| Targets |
Biotin-(Oaa)3-epoxomicin targets the proteasome, specifically the catalytic beta subunits that possess an N-terminal threonine residue essential for proteolytic activity. The epoxomicin warhead forms a covalent, irreversible morpholino adduct with the active site threonine (Thr1) of beta1, beta2, beta5 in the constitutive proteasome, and of LMP7 (beta5i), MECL1 (beta2i), and LMP2 (beta1i) in the immunoproteasome. The three Oaa (amino-oxyacetic acid) linkers provide a hydrophilic spacer that reduces steric hindrance and improves accessibility to the active site. The biotin moiety enables high-affinity binding to streptavidin, facilitating pull-down of labeled proteasome subunits. This probe allows researchers to study which proteasome subunits are catalytically active in a given cell type or condition. It operates within the ubiquitin-proteasome pathway and is a tool for investigating antigen presentation and proteasome biology.
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| ln Vitro |
In vitro, Biotin-(Oaa)3-epoxomicin does not have a traditional pharmacological activity such as an IC50; its activity is defined by its ability to specifically and covalently label active proteasome subunits. At concentrations of 0.1-10 uM, when incubated with cell lysates or purified 20S proteasome for 30-60 minutes at 37degC, the probe binds to the active subunits. The labeled subunits can be captured on streptavidin-agarose beads, eluted, and identified by mass spectrometry (LC-MS/MS) or by Western blot using subunit-specific antibodies. The probe has been used to demonstrate the selective labeling of immunoproteasome subunits LMP7, MECL1, and Z in cells treated with interferon-gamma, which induces immunoproteasome expression. It shows no labeling of inactive subunits or off-target proteins. The binding is competed by excess unlabeled epoxomicin, confirming specificity. No cytotoxicity is typically measured for the probe itself as it is used in lysates. DMSO is used as solvent (final ≤0.1% in lysate).
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| ln Vivo |
No in vivo activity data are available for Biotin-(Oaa)3-epoxomicin. This probe is designed exclusively for in vitro pull-down experiments and is not intended for administration to live animals for therapeutic or efficacy studies. While it could theoretically be used in vivo to label proteasomes, the biotin tag may lead to rapid clearance and non-specific binding, and such applications are not standard. Therefore, no in vivo animal protocols or efficacy data exist. For research use only.
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| Enzyme Assay |
For non-cellular (cell-free) proteasome labeling and pull-down, purified 20S proteasome (from human erythrocytes or rabbit muscle) is used. Prepare 50 uL of reaction mixture containing 1-5 ug proteasome in assay buffer (50 mM Tris-HCl pH 7.4, 20 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.01% NP-40). Add Biotin-(Oaa)3-epoxomicin from a 1 mM DMSO stock to final concentrations of 0.1, 0.5, 1, 5, and 10 uM (DMSO ≤1%). Incubate at 37degC for 60 minutes. Then add 10 uL of streptavidin-agarose beads (pre-equilibrated in assay buffer) and rotate at 4degC for 2 hours. Beads are collected by centrifugation (500×g, 2 min) and washed 4 times with 500 uL of assay buffer containing 0.1% Triton X-100, followed by 2 washes with PBS. Bound proteins are eluted by adding 30 uL of 2× SDS-PAGE loading buffer and boiling for 5 minutes. Eluates are resolved on 12% SDS-PAGE. For detection, either stain with Coomassie Blue or transfer to PVDF membrane for Western blot using anti-proteasome subunit antibodies (e.g., anti-beta5, anti-LMP7, anti-MECL1). For identification by mass spectrometry, the eluted proteins are digested with trypsin and analyzed by LC-MS/MS. Negative control: DMSO only. Specificity control: pre-incubate proteasome with 10-fold excess of unlabeled epoxomicin for 30 min before adding Biotin-(Oaa)3-epoxomicin.
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| Cell Assay |
For in vitro cell-based pull-down assays, cells (e.g., HeLa, B16 melanoma, or 293T) are cultured in appropriate medium. To induce immunoproteasome expression, cells may be treated with IFN-gamma (100 U/mL) for 48 hours. Cells are harvested, washed with PBS, and lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1× protease inhibitor cocktail) at 4degC for 30 minutes. Lysates are clarified by centrifugation (14,000×g, 15 min, 4degC). Protein concentration is determined by BCA. For each pull-down, 500-1000 ug of lysate protein in 500 uL volume is incubated with Biotin-(Oaa)3-epoxomicin at 1-10 uM (from DMSO stock, final DMSO ≤0.1%) at 37degC for 1-2 hours with gentle shaking. Then, 20 uL of streptavidin-agarose beads (pre-washed) is added and incubated at 4degC overnight with rotation. Beads are washed 5 times with 500 uL of lysis buffer, then 2 times with PBS. Bound proteins are eluted by boiling in SDS-PAGE loading buffer. Eluates are analyzed by Western blot or LC-MS/MS as above. Control samples: DMSO only; competition with 10-fold excess free epoxomicin. This protocol allows identification of proteasome subunits that are active and accessible to the probe.
