【作者】 张雪娇；

【导师】 齐暑华；

【作者基本信息】 西北工业大学 ， 材料学， 2019， 博士

【摘要】 有关药物手性分离和活性评价的研究一直都是分析科学领域的热门研究方向。常见的色谱分析方法包括紫外分光光度法（UV）、荧光光谱法及高效液相色谱法（HPLC）等。与这些色谱分析方法相比较,毛细管电泳法具有操作简单、样品消耗少、分离效果好及环境污染小等特点,再加上分离模式多样化,更是扩展了其在分析科学领域的应用,尤其是在药物的有效成分分析、手性药物的拆分、药物与生物体之间相互作用等方面有着非常重要的理论和现实意义。本文重点采用毛细管区带电泳（CZE）、亲和毛细管电泳（ACE）和毛细管电色谱（CEC）三种分离模式,对纳米金和聚合物等材料在药物的手性分离,以及药物与受体之间相互作用等方面的应用进行实验研究。选择氨基糖苷类抗生素链霉素作手性选择剂,采用CZE法研究其对布洛芬、扁桃酸、地匹福林、肾上腺素及异丙肾上腺素等酸性药物的手性拆分效果。链霉素结构中含有较为丰富的-NH₂、-OH等活性基团,容易与这五种药物结构中的-COOH、-OH以及-NH₂等以氢键缔合,并且链霉素以及这五种药物结构中都存在有手性中心,也会使手性选择剂与样品之间产生手性分子之间的相互作用力。重点对链霉素浓度、背景缓冲液的浓度和p H值、运行电压及分离柱温等拆分条件进行优化。结果表明,除了对地匹福林的拆分效果较差以外,其他四种药物均在一定的电泳条件下达到了基线分离。利用纳米金（GNPs）表面易于被巯基修饰这一特性,以一定浓度的L-半胱氨酸（L-Cys）对其表面进行修饰,形成金纳米复合物L-Cys-GNPs。该复合物表面因键合有较多数量的L-Cys,使其带有较多的活性基团和手性中心,满足了手性选择剂的结构特征。在随后的研究中首次以L-Cys-GNPs作为手性选择剂,采用CZE法对肾上腺素、异丙肾上腺素和去甲肾上腺素进行手性拆分研究。重点考察L-Cys的键合浓度、缓冲液p H值、运行电压及分离柱温等对分离效果的影响。结果表明,在一定的电泳条件下,三种肾上腺素类药物均达到了基线分离,其中对异丙肾上腺素的分离效果优于异丙肾上腺素和去甲肾上腺素。受柱色谱分离的启发,通过制备毛细管电色谱整体柱,考察其对手性药物的拆分效果。首先,通过微波辅助合成技术以γ-甲基丙烯酰氧丙基三甲氧基硅烷（γ-MAPS）对毛细管柱内表面进行硅烷化预处理。而后将反应单体甲基丙烯酸缩水甘油酯（GMA）、交联剂乙二醇二甲基丙烯酸酯（EDMA）、致孔剂环己醇和十二醇以及引发剂偶氮二异丁腈（AIBN）按一定的比例混合于三颈瓶中,经过通氮除氧后通入毛细管中,采用传统的原位聚合法,制备聚合物基固定相基质GMA-EDMA。然后利用聚合物固定相基质的环氧基将牛血清白蛋白键合在其表面,制备手性整体柱。重点对单体和交联剂的配比、致孔剂的选择、聚合温度及聚合时间等条件进行探讨,优化了聚合物基固定相基质的制备条件。最后以异丙肾上腺素作为样品评价其手性分离效果,为有机聚合物整体柱对手性药物的分离分析提供科学依据。在采用毛细管电色谱开管柱研究药物与β₂-肾上腺素（β₂-AR）受体之间的相互作用时,首先借助微波辅助合成技术以γ-氨丙基三甲氧基硅烷（APTS）对毛细管柱内表面进行硅烷化处理,再以β₂-AR受体为涂层材料采用化学键合法制备开管柱,并采用红外光谱、扫描电镜和荧光标记等方法对β₂-AR受体的键合效果进行表征。然后,通过控制β₂-AR受体通入毛细管柱的时间长短来改变涂层长度。最后,根据盐酸肾上腺素、重酒石酸去甲肾上腺素、盐酸普萘洛尔等三种药物在不同涂层长度的毛细管中的淌度变化,与涂层长度等数据进行线性拟合,得到三种药物与β₂-AR受体的结合常数大小次序为:盐酸肾上腺素>重酒石酸去甲肾上腺>盐酸普萘洛尔。实验结果表明,通过化学键合法固定在毛细管壁上的β₂-AR依然保持其原生物活性,能有效地与各种药物（配体）发生相互作用,可用于药物活性评价及筛选。本研究中还采用ACE法研究去甲肾上腺素（NE-B）与牛血清白蛋白（BSA）之间的相互作用。以BSA和N,N-二甲基甲酰胺（DMF）的混合物为样品,将不同浓度的NE-B添加在运行缓冲液中,分别在25℃和37℃测定BSA出峰时间,根据BSA淌度变化与NE-B浓度变化之间的关系,对浓度的倒数和BSA出峰时间进行线性拟合后,分别得到NE-B与BSA在25℃和37℃的结合常数K_b。然后又采用传统的UV法对ACE法所测得的结果进行检验,证明两种方法的测量结果具有良好的吻合性,37℃时的K_b值均大于25℃,说明结合常数在一定温度范围内随着温度的增大而增大。最后,根据结合常数对其热力学参数进行计算,结果表明△H>0,△S>0,△G<0,可知NE-B与BSA之间的作用力主要为疏水作用力且反应自发进行。更多还原

【Abstract】 In the field of analytical science,chiral separation and activity evaluation of drugs have always been a hot research direction.Common chromatographic analysis methods include ultraviolet spectrophotometry（UV）,fluorescence spectrometry and high performance liquid chromatography（HPLC）.Compared with these chromatographic methods,the capillary electrophoresis has attracted more and more attention because of simple operation,less sample consumption,high separation efficiency and low environmental pollution.In addition,the diversification of separation models has expanded its application in analytical science.Especially it has very important theoretical and practical significance in the analysis of effective components of drugs,the separation of chiral drugs and the interaction between drugs and receptors.In this paper,the Au nanoparticles and polymer materials were used to investigate their applications in chiral drug separation and the interaction between drugs and