Structure Elucidation of Verucopeptin, a HIF-1 Inhibitory Polyketide-Hexapeptide Hybrid Metabolite from an Actinomycete.

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1 Structure Elucidation of Verucopeptin, a HIF-1 Inhibitory Polyketide-Hexapeptide Hybrid Metabolite from an Actinomycete. Aya Yoshimura, Shinichi Nishimura, Saori Otsuka, Akira Hattori & Hideaki Kakeya* Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto , Japan. scseigyo-hisyo@pharm.kyoto-u.ac.jp Contents Experimental Section (page S2-S7) Supporting Figures (page S8-S17) Figure S1. Effect of verucopeptins in the HIF-1 reporter assay. Figure S2. Advanced Marfey s analysis of verucopeptin (1). Figure S3. Chemical structures of verucopeptin (1) and related natural products. Figure S4. NOE analyses of the isopropylidene derivatives 3a and 3b. Figure S5. Modified Mosher s analysis of the side chain of verucopeptin (1). Figure S6. Effect of verucopeptin (1) on the mrna levels of vegf, glut1 and bnip3. Figure S7. Effect of verucopeptin (1) on the mrna level of hif-1α. Figure S8. Comparison of the effect of verucopeptin (1) and 17-AAG. Figure S9. Effects of verucopeptin (1) on the phosphorylation status of p70s6k and S6 proteins. Figure S10. Effect of SLF on the activity of verucopeptin (1) and rapamycin. References for Supporting Information (page S18) Spectral Data (page S19-S46) Figure S11-S18. 1D and 2D NMR spectra of 1 in CDCl 3. Figure S19-S25. 1D and 2D NMR spectra of 3a in acetone-d 6. Figure S26-S32. 1D and 2D NMR spectra of 3b in acetone-d 6. Figure S33-S37. 1D and 2D NMR spectra of 4a in CDCl 3. Figure S38-S42. 1D and 2D NMR spectra of 4b in CDCl 3. Figure S43. 1 H NMR spectra of S3a in CDCl 3. Figure S44. 1 H NMR spectra of S3b in CDCl 3. S1

2 Experimental Section General. All reagents and solvents were used as received from commercial suppliers. IR spectra were measured using an FTIR spectrometer equipped with ZnSe ATR plate. Optical rotations were determined using the sodium D line (589 nm). NMR spectra were measured on a 500 MHz instrument. 1 H and 13 C chemical shift are relative to the solvent: δ H 7.26 and δ C 77.0 for CDCl 3; δ H 2.05 and δ C 29.8 for acetone-d 6. Mass spectral data were collected using FAB-MS or ESI-MS. Chemicals. Rapamycin and SLF were purchased from Cosmo Bio and Cayman Chemical, respectively. Verucopeptin was isolated as described previously. 1 Reagents were dissolved in DMF or DMSO and stored at -20 ºC. Advanced Marfey s analysis of verucopeptin (1). To a solution of 1 (1.05 mg, mmol) in AcOH (1 ml) was added PtO 2 (12.6 mg). After being stirred for 14 days in a hydrogen atmosphere, the reaction mixture was filtered through Celite to remove the catalyst. The filtrate was concentrated in vacuo and hydrolyzed in 6 N HCl (0.7 ml) for 17 h at 110 C. The reaction mixture was dried in vacuo. The obtained hydrolysate was dissolved in H 2O (50 μl), to which 1 M NaHCO 3 (20 μl) and L-FDLA (1% w/v in acetone, 100 μl) were added, and the mixture was stirred for 1 h at 37 C. The solution was neutralized with 1 N HCl (20 μl), evaporated, and then dissolved in MeCN (500 μl). The derivatives were analyzed by LC-ESI-MS or HPLC. HPLC separation was performed on a reversed-phase column (Cosmosil 5C18-AR-II, ϕ mm) with a gradient elution system of H 2O/MeCN containing 0.1% TFA (70:30 to 20:80 for 50 min). ESI-MS was performed in a positive ionization mode. Synthesis of isopropylidene derivatives 3a and 3b (3a: 24S; 3b: 24R) a (24S), 3b (24R) 20 To a stirred solution of verucopeptin (1, 8.88 mg, mmol) in CHCl 3/MeOH (1:1, 1.98 ml) was added NaBH 4 (5.62 mg, 0.15 mmol) at room temperature. After 30 min, PBS buffer was added to the reaction mixture. The organic layer was washed with PBS buffer (3 times) and concentrated in vacuo. The residue was chromatographed on an ODS column with a stepwise elution S2

