Separation of Enantiomers of Non-Protein Amino Acids by High-Performance Liquid Chromatography on a Chiral Ligand-Exchange Column

The HPLC separation of enantiomers of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Good enantiomeric separations were achieved with a variety of -amino acids carrying aliphatic or aromatic side chains, cyclic imino acids, and -amino acids, together with -methyl--amino acids, by optimizing the amount (0–20%, v/v) of 2-propanol as the organic component and the concentration (1–5 mM) of Cu²⁺ as the complexing metal ion in the aqueous-organic eluent.     

高效液相色谱手性固定相对氨基酸衍生物对映体分离研究

利用高效液相色谱手性固定相对光学异构体分离分析日益受到人们的重视。高效液相色谱氨基酸酰胺型手性固定相对拆分对映异构体具有较好的效果。本文为了探讨该类手性固定相对氨基酸的拆分影响,用L-异亮氨酸叔丁酰胺型手性固定相,对6种DL氨基酸(丙氨酸、2-氨基-n-丁酸、缬氨酸、亮氨酸、苯丙氨酸和蛋氨酸)的18种衍生物进行了拆分,并讨论了其衍生物取代基对手性拆分的影响及从立体化学角度提出了手性拆分机理。实验结果表明,这种手性固定相对外消旋氨基酸衍生物有较好的拆分能力。 1 实验部分 1.1 仪器和试剂 Varian 2010型高效液相色谱仪(美国);2050型紫外检测器及HP3394型积分仪(美国);L-异亮氨酸叔丁酰胺型手性柱(TBILS,上海药物研究所),粒度10μm,柱长100mm、内径4mm的不锈钢柱。 异丙醇(分析纯)用前重蒸,石油醚(60～90℃)分析纯,重蒸收集69℃镏分,用前全经过0.5μm薄膜并脱气。DL-氨基酸衍生物[丙氨酸(Ala),2-氨基-n-丁酸(Buty),缬氨酸(Val),亮氨酸(Leu),苯丙氨酸(Phe),蛋氨酸(Met)]用相应DL-氨基酸参考文献方法进行N-乙酰化及酯化。衍生物经元素分析合格,配制成5mg/mL溶液。     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.     

Separation of Enantiomers of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

The high-performance liquid chromatographic separation of enantiomers of N-protected non-protein amino acid esters was investigated by using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiracel OD). The effect of the N-protecting groups and the ester groups on chiral discrimination was examined. The benzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl derivatives gave good enantiomeric separations, while the formyl and t-butoxycarbonyl groups marred them. Almost all the alkyl esters examined and the benzyl ester gave enantiomeric separations better than or of the same order as the methyl ester. The N-Z-protected methyl esters of a number of non-protein -amino acids were well resolved using hexane–2-propanol as a mobile phase. The resolution of -amino acid derivatives was inferior to that of the isomeric -amino acid derivatives.     

表面等离子体共振技术用于蛋白对氨基酸手性识别的动力学研究

利用SPR技术,以牛血清白蛋白和溶菌酶为探针构筑手性识别传感膜,开展了对L-和D-苯丙氨酸以及L-和D-色氨酸手性识别的动力学研究。实验结果表明,两种蛋白在与每种氨基酸分子的L-和D-型异构体相互作用过程都存在明显的动力学差异。动力学数据进一步显示两种蛋白与每种氨基酸L-型异构体的亲和力均大于D-型。     

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。     

手性固定相高效液相色谱法拆分系列抗胆碱能药物对映体的研究

采用手性固定相高效液相色谱法研究了甲醇-磷酸盐体系、乙腈-磷酸盐体系和甲醇-乙腈-磷酸盐体系对系列抗胆碱能药物的手性拆分情况,讨论了流动相的各个因素（有机相的种类和比例、磷酸盐浓度、酸度、三乙胺用量等）对手性拆分的影响。研究表明,流动相中较大的水相比例、较高的磷酸盐浓度、pH和三乙胺浓度更有利于抗胆碱能药物的手性拆分。通过对比研究10种抗胆碱能手性药物的色谱行为,从结构上讨论了化合物中不同官能团对保留时间及手性拆分的影响,并探讨了手性拆分的内在机制。     

