Enantiomer discrimination of peptides by tandem mass spectrometry: influence of the peptide sequence on chiral recognition

The enantiomer discrimination of peptides by electrospray ionization tandem mass spectrometry is described. A cinchona alkaloid derivative, tert-butylcarbamoylquinine, is used as chiral selector. The chiral selector forms diastereomeric complexes with the peptide enantiomers in the liquid phase (methanolic solution), which are then transferred to the gas phase, where their dissociation behaviour is studied in an ion-trap mass spectrometer. Different degrees of dissociation of the diastereomeric complexes allow for the discrimination of the peptide enantiomers. The influence of the peptide sequence on enantiomer discrimination is discussed and molecular recognition information is derived by comparing the results obtained for related peptides. For dipeptides, small amino acid residues at the N-terminus and bulky side chains at the C-terminus were found to enhance chiral recognition, while for tripeptides the effects were rather irregular.     

Improvement of chiral stationary phases based on cinchona alkaloids bonded to crown ethers by chiral modification

To improve the chiral recognition capability of a cinchona alkaloid crown ether chiral stationary phase, the crown ether moiety was modified by the chiral group of (1S, 2S)‐2‐aminocyclohexyl phenylcarbamate. Both quinine and quinidine‐based stationary phases were evaluated by chiral acids, chiral primary amines and amino acids. The quinine/quinidine and crown ether provided ion‐exchange sites and complex interaction site for carboxyl group and primary amine group in amino acids, respectively, which were necessary for the chiral discrimination of amino acid enantiomers. The introduction of the chiral group greatly improved the chiral recognition for chiral primary amines. The structure of crown ether moiety was proved to play a dominant role in the chiral recognitions for chiral primary amines and amino acids.     

Preparation and evaluation of a chiral stationary phase covalently bound with a chiral pseudo-18-crown-6 ether having a phenolic hydroxy group for enantiomer separation of amino compounds

In order to develop a chiral stationary phase (CSP), which has even higher separation ability than the corresponding commercially available crown ether based CSP (OA-8000 having a pseudo-18-crown-6 ether with an OMe group as a selector), chemically bonded type CSP having a phenolic OH group on a crown ring was developed. Normal mobile phases with or without acid additive can be used with this OH type CSP in contrast to the conventional OMe type CSP which has a neutral chiral selector. Enantiomers of 25 out of 27 amino compounds, including 20 amino acids, 5 amino alcohols, and 2 lipophilic amines, were efficiently separated on a column with this CSP. Nine amino compounds out of 27 were separated with better separation factors than the corresponding OMe type CSP. It is noteworthy that the chromatography on this CSP exhibited excellent enantiomer-separations for amines and amino alcohols when triethyl amine was used as an additive in the mobile phase. Comparison of enantiomer separation ability on this OH type of CSP and on the OMe type of CSP and correlation between the enantioselectivity in chiral chromatography and that of the corresponding model compounds in solution imply that the chiral separation arose from chiral recognition in host guest interactions.     

Chiral analysis by mass spectrometry using the kinetic method in flow systems

Chiral analysis is an important task of analytical chemistry. Besides separation techniques, mass spectrometry can be applied in this field. One mass spectrometric approach is based on Cooks' kinetic method. The method was successfully applied in a static system in which the concentration of the analyte as well as the chiral selector solution was constant during the experiment. The application of the kinetic method in dynamic systems (changing concentration of analyte) is presented. Such systems allow the speeding up of the analytical process (flow injection analysis (FIA)) or the use of the kinetic method for chiral detection after liquid chromatographic separation.The influence of the concentration of the components of the chiral selector solution as well as its flow rate on the recognition of enantiomers was evaluated. A new procedure for correction for the differences between ratio of enantiomers in the liquid phase and their observed ratio in the gas phase is also described. A significant improvement in accuracy using this procedure was achieved. Applicability of the method was demonstrated in the analysis of amino acids using FIA as well as HPLC/MS. After an achiral separation of leucine and isoleucine, chiral mass spectrometric detection was successfully used for enantiomeric recognition.     

