Possible origin and suppression of apparent +56 Da impurities during peptide synthesis.

Apparent +56 Da signals present a significant challenge in therapeutic peptide characterization. Using a 36-residue carbohydrate-binding module (CBM) as a representative model, we systematically investigated the chemical origins and analytical behavior of these species. The major chromatographically separable +56 Da impurities were identified as Cys S-tert-butylation products generated during acidic cleavage and global deprotection and were effectively suppressed by replacing Cys(Trt) with Cys(StBu). Additional coeluting +56 Da species originated from Tyr aromatic tert-butylation and intramolecular O-to-N tert-butyl migration and were eliminated by employing side chain unprotected Tyr building blocks during SPPS and retaining the N-terminal Fmoc protection during cleavage. A pseudoisobaric +57 Da impurity arising from double Gly insertion during chain elongation substantially overlapped with the +56 Da tert-butylation peak under low-resolution mass spectrometric (LRMS) conditions but was minimized using lower-basicity DIC/Oxyma-mediated Gly coupling. HRMS further demonstrated that residual +56 Da-like signals partially originated from noncovalent adduct and solvent-cluster species formed during electrospray ionization. Collectively, these findings demonstrate that apparent +56 Da impurities arise from multiple mechanistically distinct sources, establish HRMS as an essential confirmatory tool for their definitive assignment, and provide practical strategies for impurity suppression in complex peptide synthesis.