Nicrophorusamides A and B, Antibacterial Chlorinated Cyclic Peptides from a Gut Bacterium of the Carrion Beetle Nicrophorus concolor

Article abstract of DOI:10.1021/acs.jnatprod.7b00506

Nicrophorusamides A and B (1 and 2) were discovered from a rare actinomycete, Microbacterium sp., which was isolated from the gut of the carrion beetle Nicrophorus concolor. The structures of the nicrophorusamides were established as new chlorinated cyclic hexapeptides bearing uncommon amino acid units mainly based on 1D and 2D NMR spectroscopic analysis. The absolute configurations of the amino acid residues 5-chloro-l-tryptophan, d-threo-β-hydroxyasparagine/d-asparagine, l-ornithine, l-allo-isoleucine, d-leucine, and d-valine were determined using Marfey's method and chemical derivatization with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate followed by LC/MS analysis. Nicrophorusamide A (1) showed antibacterial activity against several Gram-positive bacteria.

Full text of DOI:10.1021/acs.jnatprod.7b00506

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX-XXX
pubs.acs.org/jnp
Nicrophorusamides A and B, Antibacterial Chlorinated Cyclic
Peptides from a Gut Bacterium of the Carrion Beetle Nicrophorus
concolor
†
,∥
‡
§,∥
§
⊥
†
Yern-Hyerk Shin, Suhyun Bae, Jaehoon Sim, Joonseong Hur, Shin-Il Jo, Jongheon Shin,
Young-Ger Suh,§ Ki-Bong Oh, and Dong-Chan Oh*
‡
,†
†Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826,
Republic of Korea
‡
Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, 1 Gwanak-ro,
Gwanak-gu, Seoul 08826, Republic of Korea
§
College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
∥
College of Pharmacy, CHA University, 120 Haeryong-ro, Pocheon-si, Gyeonggi-do 11160, Republic of Korea
⊥
Animal Welfare Division, Seoul Zoo, Seoul Grand Park, 102 Daegongwongwangjang-ro, Gwacheon-si, Gyeonggi-do 13829, Republic
of Korea
*
S Supporting Information
ABSTRACT: Nicrophorusamides A and B (1 and 2) were discovered from a rare actinomycete, Microbacterium sp., which was
isolated from the gut of the carrion beetle Nicrophorus concolor. The structures of the nicrophorusamides were established as new
chlorinated cyclic hexapeptides bearing uncommon amino acid units mainly based on 1D and 2D NMR spectroscopic analysis.
The absolute configurations of the amino acid residues 5-chloro-L-tryptophan, D-threo-β-hydroxyasparagine/D-asparagine, L-
ornithine, L-allo-isoleucine, D-leucine, and D-valine were determined using Marfey’s method and chemical derivatization with
2
,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate followed by LC/MS analysis. Nicrophorusamide A (1) showed
antibacterial activity against several Gram-positive bacteria.
nvestigating the secondary metabolites produced by bacteria
entomopathogenic bacteria originating from carrion have not
yet been clearly elucidated. Previous chemical studies of
symbionts in beetle-type insects (order: Coleoptera) led to
the discovery of structurally novel bioactive compounds,
including antimicrobial compounds. For example, an antifungal
polyene peroxide was isolated from a symbiotic Streptomyces sp.
I
associated with insect hosts has become a promising method
1
of discovering novel bioactive small molecules. Carrion beetles
(
Coleoptera, Silphidae) are ecologically interesting because
2
they utilize vertebrate carrion to rear their offspring. Carrion
beetles are, thus, exposed to carrion-borne bacteria, which may
3
be pathogenic to them during development. A recent analysis
4
in the southern pine beetle Dendroctonus frontalis. Addition-
of the gut microbiome of six carrion beetle species belonging to
the genus Nicrophorus indicated that these carrion-feeding
beetles harbor gut microbial communities that are distinctively
different from those of herbivorous, xylophagous, humivorous,
ally, bacterial strains associated with the dung beetle Copris
tripartitus produced a dichlorinated indanone that inhibits
5
histone demethylase and the highly modified cyclic peptides
6
2
coprisamides A and B. In this context, we assumed that the gut
omnivorous, and predatory beetles. Although that study
proposed hypothetical roles of carrion beetles’ gut symbionts,
2
such as carcass degradation, detoxification, and defense, the
Received: June 13, 2017
mechanisms by which carrion beetles defend themselves against
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00506
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
symbiotic bacteria of the carrion beetle Nicrophorus concolor
may be a potential source of antimicrobial compounds.
