Structure-Activity Relationship Study on Isothiocyanates: Comparison of TRPA1-Activating Ability between Allyl Isothiocyanate and Specific Flavor Components of Wasabi, Horseradish, and White Mustard

Article abstract of DOI:10.1021/acs.jnatprod.5b00272

Allyl isothiocyanate (ITC) (4) is the main pungent component in wasabi, and it generates an acrid sensation by activating TRPA1. The flavor and pungency of ITCs vary depending on the compound. However, the differences in activity to activate TRPA1 between ITCs are not known except for a few compounds. To investigate the effect of carbon chain length and substituents of ITCs, the TRPA1-activiting ability of 16 ITCs was measured. Since most of the ITCs showed nearly equal TRPA1-activiting potency, the ITC moiety is likely the predominant contributor to their TRPA1-activating abilities, and contributions of other functional groups to their activities to activate TRPA1 are comparatively small. (Figure Presented).

Full text of DOI:10.1021/acs.jnatprod.5b00272

Article
pubs.acs.org/jnp
Structure−Activity Relationship Study on Isothiocyanates:
Comparison of TRPA1-Activating Ability between Allyl
Isothiocyanate and Specific Flavor Components of Wasabi,
Horseradish, and White Mustard
†
§
,†
Yuko Terada, Hideki Masuda, and Tatsuo Watanabe*
†Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526,
Japan
§
Maihama Research Center, Ogawa & Company, Ltd., 15-7 Chidori, Urayasu-shi, Chiba 279-0032, Japan
*
S Supporting Information
ABSTRACT: Allyl isothiocyanate (ITC) (4) is the main
pungent component in wasabi, and it generates an acrid
sensation by activating TRPA1. The flavor and pungency of
ITCs vary depending on the compound. However, the
differences in activity to activate TRPA1 between ITCs are
not known except for a few compounds. To investigate the
effect of carbon chain length and substituents of ITCs, the
TRPA1-activiting ability of 16 ITCs was measured. Since most
of the ITCs showed nearly equal TRPA1-activiting potency,
the ITC moiety is likely the predominant contributor to their
TRPA1-activating abilities, and contributions of other functional groups to their activities to activate TRPA1 are comparatively
small.
asabi (Wasabia japonica Matsumura), horseradish
(Cochlearia armoracia L.), and white mustard (Sinapis
Compound 4 induces an acrid sensation by activating
transient receptor potential ankyrin 1 (TRPA1). TRPA1 is a
7
W
alba L.) belong to the Cruciferae family. When tissues of these
nonselective cation channel and is mainly expressed in primary
8
plants are damaged, isothiocyanates (ITCs) are liberated from
sensory neurons. In the beginning, TRPA1 was identified as a
1
8
their precursors, glucosinolates. The characteristic flavors of
cold receptor. However, whether mammalian TRPA1 is a
grated wasabi, horseradish, and white mustard are attributed to
the ITC components.
receptor for noxious cold is highly controversial, as channel
activity by cold was observed by some groups but not by
9
Allyl ITC (4) is the main spicy component of wasabi,
horseradish, and white mustard. It is highly pungent and elicits
a lachrymatory sensation. Alkyl ITCs [isopropyl ITC (1), sec-
butyl ITC (2), and isobutyl ITC (3)] are found in both grated
wasabi and horseradish and have a chemical-like odor. ω-
Alkenyl ITCs [3-butenyl ITC (5), 4-pentenyl ITC (6), 5-
hexenyl ITC (7), and 6-heptenyl ITC (8)] are important for
green notes of wasabi. ω-Methylthioalkyl ITCs [5-methyl-
thiopentyl ITC (14), 6-methylthiohexyl ITC (15), and 7-
methylthioheptyl ITC (16)] give wasabi a radish-like, sweetish
smell. In particular, 15 is an important contributor to the
radish-like odor of wasabi and to the richness in flavor. While 4
is a strong irritant, ω-alkenyl ITCs (5−8) and ω-methyl-
thioalkyl ITCs (14−16) have milder flavors. Benzyl ITC (9)
and phenylethyl ITC (10) are characteristic components of
grated horseradish. Compound 9 has a chemical-like smell, and
others. Recently, it is reported that human TRPA1 is cold
10
insensitive. TRPA1 is activated by pungent compounds, such
as cinnamaldehyde (derived from cinnamon), allicin (found in
8
,11−13
crushed garlic), and piperine (found in pepper).
