Highly Pure Combinatorial Peptide Libraries by Resin-Enabled Catch and Release.

For peptide-drug conjugate (PDC) development, high-quality peptide ligands with easy functionalization are essential. Combinatorial chemical peptide libraries are key for discovering high-affinity non-natural ligands, yet their utility has long been hindered by low-purity libraries that cause false positives. To address this, we developed an N-terminal cysteine-based dynamic catch-and-release (CbDCR) platform, leveraging its unique 1,2-aminothiol motif as a privileged handle for site-selective orthogonal conjugation, enabling efficient library purification while retaining a universal anchor for downstream functionalization. A recyclable 2-formylphenylboronic acid resin (2FPBA resin) was designed to catch and release N-terminal cysteine-containing peptides in a pH-responsive manner, eliminating the need for complex tags and providing simplicity, scalability, and high purification efficiency through optimized workflows. The platform effectively purifies SPPS-synthesized peptides, enriches N-terminal cysteine-containing peptides from protein lysates, supports high-purity split-and-pool library preparation, and integrates with microplate-based high-throughput workflows. Using a highly pure "RGD"-focused nonstandard library, high-affinity integrin αvβ6-targeting peptides were identified, and three peptide-radionuclide conjugates with potential diagnostic value for pancreatic cancer were constructed via N-terminal cysteine. Overall, CbDCR streamlines library purification, affinity screening, and conjugate construction, accelerating the development of high-value peptide ligands and PDCs.