electronic supplementary information b, a, zou,a jun xie,a ... · rmsd root-mean-square deviation...

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Electronic Supplementary Information Guo-Dong Chen,a,† Dan Hu,a,† Mei-Juan Huang,a,† Jia Tang,a Xiao-Xia Wang,a Jian Zou,a Jun Xie,a Wei-Guang Zhang,c Liang-Dong Guo,d Xin-Sheng Yao,a,* Ikuro Abe,b,* and Hao Gaoa,*

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020

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Table of Contents

1.The list of abbreviations ...........................................................................................32.Structural elucidations of isolated compounds.......................................................43. Structural characterizations of 1-5.........................................................................94. Materials and methods ..........................................................................................10

4.1 General experimental procedures................................................................................104.2 Fungal materials and fermentation..............................................................................104.3 Extraction and isolation ..............................................................................................114.4 X-ray crystallographic analyses ..................................................................................11

5. Chiral separations of 1 and 5 ................................................................................156. Quantum chemical calculations of 1–5.................................................................16

6.1 Quantum chemical VCD calculations of 1 .................................................................166.2 Quantum chemical ECD calculations of 1-5...............................................................18

7. HPLC-ECD coupling analyses of 1-5 ...................................................................257.1 HPLC-ECD coupling analyses of 1 and 5 ..................................................................257.2 HPLC-ECD coupling analysis of 2 .............................................................................277.3 HPLC-ECD coupling analysis of 3 .............................................................................287.4 HPLC-ECD coupling analysis of 4 .............................................................................29

8. The process of obtaining unlabeled and labeled 1...............................................309. Biosynthetic investigation of 1...............................................................................32

9.1 Whole genome sequencing and analysis of Sporormiella sp.40-1-4-1.......................329.2 RNA preparation and reverse transcription PCR........................................................349.3 Construction of gene inactivation mutants..................................................................369.4 Heterologous expression of g7393 in Aspergillus oryzae NSAR1 .............................39

10. Short-term memory assays of 1-5 on the AD fly model ....................................4211.The 1D and 2D NMR spectra of 1-5 ....................................................................45

11.1 The 1D and 2D NMR spectra of 1 ............................................................................4511.2 The 1D and 2D NMR spectra of 2 ............................................................................4911.3 The 1D and 2D NMR spectra of 3 ............................................................................5211.4 The 1D and 2D NMR spectra of 4 ............................................................................5511.5 The 1D and 2D NMR spectra of 5 ............................................................................58

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1.The list of abbreviations

Abbreviation Full nameAD Alzheimer’s diseaseCE Cotton effect

CRISPR-Cas9 clustered regularly interspaced palindromic repeats-Cas9DFT density functional theoryECD Electronic circular dichroismELSD evaporative light scattering detector

GAPDH glyceraldehyde-3-phosphate dehydrogenaseHPLC high-performance liquid chromatography

HRESIMS high resolution electrospray ionization mass spectroscopymaxconfs the maximum number of conformers

NMR nuclear magnetic resonanceNR-PKS non-reduced polyketide synthase

o-QM ortho-quinone methidePI performance indexPT product template

RNA-seq RNA-sequencingRT-PCR reverse transcription PCRRMSD root-mean-square deviationVCD vibrational circular dichroism

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2.Structural elucidations of isolated compoundsSporormielone A (1) was obtained as yellow needle-like crystals. The quasi-molecular ion

peak at m/z 305.1024 [M + H]+(calcd. for C16H17O6, 305.1025) by HRESIMS indicated that the molecular formula of 1 was C16H16O6 with nine degrees of unsaturation. The 13C NMR spectrum and DEPT-135 spectrum showed 16 carbon signals, ascribable to three carbonyl carbons, six aromatic or olefinic carbons, two sp3 quaternary carbons[including one oxygenated (δC 86.8)], one sp3methine carbon, and four methyl carbons. All non-exchangeable proton resonances were assigned to the relevant carbon atoms by HSQC experiment (Table S1). The HMBC correlations from H3-1 to C-2/C-3/C-7, from H3-8 to C-2/C-6/C-7/C-6', from H3-1' to C-2'/C-3'/C-7', from H-6' to C-6/C-7/C-8/C-2'/C-4'/C-5'/C-7', and from H3-8' to C-2'/C-6'/C-7' revealed the sectional structure. Based on molecular formula, degrees of unsaturation, chemical shifts of 1H and 13C, and the single-crystal X-ray crystallographic analysis, the planar structure of 1 was established. The relative configuration of 1 was determined as 7R*, 5'R*, 6'R* based on the single-crystal X-ray crystallographic analysis.

1

1

3

5

7

8 1'8'

7'

5'

3'

4'O

O

O

HO

OH

OH

H

Table S1 NMR data of 1 in DMSO-d6 (600 MHz for 1H; 150 MHz for 13C)

No. δC, mult. δH(J in Hz) 1H-1HCOSY HMBC ROESY

1 14.5, CH3 2.11, s 2, 3, 7 8, 6′, 8′2 136.1, C3 145.5, C4 177.0, C5 147.4, C6 125.8, C7 46.8, C8 29.1, CH3 1.50, s 2, 6, 7, 6′ 1, 6′1′ 8.2, CH3 1.54, br s 6′, 8′ 2′, 3′, 7′ 8′2′ 136.3, C3′ 199.9, C4′ 196.8, C5′ 86.8, C6′ 59.4, CH 3.45, br s 1′ 6, 7, 8, 2′, 4′, 5′, 7′ 1, 8, 8′, 5′-OH7′ 167.8, C8′ 16.6, CH3 1.89, br s 1′ 2′, 6′, 7′ 1, 6′

5′-OH 6.68, brs 6′OH 8.88, brs

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Sporormielone B (2) was obtained as yellow needle-like crystals. The quasi-molecular ion peak at m/z 327.0847 [M + Na]+ (calcd. for C16H16O6Na, 327.0845) by HRESIMS indicated that the molecular formula of 2 was C16H16O6 with nine degrees of unsaturation. The 13C NMR spectrum and DEPT-135 spectrum showed 16 carbon signals, ascribable to two carbonyl carbons, eight aromatic or olefinic carbons, one sp3 oxygenated quaternary carbon, one sp3quaternary carbon, one sp3 methylene carbon, three methyl carbons. All non-exchangeable proton resonances were assigned to the relevant carbon atoms by HSQC experiment (Table S2). The HMBC correlations from H3-1 to C-2/C-3/C-7, from H-8ato C-7/C-2′/C-7′, from H-8b to C-2/C-6/C-7/C-2′/C-3′, from H3-1' to C-8/C-2′/C-3′/C-7′, from H-6′ to C-2′/C-3′/C-5′/C-7′/C-8′, and from H3-8′ to C-2′/C-6′/C-7′ revealed sectional structure. Based on molecular formula, degrees of unsaturation, chemical shifts of 1H and 13C, and the single-crystal X-ray crystallographic analysis, the planar structure of 2 was established. The relative configuration of 2 was determined as 2'S*, 3'R* based on the single-crystal X-ray crystallographic analysis.

OOHHO

HO

OOH

2

7 83'

1'

5'

7'

8'

4'

1


No. δC, mult. δH (J in Hz) 1H-1H COSY HMBC

1 10.6, CH3 2.07, s 2, 3, 72 113.6, C

3 153.8, C

4 130.2, C

5 149.9, C

6 107.0, C

7 131.1, C

2.60, d, (16.5), a 8b 7, 2′, 7′8 29.5, CH2

3.12, d, (16.5), b 8a 2, 6, 7, 2′, 3′

1′ 24.5, CH3 1.17, s 8, 2′, 3′, 7′

2′ 48.9, C3′ 85.6, C

4′ 197.3, C

5′ 200.9, C

6′ 127.9, CH 5.95, br s 8′ 2′, 3′, 5′, 7′, 8′

7′ 182.4, C8′ 14.7, CH3 1.90, br s 6′ 2′, 6′, 7′

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Sporormielone C (3) was obtained as yellow needle-like crystals. The quasi-molecular ion peak at m/z 305.1022 [M + H]+ (calcd. for C16H17O6, 305.1025) by HRESIMS indicated that the molecular formula of 3 was C16H16O6 with nine degrees of unsaturation. The 13C NMR spectrum and DEPT-135 spectrum showed 16 carbon signals, ascribable to two carbonyl carbons, eight aromatic or olefinic carbons, two sp3 oxygenated quaternary carbons, four methyl carbons. All non-exchangeable proton resonances were assigned to the relevant carbon atoms by HSQC experiment (Table S3). The HMBC correlations from H3-1 to C-2/C-3/C-7, from H-8 to C-2/C-6/C-7, from H3-1′ to C-2′/C-3′/C-7′, from H-6' to C-2′/C-3′/C-5′/C-7′/C-8′, and from H3-8′ to C-2′/C-6′/C-7′ revealed sectional structure. Based on molecular formula, degrees of unsaturation, chemical shifts of 1H and 13C, and the single-crystal X-ray crystallographic analysis, the planar structure of 3 was established. The relative configuration of 3 was determined as 2′S*, 3′R* based on the single-crystal X-ray crystallographic analysis.

