Oryzamides A-E, Cyclodepsipeptides from the Sponge-Derived Fungus Nigrospora oryzae PF18

Article abstract of DOI:10.1021/acs.jnatprod.6b00349

Three new cyclohexadepsipeptides, oryzamides A-C (1-3), two isolation artifacts, oryzamides D (4) and E (5), and the known congener scopularide A (6), all possessing a rare 3-hydroxy-4-methyldecanoic acid (HMDA) substructure, were isolated from the mycelial extract of the sponge-derived fungus Nigrospora oryzae PF18. Their planar structures were elucidated by spectroscopic analysis and comparison with the literature data. The absolute configurations were determined using the advanced Marfey's method and single-crystal X-ray diffraction analysis. Among them, oryzamides D (4) and E (5) were a pair of diastereomers at the sulfur atom of the l-methionine sulfoxide residue, which showcased the possible separation of a pair of methionine sulfoxide diastereomers. The X-ray crystal structure of scopularide A (6) was obtained for the first time, thereby establishing its relative and absolute configuration at C-4 of the HMDA residue. Oryzamides A-C (1-3) did not display cytotoxic, antibacterial, antiparasitic, and NF-κB inhibitory activities.

Full text of DOI:10.1021/acs.jnatprod.6b00349

Article
pubs.acs.org/jnp
Oryzamides A−E, Cyclodepsipeptides from the Sponge-Derived
Fungus Nigrospora oryzae PF18
Li-Jian Ding,†,‡ Wei Yuan,† Xiao-Jian Liao,‡ Bing-Nan Han,† Shu-Ping Wang,† Zhi-Yong Li,§
Shi-Hai Xu,*,‡ Wei Zhang,*,⊥ and Hou-Wen Lin*,†
†Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji
Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, People’s Republic of China
‡College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China
§Key Laboratory of Microbial Metabolism, Marine Biotechnology Laboratory, School of Life Sciences and Biotechnology, Shanghai
Jiao Tong University, Shanghai 200240, People’s Republic of China
⊥Center for Marine Bioproducts Development, Flingers University, Adelaide 5001, Australia
S
* Supporting Information
ABSTRACT: Three new cyclohexadepsipeptides, oryzamides A−C
(1−3), two isolation artifacts, oryzamides D (4) and E (5), and the
known congener scopularide A (6), all possessing a rare 3-hydroxy-4-
methyldecanoic acid (HMDA) substructure, were isolated from the
mycelial extract of the sponge-derived fungus Nigrospora oryzae PF18.
Their planar structures were elucidated by spectroscopic analysis and
comparison with the literature data. The absolute configurations were
determined using the advanced Marfey’s method and single-crystal X-
ray diffraction analysis. Among them, oryzamides D (4) and E (5)
were a pair of diastereomers at the sulfur atom of the L-methionine
sulfoxide residue, which showcased the possible separation of a pair of
methionine sulfoxide diastereomers. The X-ray crystal structure of
scopularide A (6) was obtained for the first time, thereby establishing
its relative and absolute configuration at C-4 of the HMDA residue.
Oryzamides A−C (1−3) did not display cytotoxic, antibacterial, antiparasitic, and NF-κB inhibitory activities.
new oryzamides A−E (1−5) and one known analogue,
scopularide A (6), were isolated from the CH2Cl2 extract of
Nigrospora oryzae PF18 obtained from the marine sponge P.
fusca. Apart from an amino acid residue difference, cyclo-
hexadepsipeptides 1−6 bear the same four amino acid and
unusual HMDA residues. Scopularide A (6), whose complete
stereostructure was not determined before, was previously
isolated from a marine sponge-associated fungus.18 It is
noteworthy that the X-ray crystal structures of 1 and 6 enabled
the rigorous assignments of the corresponding absolute
configurations. Furthermore, a pair of methionine sulfoxide
diastereomers, oryzamides D (4) and E (5), were readily
separated from each other. Herein, we describe the detailed
isolation process, structure elucidation, and biological activities
of these cyclodepsipeptides.
