Biosurfactants from Marine Cyanobacteria Collected in Sabah, Malaysia

Article abstract of DOI:10.1021/acs.jnatprod.0c00164

Chemical investigation of the organic extract from Moorea bouillonii, collected in Sabah, Malaysia, led to the isolation of three new chlorinated fatty acid amides, columbamides F (1), G (2), and H (3). The planar structures of 1-3 were established by a combination of mass spectrometric and NMR spectroscopic analyses. The absolute configuration of 1 was determined by Marfey's analysis of its hydrolysate and chiral-phase HPLC analysis after conversion and esterification with Ohrui's acid, (1S,2S)-2-(anthracene-2,3-dicarboximido)cyclohexanecarboxylic acid. Compound 1 showed biosurfactant activity by an oil displacement assay. Related known fatty acid amides columbamide D and serinolamide C exhibited biosurfactant activity with critical micelle concentrations of about 0.34 and 0.78 mM, respectively.

Full text of DOI:10.1021/acs.jnatprod.0c00164

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Article
Biosurfactants from Marine Cyanobacteria Collected in Sabah,
Malaysia
Jakia Jerin Mehjabin, Liang Wei, Julie G. Petitbois, Taiki Umezawa, Fuyuhiko Matsuda,
Charles S. Vairappan, Masaaki Morikawa, and Tatsufumi Okino*
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ABSTRACT: Chemical investigation of the organic extract from
Moorea bouillonii, collected in Sabah, Malaysia, led to the isolation of
three new chlorinated fatty acid amides, columbamides F (1), G (2),
and H (3). The planar structures of 1−3 were established by a
combination of mass spectrometric and NMR spectroscopic
analyses. The absolute configuration of 1 was determined by
Marfey’s analysis of its hydrolysate and chiral-phase HPLC analysis after conversion and esterification with Ohrui’s acid, (1S,2S)-2-
(anthracene-2,3-dicarboximido)cyclohexanecarboxylic acid. Compound 1 showed biosurfactant activity by an oil displacement assay.
Related known fatty acid amides columbamide D and serinolamide C exhibited biosurfactant activity with critical micelle
concentrations of about 0.34 and 0.78 mM, respectively.
iosurfactants are promising amphipathic compounds
Bderived from microorganisms and can exhibit a diversity
of bioactivities such as cytotoxic, antimicrobial, antiadhesive,
and antibiofilm activities against human pathogens.1 Due to
their chemical diversity and surface activities, biosurfactants are
widely used in different fields including food, agriculture,
cosmetics, and petrochemical industries.2−6 According to a
2017 market research report, the global market for
biosurfactants was valued at USD 3.99 billion in 2016 and is
expected to reach USD 5.52 billion by 2022.7 A number of
studies have reported biosurfactant properties of known
natural product structures including the cytotoxic somocysti-
namide A and apratoxins,1 2-acyloxyethylphosphonate,8 and
exopolysaccharides9 from cyanobacteria. The high structural
diversity and bioactivities of cyanobacterial secondary metab-
olites present them as potential candidates for novel
biosurfactants. As part of our continuing study of bioactive
compounds from marine cyanobacteria collected in Sabah,
Malaysia,10 here we report the biosurfactant-assay-guided
isolation and structure elucidations of the three new
compounds columbamides F (1), G (2), and, H (3) from
Moorea bouillonii. Marfey’s analysis of the N,O-dimethylserinol
moiety and Ohrui’s method followed by chiral-phase HPLC
were executed to determine the absolute configuration. The
biosurfactant activities were measured by the oil displacement
assay for 1 and by the ring method for the previously isolated
compounds from cyanobacteria. Cytotoxicities of these
compounds were evaluated using MCF-7 breast cancer cells.
