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59-92-7 Usage

Description

Levodopa is an amino acid precursor of dopamine with antiparkinsonian properties. It is a prodrug that is converted to dopamine by DOPA decarboxylase and can cross the blood-brain barrier. When in the brain, levodopa is decarboxylated to dopamine and stimulates the dopaminergic receptors, thereby compensating for the depleted supply of endogenous dopamine seen in Parkinson's disease. To ensure that adequate concentrations of levodopa reach the central nervous system, it is administered with carbidopa, a decarboxylase inhibitor that does not cross the blood-brain barrier, thereby diminishing the decarboxylation and inactivation of levodopa in peripheral tissues and increasing the delivery of dopamine to the CNS.

Uses

Used in Pharmaceutical Industry:
Levodopa is used as an antiparkinsonian agent for the treatment of Parkinson's disease. It is most often administered in combination with peripheral decarboxylase inhibitors such as benserazide and carbidopa to increase the delivery of dopamine to the central nervous system and reduce the decarboxylation and inactivation of levodopa in peripheral tissues. Levodopa serves as an immediate precursor of dopamine, helping to compensate for the depleted supply of endogenous dopamine in Parkinson's disease.

Originator

Larodopa,Roche,US,1970

Manufacturing Process

Levodopa can be prepared from 1-3-dinitrotyrosine, 3-(3,4-methylenedioxyphenyl)-l-alanine, and l-tyrosine, and by fermentation of l-tyrosine.A charge of 1,000 g of ground velvet beans was extracted with 9 liters of 1% aqueous acetic acid at room temperature over a 20-hour period with occasional stirring during the first 4 hours. The liquor was decanted and thebean pulp slurry was vacuum filtered through a cake of acid-washed diatomaceous earth in a Buechner funnel. The decanted liquor was combined with the filtrate and concentrated under vacuum and a nitrogen atmosphere to a volume of 900 ml. After treating with acid-washed activated carbon, the concentrate was then filtered through acid-washed diatomaceous earth.After concentrating the filtrate to approximately 400 ml, solids started crystallizing out at which time the filtrate was cooled by refrigerating at 5°C for several hours. Filtration gave 18.7 g of L-Dopa, MP 284° to 286°C (dec.); [α]D 8.81° (1% solution in aqueous 4% HCl). The infrared spectrum and paper chromatography indicated very good L-Dopa according to US Patent 3,253,023.Various synthetic routes are also described by Kleeman and Engel.

Therapeutic Function

Antiparkinsonian

Biological Functions

Levodopa (L-DOPA), the most reliable and effective drug used in the treatment of parkinsonism, can be considered a form of replacement therapy. Levodopa is the biochemical precursor of dopamine. It is used to elevate dopamine levels in the neostriatum of parkinsonian patients. Dopamine itself does not cross the blood-brain barrier and therefore has no CNS effects. However, levodopa, as an amino acid, is transported into the brain by amino acid transport systems, where it is converted to dopamine by the enzyme L-aromatic amino acid decarboxylase. If levodopa is administered alone, it is extensively metabolized by L-aromatic amino acid decarboxylase in the liver, kidney, and gastrointestinal tract. To prevent this peripheral metabolism, levodopa is coadministered with carbidopa (Sinemet), a peripheral decarboxylase inhibitor. The combination of levodopa with carbidopa lowers the necessary dose of levodopa and reduces peripheral side effects associated with its administration. Levodopa is widely used for treatment of all types of parkinsonism except those associated with antipsychotic drug therapy. However, as parkinsonism progresses, the duration of benefit from each dose of levodopa may shorten (wearing-off effect). Patients can also develop sudden, unpredictable fluctuations between mobility and immobility (on-off effect). In a matter of minutes, a patient enjoying normal or nearly normal mobility may suddenly develop a severe degree of parkinsonism. These symptoms are likely due to the progression of the disease and the loss of striatal dopamine nerve terminals.

