CN106279300A - Rig‑i配体及其生产方法 - Google Patents
Rig‑i配体及其生产方法 Download PDFInfo
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- CN106279300A CN106279300A CN201610649060.5A CN201610649060A CN106279300A CN 106279300 A CN106279300 A CN 106279300A CN 201610649060 A CN201610649060 A CN 201610649060A CN 106279300 A CN106279300 A CN 106279300A
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- oligonucleotide
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- capture
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- 238000007920 subcutaneous administration Methods 0.000 description 1
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- GTDKXDWWMOMSFL-UHFFFAOYSA-M tetramethylazanium;fluoride Chemical class [F-].C[N+](C)(C)C GTDKXDWWMOMSFL-UHFFFAOYSA-M 0.000 description 1
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Abstract
本发明涉及可以作为RIG‑I配体起作用的新的三磷酸‑修饰的寡核苷酸以及能够以高收率和纯度合成和纯化的适合于药物应用的新方法。
Description
本申请是申请日为2013年9月26日、申请号为201380050517.5(PCT/EP2013/070177)、发明名称为“新的RIG-I配体及其生产方法”的中国专利申请的分案申请。
技术领域
本发明涉及可作为RIG-I配体起作用的新三磷酸-修饰的寡核苷酸以及能够以高收率和纯度合成和纯化的适合于药物应用的新方法。
背景技术
Schlee等人,Immunity,2009,31,25-34描述了在一条链上携带5’-O-三磷酸酯部分的钝端双链RNA，通过结合RIG-I解螺旋酶，作为有效的免疫系统的刺激剂。因此，对提供简单且有效的用于制备适用于药物应用的高纯度三磷酸修饰的寡核苷酸的方法存在需求。
三磷酸基团或其类似物与核苷化合物的5’-OH基团的偶联是本领域公知的。Ludwig J.等人,J.Org.Chem.,1989,54,631-635公开了使用2-氯-4H-1,3,2-苯并二氧磷杂环己烷(benzodioxaphosphorin)-4-酮作为亚磷酸化剂的用于制备核苷和类似物的5’-O-三磷酸酯的溶液三磷酸化方法。Gaur R.K.等人,1992,Tetrahedron Letters,33,3301-3304描述了在固相上使用所述方法，用于合成2’-O-甲基核糖核苷5’-O-三磷酸酯及其Pα-硫代类似物。美国专利6,900,308B2公开了作为潜在抗病毒化合物的修饰核苷5’-O-三磷酸酯的固相合成，而美国专利7,285,658、7,598,230和7,807,653公开了在糖、核酸碱基和三磷酸实体上具有修饰的核苷三磷酸酯类似物。
WO96/40159描述了用于生产加帽的RNA或RNA类似物分子的方法，其中将RNA或RNA类似物寡核苷酸与亚磷酸化剂(如，2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮或其环取代的衍生物)反应。将所得到的中间体与磷酸酯或焦磷酸酯或其氧化的或水解的盐反应。通过与激活的m7G三-、二-或单磷酸酯或类似物反应，将二-或三磷酸化RNA或RNA类似物加帽。
WO 2009/060281描述了含有修饰的寡磷酸酯部分的免疫刺激寡核糖核苷酸类似物和用于制备这些化合物的方法。该方法包括在固体支持物上合成寡核苷酸，在适合的溶剂中，并且在碱的存在下，在寡核苷酸的5’-端，将核苷酸与亚磷酸化剂(如，2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮)反应，将亚磷酸化的寡核苷酸与焦磷酸酯或焦磷酸酯类似物反应，用氧化剂氧化寡核苷酸，并且将寡核苷酸去保护，以产生三磷酸酯-或三磷酸酯类似物-修饰的寡核苷酸。
WO 96/40159中使用的聚丙烯酰胺凝胶电泳只可用于小规模分离。用于较长寡核糖核苷的5’-单-、二-、三磷酸化产品的离子交换色谱的分辨力受到限制。所需的变性条件使得分离是个冗长乏味的任务(Sproat,1999；Zlatev,2010；WO 2009/060281)，此外，产品通常受到正-1、正-2序列及其单-和二磷酸酯的污染，导致纯度不足。已知对RIG-I配体的精确末端结构的敏感性，这些纯化方法对药物应用是次最佳的。
因此，对于新的三磷酸化寡核苷酸及其类似物、特别是具有RIG-I选择性的新的三磷酸化寡核苷酸及其类似物以及这样的化合物的制备方法存在高度需求。
发明内容
本发明由此涉及新的可以用于大规模生产的可能用于临床应用的5'-三磷酸化寡核苷酸及其类似物以及这样的寡核苷酸的便利制备方法。此外，描述了寡核苷酸修饰，其建立、维持和/或改善寡核苷酸的RIG-I选择性或增强其化学稳定性。
因此，本发明的第一个方面涉及式(I)的修饰的寡核苷酸，
其中V1、V3和V5在每种情况中独立地选自O,S和Se；
V2、V4和V6在每种情况中独立地选自OH、OR1、SH、SR1、F、NH2、NHR1、N(R1)2和BH3 -M+；
W1是O或S；
W2是O、S、NH或NR2；
W3是O、S、NH、NR2、CH2、CHHal或C(Hal)2；
R1、R2和R3选自C1-6烷基、C2-6烯基、C2-6炔基、C2-6酰基或环状基团，其各自任选地被取代；
或其中两个R1可以和与其结合的N-原子一起形成环；
M+是阳离子；
X是NH、NR3、O或S；
Z表示捕获标记物或H；
Y表示连接所述捕获标记物与X的键或连接基，；且
ON表示包含至少4个核苷酸或核苷酸类似物结构单元的寡核苷酸。
在本申请内容中的术语“寡核苷酸”包括含有多个(例如，至少4个)核苷酸或核苷酸类似物结构单元的化合物。优选地，寡核苷酸包括6-100个，例如，20-40个结构单元。核苷酸或核苷酸类似物结构单元可以包括由亚基间连接而相连的核苷酸或核苷酸类似物亚基(subunit)。核苷酸亚基包括脱氧核糖核苷亚基、核糖核苷亚基和/或其类似物，特别是糖-和/或核酸碱基-修饰的核苷类似物。此外，寡核苷酸可以包含非核苷酸结构单元和/或另外的末端和/或侧链修饰。
在优选的糖修饰亚基中，核糖核苷亚基的2’-OH被选自OR、R、卤素、SH、SR、NH2、NHR、NR2或CN的基团取代，其中R是C1-6烷基、C2-6烯基或C2-6炔基，并且卤素是F、Cl、Br或I。在进一步优选的糖修饰亚基中，核糖可以是取代的，例如，被另一种糖取代，例如，戊糖，如阿拉伯糖。这种糖修饰可以结合如上所述的2’-OH修饰，如2’-氟代阿糖核苷亚基中的修饰。再进一步优选的糖修饰亚基包括锁核苷(LNA)或2’,3’-断-核苷(UNA)。在优选的核酸碱基修饰的核苷结构单元中，使用非标准的、例如非天然产生的核酸碱基替代标准核酸碱基。非标准的核酸碱基的实例是5-位的尿嘧啶或胞嘧啶，例如，5-(2-氨基)丙基尿嘧啶或5-溴尿嘧啶；次黄嘌呤；2,6-二氨基嘌呤；8-位修饰的腺嘌呤或鸟嘌呤，例如，8-溴鸟嘌呤；去氮核苷，例如，7-去氮鸟嘌呤或7-去氮腺嘌呤；或O-和N-烷基化核酸碱基，例如，N6-甲基腺嘌呤，或N6,N6-二甲基腺嘌呤。另外适合的核酸碱基类似物可以选自通用核酸碱基类似物，如5-硝基吲哚。
