Hydantoin Impurity Formation During Peptide N-Terminal Acetylation with Acetic Anhydride

Abstract Previously unreported N-terminal hydantoin and N-acetyl hydantoin impurities were observed during peptide N-terminal acetylation with acetic anhydride under commonly used base/solvent conditions. The impurities were isolated and structurally characterized by HRMS and NMR. Systematic studies revealed pronounced solvent dependence and sequence sensitivity and identified conditions that suppressed their formation. Isotope-labeling experiments excluded acetic anhydride as the source of the carbonyl moiety incorporated into the hydantoin and supported solvent participation in the byproduct-forming pathway. Collectively, these results define an unexpected side reaction in widely used acetylation chemistry and provide practical guidance for impurity control in peptide synthesis and process development.