1

3 5

3

OH OO

OHHO O

8

4'3'

1' 8'

5'



1 11.4, CH3 1.97, br s 8 2, 3, 7

2 119.7, C

3 150.7, C

4 130.2, C

5 145.6, C

6 113.2, C

7 128.7, C

8 15.5, CH3 2.13, br s 1 2, 6, 7

1′ 20.9, CH3 1.52, s 2′, 3′, 7′

2′ 86.8, C

3′ 89.0, C

4′ 188.9, C

5′ 197.3, C

6′ 130.7, CH 6.22, br s 8′ 2′, 3′, 5′, 7′, 8′

7′ 178.0, C

8′ 13.3, CH3 2.02, br s 6′ 2′, 6′, 7′

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Sporormielone C (4) was obtained as yellow needle-like crystals. The quasi-molecular ion peak at m/z 305.1029 [M + H]+ (calcd. for C16H17O6, 305.1025) by HRESIMS indicated that the molecular formula of 4 was C16H16O6 with nine degrees of unsaturation. The 13C NMR spectrum and DEPT-135 spectrum showed 16 carbon signals, ascribable to two carbonyl carbons, eight aromatic or olefinic carbons, one sp3 oxygenated quaternary carbon, one sp3 oxygenated methane carbon, four methyl carbons. All non-exchangeable proton resonances were assigned to the relevant carbon atoms by HSQC experiment (Table S4). The HMBC correlations from H3-1 to C-2/C-3/C-7, from H-8 to C-2/C-6/C-7, from H3-1′ to C-2′/C-3′/C-7′, from H-6′ to C-5/C-2′/C-4′/C-5′/C-7′/C-8′, and from H3-8′ to C-2′/C-6′/C-7′ revealed sectional structure. Based on molecular formula, degrees of unsaturation, chemical shifts of 1H and 13C, and the single-crystal X-ray crystallographic analysis, the planar structure of 4 was established. The relative configuration of 4 was determined as 5′R*, 6′S* based on the single-crystal X-ray crystallographic analysis.

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1

3 5

4

OHOOH

O OOH

6

7

8

4' 5'6'

8'

2' 1'

3'

7'H



1 11.3, CH3 1.97, s 2, 3, 7

2 119.4, C

3 150.4, C

4 131.2, C

5 145.2, C

6 113.6, C

7 128.7, C

8 15.5, CH3 2.13, s 2, 6, 7

1′ 8.2, CH3 1.60, br s 6′, 8′ 2′, 3′, 7′

2′ 138.8, C

3′ 197.9, C

4′ 188.6, C

5′ 85.0, C

6′ 84.1, CH 5.23, br s 1′, 8′ 5, 2′, 4′, 5′, 7′, 8′

7′ 166.0, C

8′ 12.9, CH3 2.04, brs 1′ 2′, 6′, 7′

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Sporormielone E (5) was obtained as yellow needle-like crystals. The quasi-molecular ion peak at m/z 305.1038 [M + H]+ by HRESIMS indicated that the molecular formula of 5 was C16H16O6 with nine degrees of unsaturation. The 13C NMR spectrum and DEPT-135 spectrum showed 15 carbon signals (one carbon was obtained from HMBC), ascribable to two carbonyl carbons, seven aromatic or olefinic carbons, one sp3oxygenated quaternary carbon, one sp3oxygenatedmethine carbon, four methyl carbons. All non-exchangeable proton resonances were assigned to the relevant carbon atoms by HSQC experiment (Table S5). The HMBC correlations from H3-1 to C-2/C-3/C-7, from H3-8 to C-2/C-6/C-7, from H3-1′ to C-2′/C-3′/C-7′, from H-6′ to C-2′/C-4′/C-5′/C-7′, and from H3-8′ to C-2′/C-6′/C-7′ revealed sectional structure. Based on molecular formula, degrees of unsaturation, chemical shifts of 1H and 13C, and the single-crystal X-ray crystallographic analysis, the planar structure of 5 was established. The relative configuration of 5 was determined as 5′R*, 6′S* based on the single-crystal X-ray crystallographic analysis.

O

O

OHO

OH

OH1

3 5

7

8

3'6'4'

5'

1'

8'7'

5

Table S5 NMR data of 5 in DMSO-d6 (600 MHz for 1H;150 MHz for 13C)

No. δC, mult δH (J in Hz) 1H-1H COSY HMBC

1 11.0, CH3 2.00, br s 8 2, 3, 7

2 119.2, C

3 153.9*, C

4 126.8, C

5 160.8, C

6 110.2, C

7 128.1, C

8 12.9, CH3 2.31, br s 1 2, 6, 7

1′ 8.2, CH3 1.70, br s 6′, 8′ 2′, 3′, 7′

2′ 135.7, C

3′ 194.9, C

4′ 191.7, C

5′ 97.7, C

6′ 75.6, CH 4.87, br s 1′, 8′ 2′, 4′, 5′, 7′

7′ 170.7, C

8′ 13.4, CH3 2.10, br s 1′, 6′ 2′, 6′, 7′

*The data was obtained from HMBC.

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3. Structural characterizations of 1-5Sporormielone A (1): yellow needle-like crystals (MeOH); m.p. 249~251°C; [α]29 D +10.7

(c 0.5, MeOH); UV (MeOH) λmax (log ε) 234 (4.26), 306 (3.96) nm;IR (KBr) νmax 3208, 2971, 2870, 1700, 1621, 1370, 1127 cm-1;ESI-MS (positive): m/z 305 [M + H]+, m/z 327 [M + Na]+; HRESIMS (positive) m/z 305.1024 [M + H]+ (calcd. for C16H17O6, 305.1025); 1H and 13C NMR see Table S1.

(+) (7R, 5′R, 6′R) sporormielone A (1a): []25 D +222.6 (c 0.1, MeOH); ECD (3.3 104 M,MeOH) λmax (△ε): 239 (+14.37), 264 (-18.49), 299 (+8.16)nm.

() (7S, 5′S, 6′S) sporormielone A (1b): []25 D -188.7 (c 0.1, MeOH); ECD (3.3 104 M,MeOH) λmax (△ε): 239 (-11.05), 264 (+15.57), 299 (-6.59) nm.

Sporormielone B (2): yellow needle-like crystals (MeOH); m.p.250~253°C; [α]27 D -4.7 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 222 (3.62), 321 (3.36) nm; IR (KBr) νmax 3490, 3050, 2961, 2850, 1708, 1630, 1607, 1583, 1457, 1380, 1140, 850cm-1; ESI-MS (positive): m/z 305 [M + H]+, m/z 631 [2M + Na]+; HRESIMS (positive) m/z 327.0847 [M + Na]+ (calcd. for C16H16O6Na, 327.0845); 1H and 13C NMR see Table S2.

Sporormielone C (3): yellow needle-like crystals (MeOH); m.p. 252~254°C; [α]27 D-5.7 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 225 (4.15), 306 (3.71) nm; IR (KBr) νmax 3447, 3020, 2989, 2831, 1718, 1644, 1601, 1570, 1378, 1250, 1083, 825 cm-1; ESI-MS (positive): m/z 305 [M + H]+, m/z 631 [2M +Na]+; HRESIMS (positive) m/z 305.1022 [M +H]+ (calcd. for C16H17O6, 305.1025); 1H and 13C NMR see Table S3.

Sporormielone D (4): yellow needle-like crystals (MeOH); m.p. 260~262°C; [α]25 D +11.3 (c 0.031, MeOH); UV (MeOH) λmax (logε) 217 (3.92), 297 (3.29) nm;IR (KBr) υmax 3427, 2952, 2813, 1716, 1643, 1603, 1580, 1384, 1266, 1080 cm-1; ESI-MS (positive): m/z 305 [M + H]+, m/z 631 [2M + Na]+; HRESIMS (positive) m/z305.1029 [M +H]+ (calcd. for C16H17O6, 305.1025); 1H and 13C NMR see Table S4.

Sporormielone E (5): yellow needle-like crystals (MeOH); m.p. 220~223 °C; [α]28 D-19.6 (c0.5, MeOH); UV (MeOH) λmax (log ε) 222 (4.14), 297 (3.77) nm; IR (KBr) νmax 3287, 2922, 2856, 1712, 1636, 1601, 1511, 1381, 1245, 1023 cm-1; ESI-MS (positive): m/z 305 [M + H]+, m/z 631 [2M +Na]+; HRESIMS (positive) m/z 305.1038 [M +H]+ (calcd. for C16H17O6, 305.1025); 1H and 13C NMR see Table S5.

(+) (5′S, 6′R) Sporormielone E (5a): []29 D +96.0 (c 0.02, MeOH); ECD (1.9 104 M, MeOH) λmax (△ε): 225 (+11.57), 243 (-19.64), 314 (+3.51) nm.

() (5′R, 6′S) Sporormielone E (5b): []29 D -150.0 (c 0.02, MeOH); ECD (1.9 104 M, MeOH) λmax (△ε): 225 (-9.92), 242 (+15.70), 319 (-2.89) nm.

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4. Materials and methods

4.1 General experimental procedures

Methanol (MeOH) was purchased from Yuwang Industrial Co. Ltd (Yucheng, China). Acetonitrile (MeCN) was obtained from Oceanpak Alexative Chemical Co. Ltd (Gothenburg, Sweden).Ethyl acetate (EtOAc) and Cyclohexane were analytical grade from Fine Chemical Co. Ltd (Tianjin, China).

