Some mechanistic aspects on chiral discrimination of organic acids by immobilized bovine serum albumin (BSA)

Although chiral anionic compounds, notably a large number of organic acids, have been found to be readily separated into enantiomers on BSA-based columns, the structural requirements for an efficient enantiomer discrimination by the protein is still not very well known. Since it is often observed that very hydrophobic acids, like many of the antiinflammatory “profens,” can be resolved with large separation factors for the enantiomers, a systematic study of a series of racemic α-substituted alkanoic acids was made. The series of analytes was prepared from α-amino acids, RCH(NH₂)CO₂H (where R = C₁-C₆), by reaction with N-(chloroformyl)-carbazole. A rapid increase in the capacity ratios of both enantiomers was found with increasing length of R. The effect, however, was larger for the last eluted enantiomer, leading to a substantial increase in the separation factor; this being 7.3 for R = C₆ in 20 mM phosphate buffer (pH 8.0) with 30% of acetonitrile. Further, the separation factor also increased with decreasing organic modifier content. Thus when the R = C₆-analyte was run at a mobile phase concentration of 20% acetonitrile and a flow rate of 1.5 ml/min, the time difference between the two eluted enantiomers exceeded 20 hr. A reasonable interpretation of our results seems to be that enantioselectivity is promoted by increased hydrophobic interaction. Since the anionic charge of the analyte is also taking part in the retention mechanism, a tight binding of the analyte will result from simultaneous electrostatic and hydrophobic interaction. When the latter is increased, less conformational freedom will be left for the analyte and the steric configuration at the α-carbon atom will become more and more important. Steric hindrance by the α-substituent in the first eluted enantiomer will counteract the tight binding caused by the combined binding interactions and lead to a smaller increase in the capacity ratio.     

Chiral α-substituted α-aryloxy acetic acids: Synthesis,absolute configuration,chemical resolution,and direct separation by hplc

Several α-monoalkyl-α-aryloxyacetic acids have been synthesized and resolved into their optical antipodes; their absolute configuration was also established by chiroptical and chemical methods. The two enantiomers of a series of these compounds show opposite effects on skeletal muscle fibers chloride conductance. Therefore a HPLC procedure was developed for the direct determination of the optical purity of the antipodes before submitting them to biological tests. The chromatographic study was performed on DACH-DNB chiral stationary phase which shows a remarkable enantioselectivity for the considered compounds as free acids, esters and amides under different conditions with essentially the same chiral mechanism of separation. © 1992 Wiley-Liss, Inc.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part II. Optical resolution of racemic (thio)glycidyl esters on modified cellulose chiral stationary phases

Chiral chromatography on cellulose tris(3,5-dimethylphenyl carbamate) (Chiralcel OD) and cellulose tribenzoate (Chiralcel OB) coated stationary phases has been successfully used for the optical resolution of rac-(thio)glycidyl esters (acetate, propionate, butyrate). Glycidyl esters could sufficiently be resolved on the OD column whereas for the thio analogues baseline resolution is obtained on CSP OB using hexane/2-propanol mobile phases. The separation factor (α) and resolution (R_S) depend on column temperature, eluent composition, and flow rate, respectively. Best results were obtained for the butyrates and at low temperatures in general. © 1993 Wiley-Liss, Inc.     

Chiral separation of tryptophan enantiomers by liquid chromatography with BSA-silica stationary phase

The separation of tryptophan enantiomers was carried out with medium-pressure liquid chromatography using BSA (bovine serum albumin)-bonded silica as a chiral stationary phase. The influence of various experimental factors such as pH and ionic strength of mobile phase, separation temperature, and the presence of organic additives on the resolution was studied. In order to expand this system to preparative scale, the loadability of sample and the stability of stationary phase for repeated use were also examined. The separation of tryptophan enantiomers was successful with this system. The data indicated that a higher separation factor (α) was obtained at a higher pH and lower temperature and ionic strength in mobile phase. Addition of organic additives (acetonitrile and 2-propanol) in mobile phase contributed to reduce the retention time of L-tryptophan. About 30% of the separation factor was reduced after 80 days of repeated use.     

Chiral discrimination of the enantiomers of δ-phenyl-δ-valerolactone by cellulose triacetate: A chromatographic and microcalorimetric study of the thermodynamics

The resolution of racemic δ-phenyl-δ-valerolactone by chromatography on cellulose triacetate CTA I results in one of the best separations of optical antipodes observed so far on this chiral stationary phase. The thermodynamics of the stereoselective interaction of the enantiomers of δ-phenly-δ-valerolactone have been studied by chromatography at different temperatures and by direct microcalorimetric investigations of the complexation with CTA I. This analysis suggests that the separation process is mainly controlled thermodynamically and that kinetic effects, if any, play a minor role. Microcalorimetric titration experiments indicate that specific (optimum) complexation sites on CTA I for the stronger retained enantiomer of δ-phenly-δ-valerolactone are rapidly saturated, whereas the first eluted enantiomer seems to interact much less selectively with defined interaction sites on the chiral polymer matrix. © 1993 Wiley-Liss, Inc.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Chiral nuclear magnetic resonance shift reagents: Lanthanide mixtures with 1-(1-naphthyl) ethylurea derivatives of amino acids