done on the basis of its morphology11 and phylogenetic studies
based on 16S rRNA gene sequencing analysis (Supporting
Information S1, S2, and S3). The cyanobacterial sample was
extracted with MeOH, and solvent partitioning was done by
using EtOAc, H2O, and BuOH. The chemical profiling of the
EtOAc fraction by LC-MS exhibited the presence of a number
of known compounds including apratoxins A and C,
wewakazole, lyngbyabellin A, laingolides, and additional
possibly new chlorinated compounds. The EtOAc fraction
was subjected to normal-phase silica gel column chromatog-
raphy and divided into 12 fractions. The silica gel fractions of
hexane/EtOAc (50:50 v/v), 100% EtOAc, and EtOAc/MeOH
(90:10 v/v) exhibited a higher value for the diameter of the
clear zone (∼140 mm) compared to other fractions in an oil
displacement assay.12 On the basis of their bioactivity and
chemical profile these fractions were chosen for the isolation of
new halogenated compounds. The compounds that eluted with
a 50:50 (v/v) mixture of hexane/EtOAc were determined as
columbamides F (1) and G (2) with di- and trichlorinated
patterns, respectively (Figure S4). Another new compound,
columbamide H (3), was purified from the 100% EtOAc
fraction along with known compounds apratoxin A13 and
columbamide D,10 confirmed by comparing their H and 13C
1
NMR chemical shift data.
Received: February 13, 2020
RESULTS AND DISCUSSION
■
The mat-forming M. bouillonii sample was collected from
Mantanani Island, Sabah, Malaysia, and the identification was
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Chart 1
The planar structure of 1 was established by analyzing NMR
and MS data (Figure 1 and Table 1). The molecular formula of
C25H44Cl3NO4, indicating three degrees of unsaturation. The
mass difference between columbamide G (2) and colum-
bamide F (1) was 34 Da, suggesting the presence of an
additional chlorine. The position of the additional chlorine was
confirmed by the triplet proton signal at δH 5.75 and associated
carbon signal at δC 73.6 ppm, which indicated a terminal
dichloromethine at C-18, similar to columbamide E.10
The molecular formula of columbamide H (3) was
determined as C23H44ClNO3 by ESITOFMS, indicating two
degrees of unsaturation. The NMR signals for columbamide H
(3) are similar to columbamide F (1) except for the
replacement of the acetoxy group at C-22 by a hydroxy
group (δH 3.02), resulting in the shielded shift of the adjacent
methylene group to H-22 (δH 3.79) and the lack of a terminal
chlorine at position C-18, confirmed by the triplet signal for
the methyl group at H-18 (δH 0.88 and δC 14) (Table 1).
An E configuration of the double bond for compounds 1−3
was obtained by measuring the coupling constant (3JH,H = 16−
17 Hz) from the 13C satellites observed by nondecoupled
HSQC analysis15 (Figure S28). The absolute configuration of
the stereogenic center of the dimethylated and acetylated
serinol moiety was determined by Marfey’s analysis.16 The two
standards, (R)- and (S)-N,O-dimethylserinol (4), were
synthesized17 (Figure S29) and derivatized with Marfey’s
reagent. In a similar way derivatization was done for the
hydrolysates of compounds 1−3. The R-configurations for
N,O-dimethylserinol residues were established (Figure S30) by
comparing the retention times.
In our previous study, the absolute configuration of the
chloromethine of columbamide D was determined by total
synthesis of all four isomers followed by chiral-phase HPLC
analysis. For this study we examined the potential of a chiral
labeling reagent for determining the configuration of the
chloromethine on the long alkyl chain of columbamide F (1).
The absolute configuration of the chloromethine at C-10 was
determined by chemical conversion of columbamide D (5)
standards and natural compound 1 followed by esterification
with Ohrui’s acid (6).18 The use of the highly potent
fluorescent chiral labeling reagent (R,R)- and (S,S)-2-
(anthracene-2,3-dicarboximido)cyclohexanecarboxylic acid
(Figure 2) has been well reported for the discrimination of
compounds having remote stereocenters.19 Because our team
previously synthesized columbamide D stereoisomers,10
(10S,20R)- and (10R,20R)-(5) were used as standards in the
Ohrui reaction for columbamide F (1). Synthesized diaster-
eomers of columbamide D and natural 1 were converted to
diol 7 by dihydroxylation and then cleaved oxidatively with
sodium periodate (NaIO4) to form two aldehydes (8, 9). After
separation, 8 was reduced with NaBH4 to furnish a linear
Figure 1. 2D NMR correlations for 1.
