In the pursuit of more complex protein targets, sequential NCL steps will require orthogonal N-terminal cysteine protecting groups for middle segments (Table , Entries 8-12). A common and reliable choice is the protection of cysteine in a thiazolidine ring (Thz), due to the facile conversion into cysteine, compatibility with ligation conditions and its commercial availability in both enantiomeric forms. However, Thz was found to be unstable under the oxidation conditions required for peptide hydrazide oxidation. Numerous efforts have been directed to the development of other orthogonal, N-terminal cysteine protecting groups to facilitate sequential peptide hydrazide ligations (Table , Entries 9-12). Preparation of their enantiomeric counterpart is theoretically simple. Indeed, TFA-Thz, which is stable to hydrazide oxidation, was used in the preparation of enantiomeric polymerase D-Dpo4 and ubiquitin.