Figure S1. Analytical HPLC of the synthetic lactam-bridged peptides used in this work (see Table S1 for tR values and gradient).

Figure S2. MALDI-TOF-MS of the synthetic lactam-bridged peptides used in this work (see Table S1 for Mfound).

Figure S3. CD spectra of the lactam-bridged peptides in (a, b) phosphate buffer (50 mM, pH 7.3) and (c, d)

water (pH range 3-4). The dashed boxes show the maximal CD contribution below 200 nm, which is higher than 6000 deg cm2 dmol-1 for 1Y, 2-5, 7 (a, c), and smaller than 4000 deg cm2 dmol-1 for 6, 8-10 (b, d). Difference spectra (peptide – 1Y) in (e) phosphate buffer and (f) water

Figure S4. Comparison of the CD spectra of the lactam-bridged peptides in phosphate buffer (50 mM, pH 7.3) and water (pH range 3-4).

Figure S5. Comparison of the CD spectra of the lactam-bridged peptides 2 and 5 in phosphate buffer (50 mM, pH 7.3), acetate buffer (10 mM, pH 4.5) and water (pH range 3-4).

Figure S6. Chemical shifts differences (ppm) between the measured and random coil1 HN for each residue of the peptides in water (pH range 3-4)

Figure S7. NOE pattern of the lactam-bridged peptides in water (pH range 3-4)

Figure S8. 1D-NMR spectra of the lactam-bridged peptides in water (pH range 3-4)

Figure S9. Effect of the lactam-bridged peptides 1Y and 2-10 (100 μM) on cancer-cell viability. MCF-7: human breast adenocarcinoma; A549, SKLU-1, H1975 und A427: human lung non-small cell adenocarcinoma; HCC827: human lung non-small cell adenocarcinoma with an EGFR mutation; H520: human lung squamous cell carcinoma; H460: human lung large cell adenocarcinoma. Human primary lung fibroblasts were used as control. p-Values (referred to 1Y): ***: <0.001; **: <0.01; *: <0.05. In previous reports we showed that 1Y reduces cancer-cell viability in the low micromolar concentration range2-4. Here, we show that none of the new analogs was more effective than 1Y, displaying similar or reduced efficiency depending on the cell line. About the three analogs with the highest sequence similarity (2-4), the Tyr-4-containing analog 3 was less effective than 1Y in all tested cell lines, whereas the Nle-4-containing analog 2 and the Ile-7-containing analog 3 were comparable to 1Y only in four and three cell lines, respectively.

Table S1. Analytical characterization of the synthetic lactam-bridged peptides used in this work (X = Nle)

Table S2. Statistics of the NMR-derived structures of the lactam-bridged peptides

Table S3. Random coil chemical shifts of norleucine in the reference peptide Ac-GGXGG-NH2 measured in 7 M urea in D2O at pH 2.3 or 7.4 (the peptide was synthesized by using the protocol reported above for the linear precursors of the lactam-bridged peptides 1Y, 2-10. Purity based on analytical HPLC: 95%. MStheor.: 400.44, MSfound for M+Na+: 423.602 Da).

Table S4. Chemical shifts of the backbone amide protons (HN) and 3JHNa coupling constants of the lactambridged peptides in water (pH range 3-4). Values extrapolated from partially overlapped signals are in brackets (n.e.: not extractable. X = norleucine).

Table S4. Chemical shifts of the backbone amide protons (HN) and 3JHNa coupling constants of the lactambridged peptides in water (pH range 3-4). Values extrapolated from partially overlapped signals are in brackets (n.e.: not extractable. X = norleucine).

Table S6. Tilt angles q and residues per turn calculated for the lactam-bridged cyclized motifs from the crystal structures of Ac-(cyclo-2,6)-F(p-NO2)KLLLDF(p-NO2)-NH2 (CCDC deposition number 19410686), Ac-(cyclo- 6,10)-HKILHKLLQDS-NH2 (PDB ID: 5WGD7), and Ac-(bicyclo-3,7+6,10)-HKS5LHKS5LQDS -NH2 with S5 = (S)-2-(4-pentenyl)Ala (PDB ID: 5WGQ7).