Synthetic dinucleotide mRNA cap analogs with tetraphosphate 5',5' bridge containing methylenebis(phosphonate)
Anna Maria Rydzik1, Maciej Lukaszewicz, Joanna Zuberek
1Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.
Abstract:
An effective and facile synthesis of six novel tetraphosphate cap analogs modified with a methylenebis(phosphonate) moiety (1-6) is presented. Analogs have been rationally designed to bind tightly to the eukaryotic initiation factor 4E (eIF4E) responsible for cap binding during the initiation of translation, and have increased stability owing to resistance to enzymatic degradation. Final compounds turned out to have significantly higher association constant values (K(AS)) for binding to eIF4E (5-9 fold higher than standard). Four of the analogs were resistant towards enzymatic degradation by human Decapping Scavenger enzyme (DcpS). Binding studies of non-hydrolyzable analogs with DcpS revealed a broad range of K(AS) values for different analogs. All of the analogs were potent inhibitors of translation in a rabbit reticulocyte lysate system (RRL) and those resistant to DcpS turned out to be stable under an elongated time of preincubation while the inhibitory potency of standard was diminished in these conditions. For Anti Reverse Cap Analog (ARCA) dinucleotides (4-6), we have shown that they are effectively incorporated into mRNA and transcripts capped with these analogs undergo translation in vitro.
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