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Encoding small-molecule information in DNA has been leveraged to accelerate the discovery of ligands for therapeutic targets such as proteins. However, oligonucleotide-based encoding is hampered by inherent limitations of information stability and density. In this study, we establish abiotic peptides for next-generation information storage and apply them for the encoding of diverse small-molecule synthesis. The chemical stability of the peptide-based tag allows the use of palladium-mediated reactions to efficiently synthesize peptide-encoded libraries (PELs) with broad chemical diversity and high purity. We demonstrate the successful de novo discovery of small-molecule protein ligands from PELs by affinity selection against carbonic anhydrase IX and the oncogenic protein targets BRD4(1) and MDM2. Collectively, this work establishes abiotic peptides as carriers of information for the encoding of small-molecule synthesis, leveraged herein for the discovery of protein ligands. Description Peptide tags for small molecules During the early stages of drug discovery, chemists often expose target proteins to vast libraries of small molecules in the hope of finding one that binds tightly. Tagging the molecules with small fragments of DNA has proven a convenient means of interpreting the screen at concentrations where it would otherwise be hard to determine which of them were bound. However, nucleotide structure constrains the chemistry applicable to making the drug candidates. Rössler et al. now showcase an alternative method in which the tags consist of peptides in place of oligonucleotides, expanding the scope of compatible chemistry (see the Perspective by Haap). —JSY Peptides are used in place of oligonucleotides to tag libraries of small-molecule drug candidates for affinity selection. INTRODUCTION The discovery of therapeutics and biochemical probes hinges on the ability to identify molecules that interact with biological targets of interest. Technologies such as DNA-encoded libraries have transformed the process of drug discovery by enabling the rapid synthesis of vast collections of molecules, each encoded with a unique appendant DNA tag, and their subsequent screening in affinity selection experiments. However, the use of DNA to encode information confers synthetic limitations owing to the incompatibility of oligonucleotides with various chemical reaction conditions that result in the loss of stored information. As a consequence, chemical transformations developed for DNA-encoded library synthesis have to be optimized for oligonucleotide compatibility rather than reaction efficiency and scope. Given the vast potential of encoded library technologies in drug discovery, complementary platforms addressing the limitations of DNA encoding by leveraging carriers of information with higher stability and versatility are desirable. RATIONALE Molecular encoding can in principle be achieved in any polymer with at least two distinguishable monomers. Peptides constitute a biopolymer that is routinely sequenced for elucidation of protein identity through the use of tandem mass spectrometry. Accordingly, synthetic peptides can serve as carriers of information decoded through determination of their amino acid sequence. We hypothesized that the excellent chemical stability of peptides and their compatibility with a broad range of reaction conditions would render them particularly suited for the encoding of small-molecule synthesis. Peptides, which can be efficiently prepared by solid-phase synthesis, offer potential as carriers of information for a next-generation discovery platform with combinatorial libraries of small molecules. RESULTS We describe the design of abiotic peptides as carriers of information for the encoding of small-molecule synthesis. Therein, the identity of a small molecule was stored in an appendant peptide that is elongated in accordance with the synthetic elaboration of the small molecule. The encoding peptide featured a hexadecimal encoding alphabet of nonisobaric amino acids, resulting in high information density and chemical stability. The sequence of the encoding peptide was optimized through the systematic inclusion of selected amino acids to fine-tune polarity and ease of sequencing, resulting in high-fidelity decoding by tandem mass spectrometry. The chemical stability of the peptide tag enabled synthetic versatility for small-molecule transformations, including acidic conditions or transition metal catalysis with reported incompatibility with DNA tags. This broad compatibility allowed the implementation of palladium-mediated cross-coupling reactions characterized by a diverse scope and high reaction efficiency. The encoding of small molecule synthesis in peptides was leveraged for the split-and-pool synthesis of combinatorial libraries called peptide-encoded libraries (PELs) characterized by high purity. PELs featuring tens of thousands of drug-like small molecules resulting from optimized palladium-mediated C–C and C–N cross-coupling reactions were used in affinity selections against oncogenic proteins. The peptide sequences of enriched conjugates were decoded, and the corresponding small molecules were rapidly prepared by solid-phase synthesis and subsequently confirmed to exhibit affinity for their target protein. The PEL discovery platform is characterized by high efficiency and has afforded diverse, previously unknown small-molecule binders for the target proteins. CONCLUSION The results demonstrate that abiotic peptides can be used to encode and decode information to discover small molecules with affinity to proteins of interest. The PEL discovery platform establishes a starting point for the next generation of encoded library technology with broad implications for therapeutics discovery and biochemical research. Peptide-encoded libraries of small molecules enable the discovery of hit compounds with affinity for protein targets of interest. Split-and-pool synthesis of small molecules generates libraries encoded by unique peptide tags. The peptide-encoded libraries are used in affinity selections with targets of interest. After releasing the encoding tag, the information is decoded by tandem mass spectrometry to identify the hit molecule. nLC-MS/MS, nanoscale liquid chromatography–tandem mass spectrometry.