Determination of the Absolute Configuration of a Monoglyceride Antibolting Compound and Isolation of Related Compounds from Radish Leaves (Raphanus sativus)

Article abstract of DOI:10.1021/acs.jnatprod.6b00746

A monoglyceride (1) has been reported to possess an antibolting effect in radish (Raphanus sativus), but its absolute configuration at the C-2 position was not determined earlier. In this work, the absolute configuration of 1 was determined to be (2S), and it was also accompanied by one new (2) and two known monoglycerides (3 and 4). The chemical structure of 2 was determined as β-(7′Z,10′Z,13′Z)-hexadecatrienoic acid monoglyceride (β-16:3 monoglyceride). Qualitative and quantitative analytical methods for compounds 1-4 were developed, using two deuterium-labeled compounds (8 and 9) as internal standards. The results revealed a broader range of distribution of 1-4 in several annual winter crops. It was also found that these isolated compounds have an inhibitory effect on the root elongation of Arabidopsis thaliana seedlings at concentrations of 25 and 50 μM in the medium. However, the inhibitory effect of 1 was not dependent on coronatin-insensitive 1 (COI1) protein, which may suggest the involvement of an unidentified signaling system other than jasmonic acid signaling.

Full text of DOI:10.1021/acs.jnatprod.6b00746

Article
pubs.acs.org/jnp
Determination of the Absolute Configuration of a Monoglyceride
Antibolting Compound and Isolation of Related Compounds from
Radish Leaves (Raphanus sativus)
Tsuyoshi Ogihara,† Naruki Amano,† Yuki Mitsui,‡ Kaien Fujino,† Hiroyuki Ohta,§ Kosaku Takahashi,†
and Hideyuki Matsuura*,†
†Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
‡Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
§School of Life Science and Technology, Tokyo Institute of Technology, 4259-B65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501,
Japan
S
* Supporting Information
ABSTRACT: A monoglyceride (1) has been reported to possess an antibolting effect in radish (Raphanus sativus), but its
absolute configuration at the C-2 position was not determined earlier. In this work, the absolute configuration of 1 was
determined to be (2S), and it was also accompanied by one new (2) and two known monoglycerides (3 and 4). The chemical
structure of 2 was determined as β-(7′Z,10′Z,13′Z)-hexadecatrienoic acid monoglyceride (β-16:3 monoglyceride). Qualitative
and quantitative analytical methods for compounds 1−4 were developed, using two deuterium-labeled compounds (8 and 9) as
internal standards. The results revealed a broader range of distribution of 1−4 in several annual winter crops. It was also found
that these isolated compounds have an inhibitory effect on the root elongation of Arabidopsis thaliana seedlings at concentrations
of 25 and 50 μM in the medium. However, the inhibitory effect of 1 was not dependent on coronatin-insensitive 1 (COI1)
protein, which may suggest the involvement of an unidentified signaling system other than jasmonic acid signaling.
summer; (ii) the plants develop biomass in the rosette form to
lants respond to many environmental factors, such as
Ptemperature, the length of day, and availability or excess of transport assimilated products into the storage organs and
survive the winter by leaving the storage organs underground;
and (iii) in the spring, the plants develop initial stems that are
elongated to bear flowers at the top of the bolted stem. These
flowers attract pollinators and then scatter the seeds in the
summer. To perform the above-mentioned life cycle, plants
must appropriately control growing and flowering times to
ensure the formation of the next generation. Thus, biological
regulation to inhibit bolting is a pivotal step for the plant to
complete its life cycle, and extensive work has been conducted
to uncover the mechanism of this process. In many cold-
requiring LD plants, the exogenous application of gibberellic
acid (GA) under short-day conditions induces bolting.
water. In the autumn, radish (Raphanus sativus L.; Brassica-
ceae), spinach (Spinacia oleracea), beet (Beta vulgaris), and
carrot (Daucus carota) grow leaves on a dwarf stem, at a level
close to the ground. These structures are called rosettes. They
accumulate their assimilated products in a thick root under
short-day (SD) conditions and are categorized as cold-requiring
long-day (LD) plants because a certain chilling period in the
winter season is needed for initial stem growth. This effect
occurs in response to the LD conditions of the next spring, in
which the plant finally bears flowers on an elongated stem. This
biological phenomenon, in which exposure to cold conditions is
required for developing the initial stem from the storage organ,
is known as vernalization,1 and the elongation is known as
bolting.2,3 Thus, the life cycle of the cold-requiring LD plants
can be summarized as follows: (i) the seeds germinate in the
Received: August 12, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chailakh4 noted that the endogenous amount of GA in rosette
plants under SD conditions is relatively low, and LD conditions
should raise the level of endogenous GA to stimulate bolting.