3 of H 2O/MeOH (from 100:0 to 0:100). Fractions eluted with H 2O/MeOH (10:90 and 0:100) were combined and subjected to ODS HPLC on Cosmosil 5C18-AR-II (ϕ mm) with H 2O/MeCN (40:60) to afford the reduced derivative 2a (4.92 mg, 55%) and its diastereomer 2b (1.98 mg, 22%) as a colorless amorphous solid, respectively. Reduced derivative 2a (3.62 mg, mmol) was mixed with 2,2-DMP (98.76 μl, mmol) and PPTS (5.06 mg, mmol) in CH 2Cl 2 (1.34 ml), which was stirred at room temperature. After 1 h, satd aq NaHCO 3 was added to the reaction mixture. The organic layer was washed with satd aq NaHCO 3 (3 times) and concentrated in vacuo. The obtained residue was chromatographed on an HP20 column with a stepwise elution of H 2O/MeOH (from 100:0 to 0:100) and CHCl 3. Fractions eluted with H 2O/MeOH (50:50 to 0:100) and CHCl 3 were combined and subjected to ODS HPLC on Cosmosil 5C8-MS (ϕ mm) with H 2O/MeCN (35:65) to afford 3a (2.43 mg, 64%): [α] 20 D (c 0.19, CHCl 3); IR (neat) 3339, 2958, 1650, 1489, 1192, 753 cm -1 ; 1 H NMR (acetone-d 6, 500 MHz) δ 4.1/3.44 (Gly or Me-Gly) *, 5.20/3.80 (Gly or Me-Gly) *, 5.27 (1H)/3.55 (1H, d, J = 17.5 Hz) (Gly or Me-Gly) *, 4.85 (H4), 3.80 (1H, H4), 7.59 (br, 4-NH), 5.14 (1H, H10), 1.89 (H11), 1.70 (H11), 2.22 (1H, H12), 1.54 (H12), 3.13 (H13), 2.72 (H13), 4.85 (13-NH), 6.13 (H15), 6.97 (br, 15-NH), 4.78 (H16), 1.83 (H17), 0.87 (H18), 1.12 (d, J = 7.4 Hz, H19), 3.15 (s, H20 or H21) *, 2.85 (s, H20 or H21) *, 3.93 (1H, H24), 1.87 (H25), 1.53 (H25), 1.70 (H26), 3.36 (1H, H27), 4.11 (H28), 5.22 (1H,d, J = 9.6 Hz, H30), 2.57 (1H, H31), 1.24 (H32), 1.08 (H32), 1.55 (H33), 1.07 (H34), 1.06 (H34), 1.42 (H35), 1.30 (H36), 1.14 (H36), 0.86 (H37), 1.49 (s, H38), 3.39 (3H, s, H39), 1.67 (s, H40), 0.92 (H41), 0.82 (H42), 0.83 (H43), 1.56 (s, H1 ), 1.37 (s, H2 ) (Chemical shifts were assigned on the basis of the COSY data; *signals could be exchangeable); HRMS (ESI) m/z [M+Na] + calcd for C 46H 79N 7NaO 13, b (2.16 mg) was mixed with 2,2-DMP (59.06 μl, mmol) and PPTS (3.03 mg, mmol) in CH 2Cl 2 (0.80 ml), which was stirred at room temperature. Except for the HPLC condition (Cosmosil 5C8-MS (ϕ mm) with H 2O/MeCN (38:62)), the reaction mixture was 20 treated as described above to afford 3b (1.08 mg, 52%): [α] D -7.5 (c 1.11, CHCl 3); IR (neat) 3338, 2958, 1651, 1243, 754 cm -1 ; 1 H NMR (acetone-d 6, 500 MHz) δ 5.24 (1H, d, J = 14.8 Hz)/3.76 (Gly or Me-Gly) *, 4.58 (1H, d, J = 18.5 Hz)/3.72 (Gly or Me-Gly) *, 4.83/3.78 (Gly or Me-Gly) *, 5.13 (1H, H10), 2.23 (1H, H11), 1.88 (H11), 1.57 (H12), 1.45 (H12), 3.11 (H13), 2.72 (1H, H13), 6.08 (1H, H15), 4.79 (H16), 1.84 (H17), 0.93 (H18), 1.12 (d, J = 6.9 Hz, H19), 3.15 (3H, s, H20 or H21) *, 2.84 (s, H20 or H21) *, 3.95 (H24), 1.96 (H25), 1.53 (H25), 1.83 (H26), 3.27 (1H, H27), 4.02 (H28), 5.18 (1H,d, J = 9.8 Hz, H30), 2.58 (1H, H31), 1.24 (H32), 1.07 (H32), 1.55 (H33), 1.06 (H34), 1.42 (H35), 1.31 (H36), 1.15 (H36), 0.87 (H37), 1.24 (s, H38), 3.36 (3H, s, H39), 1.69 (3H, s, H40), 0.93 (d, J = 7.3 Hz, H41), 0.83 (H42), 0.84 (H43), 1.48 (s, H1 ), 1.41 (s, H2 ) (Chemical sifts were assigned on the basis of the COSY data; *signals could be exchangeable); HRMS (ESI) m/z [M+Na] + calcd for C 46H 79N 7NaO 13, S3

