CN104475066B - 一种适用于氨基酸手性拆分的高效液相色谱分离柱 - Google Patents

一种适用于氨基酸手性拆分的高效液相色谱分离柱 Download PDF

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CN104475066B
CN104475066B CN201410741345.2A CN201410741345A CN104475066B CN 104475066 B CN104475066 B CN 104475066B CN 201410741345 A CN201410741345 A CN 201410741345A CN 104475066 B CN104475066 B CN 104475066B
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袁黎明
伍鹏
路振宇
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Yunnan Normal University
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Abstract

本发明公开了一种适用于氨基酸手性拆分的高效液相色谱分离柱。取R(或者S)‑(3,3'‑溴基‑1,1'‑二萘基)‑20‑冠‑6溶于二氯甲烷中，将冠醚溶液均匀分散到C18硅胶上，旋转蒸发除去溶剂制成手性固定相。取手性固定相混合于甲醇水溶液中，搅拌成匀浆液，用甲醇水溶液做顶替液，采用湿法装填色谱柱。该分离柱能在常温条件下对19个具有手性位点的蛋白质氨基酸全部达到有效分离，能对手性位点上有伯胺的手性药物达到有效分离。本发明具有高分辨率、分离速度快、重现性高、可反复使用、制柱成本相对较低等显著特点。其对氨基酸的分离效果要明显优于国内外同类产品。