1 was determined to be C25H45Cl2NO4 by ESITOFMS,
suggesting three degrees of unsaturation. The presence of
carbonyls (δC 173.3, 170.7), two olefinic methines (δC 129.1,
130.9) with identical protons (δH 5.46), a chlorinated methine
(δC 64.2, δH 3.88), an N-methyl (δC 31.7, δH 2.95), an N-
methine (δC 51.9, δH 4.90), a chlorinated methylene (δC 45.1,
δH 3.53), a methoxy (δC 58.9, δH 3.34), two oxo-methylenes
(δC 70.9, δH 3.57 and δC 62.0, δH 4.26), and other methylenes
(δC 26.4−38.4, δH 1.31−2.42) revealed the similarities of this
compound to the columbamides.10,14 The mass difference
between columbamide D and 1 is 42, indicating the possibility
of the acetylation of columbamide D for 1. Columbamide A
has an acetyl function as well. An acetylated N,O-dimethylser-
inol moiety was elucidated by HMBC correlations from both
the methyl singlet H3-24 and the deshielded methylene
protons H2-22 to an acetoxy carbonyl at δC 170.7, H-20 to
H-21 and H2-22 by COSY, H-20 to C-19 and H3-25 to C-21
by HMBC, and H-20 to C-21 and C-22 by H2BC. The
connection between C-1 and H-19 was also made by HMBC
correlations. COSY and H2BC correlations allowed the
assignment from H-3 to H-4. The double bond between C-4
and C-5 was established by H2BC signals from H-4/5 to C-3
and C-6 and corresponding HMBC signal from H-5 to C-4.
COSY signals from H-5 to H-6, H-6 to H-7, and H-8 to H-9
allowed the connections of these protons to the adjacent
methylenes. The position of the chlorine atom at position C-10
was established by the following signals: COSY correlations
from H-10 to H-9 and H-11, H2BC correlations from H-10 to
C-9 and C-11, and HMBC correlations from H-10 to C-8, C-9,
C-11, and C-12. The assignment of methylenes from C-11 to
C-17 was confirmed by COSY, HMBC, and H2BC
correlations. The position of the second chlorine at the
terminal C-18 was supported by COSY signals from H-17 to
H-18 and vice versa. The corresponding H2BC and HMBC
signals completed the planar structure of columbamide F (1).
ESITOFMS analysis of 2 showed the [M + H]+ ion at m/z
528.2451, corresponding with the molecular formula
B
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Table 1. NMR Spectroscopic Data for Columbamides F (1), G (2), and H (3) in CDCl3
columbamide F (1)
columbamide G (2)
columbamide H (3)
COSY/
HMBC/
b
a
b
b
c
b
c
b
position
δC, type
δH (J in Hz)
TOCSY
H2BC
δC, type
δH (J in Hz)
δC, type δH (J in Hz)
1
2
3
4
5
6
7
173.3, C
34.02, CH2
28.0, CH2
129.1, CH
130.9, CH
32.6, CH2
28.8, CH2
173.2, C
174.3, C
2.38, m
2.33, m
5.46, m
5.46, m
2.00, m
3
4
3, 3′
6
5, 7
6
1, 3′
2′, 4′
3′, 5′, 6
3, 4, 4′, 6′
4, 5, 7, 8
34.02, CH2
28.0, CH2
129.1, CH
130.9, CH
32.3, CH2
28.9, CH2
2.38, m
2.33, m
5.45, m
5.45, m
2.00, m
1.33, m
33.9, CH2
28.0, CH2
128.9, CH
131.1, CH
32.3, CH2
2.40, m
2.34, m
5.46, m
5.46, m
2.0, m
d
1.31, m
28.04,
CH2
1.32, m
8
9
10
25.9, CH2
38.5, CH2
64.2, CH
