Supporting Information Acaulins A and B, Trimeric Macrodiolides from Acaulium sp. H-JQSF Ting Ting Wang, Ying Jie Wei, Hui Ming Ge, Rui Hua Jiao, Ren Xiang Tan* * Corresponding author. E-mail: rxtan@nju.edu.cn Table of Contents Experimental Procedures...2 4 References...5 Supplementary tables...6 8 Supplementary figures...9-35 1
Experimental Procedures General As described. 1 Cultivation and extraction The fungus Acaulium sp. H-JQSF researched in this work has been described previously. 1 The strain was inoculated in 1 L Erlenmeyer flasks containing 400 ml Martin medium (10.0 g/l peptone, 20.0 g/l yeast extract, 10.0 g/l sucrose, 1.0 g/l KH 2 PO 4, 0.5 g/l MgSO 4 ) followed by 16-day fermentation at 28 C (140 rpm) for 200-L scale. The broth was extracted with EtOAc and concentrated under the reduced pressure to yield crude extract (270 g). Isolation of 1 3 The obtained crude extract was separated by column chromatography (CC) over silica gel to afford 6 groups (Fr. 1 Fr. 6). Further purification of Fr. 3 by CC over ODS-A eluted with MeOH/H 2 O mixtures (v/v, 25:100 0:100) yielded eighteen subfractions (Fr. 3.1 ~ Fr. 3.18). Fr. 3.1 was separated over Sephadex LH-20 (MeOH) followed by purification via RP-HPLC (CH 3 CN/H 2 O, 18:82) to yield 3 (1.4 mg). Fr. 6 was separated successively by CC over ODS-A and Sephadex LH-20 (MeOH) to afford fractions Fr. 6.18.2 and Fr. 6.20.2. RP-HPLC of Fr. 6.18.2 with CH 3 CN/H 2 O (45:55) gave 1 (3.0 mg), that of Fr. 6.20.2 with CH 3 CN/H 2 O (32:68) supplied 2 (8.0 mg). Physio-chemical data of 1 3 Acaulin A (1). Colorless powder, [α] 25 D = +60 (c = 0.05, MeOH); UV (MeOH) λ max (log ε) = 212 (3.82) nm; for 1 H and 13 C NMR data see Table S1; HR-ESI-MS [M + Na] + m/z 885.3143 (calcd for C 42 H 54 O 19 Na, 885.3152). Acaulin B (2). Colorless prism, [α] 25 D = +45 (c = 0.05, MeOH); UV (MeOH) λ max (log ε) = 215 (3.99) nm; for 1 H and 13 C NMR data see Table S1; HR-ESI-MS [M + Na] + m/z 903.3261 (calcd for C 42 H 56 O 20 Na, 903.3257). 2
Acaudiolic acid (3). Colorless oil, [α] 25 D = +44 (c = 0.05, MeOH); UV (MeOH) λ max (log ε) = 218 (3.51) nm; for 1 H and 13 C NMR data see Table S2; HR-ESI-MS [M + Na] + m/z 323.1092 (calcd for C 14 H 20 O 7 Na, 323.1101). Single crystal X-ray diffraction The crystals of acaulin B (2) were crystallized from 800 μl MeOH with 40 μl H 2 O in 10 ml penicillin bottle at 4 C. The crystal X-ray diffraction data of 2 were recorded on a Bruker APEX DUO diffractometer through Cu Kα (λ = 1.54178 Å) radiation at 153(2) K. Data reduction and cell refinement were completed by Bruker SAINT. The structure of 2 was solved via direct methods (SHELXT-2014) and refined via full-matrix least-squares difference Fourier techniques (SHELXL-2016). Crystallographic data for 2 has been deposited in the Cambridge Crystallographic Data Centre (CCDC), with copies of the data can be obtained at http://www.ccdc.cam.ac.uk/ data_request/cif, or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; or e-mail: data_request@ccdc.cam.ac.uk. Crystal data of 2 Acaulin B (2). C 42 H 56 O 20, M r = 880.86, space group P2 1 2 1 2 1, a = 10.4486(7) Å, b = 15.2134(10) Å, c = 28.9143(17) Å, V = 4596.2(5) Å 3, Z = 4, µ = 0.861 mm -1, F(000) = 1872.0; crystal size: 0.270 0.250 0.220 mm 3 ; 7118 unique reflections with 6554 obeying the I > 2 (I); R = 0.0527, wr2 = 0.1505, S = 1.023; flack parameter = 0.06(9); supplementary publication No. CCDC-1565521. Macrolactonization As described, 2 within a 50-minute duration, 2 (3.0 mg, 3.41 µmol) diluted in 100 µl N,N-dimethylformamide (DMF) was dropwisely added to the solution of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl) (0.69 mg, 3.60 µmol) and 4-dimethylaminopyridine (DMAP) (0.52 mg, 4.26 µmol) in 400 µl DMF, followed by stirring for the ensuing 5 hours at room temperature (rt). The macrolactonization product was purified by RP-HPLC. As described, 2 EDC HCl (0.20 mg, 1.05 µmol) and DMAP (0.15 mg, 1.25 µmol) in 20 µl DMF were mixed at rt, 3 (0.3 mg, 1.0 µmol) in 10 µl DMF was slowly added during 15 min followed by shaking for another 30 min. Stable isotope labeling experiment 3
Acaulium sp. H-JQSF was re-cultured in 1 L Erlenmeyer flask with 400 ml modified culture medium (25.0 g/l peptone, 50.0 g/l yeast extract, 25.0 g/l sucrose, 2.5 g/l KH 2 PO 4, 1.25 g/l MgSO 4 ). As mentioned previously, 3 [1-13 C]-sodium acetate (200 mg) dissolved in 1 ml sterile water was filtrated through Millipore filter and subpackaged in 4 Eppendorf tubes, which were respectively added into the fungal culture at 3 d, 4 d, 5 d, and 6 d after inoculation. After cultivation for 7 more days, the labeled acaudiol was purified (6.5 mg) by RP-HPLC (CH 3 CN/H 2 O, 24:76). Evaluation of anti-osteoporotic effect in zebrafish As described, 1,4 compounds 1 and 2 (0.4, 2.0, and 10 µm) combined with prednisolone (PN, 25.0 µm) were added into the 24-well plates (6 fish per well) to treat the 3 dpf (3 days post-fertilization) wild-type zebrafish with the DMSO (0.4%)- and PN (25.0 µm)-added wells serving as controls in three independent experiments. 4
References (1) Wang, T. T.; Wei, Y. J.; Ge, H. M.; Jiao, R. H.; Tan, R. X. Org. Lett. 2018, 20, 1007 1010. (2) Hanessian, S.; Ma, J. G.; Wang, W. G. J. Am. Chem. Soc. 2001, 123, 10200 10206. (3) Han, W. B.; Lu, Y. H.; Zhang, A. H.; Zhang, G. F.; Mei, Y. N.; Jiang, N.; Lei, X. X.; Song, Y. C.; Ng, S. W.; Tan, R. X. Org. Lett. 2014, 16, 5366 5369. (4) (a) Jeong, Y. T.; Baek, S. H.; Jeong, S. C.; Yoon, Y. D.; Kim, O. H.; Oh, B. C.; Jung, J. W.; Kim, J. H. J. Med. Food. 2016, 19, 805 814. (b) Fleming, A.; Sato, M.; Goldsmith. P. J. Biomol. Screen. 2005, 10, 823 831. (c) Barrett, R.; Chappell, C.; Quick, M.; Fleming, A. Biotechnol. J. 2006, 1, 651 655. 5
