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| Targets |
H-Leu-pNA·HCl targets the active sites of aminopeptidases and other proteases that cleave N-terminal leucine residues. The compound is a synthetic substrate designed to be recognized and hydrolyzed by these enzymes. It does not target a specific receptor or therapeutic target but rather serves as a tool for measuring enzyme activity. The p-nitroaniline group acts as a chromophore that is released upon enzymatic cleavage, allowing for colorimetric detection. This substrate is particularly useful for studying leucine aminopeptidase, which is involved in protein degradation and turnover, as well as other proteases that have specificity for leucine or similar hydrophobic amino acids at the P1 position.
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| ln Vitro |
Commercial supplements containing amino acids and their derivatives have been utilized for ergogenic purposes. They affect the release of anabolic hormones, the availability of food for activity, the ability to think clearly under pressure, and the prevention of muscle damage brought on by exercise. As ergogenic food ingredients, they are known to be advantageous[1].
In vitro, H-Leu-pNA·HCl is not a pharmacologically active compound but rather a chromogenic substrate for enzyme assays. Its activity is measured by the rate of p-nitroaniline release upon enzymatic cleavage, which produces a yellow color that can be detected at 405 nm. The compound is used to determine the kinetic parameters of aminopeptidases, including Vmax, Km, and kcat. It is also employed in inhibitor screening assays to identify compounds that modulate protease activity. The compound's utility lies in its sensitivity and specificity for certain proteases, making it a standard reagent in enzymology laboratories. It does not exhibit cytotoxic or receptor-binding activities at concentrations used in these assays. |
| ln Vivo |
H-Leu-pNA·HCl is not a pharmacologically active drug and does not have defined in vivo activity as a therapeutic agent. Its primary use is as a research reagent for in vitro enzyme assays. In vivo, the compound would likely be rapidly metabolized or cleared, and its chromogenic properties are not relevant in living systems. The compound is not used in animal studies as a therapeutic intervention, although it may be employed in ex vivo assays to measure enzyme activity in tissue homogenates or biological fluids. Its value is almost exclusively confined to in vitro biochemical applications, where it serves as a sensitive and specific substrate for protease activity measurements.
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| Enzyme Assay |
In vitro enzyme assays using H-Leu-pNA·HCl typically follow a standardized protocol. The substrate is dissolved in a suitable buffer, such as Tris-HCl or phosphate buffer at physiological pH. A biological sample containing the enzyme of interest, such as purified aminopeptidase, cell lysate, or serum, is added to the substrate solution. The reaction mixture is incubated at an appropriate temperature, typically 37°C, and the release of p-nitroaniline is monitored continuously or at fixed time points by measuring absorbance at 405 nm. The initial velocity of the reaction is calculated from the linear portion of the absorbance versus time curve. Kinetic parameters are determined by performing assays at various substrate concentrations. Inhibitors can be evaluated by including them in the reaction mixture and comparing the activity to control samples.
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| Cell Assay |
In vitro cellular assays using H-Leu-pNA·HCl are not commonly performed because the compound is not a biologically active molecule. However, it can be used to measure aminopeptidase activity in cell lysates or culture supernatants. In such experiments, cells are lysed, and the lysate is incubated with the substrate in a suitable buffer. The release of p-nitroaniline is measured spectrophotometrically at 405 nm, and enzyme activity is calculated. This approach can be used to assess changes in aminopeptidase expression or activity in response to cellular treatments, such as drug exposure or genetic manipulation. The assay is simple, rapid, and sensitive, making it suitable for high-throughput screening of enzyme activity in cellular samples.
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| Animal Protocol |
In vivo animal studies using H-Leu-pNA·HCl are not typical, as the compound is a chromogenic substrate rather than a pharmacologically active agent. However, it may be used in ex vivo studies where tissues or biological fluids collected from animals are assayed for aminopeptidase activity. In such experiments, animals may be treated with compounds that modulate protease activity, and tissue homogenates or plasma samples are then incubated with the substrate to measure enzyme activity. These studies help to understand the regulation of proteases in physiological and pathological conditions. The compound is not administered to animals as a therapeutic intervention, and its use is limited to biochemical analysis of biological samples.
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| ADME/Pharmacokinetics |
As a peptide substrate, H-Leu-pNA·HCl is not a drug candidate and therefore lacks specific pharmacokinetic data. If administered in vivo, the compound would likely be rapidly cleared from the circulation by hydrolysis or excretion. The p-nitroaniline moiety could be released by aminopeptidases, and this metabolite may have its own pharmacokinetic properties. However, the compound is not designed for systemic administration, and its use is confined to in vitro assays. For research purposes, the compound is typically stored as a powder at -20°C for long-term stability. Its stability in solution depends on buffer composition and temperature.
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| Toxicity/Toxicokinetics |
The toxicity of H-Leu-pNA·HCl is primarily associated with the p-nitroaniline moiety, which can be released upon hydrolysis. p-Nitroaniline is known to have toxic effects, including methemoglobinemia and potential carcinogenicity. However, at the low concentrations used in enzyme assays, the compound is generally considered safe for laboratory use. Standard laboratory safety precautions, including the use of personal protective equipment and working in a fume hood, are recommended. The compound should be handled with care to avoid inhalation, ingestion, or skin contact. It is not classified as a highly toxic substance, but appropriate safety measures should be followed.
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| References | |
| Additional Infomation |
L-Leucine p-nitroanilide hydrochloride (H-Leu-pNA·HCl, CAS 16010-98-3) is a chromogenic substrate used for measuring aminopeptidase and protease activity. Its chemical formula is C₁₂H₁₈ClN₃O₃ and molecular weight is 287.74. Upon enzymatic cleavage by leucine aminopeptidase or related proteases, the compound releases p-nitroaniline, which produces a yellow color measurable at 405 nm. This property makes it a valuable reagent for enzyme kinetics, inhibitor screening, and diagnostic applications. The compound is intended for research use only and is not for clinical or therapeutic purposes. It is typically stored as a powder at -20°C for long-term stability.
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| Molecular Formula |
C12H18CLN3O3
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| Molecular Weight |
287.7426
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| Exact Mass |
287.103
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| CAS # |
16010-98-3
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| PubChem CID |
16219558
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| Appearance |
Off-white to light yellow solid powder
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| Boiling Point |
477.8ºC at 760 mmHg
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| Melting Point |
279-280℃
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| Flash Point |
242.7ºC
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| LogP |
4.005
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
19
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| Complexity |
293
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| Defined Atom Stereocenter Count |
1
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| SMILES |
Cl[H].O=C([C@]([H])(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])N([H])[H])N([H])C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-]
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| InChi Key |
PFOYXOWPGRWPLS-MERQFXBCSA-N
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| InChi Code |
InChI=1S/C12H17N3O3.ClH/c1-8(2)7-11(13)12(16)14-9-3-5-10(6-4-9)15(17)18;/h3-6,8,11H,7,13H2,1-2H3,(H,14,16);1H/t11-;/m0./s1
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| Chemical Name |
(2S)-2-amino-4-methyl-N-(4-nitrophenyl)pentanamide;hydrochloride
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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 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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4754 mL | 17.3768 mL | 34.7536 mL | |
| 5 mM | 0.6951 mL | 3.4754 mL | 6.9507 mL | |
| 10 mM | 0.3475 mL | 1.7377 mL | 3.4754 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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