Isolation and Total Synthesis of Mabuniamide, a Lipopeptide from an Okeania sp. Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.jnatprod.9b00749

The bioassay-guided fractionation of an Okeania sp. marine cyanobacterium collected in Okinawa led to the isolation of the lipopeptide mabuniamide (1). The gross structure of 1 was determined by spectroscopic analyses, and its absolute configuration was determined using Marfey's analysis of the acid hydrolysate of 1. The absolute configuration of 1 was confirmed by total synthesis. Mabuniamide (1) stimulated glucose uptake in cultured rat L6 myotubes. In addition, mabuniamide (1) and its stereoisomer (2) exhibited moderate antimalarial activity.

Full text of DOI:10.1021/acs.jnatprod.9b00749

Article
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Isolation and Total Synthesis of Mabuniamide, a Lipopeptide from
an Okeania sp. Marine Cyanobacterium
Kaori Ozaki,† Arihiro Iwasaki,‡ Dai Sezawa,‡ Haruka Fujimura,‡ Tomoyoshi Nozaki,§
Yumiko Saito-Nakano,⊥ Kiyotake Suenaga,*,‡ and Toshiaki Teruya*,†
†Graduate School of Engineering and Science, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
‡Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa
223-8522, Japan
§Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-0033, Japan
⊥Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
S
* Supporting Information
ABSTRACT: The bioassay-guided fractionation of an Okeania sp. marine cyanobacterium collected in Okinawa led to the
isolation of the lipopeptide mabuniamide (1). The gross structure of 1 was determined by spectroscopic analyses, and its
absolute configuration was determined using Marfey’s analysis of the acid hydrolysate of 1. The absolute configuration of 1 was
confirmed by total synthesis. Mabuniamide (1) stimulated glucose uptake in cultured rat L6 myotubes. In addition,
mabuniamide (1) and its stereoisomer (2) exhibited moderate antimalarial activity.
we report the isolation, structure elucidation, synthesis, and
biological activity of 1.
arine organisms are well known to be a rich source of
1,2
Mbioactive substances.
Marine cyanobacteria also are
good producers of secondary metabolites possessing various
bioactivities with structural diversity.3 In the past 10 years,
numerous marine cyanobacteria-derived peptides have been
discovered.4 Most of the linear peptides isolated from marine
cyanobacteria exhibit biological activities, such as antitumor,
antimicrobial, antimalarial, and enzyme inhibition properties.4,5
For example, bisebromoamide, isolated from a Lyngbya sp.,
showed potent cytotoxicity against HeLa S3 cells.6 Almiramides
B and C, isolated from a Lyngbya majuscula, showed strong in
vitro antiparasitic activity against Leishmania donovani.7
Meanwhile, some peptides that show bioactivity related to
metabolic disease such as type 2 diabetes and osteoporosis
have been isolated from marine cyanobacteria. For example,
largazole, isolated from a Symploca sp., exhibited in vitro and in
vivo osteogenic activity.8,9 Ypaoamide B, isolated from an
Okeania sp., stimulated glucose uptake in cultured L6
myotubes by activation of AMP-activated protein kinase
(AMPK).10
RESULTS AND DISCUSSION
■
An Okeania sp. marine cyanobacterium (392.1 g, wet weight)
was collected from the coast of Odo, Okinawa, Japan, and
extracted with MeOH. The extract was subjected to
fractionation guided by glucose uptake activity in L6 myotubes
with solvent partitioning, ODS silica gel column chromatog-
In our search for novel bioactive substances, we recently
isolated mabuniamide (1), which stimulated glucose uptake in
cultured L6 myotubes and showed antimalarial activity. Here,
Received: August 6, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00749
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Table 1. NMR Data for Mabuniamide (1) in CDCl3
a
b
a
b
unit
Pro1
no.
δC, type
δH mult (J in Hz)
unit
no.
δC, type
δH mult (J in Hz)
1
2
172.5, C
59.0, CH
28.9, CH2
Leu
1
2
175.0, C
49.4, CH
39.7, CH2
4.34, dd (8.6, 5.6)
2.09, m
4.69, m
1.81, m
1.36, m
1.71, m
0.90, d (6.6)
0.87, d (6.6)
8.78, d (6.3)
3a
3b
4a
4b
5
3a
3b
4
1.81, m
1.86, m
1.72, m
3.23, m
25.1, CH2
24.8, CH
23.5, CH3
21.3, CH3
5
6
46.8, CH2
51.3, CH3
168.0, C
O-Me
1
3.68, s
NH
1
c
N-Me-Phe1
N-Me-Phe2
170.16, C
2
56.4, CH
35.2, CH2
5.52, dd (8.7, 6.1)
3.27, m
2.73, dd (13.4, 6.1)
2
62.9, CH
33.8, CH2
4.95, dd (11.8, 2.8)
3.22, dd (14.2, 2.8)
2.87, m
3a
3b
4
3a
3b
4
137.3, C
138.1, C
05/9
06-/8
7
N-Me
1
129.5, CH
128.4, CH
127.0, CH
29.8, CH3
168.2, C
7.11, m
7.21. m
7.19, m
2.86, s
05/9
06/8
7
N-Me
1
129.6, CH
129.0, CH
126.7, CH
29.3, CH3
173.3, C
7.20, m
7.25, m
7.16, m
2.82, s
Gly
Val
Pro2
2a
2b
NH
1
2
3
4
5
NH
1
2
41.4, CH2
4.16, dd (17.5, 5.3)
3.91, dd (17.5, 3.4)
6.93, brt (5.3, 3.4)
2
3
55.2, CH
28.3, CH2
25.4, CH2
4.33, dd (7.9, 5.2)
0.76, m
1.94, m
1.54, m
3.53, m
4a
4b
5a
5b
1
171.2, C
59.1, CH
30.2, CH
19.6, CH3
18.4, CH3
4.24, dd (8.4, 7.0)
2.20, m
0.93, m
0.93, m
6.86, m
47.6, CH2
3.47, m
N-Me-Ala
170.1, C
2
3
49.6, CH
14.3, CH3
30.6, CH3
173.2, C
35.7, CH2
18.4, CH2
14.0, CH3
5.39, q (6.9)
1.18, d (6.9)
2.91, s
c
N-Me-Val
170.19 , C
N-Me
1
2
3
4
63.2, CH
26.0, CH
20.4, CH3
18.7, CH3
31.5, CH3
4.64, m
2.36, m
1.01, d (6.4)
0.83, d (6.8)
3.10, s
Ba
3
4
5
2.26, t (7.5)
1.63, m
0.93, t (7.1)
0.90, d (6.6)
N-Me
a
b
c
Measured at 125 MHz. Recorded at 500 MHz. These signals are interchangeable.
raphy (MeOH−H2O), and reversed-phase HPLC to give
mabuniamide (1).
(δH 2.82)/C-1 of Pro2 (δC 173.3), and N-Me of N-Me-Ala (δH
2.91)/C-1 of Ba (δC 173.2) and the NOESY correlation H2-5
of Pro2 (δH 3.53 and 3.47)/H-2 of N-Me-Ala (δH 5.39)
established the linear sequence Pro1-N-Me-Phe1-Gly-Val-N-
Me-Val-Leu-N-Me-Phe2-Pro2-N-Me-Ala-Ba. Thus, the gross
structure of 1 was determined and is shown in Figure 1a. The
sequence of residues in 1 was further confirmed by analysis of
ESIMS/MS fragmentation patterns shown in Figure 1b. Thus,
the gross structure of 1 was determined and is shown in Figure
1a.
The configurations of the Pro1, Val, N-Me-Val, Leu, Pro2,
and N-Me-Ala units in 1 were L, L, D, L, L, and L, respectively, as
determined by hydrolysis of 1 following Marfey’s method.11
The analysis described above revealed that 1 contained N-Me-
L-Phe and N-Me-D-Phe. To distinguish them from one another,
the partial acid hydrolysate of 1 was subjected to reversed-
phase HPLC-guided fractionation. Treatment of 1 with 4 M
HCl for 30 min at 100 °C afforded the partial acid hydrolysate
of 1.12,13 The ESIMS analysis of each fraction allowed us to
identify fragments of N-Me-Phe-Gly-Val (fragment A) and
Leu-N-Me-Phe-Pro (fragment B). Complete acid hydrolysis of
their fragments and reversed-phase HPLC-guided fractionation
generated the N-Me-Phe from each fragment, respectively.
Marfey’s analysis of each N-Me-Phe revealed that the absolute
configuration of the N-Me-Phe adjacent to Leu and Pro was L
Mabuniamide (1) was obtained as a colorless oil, and its
molecular formula was determined to be C58H87N9O11 by
HRESIMS. The NMR data for 1 are summarized in Table 1.
1
The H NMR spectra suggested the presence of three NH
groups (δH 8.78, 6.93, 6.86), four N-methylamide groups (δH
3.10, 2.91, 2.86, 2.82), and one methyl ester moiety (δH 3.68).
In addition, the 1H and 13C NMR spectra revealed the
presence of several α-protons (∼δH 4.24−5.52) and several
1
carbonyl carbons (∼δC 168.0−175.0). Thus, the H and 13C
NMR spectra suggested that 1 was a peptidic compound
(Table 1). The analyses of COSY, HSQC, and HMBC spectra
recorded in CDCl3 revealed the presence of two prolines (Pro1
and Pro2), two N-methyl-phenylalanines (N-Me-Phe1 and N-
Me-Phe2), glycine (Gly), valine (Val), N-methyl-valine (N-
Me-Val), leucine (Leu), N-methyl-alanine (N-Me-Ala), and
butanoic acid (Ba).
