Natalenamides A–C, cyclic tripeptides from the termite-associated Actinomadura sp. RB99

Article abstract of DOI:10.3390/molecules23113003

In recent years, investigations into the biochemistry of insect-associated bacteria have increased. When combined with analytical dereplication processes, these studies provide a powerful strategy to identify structurally and/or biologically novel compounds. Non-ribosomally synthesized cyclic peptides have a broad bioactivity spectrum with high medicinal potential. Here, we report the discovery of three new cyclic tripeptides: natalenamides A–C (compounds 1–3). These compounds were identified from the culture broth of the fungus-growing termite-associated Actinomadura sp. RB99 using a liquid chromatography (LC)/ultraviolet (UV)/mass spectrometry (MS)-based dereplication method. Chemical structures of the new compounds (1–3) were established by analysis of comprehensive spectroscopic methods, including one-dimensional (¹H and¹³C) and two-dimensional (¹H-¹H-COSY, HSQC, HMBC) nuclear magnetic resonance spectroscopy (NMR), together with high-resolution electrospray ionization mass spectrometry (HR-ESIMS) data. The absolute configurations of the new compounds were elucidated using Marfey’s analysis. Through several bioactivity tests for the tripeptides, we found that compound 3 exhibited significant inhibitory effects on 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production. The effect of compound 3 was similar to that of kojic acid, a compound extensively used as a cosmetic material with a skin-whitening effect.

Full text of DOI:10.3390/molecules23113003

molecules
Article
Natalenamides A–C, Cyclic Tripeptides from the
Termite-Associated Actinomadura sp. RB99
1
1
1
2
3
4
Seoung Rak Lee , Dahae Lee , Jae Sik Yu , René Benndorf , Sullim Lee , Dong-Soo Lee ,
5
6
7
2
Jungmoo Huh , Z. Wilhelm de Beer
Ki Sung Kang 4 and Ki Hyun Kim *
, Yong Ho Kim , Christine Beemelmanns
,
1,
1
School of Pharmacy, Sungkyunkwan University, Suwon 16419, Korea; davidseoungrak@gmail.com (S.R.L.);
pjsldh@naver.com (D.L.); jsyu@bu.edu (J.S.Y.)
Leibniz Institute for Natural Product Research and Infection Biology—Hans-Knöll-Institute,
Beutenbergstraße 11a, 07745 Jena, Germany; rene.benndorf@hki-jena.de (R.B.);
Christine.beemelmanns@hki-jena.de (C.B.)
College of Bio-Nano Technology, Gachon University, Seongnam 13120, Korea; sullimlee@gachon.ac.kr
College of Korean Medicine, Gachon University, Seongnam 13120, Korea; vet4animal@hotmail.com (D.-S.L.);
kkang@gachon.ac.kr (K.S.K.)
2
3
4
5
6
7
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University,
Gwanak-gu, Seoul 08826, Korea; goodhjm112@snu.ac.kr
Forestry and Agriculture Biotechnology Institute, University of Pretoria, Pretoria 0028, South Africa;
wilhelm.debeer@fabi.up.ac.za
SKKU—Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 16419, Korea;
yhkim94@skku.edu
*
Correspondence: khkim83@skku.edu; Tel.: +82-31-290-7700
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 1 October 2018; Accepted: 12 November 2018; Published: 16 November 2018
Abstract: In recent years, investigations into the biochemistry of insect-associated bacteria have
increased. When combined with analytical dereplication processes, these studies provide a
powerful strategy to identify structurally and/or biologically novel compounds. Non-ribosomally
synthesized cyclic peptides have a broad bioactivity spectrum with high medicinal potential. Here,
we report the discovery of three new cyclic tripeptides: natalenamides A–C (compounds 1–3).
These compounds were identified from the culture broth of the fungus-growing termite-associated
Actinomadura sp. RB99 using a liquid chromatography (LC)/ultraviolet (UV)/mass spectrometry
(
MS)-based dereplication method. Chemical structures of the new compounds (
1–3) were established
1
13
by analysis of comprehensive spectroscopic methods, including one-dimensional ( H and C)
1
1
and two-dimensional ( H- H-COSY, HSQC, HMBC) nuclear magnetic resonance spectroscopy
NMR), together with high-resolution electrospray ionization mass spectrometry (HR-ESIMS) data.
The absolute configurations of the new compounds were elucidated using Marfey’s analysis.
Through several bioactivity tests for the tripeptides, we found that compound exhibited significant
inhibitory effects on 3-isobutyl-1-methylxanthine (IBMX)-induced melanin production. The effect of
compound was similar to that of kojic acid, a compound extensively used as a cosmetic material
(
3
3
with a skin-whitening effect.
Keywords: fungus-growing termite; Actinomadura sp.; tripeptides; natalenamides A–C; skin-whitening
effects
1
. Introduction
The chemical analysis of protective bacterial symbionts of farming insects has attracted much
attention among natural product chemists in recent years [
1
–5
]. Using state-of-the-art nuclear magnetic
Molecules 2018, 23, 3003; doi:10.3390/molecules23113003
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3003
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resonance (NMR)/mass spectrometry (MS)-based dereplication processes and ecologically relevant
bioassays, an impressive amount of structurally intriguing natural products, including several
non-ribosomally synthesized cyclic peptides, have been uncovered from microbial symbionts [
The most prominent examples include the antifungal dentigerumycin, a cyclic depsipeptide isolated
from a fungus-growing-ant-associated Pseudonocardia sp. [ ], and the gerumycins A–C, piperazic
6].
