CN105001307B - 一种溶解难溶多肽的偶联肽链及其在液相色谱中分离纯化的应用 - Google Patents
一种溶解难溶多肽的偶联肽链及其在液相色谱中分离纯化的应用 Download PDFInfo
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- CN105001307B CN105001307B CN201510466145.5A CN201510466145A CN105001307B CN 105001307 B CN105001307 B CN 105001307B CN 201510466145 A CN201510466145 A CN 201510466145A CN 105001307 B CN105001307 B CN 105001307B
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- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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Abstract
本发明公开了一种溶解难溶多肽的偶联肽链及其在液相色谱中分离纯化的应用，属于生物化学领域。利用特殊连接臂将亲水多肽链与难溶多肽链连接，解决了难溶多肽链在液相色谱中无法操作的问题，优化了亲水氨基酸的组合，然后利用酯键水解断开难溶多肽链和亲水多肽链，使目标肽链直接析出，具有简易、高效的特点，用该方法获得的难溶多肽产品完全可以达到客户要求标准。
Description
技术领域
本发明涉及生物工程和多肽生产领域，具体的说涉及一种溶解难溶多肽的偶联肽链及其在液相色谱中分离纯化的应用。
背景技术
溶解性是研究蛋白质和多肽会遇到比较重要的问题，每个氨基酸都有其固有的化学特性。如亮氨酸、异亮氨酸、颉氨酸是疏水性的，而赖氨酸、组氨酸、精氨酸是亲水性的。难溶多肽主要是其包含的难溶氨基酸引起的，一般来说，难溶氨基酸占多肽比例超过75％就会造成多肽的难溶。
在多肽生产和制备方面，难溶性一直是多肽分离纯化步骤的一大难题。多肽主要的纯化手段是反相高效液相色谱，其具有分离效果好，分辨率高回收率高的特点。反相高效液相色谱在对多肽进行分离纯化时，需要将多肽完全溶解到溶剂中，最适合的常规溶剂为H2O、ACN，这样才能利用反相高效液相色谱进行有效的分离纯化，但难溶多肽给广大科研人员在该操作带来极大麻烦。
现有解决难溶多肽纯化的方法只能选择优良的有机溶剂(如：DMSO，DMF)去溶解多肽，然后进行反相高效液相色谱分离纯化。而DMF或DMSO是通过破坏多肽的二级结构助溶，会导致高效液相色谱分离效果大大降低，且对难容肽链的溶解能力有限。
发明内容
发明目的：本发明的目的是提供一种用于溶解难溶多肽的的偶联肽链；本发明还有一个目的是提供前述偶联肽链在溶解难溶多肽上的应用。
技术方案：为了实现上述发明目的，本发明的一种溶解难溶多肽的偶联肽链如式(I)所示：
X-Ph-Y-(B)n (I)，
其中，Ph表示1,4位被取代的苯环；
X表示羟基或C1-C4的单羟基烷基；
Y表示与(B)n发生脱水缩合反应后形成的羰基或C2～C4羰基烷基；
(B)n表示n个相同或不同的亲水氨基酸缩合组成的亲水多肽链，n为难溶多肽氨基酸数的1.2～2倍。
