Cyclic tripeptides from the halotolerant fungus Aspergillus sclerotiorum PT06-1

Article abstract of DOI:10.1021/np100198h

Eleven new aspochracin-type cyclic tripeptides, sclerotiotides A-K (1-11), together with three known compounds, JBIR-15 (12), aspochracin (13), and penicillic acid, were isolated from the ethyl acetate extract of the fermentation broth of the halotolerant Aspergillus sclerotiorum PT06-1 in a hypersaline nutrient-rich medium. Their structures were elucidated by spectroscopic analysis and chemical methods. Chemical transformations of 12 and 13 proved that sclerotiotides D-K (4-11) were artifacts probably formed during the fermentation or subsequent isolation steps. All 13 cyclic tripeptides have been evaluated for their antimicrobial and cytotoxic effects. Only sclerotiotides A (1), B (2), F (6), and I (9) and JBIR-15 (12) showed selective antifungal activity against Candida albicans with MIC values of 7.5, 3.8, 30, 6.7, and 30 μM, respectively.

Full text of DOI:10.1021/np100198h

J. Nat. Prod. 2010, 73, 1133–1137
1133
Cyclic Tripeptides from the Halotolerant Fungus Aspergillus sclerotiorum PT06-1
Jinkai Zheng,† Zhihong Xu,† Yi Wang,† Kui Hong,‡ Peipei Liu,† 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, and Institute of Tropical Biological Sciences and Biotechnology, Chinese Academy of Tropical
Agricultural Science, Haikou 571101, People’s Republic of China
ReceiVed March 23, 2010
Eleven new aspochracin-type cyclic tripeptides, sclerotiotides A-K (1-11), together with three known compounds,
JBIR-15 (12), aspochracin (13), and penicillic acid, were isolated from the ethyl acetate extract of the fermentation
broth of the halotolerant Aspergillus sclerotiorum PT06-1 in a hypersaline nutrient-rich medium. Their structures were
elucidated by spectroscopic analysis and chemical methods. Chemical transformations of 12 and 13 proved that
sclerotiotides D-K (4-11) were artifacts probably formed during the fermentation or subsequent isolation steps. All
13 cyclic tripeptides have been evaluated for their antimicrobial and cytotoxic effects. Only sclerotiotides A (1), B (2),
F (6), and I (9) and JBIR-15 (12) showed selective antifungal activity against Candida albicans with MIC values of 7.5,
3.8, 30, 6.7, and 30 µM, respectively.
Hypersaline environments might induce biosynthetic pathways
of halotolerant microbes to produce structurally unique compounds.1,2
In our previous studies, novel alkaloids were identified as secondary
metabolites of halotolerant microorganisms.3-5 Recently, two novel
cyclic hexapeptides, sclerotides A and B, with antibiotic activity
were obtained from the marine-derived halotolerant fungus As-
pergillus sclerotiorum PT06-1 in a hypersaline nutrient-limited
medium.6 In order to further explore the effects of high-salt stress
on the production of secondary metabolites of this strain, A.
sclerotiorum PT06-1 was cultured in a nutrient-rich medium with
10% salt concentration. The fermentation broth exhibited distinct
TLC and HPLC profiles from those in the nutrient-limited medium
(Figure 1). Chemical investigation resulted in the identification of
11 new aspochracin-type cyclic tripeptides, sclerotiotides A-K
(1-11), and three known compounds, JBIR-15 (12),7 aspochracin
(13),8 and penicillic acid.9 Chemical transformations of 12 and 13
proved that sclerotiotides D-K (4-11) were artifacts probably
formed during the fermentation or subsequent isolation steps. The
chemical diversity of these cyclic tripeptides was represented in
the unsaturated fatty acid side chain, the constitution of amino acids,
and N-methyl substitution in the amino acid moieties. These
compounds did not show cytotoxicity against HL-60 and A549 cell
lines, but selectively inhibited the growth of Candida albicans with
MIC values of 7.5, 3.8, 30, 6.7, and 30 µM for sclerotiotides A
(1), B (2), F (6), and I (9) and JBIR-15 (12), respectively. In
addition, the 13C NMR data of aspochracin (13) are reported here
for the first time.
Sclerotiotide A (1), a pale yellow powder, had a molecular
formula of C22H34N4O4 from the [M + H]+ ion peak at m/z
419.2664 in the HRESIMS spectrum. A UV absorption at λmax 296
nm revealed the presence of a conjugated substructure. The 1H and
13C NMR spectra (Tables 1 and 2) of 1 showed four amide
carbonyls and one N-methyl, indicating its peptide nature. Three
characteristic R-methine signals at δH/C 4.77/55.9, 4.59/54.2, and
4.48/53.0 further supported a tripeptide structure. Except for the
lack of an N-methyl group, the 1D NMR spectrum of 1 was similar
to that of aspochracin (13),8 indicating 1 as an analogue of 13.