4 Synthesis of bis-mtpa derivatives 4a and 4b a: (R)-MTPA 4b: (S)-MTPA To a stirred solution of 2a (1.85 mg, mmol) in CHCl 3 (82.45 μl) was added MeI (2.60 μl, mmol), TBAB (6.64 mg, mmol) and 1M K 2CO 3 (41.2 μl, mmol). After being stirred at room temperature for 30 min, water was added to the reaction mixture. The mixture was chromatographed on an ODS column with a stepwise elution of H 2O/MeCN (from 100:0 to 0:100). Fractions eluted with H 2O/MeCN (10:90 to 0:100) were combined and subjected to ODS HPLC on Cosmosil 5C18-AR-II (ϕ mm) with H 2O/MeCN (50:50) to afford methylated 2a (0.81 mg, 43%). The methylated 2a was split into two portions. One portion of the material (0.55 mg, mmol) was mixed with DMAP (1.48 mg, mmol) and (S)-MTPACl (3.05 mg, mmol) in CH 2Cl 2 (60.34 μl), which was stirred at room temperature. After 1 h, satd aq NH 4Cl was added to the reaction mixture. The organic layer was washed with satd aq NH 4Cl (3 times) and concentrated in vacuo. The obtained residue was chromatographed on an ODS column with a stepwise elution of H 2O/MeCN (from 100:0 to 0:100). Fractions eluted with H 2O/MeCN (0:100) were combined and subjected to ODS HPLC on Cosmosil 5C18-AR-II (ϕ mm) with H 2O/MeCN 20 (10:90) to afford 4a (0.67 mg, 83%): [α] D (c 0.12, CHCl 3); IR (neat) 3343, 2958, 1745, 1651, 1168 cm -1 ; 1 H NMR (CDCl 3, 500 MHz) δ 4.35 (d, J = 17.0 Hz)/3.66 (d, J = 19.3 Hz) (Gly or Me-Gly) *, 5.05/3.80 (Gly or Me-Gly) *, 4.56/3.51 (1H) (Gly or Me-Gly) *, 4.49 (d, J = 20.4 Hz, H4), 3.99 (H4), 6.81 (4-NH), 5.14 (H10), 2.38 (H11), 1.83 (H11), 1.53 (H12), 1.32 (H12), 3.03 (H13), 2.66 (H13), 4.51 (13-NH), 5.95 (H15), 4.86 (dd, J = 10.3, 2.7 Hz, H16), 1.94 (H17), 0.88 (H18), 1.01 (d, J = 6.0 Hz, H19), 3.15 (s, H20 or H21) *, 3.00 (s, H20 or H21) *, 3.84 (s, N-OMe), 5.49 (1H, H24), 2.04 (H25), 1.75 (H25), 1.63 (H26), 3.44 (H27), 5.52 (H28), 5.09 (H30), 2.41 (H31), 1.04 (H32), 0.85 (H37 or 42 or 43) *, 1.27 (s, H38), 3.35 (s, H39), 1.51 (s, H40), 0.82 (H41), 0.79 (H37 or 42 or 43) *, 0.77 (H37 or 42 or 43) *, 3.50 (s, H 3-1 or H 3-2 ) *, 3.54 (s, H 3-1 or H 3-2 ) *, (phenyl in bis-mtpa) (Chemical shifts were assigned on the basis of the COSY data; *signals could be exchangeable.); HRMS (ESI) m/z [M+Na] + calcd for C 64H 91F 6N 7NaO 17, S4

