Many critical disease-related proteins, including cancer drivers like KRAS, β-catenin, and MYC, are difficult drug targets because they lack the binding pockets that traditional small molecule drugs require. Small peptides composed of mostly non-natural amino acids offer a promising solution, as these "beyond Rule of 5" compounds can bind flat protein surfaces while maintaining the ability to cross cell membranes.¹ However, successfully developing these molecules requires robust methods for generating and screening large combinatorial libraries.

DNA-encoded libraries (DELs) are a powerful platform for drug discovery, yet the success of their application to peptides has been limited. As many coupling steps are needed for the construction of peptides from amino acids, the quality of the final DELs is usually very low, distorting the genotype-phenotype linkage.² Here, we report the integration of solid phase peptide-synthesized (SPPS) building blocks into a DEL synthesis workflow to generate high-purity library in only two cycles. For this purpose, a minimal workup procedure for SPPS products without purification is employed, enabling the parallel synthesis of hundreds to thousands of building blocks in high quality. The coupling efficiencies of the synthesized tri- and tetramers were comparable to those of Fmoc-amino acids. A proof-of-concept 71 x 96 membered library featuring validated binders for MDM2 was synthesized. After affinity selection against MDM2, clear enrichment of the consensus sequence was observed, while the enrichment could be directly linked to the quality of the genotype-phenotype linkage.

[1] X. Ji, A. L. Nielsen, C. Heinis, Angew. Chem. Int. Ed., 2024, 136 (3), e202308251.

[2] M. Keller, D. Petrov, A. Gloger, B. Dietschi, K. Jobin, T. Gradinger, A. Martinelli, L. Plais, Y. Onda, D. Neri, J. Scheuermann, Science, 2024, 384 (6701), 1259–1265.