Janadolide, a Cyclic Polyketide-Peptide Hybrid Possessing a tert-Butyl Group from an Okeania sp. Marine Cyanobacterium

Article abstract of DOI:10.1021/acs.jnatprod.6b00171

Janadolide, a new cyclic polyketide-peptide hybrid possessing a tert-butyl group, was isolated from an Okeania sp. marine cyanobacterium. The gross structure was elucidated by spectroscopic analyses, and the absolute configurations of the amino acid moieties were determined by acid hydrolysis and chiral-phase HPLC analyses. The absolute configuration of the two stereogenic centers in the polyketide moiety was elucidated based on a combination of degradation reactions and spectroscopic analyses including the phenyl-glycine methyl ester method. Janadolide showed potent antitrypanosomal activity with an IC₅₀ value of 47 nM without cytotoxicity against human cells at 10 μM.

Full text of DOI:10.1021/acs.jnatprod.6b00171

Note
pubs.acs.org/jnp
Janadolide, a Cyclic Polyketide−Peptide Hybrid Possessing a tert-
Butyl Group from an Okeania sp. Marine Cyanobacterium
Hidetoshi Ogawa,† Arihiro Iwasaki,† Shinpei Sumimoto,† Yuki Kanamori,† Osamu Ohno,‡
Masato Iwatsuki,§,⊥ Aki Ishiyama,§,⊥ Rei Hokari,§ Kazuhiko Otoguro,§ Satoshi Omura,§
̅
and Kiyotake Suenaga*,†
†Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
‡Department of Chemistry and Life Science, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015, Japan
⊥
§Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, and Graduate School of Infection Control Sciences,
Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8641, Japan
S
* Supporting Information
ABSTRACT: Janadolide, a new cyclic polyketide−peptide hybrid possessing a tert-
butyl group, was isolated from an Okeania sp. marine cyanobacterium. The gross
structure was elucidated by spectroscopic analyses, and the absolute configurations of
the amino acid moieties were determined by acid hydrolysis and chiral-phase HPLC
analyses. The absolute configuration of the two stereogenic centers in the polyketide
moiety was elucidated based on a combination of degradation reactions and
spectroscopic analyses including the phenyl-glycine methyl ester method. Janadolide
showed potent antitrypanosomal activity with an IC50 value of 47 nM without
cytotoxicity against human cells at 10 μM.
arine prokaryotes produce diverse secondary metabo-
lites, and many interesting compounds have been
An Okeania sp. marine cyanobacterium was collected at the
coast near Janado, Okinawa. The collected cyanobacterium
(700 g, wet weight) was extracted with MeOH, and the extract
was filtered and concentrated. The residue was partitioned
between EtOAc and H2O. The material obtained from the
organic layer was partitioned between 90% aqueous MeOH and
hexanes. The aqueous MeOH fraction was separated by
reversed-phase column chromatography (ODS silica gel,
MeOH−H2O) and reversed-phase HPLC (Cosmosil 5C18AR-
II, MeOH−H2O) to give janadolide (1) (6.9 mg). The
molecular formula of janadolide (1) was determined on the
basis of the positive HRESIMS data. Table 1 shows the NMR
M
discovered from them to date.1 In particular, marine
cyanobacteria have been recognized as prolific producers of
compounds with interesting structures.2 Against this backdrop,
we have isolated several natural compounds possessing new
skeletons such as jahanyne,3 bisebromoamide,4 and kurahyne5
from marine cyanobacteria. In our efforts to discover substances
with unique structures, we investigated the constituents of an
Okeania sp. marine cyanobacterium and isolated a new cyclic
polyketide−peptide hybrid possessing a tert-butyl group,
janadolide (1). So far, several tert-butyl-containing PKS−
NRPS hybrid compounds, such as the antillatoxins6,7 and the
apratoxins,8 have been isolated from marine cyanobacteria.
Here, we report the isolation, structure elucidation, preliminary
biological characterization, and molecular modeling of
janadolide (1).
