人血清白蛋白与手性药物相互作用的毛细管电泳研究Ⅱ.药物对映体竞争结合参数的测定

运用毛细管电泳（CE）技术,在对碱性药物Verapamil（VER）手性拆分的基础上对Verapamil与人血清白蛋白（HSA）平行体系进行了相互作用研究。通过定量HSA－VER体系中VER对映体的浓度,建立对映体对结合位点竞争的理论方程,获得了R和S型药物对映体与HSA的结合常数,其值分别为K（R）-VER=2．7×103×（±4．4×102）和K（S）-VER=8．5×102（±1.0×102）。实验证实,HSA具有手性选择性,与（R）－VER的结合强于与（S）-VER的结合,结合比随着HSA与（±）VER的浓度比而变化。     

人血清白蛋白与手性药物相巨作用的毛细管电泳研究Ⅰ.液相预柱毛细管电泳技术定量可靠性的考察

以药物Verapamil（VER）与人血清白蛋白（HSA）相互作用体系中游离的药物对映体浓度定量测定为目标,建立了一项适用于相互作用研究的液相预柱毛细管电泳（LPC－CE）技术。通过对该技术的考察,确定了这项技术的定量可靠性。在生理pH值条件下（pH7.4,离子强度I=0．17）,使药物与人血清白蛋白达到结合平衡。在毛细管电泳手性拆分[pH2.5缓冲浪;三甲基-β-环糊精（TM－β-CD）浓度为45mmol／L]柱（32cm×50μm）内预先注入一段生理pH缓冲液,形成一段液相预柱（2．8cm）。     

液相预柱毛细管电泳技术在手性药物对映体与白蛋白相互作用研究中的应用

在对碱性药物吡啶茚胺、四氢萘唑啉、去甲肾素茶碱和维拉帕米(verapamil)等药物手性拆分的基础上,运用液相预柱毛细管电泳（LPC-CE）技术,建立了对药物对映体与人血清白蛋白（HSA）相互作用体系中对映体浓度的检测方法.　该技术利用HSA与药物在生理pH下的电泳特性差异,使HSA留在预柱内或反向流出,不进入手性拆分区域,从而消除白蛋白对药物对映体拆分及浓度检测的干扰.　对维拉帕米对映体与HSA结合参数以及多药物组分竞争结合的研究表明,该技术为多种药物与蛋白共存的复杂体系研究提供了一条有效的途径.     

疏水性L-酒石酸异丁酯立体选择性萃取拆分氰戊菊酸对映体

合成了萃取拆分氰戊菊酸(FA)对映体的手性选择体L-酒石酸异丁酯.研究了氰戊菊酸对映体在含有手性选择体L-酒石酸异丁酯的水-有机相双相体系中的萃取分配行为.考察了有机稀释剂类型、L-酒石酸异丁酯浓度、pH值和磷酸盐浓度诸因素对分配系数(K)和分离因子(α)的影响.研究结果表明:L-酒石酸异丁酯与S对映体形成的复合物稳定性比与R对映体形成的复合物要好;1,2-二氯乙烷作为有机稀释剂更有利于萃取分离;随着L-酒石酸异丁酯浓度的增大,K逐渐增大,α先增大后减小,当L-酒石酸异丁酯浓度为0.30 mol\5L-1时,α达最大;pH值增大,K和α都降低;磷酸盐浓度对分配系数和分离因子也有较大影响.     

手性配体的空间结构与产物对映选择性的关系

首次采用4－烷氧羰基噻唑烷、E唑烷作为手性配体，诱导烷基锌对醛类的亲核加成反应，获得产物是烷基化的醛，最高达90％ee，产率98％的（S）－二级醇。系统地考察了该类手性配体三维空间结构变化与产物对映选择性的关系，当配体4－位烷氧羰基上的R、2－位R′基和环体上原子X发生变化时都会引起产物（S）－二 级醇的对映选择性发生规律性变化。对五种不同结构的底物醛在同一手性配体催化下，诱导烷基锌对醛类的亲核加成反应，底物结构变化也会引起（S）－二级醇对映选择性变化。     

