A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability

Linkers that enable the site-selective synthesis of chemically modified proteins are great interest to field chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route site-selectively modify proteins, many successfully approved therapeutics make use cysteine directed conjugation reagents. However, commonly used linkers, including maleimide–thiol conjugates, not stable low concentrations thiol present blood. Furthermore, only few cysteine-targeting reagents attachment multiple functionalities: useful tool fields theranostics therapeutic blood half-life extension. Herein, we demonstrate application pyridazinedione motif three functionalities protein bearing single residue. Extending upon previously documented dual modification work, here by exploiting bromide leaving group an additional reactive point on scaffold, or aniline derivative can be added protein, post-conjugation. Thiol cleavability appraisal resultant C–S C–N linked thio-bioconjugates demonstrated functionalized linkers cleavable noncleavable when incubated excess glutathione. The plug-and-play trifunctional platform was exemplified attaching clinically relevant motifs: biotin, fluorescein, polyethylene glycol chain, model peptide. This provides rare opportunity combine up fashion. selecting amine for functionalization, provide unique control over linker toward thiols, allowing this novel applied range physiological environments.