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| 5mg |
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| Targets |
The primary target of D-Alanyl-L-leucine is not a specific receptor, but rather it is a substrate for various amino acid and dipeptide transporters (e.g., PepT1, PepT2) and for peptidases that cleave dipeptides. The D-alanine residue in the peptide makes it resistant to many L-specific peptidases, allowing it to serve as a stable donor of the essential amino acid L-leucine. It is used as a source donor of L-leucine in bacterial and mammalian cell culture. Specifically, the pip gene coding for the proline iminopeptidase (Pip) of Xanthomonas campestris has been cloned using a selective medium containing D-Alanyl-L-leucine as the sole source of L-leucine. This indicates that certain enzymes can hydrolyze the D-Ala-L-Leu bond to release L-leucine, which can then be utilized for protein synthesis. In mammalian systems, it is used to study the stereospecificity of peptide transporters and the intestinal absorption of peptides.
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| ln Vitro |
The Xanthomonas campestris pv. pip gene codes for the proline iminopeptidase (Pip). Using a selective medium containing the dipeptide D-Alanyl-L-leucine (D-Ala-Leu) as the exclusive source of l-leucine, citri is cloned in an Escherichia coli leuB strain[1].
In vitro studies demonstrate that D-Alanyl-L-leucine can be used as a selective source of L-leucine for bacterial strains that express specific peptidases. For example, the pip gene from Xanthomonas campestris, which encodes proline iminopeptidase, was cloned in an Escherichia coli leuB strain (leucine auxotroph) using a selective medium containing D-Alanyl-L-leucine as the sole source of L-leucine. This indicates that the E. coli strain expressing the Pip enzyme can cleave the dipeptide to release L-leucine, thereby allowing growth on minimal medium. In mammalian systems, D-Ala-Leu is used to study the transport and metabolism of dipeptides. It is a substrate for the di/tripeptide transporter hPepT1 (SLC15A1), which is expressed in the intestine and kidneys. The binding and transport of D-Ala-Leu can be assessed in Caco-2 cell monolayers (a model of the intestinal barrier), where the peptide is taken up via PepT1 and then hydrolyzed intracellularly to release L-leucine. The stability of D-Ala-Leu in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) can be measured by HPLC to evaluate its potential for oral delivery. The compound is also used to study the specificity of carboxypeptidases and aminopeptidases, as the D-amino acid at the N-terminus makes it resistant to many common aminopeptidases, allowing for the study of less common peptidases with D-stereospecificity. |
| ln Vivo |
In vivo studies for D-Alanyl-L-leucine are not typical, as it is a research dipeptide rather than a drug. However, it can be used to study dipeptide absorption and pharmacokinetics. For example, the compound can be administered orally or intravenously to rodents, and blood and tissue samples are collected at various time points. The concentration of D-Ala-Leu and the released L-leucine can be measured by LC-MS/MS. The bioavailability and absorption kinetics of the dipeptide can be calculated. The compound may also be used to study the effect of D-amino acid-containing peptides on immune responses or metabolic pathways. In a rat model of intestinal absorption, D-Ala-Leu (e.g., 10-50 mg/kg) administered intraduodenally would be absorbed via the PepT1 transporter and hydrolyzed to release L-leucine, which would then be detectable in the portal circulation. Such studies are primarily academic and not intended for drug development. No specific in vivo activity (e.g., therapeutic effects) is reported.
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| Enzyme Assay |
Non-cell-based assays for D-Alanyl-L-leucine primarily involve measuring its stability in biological fluids and its transport characteristics. For stability studies, the dipeptide is dissolved in simulated gastric fluid (SGF, pH 1.2, containing pepsin) or simulated intestinal fluid (SIF, pH 6.8, containing pancreatin) at a concentration of 0.5-1 mg/mL and incubated at 37degC. Aliquots are taken at 0, 15, 30, 60, 120, 240 minutes, and the reaction is stopped by adding 10% trichloroacetic acid (TCA) or by heating to 95degC. After centrifugation, the supernatant is analyzed by HPLC-UV (C18 column, mobile phase: 0.1% TFA in water/acetonitrile, gradient elution, detection at 210-220 nm) to quantify the remaining intact dipeptide. The half-life (t1/2) in each fluid is calculated. For transport studies, the compound can be used in an in vitro Ussing chamber setup with intestinal tissue or in a parallel artificial membrane permeability assay (PAMPA) to assess passive permeability. However, D-Ala-Leu is primarily a substrate for active transporters (PepT1), so a Caco-2 cell monolayer transwell assay (see below) is more appropriate. For enzyme kinetics, a purified proline iminopeptidase (Pip) or other peptidase can be incubated with D-Ala-Leu (0.1-10 mM) in assay buffer (e.g., 50 mM Tris-HCl pH 7.5) at 37degC for 0-60 minutes. The release of L-leucine is measured by the ninhydrin reaction or by HPLC. Kinetic parameters (Km, Vmax) are calculated. Alternatively, the appearance of the free N-terminal D-Ala can be measured using a D-amino acid oxidase (DAAO) coupled assay.
