Resolution Of Amino Acid Enantiomers Research Articles

Development of a diphenyl sulfide structure derivatization reagent for amino acid enantiomers analysis: Application of dynamic monitoring in human urine after drinking wine

We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71–5.42, and an excellent linear relationship was obtained in the range of 0.25–500 pmol (R2 ≥0.9993). The detection limit was 0.05–0.25 pmol. The intra- and inter-day precisions were 0.51–5.76% and 0.78–5.18%, respectively, and the average recovery was 90.03–99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.

Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Study of the enantioselectivity and recognition mechanism of sulfhydryl-compound-functionalized gold nanochannel membranes.

Two new chiral membranes were prepared by modification of gold nanochannel membranes with D-penicillamine and N-acetyl-L-cysteine and were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. The effects of key factors such as the gold deposition time, the pH, and the concentration of sodium dihydrogen phosphate on the separation factor are discussed. Chiral resolution of amino acid enantiomers by the chiral membranes was investigated. The experimental results show that the D-penicillamine-modified membrane has good enantioselectivity toward tyrosine and phenylalanine enantiomers, whereas the N-acetyl-L-cysteine-modified membrane has good enantioselectivity toward tyrosine and tryptophan enantiomers. Furthermore, the chiral recognition mechanism was studied by density functional theory. The calculation results indicate that the basic chiral recognition system of D-penicillamine complexes involves only one chiral selector and one selected enantiomer, whereas that of N-acetyl-L-cysteine complexes involves two chiral selectors and one selected enantiomer. Finally, the NH3+ group of D-penicillamine is proved to play an important role in enhancing interactions between complexes and improving enantioselectivity. Graphical abstract Enantioselective interactions between amino acid enantiomers and sulfhydryl-compound-functionalized gold nanochannel membranes.

Derivatization and Multidimensional Gas‐Chromatographic Resolution of α‐Alkyl and α‐Dialkyl Amino Acid Enantiomers

AbstractThe quantitative resolution of amino acid enantiomers is crucial to a number of fields of bioanalytical chemistry, including food and plant analysis, metabolomics, medicinal chemistry, and archeochemistry. The enantioselective analysis of α‐dialkyl amino acids, however, has proven more difficult than for proteinaceous α‐protonated amino acids. Herein, the development of an analytical method capable of base‐line resolving enantiomers of α‐alkyl and α‐dialkyl amino acids in two chromatographic dimensions is reported. It was found that enantiomers of α‐dialkyl amino acids can be readily resolved with the use of an enantioselective Chirasil‐Val stationary phase in the first chromatographic dimension and a polyethylene glycol stationary phase in the second dimension. These data offer new possibilities for the enantioselective resolution and precise quantification of non‐biological α‐dialkyl amino acids. This methodology will subsequently be applied particularly towards enantioselective analyses of samples relevant for understanding of the origin of homochirality on Earth such as meteorites, comets, and interstellar ices.

Recent Patents on the Optical Resolution of Amino Acid Enantiomers by Crystallization from Solution

Recent Patents on the Optical Resolution of Amino Acid Enantiomers by Crystallization from Solution

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of D-amino acids typically found in biological fluids and tissues.

Recent Patents on the Optical Resolution of Amino Acid Enantiomers by Crystallization from Solution

Amino acids are a significant class of compounds in a number of different application areas, including pharmaceuticals, nutrition, and nonlinear optical materials. Nearly all amino acids are chiral molecules, and appear in Dand L-forms (enantiomers) which are mirror images of each other and thus share the same chemical properties (if they do not interact with another chiral molecule), apart from their optical rotation. Although the α-amino acids are ubiquitous in natural materials, industrially it is more convenient to produce them with chemical syntheses, which typically are not specific to a desired enantiomer and therefore produce a racemic mixture having equal quantities of the two enantiomers. In most applications of the materials a pure enantiomeric form is required and thus the racemic mixture must be separated into its component enantiomers, a process which is typically known as optical resolution. Optical resolution is often a difficult process because the components separated have identical properties, however a range of ingenious techniques has been developed to perform the resolution. This article reviews the patented techniques to achieve optical resolution of amino acids using methods involving crystallization from solution. These methods represent the most industrially significant resolution methods, in particular preferential crystallization and crystallization of diastereomer salts. A short summary of several types of techniques not using crystallization is also presented. Keywords: Amino acid, Optical resolution, Enantiomer, Preferential crystallization, Resolution via diastereomer salts, Dutch resolution, α-amino acid, β-amino acid

Indirect TLC resolution of amino acid enantiomers after derivatization withMarfey’sreagent and its chiral variants

A simple and rapid method has been established for indirect separation of the optical isomers of seventeen DL amino acids by reversed-phase and normal-phase TLC. Amino acids derivatized with 1-fluoro-2,4-dinitrophenyl-5-L -alaninamide (FDNP-L -Ala-NH 2 ), 1-fluoro-2,4-dinitrophenyl-5-L -phenylalaninamide (FDNP-L -Phe-NH 2 ), or 1-fluoro-2,4-dinitrophenyl-5-L -valinamide (FDNP-L -Val-NH 2 ) were spotted on precoated plates. Diastereomers of all the DL amino acids were separated most effectively by normal-phase TLC with phenol-water, 3:1 ( v / v ), as mobile phase. In reversed-phase TLC the diastereomers were separated most effectively by use of mobile phases containing acetonitrile and triethylamine-phosphate buffer (50 mM, pH 5.5). The results obtained by use of the classical Marfey’s reagent (FDNP-L -Ala-NH 2 ) were compared with those obtained by use of FDNP-L -Phe-NH 2 and FDNP-L -Val-NH 2 . The effects of buffer concentration, pH, and concentration of organic modifier were studied. This indirect metho...