1.51, 1.39, m
1.69, m
3.88, m
9
25.8, CH2
38.5, CH2
64.2, CH
1.39, m
1.70, m
3.88, m
25.9, CH2
38.3, CH2
64.3, CH
1.38, m
1.70, m
3.88, m
8, 10′
9, 9′, 11
8, 9, 9′, 11′,
11, 12
11
12
38.4, CH2
26.4, CH2
1.69, m
10, 12
11
10, 10′, 12′,
38.5, CH2
26.4, CH2
1.70, m
38.3, CH2
26.5, CH2
1.70, m
12, 13
1.51, 1.39, m
11, 11′, 13′
11, 12, 14
14′, 16′
1.54, m 1.38, m
1.51, m
1.38, m
1.28, m
1.29, m
1.29, m
1.38, m
d
13
14
15
16
28.8, CH2
29.0, CH2
28.7, CH2
26.8, CH2
1.31, m
28.4, CH2
29.2, CH2
28.7, CH2
26.8, CH2
1.32, m
1.29, m
1.32, m
1.42, m
28.0, CH2
29.2, CH2
28.0, CH2
26.5, CH2
d
1.31, m
d
1.31, m
1.40, m
1.76, m
17
16, 18, 18′
17
15, 17, 15′,
17′
17
32.5, CH2
16, 16′, 18,
18′
16, 17, 17′
43.6, CH2
2.2, m
22.6, CH2
1.27, m
18
19
45.1, CH2
31.7 (27.3),
CH3
3.53, t (6.8)
2.95, s (2.82, s)
73.6, CH
31.5 (27.2),
CH3
5.75, t (6.1)
2.95, s (2.82, s)
14.0, CH3 0.88, t (6.8)
31.8, CH3 3.02, s (2.84,
s)
1
20
21
51.9, CH
70.9, CH2
4.90, m
3.57, dd (10.3, 6.8) 3.48, dd
(10.3, 5.5)
21, 22
20
19, 21′, 22′
20′, 20, 22, 25
51.9, CH
70.9, CH2
4.90, m
57.9, CH
4.39, m
3.59, dd (10.3, 6.8) 3.48, 71.0, CH2 3.57, dd (10.3,
dd (9.6, 4.8)
4.8)
22
62.0, CH2
4.26, dd (11.0, 7.5) 4.20, dd
(12.3, 4.8)
20
20, 21, 23
62.0, CH2
4.26, dd (11.6, 7.5) 4.20, 62.3, CH2
dd
3.79, m
22-OH
23
24
3.02, bs
3.34, s
170.7, C
20.8, CH3
58.9, CH3
170.7, C
20.8 CH3
59.0, CH3
58.9, CH3
2.04, s (2.05, s)
2.04, s (2.05, s)
3.32, s 3.34, s
23
21
25
3.32, s
3.34, s
a
b
c
Measured at 150 MHz. Measured at 600 MHz. Values in parentheses are from TOCSY and H2BC experiments. Derived from HSQC, HMBC,
d
and H2BC data. Overlapping signals.
Figure 2. Synthetic standards and Ohrui’s acid for determining the configuration at a remote stereocenter.
alcohol (10), which was esterified with (S,S)-2-(anthracene-
2,3-dicarboximido)cyclohexanecarboxylic acid (6) in the
presence of EDCI and DMAP to afford 11. Natural
columbamide F (1) was converted and esterified with 6 in a
similar way (Figure 3). Chiral-phase HPLC analysis of
synthetic 10S and 10R standards revealed that a co-injection
experiment can separate both standards. Then by a coelution
experiment, natural 1 gave a single peak with the synthetic 10R
isomer, while two peaks were observed with the synthetic 10S
isomer, confirming the R-configuration for the chloromethine
of 1 (Figure S32). Although in Ohrui’s original method, the
HPLC separation of the diastereomers was done by using a
C18 or C30 column and fluorescence detection using different
temperatures ranging from 40 to −50 °C,19−21 here we report
the significance of chiral-phase HPLC for the separation of
diastereomers using a UV detector at room temperature.
Ohrui’s acid was used to determine the absolute chloromethine
using a small amount of sample, and this can be a powerful
approach for determining the absolute configuration of natural
products having a remote stereogenic center.