Gas-Phase Binding of Noncovalent Complexes Between α-cyclodextrin and Amino Acids Investigated by Mass Spectrometry

Noncovalent complexes between cyclodextrins and small molecules have been extensively studied recently because of their widespread application in the pharmaceutical industry for chiral and molecular recognition. To date, gas phase noncovalent binding affinities between α-cyclodextrin and amino acids have not been widely investigated. In this study, gas-phase binding of noncovalent complexes between α-CD and amino acids was investigated by electrospray ionization mass spectrometry (ESI-MS), demonstrating the formation of 1:1 stoichiometric noncovalent complexes. The binding of the complexes were further confirmed by collision-induced dissociation by tandem mass spectrometry. Mass spectrometric titrations between α-cyclodextrin and phenylalanine, glutamic acid, and arginine were performed to provide binding constants (lgK_a) as references for competitive ESI-MS. Calibration curves for the complexes of α-cyclodextrin with phenylalanine, glutamic acid, and arginine were plotted. Through competitive ESI-MS, the lgK_a for the complexes of α-CD with aspartic acid, lysine, proline, glycine, alanine, asparagine, cystine, glutamine, histidine, leucine, isoleucine, methionine, serine, threonine, and valine were measured directly. By comparison, it is seen that the measured binding constants for the complexes of α-cyclodextrin with basic amino acids such as arginine and lysine are lower than those for most complexes of neutral amino acids. The chiral selectivity of α-cyclodextrin for L- and D-isomers of methionine, threonine, asparagine, and phenylalanine determined by ESI-MS revealed its application as a chiral selector.     

奎宁-冠醚组合型手性固定相直接拆分氨基酸的机理

合成了一种新型奎宁-冠醚组合型手性固定相(QN-CR CSP)并用于氨基酸手性对映体的直接拆分,该固定相对12种氨基酸对映体有良好的手性拆分能力。基于氨基酸手性识别中离子交换和络合的协同作用,建立了一种新型的等温吸附模型。通过迎头特殊点洗脱法(FACP)测定色氨酸(Trp)在不同金属离子添加剂条件下的等温吸附线,验证了模型的合理性。流动相中的Li⁺、Na⁺、K⁺等金属离子与氨基酸竞争固定相中的冠醚络合位点,随着金属离子与冠醚的络合作用力和络合吸附平衡常数增大,固定相对Trp的手性拆分能力下降。该模型的建立对理解氨基酸在此类固定相中的手性保留行为以及固定相结构的进一步优化具有重要意义。     

Enantioseparation of dansyl amino acids by HPLC on a monolithic column dynamically coated with a vancomycin derivative

In this work a chiral stationary phase was prepared by dynamically coating a monolithic reversed-phase HPLC column with a vancomycin-derivative as chiral selector. A hydrophobic alkyl-chain was attached to the vancomycin molecule, providing the immobilization of the chiral selector on the reversed-phase material. Dansyl amino acids were chosen as model analytes for testing the separation power of the dynamically coated phase. All investigated compounds were separated into their enantiomers. Compared with a conventionally packed vancomycin-CSP, a reversal of the enantiomer elution order was obtained.     

Enantioseparation of DPP-4 Inhibitors on Immobilized Crown Ether-Based Chiral Stationary Phase

A group of DPP-4 inhibitors such as alogliptin, linagliptin and saxagliptin were selected for enantioseparation for this study. Crown ether columns contain a chiral crown ether ring as a chiral selector is suitable for the compounds having primary amino groups. Since the selected compounds contain free amino groups, a crown ether column was selected for enantiomeric separation of dipeptidyl peptidase-4 inhibitors. Dipeptidyl peptidase-4 inhibitors are a class of oral hypoglycemics that block dipeptidyl peptidase-4 enzyme and can be used to treat diabetes mellitus type 2. A sensitive HPLC method was developed to separate the enantiomers of each dipeptidyl peptidase-4 inhibitor. Effects of various parameters such as type of solvents, selection of additives, effect of pH and column temperature on chromatographic results were studied. The optimal conditions for the chiral separation of dipeptidyl peptidase-4 inhibitors without any derivatization were investigated.     

New chiral crown ether stationary phase for the liquid chromatographic resolution of alpha-amino acid enantiomers

A new chiral stationary phase (CSP) for the liquid chromatographic separation of enantiomers was prepared by bonding a novel enantiopure (diphenyl-substituted 1,1'-binaphthyl) crown ether to 5 microm silica gel. The resulting CSP was applied to the separation of the enantiomers of various natural and unnatural alpha-amino acids. All alpha-amino acids tested were resolved very well on the new CSP, with the exception of proline, which does not contain a primary amino group. The resolution of alpha-amino acid enantiomers on this new CSP was found to be dependent on the type and amounts of organic and acidic modifiers, and on column temperature.     