1
data for 1. An analysis of the H NMR spectrum revealed the
presence of two N-methyl groups (δH 2.30 and 2.82) and six
protons corresponding to the α-positions of amino acids (δH
3.47, 4.02, 4.236, 4.241, 4.68, and 5.69). In addition, the
presence of a tert-butyl group (δH 0.83), a vinyl methyl group
(δH 1.75), and an olefinic proton (δH 5.20) was also clarified.
The 13C NMR spectrum indicated the existence of six carbonyl
groups (δC 175.2, 172.7, 170.6, 170.1, 169.8, and 167.1) and
one double bond (δC 144.3 and 120.0). Intensive interpretation
of the COSY, HMQC, and HMBC spectra established five
amino acid residues: N-methylleucine (N-Me-Leu), N-methyl-
alanine (N-Me-Ala), glycine (Gly), valine (Val), and proline
Received: February 26, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00171
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
1
Table 1. H and 13C NMR Data for Janadolide (1) in C6D6
a
b
a
b
position
Pro
δC
δH (J in Hz)
position
N-Me-Leu
δC
δH (J in Hz)
1
172.7, C
62.4, CH
31.8, CH2
1
170.6, C
2
4.236, m
2
58.2, CH
39.1, CH2
4.68, dd (10.7, 4.3)
1.87, m
3a
1.89, m
3a
3b
1.72, m
3b
1.64, m
4a
22.9, CH2
46.8, CH2
1.56, m
4
25.2, CH
23.4, CH3
21.7, CH3
29.7, CH3
1.42, m
4b
1.234, m
5
0.82, d (6.7)
1.04, d (6.5)
2.82, s
5a
3.76, ddd (12.0, 8.5, 3.5)
3.39, m
6
5b
N-Me
Val
polyketide moiety
1
169.8, C
1
175.2, C
39.7, CH
35.4, CH2
2
58.0, CH
33.0, CH
19.0, CH3
18.5, CH3
4.241, m
2
2.40, m
2.43, m
1.01, m
2.15, m
1.66, m
3
2.05, m
3a
3b
4a
4b
4
0.92, d (6.6)
0.91, d (6.7)
7.00, d (6.9)
5
40.6, CH2
NH
Gly
1
167.1, C
5
6
7
8
144.3, C
120.0, CH
79.3, CH
35.8, C
2a
41.8, CH2
4.02, dd (18.3, 5.4)
3.47, dd (18.3, 2.1)
6.82, dd (5.4, 2.1)
5.20, dq (10.1, 0.9)
5.43, d (10.1)
2b
NH
N-Me-Ala
1
170.1, C
49.8, CH
14.9, CH3
9
25.8, CH3 × 3
19.2, CH3
0.83, s
2
5.69, q (6.8)
1.232, d (6.8)
2.30, s
10
11
0.98, d (6.7)
1.75, d (0.9)
3
16.7, CH3
N-Me
28.5, CH3
a
b
Measured at 100 MHz. Measured at 400 MHz.
(Pro). Moreover, the presence of a residue derived from 7-
hydroxy-2,5,8,8-tetramethylnon-5-enoic acid (polyketide moi-
ety) was elucidated. The configuration of the C-5−C-6 olefinic
bond in the polyketide moiety was determined to be E on the
basis of the two NOESY correlations in the polyketide moiety
(H-4b/H-6 and H-7/H-11). The chemical shift of the vinyl
methyl carbon at C-11 (δC 16.7) also supported this result.9
The connectivity of these partial structures was determined
based on the following six HMBC correlations: H-7 of the
polyketide moiety/C-1 of N-Me-Leu, N-Me of N-Me-Leu/C-1
of N-Me-Ala, N-Me of N-Me-Ala/C-1 of Gly, NH of Gly/C-1
of Val, NH of Val/C-1 of Pro, and H-2 of Pro/C-1 of the
polyketide moiety. As a result, the gross structure of 1 was
established as shown in Figure 1.
