Fig. 2. Chemical synthesis of linker histone H1.2 using the Ru catalyst. (A) Synthetic strategy. / represents the cleavage sites. (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. (B) Reaction tracking of the one-pot ligation involving catalytic amount of Ru-4 (20 mol%) for the alloc deprotection by analytical HPLC (gradient: 10–46% for 30 min) at 220 nm. Compounds 5′, 7′, and 9′ are the alloc-protected peptides 5, 7, and 9, respectively. # = MPAA. * = allyl-transferred MPAA. (a) 1st NCL (t = 2 min). (b) 1st NCL (t = 2 h). (c) 1st deprotection. (d) 2nd NCL (t = 2 h). (e) 2nd deprotection. (f) 3rd NCL (t = 2 h). (g) 3rd deprotection. (h) 4th NCL (t = 2 h). (C) HPLC profile (left, gradient 20–50% for 30 min) and MALDI-TOF mass spectrum (right) of purified peptide 12. Calculated mass of 12 [M + H]⁺: 21 276.6; mass found [M + H]⁺: 21 277.8.