Extension of chromatographically derived chiral recognition systems to chiral recognition and enantiomer analysis by electrospray ionization mass spectrometry

Chiral recognition by positive ion electrospray ionization (ESI) mass spectrometry is demonstrated through the adaptation of chromatographically derived chiral recognition systems. Solutions of soluble analogues of chiral selectors used in Pirkle-type chiral stationary phases, when mixed with a chiral analyte, whose enantiomers are known to be resolved on the analogous chiral stationary phase, are shown to afford selector–analyte complexes in the mass spectrum. Pseudo-enantiomeric chiral selectors, where each pseudo-enantiomer has a different mass and a higher affinity for the opposite analyte enantiomer of its pseudo-antipode, were prepared. When mixed with a chiral analyte, solutions of these pseudo-enantiomeric selectors afford selector–analyte complexes in the ESI-mass spectrum where the relative intensities of the selector–analyte complexes are dependent on the enantiomeric composition of the analyte. Additionally, the sense of the observed chiral recognition is in agreement with the sense of chiral recognition observed chromatographically.     

C18柱串联冠醚手性柱高效分离3种芳香族氨基酸对映体

将C18柱与手性冠醚柱串联,建立了一种反相高效液相色谱法用于3种芳香族氨基酸对映体同时拆分的方法.考察了反相色谱流动相的组成、pH值、柱温、流速对对映体拆分的影响.实验结果表明,当流动相为HClO4-乙睛溶液(86:14,V/V,pH 2.0)、柱温20℃、流速0.4 mL/min时,3种氨基酸对映体可获得基线分离.进一步对比了C18柱、冠醚手性柱和串联顺序不同的4种分离模式,结果表明,C18柱不能拆分氨基酸对映体,仅能分离不同种类氨基酸;冠醚手性柱可分离氨基酸映体,但不同种类氨基酸色谱峰出现重叠;串联模式能实现3种氨基酸对映体的基线分离,实现双柱优势互补,而串联顺序对分离影响不大,仅影响色谱峰的峰形.     

Chiral separation of gemifloxacin in sodium-containing media using chiral crown ether as a chiral selector by capillary and microchip electrophoresis

Chiral crown ether, (+)-(18-crown-6)-tetracarboxylic acid (18C6H(4)), is an effective chiral selector for resolving enantiomeric primary amines owing to the difference in affinities between 18C6H(4) and each of the amine enantiomers. In addition to the destacking effect of sodium ion in the sample solution, the strong affinity of sodium ion to the polyether ring of crown ether is unfavorable to chiral capillary electrophoresis using 18C6H(4) as a chiral selector. In this report, the chiral separation of gemifloxacin dissolved in a saline sample matrix using 18C6H(4) was investigated. Adding a chelating agent, ethylenediaminetetraacetic acid (EDTA), to the run buffer greatly improved the separation efficiencies and peak shapes. The successful chiral separation of gemifloxacin in a urinary solution was demonstrated for both capillary and microchip electrophoresis.     

手性配位体交换流动相添加剂法拆分对映体

综述了手性配合基交换色谱法通常采用三种手性相系统中的流动相添加剂方法。主要内容有：（Ａ）手性配合基交换机制,给出了描述对映体对在色谱系统中的保留时间和分离选择性的公式,包括手性选择剂在固定相和流动相中的各种不同情况。公式表明整个色谱往系统的对映体选择性不同于溶液中所存在的选择剂与被选择物作用的情况;（Ｂ）影响配合交换的参数,讨论了金属离子、金属离子／配位体比率、金属离子络合物浓度、固定相、流动相ｐＨ、洗脱顺序、有机调节剂、离子对试剂、流动相离子强度、温度、立体选择性和手性交互识别;（Ｃ）应用。     

Liquid chromatographic enantiomer separation of racemic amine using chiral crown ether stationary phase

Direct enantioseparation of racemic amine, amino-thiophene-2-yl-acetonitrile (TAN), on chiral crown ether stationary phase [Crownpak CR (+)] is described in this study. The elution behavior and the effect of acid additives on resolution of racemic amine, TAN, is intensely investigated. Moreover, the chiral recognition mechanism in this specific system is proposed based on computational methods with the density functional theory. Diastereomeric complexation of the ammonium ion of racemic amine inside the cavity of chiral crown ether appears essential for the chiral discrimination. The pH of the mobile phase containing acid additives also acts as an important factor for the chiral recognition.     

