Side Reaction Analysis in Solid‐Phase Peptide Synthesis: A Case Study in the Glu–Asp–Tyr Motif

ABSTRACT Peptides are important tools in biological, medical, and pharmaceutical research. Solid‐phase peptide synthesis (SPPS) is the primary method for their preparation in high yields and purity. However, SPPS often encounters difficulties due to the occurrence of side reactions, which can generate by‐products that are difficult to remove. Side reactions can occur under both basic and acidic conditions, resulting in reduced yields, costly purification processes, and sometimes potential inaccessibility of the target peptides. By‐products include the formation of aspartimide, glutarimide, and pyroglutamic acid. The problem of unwanted intramolecular cyclizations is well known and unavoidable, in some cases minimized, as it depends on the intrinsic thermodynamics of the final structures. In this context, we report a systematic analysis of the side reactions that occur during SPPS in peptides containing the Glu‐Asp‐Tyr motif. This study is the first to investigate the formation of three different by‐products within the same peptide sequence and to provide valuable insights into the experimental conditions that favor one reaction over the others. The study aims to identify the optimal experimental conditions to mitigate these by‐products' formation. Our results highlight that the steric and conformational effects of the growing protected peptide chain play a role that is often overlooked or misinterpreted.