Development of a red-light-controllable nitric oxide releaser to control smooth muscle relaxation in vivo

Article abstract of DOI:10.1021/acschembio.0c00601

We designed and synthesized a novel Si-rhodamine derivative, NORD-1, as a red-light-controllable nitric oxide (NO) releaser, on the basis of photoredox parameter analysis. Red-light-responsive NO release from NORD-1 was confirmed by ESR spin trapping and quantified with an NO electrode and by means of Griess assay. The NO release cross section (ε_{656 nm}·Φ_NO) of NORD-1 was calculated to be 3.65 × 10², which is larger than that of a previously reported yellowish-green-light-controllable NO releaser, NO-Rosa5. The photoresponsiveness of NO release from NORD-1 was precise and efficient enough to induce vasodilation ex vivo under Magnus test conditions. Finally, we showed that intracavernous pressure (ICP) could be controlled in rats in vivo with the combination of NORD-1 and a red-light source without increasing systemic blood pressure, which is a serious side effect of usual NO releasers, such as nitroglycerin and isopentyl nitrite. NORD-1 is expected to be a useful chemical tool for NO research, as well as a candidate agent to control the circulatory system.

Full text of DOI:10.1021/acschembio.0c00601

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Articles
Development of a Red-Light-Controllable Nitric Oxide Releaser to
Control Smooth Muscle Relaxation in Vivo
Naoya Ieda,* Yuji Hotta, Ayaka Yamauchi, Atsushi Nishikawa, Takahiro Sasamori, Daisuke Saitoh,
Mitsuyasu Kawaguchi, Kazunori Kimura, and Hidehiko Nakagawa*
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ABSTRACT: We designed and synthesized a novel Si-rhodamine
derivative, NORD-1, as a red-light-controllable nitric oxide (NO)
releaser, on the basis of photoredox parameter analysis. Red-light-
responsive NO release from NORD-1 was confirmed by ESR spin
trapping and quantified with an NO electrode and by means of
Griess assay. The NO release cross section (ε656 nm·ΦNO) of NORD-
1 was calculated to be 3.65 × 102, which is larger than that of a
previously reported yellowish-green-light-controllable NO releaser,
NO-Rosa5. The photoresponsiveness of NO release from NORD-1
was precise and efficient enough to induce vasodilation ex vivo under
Magnus test conditions. Finally, we showed that intracavernous pressure (ICP) could be controlled in rats in vivo with the
combination of NORD-1 and a red-light source without increasing systemic blood pressure, which is a serious side effect of usual
NO releasers, such as nitroglycerin and isopentyl nitrite. NORD-1 is expected to be a useful chemical tool for NO research, as well as
a candidate agent to control the circulatory system.
(Scheme 1a). Photoexcitation of the antenna moiety results in
electron transfer from the NO-releasing moiety to the antenna
moiety. In the charge-separated form, the phenoxyl radical
readily undergoes homolysis of the N−N bond to release
relatively stable NO and quinoneimine. On the basis of this
mechanism, we have developed various NO releasers that are
controllable with low-energy blue or yellowish-green light.20−23
This strategy to utilize N-nitrosoaniline as a NO-releasing
moiety in low-energy-light-controllable NO releasers has been
recognized and utilized by various groups.24−26 On the basis of
our previous results and the fact that the wavelength is longer
and the light is more permeable and more biologically
applicable,27 in the present work, we designed a red-light-
controllable PeT-type NO releaser, NORD-1, which is
controllable by the longest wavelength light in the PeT-type
NO releasers. Photoresponsive NO release from NORD-1 was
confirmed by ESR spin trapping, as well as with an NO
electrode and by means of Griess assay. NO release was
controllable by red-light irradiation not only in vitro but also ex
vivo (vasodilation of rat aorta strips). Finally, we applied
NORD-1 for the photomanipulation of intracavernous
INTRODUCTION
■
Nitric oxide (NO) is a gaseous transmitter that is involved in
vasodilation, neuronal signaling, and immune response.1−4
Currently, NO donors are used clinically for the symptomatic
treatment of cardiac infarction and they are also considered to
be candidate therapeutic agents for circulatory system diseases,
such as erectile dysfunction and pulmonary hypertension.5
Although various caged NOs have been developed to control
NO’s bioactivity precisely, their biological application is
difficult because NO release in some caged NOs requires
high-energy, ultraviolet light6−9 and some are triggered by two-
photon excitation using a pulse laser, which is expensive and
less readily available for use.10,11 Not only synthetic small
molecules but also S-nitrosothiol derivatives, such as S-
nitrosoglutathione, have been reported to release NO in
response to low-energy red light.12,13 However, S-nitrosothiol
could induce nitrosyl transfer to other thiols of cysteine residue
in proteins, peptide, and coenzyme A to induce various signals
without light stimulation.14,15 To avoid this drawback, we have
focused on photoinduced electron transfer (PeT) as a trigger
for low-energy-light-controllable NO releasers. Although PeT
has usually been utilized for fluorescence quenching in
fluorescent probes, we anticipated that PeT could be an
efficient release trigger for caged compounds, because it is
applicable to a broad range of light-harvesting dyes.16−19 On
the basis of this idea, we have developed PeT-type NO
releasers composed of two functional groups, a light-harvesting
antenna moiety and an NO-releasing N-nitrosoaniline moiety
Received: July 21, 2020
Accepted: October 27, 2020
https://dx.doi.org/10.1021/acschembio.0c00601
© XXXX American Chemical Society
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Scheme 1. (a) Plausible Mechanism of Photoinduced NO Release from PeT-Type NO Releasers, (b) Structures of a Yellowish-
Green-Light-Controllable NO Releaser, NO-Rosa5, a Red-Light-Controllable One, NORD-1, and an NIR Fluorescence Dye,
SiR650, and (c) Synthetic Scheme of NORD-1
pressure in rats in vivo. The results indicate that NORD-1 will
be a useful chemical tool for NO research, as well as a
candidate agent to control circulatory systems.
