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| Targets |
3-Nitro-L-tyrosine (non-deuterated) is a post-translational modification of tyrosine residues in proteins resulting from nitrative stress. It serves as a marker of protein damage by reactive nitrogen species but does not have a specific drug target. Nitration of tyrosine residues can alter protein function, leading to enzyme inactivation, altered signal transduction, and accumulation of damaged proteins. 3-Nitro-L-tyrosine has been shown to act as an oxidant and cytotoxic agent. The deuterated version (3-Nitro-L-tyrosine-d3) shares the same chemical properties but is used as an internal standard for quantification, not for activity studies.
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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].
3-Nitro-L-tyrosine-d3 is not used for in vitro activity studies; it serves exclusively as an analytical internal standard. The non-deuterated parent compound, 3-nitro-L-tyrosine, is used in cell culture models to study the effects of protein nitration on cellular function. Treatment of neuronal cells (e.g., SH-SY5Y), endothelial cells, or macrophages with 3-nitro-L-tyrosine (10-500 uM) for 24-48 hours induces cytotoxicity, oxidative stress, and apoptosis. It is also used to study the inhibition of tyrosine kinase activity (e.g., inactivation of protein tyrosine phosphatases) and the formation of protein aggregates. The compound can be detected in cell lysates and culture medium by LC-MS using the deuterated standard for quantification. |
| ln Vivo |
In vivo, 3-nitro-L-tyrosine (non-deuterated) is an endogenous biomarker of nitrative stress and is elevated in many inflammatory and degenerative diseases. It can serve as a biomarker for nitrogen-free radical-modified proteins under systemic autoimmunogenic conditions. In animal models of disease (e.g., atherosclerosis, Alzheimer‘s disease, Parkinson's disease, multiple sclerosis), levels of 3-nitro-L-tyrosine in tissues (brain, aorta, spinal cord) and biological fluids (plasma, urine, cerebrospinal fluid) are elevated compared to healthy controls. The deuterated version (3-Nitro-L-tyrosine-d3) is not used for in vivo efficacy studies; it is used as an internal standard to quantify 3-nitro-L-tyrosine levels in these samples.
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| Enzyme Assay |
For non-cellular assays (analytical quantification), 3-Nitro-L-tyrosine-d3 is prepared as a stock solution in 0.1 M HCl or methanol (1 mg/mL). For LC-MS/MS analysis, a calibration curve for 3-nitro-L-tyrosine is prepared in human plasma, urine, or cerebrospinal fluid (0.1-100 ng/mL) with a fixed concentration of 3-nitro-L-tyrosine-d3 (e.g., 5-25 ng/mL). Sample preparation: 100 uL plasma + 20 uL internal standard + 380 uL methanol for protein precipitation. After centrifugation, the supernatant is injected onto a C18 column (2.1×100 mm, 1.8 um) with a mobile phase of 0.1% formic acid in water and methanol (gradient elution). MRM transitions: 3-nitro-L-tyrosine 225→179 (loss of COOH) and 225→136; 3-nitro-L-tyrosine-d3 228→182 and 228→139.
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| Cell Assay |
For cell-based assays, neuronal cells (e.g., SH-SY5Y, PC12 cells), endothelial cells (e.g., HUVEC), or macrophages (e.g., RAW 264.7) are seeded in 6-well plates (1×10⁶ cells/well) in DMEM with 10% FBS. To induce endogenous nitrative stress, cells are treated with inflammatory stimuli such as lipopolysaccharide (LPS, 1 ug/mL) + interferon-gamma (IFN-gamma, 100 U/mL) for 24-48 hours. Alternatively, cells are treated with peroxynitrite donors (e.g., SIN-1, 100-500 uM) for 1-6 hours. Protein lysates are prepared in RIPA buffer, and proteins are digested with trypsin. 3-Nitro-L-tyrosine levels are measured by LC-MS/MS using 3-Nitro-L-tyrosine-d3 as internal standard. Cell viability is assessed by MTT assay, and apoptosis is measured by caspase-3/7 activity. For external treatment studies, cells are treated with synthetic 3-nitro-L-tyrosine (10-500 uM) for 24-48 hours.
