Bulbimidazoles A-C, Antimicrobial and Cytotoxic Alkanoyl Imidazoles from a Marine Gammaproteobacterium Microbulbifer Species

Article abstract of DOI:10.1021/acs.jnatprod.0c00082

Three new alkanoyl imidazoles, designated bulbimidazoles A-C (1-3), were found from the culture extract of the gammaproteobacterium Microbulbifer sp. DC3-6 isolated from a stony coral of the genus Tubastraea. The absolute configuration of the anteiso-methyl substitution in 1 was established to be a mixture of (R)- and (S)-configurations in a ratio of 9:91 by applying the Ohrui-Akasaka method. Compounds 1-3 displayed unique broad-spectrum antimicrobial activity against Gram-positive and -negative bacteria and fungi with MICs ranging from 0.78 to 12.5 μg/mL. They also exhibited cytotoxicity toward P388 murine leukemia cells with IC₅₀ in the micromolar range.

Full text of DOI:10.1021/acs.jnatprod.0c00082

pubs.acs.org/jnp
Note
Bulbimidazoles A−C, Antimicrobial and Cytotoxic Alkanoyl
Imidazoles from a Marine Gammaproteobacterium Microbulbifer
Species
Md. Rokon Ul Karim, Enjuro Harunari, Naoya Oku, Kazuaki Akasaka, and Yasuhiro Igarashi*
Cite This: https://dx.doi.org/10.1021/acs.jnatprod.0c00082
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı Supporting Information
ABSTRACT: Three new alkanoyl imidazoles, designated bulbimi-
dazoles A−C (1−3), were found from the culture extract of the
gammaproteobacterium Microbulbifer sp. DC3-6 isolated from a
stony coral of the genus Tubastraea. The absolute configuration of
the anteiso-methyl substitution in 1 was established to be a mixture of
(R)- and (S)-configurations in a ratio of 9:91 by applying the Ohrui−
Akasaka method. Compounds 1−3 displayed unique broad-spectrum
antimicrobial activity against Gram-positive and -negative bacteria
and fungi with MICs ranging from 0.78 to 12.5 μg/mL. They also
exhibited cytotoxicity toward P388 murine leukemia cells with IC50
in the micromolar range.
arine bacteria associating with marine invertebrates are
regarded as a promising yet underexplored source of new
nm were detected in a culture extract of a strain of the genus
Microbulbifer isolated from a scleractinian (stony) coral,
Tubastraea sp., collected off the south shore of central Japan.
As the UV spectrum was not present in our in-house UV
database, HPLC/UV-guided purification was conducted, and
three new alkanoyl imidazoles designated bulbimidazoles A−C
(1−3) were identified. These compounds are the deamino
congeners of nocarimidazoles A (4) and B (5) previously
M
chemical entities. Compared to the marine animals, the number
of new compounds from microorganisms associated with or
1
symbiotic to the host invertebrates is still small. However,
marine animal-associated bacteria are attracting attention
because of the accumulating evidence that bacteria are the
2
true producers of a number of marine natural products. One of
12
the striking examples of marine natural products isolated from
both invertebrates and bacteria is the anticancer cyclic
isolated from a marine actinomycete of the genus Nocardiopsis.
Herein, we report the isolation, structure determination, and
biological activities of 1, 2, and 3.
3
depsipeptide didemnin B. It was first isolated from a tunicate
and was reisolated from a cultured Tistrella bacterium collected
4
from a sediment sample.
Besides sponges and tunicates, corals are another rich source
5
of natural products. Natural products obtained from corals have
unique structures and potent biological activities, while coral-
associated microorganisms are underexplored in natural product
6
discovery. Bacteria of the genus Microbulbifer are marine
obligate, Gram-negative, strictly aerobic, and rod-shaped, belong
7
to the class Gammaproteobacteria, and are commonly found in
marine habitats such as salt marshes, intertidal sediments, solar
8
salterns, marine sediments, and marine algae. According to the
latest genomic data, some Microbulbifer species possess
biosynthetic genes for the production of nonribosomal peptides
9
and siderophores, but there are merely two reports to date
Received: January 21, 2020
regarding the compounds produced by this genus: benzoate
10
derivatives from a sediment-derived strain and an unsaturated
11
fatty acid from a coral-derived strain.
In the course of our continuing search for new bioactive
compounds with therapeutic potential from marine microbes,
HPLC peaks displaying UV absorption maximum around 254
©
XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00082
A
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Note
1
13
Table 1. H and C Spectroscopic Data for Bulbimidazoles A−C (1−3) in DMSO-d with TFA
6
1
2
3
a
b
b
,
c
a
b
a
b
no.
