Coprisamides A and B, new branched cyclic peptides from a gut bacterium of the dung beetle Copris tripartitus

Article abstract of DOI:10.1021/acs.orglett.5b00249

Coprisamides A and B (1 and 2) were isolated from a bacterium in the gut of the dung beetle Copris tripartitus. Spectroscopic analysis revealed that the planar structures of 1 and 2 are novel cyclic heptapeptides bearing unusual units, such as β-methylaspartic acid and 2,3-diaminopropanoic acid branched to valine and 2-heptatrienyl cinnamic acid. Absolute configurations were established by chemical derivatization and chiroptical spectroscopy. The coprisamides displayed significant activity for induction of quinone reductase.

Full text of DOI:10.1021/acs.orglett.5b00249

Letter
pubs.acs.org/OrgLett
Coprisamides A and B, New Branched Cyclic Peptides from a Gut
Bacterium of the Dung Beetle Copris tripartitus
Soohyun Um,† So Hyun Park,† Jihye Kim,† Hyen Joo Park,† Keebeom Ko,† Hea-Son Bang,‡
Sang Kook Lee,† Jongheon Shin,† and Dong-Chan Oh*,†
†Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul, 151-742, Republic of Korea
‡Department of Agricultural Environment, National Academy of Agricultural Science, Jeonju, 560-500, Republic of Korea
S
* Supporting Information
ABSTRACT: Coprisamides A and B (1 and 2) were isolated from a bacterium in the
gut of the dung beetle Copris tripartitus. Spectroscopic analysis revealed that the
planar structures of 1 and 2 are novel cyclic heptapeptides bearing unusual units, such
as β-methylaspartic acid and 2,3-diaminopropanoic acid branched to valine and 2-
heptatrienyl cinnamic acid. Absolute configurations were established by chemical
derivatization and chiroptical spectroscopy. The coprisamides displayed significant
activity for induction of quinone reductase.
B (1 and 2), which incorporate unusual amino acids and a new
2-alkenyl cinnamic acid acyl chain.
acteria associated with insect symbiotic systems are a
Bstrategic source for the discovery of new bioactive small
molecules.1 Structurally novel compounds from the symbiotic
bacteria of insects include the polyene peroxide mycangimycin
from a Streptomyces symbiont of the southern pine beetle
Dendroctonus frontalis2 and the cyclic peptide dentigerumycin
from Pseudonocardia sp. on the exoskeleton of the fungus-
growing ant Apterostigma dentigerum.3 Because the biological
diversity of insects is tremendously high,4 it could be promising
to study diverse insects and associated bacteria for chemical
discovery. Our recent studies have demonstrated that bacteria
in the dung beetle ecosystem are also a promising source of
novel organic compounds.5−8 For example, we have reported
tripartilactam, a cyclobutane-bearing macrocyclic lactam, as a
Na+/K+ ATPase inhibitor5 and tripartin, a dichlorinated
indanone, as a specific histone demethylase inhibitor6 from
actinomycete strains isolated from the ecosystem of the dung
beetle Copris tripartitus Waterhouse. However, chemical
investigations of insect gut bacteria have not been performed.
The insect gut is very densely colonized, and gut microbiota
play crucial roles in an insect’s life, such as in digestion.9 To
investigate the potential of insect gut microbiota to harbor
chemically interesting bacteria, we isolated bacterial strains
from the dung beetle Copris tripartitus. Then, the secondary
metabolites produced by the cultivated bacterial strains were
analyzed by LC/MS. In the chemical analysis, we identified a
Streptomyces strain, SNU533, isolated from the gut of a male
adult specimen of C. tripartitus collected in Jeju Island, Republic
of Korea, that produced a previously unreported compound
based on UV (λmax 282 nm) and mass spectra ([M + H]+ at m/
z 907). Further scale-up of the culture of SNU533 and isolation
of the compound and its analog resulted in the discovery of the
structurally novel branched cyclic peptides coprisamides A and
Coprisamide A (1) was obtained as a yellow powder with the
molecular formula C46H66N8O11 and 18 degrees of unsatura-
1
tion, as determined by H and 13C NMR spectroscopy (Table
1) and high-resolution FAB mass spectrometry ([M + H]+ m/z
1
907.4927, calculated 907.4929). The H NMR spectrum (in
CD3OH) included seven exchangeable amide proton signals
[δH 8.78, 8.55, 8.54, 8.50, 8.43, 8.37, and 7.49] and seven α-
amino proton signals [δH 4.80, 4.52, 4.36, 4.32, 4.28, 3.87, and
3.66], consistent with a peptide-derived compound. The 13C
NMR spectrum also consistently showed nine amide/ester/
carboxylic acid carbonyl carbon signals [δC 178.5, 176.3, 174.5,
172.8, 171.3, 170.9, 170.9, 169.6, and 168.7] and seven α-
carbon resonances [δC 60.2, 60.0, 56.6, 54.9, 52.9, 52.5, and
1
51.3]. Further analysis of the H NMR spectrum revealed that
coprisamide A possesses eight olefinic signals [δH 7.75, 7.05,
6.52, 6.39, 6.35, 6.24, 6.01, and 5.77] and ortho-substituted
aromatic ring protons [δH: 7.62 (d, J = 7.5), 7.34 (dd, J = 7.5,
7.5), 7.28 (d, J = 7.5), and 7.25 (dd, J = 7.5, 7.5)], which are
atypical for an ordinary peptide.
Analysis of the HSQC NMR spectrum readily assigned all
1
the one-bond H−13C correlations. The subsequent interpre-
tation of the COSY and HMBC NMR correlations resulted in
eight partial structures dissected by amide or ester carbonyl
carbons. These included five common amino acids: two valines,
a leucine, a serine, and an alanine. The other three partial
structures were uncommon amino acids and an unusual acyl
chain.
First, the COSY NMR spectrum showed one spin system
composed of 2-NH [δH 8.43], H-2 [δH 3.87], H-3 [δH 3.10],
Received: January 26, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b00249
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Letter
a
Table 1. NMR Data for Coprisamide A (1) in CD3OH
no.
δH
mult (J in Hz)
δC
type
1
2
170.9
56.6
C
3.87
8.43
3.10
1.05
dd (10.0, 7.0)
d (7.0)
CH
2-NH
3
qd (10.0, 7.0)
d (7.0)
40.0
CH
CH3
C
4
15.4
5
178.5
170.9
51.3
6
C
b
7
4.80
8.50
4.39
2.95
8.54
m
CH
7-NH
8a
d (9.5)
m
41.0
CH2
8b
ddd (12.5, 6.0, 3.0)
d (6.0)
8-NH
9
and H3-4 [δH 1.05]. HMBC correlations from H3-4 to the
carboxylic acid carbonyl carbon C-5 [δC: 178.5] and from H-2
to C-1 [δC 170.9] identified β-methylaspartic acid (Figure 1a).
172.8
60.0
C
10
4.36
8.55
2.48
0.94
0.87
m
CH
10-NH
11
d (6.0)
m
29.5
19.7
16.9
176.3
54.9
CH
CH3
CH3
C
12
d (7.0)
d (7.0)
13
14
15
4.28
8.78
1.64
1.50
1.57
0.95
0.91
m
CH
15-NH
16a
16b
17
d (3.0)
m
40.1
CH2
m
m
25.6
22.3
23.1
171.3
52.9
CH
CH3
CH3
C
Figure 1. Determination of the unusual partial structures in 1. (a) β-
Methylaspartic acid, (b) 2,3-diaminopropanoic acid, (c) 2-heptatrienyl
cinnamic acid (HTCA).
18
d (6.5)
d (6.5)
19
20
b
21
4.52
7.49
5.29
3.94
br dd (7.5, 2.0)
CH
Moreover, the COSY correlations from H-7 [δH 4.80] to 7-NH
[δH 8.50] and H2-8 [δH 4.39, 2.95] identified 7-NH-C-7-C-8
connectivity. This unit was further extended to 8-NH [δH 8.54]
by the 1H−1H coupling between H2-8 and 8-NH and to C-6 by
the HMBC correlation from H-7 to C-6 [δC 170.9], assigning
the 2,3-diaminopropanoic acid (Dpr) (Figure 1b). The initially
identified olefinic and aromatic signals suggested an unusual
acyl chain. The COSY correlations of H-27 to H-28 and H-35
21-NH
22a
22b
23
d (7.5)
dd (11.0, 2.0)
br d (11.0)
64.4
CH2
174.5
52.5
C
24
4.32
8.37
1.45
qd (7.0, 6.5)
d (6.5)
CH
24-NH
25
d (7.0)
16.8
CH3
C
26
168.7
122.3
140.3
134.3
127.5
128.5
130.3
131.6
139.1
127.7
133.4
127.0
137.3
132.9
132.2
18.1
1
to H-41 indicated two olefinic moieties. The H−1H coupling
27
7.05
7.75
d (16.0)
d (16.0)
CH
CH
C
of the aromatic protons H-30, H-31, H-32, and H-33 and their
HMBC correlations identified an ortho-substituted aromatic
ring and assigned this ring to the middle of the two olefinic
partial structures. Thus, the last unit was revealed as 2-
heptatrienyl cinnamic acid (HTCA), which has not been
previously reported (Figure 1c).
28
29
30
7.62
7.25
7.34
7.28
d (7.5)
CH
CH
CH
CH
C
31
dd (7.5, 7.5)
dd (7.5, 7.5)
d (7.5)
32
33
The geometry of the double bond at C-27 in HTCA in 1 was
34
1
assigned as E by the H−1H coupling constant (16.0 Hz)
35
6.52
6.39
6.24
6.35
6.01
5.77
1.73
d (11.0)
CH
CH
CH
CH
CH
CH
CH3
C
between H-27 and H-28. The cis-coupling constant (11.0 Hz)
between H-35 and H-36 assigned the 35Z geometry. The large
36
dd (11.0, 11.0)
dd (15.0, 11.0)
dd (15.0, 11.0)
dd (15.0, 11.0)
qd (15.0, 7.0)
d (7.0)
37
1
vicinal H−1H coupling constants (C-37 and C-38, JHH = 15.0
38
Hz; C-39 and C-40, JHH = 15.0 Hz) confirmed the geometries
of 37E and 39E. All geometry assignments were further
supported by ROESY correlations.
39
40
41
The connectivity of the eight partial structures (two valines, a
leucine, a serine, an alanine, 2,3-diaminopropanoic acid, β-
methylaspartic acid, and HTCA) was established by analysis of
the HMBC and ROESY correlations (Figure 2). The HMBC
correlation from the 2-NH [δH 8.43] of β-methyl-Asp to the C-
6 [δC 170.9] belonging to Dpr established the connectivity
between β-methyl-Asp and Dpr. The connectivity between Dpr
and Val-1 was deduced by long-range heteronuclear couplings
from the 7-NH [δH 8.50] of Dpr and the α-proton [H-10; δH
42
169.6
60.2
43
3.66
2.19
1.07
1.02
d (4.5)
m
CH
CH
CH3
CH3
44
31.7
45
d (7.0)
d (7.0)
18.7
46
17.2
a
1H and 13C NMR were recorded at 600 and 150 MHz, respectively.
Measured in CD3OD.
b
1
4.36] of Val-1 to C-9 [δC 172.8]. The long-range H−13C
B
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Figure 3. (a) ΔδS‑R values in ppm for the S- and R-PGME amide
products (3a and 3b) in CD3OD. (b) J-based configuration analysis of
C-2 and C-3.
Coprisamide B (2) was isolated as a yellow powder along
with coprisamide A (1) during HPLC purification. Its
molecular formula was determined as C46H66N8O11, which is
identical to that of 1. The HR-FAB mass spectrum ([M + H]+
m/z 907.4919, calculated 907.4929) and 1H and 13C NMR data
(Table S2) were obtained. The analysis of the 1D and 2D
NMR data revealed that the gross structure of 2 is identical to
Figure 2. Key HMBC and ROESY correlations of 1.
coupling from the amide proton 10-NH [Val-1; δH 8.55] to C-
14 [Leu; δC 176.3] connected Val-1 to Leu. Both the 15-NH
[δH 8.78] of Leu and H-21 [δH 4.52], the α-proton of Ser,
exhibited HMBC correlations with C-20 [δC 171.3], establish-
ing the connection of Leu to Ser. Based on the HMBC
correlations of 21-NH [δH 7.49] and H-24 [δH 4.32] to C-23,
the connectivity of Ser to Ala was assigned. The HTCA was
1
that of 1. Further analysis of the H−1H coupling constants
revealed that coprisamide B is a geometric isomer of 1
possessing the 35E configuration (JH35H36 = 15.0 Hz), in
contrast to the 35Z geometry of 1. Comparison of the CD
spectra of 1 and 2 indicated that the absolute configuration of 2
was identical to that of 1 (see Supporting Information Figure
S14).
Coprisamides A and B (1 and 2) were primarily tested for
growth inhibition activity against human pathogenic bacteria
(Staphylococcus aureus, Bacillus subtilis, Kocuria rhizophila,
Salmonella enterica, Proteus hauseri, and Escherichia coli) and
fungi (Candida albicans and Aspergillus fumigatus). However,
these compounds did not exhibit antibacterial or antifungal
activity. The cytotoxicities of 1 and 2 were evaluated against
various cancer cell lines (HCT 116, A549, SNU-638, SK-HEP-
1, and MDA-MB-231), but no significant cytotoxicity was
observed.
The induction of phase II detoxification enzymes is an
attractive, safe, and promising strategy for decreasing the risk of
developing cancer. Because quinone reductase (QR), a
representative phase II detoxification enzyme, is widely
distributed in mammalian tissues and is easily measured, QR
is considered to have significance in cancer chemoprevention.
The biological activity of the coprisamides was evaluated using
a QR induction assay. In the assay, the Hepa-1c1c7 murine
hepatoma cell line was utilized because the induction of QR in
this cell line is highly associated with the induction of phase II
enzymes in vivo.13
Coprisamide A (1) significantly induced QR activity by 2.4-,
2.6-, and 3.3-fold at concentrations of 5, 10, and 20 μM,
respectively (Figure 4a), while coprisamide B enhanced QR
activity by 1.7-, 2.1-, 2.8-, and 3.2-fold at concentrations of 5,
10, 20, and 40 μM, respectively (Figure 4b). The significant
effects of 1 and 2 QR induction suggest that the coprisamides
may represent a chemotype for cancer chemopreventive agents.
The effects of coprisamides A and B on melanogenesis were
also evaluated in cell-based assay systems. Coprisamides A and
B inhibited the production of melanin content in mouse melan-
a cells by 51.3% and 41.9%, respectively, at 20 μM. These data
indicate that coprisamides A and B have inhibitory activity in
melanin formation (see Supporting Information Figure S15).
Therefore, coprisamides A and B may be applicable as potential
candidates for treating or preventing hyperpigmentation and as
skin whitening agents.
1
positioned next to Ala by the H−13C long-range correlations
from the NH-24 [δH 8.37] of Ala and the H-28 [δH 7.75] of
HTCA to C-26 [δC 168.7]. The HMBC correlations from H-
22b [β-proton of Ser; δH 3.94] and H-2 [α-proton of β-methyl-
Asp; δH 3.87] to C-1 [δC 170.9] secured the ring closure,
satisfying the 18 degrees of unsaturation deduced from the
molecular formula. Finally, the NH proton [8-NH; δH 8.54]
and the β-proton [H-8b; δH 2.95] of Dpr exhibited
heteronuclear correlations to C-42 [δC 169.6], connecting the
branched amino acid unit Val-2 to Dpr. The ROESY
correlation between 8-NH and H-43 [δH 3.66] also supported
the linkage of these two units.
The absolute configurations at the α-carbons of the amino
acids in coprisamide A (1) were established by the advanced
Marfey’s method with FDAA (1-fluoro-2,4-dinitrophenyl-5-
alanine amide).10 After acid hydrolysis of 1 for 2 h, the free
amino acids in the hydrolysate were derivatized with L- and D-
FDAA. LC/MS analysis of the derivatives revealed two L-Val, β-
methyl-L-Asp, D-Ser, D-Ala, and D-Leu. In addition, by
comparison of the retention times of the authentic standard
S-Dpr coupled with L- and D-FDAA (di, [M + H]+ m/z 609),
Dpr in 1 was assigned as S (see Supporting Information Table
S1).
To establish the absolute configuration of the additional
stereogenic center at C-3 of the β-methyl-Asp-bearing
carboxylic acid, 1 was subjected to phenylglycine methyl ester
(PGME) derivatization.11 The derivatization was performed in
the dark at rt for 40 min to avoid degradation. After careful
analysis of the 1H and COSY NMR data of the S- and R-PGME
amides (3a and 3b, [M - H]− m/z 1052), the absolute
configuration at the β-carbon of β-methyl-L-Asp in coprisamide
A (1) was determined as S (Figure 3a). This configuration was
further supported by J-based configuration analysis of the C-2
and C-3 consecutive stereogenic centers.12 The large coupling
constant (10.0 Hz) between H-2 and H-3 established their anti-
relationship. The H3-4/2-NH ROESY correlation established
the rotamer in Figure 3b. Because the absolute configuration of
the α-carbon of β-methyl-L-Asp was already determined as S by
the advanced Marfey’s method (vide supra), the established
rotamer possesses a 3S configuration, supporting the PGME
analysis result.
C
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o f S c i e n c e , I C T a n d F u t u r e P l a n n i n g )
(2014R1A2A1A11053477 and 2009-0083533) and the HHMI
International Early Career Scientist Program.
REFERENCES
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Figure 4. Effects of (a) coprisamide A and (b) coprisamide B on the
induction of quinone reductase in murine Hepa-1c1c7 cells. Cells were
cultured for 24 h and then exposed to coprisamide A or B for 24 h.
Quinone reductase activities in the cell lysates were measured by
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The structures of the coprisamides are unique in several
aspects. First, the amino acid sequence incorporating mostly
the unusual amino acids S-β-methyl-L-Asp and S-Dpr as well as
D-Leu, D-Ser, and D-Ala is unprecedented among the numerous
reported nonribosomal peptides.14 In particular, β-methyl-L-
Asp is infrequently encountered, with the rare examples of the
friulimicin class of peptides15 and the skyllamycins.16 The
branched feature of 1 and 2 at Dpr extended to Val-2 is also
unique in nature. Moreover, the acyl chain, which is composed
of a 2-heptatrienyl cinnamic acid (HTCA), has not been
previously reported. Such 2-alkenyl cinnamic acids are
extremely rare. Based on our exhaustive literature search, only
four classes of natural products bearing a 2-alkenyl cinnamic
acid have been reported. WS9326s, the mohangamides, and the
pepticinnamins incorporate a 2-pentenyl cinnamic acid,17
whereas the skyllamycins possess a 2-propenyl cinnamic
acid.16 Based on the study of skyllamycin biosynthesis,18 the
HTCA unit could be biosynthesized from the putative
precursor C16-polyene by an enzymatic 6π-electrocyclization
(see Supporting Information Figure S18).
The coprisamides are the first secondary metabolites to be
isolated from insect gut symbionts. Insect gut microbiota are
likely determined by the environmental habitat, diet, devel-
opmental stage, and phylogeny of the host, resulting in the
tremendous biodiversity of insect gut-associated microbes.19
Our discovery of the coprisamides from a dung beetle gut
symbiont signifies the huge chemical potential of insect
microbiota for bioactive small molecules.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The detailed experimental procedures and NMR data for
compounds 1 and 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: dongchanoh@snu.ac.kr.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation
of Korea Grants funded by the Korean Government (Ministry
■
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