Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ¶β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol.     

Comparison of enantioseparation of amino acid derivatives in aqueous and mixed aqueous-organic media by capillary zone electrophoresis

The chiral separation of dansyl-amino acids has been performed by capillary zone electrophoresis using ?β-cyclodextrin as a chiral selector, urea as an additive and 2-propanol and methanol as organic modifiers. The enantiomeric separations of dansyl-amino acids were investigated in aqueous medium and compared with the separation in mixed aqueous-organic medium as background electrolytes. The separation conditions, (concentration of buffer, β-cyclodextrin, methanol, urea and the pH value of buffer) were optimized. In the absence of organic modifier, only five pairs of 8 separated dansyl-amino acids were resolved when run separately. A mixture of up to eight chiral amino acids can be baseline resolved in less than 19 min by β-cyclodextrin-modified capillary zone electrophoresis with a buffer of 60 mmol L^–1 H₃BO₃-KCl/40 mmol L^–1 NaOH (pH 9.0), 4 mol L^–1 urea, 100 mmol L^–1β-cyclodextrin and 10% (v/v) methanol. Received: 15 March 1999 / Revised: 10 May 1999 / Accepted: 12 May 1999     

Coelectroosmotic capillary electrophoresis of phenolic acids and derivatized amino acids using N,N-dimethylacrylamide-ethylpyrrolidine methacrylate physically coated capillaries

Two different families of compounds, i.e., phenolic and amino acids have been separated by capillary electrophoresis using a physically adsorbed polymer as capillary coating. The polymer used was N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EpyM) and it provided an stable coating by only flushing the capillary with a DMA-EpyM aqueous solution for 2 min between runs. The usefulness of this procedure has been demonstrated through the fast analysis of different families of solutes. Two different detection systems, diode-array detector and laser-induced fluorescence, have been used to determine phenolic acids and derivatized amino acids with fluorescein isothiocyanate, respectively. The main factors affecting reversal of electroosmotic flow (EOF) such as pH, type and concentration of buffer, and concentration and influence of organic solvents, as well as all the instrumental conditions were studied and optimized for both families of compounds.     

A composite amperometric tyrosinase biosensor for the determination of the additive propyl gallate in foodstuffs

The performance of a graphite-Teflon composite amperometric tyrosinase biosensor for the determination of the food additive propyl gallate (PG) in different types of foodstuffs is reported. The enzyme reaction involves the catalytic oxidation of PG to the corresponding o-quinone, and the electrochemical reduction of this o-quinone was employed to monitor the enzyme reaction. Depending on the nature of the food sample analysed and on the presence of other phenolic antioxidants in these samples, aqueous buffer solutions or predominantly nonaqueous acetonitrile-Tris buffer mixtures were employed as working media. Experimental conditions such as the aqueous solution percentage in the predominantly nonaqueous medium, pH, and the potential to be applied were optimised. Control charts constructed showed a useful lifetime for the biosensor of 40 days when working in phosphate buffer of pH 6.5, and of 50 days in 80:20 acetonitrile-Tris buffer (pH 7.4) mixture. The limits of detection obtained for PG in these media were 9.0×10⁻⁷ and 1.1×10⁻⁶ mol L⁻¹, respectively. The composite bioelectrode also performed well in the flow-injection mode. PG was determined in dehydrated broth bars using the phosphate buffer solution of pH 6.5 as working medium. However, PG was determined in spiked olive oil in the working medium formed by the 80:20 acetonitrile-Tris buffer mixture, because a liquid-liquid extraction step was carried out. Comparison of the results with those obtained by applying reference methods showed that no significant differences existed at a significance level of 0.05.     

Protease-catalyzed peptide synthesis on solid support

The direct enzymatic synthesis of peptides from amino acids is widely used as a useful alternative to chemical synthesis. However, good yields of such enzyme-catalyzed reactions require altered reaction conditions to overcome the bias for hydrolysis in aqueous medium. We argue that the synthesis/hydrolysis equilibrium can be shifted toward synthesis in aqueous medium by immobilizing the amine on solid support. In this report, we show the first examples of solid-phase peptide synthesis catalyzed by a protease in bulk aqueous buffer.     

