CN109836455B - 基于磷或亚磷酰氧基二苯甲醇及其衍生物及辅助的胸腺五肽液相合成方法 - Google Patents
基于磷或亚磷酰氧基二苯甲醇及其衍生物及辅助的胸腺五肽液相合成方法 Download PDFInfo
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Abstract
本发明涉及一种基于多芳基磷或亚磷酰氧基苯甲醇(POB)类化合物及其衍生物及辅助的胸腺五肽(TP‑5)液相合成方法,以磷或亚磷酸酯类载体替代固相树脂,在偶联脱水剂的作用下与Fmoc保护酪氨酸的C‑端连接;分离纯化后脱除N‑端Fmoc;再依次与Fmoc和侧链保护的缬氨酸、天门冬氨酸、赖氨酸和精氨酸进行偶联和脱Fmoc反应,制备胸腺五肽的前体C;侧链脱保护并剪除载体,得到胸腺五肽的固体。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、高效地大规模合成制备胸腺五肽,而且POB辅助基团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
本发明属于生物有机化学领域中多肽的合成方法,涉及一种基于磷或亚磷酰氧基二苯甲醇及其衍生物及辅助的胸腺五肽液相合成方法。
背景技术
胸腺激素是由胸腺所分泌的一族多肽类蛋白质激素。这类激素在个体发育和免疫系统功能调节方面起着至关重要的作用。胸腺生成素(Thymopoientin,TP)是由胸腺上皮细胞分泌的激素之一,从人胸腺组织中分离出的一种49个氨基酸组成的多肽,具有促进胸腺细胞和外周T细胞及B细胞分化发育、调节机体免疫功能等生物活性,见文献:蒋定文,李楚芳,国外医学:预防、诊断、治疗用生物制品分册,1999,2(2):69-72。1974年由Goldsfein G从牛胸腺中分离并确定TP的一级结构,发现胸腺生成素II(thymopoletin II)的免疫活性中心是一个五肽片段,位于多肽的第32~36位,氨基酸序列为氨基酸序列为精氨酸-赖氨酸-天门冬氨酸-缬氨酸-酪氨酸(H-Arg-Lys-Asp-Val-Tyr-OH),被称作胸腺五肽(Thymopentin,TP-5)。1979年Goldsfein G通过固相合成胸腺生成素的32~36位氨基酸片段,人工合成的胸腺五肽(Thymopentin,TP-5)与TP的32~36位氨基酸残基顺序相同,即H-Arg-Lys-Asp-Val-Tyr-OH,见文献:Gonser S,Crompton EAN,Folkers G,et al.MutationRes,2004,558:19-26。胸腺五肽于1981年首先由美国ORTHO-PHPRIVl公司研制成功上市,国内在1995年成功上市。
胸腺五肽是一个碱性短肽,英文名称为Thymopentin,简称TP-5,其化学名称是L-精氨酰-L-赖氨酰-L-天门冬氨酰-L-缬氨酰-L-酪氨酸,结构式为H-Arg-Lys-Asp-Val-Tyr-OH,分子式C30H49N909,分子量679.77,等电点pI=8.59,TP-5是由两个碱性氨基酸、两个中性氨基酸及一个酸性氨基酸组成的多肽类药物,具有确定的分子式及分子量,呈微碱性,溶于水及乙酸,不溶于乙醚及乙酸乙酯等有机溶剂,见文献:Cascinelli N,Clemente C,Bufalino R,et a1.Melanoma Res,1993,3(6):471-476。
目前TP-5的主要制备路线有两种:生物技术法和化学合成法。1.生物技术法:以生物技术制造TP-5的研究虽然非常活跃,但所面临的主要问题是一方面技术较为复杂,技术也尚不成熟,而且受到原料等因素的制约,另一方面由于原料的原因还可能会造成制品的污染,难以大规模生产,发展前景非常黯淡。2.化学合成法:TP-5的化学合成包括液相合成和固相合成两种。现有的液相合成法,见文献:
1、王锐,魏洁,赵倩予,等,一种胸腺五肽液相合成法,中国专利,03134355.4,2003.6.10
2、彭师奇,赵明,王超,等,胸腺五肽的制备方法,中国专利,200410022741.6,2004.6.1
3、葛存惠,梁胜,任仲辉,等,一种胸腺五肽的制备方法,中国专利,200810064651.1,2008.6.3
4、刘大学,一种多肽液相合成方法,中国专利,201110051933.X,2011.3.4
5、何平,邹倩儒,一种酶合成胸腺五肽的方法,中国专利,201310024160.5,2013.1.22;
主要有(1)采用叔丁氧羰基(Boc)和9-芴甲氧羰基(Fmoc)策略相结合的液相方法合成TP-5,保护氨基酸原料分别为HCl·H-Tyr(Bzl)-OBzl、Boc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Lys(Boc)-OH和Fmoc-Arg(Pbf)-OH,缩合试剂为BOP-HOBt,缩合时间2h,反应溶剂为三氯甲烷,碱中和试剂采用N-甲基吗啉(NMM)。最后保护基的脱除系统采用三氟甲磺酸/三氟醋酸(TFMSA/TFA)+苯甲硫醚,0℃反应2h。(2)采用Boc氨基保护策略和碳链逐步增长法液相合成TP-5:氨基采用Boc-HCl/二噁烷保护系统,DCC-HOBt缩合得到全保护的Boc-Arg(Tos)-Lys(Z)-Asp(OBz1)-Val-Tyr(Bz1)-Ome,皂化后,采用TFA/TFMSA+苯甲醚、茴香硫醚和邻甲基苯酚,室温反应1h,脱除侧链得白色结晶,纯化后总收率30%。