5 The another portion of the methylated 2a (0.50 mg) was mixed with DMAP (1.34 mg, mmol) and (R)-MTPACl (2.78 mg, mmol) in CH 2Cl 2 (54.90 μl), which was stirred for 1 h at room temperature. The reaction mixture was fractionated as described above to afford 4b (0.57 mg, 77%): [α] 20 D (c 0.06, CHCl 3); IR (neat) 3356, 2918, 1745, 1652, 1184, 718 cm -1 ; 1 H NMR (CDCl 3, 500 MHz) δ 5.06/3.81 (Gly or Me-Gly) *, 4.56/3.50 (Gly or Me-Gly) *, 4.38/3.76 (Gly or Me-Gly) *, 4.50 (H4), 3.95 (H4), 6.71 (4-NH), 5.12 (H10), 2.38 (H11), 1.84 (H11), 1.56 (H12), 1.33 (H12), 3.04 (H13), 2.65 (H13), 5.96 (H15), 4.86 (dd, J = 10.5, 3.3 Hz, H16), 1.94 (H17), 0.89 (d, J = 6.4 Hz, H18), 1.02 (H19), 3.05 (s, H20 or H21) *, 3.14 (s, H20 or H21) *, 3.84 (s, N-OMe), 5.47 (H24), 1.93 (H25), 1.75 (H25), 1.61 (H26), 3.38 (H27), 5.45 (H28), 5.26 (H30), 2.50 (H31), 1.10 (H32), 1.43 (H33), 1.01 (H34), 1.36 (H35), 1.24 (H36), 1.15 (H36), 0.84 (H37), 1.42 (s, H38), 3.24 (s, H39), 1.67 (s, H40), 0.89 (H41), 0.79 (H42), 0.78 (H43), 3.50 (s, H1 or H2 ) *, 3.48 (s, H1 or H2 ) *, (phenyl in bis-mtpa) (Chemical shifts were assigned on the basis of the COSY data; *signals could be exchangeable); HRMS (ESI) m/z [M+Na] + calcd for C 64H 91F 6N 7NaO 17, Synthesis of the MTPA derivatives S3a and S3b. S3a: (R)-MTPA S3b: (S)-MTPA To a stirred solution of verucopeptin (1, 2.4 mg, mmol) in CH 2Cl 2 (1.35 ml) was added NaIO 4 (48.55 mg, 0.23 mmol) and silica gel in H 2O (0.20 ml) at room temperature. After 15 h, the reaction mixture was filtrated. The organic layer was washed with H 2O (3 times) and concentrated in vacuo. The residue was chromatographed on a PLC plate (CHCl 3/MeOH = 15/1), followed by extraction and concentration in vacuo. The residue (1.52 mg) was mixed with NaOMe in MeOH (0.70 ml), which was stirred at room temperature. After 2.5 h, the reaction mixture was added to 1N HCl and concentrated in vacuo. The residue was chromatographed on an ODS column with H 2O and MeOH. The fraction eluted by MeOH was concentrated in vacuo. The residue (1.61 mg) was reacted with TMSCHN 2 (0.10 ml in n-hexane, ca. 0.6 M) in MeOH (0.8 ml) and 1N HCl at room temperature. After 1.5h, H 2O was added to the reaction mixture and concentrated in vacuo. The residue was chromatographed on an ODS column with H 2O and MeOH. The fraction eluted by MeOH was concentrated in vacuo and chromatographed on a PLC plate (CHCl 3/MeOH= 20/1). The material (Rf = 0.94 in CHCl 3/MeOH S5

