Psychrophilins E-H and versicotide C, cyclic peptides from the marine-derived fungus aspergillus versicolor ZLN-60

Article abstract of DOI:10.1021/np500469b

Four new cyclic peptides, psychrophilins E-H (1-4), possessing a rare amide linkage between the carboxylic acid in anthranilic acid (ATA) and the nitrogen from an indole moiety, along with a new ATA-containing hexapeptide, versicotide C (5), were obtained from the culture of the marine-derived fungus Aspergillus versicolor ZLN-60. The structures, including absolute configurations, were elucidated by a combination of HRESIMS, NMR, X-ray crystallography, TDDFT ECD calculations, and Marfeys method. Versicotide C (5) is the first natural cyclic hexapeptide containing two anthranilic acids. Compounds 1-5 were not cytotoxic, and compound 3 showed potent lipid-lowering effects.

Full text of DOI:10.1021/np500469b

Article
pubs.acs.org/jnp
Psychrophilins E−H and Versicotide C, Cyclic Peptides from the
Marine-Derived Fungus Aspergillus versicolor ZLN-60
†
†
†
‡
‡
†
‡
Jixing Peng, Huquan Gao, Xiaomin Zhang, Shuai Wang, Chongming Wu, Qianqun Gu, Peng Guo,
,†
,†
Tianjiao Zhu,* and Dehai Li*
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao, 266003, People’s Republic of China
‡
Pharmacology and Toxicology Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences,
Peking Union Medical College, Beijing, 100193, People’s Republic of China
*
S Supporting Information
ABSTRACT: Four new cyclic peptides, psychrophilins E−H
1−4), possessing a rare amide linkage between the carboxylic
(
acid in anthranilic acid (ATA) and the nitrogen from an indole
moiety, along with a new ATA-containing hexapeptide,
versicotide C (5), were obtained from the culture of the
marine-derived fungus Aspergillus versicolor ZLN-60. The
structures, including absolute configurations, were elucidated
by a combination of HRESIMS, NMR, X-ray crystallography,
TDDFT ECD calculations, and Marfey’s method. Versicotide
C (5) is the first natural cyclic hexapeptide containing two
anthranilic acids. Compounds 1−5 were not cytotoxic, and
compound 3 showed potent lipid-lowering effects.
he OSMAC (one strain many compounds) approach is an
T
effective way to expand the structural diversity of
metabolites of a single fungal strain by systematic alteration
of its cultivation parameters including media composition, pH,
temperature, oxygen supply, quality and quantity of light,
bioreactor platform, and the addition of precursors or enzyme
1
−3
inhibitors.
Our laboratory has successfully applied these
methods to maximize the production of new secondary
metabolites from marine-derived fungi. Examples of these
4
were cytochalasins from Spicaria elegans KLA03 and melegarin
alkaloids and nitrogen-containing sorbicillinoids from Penicil-
5
lium sp. F23-2.
In our preliminary screening using the OSMAC strategy, the
CHCl −MeOH to give three fractions. These were further
3
fungus Aspergillus versicolor ZLN-60, which produces anthranilic
fractionated by Sephadex LH-20 column chromatography and
semipreparative HPLC to give compounds 1−5.
6
acid (ATA)-containing pentapeptides (versicotides A, B) and
7
prenylated diphenyl ethers (diorcinols B−E), was selected for
Psychrophilin E (1) was obtained as colorless crystals. Its
molecular formula of C H N O was established on the basis
further study. The HPLC-UV profile of the culture changed
2
5
24
4
4
6
+
dramatically upon alteration of the original liquid medium to a
of the HRESIMS ion at m/z 445.1870 [M + H] , requiring 12
degrees of unsaturation. Analysis of the H NMR and
1
13
rice-based solid medium. Further fractionation of the extract of
the solid-phase culture led to the discovery of five new cyclic
peptides, psychrophilins E−H (1−4) and versicotide C (5).
