Cyclic heptapeptides, cordyheptapeptides C-E, from the marine-derived fungus Acremonium persicinum SCSIO 115 and their cytotoxic activities

Article abstract of DOI:10.1021/np300152d

Three new cycloheptapeptides, cordyheptapeptides C-E (1-3), were isolated from the fermentation extract of the marine-derived fungus Acremonium persicinum SCSIO 115. Their planar structures were elucidated on the basis of extensive MS, as well as 1D and 2

Full text of DOI:10.1021/np300152d

Note
pubs.acs.org/jnp
Cyclic Heptapeptides, Cordyheptapeptides C−E, from the Marine-
Derived Fungus Acremonium persicinum SCSIO 115 and Their
Cytotoxic Activities
Ziming Chen,† Yongxiang Song,† Yuchan Chen,‡ Hongbo Huang,† Weimin Zhang,‡ and Jianhua Ju*,†
†CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, Guangdong Key Laboratory of Marine Materia Medica, RNAM
Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301, People's Republic of China
‡Guangdong Institute of Microbiology, 100 Central Xianlie Road, Guangzhou 510070, People's Republic of China
S
* Supporting Information
ABSTRACT: Three new cycloheptapeptides, cordyheptapep-
tides C−E (1−3), were isolated from the fermentation extract
of the marine-derived fungus Acremonium persicinum SCSIO
115. Their planar structures were elucidated on the basis of
extensive MS, as well as 1D and 2D (COSY, HMQC, and
HMBC) NMR spectroscopic data analyses. The absolute
configurations of the amino acid residues were determined by
single-crystal X-ray diffraction, Marfey’s method, and chiral-
phase HPLC analysis. Compounds 1 and 3 displayed
cytotoxicity against SF-268, MCF-7, and NCI-H460 tumor
cell lines with IC50 values ranging from 2.5 to 12.1 μM.
arine fungi have attracted increasing attention from
those in search of new pharmaceutically useful natural
M
compounds in the past decade. So far, more than one thousand
structurally unique and biologically active compounds have
been isolated from marine-derived fungi.1 Accordingly, marine
fungi have been considered as an emerging resource for drug
discovery.2 For instance, plinabulin (NPI-2358), a synthetic
analogue of halimide deriving from the marine fungus
Aspergillus sp. CNC-139, is now in phase II clinical trial for
the treatment of cancer.3 In our efforts to identify novel
structures and bioactive metabolites from South China Sea-
derived fungi, we screened the fermentation extracts of 180
fungi for cytotoxic activity and have reported that the fungus
Xylaria sp. SCSIO 156 produces new cytotoxic cytochalasins.4
Another fermentation extract of strain SCSIO 115, identified as
Acremonium persicinum, showed strong lethality against brine
shrimp (Artemia salina) and cytotoxicity toward a panel of
tumor cell lines. Chemical investigation of an A. persicinum
SCSIO 115 extract has led to the identification of three new
cycloheptapeptides, designated cordyheptapeptides C−E (1−
3). Here we report the fermentation, isolation, structure
elucidation, and cytotoxic activities of these compounds against
three cancer cell lines, SF-268, MCF-7, and NCI-H460.
signals for a heptapetide. Seven carbonyl resonances at δC
174.1, 172.3, 170.9, 170.5, 170.4, 168.4, and 168.2 together with
the seven α-amino acid carbon resonances between δC 69.2 and
47.6 in the 13C NMR spectrum indicated the presence of seven
1
amino acid residues. The H NMR spectrum of 1 displayed
seven methyl signals in the upfield region, three of which were
assigned as N-Me groups at δH 3.03, 2.61, and 2.91.