Biological Activity

Immediate precursor of dopamine, produced by tyrosine hydroxylase. Displays antiParkinsonian activity.

Biochem/physiol Actions

3,4-Dihydroxy-L-phenylalanine or L-DOPA is a natural isomer of the immediate precursor of dopamine that crosses the blood-brain barrier. It is used for the treatment of Parkinson′s disease and is a product of tyrosine hydroxylase.

Pharmacology

In a number of attempts to fix the deficit of dopamine in Parkinsonism, the introduction of a direct precursor of dopamine—levodopa—into the patient is considered a very logical therapy since levodopa diffuses across the blood–brain barrier, where it turns into dopamine and normalizes the level of dopamine. In this manner, levodopa stops or slows the development of Parkinsonism. Levodopa belongs to a group of the most effective drugs for treating the type of Parkinsonism not caused by medicinal agents.

Safety Profile

Poison by ingestion. Moderately toxic by intravenous and intraperitoneal routes. Human systemic effects by ingestion: somnolence, hallucinations and distorted perceptions, toxic psychosis, motor activity changes, ataxia, dyspnea. Experimental teratogenic and reproductive effects. Questionable human carcinogen producing skin tumors. Human mutation data reported. An anticholinergic agent used as an anti Parhnsonian drug. When heated to decomposition it emits toxic fumes of NOx

Synthesis

Levodopa, (-)-3-(3,4-dihydroxyphenyl)-L-alanine (10.1.1), is a levorotatory isomer of dioxyphenylalanine used as a precursor of dopamine. There are a few ways of obtaining levodopa using a semisynthetic approach, which consists of the microbiological hydroxylation of L-tyrosine (10.1.1), as well as implementing a purely synthetic approach. Oxidation of L-tyrosine, for selective introduction of a hydroxyl group at C3 of the tyrosine ring, can be accomplished in a purely synthetic manner by using a mixture of hydrogen peroxide and iron(II) sulfate mixture in water as an oxidant with permanent presence of oxygen. The third method of levodopa synthesis consists of the acetylation of tyrosine using acetylchloride in the presence of aluminum chloride and the subsequent oxidative deacylation of the formed 3-acetyltyrosine (10.1.2) using hydrogen peroxide in sodium hydroxide solution.

Purification Methods

Likely impurities are vanillin, hippuric acid, 3-methoxytyrosine and 3-aminotyrosine. DOPA recrystallises from large volumes of H2O forming colourless white needles; its solubility in H2O is 0.165%, but it is insoluble in EtOH, *C6H6, CHCl3, and EtOAc. Also crystallise it by dissolving it in dilute HCl and adding dilute ammonia to give pH 5, under N2. Alternatively, crystallise it from dilute aqueous EtOH. It is rapidly oxidised in air when moist, and darkens, particularly in alkaline solution. Dry it in vacuo at 70o in the dark, and store it in a dark container preferably under N2. It has at 220.5nm (log 3.79) and 280nm (log 3.42) in 0.001N max HCl. [Yamada et al. Chem Pharm Bull Jpn 10 693 1962, Bretschneider et al. Helv Chim Acta 56 2857 1973, NMR: Jardetzky & Jardetzky J Biol Chem 233 383 1958, Beilstein 4 IV 2492, 2493.]

Check Digit Verification of cas no

The CAS Registry Mumber 59-92-7 includes 5 digits separated into 3 groups by hyphens. The first part of the number,starting from the left, has 2 digits, 5 and 9 respectively; the second part has 2 digits, 9 and 2 respectively.
Calculate Digit Verification of CAS Registry Number 59-92:
(4*5)+(3*9)+(2*9)+(1*2)=67
67 % 10 = 7
So 59-92-7 is a valid CAS Registry Number.
InChI:InChI=1/C9H11NO4/c10-6(9(13)14)3-5-1-2-7(11)8(12)4-5/h1-2,4,6,11-12H,3,10H2,(H,13,14)/t6-/m0/s1

