Synthesis and Incorporation of the Phosphoramidite Derivative of 2′‑O‑Photocaged 3′‑S‑Thioguanosine into Oligoribonucleotides: Substrate for Probing the Mechanism of RNA Catalysis

ABSTRACT: Oligoribonucleotides containing 3′-S-phosphorothiolate linkages possess properties that can reveal deep mechanistic insights into ribozyme-catalyzed reactions. “Photocaged ” 3′-S- RNAs could provide a strategy to stall reactions at the chemical stage and release them after assembly steps have occurred. Toward this end, we describe here an approach for the synthesis of 2′-O-(o-nitrobenzyl)-3′-thioguanosine phosphoramidite starting from N2-isobutyrylguanosine in nine steps with 10.2 % overall yield. Oligonucleotides containing the 2′-O-(o-nitrobenzyl)-3′-S-guanosine nucleotide were then constructed, characterized, and used in a nuclear pre-mRNA splicing reaction. Oligonucleotides containing phosphorothiolate linkages, inwhich sulfur replaces the 3′- or 5′-bridging oxygen connected to the ribofuranose ring (Figure 1, II and III), serve as powerful biochemical probes to investigate fundamental features of enzyme catalysis.1−4 RNAs containing a 3′-S-phosphorothiolate linkage have been used in metal ion rescue experiments to elucidate the mechanism of metalloribozymes such as the group I intron, the group II intron, and the spliceosome.5−8 These studies rely on careful comparison of the 3′-O and 3′-S substrates and must include assays tha