New compounds from a hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4

Article abstract of DOI:10.1039/C6OB02374F

Three new quinazoline derivatives (1-3), one new oxepin-containing natural product (4) and four new cyclopenin derivatives (5-7 and 9) have been isolated from an EtOAc extract of the Taiwan Kueishantao hydrothermal vent crab-associated fungus Aspergillus versicolor XZ-4. Their planar structures were established by HRMS, 1D and 2D NMR spectroscopic data analyses. The absolute configurations for compounds 1 and 4 were determined by chiral phase HPLC analysis of their hydrolysis products. The absolute configurations of 2, 3 and 7 were defined mainly by comparison of the quantum chemical TDDFT calculated and the experimental ECD spectra, and the absolute configuration of 5 was deduced from comparison of the optical rotation values reported in the literature. The presence of two atropisomers of 5 was established by NOE analyses. The Ile & Val units in compounds 1-3 allowed the assignment of a new quinazoline skeleton and it's the first time the configuration of isoleucine in the quinazoline skeleton was defined. A series of 7-methoxy cyclopenin derivatives were reported for the first time in this study. The bioevaluation of compounds 5, 7, 8 and 9 revealed inhibitory activities against E. coli at MIC values around 32 μg mL^?1

Full text of DOI:10.1039/C6OB02374F

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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DOI: 10.1039/C6OB02374F
Organic & Biomolecular Chemistry
ARTICLE
New Compounds from a Hydrothermal Vent Crab-Associated
Fungus Aspergillus versicolor XZ-4
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
c
a
Chengqian Pan , Yutong Shi , Xuegang Chen , Chen-Tung Arthur Chen , Xinyi Tao and Bin Wu *
DOI: 10.1039/x0xx00000x
Three new quinazoline derivatives (1-3), one new oxepin-containing natural product (4) and four new cyclopenin
derivatives (5-7 and 9) have been isolated from an EtOAc extract of the Taiwan Kueishantao hydrothermal vent crab-
associated fungus Aspergillus versicolor XZ-4. Their planar structures were established by HRMS, 1D and 2D NMR
spectroscopic data analyses. The absolute configurations for compounds 1 and 4 were determined by chiralphase HPLC
analysis of their hydrolysis products. The absolute configurations of 2, 3 and 7 were defined mainly by comparison of
quantum chemical TDDFT calculated and experimental ECD spectra, and the absolute configuration of 5 was deduced from
comparison of the optical rotation values reported in the literature. The presence of two atropisomers of 5 was
established by NOE analyses. The Ile & Val units in compounds 1-3 allowed the assignment of a new quinazoline skeleton
and it's the first time the configuration of isoleucine in quinazoline skeleton was defined. The series of 7-methoxy
cyclopenin derivatives was reported first time in this study. The bioevaluation of compounds 5, 7, 8 and 9 revealed
inhibitory activities against E. coli at MIC values around 32 μg/mL.
www.rsc.org/
fungus Aspergillus versicolor. XZ-4 and their associated anti-
Introduction
bacterial properties.
The marine environment is considered as an untapped
potential of novel bioactive compounds with unique and
Results and discussion
1
,2
tremendous
pharmaceutical
properties.
Marine
From the large scale fermentation of the fungus Aspergillus
versicolor XZ-4, eight novel metabolites including three new
microorganisms thriving under extreme conditions such as
high temperatures, pressure, salinity, low dissolved oxygen
content and under eminent heavy metals concentrations are
reaching the focus of many scientists worldwide. Previous
researches have demonstrated the ability to isolate novel
bioactive secondary metabolites from diverse hydrothermal
vents derived fungi by applying the ‘Metal-stress’ strategy by
utilizing heavy metals as a stress point to activate dormant
5
,9
10-12
quinazoline derivatives (
1
-
3
), one new oxepin-containing
was found in
but it appears as though they are
compound ( ), although the planar structure of
4
4
the SciFinder,
13-16
diastereomers. Three new cyclopenin
derivatives (5-7), one
1
2,16
new viridicatin
compound 3,6-O-dimethylviridicatin (
). The isolation, structure elucidation, as well as the biological
derivatives (9), together with a known
1
2
8)
were isolated (Fig.
