Site-Specific Fluorogenic Protein Labelling Agent for Bioconjugation.

Many clinically relevant therapeutic agents are formed from the conjugation of small molecules to biomolecules through conjugating linkers. In this study, two novel conjugating linkers were prepared, comprising a central coumarin core, functionalized with a dimaleimide moiety at one end and a terminal alkyne at the other. In our first design, we developed a protein labelling method that site-specifically introduces an alkyne functional group to a dicysteine target peptide tag that was genetically fused to a protein of interest.

Mechanisms of Alkyl and Aryl Thiol Addition to N-Methylmaleimide.

A mechanistic study was undertaken to elucidate the reaction pathways for thiol addition to N-methylmaleimide in water. We used linear free energy relationships, solvent kinetic isotope effects (SKIEs), activation parameters, and ionic strength effects to probe the nature of the rate-limiting transition states. Calculations were also employed and assisted in illuminating three possible mechanistic pathways: (1) stepwise addition with rate-limiting nucleophilic attack, (2) stepwise addition with rate-limiting proton transfer, and (3) concerted addition with nucleophilic attack and proton transfer occurring concurrently.