The HPLC Enantioresolution of Phenyl Isothiocyanated Amino Acids on Vancomycin Bonded Phase Using the Acetonitrile‐Based Mobile Phase: A Comparison to the Teicoplanin Phase

The phenyl isothiocyanate, an electrophilic reagent for peptide chain sequencing, is used to pre‐column derivatize a variety of α‐amino acids in alkaline medium before their enantioresolution on a vancomycin bonded chiral phase using the acetonitrile‐based mobile phase. The observed resolution is believed to be due to the re‐location of the hydrogen receptor site from sulfur to nitrogen on the isothiocyanyl fragment of derivatizing reagent, which in turn changes the enantioselectivity. Under the same chromatographic conditions, the resolution for N‐benzoylated, 3,5‐dinitrobenzoylated and N‐carbobenzyloxylated amino acids is either not found or unsatisfactory. Also, no resolution is obtained in the reversed‐ or normal phase mode for all phenyl isothiocyanated amino acids examined in this study.     

The Enantioresolution of n-Benzoyl and Its Analogs Derivatized Amino Acids on Cyclodextrin Bonded Chiral Stationary Phases Using a Nonaqueous Acetonitrile-Based Mobile Phase

The separation of amino acids into enantiomers while derivatizing with benzoyl chlorides is demonstrated. It was found that enantioselectivity among these reagents tagging with the same amino acid is quite different and is very sensitive to its structural variations. A noninclusion complexation mechanism i.e. external association is thought to involve in the separation carried out with a nonaqueous polar organic mobile phase. Results also indicate that the chiral recognition seems to be controlled either by moieties from tagging reagents or functional groups on all amino acids examined in this study on the basis of competition. The polarity of side-chain group on peptide backbone can not be ignored in achieving a successful separation. Furthermore, chiral recognition appears to closely rely upon the location of stereogenic center that is simultaneously attached by amino and carboxyl groups. Finally, mechanism involved will be explored structurally with these tagging reagents derivatized amino acids which structures are highly ordered and amine-containing compounds with structures similar to that of amino acid.     

The Enantioresolution of Methylthiohydantoin-Amino Acids on R,S-2-hydroxypropyl Derivatized β-Cyclodextrin Bonded Stationary Phase Using the Water-Based Mobile Phase

The enantioresolution of thirteen methylthiohydantoin-amino acids (MTH-amino acids), which are substrates for producing D-amino acids through enzyme-catalyzed hydrolysis, is described on R, S-2-hydroxypropyl derivatized β-cyclodextrin bonded stationary phase (RSP-β-CD CSP) with a water-based mobile phase. The enantioresolution is relatively sensitive to the structural variation of group which is attached to the carbon at position five on methylthiohydantoin moiety and subsequently turns into side-chain group of corresponding D-amino acid produced after hydrolysis. The inclusion complexation is believed to be the mechanism responsible for the observed enantioresolution that cannot be reproduced either on native β-cyclodextrin CSP under the same chromatographic conditions or on both CSPs using the acetonitrile-based mobile phase. Approaches for enantioimprovement include varying the percentage of organic modifier in the mobile phase and using the buffer-typed mobile phase such as triethylammonium acetate (pH 4.1).     

The HPLC Stereoselective Resolution of N-2,4-Dinitrophenylated (DNP) Amine-Containing Enantiomers on Teicoplanin Bonded Phase Using the Methanol-Based Mobile Phase

A variety of compounds containing an amine group (i.e., amino acids and amino alcohols) are fast and much better than baseline enantioresolved on a teicoplanin bonded chiral phase using the methanol-based mobile phase after their pre-column derivatization with 2,4-dinitrofluorobenzene (DNFB) in alkaline medium. The resolution is found relatively insensitive to the structural variations of the analyte and is enhanced as the stereogenic center of analyte is close to the nitro group substituted aromatic moiety of the tagging reagent for stronger - interaction. The resolution is either not observed or unsatisfactory using the acetonitrile-based mobile phase.     