Information S16−19). The absolute configuration of the two
stereogenic centers in the polyketide moiety was elucidated
based on a combination of degradation reactions and
spectroscopic analyses as follows. Ozonolysis and oxidative
workup of 1 followed by acid hydrolysis afforded 2-methyl-5-
oxohexanoic acid and 2-hydroxy-3,3-dimethylbutanoic acid
derived from the polyketide moiety. Condensation of both
compounds with (S)- or (R)-PGME gave the corresponding
phenyl-glycine methyl ester (PGME) amides 2a and 2b from 2-
methyl-5-oxohexanoic acid and 3a and 3b from 2-hydroxy-3,3-
1
dimethylbutanoic acid, respectively. The H NMR chemical
shifts of 2a and 2b were compared, and calculated Δδ values
revealed that the absolute configuration of C-2 was 2R (Scheme
1; S15).11 To determine the absolute configuration of the
remaining stereogenic center at C-7, we synthesized the (R)-
PGME amide of (S)-2-hydroxy-3,3-dimethylbutanoic acid12 as
The absolute configurations of the amino acids were
determined by chiral-phase HPLC analyses and Marfey’s
analysis10 of the acid hydrolysate of 1. These analyses revealed
that all of the amino acids had L-configurations (Supporting
1
an authentic sample, and its H NMR spectrum was compared
with those of 3a and 3b. As a result, the spectrum of the (S)-
PGME amide (3a) matched with that of the authentic sample,
and it was clarified that the stereogenic center at C-2 of the 2-
hydroxy-3,3-dimethylbutanoic acid from 1 was R (S11−13).
Therefore, the absolute configuration of C-7 was determined to
be 7S, and the absolute configuration of janadolide was
established as shown in 1. On the basis of the NMR data, we
conducted preliminary molecular modeling of janadolide and
proposed its lowest energy conformation (S23−24).
With regard to the biological activity of janadolide (1), it
showed potent antitrypanosomal activity against Trypanosoma
brucei brucei GUTat 3.1 strain (causative agent of Nagana
disease in animals) with an IC50 value of 47 nM, which was
more potent than a commonly used therapeutic drug, suramin
(IC50 1.2 μM). On the other hand, 1 did not inhibit the growth
Figure 1. Gross structure of janadolide (1), based on 2D NMR
correlations.
B
DOI: 10.1021/acs.jnatprod.6b00171
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Journal of Natural Products
Note
Scheme 1. Preparation of the PGME Amides Derived from the Polyketide Moiety of Janadolide (1)
fraction (778 mg) was separated by column chromatography on ODS
(7.8 g) eluted with 40% MeOH, 60% MeOH, 80% MeOH, MeOH,
and CHCl3−MeOH (1:1). The fraction (366 mg) that eluted with
80% MeOH was subjected to HPLC [Cosmosil 5C18AR-II (ϕ 20 ×
250 mm); flow rate 5 mL/min; detection, UV 215 nm; solvent 64%
MeOH] in eight batches to give a fraction containing janadolide (288
mg, tR = after 88.2 min, last collected fraction). The fraction was
further separated by HPLC [Cosmosil 5C18AR-II (ϕ 20 × 250 mm);
flow rate 5 mL/min; detection, UV 215 nm; solvent 75% MeOH] in
of human cells, such as MRC-5, HL60, and HeLa cells, even at
10 μM.
Janadolide (1), a new cyclic polyketide−peptide hybrid
possessing a tert-butyl group, was isolated from an Okeania sp.
marine cyanobacterium. The gross structure was elucidated by
spectroscopic analyses, and the absolute configuration of the
amino acid moieties was determined by acid hydrolysis and
chiral-phase HPLC analyses. The absolute configuration of two
stereogenic centers in the polyketide moiety was elucidated
based on a combination of degradation reactions and
spectroscopic analyses including the PGME method.
So far, approximately 20 tert-butyl-containing metabolites,
such as bastimolide A,13 nuiapolide,14 and the amantelides,15
have been discovered from marine cyanobacteria, and some of
these compounds are classified as PKS−NRPS hybrids,
including the antillatoxins6,7 and the apratoxins.8 The discovery
of these tert-butyl-containing compounds including janadolide
(1) indicates the usefulness of marine cyanobacteria as good
sources of natural products with interesting structures.
