Motobamide, an Antitrypanosomal Cyclic Peptide from a Leptolyngbya sp. Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.jnatprod.1c00234

Motobamide (1), a new cyclic peptide containing a C-prenylated cyclotryptophan residue, was isolated from a marine Leptolyngbya sp. cyanobacterium. Its planar structure was established by spectroscopic and MS/MS analyses. The absolute configuration was elucidated based on a combination of chemical degradations, chiral-phase HPLC analyses, spectroscopic analyses, and computational chemistry. Motobamide (1) moderately inhibited the growth of bloodstream forms of Trypanosoma brucei rhodesiense (IC50 2.3 μM). However, it exhibited a weaker cytotoxicity against normal human cells (IC50 55 μM).

Full text of DOI:10.1021/acs.jnatprod.1c00234

pubs.acs.org/jnp
Article
Motobamide, an Antitrypanosomal Cyclic Peptide from a
Leptolyngbya sp. Marine Cyanobacterium
Hiroki Takahashi, Arihiro Iwasaki, Naoaki Kurisawa, Ryota Suzuki, Ghulam Jeelani, Teruhiko Matsubara,
Toshinori Sato, Tomoyoshi Nozaki, and Kiyotake Suenaga*
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ABSTRACT: Motobamide (1), a new cyclic peptide containing a
C-prenylated cyclotryptophan residue, was isolated from a marine
Leptolyngbya sp. cyanobacterium. Its planar structure was
established by spectroscopic and MS/MS analyses. The absolute
configuration was elucidated based on a combination of chemical
degradations, chiral-phase HPLC analyses, spectroscopic analyses,
and computational chemistry. Motobamide (1) moderately
inhibited the growth of bloodstream forms of Trypanosoma brucei
rhodesiense (IC50 2.3 μM). However, it exhibited a weaker
cytotoxicity against normal human cells (IC50 55 μM).
frican sleeping sickness, one of the neglected tropical
diseases, is caused by the protozoa Trypanosoma brucei
A
rhodesiense and Trypanosoma brucei gambiense and threatens
human health, mainly in sub-Saharan Africa.1 As African
sleeping sickness is a fatal disease without any therapy, effective
antitrypanosomal drugs are essential for people living in areas
where this disease is endemic. However, existing antitrypano-
somal drugs have several problems, such as serious side effects
and emergence of drug-resistant strains.1 Therefore, the
discovery of new lead compounds is desired for the
development of more effective drugs.
Marine cyanobacteria have gained attention as rich sources
of bioactive secondary metabolites. In fact, several antiparasitic
natural products, such as almiramides B and C, which exhibit
antileishmanial activity,2 and ikoamide, which shows anti-
malarial activity,3 have been isolated from marine cyanobac-
teria. Hence, we investigated the secondary metabolites of
marine cyanobacteria as a source for antitrypanosomal drugs.
As a result, we isolated a new antitrypanosomal compound,
motobamide (1), from a Leptolyngbya sp. marine cyanobacte-
rium. Structurally, 1 is a cyclic decapeptide that contains an
unusual C-prenylated cyclotryptophan residue. Motobamide
(1) showed stronger antitrypanosomal activity than cytotox-
icity against normal human cells, WI-38. Here we report the
isolation, structure determination, and biological activities of
motobamide (1).
concentrated, and partitioned between EtOAc and H2O. The
EtOAc-soluble material was further partitioned between 90%
aqueous MeOH and hexane. The aqueous MeOH fraction was
subjected to fractionation by reversed-phase column chroma-
tography (ODS silica gel, MeOH−H2O) and repeated
reversed-phase HPLC to give motobamide (1, 0.6 mg).
