Highly improved enzymatic peptide synthesis by using biphasic reactors

The synthesis of hydrophobic peptide as Leu-enkephalin derivatives (N-acetyl phenyl-l leucinamide) from hydrophobic esters (N-acetyl phenylalanine methyl ester) plus a high excess hydrophilic nucleophile (l-leucinamide) catalyzed by immobilized chymotrypsin (-CT) was studied. By using biphasic systems, the biocatalyst and the necessary high excess of nucleophile remain in the aqueous phase. The hydrophobic acyl donor in the organic phase is partially partitioned into the aqueous phase allowing the synthesis of the peptide. Then the highly hydrophobic reaction product is completely partitioned to the organic phase and it cannot be hydrolyzed by the biocatalyst. Under these conditions, synthetic yields of 95% with respect to the acyl donor were obtained. The excess of nucleophile does not need to be recovered at the end of the synthesis because it remains immobilized in the aqueous phase and it can be re-used as well as the immobilized biocatalyst. In such biphasic systems, in each reaction cycle, the synthesis proceeded according to this interesting mass balance: 50mM N-acetyl phenylalanine methyl ester plus 47.5mM l-leucinamide were converted into 47.5mM peptide derivative, that precipitated in the organic phase as a fluffy solid, and 2.5mM N-acetyl phenyl acid as side product in the aqueous phase. The immobilized biocatalyst (inside a porous structure) is not in contact with the organic phase. Three consecutive reaction cycles were performed, and 95% of peptide was always obtained.