Liquid Chromatographic Enantioseparation of Newly Synthesized Fluorinated Tryptophan Analogs Applying Macrocyclic Glycopeptides-Based Chiral Stationary Phases Utilizing Core-Shell Particles.

Due to the favorable features obtained through the incorporation of fluorine atom(s), fluorinated drugs are a group with emerging pharmaceutical importance. As their commercial availability is still very limited, to expand the range of possible candidates, new fluorinated tryptophan analogs were synthesized. Control of enantiopurity during the synthesis procedure requires that highly efficient enantioseparation methods be available. In this work, the enantioseparation of seven fluorinated tryptophans and tryptophan was studied and compared systematically to (i) develop analytical methods for enantioselective separations and (ii) explore the chromatographic features of the fluorotrytophans. For enantioresolution, macrocyclic glycopeptide-based selectors linked to core-shell particles were utilized, applying liquid chromatography-based methods. Application of the polar-ionic mode resulted in asymmetric and broadened peaks, while reversed-phase conditions, together with mobile-phase additives, resulted in baseline separation for all studied fluorinated tryptophans. The marked differences observed between the methanol and acetonitrile-containing eluent systems can be explained by the different solvation abilities of the bulk solvents of the applied mobile phases. Among the studied chiral selectors, teicoplanin and teicoplanin aglycone were found to work effectively. Under optimized conditions, baseline separations were achieved within 6 min. Ionic interactions were semi-quantitatively characterized and found to not influence enantiorecognition. Interestingly, fluorination of the analytes does not lead to marked changes in the chromatographic characteristics of the methanol-containing eluents, while larger differences were noticed when the polar but aprotic acetonitrile was applied. Experiments conducted on the influence of the separation temperature indicated that the separations are enthalpically driven, with only one exception. Enantiomeric elution order was found to be constant on both teicoplanin and teicoplanin aglycone-based chiral stationary phases (L < D) under all applied chromatographic conditions.

Cinchona-alkaloid-based zwitterionic chiral stationary phases as potential tools for high-performance liquid chromatographic enantioseparation of cationic compounds of pharmaceutical relevance.

Enantiomers of cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs, were separated by high-performance liquid chromatography. Separations were performed on Cinchona-alkaloid-based zwitterionic ion exchanger type chiral stationary phases applied as cation exchangers using mixtures of methanol and acetonitrile or tetrahydrofuran as bulk solvent components containing triethylammonium acetate or ammonium acetate as organic salt additives. On the zwitterionic ZWIX(+) and ZWIX(-) columns investigated, retention and enantioseparation of the studied basic analytes were influenced by the nature and concentration of the organic components of the mobile phase. The effect of organic salt additives on the retention behavior of the studied analytes can be described by the stoichiometric displacement model related to the counterion concentration. Investigations on the structure-retention relationships were performed applying different mobile phase systems for the two types of cationic analytes. For the thermodynamic characterization, parameters such as changes in standard enthalpy (Δ(ΔH°)), entropy (Δ(ΔS°)), and free energy (Δ(ΔG°)) were calculated on the basis of van't Hoff plots derived from the ln α versus 1/T curves. In most cases, enthalpy-driven enantioseparations were observed, with a consistent dependence of the calculated thermodynamic parameters on the mobile phase composition. Elution sequences of the studied compounds were determined in all cases.

Liquid Chromatographic Enantioseparations Utilizing Chiral Stationary Phases Based on Crown Ethers and Cyclofructans.

Natural compounds can exist in different forms, where molecules possessing chirality play an essential role in living organisms. Currently, one of the most important tasks of modern analytical chemistry is the enantioseparation of chiral compounds, in particular, the enantiomers of compounds having biological and/or pharmaceutical activity. Whether the task is to analyze environmental or food samples or to develop an assay for drug control, well-reproducible, highly sensitive, stereoselective, and robust methods are required. High-performance liquid chromatography best meets these conditions. Nevertheless, in many cases, gas chromatography, supercritical fluid chromatography, or capillary electrophoresis can also offer a suitable solution. Amino acids, proteins, cyclodextrins, derivatized polysaccharides, macrocyclic glycopeptides, and ion exchangers can serve as efficient selectors in liquid chromatography, and they are quite frequently applied and reviewed. Crown ethers and cyclofructans possessing similar structural characteristics and selectivity in the enantiodiscrimination of different amine compounds are discussed less frequently. This review collects information on enantioseparations achieved recently with the use of chiral stationary phases based on crown ethers or cyclofructans, focusing on liquid chromatographic applications.

