N-(n-Butylamide) of (S)-2-(phenylcarbamoyloxy)propionic acid: A new chiral solvating agent,derived from L-lactic acid,for the enantiomeric purity determination of derivatized amino acids

The N-(n-butylamide) of (S)-2-(phenylcarbamoyloxy)propionic acid, easily prepared starting from the inexpensive L -ethyl lactate, can be used as convenient chiral solvating agent (CSA) to determine the enantiomeric composition of N-(3,5-dinitrobenzoyl)amino acid methyl esters.     

Direct separation of albendazole sulfoxide enantiomers by liquid chromatography on a chiral column deriving from (S)-N-(3,5-dinitrobenzoyl) tyrosine: application to enantiomeric assays on plasma samples

The direct enantiomeric resolution of albendazole sulfoxide (SOABZ), an anthelmintic drug belonging to the benzimidazole class, is reported on a chiral stationary phase (CSP) synthesized by covalent binding of (S)-N-(3,5-dinitrobenzoyl)tyrosine-O-(2-propen-1-yl) methyl ester on a gamma-mercaptopropyl-silanized silica gel. A comparison with the resolution achieved on commercially available Pirkle-type CSPs obtained from N-(3,5-dinitrobenzoyl) derivatives of (R)-phenyglycine or (S)-phenylalanine is described. Some structurally related chiral sulfoxides including oxfendazole (SOFBZ) are also studied. Optimization of the mobile phase nature and composition is investigated showing that a hexane-dioxane-ethanol ternary mixture affords an almost baseline resolution (Rs = 1.25); however, in this case, albendazole sulfone (SO2ABZ) is eluted between the two sulfoxide enantiomers; accordingly, a hexane-ethanol mobile phase would be preferred for biological samples containing both metabolites. The influence of temperature on the resolution is depicted with a hexane-ethanol mobile phase. Finally, application to the enantiomeric assays of SOABZ in plasmatic extracts of rat, sheep, bovin, and man after oral administration of albendazole (sulfoxidized to SOABZ and SO2ABZ) is reported. Some distortions in the enantiomeric ratios are evidenced depending on the species.     

Determination of optical purity of 3,5-dimethoxybenzoyl-leucine diethylamide by chiral chromatography and 1H and 13C NMR spectroscopy

(R)-N-3,5-dinitrobenzoyl (DNB) leucine derived chiral selector was used as an HPLC chiral stationary phase for the resolution of various racemic amino acids derivatives. In this study, determination of optical purity of an amino acid derivative was performed by chiral high performance liquid chromatography and 1H and 13C NMR spectroscopy by using the DNB leucine derived chiral selector. The accuracy and precision of each optical purity value are calculated and the data are compared to each other.     

Enantioseparation of N-protected α-amino acid derivatives by liquid-liquid extraction technique employing stereoselective ion-pair formation with a carbamoylated quinine derivative

Two-phase liquid-liquid extraction experiments were undertaken to study the enantioselective transport of the chiral N-protected α-amino acid derivatives from an aqueous buffer solution into an organic phase employing highly lipophilic carbamoylated quinine as chiral selector and phase transfer carrier, respectively. The chiral separation, derived from enantioselective ion-pair formation and differential solubility in the aqueous and organic phases of diastereomeric associates thus formed has been shown to be primarily dependent on the structure of the selectand, the nature of the organic solvent, the molar ratio of a given chiral selector to selectand in the two phases, and the pH of the aqueous phase. Extracted enantiomers were recovered by back-extraction using a relatively polar acidic medium in which the selector is barely insoluble. Thus, the enantiomeric purity of N-(3,5-dinitrobenzoyl)-leucine exceeded 95% enantiomeric excess with 70% overall yield with a single extraction and back-extraction step. Chirality 9:268–273, 1997. © 1997 Wiley-Liss, Inc.     

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.     

A high-performance liquid chromatographic method for the enantiomeric analysis of the enzymatically synthesized coenzyme A ester of 2-tetradecylglycidic acid

The enantiomeric composition of an enzymatically synthesized sample of the coenzyme A ester of 2-tetradecylglycidic acid (TDGA-CoA) was determined by the use of high-performance liquid chromatography with a chiral stationary phase. The stationary phase was commercially available and consisted of (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to aminopropyl silica gel. Analysis was performed using the phenacyl derivative of 2-tetradecylglycidic acid (TDGA), obtained by mild hydrolysis of the TDGA-CoA followed by reaction of the extracted TDGA with phenacyl chloride. Chromatography showed the enantiomeric purity of TDGA-CoA, synthesized in a rat liver microsomal enzyme mixture over a 2-h period, to be a 15.6:1 ratio of the R:S enantiomers (88% ee). The result demonstrates the steroselectivity of the long-chain fatty acid-coenzyme A synthetase for chiral fatty acid epoxide, TDGA.     

