Department of Medicinal Chemistry, Isis Pharmaceuticals, 1896 Rutherford Road, Carlsbad, California 92008, USA.
J Org Chem. 2010 Mar 5;75(5):1569-81. doi: 10.1021/jo902560f.
We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.
我们最近发现，将 2'O-甲氧基乙基（MOE）和锁核酸（LNA）核苷的结构元件结合起来，得到了一系列核苷修饰物（cMOE，2'，4'-约束 MOE；cEt，2'，4'-约束乙基），与 MOE 相比，这些修饰物具有更高的效力，与 LNA 反义寡核苷酸相比，具有更好的治疗指数。在本报告中，我们介绍了有关 cMOE 和 cEt 核苷膦酰胺的合成以及含有这些核苷修饰物的寡核苷酸的物理化学评估的详细信息。从廉价的商业可得的二丙酮阿洛呋喃糖起始，高效地完成了 cMOE 和 cEt 核苷膦酰胺的合成。该合成的特点是使用一种很少使用的 2-萘甲基保护基，该保护基在合成过程中提供了结晶中间体，并可以在温和条件下进行清洁脱保护。关键的单 TBDPS 保护二醇中间体的结晶性极大地促进了合成。对于 cEt 核苷，以立体选择性方式引入任一种构型的甲基。在非常温和的条件下，通过将 2'-羟基环合到次级甲磺酸酯离去基团上，以立体化学完全反转的方式，实现了立体化学的环合。对于 S-cEt 修饰物，在多克规模上完成了所有四个（胸腺嘧啶、5-甲基胞嘧啶、腺嘌呤和鸟嘌呤）碱基修饰的膦酰胺的合成。对含有 cMOE 和 cEt 的寡核苷酸的物理化学评估表明，它们具有与 LNA 相似的杂交和错配区分属性，但对核酸外切酶的消化具有大大提高的抗性。
