Coelenterazine e , CAS No.114496-02-5

CAS: 114496-02-5 Cat. No.: C944463 PubChem CID: 135845004

Storage

Room temperature

Specifications

Storage

Room temperature

Names and Identifiers

Canonical Smiles

C1CC2=C(C3=C1C=C(C=C3)O)N=C(C4=NC(=C(N24)O)CC5=CC=C(C=C5)O)CC6=CC=CC=C6

IUPAC Name

16-benzyl-13-[(4-hydroxyphenyl)methyl]-11,14,17-triazatetracyclo[8.7.0.02,7.011,15]heptadeca-1(10),2(7),3,5,12,14,16-heptaene-5,12-diol

InChIKey

XZCAVVZKPQNLCZ-UHFFFAOYSA-N

INCHI

1S/C28H23N3O3/c32-20-9-6-18(7-10-20)15-24-28(34)31-25-13-8-19-16-21(33)11-12-22(19)26(25)29-23(27(31)30-24)14-17-4-2-1-3-5-17/h1-7,9-12,16,32-34H,8,13-15H2

Isomeric SMILES

C1CC2=C(C3=C1C=C(C=C3)O)N=C(C4=NC(=C(N24)O)CC5=CC=C(C=C5)O)CC6=CC=CC=C6

Alternate CAS

114496-02-5

PubChem CID

135845004

Taxonomic Classification

Taxonomy Tree

• Kingdom Organic compounds
  • Superclass Benzenoids
    • Class Naphthalenes

• Kingdom: Organic compounds
• Superclass: Benzenoids
• Class: Naphthalenes
• Subclass: Not available
• Intermediate Tree Nodes: Not available
• Direct Parent: Naphthalenes
• Alternative Parents: Imidazopyrazines 1-hydroxy-2-unsubstituted benzenoids Pyrazines N-substituted imidazoles Benzene and substituted derivatives Heteroaromatic compounds Azacyclic compounds Organopnictogen compounds Organooxygen compounds Organonitrogen compounds Hydrocarbon derivatives
• Molecular Framework: Aromatic heteropolycyclic compounds
• Substituents: Naphthalene - Imidazopyrazine - 1-hydroxy-2-unsubstituted benzenoid - Phenol - Pyrazine - N-substituted imidazole - Monocyclic benzene moiety - Heteroaromatic compound - Imidazole - Azole - Azacycle - Organoheterocyclic compound - Organic nitrogen compound - Organic oxygen compound - Organopnictogen compound - Hydrocarbon derivative - Organooxygen compound - Organonitrogen compound - Aromatic heteropolycyclic compound
• Description: This compound belongs to the class of organic compounds known as naphthalenes. These are compounds containing a naphthalene moiety, which consists of two fused benzene rings.
• External Descriptors: Not available

Data sources

1. Djoumbou Feunang Y, Eisner R, Knox C, Chepelev L, Hastings J, Owen G, Fahy E, Steinbeck C, Subramanian S, Bolton E, Greiner R, and Wishart DS. ClassyFire: Automated Chemical Classification With A Comprehensive, Computable Taxonomy. Journal of Cheminformatics, 2016, 8:61.

DOI: 10.1186/s13321-016-0174-y

Chemical and Physical Properties

• Molecular Weight: 449.500 g/mol
• XLogP3: 5.900
• Hydrogen Bond Donor Count: 3
• Hydrogen Bond Acceptor Count: 5
• Rotatable Bond Count: 4
• Exact Mass: 449.174 Da
• Monoisotopic Mass: 449.174 Da
• Topological Polar Surface Area: 90.900 Ų
• Heavy Atom Count: 34
• Formal Charge: 0
• Complexity: 680.000
• Isotope Atom Count: 0
• Defined Atom Stereocenter Count: 0
• Undefined Atom Stereocenter Count: 0
• Defined Bond Stereocenter Count: 0
• Undefined Bond Stereocenter Count: 0
• The total count of all stereochemical bonds: 0
• Covalently-Bonded Unit Count: 1

Application Protocols

No item-specific, validated protocols are provided in the Product Data. The following outlines general, literature-based procedures for coelenterazine-class substrates:

• Endpoint luciferase reporter assay (cells expressing a CTZ-dependent luciferase)

  1. Prepare 1–10 mM stock of coelenterazine e in anhydrous DMSO or ethanol (amber vial). Optionally sparge with N2/Ar.
  2. Dilute to working solution in assay buffer immediately before use to a final 1–10 µM substrate concentration; keep organic solvent ≤1% v/v.
  3. Add equal volume to wells, incubate 1–5 minutes in the dark, and read luminescence with appropriate integration time and emission filter if needed.

• BRET assay

  1. Prepare donor–acceptor fusion constructs; express in cells.
  2. Add coelenterazine e working solution; acquire dual-emission signals to compute BRET ratios. Optimize substrate concentration to balance donor brightness and acceptor saturation.

• Aequorin calcium assay

  1. Reconstitute apo-aequorin with coelenterazine e per photoprotein protocol.
  2. Challenge with defined Ca2+; capture flash emission kinetics.

Adjust concentrations and timings empirically; validate against positive/negative controls. Refer to the CoA/Spec Sheet for any item-specific recommendations.

Biological Roles

• General role in nature (literature)

  • Coelenterazine and its analogs are luciferins used by various marine organisms (ctenophores, copepods, cnidarians) in bioluminescent systems. The imidazopyrazinone core undergoes oxygenation in luciferase active sites to yield an excited-state product that emits visible light.

• Laboratory role

  • Serves as a chemiluminescent substrate for CTZ-dependent luciferases and aequorin-type photoproteins. The “e” analog modulates emission color and kinetics, enabling assay customization and spectral separation in multiplexed reporter experiments.

• Biochemical notes

  • The reaction consumes molecular oxygen and typically generates CO2 and an excited coelenteramide species that relaxes with photon emission. Calcium is not directly required for luciferase activity but is essential for photoproteins like aequorin that use CTZ analogs.

• Cell compatibility

  • The relative lipophilicity of CTZ analogs facilitates membrane permeability, allowing live-cell assays without permeabilization. Background autoluminescence is controlled by fresh working solutions, low-light handling, and appropriate cosolvent control.

No organismal/clinical function is claimed for this product; it is intended strictly for research use.

Buffer Applications

This compound is not a buffer component. However, practical buffer considerations apply for assays using coelenterazine e (literature guidance):

• Assay buffers

  • Use neutral pH (≈6.8–7.6) phosphate- or HEPES-based buffers to maintain luciferase activity while minimizing base-catalyzed autoxidation.
  • Include ≤0.5–1% v/v cosolvent (DMSO or ethanol) from the substrate stock; verify enzyme tolerance.
  • Optional additives: 0.01–0.1% BSA to reduce surface adsorption; chelators should be minimized for metallo-luciferases if applicable.

• Preparation tips

  • Prepare substrate working solutions immediately before use, under low light. Filter (0.2 µm PTFE) if particulates form upon dilution from organic stock.

Item-specific buffer composition or recipes are not provided; refer to your assay protocol and CoA/Spec Sheet.

Green Alternatives

While coelenterazine e is a specialized bio-reagent without direct “green” substitutes for its bioluminescent function, greener choices can be made around solvent and handling.

• Solvent considerations (general)

  • Prefer ethanol or water/ethanol cosolvent systems over DMSO when compatible with the biology and signal quality.
  • Minimize solvent volumes and use concentrated stocks to reduce waste.

• Packaging and stability

  • Single-use amber aliquots reduce repeated opening and waste from degradation, improving overall material efficiency.

• Comparison (general guidance; not item-specific)

  • DMSO vs Ethanol
    • Environmental profile: ethanol is renewable and lower-toxicity; DMSO is biodegradable but can enhance dermal absorption—use carefully.
    • Assay impact: DMSO typically offers superior solubility; ethanol can be preferable for sensitive cells if solubility allows.