Enantioselective Liquid Chromatographic Separations Using Macrocyclic Glycopeptide-Based Chiral Selectors.

Numerous chemical compounds of high practical importance, such as drugs, fertilizers, and food additives are being commercialized as racemic mixtures, although in most cases only one of the isomers possesses the desirable properties. As our understanding of the biological actions of chiral compounds has improved, the investigation of the pharmacological and toxicological properties has become more and more important. Chirality has become a major issue in the pharmaceutical industry; therefore, there is a continuous demand to extend the available analytical methods for enantiomeric separations and enhance their efficiency. Direct liquid chromatography methods based on the application of chiral stationary phases have become a very sophisticated field of enantiomeric separations by now. Hundreds of chiral stationary phases have been commercialized so far. Among these, macrocyclic glycopeptide-based chiral selectors have proved to be an exceptionally useful class of chiral selectors for the separation of enantiomers of biological and pharmacological importance. This review focuses on direct liquid chromatography-based enantiomer separations, applying macrocyclic glycopeptide-based chiral selectors. Special attention is paid to the characterization of the physico-chemical properties of these macrocyclic glycopeptide antibiotics providing detailed information on their applications published recently.

Cyclodextrin-mediated capillary electrophoresis enantioseparation of dansylated ß-amino acids with bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures.

The present study investigated the separation of bicyclic ß-amino acids with bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures by capillary electrophoresis using native cyclodextrins as well as neutral and charged derivatives as chiral selectors. The amino acids were derivatized with dansyl chloride to provide a UV chromophore. Separations were carried out at 20°C in a 48.5/40 cm, 50 µm fused-silica capillary at an applied voltage of 20 kV. Fifty millimolar sodium phosphate background electrolytes pH 2.5 and 7.2 containing either 5 or 30 mg/mL of the CDs were used. For the majority of the investigated CDs, enantioseparations could only be achieved at pH 2.5 when the analytes are positively charged. Successful enantioseparations as negatively charged analytes at pH 7.2 were only observed for few compounds. In the case of methyl-γ-cyclodextrin, opposite enantiomer migration order was observed in pH 2.5 or 7.2 background electrolytes. Dependence of the enantiomer migration order on the size of the cavity of the cyclodextrins was also found. Furthermore, the degree of methylation of ß-cyclodextrin derivatives affected the migration order of several analyte enantiomers.

High-performance liquid chromatographic and subcritical fluid chromatographic separation of α-arylated ß-carboline, N-alkylated tetrahydroisoquinolines and their bioisosteres on polysaccharide-based chiral stationary phases.

New, pharmacologically interesting chiral amino compounds, namely, stereoisomers of α-hydroxynaphthyl-ß-carboline, benz[d]azepine and benz[c]azepine analogs as well as N-α-hydroxynaphthylbenzyl-substituted isoquinolines were enantioseparated by high-performance liquid chromatographic and subcritical fluid chromatographic methods on polysaccharide-based chiral stationary phases. Separation of the stereoisomers was optimized in both subcritical fluid chromatography and normal phase liquid chromatographic modes by investigating the effects of the composition of the bulk solvent, temperature, and the structures of the analytes and selectors. Both normal phase liquid chromatography and subcritical fluid chromatography exhibited satisfactory performance, albeit with somewhat different effectiveness in the separation of the stereoisomers studied. The optimized methods offer the possibility to apply preparative-scale separations thereby enabling further pharmacological investigations of the enantiomers.

Chiral high-performance liquid and supercritical fluid chromatographic enantioseparations of limonene-based bicyclic aminoalcohols and aminodiols on polysaccharide-based chiral stationary phases.