The HMBC correlations (Figure 2) between N-Me (δ 2.96) and
C-2 and C-4 indicated that the N-methyl was still present at the
alanine residue, while the N-methyl group in the valine residue was
absent. The absolute configurations of the amino acid residues of
1 were determined by Marfey’s method.10 HPLC analyses of
derivatives of the hydrolysates with authentic samples (co-injection)
revealed that the amino acids were L-NMe-Ala, L-Val, and L-Orn
(Figure S31). Thus, sclerotiotide A (1) was thus elucidated as
(2E,4E,6E)-cyclo-[(NMe-L-Ala)-L-Val-(NR-octa-2,4,6-trienoyl-L-
Orn)].
The molecular formula of sclerotiotide B (2) was determined as
C21H34N4O4 on the basis of the molecular ion peak [M + H]+ at
m/z 407.2646 in the HRESIMS spectrum. Except for the fatty acid
1
side chain, the H NMR spectrum of 2 was similar to that of the
known aspochracin (13).8 There are only two double bonds in the
side chain of 2 and both are E on the basis of the large coupling
constants (J ) 15.3, 15.0 Hz). Amino acid analysis disclosed
L-NMe-Ala, L-NMe-Val, and L-Orn (Figures S29 and S30).10
Sclerotiotide B (2) was thus elucidated as (2E,4E)-cyclo-[(NMe-
L-Ala)-(NMe-L-Val)-(NR-hexa-2,4-dienoyl-L-Orn)].
The molecular formula of sclerotiotide C (3) was deduced as
C24H38N4O4 on the basis of the HRESIMS data, corresponding to
an extra CH2 compared to 13. However, the chemical shifts of the
ornithine unit in 13 were not duplicated in the spectra of 3. These
data implied that the ornithine unit in 13 was replaced by a lysine
unit in 3. Furthermore, the acidic hydrolysis experiment established
the presence of L-Lys, L-NMe-Ala, and L-NMe-Val (Figure S32).10
Therefore, sclerotiotide C (3) was identified as (2E,4E,6E)-cyclo-
[(NMe-L-Ala)-(NMe-L-Val)-(NR-octa-2,4,6-trienoyl-L-Lys)].
Sclerotiotides D (4) and E (5) were assigned the molecular
formulas C22H34N4O4 and C23H36N4O4, respectively, on the basis
of the HRESIMS data, indicating 4 and 5 as isomers of 12 and 13.
The NMR differences between 4 and 12 and between 5 and 13
were in the terminal double-bond region of the side chain, indicating
3
that they were pairs of geometric isomers. The smaller J(H6′-H7′)
values of 11.4 Hz in 4 and 10.8 Hz in 5 indicated that they were
the 6′-Z isomers of 12 and 13, respectively. The deductions were
confirmed by photoisomerization of 12 and 13 to 4 and 5,
respectively (Figures 3 and S34). Accordingly, sclerotiotides D (4)
and E (5) were determined as (2E,4E,6Z)-cyclo-[L-Ala-(NMe-L-
Val)-(NR-octa-2,4,6-trienoyl-L-Orn)] and (2E,4E,6Z)-cyclo-[(NMe-
L-Ala)-(NMe-L-Val)-(NR-octa-2,4,6-trienoyl-L-Orn)], respectively.
The HRESIMS peak of sclerotiotide F (6) at m/z 421.2455 [M
+ H]+ corresponded to the molecular formula C21H32N4O5. The
differences in the NMR data from 13 occurred in the fatty acid
side chain. An aldehyde signal at δH/C 9.62 (d, 8.2)/194.6 was
present instead of a propenyl group in 13, suggesting that the side
chain of 6 was (2E,4E)-6-oxohexa-2,4-dienoic acid. The molecular
formula of sclerotiotide G (7) was deduced as C23H36N4O6 from
* To whom correspondence should be addressed. Tel: 0086-532-
82031268. Fax: 0086-532-82031268. E-mail: weimingzhu@ouc.edu.cn.
† Ocean University of China.
‡ Institute of Tropical Biological Sciences and Biotechnology.
10.1021/np100198h  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/26/2010

1134 Journal of Natural Products, 2010, Vol. 73, No. 6
Zheng et al.
