RUI: Atranyl-Nucleosides as Ribozyme Probes

目前关于蛋白质酶催化机制的模型要比RNA酶（核酶）的模型先进得多。该项目将有助于了解核酶与蛋白酶的催化机制的比较。该项目的具体目标是：(1)合成atranyl-nucleosides 1-3；(2)表征并确定阿特拉基核苷的水稳定性；(3)利用阿特拉基核苷作为过渡态类似物（TSAs）研究锤头核酶和铅依赖核酶的协同作用；(4)培养本科生合成有机技术，为研究生学习和/或从事科学工作做准备。tsa对酶的研究很有用；然而，缺乏适合于磷酰转移反应的tsa。化合物1-3的无烷部分与核糖核苷结合。原子包含一个三角形双锥体硅、锗、锡或钛原子；因此，1-3模拟核酶催化反应的氧磷过渡态。化合物2是一种受保护的磷酸酰胺，含有尿苷和脱氧腺苷，通过胞嘧啶段连接。将评价几种以5'-O-DMT-2'-氨基-2'-脱氧尿苷为起始原料合成2的方法。加州大学圣克鲁兹分校的William G. Scott教授将通过固相合成的方法将2掺入锤头状底物中，以获得锤头状核酶- tsa晶体结构。科罗拉多大学博尔德分校的Arthur Pardi教授也将使用核磁共振波谱法来确定溶液中的结构。类似的方法将用于铅酶的结构研究后合成的磷酰胺3。这些实验将阐明每种小RNA催化剂达到过渡态所需的构象重排的大小。本研究还将有助于确定特定RNA官能团和金属阳离子催化的作用。这一知识对于理解生命起源前的“RNA世界”以及我们现在的生命形式是如何从中进化而来的至关重要。

Current models for the catalytic mechanisms of protein enzymes are far more advancedthan those for RNA enzymes (ribozymes). This project will contribute to understanding how thecatalytic mechanisms of ribozymes compare with those of protein enzymes. The specificobjectives of this project are to: (1) synthesize atranyl-nucleosides 1-3; (2) characterize anddetermine the aqueous stability of atranyl-nucleosides; (3) use atranyl-nucleosides as transitionstate analogues (TSAs) to study the hammerhead and lead-dependent (leadzyme) ribozymes viacollaborations; and (4) train undergraduate students in synthetic organic techniques inpreparation for graduate study and/or careers in science. TSAs are useful for enzyme studies;however, suitable TSAs for phosphoryl transfer reactions are lacking. Compounds 1-3 have anatrane moiety incorporated into a ribonucleoside. The atrane contains a trigonal bipyramidalsilicon, germanium, tin, or titanium atom; and thus, 1-3 mimic the oxyphosphorane transitionstate of the ribozyme-catalyzed reaction. Compound 2 is a protected phosphoramidite thatcontains uridine and deoxyadenosine connected through the atrane moiety. Several syntheses of2 starting from 5'-O-DMT-2'-amino-2'-deoxyuridine will be evaluated. Prof. William G. Scott ofUC-Santa Cruz will incorporate 2 into the hammerhead substrate by solid-phase synthesis toobtain a hammerhead ribozyme-TSA crystal structure. Prof. Arthur Pardi of the University ofColorado-Boulder will similarly determine the structure in solution using NMR spectroscopy.Analogous approaches will be taken for leadzyme structural studies following the synthesis ofphosphoramidite 3. These experiments will elucidate the magnitude of conformationalrearrangement required to reach the transition state in each of these small RNA catalysts. Thisresearch will also help identify the role of specific RNA functional groups and metal cations incatalysis. This knowledge is paramount for understanding the pre-biotic "RNA world" and howour current life forms evolved from it.