title: Synthesis and biochemical application of 2'-O-methyl-3'-thioguanosine as a probe to explore group I intron catalysis.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry & Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Nan-Sheng
authors.initials: NS
authors.lastname: Li
authors.forename: Raghuvir N
authors.initials: RN
authors.lastname: Sengupta
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Piccirilli
mesh_terms: D000818:Animals; D002384:Catalysis; D006151:Guanosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D006860:Hydrogen Bonding; D007438:Introns; D008968:Molecular Conformation; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods; D013768:Tetrahymena / Q000201:enzymology; D013872:Thionucleosides / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: C530736:2'-O-methyl-3'-thioguanosine; D015335:Molecular Probes; D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D016337:RNA, Catalytic; D013872:Thionucleosides; C434331:phosphoramidite; D006151:Guanosine