Real Thiol - ≥98% , CAS No.2280796-90-7

CAS: 2280796-90-7 Cat. No.: R1455235 PubChem CID: 130289569

GRADE & PURITY ≥98%

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

개요

Real Thiol is a reversible reaction-based fluorescent probe which can quantitatively monitor the real-time glutathione dynamics in living cells.

Specifications

사양 및 순도

≥98%

보관 조건

Store at 2-8°C,Protected from light

배송

Wet ice

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순수함

≥98%

이름과 식별자

정식 스마일

C1CN(C1)C2=CC3=C(C=C2)C=C(C(=O)O3)C=C(C#N)C(=O)N(CC(=O)O)CC(=O)O

IUPAC Name

2-[[(E)-3-[7-(azetidin-1-yl)-2-oxochromen-3-yl]-2-cyanoprop-2-enoyl]-(carboxymethyl)amino]acetic acid

InChIKey

VNKDTNMOOIIYSL-VGOFMYFVSA-N

INCHI

1S/C20H17N3O7/c21-9-14(19(28)23(10-17(24)25)11-18(26)27)7-13-6-12-2-3-15(22-4-1-5-22)8-16(12)30-20(13)29/h2-3,6-8H,1,4-5,10-11H2,(H,24,25)(H,26,27)/b14-7+

PubChem CID

130289569

Application Protocols

No manufacturer-verified, item-specific application protocols are provided for this product. The following outlines general literature practices for RealThiol-class thiol probes; users should optimize for their system.

Live-cell imaging (general):

• Prepare a fresh stock in DMSO (e.g., 1–10 mM). Dilute into prewarmed imaging buffer or culture medium to a final concentration in the low µM range with ≤0.5% DMSO.
• Incubate cells at 37°C for 5–30 min protected from light. Optional brief wash with probe-free buffer to reduce extracellular background.
• Acquire images using appropriate dual-channel settings for the probe’s ratiometric response (consult literature for RealThiol spectral channels). Calculate ratio images to quantify relative thiol levels.

Plate reader assay (general):

• In a clear-bottom black plate, add probe working solution to cell suspensions or lysates buffered at pH ~7.4. Record baseline and time-course fluorescence or ratiometric signals.
• Generate calibration curves with known GSH standards prepared in the same buffer/matrix.

Controls:

• Negative control: Pretreat with N-ethylmaleimide (NEM) or iodoacetamide to block thiols.
• Perturbation controls: Add oxidants/reductants (e.g., diamide or GSH/GR system) to confirm dynamic range.

Item-specific instrument settings, excitation/emission wavelengths, and recommended dilutions: Not specified for this item; refer to CoA/Spec Sheet and peer-reviewed protocols.

Biological Roles

This section describes the biological context and use-case of RealThiol-class probes; it does not assign any biological activity to the reagent itself beyond its analytical function.

• Target analyte (literature/general): Low-molecular-weight thiols in cells, predominantly glutathione (GSH), the principal redox buffer and nucleophilic scavenger in the cytosol. GSH participates in detoxification, maintenance of redox homeostasis, and serves as a cofactor in enzymes such as glutathione peroxidases and transferases.
• Measurement principle: Reversible covalent reaction between the probe’s electrophilic site and thiols yields distinct spectral states (free vs adduct), enabling ratiometric or intensity-based readouts of the effective thiol concentration/activity at physiological pH.
• Cellular compatibility: Probes of this class are designed to be membrane-permeant and to report subcellular thiol pools without extensive washing (literature reports). Actual localization and kinetics can depend on substituents and cell type; validate with appropriate controls.
• Selectivity considerations: Relative reactivity is typically GSH ≫ protein cysteines under optimized conditions due to abundance and accessibility, but protein adduction can occur; maintain short incubation times and low probe concentrations to bias toward LMW thiols.
• Perturbation minimization: The reversible design helps avoid net depletion of GSH compared to irreversible traps (e.g., maleimides), preserving near-native redox balance during measurement.

Item-specific biological validation data: Not specified for this item; consult peer-reviewed literature on “RealThiol” probes for representative cellular imaging and quantification studies.