The
TRPA1-activating ability of 4, 9, 10, and 15 has been
14,15
reported.
Furthermore, it has been shown that 4 activates
capsaicin (CAP) receptor TRP vanilloid (TRPV) 1 at a high
1
4,15
concentration.
TRPA1 and menthol receptor TRP
melastatin (TRPM8) have common agonists, such as menthol
16
and icilin. But it has been shown that 4 and 15 do not have an
11,15
agonist activity on TRPM8.
ITC flavors have been well analyzed, and their odors and
pungencies depend on the ITC structure. However, the
differences in a TRPA1-activating ability between ITC
compounds are not clear. To investigate whether the carbon
chain length, benzene ring, and substituents of ITCs affect their
TRPA1-activating potencies, a chemical structure and a
TRPA1-activiting ability relationship study was performed. In
1
0 possesses a strong radish-like flavor. Since the content of 10
in horseradish is relatively high, it is considered an important
contributor to the radish-like flavor of horseradish. p-
Hydroxybenzyl ITC (11), a component specific to white
Received: March 31, 2015
2
−6
mustard, is characterized by its mild pungency.
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00272
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
addition, activities to activate TRPV1 and TRPM8 of the ITCs
were measured. In this study, 16 ITCs derived from wasabi,
horseradish, and white mustard were examined.
Activation of TRPA1 by Compounds 1−16. A 1 μM
concentration of compounds 1−10 and 12−16 and 30 μM p-
2
+
hydroxybenzyl ITC (11) elicited Ca influx on TRPA1-
expressing HEK cells. These responses were significantly
attenuated by cotreatment with TRPA1 antagonist HC-
2
+
0
30031, and these compounds did not cause a Ca response
in TREx HEK cells, which do not express TRPA1 (Figure 1A).
These results show that compounds 1−16 have a TRPA1
activation potency. Figure 1B shows the concentration−
response curves of compounds 1−16. Table 1 shows the
EC value and maximum response (MAX) of each compound.
Figure 1. TRPA1-activating potency of compounds 1−16. Each ITC
group is colored as follows: alkyl ITCs, orange; allyl ITC, black; ω-
alkenyl ITCs, green; aromatic ITCs, blue; ω-methylthioalkyl ITCs,
pink. (A) 1 μM compounds 1−10 and 12−16 and 30 μM 11 were
administered to TRPA1-expressing HEK cells. y-Axis: relative activity
50
Among the tested compounds, 14 of them (1−3, 5−10, and
2−16) showed a TRPA1 activation potency that was either
stronger than or equal to that of allyl ITC (4) (EC : 0.58 μM,
1
2+
5
0
to 5 μM ionomycin (%). Solid column, Ca response by ITCs in
MAX: 97%). Benzyl ITC (9), the horseradish-specific volatile,
TRPA1-expressing cells; slant-lined column, TRPA1 antagonist HC-
030031 was cotreated with compounds 1−16 and administered to
TRPA1-expressing cells; unfilled column, compounds 1−16 were
added to TREx HEK cells not expressing TRPA1. (B) Concentration−
response curves of 1−16. x-Axis: agonist concentration (log M); y-
axis: relative activity to 100 μM 4. Each data point represents the mean
showed the strongest activity to activate TRPA1 (EC : 0.25
50
μM, MAX: 95%). Its EC value was approximately half that of
5
0
4, and its maximum response was almost equal to that of 4. p-
Hydroxybenzyl ITC (11), a characteristic component of white
mustard, had the lowest TRPA1-activating ability among the
compounds tested (EC : 20 μM, MAX: 96%). Its EC value
±
SEM, n = 3−8 [*** indicates p < 0.0005 (unpaired t-test)].
5
0
50
was 40 times higher than that of 4, and its TRPA1-activating
potency was quite weak compared to those of other ITCs.
Among ITCs with similar functional groups, it was found that
the TRPA1-activating ability decreased slightly as the carbon
chain length increased. However, since the TRPA1 activation
potencies of most ITCs were nearly equal, their ITC moieties
are likely the predominant contributors to their TRPA1-
activating abilities, and contributions of other structure features
Compounds 1 (EC : 100 μM, MAX: 50%) and 2 (EC : 100
50
50
μM, MAX: 62%) had slightly lower EC50 values and maximum
responses that were 2 to 3 times larger than those of 4 (EC :
5
0
160 μM, MAX: 25%). The aromatic ITCs, 9 (EC : 223 μM,
50
MAX: 49%), 10 (EC : 180 μM, MAX: 43%), and 11 (EC :
50
50
170 μM, MAX: 63%), showed slightly higher EC50 values than
did 4, but their maximum responses were 2 to 3 times larger.
Agonist Activity of Compounds 1−16 on TRPM8.
Compounds 1−16 (300 μM) were each used to treat HEK
cells stably expressing TRPM8. As a result, all compounds did
not elicit any Ca2+ responses (data not shown). It has been
reported that 4 and 15 do not activate TRPM8, and our result
(
carbon chain length, thiol moiety, and double bond) to their
TRPA1 activation potencies are comparatively small.
Activity to Activate TRPV1 of Compounds 1−16.
Compounds 1−16 were each administered at 300 μM to HEK
cells stably expressing TRPV1. Six of them, two alkyl ITCs (1
and 2), allyl ITC (4), and aryl ITCs (9−11), elicited relatively
strong responses in TRPV1-expressing cells. Responses to the
ω-alkenyl ITCs (5−8) and ω-methylthioalkyl ITCs (12−16)
11,15
was consistent with the previous reports.
Except for 11 (EC : 20 μM), all ITCs had high TRPA1
50
activation potencies (EC : 0.25−1 μM; MAX: 80−95%).
50
2
+
were quite weak (Figure 2A). The Ca influx induced by the
six aforementioned ITCs (1, 2, 4, 9−11) was significantly
inhibited by addition of TRPV1 antagonist capsazepine (CPZ),
and the responses in TREx HEK cells were significantly weaker
than those in TRPV1-expressing HEK cells (Figure 2B). These
results indicated that these six ITCs activate TRPV1. Figure 2C
shows the concentration−response curves of these compounds.
Regarding their TRPV1-activating abilities, most of them did
not evoke a response in TRPV1-expressing cells, and only six of
them (1, 2, 4, and 9−11) weakly activated TRPV1. Their EC
5
0
values were between 100 and 200 μM, and they had low
maximum responses. Accordingly, the ITCs activate TRPA1
much more strongly than they activate TRPV1. Although the
odors and irritancies of the ITCs vary depending on the
B
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Table 1. TRPA1 and TRPV1-Activating Abilities of
Compounds 1−16
TRPA1
activation
potency
TRPV1 activation
potency
EC50
Top
EC50
Top
b
a
compound name
isopropyl ITC (1)
(μM)
(%)
(μM)
(%)
0.53
0.68
0.69
0.58
0.36
0.54
1.04
1.05
0.25
0.37
20
82
88
89
97
95
90
93
90
95
94
96
94
97
92
92
90
100
100
50
62
sec-butyl ITC (2)
isobutyl ITC (3)
allyl ITC (4)
c
−
160
25
3-butenyl ITC (5)
4-pentenyl ITC (6)
5-hexenyl ITC (7)
6-heptenyl ITC (8)
−
−
−
−
benzyl ITC (9)
223
180
170
49
43
63
phenylethyl ITC (10)
p-hydroxybenzyl ITC (11)
3-methylthiopropyl ITC (12)
4-methylthiobutyl ITC (13)
5-methylthiopentyl ITC (14)
6-methylthiohexyl ITC (15)
7-methylthioheptyl ITC (16)
0.45
0.42
0.5
−
−
−
−
−
0.58
0.61
a
b
Percent response to 100 μM allyl ITC (4). Percent response to 10
μM CAP (capsaicin). − indicates no TRP-activating ability.
Figure 2. TRPV1-activating ability of compounds 1−16. Each ITC
group and positive controls were classified with the following different
colors: alkyl ITCs, orange; allyl ITC, black; ω-alkenyl ITCs, green;
aromatic ITCs:, blue; ω-methylthioalkyl ITCs, pink; capsaicin (CAP),
gray. (A) Ca2 influx elicited by 300 μM 1−16 on TRPV1-expressing
HEK cells. (B) Solid column: responses by 300 μM 1, 2, 4, and 9−11
in TRPV1-expressing cells; lined column: TRPV1 antagonist
capsazepine (CPZ); each ITC compound was coadded to TRPV1-
expressing cells; unfilled column: responses by 1, 2, 4, and 9−11 in
TREx HEK cells not expressing TRPV1. (C) Concentration−response
curves of 1, 2, 4, and 9−11. x-Axis: agonist concentration (log M); y-
axis: relative activity to 10 μM CAP. Data are expressed as percent
responses to the ionophore ionomycin (5 μM; A and B) and 10 μM
CAP (C). Each data point represents the mean ± SEM, n = 3−7 [***
means p < 0.0005 (unpaired t-test)].
c
structures of the compounds, most of them showed nearly
equal activity to activate TRPA1. Therefore, the ITC moiety is
considered the most important factor for their TRPA1-
activating potencies, and the effects of their other functional
groups (carbon chain length, double bond, benzene ring, and
thiol moiety) against TRPA1-activating abilities are small in
comparison.
+
Alkyl ITCs (1 and 2) are found in both wasabi and
3
,5
horseradish. Their activities to activate TRPA1 were almost
equal to that of 4, and their TRPV1 activation potencies were
stronger than that of 4. ω-Alkenyl ITCs (5−8) and ω-
methylthioalkyl ITCs (12−16) are wasabi-specific components
and showed the same ability to activate TRPA1 as did 4, but did
not elicit TRPV1 activation. Benzyl ITC (9) and 10 are
horseradish-specific volatiles, and their EC50 values on TRPA1
are half that of 4. In terms of activity to activate TRPV1, these
ITCs had almost equal activation potencies to that of 4.
We compared the contents of each ITC compound with that
of 4 in wasabi, and their contents were 100 times lower than
activated by an alkaline pH, and cornea and nasal mucosa pains
induced by ammonia vapors are considered the result of
TRPA1 activation expressed in the trigeminal nerve. Since 4 is
highly volatile and causes a painful sensation similar to that
caused by vaporized ammonia, it is thought that 4 induces a
noxious stimulus in the same way that ammonia does; 4
evaporates to the nasal cavity from the oral cavity and generates
a lachrymatory sensation by activating TRPA1 expressed in the
2
that of 4. Accordingly, the main TRPA1 agonist of wasabi is 4.
19
In horseradish, the content of 10 is relatively high and is one-
trigeminal nerve innervating the nasal cavity. Accordingly, the
volatility of the ITC compounds may be directly related,
important for generating their irritancy. Since vapor pressure is
an index for the volatility of chemical compounds, we
compared the vapor pressure of 4 with those of the flavor
components of wasabi (7, 8, 15, and 16). The vapor pressure
values, quoted from SciFinder, of 7, 8, 15, 16, and 4 were 0.25,
0.09, 0.005, 0.002, and 4.57 mmHg, respectively. A higher
vapor pressure corresponds to higher volatility, and the vapor
pressures of compounds 7, 8, 15, and 16 were 100 to 1000
times lower than that of 4. This indicates that those wasabi-
specific components are much less volatile than 4. It was
believed that the ability of ω-methylthioalkyl ITCs and ω-
alkenyl ITCs to activate TRPA1 was equal to that of 4.
However, because of their low volatility, they cannot reach the
nasal cavity from the oral cavity to activate TRPA1 expressed in
the primary nerve and do not elicit an acrid sensation.
4
fifth that of 4. The TRPA1 activation potency of 10 was
slightly higher than that of 4; therefore, it was speculated that
1
0 has some contribution to the activation of TRPA1 by
horseradish.
