Acaulins A and B, Trimeric Macrodiolides from Acaulium. Table of Contents

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1 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. rxtan@nju.edu.cn Table of Contents Experimental Procedures References...5 Supplementary tables Supplementary figures

2 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 and Fr RP-HPLC of Fr with CH 3 CN/H 2 O (45:55) gave 1 (3.0 mg), that of Fr 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 (calcd for C 42 H 54 O 19 Na, ). 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 (calcd for C 42 H 56 O 20 Na, ). 2

3 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 (calcd for C 14 H 20 O 7 Na, ). 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 = 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 data_request/cif, or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: ; or data_request@ccdc.cam.ac.uk. Crystal data of 2 Acaulin B (2). C 42 H 56 O 20, M r = , space group P , a = (7) Å, b = (10) Å, c = (17) Å, V = (5) Å 3, Z = 4, µ = mm -1, F(000) = ; crystal size: mm 3 ; 7118 unique reflections with 6554 obeying the I > 2 (I); R = , wr2 = , S = 1.023; flack parameter = 0.06(9); supplementary publication No. CCDC 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

4 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

5 References (1) Wang, T. T.; Wei, Y. J.; Ge, H. M.; Jiao, R. H.; Tan, R. X. Org. Lett. 2018, 20, (2) Hanessian, S.; Ma, J. G.; Wang, W. G. J. Am. Chem. Soc. 2001, 123, (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, (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, (b) Fleming, A.; Sato, M.; Goldsmith. P. J. Biomol. Screen. 2005, 10, (c) Barrett, R.; Chappell, C.; Quick, M.; Fleming, A. Biotechnol. J. 2006, 1,

6 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) dd ( ) dd ( ) dd ( ) dd ( ) m m 4-OH 4.45 d (6.1) qd (6.7, 2.0) m d (6.7) d (6.4) d (10.0) d (10.0) d (10.0) d (10.0) a m m 11b 2.94 m 2.98 m 12a m m 12b 2.65 m 2.63 m m m d (6.5) d (6.5) 1' ' dd ( ) dd ( ) 3' dd ( ) dd ( ) 4' m m 4'-OH 4.76 d (6.2) 5' dq ( ) m 6' d (6.1) d (6.4) 7' ' dd ( ) m 9'a dd ( ) m 9'b 3.42 dd ( ) 3.00 m 6

7 10' 'a m m 11'b 2.81 m 2.74 m 12'a m m 12'b 2.33 m 1.89 m 13' m m 14' d (6.5) d (6.4) 1'' '' dd ( ) dd ( ) 3'' dd ( ) dd ( ) 4'' m m 4''-OH 4.96 d (5.9) 5'' dq ( ) dq ( ) 6'' d (6.3) d (6.2) 7'' '' '' '' ''-OH 3.55 d (1.6) 11''a m m 11''b 2.57 m 2.55 m 12''a m m 12''b 2.47 m 2.66 m 13'' m m 14'' d (6.1) 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) dd ( ) 7

8 dd ( ) m m d (6.4) d (16.0) d (16.0) m m m d (6.2) Table S3 13 C enrichment in acaudiol after feeding [1-13 C]-acetate. position δ C relative intensity a 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

9 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

10 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

11 A B 1 i [M + Na] + m/z 885 ii [M + Na] + m/z 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

12 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

13 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

14 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

15 Figure S10. 1 H NMR spectrum of acaulin A (1, 600 MHz, acetone-d 6 ) 15

16 Figure S C NMR spectrum of acaulin A (1, 150 MHz, acetone-d 6 ) 16

17 Figure S12. DEPT135 spectrum of acaulin A (1, 150 MHz, acetone-d 6 ) 17

18 Figure S13. 1 H 1 H COSY spectrum of acaulin A (1, acetone-d 6 ) 18

19 Figure S14. HSQC spectrum of acaulin A (1, acetone-d 6 ) 19

20 Figure S15. HMBC spectrum of acaulin A (1, acetone-d 6 ) 20

21 Figure S16. NOESY spectrum of acaulin A (1, acetone-d 6 ) 21

22 Figure S17. 1 H NMR spectrum of acaulin B (2, 600 MHz, acetone-d 6 with 0.2% NaOD) 22

23 Figure S C NMR spectrum of acaulin B (2, 150 MHz, acetone-d 6 with 0.2% NaOD) 23

24 Figure S19. DEPT135 spectrum of acaulin B (2, 150 MHz, acetone-d 6 with 0.2% NaOD) 24

25 Figure S20. 1 H 1 H COSY spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 25

26 Figure S21. HSQC spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 26

27 Figure S22. HMBC spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 27

28 Figure S23. NOESY spectrum of acaulin B (2, acetone-d 6 with 0.2% NaOD) 28

29 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

30 Figure S C NMR spectrum of acaudiolic acid (3, 150 MHz, methanol-d 4 ) 30

31 Figure S26. DEPT135 spectrum of acaudiolic acid (3, 150 MHz, methanol-d 4 ) 31

32 Figure S27. 1 H 1 H COSY spectrum of acaudiolic acid (3, methanol-d 4 ) 32

33 Figure S28. HSQC spectrum of acaudiolic acid (3, methanol-d 4 ) 33

34 Figure S29. HMBC spectrum of acaudiolic acid (3, methanol-d 4 ) 34

35 Figure S30. NOESY spectrum of acaudiolic acid (3, methanol-d 4 ) 35