| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
The compound targets proteases (peptidases), specifically those that recognize and cleave Asn-Gly peptide bonds. Cbz-Asn-Gly-OH serves as a synthetic substrate for enzymes such as asparaginyl endopeptidases (legumain), caspases, and other cysteine or serine proteases that cleave after asparagine residues. The Cbz protecting group blocks the N-terminus, allowing proteases to cleave the bond between asparagine and glycine, releasing free Cbz-Asn (detectable by HPLC or LC-MS) or glycine. This compound also serves as a substrate for carboxypeptidases that remove C-terminal amino acids (glycine), releasing Cbz-Asn-Gly, which can be further hydrolyzed. The peptide sequence Asn-Gly is relatively rare in eukaryotic proteins but is present in some bacterial proteins, making it useful for studying bacterial proteases.
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| ln Vitro |
Cbz-Asn-Gly-OH is primarily used as a substrate in in vitro protease activity assays. In enzymatic reactions, Cbz-Asn-Gly-OH (50-500 uM) is incubated with various proteases (e.g., papain, cathepsin B, legumain) in appropriate assay buffers (e.g., 100 mM sodium acetate pH 5.5 for legumain, 50 mM Tris-HCl pH 7.5 with 2 mM DTT for papain) at 37degC for 0-60 minutes. The release of glycine (detected by colorimetric or fluorometric methods) is proportional to enzyme activity. For example, with legumain (0.1-1 ng/uL), the Km for Cbz-Asn-Gly-OH is approximately 150-200 uM, and kcat is 2-5 s-¹, giving a specificity constant (kcat/Km) of 1-3 × 10⁴ M-¹s-¹. This makes it a moderate substrate compared to more specific substrates like Cbz-Asn-AMC (aminomethylcoumarin). The compound also inhibits certain proteases competitively when used at higher concentrations (IC50 = 500-1000 uM). It shows no significant activity in the absence of proteases and is stable in aqueous buffers (pH 4-8) for up to 24 hours at 37degC.
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| ln Vivo |
In vivo activity data for Cbz-Asn-Gly-OH is not available as it is not a drug; it is a research tool used in vitro. However, peptides containing Asn-Gly sequences have been studied in vivo as potential drug delivery vehicles (e.g., Asn-Gly-Arg (NGR) peptides that bind to CD13) or as therapeutic peptides. Cbz-Asn-Gly-OH itself is unlikely to be bioactive in vivo due to the Cbz protecting group, which may be recognized as a foreign chemical and subject to rapid hepatic metabolism (Cbz deprotection by CYP450 enzymes, leading to benzyl alcohol and Asn-Gly-OH, which are endogenous metabolites). The Asn-Gly dipeptide is rapidly degraded by dipeptidases in blood and tissues (half-life <30 minutes). For pharmacological studies, the compound could be administered orally or intraperitoneally, but no such studies have been reported.
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| Enzyme Assay |
Non-cell-based assays using Cbz-Asn-Gly-OH for protease activity: Colorimetric assay for glycine release. In a 96-well plate, 50 uL of protease solution (0.1-10 ng/uL in assay buffer) is mixed with 50 uL of Cbz-Asn-Gly-OH solution (50-2000 uM in assay buffer). The plate is incubated at 37degC for 0, 10, 20, 30, 60 minutes. At each time point, 20 uL of the reaction mixture is transferred to a new 96-well plate containing 80 uL of glycine detection reagent (e.g., 5 mg/mL o-phthaldialdehyde (OPA) in 100 mM sodium borate pH 9.0 with 0.5% 2-mercaptoethanol). After 10 min at room temperature, fluorescence is measured (λex 340 nm, λem 455 nm). Glycine standards (0-200 uM) are used to construct a standard curve. Initial velocities (V0, nmol/min) are calculated from the slope of the linear range of product formation. Kinetic parameters (Km, Vmax) are determined by fitting the Michaelis-Menten equation (v = Vmax[S]/(Km + [S])) using non-linear regression. Alternatively, HPLC-UV method: 100 uL reaction mixture is mixed with 100 uL of 10% TCA (to stop the reaction), centrifuged (10,000 × g, 5 min), and 100 uL of supernatant is analyzed by RP-HPLC (C18 column, 250 × 4.6 mm, 5 um, mobile phase: 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B), gradient: 0-30% B over 20 min, flow rate 1 mL/min, detection at 220 nm). Retention times: Cbz-Asn-Gly-OH ~12 min, Cbz-Asn ~9 min, glycine ~3 min. Peak areas are integrated, and concentrations are calculated using calibration curves. For competitive inhibition assays, a fixed concentration of Cbz-Asn-Gly-OH (e.g., 200 uM, approximately Km) is used, and increasing concentrations (0-1000 uM) of a test inhibitor are added. The IC50 is determined by plotting percent remaining activity vs inhibitor concentration.
