Luminescent enzyme substrate

Replacement of the methylene group at the C-8 position with an extended electronic conjugation is a new promising method to develop red-shifted coelenterazine derivatives. In this paper, we have described an oxygen-containing coelenterazine derivative with a significant red-shifted (63 nm) bioluminescence signal maximum relative to coelenterazine 400a (DeepBlueC™, 1). In cell imaging, the sulfur-containing coelenterazine derivative displayed a significantly (1.77 ± 0.09; P ≤ 0.01) higher luminescence signal compared to coelenterazine 400a and the oxygen-containing coelenterazine derivative exhibited a slightly (0.74 ± 0.08; P ≤ 0.05) lower luminescence signal. It is beneficial to understand further the underlying mechanisms of bioluminescence.[3]

Relative quantum yield (RQY) and kinetics of in vitro luminescence[3]
The RQY study and kinetic analysis were performed using an IVIS Kinetic which consisted of a cooled charge-coupled device (CCD) camera mounted on a light-tight specimen chamber (dark box), a camera controller, a camera cooling system, and controlled using a computer. The data are represented as pseudocolor images (in photons/s/cm2/scr) of light intensity (blue—least intense, red—most intense) superimposed over the grayscale reference images. Circular specified regions of interest (ROIs) were drawnon the areas, and the light output were quantified as the total number of photons emitted per second using Living Image software. To determine the appropriate unsaturated amount of substrate, 10 μL of coelenterazine (CTZ)  derivatives 1–3 between 1 and 100 μmol/L and 90 μL of either DMSO or Rluc enzyme at a final concentration of 15 nmol/L (bioluminescence) were used. The RQY and reaction kinetics was determined by mixing 10 μL of coelenterazine (CTZ)  derivatives (final concentration of 5 μmol/L) with 90 μL of either DMSO or Rluc enzyme (15 nmol/L) onto wells of 96-well black plates to prevent light reflection from well to well. Luminescent signals were measured immediately after mixing and monitored over a period of 25–30 min (luminescence had almost decayed to near-background levels) using the IVIS. Light output was recorded every 5 min with an exposure time of 30 s for chemiluminescence and every 1 min with an exposuretime of 5 s in the first 15 min for bioluminescence. The collected data was analyzed by employing the Prism 5.0 GraphPad software to compute the total light output. As a corresponding blank control, Tris–HCl buffer was added instead of coelenterazine (CTZ)  derivatives solution under the same conditions. All assays were performed in triplicate.

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Assay for luminescence activity in live cell[3]
ES-2 cells (human ovarian cancers cell line) expressing Rluc were supplied by BioDiagnosis. The ES-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; high glucose with l-glutamine) containing 10% fetal bovine serum (FBS) and 0.5 μg/mL puromycin at 37 °C in a humidified atmosphere in a 5% CO2 incubator.coelenterazine (CTZ)  derivatives was dissolved in ethanol to make a 1 mmol/L stock solution, and diluted with Tris–HCl buffer to gradient concentration (5, 10, 20, 40, 60, 80, 100 μmol/L). The ES-2-Rluc cells were grown in black 96-well plates (4 × 104 cells per well). After a 24-h incubation period, the medium was removed. Then cells were washed with Tris–HCl buffer twice and treated with 100 μL of a series of concentrations of coelenterazine (CTZ)  derivatives solutions (ranging from 0 to 100 μmol/L). Bioluminescent signals were then immediately determined using the IVIS. Light output was recorded every 1 min with an exposure time of 30 s until luminescence almost decayed to near-background levels. Luminescent signal (photons per second) for each well was measured and plotted as average values. All experiments were performed in triplicate.

[1]. Bisdeoxycoelenterazine derivatives for improvement of bioluminescence resonance energy transfer assays. J Am Chem Soc. 2007 Oct 3;129(39):11900-1.

[2]. The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS). J Recept Signal Transduct Res. Feb-Nov 2002;22(1-4):533-41.