δC
δH mult (J in Hz)
HMBC
4, 5, 6
2, 5, 6
δC
δH mult (J in Hz)
δC
δH mult (J in Hz)
2
4
5
6
7
8
9
137.6, CH
125.2, CH
131.8, C
9.15, d (0.6)
8.55, d (0.6)
137.5, CH
125.1, CH
131.5, C
9.24, d (0.6)
8.61, d (0.6)
137.5, CH
125.1, CH
131.5, C
9.20, d (0.6)
8.57, d (0.6)
190.6, C
190.5, C
190.5, C
38.6, CH2
23.5, CH2
28.6, CH2
29.2, CH2
26.4, CH2
36.0, CH2
33.8, CH
29.0, CH2
11.3, CH3
19.1, CH3
2.89, t (7.3)
1.59, quint (7.3)
1.29, m
5, 6, 8, 9
38.5, CH2
23.5, CH2
28.5, CH2
28.7, CH2
28.8, CH2
2.89, t (7.3)
38.5, CH2
23.5, CH2
28.5, CH2
29.0, CH2
26.7, CH2
38.4, CH2
27.4, CH
22.5, CH3
22.5, CH3
2.89, t (7.3)
1.60, quint (7.3)
1.28, m
6, 7, 9, 10
8, 10
1.60, quint (7.2)
e
1.22−1.30
d
e
10
11
12
13
14
15
16
1.23, m
1.22, m
1.22−1.30
1.24, m
1.24, m
1.11, m
1.47, sept (6.7)
0.82, d (6.7)
0.82, d (6.7)
d
e
13
1.22−1.30
d
e
1.05, m; 1.24, m
1.26, m
1.09, m; 1.27, m
0.80, t (7.3)
0.79, d (6.7)
11, 13
12, 15, 16
12, 13, 15, 16
13, 14
28.9, CH2
1.22−1.30
31.3, CH2
22.1, CH2
14.0, CH3
1.22, m
1.23, m
0.83, t (6.9)
12, 13, 14
a
b
c
Recorded at 125 MHz (reference δ 39.5). Recorded at 500 MHz (reference δ 2.49). HMBC correlations are from proton(s) stated to the
C
H
d
e
indicated carbon. Assignment may be interchanged. Overlapping signals.
The producing strain, coded DC3-6, was cultured in A11M
seawater medium at 30 °C for 5 days. The extract, obtained by 1-
butanol extraction of the whole culture broth, was subjected to
consecutive fractionation using silica gel and ODS column
chromatographies, followed by reversed-phase HPLC purifica-
tion, yielding bulbimidazoles A (1, 11.3 mg), B (2, 3.2 mg), and
C (3, 2.5 mg) from 3 L of culture.
11) (Figure 1). These aliphatic fragments were joined via a
methylene group at C-10 by a series of HMBC correlations from
The molecular formula of 1 was determined to be C H N O
1
4
24
2
+
based on a protonated molecule [M + H] at m/z 237.1964 by
an HR-ESITOFMS measurement. The calculated four degrees
of unsaturation was in good agreement with an observed UV
absorption maximum at 253 nm, implying the presence of a
conjugated system with three or more double bonds. Moreover,
the IR spectrum showed absorption bands at 3300 and 1665
Figure 1. COSY and key HMBC correlations for 1−3.
−
1
cm , corresponding to NH group(s) and a carbonyl
H -8 to C-10 and H -9 to C-10 and C-11. Furthermore, long-
2
2
1
functionality, respectively. The H NMR spectrum showed
range correlations were observed from H -7 and H -8 to the
2
2
2
two distinctive doublet resonances of sp protons at δ 8.55 and
H
carbonyl carbon (C-6), thereby establishing an 8-methyldeca-
noyl moiety. The last spin-system consisted of the two aromatic
protons, H-2 and H-4, which were long-range coupled with J =
9
0
.15, one doublet and one triplet methyl proton at δ 0.79 and
H
.80, one aliphatic methine at δ 1.26, and several methylene
H
multiplets, confirming the presence of an aromatic group and a
0
.6 Hz, exchanged HMBC correlations with the corresponding
methyl-branched alkyl chain in 1. However, a 1 C NMR
3
carbons, and were further correlated with the nonprotonated
spectrum recorded in DMSO-d exhibited only 10 aliphatic
carbon at δ 131.5 (C-5). Considering the remaining elemental
6
C
carbons and failed to detect the rest of the resonances (Figure
composition HN2 with two double-bond equivalents, an
imidazole ring was the only option to satisfy these requirements.
Finally, the alkanoyl moiety and imidazole were connected by
HMBC correlations from the imidazole protons H-2 and H-4 to
the carbonyl carbon C-6 and from the alkanoyl α-proton H -7 to
the imidazole carbon C-5, thus completing the planar structure
2
S6), which were expected to be of the sp carbons. These
carbons were detected in HSQC and HMBC spectra as cross-
2
peaks from the aforementioned sp protons and some of the
aliphatic protons (Figures S9 and S11). We suspected that the
2
2
13
lack of resonances for sp carbons in the C NMR spectrum was
due to the signal broadening and splitting caused by tautomeric
exchange in the unidentified functional group. To convert the
envisaged tautomers into a (few) resonating structure(s), a trace
amount of trifluoroacetic acid (TFA) was supplemented to the
NMR solvent. Gratifyingly, the anticipated four resonances
1
3,14
of 1. The carbon chemical shifts of C-2, C-4, C-5, and C-6
as
14
well as the UV maximal absorption were in good accordance
with those reported for synthetic alkanoyl imidazoles.
In order to establish the absolute configuration of the single
stereogenic center C-13, the Ohrui−Akasaka method was
13
15
appeared at δ 190.6, 137.5, 131.8, and 125.2 in the C NMR
employed (Figure 2). This method is one of the few analytical
C
spectrum (Figure S5). In combination with HSQC data, the
assignment of all 14 carbon signals was made as one deshielded
means to elucidate the absolute configuration of an isolated
stereogenic center with a methyl substituent in a long alkyl chain
system. To generate a carboxyl group for derivatization, the
imidazole ring was oxidized with RuCl −NaIO in CCl −
2
2
carbonyl carbon, one nonprotonated sp carbon, two sp
3
3
methines, seven sp methylenes, one sp methine, and two
methyl groups (Table 1). Analysis of the COSY spectrum
elucidated three spin-systems, two of which were a trimethylene
unit (H -7−H -8−H -9) and a six-carbon fragment with an
3
4
4
16
MeCN−H O, which yielded 8-methyldecanoic acid (nat-4).
2
This was derivatized with (R)-2-(anthracene-2,3-
dicarboximido)propanol [(R)-2A1P] to give ester nat-4-(R)-
2A1P (Figure 2c). The authentic (S)-4-(R)-2A1P and (S)-4-
2
2
2
anteiso-branching (H -15−H -14−H-13(H -16)−H -12−H -
3
2
3
2
2
B
https://dx.doi.org/10.1021/acs.jnatprod.0c00082
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Note
1
2.5 μg/mL. Compounds 1−3 also showed relatively potent
activity against three fungi tested in this study with MIC values
ranging from 0.78 to 12.5 μg/mL. Overall, compounds 1 and 2
showed better activity than 3, implying a key role of the alkyl
chain length in exerting antimicrobial activity. In addition, 1−3
exhibited moderate cytotoxicity against P388 murine leukemia
cells with IC values of 5.0, 5.8, and 7.0 μM, respectively.
5
0
Bulbimidazoles A−C (1−3) are new members of imidazole-
containing natural products in which an alkyl chain and an
imidazole ring are coupled through a keto group. These
compounds, together with the 4-amino-substituted congeners,
nocarimidazoles A (4) and B (5) from a marine-derived
12
actinomycete Nocardiopsis sp., are the only natural products of
this class. As synthetic compounds, alkanoyl imidazoles were
reported as synthetic intermediates or structural motifs of a
variety of drugs/drug candidates such as adrenoceptor agonists
Figure 2. Results of Ohrui−Akasaka analysis. (a) Standard (S)-4-(R)-
2
A1P and (S)-4-(S)-2A1P. (b) nat-4-(R)-2A1P derived from 1. (c)
Oxidative degradation of 1 and derivatization with (R)-2A1P.
13,17
or HIV-1 protease inhibitors.
(
4
S)-2A1P was prepared from (S)-8-methyldecanoic acid [(S)-
], which was synthesized from commercially available (S)-6-
Bacterial anteiso-fatty acids have been regarded to have an (S)-
configuration based on the biosynthesis of the starter unit, 2-
18,19
methyloctanol in four steps (Scheme S1). Retention times of
these reference compounds were 243 min for (S)-4-(R)-2A1P
and 234 min for (S)-4-(S)-2A1P, which is chromatographically
equivalent to (R)-4-(R)-2A1P, whereas nat-4-(R)-2A1P showed
two peaks for (R)-4-(R)-2A1P and (S)-4-(R)-2A1P in a ratio of
methylbutanoyl-CoA, from L-isoleucine.
However, this idea
seems not applicable to secondary metabolites: nocapyrone L, a
metabolite of Nocardiopsis, is a mixture of (R)- and (S)-
20
enantiomers in a ratio of 2:3; compound 1 is the second
example of this kind. Both of these compositions were
unambiguously elucidated by a sound analytical technique.
The occurrence of (R)-anteiso enantiomers implies that the
enantiospecificity of chain elongation enzymes toward the
substrate, 2-methylbutanoyl CoA, is lower than expected, or
9
.1:90.9 (Figure 2). Therefore, 1 was concluded to be an
enantiomeric mixture consisting of 9% (R)- and 91% (S)-
isomers.
