Tripartilactam, a cyclobutane-bearing tricyclic lactam from a Streptomyces sp. in a dung beetle's brood ball

Article abstract of DOI:10.1021/ol300108z

Tripartilactam, a structurally unprecedented cyclobutane-bearing tricyclic lactam metabolite, was discovered from Streptomyces sp. isolated from a brood ball of the dung beetle, Copris tripartitus. The structure of this compound was elucidated by the combination of NMR, MS, UV, and IR spectroscopy and multistep chemical derivatization. Tripartilactam was evaluated as a Na⁺/K ⁺ ATPase inhibitor (IC₅₀ = 16.6 μg/mL).

Full text of DOI:10.1021/ol300108z

ORGANIC
LETTERS
2
012
Vol. 14, No. 5
258–1261
Tripartilactam, a Cyclobutane-Bearing
Tricyclic Lactam from a Streptomyces sp.
in a Dung Beetle’s Brood Ball
1
†
Seon-Hui Park, Kyuho Moon, Hea-Son Bang, Seong-Hwan Kim, Dong-Gyu Kim,
†
§
†
†
†
Ki-Bong Oh, Jongheon Shin, and Dong-Chan Oh*
,†
Natural Products Research Institute, College of Pharmacy, Seoul National University, 1
Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea, Department of Agricultural
Environment, National Academy of Agricultural Science, Suwon 441-707, Republic of
Korea, and Department of Agricultural Biotechnology, College of Agriculture and Life
Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic
of Korea
dongchanoh@snu.ac.kr
Received January 16, 2012
ABSTRACT
Tripartilactam, a structurally unprecedented cyclobutane-bearing tricyclic lactam metabolite, was discovered from Streptomyces sp. isolated
from a brood ball of the dung beetle, Copris tripartitus. The structure of this compound was elucidated by the combination of NMR, MS, UV, and IR
þ
þ
spectroscopy and multistep chemical derivatization. Tripartilactam was evaluated as a Na /K ATPase inhibitor (IC50 = 16.6 μg/mL).
Diverse insect ecosystems have recently drawn signifi-
cant attention as a new source of natural products with
the mud dauber, Sceliphron caementarium, led to the
discovery of a novel antifungal polyene macrocyclic lac-
tam, sceiliphrolactam, although its ecological role has not
been completely determined. Biomedical investigation of
the gut microbiota of a mantis isolated a fungal species,
Daldinia eschscholzii, that produces new immunosuppres-
5
sive polyketides known as dalesconols. Even a fungal
1
pharmaceutical potential. This attention is partly due to
4
insectꢀmicrobial mutualisms being mediated by microbial
secondary metabolites with selective activity, as shown in
the ecologically well-defined symbiotic systems of the
2
southern pine beetle (Dendroctonus frontalis) and the
3
fungus-growing ant (Apterostigma dentigerum). However,
strain isolated from the nest of the fungus-growing
ant (A. dentigerum) yielded a new polyketide
the potential of microorganisms discovered in insect eco-
systems isnow being explored because mostsuchmicrobial
communities are unexplored niches. The recent study of
6
glycoside.
The dung beetle, Copris tripartitus, is a soil-dwelling
insect, the life cycle of which is tightly dependent on the
7
feces of herbivores. The brood balls mainly composed of
†
Natural Products Research Institute, College of Pharmacy, Seoul Na-
tional University.
feces are expected to harbor extensive microbial commu-
nities. Our recent selective isolation of actinomycetes from
§
National Academy of Agricultural Science.
Department of Agricultural Biotechnology, College of Agriculture and
Life Science, Seoul National University.
(
1) (a) Crawford, J. M.; Clardy, J. Chem. Commun. 2011, 47, 7559. (b)
(4) (a) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Org. Lett.
2011, 13, 752. (b) Poulsen, M.; Oh, D.-C.; Clardy, J.; Currie, C. R. PLoS
One 2011, 6, e16763.
Bode, H. B. Angew. Chem., Int. Ed. 2009, 48, 6394. (c) Kaltenpoth, M.
Trends Microbiol. 2009, 17, 529.