Received: March 12, 2021
Published: May 13, 2021
RESULTS AND DISCUSSION
■
A Leptolyngbya sp. marine cyanobacterium (1.1 kg, wet weight)
was collected at Bisezaki, Okinawa, Japan, in April 2018. The
sample was extracted with EtOH, and the extract was filtered,
© 2021 American Chemical Society and
American Society of Pharmacognosy
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Table 1. NMR Data for Motobamide (1) in CD3OD
a
b
unit
Leu-1
position
δC, type
δH (J in Hz)
COSY correlations
selected HMBC correlations selected NOESY correlations
c
1
173.2, C
2
3
4
5
6
7
50.6, CH
41.7, CH2
25.1, CH
24.2, CH3
21.5, CH3
175.6, C
4.76, dd (9.2, 5.5)
1.58, m
1.73, m
0.98, d (7.2)
0.96, d (6.7)
3
2, 4
3, 5, 6
4
4
1
63a, 63b
Prenyl-Trp
8
9a
63.1, CH
42.7, CH2
4.13, m
2.79, dd (13.3, 8.2)
2.36, m
9a, 9b
8, 9b
8, 9a
7
25a, 25b
10, 16
10
9b
10
11a
11b
12
13
14
15
16
17
18
19
20
21
22
23
24
25a
25b
26a
26b
27
28
29
30
31
32
33
34
35a
35b
36
37
38
39
40
41a
41b
42
43
44
45
46a
46b
47a
47b
48a
48b
49
50
51
52
56.9, C
35.1, CH2
2.42, m
2.34, m
5.19, t (6.9)
11b, 12
11a, 12
11a, 11b, 14, 15
10
10
14, 15
120.1, CH
136.9, C
14, 16
12
d
26.2, CH3
1.72, s
1.57, s
5.53, s
12
12
13
12, 13
8, 11, 17, 22
18.2, CH3
84.8, CH
133.3, C
12
124.1, CH
120.2, CH
129.6, CH
110.5, CH
149.6, C
7.13, d (7.8)
6.74, t (7.8)
7.05, t (7.8)
6.64, d (7.8)
19, 20, 21
18, 20, 21
18, 19, 21
18, 19, 20
10
17
22
e
Pro-1
173.5, C
61.2, CH
29.13, CH2
4.15, m
2.06, m
1.35, m
2.09, m
1.96, m
3.73, t (6.9)
25
24, 25b, 26a, 26b
24, 25a, 26a, 26b
25a, 25b, 26b, 27
25a, 25b, 26a, 27
26a, 26b
23
23
8
8
f
25.6, CH2
49.1, CH2
29
27
c
Val-1
Leu-2
172.9, C
57.4, CH
33.5, CH
20.0, CH3
18.5, CH3
175.0, C
4.63, d (7.4)
2.16, m
1.13, d (6.9)
1.06, d (6.9)
30
28, 33
29, 31, 32
30
30
56.6, CH
41.2, CH2
4,16, m
2.00, m
1.59, m
1.79, m
1.04, d (6.4)
0.96, d (6.7)
35a, 35b
34, 35b, 36
34, 35a, 36
35a, 35b, 37, 38
36
33
d
26.1, CH
23.9, CH3
21.3, CH3
36
e
Met
174.2, C
53.2, CH
not detected
3.78, dd (11.0, 3.1)
2.20, m
2.04, m
2.44, m
1.93, s
41a, 41b
39
46a, 46b
40, 41b, 42
40, 41a, 42
41a, 41b
31.0, CH2
14.8, CH3
42
44
c
Pro-2
173.0, C
61.7, CH
30.0, CH2
4.44, m
1.87, m
1.58, m
2.05, m
1.68, m
3.63, m
3.50, m
46
45, 46b, 47a, 47b
45, 46a, 47a, 47b
46a, 46b, 47b, 48a, 48b
46a, 46b, 47a, 48a, 48b
47a, 47b, 48b
40
40
26.1, CH2
49.0, CH2
50
50
47a, 47b, 48a
Val-2
170.7, C
58.1, CH
31.4, CH
20.0, CH3
4.53, d (10.5)
2.22, m
0.912, d (6.9)
51
49
48a, 48b
50, 52, 53
51
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Table 1. continued
a
b
unit
position
δC, type
δH (J in Hz)
COSY correlations
selected HMBC correlations selected NOESY correlations
53
54
18.8, CH3
173.4, C
0.907, d (6.9)
51
e
Pro-3
55
62.4, CH
33.1, CH2
4.47, m
2.33, m
2.23, m
1.96, m
1.80, m
3.68, m
3.49, m
56a, 56b
54
60
56a
56b
57a
57b
58a
58b
59
55, 56b, 57a, 57b
55, 56a, 57a, 57b
56a, 56b, 57b, 58a, 58b
56a, 56b, 57a, 58a, 58b
57a, 57b, 58b
23.2, CH2
48.3, CH2
57a, 57b, 58a
c
Pro-4
173.1, C
60
60.1, CH
29.13, CH2
4.23, dd (8.8, 4.6)
2.30, m
1.87, m
2.06, m
2.02, m
61a, 61b
55
61a
61b
62a
62b
63a
63b
60, 61b, 62a, 62b
60, 61a, 62a, 62b
61a, 61b, 62b, 63a, 63b
61a, 61b, 62a, 63a, 63b
62a, 62b, 63b
f
25.7, CH2
48.2, CH2
3.91, m
3.64, m
2
2
62a, 62b, 63a
a
b
c−f
Measured at 100 MHz. Measured at 400 MHz.
