Cyclopeptides and polyketides from coral-associated fungus, Aspergillus versicolor LCJ-5-4

Article abstract of DOI:10.1016/j.tet.2011.07.003

Three new cyclopentapeptides, versicoloritides A-C (1-3), a new orcinol tetramer, tetraorcinol A (4), and two new lactones, versicolactones A and B (5 and 6) together with three known metabolites, diorcinol, glyantrypine, and cordyol C were isolated from the fermentation broth of the coral-associated fungus Aspergillus versicolor LCJ-5-4. Their structures were elucidated by spectroscopic and chemical methods. The new compounds 1-4 were evaluated for their radical-scavenging activity and antimicrobial activity against Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Candida albicans and cytotoxicity against P388 and Hela cell lines. Compound 4 showed weak radical-scavenging activity against the DPPH radical with an IC₅₀ value of 67 μM.

Full text of DOI:10.1016/j.tet.2011.07.003

Tetrahedron 67 (2011) 7085e7089
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Cyclopeptides and polyketides from coral-associated fungus, Aspergillus versicolor
LCJ-5-4
y
y
*
Yibin Zhuang , Xiancun Teng , Yi Wang, Peipei Liu, Hui Wang, Jing Li, Guoqiang Li, Weiming Zhu
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
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new cyclopentapeptides, versicoloritides AeC (1e3), a new orcinol tetramer, tetraorcinol A (4), and
two new lactones, versicolactones A and B (5 and 6) together with three known metabolites, diorcinol,
glyantrypine, and cordyol C were isolated from the fermentation broth of the coral-associated fungus
Aspergillus versicolor LCJ-5-4. Their structures were elucidated by spectroscopic and chemical methods.
The new compounds 1e4 were evaluated for their radical-scavenging activity and antimicrobial activity
against Staphylococcus aureus, Escherichia coli, Enterobacter aerogenes, Bacillus subtilis, Pseudomonas
aeruginosa, and Candida albicans and cytotoxicity against P388 and Hela cell lines. Compound 4 showed
Received 21 April 2011
Received in revised form 22 June 2011
Accepted 1 July 2011
Available online 6 July 2011
Keywords:
Coral-associated fungus
Cyclopentapeptides
Polyketides
weak radical-scavenging activity against the DPPH radical with an IC50 value of 67 mM.
Ó 2011 Elsevier Ltd. All rights reserved.
Aspergillus versicolor
Cladiella sp.
1. Introduction
reports the isolation, structure elucidation, and bioactivity of these
new compounds.
Coral is an important source of novel, biologically active com-
pounds.1 For example, pseudopterosin A, a diterpene glycoside de-
rived from the marine octocoral Pseudopterogorgia elisabethae, is in
Phase II trials as a wound healing agent.2 Moreover, marine surface-
associated microorganisms have proven to be a rich source for novel
bioactive because of the necessity to evolve allelochemicals capable
of protecting the producer from the fierce competition.3 However,
researches on secondary metabolites of coral-associated microor-
ganisms are rarely reported. During our ongoing pursuit for novel
and bioactive natural products from marine surface-associated
microorganisms, a fungal strain LCJ-5-4 authenticated as Aspergil-
lus versicolor, widely found in human and animal foodstuffs, was
firstly isolated from the soft coral Cladiella sp. collected from the
South China Sea. Previous study had been identified three new
quinazolinone alkaloids from A. versicolor LCJ-5-4.4 Continuous
chemical study on this fungus resulted in the isolation and identi-
fication of three new cyclopentapeptides, versicoloritides AeC
(1e3), a new orcinol tetramer, tetraorcinol A (4), and two new lac-
tones, versicolactones A and B (5 and 6), together with three known
metabolites, diorcinol,5 cordyol C,6 and glyantrypine.7 This paper
2. Results and discussion
Versicoloritide A (1) was obtained as a white amorphous pow-
der. Its molecular formula was determined as C31H37N5O5 accord-
ing to the HRESIMS at m/z 560.2869 [MþH]þ, requiring 16 degrees
of unsaturation. Analysis of the NMR data (Table 1) revealed the
presence of five carbonyl signals (dC 170.6, 169.5, 169.5, 170.6, and
170.2), five normal a-amino acid methine carbon resonances (dC
49.7, 60.8, 52.4, 60.9, and 51.7), and three amide protons (dH 7.50,
6.38, and 8.31), indicating its peptide nature. 1He1H COSY experi-
ment (Fig. 1) constructed the five amino acid residues as an alanine,
two phenylalanines, and two prolines. These residues accounted for
* Corresponding author. Tel./fax: þ86 532 82031268; e-mail address:
weimingzhu@ouc.edu.cn (W. Zhu).