Tables and Figures Table S1. 1 H and 13 C NMR data for acaulins A (1) and B (2). 1 2 a position δ C δ H (mult, J, Hz) δ C δ H (mult, J, Hz) 1 165.6 166.3 2 122.3 5.91 dd (15.7 3.4) 121.6 5.96 dd (15.7 1.6) 3 147.0 6.98 dd (15.7 3.4) 148.0 7.06 dd (15.7 2.9) 4 73.1 4.24 m 72.9 4.29 m 4-OH 4.45 d (6.1) 5 77.6 4.98 qd (6.7, 2.0) 76.9 5.00 m 6 18.0 1.20 d (6.7) 18.0 1.25 d (6.4) 7 173.3 172.5 8 46.8 4.70 d (10.0) 47.8 4.30 d (10.0) 9 60.5 3.67 d (10.0) 61.0 3.56 d (10.0) 10 203.0 205.4 11a 37.2 2.90 m 36.9 2.90 m 11b 2.94 m 2.98 m 12a 25.5 1.42 m 25.2 1.49 m 12b 2.65 m 2.63 m 13 69.2 5.13 m 69.3 5.14 m 14 19.2 1.30 d (6.5) 19.1 1.31 d (6.5) 1' 165.0 167.0 2' 120.7 5.88 dd (15.8 1.7) 121.8 6.08 dd (15.7 1.3) 3' 149.5 6.80 dd (15.8 3.7) 149.4 7.10 dd (15.7 4.7) 4' 72.6 4.22 m 75.4 4.14 m 4'-OH 4.76 d (6.2) 5' 77.5 4.33 dq (9.2 6.1) 70.4 3.76 m 6' 18.3 1.34 d (6.1) 19.0 1.17 d (6.4) 7' 174.6 176.8 8' 45.8 3.96 dd (5.8 2.9) 51.7 4.28 m 9'a 42.0 3.31 dd (19.0 5.8) 43.5 2.67 m 9'b 3.42 dd (19.0 2.9) 3.00 m 6
10' 207.0 210.4 11'a 35.4 2.46 m 38.9 2.62 m 11'b 2.81 m 2.74 m 12'a 26.4 1.44 m 30.3 1.86 m 12'b 2.33 m 1.89 m 13' 68.8 5.09 m 71.2 4.97 m 14' 19.0 1.19 d (6.5) 20.2 1.26 d (6.4) 1'' 166.0 166.8 2'' 120.6 5.93 dd (15.6 1.9) 120.0 5.92 dd (15.6 1.7) 3'' 148.9 6.65 dd (15.6 2.9) 150.0 6.88 dd (15.6 2.7) 4'' 72.5 4.23 m 72.3 4.19 m 4''-OH 4.96 d (5.9) 5'' 76.0 4.77 dq (9.4 6.3) 75.0 4.72 dq (9.4 6.2) 6'' 18.5 1.53 d (6.3) 18.4 1.38 d (6.2) 7'' 167.2 168.4 8'' 66.3 66.9 9'' 212.1 210.9 10'' 79.1 78.8 10''-OH 3.55 d (1.6) 11''a 37.1 1.45 m 36.8 1.43 m 11''b 2.57 m 2.55 m 12''a 29.4 1.67 m 29.6 1.65 m 12''b 2.47 m 2.66 m 13'' 71.5 4.60 m 71.8 4.62 m 14'' 21.1 1.23 d (6.1) 21.0 1.25 d (6.4) a Approximately 2 L of 0.2% NaOD in D 2 O was added into the NMR tube to prevent the intramolecular hydrogen bonding of 7'-carboxylic acid with carbonyls for a better resolution of the 1 H and 13 C NMR signals. Table S2. 1 H and 13 C NMR data for acaudiolic acid (3) position δ C δ H (mult, J, Hz) 1 167.9 2 122.6 6.04 dd (15.7 1.7) 7
3 149.7 7.04 dd (15.7 4.9) 4 76.2 4.05 m 5 71.3 3.70 m 6 19.2 1.18 d (6.4) 7 173.8 8 137.2 6.80 d (16.0) 9 139.6 6.72 d (16.0) 10 202.6 11 37.4 2.74 m 12 31.1 1.91 m 13 71.8 4.98 m 14 20.4 1.27 d (6.2) Table S3 13 C enrichment in acaudiol after feeding [1-13 C]-acetate. position δ C relative 1 167.0 5.03 2 125.2 0.96 3 148.5 5.20 4 77.2 0.99 5 73.1 5.22 6 17.9 1.14 7 8 9 10 11 12 13 14 167.3 121.9 153.5 70.7 29.8 27.1 71.2 17.8 intensity a 4.91 0.99 5.49 0.89 5.26 0.97 4.86 1.14 a The ratio of the 13 C-labeled carbon signals to the unlabeled counterparts with the bold number highlighting the substantial 13 C- incorporation. 8
A B C Figure S1. A) Possible intramolecular hydrogen bonds formed via 7'-carboxylic acid with 7- (i) and 7''-esteric (ii) and 9''-ketonic (iii) carbonyls. B) Selected NOESY correlations of 2. C) 2 converts to its sodium salt 2a upon addition of NaOD to the acetone-d 6 solution in the NMR tube. 9