Enantioseparation of limonene-based bicyclic 1,3-aminoalcohols and 1,3,5- and 1,3,6-aminodiols was performed by normal-phase high-performance liquid chromatographic and supercritical fluid chromatographic (SFC) methods on polysaccharide-based chiral stationary phases. The effects of the composition of the mobile phase, the column temperature and the structures of the analytes and chiral selectors on retention and selectivity were investigated by normal-phase LC and SFC technique. Thermodynamic parameters derived from selectivity-temperature-dependence studies were found to be dependent on the chromatographic method applied, the nature of the chiral selector and the structural details of the analytes. Enantiorecognition in most cases was enthalpically driven but an unusual temperature behavior was also observed: decreased retention times were accompanied by improved separation factors with increasing temperature, i.e. some entropically driven separations were also observed. The elution sequence was determined in all cases. The separation of the stereoisomers was optimized in both chromatographic modalities.

Comparative study on the liquid chromatographic enantioseparation of cyclic ß-amino acids and the related cyclic ß-aminohydroxamic acids on Cinchona alkaloid-based zwitterionic chiral stationary phases.

The enantiomeric pairs of cis and trans stereoisomers of cyclic ß-aminohydroxamic acids and their related cis and trans cyclic ß-amino acids containing two chiral centers were directly separated on four structurally related chiral stationary phases derived from quinine and quinidine modified with (R,R)- and (S,S)-aminocyclohexanesulfonic acids. Applying these zwitterionic ion-exchangers as chiral selectors, the effects of the composition of the bulk solvent, the acid and base additives, the structures of the analytes, and temperature on the enantioresolution were investigated. To study the effects of temperature and obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range 5-50°C. The differences in the changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) were calculated from the linear van't Hoff plots derived from the ln α versus 1/T curves in the studied temperature range. Results thus obtained indicated enthalpy-driven separations in all cases. The sequence of elution of the enantiomers was determined and found to be reversed when ZWIX(-)™ was changed to ZWIX(+)™ or ZWIX(-A) to ZWIX(+A).

Exploring the enantiorecognition mechanism of Cinchona alkaloid-based zwitterionic chiral stationary phases and the basic trans-paroxetine enantiomers.

The enantiomers of trans-paroxetine (the selectand) were separated on four chiral stationary phases incorporating either quinine [ZWIX(+), ZWIX(+A)] or quinidine [ZWIX(-), ZWIX(-A)] and (R,R)-aminocyclohexanesulfonic acid [in ZWIX(-), and ZWIX(+A)] or (S,S)-aminocyclohexanesulfonic acid [in ZWIX(+), and ZWIX(-A)] chiral selectors. The zwitterion nature of the phases is due to the presence of either (R,R)- or (S,S)-aminocyclohexanesulfonic acid in the selector structure bearing the quinuclidine moiety. ZWIX(+) and ZWIX(-) phases are available on the market with the commercial names CHIRALPAK ZWIX(+) and CHIRALPAK ZWIX(-), respectively. With the aim of rationalizing the enantiomer elution order with the above chiral stationary phases, a molecular dynamic protocol was applied and two energetic parameters were initially measured: selectand conformational energy and selectand interaction energy. In the search for other descriptors allowing a better fitting with the experimental evidences, in the present work we consider an energetic parameter, defined as the selector conformational energy, which resulted to be relevant in the explanation of the experimental elution order in most of the cases. Very importantly, the computational data produced by the present study strongly support the outstanding role of the conformational energy of the chiral selector as it interacts with the analytes.

Liquid and subcritical fluid chromatographic enantioseparation of Nα -Fmoc proteinogenic amino acids on Quinidine-based zwitterionic and anion-exchanger type chiral stationary phases. A comparative study.