Comprehensive analysis of the 1D (1H and 13C, DEPT) and
2D (COSY, HMQC, and HMBC) NMR spectroscopic data
revealed that 1 was a heptapeptide containing N-MeTyr, Phe,
N-MeGly, Pro, N-MePhe, Leu, and Val residues (Table 1). The
The organic extract of the fungal culture was subjected to
column chromatography on silica gel, reversed-phase silica gel
(ODS), and Sephadex LH-20 guided by brine shrimp lethality
assays4 to yield compounds 1−3. Compound 1 was isolated as
colorless crystals. Its molecular formula, C48H63N7O8, was
established by HRESIMS, indicating 21 degrees of unsatura-
tion. The 1H and 13C NMR spectra of 1 exhibited characteristic
Received: March 2, 2012
Published: May 29, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Note
1
Table 1. Summary of H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for Compounds 1−3
a
b
a
1
2
3
position
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
δC
δH, mult. (J in Hz)
NMeTyr
NMeTyr1
NMeTyr1
d
CO
α
170.4, C
170.7, C
170.4, C
69.2, CH
32.4, CH2
3.40, dd (11.5, 3.5)
3.16, m
69.0, CH
32.4, CH2
3.40, m
3.13, m
2.72, m
69.3, CH
32.1, CH2
3.39, m
β
3.16, d (11.5)
2.70, m
2.72, m
e
1
127.0, C
126.7, C
127.0, C
130.0, CH
115.8, CH
155.7, C
40.5, CH3
Phe
2,6
3,5
4
129.8, CH
115.7, CH
6.22, d (8.1) 155.3, C
6.53, d (8.1)
129.7, CH
115.6, CH
155.3, C
40.3, CH3
Phe
6.21, d (8.4)
6.47, d (8.4)
6.21, d (6.5)
6.53, d (6.5)
NMe
Phe
CO
α
40.4, CH3
2.61, s
2.59, br s
2.56, s
d
170.5, C
170.2, C
49.8, CH
38.0, CH2
170.3, C
50.2, CH
38.1, CH2
50.1, CH
38.1, CH2
5.36, m
5.26, m
5.29, m
β
3.05, dd (12.3, 11.7)
2.82, dd (12.3, 3.0)
3.04, dd (12.5, 11.5)
2.74, dd (12.5, 3.0)
3.06, d (12.5)
2.71, d (12.5)
1
137.3, C
137.3, C
137.4, C
2, 6
3, 5
4
128.6, CH
130.1, CH
126.8, CH
7.31, br d (7.3)
7.40, m
130.1, CH
128.5, CH
7.29, m
130.1, CH
128.6, CH
126.7, CH
7.25, t (6.5)
7.33, t (6.5)
7.31, m
7.36, m
e
7.36, m
126.6, CH
7.30, m
NH
NMeGly
CO
α
8.61, d (9.5)
8.55, d (8.0)
8.59, d (10.0)
NMeGly
NMeGly
168.2, C
168.1, C
168.1, C
50.8, CH2
5.40, d (18.0)
3.33, d (18.0)
2.91, s
50.8, CH2
5.29, d (17.5)
3.28, d (17.5)
2.85, s
50.8, CH2
3.30, d (17.5)
5.34, d (17.0)
2.87, s
NMe
35.5, CH3
35.5, CH
Pro
35.8, CH3
Pro
Pro
CO
172.3, C
172.5, C
58.1, CH
31.4, CH2
22.0, CH2
48.4, CH2
172.6, C
57.7, CH
31.5, CH2
22.2, CH2
48.6, CH2
α
57.8, CH
31.4, CH2
22.0, CH2
48.4, CH2
4.39, m
4.38, dd (9.2, 3.2)
2.40, m; 1.98, m
1.84, m; 1.77, m
3.70, m; 3.55, m
4.37, dd (9.0, 2.5)
2.42, m; 2.02, m
1.87, m; 1.77, m
3.75, m; 3.58, m
β
2.42, m; 2.04, m
1.78, m; 1.85, m
3.60, m; 3.77, m
γ
δ
NMePhe
NMeTyr2
NMeTyr2
CO
α
168.4, C
54.5, CH
35.2, CH2
168.7, C
54.6, CH
34.1, CH2
168.6, C
54.9, CH
34.3, CH
5.56, dd (12.0, 4.5)
3.28, dd (14.7, 12.0)