These carbon signals are interchangeable.
Motobamide (1) was obtained as a colorless oil. The
molecular formula of 1 was found to be C63H95N11O10S by
HRESIMS (m/z 1220.6864). In the 1H NMR spectrum (Table
1), one triplet signal at 5.19 ppm and two singlet methyl signals
at 1.72 and 1.57 ppm indicated the presence of one carbon−
carbon double bond and two vinyl methyl groups. In the 13C
NMR spectrum (Table 1), 10 signals (δC 175.6, 175.0, 174.2,
173.5, 173.4, 173.2, 173.1, 173.0, 172.9, and 170.7), considered
to be amide or ester carbonyl carbons, were detected. In
addition, detailed analyses of the 2D NMR spectra revealed the
presence of nine proteogenic amino acids: two leucines (Leu),
four prolines (Pro), two valines (Val), and a methionine
(Met). Although we could not detect the carbon signal of C-
41, the presence of a methylene group at C-41 was verified
based on the COSY correlations (H-40/H-41a, H-40/H-41b,
H-41a/H-42, and H-41b/H-42). In addition, we also clarified
the presence of an unusual amino acid, C-prenylated
cyclotryptophan (Prenyl-Trp), as follows. The COSY
correlations (H-8/H-9a, H-8/H-9b, H-18/H-19, H-19/H-20,
and H-20/H-21) revealed two partial structures (C-8−C-9 and
C-18−C-19−C-20−C-21). In addition, two COSY correla-
tions (H-11a/H-12 and H-11b/H-12), five HMBC correla-
tions (H-12/C-14, H-12/C-15, H-14/C-13, H-15/C-12, and
H-15/C-13), and their chemical shifts established the presence
of a prenyl group. These three partial structures and four
carbons (C-10, C-16, C-17, and C-22) were connected based
on the HMBC correlations (H-9a/C-10, H-9b/C-10, H-9a/C-
16, H-11a/C-10, H-11b/C-10, H-16/C-8, H-16/C-11, H-16/
C-17, H-16/C-22, H-18/C-10, H-19/C-17, and H-20/C-22).
Finally, we clarified the location of the two nitrogen atoms
based on the deshielded chemical shifts of C-8 (δC 63.1, δH
4.13), C-16 (δC 84.8, δH 5.53), and C-22 (δC 149.6). As a
result, these moieties formed a cyclic tryptophan residue with a
prenyl side chain.
Figure 1. Gross structure of motobamide (1) based on 2D NMR
analyses.
correlations (H-2/H-63a, H-2/H-63b, and H-55/H-60)
established the sequence: Pro-3-Pro-4-Leu-1. As we could
not get any additional structural information from NMR data,
we carried out an MS/MS analysis of 1 to connect these three
partial sequences (Figure 2). The MS/MS analysis showed
three fragmentation patterns as shown in Figure 2, suggesting
structure 1 for motobamide (1).