7
acid-bearing cyclic depsipeptides identified from Pseudonocardia sp. [8].
Cyclic peptides have been shown to exhibit a broad range of biological activities, fostering several
synthetic approaches toward these pharmacologically promising structures [9–12]. Over 40 cyclic
peptide drugs are currently marketed and widely utilized in clinical environments, and approximately
one new cyclic peptide drug enters the market every year, on average [13].
As part of our continuing endeavor to identify structurally novel and/or bioactive secondary
metabolites from termite-associated microbes [14–17], we focused on the termite-associated
Actinomadura sp. RB99, isolated from the fungus-growing termite, Macrotermes natalensis. Our liquid
chromatography (LC)/ultraviolet (UV)/mass spectrometry (MS)-based dereplication strategy yielded
potentially novel bioactive natural products. In this study, we report the isolation and chemical
identification of three new cyclic tripeptides, natalenamide A–C (compounds 1–3, Figure 1), using
a LC/UV/MS-based dereplication method as well as their biological evaluations for cytotoxic,
anti-inflammatory, and skin-whitening activities.
Figure 1. The chemical structures of natalenamides A–C (compounds 1–3).
2
. Results and Discussion
2.1. Liquid chromatography (LC)/Ultraviolet (UV)/Mass Spectrometry (MS)-Based Isolation of Compounds 1–3
Actinomadura sp. RB99 was isolated from the surface of a termite worker (M. natalensis).
Phylogenetic analysis of a nearly complete 16S ribosomal ribonucleic acid (rRNA) sequence suggests
that strain RB99 belongs to the genus Actinomadura, with the closest neighbor Actinomadura nitritigenes
T
NBRC 15918 . Subsequent LC/UV/MS-based analysis of enriched culture extracts revealed a
set of characteristic UV absorptions with unique molecular ion peaks containing nitrogen atoms.
To identify the respective metabolites and investigate their activity, large-scale fermentation using
◦
optimized growth conditions (100 agar plates from 2 L ISP2 agar, pH 6, 10 days at 30 C) and an
established work-up and pre-purification procedure were applied [17]. Subsequent LC-UV/MS-guided
fractionation and repetitive semi-preparative high performance liquid chromatography (HPLC)
resulted in isolation of three metabolites with a unique NMR/MS pattern. We performed growth and
media studies using different salt (NaCl, KBr 1–3%) and media compositions (ISP1-6 media) to mimic
the natural environment and stimulate metabolite production, which also led to the presence of the
three new metabolites (see Supplementary Figure S19).

Molecules 2018, 23, 3003
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2
.2. Structural Elucidation of the Compounds
Natalenamide A ( ) was acquired as an amorphous powder. The molecular formula of
1
1
was
+
determined to be C H N O from the sodium-adducted molecular ion at m/z 398.1691 [M + Na]
19
25
3
5
(
calculated for C H N O Na, 398.1692) using positive-ion mode high-resolution electrospray
19 25 3 5
ionization mass spectrometry (HR-ESIMS) data. The infrared (IR) spectrum showed a strong absorption
−
1
band at 1670 cm , suggesting the presence of amide groups in the molecule. The detailed analysis of
1
H NMR data of
1 (Table 1) indicated the typical signals of a peptide skeleton, displaying two methyl
signals (
δ
0.91 (3H, d, J = 7.0 Hz, H-3) and 0.92 (3H, d, J = 7.0 Hz, H-4)), three methylene signals ( H
δ
H
1
8
4
.95 (1H, m, H-7a), 2.27 (1H, m, H-8a), 2.36 (1H, m, H-8b), 2.48 (1H, m, H-7b), 2.97 (1H, dd, J = 14.0,
.0 Hz, H-12a), and 3.20 (1H, dd, J = 14.0, 5.0 Hz, H-12b)), four methine signals ( H 2.02 (1H, m, H-2),
.16 (1H, d, J = 7.5 Hz, H-1), 4.20 (1H, dd, J = 8.5, 4.5 Hz, H-6), and 4.63 (1H, dd, J = 8.0, 5.0 Hz, H-11)),
7.18 (1H, m, H-16), 7.23 (2H, m, H-14/18), and 7.24 (2H,
δ
and five overlapped aromatic protons (
δ
H
m, H-15/17)). The 13C NMR spectrum (Table 1), with assistance from the HSQC and HMBC spectra,
showed a total of 19 carbon resonances responsible for two methyl signals ( C 18.9 and 19.9), three
methylene signals ( 27.0, 30.6, and 38.8), nine methine signals ( C 32.1, 55.6, 58.0, 60.4, 127.8, 129.5
2)), including five aromatic carbons, one olefinic carbon signal ( C 138.8), and four
carbonyl carbon signals ( 173.2, 173.3, 174.8, and 181.3). Combined with the above spectroscopic data,
δ
δ
δ
C
(
×
2), and 130.6 (
×
δ
δ
C
1
1
detailed inspection of two-dimensional (2D) NMR ( H- H COSY, HSQC, and HMBC experiments)
revealed the presence of three amino acids in : glutamate (Glu), phenylalanine (Phe), and valine
Val) (Figure 2). The connectivity of these amino acids was determined by the HMBC correlations of
H-1/C-5 and C-19, H-11/C-10 and C-19, and H-6/C-5 and C-10 (Figure 2). To identify the absolute
configuration of , Marfey’s method was applied to determine the stereochemistry of -H multiples
C-1, C-6, and C-11). The acid hydrolysates of and standard amino acids (L/D-Glu, Phe, and Val) were
derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (L-FDAA). Successively, the derivatized
mixture of and the standard amino acids were analyzed by LC/MS to examine their retention time.
