Evaluation of ligation positions on insulin. We first used published procedures to synthesize insulin cores with an ester on the C-terminal region of the B chain described in this section. To determine the location of ligation, the ester linkage was first employed between B22-B23 of insulin core (1a) to test the ligation activity (table 1, entry 1). No ligation was observed after treated with omniligase-1 and the peptide of insulin B23-B30 sequence, GFFYTPKT. The ester of 1a was hydrolyzed under the basic reaction condition to the same degree regardless of the presence of omniligase-1. According to the sequence recognition preference of omniligase-1, the GluB21 at P2 position of 1a is a suboptimal residue for omniligation-1 ligation. We mutated GluB21 to Ala to address this issue (Table , entry 2) but the ligation product was still not observed for 1b. We argued that since omniligase-1 needs to recognize P1-P4, the location of ester between B22-B23 may be too close to the bulky disulfide branch at B19, which prevent the ester from entering the catalytic center of omniligase-1. We then moved the ester linkage between B23-B24 (1c), which brought two Gly in the sequence to sit at P1 and P4. Both cases are unfavorable for omiligase-1 ligation (Table , entry 3). Consequently, the substrate having an ester between B23-B24 (1c) was omitted in this study. We next turned to an ester linkage between B24-B25. The insulin ester with human sequence and an ester between B24-B25 (1d) was predicted no ligation activity because B21Glu at P4 was reported forbidden for omniligase-1 ligation. As predicted, no ligation was observed for 1d (table 1, entry 4). Mutation of GluB21 to Ala of insulin was reported having low influence on the binding affinity to IR and, importantly, B21Ala at P4 is an allowed residue for omniligase-1 ligation. The insulin ester with B21Ala mutation and an ester between B24-B25 (1e) was successfully ligated with the peptide of insulin B25-B30 sequence, FYTPKT, by omniligase-1 to give the B21Ala insulin analog (2) at 37°C at 3 hours with a 70% ligation rate (table 1, entry 5). However, 1e was not completely soluble at 0.5 mM working concentration in the ligation buffer. 1e could be completely dissolved in water and prepared as a stock solution in a high concentration (3-5 mM) but was immediately precipitated after added into the ligation buffer at pH 8.0. The precipitant 1e was later consumed gradually along with the reaction proceeding. We proposed that the Glu to Ala mutation led to reduced hydrophilicity. This mutation also increased the isoelectric point (pI) of insulin to be closer to the buffer pH and therefore made 1e less soluble than 1d. Adding an extra Asp at the C-terminus of 1e (1f) could decrease the pI and compensate the loss of hydrophilicity to rescue the solubility and yield the same ligation product 2. 1f was completely soluble in the ligation buffer as a 0.5 mM solution and could be ligated with the peptide of insulin B25-B30 sequence, FYTPKT, by omniligase-1 to give insulin analog 2 with a mgscale isolation yield of 77% (Table , entry 6, Figure ). Due to the excellent result, insulin core 1f was selected for subsequent studies using omniligase-1. a 0.5 mM. b n.d. = not detected at 24 h. c 1c was not tested because sequence X (GERG) is not compatible with omniligase-1. d Solubility < 0.2 mM. e Isolation yield for mg-scale.