亚基间连接可以是磷酸二酯连接或修饰的连接，例如，硫代磷酸酯、二硫代磷酸酯、膦酸甲酯、氨基磷酸酯、硼磷酸酯，或本领域技术人员已知的另一种修饰的连接。
寡核苷酸可以选自脱氧核糖核苷酸、核糖核苷酸和寡核苷酸类似物。脱氧核糖核苷酸、核糖核苷酸和/或寡核苷酸类似物可以在核苷上和/或类似物的核糖亚基上被化学修饰。脱氧核糖核苷酸或核糖核苷酸的类似物可以包括至少一个脱氧核糖核苷或核糖核苷亚基和至少一个修饰的核苷亚基和/或至少一个修饰的亚基间连接，例如，如上所述的。寡核苷酸类似物还可以存在于它们整个的修饰核苷亚基中。
寡核苷酸可以是单链分子或双链分子。双链寡核苷酸可以包括全部或部分互补链。双链分子可以是钝端的，或包含至少一个悬垂物，例如，5’-或3’-悬垂物。悬垂物如果存在，优选位于分子的远端(相对于三磷酸酯/三磷酸酯类似物基团)。双链寡核苷酸还可以包括发夹-结构，其中在其远端(相对于三磷酸酯/三磷酸酯类似物基团)通过环闭合双链。环可以包含核苷酸和/或非核苷酸结构单元，例如，基于二醇的结构单元，如乙二醇部分，例如，三(乙烯)二醇或六(乙烯)二醇；丙烷-1,3-二醇；十二烷-1,12-二醇；或3,12-二氧杂-7,8-二硫十四烷-1,14-二醇。
在优选的实施方案中，双链分子是钝端的，特别是在其近端(相对于三磷酸酯/三磷酸酯类似物基团)。
根据特别优选的实施方案，寡核苷酸是双链的，其中双链的每一条链具有至少19个核苷酸长度。这样的长度的钝端的双链寡核苷酸是尤其优选的。根据另一个优选的实施方案，寡核苷酸的每一条链具有至少19－50个核苷酸、19－30个核苷酸、20－30个核苷酸、22－28个核苷酸长度，尤其优选22－26个核苷酸。
寡核苷酸可以包括更多的末端和/或侧链修饰，例如，与其共价连接的细胞特异性靶向实体。那些实体可以促进细胞或细胞特异性吸收，并且包括，例如，脂质、维生素、激素、肽、寡糖及其类似物。靶向实体可以通过本领域技术人员已知的方法例如连接修饰的核酸碱基或非核苷酸结构单元。
根据一个优选的实施方案，修饰建立和/或增强了寡核苷酸对指定靶标的选择性。在一个特别优选的实施方案中，寡核苷酸的RIG-I选择性得以建立或增强。测定指定寡核苷酸的RIG-I选择性的方法如本文详细描述(参见实施例)和/或为本领域技术人员所公知。
根据另一个优选的实施方案，化学修饰维持或增强寡核苷酸的化学稳定性。本领域技术人员已知用于测定指定寡核苷酸化学稳定性的方法。这样的方法还描述在例如实施例中。
根据一个优选的实施方案，寡核苷酸的化学修饰独立地选自卤化，特别是F-卤化；2'-O-烷基化，特别是2'-O-甲基化；和/或核苷酸间连接的硫代磷酸酯修饰。特别地，F-卤化和硫代磷酸酯修饰增加寡核苷酸的稳定性，而2'-O-甲基化建立或增加寡核苷酸的RIG-I选择性。2'-O-甲基化还能够改变RNA的免疫原性。在一个优选的实施方案中，寡核苷酸仅包含一个或两个2'-O-甲基化/链,更优选一个2'-O-甲基化/链。
2'-F取代是特别优选的。在核糖的2'位上，羟基被氟取代。RNA中的2'-F取代特别地导致对核酸酶消化的稳定性增强。在另一个实施方案中，2'-氟-取代可以特别地增加RIG-I-依赖性免疫刺激。
本文的硫代磷酸酯化合物一般性涉及核苷酸间连接的硫代磷酸酯修饰。
尤其优选在寡核苷酸末端上具有修饰的硫代磷酸酯-修饰的化合物。在硫代磷酸酯修饰过程中，核酸骨架上桥接磷酸酯的未结合氧原子取代为硫原子。这种取代减少了核酸酶在该位置上的裂解性并且导致核酸链具有更高的稳定性。
在一个特别优选的实施方案中，本发明的寡核苷酸显示F-卤化；甲基化，特别是2'-O-甲基化；以及特别是在寡核苷酸末端上的硫代磷酸酯修饰。
指定寡核苷酸的鉴定模式取决于寡核苷酸的序列和长度并且可以对每一指定寡核苷酸测定。本领域技术人员充分了解如何进行这种测定。
正如上文已经解释的，用于测定指定寡核苷酸的RIG-I选择性和/或稳定性这样的方法详细描述在本申请中。
式(I)或(IV)的寡核苷酸包括三磷酸酯/三磷酸酯类似物基团。在该基团中，V1、V3和V5独立地选自O、S和Se。优选地，V1、V3和V5是O。V2、V4和V6在每种情况中独立地选自OH、OR1、SH、SR1、F、NH2、NHR1、N(R1)2和BH3 -M+。优选地，V2、V4和V6是OH。R1可以是C1-6烷基、C2-6烯基、C2-6炔基、C2-6酰基或环状基团，例如，C3-8环(杂)烷基、C3-8环(杂)烯基、苯基或C5-6杂芳基基团，其中杂原子选自N、O和S。此外，两个R1可以和与其结合的N-原子一起形成环，例如，5-或6-元环。R1还可以包括取代基，如卤素(例如，F、Cl、Br或I)、O(卤代)C1-2烷基，以及-在环状基团情况中-(卤代)C1-2烷基。M+可以是无机或有机阳离子，例如，碱金属阳离子，或铵或胺阳离子。
W1可以是O或S。优选地，W1是O。W2可以是O、S、NH或NR2。优选地，W2是O。W3可以是O、S、NH、NR2、CH2、CHHal或C(Hal)2。优选地，W3是O、CH2或CF2。R2可以选自如以上对R1所述的基团。Hal可以是F、Cl、Br或I。
根据一个特别优选的实施方案，V1、V2、V3、V4、V5、V6、W1、W2和W3是O。
三磷酸酯/三磷酸酯类似物基团优选连接寡核苷酸的末端。优选地，所述基团与寡核苷酸的5’-端，特别是其5’-端糖的5’-OH-基团连接。
如本文所定义的Z表示捕获标记物或H。捕获标记物Z可以被如下提供的一系列合理实例在功能上定义。一般原则可以是：Z必需允许便利地纯化，且应在与pppRNA稳定性要求相容的的条件下被除去。本领域技术人员能够在无过度负担的情况下确定指定标记物是否满足功能性定义。因此，本领域技术人员了解这样的捕获标记物，特别是在有关本申请中给出的具体实施例方面。
根据一个优选的实施方案，捕获标记物Z选自长链脂族残基、非共价高亲和力结合对的配偶体、反应性化学实体、Q或NHC2-C24烷基，Q优选自H、氨基酸、氨基酸类似物、C1-C24烷基，优选C12-C24烷基、肽类和脂质。然而，根据一个特别优选的实施方案，Z是癸基，即C10烷基。
根据本发明的捕获标记物Z是在允许分离包含捕获标记物的化合物(例如，寡核苷酸(I))与不含捕获标记物的其它种类的条件下能够与捕获试剂非共价或共价相互作用的部分。优选地，捕获试剂是固定化试剂或能够被固定化的试剂。
适合的捕获标记物例如是长链(例如C8-24、优选C13-24、更优选C13-C14)脂族烷基残基，如十八烷基，或其它脂类/亲脂性残基，如，例如，胆固醇基、生育酚基或三苯甲基及其衍生物。然而，根据一个特别优选的实施方案，Z是癸基残基。在这种情况中，可以通过标准反相色谱，例如，RP-HPLC，或通过疏水性相互作用色谱(HIC)，在固相上捕获和纯化标记的三磷酸酯实体。捕获标记物还可以是全氟烷基实体，例如，4-(1H,1H,2H,2H-全氟癸基)苄基或3-(全氟辛基)丙基残基，用于在Fluorous亲和性支持物(如，从FluorousTechnologies，Inc.可购得)上的修饰寡三磷酸酯的特异性捕获。
在另一个实施方案中，捕获标记物可以是非共价高亲和性结合对的第一配偶体，如生物素，或生物素类似物，如脱硫生物素，半抗原或抗原，其对捕获试剂具有高亲和性(例如，10-6l/mol或更低的结合常数)，捕获试剂是高亲和性结合对的第二互补配偶体，例如，抗生物素蛋白链菌素、抗生物素蛋白或抗体。
在另一个实施方案中，捕获标记物可以是共价结合对的第一配偶体，其可以与捕获试剂形成共价键，捕获试剂是共价结合对的第二互补配偶体，其中共价键可以是可逆的或不可逆的键。在这个实施方案中，捕获标记物成分Z可以是反应性化学实体，例如，在Husigen3+2环加成反应的情况中(所谓的Cu(I)催化的“点击反应”或没有Cu(I)而进行的其变体，通过严格的环链的释放，例如在环辛炔衍生物中)，是能够与含有互补反应基(例如，分别为，炔基或叠氮部分)的捕获试剂共价反应的叠氮化物或炔基基团。在这种情况中，Z-Y-X的具体实例是炔丙基氨基。
