Solvent-free enzymatic synthesis of alitame precursor using eutectic substrate mixtures

Area of Science:

• Biocatalysis
• Enzymatic synthesis
• Peptide chemistry

Background:

• Alitame is a high-intensity sweetener.
• Enzymatic synthesis offers a greener alternative to traditional chemical methods.
• Optimizing reaction conditions is crucial for efficient biocatalysis.

Purpose of the Study:

• To synthesize N-benzyloxycarbonyl-L-aspartic acid ethyl ester-D-alanine amide, a derivative of alitame.
• To identify optimal conditions for enzymatic synthesis using alpha-chymotrypsin.
• To investigate the role of eutectic mixtures and adjuvants in substrate preparation.

Main Methods:

• Enzymatic synthesis using alpha-chymotrypsin.
• Preparation of a eutectic substrate mixture with N-benzyloxycarbonyl-L-aspartic acid diethyl ester and D-alanine amide.
• Screening of hydrophilic solvents (DMSO, MEA) as adjuvants.
• Optimization of temperature, substrate ratio, and solvent composition.

Main Results:

• DMSO and MEA were identified as effective adjuvants for eutectic mixture formation.
• A eutectic temperature of 27°C was achieved with 9% DMSO, 18% MEA, and 12% water.
• A conversion yield of 70.3% (mol/mol) was obtained at 37°C.
• The optimal molar ratio of D-alanine amide to N-benzyloxycarbonyl-L-aspartic acid diethyl ester was 1:1.

Conclusions:

• Enzymatic synthesis using alpha-chymotrypsin is effective for producing alitame derivatives.
• Hydrophilic solvents and eutectic mixtures significantly enhance substrate solubility and reaction efficiency.
• Optimized conditions provide a high yield, suggesting a viable industrial application.