CN111285921B - Bdk辅助基团及基于bdk辅助基团的普卡那肽及类似物的液相全合成方法 - Google Patents
Bdk辅助基团及基于bdk辅助基团的普卡那肽及类似物的液相全合成方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及一种BDK辅助基团及基于BDK辅助基团的普卡那肽及其类似物的液相全合成方法,合成步骤:BDK辅助基团替代固相树脂,在偶联脱水剂的作用下与N‑端Fmoc保护的氨基酸的C‑端连接;然后分离纯化、脱除N‑端Fmoc、多肽偶联;重复以上依次偶联上相应的氨基酸,构建普卡那肽及其类似物前体的氨基酸序列;再侧链脱保护、分离纯化得到普卡那肽及其类似物。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再生并循环利用,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BDK辅助基团及基于BDK辅助基团的普卡那肽及其类似物的液相全合成方法。尤其涉及合成普卡那肽及其类似物的一种基于小分子载体,4,4’-双(二苯基次磷酰氧基)二苯甲酮衍生物(4,4’-Bisdiphenylphosphinyloxyl Diphenyl Ketone,BDK结构式如图2中的A)的普卡那肽及其类似物(结构见图2)的液相全合成方法的普卡那肽及其类似物全合成方法(合成路线如图2所示)。本发明以磷酸酯类小分子BDK衍生物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、高效地合成制备普卡那肽及其类似物,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
普卡那肽(Plecanatide,结构见图2),其他译名为皮卡那肽,研究代号SP-304和GCRA等。最初由美国Callisto制药公司研制,然后转让给Synergy Pharmaceuticals开发的口服鸟苷酸环化酶-C(GC-C)激动剂,于2017年1月19日首先获得美国食品药品管理局(FDA)批准上市,商品名为Trulance。普卡那肽是促尿钠排泄的鸟苷酸环化酶C(Guanylatecyclase-C,GC-C)受体激动药,能调节胃肠道中的酸碱离子,诱导液体转运进入胃肠道,增加胃肠道的蠕动,用于治疗成人胃肠道疾病,如慢性特发性便秘(Chronicidiopathicconstipation,CIC)和便秘型肠易激综合征(IBS-C)。它是一种合成的人类尿鸟苷蛋白(Uroguanylin,UG)的类似物,含有双二硫键连接的16个氨基酸的环状多肽,英文化学名为:L-asparaginyl-L-α-aspartyl-L-α-glutamyl-L-cysteinyl-L-α-glutamyl-L-leucyl-L-cysteinyl-L-valyl-L-asparaginyl-L-valyl-L-alanyl-L-cysteinyl-L-threonyl-L-glycyl-L-cysteinyl-L-leucine cyclic(4→12),(7→15)-bis(disulfide),中文化学名为(第4与12位L-半胱氨酸),(第7与15位L-半胱氨酸)环状双二硫键结合的L-天冬酰胺酰基-L-α-天冬氨酰基-L-α-谷氨酰基-L-半胱氨酰基-L-α-谷氨酰基-L-亮氨酰基-L-半胱氨酰基-L-缬氨酰基-L-天冬酰胺酰基-L-缬氨酰基-L-丙氨酰基-L-半胱氨酰基-L-苏氨酰甘氨酰-L-甘氨酰基-L-半胱氨酰基-L-亮氨酸。普卡那肽的氨基酸序列与尿鸟苷的相似,只是在尿鸟苷N-端的3位用谷氨酸(Glu)取代天冬氨酸(Asp)。普卡那肽的结构与多卡那肽(Dolcanatide)相似,只是两端的氨基酸被它们各自的D-立体异构体所取代。普卡那肽及其类似物具有四个半胱氨酸(Cys),能够形成两个分子内二硫键,见文献1:Shailubhai K.,Comiskey S.,Foss J.A.,Feng R.,Barrow L.,Comer G.M,JacoG.S.b.Plecanatide,an Oral Guanylate Cyclase C Agonist Acting Locally in theGastrointestinal Tract,Is Safe and Well-Tolerated in SingleDoses.Dig.Dis.Sci.,2013,58:2580–2586.DOI10.1007/s10620-013-2684-z.和文献2:Shailubhai K.,Palejwala V.,Arjunan K.P.,Saykhedka S.r,Nefsky B.,J.AFoss,Comiskey S.,Jacob G.S.,S.E.Plevy.Plecanatide and dolcanatide,novel guanylatecyclase-C agonists,ameliorate gastrointestinal inflammation in experimentalmodels of murine colitis.World J.Gastrointest Pharmacol Ther.,2015,6(4):213-222.。Plecanatide的分子量为1.682kDa,与天然尿鸟苷蛋白(Uroguanylin)有1个氨基酸区别,使得其稳定性更好、与GC-C受体的结合活性增强了8倍,Dolcanatide为Synergy公司开发的第二个Uroguanylin类似物,目前处于溃疡性结肠炎(UC)的Phase 1临床研究阶段,结构如图2。
普卡那肽(Plecanatide)的氨基酸序列为:
普卡那肽类似物尿鸟苷蛋白(Uroguanylin)的氨基酸序列为:
普卡那肽类似物多卡那肽(Dolcanatide)的氨基酸序列为:
获批上市的另一种有效治疗CIC的药物利那洛肽(Linaclotide)的氨基酸序列为:
慢性特发性便秘(CIC)属于下消化道动力学障碍性疾病,其发病与结肠、肛门直肠动力学及精神心理异常有关,也是困扰中、老年人群的常见多发病,不同程度影响现代人的工作和生活质量。流行病学调查结果显示,世界各国和地区因饮食结构和生活习惯各异,CIC的发病率有较大差异。据不完全统计,欧美等发达国家为2.0%~28.0%;中国南北方地区为9.0%~20.3%。CIC是排除器质性疾病后的慢性便秘。估计全球有>1亿的人群遭受CIC和IBS-C的痛苦,其中美国人群的患病率约为15%即4500万(其中的CIC约为3300万)。CIC的常见症状为便秘(每周排便少于3次,持续3个月以上),硬或块状的粪便,排便不完全,排便费力。IBS-C的常见症状为腹痛,便秘,排便不完全。慢性便秘的治疗药物有溶剂性泻药、渗透性泻药、刺激性泻药、促动力药、促分泌药、灌肠药和栓剂(国内常用药物可参见本文后述引用的“便秘治疗药物循证医学证据表”),见文献3:Chang W-C L,Masih S.,Thadi A.,Patwa V.,Joshi A.,Cooper H S.,Palejwala V A.,Clapper M L.,Shailubhai K.,Plecanatide-mediated activation of guanylate cyclase-C suppressesinflammation-induced colorectal carcinogenesis in Apc+/Min-FCCC mice.WorldJ.Gastrointest Pharmacol Ther.,2017,8(1):47-59.