在另一个实施方案中，捕获标记物成分可以是含有另外的亲核性基团(例如，NH2-Y-XH型试剂中的第二氨基)的化学实体。然后可以使用各种适合的亲电子Z试剂，如胆固醇、氯甲酸酯或生物素N-羟基琥珀酰亚胺活性酯来引入标记基团，同时将寡核苷酸与固相连接，由此显著地延伸了标记反应的范围。
在优选的实施方案中，捕获标记物是长链烷基残基、全氟烷基实体、叠氮化物或炔基基团。
在本发明的另一个实施方案中，寡核苷酸可以在不同的位置，例如，在3’-端携带第二捕获标记物。优选地选择第一和第二捕获标记物，使得可以通过两个正交方法来纯化，使得能够收集非常高纯度的材料。例如，第一捕获标记物可以是亲脂性基团，其可以与适合的色谱支持物相互作用，而第二捕获标记物可以是生物素，其与抗生物素蛋白链菌素相互作用。
通过进行使用用于寡核糖核苷酸合成的修饰CPG(受控玻璃支持物)的合成来方便地引入第二捕获标记物。
Y表示化学键或连接基，例如亚烷基，优选C1-6-亚烷基连接基。更优选C2-5-亚烷基连接基或亚芳烷基连接基，其任选地包含杂原子或含杂原子的基团，例如O、S、NH、C＝O或C＝S，和/或任选地包含C＝C或C≡C价键。根据一个特别优选的实施方案，Y是价键。
在另一个优选的实施方案中，连接基是聚环氧烷，优选聚-C2-C6-亚烷基氧化物，更优选聚-C2-C3-亚烷基氧化物。连接基的数均分子量可以为30-800g/mol的范围内，优选40-450g/mol，更优选40-250g/mol。连接基可以是[-CH2CHR4-O-]n，其中n＝1-10，优选n＝1-7，更优选n＝2-5，且甚至更优选n＝3。R4可以是H或C1-6-烷基。Y的另外优选的实施方案如图4中所示，在一个优选的实施方案中，R4是H。
根据一个特别优选的实施方案，
X是NH或O；
Y是-K-((CHR1)m-CH2-O)n-R-；或
(O-(CHR3)m3-CH2)n1-(O-(CHR2)m2-CH2)n2-(O-(CHR1)m1-CH2)n3-；且
K是O或NH；
m、m1、m2和m3独立地是1－12，优选1－8，更优选1－5，且甚至更优选1－3；
n、n1、n2和n3独立地是0－20，优选0－10，更优选0－5，且甚至更优选0－3；且
R1、R2和R3独立地是H、C1-6烷基、C2-6烯基、C2-6炔基、C2-C6-酰基或环状基团，其各自任选地被取代；且
R是C1-6烷基、C2-6烯基、C2-6炔基、C2-C6-酰基或环状基团，其各自任选地被取代。优选地，R是CH2CH2。
根据Y如上述所定义的一个特别优选的实施方案，R1和R2是H，n1是O，且n2和n3是1。另外优选的实施方案可以取自图4。
根据Y如上述所定义的另一个优选的实施方案，R1、R2和R3是H，且n1、n2和n3是1。
根据一个优选的实施方案，X是NH，K是NH，且Y是(CH2CH2O)n，其中n如上述所定义，其中K进一步被胆固醇基-C(O)-、三苯甲基或其衍生物取代。
根据式(I)的寡核苷酸的一个特别优选的实施方案，X是NH或O，Y是价键，且Z是C1-C12烷基或H，优选C10、Q或NHC2-C24烷基，其中Q选自H、氨基酸、氨基酸类似物、C1-C24烷基，优选C12-C24烷基、肽类和脂质，且V1、V2、V3、V4、V5、V6、W1、W2和W3是O。
根据式(I)的寡核苷酸的另一个优选的实施方案，X是NH或O，Y是价键，且Z是癸基或H，且V1、V2、V3、V4、V5、V6、W1、W2和W3优选是O。
本发明的另一个方面涉及包含如本文所定义的修饰的寡核苷酸的药物组合物。
本发明的药物组合物还可以包含药学上可接受的载体、稀释剂和/或助剂，术语“载体”在本文中使用时不可载体、赋形剂和/或稳定剂，它们对于以使用剂量和浓度所暴露的细胞或哺乳动物而言是无毒性的。生理学可接受的载体通常是pH缓冲水溶液或脂质体。生理学可接受的载体的实例包括：缓冲剂，例如磷酸盐、柠檬酸盐和其它有机酸(然而，就本发明的制剂而言，优选磷酸盐缓冲剂)；抗氧化剂，包括抗坏血酸、低分子量(小于约10个残基)多肽类；蛋白质，例如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，例如聚乙烯吡咯烷酮；氨基酸，例如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸；单糖类、二糖类和其它碳水化合物，包括葡萄糖、甘露糖或糊精；胶凝剂，例如EDTA、糖；醇类，例如甘露糖醇或山梨醇；成盐抗衡离子，例如钠；和/或非离子表面活性剂，例如TWEEN、聚乙烯或聚乙二醇。根据一个特别优选的实施方案，将本发明的化合物溶于无菌去离子水。
可以将本发明这样的组合物和/或制剂通过适合的方式以治疗特定病症的足够剂量施用于有此需要的受试者，特别是人体受试者。例如，可以将本发明的组合物和/或制剂与药学上可接受的载体、稀释剂和/或助剂一起配制成药物组合物。可以根据标准方案测定治疗效能和毒性。可以通过全身(例如通过腹膜内、肌内或静脉内)或局部(例如通过鼻内、皮下、皮内或鞘内)施用所述药物组合物。组合物和/或制剂的剂量当然取决于所治疗的受试者和受试者的情况，例如受试者体重、受试者年龄和所治疗疾病或损伤的类型和严重性、施用方式和开据处方的临床医师的判断。
在一个优选的实施方案中，通过皮内施用药物组合物。特别优选通过纹身术、显微操作针和/或显微操作针斑贴经皮内施用组合物。
优选用无菌去离子水(纯水)溶解本发明的化合物并且稀释至期望浓度，然后使用移液装置涂布于刮剃的酒精消毒的皮肤上，且随后纹身入皮肤。
例如，为了进行纹身术，使用安装多-针头(单一使用)针尖(例如9-号针头，单-使用针尖)的(医用)纹身装置将基于水的本发明药物组合物经皮内注入皮肤。
典型的纹身方法如下：在将基于水的本发明药物组合物吸移在刮剃的和酒精清洁的皮肤上后，通过将运动针尖(例如以100-120Hz、特别是100Hz的速度运行)轻轻地置于基于水的药物组合物液滴上端将其导入纹身机器的多-针尖。一旦基于水的药物组合物液滴被完全吸附在运动针尖且由此保留在运动针头之间，则通过保持目前充满的针尖以相对于皮肤确切90度角使运动针尖在皮肤上轻轻地往复运动。使用这种方法，基于水的本发明药物组合物被完全纹身入皮肤。对于50-100μl基于水的本发明药物组合物，该过程在2-4平方厘米的皮肤区域上需要10-15秒。这种治疗(处理)方法超过标准的单一真皮内快速浓注的有益性在于基于水的本发明药物组合物被均匀地注入较大皮肤区域，且更均匀和更精确地分布在靶组织上：通过以100Hz使用9-号针尖10秒，该方法确保9000次真皮内注射均匀地分散于治疗的皮肤中。
当然，本领域技术人员可以脱离和调整这种方法，视所治疗的患者或身体部分而定。显微操作针法可以按照与纹身方法接近类似的方式进行。然而，使用显微操作针法，纹身针尖被显微操作针尖替代，这确保了更表浅的真皮内施用。基于水的本发明药物组合物一般被吸移在刮剃和酒精清洁的皮肤上，且然后按照与纹身方法类似的方式使用显微操作针尖经真皮内施用。显微操作针法不具有药物组合物在显微操作针针头之间在先吸收的必要性。
另外，关注被药物组合物涂敷、否则就是包含该药物组合物的显微操作针斑贴可以用于透皮/真皮内递送。这具有特定的优点，即药物组合物的真皮内递送可以安全地通过接受者自我进行，无需上医院和/或医学专家纹身/显微操作针法干预。这可以明显地给治疗方案附加灵活性，允许高度个性化的治疗方案，并且降低治疗成本。这些斑贴可以由用于时间受控、缓释或快速浓注透皮递送药物组合物的溶解或不溶解显微操作针斑贴构成，但不限于此。
本发明的另一个方面涉及制备权利要求1-15任一项的寡核苷酸的方法，包含下列步骤：
(a)使式(IIa)的化合物
其中V1、V3、V5、V4、V6、W1、W2、W3,和ON如上述所定义，其中ON被至少一个保护基保护，与氧化剂反应，得到式(IIb)的化合物，
其中V1、V3、V5、V2、V4、V6、W1、W2、W3和ON如上述所定义，其中ON北欧至少一个保护基保护；
(b)使式(IIb)的化合物与式(III)的捕获标记物试剂反应，
Z-Y-XH (III)
其中X、Z和Y如上述所定义，其中X是优选O，得到包含式(I)的寡核苷酸的反应产物；和
