# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 4 Conclusions  
Reactive oxygen species, or ROS, play an important role in the regulation of different signal transduction pathways required for biological development. Among the most relevant ROS species is superoxide anion, a reduced form of molecular oxygen and a primary product in the respiratory chain. Due to its involvement in inter- and intracellular signaling pathways that are associated with several cellular events, superoxide anion attracts significant biological interest. However, it can also be responsible for oxidative stress and deleterious health effects such as inflammation and cancer.  
Given this, the ability to sense superoxide anion is highly relevant but its high reactivity and low short-term survival rate make this task challenging. One promising sensing strategy is the use of chemiluminescent probes such as marine Coelenterazine. Coelenterazine exhibits selectivity toward superoxide anion, which, when added to the high signal-to-noise ratio of chemiluminescent systems, enables dynamic, sensitive, and selective sensing of this ROS.  
Here, we performed a comparative study of the superoxide anion-triggered chemiluminescent reaction of native Coelenterazine and two halogenated analogs (bearing either  
\begin{table}
\begin{tabular}{l l l l} \hline
**Compound** & **Methanol** & **Water/Acetate Buffer** & **DMF/Acetate Buffer** \\ \hline Coelenterazine & 6.4 \(\pm\) 1.3 & 1.2 \(\pm\) 0.6 & 89.2 \(\pm\) 3.0 \\ \hline CLA-1 & 2.3 \(\pm\) 0.2 & 1.5 \(\pm\) 0.3 & 134.0 \(\pm\) 5.3 \\ \hline CLA-2 & 2.7 \(\pm\) 0.2 & 1.5 \(\pm\) 0.6 & 141.9 \(\pm\) 4.0 \\ \hline \end{tabular}
\end{table}
Table 3: Light-emission half-life (in seconds, s) for chemiluminescent reactions of native Coelenterazine, CLA-1, and CLA-2 in different media: methanol, deionized water/1% acetate buffer pH 5.2, or DMF/1% acetate buffer pH 5.2. Reactions were initiated by adding 5 mg of potassium superoxide to the protic solvents. Final volume was 500 \(\mu\)L, with a compound concentration of 5 \(\mu\)M.  
a bromine or a fluorine heteroatom). Both analogs showed a significant enhancement of superoxide anion-triggered enhanced emission in aqueous solution, with an increase of up to 100 times compared to native Coelenterazine. Therefore, these compounds hold great potential as the foundation for developing novel Coelenterazine-based chemiluminescent probes for sensing superoxide anion in biological media.  
However, the difference in emission between the analogs and Coelenterazine varied with the used solvent. In methanol, the analogs only enhanced the superoxide anion-triggered emission by 2 to 4 times. Interestingly, in _n,n_-dimethylformamide, in the absence of superoxide anion, the intensity of light emitted by Coelenterazine was significantly higher than that of the analogs. Furthermore, the light-emission intensities of the analogs remained relatively constant across protic and aprotic media, whereas that of Coelenterazine was significantly decreased in aqueous solution. This indicates that the analogs did not show a superoxide anion-triggered enhanced emission, but rather that the chemiluminescent reaction of native Coelenterazine suffered a relevant quenching effect in aqueous solution, which barely affected the analogs. Finally, we also did not observe any significant differences in the studied properties of the analogs that could be attributed to a specific halogen element. Overall, these findings advance our understanding of this important type of chemiluminescent system.