A B Figure S2. 1 H NMR (A) and 13 C NMR (B) spectra of 2 without (red) and with (blue) 0.2% NaOD. 10
A B 1 i [M + Na] + m/z 885 ii [M + Na] + m/z 885 1 2 C 1 (from fungal culture, 600 MHz) 1 (lactonized from 2, 600 MHz) Figure S3. (A) Macrolactonization of 2 into 1. (B) LC-MS (EIC) analysis of the macrolactoning mixture of 2 (i) in comparison with authentic sample of 1 (ii). (C) The 1 H NMR spectra of natural (red) and synthetic (blue) 1. 11
Figure S4. CD spectra of 1 and 2. Figure S5. Key 1 H 1 H COSY, HMBC and NOESY correlations of 3. Figure S6. CD spectra of 3 and 10-ketoacaudiol. 12
A B 10-ketoacaudiol i [M + Na] + m/z 305 ii [M + Na] + m/z 305 Figure S7. (A) Macrolactonization of 3 into 10-ketoacaudiol. (B) LC-MS (EIC) analysis of the macrolactoning mixture of 3 (i) in comparison with authentic sample of 10-ketoacaudiol (ii). A B Figure S8. 13 C NMR spectra of acaudiol with (A) and without (B) [1-13 C] acetate labeling (100 MHz, methanol-d 4 ). 13
Figure S9. Ventral view of 8 dpf (8 days postfertilization) zebrafish treated with DMSO, prednisolone (PN) and 1 combined with PN. Parts of the skull opercular bone (op), ceratobranchial 5 (cb5), anterior tip of the notochord (no), and cleithrum (cl), parasphenoid (ps), and otolith (o, not bone) stained with Alizarin Red were annotated via arrows. 14
Figure S10. 1 H NMR spectrum of acaulin A (1, 600 MHz, acetone-d 6 ) 15
Figure S11. 13 C NMR spectrum of acaulin A (1, 150 MHz, acetone-d 6 ) 16
Figure S12. DEPT135 spectrum of acaulin A (1, 150 MHz, acetone-d 6 ) 17
Figure S13. 1 H 1 H COSY spectrum of acaulin A (1, acetone-d 6 ) 18
Figure S14. HSQC spectrum of acaulin A (1, acetone-d 6 ) 19
Figure S15. HMBC spectrum of acaulin A (1, acetone-d 6 ) 20
Figure S16. NOESY spectrum of acaulin A (1, acetone-d 6 ) 21
Figure S17. 1 H NMR spectrum of acaulin B (2, 600 MHz, acetone-d 6 with 0.2% NaOD) 22
Figure S18. 13 C NMR spectrum of acaulin B (2, 150 MHz, acetone-d 6 with 0.2% NaOD) 23
Figure S19. DEPT135 spectrum of acaulin B (2, 150 MHz, acetone-d 6 with 0.2% NaOD) 24
Figure S20. 1 H 1 H COSY spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 25
Figure S21. HSQC spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 26
Figure S22. HMBC spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 27
Figure S23. NOESY spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 28
Figure S35. 1 H NMR spectrum of dehydroacaudiolic acid (5, 400 MHz, methanol-d 4 ) Figure S24. 1 H NMR spectrum of acaudiolic acid (3, 600 MHz, methanol-d 4 ) 29
Figure S25. 13 C NMR spectrum of acaudiolic acid (3, 150 MHz, methanol-d 4 ) 30
Figure S26. DEPT135 spectrum of acaudiolic acid (3, 150 MHz, methanol-d 4 ) 31
Figure S27. 1 H 1 H COSY spectrum of acaudiolic acid (3, methanol-d 4 ) 32
Figure S28. HSQC spectrum of acaudiolic acid (3, methanol-d 4 ) 33
Figure S29. HMBC spectrum of acaudiolic acid (3, methanol-d 4 ) 34
Figure S30. NOESY spectrum of acaudiolic acid (3, methanol-d 4 ) 35