Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating three kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 nM and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 h and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties
