Conditionally Deprotectable Phosphoramidite Chemistry for Recording-by-Synthesis of DNA-Encoded N-Methyl Peptides

DNA-Encoded Library (DEL) enables the efficient discovery of bioactive ligands. However, conventional DELs utilize unprotected DNA barcodes, which are susceptible to damage during library construction due to chemical reactions. The instability of DNA barcodes limits the applicable reaction conditions and building blocks, and restricts the number of synthesis cycles to approximately 2-4. As a result, the application of DELs has been mainly confined to small molecules, making it challenging to extend the approach to medium-sized molecules such as peptides. In this study, we report a chemically stable DNA barcode that addresses this issue. We focused on the Recording-by-Synthesis method, which chemically constructs DNA barcodes, and developed the N -acetylpyrrolidin-2-ylmethyl (NAPM) protective group to overcome one of its challenges, the fragility of protected phosphodiester group. The NAPM protective group is stable under conditions used for the solid-phase peptide synthesis, while allowing for deprotection under neutral to weakly basic heating conditions. By integrating the NAPM protective group in short DNA barcode technology, we established a new Recording-by-Synthesis approach that enables ligand synthesis under standard organic solvent-based reaction conditions. Using this strategy, we successfully achieved 10 cycles of alternating synthesis of DNA barcode and peptide, even incorporating bulky N -methyl amino acids.