Chemical modifications of the mRNA cap structure can
enhance the
stability, translational properties, and half-life of mRNAs, thereby
altering the therapeutic properties of synthetic mRNA. However, cap
structure modification is challenging because of the instability of
the 5′-5′-triphosphate bridge and N7-methylguanosine.
The Suzuki–Miyaura cross-coupling reaction between boronic
acid and halogen compound is a mild, convenient, and potentially applicable
approach for modifying biomolecules. Herein, we describe two methods
to synthesize C8-modified cap structures using the Suzuki–Miyaura
cross-coupling reaction. Both methods employed phosphorimidazolide
chemistry to form the 5′,5′-triphosphate bridge. However,
in the first method, the introduction of the modificationviathe Suzuki–Miyaura cross-coupling reaction at
the C8 position occurs postsynthetically, at the dinucleotide level,
whereas in the second method, the modification was introduced at the
level of the nucleoside 5′-monophosphate, and later, the triphosphate
bridge was formed. Both methods were successfully applied to incorporate
six different groups (methyl, cyclopropyl, phenyl, 4-dimethylaminophenyl,
4-cyanophenyl, and 1-pyrene) into either the m7G or G moieties
of the cap structure. Aromatic substituents at the C8-position of
guanosine form a push–pull system that exhibits environment-sensitive
fluorescence. We demonstrated that this phenomenon can be harnessed
to study the interaction with cap-binding proteins,e.g., eIF4E, DcpS, Nudt16, and snurportin.