Enantioseparation of Aromatic β³-Amino acid by Precolumn Derivatization with o-Phthaldialdehyde and N-Isobutyryl-l-cysteine

In this study we describe the enantioseparation of aromatic β³-amino acids by precolumn derivatization with o-phthaldialdehyde and N-isobutyryl-l-cysteine. Derivatization conditions were studied in detail for (R,S)-β-phenylalanine and (R,S)-β-tyrosine revealing a reaction time of 1 min and a molar ratio of the reagents β^³-amino acid to o-phthaldialdehyde to N-isobutyryl-l-cysteine of 1:25:25 as optimal. The method was validated for (R,S)-β-phenylalanine in a bacterial cell extract. The analysis provided excellent specificity and reproducibility. The limit of quantification was 25 pmol per 0.5 μL injection. The method could be successfully transferred to the enantioseparation of other β^³-amino acids. Enantioseparation of all studied compounds could be achieved in 4–11 min.     

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.     

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.     

β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k _non) the enzyme at pH 5.3, which lies in the pH-independent region for k _cat and k _non. Rate enhancements (k _cat/k _non) for 4NPX, 4NPA, and X2 are 4.3?×?10¹¹, 2.4?×?10⁹, and 3.7?×?10¹², respectively, at 25 °C and increase with decreasing temperature. Relative parameters k _cat ^4NPX/k _cat ^4NPA, k _cat ^4NPX/k _cat ^X2, and (k _cat/K _m)^4NPX/(k _cat/K _m)^X2 increase and (k _cat/K _m)^4NPX/(k _cat/K _m)^4NPA, (1/K _m)^4NPX/(1/K _m)^4NPA, and (1/K _m)^4NPX/(1/K _m)^X2 decrease with increasing temperature.     

An improved FIA biosensor for the determination of aspartame in dietary food products

A flow injection analysis (FIA) biosensor system was developed for the determination of the artificial sweetener aspartame (l-aspartyl-l-phenylalanine methyl ester). The system consisted of an enzyme column of pronase immobilized on activated arylamine glass beads and al-amino acid oxidase electrode connected in series. The dipeptide bond of aspartame was cleaved by immobilized pronase to release phenylalanine, which was in turn monitored by the enzyme electrode that usedl-amino acid oxidase, immobilized on a preactivated nylon membrane in combination with an amperometric electrode (platinum vs silver/silver chloride, 700 mV). The response of the FIA biosensor was linear up to 1 mM aspartame with a lower detection limit of 25 μM and had good reproducibility (rsd 0.3%). The FIA biosensor was stable for at least 30 h of continuous use atT _r .Each assay takes 4 min giving a sample throughput of 15 h^?1 When applied to aspartame in dietary food products the results obtained agreed well with those reported by the product manufacturers.     

Ocular Pharmacokinetic Study of l-Carnosine and N-Acetyl-l-carnosine in Rabbit by Microdialysis Coupled with UPLC-MS-MS

In order to provide useful information for rational drug design, the ocular pharmacokinetics of l-carnosine (CAR) and its acetylized prodrug N-acetyl-l-carnosine (NAC) were investigated. The in vivo microdialysis sampling coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) was developed for continuously simultaneous monitoring of CAR and NAC in rabbit aqueous humor. The measured in vitro recoveries of the probe were 61.3% for CAR and 65.8% for NAC, while in vivo recoveries decreased to 43.1% for CAR and 43.0% for NAC, respectively. The method was sensitive with LLOQ 20.5 ng mL^?1 for CAR and 20.4 ng mL^?1 for NAC. The initial data indicated that the value of C _max and AUC_(0?C??) of NAC were higher than these of CAR (C _max 2305 vs. 1,802 ng mL^?1), (AUC_(0?C??) 1,337 vs. 1,891 ng h mL^?1), which indicated that the NAC exhibited better ocular bioavailability and duration. The method was rapid, specific and sensitive for continuously monitoring of aqueous humor and it was successfully applied to pharmacokinetic studies of CAR and NAC.     

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.     

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.

