Chemical Synthesis and Functional Screening of an 800 000‐Member Library Using a Peptide‐Pairing Strategy

Peptide library generation by chemical synthesis allows using a wide range of non-canonical amino acids and thus the screening of chemically and structurally highly diverse libraries. However, despite recent improvements in high-throughput synthesis such as solid-phase peptide synthesis in 384-well plates, the size of synthetic peptide libraries is typically limited to a few thousand molecules. Herein we developed a strategy in which m short peptides with a thiol functional group are combinatorially paired with n short peptides of the same format by a disulfide bridge to generate m × n different peptide sequences. Using acoustic liquid transfer for rapid reagent transfer and formation of multiple peptide pairs per well in 1536-well plates, we were able to screen over 800 000 different compounds that led to the identification of nanomolar inhibitors of the coagulation disorder protease thrombin. The concept of pairing synthetic peptide fragments might be applicable to other targets for identifying peptidic ligands below 1 kDa and thereby small enough to potentially deliver starting points for the development of membrane-permeable drugs for oral application and/or intracellular targeting.