For the CLA-1-based Coelenteramide, the quantum yield ranged from 8 to 17%, with a higher value observed in methanol.