The geometries of the peptide bonds of the proline residues
were determined based on the 13C NMR chemical shift
differences between the β and γ positions (Δδβ−γ).4 The large
difference observed in Pro-3 (Δδβ−γ = 9.9 ppm) indicated that
its peptide bond has a cis geometry. This conclusion was also
supported by the NOESY correlation, H-55/H-60. On the
contrary, the difference at Pro-1 (Δδβ−γ = 3.5 ppm), Pro-2
(Δδβ−γ = 3.9 ppm), and Pro-4 (Δδβ−γ = 3.4 ppm) indicated
that they possess a trans geometry, supported by the NOESY
correlations (H-27/H-29, H-48a/H-50, H-48b/H-50, H-2/H-
63a, and H-2/H-63b). The geometry of the peptide bond at
the C-prenylated cyclotryptophan residue was determined to
be cis on the basis of the NOESY correlations (H-8/H-25a and
H-8/H-25b).
Next, we determined the sequence of these 10 amino acid
residues (Figure 1). The HMBC correlation (H-29/C-33) and
NOESY correlations (H-27/H-29, H-8/H-25a, and H-8/H-
25b) revealed the following sequence: Prenyl-Trp-Pro-1-Val-1-
Leu-2. Based on the NOESY correlations (H-40/H-46a, H-40/
H-46b, H-48a/H-50, and H-48b/H-50), the segment of Met-
Pro-2-Val-2 was established. Furthermore, the NOESY
The absolute configurations of all the usual amino acids in
motobamide (1) were determined by acid hydrolysis followed
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Figure 2. MS/MS fragmentation of motobamide (1).
Figure 3. Experimental ECD spectrum of motobamide (1) and theoretical ECD spectra of the model compounds (8S,10S,16S)-2 and
(8S,10R,16R)-2.
by chiral-phase HPLC analyses. To make the results clear, we
isolated each amino acid from the hydrolysate before chiral-
phase HPLC analyses. These analyses revealed that they are all
of the L form. As for Prenyl-Trp, acid hydrolysis of 1 with
phenol afforded tryptophan derived from the Prenyl-Trp
residue. Acid hydrolysis in the presence of phenol5 and chiral-
HPLC analysis afforded L-Trp (from Prenyl-Trp) and
determined the absolute configuration of C-8 as S.
The NOESY correlation between H-12 and H-16 indicated
that the prenyl group and H-16 are located on the same face of
Prenyl-Trp. Therefore, there were two possible absolute
configurations of Prenyl-Trp: (8S,10S,16S) and
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calculations were run with Gaussian 16 with default grids and
convergence criteria.
(8S,10R,16R). The electronic circular dichroism (ECD)
spectrum of motobamide (1) showed a negative Cotton effect
at 295 nm, as shown in Figure 3. Considering the UV
absorption wavelength of the functional groups in 1, this
Cotton effect was derived from the aromatic ring in the Prenyl-
Trp residue. As the whole structure of 1 involves a large
number of conformers to be considered, we designed the two
simplified model compounds (8S,10S,16S)-2 and
(8S,10R,16R)-2. Next, we calculated theoretical ECD spectra
of the model compounds using several pairs of basis function
and functional, respectively, and compared them with that of
natural 1 (Figure 3). The theoretical spectra of (8S,10S,16S)-2
in all calculation conditions showed Cotton curves similar to
that of 1, suggesting the absolute configuration of the Prenyl-
Trp residue as 8S, 10S, 16S.
Next, we evaluated the biological activity of motobamide
(1). Motobamide (1) inhibited the growth of the bloodstream
form of T. b. rhodesiense, the causative organism of human
African sleeping sickness, with an IC50 value of 2.3 μM, while
the cytotoxicity of 1 against WI-38 cells, normal human
fibroblasts (IC50 = 55 μM), was more than 20-fold weaker. In
addition, 1 did not show any growth-inhibitory activity against
HeLa or HL60 cells at 10 μM.