和文献4:Miner Jr P.B.,Koltun WD,Wiener G.J.,Portilla M.De.La.,Prieto B.,Shailubhai K.,Layton M.B.,etal.ARandomized Phase III Clinical Trial of Plecanatide,a Uroguanylin Analog,in Patients With Chronic Idiopathic Constipation.Am.J.Gastroenterol.,2017;112:613–621;doi:10.1038/ajg.2016.611.。其中,除了TrulanceTM外,国外批准上市的促分泌类处方药还有2个:①(linaclotide,利那洛肽):Forest Laboratories公司(现属Allergan,Plc公司)注册产品,同为GC-C激动剂,为14个氨基酸肽(CCEYCCNPACTGCY,Cysbridge:1-6、2-10、5-13),2012年8月30日获FDA批准,适应症为成人CIC和IBS-C的治疗,见文献5:Busby R.W.,Ortiz S..Clarification of Linaclotide Pharmacology Presentedin a Recent Clinical Study of Plecanatide.Dig.Dis.Sci.,2014,59:1066–1067.DOI10.1007/s10620-013-2953-x.和文献6:Roque M V,Camilleri M.Linaclotide,asynthetic guanylate cyclase C agonist,for the treatment of functional
gastrointestinal disorders associated with constipation.ExpertRev.Gastroenterol.Hepatol.,2011,5(3):301-310.;②(lubiprostone,鲁比前列酮):Sucampo Pharma,LLC公司注册产品,为氯离子C2通道活化剂,分子式为C20H32F2O5,2006年1月31日获FDA批准,适应症为成人CIC和IBS-C等。2016年/>的销售额为9.59亿美元,/>为4.67亿美元。Synergy公司预计其TrulanceTM产品的市场机会为20亿美元,其市场需求不可小觑。
目前普卡那肽及其类似物是通过生物提取技术得到,生物提取受到现有有限生物资源的影响,提取过程较繁琐,效率低,且比较难得到高纯度的产物,并且在自然环化时无法精准定位。此外,普卡那肽及其类似物也可以采用固相多肽合成法进行制备,相比于生物提取的方法,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,见文献6:Roque M V,Camilleri M.Linaclotide,a synthetic guanylate cyclase C agonist,for the treatment of functional
gastrointestinal disorders associated with constipation.ExpertRev.Gastroenterol.Hepatol.,2011,5(3):301-310.;文献7:陈丰,刘成宝,钱君超,陈志刚.一种普卡那肽的简易合成方法.中国发明专利,CN 107383170A,2017.11.24.;文献8:陈丰,钱君超,刘成宝,陈志刚.一种通过二次环化固相合成普卡那肽的方法.中国发明专利,CN 107383171A,2017.11.24.和文献9:沈永刚,谢振亮,程益明,沈永良,付坤.一种普卡那肽的固相合成方法.中国发明专利,CN 108440652A,2018.08.24.。自1963年Merrifield教授等发展了多肽固相合成(Solid phase peptide synthesis,SPPS)这一独创性的方法,见文献10:Merrifield R.B.Solid phase peptide synthesis.I.the synthesis oftetrapeptide,J.Am.Chem.Soc.,1963,85(14):2149-2154.和文献11:Lerner R.A.,Retrospective.R.Bruce Merrifield(1921-2006),Science.,2006,313(5783):57.。由此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。
另一方面,普卡那肽及其类似物的现有生产技术都面临的挑战是在合成和纯化过程中必需使用大量溶剂,严重威胁生产安全和环境保护。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12:Bonnamour J,Métro.T-X,Martinez.J,Lamaty.F.Green Chem.,2013,15:1116-1120.。在目前已报道的普卡那肽及其类似物的固相合成方法中,见文献7~文献9,偶联反应和脱保护过程都是以非均相的形式进行的,为了让反应进行得彻底完全,通常需要使用3-5倍过量的保护氨基酸原料、偶联试剂、脱保护试剂和巨量溶剂,而且用作载体的固相树脂和用于氨基酸羧基官能团保护的基团,文献13:Kocienski,P.J.Protecting Groups;Georg Thieme Verlag:Stuttgart New York,2004.和文献14:Green,T.W.;Wuts,P.G.M.Protective Groups inOrganic Synthesis;John Wiley and Sons:New York,1999.以及文献15:Isidro-Llobet,A.;Alvarez,M.;Albericio,F.Chem.Rev.2009,109,2455–2504.,固相树脂和基团在脱除过程中都已遭到破坏或分解掉,完全不能回收再利用,这就不仅造成原料和试剂的极大浪费,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。
综上所述,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或载体仍然是值得期待的重要研究课题。我们经过系统地筛选研究发现,磷酸酯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BDK辅助基团及基于BDK辅助基团的普卡那肽及类似物的液相全合成方法,主要解决目前普卡那肽及其类似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4,4’-双(二苯基次磷酰基)二苯基酮衍生物的BDK辅助基团,其特征在于:BDK分子结构通式为
一种采用所述BDK辅助基团进行基团辅助的普卡那肽及其类似物物液相的合成方法,其特征在于步骤如下:
步骤1、辅助基团与氨基酸的偶联:以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-Xaa1-OH的C-端与BDK辅助基团连接,生成中间体化合物1,Fmoc-Xaa1-BDK;
所述氨基酸与BDK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BDK;
所述氨基酸采用芴甲氧羰基Fmoc保护的某氨酸Fmoc-Xaa1-OH的C-端与BDK辅助基团连接,生成中间体化合物1,Fmoc-Xaa1-BDK;
步骤2、分离纯化:向生成物1加入极性小的烷烃或醚类溶剂,借助BDK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;
向生成物2加入极性小的烷烃或醚类溶剂,借助BDK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,H2N-Xaa1-BDK.
