新型反义寡核苷酸化学修饰：2'F,4'-C-取代阿拉伯糖核苷酸的合成及性质评价

Chemical modification is the key to clinical application of antisense technology, various chemically modified oligonucleotides have been developed to enhance nuclease resistance, increase target affinity and specificity, improve pharmacokinetic and reduce toxicity. Learning from those chemical modifications, especially 2',4'-LAN, 2'F-ANA and 4'-substituted oligonucleotides, we propose a novel chemically synthetic 2'F, 4'-C-substitutied arabinonucleotides modification, such modification is anticipated to have beneficial effects on nuclease resistance and target binding stability through 4'-substituted steric hindrance and H•••F pseudohydrogen bond, synergistically. Starting with 2'F-arabinose and chemically introducing various substituent into suger 4' position, in this project, a series of 2'F, 4'-C-substitutied arabinonucleotide monomer will be synthesized and incorporated into oligonucleotides, then the properties of the corresponding oligonucleotide derivatives will be systemically evaluated to establish the structure-activity relationship between 4'-substituent and modified oligonucleotides, as a result, we aim to develop a novel, effective, affordable and versatile 2'F, 4'-C-substitued arabinonucleotides modification for antisense therapeutic strategy.

运用化学修饰寡核苷酸可以增强其对核酸酶的稳定性，提高对靶基因的特异性和亲和力，改善药物代谢性质，降低毒性，化学修饰是反义核酸药物临床运用的关键技术。基于各种糖基修饰策略，特别是2',4'-LAN 、2'F-ANA及4'位修饰情况，本项目提出了一种新的2'F, 4'-C-取代阿拉伯糖核苷酸修饰策略，这种2'F, 4'-C-双取代预期既可以通过F原子的H•••F假氢键作用，提高与靶基因的亲和力，又可以通过4'位取代位阻作用增强对核酸酶的稳定性，取得协同效果。以2'F-阿拉伯糖为起始原料，通过在糖基4'-位引入各种不同基团，本项目将合成一系列的2'F, 4'-C-取代阿拉伯糖核苷单体并将其整合入寡核苷酸序列中，系统地评价这些序列的性质，建立4'-取代基对反义寡核苷酸性质的影响机制，进而发展出一类新的、有效的、易获得并可广泛运用的2'F, 4'-C-取代阿拉伯糖核苷酸，支持核酸药物的发展。

化学修饰是核酸药物成药的关键技术，通过化学修饰可提高核酸与靶基因的结合亲和力，增强对核酸酶的稳定性，改善核酸药物的成药性。本项目基于2'-脱氧-2'-氟阿拉伯糖核酸 (2'-F ANA) 的独特性质，通过在2'-F ANA糖基4'-位引入取代基团，设计并合成了系列2'-F,4'-C-X ANA (X为取代基) 的修饰结构，包括2'-F,4'-C-HM-araU (UHM)， 2'-F,4'-C-MOE-araU (UMOE)，2'-F,4'-C-OMe-araU (UOMe)等，此外，为做比较研究，还合成了差向异构体2'-F,4'-C-MOE-rU (rUMOE)，2'-F,4'-C-OMe-rU (rUOMe)，随后将这些核苷单体嵌入相应的寡核苷酸序列中，再用热变性实验评价了这些修饰寡核苷酸序列与靶RNA及靶DNA的杂交性质，使用蛇毒磷酸二酯酶评价核酸酶耐受性，总结评价结果，本项目发现，相对于2'-F-araU，2'-F,4'-C-OMe-araU修饰能提高寡核苷酸对RNA的选择性，无论是单修饰还是多修饰，这些寡核苷酸均可与互补RNA形成典型的A构象。同时，与2'-F-araU及2'-F,4'-C-OMe-rU相比，2'-F,4'-C-OMe-araU修饰可增强寡核苷酸与互补RNA的结合亲和力，不过，这种增强作用具有序列依赖性，在5'-嘧啶-嘌呤-3'步序中能够形成C-H…F-C假氢键作用，效果最好 (可提高2-3 ℃)。此外，3'-端修饰2'-F,4'-C-OMe-araU能够提高寡核苷酸对3'-核酸酶的耐受性，优于2'-F,4'-C-OMe-araU，这表明2’-F,4’-C-OMe ANA修饰不仅可以通过假氢键作用提高与互补RNA的结合亲和力，而且能够提高对核酶稳定性，发挥协同作用，实现‘1＋1 > 2’ 效果，由此2'-F,4'-C-OMe-araU修饰可作为一种新型核酸结构，可用于反义核酸的化学修饰。