5.40, dd (11.5, 5.0)
3.11, m
5.46, dd (11.5, 5.0)
3.18, dd (15.0, 5.0)
2.97, dd (15.0, 5.0)
β
c
3.02
2.90, dd (15.0, 5.0)
1
136.8, C
127.3, C
130.6, CH
115.3, CH
155.3, C
29.9, CH3
Leu
127.2, C
130.7, CH
115.7, CH
155.3, C
29.8, CH3
Leu
2, 6
3, 5
4
129.7, CH
128.6, CH
126.8, CH
30.1, CH3
7.15, t (7.0)
7.12, t (7.0)
7.04, t (7.0)
3.03, s
6.93, d (8.3)
6.54, d (8.3)
6.98, d (8.0)
6.59, d (8.0)
NMe
Leu
CO
α
2.97, s
3.02, s
174.1, C
47.6, CH
39.9, CH2
174.1, C
47.6, CH
39.9, CH2
174.3, C
47.8, CH
40.0, CH2
4.92, t (9.5)
1.34, t (12.0)
0.12, t (12.0)
1.55, m
4.87, m
4.97, dd (12.5, 9.0)
1.44, t (12.5)
0.23, t (12.5)
1.56, m
β
1.39, t (11.5)
0.21, t (11.5)
1.51, m
γ
24.8, CH
23.7, CH3
20.8, CH3
24.8, CH
23.5, CH3
19.5, CH3
24.9, CH
23.8, CH3
21.0, CH3
δA-Me
δB-Me
NH
Val
CO
α
0.91, d (7.0)
0.84, d (7.0)
8.20, d (9.5)
0.91, d (7.0)
0.86, d (7.0)
8.16, d (8.0)
0.95, d (6.5)
0.85, d (6.5)
8.20, d (9.0)
Val
L-allo-IIe
170.9, C
171.1, C
58.2, CH
28.4, CH
20.7, CH3
16.2, CH3
171.4, C
58.1, CH
28.5, CH
16.3, CH3
19.6, CH3
4.40, dd (9.5, 2.5)
2.63, m
4.33, dd (9.5, 3.2)
2.60, m
56.0, CH
35.2, CH
26.6, CH2
12.0, CH3
14.4, CH3
4.57, dd (9.5, 3.0)
2.39, m
β
γ
0.80, d (7.0)
0.89, d (7.0)
0.81, d (7.0)
0.78, d (7.0)
1.22, m
δ-Me
β-Me
NH
0.89, t (7.4)
0.80, d (7.0)
5.87, d (9.5)
5.88, d (9.5)
5.94, d (9.5)
a
b
c
d,e
Recorded in CDCl3. Recorded in CDCl3−CD3OD. Overlapped with 3.03. The assignments bearing the same superscript can be interchanged.
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Note
Figure 1. COSY and HMBC correlations of compounds 1−3.
amino acid sequence of 1 was determined on the basis of
HMBC experiments (Figure 1A). Paramount HMBC correla-
tions of N-Me (N-MePhe)/CO (Leu), NH (Leu)/CO
(Val), NH (Val)/CO (N-MeTyr), N-Me (N-MeTyr)/CO
(Phe), NH (Phe)/CO (N-MeGly), and N-Me (N-MeGly)/
CO (Pro) established that the heptapeptide contained the
sequence −N-MePhe-Leu-Val-N-MeTyr-Phe-N-MeGly-Pro−,
accounting for 20 out of the 21 calculated degrees of
unsaturation. The cyclic nature of 1 accounted for the
remaining degree of unsaturation not satisfied by an acyclic
peptide. The amino acid sequence of 1 was also confirmed by
analyses of the ESIMS/MS data of the quasi-molecular ion
(Supporting Information, Figure S1c).5 Consequently, the
planar structure of 1 was elucidated as cyclo-(-N-MeTyr-Phe-
N-MeGly-Pro-N-MePhe-Leu-Val).
disclosed that the structure of 1 is very similar to those of the
reported cordyheptapeptides A and B, which were isolated from
the insect pathogenic fungi Cordyceps sp. BCC 1788 and
16176.9,10 The L-Ile residue in cordyheptapeptide A was
replaced by an L-Val in 1. Hence, compound 1 was accordingly
named cordyheptapeptide C.