Esters of 1-(1-naphthly)ethylurea derivatives of L-valine, L-leucine, L-tert-leucine, and L-proline are examined as organic-soluble chiral nuclear magnetic resonance (NMR) resolving agents. The reagents are useful for resolving the spectra of chiral sulfoxides, amines, alcohols, and carboxylic acids. Enantiomeric resolution is caused by a combination of diastereomeric effects and the different association constants of the substrates with the resolving agents. Organic-soluble lanthanide species are added to resolving agent-substrate mixtures and often enhance the enantiomeric resolution. The enhancement occurs because the substrate that exhibits weaker binding with the resolving agent is more available to bond to the lanthanide. Broadening in the spectra with lanthanides is reduced at 50°C. Enantiomeric resolution is still observed at elevated temperatures. Chirality 9:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Enantioselective recognition of carboxylic acids by novel fluorescent triazine‐based thiazoles

Hydrogen bonding and π‐π interactions take special part in the enantioselectivity task. In this regard, because of having both hydrogen acceptor and hydrogen donor groups, melamine derivatives become more of an issue for enantioselectivity. In the light of such information, triazine‐based chiral, fluorescence active novel thiazole derivatives L1 and L2 were designed and synthesized from (S)‐(?)‐2‐amino‐1‐butanol and (1S,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol. The structural establishment of these compounds was made by spectroscopic methods such as FTIR, ¹H, and ¹³C NMR. While the solution of these compounds in DMSO did not show any fluorescence emission, it was observed that the emission increased 44‐fold for L1 and 55‐fold for L2 in 95% water, similar to the aggregation‐induced emission (AIE) characterized compounds. In this regard, enantioselective capabilities of these compounds against carboxylic acids were tested, and in experiments carried out at a ratio of 40/60 DMSO/H₂O, it was determined that R‐2ClMA increased the fluorescence emission of L1 chiral receptor by 2.59 times compared to S‐isomer.     

Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

The resolution of five racemic alpha-lactams by HPLC

The resolution of five racemic alpha-lactams (1a-d,g) using HPLC is reported. Five different Pirkle-type stationary phases were tested. The enantiomers of alpha-lactams containing the trityl group (1a-d) were separated (selectivity factors ranging from 1.08 to 1.20) using a mobile phase of hexane/2-propanol:98/2 and a stationary phase consisting of the 3,5-dinitroaniline derivative of (S)-valine with a urea linkage. Among the dialkyl-substituted alpha-lactams (1e-g), only 1,3-di-tert-butylaziridinone (1g) could be resolved, but only partially (selectivity factor = 1.07), with a mobile phase of hexane/1,2-dichloroethane:95/5 and the stationary phase consisting of the 3,5-dinitrobenzoic acid derivative of (R)-1-naphthylglycine.     

Optical resolution of some biologically active compounds by chiral liquid chromatography on BSA-silica (resolvosil) columns

Optical resolution on the analytical scale of a number of racemic pharmaceuticals and some other biologically active compounds has been studied using immobilized bovine serum albumin (BSA) as the stationary phase. For some of the compounds the elution order was determined by the use of optically enriched fractions obtained from a preceding passage of a sample through a preparative column containing microcrystalline triacetylcellulose (MCTA). The reversal in the sign of optical rotation shown in the polarimetric elution profile from the latter, combined with the integrated peak area ratio obtained on resolution on the analytical column, gave directly the order of elution. For one of the benzothiadiazines studied (bendroflumethiazide), increasing the pH of the mobile phase produced opposite effects on the retention of the two enantiomers, leading to a large effect on the separation factor. For many of the compounds studied, high separation factors (α > 2) could be achieved.     

Fast liquid chromatography for the resolution of chiral compounds

A Pirkle-concept chiral stationary phase (CSP) derived from N-(1-naphthyl)leucine was evaluated for developing methods to reduce analysis times and investigating techniques in the rapid screening of a variety of chiral compounds over a given chiral selector. The effects of reduced column lengths and elevated temperatures were studied to shorten analysis times.     