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| Animal Protocol |
Not applicable. Biotin-(Oaa)3-epoxomicin is not used for in vivo animal studies. It is an in vitro biochemical probe for pull-down assays from cell lysates or purified proteasomes. No animal dosing, efficacy, or toxicology studies are performed or reported. For research use only.
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| ADME/Pharmacokinetics |
Not applicable. Biotin-(Oaa)3-epoxomicin is not a drug candidate; its pharmacokinetic parameters are not measured. It is used exclusively in test-tube assays. For storage, the lyophilized powder should be kept at -20degC for up to 3 years, protected from moisture and light. The compound is soluble in DMSO (e.g., 10 mM). Once dissolved, store aliquots at -80degC for up to 1 year, avoid freeze-thaw cycles. Molecular weight: 1078.45 g/mol. The extended Oaa linkers increase hydrophilicity compared to Biotin-epoxomicin.
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| Toxicity/Toxicokinetics |
No toxicity data are available for Biotin-(Oaa)3-epoxomicin. At the concentrations used in pull-down assays (1-10 uM), no acute toxicity is expected in the context of the assay. However, epoxomicin is a potent proteasome inhibitor and can be cytotoxic to cells if internalized. The biotinylated form may also inhibit proteasomes in living cells if administered, but this is not the intended use. Standard laboratory safety precautions should be followed: avoid inhalation, ingestion, and skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a fume hood. For research use only-not for human use. Dispose of waste according to local hazardous waste regulations.
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| References | |
| Additional Infomation |
CAS: 247068-92-4. Also known as Compound 13. Molecular formula: C54H95N9O11S, molecular weight 1078.45. Sequence: Biotin-[Oaa]-[Oaa]-[Oaa]-IIT-[Leu((2R)-2-methyloxiranyl)], where Oaa = amino-oxyacetic acid. Purity: typically >95% by HPLC. Storage: powder at -20degC. Solubility: DMSO. This probe is used for affinity purification of proteasome subunits, particularly immunoproteasome subunits LMP7, MECL1, and Z. For research only. Not for human use.
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| Exact Mass |
1077.687
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|---|---|
| CAS # |
247068-92-4
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| PubChem CID |
177746373
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| Sequence |
Biotin-{Oaa}-{Oaa}-{Oaa}-Ile-Ile-Thr-{Leu((2R)-2-methyloxiranyl)}Biotin-{Oaa}-{Oaa}-{Oaa}-IIT-{Leu((2R)-2-methyloxiranyl)}
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| Appearance |
Typically exists as solids at room temperature
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| Hydrogen Bond Donor Count |
9
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| Rotatable Bond Count |
39
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| Heavy Atom Count |
75
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| Complexity |
1880
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| Defined Atom Stereocenter Count |
11
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| InChi Key |
TWHRMHRNYSDMAU-LLLMMZAZSA-N
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| InChi Code |
InChI=1S/C54H95N9O11S/c1-10-35(5)45(50(70)61-46(37(7)64)51(71)58-38(31-34(3)4)49(69)54(8)33-74-54)60-52(72)48(36(6)11-2)63(9)44(68)27-17-14-22-30-56-42(66)25-15-12-20-28-55-41(65)24-16-13-21-29-57-43(67)26-19-18-23-40-47-39(32-75-40)59-53(73)62-47/h34-40,45-48,64H,10-33H2,1-9H3,(H,55,65)(H,56,66)(H,57,67)(H,58,71)(H,60,72)(H,61,70)(H2,59,62,73)/t35-,36-,37+,38-,39-,40-,45-,46-,47-,48-,54+/m0/s1
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| Chemical Name |
(2S,3S)-2-[[(2S,3S)-2-[6-[6-[6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]hexanoylamino]hexanoylamino]hexanoyl-methylamino]-3-methylpentanoyl]amino]-N-[(2S,3R)-3-hydroxy-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxobutan-2-yl]-3-methylpentanamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 100 mg/mL (~92.73 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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