was linked to Si-rhodamine by reductive amination. N-
Nitrosylation with NaNO2 and deprotection of the tert-
butyldimethylsilyl group under mild conditions with a NaF/
HF buffer gave NORD-1 on a >300 mg scale.31 The structure
1
and purity were confirmed by H nuclear magnetic resonance
RESULTS AND DISCUSSION
■
(NMR), 13C NMR, electrospray ionization mass spectrometry
(ESI-MS), and elemental analysis (see the Experimental
Section in the Supporting Information).
Design and Synthesis. As an antenna moiety for the red-
light-controllable NO releaser, we chose SiR650, because its
synthesis is straightforward (Scheme 1b).28 Before synthesizing
NORD-1, we first examined the photoredox parameters of
SiR650 to assess whether PeT from the NO-releasing moiety
to the antenna could be expected to take place under
photoactivated conditions. We measured the redox potential
of SiR650 and calculated the Gibbs free energy of PeT
(ΔGPeT) on the basis of the equation in the Supporting
Information (eq S1).29 Cyclic voltammetry indicated that the
reduction potential of SiR650 was −0.73 V vs SCE (Figure
S1). The oxidation potential of N-methyl-N-nitroso-4-amino-
phenol, as a model compound of the NO-releasing moiety, was
reported to be 0.97 V vs SCE (Table S1).22 In eq S1, Ered(A)
and Eox(D) correspond to the reduction potential of SiR650
and the oxidation potential of the NO-releasing moiety,
respectively. By applying these values to eq S1, ΔGPeT of the
designed NO releaser, NORD-1, was calculated to be −0.16
eV, indicating that PeT should take place.
Next, we measured the absorption and fluorescence spectra
of NORD-1 and SiR650 as a reference compound. As shown in
Figure 1, the absorption maximum (λmax) of NORD-1 (656
nm) was red-shifted by 10 nm from that of SiR650 (646 nm).
Such a red shift was also observed in the case of our yellowish-
green-light-controllable NO releaser, NO-Rosa5.22 It is
probably caused by π-stacking between the antenna and NO-
releasing moiety.32 Due to the strong absorption of NORD-1
at around 650 nm, a Xe lamp equipped with a 630−690 nm
band-pass filter (MAX-303, Asahi Spectra) was utilized as a
light source for NO release reaction. On the contrary, the
fluorescence quantum yield (ΦF) of NORD-1 (0.12) was lower
than that of SiR650 (0.31),18 implying that the excitation
energy of NORD-1 was released through a nonradiative
process, probably PeT.
Detection of NO Release from NORD-1. To detect NO
release from NORD-1 in response to red-light irradiation, the
electron spin resonance (ESR) spin-trapping method with iron
ion and N-methylglucamine dithiocarbamate complex (Fe-
MGD) was employed (Figure 2).33 Fe-MGD traps NO
efficiently to form a NO-Fe-MGD complex, which exhibits a
typical three-line signal at around 330 mT in 1 GHz ESR
NORD-1 was synthesized according to Scheme 1c. Briefly,
after lithiation of 2-(2-bromophenyl)-1,3-dioxolane (1) with
sec-BuLi, a reported Si-xanthone (2) was added to construct
the Si-rhodamine structure.30 Acetal protection was removed
under acidic conditions to obtain a formyl Si-rhodamine (3).