Melting points were measured on an uncorrected BÜCHIB-545melting point measurement (BÜCHI Labor technik AG, Flawil, Switzerland). UV data were recorded using a JASCO V-550 UV/vis spectrometer (Jasco International Co. Ltd, Tokyo, Japan). IR data were recorded on a JASCO FT/IR-480 plus spectrometer (Jasco International Co. Ltd, Tokyo, Japan). Optical rotations were measured on a JASCO P1020 digital polarimeter (Jasco International Co. Ltd, Tokyo, Japan). ECD spectra were recorded in MeOH using a Chirascan-plus qCD spectrometer (Applied Photophysics Ltd, UK) at room temperature. HRESIMS spectra were obtained on a Waters Synapt G2 TOF mass spectrometer (Waters Corporation, Milford, USA). 1D and 2D NMR spectra were acquired with Bruker AV 400 and Bruker AV 600 spectrometers (Bruker BioSpin Group, Faellanden, Switzerland) using the solvent signals (DMSO-d6: δH2.50/δC39.5) as internal standards. Column chromatography (CC) was carried out on silica gel (200-300 mesh) (Qingdao Haiyang Chemical Group Corporation, Qingdao, China), ODS (50 μm, YMC), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden). TLC was performed on precoated silica gel plates (SGF254, 0.2 mm, Yantai Chemical Industry Research Institute, China). Analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector, an Ultimate 3000 column compartment, an Ultimate 3000 autosampler (Dionex, USA), and an Alltech (Grace) 2000ES evaporative light scattering detector (Alltech USA) using a Phenomenex Gemini C18 column (4.6 × 250 mm, 5 μm). Semi-preparative HPLC was carried out on a Shimadzu LC-6AD system equipped with UV detectors, using a YMC-Pack ODS-A column (10.0 × 250 mm, 5μm). Preparative HPLC was carried out on Shimadzu LC-6AD system equipped with UV detectors, using a Marchal C18 6 μ C18 column (6 μm, 50 × 250 mm). Medium pressure liquid chromatography (MPLC) was performed on ODS (50μm) and equipped with a dual pump gradient system, a UV preparative detector, and a Dr Flash II fraction collector system (Shanghai Lisui E-Tech Co. Ltd, Shanghai, China).

4.2 Fungal materials and fermentation

The strain (40-1-4-1) was isolated from the lichen Cladoniasubulata (L.) Wigg. collected from Changbai Mountain, Jilin province, in August 2006.The strain was identified as Sporormiella sp. by Prof. L.-D. Guo and Prof. D. Hu based on its morphological characteristics and the gene sequence analysis. The ribosomal internal transcribed spacer (ITS) and the 5.8S rRNA gene sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as MK942641.

The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 3 days. Agar plugs were used to inoculate 25 Erlenmeyer flasks (500 mL), each containing 100 mL of potato dextrose broth (PDB).Fermentation was carried out in 200 Erlenmeyer flasks (500 mL), each containing 70 g of rice. Distilled H2O (105 mL) was added to each flask, and the rice was

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soaked overnight before autoclaving at 120 °C for 30 min. After cooling down to the room temperature, each flask was inoculated with 10.0 mL of the seed culture containing mycelia and incubated at 27 ℃ for 50 days.

4.3 Extraction and isolation

The culture was extracted thrice with EtOAc, and the pooled organic solvent was evaporated to dryness under vacuum to afford a crude extract (115.1 g). Then the crude extract was subjected to silica gel CC (4×15 cm) using cyclohexane-MeOH (100:0 and 0:100, v/v) to afford a cyclohexane extract (C, 70.4 g) and a MeOH extract (w, 38.5 g). The MeOH extract (w, 38.5 g) was separated by ODS CC (4 × 30 cm) eluting with MeOH-H2O (30:70, 50:50, 70:30, 100:0, MeOH-CHCl3 1 :1 v/v) to afford 5 fractions (w1-w5). Fraction w1 (19.7 g) was further subjected to MPLC on ODS CC (4 × 45 cm) eluted with a gradient of MeOH-H2O (5:95 to 100:0, v/v) for 800 min at a flow rate of 20 mL/min to afford fractions w1-1 to w1-6. Fraction w1-4 (9.0 g) was subjected to preparative HPLC using MeOH-H2O (18:82, v/v) at a flow rate of 100 mL/min to afford fractions w1-4-1, w1-4-2 (775.3 mg), w1-4-3 (264.6 mg), and 1 (tR: 32.0 min, 5.5 g). Fractions w1-4-2 (775.3 mg) and w1-4-3 (264.6 mg) were combined with w1-5 (1.53 g) were subjected to Sephadex LH-20 using MeOH to afford fractions w1-5-1 to w1-5-7. Fraction w1-5-5 (368.4mg) was subjected to semi-preparative HPLC using MeCN-H2O (18:82, v/v) at a flow rate of 3 mL/min to afford fractions w1-5-5-1 (92.1 mg), w1-5-5-2 (8.6 mg), w1-5-5-3 (18.5 mg), 2 (tR: 28.0 min, 3.0 mg), and 3 (tR: 37.0 min, 4.0 mg). Fraction w1-5-5-3 (18.5 mg) was subjected to semi-preparative HPLC using MeCN-H2O (18:82, v/v) at a flow rate of 3 mL/min to 4 (tR: 46.0 min, 2.0 mg) and 5 (tR: 36.0 min, 5.0 mg).

4.4 X-ray crystallographic analyses

4.4.1 X-ray crystallographic analysis of 1

C1'

C8'

C2'

C3'

C4'C5'

C6'

C7'C1

C2

C3

C4

C5C6

C7

C8

Figure S1 X-ray crystallographic analysis of 1Upon crystallization from MeOH using the vapor diffusion method, yellow needle-like

crystals of 1 were obtained. Data was collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using CuKα radiation (λ = 1.54184 Å). Data reduction was carried out with the diffractometer's software. Crystal data: C16H16O6, M = 304.29, space group monoclinic, P21/n; unit cell dimensions were determined to be a = 8.17188(13) Å, b = 12.40101(16) Å, c = 13.7935(2) Å, α = 90.00 °, β = 91.4883(15) °, γ = 90.00 °, V = 1397.35(4) Å3, Z = 4, Dx = 1.446 g/cm3, F (000) = 640.0, μ (Cu Kα) = 0.937 mm–1. 17789 reflections were collected (9.592° ≤ 2θ ≤ 147.448°), in which 2788 independent unique reflections (Rint= 0.0317, Rsigma= 0.0151) were used in all calculations. Using Olex2, the structure

12

was solved by direct methods using the SHELXS program and refined by the SHELXL program. In the structure refinements, hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. The final refinement gave R1 = 0.0430 (I >2σ(I)), WR2 = 0.1185 (all data), and S = 1.063. Crystallographic data for sporormielone A (1) has been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC1971064. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]).


C5C4

C3 C2 C7

C8C2'

C3'C4'C6 C5'

C6'C7'

C8'

C1'

C1


crystals of 2 were obtained. Data was collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation (λ = 1.54184 Å). Data reduction was carried out with the diffractometer's software. Crystal data: C16H16O6, M = 304.29, space group monoclinic, P21/n; unit cell dimensions were determined to be a = 9.17259(16) Å, b = 11.66700(14) Å, c = 12.81692(17) Å, α = 90.00 °, β = 98.1439(14) °, γ = 90.00 °, V = 1357.79(3) Å3, Z = 4, Dx = 1.489 g/cm3, F (000) = 640.0, μ (Cu Kα) = 0.964 mm–1. 27554 reflections were collected (10.3° ≤ 2θ ≤ 147.528°), in which 2724 independent unique reflections (Rint= 0.0451, Rsigma= 0.0179) were used in all calculations. Using Olex2, the structure was solved by direct methods using the SHELXS program and refined by the SHELXL program. In the structure refinements, hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. The final refinement gave R1 = 0.0411 (I >2σ(I)), WR2 = 0.1139 (all data), and S = 1.047. Crystallographic data for sporormielone B (2) has been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC1971065. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]).

mailto:[email protected]

13


C1 C2

C3 C4C5

C6C7

C8

C1'C2'

C3'C4'

C5'

C6'

C7'

C8'


crystals of 3 were obtained. Data was collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using CuKα radiation (λ = 1.54184 Å). Data reduction was carried out with the diffractometer's software. Crystal data: C16H16O6, M = 304.29, space group monoclinic, P21/n; unit cell dimensions were determined to be a = 8.5190(7) Å, b = 16.0695(13) Å, c = 10.3653(8) Å, α = 90.00 °, β = 102.495(8) °, γ = 90.00 °, V = 1385.4(2) Å3, Z = 4, Dx = 1.459 g/cm3, F (000) = 640.0, μ (Cu Kα) = 0.945 mm–1. 5373 reflections were collected (10.33° ≤ 2θ ≤ 146.832°), in which 2717 independent unique reflections (Rint= 0.0655, Rsigma= 0.0751) were used in all calculations. Using Olex2, the structure was solved by direct methods using the SHELXS program and refined by the SHELXL program. In the structure refinements, hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. The final refinement gave R1 = 0.0767 (I >2σ(I)), WR2 = 0.2219 (all data), and S = 1.178. Crystallographic data for sporormielone C (3) has been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC1971066. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]).


C1C2

C3 C4

C5

C6C7

C8

C1'

C2'

C3'C4' C5'

C6'

C7'

C8'


crystals of 4 were obtained. Data was collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation (λ = 1.54184 Å). Data reduction was carried out with the diffractometer's software. Crystal data:

14

C16H18O7, M = 322.30, space group monoclinic, P21/c; unit cell dimensions were determined to be a = 13.4502(10) Å, b = 7.6047(4) Å, c = 15.7124(13) Å, α = 90.00 °, β = 110.641(9) °, γ = 90.00 °, V = 1504.0(2) Å3, Z = 4, Dx = 1.423 g/cm3, F (000) = 680.0, μ (Cu Kα) = 0.952 mm–1. 9690 reflections were collected (7.022° ≤ 2θ ≤ 147.472°), in which 2968 independent unique reflections (Rint= 0.0557, Rsigma= 0.0476) were used in all calculations. Using Olex2, the structure was solved by direct methods using the SHELXS program and refined by the SHELXL program. In the structure refinements, hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. The final refinement gave R1 = 0.1378 (I >2σ(I)), WR2 = 0.3308 (all data), and S = 1.342.Crystallographic data for sporormielone D (5) has been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC1971067. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]).