6 (20/1)) was concentrated in vacuo. The material was split into two portions. A half portion of the material (0.28 mg) was mixed with DMAP (1.69 mg, mmol) and (S)-MTPACl (5.00 mg, mmol) in CH 2Cl 2 (30.00 μl), which was stirred at room temperature. After 1 h, satd aq NaHCO 3 was added to the reaction mixture. The organic layer was washed with satd aq NaHCO 3 (3 times) and concentrated in vacuo. The residue was chromatographed on an ODS column with a stepwise elution of H 2O/MeOH (from 100:0 to 0:100). Fractions eluted by H 2O/MeCN (0:100) were combined and subjected to ODS HPLC on CAPCELL PAK UG120 (ϕ mm) with H 2O/MeCN (10:90) to afford S3a (0.29 mg, 64% over 4 steps): 1 H NMR (CDCl 3, 500 MHz) δ 1.83/2.39 (H25), 1.66/1.72 (H26), 3.39 (H27), 5.52 (d, J = 4.0 Hz, H28), 5.12 (d, J = 9.7 Hz, H30), 2.47 (H31), 1.54/1.25 (H32), 1.56 (H40), 0.87 (H41) (Chemical shifts were assigned on the basis of the COSY data.); MS (ESI) m/z [M+Na] + calcd for C 64H 91F 6N 7NaO 17, The another portion of the residue (0.29 mg) was mixed with DMAP (2.34 mg, mmol) and (R)-MTPACl (5.00 mg, mmol) in CH 2Cl 2 (30.00 μl), which was stirred for 1 h at room temperature. The reaction mixture was fractionated as described above to afford S3b (0.54 mg, quant. over 4steps): 1 H NMR (CDCl 3, 500 MHz) δ 2.31 (H25), 1.55/1.59 (H26), 3.36 (H27), 5.49 (d, J = 5.2 Hz, H28), 5.31 (d, J = 10.3 Hz, H30), 5.53 (H31), 1.18 (H32), 1.68 (H40), 0.92 (H41) (Signals were assigned on the basis of the COSY data.); MS (ESI) m/z [M+Na] + calcd for C 64H 91F 6N 7NaO 17, Cell culture. HT1080 cells were cultured at 37 C under an O 2 atmosphere (21%) in DMEM (Gibco) containing 10% FCS, penicillin and streptomycin. Hypoxic condition (1% O 2) was prepared in a multi-gas incubator (SANYO). HIF-1 reporter assay. HRE-driven luciferase reporter assay was carried out as described previously. 2 Cell proliferation was measured by using Alamar Blue. Western blot analysis. Before the day of verucopeptin treatment HT1080 cells were plated at a density of cells per a 60 mm tissue culture dish. Compound-treated cells were washed with ice-cold PBS and lysed in extraction buffer A [50 mm Tris HCl (ph 7.5), 150 mm NaCl, 0.5 mm DTT, 0.2% NP-40, 0.2 mm PMSF and 20 nm MG132]. Cell extracts were cleared by centrifuging at 15,000 g for 10 min at 4 C. Total protein concentration was determined by BCA assay (Nacalai Tesque). Protein extracts (25 μg) were subjected to SDS-PAGE followed by blotting onto a PVDF membrane. The membrane was briefly washed with PBST buffer (PBS containing 0.1% Tween20) and blocked with blocking buffer (PBST buffer containing 5% non-fat milk or 3% BSA). The membrane was incubated with primary antibody in blocking buffer overnight. After washing with S6