Bulbimidazole B (2) was obtained as a pale yellow,
amorphous solid. The molecular formula was determined to
be C H N O by HR-ESITOFMS, which was one methylene
19
even is lacking. In the upstream pathway, conversion of L-
1
4
22
2
isoleucine into both of the enantiomers of 2-methylbutanoyl
2
1,22
23
(
14 amu) fewer than 1. 1D and 2D NMR data were in overall
CoA takes place in Streptomyces,
rat skin, and apple
24
similarity to those for 1, except for the absence of the doublet
(Scheme S2). It is no wonder that the downstream pathway is
methyl and a methine (H -16 and H-13 in 1) resonance and the
appearance of one additional methylene resonance (δ 31.3/δH
1
protons in an n-propyl spin-system H -15−H -14−H -13 and
HMBC correlations from H -15 to C-13 and C-14 and H -14 to
C-12 and C-13 established a nonbranching linear alkyl terminus
in 2 (Table S1, Figure 1). The H and C NMR chemical shifts
for the imidazole ring were mostly the same as those observed
for 1 (Table 1).
Bulbimidazole C (3) was also obtained as a pale yellow,
amorphous solid. It has the same molecular formula
also shaped to accept and process both enantiomers.
3
C
The wide antimicrobial spectrum of 1−3 against pathogens of
humans (S. aureus, C. albicans, and T. rubrum), fish (T.
maritimum), and plants (G. cingulata) poses a question
regarding their molecular target(s). Finally, discovery of 1−3
warrants further investigation on the metabolites of the genus
Microbulbifer, from which only two classes of compounds are
.22, CH -13). COSY correlations between neighboring
2
3
2
2
3
2
1
13
10,11
known,
and other taxonomic groups of marine bacteria,
which are not yet exploited as a source of drug discovery.
EXPERIMENTAL SECTION
■
(
C H N O) and UV maximum absorption (254 nm) as 2,
14 22 2
1
General Experimental Procedures. The specific rotation was
measured on a JASCO P-1030 polarimeter. UV spectra were obtained
on a Shimadzu UV-1800 spectrophotometer. IR spectra were recorded
on a PerkinElmer Spectrum 100 spectrophotometer. NMR spectra
were obtained on a Bruker AVANCE 500 spectrometer in DMSO-d6
supplemented with or without a trace amount of trifluoroacetic acid,
indicating that 3 is an alkyl chain isomer of 2. The H NMR
spectrum showed a doublet methyl resonance (δ 0.82 d, J = 6.7
Hz) accounting for six protons, and this methyl group was
correlated with a septet methine proton (δ 1.47, J = 6.7 Hz) in
the COSY spectrum, confirming the presence of an isopropyl
group. The intervening methylene chain part and the imidazole
moiety were connected by COSY and HMBC correlations, thus
establishing the structure of 3 (Table S1, Figure 1).
H
H
using the signals of the residual solvent protons (δ 2.49) and carbons
H
(
δ 39.5) as internal standards for compounds 1−3 or in CDCl using
C
3
the signals of the residual solvent protons (δ 7.27) and carbons (δ
H
C
Compounds 1−3 were tested against two Gram-positive
bacteria (Kocuria rhizophila ATCC9341 and Staphylococcus
aureus FDA209P JC-1), three Gram-negative bacteria (Escher-
ichia coli NIHJ JC-2, Rhizobium radiobacter NBRC14554, and
Tenacibaculum maritimum NBRC16015), and three fungi
77.0) as internal standards for other compounds. HR-ESITOFMS
spectra were recorded on a Bruker micrOTOF focus. An Agilent
HP1200 system equipped with a diode array detector was used for
analysis and purification.
Microorganism. The coral sample Tubastraea sp., collected as
fishery waste at −10 to −15 m near the coast of Minami-Ise, Mie, Japan,
was obtained through a local aquarium vendor. Strain DC3-6 was
(
Candida albicans NBRC0197, Glomerella cingulata
NBRC5907, and Trichophyton rubrum NBRC5467). Com-
pounds 1−3 inhibited the growth of K. rhizophila and S. aureus
with MICs of 0.78−3.12 μg/mL. They were inactive against E.
coli and R. radiobacter, but were active against T. maritimum, a
causative agent of fish skin ulcer, with MICs ranging from 3.12 to
25
isolated according to the method described previously and was
identified as a member of genus Microbulbifer on the basis of 99.3%
similarity in the 16S rRNA gene sequence (1455 nucleotides; DDBJ
T
accession number LC498626) to Microbulbifer echini AM134
(accession number KJ789957).