(
2) (a) Scott, J. J.; Oh, D.-C.; Yuceer, M. C.; Klepzig, K. D.; Clardy,
(5) Zhang, Y. L.; Ge, H. M.; Zhao, W.; Dong, H.; Xu, Q.; Li, S. H.;
Li, J.; Zhang, J.; Song, Y. C.; Tan, R. X. Angew. Chem., Int. Ed. 2008, 47,
5823.
J.; Currie, C. R. Science 2008, 322, 63. (b) Oh, D.-C.; Scott, J. J.; Currie,
C. R.; Clardy, J. Org. Lett. 2009, 11, 633.
(
3) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Nat. Chem. Biol.
(6) Freinkman, E.; Oh, D.-C.; Scott, J. J.; Currie, C. R.; Clardy, J.
Tetrahedron Lett. 2009, 50, 6834.
2
009, 5, 391.
1
0.1021/ol300108z r 2012 American Chemical Society
Published on Web 02/23/2012

the beetle’s brood balls revealed the diversity of the
actinomycetous population and their potential as a new
bioactive chemotype.
8
Table 1. NMR Data for Tripartilactam (1) in DMSO-d6
a
b
We have continuously investigated the chemical and
biological diversity of the actinomycetes isolated from
dung brood balls in the search for structurally novel
bioactive compounds. In our chemical analysis of the
actinomycetes’ secondary metabolites, we discovered a
C/H
δH
mult (J in Hz)
δC
1
2
2
3
4
5
6
7
8
9
1
164.4
51.4
C
a
b
3.47
2.96
d (11.5)
CH
2
d (11.5)
191.2
121.7
147.0
135.4
145.6
38.0
C
9
Streptomyces strain, SNA112, that produces a compound
6.03
7.10
d (15.5)
d (15.5)
CH
CH
C
with a distinct UV spectrum (λmax at 277 nm). Further
characterization of this compound’s structure led us to
identify an unprecedented cyclobutane-bearing tricyclic
lactam, tripartilactam (1). Herein, we report the isolation,
structural determination, stereochemistry, and biological
activity of this novel tricyclic lactam.
c
5.70
2.57
2.38
m
CH
CH
CH
C
ddd (12.0, 10.0, 10.0)
m
48.6
0
210.0
70.2
11
3.82
5.29
4.02
4.93
4.52
4.63
5.71
5.76
3.54
2.43
br s
CH
1
1
1
1
1
1
1
1
1
1
1-OH
br d (2.5)
2
m
78.7
74.2
CH
CH
2-OH
br d (4.0)
3
dd (7.5, 3.5)
br d (7.5)
3-OH
c
4
5
6
7
8
m
130.0
125.0
44.4
CH
CH
CH
CH
C
ddd (10.0, 5.0, 2.5)
m
ddd (10.0, 4.0, 2.5)
52.2
1
0
Tripartilactam (1) was obtained as a yellowish powder.
The molecular formula, C H NO , was deduced based
134.3
130.1
125.7
132.5
130.3
137.8
38.2
2
8
35
6
19
20
21
5.39
6.12
5.68
6.02
5.23
2.32
3.25
2.90
7.99
1.26
1.49
0.93
d (11.0)
CH
CH
CH
CH
CH
CH
CH
1
13
on H and C NMR spectral data (Table 1) and FAB
dd (14.5, 11.0)
þ
high-resolution mass spectrometry (observed [MþH] at
dd (14.5, 10.0)
1
3
2
2
2
2
2
2
2
dd (15.0, 10.0)
m/z: 482.2542, calculated: 482.2543). The C NMR spec-
trum displayed a polyunsaturated signature with 12 car-
bon signals from δC 147.0 to 121.7. Three carbonyl
3
dd (15.0, 9.0)
4
m
5a
5b
5-NH
m
44.1
2
carbons were observed at δ 210.0, 191.2, and 164.4. Based
C
ddd (13.0, 4.0, 4.0)
on the carbon chemical shift and the IR absorption at
13
dd (8.0, 4.0)
ꢀ1
1
asanamidecarbon. Inthe oxygenated carbonregion, three
677 cm , the carbonyl C peak at δ 164.4 was assigned
26
27
s
13.1
12.9
19.0
CH3
C
s
CH
CH
3
3
2
8
d (6.5)
carbons were identified at δ 78.7, 74.2, and 70.2. The
C
1
3
upfield region of the C NMR spectrum displayed signals
for 10 aliphatic carbons (Table 1).
a
b
c
00 MHz. 125 MHz. Overlapped.
5
1
The H NMR spectrum contained signals for a poly-
unsaturated feature consistent with 10 olefinic protons
from 7.10 to 5.23 ppm and two allylic methyl groups
group (δ 0.93) and nine protons betweenδ 3.54 and 2.32
H
H
were found.