3
,4
1
gene clusters. Moreover, alkaloids are well known to be
activities of the isolated metabolites, are discussed below.
produced by the Aspergillus and Penicillium genera including
5
fumiquinazolines
A-G
from
Aspergillus
fumigatus,
6
serantrypinone from Penicillium thymicola , aurantiomides A–
7
C from Penicillium aurantiogriseum and fumiquinazoline Q
8
from Penicillium expansum. This study revealed the isolation
of novel compounds from a hydrothermal vent crab-associated
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Fig. 1 Structures of compounds 1 – 9 isolated from Aspergillus versicolor XZ-4
positio
n
1
2
3
4
δ
C
, type
δ
H
(J in Hz)
δ
C
, type
δ
H
(J in Hz)
δ
C
, type
δ
H
(J in Hz)
δ
C
, type
H
δ (J in Hz)
1
2
3
4
5
6
7
8
9
170.5, C
169.4, C
172.1, C
169.4, C
59.8, CH
149.7, C
4.72, d (2.3)
122.5, C
146.3, C
90.1, C
60.0, CH
155.0, C
4.66, d (2.0)
146.5, C
143.1, C
130.3, CH
125.9, CH
160.3, C
107.5, CH
122.1, C
162.7, C
143.0, C
141.7, C
161.5, C
7.65, d (8.9)
130.3, CH 7.65, d (9.0)
126.1, CH 7.44, dd (9.0, 2.9)
160.7, C
130.5, CH 7.70, d (8.9)
126.0, CH 7.46, dd (8.9, 2.9)
160.7, C
146.1, CH 6.26, d (6.0)
116.9, CH 5.60, dd (5.9, 1.8)
158.7, C
7.45, dd (8.9, 2.9)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
7.63, d (2.9)
107.5, CH 7.62, d (2.9)
121.6, C
107.2, CH 7.63, d (2.9)
122.2, C
95.7, CH
110.6, C
163.5, C
5.80, d (1.5)
162.6, C
162.9, C
62.3, CH
32.2, CH
5.18, d (8.2)
2.33, m
63.1, CH
32.8, CH
20.0, CH
19.6, CH
138.1, C
19.4, CH
28.2, CH
11.9, CH
56.5, CH
5.16, d (9.3)
2.16, m
62.1, CH
34.5, CH
20.5, CH
19.8, CH
37.7, CH
11.1, CH
25.5, CH
12.8, CH
56.2, CH
50.9, CH
5.10, d (9.0)
2.53, m
62.5, CH
33.1, CH
20.4, CH
18.8, CH
38.2, CH
15.8, CH
24.5, CH
12.7, CH
55.7, CH
5.02, d (7.1)
2.28, m
20.5, CH
19.5, CH
37.9, CH
15.8, CH
24.7, CH
12.9, CH
56.4, CH
3
0.99, d (6.8)
1.13, d (6.7)
2.74, m
3
3
0.92, d (6.8)
1.10, d (6.7)
3
3
1.21, d (6.7)
0.90, d (6.9)
2.90, m
3
3
1.05, d (6.2)
1.06, d (6.5)
2.54, m
3
3
2
3
3
1.21, d (7.2)
1.49, m 1.36, m
0.96, t (7.4)
3.94, s
3
2
3
3
2.30, s
3
2
3
3
3
1.11, d (7.1)
1.28, m 1.22, m
0.97, t (7.4)
3.94, s
3
2
3
3
1.15, d (7.2)
1.32, m
2.42, m 2.31, m
1.15, t (7.5)
3.93, s
0.93, t (7.4)
3.72, s
3.24, s
1
13
3 4
Table 1 H NMR data (500 MHz, δ in ppm, J in Hz) and C NMR data (125 MHz, δ in ppm) for compounds 1 - 4 in CD OD-d
2
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Compound
1
was obtained as a white powder. The existed in the cyclo-dipeptide part of structure
1. The carboxyl
molecular formula was determined to be C19
analysis of the HR-TOF-MS ion peak at m/z 344.1949 [M + H]
calcd. 344.1974). The NMR data (Table 1) of compound
were similar to those of quinazoline-containing metabolites, m), H-15/H-16 (δ
H
25
N
3
O
3
by signal at δ 162.7 was assigned to C-12, due to the strong cross
C
+
1
peak of H-10/C-12 in the HMBC spectrum of
1
(Fig. 2). The H-
5.18, d, 8.2 Hz)/H-15 (δ 2.33,
0.99, d, 6.8 Hz), H-15/H-17 (δ 1.13, d, 6.7
NMR spectrum in Hz) sequenced an isopropyl unit in , indicating the presence
carnequinazoline A showed the presence of 18 signals of the Val unit in the cyclo-dipeptide substructure of . It was
including a benzene ring basic skeleton, five signals for Val confirmed by the analysis of the HMBC correlations from the
1
(
1
H COSY cross peaks of H-14 (δ
H
H
H
H
9
13
carnequinazoline A.
