Analysis of multicomponent mixture and simultaneous enantioresolution of proteinogenic and non-proteinogenic amino acids by reversed-phase high-performance liquid chromatography using chiral variants of Sanger’s reagent

Four chiral derivatizing reagents (CDR 1–4), namely, FDNP-l-Ala, FDNP-l-Val, FDNP-l-Phe, and FDNP-l-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with l-Ala, l-Val, l-Phe, and l-Leu (CDR 1–4). The other set of CDRs, namely, FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, and FDNP-l-Leu-NH₂, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the d-amino acid in a solution of dl-amino acid. The resolution (R _S) and selectivity (α) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of d-enantiomer.     

Intercalation of basic amino acids into layered zirconium proline-<Emphasis Type="Italic">N</Emphasis>-methylphosphonate phosphate

Intercalation of basic amino acids into layered zirconium proline-N-methylphosphonate phosphate (α-ZPMP) was investigated at room temperature. Three kinds of host-guest compounds were prepared and characterised by elemental analysis, inductively coupled plasma analysis (ICP), Fourier transform infrared spectrum (FT-IR), Raman spectrum, X-ray powder diffraction (XRD) and thermoanalysis. The interaction of amino acid guests with P-OH of α-ZPMP host was documented by FT-IR and Raman spectra. In addition, the XRD patterns indicated that l-arginine or l-lysine were intercalated into the interlayer galleries of α-ZPMP host; the interlayer distances of the Larginine and l-lysine intercalation compounds were expanded from 1.520 nm to 2.218 nm and 2.207 nm, respectively. l-arginine and l-lysine would be arranged as a mono-molecule layer in different orientations. The interlayer distance of l-histidine (d = 1.522 nm) was similar to that of α-ZPMP host (d = 1.520 nm), l-histidine might be adsorbed on the outer surface of the α-ZPMP host. Thermoanalysis showed that the intercalated l-arginine and l-lysine were removed at 110–305°C or 150–250°C, respectively, the adsorbed l-histidine was released at a temperature of up to 320°C.     

Glycoconjugates of amino acids. Preparation throughN-alkylation of amino acids withN-chloroacetyl-β-glycopyranosylamines

Monoalkylation of amino acids of different structural types withN-chloroacetyl-glycosylamines was shown to be applicable for the preparation of glycoconjugates containing β-d-galactose,N-acetyl-β-d-glucosamine, β-d-mannose, and lactose residues. The glycoconjugates were synthesized from amino acids with secondary (sarcosine,l-proline) or primary (l-2- and 4-aminobutyric acids,l-tryptophan) amino groups as well as from various amino dicarboxylic acids (N-methyl-dl-aspartic,dl-aspartic,l-glutamic, anddl-2-aminoadipic acids). The derivatives obtained may be of interest for glycotargeting of physiologically active compounds of this series. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1377–1380, July, 1999.     

Determination of the absolute configuration of selenomethionine from antarctic krill by RP-HPLC/ICP-MS using chiral derivatization agents

A fast and sensitive method was developed for the determination of the absolute configuration of selenomethionine. The enantiomers of selenomethionine were converted into diastereomeric isoindole derivatives by reaction with o-phthaldialdehyde and N-isobutyryl-l-cysteine. This easy-to-handle reaction proceeds quantitatively in a few minutes at room temperature. Separation and detection of the diastereomers was achieved by reversed-phase high-performance liquid chromatography–inductively coupled plasma-mass spectrometry (RP-HPLC/ICP-MS) using a conventional C18 reversed-phase column. Detection limits of about 4 µg L^–1 were obtained. The method was applied to the determination of the configuration of selenomethionine extracted from antarctic krill, which turned out to possess the l-configuration.     

The use of cesium dibenzyl and diphenyl phosphates for stereoselective synthesis of glycosyl phosphate derivatives

Peracetates of β-glycosyl dibenzyl phosphates are formed efficiently in the reaction of cesium dibenzyl phosphate with peracetyl-α-glycosyl nitrates derived froml-fucopyranose,d-galactopyranose, and 2-azido-2-deoxy-d-galactopyranose or with tri-O-acetyl-α-l-fucopyranosyl bromide. On the contrary, the reaction of the above-mentioned glycosyl nitrates with cesium diphenyl phosphate leads to thermodynamically more stable α-glycosyl diphenyl phosphatevia intermediate formation of the corresponding β-anomers. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 1924–1928, November, 2000.     