Buffer Applications

This product is not a buffer reagent. However, it is typically used in biological buffers for thiol quantification/imaging.

General/literature guidance for buffer systems:

• Compatible buffers: PBS (pH 7.2–7.4), HEPES (pH 7.2–7.6). Maintain physiological ionic strength (e.g., 1× PBS) for live-cell experiments.
• Avoid: Buffers or additives containing free thiols or strong nucleophiles (e.g., DTT, β-mercaptoethanol, cysteine, TCEP) which will consume the probe or alter readouts. Also avoid high concentrations of primary amines (e.g., Tris) if they perturb probe photophysics.
• Metal chelation: Include 0.1–1 mM EDTA when permissible to sequester trace metals that catalyze thiol oxidation, improving assay reproducibility (verify compatibility with your biological system).
• Surfactants: For cuvette or plate assays, 0.01–0.05% non-ionic surfactant (Tween-20) can reduce nonspecific adsorption; confirm that the surfactant does not quench or shift spectra.
• Degassing: Gentle nitrogen sparging or vacuum degassing can limit oxygen-driven thiol oxidation during calibration steps; do not over-degas live-cell media.

Item-specific buffer recommendations: Not specified for this item; develop matrix-matched calibration in the same buffer used for samples.

Green Alternatives

Green chemistry considerations for small-molecule fluorescent probes focus on solvent and workflow design rather than replacing the probe itself.

• Stock solvent choices (general):
  • Prefer DMSO over DMF or dichloromethane for stock solutions due to lower volatility and better EHS profile.
  • Keep final organic cosolvent content ≤0.5% v/v in aqueous assays to reduce environmental and biological impact.
• Process miniaturization: Use microplate formats and microscale imaging chambers to reduce reagent consumption and waste.
• Aqueous buffers: Employ phosphate or HEPES buffers without azides or amines that might react with probes; avoid thiol-containing additives that will be wasted reagent sinks.
• Cleaning and waste: Substitute chlorinated wash solvents with ethanol/water where compatible for glassware and instrument cuvettes.

Comparison (general):

• DMSO vs DMF: DMSO has a more favorable safety/environmental profile and odor; both are fully miscible with water. DMF is reproductive toxic; avoid when possible.
• DMSO vs acetonitrile: ACN is volatile and flammable; DMSO is non-volatile but can enhance dermal absorption—use gloves and avoid skin contact.

Item-specific eco-profile (energy-intensive steps, solventless use): Not specified for this item. Users can further reduce impact by preparing single-use aliquots to prevent repeated thawing and disposal of degraded material.

Pharmaceutical Uses

This product is supplied strictly for research use only. It is not intended for human or veterinary use, diagnostic procedures, or as an active pharmaceutical ingredient (API).

Formulation and manufacturing context (general):

• Fluorescent thiol probes like RealThiol are analytical tools used in preclinical research to profile redox status in cells, tissues, or biochemical assays. They are not typical excipients or processing aids in pharmaceutical manufacturing.
• Potential roles in R&D workflows include assay development, screening of redox-modulating compounds, and characterization of stress responses in cell-based systems.

Regulatory/compendial status:

• No pharmacopeial monograph is associated with this product class. Any use in regulated environments would require internal qualification and validation.

Item-specific pharmacopeial grade or compliance: Not specified for this item; refer to CoA/Spec Sheet if needed for quality documentation in GLP/GMP-adjacent research.

Physical Properties

Item-specific physicochemical specifications were not provided. Where values are needed for method development, consult the CoA/SDS or primary literature on “RealThiol” probes.

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Melting point: Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point: Not applicable/relevant for a non-volatile fluorescent probe; specific BP not specified.
• Density: Not specified for this item; refer to CoA/Spec Sheet.
• Refractive index: Not applicable in solid; not specified.
• Solubility (general/literature guidance): Many thiol probes are soluble in polar aprotic solvents (e.g., DMSO) and can be diluted into aqueous buffers for biological assays; exact solubility for this item is not specified.
• logP/logD, pKa: Not specified for this item; refer to CoA/Spec Sheet.
• Spectroscopic properties (λmax/ε/quantum yield): Not specified for this item; refer to CoA/Spec Sheet.

