Cycloaspeptides F and G, cyclic pentapeptides from a Cordyceps-colonizing isolate of Isaria farinosa

Article abstract of DOI:10.1021/np900205m

Cycloaspeptides F (1) and G (2), two new cyclic pentapeptides, and the known cycloaspeptides A (3), C (4), and bisdethiodi(methylthio)hyalodendrin (5) have been isolated from the crude extract of the fungus Isaria farinosa that colonizes Cordyceps sinensi

Full text of DOI:10.1021/np900205m

1364
J. Nat. Prod. 2009, 72, 1364–1367
Cycloaspeptides F and G, Cyclic Pentapeptides from a Cordyceps-Colonizing Isolate of Isaria
farinosa
Yonggang Zhang,†,‡ Shunchun Liu,† Hongwei Liu,*,† Xingzhong Liu,† and Yongsheng Che*,†
Key Laboratory of Systematic Mycology & Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s
Republic of China, and Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
ReceiVed April 2, 2009
Cycloaspeptides F (1) and G (2), two new cyclic pentapeptides, and the known cycloaspeptides A (3), C (4), and
bisdethiodi(methylthio)hyalodendrin (5) have been isolated from the crude extract of the fungus Isaria farinosa that
colonizes Cordyceps sinensis. The structures of 1 and 2 were elucidated primarily by NMR and MS methods. The
absolute configuration of 1 was assigned using Marfey’s method on its acid hydrolysate. Compounds 1 and 2 showed
cytotoxic effects against HeLa and MCF7 cell lines, displaying the same magnitude of activity toward the MCF7 cells
as the positive control 5-fluorouracil.
Cordyceps sinensis (Berk.) Sacc. (Anamorph: Hirsutella sinen-
sis)1 is known as Chinese caterpillar fungus. The combination of
the fungus and the dead caterpillars of the moth Hepilus spp.,
“DongChongXiaCao” (winter-worm, summer-grass), is a famous
fungal traditional Chinese medicine (FTCM) that has been widely
used as a tonic and/or medicine for hundreds of years in the Orient.
Cordyceps is a unique black, blade-shaped fungus found primarily
at high altitude on the Qinghai-Tibetan plateau and endophytically
parasitizes dead caterpillars of the moth Hepilus spp. Chemical
studies of C. sinensis have shown that the species can produce
different bioactive compounds.2 However, due to its growing
popularity, the natural fungus has been overharvested to the extent
that it is an endangered species, and the scarcity of the natural
material has limited more systematic chemical investigations of the
fungus as a source of bioactive metabolites.
Applications of fungal ecology in the search for new bioactive
natural products have proven effective.3 On the basis of ecological
considerations, we initiated chemical investigations of the fungal
species that colonize the fruiting body of C. sinensis,4-6 which we
members of the anthranilic acid (ABA)-containing cyclic pentapep-
named Cordyceps-colonizing fungi because of their ill-defined
tides initially isolated from Aspergillus sp. NE-45,8,9 whereas
relationships with C. sinensis.7 Despite claimed medical benefits
bisdethiodi(methylthio)hyalodendrin (5) is a diketopiperazine me-
including antitumor activity of C. sinensi, whether these effects
tabolite first reported from an unidentified fungus NRRL 3888.10
Cycloaspeptide F (1) was obtained as a colorless powder. It was
assigned a molecular formula of C42H53N5O11 on the basis of its
were originated from its own metabolites or those produced by its
colonizing fungi remains to be clearly defined. Our long-term goal
is to explore the living strategy of the Cordyceps-colonizing fungi
HRESIMS (m/z 826.3634 [M + Na]+; ∆ -2.5 mmu). Analysis of
and understand their relationships with C. sinensis using bioactive
1
the H, 13C, and HMQC NMR spectroscopic data of 1 (Table 1)
revealed the presence of a sugar moiety, three amide N-H protons
(δH 7.95, 8.17, and 12.14, respectively), five methyl groups
secondary metabolites as probes. During the course of our continu-
ing search for new bioactive natural products from this unique
source, the fungus Isaria farinosa (XJC04-CT-303) isolated from
(including two N-methyls), three methylenes, five methines (four
a sample of C. sinensis collected in Linzhi, Tibet, People’s Republic
of which are heteroatom-bonded), three phenyl rings, and five
of China, was grown in a solid-substrate fermentation culture. Its
1
carboxylic carbons. These data accounted for all the H and 13C
NMR resonances for 1 and are consistent with the molecular
formula C42H53N5O11.