59-92-7 Well-known Company Product Price

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  • TCI America

  • (D0600)  3-(3,4-Dihydroxyphenyl)-L-alanine  >98.0%(HPLC)(T)

  • 59-92-7

  • 5g

  • 370.00CNY

  • Detail
  • TCI America

  • (D0600)  3-(3,4-Dihydroxyphenyl)-L-alanine  >98.0%(HPLC)(T)

  • 59-92-7

  • 25g

  • 890.00CNY

  • Detail
  • TCI America

  • (D0600)  3-(3,4-Dihydroxyphenyl)-L-alanine  >98.0%(HPLC)(T)

  • 59-92-7

  • 100g

  • 2,690.00CNY

  • Detail
  • Alfa Aesar

  • (A11311)  3,4-Dihydroxy-L-phenylalanine, 98+%   

  • 59-92-7

  • 5g

  • 336.0CNY

  • Detail
  • Alfa Aesar

  • (A11311)  3,4-Dihydroxy-L-phenylalanine, 98+%   

  • 59-92-7

  • 25g

  • 1272.0CNY

  • Detail
  • Alfa Aesar

  • (A11311)  3,4-Dihydroxy-L-phenylalanine, 98+%   

  • 59-92-7

  • 100g

  • 4378.0CNY

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  • Sigma-Aldrich

  • (PHR1271)  Levodopa  pharmaceutical secondary standard; traceability to USP, PhEur and BP

  • 59-92-7

  • PHR1271-500MG

  • 804.73CNY

  • Detail
  • Sigma-Aldrich

  • (L0400000)  Levodopa  European Pharmacopoeia (EP) Reference Standard

  • 59-92-7

  • L0400000

  • 1,880.19CNY

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  • USP

  • (1361009)  Levodopa  United States Pharmacopeia (USP) Reference Standard

  • 59-92-7

  • 1361009-200MG

  • 4,662.45CNY

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  • Sigma

  • (D9628)  3,4-Dihydroxy-L-phenylalanine  ≥98% (TLC)

  • 59-92-7

  • D9628-5G

  • 486.72CNY

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  • Sigma

  • (D9628)  3,4-Dihydroxy-L-phenylalanine  ≥98% (TLC)

  • 59-92-7

  • D9628-25G

  • 1,684.80CNY

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  • Sigma

  • (D9628)  3,4-Dihydroxy-L-phenylalanine  ≥98% (TLC)

  • 59-92-7

  • D9628-100G

  • 5,631.21CNY

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59-92-7SDS

SAFETY DATA SHEETS

According to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) - Sixth revised edition

Version: 1.0

Creation Date: Aug 12, 2017

Revision Date: Aug 12, 2017

1.Identification

1.1 GHS Product identifier

Product name L-dopa

1.2 Other means of identification

Product number -
Other names l-dop

1.3 Recommended use of the chemical and restrictions on use

Identified uses For industry use only.
Uses advised against no data available

1.4 Supplier's details

1.5 Emergency phone number

Emergency phone number -
Service hours Monday to Friday, 9am-5pm (Standard time zone: UTC/GMT +8 hours).

More Details:59-92-7 SDS

59-92-7Synthetic route

(S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propanoic acid
7423-93-0

(S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propanoic acid

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
Stage #1: (S)-2-amino-3-(3-chloro-4-hydroxyphenyl)propanoic acid With sodium nitrate; 2-Methylcyclohexanol; N-bromoacetamide at 56℃; for 2.16667h;
Stage #2: With nickel dichloride at 25℃; for 4h; Temperature;
98.7%
S,S-m-methoxy-p-acetoxy-N-acetylphenylalanine α-phenylethylamide
68706-13-8

S,S-m-methoxy-p-acetoxy-N-acetylphenylalanine α-phenylethylamide

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With hydrogen bromide96%
C17H19NO5

C17H19NO5

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With 10 wt% Pd(OH)2 on carbon; hydrogen In methanol at 45℃; for 7h; Reagent/catalyst; Temperature; Solvent; Green chemistry;87%
(S)-4'-(3,4-dihydroxybenzyl)-9λ4-boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaborolidin]-5'-one

(S)-4'-(3,4-dihydroxybenzyl)-9λ4-boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaborolidin]-5'-one

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With tetrabutyl ammonium fluoride In tetrahydrofuran; water at 20℃; for 2h;86%
piruvate
57-60-3

piruvate

benzene-1,2-diol
120-80-9

benzene-1,2-diol

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With ethylenediaminetetraacetic acid; pyridoxal 5'-phosphate; Ammonium; recombinant wild-type thermophilic tyrosine phenol lyases from thermophilic microorganism Symbiobacterium toebii In aq. phosphate buffer at 37℃; pH=8; Catalytic behavior; Green chemistry; Enzymatic reaction; stereoselective reaction;65%
With ammonium acetate at 45℃; β-tyrosinase from Symbiobacterium thermophilum, 0.4 mM pyridoxal phosphate, 8 mM EDTA, 0.1 percent dithiothreitol;90 % Turnov.
(S)-2-Amino-3-(3,4-diacetoxy-phenyl)-propionic acid ethyl ester
330455-62-4

(S)-2-Amino-3-(3,4-diacetoxy-phenyl)-propionic acid ethyl ester

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With hydrogenchloride In acetone at 90℃; for 20h;60%
L-tyrosine
60-18-4

L-tyrosine

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With phosphate buffer at 37℃; Irradiation; also with m-tyrosine;
With dihydroxyfumaric acid; acetate buffer; oxygen; horse radish peroxidase at 0℃; for 3h; Product distribution; other aromatic compounds, different peroxidases;
With air; mushroom monophenol monooxygenase In water at 23℃; for 0.15h; Rate constant; pH 6.6;
dopa
63-84-3

dopa

A

levodopa
59-92-7

levodopa

B

D-Dopa
5796-17-8

D-Dopa

L-phenylalanine
63-91-2

L-phenylalanine

A

levodopa
59-92-7

levodopa

B

o-hydroxyl-phenylalanine
7423-92-9

o-hydroxyl-phenylalanine

D

L-tyrosine
60-18-4

L-tyrosine

Conditions
ConditionsYield
With oxygen; Fe(2+)-EDTA In water at 40℃; for 4h; electrochemical cell, pH 3;
With oxygen; Fe(2+)-EDTA In water at 40℃; for 4h; Product distribution; electrochem. oxidation; pH: 3;
With dihydrogen peroxide; ascorbic acid In water at 37℃; Product distribution; hydroxylation with various reaction systems;
With Tris-HCl buffer; 6,7-dimethyl-5,6,7,8-tetrahydropterin; 2-hydroxyethanethiol; catalase; phenylalanine hydroxylase at 37℃; for 0.5h; Product distribution; further enzyme;
L-tyrosine
60-18-4

L-tyrosine

A

1H-indole-5,6-diol
3131-52-0

1H-indole-5,6-diol

B

levodopa
59-92-7

levodopa

C

leucodopachromene
18766-67-1

leucodopachromene

D

(2S)-dopachrome
3571-34-4, 89762-39-0

(2S)-dopachrome

Conditions
ConditionsYield
With water; mushroom tyrosinase Rate constant; phosphat buffer, ph 6.8;
L-tyrosine
60-18-4

L-tyrosine

A

levodopa
59-92-7

levodopa

B

L-tyrosine radical
16978-66-8

L-tyrosine radical

C

2,4-dihydroxy-L-phenylalanine
89462-15-7

2,4-dihydroxy-L-phenylalanine

Conditions
ConditionsYield
With water; dinitrogen monoxide Rate constant; Irradiation;

A

levodopa
59-92-7

levodopa

B

leucodopachromene
18766-67-1

leucodopachromene

C

L-2-carboxy-2,3-dihydroindole-5,6-quinone

L-2-carboxy-2,3-dihydroindole-5,6-quinone

Conditions
ConditionsYield
With pH 8.6 In water Rate constant; pH-dependence, inhib. by cysteine;
N-acetyl-3,4-dihydroxyphenylalanine ethylester
19641-91-9