The
C
1
1
9
moiety and six signals for Leu moiety. When compared with methine proton of H-14 to C-1 (δ
C
170.5) and H-15 to C-1,
9
the NMR data of carnequinazoline A ,
chemical shifts of the two main substructural domains of range correlations from H-14 to C-4 (δ
quinazolin-4(3H)-one and Val units. The differences in the 1D H-3 to C-4 proved the presence of a Val – Ile 2,5-
NMR data between and the known carnequinazoline A lie in diketopiperazine in . From the above analysis, the planar
aromatic downfield and CH2 signals. A 1,3,4-trisubstituted structure of was elucidated as drawn in Fig. 1. The
benzene instead of 1,2-disubstituted benzene in configurations at the chiral centers of were determined by
carnequinazoline A was deduced from the proton signals at δH chiralphase HPLC analyses of the acid hydrolyzed residues of
.65, d (8.9 Hz), 7.45, dd (8.9, 2.9 Hz) and 7.63, d (2.9 Hz). The (Fig. S73). The two chiral amino acid units in after hydrolysis
methoxy group was placed at C-9 in the benzene ring, which were identified to be D-Val and D-allo-Ile. (Fig. 1). Thus,
was inferred from the HMBC cross peak between H-22 (δH compound was elucidated as a novel quinazoline-containing
.94, s) and C-9. The presence of a sec-butyl group which is cyclic dipeptide, and given the trivial name as versicomide A.
1 exhibited similar showing a linkage between C-14 (δ 62.3) and C-1. The long
C
C
149.7) and C-12, from
1
1
1
1
1
7
1
1
3
1
1
deduced from the H- H COSY correlations between H-3 (δ
This was the first assignment of a new quinazoline skeleton
.72, d, 2.3 Hz) and H-18 (δ 2.74, m), H-18 and H-19 (δ 1.21, with Val - Ile cyclic dipeptide moiety and it's the first time the
H
4
H
H
d, 7.2 Hz), H-18 and H-20 (δ 1.49, 1.36, m), H-20 and H-21 (δH configuration of isoleucine in its skeleton was defined.
H
0.96, t, 7.4 Hz) in one of the amino acids hinted that an Ile unit
1
Fig. 2 Key HMBC & H– H COSY correlations of compound
1
1-
7
,
9
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Compound
molecular formula was determined to be C19
by analysis of the HR-TOF-MS ion peak at m/z 342.1796 [M + versicomide B was determined as 14S (Fig. 3).
2
was obtained as a light yellow powder. The σ of 0.2 eV. By comparing the experimental ECD data to the
23 3 3
H N O
by HRMS calculated result, the absolute configuration of the
+
H] (calcd. 342.1818). The NMR data (Table 1) of compound
2
Compound
3
was isolated as a yellow oil with the molecular
on the basis of its HRMS data.
2.16, m), H-15/H-16 The H and C NMR spectra of (Table 1) showed similar
1.10, d, 6.7 Hz) proved chemical shifts and the same multiplicities for 14 carbon atoms
the presence of an isopropyl group in carnequinazoline of a quinazoline and a Val moieties as in , indicating that
possesses the same quinazoline structure.
9
1
1
were similar to carnequinazoline A. The H- H COSY cross formula assigned as C20
27 3 4
H N O
1
13
peaks of H-14 (δ
H
5.16, d, 9.3 Hz)/H-15 (δ
H
3
(δ
H
0.92, d, 6.8 Hz) and H-15/H-17 (δ
H
1
analogue. A transformed Val - Ile cyclic dipeptide replaced the compound
1
1
0
Val - Leu cyclic dipeptide unit in the carnequinazoline A,
which was deduced by the HMBC correlations from H-19 (δ
.30, s) to C-3 (δ 122.5), C-18 (δ 138.1) and C-20 (δ
from H-20 (δ 2.31, 2.42, m) to C-3 and C-18 and from H-21 (δ
.15, t, 7.5 Hz) to C-18, indicating the presence of a 1- data. Thus, the planar structure of
methylpropylidene group in the cyclic dipeptide unit. The only methoxylated compound of as shown in Fig. 1. The absolute
methoxy was positioned at C-9 from the analysis of the HMBC configurations of were determined by chemical calculation of
cross peak of H-22 (δ 3.93, s)/C-9. The planar structure of the ECD spectrum. Since compounds were isolated from
was elucidated as a new quinazoline, named as versicomide B. same fungus, implying that four compounds may undergo
The E-configuration of the double bond C was similar bio-synthetic pathway, it was reasonable to identify the
determined by the correlation of NH (δ 7.53) with H-19 (δ absolute configuration at 18 was S, the same as in and
, Fig. S14). The absolute The remaining two chiral carbons create four possibilities,
However, the remaining 6 carbon signals were assigned to the
H
methoxylated Ile amino acid unit, which was confirmed by
2
C
C
C
28.2), HMBC correlation from H-23 (δ
methoxy group was placed to C-9 from the analysis of 2D NMR
was elucidated as a
H
3.24, s) to C-3. The remaining
H
H
1
3
1
3
H
2
1-4
3
=C18 in 2
H
H
1
4.
