CN101166761A - 肽合成法 - Google Patents
肽合成法 Download PDFInfo
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- CN101166761A CN101166761A CNA2006800146153A CN200680014615A CN101166761A CN 101166761 A CN101166761 A CN 101166761A CN A2006800146153 A CNA2006800146153 A CN A2006800146153A CN 200680014615 A CN200680014615 A CN 200680014615A CN 101166761 A CN101166761 A CN 101166761A
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- resin
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- ser
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Abstract
本发明涉及一种用于胸腺素α1的固相合成的方法。
Description
本发明涉及一种用于通常难以合成的肽的固相肽合成法,并涉及代表性的肽-固相偶联物。
固相肽合成法的一项挑战是尽管已有建立好的例行方法存在,但是成功和有效率的合成则高度视该待合成肽的序列而定。
在对于许多‘正常’肽而言被高度成功使用之相同树脂材料上,取自于天然肽或蛋白质区段的某些肽序列就是比其他肽序列之表现远差。在文献中,鉴于防止因形成β片层所造成树脂上的链内集结作用,已提出要经常小心地对溶剂系统进行微调。此外,使用二氯甲烷、N-甲基吡咯烷酮或二甲基甲酰胺,最后佐以稳定螺旋体结构的特殊极性溶剂如三氟乙醇,对难合成的肽而言一点也无法提供普遍的解决方法。
胸腺素α1(国际非专有医药名称:胸腺法新)是一种在人体胸腺产生的N-乙酰化的28聚体肽激素,并且被用作为治疗慢性乙型肝炎的药物。
其直链全长固相合成早就已经被说明过,但是至今仍受产率不佳之苦。很不幸的,胸腺素α1的氨基酸序列并未提供在逐步合成法的传统阶段偶联方法中可用作为间隔残基的甘氨酸或脯氨酸。
Echner等人(Liebigs Ann.Chem.,1988,1095-1097)描述了在Wang-聚苯乙烯树脂上进行的直链固相合成。虽然其宣称该偶联效率在使用溶于DMF的BOP试剂进行的每一偶联作用后都用Kaiser试验检查过,但其仅在每五次偶联作用之后才利用乙酰化作用对未偶联的肽加帽。其使用浓三氟乙酸将受保护以及末端被乙酰化的肽从树脂上切下来;而后使用过滤法将树脂和液相分开并且用二乙醚进行沉淀作用以收集沉淀物。定量以此方式所收集到的沉淀物,并标注以‘粗产物’,计算其产率达76%。在此后才对该粗产物进行第一次色谱纯化,接着进行反向HPLC。由此获得的纯产物的产率则并未被指出。
使用Echner法的问题在于,在产率计算时未考虑在合成时导致缩短的不希望的序列变体的不完全链偶联。‘粗产物’等于合成期间获得的每一种肽,包括未完全去保护的/烷基化的肽。然而,当牵涉到难以合成的肽时,得自于线性合成的肽的纯度就成了一项问题;有趣的是,Echner并未指出任何因此获得的纯化产物的产率,也并未在其发表文章中给予如技术领域中会例行地完成的任何HPLC色谱图以证明该合成的品质。在我们的操作实践中,Echner的合成流程只能产生非常低产率的正确产物,各种不正确的肽产物则构成该粗产物的大部分。
还有一项作者本人提到的与Echner的方法有关的问题(第1095页,左栏,第3段)是将胸腺素α1的C-端氨基酸一其为天冬酰胺,装载到Wang树脂;Echner所达到的装载量是0.15毫摩尔/克,此为极低的装载量。Fmoc-Asn(4,4’-二甲氧基二苯基甲基-)-OH氨基酸必须被用于装载该树脂才不会产生降解副反应的风险。
本发明的目标是要设计一种改进的胸腺素α1的固相合成法。此项目标由权利要求1的方法来解决。
根据本发明,提出了一种在固相上合成胸腺素α1或者包含成熟胸腺素α1残基1-27的C-端截短形式、至少包含成熟胸腺素α 1残基19-28的N-端截短形式、或至少包含成熟胸腺素α1残基19-27的截短形式的方法,所述方法包括以下步骤:
a.将适当保护的FMOC-Glu或FMOC-Asn残基偶联到PEG树脂上,其中PEG树脂优选选自下组:聚苯乙烯-PEG混合树脂、PEG聚醚-聚酯混合树脂、或PEG聚醚-聚酰胺混合树脂以及基本上纯的PEG树脂,
b.通过FMOC合成法延长肽链,其中个别氨基酸的侧链可用适当的保护基加以衍生,
c.任选将最末一个N-端残基乙酰化,并且
d.将肽从树脂上切下。
本发明能在单一固相线性合成法中以无前例的纯度和从而得到的高产率合成胸腺法新。根据本发明,也可以在固相上仅合成C-端片段,并通过区段偶联的方式组合任何全长的肽或至少该肽的较长形式,在一项优选实施方式中是利用偶联N-端片段,该N-端片段具有C-端Ser或Thr,其经假脯氨酸二肽法保护以防止消旋(W_hr等人,J.Am.Chem.Soc.,118,9218)。
较好是待合成的肽是具有以下序列的胸腺素α1(为区别于前体肽也成为成熟的活性胸腺素α1):
1-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-28
其中的个别氨基酸链是未保护的,或如本领域的常识当给定的个别氨基酸残基需要时予以适当保护(请参考例如Bodansky,如下)。更优选地,这种肽在一个紧接于线性合成、但在从树脂上切下之前的步骤中经N-端乙酰化。亦被用作药物的天然胸腺法新(tymalfasin)即被N-端乙酰化。
应注意,本发明的发明人已发现主要是胸腺法新肽的C-端部分(大约是残基19-28)因链删除作用而导致大部分的杂质并因此造成在线性相合成期间产生的终产物产率的损失。该肽的此部分已经被发现极度难以合成。
