| The Tetrahymena Group I ribozyme catalyzes a site specific endonucleolytic cleavage of RNA or DNA substrates (S). This thesis presents our study of the structure and function of this ribozyme through chemical modifications. An abasic ribose-phosphoramidite (H) was synthesized and incorporated into the cleavage site of the oligonucleotide substrate. The ribozyme cleaves this modified substrate (rmSH) with decreased rate (∼104-fold) and affinity (∼10-fold) compared with a normal substrate (rmSU), representing an overall transition state destabilization of ∼5.9 kcal mol−1. However, similar to rmSU, rmSH still binds to the ribozyme in the closed complex as demonstrated by the existence of the strong tertiary interaction between the 2′-OH of U−3 and the ribozyme core. The decreased activity of rmSH relative to rmSU can not be rescued by exogenous addition of free nucleobases, but can be rescued by gradually putting back the functional groups that H lacks in comparison with U.; The 5′-bridging oxygen of the reactive phosphate at the cleavage site is also replaced with a 5′-methylene (-CH2-) group. The ribozyme unexpectedly catalyzes cleavage of this phosphonate linkage 103-fold faster than the unmodified linkage in substrates containing all deoxynucleotides. This effect is attributed to a 10-fold effect on docking and 100-fold effect on the chemical step. Hydrolysis experiments on two model compounds show that the methylene phosphonate reacts ∼5-fold faster than its isosteric phosphate counterpart, only partially accounting for the rate difference observed in the ribozyme reaction. However, the greater transition state stabilization of the phosphonate monoester relative to the phosphate diester could be explained by a change in solvation of the 5 ′-group during the reaction that resembles the transfer of the group from water to a hydrophobic solvent. The observation that 5′ -methylene substitution does not have a deleterious effect on the rebozyme reaction suggests that there are no important interactions with the 5′-oxygen in the transition state.; In our attempt to synthesize 2′-C-difluoromethyl ribonucleosides, we developed a general methodology to introduce the difluoromethyl group into organic molecules. This methodology can be applied to the synthesis of fluoro-containing molecules with potential as themotherapeutic agents and biochemical probes. |
