Bioorthogonal, chemoselective ligation methods are highly valuable to investigate biochemical pathways and to modify proteins and peptides for biomedical applications. The Staphylococcus aureus transpeptidase sortase A (SaSrtA) has been widely established for bioconjugation due to the mild conditions and high selectivity of the enzymatic reaction. Published applications include protein labelling, anti-body modification, cyclization of proteins and peptides and cell surface modification ^[1]. SaSrtA recognises the canonical LPXTG pentapeptide motif of its first proteinogenic substrate and conjugates it to an N-terminal polyglycine containing second substrate (acyl-acceptor substrate, AAS). Firstly, sortase A binds to the acetyl group of threonine, cleaving off the terminal glycine by forming a thioester intermediate. Secondly the N-terminal amine of the acyl-acceptor substrate releases the sortase by forming a new amide bond with the acyl group of threonine ^[2]. While the structure-function relation of the first substrate and the enzyme is well reported and crystal structures with bound intermediate are available, recognition and binding of the acyl-acceptor substrate is hitherto not described ^[3].

Here we report the successful design and employment of a variety of SaSrtA AAS variants to determine the minimal necessary properties of the N-terminal functional group of the second sortase substrate for optimization of the SaSrtA bioconjugation method.