a) saponification of ester insulin 2 to give KP-insulin 3. Conditions were 2: 0.12 mg mL−1, NaOH: 25 mM, acetonitrile-H2O (2.5:7.5), 4 °C. Reaction mixture at T = 2 h (upper panel) and T = 24 h (lower panel), #unrelated column contaminant, *derived from A-chain, **B-chain; b) purified KP-insulin 3. (Inset) On-line ESI-MS spectra taken at the top of the main peak. Chromatographic separations were performed as described in Figure 2 legend; c) binding affinities of synthetic KP-insulin 3 and authentic KP-insulin (purchased from Eli Lilly and Co) to the insulin receptor.