Immobilization of difunctional building blocks on hydroxysuccinimide activated silica: versatile in situ preparation of chiral stationary phases.

Several brush-type chiral stationary phases (CSPs) based on undecanoyl- or butanoyl-bound (R,R)-1,2-diphenylethane-1,2-diamine (DPEDA) as chiral selector were prepared by an innovative, fast, and less expensive kind of preparation. The key to this method is the immobilization of the enantiomeric pure diamine with only one amino function in a simple substitution reaction on hydroxysuccinimide ester-activated silica. No excess chiral material is lost. Loading can be easily monitored analyzing the filtrate. The free second amino function can subsequently be acylated with different acyl halogenides. Examples with benzoyl- and 3,5-dinitrobenzoyl (DNB) amides show that, based on our new approach, a library of differently acylated Pirkle-type CSPs can easily be obtained. A benzoylated analog of the commercially available ULMO CSP is shown to be very effective in separating enantiomers of N-acyl amino acids.     

Steric hindrance influence on the enantiorecognition ability of tyrosine-derived chiral stationary phases

Four chiral stationary phases (CSPs) derived from N-(3,5-dinitrobenzoyl)tyrosine have been synthesized. They differ by the substituent nature (methyl, ethyl, isopropyl, tert-butyl) of the aliphatic amide function. The enantiorecognition ability of these CSPs was evaluated with 10 racemates. For the majority of them, the stereoselectivity increases with the steric hindrance of the substituent. The chiral selector enantiomeric separation on the resulting CSPs has evidenced a reversal of elution order only for CS 4 on CSP 4 (tert-butyl substituent), suggesting a change in its conformation.     

1,3,5-Triazine multiselector systems: New tools for chiral discrimination

2-Hexylamino-4-[(S)-1-(1-naphthyl)ethylamino]-6-L-valyl-L-valyl-L-valine isopropylester-1,3,5-triazine (1), a molecule characterized by two different chiral selectors, and 2-hexylamino-4,6-bis-L-valyl-L-valyl-L-valine isopropylester-1,3,5-triazine (2) and 2-ethoxy-4-hexylamino-6-[(S)-1-(1-naphthyl) ethylamino]-1,3,5-triazine (3), systems in which a single kind of chiral selector is present, have been prepared. The enantiodiscriminating ability in solution of the three compounds toward the N-3,5-dinitrobenzoyl derivatives of 1-phenylethylamine (4) or valine methylester (5) has been investigated by ¹H nuclear magnetic resonance (NMR) spectroscopy: 1 shows an improved versatility, relative to 2 and 3, as a chiral solvating agent for NMR spectroscopy. On the basis of the indications obtained, the usefulness of 2-chloro-4-[(S)-1-(1-naphthyl)ethylamino]-6-L-val-L-val-L-valine isopropylester-1,3,5-triazine (1a), a direct precursor of 1, as chiral solvating agent for the determination by NMR of the enantiomeric compositions of derivatives of amines, amino alcohols, amino acids, and carboxyl acids bearing a 3,5-dinitrophenyl moiety, has been demonstrated. Chirality 9:113–121, 1997. © 1997 Wiley-Liss, Inc.     

NMR studies of chiral discrimination relevant to the enantioseparation of N-acylarylalkylamines by an (R)-phenylglycinol-derived chiral selector

Recently, it was reported that the chiral recognition ability of (R)-N-3,5-dinitrobenzoyl phenylglycinol derivative was examined as a new HPLC chiral stationary phase (CSP 1) for the resolution of racemic N-acylnaphthylalkylamines. However, the mechanism of chiral discrimination on the CSP remained elusive until now. In this study, a spectroscopic investigation of the chiral discrimination mechanism of CSP 1 was undertaken using mixtures of (R)-N-3,5-dinitrobenzoyl phenylglycinol-derived chiral selector (2) and each of the enantiomers of N-acylnaphthylalkylamines (3) by NMR study. First, the differences in free energy changes (DeltaDeltaG) upon diastereomeric complexation in solution between the complex of each isomer with chiral selector 2 by NMR titration were calculated. The values were then compared with those estimated by chiral HPLC. The chemical shift changes of each proton on the chiral selector and analytes were also checked and it was found that the chemical shift changes decreased continuously as the acyl group on analytes increased in length. This observation was consistent with the HPLC data. From these experimental results, the interaction mechanism of chiral discrimination between the chiral selector and the analytes is more precisely explained.     

