CN111269261A - Tsbp辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法 - Google Patents
Tsbp辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法 Download PDFInfo
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Abstract
本发明涉及一种TSBP辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法,合成步骤:以TSBP替代固相树脂与N‑端PG保护的氨基酸(PG‑Xaa‑OH,Xaa为任意氨基酸)的C‑端连接,再经过分离纯化、脱除N‑端PG、多肽偶联,重复偶联上相应的氨基酸,构建脑啡肽或其衍生物前体的氨基酸序列H2N‑Tyr(OtBu)‑Gly‑Gly‑Phe‑Xaa‑TSBP;再经过侧链脱保护、脑啡肽及其衍生物的分离纯化、得到脑啡肽及其衍生物H2N‑Tyr‑Gly‑Gly‑Phe‑Xaa‑ZH(Z=O,S,NH等)。与目前已有的合成方法相比,本发明兼备了液相和固相合成法的优点,可以更加简便、快捷、节约、高效地合成制备谷胱甘肽,而且磷酸酯类载体可以回收并直接再利用,降低原材料浪费,减少废弃物污染,节约成本,利于环保。
Description
技术领域
本发明属于有机化学与多肽合成领域,涉及一种TSBP辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法。尤其涉及磷酸三[4-(α-取代苄基)苯基]酯TSBP的辅助基团,以及以TSBP辅助基团进行基团辅助的脑啡肽及其衍生物液相的合成方法。涉及合成脑啡肽及其衍生物的一种基于磷酸酯类载体三[4-(α-取代苄基)苯基]磷酸酯(TSBP,结构式A)的脑啡肽及其衍生物全合成方法(合成路线如图0所示)。本发明以磷酸酯类小分子TSBP为载体替代固相树脂载体,辅助分离纯化,兼备了液相和固相合成法的优点,可以更加简便、快捷、节约、高效地合成制备脑啡肽及其衍生物,而且磷酸酯类载体可以回收并再生利用,降低原材料浪费,减少废弃物污染,节约成本,利于环保。
背景技术
动物脑内到处都存在着氨基酸。过去只认为它们是合成蛋白质的原料,或是蛋白质分解的产物。近年来,注意到某些氨基酸在中枢的突触传递中起着递质的作用。而且发现,凡是中性氨基酸,如γ-氨基丁酸、甘氨酸、β-丙氨酸等对中枢神经元表现抑制作用,而酸性氨基酸如谷氨酸、天门冬氨酸则表现为兴奋作用。
有一些小分子肽类在中枢神经系统中也具有神经递质同的作用。1975年由阿伯丁大学的J.Hughes和M.Kosterlitz从猪大脑中发现了一种衍生吗啡的肽类物质,具有强力鸦片剂性质。自从发现这一物质之后,许多研究者对它做了进一步分离、提纯和鉴定,它天然地存在于脑中,后又发现胃内也有它的存在,是列为神经肽类(2-29个氨基酸组成)的一种,作为医用药品应用于临床上具有镇痛、解痛和止痛的作用,只需要很少的量便能发挥其功效,据称这是一类最新的最激动人心的良好镇痛药物。已从猪和牛的脑中分离出两种与吗啡样活性相似的五肽,并测定了氨基酸排列顺序的结构。经研究发现的脑啡肽(Enkephalin)是由五个氨基酸残基构成的寡肽(YGGFX),是由脑细胞内合成的具有吗啡样作用的肽,故名脑啡肽,属于内啡肽。有两种天然脑啡肽存在于脑、脊髓和肠,两者的区别仅在于C-端分别是亮氨酸和甲硫氨酸,其前体(前脑啡肽)相同。内啡肽和脑啡肽的N端4肽序列相同,见文献:张叔平,高允生.《药理学》:科学出版社,2012。从猪脑中分离出来的脑啡肽又分为两型。亮氨酸型(亮脑啡肽,Leu-enkephalin):H-酪-甘-甘-苯丙-亮-OH,即Tyr-Gly-Gly-Phe-Leu;蛋氨酸型(蛋脑啡肽,Met-enkephalin):H-酪-甘-甘-苯丙-蛋-OH,即Tyr-Gly-Gly-Phe-Met。它们都是中枢神经系统中的类吗啡性神经递质。猪脑中所含的蛋氨酸脑啡肽比亮氨酸脑啡肽约大4倍;而牛脑中所含亮氨酸脑啡肽多,其比例与猪脑中相反。它们结合于细胞表面受体似同阿片一样。含脑啡肽的神经元存在于脑和脊髓灰质中。它在脊髓的主要功能是调节痛的感觉,在脑部的功能不明,但也能镇静和提高痛阈,见文献:a)Salvadori S,et al.Structure-activity relationship of enkephalin tetrapeptideanalogues containingβ-alanine.Farmaco(Sci)1983,38(9):640.b)Sarto G,et al.Theanalgesic activity of a powerful derivative of endorphin.Farmaco(Sci)1983,38(9):647.c)王鸿利,叶裕春主编,中华检验医学大辞典(第1版),上海科学技术出版社,2000:648.。
脑中吗啡类的发现,对镇痛原理的研究进入了一个新的领域,特别对我国研究针麻的原理给予了新的启示。已知吗啡(Morphine)要先与脑内的吗啡受体结合才能产生镇痛、欣快作用,而脑啡肽之所以有吗啡样作用也是因为它们能与吗啡受体结合。吗啡是外来的物质,而脑啡肽是内源性的。此外,已知脑内还有一些能与吗啡受体结合并产生吗啡样作用的其它的肽类,称之为内啡肽(Endorphin),虽然还不能十分肯定这些肽类都是真正的神经递质(即完全符合前述神经递质的条件),但是,研究它们的作用对阐明脑的功能,特别对阐明痛觉原理是很有意义的。脑啡肽及其衍生物除镇痛作用外,见文献:Walker J M,Berntson G G,Sandman C A,Coy D H,Schally A V,Kastin A J.Science,1977,196:85-87;还可参与神经内分泌与免疫系统间的循环,免疫调节,见文献:Wybran J,Appelboom T,Famaey J P,Govaerts A.J.Immunol.1979,123(3):1068-1070;感情行为的控制,见文献:Nieto M M,Guen S L,Kieffer B L,Roques B P,Noble F,Neuroscience,2005,135(2):305-313;促进伤口恢复,见文献:Yang D J,Lee K S,Ko C M,Moh S H,Song J,Hur L C,Cheon Y W,Yang S H,Choi Y H,Kim K W.Peptides,2016,76:57-64;辅助抗癌作用等,见文献:Srisuchart B,Fuchs B A,Sikorski E E,Munson A E,Loveless SE.Int.J.Immunopharmacol.1989,11(5):487-500。
脑啡肽种类脑啡肽并非唯一的吗啡样肽。在垂体中有人曾测得,不同结构的吗啡样肽类,对平滑肌具有拟似吗啡的作用,并能与吗啡受体竞争相结合。如肛趋膳素是一种含有91个氨基酸的垂体肽,其中的第61-65的氨基酸与蛋氨酸脑啡肽的氨基酸顺序相同。因此,在IIB一趋脂素的几个片段中,61~69氨基酸片段称为肛内啡肽,它在与吗啡受体结合上,以及在影响平滑肌和镇痛上,都具有强烈的吗啡样作用。脑啡肽的作用许多证据表明,脑啡肽是脑中特殊神经元系统的神经递质。这种特殊神经元系统,控制着属于痛觉和情感行为的感觉信息的整合,以及其他功能。因为发现脑啡肽水平的区域性差别与吗啡类受体相平行。如脑啡肽富集于包含神经末梢的部分,这符合对一种神经递质的预测,显示脑啡肽神经末梢与轴索系统的免疫组织化学图谱,也支持它作为一种神经递质的作用。含脑啡肽神经元末梢的存在,脑啡肽及吗啡对于具有吗啡类受体的细胞的冲动发放的特异性和选择性作用,使脑啡肽获得比其他多数脑内递质更充分的支持。
另外,在多种动物的胃肠道中也测得到脑啡肽,但在其他任何组织中并未发现。脑啡肽严格限制于胃肠遭及脑内,这点很象其他肽类如P物质和生长激素释放抑制素等物质,看来它是在外周起着激素样作用,而在脑内作为神经递质可能与脑啡肽相同。垂体吗啡样肽类不可能透入脑内,它们能调节吗啡类所能改变的垂体功能。至于较长肽链的吗啡样肽如趋脂素等与脑啡肽之间有无关系,从某些具有生物活性的肽是由较长的肽断离下来的片段来看,可以设想,趋脂素可能作为脑啡肽的前体物。如肛内啡肽能在脑中测得,而趋脂素本身则在脑中未发现。有可能,较大的肽如肛内啡肽是垂体所独有,而脑啡肽则为脑特有,见文献:李恩编著,前列腺素与现代医学(第1版),人民卫生出版社,1985:139.。
多肽是由氨基酸通过酰胺键连接形成的、大小介于氨基酸与蛋白质之间的重要生理活性物质。多肽既是蛋白质的重要组成部分,同时作为生命物质,其自身也参与生物体内众多的分子事件,并发挥着重要的生理生化功能。多肽在生物体内可作为神经递质、神经调质和激素等,通过与受体相互作用影响细胞间的信号传导与信息交流。以多肽为识别功能单元的分子探针可被应用于生命相关活性分子的检测,见文献:1、Weinstain R,SavariarE N,Felsen C N,Tsien R Y,J.Am.Chem.Soc,2014,136(3):874-877.;2、Wu J C,Zou Y,LiC Y,Sicking W,Piantanida I,Tao Y,Schmuck C.J.Am.Chem.Soc,2012,134(4):1958-1961.;3、Dai N,Guo J,Teo Y N,Kool E T.Angew.Chem.Int.Ed.2011,50(22):5105-5109.;4、Wang W Z,Wei Z W,Zhang D,Ma H L,Wang Z H,Bu X L,Li M L,Geng L L,Lausted C,Hood L,Fang Q J,Wang H,Hu Z Y.Anal.Chem,2014,86(23):11854-11859;利用多肽特异性识别靶分子的特点,发展了基于多肽识别的靶向药物运输体系,见文献:Rodriguez P L,Harada T,Christian D A,Pantano D A,Tsai R K,Discher DE.Science,2013,339(6122):971-975.和Pan L,He Q,Liu J,Chen Y,Ma M,Zhang L L,ShiJ L,J.Am.Chem.Soc,2012,134(13):5722-5725.。此外,多肽在医药、材料领域也得到越来越广泛的应用,可构建诸如纳米笼、纳米管、纳米纤维等结构的自组装体,具有催化、模拟生物分子结构等功能,见文献:Fletcher J M,Harniman R L,Barnes F R H,Boyle A L,Collins A,Mantell J,Sharp T H,Antognozzi M,Booth P J,Linden N,Miles M J,Sessions R B,Verkade P,Woolfson D N,Science,2013,340(6132):595-599.和Rufo CM,Moroz Y S,Moroz O V,Stohr J,Smith T A,Hu X Z,DeGrado W F,Korendovych I V,Nat.Chem,2014,6(4):303-309.;多肽也因其生物相容性好、毒副作用小,被应用于抗癌、抗病毒、抗心脑血管等疾病的药物,并显示出诱人的发展前景。无论是多肽的功能研究还是多肽的生物化学应用,均离不开对多肽纯品的需求。相比于生物制备方法,多肽的化学合成方法具有制备简单、合成效率高、易于自动化等优势。自1963年Merrifield教授等,见文献:Merrifield R B,J.Am.Chem.Soc,1963,85(14):2149-2154.和Lerner R A,Science,2006,313(5783):57.,发展了多肽固相合成(Solid phase peptide synthesis,SPPS)这一独创性的方法以来,引起了多肽合成领域的革命性转变,Merrifield教授也因此获得了1984年诺贝尔化学奖。目前多肽的固相化学合成已成为最重要的多肽制备方法之一,SPPS除了可以合成含有天然氨基酸的多肽之外,还能够合成含有非天然氨基酸的多肽,大大增加了合成多肽的多样性和信息量。然而由于多肽的基本结构单元除20种天然氨基酸外还有众多的衍生物,其侧链基团结构差异大,且性质各异,极易造成制备过程中偶联效率低,以及氨基酸残基的消旋、敏感位点的修饰等副反应,因此多肽的高效合成与纯化鉴定仍然是亟需解决的关键问题。
由于替代给药途径的兴起和提高其体内稳定性和生物利用度新策略的发展,合成治疗药用多肽作为有希望的候选药物最近重新引起了人们的兴趣和关注。除了这一高潜力之外,多肽的生产技术来自于合成和纯化过程中所需的大量溶剂。为了使反应物能够合适地溶解和提高反应速度,通常使用溶剂如DMF、DMAc、NMP或DCM。不幸的是,这些溶剂具有高沸点或高毒性。此外,多肽合成通常需要使用可溶的有机碱,如三乙胺、N、N-二异丙基乙胺或哌啶,用于脱质子铵盐或去除Fmoc保护基。在化学性质上,这些碱是易燃的、腐蚀性的和有毒的。因此,寻找一种方便、高效和环境友好的方法具有很高的兴趣,见文献:BonnamourJ,Métro T-X,Martinez J,Lamaty F,Green Chem.,2013,15:1116-1120。