Nonlinear effects in enantioselective chromatography: prediction of unusual elution profiles of enantiomers in non-racemic mixtures on an achiral stationary phase doped with small amounts of a chiral selector

Nonlinear effects caused by molecular association of enantiomers in non-racemic mixtures can cause unexpected effects in chiroptics, NMR spectroscopy, homogeneous catalysis, and chromatography. Herein we present a theoretical model to simulate and verify unusual elution orders of enantiomers on an achiral stationary phase doped with a small amount of a chiral selector or achiral columns coupled with columns doped with a chiral selector. Scenarios with strong, medium, and weak associations of enantiomers, different separation efficiencies typical for flash chromatography and liquid chromatography, and the influence of the enantioselectivity of the chiral selector on the complex equilibria have been investigated. The findings presented here are of importance for the validation of the determination of enantiomeric ratios in not fully separated elution zones as well as for the preparative separation of non-racemic enantiomeric mixtures on chiral stationary phases bonded to achiral matrices.     

HPLC enantioseparation of β2‐homoamino acids using crown ether‐based chiral stationary phase

RP high‐performance liquid chromatographic methods were developed for the enantioseparation of eleven unusual β²‐homoamino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid as chiral selector. The effects of organic (alcoholic) and acidic modifiers, the mobile phase composition and temperature on the separation were investigated. The structures of the substituents in the α‐position of the analytes substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.     

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.     

Existence of a low isoenantioselective temperature in complexation gas chromatography. Profound change of enantioselectivity of a nickel(II) chiral selector either bonded to, or dissolved in, poly(dimethylsiloxane)

The gas chromatographic enantiorecognition of the two enantiomeric pairs of Z- and E-2-ethyl-dioxaspiro[4,4]nonane (chalcogran), respectively, critically depends on whether the chiral selector nickel(II) bis[3-(heptafluorobutanoyl)-(1R)-camphorate] is chemically linked via a methylene spacer to poly(dimethylsiloxane) (Chirasil-Nickel 1), or is only dissolved in poly(dimethylsiloxane) (Chira-Nickel 2a). On 2a, the enantiomers are separated at ambient temperature with a large apparent enantioseparation factor alpha(app) whereas on Chirasil-Nickel 1 only a low alpha(app) is observed whereby the elution order of the enantiomers is reversed. Concise temperature-dependent studies show that on Chirasil-Nickel 1 a low isoenantioselective temperature, T(isoenant), of 80 degrees C is experimentally observed as the result of enthalpy/entropy compensation. For thermodynamic measurements, the method of the retention-increment R' has been employed. This concept separates achiral contributions to retention due to the poly(dimethylsiloxane) matrix, which are identical for enantiomers, from enantioselective contributions to retention, which are due to complexation of enantiomers with the chiral selector. From Van't Hoff plots, concise thermodynamic data of enantioselectivity, -Delta(D),(L)(DeltaG), -Delta(D,L)(DeltaH), Delta(D,L)(DeltaS) and T(isoenant) were obtained. It was found that minute changes in the structure of the spiroketals as well as miniscule differences of the nature of the chiral selector present in the stationary phase (chemically bonded versus physically dissolved) led to profound differences in enantioselectivity. The observation of a low isoenantioselective temperature T(isoenant) represents are rare phenomenon in enantioselective gas chromatography which both complicates the study of chiral recognition mechanisms and the correlation of the absolute configuration and retention due to the temperature-dependent reversal of the elution order.     

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H₄) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H₄ chiral stationary phase to study chiral recognition by 18C6H₄ of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H₄, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H₄. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.     

Thin-layer chromatographic enantiomeric resolution via ligand exchange

A thin-layer chromatographic technique for the separation of proteinogenic and non-proteinogenic amino acids, dipeptides and alpha-hydroxy acids is described. Other examples are given from the field of alpha-methyl, N-alkyl and halogenated amino acids. The separation of the enantiomers is achieved, without derivatization, by means of ligand exchange on a reversed-phase silica gel as stationary phase, which is covered with a chiral selector (proline derivative). The resolution is so good that the respective enantiomers can be determined at trace levels (greater than or equal to 0.25%). The proposed method is simple, inexpensive and needs no sophisticated instruments.