organic solvent extract displayed cytotoxicity against two human
tumor cell lines, HeLa and MCF7. Fractionation of the extract led
to the isolation of two new cyclic pentapeptides, which we named
Interpretation of the 1H, 13C, and 2D NMR data of 1, especially
cycloaspeptides F (1) and G (2), together with the known cy-
cloaspeptides A (3) and C (4)8,9 and bisdethiodi(methylthio)hyalo-
HMBC, revealed its peptidic nature and structural similarity to those
of 3 and 4. Five 13C NMR resonances, at δC 174.0, 170.5, 169.9,
and 169.1 (2), were characteristic of amide carbonyls with four
methine peaks at δH/δC 5.43/63.8, 4.68/49.0, 4.56/45.0, and 4.06/
69.2 being indicative of four amino acid R-CH groups. The 1H NMR
resonances for a cluster of five aromatic protons at δH 7.23-7.30
suggested the presence of a monosubstituted benzene ring, which
was connected to C-19 by HMBC correlations from H2-19 to C-20,
C-21, and C-25. Correlations from H-18 to C-17 and C-19, from
H-18 to C-26, and from H3-26 to C-18 established the N-
methylphenylalanine (N-Me-Phe) unit, like that found in cy-
cloaspeptide A (3). Analysis of the1H-1H coupling patterns for
dendrin (5).10,11 Details of the isolation, structure elucidation, and
biological activities of these compounds are reported herein.
The known metabolites isolated from the crude extract were
identified as cycloaspeptides A (3) and C (4) and bisdethiodi(m-
ethylthio)hyalodendrin (5) by comparison of their NMR and MS
data with those reported.8-11 Cycloaspeptides A (3) and C (4) are
* To whom correspondence should be addressed. Tel/Fax: +86 10 82618785.
E-mail: (Y.C.) cheys@im.ac.cn; (H.L.) liuhongwei60@hotmail.com.
† Institute of Microbiology.
‡ Graduate School of Chinese Academy of Sciences.
10.1021/np900205m CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/02/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 7 1365
Table 1. NMR Spectroscopic Data of Cycloaspeptide F (1) in
Acetone-d6
a
residue position
δH (J in Hz)
δCb, mult. HMBC (H f C#)
N-Me-Tyr
1
169.1, qC
2
4.06, m
69.2, CH3
3
4
3.35, m; 3.45, m
32.8, CH2 2, 4, 5, 9
133.4, qC
5
6
7
7.14, d (8.5)
7.05, d (8.5)
131.0, CH 3, 4, 6, 7, 9
117.5, CH 4, 5, 7, 8
157.6, qC
8
9
7.05, d (8.5)
7.14, d (8.5)
2.78, s
117.5, CH 4, 6, 7, 9
131.0, CH 3, 4, 5, 7, 8
39.2, CH3 2, 11
170.5, qC
49.0, CH 11, 14, 17
42.2, CH2 11
25.4, CH
22.7, CH3 13, 14, 16
23.5, CH3 13, 14, 15
10
11
12
13
14
15
16
NH
17
18
19
Leu
4.68, t (8.0)
1.35, m; 1.76, m
1.65, m
0.97, d (6.5)
0.97, d (6.5)
8.17, d (8.0)
Figure 1. Selected NOESY correlations for cycloaspeptide F (1).