Production of Fumaric Acid by <Emphasis Type="BoldItalic">Rhizopus oryzae</Emphasis>: Role of Carbon–Nitrogen Ratio

Cytosolic fumarase, a key enzyme for the accumulation of fumaric acid in Rhizopus oryzae, catalyzes the dehydration of l-malic acid to fumaric acid. The effects of carbon–nitrogen ratio on the acid production and activity of cytosolic fumarase were investigated. Under nitrogen limitation stress, the cytosolic fumarase could keep high activity. With the urea concentration decreased from 2.0 to 0.1 g l⁻¹, the cytosolic fumarase activity increased by 300% and the production of fumaric acid increased from 14.4 to 40.3 g l⁻¹ and l-malic acid decreased from 2.1 to 0.3 g l⁻¹. Cytosolic fumarase could be inhibited by substrate analog 3-hydroxybutyric acid. With the addition of 3-hydroxybutyric acid (50 mM) in the fermentation culture, fumaric acid production decreased from 40.3 to 14.1 g l⁻¹ and l-malic acid increased from 0.3 to 5.4 g l⁻¹.     

Glycosylation of betulin acetates with glycals

Betulin 2-deoxy-α-d-, 2-deoxy-α-l-, and 2,6-dideoxy-α-l-arabino-hexopyranosides were synthesized by acid-catalyzed glycosylation (cationite in the H⁺ form, LiBr) of betulin 3- and 28-monoacetates with glycal acetates. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 531–534, March, 1998.     

Enantiomeric Recognition of d- and l-Amino Acid Methyl Ester Hydrochlorides by New Chiral Bis-pyridino-18-crown-6 Substituted with Urea, and Diphenyl Groups

The article reports the synthesis and chiral recognition properties of a new chiral bis-pyridino-18-crown-6 (7), having urea, diphenyl, and allyloxy groups. The chiral bis-pyridino-18-crown-6 was prepared by a thirteen-steps procedure from the commercially available (S)-(+)-mandelic acid and chelidamic acid. The association constants (K _a) (1.33 × 10³–3.20 × 10³) for enantiomeric recognition of d- and l-amino acid methyl ester hydrochlorides using the chiral bis-pyridino-18-crown-6 have been examined by ¹H-NMR titration method in CDCl₃ at 25 °C. The chiral bis-pyridino-18-crown-6 showed higher association constants for the d-series amino acid methyl ester (d-AlaOMe, d-LeuOMe, d-MetOMe) hydrochlorides as compared to the corresponding l-series (l-AlaOMe, l-LeuOMe, l-MetOMe) hydrochlorides.     

N-glycyl-β-glycopyranosylamines, derivatives of mono- and disaccharides, and their use for the preparation of carboxylic acid glycoconjugates

A convenient preparative procedure was developed for the synthesis ofN-glycyl-β-glycopyranosylamines, derivatives of monosaccharides (d-galactose,d-mannose,l-fucose, andN-acetyl-d-glucosamine) and disaccharides (lactose, melibiose, cellobiose, and maltose). These compounds were demonstrated to be useful for the preparation of glycoconjugates of biologically active compounds containing the carboxy group (nicotinic, orotic, kynurenic, and indoleacetic acids). Synthetic pathways were developed for conversions ofN-glycyl-β-glycopyranosylamines into derivatives containing the carboxy group with the use of malonic andl-tartaric acid derivatives. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1461–1466, August, 2000.     