Compound 2 was obtained as white needles. The molecular
formula of 2 was determined by HRESIMS as C48H63N7O9,
1
suggesting that 2 had one more oxygen atom than 1. The H
NMR data of 2 resembled those of 1, except that two pairs of
ortho-coupled aromatic proton signals at δH 6.93 and 6.54 (d, J
= 8.3 Hz) were observed instead of five aromatic proton signals
between δH 7.04 and 7.15. The 13C NMR spectrum of 2 was
characterized by two oxygen-bearing quaternary carbon signals
at δC 155.3. These data suggested the presence of two Tyr
residues in 2. Detailed interpretation of the COSY, HMQC,
and HMBC spectra revealed that the N-MePhe in 1 was
replaced by a N-MeTyr in 2 (Figure 1B). The amino acid
sequence of 2 was also supported by analyses of the ESIMS/
MS data of the quasi-molecular ion (Supporting Information,
Figure S2c). The absolute configurations of the amino acid
residues composing cyclopeptide 2 were determined by
Marfey’s method and found to be identical to those identified
in 1. Therefore, the structure of 2 was established as cyclo-(-N-
Me-L-Tyr1-L-Phe-N-MeGly-L-Pro-N-Me-D-Tyr2-L-Leu-L-Val-)
and named cordyheptapeptide D.
The absolute configuration of 1 was determined by single-
crystal X-ray crystallography using Cu Kα radiation (Figure 2).
Compound 3 was determined by HRESIMS to have the
molecular formula C49H66N7O9, possessing one CH2 beyond
that of 2. A comparison of the 1H and 13C NMR spectroscopic
data for 3 with those of 2 revealed close structural similarity
with the exception of one additional methylene (δH 1.22 and δC
26.6) in 3. Furthermore, the 13C NMR signal of a Val methine
in 2 was shifted downfield from δC 28.4 to δC 35.2 in 3, and two
Val methyl signals at δC 20.7 and 16.2 in 2 were shifted to δC
12.0 and 14.4 in 3, respectively. These data suggested that the
Val residue in 2 was substituted by an Ile in 3. Detailed analyses
of the COSY, HMQC, and HMBC spectroscopic data of 3
confirmed this hypothesis (Figure 1C). Evidence in support of
the amino acid sequence of 3 was also obtained from the
ESIMS/MS data of the quasi-molecular ion (Supporting
Information, Figure S3c). It was noticed that the 13C NMR
spectroscopic data of the Ile residue in 3 were significantly
different from those of the L-Ile in cordyheptapeptides A and
B,9,10 but very similar to those reported for L-allo-Ile unit,11
thereby suggesting the presence of an L-allo-Ile residue in 3.
Through Marfey’s analysis, the L-Ile and L-allo-Ile residues
Figure 2. X-ray crystal structure of cordyheptapeptide C (1).
The X-ray diffraction pattern not only confirmed the amino
acid sequence but also allowed assignment of the six chiral
amino acids as N-Me-D-Phe, L-Leu, L-Val, N-Me-L-Tyr, L-Phe,
and L-Pro through refinement of Flack’s parameter [x =
0.05(15)].6 These results were in agreement with HPLC
analyses of the acid hydrolysates of 1 generated using Marfey’s
method and chiral-phase HPLC analyses.7,8 A literature search
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Journal of Natural Products
Note
collected with an Oxford Xcalibur Onyx Nova diffractometer using Cu
Kα radiation. The common amino acids were purchased from Sangon
Biotech Co., Ltd (Shanghai). N-Me-D-Phe and N-Me-D-Tyr were
synthesized by Binhai Hanhong Biochemical Co., Ltd (Shanghai), and
N-Me-L-Phe and N-Me-L-Tyr were purchased from Sigma-Aldrich Co.