The absolute configurations of the Glu, Phe, and Val moieties were all determined to be L-forms, based
on the retention times of the L-FDAA derivatives of compared with those of the standard amino
acids. Thus, the structure of 1 was elucidated as the cyclic tripeptide shown in Figure 1.
1
(
1
α
(
1
1
1
Table 1. H (800 MHz) and C (200 MHz) NMR data of compounds 1–3 in d -MeOH. a,b.
1
13
4
1
2
3
Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
Position
δC
δH (J in Hz)
1
2
3
4
5
6
60.4 d
32.1 d
18.9 q
19.9 q
174.8 s
58.0 d
27.0 t
4.16 d (7.5)
2.02 m
0.91 d (7.0)
0.92 d (7.0)
1
2
3
4
5
53.1 d
42.4 t
26.2 d
22.1 q
23.7 q
174.9 s
58.1 d
26.9 t
-
4.42 m
1.63 m
1.69 m
0.92 d (6.5)
1
2a
2b
56.3 d
38.5 t
-
4.53 dd (7.5, 5.0)
3.02 dd (14.0, 7.5)
3.24 dd (14.0, 5.0)
3
138.9 s
130.7 d
129.7 d
127.8 d
175.0 s
57.9 d
26.6 t
-
30.0 t
181.7 s
174.0 s
55.8 d
38.4 t
-
-
-
0.95 d (6.5)
4/8
5/7
6
9
10
11a
11b
12
13
14
7.21 m
7.23 m
7.17 m
-
4.20 dd (8.5, 4.5)
6
7
-
7
a
1.95 m
2.48 m
2.27 m
2.36 m
-
-
4.09 dd (9.0, 4.5)
7
b
8a
8b
9a
9b
10
11
12
13a
13b
14
15/19
16/18
17
20
-
1.82 m
2.34 m
2.16 m
2.19 m
-
-
8
a
30.6 t
4.04 dd (9.0, 4.5) 8
8
b
30.3 t
-
1.74 m
2.29 m
2.15 m
-
9
181.8 s
173.3 s
55.6 d
38.8 t
-
138.8 s
130.6 d
129.5 d
127.8 d
173.2 s
-
1
1
0
1
181.6 s
173.4 s
55.8 d
38.9 t
-
138.6 s
130.5 d
129.4 d
127.6 d
173.3 s
-
4.63 dd (8.0, 5.0)
2.97 dd (14.0, 8.0)
3.20 dd (14.0, 5.0)
1
2a
4.72 dd (10.0, 5.0)
2.89 dd (14.0, 10.0)
3.23 dd (14.0, 5.0)
-
1
2b
15
4.63 dd (10.0 4.5)
2.80 dd (14.0, 10.0)
3.23 dd (14.0, 4.5)
1
3
-
16a
16b
17
18/22
19/21
20
1
1
4/18
5/17
7.23 m
7.24 m
7.18 m
-
-
-
-
7.21 m
7.25 m
7.19 m
-
138.4 s
130.2 d
129.3 d
127.4 d
173.8 s
-
1
1
6
9
7.21 m
7.23 m
7.17 m
-
-
-
-
-
23
a
Coupling constants (J, in parentheses) are in Hz, chemical shifts (δ
) are in ppm; b 13C NMR data were assigned
based on the HSQC and HMBC experiment.

Molecules 2018, 23, 3003
4 of 12
Figure 2. Key COSY (
)) and HMBC (→) correlations for compounds 1–3.
Natalenamide B (
2) was isolated as an amorphous powder. Its molecular formula (C H N O )
20
27
3
5
+
was deduced from hydrogen and sodium adducted molecular ion peaks at 390.2032 [M + H]
+
(
calculated for C H N O , 390.2029) and 412.1849 [M + Na] (calculated for C H N O Na,
20
28
3
5
20 27
3
5
4
12.1848), respectively. Compared with the molecular formula and NMR spectral data of 1, compound 2
appeared to have an additional CH group in the peptide skeleton. Comprehensive scrutiny of
2
1
13
1
1
the one-dimensional (1D) ( H and C NMR) and 2D NMR ( H– H COSY, TOCSY, HSQC, and
HMBC) spectra of revealed the existence of three amino acid residues: Glu, Phe, and Leu (leucine).
2
The sequence of these amino acids was constructed based on the HMBC correlations of H-1/C-6 and
C-20, H-12/C-11 and C-20, and H-7/C-6 and C-11 (Figure 2). To determine the absolute configuration
of
by LC/MS. In the LC/MS data, L-Glu, L-Phe, and L-Leu were absolutely detected by comparing
the retention times for the L-FDAA derivatives of with those of the standard amino acids. Thus,
the chemical structure of 2 was established as the cyclic tripeptide shown in Figure 1.
