【作者】 童胜强；

【导师】 关怡新；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 博士

【摘要】 本论文主要考察了两类手性试剂在高速逆流色谱(HSCCC)手性分离中的应用,对逆流色谱以及pH区带精制逆流色谱在手性药物以及异构体分离中的色谱保留机制以及相关理论进行了探讨。两类手性试剂是指水溶性手性试剂羟丙基-β-环糊精(HP-β-CD)以及脂溶性手性试剂酒石酸酯类化合物。选择了共10个外消旋体药物以及药物中间体作为手性拆分中的研究对象,包括药物中间体苯基琥珀酸、α-环已基扁桃酸,2-芳基丙酸类药物萘普生、布洛芬、酮基布洛芬、氟比洛芬、舒洛芬以及卡洛芬,p-受体阻滞剂类药物普萘洛尔和吲哚洛尔外消旋体。首先,通过手性萃取拆分研究确定了高速逆流色谱拆分苯基琥珀酸(PSA)的两相溶剂体系为：正已烷：甲基叔丁基醚：水相(0.5：1.5：2,v/v),其中水相为0.1mol/L磷酸盐缓冲溶液pH2.51并含0.05mol/L HP-β-CD。考察了影响对映体分布系数与分离因子的主要因素。计算了环糊精对PSA对映体手性识别过程中的焓变和熵变。测定了PSA对映体在两相溶剂中的Langmuirian等温分布图。采用制备型高速逆流色谱手性拆分了712mgPSA外消旋体。通过分析型高速逆流色谱实验探讨了色谱保留机制并计算了手性试剂-对映体包结常数。同时,提出了双相识别(S/M)逆流色谱手性分离模式,采用该方法拆分α-环已基扁桃酸(a-CHMA)对映体。推导出双相识别中分离因子的表达式,表明在双相识别体系中其对映体的选择性分离因子与两种手性试剂形成的识别能力成乘积关系,而非加和关系。合成了14种酒石酸酯,通过手性萃取拆分研究确定了高速逆流色谱拆分α-CHMA的两相溶剂体系：正已烷：甲基叔丁基醚：水相(9：1：10,v/v),其中水相为0.1mol/L磷酸盐缓冲溶液pH2.51并含0.1mol/L手性试剂HP-β-CD,双相识别过程中同时在有机相中添加0.3mo1/L的D-酒石酸-2-乙基已酯。对比研究了单相识别与双相识别模式中影响对映体分布系数与分离因子的主要因素。测定了双相识别过程中α-CHMA对映体在两相溶剂中的Langmuirian等温分布图。分别在单相识别与双相识别模式中采用制备型高速逆流色谱手性分离α-CHMA对映体。探讨了单相识别模式逆流色谱手性分离α-CHMA的色谱保留机制以及手性试剂-对映体包结常数的计算,提出了在逆流色谱中测定第二个平衡包结常数的方法。经过研究发现HP-β-CD在对CHMA对映体进行手性识别过程中以2：1的形式结合,并非通常认为的1：1结合模式。其次,将闭路循环洗脱模式(CLRM)应用于HSCCC手性拆分中。经3次循环洗脱,将2-芳基丙酸类药物萘普生(NAP)对映体分离度由原来的0.7提高至1.4,基本达到完全分离。选择的溶剂体系为正已烷：乙酸乙酯：水相(8：2：10,v/v),其中水相为0.1mol/L磷酸盐缓冲液pH2.6并含有0.1mol/L手性试剂HP-β-CD。考察了溶剂体系中影响萘普生对映体分布系数与分离因子的主要因素。环糊精在水相中不但起到手性识别作用,更重要的是通过环糊精的包结作用提高了有机分子在水相中的溶解度,这对于逆流色谱溶剂体系的选择是非常重要的。同时研究了其它5个2-芳基丙酸类药物外消旋体的拆分工作,但是均未获得成功。对HSCCC分离机理进行了探讨,特别是针对HSCCC分离度方程进行了深入的讨论,以分离NAP对映体为例探讨了如何提高对映体之间分离度问题,对逆流色谱手性分离具有指导性意义。然后,采用氯仿：水相(1：1,v/v)为溶剂体系,其中水相为0.05mol/L醋酸盐缓冲溶液pH4.4(三乙胺调节)并含0.1mol/L硼酸,有机相为氯仿含有0.1mol/L的L-酒石酸正已酯,采用HSCCC成功拆分了β-受体阻滞剂类药物普萘洛尔与吲哚洛尔。拆分原理主要基于在有硼酸的作用下,通过配位键等作用提高了对普萘洛尔和吲哚洛尔对映体的识别能力。另外,采用溶剂体系氯仿：水相(1：l,v/v),其中水相为0.1mol/L硼酸并添加5mmol/L的盐酸作为洗脱酸,有机相为氯仿含有0.1 mol/L的L-酒石酸正丁酯并添加20mmol/L的三乙胺作为保留碱,实现了pH区带精制逆流色谱拆分普萘洛尔对映体。最后,理论分析表明pH区带逆流色谱对于分离离子型化合物来说若有分离区带的交叉现象,主要有以下三个原因引起：一是进样量的增加；二是如果被分离的溶质疏水性大小与酸碱性pKa值类似会引起重叠,第三种情况是疏水性大小与酸碱性相差都比较大,但是这两种作用互相抵消,则也是引起峰型的交叉的原因。对于后两种情况,解决这个问题的方法是同时提高保留酸(碱)与洗脱碱(酸)的浓度。通过pH区带分离咖啡酰基奎尼酸类3个立体异构体组分包括3-咖啡酰基奎尼酸、3,5-二咖啡酰基奎尼酸和3,4-二咖啡酰基奎尼酸的实验验证了上述结果。HSCCC是一种良好的手性分离制备色谱,其主要缺陷是理论塔板数较低,本文建立了双相识别基础上的HSCCC手性分离方法,该模式可大大提高HSCCC手性分离中的分离因子；通过采用灵活多样的HSCCC洗脱模式如闭路循环洗脱模式以及pH区带精制逆流色谱模式也可以在一定程度上弥补HSCCC技术分离效率较低的缺点从而提高对映体之间的分离度；为提高对映体之间的分离度,考察了HSCCC色谱分离度方程,在HSCCC仪器以及手性试剂确定的情况下,主要可以通过提高对映体分布系数以及提高分离柱内固定相保留值的方法来获取相对较高的分离度。上述研究对逆流色谱手性分离具有指导性意义。更多还原

【Abstract】 This work concentrates on the application of two chiral selectors including hydrophilic chiral selector hydroxypropyl-β-cyclodextrin (HP-β-CD) and lipophilic chiral selector dialkyl tartrate in enantioseparation of ten racemic drugs or intermediates by high speed counter-current chromatography (HSCCC). The chromatographic retention mechanism of racemates by HSCCC with HP-β-CD as chiral selector was proposed and discussed. Three stereoisomeric caffeoylquinic acids were selected and separated by pH-zone-refining counter-current chromatography (CCC) to study on elution sequence and zone overlap phenomena, which were frequently observed during pH-zone-refining CCC separation. The ten racemic drugs or intermediates were as follows:intermediates phenylsuccinic acid and a-cyclohexylmandelic acid,2-arylpropionic acid drugs including naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen and carprofen, andβ-blocker drugs propranolol and pindolol.Firstly, HSCCC was applied to resolution of phenylsuccinic acid (PSA) enantiomers with HP-β-CD as chiral selector and chromatographic retention mechanism was proposed. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-0.1 mol/L phosphate buffer solution with pH=2.51 containing 0.05 mol/L HP-β-CD (0.5:1.5:2, v/v). Influence factors involved in the chiral separation were investigated, including concentration of chiral selector, pH of aqueous phase and separation temperature. Thermodynamic parameters of inclusion complex were calculated. The correlation between both separation factor (a) and peak resolution (Rs) and the concentration of HP-β-CD was investigated. The chromatographic retention mechanism of PSA by HSCCC with HP-P-CD as chiral selector was discussed. A mathematical model showing the relationship of capacity factor (k’) with HP-β-CD concentration, hydrogen ion concentration, complex formation constant (Kf) and dissociation constants (Ka) of PSA was proposed, in which the complex formation constants were calculated. Loading limits for HSCCC separation run with a given value of chiral selector was determined and Langmuirian isotherm for the enantiomers in the two phase solvent system was investigated. Under optimum separation conditions,712 mg of PSA racemate was separated using preparative apparatus. Meanwhile, a novel chiral separation technology named biphasic chiral recognition HSCCC was proposed and applied to resolution of racemic a-cyclohexylmandelic acid (CHMA). The biphasic chiral recognition HSCCC was performed by adding lipophilic (-)-2-ethylhexyl tartrate in the organic stationary phase and hydrophilic HP-β-CD in the aqueous mobile phase, which preferentially recognized the (-)-enantiomer and (+)-enantiomer, respectively. The two-phase solvent system composed of n-hexane-methyl tert-butyl ether-0.1 mol/L phosphate buffer solution with pH=2.51 (9:1:10, v/v/v) was selected. Totally 14 dialkyl tartrate derivatives were synthesized. Factors affecting the chiral separation were investigated. The mechanism involved in this biphasic recognition chiral separation by HSCCC was discussed. Loading limits for a given value of chiral selectors were estimated as for HSCCC separation. Under optimum separation conditions,365 mg of sample was separated using the preparative HSCCC. Enantioseparation of racemic CHMA by HSCCC using only HP-β-CD as chiral selector in the aqueous phase was also compared. The chromatographic retention mechanism was studied and an efficient method was developed for calculation of complex formation constant between HP-β-CD and CHMA enantiomer. Experimental results showed that a 2:1 stoichiometry between the cyclodextrin host and the guest molecule was found during chiral recognition instead of 1:1 stoichiometry.In the second place, closed loop recycling mode (CLRM) HSCCC was successfully applied to resolution of 2-arylpropionic acid drug (R,S)-naproxen (NAP) using HP-β-CD as chiral selector. The two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol/L phosphate buffer solution (8:2:10, v/v/v) was selected. Influence factors for the chiral