Photoactivatable Odorants for Chemosensory Research

Article abstract of DOI:10.1021/acschembio.0c00541

The chemosensory system of any animal relies on a vast array of detectors tuned to distinct chemical cues. Odorant receptors and the ion channels of the TRP family are all uniquely expressed in olfactory tissues in a species-specific manner. Great effort has been made to characterize the molecular and pharmacological properties of these proteins. Nevertheless, most of the natural ligands are highly hydrophobic molecules that are not amenable to controlled delivery. We sought to develop photoreleasable, biologically inactive odorants that could be delivered to the target receptor or ion channel and effectively activated by a short light pulse. Chemically distinct ligands eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365 and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate (AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.

Full text of DOI:10.1021/acschembio.0c00541

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Photoactivatable Odorants for Chemosensory Research
Sangram Gore,¶ Kirill Ukhanov,¶ Cyril Herbivo, Naeem Asad, Yuriy V. Bobkov, Jeffrey R. Martens,*
and Timothy M. Dore*
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ABSTRACT: The chemosensory system of any animal relies on a
vast array of detectors tuned to distinct chemical cues. Odorant
receptors and the ion channels of the TRP family are all uniquely
expressed in olfactory tissues in a species-specific manner. Great
effort has been made to characterize the molecular and
pharmacological properties of these proteins. Nevertheless, most
of the natural ligands are highly hydrophobic molecules that are
not amenable to controlled delivery. We sought to develop
photoreleasable, biologically inactive odorants that could be
delivered to the target receptor or ion channel and effectively
activated by a short light pulse. Chemically distinct ligands
eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-
thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-
hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365
and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels
expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination
with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels
was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently
activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate
(AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.
molecules from different chemical classes, such as ketones,
carboxylic acids, alcohols, aldehydes, sulfur-containing com-
pounds, and others.21,22 One OR recognizes multiple odorants,
and one odorant is recognized by multiple ORs, and
interestingly, different odorants are recognized by different
combinations of ORs.23 Most of the natural ligands are highly
hydrophobic molecules that are difficult to deliver to the
immediate vicinity of the ORs in native tissue, which is
important for high resolution imaging and study of the
signaling pathways. The process of olfactory transduction takes
place at the fine cilia, which makes the experimental
manipulations for studying olfactory receptors difficult.
To address this challenge, we designed and synthesized
photoactivatable odorants that can be delivered to the target
receptor or ion channel in intact olfactory tissues and
effectively activated by a brief light pulse to enable the study
of this chemosensory system. We used (8-cyano-7-hydrox-
INTRODUCTION
■
The olfactory system has a central function in the detection
and interpretation of chemical cues present in the environ-
ment.1−4 Odorants are low molecular weight volatile
compounds that can be detected by the human or animal
olfactory system. In all living organisms, odorants play
important roles, particularly in environmental threat detec-
tion,5,6 food scavenging,7 disease detection,8,9 navigation,10,11
mate recognition,12,13 and appetite stimulation.14−16 In
terrestrial vertebrates, odorants are inhaled into the nasal
cavity where they are recognized by olfactory sensory neurons
(OSNs) in the olfactory epithelium (OE). Odorant binding to
the receptors on these neurons activates signaling pathways in
a ligand-dependent manner that transmit information directly
to glomeruli in the olfactory bulb and further to the brain,
where odor perception is constructed.17,18
There are about 400 human and more than 1000 mouse
genes that encode olfactory receptors (ORs), which comprise
one of the largest families of G-protein coupled receptors
(GPCRs).19,20 ORs along with ion channels of the transient
receptor potential (TRP) family are uniquely expressed in a
tissue-specific manner. As one of the most significant elements
in smell sensory neurons, ORs greatly contribute to a high
performance olfactory system that can identify various odor
Received: July 6, 2020
Accepted: August 14, 2020
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yquinolin-2-yl)methyl (CyHQ)24−27 as the photoremovable
protecting group (PPG) to inactivate the odorants because of
its excellent photochemical properties and biocompatibility,
such as good solubility in aqueous media, high quantum yields,
and fast release kinetics in a clean reaction that releases the
attached biological effectors in high yield along with bio-
logically benign byproducts. We prepared CyHQ-protected
versions of the odorants benzaldehyde (CyHQ-BZA), 2-
phenethylamine (CyHQ-2PEA), eugenol (CyHQ-EG), etha-
nethiol (CyHQ-(SEt)2), butane-1-thiol (CyHQ-(SBu)2), and
2,2-dimethylethane-1-thiol (CyHQ-(St-Bu)2) (Figure 1). A
CyHQ-thioacetals to mediate the photoactivation of alka-
nethiols.
Synthesis. CyHQ-BZA was prepared from the previously
reported aldehyde 2,24 which we synthesized from 1 using an
improved procedure (Scheme 1).26 Wittig olefination provided
Scheme 1. Preparation of CyHQ-Protected Benzaldehyde
alkene 3, which was oxidized by osmium(VIII) oxide to diol 4
in 73% yield. The reaction of 4 with (dimethoxymethyl)-
benzene in the presence of iron(III) chloride in refluxing THF
afforded CyHQ-BZA.
CyHQ-2PEA was prepared by first activating the primary
alcohol on MOM-CyHQ-OH (prepared as previously
described24,26) with 1,1′-carbonyldiimidazole (CDI) followed
by displacement of the remaining imidazole with phenethyl
amine (Scheme 2). CyHQ-EG was prepared in moderate yield
by heating eugenol with MOM-CyHQ-OMs (prepared as
previously described26) in DMF in the presence of potassium
carbonate (Scheme 2). The CyHQ-protected thiols CyHQ-
(SEt)2, CyHQ-(SBu)2, and CyHQ-(St-Bu)2 were synthesized
in moderate to good yields from aldehyde 2 (Scheme 1) using
the corresponding thiols (ethanethiol, butane-1-thiol, and 2,2-
dimethylethane-1-thiol) (Scheme 2) and concentrated hydro-
chloric acid. CyHQ-VNA (Figure 1) was prepared as
previously described.25
Photochemistry. Each of the CyHQ-protected odorants
photolyzed efficiently when exposed to 365 or 405 nm light
under simulated physiological conditions (pH 7.2 KMOPS
buffer) (Scheme 3, Figure 2, and Table 1). The 405 nm
wavelength was tested because this laser line is commonly
found on commercially available turn-key confocal microscope
systems and can be used for photoactivation. The photo-
chemistry of CyHQ-VNA has been reported previously.25
CyHQ-BZA released benzaldehyde reasonably efficiently at
365 and 405 nm light, although prolonged exposure shows
significant disappearance of benzaldehyde under the reaction
conditions. Benzaldehyde does not absorb light above 300 nm,
so the decomposition is likely the result of nonphotochemical
air oxidation to benzoic acid. CyHQ-2PEA photolyzed
efficiently, resulting in a ∼70% yield of 2-phenethylamine.
CyHQ-EG photolyzed with moderate quantum efficiencies at
365 and 405 nm, but the yield of eugenol was much lower than
expected. The expected products CyHQ-OH and eugenol were
readily observed in the HPLC and LC-MS traces, but no other
discrete products (e.g., isoeugenol) were detected, only
nonspecific decomposition.