Herein, we report the details of the isolation, structure
elucidation, and biological activities of these new cyclic
peptides.
C
NMR data (Tables 1 and 2) revealed the presence of 24 proton
and 25 carbon resonances including four amide carbonyls (δ
1
C
66.6, 168.2, 169.9, 171.7), two sp3 methines, four sp
3
methylenes, one methyl, two exchangeable protons, and 14
aromatic carbons including two ortho-substituted benzenes with
one of them in an indole moiety, suggesting a peptide with an
anthranilic acid and a tryptophan-derived unit, which were
RESULTS AND DISCUSSION
■
The EtOAc extract (6 g) of the 30-day static fermentation in
rice medium was subjected to vacuum liquid chromatography
over silica gel using a gradient elution with petroleum ether−
Received: June 9, 2014
Published: September 23, 2014
©
2014 American Chemical Society and
American Society of Pharmacognosy
2218
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1
Table 1. H NMR Spectroscopic Data (600 MHz, DMSO-d ) for Compounds 1−4
6
position
1
2
3
4
2
3
4.65, m
3.16, m; 2.87 (dd, 12.4, 4.5)
.88, m
5.35, dd (12.5, 4.5)
5.44, d (10.1)
5.18, d (10.1)
5.37, d (10.1)
5.25, d (10.1)
3.26, t (12.4); 3.02 (dd, 12.4, 4.5)
3.02, dd (12.1, 4.5)
2
3
6
7
8
9
1
1
1
1
1
1
2
2
2
2
2
2
-OH
5.94, br s
not det.
a
7.74, m
7.67, br d (6.8)
7.93, br d (7.7)
7.34, ddd (7.7, 7.7, 1.0)
7.39, ddd (7.7, 7.7, 1.2)
8.47, br d (7.7)
6.86, s
a
7.35, ddd (7.7, 7.7, 1.0)
7.39, ddd (7.7, 7.7, 1.3)
8.46, br d (7.7)
7.36, m
6.74, d (8.2)
7.25, t (8.2)
7.98, d (8.2)
6.84, s
a
7.41, m
8.49, d (6.8)
6.82, s
1
6.77, s
a
4
7.72, m
7.72, br d (7.6)
7.65, br d (7.6)
7.65, ddd (7.6, 7.6, 0.8)
7.40, ddd (7.6, 7.6, 1.1)
7.73, br d (7.6)
9.32, s
7.72, br d (7.7)
a
5
7.39, ddd (7.7, 7.7, 0.9)
7.65, ddd (7.7, 7.7, 1.0)
7.40, m
7.40, ddd (7.7, 7.7, 0.8)
7.65, ddd (7.7, 7.7, 0.9)
7.60, br d (7.7)
9.40, s
6
7.64, ddd (7.6, 7.6, 1.0)
7.83, br d (7.6)
9.30, s
a
7
7.71, m
8-NH
9.36, s
0
1
2
3
5
6
4.53, dd (8.3, 3.9)
1.82, m; 1.62, m
1.45, m; 1.25, m
3.37, m; 2.02, m
1.87, s
4.50, dd (8.0, 4.4)
1.83, m; 1.67, m
1.43, m; 1.35, m
3.51, m; 1.93, m
2.08, s
4.49, dd (8.3, 3.8)
1.81, m; 1.60, m
1.40, m; 1.28, m
3.49, m; 1.90, m
2.08, s
4.52, dd (7.7, 3.2)
1.83, m; 1.59 m
1.44, m; 1.26 m
3.51, m; 2.12 m
2.08, s
8.55, d (6.1)
3.18, s
3.27, s
3.28, s
a
Overlapped signals.