C1 C2

C3 C4C5

C7

C8

C6

C3'

C7'

C8'

C2' C1'C6'

C5'

C4'


crystals of 5 were obtained. Data was collected at 100 K on a Rigaku Oxford Diffraction Supernova Dual Source, Cu at Zero equipped with an AtlasS2 CCD using Cu Kα radiation (λ = 1.54184 Å). Data reduction was carried out with the diffractometer's software. Crystal data: C16H16O6, M = 304.29, space group orthorhombic, P212121; unit cell dimensions were determined to be a = 9.1094(2) Å, b = 10.3354(2) Å, c = 14.8756(3) Å, α = 90.00 °, β = 90.00 °, γ = 90.00 °, V = 1400.53(5) Å3, Z = 4, Dx = 1.443 g/cm3, F (000) = 680.0, μ (Cu Kα) = 0.935 mm–1. 9339 reflections were collected (10.422° ≤ 2θ ≤ 147.356°), in which 2783 independent unique reflections (Rint= 0.0344, Rsigma= 0.0281) were used in all calculations. Using Olex2, the structure was solved by direct methods using the SHELXS program and refined by the SHELXL program. In the structure refinements, hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. The final refinement gave R1 = 0.0376 (I >2σ(I)), WR2 = 0.1044 (all data), and S = 1.056. Crystallographic data for sporormielone E (5) has been deposited in the Cambridge Crystallographic Data Center as supplementary publication No. CCDC1971068. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033, or e-mail: [email protected]).

15

5. Chiral separations of 1 and 5

The chiral HPLC separation of compound 1 was separated successfully to obtain 1a [tR: 13.7 min []25 D= +222.6 (c 0.1, MeOH)] / 1b (tR: 18.5 min []27 D = -188.7 (c 0.1, MeOH) by using an EnantioPak OZ-3(5 μm,4.6×250 mm)at the rate of 1.0 mL/min.

Detection wavelength: 254 nmMobile phase: MeCN-H2O 15:85Flow: 1.0 mL/minRelative peak area: 1a = 53.4%

1b = 46.6%

Figure S6 The chiral HPLC chromatogram of 1

The chiral HPLC separation of compound 5 was separated successfully to obtain 5a [tR: 11.7min []29 D= +96.0 (c 0.02, MeOH)] / 5b (tR: 20.0 min []29 D = -150.0 (c 0.02, MeOH) by using an EnantioPak OZ-3 (5 μm,4.6×250 mm) at the rate of 1.0 mL/min.

5a 5b 5


5b = 52.7%

Figure S7 The chiral HPLC chromatograms of 5

16

6. Quantum chemical calculations of 1–5

6.1 Quantum chemical VCD calculations of 1

(7R, 5'R, 6'R)-1 (7S, 5'S, 6'S)-1

1

3

5

7

8 1'8'

7'

5'3'

4'

6'

OO

O

OH

HO

OH OO

O

OH

OH

HO5'

6'7

H H

Figure S8 Structures of (7R, 5′R, 6′R)-1 and (7S, 5′S, 6′S)-1

The IR and VCD spectra of 1a and 1b in CD3OD solution were measured at a resolution of 4 cm-1 using Synchro cell (2.75 sec per cycle) on a BioTools ChiralIR-2X VCD spectrometer. The CD3OD solution of 1a and 1b were injected into a cell (150 mm in optical length) with BaF2 windows. The Zn Sephotoelastic modulator of the instrument was set to 1400 cm-1. In order to improve the signal-to-noise ratio, these spectra were measured for 5 h. A baseline correction by subtracting the solvent reference spectrum collected under the same conditions was carried out for the IR spectra. The molecule of (7R, 5′R, 6′R)-1 was converted into SMILES codes before their initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09. Further optimization at the B3LYP/6-31G(d) level determined the dihedral angles. From this, three stable conformers of (7R, 5′R, 6′R)-1 were determined. These three stable conformers were used for the VCD calculations, which were performed with Gaussian09 [B3LYP/6-311++G(2d,p)]. The solvent effect was accounted by the polarizable-conductor calculation model (PCM, MeOH as the solvent). Calculated frequencies were empirically scaled using a 0.9984 scaling factor, and the calculated spectra were obtained with Boltzmann weighting. Comparisons of the experimental and calculated spectra were performed with the Origin software (Table S6). [1]

17

1900 1800 1700 1600 1500 1400 1300 1200

0.0

0.5

1.0

A

Expt. IR-1a

Expt. IR-1b

-1.5

-0.5

0.0

0.5

1.0

-1.0

ΔA*1

04

Expt. VCD-1b

Expt. VCD-1a

Wavenumber/cm-1

Figure S9 The experimental IR and VCD spectra of 1a and 1b in the CD3OD solution

1900 1800 1700 1600 1500 1400 1300 1200

A

Wavenumber/cm-1

1

2

3

4 5 67

8

9

10

11

2 3

4 5 67

8

9

10

11

Expt. VCD-1aCalcd. VCD-7R, 5′R, 6′R

1

(A)

Wavenumber/cm-1

Calcd. IR-7R, 5′R, 6′RExpt. IR-1a

A

1

2

3

4

56

1

23

4

56

1900 1800 1700 1600 1500 1400 1300 1200

1900 1800 1700 1600 1500 1400 1300 1200

Expt. VCD-1bCalcd. VCD-7S, 5′S, 6′S

Wavenumber/cm-1

1

1

23

4 5

6

78

9

23

4 5 6

78

9

A

(B)

Wavenumber/cm-1

Calcd. IR-7S, 5′S, 6′SExpt. IR-1b

A

1

2

3

4

56

1

23

4

56

1900 1800 1700 1600 1500 1400 1300 1200

Figure S10 (A) Expt. VCD-1a vs Calcd. VCD-(7R, 5′R, 6′R)-1 and Expt. IR-1a vs Calcd. IR-(7R, 5′R, 6′R)-1; (B) Expt. VCD-1b vs Calcd. VCD-(7S, 5′S, 6′S)-1 and Expt. IR-1a vs Calcd. IR-(7S, 5′S, 6′S)-1

18

Table S6 Stable conformers of (7R, 5′R, 6′R)-1 at the B3LYP/6-31G(d) level in MeOHConformers Contribution %

C1 87.61C2 12.38C3 0.01

conformer 2 (12.38%)conformer 1 (87.61%) conformer 3 (0.01%)

Figure S11 Stable conformers of (7R, 5′R, 6′R)-1

6.2 Quantum chemical ECD calculations of 1-5

6.2.1 Quantum chemical ECD calculations of 1The molecules of (7R, 5′R, 6′R)-1 and (7S, 5′S, 6′S)-1 were converted into SMILES codes

before their initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09.[2] Further optimization at three levels [B3LYP/6-31g(d), B3LYP/6-311+G**, APFD/6-31g(d)] determined the dihedral angles, respectively. From this, three stable conformers were determined as shown in Tables S6-S8. The optimized conformers were used for the ECD calculations, which were performed with Gaussian09 using B3LYP/TZVP, B3LYP/def2TZVP, APFD/6-311++g(2d,p), respectively. The solvent effect wasaccounted by the polarizable-conductor calculation model (IEFPCM, methanol as the solvent). Comparisons of the experimental and calculated spectra were performed with SpecDis software.[3-4] This was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and Boltzmann weighting of the spectra, Figure S14.

Table S7 Stable conformers of (7R, 5′R, 6′R)-1 at the B3LYP/6-311+G** level in methanol

Conformers Contribution %

C1 64.40C2 35.55C3 0.05

19

conformer 2 (35.55%)conformer 1 (64.40%) conformer 3 (0.05%)

Figure S12 Stable conformers of (7R, 5′R, 6′R)-1 (the relative populations are in parentheses)

Table S8 Stable conformers of (7R, 5′R, 6′R)-1 at the APFD/6-31g(d) level in methanol


C1 72.32C2 27.63C3 0.05

conformer 1 (72.32%) conformer 2 (27.63%) conformer 3 (0.05%)

Figure S13 Stable conformers of (7R, 5′R, 6′R)-1 (the relative populations are in parentheses)

200 300 400

-30

-20

-10

0

10

20

30

wavelength(nm)

Expt. 1a Expt. 1b Method1 (7R,5R',6R')-1 Method2 (7R,5R',6R')-1 Method3 (7R,5R',6R')-1

200 300 400

-30

-20

-10

0

10

20

30

Expt. 1a Expt. 1b Method1 (7S,5S',6S')-1 Method2 (7S,5S',6S')-1 Method3 (7S,5S',6S')-1

wavelength(nm)

Figure S14 Experimental ECD of 1a and 1b and calculated ECD of (7R, 5′R, 6′R)-1 and (7S, 5′S,

6′S)-1 using B3LYP/TZVP // B3LYP/6-31G(d) (method 1), B3LYP/def2TZVP // B3LYP/6-

311+G** (method 2), and APFD/6-31g(d)/6-311++G(2d,p) // APFD/6-31g(d) (method 3) three

DFT-methods (UV correction = -23 nm, bandwidth σ = 0.3 eV)

20

6.2.2 Quantum chemical ECD calculations of 2The molecules of (2′S,3′R)-2 and (2′R,3′S)-2 were converted into SMILES codes before their

initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09. Further optimization at the B3LYP/6-31g(d) level determined the dihedral angles. From this, only one stable conformer was determined. The optimized conformer was used for the ECD calculations, which was performed with Gaussian09using B3LYP/TZVP.[2] The solvent effect was accounted by the polarizable-conductor calculation model (IEFPCM, acetonitrile as the solvent). Comparisons of the experimental and calculated spectra were performed with SpecDis software.[3-

4] This was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and Boltzmann weighting of the spectra.

