Novel cyclic hexapeptides from marine-derived fungus, aspergillus sclerotiorum PT06-1

Article abstract of DOI:10.1021/ol902197z

Two novel cyclic hexapeptides containing both anthranilic acid and dehydroamino acid units, sclerotides A (1) and B (2), were isolated from the marine-derived halotolerant Aspergillus sclerotiorum PT06-1 in a nutrient-limited hypersaline medium. Both 1 an

Full text of DOI:10.1021/ol902197z

ORGANIC
LETTERS
2009
Vol. 11, No. 22
5262-5265
Novel Cyclic Hexapeptides from
Marine-Derived Fungus, Aspergillus
sclerotiorum PT06-1
Jinkai Zheng,†,⊥ Huajie Zhu,‡,⊥ Kui Hong,§ Yi Wang,† Peipei Liu,† Xin Wang,†
Xiaoping Peng,† and 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, State Key Laboratory of Phytochemistry and Plant Resources, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic
of China, and Institute of Tropical Biological Sciences and Biotechnology, Chinese
Academy of Tropical Agricultural Science, Haikou 571101, People’s Republic of China
weimingzhu@ouc.edu.cn
Received September 22, 2009
ABSTRACT
Two novel cyclic hexapeptides containing both anthranilic acid and dehydroamino acid units, sclerotides A (1) and B (2), were isolated from
the marine-derived halotolerant Aspergillus sclerotiorum PT06-1 in a nutrient-limited hypersaline medium. Both 1 and 2 are photointerconvertible
and could be interconverted via a radical reaction initiated by direct photoisomerization. Both compounds showed moderate antifungal activity.
Compound 2 also showed weak cytotoxicity and antibacterial activity.
Cyclopeptides derived from natural sources have been chiefly
found in plants, in lower sea animals, and in microorganisms,
possessing many interesting bioactivities, such as antimi-
crobial, insecticidal, antimalarial, estrogenic, sedative, ne-
maticidal, cytotoxic, and immunosuppressive activities.1,2 As
part of our ongoing efforts to discover structurally novel and
bioactive natural compounds from microorganisms, a marine-
derived halotolerant fungal strain PT06-1, identified as
Aspergillus sclerotiorum, was isolated from the Putian Sea
Salt Field, Fujian, China. Only five secondary metabolites,
sclerotiamide,3 scleramide,4 penicillic acid,5 ochratoxin A,6
and Ro 09-1469,7 had been previously reported from A.
sclerotiorum. The metabolite pattern of this fungus exhibited
distinct different TLC and HPLC profiles in different salty
media. Two novel cyclic hexapeptides containing both
anthranilic acid and dehydrotryptophan residues, sclerotides
(3) Whyte, A. C.; GIoer, J. B. J. Nat. Prod. 1996, 59, 1093–1095.
(4) Whyte, A. C.; Joshi, B. K.; Gloer, J. B.; Wicklow, D. T.; Dowd,
P. F. J. Nat. Prod. 2000, 63, 1006–1009.
† Ocean University of China.
(5) Kang, S. W.; Park, C. H.; Hong, S. I.; Kim, S. W. Bioresour. Technol.
2007, 98, 191–197.
‡ Kunming Institute of Botany.
§ Institute of Tropical Biological Sciences and Biotechnology.
⊥ These authors contributed equally to this work.
(1) Pomilio, A. B.; Battista, M. E.; Vitale, A. A. Curr. Org. Chem. 2006,
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10.1021/ol902197z CCC: $40.75
Published on Web 10/14/2009
 2009 American Chemical Society

A (1) and B (2), were identified from the metabolites in a
nutrient-limited medium containing 10% NaCl. To the best
of our knowledge, this is the first example of cyclopeptides
containing both anthranilic acid and dehydroamino acid
residues in natural products, and the microbial secondary
metabolites under high salt culture conditions were rarely
reported.
due to the deshielding effect of the 27-carbonyl,8,9 while 2
was E-configuration. The absolute configurations of 1 and 2
were determined by amino acids analyses of acidic hydroly-
sates through a chiral Crownpak CR(+) HPLC column.9,10
Mix HPLC analyses of the hydrolysates with authentic
samples (co-injection) confirmed that the amino acids both
in 1 and 2 were L-Thr, L-Ala, D-Phe, and D-Ser.