2.83) showed an HMBC correlation to the carboxylic carbon of
Ala (C-27; δC 174.0), indicating that it is acylated by Ala, and this
connection was also supported by NOESY correlation of H-18 with
H-28. An HMBC cross-peak from the N-methyl proton of N-Me-
Tyr (H3-10; δH 2.78) to the carboxyl carbon of Leu (C-11; δC 170.5)
led to the connection of these two residues, which was supported
by NOESY correlations of H3-10 with H-2 and H-12. The R-proton
of Leu (H-12; δH 4.68) was correlated to the carboxylic carbon of
N-Me-Phe (C-17; δC 169.1). These correlations enabled assignment
of the partial sequence Ala f N-Me-Phe f Leu f N-Me-Tyr.
Although no further sequence-relevant HMBC correlations were
observed, the only remaining ABA unit must be placed between
N-Me-Tyr and Ala, thereby completing the sequence of 1 as shown.
The relative configurations between the R-protons of Ala and
N-Me-Phe, and Leu and N-Me-Tyr glycoside were assigned by
analysis of NOESY correlations (Figure 1). In the NOESY spectrum
of 1, the R-protons of N-Me-Phe (H-18; δH 5.43) and Ala (H-28;
δH 4.56) were correlated, indicating that these protons have the
same orientations with respect to the macrocycle, whereas correla-
tions of the ꢀ-protons of Ala (H3-29; δH 0.37) with H-24 (δH 7.29)
and H-25 (δH 7.30) of N-Me-Phe were used to place them on the
opposite face. The N-methyl signal of N-Me-Tyr glycoside (H3-
10; δH 2.78) was correlated to the R-protons of Leu (H-2; δH 4.06)
and itself (H-12; δH 4.68), indicating that these protons are all
cofacial, and this assignment was supported by correlations from
H3-15 (δH 0.97) to H-8 (δH 7.05) and H-9 (δH 7.14).
N-Me-Phe
169.1, qC
63.8, CH 17, 19, 26
5.43, dd (12, 3.0)
2.97, dd (14, 3.0); 34.8, CH2 18, 20, 21, 25
3.48, dd (14, 12)
20
21
22
23
24
25
26
27
28
29
NH
30
31
32
33
34
35
36
NH
1′
139.5, qC
7.30, m
7.29, m
7.23, t (7.0)
7.29, m
7.30, m
2.83, s
130.5, CH 19, 20, 22, 23, 25
129.6, CH 20, 21, 23, 24
127.5, CH 21, 22, 24, 25
129.6, CH 22, 23, 25, 20
130.5, CH 19, 20, 21, 23, 24
30.6, CH3 18, 27
174.0, qC
45.0, CH 29
16.4, CH3 27, 28
Ala
4.56, q (6.5)
0.37, d (6.5)
7.95, br s
ABA
169.9, qC
117.0, qC
7.89, d (8.5)
7.01, t (8.5)
7.49, t (8.5)
8.89, d (8.5)
128.9, CH 30, 34, 36
122.5, CH 31, 32, 34, 35
134.0, CH 32, 36
120.3, CH 31, 33
142.4, qC
12.14, s
4.94, d (7.0)
3.47, m
3.51, m
3.49, m
3.51, m
3.88, dd (12,2.0);
3.72, dd (12, 5.0)
Glu
102.2, CH
7
2′
74.6, CH 1′, 3′
77.8, CH 2′, 4′
71.2, CH 2′, 5′
77.6, CH 4′, 6′
62.5, CH2 4′
3′
4′
5′
6′
Marfey’s method12 was applied to assign the absolute configu-
ration of the Ala and Leu residues resulting from acid hydrolysis
of cycloaspeptide F (1). HPLC analysis of the 1-fluoro-2,4-
dinitrophenyl-5-L-alanine amide (FDAA) derivatives of the acid
hydrolysate of 1 gave the same retention times as those prepared
from samples of authentic L-Ala and L-Leu. Therefore, the Ala and
Leu residues in 1 were both assigned the L-configuration. Consider-
ing the relative configurations established between Ala and N-Me-
Phe, and Leu and N-Me-Tyr glycoside by NOESY data, N-Me-
Phe and N-Me-Tyr were both deduced to have the L-configuration.
