Protease-catalyzed enzymatic synthesis of antifreeze peptide oligomers and their cryopreservation performance

Sub-zero preservation is essential in biomedical fields like tissue engineering, gene therapy, and transplantation. Traditional cryoprotectants like dimethyl sulfoxide are toxic and can alter cell epigenetics, necessitating safer alternatives. Herein, an enzymatic synthesis of antifreeze peptide oligomers (AFPOs) using protease-catalyzed polymerization is introduced. Employing proteases as catalysts, AFPOs were synthesized by condensing <i>L</i>-alanyl-alanyl-threonine (<i>L</i>-AAT) as a model monomer, and this method was extended to produce two additional sequences, poly(<i>L</i>-alanyl-threonine-alanyl) (<i>L</i>-PATA) and poly(<i>L</i>-threonine-alanyl-alanyl) (<i>L</i>-PTAA). Notably, using <i>L-</i>alanyl-threonyl-alanine (<i>L</i>-ATA) as the monomer, a degree of polymerization up to 14 was achieved. Although identical amino acid compositions were shared by all AFPOs, measurable antifreeze activity was exhibited only by <i>L</i>-PTAA, indicating that monomer sequence-and thus detailed backbone architecture-rather than composition alone governs antifreeze performance. Excellent antifreezing properties were demonstrated by <i>L</i>-PTAA, marking it as safe, sustainable alternatives to conventional cryoprotectants. These findings highlight the effectiveness of enzymatic synthesis methods for producing functional AFPOs.