A. sclerotiorum PT06-1 was grown under static conditions
at 28 °C for 30 days and then was harvested by extraction
with EtOAc. The EtOAc extract was concentrated under
reduced pressure to give a dark brown gum (8.0 g). The crude
residue was separated into five fractions on a silica gel
vacuum liquid chromatograph using a step gradient elution
with MeOH-CHCl3 (0-100%). Fraction 3 (1.2 g) from the
20:1 of CHCl3/MeOH eluents was subjected to silica gel
column chromatography using a step gradient elution of
acetone/petroleum ether (40-100%) to afford three subfrac-
tions (Fr.3-1∼3-3). Fr.3-3 (73 mg) was combined with
fraction 4 (0.8 g) from the 10:1 of CHCl3/MeOH eluents,
and the mixture was further separated into four subfractions
(Fr.4-1∼4-4) by Sephadex LH-20, eluting with MeOH.
Fr.4-1 (240 mg) was then purified by semipreparative HPLC
(57% MeOH/H2O, 4.0 mL/min) to yield compounds 2 (14
mg, tR ) 13 min) and 1 (90 mg, tR ) 15 min). Both compounds
1 and 2 were kept away from radiation by silver paper.
Figure 1.
Key 1H-1H COSY and HMBC correlations of 1 and 2.
To further confirm the geometry of the double bond,
conformational searches were performed by the HyperChem
package using an Amber force field. The conformations with
low energy from 0 to 3 kcal/mol were optimized again at
the B3LYP/3-21G* level.11 The B3LYP/3-21G*-optimized
geometries with energy from 0 to 1.5 kcal/mol were
optimized at the B3LYP/6-31G(d) level to look for the most
stable geometries.12 Both structures were found that have
1.0 kcal/mol energy lower than the second stable conforma-
tion. Thus, only two conformations were used in 13C NMR
computations at the B3LYP/6-311+G(2d,p) level for 1 and
2. The magnetic shielding values were converted into
chemical shifts after the corrections using slope and intercept
of the linear-square functions.13 The relative errors of
chemical shifts were computed by subtracting the calculated
13C NMR from the experimental shifts. Two cases should
be considered in the data treatments. The first case is that
Compound 1, [R]25 -73 (c 0.3, MeOH), UV (MeOH)
D
λmax (log ε) 203 (3.9), 222 (3.8), 268 (3.3), 350 (3.6) nm,
was obtained as a yellow amorphous powder. Its molecular
formula was determined as C37H39N7O8 according to the
HRESIMS at m/z 710.2962 [M + H]+, requiring 22 degrees
1
of unsaturation. Analysis of H and 13C NMR data (Table
1) revealed the presence of six carbonyl signals (δC 172.6,
171.7, 170.4, 170.3, 169.0, and 163.6) and six amide protons
(δH 7.37, 8.37, 8.62, 8.76, 8.77, and 10.92) coupling with
signals between 3.6 and 4.6 ppm, indicating the hexapeptide
nature of 1. 1H-1H COSY experiment (Figure 1) constructed
the six amino acid residues as threonine, alanine, phenyla-
lanine, serine, anthranilic acid (AA), and dehydrotryptophan
(∆-Trp). These residues accounted for 21 of the 22 degrees
of unsaturation, indicating that 1 is a cyclopeptide. The amino
acid sequence was determined mainly by HMBC correlations
of the carbonyl carbon of one amino acid residue with the
amide protons of the neighboring residue, and the carbonyl
carbon signals in each amino acid unit were deduced by their
HMBC correlations with the ꢀ-proton of respective amino
acids (Figure 1). Thus, the constitution of 1 was assigned as
(8) Marchelli, R.; Dossena, A.; Pochini, A.; Dradi, E. J. Chem. Soc.,
Perkin Trans. 1 1977, 713–717
(9) Wang, W. L.; Lu, Z. Y.; Tao, H. W.; Zhu, T. J.; Fang, Y. C.; Gu,
Q. Q.; Zhu, W. M. J. Nat. Prod. 2007, 70, 1558–1564
(10) Gunasekera, S. P.; Ross, C.; Paul, V. J.; Matthew, S.; Luesch, H.
J. Nat. Prod. 2008, 71, 887–890
.
.
.