The Glc unit in 1 was connected to the N-Me-Tyr via a ꢀ-linkage
on the basis of the downfield chemical shift of the anomeric carbon
(C-1′; δC 102.2), as well as the 1H-1H coupling constant (7.0 Hz)
observed for the anomeric proton (H-1′). Acid hydrolysis was
performed to separate the Glc unit from the N-Me-Tyr glycoside
portion of 1. Treatment of the liberated Glc with L-cysteine methyl
ester, followed by trimethylsilylation, afforded a derivative matching
that of the D-Glc by GC-MS analysis in comparison with the D-
and L-standards.13,14
a Recorded at 500 MHz. b Recorded at 150 MHz.
the aromatic protons H-32 (d; J ) 8.5 Hz), H-33 (t; J ) 8.5 Hz),
H-34 (t; J ) 8.5 Hz), and H-35 (d; J ) 8.5 Hz) revealed a
substructure for an ortho-substituted aryl ring, which was supported
by relevant H-1H COSY and HMBC correlations. An HMBC
1
cross-peak from H-32 to C-30 led to the connection of C-30 to
C-31, completing the anthranilic acid (ABA) unit. Similarly,
interpretation of the 1H-1H COSY and HMBC data for 1
established the structures of leucine (Leu), alanine (Ala), and
N-methyltyrosine (N-Me-Tyr) residues. Due to the lack of HMBC
correlation from H-1 and/or H2-3 to C-1, the C-1 amide carbon
was assigned to N-Me-Tyr by default. The sugar moiety in 1 was
determined as glucose (Glc) on the basis of its H and 13C NMR
1
data (Table 1). A key HMBC correlation from the anomeric proton
of Glc (H-1′; δH 4.94) to the oxygenated aromatic carbon of N-Me-
Tyr (C-7; δC 157.6) connected the Glc unit to N-Me-Tyr at C-7.
Upon extensive analysis of these data, cycloaspeptide F (1) was
assigned as a cyclic pentapeptide containing one equivalent each
of Leu, Ala, N-Me-Phe, ABA, and N-Me-Tyr glycoside.
The sequence of 1 was determined by HMBC correlations of
the N-methyl groups and various R- and ꢀ-protons with neighboring
carboxylic carbons and was supported by relevant NOESY cor-
relations (Figure 1). The N-methyl proton of N-Me-Phe (H3-26; δH
The molecular formula of cycloaspeptide G (2) was determined
to be C36H43N5O7 on the basis of its HRESIMS (m/z 680.3047 [M
+ Na]+; ∆ -0.8 mmu). Analysis of the 1H and 13C NMR
spectroscopic data of 2 (Table 2) revealed structural features similar
as those found in 1, except that the signals for the Glc moiety were
absent, and those for the N-Me-Phe unit were replaced by an N-Me-

1366 Journal of Natural Products, 2009, Vol. 72, No. 7
Notes
Table 2. NMR Spectroscopic Data of Cycloaspeptide G (2) in
Acetone-d6
several Penicillia and a Tricothecium strain.15 Cycloaspeptides F
(1) and G (2) are new members of this class of metabolites, and
the presence of a Glc unit in 1 is unprecedented. Cycloaspeptide G
(2) is closely related to C (3), but differs in having an N-Me-Tyr
unit rather than an N-Me-Phe moiety.
