Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Graduate School of Natural Science and Technology, Gifu University, Gifu, Japan.
Curr Protoc. 2022 Sep;2(9):e501. doi: 10.1002/cpz1.501.
Antisense oligonucleotide (ASO) therapeutics target the pathogenic mRNA directly and modulate protein expression. Novel chemical modifications help to improve the action of ASOs with better thermal stability and resistance against nucleases. Oligodeoxynucleotides (ODNs) containing 4'-C-(aminoethyl)thymidine modifications exhibit efficient and stable hybridization with complementary DNA as well as RNA strands showing remarkably improved resistance against nucleolytic hydrolysis, which makes them promising candidates for antisense therapeutics. This article describes the synthesis of a novel nucleoside analog, 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T), 3, and previously reported 4'-C-aminoethyl-thymidine (4'-AE-T), 2, through a newly designed synthetic route to obtain a high overall yield. This has been established by changing the starting material from thymidine to diacetone-D-glucofuranose and synthesizing the known 4-C-hydroxyethyl pentofuranose. Conversion of the hydroxy group to an azide functional group through Mitsunobu azidation and performing acetolysis, provide the common intermediate 4-C-(2-azidoethyl)-ribofuranose. Subsequent coupling of the thymine nucleobase with the common intermediate under Vorbrüggen glycosylation conditions provides the corresponding modified nucleoside in high yield. It was subjected for conversion of the azide to an amine by Staudinger reaction and 2'-deoxygenation using Barton-McCombie conditions. Debenzylation with Lewis acid and mono-dimethoxytritylation of the 5'-OH afforded a fully protected 3'-OH intermediate for phosphitylation to give the corresponding phosphoramidites. In the case of 4'-MAE-T, benzyloxymethyl protection of the N -position and methylation were carried out prior to debenzylation. These phosphoramidite monomers were suitable with conventional oligonucleotide synthesis, and imparted ameliorated nuclease resistance, and competent RNase H activity, suggesting its potential utilization in ASO drugs. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 4-C-(2-azidoethyl)-ribofuranose (6) Basic Protocol 2: Synthesis of 4'-C-aminoethyl thymidine phosphoramidite (15) Basic Protocol 3: Synthesis of 4'-C-(N-methyl)aminoethyl thymidine phosphoramidite (20).
反义寡核苷酸（ASO）疗法直接针对致病 mRNA，并调节蛋白质表达。新型化学修饰有助于提高 ASO 的作用，使其具有更好的热稳定性和对核酸酶的抗性。含有 4'-C-(氨基乙基)胸腺嘧啶修饰的寡脱氧核苷酸 (ODN) 与互补的 DNA 以及 RNA 链具有高效且稳定的杂交作用，表现出显著提高的抗核酸酶水解能力，使其成为有前途的反义治疗候选物。本文描述了一种新型核苷类似物 4'-C-[(N-甲基)氨基乙基]胸腺嘧啶 (4'-MAE-T)，3 的合成，以及之前报道的 4'-C-氨基乙基-胸腺嘧啶 (4'-AE-T)，2，通过新设计的合成路线获得高总产率。这是通过将起始材料从胸腺嘧啶改为二丙酮-D-葡萄糖呋喃糖，并合成已知的 4-C-羟乙基戊呋喃糖来实现的。通过 Mitsunobu 叠氮化物使羟基转化为叠氮基官能团，并进行乙酰解，提供共同的中间体 4-C-(2-叠氮乙基)-核糖呋喃糖。随后，在 Vorbrüggen 糖苷化条件下，嘧啶核苷碱基与共同中间体偶联，以高产率提供相应的修饰核苷。它通过斯塔德勒反应将叠氮化物转化为胺，并在巴顿-麦科姆比条件下进行 2'-脱氧。用路易斯酸脱苄基和 5'-OH 的单二甲氧基三苯甲基化，得到完全保护的 3'-OH 中间体进行膦酰化，得到相应的磷酰胺。对于 4'-MAE-T，在脱苄基之前进行 N-位的苄氧甲基保护和甲基化。这些磷酰胺单体适用于常规寡核苷酸合成，并赋予改善的核酸酶抗性和有效的 RNase H 活性，表明其在 ASO 药物中的潜在用途。 © 2022 威利父子公司。 基本方案 1：制备 4-C-(2-叠氮乙基)-核糖呋喃糖（6） 基本方案 2：合成 4'-C-氨基乙基胸腺嘧啶磷酰胺（15） 基本方案 3：合成 4'-C-(N-甲基)氨基乙基胸腺嘧啶磷酰胺（20）。