yclodepsipeptides are a large group of cyclic peptides that
Care characterized by the presence of at least one ester
bond as part of their backbone.1,2 They have received enduring
attention for a wide range of bioactivities, such as anticancer,3
immunosuppressant,4 anthelmintic,5 antibiotic,6,7 anti-inflam-
matory,8 and antiviral9 properties. Remarkably, several
representative members have been approved for pharmaceutical
use, such as emodepsin,10 or are in clinical trials, as exemplified
by kahalalide F11 and aplidin.12 Structurally, the diversity of
hydroxy acid building blocks has been well characterized in
cyclodepsipeptides.13 As a rare member, the 3-hydroxy-4-
methyldecanoic acid (HMDA) moiety was only encountered in
beauverolides A, B, Ba, L, and La,14−16 beauveriolide VIII,17 and
scopularide A18 from fungi, as well as in arenamides A and C
from an actinomycete strain.19
Our previous chemical investigation of the marine sponge
Phakellia fusca collected from Yongxing Island led to the
isolation of four new cyclopeptides, phakellistatins 15−18.20
That result prompted us to examine and identify the presence
of cyclopeptides from P. fusca-derived microbes, allowing for
the fact that sponge-derived natural products are typically
associated with microbial symbiotic consortia.21,22 Conse-
quently, six HMDA-containing cyclodepsipeptides, including
RESULTS AND DISCUSSION
■
Oryzamide A (1) was isolated as a white crystal. The molecular
formula C33H59N5O7 was deduced from the HRESIMS data
(m/z 638.4504 [M + H]+) in combination with the 1D and 2D
Received: April 20, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00349
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Chart 1
NMR spectra, implying seven degrees of unsaturation. The IR
spectrum of 1 showed strong absorption bands at 1724 and
1667 cm−1, revealing the presence of ester and amide
The major differences between the 1H NMR spectra of 2 and 1
were the resonances associated with aromatic protons.
Interpretation of the 2D NMR data further demonstrated
that the Leu1 residue in 1 was replaced by the Tyr residue in 2,
and the latter residue accounted for those aromatic signals.
Therefore, oryzamide B (2) encompassed the Ala, Gly, HMDA,
Leu, Val, and Tyr residues. The connection of the residues was
established as cyclo-(Tyr−Ala−Leu−Val−Gly−HMDA) (Fig-
ures 2 and S18), according to the COSY and HMBC
correlations in the same manner as 1 (Figure 1), which was
further supported by the corresponding ESIMS/MS fragments.
The absolute configurations of the amino acid residues were
deciphered by the advanced Marfey’s method.23−25 The acid
hydrolysates of 2 were derivatized with 1-fluoro-2,4-dinitro-
phenyl-5-L-leucinamide (L-FDLA) and L-/D-FDLA, and the
amino acid derived products were detected by UPLC-MS. The
absolute configurations of all of the amino acid residues were
assigned to be L, except for D-Leu (Figure S21), based on the
retention times of the L-FDLA derivatives with L-/D-FDLA
derivatives. Regarding the HMDA unit of 2, the absolute
configuration was deduced as the same as that of 1, owing to a
shared biogenesis.
1
functionalities. The H spectrum displayed resonances for five
NH doublets (δH 7.72, 7.96, 8.65, 7.79, and 7.95) and six α-H
multiplets (δH 3.38−4.18), indicating the peptidic nature of 1
(Table 1). In addition, the 13C NMR spectrum exhibited signals
for six ester/amide-type carbonyls (δC 169.1−171.9) and one
oxygenated sp3-hybridized carbon (δC 74.7), thus identifying 1
as a depsipeptide (Table 1). A detailed analysis of the COSY,
HSQC, and HMBC data allowed the construction of an alanine
(Ala), a glycine (Gly), a valine (Val), a 3-hydroxy-4-
methyldecanoic acid (HMDA), and two leucine (Leu1 and
Leu2) residues. The residue sequence of Leu1−Ala−Leu2−Val−
Gly−HMDA in 1 was established by the HMBC correlations of
Leu1-NH (δH, 7.72)/Ala C-7 (δC, 171.9), Ala-NH (δH, 7.96)/
Leu2 C-10 (δC, 171.1), Leu2-NH (δH, 8.65)/Val C-16 (δC,
171.7), Val-NH (δH, 7.79)/Gly C-21 (δC, 169.1), and Gly H2-
22 (δH, 4.10 and 3.38)/HMDA C-23 (δC, 169.9) (Figure 1).
The assignments were further confirmed by the MS/MS
fragment ion series at m/z 525, 454, and 341, corresponding to
cleavages of the amide bonds between Leu1/Ala, Ala/Leu2, and
Leu2/Val, respectively (Figures 2 and S8). Given that six of the
seven degrees of unsaturation were attributed to six carbonyl
carbons, a monocyclic structure of 1 was inferred from the
remaining degree of unsaturation, suggesting that the Leu1
residue was attached to the HMDA moiety by an ester bond.
