RUI: Atranyl-Nucleosides as Ribozyme Probes

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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.