Rational design of transglutaminase substrate peptides for rapid enzymatic formation of hydrogels

Short peptide substrates with high specificity toward transglutaminase (TGase) enzyme were designed, characterized, and coupled to a biocompatible polymer, allowing for rapid enzymatic cross-linking of peptide-polymer conjugates into hydrogels. Eight acyl acceptor Lys-peptide substrates and three acyl donor Gln-peptide substrates were rationally designed and synthesized. The kinetic constants of these peptides toward tissue transglutaminase were measured by enzyme assay using RP-HPLC analysis with the aid of LC-ESI/MS. Several acyl donor and acyl acceptor peptides with high specificities toward TGase were identified, including a few containing the unusual amino acid l-3,4-dihydroxylphenylalanine (DOPA), which is found in the adhesive proteins secreted by marine and freshwater mussels. Acyl donor and acyl acceptor peptides with high substrate specificities were separately coupled to branched poly(ethylene glycol) (PEG) polymer molecules. Equimolar solutions of these polymer-peptide conjugates rapidly formed hydrogels in less than 2 min in the presence of transglutaminase under physiological conditions. The use of biocompatible building blocks, their rapid solidification from a liquid precursor under physiologic conditions, and the ability to incorporate adhesive amino acid residues using biologically benign enzymatic cross-linking are advantageous properties for the use of such materials for tissue repair, drug delivery, and tissue engineering applications.