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A New Synthetic Method for Oligoribonucleotides by the Phosphite Triester Approach

亚磷酸三酯法合成寡核糖核苷酸的新方法

基本信息

批准号：
61470150
负责人：
UESUGI Seiichi
金额：
$ 4.29万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

1. Synthesis of ribooligonucleotides using the phosphoramidite derivativesRibooligonucleotide derivatives (1), which are protected with an o-nitrobenzyl group at the 2'-hydroxyl, a methoxytrityl group at the 5'-hydroxyl and an acyl group at the base amino group, were synthesized. Methyl or cyanoethylidiisopropylamidite derivatives(2) were synthesized by phosohitylation at the 3'-hydroxyl group. On the other hand, nucleoside resin derivatives (3) were synthesized by coupling 1 with the controlled pore glass support. The oligomers were synthesized by repeated coupling of 2 with 3. When 5-(P-nitrophenyl)tetrazole is used for activation of the amidite, the coupling reaction is usually completed within one miunte.2. Synthesis of ribooligonucleotides using the H-phosphonate derivativesH-Phosphonate derivatives (4) were synthesized from 1 by treatment with PC1_3. The oligomers were synthesized by repeated coupling of 4 with 3 using pivaloyl chloride as the condensing agent. tRNA fragments with chain lengths up to 34 were synthesized by using a mucleic acid synthesizer.3. Synthesis of ribooligonucleotides in quantityA method for synthesis of the oligomers with chain lengths 10-20 by solid-phase phosphoramidite approach was established. Thus the RNA oligamers of abaout 100 absorbance units (260 nm), which can be used for NMR measurement, are easily synthesized in a short time in 10-20%yields.4. Conformational studies on the ribooligonucleotides by NMR spectroscopyOligomer complexes having a catalytic activity or partial base sequence of an RNA enzyme and oligomers with extraordinary stable hairpin structures were synthesized by the above method and their conformation was examined by NMR.

1.用亚磷酰胺衍生物合成核糖寡核苷酸合成了核糖寡核苷酸衍生物（1），其在2 ′-羟基上用邻硝基苄基保护，在5 ′-羟基上用甲氧基三苯甲基保护，在基础氨基上用酰基保护。通过3 ′-羟基的膦酰化反应合成了甲基或氰乙基二异丙基酰胺衍生物（2）。另一方面，核苷树脂衍生物（3）通过偶联1与可控孔玻璃载体合成。通过2与3的重复偶联合成低聚物。用5-（对硝基苯基）四氮唑活化亚酰胺时，偶联反应通常在1分钟内完成.使用H-膦酸酯衍生物的核糖寡核苷酸的合成H-膦酸酯衍生物（4）通过用PCl_3处理由1合成。以特戊酰氯为缩合剂，通过4与3的重复偶联合成了低聚物。利用核酸合成仪合成了链长为34的tRNA片段.核糖寡核苷酸的大量合成建立了固相亚磷酰胺法合成链长为10-20的寡核苷酸的方法。因此，可以在短时间内以10- 20%的产率容易地合成约100个吸光度单位（260 nm）的RNA寡聚体，其可以用于NMR测量.用核磁共振波谱法研究核糖寡核苷酸的构象用上述方法合成了具有RNA酶催化活性或部分碱基序列的寡聚物复合物和具有非常稳定的发夹结构的寡聚物，并用核磁共振波谱法研究了它们的构象。