综上所述,在目前已报道的脑啡肽及其衍生物合成方法,见文献:1、a)嵇汝运,殷敦祥,华家怪,宗汝实,药学学报,1979,14(12):742-745.b)瑙甘,金钰龙,黄嫣嫣,赵睿.分析测试学报,2017,36(2):190-195.;2、a)Ye Y-H,Tian G-L,Xing G-W,Dai D-C,Chen G,LiC-X,Tetrahedron,1998,54:12585-12596.b)Clapts P,Torresa J-L,Adlercreutz P,Bioorg.Med.Chem.,1995,3(3):245-255.;3、a)Polt R,SzabóL,Treiberg J,Li Y S,HrubyV J.J.Am.Chem.Soc.,1992,114:10249-10258.b)Weltrowska G,Berezowska I,LemieuxC,Chung N N,Wilkes B C,Schiller P W.Chem.Biol.Drug Des.2010,75:182-188.三篇文献中,用于氨基酸羧基官能团保护的基团在脱除过程中都已遭到破坏或分解掉了,完全不能回收再利用,这就大大消耗了生产成本,而且废料的排放必然会造成严重的环境污染问题,无论从经济成本还是从社会效益来看,都还谈不上是最佳方案,见文献:1、Kocienski,P.J.Protecting Groups,Georg Thieme Verlag:Stuttgart New York,2004.2、Green,T.W.;Wuts,P.G.M.Protective Groups in Organic Synthesis;John Wiley and Sons:New York,1999.3、Isidro-Llobet,A.Alvarez,M.Albericio,F.Chem.Rev.2009,109,2455–2504.。
所以,在日益提倡经济社会绿色可持续发展理念的今天,探索和发现新的可回收并循环利用的氨基酸羧基保护基团仍然是值得期待重要研究课题。我们经过系统地筛选研究发现,磷酸酯TSBP这类小分子在碱性条件和常温常压下经过偶联剂的介导可与氨基酸的羧基发生反应,可高产率地生成稳定的氨基酸衍生物,用于氨基酸的羧基保护,而且发现磷酸酯类载体TSBP及其氨基酸或多肽衍生物在非极性溶剂中很容易结晶而沉淀出来,经简单地分离纯化和碱性水解就可以从其氨基酸或多肽衍生物上将其脱除,回收纯化后可以直接循环利用,实现规模化可持续的绿色生成过程。另一方面,从现有的文献调研和分析来看,这种利用本发明中所涉及的磷酸酯类载体TSBP进行氨基酸羧基保护与辅助纯化的策略尚属首创。与已有的氨基酸羧基保护基团或树脂载体相比,具有原料丰富易得并可回收再利用、操作简便、条件温和、设备成本低、三废少的绿色环保优势和特色。
发明内容
要解决的技术问题
为了避免现有技术的不足之处,本发明提出一种TSBP辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法,主要解决目前脑啡肽及其衍生物化学合成方法的液相反应比较复杂,分离步骤多、耗时周期长、纯化规模小、生产成本高,而固相反应的生产规模小,原料价格贵、浪费大,树脂类废弃物多和环境污染严重的问题。
技术方案
一种涉及磷酸三[4-(α-取代苄基)苯基]酯TSBP辅助基团,其特征在于:分子结构通式为
一种采用所述TSBP辅助基团进行基团辅助的脑啡肽及其衍生物液相的合成方法,其特征在于步骤如下:
步骤1、辅助基团与氨基酸的偶联:辅助基团在脱水偶联剂的作用下与氨基酸在0~50℃下搅拌反应1~3小时;氨酸Fmoc-Xaa-OH的C-端与TSBP辅助基团连接,生成中间体化合物1,(Fmoc-Xaa)3-TSBP;
所述氨基酸与TSBP辅助基团的摩尔比为1~3:1;所述脱水偶联剂为1︰1摩尔比的脱水偶联活化剂和碱性物质;
所述辅助基团为磷酰氧基苯甲醇TSBP;
所述氨基酸采用芴甲氧羰基Fmoc保护的某氨酸Fmoc-Xaa-OH;
步骤2、分离纯化:向生成物A加入极性小的烷烃或醚类溶剂,借助TSBP辅助基团在溶剂系统中易结晶沉淀的特性,将生成物1与其他杂质分离;
对分离后的生成物1进行过滤和洗涤或重结晶操作得到纯化的生成物1;
步骤3、脱除N-端Fmoc:将纯化的生成物1采用脱Fmoc试剂处理,在10~50℃下搅拌反应0.5~2小时得到生成物2,(H2N-Xaa)3-TSBP;
向生成物2加入极性小的烷烃或醚类溶剂,借助TSBP辅助基团在溶剂系统中易结晶沉淀的特性,将生成物2与其他杂质分离;
对分离后的生成物2进行过滤和洗涤或重结晶操作得到纯化的生成物2;
步骤4:以纯化的含有辅助基团TSBP的生成物2作为原料,替代步骤1的辅助基团,以芴甲氧羰基Fmoc保护的苯丙氨酸Fmoc-Phe-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Phe-Xaa)3-TSBP;
以第一次循环重复后纯化的生成物(H2N-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的甘氨酸Fmoc-Gly-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Gly-Phe-Xaa)3-TSBP;
以第二次循环重复后纯化的生成物(H2N-Gly-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的甘氨酸Fmoc-Gly-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Gly-Gly-Phe-Xaa)3-TSBP;
以第三次循环重复后纯化的生成物(H2N-Gly-Gly-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的酪氨酸Fmoc-Tyr(OtBu)-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到脑啡肽或其衍生物的前体化合物[H2N-Tyr(OtBu)-Gly-Gly-Phe-Xaa]3-TSBP;