The sequence for 1 was determined by HMBC and NOESY
analyses. The HMBC signals H2-5 of Pro1 (δH 3.23)/C-1 of N-
Me-Phe1 (δC 168.0), N-Me of N-Me-Phe1 (δH 2.86)/C-1 of
Gly (δC 168.2), NH of Gly (δH 6.93)/C-1 of Val (δC 171.2),
NH of Val (δH 6.86)/C1 of N-Me-Val (δC 170.19), N-Me of
N-Me-Val (δH 3.10)/C-1 of Leu (δC 175.0), NH of Leu (δH
8.78)/C-1 of N-Me-Phe2 (δH 170.16), N-Me of N-Me-Phe2
B
DOI: 10.1021/acs.jnatprod.9b00749
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Figure 1. (a) Key 2D NMR correlations for mabuniamide (1). (b) ESIMS/MS fragmentation patterns of mabuniamide (1).
Scheme 1. Total Synthesis of Mabuniamide (1) and its Stereoisomer 2
and that the other was D. This completed the structural
assignments of mabuniamide (1).
and N-Me-L-Pro, sequentially. The addition of L-alanine to the
resulting octapeptide 9a followed by the condensation of
butanoic acid gave mabuniamide (1, 12% through 16 steps).
Its stereoisomer 2, which possessed the inverted configurations
regarding the two N-Me-Phe residues, was synthesized from
known dipeptide 3b15 in a similar manner, as shown in Scheme
1 (14% through 16 steps). The spectral data of natural
mabuniamide were consistent with those of synthetic 1, but
To confirm the absolute configuration of 1, we synthesized
mabuniamide (1) and its stereoisomer 2, whose configurations
of the two N-Me-Phe residues were exchanged (Scheme 1).
Known dipeptide 3a14 was condensed with the following
amino acids, Gly, L-Val, and N-Me-D-Val, one by one, affording
pentapeptide 6a. We further extended the sequence of 6a by
the condensation of the following residues, L-Leu, N-Me-L-Phe,
C
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not 2. Therefore, we confirmed the correctness of our structure
determination.
1.4 0.2 and 2.8 0.1 μM, respectively. However, the IC50
value of chloroquine as a positive control, 7.6 0.5 nM, was
much lower than those of mabuniamide (1) and its analogue
(2).
Mabuniamide (1) is structurally similar to malevamide A16
with the replacement of several amino acid moieties and a 2-
methylhexanoic acid residue (Mha). The proton and carbon
chemical shifts of 1 also were very similar to those of
malevamide A. However, the configurations of two N-Me-Phe
residues and the Mha moiety in malevamide A have not been
determined. Therefore, the similar spectroscopic data of 1 may
suggest the absolute configuration of malevamide A.
The biological activity of mabuniamide (1) was evaluated
using a glucose uptake assay in cultured rat L6 myotubes.7,17
Mabuniamide (1) showed no cytotoxicity at 10−40 μM
(Figure 2a) and stimulated glucose uptake in a dose-dependent
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotation was
measured on a JASCO P-1010 polarimeter or DIP-1000. UV spectra
were measured on a JASCO V-660 UV visible spectrometer. IR
spectra were measured on a JASCO FT/IR-6100 or RT/IR-4200
1
spectrometer. H NMR and 13C NMR spectra were recorded on a
Bruker AVANCE III 500 (500 MHz) or JEOL JNM-ECX400 (400
MHz). Chemical shifts were reported relative to the residual solvent
signals (CDCl3: δH 7.26, δC 77.2). ESIMS data were obtained using a
Waters Quattro micro API mass spectrometer; HRESIMS was
performed on a Waters Micromass Q-TOF or LC Premier EX
spectrometer. HPLC was carried out with a JASCO PU-2080 Plus
Intelligent HPLC pump and a JASCO UV-2075 Plus Intelligent UV/
vis detector. The absorbance of the assay mixture was determined
using a BioTek ELx 800 absorbance microplate reader. Chemicals and
solvents were the best grade available and used as received from
commercial sources.
Identification of the Marine Cyanobacterium. A cyanobacte-
rial filament collected in May 2018 was isolated under a microscope
and crushed with freezing and thawing. The 16S rDNA genes were
PCR-amplified from isolated DNA using the primer set CYA106F, a
cyanobacterial-specific primer, and 16S1541R, a universal primer. The
PCR reaction contained DNA derived from a cyanobacterial filament,
0.5 μL of KOD-Multi & Epi- (Toyobo), 1.0 μL of each primer (50
pM, 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,
amplification by 40 cycles of 10 s at 98 °C, 10 s at 58 °C, 1 min at 68
°C, and final elongation for 7 min at 68 °C. PCR products were
analyzed on agarose gel (1%) in Tris-borate-EDTA buffer and
visualized by ethidium bromide staining. The obtained DNA was
sequenced with CYA106F and 16S1541R primers. This sequence is
available in the DDBJ/EMBL/GenBank databases under the
accession number LC466966. The nucleotide sequence of the 16S
rRNA gene obtained in this study was used for phylogenetic analysis
with the sequences of related cyanobacterial 16S rRNA genes.18 All
sequences were aligned by SINA Web service (version 1.2.11)19 with
default settings. The poorly aligned positions and divergent regions
were removed by Gblocks Server (version 0.91b),20 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 1190 nucleotide
positions have been used for phylogenetic analysis. JModeltest
(version 2.1.7)21 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),22 using the GTR+I+G model with a gamma shape
parameter of 0.4830, a proportion of invariant sites of 0.5300, and
nucleotide frequencies of F(A) = 0.2465, F(C) = 0.2272, F(G) =
0.3151, F(T) = 0.2112. Bootstrap resampling was performed on 1000
replicates. The ML tree was visualized with Njplot (version 2.3).23
The Bayesian analysis was conducted by MrBayes (version 3.2.5)24
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 formed a clade with Okeania. Therefore,
the cyanobacterium was classified into the genus Okeania.
Figure 2. Effects of natural 1, synthetic 1, and its stereoisomer 2 on
cell viability and glucose uptake in cultured L6 myotubes. (a) Cells
were treated with the indicated concentrations of compounds. After
incubation for 22 h, cell viability was determined based on an MTT
assay. Values are the mean SD of quadruplicate determinations. (b)
Cells were preincubated in Krebs-Henseleit-HEPES buffer (KHH
buffer) without glucose for 2 h. They were then incubated in KHH
buffer containing 11 mM glucose with the indicated concentrations of
compounds for 22 h. Glucose uptake was measured using a Glucose
CII test kit. Values are the mean
SD of quadruplicate
determinations.
and insulin-independent manner (Figure 2b). Synthetic 1 also
stimulated glucose uptake in a dose-dependent and insulin-
independent manner. The effect of synthetic 1 was slightly
stronger than that of 1 (Figure 2b). In addition, we assessed
the antimalarial activities of 1 and 2 against the asexual
erythrocytic stage of the Plasmodium falciparum 3D7 clone,
which is a standard reference that is sensitive to most
antimalarials. As a result, mabuniamide (1) and its stereo-
isomer 2 exhibited antiplasmodial activity with IC50 values of
Extraction and Isolation. The Okeania sp. cyanobacterium
(392.1 g, wet weight) was collected at Odo, Okinawa, Japan (26°09′
N, 127°71′ E), in May 2018. The cyanobacterium was extracted with
D
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MeOH (1.5 L) at room temperature for 4 days. The extract was
filtered, and the filtrate was concentrated. The residue was partitioned
between H2O (0.2 L) and EtOAc (0.2 L × 3). The material obtained
from the organic layer was further partitioned between 90% aqueous
MeOH (0.1 L) and n-hexane (0.1 L × 3). The aqueous 90% MeOH
fraction (0.25 g) was separated by column chromatography on ODS
(4.0 g) using 40% aqueous MeOH, 60% aqueous MeOH, 80%
aqueous MeOH, and MeOH. The fraction (47.2 mg) that eluted with
80% aqueous MeOH was subjected to reversed-phase HPLC
[Cosmosil 5C18-AR-II (20 × 250 mm), 80% MeOH at 5.0 mL/
min, UV detection at 215 nm] to yield mabuniamide (1, 32.0 mg, tR =
40.1 min). The purity of 1 was determined as >95% by HPLC
analysis.
follows: N-Me-L-Phe (8.3) and N-Me-D-Phe (10.3) in solvent B. The
retention time and ESIMS product ion (m/z [M + Na]+) of the L-
DAA derivative of N-Me-Phe from the hydrolysate of fragment B was
8.3 min (454.1) in solvent B. The retention times and ESIMS product
ion (m/z [M + Na]+) of the L-DAA derivative of N-Me-Phe from the
hydrolysate of fragment A was 10.3 min (454.0) in solvent B.
