CN101289486B - 一种合成rna单体的方法 - Google Patents
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Abstract
本发明公开了一种合成RNA单体的方法。该方法中所采用的RNA合成单体以其独特的2’-保护基为出发点，RNA合成单体结构不同于以往的RNA合成单体。其特点在于该保护基在酸性脱保护条件下稳定，在硷性脱保护条件下也很稳定。在固相合成完成后，在把5’-DMT保护基脱掉而2’-保护基仍在链上的情况下可以进行高压液相纯化，一级纯化后的产品可以在适当的条件下将2’-保护基完全脱下来，进一步纯化后即可与另一条互补链退火成双链。由此合成及纯化获得的RNA寡核苷酸纯度可达97％以上，纯化回收率可达55％以上。
Description
本发明公开了一种合成RNA单体的方法。该方法中所采用的RNA合成单体以其独特的2’-保护基为出发点，RNA合成单体结构不同于以往的RNA合成单体。其特点在于该保护基在酸性脱保护条件下稳定，在硷性脱保护条件下也很稳定。在固相合成完成后，在把5’-DMT保护基脱掉而2’-保护基仍在链上的情况下可以进行高压液相纯化，一级纯化后的产品可以在适当的条件下将2’-保护基完全脱下来，进一步纯化后即可与另一条互补链退火成双链。由此合成及纯化获得的RNA寡核苷酸纯度可达97％以上，纯化回收率可达55％以上。
技术领域
本发明涉及一种RNA单体的制备方法。
背景技术
RNA单体目前由Glen Research，Transgenomic，ChemGene等少数几个大公司垄断生产，国内无任何RNA单体生产厂家；我们依靠自有技术生产的RNA单体质量指标高于现有厂家生产产品；
发明内容
本发明的目的在于采用独特的2’-保护基为出发点，合成RNA单体。本发明的上述目的是通过下述技术方案实现的：
A.制备2’-保护基1-(3-氯苯)-4-哌啶烯(CPP)；
B.尿苷单体合成；
C.胞苷单体合成；
D.腺苷单体合成；
E.鸟苷单体合成；
所述的RNA单体合成方法，其特征在于：
采用独特的2’-保护基1-(3-氯苯)-4-哌啶烯(CPP)为出发点，合成RNA单体。
附图说明
图1为CPP制备方法结构图：
图2为尿苷单体合成流程图：
图3为胞苷单体合成流程图：
图4为腺苷单体合成流程图：
图5为鸟苷单体合成流程图：
具体实施方式
2’-保护基CPP的制备方法：
3-氯丙酰氯与乙烯在刘易斯酸的作用下生产1，5-二氯戊酮，再与3-氯苯胺缩合形成N-(3-氯苯)-哌啶酮，后者再与甲醇缩合生成二甲基-N-(3-氯苯)-哌啶缩酮，最后再在减压加热条件下脱掉一分子甲醇生成所要的1-(3-氯苯)-4-哌啶烯(CPP)；
RNA合成单体的制备：
尿苷单体：3’，5’-氢氧基用Marcwicz保护基进行保护，然后在酸性条件下引入2’-CPP保护基，随后脱去Marcwicz保护基，5’-氢氧基用1，4-二甲氧基三苯甲烷氯(DMTrCl)进行保护，最后在3’-位用磷酸化试剂进行亚磷酰胺化，制得尿苷单体。
胞苷单体、腺苷单体、鸟苷单体制备：将胞苷/腺苷/鸟苷的碱基先用苯甲酰基保护，后继步骤与鸟苷相同。
Claims (2)
1.一种RNA单体的合成方法，其特征在于独特的2’-保护基为出发点，RNA合成单体结构不同于以往的RNA合成单体，合成方法包括如下步骤：
A.制备2’-保护基1-(3-氯苯)-4-哌啶烯；
B.尿苷单体合成：3’，5’-氢氧基用Marcwicz保护基进行保护，然后在酸性条件下引入2’-1-(3-氯苯)-4-哌啶烯保护基，随后脱去Marcwicz保护基，5’-氢氧基用1，4-二甲氧基三苯甲烷氯进行保护，最后在3’-位用磷酸化试剂进行亚磷酰胺化，制得尿苷单体；
C.胞苷单体合成：将胞苷的碱基先用苯甲基保护，然后3’，5’-氢氧基用Marcwicz保护基进行保护，然后在酸性条件下引入2’-1-(3-氯苯)-4-哌啶烯保护基，随后脱去Marcwicz保护基，5’-氢氧基用1，4-二甲氧基三苯甲烷氯进行保护，最后在3’-位用磷酸化试剂进行亚磷酰胺化，制得胞苷单体；
D.腺苷单体合成：将腺苷的碱基先用苯甲基保护，然后3’，5’-氢氧基用Marcwicz保护基进行保护，然后在酸性条件下引入2’-1-(3-氯苯)-4-哌啶烯保护基，随后脱去Marcwicz保护基，5’-氢氧基用1，4-二甲氧基三苯甲烷氯进行保护，最后在3’-位用磷酸化试剂进行亚磷酰胺化，制得腺苷单体；
E.鸟苷单体合成：将鸟苷的碱基先用苯甲基保护，然后3’，5’-氢氧基用Marcwicz保护基进行保护，然后在酸性条件下引入2’-1-(3-氯苯)-4-哌啶烯保护基，随后脱去Marcwicz保护基，5’-氢氧基用1，4-二甲氧基三苯甲烷氯进行保护，最后在3’-位用磷酸化试剂进行亚磷酰胺化，制得鸟苷单体。
2.如权利要求1所述的RNA单体的合成方法，其特征在于，所述获得的RNA单体的化学结构为：
(1)尿苷单体：
(2)胞苷单体：
(3)腺苷单体：
(4)鸟苷单体：
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007100395684A CN101289486B (zh) | 2007-04-18 | 2007-04-18 | 一种合成rna单体的方法 |
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| CN2007100395684A CN101289486B (zh) | 2007-04-18 | 2007-04-18 | 一种合成rna单体的方法 |
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| CN101289486B true CN101289486B (zh) | 2012-05-30 |
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| EP2508530A1 (en) * | 2011-03-28 | 2012-10-10 | Rheinische Friedrich-Wilhelms-Universität Bonn | Purification of triphosphorylated oligonucleotides using capture tags |
| EP2712870A1 (en) * | 2012-09-27 | 2014-04-02 | Rheinische Friedrich-Wilhelms-Universität Bonn | Novel RIG-I ligands and methods for producing them |
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