• Process tips

  • Implement inert-atmosphere handling to extend shelf life of solutions, decreasing the frequency of remakes and associated solvent waste.

No validated green-chemistry alternative reproduces the same enzyme–substrate photochemistry as coelenterazine e; optimization focuses on solvent, scale, and handling efficiency.

Pharmaceutical Uses

Not applicable as a pharmaceutical or clinical agent. Coelenterazine e is supplied for research use only (per Product Data). In the context of pharmaceutical R&D laboratories (non-clinical):

• Typical roles (general)

  • Reporter assays for pathway activation/inhibition using CTZ-dependent luciferases.
  • BRET donor substrate in target engagement and protein–protein interaction studies.
  • High-throughput screening readouts where emission wavelength/kinetics of the “e” analog provide advantages over native CTZ.

• Formulation/excipient status

  • No pharmacopeial monograph or excipient designation is implied. Any on-instrument or on-plate handling should follow internal GLP where applicable.

No therapeutic, diagnostic, or clinical claims are made for this item.

Physical Properties

• Item-specific properties

  • Appearance: Not specified for this item; refer to CoA/Spec Sheet.
  • Purity/grade: Not specified for this item; refer to CoA/Spec Sheet.
  • Density, refractive index, UV cutoff, metal/impurity limits: Not specified for this item; refer to CoA/Spec Sheet.

• General/literature expectations for coelenterazine-class luciferins (for context; not item specifications)

  • Physical state: typically a light-sensitive, air-oxidizable solid; often off-white to pale yellow in appearance.
  • Solubility: sparingly soluble in water; readily soluble in polar aprotic organics (DMSO, DMF) and lower alcohols (ethanol, methanol). Commonly prepared as concentrated stocks (1–10 mM) in anhydrous DMSO or ethanol.
  • Stability: prone to autoxidation in the presence of oxygen, light, and trace bases; stabilizes under inert atmosphere and light protection.
  • Partitioning: moderate-to-high lipophilicity is typical for coelenterazine analogs, promoting membrane permeability in cell-based assays.

• Thermal properties (literature)

  • Melting point, boiling point: specific values vary by analog and are not robustly standardized for coelenterazine e; consult primary literature or the CoA/Spec Sheet for definitive values.

Quality & Grades

• Item-specific status

  • Grade/Purity: Not specified for this item; refer to CoA/Spec Sheet.

• Interpreting grades for bioluminescent substrates (general guidance)

  • Research/assay grade typically emphasizes low background autoluminescence and controlled impurity profile (e.g., minimal oxidized byproducts) to ensure high signal-to-noise.
  • HPLC verification is commonly used to confirm identity/purity and to monitor trace oxidative degradants that elevate assay background.
  • Low-peroxide and low-metal content are beneficial because peroxides/metals can catalyze off-path oxidation; where applicable, suppliers specify such limits—consult the CoA for this item.

• Stabilizers and formulation

  • Some formats include trace antioxidants or are packaged under inert gas to preserve potency. If present, stabilizers will be disclosed on the CoA/Spec Sheet.

• Practical QC notes

  • For quantitative reporter assays, lot-to-lot comparison using a standard luciferase or photoprotein is recommended to benchmark specific activity and emission kinetics.
  • If using in HTP screening, verify background counts in blank matrices (buffer/medium) before large-scale use.

Reaction & Applications

• Principal use (literature/general)

  • Coelenterazine e is a bioluminescent substrate for select luciferases and photoproteins, engineered to modulate emission wavelength, intensity, and kinetics relative to native coelenterazine. It is used in reporter gene assays, live-cell bioluminescence imaging, BRET assays, and calcium measurements with aequorin-type photoproteins.

