Fig. 3: Chemical synthesis of D-monobody.

A Target sequence derived from Mb5−11. BC- and FG-loop and Cys substituted sites (ligation sites) are highlighted in blue, pink, and green, respectively. B Synthetic scheme of D-monobody, 9D via C-to-N 3 segment ligation. Reaction conditions: (a) 100 mM MPAA, 50 mM TCEP, 6 M Gn·HCl, 0.2 M phosphate (pH 6.8), 37 °C; (b) 200 mM methoxyamine (pH 4.0) in addition to the NCL condition. C HPLC monitoring of the one-pot reaction of the 1st NCL and subsequent thiazolidine deprotection. HPLC peaks were monitored at 220 nm in the linear gradient with water/acetonitrile containing 0.1% TFA. The gradient of HPLC: acetonitrile 10–60% for 30 min. D HPLC monitoring of the 2nd NCL. HPLC peaks were monitored at 220 nm in a linear gradient with water/acetonitrile containing 0.1% TFA. The gradient of HPLC: acetonitrile 10–60% for 30 min. E MALDI-TOF/MS spectrum of peptide 9D after purification. MS(MALDI-TOF) m/z: [M + H]⁺ Calcd for C₅₇₆H₈₄₉N₁₆₁O₁₇₅S₃ 12926.1; Found 12927.3. F SDS-PAGE analysis of 9D and 9 L, whose synthetic data are shown in Supplementary Fig. 21 and 22. MK: molecular weight ladder marker. Source data are provided as a Source Data file.