y
These authors made equal contribution to this paper.
15 of the 16 degrees of unsaturation, indicating that 1 is
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.07.003

7086
Y. Zhuang et al. / Tetrahedron 67 (2011) 7085e7089
Table 1
1H and 13C NMR data for 1e3 (600, 150 MHz, DMSO-d6, TMS,
d
ppm)
Position
1
2
3
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1
2
170.6, qC
49.7, CH
170.7, qC
42.5, CH2
168.8, qC
56.4, CH
4.22 (dq, 10.1, 7.3)
0.85 (d, 7.3)
3.91 (dd, 13.7, 10.3); 3.28
(d, 12.4)
4.22 (m)
3
19.4, CH3
3.17 (m), 3.24 (m)
4.94 (t, 6.1)
7.51 (d, 10.1)
62.0, CH2
3-OH
NeH
7.50 (d, 10.1)
7.85 (d, 9.6)
Proa
5
6
7
8
4
169.5, qC
60.8, CH
31.1, CH2
22.5, CH2
46.5, CH2
169.4, qC
60.1, CH
30.9, CH2
22.1, CH2
46.6, CH2
170.2, qC
60.8, CH
31.2, CH2
22.4, CH2
46.5, CH2
3.25 (t, 6.4)
3.01 (m)
3.21 (m)
1.67 (m), 1.65 (m)
1.54 (m), 1.49 (m)
3.30 (m)
1.70 (m), 1.37 (m)
1.53 (m), 1.48 (m)
3.23 (m)
1.72 (m), 1.62 (m)
1.64 (m), 1.48 (m)
3.42 (m)
Phea
10
11
9
169.5, qC
52.4, CH
37.9, CH2
169.4, qC
53.6, CH
38.0, CH2
169.4, qC
52.7, CH
38.8, CH2
4.30, m
2.87 (dd, 12.4, 5.0); 2.78
(dd, 12.4, 5.0)
4.06 (m)
2.97 (dd, 12.8, 5.0); 2.79
(dd, 12.5, 6.8)
4.26 (m)
2.91 (dd, 12.8, 5.0); 2.79
(dd, 12.3, 4.0)
12
13
14
15
16
17
NH
136.1, qC
129.6, CH
128.5, CH
127.0, CH
128.5, CH
129.6, CH
135.9, qC
129.3, CH
128.6, CH
127.2, CH
128.6, CH
129.2, CH
136.1, qC
129.1, CH
128.5, CH
127.0, CH
128.5, CH
129.1, CH
7.11 (d, 7.3)
7.30 (t, 7.3)
7.24 (m)
7.30 (t, 7.3)
7.11 (d, 7.3)
6.38 (d, 7.3)
7.15 (m)
7.32 (t, 7.3)
7.28 (m)
7.32 (t, 7.3)
7.15 (m)
7.71 (br s)
7.12 (d, 7.3)
7.31 (d, 7.8)
7.24 (m)
7.31 (d, 7.8)
7.12 (d, 7.3)
6.61 (d, 6.4)
Prob 18
19
20
21
22
170.6, qC
60.9, CH
31.3, CH2
21.5, CH2
46.9, CH2
170.8, qC
60.3, CH
32.0, CH2
22.1, CH2
46.7, CH2
7.12 (d, 7.3)
4.11 (m)
2.01 (m), 1.83 (m)
1.74 (m), 1.68 (m)
3.45 (m)
170.5, qC
60.8, CH
31.4, CH2
21.6, CH2
46.8, CH2
4.14 (dd, 7.8, 1.5)
2.01 (m), 1.81 (m)
1.77 (m), 1.68 (m)
3.48 (m)
4.23, dd (7.8, 2.8)
2.09 (m), 1.92 (m)
1.68 (m), 1.62 (m)
3.44 (m)
Pheb 23
24
25
170.2, qC
51.7, CH
39.4, CH2
168.2, qC
51.8, CH
38.2, CH2
169.9, qC
52.2, CH
38.1, CH2
4.82 (dt, 15.5, 7.8)
3.09 (dd, 13.7, 6.9); 2.76
(dd, 13.7, 7.7)
4.62 (dt, 15.6, 7.3)
3.01 (m);
2.76 (dd, 13.2, 6.8)
4.72 (dt, 13.9, 6.2)
3.05 (dd, 13.7, 7.7); 2.72
(dd, 13.3, 4.8)
26
27
28
29
