Multiple linkages via sortase-mediated ligation with the example of protein-polymer conjugates

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Sortases are enzymes occurring in the cell wall of gram-positive bacteria that covalently link surface proteins to the cell wall. Sortase A forms a peptide bond between the peptide motif LPXTG and an oligoglycine. This ligation technique is known as sortase-mediated ligation (SML) or sortagging and developed to a frequently used tool in basic research. Using sortase A, a protein can be ligated to another biomolecule, a small synthetic molecule, a polymer or a surface. Therefore, the substrates only need to be equipped with a C-terminal LPXTG and an N-terminal Gx sequence. A weakness of SML is that a formed bond (LPXTGGG) represents again a substrate for the enzyme. The reaction is reversible and, consequently, a formed bond can be cleaved again by the enzyme. Nowadays, several possibilities allow to shift the equilibrium of the reaction enabling ligations in nearly quantitative yield. However, the formation of consecutive bonds by one sortase is still impossible or can be only reached via demanding strategies. Therefore, chains of proteins, multiple layers of proteins on surfaces, and proteins modified at both C- and N-termini are difficult to access. In the course of this project, an efficient approach should be developed to link several building blocks by means of sortase A. This should be shown with the example of protein-polymer conjugates and give access to a type of conjugates with two different polymer chains linked to a protein. Furthermore, protein oligomers should be generated in a procedure similar to the Merrifield peptide synthesis.The project consists of the synthesis of polymer blocks for the ligation to both C- and N-terminus of a protein. On this basis, protein-polymer conjugates will be synthesized by SML. Consecutive ligations using sortase A, where the formed bonds could not be cleaved by the enzyme, have not been shown yet. This should be reached by utilizing amino acid sequences that form β-hairpin structures which are not a substrate for sortase A anymore. The approach will be demonstrated first with the multiple (uncontrolled) ligation of a protein. To ensure a controlled ligation of three or more building blocks, one protein terminus has to be blocked at first. This procedure will be developed using masked oligoglycines and shown with the ligation of several protein blocks. Subsequently, the strategy should be transferred to the linkage of two polymer chains to one protein.