SUPPLEMENTARY MATERIAL
Antifungal and antibacterial metabolites from an endophytic Aspergillus
sp. associated with Melia azedarach
Jian Xiao, Qiang Zhang, Yu-Qi Gao, Xin-Wei Shi, Jin-Ming Gao*
Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College
of Science, Northwest A&F University, Yangling 712100, Shaanxi, PR China
Email: jinminggao@nwsuaf.edu.cn
1
Experimental
General
1D, 2D-NMR spectra: Bruker Avance-500; ESI-MS: Thermo Fisher LTQ Fleet
instrument spectrometer; HPLC: Waters 1525; Column chromatography (CC): silica gel
(SiO2: 200-300 or 300-400 mesh, Qingdao); Sephadex LH-20 (Pharmacia Co.); Hypersil
BDS 5 µm C18 (250×4.6 or 250×10; Thermo). Fractions were monitored by TLC, and
spots were visualized by spraying with 5% H2SO4 in ethanol, followed by heating. All
other chemicals used in this study are of analytical grade.
Fungal material and cultivation
The endophytic fungus KJ-9 was isolated from the symptomless tissue of stem bark of
Melia azedarach L., collected at Yangling, Shaanxi province, China, in August 2011. It
was identified as Aspergillus sp. based on the analysis of morphological characteristics
and deposited at the College of Science, Northwest A&F University. This fungus was
incubated into the PDA medium (150 mL each in 500 mL Erlenmeyer flasks) and
fermented at 28 ± 0.5 °C for 4 weeks, in total 30 L.
Extraction and Isolation
The fermented cultures were ultrasonically extracted with methanol, and then the
methanol layer was extracted by EtOAc to give an EtOAc extract (22 g). This crude
extract was separated by silica gel CC using CHCl3-MeOH gradient elution to provide
fractions (Fr.1–Fr.4). Fr. 2 (3 g) was separated by repeated CC (silica gel) to yield
compound 1 (30 mg). Fr. 1 (2.8 g) was subjected to repeated CC (Sephadex LH-20, silica
gel, RP-C18 and HPLC) to yield compounds 2 (13 mg), 3 (1.7 mg) and 6 (9 mg). Fr. 3
(1.1 g) was separated by CC (silica gel and HPLC) to yield compound 4 (3.7 mg). Fr. 4
(0.9 g) was separated by repeated CC (silica gel, Sephadex LH-20 and HPLC) to yield
compounds 5 (10 mg) and 7 (4 mg).
Compound (1): Yellow amorphous powder; [α]D23 -7.50 (c 0.1, DMSO); 1H NMR (400
MHz, DMSO-d6): δ 15.10 (1H, s, 5'-OH), 15.01 (1H, s, 5-OH), 10.25 (1H, s, 8-OH), 7.27
(1H, s, H-10), 7.12 (1H, s, H-9), 6.55 (1H, d, J = 2.19 Hz, H-9'), 6.22 (1H, s, H-3), 6.21
(1H, s, H-3'), 6.15 (1H, d, J = 2.17 Hz, H-7'), 3.93 (3H, s, 6'-OMe), 3.58 (3H, s, 8'-OMe),
3.39 (3H, s, 6-OMe), 2.41 (3H, s, 2-Me), 2.14 (3H, s, 2'-Me);
2
13
C NMR (100 MHz,
DMSO-d6): δ 184.0 (C-4'), 184.0 (C-4), 168.9 (C-2), 168.6 (C-2'), 162.0 (C-5'), 161.3
(C-5), 161.2 (C-8'), 160.8 (C-6'), 158.5 (C-8), 158.3 (C-6), 152.5 (C-10a), 150.3 (C-10'a),
140.2 (C-9a), 140.1 (C-9'a), 116.8 (C-7), 109.8 (C-5a), 107.6 (C-5'a), 106.8 (C-3), 106.7
(C-3'), 105.4 (C-10'), 105.3 (C-9), 103.4 (C-4'a), 103.4 (C-4a), 100.5 (C-10), 96.8 (C-7'),
96.5 (C-9'), 61.5 (6-OMe), 56.1 (6'-OMe), 55.1 (8'-OMe), 20.3 (2-Me), 20.2 (2'-Me).
