4'-C-(2-甲氧乙氧基)修饰的2'β-F-/2'α-F-2'-脱氧尿苷的合成及其修饰的反义核酸性质评价  

Synthesis and antisense properties of 2'β-F- and 2'α-F-2'-deoxy-uridines modified oligonucleotides with 4'-C-(2-methoxyethoxy) substituent

作　　者：马薇[1,2] 王靖[2,3] 鲁丹丹[2] 杨静[2] 秦炳杰[4] 何小羊[2] 王升启[2] 

机构地区：[1]天津中医药大学研究生院,天津300193 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]郑州大学药学院,河南郑州450001 [4]军事医学科学院毒物与药物研究所,北京100850

出　　处：《药学学报》2016年第8期1271-1280,共10页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81402841);国家"863"计划资助项目(2012AA022501);国家科技重大专项资助项目(2013ZX09-304-102;2012ZX09301003-001-004)

摘　　要：核酸的化学修饰是反义寡核苷酸分子成药的关键技术,本文以相应的2'-氟阿拉伯糖尿苷(2'-F-ara U)及2'-氟尿苷(2'-F-r U)为起点,通过在糖基的4'-位引入2-甲氧乙氧基(MOE),合成了新的单体2'-F-4'-C-MOE-ara U及其差向异构体2'-F-4'-C-MOE-r U,并将它们转化为相应的亚磷酰胺,用于所需DNA序列的合成。它们对于ds DNA和DNA-RNA双螺旋的热稳定性的影响显示,与2'-F-4'-C-MOE-r U及2'-F-ara U修饰的寡核苷酸相比,2'-F-4'-C-MOE-ara U修饰,特别是在5'-嘧啶-嘌呤-3'(5'-pyrimidine-purine-3')步序上形成C-H…F-C假氢键时,可显著增强寡核苷酸对互补RNA的亲和力,并能够保持对互补DNA的亲和力。同时,2'-F-4'-C-MOE-ara U修饰后的寡核苷酸对完全配对的RNA/DNA单链均具有良好的结合特异性;而2'-F-4'-C-MOE-r U仅对互补RNA具有较好的杂交能力。不过,3'-末端2'-F-4'-C-MOE-ara U修饰的寡核苷酸对核酸酶水解的稳定性却低于2'-F-4'-C-MOE-r U修饰。这些结果表明了在2'-F-ANA单元上进一步引入4'-位修饰的可行性,以及2'-F取代单体对于双螺旋结构的稳定性具有显著影响,为进一步的核酸化学修饰和反义核酸药物的开发提供了新的技术支持。Chemical modification is critical for the therapeutic applications of antisense oligonucleotides. Novel 4＇-C-MOE and 2＇-fluoro- modified monomer 2＇-F-4＇-C-MOE-ara U and its epimeric 2＇-F-4＇-C-MOE-r U were synthesized from 2＇-fluorinated arabinourine（2＇-F-ara U） and 2＇-fluorouridine（2＇-F-r U）, respectively. Their phosphoramidites were synthesized and successfully incorporated into oligodeoxynucleotides. The mismatch discrimination ability of these unnatural monomers and their effect on thermal stability were evaluated in the context of ds DNA and DNA-RNA chimeras. The thermal denaturation studies showed that the incorporation of 2＇-F-4＇-C-MOE-ara U led to enhanced binding affinity to complementary RNA strand and almost equivalent binding ability to complementary DNA, when compared with 2＇-F-4＇-C-MOE-r U and 2＇-F-ara U modified duplexes. Especially a C-H…F-C pseudohydrogen bond was supposed to contribute more binding affinity at uridine-purine steps, meanwhile, 2＇-F-4＇-C-MOE-ara U had almost the same base discriminatory ability as uridine in ds DNA and DNA-RNA chimeras, while 2＇-F-4＇-C-MOE-r U was found to have only moderate RNA hybridization ability. However, 2＇-F-4＇-C-MOE-ara U at 3＇-end of oligonucleotide could not led to more nuclease hydrolytic stability than that with 2＇-F-4＇-C-MOE-r U modification. These results demonstrated the feasibility of C4＇-MOE modification on 2＇-F-ANA and the dramatic effects of the 2＇-F substituent, which provides a new approach for further chemical modification of antisense drugs.

关 键 词：氟 假氢键作用 化学修饰 反义核酸 性质评价 

分 类 号：R916[医药卫生—药学]