(
δ 1.49 and 1.26), accounting for six double bonds indi-
H
1
3
1
1
3
The overall analysis of the C and H NMR spectra and
the IR spectrum revealed that this compound bears six
double bonds and three carbonyl groups, which account
for 9 out of 12 unsaturation equivalents calculated from
the molecular formula. Thus, tripartilactam (1) must be a
tricyclic compound.
cated by the 12 olefinic carbons in the C NMR spectrum.
The amide functionality was also confirmed by the ob-
servation of the NH signal at δ 7.99. In addition, tripar-
H
tilactam showed three carbinol protons (δH 4.52, 4.02,
3
.82) along with three D O exchangeable proton signals
2
(
δ 5.29, 4.93, 4.63). The presence of hydroxy moieties was
H
ꢀ
1
also supported by the broad IR absorption at 3361 cm .
In the aliphatic region, one doublet representing a methyl
Interpretation of the gHSQC and gCOSY NMR spectra
established the isolated spin system from C-19 to 25-NH,
including the connectivity between C-28 (aliphatic methyl
group; δ 19.0ꢀδ 0.93) and C-24 (methine; δ 38.2ꢀδ
(7) Fincher, G. T. J. Ga. Entomol. Soc. 1981, 16, 316.
(8) Kim, S.-H.; Ko, H.; Bang, H.-S.; Park, S.-H.; Kim, D.-G.; Kwon,
C
H
C
H
2.32) (Figure 1). The COSY correlations among the ali-
H. C.; Kim, S. Y.; Shin, J.; Oh, D.-C. Bioorg. Med. Chem. Lett. 2011, 21,
715.
9) The bacterial strain SNA112 was isolated from a dung brood ball
5
phatic methines (C-8 δ 38.0ꢀδ 2.57; C-9 δ 48.6ꢀδ
C
H
C
H
(
2
.38; C-16 δC 44.4ꢀδ 3.54; C-17 δ 52.2ꢀδ 2.43)
H
C
H
of C. tripartitus on Czapek-Dox agar supplemented with cycloheximide.
The 16S rDNA analysis resulted in placing SNA112 in the genus
Streptomyces because it is most closely related to Streptomyces corchorusii
surprisingly revealed the existence of a cyclobutane ring.
Further extension of this spin system includes C-7, C-15,
and C-14 according to the COSY and the HMBC correla-
tions. The triol moiety (C-11 to C-13), a discrete double
bond (C-4 and C-5), and an isolated methylene (C-2) were
(
99% identity).
10) Tripartilactam (1): yellow powder; [R]
IR (neat) νmax 3361, 2922, 1723, 1677, 1581 cm ; UV (MeOH) λmax
(
D
= ꢀ128 (c 0.20, MeOH);
ꢀ
1
(
log ε) 277 (4.51) nm; NMR spectral data, see Table 1; HR-FAB MS
þ
þ
[
MþH] m/z 482.2542 (C28
H
35NO
6
) calcd [MþH] 482.2543.
Org. Lett., Vol. 14, No. 5, 2012
1259

Figure 1. Key ROESY, COSY, and HMBC correlations estab-
lishing the planar structure of tripartilactam (1).
Figure 2. Key ROESY correlations of the cyclobutane and
cyclooctene ring in tripartilactam (1).
1
1
also identified, based on the Hꢀ H couplings in the
COSY spectrum (Figure 1).
The ROESY correlations in the cyclobutane and cyclo-
octene rings indicated that the relative configurations of
C-8, C-9, C-11, C-12, C-13, C-16, and C-17 were 8R*, 9R*,
The long-range heteronuclear correlations in the gHMBC
spectrum verified the connectivities of these spin systems.