ESI-MS (negative) m/z: 555.42 [M-H]-.
Compound 2: Yellow amorphous powder; 1H-NMR (400 MHz, CDCl3) δ 7.16 (1H, s,
H-10), 6.98 (1H, s, H-9), 6.42 (1H, d, J = 2.07 Hz, H-7'), 6.22 (1H, d, J = 2.07 Hz, H-9'),
6.07 (1H, s, H-3), 6.00 (1H, s, H-3'), 4.03 (3H, s, 6'-OMe), 3.79 (3H, s, 8-OMe), 3.62 (3H,
s, 8'-OMe), 3.47 (3H, s, 6-OMe), 2.42 (3H, s, 2-Me), 2.13 (3H, s, 2'-Me); 13C-NMR (100
MHz, CDCl3) δ 184.57 (C-4'), 184.42 (C-4), 167.71 (C-2), 167.65 (C-2'), 162.62 (C-5'),
161.91 (C-5), 161.38 (C-8'), 160.99 (C-6'), 160.12 (C-8), 158.50 (C-6), 153.30 (C-10a),
150.77 (C-10'a), 140.67 (C-9a), 140.51 (C-9'a), 117.55 (C-7), 111.37 (C-5a), 108.50
(C-5'a), 107.37 (C-3), 107.18 (C-3'), 105.14 (C-10'), 104.67 (C-4a), 104.20 (C-4'a),
101.34 (C-9), 101.22 (C-10), 96.83 (C-7'), 96.43 (C-9'), 62.01 (6-OMe), 56.17 (6'-OMe),
55.89 (8-OMe), 55.11 (8'-OMe), 20.75 (2-Me), 20.69 (2'-Me). ESI-MS (positive) m/z:
571.26 [M+H]+.
H3 C
O
OH
6'
7'
H3 C
5'
5'a
8'
O
O
9'a
9'
4'
4'a
3'
10'a
2'
O
10'
CH3
1'
HO
O
9
8
10
7
10a
6a
6
CH3
O
10b 1
2
4a
3
5
4
OH
O
CH3
Compound 3: Yellow amorphous powder; 1H-NMR (400 MHz, DMSO-d6) δ 15.10 (1H, s,
5'-OH), 12.97 (1H, s, 5-OH), 7.33 (1H, s, H-7), 7.14 (1H, s, H-6), 6.55 (1H, d, J = 2.14
Hz, H-7'), 6.54 (1H, s, H-3), 6.21 (1H, s, H-3'), 6.09 (1H, d, J = 2.14 Hz, H-9'), 3.93 (3H,
s, 6'-OMe), 3.75 (3H, s, 8-OMe), 3.57 (3H, s, 8'-OMe), 3.43 (3H, s, 10-OMe), 2.48 (3H, s,
2-Me), 2.12 (3H, s, 2'-Me);
13
C-NMR (100 MHz, DMSO-d6) δ 183.94 (C-4'), 182.49
(C-4), 168.55 (C-2), 168.29 (C-2'), 162.10 (C-5'), 161.28 (C-8'), 160.84 (C-6'), 159.57
3
(C-8), 156.32 (C-10), 155.93 (C-5), 154.52 (C-10b), 150.16 (C-10'a), 140.37 (C-6a),
140.00 (C-9'a), 116.45 (C-9), 110.10 (C-3), 108.56 (C-4a), 107.58 (C-5'a), 107.35
(C-10a), 106.82 (C-3'), 105.48 (C-6), 104.88 (C-10'), 103.27 (C-4'a), 102.10 (C-7), 96.58
(C-7'), 96.56 (C-9'), 61.09 (10-OMe), 56.12 (6'-OMe), 56.09 (8-OMe), 55.09 (8'-OMe),
20.15 (2'-Me), 19.96 (2-Me). ESI-MS (positive) m/z: 571.28 [M+H]+.