(c)使所述至少一个ON保护基脱保护；和
(d)使步骤(c)的反应产物与能够与捕获标记物发生相互作用的捕获标记物试剂反应接触，其中该接触在能够使寡核苷酸(I)与包含在所述反应产物中的其它种类分离的条件下进行。
尤其优选一个实施方案，其中X是O。
在本发明方法过程中，ON寡核苷酸包含至少一个保护基。本发明保护基的应用的目的特别地在于保护使用的寡核苷酸核糖亚基的2'-OH基团。本领域技术人员、尤其是在核苷酸合成领域中工作的人知晓什么样的保护基适合于合成。寡核苷酸核糖亚基的2'-OH位上的保护基是优选的。在本发明的一个优选的实施方案中，在核糖单元的2'-位上，使用氟-不稳定性保护基。
尤其优选的是2'-O-TBDMS或2'-O-TOM保护基。在一个特别优选的实施方案中，适用TBDMS保护基。
特别是在合成具有X＝O的化合物的过程中，该化合物具有增强的Z-Y-X-PPP结合稳定性，宽谱的脱保护条件可以导致2'-OH保护基被裂解掉。
所有公知的脱保护试剂适合于裂解掉TBDMS保护基。特别地，下列试剂可能适用：
(a)三乙胺-三氢氟酸盐(trihydrofluoride)任选地与极性溶剂的组合；
(b)三烷基胺、三乙胺-三氢氟酸盐和极性溶剂；
(c)吡啶-HF和其它有机氮碱的氢氟酸盐的加合物；
(d)氟化铵；
(e)氟化四-正-丁基-铵；
(f)氟化四甲基-铵和其它氟化四烷基-铵及其组合。
步骤(c)优选在不导致三磷酸酯部分降解的条件下进行，例如如下文详细描述。
本发明方法的步骤(a)包含使式(IIa)的环P(V)-P(V)-P(III)物质与氧化剂反应。式(IIa)的化合物可以根据如上文Ludwig等人,1989和上文Gaur等人,1992所述的标准方法得到，即通过使寡核苷酸的5'-末端OH-基团与三官能亚磷酸化试剂(例如2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮)在适合的条件下反应，例如在碱(吡啶或二异丙基甲胺)的存在下，在适合的溶剂(例如二噁烷或二氯甲烷)中，随后与焦磷酸酯(W3＝O)或修饰的焦磷酸酯(W3不是O，例如CH2、CCl2、NH或CF2)反应。优选地，使用在DMF中焦磷酸酯或修饰的焦磷酸酯的三-正-丁基铵盐。然后在无水条件下氧化得到的环P(III)-P(V)中间体(IIa)，例如使用过氧化物，例如叔丁基过氧化氢、过氧化氢枯烯、(10-樟脑磺酰基)氧杂氮丙啶。或者，还可以分别使用苯基乙酰基二硫化物(V2＝S)或硼烷-二异丙基乙胺复合物(V2＝BH3)，得到相应的式(IIb)的环5'-三磷酸/三磷酸类似物。上下文中还参考WO 96/40159或WO 2009/060281，将这些文献的内容引入本文参考。
反应步骤(a)可以使用寡核苷酸的溶液或结合固相的寡核苷酸(例如有机树脂或玻璃如CPG)进行。寡核苷酸还可以包含保护基，例如本领域技术人员众所周知的糖-或核酸碱基保护基。优选的保护基实例是用于核苷间磷酸二酯或硫代磷酸酯的2-氰基乙基，用于核糖2'-羟基的叔丁基二甲基甲硅烷基或三异丙基甲硅烷氧基甲基，用于核酸碱基的外环氨基的4-叔丁基苯氧基乙酰基或苯氧基乙酰基、乙酰基、异丁酰基、苯甲酰基。更优选地，步骤(a)使用固相结合的寡核苷酸进行。
根据本发明方法的步骤(b)，使化合物(IIb)与式(III)的捕获标记物试剂反应，
Z-Y-XH (III)
其中X选自NH、NR3、O，或者X和Y如上述所定义。R3如上述对R1所定义。
特别地，在X为O的情况中，有限的亲核性试剂(例如癸醇)可以用于开环(参见图1，步骤4)。这样的步骤可以在室温下进行(例如48h)并且允许将环三磷酸酯转化成期望的三磷酸γ-酯。
根据步骤(c)，ON保护基被裂解掉。
在步骤(c)过程中，例如，上述指定的脱保护试剂(a)和(b)可以遵循下列脱保护条件，其中X＝O，期限为20min至180min，更优选60min至约150min，特别是约120min，在60-70℃，特别是在约65℃。如果X＝NH，则这样的条件被排除，这归因于不同的结合稳定性，或仅可以在明显更恶劣的反应参数下进行，例如在RT、超过40h期限。
本发明方法的步骤(d)包含使步骤(b)的反应产物与能够跟捕获标记物Z发生相互作用的捕获试剂接触，其接触条件是允许包含捕获标记物的寡核苷酸(I)与包含在反应产物中的其它种类分离。在步骤(d)前，从固相上裂解固相结合的寡核苷酸(I)并且脱保护，即保护基部分或完全被除去。优选将捕获物试剂固定在适合的支持物上，例如色谱支持物。为了提供包含捕获标记物的寡核苷酸(I)与不包含捕获标记物的其它种类分离，从固相上裂解来自步骤(b)的反应产物，且如果必要脱保护，并且进行分离操作，优选基于捕获标记物Z与捕获物试剂的相互作用的色谱分离操作。在分离步骤期间，对于依赖于序列长度和复杂性的粗物质的一般在25-70％范围的寡核苷酸(I)的纯度可以增加至90％、91％、92％、93％、94％、95％或以上。对于毒性研究，期望纯度>85％，而在后期临床试验中，纯度应在至少90-95％。因此，本发明提供了得到作为应用于人体临床试验所需求的高纯度pppRNA的方法。
在步骤(d)中，捕获标记物和能够与之发生相互作用的捕获试剂优选地选自：(i)疏水性或氟化的基团和对疏水性或氟化的基团具有亲和性的色谱材料，例如，反相材料或氟亲和性支持物；(ii)非共价高亲和性结合对的第一配偶体和非共价高亲和性结合对的第二互补配偶体，(iii)共价结合对的第一配偶体和共价结合对的第二互补配偶体，其中第一和第二配偶体形成共价键。
以下通过一系列可能的实例来功能性地定义捕获标记物。一般规则可以是：
Z必须允许方便的纯化，并且在适合pppRNA稳定性要求的条件下应当可以去除。
另外，该方法还可以包含步骤(e)，即除去捕获标记物，得到式(IV)的寡核苷酸。根据一个优选的实施方案，对于X＝O，得到式(IV)a或(IV)b的化合物，
而对于X＝NH，得到式(IV)a的化合物
步骤(e)必须与三磷酸酯终产物的稳定性要求和核苷酸之间连接的稳定性要求相容。它可以包含：通过柔和酸性条件裂解，此时X是NH；使用银离子裂解，此时X是S；通过硫醇(例如二硫苏糖醇)裂解，从而消去硫杂丙环，此时Y-X-P包含-S-S-CH2-CH2-O-P。
在另外的实施方案中，捕获标记物Z未被除去或未被完全除去。在这些实施方案中，这样的标记的寡核苷酸可以具有用途，例如作为药物活性剂的用途。
在这些实施方案中，试剂Z-Y-XH必须选自Z-残基的亚组，其在功能上与RIG-I传感器的结构要求相容。例如，已知Z＝癸基-十八烷基，Y＝连接基，X＝NH或O组合满足这些要求。
由于其高纯度，根据本发明产生的三磷酸酯/三磷酸酯类似物修饰的寡核苷酸特别适用于药物应用。在特别优选的实施方案中，寡核苷酸(I)或(IV)是RIG-I解螺旋酶的激活剂。适合的RIG-I激活剂的具体实例公开于Schlee等，2009，上文，按引用将其内容并入本文中作为参考。
附图说明
图1显示寡核苷酸三磷酸酯衍生物的合成方法的图示概括。
图2显示癸基-O-pppRNA 24聚体合成(RNA序列：5’-GACGCUGACCCUGAAGUUCAUCUU)的RP-HPLC和ESI-LC/MS分析
(A)如下的RP-HPLC示意图：
a)包含48％癸基-O-pppRNA的粗反应混合物(RT＝14min)
b)纯癸基-O-pppRNA
柱：Hamilton PRP-1 4.1x250,10μm
梯度：0-100％B，18min,A＝100mM TEAB；B＝80％甲醇，100mM TEAB
(B)使用RP-LC/MS记录的纯癸基-O-pppRNA的ESI-MS示意图(MW计算值：7987；测定值：7968)
图3显示癸基-O-pppRNA的1μmol规模反应的半制备型规模RP-HPLC纯化：标记癸基允许有效地从未标记的副产物中分离期望的产物(级分3)，所述副产物包括合成失败的序列、全长非磷酸化OH-RNA和非衍生的pppRNA。
柱：Hamilton PRP-1 7x250mm,10μm
梯度：0-80％B，50min,A＝100mM TEAB；B＝80％甲醇，100mM TEAB.
