Synthesis of a Nonhydrolyzable Nucleotide Phosphoroimidazolide Analogue That Catalyzes Nonenzymatic RNA Primer Extension

We report the synthesis of guanosine 5′-(4-methylimidazolyl)phosphonate (ICG), the third member of a series of nonhydrolyzable nucleoside 5′-phosphoro-2-methylimidazolide (2-MeImpN) analogues designed for mechanistic studies of nonenzymatic RNA primer extension. The addition of a 2-MeImpN monomer to a primer is catalyzed by the presence of a downstream activated monomer, yet the three nonhydrolyzable analogues do not show catalytic effects under standard mildly basic primer extension conditions. Surprisingly, ICG, which has a pK_a similar to that of 2-MeImpG, is a modest catalyst of nonenzymatic primer extension at acidic pH. Here we show that ICG reacts with 2-MeImpC to form a stable 5′-5′-imidazole-bridged guanosine-cytosine dinucleotide, with both a labile nitrogen-phosphorus and a stable carbon-phosphorus linkage flanking the central imidazole bridge. Cognate RNA primer-template complexes react with this GC-dinucleotide by attack of the primer 3′-hydroxyl on the activated N-P side of the 5′-5′-imidazole bridge. These observations support the hypothesis that 5′-5′-imidazole-bridged dinucleotides can bind to cognate RNA primer-template duplexes and adopt appropriate conformations for subsequent phosphodiester bond formation, consistent with our recent mechanistic proposal that the formation of activated 5′-5′-imidazolium-bridged dinucleotides is responsible for 2-MeImpN-driven primer extension.