Highly concentrated water-in-oil emulsions as novel reaction media for protease-catalysed kinetically controlled peptide synthesis

High-internal-phase-ratio-emulsions (HIPREs) or gel emulsions, formulated with a large amount of water (80.0-99.5% w/w), were investigated as reaction media for alpha -chymotrypsin-catalysed peptide synthesis under kinetic control using Ac-L-Phe-OEt and H-L-Leu-NH2 as model substrates. Both the initial reaction rate and dipeptide yield were examined as a function of the structure of the non-ionic polyoxyethylene alkyl ether type surfactant, alkyl chain length of the oil component, temperature and aqueous buffer content. Dipeptide yields of 70% were achieved in gel emulsions formulated with 90% w/w aqueous buffer. In these systems, the reaction performance was found to be independent of the gel emulsion system (i.e. surfactant and oil) and therefore of the water-oil interfacial tension. Interestingly, alpha -chymotrypsin showed superactivity at surfactant concentrations ranging between 0.2 and 0.8% w/w, that is, at 99.5and 98.0% w/w water content, respectively. Furthermore, high dipeptide yields (90-94%) were achieved in the gel emulsions studied at very high substrate concentrations and thus with undissolved reactants. Under these conditions, examples of alpha -chymotrypsin-catalysed dipeptide synthesis on an analytical and preparative scale were conducted.