Table 2. 13C NMR Spectroscopic Data (150 or 100 MHz,
DMSO-d ) for Compounds 1−4
6
a
a
a
b
position
1
2
3
4
1
2
3
4
5
6
7
8
9
171.7, C
170.3, C
169.3, C
169.4, C
51.8, CH
28.0, CH2
116.4, C
52.7, CH
25.8, CH2
115.9, C
58.0, CH
66.2, CH
119. 8, C
128.7, C
59.4, CH
65.6, CH
118.5, C
130.2, C
129.8, C
117.4, C
119.1, CH
124.3, CH
126.0, CH
116.8, CH
135.4, C
118.4, CH
123.8, CH
125.0, CH
116.4, CH
135.0, C
120.6, CH
123.4, CH
125.3, CH
116.0, CH
135.2, C
149.2, C
109.6, CH
126.8, CH
107.8, CH
137.0, C
Figure 1. Key COSY and HMBC correlations of 1 and 2.
the known cyclic nitropeptide psychrophilin A, which contains
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
8
a nitro group at C-2. Finally, the planar structure of 1 was
125.4, CH
166.6, C
125.6, CH
166.1, C
124.0, CH
166.1, C
123.1, CH
166.3, C
confirmed by attaching an acetamide on C-2 according to the
H
COSY correlation between H-2 (δ 4.65, m) and H-26 (δH
127.4, C
126.5, C
126.7, C
127.2, C
8
1
Z
.55, d, J = 6.06 H ) and HMBC correlations from H-25 (δH
132.1, CH
126.0, CH
132.9, CH
123.5, CH
134.3, C
131.6, CH
125.2, CH
132.3, CH
122.6, CH
134.0, C
131.4, CH
124.7, CH
132.2, CH
123.0, CH
133.6, C
131.6, CH
125.7, CH
132.5, CH
123.5, CH
133.7, C
.87, s) to C-24 (δ 169.9) and from H-26 to C-24 (Figure 1
C
and Table 1). To determine the absolute configuration of 1,
crystallographic quality crystals were successfully grown and
analyzed by X-ray diffraction with Cu Kα irradiation (Flack
parameter = −0.08), revealing the absolute configurations at C-
168.2, C
168.0, C
167.2, C
167.3, C
2
and C-20 to be 2S and 20S (Figure 2).
HRESIMS and 1D NMR data for psychrophilin F (2)
59.3, CH
24.8, CH2
25.9, CH2
46.7, CH2
169.9, C
59.2, CH
24.2, CH2
25.5, CH2
46.0, CH2
170.8, C
58.5, CH
24.7, CH2
24.0, CH2
45.9, CH2
171.2, C
58.2, CH
25.0, CH2
24.2, CH2
46.2, CH2
171.4, C
established the molecular formula C H N O , a 14 amu
26 26
4
4
increase in the molecular weight compared with 1. The 1D
NMR data indicated the appearance of an N-Me (δ 3.18, δ
H
C
3
1.7) and was assigned by the HMBC correlations from H -26
22.5, CH3
22.1, C
22.0, CH3
31.2, CH3
22.2, CH3
31.7, CH3
3
to C-2 and C-24 (Tables 1 and 2 and Figure 1). The
31.7, CH3
a
b
configuration of the L-proline in 2 was determined by the
advanced Marfey’s method. Biogenetically, the absolute
Measured at 150 MHz. Measured at 100 MHz.
9
configuration of C-2 was proposed to be the same as 1,
further identified by 2D NMR data (Figure 1). A proline unit
which was also supported by the similar CD spectra (Figure
8
was deduced by COSY correlations between H-20 and H -21,
3).
2
between H -21 and H -22, and between H -22 and H -23,
The HRESIMS adduct peaks indicated the molecular
formulas for psychrophilins G (3) and H (4) to be
C H N O and C H N O , with one and two more oxygen
2
2
2
2
together with the HMBC correlations from H -21 to C-19. The
2
amino acid sequence of 1 was established by the HMBC
correlations (Figure 1) and the comparison of the NMR data to
26
26
4
5
26 26
4
6
1
13
atoms than compound 2, respectively. Their H and C NMR
2
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Figure 4. Key COSY and HMBC correlations of 3 and 4.