Figure 1. CyHQ-protected odorants and CyHQ-VNA.
photoactivatable form of the equipotent capsaicin analogue N-
(4-hydroxy-3-methoxybenzyl)nonanamide, more commonly
known as N-vanillyl-nonanoylamide (VNA),28 which activates
TRPV1 channels,29−32 CyHQ-VNA,25 was used as a positive
control to evaluate the odorants’ ability to activate TRPV1
channels. Other photoactivatable forms of capsaicin33 or
VNA34−37 exist, but none have the high sensitivity and rapid
capsaicinoid release kinetics of CyHQ-VNA. The CyHQ-
protected odorants were evaluated for their ability to activate
TRPA1 channels and induce a dose-dependent electroolfacto-
gram (EOG) in native mouse OE.
RESULTS AND DISCUSSION
■
Design. Creation of the CyHQ-protected versions of the
odorants required the development of some new photo-
chemistry. The design of CyHQ-BZA was inspired by previous
work that identified a bromohydroxycoumarin derivative, Bhc-
diol,38 as a PPG suitable for mediating the photorelease of
ketones and aldehydes. Because primary amines are not
sufficiently good leaving groups, a carbamate linkage was used
to prepare CyHQ-2PEA. CyHQ-EG was designed based on
previous work that showed that quinoline-derived PPGs
effectively mediated the release of phenols under physiological
conditions.25,39 Directly releasing alkanethiols from PPGs is
challenging because the photochemistry often generates sulfur-
based radicals that react with the PPG and other species in the
system. Nevertheless, some creative solutions to the problem
have been reported.40−46 Quinoline-based PPGs do not release
thiols directly from the thioether. To overcome this, we were
inspired by the ability of the (8-bromo-7-hydroxyquinolin-2-
yl)methyl (BHQ) PPG to release diols.47 Our design uses
The photolysis of the CyHQ-protected thiols yielded a
variety of quinoline-based photoproducts, including CyHQ-H,
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Scheme 2. Synthesis of CyHQ-Protected 2-Phenethylamine, Eugenol, and Alkyl Thiols
Scheme 3. Photolysis Reactions of CyHQ-Protected Odorants
CyHQ-OH, CyHQ-SR, and thioether 5 (Scheme 3) in
addition to the alkanethiol. Release of the alkanethiol was
evident because of the distinct foul smell that emanated from
the reaction vessel within seconds of light exposure and
increased in intensity as the photolysis reaction progressed.
Ethanethiol and 2,2-dimethylethane-1-thiol are used to tag
liquid petroleum gas (LPG) and natural gas, respectively, and
butane-1-thiol is used in “stink bombs”. The human olfactory
system detects these odorants at 0.36, 0.33, and 1.4 ppb levels,
respectively.48 CyHQ-(SEt)2 and CyHQ-(SBu)2 photolyzed
with quantum efficiencies, Φu = 0.34 (365 nm) and 0.37 (405
nm) and Φu = 0.26 (365 nm) and 0.20 (405 nm), respectively,
whereas CyHQ-(S-t-Bu)2 displayed much lower quantum
efficiencies.
CyHQ-BZA, CyHQ-2PEA, CyHQ-EG, CyHQ-(SEt)2,
CyHQ-(SBu)2, CyHQ-(S-t-Bu)2, and CyHQ-VNA25 had
sufficient solubility in the KMOPS buffer and adequate
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Figure 2. Time courses of the photolysis reactions at (A, B) 365 nm and (C, D) 405 nm. Percentage remaining was determined by HPLC analysis
and is the average of three runs. Solid lines are least-squares fits of the data to a simple exponential decay. Dashed lines are least-squares fits of the
data to an exponential rise to max or a spline curve in the case of benzaldehyde. Error bars represent the standard deviation of the mean.
a
Table 1. Photochemical Properties of CyHQ-Protected Odorants
b
b
ε365
ε405
sensitivity
(ε365Φu)
sensitivity
(ε405Φu)
yield (365 nm) yield (405 nm) dark stability
c
compound
λmax(nm) (M−1 cm−1
)
(M−1 cm−1
)
Φu(365) Φu(405)
(%)
(%)
(h)
CyHQ-BZA
CyHQ-2PEA
CyHQ-EG
CyHQ-(SEt)2
CyHQ-
(SBu)2
365
365
365
368
368
5500
7460
7340
3340
5350
470
280
798
1000
710
0.13
0.34
0.21
0.34
0.26
0.11
0.31
0.15
0.37
0.20
694
2487
1541
1121
1378
44
88
121
370
140
35
70
25
30
70
19
24
48
24
24
24
CyHQ-(S-t-
387
368
6650
6100
6200
0.09
0.41
0.07
567
444
48
Bu)2
d
CyHQ-VNA
2501
65
100
a
b
c
0.1 mM solution in KMOPS buffer, pH 7.2. Yield of benzaldehyde, 2-phenethylamine, or eugenol as measured by HPLC. No spontaneous
d
hydrolysis in the dark observed within the time given. Data taken from the literature on CyHQ-protected N-(4-hydroxy-3-methoxybenzyl)-
acetamide, commonly known as N-vanillyl-acetamide (VAA), an analogue of CyHQ-VNA that is more soluble in aqueous buffers to the extent
needed for analyzing photochemical reactions.25
stability toward spontaneous hydrolysis in the dark, making
them fit for studies in biological systems.
allicin, allyl isothiocyanate, and vanillin.51,52 Eugenol is a
known odorant ligand for several mammalian and insect
olfactory receptors,53,54 and it is unique in its anti-
inflammatory potential and antibacterial activity against
human pathogens.55,56 The CyHQ-protected odorants were
tested for their ability to activate TRPV1 channels on cultured
HEK 293 cells that expressed human TRPV1 on the cell
membranes in comparison to CyHQ-VNA.25
Activation of TRPV1 Channels Using CyHQ-Protected
Odorants and VNA. Several odorant and flavor molecules,
including eugenol, have been characterized as activators of ion
channels belonging to the TRP family, including TRPA1 and
TRPV1.31,49,50 Both TRPA1 and TRPV1 channels can be
activated by electrophilic pungent molecules like capsaicin,
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HEK 293 cells were transiently cotransfected with cDNA
encoding human TRPV1 and GFP. Only cells visible by their
green fluorescence were used for patch clamp recording.
Inward ionic current was measured in a whole-cell voltage
clamp configuration in a single cell superfused with either
control physiological solution or supplemented with the
indicated concentration of the CyHQ-protected odorant. For
photolysis, we used 365 nm light to most closely match the
maximum wavelength of excitation of the CyHQ-protected
odorants (see Supporting Information for UV−vis spectra) and
VNA. In the presence of CyHQ-VNA (10 μM), a 100 ms pulse
of 365 nm light from an LED at 50% of its maximal output
robustly activated inward ionic current with a latency of 69.5
10.9 ms (3 cells) and a current−voltage dependence that is
typical for TRPV1 (Figure 3).51 A TRPV1-dependent ion
confocal microscopes. To demonstrate, we used 405 nm light
and the CyHQ-protected odorants to activate TRPV1 channels
stably expressed in cultured HEK 293 cells. Photochemical
release of the capsaicinoid VNA from CyHQ-VNA25 served as
a positive control. The TRPV1-experessing HEK 293 cells
were incubated with the Ca2+ sensitive dye Fluo-4 AM and the
CyHQ-protected odorants or CyHQ-VNA. Using a confocal
microscope, the Ca2+ influx into the cells was measured by
imaging at 488 nm before and after a brief pulse of 405 nm
light directed close to the external side of the cell membrane
(Figure 4). The concentration of each CyHQ-protected
Figure 4. Calcium influx measured in HEK 293 cells stably expressing
TRPV1 channels after activation of CyHQ-protected odorants and
VNA (10 μM) with 405 nm light (green arrow). The laser was set to
100% output, which measured 1.4 mW at the back focal plane of the
objective. Each trace is the average of three different experiments. See
Figures S1−S7 in the Supporting Information for images of cells
before and after light exposure.
odorant and the laser power were held constant, and the
duration of the light exposure varied to maximize the
fluorescence signal with minimal toxicity to cells.
Benzaldehyde, 2-phenethylamine, eugenol, ethanethiol,
butane-1-thiol, and 2,2-dimethylethane-1-thiol were not as
potent activators of the TRPV1 channel as VNA, which gave a
robust response with a 25 ms pulse of 405 nm light that could
be repeated to give a consistent Ca2+ response (Figure S8).
The fluorescent signal from release of VNA from CyHQ-VNA
depended on concentration and light intensity (Figure S9).
Among the odorants, CyHQ-(SBu)2 was the most potent
because it gave a response comparable to that from CyHQ-
VNA with the shortest duration of light pulse (100 ms)
compared to 250 ms for CyHQ-(SEt)2 and CyHQ-(S-t-Bu)2.
The rise times of the fluorescent signals for the CyHQ-
protected odorants were slightly longer than those of CyHQ-
VNA except that of CyHQ-2PEA, which was significantly
longer (Table 2), indicating rapid channel opening after
photolysis of most of the CyHQ-protected odorants and VNA.
The delayed channel activation with CyHQ-2PEA is likely due
to the fact that a slow decarboxylation step after photocleavage
of the carbamate is required to reveal the active amine. The
lower potency of the odorants might explain the slightly longer
rise times compared to VNA. The fluorescent signal did not
return to its original level prior to light exposure when the
odorants were released, whereas the signal did return to
baseline when the TRPV1 channels were photoactivated by
Figure 3. Photoactivation of TRPV1 transiently expressed in HEK
293 cells. (A) A representative whole-cell patch clamp recording of
the inward ionic current evoked by photolysis of CyHQ-VNA using a
brief 100 ms pulse of 365 nm light from an LED at 50% power output.
A single cell transiently expressing TRPV1 was identified by
coexpression of GFP. CyHQ-VNA (10 μM) in Ringer solution was
superfused on the cell 10−15 s before photoactivation. The LED was
triggered at the time point marked with a square pulse (purple bar).
The baseline was corrected for the leak current. (B) Current−voltage
relationship of the current measured in a different cell at the decaying
phase of the evoked current and corrected for leak current. A
continuous voltage ramp spanning −100 to +100 mV in 500 ms was
applied to measure the current.
current was evoked multiple times in the same cell, suggesting
very little inactivation. Importantly, the 475 nm light used to
visualize GFP did not activate any inward current prior to the
photoactivation pulse, supporting the utility of a Ca2+ imaging
assay to perform higher throughput screening of the photo-
activatable odorants.
Photolysis of the CyHQ-protected odorants with 405 nm
light produced active compounds (Figure 2), which suggested
that photoactivation can be conveniently accomplished using a
laser line that is standard for most commercially available
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resolution multicolor microscopy along with photorelease of
odorants.
Table 2. Rise Times for Photoactivation of TRPV1
Channels after Photoactivation of CyHQ-Protected
Odorants and VNA
a
Activation of Human TRPA1 Ion Channels Using
CyHQ-Protected Odorants. We tested whether CyHQ-EG
could be used as a prototypic photoprotected odorant relevant
for chemosensory biology. Whole-cell ionic currents were
recorded from HEK 293 cells transiently coexpressing
recombinant human TRPA1 channels and GFP. Bath
application of eugenol, using a rapid superfusion system that
enabled the solution exchange in less than 10 ms, activated an
inward current following a significant delay (Figure 5A).
Illumination of TRPA1-expressing cells with 365 nm light or
bath application of CyHQ-EG (250 μM) alone did not activate
any inward current (Figure 5A). However, following a short
waiting period after the solution exchange from normal Ringer
to that containing CyHQ-EG, a 100 ms pulse of 365 nm light
from an LED at 50% output robustly activated inward ionic
current with fast kinetics (Figure 5B). Photolysis-evoked
inward current was detected with a latency of 87.8 10.8 ms
and a time to reach 90% of the peak magnitude of 1.7 0.2 s
(4 cells, n = 5). The inward ionic current had a current−
voltage relationship typical for TRPA1 (Figure 5C).51,57
Because cells were superfused with the solution containing a
given concentration of CyHQ-EG, we measured the
concentration dependence of the TRPA1-mediated current
compound
rise time (s)
CyHQ-BZA
CyHQ-2PEA
CyHQ-EG
CyHQ-(SEt)2
CyHQ-(SBu)2
CyHQ-(S-t-Bu)2
CyHQ-VNA
7.8
49.8
8.9
7.0
7.8
8.9
4.0
a
Rise time = t90% − t10%, where t90% and t10% are the times at 90% and
10% of max response, respectively, taken from the data in Figure 4.
CyHQ-VNA. This might have resulted from saturating
stimulation of the TRPV1 channels that prolonged recovery,
left some channels open, or both. Further, the odorants are
small and lipophilic and could have been absorbed into the
membrane to create a reservoir of activator ligands that slowly
released to interact with TRPV1. We confirmed that even
prolonged illumination with 488 nm light used for Fluo-4
imaging was not able to activate any TRPV1-mediated Ca2+
influx. We conclude that CyHQ photoprotection provided
excellent spectral selectivity essential to perform high-
Figure 5. Photoactivation of human TRPA1 channels transiently expressed in HEK 293. (A) Prolonged 20 s illumination with 365 nm light with no
added CyHQ-EG as well as application of CyHQ-EG (250 μM) in the absence of light did not activate any current. A 20 s superfusion with
eugenol (250 and 2500 μM) activated ionic current with typical delayed TRPA1 kinetics. (B) A whole-cell inward current measured in a single
HEK293T cell transiently expressing TRPA1 and superfused with increasing concentrations of CyHQ-EG prior to illumination with a 100 ms pulse
of 365 nm light from an LED at 50% power output given at the indicated time. For comparison, photolysis of CyHQ-(SBu)2 (50 μM) also robustly
activated TRPA1. (C) A current−voltage relationship of the TRPA1 mediated current, corrected for leak current, was measured at the decaying
phase. A continuous voltage ramp spanning −100 to +100 mV in 500 ms was applied to measure the current. (D) Concentration dependence of
the TRPA1 current evoked by photolysis of CyHQ-EG superfused in cells at the indicated concentration and illuminating with a 100 ms pulse of
365 nm light from an LED at 50% output power. The amplitude was normalized to the maximum of the inward current in each series. Data points
were fitted to a Hill equation yielding a Hill slope of 2.1 and EC50 = 12.6 μM.
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evoked by light of the same intensity and duration (Figure
5D). Notably, as little as 1 μM CyHQ-EG was sufficient to
photoactivate TRPA1 channels (Figure 5B). Photoprotected
thiol CyHQ-(SBu)2 (50 μM) was also an effective photo-
activator of the TRPA1 channel (Figure 5B and S10). Other
CyHQ-protected odorants, 2-phenethylamine (nonrelevant
TRPA1 ligand) and benzaldehyde (a weak agonist with EC50
> 300 μM),58 did not photoactivate TRPA1 channels as
strongly as CyHQ-(SBu)2 (Figure S10).
that the photolysis reaction byproduct CyHQ-OH was
biologically inert in this assay (Figure S11B).