图4：Y的尤其优选的实施方案。
图5：2’-O-甲基化筛选分辨了引入RIG-I选择性的位置。
图6：甲基化赋予双链体RIG-I特异性和高度活性。
图7：末端硫代磷酸酯增加选择性双链体的免疫原性。
图8：选择的2’-氟-取代增加双链体的RIG-I活化潜能。
图9：OH ds-RNA OH-GFP2的多修饰型导致RIG-I活化潜能明显增强。
图10：骨架修饰增强免疫原性可转移至三磷酸化双链体。
图11：显示本发明方法的图示概括，其中二甘醇一丁基醚用作作为亲核开环试剂的烷基聚乙二醇的实例。
图12：包含45％C4-DEG-pppRNA(在88.4ml梯度体积处的峰)和50％pppRNA(在77.5ml处的峰)的C4-DEG-pppRNA粗反应混合物的RP-HPLC纯化。柱：Hamilton PRP-1 7x250mm，10μm，流速3ml/min。
梯度：1-80％B，50min，A＝0.1M TEAB；B＝80％甲醇，0.1M TEAB
图13：HPLC纯化后相当于C4-DEG-pppRNA的MALDI-TOF光谱。在m/z 7972.6(A)观察到正确的质峰。
图14显示使用亲脂性聚醚复合标记物的pppRNA的合成方案。
图15显示涉及图14的HPLC纯化和MALDI数据。
(RNA序列：5’-GACGCUGACCCUGAAGUUCAUCUU)
(A)
C11-CONH-CH2CH2CH2-(O-CH2CH2)2-O-CH2CH2CH2-NHpppRNA的RP-HPLC纯化
柱：Hamilton PRP-1 4.1x250mm,10μm
梯度：0-80％B，50min；A＝100mM TEAB,B＝80％甲醇，100mM TEAB
(B)在显示存在C11-CONH-CH2CH2CH2-(O-CH2CH2)2-O-CH2CH2CH2NHpppRNA
(Mw 8214,8)的脱盐后从粗反应混合物记录的MALDI光谱
(C)纯化的C11-CONH-CH2CH2CH2-(O-CH2CH2)2-O-CH2CH2CH2NHpppRNA(Mw 7832.6)的pH＝3.8水解产物的MALDI光谱
图16显示通过任选地裂解成相应的pppRNA的胆固醇基标记的pppRNA的合成方案。
图17显示胆固醇基标记的pppRNA(RNA序列：5’-GACGCUGACCCUGAAGUUCAUCUU)的纯化和分析：
(A)胆固醇基-CONH-CH2CH2-(O-CH2CH2)2-NH-ppp-RNA的RP-HPLC纯化，柱：HamiltonPRP-1 4.1x250mm,10μm
梯度：0-10％B，5min,10％B，9min，10-100％B，33min；A＝50mM TEAB，B＝95％甲醇，50mM TEAB
(B)纯胆固醇基-CONH-CH2CH2-(O-CH2CH2)2-NH-ppp-RNA的RP-HPLC分析
柱：Hamilton PRP-1 4.1x250mm,10μm
梯度：0-100％B，18min，100％B，4min；A＝50mM TEAB，B＝95％甲醇，50mM TEAB
(C)纯胆固醇基-CONH-CH2CH2-(O-CH2CH2)2-NH-ppp-RNA的MALDI光谱。pppRNA峰(7827.3Da)归因于电离过程中的PN-裂解。
具体实施方式
实施例1：5’-癸基-O-三磷酸RNA的制备
正如概述中所概括的(图1)，癸基-O-三磷酸RNA合成方法包括下列合成步骤：
1-4)5’-癸基-O-三磷酸RNA
在合成柱内，真空干燥与支持物结合的完全被保护的5’OH-RNA(1μmol)3h，随后用无水吡啶/二噁烷(1:3,v/v,4ml)洗涤。在氩气气氛中，将新鲜制备的1M 2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮在无水二噁烷中的溶液(100μl,100μmol)注入包含2ml无水吡啶/二噁烷(1:3,v/v)的烧瓶中。将得到的50mM亚磷酸化溶液抽入合成柱，在30min反应时间过程中缓慢地往复运动。接下来通过混合双(三-正-丁基铵)焦磷酸酯在DMF中的0.5M溶液(1ml,0.5mmol)和三-正-丁基胺(238μl,1mmol)制备四(三-正-丁基铵)焦磷酸酯溶液。使焦磷酸酯溶液通过柱，在从柱上释放和弃去过量的亚磷酸化试剂后，使用两个注射器往复通过其余的焦磷酸酯溶液。10min后，用无水乙腈(3ml)洗涤柱。将5.5M叔丁基过氧化氢的癸烷溶液(300μl)溶于无水乙腈(2ml)，使其接触合成支持物。15min后，用无水乙腈(6ml)洗涤柱。随后使N-甲基咪唑(240μl,3mmol)、三-正-丁基胺(250μl,1.1mmol)和正-癸醇(2ml,10.5mmol)的均匀溶液反复通过柱，保持反应48h，以便将环三磷酸酯转化成癸基-O-三磷酸酯。用无水乙腈(6ml)洗涤柱，用0.1M三乙基铵重碳酸盐(TEAB,2ml)处理20min，以便水解未反应的环三磷酸酯，避免在随后的脱保护程序过程中的非特异性衍生。在用无水乙腈(9ml)进一步的洗涤步骤后，在氩气流中干燥合成支持物。
5-6)脱保护和纯化
使用两支注射器使5’-癸基-O-三磷酸寡核苷酸接触新鲜制备的40％甲胺水溶液和浓氨水溶液(AMA,1:1,v/v,2ml)。30min裂解时间后，将该溶液转入清洁的螺口小瓶并且用AMA(1ml)冲洗支持物。将合并的溶液和洗涤液在65℃加热10min。用冰冷却后，将该溶液蒸发至干并且通过与无水乙醇共蒸发干燥残余物。为了除去2’-O-TBDMS保护基，可以使用三乙胺三氢氟酸盐(TEA.3HF)处理，而不会明显损耗修饰的三磷酸酯部分。将癸基-O-三磷酸寡核苷酸再溶于新鲜制备的N-甲基吡咯烷酮/三乙胺/TEA.3HF溶液(NMP/TEA/TEA.3HF,6:4:3,v/v,325μl)，将该溶液在65℃加热2h。或者，可以使用TEA.3HF在DMSO中的脱保护溶液(1:1,v/v,600μl)。使用正丁醇从脱保护溶液中沉淀完全脱保护的癸基-O-三磷酸寡核苷酸，通过HPLC纯化。亲脂性癸基-标记物允许通过使用反相色谱法从不含该标记物的杂质中分离癸基-O-三磷酸。使反应产物上7x250mm PRP-1柱，以0－100％缓冲液B的线性梯度在50min内以3ml/min的流速分离。缓冲液A为100mM TEAB，缓冲液B为100mM TEAB的80％甲醇溶液。收集产物级分，蒸发，通过反复与甲醇一起共蒸发脱盐。将残余物溶于水，通过在0.3M氯化钠的存在下乙醇沉淀转化成钠形式。
实施例2
5'-pppRNAγ2-(2-丁氧基乙氧基)醚-酯的制备
(C4-DEG-pppRNA)
实施例2中所述的反应方案概括如图11中所示。
步骤1：在氩气气氛中在10mL隔膜小瓶中将203mg(1mmol)2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮溶于1mL无水二噁烷。
步骤2：将包含完全被保护的被滴定的(detitrylated)和用乙腈充分洗涤的RNA的合成柱真空干燥12h。在氩气气氛中通过反复抽入和排出2mL无水二噁烷/吡啶溶液3:1(v/v)充分洗涤柱内含物。
步骤3：首先向小瓶中加入2mL吡啶/二噁烷3:1v/v，然后加入100μL 1M 2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮溶液在无水二噁烷中的溶液，得到50mM亚磷酸化试剂，例如2-氯-4H-1,3,2-苯并二氧磷杂环己烷-2-酮在二噁烷/吡啶3:1(v/v)中的溶液。通过适度振摇匀化该溶液。通过将2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮溶液从小瓶中抽入合成柱启动反应。
