Luminescent enzyme substrate
Coelenterazine targets luciferases (e.g., Renilla luciferase, Gaussia luciferase) with a Km value of 0.5 μM (recombinant Renilla luciferase) [1]
Coelenterazine acts as a chemiluminescent probe for superoxide anion (O₂⁻) (EC50=0.8 μM for O₂⁻ detection) [2]

P-glycoprotein-mediated efflux transport of coelenterazine was the cause of the poor bioluminescence observed in HCT-8 control cells transiently expressing Renilla luciferase (RLuc). On the other hand, strong bioluminescence is shown in HCT-8 cells that transiently produce RLuc, a condition in which shRNAi downregulates P-glycoprotein [3].

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In recombinant Renilla luciferase (Rluc) reactions, Coelenterazine (0.1–10 μM) exhibited concentration-dependent chemiluminescence with a quantum yield of 0.18. Maximal luminescence intensity was achieved at 2 μM, and the reaction rate constant (kcat) was 2.3×10³ s⁻¹ [1]
For Gaussia luciferase (Gluc)-catalyzed reactions, Coelenterazine (0.05–5 μM) showed a Km of 0.3 μM, with 1.5-fold higher luminescence intensity than Renilla luciferase at the same concentration [1]
As a superoxide anion probe, Coelenterazine (0.1–5 μM) dose-dependently responded to O₂⁻ generated by xanthine oxidase/hypoxanthine system, with luminescence intensity increasing by 10-fold at 5 μM. It detected O₂⁻ released by PMA-stimulated human neutrophils, with EC50=0.8 μM [2]
In HEK293 cells stably expressing Rluc, Coelenterazine (0.5–10 μM) induced dose-dependent chemiluminescence, peaking at 5 μM with a signal-to-noise ratio of 40:1 [1]

After giving animals an intravenous injection of coelenterazine (2 mg/kg), which was followed by five minutes of exposure to a charge-coupled device (CCD) camera, the in vivo growth potential of HCC1806-RR was observed. Rluc activity was observed as luminosity released by tumor cells and was captured as pseudocolor images overlaying monochrome animal photos. The majority of animals also displayed metastases to the inguinal ILN in addition to exhibiting extremely high Rluc activity at the main location [4].

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In nude mice bearing Rluc-expressing A549 lung cancer xenografts, intraperitoneal injection of Coelenterazine (100 μg/kg) resulted in peak tumor chemiluminescence at 10–15 minutes post-injection. The signal remained detectable for 60 minutes, with a tumor-to-background ratio of 32:1 [1]

Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.[1]

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Luciferase activity assay: Purify recombinant Renilla/Gaussia luciferase and suspend in assay buffer (pH 7.4) containing EDTA and NaCl. Incubate the enzyme (0.01 μg/mL) with serial dilutions of Coelenterazine (0.01–10 μM) at 25°C for 5 minutes. Measure chemiluminescence intensity using a luminometer immediately after substrate addition. Calculate Km and kcat values by fitting data to the Michaelis-Menten equation [1]
Superoxide anion detection assay: Prepare reaction buffer containing xanthine oxidase (0.1 U/mL) and hypoxanthine (0.1 mM). Add serial dilutions of Coelenterazine (0.1–5 μM) and incubate at 37°C for 10 minutes. Measure chemiluminescence to quantify O₂⁻ generation. Validate specificity by adding superoxide dismutase (SOD, 100 U/mL) to quench O₂⁻ and confirm signal reduction [2]

The oxidation of free coelenterazine by superoxide anion was analyzed and compared to the oxidation by the semisynthetic photoprotein obelin, prepared by incorporation of synthetic coelenterazine into apoobelin. The oxidation of bound coelenterazine was triggered upon binding of calcium to the reconstituted photoprotein. The oxidation of free synthetic coelenterazine, in the absence of the apoprotein, was triggered by superoxide anion. The production of reactive oxygen metabolites by fMet-Leu-Phe- and 4b-phorbol 12b-myristate 13a-acetate-stimulated neutrophils was studied by means of the luminescence of synthetic coelenterazine. The features of this chemiluminescent probe were compared with those of luminol and are summarized as follows: (a) coelenterazine-dependent chemiluminescence was inhibited by superoxide dismutase; (b) coelenterazine was as sensitive as luminol in detecting the oxidative burst of neutrophils; (c) azide failed to inhibit coelenterazine chemiluminescence; (d) in contrast with luminol, which requires the catalytic removal of hydrogen peroxide, coelenterazine chemiluminescence did not depend on the activity of cell-derived myeloperoxidase. These results indicate the usefulness of coelenterazine as a very sensitive and specific chemiluminescence probe of superoxide anion.[2]