30
31
NH
137.6, qC
129.1, CH
128.0, CH
126.3, CH
128.0, CH
129.1, CH
137.8, qC
129.2, CH
128.2, CH
126.3, CH
128.2, CH
129.2, CH
137.8, qC
129.1, CH
128.0, CH
126.2, CH
128.0, CH
129.1, CH
7.18 (d, 7.3)
7.20 (t, 7.3)
7.15 (m)
7.20 (t, 7.3)
7.18 (d, 7.3)
8.31 (d, 8.7)
7.18 (d, 7.3)
7.23 (t, 7.3)
7.14 (m)
7.23 (t, 7.3)
7.18 (d, 7.3)
8.34 (d, 8.7)
7.19 (d, 8.2)
7.23 (d, 7.3)
7.16 (m)
7.23 (t, 7.3)
7.19 (d, 8.2)
8.32 (d, 8.3)
H), which then lost 71 amu (Ala), affording m/z 342 (ePro-Phe-Pro
plus H). The latter fragment lost 97 amu (Pro), yielding m/z 245
(ePhe-Pro plus H) (Fig. 2). Another pathway left the major
fragment m/z 463 [MþHePro]þ after loss of 97 amu (Pro) from m/z
560 [MþH]þ, which then lost sequentially 147 amu (Phe), leaving
m/z 316 [MþHꢀPro-Phe]þ (Fig. S30, Supplementary data). Fur-
thermore, The geometry of the amide bonds of both Proa and Prob
were cis, on the basis of the large difference in the 13C NMR
HO
O
O
O
OH
O
N
HN
O
O
4
NH
R
OH
HN
O
COSY
HMBC
OH
N
O
O
O
chemical shifts of Proa
(
DdC6(b)eC7( ¼8.6 ppm) and Prob
g
)
(
DdC20(b)eC21( ¼9.8 ppm).8,9 The absolute configuration of the
g)
1:
2:
3:
R = CH3; R = H; R = CH2OH
5
hydrolyszaes with authentic samples (co-injection) revealed that
Fig. 1. The key 1He1H COSY and HMBC correlations of 1e5.
the amino acids were
L-Ala,
L-Pro, and
L-Phe (Fig. S26,
Supplementary data). The structure of versicoloritide A (1) was
consequently elucidated as cyclo-(L-Ala-cis-L-Pro-L-Phe-cis-L-Pro-L-
a cyclopeptide. Diagnostic 2D NMR (DMSO-d6) HMBC correlations
(Fig. 1) from 24-NH to C-1, 2-NH to C-4, and 17-NH to C-18 estab-
lished the sequence Pheb-Ala-Proa and Phea-Prob that combined
together by cyclization between the Pro nitrogen and the Phe car-
boxyl from the molecular formula. The complete amino acid se-
quence of 1 was also confirmed on the basis of the results of Q-TOF-
MS2 experiments. Although there was more than one possible ring-
opening position for the peptide, the preferred ring-opening of 1
occurred at the ProePhe amide bond. One ion series started with
loss of 147 amu due to Pre, leaving m/z 413 (eAla-Pro-Phe-Pro plus
Phe).
The molecular formula of versicoloritide B (2) was established as
C30H35N5O5 from the pseudomolecular ion peak at m/z 546.2703
[MþH]þ in the HRESIMS, indicative of a CH2 homologue of 1.