receptors,with three analysis models,capillary zone electrophoresis（CZE）,affinity capillary electrophoresis（ACE）and capillary electro chromatography（CEC）.In the study of enantiomeric separation by CZE method,five acidic drugs,ibuprofen,mandelic acid,dipivlin,epinephrine and isoprenaline,were investigated using streptomycin as a new chiral selector.Streptomycin,as an aminoglycoside antibiotic,contains abundant active groups,such as amino and hydroxyl groups.It is easy to associate with carboxyl,hydroxyl and amino groups in the five drugs’structures by hydrogen bond.And there are many chiral centers in streptomycin and the five drugs,which also cause interaction between streptomycin and durgs.The experimental parameters such as the concentration of streptomycin,buffer concentration and p H value,operating voltage and column temperature were all investigated systematically in order to optimize the chiral sepatation.Except for the poor resolution of dipivlin,the results showed that the baseline separation of the other four drugs was achieved using streptomycin as chiral selector by CZE method.Gold nanoparticles（GNPs）have the characteristic that is easily modified by sulfhydryl groups.Therefore,the GNPs were modified with chiral molecular L-cysteine（L-Cys）,which the complex of L-Cys-GNPs was synthesized.There was a large amount of L-Cys on the surface of the complex,which makes it have more active groups and chiral centers,and possess the structural characteristics of chiral selectors.In the next steps,the enantiomers of epinephrine,isoproterenol and norepinephrine were separated excellently by CZE using L-Cys-GNPs as a chiral selector for the first time.The experimental parameters such as the L-Cys bonding concentration,p H value of buffer solution,operating voltage and column temperature were all investigated systematically in order to optimize the chiral sepatation.The results showed that all three adrenaline drugs reached baseline separation using L-Cys-GNPs as chiral selector by CZE method.And the separation effect of isoproterenol was better than that of isoproterenol and norepinephrine.Enlightened by column chromatographic separation,a monolithic capillary electrochromatography column was prepared to investigate the resolution effect of chiral drugs.Firstly,the inner surface of capillary column was silylated byγ-methacrylate oxypropyltrimethoxysilane（γ-MAPS）under the technology of microwave-assisted.Then the reaction monomer glycidyl methacrylate（GMA）,crosslinking agent ethylene glycol dimethyl acrylate（EDMA）,mixed porogen cyclohexanol and dodecanol,and initiator AIBN were mixed in a three-neck bottle in a certain proportion.After removing oxygen by feeding nitrogen,the mixture was pushed into the capillary.As a result,the polymer-based GMA-EDMA stationary phase matrix was prepared by traditional in-situ polymerization.Thirdly,the bovine serum albumin（BSA）was bonded onto the surface of GMA-EDMA stationary phase matrix with epoxy group,and the