具体地说，式(I)所述X为直链或支链的–CH2OH、–CH2CH2OH、–CH2CH2CH2OH、–CH2CH2CH2CH2OH的任意一种；所述Y为直链或支链的–CH2CO–、–CH2CH2CO–或–CH2CH2CH2CO–的任意一种。
X-Ph-Y原本来自于1,4位分别被单羟基烷基和单羧基烷基取代的苯环。羧基的-OH与(B)n的氨基脱水缩合形成肽链，因此通式(I)的Y为直链或支链的–CH2CO–、–CH2CH2CO–或–CH2CH2CH2CO–的任意一种。
本发明的耦合肽链中，X–Ph–Y是将亲水多肽链和难溶多肽链耦合的“连接臂”，该“连接臂”来自4-羟基苯甲酸、对羟甲基苯甲酸、4-(2-羟乙基)苯甲酸、4-(3-羟基丙基)苯甲酸、4-(4-羟基丁基)苯甲酸、对羟基苯乙酸、4-(羟甲基)苯乙酸、对羟基苯丙酸、3-[4-(羟基甲基)苯基]丙酸、4-(4-羟基苯基)丁酸、2-(4-羟基苯)丙酸、3-(4-羟基苯基)丁酸、2-[(4-羟基苯基)甲基]氨基丁酸、4-(1-羟基乙基)-苯甲酸的任意一种，通过脱水缩合反应与亲水多肽链的N端连接。
另外，本发明的耦合肽链中，亲水氨基酸包括但不限于精氨酸、赖氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸、脯氨酸中的任意一种，这些氨基酸如下表1所示，疏水参数很小；而所述的难溶多肽链，可以理解为包含难溶氨基酸且在液相色谱中溶解度很低或难溶的多肽链，难溶氨基酸包括丙氨酸、蛋氨酸、半胱氨酸、苯丙氨酸、亮氨酸、缬氨酸、异亮氨酸的任意一种，这些氨基酸如表2所示，疏水参数大，含有这些氨基酸的多肽链一般溶解度都非常低。
表1 亲水氨基酸列表
| 氨基酸名称 | 缩写 | 代码 | 疏水参数 |
| 精氨酸 | Arg | R | -4.5 |
| 赖氨酸 | Lys | K | -3.9 |
| 天冬酰胺 | Asn | N | -3.5 |
| 天冬氨酸 | Asp | D | -3.5 |
| 谷氨酰胺 | Gln | Q | -3.5 |
| 谷氨酸 | Glu | E | -3.5 |
| 组氨酸 | His | H | -3.2 |
| 脯氨酸 | Pro | P | -1.6 |
表2 难溶氨基酸列表
| 氨基酸名称 | 缩写 | 代码 | 疏水参数 |
| 丙氨酸 | Ala | A | 1.8 |
| 蛋氨酸 | Met | M | 1.9 |
| 半胱氨酸 | Cys | C | 2.5 |
| 苯丙氨酸 | Phe | F | 2.8 |
| 亮氨酸 | Leu | L | 3.8 |
| 缬氨酸 | Val | V | 4.2 |
| 异亮氨酸 | Ile | I | 4.5 |
上述难溶多肽链一般是肽链合成中的目标产物，而亲水多肽链和连接臂可结合生产实践进行优选。作为本发明的进一步优化，所述X-Ph-Y来自4-羟基苯甲酸、对羟甲基苯甲酸、4-(2-羟乙基)苯甲酸、对羟基苯乙酸、4-(羟甲基)苯乙酸的任意一种，通过脱水缩合反应与亲水多肽链的N端连接。亲水多肽链中的亲水氨基酸优选谷氨酸或天冬氨酸。当谷氨酸和/或天冬氨酸总数目占总亲水氨基酸数目的30％～80％时，利用X-Ph-Y-(B)n在液相色谱中分离纯化，最后再水解酯键的效果最好。
本发明利用上述偶联肽链进行分离纯化的步骤如下：
(1)通过固相合成法先合成亲水多肽链，再合成连接臂和难溶多肽链，经过处理后获得难溶多肽链-连接臂-亲水多肽链粗品；
(2)将上述产品用水/乙腈溶解，于高效液相色谱装样进行分离提纯，真空冷冻干燥，获得纯品；
