万古霉素键合手性固定相的制备与评价

大环抗生素作为一种新型的手性选择器,与高效液相色谱(HPLC)、毛细管电泳(CE)和毛细管电色谱(CEC)等联用,成功分离各类手性化合物~([1～3]).自1994年Armstrong等~([4])首次将大环糖肽抗生素作为手性选择器合成手性固定相以来,适用于手性分离的大环糖肽抗生素键合固定相的制备与应用得到飞速发展.本研究以万古霉素为手性选择剂,制备了万古霉素键合手性固定相液相色谱柱.采用反相高效液相色谱法对谷氨酸对映体进行了拆分,并考察了流动相条件对对映体拆分的影响.     

去甲万古霉素键合手性固定相的制备及其对磷酸苯并哌啉对映异构体的拆分

制备了去甲万古霉素键合手性固定相以及由苯基异氰酸酯衍生化去甲万古霉素键合手性固定相,并在极性有机相模式下对磷酸苯并哌啉进行了手性拆分的研究。发现在此模式下,衍生化去甲万古霉素键合手性固定相对磷酸苯并哌啉的手性选择性要优于没有衍生的去甲万古霉素键合手性固定相;在衍生化去甲万古霉素键合手性固定相上详细考察了流动相组成、酸碱添加剂用量、温度以及流速对分离的影响,优化了分离的条件。在优化的实验条件下,磷酸苯并哌啉可获得基线分离。     

一种手性中间体在键合型和涂覆型纤维素手性固定相上的拆分

制备了涂覆型和键合型纤维素-(3, 5-二甲基苯基氨基甲酸酯)固定相, 分别在制备的纤维素手性固定相上成功地拆分了一种手性中间体, 通过考察流动相中的改性剂(醇、四氢呋喃、三氯甲烷)对手性拆分的影响, 优化了手性中间体在两种手性固定相上的色谱分离条件, 并比较了手性中间体在涂覆和键合型纤维素手性固定相上的拆分. 结果表明, 涂覆型和键合型手性固定相对这种手性中间体均有较好的拆分效果, 在150 mm的色谱柱上, 这两种手性固定相对这种手性中间体的拆分能力相差不大, 但键合型固定相上可选择的流动相范围更广.     

伊瑞霉素键合手性毛细管整体柱的制备与对映体分离

以具有22个不同种类手性中心的新型大环抗生素伊瑞霉素为手性选择器,基于环氧基团高反应活性的特征,将伊瑞霉素用一步法键合到甲基丙烯酸酯整体柱表面制备伊瑞霉素键合手性毛细管整体柱。通过对制备条件进行优化,证实该制备方法可在较宽的pH范围(6.0～9.0)内进行,方法简单易行,反应条件温和。应用制备的手性毛细管整体柱在毛细管电色谱模式下,对5种手性氨基酸对映体和手性药物罗格列酮对映体进行拆分,均得到了基线分离,说明伊瑞霉素手性固定相具有较强的手性拆分能力。在优化的色谱条件下,6种对映体的分析时间均小于4 min,分析速度快。通过对有机调节剂、缓冲液pH值和缓冲盐浓度等分离条件进行系统考察,初步探讨了该手性毛细管整体柱对不同溶质的手性识别机理。     

替考拉宁和万古霉素高效液相色谱手性固定相的研究

分别使用四种不同的方法将替考拉宁和万古霉素键合在硅胶表面,制备了八种不同的高效液相色谱柱固定相,并在反相模式和极性有机相模式下考察了这八种手性柱的性能.实验表明,固定相制备方法的不同和色谱条件的改变对色谱柱拆分性能有一定的影响,不相同的键合臂,能拆分的对映异构体可能不一样,这些柱之间具有一定的互补性.八种手性固定相均有...     

