N, S-Double Labeling of N-Terminal Cysteines via an Alternative Conjugation Pathway with 2-Cyanobenzothiazole

Conjugation of 2-cyanobenzothiazole (CBT) with N-terminal cysteines (NCys) typically gives a luciferin product. We herein report an alternative reaction pathway leading to an N-terminal amidine rendering the side chain thiol available for further modification. Examination of peptide sequence dependence of this amidine conjugation reveals a tripeptide tag CIS that allows facile N, S-double labeling of a protein of interest with >90% yield. This alternative reaction pathway of CBT-NCys condensation presents a significant addition to the toolbox for site-specific protein modifications.