Massively parallel, single-molecule assessment of synthetic fidelity and drug-like properties in a DNA-encoded library

DNA-encoded libraries (DELs) have emerged as a promising drug discovery strategy, but successful translation of hits is often impeded by synthetic inefficiency and enrichment of poorly permeable compounds. Here we introduce a novel sequencing-based separation strategy, LC-seq, that simultaneously evaluates synthetic fidelity and permeability-relevant lipophilicity for individual DNA-encoded library members. Using a 120,000-member peptide library, we mapped reaction efficiency across all synthetic cycles and identified structure-reactivity trends. The on-DNA lipophilicities for resynthesized library members correlate strongly with their off-DNA lipophilicities and passive permeability in artificial membranes and MDCK cells. This approach enables direct assessment of compound quality and drug-like properties at unprecedented scale, potentially transforming DEL-based drug discovery.