LC–MS–MS Simultaneous Determination of l-Dopa and Its Prodrug l-Dopa n-Pentyl Ester Hydrochloride in Rat Plasma

A sensitive, simple and rapid LC–MS–MS method has been developed and validated for the simultaneous determination of l-dopa and l-dopa n-pentyl ester hydrochloride in rat plasma in the present study. The analytes were separated on a C₁₈ column (5 μm, 2.1 × 150 mm) with a security guard C₁₈ column (5 μm, 4 × 20 mm) and a triple-quadrupole mass spectrometer was applied for detection. The method was linear over the concentration ranges of 25–5,000 ng mL^?1 for l-dopa and 12.5–2,500 ng mL^?1 for l-dopa n-pentyl ester hydrochloride. Finally, the method was successfully applied to support the pharmacokinetic study.     

A voltammetric sensor based on graphene-modified electrode for the determination of trace amounts of l-dopa in mouse brain extract and pharmaceuticals

l-Dopa is the intermediate precursor of the neurotransmitter dopamine. Unlike dopamine, l-dopa easily enters the central nervous system. l-Dopa, as one of the catecholamines, is widely used as a source of dopamine in the treatment of most patients with Parkinson’s disease and epilepsy. Graphene (GR) is ideally suited for implementation in electrochemical applications due to its reported large electrical conductivity, large surface area, unique heterogeneous electron transfer rate, and low production costs. This work reports the synthesis of GR using a modified Brodie method and its application for the electrochemical determination of l-dopa in real samples. Electrochemical measurements were performed at glassy carbon electrode modified with graphene (GR/GCE) via drop casting method. Cyclic voltammograms of l-dopa at GR/GCE showed an increased current intensity compared with GCE. All the measurements were done in phosphate buffer solution 0.1 M (pH 6.2) and the oxidation peak was observed at 0.27 V vs. Ag/AgCl. The effect of scan rate showed that oxidation of l-dopa on GR/GCE was surface controlled. The oxidation peak current of l-dopa gradually increased with increasing accumulation time from 0 to 300 s and accumulation potential from 0.0 to 0.3 V and reached the maximum current response at 240 s and 0.2 V for the accumulation time and accumulation potential, respectively. Voltammetric peak currents showed a linear response for l-dopa concentration in the range of 0.04 to 79 μM and a detection limit of 0.022 μM (22 nM). The relative standard deviation for five determinations of 50 μM l-dopa was 0.52 %.     

The yjjN of E. coli codes for an l-galactonate dehydrogenase and can be used for quantification of l-galactonate and l-gulonate

Escherichia coli is able to utilize l-galactonate as a sole carbon source. A metabolic pathway for l-galactonate catabolism is described in E. coli, and it is known to be interconnected with d-galacturonate metabolism. The corresponding gene encoding the first enzyme in the l-galactonate pathway, l-galactonate-5-dehydrogenase, was suggested to be yjjN. However, l-galactonate dehydrogenase activity was never demonstrated with the yjjN gene product. Here, we show that YjjN is indeed an l-galactonate dehydrogenase having activity also for l-gulonate. The K _m and k _cat for l-galactonate were 19.5?±?0.6 mM and 0.51?±?0.03 s^?1, respectively. In addition, YjjN was applied for a quantitative detection of the both of these substances in a coupled assay. The detection limits for l-galactonate and l-gulonate were 1.65 and 10 μM, respectively.     

l-Ribose Production from l-Arabinose by Immobilized Recombinant Escherichia coli Co-expressing the l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase Genes from Geobacillus thermodenitrificans

l-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the l-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l l-ribose from 300 g/l l-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co²⁺, with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h^?1 produced an average of 100 g/l l-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for l-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from l-arabinose as the substrate.     

Interactions of some protonated α-amino acid methyl esters with hexaethyl p-tert-butylcalix[6]arene hexaacetate in nitrobenzene saturated with water

The exchange extraction constants corresponding to the general equilibrium C⁺(aq) + 1·Cs⁺(nb) ? 1·C⁺ (nb) + Cs⁺(aq) occurring in the two-phase water–nitrobenzene system (C⁺ = protonated α-amino acid methyl ester, 1 = hexaethyl p-tert-butylcalix[6]arene hexaacetate; aq = aqueous phase, nb = nitrobenzene phase) were evaluated on the basis of extraction experiments and γ-activity measurements. Further, the stability constants of the 1·C⁺ cationic complex species in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: protonated l-tryptophan methyl ester < protonated l-phenylalanine methyl ester < protonated l-leucine methyl ester < protonated l-methionine methyl ester < protonated l-valine methyl ester.     