步骤4:以纯化的含有辅助基团BDK的生成物2和H2N-Xaa1-BDK作为原料,以再与保护的半胱氨酸Fmoc-Cys(Trt)-OH进行偶联反应;重复步骤1~步骤3,得到化合物H2N-Cys(Trt)-Xaa1-BDK;
以第一次循环重复后纯化的生成物H2N-Cys(Trt)-Xaa1-BDK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-Cys(Trt)-Xaa1-BDK;
以第二次循环重复后纯化的生成物H2N-Gly-Cys(Trt)-Xaa1-BDK作为原料,替代步骤1的辅助基团,以保护的苏氨酸Fmoc-Thr(tBu)-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-Thr(tBu)-Gly-Phe-Xaa1-BDK;
以上次循环重复后纯化的生成物H2N-Gly-Cys(Trt)-Xaa1-BDK作为原料,替代步骤1的辅助基团,依次以Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Leu-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Xaa2-OH[Fmoc-Glu(tBu)-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到普卡那肽及其类似物的前体化合物H2N-X3-Asp(tBu)-X2-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Cys(Trt)-Xaa1-BDK;
或依次以Fmoc-Asp(tBu)-OH]、Fmoc-Asp(tBu)-OH和Fmoc-Xaa3-OH[Fmoc-Asn-OH替代步骤1的氨基酸进行偶联反应;
或依次以Fmoc-DAsn-OH]替代步骤1的氨基酸进行偶联反应;
步骤5、侧链脱Trt保护基团:采用25%TFA/DCM处理化合物4,脱除侧链上的保护基团Trt,经辅助沉淀分离,纯化得到化合物5,其氨基酸序列如下:H2N-Xaa3-Asp(tBu)-Xaa2-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Cys-Xaa1-BDK;
步骤6、第一次氧化环合:将氧化试剂加入上述化合物5的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物6,其氨基酸序列如下:
步骤7、第二次氧化环合:将适量氧化试剂碘加入上述化合物6的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物7,其氨基酸序列如下:
步骤8、脱除全部侧链保护并剪除载体:向上述所得产物7中加入足量的脱保护试剂(TFA:Tis:H2O=95:2.5:2.5),室温下搅拌3-5小时。反应完毕后,旋蒸回收三氟乙酸TFA,残留溶液用适量的乙醇溶解,加入乙酸乙酯,普卡那肽及其类似物因溶解度小而以沉淀形式析出,过滤并用适量乙酸乙酯洗涤沉淀,收集沉淀并干燥后即得普卡那肽及其类似物8,其氨基酸序列如下(Z=O,S,NH等):
所述氧化试剂:3.6g的过氧化尿素加入100mL的DMSO溶解溶液。
一种所述方法制备过程中得到的Fmoc保护某氨酰-BDK化合物,其特征在于:所述Fmoc-Xaa1-BDK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-BDK化合物,其特征在于:所述H2N-Xaa1-BDK的分子结构通式为
一种所述方法制备过程中得到的Fmoc保护半胱氨酰-某氨酰-BDK化合物,其特征在于:所述Fmoc-Cys(Trt)-Xaa)3-BDK分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护半胱氨酰-某氨酰-BDK化合物的结构通式,其特征在于:所述H2N-Cys(Trt)-Xaa)3-BDK分子结构通式为
一种所述方法制备过程中得到的Fmoc保护苏氨酰-半胱氨酰-某氨酰-BDK化合物,其特征在于:所述Fmoc-Thr(tBu)-Cys(Trt)-Xaa1-BDK分子结构通式为
一种所述方法制备过程中得到的脱Fmoc保护苏氨酰-半胱氨酰-某氨酰-BDK化合物,其特征在于:所述H2N-Thr(tBu)-Cys(Trt)-Xaa1-BDK分子结构通式为
一种所述方法制备过程中得到Fmoc保护的十六肽-BDK中间体化合物,其特征在于:所述Fmoc-Xaa3-Asp(tBu)-Xaa2-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Cys(Trt)-Xaa1-BDK分子结构通式为
一种所述方法制备过程中得到脱Fmoc保护的十六肽-BDK中间体化合物,其特征在于:所述Fmoc-Xaa3-Asp(tBu)-Xaa2-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Cys(Trt)-Xaa1-BDK分子结构通式为
一种所述方法制备过程中得到脱Trt保护的十六肽-BDK化合物,其特征在于:所述脱Trt保护的十六肽-BDK中间体分子结构通式为
其第一次氧化环合产物中间体的分子结构通式为
其第二次氧化环合产物的分子结构通式为
一种所述方法制备过程中最后脱侧链保护并剪除载体BDK,经分离纯化得到的普卡那肽及其类似物,其特征在于:所述普卡那肽及其类似物分子结构通式为
其中,Xaa1=Leu或DLeu,Xaa2=Glu或Asp,Xaa1=Asn或DAsn,Z=O,S,NH。
一种所述基团辅助的脑啡肽及其衍生物液相的合成中BDK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BDK在不同溶剂系统中易结晶沉淀的特性,将BDK载体残留与其他杂质分离;对分离后的BDK载体残留进行过滤和洗涤或重结晶操作得到纯化的BDK载体残留,回收再生转化后直接作为辅助基团再利用;所述BDK载体残留的分子结构通式为
有益效果
本发明提出的一种BDK辅助基团及基于BDK辅助基团的普卡那肽及其类似物的液相全合成方法,主要解决目前普卡那肽及其类似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,4,4’-双(二苯基次磷酰基)二苯基酮衍生物(4,4’-Bis-diphenylphosphatylDiphenyl Ketone,BDK结构式A),替代固相树脂,在偶联脱水剂的作用下与N-端Fmoc保护的氨基酸(Fmoc-X1-OH,其中X1为Leu或其对映异构体DLeu)的C-端连接;(2)分离纯化:反应完成后,借助磷酸酯类载体BDK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(Fmoc-X1-BDK);(3)脱除N-端Fmoc:将中间体1采用脱Fmoc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-X1-BDK);(4)多肽偶联:采用保护的氨基酸如Fmoc-Cys(Trt)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Leu-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(tBu)-OH、Fmoc-X2-OH[Fmoc-Glu(tBu)-OH或Fmoc-Asp(tBu)-OH]、Fmoc-Asp(tBu)-OH和Fmoc-X3-OH[Fmoc-Asn-OH或Fmoc-DAsn-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,构建普卡那肽及其类似物前体的氨基酸序列;(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BDK。(6)普卡那肽及其类似物的分离纯化: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,即得到普卡那肽及其类似物。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再生并循环利用,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1:合成路线示意图
图2:普卡那肽及其类似物的氨基酸序列与结构
图3:本发明的合成路线
图4:BDK载体的合成方案(Qin et al.China Patent CN109503655A).
图5:普卡那肽Plecanatide(TrulanceTM)的合成路线
i:Fmoc-Leu-OH,EDC·HCl/DMAP,DCM,r.t.,2h;ii:DEA/MeCN(3:1),r.t.,0.5h;iii:Fmoc-AA-OH[Fmoc-Cys(Trt)-OH,Fmoc-Gly-OH,Fmoc-Thr(tBu)-OH,Fmoc-Cys(Acm)-OH,Fmoc-Ala-OH,Fmoc-Val-OH,Fmoc-Asn(Trt)-OH,Fmoc-Val-OH,Fmoc-Cys(Trt)-OH,Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH,Fmoc-Cys(Acm)-OH,Fmoc-Glu(tBu)-OH,Fmoc-Asp(tBu)-OH,Fmoc-Asn(Trt)-OH],EDC·HCl/HOBt/DIEA,DCM,r.t.,2h;iv:TFA:Tis:H2O(95:2.5:2.5),r.t.,3h;v:5%DMSO/H2O,THF,NH4OH,r.t,24h;vi:I2/CH3OH,50%AcOH/H2O,Ascorbicacid quenching,r.t.,1h;
图6:HRMS(ESI)of BDK attached linear Plecanatide
图7:TLC analysis of BDK attached peptide chain(CHCl3:CH3OH=10:1)
图8:Optical photos of(A)BDK-attached Plecanatide precipitation and(B)linear Plecanatide precipitation by diethyl ether after shearing from the BDKsupport.