Thus, 1 was identified as cyclo-(Leu1−Ala−Leu2−Val−Gly−
HMDA). The absolute configuration of 1 was determined by a
single-crystal X-ray diffraction experiment using Cu Kα
radiation, which allowed the assignments of amino acid
residues as L-Leu1, L-Ala, D-Leu2, and L-Val and gave a 3S,4S
configuration for the HMDA moiety (Figure 3)
Oryzamide C (3) has the molecular formula C32H57N5O7S,
as indicated by the HRESIMS data. Signals observed at 169.0,
169.6, 170.7, 171.0, 171.5, and 171.7 ppm in its 13C NMR
spectrum (Table 1) accounted for the presence of six amide/
ester-type carbonyls, revealing that the structure of 3 was
similar to that of 1. This was further supported by the strong IR
1
absorption bands of ester and amide functionalities. The H
and COSY NMR spectra of 3 revealed the presence of a spin
system containing a two-proton doublet of triplets at δH 1.97, a
two-proton multiplet at δH 2.54, and a methyl singlet at δH 2.04,
consistent with a −CH2CH2SCH3 unit of methionine.16
Considering that the resonances for the Leu1 group were
missing, oryzamide C (3) was composed of a methionine
moiety instead of the Leu1 counterpart in 1 (Figure 1). This
planar structure was further confirmed by the analysis of the
ESIMS/MS fragments of m/z 525, 454, and 341 (Figures 2 and
S29), arising from the cleavage of the amide bonds between
The molecular formula of oryzamide B (2) was determined
to be C36H57N5O8 by the HRESIMS data, establishing seven
1
degrees of unsaturation. The H NMR spectrum of 2 was
similar to that of 1, with five NH protons (δH 7.78, 7.88, 7.90,
8.01, and 8.64) and six α-H protons (δH 3.42−4.24),
confirming the cyclic hexadepsipeptide nature of 2 (Table 1).
B
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1
Table 1. H (600 MHz) and 13C (150 MHz) NMR Data for Oryzamides A−C (1−3) in DMSO-d6
oryzamide A (1)
oryzamide B (2)
oryzamide C (3)
unit
position
δC, type
δH (J in Hz)
unit
position
δC, type
δH (J in Hz)
unit
position
δC, type
δH (J in Hz)
L-Leu1
1
2
171.7, C
51.0, CH
L-Tyr
1
2
170.9, C
54.9, CH
L-Met
1
2
170.7, C
51.4, CH
4.14, m
4.24, td (7.2,
6.8)
4.30, dt (7.4,
7.1)
3
39.5, CH2 1.62, m
3
36.0, CH2 2.85, m
3
30.2, CH2 1.97, td (7.4,
7.1)
1.47, m
4
127.1, C
4
29.4, CH2 2.54, m
14.3, CH3 2.04, s
7.54, d (7.2)
4
24.1, CH
1.68, m
5/9
6/8
7
130.0, CH
115.0, CH
156.0, C
7.03, d (8.4)
5
5
21.5, CH3 0.83, d (6.6)
22.7, CH3 0.88, d (6.3)
7.72, d (7.0)
6.64, d (8.4)
2-NH
6
L-Ala
6
7
171.7, C
2-NH
2-NH
8.01, d (6.8)
47.6, CH
4.19, dq (8.2,
7.1)
L-Ala
7
171.9, C
L-Ala
10
171.8, C
47.6, CH
8
16.8, CH3 1.24, d (7.1)
8.03, d (8.2)
8
47.6, CH
4.18, m
11
4.19, m
7-NH
9
9
17.4, CH3 1.20, d (7.0)
7.96 d (4.8)
12
17.8, CH3 1.15, d (7.0)
7.78, d (8.1)
D-Leu
171.0, C
8-NH
10
11-NH
13
10
52.2, CH
38.7, CH2 1.50, m
24.0, CH 1.66, m
3.98, m
D-Leu2
171.1, C
D-Leu
171.0, C
11
11
52.2, CH
38.7, CH2 1.47, m
24.2, CH 1.64, m
3.98, m
14
51.9, CH
38.7, CH2 1.46, m
24.1, CH 1.63, m
4.05, m
12
12
15
13
22.6, CH3 0.91, d (6.5)
21.0, CH3 0.84, d (7.0)
8.52, d (5.9)
13
16
14
14
23.0, CH3 0.89, d (6.4)
21.1, CH3 0.81, d (6.6)
8.65, d (5.9)
17
23.0, CH3 0.89, d (6.6)
21.1, CH3 0.82, d (6.6)
8.64, d (6.3)
10-NH
15
15
18
L-Val
171.5, C
11-NH
16
14-NH
19
16
58.2, CH
29.8, CH
4.09, t (8.0)
1.89, m
L-Val
171.7, C
L-Val
171.7, C
17
17
58.3, CH
30.0, CH
4.09, m
1.84, m
20
58.3, CH
29.8, CH
4.10, t (8.6)
1.87, m
18
18.7, CH3 0.90, d (6.6)
18.5, CH3 0.85, d (6.8)
7.69, d (7.6)
18
21
19
19
19.0, CH3 0.88, d (6.7)
18.7, CH3 0.83, d (6.6)
7.79, d (7.9)
22
19.0, CH3 0.88, d (7.4)
18.8, CH3 0.84, d (6.8)
7.90, d (8.5)
16-NH
20
20
23
Gly
169.0, C
17-NH
20-NH
21
42.2, CH2 4.13, dd (16.4,
7.1)
Gly
21
22
169.1, C
Gly
24
25
168.9, C
3.38, dd (16.3,
4.1)
42.4, CH2 4.10, m
3.38, m
42.3, CH2 4.05, m
21-NH
22
7.89, dd (7.0,
4.2)
3.42, dd (16.7, HMDA
4.2)
169.6, C
22-NH
23
7.95, d (2.9)
169.9, C
25-NH
26
7.88, d (6.3)
23
36.9, CH2 2.50, m
HMDA
HMDA
169.8, C
2.27, dd (15.0,
2.2)
24
36.9, CH2 2.50, d (15.0)
27
37.2, CH2 2.43, d (9.3)