Fatty acid amides columbamides A and B14 isolated from
cultured M. bouillonii showed cannabinomimetic activity
C
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Figure 3. Chemical conversion to apply Ohrui’s method for determining the configuration of the chloromethine of columbamide F (1).
similar to other fatty acid amides.22,23 Here we report the
surface tension lowering activity of fatty acid amides
columbamide D (5) and serinolamide C (12)24 (isolated by
our group). Because of the amphiphilic properties, these
compounds reduced the surface tension of aqueous solutions
at concentrations of 10−1200 μg/mL, as determined by the
ring method. The critical micelle concentrations (CMCs) of 5
and 12 were 0.34 and 0.78 mM, respectively, which are lower
than the CMC value of the synthetic surfactant SDS (8.2
mM)25 and comparable to pluronic F-68 (0.07−0.35 mM)26
and triton X-100 (0.33 mM).27 Serinolamide C (12) isolated
from Okeania sp. showed potent antifouling activity (0.88 μg/
mL) against Amphibalanus amphitrite larvae. The biosurfactant
properties of serinolamide C could reduce the surface tension
of seawater in the microplate well, and therefore the larvae
cannot adhere easily. Because of the small amount of sample,
the biosurfactant activity of columbamide F (1) was checked
by an oil displacement assay, which is an indirect measurement
of surface activity. The diameter of the clear zone was
measured as ∼90 mm for a concentration of 10 mg/mL, which
is a slightly higher displacement area compared to SDS (∼84
mm) and pluronic F-68 (∼54 mm) for the same concentration.
In addition columbamides F−H (1−3) were tested for the
cytotoxicity against the MCF-7 breast cancer cell line but were
inactive (IC50 > 10 μg/mL).
In summary, the new columbamides F (1), G (2), and H (3)
were isolated from M. bouillonii collected in Sabah, Malaysia.
In combination with NMR and MS data the planar structures
of these three new columbamides were established. The
absolute configuration of columbamide F (1) has been
determined to be (10R,20R) by Marfey’s analysis and chiral-
phase HPLC analysis of synthetic standards and columbamide
F (1) after derivatization with the Ohrui’s acid. Ohrui’s
method combined with chiral-phase HPLC analysis at room
temperature may increase its application to determine the
absolute configuration of natural compounds having a remote
stereogenic center. Because of having surface-tension-lowering
properties with a low CMC value and weak cytotoxicity, the
fatty acid amides columbamide D (5) and serinolamide C (12)
may have potential for applications in various fields such as
healthcare, food, and cosmetics. The current work reports the
discovery of new potential biosurfactants and has uncovered
greater diversity in the columbamide family of compounds.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotation data were
measured on a Horiba SEPA-300 polarimeter using chloroform as a
solvent. IR spectra were measured on a JASCO FT/IR-4100 type A.
The 1H NMR, 13C NMR, and 2D NMR spectra for compounds 1, 2,
and 3 were acquired using a JEOL 600 MHz spectrometer. For all
NMR experiments, CDCl3 (Cambridge Isotope Laboratories, Inc.)
was used as solvent as an internal standard (δH 7.26 and δC 77.0). LC-
MS data were acquired on an Agilent 1100 series HPLC system
coupled with a Bruker Daltonics microTOF-HS mass spectrometer.
The HPLC system was equipped with a Cadenza CD-C18 column (2
× 150 mm, 3 μm, flow rate 0.2 mL/min at 25 °C) and operated under
the following conditions: 0−15 min 50−80% MeCN with 0.1% (v/v)
formic acid (FA) in Milli-Q H2O; 15−30 min, isocratic 80% MeCN
with 0.1% (v/v) FA. Preparative HPLC systems used a Cosmosil
Cholester column (10 × 250 mm, 5 μm) at a flow rate of 3 mL/min,
with UV detection at 210 nm.
