Product classification

59-92-7

Product Name：Levodopa
Molecular Formula：C₉H₁₁NO₄
Purity：99%
Molecular Weight：

Inquiry

Product Details;

CasNo： 59-92-7

Molecular Formula： C₉H₁₁NO₄

Appearance： colorless crystalline powder

59-92-7 Properties

Molecular Formula:C9H11NO4
Molecular Weight:197.191
Appearance/Colour:colorless crystalline powder
Vapor Pressure:7.97E-09mmHg at 25°C
Melting Point:276-278 °C(lit.)
Refractive Index:-12 ° (C=5, 1mol/L HCl)
Boiling Point:448.4 °C at 760 mmHg
PKA:2.32(at 25℃)
Flash Point:225 °C
PSA：103.78000
Density:1.468 g/cm³
LogP:0.75250

59-92-7 Usage

Description

Levodopa is an amino acid precursor of dopamine with antiparkinsonian properties. Levodopa 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 assure 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.

Chemical Properties

L-Dopa [59-92-7], levodopa, crystallizes as colorless, odorless, and tasteless needles from water, mp 276-278℃(decomp.). It is freely soluble in dilute hydrochloric and formic acids but practically insoluble in ethanol, benzene, chloroform, and ethyl acetate. Solubility in water is 66 mg/40 mL. In the presence of moisture, l-dopa is rapidly oxidized by atmospheric oxygen, with darkening.

Originator

Larodopa,Roche,US,1970

Uses

Levodopa is an immediate precursor of dopamine and product of tyrosine hydroxylase. It derived from vanillin is widely used for treatment of Parkinson’s disease, most often in combination with peripheral decarboxylase inhibitors such as benserazide and carbidopa.

Definition

ChEBI: Levodopa is an optically active form of dopa having L-configuration. Used to treat the stiffness, tremors, spasms, and poor muscle control of Parkinson's disease.

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.

Brand name

Bendopa (Valeant); Dopar (Shire); Larodopa (Roche).

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.

General Description

The first significant breakthrough in the treatment of PDcame about with the introduction of high-dose levodopa.Fahn referred to this as a revolutionary development intreating parkinsonian patients. The rationale for the use oflevodopa for the treatment of PD was established in theearly 1960s. Parkinsonian patients were shown to have decreasedstriatal levels of DA and reduced urinary excretionof DA. Since then, levodopa has shown to be remarkablyeffective for treating the symptoms of PD.Because ofenzymatic action of MAO-A in the gastrointestinal (GI)tract and AADC in the periphery, only a small percentage(1%–2%) of levodopa is delivered into the CNS.Coadministration of levodopa with the AADC inhibitor,carbidopa, prevents decarboxylation of levodopa outside ofthe CNS. The combination of levodopa and carbidopa resultsin a substantial increase in DA delivery to the CNSwith a decrease in peripheral side effects. Long-term therapywith levodopa leads to predictable motor complications.These include loss of efficacy before the next dose(“wearing off”), motor response fluctuations (“on/off”), andunwanted movements (dyskinesias).These effects arethought to be caused by the inability of levodopa therapyto restore normal DA levels in the CNS.As a result, theuse of longer-acting DA agonists may benefit parkinsonianpatients.

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.]

Description

Chemical Properties

Originator

Larodopa,Roche,US,1970

Uses

Definition

ChEBI: Levodopa is an optically active form of dopa having L-configuration. Used to treat the stiffness, tremors, spasms, and poor muscle control of Parkinson's disease.

Manufacturing Process

Brand name

Bendopa (Valeant); Dopar (Shire); Larodopa (Roche).

Therapeutic Function

Antiparkinsonian

Biological Functions

General Description

Biological Activity

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

Biochem/physiol Actions

Pharmacology

Safety Profile

Synthesis

Purification Methods

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 Relevant articles

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

Detection of tyrosine and monitoring tyrosinase activity using an enzyme cascade-triggered colorimetric reaction

Chen, Huei-Yu,Yeh, Yi-Chun

, p. 29745 - 29750 (2020)

The aromatic amino acid tyrosine is an essential precursor for the synthesis of catecholamines, including l-DOPA, tyramine, and dopamine. A number of metabolic disorders have been linked to abnormal tyrosine levels in biological fluids. In this study, we developed an enzyme cascade-triggered colorimetric reaction for the detection of tyrosine, based on the formation of yellow pigment (betalamic acid) and red fluorometric betaxanthin. Tyrosinase converts tyrosine to l-DOPA, and DOPA-dioxygenase catalyzes oxidative cleavage of l-DOPA into betalamic acid. Response is linear for tyrosine from 5 to 100 μM, and the detection limit (LOD) is 2.74 μM. The enzyme cascade reaction was applied to monitor tyrosinase activity and tyrosinase inhibition assays. Lastly, the performance of the proposed biosensor proved successful in the analysis of urine samples without the need for pre-treatment. This journal is

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.

Measurement of intrinsic rate constants in the tyrosine hydroxylase reaction

Eser, Bekir E.,Fitzpatrick, Paul F.