Total Synthesis Procedures. Methyl N-((tert-Butoxycarbonyl)-
glycyl)-N-methyl-D-phenylalanyl-L-prolinate (4a). To a stirred
solution of known dipeptide 3a (395 mg, 1.01 mmol) in CH2Cl2 (2
mL) was added TFA (1 mL) at 0 °C. After stirring at rt for 1 h, the
mixture was diluted with benzene (4 mL) and concentrated. The
trifluoroacetate salt of deprotected amine 3a was afforded and used in
the next reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 3a, N-Boc-glycine (212 mg,
1.21 mmol), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU)
(462 mg, 1.22 mmol) in dimethylformamide (DMF, 2 mL) was
added N,N-diisopropylethylamine (DIPEA) (0.85 mL) at room
temperature. After stirring for 3 h, the mixture was diluted with
EtOAc (15 mL), washed with 10% aqueous citric acid (10 mL),
saturated aqueous sodium hydrogen carbonate (10 mL), and brine
(10 mL), dried (Na2SO4), and concentrated. The residual oil was
purified by column chromatography on silica gel (hexane−EtOAc, 1:1
v/v) to give compound 4a as a colorless oil (339 mg, 0.76 mmol,
75%): [α]24D +63 (c 1.0, CHCl3); IR (neat) 3325, 2965, 1744, 1640,
Mabuniamide (1): colorless oil; [α]26D −4.86 (c 1.00, CHCl3), UV
(MeOH) λmax (log ε) 200, 278 nm (4.93); IR (neat) 3285, 2960,
1
1747, 1642, 754 cm−1; H NMR, 13C NMR, and HMBC data, Table
1; HRESIMS m/z 1086.6572 [M + H]+ (calcd for C58H88N9O11,
1086.6603).
Total Hydrolysis and Derivatization with L-FDAA. Mabunia-
mide (1, 1.8 mg) was treated with 6 M HCl (500 μL) for 18 h at 110
°C. The hydrolyzed product was evaporated to dryness and subjected
to HPLC [Cosmosil HILIC (4.6 × 250 mm), MeCN/10 mM
AcONH4 = 85:15 at 1.0 mL/min, UV detection 215 nm] to yield Pro,
N-Me-Phe, Val, N-Me-Val, Leu, and N-Me-Ala. Each amino acid was
mixed with a 0.1% solution of Nα-(6-fluoro-2,4-dinitrophenyl)-L-
alaninamide (L-FDAA, Marfey’s reagent, 100 μL) in acetone and 0.5
M NaHCO3 (300 μL) followed by heating at 40 °C for 90 min. After
cooling to room temperature (rt), the reaction mixture was
neutralized with 2 M HCl (75 μL) and diluted with MeOH (1.0
mL). The solution was subjected to reversed-phase HPLC [Cosmosil
5C18-AR-II (4.6 × 250 mm), MeOH/20 mM AcONa = 60:40
(solvent A), 55:45 (solvent B), 50:50 (solvent C), or 45:65 (solvent
D) at 1.0 mL/min, UV detection at 340 nm]. The L-DAA derivatives
of standard amino acids were prepared by the same procedure. The
retention times (min) of the derivatives of authentic standards were as
follows: L-Leu (6.8) and D-Leu (14.6) in solvent A, N-Me-L-Phe (8.3),
N-Me-D-Phe (10.3), N-Me-L-Val (8.2), N-Me-D-Val (15.2), N-Me-L-
Ala (6.8), and N-Me-D-Ala (9.3) in solvent B, L-Val (7.6) and D-Val
(21.5) in solvent C, L-Pro (6.4) and D-Pro (10.5) in solvent D. The
retention time and ESIMS product ion (m/z [M + Na]+) of the L-
DAA derivative of Leu from hydrolysate was 6.8 min (406.1) in
solvent A. The retention times and ESIMS product ions (m/z [M +
Na]+) of the L-DAA derivatives of N-Me-Phe from hydrolysate were
8.3 min (454.1) and 10.3 min (454.0) in solvent B, respectively. The
retention times and ESIMS product ions (m/z [M + Na]+) of the L-
DAA derivatives of N-Me-Val and N-Me-Ala from hydrolysate were
15.2 min (406.1) and 6.8 min (378.2) in solvent B, respectively. The
retention time and ESIMS product ion (m/z [M + Na]+) of the L-
DAA derivative of Val from hydrolysate was 7.6 min (392.1) in
solvent C. The retention time and ESIMS product ion (m/z [M +
Na]+) of the L-DAA derivative of Pro from hydrolysate was 6.4 min
(390.2) in solvent D.
Partial Hydrolysis and Derivatization with L-FDAA. Mabu-
niamide (1, 1.0 mg) was treated with 4 M HCl (600 μL) for 30 min at
100 °C. The hydrolysate was evaporated to dryness and subjected to
HPLC [Cosmosil 5C18-AR-II (4.6 × 250 mm), 45% MeOH
containing 0.1% trifluoroacetic acid (TFA) at 1.0 mL/min, UV
detection 215 nm]. Eight fractions were analyzed by ESIMS and
assigned peptide sequences. After the assignments of each peptide
sequence, two fractions were identified to contain N-Me-Phe-Gly-Val
(fragment A) and Leu-N-Me-Phe-Pro (fragment B). Fragment A and
fragment B were treated with 6 M HCl (600 μL) for 12 h at 110 °C.
The hydrolysates were dried and mixed with a 0.1% solution of L-
FDAA (100 μL) in acetone and 0.5 M NaHCO3 (200 μL) followed
by heating at 40 °C for 90 min. After cooling to rt, the reaction
mixture was neutralized with 2 M HCl (50 μL) and diluted with
MeOH (1.0 mL). The solution was subjected to reversed-phase
HPLC [Cosmosil 5C18-AR-II (4.6 × 250 mm), MeOH/20 mM
AcONa = 55:45 (solvent B) at 1.0 mL/min, UV detection at 340
nm]. The retention times (min) of the authentic standards were as
1
754 cm−1; H NMR (400 MHz, CDCl3) δ 7.30−7.18 (m, 5H), 5.58
(dd, J = 8.4, 7.2 Hz, 1H), 5.36 (brs, 1H, NH), 4.42 (dd, J = 8.8, 5.2
Hz, 1H), 4.01 (dd, J = 17.6, 4.8 Hz, 1H), 3.77 (dd, J = 17.6, 3.6 Hz,
1H), 3.72 (s, 3H), 3.44−3.33 (m, 2H), 3.28 (dd, J = 14.0, 8.4 Hz,
1H), 2.96 (s, 3H), 2.83 (dd, J = 14.0, 7.2, 1H), 2.17 (m, 1H), 1.98−
1.79 (m, 3H), 1.44 (s, 9H); 13C NMR (100 MHz, CDCl3) δ 172.6,
168.6, 168.1, 155.8, 137.2, 129.6, 128.5, 126.8, 79.8, 59.1, 56.4, 52.4,
47.0, 42.4, 35.1, 29.7, 29.0, 28.5, 25.2; HRMS (ESI-TOF) m/z
448.2434 [M + H]+ (calcd for C23H34N3O6 448.2448).
Methyl N-(tert-Butoxycarbonyl)-L-valylglycyl-N-methyl-D-phe-
nylalanyl-L-prolinate (5a). To a stirred solution of tripeptide 4a
(255 mg, 0.57 mmol) in CH2Cl2 (2 mL) was added TFA (1 mL) at 0
°C. After stirring at rt for 1 h, the mixture was diluted with benzene (4
mL) and concentrated. The trifluoroacetate salt of deprotected amine
4a was afforded and used in the next reaction without purification. To
a stirred solution of the trifluoroacetate salt of deprotected amine 4a,
N-Boc-L-Val (149 mg, 0.69 mmol), and HATU (261 mg, 0.69 mmol)
in DMF (0.9 mL) was added DIPEA (0.5 mL) at rt. After stirring for
2 h, the mixture was diluted with EtOAc (15 mL), washed with 10%
aqueous citric acid (10 mL), saturated aqueous sodium hydrogen
carbonate (10 mL), and brine (10 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 100:1 v/v) to give compound 5a
as a colorless oil (310 mg, 0.57 mmol, quant.). The ratio of major and
minor rotamers is 10:1; [α]24 +46 (c 1.0, CHCl3); IR (neat) 3325,
D
1
2967, 1747, 1638, 754 cm−1; H NMR (400 MHz, CDCl3) for the
major rotamer δ 7.30−7.18 (m, 5H), 6.79 (brs, 1H, NH), 5.57 (dd, J
= 7.6, 7.6 Hz, 1H), 5.03 (brs, 1H), 4.42 (dd, J = 8.8, 5.2 Hz, 1H), 4.09
(dd, J = 18.0, 4.0 Hz, 1H), 4.03 (m, 1H), 3.91 (dd, J = 18.0, 3.6 Hz,
1H), 3.72 (s, 3H), 3.42−3.33 (m, 2H), 3.27 (dd, J = 14.0, 8.0 Hz,
1H), 2.99 (s, 3H), 2.83 (dd, J = 14.0, 7.2, 1H), 2.24−2.11 (m, 2H),
2.00−1.79 (m, 3H), 1.44 (s, 9H), 0.95 (d, J = 7.2 Hz, 3H), 0.87 (d, J
= 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) for the major rotamer δ
172.6, 171.6, 168.1, 168.0, 155.9, 137.1, 129.5, 128.5, 126.8, 80.0,
59.8, 59.1, 56.4, 52.4, 46.9, 41.3, 35.1, 31.2, 29.8, 28.9, 28.4, 25.2,
19.4, 17.5; HRMS (ESI-TOF) m/z 547.3124 [M + H]+ (calcd for
C28H43N4O7 547.3132).