C
https://dx.doi.org/10.1021/acs.jnatprod.0c00082
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Note
Fermentation. Strain DC3-6 was maintained on Marine Agar 2216
Difco). A loopful of strain DC3-6 was inoculated into a 500 mL K-1
flask containing 100 mL of Marine Broth 2216 as a seed culture. The
seed culture was incubated at 30 °C on a rotary shaker at 200 rpm for 2
days. Then 3 mL each of seed culture was inoculated into 30 500 mL K-
flasks containing 100 mL of A11M production medium, which
consists of glucose 0.2%, soluble starch 2.5%, yeast extract 0.5%,
polypeptone (Wako Pure Chemical Industries, Ltd.) 0.5%, NZ-amine
A solution of methyl (S,E)-8-methyl-2-decenoate (12 mg, 60 μmol)
in MeOH (3 mL) was vigorously stirred with Pd/C (20 mg) under a H2
atmosphere at rt. After stirring for 8 h, the reaction mixture was passed
through Celite and the eluent was concentrated under reduced pressure
to give methyl (S)-8-methyldecanoate (10 mg, 50 μmol), which was
further subjected to hydrolysis in MeOH−THF (2 mL each)
containing 1 M NaOH (2 mL). After stirring at rt for 12 h, the
solution was acidified with 2 M HCl and extracted with EtOAc. The
(
1
(
Wako Pure Chemical Industries, Ltd.) 0.5%, CaCO 0.3%, and Diaion
EtOAc layer was washed with H
Na SO , and concentrated in vacuo. The residue was purified on a silica
gel column (n-hexane−EtOAc = 1:0−1:1) to afford (S)-8-methyl-
2
O and brine, dried over anhydrous
3
HP-20 (Mitsubishi Chemical Co.) 1% in natural seawater (collected
from Toyama Bay, Japan). The pH of the medium was adjusted to 7.0
before sterilization. The inoculated flasks were incubated at 30 °C for 5
days with rotational shaking at 200 rpm.
Extraction and Isolation. After fermentation, 100 mL of 1-butanol
was added to each flask, and the flasks were shaken for 1 h. The
emulsified mixture was centrifuged at 6000 rpm for 10 min, and the
organic layer was separated from the aqueous layer. Then, the organic
layer was concentrated in vacuo to afford 4.3 g of extract from 3 L of
production culture. The extract was chromatographed on a silica gel
2
4
1
decanoic acid (10 mg, 88% yield): H NMR (CDCl
(3H, d, J = 6.3 Hz), 0.85 (3H, t, J = 7.2 Hz), 1.06−1.16 (2H, m), 1.24−
, 500 MHz) δ 0.83
3
1
3
1.37 (9H, m), 1.63 (2H, quint, J = 7.6 Hz), 2.34 (2H, t, J = 7.6 Hz); C
NMR (CDCl , 125 MHz) δ 179.2, 36.5, 34.3, 33.9, 29.5, 29.4, 29.0,
6.8, 24.6, 19.1, 11.3.
Preparation of (R)- and (S)-2-(Anthracene-2,3-
dicarboximido)propyl ester of (S)-8-Methyldecanoic acid [(S)-
-(R)-2A1P and (S)-4-(S)-2A1P]. (S)-8-Methyldecanoic acid (4, 5.0
3
2
4
mg, 26 μmol) was treated with (R)-2-(anthracene-2,3-dicarboximido)-
propanol [(R)-2A1P] (8.0 mg, 26 μmol), EDAC (6.0 mg, 34 μmol),
and DMAP (trace amount) in dry CH Cl (4 mL) at rt for 17 h. The
column with CHCl −MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/
3
v). Fraction 3 (10:1) was concentrated to provide 0.35 g of brown oil,
which was then fractionated by ODS column chromatography with a
2
2
reaction was quenched with ice−water, and the mixture was extracted
with EtOAc. The organic layer was concentrated in vacuo, and the
residue was chromatographed over a silica gel column (n-hexane−
EtOAc = 1:0−1:1) to give (R)-2-(anthracene-2,3-dicarboximido)-
gradient of MeCN−0.1% HCO H aqueous solution (2:8, 3:7, 4:6, 5:5,
2
6
:4, 7:3, and 8:2 v/v). Fraction 4 (5:5) was concentrated in vacuo, and
the remaining aqueous layer was extracted with EtOAc. The organic
layer was dried over anhydrous Na SO , filtered, and concentrated to
2
4
propyl ester of (S)-8-methyldecanoic acid [(S)-4-(R)-2A1P, 3.5 mg]:
give 83 mg of semipure material. Final purification was achieved by
preparative HPLC (Cosmosil AR-II, Nacalai Tesque Inc., 10 × 250
mm, 4 mL/min, UV detection at 254 nm) with an isocratic elution of
1
H NMR (CDCl , 500 MHz) δ 0.74 (3H, d, J = 6.3 Hz), 0.78 (3H, t, J =
3
7
2
1
.3 Hz), 0.98−1.22 (9H, m), 1.49 (2H, m), 1.57 (3H, d, J = 7.1 Hz),
.22 (2H, t, J = 7.5 Hz), 4.43 (1H, dd, J = 11.2, 5.0 Hz), 4.63 (2H, dd, J =
MeCN/0.1% HCO H aqueous solution (46:54) to afford 1 (11.3 mg,
2
1.2, 9.8 Hz), 4.74 (1H, m), 7.63 (2H, m), 8.09 (2H, m), 8.51 (2H, s),
t 15.9 min), 2 (3.2 mg, t 11.2 min), and 3 (2.5 mg, t 10.5 min).
R
R
R
+
2
3
D
8.64 (2H, s); HR-ESITOFMS m/z 496.2451 [M + Na] (calcd for
Bulbimidazole A (1): pale yellow, amorphous solid; [α] +1.2 (c
C H NO Na, 496.2458).
3
0
35
4
0
3
.10, MeOH); UV (MeOH) λ (log ε) 253 (4.32) nm; IR (ATR) ν
max max
−
1
1
13
(S)-4-(S)-2A1P was prepared from (S)-4 and (S)-2A1P in a similar
125, 2957, 2925, 1665 cm ; H and C NMR data, Table 1; HR-
1
+
manner to that described above: H NMR (CDCl
(
(
3
, 500 MHz) δ 0.73
3H, d, J = 6.3 Hz), 0.78 (3H, t, J = 7.3 Hz), 0.98−1.25 (9H, m), 1.49
2H, m), 1.57 (3H, d, J = 7.2 Hz), 2.22 (2H, t, J = 7.6 Hz), 4.43 (1H, dd,
ESITOFMS m/z 237.1964 [M + H] (calcd for C H N O,
1
4
25
2
2
37.1961).
Bulbimidazole B (2): pale yellow, amorphous solid; UV (MeOH)
λmax (log ε) 254 (4.32) nm; IR (ATR) ν 3127, 2957, 2925, 1667
J = 11.2, 4.9 Hz), 4.63 (1H, dd, J = 11.2, 9.7 Hz), 4.74 (1H, m), 7.63
(2H, m), 8.09 (2H, m), 8.50 (2H, s), 8.64 (2H, s); HR-ESITOFMS m/
max
−
1
1
13
cm ; H and C NMR data, Table 1; HR-ESITOFMS m/z 223.1809
M + H] (calcd for C H N O, 223.1804).
+
z 496.2454 [M + Na] (calcd for C H NO Na, 496.2458).
+
30 35
4
[
13 23 2
Oxidative Degradation of 1 and Derivatization with (R)-
A1P. A solution of bulbimidazole A (1, 0.5 mg, 2 μmol) in a mixture of
Bulbimidazole C (3): pale yellow, amorphous solid; UV (MeOH)
2
λmax (log ε) 254 (4.28) nm; IR (ATR) ν 3260, 2958, 2929, 1668
max
MeCN (80 μL) and deionized H O (60 μL) was stirred with NaIO
−
1
1
13
2
4
cm ; H and C NMR data, Table 1; HR-ESITOFMS m/z 223.1809
M + H] (calcd for C H N O, 223.1804).
(
4.8 mg, 22 μmol) until the salt was dissolved. To this solution were
+
[
13 23 2
added CCl (80 μL) and a solution of RuCl hydrate in 0.1 M sodium
4
3
Synthesis of (S)-8-Methyldecanoic Acid (4). To a solution of (S)-6-
phosphate buffer (1 mg/mL, 60 μL, pH 7.6), and the biphasic mixture
was vigorously stirred at rt for 18 h. The reaction mixture was passed
through Celite, and the filter cake was washed with MeCN. After
evaporation of the organic solvent from the filtrate, the aqueous
solution was acidified with 2 M HCl and extracted with EtOAc. The
methyl-1-octanol (50 mg, 0.35 mmol, Wako Pure Chemical Industries,
Ltd.) in dry CH Cl (5 mL) was added Dess-Martin periodinane (200
2
2
mg, 0.47 mmol) at room temperature (rt), and the resultant mixture
was stirred for 2 h. The reaction mixture was quenched by adding
saturated a NaHCO solution and Na SO solution and extracted with
3
2
3
EtOAc layer was washed with H O and brine, dried over anhydrous
2
EtOAc. The organic layer was washed with H O and brine, dried over
2
Na SO , and concentrated in vacuo to give (S)-8-methyldecanoic acid
2
4
anhydrous Na SO , and concentrated in vacuo to afford (S)-6-
2
4
(0.8 mg), which was reacted with (R)-2A1P in a similar manner to that
described for (S)-4-(R)-2A1P to give the ester derivative of 8-
methyldecanoic acid derived from 1 [nat-1-(R)-2A1P, 0.4 mg]: HR-
methyloctanal (30 mg), which was used for the next reaction without
further purification.
(
S)-6-Methyloctanal (30 mg, 0.21 mmol) was then reacted with
+
ESITOFMS m/z 496.2457 [M + Na] (calcd for C H NO Na,
3
0
35
4
methyl (triphenylphosphoranylidene)acetate (140 mg, 0.42 mmol) in
4
96.2458).
dry CH Cl (1 mL) at rt. After stirring for 2 h, ice−water was added to
2
2
Determination of the Absolute Configuration at the anteiso-
the reaction mixture, which was then extracted with EtOAc. The
Methyl Branching in 1. nat-4-(R)-2A1P and synthetic (S)-4-(R)-
2A1P and (S)-4-(S)-2A1P were analyzed by HPLC under the following
conditions. Column: tandemly connected Develosil ODS-HG-3 (3.0
mm i.d. × (250 + 150) mm, Nomura Chemical); mobile phase:
MeCN−MeOH−THF = 3:1:1; column temperature: −42.5 °C; flow
rate: 0.10 mL/min. The column was cooled by using Cryocool CC-100
(Neslab Instruments Inc.). HPLC peaks were detected by monitoring
fluorescence intensity at 460 nm with the excitation at 298 nm by using
an FP-4025 fluorescence detector (JASCO Corporation). Retention
times were 234 min for (S)-4-(S)-2A1P and 243 min for (S)-4-(R)-
2A1P, while nat-4-(R)-2A1P gave peaks at 235 and 244 min in a ratio of
9.1:90.9.
organic layer was washed with H O and brine, dried over anhydrous
2
Na SO , and concentrated under reduced pressure. The residue was
2
4
chromatographed over a silica gel column (n-hexane−EtOAc = 1:0−
1
1
:1) to give methyl (S,E)-8-methyl-2-decenoate (12 mg, 17% yield): H
NMR (CDCl , 500 MHz) δ 0.83 (3H, d, J = 6.3 Hz), 0.85 (3H, t, J = 7.2
3
Hz), 1.08−1.15 (2H, m), 1.24−1.37 (5H, m), 1.40−1.45 (2H, m), 2.20
(
2H, ddt, J = 1.7, 7.1, 7.1 Hz), 3.72 (3H, s), 5.81 (1H, dt, J = 15.6, 1.7
1
3
Hz), 6.97 (1H, dt, J = 15.6, 7.0 Hz); C NMR (CDCl , 125 MHz) δ
3
1
67.2, 149.8, 120.8, 51.4, 36.3, 34.3, 32.3, 29.4, 28.3, 26.6, 19.2, 11.4;
+
HR-ESITOFMS m/z 221.1648 [M + Na] (calcd for C H O Na,
1
2
22
2
2
21.1644).
D
https://dx.doi.org/10.1021/acs.jnatprod.0c00082
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Note
AUTHOR INFORMATION
Corresponding Author
■
Yasuhiro Igarashi − Biotechnology Research Center, Toyama
Prefectural University, Imizu, Toyama 939-0398, Japan;
orcid.org/0000-0001-5114-1389; Phone: +81-766-56-
7500; Email: yas@pu-toyama.ac.jp; Fax: +81-766-56-2498
Authors
maritimum NBRC16015, respectively. Compounds 1−3, reference
drugs kanamycin sulfate for bacteria, sulfamethoxazole for R. radio-
bacter NBRC14554 and T. maritimum NBRC16015, and amphotericin
B for fungi were made in 2-fold dilution series along the longer side of
the plates by sequential transfer of 100 μL aliquots between the adjacent
wells, to which the same amount of medium was predispensed. To each
well was added a 100 μL suspension of the indication strains prepared at
Md. Rokon Ul Karim − Biotechnology Research Center, Toyama
Prefectural University, Imizu, Toyama 939-0398, Japan
Enjuro Harunari − Biotechnology Research Center, Toyama
Prefectural University, Imizu, Toyama 939-0398, Japan;
orcid.org/0000-0001-8726-0865
Naoya Oku − Biotechnology Research Center, Toyama Prefectural
University, Imizu, Toyama 939-0398, Japan
4
5
1
× 10 −10 cfu/mL from a culture at the logarithmic growth phase.
Kazuaki Akasaka − Shokei Gakuin University, Natori, Miyagi
The solvent vehicle added to the top rows was set at 0.5% of the final
culture volume to avoid the effect on the growth of microbes. The plates
were incubated at 37 °C for 20 h for bacteria, at 24 °C for T. maritimum
NBRC16015, and at 25 °C for fungi. The experiments were done in
triplicate, and the absorbance at 650 nm was measured using a
microplate reader.
9
81-1295, Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00082
Notes
Cytotoxicity Assay. P388 murine leukemia cells were maintained
in RPMI-1640 medium containing L-glutamine (product no. 186-
The authors declare no competing financial interest.
0
2155) supplemented with 10% fetal bovine serum and 0.1 mg/mL
ACKNOWLEDGMENTS
gentamicin sulfate. Compounds 1−3 and doxorubicin as a reference
were used to validate cytotoxicity in a 96-well round-bottom microtiter
plate. To each well were seeded the cells at a final density of 1 × 10
cells/well, and 200 μL cultures thus made were incubated for 72 h at 37
■
Authors are in debt to Prof. D. Urabe at Toyama Prefectural
University for his technical advice on the synthesis of (S)-8-
methyldecanoic acid. P388 cells were obtained from JCRB Cell
Bank under accession code JCRB0017 (Lot. 06252002).
