申请人:Illumina Cambridge Limited
公开号:EP2607369A1
公开(公告)日:2013-06-26
The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached thereto, such that the 3' carbon atom has attached a group of the structure
-O-Z
wherein Z is any of -C(R')2-O-R", -C(R')2-N(R")2,-C(R')2-N(H)R", -C(R')2-S-R" and -C(R')2-F,
wherein each R" is or is part of a removable protecting group;
each R' is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R')2 represents an alkylidene group of formula =C(R"')2 wherein each R''' may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and
wherein said molecule may be reacted to yield an intermediate in which each R" is exchanged for H or, where Z is -C(R')2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3'OH;
with the proviso that where Z is -C(R')2-S-R", both R' groups are not H.
本发明提供了修饰的核苷酸或核苷分子,该分子包含一个
嘌呤或
嘧啶碱基和一个
核糖或脱氧
核糖糖基,
核糖或脱氧
核糖糖基上共价连接有一个可移动的 3'-OH 封闭基团,从而使 3'碳原子上连接有一个结构如下的基团
-O-Z
其中 Z 是-C(R')2-O-R"、-C(R')2-N(R")2、-C(R')2-N(H)R"、-C(R')2-S-R "和-C(R')2-F 中的任意一个
其中每个 R "是可拆卸保护基团的一部分;
每个 R'独立地为氢原子、烷基、取代烷基、芳烷基、烯基、炔基、芳基、杂芳基、杂环基、酰基、
氰基、烷氧基、芳氧基、杂芳氧基或
氨基,或通过连接基团连接的可检测标签;或 (R')2 代表式 =C(R"')2的亚烷基,其中每个 R''可以相同或不同,并选自包括氢原子、卤素原子和烷基在内的基团;及
其中,所述分子可通过反应生成一种中间体,在该中间体中,每个 R "被交换为 H,或者,当 Z 为-C(R')2-F 时,F 被交换为 OH、SH 或 NH2,优选 OH,该中间体在
水性条件下解离,生成具有游离 3'OH 的分子;
但在 Z 为-C(R')2-S-R "的情况下,两个 R'基团都不是 H。