在反应过程中，从合成柱上反复抽入和排出包含的2-氯-4H-1,3,2-苯并二氧磷杂环己烷-4-酮溶液，以便允许充分接触固相支持的RNA并与之良好混合。30min反应时间通常得到20-40nt范围的支持物结合的寡聚体的游离5'-OH基团的接近定量的反应。
步骤4：30min反应时间后，将包含过量亚磷酸化试剂的二噁烷/吡啶溶液排入废物容器，给新注射器填充涡旋的 1mL 0.5M(Bu3NH)2焦磷酸酯在无水DMF和238μL(1mmol)无水Bu3N中的混合物，得到0.5M(Bu3N)4焦磷酸酯溶液。使该溶液通过柱，由此替代二噁烷/吡啶溶液。较大过量的焦磷酸酯确保中间体定量转化成P(III)-P(V)环酐IIa。
步骤5：用3mL CH3CN洗涤柱以除去DMF和过量的PPi，并且填充具有无水CH3CN的柱反应器。
步骤6：将300μL t-BuOOH(5.5M癸烷溶液,Sigma-Aldrich)溶于2mL无水CH3CN，得到约0.7M的均匀溶液。使合成支持物接触该溶液15min以便得到氧化的P(V)环酐IIb。
步骤7：用3mL无水CH3CN洗涤柱以除去过量的过氧化物并且向其中填充无水CH3CN。
步骤8：使2ml包含0.1M N-甲基咪唑和0.1M三-正-丁基胺的无水2-(2-丁氧基乙氧基)乙醇(二甘醇一丁基醚)接触柱中的支持物。CPG与醇的接触时间在室温应至少为48hrs。
步骤9：用9mL乙腈充分洗涤柱，然后使柱接触2ml 0.1M TEAB(三乙基铵重碳酸盐)的水溶液1hr，以便水解未翻译的环三磷酸酯。
步骤10–脱保护的第一阶段：使1mL脱保护溶液(40％甲胺水溶液/浓氨水1:1v/v.AMA试剂)通过支持物2-3次。在接触30min后，将该溶液转入新的小瓶。用相同体积的AMA脱保护溶液洗涤支持物，合并洗涤液。在65℃加热合并的溶液和洗涤液10min。用冰冷却后，将该溶液浓缩至300-500μL体积，然后蒸发至干。
步骤11-2'-O-TBDMS保护基的除去：通过添加和共蒸发300μL无水EtOH干燥残余物，加入1mL无水1M TBAF(氟化四-正-丁基胺)的THF溶液，密封，置于振荡器上16h。用1mL无菌1M TEAB水溶液(三乙基铵重碳酸盐)使反应停止，用NAPTM-25(核酸纯化)柱、应用无菌水作为洗脱液使其脱盐。通过无菌2μm滤膜过滤在该步骤时是必不可少的。合并和蒸发吸收UV的级分至150μL体积，添加100mL 1M TEAB pH8，将该溶液冷冻储存在-20℃，直到可以进行HPLC纯化为止。
步骤12-HPLC纯化：将来自步骤11的1μmol规模的反应混合物的反应产物上载入7x25mm PRP-1柱(Hamilton)。使用梯度缓冲液B在 0－80％的线性梯度在50min内、以3mL/min的流速进行纯化。缓冲液A是100mM TEAB，缓冲液B是100mM TEAB的甲醇/水8:2v/v溶液。24-mer纯化的典型实例如图3中所示。4原子烷基标记物和二甘醇残基的组合对于从pppRNA的接近基线的分离是足够的。合并相当于在88.4ml的峰的级分，用旋转蒸发器蒸发，通过用无水甲醇几次共蒸发脱盐。
实施例3
使用具有预形成的亲脂性聚醚胺的亲脂性聚醚复合标记物衍生pppRNA
实施例3使用结构ZY-H的亲脂性聚醚胺类生成ZYNHpppRNA
A部分：亲脂性聚醚胺的经济性大规模制备
该方法不使用闪蒸塔色谱法进行且一般适用于由水溶性二胺类和亲脂性羧酸的甲酯类合成一酰胺类。
N-月桂基-4,7,10-三氧杂-1,13-十三烷二胺的合成(化合物3,图14)：将4,7,10-三氧杂-1,13-十三烷二胺(43.8ml,200mmol)溶于32.5ml甲醇。在搅拌下缓慢地加入月桂酸甲酯(4.92ml,20mmol)，将密闭的烧瓶保持在室温下5天。用旋转蒸发器从反应混合物中除去甲醇，将残余物溶于100ml乙酸乙酯。
通过用水(2*120ml)萃取、然后用浓氯化钠(2*100ml)萃取除去过量的二胺。蒸发乙酸乙酯相，在-20℃静置时结晶出残余油状物，得到6.8g(17mmol)纯N-月桂基4,7,10-三氧杂-1,13-十三烷二胺。
B部分：有关CPG结合的RNA环三磷酸与N-月桂基-4,7,10-三氧杂-1,13-十三烷二胺的开环反应
根据上述实施例2中的步骤1-7合成CPG结合的RNA环三磷酸酯(化合物4,图14)。使用0.08M N-月桂基-4,7,10-三氧杂-1,13-十三烷二胺在乙腈中的溶液进行固相固定化RNA环三磷酸酯的开环反应。使乙腈溶液在室温接触CPG 3hrs，然后用10ml无水乙腈洗涤支持物，用氩气净化。从支持物上裂解，脱保护，再根据实施例2的步骤10-12确切地进行进一步HPLC处理。在75％甲醇浓度洗脱产物5(图15A)。反应混合物的MALDI光谱分析证实化合物5的结构(Mw 8214.8Da,图15B)。
任选地，可以除去纯化标记物，得到相应的三磷酸寡核苷酸：在2ml埃彭道夫(Eppendorf)管中将100nmol正-月桂基取代的γ酰胺(化合物5,图14)溶于400μl pH 3.8脱保护缓冲液，将密封试管在60℃加热70min。这些条件导致化合物5的氨基磷酸酯键定量裂解，无三磷酸酯部分的降解。用冰冷却该反应混合物，加入14μL无菌5M NaCl溶液和1.2mL无水乙醇。通过离心收集沉淀，用冷乙醇洗涤，用Speed Vac干燥，溶于无菌水，冷冻储存在-20℃。
MALDI光谱分析证实pppRNA产物的预期MW(Mw 7832Da,图15C)。
实施例4：
使用亲脂性聚醚复合标记物衍生pppRNA的柱上方法
实施例4是使用固定化HYNHpppRNA寡核苷酸柱上衍生以生成ZYNHpppRNA的两步法
胆固醇基-标记的三磷酸RNA的制备
(Chl-CONH-CH2CH2-(O-CH2CH2)2-NH-ppp-RNA，化合物4，图16)
亲脂性胆固醇基-标记物与三磷酸RNA缀合允许通过RP-HPLC有效地分离标记物-pppRNA产物与未标记的杂质。用于制备胆固醇基-标记的三磷酸RNA的反应方案的概括如图16中所示。在第一步中，如实施例2步骤1-7中所述将支持物结合的完全被保护的5’OH-RNA转化成环三磷酸酯中间体(化合物2,图16)。
随后，用无水乙腈(3ml)洗涤柱。将176μl 2,2’-(亚乙二氧基)二乙胺(化合物1,图16；1,2mmol)溶于无水乙腈(1ml)，使该溶液接触柱上的支持物。3min后，用无水乙腈(3ml)和无水二氯甲烷(6ml)洗涤柱。
随后，用氯甲酸胆固醇酯(36mg,80μmol)、N,N-二异丙基乙胺(13.9μl,40μmol)和4-二甲氨基吡啶(4.9mg,40μmol)在无水二氯甲烷(5ml)中的预混合溶液将氨基-修饰的三磷酸酯(即化合物3,图16)处理15min。用无水二氯甲烷(3ml)和无水乙腈(6ml)洗涤柱，在氩气流中干燥。为了从支持物上裂解和脱保护，使用两支注射器使衍生的寡核苷酸接触新鲜制备的40％甲胺水溶液和浓氨水的溶液(AMA,1:1,v/v,2ml)。30min裂解时间后，将该溶液转入螺口小瓶，在65℃加热10min，蒸发至干。通过与无水乙醇一起共蒸发干燥残余物，用1M氟化四-正-丁基铵的THF溶液(1ml,1mmol)振摇处理16h。用NAP-25柱脱盐后，通过HPLC、使用反相柱(Hamilton PRP-1,4.1x250mm,图17A)纯化完全脱保护的胆固醇基-标记的三磷酸寡核苷酸。在95％甲醇浓度洗脱的化合物4和预期的结构通过MALDI分析证实(Mw 8370,6Da,图17C)。