人血清白蛋白柱上药物的手性拆分

考察了４种酸性药物和１种中性药物对映体在人血清白蛋白手性固定相上的保留行为。这５种药物与人血清白蛋白结合的亲和力高，难于实现快速分离，作者提出在流动相中加入短链脂肪酸－正己酸，可快速手性拆分非诺洛芬、萘普生和布洛芬。酮基布洛芬对映体分离选择性随乙腈浓度升高而增大，流动相中加入适量异丙醇可使对映体选择性大大增加（α～１．２３），华法令同样可取得很好分离。     

安非他明类毒品的手性对映体气相色谱-质谱分析

采用手性衍生化试剂：（S）（－）N－三氟乙酰－1－脯胺酰氯（TPC）和（R）（＋）－α-甲氧基－α－三氟甲基苯乙酸（MTPA）与安非他明类对映体衍生化产物,通过常规的GC／MS方法将其分离,本文较系统地考察了这两种手性试剂衍生化反应中溶剂、手性试剂用量、加热温度、反应时间等因素对安非他明类在体衍生化结果的影响。实现了Am、MAm、MDA、MDMA、MDEA、MBDB等几种毒品对映体间的良好分离。     

中枢神经系统药物树脂制备及其静态交换特性研究

以中枢神经系统药物氢溴酸美沙芬（ＤＭ）、盐酸伪麻黄碱（ＰＥ）、盐酸苯丙醇胺（ＰＭ）作为模型药物,以００１×７离子交换树脂作为药物载体,在静态条件下,考察了该树脂与上述药物的交换反应动力学和热力学．结果表明,２５℃时树脂与ＤＭ、ＰＥ和ＰＭ的反应速率常数ｋ（ｍｉｎ－１）分别为１．０４１×１０－３±２．５６×１０－５,２．２９０×１０－２±１．２４×１０－４,２．４０×１０－２±１．０２×１０－４,且随温度的升高而增加,反应活化能Ｅａ（ｋＪ／ｍｏｌ）分别为５０．２６,２１．６８,２０．８３;在２５℃反应达平衡时,表观交换反应平衡常数Ｋｅ分别为３．２３５±０．２５２,３．６８０±０．２１４,４．５１±０．３２８;其自由能变化ΔＧ°（ｋＪ／ｍｏｌ）分别为－２．９０９±０．０２０５,－３．２２８±０．０１８１,－３．７３２±０．０１２７,表明交换反应是自发的;反应热ΔＨ°（ｋＪ／ｍｏｌ）分别为８８．４４±５．５４８,４８．２９±３．２１４,４８．６６±３．１５８,即正反应为吸热反应,温度升高有利于向正反应方向进行     

吸附固定相电色谱和动态改性电色谱的手性分离

对动态改性电色谱手性分离进行了研究。电色谱柱填充强阴离子交换固定相（SAX0,添加在流动相中的磺化β－环糊精（S－CD）动态地吸陵于SAX填料表面,形成一层准手性固定相。色氨酸、阿托品和异博定对映体在本体系获得了很好的分离,它们的分离分别为2．06,10．1和1．96,对映体峰的柱效价于85,000塔板数／米和412,000塔板数／米之间。连续运行17次,死时间和色氨酸对映体的电色谱保留因子的相对标准偏差分别为0．53％,0．62％和0．69％。此外,以吸附于SAX填料的牛血清白蛋白和S－CD为手性固定相进行了电色谱手性分离的研究。在这两种体系下分离色氨酸对映体的分离度分别为3．86和2．97。吸附S－CD柱电色谱和动态改性电谱的重现性进行子比较,发现动态改性电色谱有更好的重现性。     

2,4,5-涕丙酸的NMR手性分离条件

以1，2－二苯基二氨基乙烷（简称DPEDA，化合物1）的（1R，2R）体（简称（R）－1）为手性溶剂（CSA），解决了外消旋2，4，5－涕丙酸（化合物2）在氯仿溶剂中溶解性不良的问题，并且化合物2的^1H谱获得了出色的手性分离效果；通过研究包括温度、浓度、底物与CSA的摩尔比、CSA结构等影响化学位移不等价的因素得出结论，对于手性中心CH，在化合物2与（R）－1的摩尔比为2：1时，提高底物浓度和降低测试温度有利于增加手性分离度；而对于与手性中心相连的CH3，增大（R）－1的比例，减小底物浓度，提高测试温度，有利于增大△δH；化合物1的（1S，2S）体（简称（S）－1）与（R）－1具有相同的手性分离作用；另外还对各影响因素的作用机理进行了探讨。     

