A century has passed since the discovery of insulin in the 1920s, which clarified the physiological roles of insulin and resulted in a tremendous impact on the treatment of diabetes (Figure ). Following determination of insulin's sequence, a myriad of attempts to synthesize insulin resulted in the continuous advance of peptide chemistry. The chemical synthesis of insulin has remained a landmark that continues to define the state-of-the-art in peptide/protein chemistry. Insulin has two interchain disulfide bonds between the A and B chains and one intrachain linkage in the A chain. Furthermore, the A chain shows lower solubility in aqueous buffer, which leads to difficulty in manipulating the peptide. In this context, an efficient regioselective disulfide bond-forming reaction and solubilizing technology are still required to elevate the chemical synthesis of insulin. Our group has recently accomplished tryptophan (Trp)-selective C-H sulfenylation by utilizing an acid-activated S-protected cysteine sulfoxide in the presence of chloride anion (Figure ). This discovery provides insight into revising the mechanism involved in the sulfoxide-mediated disulfide bonding reaction previously reported by our group. The Trp sulfenylation requires the formation of S-chlorocysteine from S-protected cysteine sulfoxides aided by acid and chloride anion with the resulting S-chlorocysteine participating in aromatic electrophilic substitution reaction (S E Ar) with the indole. The presence of Trp and methionine (Met) in peptides allows the Schlorocysteine to react not only with Trp but also with Met in a Met-preferential manner. The reaction with Met reversibly gives S-sulfenylsulfonium Met cation due to the difficulty of releasing the methyl cation from the resulting sulfonium cation. Otherwise, the sulfonium cation could participate in the S E Ar with Trp as an electrophile (Figure ). In 1988, we reported a disulfide bond-forming reaction using acid-activated S-protected cysteine sulfoxides and proposed that nucleophilic attack of the free thiol group regenerated from a protected cysteine to an acid-activated sulfoxide gives the disulfide with the release of protection as a cation. (Figure ). Consequently, we and DiMarch's group have successfully applied this to discontinuous disulfide formation by synthesizing several disulfide-containing peptides. However, stepwise disulfide bond formation using different sequential pairings of an S-protected cysteine sulfoxide and the corresponding Sprotected cysteine has yet to be reported. Attempts based on the initially proposed mechanism failed to achieve the sequential disulfide formation. The involvement of the Met sulfide in the S-chlorocysteinemediated Trp-sulfenylation (Figure ) suggests that the reaction of S-chlorocysteine with an S-protected cysteine could also yield the corresponding S-sulfenylsulfonium cation. The resulting cation could then either be converted to the disulfide or revert back to starting materials depending on the ease of releasing an S-protecting group as a cation (Figure , steps ii vs iii). This rationale encouraged us to synthesize insulin by utilizing S-protected cysteine sulfoxide-mediated stepwise disulfide bond formation. We anticipated that incorporating the sulfoxides into the A chain would also improve its solubility. We report herein an advanced insulin synthesis that uses acidactivated S-protected cysteine sulfoxides for one-pot/stepwise disulfide bond formation as well as a subsequent lipidation sequence.