Contrastive analysis of NMR spectra (Table 1) between 1 and 2
revealed that a methylene signal (dH/C 3.91 and3.28/42.5) replaced
a methyl signal (dH/C 0.85/19.4) and a methine signal (dH/C 4.22/
49.7) in 1, indicating that 2 is the Gly-substituted analogue for Ala
of 1. This deduction was further supported by analysis of 1He1H
COSY correlations (Fig. 1) and the Q-TOF-MS2 experiments (Fig. 2)

Y. Zhuang et al. / Tetrahedron 67 (2011) 7085e7089
7087
+
560 M H
[
Supplementary data).10 The absolute configuration of Ser was de-
termined as
- by amino acids analysis on a chiral Crownpak CR(þ)
HPLC column (Fig. S28, Supplementary data).11,12 The structure of
versicoloritide C (3) was accordingly determined to be cyclo-( -Ser-
cis- -Pro- -Phe-cis- -Pro- -Phe).
Tetraorcinol (4) was assigned
+
m z =
/
]
463
L
O
O
H
316
H
N
L
N
N
N
H
O
N
L
L
L
L
H
O
A
a molecular formula of
O
C28H26O5, according to HREIMS at m/z 442.1785 [MþH]þ, requiring
16 sites of unsaturation. NMR spectra only showed 14 carbon and
13 proton signals including six aromatic methines, six aromatic
quaternary carbons (four oxygenated), two methyl groups (dH/C
2.18/21.1 and 2.24/21.2), and one exchangeable proton (dH 9.47),
indicating a symmetrical structure. The 1H NMR data also showed
two sets of coupled 1H NMR signals at dH 6.16, 6.25 (br s), 6.34 (br s)
and 6.50 (br s), 6.62 (br s), 6.75 (br s) assigned by 1He1H COSY,
revealing the presence of two 1,3,5-trisubstituted benzene nucleus
moieties similar to those of diorcinol.5 These data revealed 4 as
a diorcinol dimer via CeOeC bridge that was further confirmed by
HMBC correlations from H3C-7/70 to C-4/40, C-5/50, and C-6/60, from
HO-3 to C-2/20, C-3/30, and C-4/40, and from H-2/20 to C-6/60.
Therefore, the structure of 4 was elucidated as 3,30-{[oxybis(5-
methyl-1,3-phenylene)]bis (oxy)} bis(5-methylphenol).
342
245
413
+
+
m z =
/
546 M H
[
]
449
O
O
O
H
N
302
H
N
N
N
H
O
N
H
O
O
245
342
399
+
+
479
Hm/z =
576 M H
[
]
O
O
H
332
H
N
N
N
N
H
O
N
H
Versicolactone A (5) owned molecular formula C8H10O4 from
the HRESIMS peak at m/z 193.0486 [MþNa]þ, requiring four sites of
unsaturation. 1H and 13C NMR (Table 2) revealed the presence of
a carbonyl carbon (dC 169.6), a sp2 quaternary carbon (dC 169.6),
three sp2 methine (dH/C 6.43/119.3, 7.87/145.5 and 5.47/117.2), to-
O
O
245
342
429
Fig. 2. Q-TOF-MS2 sequence ions (m/z) for protonated molecular [MþH]þ ions of 1e3.
gether with
a methyl (dH/C 1.02/19.0), and two oxygenated
methines dH/C 3.60/69.4 and 4.26/70.0). Two fragments,
(
CH3eCH(OH)eCH(OH)eCH and CH]CH, could be deduced from
the 1He1H COSY correlations of H-8/H-7/H-6/H-5, and H-2/H-3
(Fig. 1). Furthermore, 5-(2,3-dihydroxybutylidene)furan-2(5H)-one
skeleton was established on the base of the key HMBC correlations
and the acidic hydrolysis experiment (Fig. S27, Supplementary
data).10 Versicoloritide B (2) was thus identified as cyclo-(Gly-cis-
L-Pro-L-Phe-cis-L-Pro-L-Phe).