separation process were investigated and several thermodynamic parameters of NAP-HP-β-CD inclusion complex were calculated.29 mg of (R,S)-NAP was separated using preparative recycling HSCCC. Resolution factor for NAP enantiomers could be improved from 0.7 to 1.2 when CLRM HSCCC was used. The successful enantioseparation of other five 2-arylpropionic acid drugs were not be achieved by HSCCCC using HP-[3-CD as chiral selector because enantioseparation factor was too small as for HSCCC. Theoretical aspects of HSCCC were demonstrated in order to improve resolution factor of NAP enantiomers. Exact equation for resolution factor of HSCCC was discussed in detail, which might be helpful for chiral separation by HSCCC.Then, dialkyl L-tartrate was used as chiral selector for enantioseparation ofβ-blockers propranolol and pindolol by HSCCC as well as pH-zone-refining counter-current chromatography. Dialkyl L-tartrate exhibited a distinctive chiral discrimination with respect to racemic P-amino-alcohols drugs propranolol and pindolol in the liquid-liquid two phase system in the presence of boric acid because a borate complex of the 1,2-diol group of the tartrate and the amino-alcohol was formed in the system. A two-phase system chloroform:aqueous (1:l,v/v) consisting of a chloroform solution of 0.1 mol/L dihexyl L-tartrate and an aqueous solution of 0.1 mol/L boric acid was successfully used for enantioseparation of propranolol and pindolol by HSCCC. pH-zone-refining CCC was also successfully used for preparative enantioseparation of racemic propranolol and 356 mg of propranolol racemate was separated by this method.Finally, principles about the elution sequence and zone overlaps during pH-zone-refining CCC were discussed. The chances of obtaining zone overlaps increase when the amount of injection in the CCC machine increase, the difference between the compounds to be separated is low (similar pKa and similar distribution coeffincients). The typical way to solve the problem is to increase both retainer and eluter concentrations. Two isomeric dicaffeoylquinic acids, including 3, 5-dicaffeoylquinic acid and 3,4-dicaffeoylquinic acid along with 3-caffeoylquinic acid were studied using pH-zone-refining CCC. The elution sequence of the isomeric dicaffeoylquinic acids was discussed in terms of their acidities, hydrophilicity, and steric configuration. The possible explanations were offered for the mechanism of separation.Although HSCCC is a powerful preparative technique with its high capacity, low cost of stationary phases and low solvent consumption, it yields low efficiency compared to classic liquid chromatography. The current studies demonstrated that biphasic chiral recognition would give much higher separation factor for enantiomers than monophasic chiral recognition during HSCCC enantioseparation, which could make up its disadvantage to some extent. Closed loop recycling elution mode and pH-zone-refining counter-current chromatography could also be applied to improve resolution factor. Under constant theoretical plates and the same chiral selector, better resolution factor could be obtained by improving distribution factor or retention of stationary phase in the separation column.更多还原