Effect of bromide salts on the acid hydrolysis of anti-bacterial hydrophilic Schiff base amino acid iron(II) complexes

Salt effects on the kinetics of acid hydrolysis of some novel hydrophilic iron(II) complexes have been investigated in aqueous medium. The ligands are derived from the condensation of amino acids (glycine, L-alanine, L-leucine, L-isoleucine, DL-methionine, DL-serine or L-phenylalanine) and sodium 2-hydroxybenzaldehyde-5-sulfonate. The reaction was studied under conditions of pseudo first order kinetics. The general rate equation was suggested as follows: rate = k _obs[complex], where k _obs = k ₂[H⁺]. The reaction rate decreases with an increase of the salt concentration.     

Densities of aqueous solutions containing model compounds of amino acids and ionic salts at T = 298.15 K

Densities of amino acids in aqueous and in aqueous electrolyte solutions have been measured by a high precision vibrating tube digital densitometer at T = 298.15 K under atmospheric pressure. The investigated systems contained amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with electrolytes of potassium chloride (KCl), potassium bromide (KBr) and potassium acetate (KAc). In this series of measurements, the aqueous samples were prepared with various concentrations of the amino acids, up to saturated conditions, and over salt concentrations from 1 to 4 M. The density increments resulting from the addition of the different model compounds of amino acids and the ionic salts were investigated, respectively. An empirical linear combination equation with an augmented term to account the interactions between amino acid and ionic salt was used to quantitatively correlate the experimental densities over the entire concentration ranges.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Acylation of Amino Acids with Furancarboxylic Acid Chlorides

Acylation of aromatic amino acids with furancarboxylic acid chlorides effectively proceeds in water-acetone medium at pH 8-9. Aliphatic amino acids are acylated at higher pH values, but under these conditions hydrolysis of acid chlorides becomes the main process. Acylation of chlorohydrates of methyl esters of aliphatic amino acids proceeds smoothly in chloroform in the presence of triethylamine. Alkaline hydrolysis of obtained products leads to N-acylated amino acids containing furan heteroring in the acyl radical.     

Simultaneous purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis on a hydrophobic support

Purification and reversible immobilization of d-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 mM pyrophosphate buffer (pH 8.5). The presence of a high salt concentration of 2M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization. The enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0. The immobilized preparation could be reused in a batch process for the conversion of d-amino acids to α-keto acids. When the activity of the preparation dropped below practical limits, the gel could be regenerated by water wash and recharged with fresh crude extract from yeast.     

Mixed-Ligand Complex Formation Equilibria of Vanadium(III) with 2,2′-Bipyridine and the Amino Acids Glycine, Proline, α-Alanine and β-Alanine Studied in 3.0 mol?dm?3 KCl at 25 °C

We studied speciation of the mixed-ligand complex formation equilibria of vanadium(III) with both 2,2??-bipyridine (Bipy) and the amino acids glycine (HGly), proline (HPro), ??-alanine (H??Ala), and ??-alanine (H??Ala) by means of electromotive forces measurements emf(H) using 3.0?mol?dm^?3 KCl as the ionic medium at 25 °C. The experimental data were analyzed by means of the computational least-squares program LETAGROP, taking into account the hydrolysis of the vanadium(III) cation, the respective stability constants of the binary complexes, and the acid/base reactions of the ligands which were kept fixed during the analysis. In all four amino acid systems studied we observed the complexes [V₂O(Bipy)(B)]³⁺, [V₂O(Bipy)₂(B)₂]²⁺, [V(OH)(Bipy)(B)₂] and [V(OH)₂(Bipy)(B)], where B represents the deprotonated form of the amino acids studied in this work. The respective stability constants were determined and the species distribution diagrams as a function of pH are briefly discussed.     