The exogenous application of inhibitor upon GA biosynthesis
has led to adverse biological effects.5,6 Yoshida et al.7 predicted
the existence of an anti-GA compound, or in other words, an
antibolting compound (1), which is synthesized under SD
conditions to inhibit stem elongation. They isolated α-
(7′Z,10′Z,13′Z)-hexadecatrienoic acid monoglyceride (α-16:3
monoglyceride) (1) as the antibolting compound. However,
they determined neither its absolute configuration nor its anti-
GA activity. In this contribution, the absolute configuration of
compound 1 is reported together with isolation of one new (2)
and two known (3 and 4) monoglycerides from leaves of the
radish (R. sativus). Also, qualitative and quantitative analytical
methods were determined for compounds 1−4, and their
biological activities were evaluated against GA.
naturally derived 1 (10.8 mg) was isolated from radish (R.
sativus) leaves (Figure S1, Supporting Information). Addition-
ally, three monoglycerides (2−4) were also isolated, which had
retention times close to 1, as shown by the corresponding
features in the HPLC chromatogram (Figure S2, Supporting
Information).
The isolated natural compound 1 showed an optical rotation
value of [α]26 +2.4 (c 0.82, MeOH). (2S)- and (2R)-1 were
D
synthesized according to a previously reported method,9 with
the exception that 5 was used as the fatty acid moiety for
conjugation with (R)-2,2-dimethyl-1,3-dioxolane-4-methanol
(6, Scheme 1) and (S)-2,2-dimethyl-1,3-dioxolane-4-methanol
(7, Scheme S1, Supporting Information). The optical rotation
values of (2S)-α-(7′Z,10′Z,13′Z)-hexadecatrienoic acid mono-
glyceride (1a, Scheme 1) and (2R)-α-(7′Z,10′Z,13′Z)-
hexadecatrienoic acid monoglyceride (1b, Scheme S1, Support-
ing Information) were measured as [α]26 +4.4 (c 0.75,
D
MeOH) and −4.5 (c 0.75, MeOH), respectively, which
revealed that the absolute configuration of the C-2 position
of natural 1 might be in the S configuration. However, it was
considered that natural 1 occurs in a racemic mixture to result
into its lower optical rotation than that of the synthetic
compound 1a. In order to check the possibility that naturally
derived 1 was obtained as a racemic mixture, the generation of
(S)-MTPA esters composed of naturally derived 1, 1a, and 1b
(Figures S3−S5, Supporting Information) was conducted to
evaluate the optical purity. It was observed that there were
distinct differences in the resonances of 1H NMR spectra
between the (S)-MTPA esters of 1a and 1b, and the resonances
of the 1H NMR spectrum of the (S)-MTPA ester of 1
contained signals corresponding to the (S)-MTPA esters of 1a
and 1b (Figure S6, Supporting Information). It was revealed
that naturally derived 1 was a racemic mixture having an optical
purity of 55%.
Compounds 2 (8.7 mg), 3 (21 mg), and 4 (6.9 mg) were
isolated from an EtOH extract of radish leaves (2 kg).
Compound 2 was isolated as a colorless oil, and its elemental
formula, as determined by HRFDMS (Figure S7, Supporting
Information), was found to be C19H32O4, indicating four
RESULTS AND DISCUSSION
■
First, the optically active antibolting compound 1 was obtained,
using a synthetic compound as a standard for the purification
steps. The fatty acid moiety of the standard compound,
(7′Z,10′Z,13′Z)-hexadeca-7,10,13-trienoic acid (5), was syn-
thesized according to a reported method,8 but having 6-
bromohexan-1-ol as a starting material. Then, compound 5 was
coupled with an excess amount of propane-1,2,3-triol (6) to
give synthetic 1 in the racemic mixture form. Subsequently, the
1
double-bond equivalents. The H NMR spectrum (Figure S8,
Supporting Information) showed a signal for one methyl group
at δH 0.95 (3H, t, J = 7.6 Hz), six olefinic protons at δH 5.42−
5.24 (6H, m), and five alkyl protons of glycerol at δH 3.81 (4H,
d, J = 7.6 Hz) and 4.90 (1H, quintet, J = 4.6 Hz). One methyl
Scheme 1. Synthesis of (2S)-α-(7′Z,10′Z,13′Z)-Hexadecatrienoic Acid Monoglyceride (1a)
B
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carbon, three downfield oxygen-connected carbons including
one methine and two equivalent methylene carbons, seven sp2
carbons including one carbonyl group, and eight methylene
carbons were observed in the 13C NMR and DEPT spectra.
