Supporting Information

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1 Supporting Information Asperphenins A and B, Lipopeptidyl Benzophenones from a Marinederived Aspergillus sp. Fungus Lijuan Liao, Song Yi Bae, Tae Hyung Won, Minjung You, Seong-Hwan Kim, Dong-Chan Oh, Sang Kook Lee, Ki-Bong Oh,,* and Jongheon Shin,* Natural Products Research Institute, College of Pharmacy, Seoul National University, San 56-1, Sillim, Seoul , Korea Department of Agricultural Biotechnology, College of Agricultural and Life Science, Seoul National University, San 56-1, Sillim, Gwanak, Seoul , Korea *Corresponding Author: Jongheon Shin Seoul National University, San 56-1, Sillim, Gwanak, Seoul , Korea Tel: Fax: shinj@snu.ac.kr *Co-corresponding Author: Ki-Bong Oh Seoul National University, San 56-1, Sillim, Gwanak, Seoul , Korea Tel: ohkibong@snu.ac.kr 1

2 Experimental Section General experimental procedures S5 Isolation and identification of the fungal strain. S5 Fermentation and isolation... S5 Asperphenin A (1) S6 Asperphenin B (2) S6 Cyclization of compounds 1 and 2... S6 MTPA-Esterifications of compounds 3 and 4. S7 Advanced Marfey s analysis of compounds 1 and 2. S9 Reduction of 7-ketone group of compounds 1 and 2. S10 Reduction of 15-ketone group of compounds 5 and 7 S12 Computational ECD analysis S14 Tables Table S1. Inhibition of cancer cell proliferation by asperphenins.... S15 Figures Figure S1. The δ H (4aS-4aR) values of MTPA esterifications for triacetyl-cycloasperphenin B (4a)... S15 Figure S2. The LC/MS spectra of advanced Marfey s analysis of 1 and 2 S17 Figure S3. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin A (9) at C-15 and C S18 Figure S4. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin A (9) at C-16 and C-17. S19 Figure S5. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin A (10) at C-15 and C- 16 S20 Figure S6. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin A (10) at C-16 and C- 17 S21 Figure S7. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin B (11) at C-15 and C- 16 S22 Figure S8. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin B (11) at C-16 and C- 17 S23 Figure S9. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin B (12) at C-15 and C- 16 S24 Figure S10. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin B (12) at C-16 and C- 2

3 17 S25 Figure S11. Experimental CD spectra of asperphenins A (1) and B (2)... S26 Figure S12. The 1 H NMR spectrum of asperphenin A (1) S27 Figure S13. The 13 C NMR spectrum of asperphenin A (1) S28 Figure S14. The DEPT135 spectrum of asperphenin A (1)... S28 Figure S15. The HSQC spectrum of asperphenin A (1) S29 Figure S16. The COSY spectrum of asperphenin A (1) S29 Figure S17. The TOCSY spectrum of asperphenin A (1). S30 Figure S18. The HMBC spectrum of asperphenin A (1)... S30 Figure S19. The 1 H NMR spectrum of asperphenin B (2) S31 Figure S20. The 13 C NMR spectrum of asperphenin B (2)... S32 Figure S21. The DEPT135 spectrum of asperphenin B (2) S32 Figure S22. The HSQC spectrum of asperphenin B (2) S33 Figure S23. The COSY spectrum of asperphenin B (2) S33 Figure S24. The TOCSY spectrum of asperphenin B (2). S34 Figure S25. The HMBC spectrum of asperphenin B (2). S34 Figure S26. The 1 H NMR spectrum of cycloasperphenin A (3) S35 Figure S27. The 13 C NMR spectrum of cycloasperphenin A (3) S35 Figure S28. The HSQC spectrum of cycloasperphenin A (3) S36 Figure S29. The COSY spectrum of cycloasperphenin A (3) S36 Figure S30. The HMBC spectrum of cycloasperphenin A (3) S37 Figure S31. The 1 H NMR spectrum of cycloasperphenin B (4). S37 Figure S32. The 13 C NMR spectrum of cycloasperphenin B (4) S38 Figure S33. The HSQC spectrum of cycloasperphenin B (4) S38 Figure S34. The COSY spectrum of cycloasperphenin B (4) S39 Figure S35. The HMBC spectrum of cycloasperphenin B (4) S39 Figure S36. The 1 H NMR spectrum of (S)-MTPA ester of triacetyl-cycloasperphenin A (3aS) S40 Figure S37. The 1 H NMR spectrum of (R)-MTPA ester of triacetyl-cycloasperphenin A (3aR)... S40 Figure S38. The 1 H NMR spectrum of (S)-MTPA ester of triacetyl-cycloasperphenin B (4aS) S41 Figure S39. The 1 H NMR spectrum of (R)-MTPA ester of triacetyl-cycloasperphenin B (4aR)... S41 Figure S40. The 1 H NMR spectrum of 7-hydroxyasperphenin A (5). S42 Figure S41. The 13 C NMR spectrum of 7-hydroxyasperphenin A (5) S42 Figure S42. The 1 H NMR spectrum of 7-epi-hydroxyasperphenin A (6). S43 3

4 Figure S43. The 13 C NMR spectrum of 7-epi-hydroxyasperphenin A (6). S43 Figure S44. The 1 H NMR spectrum of 7-hydroxyasperphenin B (7) S44 Figure S45. The 13 C NMR spectrum of 7-hydroxyasperphenin B (7) S44 Figure S46. The 1 H NMR spectrum of 7-epi-hydroxyasperphenin B (8). S45 Figure S47. The 13 C NMR spectrum of 7-epi-hydroxyasperphenin B (8) S45 Figure S48. The 1 H NMR spectrum of 7,15(S)-dihydroxyasperphenin A (9) S46 Figure S49. The 13 C NMR spectrum of 7,15(S)-dihydroxyasperphenin A (9) S46 Figure S50. The HETLOC NMR spectrum of 7,15(S)-dihydroxyasperphenin A (9) S47 Figure S51. The ROESY NMR spectrum of 7,15(S)-dihydroxyasperphenin A (9) S47 Figure S52. The 1 H NMR spectrum of 7,15(R)-dihydroxyasperphenin A (10). S48 Figure S53. The 13 C NMR spectrum of 7,15(R)-dihydroxyasperphenin A (10). S48 Figure S54. The HETLOC NMR spectrum of 7,15(R)-dihydroxyasperphenin A (10). S49 Figure S55. The ROESY spectrum of 7,15(R)-dihydroxyasperphenin A (10). S49 Figure S56. The 1 H NMR spectrum of 7,15(S)-dihydroxyasperphenin B (11). S50 Figure S57. The 13 C NMR spectrum of 7,15(S)-dihydroxyasperphenin B (11). S50 Figure S58. The HETLOC NMR spectrum of 7,15(S)-dihydroxyasperphenin B (11). S51 Figure S59. The ROESY NMR spectrum of 7,15(S)-dihydroxyasperphenin B (11). S51 Figure S60. The 1 H NMR spectrum of 7,15(R)-dihydroxyasperphenin B (12) S52 Figure S61. The 13 C NMR spectrum of 7,15(R)-dihydroxyasperphenin B (12) S52 Figure S62. The HETLOC NMR spectrum of 7,15(R)-dihydroxyasperphenin B (12)... S53 Figure S63. The ROESY NMR spectrum of 7,15(R)-dihydroxyasperphenin B (12) S53 4

