Monomer Choice Influences N-Acryloyl Amino Acid Grafter Conversion via Protease Catalysis

End-capped peptides modified with reactive functional groups on the N-terminus provide a route to prepare peptide-polymer conjugates for a broad range of applications. Unfortunately, current chemical methods to construct modified peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks green preparative characteristics and is costly, thus limiting its applicability to specialty applications such as regenerative medicine. This work evaluates N-terminally modified N-acryloyl-glutamic acid diethyl ester, N-acryloyl-leucine ethyl ester, and N-acryloyl-alanine ethyl ester as grafters and papain as the protease for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease-catalyzed peptide synthesis (PCPS) and the corresponding formation of N-acryloyl-functionalized oligopeptides in a one-pot aqueous reaction. It was hypothesized that by building N-acryloyl grafters from AA-OEt monomers that are known to be good substrates for papain in PCPS, the corresponding grafters would yield high grafter conversions, high ratio of grafter-oligopeptide to free NH₂-oligopeptide, and high overall yield. However, this work demonstrates based on the grafter/monomers studied herein that the dominant factor in N-acryloyl-AA-OEt grafter conversion is the co-monomer used in co-oligomerizations. Computational modeling using Rosetta qualitatively recapitulates the results and provides insight into the structural and energetic bases underlying substrate selectivity. The findings herein expand our knowledge of factors that determine the efficiency of preparing N-acryloyl-terminated oligopeptides by PCPS that could provide practical routes to peptide macromers for conjugation to polymers and surfaces for a broad range of applications.