24
25
74.6, CH
36.1, CH
4.98, m
1.65, m
2.24, dd (15.2,
1.9)
2.24, dd (14.9,
2.1)
25
26
27
74.7, CH
36.3, CH
4.95, m
1.63, m
28
29
30
75.0, CH
36.1, CH
4.90, m
1.51, m
26
27
31.6, CH2 1.35, m
1.01, m
31.7, CH2 1.33, m
0.99, m
31.5, CH2 1.21, m
0.90, m
26.3, CH2 1.30, m
1.21, m
28
26.6, CH2 1.28, m
1.18, m
31
26.5, CH2 1.21, m
1.11, m
28
29
30
31
32
28.7, CH2 1.24, m
30.9, CH2 1.25, m
21.8, CH2 1.27, m
13.6, CH3 0.87, t (6.8)
14.5, CH3 0.83, d (7.7)
29
30
31
32
33
29.0, CH2 1.23, m
31.2, CH2 1.23, m
22.2, CH2 1.25, m
14.0, CH3 0.85, t (6.9)
14.8, CH3 0.81, d (6.9)
32
33
34
35
36
29.0, CH2 1.23, m
31.2, CH2 1.24, m
22.1, CH2 1.26, m
14.0, CH3 0.86, t (7.4)
14.6, CH3 0.69, d (6.8)
Met/Ala, Ala/Leu, and Leu/Val, respectively. Thus, oryzamide
C (3) was identified as cyclo-(Met−Ala−Leu−Val−Gly−
HMDA), which possesses the same planar structure as
arenamide C.19 The absolute configurations of the amino acid
residues in 3 were determined as L-Met, L-Ala, D-Leu, and L-Val
(Figure S32) via the advanced Marfey’s method. Accordingly,
the structure of oryzamide C (3) was different from that of
arenamide C owing to the opposite absolute configuration of
the Leu residue. The stereogenic centers (C-3 and C-4) in the
HMDA substructure probably possess the same configurations
as those of 1, allowing for its co-occurrence with 1.
Oryzamides D (4) and E (5), two minor metabolites, were
found to have the same molecular formula of C32H57N5O8S via
HRESIMS measurements, possessing an additional oxygen
1
atom compared to oryzamide C (3). The H and 13C NMR
spectra (Table 2) of 4 and 5 were almost identical to each other
C
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Figure 1. Key COSY and HMBC correlations of oryzamides A−E (1−5).
Figure 2. ESIMS/MS fragment ions (m/z) of oryzamides A−E (1−5).
D
DOI: 10.1021/acs.jnatprod.6b00349
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Figure 3. ORTEP drawings of 1 and 6 (Cu Kα).
and very similar to those of 3, except for the deshielded
resonances of C-4 at δC 48.5 or 49.1 and C-5 at δC 37.5 or 37.9,
respectively. This deshielding effect indicated the presence of a
methionine sulfoxide group,26 which was also confirmed by the
strong IR absorption bands at 1022 and 1025 cm−1,24
respectively. Careful comparisons of the 1D and 2D NMR
data between 4 and 5 showed the overall similarity but only
slight differences in the chemical shifts of C-3, C-4, and C-5,
implying that 4 and 5 are two diastereomers at a methionine
sulfoxide residue, which was further supported by the same
ESIMS/MS fragments (Figures 2, S40, and S51). The methione
sulfoxide residue is generally recognized as an artifact formed
via oxidation of methionine-containing natural products,
resulting from the multistep purification process.27,28 Com-
pound 3 was treated with a mild oxidant (m-chloroperbenzoic
acid), resulting in compounds 4 and 5. Thus, compounds 4 and
5 were oxidative products of compound 3. Interestingly, R and
S sulfoxide diastereomers are typically isolated as a mixture,28
but methionine sulfoxide diastereomers are not inherently
resistant to separation.29 Compounds 4 and 5 were easily
obtained in pure form. The absolute configurations of
compounds 4 and 5 were partially determined as L-Ala, L-Val,
and D-Leu by the advanced Marfey’s method, and the α-carbon
C-2 of the methionine sulfoxide was assigned the same
configuration as compound 3, considering that compounds 4
and 5 originated from compound 3.
Fortunately, scopularide A (6) yielded a crystal from 80%
MeOH/H2O, which was suitable for X-ray crystal structure
determination. The stereocenters of scopularide A except at C-
4 of the HMDA residue were determined previously by
Marfey’s and Mosher’s methods. Those identified stereocenters
are in agreement with the X-ray diffraction data. Notably, the
crystallographic analysis of 6 allowed the unambiguous
assignment of a 4S configuration of the HMDA unit for the
first time (Figure 3).