A Validated Chiral LC Method for the Enantiomeric Separation of Nateglinide and the Quantitative Determination of Its l-Enantiomer

A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R _s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min^?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL^?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.     

Enantioselective extraction of fenvaleric acid enantiomers by two-phase (W/O) recognition chiral extraction

To establish an extraction method for fenvaleric acid (FA) enantiomers using l-iso-butyl-l-tartaric esters and hydroxypropyl-β-cyclodextrin (HP-β-CD) as chiral selector, the distribution of FA enantiomers was examined in methanol aqueous solution containing HP-β-CD and 1,2-dichloroethane organic solution containing l-iso-butyl-l-tartaric esters. The influences of the concentration of l-iso-butyl-l-tartaric esters and HP-β-CD, organic diluent, pH, extraction temperature and the concentration of methanol aqueous solution on the partition coefficient (k) and separation factor (α) of FA were investigated. The experiment results showed that the complex formed by l-iso-butyl-l-tartaric esters with S-enantiomer is stabler than that with R-enantiomer. With the increase of the concentration of l-iso-butyl-l-tartaric ester, k and α increased; With the increase of the concentration of HP-β-CD, k increased firstly, and then decreased, but α increased all the while, k was the highest when the concentration of HP-β-CD was 4 mmol L^?1. 1,2-dichloroethane organic diluent was better than the others. With the increase of pH, k and α decreased; with further increasing concentration of methanol aqueous solution, k and α decreased, k and α were the highest when the concentration of methanol aqueous solution was 10%. The extraction temperature had a great influence on k and α, too.     

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm^?3 (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄.

Enantioselective Separation and Determination of Carnosine in Rat Plasma by Fluorescence LC for Stereoselective Pharmacokinetic Studies

A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λ_ex = 350 nm, λ_em = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL^?1 for l-carnosine, 4,000 ng mL^?1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL^?1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg^?1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (C _max 5,344 vs. 1,914 ng mL^?1), and that of l-carnosine had a lower value of AUC_(0?∞) and t _1/2(h) (AUC_(0?∞) 5,306 vs. 6,321 ng h mL^?1, t _1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.     

Solid state radiolysis of non-proteinaceous amino acids in vacuum: astrochemical implications

The analysis of the amino acids present in Murchison meteorite and in other carbonaceous chondrites has revealed the presence of 66 different amino acids. Only eight of these 66 amino acids are proteinaceous amino acids used by the present terrestrial biochemistry in protein synthesis, the other 58 amino acids are somewhat “rare” or unusual or even “unknown” for the current terrestrial biochemistry. For this reason in the present work a series of “uncommon” non-proteinaceous amino acids, namely, l-2-aminobutyric acid, R(?)-2-aminobutyric acid, 2-aminoisobutyric acid (or α-aminoisobutyric acid), l-norleucine, l-norvaline, l-β-leucine, l-β-homoalanine, l-β-homoglutamic acid, S(?)-α-methylvaline and dl-3-aminoisobutyric acid were radiolyzed in vacuum at 3.2 MGy a dose equivalent to that emitted in 1.05 × 10⁹ years from the radionuclide decay in the bulk of asteroids or comets. The residual amount of each amino acid under study remained after radiolysis was determined by differential scanning calorimetry in comparison to pristine samples. For optically active amino acids, the residual amount of each amino acid remained after radiolysis was also determined by optical rotatory dispersion spectroscopy and by polarimetry. With these analytical techniques it was possible to measure also the degree of radioracemization undergone by each amino acid after radiolysis. It was found that the non-proteinaceous amino acids in general do not show a higher radiation and radioracemization resistance in comparison to the common 20 proteinaceous amino acids studied previously. The unique exception is represented by α-aminoisobutyric acid which shows an extraordinary resistance to radiolysis since 96.6 % is recovered unchanged after 3.2 MGy. Curiously α-aminoisobutyric acid is the most abundant amino acid found in carbonaceous chondrites. In Murchison meteorite α-aminoisobutyric acid represents more than 20 % of the total 66 amino acids found in this meteorite.     