Therefore, we extracted the intestinal parts from an N. concolor
specimen and isolated the bacterial strains, targeting chemically
prolific actinobacteria. The isolated actinobacterial strains were
cultivated and subjected to chemical analysis. During our
chemical analysis, a rare actinomycete strain (UTG9) belonging
to the genus Microbacterium was found to produce a series of
previously unreported compounds based on ultraviolet (UV)
spectroscopic data and low-resolution electrospray ionization
mass spectrometry (LRESIMS) data with a characteristic
isotopic pattern corresponding to chlorination. This initial
chemical evaluation prompted a large-scale cultivation (72 L)
and deeper chemical investigation by chromatographic
purification and spectroscopic analysis of the two major
compounds: nicrophorusamides A and B (1 and 2). Here, we
report the structures and antibacterial activity of nicrophor-
usamides A and B.
NMR data of nicrophorusamide A (1) also displayed 15
aliphatic carbon resonances at δ
39.6−11.3, including six
C
methyl carbons (δ 22.7, 21.8, 18.8, 18.5, 14.5, and 11.3). All
C
1
13
the one-bond H− C correlations were established by
1
13
analyzing the H, C, and HSQC NMR spectral data together.
Because nicrophorusamide A (1) was revealed to be a
peptide with several amino acid units, individual amino acid
moieties were elucidated by interpreting COSY, TOCSY, and
HMBC NMR data. A 2-NH (δ 7.98)/H-2 (δ 4.62) COSY
H
H
correlation connected the nitrogen to the C-2 α-carbon (δC
5
2
3.5). The COSY correlations from H-2 to H -3 (δ 3.11 and
.89) showed connectivity between the α-carbon and β-carbon
2 H
of the amino acid unit. The HMBC correlations from H -3 to
2
C-4 (δ 109.8), C-5 (δ 125.4), and C-11 (δ 128.4) revealed
C
C
C
that this amino acid has an aromatic ring structure in its side
chain group.
The aromatic ring was constructed by analyzing COSY and
1
1
HMBC NMR data. A H− H coupling between H-7 (δ 7.32
H
[
d, J = 8.5 Hz]) and H-8 (δH 7.05 [dd, J = 8.5, 2.0 Hz])
established the C-7−C-8 connectivity. The J
3
value (8.5
H7H8
Hz) indicated that this spin system belongs to a six-membered
aromatic ring. This six-membered aromatic ring was assigned
based on three-bond HMBC correlations from H-7 to C-9 and
C-11, from H-8 to C-6 and C-10, and from H-10 to C-6 and C-
8
. The other aromatic proton, H-5 (δ 7.15), correlated with 5-
H
NH (δ 11.03) in the COSY NMR data, allowing for C-5-N
H
connectivity. The HMBC correlations from H-5 to C-4, C-6
(
δC 134.5), and C-11 and from 5-NH to C-4, C-6, and C-11
indicated an indole ring structure, thereby identifying a
tryptophan moiety (Figure 1a). An array of COSY correlations
RESULTS AND DISCUSSION
Nicrophorusamide A (1) was purified as a white powder, and
the molecular formula was deduced as C H ClN O , which
■
3
7
56
9
8
Figure 1. Structure determination of the uncommon amino acid units
in 1 based on COSY and HMBC correlations: (a) 5-chlorotryptophan,
has an unsaturation number of 14, based on high-resolution fast
atom bombardment MS (HRFABMS) data. The H and
HSQC NMR spectra of 1 identified 11 exchangeable protons
1
(b) β-hydroxyasparagine, and (c) ornithine.
(
7
δH 11.03, 8.43, 7.98, 7.87, 7.81, 7.66 (2H), 7.62, 7.45, 7.31, and
.28) and four aromatic protons (δ 7.57, 7.32, 7.15, and 7.05)
H
in the downfield region below δ 7.0. One more heteroatom-
between 13-NH (δ
and 14-OH (δ 5.85) showed 13-NH-C-13-C-14-OH con-
nectivity. This spin system was also confirmed by their TOCSY
H H H
7.62), H-13 (δ 4.60), H-14 (δ 4.47),
H
bound proton was detected at δ 5.85, and six α-amino proton
H
H
resonances were observed at δ 4.7−3.5 (δ 4.62, 4.60, 4.24,
H
H
1
13
4
.23, 4.01, and 3.75). This initial analysis indicated that
correlations. The two-bond H-13/C-12 (δ
coupling along with HMBC correlations from 15-NH
7.28) and H-14 to C-15 (δ 173.2) indicated β-hydroxyaspar-
C
169.4) H− C
nicrophorusamide A is likely a peptide-derived compound with
an aromatic ring structure. In addition, the H NMR and
2
b (δ
H
1
C
HSQC data revealed the existence of a methine proton (δH
agine (Figure 1b). An ornithine unit was assigned based on the
1
1
4
.47) directly bonded to an oxygen-bearing carbon (δ 70.8).
H− H couplings of 17-NH (δ
H
8.43), H-17 (δ
H
4.01), H
2
-18
C
1
Further analysis of the H NMR and multiplicity-edited HSQC
spectra of 1 identified six aliphatic methylene and three
methine protons between 3.11 and 1.16 ppm and six methyl
(δ 1.84 and 1.64), H -19 (δ 1.50), H -20 (δ 2.79), and 20-
H 2 H 2 H
NH (δ 7.66) in the COSY and TOCSY NMR data, along
2
H
with an H-17/C-16 (δ 170.6) HMBC correlation (Figure 1c).
C
groups (δ 0.87, 0.86, 0.85, 0.83, 0.72, and 0.69).
Other amino acid units, including valine, leucine, and
isoleucine, were identified by further analyzing COSY,
TOCSY, HSQC, and HMBC NMR data. Based on these
results, nicrophorusamide A is a peptide with six amino acid
units. After elucidating the six amino acids, the chlorine atom
was assigned at the carbon C-9 in the tryptophan unit based on
the molecular formula and the 13C chemical shift, correspond-
ing to 5-chlorotryptophan (Figure 1a).
H
13
The C NMR data of 1 (in DMSO-d at 125 MHz) revealed
6
seven carbonyl carbon signals (δ 173.2, 172.1, 171.7, 171.1,
C
1
1
70.6, 170.4, and 169.4), eight aromatic carbon resonances (δC
34.5, 128.4, 125.4, 122.9, 120.7, 117.5, 112.7, and 109.8), one
oxygenated carbon (δC 70.8), and six amino acid α-carbon
peaks (δ 60.4, 56.5, 55.8, 53.5, 53.1, and 51.5), reflecting the
C
13
characteristic features of a peptide-class compound. The
C
B
DOI: 10.1021/acs.jnatprod.7b00506
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Journal of Natural Products
Article
The discrete structures of 5-chlorotryptohan, β-hydroxyas-
paragine, ornithine, valine, leucine, and isoleucine bearing seven
carbonyl carbons, four double bonds, and two rings explain 13
out of 14 double-bond equivalents. Therefore, nicrophorusa-
mide A (1) was deduced to possess an additional ring. The last
ring was constructed by connecting these six amino acid partial
structures based on HMBC and ROESY correlations (Figure
isoleucine and L-allo-isoleucine with GITC and subsequently
comparing their retention times revealed that isoleucine in 1 is
L-allo-isoleucine (Figure S20).
Nicrophorusamide B (2) was isolated as a white powder, the
molecular formula of which was determined to be
1
13
C H ClN O by HRFABMS and 1D H and C NMR
37
56
9
7
spectral data (Table 1). Based on its molecular formula,
nicrophorusamide B (2) has one fewer oxygen atom than 1.
The NMR and UV spectra of 2 displayed high degrees of
similarity to those of 1, indicating that this compound is
analogous to nicrophorusamide A (1). Comparison of 1D and
2). The HMBC correlations from 2-NH of 5-chlorotryptophan
2
D NMR spectroscopic data with those of 1 revealed that D-
threo-β-hydroxyasparagine was replaced with D-asparagine,
whereas the other amino acids were identical. The absolute
configurations of the amino acid units in 2 were deduced to be
identical to those in 1 based on their identical circular
dichroism (CD) data (Figure S16) and the common
biosynthetic origin.
Other cyclic hexapeptides sharing similar amino acid units to
the nicrophorusamides have been reported. For example,
desotamides A−D, discovered from deep-sea-derived Strepto-
myces scopuliridis, contain L-tryptophan, glycine, L-asparagine, L-
9
allo-isoleucine/L-valine, D-leucine, and L-leucine. Their bio-
Figure 2. Identification of the amino acid sequence in 1 based on
HMBC and ROESY NMR spectra.
synthetic gene cluster was also recently reported along with a
10
new analogue produced by heterologous expression. How-
ever, the desotamides and nicrophorusamide A share only L-
allo-isoleucine and D-leucine both in different positions in their
hexapeptide sequences. Another series of cyclic hexapeptides,
wollamides A and B, which were isolated from an Australian
soil Streptomyces, are more similar to the desotamides rather
than the nicrophorusamides; these antimycobacterial com-
pounds contain D-ornithine in place of the glycine found in the
to C-12 of β-hydroxyasparagine showed a linkage between the
5
-chlorotryptophan and β-hydroxyasparagine units. The
HMBC correlation of 13-NH to C-16 revealed the connectivity
between β-hydroxyasparagine and ornithine. Isoleucine was
1
13
located next to ornithine by the H− C long-range correlation
from 17-NH of ornithine to the amide carbon C-21 (δ 171.7)
C
11
of isoleucine. The HMBC signal between 22-NH (δ 7.87), the
destoamides. The most closely related cyclic hexapeptide to
nicrophorusamides (1 and 2) is longicatenamycin A, an
antibacterial compound previously discovered from Streptomy-
H
amide proton of isoleucine, and C-27 (δ 172.1), the carbonyl
C
carbon of leucine, indicated that leucine can have connectivity
with isoleucine. The valine unit was placed adjacent to leucine
12
ces diastaticus strain S-520. Longicatenamycin A is a cyclic
hexapeptide containing 5-chlorotryptohan and ornithine as
found in 1 and 2, but with glycine, homohomoleucine, valine,
and β-hydroxyglutamic acid replacing the valine, leucine,
isoleucine, and β-hydroxyasparagine of the nicrophorusamide
A. The absolute configuration of the amino acids in 1 is
opposite that of the amino acids in longicatenamycin A, of
1
13
by long-range H− C couplings from 28-NH (δ 7.45) to C-
H
3
3 (δ 170.4). A macrocyclic ring was closed by an HMBC
C
correlation from 34-NH (δ 7.81) to C-1, completing the cyclic
H
hexapeptide structure of nicrophorusamide A (1). ROESY
correlations between amide NH’s and α-protons further
confirmed the amino acid sequence of 1 identified based on
the HMBC correlations (Figure 2).
12b
which the structure was confirmed by total synthesis.
The absolute configurations at the α-carbons of the six amino
acid units were determined by applying the advanced Marfey’s
method with the L- and D-forms of 1-fluoro-2,4-dinitrophenyl-5-
Nicrophorusamide A (1) has the sequence L-D-D-L-L-D, whereas
longicatenamycin A possesses the sequence D-NA-L-D-D-L.
The biological activities of the nicrophorusamides were
evaluated against pathogenic bacterial strains (Table 2).
Nicrophorusamide A (1) showed antibacterial activity against
Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC
19433, Enterococcus faecium ATCC 19434, and Salmonella
enterica ATCC 14028 with minimum inhibitory concentration
(MIC) values of 8−16 μg/mL. Nicrophorusamide B (2), which
bears D-asparagine instead of D-threo-β-hydroxyasparagine,
displayed 8 times lower activity against these bacteria than 1,
implying that D-threo-β-hydroxyasparagine could play a
significant role in determining bioactivity. The structural
importance of the hydroxy group in nicrophorusamide A (1)
can be rationalized by the conformational difference between
nicrophorusamides A and B. Careful analysis of the ROESY
NMR spectra and density functional theory (DFT) modeling
for the energy minimization of nicrophorusamide A (1) clearly
reveals a ROESY correlation between the hydroxy proton (14-
OH) of D-threo-β-hydroxyasparagine and the terminal amine
7
alanine amide (FDAA). Nicrophorusamide A (1) was
hydrolyzed to obtain free amino acids, which were derivatized
with L- and D-FDAA and analyzed by LC/MS. Comparing the
LC/MS analysis results revealed 5-chloro-L-tryptophan, D-β-
hydroxyasparagine, L-ornithine, L-isoleucine, D-leucine, and D-
valine (Table S1, Figures S17 and S18).
An additional chiral center at C-14 of D-β-hydroxyasparagine
had to be subjected to further stereochemical analysis. As β-
hydroxyasparagine is converted to β-hydroxyaspartic acid
during acid hydrolysis, samples of both threo-β-hydroxyaspartic
acid and erythro-β-hydroxyaspartic acid (see the Supporting
Information) were prepared and compared to the product from
1
using LC/MS; the latter was shown to be D-threo-β-
hydroxyaspartic acid, thus determining a 14R configuration. To
elucidate the absolute configuration at C-23 of the L-isoleucine
unit, 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate
8
(
GITC) derivatization was used. Derivatizing authentic L-
C
DOI: 10.1021/acs.jnatprod.7b00506
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Journal of Natural Products
Article
Table 1. H and 13C NMR Data for Nicrophorusamide A and B (1 and 2) in DMSO-d6
1
a
b
1
2
position
δ ,
C
type
δ ,
H
mult (J in Hz)
δ ,
C
type
δ ,
H
mult (J in Hz)
1
171.1,
C
170.8,
C
2
2
3
3
4
5
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
53.5,
CH
4.62,
7.98,
3.11,
2.89,
ddd (7.5, 7.5, 7.5)
d (7.5)
53.4,
CH
4.51,
7.80,
3.18,
2.88,
ddd (7.5, 7.5, 7.5)
d (7.5)
-NH
a
26.7,
CH2
dd (14.5, 7.5)
dd (14.5, 7.5)
26.5,
CH2
dd (14.5, 7.5)
dd (14.5, 7.5)
b
109.8,
125.4,
C
109.8,
125.7,
C
CH
7.15,
d (2.0)
d (2.0)
CH
7.18,
d (2.0)
d (2.0)
-NH
11.03,
11.06,
134.5,
112.7,
120.7,
122.9,
117.5,
128.4,
169.4,
55.8,
C
134.5,
112.8,
120.7,
123.0,
117.5,
128.4,
170.6,
49.3,
C
CH
CH
C
7.32,
7.05,
d (8.5)
CH
CH
C
7.32,
7.04,
d (8.5)
dd (8.5, 2.0)
dd (8.5, 2.0)
0
CH
C
7.57,
d (2.0)
CH
C
7.54,
d (2.0)
1
2
C
C
3
CH
4.60,
7.62,
4.47,
5.85,
dd (8.5, 2.5)
d (8.5)
CH
4.55,
7.95,
2.56,
dd (8.0, 6.0)
d (8.0)
3-NH
4
70.8,
CH
C
dd (6.0, 2.5)
d (6.0)
36.0,
CH2
C
d (6.0)
4-OH
5
173.2,
171.8,
5-NH2a
7.31,
7.28,
d (2.0)
d (2.0)
5-NH2b
6
170.6,
53.1,
C
170.5,
52.3,
C
7
CH
4.01,
8.43,
1.84,
1.64,
1.50,
2.79,
7.66,
ddd (7.5, 7.5, 6.0)
CH
4.19,
7.81,
1.85,
1.60,
1.52,
2.79,
7.76,
ddd (7.5, 7.5, 6.0)
7-NH
d (6.0)
m
d (7.5)
m
8a
26.8,
CH2
28.0,
CH2
8b
m
m
9
23.7,
38.4,
CH2
CH2
m
23.8,
38.5,
CH2
CH2
m
0
br s
br s
m
0-NH2
t (7.0)
1
171.7,
56.5,
C
171.2,
57.1,
C
2
CH
4.24,
7.87,
1.89,
1.35,
1.16,
0.86,
0.85,
dd (7.5, 6.0)
CH
4.11,
8.05,
1.92,
1.29,
1.19,
0.84,
0.85,
dd (7.5, 7.0)
2-NH
d (7.5)
m
d (7.0)
m
3
36.3,
25.6,
CH
35.9,
25.7,
CH
4a
CH2
m
CH2
m
4b
m
m
5
11.3,
14.5,
CH3
CH3
C
d (7.5)
d (7.5)
11.5,
14.6,
CH3
CH3
C
d (7.0)
d (7.0)
6
7
172.1,
51.5,
172.8,
51.4,
8
CH
4.23,
7.45,
1.57,
1.47,
0.87,
0.83,
dd (7.5, 7.5)
d (7.5)
CH
4.43,
7.61,
1.50,
1.49,
0.90,
0.85,
dd (7.0, 6.5)
d (7.0)
m
8-NH
9
39.6,
24.3,
22.7,
21.8,
170.4,
60.4,
CH2
CH
CH3
CH3
C
dd (7.5, 7.5)
m
41.0,
24.2,
22.5,
22.6,
170.4,
59.8,
CH2
CH
CH3
CH3
C
0
m
1
d (6.5)
d (6.0)
d (7.0)
2
d (6.5)
3
4
CH
3.75,
7.81,
1.84,
0.72,
0.69,
dd (7.0, 7.0)
d (7.0)
m
CH
3.84,
7.92,
1.90,
0.70,
0.64,
dd (8.0, 7.5)
d (8.0)
m
4-NH
5
6
7
29.1,
18.5,
18.8,
CH
29.8,
19.0,
18.1,
CH
CH3
d (6.5)
d (6.5)
CH3
CH3
d (6.5)
d (6.5)
CH3
a
1H 600 MHz, 13C 125 MHz. 1H 500 MHz, 13C 125 MHz.
b
protons (20-NH ) of L-ornithine, which enables an additional
Nicrophorusamides A and B did not inhibit the pathogenic
2
ring to form that is closed by a 14-O−20-NH hydrogen bond
fungi Candida albicans ATCC 10231, Aspergillus fumigatus HIC
(
Figure 3a). In contrast, nicrophorusamide B (2), which lacks
6
094, Trichophyton rubrum NBRC 9185, or T. mentagrophytes
the hydroxy group at C-14, is unable to make a rigid
conformation with the corresponding hydrogen bond (Figure
IFM 40996. In cytotoxicity tests against various human cancer
cell lines, such as A-549, HCT-116, SNU-638, SK-HEP-1,
3b).
D
DOI: 10.1021/acs.jnatprod.7b00506
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Journal of Natural Products
Article
carrion beetles were expelled with a sterilized surgical blade and
diluted with 40 mL of sterilized water to extract the bacterial strains
from the intestines. For bacterial isolation from the guts of the carrion
beetles, the bacterial suspension was spread on various isolation agar
media (actinomycetes isolation agar medium, YPM agar medium, YPG
agar medium, A1 agar medium, K agar medium, Czapek-Dox agar
medium, ISP1 agar medium, ISP4 agar medium, starch-casein agar
medium, chitin-based agar medium, and R2A agar medium), and the
plates were cultivated at 25 °C for more than 14 days. The
actinobacterial strain UTG9, which produces nicrophorusamides A
and B (1 and 2), was separated on starch-casein agar medium (10 g of
soluble starch, 1 g of casein, 0.5 g of K HPO , 18 g of agar, and 100 mg
Table 2. Inhibitory Activities of Nicrophorusamides A and B
(1 and 2) against Bacterial Strains (MIC values in μg/mL)
strain
1
2
ampicillin
S. aureus
E. faecalis
E. faecium
S. enterica
E. coli
8
64
0.13
0.5
16
16
16
128
128
0.25
0.13
8
not active
not active
not active
2
4
MDA-MB-231, and K-562, nicrophorusamides A and B
exhibited no significant cytotoxicity.
of cycloheximide per 1 L of sterilized water). The actinomycete strain
was identified as Microbacterium sp. (97% identity with Microbacterium
paraoxydans) by analyzing its 16S rDNA sequence (GenBank
accession number: MF000987).
The rare actinomycete genus Microbacterium has not been
the subject of many chemical investigations, with most such
13
studies focusing on simple sugars. The only peptide-derived
metabolites isolated from this genus are cytotoxic micro-
Cultivation and Extraction. To obtain nicrophorusamides A and
B (1 and 2) produced by the Microbacterium sp. UTG9 strain, a spore
of the bacterial strain was transferred and inoculated into a 500 mL
flask containing 125 mL of YPM liquid medium (2 g of yeast extract, 2
g of peptone, and 4 g of mannitol per 1 L of sterilized water) and
cultivated on a rotary shaker at 200 rpm and 27 °C. After 2 days of
fermentation, 10 mL of the liquid culture was moved to a 2.8 L large
flask with 1 L of YPM liquid medium for scale-up and incubated on a
rotary shaker at 160 rpm and 27 °C for 6 days. The entire culture (72
L) was extracted with an equivalent volume of ethyl acetate (EtOAc)
in a separation funnel. The EtOAc layer was segregated from the water
layer and dried by a rotary evaporator in vacuo to acquire 10 g of the
crude extract.
Purification of Nicrophorusamides A and B (1 and 2). The
entire extraction was absorbed on Celite and loaded onto 20 g of C18
resin for fractionation. Then, 200 mL aliquots of 20%, 40%, 60%, 80%,
and 100% aqueous methanol (MeOH/aq) were collected, and a
portion of each fraction was analyzed by LC/MS under a gradient
solvent system (flow rate: 0.7 mL/min; UV detection: 210, 230, 254,
14
bacterins A and B from deep-sea-inhabiting M. sediminis. The
nicrophorusamides constitute the second example of peptide-
derived metabolites from the rare actinomycete genus Micro-
bacterium, reflecting the untapped chemical potential of this
relatively unstudied actinomycete.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
obtained at 25 °C using a JASCO P-2000 polarimeter with a 1 cm
cell. UV spectra were obtained using a PerkinElmer LAMBDA 25 UV/
vis spectrophotometer with a 1 cm cuvette. CD spectra were recorded
at 25 °C using an Applied Photophysics Chirascan-Plus circular
dichroism spectrometer and a 1 mm CD cell. Infrared (IR) spectral
1
data were acquired by a JASCO FT/IR-4200 FT-IR spectrometer. H,
C, and 2D NMR experiments were conducted with Bruker Avance
1
3
5
00 and 600 MHz spectrometers. LC/MS data and LRESIMS data
were obtained by an Agilent Technologies 1200 series high-
performance LC (HPLC) coupled with an Agilent Technologies
280, and 360 nm; 10% to 100% aqueous acetonitrile [CH CN/H O]
3 2
with 0.1% formic acid over 20 min) with a Phenomenex column
(Luna, 100 × 4.6 mm, C18, 5 μm). Analyzing the LC/MS data
confirmed that nicrophorusamides A and B (1 and 2) were present in
the 80% and 100% MeOH/aq fractions. To acquire the nicrophor-
usamides, the fractions were purified by semipreparative reversed-
phase HPLC with a YMC column (250 × 10 mm, C18, 5 μm) under
gradient solvent conditions (flow rate: 2 mL/min; UV detection: 280
6
7
130 quadrupole MS. HRFABMS data were acquired on a JEOL JMS-
00 high-resolution MS. The UV/vis spectrophotometer, NMR
spectrometer, and HRFABMS were located at the National Center
for Interuniversity Research Facilities (NCIRF) in Seoul National
University.