Fungal Material. Fungus SCSIO 115 was isolated from a marine
sediment sample collected in the South China Sea at E 1114°47.802′,
N 18°47.922′ and at a depth of 205 m. This fungus was characterized
as Acremonium persicinum SCSIO 115 on the basis of morphology12
and analysis of the ITS region13 sequence with Genbank accession
number JQ599382. This fungus was deposited in RNAM Center for
Marine Microbiology, South China Sea Institute of Oceanology,
Chinese Academy of Sciences (Guangzhou, China).
could not be differentiated because the retention times of their
FDAA derivatives were identical. Subsequently, chiral-phase
HPLC analysis of the acid hydrolysates of 3 was carried out,
which successfully clarified the configuration of the Ile residue
in 3 as L-allo-Ile. The absolute configurations of the other
amino acid residues were determined to be the same as those in
2 using Marfey’s method. Thus, compound 3 was identified as
cyclo-(-N-Me-L-Tyr1-L-Phe-N-MeGly-L-Pro-N-Me-D-Tyr2-L-
Leu-L-allo-Ile-), possessing an L-allo-Ile residue instead of the L-
Ile residue as well as an N-Me-D-Tyr residue instead of the N-
Me-D-Phe residue in cordyheptapeptide A. Compound 3 was
named cordyheptapeptide E.
Fermentation and Isolation. A. persicinum SCSIO 115 was
maintained on potato dextrose agar at 25 °C. Agar plugs were
inoculated into a 250 mL Erlenmeyer flask containing 50 mL of potato
dextrose broth supplemented with 3% sea salt. Flask cultures were
incubated at 28 °C on a rotary shaker at 200 rpm for two days as seed
culture. Thirty 1 L Erlenmeyer flasks, each containing 250 mL of liquid
medium (20% peeled potatoes, 2% glucose, 3% sea salt, and 0.5%
bacterial peptone), were individually inoculated with 25 mL of seed
culture and then incubated at 28 °C on a rotary shaker at 200 rpm for
seven days. The fermented broth was filtered through cheesecloth to
separate the supernatant and mycelia. The supernatant was processed
with XAD-16 resin three times, and the XAD-16 resin was eluted with
EtOH to afford the broth extract. The mycelia were extracted with
acetone (3 × 1 L) to afford the mycelial extract. These two extracts
were combined (affording 8.28 g) and applied to Si CC; gradient
elution with CHCl3−MeOH afforded six fractions (Fr.1−Fr.6). Fr.2
(1.60 g), eluted with CHCl3−MeOH (95:5), was further refined by
Sephadex LH-20 CC eluted with CHCl3−MeOH (1:1) to give four
fractions (Fr.2-1−Fr.2-4). Fr.2-1 was purified by Si CC, eluted with
gradient ratios of CHCl3−MeOH (100:0, 99:1, 98:2, 97:3, 96:4, and
95:5), to give seven fractions (Fr.2-1-1−Fr.2-1-7). Fr.2-1-6, eluted by
CHCl3−MeOH (96:4), was further purified by MPLC with an ODS
column eluted with MeOH−H2O (30:70 to 80:20 over 50 min, 15
mL/min) to yield 1 (15.6 mg). Fr.2-1-7, eluted with CHCl3−MeOH
(95:5), was subjected to Si CC eluted with EtOAc−MeOH to give
four fractions (Fr.2-1-7-1−Fr.2-1-7-4). Fr.2-1-7-2, eluted with EtOAc−
MeOH (97:3), was isolated by semipreparative HPLC with an ODS
column eluted with MeCN−H2O (50:50 to 100:0 over 30 min, 2.5
mL/min) to yield compound 3 (6.7 mg, tR 14.9 min). Fr.2-1-7-3,
eluted with EtOAc−MeOH (96:4), was further purified by MPLC
with an ODS column eluted with MeOH−H2O (40:60 to 100:0 over
60 min, 15 mL/min) to yield compound 2 (86.2 mg).
