Having succeeded in the one-pot/stepwise synthesis of insulin using the acid-activated sulfoxide system, we next undertook a one-pot/stepwise disulfide formation-lipidation sequence for preparing a fatty acid-modified insulin. Lipidated insulins, such as insulin degludec or insulin detemir, have been widely used as long-acting basal insulin. These modified insulins have a fatty acid unit on their LysB29 residue. Therefore, we planned to replace the LysB29 with Trp, on which the acid-activated sulfoxide-mediated sulfenylation of the indole ring would permit incorporation of a fatty acid moiety. Our initial attempt at constructing disulfide bonds of the TrpB29 insulin encountered problems, when during HPLC examination of the second-step reaction we observed co-elution of the desired peptide and anisole. To circumvent this, we performed a proof-of-concept experiment using a C-terminally TrpB31-amide-extended B chain (Figure ). The requisite Trp31 B-chain 20 was prepared by Fmoc SPPS protocols (Figure ). Reaction of 20 with 17 under the conditions identical to those employed for the one-pot synthesis of human insulin unambiguously afforded the desired TrpB31 insulin analogue 22 as a main component on HPLC analysis through the intermediacy 21 (Figures and). At this stage, the insulin analogue was obtained in 16% isolated yield (Figures S17 and 18). Subsequent addition of a TFA solution of the Cys(Acm)(O)-modified fatty acid derivative 23 (Figure ) into the reaction mixture gave the desired TrpB31-lipidated insulin 24 in a one-pot manner in 12% isolated yield (Figures and).