Chiral resolution of l- and d-alanine and a racemic macrocyclic nickel(II) complex: synthesis and crystal structures

The reactions of a racemic four-coordinate Ni(II) complex [Ni(rac-L)](ClO₄)₂ with l- and d-alanine in acetonitrile/water gave two six-coordinate enantiomers formulated as [Ni(RR-L)(l-Ala)](ClO₄)·2CH₃CN (1) and [Ni(SS-L)(d-Ala)](ClO4) (2) (L = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclo-tetradecane, Ala^? = alanine anion), respectively. Evaporation from the remaining solutions gave two four-coordinate enantiomers characterized as [Ni(SS-L)](ClO₄)₂ (S-3) and [Ni(RR-L)](ClO₄)₂ (R-3), respectively. Single-crystal X-ray diffraction analyses of complexes 1 and 2 revealed that the Ni(II) atom has a distorted octahedral coordination geometry, being coordinated by four nitrogen atoms of L in a folded configuration, plus one carboxylate oxygen atom and one nitrogen atom of l- or d-Ala^? in mutually cis-positions. Complexes 1 and 2 are supramolecular stereoisomers, constructed via hydrogen bonding between [Ni(RR-L)(l-Ala)]⁺ or [Ni(SS-L)(d-Ala)]⁺ monomers to form 1D hydrogen-bonded zigzag chains. The homochiral natures of complexes 1 and 2 have been confirmed by CD spectroscopy.     

Transformation of l-phenylalanine to (S)-indoline-2-carboxylic acid without group-protection

(S)-Indoline-2-carboxylic acid was synthesized by use of a nitro amination approach with l-phenylalanine as chiral pool. The first step of the synthesis was nitration of l-phenylalanine, with urea nitrate (UN)/H₂SO₄ as nitrating reagent, to give 2,4-dinitro-l-phenylalanine in 75.7 % yield in one-pot synthesis and 69.1 % yield by step-wise nitration. Intramolecular nitro amination of 2,4-dinitro-l-phenylalanine gave (S)-6-nitro-indoline-2-carboxylic acid in 65.7 % yield and more than 99.5 % enantiomeric excess (ee). The title compound, (S)-indoline-2-carboxylic acid, was obtained in 85.9 % yield and high ee by one-pot transformation of (S)-6-nitroindoline-2-carboxylic acid. The total synthesis consisted of three operations and gave the title compound in 42 % yield and more than 99.5 % ee.     

Enhanced l-Lactic Acid Production from Biomass-Derived Xylose by a Mutant Bacillus coagulans

Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient l-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for l-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l^?1? l-lactic acid was obtained from 100 g l^?1 xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l^?1? l-lactic acid was obtained in 36 h and the yield was 83.09 %.     

Characteristics of α-l-Arabinofuranosidase from Streptomyces sp I10-1 for Production of l-Arabinose from Corn Hull Arabinoxylan

Streptomyces sp I10-1 α-l-arabinofuranosidase efficiently produced l-arabinose from high arabinose-content corn hull arabinoxylan (ratio of arabinose to xylose, 0.6). The optimum pH at 40 °C was around 6, and the enzyme was stable from pH 5 to 11. The optimum temperature was 50 °C at pH 5, and the activity was stable at 40 °C. The enzymatic activity against corn hull arabinoxylan was 2.3 times higher than towards p-nitrophenyl-α-l-arabinofuranoside. Approximately 45 % l-arabinose recovery was achieved from corn hull arabinoxylan. It was considered that l-arabinose residues not removed by the enzyme were attributable to those linked with ferulic acid. The open reading frame of the enzyme gene consisted of 1,224 bp, and the predicted peptide was 408 amino acids, which corresponded to a molecular size of 45, 248 Da. It was presumed that the smaller molecular size (31,000 Da) estimated on SDS-PAGE resulted from proteolysis by proteases. I10-1 α-l-arabinofuranosidase belongs to the Alpha-l-AF C superfamily, which is associated with glycoside hydrolase family 51, but the properties were unique.     