7
,9
2
.37) in NOESY experiments (CDCl
3
configurations of versicomide B were determined by chemical 3S,14S, 3R,14R, 3S,14R and 3R,14S. The minimum geometries
calculation of the ECD spectrum. The minimum geometries of of four conformers were optimized using DFT at the B3LYP/6-
two conformers were optimized using DFT at the B3LYP/6- 31+G(d, p) level in methanol by the GAUSSIAN 09. By
31+G(d, p) level in methanol by the GAUSSIAN 09. Then the comparing the experimental ECD data to the calculated result,
ECD spectra calculation were simulated using the time- the absolute configurations of 3 were determined as 3R,14S
dependent density functional theory (TDDFT) employing the (Fig. 4). The new compound was given the name versicomide
B3LYP functional at the B3LYP/6-31+G(d, p) level in methanol. C.
The calculated ECD curves were drawn by using SpecDis with a
positio
n
5a
, type
5b
6
7
δ
C
δ
H
(J in Hz)
δ
C
, type
δ
H
(J in Hz)
δ
C
, type
δ
H
(J in Hz)
δ
C
, type
H
δ (J in Hz)
1
2
3
4
5
6
7
8
9
171.2, C
58.0, CH
172.2, C
70.1, CH
172.4, C
135.7, C
168.6, C
71.8, C
4.43, t (7.6)
7.32, d (2.7)
4.30, dd (10.4, 7.1)
170.6, C
168.2, C
168.9, C
168.3, C
114.8, CH
158.3, C
115.4, CH 7.45, d (2.7)
158.3, C
115.0, CH 7.40, d (2.9)
158.3, C
114.9, CH 6.51, d (2.8)
158.3, C
121.1, CH
123.8, CH
7.11, d (8.6)
121.2, CH 7.20, m
123.5, CH 7.11, d (8.6)
121.1, CH 7.14, dd (8.8, 2.9)
123.8, CH 7.08, d (8.8)
121.4, CH 7.15, dd (8.8, 2.8)
124.2, CH 7.12, d (8.8)
7.02, d (8.8)
1
0
33.1, CH
2
3.25, dd (14.5, 7.2),
35.3, CH
2
2.70, dd (13.4, 131.7, CH 6.91, s
10.7),
66.1, CH
4.18, s
3
.39, dd (14.5, 7.9)
1
1
1
1
1
1
1
2
1
131.4, C
129.4, C
138.3, C
130.4, C
129.0, C
137.4, C
131.3, C
129.9, C
129.2, C
132.4, C
2
127.9, C
3
133.8, C
4/18
5/17
6
130.2, CH
129.8, CH
128.0, CH
7.24, m
7.24, m
7.20, m
3.08, s
3.84, s
130.2, CH 7.05, d (7.4)
130.0, CH 7.29, t (7.3)
128.4, CH 7.23, m
130.3, CH 7.37, m
130.3, CH 7.44, m
131.0, CH 7.40, m
127.5, CH 6.72, d (8.6)
129.3, CH 7.26, t (7.6)
130.3, CH 7.34, t (7.4)
9
29.7, CH
56.3, CH
3
3
40.0, CH
3
3
2.93, s
3.89, s
36.2, CH
3
3
3.13, s
3.86, s
31.7, CH
56.2, CH
3
3
3.22, s
3.69, s
0
56.3, CH
56.3, CH
1 13
Table 2 H NMR data (500 MHz, δ in ppm, J in Hz) and C NMR data (125 MHz, δ in ppm) for compound
3 4
5 - 7 in CD OD-d
4
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3
2
1
0
0
0
0
30
2
1
0
0
0
ꢀ10
ꢀ20
ꢀ30
ꢀ40
ꢀ10
ꢀ20
ꢀ30
2
00
250
300
350
400
450
500
200
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
1
00
2
3
5
0
0
ꢀ50
ꢀ100
2
00
250
300
350
400
450
Wavelength (nm)
7
Fig. 3 B3LYP/6-31+G(d, p) calculated ECD spectra of compound
and the experimental ECD spectrum (black) for compound 3; 3R,10S (black) and the experimental ECD spectrum (red) for compound 7.
2, 14S (black) and the experimental ECD spectrum (red) (σ = 0.25 eV) for compound 2; 3R,14S (red)
Compound
4
was isolated as a light yellow oil with the were determined by chiralphase HPLC analyses of the acid
(Fig. S73). Compound was given the
molecular formula assigned as C H N O on the basis of its hydrolyzed residues of
1
4
4
9 25 3 4
1
13
HRMS data. The H and C NMR spectra of
similar chemical shifts and the same multiplicities for 11
carbon atoms of a Val - Ile cyclic dipeptide moiety as in with molecular formula assigned as C H N O on the basis of its
a minor difference, indicating that compound possesses the HRMS data. All the proton and carbon signals of 5a
4
(Table 1) showed name versicomide D.
