Proteins with functionalized C-termini are powerful and versatile tools that provide scientists shortcuts to discover and understand biological questions. Although many powerful methods have been reported to obtain functionalized proteins, they all suffer from many drawbacks like protein length limitation or premature hydrolysis issue. To totally bypass the involvement of an enzyme which leads to some significant limitations for expressed protein ligation, we designed an activated cysteine-based protein ligation (ACPL) strategy that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its N-side amide for undergoing nucleophilic acyl substitution with small amines molecules in order to form a newly obtained amide bond. In this talk, I will introduce how a commercially available chemical called 2-nitro-5-thiocyanatobenzoic acid (NTCB) which was originally used as a universal cysteine-based cleavage reagent was extended to perform amino-containing compounds substitution reaction. The feasibility of this newly invented approach was proved by both dipeptide-based and protein-based reactions. Furthermore, by applying different compounds such as hydrazine, unnatural amino acids or Gly-AMC, any desired functional group can be added at protein C-termini to obtain activity-based probes, fluorogenic probes, ncAA incorporated proteins, protein hydrazide ligation precursors and commercial peptide drugs with post-translational modified amino acids. The technique, which is exceedingly simple and controllable but highly versatile, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.

Activated-cysteine directed protein ligation: Expressed protein ligation without intein

ACS Spring Meeting 2021

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