Phanquinone as a suitable derivatization reagent in micellar electrokinetic chromatography and HPLC analysis of amino acids

Phanquinone (chemically: 4,7-phenanthroline-5,6-dione) was applied as an original precolumn derivatization reagent for amino acids followed by separation using MEKC with UV detection (240 nm). The derivatization reaction was carried out at 68 degrees C in the presence of aqueous phosphate buffer (pH 8.0) and it was found to be complete after 30 min. Twelve derivatized standard amino acids were separated in about 22 min under MEKC conditions using sodium cholate (250 mM) as the surfactant in phosphate buffer (20 mM, pH 9.0). The developed method was validated for the analysis of D,L-phosphoserine (D,L-p-Ser) and L-glutamine (L-Gln); good linearity (r > 0.999) was achieved in the calibration range of 0.25-2.5 micromol/mL. The sensitivity of the MEKC method (LOD 0.1 micromol/mL; LOQ 0.25 micromol/mL, RSD% <5.0%, n = 3) was found to be adequate for quantitation of amino acids in pharmaceuticals. Quantitative applications of the validated MEKC method were carried out by the analysis of commercially available oral polyaminoacid formulations (tablets and extemporaneous solutions) containing L-Gln and D,L-p-Ser; the obtained results were found to be in agreement with those from a validated reference RP-HPLC method.     

Interaction of iron(II) and the simultaneous spectrophotometric determination of Fe, Cu, Zn, Co and Ni with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol

Summary The reaction of Fe(II) with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (Br-PADAP) is studied in detail and procedures for the sensitive determination of Fe(II) at pH 4.7 (acetate buffer), pH 9.0 (borate buffer) and in the presence of EDTA are optimized. A simultaneous determination of Fe, Cu, Zn, Co and Ni in aqueous medium and of Fe, Cu and Zn in blood serum with Br-PADAP at pH 9.0 using multivariate calibration with PLS evaluation of absorbance data also give satisfactory results.     

L-谷氨酸与维多利亚绿S作用机理及应用研究

用紫外-可见光度法、共振散射光谱法探讨了在弱酸性介质中,碱性染料维多利亚绿S与酸性氨基酸L-谷氨酸相互作用机理,认为L-谷氨酸分子通过盐键和氢键相互结合成含有亲水性基团的分子链,这条分子链可以将维多利亚绿S的疏水性基团包在里面,使其亲水性基团朝外,并通过盐键和氢键与维多利亚绿S作用形成大的聚合体,其聚合体体积远远大于染料自身聚集体的体积,使溶液颜色变深,分子光谱吸收值增加.并优化了反应体系酸度、反应物用量、反应时间、加试剂顺序等影响反应速度的因素,确定了最佳实验条件,建立了测定L-谷氨酸的方法.线性范围为10mg/L～100 mg/L,相关系数为0.9989.直接测定样品中谷氨酸获得满意结果.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic-mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-Sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using 18O-labelled amino acids and gas chromatography-mass spectrometry.     

Preparation and some properties of maleimido acids and maleoyl derivatives of peptides.

N-Alkoxycarbonylmaleimides 3 have been prepared and used to convert amino acids to maleimido acids (6–8) in aqueous solution. The carboxyl group of maleimido acids can be activated for amide or peptide synthesis (e.g., in the N-succinimidyl esters 10); t-butyl-based protecting groups can be cleaved without damage to the maleimide moiety. Peptides carrying maleimide groups are accessible either from the maleimido acids (e.g., 11b, 15) or by direct maleoylation (e.g., 16b). The maleoyl group can be cleaved off by successive mild alkaline and acid hydrolysis or by hydrazinolysis. The reactivity of maleimides toward thiol groups suggests the use of maleimido acids and maleoylpeptides for preparing a wide range of conjugates of biochemical interest.     

