Synthesis of Puromycin Tethered RNA and It's Application for Molecular Evolution

本文介绍了一种合成3‘端嘌呤霉素连接的寡核苷酸的简便方法。该方法利用了一种从商业上可获得的嘌呤霉素衍生的新型CPG载体。普鲁霉素(Pu)是3‘-氨基-3’-脱氧腺苷抗生素家族中的一员，被认为是一种非常有效的蛋白质生物合成抑制剂。Pu对蛋白质生物合成的抑制导致Pu与中断的早熟多肽之间产生共价加合物。为了完成蛋白质的分子进化系统，我们尝试了利用3‘端的Pu连接的信使核糖核酸将延长的多肽与信使核糖核酸的3’端进行交联。因此，我们开发了一种简单的合成Pu-连接的RNA或DNA的方法，通过制备一种新型的Pu连接的CPG载体。利用这种载体，我们合成了不同长度的3‘端到40聚体的Pu-拴系寡核苷酸。以市售的二盐酸钚为原料，经四步反应合成了负载Pu的CPG载体。Pu的氨基和5‘-羟基分别被保护在基团上，分别为亚甲氧基和二甲氧基三苯基。2‘-羟基与丁二酸酐发生酯化反应。为了防止Fmoc基团的不必要释放，通过最近的Fraser方法，在中性条件下将所得产品连接到LCAA-CPG载体上。通过监测在酸性条件下释放的DMTR阳离子，发现CPG载体的核苷结合力为25j.umole/g。

A facile method for the synthesis of puromycin-tethered oligonucleotides at the 3'-end is described. The method utilizes a novel CPG support derived from commercially available puromycin. Puromycin-tethered DNA and RNA oligomers were synthesized using a puromycin-tethered CPG support by the usual protocol for automated DNA and RNA synthesis.Puromycin (Pu) is a member of the 3'-amino-3'-deoxyadenosine family of antibiotics and is known as an extremely powerful inhibitor for protein biosynthesis. Inhibition of protein biosynthesis by Pu is shown to result in the production of a covalent adduct between Pu and the interrupted premature peptide. The cross-linking of an elongated peptide and the 3'-end of mRNA was attempted using Pu-tethered mRNA at the 3'-end in order to accomplish a molecular evolution system of proteins. Thus, we developed a facile synthetic method of Pu-tethered RNA or DNA by preparing a novel Pu-attached CPG support. Using this support, various lengths of Pu-tethered oligonucleotides at the 3'-end up to 40 mer were synthesized. The Pu-attached CPG support was synthesized from commercially available Pu dihydrochloride in four steps. The amino group and 5'-hydroxyl group of Pu were protected by fluorenylmethoxycarbonyl and dimethoxytrityl groups, respectively. A 2'-Hydroxyl group was esterified with succinic anhydride. The resulting product was attached to an LCAA-CPG support under neutral conditions by the recent Fraser method in order to prevent unwanted release of the Fmoc group. The nucleoside attachment for CPG support was found to be 25 j.umole/g by monitoring the released DMTr cation under acidic conditions.