Reverse proteolysis promoted by in situ generated peptide ester fragments

Area of Science:

• Biocatalysis and Peptide Synthesis
• Organic Chemistry
• Enzymology

Background:

• Protease-mediated peptide synthesis requires specific reactants.
• Existing methods for preparing these reactants can be complex or inefficient.
• Methyl thioesters offer potential as versatile precursors for peptide coupling.

Purpose of the Study:

• To present a general synthesis concept for in situ preparation of protease-specific reactants.
• To utilize methyl thioesters as universal precursors for enzymatic peptide synthesis.
• To develop a method for generating substrate mimetics for enhanced protease-mediated reactions.

Main Methods:

• Methyl thioesters were employed as universal precursors for protease-specific reactants.
• A one-pot spontaneous transthioesterification reaction was utilized for in situ substrate mimetic preparation.
• Model peptide syntheses were performed using trypsin, alpha-chymotrypsin, and V8 protease.

Main Results:

• Methyl thioesters were successfully used directly as reactants for protease-mediated peptide coupling.
• In situ preparation of substrate mimetics via transthioesterification was achieved efficiently.
• The synthesized substrate mimetics acted as effective acyl donors, enabling irreversible reactions.
• The approach demonstrated compatibility with solid-phase peptide ester synthesis.

Conclusions:

• A general and efficient synthesis concept for protease-specific reactants using methyl thioesters was established.
• The in situ preparation of substrate mimetics streamlines protease-mediated peptide synthesis.
• This method offers synthetic value and broad applicability in biocatalytic peptide chemistry.