反之亦然,在线性合成期间,使用这种如W_hr等人(上述)所述的_唑烷二肽亦可行。使用这种二肽而不使用受到(较好是Fmoc)保护的个别氨基酸的好处可由胸腺法新的序列推知,尤其是对于Asp-Thr二肽而言;使用_唑烷二肽衍生物在本发明中能帮助避免合成期间形成天冬酰亚胺。
类似的,防止戊二酰亚胺形成可能需要使用到特殊保护基。
虽然本发明强烈优选使用Fmoc化学进行偶联反应,但是Boc化学类似地在另一实施方式中可被用于执行本发明。
肽合成所使用之偶联剂在本领域中是已经熟知的(见Bodansky,M.的《肽合成原理》(Principles of Peptide Synthesis),第二版,Springer Verlag Berlin/德国海德堡,1993;也可参见其中关于偶联添加物或佐剂作用的讨论)。偶联剂可为混合的酸酐(例如T3P:丙烷膦酸酐)或其他酰化剂如活化的酯类或酸性卤化物(例如ICBF,氯甲酸异丁酯),或其可为碳二亚胺(例如1-乙基-3-(3-二甲氨基丙基)-碳二亚胺)、活化的苯并三嗪衍生物(DEPBT:3-(二乙氧基磷酰氧基)-1,2,3-苯并三嗪-4(3H)-酮)或苯并三唑的尿鎓盐或鏻盐衍生物。
鉴于最佳产率、短的反应时间、与防止链延伸期间的消旋作用,更优选该偶联剂选自能够活化游离羧酸官能团的苯并三唑的尿鎓盐或鏻盐,并且该反应在碱存在下进行。这种尿鎓或鏻偶联盐类的适当的和相似的优选实例为例如:HBTU(O-1H-苯并三唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯)、BOP(六氟磷酸苯并三唑-1-基-氧基-三-(二甲基氨基)鏻)、PyBOP(六氟磷酸苯并三唑-1-基氧基-三吡咯烷基鏻)、PyAOP、HCTU(O-(1H-6-氯-苯并三唑-1-基)-1,1,3,3-四甲基脲六氟磷酸酯)、TCTU(O-1H-6-氯苯并三唑-1-基)-1,1,3,3-四甲基脲四氟硼酸酯)、HATU(O-(7-偶氮苯并三唑-1-基)-1,1,3,3-四甲基脲六氟磷酸酯)、TATU(O-(7-偶氮苯并三唑-1-基)-1,1,3,3-四甲基脲四氟硼酸酯)、TOTU(O-[氰基(乙氧羰基)亚甲基氨基]-N,N,N’,N’-四甲基脲四氟硼酸酯)、HAPyU(O-(苯并三唑-1-基)氧基双-(吡咯烷基)脲六氟磷酸酯)。
优选是在使用DEPBT或类似的尿鎓盐或鏻盐试剂时,需要有再一种或第二种弱碱试剂来进行该偶联步骤。这是通过共轭酸具有7.5-15,更优选7.5-10的pKa值的碱来配合的,但肽或氨基酸或氨基酸衍生物的α氨基官能团除外,并且该碱优选为立体结构上受到阻碍的叔胺。此类及其更佳的实例为Hünig-碱(N,N-二异丙基乙胺)、所具有的烷基为直链或支链C1-C4烷基的N,N’-二烷基苯胺、2,4,6-三烷基吡啶、或N-烷基吗啉,更优选为N-甲基吗啉或可立啶(2,4,6-三甲基吡啶),最优选为可立啶。
使用偶联添加物尤其是苯并三唑型偶联添加物也是已知的(请参考Bodansky,同上)。当使用高度活化、之先前说过的尿鎓盐或鏻盐偶联剂时,使用其等尤佳。因此更佳系该偶联剂添加物系能够形成活化酯的亲核的羟基化合物,更佳是具有酸性的、亲核的N-羟基官能者,其中N是酰亚胺或是N-酰基或N-芳基取代的三氮烯,最优选系该偶联添加物为N-羟基-苯并三唑衍生物(或1-羟基-苯并三唑衍生物)或为N-羟基-苯并三嗪衍生物。这种偶联添加物N-羟基化合物已被大量且广泛地说明在WO94/07910和EP-410182中,且其个别的揭示内容则以引用方式掺入本文中。实例为例如N-羟基-琥珀酰亚胺、N-羟基-3,4-二氢-4-氧-1,2,3-苯并三唑(HOOBT)、1-羟基-7-氮杂苯并三唑(HOAt)和N-羟基-苯并三唑(HOBt)。特别优选N-羟基苯并三嗪衍生物,首先且尤其是HOOBt。
偶联添加物的铵盐化合物是已知的,且其在偶联化学上的用途已经在例如US4806641中描述过。
在一项更尤其优选的实施方式中,该尿鎓盐或鏻盐偶联剂是一种尿鎓盐试剂,优选其为HCTU、TCTU或HBTU,更优选其为HCTU或TCTU,且最优选是与N-羟基-3,4-二氢-4-氧-1,2,3-苯并三嗪或其盐一起用于反应中。
在本发明的上下文中,应注意到HCTU和TCTU被定义涵盖于术语‘尿鎓盐试剂’中,尽管利用晶体结构分析这些化合物和可能的类似物已经显示其包含异亚硝基部分而非尿鎓基部分(O.Marder,Y.Shvo和F.Albericio,《HCTU和TCTU:新颖的偶联剂:研发与工业应用》(HCTUand TCTU.:New Coupling Reagents:Development andIndustrial Applications),Presentation Gordon Conference 2002-02和Chimica Oggi 2002,20:37-41),该部分是一种由于胍结构取代而在杂环核心上形成的N-脒基取代基。在本说明书之上下文中,此类化合物被称作本发明所述的尿鎓盐试剂的‘胍型亚类’。
可用本领域的常规方法对碱不稳定性Nα进行去保护,例如在Fmoc化学的情形中使用溶于N-甲基吗啉的20%哌啶来实行。最后一个待添加的氨基酸,例如通常为成熟胸腺法新的N-端丝氨酸,当然可以携带一个除Fmoc以外的N-端保护基,例如其可携带Boc或方便地携带一个乙酰基保护基,虽然后者通常可通过偶联后使该肽去保护的N端氨基酸乙酰化而能较方便地被导入。优选是可使用除Fmoc外的正交保护基以保护最后的N-端残基。实例为Alloc保护基(例如用于保护伯胺),都可通过Pd(0)催化的转酰化作用从肽上选择性移除(Gomez-Martinez,《在固相肽合成中Nα-Alloc的暂时保护作用一使用胺-硼烷复合物做为丙烯基清除剂》(Nα-Alloc temporaryprotection in solid-phase peptide synthesis,use of amine-borane complexes as allyl groupscavengers),J.Chem.Soc.Perkin Trans.,1,1999,2871-2874)、Dde基团(Bycroft等人,《一种新颖之用于分支肽之SPPS的离胺酸保护方法》(A novel lysine protectingprocedure for SPPS of branched peptides),J.Chem.Soc.Chem.Commun.,1993,778-779)、可通过肼解作用移除的双甲酮(dimedon)衍生物、及其功能性同源物如Nde基团(N-1-(4-硝基-1,3-二氧茚满-2-基)-乙基,Kellam等人,Tetrahedron,54,1998,6817-6832;N-1-(4,4-二甲基-2,6-二氧环亚己基)-3-甲基丁基,Chan,1995)。Dde、其衍生物及同源物都是Dde型保护基,它们在未处理的游离状态时共同具有式III所示的二氧亚烷基官能团部分:
III (-CO)2C=C(-R)(-OH)
其中R是取代的或经取代的烷基,并优选两个羰基官能团形成一个由-CH2-CR’CR”-CH2-、-NR’-CO-NR”或-CR’=CR”-骨架连接的环状结构。优选是R’、R”为烷基,或是连接在一起成为芳基。
使用经过修饰或标记的正交保护基也很容易实行,其另外的好处为能够通过紧接的亲和层析或使用这种可逆亲和标记的亲和力经过改进的‘标准’RP HPLC进行简易纯化。在此情形中,所合成的肽是适合地在避免整体去保护的温和条件下从树脂上切下、进行纯化、选择性的将N-端的探针移除以允许紧接的N-端乙酰化作用,和最终使该肽整体去保护。一项这种实例为Ball等人在Int.J.Peptide Protein Res.,1992,40:370-379和Ball等人在J.Chromatography,1994,686:73-88中描述的Fmoc衍生物。
对于本发明而言,特别优选用20%哌啶-DMF(v/v)来进行Fmoc基团的移除,例如2×1分钟,2×10分钟,1×5分钟。类似优选的是,Fmoc-aa-OH(5当量)的偶联是用以上提到的偶联剂在DMF或类似溶剂中进行60-120分钟。为了使合成以及尤其对个别序列位置的偶联时间达到最优选,因此在偶联之后进行茚三酮试验,若试验结果为阳性,则在同样的条件下重复偶联作用,否则就继续进行该方法。若在第二次偶联作用之后,茚三酮试验仍为阳性,则用Ac2O和DIEA或类似方法进行乙酰化步骤以替删除之肽链加帽。然后在相同的条件下将这种改进的偶联和/或加帽法应用到特定给定序列位置的重复合成上。值得注意的是,我们发现树脂的选择会强烈影响如在实验部分所举例的特定于序列位置的偶联问题。
根据本发明,固相包含了PEG树脂。根据本发明,其等能使胸腺法新或胸腺法新的难以合成的核心肽部分以无前例的产率和纯度被合成。在本说明书的上下文中,‘固相’的定义包括(除了惰性树脂基质以外)在惰性基质材料上存在以连接肽或是与进一步的接枝接头或把手连接的整合接头。
这种树脂已经被说明在例如美国专利案第2003078372A1号中,借着掺入固相中的PEG,这种树脂获得使其异于传统树脂材料的两性性质。已知它们在膨胀之后具有一种‘类似胶体’的行为而非传统上例如Merrifield树脂之类似固体的行为。这种特别的特性被认为能因为例如洗涤、偶联、去保护步骤等等期间之扩散/液体交换而提供降低循环时间的好处。值得注意的是,根据本发明这种PEG树脂也被发现能显著增进困难且具肽特定性之肽偶联步骤的效率,并且以未曾预期的协同促进的方式增进纯度和产率。‘PEG树脂’一词在本说明书的上下文中普遍地应理解为至少包含一种聚醚共聚性的或嵌段共聚物部分(polymer share)。此型树脂有不同的实施方式存在,碳单体(包括环氧乙烷之外的单体)的聚合作用并不仅限于聚醚化学。因而,该固相的树脂基质可由例如两性的聚苯乙烯-PEG混合树脂所组成(例如天塔胶(Tentagel),请参考美国专利案第4908405号)或PEG-聚酰胺或PEG-聚酯之混合树脂,例如Kempe等人,J.Am.Chem.Soc.,1996,118,7083;亦于美国专利案第5,910,554A号。通常,树脂的装载较无效率且/或化学稳定性(尤其是在酸性基质中者)经常无法与传统的树脂-例如传统的Merrifield聚苯乙烯-DVB相比。聚苯乙烯-PEG的混合树脂能达到较高的装载量,但却得到较低的两性性质,因为PEG的含量降低。
亦可使用非PEG树脂基质与一层适当的PEG树脂材质接枝所得到的PEG树脂,例如Kates等人,《用于固相合成之高装载量之聚乙二醇-聚苯乙烯(PEG-PS)接枝支持物》(High-load polyethylene glycol-polystyrene(PEG-PS)graft supports for solid-phasesynthesis),Biopolymers,1998,47:365-380。
在一项根据本发明的极佳的实例中,PEG树脂是一种基本纯的PEG-聚醚树脂,其较好是不含任何(最终进一步被取代)聚苯乙烯共聚性或嵌段共聚性部分,且更佳是其亦尚且不包含任何内部的酯或酰胺官能团而仅有聚醚官能团。最优选是其为纯的PEG树脂或PEG-聚醚树脂。优选地,‘基本纯的’的意义为以上纯的PEG树脂的结构定义达到固相材质干重的至少70%,使其他类型聚合物添加占较少部分。这种理想的纯PEG树脂材质提供最优选的化学稳定性和良好的相容性,且在一般的Fmoc合成中容易用标准的溶剂处理。值得注意的是在较后的定义中,直接或经由整合和/或接枝接头使树脂与肽桥接的末端酯或酰胺键结并未计入其中,因为它们不是内部结构、交联的特性,因而在这方面不会影响‘纯的PEG’的定义。这种‘纯的’、高度两性的PEG树脂由不同的公司所提供,例如加拿大魁北克省的Matrix InnovationsInc.(‘ChemMatrix’品牌)或丹麦的VersamatrixA.S.,并且说明在WO02/40559中。它们可提供大于0.5毫摩尔/克的装载容量,请参考例如先前所说之WO’559中说明的ChemMatrix品牌树脂。为了连接肽,这种树脂可具有末端整合接头,如羟甲基或从其衍生的例如‘准天然’氨基甲基、羧基或溴甲基或碘甲基,或其可用已知的固相连接的整合或接枝接头或把手(handle)衍生,例如Wang、PAL、4-烷氧基-苯甲醛或Rink或Sieber酰胺接头。