, p. 645 - 652 (2010)

Tyrosine hydroxylase (TyrH) is a pterin-dependent mononuclear non-heme aromatic amino acid hydroxylase that catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Chemical quench analyses of the enzymatic reaction show a burst of DOPA formation, followed by a linear rate equal to the kcat value at both 5 and 30 °C. The effects of increasing solvent viscosity confirm that kcat is ～84% limited by diffusion, most probably due to slow product release, and that tyrosine has a commitment to catalysis of 0.45. The effect of viscosity on the kcat/Km for 6-methyltetrahydropterin is greater than the theoretical limit, consistent with the coupling of pterin binding to the movement of a surface loop. The absorbance changes in the spectrum of the tetrahydropterin during the first turnover, the kinetics of DOPA formation during the first turnover, and the previously described kinetics for formation and decay of the Fe(IV)O intermediate [Eser, B. E., Barr, E. W., Frantom, P. A., Saleh, L., Bollinger, J. M., Jr., Krebs, C., and Fitzpatrick, P. F. (2007) J. Am. Chem. Soc. 129, 11334-11335] were analyzed globally, yielding a single set of rate constants for the TyrH reaction. Reversible binding of oxygen is followed by formation of Fe(IV)O and 4a-hydroxypterin with a rate constant of 13 s-1 at 5 °C. Transfer of oxygen from Fe(IV)O to tyrosine to form DOPA follows with a rate constant of 22 s-1. Release of DOPA and/or the 4a-hydroxypterin with a rate constant of 0.86 s-1 completes the turnover.

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.

Novel strategy for enhancing productivity in l-DOPA synthesis: The electroenzymatic approach using well-dispersed l-tyrosine

Min, Kyoungseon,Kathavarayan, Thenmozhi,Park, Kyungmoon,Yoo, Young Je

, p. 87 - 90 (2013)

Although l-DOPA (l-3,4-dihydroxyphenylalanine) is widely used as a drug for Parkinson's disease, there are critical drawbacks in the commercial synthetic method such as low conversion rate, poor productivity, and long operational time. In order to overcome these limitations, a novel electroenzymatic system using tyrosinase/carbon nanopowder/polypyrrole composite as a working cathode was reported with the outstanding conversion rate up to 95.9%. However, the productivity was still limited due to a low solubility of the substrate l-tyrosine in aqueous phase. Herein, we demonstrated a novel strategy for enhancing the productivity by employing well-dispersed l-tyrosine as the substrate. When using well-dispersed l-tyrosine, not only the concentration of the substrate was increased to 90.6 gL-1 in aqueous phase but also the productivity was enhanced up to 15.3 gL-1 h-1. We also determined kinetic parameters in the electroenzymatic system and the kinetic results revealed that the outstanding conversion rate was based on the fast electrical reduction of the by-product to l-DOPA. Thus the electroenzymatic synthesis using well-dispersed l-tyrosine can be a potential candidate as a novel process for l-DOPA synthesis.

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.

Daedalin A, a metabolite of daedalea dickinsii, inhibits melanin synthesis in an in vitro human skin model

Morimura, Keiji,Hiramatsu, Kenji,Yamazaki, Chihiro,Hattori, Yasunao,Makabe, Hidefumi,Hirota, Mitsuru

, p. 627 - 632 (2009)

The culture broth of Daedalea dickinsii was found to predominantly contain the tyrosinase inhibitor, (2R)-6- hydroxy-2-hydroxymethyl-2-methyl-2H-chromene, daedalin A (1). Ongoing research into bioactive metabolites resulted in the identification of two new 2H-chromenes, 6-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H- chromene (3) and 6-hydroxy-2-hydroxymethyl-5-methoxy-2-methyl- 2H-chromene (4), together with 6-hydroxy-2,2-dimethyl- 2H-chromene (2). Comparative studies of isolated compounds 1-4 and related compounds (±)-1 and 1a-1c showed 1 to have the strongest tyrosinase inhibitory activity. These results suggest that the hydroxyl groups at positions 6 and 9 of 1 were important for the potent activity. A Lineweaver-Burk plot for a kinetic analysis indicates that 1 competed with L-tyrosine for tyrosinase. Compound 1 also suppressed melanogenesis in a human skin model (up to 49% at 2.8μmol/tissue application) without affecting the cell viability. Compounds 1, 1b and 1c also showed 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity comparable to that of α-tocopherol.

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.

Self-assembled adhesive biomaterials formed by a genetically designed fusion protein

Aich, Pulakesh,An, Jaeyeon,Yang, Byeongseon,Ko, Young Ho,Kim, Junghyun,Murray, James,Cha, Hyung Joon,Roh, Joon Ho,Park, Kyeng Min,Kim, Kimoon

, p. 12642 - 12645 (2018)

Here we report a recombinant protein (MS) obtained by genetic fusion of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The MS not only self-assembled into a supramolecular fibre, as does the parent MaSp1, but also showed enhanced adhesiveness resulting from the DOPA-containing Mfp3 portion. The successful incorporation of the wet adhesiveness of Mfp3 into the well-structured assembly of MaSp1 may provide a new insight for the genetic design of underwater adhesive recombinant proteins by utilizing the structural features of a spidroin protein.