• Enzyme compatibility (examples from literature)

  • Renilla-type luciferases (e.g., RLuc variants) often display altered brightness and emission spectra with coelenterazine e versus native CTZ, enabling spectral multiplexing.
  • Aequorin and engineered Ca2+-binding photoproteins: coelenterazine analog selection tunes sensitivity and decay kinetics; “e” is one such analog used to shift kinetics/emission.
  • Gaussia luciferase and other secreted CTZ-dependent luciferases may exhibit distinct preferences; empirical optimization is advised.

• Practical tips

  • Minimize pre-incubation in aqueous media; add substrate immediately before measurement to limit autoxidation and background.
  • Use low-light conditions and oxygen-controlled handling when feasible.
  • For kinetic assays, standardize injection timing and temperature; record full emission decay for model fitting (flash vs glow behavior depends on analog/enzyme pair).

• Controls

  • Include no-enzyme and no-substrate blanks to quantify background chemiluminescence.
  • Calibrate instrument gain to avoid detector saturation at peak emission.

Item-specific “Manufacturer Applications” were not provided beyond the title; the above expands on established literature practice for coelenterazine-class substrates.

Reaction Conditions

This section is interpreted for biochemical assay conditions rather than preparative organic reactions (coelenterazine e is a substrate, not a reagent):

• Luciferase assays (literature guidance)

  • Buffer: neutral pH (phosphate, Tris, HEPES), low ionic strength to moderate salt (50–150 mM NaCl typical).
  • Temperature: ambient (20–25°C) or physiological (37°C) depending on enzyme/source and cell system.
  • Substrate: prepare fresh working concentrations typically in the 0.5–20 µM range; exact values depend on enzyme expression and detector sensitivity.
  • Oxygen: required for luminescent reaction; avoid deoxygenation during measurement.
  • Kinetics: coelenterazine e may display altered flash/glow profiles vs native CTZ; capture time course or use injectors for reproducibility.

• Aequorin/photoprotein assays

  • Incorporation: photoproteins are reconstituted with the CTZ analog prior to measurement; calcium triggers emission.
  • Ca2+ handling: maintain defined free [Ca2+] using EGTA/Ca2+ buffers; record emission upon calcium addition.

• Controls and standards

  • Include no-substrate and heat-inactivated enzyme controls to quantify background.
  • Use a standard luciferase–substrate pair to normalize day-to-day instrument drift.

These are literature-driven guidelines; optimize empirically for your enzyme and instrument. No item-specific conditions are claimed.

Safety & Handling

• Item-specific hazard data

  • GHS classification: Not specified for this item; refer to SDS.
  • Signal word / pictograms / H-statements: Not specified for this item; refer to SDS.

• General handling guidance for coelenterazine-class luciferins (literature/best practice)

  • Protect from light and air to minimize autoxidation and loss of activity. Work under subdued light; use amber vials and foil wrapping.
  • Avoid strong oxidizers, bases, and metal ions that may catalyze oxidative degradation.
  • Use analytical-grade, anhydrous solvents. Degassing/blanketing with inert gas (N2/Ar) can improve stability.
  • PPE: laboratory coat, nitrile gloves, safety glasses. Handle powders in a fume hood to avoid dust inhalation.

• First-aid overview (general)

  • Inhalation: move to fresh air; seek medical attention if symptoms occur.
  • Skin/eye contact: rinse with water for at least 15 minutes; remove contaminated clothing; obtain medical evaluation if irritation persists.
  • Ingestion: rinse mouth; do not induce vomiting; seek medical attention.

• Waste and spill response

  • Collect spills with inert absorbent; place waste in light-protected, sealed containers. Dispose in accordance with institutional and local regulations. Always defer to the product SDS for authoritative safety and regulatory information.

Solvent Selection

• Polarity and miscibility (general/literature for coelenterazine-class luciferins)

  • Water solubility: low; tends to precipitate in purely aqueous media at >1–10 µM unless aided by co-solvent or carrier protein.
  • Organic solubility: high in DMSO, DMF; good in ethanol and methanol. Limited in nonpolar hydrocarbons.