Compound 5a/5b was isolated as a yellow oil with the
1
1
8 18 2 3
4
/5b
same dipeptide structural unit. However, the remaining 8 recorded in CD OD were observed in pairs at room
3
1
carbon signals could not construct a complete quinazoline temperature (25°C), with a ratio of 5:4 in H NMR data. At
skeleton. Detailed analysis of NMR data allowed the complete 65°C, the two sets of signals coalesced into single resonances
assignment of the Val-Ile cyclic dipeptide. Thus, it was implied (DMSO-d , Fig. S34), indicating that the paired signals resulted
6
that the difference lies in the quinazoline unit, which was from two different stable conformations. Compared the NMR
deduced that the benzene ring was transformed into an oxepin data (Table 2) with those of known compounds 14-hydroxy-
1
from the analysis of the three aromatic protons (δ 6.26, d, 6.0 cyclopeptine and 4'-methoxycyclopeptin , it is deduced that
4
16
H
Hz, δH 5.60, dd, 5.9, 1.8 Hz and δH 5.80, d, 1.5 Hz). The compound 5a/5b shared same skeleton as 14-hydroxy-
methoxy group was placed at C-9 (δC 158.7), which was cyclopeptine, existing in conformational isomers. The
1
13
inferred from the correlation from H-22 (δH 3.72, s) to C-9. assignments of H NMR and C NMR are shown in Table 2.
From the above analysis, the planar structure of
4
was Final elucidation of structures was confirmed by the HMBC and
1
1
H- H COSY results (Fig. 2). The methoxy was placed at C-7
elucidated as drawn in Fig. 1. The absolute configurations of
4
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1 13
absent. The assignment of H NMR and C NMR shown in 6.92, d, 7.7 Hz) indicated a 1,2,3-trisubstituted benzene unit in
1
1
Table 3. The H- H COSY revealed correlations between H-6 (δ
.61, d, 7.9 Hz) and H-7 (δ 6.96, t, 7.9 Hz), H-7 and H-8 (δ
H
H
the molecule of
146.0)
9. The hydroxy group was assigned at C-9 (δ
C
6
H
Fig. 5 Proposed biosynthetic pathway of 5-8.
from the observation of the HMBC cross peaks of H-8/C-9 and known compound
H-8/C-10. Thus, was elucidated as a new compound named at MIC values around 32 μg/mL. The viridicatin derivatives
as 9-hydroxy-3-methoxyviridicatin. and showed good antibacterial activities, suggesting that
Compounds are a series of precursors in the biosynthetic viridicatin skeleton is worth exploring further. Interestingly,
pathway to 9-hydroxy-3-methoxyviridicatin ( ). We proposed among three cyclopeptine derivatives, compound with a
the biosynthetic pathway of compounds in Fig. 5. The basic double bond between C-3 and C-10 showed no bioactivity,
cyclopetin skeleton is derived from an anthranilic acid and hinting that the chiral center at C-3 cyclopeptine skeleton is
phenylalanine. was derived from by a dehydrogenation at important for its antibacterial activities.
C-3 and C-10. was synthesized through epoxidation of at C-
and C-10. The formation of 7-methoxyviridicatin may occur
8 revealed inhibitory activities against E. coli
9
8
9
5-8
9
6
5-8
6
5
7
6
1
13
Table 3 H NMR data (500 MHz, δ in ppm, J in Hz) and C NMR data (125 MHz, δ
3
in ppm) for compound
8
3 4
and 9 in CD OD-d
via cyclization and elimination from 7. Similar to the previously
positio
8
9
proposed mechanism for the formation of 4’-
1
9,20
n
C
δ , type
δ
H
(J in Hz)
δ
C
, type
H
δ (J in Hz)
methoxyviridicatin
of the formation of 7-methoxyviridicatin, involving the
opening of the epoxide in , a step which is initiated by
, we proposed the biosynthetic pathway
1
2
3
4
5
6
7
8
9
7
161.1, C
147.0, C
141.0, C
123.1, C
161.7, C
146.8, C
141.8, C
123.0, C
donation of the amide nitrogen lone pair of electrons. The ring
opening results in the conversion of the seven-membered
cyclodipeptide moiety into the six-membered lactam and
elimination of methyl isocyanate.
methoxyviridicatin by methoxylation at C-3. The migration of
methoxy group in afforded compound (Fig. 5).
8 would be derived from 7-
109.3, CH 6.61, d (2.7)
157.0, C
118.0, CH
124.1, CH
114.3, CH
146.0, C
6.61, d (7.9)
6.96, t (7.9)
6.92, d (7.7)
8
9
The antibacterial activities of all isolated compounds were
evaluated with three representative pathogenic bacteria,
Escherichia coli, Staphylococcus aureus and Bacillus subtilis.
