Chiral separation of amino acids and glycyl dipeptides by chiral ligand-exchange capillary electrophoresis comparing Cu(II), Co(II), Ni(II) and Zn(II) complexes of three different sugar acids

This paper deals with comparative studies on the use of copper(II), cobalt(II), nickel(II) and zinc(II) complexes of d-gluconic acid, d-saccharic acid and l-threonic acid as chiral selectors for the enantioseparation of aromatic amino acids and glycyl dipeptides using the principle of ligand-exchange capillary electrophoresis. Although copper(II) is the most frequently used central ion in ligand-exchange capillary electrophoresis, in the case of d-gluconic acid cobalt(II) was shown to be an alternative for the enantioseparation of amino acids. Glycyl dipeptides, however, were resolved only with copper(II) complexes. Zn(II) as a central ion was not effective in all cases and with Ni(II) only some partial separations were achieved.     

Influence of structure and chirality of the selector on the chiral recognition of amino acids using ligand-exchange capillary electrophoresis

The dependence of the chiral recognition ability and enantiomer migration order on the structure, substitution pattern and chirality of chiral selectors used in ligand-exchange capillary electrophoresis is investigated. As chiral selectors different N-alkyl derivatives of proline and hydroxyproline as their copper(II) complexes are used. The influence of the position and conformation of the hydroxy group in the hydroxyproline derivatives and of the structure and chirality of the side chain on enantioselectivity is investigated. Furthermore, the effect of surfactants such as sodium dodecyl sulfate and cetyl trimethyl ammonium bromide on resolution and enantiomer migration order is studied. The investigations were carried out with three aromatic amino acids as model compounds.     

Complex Formation Reactions of Promethazine Copper(II) and Various Biologically Relevant Ligands. Synthesis,Equilibrium Constants,Spectroscopic Characterization and Biological Activity

Binary and ternary complexes of copper(II) involving promethazine, N,N-dimethyl-3-(phenothiazin-10-yl)propylamine (Prom) and various biologically relevant ligands containing different functional groups, were investigated. The ligands (L) are dicarboxylic acids, amino acids, amides and DNA constituents. The ternary complexes of amino acids, dicarboxylic acids or amides are formed by simultaneous reactions. The results showed the formation of Cu(Prom)(L) complexes with amino acids and dicarboxylic acids. The effect of chelate ring size of the dicarboxylic acid complexes on their stability constants was examined. Amides form both Cu(Prom)(L) complexes and the corresponding deprotonated species Cu(Prom)(LH₋₁). The ternary complexes of copper(II) with (Prom) and DNA are formed in a stepwise process, whereby binding of copper(II) to (Prom) is followed by ligation of the DNA components. DNA constituents form both 1:1 and 1:2 complexes with Cu(Prom)²⁺. The stability of these ternary complexes was quantitatively compared with their corresponding binary complexes in terms of the parameters Δlog₁₀ K. The values of Δlog₁₀ K indicate that the ternary complexes containing aromatic amino acids were significantly more stable than the complexes containing alkyl- and hydroxyalkyl-substituted amino acids. The concentration distribution of various complex species formed in solution was also evaluated as a function of pH. The solid complexes [Cu(Prom)L)] where L=1,1-cyclobutanedicarboxylic acid (CBDCA), oxalic and malonic acid were isolated and characterized by elemental analysis, infrared, TGA, and magnetic susceptibility measurements. Spectroscopic studies of the complexes revealed that the complexes exhibits square planar coordination with copper(II). The isolated solid complexes have been screened for their antimicrobial activities using the disc diffusion method against some selected bacteria and fungi. The activity data show that the metal complexes are found to have antibacterial and antifungal activity.     

Chiral separation of dansyl-DL-amino acids with micellar systems containing copper (II) ion and N-n-dodecyl-L-proline in electrokinetic capillary chromatography.

Enantiomers of dansylated DL-amino acids were resolved by chiral copper (II)-N-n-dodecyl-L-proline (1) complexes incorporated in micelles of sodium dodecyl sulfate (SDS) in electrokinetic capillary chromatography (EKC). This resolution is caused by formation of diastereomeric ternary complexes consisting of chiral ligand 1, central copper (II) ion and enantiomeric amino acid derivatives in micellar phase. However, the resolution was not observed when SDS with an anionic polar head grop was replaced with dodecyl trimethylammonium brode (DTMAB) with a cationic polar head group. The ratio between copper (II) ion and 1 in the complex in either SDS or DTMAB was measured by UV-visible spectra, which respond to the d-d transition of copper (II). Mechanism of separation should be discussed in terms of effect of surfactant structures on constitution of copper (II) ion and 1 in the micellar phase and that of arene substituent structures linked to sulfonamide units in amino acid derivatives to be separated.     

Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application to a cell extract

Ligand-exchange micellar electrokinetic capillary chromatography was used for the chiral resolution of underivatized and dansyl amino acid enantiomers simultaneously. The separation was achieved by chiral copper(II)-L-valine complexes incorporated in micelles of sodium dodecyl sulfate (SDS). The enantioresolution was strongly affected by SDS and a concentration of 20 mM SDS was shown to be necessary for the separation. Other impacting factors were investigated including pH, the molar ratio of copper(II) to L-valine and the total concentration of complex. Using the proposed method, 11 different dansyl amino acids and two underivatized amino acids were separated successfully with a running electrolyte of 20 mM NH4OAc, 4 mM CuSO4, 8 mM L-valine and 20 mM SDS at pH 9.0 in less than 25 min. Experiments were also performed with other amino acid ligands in order to vary the stability and the sterical arrangement of the copper(II) complexes and the possible chiral recognition mechanism was also discussed briefly.     

Molecular Recognition of Amino Acids by Copper(II) Complexes of 6(A),6(X)-Diamino-6(A),6(X)-dideoxy-beta-cyclodextrin (X = B, C, D)

The three regioisomers of beta-cyclodextrin 6-difunctionalized with NH(2) groups (6(A),6(X)-diamino-6(A),6(X)-dideoxy-beta-cyclodextrin, A,X-CDNH(2), X = B, C, or D) were synthesized. Their binary and ternary copper(II) complexes with amino acids were characterized by ESR and electronic spectroscopy. Furthermore, the binary copper(II) complexes were used as eluent in ligand exchange chromatography (LEC), to resolve racemates of unmodified amino acids. HPLC separation of enantiomers of aromatic amino acids was obtained only when the complex [Cu(A,B-CDNH(2))](2+) was used as eluent. The two complexes with the other two regioisomers did not show chiral recognition ability. Circular dichroism (c.d.) spectroscopy studies of the ternary complexes with D- and L-amino acids carried out in the presence and in the absence of 1-adamantanol, suggested a recognition mechanism that involves the cyclodextrin cavity, only in the case of ternary A,B-CDNH(2) complexes.     

Amino Acid Ionic Liquids as Chiral Ligands in Ligand‐Exchange Chiral Separations

Recently, amino acid ionic liquids (AAILs) have attracted much research interest. In this paper, we present the first application of AAILs in chiral separation based on the chiral ligand exchange principle. By using 1‐alkyl‐3‐methylimidazolium L ‐proline (L ‐Pro) as a chiral ligand coordinated with copper(II), four pairs of underivatized amino acid enantiomers—dl ‐phenylalanine (dl ‐Phe), dl ‐histidine (dl ‐His), dl ‐tryptophane (dl ‐Trp), and dl ‐tyrosine (dl ‐Tyr)—were successfully separated in two major chiral separation techniques, HPLC and capillary electrophoresis (CE), with higher enantioselectivity than conventionally used amino acid ligands (resolution (R_s)=3.26–10.81 for HPLC; R_s=1.34–4.27 for CE). Interestingly, increasing the alkyl chain length of the AAIL cation remarkably enhanced the enantioselectivity. It was inferred that the alkylmethylimidazolium cations and L ‐Pro form ion pairs on the surface of the stationary phase or on the inner surface of the capillary. The ternary copper complexes with L ‐Pro are consequently attached to the support surface, thus inducing an ion‐exchange type of retention for the dl ‐enantiomers. Therefore, the AAIL cation plays an essential role in the separation. This work demonstrates that AAILs are good alternatives to conventional amino acid ligands for ligand‐exchange‐based chiral separation. It also reveals the tremendous application potential of this new type of task‐specific ILs.     

Elucidation of chiral recognition processes of macrocyclic antibiotic vancomycin

A theoretical investigation was carried out on the retention and separation of enantiomeric molecules including nonsteroidal anti-inflammatory drugs, anti-neoplastic compounds and N-derivatized amino acids by capillary electrophoresis using macrocyclic antibiotics, a new class of chiral selectors, as stationary phase. Firstly docking methods were used to study the enantiorecognition in chiral electrophoresis. The molecular dynamics simulations of the two diastereoisomer complexes were then performed in order to understand how these antibiotics recognize the enantiomers. Another approach was applied in this study to establish a quantitative structure-enantioselectivity relationship (QSER) model, able to describe the resolution of a series of chiral compounds in capillary electrophoresis using vancomycin as the resolving agent.     