Figure 2. X-ray crystal structure of compound 1.
data (Tables 1 and 2) revealed that the structures of 3 and 4
closely resembled compound 2, sharing the same cyclic peptide
skeleton. The differences were attributed to the appearance of a
C-3 hydroxy group in 3 and at C-3 and C-6 in 4, which were
supported by the chemical shifts (δ 5.18 and δ 66.2, CH-3 in
H
C
3
; δ 5.25 δ 65.6, CH-3, and δ 149.2, C-6 in 4), together with
H C C
COSY and HMBC correlations (Tables 1 and 2 and Figure 4).
The configurations at C-20 of psychrophilins G (3) and H
4) were determined as S from a Marfey’s analysis of the L-
proline unit. The anti relationships of H-2 and H-3 were
(
Figure 5. Selected NOE correlations of 3 and NOESY correlations of
4
.
suggested by the vicinal coupling constants between them
3
(
JH‑2,H‑3 = 10.1 Hz for both of 3 and 4), leading to two possible
The molecular formula of C H N O for versicotide C (5)
28 34 6 6
absolute configurations, (2S, 3R, 20S) and (2R, 3S, 20S), for
compounds 3 and 4. The NOEs of H-6/H-2 and H-26/H-3/H-
was provided by its HRESIMS and also supported by NMR
1
data (Table 3), indicating 15 degrees of unsaturation. The H
1
1 in 3 supported the 2S/3R configurations (Figure 5). The
TDDFT-ECD spectra for the two epimers (2S,3R,20S)-3 and
2R,3S,20S)-3 were calculated (Figure 6), and the absolute
configuration of 3 was unambiguously determined as (2S, 3R,
0S), because the calculated curves for (2S,3R,20S)-3 agreed
and 13C NMR spectra indicated the presence of four NH
doublets (δ 12.33, 11.89, 9.13, and 8.28), four characteristic α-
H
(
CH groups (δ /δ 5.19/45.2, 5.19/44.8, 4.57/54.9, and 4.01/
H
C
61.3), and six amide carbonyl signals (δ 171.5, 170.5, 169.7,
C
2
169.2, 166.9, and 166.8).
with the experimental one. The absolute configuration of
compound 4 was also inferred to be the same as 3 based on the
similarity of its experimental ECD spectrum (Figure 3).
Analysis of the 1D and 2D NMR data (Table 3 and Figure 7)
of 5 established six amino acid residues: two alanines, two N-
methylalanines, and two ATA residues. The sequence of the six
Figure 3. Experimental ECD spectra of compounds 1, 2, 3, and 4.
2
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Figure 7. Key COSY and HMBC correlations of 5.
Figure 6. B3LYP/6-31+G(d)-calculated ECD spectra of (2S, 3R,20S)-
3
(red) and (2R,3S,20S)-3 (blue) and the experimental ECD spectrum
of 3 (black) (σ = 0.40 eV).
planar structure of versicotide C (5) was thus elucidated as a
cyclo-[(Me-Ala)-Ala-ATA-(Me-Ala)-Ala-ATA]. Similar to the
previously isolated versicotides A and B, compound 5 was also
6
Table 3. NMR Spectroscopic Data (400 MHz, DMSO-d ) for
6
Compound 5
composed of ATA, Ala, and Me-Ala units.
The amino acid residues in versicotide C (5) were identified9
position
1
δC, type
δH (J in Hz)
as L-Ala and N-Me-L-Ala by the advanced Marfey’s method,
169.7, C
applied to the acid hydrolysate of 5.