In addition, janadolide (1) showed potent antitrypanosomal
activity without cytotoxicity against human cells, and this
indicates that further investigation should be undertaken to
evaluate the possibility of janadolide and its synthetic analogues
being used to develop new antitrypanosomal drugs.
six batches to give a fraction containing janadolide (107 mg, tR
=
before 116 min, first collected fraction). The fraction was further
separated by HPLC [Cosmosil 5C18AR-II (ϕ 20 × 250 mm); flow rate
5 mL/min; detection, UV 215 nm; solvent 78% MeOH] in three
batches to give janadolide (1) (6.9 mg, tR = 41.1 min, total yield
0.000 99% based on wet weight).
Janadolide (1): colorless oil; [α]28D −88 (c 0.05, MeOH); IR (film)
1
νmax 2959, 1733, 1652, 1635, 1456 cm−1; H NMR and 13C NMR,
Table 1; HRESIMS m/z 676.4650 (calcd for C36H62N5O7, 676.4649).
Identification of the Marine Cyanobacterium. The marine
cyanobacterium was morphologically classified into the genus Okeania
sp.,16 because the cells were 14.5 1.3 μm wide (n = 10) and 3.3
0.7 μm long (n = 10), the sheaths were 1.8 0.2 μm thick (n = 10),
and the apical cells were sometimes attenuated with calyptra. A
voucher specimen, named 1305-10, has been deposited at Keio
University. In addition, we tried to identify the cyanobacterium based
on the 16S rRNA gene. Despite our efforts, we could not obtain any
sequences corresponding to marine cyanobacteria.
To verify the fact that janadolide was produced by an Okeania sp.
marine cyanobacterium, we collected a janadolide-producing cyano-
bacterium, named 1504-15, at Minnajima Island, Okinawa, Japan, at a
depth of 0−1 m in April 2015. The morphological features of this
sample perfectly matched those of the previous sample collected at
Janado, and janadolide was isolated from this sample by the same
procedures described above. Therefore, the identification of this
cyanobacterium based on the 16S rRNA gene was carried out as
follows.
A cyanobacterial filament was isolated under a microscope and
crushed with freezing and thawing. The 16S rRNA genes were PCR-
amplified from isolated DNA using the primer set CYA 106F, a
cyanobacterial-specific primer, and 23S 30R, a cyanobacterial-specific
primer. The PCR reaction contained DNA derived from a
cyanobacterial filament, 0.5 μL of KOD-Multi & Epi- (TOYOBO),
12.5 μL of 2× PCR buffer for KOD-Multi & Epi- (TOYOBO), 1.0 μL
of each primer (10 pM), and H2O for a total volume of 25 μL. The
PCR reaction was performed as follows: initial denaturation for 10 min
at 94 °C, amplification by 40 cycles of 10 s at 98 °C, 10 s at 60 °C, and
1 min at 68 °C, and final elongation for 7 min at 68 °C. PCR products
were analyzed on agarose gel (1%) in TBE buffer, visualized by
ethidium bromide staining, and purified by a PCR Advanced PCR
clean up system (VIOGENE). Sequences were determined with CYA
106F, 16S 1541R, and 23S 30R primers by a commercial firm
(Macrogen Japan Corp.). These sequences are available in the DDBJ/
EMBL/GenBank databases under accession number LC149728. From
the phylogenetic tree inferred from 573 bp of the 16S-23S internal
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO DIP-1000 polarimeter. IR spectra were
recorded on a JASCORT/IR-4200 instrument. Chemicals and solvents
were the best grade available and used as received from commercial
sources. All NMR spectroscopic data were recorded on a JEOL ECX-
1
1
400 spectrometer for H (400 MHz) and 13C (100 MHz). H NMR
chemical shifts (referenced to residual C6HD5 observed at δH 7.16 and
CHD2OD observed at δH 3.31) were assigned using a combination of
data from COSY and HMQC experiments. Similarly, 13C NMR
chemical shifts (referenced to residual C6D6 observed at δC 128.06 and
CD3OD observed at δC 49.00) were assigned based on HMBC and
HMQC experiments. ESI mass spectra were obtained on an LCT
premier EX spectrometer (Waters). Chromatographic analyses were
performed using an HPLC system consisting of a pump (model PU-
2080, Jasco) and a UV detector (model UV-2075, Jasco). Semi-
preparative HPLC was performed on a Cosmosil series (Nacalai
Tesque).
