L,L-Dityrosine does not possess traditional pharmacological targets; rather, it is a stable compound generated by oxidative stress and serves as a biomarker of oxidative protein damage. It forms covalent cross-links between tyrosine residues in proteins primarily through enzymatic or free radical-mediated reactions. Enzymes that mediate its formation include myeloperoxidase (MPO) and CYP56A1. In the context of protein aggregation, dityrosine cross-links can alter the structural stability of target proteins. For example, in Parkinson’s disease, dityrosine cross-links are involved in the aggregation process of α-synuclein, impacting Lewy body formation.

The fluorescence of the rare amino acid LL-dityrosine, which is found in insoluble biological materials with structural features, was recently shown to decay non-exponentially (Kungl et al. (1992) J. Fluorescence 2, 63-74). Here we investigated the time-resolved fluorescence of a dityrosine-containing peptide (DCP) to study the influence of side chains on the fluorescence decay of the chromophore. The fluorescence decay of DCP was best fitted by three exponential terms including a sub-nanosecond rise term, the values of which are quite similar to the parameters obtained for the decay of free dityrosine. They were found to depend on the pH of the aqueous solution but not on the temperature. Analysis by an exponential series method revealed broad fluorescence lifetime distributions for DCP. Compared to the corresponding analysis of dityrosine transients, similar lifetime centers were found whereas the widths of the distributions were found broader for DCP. Molecular dyamics (MD) simulations of dityrosine at 300 K show that chi 1 and chi 2 side chain conformers (rotamers) of both tyrosine subunits interconvert on a picosecond timescale. The rates of interconversion were shown to depend critically upon the MD technique applied: in vacuo simulations yielded lower interconversion rates compared to stochastic dynamics (SD) and full MD (water explicitly included). However, MD simulations of the dityrosine-containing peptide revealed no interconversions of the chi 1 and chi 2 side chain rotamers of both tyrosine subunits within a 400 ps trajectory. Interconversions could be induced by raising the temperature of the system (DCP plus solvent) to 340 K. Side chain rotamers of dityrosine are not stable on a fluorescence time scale but are stable when a dityrosine-containing peptide is regarded. Nevertheless both molecules yield similar fluorescence decay patterns. We therefore conclude that the rotamer model proposed for the fluorescence decay of tyrosine and tryptophan cannot be applied to the fluorescence decay of dityrosine and peptides containing this chromophore. This should be of future interest when dityrosine is used as an intrinsic sensor to study complex dityrosine-containing macromolecules by fluorescence spectroscopy [1].

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In vitro studies demonstrate that L,L-dityrosine is a stable end product formed when tyrosyl radicals and reactive nitrogen species attack tyrosine residues. In fluorescence studies of dityrosine-containing peptides, the fluorescence decay is best fitted by three exponential terms, with values similar to those obtained for free dityrosine decay, and is dependent on the pH of the aqueous solution. Furthermore, in vitro experiments confirm that tyrosyl radicals, generated by the heme protein myeloperoxidase, selectively increase L,L-dityrosine levels; in contrast, hydroxyl radical treatment of proteins primarily increases ortho-tyrosine levels. These findings indicate that distinct reactive species produce characteristic patterns of tyrosine modification.

Mice treated with MPTP showed an increase in L,L-dityrosine hydrochloride (o,o'-dityrosine hydrochloride) in the ventral midbrain and striatum [2].

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In vivo studies show that L,L-dityrosine levels are elevated in various disease models, primarily serving as a biomarker of oxidative stress. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson‘s disease, L,L-dityrosine levels are markedly increased in the striatum and ventral midbrain, but not in brain regions resistant to MPTP. This indicates that tyrosyl radicals and reactive nitrogen species are involved in MPTP neurotoxicity, while hydroxyl radicals play a lesser role in this model. In patients with interstitial lung disease, plasma dityrosine levels are also significantly elevated and correlate with disease severity. Furthermore, L,L-dityrosine has been shown to inhibit the growth of atherosclerotic lesions in experimental models.

Sample Preparation: Subject biological samples containing target proteins to acid hydrolysis (e.g., 6 M HCl, 110°C, 24 hours) to release protein-bound L,L-dityrosine. Extraction and Purification: Remove impurities from the hydrolysate by solid-phase extraction or centrifugal filtration, dry, and reconstitute in mobile phase (e.g., 0.1% formic acid in water). Chromatographic Separation: Perform separation using reverse-phase high-performance liquid chromatography (RP-HPLC) or hydrophilic interaction chromatography (HILIC), typically using a C18 column. Mass Spectrometry Detection: Quantify using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ion mode, monitoring characteristic ion transitions (e.g., m/z 361 → m/z 165). Fluorescence Detection: L,L-Dityrosine possesses natural fluorescence and can be detected using a fluorescence detector at excitation ~320 nm and emission ~410 nm. Data Analysis: Calculate L,L-dityrosine concentrations in samples using standard curves, normalizing to protein content or creatinine levels.

