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| Targets |
L-Asparagine-13C4,15N2,d8 targets the same pathways as unlabeled L-Asparagine. L-Asparagine is an essential amino acid for leukemic cells (especially acute lymphoblastic leukemia, ALL) because they lack asparagine synthetase (ASNS) and depend on exogenous asparagine for growth. This forms the basis for the use of L-Asparaginase in ALL therapy, which depletes plasma asparagine, selectively killing leukemic cells. L-Asparagine is also a substrate for L-Asparaginase, the enzyme used in chemotherapy, and is involved in protein synthesis, nitrogen transport, and the metabolic control of nerve and brain tissue cell functions. As a tracer, the multiple stable isotopes allow precise tracking of asparagine metabolism.
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| ln Vitro |
Drug compounds have included stable heavy isotopes of carbon, hydrogen, and other elements, mostly as quantitative tracers while the drugs were being developed. Because deuteration may have an effect on a drug's pharmacokinetics and metabolic properties, it is a cause for concern [1].
As a stable isotope tracer, L-Asparagine-13C4,15N2,d8 is not used to measure biological activity in the traditional sense. Instead, it is added to cell culture media to trace asparagine metabolism, protein synthesis, and the activity of asparagine synthetase (ASNS). Leukemic cells (e.g., Jurkat, NALM-6) are cultured in medium containing the labeled asparagine (10-100 uM) for 6-48 hours. The incorporation of 13C, 15N, and 2H into cellular proteins, other amino acids (aspartate, glutamate), and metabolites is analyzed by LC-MS or NMR. L-Asparagine (unlabeled) is known to be a critical nutrient for leukemic cell proliferation, and its depletion by L-Asparaginase induces apoptosis. The labeled version allows the study of ASNS upregulation as a mechanism of resistance to L-Asparaginase therapy. |
| ln Vivo |
In vivo, L-Asparagine-13C4,15N2,d8 is administered to animals (e.g., mice) to trace asparagine metabolism, protein synthesis, and the in vivo activity of L-Asparaginase therapy. The compound is used in pharmacokinetic and pharmacodynamic studies of L-Asparaginase (a chemotherapeutic agent) to measure asparagine depletion and repletion kinetics in plasma and tissues. In xenograft models of ALL, mice bearing human leukemic tumors are administered L-Asparaginase (e.g., 1,000 IU/kg, IP), and L-Asparagine-13C4,15N2,d8 is used as an internal standard for LC-MS quantification of endogenous asparagine levels. The labeled version can also be infused intravenously to measure the rate of asparagine turnover and its incorporation into newly synthesized proteins. Unlabeled L-Asparagine is critical for leukemic cell survival; depletion is the therapeutic mechanism of L-Asparaginase.
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| Enzyme Assay |
For non-cellular assays (analytical quantification), L-Asparagine-13C4,15N2,d8 is prepared as a stock solution in water or 0.1 M HCl (1 mg/mL). For LC-MS/MS analysis, a calibration curve for L-Asparagine is prepared in human plasma (0.1-1000 ng/mL) with a fixed concentration of the triple-labeled internal standard (e.g., 50 ng/mL). Sample preparation: 100 uL plasma + 20 uL internal standard + 380 uL acetonitrile for protein precipitation. After centrifugation, the supernatant is diluted with water (1:1) and injected onto a C18 column or a HILIC column with a mobile phase of 0.1% formic acid in water and acetonitrile (gradient elution). MRM transitions: L-Asparagine 133→87 (loss of H2O) and 133→74; L-Asparagine-13C4,15N2,d8 146→100 and 146→81. For L-Asparaginase activity assays, the enzyme is incubated with unlabeled L-asparagine in buffer, and the product (aspartic acid) is quantified by LC-MS/MS using the labeled internal standard.
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| Cell Assay |
For cell-based assays, leukemic cells (e.g., Jurkat, NALM-6, Reh, or primary ALL cells) are seeded in 6-well plates (1×10⁶ cells/well) in RPMI-1640 with 10% FBS. For metabolic labeling, cells are cultured in medium containing L-Asparagine-13C4,15N2,d8 (10-100 uM) for 6-48 hours. Cell lysates are prepared in 80% methanol. 13C, 15N, and 2H enrichment in asparagine, aspartate, glutamate, and other metabolites is analyzed by LC-MS/MS. For L-Asparaginase resistance studies, cells are treated with L-Asparaginase (0.0001-10 IU/mL) for 24-72 hours, and ASNS (asparagine synthetase) mRNA expression is quantified by qPCR. Asparagine levels in culture medium and cell lysates are quantified by LC-MS using the labeled internal standard. Cell viability is assessed by MTT or trypan blue exclusion.