Collection, Extraction, and Isolation. The marine cyanobacte-
rium was collected at the coast near Janado, Okinawa Prefecture,
Japan, at a depth of 0−1 m in May 2013. The collected
cyanobacterium (700 g, wet weight) was extracted with MeOH (5
L) for a month. The extract was filtered and 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 hexanes (3 × 0.3 L). The aqueous MeOH
C
DOI: 10.1021/acs.jnatprod.6b00171
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Journal of Natural Products
Note
1
transcribed spacer (ITS) gene sequences revealed that the present
cyanobacterium (accession no. LC149728) formed a clade with
Okeania (S28, Figure S3). Therefore, the cyanobacterium was
classified into the genus Okeania.
Acid Hydrolysis of Janadolide (1). Janadolide (1) (0.5 mg, 0.7
μmol) and 9 M HCl (0.1 mL) were charged in a reaction tube, which
was sealed under reduced pressure and heated at 110 °C for 24 h. The
mixture was evaporated to dryness and was separated into each
component by HPLC. The retention times of components were as
follows: N-Me-Ala (tR = 3.0 min), N-Me-Leu (tR = 6.0 min), Pro (tR =
3.2 min), Val (tR = 3.4 min) [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].
Chiral-Phase HPLC Analysis of Amino Acid Components, N-
Me-Leu, Pro, Val. Each fraction that contained amino acids except for
N-Me-Ala was dissolved in H2O (50 μL) and analyzed by chiral-phase
HPLC, and the retention times were compared to those of authentic
standards. The retention times of N-Me-Leu, Pro, and Val in the
hydrolysate matched those of N-Me-L-Leu (tR = 17.9 min), L-Pro (tR =
4.8 min), and L-Val (tR = 6.0 min), but not N-Me-D-Leu (tR = 10.3
min), D-Pro (tR = 2.7 min), and D-Val (tR = 3.4 min) [DAICEL
CHIRALPAK (MA+) (ϕ 4.6 × 50 mm); flow rate 1.0 mL/min;
detection 254 nm; solvent 2.0 mM CuSO4].
2a: H NMR (CD3OD, 400 MHz) δH 7.40−7.33 (5H, m), 5.48
(1H, s), 3.71 (3H, s), 2.46 (1H, m), 2.37 (1H, t, J = 7.8 Hz), 2.36 (1H,
dd, J = 8.4, 6.3 Hz), 2.03 (3H, s), 1.75 (1H, m), 1.63 (1H, m), 1.14
(3H, d, J = 6.8 Hz); HRESIMS m/z 292.1553 (calcd for C16H22NO4,
292.1549).
1
3a: H NMR (CD3OD, 400 MHz) δH 7.40−7.35 (5H, m), 5.47
(1H, s), 3.71 (3H, s), 3.69 (1H, s), 0.99 (9H, s); HRESIMS m/z
280.1540 (calcd for C15H22NO4, 280.1549).
A portion of the crude product containing 2-methyl-5-oxohexanoic
acid and 2-hydroxy-3,3-dimethylbutanoic acid (0.5 mg) was mixed
with HATU (3.6 mg, 90 μmol), HOAt (1.9 mg, 14 μmol), (R)-
PGME·HCl (1.2 mg, 5.9 μmol), DIEA (50 μL), and DMF (50 μL),
which was allowed to stir at rt. After 4 h, the reaction mixture was
separated as described above to give the (R)-PGME amide of the
natural 2-methyl-5-oxohexanoic acid (2b) (0.1 mg, 0.3 μmol, tR = 11.3
min) and (R)-PGME amide of the natural 2-hydroxy-3,3-dimethylbu-
tanoic acid (3b) (0.1 mg, 0.3 μmol, tR = 12.7 min), respectively.
1
2b: H NMR (CD3OD, 400 MHz) δH 7.40−7.33 (5H, m), 5.42
(1H, s), 3.71 (3H, s), 2.58 (2H, t, J = 7.6 Hz), 2.47 (1H, m), 2.17 (3H,
s), 1.77 (1H, m), 1.69 (1H, m), 1.08 (3H, d, J = 7.0 Hz); HRESIMS
m/z 292.1552 (calcd for C16H22NO4, 292.1549).
