Synthesis of Novel C4-Linked C2-Imidazole Ribonucleoside Phosphoramidite and Its Application to Probing the Catalytic Mechanism of a Ribozyme

The synthesis of a novel C4-linked C-2-imidazole ribonucleoside phosphoramidite (ICN-C-2-PA 1) with a two-carbon linker between imidazole and ribose moieties is described. In the phosphoramidite, POM and 2-cyanoethyl groups were selected to protect the endocyclic amine function of imidazole and the 2'-hydroxyl function of D-ribose, respectively. The C-2-imidazole nucleoside, a flexible structural mimic of a purine nucleobase, was successfully incorporated using ICN-C-2-PA 1 into position 638 of the VS ribozyme through 2'-TBDMS chemistry to study the role of G638 in general acid-base catalysis. The modified VS ribozyme (G638C(2)Imz) exhibited significantly greater catalytic activity than observed with the C-0-imidazole that has no carbon atoms linking the ribose and the C4-imidazole. Imidazole nucleoside analogues with variable spacer lengths could provide a valuable general methodology for exploring the catalytic mechanisms of ribozymes.