值得注意的是,根据本发明的树脂还有一项令人惊异的性质要素,那就是在与传统的树脂(例如PS-DVB或CTC-PS-DVB)比较时,第二氨基酸第27号的Glu的偶联骤然被发现成为严重困难的步骤,尤其是当使用侧链锚定末端的Asp/Asn残基时如此,然而在序列19-28之其他重要残基的偶联效率则被发现有增进,但却未导致无价值物。因而当使用PEG树脂且尤其是纯的或基本纯的PEG-聚醚树脂时,延长和/或重复偶联第27号残基到固定在树脂上的Asn部分是需要的。‘延长’系关于相较于平均的残基而言使用更多当量之Fmoc氨基酸或较长的偶联时间。
本发明进一步的目标为以此方式获得的各种肽-固相偶联物。先前所提出的定义与解释同样适用于这些目标。
这种目标为式I的肽偶联物:
R1-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn_R2_xR3
I
其中,每一个氨基酸残基都单独被保护或者未保护,R3是包含整合接头的PEG树脂固相,R2是接枝的接头,x是0或1,R1是氢、保护基或肽基,优选数目小于50而更优选少于25个氨基酸残基的肽基,且其中如果R1是肽基,则所述基团上的各个氨基酸残基单独被保护或未保护,且所述基团的N端是游离的、被乙酰化或被保护基Y保护。
因此,优选R1是以下肽残基:Y-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-、Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu或H-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Leu-Lys-Glu,Y具有以上所提到的意义。
由Guillier、Orian和Bradley,Chem.Rev.,100,2091-2157,2000所定义的接头被仅仅视为固定化的保护基,并且被分类成两种型式中的一种:(a)整合接头(Integrallinker),其中固相核心材质部分形成或即为接头不可分开的部分,两者共同组成R3,和(b)非整合或接枝接头(Nonintegral or grafted linker)R2,其中该接头另外连接到位在整合接头/固相R3上的固体支持物。整合接头的实例为对-甲基二苯甲胺(MBHA)、2-氯三苯甲基(CTC)、氨基-甲基等。整合接头是必须的而接枝的接头R2是任选的。接枝接头的典型例子为4-羟基甲基苯氧基乙酰基-或4-羟基甲基苯甲酸。
通常,该肽是经由其C端残基通过其1-羧酸官能团分别连接到固相或接枝接头。
优选地,根据本发明,胸腺法新或其先前提过的片段和变体的合成系藉倒数第二个Glu或C端Asp或Asn残基之侧链锚定来达成。‘倒数第二个Glu’在本说明书的上下文中可意指一种固定在固相上之Glu-Asn被保护的二肽,或其可意指一种不含天然胸腺法新C端Asn之胸腺法新变体的合成是从该Glu残基开始的。没有预期到的是,侧链的锚定被发现会造成更有效率的合成。从树脂上切下之后,可对以其β-羧基官能团固定到固相或接枝接头之末端Asp残基进行合成后的酰胺化。然而更佳地,C羧基官能团经适当保护(优选通过叔丁基保护基)的Fmoc-Asp是侧链锚定到一个能产生酰胺的接头,优选能产生酰胺的接枝接头上;就从树脂上切割后的最终产物而言,可考虑这种偶联物,且由于使用了能产生酰胺的结构而被称为‘侧链锚定的Asn’。这种能产生酰胺的结构的例子和尤其优选的实施方式是,例如‘Rink酰胺’4-(2’,4’-二甲氧基苄基-氨基甲基)苯氧基-树脂、Sieber树脂(Tetrahedron Lett.,1987,28,2107-2110)或类似的9-氨基-呫吨基-型树脂、PAL树脂(Albericio等人,1987,Int.J.Pept.Protein Research,30,206-216)。能产生酰胺的接头不太优选的例子是如Meisenbach等人(1997,Chem.Letters,1265开始)所述的经过特别取代的三苯甲胺衍生物。这些是(Fmoc化学相容性)接头基团的实例,在肽从树脂被切下时能从该等基团会产生或释放出Cα-羧酰胺。不用说,这种酰胺接头的使用当然要视所实行的固相合成法的型式而定,亦即其为传统的Boc或为目前常用之用于偶联的正交Fmoc保护化学,即(例如)是否系传统Boc或用于于偶联之目前惯用的、正交Fmoc保护化学;一种特定于Boc的酰胺树脂接头是PAM。因此,在本说明书的上下文中包含这种接头基团的固相即称为‘能产生酰胺的固相’。
利用偶联剂将Fmoc-Asp-OtBu合并到含有一种或两种接头的固相上(Albericio,van Abel,Barany,Int.J.Peptide Protein Res.,35,284-286,1990)已经被说明并且可以适用于本说明书的上下文。通常,尿鎓型偶联剂对于达成此而言为优选者。
这种侧链锚定策略的优点为:
(i)第一个氨基酸掺入固相支持物系经由酰胺键以较为定量的产率来实行,因此可使用用于肽键结形成所之同种试剂。此外,将天冬酰胺衍生物掺入Wang型树脂系以低的产率发生,若是侧链酰胺未保护则会有消旋作用和形成β-氰基丙氨酸的风险(Katsoyannis等人,1958年,Am.chem.soc.,80:2558)。而且,侧链未保护的天冬酰胺可能经由1-羧基和羧酰胺官能团二者而被掺入卤素树脂,这需要使用例如适当地保护的Fmoc-Asn(Mbh)-OH氨基酸,而其会在最终对肽进行去保护的阶段产生更多问题。
(ii)仅涉及对Lys侧链采用酸不稳定性t-Bu基保护基(对第一个残基的1-羧酸和Asp与Glu的ω-羧酸之tBu酯,Ser和Thr侧链的tBu酯)和酸不稳定性Boc的侧链锚定策略有助于最终的整体去保护,这是由于简单的清除剂如H2O可被使用。相对而言,若使用三苯甲基、xantyl基、2,4,6-三甲氧基苄基保护基,则需要另外的清除剂存在。