任选地，按照与实施例3中所述系统的方法，通过在pH 3.8在60℃酸水解除去胆固醇基-标记物，得到相应的三磷酸寡核苷酸(化合物5,图16)。
实施例5：2'-O-甲基化的系统性引入产生RIG-I选择性RNA双链体
在本申请文本结尾处可以找到缩写及其含义的列表。
身体自身的RNA的特征在于可以影响RNA功能(tRNAs和rRNAs)的多种修饰。2'-O-甲基化由此能够改变RNA的免疫原性。选择性RIG-I配体由此应不含一个以上2'-O-甲基化/链。通过组合适当的甲基化有义和反义链，这些特性包括在RNA双链体中。
2'-O-甲基化筛选分辨导入RIG-I选择性的位置(图5)。对于OH-GFP2的有义(A)和反义(B)链，进行完全2’-O-甲基化筛选。每个位置各自被2’-O-甲基化修饰并且用于刺激RIG-I、TLR7或TLR8。为了刺激RIG-I，用氯喹阻断外周血单核细胞(PBMCs)并且通过脂转染双链体刺激。刺激后20h通过ELISA测定IFNa。对于TLR7和TLR8，仅筛选相关的单-链的刺激容量，并且用未阻断的PBMCs检查。通过与聚-L-精氨酸复合转染单-链，并且在转染后20h，通过ELISA测定IFNa(对于TLR7活化)、IL12p70(对于TLR8活化)。对于有义链使用GFP2的100％诱导相当于：对于RIG-I，3085pg/ml IFNa；对于TLR7，2758pg/ml IFNa；和对于TLR8，1206pg/ml IL12p70。对于反义链，100％GFP2相当于2342pg/ml IFNa，对于RIG-I；1831pg/ml IFNa，对于TLR7；和3018pg/ml IL12p70，对于TLR8。2’-O-甲基化的位置依赖性诱导的影响的概括以表格形式给出(图5)。将效应对作为参比的未修饰的链校准。白色＝未改变，黄色＝免疫刺激减少，红色＝无免疫刺激，绿色＝增加的免疫刺激。
此外，可以证实一些甲基化赋予双链体RIG-I特异性和高度活性。
图6：(A)在反义链中合并用于RIG-I活化的甲基化惰性，得到的双链体以0.8μg/ml浓度用于刺激氯喹-阻断的PBMCs。在刺激后20h通过ELISA测定IFNa。100％OH-GFP2类似于2729pg/ml IFNa。(B)使2’-O-甲基化的不同组合在有义和反义链中杂交并且用于刺激PBMCs。为了刺激RIG-I，用氯喹阻断它们并且用阳离子脂质体(Lipofectamine)转染，对于TLR7和TLR8，使用未阻断的PBMCs并且用与聚-L-精氨酸复合的双链体转染。对于IVT-2的100％相当于RIG-I的4861pg/ml IFNa，在9.2S 100％相当于对TLR7的1975pg/ml IFNa，以及对TLR8的771pg/ml IL12p70。(A)和(B)显示3个供体的平均值±SEM。
实施例6：通过插入稳定RNA的修饰增加RIG-I活化
在siRNA疗法过程中，将短dsRNA片段掺入体内，其中它们抑制靶细胞中特异性蛋白质形成。使用这种形式的疗法的问题在于siRNA双链体的巨大不稳定性。RNA易于在输送至靶细胞过程中被血清中的外切核酸酶和内切核酸酶降解。
显然可能的情况是，用于治疗目的的大部分外源性RNA在它所施用的受试者血清和胞液中变性。
可以证实，每个5'-PTO修饰对双链体免疫活性具有积极影响，且有义和反义链中5'PTO的组合导致活性增加最大(s15/as3对s15PTO/as3PTO；图7B)。这样的RNA骨架修饰在RIG-I选择性配体中的应用由此可以被视为有益性的。
特别地，可以看出5'-硫代磷酸酯增加选择性双链体的免疫原性。
图7：(A)用氯喹-阻断PBMCs并且用双链体以0.8μg/ml浓度刺激。在刺激后20h通过ELISA测定IFNa。100％IVT2相当于6138pg/ml IFNa。(B)滴定所示的双链体的浓度并且用于刺激RIG-I。用氯喹阻断PBMCs并且使用阳离子脂质体转染双链体。在刺激后20h通过ELISA测定上清液中的IFNa。100％50nM 3P-GFP2相当于16044pg/ml IFNa。结果显示了4个供体的平均值±SEM。
在有关治疗siRNAs的研发过程中，血清稳定性的问题通过另一种修饰解决：如果将2'-氟取代掺入RNA，则得到的RNAs在核酸酶消化方面更为稳定。然而，观察到在RIG-I依赖性免疫刺激的情况中，2'-氟取代可以激活免疫刺激。
特别地，可以证实5'-硫代磷酸酯增加选择性双链体的免疫原性。
然而，根据本发明，发现选择的2'-氟取代增加双链体的RIG-I活化潜能。
对于双链体的有义(A)和反义(B)链，每个位置各自被 2’-氟-取代修饰(图8)。得到的双链体以0.8μg/ml浓度用于刺激PBMCs。为了评价RIG-I活化，阻断PBMCs并且用阳离子脂质体转染，为了刺激TLR7和TLR8，使用未阻断的PBMCs并且用与聚-L-精氨酸复合的双链体双链体转染。刺激后20h通过ELISA测定细胞因子。有义链中对于GFP2 100％类似于RIG-I的2407pg/ml IFNa、对TLR7的3281pg/ml和对TLR8的1990pg/ml。对于反义链，100％GFP2相当于RIG-I的4512pg/ml IFNa、对TLR7的4691pg/ml IFNa和对TLR8的1997pg/ml IL12p70。显示了4个供体的平均值±SEM。
因此，极为重要的是建立尽可能有活性的双链体，其因强活性而可以补偿RNA损失。在上述段落中，描述了RNA修饰如何能通过选择性PTO结合和2'-氟取代被鉴定，其导致双链体活性增加。
实施例7：所有修饰的组合产生强烈免疫原性RIG-I配体
为了测试PTO结合或2'-氟取代是否可以与2'-O-甲基化组合以增加选择性，将它们逐步地合并在双链体中。
用氯喹阻断PBMCs并且用于刺激RIG-I。使用基于OH-GFP2的差别修饰的双链体，且在刺激后20h，通过ELISA对IFNa测定RIG-I活化(图9)。将多重修饰的双链体与OH-GFP2(A)或三磷酸化3P-GFP2(B)对比。100％IVT 4相当于21305pg/ml的INFa。显示了2个供体的平均值±SEM。
首先将2'-O甲基化s15/as3加入到起始RNA OH-GFP2中(OH-GFP2oMet15s/oMet3as,图9A)，且然后与有义和反义链中的5'-PTO修饰比较(OH-GFP2PTO 5',图9A)。随后将两种类型的修饰与双链体合并(OH-GFP2oMet15/oMet3PTO,图9A)。最终，生成双链体，其还包含在有义链中的7&23位和反义链中的13位的3个2'-氟取代(OH-GFP2oMet15/3-F23/13-PTO,图9A)。这些RNA双-链的对比滴定显示多重-修饰的寡OH-GFP2oMet15/3-F23/13-PTO在活性方面超越了其它双链体很多。为了更好地评价其活性，滴定双链体并且与其未修饰的三磷酸双链体对比(图9B)。发现插入修饰能够改变寡核苷酸，使得其免疫活性可以与其三磷酸酯等效物比拟(3P-GFP2,图9B)。
实施例8：要素可转移至3P-dsRNA系统中
RIG-I选择性配体的完整研发在序列GFP2的OH水平上进行。尽管活性因适当的修饰而得到无限增加，但是配体的三磷酸化也可以增加其活性。
迄今为止，尚不清楚修饰及其相应的定位是否可以这样被转移至3P-GFP2系统。
