We report a solid‐phase strategy for total synthesis of the peptidic natural product yaku'amide B (1), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four β,β‐dialkylated α,β‐dehydroamino acid residues and is capped with an N‐terminal acyl group (NTA) and a C‐terminal amine (CTA). To realize the Fmoc‐based solid‐phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C‐terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)3 led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin‐cleavage and C‐terminus modification were simultaneously achieved with an ester–amide exchange reaction using CTA and AlMe3 to deliver 1 in 9.1 % overall yield (24 steps from the resin).

Yaku′amide B (1) inhibits cancer cell growth through a unique mechanism of action. Compound 1 binds to mitochondrial FoF1‐ATP synthase, inhibits ATP production, and enhances ATP hydrolysis. The presence of one (E)‐ and two (Z)‐α,β‐dehydroisoleucines (ΔIle) in the linear 13‐mer sequence is the most unusual structural feature of 1. To uncover the biological importance of these residues, we synthesized 1 and its seven E/Z isomers 2–8 by devising a new divergent solid‐phase strategy. Both the (E)‐ and (Z)‐ΔIle residues were stereoselectively constructed by traceless Staudinger ligation on resin to ultimately deliver 1–8. All isomers 2–8 displayed effects on the inhibition of cell growth and ATP production, and enhanced ATP hydrolysis, thus indicating that 2–8 share the same mode of action as 1. The least potent isomer, 8, was isomeric at three ΔIle residues of the most potent 1. These findings together indicate that the E/Z stereochemistry of the three ΔIle residues controls the magnitude of the biological functions of 1.

We report a solid‐phase strategy for total synthesis of the peptidic natural product yaku'amide B (1), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four β,β‐dialkylated α,β‐dehydroamino acid residues and is capped with an N‐terminal acyl group (NTA) and a C‐terminal amine (CTA). To realize the Fmoc‐based solid‐phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C‐terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)3 led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin‐cleavage and C‐terminus modification were simultaneously achieved with an ester–amide exchange reaction using CTA and AlMe3 to deliver 1 in 9.1 % overall yield (24 steps from the resin).

Polytheonamide B (1) is an exceptionally large peptide that forms a transmembrane ion channel. The potent cytotoxicity of 1 against MCF-7 cancer cells originates from its two ion transport functions....