Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity

Area of Science:

• Biochemistry
• Enzymology
• Organic Chemistry

Background:

• Proteases like alpha-chymotrypsin and subtilisins are widely used in peptide synthesis.
• Understanding enzyme specificity is crucial for optimizing synthetic yields and product purity.
• Hydrophilic organic solvents offer unique environments for enzymatic reactions.

Purpose of the Study:

• To investigate the substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis.
• To compare enzyme behavior in hydrophilic organic solvents with their known specificities in aqueous solutions.
• To explore the influence of solvent composition and substrate structure on enzyme activity.

Main Methods:

• Enzymatic peptide synthesis using alpha-chymotrypsin, subtilisin Carlsberg, and subtilisin BPN'.
• Varying acyl donors (aromatic amino acids) and acyl acceptors (hydrophilic amino acid amides).
• Modifying water-organic solvent composition and evaluating nucleophile reactivity.

Main Results:

• Chymotrypsin showed high specificity for aromatic amino acids as acyl donors.
• Subtilisins recognized aromatic and neutral aliphatic amino acids, mirroring their hydrolysis specificities.
• Chymotrypsin favored hydrophilic amino acid amides as acyl acceptors, differing from aqueous solution behavior.
• Nucleophile specificity shifted with water content, altering relative reactivity of leucinamide and alaninamide.
• Amidation of alanine's carboxyl group was more effective than esterification for nucleophile reactivity.

Conclusions:

• Enzyme substrate specificities are adaptable to organic solvent environments for peptide synthesis.
• Nucleophile choice and protection strategy significantly impact peptide synthesis efficiency.
• Water content in organic solvents plays a critical role in modulating enzyme nucleophile specificity.