Enantioseparation of Novel Amino Analogs of Indole Phytoalexins on Macrocyclic Glycopeptide-Based Chiral Stationary Phase

Direct chiral separation of the enantiomers of spirobrassinin, 1-methoxyspirobrassinin and ten novel cis- and trans-diastereoisomers of 2-amino analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether on macrocyclic glycopeptide-based chiral stationary phase (CSP) with teicoplanin (Chirobiotic T) was studied. Normal phase eluents containing n-hexane with modifiers ethanol and 2-propanol were used. The effects of mobile phase composition, structure of the analytes and temperature were investigated. Chiral resolution on teicoplanin CSP was achieved only in the case of trans-diastereoisomers. The van’t Hoff plots were found to show linear behavior in all cases. It was found that studied normal phase enantioseparations were enthalpy driven. The elution order of the enantiomers was determined in some cases.

     

High-performance liquid chromatographic enantioseparation of monoterpene-based 2-amino carboxylic acids on macrocyclic glycopeptide-based phases

The enantiomers of five monoterpene-based 2-amino carboxylic acids were directly separated on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Astec Chirobiotic T and T2) and teicoplanin aglycone (Chirobiotic TAG) as chiral selectors. The effects of pH, the mobile phase composition, the structure of the analyte and temperature on the separations were investigated. Experiments were performed at constant mobile phase compositions in the temperature range 10–40 °C to study the effects of temperature and thermodynamic parameters on separations. Apparent thermodynamic parameters and T_iso values were calculated from plots of ln k or ln α versus 1/T. Some mechanistic aspects of the chiral recognition process are discussed with respect to the structures of the analytes. It was found that the enantioseparations were in most cases enthalpy driven. The sequence of elution of the enantiomers was determined in all cases.     

Efficient enantiorecognition of ruthenium(II) complexes by silica-bound teicoplanin

A series of chiral tris-diimine ruthenium(II) complexes have been resolved by HPLC on a chiral stationary phase. The stationary phase (CSP1) was prepared by covalent attachment of the glycopeptide antibiotic teicoplanin to isocyanate activated silica gel. CSP1 selectively retains the enantiomers of [Ru(L)₃]²⁺ (L=2,2′-bypyridine (bpy), 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline), with a preference for the Δ isomer. For the mixed-ligand complexes [Ru(bpy)₂pztr]⁺ and [Ru(bpy)₂pytr]⁺ (Hpztr=3-(pyrazin-2-yl)-1,2,4-triazole, Hpytr=3-(pyridin-2-yl)-1,2,4-triazole), where the triazole unit is bound to the metal centre either through the N² or the N⁴ nitrogen of the ring, CSP1 discriminates both the enantiomers and the regioisomers. Diastereo- and enantioselective association was also observed between CSP1 and the stereoisomers of the dinuclear complex ((Ru(bpy)₂)₂bpt]³⁺ (Hbpt=3,5-bis(pyridin-2-yl)-1,2,4-triazole), with differences in binding affinities of 1.4 kJ/mol between the homochiral enantiomers.     

Evaluation and Comparison of a 3,5‐Dimethylphenyl Isocyanate Teicoplanin with Phenyl Isocyanate Teicoplanin Chiral Stationary Phase Using RP‐HPLC

HPLC enantiomeric separations of 8 α‐amino acids were achieved using two self‐made chiral stationary phases (CSP)–phenyl isocyanate teicoplanin (Phe‐TE) and 3,5‐dimethylphenyl isocyanate teicoplanin (DMP‐TE), using reversed phase mobile phases. The Phe‐TE or the DMP‐TE CSP was prepared from the TE using derivative agents, phenyl isocyanate or 3,5‐dimethylphenyl isocyanate, respectively. The chromatographic results were given as the retention, selectivity, resolution factor and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The effect of pH, organic modifier type and amount were discussed, and the stereoselectivities for two TE‐based CSPs were compared. The chiral selectivity factor for six α‐amino acids on DMP‐TE is somewhat bigger than that on Phe‐TE CSP under reversed phase (RP) mode. Comparison of the enantiomeric separations using self‐made Phe‐TE and DMP‐TE was conducted to gain a better understanding of the chiral recognition mechanism of the macrocyclic glycopeptide CSP.     

