Scheme 1. Synthesis of GccF (1) and [²⁷d-His]-GccF, with the problematic His highlighted in red. Reagents and conditions: (i) 6 M Gn·HCl, 0.2 M Na₂HPO₄, 100 mM MPAA, 20 mM TCEP, pH 6.8, r.t, 2 h; (ii) 6 M Gn·HCl, 0.2 M Na₂HPO₄, methoxylamine·HCl, pH 4, r.t, 16 h; (iii) 6 M Gn·HCl, 0.2 M Na₂HPO₄, 100 mM MPAA, 20 mM TCEP, pH 6.8, r.t, 4 h; (iv) 6 M Gn·HCl, 1 M HEPES, hydrazine, 2-mercaptoethanol, 0 °C, 30 min; (v) 1.5 M Gn·HCl, 50 mM Na₂HPO₄, 2 mM cysteine, 0.25 mM cystine, 0.025 mM EDTA, 0.25 mM peptide, pH 8.2, 4 °C, 16 h.