In conclusion, we discovered a new modified cyclic
decapeptide, motobamide (1), from a Leptolyngbya sp. marine
cyanobacterium collected in Okinawa, Japan. The planar
structure of 1 was established based on the analyses of 2D
NMR and MS/MS spectra. The absolute configurations of the
usual amino acids were established by acid hydrolysis followed
by chiral-phase HPLC analyses. The absolute configuration of
the unusual amino acid Prenyl-Trp was determined by a
combination of experimental and computational calculation
chemistry. Since some cyanobacteria produce Prenyl-Trp-
containing compounds, i.e., kawaguchipeptin A5 and trikor-
amide A,6 the calculated ECD spectra of the two model
compounds (8S,10S,16S)-2 and (8S,10R,16R)-2 can be useful
for determining the absolute configuration of Prenyl-Trp units
of these compounds. Motobamide (1) moderately inhibited
the growth of bloodstream forms of T. b. rhodesiense, but did
not show any cytotoxicity against human cells at 10 μM.
Further evaluations of the antiparasitic potency of 1 are
ongoing in our laboratory.
Identification of the Cyanobacterium. A cyanobacterial
filament was isolated under the microscope and crushed by freezing
and thawing. The 16S rDNA genes were PCR-amplified from the
isolated DNA using the primer set CYA359F (a cyanobacterial-
specific primer) and CYA1371R (a universal primer). The PCR
reaction contained DNA derived from the cyanobacteria filament, 0.5
μL of KOD-Multi & Epi- (Toyobo), 0.8 μL of each primer (0.4 μM,
respectively), 12.5 μL of 2× PCR buffer for KOD-Multi & Epi-, and
H2O for a total volume of 25 μL. The PCR reaction was performed as
follows: initial denaturation for 2 min at 94 °C and amplification by
40 cycles of 10 s at 98 °C and 1.5 min at 66 °C. PCR products were
analyzed on agarose gel (1%) in TBE buffer and visualized by
ethidium bromide staining. The obtained DNA was sequenced with
CYA359F and CYA1371R primers. This sequence is available in the
DDBJ/EMBL/GenBank databases under accession number
LC602971. The nucleotide sequence of 16S rRNA gene obtained in
this study was used for phylogenetic analysis with the sequences of
related cyanobacterial 16S rRNA genes.7 All sequences were aligned
by the SINA web service (version 1.2.11)8 with default settings. The
poorly aligned positions and divergent regions were removed by
Gblocks Server (version 0.91b),9 implementing the options for a less
stringent selection, including the ‘Allow smaller final blocks’, ‘Allow
gap positions within the final blocks’, and ‘Allow less strict flanking
positions’ options. The obtained 802 nucleotide positions were used
for phylogenetic analysis. JModeltest (version 2.1.7)10,11 with default
settings was used to select the best model of DNA substitution for the
Maximum Likelihood (ML) analysis and Bayesian analysis according
to the Akaike information criterion (AIC). The ML analysis was
conducted by PhyML (version 20131016),11 using the TIM2+I+G
model with a gamma shape parameter of 0.4280, a proportion of
invariant sites of 0.4750, and nucleotide frequencies of F(A) = 0.2566,
F(C) = 0.2155, F(G) = 0.3054, and F(T) = 0.2225. Bootstrap
resampling was performed on 1000 replicates. The ML tree was
visualized with Njplot (version 2.3).12 The Bayesian analysis was
conducted by MrBayes (version 3.2.5)13 using the GTR+I+G model.
The Markov chain Monte Carlo process was set at 2 chains, and
1 000 000 generations were conducted. Sampling frequency was
assigned at every 500 generations. After analysis, the first 100 000
trees were deleted as burn-in, and the consensus tree was constructed.
The Bayesian tree was visualized with FigTree (version 1.4.0, http://
tree.bio.ed.ac.uk/software/figtree). As a result, the cyanobacterium
(accession no. LC602971) formed a clade with Leptolyngbya sp.
Therefore, the cyanobacterium was classified into Leptolyngbya sp.
Collection, Extraction, and Isolation. The Leptolyngbya sp.
cyanobacterium (cell mass 1.1 kg, wet weight) was collected at Bise,
Okinawa Island, Okinawa Prefecture, Japan, during April 2018. The
collected cyanobacterium was extracted with EtOH (2 × 2 L) for 5
days. The extract was filtered, and the filtrate was concentrated. The
residue was partitioned between EtOAc (3 × 0.3 L) and H2O (0.3 L).