Study on the intermolecular complexation behavior between p-sulfonatocalix[4]arene with l-tyrosine

The formation of the complexation behavior of host molecules water-soluble p-sulfonatocalix[4]arene (p-SCX4) with l-tyrosine (l-Tyr) guest molecule has been studied by spectrophotometric including fluorescence and nuclear magnetic resonance (NMR) spectroscopy. Experimental conditions including concentration of p-SCX4 and medium acidity were investigated in detail. The results showed that p-SCX4 forms 1:1 complexes with l-Tyr in water. Their stability constant determined by spectrofluorometric titration is 15761 L mol. Moreover, to obtain information about the binding mechanism of the interaction, ¹H NMR studies were carried out showing that the water-soluble p-SCX4 was found to be able to complex the aromatic l-Tyr, and the part of benzene ring of amino acid penetrated into the hydrophobic cavity of calix[4]arene and charged aliphatic chain of l-Tyr stick out of the cavity. In addition, the thermodynamic parameters for the complexation of p-SCX4 with l-Tyr were determined through Van’t Hoff analysis. The obtained data further confirmed the binding mode of p-SCX4 with l-Tyr. The related mechanism is proposed to explain the complexation processes.     

Efficient Microbial Conversion of l-Tyrosine to l-DOPA by Brevundimonas sp. SGJ

l-DOPA (3,4-dihydroxyphenyl-l-alanine), the most widely used drug for the treatment of Parkinson??s disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the l-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH?8, 2?g?l^?1 cell mass, 2?g?l^?1 l-tyrosine, 0.04?g?l^?1 CuSO₄, 0.02?g?l^?1 l-ascorbic acid, 0.5?g?l^?1 carrageenan, and 40?°C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81?g?l^?1 (95.2%) of l-DOPA with utilization of 4?g?l^?1 l-tyrosine, and the highest tyrosinase activity (9,201?U?mg^?1) was observed at 18?h of incubation. Furthermore, the produced l-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography?Cmass spectroscopy. Kinetic studies showed significant values of Y _p/s, Q _s, and q _s after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of l-DOPA.     

Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-l-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-l-methionine sulfoxide and the product Fmoc-l-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-l-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-l-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis–Menten constant, K _m, and the maximum velocity, v _max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

Synthesis and characterization of polylactic acid (PLA) using a solid acid catalyst system in the polycondensation method

In this study, synthesis of poly(l(+) lactic acid) was carried out by using an acid catalyst. Neat chlorosulfonic acid reacts with cellulose to give cellulose sulfuric acid in which sulfuric acid is immobilized on the cellulose surface via bond formation. Cellulose sulfuric acid was used as a catalyst in a quantity of 1.0 wt.% calculated on the monomer. Polycondensation was carried out over a period of 24 h. The resulting poly(l(+) lactic acid) was characterized by Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) thermogravimetric analysis (TGA), ¹H and ¹³C nuclear magnetic resonance (NMR), and gel permeation chromatography. The molecular weight of poly(l-lactic acid) reached as high as 14,875.     

Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

An extracellular l-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg^?1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified l-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K _m, V _max, and k _cat values of the enzyme were 0.257 mM, 1.537 U μg^?1, and 993.93 s^?1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α?+?β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified l-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the l-asparaginase has significant antineoplastic properties.     

Carbohydrate recognition of symmetrical tripodal receptor type tris(2-aminoethyl)amine derivatives

Carbohydrate recognition of some bioactive symmetrical tripodal receptor type tris(2-aminoethyl)amine (TAEA) derivatives was investigated. In calorimetric experiments, the highest binding constant (Ka) of compound C (C₃₅H₄₉N₅O₄S) with methyl α-d-mannopyranoside was Ka = 858 M^?1 with 1:1 stoichiometry. Formation of hydrogen bonds in binding between symmetrical tripodal receptor type compound C and sugars was suggested by the large negative values of ?H° (=?34 to ?511 kJ mol^?1). In a comparison of each set of α- and β-anomers of some monosaccharides (methyl α/β-d-galactopyranoside, methyl α/β-d-glucopyranoside, and methyl α/β-l-fucopyranoside), compound C showed that the binding constant of β-anomer was larger than that of the corresponding α-anomer, indicating higher β-anomer selectivity. The calculated energy-minimized structure of the complex of compound C with guest methyl α-d-mannopyranoside is also presented. The experimental results obtained from this work indicated that symmetrical tripodal receptor type TAEA derivative C has a lectin-like carbohydrate recognition property.