Methyl N-N-(tert-Butoxycarbonyl)-N-methyl-D-valyl-L-valylglyc-
yl-N-methyl-D-phenylalanyl-L-prolinate (6a). To a stirred solution
of tetrapeptide 5a (261 mg, 0.48 mmol) in CH2Cl2 (2 mL) was added
TFA (1 mL) at 0 °C. After stirring at rt for 1 h, the mixture was
diluted with benzene (4 mL) and concentrated. The trifluoroacetate
salt of deprotected amine 5a was afforded and used in the next
reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 5a, N-Boc-N-Me-D-Val
E
DOI: 10.1021/acs.jnatprod.9b00749
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(133 mg, 0.58 mmol), and HATU (218 mg, 0.57 mmol) in DMF (0.7
mL) was added DIPEA (0.41 mL) at rt. After stirring for 3 h, the
mixture was diluted with EtOAc (10 mL), washed with 10% aqueous
citric acid (8 mL), saturated aqueous sodium hydrogen carbonate (8
mL), and brine (8 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(CHCl3−MeOH, 140:1 v/v) to give compound 6a as a colorless oil
(brt, J = 3.7 Hz, 1H, NH), 5.56 (dd, J = 8.4, 6.4 Hz, 1H), 5.00−4.78
(m, 2H), 4.56 (m, 1H), 4.38 (dd, J = 8.8, 5.6 Hz, 1H), 4.29−4.07 (m,
2H), 3.84 (dd, J = 18.0, 3.6 Hz, 1H), 3.70 (s, 3H), 3.40−3.20 (m,
3H), 3.07−2.87 (m, 9H), 2.80 (dd, J = 14.0, 6.8 Hz, 1H), 2.72 (s,
3H), 2.34 (m, 1H), 2.22−2.07 (m, 2H), 1.94−1.74 (m, 3H), 1.54 (m,
1H), 1.35 (s, 9H), 1.01−0.87 (m, 15H), 0.83 (d, J = 6.8 Hz, 3H);
some signals overlapped with a water signal; 13C NMR (100 MHz,
CDCl3) for the major rotamer δ 173.9, 172.5, 170.8, 170.5, 170.3,
169.8, 168.0, 156.1, 137.7, 137.2, 129.6, 129.0, 128.4, 126.7, 80.8,
63.2, 59.0, 58.5, 56.3, 52.3, 48.2, 46.8, 42.1, 41.7, 41.3, 35.1, 34.1,
30.8, 30.7, 29.7, 28.9, 28.3, 25.8, 25.1, 24.9, 23.2, 22.1, 19.9, 19.5,
18.6, 17.9, 17.7; HRMS (ESI-TOF) m/z 934.5637 [M + H]+ (calcd
for C50H76N7O10 934.5654).
(195 mg, 0.30 mmol, 63%): [α]24 +80 (c 1.0, CHCl3); IR (neat)
D
1
3319, 2965, 1748, 1638, 754 cm−1; H NMR (400 MHz, CDCl3) δ
7.30−7.18 (m, 5H), 7.03 (brs, 1H, NH), 6.79 (brs, 1H, NH), 5.57
(dd, J = 7.2, 7.2 Hz, 1H), 4.41 (dd, J = 8.8, 5.6 Hz, 1H), 4.30 (m,
1H), 4.06 (brd, J = 15.2 Hz, 1H), 4.03 (m, 1H), 3.90 (brd, J = 15.2
Hz, 1H), 3.72 (s, 3H), 3.42−3.33 (m, 2H), 3.28 (dd, J = 14.0, 8.4 Hz,
1H), 2.98 (s, 3H), 2.82 (m, 1H), 2.81 (s, 3H), 2.32 (m, 1H), 2.24−
2.11 (m, 2H), 2.00−1.79 (m, 3H), 1.47 (s, 9H), 0.96 (d, J = 7.2 Hz,
3H), 0.94 (d, J = 8.0 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H), 0.88 (d, J =
6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 172.6, 171.3, 171.0,
168.0, 157.3, 137.1, 129.6, 128.5, 126.8, 80.5, 65.7, 59.1, 58.4, 56.3,
52.4, 46.9, 41.3, 35.1, 31.1, 30.6, 29.8, 29.0, 28.5, 26.3, 25.2, 20.0,
19.6, 18.8, 17.7; HRMS (ESI-TOF) m/z 660.3979 [M + H]+ (calcd
for C34H54N5O8 660.3972).
tert-Butyl (S)-2-(((2R,8S,11R,14S,17S)-2-Benzyl-14-isobutyl-8,11-
diisopropyl-1-((S)-2-(methoxycarbonyl)pyrrolidin-1-yl)-3,12-di-
methyl-1,4,7,10,13,16-hexaoxo-18-phenyl-3,6,9,12,15-pentaazaoc-
tadecan-17-yl)(methyl)carbamoyl)pyrrolidine-1-carboxylate (9a).
To a stirred solution of heptapeptide 8a (148 mg, 0.16 mmol) in
CH2Cl2 (1.5 mL) was added TFA (0.8 mL) at 0 °C. After stirring at rt
for 1 h, the mixture was diluted with benzene (4 mL) and
concentrated. The trifluoroacetate salt of deprotected amine 8a was
afforded and used in the next reaction without purification. To a
stirred solution of the trifluoroacetate salt of deprotected amine 8a, N-
Boc-L-Pro (41 mg, 0.19 mmol), and HATU (72 mg, 0.19 mmol) in
DMF (0.25 mL) was added DIPEA (0.14 mL) at rt. After stirring for
5 h, the mixture was diluted with EtOAc (8 mL), washed with 10%
aqueous citric acid (6 mL), saturated aqueous sodium hydrogen
carbonate (6 mL), and brine (6 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 100:1 v/v) to give compound 9a
as a colorless oil (75 mg, 72 μmol, 45%): [α]24D +48 (c 1.0, CHCl3);
Methyl N,N-((tert-Butoxycarbonyl)-L-leucyl)-N-methyl-D-valyl-L-
valylglycyl-N-methyl-D-phenylalanyl-L-prolinate (7a). To a stirred
solution of pentapeptide 6a (169 mg, 0.26 mmol) in CH2Cl2 (1.5
mL) was added TFA (0.8 mL) at 0 °C. After stirring at rt for 1 h, the
mixture was diluted with benzene (4 mL) and concentrated. The
trifluoroacetate salt of deprotected amine 6a was afforded and used in
the next reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 6a, N-Boc-L-Leu·H2O (77
mg, 0.31 mmol), and HATU (178 mg, 0.47 mmol) in DMF (0.35
mL) was added DIPEA (0.22 mL) at rt. After stirring for 2 h, the
mixture was diluted with EtOAc (8 mL), washed with 10% aqueous
citric acid (6 mL), saturated aqueous sodium hydrogen carbonate (6
mL), and brine (6 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(CHCl3−MeOH, 120:1 v/v) to give compound 7a as a colorless oil
1
IR (neat) 3304, 2962, 1748, 1650, 754 cm−1; H NMR (400 MHz,
CDCl3) δ 8.53 (d, J = 6.8 Hz, 1H, NH), 7.25−7.07 (m, 10H), 6.94−
6.76 (m, 2H, NH), 5.54 (dd, J = 9.2, 6.4 Hz, 1H), 4.99 (d, J = 11.2
Hz, 1H), 4.76 (m, 1H), 4.65 (m, 1H), 4.37 (dd, J = 8.4, 5.2 Hz, 1H),
4.25−4.14 (m, 3H), 3.92 (dd, J = 17.6, 3.6 Hz, 1H), 3.71 (m, 1H),
3.69 (s, 3H), 3.41−3.19 (m, 6H), 3.11 (s, 3H), 2.94 (m, 1H), 2.89 (s,
3H), 2.86 (s, 3H), 2.75 (dd, J = 14.0, 6.0 Hz, 1H), 2.39 (m, 1H), 2.21
(m, 1H), 2.11 (m, 1H), 1.93−1.78 (m, 4H), 1.48 (m, 1H), 1.42 (s,
9H), 1.02 (d, J = 6.4 Hz, 3H), 0.98−0.82 (m, 15H), 0.74 (m, 1H);
some signals overlapped with a water signal; 13C NMR (100 MHz,
CDCl3) δ 174.9, 174.2, 172.6, 171.3, 170.3, 168.2, 168.1, 154.7, 138.1,
137.3, 129.6, 129.0, 128.5, 127.0, 126.7, 80.2, 62.6, 59.0, 56.4, 54.3,
52.3, 49.3, 47.3, 46.9, 41.4, 39.8, 35.2, 34.0, 30.2, 29.8, 29.4, 28.9,
28.6, 25.9, 25.1, 24.9, 24.8, 23.2, 21.5, 20.3, 19.6, 18.7, 18.4; HRMS
(ESI-TOF) m/z 1031.6198 [M + H]+ (calcd for C55H83N8O11
1031.6181).
