Fig. 2: Electrostatic assistance of peptide thioester aminolysis.

a The reaction of peptide thioester 1a (5 mM) with glycyl peptide 2a (5 mM) shows the formation of target peptide 3a,a as the major product. The reaction was run in sodium hydrogen carbonate/carbon dioxide pH 7 buffer at 37 °C, which was obtained by equilibrating sodium hydrogen carbonate (50 mM) in a cell incubator (5% partial CO₂ pressure). b The reaction of peptide thioester 4 (1.6 mM) with Gly-(Arg)₆-I₂₇ titin protein 5 (0.8 mM) yielded target protein 6 as the major product. Conditions: Sodium bicarbonate/CO₂ buffer (20 mM), 37 °C, TCEP ∙ HCl 1 mM, n-octylglucoside 10 mM, 20 h. c Kinetic monitoring of the formation of target protein 6. d LC-MS of the crude mixture corresponding to the reaction shown in b. *peptide thioester hydrolysis byproduct, **peptide thioester cyclization byproduct (see Supplementary Methods for details). e SDS-PAGE analysis of purified target conjugate 6 (Coomassie blue staining).