7 PBST buffer three times, the membrane was incubated with secondary antibody for 1 h. Antibodies used were as follows: anti-human HIF-1α mab (clone54, Becton Dickson), anti-human HIF-1β mab (D28F3, Cell Signaling Technology), anti-human p70 ribosomal S6 kinase mab (32D7, Cell Signaling Technology), anti-human phospho-p70 ribosomal S6 kinase (Ser380) mab (9D9, Cell Signaling Technology), anti-human S6 ribosomal protein mab (5G10, Cell Signaling Technology), anti-human phosphor-s6 ribosomal protein (Ser235/236) mab (D57.2.2E, Cell Signaling Technology), anti-human c-raf pab (Santa Cruz), anti-human α-tubulin mab (11H10, Cell Signaling Technology) and alkaline phosphatase or horse radish peroxidase-conjugated goat IgG against mouse or rabbit IgG (Promega). The results were visualized by the nitro-blue tetrazolium chloride/5-bromo-4-chloro-3 -indolyphosphate p-toluidine salt (BCIP/NBT) colorimetric detection or enhanced chemiluminescence. Quantitative RT-PCR. Total RNA was isolated using Total RNA Extraction Miniprep System (Viogene) according to the manufacturer s instruction. cdna was synthesized using ReverTra Ace α (Toyobo). PCR reactions were performed using FastStart Universal SYBR Green Master (Roche Diagnostic) in ABI Step One Plus (Applied Biosystems). 18S ribosomal RNA was used as an internal control. The following primers were used: 5 -GCAATTATTCCCCATGAACG-3 (18S sense); 5 -GGACTTAATCAACGCAAGC-3 (18S antisense); 5 -GCAGCTTGAGTTAAACGAACG-3 (VEGF sense); 5 -GGTTCCCGAAACCCTGAG-3 (VEGF anisense); 5 -GGTTGTGCCATACTCATGACC-3 (GLUT1 sense); 5 -CAGATAGGACATCCAGGGTAGC-3 (GLUT1 antisense); 5 -TGCTGCTCTCTCATTTGCTG-3 (BNIP3 sense); 5 -GACTCCAGTTCTTCATCAAAAGGT-3 (BNIP3 antisense); 5 -TTTTTCAAGCAGTAGGAATTGG-3 (HIF-1α sense); 5 -GTGATGTAGTAGCTGCATGATC-3 (HIF-1α antisense). S7

8 luciferase activity (%) viability (%) : luciferase : viability verucopeptin (1) (μm) derivative 2a (μm) Figure S1. Effect of verucopeptins in the HIF-1 reporter assay. Verucopeptin (1) inhibited the luciferase activity with an IC 50 value of 0.22 μm (left). The cytotoxicity was detected at concentrations above 1.1 μm. The inhibitory activity of the reduced derivative of verucopeptin (2a) was significantly lower than that of verucopeptin (1) (right). S8

9 Figure S2. Advanced Marfey s analysis of verucopeptin (1). (a) Analysis of the configuration of the piperazic acid. The retention time of the verucopeptin-derived L-FDLA derivative (top) was compared with those of L-FDLA-D-ornithine (middle) and L-FDLA-L-ornithine (bottom) using LC-ESI-MS. Verucopeptin-derived derivative showed a retention time same as that of D-ornithine. Mass chromatogram at m/z 721 are shown. (b, c) Analysis of the configuration of β-hydroxyleucine. b. The retention time of the verucopeptin-derived L-FDLA derivative (top) was compared with those of L-FDLA-labeled synthesized β-hydroxyleucine (2S,3S and 2R, 3R: middle, 2S,3R and 2R,3S: bottom) by ODS HPLC. The retention time of the verucopeptin-derived derivative matched that of synthesized (2S, 3R)-β-hydroxyleucine. c. Co-injection analysis of the L-FDLA derivatives. The L-FDLA derivative of synthesized (2S, 3S)-β-hydroxyleucine was co-injected with that of the verucopeptin hydrolysate (top) or that of synthesized (2S, 3R)-β-hydroxyleucine (bottom). The FDLA derivative of the verucopeptin hydrolysate was co-eluted with that of synthesized (2S, 3S)-β-hydroxyleucine. Chromatograms at 340 nm are shown. Standard materials of β-hydroxyleucine were synthesized as reported previously. 3 The elution order of the L-FDLA conjugates of synthesized β-hydroxyleucine was same as that reported. 4 S9