The material obtained from the organic layer was partitioned between
90% aqueous MeOH (0.3 L) and hexane (3 × 0.3 L) to give an
aqueous MeOH fraction (593 mg). The aqueous MeOH fraction was
separated by column chromatography on ODS (6.0 g) eluted with
40% MeOH, 60% MeOH, 80% MeOH, MeOH, and CHCl3−MeOH
(1:1). The fraction eluted with 80% MeOH (143 mg) was subjected
to HPLC [Cosmosil 5C18MS-II (ϕ 20 × 250 mm); flow rate 5 mL/
min; detection, UV 215 nm; solvent 80% aqueous MeOH] in five
batches to give a fraction that contained motobamide (1) (58.8 mg,
the last collected fraction). This fraction was further purified by
HPLC [Cosmosil 5C18MS-II (ϕ 20 × 250 mm); flow rate 5 mL/min;
detection, UV 215 nm; solvent 85% aqueous MeOH] in two batches
to give a motobamide (1)-containing fraction (8.9 mg, tR = 44.8 min).
Moreover, this fraction was subjected to HPLC [Cosmosil Cholester
(ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215 nm;
solvent aqueous 70% MeCN] to give a fraction that contained 1 (2.0
mg, tR = 32.0 min). Finally, this fraction was purified by HPLC
[Cosmosil 5PE-MS (ϕ 20 × 250 mm); flow rate 5 mL/min;
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO DIP-1000 polarimeter. UV spectra were
recorded on a JASCO V730-BIO instrument. ECD spectra were
measured with a JASCO J-720 W spectropolarimeter. IR spectra were
recorded on a Bruker ALPHA instrument. All NMR data were
1
recorded on a JEOL JNM-ECS400 spectrometer for H (400 MHz)
and 13C (100 MHz). 1H NMR chemical shifts (referenced to residual
CD3OD observed at δH 3.31) were assigned using a combination of
data from COSY and HMQC experiments. Similarly, 13C NMR
chemical shifts (referenced to CD3OD observed at δC 49.0) were
assigned based on HMBC and HMQC experiments. HRESIMS
spectra were obtained on a Waters LCT Premier XE time-of-flight
(TOF) mass spectrometer. MS and MSn spectra were collected in
positive mode by using a Bruker Daltonics amaZon SL ion trap mass
spectrometer equipped with an ESI source. Chromatographic analyses
were performed using an HPLC system consisting of a pump (model
PU-2080, JASCO) and a UV detector (model UV-2075, JASCO). All
chemicals and solvents used in this study were the best grade available
and obtained from a commercial source. Density functional theory
(DFT) and time-dependent density functional theory (TDDFT)
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detection, UV 215 nm; solvent aqueous 63% MeCN] to give
h. The plates were read in a SpectraMax Gemini XS microplate
fluorescence scanner (Molecular Devices) using an excitation
wavelength of 536 nm and an emission wavelength of 588 nm.
Conformational Search for the Two Model Compounds of
the Prenyl Tryptophan Moiety, (8S,10S,16S)-2 and
(8S,10R,16R)-2. The conformational search for the two model
compounds (8S,10S,16S)-2 and (8S,10R,16R)-2 was carried out using
the MMFF force field and torsional sampling of the Macro Model
program.17 An energy window cutoff of 10.0 kcal/mol was used
during the conformational search. Redundant conformers were
eliminated using an RMSD cutoff of 1.0 Å. Then, all conformers
were subjected to geometry optimization using the Gaussian 16
package18 in the gas phase at the B3LYP/6-31G* level. The resulting
conformers were further optimized at the B3LYP level of theory using
the def2-SVP basis set with PCM19 in MeCN using the Gaussian 16
package.18 As a result, we obtained seven conformers for
(8S,10S,16S)-2 and 15 conformers for (8S,10R,16R)-2 as the
conformers that have a calculated Boltzmann weight of more than 1%.
ECD Calculation Methods. The optimized conformers of
(8S,10S,16S)-2 and (8S,10R,16R)-2 that showed >1% Boltzmann
population were used for ECD calculations using TDDFT20 with
PCM19 in MeCN. We used the four different combinations of
functional and basis set as follows: B3LYP/def2-SVP, ωB97X-D/def2-
SVP, cam-B3LYP/def2-SVP, and ωB97X-D/6-311+G*, respectively.
ECD curves were generated with Spec Dis software using a half-
bandwidth of 0.16 eV.21 The relative populations of each conformer
were calculated based on the Boltzmann weighting factor at 298.15 K.
UV correction of +20 nm for B3LYP/def2-SVP, +35 nm for ωB97X-
D/def2-SVP and cam-B3LYP/def2-SVP, and +25 nm for ωB97X-D/
6-311+G* were applied for proper comparison. All calculated spectra
were scaled by a factor of 1/2 to enable comparison with the
experimental spectrum.
motobamide (1, 0.6 mg, tR = 35.3 min).
Motobamide (1): colorless oil; [α]25 −243 (c 0.17, MeOH); UV
D
(MeOH) λmax (log ε) 238 (4.05), 295 (3.56) nm; ECD (0.83 mM,
MeCN) λmax (Δε) 295 (−2.52) nm; IR (neat) 3307, 2960, 1641,
1527, 1446, 1310 cm−1; 1H NMR, 13C NMR, COSY, HMBC,
NOESY, and TOCSY data, Table 1; HRESIMS m/z 1220.6864 [M +
Na]+ (calcd for C63H95N11O10SNa, 1220.6882).
Determination of the Absolute Configuration of Motoba-
mide (1). Motobamide (1, 0.2 mg) was treated with 6 M HCl (100
μL) for 18 h at 110 °C. The reaction mixture was evaporated to
dryness and separated on an analytical HPLC column [conditions for
HPLC separation: column, Cosmosil 5C18-PAQ (ϕ 4.6 × 250 mm);
flow rate, 1.0 mL/min; detection at 215 nm; solvent H2O; retention
times (min) of components: Pro (3.5), Val (3.7), Met (4.7), and Leu
(5.4)].
Each fraction was dissolved in H2O (50 μL) and analyzed by chiral-
phase HPLC, and the retention times were compared to those of
authentic standards [column, DAICEL CHIRALPAK MA (+) (ϕ 4.6
× 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent 2
mM CuSO4]. The retention times for authentic standards were 2.9
min (D-Pro), 5.8 min (L-Pro), 3.8 min (D-Val), 6.7 min (L-Val), 6.8
min (D-Met), 11.2 min (L-Met), 8.1 min (D-Leu), and 15.3 min (L-
Leu). The retention times of each amino acid from natural 1 were 5.8,
6.7, 11.2, and 15.3 min, indicating the presence of L-Pro, L-Val, L-Met,
and L-Leu, respectively.
Motobamide (1, 0.1 mg) was treated with 1% phenol in 6 M HCl
(100 μL) for 4 h at 110 °C.5 The hydrolyzed product was evaporated
to dryness, and Trp was isolated as a derivative of the Prenyl-Trp
residue of 1 by HPLC separation [conditions for HPLC separation:
column, Cosmosil 5C18-PAQ (ϕ 4.6 × 250 mm); flow rate, 1.0 mL/
min; detection at 215 nm; solvent 20% MeOH; retention time of Trp
is 9.2 min]. The fraction was dissolved in H2O (50 μL) and analyzed
by chiral-phase HPLC, and the retention time was compared to those
of authentic standards [column, DAICEL CHIRALPAK MA(+) (ϕ
4.6 × 50 mm); flow rate, 1.0 mL/min; detection at 254 nm; solvent
15% MeCN 2 mM CuSO4]. The retention times for authentic
standards were 8.8 min (D-Trp) and 10.8 min (L-Trp). The retention
time of Trp from Prenyl-Trp of natural 1 was 10.8 min, indicating the
presence of Prenyl-L-Trp.
Cell Growth Analysis. WI-38 cells and HeLa cells were cultured
at 37 °C with 5% CO2 in DMEM (Nissui) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin,
100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 300 μg/mL L-
glutamine, and 2.25 mg/mL NaHCO3. HL60 cells were cultured at 37
°C with 5% CO2 in RPMI (Nissui) supplemented with 10% heat-
inactivated FBS, 100 units/mL penicillin, 100 μg/mL streptomycin,
0.25 μg/mL amphotericin, 300 μg/mL L-glutamine, and 2.25 mg/mL
NaHCO3. WI-38 cells were seeded at 4 × 103 cells/well in 96-well
plates (Iwaki) and cultured overnight. HeLa cells were seeded at 2 ×
104 cells/well in 96-well plates (Iwaki) and cultured overnight. HL60
cells were seeded at 10 × 104 cells/well in 96-well plates. Various
concentrations of compounds were then added, and cells were
incubated for 72 h. Cell proliferation was measured by the MTT
assay.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.1c00234.
■
sı
NMR spectra for motobamide (1); HPLC chromato-
grams for determination of the absolute configurations;
phylogenetic tree of the cyanobacterial sample, detailed
results of the computational chemistry (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Kiyotake Suenaga − Department of Chemistry, Faculty of
Science and Technology, Keio University, Yokohama,
Kanagawa 223-8522, Japan; Email: suenaga@
chem.keio.ac.jp
Authors
Hiroki Takahashi − Department of Chemistry, Faculty of
Science and Technology, Keio University, Yokohama,
Kanagawa 223-8522, Japan
Arihiro Iwasaki − Department of Chemistry, Faculty of Science
and Technology, Keio University, Yokohama, Kanagawa 223-
8522, Japan; orcid.org/0000-0002-3775-5066
Naoaki Kurisawa − Department of Chemistry, Faculty of
Science and Technology, Keio University, Yokohama,
Kanagawa 223-8522, Japan
Ryota Suzuki − S&E Simulation, Yokohama, Kanagawa 234-
0052, Japan
Ghulam Jeelani − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan
In Vitro Antitrypansomal Assay. The bloodstream form of
Trypanosoma brucei rhodesiense strains IL-150114 was cultured at 37
°C under a humidified 5% CO2 atmosphere in HMI-9 medium15
supplemented with 10% heat-inactivated FBS. For in vitro studies,
compounds were dissolved in DMSO and diluted in culture medium
prior to being assayed. The maximum DMSO concentration in the in
vitro assays was 1%. The compounds were tested in an AlamarBlue
serial drug dilution assay16 to determine the 50% inhibitory
concentrations (IC50). Serial drug dilutions were prepared in 96-
well microtiter plates containing the culture medium, and wells were
inoculated with 2.0 × 104 cells/mL T. b. rhodesiense IL-1501 parasites.
Cultures were incubated for 69 h at 37 °C under a humidified 5%
CO2 atmosphere. After this time, 10 μL of resazurin (12.5 mg of
resazurin [Sigma] dissolved in 100 mL of phosphate-buffered saline)
was added to each well. The plates were incubated for an additional 3
Teruhiko Matsubara − Department of Biosciences and
Informatics, Faculty of Science and Technology, Keio
1654
https://doi.org/10.1021/acs.jnatprod.1c00234
J. Nat. Prod. 2021, 84, 1649−1655

Journal of Natural Products
pubs.acs.org/jnp
Article
Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov,
V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.;
Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J.
Gaussian 16, Revision C.01; Gaussian, Inc.: Wallingford, CT, 2016.
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2999−3094.
University, Yokohama, Kanagawa 223-8522, Japan;
orcid.org/0000-0002-8006-4324
Toshinori Sato − Department of Biosciences and Informatics,
Faculty of Science and Technology, Keio University,
Yokohama, Kanagawa 223-8522, Japan; orcid.org/0000-
0002-4429-6101
Tomoyoshi Nozaki − Department of Biomedical Chemistry,
Graduate School of Medicine, The University of Tokyo,
Tokyo 113-0033, Japan
(20) Autschbach, J. ChemPhysChem 2011, 12, 3224−3235.
(21) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G.
Chirality 2013, 25, 243−249.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.1c00234
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI (Grant Numbers
18K14346 and 20H02870).
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