4
°C in an atmosphere of 5% CO in air with 100% humidity. Viability of
2
the cells was visualized by addition of 50 μL of medium containing XTT
1 mg/mL) and PMS (40 μg/mL) to each well. After incubating for 4 h
(
REFERENCES
1) Rizzo, C.; Giudice, A. L. Diversity 2018, 10, 52.
at 37 °C, medium was carefully removed by a suction aspirator, and
formazan dye formed by respiratory reduction by living cells was
quantified by measurement of absorption at 450 nm and read by a
microplate reader to calculate the rate of cell growth inhibition at each
concentration. The results of triplicate experiments were plotted on a
graph to deduce IC50 values.
■
(
(
(
2) Tobias, A. M.; et al. Curr. Opin. Microbiol. 2009, 12, 252−260.
3) Hossain, M. B.; et al. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 4118−
4
(
(
(
(
(
122.
4) Tsukimoto, M.; et al. J. Nat. Prod. 2011, 74, 2329−2331.
5) Sang, V. T.; et al. Mar. Drugs 2019, 17, 468.
6) Raimundo, I.; et al. Mar. Drugs 2018, 16, 485.
7) Gonzalez, J. M.; et al. Int. J. Syst. Bacteriol. 1997, 47, 369−376.
8) (a) Yoon, J.-H.; Kim, I.-G.; Shin, D.-Y.; Kang, K. H.; Park, Y.-H.
Table 2. Antimicrobial Activity of Bulbimidazoles A−C (1−
3
)
Int. J. Syst. Evol. Microbiol. 2003, 53, 53−57. (b) Yoon, J.-H; Kim, I.-G.;
et al. Int. J. Syst. Evol. Microbiol. 2004, 54, 1111−1116. (c) Yoon, J.-H.;
et al. Int. J. Syst. Evol. Microbiol. 2007, 57, 2365−2369. (d) Zhang, D.-S.;
et al. Int. J. Syst. Evol. Microbiol. 2012, 62, 505−510. (e) Nishijima, M.;
et al. Int. J. Syst. Evol. Microbiol. 2009, 59, 1696−1707.
MIC (μg/mL)
microorganism
1
2
3
Kocuria rhizophila ATCC9341
Staphylococcus aureus FDA209P JC-1
Escherichia coli NIHJ JC-2
Rhizobium radiobacter NBRC14554
Tenacibaculum maritinum NBRC16015
Candida albicans NBRC0197
0.78
1.56
>100
>100
3.12
6.25
1.56
0.78
0.78
1.56
>100
>100
6.25
12.5
1.56
1.56
1.56
3.12
>100
>100
12.5
6.25
3.12
3.12
(9) https://antismash.secondarymetabolites.org (accessed on Dec.
2
(
6, 2019).
10) Jayanetti, D. R.; et al. J. Nat. Prod. 2019, 82, 1930−1934.
11) Sharma, A. R.; et al. Beilstein J. Org. Chem. 2019, 15, 2327−2332.
12) Leutou, A. S.; et al. J. Nat. Prod. 2015, 78, 2846−2849.
13) Kawakami, J.-I.; et al. Synthesis 2003, 5, 0677.
(
(
(
(
(
(
(
Glomerella cingulata NBRC5907
Trichophyton rubrum NBRC5467
14) Iwasaki, S. Helv. Chim. Acta 1976, 59, 2738−2752.
15) Akasaka, K.; et al. Tetrahedron Lett. 1997, 38, 6853−6856.
16) Carlsen, P. H. J.; et al. J. Org. Chem. 1981, 46, 3936−3938.
17) Thompson, S. K.; Murthy, K. H. M.; et al. J. Med. Chem. 1994, 37,
3
100−3107.
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00082.
■
(18) Kaneda, T. Microbiol. Rev. 1991, 55, 288−302.
(19) Challis, G. L. Microbiology 2008, 154, 1555−1569.
(20) Kim, Y.; et al. Mar. Drugs 2014, 12, 4110−4125.
*
(
21) Yajima, T.; et al. Antimicrob. Agents Chemother. 1976, 9, 224−
232.
UV, IR, and 1D and 2D NMR spectra of 1, 2, and 3;
COSY and key HMBC correlations for 2 and 3;
(
(
(
22) Li, Q.; et al. J. Am. Chem. Soc. 2016, 138, 408−415.
23) Oku, H.; et al. Biosci., Biotechnol., Biochem. 1996, 60, 1724−1725.
24) Matich, A.; Rowan, D. J. Agric. Food Chem. 2007, 55, 2727−2735.
1
13
assignment of H and C NMR chemical shifts with
HMBC correlations for 2 and 3; synthesis of (S)-8-
methyldecanoic acid; possible biosynthetic routes to (R)-
and (S)-2-methylbutanoyl CoA from L-isoleucine (PDF)
(25) Raj Sharma, A.; et al. J. Antibiot. 2019, 72, 634−639.
E
https://dx.doi.org/10.1021/acs.jnatprod.0c00082
J. Nat. Prod. XXXX, XXX, XXX−XXX