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.     

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.     

Absolute configuration of cis-5,6-dihydrodiol enantiomers derived from helical conformers of 1,12-dimethylbenz[a]anthracene

1,12-Dimethylbenz[a]anthracene (1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBA with osmium tetroxide in pyridine in low yield (less than or equal to 3%) and was purified by sequential use of reversed-phase and normal-phase HPLC. Two pairs of 1,12-DMBA cis-5,6-dihydrodiol enantiomers, derived from P (right-handed helix) and M (left-handed helix) conformers, were eluted as a single chromatographic peak on both reversed-phase and normal-phase HPLC. However, these four enantiomers were resolved by sequential use of two chiral stationary phase (CSP) HPLC columns. CSP (Pirkle type I) columns were packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine, which is ionically bonded to gamma-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6-dihydrodiol enantiomers of 4-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene with known absolute stereochemistry.     

Diphenylethanediamine (DPEDA) derivatives as chiral selectors: IV. A comparison of 3,5-dinitrobenzoylated (S,S)- and (S,R)-DPEDA-derived chiral stationary phases with Pirkle's standard (R)-phenylglycine-derived phase in normal phase HPLC

Undecanoyl bound 3,5-dinitrobenzoyl-(S,R)-1,2-diphenylethane-1,2-diamine [(1S,2R)-DNB-DPEDA] as chiral selector (SO) has been synthesized and used as a chiral stationary phase (CSP II) for normal-phase enantioselective HPLC. It is compared with the already published diastereomeric (1S,2S)-DNB-DPEDA-derived CSP I and with the “standard” Pirkle DNB-(R)-phenylglycine-derived CSP III. Chromatographic data for about 100 racemic analytes reveal that CSP II is able to separate especially well enantiomers of derivatized aromatic carboxylic acids and analytes having a benzyl substituent bound at the chiral center. However, CSP I was found to be superior to CSP II and III in its general applicability and its ability to resolve enantiomers of heterocyclic drugs. © 1994 Wiley-Liss, Inc.     

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.     

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.     

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.     

A new model to account for the order in which enantiomers of alkylarylcarbinols elute from a Pirkle chiral HPLC column: preparation, absolute stereochemistry, and chromatographic properties of (+)-1,2-benzocyclononen-3-ol and (+)-1,2-benzocyclodecen-3-ol

Samples enriched in (-)- and (+)-1,2-benzocyclononen-3-ol were prepared by microbially mediated reactions. An enriched sample of (+)-1,2-benzocyclodecen-3-ol was prepared by fractional crystallization of the diastereoisomeric camphanates, followed by hydrolysis. The absolute stereochemistry of both alcohols was established by chemical transformations. The elution order of their enantiomers from a chiral Pirkle HPLC column [(R)-N-(3,5-dinitrobenzoyl)phenyl glycine ionically bound to gamma-aminopropyl silanized silica] was determined. The information in conjunction with other data was used to formulate a rule to predict the configuration of an enantiomer of an alkylarylcarbinol from its elution order from this column.     

Evaluation of chiral discrimination of highly negatively charged enantiomers by quaternary ammonium beta-cyclodextrin using 1H NMR

The positively charged quaternary ammonium cyclodextrin, QA-beta-CD, was previously used as a chiral selector to achieve baseline resolution of two of the dianionic enantiomers of disodium 3-(p-isothiocyanatophenoxy)-3-(p-isothiocyanatophenyl)propane-1,2-disulfate by capillary electrophoresis. The basis of the chiral discrimination between QA-beta-CD and the enantiomers was investigated by (1)H NMR spectroscopy. COSY and NOESY spectra were used to infer the role that molecular interactions and the stereocenters have upon association of QA-beta-CD with the enantiomers. A parallel two-step complexation model is used to rationalize the NMR and the chiral discrimination observed during separation of the enantiomers.     

Chiral recognition of binaphthyl derivatives: a chiral recognition model on the basis of chromatography, spectroscopy, and molecular mechanistic calculations for the enantioseparation of 1,1'-binaphthyl derivatives on cholic acid-bonded stationary phases.