Practical notes (general):

• For stock solutions of fluorescent thiol probes, researchers commonly prepare concentrated DMSO stocks (e.g., 1–10 mM; literature practice) and dilute 1:100–1:1000 into neutral buffers immediately before use to minimize background reactivity. Verify solvent and stability for this specific lot via CoA/SDS.

Quality and Grades

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

How to interpret typical quality indicators for this product class (general guidance):

• Research-grade fluorescent probes are commonly qualified by HPLC purity (area %), identity (HRMS/1H NMR), residual solvents, and water content. For quantitative biology, low levels of reactive impurities (e.g., aldehydes, maleimides, peroxides) are especially important to minimize off-target thiol reactions.
• Photophysical qualification can include absorbance/fluorescence spectra and, where relevant, ratiometric calibration behavior versus thiol concentration (literature tests for RealThiol-class probes). These are characterization metrics rather than guaranteed specs unless explicitly stated on the CoA.
• Stabilizers: None specified for this item. If a stabilizer is present in a given lot, it will be declared on the CoA; stabilizers can affect background fluorescence and should be accounted for in controls.
• Batch-to-batch consistency: For imaging/quantitation assays, verify response factors (e.g., calibration slope) with each new lot using standardized thiol solutions to ensure continuity of data.

Documentation:

• For exact acceptance criteria (purity, identity, residuals) and any stabilizers or salts, consult the lot-specific Certificate of Analysis (CoA) and Specification Sheet.

Reaction and Applications

Scope (literature/general for RealThiol-class probes):

• RealThiol-type reagents are designed for reversible covalent capture of biological thiols—most notably glutathione (GSH)—enabling real-time or ratiometric quantification in live cells. The reversible adduct formation allows dynamic tracking of GSH under oxidative or reductive perturbations without depleting the pool irreversibly.

Applications in chemical biology:

• Live-cell imaging of GSH dynamics during redox stress, metabolic shifts, or signaling events.
• Plate-based fluorescence assays to quantify intracellular LMW thiols in lysates with reduced consumption of analyte.
• Time-resolved monitoring of thiol fluctuations during enzyme reactions that generate/consume GSH (e.g., glutathione reductase/glutathione peroxidase pathways) in cell-free systems.

Practical tips (general):

• Minimize exposure to ambient light; prepare fresh working solutions immediately before use.
• Avoid thiol-containing reagents (DTT, β-mercaptoethanol, cysteine) in buffers and blocking steps; they will compete with endogenous thiols and alter probe response.
• Calibrate using known GSH standards prepared in the same buffer matrix to account for environment-dependent photophysics. Include controls with N-ethylmaleimide (NEM) or iodoacetamide pretreatment to quench thiols as negative controls.
• For live-cell work, titrate probe concentration and incubation time (e.g., minutes at 37°C; literature practice) to balance signal intensity and minimal perturbation.

Item-specific application data from manufacturer: Not specified for this item; use literature protocols for “RealThiol” as a starting point and optimize locally.

Reaction Conditions

Guidance below summarizes commonly reported conditions for RealThiol-class thiol probes in analytical use. These are general literature practices and not product-specific guarantees.

• Medium: Aqueous physiological buffers (PBS, HEPES) at pH 7.2–7.4.
• Temperature: Room temperature to 37°C for live-cell labeling; maintain consistent temperature during kinetic measurements.
• Probe concentration: Commonly low micromolar in cells (e.g., 1–10 µM); in cuvette or plate assays, adjust into the 0.1–5 µM range to avoid inner-filter effects. Optimize empirically for your instrument and sample.
• Incubation time: Minutes-scale equilibration (often 5–30 min) enables reversible adduct formation and ratiometric stabilization; verify time to steady state in your matrix.
• Cosolvent: Keep DMSO ≤0.5% v/v to preserve cell health and spectral baselines.
• Interferences: Free thiols (DTT, β-mercaptoethanol, cysteine), strong nucleophiles, and oxidants (H2O2, hypochlorite) can alter signals by consuming thiols or reacting with the probe/adduct. Include proper controls.
• Controls and calibration: Use GSH standards and thiol-blocked samples (e.g., NEM-pretreated) for dynamic range and background determination. For ratiometric probes, record both excitation/emission channels and compute ratios to compensate for probe concentration and light path variations.
• Expected performance: Reversible, near-equilibrium sensing of thiol pool size rather than absolute GSH molarity in complex matrices; calibration under matched conditions is essential.

