论文题目：异核苷及其杂寡核苷酸的合成、性质和生物活性研究

作者简介：杨振军，男，1964年生，1995年师从北京医科大学张礼和教授，于1998年获博士学位。

摘 要

许多异核苷是由叠氮基对糖基上的环氧乙烷基的进攻开环, 继以还原成氨基后拼合碱基的方法而获得。为了避免冗长的合成路线, 我们合成异核苷的方法是在碱性条件下, 直接使用碱基对糖基上的环氧基进攻开环。我们首次从D-葡萄糖经7步反应, 高收率地得到了2,5:3,4-二脱水-L-呋喃塔罗糖二甲缩醛(18)。由化合物18, 位置选择性及立体选择性地合成了一系列2,5-脱水-4-脱氧-4-碱基-L-甘露糖醇, 收率较好。2,5:3,4-二脱水-6-O-对甲苯磺酰基-L-呋喃塔罗糖二甲缩醛(19)与尿嘧啶和胸腺嘧啶反应, 分别得到了2,5:3,4-二脱水-6-脱氧-6-(尿嘧啶基或胸腺嘧啶基)-L-呋喃塔罗糖二甲缩醛(26或28)。当腺嘌呤与化合物19在同样条件下反应, 除得到2,5:3,6-二脱水-4-脱氧-4-腺嘌呤基-L-呋喃甘露糖二甲缩醛(31)外, 发现该化合物在反应中发生了四氢呋喃环的重排, 主要得到了4-(S)-腺嘌呤基-5-(R)-[1’-(R)-羟基-2,2’-二甲氧基]乙基-2,3-二氢呋喃(30)。因此, 我们认为当TsO^-和环氧基共存于同一分子时, 前者对嘧啶碱基的亲核进攻更活泼。由于可能的立体化学原因, 更强的碱-腺嘌呤进攻化合物19时, 得到了环氧开环的结果, 并发生了分子内重排。此外, 探索了合成两个isoddA类似物的合成路线, 得到了两个双脱氧异腺苷类似物52和55。由计算机分子力学优化, 结合二维NMR讨论了所合成异核苷的优势构象, 主要是‘S’构象。

为提高寡核苷酸作为基因表达抑制剂的生物活性, 人们所做的努力之一是增加它们的稳定性, 以耐受核酸酶的降解。以硫代磷酸酯为骨架的寡核苷酸在几个方面优越于其它的结构形式, 包括具有相对高的核酸酶抗性和启动靶序列被核酸酶RNase H降解的能力。然而, 硫代磷酸酯寡核苷酸仍然可以被降解, 一般首先从3’-端开始; 也发现急性HIV-1感染的细胞中, 它以非序列特异性方式抑制HIV-1的增殖, 因而有一定的毒副作用。在使用这些修饰的寡核苷酸时, 另一个问题是它们不能有效地透过细胞膜。报道中提出了各种稳定寡核苷酸磷酸二酯键的方法, 如各种3’和5’羟基取代基的插入。另一个解决办法是用类似天然2-脱氧核糖的五员糖基修饰寡核苷酸, 如含有六碳糖核苷类似物的寡核苷酸也有报道, 它在保持亲和性的同时, 增加了耐受磷酸二酯酶的能力。最近, 文献中见到了一类新的称为“inverse oligonucleotide”的寡核苷酸, 其骨架是通过磷酸二酯键相连的环戊二醇, 杂环碱基通过柔性的亚乙基与类糖基相连。

异核苷代表了一类新的核苷类似物。以其组成寡核苷酸, 磷酸二酯骨架的扭转角必然较正常的寡核苷酸有很大的改变, 由此可能影响核酸酶对该类寡聚物的识别。设想如果保留正常碱基, 则该类寡聚物应该能够与正常的互补序列形成双链。因此, 我们研究了异核苷掺入的寡核苷酸的杂交性质和酶解稳定性。

此外, 化合物C, E - G的核酸酶(SVPDE)水解的结果表明, 异核苷的端基掺入提高了寡核苷酸的稳定性, 基本上不被酶水解, 而在同样条件下, H和I被部分水解, d(T)₁₄则完全被降解。

因此, 在上述结果的基础上, 我们可以继续设计合成新的异核苷, 并合成相应的反义寡核苷酸, 以期找到抗肿瘤抗病毒药物。也为今后用异核苷合成反义寡核苷酸指明了方向。

(ABSTRACT)

Ph. D Student: Zhenjun Yang; Instructor: Prof. Lihe Zhang and Prof. Jimei Min

Beijing Medical University

A number of nucleoside analogues have been found to possess anticancer and antiviral activities. In the search for effective, selective and nontoxic anticancer and antiviral agents, the discovery of new class of nucleosides is of immense importance. Isonucleoside is a new class of nucleoside analogues in which the nucleobase is linked to a position of ribose other than C-1’. Therefore isonucleosides have attracted much attention owing to their chemical and enzymatic stability and potential antiviral activities. A series of isomers of 2’,3’-dideoxynucleosides which contain a modified carbohydrate moiety have been synthesized and some of these compounds exhibited significant and selective anti-HIV activity. Recently, a number of nucleosides with the unnatural L-configuration have been reported as potent chemotherapeutic agents against HIV, HBV and certain forms of cancer. It is interesting that these L-nucleosides have potent biological activities, while some of them show lower toxicity profiles than their D-counterparts.

