CN110317257B - 一种辛卡利特的固液相合成法 - Google Patents
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Abstract
本发明涉及一种辛卡利特固液相结合的合成方法,主要解决现有技术存在的原料昂贵以及后处理繁琐的技术问题。技术方案为:一种辛卡利特的固液相合成法,包括以下步骤:(1)液相法合成Fmoc‑Asp‑Phe‑CONH2二肽片段;(2)将二肽片段装载到二氯三苯基氯树脂上,得到Fmoc‑Asp(β‑2Cl‑Trt Cl树脂)‑Phe‑NH2;(3)固相合成法依次连接余下保护氨基酸,得到辛卡利特树脂肽,Asp‑Tyr(SO3H)‑Met‑Gly‑Trp‑Met‑Asp(β‑2Cl‑Trt Cl树脂)‑Phe‑CONH2;(4)切割得到辛卡利特粗品,Asp‑Tyr(SO3H)‑Met‑Gly‑Trp‑Met‑Asp‑Phe‑CONH2;粗品再经高效液相色谱分离纯化、冻干精制得到辛卡利特。
Description
技术领域
本发明涉及多肽化合物合成技术领域,尤其涉及一种辛卡利特的固液相合成法。
背景技术
胆囊收缩素(CCK)是一种具有多种分子形式且具有双重分布的脑肠肽,由33 个氨基酸组成,具有激素和神经递质的作用。辛卡利特(sincalide)为一八肽分子,是胆囊收缩素的一个片段,具有胆囊收缩素的全部生物活性,是一种人工合成的胆囊素八肽(Octapeptide),主要用于促胆囊收缩,胆囊诊断试剂,临床用于治疗慢性胰腺炎,发现胰分泌功能明显改善,症状明显减轻。CAS 号为:25126-32-3,分子式为C49H62N10O16S3,分子量1143.27。
目前,辛卡利特的制备方法主要有酶法和化学法两种。其中,酶法合成由于涉及酶制剂种类比较多、酶制剂价格昂贵,且中间产物分离困难、制备周期长,目前已不再使用酶法制备辛卡利特。化学合成是目前常用的辛卡利特制备方法。CN201410105423.X公开了一种制备辛卡利特的方法,该方法中选择了氨基树脂,在低温条件下用三氟乙酸裂解多肽树脂,虽然能减少磺酰基脱落,但是不能完全避免,而且会导致多肽裂解不完全,降低粗品收率;CN200810043920.6公开了一种制备辛卡利特的方法,该方法选择用氨水裂解多肽树脂,其中氨水有毒,对眼、鼻、皮肤有刺激性和腐蚀性,不利于后期产业化,而且选用的Fmoc-Asp(odmab)-OH这一原料价格昂贵,增加了生产成本,没有显著的经济效益。CN201810365226.X公开了一种制备辛卡利特的方法,该方法也是选取了两肽片段树脂作为起始树脂,但是Asp1选择了Fmoc-Asp (Ome)-OH保护策略,在裂解后还需要水解去除Ome。
发明内容
本发明的目的在于提供一种高收率、低成本、反应条件温和、环境污染小、有利于实现产业化的辛卡利特的合成方法,主要解决现有技术存在的原料昂贵以及后处理繁琐的技术问题。
本发明的技术方案为:一种辛卡利特的固液相合成法,包括以下步骤:
(1)液相法合成Fmoc-Asp-Phe-CONH2二肽片段;
(2)将二肽片段装载到二氯三苯基氯树脂上,得到Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-NH2;
(3)固相合成法依次连接余下保护氨基酸,得到辛卡利特树脂肽,
Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp(β-2Cl-Trt Cl树脂)-Phe-CONH2;
(4)切割得到辛卡利特粗品,Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-CONH2;粗品再经高效液相色谱分离纯化、冻干精制得到辛卡利特。
具体步骤如下:
(1)Fmoc-Asp(Otbu)-Osu,NH2-Phe-CONH2溶于四氢呋喃中,加入DIEA,搅拌反应2小时,旋蒸除去四氢呋喃,加入体积比95%TFA溶液搅拌反应1-2小时,去除叔丁基,再加乙醚结晶得到Fmoc-Asp- Phe-CONH2二肽片段;