HMBC
a
residue
position
δH (J in Hz)
δCb, mult. (H f C#)
N-Me-Tyr1
1
169.1, qC
The species I. farinosa (formerly classified as Paecilomyces
farinosus) is a well-known entomopathogenic fungus, which was
regarded as a biocontrol agent against Rhizoecus kondonis (Ho-
moptera), Dendrolimus pini (Lepidoptera), Lymantria dispar (Lepi-
doptera), Hyloicus pinastri (Lepidoptera), and Bupalus piniarius
(Lepidoptera), as well as various pest insects, plant diseases, and
nematodes.16-18 A variety of metabolites have been reported from
different strains of the fungus as antioxidative, antitumor, and
antifungal agents.19-22 In this work, the discovery of new ABA-
containing cycloaspeptides from a Cordyceps-colonizing strain of
I. farinosa further expanded structural diversity of the secondary
metabolites produced by this fungal species.
2
3
4
5
6
7
8
9
4.02, m
3.35, m; 3.36, m
70.1, CH
32.7, CH2 5, 9
129.7, qC
131.1, CH 3, 6, 7, 9
116.1, CH
157.6, qC
7.03, d (8.5)
6.81, d (8.5)
6.81, d (8.5)
7.03, d (8.5)
2.78, s
116.1, CH
131.1, CH 3, 5, 7, 8
39.1, CH3 2, 11
170.6, qC
54.0, CH
42.2, CH2 11
25.3, CH
10
11
12
13
14
15
16
NH
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
NH
Leu
4.66, t (8.0)
1.35, m; 1.82, m
1.36, m
0.96, d (7.0)
0.96, d (7.0)
8.17, d (8.0)
22.6, CH3 13, 14, 16
23.4, CH3 13, 14, 15
Experimental Section
N-Me-Tyr2
169.2, qC
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a
Shimadzu Biospec-1601 spectrophotometer. IR data were recorded
5.34, dd (12, 3.0)
2.97, m; 3.30, m
64.0, CH 17
33.9, CH2 21, 25
130.3, qC
131.4, CH 19, 25
116.3, CH 20, 24
157.2, qC
116.3, CH 20, 22
131.4, CH 19, 21
30.5, CH3 18, 27
174.1, qC
1
using a Nicolet Magna-IR 750 spectrophotometer. H and 13C NMR
7.09, d (8.5)
6.77, d (8.5)
data were acquired with Bruker Avance-500 and -600 spectrometers
using solvent signals (acetone-d6; δH 2.05/δC 29.8, 206.0) as references.
The HMQC and HMBC experiments were optimized for 145.0 and
8.0 Hz, respectively. ESIMS data were recorded on a Bruker Esquire
3000plus spectrometer, and HRESIMS data were obtained using a Bruker
APEX III 7.0 T spectrometer. GC-MS (70 eV) data were acquired on
a Shimadzu GCMS-QP2010 instrument using a DB-5 ms capillary
column (30 m × 0.25 mm, i.d.; J & W Scientific) with He as carrier
gas and a detector and an injection temperature of 250 °C. The initial
temperature was maintained at 50 °C for 2 min and then raised to 250
°C at the rate of 15 °C/min.
Fungal Material. The culture of I. farinosa was isolated by Dr.
Mu Wang from a sample of C. sinensis (Berk.) Sacc. collected in Linzhi,
Tibet, in June 2004. The fungus was identified by one of the authors
(B.S.) based on sequence (Genbank Accession No. AB233337.1)
analysis of the ITS region of the rDNA and assigned the Accession
No. XJC04-CT-303 in X.L’s culture collection at the Institute of
Microbiology, Chinese Academy of Sciences, Beijing. The fungal strain
was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10
days. The agar plugs were used to inoculate 250 mL Erlenmeyer flasks,
each containing 50 mL of media (0.4% glucose, 1% malt extract, and
0.4% yeast extract), and the final pH of the media was adjusted to 6.5
before sterilization. Flask cultures were incubated at 25 °C on a rotary
shaker at 170 rpm for five days. Fermentation was carried out in four
500 mL Fernbach flasks each containing 75 g of rice. Spore inoculum
was prepared by suspension in sterile, distilled H2O to give a final spore/
cell suspension of 1 × 106/mL. Distilled H2O (100 mL) was added to
each flask, and the contents were soaked overnight before autoclaving
at 15 lb/in.2 for 30 min. After cooling to room temperature, each flask
was inoculated with 5.0 mL of the spore inoculum and incubated at 25
°C for 40 days.