Stereoselective high-performance liquid chromatographic and subcritical fluid chromatographic separations of 19 Nα -Fmoc proteinogenic amino acid enantiomers were carried out by using Quinidine-based zwitterionic and anion-exchanger-type chiral stationary phases Chiralpak ZWIX(-) and QD-AX. For optimization of retention and enantioselectivity, the ratio of bulk solvent components (MeOH/MeCN, H2 O/MeOH, or CO2 /MeOH) and the nature and concentration of the acid and base additives (counter- and co-ions) were systematically varied. The effect of column temperature on the enantioseparation was investigated and thermodynamic parameters were calculated from the van't Hoff plots ln α vs. 1/T. The thermodynamic parameters revealed that the enantioseparations were enthalpy-driven. The elution sequence was determined in all cases and with the exception of Fmoc-Cys(Trt)-OH, it was identical on both chiral stationary phases whereby the L-enantiomers eluted before the D-enantiomers.

Liquid chromatographic enantioseparation of limonene-based carbocyclic ß-amino acids on zwitterionic Cinchona alkaloid-based chiral stationary phases.

The eight stereoisomers of limonene-based carbocyclic ß-amino acids containing three chiral centers have been directly separated on chiral stationary phases containing Cinchona alkaloid-based zwitterionic selectors. The effects of bulk solvent composition of the mobile phase, the nature of base additives, counterion concentration, and the structure of selector on the enantiorecognition were studied. Experiments were performed at constant mobile phase composition in the temperature range 5-40°C to study the effect of temperature. Thermodynamic parameters were calculated on the basis of the plots of ln α versus 1/T curves. The enthalpically or entropically driven enantioseparations were found to depend strongly on the structures of analyte and selector. The eight stereoisomers of limonene-based carbocyclic ß-amino acids could be differentiated as well-separated peaks in a traditional 1D chromatographic system in two runs by applying the two complementary ZWIX(+)™ and ZWIX(-)™ columns.

High-performance liquid chromatographic enantioseparation of fluorinated cyclic ß(3) -amino acid derivatives on polysaccharide-based chiral stationary phases. Comparison with nonfluorinated counterparts.

The stereoisomers of five fluorinated cyclic ß(3) -amino acid derivatives and their nonfluorinated counterparts were separated on chiral stationary phases containing as chiral selectors cellulose tris-(3,5-dimethylphenyl carbamate), cellulose tris-(3-chloro-4-methylphenyl carbamate), cellulose tris-(4-methylbenzoate), cellulose tris-(4-chloro-3-methylphenyl carbamate), amylose tris-(3,5-dimethylphenyl carbamate) or amylose tris-(5-chloro-2-methylphenyl carbamate). The enantioseparations were carried out in normal-phase mode with n-hexane/alcohol/alkylamine mobile phases in the temperature range 5-40 °C. The effects of the mobile phase composition, the nature and concentration of the alcohol and alkylamine additives, the structures of the analytes and temperature on the separations were investigated. Thermodynamic parameters were calculated from plots of ln α vs. 1/T. The Δ(ΔH°) values ranged between -5.0 and +1.6 kJ/mol, while Δ(ΔS°) varied between -12.6 and +5.7 J/mol/K. The enantioseparation was enthalpically controlled, the retention factor and the separation factor decreasing with increasing temperature, but entropically controlled separation was also observed. The elution sequence was determined for all of the investigated analytes. Copyright © 2016 John Wiley & Sons, Ltd.

A Comparative Study of Enantioseparations of Nα-Fmoc Proteinogenic Amino Acids on Quinine-Based Zwitterionic and Anion Exchanger-Type Chiral Stationary Phases under Hydro-Organic Liquid and Subcritical Fluid Chromatographic Conditions.

The focus of this contribution is a comparative investigation of enantioseparations of 19 Nα-Fmoc proteinogenic amino acids on Quinine-based zwitterionic and anion-exchanger type chiral stationary phases employing hydro-organic and polar-ionic liquid and subcritical fluid chromatographic conditions. Effects of mobile phase composition (including additives, e.g., water, basis and acids) and nature of chiral selectors on the chromatographic performances were studied at different chromatographic modes. Thermodynamic parameters of the temperature dependent enantioseparation results were calculated in the temperature range 5-50 °C applying plots of lnα versus 1/T. The differences in standard enthalpy and standard entropy for a given pair of enantiomers were calculated and served as a basis for comparisons. Elution sequence in all cases was determined, where a general rule could be observed, both in liquid and subcritical fluid chromatographic mode the d-enantiomers eluted before the L ones. In both modes, the principles of ion exchange chromatography apply.