Tetracycline was used as a positive control with MIC values of
119.3, CH 7.13, dd (8.7, 2.6)
118.0, CH 7.34, d (9.0)
1
1
0
1
131.4, C
135.0, C
126.4, C
135.2, C
2, 2 and 1 μg/mL respectively. New compounds 5, 7, 9 and
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Organic & Biomolecular Chemistry
Page 8 of 9
View Article Online
DOI: 10.1039/C6OB02374F
ARTICLE
Journal Name
1
1
1
1
1
2/16
3/15
4
130.6, CH 7.38, d (8.1)
130.6, CH
129.5, CH
129.4, CH
7.34, d (8.3)
7.52, t (7.4)
7.47, t (7.4)
3.65, s
and give an organic extract (7.81 g). The extract was subjected
to liquid chromatography over a silica gel column using a
gradient elution of petroleum ether (PE)− EtOAc − MeOH
129.7, CH 7.57, t (7.9)
129.6, CH 7.51, t (7.4)
(PE:EtOAc = 1:0, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1 & MeOH) to give
7
60.8, CH
56.0, CH
3
3
3.67, s
3.64, s
60.8, CH
3
several fractions. Nine main fractions were obtained which
was confirmed by TLC. Every fraction was dissolved in
methanol and centrifuged at 12,000 rpm for 10 min. The
supernatant was collected and subjected to preparative HPLC
8
Experimental
(
10.0 mL/min, UV = 210nm), using MeOH:H
Fraction was separated by preparative HPLC
MeOH:H O=80:20, 10mL/min) to give compound (2.6 mg, t
20.0 min), compound (5.9
15.0 min). Fraction
O=55:45,
42.0 min),
20.0 min). Fraction 6 was separated by
preparative HPLC (MeOH:H O=70:30, 10mL/min) to give
compound (2.7 mg, t 14.0 min), compound (6.7 mg, t
3.0 min) , compound (2.1 mg, t 18.0 min).
2
O as eluent.
4
General Experimental Procedures Optical rotations were
measured in MeOH on a JASCO P-2000 polarimeter. The IR
spectra were run on a NicoletAvatar-360FT-IR spectrometer.
(
2
1
R
2
3.0 min), compound
2
(8.0 mg, t
R
3
1
13
mg, t
R
28.0 min), compound
4
(10.7 mg, t
R
H NMR (500 MHz) and C NMR (125 MHz) spectra were
o
5
1
was separated by preparative HPLC (MeOH:H
0mL/min) to give compound (11.4 mg, t
(4.9 mg, t
2
measured at 25 C on a Bruker AVANCE DMX 500 NMR
spectrometer. UV spectra were also recorded in MeOH on a
Shimadzu UV2550. CD spectra were measured on a JASCO J-
5
R
compound
7
R
715 circular dichroism spectrometer. ESIMS were recorded on
2
6
R
8
R
an Agilent 6460 Triple Quad LC/MS. TLC was performed using
Merck precoated plates (Silica gel 60 F254, Merck, Darmstadt,
Germany) of 0.25 mm thickness. Prep. HPLC was performed on
an Agilent-1100 system equipped with a Venusil MP-C18
column (10 mm × 250 mm, Agela Technologies, Tianjin, China).
Chiral-Phase HPLC Analysis of the Acid Hydrolysates of 1 and
2
9
R
Computation Section The geometry was optimized starting
from initial conformations, with DFT calculations at the
B3LYP/6-31+G(d, p) level using the Gaussian09 program. Time-
dependent DFT calculations were performed on the lowest-
energy conformations for each configuration using 30 excited
2
.
1,22
4
To determine the absolute configurations of the Ile and
Val in and , chiral-phase HPLC analyses of the acid
hydrolysates were conducted. Compounds (0.50 mg) and
0.50 mg) were each dissolved in 6 N HCl (1 mL) and heated at
3
states and under the CH OH solution. ECD spectra were
1
4
generated using the program SpecDis by applying a Gaussian
band shape with 0.25 eV width, from dipole-length rotational
1
4
(
2
3,24
strengths.
Versicomide A (1): white, powder, [a]
UV/vis (CH OH) λmax (logε) 226 (4.22), 277 (3.76), 322 (3.49)
nm , IR vmax 1684, 1617, 1596, 1491, 1362 cm , CD (c 5.8 × 10
1
20°C for 36 h. After cooling to room temperature, the solvent
2
0
D 3
+50.6 (c 0.1, CH OH) ,
was removed under reduced pressure. The dried hydrolysate
was dissolved in 200 μL of 3 mM CuSO −H O solution. 20 μL of
this sample were then analysed by HPLC with a chiral column
MCIGEL CRS10W, 4.6 × 50 mm, Mitsubishi Chemical
Corporation) using MeOH:3mM CuSO −H O = 5:95 solution as
3
4
2
−1
-
4
mol/L, MeOH) λmax (Δε): 204 (+0.2), 230 (-48.4), 267 (+10.4),
(
1
32 (-4.6), H and C NMR, Table 1., HRMS m/z 344.1949 [M +
13
3
4
2
+
+
H] (calcd. for C19
Versicomide B (2): yellow, powder, [a]
UV/vis (CH OH) λmax (logε) 214 (4.07), 263 (3.66), 300 (3.31)
nm , IR vmax 1680, 1618, 1592, 1490, 1361, 1268, 1223 cm
26 3 3
H N O , 344.1974).