N-acetyl-3,4-dihydroxyphenylalanine ethylester

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With sodium hydroxide at 65℃; for 3h;
L-dopasemiquinone

L-dopasemiquinone

A

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
In water Rate constant;
L-5-(3,4-dihydroxybenzyl)hydantoin

L-5-(3,4-dihydroxybenzyl)hydantoin

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With Flavobacterium sp. AJ-3940 In various solvent(s) at 43℃; for 2h;
With Flavobacterium sp. AJ-3940; hydroxylamine In various solvent(s) at 43℃; for 2h; Product distribution; pH 7.0, other reagent;
L-DOPA methyl ester
7101-51-1

L-DOPA methyl ester

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With water; sodium chloride In dimethyl sulfoxide at 37℃; Rate constant; also human plasma as reagent;
With sodium carbonate In acetonitrile for 15h;
With potassium hydroxide In methanol
3,4-dihydroxy-L-phenylalanine benzyl ester
55720-47-3

3,4-dihydroxy-L-phenylalanine benzyl ester

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With water; sodium chloride In dimethyl sulfoxide at 37℃; Rate constant; also human plasma as reagent;
L-DOPA ethyl ester
37178-37-3

L-DOPA ethyl ester

levodopa
59-92-7

levodopa

Conditions
ConditionsYield
With water; sodium chloride In dimethyl sulfoxide at 37℃; Rate constant; also human plasma as reagent;

59-92-7Relevant articles and documents

New L-dopa codrugs as potential antiparkinson agents

Sozio, Piera,Iannitelli, Antonio,Cerasa, Laura Serafina,Cacciatore, Ivana,Cornacchia, Catia,Giorgioni, Gianfabio,Ricciutelli, Massimo,Nasuti, Cinzia,Cantalamessa, Franco,Di Stefano, Antonio

, p. 412 - 417 (2008)

This paper reports the synthesis and preliminary evaluation of new L-dopa (LD) conjugates (1 and 2) obtained by joining LD with two different natural antioxidants, caffeic acid and carnosine, respectively. The antioxidant efficacy of compounds 1 and 2 was

Immobilization of polyphenol oxidase onto mesoporous activated carbons - isotherm and kinetic studies

John Kennedy,Selvi,Aruna Padmanabhan,Hema,Sekaran

, p. 262 - 270 (2007)

Investigations were carried out in batch modes for studying the immobilization behavior of polyphenol oxidase (PPO) on two different mesoporous activated carbon matrices, MAC400 and MAC200. The PPO was immobilized onto MAC400 and MAC200 at various enzyme activities 5 × 104, 10 × 104, 20 × 104, 30 × 104 U l-1, at pH 5-8, and at temperature ranging from 10 to 40 °C. The intensity of immobilization of PPO increased with increase in temperature and initial activities, while it decreased with increase in pH. Immobilization onto MAC400 followed the Langmuir model while Langmuir and Freundlich models could fit MAC200 data. Non-linear pseudo first order, pseudo second order and intraparticle diffusion models were evaluated to understand the mechanism of immobilization. The free and immobilized enzyme kinetic parameters (Km and Vmax) were determined by Michaelis-Menten enzyme kinetics. The Km values for free enzyme, PPO immobilized in MAC400 and in MAC200 were 0.49, 0.41 and 0.65 mM, respectively. The immobilization of PPO in carbon matrices was confirmed using FT-IR spectroscopy and scanning electron microscopy.