Also observed were eight methylenes, six olefinic carbons, and
one methyl carbon that showed an ω-3 fatty acid structure,
which was supported by the data from the DEPT and HSQC
experiments (Figures S9−S11, Supporting Information). The
key correlations between carbons and protons (J2,3) in the
HMBC (Figure S12, Supporting Information) revealed an
attachment between the carbonyl carbon and the primary
carbon of glycerol. Thus, the structure of 2 was determined as
β-(7′Z,10′Z,13′Z)-hexadecatrienoic acid monoglyceride (β-
16:3 monoglyceride), as shown in Figure 1, and this was
was not determined earlier. To determine the absolute
configuration, (2S)-α-(9′Z,12′Z,15′Z)-octadecatrienoic acid
monoglyceride (3a, Scheme S2, Supporting Information) and
(2R)-α-(9′Z,12′Z,15′Z)-octadecatrienoic acid monoglyceride
(3b, Scheme S3, Supporting Information) were synthesized
according to the above-mentioned method9 and showed optical
rotation values of [α]26 +3.8 (c 0.42, MeOH) and −3.9 (c
D
0.19, MeOH), respectively. Since naturally occurring 3
exhibited a reported optical rotation of [α]26 +2.8 (c 1.5,
D
MeOH), the absolute configuration of the C-2 position of 3
was determined to be S, having an optical purity of 74%, as
determined using the same procedure as compound 1 (Figures
S14−S17, Supporting Information).
To uncover the biological roles of 1−4, methods for their
qualitative and quantitative analysis were required. Deuterium-
labeled compounds 8 and 9 were synthesized using [2H2-1,2H1-
2,2H2-3]propane-1,2,3-triol. The ionic optimizations for each
compound and UPLC conditions to separate the compounds
were established, although complete separations between 1 and
2 as well as between 3 and 4 were not performed (Figure 2).
This method was applied to reveal a more widely ranging
distribution of 1−4 among several winter plants (Figure 3).
The results also revealed that the endogenous amount of each
compound was dependent on the variety of the plant.
Figure 1. HMBC correlations for compound 2.
It is generally accepted that gibberellic acid promotes bolting
in plants. Thus, a possible biological role of 1−4 could result
from an antagonistic effect on GA. Since the biological
importance of GA for developing roots was reported,10 the
inhibitory activities of root elongation using Arabidopsis
thaliana have been examined, and the results are shown in
Figure 4. The application of compounds 1−4 at a concentration
of 25 μM showed inhibitory effects against the root growth of
A. thaliana seedlings, in which 1 showed the highest inhibitory
effect, causing significant differences in root elongation (Figure
4A). Differences in the biological activity due to the absolute
substantiated by analysis of the COSY data (Figure S13,
Supporting Information). Assignments of the 1H and 13C NMR
data of 2 are given in Table 1 together with those of 1 for
comparison.
Compounds 3 and 4 were identified as α-(9′Z,12′Z,15′Z)-
octadecatrienoic acid monoglyceride (α-18:3 monoglyceride)
and β-(9′Z,12′Z,15′Z)-octadecatrienoic acid monoglyceride (β-
18:3 monoglyceride), respectively, due to good agreement with
the spectroscopic data reported.9 However, the absolute
configuration of the C-2 position of the glycerol moiety in 3
Table 1. NMR Spectroscopic Data of Compounds 1 and 2 (500 MHz, CDCl3)
1
2
position
1
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
a
a
65.7, CH2
4.16, dd (11.6, 5.9)
4.11, dd (11.6, 5.9)
3.91, qui (4.9)
62.6, CH2
3.81, d (4.6)
2
3
70.4, CH
63.5, CH2
75.2, CH
62.6, CH2
4.90, tt (4.6, 4.6)
3.81, d (4.6)
3.69, dd (11.6, 5.4)
3.59, dd (11.6, 5.4)
1′
174.4, C
174.1, C
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
13′
14′
15′
16′
34.2, CH2
25.0, CH2
28.9, CH2
29.4, CH2
27.2, CH2
130.1, CH
127.2, CH
25.8, CH2
128.5, CHc
128.3, CHc
25.7, CH2
128.2, CH
132.2, CH
20.7, CH2
14.4, CH3
2.44, t (7.6)
2.03, t (7.6)
1.33, m
34.4, CH2
25.0, CH2
28.9, CH2
29.4, CH2
27.1, CH2
130.0, CH
127.2, CH
25.8, CH2
128.5, CHe
128.3, CHe
25.7, CH2
128.2, CH
132.2, CH
20.6, CH2
14.4, CH3
2.37, t (7.3)
1.63, qua (7.3)
1.35, t (3.2)
1.35, t (3.2)
2.05, qua (6.5)
5.42−5.24, m
5.42−5.24, m
2.78, t (5.7)
5.42−5.24, m
5.42−5.24, m
2.78, t (5.7)
5.42−5.24, m
5.42−5.24, m
2.05, qua (6.5)
0.95, t (7.6)
1.33, m
2.07, qua (7.6)
5.38−5.30, m
5.38−5.30, m
2.78, t (5.9)
5.38−5.30, m
5.38−5.30, m
2.78, t (5.9)
5.38−5.30, m
5.38−5.30, m
2.07, qua (7.6)
0.95, t (7.6)
b
b
d
d
a−e
Interchangeable in each symbol.