Oryzamides A−E (1−5) and scopularide A (6) are
structurally and biogenetically related cyclohexdepsipeptides
with the L-Val, D-Leu, L-Ala, Gly, and HMDA residues, which
constitute the same sequence (−Ala−Leu−Val−Gly−
HMDA−). Prior to this study, only nine cyclodepsipeptides
with an HMDA unit have been reported.14−19 Oryzamides A−
E (1−5) are probably hybrid peptide−polyketide natural
products based on the recent identification of the scopularide
A (6) biosynthetic gene cluster.30 Therefore, the chemo-
diversity of these compounds is presumably due to the
substrate promiscuity of the adenylation domain of the
nonribosomal peptide synthetase.31,32
In the cases of the bioactivities, oryzamides A−C (1−3) were
not cytotoxic (IC50 < 10 μM) against HeLa cells, with IC50
values of 25.7, 12.4, and 31.3 μM, respectively. None of them
showed any NF-κB inhibitory, antibacterial (methicillin-
sensitive Staphylococcus aureus, methicillin-resistant Staph-
ylococcus aureus, and Escherichia coli), or antiparasitic properties
(Ichthyophthirius multifiliis). Oryzamides D (4) and E (5) were
not evaluated for the aforementioned bioactivities because of
the scarcity of material.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on an Electrothermal 9100 apparatus and are uncorrected.
Optical rotations were measured with a PerkinElmer model 341
polarimeter with a 10 cm length cell at 589 nm. UV spectra were
obtained on a Hitachi U-3010 spectrophotometer. IR spectra (film)
were acquired on a Jasco FTIR-400 spectrometer. NMR spectroscopic
data were recorded on an Agilent 600 MHz NMR instrument.
HRESIMS and ESIMS/MS spectra were determined on a Waters
Xevo G2-XS QTOF spectrometer. UPLC-MS spectra were obtained
using a Waters Acquity UPLC system equipped with a Waters Xevo
G2-XS QTOF spectrometer and a C18 Acquity UPLC BEH column
(Waters, 2.1 × 50 mm, 1.7 μm). Preparative medium-pressure liquid
chromatography (MPLC) was carried out on an Interchim Puriflash
450 Instrument. Semipreparative reversed-phased HPLC (RP-HPLC)
was performed on a system composed of a Waters 1525 pump
equipped with a 2998 photodiode array detector and a YMC-Pack Pro
C18 column (YMC, 10 × 250 mm, 5 μm). 1-fluoro-2,4-dinitrophenyl-
5-L-/D-leucinamide (L-/D-FDLA), and m-chloroperbenzoic acid were
purchased from Sigma-Aldrich Chemical Corporation.
Fungal Material. The strain PF18 was isolated from the inner
tissue of the sponge Phakellia fusca collected from Yongxing Island,
China. The fungus was identified as Nigrospora oryzae by
morphological characteristics and sequence analysis of the ITS region
(GenBank accession no. FJ941865.1). A voucher strain was deposited
at the School of Life Sciences and Biotechnology, Shanghai Jiao Tong
University, Shanghai, China.
Fermentation. After being maintained on PDA medium for 7
days, the strain PF18 was inoculated into 250 mL Erlenmeyer flasks
each containing 100 mL of the seed medium (potato 200 g/L,
dextrose 20 g/L, and artificial seawater salts33 33 g/L in tap water) on
a rotatory shaker (180 rpm) at 28 °C for 48 h. The subsequent
fermentation was performed in 48 × 2-L Erlenmeyer flasks. Each flask
contained 12 g of mannitol, 12 g of maltose, 6 g of glucose, 6 g of
E
DOI: 10.1021/acs.jnatprod.6b00349
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1
Table 2. H (600 MHz) and 13C (150 MHz) NMR Data for Oryzamides D and E (4 and 5) in DMSO-d6
oryzamide D (4)
δC, type
oryzamide E (5)
δC, type
unit
position
δH (J in Hz)
4.27, m
unit
position
δH (J in Hz)
4.23, m
L-Met (O)
1
2
3
170.4, C
51.7, CH
23.1, CH2
L-Met (O)
1
2
3
170.3, C
52.1, CH
24.0, CH2
2.07, m
2.01, m
2.87, m
2.73, m
2.53, s
2.10, m
2.00, m
2.81, m
2.78, m
2.54, s
4
48.5, CH2