The cytotoxicities of cordyheptapeptides C−E (1−3) were
evaluated using human glioblastoma (SF-268), human breast
cancer (MCF-7), and human lung cancer (NCI-H460) cell
lines and the sulforhodamine B (SRB) method (Table 2).
Table 2. In Vitro Cytotoxicities (IC50, μM) of Compounds
1−3 (x s, n = 3)
SF-268
MCF-7
NCI-H460
1
3.7 0.3
45.6 5.4
3.2 0.2
4.6 0.1
3.0 0.2
82.7 1.8
2.7 0.2
10.2 0.4
11.6 0.5
>100 μM
4.5 0.3
1.6 0.1
2
3
a
cisplatin
a
Positive control.
Cordyheptapeptide E (3) demonstrated cytotoxicity against all
three cell lines, with IC50 values of 3.2, 2.7, and 4.5 μM,
respectively. Cordyheptapeptide C (1) also was found to
possess cytotoxicity against SF-268 and MCF-7 cells with IC50
values of 3.7 and 3.0 μM, respectively, and weaker cytotoxicity
against the NCI-H460 cell line. The most polar compound,
cordyheptapeptide D (2), displayed no activity against all three
cell lines. The results of the biological assays are shown in
Table 2. In the literature, cordyheptapeptides A and B were
reported to possess cytotoxicities against KB (oral human
epidermoid carcinoma), BC (human breast cancer), NCI-H187
(human small cell lung cancer), and Vero (African green
monkey kidney fibroblasts) cell lines with IC50 values of 0.78,
0.20, 0.18, 14 μM and 2.0, 0.66, 3.1, 1.6 μM, respectively.10
These results provide a vivid demonstration of how subtle
differences in structure among cordyheptapeptides A−E
profoundly impact their biological activities.
Cordyheptapeptide C (1): colorless crystals (CHCl3−MeOH, 3:1);
mp 256−257 °C; [α]25 −80 (c 0.2, CHCl3); UV (MeOH) λmax (log
D
ε) 228 (4.10), 278 (3.24) nm; IR (KBr) νmax 3277, 2910, 1630, 1541,
1
1510 cm−1; H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,
CDCl3) data, see Table 1; HR-ESIMS m/z 864.4648 [M − H]− (calcd
for C48H63N7O8, 864.4665).
EXPERIMENTAL SECTION
■
Cordyheptapeptide D (2): white needles (CHCl3−MeOH, 1:1);
General Experimental Procedures. Melting points were
determined on an SFW-X-4 apparatus and are uncorrected. Optical
rotations were obtained with an Anton Paar MCP 300 polarimeter.
UV spectra were recorded on a U-2910 spectrometer (Hitachi). IR
spectra were measured on an IRAffinity-1 Fourier transform infrared
spectrophotometer (Shimadzu). 1D and 2D NMR spectra were
recorded with a Bruker Avance-500 spectrometer using TMS as an
internal standard. ESIMS spectra were measured using a Bruker
Esquire 3000plus spectrometer. HR-ESIMS spectra were measured with
a Waters Q-TOF-micromass spectrometer. ESIMS2 spectra were
measured using a Bruker Maxis spectrometer. TLC was performed on
precoated plates with silica gel GF254 (10−40 μm). Column
chromatography (CC) was performed using silica gel (100−200
mesh, Qingdao Marine Chemical Chemicals) and Sephadex LH-20
(Amersham Pharmacia Biotech). MPLC was performed using a
CHEETAH MP 100 (Bonna-Agela Technologies) equipped with an
ODS column (20 × 90 mm, 40−63 μm, YMC). Semipreparative
HPLC was performed using a Hitachi L-2000 with an ODS column
(250 × 10 mm, 5 μm, YMC-Pack ODS-A). Single-crystal data were
mp 207−208 °C; [α]25 −85 (c 0.2, CHCl3−MeOH, 1:1); UV
D
(MeOH) λmax (log ε) 228 (4.18), 278 (3.42) nm; IR (KBr) νmax 3291,
1
2960, 1628, 1539, 1510 cm−1; H NMR (500 MHz, CDCl3) and 13C
NMR (125 MHz, CDCl3) data, see Table 1; HR-ESIMS m/z 882.4777
[M + H]+ (calcd for C48H63N7O9, 882.4760).
