RUI: Atranyl-Nucleosides as Ribozyme Probes

RUI：Atranyl 核苷作为核酶探针

• 批准号: 0135279
• 负责人: Edward Fenlon
• 金额: $ 9.6万
• 依托单位: Xavier University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0135279&HistoricalAwards=false
• 关键词: RUI; Atranyl; Nucleosides; Ribozyme; Probes

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.

目前蛋白酶催化机制的模型比RNA酶（核酶）的模型先进得多。这个项目将有助于理解核酶的催化机制与蛋白酶的催化机制的比较。本项目的具体目标是：（1）合成蒽基核苷1-3;（2）表征和测定蒽基核苷的水稳定性;（3）以蒽基核苷为过渡态类似物（TSA）研究锤头和铅依赖性（leadzyme）核酶;（4）培养本科生有机合成技术，为研究生学习和/或从事科学事业做准备。TSA可用于酶研究;然而，缺乏用于磷酰基转移反应的合适的TSA。化合物1-3具有掺入核糖核苷中的阿纳三环部分。atrane含有三角形双金属硅、锗、锡或钛原子;因此，1-3模拟核酶催化反应的氧正膦过渡态。化合物2是一个被保护的亚磷酰胺，含有通过氮杂三环连接的尿苷和脱氧腺苷。评价了从5 ′-O-DMT-2 ′-氨基-2 ′-脱氧尿苷出发合成2的几种方法。William G.教授加州大学圣克鲁兹分校的斯科特将通过固相合成将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亚瑟帕迪教授将用核磁共振光谱法同样确定溶液中的结构。在合成亚磷酰胺3后，将采用类似的方法进行leadzyme结构研究。这些实验将阐明在这些小RNA催化剂中达到过渡态所需的构象重排的大小。本研究也将有助于确定特定RNA功能基团和金属阳离子在催化中的作用。这些知识对于了解生命前的“RNA世界”以及我们当前的生命形式如何从中进化至关重要。