(iii)将骨架肽链增长入侧链锚定策略会比在1-羧固定策略中具有更多弹性,因为该固定是经由侧链进行的。
方案I概述根据本发明在PEG树脂上合成胸腺法新(Zadaxine)的最优选模式,最优选是在纯的或基本上纯的PEG树脂上,其中接头I是一种能产生酰胺的接头而接头2则是一种整合接头:
方案I
(i)利用偶联剂将Fmoc-Asp-OtBu掺入含有一种或两种接头的固体支持物(SS)上(Albericio,van Abel,Barany,Int.J.Peptide Protein Res.,35,284-286,1990)。
(ii)用其余经Fmoc保护的氨基酸延伸肽链(对Asp、Glu、Ser和Thr是用tBu;对Lys是用Boc)。
(iii)乙酰化。
(iv)把肽从固体支持物上切下并且移除保护基,这可在一个(高酸浓度的溶液)或两个步骤(先用低酸浓度的溶液接着用高酸浓度的溶液)中实行。
实施例
一般方法。Fmoc-Sieber-PS-树脂购自NovaBiochem(瑞典,L_ufelfingen),ChemMatrix树脂购自Matrix Innovation(加拿大,魁北克省),2-Cl-TrtCl-树脂购自CBL(希腊,Patras),HCTU购自Luxembourg Industries Ltd.(以色列,Tel Aviv),保护的Fmoc-氨基酸衍生物系购自IRIS Biotech(德国,Marktredwitz)。
固相合成是在装备有滤器的玻璃漏斗或是装配有聚乙烯多孔盘的聚丙烯针筒(50毫升)中进行的。利用抽吸法移除溶剂和可溶性试剂。用哌啶-DMF(2∶8,v/v)(2×1分钟,2×10分钟,1×5分钟)进行Fmoc基团的移除。在去保护作用与偶联作用之间进行洗涤,再用DMF(5×0.5分钟)和CH2Cl2(5×0.5分钟)且每次使用10毫升溶剂/克树脂进行去保护的步骤。肽合成的转型和洗涤是在25℃进行的。在固相上进行的合成是通过对将肽-树脂等份(大约2毫克)以TFA-H2O(95∶5)切割一小时以后所得到的中间体进行HPLC来控制的。HPLC反相柱SymmetryTM C184,6×150mm,5微米(柱A)购自Waters公司(爱尔兰)。分析型HPLC是在包含两个溶剂输送泵(Waters 1525)、自动进样器(Waters 717自动取样器)、双波长检测器(Waters 2487)的Waters仪器上进行的。UV检测是在215nm或220nm,以30%到100%的CH3CN(+0.036%TFA)到H2O(+0.045%TFA)的线性梯度在15分钟内进行的。
在PerSeptive Biosystems Voyager DE RP中用ACH基质(ACH matrix)进行肽样本的MALDI-TOF和ES-MS分析。
实施例1
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asp(Rink-ChemMatrix-树脂)-OtBu
步骤1
Fmoc-Asp(Rink-ChemMatrix-树脂)-OtBu的侧链锚定
将Fmoc-Rink-ChemMatrix-树脂(0.45毫摩尔/克)(5克,2.25毫摩尔)置入配备有多孔盘的玻璃漏斗中。对该树脂进行以下洗涤/处理,使用CH2Cl2(3×0.5分钟)、DMF(3×0.5分钟),和如在一般步骤中所指定的哌啶,以及DMF(5×0.5分钟)。然后加入溶于DMF(9毫升)中的Fmoc-Asp-OtBu(4.11克,5当量)和DIEA(3.4毫升,10当量),接着加入溶于DMF(5毫升)中的HCTU(3.96克,4.8当量)。使混合物保持机械搅拌2小时并用DMF(3×0.5分钟)洗涤树脂,茚三酮试验的结果为阴性。加入溶于DMF(14毫升)中的Ac2O(0.5毫升,5毫摩尔)和DIEA(1.7毫升,5毫摩尔),保持机械搅拌15分钟,将树脂过滤并且用DMF(3×0.5分钟)洗涤。
经测定成功的装载达到0.4毫摩尔/克。
步骤2
H-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBr)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asp(Rink-ChemMatrix-树脂)-OtBu
将Fmoc基团移除并紧接着将Fmoc-aa-OH(5-10当量)和溶于DMF(9毫升)的DIEA(3.4毫升,10当量)一起加入上述肽基-树脂(步骤1),接着加入溶于DMF(5毫升)的HCTU(3.96克,4.8当量)。使混合物保持机械搅拌1-2小时,并且用DMF(3×0.5分钟)洗涤树脂,进行茚三酮试验。若试验结果为阳性(蓝色),则重复偶联反应,而若试验为阴性(褐色),则用溶于DMF(14毫升)的Ac2O(0.5毫升,5毫摩尔)和DIEA(1.7毫升,5毫摩尔)进行乙酰化步骤并予机械搅拌15分钟。在哌啶处理之后取样小部分树脂。在位置E27、K19处,使用10当量的Fmoc氨基酸对于每一个位置进行双偶联反应,每单次偶联的反应时间至少是35分钟。
步骤3
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asp(Rink-ChemMatrix-树脂)-OtBu
使用溶于DMF(14毫升)的Ac2O(0.5毫升,5毫摩尔)和DIEA(1.7毫升,5毫摩尔)溶液以机械式搅拌15分钟进行最终的乙酰化作用,其中茚三酮试验的结果为阴性。将树脂干燥获得16.5克。
步骤4
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Zadaxin,胸腺法新)
以单步骤从树脂上切下并进行全体去保护。将得自步骤3的5克树脂部分置于装配有多孔盘的玻璃漏斗中并且用冷的TFA-H2O(95∶5)(35毫升)处理2小时,然后把溶液过滤并将树脂用TFA-H2O(95∶5)(10毫升)进一步洗涤。把合并的TFA溶液倒到冷的叔丁基甲基醚(350毫升)并将混合物离心。利用倾倒法把固体分离出来。MALDI-TOF-MS,计算值3106.50。实测值:m/z 3107.36[M+H]+。