Methyl N,N,N,N-(tert-Butoxycarbonyl)-N-methyl-L-alanyl-L-prol-
yl-N-methyl-L-phenylalanyl-L-leucyl-N-methyl-D-valyl-L-valylglycyl-
N-methyl-D-phenylalanyl-L-prolinate (10a). To a stirred solution of
octapeptide 9a (106 mg, 0.10 mmol) in CH2Cl2 (1.2 mL) was added
TFA (0.6 mL) at 0 °C. After stirring at rt for 1 h, the mixture was
diluted with benzene (4 mL) and concentrated. The trifluoroacetate
salt of deprotected amine 9a was afforded and used in the next
reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 9a, N-Boc-N-Me-L-Ala
(35 mg, 0.17 mmol), and HATU (60 mg, 0.16 mmol) in DMF (0.15
mL) was added DIPEA (0.11 mL) at rt. After stirring for 3 h, the
mixture was diluted with EtOAc (8 mL), washed with 10% aqueous
citric acid (6 mL), saturated aqueous sodium hydrogen carbonate (6
mL), and brine (6 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(CHCl3−MeOH, 100:1 v/v) to give compound 10a as a colorless oil
(103 mg, 92 μmol, 92%): [α]24D +11 (c 1.0, CHCl3); IR (neat) 3340,
2964, 1747, 1648, 754 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.76 (m,
1H, NH), 7.25−7.06 (m, 10H), 6.94−6.84 (m, 2H, NH), 5.51 (dd, J
= 8.8, 6.4 Hz, 1H), 5.00−4.93 (m, 2H), 4.75−4.60 (m, 2H), 4.44−
4.33 (m, 2H), 4.24 (dd, J = 8.0, 7.2 Hz, 1H), 4.17 (dd, J = 17.6, 5.2
Hz, 1H), 3.91 (dd, J = 17.6, 3.2 Hz, 1H), 3.68 (s, 3H), 3.54 (m, 1H),
3.42 (m, 1H), 3.33−3.20 (m, 4H), 3.09 (s, 3H), 3.05 (m, 1H), 2.93
(172 mg, 0.22 mmol, 85%): [α]24 +58 (c 1.0, CHCl3); IR (neat)
D
1
3320, 2962, 1747, 1639, 754 cm−1; H NMR (400 MHz, CDCl3) δ
7.25−7.11 (m, 5H), 6.82 (m, 2H, NH), 5.54 (m, 2H), 4.63 (dd, J =
8.4, 6.8 Hz, 1H), 4.48 (d, J = 11.6 Hz, 1H), 4.41−4.33 (m, 2H), 4.23
(dd, J = 17.6, 5.2 Hz, 1H), 3.81 (dd, J = 17.6, 3.2 Hz, 1H), 3.72 (s,
3H), 3.35−3.27 (m, 2H), 3.06 (s, 3H), 3.05 (m, 1H), 2.98 (s, 3H),
2.79 (dd, J = 14.0, 6.4 Hz, 1H), 2.38−2.12 (m, 4H), 2.00−1.79 (m,
3H), 1.51−1.41 (m, 2H), 1.40 (s, 9H), 0.99 (d, J = 6.4 Hz, 3H), 0.97
(d, J = 6.4 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H),
0.87 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 7.2 Hz, 3H); 13C NMR (100
MHz, CDCl3) δ 174.7, 172.6, 170.9, 170.2, 168.11, 168.06, 155.8,
137.2, 129.6, 128.5, 126.8, 79.6, 63.8, 59.1, 58.3, 56.5, 52.4, 49.8, 47.0,
42.3, 41.3, 35.2, 31.0, 30.5, 29.9, 29.0, 28.5, 25.8, 25.2, 24.9, 23.3,
22.1, 20.0, 19.6, 18.8, 17.6; HRMS (ESI-TOF) m/z 773.4828 [M +
H]+ (calcd for C40H65N6O9 773.4813).
Methyl N,N,N-(tert-Butoxycarbonyl)-N-methyl-L-phenylalanyl-L-
leucyl-N-methyl-D-valyl-L-valylglycyl-N-methyl-D-phenylalanyl-L-
prolinate (8a). To a stirred solution of hexapeptide 7a (152 mg, 0.20
mmol) in CH2Cl2 (1.5 mL) was added TFA (0.8 mL) at 0 °C. After
stirring at rt for 1 h, the mixture was diluted with benzene (4 mL) and
concentrated. The trifluoroacetate salt of deprotected amine 7a was
afforded and used in the next reaction without purification. To a
stirred solution of the trifluoroacetate salt of deprotected amine 7a, N-
Boc-N-Me-L-Phe (73 mg, 0.26 mmol), and HATU (100 mg, 0.26
mmol) in DMF (0.35 mL) was added DIPEA (0.2 mL) at rt. After
stirring for 6 h, the mixture was diluted with EtOAc (8 mL), washed
with 10% aqueous citric acid (6 mL), saturated aqueous sodium
hydrogen carbonate (6 mL), and brine (6 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 120:1 v/v) to give compound 8a
as a colorless oil (173 mg, 0.19 mmol, 95%). The ratio of major and
minor rotamers is 1:0.7; [α]24 +56 (c 1.0, CHCl3); IR (neat) 3303,
D
1
2963, 1748, 1632, 754 cm−1; H NMR (400 MHz, CDCl3) for the
major rotamer δ 7.25−7.11 (m, 10H), 6.99−6.78 (m, 2H, NH), 6.74
F
DOI: 10.1021/acs.jnatprod.9b00749
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Journal of Natural Products
Article
(m, 1H), 2.86 (s, 3H), 2.82 (s, 3H), 2.72 (s, 3H), 2.70 (m, 1H), 2.37
(m, 1H), 2.20 (m, 1H), 2.09 (m, 1H), 1.97 (m, 1H), 1.90−1.67 (m,
5H), 1.57 (m, 1H), 1.42 (s, 9H), 1.34 (m, 1H), 1.17 (d, J = 6.8 Hz,
3H), 1.01 (d, J = 6.4 Hz, 3H), 0.96−0.81 (m, 15H), 0.68 (m, 1H);
some signals overlapped with a water signal; 13C NMR (100 MHz,
CDCl3) δ 175.1, 173.4, 172.5, 171.3, 170.2, 170.1, 168.1, 168.0, 155.6,
138.1, 137.3, 129.6, 129.5, 129.0, 128.4, 127.0, 126.7, 80.2, 62.9, 59.0,
56.4, 55.2, 53.5, 52.3, 51.4, 49.5, 47.4, 46.8, 41.4, 39.5, 35.1, 33.8,
30.2, 29.8, 29.6, 29.3, 28.9, 28.5, 25.9, 25.4, 25.1, 24.8, 23.4, 21.3,
20.4, 19.6, 18.7, 18.4, 14.3; HRMS (ESI-TOF) m/z 1116.6685 [M +
H]+ (calcd for C59H90N9O12 1116.6709).
Mabuniamide (1). To a stirred solution of nonapeptide 10a (69
mg, 62 μmol) in CH2Cl2 (0.8 mL) was added TFA (0.4 mL) at 0 °C.
After stirring at rt for 1 h, the mixture was diluted with benzene (4
mL) and concentrated. The trifluoroacetate salt of deprotected amine
10a was afforded and used in the next reaction without purification.
To a stirred solution of the trifluoroacetate salt of deprotected amine
10a, butanoic acid (10 μL, 110 μmol), and HATU (35 mg, 93 μmol)
in DMF (1.1 mL) was added DIPEA (0.1 mL) at rt. After stirring for
2 h, the mixture was diluted with EtOAc (8 mL), washed with 10%
aqueous citric acid (6 mL), saturated aqueous sodium hydrogen
carbonate (6 mL), and brine (6 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 70:1 v/v) to give mabuniamide
(1) as a colorless oil (50 mg, 46 μmol, 74%); [α]26 −3.18 (c 0.41,
CHCl3); IR (neat) 3338, 2964, 1745, 1647, 753 cm−1D; 1H NMR (400
MHz, CDCl3) see Table 1; 13C NMR (100 MHz, CDCl3) see Table
1; HRMS (ESI-TOF) m/z 1086.6576 [M + H]+ (calcd for
C58H88N9O11 1086.6603).
minor rotamers is 5:1; [α]24 −87 (c 1.0, CHCl3); IR (neat) 3326,
D
1
2970, 1745, 1640, 754 cm−1; H NMR (400 MHz, CDCl3) for the
major rotamer δ 7.28−7.16 (m, 5H), 6.79 (brt, J = 4.0 Hz, 1H, NH),
5.63 (dd, J = 8.8, 7.6 Hz, 1H), 5.10 (brd, J = 8.4 Hz, 1H), 4.41 (dd, J
= 8.0, 4.0 Hz, 1H), 4.01 (dd, J = 18.4, 4.0 Hz, 1H), 3.96 (m, 1H),
3.82 (dd, J = 18.4, 4.0 Hz, 1H), 3.69 (s, 3H), 3.60 (m, 1H), 3.40 (m,
1H), 3.24 (dd, J = 14.4, 6.8 Hz, 1H), 2.99 (s, 3H), 2.97 (m, 1H), 2.15
(m, 1H), 2.02−1.85 (m, 4H), 1.41 (s, 9H), 0.90 (d, J = 6.8 Hz, 3H),
0.86 (d, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) for the major
rotamer δ 172.4, 171.6, 169.0, 168.5, 155.8, 136.5, 129.0, 128.6, 126.9,
79.8, 59.7, 59.1, 55.1, 52.3, 47.0, 41.4, 35.0, 31.2, 30.0, 29.1, 28.4,
24.9, 19.3, 17.7; HRMS (ESI-TOF) m/z 547.3115 [M + H]+ (calcd
for C28H43N4O7 547.3132).