1
3b: H NMR (CD3OD, 400 MHz) δH 7.40−7.34 (5H, m), 5.48
(1H, s), 3.71 (3H, s), 3.70 (1H, s), 0.93 (9H, s); HRESIMS m/z
280.1539 (calcd for C15H22NO4, 280.1549).
Marfey’s Analysis of N-Me-Ala. The fraction containing N-Me-
Ala was dissolved in H2O (100 μL). Marfey’s reagent (1.0% 1-fluoro-
2,4-dinitrophenyl-5-L-leucinamide) solution in acetone (200 μL) and
50 μL of 1 M NaHCO3 were added, and the mixture was heated at 80
°C for 3 min. The solution was cooled to room temperature (rt),
neutralized with 1 M HCl, and evaporated to dryness. The residue was
resuspended in 200 μL of MeCN−H2O (1:1), and the solution was
analyzed by reversed-phase HPLC.17 The retention time of the
derivatized N-Me-Ala in the hydrolysate matched that of the Marfey’s
derivative of the N-Me-L-Ala authentic sample (tR = 12.7 min), but not
Marfey’s derivative of the N-Me-D-Ala authentic sample (tR = 21.8
min) [Cosmosil Cholester (ϕ 4.6 × 250 mm); flow rate 1.0 mL/min;
detection at 340 nm; solvent 0.02 M NaOAc aq−MeOH (45/55)].
Ozonolysis and Acid Hydrolysis of 1 (2-Methyl-5-oxohex-
anoic Acid and 2-Hydroxy-3,3-dimethylbutanoic Acid). Jana-
dolide (1) used in this reaction was additionally isolated from the same
cyanobacterium collected at Minnajima Island by the same procedures
described above. Ozone was bubbled through a stirred solution of
janadolide (1, 9.7 mg, 14 μmol) dissolved in CH2Cl2 (1.0 mL) at −78
°C for 40 min. The reaction mixture was concentrated and treated
with 1 mL of H2O2−HCOOH (1:2) at 70 °C for 4 h. After removal of
the solvent in vacuo, the oxidized product and 9 M HCl (0.2 mL) were
charged in a reaction tube, which was sealed under reduced pressure
and heated at 110 °C for 24 h. Then, 2 mL of H2O was added to the
reaction mixture, and the mixture was extracted three times with
EtOAc. The combined organic layers were concentrated in vacuo to
afford the crude product containing 2-methyl-5-oxohexanoic acid and
2-hydroxy-3,3-dimethylbutanoic acid (2.0 mg, 2-methyl-5-oxohexanoic
acid:2-hydroxy-3,3-dimethylbutanoic acid = 3:1).
PGME Derivatives of the Natural 2-Methyl-5-oxohexanoic
Acid and 2-Hydroxy-3,3-dimethylbutanoic Acid (2a, 2b, 3a,
and 3b). A half-portion of the crude product (1.0 mg) containing 2-
methyl-5-oxohexanoic acid and 2-hydroxy-3,3-dimethylbutanoic acid
was mixed with O-(7-azabenzotriazoll-yl)-l,l,3,3-tetramethyluronium
hexafluorophosphate (HATU) (4.0 mg, 10 μmol), 1-hydroxy-7-
azabenzotriazole (HOAt) (1.9 mg, 14 μmol), (S)-PGME·HCl (2.0
mg, 9.9 μmol), diisopropylethylamine (DIEA) (50 μL), and DMF (50
μL), which was allowed to stir at rt. After 3.5 h, 1.5 mL of EtOAc was
added to the reaction mixture. The organic layer was washed with
saturated aqueous NH4Cl (3 × 1.0 mL), concentrated, and purified by
reversed-phase HPLC to give the (S)-PGME amide of the natural 2-
methyl-5-oxohexanoic acid (2a) (0.3 mg, 1 μmol, tR = 11.3 min) and
(S)-PGME amide of the natural 2-hydroxy-3,3-dimethylbutanoic acid
(3a) (0.2 mg, 0.7 μmol, tR = 12.6 min), respectively [Cosmosil
5C18MS-II (ϕ 20 × 250 mm); flow rate 5 mL/min; detection, UV 215
nm; solvent 80% MeCN].