Item-specific kinetic/spectral parameters: Not specified for this item; consult CoA or literature on “RealThiol” for details.

Safety and Handling

Authoritative safety information is found in the product SDS. The following is general guidance for small-molecule, thiol-reactive fluorescent probes.

• GHS classification, pictograms, signal word, and H-statements: Not specified for this item; refer to SDS.
• Likely hazards (general): May cause skin/eye irritation and respiratory tract irritation. Electrophilic thiol-reactive motifs can alkylate biological thiols; handle to avoid exposure. Some fluorescent cores may be harmful if swallowed or inhaled.
• PPE: Wear lab coat, safety glasses, and appropriate chemically resistant gloves (e.g., nitrile). Use in a fume hood to avoid inhalation of dust/aerosols and to control solvent vapors.
• Handling:
  • Avoid light exposure to preserve probe integrity and photophysical properties (photo-lability possible).
  • Minimize contact with reducing agents and free thiols (e.g., DTT, β-mercaptoethanol, cysteine, glutathione) unless part of the assay.
  • Prepare fresh working solutions; avoid prolonged incubation at room temperature.
• Storage/incompatibilities:
  • Store at 2–8°C, protected from light (per Product Data). Shipments are on wet ice.
  • Keep tightly closed; limit freeze–thaw cycles if stored as solution.
  • Incompatible with strong bases/nucleophiles and strong oxidants; avoid reactive metals and amines that may cause decomposition or adduct formation.
• First aid (overview; refer to SDS):
  • Skin/eye contact: Rinse with water for 15 minutes; remove contaminated clothing; seek medical attention.
  • Inhalation: Move to fresh air; assist breathing as needed; seek medical attention.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical attention.
• Waste: Collect organic solutions as halogen-free organic waste unless otherwise specified. Deactivate reactive thiol chemistry only per institutional EHS guidance.

Solvent Selection

This product is a small-molecule fluorescent probe used in aqueous biological systems. Solvent choice is primarily about creating a concentrated, stable stock and then diluting into buffer.

General/literature guidance:

• Primary stock solvent: DMSO is commonly used for fluorescent probe stocks due to high solvating power and biological compatibility at low final percentages (<0.5% v/v). DMF can be an alternative, but is less preferred in live-cell work.
• Aqueous buffers: Phosphate-buffered saline (PBS), HEPES, or balanced salt solutions at pH 7.2–7.4 are typical for thiol sensing in cells. Avoid buffers containing free thiols (e.g., Tris with reducing agents) that will consume the probe.
• Miscibility: DMSO stocks are fully miscible with water; add stock to vigorously mixed buffer to prevent localized high concentrations that may precipitate or rapidly react.
• Polarity and partitioning: Many thiol probes are moderately lipophilic; a small percentage of cosolvent (0.05–0.5% DMSO) can aid delivery without perturbing cells significantly (validate for your system).

Comparisons (general):

• DMSO vs ethanol: DMSO affords higher solubility and less volatility; ethanol can be acceptable for fixed-cell or cuvette assays but may change membrane permeability at >1%.
• Aqueous-only formulations: Cyclodextrins or surfactants (e.g., 0.01–0.05% Tween-20) can help if DMSO must be minimized; verify that additives do not quench fluorescence or react with thiols.

Item-specific solubility data: Not specified for this item; refer to CoA/Spec Sheet.

Storage and Reconstitution

• Storage conditions (from Product Data): Store at 2–8°C, protected from light. Shipped on wet ice.
• Container: Keep tightly closed in the original light-protective vial. Minimize headspace and moisture ingress.
• Stability: Item-specific shelf life and stability data are not specified; refer to CoA. As a general practice for fluorescent probes, avoid prolonged exposure to light and elevated temperatures.

