The conceptual guide to the strategy for synthesizing insulin arose from our previous study on peptide heterodimerization of Trp-peptides using a linker tagged with S-acetamidomethyl (Acm) and S-p-methoxybenzyl (MBzl) cysteine sulfoxides (Cys(Acm)(O) and Cys(MBzl)(O)). We showed that Cys(Acm)(O) is converted to S-chlorocysteine under mildly acidic conditions (0.1% TFA either in AcOH or in the ionic liquid 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate (BMPy•OTf)) in the presence of magnesium chloride (MgCl 2 ), with coexisting Cys(MBzl)(O) remaining intact. In contrast, the formation of S-chlorocysteine from the Cys(MBzl)(O) occurs under more acidic conditions in the presence of guanidine hydrochloride (Gn•HCl). Additionally, the Cys(Acm)-derived sulfenylsulfonium cation, resulting from the reaction of Shalocysteine with Cys(Acm), releases the Acm group as a cation under mildly acidic or neutral conditions to lead to disulfide formation, which probably may not be the case for the Cys(MBzl)-derived cation. Such a difference in the chemical behavior between Cys(Acm) and Cys(MBzl) derivatives allowed us to envision the strategy for stepwise interchain disulfide formations as shown in Scheme 1. and and). Each pair of Acm series (1a and 2a) or MBzl series (1b and 2b) was treated under disulfide-forming conditions suitable for the pairs in the presence of Tyr, Trp, or Met-containing peptide (Ac-G-Xaa-GAL-NH 2 : Xaa = Tyr (9a), Trp (9b), Met (9c)), and the results are summarized in Table . Reactions using the Cys(Acm)(O)/Cys(Acm) pair under mild acidic conditions proceeded in almost completely disulfide selectivity (entries 1-3 and Figure ). In contrast, selectivity in reactions of the MBzl pair were slightly decreased, but no significant side products, including peptides 10a and 10c, were observed on HPLC analysis of the reactions in the presence of 9a and 9c. Reaction of the Trp peptide 9b was accompanied a small amount of 10b and MBzl-modified 9b (entries 4-6 and Figure ). Reaction conditions Selectivity (%) or in TFA in the presence of 4 M Gn•HCl and 50 mM anisole at 37 C° for 1 h. The selectivity ratio of the reactions of the sulfoxides with S-protected cysteines vs Tyr, Trp, or Met was estimated by comparing the reduction ratio of the starting sulfide peptides (2a or 2b) (%) between in the presence and absence of peptides 9. Each reduction ratio (%) was calculated using the equation (100-100*((integ. 2a at 7 h (or 2b at 1 h)/integ. IS)/(integ. 2a (or 2b) at 0 h/integ. IS)). The reduction ratio in the absence of peptides 9 was set to 100% and the selectivites were calculated, where integ. = integration of peak area of the UV absorption and IS = internal standard (Phe). Negligible amount of 10b was detected on HPLC analysis.