Reductase-catalyzed tetrahydrobiopterin regeneration alleviates the anti-competitive inhibition of tyrosine hydroxylation by 7,8-dihydrobiopterin

Ding, Zhongyang,Li, Leyun,Li, Youran,Shi, Guiyang,Xu, Yinbiao,Zhang, Liang

, p. 3128 - 3140 (2021)

l-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical and pharmaceutical chemicals. Two major challenges in constructing these pathways in bacteria are the improvement of hydroxylase catalytic efficiency and the production of cofactor tetrahydrobiopterin (BH4). In this study, we analyzed the evolutionary relationships and conserved protein sequences between tyrosine hydroxylases from different species by PhyML and MAFFT. Finally, we selected 7 tyrosine hydroxylases and 6 sepiapterin reductases. Subsequently, the function of different groups was identified by a combined whole-cell catalyst, and a series of novel tyrosine hydroxylase/sepiapterin reductase (TH/SPR) synthesis systems were screened including tyrosine hydroxylase (from Streptosporangium roseum DSM 43021 and Thermomonospora curvata DSM 43183) and sepiapterin reductase (from Photobacterium damselae, Chlorobaculum thiosulfatiphilum and Xenorhabdus poinarii), namely as SrTH/PdSPR, SrTH/CtSPR, SrTH/XpSPR and TcTH/PdSPR, which can synthesize l-Dopa by hydroxylating l-tyrosine in Bacillus licheniformis. Furthermore, we analyzed the characterization of SrTH by enzyme catalysis and demonstrated that 7,8-dihydrobiopterin (BH2) formed by BH4 autooxidation was an anticompetitive inhibitor on SrTH. Finally, pure dihydropteridine reductase from Escherichia coli (EcDHPR) was added to the solution, and l-Dopa could be continually synthesized after 3 h, which was improved by 86% at 6 h in the catalytic reaction by SrTH. This indicates that BH4 regeneration can alleviate the inhibition by BH2 during tyrosine hydroxylation. This study provides a good starting point and theoretical foundation for further modification to improve the catalytic efficiency of tyrosine hydroxylation by tyrosine hydroxylase.

Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities

Choi, Hye Won,Hong, Sungguan,Jo, Hyun-Joo,Kong, Kwang-Hoon,Lee, Sung Jun,Noh, Hyangsoon

, p. 726 - 732 (2020)

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase. Seven histidine mutants in the two copper-binding sites were generated, and catalytic activities were characterised. The tyrosine hydroxylase activities of the CuA site mutants were approximately 50% lower than those of the wild-type tyrosinase, while the dopa oxidation activities of the mutants were not significantly different from that of wild-type tyrosinase. By contrast, mutations at CuB significantly decreased both tyrosine hydroxylation and dopa oxidation activities, confirming that the catalytic sites for these two activities are at least partially distinct. These findings provide a useful resource for further structural determination and development of tyrosinase inhibitors in the cosmetic and pharmaceutical industries.

Singlet oxygen-mediated protein oxidation: Evidence for the formation of reactive side chain peroxides on tyrosine residues

Wright, Adam,Bubb, William A.,Hawkins, Clare L.,Davies, Michael J.

, p. 35 - 46 (2002)

Singlet oxygen (1O2) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin can

Pulsed EPR study of amino acid and tetrahydropterin binding in a tyrosine hydroxylase nitric oxide complex: Evidence for substrate rearrangements in the formation of the oxygen-reactive complex

Krzyaniak, Matthew D.,Eser, Bekir E.,Ellis, Holly R.,Fitzpatrick, Paul F.,McCracken, John

, p. 8430 - 8441 (2013)