1
2
3
4
4
5
6
7
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
-NH
-NMe
-NH
12.33, s
Although the planar structure of 5 is symmetrical, the
distinguishing NMR signals of the two N-Me (Table 3)
indicated the different configurations of the two corresponding
amide bonds considering that they are both formed from N-
54.9, CH
16.8, CH3
170.5, C
4.57, q (7.0)
1.56, d (7.0)
29.6, CH3
44.8, CH
17.6, CH3
166.8, C
2.91, s
10
Me-L-Ala. Consistent with previous reports, the N-methyl
5.19, m
resonance of the cis conformer (syn to carbonyl group) was
upfield relative to the trans due to the shielding effect of the
carbonyl group, whereby the 4-NMe (δ 2.91, δ 29.6) was
1.22, d (6.5)
H
C
9.13, d (9.1)
deduced as cis, while 17-NMe (δ 3.35, δ 37.6) was trans. The
H
C
118.3, C
cis/trans conformation was further confirmed by a variable-
127.7, CH
121.7, CH
132.4, CH
118.4, CH
139.9, C
7.92, d (8.4)
7.01, br t (8.4)
7.43, br t (8.4)
8.60, d (8.4)
1
temperature H NMR experiment, which showed a significant
0
1
decrease in the number of H NMR resonances measured at 80
1
°
C compared to that measured at 25 °C (Figure S40,
2
Supporting Information), based upon the rotation of the C−
3
N amide bond at higher temperature.
4
169.2, C
The new cyclic peptides 1−5 were evaluated for cytotox-
icities against A-549, HeLa, and SMMC-7721 cancer cells with
4-NH
11.89, s
5
61.3, CH
12.3, CH3
171.5, C
4.01, q (6.8)
1.37, d (6.8)
11
the sulforhodamine B (SRB) method, but none of them were
6
toxic (IC50 >100 μM). In a test for lipid-lowering activity in
HepG2 hepatocarcinoma cells, compound 3 exhibited potent
lipid-lowering effects at a dose of 10 μM as assessed by Oil Red
7
7-NMe
37.6, CH3
45.2, CH
17.1, CH3
166.9, C
3.35, s
8
5.19, m
12
O staining (Figure 8).
9
1.33, d (7.1)
In summary, five new cyclic peptides were disclosed from A.
versicolor ZLN-60 guided by the OSMAC approach, indicating
that the Aspergillus genus is a pliable source of secondary
metabolites with high structural diversity and interesting
0
0-NH
8.28, d (7.3)
1
2
3
4
5
6
115.5, C
128.2, CH
121.4, CH
132.2, CH
118.3, CH
140.1, C
7.91, d (7.6)
6.85, br t (7.6)
7.01, br t (7.6)
7.91, d (7.6)
13
bioactivities. Psychrophilins represent rare fungal cyclic
peptides containing amide groups composed of anthranilic
acid and indole motifs in the macrocycle. Only four prior
examples of such were reported to date (psychrophilins A−D
8
from Penicillium sp.). Compounds 1−4 extend the psychro-
philin class from four compounds to the present eight. It is
worth noting that all of the compounds isolated contain one or
two anthranilic acid residues, which are relatively rare in cyclic
peptides, and versicotide C (5) is the first natural cyclic
hexapeptide containing two anthranilic acids. Biogenetically,
anthranilic acid can be produced by two possible pathways, the
shikimic acid pathway, as an intermediate during the synthesis
of tryptophan, and the kynurenine pathway, in which
tryptophan is first metabolized to kynurenine and subsequently
amino acid units was confirmed on the basis of the observed
HMBC correlations from alpha and N-methyl protons, as well
as amide NH signals to the carbonyl carbons including 1-NH
6
1
(
ATA ) with the carbonyl carbon C-1 (Me-Ala ), H-2 and N-
1
methyl protons (4-NMe, Me-Ala ) with amide carbon C-4
2
2
3
(
(
(
1
Ala ), H-5 (Ala ) with carbonyl carbon C-7 (ATA ), 14-NH
3
4
ATA ) with amide carbon C-14 (Me-Ala ), N-methyl protons
4
5
17-NMe, Me-Ala ) with carbonyl carbon C-17 (Ala ), and H-
8 (Ala ) with carbonyl carbon C-20 (ATA ) (Figure 7). The
5
6
14
to ATA by kynureninase. Further studies are needed to
2
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Psychrophilin D (1): colorless crystals; mp 190 °C; [α]25 +96 (c
D
0
(
(
2
.1, MeOH); UV (MeOH) λ (log ε) 214 (4.2), 244 (4.0), 302
max
−3
3.7); ECD (2.25 × 10 M, MeOH) λmax (Δε) 374 (+0.06), 316
−1.26), 288 (+2.81), 273.5 (+2.24), 256.5sh (+0.20), 233 (−12.97),
25 (−11.82), 220 (−12.76) nm; IR (KBr) ν 3375, 1699, 1636,
−1 1 13
max
1
449, 1363 cm ; H NMR and C NMR (see Tables 1 and 2);
+
HRESIMS [M + H] m/z 445.1875 (calcd for C H N O ,
2
5
25
4
4
4
45.1870).
