NMR‐Guided Fragment Screening Identifies Privileged Noncanonical Amino Acids for mRNA Display

Fragment‐based strategies have transformed small‐molecule drug discovery, yet analogous approaches for selecting noncanonical amino acids (ncAAs) in peptide drug discovery remain largely unexplored. Here we introduce an NMR‐guided screening workflow that identifies individual ncAAs as “fragments” to identify privileged monomers prior to peptide library construction. Using the p53–MDM2 interaction as a benchmark system, we screened a panel of tryptophan analogs and identified 6‐bromo and 6‐chloro tryptophan (6Br‐Trp and 6Cl‐Trp) as a higher‐affinity fragments relative to native Trp. Incorporation of 6Br‐Trp into a model peptide improved binding to MDM2, demonstrating that amino acid affinity translates directly to peptide‐level binding. We further show that 6Br‐Trp and 6Cl‐Trp can be efficiently charged onto tRNA and incorporated into peptides using the PURE translation system. In an mRNA display selection, peptides containing 6Br‐Trp and 6Cl‐Trp exhibited enhanced enrichment compared to their Trp‐containing counterparts. These results show ncAA fragment screening to be a front‐end strategy for guiding monomer selection in high‐throughput peptide discovery via mRNA display. ncAA screening also provides a route to bias peptide libraries toward more productive chemical space to accelerate the identification of high‐affinity ligands for challenging protein targets.