10 verucopeptin (1) azinothricin A83596C kettapeptin IC101 Figure S3. Chemical structures of verucopeptin (1) and related natural products. S10

11 (a) (b) a (ppm) (c) (d) b (ppm) Figure S4. NOE analyses of the isopropylidene derivatives 3a and 3b. (a, b) 1 H control 1D spectrum (upper, 500 MHz, in acetone-d 6) and NOE difference spectrum (lower) for the derivative 3a. One methyl group in the isopropylidene group (arrow), H-24 (yellow circle), and Me-38 (green circle) mutually showed NOE correlations, indicating that Me-38 and H-24 in 3a are positioned in a syn configuration. (c,d) 1 H control 1D spectrum (upper, 500 MHz, in acetone-d 6) and NOE difference spectrum (lower) for the derivative 3b. Me-38 (black arrow) showed NOE correlations with H 2-25 (yellow circle) and one of two methyl groups in the isopropylidene function (green circle). The other methyl group in the isopropylidene function showed a NOE correlation with H-24. These results revealed that Me-38 and H-24 in the derivative 3b was located in an anti configuration. S11

12 Figure S5. Modified Mosher s analysis of the side chain of verucopeptin (1). (a) Preparation of the MTPA derivatives S3a and S3b. (b) Δδ (δ (S)-MTPA - δ (R)-MTPA) values for the MTPA derivatives S3a and S3b. Δδ values are shown in ppm. S12

13 relative mrna level : 21% O 2 : 1% O 2 : 1% O (10 nm) : 1% O (100 nm) 0 vegf glut1 bnip3 Figure S6. Effect of verucopeptin (1) on the mrna levels of vegf, glut1 and bnip3. HT1080 cells were treated with verucopeptin (1) for 24 h in a hypoxic condition. Cells were harvested, and the mrna levels of vegf, glut1 and bnip3 were quantitatively analyzed. Verucopeptin (1) decreased the expression levels of gult1 and bnip3 in a dose-dependent manner (Figure 4a), but not that of vegf. Means and SD are shown (n = 4). S13

14 Figure S7. Effect of verucopeptin (1) on the mrna level of hif-1α. Cells were treated with or without verucopeptin (veru; 1) (100 nm) for 4 h. The mrna level was measured quantitatively. The mrna level of HIF-1α was not affected by the concentration of O 2 as reported previously. 5 Verucopeptin (1) did not change the amount of hif-1α mrna. Means and SD are shown (n = 4). S14

15 Figure S8. Comparison of the effect of verucopeptin (1) and 17-AAG on the protein levels of HIF-1α and c-raf. HT1080 cells were incubated in the presence of verucopeptin (veru; 1) (100 nm) or 17-AAG (20 μm) for 24 h in a hypoxic condition. 17-AAG, an HSP90 inhibitor, decreased the protein levels of HIF-1α and c-raf, both of which are client proteins of HSP90. In contrast, verucopeptin (1) did not decrease the protein level of c-raf. S15

16 Figure S9. Effects of verucopeptin (1) on the phosphorylation status of p70s6k and S6 proteins. Phosphorylation level of p70s6k and S6 proteins after treatment with verucopeptin (1). HT1080 cells were treated with verucopeptin (veru; 1) (100 nm) for 4, 12 and 24 h, or with rapamycin (rapa; 0.5 nm) for 1 h. S16