Description

一种适用于氨基酸手性拆分的高效液相色谱分离柱
技术领域
本发明属于高效液相色谱分离技术领域，具体是涉及一种特别适用于氨基酸手性拆分的高效液相色谱分离柱。
背景技术
高效液相色谱具有高压、高速、高效、高灵敏度、应用范围广、样品不被破坏等优点，已广泛应用于核酸、肽类、内酯、稠环芳烃、表面活性剂，抗氧化剂、医用药物等物质的分析。而运用手性高效液相柱分离手性药物是最有效的方法之一，但目前专门用于氨基酸手性拆分的高效液相色谱分离柱还存在以下问题，如：对混合氨基酸的手性拆分效果不好、在对部分单个氨基酸进行手性拆分时的温度条件较为苛刻、常温条件下没有分离效果等。这些问题阻碍了高效液相色谱分离技术的进一步应用，但现有技术中尚未见有很好地解决办法。
发明内容
本发明的目的在于针对现有技术的不足，提供一种适用于氨基酸手性拆分的高效液相色谱分离柱。该分离柱能够在常温下对组成蛋白质的19种手性氨基酸以及一些手性伯胺进行拆分。
本发明的目的通过以下技术方案予以实现。
除非另有说明，本发明所采用的百分数均为质量百分数。
一种适用于氨基酸手性拆分的高效液相色谱分离柱，由下述方法制备得到：
(1)取R(或者S)-(3,3'-二溴基-1,1'-二萘基)-20-冠-6溶于二氯甲烷中，将冠醚溶液均匀分散到8～12倍的C18支撑体上，旋转蒸发除去溶剂得到手性固定相；
(2)取制备好的手性固定相混合于甲醇水溶液中，甲醇和水的体积比为1：1～2.5，搅拌成匀浆液，采用湿法装填色谱柱，即得到所需的高效液相色谱分离柱。
相对于现有技术，本发明具有以下优点：
1、R(或者S)-(3,3'-二溴基-1,1'-二萘基)-20-冠-6对组成蛋白质的手性伯胺有特殊识别能力，因此本发明的高效液相色谱分离柱能在常温条件下对19个具有手性位点的蛋白质氨基酸(具有手性的蛋白质氨基酸只有19种)全部达到有效分离；
2、R(或者S)-(3,3'-二溴基-1,1'-二萘基)-20-冠-6对在手性位点上的伯胺具有特殊的识别能力，因此本发明色谱柱能对手性位点上有伯胺的手性药物达到有效分离；
3、本发明色谱柱具有高分辨率、分离速度快、重现性高、可反复使用、制柱成本相对较低等显著特点。其对氨基酸的分离效果要明显优于国内外同类产品。
附图说明
图1为R(或者S)-(3,3'-二溴基-1,1'-二萘基)-20-冠-6的合成路线图；
图2为分别利用本发明色谱柱和CR(+)手性商品高效液相色谱分离柱(日本，大赛璐Daicel公司)，对DL-谷氨酸、DL-蛋氨酸、DL-苯甘氨酸和DL-酪氨酸进行高效液相色谱手性拆分的效果对比图；
图3为25℃条件下利用本发明色谱柱对DL-天冬酰胺和DL-谷氨酰胺(两种非蛋白质组成的α-手性伯胺)的高效液相色谱手性拆分图。
具体实施方式
以下结合附图和实施例对本发明作进一步的详细说明，但附图和实施例并不是对本发明技术方案的限定，所有基于本发明教导所做出的变换，均属于本发明的保护范围。
实施例1
合成R-(3,3'-二溴基-1,1'-二萘基)-20-冠-6的方法(如图1所示)：将R-联萘酚10g加入180mL乙腈中,加入20g K2CO3和0.5g CsCO3做催化剂,在70℃磁力回流搅拌,在回流过程中缓慢滴加碘甲烷6.52mL,反应4h。混合物用二氯甲烷萃取,干燥有机层并用硅胶柱纯化[洗脱剂V(乙酸乙酯):V(石油醚)＝1:5],减压除去洗脱剂得白色晶体B 10.54g；在氮气的保护下,将37.6mL的正C4H9Li(1.6mol/L)加入到四甲基二乙胺(6.1mL)的200mL乙醚溶液中,25℃搅拌15min加入8.0g产物B搅拌3h。然后将反应物冷却到-75℃,在10min内加入Br2(3.9mL)的戊烷(20mL)溶液,然后将反应物升到室温搅拌过夜,最后加入160mL饱和亚硫酸钠溶液搅拌4h。混合物用二氯甲烷萃取,干燥有机层并用硅胶柱纯化(洗脱剂V(丙酮):V(环己烷)＝1:40),减压除去洗脱剂得淡黄色晶体C 6.97g；在氮气保护下,将5g的产物C加入到150mL的无水二氯甲烷溶液中,将反应物降温到0℃缓慢的加入10mL BBr3搅拌15min升温至25℃搅拌24h,然后再将反应物降温至0℃加水除去过量的BBr3。混合物用二氯甲烷萃取,干燥有机层并用硅胶柱纯化[洗脱剂V(乙酸乙酯):V(石油醚)＝1:10],减压除去洗脱剂得白色晶体D3.90g,在氮气保护下,将3g产物D和2.5g二对甲苯磺酸戊乙二醇加入到180mL无水四氢呋喃溶液中,再加入0.88g KOH在60℃条件下搅拌回流72h。混合物用二氯甲烷萃取,干燥有机层并用硅胶柱纯化[洗脱剂V(乙酸乙酯):V(环己烷)＝1:2]得亮白色晶体E 2.58g。晶体E即为R-(3,3'-二溴基-1,1'-二萘基)-20-冠-6。
