Flexizyme‐Based Strategy for the Synthesis of Stable, Non‐Isomerizable Amide‐Linked 2’‐Aminoacyl‐tRNAs and their Shortened Analogs

The study of the regiospecificity of aminoacyl-tRNA (AA-tRNA)-dependent enzymes and their structural characterization with AA-tRNAs are limited by rapid hydrolysis of the ester bond linking amino acid to tRNA. To overcome this limitation, stable AA-tRNA analogs bearing hydrolysis-resistant linkages, such as amide bonds or ester bioisosteres, have been developed. These analogs are valuable tools for investigating interactions between AA-tRNAs and various enzymes or ribonucleoproteins, including elongation factors, ribosomes, Fem-family transferases, and cyclodipeptide synthases. However, their synthesis remains technically challenging. Recently, flexizymes-engineered ribozymes capable of aminoacylating tRNAs with diverse amino acids or analogs-have enabled the synthesis of 3'-amide-linked AA-NH-tRNAs. Due to their inherent specificity for 3'-OH acylation, flexizymes have not been used to generate 2'-amide-linked analogs, and such regioisomers have remained unexplored. In this study, we demonstrate that while flexizymes cannot directly aminoacylate the 2' position, they can nevertheless mediate the synthesis of 2'-aminoacyl-NH-tRNAs via a two-step regioisomerization mechanism with excellent yields. This finding provides new insights into the binding mode of AA-tRNAs to flexizymes and expands the chemical space of stable AA-tRNA analogs. Access to both 3'- and 2'-amide regioisomers will enable more precise studies of AA-tRNA recognition and catalysis by various AA-tRNA-dependent systems.