(Figures and). Treatment of 11 (1 mM) with 12 (1.2 mM) in 0.1% TFA/AcOH in the presence of 30 mM MgCl 2 at 37 °C for 5 h allowed the regioselective intermolecular disulfide formation between the Cys(Acm)(O) and Cys(Acm) residues to give heterodimeric mono disulfide peptide 13 quantitatively. Upon addition of an equal volume of 4 M Gn•HCl-100 mM anisole in TFA to the first reaction mixture, the second disulfide formation occurred between the Cys(MBzl)(O) and Cys(MBzl) residues to afford the bis(disulfide) peptide 14 after 2 h reaction at 37 °C in 2 M Gn•HCl-50 mM anisole in TFA/AcOH (1:1).No detectable disulfide shuffling was observed. Tryptic digestion of 14 followed by peptide mapping indicated that the resulting 14 has the correct disulfide pairings (Figure ). Based on the success of one-pot/stepwise regioselective disulfide formation using the model peptide, we undertook synthesis of human insulin using the stepwise disulfide formation protocol (Figure ). This work began by assembling the protected peptide resin for A and B chains using 9-fluororenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis (Fmoc SPPS). Chain assembly employed S-triphenylmethyl (Trt) group for CysA6 and CysA11, S-Acm for CysA7 and CysB7, and S-MBzl for CysA20 and CysB19. For the preparation of the hydrophobic A chain, an Oacyl isopeptide strategy was employed to address the solubility issue. The resulting oxidized A chain 17 was then subjected to the first disulfide formation step induced by MgCl 2 under weakly acidic conditions, where the less soluble character of the A chain peptide necessitated the addition of an ionic liquid (BMPy•OTf) and LiCl as solubility-enhancing additives. The first disulfide formation between the oxidized A chain 17 (1.0 mM) and B chain 18 (1.2 mM) in 0.1% TFA/AcOH in the presence of 30 mM MgCl 2 , 5% BMPy•OTf and 0.4 M LiCl at 37 °C for 7 h proceeded in a highly Cys(Acm)(O)A7/Cys(Acm)B7 selective manner between the A and B chains to yield heterodimeric mono disulfide peptide 19 (Figure ). Subsequent addition of 4 M Gn•HCl-100 mM anisole in TFA to the first reaction mixture, followed by reaction at 37 °C for 2 h, initiated the second disulfide formation between Cys(MBzl)(O)A19 and Cys(MBzl)B20 to give the desired insulin (Figure ). HPLC analyses of the reaction mixtures indicated that the stepwise disulfide-bonding steps proceeded in a highly regioselective manner to yield insulin efficiently. Finally, HPLC purification of the crude mixture gave homogeneous human insulin with an isolated yield of 21%, calculated based on oxidized A chain 17 (Figures and). This result falls short of the best data (38 or 47%) obtained from a one-pot procedure that utilizes the wellestablished conventional disulfide-forming reactions. Peptide mappings of the digest of synthetic material or an authentic sample by endoproteinase Glu-C accorded well together and the disulfide pairing of fragments was also confirmed by the synthesis of possible three disulfide isomers (Figures S11-15). It is worth noting that the achieved stepwise disulfide formation relies on only a single oxidation step for the conversion of 15 to 16. In contrast, conventional insulin synthesis requires three stepwise oxidative manipulations for disulfide formation. The application of the isopeptide 16 to stepwise disulfide formation could have overcome solubility issues of the oxidized A chain 17 that required the addition of the BMPy•OTf and LiCl, however no drastic improvement was observed. More importantly, the use of the isopeptide encountered a side reaction during the first disulfide-forming step (data not shown).