Extraction and Isolation. The fermented rice substrate was extracted
repeatedly with EtOAc (3 × 500 mL), and the organic solvent was
evaporated to dryness under vacuum to afford the crude extract (3.3
g). The extract was fractionated by Si gel vacuum liquid chromatog-
raphy (VLC) using n-hexanes-CH2Cl2-MeOH gradient elution. The
fraction (110 mg) eluted with CH2Cl2-MeOH (100:2) was fractionated
by Sephadex LH-20 column chromatography (CC) using CH3OH as
eluent. The subfraction (25 mg) was further separated by semiprepara-
tive reversed-phase HPLC (RP HPLC; Agilent Zorbax SB-C18 column;
5 µm; 9.4 × 250 mm; 20% CH3OH in H2O for 5 min, and followed by
20-100% CH3OH in H2O for 25 min; 2 mL/min) to afford cycloaspep-
tide A (3; 4.0 mg, tR 23.3 min) and bisdethiodi(methylthio)hyalodendrin
(5; 1.5 mg, tR 17.5 min). The fractions (135 mg) eluted with 100:4
CH2Cl2-CH3OH were combined and separated again by Sephadex LH-
20 CC eluting with CH3OH. Purification of the resulting subfractions
by RP HPLC (20% CH3OH in H2O for 5 min, followed by 20-100%
CH3OH in H2O for 40 min) afforded cycloaspeptides G (2; 1.5 mg, tR
36.6 min) and C (4; 2.5 mg, tR 38.0 min). The fractions (225 mg) eluted
with 100:6 and 100:8 CH2Cl2-CH3OH were combined and separated
6.77, d (8.5)
7.09, d (8.5)
2.83, s
Ala
4.59, q (7.0)
0.50, d (7.0)
45.2, CH
16.3, CH3 27, 28
171.8, qC
ABA
118.5, qC
7.91, d (8.5)
7.02, t (8.5)
7.49, t (8.5)
8.89, d (8.5)
128.9, CH 30, 34, 36
122.5, CH 35
134.0, CH 32, 36
120.3, CH 31, 33
142.1, qC
12.14, s
a Recorded at 500 MHz. b Recorded at 150 MHz.
Table 3. Cytotoxic Effects of Compounds 1-5
GI50 (µM)
compound
HeLa
MCF7
1
2
3
4
5
49.8
30.4
31.2
>63.4
>112.9
10.0
18.7
15.2
23.4
31.9
112.9
15.0
5-fluorouracil
Tyr in the spectra of 2. These observations were confirmed by
relevant H-1H COSY and HMBC correlations. Therefore, the
1
gross structure of cycloaspeptide G was established as shown. The
absolute configuration of 2 was deduced by analogy to 1.
Cycloaspeptides F (1) and G (2) and the known compounds
cycloaspeptides A (3) and C (4) and bisdethiodi(methylthio)hyalo-
dendrin (5) were evaluated for cytotoxic activity against two human
tumor cell lines, HeLa and MCF7 (Table 3). Cycloaspeptides F
(1) and G (2) showed inhibitory effects on the growth of MCF7
cells that are comparable to the positive control 5-fluorouracil, with
GI50 values of 18.7 and 15.2 µM, respectively (5-Fu showed a GI50
value of 15.0 µM). Compounds 1 and 2 also displayed modest
cytotoxic effects against the HeLa cells, with GI50 values of 49.8
and 30.4 µM, respectively (5-Fu showed a GI50 value of 10.0 µM).