Performance of brush-type HPLC chiral stationary phases with tertiary amide in the connecting tether

The replacement of the N-H hydrogen of the secondary amide-tethered Pirkle-concept N-(3,5-dinitrobenzoyl)-L-leucine derived chiral stationary phase with various pi-basic or aliphatic groups improved the chiral discrimination ability of new chiral stationary phases, based on the leucine- or alanine-derived chiral selector, for the enantiomers of various racemic neutral analytes with amide functional groups. Retention times decreased while separation and resolution factors increased, thus proving the role of pi-donor aromatic unit as an electron-rich shield in the front of a silica surface. In general, chiral stationary phase (CSP) 5 with the 3,5-dimethylphenyl unit showed best performance, while CSP 3, with phenyl unit, and CSP 7, with 1-naphthyl unit in the tertiary amide connecting tether, were less efficient.     

Liquid chromatographic resolution of N-acyl-alpha-amino acids as their anilide derivatives on a chiral stationary phase based on (S)-leucine

A chiral stationary phase (CSP 1) derived from N-(3,5-dinitrobenzoyl)leucine N-phenyl N-alkylamide was used for the liquid chromatographic resolution of anilide derivatives of N-acyl-alpha-amino acids and the chromatographic resolution results were compared with those from four other commercial CSPs. The chromatographic resolution results showed that CSP 1 was most effective among five CSPs used in this study. The chiral recognition mechanism exerted by CSP 1 for the resolution of anilide derivatives of N-acyl-alpha-amino acids is proposed to involve a face-to-face pi-pi interaction and two hydrogen bonding interactions between the CSP and the analytes from the chromatographic resolution behaviors of slightly modified anilide derivatives of N-acyl-alpha-amino acids. The chiral recognition mechanism proposed is quite similar to that advanced previously for the resolution of N-(3,5-methoxybenzoyl)-alpha-amino acids on CSP 1, even though the interaction sites of the two types of analytes were totally different from each other. The apparent similarity of the two chiral recognition mechanisms was assumed to stem from the identical interaction modes of the two types of analytes with the CSP. In addition, the dependence of the enantioselectivity of anilide derivatives of N-acyl-alpha-amino acids on the length of the alkyl tail of the N-acyl group of analytes was rationalized to stem from the intercalation of the N-acyl group of the (R)-enantiomer of analytes between the tethers of the CSP.     

High‐performance liquid chromatography separation of enantiomers of mandelic acid and its analogs on a chiral stationary phase

The enantiomers of mandelic acid and its analogs have been chromatographically separated on a chiral stationary phase (CSP) derived from 4‐(3,5‐dinitrobenzamido) tetrahydrophenanthrene. The rationale of separations of these compounds is discussed with respect to the method development for determining enantiomeric purity and possibility of obtaining enantiomerically pure materials by high‐pressure liquid chromatography. The relationship of analyte structure to the extent of enantiomeric separation has been examined and separation factors (α) are presented for various groups of structurally related compounds. Chiral recognition models have been suggested to account for the observed separations. These models provide mechanistic insights into the chiral recognition process. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Evaluation of π-acid chiral stationary phases deriving from tyrosine and related amino acids for the chromatographic resolution of racemates: Specific requirements for enantiorecognition ability

Chromatographic applications of three novel chiral stationary phases (CSPs) deriving from (S)-(N)-(3,5-dinitrobenzoyl)tyrosine are reported, under liquid chromatographic (LC) and subscritical fluid chromatographic (SubFC) conditions. Two grafting modes of the chiral moiety have been experimented starting either from γ-mercaptopropyl-silanized (type 1) or γ-aminopropyl-silanized (type 2) silica gels. For type 2 CSPs an evaluation of the stability of the amide linkage was achieved by means of SubFC; the relative contriution of ionic and covalent bindings to the ciral recognitio aility was then outlined. The chromatographic properties of these CSPs were compared with those of the corresponding CSPs deriving from phenylglycine, p-hydroxyphenylglycine, and phenylalanine for the resolution of some tertiary phosphine oxide, naphthoyl amide, and α-methylene γ-lactam enantiomers. Some simple requirements regarding the solute and CSP structures for chiral recognition ability can be inferred from these results. In addition, the resolutio of π-acid α-N-(3,5-dinitrobenzoyl)amino esters was investigated on these π-acid CSPs. An example of preparative scale chromatography is also presented.     