A Cyclodextrin Self-Assembled Monolayer (SAM) Based Surface Plasmon Resonance (SPR) Sensor for Enantioselective Analysis of Thyroxine

Heptakis 6-deoxy-6-[12-(thiododecyl) undecanamido-β-cyclodextrin has been produced by reaction of Heptakis(6-deoxy-6-amino)-β-cyclodextrin and 12-(thiododecyl)undecanoic acid using O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) as activating agent. Self-assembled monolayers of this macrocycle have been used in a surface plasmon resonance (SPR) sensor; it has been shown that this system is suitable to discriminate between d and l enantiomers of thyroxine, with a greater affinity for the d-enantiomer. Received in final form: 6 January 2005     

Calorimetric, NMR, and UV Investigations of Aliphatic l-Amino Acids Complexation by Calix[4]arene Bis-hydroxymethylphosphous Acid

The stability constants, enthalpy ΔH ⁰, entropy ΔS ⁰, and Gibbs energy ΔG ⁰ were determined for the host–guest complexes (1:1) of calix[4]arene bis-hydroxymethylphosphous acid with glycine, l-alanine, l-valine, l-leucine, l-isoleucine residues in methanol solution with the aid of the titration experiments followed by calorimetric and spectroscopic (¹H NMR, UV) methods. The experimental data indicated that the host–guest complexation was under control of the direct electrostatic interaction between negatively charged calixarene phosphoryl group and amino acid residue NH ₃ ⁺ group, modulated by the hydrophobic interaction, which drive the inclusion of the residue alkyl side-chain into the calixarene cavity. The stability of the inclusion complexes was found correlated with the size of the aliphatic amino acid’s side-chain. The experimental data were additionally analyzed in the terms of the three state model corresponding to coexistence of 2:1 and 1:1 complexation equilibria.     

Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal d-Ala-d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two d-Ala-d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala-d-Ala-d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10⁻⁶ and 3.0 × 10⁻⁶ mol L⁻¹, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic interaction.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

Preparation of biodegradable microspheres by anionic dispersion polymerization with PLA copolymeric dispersion stabilizer

We prepared poly(d,l-lactide) (PLA) microspheres by anionic dispersion polymerization of d,l-lactide. The polymerization was carried out in xylene/heptane (1:2 in v/v) mixture solution at 368 K for 9 h, with poly(dodecyl methacrylate)-co-poly[α-methacryloxyethoxy-poly(l-lactide)] (PDMA-co-P(MA-PLLA)) synthesized in this study, as a dispersion stabilizer. The number-averaged diameter and diameter distribution (coefficient of variation) of obtained PLA microspheres ranged from 180 to 800 nm and 14–40%, respectively, depending on the preparation condition. Furthermore, the time courses of monomer conversion, particle diameter, and particle number were investigated to clarify the formation mechanism of microspheres with PDMA-co-P(MA-PLLA) as a dispersion stabilizer. From this experiment, we found that the aggregation of primary particles occurred in anionic dispersion polymerization, and the particle diameter of obtained PLA microspheres decreased with increasing PDMA-co-P(MA-PLLA) concentration. In conclusion, we clarified that PDMA-co-P(MA-PLLA) effectively contributed to the stability of primary particles.     

New class of carbohydrate-based nonionic surfactants: diblock co-oligomers of tri-<Emphasis Type="Italic">O</Emphasis>-methylated and unmodified cello-oligosaccharides

Mixtures of diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides have been found to be amphiphilic, as reported before. In order to clarify their accurate amphiphilic property, diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides with monodispersity, methyl β-d-glucopyranosyl-(1→4)-2,3,6–tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6–tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-d-glucopyranoside (1, pentamer), methyl β-d-glucopyranosyl-(1→4)- β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-d-glucopyranoside (2, hexamer), and methyl β-d-glucopyranosyl-(1→4)-2,3,6-tri-O-methyl-β-d-glucopyranosyl-(1→4)- 2,3,6-tri-O-methyl-d-glucopyranoside (3, trimer) were synthesized independently. These compounds had higher surface activities compared to the mixture of diblock co-oligomers of tri-O-methylated and unmodified cello-oligosaccharides and commercially available methylcellulose (MC) SM-4. This paper describes the methods of synthesis of these compounds, and the influence of amphiphilic character on their surface activity. A new class of carbohydrate-based nonionic surfactant without long alkyl chain was discovered.     

