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| 5mg |
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| Targets |
Cbz-Ala-Ala-Asn TFA serves as a substrate for the cysteine protease legumain (asparaginyl endopeptidase, AEP). Legumain specifically cleaves peptide bonds C-terminal to asparagine residues. This peptide has no intrinsic biological activity but is used as a tool to study legumain activity and as a scaffold for drug delivery systems.
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| ln Vitro |
In vitro, Cbz-Ala-Ala-Asn TFA has no direct activity on cells. Its utility is demonstrated when conjugated to fluorophores (e.g., AMC) to create fluorogenic substrates for measuring legumain activity. The peptide itself is used to compete with fluorogenic substrates or as a building block for legumain-cleavable prodrugs. It can be applied as a scaffold for drug delivery, enabling targeted release at sites of high legumain expression.
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| ln Vivo |
As a peptide scaffold, Cbz-Ala-Ala-Asn TFA is not administered directly in animal studies. However, it can be incorporated into prodrugs or drug delivery systems that are cleaved by legumain. In mouse tumor models, legumain-cleavable prodrugs containing this sequence show enhanced antitumor efficacy and reduced systemic toxicity compared to non-cleavable controls.
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| Enzyme Assay |
For in vitro binding studies with legumain, the peptide substrate (conjugated to a fluorophore such as AMC) is used. The assay is performed in 96-well plates with legumain enzyme (activated at pH 4.0-5.5) and varying concentrations of the peptide substrate. Fluorescence is measured at excitation 380 nm and emission 460 nm over 30-60 minutes to calculate kinetic parameters (Km and Vmax).
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| Cell Assay |
No direct cellular assays are performed with Cbz-Ala-Ala-Asn TFA as it is a peptide substrate not designed to enter cells. To study legumain activity in cells, a cell-permeable fluorogenic substrate (e.g., Cbz-Ala-Ala-Asn-AMC) can be used. Cells are incubated with the substrate for 1-4 hours, and AMC release is measured in the culture medium or cell lysates, with legumain activity proportional to fluorescence.
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| Animal Protocol |
For in vivo studies using legumain-cleavable prodrugs, female BALB/c nude mice (6-8 weeks) are implanted subcutaneously with legumain-positive tumor cells (e.g., MDA-MB-231 or HT-1080). When tumors reach ~100 mm3, mice are treated intravenously with prodrug (5-20 mg/kg) or control every 3-4 days for 2-3 weeks. Tumor volumes and body weights are measured twice weekly.
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| ADME/Pharmacokinetics |
As a peptide scaffold, Cbz-Ala-Ala-Asn TFA is not administered as a therapeutic agent and has no independent pharmacokinetic properties. When incorporated into a drug conjugate, the TFA salt improves water solubility, aiding formulation for parenteral administration. In vivo stability is enhanced by the Cbz protecting group, which prevents non-specific proteolysis in circulation, while the Asn residue remains cleavable by legumain.
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| Toxicity/Toxicokinetics |
The TFA counterion is considered relatively non-toxic at the low concentrations encountered in research applications. As a synthetic peptide, Cbz-Ala-Ala-Asn TFA is not intended for human use. Standard safety precautions for handling peptides (e.g., avoiding inhalation, skin contact) should be followed. No significant toxicity is expected at typical research doses.
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| References |
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| Additional Infomation |
Cbz-Ala-Ala-Asn TFA (C20H25F3N4O8, MW 522.43) is a protected tripeptide used in legumain research. The Cbz group provides stability against non-specific proteases, while the TFA salt improves solubility. It serves as a scaffold for targeted drug delivery to legumain-overexpressing tumors (e.g., breast, prostate, and glioma) and is also used to develop legumain-activated prodrugs and imaging probes. Purity is ≥98% by HPLC.
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| Molecular Formula |
C20H25F3N4O9
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| Molecular Weight |
522.43
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| Appearance |
White to off-white solid powder
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 1.9141 mL | 9.5707 mL | 19.1413 mL | |
| 5 mM | 0.3828 mL | 1.9141 mL | 3.8283 mL | |
| 10 mM | 0.1914 mL | 0.9571 mL | 1.9141 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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