Methyl N,N-(tert-Butoxycarbonyl)-N-methyl-D-valyl-L-valylglyc-
yl-N-methyl-L-phenylalanyl-L-prolinate (6b). To a stirred solution
of tetrapeptide 5b (518 mg, 0.95 mmol) in CH2Cl2 (3.5 mL) was
added TFA (1.8 mL) at 0 °C. After stirring at rt for 1 h, the mixture
was diluted with benzene (5 mL) and concentrated. The
trifluoroacetate salt of deprotected amine 5b was afforded and used
in the next reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 5b, N-Boc-N-Me-D-Val
(263 mg, 1.14 mmol), and HATU (432 mg, 1.14 mmol) in DMF (1.8
mL) was added DIPEA (0.66 mL) at rt. After stirring for 4 h, the
mixture was diluted with EtOAc (20 mL), washed with 10% aqueous
citric acid (15 mL), saturated aqueous sodium hydrogen carbonate
(15 mL), and brine (15 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(CHCl3−MeOH, 140:1 v/v) to give compound 6b as a colorless oil
(397 mg, 0.60 mmol, 63%). The ratio of major and minor rotamers is
5:1; [α]24 +9.0 (c 1.0, CHCl3); IR (neat) 3316, 2965, 1746, 1640,
Methyl N-((tert-Butoxycarbonyl)glycyl)-N-methyl-L-phenylalan-
yl-L-prolinate (4b). To a stirred solution of known dipeptide 3b
(925 mg, 2.37 mmol) in CH2Cl2 (5 mL) was added TFA (2.5 mL) at
0 °C. After stirring at rt for 2 h, the mixture was diluted with benzene
(6 mL) and concentrated. The trifluoroacetate salt of deprotected
amine 3b was afforded and used in the next reaction without
purification. To a stirred solution of the trifluoroacetate salt of
deprotected amine 3b, N-Boc-glycine (498 mg, 2.84 mmol), and
HATU (1.08 g, 2.84 mmol) in DMF (5 mL) was added DIPEA (2
mL) at rt. After stirring for 2 h, the mixture was diluted with EtOAc
(30 mL), washed with 10% aqueous citric acid (25 mL), saturated
aqueous sodium hydrogen carbonate (25 mL), and brine (25 mL),
dried (Na2SO4), and concentrated. The residual oil was purified by
column chromatography on silica gel (hexane−EtOAc, 1:1 v/v) to
give compound 4b as a colorless oil (950 mg, 2.12 mmol, 89%). The
ratio of major and minor rotamers is 5:1; [α]24D −33 (c 1.0, CHCl3);
D
1
754 cm−1; H NMR (400 MHz, CDCl3) for the major rotamer δ
7.28−7.16 (m, 5H), 6.98 (brd, J = 8.4 Hz, 1H, NH), 6.73 (brs, 1H,
NH), 5.64 (dd, J = 7.6, 7.6 Hz, 1H), 4.41 (dd, J = 9.2, 4.0 Hz, 1H),
4.24 (m, 1H), 4.00 (m, 1H), 3.78 (m, 1H), 3.69 (s, 3H), 3.65−3.50
(m, 2H), 3.40 (m, 1H), 3.23 (dd, J = 14.4, 7.6 Hz, 1H), 2.98 (s, 3H),
2.97 (m, 1H), 2.77 (s, 3H), 2.27 (m, 1H), 2.15 (m, 1H), 2.06−1.89
(m, 4H), 1.44 (s, 9H), 0.95−0.84 (m, 12H); 13C NMR (100 MHz,
CDCl3) for the major rotamer δ 172.4, 171.2, 170.9, 169.1, 168.5,
157.3, 136.5, 129.1, 128.6, 126.9, 80.5, 65.5, 59.2, 58.4, 55.1, 52.3,
47.1, 41.5, 35.1, 31.0, 30.7, 30.0, 29.1, 28.4, 26.2, 24.9, 20.0, 19.5,
18.8, 17.8; HRMS (ESI-TOF) m/z 660.3977 [M + H]+ (calcd for
C34H54N5O8 660.3972).
Methyl N,N-((tert-Butoxycarbonyl)-L-leucyl)-N-methyl-D-valyl-L-
valylglycyl-N-methyl-L-phenylalanyl-L-prolinate (7b). To a stirred
solution of pentapeptide 6b (312 mg, 0.47 mmol) in CH2Cl2 (2 mL)
was added TFA (1 mL) at 0 °C. After stirring at rt for 1.5 h, the
mixture was diluted with benzene (5 mL) and concentrated. The
trifluoroacetate salt of deprotected amine 6b was afforded and used in
the next reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 6b, N-Boc-L-Leu·H2O (142
mg, 0.57 mmol), and HATU (225 mg, 0.59 mmol) in DMF (1.2 mL)
was added DIPEA (0.48 mL) at rt. After stirring for 1.5 h, the mixture
was diluted with EtOAc (15 mL), washed with 10% aqueous citric
acid (10 mL), saturated aqueous sodium hydrogen carbonate (10
mL), and brine (10 mL), dried (Na2SO4), and concentrated. The
residual oil was purified by column chromatography on silica gel
(CHCl3−MeOH, 140:1 v/v) to give compound 7b as a colorless oil
(257 mg, 0.33 mmol, 70%). The ratio of major and minor rotamers is
10:2:1; [α]24D +7.7 (c 1.0, CHCl3); IR (neat) 3316, 2962, 1745, 1638,
1
IR (neat) 3325, 2975, 1744, 1647, 752 cm−1; H NMR (400 MHz,
CDCl3) for the major rotamer δ 7.33−7.19 (m, 5H), 5.66 (dd, J = 8.1,
7.6 Hz, 1H), 5.33 (brs, 1H, NH), 4.42 (dd, J = 8.8, 4.0 Hz, 1H), 3.93
(dd, J = 17.6, 4.4 Hz, 1H), 3.74 (m, 1H), 3.71 (s, 3H), 3.60 (m, 1H),
3.41 (m, 1H), 3.25 (dd, J = 14.8, 7.6 Hz, 1H), 3.00 (m, 1H), 2.99 (s,
3H), 2.15 (m, 1H), 2.05−1.89 (m, 3H), 1.42 (s, 9H); 13C NMR (100
MHz, CDCl3) for the major rotamer δ 172.3, 169.1, 168.0, 155.7,
136.5, 129.0, 128.5, 126.8, 79.6, 59.1, 55.0, 52.2, 47.0, 42.4, 35.0, 29.8,
29.0, 28.3, 24.8; HRMS (ESI-TOF) m/z 448.2430 [M + H]+ (calcd
for C23H34N3O6 448.2448).
Methyl N-(tert-Butoxycarbonyl)-L-valylglycyl-N-methyl-L-phenyl-
alanyl-L-prolinate (5b). To a stirred solution of tripeptide 4b (715
mg, 1.60 mmol) in CH2Cl2 (4 mL) was added TFA (2 mL) at 0 °C.
After stirring at rt for 1 h, the mixture was diluted with benzene (6
mL) and concentrated. The trifluoroacetate salt of deprotected amine
4b was afforded and used in the next reaction without purification. To
a stirred solution of the trifluoroacetate salt of deprotected amine 4b,
N-Boc-L-Val (417 mg, 1.92 mmol), and HATU (730 mg, 1.92 mmol)
in DMF (2.2 mL) was added DIPEA (1.1 mL) at rt. After stirring for
2 h, the mixture was diluted with EtOAc (30 mL), washed with 10%
aqueous citric acid (10 mL), saturated aqueous sodium hydrogen
carbonate (25 mL), and brine (25 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 140:1 v/v) to give compound 5b
as a colorless oil (616 mg, 1.13 mmol, 71%). The ratio of major and
1
754 cm−1; H NMR (400 MHz, CDCl3) for the major rotamer δ
7.31−7.13 (m, 5H), 6.75 (m, 1H, NH), 5.66 (dd, J = 8.0, 8.0 Hz,
1H), 5.47 (d, J = 8.4 Hz, 1H), 4.63 (m, 1H), 4.49 (d, J = 11.6 Hz,
1H), 4.42 (dd, J = 8.8, 4.0 Hz, 1H), 4.29 (dd, J = 8.4, 5.2 Hz, 1H),
3.92 (d, J = 4.0 Hz, 1H), 3.70 (s, 3H), 3.66−3.53 (m, 2H), 3.41 (m,
1H), 3.26 (dd, J = 14.4, 7.6 Hz, 1H), 3.01 (s, 3H), 2.98 (s, 3H), 2.97
(m, 1H), 2.33 (m, 1H), 2.22−2.11 (m, 2H), 2.03−1.89 (m, 3H),
1.51−1.43 (m, 2H), 1.39 (s, 9H), 1.01−0.82 (m, 18H); some signals
overlapped with a water signal; 13C NMR (100 MHz, CDCl3) for the
major rotamer δ 174.5, 172.4, 170.7, 170.1, 168.9, 168.5, 155.8, 136.6,
129.2, 128.6, 126.9, 79.6, 63.7, 59.2, 58.4, 55.1, 52.3, 49.7, 47.1, 42.3,
G
DOI: 10.1021/acs.jnatprod.9b00749
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Journal of Natural Products
Article
41.5, 35.2, 30.9, 30.7, 30.0, 29.8, 29.1, 28.5, 25.8, 24.9, 23.3, 22.1,
20.0, 19.5, 18.7, 17.8; HRMS (ESI-TOF) m/z 773.4837 [M + H]+
(calcd for C40H65N6O9 773.4813).
(ESI-TOF) m/z 1053.5984 [M + Na]+ (calcd for C55H82N8O11Na
1053.6001).