Cordyheptapeptide E (3): white needles (CHCl3−MeOH, 1:1); mp
189−190 °C; [α]25 −60 (c 0.2, CHCl3); UV (MeOH) λmax (log ε)
D
225 (4.11), 281 (3.39); IR (KBr) νmax 3292, 2957, 1628, 1539, 1505
cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,
CDCl3) data, see Table 1; HR-ESIMS m/z 896.4925 [M + H]+ (calcd
for C49H65N7O9, 896.4917).
X-ray Structure Determination of 1. A colorless crystal of 1 was
obtained in CHCl3−MeOH (3:1). The crystal data of 1 were recorded
on an Oxford Xcalibur Onyx Nova single-crystal diffractometer with
Cu Kα radiation (λ = 1.5418 Å). The structure was solved by direct
methods (SHELXS-97) and refined using full-matrix least-squares
difference Fourier techniques.14 Crystallographic data for 1 have been
deposited in the Cambridge Crystallographic Data Center with the
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dx.doi.org/10.1021/np300152d | J. Nat. Prod. 2012, 75, 1215−1219

Journal of Natural Products
Note
deposition number CCDC 853793. A copy of the data can be
obtained, free of charge, on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK [fax: +44(0)-1233-336033 or
e-mail: deposit@ccdc.cam.ac.uk].
Crystal data of 1: monoclinic, C49H69N7O10, space group P21, a =
12.4995(14) Å, b = 13.3874(14) Å, c = 14.8616(15) Å, α = 90°, β =
99.9697(11)°, γ = 90°, V = 2449.31(5) Å3, Z = 2, Dcalcd = 1.242 g/cm3,
μ = 0.711 mm−1, and F(000) = 984. Crystal size: 0.34 × 0.23 × 0.18
mm3. Independent reflections: 8779 [Rint = 0.0881]. The final indices
were R1 = 0.0426, wR2 = 0.1061 [I > 2σ(I)].
bound dye was dissolved in 10 mM Tris base solution (200 μL) for
OD determination at 570 nm using a microplate reader. Cisplatin was
used as a positive control, possessing potent cytotoxic activity. All data
were obtained in triplicate and are presented as means
SD. IC50
values were calculated with the SigmaPlot 10.0 software using a
nonlinear curve-fitting method.
ASSOCIATED CONTENT
■
S
* Supporting Information
1D and 2D NMR, HRESIMS, and ESIMS2 spectra of
compounds 1−3. This material is available free of charge via
the Internet at http://pubs.acs.org.