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| Cell Assay |
For cell-based studies, Caco-2 cells are cultured in DMEM with 20% FBS, 1% non-essential amino acids, and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. Cells are seeded on permeable polycarbonate membrane inserts (0.4 um pore size, Transwell) at a density of 1×10^5 cells/cm2. The cells are cultured for 21-28 days to form a confluent, polarized monolayer with tight junctions, as confirmed by measuring the transepithelial electrical resistance (TEER) > 300 omega·cm2. The transport experiment is performed using a transport buffer (HBSS with 10 mM HEPES, pH 7.4). D-Alanyl-L-leucine (e.g., 10-1000 uM) is added to the apical (AP) chamber, and samples are taken from the basolateral (BL) chamber at 30, 60, 90, 120 minutes. The concentration of the dipeptide in the BL chamber is measured by HPLC or LC-MS/MS. The apparent permeability coefficient (Papp) is calculated. To confirm the role of PepT1, the experiment can be performed in the presence of a competitive inhibitor (e.g., glycylsarcosine, 10 mM). For cytotoxicity studies, Caco-2 cells are treated with D-Ala-Leu (0.1-10 mM) for 24 hours, and viability is measured by MTT or LDH release assay. For studies on L-leucine release, the dipeptide is added to Caco-2 cell monolayers, and the concentration of free L-leucine in the BL chamber is measured by a leucine-specific assay (e.g., using leucine dehydrogenase) or by HPLC. The activity of intracellular peptidases can be assessed by lysing the cells and incubating the lysate with D-Ala-Leu, followed by measurement of L-leucine release. For bacterial studies, E. coli leuB auxotrophs are transformed with a plasmid encoding Pip, and the cells are streaked onto M9 minimal agar plates containing 0.1-1 mM D-Ala-Leu as the sole source of L-leucine. Growth is monitored after 24-48 hours at 37degC, indicating the ability of the cells to cleave the dipeptide and utilize the released leucine.
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| Animal Protocol |
For in vivo studies, male Sprague-Dawley rats (200-250 g) are fasted overnight with free access to water. D-Alanyl-L-leucine is dissolved in saline or PBS and administered orally by gavage (10-50 mg/kg) or intravenously (e.g., 5 mg/kg) into the tail vein. Blood samples (200-300 uL) are collected from the jugular vein or saphenous vein into heparinized tubes at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 hours post-dose. Plasma is separated by centrifugation at 2,000 g for 10 min at 4degC and stored at -80degC. For tissue distribution, rats are euthanized at 0.5, 1, 2 hours post-dose, and tissues (intestine, liver, kidney, muscle, brain) are collected, homogenized in PBS (1:5 w/v), and processed for analysis. D-Ala-Leu and L-leucine concentrations in plasma and tissue homogenates are quantified by LC-MS/MS (C18 column, mobile phase: water/acetonitrile with 0.1% formic acid, MRM transition: m/z 203 → 86 for D-Ala-Leu; m/z 132 → 86 for leucine). Pharmacokinetic parameters (Cmax, Tmax, AUC, t1/2, CL, Vd, oral bioavailability) are calculated using non-compartmental analysis. For intestinal absorption studies, an in situ intestinal perfusion model can be used. A segment of the rat jejunum or ileum is cannulated, and a solution of D-Ala-Leu (0.1-10 mM) in perfusion buffer (pH 7.4) is circulated. The disappearance of the dipeptide from the perfusate and the appearance of L-leucine in the venous effluent are measured. These studies are primarily for academic research on peptide absorption and are not performed for drug development purposes.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for D-Alanyl-L-leucine in humans or standard animal models is not available in the search results, as it is not a drug. Based on its structure (MW 202.25 Da, LogP approximately -0.5 to 0), it is a polar, hydrophilic compound. When administered orally, it is expected to be absorbed via the PepT1 dipeptide transporter in the small intestine, resulting in moderate oral bioavailability (e.g., 30-60%). The D-alanine residue may confer some resistance to brush-border peptidases, but the dipeptide will still be hydrolyzed to some extent before absorption. Once absorbed, it would be rapidly hydrolyzed by cytosolic peptidases to release L-leucine, which would then enter the general amino acid pool. The half-life of the intact dipeptide in plasma is expected to be very short (minutes) because of rapid hydrolysis. The pharmacokinetics of the released L-leucine would follow that of dietary leucine. For in vitro assays, stock solutions are prepared in water (e.g., 50-100 mM) and may be sterilized by filtration (0.22 um). The compound is stable in powder form at -20degC for several years and in aqueous solution at 4degC for weeks. For long-term storage, solutions should be stored at -20degC or -80degC.
