Protease-Catalyzed Oligomerization of l -Lysine Ethyl Ester in Aqueous Solution

This paper describes the protease-catalyzed synthesis of oligo( l -Lys) from l -Lys ethyl ester ( l -Lys-Et) in an aqueous reaction medium at controlled pH using a pH-stat. Four proteases (papain, bromelain, α-chymotrypsin, and trypsin) were studied to determine their activity for l -Lys ethyl ester oligomerization at pH values ranging from 6 to 11. Bromelain was found to be preferred relative to the other protease catalysts because it gave the highest values of oligo( l -Lys) yield and optimal average chain length (DP avg ). To evaluate reaction progress and product structure, 1 H NMR and HPLC–UV-MS methods were developed. A series of model oligo( l -Lys) compounds with chain lengths 2, 3, and 4 was obtained and analyzed to assign 1 H NMR signals. All components of the mixture were successfully resolved and analyzed by HPLC–UV-MS by ion-pairing reversed-phase chromatography using heptafluorobutyric acid as the ion-pairing agent. The effects of medium pH, l -Lys-Et concentration, bromelain concentration, reaction time, and reaction temperature on oligo( l -Lys) synthesis was evaluated. Oligomers with chain lengths more than 10 are formed within 5 min. By 30 min, the DP avg and longest oligomer chain lengths reached maximum values of ∼3.6 and 12.0, respectively. Residual l -Lys-Et was only 2% by 40 min. Products formed by 30 min remained relatively unchanged as the reaction was continued for 2.5 h. The competitive reaction pathways during oligomer initiation, propagation and transamidation/hydrolysis pathways are presented and discussed relative to the results obtained herein.