H3C
O
OH
6'
5'
7'
H3 C
5'a
8'
O
4'
4'a
9'a
9'
O
3'
10'a
2'
O
10'
CH3
1'
H3 C
O
O
9
8
10
7
10a
6a
6
CH3
O
10b 1
2
4a
3
5
4
OH
O
CH3
Compound 4: Yellow amorphous powder; 1H-NMR (400 MHz, DMSO-d6) δ 15.11 (1H, s,
5'-OH), 12.92 (1H, s, 5-OH), 10.22 (1H, s, 8-OH), 7.08 (1H, s, H-7), 6.98 (1H, s, H-6),
6.56 (1H, d, J = 2.07 Hz, H-7'), 6.52 (1H, s, H-3), 6.22 (1H, s, H-3'), 6.14 (1H, d, J = 2.19
Hz, H-9'), 3.93 (3H, s, 6'-OMe), 3.58 (3H, s, 8'-OMe), 3.40 (3H, s, 10-OMe), 2.47 (3H, s,
2-Me), 2.14 (3H, s, 2'-Me);
13
C-NMR (100 MHz, DMSO-d6) δ 183.99 (C-4'), 182.39
(C-4), 168.54 (C-2'), 168.02 (C-2), 162.08 (C-5'), 161.16 (C-8'), 160.84 (C-6'), 158.48
(C-8), 156.90 (C-10), 155.99 (C-5), 154.79 (C-10b), 150.29 (C-10'a), 140.18 (C-6a),
140.10 (C-9'a), 116.26 (C-9), 110.06 (C-3), 108.12 (C-4a), 107.61 (C-5'a), 106.79 (C-3'),
106.60 (C-10a), 105.27 (C-7), 105.21 (C-10'), 104.48 (C-6), 103.36 (C-4'a), 96.84 (C-9'),
96.42 (C-7'), 61.04 (10-OMe), 56.12 (6'-OMe), 55.09 (8-OMe), 20.21 (2'-Me), 19.93
(2-Me). ESI-MS (negative) m/z: 555.06 [M-H]-.
H3 C
O
OH
6'
5'
7'
H3 C
5'a
8'
O
4'
4'a
9'a
9'
O
3'
10'a
2'
O
10'
CH3
1'
HO
O
9
8
CH3
10
7
O
10a
6a
6
4
10b 1
2
4a
3
5
4
OH
O
CH3
Compound 5: Pale yellow amorphous powder; 1H-NMR (400 MHz, CDCl3) δ 8.38 (1H, d,
J = 1.70 Hz, 30-NH), 7.44 (1H, d, J = 7.43 Hz, H-27), 7.28 (2H, m, H-20, 22), 7.27 (2H,
m, H-19, 23), 7.22 (1H, m, H-21), 7.15 (1H, m, H-7), 7.12 (7H, m, H-25, 26, 42, 43, 44,
45, 46), 6.88 (1H, d, J = 7.30 Hz, H-31), 6.84 (1H, d, J = 2.31 Hz, 38-NH), 6.77 (1H, d,
J = 7.91 Hz, H-8), 6.73 (1H, m, H-6), 6.34 (1H, d, J = 1.70 Hz, 14-NH), 6.04 (1H, s,
35-NH), 5.99 (1H, d, J = 1.83 Hz, 1-NH), 5.62 (1H, d, J = 1.95 Hz, H-2), 4.22 (1H, m,
H-37), 3.90 (1H, br. s, H-15), 3.59 (1H, m, H-34), 3.39 (1H, m, H-33a), 3.31 (1H, m,
H-12a), 3.19 (1H, m, H-40a), 3.16 (1H, m, H-17a), 3.13 (1H, m, H-11), 3.01 (1H, m,
H-17b), 2.98 (1H, m, H-33b), 2.93 (1H, m, H-40b), 2.51 (1H, dd, J = 13.51, 10.59 Hz,
H-12b);
13
C NMR (100 MHz, CDCl3) δ 168.91 (C-13), 168.21 (C-36), 167.51 (C-39),
166.94 (C-16), 145.82 (C-9), 134.96 (C-29), 134.90 (C-18), 134.46 (C-41), 131.88 (C-4),
129.70 (C-20, 22, 43, 45), 129.23 (C-44), 128.97 (C-19, 23), 128.94 (C-42, 46), 128.06
(C-28), 127.74 (C-7), 127.56 (C-21), 124.90 (C-24), 124.24 (C-6), 124.13 (C-25), 120.76
(C-26), 119.78 (C-31), 118.98 (C-5), 118.63 (C-27), 110.26 (C-8), 109.27 (C-32), 83.18
(C-2), 59.06 (C-37), 57.36 (C-3), 56.23 (C-11), 56.17 (C-15), 54.15 (C-34), 40.13 (C-40),
39.96 (C-17), 38.91 (C-12), 29.67 (C-33). ESI-MS (positive) m/z: 665.18 [M+H]+, 687.46
[M+Na]+.
