Mechanism Studies of Catalytic RNA, and RNA and DNA Model Compounds

Abstract: This thesis describes mechanism studies of transesterifications of phosphate esters. The aim of the studies concerns two fields. First, the catalytic mechanism that group I introns use in self-splicing. The focus was on metal ion coordinating groups at the catalytic site. Studies were performed on a (almost) full-length group I intron. A 2'-amino modification was introduced in the co-substrate and metal ion switch experiments were carried out. In the presence of magnesium ions the reaction rate decreased but introducing nitrogen preferring metal ions as for example manganese, this negative trend could be reversed. Further studies of the catalytic mechanism were performed on the same group I intron but converted to a ribozyme by deleting the substrate site. We introduced a 2'-amino group in the leaving group nucleoside, in a chemical synthesized substrate and metal ion switch experiments were carried out. At most we could study the cleavage reaction with two 2'-amino modifications at the catalytic site, in the co-substrate and in the substrate. In the presence of two 2'-amino modifications we obtained more than additive effects on the rate, in the presence of manganese ions. These experiments showed how one catalytic metal ion coordinated to the 2'-position in the co-substrate and a second one to the 2'-position in the leaving sugar nucleoside. An alternative model of the earlier proposed two metal ion mechanism used by group I introns, is presented.Five RNA model compounds, methyl furanosides with a 5-diphenyl phosphate group were synthesized. The metal ion promoted intramolecular cyclisation reaction, where the 3-hydroxyl attacks the 5-diphenyl phosphate was studied in the presence of different divalent metal ions. Two derivatives contained a 2-amino group and higher reaction rates were obtained in experiments with nitrogen preferring metal ions. These studies showed the effect of an adjacent position, on the hydroxy nucleophile, via coordination of a metal ion and also give data supporting the mechanism model for the catalytic RNA. The other field in this thesis concerns investigations on why ethylene oxide causes more DNA damages, than propylene oxide. To exclude that the intramolecular phosphate transesterification reaction was responsible for the observed differences, two dithymidine alkyl phosphates were synthesized and studied. Further, to see that the respective epoxide did not have different alkylation rates toward a phosphate center, the alkylation rate for the respective epoxide was studied. Neither of these events could be ascribed to be responsible for the earlier observations concerning ethylene oxide and propylene oxide.