5 EXPERIMENTAL SECTION General Experimental Procedures. The optical rotations were measured on a JASCO P-2000 polarimeter using a 1 cm cell. The UV spectra were acquired with a Hitachi U-3010 spectrophotometer. The CD spectra were recorded using an Applied Photophysic Chirasscan-plus circular dichroism detector. The IR spectra were recorded on a JASCO 4200 FT-IR spectrometer, using a ZnSe cell. The NMR spectra were recorded on Bruker Avance 500 and 600 MHz spectrometer at the NCIRF (National Center for Interuniversity Research Facilities at Seoul National University), and on Bruker Avance II 800 MHz and 900 MHz at the KBSI (Korea Basic Science Institute) at Ochang, Korea. HRFABMS data were acquired using a Jeol JMS 700 mass spectrometer with metanitrobenzyl alcohol (NBA) as the matrix at the KBSI (Korea Basic Science Institute) at Daegu, Korea. The HPLC analyses were performed on a Spectrasystem p2000 equipped with a refractive index detector (Spectrasystem RI-150). All of the solvents used were spectroscopic grade or distilled from glass prior to use. Isolation and Identification of the Fungal Strain. The fungal strain Aspergillus sp. (strain number F452) was isolated from submerged decaying wood off of the shore of Jeju Island, Korea, in November The strain was identified using standard molecular biological protocols by DNA amplification and sequencing of the ITS region. The genomic DNA extraction was performed using Intron s i-genomic BYF DNA Extraction Mini Kit according to the manufacturer s protocol. The nucleotide sequence of F452 has been deposited in the GenBank database under the accession number KF The 18S rdna sequence of this strain showed a 99% identity with Aspergillus versicolor Ppf48 (GenBank accession number GU586852). Fermentation and Isolation. The isolated strain was cultivated on a YPG agar plate (5 g yeast extract, 5 g peptone, 10 g glucose, and 16.0 g agar in 1 L artificial seawater) for 4 days. The agar plugs (1 cm 1 cm, 5 pieces each) were inoculated into 100 ml of the YPG media in a 250 ml Erlenmeyer flask for 5 days, then separately transferred to 2.8 L glass Fernbach flasks with rice media (2 g peptone, 2 g yeast extract, 200 g rice with 200 ml artificial seawater in each flask, boiled in an autoclave for 20 min at 120 C; 24 flasks in total). The fermentation in the rice media was conducted under static conditions for 6 weeks and then was extracted by MeOH (1 L 3) and CH 2Cl 2 (1 L 3). The solvent was evaporated to obtain an organic extract. The combined extracts (62.6 g) were successively partitioned between n-buoh (42.5 g) and H 2O (16.8 g); the former fraction was repartitioned using H 2O-MeOH (15:85) (20.3 g) and n- hexane (19.2 g). The H 2O-MeOH fraction was separated by C 18 reversed-phase vacuum flash chromatography using a sequential mixture of MeCN and H 2O as eluents (five fractions in gradient, 5

6 H 2O-MeCN, from 80:20 to 0:100), MeOH, acetone, and finally EtOAc. On the basis of the results of LC-MS analysis, the fractions eluted with H 2O-MeCN (40:60) (6.3 g) was separated by C 18 reversedphase vacuum flash chromatography again using a sequential mixture of MeCN and H 2O as eluents (six fractions in gradient, H 2O-MeCN, from 50:50 to 0:100), and 100% MeOH. The fraction eluted with H 2O-MeCN (40:60) (2.1 g) was separated by semi-preparative reversed-phase HPLC (YMC- ODS column, mm; H 2O-MeOH, 30:70, 2.0 ml/min) to afford compounds 1 (t R 50.2 min, 43.2 mg) and 2 (t R 58.5 min, 46.7 mg). Asperphenin A (1): yellow amorphous solid, [ ] 25 D (c 0.1, MeOH); UV (MeOH) max (log ) 211 (4.65), 268 (4.25), 322 (3.95) nm; CD (MeOH) λ ( ε) 217 (+2.26), 235 (-1.79), 331 (-0.30) nm; IR (ZnSe) max 3309, 1750, 1671 cm -1 ; 1 H and 13 C NMR data, see Table S1; HRFABMS, m/z [M+H] + (Calcd for C 42H 62N 5O 11, ). Asperphenin B (2): yellow amorphous solid, [ ] 25 D (c 0.1, MeOH); UV (MeOH) max (log ) 211 (4.65), 268 (4.25), 322 (3.95) nm; CD (MeOH) λ ( ε) 224 (-0.80), 242 (-1.10), 280 (+0.66), 337 (-0.08), 448 (+0.10) nm; IR (ZnSe) max 3309, 1750, 1670 cm 1 ; 1 H and 13 C NMR data, see Table S1; HRFABMS, m/z [M+H] + (Calcd for C 42H 62N 5O 11, ). Cyclization of Compounds 1 and 2. To a solution of K 2CO 3 (4.3 mg) in DMF (3 ml) was added asperphenin A (1, 21.2 mg, 26 M) and stirred at room temperature for 7 h. The solution was extracted with EtOAc, evaporated under vacuum and the residue was purified by reverse-phase HPLC (YMC-ODS column, mm; H 2O-MeCN, 50:50, 1.0 ml/min) to afford cycloasperphenin A (3, t R 17.5 min, 4.2 mg, 5.2 M). Cycloasperphenin B (4, t R 19.5 min, 4.3 mg, 5.4 M) was prepared in the same manner from asperphenin B (2, 22.8 mg, 28 M). O O 1 K 2 CO 3 DMF, rt, 7h N H HO OH OH 2 K 2 CO 3 DMF, rt, 7h N H HO OH OH 3 4 Cycloasperphenin A (3): [ ] 25 D (c 1.0, MeOH); UV (MeOH) max (log ) 212 (4.18), 248 (4.03), 347 (3.22), 445 (2.92) nm; CD (MeOH) λ ( ε) 248 (+1.99), 334 (-0.44), 448 (+0.10) nm; IR (ZnSe) max 3309, 1748, 1671 cm 1 ; 1 H NMR (600 MHz, MeOH-d 4) H 7.02 (1H, t, J = 8.3 Hz, H-11), 6

