Pilot-scale enzymatic synthesis of bioactive oligopeptides in eutectic-based media

The bioactive peptide precursors N-alpha-Cbz-L-Lys(N-zeta-Cbz)-Gly-OEt (1), N-alpha-Cbz-L-Lys(N-zeta-Cbz)-Gly-L-Asp(OAII)-OAII (2), and N-alpha-Cbz-L-Lys(N-zeta-Cbz)-Gly-L-Asp(OAII)-L-Glu(OAII)OEt (3) were synthesized via a sequential N-to-C strategy in heterogeneous solid-liquid mixtures of substrates using Celite-deposited chymopapain and subtilisin as catalysts. The substrate pairs displayed eutectic formation, and in the presence of 16-20% w/w of water and ethanol furnished low-melting point systems that provided substrate concentrations of 0.81-1.01 M, and sustained protease catalysis. The di-, tri-, and tetra-peptide were obtained in yields of 73, 74, and 67% (yield per step) respectively, and product concentrations of 0.34, 0.48, and 0.45 g per gram of reaction mixture were achieved with these systems. The syntheses were scaled up using wiped-blade and rotating-drum reactors, and 1, 2, and 3 were produced in yields of 71, 73, and 71 %, at product concentrations of 0.36, 0.49, and 0.48 kg kg(-1), on a reaction scale of 11.34, 6.30, and 5.83 kg (11.54, 6.17, and 4.96 mol), respectively. The corresponding space-time yields were 0.30, 0.64, and 0.30 kg kg(-1) d(-1), and biocatalyst reuse provided productivities of 166, 235, and 312 kg-product kg-enzyme(-1). Furthermore, a sequential one-pot, three-step protocol was readily applied to the synthesis of the tetrapeptide 3 which was obtained with an overall yield of 34%, product concentration of 0.25 kg per kg, and space-time yield of 0.07 kg kg(-1) d(-1).