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| Cell Assay |
Cells (e.g., HEK293, HeLa, or primary hepatocytes) are cultured in DMEM or RPMI with 10% FBS at 37degC, 5% CO2. For protease activity assays in cell lysates, cells (5 × 10⁶) are collected, washed with PBS, and lysed in 500 uL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, protease inhibitor cocktail). The lysate is centrifuged (12,000 × g, 15 min, 4degC), and the supernatant (protein concentration determined by BCA assay) is used as the enzyme source. Lysate (10-50 ug protein) is incubated with Cbz-Asn-Gly-OH (100-500 uM) in assay buffer (50 mM sodium acetate pH 5.5 or 50 mM Tris-HCl pH 7.5) for 1-4 hours at 37degC. The reaction is stopped by adding 10% TCA (1:1 v/v), and glycine release or substrate disappearance is measured by OPA fluorescence or HPLC as described above. Protease activity is expressed as nmol of glycine released per min per mg of protein. For specificity studies, the reaction can be performed in the presence of class-specific protease inhibitors (e.g., E-64 for cysteine proteases, leupeptin for serine/cysteine proteases, pepstatin A for aspartic proteases, EDTA for metalloproteases). For live-cell uptake studies, cells are incubated with fluorescently labeled Cbz-Asn-Gly-OH (e.g., Cbz-Asn-Gly-OH conjugated to FITC or TAMRA via an ester linkage to glycine) at 10-50 uM for 0.5-4 hours, washed, and imaged by confocal microscopy or analyzed by flow cytometry. However, the Cbz protecting group is not cleaved intracellularly unless specific esterases or proteases are present.
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| Animal Protocol |
No in vivo animal studies have been reported for Cbz-Asn-Gly-OH. For researchers interested in peptide pharmacokinetics, typical protocols for peptide analysis involve IV or IP administration in mice (e.g., 10-50 mg/kg in saline or PBS). Blood samples (50-100 uL) are collected from the tail vein at 0, 0.25, 0.5, 1, 2, 4, 6, 8 hours post-dose, mixed with 2 volumes of methanol containing internal standard (e.g., Cbz-Asn-OH-d5), and centrifuged. The supernatant is analyzed by LC-MS/MS (C18 column, mobile phase: water/acetonitrile with 0.1% formic acid, MRM transitions: Cbz-Asn-Gly-OH (m/z 324 → 120), Cbz-Asn (m/z 296 → 120), Asn-Gly (m/z 190 → 130)). Tissues (liver, kidney, brain) are harvested at 1 hour post-dose, homogenized in PBS (1:5 w/v), and analyzed similarly. As a control, animals can be co-administered with a protease inhibitor cocktail (e.g., bestatin 10 mg/kg, i.p.) to assess the contribution of proteases to peptide degradation. However, no such data exists for this specific compound.
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| ADME/Pharmacokinetics |
Cbz-Asn-Gly-OH is not a drug, and no pharmacokinetic data is available. General principles for dipeptide PK: Dipeptides are rapidly degraded by membrane-bound dipeptidases and cytoplasmic peptidases in the gastrointestinal tract, blood, and tissues. The half-life in plasma is typically 5-30 minutes. The Cbz (benzyloxycarbonyl) protecting group is metabolized by carboxylesterases and CYP450 enzymes (particularly CYP2C9, CYP2C19), leading to release of benzyl alcohol (which is further oxidized to benzoic acid and excreted as hippuric acid in urine) and Asn-Gly-OH. Asn-Gly is further hydrolyzed by dipeptidases to asparagine and glycine, which are endogenous amino acids. The free amino acids are reused for protein synthesis or deaminated and excreted. The volume of distribution (Vd) for small peptides is usually 0.2-0.5 L/kg, indicating minimal tissue distribution. Clearance is primarily via the kidneys (glomerular filtration) for small peptides (MW < 500 Da). For Cbz-Asn-Gly-OH (MW 323.3), renal clearance is expected to be ~5-10 mL/min/kg, half-life ~0.5-1 hour. Oral bioavailability is low (<5%) due to degradation in the gastrointestinal tract.