Comparison of the separation efficiencies of Chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained α-amino acids, Phe and Tyr analogs, and 12 β-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol −1 for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.

Reversal Behaviors of the Enantiomer Migration Order and the Stereo-selectivity of Cu(II) Complex with Amino Acid Enantiomers in Ligand Exchange-Micellar Electrokinetic Chromatography

The dependence of the enantioselectivity and enantiomer migration order (EMO) on the chirality and stereo-conformation of ligands used for the chiral selectors of Cu(II) complexes was investigated. It was interestingly observed that EMOs were reversed not only by a change of the ligand chirality of selector, but also by the trans- or cis-conformation of a 4-hydroxy substituent in 4-hydroxyproline. In the presence of the sodium dodecyl sulfate (SDS) micellar phase, the EMOs were reversed when both the bidentate and tridentate ligands of the proline family were used. The resolution (Rs) of enantiomers decreases when the cis-hydroxy substituent exists in d- or l-hydroxyproline. SDS micellar phase improves the resolution of amino acid enantiomers. The relationships among the EMO, ligand chirality, complex stability and stereo-selectivity are discussed.

Highly effective separation and highly sensitive detection for clinical chemistry and biochemistry

Highly effective separation and highly sensitive detection reagents for clinical chemistry and biochemistry were developed and their applications were investigated. The sensitive detection of carboxylic acids was accomplished using 9-anthryldiazomethane (ADAM) which gave intensely fluorescent derivatives from carboxylic acids without catalysts or heating. 1-Pyrenyldiazomethane was then synthesized and proved to react also readily with carboxylic acid and more sensitive than ADAM. The optical resolution of amino acid enantiomers was achieved using 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). GITC derivatized enantiomeric amino acids under mild conditions to give highly hydrophobic diastereomers which could be resolved on conventional reversed phase columns and easily detected by the absorption based on the thiourea structure. Then we devised an o-phthalaldehyde-N-acetylcystein reagent (OPA/NAcCys) giving diastereomers which were also resolved on a reversed phase column and detected fluorometrically with excellent sensitivity. OPA/NAcCys was useful for the assay of D-amino acids in the blood of uremic patients. The hypochlorite-thiamine method for the assay of proteins and peptides was established providing sensitive fluorometry, which well reflected the number of peptide groups in the molecule. This principle was applied to the assay using N-chlorodansylamide, designed for the fluorometry of peptides. The alkaline ninhydrin method was applied to the detection of guanidino compounds in the blood of uremic patients. Several fluorometric methods for the simultaneous detection of reducing and non-reducing carbohydrates, and guanidines were found to be useful reagents for this purpose because these compounds were resistant to the oxidation with periodate. Then protamine-bound columns were prepared for the separation of carbohydrates on HPLC, which showed excellent recovery of reducing carbohydrates in comparison with conventional alkylamino columns.

Synthesis of crosslinked poly(vinyl alcohol) with l‐proline pendant as the chiral stationary phase for resolution of amino acid enantiomers

Synthesis of crosslinked poly(vinyl alcohol) with l‐proline pendant as the chiral stationary phase for resolution of amino acid enantiomers

Synthesis of crosslinked poly(vinyl alcohol) withl-proline pendant as the chiral stationary phase for resolution of amino acid enantiomers

The porous crosslinked poly(vinyl alcohol) beads with the L-proline pendant was synthesized as the chiral stationary phase (CSP) for ligand-exchange chromatography of amino acid racemates via suspension polymerization of vinyl acetate and triallyl isocyanurate as a crosslinker, methanolysis of the copolymer, glycidylation of the formed crosslinked poly(vinyl alcohol), and final functionalization with L-proline. After the polymer with the chiral ligand was complexed with copper(II) cations, it was employed as the CSP to resolve the enantiomers of 15 amino acids. The results showed that the CSP possessed powerful enantioselectivity and all the tried amino acid racemates were completely separated. The large loadability (up to 60 mg per injection) permitted its application on a large scale. © 1996 John Wiley & Sons, Inc.

