New PEGs for Peptide and Protein Modification, Suitable for Identification of the PEGylation Site

摘要

New PEG derivatives were studied for peptide and protein mdofication, based upon an amino acid arm, Met-Nle or Met-#beta#Ala, activated as succinimidyl ester. PEG-Met-Nle-OSU or PEG-Met-#beta#Ala-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leavying Nle or #beta#Ala as reporter amino acid, as the site where PEG was bound. The conjugatoin of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterizaation of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reported norleucine that corresponds to the position of PEG binding.