LC Enantioseparation of β-Lactam and β-Amino Acid Stereoisomers and a Comparison of Macrocyclic Glycopeptide- and β-Cyclodextrin-Based Columns

Direct reversed-phase high-performance liquid chromatographic methods were developed for the separation of the enantiomers of tricyclic β-lactams, cis-3,4-benzo-6-azabicyclo[3.2.0]heptan-7-one, cis-4,5-benzo-7-azabicyclo[4.2.0]-octan-8-one, cis-5,6-benzo-8-azabicyclo[5.2.0]nonan-9-one and new bicyclic β-amino acids, the six- and seven-membered homologues of cis-1-amino-4,5-benzocyclopentane-2-carboxylic acid (benzocispentacin), cis-1-amino-5,6-benzocyclohexane-2-carboxylic acid and cis-1-amino-6,7-benzocycloheptane-2-carboxylic acid. The direct separations of the analytes were performed on chiral stationary phase (CSP) columns containing the macrocyclic glycopeptide antibiotic teicoplanin (Chirobiotic T), teicoplanin aglycone (Chirobiotic TAG), vancomycin (Chirobiotic V), vancomycin aglycone (Chirobiotic VAG), ristocetin A (Chirobiotic R) or a new dimethylphenyl carbamate-derivatized β-cyclodextrin-based Cyclobond DMP. The results achieved with the different methods were compared in systematic chromatographic examinations. The effects of an organic modifier and of the mobile phase composition on the separation and the separation efficiency of different columns were investigated. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these β-lactams and β-amino acids ranged between 0.3 and −1.1 kJmol⁻¹. Better enantioseparations were attained in most cases on the aglycone CSP.

     

Natural terpene derivatives as new structural task‐specific ionic liquids to enhance the enantiorecognition of acidic enantiomers on teicoplanin‐based stationary phase by high‐performance liquid chromatography

We present the specific cooperative effect of a semisynthetic glycopeptide antibiotic teicoplanin and chiral ionic liquids containing the (1R ,2S ,5R )‐(–)‐menthol moiety on the chiral recognition of enantiomers of mandelic acid, vanilmandelic acid, and phenyllactic acid. Experiments were performed chromatographically on an Astec Chirobiotic T chiral stationary phase applying the mobile phase with the addition of the chiral ionic liquids. The stereoselective binding of enantiomers to teicoplanin in presence of new chiral ionic liquids were evaluated applying thermodynamic measurements and the docking simulations. Both the experimental and theoretical methods revealed that the chiral recognition of enantiomers in the presence of new chiral ionic liquids was enthalpy driven. The changes of the teicoplanin conformation occurring upon binding of the chiral ionic liquids are responsible for the differences in the standard changes in Gibbs energy (ΔG ⁰) values obtained for complexes formed by the R and S enantiomers and teicoplanin. Docking simulations revealed the steric adjustment between the chiral ionic liquids cyclohexane ring (chair conformation) and the β‐d ‐glucosamine ring of teicoplanin and additionally hydrophobic interactions between the decanoic aliphatic chain of teicoplanin and the alkyl group of the tested salts. The obtained terpene derivatives can be considered as “structural task‐specific ionic liquids” responsible for enhancing the chiral resolution in synergistic systems with two chiral selectors.     

Chromatographic enantioseparation of amino acids using a new chiral stationary phase based on a macrocyclic glycopeptide antibiotic

The separation of the enantiomers of several a-amino acids was studied on a new chiral stationary phase (CSP) which is based on the macrocyclic glycopeptide antibiotic eremomycin attached to silica particles. Retention and separation factors were determined under analytical conditions at ambient temperature for different mobile phase compositions. In order to evaluate the potential with respect to preparative separations the adsorption isotherms of D- and L-methionine were determined for one mobile phase composition applying the elution by characteristic point method. The isotherms were validated by comparing experimentally determined elution profiles with predictions based on the equilibrium dispersive model. Finally, the performance of the eremomycin CSP was compared with a commercially available CSP based on the macrocyclic antibiotic teicoplanin. After determining the isotherms of D- and L-methionine also for the teicoplanin phase, the equilibrium dispersive model was used for both CSP to identify optimal operating conditions. For the separation and conditions considered the new eremomycin CSP revealed a better performance compared to the teicoplanin CSP.     