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BDK衍生物替代固相树脂,在脱水偶联剂的作用下与第一个Fmoc保护任意氨基酸(Fmoc-Xaa1-OH,其中Xaa1=Leu或其对映异构体DLeu)的C-端连接;
b)分离纯化:反应完成后,借助磷酸酯类载体BDK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,即为Fmoc-Xaa1-BDK;
c)脱除N-端Fmoc:将生成物1用脱Fmoc试剂处理后,再借助磷酸酯类载体BDK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,即为H2N-Xaa1-BDK;
d)多肽延长:重复以上步骤a、b、c,依次偶联上侧链保护的半胱氨酸Fmoc-Cys(Trt)-OH、Fmoc-Gly-OH、Fmoc-Thr(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn(Trt)-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Leu-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Xaa2-OH[Fmoc-Glu(tBu)-OH或Fmoc-Asp(tBu)-OH]、Fmoc-Asp(tBu)-OH和Fmoc-Xaa3-OH[Fmoc-Asn(Trt)-OH或Fmoc-DAsn(Trt)-OH],经辅助沉淀分离,得到纯化的化合物3,其氨基酸序列如下:
Fmoc-Xaa3-Asp(tBu)-Xaa2-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Xaa1-BDK,脱Fmoc后,经辅助沉淀分离,纯化得到化合物4,其氨基酸序列如下:
H2N-Xaa3-Asp(tBu)-Xaa2-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Xaa1-BDK;
e)侧链脱Trt保护基团:采用25%TFA/DCM处理化合物4,脱除侧链上的保护基团Trt,经过辅助沉淀分离,纯化得到化合物5,其氨基酸序列如下:
H2N-Xaa3-Asp(tBu)-Xaa2-Cys(Acm)-Glu(tBu)-Leu-Cys-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys-Xaa1-BDK;
f)第一次氧化环合:将适量氧化试剂(3.6g的过氧化尿素加入100mL的DMSO溶解溶液)加入上述化合物5的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物6,其氨基酸序列如下:
g)第二次氧化环合:将适量氧化试剂碘加入上述化合物6的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物7,其氨基酸序列如下:
h)脱除全部侧链保护并剪除载体:向上述所得产物7中加入足量的脱保护试剂(TFA:Tis:H2O=95:2.5:2.5),室温下搅拌3-5小时。反应完毕后,旋蒸回收三氟乙酸TFA,残留溶液用适量的乙醇溶解,加入乙酸乙酯,普卡那肽及其类似物因溶解度小而以沉淀形式析出,过滤并用适量乙酸乙酯洗涤沉淀,收集沉淀并干燥后即得普卡那肽及其类似物8,其氨基酸序列如下(Z=O,S,NH等):
i)载体的回收与再生:将h所得乙酸乙酯的有机相溶液浓缩至适当体积后,向溶液中加入适量的石油醚等烃类溶剂,BDK衍生物载体的残余部分会沉淀析出,经过滤、重结晶、洗涤、干燥等操作,即回收得到BDK衍生物载体的残余部分,经适当的再生反应后,可以循环再用。
本发明的合成路线见图3
本发明中一些常用的缩写具有以下含义:
BDK:4,4’-双(二苯基次磷酰基)二苯基酮及其衍生物
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
BDK:绿色多肽合成载体
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:
以下将参照实例对本发明作进一步的详细描述,本发明适用于普卡那肽及其类似物的合成制备与水解脱除。
化合物(2)的合成:准确称取4,4’-羟基二苯甲酮(2.14g,10mmol,1equiv)于150mL的反应瓶中,加入60mL的四氢呋喃THF并置于冰浴中搅拌30min,反应体系中滴加加入缚酸剂三乙胺Et3N(3.4mL,24mmol,2×1.2=2.4equiv),准确量取取二苯基次磷酰氯Ph2POCl(4.6mL,24mmol,2×1.2=2.4equiv)滴加加入上述反应体系,冰浴条件下反应30min后移去冰浴TLC检测反应体系室温反应2.0h,加入稀硫酸(0.1mol/L,10mL)淬灭反应,旋转蒸发仪浓缩除去THF溶剂并加入20mL去离子水,加入乙酸乙酯萃取得到有机相,无水硫酸镁干燥,旋干后加入3mL的乙酸乙酯是样品充分溶解,滴加加入正己烷15mL(VEA/VPE=1:5),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(2),产率约99%。
化合物(3)的合成:准确称取(2)(1.23g,2mmol,1equiv)于100mL的反应瓶中,加入甲醇溶液40mL置于冰浴下搅拌30min,反应体系分三批次加入硼氢化钠NaBH4(92mg,2.4mmol,1.2equiv),加上气球转入室温密封反应2h,TLC检测反应至原料消耗完毕后加入饱和氯化铵淬灭反应,浓缩除去甲醇溶液后加入去离子水10mL,乙酸乙酯萃取得到有机相,无水硫酸镁干燥,旋干后加入1mL的乙酸乙酯使样品充分溶解,滴加加入正己烷9mL(VEA/VPE=1:9),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(3),产率约97%。
Fmoc-Leu-BDK的合成:准确称取(3)(616mg,1.0mmol,1equiv)于100mL的反应瓶中,加入30mL DCM溶解,反应体系依次加入Fmoc-Leu-OH(423mg,1.2mmol,1.2equiv),4-二甲氨基吡啶DMAP(14.64mg,0.12mmol,0.12equiv),EDC·HCl(230mg,1.2mmol,1.2equiv),室温下反应2h,TLC检测反应结束后浓缩加入乙酸乙酯30mL溶解,加入饱和NaHCO3溶液洗涤,无水硫酸镁干燥,浓缩后用0.5mL的乙酸乙酯溶解样品,逐滴加入正己烷7mL(VEA/VN-Hexane=1:14),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-BDK,产率约98%,经过一次沉淀可作为原料进行下一步投料。
0.88-0.84(dd,J=8.0Hz,6H)ppm;31P NMR(162MHz,CDCl3),δ29.75ppm;13C NMR(100MHz,CDCl3),δ172.0,155.9,150.6,143.7,141.3,135.5,132.5,131.8,131.7,130.2,128.7,128.6,128.5,127.7,127.1,125.1,120.8,120.0,76.6,67.0,52.6,47.2,41.4,24.7,22.8,21.8ppm;HRMS(ESI)m/z calcd for C58H52NO8P2 +(M+H)+952.31627,found 952.31641.