Differential-pulse polarography of amino acids in acetaldehyde-borax medium

A differential-pulse polarographic method for the determination of amino acids is reported, based on the formation of Schiff's base compounds in borax buffer solution (pH 10.10) containing acetaldehyde. The compounds are reduced at a mercury electrode with peak potentials of about ?1.5 V (vs. SCE) and well defined polarographic waves are obtained which can be used to determine amino acids in borax medium. The differential-pulse polarographic method was found to be the most sensitive and suitable for the determination of amino acids in the concentration range 1 × 10^?6–8 × 10^?4 M (lysine) and 2.8 × 10^?6–1 × 10^?3 M (arginine). The polarographic characteristics of these waves were studied by differential-pulse polarographic and cyclic voltammetric methods. The waves are ascribed to the reduction of the imido group in the Schiff's base compounds. The procedure was applied to the determination of lysine and arginine in foodstuffs and the total proteins in serum samples.     

Investigation on the pH-dependent binding of vitamin B12 and lysozyme by fluorescence and absorbance

The binding reaction between vitamin B12 (B12, cyanocobalamin) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet–vis (UV) absorbance, and three-dimensional fluorescence. The intrinsic fluorescence of Lys was strongly quenched by the addition of B12 in different pH buffer solutions (pH 3.4, 7.4, and 9.0) and the spectroscopic observations are mainly rationalized in terms of a static quenching process at lower concentration of B12 (C_B12/C_Lys < 5) and a combined quenching process at higher concentration of B12 (C_B12/C_Lys > 5). The structural characteristics of B12 and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.4, 7.4, and 9.0). The effect of B12 on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of B12 to Lys causes apparent change in the secondary or tertiary structures of Lys. Furthermore, the effect of Zn²⁺ on the binding constant of B12 with Lys under various pH conditions (pH 3.4, 7.4, and 9.0) was also studied.     

Fatty acids and amino acids of entomopathogenic fungus <Emphasis Type="Italic">Conidiobolus coronatus</Emphasis> grow on minimal and rich media

Entomopathogenic fungi are referred to as potential candidates as insect pest control agents. The objective of the study was to identify fatty acids and amino acids from Conidiobolus coronatus cultured on two different media. Each medium was extracted with ethyl acetate and its mixtures with isopropanol, acetonitrile and methanol. Analyses of fatty acids and amino acids of entomopathogenic fungus C. coronatus were performed by means of gas chromatography coupled with mass spectrometry. The analysis showed that the fungus C. coronatus produces the following groups of compounds: fatty acids and amino acids; α- and β-glucopyranose were also identified. The identified fatty acids included 12–20, 22 and 24 carbon atoms per chain. The highest content of fatty acids was detected in a mycelium sample cultured in a liquid minimal medium extracted with ethyl acetate. The lowest content of these organic compounds was identified in mycelium cultured in a liquid nutrient-rich medium extracted with ethyl acetate–methanol mixture. Fatty acids were found to account for 62.0 mass % to 94.4 mass % of all organic compounds in the analyzed mycelia. C18:1 acids were detected in the highest amounts when ethyl acetate was used as the extracting agent. The identified amino acids accounted for 4 mass % to 21 mass % of all organic compounds. Upon extraction of C. coronatus mycelium samples with the ethyl acetate—methanol mixture, two anomeric forms of glucose were also identified. An analysis of the studied material confirmed, that the entomopathogenic fungus C. coronatus is a very rich source of organic compounds, which might encourage its further research so as to identify an even larger number of compounds being produced by this species.     

N-Substituted tetrahydro-1,3-oxazines and oxazolidines. 1. A new version of the Mannich reaction involving amino alcohols

A new version of the Mannich reaction involving amino alcohols (3-aminopropan-1-ol and aminoethanol) was developed. These compounds react with formaldehyde and CH or NH acids to give N-substituted tetrahydro-1,3-oxazines or oxazolidines.