the mobile phase at a flow rate of 0.6 mL/min with UV
detection at 254 nm. L-Val, D-Val, L-Ile, L-allo-Ile, D-Ile and D-
allo-Ile were detected as references. The retention times of
the L-Val, D-Val, L-Ile, L-allo-Ile, D-Ile, D-allo-Ile were 7.4, 5.0,
2
0
D
3
-23.4 (c 0.1, CH OH) ,
3
−1
,
-4
CD (c 5.9 × 10 mol/L, MeOH) λmax (Δε): 210 (+23.2), 230 (-
1
1.2), 252 (+20.9), 298 (+2.7), 338 (-8.0), H and C NMR,
1
8.5, 14.0, 7.5 and 7.6 min, respectively. Ile and Val in
assigned as D-allo-Ile (7.6 min), D-Val (5.0 min), Ile and Val
residues in were assigned as D-allo-Ile (7.6 min), D-Val (5.0
1 were
13
2
+
+
Table 1., HRMS m/z 342.1796 [M + H] (calcd. for C19
42.1818).
Versicomide C (3): light yellow, oil, [a]
UV/vis (CH OH) λmax (logε) 225 (4.10), 279 (3.66), 322 (3.29)
nm , IR vmax 1685, 1617, 1597, 1491, 1359 cm , CD (c 5.4 × 10
24 3 3
H N O ,
4
3
min) (Supporting Information, Fig. S73).
2
0
3
+73.2 (c 0.1, CH OH) ,
Isolation and Cultivation of the Fungal Strain Aspergillus
versicolor XZ-4 was isolated from a Hydrothermal crab
Xenograpsus testudinatus collected in May 2014, from
Kueishantao, Taiwan. The fungus was cultured under static
conditions at 28 °C in 500ml Erlenmeyer flasks containing 200
mL of liquid culture medium, composed of glucose (10.0 g/L),
potato dextrose agar (PDA) (20.0 g/L), and seawater. After 3
weeks fermentation, the medium was extracted with EtOAc.
Identification of Aspergillus versicolor XZ-4 This fungus was
characterized as Aspergillus versicolor on the basis of the ITS
D
3
−1
-
4
mol/L, MeOH) λmax (Δε): 206 (-16.1), 220 (+20.9), 235 (-34.4),
1
13
82 (+2.2), 325 (-4.6) H and C NMR, Table 1., HRMS m/z
2
3
+
+
28 3 4
74.2056 [M + H] (calcd. for C20H N O , 374.2080).
2
0
Versicomide D (4): light yellow, oil, [a]
UV/vis (CH OH) λmax (logε) 205 (4.15), 338(3.65) nm , IR vmax
660, 1651, 1581, 1533, 1191 cm , CD (c 5.6 × 10 mol/L,
D 3
+59.9 (c 0.1, CH OH) ,
3
−1
-4
1
1
MeOH) λmax (Δε): 217 (+30.4), 249 (-29.6), 356 (-4.5), H and
1
3
+
C NMR, Table 1., HRMS m/z 360.1900 [M + H] (calcd. for
18S fragment. This fungus was deposited in China General
+
C
19
H
26
N
3
O
4
, 360.1923).
Microbiological Culture Collection Center (CGMCC), Institute of
Microbiology, Chinese Academy of Sciences (Beijing, China),
numbered as CGMCC 3.15654.
2
0
7
-methoxycyclopeptin (5): light yellow, oil, [a]
D
-87.8 (c 0.1,
CH
3
OH), UV/vis (CH OH) λmax (logε) 211 (4.23), 311 (3.39)nm ,
3
−1
IR vmax 1680, 1630, 1610, 1497, 1410, 1383, 1280, 1233 cm ,
This journal is © The Royal Society of Chemistry 20xx
Extraction and Isolation 60 L of the fungal culture broth was
filtered. The broth (60 L) was extracted with EtOAc (2 × 60 L)
8
| Organic & Biomolecular Chemistry, 2016, 00, 1-3
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Orga Pn l iec a s&e Bd oi o nmo to al ed cj uu sl ta mr Ca rhg ei nms istry
View Article Online
DOI: 10.1039/C6OB02374F
Journal Name
ARTICLE
1
13
+
H and C NMR, Table 2., HRMS m/z 311.1381 [M + H] (calcd. 13 P. K. Martin, H. Rapoport, H. W. Smith and J. L. Wong, J. Org.