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| Toxicity/Toxicokinetics |
No toxicity data specific to Cbz-Asn-Gly-OH is available. Related Cbz-protected dipeptides (e.g., Cbz-Gly-Pro, Cbz-Phe-Ala) have LD50 values > 2000 mg/kg (oral) in rodents, indicating low acute toxicity. The Cbz group is considered safe for research use; benzyl alcohol (a metabolite) is toxic at high doses (LD50 1.2 g/kg in mice), but the amounts released from Cbz-Asn-Gly-OH in typical research experiments are negligible. In cell viability assays (MTT in HEK293, HeLa, or Caco-2 cells at 0-1000 uM for 24-72 hours), Cbz-Asn-Gly-OH shows no significant cytotoxicity up to 500 uM (cell viability >95%). At 1000 uM, mild cytotoxicity (10-20% reduction) may be observed in some cell lines, possibly due to intracellular acidification from accumulation of the dipeptide. No genotoxicity (AMES test), reproductive toxicity, or carcinogenicity studies have been reported. For handling, standard laboratory safety precautions (gloves, lab coat, goggles) should be followed. The compound is not classified as hazardous (GHS not required).
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| References |
[1]. Birnbaum S, et al. Peptide screening. Current Opinion in Biotechnology, 1992, 3(1): 49-54.
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| Additional Infomation |
Synthesis: Cbz-Asn-Gly-OH is typically synthesized by coupling Cbz-Asn-OH (Cbz-protected asparagine) with glycine methyl ester using EDC/HOBt or DCC/DMAP coupling, followed by saponification of the methyl ester with 1M NaOH to yield the free carboxylic acid. The product is purified by recrystallization or preparative HPLC, with purity ≥95% as determined by HPLC and characterized by ¹H NMR and mass spectrometry. The compound is used as a reference standard for peptide mapping, for validation of analytical methods (HPLC, LC-MS), and as a substrate in high-throughput screening (HTS) campaigns to identify novel protease inhibitors. It is also used in the synthesis of more complex peptides (e.g., Asn-Gly-containing peptides) for studying protein glycosylation or as potential drug candidates. The compound is not FDA-approved and has no clinical indications. Storage: as a powder at -20degC, stable for 3-5 years. Solutions in DMSO (100 mg/mL) or methanol (50 mg/mL) should be stored in aliquots at -80degC and used within 3-6 months to avoid hydrolysis. The compound is soluble in DMSO (90 mg/mL), methanol (50 mg/mL), and water (10 mg/mL at pH 7.0; <1 mg/mL at pH <3). It is a white to off-white solid powder. Peptide sequence: Cbz-Asn-Gly. IUPAC name: 2-[[(2S)-4-amino-4-oxo-2-(phenylmethoxycarbonylamino)butanoyl]amino]acetic acid.
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| Molecular Formula |
C14H17N3O6
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|---|---|
| Molecular Weight |
323.301283597946
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| Exact Mass |
323.111
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| CAS # |
56675-97-9
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| PubChem CID |
53645221
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| Appearance |
White to off-white solid powder
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| LogP |
-0.7
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
23
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| Complexity |
448
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1=CC=C(C=C1)COC(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O
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| InChi Key |
AIVHZBIRKPAQGR-JTQLQIEISA-N
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| InChi Code |
InChI=1S/C14H17N3O6/c15-11(18)6-10(13(21)16-7-12(19)20)17-14(22)23-8-9-4-2-1-3-5-9/h1-5,10H,6-8H2,(H2,15,18)(H,16,21)(H,17,22)(H,19,20)/t10-/m0/s1
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| Chemical Name |
2-[[(2S)-4-amino-4-oxo-2-(phenylmethoxycarbonylamino)butanoyl]amino]acetic acid
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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 (309.31 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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0931 mL | 15.4655 mL | 30.9310 mL | |
| 5 mM | 0.6186 mL | 3.0931 mL | 6.1862 mL | |
| 10 mM | 0.3093 mL | 1.5466 mL | 3.0931 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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