Application of empirical design methodologies to the study of the influence of reaction conditions and N-α-protecting group structure on the enzymatic X-Phe-Leu-NH2 dipeptide synthesis in buffer/dimethylformamide solvents systems

• Published in: Biotechnology and bioengineering 1992-03, Vol.39 (5), p.539-549
• Main Authors: Calvet, S., Clapés, P., Vigo, J. P., Xaus, N., Jorba, X., Mas, R. M., Torres, J. L., Valencia, G., Serralheiro, M. L., Cabral, J. M. S., Empis, J. M. A.
• Format: Article
• Language: English
• Subjects: enzymatic peptide synthesis; experimental design; log P; molecular refractivity; N-terminal protecting groups; partition constant; reaction rate; response surfaces; α-chymotrypsin
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.260390509

The influence of five different N‐terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the α‐chymotrypsin‐catalyzed synthesis of the dipeptide derivative X‐Phe‐Leu‐NH2 was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N‐α protection choice. Boc and Z were the most reactive N‐α groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.