Synthesis of the Authentic Sample of (R)-PGME Amide of
(S)-2-Hydroxy-3,3-dimethylbutanoic acid. (S)-2-Hydroxy-3,3-di-
methylbutanoic acid12 (1.1 mg, 8.3 μmol) was mixed with HATU (3.6
mg, 90 μmol), HOAt (1.5 mg, 11 μmol), (R)-PGME·HCl (1.6 mg, 7.9
μmol), DIEA (50 μL), and DMF (50 μL), which was allowed to stir at
rt. After 4 h, 1.5 mL of EtOAc was added to the reaction mixture. The
organic layer was washed with saturated aqueous NH4Cl (3 × 1.0 mL),
concentrated, and purified by reversed-phase HPLC to give the (R)-
PGME amide of (S)-2-hydroxy-3,3-dimethylbutanoic acid (0.2 mg, 0.7
μmol, 9%, tR = 12.4 min) [Cosmosil 5C18MS-II (ϕ 20 × 250 mm);
flow rate 5 mL/min; detection, UV 215 nm; solvent 80% MeCN].
1
(R)-PGME amide of (S)-2-hydroxy-3,3-dimethylbutanoic acid: H
NMR (CD3OD, 400 MHz) δH 7.40−7.34 (5H, m), 5.47 (1H, s), 3.71
(3H, s), 3.69 (1H, s), 0.99 (9H, s); HRESIMS m/z 280.1544 (calcd
for C15H22NO4, 280.1549).
Cell Growth Analysis. Cell proliferation of HeLa cells and HL60
cells was measured by the MTT assay as described previously.5
Measurement of cytotoxic activity against human fetal lung fibroblast
MRC-5 cells was carried out as described previously.18
In Vitro Antitrypanosomal Assay. The bloodstream forms of T.
b. b. strain GUTat 3.1 parasites were used for experimentation, as
described previously.19 The strain GUTat 3.1 was cultured in IMDM
with various supplements and 10% heat-inactivated fetal bovine serum
at 37 °C under 5.0% CO2−95% air. Subsequently, 95 μL of the
parasite suspension ((2.0−2.5) × 104 trypanosomes mL−1) was
transferred to a 96-well microtiter plate, and 5.0 μL of a 25% MeOH
solution of 1 was added, followed by incubation for 72 h at 37 °C.
Then, 10 μL of Alamer Blue solution was added to each well. After
incubation for 3−6 h, the resulting solution was read at 528/620 nm
excitation wavelengths and 590/630 nm emission wavelengths by an
FLx800 fluorescence microplate reader (BioTek Instruments, Inc.).
The IC50 value was determined using fluorescent plate reader software
(KC-4, BioTek Instruments, Inc.).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00171.
1H, 13C, COSY, HMQC, HMBC, and NOESY NMR
1
1
spectra in C6D6 and H, H−1H J resolved and NOESY
1
NMR spectra in CDCl3 for janadolide (1); H NMR
spectra in CD3OD for 2a, 2b, 3a, 3b, and the authentic
sample of (R)-PGME amide of (S)-2-hydroxy-3,3-
D
DOI: 10.1021/acs.jnatprod.6b00171
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
(19) Otoguro, K.; Ishiyama, A.; Namatame, M.; Nishihara, A.;
Furusawa, T.; Masuma, R.; Shiomi, K.; Takahashi, Y.; Yamada, H.;
Omura, S. J. Antibiot. 2008, 61, 372−378.
̅
dimethylbutanoic acid; HPLC chromatograms for
determination of the absolute configurations; molecular
modeling based on NMR data of janadolide (1) (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: suenaga@chem.keio.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by Grants-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan (24310160 and 20436992), for the Promotion of
Science, the Uehara Memorial Foundation, and the Keio
Gijuku Fukuzawa Memorial Fund for the Advancement of
Education and Research.