The TRPA1 activation mechanism of 4 has been reported. Its
ITC moiety covalently binds to a cysteine residue located in the
ankyrin repeat in the cytosolic N-terminus of TRPA1 and
1
7,18
triggers TRPA1 activation.
It has also been shown that 15
15
activates TRPA1 in the same manner that 4 does.
Accordingly, ITC compounds used in this study are speculated
to activate TRPA1 through the same mechanism that 4 does.
It has been reported that 4 elicits a strong acrid sensation,
while ω-alkenyl ITCs (5−8) and ω-methylthioalkyl ITCs (12−
2
1
6) have milder flavors. Although they have low pungency,
15
this study and a study by Uchida et al. showed that they
activate TRPA1 to the same extent that 4 does. TRPA1 is also
C
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Naturally occurring TRPA1 and TRPV1 agonists have been
mainly identified from spices, and most of them possess strong
pungency. Allyl ITC (4), cinnamaldehyde (found in
cinnamon), CAP (found in chili pepper), and piperine (derived
from pepper) are representative agonists for TRPA1 and
mixture was distilled to afford pure 3-butenyl ITC (5) (25.2 g, 0.22
mol, 19%).
Other alkenyl ITCs, 4-pentenyl ITC (6), 5-hexenyl ITC (7), and 6-
heptenyl ITC (8), were synthesized from bromo-1-alkenes through
the same process utilized for 5. 5-Bromo-1-pentene (140 g, 0.94 mol)
was used to prepare 6 (25.0 g, 0.20 mol, 21%), 6-bromo-1-hexene
7
,11,13,20,21
TRPV1.
It has been shown that ingestion of TRPA1
(120 g, 0.74 mol) was used to synthesize 7 (18.8 g, 0.13 mol, 18%),
and TRPV1 agonists (e.g., compound 4, piperine, cinnamalde-
hyde, and CAP) induces adrenaline secretion from adrenal
medulla and suppresses body fat accumulation by enhancing
and 7-bromo-1-heptene (100 g, 0.56 mol) was used to obtain 8 (16.6
g, 0.11 mol, 19%).
3
2
Synthesis of 3-Methylthiopropyl ITC (12). Tetra-n-butylam-
monium bromide (50% in water) (39.4 g) was added to a mixture of
1-bromo-3-chloropropane (100 g, 0.635 mol) and methyl mercaptan
sodium salt (15% in water) (298 g). The mixture was stirred for 1 h
and then extracted with ethyl acetate. The extract was dried with
anhydrous sodium sulfate and concentrated under reduced pressure,
yielding crude 1-chloro-3-methylthiopropane (88.2 g). Next, DMF
22−25
energy expenditure.
Interestingly, some low-stimulant
TRPA1 and TRPV1 agonists have been identified, including
capsiate (isolated from CH-19 Sweet), miogatrial (a low-
pungency component of Japanese ginger), 10-shogaol (found in
26−29
steamed ginger), and monoacylglycerols (found in wheat).
Although these agonists have little or no stimulant effects, they
(176 g, 2.408 mol) and potassium thiocyanate (85.3 g, 0.877 mol)
activate TRP channels and increase energy consumption
24,30
were heated for 1.5 h at 100 °C with stirring. The reaction mixture was
extracted with ethyl acetate, dried with anhydrous sodium sulfate, and
concentrated under reduced pressure to afford crude 3-methylthio-
propyl thiocyanate (65.8 g). Then, DMF (197 g, 2.70 mol) and tetra-
n-butylammonium bromide (50% in water) (17.3 g) were added to the
residue and refluxed for 1 h. The reaction mixture was extracted with
ethyl acetate, dried with anhydrous sodium sulfate, and concentrated
under reduced pressure to give 3-methylthiopropyl ITC (12) (32.9 g,
0.22 mol, 35%).
similarly to the spicy agonists.
Hence, the activities to
activate TRPA1 of wasabi-specific volatiles (ω-methylthioalkyl
ITCs and ω-alkenyl ITCs) were nearly equal to that of 4; it is
speculated that these ITCs would also promote energy
metabolism as 4 does. As weak stimulant TRP agonists
would be taken in more easily than irritant agonists would,
these low-pungency agonists could be effectively utilized to
combat obesity. We hope that this study will also contribute to
the development of low-stimulant TRPA1 agonists with
antiobesity effects.