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     

High Performance Liquid Chromatographic Enantioresolution of Benzoyl Amino Acid,Dipeptides and Tripeptide on β-Cyclodextrin Bonded Stationary Phase Using Polar-Organic Acetonitrile as the Mobile Phase

The enantiomeric resolution of several dipeptides, amino acid (i.e., isoleucine) and tripeptide (i.e., Leu-Gly-Phe) with two stereogenic centers on β-cyclodextrin bonded chiral stationary phase (β-CD CSP) using polar-organic acetonitrile as the mobile phase is examined through pre-column chemical derivatization with a series of tagging reagents such as benzoyl chloride, benzenesulfonyl chloride and 1-naphthalenesulfonyl chloride. These tagging reagents are similar in structure; however, the enantioselectivity for the same analyte derivatized with these tagging reagents is quite different and found to be the best with benzoyl chloride. In the reversed-phase mode or on the γ-CD CSP under the same chromatographic conditions, the enantioresolution diminishes for all tagged enantiomers that were examined in this study. Dipeptides derivatized by benzoyl chloride appear to be better resolved than by dansyl chloride as reported previously. Interestingly, no enantioresolution for most derivatized amino acids with single stereogenic center was observed. Finally, enantioresolution can be enhanced by replacing the basic additive such as triethylamine with tripropylamine in the polar-organic mobile phase.     

The HPLC Enantioresolution of Phenyl Isocyanated Amino Acids, Peptides and Amino Alcohols on Cyclodextrin Bonded Phases Using the Acetonitrile-Based Mobile Phase

A variety of amino acids (primary and secondary), peptides and amino alcohols are pre-column phenyl isocyanated in alkaline medium and enantioresolved on the naphthylethylcarbamated β-cyclodextrin (i.e., RN- and SN-β-CD) bonded chiral phases (CSPs) using the acetonitrile-based mobile phase and on a native β-cyclodextrin (β-CD) phase for comparison. The resolution is believed to be a result of the hydrogen bonding between the secondary hydroxyl groups of cyclodextrin and the functional groups of analyte and is enhanced as the amino and the carboxyl groups are attached to the stereogenic center of analyte. Also, the enhancement is observed if the steric hindrance between the side-chain group of amino acid and the chiral selector exists. However, the resolution is deteriorated in the case that the side-chain group close to the stereogenic center of amino acid becomes bulky or is capable of forming hydrogen bonding with chiral selector. The aromatic moiety of the tagging reagent not only contributes the retention, but also benefits the resolution in some cases on the RN- and SN-β-CD phases through π-π interaction. The resolution is either not observed or unsatisfactory in the reversed-phase mode.     

The HPLC Resolution of Nucleic‐Acid Bases and An a Log Hypoxanthine on R,S‐2‐Hydroxypropyl Derivatized β‐Cyclodextrin Bonded Chiral Stationary Phase Using a Water‐Based Mobile Phase

A HPLC approach using R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin packed column as the stationary phase was developed to resolve five nucleic‐acid bases and an a log hypoxanthine in the reversed‐phase mode. These bases are not only similar in structure but also very close in basicity. However, the resolution can be completed in less than ten minutes and is considered to be better carried out on the R,S‐2‐hydroxypropyl derivatized β‐cyclodextrin phase than that obtained on the native β‐cyclodextrin phase under the same chromatographic conditions. The mechanism involved in the resolution is believed to be inclusion complexation between the analyte and the cavity of cyclodextrin in the reversed‐phase mode. The retention time was found relevant to the size of the analyte. The number of groups on analyte that is available to form hydrogen bonding with hydroxyl groups on CDs also affects the retention scale. Factors of introducing organic acid and base or organic modifier such as methanol to the water‐based mobile phase or increasing their percent ages in the mobile phase decreases the retention time without de grading the resolution significantly.     

Reanalysis of Chiral Discrimination of Phenoxypropionic Acid Herbicides on a Teicoplanin Phase Using a Bi-Langmuir Approach

Summary The chiral discrimination of phenoxypropionic acid herbicides by reversed-phase chromatography on a teicoplanin phase has been re-examined using the perturbation method to calculate the solute distribution isotherms. The effects of both temperature and the methanol (organic modifier) content of the mobile phase on the chiral discrimination mechanism were well described by the bi-Langmuir model. The method confirmed a change in the mechanism of enantiomer retention at a critical temperature, T^*, and showed that the mechanism was independent of (i) herbicide molecular structure, i.e. the position of the chloro group on the phenol ring, and (ii) the absolute configuration of the carbon atom. Enantioselectivity was enhanced by increasing the mobile phase methanol content. Use of this approach also revealed that secondary sites on the teicoplanin surface were involved in the processes determining both retention and selectivity. It was clearly demonstrated that these secondary sites of low affinity were not affected by the temperature change and were not involved in the chiral recognition mechanism.     

柱前手性衍生色谱法拆分DL—氨基酸时流动相的影响

本文利用邻苯二甲醛和Ｎ－乙酰－Ｌ－半胱氨酸作柱前手性衍生化试剂，经反相高效液相色谱法拆分ＤＬ－氨基酸对映体，研究了流动相组成对出峰顺序和分离效果的影响。     

Chiral HPLC of Amino Acids Using Chemically Modified Chitosan as the Stationary Phase

The chiral recognition ability of chitosan is enhanced by chemical modifications. For example, chitosans modified with phenylcarbamate or cyclodextrin were used as good stationary phases for the chiral HPLC separation of aromatic alcohols or amino acid derivatives2>, respectively. We report here the preparation of chitosans having an a -amino acid moiety in the glucosamine unit and their chiral recognition behavior toward a -amino acids under ligand-exchange chromatographic separation mode.     

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.     

Enantioseparation of Amino Acid Derivatives with a Cellulose-Based Chiral Stationary Phase

Five structurally related amino acid derivatives were enantioseparated by HPLC with a commercially available chiral stationary phase, Chiralcel OD-H. The chromatographic experiments were performed in the normal phase mode. n-Hexane/polar alcohol was used as mobile phase. Excellent baseline enantioseparations could be obtained for all these solutes. The effects of the concentration of polar alcohol and the column temperature on the retentions and enantioseparations were studied in detail. From the van't Hoff plots the corresponding apparent thermodynamic parameters were derived. Mechanism aspects of chiral recognition were discussed based on the relationship between the thermodynamic parameters and the structures of the solutes. It was found that the substituent of the phenyl group on the residual group of the amino acid derivatives was close relevant to thermodynamic origin of enantioseparation. Much better enthalpy–entropy compensation effect was obtained by plotting the differential, rather than the original, thermodynamic parameters.     

大环糖肽抗生素键合相高效液相色谱法拆分7种氨基带保护基的氨基酸对映体

采用高效液相色谱法在装有大环糖肽抗生素键合相的手性柱上拆分了7种氨基带有芴甲氧羰基(fluorenylmethoxycarbonyl,Fmoc)保护的氨基酸对映体。比较了Fmoc-缬氨酸和相应的不带保护基的缬氨酸对映体在不同流动相体系中的色谱保留行为;考察了甲醇-醋酸-三乙胺流动相体系中醋酸和三乙胺的浓度以及它们二者的浓度之比对N-Fmoc氨基酸对映体拆分效果的影响。实验结果表明分离温度及流动相流速的变化也会对分离结果产生影响。该法简便快速,已成功地用于这类氨基酸的光学纯度测定。     

Enantioseparation of Amino Acids on a Chiral Stationary Phase Derived from L‐α‐Norleucinyl‐ and Pyrrolidinyl‐disubstituted Cyanuric Chloride by High‐Performance Liquid Chromatography

A silica‐based chiral stationary phase (CSP) derived from L‐α‐norleucinyl‐ and pyrrolidinyl‐disubstituted cyanuric chloride was prepared for the enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl) amino acids by high‐performance liquid chromatography. The chromatographic results show that effective enantioseparation of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids, except for proline, was achieved on this chiral stationary phase. The chromatographic resolution of racemic n‐propyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B is better than that of racemic methyl ester of N‐(3,5‐dinitrobenzoyl)valine on CSP‐B or CSP‐A reported previously (J. Chromatogr. A, 676 (1994) 303). The comparison of the chromatographic results obtained in this study with those on CSP‐A reported previously reveals that steric effect, instead of hydrophobic interaction, between the n‐butyl group attached to the chiral center of the chiral selector and the alkyl group attached to the chiral center of the chiral selectand plays a significant role in chiral discrimination. The increase in the selectivity factor of methyl esters of N‐(3,5‐dinitrobenzoyl)amino acids with bulky alkyl groups was examined on CSP‐B.     

