Protease-catalyzed synthesis of α-poly-L-Lysine and amphiphilic poly(L-lysine-co-L-phenylalanine) in a neat non-toxic organic solvent.

Subtilisin Carlsberg (alkaline protease from Bacillus licheniformis) catalyzes the syntheses of high molecular weights (ca. 20 KDa) cationic α-poly-L-lysine and amphiphilic poly(α-L-lysine-co-L-phenylalanine) in neat organic solvent. The synthesis is conducted in liquid 1,1,1,2-tetrafluoroethane solvent, which is a hydrophobic non-toxic gas that does not deplete the ozone layer and approved for pharmaceutical applications. Solubility of substrates and adequate protease activity in this system with low water environment limits the reaction of hydrolysis of the growing peptide chains. The pressurization of this organic compressed fluid to liquid has low-pressure requirements (25 bar, 40 ºC), and its complete evaporation at atmospheric pressure after completing the reaction ensures solvent-free residues in products. The resulting polypeptides present null cytotoxicity according to MTT and NR analyses, as well as Calcein/EthD-1 assay in human cells.