(11) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.;
Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.;
Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li,
X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.;
Gomperts, R.; Stratemann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels,
A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuk, A. D.; Raghavachari,
K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
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Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03 User’s
Reference; Gaussian Inc.: Wallingford, CT, 2003.
cyclo (Thr-Ala-Phe-Ser-AA-∆-Trp). Compound 2, [R]25
D
-111 (c 0.3, MeOH), UV (MeOH) λmax (log ε) 203 (3.9),
222 (3.8), 274 (3.3), 352 (3.5) nm, showed a pseudomo-
lecular ion peak at m/z 710.2929 [M + H]+, indicating that
2 was an isomer of 1. As expected, UV absorption and 1D
NMR data (Table 1) of 2 were very similar to those of 1.
Further analysis of 2D NMR data of 2 (Figure 1) revealed
that 2 shared the same constitution as 1. The obvious
differences between H-29, H-38, and NH in dehydrotryp-
tophan residue revealed that 1 and 2 are a pair of geometric
isomers of C28dC29. The downfield chemical shift of H-28
(δH 7.96 vs 7.03) implied Z-configuration of C28dC29 in 1
(12) (a) Timmons, C.; Wipf, P. J. Org. Chem. 2008, 73, 9168–9170.
(b) Nielsen, D. K.; Nielsen, L. L.; Jones, S. B.; Toll, L.; Asplund, M. C.;
Castle, S. L. J. Org. Chem. 2009, 74, 1187–1199.
(13) Liu, D. Z.; Wang, F.; Liao, T. G.; Tang, J. G.; Steglich, W.; Zhu,
H. J.; Liu, J. K. Org. Lett. 2006, 8, 5749–5752.
Org. Lett., Vol. 11, No. 22, 2009
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1
Table 1. H and 13C NMR Data for 1 and 2 (600, 150 MHz, DMSO-d6, TMS, δ ppm)
sclerotide A (1)
sclerotide B (2)
δH (J in Hz)
position
δC
δH (J in Hz)
δC
Thr
1
2
3
4
170.3, qC
59.0, CH
65.6, CH
20.8, CH3
170.5, qC
59.4, CH
65.8, CH
20.5, CH3
4.31, dd (3.2, 8.7)
4.43, m
1.16, d (6.4)
7.37, d (8.2)
5.14, br s
4.13, dd (2.2, 7.0)
4.33, m
1.12, d (6.2)
7.19, br s
NH
3-OH
5.06, d (5.9)
Ala
5
6
7
172.6, qC
49.6, CH
16.6, CH3
172.2, qC
49.8, CH
16.7, CH3
4.20, dq (6.3, 7.3)
1.26, d (7.3)
4.03, dq (6.1, 7.3)
1.21, d (7.3)
NH
8
9
8.77, d (6.4)
8.86, d (5.9)
Phe
171.7, qC
54.5, CH
172.1, qC
54.1, CH
4.57, “q” like (7.6)
2.93, dd (7.3, 13.7); 2.87,
dd (8.2, 13.7)
4.65, “q” like (7.6)
2.89, dd (7.2, 13.2); 2.86,
dd (8.1, 13.2)
10
11
12
13
14
15
16
NH
17
18
35.6, CH2
137.5, qC
129.1, CH
128.1, CH
126.4, CH
128.1, CH
129.1, CH
36.7, CH2
137.2, qC
129.1, CH
128.1, CH
126.4, CH
128.1, CH
129.1, CH
7.20, d (7.8)
7.24, t (7.3)
7.17, t (7.8)
7.24, t (7.3)
7.20, t (7.8)
8.37, d, (7.3)
7.20, d (8.0)
7.25, t (7.7)
7.19, t (8.0)
7.25, t (7.7)
7.20, d (8.0)
8.09, d (7.7)
Ser
170.4, qC
58.0, CH
169.9, qC
57.8, CH
4.23, “q” like (7.0)
4.28, “q” like (7.2)
3.64, ddd (3.7, 6.2, 12.1); 3.61,
ddd (5.5, 6.2, 12.1)
8.62, d (7.3)
19
60.9, CH2
3.67, m
8.62, br s
5.15, br s
61.3, CH2
NH
19-OH
20
21
22
23
24
25
26
5.12, t (6.2)
anthranilic acid
169.0, qC
122.0, qC
129.2, CH
122.9, CH
132.1, CH
121.2, CH
138.4, qC
168.7, qC
123.4, qC
128.9, CH
122.9, CH
131.8, CH
121.4, CH
137.7, qC
7.85, dd (1.4, 7.8)
7.20, dt (1.1, 7.8)
7.58, ddd (1.4, 7.8, 8.2)
8.53, d (8.2)
7.79, d (7.7)
7.24, dt (1.1, 7.7)
7.57, ddd (1.4, 7.7, 8.2)
8.37, d (8.2)
NH
27
28
29
30
31
32
33
34
10.92, s
7.96, s
10.79, s
7.03, s
∆-Trp
163.6, qC
122.5, qC
126.0, CH
108.6, qC
127.4, qC
117.7, CH
120.4, CH
122.2, CH
112.0, CH