OOHO

HOOH

HO 7'8'1'

3' 6'5'4'

87

53

1OH

OHOH

OOHO

2'S, 3'R 2'R, 3'SFigure S15 Structures of (2′S, 3′R)-2 and (2′R,3′S)-2

Table S9 Stable conformer of (2′S,3′R)-2 at the B3LYP/6-31G(d) level in acetonitrile

Conformer Contribution %

C1 100.00

conformer 1

Figure S16 Stable conformer of (2′S,3′R)-2

21

200 300 400-60

-30

0

30

60

wavelength(nm)

Calc.2'S, 3'R-2 Calc.2'R, 3'S-2

Calcu

late

d va

luae

Figure S17 Calculated ECD of (2′S,3′R)-2 and (2′R,3′S)-2 (UV correction = -5.8 nm, bandwidth σ

= 0.3 eV)

6.2.3 Quantum chemical ECD calculations of 3The molecules of (2′S,3′R)-3 and (2′R,3′S)-3 were converted into SMILES codes before their

initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09.[2] Further optimization at the B3LYP/6-31g(d) level determined the dihedral angles. From this, one stable conformer was determined. The optimized conformer was used for the ECD calculation, which was performed with Gaussian09 using B3LYP/TZVP. The solvent effect was accounted by the polarizable-conductor calculation model (IEFPCM, acetonitrile as the solvent). Comparisons of the experimental and calculated spectra were performed with SpecDis software.[3-4] This was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and Boltzmann weighting of the spectra.

5'

7'8'1'

3'4'

87 OH OO

OHHO O

53

1

O OHOH

OO HO

2'S, 3'R 2'R, 3'S

Figure S18 Structures of (2′S, 3′R)-3 and (2′R,3′S)-3

Table S10 Stable conformer of (2′S,3′R)-3 at the B3LYP/6-31G(d) level in acetonitrile

Conformer Contribution %

C1 100.00

22

conformer 1

Figure S19 Stable conformer of (2′S,3′R)-3

200 300 400 500

-20

-10

0

10

20

wavelength(nm)

Calc.2S3R Calc.2R3S

Calcu

late

d Va

lue

Figure S20 Calculated ECD of (2′S,3′R)-3 and (2′R,3′S)-3 (UV correction =0 nm, bandwidth σ =

0.3 eV)

6.2.4 Quantum chemical ECD calculations of 4The molecules of (5′R,6′S)-4 and(5′S,6′R)-4 were converted into SMILES codes before their

initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09.[2] Further optimization at the B3LYP/6-31g(d) level determined the dihedral angles. From this, the most stable conformer was determined. The optimized conformers were used for the ECD calculations, which were performed with Gaussian09 using B3LYP/TZVP. The solvent effect wasaccounted by the polarizable-conductor calculation model (IEFPCM, acetonitrile as the solvent). Comparisons of the experimental and calculated spectra were performed with SpecDis software.[3-4] This was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and Boltzmann weighting of the spectra.

23

24

1

3 5OHO

OH

O OOH

6

7

8

4' 5'

8'

2' 1'

3'

7'

5'R, 6'S

O OHOH

OO HO

5'S, 6'R

H H

Figure S21 Structures of (5′R, 6′S)-4 and (5′S,6′R)-4

Table S11 Stable conformer of (5′R,6′S)-4 at the B3LYP/6-31G(d) level in acetonitrile


C1 100.00

conformer 1

Figure S22 Stable conformer of (5′R,6′S)-4

200 300 400 500

-100

-50

0

50

100

wavelength(nm)

Calc.5R6S Calc.5S6R

Calcu

late

d Va

lue

Figure S23 Calculated ECD of (5′R,6′S)-4 and (5′S,6′R)-4 (UV correction =-3.6 nm, bandwidth σ

= 0.3 eV)

6.2.5 Quantum chemical ECD calculations of 5The molecules of (5′R,6′S)-5 and (5′S,6′R)-5 were converted into SMILES codes before their

initial 3D structures were generated with CORINA version 3.4. Conformer databases were generated in CONFLEX version 7.0 using the MMFF94s force-field, with an energy window for acceptable conformers (ewindow) of 5 kcal/mol above the ground state, a maximum number of conformations per molecule (maxconfs) of 100, and an RMSD cutoff (rmsd) of 0.5Å. Then each acceptable conformer was optimized with HF/6-31G(d) method in Gaussian09. Further

24

optimization at the B3LYP/6-31g(d) level determined the dihedral angles. From this, two most stable conformers were determined. The optimized conformers were used for the ECD calculations, which were performed with Gaussian09 using B3LYP/TZVP.[2] The solvent effect was accounted by the polarizable-conductor calculation model (IEFPCM, methanol as the solvent). Comparisons of the experimental and calculated spectra were performed with SpecDis software.[3-

4] This was also used to apply a UV shift to the ECD spectra, Gaussian broadening of the excitations, and Boltzmann weighting of the spectra.

7'8'

1'

4' 6'3'

8

7

53

1 OH

OHHO

O

O

O O

O

OOH

OH

OH

5'R, 6'S 5'S, 6'R

Figure S24 Structures of (5′R, 6′S)-5 and (5′S,6′R)-5

Table S12 Stable conformers of (5′R,6′S)-5 at the B3LYP/6-31G(d) level in MeOH


C1 98.69C2 1.31

conformer 1 (98.69%) conformer 2 (1.31%)

Figure S25 Stable conformers of (5′R,6′S)-5 (the relative populations are in parentheses)

200 220 240 260 280 300 320 340 360 380 400

-20

-15

-10

-5

0

5

10

15

20

wavelength(nm)

Expt. 5a Expt. 5b Calc. 5'R,6'S Calc. 5'S,6'R

Figure S26 Experimental ECD of 5a and 5b and calculated ECD of (5′S,6′R)-5 and (5′R,6′S)-5

(UV correction = - 3.6 nm, bandwidth σ = 0.3 eV)

25

7. HPLC-ECD coupling analyses of 1-5

The HPLC-ECD coupling analysis was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector, an Ultimate 3000 column compartment, an Ultimate 3000 autosampler (Dionex, USA), and an ECD detector (JASCO CD-4095). The ECD detector was operated in single wavelength mode and the procedure forHPLC-ECD coupling analysis was the following: 1) Two wavelengths (> 250 nm) were selected as the ECD detections according to the predicted ECD curves of relevent enantiomers, which were obtained using the same ECD calculation method as compounds 1 and 5; 2) The corresponding enantiomeric absolute configurations were determined based on the comparasion of the change of Cotton effects (CEs) signs of the experimental trends and that of the calculated ECD curve at selective wavelengths.

7.1 HPLC-ECD coupling analyses of 1 and 5

Since the absolute configurations of compounds 1a/1b and 5a/5b have been determined, HPLC-ECD coupling analyses of 1 and 5could be used to evaluate the accuracy of this system. Comparing the calculated and measured ECD curves (Figures S14 and S26), the calculated ECD curves of 1 and 5 obtained from B3LYP/TZVP // B3LYP/6-31G(d) method were similar to those of experimental data (especially in the ≥ 250 nm region), suggesting that this calculated method was appropriate for ECD calculation of these compounds. Thus, 275 nm and 325 nm were selected as the detected wavelengths in HPLC-ECD coupling analysis of 1 (Figure S27), and 250 nm and 300 nm were selected as the detected wavelengths in HPLC-ECD coupling analysis of 5 (Figure S28), respectively. The results were consistent with the VCD and ECD analyses of 1a/1b and 5a/5b.

275 nm

1a

1b 325 nm 1a

1b(-) CE

(+) CE (+) CE

(-) CE

(A)

(B)

Cal

cula

t ed

Valu

e

Figure S27(A): Calculated ECD spectra of (7R, 5′R, 6′R)-1 and (7S, 5′S, 6′S)-1 using B3LYP/TZVP// B3LYP/6-31g(d) (UV correction = -23 nm, bandwidth σ = 0.3 eV) (B): HPLC-

ECD coupling analysis of 1 at 275 nm and 325 nm.

26

200 300 400-20

-15

-10

-5

0

5

10

15

20

wavelength(nm)

Calc.5'R, 6'S Calc.5'S, 6'R

250 nm

5a

5b 300 nm 5a

5b(-)CE

(+)CE(+)CE

(-)CE

(A)

(B)

250 nm300 nm

Ca l

cula

ted

Valu

e

Figure S28 (A): Calculated ECD spectra of (5′R,6′S)-5 and (5′S,6′R)-5 using B3LYP/TZVP// B3LYP/6-31g(d) (UV correction = - 3.6 nm, bandwidth σ = 0.3 eV); (B): HPLC-ECD coupling

analysis of 5 at 250 nm and 300 nm.

27

7.2 HPLC-ECD coupling analysis of 2

The chiral HPLC analysis of compound 2 was analyzed and showed two peaks [2a (tR: 12.5 min) / 2b (tR: 16.7 min)] by using an EnantioPak OZ-3(5 μm, 4.6 × 250 mm) at the rate of 1.0 mL/min (Figure S29).

Detection wavelength: 254 nmMobile phase: MeCN-H2O 25:75Flow: 1.0 ml/minRelative peak area: 2a = 49.5%

2b = 50.5%

2a

2b


Based on the analysis of calculated ECD curves, 260 nm and 300 nm were selected as the detected wavelengths in HPLC-ECD coupling analysis of 2 (Figure S30).

260 nm 300 nm

2a

2b 2a

2b(-) CE

(+) CE (+) CE

(-) CE

(A)

(B)

Cal

cual

ted

V alu

e

Figure S30 (A): Calculated ECD spectra of (2′S,3′R)-2 and (2′R,3′S)-2 (UV correction = -5.8 nm, bandwidth σ = 0.3 eV) (B): HPLC-ECD coupling analysis of 2 at 260 nm and 300 nm.

28


The chiral HPLC analysis of compound 3 was analyzed and showed two peaks [3a (tR: 32.1 min) / 3b (tR: 36.2 min)] by using an EnantioPak OZ-3 (5 μm, 4.6 × 250 mm) at the rate of 1.0 mL/min (Figure S31).