(3)将上述纯品用LiOH饱和溶液进行酯键水解，常温下反应3小时后，目标多肽链析出，过滤干燥后获得成品。
由于X-Ph-Y-(B)n的亲水性强，且肽链多呈酸性，在溶解在LiOH中的效果非常突出。而亲水多肽链中谷氨酸、天冬氨酸含量高的话，利于亲水多肽链在碱性溶液中的溶解，提高目标多肽链的析出效果。
本发明中一些常用缩写及其含义如下：
HMBA 4-羟甲基苯甲酸，连接臂；
DIC N,N'-二异丙基碳二亚胺，缩合剂；
DMAP 4-二甲氨基吡啶，作为超强亲核催化剂，增加羟基与羧基链接效率；
HOBT 1-羟基苯并三唑，用于防止消旋反应，减少副反应；
DMF 二甲基甲酰胺，反应溶剂；
TFA 三氟乙酸，用于裂解多肽与树脂；
TIS 三异丙基硅烷，用于更好地去除氨基酸保护基团；
EDT 乙二硫醇，用于更好地去除氨基酸保护基团；
ACN 乙腈，增加多肽在水溶液的溶剂度；
Fmoc N-芴甲氧羰基，官能团保护基团；
Pbf [(2,3-二氢-2,2,4,6,7-五甲基苯并呋喃-5-基)磺酰基，α-氨基保护基团；
Boc 叔丁氧羰基，α-氨基保护基团；
Trt 三苯甲基，α-氨基保护基团；
Otbu 叔丁酯，α-氨基保护基团
有益效果：本发明提供了一种利用特殊连接臂将亲水多肽链与难溶多肽链连接的方案，解决了难溶多肽链在液相色谱中无法操作的问题。本发明的耦合肽链既可以在生产、试验中通过固相合成法合成，也可作作为定制产品为目标肽链的生产提供服务。尤其是本发明针对液相色谱分离提纯环境的特点，优化了亲水氨基酸的组合，最后利用酯键水解断开难溶多肽链和亲水多肽链，使目标肽链直接析出，具有简易、高效的特点，用该方法获得的难溶多肽产品完全可以达到客户要求标准。
附图说明
图1是本发明实施例1的多肽合成步骤流程示意图；
图2是本发明实施例2的多肽合成步骤流程示意图。
具体实施方式
下面结合具体实施例对本发明进行进一步说明。
实施例1
本实施例针对难溶多肽ILVLLIII进行分离纯化，连接臂选用4-羟甲基苯甲酸(HMBA)，通过连接臂连接DDDDDEEKEEEE亲水多肽链。其中，D表示天冬氨酸，疏水参数-3.5，利用OtBu保护官能团；E表示谷氨酸，疏水参数-3.5，利用OtBu保护官能团，K表示赖氨酸，疏水参数-3.9，利用Boc保护官能团；这些氨基酸都采用Fmoc保护α-氨基进行固相合成。如图1所示，具体合成步骤如下：
称取1g Fmoc-Glu(OtBu)-Wang resin树脂，执行[操作A]，即：加入哌啶：DMF＝1:4(体积比)移除N端的Fmoc保护基团，温度控制在30℃，反应20分钟，反应后利用DMF/甲醇各洗涤3次，利用茚三酮检测试剂检测，颜色为蓝色表示反应完全。
然后执行[操作B]，即：加入初始树脂摩尔数两倍的DIC和HOBT，以及两倍量的具有保护基团的氨基酸反应1小时，温度控制在30℃，反应结束后用DMF洗涤3次后，用茚三酮检测颜色为无色，则说明反应完全。
后续以[操作A]、[操作B]交替进行，只是在[操作B]中所加入的亲氨基酸随着合成顺序的进行更换。其中具有保护基团的谷氨酸、天冬氨酸和赖氨酸分别为Fmoc-Glu(OtBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH。如此反应直到连接完HMBA，用茚三酮溶液验色为无色即说明反应完成。以上反应即获得X-Ph-Y-(B)n的亲水偶联肽链。
基于上述获得的肽链，直接进行[操作B]，并在后续步骤与[操作A]交替，直至获得王氏树脂-难溶多肽链-连接臂-亲水多肽链。