High-performance liquid chromatographic separation of unusual ß(3)-amino acid enantiomers in different chromatographic modes on Cinchona alkaloid-based zwitterionic chiral stationary phases.

Polar-ionic and hydro-organic mobile phase mode of high-performance liquid chromatographic separations of 23 sterically constrained primary ß(3)-amino acid enantiomers containing, alkyl, aryl or heteroaryl side-chains were carried out by using newly developed Cinchona alkaloid-based zwitterionic chiral selectors and the stationary phases Chiralpak ZWIX(+)™ and ZWIX(-)™. In the polar-ionic mode, the effects of the composition of the bulk solvent and the natures of the co- and counter-ions, while in the hydro-organic mode, the effects of the pH, the counter-ion concentration and the structures of the analytes were investigated. The separations of the enantiomers of these 23 primary ß(3)-amino acids, which can be classified as a series of quasi- (pseudo-) homologs, were optimized in both chromatographic modes. The elution sequence was determined in most cases and a reversal of elution order on ZWIX(+)™ and ZWIX(-)™ column was observed. On the basis of this intermolecular recognition model between the selectors and the given enantiomers an indirect assignment of the resolved enantiomer via chromatography is proposed.

High-performance liquid chromatographic enantioseparation of cationic 1,2,3,4-tetrahydroisoquinoline analogs on Cinchona alkaloid-based zwitterionic chiral stationary phases.

The stereoisomers of 1,2,3,4-tetrahydroisoquinoline analogs were resolved for the first time by applying a polar ionic mobile phase on a quinine or a quinidine moiety fused with a chiral sulfonic acid-type chiral selector immobilized on silica [Chiralpak ZWIX(+)™ and Chiralpak ZWIX(-)™]. The effects of the nature and concentrations of the mobile phase components and additives and temperature on the retention and enantioseparation on the investigated chiral columns were studied. Experiments were performed in the temperature range 10-50 °C. Thermodynamic parameters were calculated from plots of ln α versus 1/T. The separations were generally enthalpy-controlled, but entropy-controlled separation was also observed below 30 °C. The enantiomer elution order was determined in some cases and was observed to be opposite on the ZWIX(+)™ and ZWIX(-)™ columns. Our results contribute to a better understanding of the enantiorecognition mechanism of chiral bases with chiral zwitterionic selectors.

High-Performance Liquid Chromatographic Enantioseparation of Cyclic ß-Amino Acids on Zwitterionic Chiral Stationary Phases Based on Cinchona Alkaloids.

Stereoselective high-performance liquid chromatographic separations of eight sterically constrained cyclic ß-amino acid enantiomer pairs were carried out using the newly developed Cinchona alkaloid-based zwitterionic chiral stationary phases Chiralpak ZWIX(+) and ZWIX(-). The effects of the mobile phase composition, the nature and concentrations of the acid and base additives, the counterions and temperature on the separations were investigated. The changes in standard enthalpy, Δ(ΔH°), entropy, Δ(ΔS°), and free energy, Δ(ΔG°), were calculated from the linear van't Hoff plots derived from the ln α vs. 1/T curves in the studied temperature range (10-50°C). The values of the thermodynamic parameters depended on the nature of the selectors and the structures of the analytes. Unusual temperature behavior was observed on the ZWIX(-) column: decreased retention times were accompanied by increased separation factors with increasing temperature. On the ZWIX(+) column only enthalpically, whereas on the ZWIX(-) column both enthalpically and entropically driven separations were observed. The elution sequence was determined in all cases and was observed to be the opposite on ZWIX(+) and on ZWIX(-).

High-performance liquid chromatographic enantioseparation of amino alcohol analogues possessing 1,2,3,4-tetrahydroisoquinoline skeleton on polysaccharide-based chiral stationary phases.

