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

Area of Science:

• Biochemistry
• Organic Chemistry
• Enzymology

Background:

• Traditional alpha-amino protecting groups (e.g., Z, Fmoc, Boc) can limit water solubility and reaction efficiency in peptide synthesis.
• Developing novel protecting groups is crucial for improving solubility and catalytic activity in enzymatic peptide coupling.

Purpose of the Study:

• To synthesize and evaluate novel, negatively charged N alpha-protecting groups, Maleyl (Mal) and Citraconyl (Cit), for enzyme-catalyzed peptide synthesis.
• To assess the impact of these charged groups on the solubility of amino acid derivatives and the efficiency of enzymatic reactions.
• To demonstrate the utility of these groups in large-scale peptide production using kyotorphin as a model.

Main Methods:

• Synthesis of amino acid derivatives functionalized with Maleyl and Citraconyl protecting groups.
• Enzyme-catalyzed peptide synthesis using alpha-chymotrypsin.
• Kinetically controlled synthesis of kyotorphin (tyrosyl-arginine) with Mal-Tyr-OEt and Arg-OEt.
• Scale-up of the synthesis to a 300 L batch experiment.

Main Results:

• Maleyl and Citraconyl groups significantly increased the water solubility of aromatic amino acid derivatives.
• These charged protecting groups enhanced the activity of enzymatic synthesis reactions.
• Successful production of approximately 12 kg of tyrosyl-arginine with a 50.4% overall yield in a large-scale batch experiment.

Conclusions:

• Negatively charged Maleyl and Citraconyl protecting groups are effective in improving solubility and enzyme-catalyzed peptide synthesis.
• These novel protecting groups facilitate efficient, large-scale production of peptides like kyotorphin.
• The findings offer a promising strategy for advancing enzymatic peptide synthesis through enhanced solubility and reactivity.