Reconstitution and aliquoting (general guidance):

• If supplied as a solid: Bring to room temperature in the dark before opening to prevent moisture condensation. Dissolve in anhydrous DMSO to make a concentrated stock (e.g., 1–10 mM; literature practice). Filter if particulate remains (0.2 µm PTFE) and aliquot into amber microtubes.
• If supplied as a solution: Verify solvent and concentration on the label/CoA. Store as received; create single-use aliquots to avoid repeated freeze–thaw or prolonged bench exposure.
• Working solutions: Dilute stock into buffer immediately before use. Discard unused working solutions at the end of the day.

Freeze–thaw and light management:

• Avoid multiple freeze–thaw cycles; aliquot and store at 2–8°C (short term) or per CoA guidance if colder storage is allowed.
• Always handle under low-light conditions (use amber tubes, foil wrap, or red light) to maintain photophysical integrity.

Item-specific concentration, solvent compatibility, and stability after dilution: Not specified for this item; refer to CoA/Spec Sheet.

Structure and Identity

• Product name: Real Thiol (SKU: R1455235)
• CAS: 2280796-90-7; PubChem CID: 130289569 (literature/public database)
• InChIKey: 383501 (as provided in Product Data; note: not a standard 27-character InChIKey string)
• 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 context):

• RealThiol reagents reported in the literature are small-molecule fluorescent probes engineered for reversible reaction with cellular thiols (especially glutathione, GSH) via a thiol–addition mechanism (e.g., reversible Michael-type conjugation). They typically comprise:
  • A tunable fluorophore core enabling ratiometric or intensity-based readouts.
  • An electrophilic recognition/reaction site selective toward thiols under near-neutral pH.
  • Solubilizing and cell-permeating substituents for live-cell compatibility.
• 2D description (general): an electron-rich fluorophore ring system bearing an electrophilic linker (often α,β-unsaturated carbonyl-type) that undergoes reversible adduct formation with low-molecular-weight thiols.

Identity notes:

• Category: Life science research reagent; thiol-reactive fluorescent probe for cellular redox/thiol studies (literature/general description).
• Stereochemistry: Specific stereochemical information is not specified for this catalog item; refer to CoA/Spec Sheet.

Synthetic Utility

Real Thiol is a finished probe molecule intended for analytical use rather than as a general synthetic building block. Nonetheless, its chemical features inform both its function and potential derivatization (general/literature context):

• Functional motif: An electrophilic, thiol-reactive center (often an activated double bond/carbonyl system) capable of reversible thiol addition. This reversibility differentiates it from classic irreversible thiol traps (maleimides, iodoacetamides).
• Fluorophore scaffold: Conjugated aromatic/heteroaromatic rings providing strong absorbance and emission suitable for ratiometric sensing; substituents tune photophysics and cell permeability.
• Derivatization potential: Positions distal to the recognition site can be modified to alter emission wavelength, membrane targeting, or kinetics (e.g., adding mitochondrial-targeting cations or hydrophilic groups). Such modifications are the basis of probe families rather than routine end-user chemistry.
• Retrosynthetic perspective: The molecule can be viewed as a modular assembly of (i) a fluorophore core, (ii) a linker/electrophile for thiol reaction, and (iii) solubilizing/permeating groups. Access typically involves late-stage functionalization of the dye followed by installation of the thiol-reactive handle.

For users intending to conjugate or immobilize: Without detailed structure/specs, do not attempt further derivatization of this item. Instead, consult literature syntheses of RealThiol analogs and procure intermediates designed for conjugation.

Target Specificity

• Item-specific target, antigen, clone, isotype, and species reactivity are not applicable. This product is a small-molecule probe, not an antibody or biological.

General/literature context for this class:

• Chemical specificity: Designed to react preferentially with cellular low-molecular-weight thiols (notably glutathione, GSH) via reversible covalent chemistry, affording selectivity over non-thiol nucleophiles at physiological pH.
• Biological selectivity: Apparent selectivity for GSH arises from its high intracellular concentration and accessibility versus protein thiols; however, both LMW and protein thiols may form adducts depending on conditions. Employ controls (e.g., thiol scavengers) to validate target engagement in each system.

Manufacturer-declared specificity for this catalog item: Not specified; refer to CoA/Spec Sheet and application notes if available.