The stereoisomers of 1,2,3,4-tetrahydroisoquinoline amino alcohol analogues and derivatives thereof were separated in normal-phase mode on chiral stationary phases based on preprepared silica coated with cellulose tris-(3,5-dimethylphenyl carbamate), cellulose tris-(3-chloro-4-methylphenyl carbamate), cellulose tris-(4-methylbenzoate) or cellulose tris-(4-chloro-3-methylphenyl carbamate). On all the investigated chiral columns, the retention and the enantioseparation were influenced by the nature and the concentrations of the mobile phase components and additives, and also the temperature. Experiments were performed in the temperature range 10-50°C. Thermodynamic parameters were calculated from plots of lnα vs 1/T. On these polysaccharide-based chiral columns, both enthalpy-driven separations and entropy-controlled enantioseparations were observed. The latter was advantageous with regard to the shorter retention and greater selectivity at high temperature. The sequence of elution of the stereoisomers was determined in all cases.

Cyclodextrin-mediated enantioseparation of phenylalanine amide derivatives and amino alcohols by capillary electrophoresis-role of complexation constants and complex mobilities.

The separation of the enantiomers of phenylalanine amide and N-methyl derivatives as well as some amino alcohols was studied by CE in acidic BGEs using CDs as chiral selectors. The native CDs displayed only low chiral recognition ability at a concentration of 15 mg/mL in 20 mM sodium phosphate buffer, pH 2.5. In contrast, the analyte enantiomers were separated in the presence of randomly sulfated CDs under reversed polarity of the applied voltage. Sulfated ß-CD proved to be the most universal selector resulting in the enantioseparation of all analytes. Opposite enantiomer migration order depending on the size of the CD cavity was observed for phenylalanine amide and 2-amino-2-phenylethanol. The R-enantiomers migrated first in the presence of sulfated α-CD and γ-CD while the S-enantiomers were detected first in the presence of sulfated ß-CD. The enantioseparations could be rationalized based on analyte complexation by the respective CDs except for 2-amino-2-phenylethanol and sulfated ß-CD where essentially equal complexation constants were derived for the enantiomers. In this case, the migration behavior could be attributed to the mobilities of the enantiomer-CD complexes adding another example to the CE-specific phenomenon of enantioseparations based primarily on complex mobilities.

Unusual temperature-induced retention behavior of constrained ß-amino acid enantiomers on the zwitterionic chiral stationary phases ZWIX(+) and ZWIX(-).

The effects of temperature on the chiral recognition of cyclic ß-amino acid enantiomers on zwitterionic [Chiralpak ZWIX(+) and ZWIX(-)] chiral stationary phases were investigated. Experiments were performed at different mobile phase compositions and under 10°C column temperature increments in the temperature range 10-50°C. Apparent thermodynamic parameters and T(iso) values were calculated from plots of ln k and ln α versus 1/T, respectively. Unusual temperature behavior was observed, especially on the ZWIX(-) column, where the application of MeOH/MeCN (50/50 v/v) containing 25 mM triethylamine and 50 mM formic acid as mobile phase led to nonlinear van't Hoff plots and increasing retention time with increasing temperature. On both columns, both enthalpically and entropically driven separations were observed.

Enantiomeric separation of bicyclo[2.2.2]octane-based 2-amino-3-carboxylic acids on macrocyclic glycopeptide chiral stationary phases.

Direct high-performance liquid chromatographic (HPLC) separation of four bicyclo[2.2.2]octane based 2-amino-3-carboxylic acid enantiomers were developed on chiral stationary phases (CSPs) containing different macrocyclic glycopeptide antibiotic selectors. The analyses were performed under reversed-phase, polar organic and polar ionic mode on macrocyclic-glycopeptide-based Chirobiotic T, T2, TAG, and R columns. The effects of the mobile phase composition including the acid and base modifier, the structure of the analytes, and the temperature on the separations were investigated. Experiments were achieved at constant mobile phase compositions on different stationary phases in the temperature range 5-40°C. Thermodynamic parameters were calculated from plots of ln k or ln α versus 1/T. It was recognized that the enantioseparations in reversed-phase and polar organic mode were enthalpically driven, but under polar-ionic conditions entropically driven enantioseparation was observed as well. Baseline separation and determination of elution sequence were achieved in all cases.