RP-HPLC分析粗制肽显示该产物主要是纯的,达到90%的产率(图1)。可通过RP-HPLC在C18 Hypersil上用10-30%乙腈/水经过45分钟将两个较小的污染峰很容易的移除,产生正确产物峰几乎被完全回收的纯产物。(图2)
实施例2:比较性实施例
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Zadaxin,胸腺法新)
使用如实施例1和2所述的实验方法,但用Fmoc-Sieber-PS树脂(0.59毫摩尔/克)(3.33克,2毫摩尔)代替Fmoc-Rink-ChemMatrix-树脂。
MALDI-TOF-MS,计算值3106.50。实测值:m/z 3107.24[M+H]+。RP-HPLC分析粗产物之混合物的纯度与产率以峰面积所测定仅达40%的产率(图3),具有广泛范围的杂质。
实施例3:比较性实施例
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-O-(2-Cl-Trt)-树脂
步骤1
把Cα端锚定在CTC树脂上,产生Fmoc-Asn(Trt)-O-TrtCl-树脂
将2-Cl-TrtCl树脂(0.2克,1.64摩尔/克;AmbersynthTM CTC,购自法国巴黎的Rohm&Haas公司,其基质由聚苯乙烯-1%二乙烯苯制造)置入一个配备有聚乙烯滤盘的10毫升聚丙烯针筒。然后用CH2Cl2(5×0.5分钟)洗涤树脂,并且加入Fmoc-Asn(Trt)-OH(0.7当量)和DIEA(241微升)的CH2Cl2(2.5毫升)溶液,将混合物搅拌15分钟,在加入更多DIEA(121微升,总共7当量)时将该混合物搅拌45分钟。搅拌10分钟以后,通过加入MeOH(160微升)使反应终止。将Fmoc-Asn(Trt)-O-TrtCl-树脂进行以下采用CH2Cl2(3×0.5分钟)和DMF(3×0.5分钟)的洗涤。通过Fmoc测定所计算出来的装载量为0.8毫摩尔/克。
步骤2
H-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-ClTrt-PS-树脂
将Fmoc基团移除并依序将Fmoc-aa-OH’s(10当量)和溶于DMF(9毫升)的DIEA(20当量)加入到上述肽-树脂(步骤1)上;接着加入溶于DMF中的HCTU(9.6当量),使用ABI自动合成仪433A以60分钟的偶联时间进行。
步骤3
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-ClTrt-PS-树脂
最终的乙酰化用溶于DMF(2毫升)的Ac2O(0.5毫摩尔)和DIEA(5毫摩尔)进行15分钟,期间辅以间歇式手动搅拌,其中茚三酮试验的结果为阴性。
步骤4
Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-OH
用TFA-Et3SiH-CH2Cl2(1∶1∶98)(5×30秒)将得自以上步骤3的保护的肽切下。在H2O(4毫升)上收集滤液并且将H2O部分减压移除掉。然后加入乙腈(ACN)以溶解在去除H2O期间所出现的固体,并将该溶液冻干。
步骤5
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Zadaxin,胸腺法新)
将得自步骤4的保护的肽溶于TFA-H2O(95∶5,5毫升)并将混合物搅拌1小时。然后把溶液滤掉并将树脂进一步用TFA-H2O(95∶5)(1毫升)洗涤。把合并的TFA溶液倒入冷的叔丁基甲基醚(25毫升)并将混合物离心。用倾倒法分离出固体。然后,加入H2O(5毫升)并予冻干。
MALDI-TOF-MS,计算值3106.50。实测值:m/z[M+H]+3107.98。
以RP-HPLC峰面积所测定出的粗纯度和产率达到令人失望的20%正确产物,其具有多种不正确链变体的强烈背景模糊带(图4)。以手动偶联小心重复缩短片段(Lys19到Asn28)的固相合成并且在每一步之后进行茚三酮试验,显示链合成经常在加入V22之后有效的终止;E21、K20和K19这三个连续偶联被证明是高度无效的,需要对未反应的链加帽。双重偶联、延长偶联时间和使用更多当量的Fmoc氨基酸并无法令人满意地克服该问题,因而无法以这种方式建立显著的更为有效的合成(图5)。
实施例4:比较性实施例
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(Zadaxin,胸腺法新)
实验步骤基本上如实施例2,除了Fmoc-Rink把手被加到MBHA树脂(4-甲基二苯甲基胺树脂LL,在聚苯乙烯-1%二乙烯苯基底上,NovaBiochemTM,Merck/德国)上。如实施例1步骤1的方法在将Rink部分的Fmoc基团移除后将Fmoc-Asp-Otbu侧链锚定到Rink氨甲基接枝的接头上,装载量经测定达0.66毫摩尔/克。用HCTU(10当量)进行偶联35分钟,而在D28(亦即N28)、E27、E21、K20、K19的位置上进行双重偶联。如上所述进行乙酰化作用之后用TFA-H2O(95∶5)进行单步切割并去保护。以RP-HPLC测定所得到的粗纯度和产率为27%(图6)。
实施例5
NH2-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(胸腺法新的片段19-28)
基本上如实施例1中所说明的方式在Rink-Chemmatrix树脂上进行侧链锚定和合成,除了未在实施例1所指出的序列位置上进行双重偶联而是进行一致的经35分钟的单一偶联。第27号Glu残基的删除是在HPLC图谱(图7)中观察到的唯一但主要的链终止反应副产物。如实施例1所示,这种副产物可以通过在E27进行延长、重复偶联成功地抑制。
实施例6
NH2-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH(胸腺法新的片段19-28)