Transport of Tb3+in Dispersion Supported Liquid Membrane System with Carrier P507

The Tb³⁺ transport in dispersion supported liquid membrane (DSLM) consisting of polyvinylidene fluoride membrane (PVDF) as the liquid membrane support and dispersion solution including HCl solution as the stripping solution and 2‐ethyl hexyl phosphonic acid‐mono‐2‐ethyl hexyl ester (P507) dissolved in kerosene as the membrane solution, has been studied. The effects of pH value, initial concentration of Tb³⁺ and different ionic strength in the feed phase, volume ratio of membrane solution and stripping solution, concentration of HCl solution, concentration of carrier, different stripping agents in the dispersion phase on transport of Tb³⁺ has also been investigated, respectively. As a result, the optimum transport conditon of Tb³⁺ was that concentration of HCl solution was 4.0 mol/L, concentration of P507 was 0.10 mol/L, and volume ratio of membrane solution and stripping solution was 1.0 in the dispersion phase, and pH value was 5.2 in the feed phase. Ionic strength had no obvious effect on transport of Tb³⁺. Under the optimum condition studied, when initial concentration of Tb³⁺ was 1.0×10^?4 mol/L, the transport rate of Tb³⁺ was up to 95.2% during the transport time of 95 min. The kinetic equation was developed in terms of the law of mass diffusion and the theory of interface chemistry. The results were in good agreement with the literature data.     

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.     

High-Pressure Solubility of l-Methionine in Water

The solubility (m _S) of l-methionine in water was measured at 298.2 K and pressures up to 200 MPa. The data were fitted to the equation ln(m _S/mol·kg^?1) = ?4.62 × 10^?6 (p/MPa)² + 2.65 × 10^?3 (p/MPa) ? 0.970 with a standard deviation of σ(ln m _S) = 0.002. The pressure coefficient of the logarithm of solubility (?ln m _S/?p) _T was thermodynamically estimated to be (2.62 ± 0.34) × 10^?3 MPa^?1 at 0.10 MPa using several parameters such as partial molar volume and activity coefficient of l-methionine in water and molar volume of solid l-methionine. The resulting value agrees well with the second term on the right-hand side of the fitted equation above, indicating the reliability of the high-pressure solubility measurements. The value of (?ln m _S/?p) _T also was compared with those of other amino acids.     

Separation of 99mTc from 99Mo by Using TOPO — Kerosene Supported Liquid Membrane

Transport of ^99mTcO₄ ^– ions across TOPO-kerosene based supported liquid membrane was investigated at different concentrations of phosphoric acid as a feed solution and different concentrations of TOPO in the membrane, where 0.9% NaCl aqueous solution was used as a stripping solution. The flux of TcO₄ ^– ions across this liquid membrane varied with the concentration of both H₃PO₄ and TOPO. The best permeability coefficient was obtained at concentrations, [H₃PO₄] = 3 mole·l^–1 and [TOPO] = 0.5 mole·l^–1 (P = 2.08·10^–9 m²·s^–1). The results were utilized for the separation of ^99mTc from ⁹⁹Mo, where a selective and effective separation was obtained since no ⁹⁹Mo transport across this liquid membrane was noticed while a high rate of ^99mTc transport took place.     

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.     

Amperometric Detection of 3-(3,4-Dihydroxyphenyl)-l-alanine (l-Dopa) in Raw and Cooked Mucuna Bean Seeds Employing Micro-HPLC

Amperometric detection of 3-(3,4-dihydroxyphenyl)-l-alanine (l-dopa) on a glassy carbon electrode at oxidation potential of +0.70 V in Mucuna pruriens after micro-high performance liquid chromatography separation is reported. Optimised eluent consisted of 0.87 mM 1-octane sulphonic acid sodium salt, 18.2 mM citric acid, and 82.8 mM sodium acetate with pH adjusted to 2.18 using 85% orthophosphoric acid. Detection of low concentrations of l-dopa up to 5.12 ng mL^?1 was achieved. The method was employed to determine l-dopa in raw and cooked beans after water extraction through a 0.45 μm membrane with no further sample treatment.     