Illuminating the OE with a 100 ms pulse of 365 nm light
from an LED at 50% power after preapplication of 50 μL of
CyHQ-EG (250 μM) immediately activated a transient EOG
response with a delay of 116.7 6.3 ms and reached a peak
amplitude in 344.6
21.9 ms (n = 16, 2 animals) (Figure
S12), which then returned to its baseline within 5 s, typical of
an odor-evoked EOG (Figure 6A).64 The delay and rise time
of the EOG evoked by a 100 ms pulse of AAc was 104.1 5.9
and 228.7 3.7 ms, respectively (n = 7, 3 animals) (Figure
S12). Following the first 100 ms pulse, a longer 5 s pulse of
365 nm light evoked a larger response, reaching its peak and
then slowly relaxing to a steady-state plateau, again typical of
an odor evoked EOG (Figure 6A). These data show that
photoreleased eugenol induced an EOG indistinguishable from
the vapor-phase odorant evoked response. Importantly, no
additionally added CyHQ-EG was required to elicit the second
response to a longer 5 s light pulse, suggesting that the pool of
CyHQ-EG was not completely depleted during the first light
pulse and remained available for subsequent photolysis.
Photoreleased eugenol diffused away or was metabolized65
fast enough to minimize sensory adaptation and enable
multiple stimulations.
Irradiation with a longer wavelength of light (405 nm) at
50% LED power also evoked an EOG using CyHQ-EG (250
μM) (Figure 6B). This outcome was similar to the photo-
activation of TRPV1 with 405 nm light (Figure 4). EOG
amplitudes were normalized to the EOG evoked by a vapor-
phase of AAc (1 mM) presented at the end of each experiment
(Figure 6C). A short 100 ms light pulse at 405 nm evoked a
significantly smaller EOG than that at 365 nm, 0.144 0.022
(n = 9) compared to 0.372 0.064 (n = 9, p = 0.0013, Mann−
Whitney test). The EOG activated with a longer 5 s pulse,
however, was not significantly different between 405 and 365
nm, suggesting in both cases that the response was nearly
saturated (Figure 6C). Consistent with a larger amplitude of
the EOG evoked by 365 nm light, a rise time of the response
was also significantly larger than at 405 nm, 344 22 ms (n =
16, p = 0.048, Mann−Whitney t test) compared to 262 15
ms (n = 7) (Figure S12). Surprisingly, delay of the EOG
evoked at 365 and 405 nm was not significantly different, 116.7
One important caveat emerged from comparing the kinetics
of TRPV1 and TRPA1 channel activation by bath applied
odorants and by photolysis of the CyHQ-protected odorants.
In our study and previously published work from other
laboratories, bath applied TRPA1 and TRPV1 agonists took a
relatively longer time to activate the ion channel, suggesting it
takes time to diffuse to the cell from the point of introduction
into the culture and interact with the channels. The binding
site for capsaicinoids on TRPV1 is outside the cell,59−61
whereas electrophilic TRPA1 agonists covalently modify
cysteine residues on the cytosolic side of the membrane,62,63
so odorants would have the added step of permeating the
membrane before interacting with TRPA1. CyHQ-protected
odorants significantly shorten the latency time relative to bath
application of the odorants because they deliver the inactive
odorant to the vicinity of the channel protein moiety, which
makes them available for fast chemical interaction with the
receptor channel upon photolysis. The CyHQ-odorant
conjugates are small, relatively lipophilic molecules that cross
cell membranes, similarly to many other so-called “caged”
compounds, so it would be reasonable to expect that they
would be present inside the HEK293 cells and facilitate short
latency times for TRPA1 activation. We conclude that CyHQ-
protected odorants represent a novel class of agonists enabling
tight control of the dynamics of TRPA1 and TRPV1 activation.
Photoactivation of Odorants Induces Response in
Mouse Olfactory Epithelium. To test if CyHQ-protected
odorants can activate native sensory neurons, we measured the
EOG evoked by the photolysis in mouse OE. An EOG is a
transepithelial voltage evoked by activation of multiple
receptors in cells at the site of the recording. It is a standard
method to assess the net sensitivity of the OE to odorants.
Because CyHQ-protected odorants should be delivered to the
EOG recording site in solution, we first ensured survival and
retention of the odor sensitivity of the tissue after superfusing
with a standard phosphate-buffered saline (PBS) solution. An
aliquot of PBS (100 μL) was applied on the mouse OE, and
then a short 100 ms pulse of amyl acetate (AAc, 1 mM) in the
vapor phase was applied after each set of recordings (Figure
S11A). The EOG evoked by AAc (1 mM) was used as a
reference because it corresponds to the approximate EC50 for
activation of EOGs and was used in many studies as a potent
olfactory stimulus.64 Eugenol as a reference is less suited for
this purpose due to its much lower volatility (Pv = 0.01 Torr)
compared to that of AAc (Pv = 4.2 Torr). AAc (1 mM)
reproducibly evoked on average 3.51 0.42 mV (n = 22, 7
animals). Application of 50−100 μL of either CyHQ-OH or
CyHQ-EG (both at 250 μM working concentration in PBS)
on the OE surface did not affect or desensitize the OE prior to
illumination, as evidenced by a strong EOG response from
AAc (1 mM) after the trial (Figure S11B). Illumination of
CyHQ-OH treated tissue for up to 5 s with 365 nm light from
an LED at 50% power also did not evoke an EOG, confirming
6.3 ms (n = 16) vs 130.4
10.9 ms (n = 9, p = 0.242,
Mann−Whitney t test) (Figure S12).
To better characterize photoactivation, we measured the
light intensity dependence of the EOG evoked by light pulses
of varying intensity. Following initial treatment with 50 μL of
CyHQ-EG (250 μM), 50 ms pulses of 365 nm light from an
LED at increasing power output (4−64%) were given at 10 s
intervals to allow full recovery (Figure 7A) prior to the next
pulse. After adding a fresh 50 μL aliquot of CyHQ-EG (250
μM), the same series of light pulses was given again, but using
405 nm light instead (Figure 7B). Both series of EOGs were
recorded at the same spot of the OE. The photoactivated
response was normalized to maximal EOG in each series and
plotted as a function of relative LED power because we did not
measure absolute irradiance at both wavelengths (Figure 7C).
Notably, 5-fold more light was required to evoke the same
amplitude of the response at 405 nm compared to 365 nm,
owing to the fact that photolysis at 365 nm is much more
efficient than at 405 nm (Figure 7C) and in line with the
absorbance maximum of the CyHQ-protected odorants (see
UV−vis spectra in the Supporting Information).
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animals) for CyHQ-(SBu)2) vs 126.1 12.2 and 228.7 3.7
ms (n = 11, 2 animals) for CyHQ-(SEt)2), respectively (Figure
9, Figure S12). The small EOG evoked by the photolysis of
CyHQ-BZA prevented analysis of the delay and rise time. The
difference in the magnitude of the EOG evoked by photolysis
of CyHQ-(SBu)2 and CyHQ-(SEt)2 was further delineated
with a light intensity-response series (Figure S13). Interest-
ingly, the delay between the light pulse and the beginning of
the EOG was similar for all the CyHQ-protected odorants,
suggesting the availability of odorants in the vicinity of the
olfactory receptors, similarly to the case of TRPA1 and TRPV1
channels. The CyHQ-photoprotected odorants can be
effectively used to activate a functional response in the
mouse OE.
CONCLUSIONS
■
A set of photoactivatable odorants, CyHQ-EG, -BZ, -2PEA,
-(SEt)2, -(SBu)2, and -(S-t-Bu)2, was designed and synthesized
for use in olfactory tissue preparations. These reagents released
the respective odorant upon photolysis at 365 or 405 nm to
varying degrees but in sufficient amounts to elicit a
physiological response in cell or tissue culture assays. CyHQ-
2PEA was the most sensitive to light, whereas CyHQ-(S-t-Bu)2
was the least. Photorelease at 405 nm is important because this
wavelength is typically available on most turn-key confocal
microscope systems. All of the CyHQ-odorant conjugates
activated TRPV1 channels after a brief pulse of light. CyHQ-
(SBu)2 was nearly as effective as the potent capsaicinoid VNA
photoreleased from CyHQ-VNA. Only CyHQ-EG and CyHQ-
(SBu)2, however, strongly activated TRPA1 channels. In
mouse olfactory epithelial tissue, CyHQ-EG and CyHQ-
(SBu)2 strongly elicited a dose-dependent EOG response
rapidly after brief light exposure that will enable precise control
of OSN activation for the study of the kinetics and
pharmacology of olfaction. These tools will be valuable for
the study of chemosensory systems in model organisms.
Figure 6. Representative recordings of EOGs measured in mouse
olfactory tissue in the presence of 50 μL of CyHQ-EG (250 μM)
following exposure to light from an LED at 50% power delivered via a
fiber light guide or a 100 ms pressure pulse of AAc (1 mM, black
arrowhead) as a positive control. (A) A 100 ms pulse of 365 nm light
evoked a transient EOG response, whereas a 5 s light pulse evoked
EOG response typical to prolonged odor stimulation. (B) Longer
wavelength light (405 nm) also evoked an EOG in the same area of
the mouse OE. (C) EOG evoked by 100 ms (empty bars) and 5 s
light (gray bars) pulses were normalized to the EOG evoked by AAc
(1 mM). The EOG amplitude was significantly larger at 365 nm than
at 405 nm, 0.372 0.064 (n = 9) compared to 0.144 0.022 (n = 9,
p = 0.0013, Mann−Whitney test). The EOGs evoked by a 5 s pulse of
light were not different (n = 3, p = 0.24, Mann−Whitney test).
Adding CyHQ-EG to the tissue prior to illumination did not evoke
any significant response or desensitize the tissue, as confirmed by
comparing the odor response before and after the addition with AAc.
Each data set was collected in different areas of the OE (value of n
indicated at each bar) in at least three independent preparations.
METHODS
■
Synthesis. General. All reagents and solvents were purchased
from commercial sources and used without further purification. H
1
NMR and 13C NMR were recorded on a Bruker Avance III HD 500
or 600 MHz NMR spectrometer. HPLC and uHPLC (analytical and
preparative) were performed on an Agilent Infinity series system with
an autosampler and diode array detector using Zorbax eclipse C-18
revers phase columns. HRMS was performed on an Agilent 6540 HD
Accurate Mass QTOF/LC/MS with electrospray ionization (ESI) or
a Micromass QTOF-Ultima with ESI. KMOPS buffer consisted of
100 mM KCl and 10 mM MOPS titrated to pH 7.2 with KOH. Flash
chromatography was performed on an Isolera Spektra 4 with Biotage
SNAP cartridges packed with KPSIL silica.
7-(Methoxymethoxy)-2-vinylquinoline-8-carbonitrile (3). Meth-
yltriphosphonium bromide (1.32 g, 3.7 mmol, 1.2 equiv) and
potassium carbonate (0.685 g, 5 mmol, 1.6 equiv) was added to
dry THF (15 mL) and stirred at RT for 15 min. Aldehyde 2 (0.750 g,
3.1 mmol, 1 equiv), was dissolved in dry THF and added dropwise to
the mixture over 10 min. The mixture was heated to reflux for 6 h.
The mixture was cooled then concentrated in vacuo, and the resulting
residue was subjected to flash column chromatography (25% EtOAc
in hexane) to yield alkene 3 as a pale yellow solid (0.469 g, 63%): 1H
NMR (500 MHz, methanol-d4, δ): 8.31 (d, J = 8.5 Hz, 1H), 8.15 (d, J
= 9.2 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 9.1 Hz, 1H), 7.08
(dd, J = 17.6, 10.8 Hz, 1H), 6.54 (dd, J = 17.6, 1.1 Hz, 1H), 5.76 (dd,
J = 10.8, 1.1 Hz, 1H), 5.54 (s, 2H), 3.60 (s, 3H); 13C NMR (126
MHz, methanol-d4, δ): 162.5, 158.3, 148.4, 137.0, 136.8, 133.8, 122.8,
The EOG evoked by photolysis of CyHQ-protected
benzaldehyde and two thiols was analyzed in a similar manner
(Figure 8). Essentially, these photoprotected odorants showed
dynamics of the EOG similarly to eugenol. However, CyHQ-
BZA was the least potent odorant, activating the smallest
response to a 100 ms and 5 s pulse of 365 nm light (Figure 8A,
Figure 9). Photoreleased butane-1-thiol was a stronger odorant
compared to benzaldehyde and ethanethiol with a latency and
rise time to peak of 98.2 5.0 and 250.1 15.9 ms (n = 17, 3
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Figure 7. Irradiation dependence of the mouse EOG evoked by photolysis of CyHQ-EG at (A) 365 and (B) 405 nm. Representative data collected
from the same preparation. An aliquot (50 μL) of CyHQ-EG (250 μM) was applied once to the OE before running a series of pulses at either
wavelength. A short 100 ms pulse of both wavelengths irradiated the same area of the tissue, increasing LED power from 4 to 64% of the maximum.
A recovery period of 20 s was allowed between each consecutive light pulse. (C) Dependence of the EOG evoked as a function of irradiation LED
power. Individual EOG amplitudes were normalized to the maximum value in each series. Each data point was calculated from at least three
replicates in two animals and different areas of the OE. Data are presented as the mean SEM.
Figure 9. Summary of EOGs evoked by photolysis of three different
CyHQ-protected odorants. EOG amplitudes evoked by 100 ms and 5
s light pulses were normalized to the EOG evoked by AAc (1 mM).
Each data set was collected in different areas of the OE (value of n
indicated at each bar) in at least three independent preparations.
Mean normalized EOG amplitudes were significantly different in both
groups (100 ms set: p = 0.045, one-way ANOVA, Kruskal−Wallis
test; 5 s set: p = 0.0143, one-way ANOVA, Kruskal−Wallis test).
120.8, 118.4, 115.2, 114.2, 98.4, 95.0, 55.7; HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C14H12N2O2 241.0972; found 241.0977.
