Hawaiienols A D, Highly Oxygenated p-terphenyls from an Insect-Associated Fungus Paraconiothyrium hawaiiense Fengxia Ren,, Shenxi Chen,, Yang Zhang, Shuaiming Zhu, Junhai Xiao, Xingzhong Liu, Ruibin Su,*, and Yongsheng Che*, State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People s Republic of China State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People s Republic of China State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 300350, People s Republic of China Contents 1) Figure S1. 2) Figure S2. 1 H NMR spectrum of hawaiienol A (1; 600 MHz, acetone-d 6 ) 3 13 C NMR spectrum of hawaiienol A (1; 150 MHz, acetone-d 6 ) 4 Page 3) Figure S3. HSQC spectrum of hawaiienol A (1; 600 MHz, acetone-d 6 ) 5 4) Figure S4. 1 H 1 H COSY spectrum of hawaiienol A (1; 600 MHz, acetone-d 6 ) 6 5) Figure S5. HMBC spectrum of hawaiienol A (1; 600 MHz, acetone-d 6 ) 7 6) Figure S6. NOESY spectrum of hawaiienol A (1; 600 MHz, acetone-d 6 ) 8 7) Figure S7. 8) Figure S8. 1 H NMR spectrum of hawaiienol B (2; 600 MHz, acetone-d 6 ) 9 13 C NMR spectrum of hawaiienol B (2; 150 MHz, acetone-d 6 ) 10 9) Figure S9. HSQC spectrum of hawaiienol B (2; 600 MHz, acetone-d 6 ) 11 10) Figure S10. 1 H 1 H COSY spectrum of hawaiienol B (2; 600 MHz, acetone-d 6 ) 12 11) Figure S11. HMBC spectrum of hawaiienol B (2; 600 MHz, acetone-d 6 ) 13 12) Figure S12. NOESY spectrum of hawaiienol B (2; 600 MHz, acetone-d 6 ) 14 13) Figure S13. 14) Figure S14. 1 H NMR spectrum of hawaiienol C (3; 600 MHz, acetone-d 6 ) 15 13 C NMR spectrum of hawaiienol C (3; 150 MHz, acetone-d 6 ) 16 15) Figure S15. HSQC spectrum of hawaiienol C (3; 600 MHz, acetone-d 6 ) 17 16) Figure S16. 1 H 1 H COSY spectrum of hawaiienol C (3; 600 MHz, acetone-d 6 ) 18 17) Figure S17. HMBC spectrum of hawaiienol C (3; 600 MHz, acetone-d 6 ) 19 1
18) Figure S18. NOESY spectrum of hawaiienol C (3; 600 MHz, acetone-d 6 ) 20 19) Figure S19. 20) Figure S20. 1 H NMR spectrum of hawaiienol D (4; 600 MHz, acetone-d 6 ) 21 13 C NMR spectrum of hawaiienol D (4; 150 MHz, acetone-d 6 ) 22 21) Figure S21. HSQC spectrum of hawaiienol D (4; 600 MHz, acetone-d 6 ) 23 22) Figure S22. 1 H 1 H COSY spectrum of hawaiienol D (4; 600 MHz, acetone-d 6 ) 24 23) Figure S23. HMBC spectrum of hawaiienol D (4; 600 MHz, acetone-d 6 ) 25 24) Figure S24. NOESY spectrum of hawaiienol D (4; 600 MHz, acetone-d 6 ) 26 25) Figure S25. Experimental CD spectrum of 1 in MeOH 27 26) Figure S26. Relative configurations of 2 (the enantiomers were not shown) 28 27) Figure S27. Relative configurations of 4 (the enantiomers were not shown) 29 28) Figure S28. The optimized conformers for 2 30 29) Figure S29. The optimized conformers for 3 32 30) Figure S30. The