Concerned with the potential of Cu(I) salts to oxidize certain amino acids, particularly Met and His, we examined the compatibility of the DBCO protection strategy using the more diverse model peptide H-C(StBu)DEAFGHIK(DBCO)LMNPQRSTVWYK-NH2 3a (and without DBCO, 3b), which contains every canonical amino acid. We found that thioanisole and 1,2ethanedithiol, two commonly used TFA cleavage scavengers that prevent Cys alkylation, diminished the protective properties of the Cu(I) additive with 3a (Figures ). Furthermore, we noted that ammonium iodide (NH4I), a cleavage additive used to reverse Met oxidation, was also capable of completely removing the StBuprotecting group on 3a and 3b (Figures ). When 3b was cleaved in the presence or absence of ~3 equiv. (MeCN)4CuBF4, the analytical HPLC and LC/MS chromatograms were similar (Figures and). However, the presumed oxidation side-product (+16 Da) increased from 6% to 12.5% in Cu(I)-treated 3b (Figure ). Using trypsin digestion and high-resolution LC/MS/MS on 3b ± Cu(I) during cleavage, Met was identified as the oxidation-prone residue (Figure ). Furthermore, when the Cu(I) salt was used in the cleavage of model peptides H-C(StBu)GKENTWYX-NH2 4a (X=H) and 4b (X=M), oxidation was only observed in the Met-containing peptide 4b (Figures ). While increased Met oxidation is a potential limitation of the DBCO protection method, all other canonical amino acids are fully compatible. In addition, Met is one of the least abundant amino acids and can typically be substituted with an isosteric norleucine (Nle) residue without affecting protein activity and folding. We next investigated the ideal concentration of the (MeCN)4CuBF4 additive.