7 6.77 (1H, br s, H-2), 6.57 (1H, br s, H-4), 6.45 (1H, d, J = 8.3 Hz, H-10), 6.41 (1H, d, J = 8.3 Hz, H- 12), 4.70 (1H, dd, J = 7.2, 5.4 Hz, H-28), 4.65 (1H, dd, J = 8.0, 7.2 Hz, H-17), 4.25 (1H, dd, J = 9.5, 4.4 Hz, H-23), 3.97 (1H, m, H-33), 2.81 (1H, dd, J = 15.4, 5.4 Hz, H-29), 2.75 (1H, dd, J = 15.4, 7.2 Hz, H-29), 2.45 (1H, dd, J = 14.3, 4.1 Hz, H-32), 2.32 (1H, dd, J = 14.3, 8.4 Hz, H-32), 2.26 (1H, overlap, H-25), 2.23 (3H, s, H-14), 2.14 (1H, dddd, J = 14.2, 7.8, 4.4, 3.8 Hz, H-24), 1.84 (1H, m, H- 24), 1.56 (1H, m, H-19), 1.48 (3H, m, H-18, H-34), 1.28 (15H, m, H-34 H-41), 0.88 (3H, t, J = 6.8 Hz, H-42), 0.71 (3H, d, J = 6.8 Hz, H-20), 0.70 (3H, d, J = 6.8 Hz, H-21); 13 C NMR (150 MHz, MeOH-d 4) C (C-15), (C-26), (C-27), (C-30), (C-31), (C-22), (C-9), (C-13), (C-5), (C-7), (C-3), (C-1), (C-16), (C- 11), (C-6), (C-4), (C-2), (C-8), (C-12), (C-10), 69.9 (C-33), 54.4 (C-23), 51.7 (C-28), 45.5 (C-17), 44.6 (C-32), 43.5 (C-18), 38.3 (C-34), 37.3 (C-29), 33.1 (C-40), 32.5 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.9 (C-24), 26.8 (C-35), 25.6 (C-19), 23.8 (C-41), 23.2 (C-21), 22.5 (C-20), 21.2 (C-14), 14.5 (C-42); HRTOFMS m/z , [M+H] + (Calcd for C 42H 60N 5O 10, ). Cycloasperphenin B (4): [ ] 25 D (c 1.0, MeOH); UV (MeOH) max (log ) 212 (4.18), 248 (4.03), 347 (3.22), 445 (2.92) nm; CD (MeOH) λ ( ε) 241 (-2.53), 318 (+0.18), 470 (-0.47) nm; IR (ZnSe) max 3310, 1748, 1671 cm 1 ; 1 H NMR (600 MHz, MeOH-d 4) H 7.03 (1H, t, J = 8.3 Hz, H-11), 6.76 (1H, br s, H-2), 6.57 (1H, br s, H-4), 6.46 (1H, d, J = 8.3 Hz, H-10), 6.40 (1H, d, J = 8.3 Hz, H- 12), 4.71 (1H, dd, J = 7.3, 5.4 Hz, H-28), 4.65 (1H, t, J = 7.8 Hz, H-17), 4.26 (1H, dd, J = 9.5, 4.5 Hz, H-23), 3.98 (1H, m, H-33), 2.81 (1H, dd, J = 15.4, 5.4 Hz, H-29), 2.75 (1H, dd, J = 15.4, 7.3 Hz, H- 29), 2.47 (1H, dd, J = 14.3, 4.1 Hz, H-32), 2.34 (1H, dd, J = 14.3, 8.1 Hz, H-32), 2.26 (2H, overlap, H-25), 2.23 (3H, s, H-14), 2.14 (1H, dddd, J = 14.1, 7.2, 5.6, 4.5 Hz, H-24), 1.85 (1H, dddd, J = 14.2, 9.5, 5.3, 3.2 Hz, H-24), 1.56 (1H, m, H-19), 1.47 (3H, m, H-18, H-34), 1.28 (15H, m, H-34 H-41), 0.89 (3H, t, J = 6.8 Hz, H-42), 0.71 (3H, d, J = 6.8 Hz, H-20), 0.69 (3H, d, J = 6.8 Hz, H-21); 13 C NMR (150 MHz, MeOH-d 4) C (C-15), (C-26), (C-27), (C-30), (C-31), (C-22), (C-9), (C-13), (C-5), (C-7), (C-3), (C-1), (C- 16), (C-11), (C-6), (C-4), (C-2), (C-8), (C-12), (C-10), 69.8 (C-33), 54.5 (C-23), 51.9 (C-28), 45.7 (C-17), 44.6 (C-32), 43.5 (C-18), 38.3 (C-34), 37.4 (C-29), 33.1 (C-40), 32.5 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.9 (C-24), 26.7 (C-35), 25.7 (C-19), 23.8 (C-41), 23.1 (C-21), 22.5 (C-20), 21.2 (C-14), 14.5 (C-42); HRTOFMS m/z , [M+H] + (Calcd for C 42H 60N 5O 10, ). MTPA-Esterifications of Compounds 3 and 4. To a solution of cycloasperphenin A (3, 2.6 mg) in dry pyridine (0.5 ml) in ice cooling water bath was added acetic anhydride (3 µl). After stirring at room temperature for 2 h, the reaction was quenched by adding ice water, evaporated under vacuum, 7

8 and the residue was divided into two (1.3 mg each). To a solution of the residue in dry pyridine (1.0 ml), were added DMAP (0.5 mg) and (-)-(R)-MTPA chloride (10 μl). The mixture was stirred under N 2 at room temperature for 2 h. After the consumption of the starting material was confirmed by HPLC-DAD, the solvent was removed under vacuum and the residue was purified by reverse-phase HPLC (YMC-ODS column, mm; H 2O-MeCN, 20:80, 1.0 ml/min) to afford 3aS, the (-)- (S)-MTPA ester of cycloasperphenin A-triacetate. Compound 3aR, the corresponding (+)-(R)-MTPA ester was also obtained from the same esterification reaction with (+)-(S)-MTPA chloride. Compounds 4aS and 4aR, the MTPA-esters of cycloasperphenin B-triacetate were obtained from 4 using the same procedure (See Figure S1 and S2). 3 Ac 2 O pyr, rt, 2h (R)-MTPA-Cl or (S)-MTPA-Cl DMAP, pyr, rt, 7h 3aS or 3aR 4 Ac 2 O pyr, rt, 2h (R)-MTPA-Cl or (S)-MTPA-Cl DMAP, pyr, rt, 7h 4aS or 4aR 3aS: 1 H NMR (600 MHz, MeOH-d 4) H 7.53 (1H, t, J = 8.3 Hz, H-11), (5H, m, Ar- MPTA), 7.30 (1H, d, J = 8.3 Hz, H-12), 7.29 (1H, d, J = 8.3 Hz, H-10), 7.18 (1H, br s, H-2), 6.96 (1H, br s, H-4), 5.55 (1H, m, H-33), 4.65 (1H, dd, J = 10.2, 4.3 Hz, H-17), 4.49 (1H, dd, J = 6.8, 6.0 Hz, H- 28), 4.32 (1H, dd, J = 9.6, 4.2 Hz, H-23), 3.51 (3H, s, OMe-MTPA), 2.76 (1H, dd, J = 16.9, 6.0 Hz, H-29), 2.72 (1H, dd, J = 16.9, 6.8 Hz, H-29), 2.65 (2H, t, J = 6.2 Hz, H-32), 2.46 (2H, t, J = 7.9 Hz, H-25), 2.34 (3H, s, H-14), 2.15 (1H, dddd, J = 14.2, 7.9, 7.9, 4.2 Hz, H-24), 2.04 (3H, s, 9-OAc), 2.02 (3H, s, 13-OAc), 1.85 (1H, dddd, J = 14.2, 9.6, 7.9, 7.9 Hz, H-24), 1.76 (1H, ddd, J = 15.1, 10.2, 4.8 Hz, H-18), 1.73 (2H, m, H-34), 1.55 (3H, s, 5-OAc), 1.50 (1H, m, H-19), 1.36 (2H, m, H-35), 1.32 (1H, m, H-18), (12H, m, H-36 H-41), 0.89 (3H, t, J = 7.1 Hz, H-42), 0.79 (3H, d, J = 6.9 Hz, H-20), 0.73 (3H, d, J = 6.5 Hz, H-21); ESIMS m/z , [M+H] + (Calcd for C 58H 73F 3N 5O ). 3aR: 1 H NMR (600 MHz, MeOH-d 4) H 7.53 (1H, t, J = 8.3 Hz, H-11), (5H, m, Ar- MPTA), 7.30 (1H, d, J = 8.3 Hz, H-12), 7.29 (1H, d, J = 8.3 Hz, H-10), 7.18 (1H, br s, H-2), 6.96 (1H, br s, H-4), 5.53 (1H, m, H-33), 4.65 (1H, dd, J = 10.2, 4.3 Hz, H-17), 4.63 (1H, dd, J = 7.6, 5.6 Hz, H- 28), 4.33 (1H, dd, J = 9.6, 4.4 Hz, H-23), 3.55 (3H, s, OMe-MTPA), 2.95 (1H, dd, J = 17.2, 5.6 Hz, H-29), 2.88 (1H, dd, J = 17.2, 7.6 Hz, H-29), 2.70 (2H, t, J = 6.4 Hz, H-32), 2.47 (2H, t, J = 7.9 Hz, H-25), 2.34 (3H, s, H-14), 2.15 (1H, dddd, J = 14.2, 8.0, 6.9, 44 Hz, H-24), 2.04 (3H, s, 9-OAc), 2.02 (3H, s, 13-OAc), 1.86 (1H, dddd, J = 14.2, 9.6, 8.0, 6.9 Hz, H-24), 1.76 (1H, ddd, J = 15.0, 10.2, 4.8 Hz, H-18), 1.63 (2H, m, H-34), 1.55 (3H, s, 5-OAc), 1.50 (1H, m, H-19), 1.32 (1H, m, H-18), (12H, m, H-36 H-41), 1.19 (2H, m, H-35), 0.89 (3H, t, J = 7.1 Hz, H-42), 0.78 (3H, d, J = 6.9 8

