Papain-Catalyzed Synthesis of Oligolysine in Low-Water Organic Reaction Media

Oligopeptides of l-lysine have the potential for applications in various scientific and technical areas. The number of residues in polycationic compounds such as oligolysine is also reported to have an effect on its biological properties. Hence, there is a necessity for developing efficient oligolysine synthesis methods where the oligopeptide dispersity can be tailored, along with optimum yield values. The ability of proteases to reverse their proteolytic activity to synthesize peptides has been reported in the literature. However protease-catalyzed synthesis of oligopeptides of basic amino acids such as lysine in aqueous buffers is hindered by unfavorable thermodynamics. In this work, a low-water organic system comprising an aqueous phase in contact with a bulk immiscible toluene solvent system has been demonstrated for efficient papain-catalyzed synthesis of oligolysine. The oligolysine mixture was separated with ion-pair LC. The LC peaks were identified with semipreparatory LC separation followed by solid-phase extraction purification and LC-MS analysis. The Plackett-Burman design method used to screen for significant variables showed that the oligolysine yield is strongly dependent on five variables, namely, the substrate concentration, the aqueous phase composition, the compositions of the additives 2-mercaptoethanol and N,N-diisopropylethylamine, and the duration of incubation. Using the one-variable-at-a-time approach, the effect of system variables on oligolysine dispersity was investigated. The results show that the dispersity profile can be tailored by modifying the magnitudes of these variables. The effect of these variables against oligolysine yield was investigated with response surface methodology. The results show that these variables interact with each other to produce a maximum yield of similar to 92% with dispersity ranging from 2-10 lysine residues.