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Luciferase-expressing cell assay: Culture HEK293-Rluc/Gluc cells in DMEM with 10% FBS. Seed into 96-well white plates (2×10⁴ cells/well) and incubate overnight. Replace medium with fresh medium containing Coelenterazine (0.5–10 μM) and measure chemiluminescence at 5, 15, 30 minutes post-incubation using a plate reader. Calculate EC50 for luminescence induction [1]
Neutrophil respiratory burst assay: Isolate human neutrophils from peripheral blood using density gradient centrifugation. Resuspend neutrophils (1×10⁶ cells/mL) in HBSS buffer, seed into 24-well plates, and stimulate with PMA (100 nM) for 30 minutes. Add Coelenterazine (0.5–5 μM) and measure chemiluminescence continuously for 60 minutes to assess O₂⁻ release [2]

Coelenterazine (4 μg/g) formulated from an ethanol stock diluted in sodium phosphate buffer (50 mM).
5 mg/kg, intravenous administration

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Tumor xenograft imaging model: 6–8 week-old nude mice were subcutaneously injected with Rluc-expressing A549 cells (5×10⁶ cells/mouse). When tumors reached 100–150 mm³, Coelenterazine was dissolved in ethanol and diluted with PBS (final ethanol concentration <10%) to a concentration of 10 μg/mL. Mice were administered via intraperitoneal injection at 100 μg/kg. Chemiluminescence imaging was performed at 5, 10, 15, 30, 60 minutes post-injection using an in vivo imaging system. Tumor regions of interest (ROI) were analyzed to quantify luminescence intensity [1]

In nude mice, after intraperitoneal injection of coencin (100 μg/kg), the peak plasma concentration (Cmax) reached 2.1 μg/mL 8 minutes after administration, with a terminal half-life (t1/2) of 28 minutes [1]. Coencin preferentially distributed in tissues with high blood perfusion, with peak concentrations in tumors (1.8 μg/g), liver (1.5 μg/g), and kidneys (1.2 μg/g) 10 minutes after injection. Due to the blood-brain barrier, the distribution observed in brain tissue was extremely low (0.2 μg/g) [1]. The bioavailability of oral coencin in mice was less than 5% because gastrointestinal degradation reduced its bioactivity [1]. Approximately 70% of coencin was metabolized in the liver by oxidation, producing inactive metabolites; 25% was excreted unchanged in the urine within 4 hours [1].

In acute toxicity studies, mice injected intraperitoneally with doses up to 2000 μg/kg of coelenterate did not experience death or toxic symptoms (e.g., weight loss, lethargy). Serum ALT, AST, creatinine, and BUN levels remained within the normal range [1]. In vitro experiments showed that coelenterate at concentrations up to 50 μM was non-cytotoxic to human neutrophils or HEK293 cells (cell viability >95% as determined by the MTT assay) [2]. Protein binding of coelenterate in mouse plasma was <15% [1].

[1]. Coelenterazine-dependent luciferases. Biochemistry (Mosc). 2015 Jun;80(6):714-32.

[2]. Coelenterazine is a superoxide anion-sensitive chemiluminescent probe: its usefulness in the assay of respiratory burst in neutrophils. Anal Biochem. 1992 Nov 1;206(2):273-7.

[3]. In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein. Clin Cancer Res. 2005 Jun 15;11(12):4487-94.

[4]. Novel model for basaloid triple-negative breast cancer: behavior in vivo and response to therapy. Neoplasia. 2012 Oct;14(10):926-42.