optimized conformers for 4 33 31) Figure S31. The optimized conformers for 3a 37 32) Figure S32. Experimental CD spectrum of 2 in MeOH and the calculated ECD spectra of 2a 2h and 2a 2h 38 33) Figure S33. Experimental CD spectrum of 4 in MeOH and the calculated ECD spectra of 4a 4p and 4a 4p 39 34) Figure S34. Linear regression fitted 13 C NMR chemical shifts of 4 with four sets of experimental values (4i, 4l, 4n, and 4o) 40 35) Table S1. The experimental and computed 13 C NMR data of 3 41 36) Table S2. Statistics of Ordinary Least Squares (OLS) Linear Regression of the experimental and computed 13 C NMR chemical shifts of 3 42 37) Table S3. The experimental and computed 13 C NMR data of 4 43 38) Table S4. Statistics of Ordinary Least Squares (OLS) Linear Regression of the experimental and computed 13 C NMR chemical shifts of 4 44 39) Scheme S1. Plausible biosynthetic pathways for 1 4 45 Contributed equally to this work. * Corresponding authors. 2
Figure S1. 1 H NMR Spectrum of Hawaiienol A (1; 600 MHz, Acetone-d 6 ) 3
Figure S2. 13 C NMR Spectrum of Hawaiienol A (1; 150 MHz, Acetone-d 6 ) 4
Figure S3. HSQC Spectrum of Hawaiienol A (1; 600 MHz, Acetone-d 6 ) 4-OCH 3 3 -OCH 3 H-2,6 H-2,6 H-3,5 H-3,5 H-6 H-5 H-2 3 -OCH 3 4-OCH 3 C-2 C-6 C-5 C-3,5 C-3,5 C-2,6 C-2,6 5
Figure S4. 1 H 1 H COSY Spectrum of Hawaiienol A (1; 600 MHz, Acetone-d 6 ) H-2,6 H-2,6 H-3,5 H-3,5 H-6 H-5 H-2 OH-2 OH-2 H-2 H-5 H-6 H-3,5 H-3,5 H-2,6 H-2,6 6
Figure S5. HMBC Spectrum of Hawaiienol A (1; 600 MHz, Acetone-d 6 ) 4-OCH 3 3 -OCH 3 OH-4 H-2,6 H-2,6 H-3,5 H-3,5 H-6 H-5 OH-4 H-2 OH-2 3 -OCH 3 4-OCH 3 C-2 C-4,6 C-5 C-1 C-3,5 C-3,5 C-1,2,6 C-2,6,1 C-3 C-4 C-4 7
Figure S6. NOESY Spectrum of Hawaiienol A (1; 600 MHz, Acetone-d 6 ) 4-OCH 3 3 -OCH 3 OH-4 H-2,6 H-2,6 H-3,5 H-6 OH-4 H-3,5 H-5 H-2 OH-2 OH-2 3 -OCH 3 4-OCH 3 OH-4 H-2 H-5 H-6 H-3,5 H-3,5 H-2,6 H-2,6 OH-4 8
Figure S7. 1 H NMR Spectrum of Hawaiienol B (2; 600 MHz, Acetone-d 6 ) 9
Figure S8. 13 C NMR Spectrum of Hawaiienol B (2; 150 MHz, Acetone-d 6 ) 10
Figure S9. HSQC Spectrum of Hawaiienol B (2; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 3 -OCH 3 H-2,6 H-3,5,3,5 H-2,6 H-6 H-5 3 -OCH 3 4-OCH 3,4 -OCH 3 C-6 C-5 C-3,5 C-3,5 C-2,6 C-2,6 11
Figure S10. 1 H 1 H COSY Spectrum of Hawaiienol B (2; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 3 -OCH 3 H-2,6 H-2,6 H-3,5,3,5 H-6 H-5 OH-5 3 -OCH 3 4-OCH 3,4 -OCH 3 OH-5 H-5 H-6 H-3,5,3,5 H-2,6 H-2,6 12
Figure S11. HMBC Spectrum of Hawaiienol B (2; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 3 -OCH 3 H-2,6 H-2,6 H-3,5,3,5 OH-1 or4 H-6 H-5 OH-1 or4 OH-5 3 -OCH 3 4-OCH 3,4 -OCH 3 C-4 C-6,1 C-5 C-3,5,3,5,3 C-2,6,1,1,2,6 C-4,4 C-2 13