2, derivatization of the Glu, Phe, and Leu residues with L-FDAA was performed and then analyzed
2
The positive-mode HR-ESIMS data of natalenamide C (
3
) displayed hydrogen and sodium
+
adducted molecular ions at 424.1870 [M + H] (calculated for C H N O , 424.1872) and 446.1694
23
26
3
5
+
[
M + Na] (calculated for C H N O Na, 424.1692). This suggested that its molecular formula was
23 25 3 5
C H N O . A detailed analysis of the 1D and 2D NMR spectra of
3
indicated that its chemical
. The connectivity of the
23
25
3
5
structure was similar to that of
three identified amino acids in
2
3
, except Leu was substituted with Phe in 3
was verified using the HMBC correlations of H-1/C-9 and C-23,
H-15/C-14 and C-23, and H-10/C-9 and C-14 (Figure 2). By applying Marfey’s method to compound
the absolute configurations of the -H multiples (C-1, C-10, and C-15) were elucidated to be one L-Glu
and two L-Phe moieties. Accordingly, the complete structure of was determined as shown in Figure 1.
3,
α
3
Natalenamides A–C are tricyclic peptides, presumably of non-ribosomal origin. Currently, several
bioactive cyclic tripeptides have been characterized as natural products: cytotoxic 17-membered cyclic
tripeptides (e.g., OF4949 I–IV) isolated from Penicillium rugulosum [18]; antifungal sclerotiotides A K,
−
identified from the fermentation broth of halotolerant bacteria [10]; and antiproliferative psychrophilin
E, isolated from a mixed culture of two marine alga-derived fungal strains of the genus Aspergillus [19].
2
.3. Biological Activities of the Compounds 1–3
Since cyclic peptides have been reported to exhibit a wide range of biological activities,
the biological effects of the isolated cyclic tripeptides (
tests. The cytotoxicity of compounds was investigated against four cancer cell lines (MCF7 breast
cancer cells, HeLa cervical cancer cells, A549 human lung cancer cells, and HepG2 liver cancer cells) at
different concentrations (0, 6.25, 12.5, 25, 50, and 100 M). Compound did not affect the cell viability
of MCF-7, HeLa, or A549 cells. However, the treatment of HepG2 cells with 100 M of compound
decreased cell viability to 78.5 3.2% (Figure 3). Compound reduced the cell viabilities of HeLa and
A549 cells to 82.9 2.1% and 73.5 3.0%, respectively, but did not affect the viability of MCF-7 or
HepG2 cells (Figure 3). Compound 3 did not affect the viability of any of the cell lines (Figure 3).
1–3) were evaluated using three bioactivity
1–3
µ
1
µ
1
±
2
±
±

Molecules 2018, 23, 3003
5 of 12
Figure 3. Cytotoxicity of compounds 1–3 against human cancer cell lines (MCF7 breast cancer cells,
HeLa cervical cancer cells, A549 lung cancer cells, and HepG2 liver cancer cells). Cells were treated
with compounds at the indicated concentrations. After 72 h of incubation, cell viability was analyzed
according to the manufacturer’s instructions using the EZ-CyTox cell viability assay kit.
Next, RAW264.7 macrophages were used to investigate the anti-inflammatory effects of the
isolated compounds. To eliminate error in the production of NO caused by changing cell survival rates,
non-toxic concentrations of each compound were examined. As shown in Figure 4, compounds 1–3
had few cytotoxic effects in RAW264.7 cells (Figure 4A–C) and exerted no inhibitory effects on NO
production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells (Figure 4D–F).
Figure 4. Inhibitory effects of compounds
RAW264.7 cells. (A–C) Effects of compounds
–3 on LPS-induced NO production in RAW264.7 cells.
1–3
on lipopolysaccharide (LPS)-induced NO production in
1–3 on cell viability. (D–F) Inhibitory effects of compounds
1
The inhibitory effects of compounds
as evidence of their skin-whitening activities (Figure 5). Since changes in cell viability cause errors
in the production and measurement of melanin, we first examined the effects of compounds 1–3
1–3 on the melanin content in B16F10 cells were examined

Molecules 2018, 23, 3003
6 of 12
on B16F10 cell survival. Compounds
1–
3
exerted no cytotoxic effects in B16F10 cells at any of the
concentrations used (Figure 5A–C). We then assessed the inhibitory effects of the compounds on
-isobutyl-1-methylxanthine (IBMX)-induced melanin production in B16F10 cells (Figure 5D–F). IBMX,
a well-known stimulator of melanogenesis, induces a significant increase in melanin production
following a single treatment in melanoma cells. Among the compounds evaluated, compound
at 5–100 M) exhibited significant inhibitory effects on IBMX-mediated melanin synthesis in a
dose-dependent manner. Kojic acid, our positive control, has been extensively used as a cosmetic
material with skin-whitening effects [20]. The inhibitory effect of compound on IBMX-induced
melanin production was similar to that of kojic acid (Figure 5F), suggesting that compound functions
as a potent inhibitor of IBMX-induced melanin production in B16F10 melanoma cells. Furthermore,
compound was contaminated with a small amount of impurities, which were determined to
be fatty acid analogues by the interpretation of NMR spectroscopic data and LC/MS analysis
Supplementary Materials, Figures S11–S15 and S23). To identify the activity of the impurities,
3
3
(
µ
3
3
3
(
additional experiments with the fatty acid analogues were conducted for their inhibitory effects
on IBMX-induced melanin production in B16F10 cells, which revealed that the tested compounds
had no whitening effect (Supplementary Materials, Figure S24). Thus, although we cannot absolutely
exclude that the impurities in compound
melanin production, this seems unlikely.