(2)将二氯三苯基氯树脂置于圆底烧瓶中,加入Fmoc-Asp-Phe-CONH2二肽片段,加入二氯甲烷,DIEA,搅拌反应2小时,再加入甲醇震荡反应30分钟,过滤除去滤液;用二氯甲烷洗涤树脂三次,干燥,得到Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2;
(3)将Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2置于多肽反应器中,加入脱保护试剂,氮气搅拌反应30分钟,抽干,再用DMF洗涤5次,加入相应的保护氨基酸,HBTU/NMM为缩合剂,DMF做溶剂,反应30-60分钟,茚三酮检测反应终点,反应结束后抽真空排干反应液,用DMF洗涤3次,再加入脱保护试剂,依次循环,直至连完最后一个天冬氨酸,脱保护,得到辛卡利特树脂肽;
脱保护试剂为体积比20%哌啶/DMF溶液,反应中所提到保护氨基酸依次是Fmoc-Met-OH,Fmoc-Trp-OH,Fmoc-Gly-OH,Fmoc-Met-OH,Fmoc-Tyr(SO3h)-OH,Fmoc-Asp(Ofm)-OH;
(4)将辛卡利特树脂肽置于圆底烧瓶中,加入裂解试剂,常温条件下反应1小时,抽滤滤去树脂颗粒,收集滤液,减压浓缩旋去溶剂,得到辛卡利特中间体;再经高效液相色谱分离纯化、冻干后得到辛卡利特精品。
裂解试剂的配比为醋酸:三氟乙酸/二氯甲烷=2/98(V/V);
本发明中一些常用缩写具有以下含义:
HBTU:O-苯并三唑-N,N,N,N-四甲基脲鎓六氟磷酸盐;
NMM:N-甲基吗啉;
Fmoc:芴甲氧羰基;
Trp:色氨酸;
Gly:甘氨酸;
Asp:天冬氨酸;
Phe:苯丙氨酸;
Met:蛋氨酸;
Tyr:酪氨酸;
HSO3:磺酰基;
Ofm:芴甲氧基;
DIEA:N,N-二异丙基乙胺;
DMF:N,N-二甲基甲酰胺;
2Cl-Trt Cl Resin:二氯三苯基氯树脂。
本发明的有益效果是:本发明中策略性的选择了一个二肽片段中的Asp侧链羧基装载到二氯树脂上,在弱酸条件下就能将多肽从树脂上切割下来,避免了强酸条件下磺酸基掉落的副反应,同时Asp1选择Fmoc-Asp(ofm)-OH保护,去Fmoc过程中一并脱除ofm保护基;省去了一个反应步骤,提高了粗品纯度,通过一步切割就得到辛卡利特粗品,该方法操作简单,反应条件温和,无毒害,收率高,并解决了水解过程中产生的废水问题,利于后期工业化放大,具有极大的应用价值。
附图说明
图1为本发明产物色谱图。
图2为本发明产物质谱图。
具体实施方式
以下将参照实例对本发明做进一步的详细描述,但本发明不限于这具体实例。
实施例
(1)制备Fmoc-Asp-Phe-CONH2
称取Fmoc-Asp (Otbu)-OSU 10.2g(MW:508.5,20mmol),NH2-Phe-CONH2 3.61g(MW:164.2,22mmol)加入200ml四氢呋喃,10mlDIEA,搅拌反应2小时,再经萃取,洗涤,旋蒸除去溶剂,得到油状物12.1g,再加入100ml 配比为95%TFA/5%H2O(V/V)去保护基,搅拌反应1-2小时,加乙醚结晶,抽滤除去滤液,得到白色固体滤饼即为二肽片段Fmoc-Asp-Phe-CONH2 ,干燥后得到二肽片段10.5g。
(2)制备Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2
称取二氯三苯基氯树脂20g,加入Fmoc-Asp-Phe-CONH2 10.5g(MW:501.5,20mmol),加入300ml二氯甲烷,30mlDIEA,搅拌反应2小时,加入200ml甲醇,震荡反应30分钟,过滤除去滤液,树脂用二氯甲烷洗涤3次,得到Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2;
(3)制备辛卡利特树脂肽
将Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2置于多肽反应器中,加入300ml脱保护试剂,氮气搅拌反应30分钟,抽干,DMF洗涤5次,称取保护氨基酸,缩合剂,加入到反应器中,再加入200mlDMF,氮气搅拌反应30-60分钟,茚三酮检测反应终点,反应结束后排干反应液,DMF洗涤3次,反复循环,直至连完最后一个氨基酸,脱保护,得到辛卡利特树脂肽;
每一步缩合反应所加入的氨基酸的量分别为:
Fmoc-Met-OH (7.42g),
Fmoc-Trp-OH (8.52g),
Fmoc-Gly-OH(5.95g),
Fmoc-Met-OH(7.43g),
Fmoc-Tyr(SO3H)-OH(9.66g),
Fmoc-Asp(Ofm)-OH(10.67g);
每一步缩合反应所采用的缩合剂的量为:HBTU(3.6g),NMM(2.27ml)
脱帽试剂为体积比20%的哌啶/DMF混合溶液;
(4)制备辛卡利特
取步骤(3)中的辛卡利特树脂肽共32g置于500ml圆底烧瓶中,加入320ml裂解液,配比为三氟乙酸/二氯甲烷=2/98(V/V),置于摇床中常温震荡反应1小时,抽滤滤掉树脂颗粒,收集滤液,减压浓缩旋去溶剂,得到辛卡利特粗品共11.5g;再经高效液相色谱分离纯化、冻干后得到纯度99%的辛卡利特精品7.6g,收率达到66%。见图1、2。
Claims (4)
1.一种辛卡利特的固液相合成法,其特征在于:包括以下步骤
(1)液相法合成Fmoc-Asp-Phe-CONH2二肽片段:Fmoc-Asp(Otbu)-Osu,NH2-Phe-CONH2溶于四氢呋喃中,加入DIEA,搅拌反应2小时,旋蒸除去四氢呋喃,加入体积比95%TFA溶液搅拌反应1-2小时,去除叔丁基,再加乙醚结晶得到Fmoc-Asp- Phe-CONH2二肽片段;
(2)将二肽片段装载到二氯三苯基氯树脂上,得到Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2:将二氯三苯基氯树脂置于圆底烧瓶中,加入Fmoc-Asp-Phe-CONH2二肽片段,加入二氯甲烷,DIEA,搅拌反应2小时,再加入甲醇震荡反应30分钟,过滤除去滤液;用二氯甲烷洗涤树脂三次,干燥,得到Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2;
(3)固相合成法依次连接余下保护氨基酸,得到辛卡利特树脂肽,Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp(β-2Cl-Trt Cl树脂)-Phe-CONH2;脱保护试剂为体积比20%哌啶/DMF溶液,反应中所提到保护氨基酸依次是Fmoc-Met-OH,Fmoc-Trp-OH,Fmoc-Gly-OH,Fmoc-Met-OH,Fmoc-Tyr(SO3h)-OH,Fmoc-Asp(ofm)-OH;
(4)切割得到辛卡利特粗品,Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-CONH2;粗品再经高效液相色谱分离纯化、冻干精制得到辛卡利特。
2.根据权利要求1所述的一种辛卡利特的固液相合成法,其特征在于:步骤(3)具体内容为:将Fmoc-Asp(β-2Cl-Trt Cl 树脂)-Phe-CONH2置于多肽反应器中,加入脱保护试剂,氮气搅拌反应30分钟,抽干,再用DMF洗涤5次,加入相应的保护氨基酸,HBTU/NMM为缩合剂,DMF做溶剂,反应30-60分钟,茚三酮检测反应终点,反应结束后抽真空排干反应液,用DMF洗涤3次,再加入脱保护试剂,依次循环,直至连完最后一个天冬氨酸,脱保护,得到辛卡利特树脂肽。
3.根据权利要求1所述的一种辛卡利特的固相合成法,其特征在于:步骤(4)具体内容为:将辛卡利特树脂肽置于圆底烧瓶中,加入裂解试剂,常温条件下反应1小时,抽滤滤去树脂颗粒,收集滤液,减压浓缩旋去溶剂,得到辛卡利特中间体;再经高效液相色谱分离纯化、冻干后得到辛卡利特精品。
4.根据权利要求3所述的一种辛卡利特的固液相合成法,其特征在于:所述裂解试剂为下述体积比的混合物:三氟乙酸:二氯甲烷=2:98。
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