Figure S12. NOESY Spectrum of Hawaiienol B (2; 600 MHz, Acetone-d 6 ) H-2,6 H-3,5,3,5 H-2,6 4-OCH 3,4 -OCH 3 3 -OCH 3 H-6 H-5 OH-1 or4 OH-1 or4 OH-5 3 -OCH 3 4-OCH 3,4 -OCH 3 OH-5 OH-1 or4 H-5 H-6 OH-1 or4 H-3,5,3,5 H-2,6 H-2,6 14
Figure S13. 1 H NMR Spectrum of Hawaiienol C (3; 600 MHz, Acetone-d 6 ) 15
Figure S14. 13 C NMR Spectrum of Hawaiienol C (3; 150 MHz, Acetone-d 6 ) 16
Figure S15. HSQC Spectrum of Hawaiienol C (3; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 H-2,6,2,6 H-3,5,3,5 H-2,5 H-3,6 4-OCH 3,4 -OCH 3 C-2,5 C-3,6 C-3,5,3,5 C-2,6,2,6 17
Figure S16. 1 H 1 H COSY Spectrum of Hawaiienol C (3; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 H-2,6,2,6 H-3,5,3,5 H-2,5 OH-3,6 H-3,6 OH-2,5 4-OCH 3,4 -OCH 3 OH-2,5 H-3,6 H-2,5 OH-3,6 H-3,5,3,5 H-2,6,2,6 18
Figure S17. HMBC Spectrum of Hawaiienol C (3; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 H-2,6,2,6 H-3,5,3,5 H-2,5 OH-3,6 H-3,6 OH-2,5 4-OCH 3,4 -OCH 3 C-2,5 C-3,6 C-1,4 C-3,5,3,5 C-2,6,2,6 C-1,1 C-4,4 19
Figure S18. NOESY Spectrum of Hawaiienol C (3; 600 MHz, Acetone-d 6 ) 4-OCH 3,4 -OCH 3 H-2,6,2,6 H-3,5,3,5 H-2,5 OH-3,6 H-3,6 OH-2,5 OH-2,5 4-OCH 3,4 -OCH 3 H-3,6 H-2,5 OH-3,6 H-3,5,3,5 H-2,6,2,6 20
Figure S19. 1 H NMR Spectrum of Hawaiienol D (4; 600 MHz, Acetone-d 6 ) 21
Figure S20. 13 C NMR Spectrum of Hawaiienol D (4; 150 MHz, Acetone-d 6 ) 22
Figure S21. HSQC Spectrum of Hawaiienol D (4; 600 MHz, Acetone-d 6 ) 4 -OCH 3,4-OCH 3 H-2,6,2,6 H-3,5,3,5 H-3 H-6 H-5 4 -OCH 3,4-OCH 3 C-3 C-5 C-6 C-3,5,3,5 C-2,6,2,6 23
Figure S22. 1 H 1 H COSY Spectrum of Hawaiienol D (4; 600 MHz, Acetone-d 6 ) 4 -OCH 3,4-OCH 3 H-2,6,2,6 H-3,5,3,5 OH-1 H-3 OH-5 OH-4 OH-6 OH-3 H-6 H-5 4 -OCH 3,4-OCH 3 H-6,OH-3 H-5 OH-6 OH-4 OH-5 H-3 OH-1 H-3,5,3,5 H-2,6,2,6 24
Figure S23. HMBC Spectrum of Hawaiienol D (4; 600 MHz, Acetone-d 6 ) 4 -OCH 3,4-OCH 3 H-2,6,2,6 H-3,5,3,5 OH-1 H-3 OH-5 OH-4 OH-6 OH-3 H-6 H-5 4 -OCH 3,4-OCH 3 C-5,3 C-1, 6 C-4 C-3,5,3,5 C-1,2,6,2,6 C-1 C-4,4 C-2 25
Figure S24. NOESY Spectrum of Hawaiienol D (4; 600 MHz, Acetone-d 6 ) 4 -OCH 3,4-OCH 3 H-2,6,2,6 H-3,5,3,5 OH-5 OH-3 OH-1 H-3 OH-4 OH-6 H-5 H-6 4 -OCH 3,4-OCH 3 OH-3 H-6 H-5 OH-6 OH-4 OH-5 H-3 OH-1 H-3,5,3,5 H-2,6,2,6 26
Figure S25. Experimental CD Spectrum of 1 in MeOH 27
Figure S26. Relative Configurations of 2 (the Enantiomers Were Not Shown) 28
Figure S27. Relative Configurations of 4 (the Enantiomers Were Not Shown) 29
Figure S28. The Optimized Conformers for 2 2a 2a 2b 2b 2c 2c 2d 2d 30
2e 2e 2f 2f 2g 2g 2h 2h 31
Figure S29. The Optimized Conformers for 3 3a 3b 32
Figure S30. The Optimized Conformers for 4 4a 4a 4b 4b 4c 4c 4d 4d 33
4e 4e 4f 4f 4g 4g 4h 4h 34
4i 4i 4j 4j 4k 4k 4l 4l 35
4m 4m 4n 4n 4o 4o 4p 4p 36
Figure S31. The Optimized Conformer for 3a 37