步骤5、侧链脱保护与TSBP辅助基团的剪除:以三氟乙酸的鸡尾酒溶液为侧链脱保护剂,分离纯化脑啡肽或其衍生物的前体化合物[H2N-Tyr(OtBu)-Gly-Gly-Phe-Xaa]3-TSBP的混合液;反应条件为5~30℃下,搅拌1~3小时,脱除侧链上的保护基团tBu、Boc和Pbf,同时剪除TSBP辅助基团,经分离纯化得到脑啡肽或其衍生物的三氟乙酸盐,[TFA*H-Try-Gly-Gly-Phe-Xaa-ZH];所述三氟乙酸的鸡尾酒溶液中的组分比例为:TFA/TIPS/H2O=95:2.5:2.5;
步骤6、脑啡肽及其衍生物的分离纯化:对步骤5得到混合液通过旋蒸去除三氟乙酸盐,再用乙酸乙酯萃取,沉淀析出,经过滤、乙酸乙酯洗涤、干燥得到纯化的脑啡肽或其衍生物,[H-Tyr-Gly-Gly-Phe-Xaa-ZH]。
一种所述方法制备过程中得到的Fmoc保护某氨酰-TSBP化合物,其特征在于:所述(Fmoc-Xaa)3-TSBP的分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护酪氨酰-TSBP化合物,其特征在于:所述(H2N-Xaa)3-TSBP的分子结构通式为
一种所述方法制备过程中得到的Fmoc保护苯丙氨酰-某氨酰-TSBP化合物,其特征在于:所述(Fmoc-Phe-Xaa)3-TSBP分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护苯丙氨酰-某氨酰-TSBP化合物的结构通式,其特征在于:所述(H2N-Phe-Xaa)3-TSBP分子结构通式为
一种所述方法制备过程中得到的Fmoc保护甘氨酰-苯丙氨酰-某氨酰-TSBP化合物,其特征在于:所述(Fmoc-Gly-Phe-Xaa)3-TSBP分子结构通式为
一种2所述方法制备过程中得到的脱Fmoc保护甘氨酰-苯丙氨酰-某氨酰-TSBP化合物,其特征在于:所述(H2N-Gly-Phe-Xaa)3-TSBP分子结构通式为
一种所述方法制备过程中得到的Fmoc保护甘氨酰-甘氨酰-苯丙氨酰-某氨酰-TSBP化合物,其特征在于:所述(Fmoc-Gly-Gly-Phe-Xaa)3-TSBP分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护赖氨酰-天门冬氨酰-缬氨酰-酪氨酰-TSBP化合物,其特征在于:所述(H2N-Gly-Gly-Phe-Xaa)3-TSBP分子结构通式为
一种所述方法制备过程中得到的Fmoc保护酪氨酰-甘氨酰-甘氨酰-苯丙氨酰-某氨酰-TSBP化合物,其特征在于:所述[Fmoc-Tyr(OtBu)-Gly-Gly-Phe-Xaa]3-TSBP分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护精氨酰-赖氨酰-天门冬氨酰-缬氨酰-酪氨酰-TSBP化合物,其特征在于:所述(H2N-Gly-Gly-Phe-Xaa)3-TSBP分子结构通式为
一种所述基团辅助的脑啡肽及其衍生物液相的合成中TSBP辅助基团的回收再利用的方法,其特征在于:将步骤6中所得的乙酸乙酯萃取溶液旋蒸浓缩至原体积的1/3~1/4,加入极性小的烷烃或醚类溶剂,借助TSBP在不同溶剂系统中易结晶沉淀的特性,将TSBP与其他杂质分离;对分离后的TSBP进行过滤和洗涤或重结晶操作得到纯化的TSBP,回收再生转化后直接作为辅助基团再利用。
有益效果
本发明提出的一种TSBP辅助基团及基团辅助的脑啡肽及其衍生物的液相全合成方法,主要解决目前脑啡肽及其衍生物化学合成方法的分离步骤多、耗时周期长、纯化规模小、生产成本高,而固相反应的生产规模小,原料价格贵、浪费大,树脂类废弃物多和环境污染严重的问题。主要合成步骤是:(1)载体偶联:采用我们发展的新型磷酸酯类载体,三[4-(α-取代苄基)苯基]磷酸酯(TSBP,结构式A),替代固相树脂,在偶联脱水剂的作用下与N-端PG保护的氨基酸(PG-Xaa-OH,Xaa为任意氨基酸)的C-端连接;(2)分离纯化:反应完成后,借助磷酸酯类载体TSBP在不同溶剂系统中易结晶沉淀的特性,可将生成物与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的中间体1(PG-Xaa-TSBP);(3)脱除N-端PG:将中间体1采用脱PG试剂处理后,再借助磷酸酯类载体在不同溶剂系统中易结晶沉淀的特性,可将生成物与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的中间体2(H2N-Xaa-TSBP);(4)多肽偶联:采用保护的氨基酸如PG-Phe-OH、PG-Gly-OH和[PG-Tyr(OtBu)-OH]等为原料,重复以上步骤(1)、(2)、(3),依次偶联上相应的氨基酸,构建脑啡肽或其衍生物前体的氨基酸序列H2N-Tyr(OtBu)-Gly-Gly-Phe-Xaa-TSBP;(5)侧链脱保护,并同时剪除载体:采用鸡尾酒式混合试剂(TFA:Tis:H2O=95:2.5:2.5)处理,脱除侧链上的保护基团如tBu、Pbf、Boc或Trt等,并同时剪除载TSBP。(6)脑啡肽及其衍生物的分离纯化:将(5)所得的溶液进行旋转蒸发,脱除TFA后,加入有机溶剂,沉淀析出,经过滤、洗涤、干燥等操作,即得到脑啡肽及其衍生物H2N-Tyr-Gly-Gly-Phe-Xaa-ZH(Z=O,S,NH等)。与目前已有的合成方法相比,本发明兼备了液相和固相合成法的优点,可以更加简便、快捷、节约、高效地合成制备谷胱甘肽,而且磷酸酯类载体可以回收并直接再利用,降低原材料浪费,减少废弃物污染,节约成本,利于环保。
附图说明
图0:本发明的脑啡肽及其衍生物合成路线图
图1:一种脑啡肽及其衍生物的液相全合成方法实例流程图
图2:Fmoc策略合成亮氨酸-脑啡肽及其衍生物
Leu-Enkephalin,RGD-Enkephalin,and RRRGD-Enkephalin i:Fmoc-Leu-OH,EDC·HCl/DMAP,DCM,r.t.,2h;ii:Fmoc-Phe-OH,EDC·HCl/HOBt/DIEA,DCM,r.t.,2h,[肽链延伸:Fmoc-Gly-OH,Fmoc-Gly-OH,Fmoc-Tyr(tBu)-OH,Fmoc-Asp(tBu)-OH,Fmoc-Gly-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH];iii:TFA:Tis:H2O=95:2.5:2.5,r.t.,3h;
图3:乙醚沉淀多肽RGD-Enkephalin(RGDYGGFL)
图4:亮氨酸-脑啡肽及其衍生物d的高效液相色谱分析
HPLC analysis of peptides,(A)Leu-Enkephalin,(B)RGD-Leu-Enkephalin and(C)RRRGD-Leu-Enkephalin.
