Coelenteramine 400a  (Synonyms: Coelenterazine 400a; Bisdeoxycoelenterazine)

Coelenteramine 400a (Coelenterazine 400a), a derivative of Coelenterazine, is a Renilla luciferase (RLuc) substrate. In the presence of Coelenteramine 400a, RLuc can emit blue light at 395 nm^[1][2]. Coelenterazine 400a will causes color change in the bioluminescence reaction of Rluc by replacing the sulfur and oxygen heteroatoms of the methylene bridge. Coelenterazine 400a provides higher signal resolution and can be used in the research of bioluminescence resonance energy transfer (BRET)^[3].

Guidelines (The following is our recommended protocol. This protocol is provided for reference only and should be modified according to your specific needs).
Luminescence reaction^[4]
1. Solution preparation:
1) Prepare 10 μL of 1.0-2.0 mM Coelenteramine 400a solution.
2) Prepare 10 μL of 2.01 mg/mL Renilla luciferase (RLuc8.6-53) solution.
3) Mix the above two solutions with 200 μL buffer.
2. Specific staining steps:
1) Record the bioluminescence spectrum of the mixed solution using a multi-channel spectrometer equipped with a liquid nitrogen-cooled CCD detector. The absolute spectral sensitivity of the spectrometer must be calibrated in advance using a 500 W spectral irradiance standard lamp.
2) A custom-made photometer equipped with a photomultiplier tube (PMT, H11890-01, Hamamatsu Photonics, Japan) was used to measure the relevant parameters of the bioluminescence reaction. The absolute response of the photometer is determined by plotting a linear graph of the relative counts measured by the photometer and the absolute counts measured by the integrating sphere.
3) In a test tube, 5-15 μL of a 10 nM solution of Coelenteramine 400a is pre-placed in the photometer, and then 150 μL of a 20 μg/mL luciferase solution (in 0.1 M GTA buffer, pH 6.0, 7.0, and 8.0) is injected to start the reaction. The reaction is monitored until the luminescence reaction is complete, and the quantum yield of the bioluminescence reaction is calculated from the total number of photons obtained and the number of luciferin molecules.
4) The Michaelis constant (K_m) of the substrate Coelenteramine 400a is calculated using the Lineweaver-Burk plot. The catalytic constant (k_cat) is calculated. The fluorescence spectrum and absolute fluorescence quantum yield of the corresponding oxidation product, Coelenteramide 400a (CTMD 400a), were measured using Quantaurus-QY with the excitation light set to 300 nm. The concentration of CTMD 400a solution in DMSO was 20 μM during the measurement.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

391.46

C₂₆H₂₁N₃O

70217-82-2

Solid

Yellow to brown

O=C1C(CC2=CC=CC=C2)=NC3=C(NC(C4=CC=CC=C4)=CN31)CC5=CC=CC=C5

Room temperature in continental US; may vary elsewhere.

-20°C, protect from light

*The compound is unstable in solutions, freshly prepared is recommended.

* Please refer to the solubility information to select the appropriate solvent. The compound is unstable in solutions, freshly prepared is recommended.

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

  [2]. Bertrand L, et, al. 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.  [Content Brief]

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

  [4]. Ikeda Y, et al. Quantitative evaluation of luminescence intensity from enzymatic luminescence reaction of coelenterazine and analogues. Journal of Photochemistry and Photobiology A: Chemistry, 2020, 394: 112459.