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.     

Role of the weak interactions in enantiorecognition of racemic dihydropyrimidinones by novel brush-type chiral stationary phases

The chiral discrimination ability of two recently prepared chiral stationary phases (CSP 1 and CSP 2), based on a leucine derived chiral selector, was tested for the enantiomers of dihydropyrimidone (DHPM) derivatives and compared with the commercially available Hyun-leucine CSP 3 and classical Pirkle-leucine CSP 4. By combining all of these CSPs, the enantiomers of all DHPM derivatives used in this study can be properly resolved. Particularly good enantioresolutions were achieved for thioureide derivatives, such as Monastrol. The results presented show that sulfur-aromatic interactions are meritorious for these very good separations.     

Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

The mixed chloro‐ and methyl‐ functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs (i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on column temperature was studied in both normal‐phase and reversed‐phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3‐CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.     

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.     

Nanocellulose 3, 5‐Dimethylphenylcarbamate Derivative Coated Chiral Stationary Phase: Preparation and Enantioseparation Performance

Nanocrystalline cellulose (NCC) with high surface area and high ordered crystalline structure was prepared from microcrystalline cellulose (MCC) under the hydrolysis of sodium hypochlorite. NCC was further reacted with 3,5‐dimethylphenyl isocyanate to obtain the nanocellulose derivative, and then coated successfully on the surface of silica gel to a prepared NCC‐coated chiral stationary phase (CSP) as a new kind of chiral separation material. Similarly, MCC derivative‐coated CSP was also prepared as contrast. The chiral separation performance of NCC‐based CSP was evaluated and compared with MCC‐based CSP by high‐performance liquid chromatography. Moreover, the effects of the alcohol modifiers, mobile phase additives, and flow rates on chiral separations were investigated in detail. The results showed that 10 chiral compounds were separated on NCC‐based CSP with better peak shape and higher column efficiency than MCC‐based CSP, which confirmed that NCC‐based CSP was a promising packing material for the resolution of chiral compounds.Chirality 28:376–381, 2016. © 2016 Wiley Periodicals, Inc.     

Detailed experimental and theoretical analysis of chiral discrimination: Enantioselective binding of R/S methyl mandelate by β-cyclodextrin

Enantiodifferentiation of methyl mandelate by β-Cyclodextrin in the liquid phase is explored in detail. Temperature dependent studies using a β-cyclodextrin chiral stationary phase provided differential binding enthalpies and entropies. NMR studies revealed where around the host molecule the guest tends to reside. Molecular dynamics simulations correctly predict the retention order and provide an atomistic account of how chiral discrimination takes place. It is found that short range dispersion forces rather than long range coulombic forces are responsible for both complexation and for enantiodiscrimination. The intermolecular hydrogen bonds are not discriminating and the idea that a tight fit of included species within a cyclodextrin cavity be a requirement for chiral discrimination is questioned. © 1996 Wiley-Liss, Inc.     

Monte Carlo simulations of the chiral recognition of fenoprofen enantiomers by cyclomaltoheptaose (beta-cyclodextrin)

Differential complexation of fenoprofen enantiomers by cyclomaltoheptaose (beta-cyclodextrin) was investigated by Monte Carlo docking simulations. The chiral discrimination of (R)- and (S)-fenoprofen by beta-cyclodextrin was discussed in terms of the difference in the interaction energies and the patterns of molecular interactions. The interaction energies between each enantiomer of fenoprofen and beta-cyclodextrin were consistent with the reported experimental results that showed that the S isomer interacted preferentially with beta-cyclodextrin and was retained longer in a separation process than the R isomer. The thermodynamic preference of inclusion complex formation of (S)-fenoprofen could be explained by the orientation of the phenyl group attached to the chiral carbon, which provided closer contact and thus more favorable intermolecular interactions between the host and guest molecule. The results presented here would be very useful for the prediction of chiral recognition ability of beta-cyclodextrin.     

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.     

HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: Influences of pore size of silica gel,coating amount,coating solvent,and column temperature on chiral discrimination

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was coated on large-pore silica gels and used as a chiral stationary phase (CSP) for high-performance liquid chromatographic separation of enantiomers. The influences of pore size of silica gel, coating amount of CDMPC, coating solvent, and column temperature on chiral discrimination were investigated. CSPs prepared with a large-pore silica gel having a small surface area showed higher chiral recognition. The amount of CDMPC adsorbed on the silica gel influenced the chiral recognition of some racemates. Loading capacity of racemates increased with an increase of the amount of CDMPC supported on the silica gel, and a CSP coated with 45% CDMPC by weight can be used for both analytical and semi-preparative scale separations. The CDMPC, coated using acetone as the coating solvent, exhibited, in many cases, higher enantioselectivity than that obtained with tetrahydrofuran F as the coating solvent. © 1996 Wiley-Liss, Inc.     

Vancomycin as chiral selector for enantioselective separation of selected profen nonsteroidal anti-inflammatory drugs in capillary liquid chromatography

The chiral selector vancomycin was used either as mobile phase additive or bound as a chiral stationary phase (CSP) for the stereoselective separation of seven racemic nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen, carprofen, flurbiprofen, indoprofen, flobufen, ketoprofen, and suprofen, by capillary liquid chromatography. The effect of the type of stationary phase, the chiral column Chirobiotic V or the achiral stationary phases Nucleosil 100 C8 HD and Nucleosil 100 C18 HD, and the concentration of vancomycin in the mobile phase on separation of the drug enantiomers were evaluated. All the drugs, except flobufen, were successfully enantioseparated on Nucleosil 100 C8 HD with 4 mM vancomycin present in the mobile phase (composed of methanol and buffer) in the reversed phase mode. On the vancomycin-bonded chiral stationary phase, it was difficult to get enantioseparations of the profen NSAIDs. However, flobufen gave better enantioseparation on the vancomycin CSP. The better enantioresolution of the majority of profen derivatives on the achiral columns with vancomycin added to the mobile phase can be attributed in particular to the higher separation efficiency of this capillary chromatographic system. In addition, vancomycin dimers, formed in the mobile phase, seem to offer a better steric arrangement for stereoselective interaction to these analytes than the vancomycin bonded on the CSP. These substantial differences in the CS structure significantly influence the chiral discrimination mechanism.     

Chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether for enantiomer separation of amino compounds using a normal mobile phase

In order to apply the excellent chiral recognition ability of chiral pseudo-18-crown-6 ethers that we developed to chiral separation, we prepared a chiral stationary phase (CSP) by immobilizing a chiral pseudo-18-crown-6-type host on 3-aminopropyl silica gel. A chiral column was prepared by the slurry-packing method in a stainless steel HPLC column. A liquid chromatography system using this CSP combined with the detection by mass spectrometry was used for enantiomer separation of amino compounds. A normal mobile phase can be used on this CSP as opposed to conventional dynamic coating-type CSPs. Enantiomers of 18 common natural amino acids were efficiently separated. The chiral separation observed for amino acid methyl esters, amino alcohols, and lipophilic amines was fair using this HPLC system. In view of the correlation between the enantiomer selectivity observed in chromatography and the complexion in solution, the chiral recognition in host-guest interactions might contribute to this enantiomer separation.     

Enantioseparation and molecular modeling study of chiral amines as three naphthaldimine derivatives using amylose or cellulose trisphenylcarbamate chiral stationary phases

This study describes the enantioseparation of three chiral amines as naphthaldimine derivatives, using normal phase HPLC with amylose and cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (CSPs). Three chiral amines were derivatized using three structurally similar naphthaldehyde derivatizing agents, and the enantioselectivity of the CSPs toward the derivatives was examined. The degree of enantioseparation and resolution was affected by the amylose or cellulose-derived CSPs and aromatic moieties as well as a kind of chiral amine. Especially, efficient enantiomer separation was observed for 2-hydroxynapthaldimine derivatives on cellulose-derived CSPs. Molecular docking studies of three naphthaldimine derivatives of leucinol on cellulose tris(3,5-dimethylphenylcarbamate) were performed to estimate the binding energies and conformations of the CSP–analyte complexes. The obtained binding energies were in good agreement with the experimentally determined enantioseparation and elution order.     