取0.4g制得的R-(3,3'-二溴基-1,1'-二萘基)-20-冠-6溶于10mL二氯甲烷中，取冠醚溶液加到装有3.6g的5μm的C18硅胶的小烧瓶中，使冠醚溶液均匀分散到C18硅胶上，旋转蒸发除去溶剂使冠醚全部涂覆在C18表面上。制得手性固定相4.0g，将其混合于24mL甲醇/水(1/2,v/v)溶液中，搅拌成匀浆液，采用湿法装填色谱柱，用甲醇/水(1/2,v/v)做顶替液，在高压(40MP)下装入不锈钢空色谱柱:250mm×4.6mm i.d，制成手性冠醚高效液相柱。
分别利用所制得的手性冠醚高效液相柱和CR(+)手性商品高效液相色谱分离柱，用高效液相色谱仪(美国，LC600，Labtech)，在紫外检测器检测波长为210nm，流动相为pH＝1，经过0.45μm滤膜过滤超声脱气的高氯酸溶液，流速为0.5mL min-1，液相柱的柱温为25℃的条件下，测试对DL-谷氨酸、DL-蛋氨酸、DL-苯甘氨酸和DL-酪氨酸的液相色谱手性拆分效果。具体见图2。
由图2可知：本发明所制得的手性冠醚高效液相柱和CR(+)手性商品高效液相色谱分离柱，在相同条件下对DL-谷氨酸、DL-蛋氨酸、DL-苯甘氨酸和DL-酪氨酸四种混合氨基酸有着不同的拆分效果。其中CR(+)柱未能拆分开混酸中的DL-蛋氨酸，未能完全拆分开DL-酪氨酸。而本发明所制得的手性冠醚高效液相柱对四种混酸都达到了完全拆分(基线分离)。本发明明显优于CR柱。
实施例2
用实施例1所得色谱柱，在紫外检测器检测波长为210nm，流动相为pH＝2，经过0.45μm滤膜过滤超声脱气的高氯酸溶液，流速为0.5mL min-1，液相柱的柱温为25℃的条件下，测试对DL-谷氨酰胺和DL-天冬酰胺的液相色谱手性拆分效果，具体见图3。
由图3可知：实施例1所得色谱柱对DL-谷氨酰胺能达到基线分离，对DL-天冬酰胺达到部分分离，说明其对非蛋白质组成的α-手性伯胺也具有一定的分离效果。
实施例3
用本发明色谱柱a(实施例1所得)以及商品CR(+)柱b手性柱对21种α-氨基酸对映体(前19种为组成蛋白质的氨基酸，后两种为其它手性伯胺)在紫外检测器检测波长为210nm，流动相为pH＝1，经过0.45μm滤膜过滤超声脱气的高氯酸溶液，流速为0.5mL min-1，液相柱的柱温为25℃的条件下的色谱手性拆分效果。保留因子k1＝(t1－t0)/t0,k2＝(t2－t0)/t0；分离因子α＝k2/k1；分离度Rs＝1.18(t2－t1)/(W1/2(1)+W1/2(2))；其中t1、t2分别为样品第一个流出峰和第二个流出峰的保留时间；W1/2(1)、W1/2(2)分别为两个对映体色谱峰的半峰宽.具体见表1。
表1手性柱对α-氨基酸对映体的拆分结果
Table 1 The separation results ofα-amino acid enantiomers on chiralcolumns
a色谱条件:流动相为pH＝2的高氯酸,流速为0.5mL min-1,柱温为25℃.柱规格:250mm×2mm；
b色谱条件:流动相为pH＝2的高氯酸,流速为0.5mL min-1,柱温为25℃.柱规格:150mm×4mm；
α＝1，Rs＝/d:表示样品没有得到拆分。
由表1可知：通过比较判断本图表中的α以及Rs值可知本发明色谱柱与商品CR(+)相比在对α-氨基酸对映体的拆分上具有明显的优势。本发明色谱柱对：苯丙氨酸、对羟基苯甘氨酸、精氨酸、半胱氨酸和丙氨酸的分离效果要明显优于商品CR(+)柱；更重要的是商品柱没能对：脯氨酸、丝氨酸、苏氨酸、天冬酰胺、组氨酸和缬氨酸达到拆分的目的，但本发明色谱柱对这六种手性物质都达到了有效的拆分。

Claims (1)

1.一种适用于氨基酸手性拆分的高效液相色谱分离柱，由下述方法制备得到：
（1）取R或者S-(3,3'-溴基-1,1'-二萘基)-20-冠-6溶于二氯甲烷中，将冠醚溶液均匀分散到8～12倍的C18支撑体上，旋转蒸发除去溶剂得到手性固定相；
（2）取制备好的手性固定相混合于甲醇水溶液中，甲醇和水的体积比为1：1～2.5，搅拌成匀浆液，采用湿法装填色谱柱，即得到所需的高效液相色谱分离柱。
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