Investigating Multicomponent Approaches for the Site-Selective Conjugation of Native Proteins

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. While mutant proteins take most of the spotlight, native and natural proteins have been left in the shadow and site-selective methods to conjugate these are underexplored. In addition, while these few methods give good results on small to medium-sized proteins, most of them tend to fall short whenever applied to bigger constructs such as antibodies. To address this limitation, we reasoned that aiming at the simultaneous conjugation of two amino acid residues should give higher chances of developing a site-selective strategy compared to the large majority of existing methods that solely target a single residue. We opted for the Ugi four-center three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable conjugates bearing several different payloads, and limits the potential conjugation sites to only six on the model antibody trastuzumab.