Fmoc-Cys(Trt)-Leu-BDK的合成:准确称取Fmoc-Leu-BDK(950mg,1.0mmol)于50mL的反应瓶中,加入6mL MeCN溶解,室温条件下搅拌条件下滴加入DEA2mL(DEA/MeCN=1:3,25%DEA脱Fmoc保护基),室温条件搅拌50min后TLC检测原料消失,浓缩除去MeCN及DEA,加入DCM,继续浓缩,连续操作4次除净DEA,加入乙酸乙酯1.5mL,滴加石油醚15mL,震荡(VEA/VN-Hexane=1:10),出现大量白色固体,静置5min后除去上清液,得到脱保护后的产物H2N-Leu-BDK(产率约99%)备用;取100mL圆底烧瓶,加入30mL DCM置于冰浴条件,体系依次加入上述脱保护产物BDK-Leu-NH2,Fmoc-Cyst(Trt)-OH(702mg,1.2mmol,1.2equiv),HOBt(162mg,1.2mmol,1.2equiv),冰浴条件下搅拌30min,然后加入EDC·HCl(230mg,1.2mol,1.2equiv),DIEA(330μL,2.0mmol,2.0equiv),继续冰浴搅拌10min后转至室温下反应2.0h,TLC检测反应结束浓缩后加入乙酸乙酯40mL溶解,用饱和Na2CO3溶液洗涤,无水硫酸镁干燥,浓缩后用3mL的乙酸乙酯溶解样品,逐滴加入石油醚18mL(VEA/VPE=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-Cys(Trt)-Leu-BDK,产率约98%,经过一次沉淀可作为原料进行下一步投料。
Plecanatide肽链的延伸:使用上述偶联试剂系统EDC·HCl/HOBt/DIEA和脱Fmoc试剂25%DEA/MeCN试剂系统,延伸Plecanatide肽链并获得各个中间体肽产物,如下所示:
(2) Fmoc-Cys(Trt)-Leu-BDK:White solid,Rf=0.42(CH2Cl2:MeOH=60:1),(97%yield).
2H),4.18-4.15(m,1H),3.72-3.70(m,1H),2.72-2.51(m,2H),1.53-1.36(m,3H),0.78-0.74(dd,J=4.0Hz,6H)ppm;31P NMR(162MHz,CDCl3),δ29.95ppm;13C NMR(100MHz,CDCl3),δ171.1,169.8,150.7,144.3,143.7,141.3,135.5,132.6,131.7,130.2,129.6,128.7,128.6,128.1,127.8,126.9,125.1,120.8,120.0,76.5,67.4,67.1,54.0,50.9,47.1,41.0,24.7,22.7,21.9ppm;HRMS(ESI)m/z calcd for C80H71N2O9P2S+(M+H)+1297.43500,found1297.43579.
(3)Fmoc-Gly-Cys(Trt)-Leu-BDK:White solid,Rf=0.40(CH2Cl2:MeOH=60:1),(98%
1H),4.62-4.57(m,1H),4.26-3.94(m,4H),3.65-3.64(m,J=4.0Hz,2H),2.97-2.92(m,1H),2.44-2.40(m,1H),1.59-1.52(m,3H),0.82-0.80(m,6H)ppm;31PNMR(162MHz,CDCl3),δ29.85ppm;13C NMR(100MHz,CDCl3),δ170.9,169.4,168.9,157.1,150.5,144.3,143.8,141.2,135.7,132.5,131.6,130.1,129.5,128.6,128.1,127.6,126.9,125.2,120.7,119.9,76.3,67.2,52.1,50.8,46.9,45.0,40.4,33.3,24.6,22.9,21.7ppm;HRMS(ESI)m/zcalcd for C82H74N3O10P2S+(M+H)+1354.45647,found 1354.45630.
(4)Fmoc-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:White solid,Rf=0.40(CH2Cl2:MeOH=50:1),
4.22-4.13(m,3H),3.90-3.73(m,2H),3.65(s,1H),2.85-2.80(m,1H),2.50-2.45(m,1H),1.54-1.41(m,3H),1.24(s,9H),1.02-1.01(d,J=4.0Hz,3H),0.80-0.79(m,6H)ppm;31P NMR(162MHz,CDCl3),δ29.99ppm;13C NMR(100MHz,CDCl3),δ171.1,170.3,169.4,168.3,156.1,150.5,144.4,143.7,141.3,135.7.132.6,131.7,130.2,129.6,128.7,128.5,128.1,127.7,126.9,125.2,120.7,120.0,77.3,76.4,75.6,67.3,64.5,59.0,52.4,51.1,47.2,43.4,40.9,33.2,28.2,24.7,22.8,21.9,17.5ppm;HRMS(ESI)m/z calcd forC90H88N4O12P2SNa+(M+Na)+1533.54869,found1533.54834.
(5)Fmoc-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:White solid,Rf=0.38(CH2Cl2:
(m,1H),6.66(s,1H),6.47-6.42(m,2H),4.61-4.59(m,1H),4.47-4.12(m,8H),3.95-3.75(m,2H),3.66(s,1H),3.02-2.80(m,3H),2.55-2.51(m,1H),1.92(s,3H),1.49-1.47(m,3H),1.17(s,9H),1.09-1.07(d,J=8.0Hz,3H),0.80-0.77(m,6H)ppm;31P NMR(162MHz,CDCl3),δ29.49ppm;13C NMR(100MHz,CDCl3),δ171.5,171.1,170.6,169.6,168.6,156.7,150.5,144.4,143.8,141.3,135.7.132.6,131.8,130.1,129.6,128.8,128.1,127.8,127.1,126.9,125.3,120.7,120.0,77.3,76.3,75.1,67.2,66.2,64.6,63.8,59.1,54.7,52.8,51.3,47.1,43.2,40.6,33.4,28.2,24.6,23.1,22.8,21.8,19.2ppm;HRMS(ESI)m/z calcdfor C96H99N6O14P2S2 +(M+H)+1685.61304,found 1685.61365.
1H),8.32-8.26(m,4H),7.90-7.84(m,10H),7.76-7.72(m,2H),7.63-7.53(m,13H),7.43-7.40(m,2H),7.35-7.10(m,25H),6.64(s,1H),4.66-4.63(m,1H),4.52-4.46(m,1H),4.34-4.14(m,9H),3.98-3.80(m,2H),2.99-2.71(m,2H),2.42-2.29(m,2H),1.85(s,3H),1.52-1.42(m,3H),1.28-1.26(m,J=8.0Hz,3H),1.10(s,9H),1.00-0.99(d,J=4.0Hz,3H),0.78-0.71(dd,J=8.0Hz,6H)ppm;31P NMR(162MHz,CDCl3),δ29.45 ppm;13C NMR(100MHz,DMSO-d6),δ173.3,171.1,170.7,170.3,170.1,169.7,168.6,156.1,150.5,144.7,144.2,141.2,136.7,133.3,131.9,130.6,129.5,128.5,128.1,127.5,127.2,125.8,120.9,120.6,75.9,74.3,67.3,66.3,66.2,65.5,63.3,58.0,53.0,51.7,51.1,50.4,47.1,28.4,24.5,23.1,23.0,21.8,19.4,18.9ppm;HRMS(ESI)m/z calcd for C99H104N7O15P2S2 +(M+H)+1756.65016,found 1756.65027.
(7)Fmoc-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:White solid,Rf=0.40
1H),7.90-7.86(m,10H),7.77-7.73(m,2H),7.61-7.40(m,15H),7.34-7.10(m,25H),6.64(s,1H),4.68-4.62(m,1H),4.51-4.49(m,2H),4.43-4.20(m,8H),3.98-3.80(m,3H),2.99-2.69(m,2H),2.42-2.30(m,2H),2.04-1.99(m,1H),1.86(s,3H),1.52-1.42(m,3H),1.26-1.25(m,3H),1.10(s,9H),1.01-0.99(d,J=4.0Hz,3H),0.89-0.85(m,6H),0.78-0.71(m,6H)ppm;31P NMR(162MHz,CDCl3),δ29.47ppm;13C NMR(100MHz,DMSO-d6),δ172.8,171.2,171.1,170.6,170.3,170.1,169.7,168.6,156.6,150.5,144.7,141.2,139.9,137.9,136.7,133.2,131.9,130.6,129.5,128.5,128.1,127.7,127.5,127.2,125.8,121.8,120.9,120.5,75.9,74.3,67.3,66.3,65.5,63.3,60.5,58.0,51.7,51.1,48.4,47.2,32.5,30.9,28.4,24.5,23.0,21.8,19.8,19.4,19.1,18.6ppm;HRMS(ESI)m/z calcd forC104H113N8O16P2S2 +(M+H)+1855.71857,found 1855.71753.
(8)Fmoc-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:Whitesolid,Rf=
11H),7.74-7.72(m,3H),7.61-7.53(m,13H),7.42-7.41(m,2H),7.26-7.16(m,38H),6.64(s,1H),4.65-4.64(m,1H),4.48-4.24(m,11H),3.97-3.81(m,3H),2.96-2.60(m,4H),2.39-2.32(m,2H),2.00-1.98(m,1H),1.85(s,3H),1.48-1.44(m,3H),1.26-1.21(m,3H),1.10(s,9H),1.00-0.99(d,J=4.0Hz,3H),0.86-0.71(m,12H)ppm;31PNMR(162MHz,CDCl3),δ29.77ppm;13C NMR(100MHz,DMSO-d6),δ172.8,171.6,171.1,170.8,170.6,170.3,170.1,169.7,169.3,168.6,156.3,150.5,145.2,144.7,144.2,141.2,136.8,133.2,131.9,131.8,130.6,129.5,129.3,129.0,128.5,128.1,127.9,127.6,127.2,126.8,125.8,120.9,120.6,75.9,74.3,69.9,67.2,66.3,65.5,63.3,57.9,57.7,52.8,51.7,51.1,48.5,47.1,31.3,28.4,24.5,23.1,21.8,19.7,19.4,18.8,18.3ppm;HRMS(ESI)m/z calcd forC127H133N10O18P2S2 +(M+H)+2211.87105,found 2211.87280.
(9)Fmoc-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,
Rf=0.40(CH2Cl2:MeOH=30:1),(91%yield).1H NMR(400MHz,DMSO-d6),δ8.63-8.51(m,2H),8.39-8.13(m,8H),7.90-7.86(m,11H),7.76-7.74(m,2H),7.61-7.54(m,13H),7.42-7.40(m,3H),7.26-7.16(m,38H),6.64(s,1H),4.65-4.63(m,1H),4.49-4.20(m,12H),4.02-3.81(m,4H),2.96-2.66(m,4H),2.39-2.32(m,2H),2.01-1.95(m,2H),1.85(s,3H),1.48-1.44(m,3H),1.23-1.19(m,3H),1.11(s,9H),1.00-0.99(d,J=4.0Hz,3H),0.90-0.71(m,18H)ppm;31P NMR(162MHz,CDCl3),δ29.97ppm;13C NMR(100MHz,DMSO-d6),δ172.8,171.6,171.1,170.8,170.6,170.4,170.3,170.1,169.7,169.3,168.6,156.3,150.5,145.2,144.7,144.2,141.2,136.7,133.2,131.9,131.8,130.6,129.5,129.3,129.0,128.5,128.1,127.9,127.5,127.2,126.7,125.9,120.9,120.5,75.9,74.3,69.9,67.3,66.3,65.5,63.3,58.0,57.6,52.8,51.7,51.1,48.5,47.1,31.3,28.4,24.5,23.0,21.8,19.6,19.4,18.7,18.5,18.3ppm;HRMS(ESI)m/z calcd forC132H142N11O19P2S2 +(M+H)+2310.93946,found 2310.93872.
(10)Fmoc-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.38(CH2Cl2:MeOH=20:1),(97%yield).1H NMR(400MHz,DMSO-d6),δ8.64-8.53(m,2H),8.43-8.12(m,8H),7.90-7.86(m,11H),7.73-7.71(m,2H),7.61-7.52(m,13H),7.41-7.10(m,57H),6.64(s,1H),4.67-4.47(m,3H),4.34-4.16(m,10H),3.98-3.80(m,4H),2.98-2.93(m,1H),2.74-2.54(m,3H),2.46-2.30(m,3H),2.00-1.85(m,5H),1.55-1.42(m,3H),1.23-1.17(m,4H),1.11(s,9H),1.01-0.99(d,J=8.0Hz,3H),0.83-0.69(m,18H)ppm;31P NMR(162MHz,CDCl3),δ29.91ppm;13C NMR(100MHz,DMSO-d6),δ172.8,171.1,170.8,170.7,170.6,170.3,170.1,169.9,169.7,169.2,168.6,156.2,150.5,145.1,144.7,144.3,141.2,136.8,133.2,131.9,130.6,129.5,129.3,129.0,128.5,128.1,127.9,127.5,127.2,126.7,125.7,120.9,120.5,75.9,74.3,69.8,67.3,66.5,66.3,58.0,51.8,47.1,32.2,31.3,28.4,24.5,23.1,21.8,19.8,19.6,19.4,18.8,18.3,17.6ppm;HRMS(ESI)m/z calcd for C154H160N12O20P2S3Na+(M+Na)+2678.04014,found2678.03564.
