# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 1 Introduction  
Reactive oxygen species (ROS) are crucial in the regulation of several signal transduction pathways essential for biological development. They can directly react or modify the structure of transcription factors, genes, and proteins [1; 2; 3; 4]. Examples of ROS include singlet oxygen, hydrogen peroxide, hydroxyl radical, and superoxide anion. Superoxide anion is a reduced form of molecular oxygen that consists of 2 oxygen atoms with a negative electrical charge and 17 electrons [3]. Superoxide anion is primarily produced in the respiratory chain and results from the one-electron reduction of molecular oxygen [2; 3]. It is also one of the first species generated by various cellular systems [3]. Its production can lead to the formation of different ROS, which can be both damaging and involved in cell signaling [2; 3]. Superoxide anion can react with reducing compounds or biomacromolecules, allowing for the control of the expression of target substances and the regulation of associated redox reactions [1]. However, its imbalance can lead to deleterious effects [5]. So, superoxide anion is involved in inter- and intracellular signaling pathways associated with different cellular events [6] and is one of the most important ROS responsible for oxidative stress [3], leading to detrimental health effects such as inflammation and cancer [7; 8; 9; 10]. The sensing of superoxide anion is particularly important but its determination is challenging due to its high reactivity and low short-term survival rate [1]. Direct determination can be achieved using electron paramagnetic resonance (EPR) spectroscopy [11; 12], which is not suitable for routine analysis [1]. Indirect determination can be achieved through different approaches, including the use of electrochemical and fluorescent (bio)sensors and surface-enhanced Raman spectroscopy (SERS) [1]. Among the possible indirect approaches, the use of chemiluminescent probes has significant potential [13; 14; 15]. Chemiluminescence involves the emission of radiation that results from chemiexcitation due to a chemical reaction [16; 17; 18]. Chemiluminescent systems do not require photoexcitation for generating the light-emitting excited state, resulting in a reduced probability of autofluorescence from the background signal [19]. Consequently, luminescent signals can be generated with high sensitivity and almost no background noise [18]. This remarkable feature makes chemiluminescent systems ideal for the sensitive, dynamic, real-time, and non-invasive determination of target analytes in intact biological systems [13; 20; 21]. Marine Coelenterazine (Figure 1) and related imidazopyrazinone-based analogs are considered the most promising chemiluminescent systems for detecting superoxide anion (Scheme 1). They are capable of both chemi- and bioluminescence (in the absence/presence of either luciferase enzymes or photoproteins, respectively) [22; 23; 24; 25] and are present in many bioluminescent organisms present in the sea [26]. One of the attractive features of Coelenterazine is that its chemiluminescent reaction can be triggered selectively by superoxide anion, leading to light emission from the chemiexcited chemiluminophore, Coelenteramide (Scheme 1) [13; 14; 15; 27; 28; 29; 30]. Moreover, unlike other chemiluminescent probes such as lucigenin, [31] Coelenterazine and its analogs do not enhance superoxide production, which is an important advantage for the sensing of this ROS species. As a result, researchers have been using them as sensitive and dynamic probes for the detection of superoxide anion [13; 14; 15; 27; 28; 29; 30]. **Scheme 1. ** Schematic representation of the overall superoxide anion-triggered chemiluminescent reaction of Coelenterazine [13; 14; 15; 27; 28; 29; 30]. The interaction between Coelenterazine and superoxide anion ultimately leads to the formation of a high-energy peroxide intermediate, dioxetanone.