Aniline derivative (4), a precursor of the NO-releasing moiety,
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Figure 1. Absorption (solid) and fluorescence (dash) spectra of
NORD-1 (red) and SiR650 (gray). The absorption spectrum of each
compound (10 μM) in HEPES buffer (100 mM, pH 7.3, 0.1%
DMSO) was measured with an Agilent 8453 spectrometer. The
fluorescence spectrum of the same solution was measured with an
RF5300-PC fluorescence spectrometer (Shimadzu). Ex. 580 nm. The
fluorescence quantum yield of NORD-1 was calculated from the
reported value of SiR650.
Figure 3. (a) Measurement of NO release from NORD-1 with an
ISO-NOP NO electrode (World Precision Instruments). A solution
of NORD-1 (10 μM) in 100 mM HEPES buffer (pH 7.3, 0.1%
DMSO) was irradiated with a MAX-303 (Asahi Spectra) equipped
with a 630−690 nm band-pass filter (light intensity: 36 mW cm−2).
(b) Temporal control of NO release from NORD-1 under the above
conditions.
To determine the total amount of NO release, we utilized a
Griess assay kit (Dojindo, Figure S3).2 Under ambient
conditions, NO is easily oxidized to nitrite or nitrate anion,
which was quantified by Griess assay using sulfanilamide, 1-
naphthylethylenediamine, and nitrate reductase. After com-
plete photodecomposition of a 10 mL solution of NORD-1
(10 μM) in HEPES buffer (100 mM, pH 7.3, 0.1% DMSO),
the solution was subjected to Griess assay. As a result, it was
Figure 2. ESR spectra of a solution containing NORD-1 (100 μM),
FeSO4 (1.5 mM), and N-methylglucamine dithiocarbamate (6 mM)
in HEPES (100 mM, pH 7.3, containing 1% DMSO) after
photoirradiation with red light (630−690 nm, 56 mW cm−2) for 15
min.
−
found that 10 μM NORD-1 released 6.87 μM NO2− and NO3
after complete photodecomposition.
We also determined the NO release quantum yield (ΦNO
)
by using an NO electrode and Reinecke’s salt (NH4[Cr-
(NH3)2(SCN)4]) as a chemical actinometer, which can count
photons over a wide wavelength range in the visible-light
region (Table S2).35 The Xe lamp of a fluorescence
spectrometer (RF5300-PC, Shimadzu) was employed as a
light source (600 nm, bandwidth: 15 nm). A solution of
NORD-1 in HEPES buffer was irradiated for one minute while
the NO concentration was monitored with the NO electrode.
Likewise, a solution of Reinecke’s salt was irradiated under the
same conditions to determine the number of photons. The
spectrometry. After red-light irradiation (630−690 nm, 56 mW
cm−2) of a HEPES-buffered solution containing Fe-MGD and
NORD-1 (100 μM) for 15 min, the ESR spectrum showed a
typical triplet signal assigned to the NO-Fe-MGD complex. In
the absence of red-light irradiation or NORD-1, this signal was
not observed (Figure S2).
To quantify NO, we employed a NO electrode (ISO-NOP,
World Precision Instruments).34 A solution of NORD-1 (10
μM) in HEPES buffer was stirred at 37 °C, and the NO
concentration was monitored. As shown in Figure 3a, the NO
concentration was increased in response to red-light
irradiation. Under these irradiation conditions (10 μM
NORD-1, 630−690 nm, 36 mW cm−2), the NO concentration
reached a plateau at 2.31 μM after 5 min under aerobic
conditions. Further, we carried out repeated light on/off
switching to investigate the photoresponsivity and repeatability
of the NO release (Figure 3b). The NO concentration
increased only during light irradiation and decreased when the
light was turned off, and these changes were reproducible.
These results indicated that the release of NO from NORD-1
is precisely controllable by a red-light source.
Φ
NO of NORD-1 was calculated to be 3.85 × 10−3. This value
is greater than that of NO-Rosa5 (1.01 × 10−3), which was
sufficient enough to induce a biological response.23 The
uncaging cross section, ε656 nm·ΦNO, was calculated to be 3.65
× 102, which is also greater than that of NO-Rosa5 (98.2).23
The photodecomposition products of NORD-1 were
explored by LC−ESI-MS examination of an irradiated solution
of NORD-1, and two main products were detected (Figure
S4). One was compound 3 (Figure S4c-2), and the other one
was an aminophenol derivative that has a five membered ring
(Figure S4c-4). The retention time and mass number of 3 were
matched with those of the authentic one. On the basis of the
ion mass numbers and structure of NORD-1, these products
appeared to have been generated via dismutation (Scheme S1).
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After NO release to form S1, the back electron transfer to
quinonimine would take place to form semiquinonimine S2.
Then, two of S2 were dismutated into an aminophenol S3 and
a quinonimine S4. In S3, aniline’s nitrogen would attack
xanthene to form a five membered ring product S5, while S4
was isomerized to S6 followed by hydrolysis to form 3.
