Qigui Nie, Xufeng Li, Xianfu Fang, Junshan Fan, Xia Yang, Gong Zhang, Yangfeng Li, Yizhou Li

Chinese Journal of Chemistry

DOI: 10.1002/cjoc.70673

Abstract

Comprehensive Summary DNA‐encoded library (DEL) technology has emerged as a powerful platform for peptide drug discovery by enabling the rapid construction and screening of vast chemical space through on‐DNA amino acid coupling. However, despite the critical role of unnatural amino acids (UAAs) in improving peptide stability, bioavailability, and target specificity, the limited availability of structurally diverse DNA‐compatible UAA building blocks remains a major bottleneck for expanding the chemical diversity of DNA‐encoded peptide libraries. Herein, we report a robust and DNA‐compatible strategy for the on‐DNA generation of dehydroalanine (Dha) through the mild elimination of DNA‐conjugated cysteine under biocompatible conditions. Acting as a highly versatile synthetic linchpin, Dha enables the divergent installation of a broad spectrum of UAAs featuring sulfur‐, nitrogen‐, carbon‐, phosphorus‐containing, and cyclic side chains, thereby substantially enriching accessible peptide chemical space. Beyond serving as a diversification handle, the utility of this platform was demonstrated in multiple settings, including mock combinatorial peptide library synthesis, late‐stage functionalization of bioactive peptide motifs, and on‐DNA peptide macrocyclization, highlighting its broad synthetic scope and operational flexibility. Importantly, Dha also functions as an electrophilic covalent warhead for ligand‐directed protein crosslinking, expanding its application beyond library diversification. Leveraging this unique reactivity, we further established a cysteine “unmasking” strategy that enables crosslinking‐assisted DEL selection. As a proof of concept, a Dha‐containing DEL screened against carbonic anhydrase II successfully identified target binders through covalent capture. Collectively, this work introduces a generalizable approach for expanding the structural diversity and functional complexity of DNA‐encoded peptide libraries while integrating crosslinking‐assisted selection capabilities, thereby providing new opportunities for peptide ligand discovery and covalent probe development.