C
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reasonable to hypothesize that 1a and 3a may be able to
produce (7Z,10Z,13Z)-hexadecatrienoic acid and α-linolenic
acid, respectively, resulting in the generation of jasmonic acid
and jasmonoyl isoleucine (JA-Ile), which have shown biological
activities via coronatin-insensitive 1 (COI1) protein.13−16
(−)-JA-L-Ile (25 μM) and 1a (25 μM) were applied to coi1-
16s A. thaliana seedlings that had received a disrupted
coronatin-insensitive 1 gene,17 and the resulting activities were
observed, which revealed that 1a showed biological activity
independent of COI1, but (−)-JA-L-Ile needed COI1 to show
activity (Figure 4D). Several investigators have used (−)-JA-L-
Ile at a 50 μM concentration to check its inhibitory effect on
A. thaliana seedlings, and it was determined that compound 1
and its related analogues 2−4 exhibited activity at 25 and 50
μM concentrations. Thus, it might be possible to categorize
these compounds as representative of a new type of plant
growth inhibitor, such as those that act by regulating GA
activity. Further studies should be performed, such as those
examining the organ distribution, biosynthetic pathways, and
transportation of the compounds as well as target proteins to
suppress GA activity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
obtained with a JASCO P-2200 polarimeter. NMR spectra were
recorded in CDCl3 and benzene-d6, using a JNM-EX 270 FT-NMR
spectrometer (JEOL) [1H NMR: 270 MHz, 13C NMR: 67.5 MHz]
and AMX 500 (Bruker) [1H NMR: 500 MHz, 13C NMR: 126 MHz].
FDMS and FIMS analyses were performed on a JMS-T100GCV
(JEOL) instrument, and CIMS was performed on a JMS-SX102A
(JEOL) instrument. Ultraperformance liquid chromatography
(UPLC) was performed on a Waters ACQUITY UPLC system
(Waters), which was equipped with a binary solvent delivery manager
and a sample manager. MS was performed on a Waters Micromass
Quattro Premier Tandem Quadrupole mass spectrometer or a Waters
Figure 2. Representative UPLC MS/MS features of the detection of
1−4 derived from radish. Compounds 8 and 9 represent the
deuterium-labeled compounds of 1 and 3 (internal standard).
configuration at the C-2 position in the monoglyceride moiety
were examined, and it was found that the absolute configuration
of the compounds did not change the inhibitory effect (Figure
4B and C). It has been reported that jasmonic acid (JA)
possesses antibolting and anti-GA activities,11,12 and thus, it is
Figure 3. Broader range of distribution of 1−4 in annual winter plants. (a) Carrot seeds were sown, and the plants grown in a greenhouse for 3
weeks in early May. (b) Wild plants were harvested in early April in Sapporo, Hokkaido. (c and d) Plants were purchased in a marketplace. (e) Seeds
of A. thaliana were sown, and plants were grown in a greenhouse for 3 weeks in early May. The leaves were harvested and extracted for analysis of
1−4 using UPLC MS/MS. n.d.: not detected.
D
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Figure 4. Inhibitory effects of 1−4 on root growth of A. thaliana.
LCT-Premier Time of Flight (Tof) mass spectrometer. UPLC/MS
system control was by MassLynx 4.0.