流动相的pH值和离子强度对牛血清蛋白高效液相色谱手性柱分离性能的影响

利用羰基咪唑-柱上衍生法制得牛血清蛋白(BSA)生物手性柱,并研究它在高效液相色谱中对3种对映异构体(色氨酸、匹多莫德及4-苯基-1,3-恶唑烷-2-硫酮(L-苯))的手性分离性能.实验结果表明:随着pH值从5.0上升到7.0,由于L-色氨酸与BSA有固定的作用位点,使得其保留值随着pH值的增加而大幅增大.而D-色氨酸与BSA无固定作用位点,其保留值随pH变化基本不变,分离度由1.15上升到8.91,增加了6.8倍;酸性样品匹多莫德与BSA主要是静电作用,与色氨酸相反,其对映体的保留值随着pH的增加逐渐减小,分离度逐渐下降,pH 7.0时为单峰,pH 5.0时Rs=1.27;中性样品L-苯的分离度随着pH值的增加有小幅增大.随着离子强度的减小,3对对映体的保留都增强,分离度增大,增加的幅度依次为色氨酸＞匹多莫德＞L-苯.     

手性流动相添加剂法拆分氧氟沙星对映体的热力学研究

采用配位体金属离子交换剂作为手性流动相添加剂,以L-苯丙氨酸和硫酸铜溶液为手性配体流动相,考察了293~313K温度范围内,氧氟沙星对映体在C18高效液相色谱柱上拆分的热力学性质。结果表明,在实验范围内,随着温度的升高,对映体的保留时间、分离度和选择性因子均减少;用lnk′对1/T作图,得Van’t Hoff曲线具有良好的线性关系,相关系数R0.999。计算了氧氟沙星对映体在色谱柱分离的焓变、焓变差值和熵变差值等热力学参数。在实验温度范围内,氧氟沙星对映体满足︱△S,R△H0︱︱T△S,R△S0︱,说明手性拆分过程为焓控过程。     

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。     

Chiral Resolution of Benzophenone Imine Derivatives of Amino Acid Esters on Chiral Stationary Phases

Abstract

A convenient way of resolving the enantiomers of amino acid esters after their derivatization using benzophenone imine is described. The enantiomers of benzophenone Schiff base derivatives of various amino acid ethyl esters are readily separated on three commercially available chiral stationary phases (CSPs). Among them, CSPs 2 and 3 afford generally the base-line enantioresolution for the analytes studied. From understanding of chromatographic results, a plausible chiral recognition mechanism is discussed.     

氨基酸桥联新型手性锌双卟啉配合物的合成与表征

生命系统中的金属卟啉生物大分子在卟啉的Ｓｏｒｅｔ带均表现出诱导圆二色性（ＩｎｄｕｃｅｄＦｉｇ．１Ｓｔｒｕｃｔｕｒｅｏｆａｍｉｎｏａｃｉｄｂｒｉｄｇｅｄｚｉｎｃｐｏｒ┐ｐｈｙｒｉｎｄｉｍｅｒＯ,Ｏ┐Ｃ２┐ＡＡ┐Ｃ２┐（ＴＰＰ）２Ｚｎ２Ｒ＝Ｂｎ,ＡＡ＝Ｐ...     

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.     

The effect of the copper(II) salt anion in the Chiral Ligand-Exchange Chromatography of amino acids

Due to the strong involvement in numerous biochemical processes, the separation and resolution of amino acids is a continuously challenging task. Among the experimental parameters affecting the performances in a Chiral Ligand-Exchange Chromatography (CLEC) environment, the effect of the copper(II) salt counter-ion has received very limited attention. Aimed at evaluating the Cu(II) counter-ion effect upon the overall chromatographic performances when the S-trityl-(R)-cysteine is adopted as the chiral selector for the coated stationary phase, a number of both organic (acetate, formate, trifluoromethane-sulfonate) and inorganic (bromide, chloride, nitrate, perchlorate, sulfate) salts has been engaged for the enantiomer separation of a selected set of aliphatic (Allo-Ile, Ile, Leu, Nor-Leu, Nor-Val, Val) and aromatic (AIDA, ATIDA, His, Phe, Phg, Tyr) amino acids. By varying the physico-chemical nature of the Cu(II) anion, a pronounced impact upon the resolution factor (R_S) has been observed. Even if to a lesser extent, the enantioseparation factor values (α) underwent variation, as well. A molecular modelling investigation has also been carried out as a rationalization attempt of the observed chromatographic outcomes.     