Bacterial Isolation. Two identical carrion beetles were collected
using pitfall traps from Maebong Mountain, Seoul, Republic of Korea,
in July 2015. The carrion beetles were identified as N. concolor by
morphological classification. The carrion beetle samples were soaked
in 70% aqueous ethanol (EtOH/aq) for 1 min and rinsed with
sterilized distilled water to eliminate residual EtOH. The guts of the
nm; 35% to 62% CH CN/H O with 0.2% trifluoroacetic acid over 50
3 2
min). Nicrophorusamides A (1, 12 mg) and B (2) eluted at 28.0 and
28.5 min, respectively. Nicrophorusamide B (2) was purified again
with another gradient solvent system (flow rate: 2 mL/min; UV
detection: 280 nm; 30% to 45% CH CN/H O with 0.2% trifluoro-
3
2
Figure 3. Energy-minimized conformations of nicrophorusamides A and B: (a) nicrophorusamide A (1), (b) nicrophorusamide B (2).
E
DOI: 10.1021/acs.jnatprod.7b00506
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
acetic acid over 50 min) for refinement. Finally, pure nicrophor-
usamide B (2, 4 mg) was obtained at 31.0 min after injection.
Nicrophorusamide A (1): white powder; [α] 2D 5 −18 (c 0.2, MeOH);
Phenomenex column (Gemini, 250 × 4.6 mm, C , 5 μm). The GITC
18
derivative eluted at 60.1 min. GITC derivatives of authentic L-
isoleucine (CAS No. 73-32-5, Sigma-Aldrich) and L-allo-isoleucine
(CAS No. 1509-34-8, Sigma) were prepared via the same procedure
and injected into the HPLC using the same analysis conditions. The
GITC derivatives of authentic L-isoleucine and L-allo-isoleucine eluted
at 60.7 and 60.0 min, respectively. Finally, the amino acid residue in 1
was identified as L-allo-isoleucine.
UV (MeOH) λmax (log ε) 228 (3.85), 290 (3.02) nm; IR (neat) ν
max
−
1
1
13
3
693, 3262, 2923, 1679, 1633, 1540, 1206, 1140 cm ; for H and C
NMR spectral data, Table 1; HRFABMS [M + H] m/z 790.4022
calcd for C H ClN O , 790.4019).
+
(
37
57
9
8
2
5
Nicrophorusamide B (2): white powder; [α]D −20 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 228 (3.79), 290 (2.97) nm; IR (neat) ν
Molecular Modeling of Nicrophorusamides A and B (1 and
). To identify the conformational difference between nicrophor-
max
−
1
1
13
2
3
413, 2926, 1682, 1634, 1543, 1207, 1138 cm ; for H and C NMR
spectral data, Table 1; HRFABMS [M + H] m/z 774.4066 (calcd for
+
usamides A and B, computational DFT calculations were performed.
The initial structural energy minimizations of 1 and 2 were established
C H ClN O , 774.4069).
3
7
57
9
7
1
5,16
by using Avogadro 1.2.0 with the UFF force field.
An array of
Determination of the Absolute Configurations at the α-
Carbons of the Amino Acid Units in Nicrophorusamide A (1).
One milligram of 1 was dissolved in 1 mL of 6 N HCl and heated at
energy optimizations were processed via Tmolex 4.3.1 with the DFT
settings (B3-LYP functional/M3 grid size) and geometry optimization
−6
−3
1
15 °C for 1 h. The heated suspension was cooled in a 0 °C ice bath
options (energy 10 hartree, gradient norm |dE/dxyz| = 10 hartree/
17
for 5 min, and the HCl was vaporized in vacuo. To eliminate residual
HCl in the vial, 1 mL of water was added to the vial and then
evaporated under low pressure three times. Subsequently, the
hydrolysate mixture was lyophilized for 24 h and divided into two
vials. Each hydrolysate sample was dissolved in 200 μL of 1 N
bohr).
Antibacterial Activity Assay. The antibacterial activities of
nicrophorusamides A and B (1 and 2) were determined by evaluating
their MIC values; ampicillin was used as a reference compound. Three
species of Gram-positive bacteria (S. aureus ATCC 25923, E. faecalis
ATCC 19433, and E. faecium ATCC 19434) and two species of Gram-
negative bacteria (S. enterica ATCC 14028 and E. coli ATCC 25922)
were used in this study. The bacteria were grown overnight in Mueller
Hinton broth (MHB) at 37 °C, harvested by centrifugation, and
washed twice with sterile distilled water. Each compound was
dissolved in DMSO and diluted with MBH to prepare serial 2-fold
dilutions of 128 to 0.03 μg/mL. The final DMSO concentration was
maintained at 1% by adding DMSO to the medium. In each well of a
NaHCO . Then, 100 μL of 10 mg/mL L-FDAA in acetone was added
3
to one of the two vials, and an equivalent proportion of D-FDAA in
acetone was added to the other vial. The two reaction vials were
heated at 80 °C for 3 min to accelerate the chemical derivatization.