基本上如实施例1中所说明的方式进行侧链锚定和合成,除了采用与Rink氨甲基接头接枝的购自Millipore-Waters的PEG-MBHA树脂(MeO[PEG]2000-CO-Orn-MBHA-PS-树脂,其类似于Kates等人在《用于固相合成之高装载量PEG-聚苯乙烯接枝支持物》(High-load PEG-polystyrene graft supports for solid-phase synthesis),同上,中所说明者。Rink-PEG-MBHA树脂的装载量经测定为0.55毫摩尔/克。特别地,在E27、E21、K20、K19的序列位置上进行双重偶联。以RP-HPLC测定所获得的粗纯度和产率为92%(图8)。
Claims (9)
1.式I的肽偶联物,
R1-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn_R2_xR3I
其中,每一个氨基酸残基都单独被保护或者未保护,R3是包含整合接头的PEG树脂固相,R2是接枝的接头,x是0或1,R1是氢、保护基或肽基,优选数目小于50而更优选少于25个氨基酸残基的肽基,且其中如果R1是肽基,则所述基团上的各个氨基酸残基单独被保护或未保护,且所述基团的N端是游离的、被乙酰化或被保护基Y保护。
2.如权利要求1所述的肽偶联物,其特征在于,所述保护基Y是Fmoc基团、Dde型基团、Boc基团或所述保护基的衍生物,优选Fmoc或Dde型基团的衍生物,其包含允许这种保护的肽可逆亲和结合至合适的色谱固体基质材料的部分。
3.如权利要求3所述的肽偶联物,其特征在于,所述保护基Y能够可逆亲和结合至固定的金属亲合色谱固态基质材料。
4.如上述权利要求中任一项所述的肽偶联物,其特征在于,R1是Y-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-、Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu或H-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Leu-Lys-Glu,Y具有以上所提到的意义。
5.一种在固相上合成胸腺素α1、包含成熟胸腺素α1残基1-27的胸腺素α1的C-端截短形式、至少包含成熟胸腺素α1残基19-28的胸腺素α1的N-端截短形式、或至少包含成熟胸腺素α1残基19-27的截短形式的方法,所述方法包括以下步骤:
a.将适当保护的FMOC-Glu、FMOC-Asp或FMOC-Asn残基偶联到PEG树脂固相上,其中PEG树脂优选选自:聚苯乙烯-PEG混合树脂、PEG聚醚-聚酯混合树脂、或PEG聚醚-聚酰胺混合树脂以及基本上纯的PEG树脂,
b.通过FMOC合成法延长肽链,其中个别氨基酸的侧链可用适当的保护基加以衍生,
c.任选将最后一个N-端残基乙酰化,并且
d.将肽从树脂上切下。
6.如权利要求5所述的方法,其特征在于,所合成的肽是具有以下序列的胸腺素α1:
1-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-28
其中,各氨基酸链是未保护的或是被适当保护的。
7.如权利要求5所述的方法,其特征在于,所述PEG树脂是两性树脂。
8.如权利要求7所述的方法,其特征在于,所述PEG树脂的装载容量至少为0.5毫摩尔/克,且该PEG树脂优选基本不含聚苯乙烯共聚部分。
9.如权利要求7所述的方法,其特征在于,除整合和/或接枝接头,所述PEG树脂是基本纯的聚醚-PEG树脂,优选是纯的PEG-聚醚树脂。
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EP05009758.3 | 2005-05-04 | ||
EP05009758 | 2005-05-04 | ||
US69985105P | 2005-07-18 | 2005-07-18 | |
US60/699,851 | 2005-07-18 | ||
PCT/EP2006/004192 WO2006117227A2 (en) | 2005-05-04 | 2006-05-04 | Solid phase bound thymosin alpha-1 and its synthesis |
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EP (1) | EP1891104B1 (zh) |
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ES (1) | ES2332666T3 (zh) |
IL (1) | IL186774A0 (zh) |
MX (1) | MX2007013400A (zh) |
NO (1) | NO20074623L (zh) |
PT (1) | PT1891104E (zh) |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN103242443A (zh) * | 2012-02-06 | 2013-08-14 | 长春百克生物科技股份公司 | 一种胸腺素α1及其类似物的制备方法 |
CN107001409A (zh) * | 2014-09-26 | 2017-08-01 | 株式会社钟化 | 疏水性肽的制造方法 |
CN108239147A (zh) * | 2016-12-27 | 2018-07-03 | 江苏豪森药业集团有限公司 | 胸腺素α1衍生物的制备方法 |
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ES2288118B1 (es) * | 2006-05-10 | 2008-11-01 | Bcn Peptides, S.A. | Procedimiento para sintetizar timosinas. |
