Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety-fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis

We describe synthesis and properties of eight dinucleotide mRNA 5′ cap analogs containing imidodiphosphate moiety within 5′,5′-tri- or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5′-imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying cap-dependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5′ capped mRNAs in vitro to be rather moderate.