(2S)-α-(9′Z,12′Z,15′Z)-Octadecatrienoic acid monoglyceride (3):
[α]27D +2.8 (c 1.5, MeOH); 1H NMR (270 MHz, CDCl3) δ 5.39−5.29
(6H, m), 4.18 (1H, dd, J = 11.6, 5.9 Hz), 4.11 (1H, dd, J = 11.6, 5.9
Hz), 3.91 (1H, quintet, 4.6), 3.63 (2H, ddd, J = 27, 11.6, 5.9 Hz), 2.79
(4H, t, J = 5.9 Hz), 2.33 (2H, t, J = 7.6 Hz), 2.04 (4H, m), 1.61 (2H, t,
J = 6.8 Hz), 1.29 (10H, m), 0.95 (3H, t, J = 7.6 Hz); 13C NMR (67.8
MHz, CDCl3) δ 174.4, 132.1, 130.4, 128.4, 128.4, 127.9, 127.3, 70.4,
65.3, 63.9, 34.3, 29.7, 29.3, 29.2, 27.3, 25.8, 25.7, 25.0, 20.7, 14.4;
FDMS m/z 352 [M]+ (100), 353 [M + H]+ (29.5); HRFDMS m/z
352.25962 (calcd for C21H36O4, 352.26136).
Plant Material. The leaves of Raphanus sativus L. (Brassicaceae)
cv. Kitano Haruichi Daikon radish seeds were purchased from Daigaku
Noen Co., Ltd., Japan. The radish cultivar was grown in a greenhouse
of the Faculty of Agriculture, Hokkaido University, Hokkaido, Japan,
in June 2015. The sowing date was May 2, and the leaves were
harvested approximately 40 days after sowing. The leaves of Daucus
carota subsp. sativus cv. Kohyoh Nigoh carrot seeds were purchased
from Takii & Co., Ltd., Japan. The carrot cultivar was grown in a
greenhouse of the Faculty of Agriculture, Hokkaido University,
Hokkaido, Japan, in May 2016. The sowing date was April 25, and
the leaves were harvested approximately 3 weeks after sowing. The
leaves of turnip (Brassica rapa subsp. rapa) and spinach (Spinacia
oleracea) were purchased in a supermarket in Hokkaido, Japan, in April
2016. The upper plant of Taraxacum officinale was collected from
Hokkaido University in Hokkaido, Japan, in April 2016. The plant was
identified by one of the authors (H.M.) at the Laboratory of Natural
Product Chemistry, Japan, and a voucher specimen (CK.E. 13712) was
deposited at the Laboratory of Natural Product Chemistry.
Extraction and Isolation. The harvested leaves (2 kg) were
extracted with EtOH (5 L) and then partitioned between EtOAc (500
mL) and H2O (500 mL). The EtOAc layer was washed with 1 M HCl
(500 mL) and saturated aqueous NaHCO3 (500 mL), and the organic
layer was dried over MgSO4 and concentrated under reduced pressure
to give a residue, which was purified by silica gel flash chromatography
with EtOAc−n-hexane (3:2), Sephadex LH-20 MeOH−CHCl3 (1:1),
and HPLC [YMC Pack ODS-AM, ⦶ 10 × 300 mm, 2 mL/min, 210
nm, MeOH−H2O (9:1)] to afford 1 (10.8 mg), 2 (8.7 mg), 3 (21
mg), and 4 (6.9 mg).
β-(9′Z,12′Z,15′Z)-Octadecatrienoic acid monoglyceride (4): 1H
NMR (270 MHz, CDCl3) δ 5.42−5.25 (6H, m), 4.91 (1H, quintet, J =
4.9 Hz), 3.81 (4H, t, J = 4.6 Hz), 2.79 (4H, t, J = 5.4 Hz), 2.36 (2H, t,
J = 7.6 Hz), 2.15−2.03 (4H, m), 1.62 (2H, m), 1.29 (8H, m), 0.96
(3H, t, J = 7.6 Hz); 13C NMR (67.8 MHz, CDCl3) δ 174.2, 132.1,
130.4, 128.5, 128.4, 127.9, 127.3, 75.2, 62.7, 34.5, 29.7, 29.3, 29.22,
29.21, 27.3, 25.8, 25.9, 25.1, 20.7, 14.4; FDMS m/z 352 [M]+ (100),
353 [M + H]+ (29.9); HRFDMS m/z 352.25977 (calcd for C21H36O4,
352.26136).