Chiral Separation of Racemates of Drugs and Amino Acid Derivatives by High-Performance Liquid Chromatography on a Norvancomycin-Bonded Chiral Stationary Phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) has been synthesized by use of the chiral selector norvancomycin, which differs from vancomycin because of the presence of leucine rather N-methylleucine. The enantiomers of some neutral and basic chiral drugs, for example warfarin, benzoin, bendroflumethiazide, and praziquantel, were directly separated by high-performance liquid chromatography in the reversed-phase mode. The effect of conditions such as organic modifier concentration, column temperature, pH, and mobile phase flow rate on retention and enantioselectivity were investigated. It was shown that hydrophobic, steric, and ionic interactions were present between the analyte and the macrocycle in this chromatographic system. Vant Hoff plots afforded the thermodynamic data _R,SH° and _R,SS°; the negative values obtained indicated the process of enantiomer separation was enthalpy-controlled. In an attempt to improve the resolution of some very polar acidic compounds (dansyl-amino acids) norvancomycin was used as stationary phase chiral selector and chiral mobile-phase additive simultaneously, better results were obtained as the result of a synergistic effect. It was also shown experimentally that the newly synthesized NVC-CSP behaved somewhat differently from the earlier reported vancomycin-bonded CSP, probably because of the different structures of norvancomycin and vancomycin.     

The Role of Amino Acid Esters and the Amide Group in the Chiral Induction Process by a Novel Monoamide‐Linked Monozinc Bisporphyrinate

A monoamide‐linked monozinc bisporphyrinate for monodentate chiral guests was designed and synthesized. It was used as a host to complex with amino acid esters; the resulting CD spectra showed typical bisignate shape, which suggest the ability to transfer chirality from amino acid esters. The molecular structure of the host indicates that amino acid esters function as monodentate ligands in the host‐guest complex. ¹H NMR spectroscopic studies suggest formation of a hydrogen bond between the amide NH and the carbonyl oxygen groups of the amino acid ester. The possible mechanism of the chiral induction process was further studied by DFT calculations.     

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.     

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.     

手性胺酰胺型液相色谱手性固定相的制备及几种氨基酸衍生物的拆分

用(R)-1-苯基2-对甲基苯基乙基胺(PTE)与L-异亮氨酸制得含两个手性中心的手性选择剂,以琥珀酸酐作为连接臂,将手性选择剂键合到氨基丙基硅胶上制得手性固定相。以正己烷-异丙醇为流动相,利用该固定相对氨基酸衍生物进行高效液相色谱手性拆分,并考察了流动相中异丙醇含量对手性拆分的影响。结果表明,该手性固定相对所分析的氨基酸衍生物大部分都具有一定的拆分能力。当异丙醇含量为1%时,亮氨酸与苯丙氨酸的苯甲酰甲酯衍生物获得基线分离。当异丙醇含量为20%时,亮氨酸、缬氨酸、丙氨酸、谷氨酸的3,5-二硝基苯甲酰甲酯衍生物获得基线分离。     

氨基酸桥联手性双卟啉的二阶非线性光学性质研究

采用半经验量子化学PM3方法对系列氨基酸桥联双卟啉1, 2, 3, 4及其锌配合物进行了几何结构优化, 并用TDHF/PM3方法计算了其静态二阶非线性光学系数. 计算结果表明, L型-氨基酸桥联双卟啉及其锌配合物具有右手螺旋结构特征. 在电偶极近似下, 不同手性氨基酸侧链基团R对分子的总体二阶非线性光学系数β和βHRS的影响不大, 但对二阶非线性光学系数的手性分量βxyz却有显著影响. βxyz与螺旋结构参数r2ζ/L4成正比关系, 符合手性分子二阶非线性光学响应的单电子螺旋模型理论. β张量分析表明, 此类分子表现为以八极为主、 偶极为辅的多极分子, 卟啉环与锌离子的配位有利于增加二阶非线性光学响应的偶极分量和手性分量βxyz.