Kinetic assay of the Michael addition-like thiol-ene reaction and insight into protein bioconjugation

Abstract

The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition-like thiol-ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent-exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl-substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L-cysteine was evaluated by UV absorption and high-resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent-exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.

Keywords: NMR spectroscopy; conjugation; proteins; thiol chemistry; thiol-ene reaction.