Massively Parallel, Single-Molecule Assessment of Synthetic Fidelity and Drug-Like Properties in a DNA-Encoded Library

Abstract DNA-encoded libraries (DELs) are powerful drug discovery tools, enabling rapid hit generation against immobilized protein targets. However, translating these hits is often hampered by synthetic inefficiency and, for libraries of “beyond-Rule-of-5” compounds such as macrocyclic peptides, by enrichment of poorly cell-permeable members. Here we introduce LC-seq, a sequencing-based chromatographic strategy that simultaneously assesses synthetic fidelity and permeability-relevant lipophilicity for individual library members. Applying LC-seq to a proof-of-concept 120,000-member peptide library, we mapped reaction efficiency across all synthetic cycles and measured each member’s lipophilicity from sequencing-count-derived retention times. We identified building-block-specific structure–reactivity trends, and the on-DNA lipophilicities of resynthesized members correlated strongly with their off-DNA lipophilicities and passive permeability in artificial membranes. This simple approach enables direct, per-member assessment of compound quality and lipophilicity, with projected scalability to libraries of millions.