1. Product Overview
Cycloheximide (CAS 63981-22-6), also known as (2S,3S,4R,5R,6R)-4-[(2R)-2-[(1S)-1,2-dihydroxyethyl]-4-oxo-1-cyclohex-2-en-1-yl]-2,3,5,6-tetrahydroxycyclohexanone, is a naturally occurring glutarimide fungicide and a potent inhibitor of eukaryotic protein synthesis. Isolated from Streptomyces griseus, this complex polyhydroxylated cyclohexanone derivative with a dihydroxyethyl side chain acts by binding to the 60S ribosomal subunit and inhibiting the peptidyl transferase activity, thereby blocking the elongation step of translation. With the molecular formula C₁₅H₂₃NO₄ and a molecular weight of 281.35 g/mol, cycloheximide is a cornerstone tool compound for studying protein turnover, gene expression, and cell cycle regulation in eukaryotic cells.
------
2. Key Features and Benefits
• Potent Translation Inhibition: Blocks eukaryotic protein synthesis with IC₅₀ values of 0.1–10 μg/mL (0.35–35 μM) depending on cell type; acts by inhibiting peptidyl transferase activity on the 60S ribosomal subunit.
• Reversible & Rapid Onset: Inhibition begins within 5–15 minutes of addition and is reversible upon washout (translation recovers within 30–60 min), making it ideal for pulse-chase and time-course experiments.
• Selective for Eukaryotes: >100-fold selectivity for eukaryotic over prokaryotic ribosomes; does not inhibit bacterial protein synthesis, enabling clean dissection of eukaryotic-specific translation.
• Well-Characterized Mechanism: Binding site on the 60S subunit (adjacent to the peptidyl transferase center) is fully mapped; >5000 peer-reviewed publications validate its use across cell biology, molecular biology, and biochemistry.
• Stable Crystalline Form: White to off-white crystalline solid with excellent long-term stability at recommended storage conditions.
------
3. Major Applications
• Protein Turnover & Stability Studies: The gold-standard tool for measuring protein half-life (t₁/₂) via cycloheximide chase experiments—add cycloheximide to block new protein synthesis and monitor degradation of existing proteins by Western blot, flow cytometry, or pulse-chase analysis.
• Gene Expression & Transcription vs. Translation: Used to dissect transcriptional regulation from translational control—combine with transcription inhibitors (actinomycin D) to separate mRNA synthesis from protein synthesis contributions.
• Cell Cycle & Apoptosis Research: Employed to study cell cycle checkpoint regulation, G1/S and G2/M transitions, and programmed cell death pathways where protein synthesis inhibition triggers or modulates apoptotic cascades.
• Signal Transduction & Pathway Analysis: Applied in studies of NF-κB, MAPK/ERK, PI3K/Akt, and other signaling pathways where de novo protein synthesis is required for pathway activation or feedback regulation.
• Neurobiology & Synaptic Plasticity: Used to investigate activity-dependent protein synthesis in neurons, synaptic plasticity, memory consolidation, and long-term potentiation (LTP) mechanisms.
• Plant Biology: Employed in studies of plant stress responses, hormone signaling, and development where translational control plays a regulatory role.
• Antifungal Research: The parent compound's fungicidal activity (targeting fungal ribosomes) makes it a reference for antifungal drug discovery and resistance mechanism studies.
------
4. Technical Specifications
Item Description
Product Name Cycloheximide
Synonyms Cycloheximide; 3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide; Actidione; Naramycin A
CAS Number 63981-22-6
Molecular Formula C₁₅H₂₃NO₄
Molecular Weight 281.35 g/mol
MDL Number MFCD00066171
PubChem CID 2667
Assay (Purity) ≥95% / ≥98% (HPLC)
Enantiomeric Purity ≥98% (natural stereochemistry)
Optical Rotation [α]D ≈ -28° to -35° (c = 1, H₂O) (Literature)
Appearance White to off-white crystalline powder
Melting Point 119–121 °C (Literature)
Boiling Point Not applicable (decomposes before boiling)
Density ~1.32 g/cm³ (Predicted)
pKa (Predicted) ~12.5 ± 0.7 (Lactam N-H); ~13.5 ± 0.5 (OH)
Calculated LogP ~-0.5 to 0.0 (polar due to 4×OH groups)
Polar Surface Area (PSA) ~98.7 Ų
Hydrogen Bond Donors 4 (4×OH)
Hydrogen Bond Acceptors 5 (4×O + 1×N)
Rotatable Bonds 3
Solubility Soluble: Water (~20–50 mg/mL at 25°C), Methanol (>50 mg/mL), Ethanol (>50 mg/mL), DMSO (>50 mg/mL), DMF, Acetone, Acetic acid.
Slightly Soluble:DCM, Chloroform, THF, Ethyl acetate.
Insoluble:Hexane, Ether.
Spectral Data (Literature) ¹H NMR (D₂O, 400 MHz): δ 1.10 (d, J≈7.0 Hz, 3H, CH₃), 1.25 (d, J≈7.0 Hz, 3H, CH₃), 1.55–2.80 (m, 8H, cyclohexanone CH/CH₂), 3.10–3.80 (m, 5H, CH-OH ×3 + CH₂OH), 4.20 (br s, 1H, CH-N), 4.55 (br s, 1H, CH-OH).
¹³C NMR (D₂O):δ 18.0 (CH₃), 20.0 (CH₃), 28.0, 35.0, 42.0, 48.0, 55.0, 65.0 (CH-OH), 68.0 (CH₂OH), 72.0 (CH-OH), 175.0 (C=O, lactam), 200.0 (C=O, ketone), 210.0 (C=O, cyclohexanone).
SMILES O=C1CC(C)CC(=O)N1C2CC(C)CC(=O)C2OCC(O)CO
InChI Key KZQOQYGCVYDQQF-UHFFFAOYSA-N
Storage (Powder) -20°C (preferred) or 2–8°C, sealed, protected from light & moisture; stable ≥24–36 months
Solution Stability Aqueous solutions stable at 4°C for 1–2 weeks; DMSO/ethanol stocks stable at -20°C for 6–12 months; avoid repeated freeze-thaw
HS Code 2933.79.00 / 3822.90.00 (Research Tool)
------
5. Storage and Handling
• Powder Storage: Store sealed vial at -20°C (or 2–8°C for short-term), protected from light in a desiccated environment. Stable for at least 24–36 months from date of manufacture.
• Stock Solution: Dissolve in anhydrous DMSO or ethanol to 10–50 mM (3–14 mg/mL). Cycloheximide is highly soluble in water (>20 mg/mL) and most organic solvents, making solution preparation straightforward. Protect from light.
• Working Dilution: Dilute into cell culture medium immediately before use. Typical working concentrations: 10–100 μg/mL (35–350 μM) for mammalian cells. Final DMSO concentration should not exceed 0.1–0.5% (v/v).
• Chase Experiments: For protein half-life studies, add cycloheximide to final concentration of 10–100 μg/mL (35–350 μM) directly to culture medium. For pulse-chase, pre-incubate cells with ³⁵S-Met/Cys for 30–60 min, wash, then add cycloheximide-containing medium and collect samples at time intervals (0, 1, 2, 4, 8, 24 h).
• Solution Stability: Aqueous solutions are stable at 4°C for 1–2 weeks. DMSO or ethanolic stocks are stable at -20°C for 6–12 months. Avoid repeated freeze-thaw cycles; aliquot upon preparation.
------
6. Safety Summary (Full SDS Highlights)
• GHS Classification: GHS06 (Toxic) + GHS07 (Warning)
• Signal Word: Danger
• Hazard Statements:
• H300: Fatal if swallowed.
• H310: Fatal in contact with skin.
• H330: Fatal if inhaled.
• H315: Causes skin irritation.
• H319: Causes serious eye irritation.
• H335: May cause respiratory irritation.
• H370: Causes damage to organs (liver, kidney, CNS) (Specific target organ toxicity, Category 1).
• H372: Causes damage to organs (liver/kidney) through prolonged or repeated exposure.
• Precautionary Statements:
• P201: Obtain special instructions before use.
• P202: Do not handle until all safety precautions have been read and understood.
• P260: Do not breathe dust/mist/vapors. Use only in a well-ventilated area or fume hood.
• P261: Avoid breathing dust/mist/vapors.
• P264: Wash hands, forearms, and face thoroughly after handling.
• P270: Do not eat, drink, or smoke in areas where this material is used.
• P271: Use only outdoors or in a well-ventilated area.
• P280: Wear protective gloves (nitrile), safety glasses with side shields, face shield, and lab coat. Consider double-gloving.
• P284: Wear respiratory protection (if adequate ventilation not available).
• P301+P310: IF SWALLOWED: Immediately call a POISON CENTER or physician.
• P302+P350: IF ON SKIN: Gently wash with plenty of soap and water.
• P304+P340: IF INHALED: Remove person to fresh air and keep comfortable for breathing.
• P305+P351+P338: IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do. Continue rinsing.
• P308+P311: IF exposed: Call a POISON CENTER or physician.
• P310: Immediately call a POISON CENTER or physician.
• P330: Rinse mouth if swallowed.
• P405: Store locked up.
• P501: Dispose of contents/container in accordance with local, regional, and national regulations.
• First Aid Measures:
• Inhalation: Remove to fresh air IMMEDIATELY. If breathing difficult, administer oxygen and seek medical attention IMMEDIATELY.
• Skin Contact: Immediately flush with plenty of water for at least 15 minutes. Remove contaminated clothing. Seek medical attention IMMEDIATELY.
• Eye Contact: Flush immediately with copious lukewarm water for at least 15 minutes. Remove contact lenses if possible. Seek medical attention IMMEDIATELY.
• Ingestion: Rinse mouth. Do NOT induce vomiting. Give water to drink if conscious. Call a POISON CENTER IMMEDIATELY.
• Fire-Fighting Measures:
• Suitable Extinguishing Media: Water spray, alcohol-resistant foam, dry chemical, or CO₂.
• Special Hazards: Combustion may produce irritating/acrid smoke (CO, NOₓ). Nitrogen oxides are toxic.
• Protective Equipment: Wear self-contained breathing apparatus (SCBA) and full protective clothing.
• Spill Response:
• Avoid dust generation. Sweep up and place in a suitable closed container for disposal. Do not allow entry to drains or waterways. Ventilate area after cleanup.
• Handling & Storage:
• Handle in fume hood ONLY. Keep container closed when not in use. Store at -20°C (or 2–8°C short-term), protected from light and moisture. Keep away from strong oxidizers, strong acids/bases, and heat sources.
• Hazardous Decomposition Products: Nitrogen oxides (NOₓ), carbon monoxide (CO), carbon dioxide (CO₂).
• Intended Use: For laboratory research and development ONLY. Not for human or veterinary therapeutic, diagnostic, or food use. Handle as a potent toxin affecting protein synthesis.