HPLC conditions:column,Kromasil,NC-2546-06251151;250×4.6mm;at 25℃.
具体实施方式
现结合实施例、附图对本发明作进一步描述:
本发明合成路线见图0,包括以下步骤:
a.载体偶联:采用我们发展的新型磷酸酯类载体TSBP替代固相树脂,在脱水偶联剂的作用下与第一个保护任意氨基酸(PG-Xaa-OH)的C-端连接;
b.分离纯化:反应完成后,借助磷酸酯类载体TSBP在不同溶剂系统中易结晶沉淀的特性,可将生成物1与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的1;
c.脱除N-端PG:将生成物1用脱保护试剂处理后,再借助磷酸酯类载体TSBP在不同溶剂系统中易结晶沉淀的特性,可将生成物2与其他杂质分离,经简单地过滤和洗涤或重结晶操作就可以得到纯化的2;
d.多肽偶联:重复以上步骤a、b、c,依次偶联上侧链保护的半胱氨酸PG-Phe-OH、PG-Gly-OH、PG-Gly-OH和PG-Tyr(tBu)-OH,脱除PG后得到化合物:H2N-Tyr(tBu)-Gly-Gly-Phe-Xaa-TSBP;
e.检出载体及侧链脱保护:采用三氟乙酸:三异丙基硅烷:水(TFA:Tis:H2O=95:2.5:2.5)溶液处理化合物H2N-Tyr(tBu)-Gly-Gly-Phe-Xaa-TSBP,剪切除掉TSBP载体同时脱除侧链上的保护基团tBu,浓缩得到化合物H2N-Tyr-Gly-Gly-Phe-Xaa和TSBP残基的混合物;
f.沉淀多肽:将冷乙醚加入到所得到e浓缩后的H2N-Tyr-Gly-Gly-Phe-Xaa和TSBP残基混合物中,过程出现大量多肽沉淀,并辅助以超声波震荡处理;
脑啡肽及其衍生物的分离纯化:将f所得的乙醚相和沉淀离心分离,重复f过程3次,即得到脑啡肽及其衍生物沉淀H2N-Tyr-Gly-Gly-Phe-Xaa-ZH(Z=O,S,NH等)和乙醚相的TSBP残基,TSBP残基再生后可重复使用;
本发明中一些常用的缩写具有以下含义:
DCM:二氯甲烷CH2Cl2
DMAP:4-二甲氨基吡啶
DMF:N,N-二甲基甲酰胺
EDC·HCl:1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
Fmoc:芴甲氧羰基
TSBP:磷酸酯类多肽合成载体
HATU:2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐
HOBT:1-羟基苯并三唑
HBTU:O-苯并三氮唑-四甲基脲六氟磷酸盐
NMM:N-甲基吗啉
NMP:N-甲基吡咯烷酮
PyBop:六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷
Pbf:2,2,4,6,7-五甲基苯并呋喃-5-磺酰基
tBu:叔丁基
TFA:三氟乙酸
THF:四氢呋喃
具体实施方式:
本发明适用于脑啡肽及其衍生物的合成制备与水解脱除,反应原理与技术路线如图1;图2为Fmoc策略合成亮氨酸-脑啡肽及其衍生物,其中:
Leu-Enkephalin,RGD-Enkephalin,and RRRGD-Enkephalin i:Fmoc-Leu-OH,EDC·HCl/DMAP,DCM,r.t.,2h;ii:Fmoc-Phe-OH,EDC·HCl/HOBt/DIEA,DCM,r.t.,2h,[肽链延伸:Fmoc-Gly-OH,Fmoc-Gly-OH,Fmoc-Tyr(tBu)-OH,Fmoc-Asp(tBu)-OH,Fmoc-Gly-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH];iii:TFA:Tis:H2O=95:2.5:2.5,r.t.,3h;
实际操作步骤:
(Fmoc-Leu)3-TSBP合成:准确称取TSBP(644mg,1.0mmol,1equiv)于100mL的反应瓶中,加入30mL的DCM溶解,反应体系依次加入Fmoc-Leu-OH(1.27g,3.6mmol,3.6equiv),4-二甲氨基吡啶DMAP(50mg,0.36mmol,0.36equiv),EDC·HCl(690mg,3.6mmol,3.6equiv),室温下反应2h,TLC检测反应结束后冷却至0℃后过滤得到滤液,浓缩后加入乙酸乙酯30mL溶解,依次用饱和NH4Cl水溶液、饱和Na2CO3溶液洗涤,无水硫酸镁干燥,浓缩后用5mL的乙酸乙酯溶解样品,逐滴加入正己烷35mL(VEA/VN-Hexane=1:7),体系中出现大量白色沉淀,过滤白色沉淀并干燥得到化合物(Fmoc-Leu)3-TSBP,产率约98%,经过一次沉淀可作为原料进行下一步投料,连续洗涤2~3次样品可进行NMR表征。
(Fmoc-Leu)3-TSBP:(98%yield),white solid,Rf=0.48(CH2Cl2:MeOH=60:1).1HNMR(400MHz,CDCl3),δ7.74-7.72(d,J=8.0Hz,6H),7.57-7.55(d,J=8.0Hz,6H),7.38-7.22(m,33H),7.16-7.15(m,6H),6.85(s,3H),5.23-5.21(m,3H),4.51-4.18(m,12H),1.64-1.49(m,9H),0.92-0.88(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,CDCl3),δ-17.19ppm;13CNMR(100MHz,CDCl3),δ172.2,156.0,149.9,143.7,141.3,139.2,137.2,128.9,128.7,128.2,127.1,126.9,125.1,120.0 77.3,67.0,52.7,47.1,41.5,24.7,22.9,21.8ppm;Calcd M=1649.65for C102H96N3O16P,HRMS(ESI)m/z found[M+1]+=1650.65259.
