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

本文描述了一种在3′端合成嘌呤霉素拴链寡核苷酸的简便方法。该方法利用了一种新型的CPG支持，来源于市售的嘌呤霉素。嘌呤霉素拴链DNA和RNA低聚物是利用嘌呤霉素拴链CPG载体，按照通常的自动DNA和RNA合成方案合成的。Puromycin （Pu）是抗生素3′-氨基-3′-脱氧腺苷家族的一员，是一种非常有效的蛋白质生物合成抑制剂。由Pu抑制蛋白质的生物合成被证明导致Pu和中断的过早肽之间的共价加合物的产生。为了完成蛋白质的分子进化系统，我们尝试在mRNA的3‘端使用Pu-tethered mRNA，将细长肽与mRNA的3’端交联。因此，我们通过制备一种新型的pu附着CPG载体，开发了一种简便的pu拴链RNA或DNA合成方法。利用这种载体，在3'端合成了长度不等、长度不超过40 mer的pu系链寡核苷酸。以市售的盐酸二磷酸为原料，分四步合成了Pu- CPG载体。Pu的氨基和5'-羟基分别被氟烯基甲氧羰基和二甲氧基三硝基保护。2′-羟基与丁二酸酐酯化。为了防止Fmoc基团不必要的释放，用最近的Fraser方法在中性条件下将所得产物附着在LCAA-CPG载体上。通过监测酸性条件下释放的DMTr阳离子，发现CPG载体的核苷附着为25 j.微摩尔/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.