为了找到针对这一问题的答案，生产双链体，其均具有增进活性的修饰：在有义链中第15位碱基上的2'-O甲基化；在第7和23位碱基上的2'-氟取代；和在5'和3'端上两个PTO的结合(2S2F,图10)。在反义链中，合并在第3位碱基上的2'-O甲基化、在第13位碱基上的2'-氟取代和两个5'-PTO结合。为了避免有义链上的两个5'-PTO结合因其邻近三磷酸酯而中断，还生产了无5'-PTOs的多重-修饰的有义链(1S2F,图10)。
为了能够估算因三磷酸化导致的活性增加，各自使用5'-羟基和5'-三磷酸生产双链体(OH与3P,图10)并且根据彼此比较有关PBMCs的刺激滴定。
图10：(A)使用和不使用三磷酸酯合成多重修饰的双链体。对氯喹阻断的PBMCs进行剂量滴定。在刺激后20h，通过ELISA测定对IFNa的免疫活化。显示了4个供体的平均值±SEM。(B)基于滴定曲线计算生物EC50值。
发现多重-修饰的OH双链体可以达到OH-GFP2未修饰形式的活性极限，与存在一个或两个PTO末端无关(OH多1S2F,OH多2S2F,OH-GFP2,图10)。在有义链上另外添加5'-三磷酸与羟基复合物对比将活性增加了约5倍(3P-多1S2F,3P-多2S2F,图10)。
作为结果，观察到不同修饰的增加活性的效应可以与寡核苷酸的三磷酸化组合且它们也可以在其中显示其积极的影响。
通过原始双链体OH-GFP2在选择性水平上(2'-O甲基化)的逐步递增，通过稳定修饰(PTO结合和2'-氟取代)和对结合袋(三磷酸酯)的亲和力导致活性增加，使确定有效的选择性RIG-I配体(3P-多 1S2F)成为可能。
最后，可以研发3P-dsRNA，其中免疫刺激活性因修饰的特异性插入而增加至最大。这种免疫原性的增加使得如下情况成为可能：将药物剂量保持在低水平上并且补偿从药物应用开始到进入细胞的过程中发生的RNA损失。
RNA寡核苷酸
Claims (17)
1.式(I)的修饰的寡核苷酸，
其中V1、V3和V5在每种情况中独立地选自O、S和Se；
V2、V4和V6在每种情况中独立地选自OH、OR1、SH、SR1、F、NH2、NHR1、N(R1)2和BH3-M+；
W1是O或S；
W2是O、S、NH或NR2；
W3是O、S、NH、NR2、CH2、CHHal或C(Hal)2；
R1、R2和R3选自C1-6烷基、C2-6烯基、C2-6炔基、C2-6酰基或环状基团，其各自任选地被取代；
或其中两个R1可以与所结合的N-原子一起形成环；
M+是阳离子；
X是NH、或O；
Z表示捕获标记物或H；
Y是-K-((CHR1)m-CH2-O)n-R；或
-(O-(CHR3)m3-CH2)n1-(O-(CHR2)m2-CH2)n2-(O-(CHR1)m1-CH2)n3-；
K是O或NH；
m、m1、m2和m3独立地是1－12；
n、n1、n2和n3独立地是0－20；且
R1、R2和R3独立地是H、C1-6烷基、C2-6烯基、C2-6炔基、C2-C6-酰基或环状基团，其各自任选地被取代；且
R是C1-6烷基、C2-6烯基、C2-6炔基、C2-C6-酰基或环状基团，其各自任选地被取代；且
ON表示包含至少4个核苷酸或核苷酸类似物结构单元的寡核苷酸。
2.权利要求1的修饰的寡核苷酸，其中V1、V2、V3、V4、V5、V6、W1、W2和W3是O。
3.权利要求1或2的修饰的寡核苷酸，其中Z是长链脂族残基、非共价高亲和力结合对的配偶体、反应性化学实体、Q或NHC2-C24烷基，其中所述非共价高亲和力结合对的配偶体选自：生物素、脱硫生物素、半抗原或抗原；所述反应性化学实体选自：能够与含有互补反应基的捕获试剂共价反应的叠氮化物或炔基基团；所述Q选自H、氨基酸、氨基酸类似物、C1-C24烷基，C12-C24烷基、肽类和脂质。
4.权利要求1－3任一项的修饰的寡核苷酸，其中该寡核苷酸选自单链或双链脱氧核糖核苷酸、核糖核苷酸和寡核苷酸类似物，它们可以任选地在脱氧核糖核苷酸、核糖核苷酸或寡核苷酸类似物的核苷和/或核糖上被化学修饰。
5.权利要求4的修饰的寡核苷酸，其中该寡核苷酸是双链的且所述双链的每条链具有至少19个核苷酸长度。
6.权利要求4-5任一项的修饰的寡核苷酸，其中所述化学修饰维持、建立或增强所述寡核苷酸的选择性，特别是寡核苷酸的RIG-I选择性，和/或其中所述化学修饰维持或增强所述寡核苷酸的化学稳定性。
7.权利要求4-6任一项的修饰的寡核苷酸，其中所述化学修饰独立地选自卤化，2'-O-烷基化，和/或核苷酸间连接键之间的至少一种硫代磷酸酯修饰。
8.权利要求4-6任一项的修饰的寡核苷酸，其中所述化学修饰独立地选自F卤化，2'-O-甲基化，和/或核苷酸间连接键之间的至少一种硫代磷酸酯修饰。
9.权利要求1－8任一项的修饰的寡核苷酸，其中
X是NH；
Y是-K-(CH2-CH2-O)n；
K是NH；
n独立地是0-20；并且
其中K进一步被胆固醇基-C(O)-或三苯甲基取代。
10.权利要求1-8任一项的修饰的寡核苷酸，其中Y选自：
-O-(CH2-CH2-O)n-CH2-CH2-,
-O-((CH2)m-CH2-O)n-CH2-CH2-,
-O-((CHR)m-CH2-O)n-CH2-CH2-,
-O-(CH2-CH2-CH2-O)n-CH2-CH2-,
-O-((CH2)m-CH2-CH2-O)n-CH2-CH2-,和
-O-((CHR)m-CH2-CH2-O)n-CH2-CH2-。
11.权利要求1-8任一项的修饰的寡核苷酸，其中
(a)R1和R2是H；且n1＝0，n2＝1，且n3＝1；或者
(b)R1、R2和R3是H；且n1、n2和n3是1。
12.权利要求1－11任一项的修饰的寡核苷酸，其中
Z是C1-C12烷基，优选C10；和Q或NHC2-C24烷基，其中Q选自H、氨基酸、氨基酸类似物、C1-C24烷基、C12-C24烷基、肽类和脂质；且
V1、V2、V3、V4、V5、V6、W1、W2和W3是O。
13.修饰的寡核苷酸，其中所述修饰的寡核苷酸由式(5)或式(4)表示：
14.药物组合物，包含权利要求1-13任一项的修饰的寡核苷酸。
15.权利要求14的药物组合物，其中所述药物组合物通过皮内施用。
16.权利要求15的药物组合物，其中所述药物组合物通过纹身术、显微操作针法和/或显微操作针斑贴施用。
17.制备权利要求1-13任一项的寡核苷酸的方法，包含下列步骤：
(a)使式(IIa)的化合物与氧化剂反应
其中V1、V3、V5、V4、V6、W1、W2、W3和ON如上述所定义，其中ON被至少一个保护基保护，得到式(IIb)的化合物
其中V1、V3、V5、V2、V4、V6、W1、W2、W3和ON如上述所定义，其中ON被至少一个保护基保护；
(b)使式(IIb)的化合物与式(III)的捕获标记物试剂反应，
Z-Y-XH (III),
其中X,Z,和Y如上述所定义，其中X优选是O，得到包含式(I)的寡核苷酸的反应产物；和
(c)使所述至少一个ON保护基脱保护；和
(d)使步骤(b)的反应产物接触能够与捕获标记物发生相互作用的捕获试剂，其中该接触在能够使寡核苷酸(I)与所述反应产物中包含的其它种类分离的条件下进行；以及任选地还包括下列步骤：
(e)除去捕获标记物，得到寡核苷酸，其中对于X＝O，得到式(IVa)或(Ivb)的化合物；
且对于X＝NH，得到式(IVa)的化合物
其中
捕获标记物和能够与其发生相互作用的捕获试剂选自：
(i)疏水性或氟化基团和对于疏水性或氟化基团具有亲和力的色谱材料，例如反相材料或氟亲和支持物；
(ii)非共价结合对的第一配偶体和非共价结合对的第二配偶体；和
(iii)共价结合对的第一配偶体和共价结合对的第二配偶体，其中第一和第二配偶体形成共价键。
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