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| Animal Protocol |
For in vivo animal experiments, xenograft mouse models of acute lymphoblastic leukemia (ALL) are used. NOD/SCID or NSG mice are injected intravenously with ALL cell lines (e.g., Jurkat, NALM-6) or patient-derived xenografts (PDX). When leukemia is established, mice are treated with L-Asparaginase (500-2,000 IU/kg, IP) for 7-14 days. L-Asparagine-13C4,15N2,d8 is used as an internal standard to quantify L-Asparagine levels in plasma and tissues by LC-MS. For kinetic studies, L-Asparagine-13C4,15N2,d8 can be administered intravenously (10-50 mg/kg), and blood samples are collected at multiple time points to measure the disappearance of labeled asparagine and its conversion to aspartate. For protein synthesis studies, labeled asparagine is infused over several hours to achieve steady-state labeling, and the rate of protein synthesis is measured by MS analysis of hydrolyzed tissue proteins.
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| ADME/Pharmacokinetics |
L-Asparagine-13C4,15N2,d8 has a molecular weight of 146.12, with four carbon-13 atoms (mass shift +4), two nitrogen-15 atoms (mass shift +2), and eight deuterium atoms (mass shift +8) for a total mass shift of +14 Da relative to unlabeled L-Asparagine (MW 132.12). The compound is a white to off-white crystalline powder, soluble in water and dilute acids. It should be stored as a powder at -20degC for up to 3 years, and in solution at -80degC for up to 6 months or at -20degC for up to 1 month. The multiple stable isotope labels provide excellent resolution in mass spectrometry, enabling highly sensitive and specific quantification. The compound is non-radioactive and metabolically identical to unlabeled L-Asparagine.
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| Toxicity/Toxicokinetics |
L-Asparagine-13C4,15N2,d8 is a stable isotope-labeled compound with minimal toxicity at analytical concentrations (ng-ug per sample). The non-deuterated parent compound, L-Asparagine, is an endogenous, non-essential amino acid generally recognized as safe (GRAS). At typical tracer doses (mg per sample or mg per kg body weight in animals), it poses no toxicity risk. High doses of asparagine are generally well-tolerated. The compound is non-radioactive and safe for research use with standard handling precautions for amino acids. No specific toxicity studies have been reported for the labeled version.
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| References | |
| Additional Infomation |
L-Asparagine-13C4,15N2,d8 is a research compound used as a stable isotope tracer and internal standard, not an approved drug. It is not intended for therapeutic use and has not undergone clinical trials as a drug. Its primary applications are in cancer research, specifically studying the role of L-Asparagine metabolism in acute lymphoblastic leukemia (ALL) and other asparagine-dependent cancers. The compound is used to investigate mechanisms of resistance to L-Asparaginase chemotherapy (e.g., upregulation of asparagine synthetase, ASNS), to quantify asparagine depletion in patients treated with L-Asparaginase, and to study asparagine metabolism in the central nervous system. It is also used as an internal standard for quantitative LC-MS analysis of L-Asparagine in clinical and research settings. Available for research use only.
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| Molecular Formula |
13C4D815N2O3
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|---|---|
| Molecular Weight |
146.12
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| Exact Mass |
146.111
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| CAS # |
1217464-18-0
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| Related CAS # |
L-Asparagine;70-47-3
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| PubChem CID |
16217723
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| Appearance |
White to off-white solid powder
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| Melting Point |
232 °C (dec.) (lit.)
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| LogP |
-3.4
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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 |
3
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| Heavy Atom Count |
9
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| Complexity |
134
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| Defined Atom Stereocenter Count |
1
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| SMILES |
[2H][15N]([2H])[13C]([13C]([2H])([2H])[13C@]([2H])([15N]([2H])[2H])[13C](=O)O[2H])=O
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| InChi Key |
DCXYFEDJOCDNAF-PYHFAUOHSA-N
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| InChi Code |
InChI=1S/C4H8N2O3/c5-2(4(8)9)1-3(6)7/h2H,1,5H2,(H2,6,7)(H,8,9)/t2-/m0/s1/i1+1D2,2+1D,3+1,4+1,5+1,6+1/hD5
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| Chemical Name |
deuterio (2S)-2,3,3-trideuterio-2,4-bis(dideuterio(15N)amino)-4-oxo(1,2,3,4-13C4)butanoate
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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) |
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 | 6.8437 mL | 34.2185 mL | 68.4369 mL | |
| 5 mM | 1.3687 mL | 6.8437 mL | 13.6874 mL | |
| 10 mM | 0.6844 mL | 3.4218 mL | 6.8437 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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