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 studies focus on its role as a marker of protein oxidation by reactive species (tyrosyl radical) and its photophysical properties. [1][2]

- Molecular dynamics (MD) simulations at 300K show that the χ1 and χ2 side chain conformers (rotamers) of both tyrosine subunits in dityrosine interconvert on a picosecond timescale. The rates of interconversion depend on the simulation technique: in vacuo simulations yield lower interconversion rates (6 transitions in χ1 and 5 in χ2 over 400 ps) compared to stochastic dynamics (SD) and full MD with explicit water (22 transitions in χ1 and 18 in χ2 over 400 ps). [1]
- MD simulations of a dityrosine-containing peptide (DCP) show no transitions in the χ1 and χ2 side chain rotamers of both tyrosine subunits at 300K within a 400 ps simulation period, indicating the chromophore becomes more rigid when incorporated into a peptide. Interconversions could be induced by raising the temperature to 340K (16 transitions in χ1 and χ2 dihedrals over 400 ps). [1]
- In vitro, when whole-brain proteins are exposed to tyrosyl radical generated by the myeloperoxidase-H2O2-tyrosine system, o,o'-dityrosine is the major product. The yield of o,o'-dityrosine in brain proteins oxidized in vitro by myeloperoxidase-generated tyrosyl radical was significantly increased compared to control. For example, in the ventral midbrain, levels increased from a control of ~0.18 mmol/mol tyrosine to ~0.38 mmol/mol tyrosine. No change in 3-nitrotyrosine or ortho-tyrosine levels was observed under these conditions. [2]
- Exposure of brain proteins to hydroxyl radical (generated by copper plus H2O2) also resulted in the accumulation of o,o'-dityrosine, though the increase was ~10-fold lower than that of ortho-tyrosine. [2]
- Exposure of brain proteins to peroxynitrite (ONOO-) resulted in a small (2-3 fold) increase in o,o'-dityrosine levels, which was <5% of the increase in 3-nitrotyrosine. [2]

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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].

- In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease, administration of MPTP markedly increased levels of o,o'-dityrosine in brain regions susceptible to MPTP neurotoxicity. Specifically, o,o'-dityrosine levels increased by 120% in the ventral midbrain (from ~0.17 to ~0.38 mmol/mol tyrosine) and by 170% in the striatum (from ~0.20 to ~0.55 mmol/mol tyrosine) of MPTP-treated mice compared to controls. No significant increase was observed in the frontal cortex or cerebellum, which are resistant to MPTP. This increase suggests a role for tyrosyl radical in MPTP-induced neurotoxicity. [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.
Mice treated with MPTP have increased levels of L,L-dityrosine (o,o'-dityrosine) in the ventral mid brain striatum [2].

- The study did not perform enzyme activity assays directly on dityrosine. However, the generation of tyrosyl radical to form dityrosine was studied using the myeloperoxidase enzyme system. In vitro oxidation reactions (1 mg brain protein/ml) were performed at 37°C in a buffer (50 mM sodium phosphate, pH 7.4). The reaction mixture for tyrosyl radical generation contained 0.1 mM H2O2, 20 nM myeloperoxidase, 0.2 mM L-tyrosine, and 0.1 mM diethylenetriaminepentaacetic acid (DTPA). The reaction was terminated by addition of 0.2 mM DTPA (pH 7.4), 300 nM catalase, and 0.1 mM butylated hydroxytoluene (BHT). Proteins were then precipitated, hydrolyzed, and analyzed by GC/MS. [2]

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.

- The study used male C57/BL mice (8 weeks old, 22-25 g). Animals were housed three per cage in a temperature- and light-controlled room with a 12-h/12-h light-dark cycle, with water and food provided ad libitum. On the day of the experiment, mice received four intraperitoneal injections of MPTP-HCl (20 mg/kg) in saline every 2 hours over an 8-hour period. Control mice received saline only. The dose volume and injection number were based on a standard MPTP regimen to model Parkinson's disease. [2]
- Animals were anesthetized and sacrificed 24 hours after the last injection. To minimize ex vivo oxidation, mice were perfused with ice-cold antioxidant buffer (100 µM diethylenetriaminepentaacetic acid (DTPA), 1 mM butylated hydroxytoluene (BHT), 10 mM 3-amino-1,2,4-triazole, 50 mM sodium phosphate, pH 7.4). Following perfusion, the cerebellum, ventral midbrain, striatum, and cerebral cortex were dissected out on an ice-cold plate, frozen on dry ice, and stored at -80°C until analysis. [2]

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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].

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.

- Dityrosine is a constituent of acid hydrolysates of several biological materials, including insect cuticular resilin, the fertilization membrane of the sea urchin egg, and the outermost layer of the ascospore wall of the yeast Saccharomyces cerevisiae. Its biological function is thus suggested to be cross-linking protein chains like disulfide bridges. [1]
- The fluorescence of dityrosine decays non-exponentially, interpreted not by the rotamer model (as for tyrosine/tryptophan) but by a consecutive reaction in the excited state. A primarily excited state (precursor) is transformed into a successor excited state with a rate corresponding to τ1^-1 (τ1 ≈ 200 ps). The deactivation rates of the precursor and successor are τ2^-1 (τ2 ≈ 1.6 ns) and τ3^-1 (τ3 ≈ 4.7 ns), respectively. [1]
- The authors conclude that the rotamer model proposed for tyrosine and tryptophan fluorescence decay cannot be applied to dityrosine because its side chain rotamers interconvert too rapidly on the fluorescence timescale. Conversely, in a dityrosine-containing peptide where rotamers are stable, the fluorescence decay pattern is similar, confirming the excited-state reaction model. [1]
- In the MPTP mouse model, the increase in o,o'-dityrosine, along with an increase in 3-nitrotyrosine, implicates reactive nitrogen species and tyrosyl radical (and consequently protein oxidation) as an early biochemical event in MPTP-induced brain injury, which may be significant for understanding the pathogenesis of Parkinson's disease. [2]

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LL-dityrosine is a dityrosine. It is the enantiomer of DD-dityrosine.

C18H20N2O6

360.36

360.132

63442-81-9

L,L-Dityrosine hydrochloride;221308-01-6

14554235

White to off-white solid

1.5±0.1 g/cm3

622.9±55.0 °C at 760 mmHg

330.5±31.5 °C

0.0±1.9 mmHg at 25°C

1.676

-0.31

6

8

7

26

458

2

O([H])C1C([H])=C([H])C(=C([H])C=1C1=C(C([H])=C([H])C(=C1[H])C([H])([H])C([H])(C(=O)O[H])N([H])[H])O[H])C([H])([H])C([H])(C(=O)O[H])N([H])[H]

OQALFHMKVSJFRR-KBPBESRZSA-N

InChI=1S/C18H20N2O6/c19-13(17(23)24)7-9-1-3-15(21)11(5-9)12-6-10(2-4-16(12)22)8-14(20)18(25)26/h1-6,13-14,21-22H,7-8,19-20H2,(H,23,24)(H,25,26)/t13-,14-/m0/s1

(2S)-2-amino-3-[3-[5-[(2S)-2-amino-2-carboxyethyl]-2-hydroxyphenyl]-4-hydroxyphenyl]propanoic acid

o,o'-Dityrosinel; L,L-dityrosine; 63442-81-9; 3,3'-dityrosine; 3,3'-Bityrosine; CJ9XG8HS20;

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~5.6 mg/mL (~15.5 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.)