• Practical choices

  • Stock solutions: prepare at 1–10 mM in anhydrous DMSO or ethanol; filter if necessary (PTFE). Protect from light and oxygen.
  • Working solutions: dilute into assay buffer immediately before use, targeting final organic cosolvent ≤0.5–1% v/v to maintain cell health and enzyme performance.
  • Additives: 0.01–0.1% BSA or cyclodextrins can reduce adsorption and autoxidation in aqueous media (literature practice).

• Comparison (general)

  • DMSO: highest solvating power; inert; may affect some cells above 0.5–1%.
  • Ethanol: biocompatible at low %; slightly higher volatility; can be acidified (trace HCl) in some protocols to improve stability.
  • Aqueous buffers: require co-solvent; maintain neutral pH (6.8–7.6) to limit base-catalyzed degradation.

Note: No item-specific solvent specifications are provided; refer to the CoA/Spec Sheet for definitive guidance.

Storage & Reconstitution

• Item-specific storage (from Product Data)

  • Storage Conditions: Room temperature.
  • Shipped In: Not specified for this item; refer to CoA/Spec Sheet.

• General/literature best practices

  • As supplied (solid): store in amber, tightly sealed containers, protected from light and moisture. If long-term storage is intended, some users prefer desiccation and inert gas overlay to minimize autoxidation.
  • In solution: dissolve in anhydrous DMSO or ethanol to 1–10 mM, aliquot into amber vials or low-bind tubes, protect from light, and minimize headspace. For extended use, store frozen (e.g., −20°C) and avoid repeated freeze–thaw by using single-use aliquots.
  • Handling: work under subdued light; prepare fresh working solutions immediately before assays.

• Reconstitution notes

  • If insolubility is observed, gently warm to room temperature and vortex/sonicate briefly. Avoid strong bases or aqueous alkalinity during dissolution.

Always defer to the SDS and CoA/Spec Sheet for authoritative, item-specific instructions. Research use only (per Product Data).

Structure & Identity

Coelenterazine e is a marine-type imidazopyrazinone luciferin analog used as a bioluminescent substrate in reporter assays and calcium-sensing photoproteins.

• Item-specific (from Product Data)

  • CAS: 114496-02-5
  • PubChem CID: 135845004
  • InChIKey: 431131
  • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES: Not specified for this item; refer to CoA/Spec Sheet.

• Structural features (general/literature)

  • Core scaffold: bicyclic imidazopyrazinone (2-oxo-2,3-dihydroimidazo[1,2-a]pyrazin-3-one) chromophore.
  • Substitution pattern: coelenterazine e bears modified aryl substituents relative to native coelenterazine to tune emission spectrum/kinetics with select luciferases and photoproteins.
  • Functional groups: conjugated heteroaromatic system, carbonyls (lactam-like), and phenyl/heteroaryl rings enabling delocalized excited states.

• 2D description (general)

  • A fused imidazole–pyrazinone ring situated centrally with two aryl groups attached to the imidazopyrazinone core through sp2 carbons; conjugation extends across the entire π-system, which is essential for chemiluminescence upon oxidative decarboxylation in the enzyme active site.

Synthetic Utility

Coelenterazine e is a finished bio-reagent rather than a synthetic building block. As such, it is not typically used as a starting material or intermediate in organic synthesis. For completeness (general context):

• Reactivity profile (literature)

  • The imidazopyrazinone core is sensitive to oxidation and strong base, limiting its use in routine synthetic transformations.
  • Aromatic substitutions and protecting-group strategies are employed in the multi-step synthesis of coelenterazine analogs, but post-synthesis diversification is uncommon due to chromophore sensitivity.

• When chemistry is performed

  • Derivatizations (e.g., installing solubilizing groups or spectral-tuning substituents) are generally executed on protected precursors prior to cyclization to the imidazopyrazinone.

For users, the practical focus is on assay deployment rather than chemical modification.

Target Specificity

Not applicable. This product is a small-molecule luciferin analog, not an antibody or affinity reagent. It does not have clone/isotype/epitope specifications. For enzyme compatibility, see Reaction & Applications for general literature guidance.