# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 3 Results and Discussion
Specifically, the maximum light emission was only achieved between the first 30 and 60 s of the reaction, and the decay was notably slower, with basal levels only being reached after approximately 5 min. Among the studied compounds, native Coelenterazine exhibited the fastest kinetics, reaching the light-emission maximum plateau faster and showing a faster decay. It was followed by CLA-1 and then CLA-2. We performed a quantitative analysis by calculating the chemiluminescence enhancement ratio between CLA-1/CLA-2 and native Coelenterazine in the three aforementioned media [35; 36] using Equation (1). The results are presented in Table 1.  
\[ \begin{split}\text{{Chemiluminescence}}&\text{{Enhancement}} \text{{Ratio}}\\ &=\frac{\text{{Light Emission}}\text{{Maxim}}\text{{of}} \text{{either}}\text{{CLA-1}}\text{{or}}\text{{CLA-2}}}{\text{{Light Emission}}\text{{Maxim}}\text{{of}}\text{{Coelentrazine}}}\end{split}  \tag{1}\]  
Both CLA compounds presented higher light-emission intensities than native Coelenterazine in methanol, with intensities 2 to 4 times higher. Both CLA compounds showed more enhanced light-emission intensities in aqueous solution that were about 100 times higher than that of Coelenterazine. These results suggest that CLA-1 and CLA-2 have the potential to be used as probes for superoxide anion in biological media, given their significantly enhanced chemiluminescence regarding native Coelenterazine, and following the previous validation of this latter molecule as a probe for this ROS [14; 15]. These findings are also consistent with the performance of other CLA compounds we have developed [35; 36; 37]. Nevertheless, it should be noted that other CLA compounds have shown even higher emission enhancements than Coelenterazine, with ratios of up to 1.11 \(\times\) 10\({}^{4}\)[35; 36]. Moreover, since CLA-1 and CLA-2 have exhibited similar relative light-emission intensities to Coelenterazine, it appears that the introduction of bromine heteroatoms is not a prerequisite for the enhanced emission of these compounds in protic solvents, especially in aqueous solution [35; 36; 37]. However, it should be noted that it was previously determined that the Coelenteramide versions of CLA-1 and CLA-2 presented blue-shifted emissions compared to native Coelenteramide [38]. This may limit the suitability of these compounds for certain \begin{table}
\begin{tabular}{l l l l} \hline \hline
**Compound** & **Methanol** & **Water/Acetate Buffer** & **DMF/Acetate Buffer** \\ \hline CLA-1 & \(4.01\times 10^{0}\pm 5.41\times 10^{-1}\) & \(1.09\times 10^{2}\pm 6.48\times 10^{1}\) & \(1.32\times 10^{-1}\pm 2.97\times 10^{-2}\) \\ \hline CLA-2 & \(1.98\times 10^{0}\pm 1.02\times 10^{-1}\) & \(1.05\times 10^{2}\pm 5.80\times 10^{1}\) & \(1.60\times 10^{-1}\pm 4.33\times 10^{-2}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Measured chemiluminescence enhancement ratios between the chemiluminescence reactions of CLA-1 or CLA-2, and native Coelenterazine. These ratios were obtained using Equation (1) while considering the light-emission maxima in the different solvents. Measurements were performed in either methanol, deionized water/1% acetate buffer pH 5.2, or DMF/1% acetate buffer pH 5.2. In protic solvents, the reactions were triggered by the addition of 5 mg of potassium superoxide. Final volume was 500 uL, with a compound concentration of 5 uM. biological applications, meaning that further optimization and tuning of their luminescent properties would be beneficial. Contrary to the observations in protic solvents, the chemiluminescence of CLA-1/2 appeared to be significantly reduced compared to that of native Coelenterazine (Table 1). In other words, the light-emission intensities of these compounds were not enhanced but were instead substantially decreased relative to Coelenterazine. Thus, the enhanced superoxide anion-triggered chemiluminescence of CLA compounds is not an intrinsic feature of these compounds [35; 36; 37], as it appears to be dependent on the reaction media.