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'-<i>O</i>-mesylated m⁷GpppA_mG <b>43</b> demonstrated a high capping efficiency of 97.1%, comparable to the commercial CleanCapAG(3'OMe) <b>9</b>. Furthermore, compound <b>43</b> exhibited a potent translational ability in the dual luciferase reporter assay, which was 1.8-fold higher than that of compound <b>9</b>. Moreover, compound <b>43</b> was resistant against decapping enzymes (DcpS and hDcp2), thereby revealing its stability under biological conditions. <i>In vivo</i> translational studies demonstrated that lipid nanoparticle <b>43a</b>, formulated from compound <b>43</b> with firefly luciferase mRNA, exhibited intense bioluminescence, supporting its translational competence.