2-(1,2-Dihydroxyethyl)-7-(methoxymethoxy)quinoline-8-carbon-
itrile (4). To a stirring 1:2 mixture of acetone/water (7.5 mL), N-
methylmorpholine N-oxide (0.290 g, 2.47 mmol, 1 equiv), osmium
tetraoxide (2% in water/acetone) solution (7.5 mL) was added
dropwise. Alkene 3 (0.6 g, 2.5 mmol, 1 equiv) was dissolved in
acetone and added dropwise to the mixture, followed by stirring at RT
for 12 h. Saturated sodium sulfite solution was added and the resulting
mixture was filtered and the filtrate concentrated in vacuo. The
resulting residue was subjected to flash column chromatography (90%
1
EtOAc in hexane) to give diol 4 as white solid (0.500 g, 73%): H
NMR (500 MHz, methanol-d4, δ): 8.37 (d, J = 8.5 Hz, 1H), 8.18 (d, J
= 9.2 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 9.2 Hz, 1H), 5.54
(s, 2H), 4.98 (dd, J = 6.1, 4.4 Hz, 1H), 4.03 (dd, J = 11.4, 4.4 Hz,
1H), 3.89 (dd, J = 11.4, 6.1 Hz, 1H), 3.59 (s, 3H); 13C NMR (126
MHz, methanol-d4, δ): 164.8, 162.4, 147.5, 137.0, 134.1, 122.8, 118.6,
Figure 8. EOGs measured in mouse olfactory tissue following
photolysis of other CyHQ-protected odorants by 365 nm LED light at
50% power delivered via a fiber light guide or application of vaporized
solution of AAc (1 mM, black arrowhead) as a positive control. A 100
115.4, 114.2, 98.2, 95.0, 74.7, 66.1, 55.8; HRMS (ESI-TOF) m/z: [M
+ H]+ calcd for C14H14N2O4 275.1026; found 275.1032.
7-Hydroxy-2-(2-phenyl-1,3-dioxolan-4-yl)quinoline-8-carboni-
ms pulse of 365 nm light evoked a transient EOG response, whereas a
trile (CyHQ-BZA). Diol 4 (0.050 g, 0.18 mmol, 1 equiv), anhydrous
5 s light pulse evoked a typical EOG response. CyHQ-protected
iron(III) chloride (0.059 mg, 0.36 mmol, 2 equiv), and
odorants were (A) benzaldehyde, (B) ethanethiol, and (C) butane-1-
(dimethoxymethyl)benzene (0.132 mL, 0.91 mmol, 5 equiv) was
thiol.
refluxed in dry THF for 24 h or when LCMS indicated the reaction
was complete. The mixture was cooled, then concentrated in vacuo.
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The resulting residue was subjected to flash column chromatography
(10:90 hexane/EtOAc) to yield a 2:1 mixture of CyHQ-BZA
diastereomers (0.020 g, 35%): Diastereomer 1: 1H NMR (500
MHz, methanol-d4, δ): 8.22 (dd, J = 8.4, 5.5 Hz, 1H), 7.98 (dq, J =
9.1, 4.5 Hz, 1H), 7.64−7.55 (m, 3H), 7.47−7.39 (m, 3H), 7.24 (dt, J
= 9.1, 2.9 Hz, 1H), 6.05 (d, J = 2.1 Hz, 1H), 5.49 (tdd, J = 7.6, 5.8, 2.5
Hz, 1H), 4.56 (td, J = 8.0, 2.3 Hz, 1H), 4.38 (ddd, J = 8.3, 5.0, 1.3 Hz,
1H); 13C NMR (126 MHz, methanol-d4, δ): 162.8, 148.4, 137.2,
133.8, 129.1, 128.0, 126.6, 121.6, 118.0, 116.8, 114.9, 105.1, 94.3,
78.8, 71.0, 54.6, 28.1; Diastereomer 2: 1H NMR (500 MHz,
methanol-d4, δ): 8.28 (dd, J = 8.4, 5.5 Hz, 1H), 8.05−7.99 (m,
1H), 7.67 (dd, J = 8.3, 3.9 Hz, 1H), 7.64−7.55 (m, 2H), 7.49−7.38
(m, 3H), 6.21 (d, J = 2.0 Hz, 1H), 5.49 (tdd, J = 7.6, 5.8, 2.5 Hz, 2H),
4.70 (ddd, J = 8.9, 6.9, 2.1 Hz, 1H), 4.26 (ddd, J = 8.3, 6.5, 1.8 Hz,
1H); 13C NMR (126 MHz, methanol-d4, δ): 164.7, 148.7, 137.4,
133.8, 129.1, 128.0, 126.4, 121.6, 118.0, 116.7, 114.9, 104.8, 94.3,
78.4, 70.7, 54.6, 30.7; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C19H14N2O3 319.1077; found 319.1083.
(8-Cyano-7-hydroxyquinolin-2-yl)methyl phenethylcarbamate
(CyHQ-2PEA). 2-Phenethylamine (0.1 g, 0.83 mmol, 1 equiv),
triethylamine (0.690 mL, 4.95 mmol, 6 equiv), and 1,1 ′-carbon-
yldiimidazole (0.401 g, 2.47 mmol, 3 equiv) were stirred together at
RT. Water was added and the mixture was filtered, dried over
anhydrous sodium sulfate, and concentrated. The resulting residue
was dissolved in dichloromethane (4 mL) followed by addition of
triethylamine (0.432 mL, 3.1 mmol, 5 equiv), MOM-CyHQ-OH
(0.151 g, 0.62 mmol, 1 equiv). Once TLC indicated the reaction to be
complete, water was added and the resulting mixture was
concentrated in vacuo. The resulting residue was treated with a 1:1
solution of TFA/CH2Cl2 (5 mL) for 12 h and then concentrated in
vacuo. The resulting residue was subjected to flash column
chromatography (95:5 CH2Cl2/MeOH) to provide CyHQ-2PEA
(0.133 g, 62%): 1H NMR (500 MHz, methanol-d4, δ): 8.23 (d, J = 8.4
Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.32−
7.16 (m, 7H), 5.34 (s, 2H), 3.40 (t, J = 7.4 Hz, 2H), 2.84 (t, J = 7.4
Hz, 2H); 13C NMR (126 MHz, methanol-d4, δ): 164.2, 160.0, 157.1,
148.3, 139.1, 137.2, 133.8, 128.5, 128.1, 125.9, 121.5, 117.6, 117.0,
114.7, 94.2, 66.5, 42.1, 35.7; HRMS (ESI-TOF) m/z: [M + H]+ calcd
for C20H17N3O3 348.1343; found 348.1348.
4H), 1.26 (t, J = 7.4 Hz, 6H); 13C NMR (126 MHz, chloroform-d, δ):
163.9, 162.7, 147.5, 137.6, 133.7, 121.7, 119.0, 118.8, 115.5, 94.9,
54.1, 26.4, 14.4; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C15H16N2OS2 305.0777; found 305.0803.
2-(Bis(butylthio)methyl)-7-hydroxyquinoline-8-carbonitrile
1
(CyHQ-(SBu)2). 75% yield. H NMR (500 MHz, chloroform-d, δ):
7.93 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.9 Hz, 1H), 7.56 (d, J = 8.4 Hz,
1H), 7.23 (d, J = 9.0 Hz, 1H), 6.21 (s, 1H), 5.16 (s, 1H), 2.66 (t, J =
7.6 Hz, 4H), 1.57 (tt, J = 8.4, 6.2 Hz, 4H), 1.41−1.32 (m, 4H), 0.86
(t, J = 7.4 Hz, 6H); 13C NMR (126 MHz, chloroform-d, δ): 164.3,
162.5, 147.7, 137.2, 133.5, 121.2, 119.4, 118.3, 116.5, 94.8, 54.9, 32.0,
31.3, 22.0, 13.6; HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C19H24N2OS2 361.1403; found 361.1433.