Simultaneous analysis of enantiomeric composition of amino acids and N-acetyl-amino acids by enantioselective chromatography

Among the three chiral columns, CHIROBIOTIC T, CHIRLPAK WH, and CHIRALCEL OD-R, tested for the separation of racemic amino acids and N-acetyl-amino acids, only CHIROBIOTIC T chiral column which is based on covalently bonded amphoteric glycopeptide, teicoplanin, as the stationary phase ligand could be successfully developed to enantiomerically separate racemic amino acids and N-acetyl amino acids simultaneously. This method can be used to determine the enantiomeric composition of amino acids and N-acetyl-amino acids in the catalysis of D-aminoacylase or L-aminoacylase and the conversion rate of N-acylamino acid racemases.     

The role of pi-acidic and pi-basic chiral stationary phases in the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids

The application of 3,5-dimethylphenyl-carbamoylated-beta-cyclodextrin (Cyclobond I 2000 DMP) and 2,6-dinitro-4-trifluoromethylphenyl-ether-beta-cyclodextrin-based (Cyclobond DNP) chiral stationary phases for the high-performance liquid chromatographic enantioseparation of unusual beta-amino acids is reported. The investigated amino acids were saturated or unsaturated alicyclic beta-3-homo-amino acids and bicyclic beta-amino acids. Prior to chromatographic analyses, all amino acids were transformed to N-3,5-dinitrobenzoyl- or N-3,5-dimethylbenzoyl form to ensure a pi-acidic or pi-basic function and to enhance the pi-acidic-pi-basic interactions between analytes and chiral selectors. Chromatographic results are given as retention, separation and resolution factors. The chromatographic conditions were varied to achieve optimal separation. The sequence of elution of the enantiomers was determined in some cases.     

Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-beta-cyclodextrin as chiral selector in partial filling mode

Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.     

Species differences in the generation of the chiral sulfoxide metabolite of albendazole in sheep and rats

The prochiral anthelmintic drug albendazole was administered orally to sheep and rats. Blood samples were taken at standardized intervals during the time course of the plasma kinetics: 18 h in rats and 48 h in sheep. The enantiomeric ratio of the sulfoxide metabolite was determined by means of HPLC on a chiral stationary phase, the chiral selector of which was a N-3,5-dinitrobenzoyl derivative of (S)-tyrosine. Two enantiomers were detected in both animal species but their ratios were inverted in rat vs. sheep. The evolution of the ratio is turned from a racemate at 15 min to 60(-):40(+) at 12 h in rats, while it moved from 23(-):77(+) at 3 h to 4(-):96(+) at 36 h after administration in sheep.     

Application of L-proline derivatives as chiral shift reagents for enantiomeric recognition of carboxylic acids

Four proline-derived chiral receptors 5-8 were readily synthesized starting from L-proline. The enantiomeric recognition ability of chiral receptors was examined with a series of carboxylic acids by (1) H NMR spectroscopy. The molar ratio and the association constants of the chiral compounds with each of the enantiomers of guest molecules were determined by using Job plots and a nonlinear least-squares fitting method, respectively. The Job plots indicate that the hosts form 1:1 instantaneous complexes with all guests. The receptors exhibited different chiral recognition abilities toward the enantiomers of racemic guests. Among the chiral receptors used in this study, prolinamide 6 was found to be the best chiral shift reagent and is effective for the determination of the enantiomeric excess of chiral carboxylic acids.     

Chirality sensing with stereodynamic copper(I) complexes

Three Cu(I) complexes derived from stereodynamic diphosphine ligands were synthesized and used for chirality sensing. The coordination of diamines and amino acids to these complexes generates distinct circular dichroism signals. The chiroptical sensor response allows determination of the absolute configuration and the enantiomeric excess of the analyte at low concentrations. This method is operationally simple, fast, and attractive for high‐throughput sensing applications.