21
22
20
23
19
18
O
17
H
H
16
15
HN
14
2
N
10
11
13
O
H
N
8
1
3
9
7
4
6
12
44
45
H
24
25
29
26
28
27
H
N
30
36
35
34
32
33
39
H
40
37
HN
42
41
O
31
43
46
5
H
NH
38
O
50
Compound 6: Yellow amorphous powder; H-NMR (400 MHz, CD3OD:CDCl3 = 1:1) δ
1
7.14 (1H, s, H-10), 6.77 (1H, d, J = 1.95 Hz, H-9), 6.56 (1H, d, J = 2.07 Hz, H-7), 6.17
(1H, s, H-3), 4.13 (3H, s, 8-OMe), 4.08 (3H, s, 6-OMe), 2.56 (3H, s, 2-Me);
13
C-NMR
(100 MHz, CD3OD:CDCl3 = 1:1) δ 183.89 (C-4), 167.88 (C-2), 161.15 (C-5), 161.06
(C-8), 159.71 (C-6), 152.51 (C-11), 140.68 (C-14), 107.50 (C-13), 106.23 (C-3), 103.42
(C-12), 100.95 (C-10), 97.31 (C-9), 96.82 (C-7), 55.04 (8-OMe), 54.55 (6-OMe), 19.55
5
(2-Me). ESI-MS (negative) m/z: 285.17 [M-H]-.
H3 C
O
OH
6
5
7
H3 C
4
13
8
12
14
O
O
3
11
9
2
O
10
CH3
1
(R)-3-Hydroxybutanonitrile (7). Pale yellow liquid; [α]D21 + 5.70 (c 0.1, CHCl3), lit.
[α]D20 + 4.1 (c 3.0, EtOH) (Itoh, Takagi, & Nishiyama, 1991); 1H NMR (400 MHz,
CD3OD): δ 4.36 (1H, m, H-3), 2.73 (1H, dd, J = 5.4, 16.1 Hz, H-2a), 2.52 (1H, dd, J =
5.4, 16.5 Hz, H-2b), 1.81 (3H, d, J = 5.3 Hz, H-4);
C NMR (100 MHz, CD3OD): δ
13
116.5 (C-1), 67.6 (C-3), 29.9 (C-2), 23.0 (C-4). ESI-MS (negative) m/z: 84.13 [M-H]-.
OH
3
4
1
2
N
Itoh, T., Takagi, Y., & Nishiyama, S. (1991). Enhanced enantioselectivity of an
enzymatic reaction by the sulfur functional group. A simple preparation of optically
active. beta.-hydroxy nitriles using a lipase. The Journal of Organic Chemistry, 56,
1521–1524.
Dehli, J.R., & Gotor, V. (2000). Enantio- and chemoselective bioreduction of β-keto
nitriles by the fungus Curvularia lunata. Tetrahedron, 11, 3693–3700.
Bioactivity assays
Antimicrobial assays were performed by our reported method (Li et al. 2012). All test
phytopathogenic fungi and bacteria were provided by the College of plant protection,
Northwest A&F University. All metabolites were tested in vitro for the antifungal activity
against five phytopathogenic fungi Gibberella saubinetti, Magnaporthe grisea, Botrytis
cinerea, Colletotrichum gloeosporiodes and Alternaria solani, as well as for the
antibacterial activity against four pathogenic bacteria Escherichia coli, Bacillus subtilis,
Staphyloccocus aureus, and Bacillus cereus. All these tested phytopathogenic fungi were
deposited at the College of Science, Northwest A&F University.