High-performance liquid chromatographic resolution of amino acid enantiomers derivatized with fluorescent chiral Edman reagents

The fluorescent chiral Edman reagents, 4-(3-isothiocyanotopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [ R(−)- and S(+)-NBD-PyNCS] and 4-(3-isothiocyanatopyrrolidin-1-yl-)7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [ R(−)- and S(+)-BDP-PyNCS], have been utilized for the resolution of amino acid enantiomers as diastereomeric derivatives. These reagents react with amino acid enantiomers in the presence of base catalyst (triethylamine, 1-azabicyclo[2.2.2]octane and 1,8-diazabicyclo[5.4.0]-7-undecene) to produce the corresponding thiocarbamoyl-amino acid diastereomers under the mild reaction conditions of 55°C for 10 min. The resulting derivatives are relatively stable, not only in alkaline reaction solution but also in acidic medium, with no measurable conversion to the thiohydantoin derivatives. The reactivities of both enantiomers of the derivatization reagents with the amino acid enantiomers ( l- and d-isoleucine) are comparable. The fluorescence properties (maximal wavelengths and intensities) of the thiocarbamoyl-amino acid derivatives are dependent upon the solvents in the medium. Some amino acids labeled with the proposed reagents are efficiently resolved by an ODS column with water-acetonitrile containing 0.05% trifluoroacetic acid. The R s values are in the ranges 3.57-0.55 (13 amino acids) for the diastereomers obtained with NBD-PyNCS, and 2.57-0.68 (14 amino acids) for those with DBD-PyNCS. The R s values obtained from neutral and/or aromatic amino acids are larger than those of basic and acidic amino acids.

AOT reversed micelles as carriers of amino acids across liquid membranes. Search for selectivity and chirality effect

Reversed micelles of diethylhexyl sodium sulphosuccinate (AOT) are used in this work as mobile carriers to transport amino acids between a source (S) and a receiving (R) aqueous phase across an organic liquid membrane (M) (isooctane). For most experiments the aqueous and organic phases were those of a pre-equilibrated Winsor II system. Semipermeable membranes have been introduced between the S and M phases as well as between the M and R phases for technical reasons. The rates of transport of p-iodophenylalanine and tryptophan were compared and discussed in relation to their partition coefficients between water and the AOT palisade layer, showing completely different behaviour between the two amino acids. As the selectivity of the transport process may depend on the water content of the membrane phase and also on the carrier/solute interactions, we have investigated the rate of water uptake by the AOT reversed micelles as well as the influence of the ionization state of amino acids. The last part of the work is concerned with an attempt to perform enantioselective transports using chiral AOT. The analysis of the receiving phase required in that case a chromatographic resolution of amino acid enantiomers. This resolution was affected by the nature of the background salt, which had to be specifically adapted for this purpose. The failure of these experiments in producing enantiomeric enrichments suggests that the rigidity of the surfactant film is not sufficient to permit chiral recognition in that case.

Synthesis of Microporous Silica-Gel Particles in a W/O Emulsion and an Application to the Direct Optical Resolution of Amino Acid Enantiomers

Synthesis of Microporous Silica-Gel Particles in a W/O Emulsion and an Application to the Direct Optical Resolution of Amino Acid Enantiomers

Resolution of Amino Acid Enantiomers by Ligand Exchange Chromatography on a New Chiral Packing

Abstract Resolution of α—amino acid enantiomers by ligand exchange chromatography on a new chiral packing, which the chiral ligands were linked onto a hydrophilic polymeric matrix through long spacers, was studied. The packing had high enantioselectivity for most of the amino acids tested. The mechanism of the resolution was discussed. The semi- preparative scale resolution was made.

Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with l- and d-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.

New chiral derivatization reagent for the resolution of amino acids as diastereomers by TLC and HPLC

The applicability of a new chiral reagent to the resolution of amino acid enantiomers has been investigated. The new reagent, S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarbonyl chloride (NSP-C1), was synthesized by the chlorination of S(-)-N-1-(2-naphthylsulphonyl)-2-pyrrolidinecarboxylic acid which was prepared by the reaction of 2-naphthalene sulphonyl chloride with L-proline. Derivatization of the amino acids proceeds rapidly at ambient temperature and no racemization takes place during the reaction. The resolution of the diastereomeric amides was performed by TLC and normal phase HPLC. Complete resolutions were observed for the enantiomers of all amino acids examined except cysteine, cystine and histidine. The favourable UV absorption of the derivatives enabled the optical antipode to be determined down to the 0.1% level.

Chiral recognition in gas chromatographic resolution of amino acid enantiomers

Studies of selectivity of hydrogen bond formation in chiral solute-solvent systems have been performed by1H and13C nuclear magnetic resonance techniques. These data are correlated with the results of gas chromatographic investigations of the same systems. Interactions between the optically active solvent(N-(N-benzoyl-L-amino acid)-anilide) and optically active solute(N-trifluoroacetyl-L-alanyl isopropyl ester) were examined. NMR evidence indicated that hydrogen bonding interaction occurred between two N-H portion and on peptidyl carbonyl portion in stationary phase and solute molecule on three points. The association constants of solvent-solute interaction were calculated and the structure of the diastereomeric association complex between N-(N-benzoyl-L-valyl)-anilide and N-TFA-L-alanyl isopropyl ester was proposed.