3b = 49.5%

3a 3b


Based on the analysis of calculated ECD curves, 300 nm and 360 nm were selected as the detected wavelengths of HPLC-ECD coupling analysis of 3 (Figure S32).

300 nm 360 nm

3a

3b

3a

3b(-) CE

(+) CE

(+) CE

(-) CE

(A)

(B)

Cal

cual

ted

Valu

e

Figure S32 (A): Calculated ECD spectra of (2′S,3′R)-3 and (2′R,3′S)-3 (UV correction = 0 nm, bandwidth σ = 0.3 eV) (B): HPLC-ECD coupling analysis of compound 3 at 300 nm and 360 nm.

29


The chiral HPLC analysis of compound 4 was analyzed and showed two peaks [4a (tR: 9.3 min) / 4b (tR: 10.5 min)] by using an EnantioPak OZ-3(5 μm,4.6 × 250 mm)at the rate of 1.0 mL/min (Figure S33).

Detection wavelength: 254 nmMobile phase: MeCN-H2O 30:70Flow: 1.0 ml/minRelative peak area: 4a = 51.5%

4b = 48.5% 4a 4b


Based on the analysis of calculated ECD curves, 300 nm and 360 nm were selected as the detected wavelengths of online-ECD of compound 4 (Figure S34).

(+) CE(+) CE

(-) CE

300 nm

4a

4b 360 nm4a

4b(-) CE

(A)

(B)

Cal

cula

t ed

Valu

e

Figure S34 (A): Calculated ECD spectra of (5′R,6′S)-4 and(5′S,6′R)-4 (UV correction = - 3.6 nm, bandwidth σ = 0.3 eV) (B): HPLC-ECD analysis of compound 4 at 300 nm and 360 nm.

30

8. The process of obtaining unlabeled and labeled 1Firstly, the fungus strain was cultivated in 100 mL of PDB (potato 200 g/L, glucose 20 g/L)

at 28 °C in dark swing bed (200 rpm) for 3 days to produce the first seed culture. Then, 5 mL of the first seed culture was transferred into another 100 mL of PDB, which was cultured in the same growing condition for one day to produce the second seed culture. Next, 40 mL of the second seed culture was transferred into 30 Erlenmeyer flasks of (500 mL),each containing 100 ml of ME medium (malt extract 20 g/L, peptone 1 g/L, glucose 20 g/L) and cultured in the same conditions. After 7 days, the culture broth (3 L) was extracted with ethyl acetate and condensed by rotary evaporator. The extract (2.72 g) was then fractionated by column chromatography over ODS using a stepwise elution of MeOH-H2O [5% - 15% (30 min) - 22% (30 min) - 35% (30 min) - 50% (30 min) - 100% (30 min)] to afford 12 fractions (ME-1 to ME-12). Fraction ME-4 (379.8mg) was subjected to preparative HPLC using MeOH-H2O (20: 80, v/v) at a flow rate of 3 mL/min to obtain 1(tR: 17.0 min, 132.3 mg).

[1-13C]acetate and [2-13C]acetate were added to 100 mL of ME media (20 mg isotope-labeled substrates/100 mL of ME medium) on the first, third, and fifth days. The amount of fermentation was 2 L. The mixture was extracted with ethyl acetate and condensed by rotary evaporation on the seventh day. The labeled compound 1 was purified in the same manner as that described above. Finally, [1-13C]acetate-derived 1 and[2-13C]acetate-derived 1 were separated producing 24.2 mg and 23.1 mg, respectively.

Table S13 The NMR data of [1-13C]acetate-derived 1 in DMSO-d6 (100 MHz for 13C)

position δ(ppm)integral area

(S1)

integral area of labeled [1-13C] acetate (S2)

relative integral

area (S2/S1)Incorporation

1 14.5 2.54 8.52 3.35 2.35

2 136.0 1.04 1.09 1.04 0.05

3 145.5 1.29 4.22 3.27 2.27

4 177.3 0.81 0.86 1.06 0.06

5 148.2 0.60 2.26 3.77 2.77

6 125.4 0.81 0.83 1.02 0.02

7 46.8 1.84 6.41 3.48 2.48

8 29.3 2.68 2.84 1.06 0.06

1' 8.1 2.44 9.67 3.96 2.96

2' 136.2 1.12 1.18 1.05 0.05

3' 199.9 1.00 3.17 3.17 2.17

4'* 196.5 1.00 1.00 1.00 0.00

5' 86.9 1.54 6.37 4.14 3.14

6' 59.3 2.27 2.49 1.09 0.10

7' 167.7 1.09 3.87 3.55 2.55

8' 16.6 2.47 2.66 1.07 0.08

Integral area S1 is the signal intensity of unlabeled, natural abundance carbon.

Integral area S2 is the signal intensity resulting from [1-13C] acetate labeling.

Incorporation = (S2-S1)/S1

*the unlabeled carbon is considered the standard one (integral area S1 is defined as 1).

31

Table S14 The NMR data of [2-13C]acetate-derived 1 in DMSO-d6 (100 MHz for 13C)

position δ(ppm)integral

area（S1）

integral area of labeled

[2-13C] acetate (S2)

relative integral

area (S2/S1)Incorporation

1 14.5 2.64 2.60 0.99 -0.02

2 136.0 1.33 2.59 1.95 0.95

3 145.5 1.37 1.38 1.01 0.01

4 177.3 0.97 1.86 1.92 0.92

5 148.2 0.75 0.73 0.97 -0.03

6 125.4 0.93 2.10 2.26 1.26

7 46.8 1.98 1.81 0.91 -0.09

8 29.3 2.64 5.37 2.03 1.03

1' 8.1 2.30 2.59 1.12 0.13

2' 136.2 1.13 2.37 2.10 1.10

3'* 199.9 1.00 1.00 1.00 0.00

4' 196.5 1.00 1.71 1.71 0.71

5' 86.9 1.58 1.64 1.04 0.04

6' 59.3 2.45 5.06 2.07 1.07

7' 167.7 1.17 1.18 1.01 0.01

8' 16.6 2.57 5.21 2.03 1.03

Integral area S1 is the signal intensity of unlabeled, natural abundance carbon.

Integral area S2 is the signal intensity resulting from[2-13C] acetate labeling.

Incorporation = (S2-S1)/S1

*the unlabeled carbon is considered the standard one (integral area S1 is defined as 1).

32

9. Biosynthetic investigation of 1

9.1 Whole genome sequencing and analysis of Sporormiella sp. 40-1-4-1

The whole genome sequencing of Sporormiella sp. 40-1-4-1 was performed by Sangon Biotech Co., Ltd. (Shanghai, China) with an Illumina HiSeq 2500 system. The sequence assembly was used SOAPdenovo version2.04(http://soap.genomics.org.cn/soapdenovo.html)to produce 656 contigs covering approximately 47.3 Mb. Gene prediction was then performed with AUGUSTUS (http://bioinf.uni-greifswald.de/webaugustus/) and manually revised according to coding sequences obtained from transcriptome and homologous genes found in the NCBI database.

unknown genePKS

additional biosynthetic genetransport-related gene

g8333P450

g8446

g534

g1147

g9989

g12116

g7393

g13710

g12668

g11198

Figure S35 10 NP-PKS genes in the genome of Sporormiella sp. 40-1-4-1

33

Ⅰ

OH

HO

2

7

OH

O

orsellinic acid

ⅡOH OH

OHHO

2

71,3,6,8-THN

Ⅲ

OH O

HOO

OH6

11

Emodin

Ⅳ

OH OH

OHO

O

OH2

7YWA1

Ⅴ

OH O

HO

OH

OOH

O4

9

Norsolorinic acid

Figure S36 Analysis of PT domains of 10 NP-PKS genes in the genome of Sporormiella sp. 40-1-4-1

34

O

O

O

OH

HO

2

7zearalenone

OH

HO

2

7

OH

O

orsellinic acid

OH

HO

OH

O2

7

3,5-dimethyl orsellinic acid

OH

HO

H

O

3-methylorcinaldehyde

2

7

1

2

34

Figure S37 Analysis of PT domains of 4 NP-PKS genes with closed evolutionary relationship

9.2 RNA preparation and reverse transcription PCR

After screening the cultured media, it was found that Sporormiella sp. 40-1-4-1 can produce compound 1 using ME medium (malt extract 20 g/L, peptone 1 g/L, glucose 20 g/L) with shaking at 180 rpm at 28°C for 7 days, and does not produce compound 1 using YMG medium (malt extract 10 g/L, yeast extract 4 g/L, glucose 10 g/L) with shaking at 180 rpm at 28°C for 7 days.

Mycelia were collected from the ME medium and the YMG medium as described above, and ground in liquid nitrogen with a mortar. RNA was then isolated using an RNeasy Plant Mini Kit (QIAGEN) according to the manufacture’s protocol. To eliminate genomic DNA, RNA samples were treated with RNase-free DNase I (TaKaRa). The first strand cDNA was synthesized with the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa). To suppress interference from genomic DNA, all the primers used for RT-PCR flank an intron, so that a smaller amplicon should be generated from total RNA compared to that from genomic DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference.

35

Figure S38 Sporormiella sp. 40-1-4-1 under 1 productive (maltose medium) and non-productive (YMG medium) conditions

g833

3g8

446

g534

g114

7g9

989

g121

16g7

393

g137

10

g126

68

g111

98

-5

0

5

10

log 2

Fold

cha

nge

Figure S39 Expression of 10 NR-PKS genes in reverse transcription PCR (RT-PCR) under maltose medium and YMG medium

The expression levels were quantified using the DESeq with an FDR< 0.05 and an absolute value of fold-change≥2. M/C, ME medium (M) in comparison to YMG medium (Y); RPKM, Reads PerKilo base of exon per Million mapped reads; Non, unexpressed.