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.

Highly Stable Zr(IV)-Based Metal-Organic Frameworks for Chiral Separation in Reversed-Phase Liquid Chromatography

Jiang, Hong,Yang, Kuiwei,Zhao, Xiangxiang,Zhang, Wenqiang,Liu, Yan,Jiang, Jianwen,Cui, Yong

supporting information, p. 390 - 398 (2021/01/13)

Separation of racemic mixtures is of great importance and interest in chemistry and pharmacology. Porous materials including metal-organic frameworks (MOFs) have been widely explored as chiral stationary phases (CSPs) in chiral resolution. However, it remains a challenge to develop new CSPs for reversed-phase high-performance liquid chromatography (RP-HPLC), which is the most popular chromatographic mode and accounts for over 90% of all separations. Here we demonstrated for the first time that highly stable Zr-based MOFs can be efficient CSPs for RP-HPLC. By elaborately designing and synthesizing three tetracarboxylate ligands of enantiopure 1,1′-biphenyl-20-crown-6, we prepared three chiral porous Zr(IV)-MOFs with the framework formula [Zr6O4(OH)8(H2O)4(L)2]. They share the same flu topological structure but channels of different sizes and display excellent tolerance to water, acid, and base. Chiral crown ether moieties are periodically aligned within the framework channels, allowing for stereoselective recognition of guest molecules via supramolecular interactions. Under acidic aqueous eluent conditions, the Zr-MOF-packed HPLC columns provide high resolution, selectivity, and durability for the separation of a variety of model racemates, including unprotected and protected amino acids and N-containing drugs, which are comparable to or even superior to several commercial chiral columns for HPLC separation. DFT calculations suggest that the Zr-MOF provides a confined microenvironment for chiral crown ethers that dictates the separation selectivity.

A novel catalytic heme cofactor in SfmD with a single thioether bond and abis-His ligand set revealed by ade novocrystal structural and spectroscopic study

Shin, Inchul,Davis, Ian,Nieves-Merced, Karinel,Wang, Yifan,McHardy, Stanton,Liu, Aimin

, p. 3984 - 3998 (2021/04/06)

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A. Here, we present a 1.78 ? resolutionde novocrystal structure of SfmD, which unveils a novel heme cofactor attached to the protein with an unusualHxnHxxxCmotif (n～ 38). This heme cofactor is unique in two respects. It contains a single thioether bond in a cysteine-vinyl link with Cys317, and the ferric heme has two axial protein ligands,i.e., His274 and His313. We demonstrated that SfmD heme is catalytically active and can utilize dioxygen and ascorbate for a single-oxygen insertion into 3-methyl-l-tyrosine. Catalytic assays using ascorbate derivatives revealed the functional groups of ascorbate essential to its function as a cosubstrate. Abolishing the thioether linkage through mutation of Cys317 resulted in catalytically inactive SfmD variants. EPR and optical data revealed that the heme center undergoes a substantial conformational change with one axial histidine ligand dissociating from the iron ion in response to substrate 3-methyl-l-tyrosine binding or chemical reduction by a reducing agent, such as the cosubstrate ascorbate. The labile axial ligand was identified as His274 through redox-linked structural determinations. Together, identifying an unusual heme cofactor with a previously unknown heme-binding motif for a monooxygenase activity and the structural similarity of SfmD to the members of the heme-based tryptophan dioxygenase superfamily will broaden understanding of heme chemistry.

59-92-7 Process route

: 63-84-3,90638-38-3
dopa

: 59-92-7,90638-38-3
levodopa

: 5796-17-8,90638-38-3
D-Dopa

Conditions

Conditions	Yield

With teicoplanin; In methanol; water; Further Variations:; Reagents; pH-values; Solvents; Product distribution;
With chiral stationary phase including isopropyl-functionalized CF₆; In methanol; acetic acid; triethylamine; acetonitrile; at 0 ℃; Purification / work up;
With Merck RP-18 WF₂₅₄S plates coated with N^τ-n-decyl-L-spinacine and Cu acetate; In methanol; water; Resolution of racemate;
With (S)-2-hydroxy-2'-(3-(N-phenylcarbamoylamino)benzyl)-1,1'-binaphthyl-3-carboxaldehyde; In dimethyl sulfoxide; stereoselective reaction; Resolution of racemate;	89.2 % ee
With (R)-2-hydroxy-2'-(3-phenylurylbenzyl)-1,1'-binaphthyl-3-carboxaldehyde; In dimethyl sulfoxide; stereoselective reaction; Resolution of racemate;	89 % ee
With ammonium formate; In methanol; at 20 ℃; pH=3.6; Reagent/catalyst; Resolution of racemate;
With perchloric acid; at 25 ℃; pH=1; Resolution of racemate;

: 59-92-7,90638-38-3
levodopa

: 5796-17-8,90638-38-3
D-Dopa