(3)采用液相最小保护策略逐步合成TP-5:氨基酸保护策略为HC1·Tyr-OMe、Boc-Val-OH、Boc-Asp(OBz1)-OH、Boc-Lys(2C1Z)-OH、Z-Arg-OH,DCC-HOBt缩合形成二肽、三肽和四肽,HBTU缩合形成保护五肽,最后采用Pd/C氢解脱除侧链保护得到目的产物。在不发生较大副反应的前提下,侧链功能基尽量不采取保护,可以避免HF的强酸裂解,条件比较温和,更适合于产业化。但存在的问题是在合成过程中每一步反应后都须将中间产物用乙腈分离纯化出来,用量大,成本高:多次循环进行纯化和重结晶过程,耗时长;HF的高危险性,使肽的切割与保护基脱除需在特殊的装置中完成,使工艺过程和所用设备繁杂。传统的TP-5化学合成法是固相方法,见文献:王玲,魏浩,韦萍,等,固相合成胸腺五肽的研究进展,化工进展,2003,22(2):153-156,张春莉等,见文献:张春莉,刘照惠,王妍,等,中国生物制品学杂志,2003,16f2):87-89;采用BOP为缩合剂进行固相合成,TP-5的收率为63%;杨玉华等,见文献:杨玉华,顾涵英,胸腺五肽的制备方法,中国专利,03116102.2,2003.4.1;采用DIC法,固相接肽反应时间为24h,氨基酸及缩合剂均以5倍过量进行固相合成TP-5。固相法归纳起来主要有Boc/Bzl和Fmoc/OtBu两种合成保护策略,见文献:1、叶善明,施傅涛,俞通泰,等,胸腺五肽合成工艺方法,中国专利,200510060558.X,2005.8.30;2、崔学云,杨平,一种胸腺五肽的制备方法,中国专利,201110442546.9,2011.12.26;3、王文琪,崔颀,周逸明,一种胸腺五肽的制备方法,中国专利,201310335626.3,2013.8.5;4、张响,胡碧煌,杨顶建,一种Fmoc法固相合成胸腺五肽的方法,中国专利,201310746262.8,2013.12.30;
(1)Boc固相合成法---Boc合成法的主要策略是采用TFA可脱除的Boc为α-氨基保护基,侧链保护采用苄醇类。合成时将一个Boc-氨基酸衍生物共价交联到Merrifield或MBHA树脂上,用TFA脱除Boc,用三乙胺中和游离的氨基末端,然后通过DCC活化、偶联下一个氨基酸,最终脱保护多采用HF法或TFMSA法。(2)Fmoc固相合成法---Fmoc法与Boc法的根本区别在于采用了碱可脱除的Fmoc为α-氨基的保护基,侧链的保护采用TFA可脱除的叔丁氧基等,树脂采用90%TFA可切除的对烷氧苄醇型树脂和1%TFA可切除的二烷氧苄醇型树脂,最终的脱保护避免了强酸处理。Fmoc作为氨基保护基的优点在于其对酸稳定,用TFA等试剂处理不受影响,仅需用温和的碱处理,通过β2消除反应即可脱去,不需用三级胺中和,且Fmoc基团有特征性紫外吸收,易于检测反应的进行,给自动化合成带来方便。但Fmoc保护的氨基酸价格昂贵,合成成本也相应较高。在TP-5的Fmoc固相合成中,采用Wang树脂,DMF为溶剂,DIC-HOBt缩合,侧链保护策略为Fmoc-Arg(Pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Val-OH和Fmoc-Tyr(tBu)-OH。固相合成方法优点在于条件比较温和,工艺比较简单,便于将树脂洗涤干净。固相合成方法已经取得了长足的发展,尤其是Fmoc法更是一种简便、高效的合成方法,为TP-5以及其他多肽类药物的合成与开发起到重要的促进作用。目前,TP-5固相合成存在的主要问题是反应速度慢,粗品纯度低,后期纯化难等,这些都有待于完善。
伴随着分子生物学、生物化学、药物化学等技术的飞速发展,多肽的研究取得了惊人的、划时代的进展,受到了越来越多人的关注,它已涉及到激素、神经、细胞生长和生殖等各个领域。多肽在改善和调整人们的生活方式方面将发挥极大的作用,出现了TP-5等一批多肽类药物并显示了强大的市场前景。多肽与蛋白质类药物是当今国际市场上的一大类重要药物,正逐步成为本世纪药物发展的主流方向。一些传统欧美制药强国如美国、瑞士、瑞典、荷兰、法国、德国和英国均在大力开发活性肽类新药,我国也已将多肽药物的研究开发列入未来几年内的重点发展方向。因此,TP-5作为疗效显著的多肽药物其研究价值不言而喻。该药虽然半衰期很短,在体内只有l min左右,却能引起较长时间的免疫调节效果,而且无论是皮下注射还是静脉给药,都极少产生毒副作用,是一种非常安全的药物。
总之,目前胸腺五肽(TP-5)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。
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,本发明提出一种基于磷或亚磷酰氧基二苯甲醇及其衍生物及辅助的胸腺五肽液相合成方法,解决目前液相法和固相法合成胸腺五肽都存在困境和不足。
技术方案
一种采用磷酰氧基苯甲醇POB进行基团辅助的胸腺五肽液相的合成方法,其特征在于,步骤如下:
步骤1、辅助基团与氨基酸的偶联:以辅助基团替代固相多肽合成中的树脂,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,得到生成物A;所述氨基酸与POB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POB,分子结构通式为:
所述氨基酸采用芴甲氧羰基Fmoc保护的酪氨酸Fmoc-Tyr(OtBu)-OH,受保护的酪氨酸Fmoc-Tyr(OtBu)-OH的C-端与POB辅助基团连接,生成化合物A,Fmoc-Tyr(OtBu)-POB;