Methyl N,N,N,N-(tert-Butoxycarbonyl)-N-methyl-L-alanyl-L-prol-
yl-N-methyl-D-phenylalanyl-L-leucyl-N-methyl-D-valyl-L-valylglycyl-
N-methyl-L-phenylalanyl-L-prolinate (10b). To a stirred solution of
octapeptide 9b (159 mg, 0.15 mmol) in CH2Cl2 (0.8 mL) was added
TFA (0.4 mL) at 0 °C. After stirring at rt for 1 h, the mixture was
diluted with benzene (4 mL) and concentrated. The trifluoroacetate
salt of deprotected amine 9b was afforded and used in the next
reaction without purification. To a stirred solution of the
trifluoroacetate salt of deprotected amine 9b, N-Boc-L-Ala (38 mg,
0.19 mmol), and HATU (70 mg, 0.18 mmol) in DMF (0.25 mL) was
added DIPEA (0.13 mL) at rt. After stirring for 3 h, the mixture was
diluted with EtOAc (15 mL), washed with 10% aqueous citric acid
(10 mL), saturated aqueous sodium hydrogen carbonate (10 mL),
and brine (10 mL), dried (Na2SO4), and concentrated. The residual
oil was purified by column chromatography on silica gel (CHCl3−
MeOH, 100:1 v/v) to give compound 10b as a colorless oil (160 mg,
0.14 mmol, 93%). The ratio of major and minor rotamers is 1:0.15;
Methyl N,N,N-(tert-Butoxycarbonyl)-N-methyl-D-phenylalanyl-L-
leucyl-N-methyl-D-valyl-L-valylglycyl-N-methyl-L-phenylalanyl-L-
prolinate (8b). To a stirred solution of hexapeptide 7b (228 mg, 0.29
mmol) in CH2Cl2 (1.2 mL) was added TFA (0.6 mL) at 0 °C. After
stirring at rt for 1 h, the mixture was diluted with benzene (4 mL) and
concentrated. The trifluoroacetate salt of deprotected amine 7b was
afforded and used in the next reaction without purification. To a
stirred solution of the trifluoroacetate salt of deprotected amine 7b,
N-Boc-N-Me-D-Phe (99 mg, 0.35 mmol), and HATU (134 mg, 0.35
mmol) in DMF (0.5 mL) was added DIPEA (0.25 mL) at rt. After
stirring for 2 h, the mixture was diluted with EtOAc (15 mL), washed
with 10% aqueous citric acid (10 mL), saturated aqueous sodium
hydrogen carbonate (10 mL), and brine (10 mL), dried (Na2SO4),
and concentrated. The residual oil was purified by column
chromatography on silica gel (CHCl3−MeOH, 100:1 v/v) to give
compound 8b as a colorless oil (262 mg, 0.28 mmol, 97%). The ratio
of major and minor rotamers is 1:0.75; [α]24D +43 (c 1.0, CHCl3); IR
(neat) 3310, 2963, 1746, 1638, 755 cm−1; 1H NMR (400 MHz,
CDCl3) for the major rotamer δ 7.25−7.14 (m, 10H), 6.95−6.60 (m,
3H, NH), 5.64 (dd, J = 8.0, 8.0 Hz, 1H), 5.05 (dd, J = 10.0, 5.6 Hz,
1H), 4.89 (m, 1H), 4.57 (d, J = 10.8 Hz, 1H), 4.40 (m, 1H), 4.19 (m,
1H), 4.01−3.75 (m, 2H), 3.69 (s, 3H), 3.57 (m, 1H), 3.42−3.27 (m,
2H), 3.23 (dd, J = 14.8, 7.2 Hz, 1H), 3.07−2.93 (m, 4H), 2.92 (s,
3H), 2.82 (m, 1H), 2.75 (s, 3H), 2.31 (m, 1H), 2.20−2.05 (m, 2H),
1.60 (m, 1H), 1.54−1.38 (m, 2H), 1.32 (s, 9H), 1.00−0.87 (m, 15H),
0.81 (d, J = 6.8 Hz, 3H); some signals overlapped with a water signal;
13C NMR (100 MHz, CDCl3) for the major rotamer δ 173.9, 172.5,
[α]24 +28 (c 1.0, CHCl3); IR (neat) 3340, 2964, 1744, 1638, 753
D
1
cm−1; H NMR (400 MHz, CDCl3) for the major rotamer δ 7.24−
7.12 (m, 10H), 6.95 (brd, J = 8.8 Hz, 1H, NH), 6.90 (brt, J = 4.2 Hz,
1H, NH), 5.67 (dd, J = 12.0, 4.8 Hz, 1H), 5.62 (dd, J = 7.6, 7.6 Hz,
1H), 4.97 (brdd, J = 12.6, 6.3 Hz, 1H), 4.75 (m, 1H), 4.69 (d, J =
10.8 Hz, 1H), 4.53 (m, 1H), 4.39 (dd, J = 8.8, 4.0 Hz, 1H), 4.19 (dd,
J = 8.4, 7.2 Hz, 1H), 3.96 (dd, J = 9.2, 4.0 Hz, 1H), 3.67 (s, 3H),
3.60−3.51 (m, 3H), 3.44−3.32 (m, 2H), 3.21 (dd, J = 14.0, 7.2 Hz,
1H), 3.05 (m, 1H), 3.03 (s, 3H), 3.02 (s, 3H), 2.95−2.82 (m, 3H),
2.90 (s, 3H), 2.73 (brs, 3H), 2.30 (m, 1H), 2.18−2.07 (m, 2H),
1.98−1.85 (m, 4H), 1.80−1.68 (m, 4H), 1.45 (m, 1H), 1.43 (s, 9H),
1.20 (d, J = 6.8 Hz, 3H), 1.16 (m, 1H), 0.99 (d, J = 6.4 Hz, 3H),
0.95−0.90 (m, 12H), 0.81 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) for the major rotamer δ 175.0, 173.0, 172.5, 171.2, 171.0,
170.7, 170.0, 168.8, 168.5, 155.7, 137.6, 136.7, 129.2, 128.9, 128.5,
128.3, 126.8, 126.6, 80.1, 62.6, 59.21, 59.15, 56.5, 55.1, 53.7, 52.3,
51.4, 49.2, 47.2, 47.1, 41.6, 40.2, 35.1, 35.6, 31.3, 30.5, 29.9, 29.1,
28.5, 28.0, 25.8, 25.3, 24.9, 24.8, 23.4, 21.8, 20.3, 19.6, 18.7, 18.5,
14.3; HRMS (ESI-TOF) m/z 1116.6721 [M + H]+ (calcd for
C59H90N9O12 1116.6709).
170.8, 170.5, 170.3, 169.8, 168.0, 156.1, 137.7, 137.2, 129.6, 129.0,
128.4, 126.7, 80.2, 63.2, 59.6, 59.0, 58.5, 58.3, 56.3, 52.3, 48.2, 46.8,
42.1, 41.7, 41.3, 35.1, 34.1, 31.4, 30.8, 30.7, 29.7, 28.9, 28.3, 25.8,
25.1, 24.9, 23.3, 22.1, 19.9, 19.5, 18.6, 17.9; HRMS (ESI-TOF) m/z
934.5698 [M + H]+ (calcd for C50H76N7O10 934.5654).