37.5, CH3
4
49.1, CH2
37.9, CH3
5
5
2-NH
7.40, d (6.5)
2-NH
7.48, d (4.2)
L-Ala
6
171.8, C
47.9, CH
16.5, CH3
L-Ala
6
171.8, C
47.8, CH
16.6, CH3
7
4.16, dq (8.1, 7.0)
1.24, d (7.0)
7
4.19, dq (8.3, 7.0)
1.24, d (7.0)
8
8
7-NH
9
8.23, d (8.1)
7-NH
9
8.26, d (8.3)
D-Leu
171.5, C
52.5, CH
38.8, CH2
D-Leu
171.4, C
52.6, CH
38.8, CH2
10
11
3.94, m
10
11
3.92, m
1.52, m
1.52, m
1.44, m
1.44, m
12
24.1, CH
22.9, CH3
21.1, CH3
1.64, m
12
24.1, CH
22.9, CH3
21.1, CH3
1.64, m
13
0.90, d (6.6)
0.82, d (6.6)
8.70, d (5.8)
13
0.90, d (6.4)
0.82, d (7.0)
8.69, d (5.5)
14
14
10-NH
15
10-NH
15
L-Val
172.2, C
L-Val
172.2, C
16
58.6, CH
29.8, CH
19.1, CH3
18.8, CH3
4.03, m
16
58.5, CH
29.9, CH
19.1, CH3
18.7, CH3
4.04, m
17
1.85, m
17
1.84, m
18
0.90, d (6.6)
0.84, d (6.5)
8.03, d (7.1)
18
0.90, d (6.6)
0.84, d (7.7)
7.93, d (7.4)
19
19
16-NH
20
16-NH
20
Gly
169.3, C
Gly
169.3, C
21
42.0, CH2
4.24, m
21
42.1, CH2
4.27, dd (16.0, 7.6)
3.28, d (16.0)
3.31, m
21-NH
22
8.01, d (3.5)
21-NH
22
8.04, d (7.2)
HMDA
169.6, C
HMDA
169.6, C
23
36.6, CH2
2.55, dd (14.9, 4.4)
2.25, dd (14.8, 2.4)
5.00, m
23
36.4, CH2
2.55, m
2.25, dd (14.8, 2.1)
4.99, m
24
25
26
75.0, CH
36.1, CH
31.6, CH2
24
25
26
74.9, CH
36.0, CH
31.6, CH2
1.64, m
1.63, m
1.34, m
1.34, m
0.98, m
0.98, m
27
26.6, CH2
1.28, m
27
26.6, CH2
1.28, m
1.20, m
1.20, m
28
29
30
31
32
29.0, CH2
31.2, CH2
22.1, CH2
14.0, CH3
14.8, CH3
1.23, m
28
29
30
31
32
29.0, CH2
31.2, CH2
22.1, CH2
14.0, CH3
14.8, CH3
1.23, m
1.24, m
1.24, m
1.26, m
1.26, m
0.86, t (6.8)
0.80, d (6.8)
0.86, t (7.0)
0.80, d (6.9)
sodium glutamate, 1.8 g of yeast extract, 0.6 g of corn syrup, 0.3 g of
KH2PO4, 0.18 g of MgSO4·7H2O, 19.8 g/L artificial seawater salts, and
600 mL of tap water, where 60 mL of the seed culture was transferred
and incubated at 28 °C with shaking at 180 rpm for 12 days.
Fr.3.C.3 was purified by semipreparative RP-HPLC (YMC-Pack Pro
C18, 50% MeCN/H2O, 2.0 mL/min, UV detection at 210 nm) to
yield oryzamide D (4, 2.8 mg, tR 16.9 min) and oryzamide E (5, 2.5
mg, tR 17.6 min). Subfraction Fr.3.C.4 was further separated by
semipreparative RP-HPLC (YMC-Pack Pro C18, 60% MeCN/H2O,
2.0 mL/min, UV detection at 210 nm) to obtain oryzamide B (2, 5.8
mg, tR 31.8 min) and oryzamide C (3, 10.3 mg, tR 33.9 min).
Subfraction Fr.3.C.5 was purified by semipreparative RP-HPLC
(YMC-Pack Pro C18, 55% MeCN/H2O, 2.0 mL/min, UV detection
at 210 nm) to afford oryzamide A (1, 18.2 mg, tR 94.9 min) and
scopularide A (6, 20.1 mg, tR 95.7 min).
Extraction and Isolation. The fungal mycelium was separated
from the fermentation broth by filtration and exhaustively extracted
with CH2Cl2/MeOH (1:1, v/v). The resultant extract was suspended
in 1 L of H2O and partitioned with CH2Cl2 (4 × 1 L) to yield the
CH2Cl2 extract (5.5 g), which was subjected to column chromatog-
raphy on Sephadex LH-20 with CH2Cl2/MeOH (1:1, v/v) as eluent,
affording four fractions (Fr.1−4). Fr.3 (2 g) was subjected to VLC
over silica gel using gradients of CH2Cl2/MeOH (20:1, 10:1, 8:1, 5:1,
1:1, 0:1, v/v) to obtain six subfractions (Fr.3.A−F). Fr.3.C (100 mg)
was further separated by reversed-phase MPLC (10−100% MeOH/
H2O, 180 min, flow rate 20 mL/min, UV detection at 210 nm) to
afford six subfractions (Fr.3.C.1−6). Subsequently, the subfraction
Oryzamide A (1): white crystals; mp 230 °C; [α]2D5 −30 (c 0.25,
MeOH); UV (MeOH) λmax (log ε) 201 (4.69) nm; IR (film) νmax
3314, 3259, 3060, 2957, 2927, 2866, 1724, 1667, 1637, 1540, 1464,
1
1444, 1377, 1276, 1235, 1186, 1169 cm−1; H and 13C NMR data,
F
DOI: 10.1021/acs.jnatprod.6b00349
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1; ESIMS/MS data, Figure 2; HRESIMS m/z 638.4504 [M +
H]+ (calcd for C33H60N5O7, 638.4493).