Biological Applications of NORD-1. To confirm the
applicability of NORD-1 for cellular experiments, imaging was
conducted with a fluorescent NO probe, DAR-4M AM,36 to
visualize NO release from NORD-1. DAR-4M AM is taken up
by cells and hydrolyzed to form DAR-4M, which reacts with
NO to form a strongly fluorescent dye. After incubation of
HEK293T cells with DAR-4M AM (10 μM), the cells were
washed with D-PBS, and then, NORD-1 (10 μM) was added
and the cells were irradiated with a MAX-303 (Asahi Spectra)
equipped with a 630−690 nm band-pass filter. As shown in
Figure 4, a fluorescence increment was observed in the
presence of NORD-1, while no increase was observed in the
absence of either NORD-1 or light irradiation (Figure S5).
Figure 5. Changes in the tension of rat aorta ex vivo induced by red-
light-mediated NO release from NORD-1 in the presence of a nitric
oxide synthetase inhibitor, L-NAME (10 μM). Rat aorta was treated
with noradrenaline (NA, 10 μM), followed by NORD-1 (10 μM), in
Magnus tubes. (a) Tube was irradiated (630−690 nm, 36 mW cm−2)
for 1 or 3 min, and (b) ODQ (10 μM) was added before the addition
of NORD-1. Light irradiation was conducted for 3 min.
decreased during irradiation and immediately recovered when
the light was turned off. To confirm that this relaxation was
indeed induced through the NO signaling pathways, the aorta
was pretreated with 1H-1,2,4-oxadiazolo[4,3-a]quinoxalin-1-
one (ODQ), a sGC inhibitor.37 As expected, the relaxation of
the aorta was blocked by the ODQ treatment (Figure 5b).
Overall, these results indicate that the vasodilation could be
finely controlled by red-light-induced release of NO from
NORD-1.
Figure 4. Photocontrolled NO release in HEK293T cells.
Fluorescence imaging of NO release from NORD-1 in HEK293T
cells was performed by using DAR-4M AM. Cultured HEK293T cells
were treated with DAR-4M AM (20 μM) and NORD-1 (10 μM).
The dish was then photoirradiated with red light (650−690 nm, 40
mW cm−2 for 15 min), and cells were observed with a confocal
microscope to obtain DIC and fluorescence images: (a) before
photoirradiation and (b) after photoirradiation. Upper, fluorescence
images; lower, DIC images. The scale bar represents 40 μm.
Finally, we examined whether NORD-1 could be used to
photocontrol NO-mediated physiological events in vivo
(Figure 6). For this purpose, we focused on the intracavernous
pressure (ICP) change in rat penis, because ICP increment and
the subsequent erectile event, are mainly governed by NO.38
Under anesthesia with isoflurane, polyethylene (PE-50) tubing
was inserted into the left carotid artery of rats to record the
systemic blood pressure (BP) (Figure S6). A needle connected
to PE-50 tubing was inserted into the left penile corpus
cavernosum for ICP measurement. After injection of a solution
of NORD-1 (100 μL of 100 μM) in heparinized saline (5 U
mL−1, 1% DMSO), photoirradiation was conducted together
with weak electrostimulation (2 Hz) of the cavernous nerve. In
the vehicle experiment (Figure 6a,b, w/o NORD-1), light
irradiation had no significant effect with either 2 (weak) or 8
Hz (strong) electrostimulation. On the contrary, in the
presence of NORD-1, ICP was increased light dependently
during 2 Hz stimulation (Figure 6a,b, w/NORD-1). As shown
in Figure 6a, this increment was reversible and dependent on
the light intensity. Notably, light irradiation did not induce a
significant decrease of BP. This is important because
spontaneous NO releasers, e.g., isoamyl nitrite and nitro-
glycerin, cause clinically serious systemic side effects, such as
hypotension (low BP), headache, and arrhythmia.39 Our
results indicated that light-induced NO release and vaso-
dilation could be precisely controlled even under in vivo
NO induces vasodilation by the activation of soluble
guanylyl cyclase (sGC), which catalyzes the production of
cyclic guanosine monophosphate (cGMP).2 We applied
NORD-1 to photocontrol the vasodilation of a rat aorta ex
vivo with red-light irradiation. All animal experiments were
performed following the Guiding Principles for the Care and
Use of Laboratory Animals of the Science and International
Affairs Bureau of the Japanese Ministry of Education, Culture,
Sports, Science, and Technology. The study design was
reviewed and approved by the Animal Experimentation Ethics
Committee of Nagoya City University (No. H25-P-09). A strip
of rat aorta was placed in a Magnus tube filled with Krebs
buffer. The aorta was tensioned by exposure to noradrenaline
(NA). As shown in Figure 5a, relaxation of the aorta was not
observed before the addition of NORD-1, while red-light
stimulation induced relaxation in the presence of NORD-1 and
the extent of the relaxation was found to be dependent on the
irradiation time. Note that the tension of the aorta only
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Figure 6. Changes in intracavernous pressure (ICP) and blood pressure (BP) in vivo induced by red-light-irradiation-mediated NO release from
NORD-1. (a) (Left) Time-courses of blood pressure (BP) and intracavernous pressure (ICP) in the absence of NORD-1 and (Right) time-courses
of BP and ICP after injection of NORD-1 (100 μL, 100 μM) in heparized saline (5 U mL−1, 1% DMSO). (b) Statistical analysis of ICP changes
induced by NORD-1 and electrical stimulation. Mean arterial pressure (MAP) means the average value of BP. Data are expressed as mean SD
(shown as error bars, n = 4). Statistical significance was examined by application of a Bonferroni-type multiple t-test. *p < 0.01; ns: not significant.
conditions. To confirm that the injected NORD-1 induced
vasodilation of corpus cavernosum smooth muscle, the penis
with tunica albuginea of a NORD-1-injected rat was subjected
to Magnus test conditions. As shown in Figure S7, photo-
vasodilation was induced in the NORD-1-injected corpus
cavernous but not in the vehicle-injected one. The vasodilation
was completely blocked by ODQ. It should be noted that only
red-light irradiation (630−690 nm) did not induce vaso-
dilation at all in both the aorta strip and cavernosum as shown
in Figures 5a and 6b and Figure S7b, whereas it was reported
that endogenous S-nitrosothiol or nitrosyl-heme in tissues
could release NO by photolysis with a 670 nm LED light (up
to 100 mW cm−2) to induce vasodilation in facial arteries
segment of mice and coronary arteries segment of pigs.12,13 In
the above-mentioned papers reporting the vasodilation
induced by red light, the authors utilized segments of facial
arteries of mice or coronary arteries of pigs, while we utilized
the aorta and cavernosum of rats. These results suggest that
there is a difference in the cumulative amount of S-
nitrosothiols or nitrosyl-hemes in each tissue or animals.
Such light-induced endogenous NO release might be
abrogated under pathophysiological conditions where the
amount of S-nitrosothiols is depressed, e.g., due to loss of
NOS expression or insufficient O2 supply.40 In contrast,
NORD-1 can release enough amounts of NO to induce a
biological response in a highly controlled manner, regardless of
these factors. Overall, these results indicate that the red-light
irradiation of NORD-1 induced a release of NO, which
activated sGC in corpus cavernosum smooth muscle, followed
by an increase of blood flow locally in the cavernous body in
vivo. To our knowledge, this is the first example of the
successful application of a small-molecular photocontrollable
NO releaser to regulate vasodilation and physiological
functions in vivo.
In conclusion, we have designed a red-light-controllable NO
releaser, NORD-1, on the basis of a photoredox parameter
assessment. Red-light-induced NO release was confirmed by
ESR spin trapping, as well as with an NO electrode and by
means of Griess assay. We confirmed that the NO release was
precisely responsive to light stimulation. The ε656 nm·ΦNO was
calculated to be 3.68 × 102, which is greater than that of a
previously reported yellowish-green-light-controllable NO
releaser, NO-Rosa5. We confirmed not only that NORD-1
could photomanipulate vasodilation of rat aorta ex vivo but also
that intracavernous pressure was controllable by the combina-
tion of NORD-1 and a red-light source even in vivo. Although
Fussenegger’s group reported a similar photocontrollable
system, optogenetic guanylyl cyclase, their system requires
relatively toxic blue-light irradiation and genetic modifica-
tion.41 Our present results suggest that NORD-1 could be a
powerful chemical tool for NO research, as well as a candidate
photovasodilator to control circulatory systems.
METHODS
■
General Methods. Proton nuclear magnetic resonance spectra
(1H NMR) and carbon nuclear magnetic resonance spectra (13C
NMR) were recorded on a JEOL JNM-A500 spectrometer in the
indicated solvent. Chemical shifts (δ) are reported in parts per million
relative to the internal standard, tetramethylsilane. Elemental analysis
was performed with a Yanaco CHN CORDER NT-5 analyzer, and all
values were within 0.4% of the calculated values. Electrospray
ionization (ESI) mass spectra were recorded on a JEOL JMS-
T100LP4G mass spectrometer. Ultraviolet−visible-light absorption
spectra were recorded on an Agilent 8453 spectrometer. Irradiation
was conducted with MAX-303 (Asahi Spectra). All other reagents and
solvents were purchased from Sigma-Aldrich, Tokyo Chemical
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Industry, FUJIFILM Wako Pure Chemical Corp., Nacalai Tesque,
Kanto Chemical, Kishida Chemical, Junsei Chemical, or Dojindo and
used without purification. Flash column chromatography was
performed using silica gel 60 (particle size 0.032−0.075 mm)
supplied by Taiko Shoji. MPLC purification was performed using
YFLC-Wprep2XY-S (Yamazen).
Hz), 7.42 (1H, d, J = 7.1 Hz), 7.37 (1H, t, J = 7.5 Hz), 7.10 (2H, d, J
= 2.8 Hz), 7.00 (1H, d, J = 6.7 Hz), 6.94 (2H, d, J = 9.0 Hz), 6.69
(2H, d, J = 8.7 Hz), 6.67 (2H, d, J = 9.7 Hz), 6.42 (2H, dd, J = 2.8
Hz, 9.6 Hz), 4.91 (2H, s), 3.42 (12H, s), 0.68 (3H, s), 0.53 (3H, s).
13C NMR (CDCl3, 125 MHz, δ; ppm): 166.31, 158.54, 154.00,
147.58, 140.93, 137.81, 133.60, 131.61, 129.55, 129.47, 129.40,
127.60, 127.51, 122.73, 120.91, 116.46, 114.14, 44.87, 41.25, −0.55,
−0.80. Anal. calcd for C32H35ClN4O2Si·2H2O: C, 63.30; H, 6.47; N,
9.23. Found: C, 62.99; H, 6.28; N, 9.16. HRMS (ESI+) calcd,
535.25293; found, 535.25249 (M+, −0.81 ppm).
Measurements of Absorption and Fluorescence Spectra.
Absorption spectra of a solution of NORD-1 (10 μM) or SiR650 (10
μM) in HEPES buffer (100 mM, pH 7.3, 0.1% DMSO) were
recorded on an Agilent 8453. The fluorescence spectra of above
solution were recorded on an RF-5300PC (Shimadzu).
ESR Measurement Using Iron Dithiocarbamate Complex. A
solution (total volume 200 μL) of FeSO4·6H2O (1.5 mM), N-
methylglucamine dithiocarbamate (6 mM), and NORD-1 (100 μM)
in HEPES (pH 7.3, DMSO 1%) was photoirradiated (630−690 nm,
56 mW cm−2) at RT for 15 min to provide a sample for ESR studies.
ESR spectra were taken on a JES-RE2X spectrometer (JEOL Co.
Ltd.). The measurement conditions were as follows: microwave
power, 10 mW; frequency, 9.4200 GHz; field, 330 mT; sweep width,
7.5 mT; sweep time, 4 min; modulation width, 0.125 mT; time
constant; 0.10 s.
Photocontrol of NO Release in HEK293T cells. HEK293T cells
were plated on 3.5 cm glass dishes at 2.0 × 105 cells dish−1 with 2 mL
of Dulbecco’s modified Eagle medium (DMEM). The cells were
incubated at 37 °C in a humidified atmosphere of 5% (v/v) CO2 in air
for 2 days. The medium was washed with 2 mL of Dulbecco’s
phosphate-buffered saline (DPBS) twice. The cells were incubated
with 20 μM DAR-4M AM (DMSO 0.1%) for 1 h under the above
conditions. Then, the medium was washed with 2 mL of DPBS twice
and the cells were treated with 10 μM NORD-1 (DMSO 0.1%) or
DMSO for 30 min under the above conditions. The cells were
irradiated with a MAX-303 (630−690 nm, 40 mW cm−2 at 650 nm)
for 15 min. After irradiation, the cells were examined under a
differential interference contrast microscope (IX71, Olympus).
Photoinduced Vasodilation with NORD-1. A rat aortic strip
was placed in a Magnus tube filled with Krebs buffer (10 mL) at 37
°C. The strip was pretreated with noradrenaline (10 μM) and L-
NAME (10 μM). After equilibration, NORD-1 (10 μM) was added to
the tube. The strip was irradiated with a MAX-302 equipped with a
630−690 nm band-pass filter. After several cycles of photoirradiation,
ODQ (10 μM) was added and the photoirradiation was performed
again.