9 Hz, H-20), 0.74 (3H, d, J = 6.5 Hz, H-21); LRESIMS m/z , [M+H] + (Calcd for C 58H 73F 3N 5O ). 4aS: 1 H NMR (600 MHz, MeOH-d 4) H 7.53 (1H, t, J = 8.3 Hz, H-11), (5H, m, Ar- MPTA), 7.29 (1H, d, J = 8.3 Hz, H-12), 7.30 (1H, d, J = 8.3 Hz, H-10), 7.19 (1H, br s, H-2), 6.96 (1H, br s, H-4), 5.53 (1H, m, H-33), 4.64 (1H, dd, J = 10.1, 4.4 Hz, H-17), 4.50 (1H, dd, J = 7.2, 5.9 Hz, H- 28), 4.31 (1H, dd, J = 9.7, 4.6 Hz, H-23), 3.50 (3H, s, OMe-MTPA), 2.74 (1H, dd, J = 17.0, 5.9 Hz, H-29), 2.67 (1H, dd, J = 17.0, 7.2 Hz, H-29), 2.64 (1H, dd, J = 15.2, 5.0 Hz, H-32), 2.58 (1H, dd, J = 15.2, 8.4 Hz, H-32), 2.46 (2H, overlap, H-25), 2.34 (3H, s, H-14), 2.13 (1H, dddd, J = 14.2, 7.9, 6.4, 4.6 Hz, H-24), 2.05 (3H, s, 9-OAc), 2.03 (3H, s, 13-OAc), 1.86 (1H, dddd, J = 14.2, 9.7, 7.9, 6.4 Hz, H-24), 1.73 (1H, m, H-18), 1.73 (2H, m, H-34), 1.56 (3H, s, 5-OAc), 1.50 (1H, m, H-19), 1.35 (2H, m, H-35), 1.33 (1H, m, H-18), (12H, m, H-36 H-41), 0.89 (3H, t, J = 7.0 Hz, H-42), 0.78 (3H, d, J = 6.8 Hz, H-20), 0.73 (3H, d, J = 6.6 Hz, H-21); ESIMS m/z , [M+H] + (Calcd for C 58H 73F 3N 5O ). 4aR: 1 H NMR (600 MHz, MeOH-d 4) H 7.53 (1H, t, J = 8.3 Hz, H-11), (5H, m, Ar- MPTA), 7.29 (1H, d, J = 8.3 Hz, H-12), 7.29 (1H, d, J = 8.3 Hz, H-10), 7.19 (1H, br s, H-2), 6.96 (1H, br s, H-4), 5.51 (1H, m, H-33), 4.65 (1H, dd, J = 10.1, 4.4 Hz, H-17), 4.63 (1H, dd, J = 8.0, 5.6 Hz, H- 28), 4.31 (1H, dd, J = 9.7, 4.6 Hz, H-23), 3.56 (3H, s, OMe-MTPA), 2.91 (1H, dd, J = 17.2, 5.6 Hz, H-29), 2.82 (1H, dd, J = 17.2, 8.0 Hz, H-29), 2.69 (1H, dd, J = 15.4, 8.4 Hz, H-32), 2.62 (1H, dd, J = 15.4, 5.0 Hz, H-32), 2.48 (2H, overlap, H-25), 2.33 (3H, s, H-14), 2.14 (1H, dddd, J = 14.3, 8.0, 6.5, 4.6 Hz, H-24), 2.05 (3H, s, 9-OAc), 2.03 (3H, s, 13-OAc), 1.87 (1H, dddd, J = 14.3, 9.7, 8.0, 6.5 Hz, H-24), 1.73 (1H, ddd, J = 15.0, 10.1, 5.0 Hz, H-18), 1.62 (2H, m, H-34), 1.56 (3H, s, 5-OAc), 1.50 (1H, m, H-19), 1.32 (1H, m, H-18), (12H, m, H-36 H-41), 1.18 (2H, m, H-35), 0.89 (3H, t, J = 7.0 Hz, H-42), 0.78 (3H, d, J = 6.8 Hz, H-20), 0.73 (3H, d, J = 6.6 Hz, H-21); LRESIMS m/z , [M+H] + (Calcd for C 58H 73F 3N 5O ). Advanced Marfey s Analysis of Compounds 1 and 2. Asperphenin A (1, 1.0 mg) was dissolved in 6 N HCl (0.5 ml) and heated at 110 o C for 12 h. The solution was evaporated with distilled water three times to remove the trace of HCl under vacuum. The divided hydrolysate (0.5 mg) was treated with 1 N NaHCO 3 (100 μl) followed by 1% L- or D- FDAA (50 μl) in acetone. The mixture was stirred at 80 o C for 5 min. After the reaction was quenched by the addition of 2 N HCl (50 μl), the mixture was analyzed by ESI-LC/MS to assign the chirality of the amino acids. The retention times of the L- and D-FDAA-derivatized hydrolysates were 10.7 and 11.4 min for L- and D-aspartic acid, respectively; and the retention times of the L- and D-FDAA-derivatized hydrolysates were 11.9 and 12.9 min for L- and D-glutamic acid, respectively. Compound 2 was prepared and analyzed using the same procedure. The results demonstrated that all the amino acids in compounds 1 and 2 were L-form 9