表面分子印迹聚合物纳米线用于蛋白质的特异性识别

手性配体交换色谱是拆分手性化合物,特别是氨基酸和羟基酸对映体的一种有效方法,通常以光活性氨基酸或其衍生物为手性选择子,可通过键合及涂渍制备手性固定相,也可作为流动相添加剂来实现手性配体交换色谱分离分析,配体交换键合固定相需要完成载体和手性选择子之间的偶联,键合量因受到载体和制备条件的影响而较难控制,且柱效较低。     

功能化苯甲酰胺基-β-环糊精手性固定相的制备及其对黄烷酮对映体的拆分

为了探讨功能基团对苯甲酰胺基-β-环糊精手性固定相手性拆分性能的影响,本文采用点击化学制备了两种手性固定相:全取代-4-氯-3-甲基苯甲酰氨基-6A-三唑基-β-环糊精键合硅胶手性固定相(CSP1)和全取代-5-氯-2-甲基苯甲酰氨基-6A-三唑基-β-环糊精键合硅胶手性固定相(CSP2)。利用核磁共振、红外光谱及元素分析等手段对固定相的结构进行了表征;在反相高效液相色谱条件下,通过对9种黄烷酮消旋体的拆分,对比研究了两种固定相的手性拆分性能。研究结果表明CSP1的拆分性能优于CSP2。这两种手性固定相仅在水与甲醇体系下即可实现对黄烷酮的手性拆分,展现了较好的应用前景。     

去甲万古霉素键合手性固定相拆分β-受体阻滞剂类药物及其结构类似物的研究

以去甲万古霉素为手性选择剂制备了大环抗生素类手性固定相去甲万古霉素键合手性固定相(NVC-CSP)。在极性有机模式下对普萘洛尔、美托洛尔、阿替洛尔及烯丙洛尔等4种β-受体阻滞剂类药物及其结构类似物的外消旋体进行了手性拆分的研究,并考察了流动相组成、酸碱添加剂用量、温度以及流速对分离的影响。研究发现:在此模式下手性物质的保留均随着流动相中甲醇含量的增加而减弱,手性分离因子（α）随着流动相中甲醇含量的增加而升高;随着柱温的升高,大部分溶质在色谱柱上的保留减弱,α值降低;在一定范围内降低流速有利于对映体的分离;     

替考拉宁高效液相色谱手性固定相的制备研究

替考拉宁属于大环抗生素,具有半篮状结构和多个手性中心,是常见的手性识别材料,广泛应用于对映体的色谱手性分离分析.本文研究了以替考拉宁为手性识别剂,采用键合的方法制备得到9种高效液相色谱手性固定相,用于苯甘氨酸和对羟基苯甘氨酸的拆分研究,并且考察了重现性和稳定性及进样量对拆分结果的影响.实验结果表明,9种手性固定相均具有拆分苯甘氨酸及对羟基苯甘氨酸的能力.     

高效液相色谱法对手性药物罗格列酮与吡格列酮的拆分

采用高效液相色谱法,在多糖类手性固定相Chiralpak IC上成功拆分了2种降糖类手性药物罗格列酮与吡格列酮,考察了流动相、流速、温度、酸碱添加剂等对手性分离的影响.以正己烷-异丙醇(70:30,体积比)为流动相,在流速1.0 mL/min、检测波长254 nm、柱温35 ℃的条件下,罗格列酮与吡格列酮均得到良好分离,选择因子(α)分别为1.29、1.22,分离度(Rs)分别为7.07、5.56;该法不仅适用于此类手性药物的生产及质量控制,同时还可应用于对映体制备.     

New chiral stationary phase with macrocyclic glycopeptide antibiotic eremomycin chemically bonded to silica

A new chiral stationary phase (CSP) was prepared by attachment of macrocyclic glycopeptide antibiotic eremomycin to the epoxy-activated silica under mild conditions. In contrast to CSP with immobilized vancomycin, which is a close structural analogue of eremomycin, the prepared CSP reveals high enantioselectivity for separation of amino acids enantiomers. It was demonstrated by the example of ristocetin A CSP that method of the immobilization of macrocyclic glycopeptide antibiotics affects remarkably the resulting enantioselectivity.     

万古霉素手性固定相的制备与对映体分离研究

采用双官能团试剂4,4′-二苯基甲烷二异氰酸酯在无水二甲基甲酰胺(DMF)中直接与大环糖肽类抗生素万古霉素及γ-氨丙基硅胶键合,得到环状抗生素手性固定相(CSP)并用于高效液相色谱手性分析。实验结果证实,合成的万古霉素CSP在正相和反相条件下均有一定的拆分能力,其中在反相条件下拆分了17种对映体,显示出其较为广泛的拆分范围,且磷酸缓冲体系略优于三乙胺-乙酸缓冲体系;对一些物质,如D,L-丹酰化氨基酸的拆分有一定的规律,能给出绝对构型信息。所制备的CSP在相体系转化时不发生老化和变性,显示了一定的稳定性。对该CSP的拆分机理进行分析所得到的结果与Armstrong等的分析结果基本一致。     

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.     

