Program of Bioorganic Chemistry, Institution of Cell and Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden.
J Org Chem. 2010 Sep 17;75(18):6122-40. doi: 10.1021/jo100900v.
A new class of conformationally constrained nucleosides, α-L-ribo-carbocyclic LNA thymidine (α-L-carba-LNA-T, LNA is an abbreviation of locked nucleic acid) analogues and a novel "double-locked" α-L-ribo-configured tetracyclic thymidine (6,7'-methylene-bridged-α-L-carba-LNA-T) in which both the sugar puckering and glycosidic torsion are simultaneously constrained, have been synthesized through a key step involving 5-exo free-radical intramolecular cyclization. These α-L-carba-LNA analogues have been subsequently transformed to corresponding phosphoramidites and incorporated into isosequential antisense oligonucleotides (AONs), which have then been examined for the thermal denaturation of their duplexes, nuclease stability, and RNase H recruitment capabilities. Introduction of a single 6',7'-substituted α-L-carba-LNA-T modification in the AON strand of AON/RNA heteroduplex led to T(m) reduction by 2-3 °C as compared to the native heteroduplex, whereas the parent 2'-oxa-α-L-LNA-T modification at the identical position in the AON strand has been found to lead to an increase in the T(m) by 3-5 °C. This suggests that the 6' and 7' substitutions lead to much reduced thermal stability for the modified heteroduplex, especially the hydrophobic 7'-methyl on α-L-carba-LNA, which is located in the major groove of the duplex. All of the AONs incorporating 6',7'-substituted α-L-carba-LNA-T have, however, showed considerably improved nuclease stability toward 3'-exonuclease (SVPDE) and in human blood serum compared to the 2'-oxa-α-L-LNA-T incorporated AONs. The hybrid duplexes that are formed by 6',7'-substituted α-L-carba-LNA-T-modified AONs with complementary RNA have been found to recruit RNase H with higher efficiency than those of the β-D-LNA-T or β-D-carba-LNA-T-modified counterparts. These greatly improved nuclease resistances and efficient RNase H recruitment capabilities elevate the α-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for potential RNA targeting therapeutics.
一种新的构象受限核苷类似物，α-L-核糖碳环 LNA 胸腺嘧啶（α-L-碳桥 LNA-T，LNA 是锁定核酸的缩写）类似物和一种新型的“双锁定”α-L-核糖四环胸腺嘧啶（6,7'-亚甲基桥接-α-L-碳桥 LNA-T），其中糖的构象和糖苷扭转都同时受到限制，通过涉及 5-外消旋自由基分子内环化的关键步骤合成。这些α-L-碳桥 LNA 类似物随后转化为相应的磷酰胺，并掺入等序反义寡核苷酸（AON）中，然后检查它们的双链体热变性、核酸酶稳定性和 RNase H 募集能力。在 AON/RNA 杂种双链体的 AON 链中引入单个 6'，7'-取代的α-L-碳桥 LNA-T 修饰导致 Tm 降低 2-3°C，与天然杂种双链体相比，而在 AON 链中相同位置的母体 2'-氧杂-α-L-LNA-T 修饰导致 Tm 升高 3-5°C。这表明 6'和 7'取代导致修饰的杂种双链体的热稳定性大大降低，特别是α-L-碳桥 LNA 的疏水 7'-甲基，位于双链体的大沟中。然而，与掺入 2'-氧杂-α-L-LNA-T 的 AON 相比，所有掺入 6'，7'-取代的α-L-碳桥 LNA-T 的 AON 都表现出对 3'-外切核酸酶（SVPDE）和人血清的显著提高的核酸酶稳定性。与互补 RNA 形成的 6'，7'-取代的α-L-碳桥 LNA-T 修饰的 AON 形成的杂种双链体已被发现比β-D-LNA-T 或β-D-碳桥 LNA-T 修饰的对应物更有效地募集 RNase H。这些大大提高的核酸酶抗性和有效 RNase H 募集能力将α-L-碳桥 LNA 修饰的核苷酸提升为一类新的用于潜在 RNA 靶向治疗的锁定核酸。