Then, 100 μL of 2 N HCl was added to neutralize both vials, and the
two reaction mixtures were diluted with 300 μL of 50% CH CN/H O
3
2
solution. Ten microliters of each reaction mixture was injected into the
LC/MS under a gradient solvent system (flow rate: 0.7 mL/min; UV
detection: 360 nm; 10% to 60% CH CN/H O with 0.1% formic acid
9
6-well plate, 190 μL of MBH containing 1, 2, and ampicillin was
3
2
7
mixed with 10 μL of broth containing approximately 10 colony-
forming units (cfu)/mL of the test bacteria (final: 5 × 10 cfu/mL).
over 40 min) with a Phenomenex column (Luna, 100 × 4.6 mm, C , 5
18
5
μm) (Table S1). The retention times of the FDAA derivatives of the
free amino acids were observed via LC/MS analysis.
The plates were incubated for 24 h at 37 °C, and the MIC value was
defined as the lowest concentration of the test compound that
inhibited bacterial growth.
Determination of the Absolute Configuration at the β-
Carbon of D-β-Hydroxyasparagine in Nicrophorusamide A (1).
Of the four diastereomers of β-hydroxyaspartic acid, only L-threo-β-
hydroxyaspartic acid [(2S,3R)-2-amino-3-hydroxysuccinic acid, CAS
No. 7298-99-9, Tocris] is commercially available. L-erythro-β-
Hydroxyaspartic acid [(2S,3S)-2-amino-3-hydroxysuccinic acid] was
synthesized via several reaction steps as described in the Supporting
Information. These two synthetic amino acids were derivatized with L-
FDAA and injected into LC/MS under a gradient solvent system (flow
rate: 0.7 mL/min; UV detection: 360 nm; 10% to 30% CH CN/H O
ASSOCIATED CONTENT
■
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00506.
3
2
1
D and 2D NMR spectra, CD spectra, HRMS data,
in water solution with 0.1% formic acid over 40 min) to compare
retention times. The retention times of the L-FDAA derivatives of L-
threo-β-hydroxyaspartic acid (2S,3S-L) and L-erythro-β-hydroxyaspartic
acid (2S,3R-L) were 19.3 and 26.8 min, respectively. To elucidate the
absolute configuration at the β-carbon of D-β-hydroxyasparagine in 1,
these L-FDAA derivatives were co-injected with the D-FDAA derivative
of D-β-hydroxyaspartic acid in the hydrolysate of 1. The LC/MS
results revealed that the D-FDAA derivative of D-β-hydroxyaspartic acid
from the hydrolysate of 1 showed the identical retention time with the
L-FDAA derivative of authentic L-threo-β-hydroxyaspartic acid (Figure
S19). This result demonstrated that the D-FDAA derivative of D-β-
hydroxyaspartic acid (2R-2-amino-3-hydroxysuccinic acid) from the
hydrolysate of 1 is an enantiomer of the L-FDAA derivative of
authentic L-threo-β-hydroxyaspartic acid, thus possessing a 3R
configuration. Therefore, D-β-hydroxyasparagine in nicrophorusamide
A (1) was elucidated as D-threo-β-hydroxyasparagine.
energy-minimized coordinates for 1 and 2, synthesis of L-
β-hydroxyaspartic acid, detailed procedure for determi-
nation of absolute configurations of amino acid units
(PDF)
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: +82-2-880-2491. Fax: +82-2-762-8322. E-mail:
dongchanoh@snu.ac.kr.
ORCID
Jongheon Shin: 0000-0002-7536-8462
Young-Ger Suh: 0000-0003-1799-8607
Dong-Chan Oh: 0000-0001-6405-5535
Determination of the Absolute Configuration of the β-
Carbon of L-Isoleucine in Nicrophorusamide A (1). The
hydrolysate of 1 was obtained by the same procedures described
above and dissolved in 1 mL of water. For the chemical derivatization
of the L-isoleucine residue in 1, 100 μL of triethylamine and 100 μL of
Notes
The authors declare no competing financial interest.
1
% GITC solution in acetone were added to the reaction vial. The
ACKNOWLEDGMENTS
■
reaction was processed at room temperature (25 °C) for 15 min and
quenched with 100 μL of 5% acetic acid solution. Then, 20 μL of the
reaction mixture was analyzed by HPLC under a gradient solvent
system (flow rate: 0.3 mL/min; UV detection: 254 nm; 35% to 50%
This work was supported by the National Research Foundation
of Korea Grants funded by the Korean Government (Ministry
of Science and ICT) (2014R1A2A1A11053477 and
2017R1A2B2009393).
CH CN in water with 0.1% formic acid over 80 min) with a
3
F
DOI: 10.1021/acs.jnatprod.7b00506
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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DOI: 10.1021/acs.jnatprod.7b00506
J. Nat. Prod. XXXX, XXX, XXX−XXX