Versicoloritide C (3) showed a pseudomolecular ion peak at m/z
from H-2 (
d
6.43) to C-1 (
d
169.6) and C-4 ( 7.87)
d
149.1), from H-3 (
d
576.2797 [MþH]þ, indicative of a molecular formula of C31H37N5O6,
with one oxygen more than 1. Compared the NMR data with 1,
compound 3 showed an additional oxygenated methylene signal
to C-1, and from H-5 (
d
5.47) to C-3 (
d
145.5), C-4 and C-7 (d 69.4).
The double bond in the side chain was assigned as Z-configuration
based on the NOE difference experiments. When H-3 and H-5 were
radiated, the signals of H-5 and H-3 were enhanced by 5.1% and
7.0%, respectively, indicating H-3 and H-5 was in the same side.
Versicolactone B (6) was the isomer of 5, with the same molecular
formula C8H10O4 from the HRESIMS peak at m/z 193.0468
[MþNa]þ. Its NMR data were almost identical to those of 5. The
differences were that H-3 and H-5 and C-3 undergo large downfield
shifts and large upfield shift (Table 2) for de-steric and steric effects
(dH/C 3.17 and 3.24/62.0), while the methyl signal (dH/C 0.85/19.4) in
1 disappeared. These data indicated that the Ser residue in 3
replaced the Ala residue in 1, which was confirmed by HMBC cor-
relations between NH protons and neighboring residue carbonyls
and 1He1H COSY correlations (Fig. 1) and further by the Q-TOF-MS2
experiments (Fig. 2). The absolute configurations of both Pro and
Phe were determined by Marfey’s analysis as
L- (Fig. S28,
Table 2
1H and 13C NMR data for 4e6 (600, 150 MHz, DMSO-d6, TMS,
d ppm)
Position
4
5
6
dH (J in Hz)
dC
dH (J¼Hz)
dC
dH (J¼Hz)
dC
1 (1000
2 (2000
3 (3000
4 (4000
5 (5000
6 (6000
7 (7000
8
)
)
)
)
)
)
)
157.7, qC
102.9, CH
158.6, qC
111.3, CH
140.2, qC
109.9, CH
21.1, CH3
169.6, qC
119.3, CH
145.5, CH
149.1, qC
117.2, CH
70.0, CH
69.4, CH
19.0, CH3
169.6, qC
120.1, CH
142.8, CH
150.3, qC
117.0, CH
71.2, CH
69.6, CH
19.6, CH3
6.16, br s
6.34, br s
6.43, dd (0.6, 5.4)
7.87,.d (5.4)
6.46, dd (1.7, 6.1)
8.16, d (6.1)
5.47, d (9.2)
5.78, dd (1.2, 8.6)
4.16, ddd (5.3, 5.4, 8.6)
3.57, ‘dq’ like (5.4, 6.3)
1.06, d (6.3)
6.25, br s
2.18, s
4.26, ddd (5.1, 5.1, 5.1)
3.60, ‘dq’ like (5.1, 9.2)
1.02, d (6.4)
10 (100)
20 (200)
30 (300)
40 (400)
50 (500)
60 (600)
70 (700)
3 (3000)-OH
6-OH
154.6, qC
108.3, CH
156.8, qC
114.1, CH
139.5, qC
116.1, CH
21.2, CH3
6.62, br s
6.50, br)
6.75, br s
2.24, br s
9.47, br s
5.16, d (5.1)
4.66, d (5.1)
5.23, d (5.2)
4.66, d (5.3)
7-OH

7088
Y. Zhuang et al. / Tetrahedron 67 (2011) 7085e7089
between H-3 and H-5, and between CH(OH)-6 and C-3, re-
Factory, Qingdao, China). Semiprepartive HPLC was performed
spectively, indicating the double bond in the side chain of 6 was E-
form. The large coupling constants of J6,7 (5.1, 5.4 Hz) and down-
using an ODS column [Shin-pak ODS (H), 20ꢁ250 mm, 5
mm,
3
4 mL/min].