135.6, qC
163.8, qC
123.1, qC
124.3, CH
108.5, qC
127.7, qC
117.5, CH
120.0, CH
121.9, CH
112.0, CH
135.6, qC
7.75, d (7.8)
7.60, d (7.7)
7.16, dt (1.3, 7.8)
7.18, dt (1.4, 7.8)
7.42, d (7.8)
7.13, dt (1.1, 7.7)
7.17, dt (1.5, 7.7)
7.44, d (7.7)
35
36
37
38
NH
11.96, s
7.98, d (2.3)
8.76, s
11.61, s
8.39, br s
9.07, s
128.7, CH
128.9, CH
the structure 1 calculated is the experimental structure 1; the
second case is that the computed 1 is the experimental
structure 2. In the first case, all of the maximum of relative
errors of chemical shift were below 8.0 ppm. However, in
the second case, the maximum errors at C-21 were 8.9 ppm.
Thus, the second assumption was wrong. The calculated
results agreed with the experimental structures 1 and 2. Thus,
the structures of compounds 1 and 2 were determined as
cyclo (L-Thr-L-Ala-D-Phe-D-Ser-AA-Z-∆-Trp) and cyclo (L-
Thr-L-Ala-D-Phe-D-Ser-AA-E-∆-Trp), respectively.
Compounds 1 and 2 were assayed for their cytotoxic
effects on HL-60 cell lines by the MTT method14 and A-549
cell lines using the SRB method.15 The antimicrobial
activities against Staphylococcus aureus, Escherichia coli,
Enterobacter aerogenes, Bacillus subtilis, Pseudomonas
(14) Mosmann, T. Immunol. Methods 1983, 65, 55–63.
Org. Lett., Vol. 11, No. 22, 2009
5264

aeruginosa, and Candida albicans were evaluated by an agar
dilution method.16 Both 1 and 2 exhibited moderate anti-
fungal activity against Candida albicans with the MIC values
of 7.0 and 3.5 µM, respectively. Compound 2 also showed
weak cytotoxic activity against the HL-60 cell line (IC50,
56.1 µM) and selective antibacterial activity against Pseudomo-
nas aeruginosa (MIC, 35.3 µM).
photoequilibrium. The time for 1 achieving the equilibrium
was only several hours, but it was several days for 2. Thus, the
photointerconversion was a radical mechanism. The foregoing
results also suggested that the Z-isomer (1) was lower in energy
and the main component. It was possible that the sclerotide A
was really produced by A. sclerotiorum, while sclerotide B was
formed from the photoreaction of sclerotide A during the
fermentation or subsequent isolation steps.17
Acknowledgment. This work was supported by the
National Natural Science Foundation of China (Nos. 30470196
and 30670219) and the Project of Chinese National Programs
forHighTechnologyResearchandDevelopment(2007AA091502).
The cytotoxicity assay was performed by Prof. M. Geng’s
Group, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences.
Scheme 1. Photointerconversion of 1 and 2
Supporting Information Available: Experimental details,
NMR spectra of 1 and 2, HPLC profiles of acidic hydroly-
sates and photointerconverting mixtures of 1 and 2, 18S
rRNA and ITS sequences of A. sclerotiorum PT06-1, and
the calcd 13C NMR by the HyperChem package. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Compounds 1 and 2 were stable in the dark, while they
changed into each other in light, indicating that 1 and 2 are
photointerconvertible (Scheme 1). HPLC analysis showed
that the ratio of 1 and 2 was 87:13 in the equilibrium, and
both temperature and solvent had little influence on the
OL902197Z
(15) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.; Tan,
N. H.; Zhou, J. J. Natl. Cancer Inst. 1990, 82, 1107–1112.
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(17) Amir-Heidari, B.; Thirlway, J.; Micklefield, J. Org. Lett. 2007, 8,
1513–1516.
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