Fig. 4. Chemical synthesis of HP1α using the Ru catalyst. (A) Synthetic strategy. / represents the cleavage cites. Red region represents chromodomain, underlined region represents hinge region, and blue region represents chromo shadow domain of HP1α. (1) NCL condition: peptides (2 mM), MPAA (100 mM) in denaturing buffer at pH 7.0, 37 °C. Removal condition: Ru-4 (20 mol%), 37 °C, 30 min. (2) Peptide (0.8 mM), TCEP (300 mM), GSH (150 mM), VA-044 (20 mM) in denaturing buffer at pH 7.0, 37 °C, overnight. (3) Peptide (0.5 mM), AgOAc (30 mM), H₂O/AcOH (1 : 1), 37 °C, overnight. (4) NCL condition: peptides (1.5 mM), MPAA (100 mM) in denaturing buffer at pH 7.0, 37 °C. Removal condition: Ru-4 (20 mol%), 37 °C, 30 min. (B) Reaction tracking of the one-pot ligation with a catalytic amount of Ru-4 (20 mol%) for the alloc deprotection by analytical HPLC (gradient: 15–51% for 30 min) at 220 nm. (a) 1st NCL (t = 4 h). (b) 3rd NCL after overnight reaction. (C) Reaction tracking of NCL between peptides 19 and 20, followed by the allyl removal with Ru-4 (20 mol%) by analytical HPLC (gradient: 15–51% for 30 min) at 220 nm. (a) NCL (t = 3 min). (b) NCL (t = 1.5 h). (c) Allyl deprotection with Ru-4. 21′′ = 21 bearing allyl-protected Asp 58. (D) HPLC profile (gradient 15–51% for 30 min) and MALDI-TOF mass spectrum of purified peptide 21. Calculated mass of 21 [M + H]⁺: 22 225.7; mass found [M + H]⁺: 22 226.0. (E) CD spectrum of 21. (F) Chemically synthesized HP1α with PTMs in this study. 21: HP1α without PTMs. 21a and 21b: HP1α with PTMs at N-terminus. 21c, 21d, and 21e: HP1α with PTMs at HR. 21f: HP1α with Ub at K154 at CSD.