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POB辅助基团在溶剂系统中易结晶沉淀的特性,将生成物A与其他杂质分离;
对分离后的生成物A进行过滤和洗涤或重结晶操作得到纯化的生成物A;
步骤3、脱除N-端Fmoc:将纯化的生成物A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B,H-Tyr(OtBu)-POB;
向生成物B加入极性小的烷烃或醚类溶剂,借助POB辅助基团在溶剂系统中易结晶沉淀的特性,将生成物B与其他杂质分离;
对分离后的生成物B进行过滤和洗涤或重结晶操作得到纯化的生成物B;
步骤4:以纯化的含有辅助基团POB的生成物B作为原料,再与芴甲氧羰基(Fmoc)保护的缬氨酸Fmoc-Val-OH进行偶联反应;重复步骤1~步骤3,得到化合物H-Val-Tyr(OtBu)-POB;
以第一次重复后纯化的生成物H-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的天门冬氨酸Fmoc-Asp(OtBu)-OH进行偶联反应;重复步骤1~步骤3,得到化合物H-Asp(OtBu)-Val-Tyr(OtBu)-POB;
以第二次重复后纯化的生成物H-Asp(OtBu)-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的赖氨酸Fmoc-Lys(Boc)-OH进行偶联反应;重复步骤1~步骤3,得到化合物H-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB;
以第三次重复后纯化的生成物Tyr H-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的精氨酸Fmoc-Arg(Pbf)-OH进行偶联反应;重复步骤1~步骤3,得到胸腺五肽的前体化合物C,,即H-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB;
步骤5、侧链脱保护与POB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,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POB辅助基团,经分离纯化得到胸腺五肽的三氟乙酸盐,[TFA*H-Arg-Lys-Asp-Val-Tyr-OH];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、胸腺五肽的分离纯化:将三氟乙酸通过旋蒸回收,残留溶液用碳酸氢钠中和,调节pH=8~9,用乙酸乙酯萃取并与水相分离,合并乙酸乙酯,沉淀析出,经过滤、乙酸乙酯洗涤,干燥得到纯化的胸腺五肽(TP-5),[H-Arg-Lys-Asp-Val-Tyr-OH]。
一种所述方法制备过程中得到的Fmoc保护酪氨酰-POB化合物,其特征在于:所述Fmoc-Tyr(OtBu)-POB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-POB化合物,其特征在于:所述H-Tyr(OtBu)-POB的分子结构通式为:
一种所述方法制备过程中得到的Fmoc保护缬氨酰-酪氨酰-POB化合物,其特征在于:所述Fmoc-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述方法制备过程中得到的脱Fmoc保护缬氨酰-酪氨酰-POB化合物的结构通式,其特征在于:所述H-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述方法制备过程中得到的Fmoc保护天门冬氨酰-缬氨酰-酪氨酰-POB化合物,其特征在于:所述Fmoc-Asp(OtBu)-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述方法制备过程中得到的脱Fmoc保护天门冬氨酰-缬氨酰-酪氨酰-POB化合物,其特征在于:所述H-Asp(OtBu)-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述方法制备过程中得到的Fmoc保护赖氨酰-天门冬氨酰-缬氨酰-酪氨酰-POB化合物,其特征在于:所述Fmoc-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB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-POB化合物,其特征在于:所述H-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述方法制备过程中得到的Fmoc保护精氨酰-赖氨酰-天门冬氨酰-缬氨酰-酪氨酰-POB化合物,
其特征在于:所述Fmoc-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB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-POB化合物,其特征在于:所述H-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB分子结构通式为:
一种所述基团辅助的胸腺五肽液相的合成中POB辅助基团的回收再利用的方法,其特征在于:将步骤6中合并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POB在不同溶剂系统中易结晶沉淀的特性,可将POB与其他杂质分离;对分离后的POB进行过滤和洗涤或重结晶操作得到纯化的POB,回收后可以直接作为辅助基团再利用。
有益效果
本发明提出的一种基于磷或亚磷酰氧基二苯甲醇及其衍生物及辅助的胸腺五肽液相合成方法。以代固相树脂,在偶联脱水剂的作用下与保护酪氨酸(Fmoc-Tyr(OtBu)-OH)的C-端连接;分离纯化后脱除N-端Fmoc;再依次与侧链保护的缬氨酸[Fmoc-Val-OH]、天门冬氨酸[Fmoc-Asp(OtBu)-OH]、赖氨酸[Fmoc-Lys(Boc)-OH]和精氨酸[Fmoc-Arg(Pbf)-OH]进行偶联和脱Fmoc反应,制备胸腺五肽的前体C[H-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB];侧链脱保护并剪除载体,得到胸腺五肽[H-Arg-Lys-Asp-Val-Tyr-OH]的固体。
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TP-5,而且POB辅助基团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1:本发明基团辅助的胸腺五肽液相合成方法实例流程图
具体实施方式
现结合实施例、附图对本发明作进一步描述:
本发明实施例包括以下步骤:
(1)载体偶联:采用我们发展的新型磷酸酯类载体(POB)替代固相树脂,在偶联脱水剂的作用下与保护酪氨酸(Fmoc-Tyr(OtBu)-OH)的C-端连接;
(2)分离纯化:反应完成后,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A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A;
(3)脱除N-端Fmoc:将生成物A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B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B;
(4)多肽偶联:重复以上步骤(1)、(2)、(3),依次与侧链保护的缬氨酸[Fmoc-Val-OH]、天门冬氨酸[Fmoc-Asp(OtBu)-OH]、赖氨酸[Fmoc-Lys(Boc)-OH]和精氨酸[Fmoc-Arg(Pbf)-OH]进行偶联和脱Fmoc反应,制备胸腺五肽的前体C[H-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB];
(5)侧链脱保护并剪除载体:采用鸡尾酒型脱保护试剂TFA/TIS/H2O=95:2.5:2.5处理胸腺五肽的前体化合物C,脱除侧链上的保护基团tBu、Boc、Pbf以及载体辅助基团POB。反应完毕后,旋蒸回收三氟乙酸(TFA),残留溶液用碳酸氢钠调节pH=8-9,乙酸乙酯萃取并与水相分离,合并有机相回收载体;析出的沉淀,经过滤、洗涤、干燥等操作,即得到胸腺五肽[H-Arg-Lys-Asp-Val-Tyr-OH]的固体。
本发明中一些常用的缩写具有以下含义:
Boc:叔丁氧羰基
DCM:二氯甲烷CH2Cl2
DCC:二环己基碳二亚胺
DEA:二乙胺
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
GPS: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
POB:磷酰氧基苄醇
tBu:叔丁基
TFA:三氟乙酸
THF:四氢呋喃
TIPS:三异丙基硅烷
TP-5:胸腺五肽
本发明适用于胸腺五肽的合成制备与水解脱除,反应原理与技术路线如图1:
实际操作步骤
4-二苯基磷酰氧基二苯甲酮(1)的合成:准确称取4-羟基二苯甲酮(5g,25mmol,1equiv)于250mL的反应瓶中,加入100mL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(4.2mL,30mmol,1.2equiv),量取取二苯基次磷酰氯(5.7mL,30mmol,1.2equiv)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1.5h,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2mL的乙酸乙酯是样品充分溶解,滴加加入正己烷14mL(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(1),产率约93%。测试结果分析:1H NMR(400MHz,CDCl3),δ=7.95-7.90(m,4H),7.76-7.73(m,4H),7.59-7.55(m,3H),7.52-7.44(m,6H),7.36-7.34(d,2H)ppm;31P NMR(162MHz,CDCl3),δ=31.51ppm;13C NMR(100MHz,CDCl3),δ=195.4,154.4,137.5,133.9,132.8,132.4,132.1,131.8,131.7,131.2,129.9,128.8,128.7,128.3,120.5ppm;HRMS(ESI)m/z calcd for C25H20O3P+[M+H]+=399.11446,found 399.11469.