(Fmoc-Phe-Leu)3-TSBP合成:准确称取(Fmoc-Leu)3-TSBP(0.80g,0.5mmol)于50mL的反应瓶中,加入6mL乙腈溶液,搅拌条件下滴加加入DEA二乙胺2mL(DEA/MeCN=1:3,25%DEA脱Fmoc保护基),搅拌30min后TLC检测原料消失,浓缩后加入2mL的DCM溶液溶解,加入正己烷8mL震荡出现大量白色固体,静置2min后除去上清液,得到白色脱保护产物,产率约99%,作为原料进行下一步投料。将(NH2-Leu)3-TSBP(440mg,0.45mmol)于100mL的反应瓶中,加入30mL的DCM溶解,反应体系依次加入Fmoc-Phe-OH(620mg,1.6mmol,3.6equiv),HOBt(215mg,1.6mmol,1.2equiv),EDC·HCl(305mg,1.6mol,3.6equiv),DIEA(265μL,1.6mol,3.6equiv)室温下反应2h,TLC检测反应结束后依次用饱和Na2CO3溶液洗涤2次,无水硫酸镁干燥,浓缩后用3.0mL的乙酸乙酯溶解样品,逐滴加入正己烷18mL(VEA/VN-Hexane=1:6),体系中出现大量白色沉淀,过滤白色沉淀并干燥得到化合物(Fmoc-Phe-Leu)3-TSBP,产率约97%,经过一次沉淀可作为原料进行下一步投料,连续洗涤2~3次样品可进行NMR表征。
TSBP-肽链的延长:肽链延长及脱Fmoc保护基团和上述方法一样,DCM作为反应溶剂使用氨基酸偶联试剂EDC·HCl/HOBt/DIEA作为缩合试剂,多肽的脱Fmoc保护采用25%DEA/MeCN(二乙胺DEA:乙腈MeCN=1:3)体系,每一步反应结束后通过沉淀方法得到多肽粗产品直接进行下一步投料反应,经过2~3次沉淀样品直接进行每一步产物的NMR测试,分别得到以下一系列化合物:
[Fmoc-Phe-Leu]3-TSBP,
[Fmoc-Gly-Phe-Leu]3-TSBP,
[Fmoc-Gly-Gly-Phe-Leu]3-TSBP,
[Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP,
[Fmoc-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP,
[Fmoc-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP,
[Fmoc-Arg(Pbf)-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP,
[Fmoc-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP;
【备注:将与TSBP相连的肽链延伸至五肽后,使用乙腈代替原始的石油醚/乙酸乙酯系统来沉淀肽产物】
(Fmoc-Phe-Leu)3-TSBP:(97%yield),white solid,Rf=0.50(CH2Cl2:MeOH=50:1).1H NMR(400MHz,CDCl3),δ7.73-7.71(d,J=8.0Hz,6H),7.50-7.46(m,6H),7.35-7.20(m,34H),7.16-7.06(m,20H),6.81-6.69(m,6H),5.77-5.57(m,3H),4.69-4.09(m,15H),2.99-2.95(m,6H),1.56-1.26(m,9H),0.80-0.75(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,CDCl3),δ-16.81ppm;13C NMR(100MHz,CDCl3),δ171.6,171.0,156.1,150.0,143.7,141.3,139.2,137.3,136.3,129.4,129.0,128.7,128.3,127.8,127.1,125.1,120.3,120.0,77.4,67.2,56.0,51.0,47.1,41.3,38.5,24.8,22.7,22.0ppm;Calcd M=2090.86 forC129H125N6O19P,HRMS(ESI)m/z found[M+1]+=2091.85922.
(Fmoc-Gly-Phe-Leu)3-TSBP:(96%yield),white solid,Rf=0.42(CH2Cl2:MeOH=40:1).1H NMR(400MHz,CDCl3),δ7.70-7.68(d,J=8.0Hz,6H),7.54-7.50(m,9H),7.22-7.20(m,36H),7.02(m,21H),6.78(s,3H),6.15-6.08(m,3H),4.86-4.68(m,6H),4.25-4.08(m,9H),3.79(s,6H),2.95(m,6H),1.57-1.45(s,9H),0.78-0.75(dd,J=8.0Hz,18H)ppm;31PNMR(162MHz,CDCl3),δ-16.98ppm;13C NMR(100MHz,CDCl3),δ171.4,170.9,169.3,156.7,149.7,143.7,141.1,139.1,137.3,136.2,129.2,128.9,128.6,128.4,127.7,127.0,126.7,126.5,125.1,120.1,119.9,75.2,67.2,54.3,51.0,46.9,44.2,40.9,38.3,24.6,22.6,21.9ppm;Calcd M=2261.92 for C135H133N9O22P,HRMS(ESI)m/z found[M+1]+=2262.89868.
(Fmoc-Gly-Gly-Phe-Leu)3-TSBP:(94%yield),white solid,Rf=0.35(CH2Cl2:MeOH=30:1).1H NMR(400MHz,DMSO-d6),δ8.61-8.58(m,3H),8.33(m,3H),8.23-8.08(m,6H),7.90-7.88(d,J=8.0Hz,6H),7.72-7.59(m,12H),7.45-7.14(m,48H),6.83(s,3H),4.61-4.41(m,6H),4.30-4.23(m,9H),3.78-3.46(m,12H),2.98-2.95(m,3H),2.73-2.68(m,3H),1.59(m,9H),0.87-0.81(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,DMSO-d6),δ-17.04ppm;13C NMR(100MHz,DMSO-d6),δ172.0,171.6,169.8,168.9,157.0,149.5,144.3,141.2,140.4,138.6,138.2,129.5,129.0,128.8,128.5,128.1,127.5,127.0,126.8,125.7,120.6,120.5,76.6,66.2,54.1,51.1,47.1,44.0,42.2,40.6,39.3,24.6,23.1,21.8ppm;Calcd M=2432.99 for C141H141N12O25P,HRMS(ESI)m/z found[M+Na]+=2455.94287.
[Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP:(95%yield),white solid,Rf=0.32(CH2Cl2:MeOH=30:1).1H NMR(400MHz,DMSO-d6),δ8.60-8.06(m,12H),7.87-7.67(m,12H),7.47-7.15(m,60H),6.86-6.80(m,12H),4.63-4.12(m,18H),3.79-3.68(m,12H),3.20-2.97(m,6H),2.78-2.73(m,6H),1.61(m,9H),1.17(m,27H),0.89-0.83(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,DMSO-d6),δ-17.27ppm;13C NMR(100MHz,DMSO-d6),δ172.4,172.0,171.6,169.4,168.9,156.4,153.8,144.2,141.1,140.4 138.6,138.2,133.3,130.2,129.6,129.0,128.5,128.1,127.5,127.0,126.8,125.8,123.8,120.5,79.7,78.0,76.6,66.2,56.7,54.1,51.1,49.1,47.0,42.6,37.4,28.9,24.7,23.1,21.9ppm;Calcd M=3091.37for C180H192N15O31P,HRMS(ESI)m/z found[M+1]+=3092.31071.
[Fmoc-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP:(95%yield),whitesolid,Rf=0.50(CH2Cl2:MeOH=20:1).1H NMR(400MHz,DMSO-d6),δ8.60-7.90(m,15H),7.73-7.68(m,9H),7.47-7.13(m,63H),6.86-6.79(m,12H),4.63-4.25(m,21H),3.77-3.63(m,12H),3.06-2.97(m,6H),2.82-2.51(m,9H),2.44-2.38(m,3H),1.61(m,9H),1.37(s,27H),1.21(s,27H),0.90-0.84(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,DMSO-d6),δ-17.35ppm;13C NMR(100MHz,DMSO-d6),δ170.3,170.1,170.0,169.4,168.2,167.7,167.3,154.6,152.2,148.0,142.7,139.6,138.8,137.0,136.6,131.1,128.6,127.9,127.4,127.2,126.9,126.5,125.9,125.4,125.2,124.1,122.2,119.0,79.0,76.3,75.0,64.7,53.8,52.7,52.5,50.4,49.5,45.4,40.9,40.5,36.5,36.3,35.7,27.3,26.5,23.0,21.5,20.2ppm;Calcd M=3603.63 for C204H231N18O40P,HRMS(ESI)m/z found[M+1]+=3604.54492.
[Fmoc-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP:(93%yield),whitesolid,Rf=0.45(CH2Cl2:MeOH=20:1).1H NMR(400MHz,DMSO-d6),δ8.59-7.62(m,21H),7.40-7.13(m,66H),6.85-6.80(m,15H),4.61-4.25(m,21H),3.74-3.67(m,18H),3.02-2.96(m,6H),2.84-2.59(m,9H),2.41(m,3H),1.60(m,9H),1.36-1.20(m,54H),0.89-0.83(dd,J=8.0Hz,18H)ppm;31P NMR(162MHz,DMSO-d6),δ-17.42ppm;13C NMR(100MHz,DMSO-d6),δ172.0,171.6,170.7,169.8,169.6,169.3,168.9,157.0,153.9,149.6,144.3,141.2,140.4,138.6,138.2,132.8,130.2,129.6,129.0,128.8,128.5,128.1,127.6,127.0,126.8,125.7,123.8,120.6,80.7,78.0,76.6,66.3,54.6,54.1,51.1,50.0,47.1,43.9,42.5,38.1,29.0,28.1,24.7,23.1,21.8ppm;Calcd M=3774.70 for C210H241N21O43P.HRMS(ESI)m/z found[M+1]+=3775.70493
[Fmoc-Arg(Pbf)-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP:(95%yield),white solid,Rf=0.45(CH2Cl2:MeOH=15:1).1H NMR(400MHz,CDCl3),δ8.57-7.64(m,17H),7.45-6.43(m,90H),4.59-4.02(m,16H),3.73-3.40(m,18H),3.04-2.44(m,30H),1.99(m,6H),1.36-0.82(m,147H)ppm;31P NMR(162MHz,DMSO-d6),δ-17.15ppm;13C NMR(100MHz,DMSO-d6),δ172.9,172.2,171.8,170.9,169.5,169.1,158.2,156.8,154.1,149.8,144.5,141.4,140.7,138.8,138.4,138.0,133.1,132.2,130.4,129.8,129.2,128.7,128.3,127.8,127.3,127.0,126.0,125.0,124.1,120.7,117.0,86.9,80.9,79.9,78.2,76.8,54.9,54.3,51.4,50.2,47.4,43.2,42.4,38.4,29.2,28.3,24.9,23.4,22.1,19.7,18.4,13.0ppm;Calcd M=4997.23 for C267H322N33O55PS3,HRMS(ESI)m/z found[M+H]+=4998.25431
Leu-Enkephalin:侧链保护基团tBu、Pbf的脱除及多肽链与保护基团的脱离纯化,配置三氟乙酸:三异丙基硅烷:水(TFA:Tis:H2O=95:2.5:2.5)溶液,准确称取:[NH2-Arg(Pbf)-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP
[NH2-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP
[NH2-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Gly-Asp(tBu)-Tyr(tBu)-Gly-Gly-Phe-Leu]3-TSBP样品100mg于25mL的反应瓶中,加入TFA:Tis:H2O=95:2.5:2.5体系的溶液2.5mL,室温条件下搅拌3h,TLC检测反应原料点消失,加入10mL的DCM溶剂后浓缩,再次加入DCM浓缩,连续重复进行3次至浓缩后为白色粘稠固体,加入10mL的冷乙醚沉淀,超声5min后离心,连续重复3次,乙醚相检测到TSBP-residue保护组,浓缩后乙酸乙酯溶解,加入正己烷进行沉淀,过滤沉淀得到回收产物化合物TSBP-residue,回收产率约为60%;离心后得到高纯度多肽:
Leu-Enkephalin,RGD-Enkephalin,RRRGD-Enkephalin.
Leu-Enkephalin YGGFL(H-Tyr-Gly-Gly-Phe-Leu-OH)(99%yield),whitesolid,1H NMR(400MHz,D2O),δ7.25-7.01(m,7H),6.77-6.72(m,2H),4.52-4.50(d,J=8.0Hz,1H),4.23-4.10(m,2H),3.76-3.74(m,4H),3.03-2.86(m,4H),1.48-1.44(m,3H),0.77-0.69(dd,J=8.0Hz,6H)ppm;13C NMR(100MHz,D2O),δ176.0,172.7,171.0,170.5,169.8,155.1,136.0,130.7,129.1,128.6,127.1,125.3,115.8,54.7,54.4,51.4,42.3,42.0,39.4,37.0,24.2,22.1,20.5ppm;Calcd M=555.27 for C28H37N5O7,HRMS(ESI)m/zfound[M+H]+=556.27679.
