Synthesis and Translational Assessment of Trinucleotide 5′-Cap Analogs for Messenger Ribonucleic Acid-Based Therapeutics

Capping is a natural and distinctive modification that occurs at the 5′-end of eukaryotic messenger RNA (mRNA), which regulates biological functions for gene expression events. Recently, the synthetic variation of a 5′-cap element has attracted considerable attention for improving the transcriptional efficiency and stability of mRNA. Herein, we describe the synthesis and biological evaluation of trinucleotide 5′-capping agents with 2′- and 3′-ribose modifications. The 3′-O-mesylated m7GpppAmG 43 demonstrated a high capping efficiency of 97.1%, comparable to the commercial CleanCapAG(3′OMe) 9. Furthermore, compound 43 exhibited a potent translational ability in the dual luciferase reporter assay, which was 1.8-fold higher than that of compound 9. Moreover, compound 43 was resistant against decapping enzymes (DcpS and hDcp2), thereby revealing its stability under biological conditions. In vivo translational studies demonstrated that lipid nanoparticle 43a, formulated from compound 43 with firefly luciferase mRNA, exhibited intense bioluminescence, supporting its translational competence.