In an effort to elucidate the mechanism of chiral discrimination of cholic acid-based stationary phases, the enantiomeric recognition ability of six chiral stationary phases (CSPs), prepared from differently substituted cholic acid derivatives, was evaluated in normal phase high-performance liquid chromatography (HPLC) with a series of 1,1'-binaphthyl compounds. The influence of structural variations of analytes on retention and enantioselectivity was investigated. Particularly high values of enantioselectivity were observed for the binaphthol enantiomers on a CSP prepared from the allyl 7 alpha,12 alpha-dihydroxy-3 alpha-phenylcarbamoyloxy-5 beta-cholan-24-oate. The complexes of this chiral selector with both enantiomers of binaphthol were studied as models for the interactions responsible for the enantioseparation with the cholic acid-based stationary phases. The 1:1 stoichiometry of the complex in solution was determined by UV titration. The chiral selector dissolved in chloroform exhibited a chiral discrimination for the binaphthol in (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopies. Some aromatic proton and carbon resonances of binaphthol were clearly separated into a pair of peaks due to enantiomers in the presence of the chiral selector. Moreover, on the basis of molecular mechanics calculation, a chiral discrimination model was proposed which nicely explains the relevant chromatographic behavior of the 1,1'-binaphthyl derivatives.     

Preparation of chiral statonary phase from an α-amino phosphonate

A chiral statonary phase (CSP) derived from an N-(3,5-dinitrobenzoyl)-α-aminobenzylphosphonate has been prepared and evaluated for its utility in the direct separation of enantiomers. This CSP, 2, is structurally related to earlier N-(3,5-dinitrobenzoyl)-α-acids acid-derived phases (e.g., CSP 1), but the mode of attachment to the support is different. In scope; CSP 2 is qualitaively similar to CSP 1. However, it differs quantitatively from CSP 1, showing either greater or lesser selectivity for different pairs of enantiomers.     

Evaluation of "click" binaphthyl chiral stationary phases by liquid chromatography

Two "click" binaphthyl chiral stationary phases were synthesized and evaluated by liquid chromatography. Their structures incorporate S-(-)-1,1'-binaphthyl moiety as the chiral selector and 1,2,3-triazole ring as the spacer. These chiral stationary phases (CSPs) allowed the efficient resolution for a wide range of racemic BINOL derivatives, particularly for nonpolar diether derivatives and 3-phenyl indolin-2-one analogs. The chromatographic data showed that the π-π interaction was crucial for enantiorecognition of these CSPs. Loss of enantioselectivity observed on CSP3, which are lacking the triazole ring linkage, indicated that the triazole ring linkage took part in the enantioseparation process, although it was remote from the chiral selector of the CSP. The substitution of the phenyl group at 6 and 6' positions can significantly improve the separation ability of the CSP. The chiral recognition mechanism was also investigated by tracking the elution orders and studying the thermodynamic parameters.     

Investigations of molecular recognition aspects related to the enantiomer separation of 2-methoxy-2-(1-naphthyl)propionic acid using quinine carbamate as chiral selector: An NMR and FT-IR spectroscopic as well as X-ray crystallographic study

The chiral recognition mechanism of a cinchona alkaloid-based chiral stationary phase (CSP) showing high enantiomer discrimination potential for 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid) was investigated. Conformational and structural analyses of the 1:1 complexes of 9-O-(tert-butylcarbamoyl) quinine selector (SO) and MalphaNP acid (selectand, SA) were carried out employing NMR spectroscopy in solution, Fourier-transform infrared (FT-IR) spectroscopy, and solid-state X-ray diffraction analysis. Intramolecular NOEs of a soluble analogue of the CSP afforded the conformational states of the free and complexed form of the selector. The (1)H-NMR spectra revealed that the free form of the SO constitutes anti-open as well as anti-closed and/or syn-closed conformers. Upon complexation with the (S)-MalphaNP acid enantiomer to form the more stable diastereomeric associate, a conformational transition of the selector takes place, resulting in the synthesis of the anti-open conformer nearly exclusively. FT-IR spectra reveal that, besides the primary ion-pairing interaction, stereoselective hydrogen bonding stabilizes the more stable complex via the amide hydrogen of the SO. X-ray diffraction analysis of 9-O-(tert-butylcarbamoyl)quinine and (S)-MalphaNP acid complex further revealed the occurrence of a bidentate H-bond-mediated ionic interaction between SO and SA as well as the lack of pi-pi interaction in the 1:1 complex, and corroborated the conclusions derived from spectroscopic and chromatographic studies.     

The comparison in enantioseparation ability of the chiral stationary phases with single and mixed selector--the selectors derived from two D-tartrates

(2S,3S)-2,3-Bis(3,5-dimethylphenylcarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid and (2S,3S)-2,3-bis(1-naphthalenecarbonyloxy)-3-(benzyloxycarbonyl)-propanoic acid were synthesized from D-tartaric acid. These two compounds were chlorinated to afford two chiral selectors for chiral stationary phases (CSPs). The selectors were separately immobilized on aminated silica gel to give two single selector CSPs; and were simultaneously immobilized to obtain a mixed selector CSP. Comparing to the single selector CSPs, the mixed selector CSP bears the enhanced enantioseparation ability, suggesting that the two selectors in the mixed selector CSP are consistent for chiral recognition in most mobile phase conditions.     