碱性药物与人血清白蛋白结合参数的毛细管区带电泳/前沿分析

将毛细管区带电泳／前沿分析技术用于测定碱性药物ｖｅｒａｐａｍｉｌ（ＶＥＲ）和ｐｒｏｐｒａｎｏｌｏｌ（ＰＲＯ）与人血清白蛋白（ＨＳＡ）平衡体系中结合参数的研究。通过压力进样将药物白蛋白混合液引入石英毛细管电泳柱（３２ｃｍ×５０μｍｉ．ｄ．,聚丙烯酰胺涂渍柱）,在ｐＨ值为７．４、离子强度为０．１７的磷酸缓冲液及运行电压为１０ｋＶ的条件下,未结合的碱性药物的浓度可以通过电泳平台峰的高度定量,并且具有良好的线性关系（相关系数ｒ＝０．９９９）。     

Enantioselective binding analysis of verapamil to plasma lipoproteins by capillary electrophoresis-frontal analysis

Capillary electrophoresis coupled with frontal analysis was applied to the study of enantioselective binding of verapamil (VER) to plasma lipoproteins. The drug-lipoprotein mixed solution, which had been in the binding equilibrium, was hydrodynamically introduced into a non-coated fused-silica capillary. Since VER is positively charged in the neutral run buffer (pH 7.4), the unbound VER enantiomers migrated toward the cathodic end much faster than negatively charged lipoproteins and their bound forms. Once unbound VER migrated apart from lipoprotein, the bound VER was quickly released from the protein to maintain the binding equilibrium. Thus, VER migrated as a zone through the capillary and gave a trapezoidal peak with a plateau region on the electropherogram. The VER concentration in this plateau region was equal to the unbound VER concentration in the initial sample solution. It was found that the bindings of VER to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL were not site-specific and not enantioselective. Partition-like binding to lipid part of these lipoproteins seemed to be dominant. The total binding affinities of LDL to VER were about seven-times stronger than those of HDL, and the oxidation of LDL by copper ion enhanced the binding affinities significantly.     

Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis

Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to HSA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 microM (+/-)AL was equilibrated with 300 microM HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and KS-AL, were 9910-11200 and 90200-104000 M(-1), respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site I). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.     

Capillary electrophoresis study of human serum albumin binding to basic drugs

Summary The applicability of capillary electrophoresis/frontal analysis (CE/FA) for determining the binding constants of the drugs propranolol (PRO) and verapamil (VER) to human serum albumin (HSA) was investigated. After direct hydrodynamic injection of a drug-HAS mixture solution into a coated capillary (32 cm × 50 μm i.d.), the basic drug was eluted as a zonal peak with a plateau region under condition of phosphate buffer (pH 7.4; ionic strength 0.17) at 12 kV positive running voltage. The unbound drug concentrations measured from the plateau peak heights had good correlation coefficients,r>0.999. Employing the Scatchard plot, the Klotz plot and nonlinear regression, the drug protein binding parameters, the binding constant and the number of binding sites on one protein molecule, were obtained. The binding constant obtained was compared to a reported equilibrium dialysis result and they are basically in good agreement.     