Photoinduced ICP Change in Vivo. Functional testing was
performed using intact male 10 week old Wistar-ST rats. Erectile
function was assessed on the basis of changes in intracavernous
pressure (ICP) in response to electrical stimulation of the cavernous
nerves. Briefly, under anesthesia with isoflurane, polyethylene tubing
(PE-50) was inserted into the left carotid artery in order to record the
systemic blood pressure. A 23 gauge needle connected to PE-50
tubing filled with heparinized saline (5 U heparin/ml) was inserted
into the left penile corpus cavernosum for ICP measurement. The use
of a three-way stopcock also provided a pathway to inject a solution of
NORD-1 (100 μL of 100 μM) in heparinized saline (5 U mL−1, 1%
DMSO) into the penis. Electrostimulation of the cavernous nerve was
performed at more than 1 min intervals with bipolar hook electrodes
using an electronic stimulator (Nihon Kohden) and isolator SS-202J
(Nihon Kohden) before and after the injection of NORD-1. The
stimulus conditions were as follows: voltage, 5.0 V; frequency, 2 or 8
Hz; pulse width, 5 ms; and duration, 1 min. Systemic arterial pressure
and ICP were recorded and analyzed with Chart 8 software
(ADInstruments Pty Ltd.).
Preparation of 3. To a solution of 1 (2.67 mL, 17.8 mmol, 3.3
equiv) in anhydrous THF (30 mL) was added dropwise sec-BuLi
(1.05 M in hexane, 16.0 mL, 16.8 mmol, 3.1 equiv) under an argon
balloon at −78 °C. The mixture was stirred for an hour, and then, a
solution of 2 (1.75 g, 5.40 mmol) in anhydrous THF (60 mL) was
added dropwise. The reaction mixture was warmed to room
temperature (RT), stirred for 15 min, quenched with 2 N HCl (30
mL), and further stirred for further 15 min to remove acetal
protection. The removal of acetal protection was confirmed by ESI-
MS, and then, water was added to the reaction mixture and the whole
was extracted with CH2Cl2. The combined organic layer was dried
over Na2SO4, filtered, and evaporated. The residue was purified by
silica gel flash column chromatography (CH2Cl2/MeOH = 15/1 to
10/1) to obtain a mixture of the desired aldehyde and methyl
hemiacetal. To hydrolyze the methyl hemiacetal, the mixture was
dissolved in acetonitrile (40 mL) and 2 N HCl (40 mL) was added to
it. The mixture was stirred at RT for 5 min, and then, H2O was added
and the whole was extracted with CH2Cl2. The organic layer was
dried over Na2SO4. Filtration, and concentration in vacuo gave 3 (1.55
g, 3.74 mmol, 70%) as a dark blue solid. 1H NMR (CDCl3, 500 MHz,
δ; ppm): 9.82(1H, s), 8.12 (1H, d, J = 7.6 Hz), 7.81 (1H, dd, J = 7.2
Hz, 7.2 Hz), 7.75 (1H, dd, J = 7.5 Hz, 7.5 Hz), 7.33 (1H, d, J = 7.2
Hz), 7.25 (2H, s), 6.92 (2H, d, J = 9.3 Hz), 6.64 (2H, d, J = 9.2 Hz),
3.42 (12H, s), 0.69 (3H, s), 0.65 (3H, s). 13C NMR (CDCl3, 125
MHz, δ; ppm): 190.29, 166.82, 154.10, 148.13, 141.14, 140.96,
134.88, 133.95, 130.59, 130.56, 129.76, 128.22, 121.01, 114.26, 41.38,
−0.38, −1.07. HRMS (ESI+) calcd, 413.20491; found, 413.20584
(M+, + 2.23 ppm).
Preparation of 5. To a solution of 3 (500 mg, 1.21 mmol) in
CH2Cl2 (25 mL) were added acetic acid (2.5 mL) and 4 (297 mg,
1.33 mmol, 1.1 equiv). The mixture was stirred at RT for one hour,
and then, sodium triacetoxyborohydride (769 mg, 3.63 mmol, 3.0
equiv) was added and stirring was continued for a further 5 min. The
reaction mixture was quenched with 0.1 N HCl (50 mL) and
extracted with CH2Cl2. The organic layer was dried over Na2SO4,
filtered, and evaporated. The crude product was used for the next
reaction without further purification. The residue was dissolved in
acetic acid (14 mL) and stirred on an ice−water bath. To the reaction
mixture was added a solution of NaNO2 (100 mg, 1.45 mmol, 1.2
equiv) in H2O (14 mL). Stirring was continued for a further 5 min,
and then, 0.1 N HCl (50 mL) was added. The whole was extracted
with CH2Cl2. The organic layer was dried over Na2SO4. Filtration,
concentration in vacuo, and purification by silica gel flash column
chromatography (CH2Cl2/MeOH = 15/1 to 10/1) gave 5 (611 mg,
0.941 mmol, 78%) as a dark blue solid. 1H NMR (CDCl3, 500 MHz,
δ; ppm): 7.43 (1H, d, J = 5.0 Hz), 7.42 (1H, d, J = 4.0 Hz), 7.26 (2H,
d, J = 2.9 Hz), 7.18 (2H, d, J = 8.7 Hz), 7.14 (1H, dd, J = 4.5 Hz, 4.5
Hz), 7.09 (1H, dd, J = 4.5 Hz, 4.5 Hz), 7.05 (2H, d, J = 9.7 Hz), 6.80
(2H, d, J = 8.7 Hz), 6.67 (2H, dd, J = 2.9 Hz, 9.7 Hz), 4.84 (2H, s),
3.44 (12H, s), 0.98 (9H, s), 0.65 (3H, s), 0.65 (3H, s), 0.20 (6H, s).
