Large Macrocyclic Libraries via Thioether Cyclization.

Macrocyclic peptides, known for their high specificity, binding affinity, and potential membrane permeability, represent a promising modality in drug discovery, particularly for intracellular targets deemed undruggable by conventional small molecules. Traditional methods for macrocycle synthesis often involve labor-intensive purification steps, limiting throughput and scalability. This protocol presents a streamlined approach for the synthesis of large macrocyclic peptide libraries, utilizing solid-phase peptide synthesis (SPPS) followed by purification-free cyclization of linear dithiol peptides in high-throughput array formats. The workflow incorporates automated peptide synthesis, efficient resin preparation, and thioether cyclization strategies, enabling the generation of diverse libraries suitable for screening campaigns. Key features include the use of bis-electrophilic linkers for cyclization, quality control via LC-MS, and optional concentration assays. This robust method accelerates the development of macrocyclic libraries, facilitating the discovery of drug candidates targeting challenging biomolecular interactions.