4-二苯基磷酰氧基二苯甲醇(POB,2)的合成:准确称取(1)(800mg,2mmol,1equiv)于100mL的反应瓶中,加入甲醇溶液20mL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0.5mL的乙酸乙酯使样品充分溶解,滴加加入正己烷5mL(VEA/VN-Hexane=1:10),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97%。测试结果分析:1H NMR(400MHz,CDCl3),δ=7.89-7.84(m,4H),7.56-7.52(m,2H),7.48-7.43(m,4H),7.31-7.23(m,7H),7.14-7.12(d,2H),5.74(s,1H),2.99(s,1H)ppm;31P NMR(162MHz,CDCl3),δ=30.58ppm;13C NMR(100MHz,CDCl3),δ=150.0,143.9,140.6,132.5,131.8,131.7,130.1,128.7,128.6,128.4,128.0,127.4,126.6,120.6,75.4ppm;HRMS(ESI)m/z calcd for C25H22O3P+[M+H]+=401.13011,found401.12985.
POB-O-Tyr(OtBu)-Fmoc(A)的合成:准确称取POB(2)(200mg,0.5mmol,1equiv)于100mL的反应瓶中,加入30mL DCM溶解,反应体系依次加入Fmoc-Tyr(OtBu)-OH(276mg,0.6mmol,1.2equiv),4-二甲氨基吡啶DMAP(7.32mg,0.06mmol,0.12equiv),二环己基碳二亚胺DCC(123mg,0.6mol,1.2equiv),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1mL的乙酸乙酯溶解样品,逐滴加入正己烷6mL(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POB-O-Tyr(tBu)-Fmoc(A),产率约95%,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。测试结果分析:1H NMR(400MHz,CDCl3),δ=7.93-7.88(m,4H),7.80-7.78(d,2H),7.59-7.54(m,4H),7.50-7.40(m,6H),7.33-7.20(m,11H),6.87(s,1H),6.82-6.79(d,4H),5.32-5.29(d,1H),4.79-4.74(m,1H),4.44-4.32(m,2H),4.23-4.19(m,1H),3.16-3.10(m,2H),1.34(s,9H)ppm;31P NMR(162MHz,CDCl3),δ=30.86ppm;13C NMR(100MHz,CDCl3),δ=170.6,155.6,154.5,150.5,143.8,141.3,139.1,135.7,132.6,131.8,130.2,129.8,129.1,128.7,128.6,128.1,127.7,127.1,125.1,124.1,120.8,120.0,78.4,67.0,54.8,47.2,37.4,33.9,28.9ppm;HRMS(ESI)m/z calcd for C53H49NO7P+[M+H]+=842.32412,found 842.32422.
POB-O-Tyr(OtBu)-NH2(B)的合成:准确称取POB-O-Tyr(OtBu)-Fmoc(420mg,0.5mmol)于50mL的反应瓶中,加入3mL乙腈溶液,搅拌条件下滴加加入DEA二乙胺1mL(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1mL DCM溶液溶解,加入正己烷4mL震荡出现大量白色固体,静置5min后除去上清液,得到白色脱保护产物,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。测试结果分析:1H NMR(400MHz,CDCl3),δ=7.93-7.88(m,4H),7.55-7.47(m,6H),7.33-7.19(m,9H),7.00-6.97(m,2H),6.88-6.85(m,3H),3.82-3.79(m,1H),3.10-3.03(m,1H),2.87-2.80(m,1H),1.75(s,2H),1.34(s,9H)ppm;31P NMR(162MHz,CDCl3),δ=30.80ppm;13C NMR(100MHz,CDCl3),δ=174.0,154.2,150.6,139.5,136.1,132.6,131.8,131.7,130.2,129.8,128.7,128.6,128.1,127.2,127.1,124.2,120.8,78.3,77.4,55.8,40.1,28.9ppm;HRMS(ESI)m/z calcd for C38H39NO5P+[M+H]+=620.25604,found620.25616.
POB-O-Tyr(OtBu)-Val-Fmoc:准确称取GAP-O-Tyr(OtBu)-NH2(300mg,0.48mmol)于100mL的反应瓶中,加入30mL DCM溶解,反应体系依次加入Fmoc-Val-OH(197mg,0.58mmol,1.2equiv),N-羟基琥珀酰亚胺NHS(67mg,0.58mmol,1.2equiv),二环己基碳二亚胺DCC(120mg,0.58mol,1.2equiv),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1.5mL的乙酸乙酯溶解样品,逐滴加入正己烷9mL(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POB-O-Tyr(OtBu)-Val-Fmoc,产率约95%,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。测试结果分析:1H NMR(400MHz,CDCl3),δ=7.93-7.88(m,4H),7.79-7.77(d,2H),7.63-7.54(m,4H),7.50-7.32(m,11H),7.19-7.18(m,6H),6.83-6.76(m,4H),6.39(s,1H),5.46(s,1H),4.99-4.94(m,1H),4.47-4.34(m,2H),4.26-4.22(m,1H),3.99(s,1H),3.09-3.07(m,2H),2.06-2.04(m,2H),1.30(s,9H),0.86-0.85(d,6H)ppm;31P NMR(162MHz,CDCl3),δ=30.82ppm;13C NMR(100MHz,CDCl3),δ=170.8,170.3,156.3,154.5,150.5,143.8,141.3,139.1,135.6,132.6,131.8,130.2,129.7,129.2,128.7,127.7,127.1,126.8,125.1,124.1,120.7,120.0,78.4,67.1,60.2,53.1,47.2,37.1,33.9,31.1,28.9,19.1,17.8ppm;HRMS(ESI)m/z calcd for C58H58N2O8P+[M+H]+=941.39253,found941.39270.
