Identification of Styryl Sulfonyl Fluoride (SSF) as An Efficient, Robust and Irreversible Cysteine-specific Protein Bioconjugation Reagent

Cysteine (Cys)-specific bioconjugation has found wide application in the synthesis of protein conjugates, particularly for the functionalization of antibody. Here, through direct assessment on protein substrate, we report the discovery of trans-styryl sulfonyl fluoride (SSF) as a near perfect Michael acceptor (MA) for cysteine-specific protein bioconjugation. Com-pared to predominantly used maleimides, SSF exhibited better chemoselectivity, self-stability and conjugate-stability while kept comparable reactivity. Using SSF-derived probes, proteins can be readily modified on the Cys residue(s) to install functionalities, e.g., fluorescent dyes, toxins and oligonucleotides (oligos), without the influence of activity. Fur-ther applications of SSF derived serum stable antibody-drug conjugates and PD-L1 nanobody-oligo conjugates demon-strate the great translational value of SSF-based bioconjugation in the drug development and single-cell sequencing.