然后用10ml TFA:TIS:EDT＝95％:3％:2％裂解树脂2小时，将偶联肽链脱除保护基团。然后将树脂滤除留下母液，加100ml乙醚析出多肽。用3000转/秒离心2min，沉淀得到粗品多肽，重复洗涤离心后在真空干燥机里干燥，获得难溶多肽链-连接臂-亲水多肽链粗品。
将上述粗品用水/乙腈的混合液体溶解，于高效液相色谱装样进行分离提纯，经过用H2O/0.1TFA％做水相，ACN/0.1％TFA做有机相的梯度洗脱色谱体系分离提纯，收集目标峰。把收集的目标峰用分析性高效液相色谱检测纯度。合格的样品用旋转蒸发仪浓缩，放冰箱预冻成固体。最后放入真空冷冻干燥机冻干，获得纯品ILVLLIII-HMBA-DDDDDEEKEEEE。
将上述纯品用LiOH饱和溶液进行酯键水解，常温下反应3小时后，目标多肽链ILVLLIII析出，而增加的序列HMBA-DDDDDEEKEEEE由于亲水性强且整个多肽呈酸性，将溶解在LiOH溶液中，然后将析出的目标多肽经过过滤冻干即可得到成品。
上述反应中，Fmoc氨基酸购于吉尔生化，生产批号GLS141015-4071、DIC HobtDMAP购于苏州昊帆、HMBA购于吉尔生化，TFA、TIS、EDT、乙醚、哌啶、DMF均购于南京晚晴。
实施例2
本实施例针对难溶多肽ILVLLIII进行分离纯化，连接臂选用4-(2-羟乙基)苯甲酸(HPA)，通过连接臂连接RRRRREDKKKKK亲水多肽链。其中，D表示天冬氨酸，疏水参数-3.5，利用OtBu保护官能团；E表示谷氨酸，疏水参数-3.5，利用OtBu保护官能团；K表示赖氨酸，疏水参数-3.9，利用Boc保护官能团，R表示精氨酸，疏水参数-4.5，利用Pbf保护官能团；这些氨基酸都采用Fmoc保护α-氨基进行固相合成。如图2所示，具体合成步骤如下：
称取1g Fmoc-Lys(boc)-Wang resin树脂，执行[操作A]，即：加入哌啶：DMF＝1:4(体积比)移除N端的Fmoc保护基团，温度控制在30℃，反应20分钟，反应后利用DMF/甲醇各洗涤3次，利用茚三酮检测试剂检测，颜色为蓝色表示反应完全。
然后执行[操作B]，即：加入初始树脂摩尔数两倍的DIC和HOBT，以及两倍量的具有保护基团的氨基酸反应1小时，温度控制在30℃，反应结束后用DMF洗涤3次后，用茚三酮检测颜色为无色，则说明反应完全。
后续以[操作A]、[操作B]交替进行，只是在[操作B]中所加入的亲氨基酸随着合成顺序的进行更换。其中具有保护基团的赖氨酸、天冬氨酸、谷氨酸、精氨酸分别为Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(Pbf)-OH，如此反应直到连接完HPA，用茚三酮溶液验色为无色即说明反应完成。以上反应即获得X-Ph-Y-(B)n的亲水偶联肽链。
基于上述获得的肽链，直接进行[操作B]，并在后续步骤与[操作A]交替，直至获得王氏树脂-难溶多肽链-连接臂-亲水多肽链。
然后用10ml TFA:TIS:EDT＝95％:3％:2％裂解树脂2小时，将偶联肽链脱除保护基团。然后将树脂滤除留下母液，加100ml乙醚析出多肽。用3000转/秒离心2min，沉淀得到粗品多肽，重复洗涤离心后在真空干燥机里干燥，获得难溶多肽链-连接臂-亲水多肽链粗品。