17 Figure S10. Effect of SLF on the activity of verucopeptin (1) and rapamycin. Cells were treated with verucopeptin (1, 100 nm, 4 h) or rapamycin (0.5 nm, 1 h) after pretreatment with SLF (0.1 mm, 1 h). Rapamycin decreased the phosphorylation level of S6 protein, which was abolished by the presence of SLF. In contrast, the effect of verucopeptin (1) was not affected by SLF. S17

18 References for Supporting Information (1) Yoshimura, A.; Kishimoto, S.; Nishimura, S.; Otsuka, S.; Sakai, Y.; Hattori, A.; Kakeya, H. J. Org. Chem. 2014, 79, (2) Yasuda, Y.; Arakawa, T.; Nawata, Y.; Shimada, S.; Oishi, S.; Fujii, N.; Nishimura, S.; Hattori, A.; Kakeya, H. Bioorg. Med. Chem. 2015, 23, (3) MacMillan B. J.; Molinski, F. T. Org. Lett. 2002, 4, (4) MacMillan, B. J.; Ernst-Russell, A. M.; de Ropp, S. J.; Molinski, F. T. J. Org. Chem. 2002, 67, (5) Scharte, M.; Jurk, K.; Kehrel, B.; Zarbock, A.; Aken, V.H.; Singbarti, K. FEBS Lett. 2006, 580, S18

19 Figure S11. 1 H NMR spectrum of 1 in CDCl 3 (500 MHz). Figure S C NMR spectrum of 1 in CDCl 3 (125 MHz). S19

20 Figure S13. COSY spectrum of 1 in CDCl 3. S20

21 Figure S14. TOCSY spectrum of 1 in CDCl 3. S21

22 Figure S15. HMQC spectrum of 1 in CDCl 3. S22

23 Figure S16. HMBC spectrum of 1 in CDCl 3. S23

24 Figure S17. NOESY spectrum of 1 in CDCl 3. S24

25 Figure S18. ROESY spectrum of 1 in CDCl 3. S25

26 Figure S19. 1 H NMR spectrum of 3a in acetone-d 6 (500 MHz). Figure S C NMR spectrum of 3a in acetone-d6 (125 MHz). S26

27 Figure S21. COSYspectrum of 3a in acetone-d 6. S27

28 Figure S22. HMQC spectrum of 3a in acetone-d 6. S28

29 Figure S23. HMBC spectrum of 3a in acetone-d 6. S29

30 Figure S24. NOESY spectrum of 3a in acetone-d 6. S30

31 Figure S25. 1D NOE spectrum of 3a in acetone-d 6. S31

32 Figure S26. 1 H NMR spectrum of 3b in acetone-d 6 (500 MHz). Figure S C NMR spectrum of 3b in acetone-d6 (125 MHz). S32

33 Figure S28. COSY spectrum of 3b in acetone-d 6. S33

34 Figure S29. HMQC spectrum of 3b in acetone-d 6. S34

35 Figure S30. HMBC spectrum of 3b in acetone-d 6. S35

36 Figure S31. NOESY spectrum of 3b in acetone-d 6. S36

37 Figure S32. 1D NOE spectrum of 3b in acetone-d 6. S37

38 Figure S33. 1 H NMR spectrum of 4a in CDCl 3 (500 MHz). Figure S C NMR spectrum of 4a in CDCl 3 (125 MHz). S38

39 Figure S35. COSY spectrum of 4a in CDCl 3. S39

40 Figure S36. HMQC spectrum of 4a in CDCl 3. S40

41 Figure S37. HMBC spectrum of 4a in CDCl 3. S41

42 Figure S38. 1 H NMR spectrum of 4b in CDCl 3 (500 MHz). Figure S C NMR spectrum of 4b in CDCl 3 (125 MHz). S42

43 Figure S40. COSY spectrum of 4b in CDCl 3. S43

44 Figure S41. HMQC spectrum of 4b in CDCl 3. S44

45 Figure S42. HMBC spectrum of 4b in CDCl 3. S45

46 Figure S43. 1 H NMR spectrum of S3a in CDCl 3 (500 MHz). Figure S44. 1 H NMR spectrum of S3b in CDCl 3 (500 MHz). S46