Comparison of the separation efficiencies of chirobiotic T and TAG columns in the separation of unusual amino acids

Two macrocyclic antibiotic type chiral stationary phases (CSPs), based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and Chirobiotic TAG, respectively, were evaluated for the high-performance liquid chromatographic separation of enantiomers of 15 unnatural conformationally constrained alpha-amino acids, Phe and Tyr analogs, and 12 beta-amino acids having cycloalkane or cycloalkene skeletons. The chromatographic results are given as the retention, separation and resolution factors along with the enantioselective free energy difference corresponding to the separation of the enantiomers. It is clearly established that in most cases the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.02 and 0.30 kcal mol(-1) for these particular amino acids. The resolution factors are higher with the aglycone CSP. Although the sugar units generally decrease the resolution of amino acid enantiomers, they can contribute significantly to the resolution of some unusual amino acid analogs. By application of these two CSPs excellent resolutions were achieved for most of the investigated compounds by using reversed phase or polar organic mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition.     

Direct high-performance liquid chromatographic separation of unusual secondary amino acids and a comparison of the performances of Chirobiotic T and TAG columns

Two macrocyclic glycopeptide antibiotic-type chiral stationary phases (CSPs) based on native teicoplanin and teicoplanin aglycone, Chirobiotic T and TAG, respectively, were evaluated with regard to the high-performance liquid chromatographic separation of the enantiomers of 10 secondary alpha-amino acids (imino acids). The chromatographic results are given as the retention, separation and resolution factors, together with the enantioselective free energy difference corresponding to the separation of the enantiomers. By application of these two CSPs, excellent resolutions were achieved for the investigated compounds by using reversed-phase mobile mode systems. The separation conditions were optimized by variation of the mobile phase composition. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP for these particular amino acids ranged between 0.70 and -1.83 kJ mol(-1). It was established that better enantioseparations of the secondary alpha-amino acids were attained in most cases on the aglycone CSP.     

Effect of Steric Hindrance on the Resolution of the Enantiomers of Alkyl Isothiocyanate Derivatives of Amino Acids on a Teicoplanin CSP Using a Methanol-Based Mobile Phase

The effect of steric hindrance on the resolution of the enantiomers of alkyl (i.e. methyl, ethyl, propyl, butyl, and tert-butyl) isothiocyanate derivatives of amino acids on a teicoplanin chiral stationary phase (CSP), with a methanol-based mobile phase, has been studied. Resolution was found to depend on the size of the alkyl group attached to the isothiocyanate reagent and deteriorated as the size of the group increased from methyl to tert-butyl under the same chromatographic conditions. This indicates that interaction between the isothiocyanate group and the chiral selector is important in chiral recognition. Better-than-baseline resolution was achieved for many amino acids with a basic amino or an amide group, for example histidine, lysine, arginine, and asparagine, because of increased solubility in the mobile phase after chemical derivatization. Revised: 22 September and 7 October 2005     

Analytical Separation of Closantel Enantiomers by HPLC

Closantel is an antiparasitic drug marketed in a racemic form with one chiral center. It is meaningful to develop a method for separating and analyzing the closantel enantiomers. In this work, two enantiomeric separation methods of closantel were explored by normal-phase high-performance liquid chromatography. The influences of the chiral stationary phase (CSP) structure, the mobile phase composition, the nature and proportion of different mobile phase modifiers (alcohols and acids), and the column temperature on the enantiomeric separation of closantel were investigated in detail. The two enantiomers were successfully separated on the novel CSP of isopropyl derivatives of cyclofructan 6 and n-hexane-isopropanol-trifluoroacetic acid (97:3:0.1, v/v/v) as a mobile phase with a resolution (Rs) of about 2.48. The enantiomers were also well separated on the CSP of tris-carbamates of amylose with a higher Rs (about 3.79) when a mixture of n-hexane-isopropanol-trifluoroacetic acid (55:45:0.1, v/v/v) was used as mobile phase. Thus, the proposed separation methods can facilitate molecular pharmacological and biological research on closantel and its enantiomers.     