field shifts of dCH3 (19.0, 19.6) of 5 and 6 (Table 2) indicated that
both compounds are threo-configuration. According to the litera-
tures, the threo-1-alkenylpropane-1,2-diol displayed larger cou-
pling constants of 3J1,2 (5e7 Hz) and larger dCH3 value (19e20) than
erythro-1-alkenylpropane-1,2-diol (3J1,2 3e4 Hz, dCH3 17e18).13ꢀ21
The steric hindrance among HOe, CH3e and alkenyl resulted in
upfield shift of CH3e in erythro-molecule (Fig. 3). The absolute
configurations of compounds 5 and 6 could be deduced by com-
parison of their specific rotations with those of their analogues, 7-
epi-goniobutenolides A and B in which the only difference is
the replacement of phenyl by the corresponding methyl groups.22,23
The same sign of specific rotations of 5 (ꢀ31.5) and 6 (þ19.8) as those
of 7-epi-goniobutenolide A (ꢀ246.5) and B (þ95.5) indicated both
compounds 5 and 6 are (R,R)-configurations. Thus, the structures of
versicolactones A and B (5 and 6) were deduced as (4Z,6R,7R)-5-
(2,3-dihydroxybutylidene)furan-2(5H)-one and (4E,6R,7R)-5-(2,3-
dihydroxybutylidene)furan-2(5H)-one, respectively.
3.2. Fungal material
A. versicolor LCJ-5-4 was isolated from the coral Cladiella sp.
collected from Lingao, Hainan province of China. It was identified
according to its morphological characteristics and 18S rRNA
sequences.4 The voucher specimen is deposited in our laboratory
at ꢀ80 ꢂC. The producing strain was prepared on Potato Dextrose
agar slants at 3.33% salt concentration and stored at 4 ꢂC.
3.3. Fermentation and extraction
A. versicolor LCJ-5-4 was grown under static conditions at 20 ꢂC
for 30 days in three hundred 1000 mL conical flasks containing
liquid medium (300 mL/flask) composed of sorbitol (20 g/L),
maltose (20 g/L), monosodium glutamate (10 g/L), KH2PO4 (0.5 g/L),
MgSO4$7H2O (0.3 g/L), tryptophane (0.5 g/L), yeast extract (3 g/L),
and sea salt (33.3 g/L), after adjusting its pH to 6.5. The fermented
whole broth (about 90 L) was filtered through cheesecloth to sep-
arate into filtrate and mycelia. The filtrate was concentrated under
reduced pressure to about a quarter of the original volume and then
extracted three times with EtOAc to give an EtOAc solution, while
the mycelia was extracted three times with acetone. The acetone
solution was concentrated under reduced pressure to afford an
aqueous solution. The aqueous solution was extracted three times
with EtOAc to give another EtOAc solution. Both EtOAc solutions
were combined and concentrated under reduced pressure to give
a crude extract (140 g).
H
O
H
O
H
O
H
O
H
C
H
H
H
O
H
O
H
H
O
H
H
H
O
H
H
H
H
C
H
3
H
3C
3C
3
erythro- 5 and 6
threo- 5 and 6
Fig. 3. The major conformations of threo- and erythro-5 and 6.
The new isolates 1ꢀ4 were evaluated for cytotoxicity against
P388 and Hela cells with the MTT method.24 Their antimicrobial
activities against Staphylococcus aureus, Escherichia coli, Enter-
obacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and
Candida albicans were also evaluated by an agar dilution method.25
They showed no cytotoxic effect on the tested cancer cell lines (IC50
3.4. Purification
The extract (140 g), was separated into eight fractions on a silica
gel VLC column using step gradient elution with CHCl3/petroleum
ether (0e100%) and then with MeOH/CHCl3 (0e50%). Fr. 5 (350 mg)
was further separated into three subfractions on a silica gel VLC
column using step gradient elution with MeOH/CHCl3 (0e50%). Fr.
5e3 (80 mg) was then separated into five subfractions on an RP-18
VLC column using step gradient elution with H2O/MeOH (0e100%).