REFERENCES
■
(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.;
Prinsep, M. R. Nat. Prod. Rep. 2015, 32, 116−211.
(2) Kleigrewe, K.; Gerwick, L.; Sherman, D. H.; Gerwick, W. H. Nat.
Prod. Rep. 2016, 33, 348−364.
(3) Iwasaki, A.; Ohno, O.; Sumimoto, S.; Ogawa, H.; Nguyen, K. A.;
Suenaga, K. Org. Lett. 2015, 17, 652−655.
(4) Teruya, T.; Sasaki, H.; Fukazawa, H.; Suenaga, K. Org. Lett. 2009,
11, 5062−5065.
(5) Iwasaki, A.; Ohno, O.; Sumimoto, S.; Suda, S.; Suenaga, K. RSC
Adv. 2014, 4, 12840−12843.
(6) Orjala, J.; Nagle, D. G.; Hsu, V. L.; Gerwick, W. H. J. Am. Chem.
Soc. 1995, 117, 8281−8282.
(7) Nogle, L. M.; Okino, T.; Gerwick, W. H. J. Nat. Prod. 2001, 64,
983−985.
(8) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J.; Corbett,
T. H. J. Am. Chem. Soc. 2001, 123, 5418−5423. (b) Luesch, H.;
Yoshida, W. Y.; Moore, R. E.; Paul, V. J. Bioorg. Med. Chem. 2002, 10,
́
1973−1978. (c) Gutierrez, M.; Suyama, T. L.; Engene, N.; Wingerd, J.
S.; Matainaho, T.; Gerwick, W. H. J. Nat. Prod. 2008, 71, 1099−1103.
(d) Matthew, S.; Schupp, P. J.; Luesch, H. J. Nat. Prod. 2008, 71,
1113−1116. (e) Tidgewell, K.; Engene, N.; Byrum, T.; Media, J.; Doi,
T.; Valeriote, F. A.; Gerwick, W. H. ChemBioChem 2010, 11, 1458−
1466. (f) Thornburg, C. C.; Cowley, E. S.; Sikorska, J.; Shaala, L. A.;
Ishmael, J. E.; Youssef, D. T. A.; McPhail, K. L. J. Nat. Prod. 2013, 76,
1781−1788.
(9) Carey, L.; Clough, J. M.; Pattenden, G. J. Chem. Soc., Perkin Trans.
1 1983, 12, 3005−3009.
(10) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596.
(11) Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853−1856.
(12) Boobalan, R.; Chen, C.; Lee, G.-H. Org. Biomol. Chem. 2012, 10,
1625−1638.
(13) Shao, C.-L.; Linington, R. G.; Balunas, M. J.; Centeno, A.;
Boudreau, P.; Zhang, C.; Engene, N.; Spadafora, C.; Mutka, T. S.;
Kyle, D. E.; Gerwick, L.; Wang, C.-Y.; Gerwick, W. H. J. Org. Chem.
2015, 80, 7849−7855.
(14) Mori, S.; Williams, H.; Cagle, D.; Karanovich, K.; Horgen, F. D.;
Smith, R., III; Watanabe, C. M. H. Mar. Drugs 2015, 13, 6274−6290.
(15) Salvador-Reyes, L. A.; Sneed, J.; Paul, V. J.; Luesch, H. J. Nat.
Prod. 2015, 78, 1957−1962.
(16) Engene, N.; Paul, V. J.; Byrum, T.; Gerwick, W. H.; Thor, A.;
Ellisman, M. K. J. Phycol. 2013, 49, 1095−1106.
(17) Under typical Marfey’s condition with L-FDAA, the L- and D-N-
Me-Ala derivatives are not separable on a standard C-18 column. We
resolved this problem by using L-FDLA and the Cosmosil-Cholester
column.
(18) Otoguro, K.; Kohana, A.; Manabe, C.; Ishiyama, A.; Ui, H.;
Shiomi, K.; Yamada, H.; Omura, S. J. Antibiot. 2001, 54, 658−663.
̅
E
DOI: 10.1021/acs.jnatprod.6b00171
J. Nat. Prod. XXXX, XXX, XXX−XXX