POB-O-TP-5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DCC/HOSU(NHS)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:
POB-O-Tyr(OtBu)-Val-NH2,产率约97%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.91-7.86(m,4H),7.78-7.74(m,1H),7.54-7.50(m,2H),7.47-7.44(m,4H),7.31-7.22(m,4H),7.18-7.17(m,4H),6.92-6.88(m,2H),6.81-6.78(m,3H),5.25(s,1H),4.99-4.94(m,1H),3.18-3.16(m,2H),2.98-2.92(m,1H),2.19-2.15(m,1H),1.48(s,2H),1.30(s,9H),0.88-0.86(d,3H),0.65-0.64(d,3H)ppm;31P NMR(162MHz,CDCl3),δ=30.79ppm;13C NMR(100MHz,CDCl3),δ=174.2,170.9,154.3,150.7,139.2,135.9,132.6,131.7,130.8,130.2,129.6,129.0,128.7,128.6,128.5,128.2,128.0,127.4,126.9,124.1,120.8,78.3,77.4,60.0,52.7,37.4,30.7,28.8,19.6,16.0ppm;HRMS(ESI)m/zcalcd for C43H48N2O6P+[M+H]+=719.32445,found 719.32471.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Fmoc,产率约92%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.92-7.87(m,4H),7.79-7.77(d,2H),7.62-7.17(m,21H),6.94-6.81(m,6H),6.47-6.41(m,1H),6.15-6.02(m,1H),4.94-4.92(m,1H),4.51-4.41(m,3H),4.27-4.19(m,2H),3.51(s,1H),3.10-2.99(m,2H),2.91-2.84(m,1H),2.71-2.70(m,1H),1.44(s,9H),1.33(s,9H),0.86-0.85(d,3H),0.78-0.77(d,3H)ppm;31P NMR(162MHz,CDCl3),δ=30.78ppm;13C NMR(100MHz,CDCl3),δ=171.3,170.6,170.3,156.2,154.4,150.8,143.8,141.3,139.2,135.8,132.6,131.8,131.7,130.2,129.7,129.1,128.7,128.6,127.8,127.5,127.1,126.9,125.1,124.2,120.8,120.0,82.0,78.4,67.4,58.6,53.2,51.4,49.1,47.1,37.2,37.0,33.9,28.9,28.0,25.6,24.9ppm;HRMS(ESI)m/z calcd for C66H71N3O11P+[M+H]+=1112.48207,found 1112.48279.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-NH2,产率98%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.90-7.85(m,4H),7.52-7.45(m,6H),7.28-7.15(m,8H),6.83-6.74(m,6H),5.26(s,1H),4.93-4.90(m,1H),4.24-4.20(m,1H),3.59-3.56(m,1H),3.06-2.93(m,2H),2.80-2.75(m,1H),2.54-2.47(m,1H),2.11-1.94(m,4H),1.43(s,9H),1.30(s,9H),0.88-0.80(d,6H)ppm;31P NMR(162MHz,CDCl3),δ=30.73ppm;13C NMR(100MHz,CDCl3),δ=173.7,171.1,170.8,170.4,154.3,150.7,139.1,135.8,132.6,131.8,131.7,130.4,130.3,130.2,129.8,129.1,128.7,128.6,128.5,128.3,128.0,127.6,126.9,124.1,120.8,81.1,78.3,58.1,53.1,52.2,40.3,37.1,33.9,30.7,28.9,28.1,19.2,17.8ppm;HRMS(ESI)m/z calcd for C51H61N3O9P+[M+H]+=890.41399,found 890.41425.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Lys(Boc)-Fmoc,产率约90%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.92-7.87(m,4H),7.78-7.76(d,2H),7.66-7.17(m,21H),6.93-6.78(m,6H),6.07-6.00(m,1H),4.97-4.90(m,2H),4.68-4.19(m,6H),3.50(s,1H),3.17-2.83(m,5H),2.68-2.60(m,1H),2.24-2.20(m,2H),1.94-1.90(m,2H),1.71-1.65(m,2H),1.45(s,9H),1.34-1.30(m,18H),1.15-1.03(m,2H),0.82-0.74(m,6H)ppm;31P NMR(162MHz,CDCl3),δ=30.71ppm;13C NMR(100MHz,CDCl3),δ=172.2,171.4,170.4,170.3,154.2,150.4,140.0,143.6,141.3,139.4.136.0,132.6,131.8,131.7,130.2,129.7,129.0,128.7,128.6,128.5,128.0,127.7,127.3,127.1,127.0,125.1,124.2,120.7,120.0,82.0,78.3,77.4,67.3,60.4,58.7,53.3,49.0,47.1,39.5,37.0,33.9,29.7,28.8,28.5,27.9,25.7,25.0,22.4,21.1,19.2,14.2ppm;HRMS(ESI)m/z calcd for C77H91N5O14P+[M+H]+=1340.62947,found 1340.62927.