Synthesis of insulin 1f. The synthesis of insulin core 1f was further optimized using a modified strategy (Scheme 1). The insulin A chain 3 and B chain 4 were independently synthesized through solid-phase peptide synthesis (SPPS) followed by chain combination. The A chain was processed as previously reported to give the A6-A11 disulfide, A7 Cys(Acm), and A20 free Cys A chain 3. To introduce the ester function group on the B chain 4, glycolyl (Gc) group was incorporated as a pre-made Fmoc-Phe-Gc-OH building block used in the SPPS by following the reported procedure (Scheme S2). In short, Fmoc-Phe-OH was linked with benzyl bromoacetate to give Fmoc-Phe-Gc-OBn followed by hydrogenolysis of the benzyl ether protecting group to give Fmoc-Phe-Gc-OH. However, during the preparation of the B chain 4, a significant amount of GlyB23-PheB24 dipeptide elimination was observed. This deletion has been reported through diketopiperazine (DKP) elimination occurring after the N-terminal Fmoc of GlyB23 was deprotected during the SPPS (Scheme 2A). To prevent the DKP elimination and increase the yield of 4, Fmoc-Arg(Pbf)-Gly-OH dipeptide as a building block was used to avoid creating the N-terminal amine of GlyB23 (Scheme 2B). Fmoc-Arg(Pbf)-Gly-OH was prepared from the coupling of Fmoc-Arg(Pbf)-OH and H-Gly-OBn followed by hydrogenolysis of the benzyl ether protecting group (Scheme S3). Cys(Acm) was used at B7 for the A7-B7 disulfide. Cys(Trt) was used at B19 and activated with 2,2'-dithiodipyridine (DTDP) during peptide cleavage for the A20-B19 disulfide at the A chain and B chain combination step. After the A chain 3 and B chain 4 were prepared, two chains were combined in equal amounts under pH 5.0 at room temperature for 3 hours to form the A20-B19 disulfide bond of 5. The third disulfide between A7 and B7 was formed by the treatment of iodine under an acidic condition. The Thr-Ser isoacyl linkage was finally transformed to an amide bond through an O-to-N acyl shift under pH 8.0 to give insulin ester 1f (Scheme 1).