Cycloaspeptides are a class of ABA-containing cyclic pentapep-
tides isolated from fungi. Examples include cycloaspeptides A-C
from Aspergillus sp. NE-45,8 cycloaspeptide D from the psychro-
tolerant fungus Penicillium ribeum,9 and cycloaspeptide E from

Notes
Journal of Natural Products, 2009, Vol. 72, No. 7 1367
again by Sephadex LH-20 CC using CH3OH as eluent. Further
purification of the subfractions by RP HPLC (40% CH3CN in H2O for
5 min, followed by 40-60% CH3CN in H2O for 25 min) afforded
cycloaspeptide F (1; 4.0 mg, tR 12.5 min).
changed to fresh DMEM. MTT (Sigma) was dissolved in serum-free
medium or PBS at 0.5 mg/mL and sonicated briefly. In the dark, 50
µL of MTT/medium was added into each well after the medium was
removed from wells and incubated at 37 °C for 3 h. Upon removal of
MTT/medium, 100 µL of DMSO was added to each well and shaken
at 60 rpm for 5 min to dissolve the precipitate. The assay plate was
read at 540 nm using a microplate reader.
Cycloaspeptide F (1): white powder; [R]D -18 (c 0.4, CH3OH); UV
(CH3OH) λmax (ε) 211 (100 100), 253 (17 600) nm; IR (neat) νmax 3323
1
(br), 2929, 2868, 1682, 1637, 1444, 1228, 1177, 1075 cm-1; H, 13C
NMR, and HMBC data see Table 1; NOESY correlations (acetone-d6,
600 MHz) H-18 T H-28; H3-10 T H-2, H-12; H3-15 T H-8, H-9;
Acknowledgment. We gratefully acknowledge financial support
from the Ministry of Science and Technology of China (2007AA021506
and 2009CB522302), the Chinese Academy of Sciences (KSCX2-YW-
G-013), and the National Natural Science Foundation of China
(30870057).
H3-29
T H-24, H-25; HRESIMS m/z 826.3634 (calcd for
C42H53N5O11Na, 826.3659).
Cycloaspeptide G (2): white powder; [R]D -16 (c 0.3, CH3OH);
λmax (ε) 211 (9000), 251 (12 500) nm; IR (neat) νmax 3303 (br), 2926,
2853, 1653, 1627, 1516, 1446, 1260, 1172, 1097 cm-1; 1H, 13C NMR,
and HMBC data see Table 2; NOESY correlations (acetone-d6, 600
MHz) H-18 T H-28; H3-10 T H-2, H-12; H3-15 T H-8, H-9; H3-29
T H-24, H-25; HRESIMS m/z 680.3047 (calcd for C36H43N5O7Na,
680.3055).
Supporting Information Available: 1H, 13C, and 2D NMR spectra
of cycloaspeptides F (1) and G (2). This material is available free of
charge via the Internet at http://pubs.acs.org.
Cycloaspeptide A (3): 1H, 13C NMR, and ESIMS data were consistent
with literature values.8
References and Notes
Cycloaspeptide C (4): 1H, 13C NMR, and ESIMS data were consistent
with literature values.8
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(8) Kobayashi, R.; Samejima, Y.; Nakajima, S.; Kawai, K.; Udagawa, S.
Chem. Pharm. Bull. 1987, 35, 1347–1352.
(9) Dalsgaard, P. W.; Larsen, T. O.; Frydenvang, K.; Christophersen, C.
J. Nat. Prod. 2004, 67, 878–881.
(10) DeVault, R. L.; Rosenbrook, W., Jr. J. Antibiot. 1973, 26, 532–534.
(11) Isaka, M.; Palasarn, S.; Rachtawee, P.; Vimuttipong, S.; Kongsaeree,
P. Org. Lett. 2005, 7, 2257–2260.
(12) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
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Prod. 2007, 70, 378–382.
(14) Haddad, M.; Miyamoto, T.; Laurens, V.; Lacaille-Dubois, M. A. J.
Nat. Prod. 2003, 66, 372–377.
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D. O.; Anzeveno, P. B.; Fields, S. C.; Gilbert, J. R.; Pearce, C. J.