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Lys(Boc)-NH2,产率约95%,测试结果分析:1HNMR(400MHz,CDCl3),δ=8.30-8.25(m,1H),7.91-7.86(m,4H),7.54-7.46(m,6H),7.32-7.17(m,9H),6.97-6.94(m,1H),6.89-6.78(m,5H),6.62(s,1H),4.92-4.90(m,1H),4.72-4.69(m,2H),4.22-4.20(m,1H),3.39-3.36(m,1H),3.13-2.99(m,4H),2.77-2.72(m,2H),2.17(s,2H),1.84-1.69(m,5H),1.44-1.41(d,18H),1.31(s,9H),0.91-0.87(m,2H),0.83-0.82(m,3H),0.73-0.72(m,3H)ppm;31P NMR(162MHz,CDCl3),δ=30.72ppm;13C NMR(100MHz,CDCl3),δ=171.0,170.6,170.4,170.3,156.1,154.4,139.2,135.8,132.5,131.8,131.7,131.6,130.5,130.2,129.7,129.0,128.7,128.6,128.5,128.2,128.0,127.5,126.9,124.1,120.7,81.7,79.0,78.3,58.6,55.0,53.2,49.5,40.1,36.8,34.5,31.6,30.2,29.9,28.8,28.4,28.0,22.8,19.1,17.3,14.1ppm;HRMS(ESI)m/z calcd for C62H81N5O12P+[M+H]+=1118.56139,found 1118.56189.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Lys(Boc)-Arg(Pbf)-Fmoc,产率约91%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.86-7.83(m,4H),7.77-7.75(d,2H),7.61-7.19(m,19H),7.13-7.09(m,2H),7.01-6.96(m,2H),6.85-6.81(m,2H),6.74-6.71(m,1H),6.63-6.51(m,1H),6.27(s,1H),5.97(s,1H),5.30(s,1H),4.99-4.69(m,3H),4.46-4.35(m,4H),4.20-4.11(m,2H),3.52-3.44(m,1H),3.20-2.92(m,7H),2.63-2.55(m,7H),2.27-2.18(m,2H),2.09-2.06(m,4H),1.92-1.55(m,9H),1.43-1.26(m,36H),1.15-1.04(m,3H),0.82-0.81(m,3H),0.72-0.64(d,3H)ppm;31P NMR(162MHz,CDCl3),δ=31.44ppm;13C NMR(100MHz,CDCl3),δ=171.1,170.6,170.4,158.7,157.1,156.2,154.2,150.1,143.7,141.2,138.4,132.8,132.3,131.7,131.6,129.6,128.9,128.7,128.0,127.7,127.2,127.1,125.2,124.6,124.2,120.7,120.6,119.3,117.5,86.3,81.7,78.8,78.4,67.1,60.4,53.5,50.0,49.0,47.1,43.3,40.0,36.6,33.9,29.7,29.4,28.8,28.4,28.0,25.6,25.0,23.2,21.1,19.4,19.1,18.1,14.2,12.5ppm;HRMS(ESI)m/z calcd for C96H119N9O18PS+[M+H]+=1748.81259,found 1748.81274.
POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Lys(Boc)-Arg(Pbf)-NH2(C),产率约96%,测试结果分析:1H NMR(400MHz,CDCl3),δ=7.98(s,1H),7.89-7.84(m,4H),7.71-7.65(m,1H),7.56-7.48(m,6H),7.29-7.12(m,10H),6.93-6.91(m,2H),6.82-6.74(m,3H),6.41-6.33(m,3H),4.97-4.67(m,3H),4.42-4.20(m,2H),3.47-3.45(m,1H),3.14-2.77(m,10H),2.61-2.54(m,6H),2.10-1.86(m,9H),1.73-1.57(m,5H),1.46-1.42(m,18H),1.38-1.35(d,9H),1.31-1.30(d,9H),0.81-0.79(d,3H),0.73-0.71(d,3H)ppm;31P NMR(162MHz,CDCl3),δ=31.21ppm;13C NMR(100MHz,CDCl3),δ=172.1,171.4,170.8,170.7,170.5,170.4,158.6,156.5,156.3,154.2,150.3,139.3,138.3,136.1,132.7,132.2,131.7,131.6,130.9,129.6,128.8,128.7,128.6,128.5,128.2,128.0,127.3,127.0,124.5,124.1,120.7,117.4,86.3,81.9,79.2,78.4,53.4,49.7,43.3,40.2,36.7,29.5,28.8,28.6,28.5,28.0,25.4,23.0,19.3,19.1,18.0,17.5,17.4,14.1,12.5ppm;HRMS(ESI)m/z calcd forC81H109N9O16PS+[M+H]+=1526.74451,found 1526.74438.