Synthesis of 4-Methylthiobutyl ITC (13), 5-Methylthiopentyl
ITC (14), 6-Methylthiohexyl ITC (15), and 7-Methylthioheptyl
3
3
ITC (16). The reaction mixture of 5 (17 g, 0.15 mol) was stirred for
9
h at room temperature with methyl mercaptan (30% in methanol)
EXPERIMENTAL SECTION
Test Compounds. Sixteen ITCs were used for the structure−
activity relationship study: isopropyl ITC (1), sec-butyl ITC (2),
isobutyl ITC (3), allyl ITC (4), 3-butenyl ITC (5), 4-pentenyl ITC
6), 5-hexenyl ITC (7), 6-heptenyl ITC (8), benzyl ITC (9),
phenylethyl ITC (10), p-hydroxybenzyl ITC (11), 3-methylthiopropyl
ITC (12), 4-methylthiobutyl ITC (13), 5-methylthiopentyl ITC (14),
■
(72.0 g) and tert-butyl hydroperoxide (TBHP) (0.3 g, 0.0033 mol) and
then extracted with ethyl acetate. The extract was dried with
anhydrous sodium sulfate and evaporated. The residue was distilled
and pure 4-methylthiobutyl ITC (13) (16.4 g, 0.10 mol, 68%) was
obtained.
(
The other methylthioalkyl ITCs were synthesized from alkenyl
ITCs through the same method utilized for 13. 4-Pentenyl ITC (6)
6
-methylthiohexyl ITC (15), and 7-methylthioheptyl ITC (16).
(
(
19 g, 0.15 mol) was used to prepare 5-methylthiopentyl ITC (14)
16.7 g, 0.10 mol, 64%), 5-hexenyl ITC (7) (14.1 g, 0.10 mol) was
Compounds 1−3, 9, and 10 were purchased from Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan), and compound 11 was obtained
from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Compounds
−8 and 12−16 were synthesized, and their purities were examined by
gas chromatography (GC). The purities of all ITC compounds were
used to synthesize 6-methylthiohexyl ITC (15) (13.6 g, 0.071 mol,
7
7
2%), and 6-heptenyl ITC (8) (13.0 g, 0.083 mol) was used to obtain
-methylthioheptyl ITC (16) (13.3 g, 0.065 mol, 78%).
Measurement of Activities to Activate TRPA1 and TRPV1 of
5
higher than 96%.
ITCs Using TRP Channel-Expressing HEK Cells. TRP activity of
compounds 1−16 was evaluated by a cell-based Ca imaging assay
using a FlexStation II system (Molecular Devices, Sunnyvale, CA,
HC-030031 was obtained from ChemBridge (San Diego, CA,
USA). Capsaicin, capsazepine, and 4 were purchased from Sigma-
Aldrich (St. Louis, MO, USA). (−)-Menthol was obtained from Wako
Pure Chemical Industries (Osaka, Japan), and 4-(3-chloro-2-
pyridinyl)-N-[4-(1,1- dimethylethyl)phenyl]-1-piperazinecarboxamide
BCTC) was purchased from Tocris BioScience (Bristol, UK).
Measurement of Purity of ITC Volatiles by Gas Chromatog-
raphy. An Agilent 6850 series gas chromatograph equipped with a
flame ionization detector (FID) (Agilent Technologies, Santa Clara,
CA, USA) and a DB-WAX fused silica capillary column (30 m × 0.25
mm i.d.; film thickness of 0.25 μm; J&W Scientific) was used. The
measurements were performed using an injector temperature of 210
2+
34
USA). A detailed experimental method was previously described. In
brief, TREx human embryonic kidney (HEK) cells stably expressing
human TRPV1 (hTRPV1), hTRPA1, or mouse TRPM8 were used.