Figure 1. TLC and HPLC-UV profiles of secondary metabolites of A. sclerotiorum PT06-1 in hypersaline nutrient-limited medium (A) and
hypersaline nutrient-rich medium (B).
the HRESIMS at m/z 465.2713 [M + H]+. The UV absorptions at
λmax 216 and 275 nm were very similar to those for 6, and the
NMR spectra of 7 were almost the same as those of 6 except for
the absence of the aldehyde signal in the side chain. Instead, an
R,ꢀ-unsaturated carbonyl signal at δC 201.9 and a -CH(OH)-CH3
spin system were displayed. When exposed to the air for 10 days,
13 produced 6 and 7 (Figures 3 and S34). The structures of
sclerotiotides F (6) and G (7) were thus determined as (2E,4E)-
cyclo-[(NMe-L-Ala)-(NMe-L-Val)-(NR-6-oxohexa-2,4-dienoyl-L-
Orn)] and (2E,4E)-cyclo-[(NMe-L-Ala)-(NMe-L-Val)-(NR-7-hydroxy-
6-oxoocta-2,4-dienoyl-L-Orn)], respectively.
Sclerotiotides H-K (8-11) were four isomers with the same
molecular formula, C23H38N4O6, and the NMR data closely related
to those of 7. The only difference of the NMR data from those of
7 was a hydroxymethine instead of the carbonyl in the side chain.
It is interesting that the NMR data of 8 and 9 were identical to
those of 10 and 11, respectively, suggesting that 8 and 10, and 9
and 11, were enantiotopic in the fatty acid moiety. The small
3J(H6′,H7′) of 8-11 (Table 1) indicated that all four compounds
displayed gauche-conformations in the side chain.11 The downfield
shift of CH3-8′ (δH/C 1.02/19.2) in 8 and 10 indicated the threo-
configuration, whereas the upfield shift of CH3-8′ (δH/C 0.95/18.2)
in 9 and 11 indicated the erythro-configuration due to the steric
hindrance between the methyl and the 5-amino-5-oxo-pentadienyl
group.12,13 In addition, 8-11 were produced by air oxidation of
13 (Figures 3 and S34). The structures of sclerotiotides H (8) and
J (10), and I (9) and K (11), were therefore determined as threo-
and erythro-(2E,4E)-cyclo-[(NMe-L-Ala)-(NMe-L-Val)-(NR-6,7-di-
hydroxyocta-2,4-dienoyl-L-Orn)], respectively.
using the SRB method.16 The antimicrobial activities against
Escherichia coli, Staphylococcus aureus, and Candida albicans
were also evaluated by an agar dilution method.17 Sclerotiotides A
(1), B (2), F (6), and I (9) and JBIR-15 (12) showed selective
antifungal activities against C. albicans with MIC values of 7.5,
3.8, 30, 6.7, and 30 µM, respectively, while no cytotoxicity nor
antibacterial activities were observed.
Sclerotiotides A-K (1-11) belong to aspochracin-type cyclic
tripeptides. So far, only three of these compounds have been
reported in the literature.7,8 It was proved in this paper that 4 and
5 could be formed from 12 and 13, respectively, via a radical
reaction14 initiated by direct photoisomerization, while 6-11 could
result from the air oxidation of 13 (Figure 3). Therefore, scleroti-
otides D-K (4-11) were artifacts probably formed during the
fermentation or subsequent isolation steps. It was also proved that
the nutrients, especially nitrogen and carbon sources in a culture
medium, affect the microbial secondary metabolites. Cyclic trip-
eptides were synthesized in the nutrient-rich medium, while cyclic
hexapeptides were synthesized in the nutrient-limited medium by
A. sclerotiorum PT06-1.
Experimental Section
General Experimental Procedures. Optical rotations were obtained
on a JASCO P-1020 digital polarimeter. UV spectra were recorded on
a Beckman DU 640 spectrophotometer. 1H and 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
δ-values. ESIMS 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 (10-40 µm) and
over silica gel (200-300 mesh, Qingdao Marine Chemical Factory)
Compounds 1-13 were tested for cytotoxic effects on the HL-
60 cell line using the MTT method15 and on the A549 cell line

Cyclic Tripeptides from Aspergillus sclerotiorum
Journal of Natural Products, 2010, Vol. 73, No. 6 1135

1136 Journal of Natural Products, 2010, Vol. 73, No. 6
Zheng et al.
NMR data, see Table 1; HRESIMS m/z 407.2646 [M + H]+ (calcd for
C21H35N4O4, 407.2658).
