Fig. 2. (A) RP-HPLC traces for the acyl transfer reaction of N36M(SBn)EK2 at t = 0 and 12 h. The observed MALDI-TOF mass for the product is 12 629.00 Da (calcd: 12 630.18 Da). Lys–Glu ligation of (B) N36M(SBn)EK1 and (C) N36M(SBn) at t = 0 and 12 h. The observed MALDI-TOF mass for the product is 12 658.68 Da (calcd: 12 657.02 Da) and 12 442.30 Da (calcd: 12 440.76 Da), respectively. Reactions contained 500 μM thioester-modified peptides in PBS/H₂O/CH₃CN (3 : 5 : 2 v/v) at room temperature. (D) SDS–PAGE analysis of the isopeptide bond formation at 0 h (lane 1), 4 h (lane 2), 8 h (lane 3), 12 h (lane 4), 16 h (lane 5), 20 h (lane 6), and 24 h (lane 7).