Design and Biosynthesis of Ornithine 8-Containing Semaglutide Variants with a Click Chemistry-Modifiable Position 26

Semaglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, constitutes an effective and widely used treatment for type 2 diabetes and obesity. However, challenges such as insufficient oral bioavailability, gastrointestinal side effects, and high costs persist. Overcoming these limitations is essential for improving patient compliance and semaglutide's safety profile. While advanced technologies such as oral delivery systems offer partial solutions, optimizing the peptide structure is crucial for addressing these issues. Establishing a rapid method to generate a large library of semaglutide mutants will enable high-throughput activity screening. In this study, we introduce a novel "Fits-In-All" approach that combines ribosomally synthesized and post-translationally modified peptide (RiPP) technology with amber stop codon incorporation to generate semaglutide variants. To counter dipeptidyl peptidase-4-mediated cleavage, our method strategically incorporates noncanonical amino acid ornithine at position 8 utilizing microbial modification enzyme OspR <i>in vivo</i>. Furthermore, functional groups are introduced by an orthogonal tRNA/aminoacyl-tRNA synthetase pair recognizing the amber stop codon at position 26, which enabled the click chemistry-based linkage of diverse groups. This approach allows for the generation of a broad array of semaglutide analogues that can be screened for optimal properties. In conclusion, this innovative approach opens new avenues for the design and synthesis of optimized peptide-based GLP-1 receptor agonists.