Nocardiamides A and B, two cyclohexapeptides from the marine-derived actinomycete Nocardiopsis sp. CNX037

Article abstract of DOI:10.1021/np400009a

Two new cyclic hexapeptides, nocardiamides A (1) and B (2), were isolated from the culture broth of marine-derived actinomycete CNX037 strain that was identified as a Nocardiopsis species. The planar structures of nocardiamides A (1) and B (2) were assigned on the basis of 1D and 2D NMR and HRESIMS spectroscopic analyses. Their absolute configurations were deduced by the advanced Marfey's method and chiral-phase HPLC analysis. The challenge of locating two d- and one l-valine residue in 1 and 2 was accomplished by total synthesis using solid-phase peptide synthetic methods. Both 1 and 2 showed negligible antimicrobial activities against seven indicator strains and exhibited no cytotoxicity against HCT-116.

Full text of DOI:10.1021/np400009a

Article
pubs.acs.org/jnp
Nocardiamides A and B, Two Cyclohexapeptides from the Marine-
Derived Actinomycete Nocardiopsis sp. CNX037
Zheng-Chao Wu,†,‡,§ Sumei Li,† Sang-Jip Nam,⊥ Zhong Liu,∥ and Changsheng Zhang*,†
†Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key
Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang
Road, Guangzhou 510301, People’s Republic of China
‡Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La
Jolla, California 92093-0204, United States
§University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
⊥Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, Republic of Korea
∥Guangzhoujinan Biomedicine Research and Development Center, Guangdong Key Laboratory of Bioengineering Medicine, Jinan
University, 601 West Huangpu Road, Guangzhou 510632, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two new cyclic hexapeptides, nocardiamides A (1) and B (2), were isolated from the culture broth of marine-
derived actinomycete CNX037 strain that was identified as a Nocardiopsis species. The planar structures of nocardiamides A (1)
and B (2) were assigned on the basis of 1D and 2D NMR and HRESIMS spectroscopic analyses. Their absolute configurations
were deduced by the advanced Marfey’s method and chiral-phase HPLC analysis. The challenge of locating two D- and one L-
valine residue in 1 and 2 was accomplished by total synthesis using solid-phase peptide synthetic methods. Both 1 and 2 showed
negligible antimicrobial activities against seven indicator strains and exhibited no cytotoxicity against HCT-116.
arine-derived actinomycetes are currently being explored
the AntiMarin database.10 Herein we report the isolation,
structure elucidation, synthesis, and biological evaluation of
these two new cyclohexapeptides, designated nocardiamides A
(1) and B (2).
1
Mas a valuable source for novel drug discovery. A number
of biologically active natural products with unique structures
have been discovered from marine-derived or obligate marine
microorganisms.2 Peptides or peptide-derived compounds
account for more than half (56%) of all bacterial metabolites3
and have also been shown to be a large group of secondary
metabolites from marine-derived actinomycetes.4 In the last
two decades, 35 new peptides or peptide-derived natural
products with various bioactivities have been reported from
marine-derived actinomycetes.4,5 These are exemplified by the
antibacterial salinamides,6 by the anti-inflamatory cyclomarins,5f
and by the antitumor proximicins,7 piperazimycins,8 and
marthiapeptide A.9 In the course of searching for new natural
products from marine-derived actinomycetes, a bacterium of
the genus Nocardiopsis sp., strain CNX037, was isolated from a
marine sediment sample and was observed to produce new
secondary metabolites with molecular ions at m/z 673.4292
and 687.4450, using HRLCMS dereplication analysis against
RESULTS AND DISCUSSION
■
The strain CNX037 was isolated from a sediment sample
collected at a depth of 18−30 m from the La Jolla Canyon, San
Diego, CA. It was identified as a Nocardiopsis species on the
basis of the phylogenetic tree constructed by a neighbor-joining
method (Figure S1). Purification of the EtOAc extracts of the
fermentation broth of strain CNX037 led to the isolation of
two new cyclohexapeptides, designated nocardiamides A (1)
and B (2).
Received: January 4, 2013
Published: April 15, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Nocardiamide A (1) was obtained as a white powder. The
molecular formula was determined as C36H58N6O7 by
HRESIMS (m/z 687.4450 [M + H]+, calcd 687.4440, Figure
S2), indicating 11 degrees of unsaturation. The peptide nature
1
of 1 was deduced from the H and 13C NMR and IR spectra
(Table 1). The IR spectrum of 1 showed an absorption band at
1
1635 cm−1, characteristic for the amide functionality. The H
NMR spectrum of 1 displayed six NH resonances (δH 7.0−8.5),
six α-amino acid hydrogen resonances (δH 3.5−5.0), and 10
methyl doublets or triplets (δH 0.5−1.0). The 13C NMR
spectrum of 1 exhibited six amide carbonyl signals resonating
between δC 169 and 172 ppm and six α-amino acid carbon
signals between δC 53 and 60 ppm. Detailed analysis of the 1D
1
Table 1. H (500 MHz) and 13C (75 MHz) NMR Data for Nocardiamides A (1) and B (2) in DMSO-d6
nocardiamide A (1)
δH, mult (J in Hz)
nocardiamide B (2)
amino acids
Tyr
pos.
δC, mult
HMBC
δC, mult
δH, mult (J in Hz)
1
2
170.0, C
53.9, CH
35.4, CH2
170.0, C
53.9, CH
35.4, CH2
4.44, m
1, 3a, 3b
2, 4
4.44, m
3a
2.73, dd (14.0, 10.0)
3.08, dd (14.0, 10.0)
2.73, dd (13.5, 9.5)
3.08, dd (13.5, 9.5)
3b
2, 4
4
128.0, C
128.0, C
5 or 9
6 or 8
7
129.9, CH
114.7, CH
155.7, C
6.95, d (8.5)
6.59, d (8.5)
129.9, CH
114.7, CH
155.7, C
6.95, d (8.5)
6.59, d (8.5)
NH
OH
10
7.56, d (8.0)
9.11, s
10
7.56, d (8.0)
9.11, s
Ile/Val4
170.5, C
170.5, C
11
59.0, CH
35.3, CH
15.3, CH3
23.9, CH2
3.80, t (7.0)
1.61, m
10
59.7, CH
29.1, CH
18.0, CH3
19.2, CH3
3.76, t (7.0)
1.85, m
12
13, 14
13
0.56, d (7.0)
1.03, m
11, 12, 14
12, 13, 15
0.69, d (6.5)
0.62, d (6.5)
14
1.03, m
15
10.7, CH3
0.69, t (7.5)
8.10, d (7.0)
14
16
NH
16
8.10, d (7.5)
Leu
171.8, C
51.9, CH
41.4, CH2
171.8, C
51.9, CH
41.4, CH2
17
4.46, m
16
17
17
4.46, m
18
1.55, m
1.55, m
1.48, m
1.48, m
19
24.0, CH
22.3, CH3
23.1, CH3
1.50, m
24.0, CH
22.3, CH3
23.1, CH3
1.50, m
20
0.88, d (6.0)
0.83, d (5.0)
7.62, d (7.0)
18, 19
18, 19
22
0.88, d (5.5)
0.83, d (8.0)
7.62, d (6.5)
21
NH
22
Val1
Val2
Val3
170.6, C
170.6, C
23
58.1, CH
29.6, CH
17.2, CH3
19.4, CH3
4.12, dd (9.0, 5.5)
2.20, m
22, 24, 25
23, 25, 26
58.1, CH
29.6, CH
17.2, CH3
19.4, CH3
4.12, dd (9.5, 5.5)
2.20, m
24
25
0.84, d (5.0)
0.82, overlapped
8.22, d (9.0)
0.84, d (5.0)
0.82, d (7.0)
8.22, d (9.0)
26
23, 24, 25
27
NH
27
171.4, C
171.4, C
28
59.7, CH
28.5, CH
19.1, CH3
19.1, CH3
3.98, dd (8.5, 7.0)
1.91, m
27, 29
28, 30, 31
29
59.7, CH
28.5, CH
19.1, CH3
19.1, CH3
3.98, t (7.5)
1.91, m
29
30
0.86, d (6.5)
0.92, d (7.0)
8.28, d (6.5)
0.86, d (7.0)
0.92, d (7.0)
8.28, d (6.5)
31
29
NH
32
32
170.7, C
170.7, C
33
57.3, CH
30.4, CH
18.6, CH3
18.6, CH3
4.20, t (8.0)
1.88, m
32, 34
57.3, CH
30.4, CH
18.6, CH3
18.6, CH3
4.20, t (8.0)
1.88, m
34
33, 35, 36
33, 34
35
0.76, d (7.0)
0.81, d (7.0)
7.39, d (3.5)
0.76, d (7.0)
0.81, d (7.0)
7.39, d (8.0)
36
NH
1
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(1H, 13C, DEPT) and 2D (HSQC, TOCSY, and HMBC) NMR
spectroscopic data (Figure S2) revealed that 1 was a
hexapeptide containing Tyr, Ile, Leu, and three Val residues
(Table 1). The hexapeptide 1 was supposed to have the
sequence Tyr-Ile-Leu-Val1-Val2-Val3 on the basis of the
following HMBC correlations: NH-Tyr (δH 7.56)/Ile CO
(δC 170.5), NH-Ile (δH 8.10)/Leu CO (δC 171.8), Leu-NH
(δH 7.62)/Val1 CO (δC 170.6), Val1-NH (δH 8.22)/Val2 C
O (δC 171.4), and Val2-NH (δH 8.3)/Val3 CO (δC 170.7)
(Figure 1). These six amino acids accounted for 10 degrees of
fragmentation of the isolated molecular ion m/z 687.4439 [M +
H]+ for 1 yielded MS2 ions m/z 277.1568 [M + H]+ for Tyr-Ile,
m/z 390.2385 [M + H]+ for Tyr-Ile-Leu, m/z 489.3061 [M +
H]+ for Tyr-Ile-Leu-Val, and m/z 588.3739 [M + H]+ for Tyr-
Ile-Leu-Val-Val. This fragmentation pattern was in good
agreement with the proposed structure cyclo-(Tyr-Ile-Leu-
Val1-Val2-Val3). Therefore, the planar structure of 1 was
unambiguously established as cyclo-(Tyr-Ile-Leu-Val1-Val2-
Val3).
The absolute configurations of the amino acid residues in 1
were determined by application of the advanced Marfey’s
method and by chiral-phase HPLC analysis. Compound 1 was
subjected to acid hydrolysis and derivatizated with the advanced
Marfey’s reagent D-FDLA or D-FDAA.11 Comparison of the
retention times and m/z values between the chiral derivatives of
the amino acid residues of 1 and derivatives of authentic amino
acids indicated the presence of L-Tyr, D-Leu, L-Ile or L-allo-Ile
or L-Leu, D- and L-Val (Table S1, Figure S4). The D-FDLA
derivatives of L-Ile, L-allo-Ile, and L-Leu (tR 55.7 min for all
three standards) were indistinguishable by reversed-phase
HPLC analysis. We then applied a chiral-phase HPLC method
to differentiate the isomers of L-Ile, L-allo-Ile, and L-Leu. The
chiral-phase analysis established the presence of an L-Ile (tR
15.8 min), but not an L-allo-Ile (tR 11.1 min) or an L-Leu (tR
13.3 min) in compound 1, by comparing with authentic
standards (Figure S5).
Due to an overlap between the D-Val derivative and an
unidentified m/z 314 compound from the hydrolysate reaction
mixture (Figure S4), the ratio of D- and L-Val in compound 1
could not be determined. Therefore, compound 1 was
alternatively subjected to Marfey’s reaction with the reagent
D-FDAA. Both D-FDAA derivatives of D-Val (tR 34.3 min, m/z
370 [M + H]+; tR 33.9 min for standard) and L-Val (tR 39.2
min, m/z 370 [M + H]+; tR 39.1 min for standard) were
obtained and were estimated in a 2:1 (D:L) ratio (Figure S4).
Given that both D- and L-Val enantiomers (D:L = 2:1) were
encountered, the configurations of the individual Val units in
the triple-Val subunit in 1 could not be easily established by
these analyses. Theoretically, there should be three possible
configurations for the triple-Val subunit: D-Val1-D-Val2-L-Val3, L-
Val1-D-Val2-D-Val3, or D-Val1-L-Val2-D-Val3. Subsequently, care-
ful analysis of NMR-derived distance restraints based on the
key ROESY correlations and the comparison with the three
predicted molecular models (Figure 3)12 revealed D-Val1-L-Val2-
D-Val3 as the most favorable configuration of the triple-Val
subunit. The ROESY correlations were observed between NH
(Tyr) and NH (Leu) and between NH (Tyr) and NH (Ile) in
1 (Figure 3). Three energy-minimized structure models were
constructed for the three possible configurations: D-Val1-L-Val2-
D-Val3 (Figure 3A), D-Val1-D-Val2-L-Val3 (Figure 3B), and L-
Val1-D-Val2-D-Val3 (Figure 3C). Based on these models, the
interproton distances for NH (Tyr) and NH (Leu) were found
to be 4.12 Å (model A), 5.77 Å (model B), and 6.18 Å (model
C), while the interproton distances for NH (Tyr) and NH (Ile)
were predicted to be 2.93 Å (model A), 4.43 Å (model B), and
5.23 Å (model C), respectively (Figure 3, Table S3). The
calculated interproton distances for NH (Tyr) and NH (Leu)
and for NH (Tyr) and NH (Ile) in models B and C (Figure 3)
were significantly different from the observed ROSEY
correlations. In contrast, the calculated interproton distances
in model A (Figure 3) matched well with NMR-derived
distance restraints (Figure 3, Table S3). Therefore, we assumed
Figure 1. Selected key TCOSY and key HMBC correlations for 1 and
2.
unsaturation, indicating that 1 was a monocyclic peptide. In
support of the cyclic nature of 1, the NH of the Val3 (δH 7.39)
also showed an HMBC correlation to the carbonyl of Tyr (δC
170.0). Therefore, the planar structure of 1 was deduced as
cyclo-(Tyr-Ile-Leu-Val1-Val2-Val3). In order to precisely define
the connectivity of alpha protons and amide NH protons to
specific carbonyls, especially for three carbonyls with very close
chemical shifts (δC 170.5, 170.6, and 170.7), we performed
additional HRESIMS/MS analysis (Figure 2, Figure S3). The
Figure 2. HRESIMS/MS of fragmentation patterns of nocardiamides
A (1) and B (2).
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that D-Val1-L-Val2-D-Val3 was the mostly likely configuration in
1.
In order to confirm the structure of 1 and to definitively
assign the locations of the D- and L-amino acids, the linear
precursor of 1, L-Tyr-L-Ile-D-Leu-D-Val-L-Val-D-Val (3), des-
ignated prenocardiamide A, was synthesized using a solid-phase
peptide synthetic (SPPS) approach (Scheme 1). The structure
of 3 was confirmed by interpretation of MS and 1D and 2D
NMR spectra (Table S2, Figure S6). Cyclization of compound
3 yielded a synthetic product, whose spectroscopic properties
were identical to those of the natural product 1 (Figure S7).
Therefore, the structure of nocardiamide A (1) was established
as cyclo-(L-Tyr-L-Ile-D-Leu-D-Val-L-Val-D-Val).
Nocardiamide B (2) was obtained as a white powder. The
molecular formula was determined as C35H56N6O7 by
HRESIMS, indicating 11 degrees of unsaturation. The 1H
and 13C NMR spectra of 2 are similar to those of 1 (Table 1).
Detailed comparison of the 1D and 2D NMR spectroscopic
data of 1 and 2 (Figure S8) revealed that the Ile residue in 1
was replaced by a Val residue in 2 (Figure 1). Comparing NMR
data of 1 and 2, signals for the ethyl group of Ile (δC 23.9, δH
1.03 and δC 10.7, δH 0.69) in 1 were absent in 2, which was
replaced by a methyl doublet (δC 19.2, δH 0.62) (Table 1).
Furthermore, the HMBC correlations (Figure 2) supported the
assignment of the planar structure of 2 as cyclo-(Tyr-Val4-Leu-
Val1-Val2-Val3), which was also confirmed by the HRESIMS/
MS analysis (Figure 2, Figure S9). Subsequently, the absolute
Figure 3. Key ROSEY correlations and energy-minimized models of
three possible configurations, D-Val1-L-Val2-D-Val3 (A), D-Val1-D-Val2-L-
Val3 (B), and L-Val1-D-Val2-D-Val3 (C), of nocardiamide A (1).
a
Scheme 1. Total Synthesis of Nocardiamide A (1)
a
Reagents: (a) Fmoc-L-Tyr(tBu)-OH; (b) HBTU, DIEA; (c) MeOH; (d) 20% piperdine, DMF; (e) Fmoc-L-Ile-OH; (f) Fmoc-D-Leu-OH; (g)
Fmoc-D-Val-OH; (h) Fmoc-L-Val-OH; (i) TFA/thioanisole/PhOH/EDT/DI H2O; (j) HBTU, DIEA, DMF (yield 7.2%). The yield for off resin step
(i) to get compound 3 was 31.1%, and the yield of the cyclization step (j) to convert 3 to 1 was 7.2%.
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configuration of 2 was determined by the advanced Marfey’s
method, in the same manner as described for 1. HPLC-MS
analysis of the resulting hydrolysates revealed the presence of L-
Tyr, D-Leu, and D- and L-Val (in a 1:1 ratio) (Table S1, Figure
S4). Given that the valine isomers in the triple-Val subunit of 2
exhibited the same chemical shifts as those in 1 (Table 1), the
absolute configurations of the valine residues in 2 were also
assigned as D−L−D. The remaining valine residue was thus
deduced to have an L configuration, to satisfy the equal ratio of
D- and L-Val in 2 (Table S1, Figure S4). Therefore, the
structure of compound 2 was deduced as cyclo-(L-Tyr-L-Val4-D-
Leu-D-Val1-L-Val2-D-Val3).
Compound 2 was also prepared by synthesis in a similar
fashion to the synthesis of 1 (Scheme 1). The linear
intermediate prenocardiamide B (4) was first synthesized
using SPPS methods (Table S2, Figure S10), and the
cyclization of 4 produced the target molecule 2. Synthetic 2
displayed an indistinguishable HPLC profile and identical
NMR data to natural 2 (Figure S11). Consequently, the
structure of 2 was confirmed as cyclo-(L-Tyr-L-Val4-D-Leu-D-
Val1-L-Val2-D-Val3).
malevamide B26 and kahalalide F (an anticancer drug in clinical
trial) and its analogues,27 it becomes challenging to determine
the absolute configuration for a single Val (or N-Me-Val)
residue. It was shown that four N-Me-Val residues in
malevamide B existed in a ratio of 3:1 (L:D); however, the
precise location of the single N-Me-D-Val turned out to be not
yet determined.26 In the case of the marine cyanobacterium-
derived cyclic depsipeptide ulongapeptin, the location of both
enantiomers of N-Me-Val residues was differentiated by
molecular modeling coupled with NMR-derived restraints.28
The challenge of determining the absolute configurations of
mixed L- and D-Val residues in kahalalide F was accomplished
almost 10 years after its discovery by a series of degradation
reactions29 and eventually by total synthesis.30 In this study, the
D−L−D absolute configurations of the triple-Val subunit in
nocardiamides A (1) and B (2) were deduced by structure
modeling and confirmed by total synthesis.
Cyclic hexapeptides exhibited a diverse range of bioactivities,
including antibacterial (e.g., chloptosin and mannopeptimy-
cins),18 antifungal (e.g., halolittoralin A and sclerotides),19,20
antialgal (nostocyclamide),17b cytotoxic against human cancer
cell lines (e.g., bouvardins, bistratamides, westiellamide,
comoramides, didmolamides, microcyclamides, and molla-
mides) or against sea urchin embryos (raocyclamides and
tenuecyclamides),16,17 inhibitory to nitric oxide (NO)
production (rubiyunnanins),15 and inhibitory to proteasome
(phepropeptins).18b Therefore, more bioactivities, not limited
by antibacterial and cytotoxic activities, should be screened for
nocardiamides 1 and 2 in the future.
Compounds 1−4 showed negligible antimicrobial activities
against the seven indicator strains, Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalis
ATCC 29212, Bacillus thuringensis SCSIO BT01, B. subtilis
SCSIO BS01, Micrococcus luteus SCSIO ML01, and Candida
albicans ATCC 10231. Compounds 1 and 2 failed to exhibit
cytotoxicity toward the human colon carcinoma cell line HCT-
116.
Cyclic hexapeptides are a large group of natural products that
are widely discovered in nature, from plants to microorganisms.
Representative plant-derived cyclic hexapeptides include
bouvardins,13 rubiaceae-type compounds,14 and rubiyunna-
nins.15 Ascidians of the genera Lissoclinum and Didemnum are
prolific producers of cyclic hexapeptides,12a,16 including
bistratamides, cycloxazoline, comoramides, didmolamides, and
mollamides. Also, many cyclic hexapeptides have been reported
from cyanobacteria,17 which are exemplified by westiellamide,
nostocyclamides, raocyclamides, dendroamides, tenuecycla-
mides, and microcyclamides. Various cyclic hexapeptides have
been reported from terrestrial Streptomyces species (such as
chloptosin, phepropeptins, mannopeptimycins, and NW-
G05),18 the terrestrial- and marine-derived fungus Aspergillus
sclerotiorum (scleramide and sclerotides),19 and the marine-
derived bacteria Halobacillus litoralis YS3106 (halolitoralin A).20
To our surprise, nocardiamides A (1) and B (2) represent the
first examples of cyclic hexapeptides isolated from marine-
derived actinomycetes. Unlike many cyclic hexapeptides from
ascidians and cyanobacteria with modified amino acid residues
that form thiazole, oxazole, thiazoline, or oxazoline rings,12a,16,17
nocardiamides contain only unmodified amino acids.
Nocardiamides A (1) and B (2) feature a triple-Val subunit
in their structures. The same triple-Val subunit is also found in
some other natural peptides, such as malaysiatin from a marine
sponge21 and tolybyssidin B from a cyanobacterium.22 A similar
subunit, triple-N-Me-Val, is naturally occurring in apramide G
and dragonamide from marine cyanobacteria,23 dictyonamide A
from a marine-derived fungus,24 and kendarimide A from a
marine sponge.25 The L-Val configurations of all Val (or N-Me-
Val) residues in these peptides have been easily identified by
using Marfey’s method.21−25 However, in the case where
multiple Val (or N-Me-Val) residues of both L- and D-
configurations are found in a natural peptide, such as
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotations were
measured using a Rudolph Research Autopol III polarimeter. UV
spectra were recorded on a Varian Cary UV−visible spectropho-
tometer with a path length of 1 cm, and IR spectra were recorded on a
Perkin-Elmer 1600 FT-IR spectrometer. 1H and 2D NMR
spectroscopic data were recorded at 500 MHz in DMSO-d6 with
TMS as an internal standard on Varian Inova or Bruker AV500
spectrometers. 13C NMR spectroscopic data were acquired at 75 MHz
on Varian Inova spectrometers or at 125 MHz on Bruker AV500
spectrometers. HRESIMS were provided by the Agilent 6230
Accurate-Mass time-of-flight mass spectrometry facility at the
Department of Chemistry and Biochemistry at the University of
California, San Diego, La Jolla, CA. HRESIMS/MS data were acquired
using a Bruker Maxis quadrupole-time-of-flight mass spectrometer.
Low-resolution LC-MS data were measured using a Hewlett-Packard
series 1100 LC/MS system with a reversed-phase C18 column
(Phenomenex Luna, 4.6 × 100 mm, 5 μm) at a flow rate of 0.7
mL/min. The chirality of the liberated amino acids was analyzed by
HPLC with a chiral-phase column (MCIGEL CRS10W, 4.6 × 50 mm,
0.5 mL/min, 2 mM CuSO4(aq), UV detection at 254 nm).
Collection and Phylogenetic Analysis of Strain CNX037. The
marine-derived actinomycete, strain CNX037, was isolated from a
sediment sample collected at a depth between 18 and 30 m in August
2009 near the La Jolla Canyon, San Diego (sediment sample #CA09-
129-C). The strain was identified as a Nocardiopsis sp. on the basis of
16S rRNA gene analysis (GenBank accession number KC149962)
(Figure S1).
Fermentation, Extraction, and Isolation. The strain CNX037
was fermented at 27 °C by shaking at 250 rpm in 10 × 2.8 L Fernbach
flasks each containing 1 L of the seawater-based medium A1BFe [10 g
starch, 4 g yeast extract, 2 g peptone, 0.04 g Fe2(SO4)3·4H2O, 0.1 g
KBr].31 After cultivation for 6 days, sterilized XAD-16 resin (20 g/L)
was added to the fermentation broth to absorb the organic products.
After shaking at 215 rpm for 2 h, the resin was filtered through
cheesecloth, washed with 2 L of deionized water, and eluted with 5 L
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of acetone. The acetone was removed under reduced pressure to
provide an aqueous layer, which was further extracted with 2 L of
EtOAc to afford a crude residue (220 mg). The crude residue was then
dissolved in 10 mL of MeOH, and the insoluble materials were
collected by centrifugation to obtain the organic extract (90 mg). The
extract (90 mg) was subjected to normal-phase silica gel column
chromatography (200−300 mesh, 3 g), eluting with CH2Cl2/CH3OH
(100/0, 100/1, 100/2, 100/5, 100/20, 100/100, and 0/100, v/v, each
of 20 mL) to obtain seven subfractions (Fr.1 to Fr.7). The two
fractions (Fr.5 and Fr.6, 14.0 mg) containing nocardiamide A were
further purified by semipreparative HPLC with a reversed-phase C18
column (Dynamax column, 3 mL/min, UV 210 nm; solvent A, 0.1%
formic acid in H2O; solvent B, MeCN; eluted with constant 50%
solvent B) to obtain nocardiamide A (1, 8.0 mg). Fr.7 (15.0 mg) was
further purified by semipreparative HPLC with a reversed-phase C18
column (Dynamax column, 2.5 mL/min, UV 210 nm; solvent A, 0.1%
formic acid in H2O; solvent B, MeCN; eluted with constant 45%
solvent B) to obtain nocardiamide B (2, 3.0 mg).
of the three possible configurations (D-Val1-L-Val2-D-Val3, D-Val1-D-
Val2-L-Val3, and L-Val1-D-Val2-D-Val3) were performed by ChemBio3D
(version 12.0.2) using the MM2 molecular mechanics force field with
minimum RMS gradient of 0.100.
Total Synthesis of Nocardiamide A. The SPPS method
(Scheme 1) was designed to synthesize nocardiamide A (1), in a
similar manner to that reported for the turnagainolides.32 Solid-phase
2Cl-Trt resin 0.56 g (loading amount 1 mmol/g) was placed in a clean
20 mL vessel and soaked with dimethylformamide (DMF) for 0.5 h.
The first amino acid, Fmoc-L-Tyr(tBu)-OH (0.27 g, 0.56 mmol), was
added in DMF; then 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uroniumhexafluorophosphate (HBTU, 0.21 g, 0.56 mmol) and N-
ethyldiisopropylamine (DIEA, 2 mL) were added to allow the reaction
to proceed for 1.5 h. The reaction was capped by the addition of
MeOH to make sure that there were no active resin Cl atoms left.
After 30 min, the reaction mixture was washed three times by
dichloromethane (DCM) and DMF (15 mL, 1 min each) and then
drained. The Fmoc protecting group in L-Tyr was removed by
treatment with 20% piperidine in DMF (10 mL) for 15 min under N2.
The solid-phase resin was then washed with DCM and DMF three
times (10 mL, 1 min each). The ninhydrin test was checked for blue
color to indicate the Fmoc protecting group in L-Tyr had been
removed and the NH2 was available. For coupling of the second amino
acid, Fmoc-L-Ile-OH (0.59 g, 1.68 mmol) was added in DMF; then
HBTU (0.64 g, 1.68 mmol) and DIEA (2 mL) were added to allow
the reaction to proceed for 0.5 h. The reaction mixture was washed
three times with DCM and DMF (15 mL, 1 min each) and then
drained for the ninhydrin color test. The cycle (deprotection/
washing/coupling/washing) was repeated to assemble the other four
amino acids in the sequence D-Leu, D-Val, L-Val, and D-Val by using
approximately 1.68 mmol of each reagent to yield the resin-bound
peptide (6.12 g).
The cleavage reagent mixture TFA/thioanisole/PhOH/ethanedi-
thiol (EDT)/DI H2O (82.5:5:5:2.5:5) was added to the resin-bound
peptide to remove the peptide from the resin to obtain the crude linear
hexapeptide (1.81 g). The crude linear peptide was purified by
semipreparative RP-HPLC (Biomisil ODS-P-C18, 30 × 250 mm, 5
μm; phase A 0.1% TFA/MeCN, phase B 0.1% TFA/H2O; gradient
21% A to 31% B, 20 mL/min; UV 220 nm) to yield the pure linear
hexapeptide prenocardiamide A (3) (124.4 mg, 31.1%). The
cyclization of the linear peptide was carried out as follows:
prenocardiamide A (3) (124.4 mg) was dissolved in 150 mL of
DMF to give a ca. 10−3−10−4 M solution of 3. Then HBTU (0.064 g,
0.17 mmol) was added to the solution, adjusting to pH 8−9 by DIEA.
The reaction proceeded by stirring at room temperature and was
monitored by HPLC. Crude cyclohexapeptide was obtained by
concentration in vacuo. The synthetic nocardiamide A (1) (8.7 mg,
7.2%) was finally purified by semi RP-HPLC (gradient: 23% A to 33%
B).
Nocardiamide A (1): white powder; [α]2D0 −3 (c 0.25, DMSO); UV
(MeOH) λmax (log ε) 224 (3.5), 278 (2.7) nm; IR (KBr) νmax 3668,
3645, 3440, 2962, 2928, 1682, 1635, 1555, 1538, 1435, 1205, 1139,
1026, 1001, 803, 723 cm−1; 1H and 2D-NMR (500 MHz, DMSO-d6);
13C NMR (75 MHz, DMSO-d6), see Table 1; HRESIMS m/z
687.4450 [M + H]+ (calcd for C36H59N6O7, 687.4440).
Nocardiamide B (2): white powder; [α]2D0 −2 (c 0.11, DMSO); UV
(MeOH) λmax (log ε) 224(4.1), 278 (3.4) nm; IR (KBr) νmax 3423,
3263, 1629, 1539, 1456, 520, 473 cm−1; 1H and 2D-NMR (500 MHz,
DMSO-d6); 13C NMR (75 MHz, DMSO-d6), see Table 1; HRESIMS
m/z 673.4292 [M + H]+ (calcd for C35H57N6O7, 673.4282).
Determination of the Absolute Configurations of the Amino
Acids in 1 and 2 by the Advanced Marfey’s Method and
Chiral-Phase Analysis. The FDLA- or FDAA-derivatized hydro-
lysates of compounds 1 (1.0 mg) and 2 (1.0 mg) and the standard
amino acids were prepared according to the published method.11 The
following solvent system was used to separate the FDLA or FDAA
derivatives of 1 and 2 with UV detection at 340 nm: solvent A, H2O
with 0.1% TFA; solvent B, MeCN with 0.1% TFA. The LCMS
program for detecting the derivatives was set as 10−50% solvent B
(0−50 min, linear gradient), 50−100% solvent B (50−63 min), 100%
solvent B (63−65 min), 100−10% solvent B (65−67 min), and 10%
solvent B (67−70 min). The chiral-phase HPLC with an analytical
column (MCIGEL CRS10W, 4.6 × 50 mm, Mitsubishi Chemical
Corporation) was performed using a constant solvent system (2 mM
CuSO4 in H2O) for 40 min at a flow rate of 1 mL/min, under the UV
detection of 254 nm. The retention times were determined as follows:
standard L-Ile (15.8 min), L-allo-Ile (11.1 min), L-Leu (13.3 min), the
Ile residue in 1 (15.5 min).
The retention times and ion peaks for the Marfey’s D-FDLA
derivatives of the amino acid standards were determined as follows: D-
Val (tR 43.8 min, m/z 412 [M + H]+), L-Val (tR 52.6 min, m/z 412 [M
+ H]+), D-Leu (tR 48.3 min, m/z 426 [M + H]+), L-Leu (tR 55.8 min,
m/z 426 [M + H]+), D/L-Ile mixtures (tR 47.4, 55.6 min, m/z 426 [M
+ H]+), D-allo-Ile (tR 47.4 min, m/z 426 [M + H]+), L-Ile (tR 55.6 min,
m/z 426 [M + H]+), L-allo-Ile (tR 55.6 min, m/z 426 [M + H]+), D-Tyr
(tR 57.6 min, m/z 770 [M + H]+), L-Tyr (tR = 58.7 min, m/z 770 [M +
H]+ for Tyr bis derivatives resulting from FDLA reactions with both
the amino and hydroxyphenyl groups of Tyr).11a Comparison of the
retention times and ion peaks of the D-FDLA derivatives for the
hydrolysate of compound 1, cyclo-(Tyr-Ile-Leu-Val1-Val2-Val3), with
those from the amino acid standards indicated the presence of L-Tyr
(tR 58.8 min, m/z 770 [M + H]+), L-Ile or L-allo-Ile or L-Leu (tR 55.7
min, m/z 426 [M + H]+), D-Leu (tR 48.3 min, m/z 426 [M + H]+), D-
Val (tR 44.3 min, m/z 412 [M + H]+), and L-Val (tR 52.8 min, m/z 412
[M + H]+). Similarly, LCMS analysis of the D-FDLA derivatives of the
hydrolysate of compound 2, cyclo-(Tyr-Val4-Leu-Val1-Val2-Val3),
revealed the presence of L-Tyr (tR 58.8, m/z 770 [M + H]+), D-Leu
(tR 48.3 min, m/z 426 [M + H]+), D-Val (tR 43.8 min, m/z 412 [M +
H]+), and L-Val (tR 52.8 min, m/z 412 [M + H]+).
Total Synthesis of Nocardimide B. The nocardiamide B was also
subjected to solid-phase synthesis according to the designed linear
sequence of L-Tyr-L-Val4-D-Leu-D-Val1-L-Val2-D-Val3, using the same
method as described for the synthesis of prenocardiamide A (3),
yielding 191 mg of the linear hexapeptide prenocardiamide B (4)
(47.8%). The subsequent cyclization of 4 afforded 19.9 mg of synthetic
nocardiamide B (2) (10.7%).
Bioassays. Minimum inhibitory concentration (MIC) values for
compounds 1−4 were determined against seven strains (Escherichia
coli ATCC 25922, Staphylococcus aureus ATCC 29213, Enterococcus
faecalis ATCC 29212, Bacillus thuringensis SCSIO BT01, Bacillus
subtilis SCSIO BS01, Micrococcus luteus SCSIO ML01, and Candida
albicans ATCC 10231) by previously described methods.33 Cytotox-
icities of compounds 1 and 2 were assayed against HCT-116 tumor
cell lines by previously described methods.34
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
Molecular Modeling. In order to determine the configurations of
the triple-Val subunit in nocardiamide A (1), the energy minimization
699
dx.doi.org/10.1021/np400009a | J. Nat. Prod. 2013, 76, 694−701

Journal of Natural Products
Article
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: +86 20 89023038. E-mail: czhang2006@gmail.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We sincerely appreciate Prof. W. Fenical (Scripps Institution of
Oceanography, UCSD) for providing laboratory facilities to
initiate this work, his insightful advice to support this work, and
his critical reading of the manuscript. We thank Dr. P. Jensen’s
group (SIO) for the generous gift of the strain CNX037. We
also thank C. Kauffman (SIO) for fermentation assistance and
L. Paul (SIO) for performing the HCT-116 cytotoxicity
bioassay. This study was supported in part by the Chinese
Academy of Sciences for Key Topics in Innovation Engineering
(KZCX2-YW-JC202, KSCX2-EW-G-12), the Ministry of
Science and Technology of China (2012AA091606,
2010CB833805), and National Science Foundation of China
(41006089, 31125001). Z.W. was supported by a scholarship
under the State Scholarship Fund (2010491110) from the
China Scholarship Council. C.Z. is a scholar of the “100 Talents
Project” of the Chinese Academy of Sciences (08SL111002).
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