Fabrication of modified TiO2 nanoparticle carbon paste electrode for simultaneous determination of dopamine, uric acid, and l-cysteine

A carbon paste electrode, modified with 2, 2′-[1,7-hepthandiylbis(nitriloethylidyne)]-bis-hydroquinone and TiO₂ nanoparticles, was used for the simultaneous determination of dopamine (DA), uric acid (UA), and l-cysteine. The study was carried out by using cyclic voltammetry, chronoamperometry, and square wave voltammetry (SWV) techniques. Some kinetic parameters such as the electron transfer coefficient (α) and heterogeneous rate constant (k_s) were also determined for the DA oxidation. A dynamic range of 8.0–1400 μM, with the detection limit of 8.4 × 10⁻⁷ M for DA, was obtained using SWV (pH = 7.0). The prepared electrode was successfully applied for the determination of DA, UA, and l-cysteine in real samples.     

Stereoselective effects in the solid-phase hydrogenation of unsaturatedl-hydroxyproline derivatives

The solid-phase catalytic hydrogenation of (R-4tert-butoxy-Δ¹-pyrroline-2-carboxylic) acid under the action of hydrogen spillover was studied. The reaction proceeds stereoselectively with the predominant formation of thel-amino acid. The configuration of the asymmetric center formed is determined by that of the asymmetric C(4) atom. The major portion of the isotope label is incorporated into the allylic C(3) and C(5) positions, and the β-H atoms are more mobile. Using quantum-chemical calculations, the geometric structure of thel-hydroxyproline molecule was calculated, and the spin-spin coupling constants for this tritium-labeled amino acid were determined. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1820–1823, October, 1997.     

A Validated Chiral LC Method for the Determination of Enantiomeric Purity of Pemetrexed Disodium on an Amylose-Based Chiral Stationary Phase

A simple and new isocratic normal phase chiral HPLC method has been developed for the determination of enantiomeric purity of pemetrexed disodium (l-enantiomer) in bulk drugs with a short run time of about 20 min. Chromatographic separation of l and d-enantiomers of pemetrexed disodium was achieved on an amylose based chiral stationary phase using a mobile phase consists of hexane, ethanol and trifluoro acetic acid. The resolution between the enantiomers was found to be more than 2.0. The system precision and method precision were found to be within 5% RSD for the distomer (d-enantiomer) at its specification level (i.e. not more than 1.0% w/w). The limit of detection and limit of quantification of distomer were 1.6 and 5 μg mL⁻¹, respectively for 10 μL injection volume. The percentage recovery of distomer was ranged from 90.6 to 105.7 in bulk drug samples. The test solution was found to be stable in the diluent for 48 h. The method was found to be specific for the enantiomers of pemetrexed disodium and can be conveniently used for the quantification of undesired d-enantiomer present in the bulk drug samples of pemetrexed disodium.     

Applications of nanostructured Pt-Au hybrid film for the simultaneous determination of catecholamines in the presence of ascorbic acid

Nanostructured platinum-gold (Pt–Au) hybrid film modified glassy carbon electrode (GCE) was fabricated by electro-deposition method in the presence of 2 × 10⁻⁴ mol l⁻¹ l-cysteine. To examine the surface morphological analysis, the (Pt–Au) hybrid film were electrochemically deposited on transparent semiconductor indium tin oxide (ITO) electrodes for scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) studies. From the SEM analysis, it was observed that the deposited nanoplatinum (250–400 nm) was formed as a cauliflower-shaped structure with the gold nanoparticles (30–90 nm). The concentration variation of additive l-cysteine results in the formation of cauliflower-shaped platinum nanoparticles. Further, the Pt–Au hybrid film modified GCE could be used for the detection of catecholamine neurotransmitters epinephrine (EP), norepinephrine (NEP) individually and in the presence of ascorbic acid (AA) in pH 7 phosphate-buffered solutions (PBS). Furthermore, the proposed Pt–Au hybrid film could be applied for the detection of epinephrine in injection solution and ascorbic acid from commercially available vitamin C tablets.     

Thermodynamics and the mechanism of interaction of uracil with amino acids in water

Interaction of most of the side groups of amino acids with uracil and their dehydration do not contribute significantly to the pair interaction coefficients. It has been suggested that the interaction between the terminal groups of amino acids and the side groups of uracil (NH, CO) occurs by the acid-base mechanism. The possibility was found of formation of uracil+l-proline associates, owing to favorable configurations of the components, uracil+l-tryptophan associates, owing to π-π electronic interaction between their aromatic rings, and uracil+l-lysine: HCl, owing to the side ammonium group in amino acid. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 932–934, May, 1997.