Antifungal activity was assessed by the microbroth dilution method in 96-well
flat-microtiter plates using a potato dextrose (PD) medium. The compounds were made
up to 2 mM in DMSO. Two commercial fungicides: carbendazim and hymexazol
6
(Aladdin chemistry Co. Ltd.) was used as positive controls, and the equal concentration
of DMSO was used as a negative control. The fungi were incubated in the PD medium
for 18~36 h at 28 ± 0.5° C at 150 rpm, and spores of different microorganism
concentrations were diluted to approximately 1 × 106 CFU with PD medium. In
flat-microtiter plates, tested compounds, fungal suspension and sterile water were added
to mad up final concentrations of the compounds in the range of 1.57-200 M. Cultures
then grew in the dark at 28 ± 0.5 °C for 48 h. Minimum inhibitory concentrations (MICs)
were inspected as the lowest concentrations in which no microbial growth could be
observed. Antibacterial activity was assessed by the microbroth dilution method in
96-well flat-microtiter plates using a beef-protein liquid (BP) medium. Antibacterial
activity was assessed in the same procedure as antifungal bioassay. Streptomycin sulfate
and ampicillin were used as positive control, and the solution of equal concentration of
DMSO was used as a negative control. Cultures were grown during 24 h at 37 ± 0.5 °C in
the dark without shaking and in a moist chamber.
Table S1. Inhibitory effect of compounds 1-7 against pathogenic bacteria……………8
Figure S1. 1H spectra of dianhydro-aurasperone C (1) in DMSO-d6 ............................ 9
Figure S2. 13C spectra of dianhydro-aurasperone C (1) in DMSO-d6 ........................... 9
Figure S3. HSQC spectra of dianhydro-aurasperone C (1) in DMSO-d6 .................... 10
Figure S4. COSY spectra of dianhydro-aurasperone C (1) in DMSO-d6 .................... 10
Figure S5. HMBC spectra of dianhydro-aurasperone C (1) in DMSO-d6 ................... 11
Figure S6. The CD spectrum of isoaurasperone A (2) in MeOH…………………… 11
Figure S7. The CD spectrum of asperpyrone A (4) in MeOH……………………… 12
7
Table S1. Inhibitory effect of compounds 1-7 against pathogenic bacteria.
pathogenic bacteria (MIC: M)
compound
Gram-positive bacteria (G+)
Gram-negative bacteria (G-)
B. c.
S. a.
B. s.
E. c.
1
NA
50
25
NA
2
NA
NA
NA
NA
3
100
25
25
50
4
NA
NA
NA
NA
5
NA
50
NA
NA
6
NA
50
NA
50
7
50
50
NA
NA
Streptomycin Sulfate
1.57
12.5
12.5
3.13
Ampicillin
3.13
1.57
12.5
6.25
Note: B. c., Bacillus cereus; S. a., Staphyloccocus aureus; B. s., Bacillus subtilis; E. c., Escherichia
coli. NA: inactive.
8
Figure S1. 1H spectra of dianhydro-aurasperone C in DMSO-d6
Figure S2. 13C spectra of dianhydro-aurasperone C in DMSO-d6
9
Figure S3. HSQC spectra of dianhydro-aurasperone C in DMSO-d6
Figure S4. COSY spectra of dianhydro-aurasperone C in DMSO-d6
10
Figure S5. HMBC spectra of dianhydro-aurasperone C in DMSO-d6
1
10
279, 38000
KJ-9-3, 1.20 mM MeOH, 0.02 cm
0.8
0.6
Molar CD
Absorbance
5
0.4
0
-5
KJ-9-3, 1.20 mM MeOH, 0.02 cm
KJ-9-3, 1.20 mM MeOH, 0.1 cm (5x)
0.2
0
200
250
300
350
 / nm
400
450
-10
250
500
300
350
 / nm
400
Figure S6. The CD spectrum of isoaurasperone A (2) in MeOH.
11
450
500
1.4
20
279, 20000
1.2
KJ-9-6, 1.3 mM MeOH, 0.05 cm
1
0.8
10
Molar CD
Absorbance
KJ-9-6, 1.3 mM MeOH, 0.05 cm
15
0.6
5
0
-5
0.4
-10
0.2
-15
0
-20
200
250
300
350
 / nm
400
450
500
200
250
300
350
 / nm
400
Figure S7. The CD spectrum of asperpyrone A (4) in MeOH.
12
450
500