10 (Figure S3). Reduction of 7-ketone of Compounds 1 and 2. To a solution of compound 1 (22.1 mg) in MeOH (2 ml) at 0 o C was added NaBH 4 (1.2 mg). After stirring for 30 min, the reaction was quenched by adding 1 N HCl and dried under vacuum. The residue was purified by reverse-phase HPLC (YMC- ODS column, mm; H 2O-MeOH, 45:55, 1.0 ml/min) to afford the 7-hydroxy derivatives 5 (8.1 mg) and 6 (8.7 mg). The corresponding 7-hydroxy derivatives 7 (8.2 mg) and 8 (8.0 mg) were prepared in the same procedure from compound 2 (24.5 mg). 7-Hydroxyasperphenin A (5): 1 H NMR (600 MHz, DMSO-d 6) H 8.16 (1H, d, J = 7.7 Hz, 28-NH), 8.10 (1H, d, J = 7.5 Hz, 17-NH), 7.92 (1H, d, J = 8.8 Hz, 23-NH), 7.45 (1H, br s, 30-NH), 7.24 (1H, br s, 26-NH), 7.03 (1H, br s, 30-NH), 6.77 (1H, br s, 26-NH), 6.59 (1H, t, J = 8.1 Hz, H-11), 6.37 (1H, br s, H-2), 6.35 (1H, br s, H-4), 6.05 (1H, s, H-7), 5.93 (1H, d, J = 8.1 Hz, H-12), 5.88 (1H, d, J = 8.1 Hz, H-10), 4.66 (1H, br s, 33-OH), 4.48 (1H, ddd, J =7.7, 7.0, 6.5 Hz, H-28), 4.27 (1H, m, H-17), 4.08 (1H, m, H-23), 3.77 (1H, m, H-33), 3.08 (1H, dd, J = 17.1, 9.3 Hz, H-16), 2.75 (1H, dd, J = 17.1, 3.2 Hz, H-16), 2.54 (1H, dd, J = 15.4, 7.0 Hz, H-29), 2.42 (1H, dd, J = 15.4, 6.5 Hz, H-29), 2.20 (2H, m, H-34), 2.12 (3H, s, H-14), 2.08 (2H, overlap, H-25), 1.94 (1H, m, H-24), 1.72 (1H, m, H-24), 1.59 (1H, m, H-19), 1.53 (1H, m, H-18), 1.37 (1H, m, H-18), (16H, m, H-34 H-41), 0.87 (3H, d, J = 6.8 Hz, H-20), 0.86 (3H, d, J = 6.8 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-42); 13 C NMR (600 MHz, DMSO-d 6) C (C-15), (C-26), (C-30), (C-31), (C-27), (C-22), (C-9), (C-13), (C-5), (C-3), (C-1), (C-11), (C-6), (C- 4), (C-8), (C-2), (C-12), (C-10), 67.5 (C-33), 63.4 (C-7), 52.7 (C-23), 49.8 (C-28), 48.0 (C-16), 44.1 (C-17), 43.4 (C-32), 43.2 (C-18), 37.2 (C-34), 37.0 (C-29), 31.5 (C-25), 31.3 (C-36), 29.2 (C-37), 29.2 (C-38), 29.0 (C-39), 28.8 (C-40), 27.5 (C-24), 25.2 (C-35), 24.4 (C-19), 23.6 (C-20), 22.1 (C-41), 21.4 (C-21), 20.8 (C-14), 14.0 (C-42); HRTOFMS m/z , [M- H 2O+H] + (Calcd for C 42H 62N 5O ). 7-epi-Hydroxyasperphenin A (6): 1 H NMR (600 MHz, DMSO-d 6) H 8.17 (1H, d, J = 7.6 Hz, 28- NH), 8.08 (1H, d, J = 7.5 Hz, 17-NH), 7.73 (1H, d, J = 8.5 Hz, 23-NH), 7.47 (1H, br s, 30-NH), 7.19 (1H, br s, 26-NH), 7.05 (1H, br s, 30-NH), 6.74 (1H, br s, 26-NH), 6.60 (1H, t, J = 7.8 Hz, H-11), 6.49 (1H, br s, H-2), 6.37 (1H, br s, H-4), 6.09 (1H, s, H-7), 5.93 (1H, d, J = 8.1 Hz, H-12), 5.90 (1H, d, J = 8.1 Hz, H-10), 4.63 (1H, br s, 33-OH), 4.50 (1H, ddd, J =7.6, 7.2, 5.7 Hz, H-28), 4.36 (1H, m, H-17), 4.07 (1H, m, H-23), 3.77 (1H, m, H-33), 3.19 (1H, dd, J = 16.6, 6.2 Hz, H-16), 2.86 (1H, dd, J = 16.6, 7.4 Hz, H-16), 2.56 (1H, dd, J = 15.4, 7.2 Hz, H-29), 2.44 (1H, dd, J = 15.4, 5.7 Hz, H-29), 2.20 (2H, m, H-34), 2.13 (3H, s, H-14), 2.06 (2H, overlap, H-25), 1.97 (1H, m, H-24), 1.70 (1H, m, H-24), 1.55 (1H, m, H-19), 1.53 (1H, m, H-18), 1.41 (1H, m, H-18), (16H, m, H-34 H-41), 0.86 (3H, d, J = 6.8 Hz, H-20), 0.85 (3H, d, J = 6.8 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-42); 13 C 10

11 NMR (600 MHz, DMSO-d 6) C (C-15), (C-26), (C-30), (C-31), (C-27), (C-22), (C-9), (C-13), (C-5), (C-3), (C-1), (C-11), (C-6), (C-4), (C-8), (C-2), (C-12), (C-10), 67.5 (C-33), 63.7 (C-7), 52.7 (C-23), 49.8 (C-28), 48.7 (C-16), 44.1 (C-17), 43.4 (C-32), 43.4 (C-18), 37.2 (C-34), 37.0 (C-29), 31.5 (C-25), 31.3 (C-36), 29.2 (C-37), 29.2 (C-38), 29.0 (C-39), 28.8 (C-40), 27.4 (C-24), 25.2 (C-35), 24.4 (C-19), 23.6 (C-20), 22.1 (C-41), 21.8 (C-21), 20.8 (C-14), 14.0 (C-42); HRTOFMS m/z , [M-H 2O+H] + (Calcd for C 42H 62N 5O ). 7-Hydroxyasperphenin B (7): 1 H NMR (600 MHz, DMSO-d 6) H 8.18 (1H, d, J = 7.7 Hz, 28-NH), 8.13 (1H, d, J = 7.3 Hz, 17-NH), 7.77 (1H, d, J = 8.8 Hz, 23-NH), 7.44 (1H, br s, 30-NH), 7.21 (1H, br s, 26-NH), 7.04 (1H, br s, 30-NH), 6.75 (1H, br s, 26-NH), 6.58 (1H, t, J = 8.1 Hz, H-11), 6.36 (1H, br s, H-2), 6.31 (1H, br s, H-4), 6.05 (1H, s, H-7), 5.92 (1H, d, J = 8.1 Hz, H-12), 5.88 (1H, d, J = 8.1 Hz, H-10), 4.66 (1H, br s, 33-OH), 4.50 (1H, ddd, J =7.7, 6.9, 6.3 Hz, H-28), 4.27 (1H, m, H-17), 4.07 (1H, m, H-23), 3.79 (1H, m, H-33), 3.10 (1H, dd, J = 17.7, 9.2 Hz, H-16), 2.69 (1H, dd, J = 17.7, 3.3 Hz, H-16), 2.56 (1H, dd, J = 15.6, 6.9 Hz, H-29), 2.45 (1H, dd, J = 15.6, 6.3 Hz, H-29), 2.21 (2H, d, J = 6.5 Hz, H-34), 2.11 (3H, s, H-14), 2.08 (2H, overlap, H-25), 1.95 (1H, m, H-24), 1.74 (1H, m, H-24), 1.64 (1H, m, H-19), 1.55 (1H, m, H-18), 1.40 (1H, m, H-18), (16H, m, H-34 H-41), 0.89 (3H, d, J = 6.8 Hz, H-20), 0.88 (3H, d, J = 6.8 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-42); 13 C NMR (150 MHz, DMSO-d 6) C (C-15), (C-26), (C-30), (C-31), (C-27), (C-22), (C-9), (C-13), (C-5), (C-3), (C-1), (C-11), (C-6), (C-4), (C-8), (C-2), (C-12), (C-10), 67.4 (C-33), 63.5 (C-7), 52.8 (C-23), 49.8 (C-28), 47.7 (C-16), 43.9 (C-17), 43.4 (C-32), 43.0 (C-18), 37.0 (C-34), 36.9 (C-29), 31.5 (C-25), 31.3 (C-36), 29.1 (C-37), 29.1 (C-38), 29.0 (C-39), 28.7 (C-40), 27.5 (C-24), 25.1 (C-35), 24.2 (C-19), 23.5 (C-20), 22.1 (C-41), 21.4 (C-21), 20.8 (C-14), 14.0 (C-42); HRTOFMS m/z , [M-H 2O+H] + (Calcd for C 42H 62N 5O ). 7-epi-Hydroxyasperphenin B (8): 1 H NMR (600 MHz, DMSO-d 6) H 8.13 (1H, d, J = 7.7 Hz, 28- NH), 8.09 (1H, d, J = 7.3 Hz, 17-NH), 7.64 (1H, d, J = 8.8 Hz, 23-NH), 7.44 (1H, br s, 30-NH), 7.15 (1H, br s, 26-NH), 7.09 (1H, br s, 30-NH), 6.71 (1H, br s, 26-NH), 6.59 (1H, t, J = 8.1 Hz, H-11), 6.46 (1H, br s, H-2), 6.36 (1H, br s, H-4), 6.08 (1H, s, H-7), 5.93 (1H, d, J = 8.1 Hz, H-12), 5.89 (1H, d, J = 8.1 Hz, H-10), 4.61 (1H, br s, 33-OH), 4.52 (1H, ddd, J =7.7, 7.5, 6.5 Hz, H-28), 4.36 (1H, m, H-17), 4.07 (1H, m, H-23), 3.77 (1H, m, H-33), 3.18 (1H, dd, J = 16.5, 6.5 Hz, H-16), 2.89 (1H, dd, J = 16.5, 7.2 Hz, H-16), 2.61 (1H, dd, J = 15.9, 7.5 Hz, H-29), 2.45 (1H, dd, J = 15.9, 6.5 Hz, H-29), 2.20 (2H, d, J = 6.5 Hz, H-34), 2.13 (3H, s, H-14), 2.05 (2H, overlap, H-25), 1.96 (1H, m, H-24), 1.68 (1H, m, H-24), 1.58 (2H, m, H-18, H-19), 1.42 (1H, m, H-18), (16H, m, H-34 H-41), 0.87 (3H, d, J = 6.8 Hz, H-20), 0.86 (3H, d, J = 6.8 Hz, H-21), 0.85 (3H, t, J = 7.0 Hz, H-42); 13 C NMR 11