(11)Fmoc-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.40(CH2Cl2:MeOH=20:1),(96%yield).1H NMR(400MHz,DMSO-d6),δ8.66-8.19(m,8H),7.96-7.85(m,9H),7.63-7.53(m,12H),7.30-7.16(m,60H),6.62(s,1H),4.63-3.79(m,18H),2.96-2.93(m,1H),2.68(m,3H),2.41-2.34(m,3H),1.99-1.84(m,8H),1.49-1.44(m,4H),1.23-1.16(m,8H),1.09(s,9H),0.99-0.98(m,3H),0.82-0.76(m,24H)ppm;31P NMR(162MHz,CDCl3),δ29.61ppm;HRMS(ESI)m/z calcd forC160H171N13O21P2S3Na+(M+Na)+2791.12421,found 2791.11450.
(12)Fmoc-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.50(CH2Cl2:MeOH=10:1),(95%yield).1H NMR(400MHz,DMSO-d6),δ8.63-8.60(m,2H),8.44-8.16(m,8H),7.96-7.85(m,13H),7.73-7.71(m,2H),7.63-7.52(m,14H),7.42-7.09(m,56H),6.62(s,1H),4.63(m,2H),4.47-3.79(m,17H),2.96-2.89(m,2H),2.73-2.64(m,3H),2.41-2.24(m,5H),2.00-1.84(m,7H),1.67(m,2H),1.52-1.35(m,8H),1.23-1.16(m,8H),1.09(s,9H),0.99-0.98(d,J=4.0Hz,3H),0.86-0.68(m,24H)ppm;31P NMR(162MHz,CDCl3),δ29.91ppm;HRMS(ESI)m/z calcd forC169H186N14O24P2S3Na+(M+Na)+2976.22941,found2976.22035.
(13)Fmoc-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.52(CH2Cl2:MeOH=10:1),(94%yield).1H NMR(400MHz,DMSO-d6),δ8.63-8.15(m,7H),7.90-7.83(m,11H),7.74-7.72(m,2H),7.61-7.53(m,14H),7.43-7.39(m,2H),7.29-7.15(m,56H),6.62(s,1H),4.63-3.79(m,22H),2.96-2.64(m,5H),2.39-2.33(m,6H),1.99-1.84(m,10H),1.44-1.34(m,8H),1.23-1.15(m,8H),1.09(s,12H),0.98(d,J=8.0Hz,3H),0.82-0.70(m,24H)ppm;31P NMR(162MHz,CDCl3),δ29.76ppm;HRMS(ESI)m/z calcd forC175H196N16O26P2S4Na+(M+Na)+3150.27570,found 3150.27197.
(14)Fmoc-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.50(CH2Cl2:MeOH=10:1),(93%yield).1H NMR(400MHz,DMSO-d6),δ8.66-8.17(m,4H),7.90-7.85(m,6H),7.63-7.53(m,9H),7.30-7.09(m,66H),6.62(s,1H),4.65-3.79(m,22H),2.96-2.63(m,4H),2.37-2.31(m,6H),2.02-1.84(m,12H),1.38-1.36(m,12H),1.24-1.16(m,10H),1.09(s,12H),1.00-0.98(m,3H),0.87-0.70(m,24H)ppm;31P NMR(162MHz,CDCl3),δ29.36ppm;HRMS(ESI)m/z calcd forC184H211N17O29P2S4Na+(M+Na)+3335.38090,found 3335.38548.
(16)Fmoc-Asn(Trt)-Asp(tBu)-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.60(CH2Cl2:MeOH=10:1),(95%yield).1H NMR(400MHz,DMSO-d6),δ8.64-8.15(m,6H),7.96-7.84(m,11H),7.75-7.72(m,2H),7.61-7.52(m,14H),7.44-7.35(m,5H),7.30-7.09(m,71H),6.63(s,1H),4.62(m,2H),4.48-4.20(m,16H),3.95-3.79(m,5H),2.97-2.93(m,2H),2.74-2.68(m,3H),2.39-2.19(m,8H),2.04-1.83(m,12H),1.65(m,2H),1.48-1.34(m,15H),1.24-1.09(m,27H),1.00-0.98(m,6H),0.85-0.70(m,32H)ppm;31P NMR(162MHz,CDCl3),δ29.36ppm;
(16de-Fmoc)H-Asn(Trt)-Asp(tBu)-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn(Trt)-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK:
White solid,Rf=0.30(CH2Cl2:MeOH=10:1),(99%yield).HRMS(ESI)m/z calcdfor C200H234N20O32P2S4Na+(M+Na)+3640.55483,found 3640.55884.
BDK/-Trt/-tBu保护基团的剪切:将H-NDECELCVNVACTGCL-BDK加入剪切试剂TFA:硫苯甲醚:EDT:苯酚:H2O=87.5:5:2.5:2.5:2.5试剂。在室温下搅拌反应3小时。将反应混合物减压浓缩以除去TFA,H2O。将残余物添加至冷的乙醚中三次以得到沉淀物,然后将沉淀物离心以获得BDK残基及直链多肽肽链,H-NDEC(Acm)ELCVNVAC(Acm)TGCL-OH。
H-Asn-Asp-Glu-Cys(Acm)-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys(Acm)-Thr-Gly-Cys-Leu-OH:1H NMR(400MHz,DMSO-d6),δ12.46(s,4H),8.54-7.73(m,19H),7.43-7.33(m,5H),6.97(s,1H),5.02-3.80(m,22H),3.38(m,5H),2.93-2.71(m,10H),2.28(m,5H),1.98-1.87(m,8H),1.65-1.46(m,6H),1.24(m,6H),1.05(m,3H),0.89-0.84(m,24H)ppm;HRMS(ESI)m/z calcd for C71H119N20O28S4 +(M+H)+1827.73800,found 1827.73759.
ppm;13C NMR(100MHz,CDCl3),δ148.8,137.4,132.9,131.9,130.1,128.8,120.7,40.2ppm;HRMS(ESI)m/z calcd for C37H30O4P2Na+(M+Na)+623.15115,found 623.15100.