was treated with 1 N NaHCO3 (20 μL) and 100 μL of L- or D-/L-
FDLA (10 mg/mL in acetone). Each mixture was heated to 50 °C for
1 h. The reaction was quenched with 20 μL of 1 N HCl and dried
under vacuum. The residue was redissolved in 300 μL of MeOH. The
retention times of the L- and L-/D-FDLA-derivatized amino acids were
measured by UPLC-MS using an Acquity UPLC BEH C18 column
(2.1 × 50 mm, 1.7 μm, 0.5 mL/min) with a linear gradient from 10%
to 100% MeCN/H2O containing 0.1% formic acid over 10 min.
Through comparison of the retention times of the L- and D-/L-FDLA
derivatives of corresponding amino acids, the absolute configurations
were established. The retention times of the L-FDLA amino acid
derivatives were 3.53 min (L-Ala, m/z 384 [M + H]+), 3.94 min (L-Val,
m/z 412 [M + H]+), 5.51 min (L-Tyr, m/z 770 [M + H]+), 3.92 min
(L-Met, m/z 444 [M + H]+), and 5.23 min (D-Leu, m/z 426 [M +
H]+). The retention times of the D-/L-FDLA amino acid derivatives
were 3.53 and 4.02 min (L-Ala, m/z 384 [M + H]+), 3.94 and 4.78 min
(L-Val, m/z 412 [M + H]+), 5.51 and 5.92 min (L-Tyr, m/z 770 [M +
H]+), 3.92 and 4.63 min (L-Met, m/z 444 [M + H]+), and 4.30 and
5.23 min (D-Leu, m/z 426 [M + H]+) (Figures S21, S32, S43, and
S54).
Cytotoxicity Assay. The cytotoxic activities of compounds 1−3
against HeLa cells were evaluated by the MTT method as described
previously.36 Cisplatin (IC50 = 4.2 μM) was used as the positive
control.
NF-κB Luciferase Assay. For compounds 1−3, the NF-κB
inhibitory activity was assessed with the methods described by Jiao
et al.37
Antibacterial Activity Assay. Compounds 1−3 were tested for
antibacterial activities against methicillin-sensitive Staphylococcus aureus
ATCC25923, methicillin-resistant Staphylococcus aureus ATCC43300,
and Escherichia coli ATCC25922 in 96-well microplates according to a
previously published protocol.38,39 Chloramphenicol was used as the
positive control and exhibited MICs of 2, 4, and 2 μg/mL, respectively.
Antiparasitic Activity Assay. Compounds 1−3 were evaluated
for antiparasitic activity against Ichthyophthirius multifiliis according to
the established protocol of Liang et al.40
Oryzamide B (2): white powder; [α]2D5 −33 (c 0.50, MeOH); UV
(MeOH) λmax (log ε) 206 (3.97), 229 (3.93), 277 (3.75) nm; IR
(film) νmax 3304, 3061, 2958, 2928, 2857, 1738, 1649, 1538, 1518,
1460, 1380, 1231, 1176 cm−1; 1H and 13C NMR data, Table 1;
ESIMS/MS data, Figure 2; HRESIMS m/z 688.4285 [M + H]+ (calcd
for C36H58N5O8, 688.4285).
Oryzamide C (3): white powder; [α]2D5 −33 (c 0.65, MeOH); UV
(MeOH) λmax (log ε) 214 (3.65) nm; IR (film) νmax 3403, 3285, 3059,
2958, 2924, 2854, 1737, 1669, 1641, 1531, 1501, 1464, 1448, 1426,
1
1381, 1312, 1279, 1233, 1195, 1170, 1102, 1065, 1018 cm−1; H and
13C NMR data, Table 1; ESIMS/MS data, Figure 2; HRESIMS m/z
656.4068 [M + H]+ (calcd for C32H58N5O7S, 656.4057).
Oryzamide D (4): white powder; [α]2D5 −28 (c 0.40, MeOH); UV
(MeOH) λmax (log ε) 213 (4.41) nm; IR (film) νmax 3302, 3050, 2959,
2928, 2861, 1737, 1650, 1540, 1462, 1383, 1277, 1233, 1209, 1173,
1
1022 cm−1; H and 13C NMR data, Table 2; ESIMS/MS data, Figure
2; HRESIMS m/z 672.4016 [M + H]+ (calcd for C32H58N5O8S,
672.4006).
