Hmb^off/on as a switchable thiol protecting group for native chemical ligation

A new thiol protecting group Hmb^off/on is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb^off) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb^off) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb^on), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb^off/on) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys⁹⁸–Arg¹⁶⁶]-OH through native chemical ligation.