胸腺五肽H-Arg-Lys-Asp-Val-Tyr-OH(TP-5)的分离与纯化:侧链保护基团tBu、Boc、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POB-O-Tyr(OtBu)-Val-Asp(OtBu)-Lys(Boc)-Arg(Pbf)-NH2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GPS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(2),回收产率约为50%;离心后得到白色胸腺五肽TP-5三氟乙酸盐固体(H-Arg-Lys-Asp-Val-Tyr-OH·TFA),产率约为93%,MS测试显示[M+H]+=680.62。测试结果分析:1H NMR(400MHz,D2O),δ=7.07-7.05(d,2H,J=8Hz),6.76-6.74(d,2H,J=8Hz),4.61-4.54(m,2H),4.29-4.25(m,1H),4.02-3.98(m,2H),3.17-3.08(m,3H),2.93-2.68(m,6H),1.93-1.57(m,10H),1.40-1.32(m,2H),0.77-0.74(m,6H)ppm;13C NMR(100MHz,D2O),δ=174.6,173.8,173.0,172.6,171.6,169.4,156.7,154.3,130.5,128.4,115.4,59.3,54.0,53.7,52.5,50.1,40.4,39.1,35.8,35.3,30.5,30.2,28.0,26.3,23.4,22.0,18.3,17.4ppm;MS(ESI)m/z calcd for C30H50N9O9[M+H]+=680.37,found 680.62.
Claims (11)
1.一种采用磷酰氧基苯甲醇POB进行基团辅助的胸腺五肽液相的合成方法,其特征在于,步骤如下:
步骤1、辅助基团与氨基酸的偶联:以辅助基团替代固相多肽合成中的树脂,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,得到生成物A;所述氨基酸与POB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POB,分子结构通式为:
所述氨基酸采用芴甲氧羰基Fmoc保护的酪氨酸Fmoc-Tyr(OtBu)-OH,受保护的酪氨酸Fmoc-Tyr(OtBu)-OH的C-端与POB辅助基团连接,生成化合物A,Fmoc-Tyr(OtBu)-POB;
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POB辅助基团在溶剂系统中易结晶沉淀的特性,将生成物A与其他杂质分离;
对分离后的生成物A进行过滤和洗涤或重结晶操作得到纯化的生成物A;
步骤3、脱除N-端Fmoc:将纯化的生成物A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B,H-Tyr(OtBu)-POB;
向生成物B加入极性小的烷烃或醚类溶剂,借助POB辅助基团在溶剂系统中易结晶沉淀的特性,将生成物B与其他杂质分离;
对分离后的生成物B进行过滤和洗涤或重结晶操作得到纯化的生成物B;
步骤4:以纯化的含有辅助基团POB的生成物B作为原料,再与芴甲氧羰基(Fmoc)保护的缬氨酸Fmoc-Val-OH进行偶联反应;重复步骤1~步骤3,得到化合物H-Val-Tyr(OtBu)-POB;
以第一次重复后纯化的生成物H-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的天门冬氨酸Fmoc-Asp(OtBu)-OH进行偶联反应;重复步骤1~步骤3,得到化合物H-Asp(OtBu)-Val-Tyr(OtBu)-POB;
以第二次重复后纯化的生成物H-Asp(OtBu)-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的赖氨酸Fmoc-Lys(Boc)-OH进行偶联反应;重复步骤1~步骤3,得到化合物
H-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB;
以第三次重复后纯化的生成物H-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB作为原料,再与芴甲氧羰基(Fmoc)保护的精氨酸Fmoc-Arg(Pbf)-OH进行偶联反应;重复步骤1~步骤3,得到胸腺五肽的前体化合物C,即H-Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(OtBu)-POB;
步骤5、侧链脱保护与POB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,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POB辅助基团,经分离纯化得到胸腺五肽的三氟乙酸盐,[TFA*H-Arg-Lys-Asp-Val-Tyr-OH];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、胸腺五肽的分离纯化:将三氟乙酸通过旋蒸回收,残留溶液用碳酸氢钠中和,调节pH=8~9,用乙酸乙酯萃取并与水相分离,合并乙酸乙酯,沉淀析出,经过滤、乙酸乙酯洗涤,干燥得到纯化的胸腺五肽(TP-5),[H-Arg-Lys-Asp-Val-Tyr-OH]。
11.一种权利要求1所述基团辅助的胸腺五肽液相的合成中POB辅助基团的回收再利用的方法,其特征在于:将步骤6中合并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POB在不同溶剂系统中易结晶沉淀的特性,可将POB与其他杂质分离;对分离后的POB进行过滤和洗涤或重结晶操作得到纯化的POB,回收后可以直接作为辅助基团再利用。
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