Table S15 Differential expression analysis of genes surrounding g7393

Expression (RPKM) Log2Fold Change Expression (RPKM) Log

2Fold Change

GeneM Y (M/Y)

GeneM Y (M/Y)

g7384 1287.487 0.000 11.384 g7392 809.206 0.000 12.036

g7385 912.391 0.000 10.625 g7393 1193.512 3.467 8.427

g7386 291.850 1.541 7.565 g7394 50.698 40.647 0.319

g7387 3863.891 4.045 9.900 g7395 48.284 42.766 0.175

g7388 360.342 0.000 9.493 g7396 35.945 27.740 0.374

36

g7389 1945.760 0.000 13.302 g7397 15.379 6.453 1.253

g7390 976.321 3.178 10.877 g7398 0.000 1.926 -2.844

g7391 186.788 0.000 9.921

9.3 Construction of gene inactivation mutants

For production of 1, the Cas9-expressing strain (JS1001)[5] and gene deletion strain were grown in rice (rice 70 g, H2O 105 mL) for 11 days.

Gene disruption in JS1001 was performed by CRISPR-Cas9 system established by our laboratory.[5] The gene (g7393) had been deleted.

For preparation of in vitro transcriptional gRNA, the gRNA cassettes containing T7 promoter, protospacer sequence and synthetic gRNA scaffold for targeting genes were PCR amplified from plasmid pUCm-gRNAscaffold-eGFP using the primers listed in Table S16, and inserted in pUCm-T to give the corresponding plasmids shown in Table S17. These vectors were used as templates for PCR amplification by primers pUCm-F/gRNA-R, the resulting PCR products were used for in vitro transcription of gRNA with the T7 RiboMAXTM Express Large Scale RNA Production System (Promega, China).

The linear neo maker gene cassette was amplified from plasmid pBSKII-PtrPC-neo-TtrPC using primers PtrpC-XbaІ-F/TtrpC-HindШ-R listed in Table S16.

Preparation and transformation of JS1001 protoplast, strain validation by genotyping PCR were performed according to the method reported previously.[5] All of the mutants used in the work are listed in Table S18.

Restriction sites are indicated by bold letters, target sequences of genes controlled by T7 promoter are underlines.

Table S16 Primers used for constructing recombinant plasmidsPrimer Sequence (5' to 3') UsagePtrpC-XbaІ-F GCTCTAGAGCGCAATTAACCCTCACTAA

TtrpC-HindШ-R CCCAAGCTTCAGGGCTGGTGACGGAATTTTCATAG

Amplification of PtrPC-neo-TtrPC from pBSKII-PtrPC-neo-TtrPC plasmid.

gRNA-g7393-F TAATACGACTCACTATAGGCCCTCTTCCGAGTCTGTAGTTTTAGAGCTAGAAATAGC

Cloning of gRNA cassette for construction of pUCm-gRNA-g7393

Table S17 Plasmids used in this studyPlasmids Characteristics Source

pBSKII-PtrPC-neo-TtrPC Plasmid containing PtrPC-neo-TtrPC maker cassette, (AmpR) Zheng, Y. M. et al.[5]

pUCm-T E. coli cloning vector, (AmpR)Sangon Biotech Co., Ltd.

pUCm-gRNAscaffold-eGFP PUCm-T containing gRNA scaffold, (AmpR) Zheng, Y. M. et al.[5]

pUCm-gRNA-g7393 PUCm-T containing gRNA-g7393scaffold, (AmpR) This work

37

Table S18 Strains used in the studyStrains Characteristics Source

E. coli DH5α Host for general plasmid cloning TaKaRa

E. coli BL21–Codon Plus (DE3) Host for gene expression TaKaRa

Sporormiella sp. 40-1-4-1 Wild-type strain Lab stock

JS1001 Cas9-expressing Sporormiella sp. 40-1-4-1 Zheng, Y. M. et al.[5]

Δg7393-JS1003 G7393 deletion mutant of JS1001 This work





Figure S40 Metabolite analysis of JS1001 and Sporormiella sp. 40-1-4-1 (wild type) (The chromatograms were monitored with evaporative light scattering detector (ELSD))

Figure S41 Metabolite analysis of g7393 deletion mutants (The chromatograms were monitored with evaporative light scattering detector (ELSD). (The mobile phase was composed of water with

0.1% formic acid (A) and MeOH with 0.1% formic acid (B). 20%-20% B (0-2 min), 20%-50% B (2-32 min), and 50%-100% B (32-65 min) with the flow rate of 1 mL min-1))

38

Figure S42 HPLC-ELSD and UHPLC-HRMS analyses of g7393 deletion mutant and compounds 1-5 (HPLC mobile phase was composed of water with 0.1% formic acid (A) and acetonitrile with 0.1%

formic acid (B). 20%-20% B (0-2 min), 20%-50% B (2-32 min), and 50%-100% B (32-65 min) with the flow rate of 1 mL min-1; UPLC mobile phase was composed of water with 0.1% formic acid (A)

and acetonitrile (B). 5%-5% B (0-2 min), 5%-100% B (2-15 min), 100%-100% B (15-20 min), 100%-5% B (20-21 min), and 5%-5% B (21-30 min) with the flow rate of 0.35 mL min-1)

39

Table S19 Putative functions of genes surrounding g7393

GeneAmino

acidsProtein homologue /origin Similarity/identity (%) Proposed function

g7384 315B8M9K8.1/Talaromyces

stipitatus ATCC 1050082/72

hypothetical protein

AN0524.2

g7385 265B8M9K3.1/Talaromyces

stipitatus ATCC 1050060/42 reductase (TropE)

g7386 259

XP_002481997.1/

Talaromyces stipitatus

ATCC 10500

61/46 esterase (TropF)

g7387 44XP_658132.1/Aspergillus

nidulans FGSC A460/39

aspyridones efflux protein

(ApdF)

g7388 109

XP_001243188.1/Coccidioid

es immitis

RS

65/47hypothetical protein

CIMG_07084

g7389 336XP_002487767.1/Talaromyc

es stipitatus ATCC 1050074/59

2-oxoglutarate-dependent

dioxygenase (TropC)

g7390 128XP_681577.1/Aspergillus

nidulans FGSC A456/35 predicted protein

g7391 486

XP_002487359.1/


ATCC 10500

58/41 transcription factor

g7392 481XP_002481992.1/Talaromyc

es stipitatus ATCC 1050067/49 FMO (Trop B)

g7393 2590

XP_002487778.1/


ATCC 10500

60/43 NR-PKS (Trop A)

9.4 Heterologous expression of g7393 in Aspergillus oryzae NSAR1

9.4.1 Removal of intron in g7393The g7393 was amplified from the genomic DNA of Sporormiella sp. 40-1-4-1, and then

inserted into the linearized empty vector pTAex3 under the regulation of the amyB promoter to yield the recombinant plasmid pTAex3-g7393. Genetic analysis of g7393 revealed that two introns are in g7393. To remove these introns, primers listed in Table S20 were designed at both sides of the introns and used for PCR amplification using pTAex3-g7393 as the template. After that, the linear gene segments were transformed to E. coli to generated the intronless plasmid pTAex3-g7393intronless.

40

Table S20 Primers used in this studyPrimer name Sequence(5’to 3’)

pTAex3-g7393-F TCGAGCTCGGTACCCTATCGCACGTCGACATGCCT

pTAex3-g7393-R CTACTACAGATCCCCTCATCCACGCAGAAATCCATCG

pTAex3-g7393-intronless-F1 CTCGGGATAGGAGTCCATTG

pTAex3-g7393-intronless-R1 CAATGGACTCCTATCCCGAGGCGTATACCTCAGTCCTCTC

pTAex3-g7393-intronless-F2 TCCTTCCGGACCTCGATGGAACCCTATCATGGTGTCCAGT

pTAex3-g7393-intronless-R2 TCCATCGAGGTCCGGAAG

9.4.2 Transformation of A.oryzae NSAR1The transformant strains of A. oryzae NSAR1 were obtained via PEG-mediated

transformation of protoplasts. 100 μL spore suspension of the parent strain was inoculated into 10 mL DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.5% KH2PO4) and grown at 200 rpm and 30 °C for two days, which were then transferred into 100 mL DPY medium and cultured for one more day. Mycelia were harvested by filtration and digested using the Yatalase (Takara, Japan) solution (1% Yatalase, 0.6 M (NH4)2SO4, 50 mM maleic acid, pH 5.5) to remove cell walls. After three hours of reciprocal shaking at 30 °C, the mixture was subjected to filtration and the protoplast suspension was collected. After centrifugation at 1500 rpm for 10 min, protoplasts were washed once with TF Solution 2 (1.2 M sorbitol, 50 mM CaCl2·2H2O, 35 mM NaCl, 10 mM Tris-HCl, pH 7.5) and then the concentration was adjusted to around 1.0× 107 cells mL-1 with TF Solution 2. 10 ng plasmids (~ 10-20 μL) and 200 μL protoplast suspension were gently mixed and placed on ice for 30 min, followed by addition of 1.35 mL TF Solution 3 (60% PEG4000, 50 mM CaCl2·2H2O, 10 mM Tris-HCl, pH 7.5) in three times. After incubation at room temperature for 20 min, 5 mL TF Solution 2 was added. Following centrifugation at 1500 rpm for 10 min, the precipitate was resuspended in 200 μL TF Solution 2 and spread on the under-layer selective medium, which was covered with the upper-layer selective medium. The selective medium was composed of 0.2% NH4Cl, 0.1% (NH4)2SO4, 0.05% KCl, 0.05% NaCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O, 2% glucose, 1.2 M sorbitol and 1.5% agar for under-layer (or 0.8% agar for upper-layer) supplemented with 0.15% methionine, 0.1% arginine and 0.01% adenine based on the plasmids used. And the transformants could be obtained after incubation at 30°C for 3-5 days.