二硫键的正交氧化形成:将线型肽H-NDEC(Acm)ELCVNVAC(Acm)TGCL-OH(180mg,0.1mmol)溶解在5mL DMSO/H2O(VDMSO:VH2O=0.05:0.95)中用稀NH4·OH调节至8.0。将反应混合物在室温搅拌24小时,然后冻干以备下次使用。随后,将上述冻干的粉末溶解于20ml的50%AcOH/H2O中,然后将1.5mL的I2/MeOH(0.1mol/L)滴加到反应混合物中。将反应混合物搅拌1小时,通过加入抗坏血酸(1mol/L,1.5mL)淬灭。将反应混合物浓缩并直接进行RP-HPLC,得到目标化合物Plecanatide(96mg,53%)。
H-Asn-Asp-Glu-Cys[4,12]-Glu-Leu-Cys[7,15]-Val-Asn-Val-Ala-Cys[4,12]-Thr-Gly-Cys[7,15]-Leu-OH(Plecanatide):1H NMR(400MHz,D2O),δ4.66-4.59(dd,J=28.0Hz,6H),4.55-4.47(m,2H),4.27-4.15(s,6H),4.10-4.01(m,3H),3.95-3.88(m,2H),3.81-3.77(d,J=16.0Hz,1H),3.27-2.64(m,16H),2.36-2.31(m,4H),2.08-1.90(m,7H),1.54-1.41(m,6H),1.25-1.23(d,J=8.0Hz,3H),1.08-1.06(d,J=8.0Hz,3H),0.83-0.70(m,24H),ppm;13C NMR(100MHz,D2O),177.6,177.4,177.2,175.1,174.4,174.3,173.9,173.8,173.1,172.8,172.6,172.5,172.2,171.8,171.7,171.2,168.8,66.6,60.2,59.8,54.3,53.8,53.4,53.1,52.7,52.0,50.8,49.7,42.6,41.0,39.9,39.4,38.9,37.7,36.4,36.2,34.8,30.4,30.1,29.7,25.8,25.6,24.4,24.2,22.3,22.1,20.7,20.6,18.7,18.3,17.6,17.3,15.8ppm;HRMS(ESI)m/z calcd for HRMS(ESI)m/z calcd for C65H105N18O26S4 +(M+H)+1681.63247,found 1681.63257.
Claims (9)
1.一种采用BDK辅助基团进行基团辅助的普卡那肽液相合成方法,其特征在于步骤如下:
步骤1、辅助基团与氨基酸的偶联:以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,Fmoc-Leu-OH的C-端与BDK辅助基团连接,生成中间体化合物1,Fmoc-Leu-BDK;
所述BDK辅助基团的分子结构式为:
所述氨基酸与BDK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BDK;
步骤2、分离纯化:向中间体化合物1加入极性小的烷烃或醚类溶剂,借助BDK辅助基团在溶剂系统中易结晶沉淀的特性,将中间体化合物1与其他杂质分离;
对分离后的中间体化合物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;
向生成物2加入极性小的烷烃或醚类溶剂,借助BDK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,H2N-Leu-BDK;
步骤4:以纯化的含有辅助基团BDK的生成物2作为原料,替代步骤1的辅助基团,再与保护的半胱氨酸Fmoc-Cys(Trt)-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-Cys(Trt)-Leu-BDK;
以第一次循环重复后纯化的生成物H2N-Cys(Trt)-Leu-BDK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-Cys(Trt)-Leu-BDK;
以第二次循环重复后纯化的生成物H2N-Gly-Cys(Trt)-Leu-BDK作为原料,替代步骤1的辅助基团,以tBu保护的苏氨酸Fmoc-Thr(tBu)-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-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK;
以上次循环重复后纯化的生成物H2N-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK作为原料,替代步骤1的辅助基团,依次以Fmoc-Cys(Acm)-OH、Fmoc-Ala-OH、Fmoc-Val-OH、Fmoc-Asn-OH、Fmoc-Val-OH、Fmoc-Cys(Trt)-OH、Fmoc-Leu-OH、Fmoc-Glu(tBu)-OH、Fmoc-Cys(Acm)-OH、Fmoc-Glu(tBu)-OH、Fmoc-Asp(tBu)-OH和Fmoc-Asn-OH替代步骤1的氨基酸进行偶联反应;重复步骤1~步骤3,得到普卡那肽的前体化合物4:
H2N-Asn-Asp(tBu)-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys(Trt)-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys(Trt)-Leu-BDK;
步骤5、侧链脱Trt保护基团:采用25%TFA/DCM处理前体化合物4,脱除侧链上的保护基团Trt,经辅助沉淀分离,纯化得到化合物5,其氨基酸序列如下:H2N-Asn-Asp(tBu)-Glu(tBu)-Cys(Acm)-Glu(tBu)-Leu-Cys-Val-Asn-Val-Ala-Cys(Acm)-Thr(tBu)-Gly-Cys-Leu-BDK;
步骤6、第一次氧化环合:将氧化试剂加入上述化合物5的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物6,其氨基酸序列如下:
所述氧化试剂为3.6g的过氧化尿素加入100mL的DMSO溶解溶液;
步骤7、第二次氧化环合:将适量氧化试剂碘加入上述化合物6的乙腈溶液中,搅拌反应2小时后回收乙腈,用乙酸乙酯溶解,经辅助沉淀分离,纯化得到化合物7,其氨基酸序列如下:
步骤8、脱除全部侧链保护并剪除载体:向上述所得化合物7中加入足量的脱保护试剂为TFA:Tis:H2O=95:2.5:2.5,室温下搅拌3-5小时,反应完毕后,旋蒸回收三氟乙酸TFA,残留溶液用适量的乙醇溶解,加入乙酸乙酯,普卡那肽及其类似物因溶解度小而以沉淀形式析出,过滤并用适量乙酸乙酯洗涤沉淀,收集沉淀并干燥后即得普卡那肽,其氨基酸序列如下:
2.一种权利要求1所述方法制备过程中得到的Fmoc保护亮氨酰-BDK化合物Fmoc-Leu-BDK,所述化合物的分子结构式为:
3.一种权利要求1所述方法制备过程中得到的脱Fmoc保护亮氨酰-BDK化合物H2N-Leu-BDK,所述化合物的分子结构式为:
4.一种权利要求1所述方法制备过程中得到的Fmoc保护半胱氨酰-亮氨酰-BDK化合物Fmoc-Cys(Trt)-Leu-BDK,所述化合物的分子结构式为:
5.一种权利要求1所述方法制备过程中得到的脱Fmoc保护半胱氨酰-亮氨酰-BDK化合物H2N-Cys(Trt)-Leu-BDK,所述化合物的分子结构式为:
6.一种权利要求1所述方法制备过程中得到Fmoc保护的十六肽-BDK中间体化合物,其特征在于:所述化合物的分子结构式为:
7.一种权利要求1所述方法制备过程中得到脱Fmoc保护的十六肽-BDK中间体化合物,其特征在于:所述化合物的分子结构式为
8.一种权利要求1所述方法制备过程中得到脱Trt保护的十六肽-BDK化合物,其特征在于:所述化合物的分子结构式为
其第一次氧化环合产物中间体的分子结构式为:
其第二次氧化环合产物的分子结构式为:
9.一种权利要求1所述的采用BDK基团辅助的普卡那肽液相合成方法中BDK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BDK在不同溶剂系统中易结晶沉淀的特性,将BDK载体残留与其他杂质分离;对分离后的BDK载体残留进行过滤和洗涤或重结晶操作得到纯化的BDK载体残留,回收再生转化后直接作为辅助基团再利用;所述BDK载体残留的分子结构式为:
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