Figure S32. Experimental CD Spectrum of 2 in MeOH and the Calculated ECD Spectra of 2a 2h and 2a 2h 38
Figure S33. Experimental CD Spectrum of 4 in MeOH and the Calculated ECD Spectra of 4a 4p and 4a 4p 39
Figure S34. Linear Regression Fitted 13 C NMR Chemical Shifts of 4 with Four Sets of Experimental Values (4i, 4l, 4n, and 4o) 40
Table S1. The Experimental and Computed 13 C NMR Data of 3 Position Experimental Calculated 3a-1 3a-2 Scaled Fitted 1 132.2 132.2 134.4 129.1 2 128.5 128.5 133.8 128.6 3 113.8 113.8 118.2 113.6 4 159.7 159.7 166.7 160.2 5 113.8 113.8 118.2 113.6 6 128.5 128.5 133.8 128.6 1 90.6 90.6 97.4 93.6 2 78.4 88.1 80.6 77.5 3 88.1 78.4 94.2 90.5 4 90.6 90.6 97.4 93.6 5 78.4 88.1 80.6 77.5 6 88.1 78.4 94.2 90.5 1 132.2 132.2 134.4 129.1 2 128.5 128.5 133.8 128.6 3 113.8 113.8 118.2 113.6 4 159.7 159.7 166.7 160.2 5 113.8 113.8 118.2 113.6 6 128.5 128.5 133.8 128.6 4-OCH3 55.5 55.5 56.0 53.8 4 -OCH3 55.5 55.5 56.0 53.8 41
Table S2. Statistics of Ordinary Least Squares (OLS) Linear Regression of the Experimental and Computed 13 C NMR Chemical Shifts of 3 Assignment CMAD a CLAD b R 2 R 2 adj RMSE F p value 3a-1 1.21 3.06 0.9967 0.9965 1.7768 5360.0760 < 0.01 3a-2 3.12 12.37 0.9671 0.9653 5.5723 528.7920 < 0.01 a CMAD = corrected mean absolute deviation, computed as (1/ n) δ calc δ exp. b CLAD = corrected largest absolute deviation, computed as max( δcalc δexp ). n i 42
Table S3. The Experimental and Computed 13 C NMR Data of 4 Position Experimental Calculated 4i 4l 4n 4o 1 131.1 129.7 128.4 130.4 132.4 2 130.7 129.8 129.8 130.2 129.8 3 112.9 112.9 113.5 113.8 112.3 4 159.9 159.2 160.1 159.2 158.7 5 112.9 112.9 113.5 113.8 112.3 6 130.7 129.8 129.8 130.2 129.8 1 82.0 82.8 86.7 83.9 82.2 2 208.9 212.9 209.9 210.5 212.5 3 73.6 79.2 79.1 83.4 82.4 4 86.0 87.1 81.6 80.4 83.0 5 77.0 78.9 77.8 76.3 78.0 6 80.1 76.4 78.9 80.1 80.6 1 135.2 133.5 130.1 128.0 133.5 2 129.5 129.5 131.8 131.9 128.7 3 113.8 112.5 113.2 112.7 112.5 4 160.0 158.1 160.2 159.4 158.5 5 113.8 112.5 113.2 112.7 112.5 6 129.5 129.5 131.8 131.9 128.7 4-OCH 3 55.4 55.7 54.5 54.7 54.9 4 -OCH 3 55.5 55.6 54.5 54.8 54.9 43
Table S4. Statistics of Ordinary Least Squares (OLS) Linear Regression of the Experimental and Computed 13 C NMR Chemical Shifts of 4 Configuration CMAD a CLAD b R 2 R 2 adj RMSE F p value 4i 1.38 5.58 0.9971 0.9970 2.11 6239.79 < 0.01 4l 1.83 5.45 0.9956 0.9953 2.62 4043.45 < 0.01 4n 2.01 9.83 0.9926 0.9922 3.38 2415.19 < 0.01 4o 1.53 8.75 0.9959 0.9956 2.53 4334.47 < 0.01 a CMAD = corrected mean absolute deviation, computed as (1/ n) δ calc δ exp. b CLAD = corrected largest absolute deviation, computed as max( δcalc δexp ). n i 44
Scheme S1. Plausible Biosynthetic Pathways for 1 4 Methylation 45