Resolution of 2-chloromandelic acid with (R)-(+)-N-benzyl-1-phenylethylamine: chiral discrimination mechanism

During the resolution of 2-chloromandelic acid with (R)-(+)-N-benzyl-1-phenylethylamine, the crystals of the less soluble salt were grown, and their structure were determined and presented. The chiral discrimination mechanism was investigated by examining the weak intermolecular interactions (such as hydrogen bond, CH/π, and van der Waals interactions) and molecular packing mode in crystal structure of the less soluble diastereomeric salt. A one-dimensional double-chain hydrogen-bonding network and a "lock-and-key" supramolecular packing mode are disclosed. The investigation demonstrates that hydrophobic layers with corrugated surfaces can fit into the grooves of one another to realize a compact packing, when the molecular structure of resolving agent is much larger than that of the racemate. This "lock-and-key" assembly is recognized to be another characteristic of molecular packing contributing to the chiral discrimination, in addition to the well-known sandwich-like packing by hydrophobic layers with planar boundary surfaces.     

Preparation of a New Chiral Stationary Phase Based on Macrocyclic Amide Chiral Selector for the Liquid Chromatographic Chiral Separations

A new chiral stationary phase (CSP) based on macrocyclic amide receptor was prepared starting from (1R,2R)‐1,2‐diphenylethylenediamine. The new CSP was successfully applied to the resolution of various N‐(substituted benzoyl)‐α‐amino amides with reasonably good separation factors and resolutions (α = 1.75 ~ 2.97 and R_S = 2.89 ~ 6.82 for 16 analytes). The new CSP was also applied to the resolution of 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs including bendroflumethiazide and methylchlothiazide and metolazone. The resolution results for 3‐substituted 1,4‐benzodiazepin‐2‐ones and some diuretic chiral drugs were also reasonably good. Chirality 28:253–258, 2016. © 2016 Wiley Periodicals, Inc.     

Enantioresolution of Chiral Derivatives of Xanthones on (S,S)‐Whelk‐O1 and l‐Phenylglycine Stationary Phases and Chiral Recognition Mechanism by Docking Approach for (S,S)‐Whelk‐O1

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)‐Whelk‐O1 and l ‐phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal‐phase, polar‐organic, and reversed‐phase). The (S,S)‐Whelk‐O1 CSP, under polar‐organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)‐Whelk‐O1 CSP by molecular docking technique. Data regarding the CSP–CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP–CDX interactions. Chirality 25:89–100, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution

Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.     

Preparation and application of a new doubly tethered chiral stationary phase containing N-CH3 amide linkage based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

A new doubly tethered chiral stationary phase (CSP 5) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid was developed by attaching the second tethering group to silica gel through a carbon atom of the first tethering group of the corresponding singly tethered CSP (CSP 2) containing an N-CH3 tertiary amide linkage, which was previously developed in our laboratory, in order to enhance the CSP stability without the loss of chiral recognition efficiency. The new CSP was quite effective in the resolution of various racemic alpha-amino acids, amines, and amino alcohols, and the chiral recognition efficiency of the new CSP was even greater than that of the corresponding singly tethered CSP especially in terms of the resolution factors (RS). The stability of the new CSP was greater than that of the corresponding singly tethered CSP. The chromatographic resolution behaviors of the new CSP were generally consistent with those of the corresponding singly tethered CSP.     

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.     

Direct enantioselective separation of some propranolol analogs by HPLC on normal and reversed cellulose chiral stationary phases

The enantiomeric separation of several racemic aryloxyaminopropan-2-ol derivatives related to propranolol on normal and reversed phase of cellulose tris (3,5-dimethylphenylcarbamate) chiral stationary phases known as Chiralcel OD and Chiralcel OD-R were studied. It was observed that the chiral separation depends on the substitution pattern of the aryl group, i.e., 1-naphthyl, 2-naphthyl, and phenyl group and polarity on the basic nitrogen in the side chain. In both normal and reversed phase modes the (+)-R-enantiomer eluted first in all of the analogs resolved. It can be concluded that: (1) substituents on the side chain did affect the interaction of the enantiomers with the polar carbamate moiety in the CSP; and (2) the dipole-dipole stacking between the π-donor 3,5-dimethylphenyl carbamate group pending from the glucose rings of the CSP and π-acceptor aryl group of the analyte is crucial for the efficient chiral discrimination. The chiral recognition mechanism(s) between these analogs and the chiral stationary phases are proposed. © 1996 Wiley-Liss, Inc.