2-(Bis(tert-butylthio)methyl)-7-hydroxyquinoline-8-carbonitrile
1
(CyHQ-(S-t-Bu)2). 85% yield. H NMR (500 MHz, chloroform-d, δ):
8.08 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.77 (d, J = 8.5 Hz,
1H), 7.25 (d, J = 8.9 Hz, 1H), 5.39 (s, 1H), 1.32 (s, 18H); 13C NMR
(126 MHz, chloroform-d, δ): 166.8, 162.2, 147.0, 136.8, 133.7, 121.8,
120.5, 118.0, 115.0, 95.6, 49.8, 46.7, 31.1; HRMS (ESI-TOF) m/z:
[M + H]+ calcd for C19H24N2OS2 361.1403; found 361.1408.
Photolysis Reactions. Solutions of the CyHQ-protected odor-
ants (0.1 mM) were prepared in KMOPS buffer. Solutions were
placed in a 3 mL quartz cuvette and continuously stirred with a
stirring bar. Irradiation was carried out with an LED lamp (Cairn
OptoLED Lite) at 365 and 405 nm with constant stirring. The lamp
intensity was measured by ferrioxalate actinometry.66 Aliquots (50
μL) were sampled at different time intervals and analyzed by reverse-
phase uHPLC. Three repeats were carried out for each experiment.
HPLC analysis was performed on an Agilent 1290 Infinity series
uHPLC using a Zorbax Eclipse Plus C18 column, monitoring the
AUC at 320 nm. Separations was carried out with a gradient elution
(flux rate of 0.3 mL/min) using a mobile phase composed of A =
0.1% trifluoroacetic acid in water and B = acetonitrile (starting from
5% B to 100% over 12 min and re-equilibrating to 5% B before the
next run). The quantification of the percentage of the starting material
remaining was performed by comparison of the AUC measured with
calibration curves created from known concentrations of the substrate
(external standard method). Percent remaining was plotted versus
time. The time in seconds for 90% of starting material to be
consumed (t90%) was obtained by fitting a single exponential decay
curve to the data using DeltaGraph (Red Rock Software). The
quantum efficiency (Φu) of the photolysis reaction was calculated
from the following equation:
2-((4-Allyl-2-methoxyphenoxy)methyl)-7-hydroxyquinoline-8-
carbonitrile (CyHQ-EG). To a microwave reaction vial, MOM-CyHQ-
OMs (0.065 g, 0.20 mmol, 1 equiv), eugenol (0.041 mL, 0.26 mmol,
1.3 equiv), and potassium carbonate (0.083 g, 0.60 mmol, 3 equiv)
were added to dimethylformamide (1 mL). The resulting mixture was
stirred with microwave heating at 90 °C for 30 min, then cooled and
concentrated in vacuo. The resulting residue was subjected to flash
column chromatography (90:10 CH2Cl2/MeOH) to provide CyHQ-
−1
Φ = (Iσt90%
)
u
where t90% = time required to consume 90% of the starting material, I
represents the lamp intensity in Einstein cm−2 s−1, and σ is the
decadic extinction coefficient (1000 × ε, molar extinction coefficient)
(Table S1).24,26,67 The release of benzaldehyde, phenethylamine, and
eugenol was quantified by monitoring the AUC at 280 nm and plotted
vs time, fitting an exponential rise to max curve to the data.
Determination of the Stability toward Spontaneous
Hydrolysis in the Dark. Solutions of each CyHQ-protected odorant
(0.1 mM) in KMOPS buffer were kept in the dark and sampled at
different time intervals over 7 days. The percentage of starting
material remaining was determined by HPLC analysis as described for
the photolysis reactions.
1
EG as yellow semisolid 0.040 g, 57% yield): H NMR (500 MHz,
methanol-d4, δ): 8.25 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 9.1 Hz, 1H),
7.67 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 9.1 Hz, 1H), 6.97 (d, J = 8.2 Hz,
1H), 6.76−6.65 (m, 1H), 5.96 (ddt, J = 16.8, 10.0, 6.7 Hz, 1H), 5.36
(s, 2H), 5.13−4.96 (m, 2H), 3.89 (s, 3H), 3.86−3.69 (m, 2H), 1.45−
1.17 (m, 2H); 13C NMR (126 MHz, methanol-d4, δ): 164.7, 160.7,
149.6, 148.5, 146.2, 137.7, 137.1, 134.0, 133.8, 121.5, 120.4, 118.0,
117.5, 115.0, 114.4, 114.3, 112.7, 94.1, 71.8, 55.1, 39.3; HRMS (ESI-
TOF) m/z: [M + H]+ calcd for C21H18N2O3 347.1390; found
347.1396.
General Procedure for the Preparation of CyHQ-Protected
Thiols. Aldehyde 2 (0.83 mmol, 1 equiv), 12 M hydrochloric acid
(4 mL), and alkanethiol (4.15 mmol, 5 equiv) were combined and
stirred at RT. When LCMS analysis indicated that the reaction was
complete, a saturated solution of sodium bicarbonate was added
followed by ethyl acetate. The ethyl acetate layer was concentrated in
vacuo, and the resulting residue subjected to flash column
chromatography using hexane/EtOAc as the eluent to provide the
corresponding CyHQ-protected thiol.
2-(Bis(ethylthio)methyl)-7-hydroxyquinoline-8-carbonitrile
(CyHQ-(SEt)2). 69% yield. 1H NMR (500 MHz, chloroform-d, δ): 8.08
(d, J = 8.5 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H),
7.33 (d, J = 9.0 Hz, 1H), 6.79 (s, 1H), 5.25 (s, 1H), 2.75−2.60 (m,
Cell Culture. Heterologous Expression and Transient Trans-
fection of TRPV1 and TRPA1 in HEK 293 Cells. HEK 293T cells were
grown in HEK media [DMEM, 10% FBS (MP Biomedicals), L-
glutamine (2 mM), and penicillin/streptomycin (100 μg/mL)
(Invitrogen)] at 37 °C with 5% CO2. The hTRPA1 channel was
transiently expressed in the cells from the recombinant expression
plasmid pcDNA5-FRT carrying the entire protein coding region for
hTRPA1 (gift of Dr. Gisselman, Bochum University, Germany). The
hTRPV1 channel was transiently expressed using the plasmid
pCMV6-NEO-TRPV1 plasmid (Origene). Semiconfluent HEK
293T cells in 35 mm dishes were transiently cotransfected with 1
μg of pcDNA5-FRT/hTRPA1 and 0.2 μg of a separate plasmid
(pXoon) carrying the coding sequence for green fluorescent protein
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(GFP) using Calfectin reagent (SignaGen). At 24−48 h post-
transfection, the cells were gently lifted from the dishes using 2 mM
EDTA in phosphate-buffered saline, washed with HEK media,
replated as individual cells/small clusters in HEK media without
antibiotics on 35 mm plates, and allowed to recover for at least 2 h
prior to electrophysiology.
Stably Transfected HEK 293 Cells Expressing TRPV1. HEK 293
cells were cultured in DMEM media with 10% FBS and 1% penicillin/
streptomycin in cell culture flasks at 37 °C and 5% CO2. HEK 293
cells stably expressing TRPV1 were created by transfection with a
pCMV6-NEO-TRPV1 plasmid (Origene) using TrueFect reagent
(United BioSystems) and G418 (600 μg/mL) for selection. The cells
were cultured in DMEM media with 10% FBS and 1% PEN-STREP.