UPLC Tof MS and MS/MS Conditions. The conditions were set
according to a previously reported method,18 except for multiple
reaction monitoring (MRM) optimization, which was set up to detect
1−4 and for which the conditions are shown in Table S1, Supporting
Information.
UPLC Conditions for MS/MS MRM. The UPLC separation was
performed on a Waters ACQUITY ethylene-bridged (BEH) C18
column (1.7 μm, 2.1 × 100 mm) at 38 °C. The UPLC system was
coupled to a Waters Micromass Quattro Premier tandem quadrupole
mass spectrometer. The analysis samples were eluted from the column
with a mixed solvent of 20% aqueous MeOH with 0.05% AcOH
(solvent A) and MeOH with 0.05% AcOH (solvent B), using the
linear gradient mode. To examine 1−4, the proportion of A and B was
30:70 from 0 s to 0.2 min, and from 0.2 to 4 min, the proportion of A
and B was linearly changed from 30:70 to 0:100. The proportion of
0:100 was maintained from 4 to 5.5 min.
(2S)-α-(7′Z,10′Z,13′Z)-Hexadecatrienoic acid monoglyceride (1):
1
[α]26 +2.4 (c 0.82, MeOH); H NMR (270 MHz, CDCl3) and 13C
D
NMR (67.8 MHz, CDCl3) see Table 1; FDMS m/z 324 [M]+ (100),
325 [M + H]+ (34.9); HRFDMS m/z 324.22859 (calcd for C19H32O4,
324.23006).
β-(7′Z,10′Z,13′Z)-Hexadecatrienoic acid monoglyceride (2): 1H
NMR (270 MHz, CDCl3) and 13C NMR (67.8 MHz, CDCl3) see
Table 1; FDMS m/z 324 [M]+ (100), 325 [M + H]+ (32.1);
HRFDMS m/z 324.22860 (calcd for C19H32O4, 324.23006).
Analysis of Endogenous Amounts of Compounds 1−4. Plant
material (2−3 g) was frozen using liquid nitrogen immediately after
harvest and then crushed and soaked for 24 h using a solution of
EtOH (20 mL) together with the addition of 8 (10 μg/sample) and 9
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(10 μg/sample). The mixture was filtered to give a crude extract, and
the volatile components of the extract were removed under reduced
pressure to give a residue, which was mixed with a solution of EtOAc
(10 mL). The mixture was successively washed with 0.5 M HCl (10
mL) and saturated aqueous NaHCO3 (10 mL), and the organic layer
was dried over Na2SO4. The volatile components of the organic layer
were removed under reduced pressure to give a residue, which was
mixed with a solution of EtOAc−n-hexane (3:2, 1 mL). The mixture
was loaded on a silica gel column, which was eluted into sample tubes
(20 mL each) using a solution of EtOAc−n-hexane (3:2, 100 mL).
The eluates in the second to fourth fractions were mixed, and their
volatile components were removed in vacuo to give a residue. The
residue was dissolved in a mixture of MeOH−H2O (4:1, 100 μL), and
a portion of the solution (5 μL) was subjected to UPLC MS/MS.
Synthesis of Compound 5. The synthesis of 5 was conducted
according to a previously reported method,19 with the exception that
6-bromohexan-1-ol was used as a starting material.
7.9 Hz), 3.96 (1H, d, J = 2.8 Hz), 3.91−3.74 (2H, m), 3.58 (1H, s),
3.41 (3H, s), 2.86 (4H, d, J = 15.8 Hz), 2.13−1.95 (6H, m), 1.49 (2H,
d, J = 6.6 Hz), 1.45−1.07 (23H, m), 0.91 (5H, t, J = 7.4 Hz); UPLC-
Tof MS m/z 591.2933 [M + Na]+ (calcd for C31H43O6F3Na,
591.2909).
MTPA ester of 3a: 1H NMR (500 MHz, benzene-d6) δ 7.68 (2H, d,
J = 7.6 Hz), 7.13−6.96 (3H, m), 5.57−5.34 (6H, m), 4.06 (1H, dd, J =
11.3, 6.6 Hz), 3.96 (1H, dd, J = 11.3, 4.1 Hz), 3.82 (ddd, J = 28.6, 11.7,
4.8 Hz), 3.57 (1H, d, J = 4.7 Hz), 3.41 (3H, s), 2.95−2.77 (4H, m),
2.14−1.93 (6H, m), 1.66 (1H, d, J = 3.8 Hz), 1.50 (2H, t, J = 6.8 Hz),
1.41−1.05 (9H, m), 0.91 (3H, t, J = 7.6 Hz); UPLC-Tof MS m/z
591.2896 [M + Na]+ (calcd for C31H43O6F3Na, 591.2909).