Fr. 5e3e5 (12 mg) was further purified by semipreparative HPLC
(65% MeOH/H2O) to yield diorcinol (3 mg, tR 15 min). Fr. 6 (780 mg)
from the 50:1 of CHCl3/MeOH eluent was further separated into
three subfractions on a silica gel VLC column using step gradient
elution with MeOH/CHCl3 (0e50%). Fr. 6e1 (62 mg) was then
separated into five subfractions on an RP-18 VLC column using step
gradient elution with H2O/MeOH (0e100%). Fr. 6e1e1 was further
purified by Sephadex LH-20, eluting with MeOH/CHCl3 (1:1), and
semipreparative HPLC (60% MeOH/H2O) to yield cordyol C (2.1 mg,
tR 14 min). Fr. 6e1e2 (53 mg) was further purified by Sephadex LH-
20 eluting with MeOH, and then by semipreparative HPLC (50%
MeOH/H2O) to yield 4 (15 mg, tR 10 min). Fr. 6e3 (205 mg) was
separated into five subfractions on an RP-18 VLC column using step
gradient elution with H2O/MeOH (0e100%). Fraction 6e3e1
(82 mg) was further purified by Sephadex LH-20 eluting with
MeOH, and then by semipreparative HPLC (50% MeOH/H2O) to
yield 1 (30 mg, tR 25 min), 2 (3 mg, tR 21 min), and 3 (6 mg, tR
19 min). Fr. 6e3e3 (38 mg) was further purified by Sephadex LH-20
eluting with MeOH, and then by semipreparative HPLC (55% MeOH/
H2O) to yield glyantrypine (4 mg, tR 7 min). Fr.6-2 (136 mg) was
subjected to silica gel VLC using step gradient elution with acetone/
petroleum (0e100%) to afford ten subfractions. Fr. 6e2e8 (36 mg)
was subjected to Sephadex LH-20, eluting with MeOH/CHCl3 (1:1),
to afford six subfractions. Fr. 6e2e8e6 (12 mg) was further purified
>50 mM) and antimicrobial activities (MIC >150 mM). In the radical-
scavenging assay, compound 4 showed weak antioxidative activity
against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, with IC50
value of 67
mM, ascorbic acid (vitamin C) serving as positive control
(IC50 22
m
M), as determined by the method of Chen and Ho.26
In conclusion, this work describes the discovery of six new
compounds from coral-associated fungus, A. versicolor LCJ-5-4. Al-
though about 150 compounds of A. versicolor were characterized,
researches on secondary metabolites of coral-associated A. versi-
color are rarely reported. The results showed that the coral-
associated microorganisms could be valuable organisms to allow
construction of diverse chemical space.
3. Experimental section
3.1. General experimental procedures
Optical rotations were obtained on a JASCO P-1020 digital po-
larimeter. UV spectra were recorded on a Beckman DUÒ 640
spectrophotometer. IR spectra were recorded on a Nicolet Nexus
470 spectrophotometer as KBr discs. 1H, 13C NMR, DEPT, and 2D
NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer
using TMS as internal standard and chemical shifts were recorded
as d
-values. ESI-MS and Q-TOF-MS2 were measured on a Q-TOF
ULTIMA GLOBAL GAA076 LC mass spectrometer. TLC and column
chromatography (CC) were performed on plates precoated with
silica gel GF254 (10e40 mm) and over silica gel (200e300 mesh,
Qingdao Marine Chemical Factory), and Sephadex LH-20 (Amer-
sham Biosciences), respectively. Vacuum-liquid chromatography
(VLC) was carried out over silica gel H (Qingdao Marine Chemical

Y. Zhuang et al. / Tetrahedron 67 (2011) 7085e7089
7089
by semipreparative HPLC (10% MeOH/H2O) to yield 5 (3 mg, tR
13 min) and 6 (2 mg, tR 11 min).
established as
L
-configuration. Due to the bad discrimination
between L/D-Ser, they were determined by amino acids analysis on
a chiral Crownpak CR(þ) HPLC column.11,12 Compound 3 (1 mg) was
dissolved in 1 mL of 6 N HCl and heated in a sealed tube at 110 ꢂC for
12 h. The hydrolyzate was dried and reconstituted in 1 mL of H2O.