正反相模式下键合纤维素手性固定相对手性化合物的拆分

在自行合成的键合型纤维素-(3,5-二甲基苯基氨基甲酸酯)(CDMPC-CSP)手性固定相上,在正相模式下对苯偶姻和甲霜灵外消旋体进行了拆分,考察了流动相中醇以及温度在手性识别中对手性分离的影响。同时考察了反相模式下外消旋体的拆分。     

Enantiomeric Resolution of Amino Acids by Reversed Phase High Performance Liquid Chromatography Using a New Chiral Mobile Phase

Abstract

The synthesis of a new chiral agent, (R,R) (-)N, N'-trans-1, 2-dihexylcyclohexanediamine, for the chromatographic resolution of racemates is reported. Highly selective separations of D- and L-isomers of free and Dns-amino acids were accomplished on a reversed-phase column using in the mobile phase a Cu(II) complex of the above chiral selector. The procedure was extended to resolve diastereomeric derivatives, which were obtained by reaction of an optically active amine with o-phthaldeyde in the presence of N-acetyl-L-cysteine.     

姜黄素键合硅胶固定相高效液相色谱分离取代芳香酸类化合物

研究了13种取代芳香酸类化合物在姜黄素键合硅胶固定相(CCSP)上的色谱行为,考察了甲醇含量、流动相pH值对CCSP分离取代芳香酸类化合物的影响,探讨了该固定相对芳香酸类化合物的色谱保留机理,并应用于实际样品复方水杨酸搽剂的分离分析。结果表明,在不同含量的甲醇-0.02 mol.L-1NaH2PO4(pH=2.5)流动相体系中,三组芳香酸标准混合样和实际样品中四种主要成分在CCSP上实现基线分离。与ODS柱相比,在相同条件下,CCSP对这类酸性化合物具有较高的选择性,且对13种取代芳香酸类化合物的洗脱顺序与ODS具有显著差异,其中5种溶质在CCSP上的保留强于ODS,这说明不同保留机理的存在。研究表明,CCSP具有典型的反相色谱特征,但疏水性较ODS弱;除疏水作用外,n-π和π-π作用、氢键作用、电荷转移作用、偶极-偶极作用对分离也有贡献。CCSP对极性、电离性物质如水杨酸、对硝基苯甲酸、3,4,5-三羟基苯甲酸、邻苯二甲酸和邻溴苯甲酸等具有较强的保留;由于CCSP较弱的疏水性和多种分离机制,邻氯苯甲酸和苯甲酸在CCSP上的保留不会像在ODS上那么强,且分离度明显优于ODS。     

去甲万古霉素键合手性固定相在反相条件下拆分氨基酸类衍生物

使用合成的大环抗生素-去甲万古霉素键合手性固定相(NVC-CSP),在反相条件下直接拆分了4种丹酰化氨基酸对映异构体,并系统考察了缓冲溶液pH值、有机改性剂用量、温度以及流速对对映体拆分的影响。研究结果表明,在反相条件下,疏水作用和静电作用是影响溶质保留和手性选择性的重要因素,其手性识别的机理类似于蛋白质类手性固定相。     

Enantioselective Solid‐Phase Peptide Synthesis Using Traceless Chiral Coupling Reagents and Racemic Amino Acids

The enantioselective condensing reagent 4,6‐dimethoxy‐1,3,5‐triazine (DMT)/strychnine/BF$\rm{{_{4}^{-}}}$ was obtained by treatment of 2‐chloro‐4,6‐dimethoxy‐1,3,5‐triazine (CDMT) with strychnine tetrafluoroborate. The reagent was useful under typical conditions of solid‐phase peptide synthesis (SPPS) with enantiomerically homogeneous substrates. By SPPS, desired dipeptides were obtained in 84–94% yield using 4 equiv. of racemic Fmoc‐Ala, Fmoc‐Phe, and/or Fmoc‐Tyr for 1 equiv. of Wang resin loaded with Gly, Ala, Leu, Phe, Glu(^tBu), and/or Pro, respectively. For all three Fmoc‐protected amino acids, the configuration of the enantiomer preferred under SPPS conditions was independent of the structure of the acylated component and identical to that established in condensations proceeding in solution. In all cases, the enantiomer ratios L /D (er) were in a similar range, and varied from 9 : 92 to 2 : 98 for alanine, and from 90 : 10 to 100 : 0 for aromatic amino acids. The synthesis of Ac‐L ‐Lys(Ac)‐D ‐Ala‐D ‐Ala‐OH from racemic Fmoc‐Ala gave an L /D ratio of 10 : 90 for the esterification of Wang resin, and 0 : 100 for the formation of peptide bonds.