Synthesis and biochemical application of 2'-O-methyl-3'-thioguanosine as a probe to explore group I intron catalysis

Oligonucleotides containing 3'-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here, we have synthesized 2'-O-methyl-3'-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme reaction. We developed an efficient method for the synthesis of 2'-O-methyl-3'-thioguanosine phosphoramidite in eight steps starting from 2'-O-methyl-N(2)-(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-phase synthesis, we used this new analogue to investigate whether the 3'-oxygen of the guanosine cofactor in the Tetrahymena ribozyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 2'-hydroxyl group. We show that regardless of whether the guanosine cofactor bears a 3'-oxygen or 3'-sulfur leaving group, replacing the adjacent 2'-hydroxyl group with a 2'-methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen bond donated by the guanosine 2'-hydroxyl group contributes to catalytic function in a manner distinct from the U(-1) 2'-hydroxyl group.