Development of Vancomycin-Capped β-CD-Bonded Silica Particles as Chiral Stationary Phase for LC

Vancomycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (VCD-HPS), a new type of substituted β-cyclodextrin-bonded chiral stationary phase (CSP) for liquid chromatography (LC), have been synthesized by treatment of bromoacetate-substituted-(3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (BACD-HPS) with vancomycin in anhydrous methanol. The stationary phase is characterized by elemental analysis. This new CSP has a chiral selector with two recognition sites: vancomycin and β-cyclodextrin (β-CD), which can provide multiple interactions with the solutes. The chromatographic performance of VCD-HPS was studied with several disubstituted benzenes and some chiral drug compounds as solutes in reversed-phase LC. The results show that VCD-HPS has excellent selectivity for the separation of aromatic positional isomers and enantiomers of chiral compounds.     

Comparative Enantioseparation of Seven Amino Alcohols on Teicoplanin‐Based Chiral Stationary Phases

Abstract

The macrocyclic antibiotics represent a relatively new class of chiral selectors in separation science and teicoplanin‐based chiral stationary phases (CSP) have been used successfully in a number of applications in high‐performance liquid chromatography. In the present studies, we self‐prepared two bonded CSPs–teicoplanin (TE) and teicoplanin phenyl isocyanate (TE‐Phe). Seven amino alcohols, propranolol, bisoprolol fumarate, atenolol, salbutamol, isoproterenol, metoprolol, and labetalol were enantioseparated on both self‐made CSPs using methanol as mobile phase and acetic acid (HOAc) and triethylamine (TEA) as mobile phase additives. On both CSPs, the different enantioseparation behavior of analytes with different structure was compared. The influence of the concentration of mobile phase additives (HOAc and TEA) on the enantioseparation was investigated. In all conditions, the retention factors (k′) of seven analytes on TE‐Phe CSP were larger than that on TE CSP. However, the separation factors (α) and resolutions (Rs) on TE‐Phe CSP were smaller than that on TE CSP. The results indicated that the derivatized TE‐Phe CSP is not efficient as original teicoplanin CSP. Our observations also suggested that, for teicoplanin‐based CSPs, π‐π interactions and dipole‐dipole between solutes and CSPs mainly contribute to the retention of solutes on CSPs while hydrogen bonding and steric interactions play important roles in the chiral recognition for teicoplanin‐based CSPs.     

Evaluation of the macrocyclic glycopeptide A-40,926 as a high-performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.     

Use of short-end injection capillary packed with a glycopeptide antibiotic stationary phase in electrochromatography and capillary liquid chromatography for the enantiomeric separation of hydroxy acids

A new chiral stationary phase (CSP) was prepared by reacting MDL 63,246 (Hepta-Tyr), a glycopeptide antibiotic belonging to the teicoplanin family, with 5-μm diol-silica particles. The CSP mixed with 5-μm amino silica particles (3:1) was packed into 75-μm fused-silica capillaries for only 6.6 cm and used for electrochromatographic experiments analyzing several hydroxy acid enantiomers. A reversed electroosmotic flow carried both analytes and mobile phase towards the anode in a short time (1–3 min), being baseline resolved all the studied analytes. In order to achieve the fastest enantiomeric resolution of the studied hydroxy acids, the effect of several experimental parameters such as mobile phase composition (organic modifier type and concentration, pH of the buffer and ionic strength), capillary temperature and applied voltage on enantioresolution factor, retention time, enantioselectivity were evaluated. The packed capillary column allowed the separation of mandelic acid enantiomers in less than 72 s with resolution factor R_s=2.18 applying a voltage of 30 kV and eluting with a mobile phase composed by 50 mM ammonium acetate (pH 6)–water–acetonitrile (1:4:5, v/v). The CSP was also tested in the capillary liquid chromatography mode resolving all the studied enantiomers applying 12 bar pressure to the mobile phase [50 mM ammonium acetate (pH 6)–water–methanol–acetonitrile, 1:4:2:3, v/v)], however, relatively long analysis times were observed (12–20 min).     