Psychrophilin E (2): white, amorphous powder; [α]25 +120 (c 0.1,
D
MeOH); UV (MeOH) λ (log ε) 214 (4.3), 244 (4.1), 302 (3.7);
max
−3
ECD (2.18 × 10 M, MeOH) λmax (Δε) 371 (+0.01), 320 (−1.01),
2
(
90.5 (+2.63), 273 (+5.30), 255sh (+0.10), 232.8 (−11.62) nm; IR
−
1
1
13
KBr) ν 3372, 1699, 1637, 1449, 1361 cm ; H NMR and
C
max
+
NMR (see Tables 1 and 2); HRESIMS [M + H] m/z 459.2027 (calcd
for C H N O , 459.2027).
Psychrophilin F (3): white, amorphous powder; [α] +29 (c 0.1,
26
27
4
4
2
5
D
MeOH); UV (MeOH) λ (log ε) 217 (4.4), 244 (4.2), 304 (3.7);
max
−3
ECD (2.10 × 10 M, MeOH) λmax (Δε) 367 (−0.07), 314 (−0.76),
2
3
90.5 (+1.76), 255.8sh (+0.18), 223 (−10.75) nm; IR (KBr) ν
max
−1 1
300, 1696, 1630, 1523, 1449, 1268, 1169, 1088, 754 cm ; H NMR
and C NMR (see Tables 1 and 2); HRESIMS [M + H]+ m/z
13
Figure 8. Effects of compounds on oleic acid-elicited intracellular lipid
accumulation. Positive control: simvastatin; blank: DMEM; OA: oleic
acid. Neutral lipids were determined by spectrophotometry at 358 nm
after Oil Red O staining. Bars depict the means ± SEM in triplicate.
4
75.1980 (calcd for C H N O , 475.1976).
Psychrophilin G (4): white, amorphous powder; [α] +31 (c 0.1,
26 27 4 5
2
5
D
MeOH); UV (MeOH) λ (log ε) 217 (4.3), 244 (4.1), 304 (3.7);
ECD (1.02 × 10 M, MeOH) λmax (Δε) 383 (+0.26), 329 (−0.71),
2
max
−3
##
p < 0.001, OA vs blank; **p < 0.01, test group vs OA group.
97 (+2.25), 257.3sh (−0.07), 236.5 (−8.00), 228.3 (−3.78), 222.2
(
1
−6.92) nm; IR (KBr) ν 3303, 1696, 1631, 1525, 1449, 1268, 1169,
max
13
−
1 1
086 cm ; H NMR and C NMR (see Tables 1 and 2); HRESIMS
determine which of these routes is responsible for the
biosynthesis of ATA in A. versicolor.
+
[
M + H] m/z 491.1930 (calcd for C H N O , 491.1925).