3 are responsible for the inhibitory effect on IBMX-induced
Figure 5. Inhibitory effects of compounds
1–
3
on melanin content in B16F10 cells. (A–C) Effects of
compounds on cell viability. ( ) Inhibitory effects of compounds 1–3
1
–
3
D
–
F
on IBMX-induced melanin
production in B16F10 melanoma cells (mean ± SD, * p < 0.05 compared to the IBMX-treated value).
3
. Materials and Methods
3
.1. General Experimental Procedures
Infrared spectra were acquired on a Bruker IFS-66/S FT-IR (Bruker, Billerica, MA, USA)
spectrometer. The electrospray ionization and HR-ESIMS spectra were measured on a SI-2/LCQ
DecaXP liquid chromatography (LC)-mass spectrometer (Thermo Fisher Scientific, Waltham, MA,
1
1
USA). NMR spectra, including H– H COSY, HSQC, and HMBC experiments, were performed using a
1
Varian UNITY INOVA 800 NMR spectrometer (Varian, Palo Alto, CA, USA) operating at 800 MHz ( H)
13
and 200 MHz ( C), with chemical shifts given in ppm (δ). Preparative HPLC utilized a Waters 1525
Binary HPLC pump with Waters 996 Photodiode Array Detector (Waters Corporation, Milford, CT,
USA). Silica gel 60 (230–400 mesh, Merck, Kenilworth, NJ, USA) and RP-C18 silica gel (230–400 mesh,
Merck, were used for column chromatography. Semi-preparative HPLC used a Shimadzu Prominence

Molecules 2018, 23, 3003
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HPLC System with SPD-20A/20AV Series Prominence HPLC ultraviolet–visible (UV–Vis) Detectors
(
(
Shimadzu, Tokyo, Japan). LC/MS analyses were carried out on an Agilent 1200 Series HPLC system
Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 Series
ESI mass spectrometer with an analytical Kinetex (4.6
gel F254 plates and RP-18 F254s plates were used for thin-layer chromatography (TLC). Spots were
detected on TLC under UV light or by heating after spraying with anisaldehyde-sulfuric acid solution.
×
100 mm, 3.5 µm). Merck pre-coated silica
3
.2. Microbial Material
Actinomadura sp. RB99 was isolated from the surface of a termite worker of the genus, M. natalensis,
(
colony Mn103, GPS S25 43 45.9 E28 14 08.9) in January 2010. Biomaterial was placed into clean plastic
bags and processed within one day of collection. Termites were washed in sterile deionized water,
and bacteria were isolated by plating the resulting suspensions on low-nutrient media with chitin (per
liter: 4 g chitin, 0.7 g K HPO , 0.3 g KH PO , 0.5 g MgSO
·
5H O, 0.01 g FeSO4 7H O, 0.001 g ZnSO ,
·
2
4
2
4
4
2
2 4
0
.001 g MnCl , and 20 g agar) [21]. Isolates with Actinobacteria-like morphology were transferred to
2
ISP2 agar (per liter: 10 g malt extract, 4 g yeast extract, 4 g glucose, and 20 g agar), and sub-cultured
until pure isolates were obtained.
3
.3. DNA Extraction and Polymerase Chain Reaction Amplification
Actinomadura sp. RB99 was grown in nutrient-rich liquid media ISP2 for five to seven days at 30 C.
◦
Cells were harvested, and genomic DNA was extracted using the GenJet genomic DNA purification
kit (#K0721, Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions with
the following changes: (a) lysozyme treatment was extended to 40 min, and (b) proteinase K treatment
was extended to 40 min. DNA was quantified photometrically using a Nanodrop Lite spectrometer
(
Thermo Scientific). For phylogenetic studies, the 16S rRNA gene was amplified using the primer
set, 1492R/27F. Each amplification reaction was prepared in a 25 L final reaction volume containing
L of each primer (2.5 M), 0.5 L of dNTPs (10 M),
L of Phusion High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA),
L of extracted DNA (template). Polymerase chain reaction (PCR) was performed under the
µ
7
0
.25
.25
µ
L of distilled water, 5
µL of HF buffer, 5
µ
µ
µ
µ
µ
and 2
µ
◦
◦
◦
◦
following conditions: 98 C for 38 s; 32 cycles of 98 Cfor 30 s, 52 C for 45 s, 72 C for 1 min 20 s;
and a final extension of 72 C for 8 min. The PCR product was visualized by agarose gel electrophoresis,
◦
and PCR reactions were purified using a PCR purification kit (Thermo Scientific). DNA fragments
were sequenced at GATC (Konstanz, Germany).