12 (150 MHz, DMSO-d 6) C (C-15), (C-26), (C-30), (C-31), (C-27), (C-22), (C-9), (C-13), (C-5), (C-3), (C-1), (C-11), (C-6), (C-4), (C-8), (C-2), (C-12), (C-10), 67.4 (C-33), 63.6 (C-7), 52.7 (C- 23), 49.7 (C-28), 47.2 (C-16), 44.2 (C-17), 43.6 (C-32), 43.4 (C-18), 37.0 (C-34), 36.9 (C-29), 31.7 (C-25), 31.3 (C-36), 29.1 (C-37), 29.1 (C-38), 29.0 (C-39), 28.7 (C-40), 27.6 (C-24), 25.0 (C-35), 24.2 (C-19), 23.6 (C-20), 22.1 (C-41), 21.7 (C-21), 20.8 (C-14), 14.0 (C-42); HRTOFMS m/z , [M-H 2O+H] + (Calcd for C 42H 62N 5O ). Reduction of 15-ketone of Compounds 5 and 7. To a solution of compound 5 (8.1 mg) in MeOH (2 ml) at 0 o C was added NaBH 4 (0.6 mg). Then the reaction was kept in room temperature for 5 min and quenched by adding 1 N HCl to adjust ph 7.0, and dried under vacuum. The residue was purified by reverse-phase HPLC (YMC-ODS column, mm; H 2O-MeCN, 70:30 to 50:50, 0-20 min, 1.0 ml/min) to afford the 7,15-dihydroxy derivatives 9 (2.1 mg) and 10 (2.2 mg). The corresponding dihydroxy derivatives 11 (2.4 mg) and 12 (2.5 mg) were prepared in the same procedure from 7 (8.2 mg). 7, 15(S)-Dihydroxyasperphenin A (9): 1 H NMR (900 MHz, MeOH-d 4) H 6.77 (1H, t, J = 8.0 Hz, H-11), 6.63 (1H, br s, H-2), 6.45 (1H, br s, H-4), 6.29 (1H, s, H-7), 6.24 (1H, d, J = 8.0 Hz, H-12), 6.13 (1H, d, J = 8.0 Hz, H-10), 5.53 (1H, dd, J = 10.0, 3.3 Hz, H-15), 4.64 (1H, t, J = 6.4 Hz, H-28), 4.29 (1H, dd, J = 10.0, 4.1 Hz, H-23), 4.23 (1H, dddd, J = 10.5, 7.3, 6.9, 4.0 Hz, H-17), 3.96 (1H, m, H-33), 2.80 (1H, dd, J = 15.8, 6.4 Hz, H-29), 2.75 (1H, dd, J = 15.8, 6.4 Hz, H-29), 2.45 (1H, dd, J = 14.1, 4.3 Hz, H-32), 2.33 (2H, overlap, H-25), 2.32 (1H, dd, J = 14.1, 6.8 Hz, H-32), 2.23 (1H, m, H- 24), 2.18 (3H, s, H-14), 2.02 (1H, ddd, J = 14.1, 10.0, 6.9 Hz, H-16), 1.94 (1H, m, H-24), 1.75 (1H, ddd, J = 14.1, 7.3, 3.3 Hz, H-16), 1.60 (1H, m, H-19), 1.51 (1H, ddd, J = 14.1, 10.5, 4.5 Hz, H-18), 1.45 (2H, m, H-34), 1.40 (1H, ddd, J = 14.1, 9.7, 4.0 Hz, H-18), (14H, m, H-35 H-41), 0.93 (3H, t, J = 7.1 Hz, H-42), 0.88 (3H, d, J = 6.9 Hz, H-20), 0.87 (3H, d, J = 6.9 Hz, H-21); 13 C NMR (200 MHz, MeOH-d 4) C (C-26), (C-30), (C-31), (C-27), (C-22), (C-5), (C-13), (C-9), 141.1(C-3), (C-1), (C-11), (C-6), (C- 2), (C-4), (C-8), (C-10), (C-12), 69.8 (C-33), 68.2 (C-15), 66.6 (C-7), 54.7 (C-23), 52.1 (C-28), 47.8 (C-17), 44.5 (C-32), 44.2 (C-18), 44.2 (C-16), 38.3 (C-34), 37.2 (C-29), 33.1 (C-40), 32.8 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.4 (C-24), 26.7 (C-35), 25.8 (C-19), 23.9 (C-41), 23.7 (C-21), 22.2 (C-20), 21.4 (C-14), 14.4 (C-42); HRTOFMS m/z , [M+Na] + (Calcd for C 42H 65N 5O 10 Na ). 7, 15(R)-Dihydroxyasperphenin A (10): 1 H NMR (900 MHz, MeOH-d 4) H 6.77 (1H, t, J = 8.0 Hz, H-11), 6.63 (1H, br s, H-2), 6.45 (1H, br s, H-4), 6.27 (1H, s, H-7), 6.24 (1H, d, J = 8.0 Hz, H-12), 6.11 (1H, d, J = 8.0 Hz, H-10), 5.38 (1H, dd, J = 10.6, 2.1 Hz, H-15), 4.65 (1H, dd, J = 7.2, 6.3 Hz, H- 12