26 27 4 6
25
Versicotide C (5): white, amorphous powder; [α] +100 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 216 (0.1), 290 (2.6); IR (KBr)
−1
1
13
EXPERIMENTAL SECTION
νmax 3356, 3190, 2906, 1664, 1613, 1426, 756 cm ; H NMR and C
■
+
NMR (see Table 3); HRESIMS [M + H] m/z 551.2616 (calcd for
C H N O , 551.2613).
General Experimental Procedures. Optical rotations were
measured with a JASCO P-1020 digital polarimeter. UV spectra
were recorded on a Beckman DU 640 spectrophotometer. CD spectra
were measured on a JASCO J-715 spectropolarimeter. IR spectra were
recorded on a Nicolet Nexus 470 spectrophotometer on KBr discs.
NMR spectra were recorded on JEOL JNMECP 600 and Bruker-400
spectrometers using TMS as an internal standard. Ectothermic NMR
spectra were recorded on Varian-500 spectrometers. HRESIMS
spectra were measured on a Micromass EI-4000 (Autospec-Ultima-
TOF). X-ray crystal data were measured on an Agilent Gemini Ultra
diffractometer (Cu Kα radiation). Semipreparative HPLC was
performed using an ODS column (YMC-Pack ODS-A, 5 μm, 10 ×
28 35
6
6
X-ray Crystallographic Analysis of Compound 1. Single-
crystal X-ray diffraction data were collected on an Agilent Gemini
Ultra diffractometer with Cu Kα radiation (λ = 1.541 84 Å). The
structure was solved by direct methods (SHELXS-97) and refined
using full-matrix least-squares difference Fourier techniques. Carbon,
oxygen, and nitrogen atoms were refined anisotropically. Hydrogen
atoms were either refined freely with isotropic displacement
parameters or positioned with an idealized geometry and refined
riding on their parent C atoms. Crystals suitable for X-ray diffraction
were obtained by slow evaporation of a solution in MeOH−H O.
2
2
50 mm, 4 mL/min). TLC and column chromatography (CC) were
Crystallographic data (excluding structure factors) for 1 have been
deposited with the Cambridge Crystallographic Data Centre: CCDC
reference number 973003. These data can be obtained, free of charge,
from the Cambridge Crystallographic Data Centre via http://www.
ccdc.cam.ac.uk/data_request/cif.
performed on plates precoated with silica gel GF254 (10−40 μm) and
over silica gel (200−300 mesh, Qingdao Marine Chemical Factory),
respectively. Size-exclusion chromatography was performed using
Sephadex LH-20 (GE Healthcare). The seawater was collected from
Huiquan Bay, Yellow Sea, China.
Crystal data for 1: orthorhombic, C25
2 2 with a = 12.396 10(10) Å, b = 12.968 80(10) Å, c = 13.795
1 1 1
H N O4, space group
24 4
Fungal Material. The fungal strain was isolated and identified as
P2
70(10) Å, α = 90°, β = 90°, γ = 90°, V = 2217.83(3) Å , Z = 4, T =
6
3
previously described.
3
−1
Fermentation and Extraction. The strain was cultured under
static conditions at about 28 °C in 1 L Erlenmeyer flasks containing
rice medium (rice 80 g, seawater 120 mL). After 30 days, the cultures
of 100 flasks were extracted with MeOH. The MeOH extract was
evaporated under reduced pressure to afford an aqueous solution and
then extracted with EtOAc. The EtOAc solution was concentrated
under reduced pressure to give an organic extract (6.0 g).
292(2) K, D
Crystal size: 0.32 × 0.3 × 0.24 mm . Independent reflections: 4129
with Rint = 0.0218. The final agreement factors are R = 0.0272 and
wR = 0.0768 [I > 2σ(I)]. Flack parameter = −0.08(15).
Absolute Configuration of Amino Acids. The acid hydrolysates
of 2−5 were redissolved in H O (50 μL) separately, and then 0.25 μM
L-FDAA in 100 μL of acetone was added, followed by 1 N NaHCO
c
= 1.331 mg/mm , μ = 0.752 mm , and F (000) = 936.