MTPA ester of 3b: 1H NMR (500 MHz, benzene-d6) δ 7.68 (2H, d,
J = 7.6 Hz), 7.13−6.96 (3H, m), 5.55−5.36 (6H, m), 4.05 (1H, dd, J =
11.0, 3.5 Hz), 3.97 (1H, dd, J = 11.5, 5.5 Hz), 3.92−3.77 (2H, m), 3.59
(1H, s), 3.41 (3H, s), 2.95−2.78 (4H, m), 2.10−1.97 (6H, m), 1.65
(1H, s), 1.50 (2H, t, J = 6.8 Hz), 1.44−1.06 (17H, m), 0.91 (3H, t, J =
7.4 Hz); UPLC-Tof MS m/z 591.2890 [M + Na]+ (calcd for
C31H43O6F3Na, 591.2909).
(7Z,10Z,13Z)-Hexadecatrienoic acid (5): 1H NMR (270 MHz,
CDCl3) δ 5.35 (6H, m), 2.78 (4H, t, J = 5.4 Hz), 2.33 (2H, t, J = 8.1
Hz), 2.05 (5H, m), 1.63 (3H, m), 1.50−1.20 (8H, m), 0.96 (3H, t, J =
8.1 Hz); FDMS m/z 250 [M]+ (100).
Synthesis of Deuterium-Labeled Compounds 8 and 9.
Compounds 5 (24.3 mg, 0.1 mmol) and α-linolenic acid (26.0 mg,
0.1 mmol) were prepared. A fatty acid in CH2Cl2 (5 mL) was added to
a stirred mixture of N,N′-dicyclohexylcarbodiimide (DCC, 34.0 mg,
0.17 mmol), N,N-dimethyl-4-aminopyridine (DMAP, 3.7 mg, 0.03
mmol), and glycerol-1,1,2,3,3-d5 (28.3 mg, Sigma-Aldrich, St. Louis,
MO, USA), and the reaction mixture was further stirred at room
temperature for 24 h. The usual workup was employed to give a crude
material, which was purified using silica gel column chromatography
with EtOAc−n-hexane (3:2) and HPLC [YMC Pack ODS-AM, f 10 ×
300 mm, 2 mL/min, 210 nm, MeOH−H2O (9:1)] to afford
compounds 8 (4.2 mg, 0.013 mmol) and 9 (8.0 mg, 0.022 mmol).
[2H2-1,2H1-2,2H2-3]-α-(7′Z,10′Z,13′Z)-Hexadecatrienoic acid
monoglyceride (8): HRFDMS m/z 329.26269 (calcd for
C19H27D5O4, 329.26144).
General Procedure for the Synthesis of Compounds 1a, 1b,
3a, and 3b. They were synthesized according to a reported method.20
(2S)-α-(7′Z,10′Z,13′Z)-Hexadecatrienoic acid monoglyceride
(1a): [α]26 +4.4 (c 0.75, MeOH); HRFDMS m/z 324.23118 (calcd
D
for C19H32O4, 324.23006).
(2R)-α-(7′Z,10′Z,13′Z)-Hexadecatrienoic acid monoglyceride
(1b): [α]26 −4.5 (c 0.75, MeOH); FDMS m/z 324 [M]+ (100),
D
325 [M + H]+ (30.6); HRFDMS m/z 324.23175 (calcd for C19H32O4,
324.23006).
(2S)-α-(9′Z,12′Z,15′Z)-Octadecatrienoic acid monoglyceride
(3a): [α]27 +3.8 (c 0.42, MeOH); HRFDMS m/z 352.26028 (calcd
D
for C21H36O4, 352.26136).
(2R)-α-(9′Z,12′Z,15′Z)-Octadecatrienoic acid monoglyceride
(3b): [α]27 −3.9 (c 0.19, MeOH); HRFDMS m/z 352.26039
[2H2-1,2H1-2,2H2-3]-α-(9′Z,12′Z,15′Z)-Octadecatrienoic acid
monoglyceride (9): HRFDMS m/z 357.29315 (calcd for
C21H31D5O4, 357.29274).
D
(calcd for C21H36O4, 352.26136).
