Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes.

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.

The synthesis of isopropylidene mRNA cap analogs modified with phosphorothioate moiety and their evaluation as promoters of mRNA translation.

Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the ß position of the 5',5'-triphosphate bridge (two diastereomers of 2',3'-iPr-m(7)GppSpG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed (1)H NMR conformational analysis and correlated the absolute configuration at the ß-phosphorous atom with previously synthesized m(7)GppSpG.