A glycopeptide antibiotic chiral stationary phase for the enantiomer resolution of hydroxy acid derivatives by capillary electrochromatography

Separation of hydroxy acid enantiomers was achieved by using capillary electrochromatography (CEC) employing a chiral stationary phase (CSP) based on MDL 63,246 (Hepta-Tyr), a macrocyclic antibiotic of the teicoplanin family. The chiral selector was chemically bonded to 5 num diol-modified silica particles and the CSP mixed with amino silica (3:1 w/w) was packed into a 75 num ID fused-silica capillary. The CEC experiments were carried out by using an aqueous reversed-phase mode for the enantiomeric resolution of hydroxy acid compounds. Good enantioresolution was achieved for mandelic acid (MA), m-hydroxymandelic acid (m-OH-MA), p-OH-MA, and 3-hydroxy-4-methoxymandelic acid (3-OH-4-MeO-MA). The CEC system was less enantioselective towards 2-phenyllactic acid (2-PhL) and 3-PhL while mandelic acid methyl ester (MA-Et-Est) enantiomers were not resolved. Several experimental parameters, such as organic solvent type and concentration, buffer pH, capillary temperature, on enantioresolution factor, retention time, and retention factor were studied.     

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.     

Comparison of enantioseparation of selected benzodiazepine and phenothiazine derivatives on chiral stationary phases based on β‐cyclodextrin and macrocyclic antibiotics

Enantioselective separation of some phenothiazine and benzodiazepine derivatives was studied on six different chiral stationary phases (CSPs) in HPLC. Selected CSPs, with respect to the structure of the separated compounds, were either based on β‐cyclodextrin chiral selectors – underivatized β‐cyclodextrin and hydroxypropyl ether β‐cyclodextrin, or on macrocyclic antibiotics – vancomycin, teicoplanin, teicoplanin aglycone, and ristocetin A. Measurements were carried out in a reversed‐phase separation mode. The influence of mobile phase composition on retention and enantioseparation was studied. Benzodiazepines could be enantioresolved with almost all the chiral stationary phases used, except for the vancomycin‐bonded CSP. Peak coalescence of oxazepam and lorazepam was observed if separation was carried out at laboratory temperature. Reduced temperature was required in some instances in order to avoid the on‐column racemization. Separation systems composed of teicoplanin‐bonded CSP and buffer‐methanolic or pure methanolic mobile phases were shown to be suitable even for preparative purposes due to high resolution values of the enantiomers. Enantioseparation of phenothiazine derivatives was more difficult to achieve but it was successful, at least partly, also with both types of the CSPs used (except for levomepromazine).     

Enantioseparation of protonated primary arylalkylamines and amino acids containing an aromatic moiety on a pyridino-crown ether based new chiral stationary phase

This paper reports the preparation and testing of a new pyridino-18-crown-6 ether based chiral stationary phase (CSP). The chiral crown ether was covalently bound to silica gel. Circular dichroism (CD) spectroscopy was used for probing the complex formation of the chiral crown ether with the enantiomers of protonated primary arylalkylamines. The (S,S)-dimethylpyridino-18-crown-6 ether selector having a terminal double bond was first transformed to a triethoxysilyl derivative by regioselective hydrosilylation, and then heated with spherical HPLC quality silica gel to obtain the CSP. The discriminating power of the HPLC column filled with the above CSP was tested by using the hydrogenperchlorate salts of racemic α-(1-naphthyl)ethylamine (1-NEA), α-(2-naphthyl)ethylamine (2-NEA) and the hydrochloride salts of aromatic α-amino acids and α-amino acids containing different aromatic side-chain protecting groups.