Tyrosine hydroxylase is a nonheme iron enzyme found in the nervous system that catalyzes the hydroxylation of tyrosine to form l-3,4- dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. Catalysis requires the binding of three substrates: tyrosine, tetrahydrobiopterin, and molecular oxygen. We have used nitric oxide as an O2 surrogate to poise Fe(II) at the catalytic site in an S = 3/2, {FeNO}7 form amenable to EPR spectroscopy. 2H-electron spin echo envelope modulation was then used to measure the distance and orientation of specifically deuterated substrate tyrosine and cofactor 6-methyltetrahydropterin with respect to the magnetic axes of the {FeNO}7 paramagnetic center. Our results show that the addition of tyrosine triggers a conformational change in the enzyme that reduces the distance from the {FeNO}7 center to the closest deuteron on 6,7-2H-6-methyltetrahydropterin from >5.9 A to 4.4 ± 0.2 A. Conversely, the addition of 6-methyltetrahydropterin to enzyme samples treated with 3,5-2H-tyrosine resulted in reorientation of the magnetic axes of the S = 3/2, {FeNO}7 center with respect to the deuterated substrate. Taken together, these results show that the coordination of both substrate and cofactor direct the coordination of NO to Fe(II) at the active site. Parallel studies of a quaternary complex of an uncoupled tyrosine hydroxylase variant, E332A, show no change in the hyperfine coupling to substrate tyrosine and cofactor 6-methyltetrahydropterin. Our results are discussed in the context of previous spectroscopic and X-ray crystallographic studies done on tyrosine hydroxylase and phenylalanine hydroxylase.

High-throughput assay of tyrosine phenol-lyase activity using a cascade of enzymatic reactions

Zhu, Hang-Qin,Hu, Wen-Ye,Tang, Xiao-Ling,Zheng, Ren-Chao,Zheng, Yu-Guo

, (2022/01/19)

Tyrosine phenol-lyase (TPL) exhibits great potential in industrial biosynthesis of L-tyrosine and its derivates. To uncover and screen TPLs with excellent catalytic properties, there is unmet demand for development of facile and reliable screening system for TPL. Here we presented a novel assay format for the detection of TPL activity based on catechol 2,3-dioxygenase (C23O)-catalyzed reaction. Catechol released from TPL-catalyzed cleavage of 3,4-dihydroxy-L-phenylalanine (L-DOPA) was further oxidized by C23O to form 2-hydroxymuconate semialdehyde, which could be readily detected by spectrophotometric measurements at 375 nm. The assay achieved a unique balance between the ease of operation and superiority of analytical performances including linearity, sensitivity and accuracy. In addition, this assay enabled real-time monitoring of TPL activity with high efficiency and reliability. As C23O is highly specific towards catechol, a non-natural product of microorganism, the assay was therefore accessible to both crude cell extracts and the whole-cell system without elaborate purification steps of enzymes, which could greatly expedite discovery and engineering of TPLs. This study provided fundamental principle for high-throughput screening of other enzymes consuming or producing catechol derivatives.

Genetically encoded dihydroxyphenylalanine coupled with tyrosinase for strain promoted labeling

George, Augustine,Indhu, Mohan,Ashokraj, Sundarapandian,Shanmugam, Ganesh,Ganesan, Ponesakki,Kamini, Numbi Ramudu,Ayyadurai, Niraikulam

supporting information, (2021/11/08)

Protein modifications through genetic code engineering have a remarkable impact on macromolecule engineering, protein translocation, protein–protein interaction, and cell biology. We used the newly developed molecular biology approach, genetic code engineering, for fine-tuning of proteins for biological availability. Here, we have introduced 3, 4-dihydroxy-L-phenylalanine in recombinant proteins by selective pressure incorporation method for protein-based cell labeling applications. The congener proteins treated with tyrosinase convert 3, 4-dihydroxy-L-phenylalanine to dopaquinone for strain-promoted click chemistry. Initially, the single-step Strain-Promoted Oxidation-Controlled Cyclooctyne-1,2-quinone Cycloaddition was studied using tyrosinase catalyzed congener protein and optimized the temporally controlled conjugation with (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol. Then, the feasibility of tyrosinase-treated congener annexin A5 with easily reactive quinone functional moiety was conjugated with fluorescent tag dibenzocyclooctyne-PEG4-TAMRA for labeling of apoptotic cells. Thus, the congener proteins-based products demonstrate selective cell labeling and apoptosis detection in EA.hy926 cells even after the protein modifications. Hence, genetic code engineering can be coupled with click chemistry to develop various protein-based fluorescent labels.

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