Synthesis and characterisation of the 3-amino-derivative of γ-cyclodextrin, showing receptor ability and metal ion coordination properties

The 3-hydroxy group of one glucopyranosinic ring of γ-cyclodextrin was selectively substituted with an amino moiety to obtain a new compound able to complex copper(II). Indeed, the new ligand, an altro-γ-CD, forms stable complexes with Cu(II), as the analogous 3-amino derivative β-CD previously exploited for the chiral separation of some amino acids by ligand exchange mechanism in capillary electrophoresis. Furthermore, the ligand forms a stable inclusion complex with anthraquinone-2-sulfonate.     

Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes

Simultaneous enantioseparations of 15 racemic aromatic amino acids and L-mimosine for their chiral discrimination were achieved by neutral selector-modified capillary electrophoresis (CE) and by charged selector-modified CE. Among the diverse cyclodextrins (CDs) examined, hydroxypropyl (HP)-alpha-CD as the neutral selector and highly sulfated (HS)-gamma-CD as the charged selector provided best chiral environments of different enantioselectivities. Fairly good enantiomeric resolutions were achieved with the HP-alpha-CD mode except for racemic 6-hydroxy-3,4-dihydroxyphenylalanine, threo-3,4-dihydroxyphenylserine and homophenylalanine while high-resolution separations of all the enantiomeric pairs were achieved in the HS-gamma-CD mode except that L-mimosine was not detected and a partial resolution (0.6) for threo-3,4-dihydroxyphenylserine enantiomers. Relative migration times to that of internal standard under the respective optimum conditions were characteristic of each enantiomer with good precision (% RSD: 0.7-3.8), thereby enabling to cross-check the chemical identification of aromatic amino acids and also their chiralities. The method linearity was found to be adequate (r> 0.99) for the chiral assay of the aromatic amino acids investigated. When applied to extracts of three plant seeds, nonprotein amino acids such as L-mimosine (42 nug/g) from Mimosa pudica Linné, and L-3,4-dihydroxyphenylalanine (268 nug/g) from Vicia faba were positively detected along with L-tryptophan, L-phenylalanine and L-tyrosine.     

Chiral separation of unmodified amino acids with non-aqueous capillary electrophoresis based on the ligand-exchange principle

A ligand exchange mechanism in non-aqueous capillary electrophoresis was employed for the separation of eight unmodified amino acids using chiral complexes of copper(II) with L-proline and L-isoleucine. The electrophoretic medium consisted of 25 mM ammonium acetate and 1 M acetic acid in methanol. We were able to completely separate the enantiomeric pairs of each of the investigated racemic amino acids. We also report the optimization of the separation parameters, such as pH*, composition of the complex, and concentration of the complexing agents.     

Chiral separation of NBD-amino acids by ligand-exchange micro-channel electrophoresis.

The chiral separation of amino acid derivatives by ligand-exchange electrophoresis in a microchannel chip was performed for the first time. A Cu(II) complex with L-prolinamide was used as a chiral selector. The migration behaviors of eleven NBD-DL-amino acids were investigated by ligand-exchange capillary electrophoresis (LE-CE). The enantiomer of five NBD-amino acids (Ser, Thr, Val, Phe and His) could be separated by LE-CE using a 20 mM ammonium acetate buffer (pH 9.0) containing 10 mM copper acetate, 20 mM L-prolinamide and 1 mM SDS. NBD-His was eluted in the order D-form and L-form, while the elution order of another enantiomers was L-form and D-form. Under this condition, the enantioseparation of these five NBD-amino acids by ligand-exchange microchip electrophoresis (LE-ME) was investigated using a glass microchip. The enantioseparation of NBD-Ser, -Thr and -His could be successfully accomplished by LE-ME. LE-ME was superior to LE-CE in terms of the short migration time and a good enantiomeric separation.     

Cyclodextrin‐mediated capillary electrophoresis enantioseparation of dansylated β‐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 β‐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 β‐cyclodextrin derivatives affected the migration order of several analyte enantiomers.     

Amino acid recognition of pyridine bis(oxazoline)-copper(II) complex in aqueous solvent

Enantioselective recognition of amino acids has been studied with C₂-symmetric chiral pyridine bis(oxazoline)-copper(II) complexes at physiological pH condition. UV-visible titration revealed strong binding of submillimolar dissociation constant between pyridine bis(oxazoline)-copper(II) complex and amino acids in aqueous solution. Moderate selectivity of up to 2:1 between d- and l-amino acids was achieved. The enantiomers were baseline resolved by capillary electrophoresis, using the bis(l-lysine)-copper(II) complex as a chiral selector.     