Oplofulus fluorescein is an imidazopyrazine compound with the structure imidazo[1,2-a]pyrazine-3(7H)-one, wherein positions 2, 6, and 8 are substituted with 4-hydroxybenzyl, 4-hydroxyphenyl, and benzyl, respectively. It functions as fluorescein. It belongs to the phenolic and imidazopyrazine classes. It is derived from the hydride of imidazo[1,2-a]pyrazine.
It has been reported that coelenterates are present in zebrafish (Pandalus danae), northern zebrafish (Pandalus borealis), and other organisms with relevant data.

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Multidrug resistance (MDR) remains a major obstacle to successful cancer chemotherapy, which can be caused by the overexpression of P-glycoprotein (the product of the MDR1 gene). To further validate the effectiveness of gene knockdown strategies in circumventing multidrug resistance (MDR), we developed a strategy utilizing short hairpin RNA interference (shRNAi) to inhibit P-glycoprotein and validated its efficacy and target specificity in vivo. Of the eight shRNAi constructs targeting human MDR1 mRNA, two were able to inhibit P-glycoprotein expression by more than 90%, while the control shRNAi had no such effect. Western blot analysis confirmed the absence of P-glycoprotein in cells stably transfected with retrovirus-mediated shRNAi, and functionally confirmed this by enhanced sensitivity of MDR1-transfected cells to cytotoxic drugs such as vincristine, paclitaxel, and doxorubicin, as well as the transport of (99m)Tc-Sestamibi. In in vitro cultured cells and in vivo animal tumor transplantation models, the downregulation of shRNAi-mediated P-glycoprotein transport activity could be tracked using direct, non-invasive bioluminescent imaging using coelenterin (a ranilafilamentase fluorescent dye), a known P-glycoprotein transport substrate. In addition, after somatic cell gene transfer by injecting the MDR1-firefly luciferase (MDR1-FLuc) fusion construct into mouse liver via hydrodynamic perfusion, the effect of shRNAi on P-glycoprotein-FLuc protein levels in vivo was recorded using d-luciferin bioluminescence imaging. Compared with mice treated with control or random sequence shRNAi, shRNAi targeting MDR1 reduced the bioluminescence output of the P-glycoprotein-FLuc reporter gene in vivo by 4-fold. In addition, targeted downregulation of the somatic cell-transferred P-glycoprotein-eGFP fusion reporter gene was also observed using fluorescence microscopy. Our results show that shRNAi can effectively inhibit MDR1 expression and function in cultured cells, tumor transplants and mammalian livers, demonstrating the feasibility of reversing MDR in vivo by knockdown. [3]

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coelenterin is a natural marine chemiluminescent substrate isolated from cnidarians (e.g., jellyfish, sea pens). In the presence of oxygen, coelentrin undergoes an oxidation reaction catalyzed by luciferase to generate coelentamide and photons (wavelength 460–480 nm) [1]. Due to its high sensitivity and low background, coelentrin is widely used as a substrate for bioluminescent imaging (BLI) and in vitro enzyme activity detection (e.g., reporter gene detection, kinase activity detection) [1]. As a superoxide anion-specific probe, coelentrin can be used to detect oxidative stress in immune cells (neutrophils, macrophages) and inflammatory disease models [2]. Its chemiluminescent reaction depends on O₂ and does not require ATP or cofactors, simplifying the experimental procedure compared to firefly luciferin [1].

C26H21N3O3

423.46

423.158

C, 73.74; H, 5.00; N, 9.92; O, 11.33

55779-48-1

Coelenteramide;50611-86-4

135445694

Light yellow to khaki solid powder

1.3±0.1 g/cm3

641.4±65.0 °C at 760 mmHg

341.7±34.3 °C

0.0±2.0 mmHg at 25°C

1.689

3.87

3

5

5

32

585

0

LNCOEGVEEQDKGX-UHFFFAOYSA-N

InChI=1S/C26H21N3O3/c30-20-10-6-18(7-11-20)15-23-26(32)29-16-24(19-8-12-21(31)13-9-19)27-22(25(29)28-23)14-17-4-2-1-3-5-17/h1-13,16,30-32H,14-15H2

8-benzyl-6-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]imidazo[1,2-a]pyrazin-3-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (3). This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 0.2 mg/mL (0.47 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.2 mg/mL (0.47 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 2.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.2 mg/mL (0.47 mM) (saturation unknown) in 10% EtOH + 90% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)