13 28), 4.32 (1H, dd, J = 9.4, 4.3 Hz, H-23), 4.29 (1H, dddd, J = 9.8, 9.8, 4.3, 4.3 Hz, H-17), 3.94 (1H, m, H-33), 2.79 (1H, dd, J = 15.9, 7.2 Hz, H-29), 2.68 (1H, dd, J = 15.9, 6.3 Hz, H-29), 2.43 (1H, dd, J = 14.4, 4.2 Hz, H-32), 2.33 (2H, overlap, H-25), 2.30 (1H, dd, J = 14.4, 8.6 Hz, H-32), 2.23 (1H, m, H- 24), 2.18 (3H, s, H-14), 2.03 (1H, m, H-24), 1.94 (1H, ddd, J = 14.3, 10.6, 4.3 Hz, H-16), 1.72 (1H, ddd, J = 14.3, 9.8, 2.1 Hz, H-16), 1.66 (1H, m, H-19), 1.54 (1H, ddd, J = 14.4, 10.4, 4.3 Hz, H-18), 1.41 (1H, ddd, J = 14.4, 9.8, 4.4 Hz, H-18), 1.45 (2H, m, H-34), (14H, m, H-35 H-41), 0.92 (3H, d, J = 6.7 Hz, H-20), 0.91 (3H, d, J = 6.7 Hz, H-21), 0.89 (3H, t, J = 7.2 Hz, H-42); 13 C NMR (150 MHz, MeOH-d 4) C (C-26), (C-30), (C-31), (C-27), (C-22), (C-5), (C-13), (C-9), (C-3), (C-1), (C-11), (C-6), (C- 2), (C-4), (C-8), (C-10), (C-12), 69.8 (C-33), 67.4 (C-15), 66.6 (C-7), 55.0 (C-23), 51.8 (C-28), 47.4 (C-17), 45.4 (C-32), 44.6 (C-18), 44.6 (C-16), 38.3 (C-34), 37.6 (C-29), 33.1 (C-40), 32.6 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.7 (C-24), 26.7 (C-35), 25.9 (C-19), 23.9 (C-41), 23.7 (C-21), 22.1 (C-20), 21.4 (C-14), 14.4 (C-42); HRTOFMS m/z , [M+Na] + (Calcd for C 42H 65N 5O 10 Na ). 7, 15(S)-Dihydroxyasperphenin B (11): 1 H NMR (900 MHz, MeOH-d 4) H 6.76 (1H, t, J = 8.1 Hz, H-11), 6.60 (1H, br s, H-2), 6.45 (1H, br s, H-4), 6.28 (1H, s, H-7), 6.23 (1H, d, J = 8.1 Hz, H-12), 6.18 (1H, d, J = 8.1 Hz, H-10), 5.50 (1H, dd, J = 10.7, 2.1 Hz, H-15), 4.66 (1H, t, J = 6.4 Hz, H-28), 4.38 (1H, dd, J = 10.2, 3.6 Hz, H-23), 4.28 (1H, dddd, J = 9.5, 9.4, 4.9, 4.2 Hz, H-17), 3.98 (1H, m, H-33), 2.80 (1H, dd, J = 15.7, 6.4 Hz, H-29), 2.75 (1H, dd, J = 15.7, 6.4 Hz, H-29), 2.44 (1H, dd, J = 14.3, 4.1 Hz, H-32), 2.34 (1H, dd, J = 14.3, 8.6 Hz, H-32), 2.33 (2H, overlap, H-25), 2.31 (1H, m, H- 24), 2.18 (3H, s, H-14), 1.97 (1H, ddd, J = 14.7, 10.7, 4.2 Hz, H-16), 1.95 (1H, m, H-24), 1.69 (1H, ddd, J = 14.7, 9.4, 2.1 Hz, H-16), 1.68 (1H, m, H-19), 1.64 (1H, ddd, J = 14.2, 10.4, 4.9 Hz, H-18), 1.48 (1H, m, H-34), 1.46 (1H, m, H-34), 1.44 (1H, ddd, J = 14.2, 9.5, 4.5 Hz, H-18), (14H, m, H-35 H-41), 0.94 (3H, d, J = 6.5 Hz, H-20), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.89 (3H, t, J = 6.5 Hz, H-42); 13 C NMR (150 MHz, MeOH-d 4) C (C-26), (C-30), (C-31), (C- 27), (C-22), (C-5), (C-13), (C-9), (C-3), (C-1), (C-11), (C-6), (C-2), (C-4), (C-8), (C-10), (C-12), 69.8 (C-33), 67.2 (C- 15), 66.7 (C-7), 54.8 (C-23), 51.9 (C-28), 47.3 (C-17), 45.3 (C-32), 44.9 (C-18), 44.6 (C-16), 38.4 (C- 34), 37.4 (C-29), 33.1 (C-40), 32.8 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.4 (C-24), 26.7 (C-35), 26.1 (C-19), 24.0 (C-41), 23.7 (C-21), 22.2 (C-20), 21.4 (C-14), 14.4 (C-42); HRTOFMS m/z , [M+Na] + (Calcd for C 42H 65N 5O 10 Na ). 7, 15(R)-Dihydroxyasperphenin B (12): 1 H NMR (900 MHz, MeOH-d 4) H 6.77 (1H, t, J = 8.0 Hz, H-11), 6.61 (1H, br s, H-2), 6.46 (1H, br s, H-4), 6.32 (1H, s, H-7), 6.24 (1H, d, J = 8.0 Hz, H-12), 6.13 (1H, d, J = 8.0 Hz, H-10), 5.48 (1H, dd, J = 9.2, 4.4 Hz, H-15), 4.65 (1H, t, J = 6.4 Hz, H-28), 13

14 4.26 (1H, dd, J = 9.8, 4.2 Hz, H-23), 4.15 (1H, dddd, J = 10.1, 7.2, 6.8, 4.7 Hz, H-17), 3.98 (1H, m, H-33), 2.80 (1H, dd, J = 15.8, 6.4 Hz, H-29), 2.74 (1H, dd, J = 15.8, 6.4 Hz, H-29), 2.44 (1H, dd, J = 14.3, 4.1 Hz, H-32), 2.33 (1H, dd, J = 14.3, 8.6 Hz, H-32), 2.31 (2H, overlap, H-25), 2.19 (1H, m, H- 24), 2.18 (3H, s, H-14), 2.03 (1H, ddd, J = 14.2, 9.2, 7.2 Hz, H-16), 1.91 (1H, m, H-24), 1.81 (1H, ddd, J = 14.2, 6.8, 4.4 Hz, H-16), 1.59 (1H, m, H-19), 1.50 (1H, ddd, J = 13.7, 10.1, 4.3 Hz, H-18), 1.48 (1H, m, H-34), 1.44 (1H, m, H-34), 1.40 (1H, ddd, J = 13.7, 9.1, 4.7 Hz, H-18), (14H, m, H-35 H-41), 0.89 (3H, t, J = 6.5 Hz, H-42), 0.89 (3H, d, J = 6.5 Hz, H-20), 0.86 (3H, d, J = 6.5 Hz, H-21); 13 C NMR (150 MHz, MeOH-d 4) C (C-26), (C-30), (C-31), (C- 27), (C-22), (C-5), (C-13), (C-9), (C-3), (C-1), (C-11), (C-6), (C-2), (C-4), (C-8), (C-10), (C-12), 69.8 (C-33), 68.5 (C- 15), 66.7 (C-7), 55.0 (C-23), 52.0 (C-28), 47.7 (C-17), 44.5 (C-32), 44.4 (C-18), 44.0 (C-16), 38.4 (C- 34), 37.4 (C-29), 33.1 (C-40), 32.8 (C-25), 30.8 (C-36), 30.7 (C-37), 30.7 (C-38), 30.5 (C-39), 28.6 (C-24), 26.7 (C-35), 26.0 (C-19), 24.0 (C-41), 23.7 (C-21), 22.4 (C-20), 21.4 (C-14), 14.4 (C-42); HRTOFMS m/z , [M+Na] + (Calcd for C 42H 65N 5O 10 Na ). Computational Analysis. The ground-state geometries were optimized with density functional theory (DFT) calculations using Turbomole 6.5 with the basis set def-sv(p) for all atoms at the DFT level, using the B3LYP functional; the ground states were further confirmed by a harmonic frequency calculation. The calculated ECD data corresponding to the optimized structures were obtained using TDDFT with the basis set def2-tzvpp for all atoms at the DFT level, using the B3LYP functional. The ECD spectra were simulated by overlapping for each traction, where σ is the width of the band at 1/e height. Ei and Ri are the excitation energies and rotatory strengths for transition i, respectively. In the current work, the value of σ was fixed at 0.10 ev. 14