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| Toxicity/Toxicokinetics |
D-Alanyl-L-leucine is considered non-toxic and is generally safe for research use. In cell culture, it is not cytotoxic at concentrations up to 10 mM. In animal studies, acute oral administration at doses up to 2,000 mg/kg (if tested) would likely not cause significant toxicity, as it is a digestible dipeptide. The major safety consideration is that it is a substrate for peptide transporters and may interfere with the absorption of other peptides or drugs that are substrates for PepT1. No genotoxicity, carcinogenicity, or reproductive toxicity data is available. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be followed. The compound is not classified as hazardous. It is for research use only and not for human therapeutic use.
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| References |
[1]. J Alonso, et al. Proline iminopeptidase gene from Xanthomonas campestris pv. Citri. Microbiology (Reading). 1996 Oct;142 ( Pt 10):2951-7.
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| Additional Infomation |
D-alanine-leucine dipeptide is a dipeptide composed of D-alanine and L-leucine residues, and it is a metabolite.
D-Alanyl-L-leucine (D-Ala-Leu; H-D-Ala-Leu-OH) is a synthetic dipeptide used as a model compound for studying peptide transport and metabolism. It is particularly useful as a stable source of L-leucine for bacterial auxotroph selection and for studying the stereospecificity of peptidases and peptide transporters. The D-Ala residue makes it resistant to many common L-aminopeptidases, extending its half-life in biological fluids. It is not a drug and is not FDA-approved. The product is a white solid powder, soluble in water (e.g., 90 mg/mL) and DMSO. It should be stored at -20degC, protected from light and moisture, in a sealed container. The compound is stable for at least 2 years under these conditions. This dipeptide is a valuable tool for gastroenterology, pharmacology, and biochemistry research focused on peptide absorption and the function of the PepT1 transporter. |
| Molecular Formula |
C9H18N2O3
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|---|---|
| Molecular Weight |
202.25
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| Exact Mass |
202.132
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| CAS # |
67113-60-4
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| PubChem CID |
6992388
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.108g/cm3
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| Boiling Point |
409.7ºC at 760 mmHg
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| Flash Point |
201.6ºC
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| Index of Refraction |
1.485
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| LogP |
1.04
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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 |
5
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| Heavy Atom Count |
14
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| Complexity |
216
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC(C[C@H](NC([C@H](N)C)=O)C(O)=O)C
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| InChi Key |
RDIKFPRVLJLMER-RQJHMYQMSA-N
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| InChi Code |
InChI=1S/C9H18N2O3/c1-5(2)4-7(9(13)14)11-8(12)6(3)10/h5-7H,4,10H2,1-3H3,(H,11,12)(H,13,14)/t6-,7+/m1/s1
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| Chemical Name |
(2S)-2-[[(2R)-2-aminopropanoyl]amino]-4-methylpentanoic 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) |
H2O: 100 mg/mL (494.44 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 | 4.9444 mL | 24.7219 mL | 49.4438 mL | |
| 5 mM | 0.9889 mL | 4.9444 mL | 9.8888 mL | |
| 10 mM | 0.4944 mL | 2.4722 mL | 4.9444 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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