[3]. Luminescence of coelenterazine derivatives with C-8 extended electronic conjugation. Chinese Chemical Letters, 2016, 27(4): 550-554.

In summary, we describe an oxygen-containing CTZ derivative 2 that exhibits a more significant redshift (63 nm) in its bioluminescent signal maximum compared to coelentrin 400a, but with a lower quantum yield. These results appear to support our hypothesis that the introduction of an oxygen heteroatom at the C-8 position can also produce a redshift effect. While we have proposed a possible mechanism for the CTZ luminescence response (see Supplementary Infographic S2), the enzymatic recognition mechanism of the Rluc system is not fully elucidated. We speculate that this may be due to the efficient extension of the π-electron conjugation, electronegativity, or hydrogen bond of the oxygen atom at the C-8 position. We also developed a novel measurement method using an IVIS Kinetic equipped with a CCD to detect RQY. The chemiluminescent RQY of CTZ derivative 3 agrees well with previously reported values. To obtain truly accurate bioluminescent quantum yields (RQY), we used commercially available purified Rluc enzyme instead of crude cytoplasmic extract to avoid enzyme-independent luminescence. The quantum yields of compounds 2 and 3 are low and differ significantly from previously reported values. However, we found that compound 3 had a higher affinity for Rluc than coelentin 400a. Compound 3 also showed a higher luminescence signal in cell imaging. In summary, compounds 2 and 3 are promising bright redshift CTZ derivatives, providing a new approach to improve the luminescence performance of CTZ analogs. More importantly, this helps to understand the potential mechanism of bioluminescence after Rluc reacts with coelentin. [3] In BRET2 (bioluminescent resonance energy transfer), Renal luciferase (RLuc) is used as the donor protein and green fluorescent protein (GFP2) is used as the acceptor protein. In the presence of the cell-permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If GFP2 is brought close to RLuc through specific biomolecular interactions, GFP2 absorbs the blue light energy and re-emits green light at 510 nm. Therefore, the BRET2 signal can be easily determined by measuring the ratio of green to blue light (510/395 nm) using a suitable dual-channel luminometer (e.g., Packard BioScience's Fusion Universal Microplate Analyzer). Since the BRET2 assay does not require a light source, it avoids the problems of excessive fluorescence background or photobleaching common in standard FRET assays. Based on the BRET2 technology, we developed a universal method for detecting G protein-coupled receptors (GPCRs). This method is based on the observation that most GPCRs, upon activation by agonists, lead to the interaction of β-arrestin (a protein involved in receptor desensitization and isolation) with the receptor. We constructed a cell line stably expressing the GFP2:β-arrestin 2 fusion protein and demonstrated that this cell line can be used to monitor the activation of various transiently expressed GPCRs in the BRET2/arrestin assay. Furthermore, using the HEK 293/GFP2:β-arrestin 2 cell line as the receptor, we constructed a bistable cell line that co-expresses the vasopressin 2 receptor (V2R) fused with RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in the BRET2/arrestin assay. This method can obtain real pharmacological results and supports the use of BRET2/arrestin detection as a tool that can be used in recombinant cell lines to characterize ligand-GPCR interactions, thereby enabling ligand identification of orphan receptors. [2]

C26H22CLN3O

427.925384998322

427.145

2320429-05-6

Coelenteramine 400a;70217-82-2

137700438

Typically exists as solid at room temperature

0

2

3

5

31

524

0

UVZGQDPDAXEWQO-UHFFFAOYSA-N

InChI=1S/C26H21N3O.ClH/c30-26-23(17-20-12-6-2-7-13-20)28-25-22(16-19-10-4-1-5-11-19)27-24(18-29(25)26)21-14-8-3-9-15-21;/h1-15,18,30H,16-17H2;1H

2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3-ol;hydrochloride

6-phenyl-2,8-bis(phenylmethyl)-imidazo[1,2-a]pyrazin-3(7H)-one,monohydrochloride; 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3-ol;hydrochloride; Coelenteramine 400a (hydrochloride); EX-A9184A; Coelenteramine 400a hydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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