15 Table S1. Inhibition of cancer cell proliferation by asperphenins and synthetic derivatives Compounds IC 50 ( M), 72 h RKO SNU638 SK-HEP-1 MDA-MB- Asperphenin A (1) Asperphenin B (2) Etoposide a a Etoposide was used as a positive control. Figure S1. The δ H (δ4as-δ4ar) values of MTPA esterifications for triacetyl-cycloasperphenin B (4a) 15

16 16

17 Figure S2. The LC/MS spectra of Advanced Marfey s analysis of compounds 1 and 2 (compound 1- D-FDAA (A), compound 1-L-FDAA (B), compound 2-D-FDAA (C), and compound 2-L-FDAA (D)) 17

18 Figure S3. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin A (9) at C-15 and C-16 18

19 Figure S4. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin A (9) at C-16 and C-17 19

20 Figure S5. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin A (10) at C-15 and C-16 20

21 Figure S6. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin A (10) at C-16 and C-17 21

22 Figure S7. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin B (11) at C-15 and C-16 22

23 Figure S8. J-based configuration analysis of 7,15(S)-dihydroxyasperphenin B (11) at C-16 and C-17 23

24 Figure S9. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin B (12) at C-15 and C-16 24

25 Figure S10. J-based configuration analysis of 7,15(R)-dihydroxyasperphenin B (12) at C-16 and C-17 25

26 Figure S11. Experimental CD spectra of asperphenins A (1) and B (2) 26

27 Figure S12. The 1 H NMR (600 MHz, DMSO-d 6) spectrum of asperphenin A (1) 27

28 Figure S13. The 13 C NMR (150 MHz, DMSO-d 6) spectrum of asperphenin A (1) Figure S14. The DEPT135 (125 MHz, DMSO-d 6) spectrum of asperphenin A (1) 28

29 Figure S15. The HSQC (600 MHz, DMSO-d 6) spectrum of asperphenin A (1) Figure S16. The COSY (600 MHz, DMSO-d 6) spectrum of asperphenin A (1) 29

30 Figure S17. The TOCSY (600 MHz, DMSO-d 6) spectrum of asperphenin A (1) Figure S18. The HMBC (600 MHz, DMSO-d 6) spectrum of asperphenin A (1) 30

31 Figure S19. The 1 H NMR (600 MHz, DMSO-d 6) spectrum of asperphenin B (2) 31

32 Figure S20. The 13 C NMR (150 MHz, DMSO-d 6) spectrum of asperphenin B (2) Figure S21. The DEPT135 (150 MHz, DMSO-d 6) spectrum of asperphenin B (2) 32

33 Figure S22. The HSQC (600 MHz, DMSO-d 6) spectrum of asperphenin B (2) Figure S23. The COSY (600 MHz, DMSO-d 6) spectrum of asperphenin B (2) 33

34 Figure S24. The TOCSY (600 MHz, DMSO-d 6) spectrum of asperphenin B (2) Figure S25. The HMBC (600 MHz, DMSO-d 6) spectrum of asperphenin B (2) 34

35 Figure S26. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of cycloasperphenin A (3) Figure S27. The 13 C NMR (150 MHz, MeOH-d 4) spectrum of cycloasperphenin A (3) 35

36 Figure S28. The HSQC (600 MHz, MeOH-d 4) spectrum of cycloasperphenin A (3) Figure S29. The COSY (600 MHz, MeOH-d 4) spectrum of cycloasperphenin A (3) 36

37 Figure S30. The HMBC (600 MHz, MeOH-d 4) spectrum of cycloasperphenin A (3) Figure S31. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of cycloasperphenin B (4) 37

38 Figure S32. The 13 C NMR (150 MHz, MeOH-d 4) spectrum of cycloasperphenin B (4) Figure S33. The HSQC (600 MHz, MeOH-d 4) spectrum of cycloasperphenin B (4) 38

39 Figure S34. The COSY (600 MHz, MeOH-d 4) spectrum of cycloasperphenin B (4) Figure S35. The HMBC (600 MHz, MeOH-d 4) spectrum of cycloasperphenin B (4) 39

40 Figure S36. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of (S)-MTPA ester of triacetylcycloasperphenin A (3aS) Figure S37. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of (R)-MTPA ester of triacetylcycloasperphenin A (3aR) 40

41 Figure S38. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of (S)-MTPA ester of triacetylcycloasperphenin B (4aS) Figure S39. The 1 H NMR (600 MHz, MeOH-d 4) spectrum of (R)-MTPA ester of triacetylcycloasperphenin B (4aR) 41

42 Figure S40. The 1 H NMR (600 MHz, DMSO-d 6) spectrum of 7-hydroxyasperphenin A (5) Figure S41. The 13 C NMR (150 MHz, DMSO-d 6) spectrum of 7-hydroxyasperphenin A (5) 42

43 Figure S42. The 1 H NMR (600 MHz, DMSO-d 6) spectrum of 7-epi-hydroxyasperphenin A (6) Figure S43. The 13 C NMR (150 MHz, DMSO-d 6) spectrum of 7-epi-hydroxyasperphenin A (6) 43

44 Figure S44. The 1 H NMR (500 MHz, DMSO-d 6) spectrum of 7-hydroxyasperphenin B (7) Figure S45. The 13 C NMR (125 MHz, DMSO-d 6) spectrum of 7-hydroxyasperphenin B (7) 44

45 Figure S46. The 1 H NMR (500 MHz, DMSO-d 6) spectrum of 7-epi-hydroxyasperphenin B (8) Figure S47. The 13 C NMR (125 MHz, DMSO-d 6) spectrum of 7-epi-hydroxyasperphenin B (8) 45

46 Figure S48. The 1 H NMR (800 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (9) Figure S49. The 13 C NMR (150 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (9) 46

47 Figure S50. The HETLOC NMR (800 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (9) Figure S51. The ROESY (600 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (9) 47

48 Figure S52. The 1 H NMR (900 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (10) Figure S53. The 13 C NMR (125 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (10) 48

49 Figure S54. The HETLOC NMR (900 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (10) Figure S55. The ROESY (600 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (10) 49

50 Figure S56. The 1 H NMR (900 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (11) Figure S57. The 13 C NMR (125 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (11) 50

51 Figure S58. The HETLOC NMR (900 MHz, MeOH-d 4) spectrum of 7,15(S)-dihydroxyasperphenin A (11) Figure S59. The ROESY (600 MHz, DMSO-d 6) spectrum of 7,15(S)-dihydroxyasperphenin A (11) 51

52 Figure S60. The 1 H NMR (800 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (12) Figure S61. The 13 C NMR (150 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (12) 52

53 Figure S62. The HETLOC NMR (800 MHz, MeOH-d 4) spectrum of 7,15(R)-dihydroxyasperphenin A (12) Figure S63. The ROESY (600 MHz, DMSO-d 6) spectrum of 7,15(R)-dihydroxyasperphenin A (12) 53