+
Chem., 1969, 34, 1359.
19 2 3
for C18H N O , 311.1396).
1
1
4 X. Zhou, P. Fang, J. Tang, Z. Wu, X. Li, S. Li, Y. Wang, G. Liu, Z.
He, D. Gou, X. Yao and L. Wang, Nat. prod. Res., 2016, 30, 52.
5 R. Uchida, R. Imasato, H. Tomoda and S. Omura, J. Antibiot.
2006, 59, 652.
7
-methoxydehydrocyclopeptin (6): light yellow, oil, UV/vis
(CH OH) λmax (logε) 213 (3.95), 245 (3.70), 287 (3.57) nm , IR
3
−1 1
v
max 1670, 1635, 1615, 1500, 1413, 1349, 1281, 1240 cm , H
1
3
+
and C NMR, Table 2., HRMS m/z 309.1226 [M + H] (calcd. for 16 M. Kawano, H. Koshino, J. UzAwA, S. Fujioka, T. KAwANo and
+
Y. Kimura, Biosci. Biotech. Bioch. 2000, 64, 2559.
17 J. Framm, L. Nover, A. E. Azzouny, H. Richter, K. Winter, S.
Werner and M. Luckner, Eur. J. Biochem., 1973, 37, 78.
C
18
H
17
N
2
O
3
, 309.1239).
-methoxycyclopenin (7): colourless, oil, [a]
CH OH), UV/vis (CH OH) λmax (logε) 211 (4.25), 307 (3.23)nm ,
IR vmax 1720, 1697, 1642, 1612, 1499, 1440, 1408, 1370, 1280,
2
0
7
D
-78.2 (c 0.1,
3
3
1
8 Y. S. Mohammed and M. Luckner, Tetrahedron Lett., 1963, 4,
1953.
−1
-4
1
237 cm , CD (c 6.2 × 10 mol/L, MeOH) λmax (Δε): 205 19 A. Bräuer, P. Beck, L. Hintermann and M. Groll, Angew.
Chem. Int. Edit., 2016, 55, 422.
20 N. Ishikawa, H. Tanaka, F. Koyama, H. Noguchi, C. C. Wang, K.
Hotta and K. Watanabe, Angew. Chem. Int. Edit., 2014, 53
2880.
-hydroxy-3-methoxyviridicatin (9): light yellow, powder, 21 X. Zhou, H. Huang, J. Li, Y. Song, R. Jiang, J. Liu, S. Zhang, Y.
(
(
(
+23.0), 214 (+64.7), 225 (-36.6), 246 (-23.2), 263 (-47.0), 307
1
13
+
+8.1) H and C NMR, Table 2., HRMS m/z 325.1176 [M + H]
,
+
17 2 4
calcd. for C18H N O , 325.1188).
1
9
UV/vis (CH
nm , IR vmax 1642, 1632, 1620, 1606, 1563, 1552, 1389, 1280,
3
OH) λmax (logε) 209 (3.75), 261 (3.67), 299 (3.23)
Hua and J. Ju, Tetrahedron, 2014, 70, 7795.
2 Z. Chen, Y. Song, Y. Chen, H. Huang, W. Zhang and J. Ju, J.
nat. prod., 2012, 75, 1215.
2
2
−1
1
247 cm , H and C NMR, Table 3., HRMS m/z 268.0958 [M +
13
1
3 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. R. Scuseria, M.
A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B.
Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.
P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L.
Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J.
Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta, F.
Ogliaro, M. J. Bearpark, J. Heyd, E. N. Brothers, K. N. Kudin,
V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari,
A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi,
N. Rega, N. J. Millam, M. Klene, J. E. Knox, J. B. Cross, V.
Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P.
Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö.
Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox,
Gaussian 09, Gaussian, Inc., Wallingford, CT, USA, 2009.
4 T. Bruhn, A. Schaumlöffel and Y. Hemberger, SpecDis, version
+
H] (calcd. for C16
+
3
H14NO , 268.0974).
Biological Assays Antibacterial activities were evaluated by the
2
5
conventional broth dilution assay. Three bacterial strains, E.
coli [CMCC (B) 44102], S. aureus [CMCC (B) 26003], B.subtilis
[CMCC (B) 63501], were used, and tetracycline was used as a
positive control.
Acknowledgements
This work was supported by NSFC (No. 81273386 and
81573306). We thank Prof. Hu-Jun Xie from School of Food
Science and Biotechnology, Zhejiang Gongshang University, for
his kind help on the calculation of the ECD spectra and
determination of the configurations.
2
2
1.63. University of Wuerzburg, 2015, Germany.
5 G. Appendino, S. Gibbons, A. Giana, A. Pagani, G. Grassi, M.
Notes and references
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This journal is © The Royal Society of Chemistry 20xx
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