Oryzamide E (5): white powder; [α]2D5 +4 (c 0.50, MeOH); UV
(MeOH) λmax (log ε) 213 (4.35) nm; IR (film) νmax 3299, 3049, 2959,
2929, 2868, 1737, 1651, 1538, 1463, 1383, 1277, 1231, 1174, 1025
1
cm−1; H and 13C NMR data, Table 2; ESIMS/MS data, Figure 2;
HRESIMS m/z 672.4015 [M + H]+ (calcd for C32H58N5O8S,
672.4006).
Scopularide A (6): white crystals; mp 230 °C; [α]2D5 −33 (c 0.30,
MeOH).
Crystal Structure Determination. Single crystals of oryzamide A
(1) and scopularide A (6) were obtained by slow evaporation from
80% MeOH/H2O and were measured on a Bruker APEX-II CCD
diffractometer using graphite-monochromated Cu Kα radiation (λ =
1.541 78 Å). The thermal ellipsoid diagram was generated by
ORTEP.34 The structure was solved by direct methods (Shelxs97)
and refined by the full-matrix least-squares method on F2.35 Hydrogen
atoms were located by geometric calculation and difference Fourier
methods. Crystallographic data for compounds 1 and 6 have been
deposited with the Cambridge Crystallographic Data Center as
supplementary publication no. CCDC 1474001 and 1474000,
respectively. 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: deposit@ccdc.cam.ac.
uk).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00349.
Crystal Data for Compound 1. The molecular structure comprises
six molecules of 1, three in the asymmetric unit, and four molecules of
water, one at half-occupancy. C99H184N15O24.5, M = 1976.6,
monoclinic, space group = P2(1), No. 4, a = 17.2997(3) Å, b =
16.9120(3) Å, c = 19.4202(3) Å, β = 90.6820(10)°, V = 5681.42(17)
Å3, T = 130 K; Z = 2, Dx = 1.155 mg/m3, F(000) = 2158, μ(Cu Kα) =
0.671 mm−1, crystal dimensions 0.25 × 0.1 × 0.03 mm3, 41 717 unique
reflections collected (θmax = 69.542°), 18 172 independent reflections
(Rint = 0.0529). The final R1 = 0.0620, wR2 = 0.1642 (w = 1/σ|F|2), and
S = 1.008. The Flack parameter was −0.07(15).
Crystal Data for Compound 6. The molecular structure comprises
one molecule of 6 and one molecule of water. C36H59N5O8, M =
689.88, monoclinic, space group = C2, No. 5, a = 26.172(2) Å, b =
9.2265(6) Å, c = 16.7839(13) Å, β = 107.326(5)°, V = 3869.0(5) Å3, T
= 130 K; Z = 4, Dx = 1.184 mg/m3, F(000) = 1496, μ(Cu Kα) = 0.679
mm−1, crystal dimensions 0.25 × 0.12 × 0.02 mm3, 13 510 unique
reflections collected (θmax = 69.263°), 5879 independent reflections
(Rint = 0.0570). The final R1 = 0.0595, wR2 = 0.1513 (w = 1/σ|F|2), and
S = 1.022. The Flack parameter was −0.1(15).
Acid Hydrolysis of Oryzamides B−E (2−5). Approximately 0.5
mg of each of 2−5 was hydrolyzed with 6 N HCl (1 mL) for 16 h at
110 °C. After cooling to room temperature, the hydrolysate mixtures
were evaporated to dryness, and traces of HCl were removed by
repeated drying in vacuo.
Absolute Configurations of Amino Acids by the Advanced
Marfey’s Analysis. Each acid hydrolysate was resuspended in 100 μL
of H2O and separated into two equal portions. Each portion (50 μL)
1D and 2D NMR, HRESIMS, HRESIMS/MS, UV, IR
spectra, and the advanced Marfey’s analysis for
compounds 1−5 (PDF)
X-ray crystallographic data of 1 and 6 (ZIP)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: txush@jnu.edu.cn (S. H. Xu).
*E-mail: wei.zhang@flinders.edu.au (W. Zhang).
*Tel: +86-21-68383346. Fax: +86-21-58732594. E-mail:
franklin67@126.com, hwlin@sjtu.edu.cn (H. W. Lin).
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Natural Science
Fund for Distinguished Young Scholars of China (No.
81225023) and the National Natural Science Fund of China
(Nos. 41428602, 41476121, 21172094, 41376155, 21372100,
81373321, U1301131, and 81402844). We are also grateful for
the financial support of the National High Technology
Research and Development Program of China (863 Project,
Nos. 2013AA092901 and 2013AA092902), Public Science and
G
DOI: 10.1021/acs.jnatprod.6b00349
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