RGD-Enkephalin,RGDYGGFL(H-Arg-Gly-Asp-Tyr-Gly-Gly-Phe-Leu-OH)(99%yield),white solid,1H NMR(400MHz,D2O),δ7.24-6.98(m,7H),6.71-6.69(d,J=8.0Hz,2H),4.59-4.55(m,2H),4.51-4.45(m,3H),4.23-4.21(m,2H),3.98-3.86(m,2H),3.80-3.61(m,6H),3.09-2.59(m,9H),1.81(m,2H),1.57-1.44(m,6H),0.77-0.70(dd,J=8.0Hz,6H)ppm;13C NMR(100MHz,D2O),δ175.8,173.9,173.5,172.7,172.0,171.6,170.7,170.4,170.0,156.6,154.4,136.1,130.5,129.1,128.6,127.9,127.1,115.3,55.2,54.8,52.7,51.3,49.8,42.5,42.1,40.3,39.3,36.9,35.9,35.1,27.8,24.2,23.3,22.1,20.5ppm;Calcd M=883.42 for C40H57N11O12,HRMS(ESI)m/z found[M+H]+=884.42572.
RRRGD-Enkephalin,RRRGDYGGFL(H-Arg-Arg-Arg-Gly-Asp-Tyr-Gly-Gly-Phe-Leu-OH)(98%yield),white solid,1H NMR(400MHz,DMSO-d6),δ7.22-6.97(m,7H),6.70-6.68(d,J=8.0Hz,2H),4.57-4.43(m,9H),4.23-4.19(m,5H),3.97(m,2H),3.77-3.42(m,12H),3.05-2.58(m,17H),1.80-1.48(m,15H),1.15-1.02(m,6H),0.77-0.71(dd,J=8.0Hz,6H)ppm;13C NMR(100MHz,D2O),δ175.7,173.9,173.5,173.2,172.7,172.0,171.6,170.7,169.5,156.6,154.4,136.1,130.5,129.1,128.6,127.9,127.1,115.3,55.4,55.2,54.8,53.5,52.4,51.2,49.7,42.5,42.2,40.4,40.3,39.3,36.9,35.0,28.0,24.3,24.2,23.3,22.1,20.5ppm;Calcd M=1195.62 for C52H81N19O14,HRMS(ESI)m/z found[M+H]+=1196.62402.
Claims (13)
2.一种采用权利要求所述TSBP辅助基团进行基团辅助的脑啡肽及其衍生物液相的合成方法,其特征在于步骤如下:
步骤1、辅助基团与氨基酸的偶联:辅助基团在脱水偶联剂的作用下与氨基酸在0~50℃下搅拌反应1~3小时;氨酸Fmoc-Xaa-OH的C-端与TSBP辅助基团连接,生成中间体化合物1,(Fmoc-Xaa)3-TSBP;
所述氨基酸与TSBP辅助基团的摩尔比为1~3:1;所述脱水偶联剂为1︰1摩尔比的脱水偶联活化剂和碱性物质;
所述辅助基团为磷酰氧基苯甲醇TSBP;
所述氨基酸采用芴甲氧羰基Fmoc保护的某氨酸Fmoc-Xaa-OH;
步骤2、分离纯化:向生成物A加入极性小的烷烃或醚类溶剂,借助TSBP辅助基团在溶剂系统中易结晶沉淀的特性,将生成物1与其他杂质分离;
对分离后的生成物1进行过滤和洗涤或重结晶操作得到纯化的生成物1;
步骤3、脱除N-端Fmoc:将纯化的生成物1采用脱Fmoc试剂处理,在10~50℃下搅拌反应0.5~2小时得到生成物2,(H2N-Xaa)3-TSBP;
向生成物2加入极性小的烷烃或醚类溶剂,借助TSBP辅助基团在溶剂系统中易结晶沉淀的特性,将生成物2与其他杂质分离;
对分离后的生成物2进行过滤和洗涤或重结晶操作得到纯化的生成物2;
步骤4:以纯化的含有辅助基团TSBP的生成物2作为原料,替代步骤1的辅助基团,以芴甲氧羰基Fmoc保护的苯丙氨酸Fmoc-Phe-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Phe-Xaa)3-TSBP;
以第一次循环重复后纯化的生成物(H2N-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的甘氨酸Fmoc-Gly-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Gly-Phe-Xaa)3-TSBP;
以第二次循环重复后纯化的生成物(H2N-Gly-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的甘氨酸Fmoc-Gly-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到化合物(H2N-Gly-Gly-Phe-Xaa)3-TSBP;
以第三次循环重复后纯化的生成物(H2N-Gly-Gly-Phe-Xaa)3-TSBP作为原料,替代步骤1的辅助基团,以芴甲氧羰基(Fmoc)保护的酪氨酸Fmoc-Tyr(OtBu)-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到脑啡肽或其衍生物的前体化合物[H2N-Tyr(OtBu)-Gly-Gly-Phe-Xaa]3-TSBP;
步骤5、侧链脱保护与TSBP辅助基团的剪除:以三氟乙酸的鸡尾酒溶液为侧链脱保护剂,分离纯化脑啡肽或其衍生物的前体化合物[H2N-Tyr(OtBu)-Gly-Gly-Phe-Xaa]3-TSBP的混合液;反应条件为5~30℃下,搅拌1~3小时,脱除侧链上的保护基团tBu、Boc和Pbf,同时剪除TSBP辅助基团,经分离纯化得到脑啡肽或其衍生物的三氟乙酸盐,[TFA*H-Try-Gly-Gly-Phe-Xaa-ZH];所述三氟乙酸的鸡尾酒溶液中的组分比例为:TFA/TIPS/H2O=95:2.5:2.5;
步骤6、脑啡肽及其衍生物的分离纯化:对步骤5得到混合液通过旋蒸去除三氟乙酸盐,再用乙酸乙酯萃取,沉淀析出,经过滤、乙酸乙酯洗涤、干燥得到纯化的脑啡肽或其衍生物,[H-Tyr-Gly-Gly-Phe-Xaa-ZH]。
13.一种权利要求2所述基团辅助的脑啡肽及其衍生物液相的合成中TSBP辅助基团的回收再利用的方法,其特征在于:将步骤6中所得的乙酸乙酯萃取溶液旋蒸浓缩至原体积的1/3~1/4,加入极性小的烷烃或醚类溶剂,借助TSBP在不同溶剂系统中易结晶沉淀的特性,将TSBP与其他杂质分离;对分离后的TSBP进行过滤和洗涤或重结晶操作得到纯化的TSBP,回收再生转化后直接作为辅助基团再利用。
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