# Investigation of the Superoxide Anion-Triggered Chemiluminescenceence of Coelenterazine Analogs
## 2 Materials and Methods  
Native Coelenterazine was purchased from NanoLight Technology, dissolved in methanol, and stored at \(-20\)\({}^{\circ}\)C. After their synthesis, as described below, stock solutions of CLA-1 and CLA-2 were also prepared in methanol and stored at \(-20\)\({}^{\circ}\)C.  
The synthesis of CLA compounds followed a general synthetic pathway [32; 33; 34; 35; 36; 37], which included an initial Suzuki-Miyaura cross-coupling between 5-bromopyrazin-2-amine and the appropriated organoboranes. This resulted in the formation of an aminopyrazine-based intermediate, which is generally called Coelenteramine. The target CLA was then obtained through condensation of the resulting Coelenteramine with methyl glyoxal. CLA-1 was then prepared according to the procedures described in [32]. CLA-2 was also synthesized following the same general pathway with some modifications. In short, a solution of methyl glyoxal (1.396 mmol, 1.5 equiv.) and 3-bromo-5-(4-fluorophenyl)pyrazin-2-amine (a fluorinated Coelenteramine analog) (0.931 mmol, 1 equiv) in ethanol (9 mL) was deoxygenated with N\({}_{2}\). It should be noted that the Coelenteramine analog was obtained as described in [36]. The resulting mixture was then cooled to \(0\)\({}^{\circ}\)C, HCl (37%, 3.35 mmol, 3.6 equiv) was added, and the solution was stirred until it reached room temperature. Then, the solution was stirred at \(70\)\({}^{\circ}\)C for 2.5 h and at room temperature overnight. The resulting solution was concentrated under reduced pressure to yield a brown oil, which was redissolved in the minimum amount of ethyl acetate. The solution was then precipitated with diethyl ether and vacuum-dried, resulting in the formation of CLA-2 as an ochre solid with a yield of 94%. Ice-water and silicon baths were used for reactions at low and high temperatures, respectively, with all reaction temperatures referring to the external bath. Organic extracts were dried over anhydrous Na\({}_{2}\)SO\({}_{4}\), filtered, and concentrated using a rotary evaporator (Buchi(r) R4-210, Buchi(r) B-491 Heating Bath 120V, KNF Neuberger D-79112 Vacuum Pump N 035.1.2 AN.18). Reactions were monitored by thin-layer chromatography (TLC) using aluminum-backed Merck 60 F254 silica gel plates and \(n\)-hexanes-ethyl acetate solvent systems. After visualization under ultraviolet light at 254 nm and 365 nm, the plates were developed by immersing them in a solution consisting of a mixture of \(p\)-anisaldehyde(2.5%), acetic acid (1%), and sulfuric acid (3.4%) in 95% ethanol, followed by heating. More details regarding the synthesis processes can be found in the Supplementary Materials. The structure of CLA-2 was confirmed by NMR and FT-MS analysis. NMR spectra were recorded in both acetone-d\({}_{6}\) or methanol-d\({}_{4}\) solutions on a Bruker NMR spectrometer (Bruker Advance III 400 MHz Ascend, 9.4 Tesla). The chemical shifts are reported on the \(\delta\) scale (ppm) using the residual solvent signals (\(\delta\) = 2.050 ppm (\({}^{1}\)H, qu, acetone-d\({}_{6}\)), 2.840 (\({}^{1}\)H, s, acetone-d\({}_{6}\))) or (\(\delta\) = 3.31 ppm (\({}^{1}\)H, qu, methanol-d\({}_{4}\)), 4.78 ppm (\({}^{1}\)H, s, methanol-d\({}_{4}\))) as internal standards. The coupling constants (J) are reported in Hz. FT-MS analysis was performed on an LTQ OrbitapTM XL hybrid mass spectrometer (Thermo Fischer Scientific, Bremen, Germany) controlled by LTQ Tune Plus and Xcalibur 2.1.0. The \({}^{1}\)H-NMR and high-resolution mass spectra for CLA-2 are available in the Supplementary Materials. Chemiluminescent kinetic measurements were performed using a homemade luminometer equipped with a Hamamatsu HC135-01 photomultiplier tube. This setup also included an automatic burette, a sample holder, and a PC for data acquisition. The reactions were performed at least in sextuplicate at room temperature in various media, including methanol, deionized water, and \(n\),\(n\)-dimethylformamide (DMF). Reaction mixtures in water and DMF also contained 1% of sodium acetate buffer pH 5.2.