Derivatized vancomycin stationary phases for LC chiral separations

In this work, synthetic and natural chiral selectors were combined to form two different chiral stationary phases (CSPs). These were made by bonding R- or S-(1-naphthylethyl) carbamate (R-NEC or S-NEC)-derivatized vancomycin molecules to a silica gel support. The two CSPs were evaluated using a set of 60 enantiomeric pairs. The results were compared to the ones obtained with the commercial underivatized vancomycin CSP. Three Chromatographic modes were used: (i) the normal-phase mode using a nonpolar mobile phase with different ratios of hexane and ethanol; (ii) the reversed-phase mode with hydro-organic mobile phases; and (iii) the polar aprotic organic mode with nonaqueous acetonitrile plus small amounts of methanol and an acid and/or base to control retention and selectivity. It is shown that the polarity of the underivatized vancomycin phase is higher than that of the two R- and S-NEC-derivatized CSPs. In the pH range 4-7, there is no ionization change of the chiral selector for the three CSPs. 43% of the studied compounds were resolved by the NEC-derivatized phases when they could not be resolved by the vancomycin CSP. However, the enantiorecognition for 12% of the compounds on the native vancomycin CSP was lost upon NEC derivatization. 45% of the studied compounds were resolved by the NEC-derivatized and native CSPs. The NEC derivatization procedure may block some useful active sites on the vancomycin molecule. Also, the R- and S-NEC moieties are chiral themselves and can contribute additional interaction sites not available on the native vancomycin molecule.     

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).     

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.     

Comparison of vancomycin-based stationary phases with different chiral selector coverage for enantioselective separation of selected drugs in high-performance liquid chromatography

Two vancomycin-based chiral stationary phases (CSPs) with different coverage of the chiral selector vancomycin (Chirobiotic V and Chirobiotic V2) were compared. beta-Blockers and profens, as structurally diverse groups of drugs, were chosen as analytes. Retention and enantioseparation of beta-blockers were studied in reversed-phase (RP) and polar-organic (PO) separation modes. Higher retention and better enantioresolution were obtained on the CSP with higher coverage of vancomycin in the both separation modes. Baseline separation of four beta-blockers (eight enantiomers) in the PO mode was achieved on the Chirobiotic V2 column within 15 min. The enantioseparation of profens did not bring so excellent and easy to interpret results. Higher retention of profens on the Chirobiotic V2 column was not always accompanied by an improvement of their chiral separation in the RP mode. The polar-organic mode was not suitable for these derivatives at all. The most interesting result was obtained with flobufen; its chiral center is further away from the rigid part of the molecule, which mostly causes difficulties in enantioselective recognition. Nevertheless, the enantiomers of flobufen were shown to be much better (baseline) resolved on the CSP with lower coverage of the chiral selector (Chirobiotic V).     

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous-bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 microm ID) by in situ copolymerization of N-(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2 x 10(-5) cm2 V(-1) s(-1) and the aqueous reversed-phase modes of operation (8.5 x 10(-5) cm2 V(-1) s(-1)). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120,000 plates meter(-1)) values.     

Microcolumn LC enantioseparation of chiral compounds using diol silica gel functionalized with vancomycin crystalline degradation products

Vancomycin crystalline degradation products (CDPs) have been introduced as one of the newest and most interesting derivatives of vancomycin for enantiomer separation of a wide variety of chiral compounds. In this attempt, a chiral stationary phase (CSP) has been prepared using diol silica gel based on vancomycin CDPs which led to a new chiral selector with new functionality of functional groups on a microcolumn LC. Different kinds of mobile phases were examined to realize the behavior of the chiral selector in separation of atropine, fluoxetine, amlodipine, mandelic acid, alanine, and phenylalanine which were separated successfully on this column. Good results were obtained by using a polar mobile phase containing water, methanol, and acid additives for separation of chiral acidic compounds and amino acid samples. Considerable results were obtained for analysis of basic compounds by using polar organic mobile phase (POP) containing methanol, acid and base additives. These results can be associated with the presence of the carboxylic acid groups present in new CSP by using a diol silica gel.     

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.