G418 antibiotic (300 μg/mL) was used for maintaining the stable
expression of TRPV1.
Whole-Cell Patch Clamp Recording. Human TRPA1 and
TRPV1 ion currents were investigated using whole-cell patch clamp
recording. The current was measured with a 200B patch clamp
amplifier (Molecular Devices) and a digital interface (Digidata 1320A,
Molecular Devices), lowpass filtered at 5−10 kHz, sampled at 2−20
kHz, and in most cases digitally filtered at 1−1.4 kHz. Analysis of the
data was carried out using pCLAMP 9.2 software (Molecular
Devices). Membrane voltage was held at −60 mV. Patch pipettes
were fabricated from borosilicate capillary glass (BF150−86-10, Sutter
Instrument) using a Flaming−Brown micropipette puller (P-87,
Sutter Instrument). The fire polished patch pipet had a resistance of
about 5 MΩ when filled with a solution composed of KCl (140 mM),
EGTA (1 mM), and Hepes (10 mM) adjusted to pH 7.4 with KOH.
To measure TRPA1 current, the pipet solution was supplemented
with sodium pyrophosphate (1 mM), which is known to protect the
channels from the run-down.68 Normal Ringer solution was used as a
bath solution and contained NaCl (140 mM), MgCl2 (1 mM), CaCl2
(1 mM), and HEPES (10 mM) adjusted to pH 7.4 with NaOH. To
measure maximal TRPA1 currents no Ca2+ was added to the bath
solution whereby preventing calcium-dependent desensitization of the
channels.69 Bath exchange was performed using a rapid solution
changer with a modified tube holder, RSC-160 (Bio-Logic, Claix,
France). Data were recorded under continuous superfusion with
Ringer solution alone or supplemented with indicated concentration
of CyHQ reagents for 30 s. This time period started 10 s before
applying the LED light pulse. GFP-positive HEK 293T cells were
visualized using an Axiovert 100 inverted microscope (Carl Zeiss)
equipped with a mercury arc lamp (HBO100) coupled to the GFP
filter set (Ex BP 475/40, Em 530/50, 1114−459, Zeiss). The sample
illumination time did not exceed 20 s to minimize any possible
phototoxicity and aberrant activation of TRPA1 channels.70 The LED
light source (Prizmatix) generating 365 and 405 nm light was
triggered by Clampfit 9.2 software in-line with the whole-cell current
recording
from a pipet and a defined area near a cell was irradiated with a short
(10−200 ms) pulse of 405 nm laser light while imaging at 488 nm.
Mouse Electroolfactograms (EOGs). Adult C57/B6 mice of
both sexes were used in this study approved by the University of
Florida IACUC protocol 201608162. Mice were anesthetized with
CO2, rapidly decapitated, and the head split along the cranial midline.
Septal tissue was removed to expose olfactory turbinates. AAc in the
vapor-phase was delivered as a short 100 ms pulse by a pressurized
nitrogen line connected to a sealed 100 mL glass bottle and directly
injected into a continuous stream of humidified carbogen flowing over
the tissue. AAc was prepared by diluting a 1 M stock solution into
deionized water to generate a final working concentration. EOGs were
recorded with a standard glass micropipette tip-filled with agarose and
backfilled with a standard phosphate-buffered saline (PBS) using a
Multiclamp 700A amplifier controlled by Multiclamp 700A and
Clampex 9.2 software (Molecular Devices). EOGs were measured as
the maximal peak amplitude from the prepulse baseline using
Clampfit 9.2 software (Molecular devices). An LED light source
(Prizmatix) generating 365 and 405 nm light was triggered by
Clampfit 9.2 software in-line with the whole-cell current recording.
Delay of the EOG and rise time from baseline to the peak was
measured using Clampfit 9.2 software.
Reagents for Patch Clamp Recording and Electroolfacto-
grams. CyHQ reagents were prepared as a 200 mM stock in DMSO
and kept at −20 °C until needed. Freshly made working solutions,
typically 250 μM, were made from the stock by adding directly to PBS
and sonicating. Eugenol and AAc were prepared as a 1 M stock in
DMSO. Eugenol was applied as a working solution in PBS at the
concentration indicated for each experiment. AAc was prepared as a
solution in ultrapure water at nominal concentration of 1 mM and
applied to the mouse nasal preparation as a vapor phase. All
experiments were performed under dimmed room light which did not
induce any measurable activation of either TRP channels or mouse
EOG.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschembio.0c00541.
1
Figures S1−S13, Table S1, H and 13C NMR spectra,
and UV−vis spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Timothy M. Dore − New York University Abu Dhabi, Abu
Dhabi, United Arab Emirates; Department of Chemistry,
University of Georgia, Athens, Georgia 30602, United States;
orcid.org/0000-0002-3876-5012; Email: timothy.dore@
nyu.edu
Jeffrey R. Martens − Department of Pharmacology and
Therapeutics and Center for Smell and Taste, University of
Florida, Gainesville, Florida 32610, United States;
Email: martensj@ufl.edu
Live Cell Imaging of Calcium and TRPV1 Activation. Live cell
imaging was carried out on an Olympus FluoView FV1000MPE
confocal microscope. Excitation using an argon ion laser was set at
488 nm, and emitted light was reflected through a 500−600 nm filter
from a dichroic mirror. Data capture and extraction was carried out
with FluoView 10-ASW version 4.0 (Olympus), ImageJ-Fiji, and
DeltaGraph (Red Rock Software).
Calcium Dye Loading. One day prior to the experiment, HEK 293
cells (stably expressing TRPV1 channels) were plated on 35 mm glass
bottom dish (previously coated with poly-D-Lysine). Fluo-4 AM (50
μg, Life Technologies) was dissolved in DMSO (50 μL) and added
with Pluronic F-127 (Molecular Probes) to HBSS and sonicated for 5
min to create a solution containing a 0.02% final concentration of
each. The solution was loaded onto the cells growing in the 35 mm
glass bottom dish and incubated for 30 min in a humidified CO2
incubator (37 °C, 5% CO2). The cells were washed with HBSS and
maintained in HBSS during the photoactivation experiments.
TRPV1 Activation on HEK 293 Cells. The 405 nm laser integrated
on the confocal microscope was used for excitation. Solutions of
CyHQ-protected odorants or VNA were added to the culture dish
Authors
Sangram Gore − New York University Abu Dhabi, Abu Dhabi,
United Arab Emirates; orcid.org/0000-0003-4236-7604
Kirill Ukhanov − Department of Pharmacology and
Therapeutics and Center for Smell and Taste, University of
Florida, Gainesville, Florida 32610, United States;
orcid.org/0000-0003-0633-9067
Cyril Herbivo − New York University Abu Dhabi, Abu Dhabi,
United Arab Emirates
Naeem Asad − New York University Abu Dhabi, Abu Dhabi,
United Arab Emirates; orcid.org/0000-0001-6871-5270
K
https://dx.doi.org/10.1021/acschembio.0c00541
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Yuriy V. Bobkov − Department of Pharmacology and
Therapeutics and Whitney Laboratory for Marine Bioscience,
University of Florida, Gainesville, Florida 32610, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.0c00541
Author Contributions
¶S.G. and K.U. contributed equally.
Funding
This research was supported by New York University Abu
Dhabi (T.M.D.) and by US Public Health Service Grant NIH/
NIDCD R01DC009606 (J.R.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Part of this work was carried out using Core Technology
Platform resources at New York University Abu Dhabi.
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