Preparative-scale enzyme-catalyzed peptide synthesis using solubilizing N-terminal protecting groups.

Several amino acid derivatives with the negatively charged N alpha-protecting groups Maleyl (Mal) and Citraconyl (Cit) were synthesized and used in enzyme-catalyzed peptide synthesis. Compared to commonly used alpha-amino protecting groups in chemical peptide synthesis (Z, Fmoc, Boc, etc.), these charged protecting groups strongly increase both water solubility of different aromatic amino acid derivatives and activities of synthesis reactions. As a consequence of the solubilizing effect, we used these groups in the kinetically controlled peptide synthesis choosing kyotorphin (tyrosyl-arginine) as a model peptide. With Mal-Tyr-OEt as substrate and Arg-OEt as nucleophile, we succeeded in the alpha-chymotrypsin-catalyzed production of about 12 kg of Tyr-Arg (50.4% overall yield) in a 300 l batch experiment.