The coupling from H -2 to the carbonyl carbons C-1 and
11R*, 12R*, 13R*, 16R*, and 17S*, respectively.
2
The absolute configuration of the C-24 chiral center was
determined by multistep chemical degradation (Figure 3).
C-3 located the methylene C-2 between these carbonyl
groups. Similarly, the HMBC correlationsfrom H-4 toC-3
and from H -26 to C-5, C-6, and C-7 established the
3
connectivity from C-1 to the cyclobutane ring. The triol
partial structure was found to be connected to the cyclo-
butane ring with the ketone carbon as a linker based on the
HMBC correlations between H-9 and both C-10 and C-11.
This assigned location is also further supported by the
long-range correlations from H-16 and H-17 to C-10. The
allylic methyl signal (H -27) generated strong HMBC
3
correlations to C-17, C-18, and C-19, connecting the chain
structure from C-20 to 25-NH to the cyclobutane ring. The
presence of an 18-membered macrocyclic ring was con-
firmed by the HMBC correlation between 25-NH and C-1.
Although no correlations in the COSY, HMBC, and
ROESY spectra in three different solvents (DMSO-d6,
pyridine-d , and CD OD) were observed to directly con-
5
3
nect C-13 to C-14, this linkage was deduced based on the
molecular formula; this linkage completed the planar
structure of 1 bearing a cyclooctene ring.
The double bond geometries in 1 were assigned based on
1
1
the analysis of Hꢀ H coupling constants and ROESY
correlations (Figure 1). The large coupling constant be-
tween H-4 and H-5 (15.5 Hz) established the 4E config-
uration. The 6E configuration was requiredbythe ROESY
Figure 3. Multistep chemical degradation and derivatization for
the determination of the absolute configuration of the C-24
chiral center.
1
1
correlations between H -26 and H-4. The Hꢀ H cis-
3
coupling (10.0 Hz) between H-14 and H-15 determined
Tripartilactam was subjected to ozonolysis and acid hy-
drolysis to yield 3-amino-2-methyl-propioinic acid (2).
This β-amino acid (2) was derivatized with the Sanger
reagent to generate a product (3) with improved UV
absorption and retention in reversed-phase HPLC. Com-
pound 3was further derivatized with (S)-and(R)-phenylglycine
methyl ester (PGME) to furnish (S)- and (R)-PGME
amide products (4a and 4b, respectively). Analysis of the
14Z. The H-20-H-21 trans coupling constant (14.5 Hz)
established 20E. The 22E configuration was assigned
based on the large coupling between H-22 and H-23
(
the ROESY correlation between H -27 and H-20.
15.0 Hz). Finally, the 18E configuration was deduced by
3
Because the coupling constants between the protons in
cyclobutane are not sufficiently specific to determine the
1
1
1
relative configuration in the ring, the relative configura-
tion of the cyclobutane ring was established by careful
analysis of the transannular ROESY correlations (Figure 2).
H NMR spectra allowed the assignment of the proton
chemical shifts in 4a and 4b. Calculation of the ΔδS-R
values clearly established the absolute configuration of
C-24 as R based on the consistent distribution of the signs
1
2
(Figure 4).
(
11) Pretsch, E.; B u€ hlmann, P.; Affolter, C. Structure Determination
of Organic Compounds-Tables of Spectral Data; Springer: New York,
000; p 176.
2
(12) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397.
Org. Lett., Vol. 14, No. 5, 2012
1
260

the A549 lung cancer cell line and the HCT116 colon
cancer cell line at 100 μg/mL. However, tripartilactam was
þ
þ
observed to be a moderate inhibitor of Na /K ATPase,
with an IC50 value of 16.6 μg/mL.
The structure of 1 is unique because of the existence of
cyclobutane linking the 8- and 18-membered rings. This
ring structure could be formed by a photochemically
1
4
derived [2 þ 2] cycloaddition reaction of the two double
bonds at positions 8 and 16 in a putative macrocyclic
lactam precursor (see SI Figure S3).
Figure 4. ΔδS-R values in ppm for the S- and R-PGME amide
products (4a and 4b) in CD OD.