Biological Material and Gene Sequencing. The cyanobacterial
sample (M1705) was collected from Rocky Point, Mantanani Island,
Sabah, Malaysia (06°42′16″ N; 116°19′23″ E) in November 2017 by
using scuba diving at a depth of 5−10 m. The sample was cleaned by
removing foreign particles, and the seawater was squeezed out by
hand before storing in MeOH for transportation. Small pieces of the
collected cyanobacterial sample were preserved in 8 mL of RNAlater
solution for genetic analysis. The sample was identified by 16S rRNA
gene sequence analysis (detailed procedure reported in Supporting
Information S2) as Moorea bouillonii (accession no. MN759382).
Extraction and Isolation. The M. bouillonii cyanobacterium
sample was homogenized at 10 000 rpm for 10 min with MeOH and
extracted three times. The extract was filtered and concentrated in
vacuo. The residue of the MeOH extract was partitioned between
EtOAc and H2O and then BuOH. These fractions were dried and
subjected to an oil displacement assay for biosurfactant activity and a
D
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cytotoxicity assay against the MCF7 breast cancer cell line. The
chemical profiling of EtOAc and BuOH fractions revealed the
presence of known cytotoxic compounds including apratoxins,
wewakazole, and possible new halogenated compounds according to
the MarinLit database. The EtOAc extract (600 mg) was then applied
onto a silica gel (Merck) column with the size 0.063−0.200 mm and
eluted with a stepwise gradient starting with 100% n-hexane to 100%
EtOAc (98:2, 95:5, 90:10, 80:20, 50:50 v/v) followed by 100%
EtOAc to 100% MeOH (95:5, 90:10, 80:20, 50:50) to yield 12 major
fractions (F1 to F12). The solvents were removed in vacuo using a
rotary evaporator, and the sample was moved to a clean small vial to
be weighed. The 50:50 (v/v) hexane/EtOAc fraction was then
subjected to semipreparative reversed-phase HPLC (gradient 0−30
min 50−80% MeCN; 30−40 min 80−100% MeCN, Cosmosil
cholester, 10 × 250 mm, 3 mL/min and UV detection at 210 nm)
using HPLC-grade MeCN (Wako) and Milli-Q H2O to obtain
columbamides F (1) (5 mg, tR = 43.3 min) and G (2) (2 mg, tR =
44.7 min). The silica gel fraction that eluted with 100% EtOAc was
subjected to RP-HPLC with similar conditions to obtain colum-
bamide H (3) (3.5 mg, tR = 39.1 min). Then compounds 1 and 3
were further purified using RP-HPLC (gradient 0−30 min, 80−100%
MeCN, Cosmosil cholester, 4.6 × 250 mm, 1 mL/min and UV
detection at 210 nm) to yield 1 (3.2 mg, tR = 13.9 min) and 3 (2 mg,
tR= 12 min).
was purified by silica gel column chromatography (EtOAc/hexane =
2.5:97.5) to give aldehyde 8 (1.40 mg, 0.00499 mmol, 46% in two
steps) as a colorless oil. To a solution of aldehyde 8 (1.40 mg,
0.00499 mmol) in MeOH (0.10 mL) was added NaBH4 (0.50 mg,
0.0132 mmol) at 0 °C. The mixture was stirred for 30 min, quenched
with saturated NaHCO3, extracted with EtOAc, washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The crude
alcohol 10 was used for the next step. To a solution of the crude
alcohol 10 in CH2Cl2 (0.30 mL) were added (S,S)-Orui’s acid (3.7
mg, 0.0114 mmol), EDCI (2.80 mg, 0.0146 mmol), and catalytic
DMAP at rt under an Ar atmosphere. The mixture was stirred for 18
h, quenched with saturated NH4Cl, extracted with EtOAc, washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(EtOAc/hexane = 20:80) to give 11 (4.4 mg), which was used for
HPLC analysis (HPLC conditions: 90:10 hexane/isopropanol,
Chiralcel OJ-H, 4.6 × 250 mm, 1 mL/min, UV 300 nm). The
synthetic 5-(10S,20R) (4.8 mg, 0.0106 mmol) and natural
columbamide F (1) (3 mg, 0.006 mmol) were degraded and
derivatized in a similar method to that described previously to obtain
6-ester of S-10 (4.0 mg) and 6-ester of columbamide F (1)−10 (2.0
mg), respectively. Using a chiral-phase HPLC system, the co-injection
experiments of the synthetic standards (11) were separated into two
peaks at 22.2 and 24.9 min. Again in a coelution experiment of natural
columbamide F (1) with synthetic standards, 10R gave a single peak
at 25.2 min while 10S was separated from natural 1 and gave two
peaks at 22.5 and 25.1 min.