Many isonucleosides syntheses have been achieved by use of epoxide opening by azide anion, subsequent reduction furnishing an amino group which is used to build up a heterocyclic moiety. In order to avoid lengthy synthetic routes, we synthesized isonucleosides by using an epoxide opening by a nucleobase itself in the basic conditions. The desired epoxide can be obtained from an corresponding sugar. Our strategy for the synthesis of the epoxide, 2,5:3,4-dianhydro-L-talofuranose dimethylacetal 18, which was synthesized from D-glucose in 7 steps. A series of 4-deoxy-4-nucleobase-2,5-anhydro-L-mannitols were synthesized regioselectively and stereoselctively from 18 in good yields. 6-O-p-Tolylsulfonyl-2,5:3,4-dianhydro-L-talofuranose dimethylacetal 19 reacted with uracil or thymine to give the corresponding isonucleosides 6-deoxy-6-(uracil-1-yl or thymin-1-yl)-2,5:3,4-dianhydro-L-talofuranose dimethyl acetal (26 or 28), but in the case of reaction of 19 with adenine, reformation of tetrahydrofuran ring took place, giving 4-(S)-adenyl-5-(R)-[1’-(R)-hydroxy-2’,2’-dimethoxy] ethyl-2,3-dihydrofuran (30) and 4-deoxy-4-adenyl-2,5:3,6-dianhydro-L-mannofuranose dimethylacetal (31). When two functional group, TsO^- and epoxide, exist in the same molecule, TsO^- is more active to the nucleophilic attack of pyrimidine base. Due to the feasible stereochemical reason, the preferential substitution of epoxide of 19 by the stronger nucleophile, adenine, and reformation of tetrahydrofuran ring took place. Two analogues of isoddA (52 and 55) were also synthesized. The stable conformation of the isonucleosides (‘S’ conformation is majority) was discussed by the results of the molecular mechanics minimized conformation combined with 2D NMR.

One of the efforts to enhance the biological activity of oligonucleotides as inhibitors of gene expression has been made by improvement of their stability to nuclease digestion. Antisense oligonucleotides with phosphorothioate backbones exhibit several advantages over other forms, including relatively high nuclease resistance as well as the ability to induce the degradation of the target sequence by RNase. However, phosphorothioate oligonucleotide are possibly hydrolyzed, primarily from the 3’-end and have also been shown to block the proliferation of HIV-1 in acutely infected cells in a non-sequence-specific manner. Another problem in the use of such modified antisense oligonucleotide is their inefficient cellular uptake. Several techniques for the stabilization of the phosphodiester bonds of the oligonucleotides have been proposed, such as the incorporation of various chemical substituents at the 3’ and 5’ hydroxyl group. Another approach to solve these problems have been development of altered sugar moieties along the oligonucleotide chain. Most of these modifications contain a five-membered sugar ring closely resembling the natural 2-deoxyribose. Oligonucleotides consisting of hexose nucleoside analogues were reported to posses significantly increased stability towards phosphodiesterases whereby the hybridization properties are retained. Recently, an “inverse oligonucleotide” was introduced, where the backbone of the oligonucleotide consists of a phosphorylated cyclopentanediol moiety and the heterocyclic base is bound via a flexible ethylene linkage.

Isonucleosides represent a new class of nucleoside analogues. The torsion angles in the sugar-phosphate backbones of such oligonucleotides exhibit profound changes compared to regular oligonucleotides. These alternations in torsion angles might affect the recognition of such oligomers by nucleases. It could also be anticipated that the bases in the modified oligonucleotide retain their hybridization properties with complementary sequences. This prompted us to study the hybridization properties and enzymatic stability of oligonucleotides bearing such isonucleosides.

Next, the enzymatic hydrolysis of the oligomers C, E-G and d(T)₁₄ was studied. The results show a significant resistance of the phosphodiester bonds of C, E-G towards snake venom phosphodiesterase, while H and I is partially hydrolyzed, (T)₁₄ is completely hydrolyzed under the same conditions.

Therefore, on the basis of above results, we can design new isonucleosides and may develop new type of antisense oligonucleotides for therapy of tumor and viral infection.