Study on the protein binding of ketoprofen using capillary electrophoresis frontal analysis compared with liquid chromatography frontal analysis

A method of capillary electrophoresis frontal analysis (CEFA) is developed for the first time to study the binding of ketoprofen to human serum albumin (HSA) and compared with high-performance liquid chromatography frontal analysis (LCFA). The separation is performed in an uncoated fused-silica capillary (60-cm x 75- micro m i.d., 50-cm effective length) with a phosphate buffer (pH 7.4, ionic strength of 0.17M) as the running buffer. The applied voltage is 13 kV and the detection is set at 254 nm. A trapezoidal peak of the unbound ketoprofen appears after HSA elution in the electropherogram. The plateau height of the peak is employed to determine the unbound concentration of ketoprofen in the HSA equilibrated sample solution. The CEFA method provides the advantages of small sample injection volume and rapidity and the disadvantage of low sensitivity compared with LCFA. CEFA is applicable to the binding parameter estimation of ketoprofen to the secondary binding site; an association constant (K(2)) of 0.24 x 10(6)M(-1) and the number for the binding site per molecule HSA of 2.54 is estimated. In contrast, LCFA measures parameters for both primary and secondary sites, which are 1.05 x 10(6)M(-1) and 0.94 for K(1) and n(1), respectively, and 0.12 x 10(6)M(-1) and 3.16 for K(2) and n(2), respectively. It is found that ketoprofen binds mainly at the primary site at a molecular ratio of ketoprofen versus HSA lower than 0.75, and the binding at the secondary site occurs at a higher ratio.     

酮洛芬与蛋白结合作用的高效液相色谱-迎头分析法研究及其与高效毛细管电泳-迎头分析法的比较

采用高效液相色谱-迎头分析法(HPLC-FA),以67 mmol/L (pH 7.4, I=0.17 mol/L) 的等渗磷酸盐缓冲液为流动相,Pinkerton GFF　Ⅱ-S5-80内表面反相柱(150 mm×4.6 mm i.d., 5 μm)为固定相,254 nm下检测,研究了酮洛芬与人血清白蛋白(HSA)的结合作用,通过非线性回归参数估算求得酮洛芬与HSA的结合参数。与高效毛细管电泳-迎头分析法(HPCE-FA)相比,HPLC-FA法具有高灵敏度的优势,但进样量较大,分析时间较长。HPLC-F     

Characterization of interactions between human serum albumin and tumor-inhibiting amino alcohol platinum(II) complexes using capillary electrophoresis

Platinum(II) complexes with amino alcohol ligands are of growing interest as anticancer agents capable of changing their reactivity toward biomolecules at different pH values. The binding of such compounds to the transport protein, human serum albumin (HSA), under simulated physiological conditions (pH 7.4, 100mM chloride, 37 degrees C) has been studied by capillary electrophoresis (CE), with the objective to acquire and compare their binding parameters. The association constants and stoichiometric ratios of the platinum-HSA adducts were determined by measuring the concentration changes of the peak area response of the Pt complex (after a 48 h incubation of the reaction mixture to attain equilibrium), constructing the binding isotherms, and their mathematical analysis. The investigated Pt(II) compounds were found to show moderate affinity toward HSA, with association constants ranging from 1.0 x 10(3) to 2.4 x 10(4)M(-1). Such binding behavior was attributed to a distinctive structural feature of bis(amino alcohol)platinum(II) complexes, that is, existence of an equilibrium between ring-opened and ring-closed forms in solution.     

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

Summary The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.     

Enantiomeric separation of basic drugs with partially filled serum albumin as chiral selector in capillary electrophoresis.

A reliable method is presented for the chiral separation of three basic drugs (mexiletine, chlorpheniramine and propranolol) with serum albumins (human and porcine, HSA and PSA) as chiral selectors by capillary electrophoresis in combination with the partial filling technique. Based on the systematic optimization of operation variables, the chiral separation of mexiletine, chlorpheniramine and propranolol was achieved in the pH 7.4 phosphate buffer by using HSA, PSA and PSA as selectors, respectively. The chiral recognition ability of HSA and PSA was compared. HSA and PSA show a different chiral recognition ability for each of the three drugs. In addition, the association constants between enantiomeric drugs and proteins were determined to be 2.00 and 3.80 x 10(2) M(-1) for mexiletine and HSA, 0.59 and 1.12 x 10(3) M(-1) for chlorpheniramine and PSA, and 0.87 and 1.42 x 10(3) M(-1) for propranolol and PSA. The method for the chiral separation and determination of association constants possesses the advantages of simple performance, effective avoiding of the interference of the UV detection from protein, and lowering of the reagent consumption.