tert-Butyl (S)-2-(((2S,8S,11R,14S,17R)-2-Benzyl-14-isobutyl-8,11-
diisopropyl-1-((S)-2-(methoxycarbonyl)pyrrolidin-1-yl)-3,12-di-
methyl-1,4,7,10,13,16-hexaoxo-18-phenyl-3,6,9,12,15-penta-
azaoctadecan-17-yl)(methyl)carbamoyl)pyrrolidine-1-carboxylate
(9b). To a stirred solution of heptapeptide 8b (213 mg, 0.23 mmol) in
CH2Cl2 (1 mL) was added TFA (0.5 mL) at 0 °C. After stirring at rt
for 1 h, the mixture was diluted with benzene (4 mL) and
concentrated. The trifluoroacetate salt of deprotected amine 8b was
afforded and used in the next reaction without purification. To a
stirred solution of the trifluoroacetate salt of deprotected amine 8b,
N-Boc-L-Pro (59 mg, 0.27 mmol), and HATU (104 mg, 0.27 mmol)
in DMF (0.4 mL) was added DIPEA (0.2 mL) at rt. After stirring for
3 h, the mixture was diluted with EtOAc (15 mL), washed with 10%
aqueous citric acid (10 mL), saturated aqueous sodium hydrogen
carbonate (10 mL), and brine (10 mL), dried (Na2SO4), and
concentrated. The residual oil was purified by column chromatog-
raphy on silica gel (CHCl3−MeOH, 140:1 v/v) to give compound 9b
as a colorless oil (180 mg, 0.17 mmol, 74%). The ratio of major and
Iso-Mabuniamide (2). To a stirred solution of nonapeptide 10b
(115 mg, 0.10 mmol) in CH2Cl2 (0.6 mL) was added TFA (0.3 mL)
at 0 °C. After stirring at rt for 1 h, the mixture was diluted with
benzene (3 mL) and concentrated. The trifluoroacetate salt of
deprotected amine 10b was afforded and used in the next reaction
without purification. To a stirred solution of the trifluoroacetate salt
of deprotected amine 10b, butanoic acid (15 μL, 170 μmol), and
HATU (47 mg, 0.12 mmol) in DMF (0.15 mL) was added DIPEA
(0.09 mL) at rt. After stirring for 2 h, the mixture was diluted with
EtOAc (8 mL), washed with 10% aqueous citric acid (6 mL),
saturated aqueous sodium hydrogen carbonate (6 mL), and brine (6
mL), dried (Na2SO4), and concentrated. The residual oil was purified
by column chromatography on silica gel (CHCl3−MeOH, 60:1 v/v)
to give iso-mabuniamide (2) as a colorless oil (79 mg, 73 μmol, 73%):
[α]24 +18 (c 1.0, CHCl3); IR (neat) 3338, 2963, 1745, 1651, 753
D
minor rotamers is 5:1; [α]24 +36 (c 1.0, CHCl3); IR (neat) 3339,
1
cm−1; H NMR (400 MHz, CDCl3) δ 7.24−7.12 (m, 10H), 6.94−
D
1
2964, 1744, 1654, 755 cm−1; H NMR (400 MHz, CDCl3) for the
6.79 (m, 2H, NH), 5.69 (dd, J = 11.6, 4.8 Hz, 1H), 5.64 (dd, J = 7.6,
7.6 Hz, 1H), 5.43 (q, J = 7.2 Hz, 1H), 4.79 (m, 1H), 4.71 (d, J = 10.8
Hz, 1H), 4.49 (dd, J = 9.6, 9.6 Hz, 1H), 4.40 (dd, J = 8.0, 4.0 Hz,
1H), 4.20 (dd, J = 8.0, 8.0 Hz, 1H), 3.96 (m, 1H), 3.68 (s, 3H),
3.62−3.49 (m, 4H), 3.41−3.34 (m, 2H), 3.22 (dd, J = 14.0, 7.2 Hz,
1H), 3.04 (s, 3H), 3.03 (s, 3H), 2.93 (s, 3H), 2.91 (s, 3H), 2.91−2.76
(m, 3H), 2.31 (m, 1H), 2.28 (t, J = 7.6 Hz, 2H), 2.14 (m, 2H), 1.98−
1.87 (m, 4H), 1.82−1.71 (m, 4H), 1.47 (m, 1H), 1.24 (m, 1H), 1.22
(d, J = 7.2 Hz, 3H), 1.01 (d, J = 6.4 Hz, 3H), 0.97−0.90 (m, 15H),
0.83 (d, J = 6.8 Hz, 3H); some signals overlapped with a water signal;
13C NMR (100 MHz, CDCl3) δ 174.9, 173.0, 172.9, 172.4, 171.0,
170.4, 169.9, 168.7, 168.4, 137.6, 136.7, 129.1, 128.8, 128.4, 128.3,
126.7, 126.5, 62.5, 59.1, 59.0, 56.4, 56.2, 55.0, 52.3, 49.5, 49.0, 47.4,
47.0, 41.6, 40.3, 35.6, 35.1, 33.5, 31.3, 30.9, 30.6, 30.5, 29.8, 29.1,
28.0, 25.7, 25.3, 24.9, 24.7, 23.4, 21.8, 20.3, 19.5, 18.6, 18.44, 18.36,
major rotamer δ 7.25−7.12 (m, 10H), 6.89 (m, 2H, NH), 5.72 (dd, J
= 12.4, 5.2 Hz, 1H), 5.63 (dd, J = 8.0, 8.0 Hz, 1H), 4.79 (m, 1H),
4.69 (d, J = 11.2 Hz, 1H), 4.44−4.37 (m, 2H), 4.19 (dd, J = 8.4, 6.8
Hz, 1H), 3.95 (dd, J = 8.0, 3.6 Hz, 1H), 3.67 (s, 3H), 3.57 (m, 1H),
3.45−3.35 (m, 3H), 3.35−3.27 (m, 2H), 3.21 (dd, J = 14.4, 7.2 Hz,
1H), 3.01 (s, 3H), 2.99 (s, 3H), 2.96−2.82 (m, 3H), 2.92 (s, 3H),
2.30 (m, 1H), 2.18−2.04 (m, 2H), 1.95−1.86 (m, 3H), 1.77−1.63
(m, 5H), 1.47 (m, 1H), 1.40 (s, 9H), 1.11 (m, 1H), 0.99 (d, J = 6.4
Hz, 3H), 0.95−0.89 (m, 12H), 0.80 (d, J = 6.8 Hz, 3H); 13C NMR
(100 MHz, CDCl3) for the major rotamer δ 174.8, 173.6, 172.5,
171.1, 170.0, 168.8, 168.5, 154.8, 137.6, 136.7, 129.2, 129.0, 128.5,
128.3, 126.8, 126.5, 79.7, 62.6, 59.2, 59.1, 56.4, 55.9, 55.1, 54.3, 52.3,
49.0, 47.1, 47.0, 41.6, 40.6, 35.1, 33.6, 31.2, 30.5, 29.9, 29.2, 28.9,
28.6, 25.8, 24.9, 24.7, 24.5, 23.3, 22.0, 20.3, 19.6, 18.7, 18.5; HRMS
H
DOI: 10.1021/acs.jnatprod.9b00749
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Journal of Natural Products
Article
14.3, 14.0; HRMS (ESI-TOF) m/z 1086.6626 [M + H]+ (calcd for
C58H88N9O11 1086.6603).
ACKNOWLEDGMENTS
■
We thank Y. Umeki for technical assistance and the Japanese
Red Cross Society for providing human RBCs and plasma. The
P. falciparum 3D7 line was obtained from MR4 (contributed
by D. J. Carucci, MRA-102). This work was supported in part
by a Grant-in-Aid for 15K01803, 18K05337, 18K14346, and
16H03285 from the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Culturing of L6 Myoblasts. L6 myoblasts were purchased from
the Japanese Collection of Research Bioresources. L6 myotube cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% (v/v) fetal bovine serum (FBS) and 2.0% penicillin/
streptomycin in a humidified 5% CO2 incubator at 37 °C. To
differentiate myotubes, L6 myoblasts were seeded at 5.0 × 103 cells/
well in 96-well plates and cultured to 90% confluency for 2 days. Then
the cells were cultured to form myotubes in DMEM containing 2%
FBS for 1 week. The medium was renewed every 2 days.
Determination of Glucose Uptake. L6 myotubes were
incubated in filter-sterilized Krebs-Henseleit-HEPES buffer (1.2 mM
MgSO4, 1.2 mM KH2PO4, 4.7 mM KCl, 119 mM NaCl, 2.5 mM
CaCl2, 25 mM NaHCO3, pH 7.4) containing 0.1% bovine serum
albumin, 10 mM HEPES, and 2 mM sodium pyruvate (KHH buffer)
for 2 h. The myotubes were then cultured for 22 h in KHH buffer
containing 11 mM glucose without or with mabuniamide (1, 10−40
μM). The differences in the glucose concentrations in the KHH buffer
before and after culture were determined by a commercial assay kit
(Glucose CII-Test Wako), and the absorbance at 490 nm was
measured using a microplate reader (Model ELx 800; Bio Tek,
Japan). The amounts of glucose consumed were calculated from the
differences in glucose concentrations between before and after culture.
Cell Viability. After the glucose uptake assay, an MTT solution
(10 μL, 5 mg/mL in H2O) was added to each well, and the plate was
incubated at 37 °C with 5% CO2 for 4 h. All remaining supernatant
was then removed, and DMSO (50 μL) was added to each well to
dissolve the resultant formazan crystals. Absorbance was measured by
using a microplate reader at the wavelength of 540 nm.
Growth-Inhibitory Assay against Malarial Parasites. The P.
falciparium 3D7 line was obtained from the Malaria Research and
Reference Reagent Resource Center (MR4). The P. falciparum axenic
culture25 and the drug sensitivity assay26 were described previously. In
brief, parasites were cultivated in RPMI-1640 medium containing 5%
heat-inactivated human serum and 0.25% Albumax II (Invitrogen),
200 mM hypoxanthine (Sigma), 10 μg/mL gentamicin (Invitrogen),
and human RBC (type O) at 2% hematocrit. One hundred microliter
cultures at 0.3% parasitemia containing different concentrations of
drugs were prepared in 96-well plates. After 72 h of cultivation in an
incubator containing 5% O2, 5% CO2, and 90% N2 at 37 °C, parasite
growth was monitored by measurement of the absorbance at 650 nm
using a DTX880 multimode detector (Beckman Coulter) in the
lactate dehydrogenase assay as previously described.26
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AUTHOR INFORMATION
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(24) Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D. L.;
Corresponding Authors
*(K.S.) E-mail: suenaga@chem.keio.ac.jp.
*(T.T.) E-mail: t-teruya@edu.u-ryukyu.ac.jp.
̈
Darling, A.; Hohna, S.; Larget, B.; Liu, L.; Suchard, M. A.;
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ORCID
(26) Gamo, F. J.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alvarez, E.;
Lavandera, J. L.; Vanderwall, D. E.; Green, D. V. S.; Kumar, V.;
Hasan, S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos,
J. F. Nature 2010, 465, 305−310.
Kaori Ozaki: 0000-0002-8668-5260
Arihiro Iwasaki: 0000-0002-3775-5066
Kiyotake Suenaga: 0000-0001-5343-5890
Toshiaki Teruya: 0000-0002-7018-7734
Notes
The authors declare no competing financial interest.
I
DOI: 10.1021/acs.jnatprod.9b00749
J. Nat. Prod. XXXX, XXX, XXX−XXX