A New Synthetic Method for Oligoribonucleotides by the Phosphite Triester Approach

1.亚磷酰胺类核糖核酸衍生物的合成合成了2‘-羟基邻硝基苯基、5’-羟基甲氧基三苯基和碱氨基酰基保护的核糖核酸衍生物(1)。通过3‘-羟基光甲基化反应合成了甲基或氰基乙基二异丙基酰胺类化合物(2)。另一方面，通过将1与可控孔玻璃载体偶联，合成了核苷树脂衍生物(3)。低聚物是由2和3重复偶联而成的。当用5-(对-硝基苯基)四唑活化酰胺时，偶联反应通常在一分钟内完成。用H-膦酸衍生物合成H-膦酸衍生物H-膦酸衍生物(4)是以1为原料，经PCl_3处理合成的。低聚物4与3以三氯丙烷为缩合剂，通过重复偶联反应合成。利用核酸合成器合成了长达34条的tRNA片段。核糖核酸的定量合成建立了用固相亚磷酰胺法合成链长为10~20的低聚物的方法。因此，可以很容易地在短时间内以10-20%的产率合成可用于核磁共振测量的约100个吸光度单位(260 Nm)的RNA寡聚体。核磁共振波谱对核糖核酸构象的研究用上述方法合成了具有RNA酶催化活性或部分碱基序列的低聚物和具有非常稳定的发夹结构的低聚物，并用核磁共振对其构象进行了表征。

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.