3
.4. Sequencing and Species Identification
Sequences were assessed for purity and mismatches using BioEdit [22]. The forward and
reverse sequences obtained for each strain were assembled with BioEdit and tested for chimeras
using DECIPHER (http://decipher.cee.wisc.edu/FindChimerasOutputs.html). Resulting sequences
were deposited in GenBank (accession number: KY558684). Blast analyses with almost-complete 16S
rRNA sequences (1368 bp) were performed using the National Center for Biotechnology Information
(NCBI) database (reference RNA sequences). The results indicated that strain RB99 is a member of
the genus, Actinomadura. Sequences of the first 10 hits were downloaded from the NCBI database
and aligned with the 16S rRNA sequence of Actinomadura sp. RB99 using the Sina sequences
alignment service [23]. Two different phylogenetic trees were reconstructed with neighbor-joining
or maximum-likelihood algorithms using MEGA software version 7.0.26 [24–26]. The evolutionary
distance model of Tamura and Nei was used to generate evolutionary distance matrices for the
maximum-likelihood and neighbor-joining algorithms, with deletion of complete gaps and missing
data [27]. For the maximum-likelihood algorithm, discrete gamma distribution was used (+G), and the
rate variation model allowed for some sites to be evolutionarily invariable (+I). For the neighbor-joining
algorithm, the rate variation among sites was modeled with a gamma distribution (+G). The confidence
values of nodes were evaluated by bootstrap analyses based on 1000 resampling steps [28].

Molecules 2018, 23, 3003
8 of 12
3
.5. Extraction and Isolation
◦
Actinomadura sp. RB99 was grown in 50 mL ISP2 broth for seven days at 30 C (pre-culture) and
◦
used to inoculate 100 ISP2 agar plates. Plates were incubated for 10 days at 30 C, cut into small
pieces, consolidated, and immersed overnight in MeOH. The MeOH phase was filtered and evaporated
under reduced pressure. The MeOH extract (20 g) was dissolved in distilled water (700 mL) and then
solvent-partitioned with EtOAc (700 mL) three times, affording 1.1 g of residue. The EtOAc-soluble
fraction (1.1 g) from the MeOH extract was loaded onto a silica gel (230–400 mesh) column for
chromatography and eluted with a gradient solvent system of CH Cl -MeOH (100:1 to 1:1, v/v).
2
2
This provided six fractions (A–F), which were subjected to LC-UV/MS-based analysis. Dereplication
analysis using an in-house UV spectral library suggested the existence of minor peptide analogues
in fraction E. These displayed a simple UV pattern at around 220 nm and unique molecular formula
ion peaks containing nitrogen atoms. The polar fraction E (233 mg) was fractionated by preparative
reversed-phase HPLC (Phenomenex Luna C18, 250
×
21.2 mm i.d., 5 µm, Torrance, CA, USA) using
CH CN/H O (1:9 to 9:1, v/v, gradient system, flow rate: 5 mL/min) to yield five sub-fractions (E1–E5).
3
2
Sub-fraction E2 (27 mg) was purified by a semi-preparative reversed-phase HPLC (Phenomenex Luna
C18, 250 10.0 mm i.d., 5 m) with 33% MeOH/H O (isocratic system, flow rate: 2 mL/min) to yield
compound (5.8 mg, t = 57.0 min). Compounds
were isolated from sub-fraction E3 (15 mg) by semi-preparative reverse-phase HPLC eluting 43%
MeOH/H O (isocratic system, flow rate: 2 mL/min).
×
µ
2
1
2 (1.6 mg, t = 42.0 min) and 3 (1.5 mg, tR = 55.0 min)
R
R
2
3
.5.1. Natalenamide A (1)
−1
Amorphous powder. [α] -16.4 (c 0.02, MeOH); IR (KBr) νmax 3370, 1723, 1670, 1409, 1054 cm ;
2
5
D
1
13
UV (MeOH) λmax (log
ε
) 208 (3.90) nm; H (800 MHz) and C NMR (200 MHz) data, see Table 1;
+
HR-ESIMS (positive ion-mode) m/z 398.1691 [M + Na] (calculated for C H N O Na, 398.1692).
19
25
3
5
3
.5.2. Natalenamide B (2)
−1
Amorphous powder. [α] -18.4 (c 0.05, MeOH); IR (KBr) νmax 3352, 1756, 1630, 1392, 1031 cm ;
2
5
D
1
13
UV (MeOH) λmax (log
ε
) 210 (3.78) nm; H (800 MHz) and C NMR (200 MHz) data, see Table 1;
+
HR-ESIMS (positive ion-mode) 390.2032 [M + H] (calculated for C H N O , 390.2029) and 412.1849
20
28
3
5
+
[M + Na] (calculated for C H N O Na, 412.1848).
20 27 3 5
3
.5.3. Natalenamide C (3)
−1
Amorphous powder. [α] -10.2 (c 0.02, MeOH); IR (KBr) νmax 3225, 1746, 1685, 1359, 1027 cm ;
2
5
D
1
13
UV (MeOH) λmax (log
ε
) 209 (3.90) nm; H (800 MHz) and C NMR (200 MHz) data, see Table 1;
+
HR-ESIMS (positive ion-mode) 424.1870 [M + H] (calculated for C H N O , 424.1872) and 446.1694
23
26
3
5
+
[M + Na] (calculated for C H N O Na, 424.1692).
23 25 3 5
3
.6. Acid Hydrolysis of Compounds 1–3
An amount totaling 0.4 mg of each compound (
h at 110 C. After cooling to room temperature, the hydrolysates of
1
–
3
) was hydrolyzed with 6 N HCl (500
were evaporated to remove
L) was added to the hydrolysate mixtures and then evaporated to
remove traces of HCl; this process was performed three times.
