Determination of Diketopiperazine Formation During the Solid‐Phase Synthesis of C‐Terminal Acid Peptides

Diketopiperazine formation (DKP) is one of the most serious side reactions during peptide synthesis. The driving force for DKP formation is the intrinsic stability of the six‐membered cyclic dipeptides formed by two α‐amino acids. Although the reaction can occur with C ‐terminal amide peptides, it is more prevalent when preparing C ‐terminal acid peptides or depsipeptides, as the alcohol is a much better leaving group than the amine. When C ‐terminal acid peptides are prepared using Wang resin, which is one of the solid supports employed for the preparation of such molecules, DKP formation can jeopardize the synthetic process. In this regard, it can lead to a loss of yield, as the C‐ terminal dipeptide is cleaved from the resin to form the cycle. Additionally, it may facilitate the formation of side products where the first two amino acids are missing, as the third amino acid and the subsequent ones can be anchored to the hydroxymethyl groups formed on the resin after the loss of DKP. Herein, an accurate method to determine the DKP formation in peptide synthesis on Wang resin is determined. Furthermore, strategies to minimize this side reaction, and side products lacking C‐terminal amino acids are described.