Fig. 2. (A) Scheme for the synthesis of the peptide 6. Reaction conditions: 1^st NCL, 6.0 M Gdn·HCl, 0.2 M Na₂HPO₄, 10 mM TCEP, 20 mM MPAA, pH 6.5, RT, 2 h; desulfurization, 6.0 M Gdn·HCl, 0.2 M Na₂HPO₄, 0.2 M TCEP, 5% t-BuSH, 2.5 eq. VA-044, pH 6.5, 37 °C, 30 min; Acm/Mob deprotection, 6.0 M Gdn·HCl, 0.2 M Na₂HPO₄, 150 eq. PdCl₂, pH 6.9, 37 °C, 2 h, and followed by quenching with DTT and further reducing by TCEP and sodium ascorbate for 30 min. (B) (a and b) Analytical HPLC traces of NCL at 1 min and 2 h, respectively. Inset a: SDS-PAGE analysis of the ligation reaction. Inset b: ESI-MS of peptide 4. (c) Analytical HPLC trace of desulfurization at 30 min and ESI-MS of the by-product 5′. (d) Analytical HPLC trace and ESI-MS of purified 5. (e) HPLC trace of PdCl₂-mediated Acm/Mob removal in 2 h and ESI-MS of the product 6.