US8076295B2 (en) * | 2007-04-17 | 2011-12-13 | Nanotope, Inc. | Peptide amphiphiles having improved solubility and methods of using same |
WO2009075788A1 (en) * | 2007-12-05 | 2009-06-18 | Semprus Biociences Corporation | Synthetic non-fouling amino acids |
CA2907454C (en) | 2013-03-21 | 2021-05-04 | Sanofi-Aventis Deutschland Gmbh | Synthesis of hydantoin containing peptide products |
EP2976325B1 (en) | 2013-03-21 | 2017-03-01 | Sanofi-Aventis Deutschland GmbH | Synthesis of cyclic imide containing peptide products |
CN104327181A (zh) * | 2014-09-28 | 2015-02-04 | 上海昂博生物技术有限公司 | 固相合成胸腺肽α1 |
CN111349152B (zh) * | 2018-12-20 | 2022-04-05 | 深圳翰宇药业股份有限公司 | 一种制备胸腺法新的方法 |
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ATE136551T1 (de) * | 1988-05-10 | 1996-04-15 | Alpha 1 Biomedicals Inc | Festphase-verfahren zur gewinnung von thymosin- alpha-1 |
US6262230B1 (en) * | 1994-01-28 | 2001-07-17 | Sciclone Pharmaceuticals Inc. | Analogs of thymosin α1 |
US6204360B1 (en) * | 1999-12-16 | 2001-03-20 | Usa Universe Bioengineering, Inc. | Retro-inverso thymosin alpha 1 hybrids |
US7413738B2 (en) * | 2002-08-13 | 2008-08-19 | Enzon Pharmaceuticals, Inc. | Releasable polymeric conjugates based on biodegradable linkers |
CN100355786C (zh) * | 2004-04-28 | 2007-12-19 | 江苏豪森药业股份有限公司 | 聚乙二醇修饰的胸腺肽1衍生物 |
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- 2006-05-04 WO PCT/EP2006/004192 patent/WO2006117227A2/en not_active Application Discontinuation
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Cited By (4)
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CN103242443A (zh) * | 2012-02-06 | 2013-08-14 | 长春百克生物科技股份公司 | 一种胸腺素α1及其类似物的制备方法 |
CN107001409A (zh) * | 2014-09-26 | 2017-08-01 | 株式会社钟化 | 疏水性肽的制造方法 |
CN108239147A (zh) * | 2016-12-27 | 2018-07-03 | 江苏豪森药业集团有限公司 | 胸腺素α1衍生物的制备方法 |
CN108239147B (zh) * | 2016-12-27 | 2023-11-10 | 江苏豪森药业集团有限公司 | 胸腺素α1衍生物的制备方法 |
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AU2006243344A1 (en) | 2006-11-09 |
EP1891104B1 (en) | 2009-08-26 |
ZA200708110B (en) | 2008-10-29 |
NO20074623L (no) | 2007-12-03 |
KR20080012884A (ko) | 2008-02-12 |
ATE440856T1 (de) | 2009-09-15 |
IL186774A0 (en) | 2008-02-09 |
US20090171068A1 (en) | 2009-07-02 |
DE602006008781D1 (de) | 2009-10-08 |
CA2600303A1 (en) | 2006-11-09 |
DK1891104T3 (da) | 2009-11-30 |
AU2006243344B2 (en) | 2011-04-21 |
WO2006117227A3 (en) | 2007-03-15 |
EP1891104A2 (en) | 2008-02-27 |
PT1891104E (pt) | 2009-12-07 |
JP2008543733A (ja) | 2008-12-04 |
WO2006117227A2 (en) | 2006-11-09 |
MX2007013400A (es) | 2008-04-29 |
ES2332666T3 (es) | 2010-02-10 |
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