将上述粗品用水/乙腈的混合液体溶解，于高效液相色谱装样进行分离提纯，经过用H2O/0.1TFA％做水相，ACN/0.1％TFA做有机相的梯度洗脱色谱体系分离提纯，收集目标峰。把收集的目标峰用分析性高效液相色谱检测纯度。合格的样品用旋转蒸发仪浓缩，放冰箱预冻成固体。最后放入真空冷冻干燥机冻干，获得纯品ILVLLIII-HPA-RRRRREDKKKKK。
将上述纯品用LiOH饱和溶液进行酯键水解，常温下反应3小时后，目标多肽链ILVLLIII析出，而增加的序列HPA-RRRRREDKKKKK由于亲水性强且整个多肽呈酸性，将溶解在LiOH溶液中，然后将析出的目标多肽经过过滤冻干即可得到成品。
试验例
本试验针对各组的肽链分别评判其在DMF溶液、DMSO溶液、HPLC中ACN:H2O(1:3)溶液、以及LiOH饱和溶液下，2mg/mL的溶解能力。上述实施例1、2中的难溶多肽链-连接臂-亲水多肽链分别作为组1、组2，单独的难溶多肽链ILVLLIII作为对照组。如表3所示：
表3 难溶多肽连处理后的溶解能力评价
目标肽链的序列为：ILVLLIII，在DMF、DMSO和常用HPLC纯化条件ACN:H2O＝1：3溶解后十分浑浊且有固体析出。ILVLLIII4-HMBA-DDDDDEEKEEEE在DMF、DMSO和常用HPLC纯化条件ACN:H2O(1:3)溶解后均十分清澈且无固体析出。而ILVLLIII-HPA-RRRRREDKKKKK在ACN:H2O(1:3)中完全溶解，但在DMF和DMSO 中仍有固形物析出。
对于第一组和第二组的肽链溶解在LiOH饱和溶液中，要求最好加入的时候能完全溶解，然后等反应3小时后，难溶多肽链水解析出固体。而第二组的溶解效果显然没有第一组好，会导致产品产率下降。可见第一组亲水多肽链的氨基酸结构更利于最后难溶多肽链的析出。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一种溶解难溶多肽的偶联肽链，其特征在于，该肽链如式(I)所示：
X-Ph-Y-(B)n (I)，
其中，Ph表示1，4位被取代的苯环；
X表示羟基或C1-C4的单羟基烷基；
Y表示与(B)n发生脱水缩合反应后形成的羰基或C2～C4羰基烷基；
(B)n表示n个相同或不同的亲水氨基酸缩合组成的亲水多肽链，n为难溶多肽氨基酸数的1.2～2倍；
所述亲水氨基酸中，谷氨酸和/或天冬氨酸总数目占总亲水氨基酸数目的30％～80％；所述亲水氨基酸为精氨酸、赖氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸的任意一种。
2.根据权利要求1所述的一种溶解难溶多肽的偶联肽链，其特征在于：
所述X为直链或支链的-CH2OH、-CH2CH2OH、-CH2CH2CH2OH、-CH2CH2CH2CH2OH的任意一种；
所述Y为直链或支链的-CH2CO-、-CH2CH2CO-或-CH2CH2CH2CO-的任意一种。
3.根据权利要求2所述的一种溶解难溶多肽的偶联肽链，其特征在于：所述式(I)中的X-Ph-Y来自4-羟基苯甲酸、对羟甲基苯甲酸、4-(2-羟乙基)苯甲酸、4-(3-羟基丙基)苯甲酸、4-(4-羟基丁基)苯甲酸、对羟基苯乙酸、4-(羟甲基)苯乙酸、对羟基苯丙酸、3-[4-(羟基甲基)苯基]丙酸、4-(4-羟基苯基)丁酸、2-(4-羟基苯)丙酸、3-(4-羟基苯基)丁酸、2-[(4-羟基苯基)甲基]氨基丁酸、4-(1-羟基乙基)-苯甲酸的任意一种。
4.如权利要求1所述的偶联肽链在液相色谱分离纯化上的应用。
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