Chiral Monolithic columns were prepared.The main factors affecting the polymerization,such as the ratio of monomer and crosslinking agent,the selection of pore-forming agent,the polymerization temperature and time,were discussed,and the polymer conditions of the mixture were optimized.Finally,the chiral separation effect of the monolithic column was evaluated by using isoproterenol as a sample,which provide a scientific basis for the separation of chiral drugs by CEC method.The interaction between drugs andβ₂-adrenergic receptors（β₂-AR）was also studied by open-tube column of CEC.Firstly,the inner surface of capillary column was silylated byγ--aminopropyl trimethoxysilane（APTS）under the technology of microwave-assisted.Then the open-tube column was prepared withβ₂-AR as coating material by chemical bonding method.The binding effect ofβ₂-AR was characterized by infrared spectroscopy,scanning electron microscopy and fluorescence labeling.Secondly,the length of the coating was changed from controlling the injection time ofβ₂-AR.Finally,the adrenaline hydrochloride,norepinephrine bistartrate and propranolol hydrochloride were injected into open-tube column with different coating lengths respectively,and the fluidity changes were linearly fitted with the coating length.Based on the linear equation,the binding constants K_b of three drugs and（β₂-AR）-AR were obtained,and the order of binding constants was adrenaline hydrochloride>norepinephrine bistartrate>propranolol hydrochloride.As shown in experimentals,theβ₂-AR bonded on the capillary wall by chemical bonding still maintains its original biological activity,and it also can interact with various drugs（ligands）effectively.This method can be used for drug activity evaluation and screening.In this paper,the interaction between norepinephrine（NE-B）and bovine serumalbumin（BSA）was also studied by ACE method.The mixture of BSA and N,N-dimethylformamide（DMF）was injected as sample,and the NE-B with different concentrations was added to the running buffer solution.Then the peak time of BSA was determined at 25℃and 37℃respectively.According to the relationship between the peak time of BSA and the concentrations of NE-B,linear fitting of reciprocal concentration and the peak time was carried out.Based on the linear equation,the binding constants K_b of NE-B and BSA were obtained at differret temperature.In order to further test the results that obtained by ACE method,the binding constants of NE-B and BSA were also determined by traditional UV method.The results showed that the binding constantsof the two methods are in good agreement,and the K_b value at 37℃was greater than that at 25℃.It was showen that the K_b value increases with the increase of temperature in a certain temperature range.Finally,a series of thermodynamic parameters were calculated according to the binding constants.It was concluded that the interaction between NE-B and BSA was hydrophobic mainly,and the reaction taked place spontaneously.更多还原