Mechanism of chiral recognition in the enantioseparation of 2-aryloxypropionic acids on new brush-type chiral stationary phases

New brush-type chiral stationary phases (CSP I-IV) comprising N-3,5,6-trichloro-2,4-dicyanophenyl-L-alpha-amino acids (1-4) were prepared by binding of chiral selectors 1-4 to gamma-aminopropyl silica gel. To check the role of excess free aminopropyl groups, CSP V was prepared by binding N-3,5,6-trichloro-2,4-dicyanophenyl-L-alanyl-(3-triethoxysilyl)propylamide to unmodified silica gel. The best separation of racemic 2-aryloxypropionic acids (TR-1-13) was obtained with CSP I; the -(-)-S enantiomer were regularly eluted first, as determined by a CD detector. The mechanism of chiral recognition implies a synergistic interaction of carboxylic acid analyte with the chiral selector and achiral free gamma-aminopropyl units on silica. In fact, CSP V, which is lacking an achiral aminopropyl spacer, shows a lower separation ability for 2-aryloxypropionic acids, but a similar enantioselective discrimination of esters TR-19-20, in comparison with CSP I. CSP I-IV retain unaltered separation ability after a few months of continuous work using a large number of various mobile phases.     

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.     

Enantioseparation of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidones on chiral stationary phases based on 3,5-dimethylanilides of N-(4-alkylamino-3,5-dinitro)benzoyl L-alpha-amino acids

Three novel chiral packing materials for high-performance liquid chromatography were prepared by covalently binding of (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]propan-amide (7), (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonylamino]-4-methylpentanamide (8), and (2S)-N-(3,5-dimethylphenyl)-2-[(4-chloro-3,5-dinitrophenyl)carbonyl-amino]-2-phenylacetamide (9) to aminopropyl silica. The resulting chiral stationary phases (CSPs 1-3) proved effective for the resolution of racemic 4-aryl-3,4-dihydro-2(1H)-pyrimidone derivatives (TR 1-14). The mechanism of their enantioselection, supported by the elution order of (S)-TR 13 and (R)-TR 13 and molecular modeling of the complex of the slower running (S)-TR 13 with CSP 1 is discussed.     

Determination of enantiomeric composition by negative-ion electrospray ionization-mass spectrometry using deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors

The ability to use mixtures of deprotonated N-(3,5-dinitrobenzoyl)amino acids as chiral selectors for the determination of enantiomeric composition by electrospray ionization-mass spectrometry is demonstrated. For each experiment, two N-(3,5-dinitrobenzoyl)amino acids were chosen such that each would have opposite selectivity for the enantiomers of the analyte. Electrospray ionization-mass spectrometry, monitored in the negative ion mode, of solutions containing the two N-(3,5-dinitrobenzoyl)amino acids, sodium hydroxide, and the analyte, in a one-to-one mixture of methanol and water, afford peaks in the mass spectrum that correspond to the deprotonated 1:1 analyte-selector complexes. The ratio of the intensities of the complexes in the mass spectrum can be related to the enantiomeric composition of the analyte. Additionally, the sense and extent of chiral recognition is consistent with chromatographic observations, using chiral stationary phases derived from N-(3,5-dinitrobenzoyl)amino acids. Each analysis of enantiomeric composition requires less than 10 s to complete, indicating that this method has great potential for the development of fast-/high-throughput chiral analyses.     

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.     

Stereospecific effects of benzo[d]isothiazolyloxypropanolamine derivatives at beta-adrenoceptors: synthesis, chiral resolution, and biological activity in vitro

We performed the asymmetric synthesis of four enantiopure benzo[d] isothiazo-3-or 5-yloxypropanolamine derivatives, previously described as competitive antagonists at beta-adrenoceptors. The chemical characterization of each enantiomer was accomplished by (1)H NMR and HPLC/DAD/CD. The direct chromatographic separation of the enantiomers via chiral HPLC was investigated. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). The enantiomers obtained had enantiomeric purities suitable for biological assays. Tested in isolated rat cardiac and intestinal tissues to evaluate their effects at beta(1)- and beta(3)-adrenoceptors, the (S)-enantiomers revealed a higher degree of antagonism than (R)-enantiomers at both subtypes, even though their activity was greater at the cardiac beta(1)-subtype. The potent and cardiospecific antagonistic effect exerted by the compounds tested suggests that the benzisothiazole moiety could be an interesting scaffold for discovering new chiral beta-blocking drugs.