Separation of Tryptophan Enantiomers by Ligand‐Exchange Chromatography With Novel Chiral Ionic Liquids Ligand

Chiral ionic liquids (CILs) with amino acids as cations have been applied as novel chiral ligands coordinated with Cu²⁺ to separate tryptophan enantiomers in ligand exchange chromatography. Four kinds of amino acid ionic liquids, including [L‐Pro][CF₃COO], [L‐Pro][NO₃], [L‐Pro]₂[SO₄], and [L‐Phe][CF₃COO] were successfully synthesized and used for separation of tryptophan enantiomers. To optimize the separation conditions, [L‐Pro][CF₃COO] was selected as the model ligand. Some factors influencing the efficiency of chiral separation, such as copper ion concentration, CILs concentration, methanol ratio (methanol/H₂O, v/v), and pH, were investigated. The obtained optimal separation conditions were as follows: 8.0 mmol/L Cu(OAc)₂, 4.0 mmol/L [L‐Pro][CF₃COO] ,and 20% (v/v) methanol at pH 3.6. Under the optimum conditions, acceptable enantioseparation of tryptophan enantiomers could be observed with a resolution of 1.89. The results demonstrate the good applicability of CILs with amino acids as cations for chiral separation. Furthermore, a comparative study was also conducted for exploring the mechanism of the CILs as new ligands in ligand exchange chromatography. Chirality 26:160–165, 2014. © 2014 Wiley Periodicals, Inc.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Recent advances in methods of direct optical resolution

Very great advances have been made in the field of direct optical resolution of organic compounds by chromatographic techniques. Chiral capillary gas chromatography now permits a determination of the enantiomeric composition of a few nanograms of a compound present in a mixture of many others. Coupled with high resolution mass spectrometry the technique will additionally permit structural elucidation; of great interest in pheromone research and related areas. Analytical separations of enantiomers are now also carried out by high-performance liquid chromatography (HPLC) methods based on a variety of principles. Basically, two main types are used, differing as to whether the mobile phase has to be a chiral medium or not. Two-dimensional HPLC, whereby compounds separated on a non-chiral column are progressively and automatically transferred to a chiral column for optical resolution, has been used successsfully for chiral amino acid separations. Many different chiral sorbents for preparative LC and HPLC resolutions have been prepared; some of these are now used in columns capable of producing pure enantiomers from a given racemate at a rate of the order of one gram/hour in continuous, automatic HPLC procedures. Apart from all important applications of these results of optical resolution technology, an increased knowledge of the underlying chiral recognition phenomena responsible for enantioselection has also been achieved.     

Chiral separation of flurbiprofen enantiomers by preparative and simulated moving bed chromatography

This study presents the chiral resolution of flurbiprofen enantiomers by preparative liquid chromatography using the simulated moving bed (SMB) technology. Flurbiprofen enantiomers are widely used as nonsteroidal anti‐inflammatory drugs, and although demonstrate different therapeutic actions, they are still marketed as a racemic mixture. The results presented here clearly show the importance of the selection of the proper solvent composition for the preparative separation of flurbiprofen enantiomers. Chiral SMB separation is carried out using a laboratory‐scale unit (the FlexSMB‐LSRE®) with six columns, packed with the Chiralpak AD® stationary phase (20 μm). Results presented include the experimental measurement of equilibrium and kinetic data for two very different solvent compositions, a traditional high hydrocarbon content [10%ethanol/90%n‐hexane/0.01% trifluoroacetic acid (TFA)] and a strong polar organic composition (100%ethanol/0.01%TFA). Experimental data, obtained using the two mobile phase compositions, are used to predict and optimize the SMB operation. After selecting 10%ethanol/90%n‐hexane/0.01%TFA as the most appropriate solvent composition, three feed concentrations of racemic flurbiprofen were considered. Using 40 g/l of racemic flurbiprofen feed solution, the purities for both outlet streams were above 99.4%, the productivity was 13.1 g_feed/(L_bed h), and a solvent consumption of 0.41 L_solvent/g_feed was achieved. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Chiral interaction and stochastic kinetics in stirred crystallization of amino acids

A study of chirally selective interaction in the stirred crystallization of glutamic acid and lysine is presented. The crystallization of S-glutamic acid is influenced by the presence of S-lysine but not R-lysine. Crystal nuclei in stirred systems are produced due to secondary nucleation. Secondary nucleation is an autocatalytic process in which a crystal produces secondary nuclei due to fluid motion, and due to crystal stirrer and crystal-crystal collisions. As a result of this autocatalysis, small fluctuations in the nucleation rates are amplified and the kinetics show a marked stochastic behavior. We investigate the stochastic behavior in detail and propose a kinetic mechanism that explains both the increase and the statistical distribution of the crystallization times of S-glutamic acid due to the presence of S-Lysine. Chirality 10:238–245, 1998. © 1998 Wiley-Liss, Inc.     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

Enantiomeric Separation of Underivatized Amino Acids: Predictability of Chiral Recognition on Ristocetin A Chiral Stationary Phase

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high‐performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R. Chirality 26:132–135, 2014. © 2014 Wiley Periodicals, Inc.     

Enantioselective separation of chiral arylcarboxylic acids on an immobilized human serum albumin chiral stationary phase

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II. Chirality 9:178–183, 1997. © 1997 Wiley-Liss, Inc.