Selective N-terminal Protein Bioconjugation Enabled by Conjugate Addition/ Ring Expansion (CARE) Cascades

Protein bioconjugation has revolutionised modern day biomedical and biotechnological research by enabling proteins to be engineered with properties that greatly surpass the limitations of the 20 canonical amino acids. Site-selective approaches are vital for preserving protein function and producing homogenous bioconjugates, both of which facilitate regulatory approval and improve reproducibility. In this context, protein N-termini have emerged as attractive sites for chemical modification owing to their distinct structure and accessibility. Despite recent advances, N-terminal selective bioconjugation remains limited by the need to balance chemo- and regioselectivity with conjugate stability and reagent accessibility. Even the most advanced reagents are not universally compatible with all proteins, highlighting the need for further innovation in this field to unlock its full potential.
Herein, this thesis describes the development of a novel, site-selective N-terminal protein bioconjugation strategy using conjugate addition/ ring expansion (CARE) cascade reactions. Building on the small molecule ring expansion strategy reported by the Unsworth group, we conceptualised, modified, and optimised an approach for N-terminal bioconjugation via ring expansion with cyclic acryloyl imides. The advantages of N-terminal CARE bioconjugation include high selectivity for N-termini over lysine ε-amino groups, the formation of stable macrocyclic bioconjugates via an irreversible ring expansion, and the potential for further derivatisation through pendant bioorthogonal tags on CARE-assembled bioconjugates. We prove the utility of this approach by modifying a series of medicinally relevant proteins, including nanobodies used for prostate cancer cell and tissue imaging, and human chemokines implicated in the human immune response.