9.4.3 Fermentation of the A. oryzae NSAR1 transformant and analysis of metabolites

Mycelia from solid culture in potato dextrose agar were inoculated into 10 mL DPY medium and cultured at 200 rpm and 30°C for two days as the seed broth. Then the broth was transferred into 100 mL modified CD medium and grown at 200 rpm and 30 °C for 1-4 days. The fermentation broth were collected and extracted with ethyl acetate (EtOAc). And the crude extract was resuspended in methanol for HPLC analysis. The mobile phase was composed of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). For analysis of extracellular metabolites, the samples were subjected to a linear gradient elution of 10%-10% B (0-2 min), 10%-40% B (2-32 min), and 40%-100% B (32-45 min) with the flow rate of 1 mL min-1 (Figure S43).

41

Figure S43 HPLC analysis of transformed and untransformed A.oryzae NSAR19.4.4 Isolation, purification, and identification of new metabolites vs. untransformed A. oryzae NSAR1

Mycelia from 5 L culture of the A. oryzae NSAR1 transformant harboring g7393 was harvested and extracted with EtOAc，and the pooled organic solvent was evaporated to dryness under vacuum to afford a crude extract. Then the crude extract was subjected 60% CH3OH-H2O on the semi-preparative HPLC at the rate of 3 mL/min to yield 7 (tR: 8.0 min, 3.5 mg) and 6 (tR: 12.0 min, 25.7 mg). Compound 6 was identified as 3-methylorcinaldehyde by comparing the retention time with standard sample and NMR data with reference (Table S21).[6] Compound 7 was identified as 4-hydroxy-3,6-dimethyl-2H-pyran-2-one by NMR analysis (Table S21).[7] It would be a by-product produced by g7393, which was similar to that of gene tropA.[8]

OH

HO

H

O

1 73

5

6

O

O

OH

2

46

7

Table S21 13C NMR of 6 and 7 in CD3OD (100 MHz)No. 6 Ref.[6] ǀδ ǀ 7 Ref. [7] ǀδǀ1 142.9 142.8 0.1 - -2 165.0 164.8 0.2 169.1 169.1 0.03 110.1 110.0 0.1 98.7 98.8 0.14 165.2 165.1 0.1 168.2 167.9 0.35 110.9 110.8 0.1 101.7 101.6 0.16 113.7 113.7 0.0 161.4 161.5 0.17 194.4 194.3 0.1 - -3-CH3 7.1 7.1 0.0 8.2 8.2 0.06-CH3 18.0 18.0 0.0 19.5 19.5 0.0

42

10. Short-term memory assays of 1-5 on the AD fly modelFly Stock.w1118(isoCJ1) is an isogenic line used as a control in all of the experiments. In the

experiment, we named this stock “2U” for convenience. Expression of Aβ42(UAS-Aβ42; referred to as H29.3) was driven by a widespread neuronal-expressing Gal4 line, elav-GAL4c155(P35). A behavioral assay was made from the first generation of the cross between P35*h29.3(AD flies).

Fly Culture. All flies were fed at 24°C, 40%~60% RH (relative humidity). On day 2, newly hatched P35*2u male flies and AD male flies were selected and were put into vials (each vial contains about 100 flies). These flies were placed at 29 °C, 40 ± 15% RH during the drug feeding process. The flies were transferred to new vials after 4 h of drug feeding from day 2 to day 8. All flies were placed at 29 °C, 40 ± 15% RH.

Drug Feeding Schedule. Drugs (compounds 1−5 and memantine) were prepared on the first day of eclosion, and the drug feeding was implemented on day 2. The original concentration of compounds was 10 mM, and the final concentration of compounds was 100 μM. These compounds were dissolved in DMSO. For each performance index, 2 vials of flies were fed with 50 μL of the resulting solution for 7 days (e.g., from day 2 to day 8).

Pavlovian Olfactory Learning. Briefly, in the training period, a group of 100 flies was put into a training tube decorated with copper grids. These flies would be orderly exposed to two odors (octanol or methyl cyclohexanol) for 1 min, who were subsequently surrounded by fresh air for 45 s. These flies would be subjected to electric shock for 1 min when they smell the first odor. In the test period, these trained flies would be immediately transported to the choice point, where the T-maze diverged into two containers containing two odors. The flies were allowed to choose one odor between the two for 2 min. Then the number of flies in two containers would be counted. The parameter of PI (performance index) was used for evaluating flies’ memory behavior. PI = 0 represents these flies cannot memorize the link between electric shock and odors. On the contrary, PI = 100% represents all the flies can avoid electric shock according to odors. Note: The experiment was carried out in a room with 25 °C, 70% RH.

Statistical Analysis. Data were analyzed (Table S22) and graphs were also plotted (Figure S44) with the program Graph Pad 8.01.[9]

Table S22 Performance indexes (PI) of AD flies fed with compounds1–5

Genotype/drug PI (100 μM)

WT 56.7 ± 7.1AD 30.3 ± 11.0

mematine 45.0 ± 6.41 47.7 ± 8.12 43.2 ± 10.03 41.3 ± 10.54 38.3 ± 8.95 41.0 ± 7.8

WT: wild type flies; AD: AD flies without drug treatment; PI: performance index (100 = perfect learning; 0 = no learning); All samples were tested at 100 μM

43

WT AD MEM 1 2 3 4 50

20

40

60

80

Perf

orm

ance

inde

x (P

I)#

***** * *

NS *

Figure S44 Performance index (PI) of AD flies fed with 1−5. The treated groups were AD flies treated

with memantine (MEM) or tested compounds (100 μM), and the control groups (WT represents the normal flies,

and AD represents the AD flies) were treated with a corresponding volume of DMSO. Statistical analysis results of

1−5. Each value was expressed as mean ± SEM, n = 3; #, p < 0.001, significantly different from the normal group,

***, p <0.001, significantly different from the AD group, **, p <0.01, significantly different from the AD group,*,

p < 0.05, significantly different from the AD group, and NS, not significantly different from the AD group; t-test.

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45

11.The 1D and 2D NMR spectra of 1-5

11.1 The 1D and 2D NMR spectra of 1

0123456789101112131415f1 (ppm)

3.24

3.32

3.30

3.12

1.10

1.07

1.00

1.50

1.54

1.89

2.11

3.45

6.68

8.88

Figure S45 1H spectrum of 1 (600 MHz, in DMSO-d6)

0102030405060708090100110120130140150160170180190200210220230f1 (ppm)

8.2

14.5

16.6

29.1

46.8

59.4

86.8

125.

8

136.

113

6.3

145.

514

7.4

167.

8

177.

0

196.

819

9.9

Figure S46 13C spectrum of 1 (150 MHz, in DMSO-d6)

46

0102030405060708090100110120130140150160170180190200210220230f1 (ppm)

1

2

Figure S47 13C and DEPT-135 spectrum of 1 (150 MHz, in DMSO-d6)

0123456789f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1(p

pm)

Figure S48 1H-1H-COSY spectrum of 1 (in DMSO-d6)

47

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

50

100

150

f1(p

pm)

Figure S49 HSQC spectrum of 1 (in DMSO-d6)

0.51.52.53.54.55.56.57.58.59.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)

Figure S50 HMBC spectrum of 1 (in DMSO-d6)

48

Figure S51 ROESY spectrum of 1 (in DMSO-d6)

49


0123456789101112131415f1 (ppm)

3.14

3.12

3.14

1.04

1.09

1.00

1.17

1.90

2.07

2.58

3.10

3.14

5.95


0102030405060708090110130150170190210230f1 (ppm)

10.6

14.7

24.5

29.5

48.9

85.6

107.

011

3.6

127.

913

0.2

131.

1

149.

915

3.8

182.

4

197.

320

0.9


50

0102030405060708090110130150170190210230f1 (ppm)

1

2


012345678910f2 (ppm)

1

2

3

4

5

6

7

8

9

10

f1(p

pm)


51

0123456789101112131415f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


0123456789101112131415f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


52


0123456789101112131415f1 (ppm)

3.00

2.96

3.00

3.09

1.08

1.52

1.97

2.02

2.13

6.22


0102030405060708090100110120130140150160170180190200210220230f1 (ppm)

11.4

13.3

15.5

20.9

86.8

89.0

113.

211

9.7

128.

7

130.

714

5.6

150.

7

178.

0

188.

9

197.

3


53

0102030405060708090110130150170190210230f1 (ppm)

1

2


0123456789f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1(p

pm)


54

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


55


0123456789101112131415f1 (ppm)

3.00

2.67

2.78

2.74

0.90

1.60

1.97

2.04

2.13

5.23


0102030405060708090100110120130140150160170180190200210220230f1 (ppm)

8.2

11.3

12.9

15.5

84.1

85.0

113.

611

9.4

128.

7

138.

814

5.2

150.

4

166.

0

188.

6

197.

9


56

-100102030405060708090110130150170190210230f1 (ppm)

1

2

Figure S66 DEPT-135 spectrum of 4 (150 MHz, in DMSO-d6)

0123456789101112131415f2 (ppm)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

f1(p

pm)


57

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

f1(p

pm)


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


58


0123456789101112131415f1 (ppm)

3.00

2.91

3.07

2.97

1.13

1.70

2.00

2.10

2.31

4.87


0102030405060708090100110120130140150160170180190200210220230f1 (ppm)

8.2

11.0

12.9

13.4

75.6

97.8

110.

2

119.

212

6.8

135.

7

160.

8

170.

7

191.

719

4.9


59

50130210f1 (ppm)

1

2

Figure S72 13Cand DEPT-135 spectrum of 5 (150 MHz, in DMSO-d6)

0123456789f2 (ppm)

0

1

2

3

4

5

6

7

8

9

f1(p

pm)


60

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f2 (ppm)

0

20

40

60

80

100

120

140

160

180

200

220

f1(p

pm)


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