13C NMR (CDCl3, 125 MHz, δ; ppm): 166.76, 155.50, 154.31,
148.50, 141.30, 137.51, 135.28, 132.37, 129.78, 129.47, 127.67,
127.43, 127.03, 125.95, 121.33, 120.92, 114.28, 46.14, 41.34, 40.06,
25.71, 18.29, −0.57, −4.29. HRMS (ESI+) calcd, 649.33940; found,
649.34028 (M+, +1.35 ppm).
Preparation of NORD-1. To a solution of 5 (611 mg, 0.941
mmol) in MeCN (14 mL) was added 0.1 M NaF/HF buffer (14 mL).
The mixture was stirred at RT for an hour, and water and brine were
added to it. The whole was extracted with CH2Cl2. The organic layer
was dried over Na2SO4. Filtration, evaporation of the filtrate in vacuo,
purification of the residue by silica gel flash column chromatography
(CH2Cl2/MeOH = 10/1) and MPLC (CH2Cl2/MeOH = 94/6 to
90/10 to 70/30) gave NORD-1 (312 mg, 0.510 mmol, 54%) as a dark
blue solid. 1H NMR (CDCl3, 500 MHz, δ; ppm): 7.47 (1H, t, J = 8.0
F
https://dx.doi.org/10.1021/acschembio.0c00601
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
pubs.acs.org/acschemicalbiology
Articles
ASSOCIATED CONTENT
ACKNOWLEDGMENTS
■
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschembio.0c00601.
This work was supported in part by JSPS KAKENHI grant
numbers 16H05103, 19H03354 (H.N.), 18K14873, 20K05752
(N.I.), 18K16742, and 20K09583 (Y.H.) as well as by a Grant-
in-Aid for Scientifc Research on Innovative Areas from MEXT
(26111012, H.N.), Toyoaki Scholarship Foundation (Y.H.),
and The Tokyo Biochemical Research Foundation, Takeda
Science Foundation, and Casio Science Promotion Foundation
(N.I.).
Discussions of cyclic voltammetry, Griess assay,
measurements of NO release quantum yield, LC−MS
analysis, and erectile functional measurements, figures of
NMR charts, cyclic voltammogram, ESR spectra,
standard curve for Griess assay, LC−MS spectra,
compounds detected in protonated form, DIC and
fluorescence images, experimental setup, and changes in
tension, scheme of plausible mechanism of NORD-1
photodecomposition, and tables of measured and
calculated values used to determine ΔGPeT and measure-
ments and calculation results of Reinecke’s salt chemical
actinometer and released NO from NORD-1 (PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
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Naoya Ieda − Graduate School of Pharmaceutical Sciences,
Nagoya City University, Nagoya, Aichi 467-8603, Japan;
Email: ieda@phar.nagoya-cu.ac.jp
Hidehiko Nakagawa − Graduate School of Pharmaceutical
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Authors
Yuji Hotta − Graduate School of Pharmaceutical Sciences,
Nagoya City University, Nagoya, Aichi 467-8603, Japan
Ayaka Yamauchi − Graduate School of Pharmaceutical
Sciences, Nagoya City University, Nagoya, Aichi 467-8603,
Japan
Atsushi Nishikawa − Graduate School of Pharmaceutical
Sciences, Nagoya City University, Nagoya, Aichi 467-8603,
Japan
Takahiro Sasamori − Graduate School of Natural Sciences,
Nagoya City University, Nagoya, Aichi 467-8501, Japan;
orcid.org/0000-0001-5410-8488
Daisuke Saitoh − Graduate School of Pharmaceutical Sciences,
Nagoya City University, Nagoya, Aichi 467-8603, Japan
Mitsuyasu Kawaguchi − Graduate School of Pharmaceutical
Sciences, Nagoya City University, Nagoya, Aichi 467-8603,
Japan; orcid.org/0000-0002-1919-1391
Kazunori Kimura − Graduate School of Pharmaceutical
Sciences, Nagoya City University, Nagoya, Aichi 467-8603,
Japan
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.0c00601
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Author Contributions
Research design and hypotheses were proposed by N.I., Y.H.,
and H.N. Chemical synthesis and cellular experiments were
performed by N.I. and A.Y. Physicochemical experiments were
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Notes
The authors declare no competing financial interest.
G
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