µL) for
◦
1
1–3
traces of HCl. Distilled water (500
µ
3
.7. Determination of the Absolute Configuration of Amino Acids in 1–3
The hydrolysate mixtures ( ), as well as the standard amino acids (L/D-Leu, Glu, Phe, and Val),
were dissolved in 1 N NaHCO (100 L) and then treated with 50 L of L-FDAA (10 mg/mL in
1–3
µ
µ
3
◦
acetone). Successively, each hydrolysate was heated for 10 min at 80 C. Each mixture was quenched
with 2 N HCl (50 L) and concentrated in vacuo. The residue was dissolved in 300 L of MeOH.
Each aliquot (5 L) acquired from the hydrolysate mixtures was directly injected onto the LC/MS
µ
µ
µ

Molecules 2018, 23, 3003
9 of 12
(
Phenomenex Luna C18, 4.6
×
100 mm, 3.5
µ
m, flow rate of0.3 mL/min), and a full scan in positive
and negative ion modes (scan range from m/z 100 to 1000) was applied to identify the retention times
of the L-FDAA-derivatized amino acids. The mobile phase, consisting of formic acid in distilled water
(
(
0.1% v/v) (A) and acetonitrile (B), was carried with a gradient solvent system as follows: 20–40%
B) for 10 min, 100% (B) isocratic for 5 min, and then 20% (B) isocratic for 5 min, to conduct a post-run
washing procedure for the column. The retention times of the L-FDAA derivatized amino acids used
+
+
as standards were 16.7 min (L-Glu, m/z 400 [M + H] ), 18.2 min (D-Glu, m/z 400 [M + H] ), 22.1 min
+
+
+
(
2
3
L-Val, m/z 370 [M + H] ), 25.1 min (D-Val, m/z 370 [M + H] ), 25.6 min (L-Phe, m/z 418 [M + H] ),
7.0 min (D-Phe, m/z 418 [M + H] ), 25.5 min (L-Leu, m/z 384 [M + H] ), and 28.2 min (D-Leu, m/z
84 [M + H] ). The retention times of the derivatized hydrolyzates of
+
+
+
1
–3
were L-Glu (16.9 min), L-Phe
(
25.7 min), and L-Val (22.5 min) from
1; L-Glu (16.7 min), L-Phe (25.7 min), and L-Leu (25.6 min) from
2
; and L-Glu (16.9 min) and L-Phe (25.7 min) from 3.
3
.8. Cytotoxic Assays Using Cancer Cells
MCF7, HeLa, and A549 cells were purchased from the American Type Culture Collection
(
Rockville, MD, USA). HepG2 cells were purchased from the Korean Cell Line Bank (Seoul, Korea).
MCF7 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with
0% fetal bovine serum. HeLa and A549 cells were cultured in Dulbecco’s minimal essential medium
DMEM, Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum. HepG2 cells
1
(
were cultured in Minimum Essential Medium supplemented with 10% fetal bovine serum. Culture
◦
conditions were maintained at 37 C in a humidified atmosphere containing 5% CO . Cytotoxicity was
2
tested on these four human cell lines (MCF7, HeLa, A549, and HepG2) using the EZ-CyTox cell viability
3
assay kit (Dojindo Laboratories, Kumamoto, Japan). Briefly, 5
×
10 cells /well were seeded in 96-well
plates. After 24 h, cells were treated with compounds at the indicated concentrations. After 72 h of
incubation, the EZ-CyTox cell viability assay kit was used. After 2 h of incubation, the absorbance
was measured at 450 nm with a reference at 620 nm. Cell viability was calculated as a percentage of
the control.
3
.9. Anti-Inflammatory Activity
The mouse macrophage RAW264.7 cell line was purchased from the American Type Culture
Collection. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS),
◦
100 units/mL penicillin, and 100
µ
g/mL streptomycin, and incubated at 37 C in a humidified
atmosphere with 5% CO . Cell viability was measured using the Ez-Cytox cell viability detection kit.
2
Cells were incubated in 96-well plates at a concentration of 2500 cells per well for 24 h and treated with
the indicated concentrations of compounds 1–3 for 24 h. Next, Ez-Cytox reagents were added to each
well. The optical density at 450 nm was measured after 1 h using a microplate reader (PowerWave
XS; Bio-Tek Instruments, Winooski, VT, USA) to estimate cell viability. In a separate experiment,
RAW264.7 cells were incubated in 96-well plates at a concentration of 30,000 cells per well for 24 h.
After serum starvation for 12 h, cells were pretreated with compounds
1–3 for 1 h and then stimulated
with 1 g/mL of LPS for 24 h. The absorbance of culture media in a Griess reaction was measured at
µ
5
40 nm using a PowerWave XS microplate reader to estimate the NO level.
3
.10. Measurement of Melanin Content
The mouse melanoma cell line, B16F10, was purchased from the American Type Culture Collection.
Cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100
µ
g/mL
◦
streptomycin, and incubated at 37 C in a humidified atmosphere with 5% CO . B16F10 cells
2
were incubated in 6 cm culture dishes at 250,000 cells per well in DMEM supplemented with 10%
FBS, 100
with phosphate-buffered saline (PBS) and then stimulated by IBMX and incubated with different
concentrations of compounds or kojic acid (positive control) in phenol red-free RPMI medium
µg/mL streptomycin, and 100 U/mL penicillin for 24 h. The cells were washed twice
1–3

Molecules 2018, 23, 3003
10 of 12
supplemented with 10% FBS, 100 units/mL penicillin, and 100
µg/mL streptomycin for 24 h and 48 h.