Nat. Prod. 2006, 69, 1506–1510.
(16) Jackson, M. A.; Cliquet, S.; Iten, L. B. Biocontrol Sci. Technol. 2003,
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(17) Jackson, M. A.; McGuire, M. R.; Lacey, L. A.; Wraight, S. P. Mycol.
Res. 1997, 101, 35–41.
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Bisdethiodi(methylthio)hyalodendrin (5): 1H, 13C NMR, and ESIMS
data were consistent with literature values.10,11
Absolute Configuration of Ala and Leu in 1.12 Separate solutions
of cycloaspeptide F (1; 0.5 mg) and G (2; 0.5 mg) in 6 N HCl (1.0
mL) were heated at 155 °C for 1 h. Upon removal of excess HCl under
vacuum, the hydrolysate was placed in a 1 mL reaction vial and treated
with a 1% solution of FDAA (200 µL) in acetone, followed by 1.0 N
NaHCO3 (40 µL). The reaction mixtures were heated at 45 °C for 1.5 h,
cooled to room temperature, and then acidified with 2.0 N HCl (20
µL). In a similar fashion, standard L-Leu, D-Leu, L-Ala, and D-Ala were
derivatized separately. The derivatives of the acid hydrolysate and the
standard amino acids were subjected to RP HPLC analysis (Kromasil
C18 column; 10 µm, 4.6 × 250 mm; 2.0 mL/min) at 25 °C using the
following gradient program: solvent A, H2O (0.1% TFA); solvent B,
acetonitrile; linear gradient, 10-50% of B for 40 min with UV detection
at 340 nm. The retention times for the FDAA derivatives of L-Ala,
D-Ala, L-Leu, and D-Leu were 22.37, 23.94, 31.74, and 35.49 min,
respectively, whereas those for the FDAA derivatives of Ala and Leu
in the hydrolysate of 1 were 22.43 and 31.74 min, respectively.
Determination of D-Glc.13,14 A sample of 2.5 mg of cycloaspeptide
F (1) in 300 µL of acetone was added to 700 µL of 6 N HCl in a
hydrolysis tube and heated at 100 °C for 24 h. After evaporation of
excess CH3OH, 1.0 mg of L-cysteine methyl ester hydrochloride in
100 µL of pyridine was added, and the mixture was stirred at 60 °C
for 1 h. A 3:1 mixture of HMDS-TMCS (hexamethyldisilazane-tri-
methylchlorosilane) was then added (150 µL), and the solution was
stirred at 60 °C for another 30 min. The precipitate was centrifuged
off, and the supernatant was concentrated under a stream of N2. The
residue was partitioned between n-hexanes and H2O, and the hexane
layer was directly subjected to GC-MS analysis. The resulting Glc
derivative coeluted with a derivatized D-Glc standard (tR 19.04 min),
but not with a derivatized L-Glc standard (tR 19.25 min).
MTT Assay.4 In 96-well plates, each well was plated with 104 cells.
After cell attachment overnight, the medium was removed, and each
well was treated with 50 µL of medium containing 0.2% DMSO or
appropriate concentration of test compounds (10 mg/mL as stock
solution of a compound in DMSO and serial dilutions). Cells were
treated at 37 °C for 4 h in a humidified incubator at 5% CO2 first and
then were allowed to grow for another 48 h after the medium was
(20) Mori, Y.; Tsuboi, M.; Suzuki, M.; Fukushima, K.; Arai, T. J. Chem.
Soc., Chem. Commun. 1982, 94–96.
(21) Yamashita, Y.; Saitoh, Y.; Ando, K.; Takahashi, K.; Ohno, H.; Nakano,
H. J. Antibiot. 1990, 43, 1344–1346.
(22) Lang, G.; Blunt, J. W.; Cummings, N. J.; Cole, A. L.; Munro, M. H.
J. Nat. Prod. 2005, 68, 810–811.
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