Columbamide F (1): colorless oil; [α]22D +6.2 (c 0.29, CHCl3); IR
1
(neat) λmax 2929, 2856, 1743, 1648, 1456, 1109, 1042 cm−1; H and
13C NMR data, Table 1; HRESIMS m/z 494.2816 [M + H]+ (calcd
Oil Displacement Assay. The oil displacement test was
performed by determining the diameter of the oil displacement
area.12 For this assay, 10 μL of crude oil was added to the surface of
40 mL of distilled H2O in a 15 cm glass Petri dish to form a thin oil
layer. The crude oil used in this experiment was manufactured by
Tokyo Chemical Industry Co., LTD (S0432). Extracted samples were
dissolved in H2O for hydrophilic fractions and EtOH for lipophilic
fractions to make a final concentration of 10 mg/mL. Then, 10 μL of
extracted samples was gently placed on the center of the oil layer. If
biosurfactant was present in the sample, the oil was displaced and a
clearing zone was formed. The clearing zone was observed by light,
and the diameter of this zone was measured with a vernier caliper.
The diameter of this clearing zone on the oil surface correlates to
surfactant activity, also called oil displacement activity.
for C25H46Cl2NO4, 494.2804).
Columbamide G (2): colorless oil; [α]23 +9 (c 0.07, CHCl3); IR
D
(neat) λmax 2923, 2853, 1741, 1651, 1456, 1106 cm−1; H and 13C
1
NMR data, Table 1; HRESIMS m/z 528.2451 [M + H]+ (calcd for
C25H45Cl3NO4, 528.2414).
Columbamide H (3): colorless oil; [α]23 +23.1 (c 0.06, CHCl3);
D
IR (neat) λmax 3394, 2925, 2855, 1624, 1456, 1121 cm−1; 1H and 13
C
NMR data, Table 1; HRESIMS m/z 418.3042 [M + H]+ (calcd for
C23H45ClNO3, 418.3087).
Acid Hydrolysis and Marfey’s Analysis. The absolute
configurations of the N,O-dimethylserinol moiety were determined
by comparisons with synthetic N,O-dimethylserinol standards
prepared according to the method of Gao et al.17 Columbamides F
(1) and G (2) (0.2 mg) were hydrolyzed using 12 M HCl at 120 °C
for 24 h to cleave both the amide and acetate groups. Columbamide
H (3) (0.2 mg) was hydrolyzed using 6 M HCl at 120 °C for 16 h.
The reaction was quenched with NaHCO3 and evaporated to dryness.
The hydrolysates from the columbamides and the synthesized N,O-
dimethyserinol were subjected to Marfey’s analysis. The hydrolysates
and standards were first dissolved in 100 μL of H2O, and then 200 μL
of FDAA (5-fluoro-2,4-dinitrophenyl-L-alaninamide) solution (in 1%
acetone) was added, followed by addition of 40 μL of 1 M NaHCO3.
The solutions were heated in an oven at 40 °C for 1 h, and then the
reaction was quenched with 20 μL of 2 M HCl. A 200 μL amount of
MeCN was added, and this mixture was directly analyzed by LC-MS
(gradient 0−80 min 10−50% MeCN with 0.1% FA in Milli-Q H2O,
Cosmosil 5C18-AR II column, 4.6 × 250 mm, flow rate 0.2 mL/min
at 25 °C, UV detection at 340 nm). The retention times for synthetic
(R)- and (S)-N,O-dimethylserinol (4) derivatives were 82.4 and 85.1
min, respectively, whereas the retention times for derivatized
hydrolysates from columbamide F−H (1−3) were 82.4, 83.0, and
83.1 min, respectively.