Sclerotiotide C (3): pale yellow, amorphous powder; [R]25 -57
D
1
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 206 (4.0), 296 (4.4) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 447.2957 [M
+ H]+ (calcd for C24H39N4O4, 447.2971).
Sclerotiotide D (4): pale yellow, amorphous powder; [R]25 -62
D
1
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (3.9), 296 (4.3) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 419.2650 [M
+ H]+ (calcd for C22H35N4O4 419.2658).
Figure 2. Selected two-dimensional NMR correlations for 1.
Sclerotiotide E (5): pale yellow, amorphous powder; [R]25 -84
and Sephadex LH-20 (Amersham Biosciences), respectively. Vacuum-
liquid chromatography (VLC) was carried out over silica gel H
(Qingdao Marine Chemical Factory). Semiprepartive HPLC was
performed using an ODS column [Shin-pak ODS (H), 20 × 250 mm,
5 µm, 4 mL/min].
Fungal Material. A. sclerotiorum PT06-1 was isolated from salt
sediments from the Putian Sea Salt Field, Fujian, China. It was identified
according to its morphological characteristics and 18S rRNA se-
quences.6 The voucher specimen is deposited in Dr. Zhu’s laboratory
at -80 °C. The producing strain was prepared on potato dextrose agar
slants at 10% salt concentration and stored at 4 °C.
Fermentation and Extraction. A. sclerotiorum PT06-1 was incu-
bated on a rotary shaker (160 rpm) at 28 °C for 16 days in 200 × 500
mL conical flasks containing liquid medium (150 mL/flask) composed
of maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10
g/L), glucose (10 g/L), yeast extract (3 g/L), corn steep liquor (1 g/L),
NaCl (80 g/L), MgSO4 (5 g/L), KH2PO4 (5 g/L), NH4Cl (5 g/L), KCl
(5 g/L), and tap water after adjusting its pH to 7.0. The fermented
whole broth (30 L) was filtered through cheesecloth to separate the
supernatant from the mycelia. The former was concentrated in Vacuo
to about a quarter of the original volume and then extracted three times
with EtOAc, while the latter was extracted three times with acetone.
The acetone solution was evaporated under reduced pressure to afford
an aqueous solution, which was then extracted three times with EtOAc.
Both EtOAc solutions were combined and concentrated in Vacuo to
give an extract (50.2 g).
D
1
(c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 (3.9), 296 (4.4) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 433.2802 [M
+ H]+ (calcd for C23H37N4O4, 433.2815).
Sclerotiotide F (6): pale yellow, amorphous powder; [R]25D -67 (c
0.5, MeOH); UV (MeOH) λmax (log ε) 206 (3.9), 270 (4.3) nm; 1H and
13C NMR data, see Tables 1 and 2; HRESIMS m/z 421.2455 [M +
H]+ (calcd for C21H33N4O5, 421.2451).
Sclerotiotide G (7): pale yellow, amorphous powder; [R]25 -59
D
1
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 216 (4.1), 275 (4.3) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 465.2713 [M
+ H]+ (calcd for C23H37N4O6, 465.2713).
Sclerotiotide H (8): pale yellow, amorphous powder; [R]25 -34
D
1
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 209 (4.0), 258 (4.2) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 467.2866 [M
+ H]+ (calcd for C23H39N4O6, 467.2870).
Sclerotiotides I (9): pale yellow, amorphous powder; [R]25 -48
D
1
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 209 (4.0), 258 (4.2) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 467.2850 [M
+ H]+ (calcd for C23H39N4O6, 467.2870).
Sclerotiotide J (10): pale yellow, amorphous powder; [R]25 -61
D
1
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 209 (4.0), 258 (4.2) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 467.2871 [M
+ H]+ (calcd for C23H39N4O6, 467.2870).
