Assembling branched and macrocyclic peptides on proteins
Abstract:
Developing a chemical toolbox for precise protein engineering has been challenging for a long time, due to the complexity arising from the vast array of functional groups. We demonstrate a two-step, biocompatible and site-selective approach for creating branched and macrocyclic protein-peptide conjugates.
This strategy employs chemoselective modification of surface-exposed cysteines on proteins using the small molecule 4-fluoro-2,6-dicyanopyridine (4-F-DCP) (<150 Da) by nucleophilic aromatic substitution. Further attachment of peptides bearing N-terminal cysteine by the aminothiol click reaction produced branched protein-peptide conjugates. These peptides contained an additional cysteine residue in their sequence which was further crosslinked by the addition of 1,3 bisbromomethylbenzene (BBMB), a common stapling agent. Moreover, a peptide containing two 1,2-aminothiol functionalities one being an N-terminal cysteine and the other in the form of a non-canonical pseudo-N-terminal cysteine amino acid was added to the DCP tagged protein to produce a macrocyclic architecture. All these reactions were carried under near-physiological pH conditions.1
Integrating this approach with bioconjugate chemistry enabled the creation of custom peptide-protein conjugates with potential therapeutic and diagnostic applications, establishing a strong foundation for next-generation protein therapeutics featuring advanced branched and macrocyclic architectures.