Synthesis and Hybridization Studies of 2‘-Amino-α-L-LNA and Tetracyclic “Locked LNA”^†

A convergent route to a new class of locked nucleic acids, i.e., 2‘-amino-α-L-LNA, has been developed. The optimized synthetic route to the corresponding phosphoramidite building block of thymine proceeds in 4% overall yield over 15 steps from the starting diol. Crucial synthetic steps include (a) introduction of a C2-azido group prior to nucleobase coupling, (b) Vorbrüggen glycosylation primarily affording the desired α-anomer, (c) separation of α-l-ribo- and β-l-ribo-configured bicyclic nucleosides, and (d) selection of a suitable protecting group to avoid intramolecular Michael addition of the C2‘-amino group onto the C6-position. Incorporation of a 2‘-amino-α-L-LNA monomer into oligodeoxyribonucleotides results in modest changes in thermal stability with complementary DNA, whereas significant increases in thermal stability are observed with RNA complements along with excellent Watson−Crick discrimination. These results, along with the flexibility of the synthetic strategy allowing chemoselective N2‘-functionalization at a late stage, render 2‘-amino-α-L-LNA a promising building block for nucleic acid based nanobiotechnology and therapeutics. A slight modification in strategy facilitated the synthesis of the corresponding phosphoramidite building blocks of Michael adducts, which due to their tetracyclic skeletons exhibit a conformationally restricted furanose ring and glycosidic torsion angle (anti-range). Incorporation of such a “locked LNA” monomer into oligodeoxyribonucleotides results in large decreases in thermal affinity toward DNA/RNA complements.本研究开发了一条合成新型锁核酸（locked nucleic acids, LNA）类化合物的汇聚式合成路线，即2'-氨基-α-L-LNA。以起始二醇为原料，经15步反应制备胸腺嘧啶对应的亚磷酰胺单体（phosphoramidite building block），总产率可达4%。核心合成步骤包括：(a) 在碱基偶联前引入C2-叠氮基；(b) 沃布吕根糖基化反应（Vorbrüggen glycosylation）主要生成目标α-异头物；(c) 分离α-L-核糖构型与β-L-核糖构型的双环核苷；(d) 筛选适配的保护基，以避免C2'-氨基对C6位发生分子内迈克尔加成（intramolecular Michael addition）。 将2'-氨基-α-L-LNA单体掺入寡脱氧核糖核苷酸（oligodeoxyribonucleotides）后，其与互补DNA的热稳定性仅出现小幅变化；而与互补RNA结合时，热稳定性则显著提升，且仍保持优异的沃森-克里克碱基配对特异性。上述研究结果，结合该合成策略可在后期实现化学选择性N2'-官能化的灵活性，使2'-氨基-α-L-LNA成为面向核酸基纳米生物技术与治疗领域的极具潜力的单体砌块。 对合成策略稍加调整，即可制备迈克尔加成产物对应的亚磷酰胺单体砌块；这类化合物因具有四环骨架，其呋喃糖环（furanose ring）与糖苷扭转角（glycosidic torsion angle）均受到构象限制，处于反式构象范围（anti-range）。将这类“锁合LNA”单体掺入寡脱氧核糖核苷酸后，其与DNA/RNA互补链的结合亲和力会大幅降低。