General Procedure of MTPA Esterification. A solution of (S)-
MTPACl (10 mg, 0.04 mmol) in dry pyridine (0.4 mL) was added to
1 (7.0 mg) in a two-necked flask, and the reaction mixture was stirred
at room temperature for 16 h. The reaction mixture was washed with 1
M HCl (30 mL), saturated aqueous NaHCO3 (30 mL), and brine (30
mL), and the organic layer was dried over Na2SO4. The volatile
components of the organic layer were removed under reduced
pressure to give a residue. The residue was purified by silica gel
column chromatography with EtOAc−n-hexane (1:3) to afford the
corresponding product.
Root Growth Inhibition Assay. The root growth inhibitory test
was performed according to a reported method.21
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00746.
■
S
MTPA ester of 1: 1H NMR (500 MHz, benzene-d6) δ 7.68 (2H, d, J
= 7.9 Hz), 7.14−6.97 (3H, m), 5.55−5.32 (6H, m), 4.13−4.02 (1H,
m), 4.02−3.93 (1H, m), 3.92−3.75 (2H, m), 3.59 (1H, d, J = 4.4 Hz),
3.41 (3H, s), 2.85 (4H, t, J = 5.4 Hz), 2.10−1.92 (6H, m), 1.69 (1H,
s), 1.57−1.43 (2H, m), 1.43−1.11 (12H, m), 0.92 (5H, t, J = 7.6 Hz);
UPLC-Tof MS m/z 563.2570 [M + Na]+ (calcd for C29H39O6F3Na,
563.2569).
Representative HPLC features for purification and
representative UPLC MS/MS features for compounds
1−4; FDMS, 1H NMR, 13C NMR, DEPT, COSY,
1
HSQC, and HMBC spectra for 2; H NMR spectra for
each (S)-MTPA ester; synthetic Schemes S1−S3 for 1b,
3a, 3b; and conditions of MS optimization for multiple
reaction monitoring in the positive mode for compounds
1, 3, 8, and 9 (PDF)
1
MTPA ester of 1a: H NMR (500 MHz, benzene-d6) δ 7.67 (2H,
d), 7.24−6.94 (3H, m), 5.55−5.31 (6H, m), 4.07 (1H, dd, J = 11.3, 6.6
Hz), 3.96 (1H, dd, J = 11.3, 4.1 Hz), 3.85 (1H, dd, J = 11.7, 4.4 Hz),
3.79 (1H, dd, J = 11.8, 5.8 Hz), 3.59 (1H, d, J = 5.0 Hz), 3.41 (3H, s),
2.85 (4H, t, J = 5.4 Hz), 2.07−1.95 (6H, m), 1.72 (1H, d, J = 4.7 Hz),
1.57−1.40 (2H, m), 1.38−1.10 (5H, m), 0.91 (3H, t, J = 7.6 Hz);
UPLC-Tof MS m/z 563.2649 [M + Na]+ (calcd for C29H39O6F3Na,
563.2569).
AUTHOR INFORMATION
Corresponding Author
*Tel: + 81 11 706 2495. Fax: + 81 11 706 2505. E-mail:
matsuura@chem.agr.hokudai.ac.jp.
■
MTPA ester of 1b: 1H NMR (500 MHz, benzene-d6) δ 7.68 (2H, d,
J = 7.6 Hz), 7.07 (3H, dt, J = 31.3, 7.4 Hz), 5.51−5.36 (6H, m), 4.13−
4.03 (1H, m), 4.02−3.94 (1H, m), 3.91−3.77 (2H, m), 3.60 (1H, s),
3.41 (3H, s), 2.85 (4H, t, J = 5.4 Hz), 2.08−1.96 (6H, m), 1.69 (1H,
s), 1.54−1.43 (2H, m), 1.41−1.11 (10H, m), 0.92 (3H, t, J = 7.6 Hz);
UPLC-Tof MS m/z 563.2645 [M + Na]+ (calcd for C29H39O6F3Na,
563.2569).
ORCID
Hideyuki Matsuura: 0000-0002-9605-7188
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank Mr. Y. Takata and Dr. E.
Fukushi (Faculty of Agriculture, Hokkaido University) for
■
MTPA ester of 3: 1H NMR (500 MHz, benzene-d6) δ 7.67 (2H, d, J
= 7.3 Hz), 7.12−6.95 (3H, m), 5.55−5.36 (6H, m), 4.05 (1H, d, J =
F
DOI: 10.1021/acs.jnatprod.6b00746
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
assistance in obtaining the spectroscopic data. We would also
like to thank the Research Faculty of Agriculture, Hokkaido
University, for the use of the UPLC MS/MS system.
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