Cell Culture: Culture target cells (e.g., SH-SY5Y neuronal cells, alveolar type II epithelial cells) in medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. Oxidative Stress Induction: Treat cells with hydrogen peroxide (100-500 µM), MPP⁺ (1-5 mM), or peroxynitrite donors to induce oxidative stress and tyrosine nitration/dimerization. Protein Extraction and Hydrolysis: Collect cell pellets and lyse with RIPA buffer containing protease inhibitors. After desalting, subject the lysate to acid hydrolysis to release bound L,L-dityrosine. Quantitative Detection: Measure L,L-dityrosine levels in the hydrolysate using LC-MS/MS or fluorescence methods. Immunodetection: Use anti-dityrosine specific antibodies to detect protein-bound L,L-dityrosine by immunofluorescence or Western blot. Data Analysis: Calculate the ratio of L,L-dityrosine levels between treatment and control groups to assess the extent of oxidative damage.

Oxidative stress is implicated in the death of dopaminergic neurons in Parkinson's disease and in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) model of Parkinson's disease. Oxidative species that might mediate this damage include hydroxyl radical, tyrosyl radical, or reactive nitrogen species such as peroxynitrite. In mice, we showed that MPTP markedly increased levels of o, o'-dityrosine and 3-nitrotyrosine in the striatum and midbrain but not in brain regions resistant to MPTP. These two stable compounds indicate that tyrosyl radical and reactive nitrogen species have attacked tyrosine residues. In contrast, MPTP failed to alter levels of ortho-tyrosine in any brain region we studied. This marker accumulates when hydroxyl radical oxidizes protein-bound phenylalanine residues. We also showed that treating whole-brain proteins with hydroxyl radical markedly increased levels of ortho-tyrosine in vitro. Under identical conditions, tyrosyl radical, produced by the heme protein myeloperoxidase, selectively increased levels of o,o'-dityrosine, whereas peroxynitrite increased levels of 3-nitrotyrosine and, to a lesser extent, of ortho-tyrosine. These in vivo and in vitro findings implicate reactive nitrogen species and tyrosyl radical in MPTP neurotoxicity but argue against a deleterious role for hydroxyl radical in this model. They also show that reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) represent an early and previously unidentified biochemical event in MPTP-induced brain injury. This finding may be significant for understanding the pathogenesis of Parkinson's disease and developing neuroprotective therapies [2].

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Animals and Models: Use C57BL/6 mice or Sprague-Dawley rats. Common models include the MPTP-induced Parkinson‘s disease model (mice, 20-30 mg/kg/day, intraperitoneal, for 5 consecutive days). Tissue Collection: Euthanize animals after the treatment period, quickly dissect target brain regions (striatum, ventral midbrain), lungs, or other relevant tissues, freeze in liquid nitrogen, and store at -80°C. Sample Processing: Weigh and homogenize tissues, perform acid hydrolysis, extract L,L-dityrosine, and quantitate by LC-MS/MS. Data Analysis: Compare L,L-dityrosine levels between model and control groups to assess the extent of oxidative stress.

Based on computational prediction data, L,L-dityrosine shows low oral bioavailability (predicted ~35.75%) and low human intestinal absorption (predicted 28.02%). Its predicted Caco-2 cell permeability is low (-1.438 cm/s), indicating limited intestinal absorption. Blood-brain barrier penetration is predicted to be low (logBB = -1.241). Plasma protein binding is predicted to be 29.03%, indicating weak binding capacity. The predicted steady-state volume of distribution is moderate (0.051 log L/kg). L,L-Dityrosine is not a P-glycoprotein substrate but may be an inhibitor of OATP1B1 and OATP1B3 (predicted probability >94%). This compound is primarily predicted to undergo phase II metabolism via UGT catalysis.

According to the Safety Data Sheet (SDS), L,L-dityrosine is not classified as a hazardous substance or mixture, and GHS label elements do not apply. This compound is not listed as a carcinogen by NTP, IARC, OSHA, or ACGIH. The toxicological effects of this product have not been thoroughly studied. In biological systems, L,L-dityrosine itself serves as a biomarker of oxidative stress, with elevated levels reflecting the presence of tissue damage. However, in Parkinson‘s disease research, dityrosine cross-links have been identified in α-synuclein aggregates and may be involved in the pathogenesis of neurodegenerative processes. Standard laboratory safety practices should be followed when handling. This product is intended for research use only and is not for human or veterinary use.

[1]. Molecular dynamics simulation of the rare amino acid LL-dityrosine and a dityrosine-containing peptide: comparison with time-resolved fluorescence. Biochim Biophys Acta. 1994 Dec 15;1201(3):345-52.
[2]. Mass spectrometric quantification of 3-nitrotyrosine, ortho-tyrosine, and o,o'-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease. J Biol Chem. 1999 Dec 3;274(49):34621-8.

C18H20N2O6

360.361205101013

217.050570

221308-01-6

L,L-Dityrosine;63442-81-9

Light yellow to light brown solid

OC1C=CC(=CC=1C1=C(C=CC(=C1)C[C@@H](C(=O)O)N)O)C[C@@H](C(=O)O)N

JJWFIVDAMOFNPS-QRPNPIFTSA-N

InChI=1S/C9H11NO3.ClH/c10-8(9(12)13)5-6-1-3-7(11)4-2-6;/h1-4,8,11H,5,10H2,(H,12,13);1H/t8-;/m0./s1

(2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid;hydrochloride

o,o'-Dityrosine hydrochloride; L,L-Dityrosine HCl; L,L-Dityrosine (hydrochloride); 221308-01-6; orb1706151

2934.99.9001

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, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~125 mg/mL (~288.50 mM)
DMSO : ~62.5 mg/mL (~144.25 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)