Sclerotiotide K (11): pale yellow, amorphous powder; [R]25 -42
D
1
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 209 (4.0), 258 (4.2) nm; H
Purification. The extract (50.2 g) was subjected to vacuum-liquid
chromatography on a silica gel column using step gradient elution with
MeOH-CHCl3 (0-100%). The collected materials were combined into
six fractions based on TLC properties. Fraction 3 from the 50:1
CHCl3-MeOH eluents was further separated on a Sephadex LH-20
column to give penicillic acid (402 mg). Fractions 4 and 5 were
separated by ODS column chromatography (H2O-MeOH gradient
mixtures) into five subfractions, respectively. Subfraction 4-2 (206 mg),
eluted with H2O-MeOH (3:2), was separated by HPLC (35% MeOH)
to yield compounds 6 (10 mg, tR 15 min) and 7 (3 mg, tR 16 min) and
by HPLC (30% MeOH) to give 8 (8 mg, tR 14 min), 9 (6 mg, tR 16
min), 10 (8 mg, tR 17 min), and 11 (13 mg, tR 20 min). Further
separation of Fr. 4.3 (3.2 g) and Fr. 5.3 (1.8 g) by CC (SiO2; petroleum
ether-AcOEt, 6:4) both afforded four subfractions. Fr. 4.3.3 (63 mg)
was finally separated by HPLC (54% MeOH) to yield 4 (8 mg, tR 14
min) and 12 (9 mg, tR 15 min). Fr. 5.3.2 (447 mg) was separated by
the same method to yield 5 (12 mg, tR 18 min) and 13 (40 mg, tR 19
min). Subfractions 4.4 and 5.4, eluted with H2O-MeOH (1:4), were
combined and purified by Sephadex LH-20 with CHCl3-MeOH (1:1)
and then HPLC with 60% aqueous MeOH to give 2 (4 mg, tR 8 min),
1 (12 mg, tR 13 min), and 3 (4 mg, tR 18 min). All compounds were
stored under nitrogen gas to avoid oxidation, and the vials were wrapped
with foil to prevent exposure to light.
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 467.2879 [M
+ H]+ (calcd for C23H39N4O6, 467.2870).
Determination of the Absolute Configurations of Amino Acids
by Marfey’s Method.10 Compounds 1-3, 12, and 13 (each 1 mg)
were hydrolyzed in HCl (6 M; 1 mL) for 20 h at 110 °C. The solutions
were then evaporated to dryness and redissolved in H2O (250 µL). A
1% (w/v) solution (100 µL) of L-FDAA (1-fluoro-2,4-dinitrophenyl-
5-L-alanine-amide) in acetone was added to an aliquot (50 µL) of the
acid hydrolysate solution. After addition of NaHCO3 solution (1 M;
20 µL) the mixture was incubated at 45 °C for 1 h. The reaction was
quenched by the addition of HCl (2 M, 10 µL). Analyses of the FDAA-
derivatized hydrolysates of compounds 2, 12, and 13 and standard
FDAA-derivatized amino acids were carried out by HPLC (Waters
600E; solvents: A, water + 0.2% TFA; B, MeCN; linear gradient: 0
min 25% B, 40 min 60% B, 45 min 100% B; 30 °C; 1 mL/min; UV
detection at λ 340 nm). Retention times of the amino acid derivatives
were as follows: L/D-Orn, tR 26.2/24.0 min; L/D-Ala, tR 16.4/19.2 min;
L/D-NMe-Ala, tR 18.1/17.3 min; L/D-NMe-Val, tR 26.3/28.8 min. Due
to the poor discrimination between L-Orn and L-NMe-Val, they were
eluted with the isocratic eluent (40% B), and retention times (min)
were 12.4 and 13.1, respectively. The derivatized hydrolysates of 2
and 13 showed peaks designated as L-Orn, L-NMe-Ala, and L-NMe-
Val. The hydrolysates of 12 consisted of L-Orn, L-Ala, and L-NMe-
Val. Analyses of the FDAA-derivatized hydrolysates of compounds 1
and 3 were also carried out by HPLC (Shimadzu SCL-10AVP) using
the above-mentioned gradient elution and detection method. Retention
times of the amino acids derivatives were as follows: L/D-Orn, tR 28.9/
27.1 min; L/D-Lys, tR 30.4/32.1 min; L/D-Val, tR 25.3/29.6 min; L/D-
Sclerotiotide A (1): pale yellow, amorphous powder; [R]25 -44
D
1
(c 0.5, MeOH); UV (MeOH) λmax (log ε) 206 (3.9), 296 (4.3) nm; H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 419.2664 [M
+ H]+ (calcd for C22H35N4O4, 419.2658).
Sclerotiotide B (2): pale yellow, amorphous powder; [R]25 -67
D
1
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 205 (3.9), 259 (4.2) nm; H
Figure 3. Photoisomerizations of compounds 12 and 13 and the oxidation of 13.

Cyclic Tripeptides from Aspergillus sclerotiorum
Journal of Natural Products, 2010, Vol. 73, No. 6 1137
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Acknowledgment. This work was supported by grants from National
Basic Research Program of China (No. 2010CB833800), from the
National Natural Science Foundation of China (Nos. 30670219 and
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Supporting Information Available: Bioassay protocols used, NMR
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of 12 and 13 and oxidation of 13. This material is available free of
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