DEVELOPMENT OF ARTIFICIAL RIBONUCLEASE BEARING NOVEL FUNCTIONALIZED ANTISENSE OLIGONUCLEOTIDES

以三(2-氨基乙基)胺为原料，合成了模拟天然核糖核酸酶的新型多胺衍生物(I-IV)。这些化合物与插层剂一起或不与插层剂一起或不与插层剂一起以咪唑和/或伯胺为酸碱催化中心。在这些衍生物中，含有咪唑和伯胺部分的化合物(III)以及嵌入剂显示出最强的RNA切割活性。化合物(I)中咪唑部分(III)被伯胺部分取代，也表现出显著的高活性。没有嵌入剂的化合物(II)和伯胺的连接臂比(III)更长的化合物(IV)是无效的。通过几个不同的实验研究了(I)和(III)切割RNA的机理。结果表明，化合物(III)的活性中心位于2‘-脱氧尿嘧啶的C-5位，两个伯胺基团均位于2’-脱氧尿嘧啶的C-5位。将得到的新型核苷转化为完全保护形式的3‘-亚磷酰胺，用于低聚物的合成，并制备了两种类型的寡核苷酸(DNA1和DNA2)。DNA1与5S rRNA环区互补，DNA2与茎区互补。两种齐聚物均为11-聚体，在其5‘-末端都有C-5修饰的2’-脱氧尿嘧啶。将这些寡聚体与5S rRNA长时间混合，用变性聚丙烯酰胺凝胶电泳法检测结果。结果，DNA1产生了两个新的片段，这两个片段本应是由底物的链断裂产生的。另一方面，DNA2根本没有给出新的条带。这些结果表明，由RNA裂解剂和寡核苷酸组成的杂化化合物将有可能成为一种新的功能化反义剂。

Novel polyamine derivatives (I-IV) were prepard from tris (2-aminoethyl) amine as a mimic of naturally occurring RNase. These compounds bear imidazole and/or primary amine groups as acid-base catalytic sites along with or without intercalating agent. Among the derivatives, the compound (III) which has a combination of imidazole and primary amine moiety along with the intercalator exhibited most potent RNA cleaving activity. The compound (I) in which an imidazole moiety of (III) was substituted by a primary amine moiety also exhibited remarkably high activity. The compound which lacks the intercalator (II) as well as the compound (IV) which has a longer linker arm for the primary amine compared to (III) were inactive.The mechanism of the RNA cleaving reation by (I) and (III) was investigated by several different experiments. The results suggest that the compounds cleave RNA by hydrolytic mechanism.The active sites of the compound (III), two primary amine groups, wereincorporated in C-5 position of 2'-deoxyuracil. The obtained novel nucleoside was converted to fully protected form of 3'-phosphoramidite and used for the synthesis of oligomer.Two types of oligonucleotides (DNA1 and DNA2) were prepared using the above phosphoramidite. The DNA1 is complementary to 5S rRNA loop region and DNA2 is complementary to the stem region. Both oligomers are 11-merlong and have C-5 modified 2'-deoxyuracil at their 5'-terminus. These oligomers were mixed with 5S rRNA for prolonged period and the results were assayd by denaturing polyacrilamide gel electrophoresis. As the result, DNA1 gave two new fragements which were supposed to be generated by the strand scission of the substrate. On the other hand, DNA2 gave no new bands at all. These results indicate that the hybrid compound consisted of RNA cleaving agent and oligonucleotide will be a useful candidate as a new functionallized antisense agent.