Methyl Scanning Approach for Enhancing the Biological Activity of the Linear Peptidic Natural Product, Efrapeptin C

Efrapeptin C (1a) is a large peptidic natural product comprising a 15-mer linear sequence and exerts potent anticancer activity by inhibiting mitochondrial F_oF₁-ATP synthase. Residues 1–6 and 9–15 of 1a fold into two 3₁₀-helical domains and interact with ATP synthase, while the central β-Ala-7–Gly-8 region functions as a flexible linker of the two domains. To enhance the function of 1a by minimally modifying its structure, we envisioned attaching one small methyl group to the β-Ala-7–Gly-8 and designed six methylated analogues 1b–1g, differing only in the position and configuration of the methyl group. We enabled the first solid-phase total synthesis of 1a and unified syntheses of 1b–1g. The growth inhibitory activities of 1a–1g against MCF-7 cells varied significantly: 1f with (S)-β³-hAla-7 and its epimer 1g with (R)-β³-hAla-7 were 4-fold more and 5-fold less potent, respectively, than 1a. Remarkably, the most potent 1f had the most stabilized 3₁₀-helical conformation and the highest hydrophobicity, which likely contributed to its effective transfer to the target protein within mitochondria. Moreover, 1f exhibited higher proteolytic stability than 1a. Therefore, the present methyl scanning approach provides a new strategy for changing the original properties of linear peptidic natural products to develop new pharmaceuticals.