3
The absolute configurations of the stereogenic centers in
cyclobutane and cyclooctene were determined by the
application of the modified Mosher method using (R)-
and (S)-R-methoxy-(trifluoromethyl) phenyl acetyl chlo-
ride (MTPA-Cl). Since tripartilactam bears three consecu-
tive hydroxy groups at C-11, C-12, and C-13, it was
challenging to generate mono-MTPA esters. After a sig-
nificant amount of effort on reaction optimization, we
tried a short reaction time (5 min) and succeeded in ob-
taining mono-(S)- and (R)-MTPA ester selectively at the
Figure 5. ΔδS-R values in ppm for the S- and R-MTPA esters (5a
and 5b) in DMSO-d6.
1
alcohol of C-13 (5a and 5b). Analysis of H NMR and
COSY spectral data for these MTPA esters allowed the
assignment of the ΔδS-R values, which sufficiently estab-
Four- and eight-membered bicyclic structures have been
13
1
reported in terpenoids from terrestrial plants and marine
5
lished the absolute configuration of C-13 as 13R (Figure 5).
1
6
Based on the relative configuration, the absolute config-
urations of the chiral centers C-8, C-9, C-11, C-12, C-16,
and C-17 were determined as 8R, 9R, 11R, 12R, 16R,
and 17S.
The biological activity of 1 was evaluated first in cell-
based antimicrobial and anticancer assays, in which no
significant inhibition against various pathogenic bacterial
and fungal strains was observed (see Supporting Informa-
tion (SI)). This compound was also inactive against
soft corals. However, the 4-, 8-, and 18-membered tri-
cyclic structure of 1 is an unprecedented carbon skeleton,
to the best of our knowledge.
The discovery of tripartilactam with a novel carbon
scaffold provides additional evidence that microbial com-
munities of insect habitats could be promising sources of
structurally novel bioactive natural products.
Acknowledgment. The authors thank Professors Sanghee
Kim in SNU, Yousung Jung in KAIST, and Michael
Fischbach in UCSF for valuable discussions about cyclobu-
tane ring formation. This work was supported by the National
Research Foundation of Korea (NRF) grants funded by the
Korean government (MEST) (No. 2011-0015931) and (No.
(
13) Seco, J. M.; Qui n~ o ꢀa , E.; Riguera, R. Tetrahedron: Asymmetry
001, 12, 2915.
14) We propose that this [2 þ 2] cycloaddition is photochemically
derived because we could not find any precedent example of enzymatic
2
(
[
2 þ 2] cycloaddition thus far to the best of our knowledge. However, we
could not completely exclude the possibility of the enzymatic involve-
ment in this thermally forbidden reaction. For the general mechanism of
photochemical [2 þ 2] cycloaddition, see: Carruthers, W. Cycloaddition
Reactions in Organic Synthesis; Pergamon Press: 1991; p 322. For another
possible cyclobutane formation mechanism by unusual oxidative cycli-
zation or radical cascading proposed in the ladderane biosynthesis, see:
Rattary, J. E.; Strous, M.; Op den Camp, H. J. M.; Schouten, S.; Jetten,
M. S. M.; Damst ꢀe , J. S. S. Biol. Direct 2009, 4, 8.
2011-0030635). D.C.O. is a Howard Hughes Medical Institute
International Early Career Scientist.
Supporting Information Available. Experimental section,
bioassay data, a proposed biosynthetic pathway of 1, and
NMR spectra of 1, 3, 4a, 4b, 5a, and 5b. This material is
available free of charge via the Internet at http://pubs.acs.org.
(15) (a) Jacobsson, U.; Kumar, V.; Saminathan, S. Phytochemistry
1
1
995, 39, 839. (b) Macleod, J. K.; Rasmussen, H. B. Phytochemistry
999, 50, 105.
(
16) (a) Sung, P.-J.; Chuang, L.-F.; Kuo, J.; Chen, J.-J.; Fan, T.-Y.;
Li, J.-J.; Fang, L.-S. Chem. Pharm. Bull. 2007, 55, 1296. (b) Groweiss, A.;
Kashman, Y. Tetrahedron 1983, 39, 3385.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 5, 2012
1261