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| Animal Protocol |
For in vivo animal experiments, rodent models of inflammatory or neurodegenerative diseases are used. For example, in Alzheimer‘s disease models (e.g., APP/PS1 transgenic mice), brain tissues (cortex, hippocampus) are collected and homogenized. Proteins are precipitated, hydrolyzed with 6 M HCl at 110degC for 24 hours, and 3-nitro-L-tyrosine levels are measured by LC-MS/MS using 3-nitro-L-tyrosine-d3 as internal standard. In models of atherosclerosis (e.g., ApoE-/- mice fed a high-fat diet), aortic tissues and plasma are collected. In multiple sclerosis models (e.g., experimental autoimmune encephalomyelitis, EAE), spinal cord tissues are collected. The deuterated standard is added before extraction to correct for matrix effects. No administration of the deuterated compound is required for these measurements.
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| ADME/Pharmacokinetics |
3-Nitro-L-tyrosine-d3 has a molecular weight of 229.20, with deuterium atoms at positions 2, 3, and 6 of the aromatic ring. The compound is a yellow solid with a melting point >212degC (dec.). It is soluble in DMSO, methanol, and dilute acids or bases (due to the presence of both amino and carboxyl groups). The deuterium labeling provides a mass shift of +3 Da, enabling clear differentiation from endogenous non-deuterated 3-nitro-L-tyrosine. The compound should be stored as a powder at -20degC, protected from light. In solution, it is stable at -80degC for up to 6 months and at -20degC for up to 1 month. Protect from light as nitroaromatic compounds can be light-sensitive.
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| Toxicity/Toxicokinetics |
3-Nitro-L-tyrosine-d3 is a stable isotope-labeled compound with minimal toxicity at analytical concentrations (ng-ug per sample). The non-deuterated parent compound, 3-nitro-L-tyrosine, is an endogenous post-translational modification generated in cells under inflammatory and oxidative stress conditions. At high exogenous concentrations (≥100 uM in cell culture), it induces cytotoxicity and apoptosis. As a biomarker of nitrative stress, elevated levels are associated with disease pathogenesis. However, when used as an internal standard, the amount is negligible and poses no toxicity risk. Standard laboratory safety precautions for handling organic compounds should be followed.
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| References | |
| Additional Infomation |
3-Nitro-L-tyrosine-d3 is an analytical standard and research tool, not an approved drug. No clinical trials have been conducted with the deuterated version for therapeutic purposes. The non-deuterated parent compound, 3-nitro-L-tyrosine, is not a drug but is an important clinical research biomarker for oxidative and nitrative stress in various diseases including atherosclerosis, neurodegenerative diseases (Alzheimer‘s, Parkinson's), diabetes, chronic kidney disease, and autoimmune disorders. The deuterated version is used as an internal standard for quantitative LC-MS/MS analysis of 3-nitro-L-tyrosine in clinical research settings, enabling accurate measurement of this biomarker in biological fluids and tissues. It is also used in studies of protein nitration, reactive nitrogen species biology, and the evaluation of antioxidant therapies. Available for research use only.
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| Molecular Formula |
C9H7D3N2O5
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| Molecular Weight |
229.20
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| Exact Mass |
229.077
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| CAS # |
213386-10-8
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| Related CAS # |
3-Nitro-L-tyrosine;621-44-3
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| PubChem CID |
10846994
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
431.6±45.0 °C at 760 mmHg
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| Melting Point |
>212° C (dec.)
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| Flash Point |
214.8±28.7 °C
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| Vapour Pressure |
0.0±1.1 mmHg at 25°C
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| Index of Refraction |
1.651
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| LogP |
0.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
16
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| Complexity |
278
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| Defined Atom Stereocenter Count |
1
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| SMILES |
[2H]C1=C(C(=C(C(=C1C[C@@H](C(=O)O)N)[2H])[N+](=O)[O-])O)[2H]
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| InChi Key |
FBTSQILOGYXGMD-UOCCHMHCSA-N
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| InChi Code |
InChI=1S/C9H10N2O5/c10-6(9(13)14)3-5-1-2-8(12)7(4-5)11(15)16/h1-2,4,6,12H,3,10H2,(H,13,14)/t6-/m0/s1/i1D,2D,4D
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| Chemical Name |
(2S)-2-amino-3-(2,3,6-trideuterio-4-hydroxy-5-nitrophenyl)propanoic 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 |
| 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 | 4.3630 mL | 21.8150 mL | 43.6300 mL | |
| 5 mM | 0.8726 mL | 4.3630 mL | 8.7260 mL | |
| 10 mM | 0.4363 mL | 2.1815 mL | 4.3630 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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