Separation of enantiomers of <Emphasis Type="Italic">N</Emphasis>-derivatives of amino acids by capillary electrophoresis using macrocyclic antibiotics

The potential for the application of macrocyclic antibiotic eremomycin as a chiral selector for the separation of enantiomers of N-derivatives of amino acids in capillary electrophoresis was estimated. The influence of several factors (the composition and pH of the running electrolyte, the concentration of the chiral selector, capillary geometry, the value of the applied voltage) on the migration of the derivatives of amino acids and enantioselectivity in the presence of eremomycin was studied in order to choose the separation conditions. Using the example of the dansyl derivatives of amino acids the enantioselectivities of vancomycin and eremomycin in capillary electrophoresis were compared.     

Recent advances in enantioseparations of peptides by capillary electrophoresis

The present review summarizes peptide enantioseparations by capillary electrophoresis with the focus on recent developments. These include the application of new chiral selectors and systematic studies involving series of di- and tripeptides with either common features or with a variety of structures. One section emphasizes mechanistic aspects of the migration order of the enantiomers in cyclodextrin-assisted chiral separations with respect to the complexation constants and the mobilities of the transient diastereomeric complexes. In addition, short paragraphs on the application of chiral capillary electrophoresis to the determination of the stereochemical purity of peptidomimetics and on chiral separations of peptides by capillary electrochromatography have also been included.     

Evaluation of the enantiomeric selectivity in the chiral ligand-exchange chromatography of amino acids by a computational model

The chromatographic resolution of enantiomeric amino acids is accomplished on a reversed phase column using aqueous mobile phase containing the chiral reagent N,N-dimethyl-S-phenylalanine-Cu(II). The separation is a result of the whole interaction between the diastereomeric complex surface and the mixed stationary phase realized by the dynamic coating of the RP-18 carbon chains layer. The elution order seems to be related to the different water coordination capability on copper ion in the formation of the mixed ternary complexes.     

Complex Formation Reactions of (2,2′-dipyridylamine)copper(II) with Various Biologically Relevant Ligands. The Kinetics of Hydrolysis of Amino Acid Esters

Binary and ternary copper(II) complexes involving 2,2′-dipyridylamine (DPA) and various biologically relevant ligands containing different functional groups are investigated. The ligands used are dicarboxylic acids, amino acids, peptides and DNA unit constituents. The ternary complexes of amino acids, dicarboxylic acids or peptides are formed by simultaneous reactions. The results showed the formation of 1:1 complexes with amino acids and dicarboxylic acids. The effect of chelate ring size of the dicarboxylic acid complexes on their stability constants was examined. Peptides form both 1:1 complexes and the corresponding deprotonated amide species. The ternary complexes of copper(II) with DPA and DNA are formed in a stepwise process, whereby binding of copper(II) to DPA is followed by ligation of the DNA components. DNA constituents form both 1:1 and 1:2 complexes with Cu(DPA)²⁺. The concentration distribution of the complexes in solution was evaluated. [Cu(DPA)(CBDCA)], [Cu(DPA)(malonate)] and [Cu(DPA)(oxalate)] were isolated and characterized by elemental analysis, i.r. and magnetic measurements. Spectroscopic studies of [Cu(DPA)(malonate)] revealed that the complex exhibits square planner coordination with copper(II). The hydrolysis of glycine methyl ester (MeGly) is catalyzed by the Cu(DPA)²⁺ complex. The reaction has been studied by a pH-state technique over the pH range 5.8–6.8 at 25 °C and I=0.1 mol dm⁻¹. The kinetic data fits assuming that the hydrolysis proceeds in two steps. The first step, involving coordination of the amino acid ester by the amino and carboxylic group, is followed by the rate-determining attack by the OH⁻ ion. The second step involves equilibrium formation of the hydroxo-complex, Cu(DPA)(MeGly)(OH), followed by intramolecular attack.     

Chiral separation based on ligand-exchange capillary electrophoresis using a copper(II)-L-ornithine ternary complex as selector

A ligand-exchange capillary electrophoresis was explored, with L-ornithine as the ligand and copper(II) as the central ion. Its applicability was demonstrated with underivatized and dansyl amino acids, a dipeptide, and drugs with amino alcohol structure. The enantioselectivity was found to be strongly dependent on pH and copper(II)-L-Orn complex concentration. Due to the adsorption of the positively charged species onto the capillary inner walls, the chiral separation selectivity is very high while the efficiency is relatively low. Permanent 1,3-propanediamine-coated capillaries show an improved separation efficiency and theoretical plate numbers increasing from 10(4) to 10(5). Similar phenomena were observed when sodium dodecyl sulfate (SDS) micelles were added to the copper(II) complex solution. The poor separation efficiency of chiral compounds in uncoated capillaries may result from the low rate of the ligand-exchange reactions, and the high enantioselectivity may derive from the complexing process in the adsorbed phase.