Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS
Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS
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DOI：
10.1261/rna.990208
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发表时间：
2008-06-01
期刊：
影响因子：
4.5
通讯作者：
Jemielity, Jacek
中科院分区：
文献类型：
--
作者：
Kowalska, Joanna;Lewdorowicz, Magdalena;Jemielity, Jacek
mRNA 帽的类似物广泛用于研究 mRNA 代谢过程，并可用于生物技术和医学应用。在这里，我们描述了在 5',5'-三磷酸链的 α、β 或 γ 位置带有单个硫代磷酸酯修饰的六种二核苷酸帽类似物的合成。其中三个还在 7-甲基鸟苷的 2'-O 位上用甲基进行修饰，以产生抗反向帽类似物 (ARCA)。由于硫代磷酸酯部分中存在立体异构 P 中心，每个类似物均以两种非对映异构体 D1 和 D2 的混合物形式获得。通过 RP HPLC 解析混合物，得到 12 种不同的化合物。采用荧光猝灭实验来确定新类似物与 eIF4E 的复合物的缔合常数 (K-AS)。我们发现硫代磷酸酯修饰通常可以稳定 eIF4E 和帽类似物之间的复合物。结合最牢固的硫代磷酸酯类似物（β 取代类似物 m(7) GppSpG 的 D1 异构体）的特征是 K-AS 比其未修饰的对应物 (m(7) GpppG) 高四倍多。所有在γ位修饰的类似物均能抵抗来自人和秀丽隐杆线虫来源的焦磷酸酶DcpS脱帽的清除剂的水解。使用酶消化并与鸟苷的 S-P 和 R-P 非对映体相关，将 α 位修饰的类似物的非对映体 D1 和 D2（即 m(7) Gppp(S)G 和 m(2)(7,2'-O) GpppSG）的绝对构型分别确定为 S-P 和 R-P 5'-O-(1-硫代二磷酸) (GDP α S)。对 DcpS 具有抗性的类似物可作为兔网织红细胞裂解物体外蛋白质合成的有效抑制剂。
Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K-AS) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7) GppSpG) was characterized by a K-AS that was more than fourfold higher than that of its unmodified counterpart (m(7) GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7) Gppp(S)G and m(2)(7,2'-O) GpppSG) were established as S-P and R-P, respectively, using enzymatic digestion and correlation with the S-P and R-P diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.