The HEK cells were seeded in 96-well plates 24 h before each assay,
and 1 μg/mL tetracycline was added to the cells to induce the
expression of these TRP channels. The HEK cells were loaded with 3
μM Fluo-4 AM (Dojindo Laboratories, Kumamoto, Japan) for 1 h at
(
2
+
3
7 °C in a measuring buffer, and the change of intracellular Ca
concentrations was measured by a FlexStation II at 37 °C. Cells were
treated with ionomycin (5 μM; positive control) to check viability and
to obtain the maximum fluorescence intensity of the cells. All
chemicals were administered at a concentration at which they did not
show a nonselective effect on the parent cell lines. The following
concentrations of agonists (positive controls) and antagonists were
used for each TRP channel: 100 μM ally ITC (4) and 30 μM HC-
°
C, a helium carrier gas flow rate of 1 mL/min, and an oven
temperature of 80 °C increased at 3 °C/min to 210 °C (44 min). The
amount of sample injected was 0.2 μL, and a split ratio of 1:100 was
used.
Preparation of 3-Butenyl ITC (5), 4-Pentenyl ITC (6), 5-
3
1
Hexenyl ITC (7), and 6-Heptenyl ITC (8). N,N-Dimethylforma-
mide (DMF) (480 g, 6.57 mol) was added to a mixture of 4-bromo-1-
butene (160 g, 1.19 mol) and potassium thiocyanate (114 g, 1.17
mol), and the mixture was stirred for 1.5 h at 100 °C. After removal of
insoluble matter, the mixture was evaporated and 3-butenyl
thiocyanate containing DMF (560 g) was obtained. Sodium iodide
0
30031 for TRPA1; 10 μM CAP and 30 μM CPZ for TRPV1; and 100
μM menthol and 10 μM BCTC for TRPM8. Compounds 1−16 and
other chemicals were dissolved in DMSO and added to the measuring
buffer. Because the ITC compounds are easy to break under aqueous
conditions, 1−16 were diluted with the measuring buffer just before
administration to the cells. Curve fitting and parameter estimations
were performed using Prism 5a software (Graph Pad Software, San
Diego, CA, USA).
(
208 g, 1.40 mol) and calcium carbonate (11.2 g, 0.11 mol) were
added to 3-butenyl thiocyanate, and the mixture was refluxed for 2 h
with stirring. Insoluble matter was removed, and the remaining
D
DOI: 10.1021/acs.jnatprod.5b00272
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Journal of Natural Products
Article
Calculation of Predicted Vapor Pressure of ITC Compounds.
The vapor pressures of the ITCs were quoted from SciFinder
Academic) [https://scifinder.cas.org/scifinder]. The values were
calculated using ACD/Laboratories software V11.02 (ACD/Labo-
ratories, Toronto, Ontario, Canada) and displayed in “SciFinder
predicted properties, chemical vapor pressure at 25 °C”.
(14) Everaerts, W.; Gees, M.; Alpizar, Y. A.; Farre, R.; Leten, C.;
Apetrei, A.; Dewachter, I.; van Leuven, F.; Vennekens, R.; De Ridder,
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4
(
AUTHOR INFORMATION
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*
(
Corresponding Author
Tel: +81-054-264-5543. Fax: +81-054-264-5550. E-mail:
watanbt@u-shizuoka-ken.ac.jp.
(
(
Notes
The authors declare no competing financial interest.
(
Chiba, H.; Muraki, E.; Tsunoda, N.; Watanabe, T. Biosci., Biotechnol.,
ACKNOWLEDGMENTS
Biochem. 2011, 75, 904−909.
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(25) Iwai, K.; Yazawa, A.; Watanabe, T. Proc. Jpn. Acad., Ser. B 2003,
This study was supported in part by Ogawa & Company, Ltd.,
and the Grants-in-Aid for Scientific Research Program from
JSPS (project number 2410919 to Y.T. and project number
7
(
9B, 207−212.
26) Iwasaki, Y.; Morita, A.; Iwasawa, T.; Kobata, K.; Sekiwa, Y.;
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2
4580194 to T.W.).
1
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Koizumi, K.; Okumura, Y.; Morimitsu, Y.; Tominaga, M.; Ozawa, Y.;
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DOI: 10.1021/acs.jnatprod.5b00272
J. Nat. Prod. XXXX, XXX, XXX−XXX