Pulse radiolysis studies of intermolecular charge transfers involving tryptophan and three-electron-bonded intermediates derived from methionine

The oxidation processes of the radiation-generated, three-electron-bonded intermediates AcMet₂ [S??S]⁺ and AcMet [S??Br] were investigated by pulse radiolysis via their reactions with tryptophan (TrpH). These intermediates were derived from N-acetyl-methionine amide (N-AcMetNH₂) and N-acetyl-methionine methyl ester (N-AcMetOMe). The bimolecular rate constant k of the reaction between each intermediate and l-tryptophan (TrpH) was measured. For N-AcMetNH₂, k for the reaction of AcMet₂ [S??S]⁺ with TrpH were 3.4?×?10⁸ and 2.2?×?10⁸?dm³?mol^?1?s^?1 at pH?=?1 and 4.5, respectively. For N-AcMetOMe, k for the reaction of AcMet₂ [S??S]⁺ with TrpH were 4.0?×?10⁸ and 2.8?×?10⁸?dm³?mol^?1?s^?1 at pH 1 and 4.5, respectively. The rate constants for the intermolecular transformation of Met [S??Br] into TrpH⁺ or Trp were also estimated. For N-AcMetNH₂, k for the reaction of AcMet₂ [S??Br] with TrpH were 2.6?×?10⁸ and 3.3?×?10⁸?dm³?mol^?1?s^?1 at pH 1 and 4.5, respectively. Related mechanisms were discussed.     

Pertraction of methylene blue using a mixture of D2EHPA/M2EHPA and sesame oil as a liquid membrane

An experimental study on the pertraction of methylene blue (MB) through a supported liquid membrane (SLM) using a mixture of mono-(2-etylhexyl) ester of phosphoric acid (M2EHPA) and bis-(2-etylhexyl) ester of phosphoric acid (D2EHPA) and sesame oil as the liquid membrane (LM) was performed. Parameters affecting the pertraction of MB such as initial MB concentration, carrier concentration, feed phase pH, and stripping phase concentration were analyzed. Optimal experimental conditions for MB pertraction (permeability of 5.63 × 10^?6) were obtained after a 7 h separation with the MB concentration in the feed phase of 80 mg L^?1, D2EHPA/M2EHPA concentration in membrane phase of 40 vol. %, feed pH of 6, and acetic acid concentration in the stripping phase of 0.4 mol L^?1. Kinetics of transport and stability of the SLM system were also studied and the mass transfer coefficient for this system was evaluated. Scanning electron microscopy (SEM) was used to morphologically characterize the membrane surface.     

Conformational changes of l-phenylalanine induced by near infrared radiation. ATR-FTIR studies

In our previous work the influence of water evaporation on Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectra of l-phenylalanine (l-phe) in a function of pH (Olsztynska et al. Appl. Spectrosc. 60(9):1040, (14)) was studied. The presence of symmetric dimers of hydrogen-bonded amino acid was observed when simultaneously CO₂ ^? ionised and COOH unionised forms of the amino acid appear in the solution (near pK ₁). It is suggested that Near Infrared (NIR) radiation may induce partial protonation of CO₂ ^? groups at a neutral pH and formation the same type of dimers. The aim of this work was to study this hypothesis. Therefore, ATR-FTIR spectra of l-phe aqueous solution before and after NIR radiation (15?min., 700?C2,000?nm) were obtained as a continuation of our earlier studies. Spectral characteristic bands of l-phe were described. The vibrational spectroscopic study of l-phe showed that it undergoes photochemical reactions under NIR exposure. It has been found that the irradiation process indeed induces a protonation of polar groups of l-phe at neutral pH what leads to forming of neutral forms and as a consequence hydrogen bonded dimers ?CC=O···HOOC?C. Moreover, hydrophobic interactions strongly increase, what favours aggregation of l-phe molecules. The phenomenon is probably due to modifications of water structure around l-phe molecules. Intra- and intermolecular hydrogen bonds weaken which could favour aggregation and protonation of polar groups what induces also formation of symmetrical hydrogen bonds between protonated and deprotonated carboxylic groups.     

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.     

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.     

Cis-Cycloprolylprolin-Anion-ein konfigurationsstabiles Carbanion

The racemisation ofcyclo-(l-Pro?l-Pro) (2) with metal amides in liq. ammonia was examined. The K-kation causes more extensive racemisation than Na-kation, which in turn is more effective than Li⁺. This, the racemisation of2 int-butyl alcohol with K⁺C₆H₅O^? and the data gained from corresponding deuterated medium show that the racemisation of2 proceeds in two steps: in the first, the less stabletrans-cyclo-(l-Pro?d-Pro) (3) is formed, followed by the rapid conversion of3 to a mixture ofcyclo-(l-Pro?l-Pro) andcyclo-(d-Pro?d-Pro) in the second step.