Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Abstract

Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the α, β, or γ position of the 5′,5′-triphosphate chain. Three of them were also modified with methyl groups at the 2′-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K _AS) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the β-substituted analog m⁷Gpp_SpG) was characterized by a K _AS that was more than fourfold higher than that of its unmodified counterpart (m⁷GpppG). All analogs modified in the γ position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the α position (i.e., m⁷Gppp_SG and m₂ ^7,2′-OGppp_SG) were established as S_P and R_P , respectively, using enzymatic digestion and correlation with the S_P and R_P diastereomers of guanosine 5′-O-(1-thiodiphosphate) (GDPαS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

Keywords

• phosphorothioate cap analogs
• ARCA
• decapping enzymes
• DcpS
• binding affinity for eIF4E
• resistance to enzymatic hydrolysis