3
1
2
2
3
Purification. The organic extract was subjected to vacuum liquid
chromatography over a silica gel column using a gradient elution with
(25 μL). The mixtures were heated for 1 h at 43 °C. After cooling to
room temperature, the reaction was quenched by the addition 2 N
HCl (25 μL). Finally the resulting solution was filtered through a small
4.5 μm filter and stored in the freezer until HPLC analysis. Amino acid
standards were derivatized with L-FDAA in a similar manner. The
resulting L-FDAA derivatives of compounds 2−4, L- and D-alanine, and
L- and D-Me-alanine were separately analyzed by reversed-phase HPLC
(5 × 250 mm YMC C18 column, 5 μm, with a linear gradient of MeCN
(A) and 0.05% aqueous TFA (B) from 5% to 55% A over 55 min at a
flow rate of 1 mL/min, UV detection at 320 nm). The resulting L-
FDAA derivatives of 5, L- and D-proline, were analyzed by reversed-
petroleum ether−CHCl −MeOH to give three fractions. Fraction 1
3
was subjected to Sephadex LH-20 column chromatography eluting
with MeOH and then purified by semipreparative HPLC (55:45
MeOH−H O, 4 mL/min) to give compound 1 (8 mg, t 13 min),
2
R
compound 2 (22 mg, t 18 min), and compound 3 (30 mg, t 10 min).
R
R
Fraction 2 was separated by Sephadex LH-20 eluting with CHCl −
3
MeOH (1:1) and then by semipreparative HPLC (55:45 MeOH−
H O, 4 mL/min) to afford compound 4 (6 mg, t 15 min) and
2
R
compound 5 (60 mg, t 19 min).
R
2
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dx.doi.org/10.1021/np500469b | J. Nat. Prod. 2014, 77, 2218−2223

Journal of Natural Products
Article
phase HPLC (5 × 250 mm YMC C18 column, 5 μm), with a linear
gradient of MeCN (A) and 0.05% aqueous TFA (B) from 10% to 50%
A over 60 min at a flow rate of 1 mL/min, UV detection at 320 nm.
Each chromatographic peak was identified by comparing its retention
times for the L-FDAA derivatives of the L- and D-amino acid standards.
The standards gave the following retention times (in min): 40.20 for L-
FDAA, 38.61 for L-Ala, 43.92 for D-Ala, 41.15 for L-Me-Ala, 39.63 for
D-Me-Ala, 37.51 for L-Pro, 38.73 for D-Pro. The analysis gave retention
times (in min) of 35.7, 38.61, and 41.15, establishing the S
configuration for all the amino acid residues.
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ASSOCIATED CONTENT
* Supporting Information
■
(
(
15) Spartan’14; Wavefunction Inc.: Irvine, CA, 2013.
S
16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Computational data, HPLC analysis of the fungal metabolite
under different culture conditions, Marfey’s experimental data
of 2−5, as well as NMR spectra for compounds 1−5. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
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AUTHOR INFORMATION
■
*
Corresponding Authors
(T. Zhu) Tel: 0086-532-82031619. Fax: 0086-532-82033054.
E-mail: zhutj@ouc.edu.cn.
(D. Li) E-mail: dehaili@ouc.edu.cn.
*
̈
Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;
Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
Notes
The authors declare no competing financial interest.
Revision A.1; Gaussian, Inc.: Wallingford, CT, 2009.
̈
(17) Bruhn, T.; Hemberger, Y.; Schaumloffel, A.; Bringmann, G.
SpecDis, Version 1.53; University of Wuerzburg: Germany, 2011.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (Nos. 41176120 and 21372208),
the NSFC-Shandong Joint Fund (No. U1406402), the National
High Technology Research and Development Program of
China (No. 2013AA092901), and the Program for New
Century Excellent Talents in University (No. NCET-12-0499).
2
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dx.doi.org/10.1021/np500469b | J. Nat. Prod. 2014, 77, 2218−2223