Ohrui’s Reaction and Chiral-Phase HPLC Analysis. To a
solution of synthetic 5-(10R,20R) (4.9 mg, 0.0108 mmol) in THF
(0.30 mL) were added NMO (50.0% in H2O, 3.3 μL, 0.0162 mmol)
and OsO4 (0.020 M in H2O, 108 μL, 0.00216 mmol) at room
temperature. The mixture was stirred for 20 h, quenched with
saturated aqueous Na2SO3, and extracted with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. To a solution of crude diol
7 in CH2Cl2 (0.50 mL) was added NaIO4/SiO2 (50.0 mg), at rt.28
The mixture was stirred for 30 min, then filtered through a pad of
Celite (elution with EtOAc), and concentrated in vacuo. The residue
Surface Tension Measurement. The surface tension of sample
̈
solutions was determined by the ring method using a Du Nouy
tensiometer (ITOH, No. 4334) equipped with a platinum ring at 25
°C, and measurements were done in triplicate. Calibration of the
instrument was done by measuring the surface tension of the pure
H2O before each set of experiments. The CMCs for pure compounds
were determined from the breakpoint of the surface tension versus the
log of the bulk concentration curve.29
Cytotoxicity Assay. The cytotoxicities of all compounds were
determined by the method previously reported by Lopez et al.
(2016).30
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c00164.
■
sı
Details of 16S rRNA gene sequencing and phylogenetic
analysis; 1D and 2D NMR and LC-MS spectra of
columbamides F−H (1−3), synthesis of N,O-dimethyl-
serinol standard, as well as chiral-phase HPLC
chromatograms (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Tatsufumi Okino − Graduate School of Environmental Science
and Faculty of Environmental Earth Science, Hokkaido
E
https://dx.doi.org/10.1021/acs.jnatprod.0c00164
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
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Matsuda, F.; Okino, T. Org. Lett. 2017, 19, 4231−4234.
University, Sapporo 060-0810, Japan; orcid.org/0000-
0002-8363-0467; Phone: +81 11 706 4519; Email: okino@
ees.hokudai.ac.jp; Fax: +81 11 706 4867
̌
́
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Liang Wei − Graduate School of Environmental Science,
Hokkaido University, Sapporo 060-0810, Japan
Julie G. Petitbois − Graduate School of Environmental Science,
Hokkaido University, Sapporo 060-0810, Japan
Taiki Umezawa − Graduate School of Environmental Science
and Faculty of Environmental Earth Science, Hokkaido
University, Sapporo 060-0810, Japan; orcid.org/0000-
0003-4280-6574
Fuyuhiko Matsuda − Graduate School of Environmental Science
and Faculty of Environmental Earth Science, Hokkaido
University, Sapporo 060-0810, Japan
Charles S. Vairappan − Institute for Tropical Biology and
Conservation, Universiti Malaysia Sabah, Kota Kinabalu
88450, Sabah, Malaysia
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Masaaki Morikawa − Graduate School of Environmental
Science and Faculty of Environmental Earth Science, Hokkaido
University, Sapporo 060-0810, Japan
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W. H. Lipids 2014, 49, 1127−1132.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00164
́
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Notes
The authors declare no competing financial interest.
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2019, 21, 1444−1448.
ACKNOWLEDGMENTS
■
(26) Samith, V. D.; Mino, G.; Ramos-Moore, E.; Arancibia-
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J.J.M. is grateful to the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) for the Japanese Govern-
ment Scholarship program. We are thankful to Dr. Y. Kumaki
of the High-Resolution NMR Laboratory, Hokkaido Univer-
sity, for the NMR analyses. We also thank Dr. M. Kurasaki of
the Faculty of Environmental Earth Science, Hokkaido
University, for the use of cell culture facilities. This work was
supported by JSPS KAKENHI Grant Number-16H04975. We
would also like to acknowledge the research permit given by
Sabah Biodiversity Center to the corresponding author to
conduct the research in Sabah (permit no. JKM/MBS.1000-2/
3 JLD.3 (28)).
Miranda, N. J. Appl. Polym. Sci. 2013, 138, 2159−2164.
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