The absorbance of the culture medium was measured at 405 nm using a PowerWave XS microplate
reader to estimate melanin content. Results are expressed as fold-change compared to the control (cells
not stimulated by IBMX).
3
.11. Statistical Analysis
All data including cell viability, NO, and melanin productions were presented as the average
value and standard deviation (SD). All the assays were done in triplicateand were repeated at least
three times. In this study, only a few numbers of repetitions of each cell experiment were included,
thus the non-parametric analysis method was adopted for statistical analysis. The Kruskall–Wallis
test was used for the statistical analysis of each variable. The SPSS statistical package was used for all
analyses (International Business Machines Corporation (IBM) SPSS statistics version 21, Boston, MA,
USA). Statistical significance was considered at a p-value lower than 0.05.
4
. Conclusions
Our report provides a chemical analysis of microbial isolates from a fungus-growing termite.
Three new cyclic tripeptides, natalenamides A–C ( ), were isolated and structurally characterized
1–3
from the culture broth of the fungus-growing -termite-associated Actinomadura sp. RB99 using an
LC-UV/MS-based dereplication method. All the isolated compounds were evaluated for biological
activities using cell-based assays. Compounds
HeLa/A549 cells, respectively. None of the isolated compounds showed significant inhibitory effects
on NO production in LPS-stimulated RAW264.7 cells. Compound exhibited significant inhibitory
1 and 2 exhibited weak cytotoxicity against HepG2 and
3
effects on IBMX-mediated melanin synthesis in a dose-dependent manner. In fact, the effect was
similar to than that of kojic acid, which is used as a cosmetic material with skin-whitening effects.
1
Supplementary Materials: The following are available online: Figure S1. HR-ESIMS data of
1
; Figure S2. H NMR
1
1
spectrum of
of (CD OD); Figure S5. HMBC spectrum of
spectrum of
of (CD OD); Figure S10. HMBC spectrum of
spectrum of
of (CD OD); Figure S15. HMBC spectrum of
amino acids of standards; Figure S17. Retention times of the L-FDAA derivatized amino acids from compound
Figure S18. Retention times of the L-FDAA derivatized L-Leu from compound ; Figure S19. Stimulation of
production of compounds ; Figure S20. Expanded key HMBC correlations of ; Figure S21. Expanded key
HMBC correlations of ; Figure S22. Expanded key HMBC correlations of ; Figure S23. LC/MS analysis of
compounds
1
(CD OD, 800 MHz); Figure S3. H- H COSY spectrum of
1
(CD OD); Figure S4. HSQC spectrum
3
3
1
1
1
(CD OD); Figure S6. HR-ESIMS data of
2
; Figure S7. H NMR
3
3
1
1
2
(CD OD, 800 MHz); Figure S8. H- H COSY spectrum of
2
(CD OD); Figure S9. HSQC spectrum
3
3
1
2
2
(CD OD); Figure S11. HR-ESIMS data of
3
; Figure S12. H NMR
3
3
1
1
3
(CD OD, 800 MHz); Figure S13. H- H COSY spectrum of
3
(CD OD); Figure S14. HSQC spectrum
3
3
3
3 (CD OD); Figure S16. Retention times of the L-FDAA derivatized
3
3
1;
2
1
1
–
3
2
3
1–
3
; Figure S24. Inhibitory effects of three fatty acid analogues on melanin content in B16F10 cells;
Figure S25. Neighbor-joining tree based on almost complete 16S rRNA gene sequences.
Author Contributions: Conceptualization, Y.H.K., C.B., K.S.K., and K.H.K.; validation, S.R.L., D.L., J.S.Y., R.B.,
S.L., D.-S.L. and J.H.; formal analysis, S.R.L., D.L., J.S.Y., R.B., S.L., D.-S.L., Y.H.K. and J.H.; investigation, S.R.L.,
D.L., J.S.Y., R.B., S.L., D.-S.L. and J.H.; resources, Z.W.B. and C.B.; writing—original draft preparation, S.R.L., C.B.,
K.S.K., and K.H.K.; writing—review and editing, S.R.L., C.B., K.S.K., and K.H.K.; project administration, K.H.K.;
funding acquisition, C.B. and K.H.K.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the
Korea government (MSIT) (2018R1A2B2006879) to K.H.K. We are grateful for financial support from the German
Research Foundation (CRC1127 ChemBioSys and BE 4799/3-1) and the Daimler Benz Foundation to C.B. R.B. is
generously supported by the International Leibniz Research School for Microbial and Biomolecular Interactions
(
ILRS) and the School for Microbial Communication (JSMC, DFG). This work was also supported by the Nano
Convergence Industrial Strategic Technology Development Program (20000105, Development of Cosmeceutical
Material Platform using Organo-Nano Complexes based on Natural Active Compounds) funded By the Ministry
of Trade, Industry & Energy (MOTIE, Korea).
Acknowledgments: We are deeply thankful to Michael Thomas-Poulsen (Department of Biology, University of
Copenhagen, Denmark) for supporting our field work.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 3003
11 of 12
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Sample Availability: Samples of the compounds are not available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).