HPLC Analysis of the Acid Hydrolysates of 1−3 Using
Marfey’s Method. Compounds 1 (0.88 mg), 2 (0.88 mg), and 3
(0.35 mg) were each dissolved in 6 N HCl (1 mL) and heated at 110
°C for 18 h. After cooling to room temperature (rt), the solvent was
removed under reduced pressure. The remaining hydrolysate was
resuspended in 50 μL of H2O and treated with 100 μL of 1% (w/v) 1-
fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA) in acetone and 25
μL of 1 M NaHCO3. The mixture was heated at 40 °C for 1.5 h. After
cooling to rt, the contents were neutralized with 25 μL of 1 M HCl,
and the resulting mixture was added to 300 μL of MeOH to afford a
final hydrolysate volume of 500 μL. From this sample was then
withdrawn a 50 μL aliquot, and its solvent removed followed by
redissolution in 50 μL of MeOH. Ten microliters of this sample was
then analyzed by HPLC (Alltima C18 column; 4.6 × 250 mm, 5 μm)
using a solvent gradient from 10% to 70% solvent B (solvent A: 15:85
MeCN−H2O w/0.1% TFA; solvent B: 90:10 MeCN−H2O w/0.1%
TFA) over the course of 40 min at 1 mL/min with detection at 340
and 210 nm. Amino acid standards (4 μM) were prepared by
dissolving amino acids in 10 μL of H2O followed by addition of 20 μL
of FDAA and 5 μL of 1 M NaHCO3, reaction at 40 °C for 1.5 h, and
neutralization with 5 μL of 1 M HCl. Mixtures were then processed for
HPLC in a fashion similar to that used for cyclopeptide analyses; the
retention times for FDAA derivatives of L-Pro, D-Pro, L-Val, D-Val, L-
Ile, L-allo-Ile, D-Ile, D-allo-Ile, L-Leu, D-Leu, L-Phe, D-Phe, N-Me-L-Phe,
N-Me-D-Phe, N-Me-L-Tyr, and N-Me-D-Tyr were 17.8, 18.7, 22.5, 26.7,
25.7, 25.7, 29.0, 29.0, 26.4, 29.5, 26.3, 28.6, 26.1, 26.1, 15.5, and 15.1
min, respectively. Accordingly, the amino acids were assigned in 1 as
N-Me-L-Tyr (15.5 min), L-Leu (26.4 min), L-Val (22.5 min), L-Phe
(26.3 min), L-Pro (17.8 min), N-Me-D-Phe or N-Me-L-Phe (26.1 min),
in 2 as N-Me-L-Tyr1 (15.5 min), N-Me-D-Tyr2 (15.1 min), L-Leu (26.4
min), L-Val (22.5 min), L-Phe (26.3 min), L-Pro (17.8 min), and in 3
as N-Me-L-Tyr1 (15.5 min) and N-Me-D-Tyr2 (15.1 min), L-Leu (26.4
min), L-Phe (26.3 min), L-Pro (17.8 min), L-Ile or L-allo-Ile (25.7 min),
respectively.
Chiral-Phase HPLC Analysis of the Acid Hydrolysates of 1
and 3. To determine the absolute configurations of the N-MePhe in 1
and the Ile in 3, chiral-phase HPLC analyses of the acid hydrolysates
were conducted. Compounds 1 (0.30 mg) and 3 (0.30 mg) were
hydrolyzed as mentioned above. The dried hydrolysate was dissolved
in 100 μL of 2 mM CuSO4−H2O solution. Ten microliters of this
sample were then analyzed by HPLC with a chiral column (MCIGEL
CRS10W, 4.6 × 50 mm, Mitsubishi Chemical Corporation) using 2
mM CuSO4−H2O solution as the mobile phase at a flow rate of 0.5
mL/min with UV detection at 254 nm. N-Me-L-Phe and N-Me-D-Phe,
L-Ile, and L-allo-Ile were detected as references. The retention times of
the N-Me-D-Phe, N-Me-L-Phe, L-allo-Ile, and L-Ile were 25.2, 26.3,
11.6, and 14.3 min, respectively. Hence, the N-MePhe residue in 1 was
assigned as N-Me-D-Phe (25.1 min), and the Ile residue in 3 was
assigned as L-allo-Ile (11.4 min) (Supporting Information, Figures
S19a and S19b).
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86-20-89023028. E-mail: jju@scsio.ac.cn.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the analytical facility center of South China Sea
Institute of Oceanology for recording NMR and MS data. This
work is supported, in part, by grants from the Knowledge
Innovation Programs of the Chinese Academy of Sciences
(KZCX2-YW-JC202 and KZCX2-EW-G-12), Science and
Technology Planning Project of Guangdong Province
(2010B030600010), National High Technology Research
Program of China (2012AA092104), National Basic Research
Program of China (2010CB833805), and Scientific Research
Foundation for the Returned Overseas Chinese Scholars of the
State Education Ministry. J.J. is a scholar of the “100 Talents
Project” of Chinese Academy of Sciences (08SL111001).
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