The hydrolyzate was then analyzed by chiral HPLC over Crownpak
CR(þ) column for serine, proline (flow rate 0.5 mL/min; solvent,
aqueous HClO4 (pH 1.5); detection, 201 nm; temperature 0 ꢂC),
respectively. The retention times of serine in hydrolyzates of 3 and
3.5. Characteristics of compounds
3.5.1. Versicoloritide A (1). White amorphous powder; ½a D25
ꢀ90.7
ꢃ
(c 1.7, MeOH); UV (MeOH) lmax (log 3) 216 (4.49), 258 (4.05), 265
(3.97) nm; IR (KBr) nmax 3297, 3064, 3027, 2978, 2963, 1640, 1506,
1449, 1345, 1320, 1189, 1160 cmꢀ1; 1H and 13C NMR data, see Table
1; HRESI-MS m/z 560.2869 [MþH]þ (calcd for C31H37N5O5,
560.2873).
the authentic L/D-Ser and L-Pro were tR 6.2 min, tR 6.2/5.2 min, and
tR 5.2 min, respectively. Co-injection of the authentic sample with
the hydrolyzate confirmed that the serine residue in compound 3
3.5.2. Versicoloritide B (2). White amorphous powder; ½a D25
ꢃ
ꢀ43.6
was L-Ser (Fig. S28).
(c 0.1, MeOH); UV (MeOH) lmax (log 3) 216 (4.49), 258 (4.05), 265
(3.97) nm; IR (KBr) nmax 3276, 2923, 1680, 1647, 1555, 1543, 1522,
Acknowledgements
1451, 1344, 748, 702 cmꢀ1 1H and 13C NMR data, see Table 1;
;
HRESI-MS m/z 546.2703 [MþH]þ (calcd for C30H35N5O5, 546.2716).
This work was supported by grants from National Basic Research
Program of China (No. 2010CB833800), from the National Natural
Science Foundation of China (Nos. 30973680 and 30670219), and
from PCSIRT (No. IRT0944). The working strain LCJ-5-4 was iden-
tified by Prof. C.X. Fang, China Center for Type Culture Collection.
3.5.3. Versicoloritide C (3). White amorphous powder; ½a D25
ꢀ118
ꢃ
(c 0.2, MeOH); UV (MeOH) lmax (log 3) 216 (4.49), 258 (4.02), 265
(3.97) nm; IR (KBr) nmax 3303, 1647, 1555, 1537, 1524, 1450, 1342,
758, 702 cmꢀ1 1H and 13C NMR data, see Table 1; HRESI-MS m/z
;
576.2797 [MþH]þ (calcd for C31H37N5O6, 576.2822).
Supplementary data
3.5.4. Tetraorcinol A (4). Purple oil; UV (MeOH) lmax (log 3) 223
(4.52), 280 (3.79) nm; IR (KBr) nmax 3417, 2923, 1614, 1585, 1463,
This data include bioassay protocols used, the NMR spectra of
compounds 1e6, HPLC profiles of acidic hydrolyzates of 1e3. Sup-
plementary data associated with this article can be found online
version, at doi:10.1016/j.tet.2011.07.003.
129, 1233, 1159, 1137, 1059, 1036, 971, 840, 782, 677, 662, 634,
596 cmꢀ1 1H and 13C NMR data, see Table 2; HRESI-MS m/z
;
442.1785 [MþH]þ (calcd for C28H26O5, 442.1780).
References and notes
3.5.5. Versicolactone A (5). White oil; ½a D25
ꢀ31.5 (c 0.02, EtOH); UV
ꢃ
ꢀ
(MeOH) lmax (log 3) 272 (3.72) nm; IR (KBr) nmax 1777, 1748, 1683,
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in acetone was added to an aliquot (50
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5e50 min, 15e45% B; 50e55 min, 45% B; temperature, 30 ꢂC; flow
rate, 1 mL/min; UV detection at lmax 340 nm). Retention times
m
L). A 1% (w/v) solution (100
L) of the acid hydrolyzate
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mL) of L-FDAA
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m
m
m
(min) of the amino acids derivatives were as follows:
29.6/34.4 min; -Pro, tR 30.6/32.3 min; -Phe, tR 40.8/51.2 min.
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L/D-Ala, tR
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L
/D
L/D
L
L
L