RUI: Atranyl-Nucleosides as Ribozyme Probes

目前蛋白质酶催化机制的模型比RNA酶(核酶)的模型先进得多。该项目将有助于理解核酶与蛋白质酶的催化机制的比较。该项目的具体目标是：(1)合成阿特拉尼基核苷1-3；(2)表征和测定阿特拉尼基核苷的水溶液稳定性；(3)使用阿特拉尼基核苷作为过渡态类似物(TSA)研究锤头状核酶和铅依赖(铅酶)核酶的协同作用；以及(4)为本科生提供合成有机技术方面的培训，为研究生学习和/或科学生涯做准备。TSA对酶的研究是有用的，但缺乏适用于磷酰基转移反应的TSA。化合物1-3具有并入核糖核苷的铁环部分。Atrane含有一个三方双锥硅、锗、锡或钛原子，因此，1-3模拟核酶催化反应的氧磷烷过渡态。化合物2是一种受保护的亚磷酰胺，含有通过atrane部分连接的尿苷和脱氧腺苷。将对以5‘-O-DMT-2’-氨基-2‘-脱氧尿苷为起始原料的几种2的合成方法进行评价。加州大学圣克鲁斯分校的威廉·G·斯科特教授将通过固相合成将2引入锤头底物，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教授将类似地使用核磁共振波谱来确定溶液中的结构。在合成亚磷酰胺3之后，将采用类似的方法对铅酶结构进行研究。这些实验将阐明在每个小RNA催化剂中达到过渡态所需的构象重排的大小。这项研究还将有助于确定特定的RNA官能团和金属阳离子催化的作用。这一知识对于理解生物前的“RNA世界”以及我们目前的生命形式是如何从它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.